Outgrowth by fin motor axons in wildtype and a finless mutant of the Japanese medaka fish

The outgrowth of motor axons to the developing pectoral fin of the Japanese medaka fish (Oryzias latipes) was investigated both in wildtype embryos and in the pectoral finless (pl) mutants in which adults are missing pectoral fins. Late in embryogenesis the pectoral fin is a simple limb which contains two antagonist muscles which are innervated by presumptive motor neurons from the first four spinal segments (S1-4). The pectoral fin develops from a fin bud located in S1 and S2 centered on the border between S1 and S2 and, as with other limbs, one of the earliest signs of differentiation is the apical ectodermal ridge (AER). By the time the AER is well formed the growth cones of the presumptive motor neurons have reached the base of the fin bud and formed a plexus by extending toward the fin bud upon emergence from the spinal cord. This is especially evident on the ventral surface of the metamerically arranged axial muscles. For example, growth cones from S2 extend in a diagonal direction (both anterior and lateral) towards the fin bud. One hypothesis which can account for the pattern of motor outgrowth is that growth cones are attracted to the base of the fin bud, perhaps via a long distance cue. This hypothesis was tested by examining outgrowth of segmental nerves in pl embryos in which the fin buds arrest early in development following the initial appearance of the AER. In pl, nerves from S1-4 converged to form a plexus at the base of the abnormal fin bud, but the pattern of outgrowth varied from wildtype in a way consistent with a diminished capacity of the fin bud to attract segmental nerves to it.     

FGF10 can induce Fgf8 expression concomitantly with En1 and R-fng expression in chick limb ectoderm, independent of its dorsoventral specification

The limb bud has a thickened epithelium at the dorsal-ventral boundary, the apical ectodermal ridge (AER), which sustains limb outgrowth and patterning. A secreted molecule fibroblast growth factor (FGF)10 is involved in inducing Fgf8 expression in the prospective AER and mutual interaction between mesenchymal FGF10 and FGF8 in the AER is essential for limb outgrowth. A secreted factor Wnt7a and a homeobox protein Lmx1 are involved in the dorsal patterning of the limb, whereas a homeobox protein Engrailed 1 (En1) is involved in the dorsal-ventral patterning as well as AER formation. Radical fringe (R-fng), a vertebrate homolog of Drosophila fringe was also found to elaborate AER formation in chicks. However, little is known about the molecular interactions between these factors during AER formation. The present study clarified the relationship between FGF10, Wnt7a, Lmx1, R-fng and En1 during limb development using a foil-barrier insertion experiment. It was found that a foil-barrier inserted into the chick prospective wing mesenchyme lateral to the mesonephric duct blocks AER induction. This experiment was expanded by implanting Fgf10-expressing cells lateral to the barrier and examined whether FGF10 could rescue the expression of the limb-patterning genes reported in AER formation. It was found that FGF10 is sufficient to induce Fgf8 expression in the ectoderm of the foil-inserted limb bud, concomitantly with R-fng and En1 expression. However, FGF10 could not rescue the expression of the dorsal marker genes, Wnt7a or Lmx1. Thus, it is suggested that epithelial factors of En1 and R-fng can induce Fgf8 expression in the limb ectoderm in cooperation with a mesenchymal factor FGF10. Some factor(s) other than FGF10, possibly from the paraxial structures medial to the limb mesoderm, is responsible for the initial dorsal-ventral specification of the limb bud.     

FGF7 and FGF10 directly induce the apical ectodermal ridge in chick embryos.

During vertebrate limb development, the apical ectodermal ridge (AER) plays a vital role in both limb initiation and distal outgrowth of the limb bud. In the early chick embryo the prelimb bud mesoderm induces the AER in the overlying ectoderm. However, the direct inducer of the AER remains unknown. Here we report that FGF7 and FGF10, members of the fibroblast growth factor family, are the best candidates for the direct inducer of the AER. FGF7 induces an ectopic AER in the flank ectoderm of the chick embryo in a different manner from FGF1, -2, and -4 and activates the expression of Fgf8, an AER marker gene, in a cultured flank ectoderm without the mesoderm. Remarkably, FGF7 and FGF10 applied in the back induced an ectopic AER in the dorsal median ectoderm. Our results suggest that FGF7 and FGF10 directly induce the AER in the ectoderm both of the flank and of the dorsal midline and that these two regions have the competence for AER induction. Formation of the AER of the dorsal median ectoderm in the chick embryo is likely to appear as a vestige of the dorsal fin of the ancestors.     

Alteration of pectoral fin nerves following ablation of fin buds and by ectopic fin buds in the Japanese medaka fish

The role of the pectoral fin bud for outgrowth by fin axons was assessed by ablation of pectoral fin buds and by transplantation of fin buds to ectopic sites in the embryos of the Japanese medaka fish (Oryzias latipes). Normally nerves from segments 1-4 (S1-4) and less frequently the S5 nerve converged at the base of the fin bud by extending toward the fin bud on the ventral surface of the axial muscles (H. Okamoto and J. Y. Kuwada, 1991, Dev. Biol. 146). Following ablation of the fin bud before motor growth cones have begun to extend laterally, nerves in S1-5 followed a trajectory down the middle of each segment parallel to the borders of the metamerically arranged axial muscles rather than converging. This trajectory was similar to that of more posterior segmental nerves which do not converge toward the fin bud. When fin buds were transplanted to more posterior segments, nerves from S1-5 often changed their trajectories and extended to the base of ectopic buds. Furthermore, motor nerves from segments posterior to S5, which normally do not innervate the fin bud, also extended to the ectopic fin bud. When faced with both the host and ectopic fin bud, motor nerves extended to either fin bud or branched and extended to both fin buds. These results demonstrate that the early fin bud is necessary for correct outgrowth of fin nerves and suggest that the fin bud normally attracts fin nerves to its base. One possible mechanism for the attraction of motor growth cones by the fin bud is a long distance cue emitted by the fin bud.     

Normal limb development in conditional mutants of Fgf4

Fibroblast growth factors (FGFs) mediate multiple developmental signals in vertebrates. Several of these factors are expressed in limb bud structures that direct patterning of the limb. FGF4 is produced in the apical ectodermal ridge (AER) where it is hypothesized to provide mitogenic and morphogenic signals to the underlying mesenchyme that regulate normal limb development. Mutation of this gene in the germline of mice results in early embryonic lethality, preventing subsequent evaluation of Fgf4 function in the AER. A conditional mutant of Fgf4, based on site-specific Cre/loxP-mediated excision of the gene, allowed us to bypass embryonic lethality and directly test the role of FGF4 during limb development in living murine embryos. This conditional mutation was designed so that concomitant with inactivation of the Fgf4 gene by excision of all Fgf4-coding sequences, a reporter gene was activated in Fgf4-expressing cells, allowing assessment of the site-specific recombination reaction. Although a large body of evidence led us to predict that ablation of Fgf4 gene function in the AER of developing mice would result in abnormal limb outgrowth and patterning, we found that Fgf4 conditional mutants had normal limbs. Furthermore, expression patterns of Shh, Bmp2, Fgf8 and Fgf10 were normal in the limb buds of the conditional mutants. These findings indicate that the previously proposed FGF4-SHH feedback loop is not essential for coordination of murine limb outgrowth and patterning. We suggest that some of the roles currently attributed to FGF4 during early vertebrate limb development may be performed by other AER factors in vivo.     

The dominant hemimelia mutation uncouples epithelial-mesenchymal interactions and disrupts anterior mesenchyme formation in mouse hindlimbs.

Epithelial-mesenchymal interactions are essential for both limb outgrowth and pattern formation in the limb. Molecules capable of communication between these two tissues are known and include the signaling molecules SHH and FGF4, FGF8 and FGF10. Evidence suggests that the pattern and maintenance of expression of these genes are dependent on a number of factors including regulatory loops between genes expressed in the AER and those in the underlying mesenchyme. We show here that the mouse mutation dominant hemimelia (Dh) alters the pattern of gene expression in the AER such that Fgf4, which is normally expressed in a posterior domain, and Fgf8, which is expressed throughout are expressed in anterior patterns. We show that maintenance of Shh expression in the posterior mesenchyme is not dependent on either expression of Fgf4 or normal levels of Fgf8 in the overlying AER. Conversely, AER expression of Fgf4 is not directly dependent on Shh expression. Also the reciprocal regulatory loop proposed for Fgf8 in the AER and Fgf10 in the underlying mesenchyme is also uncoupled by this mutation. Early during the process of limb initiation, Dh is involved in regulating the width of the limb bud, the mutation resulting in selective loss of anterior mesenchyme. The Dh gene functions in the initial stages of limb development and we suggest that these initial roles are linked to mechanisms that pattern gene expression in the AER.     

Disrupting the establishment of polarizing activity by teratogen exposure.

Between days 9.5 and 10, the forelimb buds of developing murine embryos progress from stage 1 which are just beginning to express shh and whose posterior mesoderm has only weak polarizing activity to stage 2 limbs with a distinguishable shh expression domain and full polarizing activity. We find that exposure on day 9.5 to teratogens that induce the loss of posterior skeletal elements disrupts the polarizing activity of the stage 2 postaxial mesoderm and polarizing activity is not subsequently restored. The ontogeny of expression of the mesodermal markers shh, ptc, bmp2, and hoxd-12 and 13, as well as the ectodermal markers wnt7a, fgf4, fgf8, cx43, and p21 occurred normally in day 9.5 teratogen-exposed limb buds. At stage 3, the treated limb apical ectodermal ridge usually possessed no detectable abnormalities, but with continued outgrowth postaxial deficiencies became evident. Recombining control, stage matched limb bud ectoderm with treated mesoderm prior to ZPA grafting restored the duplicating activity of treated ZPA tissue. We conclude that in addition to shh an early ectoderm-dependent signal is required for the establishment of the mouse ZPA and that this factor is dependent on the posterior ectoderm.     

Mechanism of pectoral fin outgrowth in zebrafish development

Fins and limbs, which are considered to be homologous paired vertebrate appendages, have obvious morphological differences that arise during development. One major difference in their development is that the AER (apical ectodermal ridge), which organizes fin/limb development, transitions into a different, elongated organizing structure in the fin bud, the AF (apical fold). Although the role of AER in limb development has been clarified in many studies, little is known about the role of AF in fin development. Here, we investigated AF-driven morphogenesis in the pectoral fin of zebrafish. After the AER-AF transition at ～36 hours post-fertilization, the AF was identifiable distal to the circumferential blood vessel of the fin bud. Moreover, the AF was divisible into two regions: the proximal AF (pAF) and the distal AF (dAF). Removing the AF caused the AER and a new AF to re-form. Interestingly, repeatedly removing the AF led to excessive elongation of the fin mesenchyme, suggesting that prolonged exposure to AER signals results in elongation of mesenchyme region for endoskeleton. Removal of the dAF affected outgrowth of the pAF region, suggesting that dAF signals act on the pAF. We also found that the elongation of the AF was caused by morphological changes in ectodermal cells. Our results suggest that the timing of the AER-AF transition mediates the differences between fins and limbs, and that the acquisition of a mechanism to maintain the AER was a crucial evolutionary step in the development of tetrapod limbs.     

Duplication and divergence of fgf8 functions in teleost development and evolution

Fibroblast growth factors play critical roles in many aspects of embryo patterning that are conserved across broad phylogenetic distances. To help understand the evolution of fibroblast growth factor functions, we identified members of the Fgf8/17/18-subfamily in the three-spine stickleback Gasterosteus aculeatus, and investigated their evolutionary relationships and expression patterns. We found that fgf17b is the ortholog of tetrapod Fgf17, whereas the teleost genes called fgf8 and fgf17a are duplicates of the tetrapod gene Fgf8, and thus should be called fgf8a and fgf8b. Phylogenetic analysis supports the view that the Fgf8/17/18-subfamily expanded during the ray-fin fish genome duplication. In situ hybridization experiments showed that stickleback fgf8 duplicates exhibited common and unique expression patterns, indicating that tissue specialization followed the gene duplication event. Moreover, direct comparison of stickleback and zebrafish embryonic expression patterns of fgf8 co-orthologs suggested lineage-specific independent subfunction partitioning and the acquisition or the loss of ortholog functions. In tetrapods, Fgf8 plays an important role in the apical ectodermal ridge of the developing pectoral appendage. Surprisingly, differences in the expression of fgf8a in the apical ectodermal ridge of the pectoral fin bud in zebrafish and stickleback, coupled with the role of fgf16 and fgf24 in teleost pectoral appendage show that different Fgf genes may play similar roles in limb development in various vertebrates.     

Increasing Fgf4 expression in the mouse limb bud causes polysyndactyly and rescues the skeletal defects that result from loss of Fgf8 function

A major function of the limb bud apical ectodermal ridge (AER) is to produce fibroblast growth factors (FGFs) that signal to the underlying mesenchyme. Previous studies have suggested that of the four FGF genes specifically expressed in the mouse AER, Fgf8 is unique not only in its expression pattern, but also because it is the only such FGF gene that causes limb skeletal abnormalities when individually inactivated. However, when both Fgf8 and Fgf4 are simultaneously inactivated in the AER, the limb does not develop. One possible explanation for these observations is that although both of these FGF family members contribute to limb development, Fgf8 has functions that Fgf4 cannot perform. To test this hypothesis, we used a novel method to substitute Fgf4 for Fgf8 expression in the developing limb bud by concomitantly activating a conditional Fgf4 gain-of-function allele and inactivating an Fgf8 loss-of-function allele in the same cells via Cre-mediated recombination. Our data show that when Fgf4 is expressed in place of Fgf8, all of the skeletal defects caused by inactivation of Fgf8 are rescued, conclusively demonstrating that FGF4 can functionally replace FGF8 in limb skeletal development. We also show that the increase in FGF signaling that occurs when the Fgf4 gain-of-function allele is activated in a wild-type limb bud causes formation of a supernumerary posterior digit (postaxial polydactyly), as well as cutaneous syndactyly between all the digits. These data underscore the importance of controlling the level of FGF gene expression for normal limb development.