Ere: . A , ' ve , > ; a 7 ' , : ‘ ; 4 ‘ : ‘ a we! ‘ ; yea : ad sg f i ; ‘ ‘ : Wik Bb eEcagnie : ; Tai tnt : FALE 7 it ‘ E, Pepe mata ¢ 3 j : . y" > Pep eer ee 5a ie 4 Pata 6 wate ee eat ah ee 4: EUR WN) te * tyes Bae bass ie We eas Eve ar pide le ¥ PEMA aA " ag ati 4 ree Se uns Seah an sae i aubsyph at a7 ’ wi nt yond a en at FOR THE PEOPLE FOR EDVCATION FOR SCIENCE LIBRARY OF THE AMERICAN MUSEUM OF NATURAL HISTORY b Tse nye, % i QUARTERLY JOURNAL OF ref er briny £3 - MICROSCOPICAL SCIENCE. EDITED BY Sm RAY LANKESTER, K.C.B., M.A., D.Sc., LL.D., F.R.S., HONOKAKY FELLOW OF EXETER COLLEGE, OXFORD; CORKKSPONDENT OF THE INSTITUTE OF ¥RANCE, AND OF THK IMPERIAL ACADEMY OF SCIKNCES OF ST. 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WITH THE CO-OPERATION OF ADAM SEDGWICK, M.A., F.RS., PROFESSOR OF ZOOLOGY AND COMPARATIVE ANATOMY IN THE UNIVERSITY OF CAMBRIDGE SYDNEY “dl 2 HICKSON, (M.As ies: BEYKR PRKOFESSOK OF ZOOLOGY IN THE UNIVERSITY OF MANCHESTER, AND K. A. MINCHIN, M.A., PROFESSOR OF PROTOZOOLOGY IN THE UNIVERSITY OF LONDON, VOLUME 53.—New SeERIEs. GMith Aithoqraphic Plates and Cext-Fiqueres ORD ON : J & A. CHURCHILL, 7, GREAT MARLBOROUGH STREET. 1909. COREEE NITES. CONTENTS OF No. 209, N.S., NOVEMBER, 1908. MEMOIRS: Early Ontogenetic Phenomena in Mammals and their Bearing on our Interpretation of the Phylogeny of the Vertebrates. By A. A.W. Husrecut, (With 160 Text-figures) 3 CONTENTS OF No. 210, N.S., JANUARY, 1909. MEMOIRS : Some Observations on the Infusoria Parasitic in Cephalopoda. By C. Cuirrorp Dosett, Fellow of Trinity College, Cambridge ; Balfour Student in the University. (With Plate 1) . Researches on the Intestinal Protozoa of Frogs and Toads. By C. CuirrorD DosExt, Fellow of Trinity College, Cambridge; Balfour Student in the University. (With Plates 2—5) Chromidia and the Binuclearity Hypotheses: a Review and a Criticism. By C. Cuirrorp Dosen, Fellow of Trinity College, Cambridge; Balfour Student in the University ; The Eyes of Chrysochloris hottentota and C. peice! By GEORGIANA Sweet, D.Sc., Melbourne yes eae tia Plate 6) On the Occurrence of Naidleas Dinorphients in a Halters dium parasitic in the Chaffinch, and the probable connection of this parasite with a Trypanosome. By H. M. Woopcocx, D.Se.Lond., Assistant to the University Professor of Protozoology Some Observations on Acinetaria. By C. H. Marvin, B.A., Demonstrator in Zoology in the University of Glasgow. I. The “Tinctin-kérper” of Acinetaria and the Conjugation of Acineta papillifera. II. The Life Cycle of Tachyblaston ephelotensis (gen. et spec. nov.), with a possible identifica- tion of Acinetopsis rara, Robin. (With Plates VII and VIII) PAGE 183 201 279 309 fe CONTENTS. CONTENTS OF No. 211, N.S., MAY, 1909. MEMOIRS: Studies on the Structure and Classification of the Digenetic Trematodes. By Wituiam Nicott, M.A., D.Se., of the Univer- sity of St. Andrews. (With Plates 9, 10) : é On the Anaspidacea, Living and Fossil. By Grorrrey SmirH, Fellow of New College, Oxford. (With Plates 11 and 12 and 62 Text-figures) On the so-called “Sexual” “Method of Snore: focuatten in tthe Disporic Bacteria. By C. Cuirrorp Dose, Fellow of Trinity College, Cambridge; Balfour Student in the University. (With Plate 13 and 3 Text-figures) : Studies on Polychet Larve. By F. H. Eyer ae MSe., Haaias Demonstrator of Zoology in the Victoria University of Man- chester. (With Plate 14 and 3 Text-figures) Some Observations on Acinetaria. By C. H. Mien, BA. Demonstrator at Glasgow University. (With Plate 15 and 6 Text-figures) CONTENTS OF No. 212, N.S., JULY, 1909. MEMOIRS: Studies on Ceylon Hematozoa. No. 1.—The Life Cycle of Try- panosoma vittate. By Murret Ropertson, M.A., Carnegie Fellow in the University of Glasgow. (With Plates 16 and 17 and 4 Text-figures) : : The Entry of Zooxanthelle into the — of Mfillepora, and some Particulars concerning the Meduse. By JosppH Manaan, M.A., A.R.C.Se.1.., Honorary Research Fellow of the University of Manchester. (With Plate 18) : Physiological Degeneration and Death in Bhs moeba ranarum. By C. Currrorp Dose tt, Fellow of Trinity College, Cambridge ; Balfour Student in the University. (With 5 Text-figures) Observations on the Ameeba in the Intestines of Persons Suffering from Goitre in Gilgit. By Ronerr McCarrison, M.D., M.R.C.P. Lond., Captain Indian Medical Service. (With 24 Text- figures) Peripatus ceramensis, n. s.p. By ink Mai and i C. Kur-— sHaw (of Ceram). (With Plate 19) : On the Eggs and Instars of Scutigerella sp. By F. Mum ana J.C. Kersaaw (of Ceram) The Development of the Parasite of Grassi Sore in Garnaen By H. Row, M.D.Lond., D.Se.Lond. (With Plate 20) : The Structure of Trypanosoma lewisi in Relation to Micro- scopical Technique. By E. A. Mincutn, Professor of Protozoo- logy in the University of London. (With Plates 21—23) Tirte, INpEx, AND ConvTENTS. PAGE 391 4.89 629 697 EARLY ONTOGENETIC PHENOMENA IN MAMMALS. t Early Ontogenetic Phenomena in Mammals and their Bearing on our Interpretation of the Phylogeny of the Vertebrates. By A. A. W. Hubrecht. With 160 ‘lext-figures. PREFACE. In the present paper I have attempted to bring together the results of new investigations and recent reflexions with such as had already been published on earlier occasions, but which, having appeared in very different periodicals and publi- cations (789, 790, 794, 795, 796, 799, 702, ’05, 07), could not be easily brought into the necessary connection with each other by the reader. I have to thank my friend Sir Ray Lankester for giving me the occasion to present this scattered material in a more concise form, and for his willingness to admit a profuse quantity of process figures into a JournaL which, under his direction, has become justly famous for its excellent litho- graphic plates. TABLE OF CONTENTS. PAGE PREFACE : : ; , i iL Cuap. I, THe Earutest CEeLi-LAYERs. Introductory ; ‘ : : 3 VOL 53, PART 1.—NEW SERIES. 1 A. A. W. HUBRECHT. bo Cuar. Monodelphian and Didelphian Mammals . 1. The Mammalian Morula 2. The Origin of the Entoderm : 3. Developmental Phases of the Embryonic Didente Shield 4. The Mammalian Gastrula : 5. Theoretical Speculations about the Origin of the Tronhes blast B. Ornithodelphian Mammals Se Sree C. Ichthyopsids ‘ : Il. FurrHer DEVELOPMENT OF THE TWO GERM-LAYERS OF THE VER- TEBRATES UP TO THE APPEARANCE OF THE SOMITES. I. Mammalia, Mono- and Di-delphia : 1. Developmental Processes in the Entoderm . a. The Protochordal Plate 4. The Annular Zone of Proliferation 2. Developmental Processes in the Ectoderm . a. The Protochordal Wedge . The Ventral Mesoblast 3: a Relations between the Centres of Proliferation II. Amphibia III. Sauropsida and Grnitiedenetia IV. Fishes V. Summary of Cliapters I and Il ’ III. DieLorrornoptast (= SeRous = SuBzonaAL AP antane Cuo- RION, AMNION, UMBILICAL VESICLE, AND ALLANTOIS IN ONTOGENY AND PHYLOGENY 1. Chorion and Amnion 2. The Umbilical Vesicle 3. The Allantois : IV. THe part PLAYED BY THE TROPHOBLAST IN THE NuTRITION AND ATTACHMENT OF THE EMBRYO 1. Didelphia Non-placentalia 2. Monodelphia a. Hedge-hog 6. Primates e. Rodents and Carnivores d. Other Insectivores, Ungulates, Bdcniates: atl ietivtes 3. Didelphia placentalia V. Dirrerent Aspects and Devaits or PLACcENTATION. 1. Embryonic (Trophoblastic) and Maternal (Trophospongian) Preparatory Processes a. Insectivora 30 33 100 102 105 107 112 115 118 121 EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 3 Cap. PAGE 6. Chiroptera, Carnivora, Rodentia . : x. 125 c. Primates : ; ‘ am 27 2. The Classificatory Value of Placentation : 72129 3. The Phylogeny of the Placenta : : 5 Lal 4, Summary of Chapters LV and V , : . 148 VI. ReEFLEXIONS ON THE PHYLOGENY AND THE SYSTEMATIC ARRANGE- MENT OF VERTEBRATES é : : . +149 CHapter I. THe EHaruiest CELL-LAYERS. INTRODUCTORY. The phenomenon of fecundation of the egg imaugurates the well-known series of cell-divisions which give rise in Amphioxus to a grouping of the first cleavage-cells into a hollow mulberry shape, whereas in cartilaginous fishes, in reptiles, and in birds the cleavage-cells are disposed in disc- shape at one point of the yolk, which latter, though origin- ally part of the egg, will soon take the aspect of an appen- dage to the embryo. Again, in Amphibia and in certain more archaic fishes the yolk is much less considerably deve- loped and the whole egg thus segmented in toto, whereas in the Teleosts there is an abundance of food-yolk, but a dispo- sition of the parts somewhat different from what we find in cartilaginous fishes and in Sauropsids. In Mammals again the whole of the egg-substance is seg- mented (holoblastic cleavage as against the meroblastic cleavage of the cartilaginous fishes and the Sauropsida), but the further development more and more resembles that of the reptiles in which a very considerable yolk is present, a fact that has given rise to the erroneous conclusion that the mammalian blastocyst was derived from the Sauropsidan by a process consisting in the gradual disappearance of the yolk, with retention of the other developmental characters. We will find occasion later on to discuss the value of this phylogenetic speculation. 4 A. A. W. HUBRECHT. We will in this chapter have to consider the earliest processes by which the cell-material consequent upon the cleavage of the egg comes to be arranged in the fundamental cell-layers out of which the different organs of the adult animal will gradually take their origin. And we must in the first place call attention to numerous and important investigations that have taken place, more particularly concerning invertebrate animals, in which the cleavage-cells were followed as far as possible up to their final destination with respect to organogenesis (Wilson, 792, 97; Conklin, 797; Casteel, 704), ; These researches concerning the “ cell-lineage,” as it has been called, have been carried on by the aid of such worms and molluses that had eggs as transparent as possible, and, notwithstanding the evident high importance of the results obtained, there is for the present little chance of success for similar attempts with the opaque and yolk-laden or deeply hidden eges of the Vertebrates. I mention this in order to point out that several questions in dispute might in this way be settled, and that more especially the mammalian egg with its holoblastic cleavage would here offer a most desirable object of study, ‘There has been a tendency to suppose that the two primary celi-layers which are encountered in all vertebrate and invertebrate animals, the ectoderm and the endoderm, already become separated from each other when the two first cleavage cells arise. Others have concluded that in this separation of the egg- cell into the two first cleavage cells the embryonic material was separated into the mother-cells of the right and the left half of the body or into the anterior and posterior half, as chance would have it (Roux). Experiments have even been carried out to prove this. At the present moment we are not in a position to say whether there is any general rule in this respect applicable to all vertebrates, and yet there seems to be hardly any doubt that both in Amphioxus and in man— the two opposite extremes in the phylum of the Chordata— the two first cleavage cells, if separated from each other, EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 5) may under favourable conditions each of them develop into a perfect, full-grown individual. However this may be, the formation of the primitive cell- layers out of the cell-material derived from the segmenting egg-cells must now, in the first place, be considered, and, inverting the order generally followed, we will begin by con- sidering the phenomena as they present themselves in the A. MonopgELepHIAN AND DiIpELPHIAN MAMMALS. As yet only a restricted number have been investigated with regard to the process of cleavage and the earliest formation of the layers, it being no easy matter to procure the material. As such I mention— (1) Certain species of Primates,! including both monkeys (Macacus, Cercopithecus, a. 0.) by Selenka (99, ’00) and by Keibel (04), and Tarsius by myself (’02). (2) Lemurs (Nycticebus) by myself (’07). (5) Carnivores (dog and cat) by Bonnet (’97) and Duval (94, 795). (4) Chiroptera (diverse species of Vespertilii) by E. van Beneden and Ch. Julin (790) and by Duval (99). (5) Insectivora (Talpa, Erinaceus, Gymnura, Sorex, T'u- paja) by Heape (83), Keibel (88), and myself (’89, 790, 795, US) (6) Rodentia (Lepus, Mus, Arvicola, Cavia, Sciurus, a. 0.) by Hensen (’76), E. van Beneden (’80), Selenka (83, 784), Fraser (’82), Masius (89), Fleischmann (791), Keibel (’80), Duval (’92), Robinson (’92), a.o. (7) Ungulata (Ovis, Sus, Cervus) by Bonnet (’82), Keibel (93), Assheton (’98), Weysse (794), a. o. (8) Dermaptera (Galeopithecus) by myself. (9) Edentata (Manis) by myself. (10) Didelphia (Opossum, a.o.) by Selenka (87). 1 Of the human subject no such early stages have as yet been brought to light, the earliest being those of Peters, von Heeukelom, Bryce and ‘Teacher. A. A. W. HUBRECHT. [or Fresh eggs have served for the observation of the cleavage- processes in the rabbit to van Beneden and in the bat to van Beneden and Julin. Most of the other authors have made use of preserved specimens and of sections. A certain number of the figures given by different observers are here reproduced (Figs. 1—36). 1. The Mammalian Morula. The compact mulberry stage (different in its compactness from the hollow mulberry of the holoblastic egg of Am- phioxus alluded to above) contains about 36—72 cells. In the case of Tupaja and—judging from Bonnet’s figure—of the dog the central cell or cells show a different reaction against staining reagents than the peripheral (Figs. 1, 2, 3). We will have occasion to discuss this phenomenon later on. Very soon fluid begins to accumulate between some of the constituent cells of this mulberry stage in mammalian de- velopment, and the solid mulberry then becomes converted into a hollow sphere, against the wall of which an accumu- lation of cells is visible which was already noticed by Bischoff (42, 45) and other early authors. Twenty-five years ago, when van Beneden published his remarkable researches above alluded to on the early develop- ment of the rabbit, the interpretation of these early pheno- mena was far from being satisfactory or uniform. The so- called metagastrula stage of mammals, first described by van Beneden (’80), has since been abandoned by that author [though taken up again by Duval (’99, p.64)1._ We may, how- ever, say that of late years a very general consensus of opinion has come to be established. In all the orders above-mentioned an early stage of the blastocyst has been observed corre- sponding to the phase just described in which an accumula- tion of cleavage-cells adheres at one point against an outer epithelial layer.' ‘ KE. van Beneden has ascribed the origin of the free space between the HUBRECHT. Fig. 1. Cleavage stage of the dog (after Bonnet, ‘97). The mother cells ck of the embryonic knob, centrally situated, have stained more deeply than the trophoblast cells (¢”). — Fig. 2 and 3. Sections through two different early cleavage stages of Tupaja javanica. In this case the trophoblast cells, ¢r are more deeply stained than the mother-cells of the embryonic knob e& — Fig. 4 and 5. Sections through early stages of the opossum (after Selenka, 87). In Fig. 4 there are thirteen trophoblast cells ¢ and one mother cell of the embryonic knob e&, in Fig. 5 the latter has given rise to a mass of cells which begins to project on the surface /e#) and in which the differentiation of entoderm cells has just commenced. — Tig. 6, 7, 8. Three sections of different developmental stages of the bat (after van Beneden, ‘99). In Fig. 6 the differentiation between tro- phoblast cells ¢ and embryonic knob is again expressed in the staining; in Fig. 7 the embryonic knob (Z) is not yet separated into ectoderm /e%) and entoderm (en) as it is in Fig. 8. EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 7 This outer layer has been termed by me the trophoblast, that inner cell-mass: the embryonic knob or “ Embry- onalknoten ” (88, p. 511; ’89, p. 298). E. van Beneden, while recognising that the trophoblast is a separate layer (’99), does not as yet apply that name but calls it somewhat more circumlocutionally the ‘“‘ couche enveloppante.”’ The degree of independence existing between trophoblast and embryonic knob is subject to considerable variation. As the two only become segregated when the change of the morula stage into a vesicle comes about, there is generally at the outset no such very sharp distinction, and even in later stages this distinction is sharper in certain genera of mammals (Tupaja, Figs. 21, 29; Galeopithecus, Fig. 18; Cervus, Figs. 13, 14) than in others (Hrinaceus, Figs. 33—36; Tarsius, Fig. 19; Cavia, Fig. 24. Still there is reason to believe that in some it may be traced up to the very early stages of cleavage as are indicated by Figs. 1—3. The embryonic knob would then be represented by one or a few central cells, the trophoblast by the surrounding cleavage cells (as was already noticed above). When the trophoblast is being distended into a vesicle the proliferation of the mother-cells of the embryonic knob is very much slower; the total number of cells of which the knob is built up rarely exceeding 24—30. For the Opossum we have the data furnished by Selenka (87), to which, however, I would put another interpretation. The central cell of Fig. 4, looked upon by him, without further proof, as a hypoblast cell, is undoubtedly the mother- cell of the embryonic knob as a comparison with Fig. 5 makes all the more evident. It is, of course, important to find this agreement between didelphic and monodelphic mammals. epithelial outer layer and the inner mass to the extension of intracellular vacuoles (99). His interpretation has found no support in the results obtained by Keibel and myself, nor in those of Selenka for the Opossum. 5 A. A. W. HUBRECHT. 2. The Origin of the Entoderm. Soon a most important process is inaugurated and from the inner cell-mass arises by delamination a separate lower layer which we designate as the entoderm of the embryo. These entoderm cells wander in radial direction along the inner surface of the trophoblast, which, in many cases, is thus soon transformed into a didermic structure. Sometimes, as for instance in Tarsius (Hubrecht, ’02) the more important part of the delaminating entoderm (viz. that which remains situated below the rest of the embryonic-knob- cells) is present as a distinct cell-layer before this migra- tion of entoderm cells towards the inner surface of the trophoblast commences (Figs. 8, 19); in other cases (Sorex, Lepus, a. 0.) the entoderm cells migrate as soon as formed ; whereas in Tupaja it is only after the entodermic vesicle has become nearly completed that the part of it which will remain in contact with the embryonic ectoderm is sepa- rated off from the latter by delamination (Fig. 29). In certain mammals (T'arsius, monkeys, man) the ento- derm cells never clothe the whole of the inner surface of the trophoblast, the entodermic vesicle remaining of smaller size than the trophoblastic sphere (Figs. 39, 40, 44—46, 62—65). To a certain extent this is explained by the fact that another vesicle (of which mention will be made later on) develops, at an uncommonly early period, fills up part of the vesicle formed by the trophoblast and prevents the entoderm cells from reaching the entire outer surface.} When the entoderm has separated off by delamination from ' It would seem that in Erinaceus a similar state of thing occurs tem po- rarily, it having been observed by me (’89, Figs. 25, 26) that a closed entodermic vesicle, far smaller than the trophoblastic sphere which encloses it, is here noticed in very early stages (Fig. 34). Shortly after this (Figs. 35—38) the hedge-hog’s blastocyst, is, however, a spherical trophoblast, against which the endoderm is everywhere adherent. The investigation of numerous early stages of the hedge-hog is, however, yet necessary to settle this point. HUBRECHT. (PULSE. Fig. Sa. Section through an early bat’s blastocyst (cf. Fig. 6 to 8); ¢# trophoblast; ez entoderm. The ectodermic shield has not yet emerged out of its trophoblastic covering (after van Beneden, 99). — Fig. 9. Seetion through early stage of the bat (after Duval) e& embryonic knob. ¢ trophoblast. — Fig. 10. Section through early stage of the shrew (after Hubrecht, ‘90). e& embryonic knob, ¢ trophoblast. — Fig. 11 and 12. Sections through early stages of the mouse, before and after fixation of the blastocyst to the uterine epithelium 2. — Fig. 13 and 14. Two sections through the embryonic knob of Cervus (after Keibel, 99) before and after the development of the entoderm by delamination. The trophoblast (¢~) is quite distinet from the embryonic ectoderm ec; in Fig. 13 ectoderm and entoderm are yet united in the embryonic knob. EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 9 the embryonic knob, the remaining cells of the latter form > which is thus situated between the entoderm and the trophoblast, and could for that reason easily deceive van Beneden (’80) in regarding it as a third, mesodermic layer (Figs. 8, 11, 12, 15, 17, 18, 21—23). the “embryonic ectoderm,’ 3. Developmental phases of the Didermic Embry- onic Shield. The portion of the mammalian blastocyst where the em- bryonic ectoderm and its subjacent entodermal layer are situ- ated may, already at this early stage, be conveniently termed the embryonic shield. This shield is sometimes slightly convex with the ectoderm on the convex side (rabbit, Fig. 23), sometimes it is bent the other way (Sus, Fig. 17; Tupaja, Figs. 29—31; Tarsius, Figs. 20, 45), sometimes first one way (Figs. 20, 37, 53), and later (Figs. 50, 38) the other (Tarsius, hedgehog). Sometimes it is (Fig. 23) quite flat (Lepus, Sorex, a. 0.). A very instructive and in my opinion very archaic case among those above-mentioned is that in which the embryonic shield remains separated from the overlaying trophoblast by a space which arises simultaneously with the growing blasto- cyst. This space is from the outset a lenticular or crescentic cavity. Its appearance in Erinaceus is elucidated by the accompanying diagrams (Figs. 36—38). It represents the most typical instance of the manner in which the earliest amnion may have arisen as a protective water-cushion between the trophoblast and the embryonic shield, and we shall later on see that the space within the hedgehog’s amnion is actually a later development of this early cavity. In the bat slight modifications of this simple arrangement occur, which seem to lead on to arrangements as we find them in Tarsius and in many Ungulates and Rodents, whereas on the other hand Pteropus (Figs. 22, 72), Galeopithecus (Figs. 41, 42), Cavia (Figs. 24, 25), monkeys and man (Figs. 59, 40) have developed 10 A. A. W. HUBRECHT. along another path, along which the amniotic cavity has from the first remained a closed vesicle. A very important case is that of Tupaja in which the embryonic shield is originally quite bent upon itself (Fig. 30), convexity inwards, and gradually expands (under rupture and dehiscence of the trophoblast) into a flat surface with no trophoblastic covering above it (Fig. 52) by successive stages as they are reproduced in the accompanying diagrams. This arrangement possesses suggestive points of comparison with what has been called by Selenka (’00 a, p. 201) the “ Enty- pie” of the embryonic shield, such as it exists in many rodents. All these cases are variations upon a_ similar theme as that of Tupaja, and not necessarily (as Selenka would have it) a consequence of the blastocyst undergoing its development in a cavity of exiguous dimensions, to the walls of which it had early adhered. ‘l'upaja at once does away with this argument (Hubr., 799 B, p. 173), because here the blastocyst, while free from any attachment to the uterine walls, has yet the appearance of Figs. 830 and 31. The causes of the folded condition of the embryonic shield can hardly be so simply mechanical as Selenka supposed. They remain obscure for the present and will come anew under considera- tion when the origin of the amnion will be discussed. The point to which the facts here brought forward have led us is the recognition that during the development of the mammalian blastocyst the trophoblast, which originally encloses the embryonic knob, behaves very differently in rarious mammals at the period that out of this knob arises the embryonic shield with its didermic arrangement of the cells out of which the embryo is going to be built up. In the hedgehog (Figs. 37, 38), in Gymnura, in Pteropus (Figs. 67, 68), and in the other bats hitherto examined (Fig. 8a), in Galeopithecus (Figs. 41, 42), in many rodents (Arvicola, Mus, Cavia, Figs. 24—28), in monkeys, and (most probably) in man the trophoblast remains an entirely closed vesicle, inside of which the ontogenetic development of the em- bryonie knob will follow its course. In other genera of HUBRECHT. PUESC: (> be — Bh J ) RS = % qe’ / Fig. 15. Section through a similar stage of Ammospermophilus. After a not yet published drawing of Prof. G. Lee of Minneapolis. Trophoblast ¢ continued oyer embryonic ectoderm cc, ex entoderm. — Fig. 16. The same for the sheep (after Assheton, “98). -— Fig. 17. The same for the pig (after Weysse, ‘O4). The ectoderm has not yet opened out on the surface of the blastocyst (cf. Fig. 29-32). — Fig. 18. The same for Galeopithecus. — Fig. 19 and 20. The same for Tarsius. In Fig. 19 the entoderm ez is in the very earliest phase of delamination (after Hubrecht, 02). In Fig. 20 there are yet remnants of the trophoblastic covering of the ectodermie shield. — Fig. 21. The same for Tupaja (after Hubrecht, 95). — Fig. 22. The same for Pteropus. In the ectodermal knob (ec) a cayity will soon appear wich becomes the amnion cavity (after Selenka and Gihre, 92). vz umbilical vesicle. EARLY ONTOGENETIC PHENOMENA IN MAMMALS. Et mammals that part of the embryonic knob which is going to be the ectoderm of the embryonic shield rises to the surface, interpolates itself between the trophoblast cells, which then form no longer a closed sphere, but one that is discontinuous by the fact that at one pole this ectodermic shield has replaced what were originally tropho- blast cells. This displacement may come about as it does in Tupaja (Figs. 29—32) where the unfolding of the embryonic shield bursts open the trophoblastic covering above the shield, thus increasing the surface of the vesicle by an area which is not trophoblast, but embryonic ecto- derm. Or it may happen that a similar but less distinct process of dehiscence interpolates embryonic ectoderm in the trophoblastic vesicle in the way it comes about in the Opossum (Fig. 5), Tarsius (Fig. 20), Cervus (Fig. 13), Sus (Fig. 17), Ovis (Fig. 16). Or finally the trophoblast may continue to cover the embryonic ectoderm as in the case first named, but without the development of any cavity between it and the embryonic shield (Fig. 15). In this latter case, of which the classical example is the rabbit, as it was so clearly figured by Kolliker (Fig. 23), the tro- phoblast cells covering the embryonic ectoderm flatten out considerably, and finally disappear. Another example of this is the shrew (Hubrecht, 790; Fig. 26). ‘These flat cells—superposed to the embryonic ectoderm—were for a long time designated as Rauber’s cells, Rauber having been the first to direct attention to them. It was, however, not observed by Rauber, as it was later so clearly noticed by Kolliker, that this layer is merely the continuation of the peripheral trophoblast cells, but it remained for a long time an accepted, though erroneous interpretation, that the embryonic ectoderm was uninterruptedly continued in the peripheral trophoblast, and that Rauber’s cells were an additional arrangement. This error was a natural conse- quence of a comparison, on a false basis, hereafter to be corrected, of the mammalian with the avian and reptilian blastocyst. The opinion of certain authors (Balfour, 12 A. A. W. HUBRECHIT. Heape) that some of the Rauber cells become incorporated into the embryonic shield has not been well established nor been confirmed of late. I incline to believe in their final dis- appearance, and wish to call attention to the transition case which we may notice, for example, in Tarsius (Hubrecht, 702 ; Figs. 49a, b, 50b), where trophoblast cells can remain for yet a considerable time attached to the embryonic ectoderm, but also finally disappear. In this case the trophoblast opens up according to the type prevalent in the second group described above, and the permanence of an isolated trophoblast cell on the embryonic ectoderm is a matter of chance. We may, in concluding this exposition of the varied rela- tions in which trophoblast and embryonic ectoderm stand to each other in mammals, insist upon the fact that—if we except the Ornithodelphia, which will be discussed hereafter, and are as yet barely known as far as their early ontogeny is concerned?! (Caldwell, ’87; Semon, ’94; Wilson and Hill, ’03) all the Didelphia and Monodelphia hitherto investigated show at a very early moment the didermic stage out of which the embryo will be built up enclosed in a cellular vesicle (the trophoblast), of which no particle ever enters into the embryonic organisation. 4. The Mammalian Gastrula. The didermic stage of the mammalian blastocyst just alluded to fully deserves the name of the “ gastrula” stage as I have elsewhere attempted to expound (702, p. 65—7%5 ; ’05, p. 408). We should bear in mind, as was noticed in the introduction, that comparative ontogeny has come to a dead- lock when attempting to fit in the mammals into the current interpretation of the early development of vertebrates. To ' Just lately the more extensive paper of Wilson and Hill (’07) has appeared, in which figures are given (pl. 2, figs. 4, 5), which allow us to accept quite similar arrangements for the Ornithodelphia (see text-figs. 66—70) Dokl 74 j / hy 7 Mee ta , ie i at y ‘- {ing 29 : A WW 1s. eo am shy" bs eae ‘a oe v4 / ord Ez y e a * 2c in uu { la at Re see Aiea uy HUBRECHT. Pl. D. tr tr en en Fig. 23. The same for the rabbit (after Kélliker “S2). The trophoblastic peripheral wall of the blastocyst continues into the Rauber’s cells zr above the ectoderm. — Fig. 24 and 25. The same for Cavia (after Selenka). The reduction of the trophoblast is yet far more considerable. @ amnion cavity. — lig. 26 and 27. Sections through two early stages of the mouse’s blastocyst (after Selenka, °S3). The trophoblast fev) is much further reduced in the second than in the first whereas that part of it (AZ) which will form the placenta has proliferated much more considerably. @ amnion cavity. EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 13 express it in O. Hertwig’s own words: “ Die gréssten Schwierigkeiten bereitet den Embryologen die Keimblattbil- dung bei den Situgethieren. . . wegen der von anderen Wirbelthieren stark abweichenden Befunde” (’06, p. 898). As soon as we separate the phenomena of notogenesis, such as they are found in all vertebrates — Amphioxus included—from the phenomenon of gastrulation, recognising that the former follow upon the latter and bring about the formation of the notochord and the mesoblastic somites, the difficulties are considerably simplified. Gastrulation is thus terminated in the mammalia when the didermic stage of the embryonic shield has come into exist- ence. We have seen that this takes place not in conse- quence of any process of invagination but by means of a most unmistakable delamination of the entoderm, out. of the embryonic knob. This delamination gastrula of the mammalia generally enters upon the later phases of ontogeny which will be described hereafter without the appearance of a distinct blastopore. Still to this there are a few notable exceptions that have gradually come to light, but have been mostly overlooked or misinterpreted in consequence of the erroneous views above alluded to. The most striking example is undoubtedly offered by the hedgehog, where the blastopore, a clearly visible perforation towards the hinder end of the embryonic shield, makes an evanescent appearance at one. particular stage of the individual development (Fig. 53). Along the lips of this opening the ectoderm and entoderm pass into each other, whereas these two layers, although genetically related, have up to this moment been separated and nowhere in confluence with each other. This latter fact is recognised by all observers. Iam inclined to believe that the formation of the blastopore in the hedgehog is not only very evanescent, but that it does not necessarily appear in all hedgehog-embryos, and that in exceptional cases the formation of notochord and somites may commence without the blastopore having become a visible opening. 14. A. A. W. HUBRECHT. In Tarsius on the other hand, where in an overwhelming number of cases the embryonic shield undergoes the changes consequent upon the first appearance of notochord and somites without any faint trace of a blastopore, one quite exceptional case came under observation (Fig. 52) in which — what was evidently an atavistic attempt in that direction was noticed; all the more important because it helps us to fix the spot in the didermic gastrula at which the blastopore n aturally oecurs. Similarly blastoporic openings, or attempts at such a perforation in these early stages, have been noticed in the rabbit by Keibel (’89; Figs. 46, 47), in the mole by Heape (Fig. 54), in the opossum by Selenka (Fig. 55), in the shrew by myself (Figs. 56 and 57). In the diagrams a few of these observations have been reproduced. The gastrula stage and the blastopore of the mammalia are thus limited to the early phases and the simple phenomena here described. The blastopore becomes closed in all the eases above noticed, and after that a series of processes are initiated in which it would be \utterly misleading further to use the word blastopore, Gastrulamund, Urmund, or Urmund- lippen. ‘These structures im the further development that have been thus termed ought to be termed differently if we wish to put an end to the confusion that obscures these points at the present moment. At the same time it should be noticed that one of the first features by which the formation of the notochord begins, viz., the formation on the embryonic shield of that median ectodermal proliferation, which I have called (’90) the protochordal wedge (Primitivknoten, Bonnet = Hensen’scher Knoten), takes place in the identical spot where the evanescent blastopore was or is situated (Fig. 52); and that from this point backwards a median region of proliferation extends which on O. Hertwig’s example has been called the homologue of the ‘“Urmund” and the ‘‘ Urmundlippen,” but which we ought to compare as I have elsewhere advocated (’02, 705) with an elongated stomodeal slit, which even in the hypo- thetical ancestral forms was no longer a blastopore, but HUBRECHT. Pl. FE. Fig. 28. Section through an early blastocyst of the mouse (after Selenka, “S3).. @ amnion, on the point of being constricted off. 2 Ectodermal shield. mes mesoblast. ex entoderm. /¢r tropho- blastic rudiment of placenta. — Fig. 29 to 32. Four successive stages in the early development of Tu- paja javanica. In Fig. 29 the trophoblast ¢- yet forms a solid closed sac round embryonic knob and entoderm, the latter only just beginning to split off from the embryonic knob as far as its embryonic portion is concerned. In Fig. 30 and 31 the bent embryonic ectoderm cc commences to free itself from its tro- phoblastic covering; in Fig. 32 it has quite flattened out, forming the embryonic shield on the top of the spherical blastocyst. EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 15 a dorsal mouthslit, a “ Riickenmund” (Fig. 160) of a vermactinial stage of development. The mammalian blastopore, rudimentary, rare, and eva- nescent as it is, still reminds us of the blastopore*of the invertebrates in this respect that in its immediate vicinity those cell proliferations commence which lead up to the formation of the so-called mesodermal structures. ~ 5. Theoretical Speculations about the Origin of the Trophoblast. The facts with which we have up to now become acquainted concerning the early development of didelphic and mono- delphic mammals (the so-called marsupials and the placental mammalia) fully justify the conclusion that the embryo already in its very earliest ontogenetic phases is provided with a larval envelope, an ‘ Embryonalhiille.” ‘To this layer of cells we have given the name of trophoblast. Later on we shall see that this layer, though it is at the outset only one cell thick, can undergo the most varied proliferations in very divergent spots, and that such proliferations are at the basis of the whole phenomenon of placentation. The fact that to these proliferations and their significance for the early nutrition of the embryo, attention was first directed (Hubrecht, ’88, ’89) before the more general significance of the layer as a larval envelope had yet been fully appreciated was the cause that the name of trophoblast has been given to it. We will return to this when the phenomena of placentation will be discussed. It cannot be denied that the consequences of considering the trophoblast as a larval envelope and of introducing this 1 It remains to be seen whether the name of “ trophoderm,” introduced by Sedgwick Minot (03) for that portion of the trophoblast which takes an active part in placentation, is a desirable innovation, or rather a synonymous encumbrance. But even in the former case Duval’s proposal of the name of * ecto-placenta ” has the priority. 16 A. A. W. HUBRECHT. eeneralisation into the developmental history of vertebrates may be far-reaching, Up to now foetal envelopes or membranes were only known in the ontogeny of reptiles, birds and mammals at somewhat later stages of their development. These membranes were respectively known aS amnion, chorion, serous membrane, subzonal membrane (and in case of the Sauropsids and certain mammals, even allantois) so that Milne Edwards’ subdivision of the vertebrates into Amniota, Allantoidea, as against the Anamnia, Anallantoidea, was based on the presence or absence of such membranes. Of the phylogenetic evolution of these foetal membranes no reasonable explanation has yet been offered, as is, for example, recognised, as far as the amnion is concerned, in an unbiassed handbook of human embryology, as is that of Sedgwick Minot (p. 344, lst edition). Now this obscure phylogeny would seem to become yet more compli- cated when we add to the already existing foetal membranes a new larval envelope, called trophoblast. The case is, how- ever, quite the contrary. This early envelope, that we have seen making its appearance soon after the very first phases of segmentation of the mammalian ovum, instead of adding new difficulties, helps to explain old ones. It throws new and unerring light on the first origin both of the amnion and the chorion (respectively : serous membrane) and may prove to be a valuable key that may lead to a reasonablejnterpretation of much that is as yet obscure and incomprehensible. Out of this very earliest larval envelope the others seem to have gradually evolved; they may be looked upon as further differ- entiations of it and we have now to look out for the first origin of the trophoblast itself and see if we can furnish a hypothesis worthy of further consideration, In that case the phylogeny of the other foetal membranes would & fortiori have been explained at the same time. Now, I believe that we have only to assume that the ances- tors of those Vertebrates in which a distinct trophoblast or the traces of it are found, were already possessed of a larval envelope in the antecedent stages of phylogeny, in order to : re. - , " Ld i ; 5 Dae & a@ “= & - i te bl . a Pra | Oya oe iy how » Sains’ A a: ai “ i, ty ’ ‘ ; hid “4 al a : : y ee oi * « Di , 7 . , aT - ‘ vy , Ds 5 Ti a »/* _ a a ca ay aan a os 7 _ ~y * way 0.4 { a 7, = PA ow ‘ i = ( * 3 e 4 4 : . @ a + . Pee Te ~ i oF okt aa ey as ha ™ J - . : ae Lard * r a ce : ie i ‘ { J \3 ’ ty é -~ Pl. F. HUBRECHT. 33 35 36 ie sigh ey, , attest UN BLE en __t7 RoR “ Ss. a) Ss ay tr CE en 34 6 8 R e468 \ = 9589 696 \ A a RS ey AE “hoe Ole @\ " Hi? CNY) \§| : bs oe (tak io 8o\ 8 le) ley S| 859% en LY 37 38 -CO a oP Fig. 33, 34 and 35. Sections of quite early stages of the hedgehog’s blastocyst. Zr trophoblast, ex entoderm, ¢c ectoderm yet firmly united with the trophoblast. — Fig. 36. A somewhat later stage in which considerable lacunae have originated in the proliferating trophoblast into which maternal blood penetrates. — Fig. 37. Section through a yet later stage in which the lacunae haye developed all round the blastocyst and in which the amnion cavity (a) has arisen as a split between trophoblast and embryonic ectoderm (cc). — Fig. 38. Yet later stage of the hedgehog’s blastocyst in which the development of the embryo is further advanced and the amnion-wall completed and externally clothed by mesoblast. 2 umbilical vesicle, co coelom. HUBRECHT. Pl. G. 39 41 ae kh 4 ,2hgh Bhd ad ge caagten Lavell i solhaertittettere tenets rdbtvedeestle Picchealstasdnntneed O52 O20 -“\ on Res e,0,09R, 285590 ‘ i) o® e @) @ Y BI8G RO OOO RR RET Fig. 39 and 40. Diagrammatic sections through two stages in the early blastocyst of man and the anthropomorphae, combined out of Selenka’s (99, 00) and Peters’ (99>) drawings. c connective stalk, som & spm somatic and splanchnic mesoblast. Amnion and trophoblast as in the hedgehog. — Fig. 41 and 42. A longitudinal and a transverse section of an early developmental stage of Galeo- pitheeus volans. In Fig. 41 the placenta is commencing to be formed on the upper surface of the blastocyst. Here too the amnion cavity (a) has arisen by dehiscence inside the embryonic knob. vm yentral mesoblast, connecting em- bryonic region with trophoblast. Fig. 42 belongs to a somewhat later stage in which a thickening in the entoderm (protochordal plate) has become visible. — Fig. 43. Transverse section through the ventral mesoblast of Galeopithecus. co coelom, ez entoderm, ¢r trophoblast. HARLY ONTOGENETIC PHENOMENA IN MAMMALS. 7 obtain such a working hypothesis. Both Sauropsids and Mammalia are, omnium consensu, phylogenetically derived from very early Protetrapods that lived about the Carboni- ferous period or even earlier, and which, in their turn, had aquatic and fish-like progenitors. These early, to us un- known, fishes have sprung from vermiform predecessors of ceelenterate pedigree. A tendency to exchange the radial for a bilateral sym- metry and to separate the ccelom from the enteron must at one time have characterised certain ccoelenterate ancestral forms, as has already been advocated by Sedgewick (784) and by myself (’05) on earlier occasions. It is not straining the imagination to assume that in this line of descent closely- related forms may have developed, some with, others without a larval envelope, temporarily ensheathing the cellular ele- ments that will build up the embryo itself and thus fore- shadowing the separation among their later, vertebrate descendants of such with and such others without a tropho- blast. We find examples of this amongst the Nemerteau worms. In some of these the egg after segmentation develops straight away into the young worm, in others, which as far as the typical Nemertean characteristics go are very closely related, the cleavage results in a disposition of the embry- onic material into (a) the first lineaments of the embryo itself and (b) a cellular temporary envelope of these, which is either more closely applied to (Desor’s larva) or more distant from (Pilidium larva) the material that goes to build up the embryo. And though I in no way want to infer that it is among the Nemertea or Gephyrea that we would have to look for the ancestral forms of the Vertebrates (uor either amongst any of the Annelids known to us) still it is an instructive fact that among different classes of worms (Gephyrea should also here be mentioned, see Fig. 129) the larval envelopes above alluded to are encountered in some but are absent in others. VoL. 93, PART 1.—NEW SERIES. 2 18 A. A. W. HUBRECHT. This particularity may have passed on in the ancestral line of the chordata. Now, if in our further phylogenetic speculations concerning the Protetrapods and their descendants that live at the pre- sent time, we were to start from an oviparous aquatic animal, whose early developmental stages are provided with a larval envelope, we understand that, when any such animal came to adapt itself to inhabit the dry land it would doubtlessly score certain advantages if at the same time it became viviparous. Its adaptation would certainly be the more complete if, for its reproduction, it were independent of the aquatic medium. And towards the efficiency of this viviparous condition the larval envelope could immediately contribute by the mere change of its protective or locomotor significance into an adhesive one. ‘This again would be facilitated if the larval envelope, increasing in surface, were to develop into a spherical vesicle precociously forestalling the further development of the mother-cells of the embryo of which this larval envelope had originally been an organ of protection and of locomotion. Subsidiarily this vesicular shape would contribute towards the retention of the deve- loping egg for a longer time in the maternal genital ducts. And at the same time the possibility would arise of intro- ducing through the wall of this swollen blastocyst not only fluid to increase the swelling, but also nutritive matter to further the growth of the didermic ‘ Anlage” contained in it. All these circumstances accompanying the transition to an atmospheric environment would at the same time be unques- tionable advantages of protection and nutrition to the embryo, such as are already sporadically obtained in certain fishes (Mustelus, Zoarces, and others). Besides this, however, another advantage might ensue, viz., the possibility of this larval and transitory layer becoming vascularized in aid of a yet more thorough system of nourishment at the expense of the maternal circulatory system. And it is this what we actually observe in the mammals + vel ’ a. ‘ why eS : ' 4 ay HUBRECAT. Pl. Hi vat i fe § 0, a vm : “oe tom Fig. 44—46. Three diagrams of the aspect of a longitudinal section through a Tarsius blastocyst. In Fig. 44 the trophoblast yet covers the embryonic ectoderm. The cayities of the umbilical vesicle (av) and of the extraembryonic coelom co in the ventral mesoblast entirely fill up the blastocyst; the connective stalk /c) is formed and it is at this spot (cf. Fig. 62) that the attachment of the blastocyst to the maternal tissue comes about. Ih Fig. 45 the embryonic ectoderm has become exposed to the surface after dehiscence of the trophoblast; the entoderm in the embryonic region has thickened. In Fig. 46 protochordal plate #f and protochordal wedge pw have become differentiated (cf. Fig. 48); under the stalk of ventral mesoblast the annular region of proliferating entoderm is once more cut longitudinally /@/; from here the vascularization of the con- nective stalk proceeds. — Fig. 47. The relative positions of ventral mesoblast (vm), trophoblast (¢~) on its way to leave the embryonic ectoderm uncovered (cf. Fig. 20) and umbilical vesicle in a stage of about the same age as the follow- ing figure. — Tie. 48. A somewhat later stage in which a distinet ventral proliferation (fw) of the ectoderm fuses with the entodermic proliferation (pf) of the entoderm. The protochordal wedge pw and the protochordal plate #p then become fused (cf. Tig. 52, 98, 99); the ventral mesoblast v7 springs from the embryonic ectoderm just behind the protochordal wedge. ¢r trophoblast, sm splanchnic mesoblast, 27 umbilical vesicle. HUBRECHT. PG Tt AY 50 Fig. 49. Longitudinal section of another Tarsius blastocyst in which the protochordal plate f has become fairly established and the protochordal wedge is just in its very earliest phase, more so than in Fig. 48. The ventral meso- blast vw arises from the ectoderm, close behind fw: the trophoblast ¢, is inde- pendent of both. — Fig. 50. Longitudinal section in about the same stage: the attachment of the blastocyst to the uterine wall commences about at the spot marked ¢r: the corresponding proliferation of trophoblast (¢) is not in- dicated in this figure (cf. Fig. 62); the ventral mesoblast 7 springing from the ectoderm shows the extraembryonic coelom the wall of which is partly splanchnic (spm), partly somatic mesoblast (som); pp protochordal plate. -—— Fig. 51. Trans- verse section of an early blastocyst of about the stage of fig. 46 showing the proliferating protochordal plate. Pl. KK. HUBRECHT. Fig. 52. Longitudinal section of a Tarsius embryonic shield in whieh at the spot where the protochordal wedge pw has proliferated downwards a rudimen- tary attempt at a blastoporic perforation has quite exceptionally arisen. Pp proto- chordal plate, pw protochordal wedge, wz ventral mesoblast, #7 umbilical vesicle. ec embryonic ectoderm; mes mesoblast, springing from protochordal plate. — Fig. 53. The early evanescent blastopore /é) of the hedgehog (after Hubrecht, 02). — Fig. 54. The same of the mole (after Heape); 2 blastopore, ez entoderm. HUBRECHT. PC. CE. al en ec uv b Fig. 55. The same of the opossum (after Selenka); 4 blastopore, av umbilical vesicle, ec ectoderm, ez entoderm, @Z albuminiferous layer, ¢7 tropho- blast. — Fig. 56, 57 and 58. Three longitudinal sections through on early blasto- cyst of the shrew (Sorex) of wich the embryonic shield is traced in the dia- gram of fig. 59. Fig. 56 and 57 are two succeeding sections through the posterior region where a rudimentary blastopore /2) pierces the embryonic shield, sepa- rating the proliferating ectodermal region pw (protochordal wedge) from the yet further posterior ectodermal region which will give rise to the ventral mesoblast (vm), in which the posterior coelom will take its origin in crescent-shape as in- dicated in diagram 61 and 100. Fig. 58 is the longitudinal section through the entodermal proliferation pp of Fig. 59. Fig. 100 gives a longitudinal section through the posterior crescentic coelom of fig. 61. — Fig. 59. Superficial aspect of the early ectodermal shield corresponding to the three preceding figures. The region of the protochordal wedge is indicated by the posterior white, that of the protochordal plate by the anterior shaded space. EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 19 from the Didelphia onwards, where either the omphaloidean or the umbilical arteries, or both, serve that purpose. This then is my interpretation of the phylogenetic phases through which the trophoblast has passed. They cannot be said to be numerous or intricate, nor can the interpretation be looked upon as strained or artificial. _The less so, because in all mono- and di-delphic mammals, which have as yet been examined, we do—as was noticed above—encounter a larval envelope—the trophoblast—which surrounds the formative cells of the embryo. Without exception the trophoblast undergoes the series of changes and physiological trans- formations here sketched, becoming first vesicular, then selective to certain nutritive matter, finally vascularised and locally strongly adhesive to and fused with maternal tissue. B. OrRNITHODELPHIAN MAMMALS AND SAUROPSIDA. The segmented egg of Ornithorhynchus and Hchidna, the two living representatives of the Ornithodelphia presents itself to us under a totally different aspect, as compared to the other mammals. The ornithodelphian egg does not cleave according to the holoblastic, but to the meroblastic type, and offers numerous points of comparison with that of reptiles and birds. However, our knowledge of it is as yet very scanty and limited to what Caldwell (87), Semon (94), and Wilson and Hill (03, 707) have taught us. The egg is enclosed in a leathery shell. There is no, or hardly any, albumen, and this makes investigation of the earliest stages all the more difficult. The formative protoplasm, accumulated at the upper pole of the yolk, breaks up into a number of cleavage-cells (Fig. 66) and at a very early stage the outer layer, already distinctly visible as such in the preceding stage (Fig. 67), has spread over the yolk as a membrane of flattened cells with flattened nuclei (Figs. 68 and 69). At the upper pole this layer covers—at the spot where the embryo is going 20 A. A. W. HUBRECHT. to be formed—the remains of the cleavage cells not as yet arranged in regular layers. I think we may safely compare this stage in the Ornithodelphian development with that of the higher mammals in which the, as yet undifferentiated embryonic knob is covered by the trophoblast, which has dilated into a vesicle. Although the interpretation here given differs from that of Semon, I feel confident that further and more detailed researches on the development of Monotremes will confirm this hypothesis, as well as the supplementary one which at present is not yet based on observation, viz. that the cells e.k. in Figs. 67 and 69, after a time arrange themselves into embryonic ectoderm and entoderm, the latter spreading out radially below the trophoblastic cell-layer, as indicated in Fig. 70. It is particularly to be regretted that the embryonic shield belonging to Semon’s Fig. 39 has come to grief, because it would no doubt have settled the point here under discussion.! The difference between the Ornithodelphia on one side and between Mono- and Di-delphia on the other would—if the interpretation here given were to be confirmed—consist in the fact that the trophoblast vesicle of the former includes besides an embryonic knob a very considerable amount of food-yolk, the development of which will have gone parallel with the change in the ancestral line from viviparity to ovi- parity. Also in the Sauropsida similar phenomena must have occurred of which, however, the traces are yet more difficult to establish than was the case in Ornithodelphia. The trophoblastic vesicle, which is in Ornithodelphia yet com- paratively distinct, though as yet imperfectly known, is in many reptiles and birds distinguished with great difficulty from the embryonic shield because the phenomenon of the trophoblastic vesicle opening up at one spot, in order to let 1 When this paragraph was first written Wilson and Hill’s latest extensive researches (’07) had not yet come into my hands, Their figures, here repro- duced in the Figs. 68 and 70, seem to-fully agree with the hypothetical interpretation here given, before these new facts had come to light. HUBRECHT. Pl. M. all OM Fig. 60 and 61. Two surface views of a yet later embryonic shield of the shrew. In fig. 60 the annular zone of proliferating entoderm az is indicated as well as the ‘primitive streak and the dotted outline mes of the mesoblast wings; in fig. 61 the notochord has begun to be formed; a neurenteric pore (72f)/ is visible as well as extra embryonic posterior coelom, co. (Vig. 56 to 61 after Hubrecht, tl) ee Fig. 62 to 65. Diagrams intended to demonstrate the gradual displacement in Tarsius of the embryonic shield from its original position (62) towards a position diametrically opposite the placenta (65). The zone a of fig. 16 is shown in fig. 63 as being at the same time the incipient allantois tube @//: in fig. 64 and 65 this becomes a posterior, cylindrical continuation of the enteric cavity V7, lengthening as the embryonic shield travels upwards. In 64 and 65 the placenta P has become a considerable trophoblastic proliferation in cushion shape (vide Hubrecht, 99), in Fig. 65 the amnion folds va, “a and the neuren- teric canal mc have made their appearance (cf. fig. 99); c extraembryonic coelom, HT connective stalk, am amnion (after Hubrecht, ‘07). PA EARLY ONTOGENETIC PHENOMENA IN MAMMALS. Dal the embryonic ectoderm come to the surface, has become indistinct. It was above shown how perfectly distinct this is in monodelphian and didelphian mammals, and how there can be no doubt of its occurrence in Ornithodelphia (Fig. 70). Still this latter group helps us to explain how it was that it became indistinct and thus unrecognised in Sauropsids. An outer trophoblastic layer has been described by Mehnert (94, p. 214), who perfectly recognised its identity with the layer for which in Mammalia I had introduced the name of trophoblast, but who has created confusion by nevertheless proposing the new name of teloderm! (Grenz- blatt), and greater confusion yet by comparing heterogenous cell-layers as I will yet further indicate. Mehnert describes in detail how in the embryo of Emys lutaria the outer germ layer becomes didermic and produces two layers that are totally different from each other, of which the deeper layer furnishes the material for the definite epithelium of the tortoise and represents the primitive epidermis, whereas the outer layer of flattened cells, the trophoblast (Mehnert’s telo- derm), should be looked upon as a supra-epithelial layer. According to Mehnert the trophoblast can be quite easily separated from the epiderm (I. c., p. 213, Pl. IX, Fig. 8). The growth of the trophoblast is said to be dissociated from that of the deeper epithelial layer. Mehnert claims to have established (on the authority of Mitsukuri’s [’93] figures) the presence of a trophoblast in Clemmys japonica and in ‘rionyx japonica, in Lacerta muralis, Tropidonotus, and for birds in the duck, the chick, Larus, Sterna, Podiceps, Buteo, Aegialitis, Hirundo, Luscinia, and others. Now I must begin 1 The reason he gives for substituting a new name and not applying the name of trophoblast is, ‘that it has not been proved that those cells partici- pate in the first place towards the nutritive processes of the embryo.” In this he is in full contradiction to Schauinsland (03, p. 83) who holds it to be “sehr wahrscheinlich” that these very cells have a nutritive significance in reptiles. In the Mammalia, where the layer is ever so much more con- spicuous, its phagocytic significance has been proved; but even if it had not, this seems hardly to justify Mehnert for over-burdening scientific nomencla- ture by the creation of a superfluous synonym. Ze, A. A. W. HUBREOCHT. by disclaiming the greater number of these cases. I feel convinced that in certain of the cases observed Mehnert and Mitsukuri have seen what is really the rudimentary plasmodi- trophoblast of reptiles, but that in others the first-named author has been misled and has confused what is really a superficial layer (distantly comparable to a mammalian epitrichial layer) of later embryonic phases with trophoblastic elements that can only be noticed in certain early phases. I have pub- lished this disclaimer more than ten years ago (95, p. 27, Anmerkung); I can here only repeat it. A real Reptilian trophoblast can, I think, be clearly detected in Mitsukuri’s (90) Fig. 59 of Clemmys, where we find a separate cell- layer of flattened elements accompanying the amnionfolds on their outer surface. This layer is not continued on the inner surface of the amnionfolds as Mehnert will have it in his case of Emys lutaria. Also in his coloured figures (l. c., 830a—37a) Mitsukuri seems to indicate, by a different tint of red, that he did not (as does Mehnert) see any con- tinuity between this outer trophoblastic layer and the inner lining of the amnion. If we were to adopt Mehnert’s view—as I have perhaps been inclined to do more than I was justified to in 1895— then we would have to look not only upon the inner layer of the amnion as trophoblastic, but also upon the covering layer he describes in the duck, which forms a continuous supra- epithelial stratum both on the back and on the ventral surface of the embryo; and a comparison with what we have above described for the mammals ought to make us diffident in accepting this view as the real interpretation.! ' It must be borne in mind that the phenomena here discussed are as yet only very partially known. And if we consider the very various methods which we have discussed above (p. 11), according to which the mammalian trophoblast disappears above the embryonic shield, we may also expect variatious in the Sauropsida. If we suppose that an arrangement like that in the rabbit and other rodents (Figs. 15 and 28) where the Rauber Deckschicht remains distinct for yet a longish time, were yet further protracted, we might obtain a state of things as that which is described by Melnert. for Emys and certain other forms, I should not mention this if it were not - yt ‘a 5 1 Py ‘ “ ie ' i ? ‘ jaf . ey rte ' oat ‘ id } \ i a ~~ ' ie” - Pe 4 fi) f - ' 7 of i = (e aa ha ad : ig! Pr hy nie af yd oy 4 as e i is ae Ret, iu . 7 4 - ¥ rt » Nari 1, AS SRA Ra ant re eo » ; ah ii ‘yr BUI CATT vier i. ie, aU Tee J ti " | ite a \ th, hia a >| ae) HUBRECHT. IPL NE Fig. 66 to 70. Five sections through the very earliest stages of Ornithorhynchus and Echidna. Fig. 66 earliest cleavage stage. Fig. 67 visible separation of trophoblast cells 7 and of mothercells of the embryonic knob, e& Fig. 68 and 69 this separation is yet far more clearly established: the trophoblast 7 having travelled much further over the yolk surface, the em- bryonic knob (ck) being partly imbedded in the yolk; Fig. 70 a yet further stage in which ecto- and entoderm (ec and ev) have become differentiated by de- Jamination and in which the ectoderm has come to the surface; ¢ trophoblast. (Fig. 66, 68, 70 after Wilson and Hill, ‘07; Fig 67, 69 after Semon, 94.) HUBRECHT. Pt 0. Fig. 71 to 73. Three figures of sections through the blastocyst of the frugiverous bat Pteropus (after Selenka, Géhre, ‘92). In fig. 71 the embryonic ectoderm is yet a solid cellmass, in fig. 72 an amnion cavity (a) has appeared within it, in fig. 73 the final relations between trophoblast /¢/, amnion a, embryo and umbilical vesicle (wv) are established. — Fig. 74. Transverse section through amnion a, embryo and umbilical vesicle wz of Hylobates (after Selenka, 00}. sp the splanchnic mesoblast on the umbilical vesicle (27) which carries a very dense net of thickened venae in which haematopoietic processes occur. — Fig. 75. Surface view of the amnion-fold of Chamaeleo. — Fig. 76. The same in transverse section, with proliferation of the anterior entoderm (4). ¢ trophoblast separated into two layers. — Fig. 77. Transverse section of an embryo of Sphenodon with amnion nearly closed. The trophoblast is double layered. Fig. 75 to 77 after Schauinsland, ‘03. EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 23 A second author in whose investigations a reptilian plas- moditrophoblast has come to light is Schauinsland (?03). In his figures of the young Chameleo (Fig. 76) and Sphenodon- embryo (Fig. 77), we notice that the rising folds of ectoderm, which are the first indications of the separate existence of amnion and serosa, are covered, externally, by a layer of varying thickness. ‘lhe presence of this layer seems to me indicative of a similar process in reptiles as was noticed in mammals, viz. a differentiation of the region outside of the ectodermal shield (as such we encounter the trophoblast after the embryonic ectoderm has been interpolated in it) into a superficial and a deeper layer (plasmodi- and cyto- trophoblast of v. Beneden and Hubrecht). And this differen- tiation arouses suspicion, further confirmed by the sharp dis- tinction at the free border of the amnion fold between outer and inner layer (Figs. 76 and 77, that. in reptiles the case may stand as in bats (Fig. 8a), and in the hedgehog (Fig. 38) where the outer surface of the amnion-fold is trophoblastic, whereas the inner is an upgrowth of the ectodermal shield (see also p. 77) and Duval (99, figs. 96, 102 and 117). The trophoblast of Sphenodon and Chameeleo would thus be more than one cell thick even before the somatic mesoderm has made a diplotrophoblast of it. This trophoblast does not con- tribute to line the inner surface of the amnion cavity. Here only the embryonic ectoderm (see pp. 76—78) comes into play. In this important respect Schauinsland thus sides, although not himself discussing the merits of the problem (which was not before his mind), with Mitsukuri and not with Mehnert. In Chameleon of which Schauinsland gives good illustrations (03, Pl. 26, figs. 184—186), which are very indifferently reproduced in Hertwig I, 2, p. 194, the same phenomenon is observed with quite as much distinctness (Fig. 76). After the amnion has closed in the very primitive fashion charac- teristic for Chameleon (Fig. 75) the ‘‘ membrana serosa”’ consists of a double layer of trophoblast (Fig. 76). desirable, from the very first, to keep an open eye for all the different possi- bilities that may help to elucidate these difficult points. 24 A. A. W. HUBRECHT. The facts above cited force us to the conclusion that, before the formation of the amnion in Sphenodon and in Chameeleo begins, there must exist on the surface of the blastocyst a circular delimitation of a central region—what would be the actual embryonic shield of mammals—from a peripheral trophoblastic region. This delimitation is clearly indicated in another of Schauinsland’s figures (Pl. 46, fig. 117) for Sphenodon not reproduced in Hertwig, but here reproduced in Fig. 78. In Schauinsland’s text (703, p. 142) this is noticed in the following words :—‘ As it was repeatedly noticed (the trophoblast-cells) do not spread over the embryo proper, and thus the extra-embryonic and the embryonic portion of the ectodermal blastoderm can be sharply distinguished from each other.” If we now restrict ourselves to the three cases here cited, a tortoise (Mitsukuri), Sphenodon, and the chameleon (Schauinsland), and purposely leave out of consideration all Mehnert’s cases, then we have three Sauropsida in which clear indications are noticeable that the mammalian tropho- blast is after all also present in the Sauropsida. Besides these indications there is, however, a strong & priori probability that views which are applicable to the embryonic membranes of mammals ought also to fit im with Sauropsids that have—because of these membranes—always stood in closer connection with the mammalia than with the lower vertebrates. And we should not lose view of the fact that the com- parison of Klasmobranch with Sauropsid ontogeny has always shown this incisive difference that there was never a mem- brana serosa nor an amnion in the former, so that a direct comparison in these two types of the process of the gradual inclosure of the yolk by radial expansion from the ectodermic shield was tainted by suspicion from the beginning: the whole of the serous membrane and the amnion being shed at birth in birds, reptiles and mammals; these being, in fact, larval layers. HUBRECHT. Pt. P. pp 80 nch UM Um Fig. 78. Another transverse section of Sphenodon (after Schauinsland, ’03) to show the differentiation of the twolayered trophoblast tr as against the ecto- dermal shield ZZ‘; pf protochordal plate. — Fig. 79 to 82. Four longitudinal sections of frog-embryos (after Brachet, ‘02). In Fig. 79 protochordal plate / and protochordal wedge pw have become differentiated; in Fig. 80 the noto- chord (xch) is further developed and the ventral mesoblast 7 makes its appea- rance; in Fig. 81 the segmentation cavity has coalesced with the enteric cavity that has become visible during notogenesis; in Fig. $2 notochord, somites and gut are formed, headfold has’ become visible, ventral mesoblast 2 developes below and behind the entoderm cells KARLY ONTOGENETIC PHENOMENA IN MAMMALS. 25 And now that the interpretation of the facts in mammals has become comparatively easy (see also Chap. III) we should not shrink from resolutely interpreting the Sauropsidan development along the same lines. A comparison of my own figures for early Erinaceus (’89) and of van Beneden’s (99) for early Vespertilio blastocysts with the figures above referred to of Schauinsland and Mitsukuri convinces us of the possibility of looking upon the double layer outside the formative ectoderm—say of Sphe- nodon—as a duplication of the trophoblast. The two mammalian genera above mentioned, as also Sorex and others, show a duplication and even a more considerable thickening yet of the trophoblast immediately outside the embryonic ectoderm. And so it would not be a very strained assump- tion to say that in reptiles and birds—in which as we have seen Schauinsland admits of a sharp line of demarcation between the trophoblast and the embryonic shield on the surface (I. c., p. 142)—both layers that are out- side of this line of demarcation are trophoblast-cells separated in an outer flat- tened and a deeper columnar layer. Hven of this differentiation in shape the mammals offer the counterpart, as is seen, to the left side in Figs. 8 and 8¢ of van Beneden’s (’99) early bats and Figs. 35—37 of the hedgehog here given. We will, moreover, see in Chap. V that the trophoblast often differentiates into two layers that are known as the cytotrophoblast and the plasmodi- trophoblast. And so the assumption here advocated would oblige us to conclude that, in birds and reptiles, a circular patch of embryonic cells was separated—not visibly but potentially—from a peripheral region of trophoblast cells just as we have established this for 'lupaja, Tarsius, and others, in which—after the embryonic shield has opened out—it is no longer possible to distinguish the line of demarcation between trophoblast cells and embryonic ectoderm cells, although we have noticed its actual existence in the successive ontogenetic stages. In most Sauropsida ontogeny would no lenger clearly reveal this difference, but still the mutual relations would be the same, and exceptionally favourable cases as here described and figured (Clemmys, Sphenodon, Chameleo) would be all the more welcome confirmations. Physiologically the outer layer of the serosa of Sauropsids is recognised to have undoubtedly (see p. 21, footnote) certain properties which we also encounter in the proliferating trophoblast of mammals. There is, for example, a very marked proliferation in the outer layer of Seps, a viviparous lizard in which Studiati, Giacomini (91), and others have described both an allantoi- dean and an omphaloidean contact (placentation) between the serosa and the maternal tissues, Similarly the action of the serosa of the chick in the region where Duval has described the ‘‘ organe placentaire ”’ gives rise to the same considerations. 26 A. A. W. HUBRECHT. But more extensive investigations ad hoe will have to be undertaken before the isolated cases of the Reptilia above noticed will have obtained sufficient lateral support to serve as a starting-point on which a theory on the modifica- tion of the trophoblast in the Sauropsida—simultaneously with the formation of an eggshell, etc.—may be based.! Of the part played by the Sauropsidan trophoblast in the formation of the amnion we will have to speak in another chapter. Suffice it to add that no data are as yet available to determine the exact moment at which the plasmodi- trophoblast becomes distinguishable in the above-mentioned genera. Neither Mitsukuri nor Schauinsland give any indications. Furthermore, it would be important to know whether ontogeny gives any clue which would permit a guess as to the question whether the trophoblast has, in the viviparous ancestors of the Sauropsida, been as early differentiated from the remaining cleavage cells as is the case in mammals,? or whether the differentiation has only set in later as we find in the case of those Amphibia and fishes in which traces of an outer larval layer are also present, and which we will more fully discuss in the last paragraph of the next chapter. ©. IcHTHYOPSIDS. In the paragraphs A and B of this chapter.we have attempted to show that beside the ectoderm and entoderm, which by delamination establish the gastrula stage of mammals and Sauropsids, there exists yet another very early cell-layer 1 Recently Eternod has published an article, “La Gastrule dans la série animale,” in the * Bull. Soc. Vaud. Se. Nat.,’ 1906. 5e sér., vol. 42, in which, in text-fig, 16 and in fig. 26 on pl. 18, he attempts to homologise parts that are in no way homologous, if we look upon the early developmental processes of Mammals and Sauropsids in the light above advocated. Kterncd’s views have already been successfully protested against by Schlater (‘ Anat. Anz.,’ Bd. 31, p. 31). The latter author himself misses the mark, however, when he says that ‘die epiblastische Schicht der Sauropsiden-keimblase der iiber die Grenzen der Keimscheibe hinausgewaclisene embryonale Epiblast ist.” The secondary degenerative stages of the trophoblast are here wholly misunder- stood. 2 The researches, above alluded to (pp. 12 and 20), of Wilson and Hill seem to imply that in Ornithodelphia we have yet an important intermediate stage, in which it is indeed possible, notwithstanding the yolk accumulation, to distinguish the trophoblast from the mother-cells of the embryonic knob. Semon’s (’94) figs. 33 and 34 allow of a similar interpretation. EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 27 to which the name of trophoblast has been given. This layer, phylogenetically subordinated to the ectoderm, was looked upon as a differentiation of the same order as the outer larval layer which in certain Nemertines, Gephyreans, and other worms often serves as a temporary envelope that is stripped off when the animal attains to a certain stage of development. In a later chapter it will be discussed whether the different foetal envelopes of the Amniota allantoidea may not be brought into genetic relation with this layer, and whether we might be justified in thus tracing the foetal envelopes of the higher vertebrates as far back as the invertebrate ancestors provided with an ectodermal larval investment (Larvenhiille). It would appear at first sight probable that in the Anamnia, Anallantoidea (i.e. in the Ichthyopsids) traces of this larval cell-layer should not be met with, and that this very absence would help to explain the fact that here no amnion develops, However, the chance that the intrinsic differences between say Amphibia and Reptiles are not so incisive as this separa- tion of the vertebrates in Amniota and Anamnia would make us believe, should also yet receive our consideration. And it is in this light that I intend to look upon the fact that in. many amphibia certain ontogenetic stages reveal the presence of what has been called the ‘“‘Deckschicht” of the larva. Numerous figures successively published by different authors show the extent to which such a layer has been actually observed. It should at the same time be noticed that in several other genera no trace of it has been found. The more remarkable circumstance is, however, this—that not only in Amphibia such a ‘‘ Deckschicht ” makes its occa- sional appearance, but that similarly it is noticed during the development of certain Dipnoi and Ganoids (Fig. 87), and both more constantly and more unquestionably during that of all the Teleosts (Fig. 89) of which up to now the early develop- ment has been traced. Of these different groups the “ Deck- schicht ” is here figured after the publications of different authors on the subject, and I will not here enter into further details, contenting myself with having shown that it is a 28 A. A. W. HUBRECHT. general feature in the development of 'Teleostomes, Dipnoi, and Amphibia. Suppose for a moment that we are justified in looking upon the Deckschicht of Amphibia and Teleostomes as being in reality homologous to the trophoblast of Mammalia and Sauropsids—homologous at least in that sense that what is a very active and most important layer during the development of the viviparous mammals is only a temporary, evanescent arrangement in the Ichthyopsids—then we must at the same time ask ourselves: is this homology, perhaps, indicative of an error into which we may have fallen when adopting Milne Edwards’ distinction of the vertebrates in Anamnia and Am- niota ? And should we not reconsider whether and how this error can be readjusted ? At all events the Elasmobranchs, the Cyclostomes and Amphioxus show in their early development no traces of a Deckschicht and—as we shall see in a later chapter—no traces of other organs which are characteristic for the other verte- brates. In this chapter I had to point to these facts ; in Chapter ITI, p. 81, they will be more fully discussed, as a'so in Chapter Wag p. 150. CHapter I]. Furraer DerveLopmMent OF THE ‘Two GeERM- LAYERS OF ‘HE VERTEBRATES UP TO THE APPEARANCE OF THE SOMITES. I. Mammatia (Mono- anp Di-peLputa). 1, Developmental Processes in the Entoderm. The participation of the entoderm towards the formation of tissue between the two primary layers in Mammals is denied by very high authorities as Kélliker, Selenka, Ziegler, Keibel, and others, who hold that material for mesoblastic HUBRECHT. Pl. O. iw 86 J Fig. 83 to 86. Four longitudinal sections of Hypogeophis (after Brauer, ‘97). In Fig. 83 the downward proliferation of the ectoderm (gw protochordal wedge) commences to fuse with the entodermic protochordal plate 4/. In Fig. S4 the notogenesis has proceeded further; in Fig. 85 the segmentation cavity has coalesced with the enteric cavity; in Fig. 86 the ventral mesoblast a has also made its appearance and the entoderm ev has spread below the notochord xc, y yolk. EARLY ONTOGENETIC PHENOMENA IN MAMMALS, 29 structures is budded off only from the primitive streak, and who—some of them at least—even wish to derive the vascular system and the blood from the same source. Mesenchyme formation, so sharply distinguished by O. Hertwig from mesoblast formation (see his ‘ Lehrbuch,’ ed. 1906, p. 218) is by many authors held to be of no significance whatever in mammals, although Bonnet, in his investigations on the sheep’s development (82, ’89), has attempted to stem that current of thought in demonstrating for the sheep that: the vascular region on the yolk-sac is a direct derivate of local proliferation of the entoderm. In his later publications on the dog, however, Bonnet has for that mammal denied the presence of a similar process, although from his plates (01, Pls. XVIII, XIX; fig. 6, and many others) another conclu- sion might certainly be drawn (Figs. 91 and 92). On the contrary for Sorex and Tupaja (as yet unpublished) the genesis of mesenchyme out of entoderm has been fully con- firmed by myself, and the region in which the participation of the entoderm towards the formation of blood-vessels and blood occurs, has been figured in detail by me (790, Figs. 58, 61). When seen from above the aspect is such as to warrant the designation of this region by the name of the annular zone of mesoblast-producing entoderm of the shrew and of Tupaja. Since then the battle has been raging concerning this very difficult and yet very important question of comparative embryology round which many problems, connected with the interpretation of the germinal layers and the significance of mesoblast, centre. Only very lately Riickert has given a remarkable digest— in co-operation with Mollier—in Hertwig’s ‘ Handbuch,’ Vol. I, p. 1244—1260, in which—starting from careful investiga- tions—he draws important conclusions concerning blood- formation in all the Vertebrates, that go far to demolish part of the theoretical views held by Rabl on mesoblast formation, which latter have been largely accepted by the great majority of embryologists. 30 A. A. W. HUBRECHT. IT need not here enter into a detailed exposition of this controversy, now that it has been so carefully done in the chapter just mentioned on “die erste Entstehung der Gefisse und des Blutes bei Wirbeltieren,” in Hertwig’s ‘ Manual.’ But I will pass on to a full description of what has already been observed and described in different mammals commenc- ing with what, in 1890, I have called— a. The Protochordal Plate.—This structure has at first been more or less ignored by many embryologists, later its significance has been recognised, but it has then been designated by a different name (Bonnet, 01, E.P.); this time I hope to establish definitely that [ was not only justified to distinguish this protochordal plate as an independent anterior source of mesoblast in mammals, but that we ought henceforth to admit its presence under varied aspects also in Sauropsids and Ichthyopsids, as I will point out hereafter. For mammals we have in the preceding paragraph described how in the didermic stage the entoderm cells under the ecto- dermal shield are considerably more massive than those that clothe the inner surface of the trophoblast, the latter being flattened and further apart. Figs. 8a, 14, 18, 30, 31 and 36 testify to this. As the didermic blastocyst increases in size there is a very marked phenomenon of further increase coupled with proliferating growth in that portion of the ento- derm that lies under what will later be the anterior portion of the embryonic shield. I here re-figure this for Sorex after my own (Figs. 58, 59) for the sheep and dog, after Bonnet’s (Figs. 91, 92) publications, and I add new figures indicating the same phenomenon in Tarsius (Figs. 48, 49, 50 and 51) Galeopithecus (Figs. 18,42). For the pig it has been figured, although not viewed in this light, by Keibel (’93, figs. 21— 23). 1 Keibel interprets his figures differently, and did not, in the paper above referred to, recognise the protochordal plate as a source of mesoblast, such as I had defined it three years before. Still the figures here cited leave no doubt of its existence in the pig. EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 31 In Sorex it was particularly interesting to be able to establish the independence of this early proliferation from any further source of mesoblast, although very soon after, the annular zone of mesoblast-producing entoderm connects this early protochordal plate with the mesoblast-producing regions at the hinder end of the embryonic shield. Seen from above this early phase is pictured in Figs. 59 and 60. The entodermal proliferation here described has, in its earliest phases, the aspect of a mere thickening of the lower germ-layer, but very soon that aspect changes, and we notice that certain of the proliferated cells break away from their place of origin, and take up a situation between the two germ-layers. ‘The extent to which the invasion by these mesenchyme cells of the space between ectoderm and ento- derm spreads cannot always be very strictly determined for two reasons. Firstly, because the annular zone of mesoblast- producing entoderm, which becomes confluent with the protochordal plate (Fig. 60), starts its activity almost simultaneously, though, as Fig. 59 shows, just a bit later; secondly, because another invasion of this space—starting from the ectoderm—also begins to form about this time, and will be described below. At a very early moment the cells derived from these three different sources intermingle, and it will prove a most intricate problem, which up to now has not yet been fully nor satisfac- torily solved, nay, not even approached, to make out from which of the three starting points the various organic tissues have ultimately been derived. In Sorex this was possible to some extent at a very early stage, because the anterior entodermal proliferation pro- ducing mesenchyme cells is inaugurated somewhat earlier than the process which starts from the posterior half of the ectodermal shield. In my paper on Sorex (’90) I have been able to sufficiently distinguish these early phenomena, although fully recognising that after a time further dis- crimination becomes impossible. This latter fact may have contributed to bring so many of the best modern embryolo- on A, Aw. W. HUBRECHT. gists to follow Koélliker in his negation of the participation of the entoderm towards the production of mesoblast. In Tarsius the distinction of the mesenchyme (derived from the entoderm) from other mesoblast-cells between the two germ layers is hardly feasible even in the earliest stages, because here the source of early ectodermic mesoblast at the hinder end of the ectodermic shield is in full flow at a very early period in consequence of the presence from the very outset of mesoblastic tissue, which I have called the ventral mesoblast. It formsasac, partly applied against the umbilical vesicle from the very first, and encloses an extra embry- onic coelomic space, which is thus present at a very much earlier moment than in other mammals with the exception of man and monkeys. Part of this ventral mesoblast will gradually become the connective stalk (Haftstiel, Bauch- stiel) by which the embryo will be in vascular connection with the placenta, and which will be fully discussed in a later chapter. But in this same Tarsius the entodermal proliferation above described for Sorex, and which I will continue to designate as the protochordal plate is all the more evident. It is figured in diagrams 48, 49, 50,and51. The entoderm has here become two or three cell-layers thick. ‘This region corresponds to what will later be the very front part of the head of the embryo, before the primordium of the heart has yet been folded in under that of the brain. As to other mammals I do not dispose of quite as exten- sive data as for Sorex and Tarsius, but there is no doubt if we also consult the results of other investigators—even of those who deny the participation of the entoderm towards the formation of mesoblast—that this thickening of the entoderm occurs in all mammals. For Hrinaceus, Gymnura, Talpa, and Tupaja I possess numerous convincing prepara- tions already mentioned above. Also for Manis, Galeopi- thecus, Sciurus, Mus, Lepus. Several of these are here figured (Figs. 18,37, 42). For the dog Bonnet gives very un- mistakable illustrations (01, Figs. 11—13, 31, 32), although i\ he substitutes the name ‘ Erganzungsplatte” for the older EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 33 one of “ protochordal plate.” Also in one of Assheton’s papers (796, Pl. 20, figs. 17 and 18) the author clearly figures the proliferating region in the entoderm here referred to. b. The Annular Zone of Proliferation.—How the hind end of this entodermal protochordal plate comes to fuse with the front portion of a median ectodermal down- growth of the ectodermal shield I have described for Tarsius in a former paper (1902). It will again be discussed further down. It is, however, necessary first to establish a fact already formerly insisted upon both by Bonnet (’84) and myself (790), viz., that when once the protochordal plate has made its appearance as a median, mesenchyme producing spot in the entoderm, the same mesenchyme producing properties become evident in peripheral regions of the entoderm. These regions have been named by Bonnet for the sheep the ‘* Mesoblasthof”’; shortly afterwards I have described them (790) for the shrew as an elongated ringshaped zone of entoderm which is situated under and somewhat outside the border of the ectodermal shield (Fig. 60), and which, slanting backwards from the protochordal plate both right and left, meets under the hinder part of the shield in the region where the mesoblast has acquired that median thickening which is known as the primitive streak, continued in the Primates into the connective stalk (Haftstiel). The presence of such an annular zone of mesenchyme producing entoderm has been very emphatically denied by such embryologists as Rabl, Keibel, and others, and in O. Hertwig’s latest manual he makes no mention whatever of it in the chapter on the ‘‘ Lehre von den Keimblattern.”’ This is all the more to be wondered at, because we shall see that also in lower vertebrates a similar participation of the entoderm towards mesenchyme formation can as little be denied. It seems to me that the energy with which these facts are ignored must have its origin in the strength of certain theoretical considerations with which a multiple origin of mesoderm! would clash. 1 For myself, I have on another occasion (02, p. 84) expressed my sym- VOL. 53, PART 1.—NEW SERIES. 3 34 A. A. W. HUBRECHT. There is no doubt that to a great extent the mesenchyme here described contributes towards the formation of blood- vessels and blood. The protochordal plate furnishes the endothelium of the heart, as I have elsewhere demonstrated for Tarsius (702, Pl. IX, fig. 73, a and 6b), the annular zone produces the material for the area vasculosa on the umbilical vesicle. To that effect mesenchyme cells, which originated at an early stage in the annular region here alluded to, migrate over the surface of the umbilical vesicle and come to be situated between the layer of entoderm which forms its inner, and of splanchnic mesoderm which sooner (Pri- mates) or later (other mammals) forms the outer wall of this vesicle. Besides these lateral portions of the annular zone the hinder portion of it, situated diametrically opposite to the protochordal plate, has yet an important part to play in the formation of blood-vessels and blood. From it the vascularisation of the Haftstiel of the Primates is derived. From the distal end of this connective stalk vessels irradiate towards the whole inner surface of the diplotrophoblast (man and anthropomorphe) or only towards a restricted circular part of it (Tarsius). This vascularisation must phylogeneti- cally have preceded (as we will discuss later on) that which comes about by means of a free allantois. The thickened entoderm in this hinder part of the ring is especially marked in Manis. After a comparatively short time the annular entodermal region has ceased to be a focus of mesenchyme production; henceforth the increase of the vasifactive tissue is left to mitoses of the cells already constituting it. We may after careful consideration of all the mammalian preparations at our disposal all the more safely conclude to the existence of such migration of vasifactive cells if we consider that in other vertebrates (‘Teleosts) this very phenomenon has been actually observed in the live embryo by Wenckebach (86), Ziegler (’87), and others. In how far yet other tissue than blood-vessels and blood pathies with Kleinenberg’s (’86) drastic expression, “Ks giebt kein mittleres Keimblatt.”’ BKARLY ONTOGENETIC PHENOMENA IN MAMMALS. 35 spring from this entodermal proliferation will require very close investigation in all the different orders of Mammals. 2. Developmental Processes in the Ectoderm. I have on purpose postponed the discussion of the processes of proliferation in the ectoderm because in modern text- books those in the entoderm are generally ignored or even denied, whereas as a matter of fact they are antecedent, at least in their very earliest appearance, to those which con- cern the ectoderm. About the latter a very stately list of investigators, including many of the foremost embryologists, have published the results _ both of observation and of reflexion. Still we cannot say that at present a general consensus of opinion concerning these processes has been arrived at. They have been very ably summarised by O. Hertwig in his “Lehre von den Keim- blattern” (703, pp. 918—940), and to that author I would direct those who are interested in the historical development of the different views held on this point. This will give me occasion to skip at the present moment all controversial matter, and will allow me to put forward my own view of the case based on the examination of numerous early stages of different mammals. The point of divergence with other authors will then be noticed only afterwards. a. The Protochordal Wedge.—At the time when the two germ-layers of the round or oval embryonic shield are not yet interlocked, but independent of each other, and when the future front region of that shield can already be distinguished by the proliferation in the endoderm noticed in the preceding paragraph, and many years ago designated by me (’90) by the name of protochordal plate, a proliferation, directed down- wards, of the ectoderm in the axis of the embryonic shield and somewhere in the posterior third of it, becomes visible. I have no hesitation in saying that this spot coincides with the anterior lip of what was described in Chapter II as the 36 A. A. W. HUBRECHT. evanescent blastopore of the didermic gastrula stage of mammals. However, only in some few mammals has this blastopore been shown to appear as an actual though very temporary and evanescent perforation of the embryonic shield (Figs.53—57). The proliferation has been known by the name of its first observer as ‘‘ Hensen’s knob;” it has also been called the primitive knob (Bonnet, ’89, pp. 38 and 40) ; for myself I wish to adhere to the name I have proposed for it many years ago (790, p. 501) and call it “ protochordal wedge,” as I have called the entodermal proliferation “ protochordal plate.” The point of importance in my wishing to stick to these names is that the next step in mammalian development is the firm fusion between these two independent prolifera- tions that have arisen in quick succession in the two independent germ-layers, and that will henceforth be no more disconnected (Figs. 47, 48, 52, 57, 97—99). ‘I'he noto- chord is built up of material situated in the axial line of these proliferations, hence the names. Already Hensen has correctly observed (’76) that below the rounded knob which he found projecting downwards from the ectoderm, the degree of firmness with which ento- derm and ectoderm cling together is at its maximum, and should be looked upon as an effective fusion of the two layers. ‘This is fully confirmed both by transverse and longi- tudinal sections. I found the same in the shrew (Fig. 57) and more lately, in a yet higher degree, in Tarsius (Figs. 48 and 52). In 'T'arsius where we have already seen on p. 32 how very massive the protochordal plate was, the protochordal wedge pushes downwards just behind it, over that part of the ento- derm which again consists of flattened cells. The fusion between the proliferated endoderm and ectoderm cells, not yet effected in the section of Fig. 49, comes about immediately afterwards (Fig. 48). There is not the slightest evidence in Tarsius that the knob-like ectodermal proliferation which we have called the protochordal wedge undergoes any extension forwards which could be identified with what German authors HUBRECHT. Pi: o Wig ee CPA ATUL ase ts Meelis ! aie pata ni 8S Fig. 87. Longitudinal section through on early embryo of Amia (after Bashford Dean, 96). This stage is comparable to that of Rana (Fig. 80) and of Hypogeophis (Fig. 85); 2% protochordal plate. cz notochord, vz ventral meso- blast, aZ portion of intestine comparable with allantois region of higher verte- brates. — Fig. 88. Longitudinal section of early muraenoid embryo (after Boeke, 03). pp protochordal plate, pw protochordal wedge, fv proliferation homolo- gous to the ventral mesoblast. — Fig. 89. A section of the hinder portion of an older muraenoid blastodisk. vz ventral mesoblast. 47 Kupffer’s vesicle; ch notochord (after Boeke, 03). — Fig. 90. Longitudinal section of the hinder part of a Salmonid embryo with Kupffer’s vesicle (after Ziegler, 02). — Fig. 91 and 92. Two sections through the anterior part of two different blastodisks of the dog (after Bonnet, ‘01).. The protochordal plate (Af) is proliferating in both of them. EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 37 have called the “ Kopffortsatz.” On the contrary, the moment the fusion with the protochordal plate has come about, a process of growth sets in of the tissues here con- sidered, not in a proximal, but in a distal direction. Asa comparison of Figs. 48, 52, 98, and 99 shows, the embryo- nic shield increases in length, and at the same time the dis- tance between the spot where the protochordal wedge has originated and the front end of the ectodermal shield becomes more considerable. But during this process the situation of the point of fusion between protochordal plate and proto- chordal wedge may be said to be more or less constant (though not actually any longer discernible), whereas both plate and wedge have increased in length at a relatively equal rate (see Figs. 95—95). And so the protochordal wedge becomes undoubtedly lengthened, not, however, by its send- ing out any “ Fortsatz,’ but by its being, so to say, “ spun out” in consequence of the backward growth of the tissue that is going to be the notochord,! thanks to new ectodermal proliferation being added to what had previously come into existence, and had fused with the entodermal protochordal plate. A thin canal is noted in mammals in the posterior part of the backward proliferation of this protochordal wedge (Figs. 98 and 99, ne, cn). “ b. The Ventral Mesoblast.—We will now for a moment leave the protochordal wedge and inquire whether, besides this, any further contribution of the shield ectoderm towards the formation of tissues between it and the endoderm takes place. In this respect T'arsius has proved to be a genus of mam- mals, which is of the utmost importance in throwing light upon these much disputed questions. Monkeys and man— 1 T am inclined to think that, if all those investigators that have stood up so decisively for a forward growth of the mammalian “ Kopffortsatz” in other genera of mammals, were once more to look closely at their preparations, they would be willing to leave the possibility open that this forward growth may also in their case be an elongation, by material being added posteriorly, con- comitantly with the inerease in length of the shield, 38 A. A. W. HUBRECHT. as soon as we come to know their development in these same early stages—will, in all probability, fully confirm what Tarsius teaches us, considering that in so many other impor- tant points ‘arsius is seen to resemble the other Primates most closely, and that in this very detail: the presence of an extra embryonic ccelom at a stage ever so much earlier than in any other mammal, there exists perfect uniformity. In Tarsius there is no doubt that before the appearance of the protochordal wedge (Hensen’s knob) in the posterior third of the ectodermal shield, another ectodermal prolifera- tion has already preceded Figs. 47—50, vm.), the products of which have important parts to play in the constitution both of embryo and blastocyst, different, however, from those of the protochordal wedge. This earlier ectodermal proliferation is primarily directed backwards (Fig. 49), whereas the protochordal wedge has a faint inclination forwards (Figs. 46 and 48). Like this it is median and unpaired. We will call this posterior proliferation the origin of the ventral mesoblast (Hubrecht, ’02, pp. 19 and 31), and we may emphasise that, whereas the wedge appeared in the hinder third of the ectodermal shield, this ventral mesoblast originates still further back (separated from the wedge by the potential blastopore) at the posterior extremity of the embryonic shield, there where the trophoblast is often quite sharply differenti- ated (cf. Figs. 48 and 49) from the embryonic ectoderm. We encounter this proliferation as soon as the entoderm after its delamination from the embryonic knob is busy forming a vesicle under the embryonic ectoderm (Figs. 44 and 62). This endodermal vesicle, as was seen. in the preceding chapter (p. 8), never fills the whole of the blastocyst. Now the proliferation at the hind end of the embryonic ectoderm, which we consider as the primordium of the ventral mesoblast, hollows out at the very earliest moment, thus forming a second vesicle enclosed inside the trophoblast. The cavity of this vesicle should be classed as extra-embryonic celom; its walls, where applied against the trophoblast HUBRECHT. amn Pl. S. 96a 96b Fig. 93 to 96. Four surface-views of the embryonic shield of Tarsius. In Fig. 93 the median concrescence of protochordal wedge and protochordal plate has come about and notogenesis has commenced; in Fig. 94 and 95 the region of the dorsal mouth (primitive streak) has become elongated simultaneously with the HUBRECHT. PU: early establishment of notochord and bilateral mesoblast; in Fig. 95 first in- dication of neurenteric pore which in Fig. 96 has travelled backwards conside- rably. — Fig. 96a, b and 96c. Two further stages of Tarsius development following upon those of Fig. 93—96. — Fig. 96a and 96b. Dorsal and ventral view of a blastodisk with about five somites. Ws Headfold seen from below; azz am- nionfold; @/ allantoidean tube, seen by transparency; @//J7 wide mouth of al- lantoidean tube in umbilical vesicle; zc neurenteric pore. — Fig. 96c. Later dorsal view, amnion nearly closed. sf connective stalk, ¢-w part of trophoblastic wall of blastocyst. — Fig. 97, 95 and 99. Longitudinal sections of the embryonic shields respectialy corresponding to Fig. 94 to 96. zr trophoblast, v7 ventral mesoblast, a@// allantoic tube (present, but not indicated in Fig. 98). fp proto- chordal plate; ch notochord; cz, o7 mc neurenteric canal; aa and ap anterior and posterior amnionfold; fev pericardium; % heart. Fig. 93 to 99 after Hub- recht (02). EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 39 (which then becomes a diplotrophoblast or chorion) thereby render the peripheral blastocyst didermic, and may be styled parietal or somatic mesoblast; where applied against the endodermal vesicle they fall under the category of visceral or splanchnic mesoblast (cf. Figs. 45 and 63). At the initial spot from whence the proliferation has started the ventral mesoblast is naturally more massive than in the peripheral, flattened portions, and may here be designated as the material out of which the primitive streak and the ventral stalk (Haftstiel, Bauchstiel) of the Tarsius embryo takes its origin. This stalk-shaped connection between embryo and trophoblast is thus present in the very earliest stages of development (Fig. 62). My conception of the ventral mesoblast in mammals has since been adopted by Riickert in his article above cited (’06, pp. 1248 and 1251). He compares it with the observations hitherto recorded of mesoblast formation in the same region in other Amniotes. From its posterior unpaired and median point of origin in 'l'arsius it gradually spreads forward right and left as the wings of mesoblast are known to do in other mammals (“ Mesoderm-sichel”’), and only later this vesicular mesoblast (vesicular, because the ccelom is there from the beginning, and does not, as far as the extra embryonic ccelom is concerned, originate by any ulterior splitting process) also appears in front of the embryonic shield, and invades the space (cf. Figs. 62 and 63) where the anterior and superior entodermal surface of the umbilical sac are yet in close oppo- sition with the trophoblast (Hubrecht, ’02, Figs. 48, 5lc, as compared to 57a, c). The posterior median portion has simultaneously further developed into the incipient, as yet extremely delicate Haftstiel which as we saw is there from the very beginning, i. e. from the didermic stage downwards. 40 A. A. W. HUBRECHT. 8, Mutual Relations between the Centres of Proliferation. We must now consider the relation in which on the embry- onic shield the centre of proliferation of the ventral meso- blast stands to that, which we have designated as the proto- chordal wedge. In general it may be said that in the earlier stages the former lies immediately behind the latter. We may add to this that if Tarsius were possessed of a blastopore in the didermic gastrula in the same way as Hrinaceus is (and as are some other mammals) the situation of this blastopore would be such as to separate these two centres of prolifera- tion. This becomes evident when we consider the exceptional case already noted above (p. 14) where the embryonic shield of a particular specimen of Tarsius was provided with a deep pit-like impression (Fig. 52) which cannot but be looked upon as an attempt at blastoporic perforation of atavistic sigmifi- cance, the very numerous stages of Tarsius of identical age which I have in my possession not revealing a trace of it. Other cases in which the contiguity, but at the same time the mutual independence of the two centres of proliferation is evident were figured by me in a former publication (’02, fis. 58b, 46d, 47, 48,52, b and c).1 Out of them all (see also Figs. 47, 48, 49, and 50) I have constructed the semische- matic diagrams, Figs, 44—46, IT need hardly explain that the presence, close to each other, of three centres of proliferation (one entodermal, two ectoder- mal) in the two germ-layers of the mammals, such as we have just described, combined with the fact that in each centre new cells are very actively developed which spread out in the only direction available to them, i.e. between these two germ-layers, 1 J will here notice that the mutual independence here insisted upon should be taken cum grano salis. The anterior and the posterior lip of the blastopore, being naturally connected by the lateral lips, it is not a material anatomical independence that is here meant, but an independent activity. On p. 44 this will be more fully entered into. EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 4] brings about a state of things in which it very soon becomes utterly impossible to say to which of the three centres a given cell or group of cells owes its origin. An intimate fusion, though withdrawing this question of cell-lineage out of the field of our powers of discrimination, does not, however, dimi- nish the significance of the existence of such a cell-lineage,! and we will in future researches have to keep our attention directed to that point, even though we must recognise at the present moment that much of the confusion and of the erro- neous notions that maintain such a hazy atmosphere round these important early phases of vertebrate development, is due to precocious generalisations on this head. It seems to me that the wish to uphold the reality of a third germinal layer, together with the ardent desire of not having to ascribe a multiple origin to it, is responsible for much theo- retical dogmatism that will henceforth prove valueless. The consequence of what we have here described for Tarsius is that the centres of proliferation which give rise to the protochordal wedge and to the ventral mesoblast are originally independent of each other. We shall by-and-bye see that there is all reason to believe that the same holds good for all other Mammalia, aye, for all other Vertebrates. The principal difference between my own and the current views consists in the distinction which I wish to make between what was considered as the front portion of the primitive streak (Hensen’s knob, of which even the anterior prolongation was called in full: ‘* Kopffortsatz des Primitivi- treifens”’) from the primitive streak material itself. This distinction, which is very soon effaced and could never be demonstrated in later stages, is, however, quite evident in the very early ones. And we will have to analyse, as acutely as we can, the differences this will call forth in our interpretation of the development of different tissues and organs concerned. The ventral mesoblast at its very earliest appearance (also in Tarsius) may be said—as it springs from the hinder end of the ectodermal shield—to be more or less crescent- or fan- 1 Vide E. B. Wilson (’92, °97), as against Driesch and others. 42 A. A. W. HUBRECHT. shaped. We will again encounter this crescent (or “ Sichel”’) shape in Sauropsids. But as the embryonic shield increases in length the centre of proliferation is equally stretched, and out of a crescent shape evolves a double wing-shape, the axis between the two symmetrical wings being in the axis of the embryo. Along this axis the ectoderm freely produces cell material penetrating downwards to the right and to the left between the germinal layers and forming what has often been designated as primitive streak-mesoblast continuing backwards in the median line as the connective stalk (Haftstiel). Here we encounter an all-important phenomenon, which will be better understood when we have also considered it phylogenetically, and which consists in the substitution of what was at the outset the blastopore by what has later developed into the dorsal mouth-slit. The lengthening of the tissue which formed the lateral lips of the early blastopore has now set in, and the further proliferation of this tissue, concomitant with a process of coalescence of the right and left halves with reminiscences of the original lumen which was the slit-like cavity of the stomodzeum (in the ccelenterate stage), brings our original centres of proliferation further apart. At the same time the continuity of the tissues is never interrupted. The accumulation of cell material which represents the lateral lips of the dorsal mouth-slit (Riickenmund) naturally causes an increase in length of the mammalian embryonic shield, during which the shape of the shield generally changes from a roundish to an oval or pear-shaped one (see Figs. 93—95). This lengthening is simultaneous with an in- crease in the extension of the lateral mesoblast wings (see Fig. 60). For Tarsius I have fully established this a few years ago (02, Figs. 54,57, 61,72). And for other mammals it has been demonstrated by Bonnet (’97, Figs. 18, 19), Keibel (?95, 795), and others. As soon as this accumulation of material that reveals itself in the increase in length of the embryonic shield has EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 43 reached a certain stage, an active process of transformation is inaugurated, which consists in the visible differentiation of all-important organs, notochord and somites, out of this matrix. ‘The differentiation becomes first visible at the front end where our ectodermal proliferation, the protochordal wedge, has grown downwards and has coalesced with the protochordal plate. From this point backwards the noto- chord is now, so to say, spun out, the so-called primitive streak tissues—lateral lips of the dorsal mouth—at the same time diminishing in extent. Phylogenetically it corresponds to the origin and the coalescence of the lateral lips, not of the blastopore, but of the dorsally-situated stomodzeum. A comparison between Figs. 983—95 and 96 will at a glance reveal the effect of the new state of things. The protochordal wedge situated well forwards on the embryonic shield of Fig. 95 is no longer visible as such. ‘The fine pore that was present just behind it (see the longitudinal section in Fig. 98) has undergone a displacement back- wards, and has in Fig. 96 attained a position not far from the hinder end of the embryonic shield. This is due to a very marked process of elongation which becomes perfectly evident on the comparison of two longitudinal sections through these two embryonic shields (Figs. 98 and 99). This process has been known to earlier observers, and has been described as the shortening of the primitive streak, going parallel to the formation of the earliest somites. How the cell material that has arisen as the paired wings of the ventral mesoblast and that which is spun out by the moving backwards of the protochordal wedge (producing the noto- chord in the median axis and the mesoblastic somites right and left of it) comes to arrange itself reciprocally and what changes are brought about in this material during this process is a very difficult and intricate question about which the various authors differ. I think we may safely say that by the rapid extension backwards of the differentiation process, as it is exemplified by Figs. 95 and 96, the dorsal region of the trunk is laid down in outlines (hence the word 4A A. A. W. HUBRECH'. notogenesis), whereas the derivates of the ventral mesoblast find employment in the construction of the posterior and postero-ventral portions of the embryo. It will here suffice to state that the extra embryonic ccelom which is present in Tarsius (and undoubtedly in monkeys and man) in the ventral mesoblast at so very early a phase (extending as was described on p. 38 behind and below the endodermic vesicle and the ectodermal shield) first makes its appearance in the other mammals at a later period, but exactly in the same position, viz. behind the embryonic shield (Figs. 43, 61, and 100). From there it eradually extends in crescent shape right and left along the hind margin of the embryonic shield. This ccelom—con- siderably less spacious and less precocious than that of the Primates—is fully homologous to it, both as regards the place where it is found, the cell material in which it appears, and the relation in which it stands to the ccelom of the somites and lateral plates, as will be described later on. Bonnet’s (82, 789), Keibel’s (93), and my own (’02) observations on the appearance of this crescent-shaped ccelom are in perfect agreement with each other, as also those concerning the fact that this ventral coelom only later fuses with the intra- embryonic ccelom (Keibel, 93, figs. 39 and 402; Hubrecht, 02, fig. 77d). The pericardial coelom arises independently along the front border of the embryonic shield, and will also be more fully discussed later on (Hubrecht, ’02, p. 87, figs. 70, 73). Summarising what we have here rapidly sketched we may agree to have seen that instead of a homogeneous median germ layer, instead of a mesoderm which has the same mor- phological importance as the two primary germ layers and ori- ginates from the coalescing lips of a blastopore, we find at least three foci of cell-activity in those primary germ layers. The appearance of these foci marks the end of the didermic stage of the blastocyst. In consequence of processes of prolifera- tion and rapid mitosis there is started from these three centres a host of new cells, which, together, spread between HUBRECHT. Pl. U: 100 101 sfitOME ER a SF 5. cy SR te i Baars ee DEO, One: (sovaus opine af Ss: Sn Oo peat ‘e} 102 103 104 Fig. 100. Longitudinal section through the posterior end of the shrew’s blastodisk with earliest appearance of posterior amnionfold (after Hubrecht, ‘90). co posterior coelom, vz yentral mesoblast, ¢ trophoblast (cf. Fig. 56 and 61). — Fig. 101, 102. Two sections through different blastodiks of Tarsius (nes 675 and 180 Utr. coll.) in the posterior region of the primitive streak (after Hubrecht, 02). Fig. 102 is in the more posteriorly situated, at the spot where the tailgut (Schwanzdarm) and the allantoidean tube @//, here situated ventrally of the for- mer and on the point of diverging. The wall of both is actively proliferating vascular tissue. Between the lower border of the allantois and the umbilical vesicle a complex of yet more actively proliferating cells is present: this conti- nues further backward (where allantois and umbilical vesicle have become further severed) as a median proliferating raphe on the umbilical vesicle. In Fig. 101 lateral wings of mesoblast arise from the entoderm. — Fig. 103. Transverse section through the hinder end of an early Tarsius embryo with tubular am- nion (am) and allantois /a//) in the already strongly vascularized connective stalk cs; wv wall of the umbilical vesicle. — Fig. 104. Longitudinal section of sparrow with early mesoblast in a stage of about Fig. 105 (after Schauinsland, ’03). EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 45 ectoderm and entoderm in the shape of what appears naturally as a flattened layer of so-called mesoderm, but what is in reality the strictly grouped material for different organs and tissues. ‘These have not sprung from the lips of any blasto- pore (Urmund), but have gradually come into existence in the same ontogenetical order as we must expect them to have arisen phylogenetically, “Lhe blastopore has lengthened out into the dorsal mouth. This lengthening has been accom- panied by a dorso-ventral proliferation of ectoderm (proto- chordal wedge) out of which the stomodeeum (notochord) arises, and during this time the dorsal mouth-slit has only been represented by vestiges. I have already discussed these processes elsewhere (05). The dorsal, elongated mouth (Riickenmund, 705, p. 363) may thus point to a vermactinian- like ancestor (Fig. 160) in which the appearance of notochord and coelomic pouches was already foreshadowed by the sto- modeeum and the enteric diverticula to which the stomodzeum gives access. It is far outside the scope of this paper to establish in detail the cell-lineages as they may ulteriorly be found to exist, and which will some day allow us to ascribe to each of the three centres of proliferation here alluded to, its part in the forma- tion of the Anlage of different organs and tissues between ecto- and entoderm. It should, however, be noticed that already in my publication of six years ago (’02, Pls. 8 and 9, figs. 59g and 7o/) I have distinctly figured the fact that in the posterior region of the embryonic shield a very considerable part is played by the entoderm in the development of the Jower half of wings of mesoblast of which the upper half springs directly from the ectoderm (Figs. 101—103). These and many other phenomena will have to be minutely studied and established before we can commence our com- parative analysis of these processes in Vertebrates. But it should be borne in mind that the processes just alluded to have already been mentioned on p. 34, when the vascularisation of the “ Haftstiel’? was discussed. And that in the diagram, Fig. 46, the posterior source of proliferating 46 A. A. W. HUBRECHT. entoderm is clearly indicated as forming part of the ring that is figured for Sorex in Fig. 60. The ultimate discussion upon this matter is postponed to a later publication, in which those stages of development which are inaugurated by the formation of the somites will be treated more fully. IJ. AMPHIBIA. After this description of the early developmental processes of the Mammalia we will skip the Sauropsida, and first describe what is noticed in the Amphibia. ‘This will after- wards afford us an occasion to compare the yolk-laden Saurop- sida all the more rapidly both ways. And, above all, it will increase our confidence in the interpretation which we have founded on the Mammaliaif we find it applicable as low down in the line of vertebrate descent as are the present Amphibia. It should, however, at the same time, be remembered that none of the three living stems of Amphibia neither the Gym- nophiones, nor the Urodeles, nor the Anura can be expected to stand in any way on the direct line of descent of our present mammals. Comparative anatomy has taught us (Fiirbringer ’00) that in very many respects the amphibious Promammalia of the Paleeozoic epoch must have been charac- terised by important points of difference from all the living remnants of that ancient stem. Still, if we find processes of early development that are in the main lines directly com- parable to what we have described in mammals, and if they fit in well with the explanation which we have ventured to give for the Mammalia, we might say that the difficulties which have so often been complained of (p. 13) when attempt- ing to establish the comparative ontogeny of the Vertebrates have greatly diminished. We will, therefore, take the more important and careful descriptions of Amphibian development (to which we have no personal investigations of our own to add), and see whether the three centres of proliferation which we have noticed in EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 47 the two germ-layers of the mammals are also present in the Amphibia, and whether the mutual relations of these pro- liferating centres and the further fate of the tissues and organs they produce also reveal close similarity. We begin with the Gymnophiones, about the early phases of which A. Brauer (’97) has published an exceedingly lucid exposition based on the study of an extensive material. We have successively to look out for a proliferation of the entoderm corresponding to our protochordal plate, for an ectodermal proliferation representing the protochordal wedge, and for another ectodermal centre of growth which gives rise to ventral mesoblast. I will by means of copies of Brauer’s figures show that all three are met with in Hypogeophis and that in their further relations and in the genesis of the organs produced by them the homology with the mammals is indubitable. It should at the outset be remembered that the Hypogeo- phis egg is so saturated with yolk-material, that there is no holoblastic cleavage, but that the results of the cleavage pro- cess—as was noted jn Chapter IJ—are found at one pole of the egg, and that a\process of delamination transforms the fragmented ovum without delay into a gastrula with an entodermic lower layer (see Brauer, ’97, Figs. A and B, pp. 405, 404). More or less simultaneously a differentiation of ectoderm and endoderm becomes visible, which shows very great simi- larity with what was figured above (Fig. 19) for Tarsius. The point at which the ectoderm has commenced to proliferate, and at which its first change was a bend downwards (Fig. 83) is directly comparable to the primitive (Hensen’s) knob on the mammalian ectodermal shield, and is no other than our pro- tochordal wedge. The point at which the endodermal pro- liferation becomes evident is situated just in front of it and the two proliferations, as is so particularly clear in Brauer’s fig. 43, here reproduced as Fig. 84, fuse in absolutely the same way as we see in the ''arsius, Fig. 48; with full confi- dence I indicate the corresponding regions in the Amphibian 48 A. A. W. HUBRECHT. by the letters Pp. (protochordal plate) and Pw. (proto- chordal wedge). Brauer’s figure leaves not the least doubt that the cells indicated by Pp. are of entodermal, and the cells Pw. of ectodermal origin, nor do his own views on this point differ from mine as he calls the former “ vegetative,” the latter ‘animale Zellen.” The later transformation of this henceforth fused region of double proliferation (see Figs. 85 and 86), fused on entirely the same plan as was noted not only in Tarsius but in very numerous other mammals, will be discussed later on. We must first look out for the third centre of proliferation. And we find this in Brauer’s fig. 59, here copied in Fig. 86 where at a short distance behind the protochordal wedge and separated from it by an interval comparable to what we notice in the Tarsius, Figs. 46 and 48 (where the interval is at its minimum), the ectoderm is seen’ to undergo a new and very marked proliferation, which will give rise to tissues closely corresponding to the ventral mesoblast which we saw origimating in this spot in mammals. The difference between the case of Tarsius and Hypogeo- phis is this, that in the former this posterior centre of proliferation is conspicuous first of all, whereas in the latter the two other centres have precedence. However, in this respect the other mammals side with the Amphibia, the protochordal plate and wedge being visible before or arising simultaneously with the proliferating centre for the ventral mesoblast. Having thus established well-founded comparisons between Brauer’s figures of early Gymnophiones (Ceecilia) and our own for mammals, we will now turn to the Anura, and take as starting-point Brachet’s figures (’03) of the frog. In his earlier publication of the year 1903 (Figs. 6, 7, 39—47) we find that Brachet describes early stages both for the Axolotl and for the frog in which the presence of a proto- chordal plate can hardly be denied by any impartial observer, One of his figures, copied here in Fig. 79, leaves little doubt about the presence in the entoderm of the frog ofa nee EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 49 particular spot of thickened entoderm situated at the very place where we would expect the protochordal plate at this earlier stage. His other figures, which are also copied here (Figs. 80—82) show the subsequent stages. And as to the annular band of entoderm from which the blood-vessels and blood originate, we find it similarly dis- posed in the Amphibia according to Brachet, who for the frog writes (03, p. 686), “les endotheliums vasculaires y compris |’endothelium endocardiaque et les futures cellules rouges du sang, procedent de la portion du mésoblaste : qui s’est séparée par délamination de la partie ventrale de l’endoblaste gastruléen.” And further (’03, p. 688), “‘ de tout le vaste manchon mésoblastique qui se délamine a la surface de l’endoblaste gastruléen, la partie ventrale, sur une largeur plus ou moins grande selon les régions, se sépare complétement du reste 4 des stades relativement peu avancés, et, poursuivant dés lors une évolution spéciale, donne nais- sance a tout lappareil vasculaire sanguin (endotheliums vasculaires et cellules rouges du sang).” The term manchon (muff) used by Brachet shows that he, too, has observed the region of the entoderm which produces the mesenchyme out of which the blood-vessels and the blood are developed in the shape of an annular peripheral invest- ment of the region out of which the mediodorsal organs will develop. With most laudable prudence Brachet does not generalise his results concerning the frog, but states expressly that for Triton he is inclined to stick to the conclusion he arrived at already in an earlier publication (’98), and according to which also in Triton the vascular system is of entodermic origin, but that he all the same thinks a further confirmation of his observations desirable, whereas for Axolotl he makes all reserves, the study of the origin of the vascular cells being here very difficult. He is careful to add, however, that he cannot exclude the possibility that after all Axolotl may prove to fit into the same plan as the two others. Other authors who, before Brachet, have come to similar VOL. 53, PART 1.—NEW SERIES. 4, 50 . A, A. W. HUBRECHT. conclusions concerning the origin of the vascular system in the Amphibia are Goette (75) and Schwink (’91). Both are convinced that all blood-cells are derived from the endoderm, as also the vessels. Brachet points out, however, that the stages on which Schwink bases his conclusions are already too far advanced. It is important that Brachet, repeating Corning’s (799) observations, finds that in front of the notochord’s anterior end the median protochordal-plate-material differentiates from behind forwards in this sense that mesoblast is seen to become isolated and to form a thin layer made up out of one or two layers of cells that are interposed between the ento- derm and the lower brain wall. In the beginning he finds that the anterior end of the notochord reaches into this median mesoblastic band... Soon, however, it 1s separated out of it and the anterior extremity of the notochord becomes quite free. Later yet the median mesoblastic band thins out, breaks up and ultimately disappears, or is reduced to a few sparse cells that are distributed at random. ‘I'he endoderm of the roof of the digestive tube is then closely pressed against the lower wall of the brain. This would, ceteris paribus, also apply to the mammalia. The next point we have to consider concerns the fusion described for mammalia by myself (90) and others, and for Hypogeophis by Brauer of what we have called the proto- chordal plate with the protochordal wedge. Neither amongst Brachet’s figures for Axolotl, nor among those for the frog, are the phenomena so self-evident as they were for Brauer’s Hypogeophis. Still if we consider Brachet’s figures of Axolotl and those for the frog no objection can reasonably be raised against my comparing the region which in all these figures I indicate by Pp. with that same region in Hypogeophis and in mammals. The fusion with the ectoblastic proliferation that is the protochordal wedge—although Brachet does not look upon it in that light—is inaugurated for Axolotl in Brachet’s (03) Figs. 4 and 5; for Rana in Fig. 79, here given. The ectodermal proliferation indicated as protochordal wedge is EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 51 thus located in the Amphibia (the same holds good for Hypo- geophis) at the spot where the so-called dorsal lip of the blastopore makes its first appearance. And this proliferating spot (as was already noticed in Mammals and as Brachet (702, 703], Bellonci [’841, and Lwoff [’94] have observed it for Amphibia), travels backwards a certain distance over the surface of the egg, spinning out at the same time both noto- chord and somites. In the frog I have in the reproduction of Brachet’s figures indicated the corresponding regions by the letters Pp and Pw. Pp points to most decided entoderm which has by delamination become separated from the ectoderm situated above it. And the proliferation of ectoderm marked Pw and fused (more markedly yet than in the somewhat earlier phase of Axolotl) at the very outset of its proliferation with the protochordal plate entoderm below it—about in the same way as we encounter the same phenomenon in Fig. 48 for Tarsius—has here already progressed a certain distance backwards. This distance has lengthened and the derivates of what was originally the protochordal wedge have increased in Fig. 80 (Rana), and yet the letters Pp leave no doubt but that they are directed towards the original entodermal pro- liferation. So does Pw show us what has become of the ectodermal proliferating centre. We can quite understand that also concerning Amphibia dissensions have existed as to whether the notochord—first and foremost derivate of Pw— was of entodermal, mesodermal or ectodermal origin. And the different authors that have successively sided for the one or for the other of these solutions have pronounced them- selves as best they could upon a material that is so far away from the extraordinary clearness with which these very early phenomena reveal themselves to usin Mammals. The con- tinuity in which from the beginning ectoderm and entoderm pass into each other (Fig. 79) all along the ring-shaped zone of delamination (marginal zone of Goette), has contributed so extraordinarily to propagate and to strengthen the error of those who upheld that the phenomena which we have just seen 52 A. A. W. HUBRECHT. inaugurated are phenomena of gastrulation, instead of phe- nomena of notogenesis, that consequently all conclusions were naturally biassed. However, the mammalia have come to show us the way out of the labyrinth and a reformation of our views must be the consequence. Without for the present entering into further details concerning protochordal plate, protochordal wedge and their derivates in the frog, we will now see whether the third centre of proliferation which we also have recognised in Brauer’s Gymnophiones is as clear in the Urodeles and the Anura. On this point Brachet’s researches, and those of other authors afterwards to be cited, leave no doubt whatever. The ectodermal centre of proliferation, hitherto known as the ventral lip of the blastopore is very clearly marked off (Figs. 80—82), and produces its mesoblastic derivates with perfect regularity, in a sequence that is immediately com- parable to what we have found in and described for the mammals. Brachet writes about this third centre (’05, p. 67) that it is: “‘ Un épaississement notable de la partie toute inférieure de Vlectoblaste.’ And further (I. c., p. 68): “Meme épaississement considérable de l’ectoblaste qui vient par une large base se continuer avec les éléments du bouchon vitellin et cela 4 une certaine distance dans la profondeur de Poeuf.” We have here before us the ventral mesoblast which in Tarsius (and the other Primates) arises so uncommonly early and stretches round the umbilical vesicle, creating a very early segregation of splanchnic as against somatic mesoblast. When we take the Figs. 81 and 82 (copied from Brachet) we can immediately recognise the homology between the region marked vm and that of Figs. 46,48, and 49, to which the same lettering is attached. We also see that if the mesoblast there produced in the Amphibia were to attain the early development it has in Tarsins and the cavity enclosed therein, this would similarly take the place of the so-called segmentation cavity, and be applied against the EARLY ONTOGENETIC PHENOMENA IN MAMMALS. 53 cavity which in the mammal is styled the umbilical vesicle, and which is the so-called archenteron in the Amphibian. The concrescence between this and the segmentation cavity is the same as is noticed at the yet earlier stage of Tarsius (Fig. 19), in which the entoderm forms the roof of the trophoblastic cavity. But we will return to these possible comparisons later on. It remains to be seen whether in other Amphibia than Rana and Triton the presence of a fourth focus from whence tissues are originated that take their place between ecto- and ento- derm can be confirmed. In other words, whether any- thing corresponding to the annular zone of mesenchyme- producing entoderm (stretching backwards right and left from the protochordai plate and reuniting in the median line posteriorly under the ventral mesoblast) as it was figured in Fig. 60 occurs in Amphibia. Although Brachet has not expressly stated that such an annular zone of entoderm was noticed by him, we may conclude from his descriptions that it does occur in his preparations. On p. 88 (’03) he writes about: “ L’intense activité que l’on pourrait appeler mésoblastogéne des cellules de la voute”’ (by which latter he means the roof of the archenteron); and on p. 89: ‘‘ Les bandes mésoblastiques sont plus épaisses dans la région blastoporale que dans la région gastrale proprement dite ... Le mésoblaste péri- stomal est beaucoup plus abondant que le mésoblaste gastral (p= 90)” From these citations I think we may conclude that the presence of an annular zone of mesenchyme-producing entoderm in Amphibia will in due time be yet more fully established. Authors who have actually figured it in the posterior median line of the embryonic Anlage are Robinson and Assheton (91, Figs. 14—17), where in the median region of the blastopore and behind it we notice an entodermal proliferation producing what the authors call ‘ hypoblastic or inner layer of mesoblast of primitive streak,” as against DA A; A. W. HUBRECHT,. the “epiblastic or outer layer of mesoblast of primitive streak.” This investigation thus authorises a direct com- parison of the phenomena figured in Figs. 101 and 102 for Tarsius, in which we have quite decisively recognised an epiblastic and a hypoblastic layer of mesoblast of the primitive streak, which is what was noticed in the frog by Robinson and Assheton. It should yet be added that the annular zone of mesen- chyme-producing entoderm may in the frog even commence as an unpaired ventral sheet, which only later becomes paired, and thus more or less annular. Brachet (’03, p. 686) expresses this as follows: “Il existe une phase du développe- ment ot les cellules vasculaires des futurs vaisseaux vitellins forment une couche continue impaire et médiane (Fig. 22) et la parité définitive est secondaire.” We have now seen that in the three subdivisions of the Amphibia we notice early processes of ectodermal and ento- dermal proliferation, which allow of direct comparison with what we have described for the Mammalia. And we may add that the continuity between the derivates of the proto- chordal plate and those of the annular zone is in Amphibia established even perhaps earlier yet, whereas the continuity of these latter derivates with those mesoblastic elements that originated right and left of the median dorsal line is again so very early established, that it cannot be wondered at that the Amphibia have not suggested to previous investigators the relative independence of these different sources from whence cells and tissues that will take their places between the two primary germ-layers arise. When we will later recapitulate what are the further lines of development of the products of the four proliferations above enumerated, the complete homology between Amphibia and Mammals will become yet more evident. EARLY ONTOGENETIC PHENOMENA IN MAMMALS. a5) III. SavrorsipA AND ORNITHODELPHIA. As to this class I have no observations of my own to offer. But we may glean from the researches hitherto published by others the following data which concern the participation of the entoderm in the formation of mesenchyme. For the sparrow Schauinsland (’03) figures both a surface view, a longitudinal and a transverse section which leave no doubt about the presence in this bird of a very clearly defined protochordal plate, arising as a proliferation in the entoderm before any process of mesoblast formation has been inaugurated in the ectoderm. I here copy five of his figures (Figs. 105— 109), adding that the region in the surface view which I have termed the protochordal plate is named by Schauinsland the “ Hntoblasthof.” Not only is its situation in perfect corre- spondence with the same region in Sorex, visible in Figs. 59 and 60, but the sections reveal the same constitution (Fig. 104 for the sparrow, Fig. 58 for the shrew), viz. a local thickening of the entoderm. And later when the ventral mesoblast (cf. p. 39) will have begun to make its appearance the surface view of bird and mammal will again be directly comparable, and the independent increase of tissues—intricately inter- woven though deriving from different germ layers— undeniable. Similarly in representatives of the reptiles there is no want of recent illustrations by various authors, showing that the median entodermal proliferation (protochordal plate) is present in early stages. I copy some of Passer (Figs. 104, 110), five of Sphenodon (Figs. 77, 78, 112—114), and two of Chameeleo (Figs. 76, 111), all seven taken from Schauinsland. And I may add that Mitsukuri (93), Mehnert (’92, 94), and Davenport (796, Pl. VII) have revealed a similar state of things for tortoises, Strahl (’82, ’84), Will (’90), and Corning (99) for lizards, Voeltzkow (’93) for crocodiles. If the presence of a protochordal plate in Sauropsids may thus be looked upon as well established we have to look a 56 A. A. W. HUBRECHT. little more closely before affirming that the same can be said of the annular mesenchyme-producing zone in the entoderm. If we consult Mehnert’s article in which he discusses the origin and the development of the hzmovasal tissue (area- vasculosa-crescent) in Hmys and Struthio (’96) we will then find that for these two Sauropsids he accepts as the final point of origin of the vascular tissue and the blood the ento- derm. But he does more than that. He gives a detailed account which in most points corresponds most exactly with what we have above indicated for the mammalia, of the origin in the hinder part of the “ primitive streak” of a decided entodermal proliferation which by many authors has been incorrectly looked upon as ectodermal. I believe that a careful re-examination of their preparations and a comparison of those with the numerous section series of Tarsius and Tupaja, which are always available for that purpose, may convince even those who have formerly stuck to the purely ectoblastic nature of the primitive streak, that in the lower half of the primitive-streak-tissue a direct and considerable proliferation of entoderm cannot possibly be denied. This proliferating region is, as we have seen in mammals, nothing else but the hinder median portion of the ring of vasifactive tissue, which was above discussed and figured (Figs. 46 and 60), and of which the protochordal plate is the median frontal portion. In the tortoise, Hmys, Mehnert (’96) gives detailed descrip- tions as to how this ring of tissue has in the first place the aspect of lateral outgrowths from the primitive streak ; later of crescent-shaped wings, and only finally of a ring. It may be here remembered that also in the embryonic shield of 'Tarsius the first origin of blood and blood-vessels is observed in the hinder part, and that we notice a similar wing-shaped advance in the distribution of the mesenchyme- producing annular zone. At the same time it should be borne in mind that once the primordium of the vascular tissue having arisen out of the entoderm its further develop- ment becomes independent of the region of its origin, so 3 ‘gh - ier we Ah iy ae | ier a a HUBRECHT. 105 Pl. V. 106 Fig. 105 to 109. Five surface views of the early aspect of the sparrow’s blastodisk (after Schauinsland, 03). In Fig. 105 there is as yet only an ento- dermal protochordal plate pf (ef. longi- tudinal section of Fig. 104); in Fig. 106 a downwards growing protochordal wedge pw begins its fusion with the protochor- dal plate; in Fig. 107 mesoblast has grown out from the borders of the elon- gated dorsal mouth; in Fig. 10S the ven- tral mesoblast vz has made its appea- rance; in Fig. 109 the sickle-shape be- comes visible in the posterior mesoblast. nch notochord. EARLY ONTOGENETIC PHENOMENA IN MAMMALS. Oo” that for example the fact that in Tarsius after a certain time we find the whole umbilical vesicle thickly covered with blood-vessels (Hubrecht, ’02, Fig. 91) does not imply that they have arisen in loco out of the entoderm. ‘They have become spread over this after they had once taken their origin in the annular zone here more particularly alluded to. I think it may here suffice to give the reference to Mehnert’s article in which he establishes the entodermal origin of a ring of vasifactive tissue both for a reptile and for a bird, and not in this place to describe the process more in full. The more so as it is well known how many differences of opinion yet exist on this head between different authors. The amount of difference can also be gathered from Mehnert’s paper, who gives a tabular summary of the different opinions held on this point by no less than thirty-six different authors, grouped under the heads of six different possibilities for the origin of blood and blood-vessels. Having so wide a divergence to choose between, it is only natural that I should feel inclined to side with Mehnert (96), O. Hertwig (83, p. 319), Goette (74, ’75), His (’00), and Rickert (’06) im respect to the origin of the vascular system now that different genera of mammals have pro- vided me with perfectly trustworthy sections from which to conclude to the existence of the annular mesenchyme- producing zone of the entoderm. ‘That for mammals, Kolliker, Keibel, Heape, and others have denied the partici- pation of the entoderm here advocated, and have derived the whole vascular system from the mesoblast of the primitive streak has no doubt its explanation in this fact that they must have consulted later stages of development than those in which the entodermic origin is evident. This latter stage is very soon followed by one in which the participation of the entoderm has come to a close, and in which the further deve- lopment of the vascular system is now going on between the two primary layers in the so-called mesoblast. ; All the points here discussed have been sifted and care- fully compared by Riickert and Mollier in the chapter which 58 A. A. W. HUBRECHT. they have contributed to Hertwig’s ‘ Handbuch of Embryo- logy. The student of their article will find no difficulty in accepting the generalisation above arrived at, that the ento- derm is the mother tissue from whence the vascular tissue and the blood have taken their origin. Riickert writes concerning Gecko (lI. ¢., p. 1172) : “Ich traf auf eine deutliche Ablésung der hier ziemlich dotterreichen ersten Gefissanlagen von dem angrenzenden hohen und ebenfalls dottergefiillten Entoblast ... Ihre entodermale Entstehung leet daher klar zu Tage.” Besides the protochordal plate and the annular zone of the entoderm from whence mesenchyme is produced we also find in the Sauropsida the ectodermal centres of mesoblast forma- tion which we have noticed in the mammals. The protochordal wedge is to some extent more marked than we found it in the Mammaha; it has of late been designated by O. Hertwig (?06) by the name of ‘ Mesoderm- sickchen,’”’ and it contains a cavity more spacious than the comparatively thin canal which we have encountered in mammals (Fig. 98). Also in this respect the exceptional ease of Fig. 52 should be considered as throwing a side-light on these intricate processes both in mammals and Saurop- sids. The confluence between the earliest ectodermal downgrowth with the protochordal plate has up to now not been specially examined in reptiles. Still we may conclude from the figures here given, which I copy from other authors that it comes about in exactly the same way as we noticed it in Tarsius for mammals, and in Hypogeophis for Amphibians. Fig. 419, Hertwig (’06), shows us the earliest protochordal wedge in a snake as it fuses with the thickened entoderm; behind the protochordal wedge we notice the ventral mesoblast as a third focus of mesoblast formation. Figs. 427 and 429 are con- tinuations of the same in somewhat later phases, and the correspondence with our Figs. 79, 80, 97, and 98, of Amphi- bians and Mammals is self-evident. In Hertwig’s Fig. 429 the source of ventral mesoblast has actually been shifted