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Abdominal-B homologs and limb development: mesenchymal - epidermal interactions

Detailed fate maps have been produced of the mesenchyme and apical ridge found in a stage 20 chick wing bud. The fate maps of the mesenchyme show that most of the wing arises from the posterior half of the bud. Subapical mesenchyme gives rise to digits. Cell populations beneath the ridge in the mid apical region fan out into the anterior tip of the handplate, while posterior cell populations extend right along the posterior margin. Subapical mesenchyme in the leg bud behaves similarly. The absence of anterior bending of posterior cell populations has implications when considering models of vertebrate limb evolution. The fate maps of the apical ridge show that there is also a marked anterior expansion; cells that were in the anterior apical ridge later become incorporated into non-ridge ectoderm along the margin of the bud. Mesenchyme and apical ridge do not expand in concert - the apical ridge extends more anteriorly. The fate maps were used to investigate the relationship between cell lineage and elaboration of Hoxd-13 and Fgf-4 domains. Hoxd-13 and Fgf-4 are initially expressed posteriorly until about the mid-point of the early wing bud in mesenchyme and apical ridge respectively. Later in development, the genes come to be expressed throughout most of the handplate and apical ridge respectively. At the proximal edge of the Hoxd-13 domain, cell populations stop expressing the gene as development proceeds; no evidence was found that changes in the extent of the domains were due to initiation of gene expression in anterior cells. Instead the changes in extent of expression fit with the fate maps and can be attributed to the expansion and fanning out of cell populations initially expressing the genes (Vargesson, 1997).

During initiation of limb-bud outgrowth in vertebrate embryos, the polarizing region (limb-bud organizer) is established upon activation of the Sonic hedgehog (Shh) signaling molecule at the posterior limb-bud margin. Another hallmark of establishing anteroposterior limb-bud identities is the colinear activation of HoxD genes located at the 5' end of the cluster (5'HoxD genes, Hoxd11 to Hoxd13). The unique and shared functions of Gli3 and formin in these determinative events were genetically analyzed using single and double homozygous Extra-toes (Xt; disrupting Gli3) and limb deformity (ld; disrupting formin) mouse embryos. The formins, proteins involved in murine limb and kidney development, contain a proline-rich region that matches consensus sequences for ligands of Src homology 3 (SH3) domains (Zuniga, 1999 and references).

The positive Shh/Fgf-4 feedback loop controlling morphogeneisis of distal limb structures is disrupted in ld homozygous limb buds, which results in reduced polarizing activity. This disruption is due to a primary mesenchymal (mesodermal) defect preventing complete differentiation of the anterior ectodermal ridge (AER) and the induction of Fgf-4 in the posterior AER. Mesenchymal formin functions primarily in establishment of the signalling interactions between the polarizing region (Shh) and the AER (Fgf-4). Most likely as a consequence of disrupting these mesenchymal AER interactions, the transition from early to late 5'HOXD domains is delayed in ld/ld limb buds. Consistent with a genetic function in establishing the SHH/FGF4 feedback loop, formin is expressed in posterior and distal limb bud mesenchymal cells. High levels of formin transcripts are also expressed by the AER during limb-bud outgrowth, but transgene mediated formin re-expression in the ld mutant AER fails to rescue the Shh/Fgf-4 feedback loop and limb bud patterning. Furthermore, targeted disruption of the predominant formin isoform in the AER does not disrupt limb morphogenesis (Zuniga, 1999 and references).

Analysis of the limb skeletal phenotypes reveals genetic interaction of Gli2 and formin. In addition to loss of digit identity and varying degrees of polydactyly, proximal skeletal elements are severely shortened in Xt;ld double homozygous limbs. The underlying molecular defects affect both establishment of the polarizing region and posterior limb-bud identity. In particular, the synergism between Gli3- and formin-mediated mesenchyme-AER interactions positions the Shh signaling center at the posterior limb-bud margin. The present study shows that establishment and positioning of the polarizing region is regulated both by restriction of Shh through Gli3 and its positive feedback regulation through formin. Concurrently, Gli3 functions independent of formin during initial posterior nesting of 5' HoxD domains, whereas their subsequent distal restriction and anterior expansion depends on genetic interaction of Gli3 and formin, which act together to reinforce the posterior nesting of the 5'HoxD domains (Zuniga, 1999).

Two aspects of Gli3 and formin interaction during limb morphogenesis are noted: (1) both proximal (humerus and scapula) and distal (digits) limb skeletal elements are more severely affected in Xt;ld double than single homozygous limb buds, and (2) molecular analysis shows that both positioning of the Shh-expressing polarizing region and spatial regulation of 5'HoxD domains are disrupted in Xt;ld double mutant limbs. As Gli3 and formin are co-expressed in the posterior limb-bud mesenchyme, these two proteins with postulated nuclear functions might directly interact, but biochemical studies fail to detect direct molecular interactions. Therefore alterations in double mutant limb buds are most likely caused by disruption of two distinct, but genetically interacting cascades (Zuniga, 1999).

Table of contents

Abdominal-B: Biological Overview | Promoter Structure | Transcriptional Regulation | Targets of activity | Protein Interactions | Developmental Biology | Effects of Mutation | References

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