Interdigital regulation of digit identity and homeotic transformation by modulated BMP signaling.

The developmental mechanisms specifying digital identity have attracted 30 years of intense interest, but still remain poorly understood. Here, through experiments on chick foot development, we show digital identity is not a fixed property of digital primordia. Rather, digital identity is specified by the interdigital mesoderm, demonstrating a patterning function for this tissue before its regression. More posterior interdigits specify more posterior digital identities, and each primordium will develop in accordance with the most posterior cues received. Furthermore, inhibition of interdigital bone morphogenetic protein (BMP) signaling can transform digit identity, suggesting a role for BMPs in this process.

FGF-2: apical ectodermal ridge growth signal for chick limb development.

The apical ectodermal ridge permits growth and elongation of amniote limb buds; removal causes rapid changes in mesodermal gene expression, patterned cell death, and truncation of the limb. Ectopic fibroblast growth factor (FGF)-2 supplied to the chick apical bud mesoderm after ridge removal will sustain normal gene expression and cell viability, and allow relatively normal limb development. A bioassay for FGFs demonstrated that FGF-2 was the only detectable FGF in chick limb bud extracts. By distribution and bioactivity, FGF-2 is the prime candidate for the chick limb bud apical ridge growth signal.

Limb-patterning activity and restricted posterior localization of the amino-terminal product of Sonic hedgehog cleavage.

BACKGROUND: Sonic hedgehog (Shh), a vertebrate homolog of the Drosophila segment polarity gene hedgehog (hh), has been implicated in patterning of the developing chick limb. Such a role is suggested by the restricted expression of Shh along the posterior limb bud margin, and by the observation that heterologous cells expressing Shh have limb-polarizing activity resembling that of cells from the polarizing region of the posterior limb bud margin. It has not been demonstrated, however, that the Sonic hedgehog protein (SHH) alone is sufficient for limb patterning. SHH has been shown to undergo autoproteolytic cleavage in vitro, yielding two smaller products. It is of interest, therefore, to determine whether processing of SHH occurs in the developing limb and how such processing influences the function of SHH. RESULTS: We demonstrate that SHH is proteolytically processed in developing chick limbs. Grafts of cells expressing SHH protein variants that correspond to individual cleavage products demonstrate that the ability to induce patterned gene expression and to impose morphological pattern upon the limb bud is limited to the amino-terminal product (SHH-N) of SHH proteolytic cleavage. We also demonstrate that bacterially synthesized and purified SHH-N, released from implanted beads, is sufficient for limb-patterning activity. Finally, we show that the endogenous amino-terminal cleavage product is tightly localized to the posterior margin of the limb bud. CONCLUSIONS: Our data show that, of the two cleavage products resulting from SHH autoproteolysis, SHH-N expressed in grafted heterologous cells or supplied in purified form is sufficient to impose pattern upon the developing limb. Moreover, the restricted localization of the endogenous amino-terminal SHH cleavage product to the posterior border of the chick limb bud makes it unlikely that its patterning activity results from it being distributed in a broad gradient across the antero-posterior axis. More consistent with the observed localization is a model in which the amino-terminal SHH cleavage product exerts its patterning effects by local induction in or near the polarizing region, initiating a cascade of gene expression that ultimately extends across the developing limb.

Fibroblast growth factors as multifunctional signaling factors.

The fibroblast growth factor (FGF) family consists of at least 15 structurally related polypeptide growth factors. Their expression is controlled at the levels of transcription, mRNA stability, and translation. The bioavailability of FGFs is further modulated by posttranslational processing and regulated protein trafficking. FGFs bind to receptor tyrosine kinases (FGFRs), heparan sulfate proteoglycans (HSPG), and a cysteine-rich FGF receptor (CFR). FGFRs are required for most biological activities of FGFs. HSPGs alter FGF-FGFR interactions and CFR participates in FGF intracellular transport. FGF signaling pathways are intricate and are intertwined with insulin-like growth factor, transforming growth factor-beta, bone morphogenetic protein, and vertebrate homologs of Drosophila wingless activated pathways. FGFs are major regulators of embryonic development: They influence the formation of the primary body axis, neural axis, limbs, and other structures. The activities of FGFs depend on their coordination of fundamental cellular functions, such as survival, replication, differentiation, adhesion, and motility, through effects on gene expression and the cytoskeleton.

Expression and regulation of chicken fibroblast growth factor homologous factor (FHF)-4 at the base of the developing limbs.

Fibroblast growth factor homologous factors (FHFs) have been implicated in limb and nervous system development. In this paper we describe the expression of the cFHF-4 gene during early chicken development. cFHF-4 is expressed in the paraxial mesoderm, lateral ridge, and, most prominently, in the posterior-dorsal side of the base of each limb bud. The expression pattern of cFHF-4 at the base of the limbs is not altered by tissue grafts containing the zone of polarizing activity (ZPA), by implants of Shh-expressing cells, or by implants of beads containing retinoic acid, nor does it depend on the distal growth of the limb as it is not altered in limb buds that are surgically truncated. In three chicken mutants affecting limb patterning - talpid(2), limbless, and wingless - altered patterns of cFHF-4 expression are correlated with abnormal nerve plexus formation and altered patterns of limb bud innervation. Similarly, ectopic expression of cFHF-4 is correlated with a local induction of limb-like innervation patterns when beads containing FGF-2 are implanted in the flank. In these experiments, both ectopic innervation and ectopic expression of cFHF-4 in the flank were observed regardless of the size of the FGF-2-induced outgrowths. By contrast, ectopic expression of Shh and HoxD13 are seen only in the larger FGF-2-induced outgrowths. Taken together, these data suggest that cFHF-4 regulates or is coregulated with early events related to innervation at the base of the limbs.

Development of the brachial lateral motor column in the wingless mutant chick embryo: motoneuron survival under varying degrees of peripheral load.

Survival of motoneurons in the lateral motor column (LMC) of the chick embryo is known to depend on the periphery. How this dependence relates to the normally occurring death of motoneurons is unknown. Analysis of the time course of LMC cell loss in the absence of varying amounts of limb musculature could help bring about an understanding of this relationship. We undertook this analysis by studying LMC development in wingless chick embryos. Grossly these embryos lack wings, but we have reported that some of them possess more than 40% of the normal volume of wing bud-derived muscles (M.E. Lanser and J.F. Fallon, Anat. Rec. 217:61-78, 1987). In the present work we compared the time course of LMC development in wingless embryos that possessed varying amounts of wing bud-derived musculature with that in normal embryos. In normal embryos little cell loss occurs from the brachial LMC prior to day 8 (15% of the total cell loss). Most of the normal cell loss occurs between 8 and 10 days (62% of the total cell loss). In the wingless LMC, anywhere from 55% to 70% of the total cell loss occurs before day 8. The death of motoneurons prior to day 8 is proportional to the amount of wing bud musculature eliminated by the mutation. Cell loss after day 8 is proportional to the amount of wing bud musculature spared by the mutation. Therefore, when the limb is missing, most motoneurons die before the major period of cell loss even begins in the normal LMC. Counts of dead cells in the LMC also support this conclusion. In addition, curves plotting the rates at which cells are lost from the brachial LMC provide a suggestion that normal cell loss is biphasic and that limb removal enhances primarily the first phase of cell loss. These data suggest that the majority of motoneurons may die for different reasons in the normal and the limb-deprived LMCs. Overall, the number of motoneurons surviving in the brachial LMC is proportional to the volume of wing bud-derived muscle present. However, as the muscle volume approaches zero, motoneuron number does not. This suggests that most, but not all, motoneurons depend on limb bud-derived muscles for survival. Finally, the decreased motoneuron number in the wingless LMC, when compared to normal after the cell death period, cannot be totally accounted for by the additional loss of cells that occurred during the cell death period in the wingless LMC.(ABSTRACT TRUNCATED AT 400 WORDS)

Formation of extra digits in the interdigital spaces of the chick leg bud is not preceded by changes in the expression of the Msx and Hoxd genes.

By in situ hybridization we studied the expression patterns of Msx and Hoxd genes during the late development of the chick leg autopodium (foot) and compared them to patterns during the experimental development of interdigital extra digits. Extra digits are induced in the third interdigital space after various experimental manipulations, such as transient isolation of the interdigit, or removal of the interdigital marginal ectoderm and mesoderm. Msx1 and Msx2 are normally expressed in the interdigital tissue programmed to die. Our experiment changes the fate of the interdigital tissue from cell death to chondrogenesis and provides a good model for studying Msx involvement in defining areas of programmed cell death. Among the proposed roles of Hoxd genes is their involvement in the specification of digit identity early in development. The induction of extra digits allows us to examine whether this new morphogenetic commitment of the interdigital tissues involves changes in the domains of expression of Hoxd genes. Our results show that extra digits develop without a previous modification of the normal pattern of expression of Msx or Hoxd genes. This observation does not support the correlation between the expression of Msx genes and programmed cell death and suggests a role for these genes in maintaining the interdigital tissue in an undifferentiated state. Our results show that an increased number of digits can be formed without modifications in the pattern of expression of the 5'-located Hoxd genes and suggest the existence of latent or residual digit organization mechanisms past the time when digits are normally determined, independent of Hoxd gene expression.

The ability of the chick wing bud to regulate positional disparity along the anterior-posterior axis.

When wedges of wing bud tissue are added to a host wing bud so there is positional disparity between graft and host, skeletal duplications result (L. E. Iten and D. J. Murphy 1980) Dev Biol. 75, 373-385. The polarity of the duplications is predictable by the polar coordinate model, leading to the interpretation that the positional disparity caused the duplications. To determine whether positional disparity alone causes duplications, without the complication of added tissue, we rotated wedges of ectoderm and mesoderm around the proximodistal axis within the wing bud. Wedges measuring 200-800 micron along the distal edge were rotated 180 degrees at stages 20-22, reversing the anteroposterior and dorsoventral axes relative to the bud. This caused positional disparity, similar to that achieved by Iten and Murphy (1980), without the addition of tissue. We found that rotations involving no polarizing zone tissue produced normal wings or wings lacking some distal parts, as did rotations of tissue lying entirely within the polarizing zone. However, when polarizing zone mesoderm was displaced, so that polarizing and nonpolarizing tissues were juxtaposed, a majority of the operations produced polarized skeletal duplications. Our data demonstrate that positional disparity alone does not cause skeletal duplications in the chick wing bud, unless polarizing zone tissue is displaced. Further, these data demonstrate that the chick wing bud can regulate to form a normal wing skeleton in the face of large positional disparity, provided that the polarizing zone is not moved. Finally, our results may be explained by the action of the proposed polarizing morphogen on the displaced cells causing repolarization.

Development of wing-bud-derived muscles in normal and wingless chick embryos: a computer-assisted three-dimensional reconstruction study of muscle pattern formation in the absence of skeletal elements.

The mechanisms whereby the normal pattern of muscles within the developing chick limb bud is generated are largely unexplored. It has been proposed that the muscle pattern is established independently of the pattern for the limb skeletal elements to which the muscles normally attach (Shellswell and Wolpert: "The Pattern of Muscle and Tendon Development in the Chick Wing."In: Vertebrate Limb and Somite Morphogenesis. Cambridge University Press, Cambridge, pp. 71-86, 1977). To further examine this possibility we studied the formation of the proximal wing muscles in normal and wingless chick embryos. The muscles of the shoulder region (including the pectoralis) arise as part of the dorsal and ventral premuscle masses of the developing limb bud. These secondarily migrate out of the limb to take origin from the pectoral girdle while inserting onto the humerus (Sullivan: Aust. J. Zool., 10:458-516, 1962). With rare exceptions, wingless embryos have complete absence of wing skeletal elements, but they may possess more than 40% of the normal volume of wing-bud-derived muscles. The muscles that remain in wingless embryos are primarily shoulder muscles, and to a varying extent, the pectoralis. The question we sought to answer was whether in wingless embryos the proximal wing muscles could form a normal pattern in the absence of the humerus and distal wing skeletal elements. By examining three-dimensional reconstructions of the proximal wing region in normal and wingless embryos, we found that the initial subdivision of the dorsal and ventral premuscle masses proceeded normally in the absence of the wing skeleton. This resulted in a grossly normal pattern of proximal wing muscles despite the absence of wing skeletal elements. However, some subsequent cleavages of individual muscles within premuscle mass divisions did not occur in wingless embryos. This suggests that the skeleton may be required for this step in muscle morphogenesis to occur. We also observed that the wing-bud-derived muscles in wingless embryos were nearly always anchored to the pectoral girdle at both ends. Sometimes this resulted in muscles making abnormal tendonous fusions with other muscles derived from the opposite (i.e., dorsal or ventral) premuscle mass. Therefore, attachment to the skeleton may be important for some facet of muscle development. Finally, the supracoracoideus muscle was absent in all but one wingless embryo we examined in the present study. In that one, it was substantially reduced in volume compared to normal. absence of this muscle, the space normally occupied by the supracoracoideus was maintained beneath the pectoralis.(ABSTRACT TRUNCATED AT 400 WORDS)

Posterior apical ectodermal ridge removal in the chick wing bud triggers a series of events resulting in defective anterior pattern formation.

The ability of the anterior apical ectodermal ridge to promote outgrowth in the chick wing bud when disconnected from posterior apical ridge was examined by rotating the posterior portion of the stage-19/20 to stage-21 wing bud around its anteroposterior axis. This permitted contact between the anterior and posterior mesoderm, without removing wing bud tissue. In a small but significant number of cases (10/54), anterior structures (digit 2) formed spatially isolated from posterior structures (digits 3 and 4). Thus, continuity with posterior ridge is not a prerequisite for anterior-ridge function in the wing bud. Nevertheless, posterior-ridge removal does result in anterior limb truncation. To investigate events leading to anterior truncation, we examined cell death patterns in the wing bud following posterior-ridge removal. We observed an abnormal area of necrosis along the posterior border of the wing bud at 6-12 h following posterior-ridge removal. This was followed by necrosis in the distal, anterior mesoderm at 48 h postoperatively and subsequent anterior truncation. Clearly, healthy posterior limb bud mesoderm is needed for anterior limb bud survival and development. We propose that anterior truncation is the direct result of anterior mesodermal cell death and that this may not be related to positional specification of anterior cells. In our view, cell death of anterior mesoderm, after posterior mesoderm removal, should not be used as evidence for a role in position specification by the polarizing zone during the limb bud stages of development. We suggest that the posterior mesoderm that maintains the anterior mesoderm need not be restricted to the mapped polarizing zone, but is more extensively distributed in the limb bud.

The formation of leg or wing specific structures by leg bud cells grafted to the wing bud is influenced by proximity to the apical ridge.

When quail or chick leg bud mesoderm was grafted to a chick wing bud, toes developed from grafts placed in direct contact with the wing apical ridge. The toes were primarily derived from quail leg cells, with variable participation of host wing cells. Donor cells also integrated into wing-specific structures, such as cartilage of the wing digits and the surrounding connective tissues. In addition to forming toes, the grafted leg mesoderm expressed its leg origin by enlarging skeletal elements in the host wing. In all cases, enlargements were derived of both quail donor and chick host cells, and were not the result of the addition of mass to the host bud. Grafts placed further than 162 microns from the ridge formed neither toes nor enlargements; rather, they integrated into wing-specific structures. Under the influence of the apical ridge, the grafted leg mesoderm cells are able to maintain their leg character and to form toes and skeletal enlargements. Grafts outside the range of ridge influence (162 microns) are affected by their surroundings to integrate into wing-specific structures. The formation of leg-specific structures by leg bud mesoderm grafted to the wing bud has been used to support the principle of nonequivalence, which states that, because of their different developmental histories, wing and leg cells are restricted to form structures specific for their respective limbs. However, we have shown that leg cells can form wing-specific structures, and therefore limb cells are not restricted in their development.

Initial limb budding is independent of apical ectodermal ridge activity; evidence from a limbless mutant.

Outgrowth of normal chick limb bud mesoderm is dependent on the presence of a specialized epithelium called the apical ectodermal ridge. This ectodermal ridge is induced by the mesoderm at about the time of limb bud formation. The limbless mutation in the chick affects apical ectodermal ridge formation in the limb buds of homozygotes. The initial formation of the limb bud appears to be unaffected by the mutation but no ridge develops and further outgrowth, which is normally dependent on the ridge, does not take place. As a result, limbless chicks develop without limbs. In the present study, which utilized a pre-limb-bud recombinant technique, limbless mesoderm induced an apical ectodermal ridge in grafted normal flank ectoderm. However, at stages when normal flank ectoderm is capable of responding to ridge induction, limbless flank ectoderm did not form a ridge or promote outgrowth of a limb in response to normal presumptive wing bud mesoderm. We conclude from this that the limbless mutation affects the ability of the ectoderm to form a ridge. In addition, because the limbless ectoderm has no morphological ridge and no apparent ridge activity (i.e. it does not stabilize limb elements in stage-18 limb bud mesoderm), the limbless mutant demonstrates that the initial formation of the limb bud is independent of apical ectodermal ridge activity.

Development of the lateral motor column in the limbless mutant chick embryo.

This is a report on the development of the lateral motor column (LMC) in the limbless mutant chick embryo. The limbless mutant was used to study the effects of the absence of a periphery on the developing nervous system. The limbless mutant provides a unique opportunity to compare the effects on the LMC of deletion of a limb caused by the genotype with those seen following surgical removal of the limb primordium. Cell counts of the total number of motoneurons in the LMC at both the brachial and lumbar levels were done in a large series of limbless embryos and on their normal siblings. In normal embryos, a substantial loss of LMC motoneurons was observed during the course of normal development. At the brachial level, 54% of the initial LMC cell population was lost between day 6 and day 18. At the lumbar level, 40% of the initial population was lost between the 6th and 12th days of development with no further loss through day 18. An even more massive cell loss was observed in the limbless mutant LMC at both brachial and lumbar spinal cord levels between day 5 and day 12. This resulted in the elimination of at least 85% of the motoneurons that were initially present in the limbless LMC. Our data demonstrate that the effects of peripheral deprivation on LMC development in the limbless mutant are similar to those seen following surgical removal of the periphery. The initial production of motoneurons and assembly of the LMC did not appear to be significantly affected by the mutation, while the subsequent degeneration of LMC motoneurons is greatly accelerated and increased in comparison to the normal.

An analysis of the fate of the chick wing bud apical ectodermal ridge in culture.

Stages 20 and 25 chick apical ectodermal ridge have been cultured in nutrient medium containing fetal bovine serum and the tissues have been examined for dying cells at 0, 6, 12, 18, and 24 hr. By 12 hr, an average of 43% of the cells were dying. By 24 hr, stage 20 ridge had lost its integrity and stage 25 ridge contained an average of 50% dying cells. These results are in agreement with the observations of R. L. Searls and E. Zwilling (1964, Dev. Biol. 9, 38-55) on isolated stage 20 ridge. In subsequent experiments, ridge ectoderm was cultured in serum-containing medium to which insulin (5 micrograms/ml), transferrin (5 micrograms/ml), and selenium (5 ng/ml) or insulin (5 micrograms/ml) had been added. Under these conditions the ectoderms remained viable even after 24 hr in vitro.

The effects of Janus Green B on the temporal and spatial pattern of feather germ morphogenesis.

The normal timing and appearance of feather germs was perturbed by injecting the dye Janus Green B into the amniotic fluid of chick embryos at late stage 28, prior to the first appearance of feather germs. This treatment prevented feather germ morphogenesis in some regions while elsewhere it delayed normal morphological development. The Janus Green B effect lasted for approximately 98 hours. Feather regions, which normally form epidermal placodes during the period of treatment, showed the longest delays in subsequent feather germ formation and were the most likely to remain featherless. These results suggest that the epidermal placode stage is critical for feather germ formation. Janus Green B appears to prevent feather germ morphogenesis by interfering with development prior to this critical stage. Since severely affected regions fail to recover their capacity to form feather germs, even after the period of sensitivity to the dye, a limited period of competence is suggested for feather germ formation.

Cell death in cultured dorsal and ventral chick wing bud epithelia.

We have examined the fate of cultured stage 20 and 25 dorsal and ventral wing bud epithelia and have found evidence that the requirements of apical ectodermal ridge and nonridge limb ectoderms for in vitro survival are different. As previously reported for the apical ectodermal ridge (Boutin and Fallon, '84), dorsal and ventral ectoderms were extensively necrotic after 12 hours of culture in serum-containing medium. The survival of dorsal and ventral limb epithelia at 18 hours was not improved by a collagen substratum, 10% Nuserum, epidermal growth factor, nerve growth factor, multiplication-stimulating activity, insulin, or insulin, transferrin, and selenium. This is in contrast to our observations on the ridge which remains vital for at least 24 hours in insulin or insulin, transferrin, and selenium.

Evidence that the ectoderm is the affected germ layer in the wingless mutant chick embryo.

We grafted normal flank ectoderm to the denuded presumptive wing bud mesoderm of stages 14-15 wingless embryos. When this was done, the wingless wing bud mesoderm was capable of inducing a ridge in the grafted ectoderm, maintaining that ridge, and growing out to form a wing. However, when stage 17-18 wingless wing bud mesoderm was combined with a normal leg bud ectodermal jacket, the recombinant bud failed to grow out to form a wing (Zwilling, '56a; and this report). When normal ectoderm was first grafted to a wingless host at stages 14-15, and the resulting stage 18 wing bud was removed and then the mesoderm recombined with a normal ectodermal jacket, the double recombinant bud could form a distally complete wing. However, these wings had some deficiencies compared to similar double recombinants made with normal mesoderm. These results show, first, that the ectoderm is affected by the wingless gene and, second, that there may be a prelimb bud stage interaction between wingless ectoderm and mesoderm such that, by stage 17, the wingless mesoderm becomes defective as a result of the ectodermally expressed mutation. Deficiencies in wingless mesoderm double recombinants indicate that the mesoderm may be sensitive to manipulation, possibly because the ectoderm has affected the mesoderm to some extent before stage 14. We believe it is not possible to determine the affected germ layer in wingless after the limb bud arises. However, after using the prelimb bud recombinant technique which we have designed, it becomes apparent that the ectoderm is affected by the wingless gene.

The stages of flank ectoderm capable of responding to ridge induction in the chick embryo.

Reports on the stages when chick flank ectoderm can respond to ridge induction are contradictory. Different results have been obtained using presumptive wing or leg bud mesoderm as the inducing tissue with flank ectoderm as the responding tissue. In addition, although incomplete outgrowths have been obtained from recombinants with stage-19 flank ectoderm in a small percentage of cases, no complete outgrowths have been obtained from recombinants with ectoderm older than stage 18. We reinvestigated when chick flank ectoderm can respond to ridge induction and promote outgrowth of complete limbs. To do this, we combined flank ectoderm with in situ chick presumptive wing bud mesoderm using a pre-limb bud recombinant technique. When presumptive wing bud ectoderm was removed from the host and not replaced, wing development was suppressed. When host ectoderm was replaced with stage-15 through -18 chick flank ectoderm, limbs grew out in all cases; 86.4% of these recombinant limbs were distally complete. Stage-19 flank ectoderm formed a ridge and promoted limb outgrowth in 80.9% of recombinants; 52.9% of these were distally complete limbs. Recombinants made by grafting early stage-20 (40-somite donor) flank ectoderm to stage-15 hosts resulted in outgrowths in 60% of the cases and 33.3% of these were distally complete. Graft ectoderm from older donors did not respond to inductive mesoderm. Our results demonstrate that chick flank ectoderm from stage-15 through early stage-20 donors can respond to inductive signals from presumptive wing bud mesoderm to form an apical ridge. This ridge can promote outgrowth of distally complete wings in a substantial proportion of recombinants. This is two stages beyond when the ability to promote outgrowth of distally complete wings appeared to be lost using other methods.

Development of the apical ectodermal ridge in the chick wing bud.

Histological examination of the stage-18 to stage-23 chick wing bud apex revealed the following. Initially, the wing bud was covered by a cuboidal to columnar epithelium with an overlying periderm. Thickening of the apical ectoderm was not obvious until late stage 18 (36 pairs of somites), after the appearance of the wing bud. At late stage 18, cells of the inner layer of ectoderm had elongated slightly along an axis perpendicular to the epithelial-mesenchymal interface. Well-defined apical ectodermal ridge morphology, i.e., pseudostratified columnar epithelium with an overlying periderm, was not apparent until stage 20. Subsequently the ridge lengthened along the anteroposterior perimeter of the wing bud. We demonstrated histologically that the apical ectodermal ridge of the wing bud was asymmetric with respect to the anteroposterior axis, in that there was more ridge associated with posterior mesoderm. Other observations include the spatial and temporal location of a groove in the base of the thickest part of the ridge. The groove can be correlated with the specification of distal wing elements. The groove was first seen at stage 20 and became more prominent through stage 23. An anteroposterior progression of ectodermal cell death was also observed. This began at late stage 18 and continued through each of the stages examined.