The origin of the segmental musculature of the tail of t,he Axolotl (Ambystomn). By PETER FORD, BSc., P1i.D. (DqmrtJnient, of Embryology, TJniverxity College, London).

[Received Kovcmhrr 6, 1!)48.]

(With Plates 1-111 and 20 figiircs in the text'.)

CONTENTS.

I. Introduction . . . . . . . . . . Page

11. Review of Previous Literature .................... :. . . . . . . 609 111. Vital Staining Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611

VI. Observations on t.he Histology of the Neural Plate and it,s Derivati . . . . . . . . . . . . . . . . .

VII. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628 VIII. Summary.. . . . . . . . . . . . . . . .

IX. Bibliography . . . . . . . . . . . . . .

I. INTRODUCTION. This research, commenced in 1939, and interrupted by the war, was originally

undertaken as a reinvestigation of the work of Rijtel and Woerdeman on the developmental potentialities of the posterior portion of the medullary plate of the Axolotl. During the six years of the war the parallel work of other writers, notably Bytinski-Salz and Munch, became known to t'he author, but i t is his belief that some addition to their results is demonstrated in this paper.

The author wishes to express his appreciation of the assistance received from Dr. E. A. Fraser, under whose guidance this work was commenced, and to acknowledge help from Mr. D. R. Newth in reading proofs. The modelled drawings were prepared by Miss E. R. Turlington and the photographs by Mr. F. J. Pittock, F.R.P.S., A.L.S. Mr. H. E. Barker assisted with the histological preparations. Finally, his special thanks are due to Professor G. R. de Beer, in whose department the work was carried out, for his unstinted kindness and help without which it could not have been complet.ed.

TI. REVIEW OF PREVIOUS LITERATURE. Brachet (1921) maintained that a t the end of gastrulation only the head

of the embryo is fully formed (acro- and cephalogenesis) and that the trunk and the tail have to be added subsequently from a zone of growth or indifferent cell mass, the tail-bud. In opposition to this Vogt (1925, 1929) postulated that these portions, trunk and tail, are present, but need putting into place by stretching and dikplacement only (Gestaltungsbewegnngen), Vogt further went on to say that in the formation of the tail, new mesoderm is added to that already laid down in the straightforward gastrulation process, from the portions of the neural folds that surround the blastopore, and that the material invaginated a t the ventral lip during the final stages of gastrulation forms the

PROD. ZOOL. SOC. LoND.--VOL. 119. 43

610 w r m I:OK.I)

lateral plate mesoderm of the tail. The posterior neurd folds by t,heir fusion also, he thought, gave rise to t,he vent'ral and dorsal fin of the tail.

Vogt therefore assumed a stretching of notochord and neural tube to provide for the tail, and t,hought that during the closure of thc blastopore (presumably the time, in Urodeles, elapsing during the formation of a circular " presumptive " anus from a slit-like blastopore) extra mesoderm is added as new material. but, that its material forms muscle for the tail appears to he incorrect.

I n addition, Vogt also st,at,ed that t.he indifferent cell mass adds to the stretching neural tube and notochord. Since, however, this mass is w e d u p during the stretching * of the tail i t cannot, he considered, he called a " tail bud * ' or a " centre of growth ".

His final results are summed up as follows :-'' Thus it seems to be proved that the posterior end of the body, and in particular the tail-bud, does not owe its formation to a local centre of growth : the shifting of material which t,akes place a t this point is a continuation of mesoderm invagination which began during gastrulation ; longitudinal stretching and convergence of material in a median-dorsal direction are also important points which underlie the formation of the posterior end and which make this process appear as a oon- tinuation of the gastrulation movements."

In 1931, H. Bijtel, whilst carrying out an investigation of the prospective fate of the various parts of t,he neural plate, noted that dyed areas of the posterior part of the medullary plate found t,heir way into the soniites of the tail, giving a very different interpretation to tail-muscle formation from that

It should be noted that these experiments were carried out not only on Urodeles but also on the Anura (Rana esculenta), and the results obtained were similar. This disposed of Brachet's argument Ohat differences between his conclusions and those of Vogt were due to the use of Anuran and Urodele material respectively.

In a recent paper Pasteels (1943) further discusses the proliferative possi- bilities of the tail-bud and of urogenesis in the vertebrates generally, and postulates two possible hypotheses to explain the morphogenesis of the tail, viz., ( a ) that head, trunk and tail regions are put into place by the same series of morphogenetic movements ( i . e . gastrulation), there existing only quantitative differences in the intensity of growth to the exclusion of qualitative ; or, (6 ) the morphogenesis of the tail is virtually a different qualitative method of growth of an indifferent tail-bud. I n supporting hypothesis (a ) he does not, however, deny entirely the proliferative possibilities of the tail-bud.

The latter theory ( b ) has been actively supported by D. E. Holmdahl (1931) in a series of papers up to 1939, basing his observations on serial sections of chick embryos without regard for the results of experimental methods and particularly of the vital staining results of Vogt, Rijtel and others.

Holmdahl, in order to clarify and restate his conception of the tail-bud, has made the following statement : '' The concept of indifference as applied to tissue which, by its appearance (presumably in microscopic sections), is semi- developed as judged by the size of its cells, their arrangement in the hlastema and their strong nucleoplasmic relation."

I n support of the gastrulation theory of urogenesis various investigators, notably Munch and Bytinski-Salz, have analysed the self-differentiating properties and inductive capacities of the rudiments of the so-called (' tail-bud " revealed by vital staining.

H. Bytinski-Salz (1936), working on Ambystoma and Molge, has shown that the anterior four-fifths of the medullary plate when homoplastically transplanted into the host belly, produces neural tissue, whereas the posterior

* The word '' stretching " is used in t,he sense of increase in length wit,llout, proliferatios Qr acqelerstcd cell division.

That this final invagination does, in fact, go on, we shall s

of Vogt .

one-fifth tMereiitia,tes into ni\-ot,oiiics wciisionaily scriall\- arranged i i i ii.

cylindrical prolongation. Such inyotomes may show aut,onomic cont.i.actions. The lateral lips of the blastopore in stage 15-16 also prdr iw some niiisc:lr. as does the material ventral to (below) t,he most posterior one-fifth of the nie(ld- lary plate. Transplants of neural folds, althongli deficiency expeihents (sev below) clearly show them to be responsihle f n r fin formation, only ~)ro(lrrce epidermal nodules. In all Bytinski-Salz‘s traiisplants (1 9 3 1 ) some c:\-idcm:e of axial structures, usually neural, can be seen.

Miinch’s (1937) work on Pleurodeles, .1 mb?ystomo and Triton, corisisted chiefly of investigations of embryos from which portions of the nciiral I’liitc. were extirpated, and included discussions of the regiilativc prnpertics of sucli operated embryos. His results show clearly t’hat the mednllary plate plays i L

large part in the formation of tail muscles. Investigations by F. K. Risley (1939), who studied anus formation in

Urodeles show that implantation of either notochortlal or mrsotlertnal tissuc. of the tail will induce secondary t,ails, arid thitt formation of an atliis itillst h preceded hy the presence of mesenchyme. The ” init.ia1 impetus .’ to tail formation is given by the axial structures and ii .‘ sccoiitlai.y impctun ’. I,- t h t mesenchyme to anus form a t‘ ion.

Leigh Hoadley’s (1931) work on iml)lantations of portions of the tail-btid, which he considers (cf. A. Brachet and l-). 15. Holrntl:ihl) to tw it proliferative h c t , studies the effect>s ohtainetl when ” Interd rnesotlrt~m “ is tr,;iiirl)liiritc.d. This forms tissue according to it,s position of iniplnntation in thc. host 1111l(LY:i neural ectoderm or gut endotlerm is inchitletl, a-he11 it n-ill tliffci*c.iiti, cx t ( h into mesoderm related to them.

With the invest,igations tlisciissetl above in mind. it was tlioiightj t l i i i~~ some further light could be thrown 111)oii the formation of the tail ;tiit1 it? ancillary st,ructures, arid the experiineiits (I? ilietl 1)rlon- were xc.c.ordiiigl). carried out. No apology seems needed for the repetition of work by Bijtel and Woerdemann (1928), which has now hecome c:lassic:il, siticc i t l e m l s naturally to some of the additional experiments descril)etl Iwlnw.

111. VITAL STAIXINQ EXPERIMENTS (a) Technique.

The vital s h i n was applied in circnmscri1)cd areas o n the fi”s;trnl:i i l l

essentially the same manner as that descri1)ed by W. V n g t ( 1 925).

Figure 1 .

up freshly from it sterile stock solution). Sina.11 glass vessels were used RS

opevating dishes, and contained a layer of moctelling clay as a plast,ic substrate. The movements of the dyed cells were followed through development by

observations on t’he living embryo, by dissecting specimens fixed in Zenker’s fluid, and by cutting serial sections in which the vital dye had been fixed b.y the phosphomolybtlic acid technique of T,. S. Stone ( 1 9 3 1 ) or hy meam of Yntema‘s (1939) dioxane method.

(b) Results. The majority of these experiments were carried out, on rmhryos of an age

rorre-ponding to stage 15 (all stages mentioned below refcr to 1C. (i. Harrison’s

Figure 2 .

S 9. 7.4 46

u a . st 16.

c . st. 33.

Diagram of experiment S.9.

unpublished figures as shown in V. Hamburger’s ‘ Textbook of Experimental Embryology ’), although later observations have shown that gastrulation does not finish until stage 16 (or even 17) is reached.

AVUSCL IATCEtE OF THE TAII, 01 AMBY\T(JMA 613

The areas btaind may best be indicated by reference to a simple map similar to that used by Bytinski-Salz (1936), Spofford (1943), and others.

(i) Area 1 stained. Example 8.9 (fig. d).-Stain placed in area (1) anterior to the posterior

fifth of the medullary plate, in stage 16, is found at stage 21 lying in the closed iieiiral folds in the post-dorsal region of the trunk and incipient tail-bud, but not exteiicling to its tip. As development, accompanied by stretching of the embryo and downward movement of tlie tail, proceeds, it is seen that the dyed area, by stage 33, reaches the tip of the fully-formed tail. stretching anteriorly a5 far as somite 11.

This indicates that the whole of the pobtcrior fifth of the medullary plate has taken up a ventral position in the embryo from the cloaca to the tip of the tail. Further development as far as stage 33 shows the &in to be pre3ent in the neural tube from the tip of the tail to home distance forward in thc trunk. Thus the growth in length of the tail is accompanied by the extension of the anterior four-fifths of tlic medullary pIate to supply the tailwith a neural tube.

(ii) drea 2 stained. Exawple 2S.1 (fig. 3).--Stain was applied to area 2 at stage 16, fig. 4 above,

and a t stage 21 remained localized in the tail-bud. By stage 26, that is, after the commencement of stretching, the stain appears in the trunk as far forward

Diagram of experinwnt 2S.1.

as somite 10. The stretching of the embryo thus carries thc neural tulm posteriorly and leaves part of the so-called " tail-bud" to form posterior trunk mesoderm from behind forwards, as the embryo stretches.

614 PETER FORD

(iii) Areas I niid 2 stnined in cmjtmct ion. L'.mmple S.n (fig. -i).--Contra,sting dyes, Kcutrnl Reti nnd Nile Blue

sulphate, were usctl in this experiment, placed as shown in the diagram (stage 1 6 ) . and their location in the embryo investigated by tlissect~ion a t stage 34. The author knows of no technique whereby Neutral Red can be fixed in microscope sections. This experiment confirms the conclusions drawn from the previous examples as the most posterior portion of the medullary plate 5iids its way into the posterior trunk rnyotomes.

Figure 4.

b . st . 35

c st 38 (olagrammatlci I h g m i n of' R disbectetl cmbryo, S.19, showirig the location of' dyes of contrasting colours.

The above three examples, illustrating the gross movements of the posterior iuednllary plate, were repeated many times with variations including areas of the " neural folds " and proctodaeal regions. Diagrams and photographs, together with graphical reconstructions, are given below with protocols for some of thc more useful examples.

Example S.40.-Stain was placed on the left side of the posterior medullary plaie, not extcnding to the edge of area 2 , and overlapping the posterior neural fold in areas 5 and 6 (stage 15). At stage 36 the stain lay in the myo- tomes dorral to the cloaca, partially in the tail, but to a greater extent in the

5.3. 7-4.46

s

a .

Ulilgmrn ot experiment 8.7.

616 PETER FORD

posterior trunk myotomes. Stain was also found in the ventral fin (area 3) and in the proctodaeum (area 6). The presence of the stain in the proctodaeum followed the final invagination of the lateral lips of the blastopore as it. assumed the final rounded form, constituting the primitive anus.

Exumple S.3 (fig. i5).-Stain in posterior modullary plate, covering the postero-lateral portion of the medullary plate ant1 overlapping areas 6 arid 3 (stage 17), but not on the lateral blastoporal lips. At stage 3-38 stain was visible in the dorsal and ventral fins, the cctoderni of the posterior end of the tail, and the posterior myotomes of the tail. No stain wa,s found in tho cloaca. indicating that the formation of the proctodaeum t'akes placc between stages 15 and 17 before the final closure of the neural folds.

Example S.l4.-Stain placed in the neural folds a t stage 18-19, immediately before their closure, results in the presence of stain in the ventral fin and lateral lips of the cloaca, but not within the proctodaeum. Tho area in which the

Figure 7 .

c .st.33 Diagram of experiment S.1.

stain was applied corresponds for the most part to area 3, most of aroa 6 having been previously invaginated in forming tail mesoderm and proctodaeum

Example S.l4a.-This experiment, similar to 5.14, had stain placed in thr neural folds in area 5 which, by stage 38, was found in the dorsal fin.

Example S.44.-Stnin placed in areas 6 and 3 shows their contributioiis to the proctodaeum and ventral fin respectively. (See graphical reconstruction

It was observed in this specimen when sections were cut, that the stain extended into the distal ends of the pronephric ducts, as shown in fig. 10, a photograph of a section of Example 5.a (see below) . The pronephric ducts growing back from the pronephros, as described by O'Connor (1940), meet sprouts of the proctodaeum growing forwards. According to O'Connor this cloes riot constitute an induction, the proctodaeum, lie claims, will pro(h~cc the sprouts irrespective of the presence of the pronephric duct.

fig. 20.)

i v ~ s C ~ r , A ' r i w; oJ 'TIIE WII, I W . 4 ~ i m ~ s ' r o > i ~ B I i

Exacnmyk t'S.2.--8taiii :tpplicd over the slit-shaped blastopore at dagP 1 ;i sliows clearly the coiitinriing i~i~-agimtion conciirrcnt with. the formation of the anus. The stain was found in~ thc ventral portions of tlic soniitcs 11-14, just anterior to the cloaca, and in the ~~roctotlacirni. S o stain appcarcd i i i

the ventral fin to which area li does not contribute. 15xwr~pk S : i (fig. Ci).-Cont,rastcd with thc Ircvioiis csamplc. s ta in was

apidietl over t,he rounding blastopore at, a litter stagc (l(j-lY), :LML ovcrlappitlg the nciiral folds Istcral to the Mastoporr.. So staiii was foiincl in tlic r~iyot~ornes. indicating that, t>he invagination of niesotlerrn has finishctl t l j r stage 17. St'aiti is found, however, in the ventral fin. 1)ell~r cctoderni arid proctodaeuni.

Example S.1 (fig. 7).-Stain plac around the slit-shaped blavtopore in this specimen, before the appearance of the medullary folds, is all invaginated, finding its way into the middle and posterior trunk inyotomes.

Example S.a (fig. 8).-A repetition of experiment 25.1 (fig. 3) in which the Nile blue sulphate was fixed by the dioxan technique of Yntema. The plioto- graph in Plate 11, fig. 4, shows the difficulty of reproducing the stain granules as distinct from melanin granules.

The results of the above experiments enable one to map on to (stages 13-17) neurulae the presumptive areas of material which will go to form the major organs of the tail, whilst microscopic examination of serial sections reveals amalller structures such as perichordal mesenchyme (Spoff ord, 1945).

618 t’1;’rk;K V I J R l )

l’revious investigators of t h u origin of the tail, with the osceptioii of I’asteels and Kakamura, have mapped its rudiments on to the medullary plate at stage 15, where, as has been demonstrated above: invagination is incomplete. Nakamura carried his maps, as a series from the commericement of gastrulation to the complebion of neurulation, t o the stage of open medullary plate, in which invagination is complete, viz., stage 17-18, arid the author is in agreement with his findings (fig. 13).

Figure 9.

Dictglritrn of an experiment (R.l l ) in which stained areas delineate the anterior extent of The brackets show the position of the dyes in the riotochord

From the experiments detailed above it is readily seen that area 1 will form the neural tube of the posterior trunk region and of the tail. Are;i 2 is fated to form the inyotomes of the posterior region of the trunk and of the tail. It should be noted, however, that the bulk of the material for the posterior trunk myotomes is invaginated, as the medullary plate passes from stage 15 to 18, in the normal way through the lateral blastoporal lips. Area 3 forms the ventral fin and neural crest mesenchyme, and area 5 the dorsal fin. Area 6, the last of the invaginating material, together with part of area 4,

the chorda-mesoderm. itself.

ivuscuLA'rum or' ' r i j G 'TA t i , or A . v m w - o l T , i 619

forins the proctotlacum as thc slit-shaped blastopore assii~nes its rounded form. 'l'ht. eetoderm lateral to t,hc iii'cas 3 and 5 will form the epidermis o f the tai1.

Most of area 4 will forrii t d y epittermis anterior to the blastopore and riot, as stated by Nakamura (l!E%), only the cpidermis of the tail.

During the preparation of the area maps described above i t wa's suggested

F i Z i i w 10.

Diagram ot experiment R.6, showing the position of the presumptive posterior trunk somites.

that some further staining experiments could usefully be carried out to cleter- mine the extent and arrangement of the somitic mesoderm and notochordal material on a map immediately prior to gastrulation. Some of the protocols obtained are detailed below.

The results showed that the lateral extent of the chordal matma1 wai less than that indicated by Vogt and resembled that shown by Kaltaruura.

Figure 11.

Diagram of experiment S.M.11, showing the lateral extent of the chorda material.

The anterior trunk somites occupy the same position allotted to them by Vogt but extend more medially, overlapping part of the area of his lateral chordal horns. The posterior trunk somites succeed the anterior ones but extend dorsally to the chordal horns with the most posterior somites almost meeting their fellows of the opposite side above the chordal material. The somites of the tail follow those of the posterior trunk region surrounding the chordal

c,20 L’1CTE:H V( JK1)

area totally. This disposition of somitic niesoderru (fig. 14) accords with the massive coiivcrgerice of material which takes place during gasttrulation and following the invagination of chordal niesoderm and anterior trunk somites through the dorsal and lateral lips of the blastopore. The first appearance of the blastopore is as a broad and shallow groove, preceding the characteristic crescentic dorsal lip. Through this h o a d groove the invagination of cephalic endoderm and prechortlal plate takes place and causes the converge~ice of anterior somites and chordal ma,terial leading t,o the crescentic blastopore.

Whilst carrying oiit these experinleiits the author was unaware of work carried out by Pasteels (1942) in which hc has remapped the Axolotl a i d Discoglossus in great dct,ail. Tn regard to the chordal and somit,ic rnesoderni the aut,hor is in complete agreement with his findings but has not attempted t,he detailed work which he describes.

Diagram sliowing an experiment H.15, atrliri placed in the area, of the presumptive somitic mesoderm of the entire embryo.

Example R.11 (fig. 9).-A series of marks was placed dorsal to the primitive blastopore and was completely invaginated and enfolded by the posterior neural plate. They came to lie in chorda and posterior trunk and tail somites thereby indicating the limit, dorsal to the blastopore, of the chordal material.

Example R.6 (fig. lo).-This specimen showed the lateral extent of the chorda. The coloured mark lay over the presumptive posterior trunk somites lateral to the horns of the chorda still visible, unlike Vogt’s map, in anterior view. I n conjunction with S.M.11 below, this example indicates the lateral extent of the chordal material.

h’xample S.M.ll (fig. ll).-This experiment is complementary to the preceding R.6, the stain lying more medially and on the lateral horn of the chorda.

Example R.15 (fig. 12).-A series of contrasting marks was placed so as to extend from the edge of the blastopore through the anterior trunk somites

It is found later to be in the notochord only.

t o thcl pohterior and tail soniite areas. examples 73 and 79 (1942). animal from front to hack.

'Phis corresponds roughly to Yaxteel's The stain come5 to lic in the somites of the entire

IV. OPERATIVE EXPERIMENTS. Thc operative methods employed may he simply classified into 4 cntcgorics :

( i i ) Simple transplantations, usually to ventral belly of a slightly older

( i i i ) Deficiency or extirpation experiments (Ex). ( iv) Simple operations on one embryo involving rotations of prcsiimp-

( i ) Keciprocal transplants between two embryos (R.T.x).

animal than the donor (Q.X.) .

tive areas and the like ( O x ) .

Techniqzce. All operations were carried out with glass needles and hair loops in sterile

conditions and under full strength (Holtfreter's solution). Transplants were held i n place, where necessary, with small glass bridges, and complete healing took place normally in 30-00 mins. when the embryos were transferred to one-tenth strength Holtfreter, or in later experiments (1947) to 0.05 per cent. aqueous sodium sulphadiazine as recommended by Uetwiler and Robinson (1945). The mortality of operated specimens was fairly high (ca. 3 0 per cent.), usually commencing with the formation of sub-wtodermal oedematous blot)s, but animals could, as a rule, be reared to swimming stages if reasonable care was taken. Rearing under sulphadiazine considerably reduced mortality, even on badly mutilated specimens, to 2 or 3per cent.

The areas transplanted or extirpated were intended to correspond to areas similar to those stained in the previous experiments.

(i) Reciprocal transpZants.-Numerous experiments were carried out with very uniform results, the area transplanted being the posterior third of the neural plate, which in the donor was replaced by belly ectoderm from the host. The transplants from this area normally sank below the host ectoderm, which then covered them over, a t the same time rolling up as if in situ (it was noted that extirpated neural plate, whether immediately posterior to thc brain or of the ultimate fifth, would proceed on its own in vitro to roll into a tube, without assistance from the stretching embryo or from the neural folds). This rolling up is a dynamic property of the medullary plate, whether its illtimate fate is neural or muscular. If embryos are immobilized in agar-agar jelly the neural folds are unable to close and fuse, but the neural plate, withont assistance, sinks below the surface forming a normal neural tube.

After sinking below the host ectoderm the graft proceeded to grow out into a Characteristic tubercle (Plate I, figs. 1 and 2) of roughly circular cross- section occasionally showing fin-like structures. The presence of these fin- like structures was probably due to portions of the neural folds or crest attached to the graft, or in some cases its proximity to the axial striictures of the host (see R.T. ( l ) , Plate I, fig. 3).

Example R.T.l (]).-The area transplanted corresponds to area 2 of the diagram. The characteristic tubercle appeared and produced fin-like structures (see photograph, Plate I, figs. 3 and 4), and on sectioning, a picture in miniature was obtained of the mechanism of mesoderm formation in a normal tail, namely, the splitting and migration upwards on either side of the post-anal endoderm and definitive neural tube and notochord. This took place despite the absence of these axial structures, although it is possible that some neural tissue was present. The photograph (Plate 11, fig. 1) illgstrates this migration.

PETER FOND

Figure 1:3.

J /

... :...:... 1 ...... I \ ’ ”

1 1 1 1 1 ...... I \ ... . . . . . . . 1:::’:: Lateral plate mesoderm ......

’ .:.. I I ! ~ ! Epidermis . . . . . . . ........ ;!I!:

Proctodaeum u Endoderm

Presumptive area maps showing the migration of the somitic mesoderm and neural areas. (Modified after Nakamurs.)

Donor,(seeRT(,,,) st.20. a Host,st.l5.

b . s t 20 c , st. 32

d st. 37

Experiment H.T.(1)2, in which posterior ncurd plate was replaced by belly ectoderrq

Exai,rplc R.T. (1) 2 (fig. 15).--Arw 2 w r ~ > cytirpated and trmsplantect to R.T.l (1) and replaced by belly ectoderni from a 5tage 22 neurula.

In this embryo the posterior neural folds wcre iinahle to fure in their entirety (see photograph, Platc 1 , fig. 5), thc belly ertoderm not posses4iig thc faculty of tube formation inherent in medullary plate. The tail produced was complete in all respects, exrept somitic mesoderm, which was absent (see 7’3. photograph, Plate 11, fig. 3), compared with normal tail, Plate 1 I , fig. 5 ) .

Figure 16.

2 Tp.,,, 24 9-46

Drawing of experiment 2Tp.(l), a transplant inchiding both neural and miisriilar areafi of the ~nedidlary plate. Note the presence of the fin-like stmcture in (d).

Fins, neural tube ant1 notochord were all present, the latter two demonstrably having grown ’back from the anterior four-fifths of the metliillary plate-notochord complex.

Where the dorsal fins were unfused it could be seen that either neural fold possesses the ability to form a complete fin.

(ii) Simple transplants. Example T.7.-A simple transplant of the posterior one-fifth, area 2, together with areas 3 and G (without cells of the nnderlying tissue present). In such transplants 11s this i t is, in the author’s opinion

MtTSCl'LATURE OF THE TATT, OF AMBYSTOMA

Figure 17.

DF, 23 1 4 7

625

c . st. 26 Drawing of experiment, Df.3.

Figure 18.

Drawing showing the result of the extirpation of both neural folds. I'ROC. ZOOL. S O C . LOND.-VOL. 119. 44

626 PETER FORD

impossi ble to be qiiite s1u-e of rcbnloving only t,lie hrilwrfic~ial tissue. 'L'he t,ul,crcle was characteristic (see photograph, 1'1atc: I , fig. a) , brlt, 110 fin was prc\sellt despite the inclusioii of area :3, which iiornially fornis tho ventral fin. Lt is suggested that the absence of axial orgaris in or near to the graft accounts for their absence. (Woerdeman (1945) has shown that neural crest also has the power of inducing fin.) The particular interest of this transplant is that i t shows very well two striking phenomena pecnliar to most grafts of this type (Plate TI, fig. 2 ) :-

(i) The tissue exhibits metameric segmentation. (ii) The cells of the tissue are cytologically extremely similiar to those

of neural tissue, and dissimilar, initially a t all events, to normal mesodermal somitic tissue.

Example 2Tp(l) (fig. 16).-Areas 1 and 2 transplant'ed from an embryo of stage 15 to a neurula stage 22.

A most interesting outgrowth was produced possessing neural tube, and metamerically segmented mesoderm which showed primary histogenesis of muscle. Fins, and an induced proctodaeum, together with a secondary gut lumen, made in all a complete tail structure lacking only notochordal tissue (see photographs of embryo and cross-sections, Plate 111, figs. 1 and 2).

The anus, which was imperforate, was ectodermal in origin, and so far as could be seen no mesenchyme took part in its formation (cp. Risley (1939)).

(iii) DeJiciency Experiments. Example Df. 3 (fig. 17).-The interest of this deficiency experiment (the embryo was one from which area 2 and its underlying tissue had been removed) lies in the growth posteriorly of a neural t,irbe, despite the absence of the tail into which it would normally grow.

Example E.T. 2 (fig. 18).-Extirpation of area 3 on both sides of a neurula produced a tail with ventral fin deficiency. This experiment shows that the lateral lips of the circular blastopore have fin-forming capacity.

Example 2E.2 (fig. 19).-Extirpation of the '' lateral mesoderm " of the " tail-bud,'' i.e., the material formed by the split and upgrowth of the ventral medullary plate material, produces an embryo without the somitic mesoderm of one side of the tail and the posterior trunk myotomes of the same side (cp. S. 1, S . 19, S. 40, etc.).

(iv) Simple operations. Examples 0 and O.lO.-A rotation through 180" of the superficial material of area 2 produces a normal tail. Compared with the rotation of area 2 and the underlying material which produces a forward growing tail (see Bijtel) this indicates the antero-posterior indifference of area 2 and the polarizing powers of the underlying material.

V. RECONSTRUCTIONS OF TAIL SOMITE FORMATION The reconstructions figured below illustrate the mechanics of the develop-

ment of the segmental musculature of the tail. Initially ( a ) the presumptive somites lie as a mass of tissue below the posterior extremity of the neural tube and notochord, with which it is continuous. As urogenesis proceeds (6) this mass of tissue splits into two, and in the form of a U migrates up on either side of the axial structures which are stretching backwards to take up their position in the tail. In (c) this process has, in the proximal portion of the tail, finished, and the laterally arranged strips of mesoderm have com- menced to segment, forming somites in which the histogenesis of muscle will shortly follow.

\TTTSCITLATI‘Rl? O F THE TdTL OF AMRYSTOR‘IA 6 2 i

Fignrc 19. 2 E e 25.9.46

Epidermal flap

a . st .29

c . st. 34. Posterior l imit of rnyotomes

d.st 40 s de

Removal of the lateral mesodermal rudiment. Experiment 2E(2).

vr. OBSERVATIONS ON THE HISTOLOGY OF THE NEURAL PLATE AND ITS DERIVATIVES.

I n a stage 15 neurula transverse sections of the medullary plate (Plate 111, figs. 3 and 4) in its neural and muscular areas show a remarkable similarity, if allowance is made for the greater pigmentation and differentiation of the anterior end. The cells are arranged as a stratified columnar epithelium with nuclei of an ovoid shape, and do not reveal on inspection any marked cyto- logical difference, area for area.

In transplants the similarity is carried even farther where ratio of nucleus to cytoplasm is identical in the neural material and the segmental mesoderm, formed by the anterior and posterior portions of the neural tube. As differentiation proceeds these similar tissues show a complete divergence of character, sharply one from the other, and shown in Plate 111, figs. 1 and 2, with the onset of muscle histogenesis and the rearrangement of nuclei within the neural tube.

The similarity is interesting in view of Holmdahl’s statement, that the “ tail-bud ” consists of tissue particularly sharply marked by the “ size of its cell8 and their strong micleoplasmic relBtion.” Hc claims that on this account

44*

628 PETER FORD

the cells exhibit an appearance of indifference such as would be found in a blastema. In the aut,hor’s opinion tlie similarity is due t.0 the fact that both groups of cells are a part of one structure within the embryo? the medullary plate, and that neural tube celis might just as easily be said to be blastemic as the rudimentary tail mnscle cells.

VII. DISCUSSION. The experiments described herein confirm the results of Woerdeman and

Bijtel and qualify the ussumptions made fly i‘ogt. Thc posterior fifth of the metlullary plate takes part in the formation of

somitic mesoderm in the tail ant1 in the posterior part of the trunk. This posterior nietlullary material, which finds its way into the ventral half of the tail-bud by tlie stretching of the anterior four-fifths and the body of the embryo generally, is by thc t>imc of the formation of the metlullary folds (st. 15), determined to form mesoderm when tri~>splanted i1it.o the belly of an host- embryo of approximately the same age. Furthermore, i t possesses the power of metameric segmentation and, in the presence of the axial structures, proceeds with the histogcnesis of muscle fibres (certain authors, e . g . Bytinski-Xalz claim that when neural tissue is also present autonomic contractions may be ob- served). This mesodermal part of the medullary plate possesses in transplants a remarkable power of stretching which may be associated with its position in the tail, an organ which is formed by stretching rather than proliferation. It i s noticeable that all the elements of the tail, including even the neural folds, possess this faculty of stretching when transplanted. It seems likely that the materials in the tail-bud do not possess greater powers of proliferation than do those of the rcst of the embryo, and mitotic coiints (Pasteels, 1913) confirm this opinion. It is clear that the organization of this posterior fifth of the medullary plate into mesoderm is an integral part of the gastrulation process, lacking only the tucking in through the dorsal lip. It may be a phenomenon in which the primary organizer exerts an effect superficially and from back to front, whilst the underlying material provides an additional stimulus in an opposite direction.

That this gastrulation of tail muscle, by the closure of the neural folds takes place, confirms, in general, the views of Vogt-but one cannot agree that, tail mesoderm is added from the lateral lips of the blastopore as this closure takes place. The neural tube and the notochord are placed in position, just as Vogt asserted, by their growth backwards from the anterior portion of the trunk, from the region of somite 10, to supply the posterior portion of the trunk and of the tail.

The author does not consider that the tail-bud plays the r61e of a prolifern- tive blastema in the laying down of the organs of the tail. The whole process is one of stretching. Indeed, the whole of the posterior trunk region from somite 10 backwards is put into position by this stretching, some eight or nine posterior trunk segments being added. At the same time tail muscle is being laid down, both processes taking place between stages 15 and 20.

The contention of Holmdahl that the tail-bud proliferates tail somites cannot explain, even if proliferation could be established by mitotic counts, the putting into place of the posterior region of the trunk, a phenomenon only to be explained by stretching of material already present.

The cytological similarity of the cells of the anterior and posterior parts of early medullary plate, and of the segmental muscle and neural tube in transplants, adds further certainty to the theory postulated above, since i t is clear that the tail musculature is truly a part of the medullary plate. The facility with which one can identify this tissue during its migration, as shown in fig. 20, is remarkable.

As already stated, a t stage 16 the medullary plate is fully determined to form segmental muscle blocks. It does not, however, have a fixed antero-

MUSCULATGHX UI" THE TAlL 0 1 7 BMBYHTOMA 629

posterior axis. Rotation through 180" of the posterior fifth and its iinderlyirig tissue a t stagc 15, as tlemonstretcd by Wocwlenutii a r i d Bijtel, prodiiccs a tail ahich growb forward o\ cr the enibryo'h licatl. Rotation of the superficial tissue alone produces it normal tail g r o ~ i n g posteriorly. It would seem, then, that the directional axis of the tail is governcd by the underlying tissues,

Figure 80. Migration o f tail mesoderm

A

I

9 diagrammatic representation of the movements involved in the formation of lateral mesodermal somites from a ventral rudiment.

or more probably by the growth back into the tail of the axial organs, neural tube and notochord.

The inductive capacity of this posterior fifth of the medullary plate is considerable, and in transplants host tissue may be caused to stretch. Thus secondary gut lumina and ectodermal ani may be produced, and, in association

630 1'"YMi PO k I )

with axial structures, fiiis produced from epidermis which would iiot' iiorinally h a ~ c liecii ual)able of so doing.

Kormally fins for t,he tail are formed froin t Iic nietlullai~y folds (the acti\-it.y of t,hc iieural crest, has not becu srprately investigated) and t'hc folds of oiie side are capable of forming a bilaterally symmetrical fin.

'I'he lateral lips of the 1,lastopore take part i n the foruiatioii of the 1)rocto- tlacuin , a i d it, has lxeii ol~servotl that' from tho proctodaeum sprouts of tulJulc grow forward to meet the tlescciiding proiiepliric duct.

The contention of Leigh Hoatlley that transplanted portions of the t,:d-Ilixl, rogarded by him as an indifferent blxstenia, form tissues similar to tlia,t ill which i t is implanted does not agrec with the author's results, since i t is fouiitl that, even without axid structures, the rudiment of tail muscle will form somitic material in transplants.

The iriduct'ion of an ectodermal proctodaeum by transplants of t'he postcrior two-fift,hs of t,he medullary plate is at odds with the results of lCisley, who claims that mesenchyme is the tissue responsible for the stimulus needed for secondary anus formation.

The last series of staining experiments carried out to delineate tlic extent in a 1)resumpt)ivc area map of the somitic a i d chordal mesodelm shows an sgrcenient wit>h the detailed survey of Pasteels and with Na,karuiira's rial) (fig. 13, which can bc seen to be similar to that of Pasteels as shown in fig. 1-1). 'l'h lat,cra,l horns of the chorda are less extensive than those shown by Vogt, and tlic tail mesoderm lies dorsal to the chorda separating the chordal aiitl neural areas.

VIII. SUMMARY (I) By means of vital stains the rudiments of the tail structures presciit

iii the posterior one-fifth of the medullary plate were delineated and the extent of the chordal and somitic mesoderm remapped.

( 2 ) The cells that will form the myotomes of the posterior part of the triunk arid the whole of the tail are present in the posterior medullary plate a t stage 15. The tail mesoderm is enclosed by the closure of the neural folds.

(3) The neural folds of the posterior two-fifths of the medullary platc arc: responsible for the formation of the dorsal and ventral tail fin (see 11 below).

(4) Perichordal and other mesenchyme of the tail has its origin in the posterior medullary plate.

(5) The lateral lips of the blastopore take part in the formation of the proctodaeum which produces sprouts growing forwards to meet the descending pronephric ducts.

(6) By operative techniques (transplants, deficiency experiments, etc.) the degree of organization and the inductive powers of the posterior medullary plate were determined.

(7) At stage 13 the posterior fifth of the neural plate is fully determined to form segmental muscular tissue. The establishment of its antero-posterior axis, however, depends on the presence below it of the axial structures, the neural tube and notochord.

(8) Extirpation of the posterior medullary plate results in deficiencies in somitic mesoderm of the tail and hind end of the trunk. Mesenchyme is present and is, no doubt, the product of the neural crest.

(9) Rledullary plate, whether its prospective fate be neural or muscular, possesses the power to form a tnl)ular structure without the assistance of the neural folds.

(10) In traiisplants tho segriicittal tissue formed by the posterior medullary plate closely resembles neural tube cytologically, relating tail muscle to its origin in the medullary plate.

By stage 16-17 the trunk material is invaginated.

(1 1) The neural fold of one side of t'he medullary plate is capable of forming

(1%) TIic presumptive rudiments uf the tail possess remarkable powers of a bilaterally syniiiietrical fin.

stretching.

IX. DIBLIOGRAPHY. BIJYEL, J. 13. (1'331).

UIJTEL, J. H. (1936).

P h r die 13ntwicklung des Schwanises bci Amphibien. Arch. Entw. ~l/ lcc/~. Ory. 125, 448.

1)ie ~lesodern~bildiiiigspoteiiseii tler hinteren ~~edull~rplattenbezirke bei AlllhptOiJL(2 ?iiezica?iw/L in bezug ituf die Schwanzbildung. Arch. BirtwMcclc. Org. 134, 262.

On t.lie development of the tail in the Amphibian embryo.

Paris : Masson et Cio.

RIJTEL, J. H., & WOERUEMAN, N. 1%'. (1928).

BRALHET, A. (1921). BYTINSKI-SSLZ, H. (1931). Untersuchungen uber die Indukt,iomfahigkeit der hinteren

Medullarplattenhezirke. BYTINSXI-SALZ, H. (1936). R. C. Accud. Lincei.

24, 34. DETWILEI~, 8. R., R: ROBINSOX, C. 0. (1945). Use of sodium sulfizdiazine in surgery on

Amphibian embryos. HanrBuku:kxt, 1'. (1942). A ~ ~ f m z U U l Of ExpCritMntnl h"IJtbry0logg. Chic~~gv. univ. of

Chicamgo Press. HOADLEY, L. (1931). Mesoblast, differentiation in implants of portions of the tail-bud

(diiibystomu puiictatum). HOLMDAHL, D. E. (1931). Primitifstreifen beziehungsweise Rumpfscliwanzknospe im

Verhiiltnis zur Korperentwicklung. HOLTFILETEI<, J. (1931). Arch.

h':ntwMcch. Org. 124, 404. SIUNCH, H. (1937). Uber Regeneration in der Friihentwicklung. Defektoperationen im

Gebiet der friihembryonalen Schwanzanltlge bei Amphibien. Arch. EntwMcch. Org. 137, 597.

Pvoc. K. Akad. WctetL. Anist. 31, 1030. TruitB d'h'ntbryologic dcs Verte'bre's.

Srch. EntwMcch. Org. 123, 615. Lo sviluppo della coda negli Snfibi.

Proc. Sac. e x p Riol., N.Y., 59, 202.

Arch. Biol. Paris, 42, 325.

2. ,ii&r.-cmut. E'orsch. 38, 409. Uber die Aufzucht isolierter Teile des Amphibienkeimes.

sAKAi.IURt.4, 0. (1938). O'CONNOR, R. J. (1939).

~ 'CONNOX. , R. J. (1940).

YASTEELS:, J. (1942).

Tail formation in the Urodele. zool. Mag. Tokyo, 53, 442. Experiments on the development of the pronephric duct.

The evolutionary significance of the Amphibian nephric system.

New observations concerning t,he maps of presumptive areas of the J . exp. Zool.

Arch.

J . Anat. Lond. 73, 145.

J . L4izat. Lond. 75, 96.

young Amphibian gastrula (A?izby~t~i~ia and Dixoglossus). 89, 255.

PASTEELS, J. (1943). Hiol. Paris, 54, 1.

RISLEY, p. 12. (1939). S~POFFORT), W. K. (1945). Observations on the posterior part of the neural plate in Ambly-

shoma. J . exp. 2001. 99, 35. STONE, L. S. (1931). Selective staining of the neural crest and its preservation for micro-

scopic study. VOGT, w. (1929). Gestaltungsanalyse am Amphibienkeim mit ortlicher Vitalfarbung.

Vorwort iiber Wege und Zie1e.-I. Teil. Methodik und Wirkungsweise der ortlichen Vitalfiirbung mit Agar als Farbtriiger. Arch. EntwMech. Org. 108, 542.

Gestaltungsanalyse am Amphibienkeim mit Brtlicher Vitalfarbung- 11. Tell. Gwtrolation U d Mesodermbildung bei Urodelen und Anuren. Arch. ZntwMech. Org. 120, 124.

Ada . need. Morph. norriz. path.. 5, 378.

Self differentiation of heterotopic em ectoderm in the embryo of Ambystoma punctatum.

Prolif6rations et croissance dam la queue des VertBbrbs.

An experimental study of &nus formation. J . czp. Zool. 80, 113.

Anat. Rec. 51, 267.

VOGT, w. (19%).

\ ~ O E R D E M ~ , M. W. (1945).

~ N ~ ~ ~ A , c. L. (1939).

On induction of a dorsal fin in Axolotl embryos.

J . exp. zed. 80, 1.

A bbreaia.tions used in Text-$gures and Plates. dJ; dorsal fin ; ect, ectoderm ; ecf.in, ectodermel ingrowth ; end, endoderm ;

N m r , hg, hindgut ; L.P., lateral plate mesoderm ; ach, notochord ; mr, neural crest

632 XUYCULATURE O F THE TAlL O F AMBYSTOMA

neural area ; rt j , neural fold ; npl. neural platc ; ut, neural tube ; oed, oedeme ; I’h.end; pharyngeal endoderm ; ph, pharynx ; pro, prortoclaeurn ; beg, segrncntal mcioderm , sow, somite ; syq. secondary gut lumen ; ty, tail gut ; (f, \eiitr,il fin ; yen, yolk cncloclcrm.

EXPLANATION OF THE PLATES.

PLATE I. Figs. 1 & 2. Photographs of two embryos showing the chariictoristic tubercles produced

Fie. 3. Photograph of embryo R.T.I(I) from the right side. The fin-like structures on the by implants of medullary plate. ( x 8.) - tub&& are well iilustrated.’ ( x 8.)

4. The same embryo R.T.l(l) from the left side. 5. Phot0graE.h of cmbryo R.T.l(%) showing tho flat phtc of cpitliclium sepratiug

( x 8.) 6. Photograph of embryo T.7, showing the transplant demonstrating segmentation.

-

( x 8.)

right and left dorsal fin rudiments.

( x 8.)

PLATE 11. Fig. 1. Photomicrograph of a part of a transverse section of embryo R.T.l(I). The

migration somitic material in the form of a U is clearly shown, as is the dorsal fin. (x75.)

2. Photomicrograph of a transverse section of embryo T.7, showing the srgmentation of the transplant into seven blocks of tissue. Note should be made of the cytological similarity of this segmental tissue mid the neural tube.

3. Photomicrograph of a transverse section of embryo R.T.l(d), demonstrating the absence of somitic mesoderm, the two fins separated by belly ectodenn arid the presence of axial structure which have grown back into tho tail.

4. Photomicrograph of a transverse section of embryo S.a, showing stain, fixed by Yntema’s method, conspicuous in the proctodaeum.

5. Photomicrograph of a transverse section of a normal tail for comparison with figs. 1 and 3.

( x 50.)

( X, 5 0 . )

( x 100.)

( x 100.)

PLATE 111. Fig. 1. Photomicrograph of a transverse section of embryo 2.Tp.(I), showing fin, neural

tube and segmental mesoderm in which muscle histogenesis inay be noted. ( x SO). 2. Photomicrograph of a transverse section of embryo 2.Tp.(l) posterior to fig. 1,

showing a secopdary gut lumen and ectodemal ingrowth. Note the similarity of the mesodermal and neural tissues.

3. Photomicrograph of a transverse section of normal, stage 16, neurula through the anterior end of the neural plate. ( x 50.)

4. Photomicrograph of a transverse section of a normal, stage 16, neurula through the posterior end of the neural plate. ( x 50.)

(,x YO.)

PROC. ZOOL. SOC. VOL. 119. FORD. PL. I.

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PROC. ZOOL. SOC. VOL. 119. FORD. PL. II.

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