The Embryonic Limb Bud of the Urodele:

The Embryonic Limb Bud of the Urodele: Morphological Studies of t h e Apex A. STURDEE and M. CONNOCK Department of Biological Sciences, The Polytechnic, Wolverhampton, WVI ILY,Staffs., U.K.

Received December 1974

Scanning electron microscopy and histological investigation of the embryonic fore-limb bud of Notophthalamus (Triturus) cristatus and N . vulgaris has revealed that an apical ectodermal ridge (AER) is not detectable in these species of newt (Urodela). The limb buds examined ranged from those just visible as a slight projection from the body surface through to buds possessing two prominent digits. These stages correspond to the stages in Anurans during which the AER first appears, reaches its maximum size and then regresses. The significance of this observation is discussed.

Introduction

The morphogenesis of the vertebrate limb depends on reciprocal interaction between the mesenchyme and the ectoderm of the limb bud to permit normal development (for reviews see Zwilling Ill, Goetinck 121, Saunders and Gasseling [31. Much of this interaction is thought to occur at the apex of the developing limb bud where a thickened crest of epidermis, known as the apical ectodermal ridge (AER), is situated. This structure runs around the tip ofthe bud at the cone stage and is later confined to the paddle. Extensive experimental analysis has indicated that in the chick a morphogenetic influence of the AERis essential for promoting the sequential outgrowth and differentiation of the limb mesenchyme and for determining the orientation ofthe paddle 11-51. Despitethis plethoraof analysis, other investigations have cast some doubt on the significance and interpretation of the role ascribed to the ridge [6-91. Nevertheless. the balance of contemporary opinion still favours the former view. An apical ectodermal ridge has been found during the cone stage of development on the limb buds of every vertebrate so far examined. These include representatives of all the major vertebrate groups: Man [lo];lower Mammals(l1, 121; Reptiles [131;Birds [4l; Amphibia[l4,151; Elasmobranchs [ 161 and Teleosts [ 16, 17). Until recently the presence of an AER on the amphibian limb bud was in contention, many autorities denying its existence. (For review see Tarin and Sturdee [151). This situation was clarified when Tarin and Sturdee [ 151 demonstrated Differentiation 3, 43~-49 (1975)

~

0 by

Springer-Verlag 1975

beyond reasonable doubt that the hind-limb bud of Xeaopus Iaevis does possess an AER, while Tschumi 1141, in 195 7, had presented experimental evidence that inXenopus the apical epidermis exercises a similar role to that of the chick. An AER has now been reported for several more species of Anura I1 8, 191 while one species in particular, A lytesobstetricans, possesses aridgewhich is as prominent as that of the chick 1191. All the Amphibiapreviously examinedfor the presence ofaridge have beenmembers ofthe order Anura. However, the embryonic limb disc of the Urodele was the subject of muchofthe classicalexperimentalwork(e.g.Harrison [201 and Swett [2 11) while in the adult, limb regeneration has been extensively investigated. Recent papers have stressed the similarity between the development of the regeneration blastema and that of the embryonic limb bud [22,231; the former process is thought to be dependent on interaction between the blastemal cells and the apical ectodermal cap, while the latter involves the interplay of the AER and the subjacent mesenchyme. It is therefore desirable to establish whether the limb bud of the Urodele possesses an AER. We report here the results of an investigation, using scanning electron microscopy and histology, of two species of Urodela: Notophthalamus (Triturus) cristatus and N. vulgaris. In addition a small number of specimens of two Anurans, Rana temporaria and Bufo bufo were studied for comparative purposes.

44

A. Sturdee and M. Connock:

Materials and Methods

Results

Spawn was obtained in the Spring from local ponds and cultured in continuously aerated 10%Holtfreter solution at 20” C. Hatched larvae were transferred to fresh medium at a concentration of one individuaV0.5 1 of 10% Holtfreter. Urodeleswerefedon freshwater plankton and the Anura on aquatic vegetation. Larvae were anaesthetised in MS : 222 (Sandoz). Notophthalumus was staged according to Harrison’s table for Ambystoma (published in Hamburger 1241) since n o suitable table exists for the former. Similarly the table of Taylor and Kollros 1251for Ranapipiens was used for Rana temporaria and Bufo bufo. Urodelelarvae were selected from stages 37-43 during which time the fore-limb bud develops from a small hemispherical projection to the “notch” stage when it possesses two prominent digits (Fig. la-e). Larvae of Rana and Bufo were chosen from hind limb bud stages I11 to VIT. These stages, for both urodele and anuranlarvae, thus correspond to the period of development of the hind limb bud of Xenopus during which the AER first appears, reaches its maximum size and then regresses f 151. For scanning electron microscopy, specimens were immersed in 4% Glutaraldehyde in 0.1M sodium cacodylate for 24 h [261 at 4O C . After complete dehydration in a graded series of alcohols, the tissues spent 2 hin 1; 1,2-trichIorotrifluoroethane(BDH)known as“Fluoriso1” 1271. Following this they were transferred to a small volumeof “Fluorisol” (0.5 ml-1.0ml) contained in shallow covered watch glass. The “Fluorisol” was allowed to evaporate into a saturated atmosphere, the rate of vaporisation being controlled by raising and lowering the lid of the watch glass. This method, employing “Fluorisol”, was preferred to enable direct comparison with previous work on Xenopus [IS1 . The dried tissues were glued to aluminium chucks, coated with gold/palladium and examined in either a Cambridge S2 or S4 scanning electron microscope operated at 10 k V . Urodele and anuran larvae chosen for the histological study were fixedinBouin’sfluidfor24h. Thespecimenswerethentranferredto70% alcohol where the fore-limb buds (Urodela) and the hind-limb buds (Anura) were dissected out and orientated in molten agar (45” C) to provide the appropriate longitudinal and transverse sections of the limb bud on subsequent sectioning. Following dehydration the agar pellets containing the limb buds wereembedded in paraffin wax, sectioned at 7 pm and stained in Erlich Haematoxylin and Eosin.

the fore-limb buds ofboth speciesofnewtwere similarboth histologically and in their external appearance. N . vulgaris differed fromN, cristatusin that the limb bud was smaller at each stage. At stage 37, the newt fore-limb bud was a smooth hemispherical projection lying caudal to the gills (Fig. 2,1a)andlateraltothepronephros(Fig. 9-Stage38). Epidermal cells on the bud’s surface were predominantly pentagonal or hexagonal, some ofthem appearing ciliated, while others were stippled suggesting the presence of microvilli (Fig. 3). The epidermis at this stage and at stage 38 was one to two cells deep, thicker than that of the flank, although thecells comprisingittendedto beflattenedrather than cuboidal or spherical in section (Fig. 9). The mesenchymal cells ofthe bud were tightly packed into the pocket formed by the epidermis (Fig. 9). Mitoses were frequently visible in both tissues at this and subsequent stages. Following stage 37 the bud elongateduntil, at stage 39, it had become cone shaped (Fig. lb), the overall length of the bud being twice its width. By stage 40 the apex had flattened, giving the bud an almost square distal outline (Fig. lc, 4). This preceded division into two digits which was just visible at stage 4 1(Figs. Id, 5); by stage43 the bud was deeply cleft (Fig. le). During this sequence of changes the apex and distal part of the bud presented an essentially smooth topography with no signof an elevation resembling a ridge (Fig. 4, 5 , 7). The external appearance of the epidermal cells remained polygonal, becoming more regular in shape at later stages (e.g. Fig. 4). The ciliated cells of stage 37 dwindledin number and were rarely seen after stage 40. Single cells, or parts of such cells, were sometimes seen bulging from the otherwise predominantly smooth surface (e.g. Figs. 3,4).

Fig. la-e. Typical representatives of“. cristatus at stages 37 (la), 39 (lb), 40(lc), 4 1 (ld)and43 (le) toshowthechangesin shapeandorientationof the fore-limb bud (arrowed). The positions of the gills (G), balancer (B) and eye (E) are also indicated. x 8

Apex of the Urodele Limb Bud

45

Fig. 2. Scanning electron micrograph of a stage 37 fore-limb bud ofN. cristutus to illustrate its symmetrical shape and smooth profile. x 200. The area within the rectangle is shown in Fig. 3

Fig. 3. An enlargement of part of Fig. 2, to display the raised cell boundaries (arrowed), ciliated cells (0,stippled cells (S) and cells presenting a “cobbled” surface(@ consisting ofseveral small bumps. x 500

The stippled surface of many cells noted in the early stages had given way to cells showing a complex folding of the membrane by stage 43 (Fig. 7). Within the bud substantial changes occurred between stages 37 and 43, but in the mesenchyme rather than the epidermis. Blood cells were seen among the mesenchymal cells at stage 39, while the first nerves to enter the base ofthe bud were detected at stage 40. The tight packing ofmesenchymal cells reported at stage 37 persisted until stage 40. No specific orientation of these cells, whether beneath the apical or more proximal epidermis could be discerned. Following stage 40 the mesenchyme was more loosely organised in parts of the bud, particularly in the interdigital zone. At stage 41 to 42 cells in the central proximal mesenchyme were elongated and stacked one above the other at right angles to the long axis of the bud (Fig. 12). Such cells had developed into tissue recognisable as cartilage, probably of the humerus, by stage 43. The blood vessels, following their appearance at stage 39-40 (Fig. 10) subsequently ramified throughout the bud, although

there was no marginal vessel curling round the apex beneath the epidermis. The epidermis became distinctly double-layered, containing cells ranging from those which were flattened to those morecircular in section (Figs. 10,ll).The free border of the outermost epidermal cells occasionally projected from the average line of the cell surface; cells undergoing mitosis were sometimes seenin this position. The apical and distal epidermis, whether viewed in transverseor longitudinal sections ofthe bud,couldnot bedistinguishedfromthat situated further proximally (Figs. 10, 11). Thus theepidermis in both regions appeared similar in terms of nuclear and cell size, shape and position, while the number of layers of cells in the distal epidermis also did not differ from that elsewhere. Observations of the anuran buds showed that the hind-limb buds of Rana temporaria possess a thickened epidermal ridge situated around the apex (Figs. 6,8). This structure was absent at stage 111,visible at stages IV and V, thereafter decreasing in size until it became undetectable at

46

Figs. 4-8. For explanation see page 48

A. Sturdee and M. Connock:

Apex of the Urodele Limb Bud

Figs. 9-12. For explanation see page 48

47

A. Sturdee and M. Connock:

48 stage VII. Beneath this ridge, and followingits course,there was a large blood vessel, the marginal sinus. The hind-limb buds of Bufo bufo, during the same series of stages, had a small indistinct elevation ofthe apical epidermis which was only visible at stage V. Discussion

There was no evidence from either scanning electron microscopy of histological examination that the fore-limb bud of the newt has any of the following characteristics:

Fig. 4. Scanning electron micrograph of a stage 40 fore-limb bud of N. cristatus. This bud was photographed looking directly down on the distalmost edgeofthe bud, Thereisno signof any structure resembling a ridge. x 630 Fig. 5. Stage 41 fore-limb bud of N. cristatus showing part of the distalmost edge as the depression (C) between the first two digits ( D ) becomes apparent. An apical ridge is not present. The asterisk marks a contaminating particle, probably a bacterium. Scanning electron micrograph. x 930 Fig. 6. Scanning electron micrograph ofthe apex of a stage V hind-limb bud of Rana temporaria showing the apical ectodermal ridge (A). x 660 Fig. 7.Thetipofasingledigitofthefore-limb budofN.cristutusatstage 43. An apical ridge is not present. The cell surface is convoluted rather than stippled as in previous stages. x 730 Fig. 8. Transverse section of the apical ectodermal ridge of a stage V hind limb bud ofR. temporaria. The columnar cells (0 of the ridge (A) can be seen above themarginal sinus(5‘). Theepidermis(E)is separated from the mesenchyme (M) by a well-defined basement membrane (arrowed). x 600 Fig. 9. Transverse section ofN. vulgaris at stage 38 to show thefore limb bud projecting from the lateral body wall. The epidermis (E)surrounding the bud mesenchyme ( M ) is thicker than that of the flank. Also visible in this micrograph are the developing pronephros (P),yolk filled on the dorsal side of the limb bud. x gut (G) and some pigment 200

(n

Fig. 10. Longitudinal sectionofa stage40limb budofN.cris~utuswhich cuts the straight distalmost edge (Fig. lc) at right angles. There is no elevation of the apical epidermis corresponding to a ridge. The epidermis (E) is separated from the mesenchyme ( M ) by an ill-defined basement membrane (arrowed). A blood vessel (B) is present proximally. x 500 Fig. 11. Longitudinal section through the tip of a developing digit of a stage 42 limb bud of A’. cristatus. The epidermis (E)and mesenchyme (M) can be easily distinguished in this picture. x 550 Fig. 12. The proximal part of a stage 42 limb bud ofN. cristatus to show the elongated cells(0, which will later form cartilage, orientated at right angles to the bud‘s long axis. The axilla is marked with an asterisk. x 300

1. a distally or apically located epidermal thickening

2. columnar cells in the apical epidermis 3. an increased number of cell layers in the apical epidermis 4. a marginal sinus in the mesenchyme beneath the apical epidermis. These are some of the typical characteristics of the AER in othervertebrates such asXenopus[151 andthechick [41.It is thus apparent that N. cristatus and N. vulgaris do not possess a morphologically distinct structure resembling the apical ectodermal ridge found on other vertebrate limbs during the equivalent developmental stages. This finding in the newt supports the observation of Karczmar and Berg [28] that in Ambystoma the apical ectoderm of the embryonic fore limb did not differ from that on the body. The techniques used to show that the newt lacks an AER are, however, well able to demonstrate its presence in Rune and Bufo; the former has a well-developed ridge, the latter an indistinct one. The observations agree with those ofStebler [ 191who hasshown histologicallythat an AERis present in several Anurans, including Rana temporaria and Bufo bufo, the latter species having only a weakly developed ridge. The developing newt limb appears to conform to the proximo-distal sequence of differentiation of the cartilagenous elements of the bones reported for other vertebrates (Zwilling [ I], for review). Similar conclusions can also be drawn from Balinsky’s [291 work on Triton (Urodela). However, the formation of digits differs from the usual pattern common to anuran and other pentadactyllimbs. In other vertebrates the digits begin development more or less simultanuously in a broad, well-defined paddle, whereas in thenewt fore-limb thereis merelya slightflattening distally, followed by precocious development of two digits. Considerably after this, the third and then the fourth digits appear [301. Possibly the absence of the AER is relatedin some way to this difference in the development of the distal elements in the newt limb. The lack of an AERinthenewt accounts for$ part, the opinions ofearlier workers who considered thatthe Amphibia, both Anura and Urodela, do not possess this structure [ 16,30,3 11. Much ofthe now classical work on amphibian limb development was performed on Urodeles rather than Anurans (see introduction) while the relatively few reports ofstudieson the anuranlimb 132-341, with theexception of Steiner’s [351 “Ektodermkappe”, do not remark on the presenceor absenceof anythickened epidermisat the apex. Thus it is not unnatural that the Amphibia as a group should have been regarded as lacking an AER. It is perhaps surprising that while embryonic development of the urodele limb proceeds without the intervention

Apex of the Urodele Limb Bud

of a morphologically distinct AER, or analogous thickening,theamputatedadult,or larva1,limbacquiresathick cap of epidermis which has properties comparable to those of the AER [22,23,361. However, the absence of this structure, together with the absence of a distinct marginal sinus, does not preclude the possibility that the apicalepidermis of the embryonic newt limb bud may be functionally equivalent to the AER even ifit is not morphologically equivalent. Both Balinsky’s I291andsteiner’s 1351experiments involving removal or destruction of the apical epidermis of the embryonic urodele limb suggest that this epidermis certainly is required to promote normal morphogenesis. However, before functional equivalence can be proven it is necessary to repeat some ofthemore definitive experiments such as those performed on the chick and Xenopus (reviewed by Zwilling 111) using the limb bud of the Urodele. Acknowledgement: We wish to thank the Centre for Materials Science of Birmingham University for the use ofthe Scanning Electron Microscope and Mrs. P. M. Boyden for the typing of the manuscript.

References I. Zwilling, E.: Adv. Morphogen. I , 301, 1961 2. Goetinck. P. F.: In: Current Topics in DevelopmentalBiology. A. A. Moscona, A. Monroy, (eds.) Vol. I, p. 253, New York: Academic Press 1966 3. Saunders. J. W.. Gasseling. M. T.: In: Epithelial-Mesenchymal Interactions. R. Fleischmajer, R. E. Billingham. (eds.) p. 78, Baltimore: Williams and Wilkins 1968 4. Saunders, J. W.: J. exp. Zool. 108, 363, 1948 5. Zwilling, E.: J. exp. 2001.132, 157. 1956 6. Bell. E., Kaighn, M. E.. Fessenden, L. M.: Devl. Biol. I, 101, 1959

49 7. Bell. E.. Gasseling,M. T.. Saunders, J . W.,Zwilling,E.:Devl. Biol. 4, 177, 1962 8. Amprino. R.: In: Organogenesis. R. L. DeHaan, H. Ursprung, (eds.) p. 255, New York: Holt, Rinehart and Winston 1965 9. Amprino. R., Bonetti. D. A,: Nature 214, 826, 1967 10. O’Rahilly. R., Gardner, E., Gray, D. J.:J. Embtyol. exp.Morph. 4, 254, 1956 11. Milaire, J.: Archs Biol. 67, 297, 1956 12. Milaire. J.: Archs Biol. 74, 129, 1963 13. Milaire. J.: Archs Biol. 68, 429. 1957 14. Tschumi. P. A , : J. Anat. 91, 149, 1957 15. Tarin, D., Sturdee. A. P.: J. Embryol. exp. Morph. 26, 169, 1971 16. Braus, H.: In: Hertwig’s Hbh. derEntwickelungslehre der Wirbeltiere 3, 167, 1906. Cited by Saunders [41 17. Bouvet, J.: Archs Anat. microsc. Morph. exp. 63, 79, 1974 18. Dawd, S. D., Nawar, G.: Libyan J. Sci. 2, 9, 1972 19. Stebler, R.: Wilhelm Roux Arch. EntwMech. Org. 172, 131, 1973 20. Harrison, R. G.: J. exp. Zool. 25, 413, 1918 21. Swett. F. H.: J. exp. 2001.47, 385, 1927 22. Faber, J.: Adv. Morphogen. 9, 127, 1971 23. Stocum, D. L., Dearlove, G. E.: J. exp. 2001.181, 49, 1972 24. Hamburger. V . : AManualofExperirnentalEmbryology.Chicago: Univ. Chicago Press 1960 25. Taylor. A. C., Kollros, J. J.: Anat. Rec. 94, 7, 1946 26. Boyde, A., in: Proceedings of the 5th Annual Scanning Electron Microscope Symposium. Part 11.0. Johari, I. Corvin, (eds.) p. 257, Chicago: IIT Research Institute 1972 27. Nott, J. A,: Mar. Bid. 2, 248, 1969 28. Karczmar, A. G., Berg. G. G.: J. Exp. Zool. 117, 139, 1951 29. Balinsky, B. 1.: 2001.Jb. (Allg. 2001.Physiol.) 54, 327, 1935 30. Ralinsky, B. I.: A n Introduction to Embryology, p. 439. Philadelphia and London: W. B. Saunders 1970 3 1. Pcter. K.: Arch. mikrosk. Anat. EntwMech. 61, 509, 1902 32. Byrnes, E. F.: J. Morph. XIV, 105, 1898 33. Tschernoff. N. D.: Anat. Anz. 30, 593, 1907 34. Taylor, A. C.: Anat. Rec. 87, 397, 1943 35. Steincr, K.: Wilhelm Roux Arch. EntwMech. Org. 113, 1, 1928 36. Thornton, C. S.: Adv. Morphogen. 7, 205, 1968