paired

EVOLUTIONARY HOMOLOGS

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PAX3 and limb development

The segmented mesoderm in vertebrates gives rise to a variety of cell types in the embryo, including the axial skeleton and muscle. A number of transcription factors containing a paired domain (Pax proteins) are expressed in the segmented mesoderm during embryogenesis. These include Pax-3 and a closely related gene, Pax-7, both of which are expressed in the segmental plate and in the dermomyotome. Signals from the notochord influence the pattern of expression for Pax-3, Pax-7 and Pax-9 in somites, and the subsequent differentiation of cell types that arise from the somitic mesoderm. The role of the Pax-3 gene in the differentiation of cell types derived from the dermomyotome is directly assessed by analyzing the development of muscle in splotch mouse embryos that lack a functional Pax-3 gene. A population of Pax-3-expressing cells derived from the dermomyotome that normally migrate into the limb are absent in homozygous splotch embryos; as a result, limb muscles are lost. No abnormalities are detected in the trunk musculature of splotch embryos indicating that Pax-3 is necessary for the development of the limb but not trunk muscle (Goulding, 1994).

Limb muscles in vertebrates originate from dermomyotomal cells, which during early development migrate from the ventrolateral region of somites into the limb buds. These progenitor cells do not express any muscle-specific marker genes or myogenic transcription factors until they reach their destination in the limbs. Myogenic cells in somites and a population of presumably migratory muscle precursor cells in somatopleural tissue as well as myoblasts in the developing limbs express Pax-3. Significantly, in homozygous splotch mutant mice, which synthesize altered Pax-3 mRNA but make no normal protein, no cells positive for Pax-3 transcripts can be detected in the region of migrating limb muscle precursors or in the limb itself. In contrast, myotomal precursor cells and axial skeletal muscles contain Pax-3 transcripts in the mutant. Interestingly, these mutant animals fail to develop limb musculature as demonstrated by the lack of hybridization with various probes for myogenic transcription factors (Myf-5, myogenin, MyoD) but make apparently normal axial muscles. These observations suggest that Pax-3 is necessary for the formation of limb muscles, affecting either the generation of myogenic precursors in the somitic dermomyotome or the migration of these cells into the limb field (Bober, 1994).

Specification of the myogenic lineage begins prior to gastrulation and culminates in the emergence of determined myogenic precursor cells from the somites. The myoD family (MDF) of transcriptional activators controls late step(s) in myogenic specification that are closely followed by terminal muscle differentiation. Genes expressed in myogenic specification at stages earlier than MDFs are unknown. The Pax-3 gene is expressed in all the cells of the caudal segmental plate, the early mesoderm compartment that contains the precursors of skeletal muscle. As somites form from the segmental plate and mature, Pax-3 expression is progressively modulated. Beginning at the time of segmentation, Pax-3 becomes repressed in the ventral half of the somite, leaving Pax-3 expression only in the dermomyotome. Subsequently, differential modulation of Pax-3 expression levels delineates the medial and lateral halves of the dermomyotome, which contain (respectively) precursors of axial (back) muscle and limb muscle. Pax-3 expression is then repressed as dermomyotome-derived cells activate MDFs. Quail-chick chimera and ablation experiments confirm that the migratory precursors of limb muscle continue to express Pax-3 during migration. Since limb muscle precursors do not activate MDFs until 2 days after they leave the somite, Pax-3 represents the first molecular marker for this migratory cell population. A null mutation of the mouse Pax-3 gene, Splotch, produces major disruptions in early limb muscle development. It is concluded, therefore, that Pax-3 gene expression in the paraxial mesoderm marks earlier stages in myogenic specification than MDFs and plays a crucial role in the specification and/or migration of limb myogenic precursors (Williams, 1994).

The limb muscles of vertebrates are derived from precursor cells that migrate from the lateral edge of the dermomyotome into the limb bud. Previous studies have shown that the paired domain-containing transcription factor Pax-3 is expressed in limb cells that are precursors for limb muscles. In splotch (Pax-3-) embryos, the limb muscles fail to develop and cells expressing Pax-3 are no longer found in the limb. By labeling somites adjacent to the prospective forelimb with the lipophilic dye DiI, it has been shown that cells derived from these somites do not migrate into the limbs of splotch mice. The failure of limb muscle precursors to invade the limb in splotch mice is associated with the absence of c-met expression in premigratory cells, together with a change in the morphology of the ventral dermomyotome. The lateral half of somites derived from day E9.25 splotch embryos can undergo muscle differentiation when grafted into the limb bud stage 20 chick host embryos. These results indicate that Pax-3 regulates the migration of limb muscle precursors into the limb and is not required for cells in the lateral somite to differentiate into muscle (Daston, 1996).

In vertebrates all skeletal muscles of trunk and limbs are derived from condensations of the paraxial mesoderm, that is from the somites. Limb muscle precursor cells migrate during embryogenesis from somites to limb buds, where migration stops and differentiation occurs. lbx1 homeobox genes (related to the Drosophila ladybird genes) have been characterized in chicken and mice. These genes are expressed in migrating limb muscle precursor cells in both species. Analysis of splotch mutant mice shows that lbx1 and c-met are differently affected by the lack of Pax-3. Limb buds of splotch (Pax-3 mutant) mice are devoid of lbx1 transcripts, while expression of c-met is still detectable at a low level. The presence of c-met-positive cells in splotch mice entering the limbs indicates that migration of cells from somites to limbs is not entirely dependent on Pax-3. Induction of epithelial to mesenchymal transition of Pax-3-positive cells by SF/HGF is not sufficient to induce ectopic lbx-1 expression at the inter-limb level, while ectopic limb formation is able to activate lbx1 expression. It is postulated that Pax-3 is necessary for lbx1 expression in the lateral tips of somites but additional, yet unknown signals derived from limb buds are needed to initiate lbx1 expression. The role of limb bud-derived signals involved in targeted muscle precursor cell migration, and lbx1 activation was further confirmed by analysis of explanted somite/limb bud co-cultures in collagen gels (Mennerich, 1998).

Myogenic differentiation can be initiated by a limited number of molecules. Overexpression of Lbx1 in vivo and in vitro leads to a strong activation of various muscle markers. Cell proliferation, which is strongly stimulated by Lbx1 and Pax3, is required for Lbx1- or Pax3-dependent myogenic activation. Inhibition of cell proliferation prevents expression of muscle differentiation markers, while the activation of other putative downstream targets of Pax3 and Lbx1 is not affected. These findings imply that a critical function of Pax3 and Lbx1 during muscle cell formation is the enlargement of muscle cell populations. The growth of the muscle precursor cell population may increase the bias for myogenic differentiation and thus enable myogenic cells to respond to environmental cues. These results might provide an explanation of how Pax3 and Lbx1 induce myogenesis by amplification of the myogenic precursor cell pool with an increasing potential for myogenic differentiation, and emphasize the critical role of the size of cell populations biased for differentiation (Mennerich, 2001).

Myogenic regulatory factors (MRFs) comprise a family of transcription factors that when expressed in a cell reflects the commitment of that cell to a myogenic fate before any cytological sign of muscle differentiation is detectable. Myogenic cells in limb skeletal muscles originate from the lateral half of the somites. Cells that migrate away from the lateral part of the somites to the limb bud do not initially express any member of the MRF family. Expression of MRFs in the muscle precursor cells starts after the migration process is completed. The extracellular signals involved in activating the myogenic program in muscle precursor cells in the in vivo limb are not known. Sonic Hedgehog (SHH) expressed in the posterior part of the limb bud (the zone of polarizing activity) could be involved in the differentiation of muscle precursor cells in the limb. Retrovirally overexpressed SHH in the limb bud first induces the extension of the expression domain of the Pax-3 gene, then that of the MyoD gene, and finally that of the myosin protein. This leads to a hypertrophy of the muscles in vivo. Addition of SHH to primary cultures of myoblasts results in an increase in the proportion of myoblasts that incorporate bromodeoxyuridine, resulting in an increase in the number of myotubes. These data show that SHH is able to activate myogenesis in vivo and in vitro in already committed myoblasts and suggest that the stimulation of the myogenic programme by SHH involves activation of cell proliferation. It is suggested that SHH may activate Pax-3 expression, which in turn activates MyoD (Duprez, 1998).

The closely related paired-related homeobox genes prx-1 and prx-2 are expressed in lateral plate and limb bud mesoderm, but targeted inactivation of these genes fails to demonstrate a limb phenotype. Mice carrying compound mutations in prx-1 and prx-2 have severe limb deformities. In the forelimb autopod, pre- and post-axial polydactyly are found most commonly, but also observed are syndactyly, oligodactyly, and abnormal digit placement affecting posterior elements. In the hindlimb, preaxial polydactyly with variable expressivity is seen in all cases. Extreme distal digit duplications are seen in both the fore- and hind-limbs. prx-1;prx-2 double-mutant mice also display extreme shortening and impaired ossification of the hindlimb zeugopods. Integrity of the forelimb apical ectodermal ridge is abnormal as determined by expression of FGF8 and BMP4. Expression of msx-1 and msx-2, markers for BMP signaling pathways, is absent in regions of the posterior handplates, while expression of Shh and patched is unaffected. The mutant phenotypes are dosage dependent, since prx-1-/-;prx-2+/- mice also display severe limb abnormalities. These data suggest that prx-1 and prx-2 cooperatively regulate handplate and hindlimb zeugopod morphogenesis through BMP-mediated signaling pathways (Lu, 1999a).

Pax genes encode evolutionarily conserved transcription factors that play critical roles in development. Pax3 and Pax7 constitute one of the four Pax subfamilies. Despite partially overlapping expression domains, mouse mutations for Pax3 and Pax7 have very different consequences. To investigate the mechanism of these contrasting phenotypes, Pax3 was replaced by Pax7 by using gene targeting in the mouse. Pax7 can substitute for Pax3 function in dorsal neural tube, neural crest cell, and somite development, but not in the formation of muscles involving long-range migration of muscle progenitor cells. In limbs in which Pax3 is replaced by Pax7, the severity of the muscle phenotype increases as the number of Pax7 replacement alleles is reduced, with the forelimb more affected than the hindlimb. This hypomorphic activity of Pax7 is due to defects in delamination, migration, and proliferation of muscle precursor cells with inefficient activation of c-met in the hypaxial domain of the somite. Despite this, overall muscle patterning is retained. It is concluded that functions already prefigured by the single Pax3/7 gene present before vertebrate radiation are fulfilled by Pax7 as well as Pax3, whereas the role of Pax3 in appendicular muscle formation has diverged, reflecting the more recent origin of this mode of myogenesis (Relaix, 2004).

In the vertebrate clade, the ancestral mechanism for appendicular muscle development involves the direct extension of the epithelial (dermo)myotome into the fin or limb bud. Subsequently, a mechanism for the delamination and migration of muscle progenitor cells may have evolved in teleost fishes, just prior to tetrapod radiation. However, there is a highly mosaic distribution of these characters in the vertebrate clade as well as the presence of an intermediate mode (with myotomal extension, followed by short range migration) in some reptiles and amphibians, suggesting that evolution of vertebrate appendicular muscle formation is complex. The observation that Pax7 only partially fulfills the functions of Pax3 in limb muscle formation, whereas it performs the role of Pax3 in somitogenesis and trunk myogenesis as well as in the neural tube and neural crest, is consistent with the later evolution of this myogenic mode and demonstrates the central importance of Pax3 in this development. Two possible hypotheses can be formulated for the hypomorphic Pax7 activity in appendicular muscle development: (1) either this Pax3 function evolved independently after Pax3/7 duplication, reusing biochemical functions already partially present in the Pax3/7 protein (e.g., the ancestral function in myotomal ventral body wall formation or myotomal extension into the fin or limb bud), or (2) this function was already present in the Pax3/7 protein before gene duplication and was partially lost during independent Pax7 evolution in vertebrates. The acquisition of molecular properties such as interaction with cofactors that distinguish Pax3 from Pax7 and the ancestral Pax3/Pax7 sequence was therefore probably an essential prerequisite for the evolution of vertebrate appendicular muscle (Relaix, 2004).

Vertebrate muscle arises sequentially from embryonic, fetal, and adult myoblasts. Although functionally distinct, it is unclear whether these myoblast classes develop from common or different progenitors. Pax3 and Pax7 are expressed by somitic myogenic progenitors and are critical myogenic determinants. To test the developmental origin of embryonic and fetal myogenic cells in the limb, Pax3⁺ and Pax7⁺ cells were genetically labeled and ablated. Pax3⁺Pax7^- cells contribute to muscle and endothelium, establish and are required for embryonic myogenesis, and give rise to Pax7⁺ cells. Subsequently, Pax7⁺ cells give rise to and are required for fetal myogenesis. Thus, Pax3⁺ and Pax7⁺ cells contribute differentially to embryonic and fetal limb myogenesis. To investigate whether embryonic and fetal limb myogenic cells have different genetic requirements beta-catenin, an important regulator of myogenesis, was conditionally inactivated or activated in Pax3- or Pax7-derived cells. β-Catenin is necessary within the somite for dermomyotome and myotome formation and delamination of limb myogenic progenitors. In the limb, beta-catenin is not required for embryonic myoblast specification or myofiber differentiation but is critical for determining fetal progenitor number and myofiber number and type. Together, these studies demonstrate that limb embryonic and fetal myogenic cells develop from distinct, but related progenitors and have different cell-autonomous requirements for beta-catenin (Hutcheson, 2009).

Therefore lineage analysis reveals that Pax3⁺ somitic cells in the limb contribute to both muscle and endothelial cell lineages. Transplantation studies had established that somitic cells contribute to both limb muscle and endothelial cells, and chick lineage studies showed that even single somitic cells are bipotential, contributing to muscle and endothelium. This study shows that Pax3⁺ somitic cells are bipotential in the limb. Thus, even though Pax3 is a member of the genetic cascade committing cells to myogenesis, in vivo expression of Pax3 in somitic cells is not sufficient to commit these cells to a myogenic fate (Hutcheson, 2009).

It was also found that Pax7⁺ cells are a subset of Pax3⁺ somitic cells and only give rise to myogenic, and not endothelial cells. Ablation studies demonstrate that the Pax3 lineage is required for the emergence of Pax7⁺ cells, even though Pax3 function is not required for the specification of Pax7⁺ cells (Pax7⁺ cells are present in axial muscles of Pax3^-/- mice). Subsequently, Pax7 somitic derivatives appear to be restricted, and therefore potentially committed, to the muscle lineage. Whether the expression of Pax7 itself commits Pax3⁺ cells to myogenesis or simply marks committed cells is unclear. Pax7 regulates Myf5 expression, and myogenesis (with the exception of the primary myotome) requires either Pax3 or Pax7 expression. Recently Pax7 has been shown to be more stable than Pax3, which is subject to monoubiquitination and proteasomal degradation. An attractive hypothesis is that Pax3 expression initially establishes intermediate, bipotential precursors. Given the right extrinsic cues, some of these Pax3⁺ cells differentiate into muscle (or endothelium). However, Pax3⁺ cells that do not differentiate subsequently express the more stable Pax7, committing these cells to a myogenic fate via Pax7 activation of Myf5 expression (Hutcheson, 2009).

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