Anat Embryol (1992) 186 : 27-32

Anatomy and E/nbryology

�9 Springer-Verlag 1992

Original articles

Interdigital chondrogenesis and extra digit formation in the duck leg bud subjected to local ectoderm removal D. Macias 1, Y. Gafian 1, and J.M. Hurle 2

1 Departamento de Ciencias Morfologicas, Facultad de Medicina, Universidad de Extremadura, Badajoz, Spain 2 Departamento de Anatomia y Biologia Celular, Facultad de Medicina, Universidad de Cantabria, Poligono de Cazofia s/n, E-39011 Santander, Spain

Accepted January 17, 1992

Summary. In the chick embryo the interdigital tissue in the stages previous to cell death exhibits in vitro a high chondrogenic potential, and forms extra digits when subjected in vivo to local ectodermal removal. In the present work we have analyzed the chondrogenic poten- tial both in vivo and in vitro of the interdigital mesen- chyme of the duck leg bud. As distinct from the chick, the interdigital mesenchyme of the duck leg bud exhibits a low degree of degeneration, resulting in the formation of webbed digits. Our results show that duck interdigital mesenchyme exhibits also a high chondrogenic potential in vitro until the stages in which cell death starts. Once cell death is finished chondrogenesis becomes negative and the interdigital mesenchyme forms a fibroblastic tis- sue. In vivo the interdigital mesenchyme of the duck leg bud subjected to ectoderm removal forms ectopic foci of chondrogenesis with a range of incidence similar to that in the chick. Unlike those of the chick the ectopic cartilages of the duck are rounded and smaller, and ap- pear to be located at the distal margin of the interdigital mesenchyme. Formation of extra digits in the duck oc- curs with a lower incidence than in the chick. It is con- cluded that ectopic chondrogenesis and formation of ex- tra digits is related to the intensity of interdigital cell death. The non-degenerating interdigital mesenchymal cells destined to form the interdigital webs of the duck appear to contribute very little to the formation of inter- digital cartilages.

Key words: Duck embryo - Limb development - Chon- drogenesis - Cell death

Introduction

The chick limb bud is one of the best studied models of morphogenesis (see Hinchliffe and Johnson (1980) for review). At early stages of development the limb bud

Offprint requests to: J.M. Hurle

consists only of a core of mesenchymal cells rich in blood vessels covered by an ectodermal jacket. The formation of the skeletal elements takes place in a proximo-distal gradient by condensation of the mesenchymal cells to form the prechondrogenic anlage of the skeletal rudi- ments. The process progresses in such a way that the distal part of the limb bud maintains the undifferentiated structural composition of the early limb bud until the establishment of the most distal skeletal elements. The formation of the digits takes place in the latest period of morphogenesis by the condensation of the digital pre- chondrogenic rays, while the interdigital spaces remain undifferentiated. In amniote embryos with free digits, in the course of development the interdigital mesenchy- mal cells undergo a massive cell death process which appears to play the role of sculpturing the digits (Saunders and Fallon 1967; Pautou 1974; Fallon and Cameron 1977; Hinchliffe 1982; Hurle 1988). In species with webbed digits interdigital cell death is less intense (Hurle and Colvee 1982) giving rise to the interdigital membranes. Interdigital cell death is also reduced or in- hibited in mutant chick species with syndactyly (Hinch- liffe and Thorogood 1974). In the last few years we have observed that the interdigital mesenchyme of the chick leg bud in the stages previous to cell death exhibits a high chondrogenic potential under culture conditions (Hurle et al. 1991). Furthermore, when the interdigital ectoderm is removed surgically or enzymatically from the limb bud in stages previous to cell death, the interdi- gital mesenchyme forms ectopic cartilages which in the course of development form fully differentiated extra digits (Hurle and Gafian 1986, 1987; Hurle et al. 1991). These investigations of digit morphogenesis induced in late stages of limb development raise important ques- tions concerning the mechanisms proposed for digit pat- terning. Current hypotheses explaining limb patterning propose a very early determination of the number and pattern of digits (Wolpert 1981). A well defined zone of the mesenchyme of the posterior margin of the limb, called the zone of polarizing activity (ZPA), appears to generate a signal (" morphogen") which diffuses towards

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the anter ior margin o f the limb, giving cues o f posi t ional informat ion to the mesenchymal cells and account ing for determinat ion o f each digit (Tickle et al. 1975, 1982; Tickle 1980; Thaller and Eichele 1987; Wolper t 1989; Brockes 1990). A precise pat tern o f gene expression ap- pears to be linked to this process (Nohno et al. 1991).

In fo rmat ion on the mechanism of extra digit fo rma- tion at advanced stages o f morphogenes is has only been obtained f rom chick embryos which display prominent interdigital necrotic zones. A ma jo r quest ion to be ans- wered is whether only prospective dying interdigital cells have in format ion to fo rm extra digits or if the distal mesenchyme destined to fo rm interdigital webs also shares this property.

In the present paper we have analyzed the chondro- genic potent ia l o f the interdigital mesenchyme o f the duck leg bud in vivo and in vitro, and its ability to form extra digits in vivo after ec toderm removal. The duck leg bud has a prominent au topod ium with large interdigital spaces in which mos t o f the mesenchymal cells survive, giving rise to the webbed foot. So this mod- el allows us to ascertain whether the p roper ty o f forming digits is restricted to the prospective dying cells, or if it is also shared by cells destined to fo rm interdigital membranes .

Material and methods

In all experiments we have employed Roayl Peking duck embryos ranging from days 7 to 11 of incubation.

In vivo surgicalprocedures. The eggs were fenestrated, and the right leg bud was exposed under the binocular dissecting microscope using microsurgery forceps. The marginal ectoderm of the third interdigit was removed with a fine tungsten needle, taking care to avoid damaging the neighboring digits, as previously described (Hurle and Gafian 1986). The left leg was employed as control. After the operation the egg window was sealed with sellotape and the eggs were returned to the incubator until 3 days later. The incidence of chondrogenesis was then determined by specific stain- ing with Alcian blue or methylene blue in whole-mount prepara- tions. The morphological changes of the experimental limbs were also analyzed by scanning electron microscopy.

Organ cultures. In a second group of embryos we performed an in vitro analysis of the cartilage-forming capacity of the interdigital mesoderm. For this purpose the third interdigit was removed from the leg bud and incubated for 16 or 20 rain in 2% trypsin at 4 ~ C. The specimens were then washed and the ectoderm removed using tungsten needles. The interdigital wedge was cut into two halves, proximal and distal, using a cataract knife. The fragments were then placed in culture dishes with 1 ml of medium containing Nu- trient Mixture F-12 Ham supplemented with 10% fetal calf serum. Penicilin (50 u/ml), Streptomicin (25u/ml) and Gentamicyne (40 Izl/ml) (all from Gibco) were added to retard the growth of possible contaminants. The organ cultures were incubated at 37 ~ C in an atmosphere of 5% CO2/95% air in a humidified incubator. Cultures received fresh medium daily until 3 days later. Cultures were washed with PBS and fixed in Khale's fixative for 20 rain. To visualize the distribution of cartilage, cultures were stained with Alcian blue pH 1.0, and the diameter of the chondrogenic areas was measured with the aid of an ocular micrometer. The intensity of chondrogenesis observed was classified as: (+ + +) when all the explants exhibited a high chondrogenic activity; (+ +) when

most but not all the explants exhibited a moderated chondrogenic response; (+) when the chondrogenic response was reduced and was present in some of the explants; ( - ) when most of the explants (if not all) were negative.

Results

In vitro analysis of interdigital chondrogenesis (Table 1)

All the explants, distal and proximal, f rom embryos at day 7 to 8 o f development showed nodules o f cartilage after 3 days o f culture. The cartilages present in explants o f the distal segments o f the interdigit were larger than those o f the proximal par t o f the interdigit (0.4 and 0.2 m m diameter respectively) (Figs. 1, 2). In some cases the proximal explants showed two cartilages (Fig. 3). This feature never occurred in distal explants. At days 8.5 and 9 o f development the distal par t o f the interdigit remained highly chondrogenic , while the prox- imal explants were in m a n y cases non-chondrogen ic or fo rmed a small cartilage o f no more than 0.1 m m diame- ter (Fig. 4). At day 9.5 o f development the distal par t o f the interdigit fo rmed a small chondrogenic nodule o f 0.2 m m diameter (Fig. 5), while the proximal par t o f the interdigit was never chondrogenic . F r o m day 10 to 11 o f development neither the proximal nor the distal par t o f the interdigit exhibited cartilage format ion. N o massive degenerat ion was detected in the explants with- out chondrogenesis . As can be seen in Fig. 6, these ex- plants consisted o f a growing mass o f fibroblastic mesen- chymal tissue.

In vivo induction of interdigital chondrogenesis

The third interdigital space o f the duck leg consists o f a core o f mesenchymal cells covered by ectoderm. Unti l day 8.5 o f development the marginal ec toderm exhibits the typical apical ec todermal ridge (AER). F r o m day 9 o f development the A E R flattens out. Mesenchymal cell death is observed in the mesenchyme underlying the marginal ec toderm f rom day 9.5 o f development . Be- tween days 10.5 and 11 bo th the marginal ec toderm and the distal mesenchyme exhibit a degenerat ion process. By day 12 the interdigital space achieves its final mor-

Table 1. Incidence of interdigital ectopic chondrogenesis in vitro and in vivo after ectoderm removal

Day of % operation or Chondrogenesis explantation in vivo (n)

Chondrogenesis in vitro

Proximal Distal

7 0 (17) + + + + + + 7.5 14.7 (34) + + + + + + 8 36.1 (36) + + + + + + 8.5 38.4 (39) + + + + + 9 36.3 (33) + + + 9.5 4.5 (22) - +

~o o (lo) - - 11 o (12) - -

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Figs. 1-6. Micrographs of interdigital tissue explants cultured for 3 days and stained with Alcian blue. Bar 67 gm

Fig. 1. Explant of the distal segment of the interdigit from a limb bud of the day 8 of development. Note the presence of a large chondrogenic nodule (0.4 mm diameter)

Fig. 2. Explant of the proximal segment of the interdigit from a limb bud of day 8 of development

Fig. 3. Explant of the proximal segment of the interdigit from a limb bud of day 8 of development showing two nodules of cartilage

Fig. 4. Explant of the proximal segment of the interdigit from a limb bud of day 9 of development showing a very small area Alcian blue-positive (arrow).

Fig. 5. Explant of the distal segment of the interdigit from a limb bud of day 9.5 of development showing a small cartilage (0.2 mm diameter) ; compare with Fig. 1

Fig. 6. Explant of the distal segment of the interdigit from a limb bud of day 11 of development lacking Alcian blue-positive nodules

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Fig. 7. Experimental duck leg bud three days after ridge removal at day 7 of development stained with Alcian blue. The pattern of chondrogenesis is normal. Note the presence of a prominent cleft in the third interdigit. Bar 300 gm

Fig. 8. Methylene blue cartilage staining showing the presence of a chondrogenic nodule in the third interdigit of a duck leg bud three days after ectoderm removal at day 8.5 of development. Bar 250 jxm

Fig. 9. Duck foot at day 11 of development after ectoderm removal at day 8 showing an ectopic digit in the third interdigit, Bar 275 gm

Fig. 10. Scanning electron micrograph showing a duck leg bud at day 12 of development after ectoderm removal at day 9. A small rounded cartilage appears in the tip of the interdigital membrane. Bar 275 gm

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phology consisting of a core of fibroblastic mesenchymal cells covered by an ectoderm of uniform morphology (see Hurle and Fernandez-Teran 1984, for details).

At day 7 of development the digital rays of the foot plate are hardly recognizable at the time of the opera- tion, and on some occasions the ectoderm of the digit was damaged in the course of the operation. When ridge removal was limited to the interdigital space the resulting leg showed prominent indentation at the level of the operation (Fig. 7). Chondrogenesis was not detected in any of the experimental embryos operated at this stage.

Between days 7.5 and 9 of development interdigital marginal ectoderm removal resulted in the formation of a chondrogenic nodule three days after the operation. The nodules were always rounded and were located in the very distal part of the interdigit at the level of the wound (Fig. 8). The experimental embryos operated at 7.5 days of development often showed a small indenta- tion at the level of the wound similar to that found at day 7. From day 8 of development the morphology of the interdigit of the experimental limbs was normal except for the presence of ectopic cartilages.

The incidence of chondrogenesis varied in the differ- ent stages (Table 1). Maximum incidence was detected between days 8.5 and 9 (38.4 and 36.3% respectively) and dropped sharply at day 9.5 (4.5% of the cases). Chondrogenesis was negative in all experimental em- bryos operated in days 10 and t t of development.

The size of this ectopic chondrogenesis also varied in the different stages at which the operation was per- formed. Maximum size of the cartilage was detected be- tween days 7.5 and 8.5 of development. At these stages, in 6 out 33 experimental embryos with positive chondro- genesis, the ectopic cartilage exhibited a morphology re- sembling an extra digit (Fig. 9).

In the embryos operated at day 9 of development most of the ectopic cartilages appeared as small rounded nodules forming a prominence in the margin of the inter- digital membranes (Fig. 10). Ectopic cartilages with the morphology of an extra digit were observed in 2 out 12 cases. Ectopic chondrogenesis in embryos operated at day 9.5 of development were observed in only 1 out 22 surviving embryos, and the cartilage appeared as a small nodule associated with the margin of the interdigi- tal membrane. As can be seen in Fig. 9, the extra digits appeared very rudimentary, showing the morphology of a distal phalange. In some cases a developing joint was detected in the extra digit. Three phalages were never present in the extra digits.

Discussion

Our observations show that the interdigital mesenchyme of the duck leg bud between days 7.5 and 9 of develop- ment is highly chondrogenic, both in vivo and in vitro, when deprived of the influence of the ectoderm.

It has been clearly established that the ectoderm of the limb bud exerts an important antichondrogenic ef- fect on the underlying mesenchyme (Solursh et al. 1981 ; Zanetti and Solursh 1986; Gregg et al. 1989). A similar

epithelial antichondrogenic effect has been also illustrat- ed in other developing structures (Tyler 1983; Frenz and Van de Water 1991). This study and our previous studies of the chick limb support Solursh's hypothesis. Further- more as proposed by Solursh (1984), the present results suggest that the ectoderm may play by this mechanism a patterning role in the formation of the limb skeleton, by confining the cartilage to the core of the limb bud. Our results contrast with the study of Martin and Lewis (1986), who failed to induce ectopic chondrogenesis in limb buds subjected to ectoderm removal at stages 17 to 19 by irradiation with ultraviolet light. Since the stages and the zone of the limb bud studied by Martin and Lewis are distinct from those analyzed by us, this difference might be linked to regional differences in limb bud and/or to temporal variations in the functional property of the ectoderm as illustrated by Solursh and Reiter (1988).

There is a large amount of evidence showing that the limb mesenchyme undergoes progressive differentia- tion as it becomes segregated proximally from the more undifferentiated subectodermal region called the "pro- gress zone" (Summerbell etal. 1973; Summerbell and Lewis 1975). The different incidence of chondrogenesis between the proximal and distal explants of the duck interdigit is in agreement with that idea.

The loss of the in vitro chondrogenic potential of the interdigit begins at day 9 of development. This stage corresponds with the onset of the interdigital cell death process of the duck (Hurle and Colvee 1982). This fact suggest that the dying cells of the interdigit of the duck constitute a population of chondrogenic mesoderm, while the surviving cells are the ones which follow a fibroblastic pathway of differentiation.

The experiments of in vivo induction of interdigital tissue chondrogenesis and extra digit formation reveal many similarities between the duck and the chick in spite of the different developmental significance of the interdi- gital tissue in the two species (degeneration or forming interdigital membranes). According to these results it can be assumed that the ability to form ectopic chondro- genesis, or eventually extra digits, is a general feature of the avian embryonic limb. The recent report of the existence of fossil fish species with more than five digits questions the idea of the pentadactyl limb as the arche- type of the vertebrate limb (Coates and Clack 1990). Our experiments in avian limbs fit very well with this new conception of the primitive limb.

Both in the chick and the duck the ability to form interdigital cartilages start at the time when the apical ectodermal ridge (AER) flattens. Also both in the chick and in the duck this phenomenon ends when interdigital cell death starts. On the basis of these facts it can be proposed that the formation of interdigital cartilages is correlated with the presence of a critical amount of healthy chondrogenic tissue in the interdigit. Experi- ments removing wedges of interdigital tissue in the chick clearly demonstrate the requirement of a critical amount of tissue to form cartilages (Hurle et al. 1989).

There are also several distinct differences in these ex- periments between the chick and the duck. While the

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incidence o f chondrogenes i s is s imilar in bo th species, in the duck embryos the ec topic car t i lages are s m a l l e r and occupy a very dis ta l pos i t i on in the interdigi t . Fur - ther, the incidence o f ex t ra digits is h igher in the chick (48% of the exper imen ta l embryos with ec topic chon- drogenesis) than in the duck (18% o f the exper imen ta l embryos with ec topic chondrogenes i s ) in spite o f the larger size o f the in te rd ig i t o f the duck leg bud. These differences cou ld well be l inked to the di f ferent pa t t e rn o f in terd ig i ta l cell dea th in b o t h species. In the chick mos t o f the in terd ig i ta l mesenchyme undergoes degener- a t ion (Saunders et al. 1962; Saunders and Fa l lon 1967; Hinchl i f fe 1974; Pau tou 1974); in the duck only the dis- tal in terd ig i ta l mesenchyme and a smal l a m o u n t o f the centra l in te rd ig i ta l t issue unde rgoes cell dea th (Hur le and Colvee 1982). These differences have been re la ted to a scu lp tur ing role o f in te rd ig i ta l cell dea th in l imb d e v e l o p m e n t (Hur le 1988). Our presen t results a p p e a r to cor re la te the r educed a m o u n t o f in terd ig i ta l cell dea th in the duck with the es tab l i shment o f a f ib rob las t ic pa th - way o f the d i f fe ren t ia t ion in the in te rd ig i ta l mesen- chyme. Ana lys i s o f differences in the p a t t e r n o f gene express ion re la ted to cell d i f fe ren t ia t ion in bo th species m a y be re levant to clar i fy this quest ion.

Acknowledgments. This work was supported by grants from the DGICYT PB 89-0493 and DGICYT PS 87-0095.

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