During embryogenesis, skeletal muscle forms in the vertebrate limb from progenitor cells originating in the somites. These cells delaminate from the hypaxial edge of the dorsal part of the somite, the dermomyotome, and migrate into the limb bud, where they proliferate, express myogenic determination factors and subsequently differentiate into skeletal muscle. A number of regulatory factors involved in these different steps have been identified. These include Pax3 with its target c-met, Lbx1 and Mox2 as well as the myogenic determination factors Myf5 and MyoD and factors required for differentiation such as Myogenin, Mrf4 and Mef2 isoforms. Mutants for genes such as Lbx1 and Mox2, expressed uniformly in limb muscle progenitors, reveal unexpected differences between fore and hind limb muscles, also indicated by the differential expression of Tbx genes. As development proceeds, a secondary wave of myogenesis takes place, and, postnatally, satellite cells become located under the basal lamina of adult muscle fibres. Satellite cells are thought to be the progenitor cells for adult muscle regeneration, during which similar genes to those which regulate myogenesis in the embryo also play a role. In particular, Pax3 as well as its orthologue Pax7 are important. The origin of secondary/fetal myoblasts and of adult satellite cells is unclear, as is the relation of the latter to so-called SP or stem cell populations, or indeed to potential mesangioblast progenitors, present in blood vessels. The oligoclonal origin of postnatal muscles points to a small number of founder cells, whether or not these have additional origins to the progenitor cells of the somite which form the first skeletal muscles, as discussed here for the embryonic limb.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1469-7580.2003.00139.x

The formation of skeletal muscle: from somite to limb.

Mutations in the tumor suppressor gene PATCHED (PTC) are found in human patients with the basal cell nevus syndrome, a disease causing developmental defects and tumors, including basal cell carcinomas. Gene regulatory relationships defined in the fruit fly Drosophila suggest that overproduction of Sonic hedgehog (SHH), the ligand for PTC, will mimic loss of ptc function. It is shown here that transgenic mice overexpressing SHH in the skin develop many features of basal cell nevus syndrome, demonstrating that SHH is sufficient to induce basal cell carcinomas in mice. These data suggest that SHH may have a role in human tumorigenesis.

https://science.sciencemag.org/content/sci/276/5313/817.full.pdf

Basal Cell Carcinomas in Mice Overexpressing Sonic Hedgehog

Interdigital cell death leads to regression of soft tissue between embryonic digits in many vertebrates. Although the signals that regulate interdigital apoptosis are not known, BMPs—signaling molecules of the transforming growth factor-β superfamily—are expressed interdigitally. A dominant negative type I BMP receptor (dnBMPR-IB) was used here to block BMP signaling. Expression of dnBMPR in chicken embryonic hind limbs greatly reduced interdigital apoptosis and resulted in webbed feet. In addition, scales were transformed into feathers. The similarity of the webbing to webbed duck feet led to studies that indicate that BMPs are not expressed in the duck interdigit. These results indicate BMP signaling actively mediates cell death in the embryonic limb.

https://science.sciencemag.org/content/sci/272/5262/738.full.pdf

Requirement for BMP Signaling in Interdigital Apoptosis and Scale Formation

Fibroblast growth factors (FGFs) have been implicated in diverse cellular processes including apoptosis, cell survival, chemotaxis, cell adhesion, migration, differentiation, and proliferation. This review presents our current understanding on the roles of FGF signaling, the pathways employed, and its regulation. We focus on FGF signaling during early embryonic processes in vertebrates, such as induction and patterning of the three germ layers as well as its function in the control of morphogenetic movements.

/pdf/fibroblast-growth-factor-signaling-during-early-vertebrate-4iv798m3te.pdf

Fibroblast growth factor signaling during early vertebrate development.

We describe a fetus with abnormalities suggestive, but not typical, of severe Smith-Lemli-Opitz syndrome (SLO). Biochemical studies demonstrated that there was a defect of cholesterol biosynthesis similar to that recently discovered in children with SLO. The findings in this fetus extend even further the wide spectrum of abnormalities of the SLO phenotype, and emphasize that a genetic pathological examination and biochemical studies should always be undertaken on atypical cases, especially fetuses.

Unusual case of Smith-Lemli-Opitz syndrome “type II”

The chicken mutant talpid3 (ta3) has polydactylous limbs with up to 7-8 morphologically similar digits. This lack of antero-posterior polarity in digit pattern is correlated with symmetrical expression of genes of the HoxD complex. We determined the distribution of polarizing activity in limb buds of the chick mutant ta3 by assessing the ability of mesenchyme from various positions along the antero-posterior axis to induce digit duplications when grafted anteriorly into a normal limb. Cells with highest polarizing activity were found at the posterior margin of the wing as in the polarizing region of normal limb buds. However, in contrast to normal limb buds, ta3 anterior mesenchyme also had low polarizing activity. Application of retinoic acid or a polarizing region graft to the anterior of ta3 limb buds changed digit morphology but did not induce digit duplications or digits with any characteristic a-p pattern. To determine which genes are associated with polarizing activity and which are associated with patterning of the digits, we examined expression of the genes Sonic hedgehog (shh), Bmp-2, and Bmp-7, whose expression is normally confined to the posterior margin of the early wing bud and is associated with the polarizing region. In addition, we determined the distribution of Fgf-4 transcripts which in normal limb buds are restricted to the posterior part of the apical ectodermal ridge. In ta3 limb buds, shh expression is restricted to the posterior limb mesenchyme, which has high polarizing activity, but is not expressed in regions which have low polarizing activity. In contrast, Bmp-2 and Bmp-7 are expressed uniformly along the a-p axis. Fgf-4 transcripts are present throughout the apical ectodermal ridge in ta3 limb buds. In the ta3 mutant, there is both an abnormal distribution of signalling activity and response to polarizing signals. In addition, the dissociation between the expression of shh and Bmps suggests distinct roles for the encoded molecules in signalling and response in a-p patterning of limb buds.

Expression of genes encoding bone morphogenetic proteins and sonic hedgehog in talpid (ta3) limb buds: Their relationships in the signalling cascade involved in limb patterning

Fibroblast growth factors (FGFs) are believed to be vital for limb outgrowth and patterning during embryonic development. Although the effect of FGFs on the formation of the skeletal elements has been studied in detail, their effect on the development of the limb musculature is still uncertain. In this study, we used Blindwell chemotactic chambers to examine the effect of FGF-2 and FGF-4 on the motility of myogenic cells obtained from the proximal region of the day 11.5 mouse forelimbs. The limb myogenic cells were found to be chemotactically attracted to FGF-2 and FGF-4 at 10-50 ng/ml. Both FGFs increased myogenic cell migration in a dose-dependent manner, with maximal responses attained at 1-50 ng/ml for FGF-2 and at 10 ng/ml for FGF-4; however, FGF-2 was found to be a more potent chemoattractant than FGF-4. It was possible to inhibit the myogenic cells' response to FGF-2 and FGF-4 by the addition of the appropriate neutralizing antibody. The effects of FGF-2 on cell migration were further investigated by loading this cytokine into Affi-Gel blue beads and transplanting them into day 11.5 forelimb buds. The results showed that FGF-2 attracted DiI-labelled proximal cells to migrate toward the implanted beads and that the migration was more extensive than that observed in the absence of FGF-2. A checkerboard assay was performed in which various concentrations of FGF-2 and FGF-4 were introduced to both the upper and lower wells of the Blindwell chambers. The results indicated that both FGF isoforms can stimulate chemokinesis as well as chemotaxis in myogenic cells. In addition, the effect of FGF-2 at 0.1-10 ng/ml stimulated a significant increase in the number of myocytes expressing sarcomeric myosin on examination after 48 hr in culture, but the effect of FGF-4 was negligible at all concentrations analyzed; however, both FGF-2 and FGF-4 inhibited myocyte fusion compared with the spontaneous fusion observed in control cultures. Finally, we used in situ hybridization and immunohistochemical techniques to determine the distribution of myogenic cells and FGF-2 protein in the day 11.5 mouse forelimbs.

/pdf/fibroblast-growth-factors-2-and-4-stimulate-migration-of-3w74370o78.pdf

Fibroblast growth factors 2 and 4 stimulate migration of mouse embryonic limb myogenic cells

Developing chicken wings homozygous for the talpid (ta3/ta3) mutation are polydactylous and have defects in the establishment of their anteroposterior polarity. We analysed the expression domains of the posteriorly restricted homeobox Hox-4 genes in such mutant wings. The Hox-4 genes are now expressed right across the anteroposterior axis instead of being expressed just posteriorly. This correlates well with the absence of clear morphological differences between the talpid3 digits and reinforces the idea that vertebrate Hox-4 genes are involved in setting up the limb anteroposterior asymmetry.

D.A. Ede

Papers

Expression of genes encoding bone morphogenetic proteins and sonic hedgehog in talpid (ta3) limb buds: Their relationships in the signalling cascade involved in limb patterning

Fibroblast growth factors 2 and 4 stimulate migration of mouse embryonic limb myogenic cells

The mis-expression of posterior Hox-4 genes in talpid (ta3) mutant wings correlates with the absence of anteroposterior polarity.