Table of contents

Pax3 and Pax7: mesoderm induction and differentiation

The most profound feature of the mature vertebrate somite is its organization into dorsal dermomyotome, intermediate myotome and ventral sclerotome. The role of potential signaling structures in this dorsoventral pattern was examined by ablating the structures or transplanting these structures to ectopic locations in chick embryos. The data suggest that the somite represents a naive tissue, entirely depending on external cues for its dorsoventral organization. Dorsalization by signals from dorsal neural tube and surface ectoderm stimulates the development of the dermomyotome. Neural tube and surface ectoderm play a crucial role in the initiation and maintenance of the dorsal pathway in the paraxial mesoderm in vivo: when both the neural tube and the surface ectoderm has been removed, the segmental plate fails to activate Pax3. Neural tube/ectoderm removal at the level of the anterior segmental plate and young somites expels expression of Pax3 from the paraxial mesoderm. In both cases, the somites appear completely mesenchymal and express Pax1 throughout their dorsoventral perimeter, indicating that they have been fully ventralized. Likewise, signals from notochord and floor plate ventralize the somite, at high levels overriding any dorsal information and inducing the sclerotome. The ventralizing function of notochord and floor plate seem to be carried out in part by the signaling molecule Sonic Hedgehog. The dorsalizing factors and lower levels of the ventralizing factors act in concert to induce the myotome. Candidates for the dorsalization of the paraxial mesoderm exerted by surface ectoderm and neural tube are members of the TGFbeta superfamily: for example, the bone morphogenetic proteins BMP4 and BMP7 are capable of dorsalizing the neural tube. The paraxial mesoderm intrinsically controls its competence to respond to the external inducers (Dietrich, 1997).

To understand how the skeletal muscle lineage is induced during vertebrate embryogenesis, an attempt has been made to identify the regulatory molecules that mediate induction of the myogenic regulatory factors MyoD and Myf-5 (See Drosophila Nautilus). Either signals from the overlying ectoderm or Wnt and Sonic hedgehog signals can induce somitic expression of the paired box transcription factors, Pax-3 and Pax-7, concomitant with expression of Myf-5 and prior to that of MyoD. Infection of embryonic tissues in vitro with a retrovirus encoding Pax-3 is sufficient to induce expression of MyoD, Myf-5, and myogenin in the neural tube as well as in both paraxial and lateral plate mesoderm in the absence of inducing tissues. Together, these findings imply that Pax-3 may mediate activation of MyoD and Myf-5 in response to muscle-inducing signals from either the axial tissues or overlying ectoderm and identify Pax-3 as a key regulator of somitic myogenesis (Maroto, 1997).

Pax-3 (splotch), Myf-5 (targeted with nlacZ), and splotch/Myf-5 homozygous mutant mice were analyzed to investigate the roles that these genes play in programming skeletal myogenesis. In splotch and Myf-5 mutant embryos, myogenic progenitor cell perturbations and early muscle defects are distinct. Remarkably, splotch/Myf-5 double homozygotes have a dramatic phenotype not seen in the individual mutants: body muscles are absent. MyoD does not rescue this double mutant phenotype since activation of this gene proves to be dependent on either Pax-3 or Myf-5. Therefore, Pax-3 and Myf-5 define two distinct myogenic pathways, and MyoD acts genetically downstream of these genes for myogenesis in the body. This genetic hierarchy does not appear to operate for head muscle formation (Tajbakhsh, 1997).

The splotch (Pax3) mouse mutant serves as a model for developmental defects of several types, including defective migration of dermomyotomal cells to form the limb musculature. Abnormalities of the ribs, neural arches, and acromion are described in Sp2H homozygous embryos, indicating a widespread dependence of lateral somite development on Pax3 function. Moreover, the intercostal and body wall muscles, derivatives of the ventrolateral myotome, are also abnormal in Sp2H homozygotes. Pax3 is expressed in the dermomyotome, but not in either the sclerotome or the myotome, raising the possibility that Pax3-dependent inductive influences from the dermomyotome are necessary for early specification of lateral sclerotome and myotome. Support for this idea comes from analysis of gene expression markers of lateral sclerotome (tenascin-C and scleraxis) and myotome (myogenin, MyoD, and Myf5). All exhibit ventrally truncated domains of expression in Sp2H homozygotes, potentially accounting for the rib and intercostal muscle truncations. In contrast, the medial sclerotomal marker Pax1 is expressed normally in mutant embryos, arguing that Pax3 is not required for development of the medial sclerotome. Most of the somitic markers show ectopic expression in anteroposterior and mediolateral dimensions, suggesting a loss of definition of somite boundaries in splotch and explaining the rib and muscle fusions. An exception is Myf5, which is not ectopically expressed in Sp2H homozygotes, consistent with the suggestion that Pax3 and Myf5 function in different pathways of skeletal myogenesis. PDGFalpha and its receptor are candidates for mediating signaling between myotome and sclerotome. Both genes are misexpressed in Sp2H embryos, suggesting that PDGFalpha/PDGFRalpha may function downstream of Pax3, accounting for the close similarities between the splotch and Patch mutant phenotypes. These findings point to additional regulatory functions for the Pax3 transcription factor, apart from those already demonstrated for development of the neural tube, neural crest, and dermomyotome (Henderson, 1999).

The migration of myogenic precursors to the vertebrate limb exemplifies a common problem in development -- namely, how migratory cells that are committed to a specific lineage postpone terminal differentiation until they reach their destination. In chicken embryos, expression of the Msx1 homeobox gene overlaps with Pax3 in migrating limb muscle precursors, which are committed myoblasts that do not express myogenic differentiation genes such as MyoD. Ectopic expression of Msx1 in the forelimb and somites of chicken embryos inhibits MyoD expression as well as muscle differentiation. Conversely, ectopic expression of Pax3 activates MyoD expression, while co-ectopic expression of Msx1 and Pax3 neutralizes one another's effects on MyoD. Msx1 represses and Pax3 activates MyoD regulatory elements in cell culture, while in combination, Msx1 and Pax3 oppose one another's trancriptional actions on MyoD. The Msx1 protein interacts with Pax3 in vitro, thereby inhibiting DNA binding by Pax3. Thus, it is proposed that Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors via direct protein-protein interaction. These results implicate functional antagonism through competitive protein-protein interactions as a mechanism for regulating the differentiation state of migrating cells (Bendall, 1999).

Activated by dorsalizing and lateralizing signals, the Pax3 gene is an early marker for the entire paraxial mesoderm and its dorsal derivative, the dermomyotome. Later, its expression becomes restricted to the lateral dermomyotome and to the migratory muscle precursors, giving rise to the hypaxial musculature. To understand better the role that Pax3 plays during development of paraxial mesoderm-derived structures, the development of the musculature and skeleton was followed in the murine Pax3 mutant Splotch. The mutant dermomyotomes and myotomes fail to organize and to elongate medially and laterally, leading to the reduction and malformation of the entire trunk musculature. Mutants lack both ventral aspects of the body wall musculature and muscles derived from migratory myoblasts, suggesting a crucial function for Pax3 in the long-range migration of muscle precursors giving rise to the ventral hypaxial musculature. In addition, severe malformations are detected in the skeleton. The axial and appendicular skeleton display malformations and in particular multiple bone fusions (Tremblay, 1998).

Differentiating P19 embryonal carcinoma (EC) cells transiently express an endogenous activity capable of inducing Pax-3 expression in adjacent P19 stem cells. In the present study, expression of this activity in mesodermal cell lineages is demonstrated in three ways.

  1. Expression of the mesodermal marker Brachyury correlates with expression of Pax-3-inducing activity.
  2. Leukemia inhibitory factor (LIF) blocks mesoderm differentiation at two different points and correlates with the inhibition of Pax-3-inducing activity.
  3. Two mesodermal cell lines that express Pax-3-inducing activity were derived from P19 EC cells. Each of these lines expresses high levels of the mesodermal marker Brachyury and high levels of Oct-3/4 (which is down-regulated at early times during mesoderm differentiation) suggesting that these lines are early mesodermal derivatives. Unlike EC or embryonic stem cell lines, each of the two mesodermal derivatives autoinduces Hox gene expression on aggregation even in the presence of LIF. Following aggregation, anterior-specific genes are expressed more rapidly than more posterior genes.

These observations directly demonstrate the ability of murine mesodermal derivatives to autoinduce Hox gene expression in the absence of signals from other cell lineages. Similar to the Pax-3-inducing activity, signals from mesodermal cell lines are sufficient to induce HOX expression in adjacent P19 stem cells in cell mixing assays. These observations are consistent with the suggestion that signals responsible for anterior-posterior organizer activity are localized to the anterior primitive streak mesoderm of the mouse embryo (Pruitt, 1994).

Cells of the cranial paraxial mesoderm give rise to parts of the skull and muscles of the head. Some mesoderm cells migrate from locations close to the hindbrain into the branchial arches where they undergo muscle differentiation. In spite of a lack of overt subdivision, the cranial paraxial mesoderm gives rise to separate cell lineages, including craniofacial muscles and some bones of the chordal skull (e.g. supra occipital, sphenoid, pars canalicularis and cochlearis of the otic capsule). The migratory pathways of the cranial paraxial mesoderm have been characterized in chick embryos either by DiI-labelling cells before migration or by grafting quail cranial paraxial mesoderm orthotopically. These experiments demonstrate that depending on their initial rostrocaudal position, cranial paraxial mesoderm cells migrate in streams to fill the core of the nearest branchial arch. A survey of the expression of myogenic genes has shown that the myogenic markers Myf5, MyoD and myogenin are expressed in branchial arch muscle, but at comparatively late stages, as compared with their expression in the somites. Pax3 is not expressed by myogenic cells that migrate into the branchial arches, despite its expression in migrating precursors of limb muscles. In order to test whether segmental plate or somitic mesoderm has the ability to migrate in a cranial location, quail trunk mesoderm was grafted into the cranial paraxial mesoderm region. While segmental plate mesoderm cells do not migrate into the branchial arches, somitic cells are capable of migrating and are incorporated into the branchial arch muscle mass. Grafted somitic cells in the vicinity of the neural tube maintain expression of the somitic markers Pax3, MyoD and Pax1. By contrast, ectopic somitic cells located distal to the neural tube and in the branchial arches do not express Pax3. These data imply that signals in the vicinity of the hindbrain and branchial arches act on migrating myogenic cells to influence their gene expression and developmental pathways (Hacker, 1998).

There is a striking parallel between the expression patterns of the Bmp4 (Drosophila homolog: decapentaplegic), Msx1 and Msx2 genes (Drosophila homolog: Muscle segment homeobox) in the lateral ridges of the neural plate before neural tube closure and later on, in the dorsal neural tube and superficial midline ectoderm. The spinous process of the vertebra is formed from Msx1- and 2-expressing mesenchyme and that the dorsal neural tube can induce the differentiation of subcutaneous cartilage from the somitic mesenchyme. Mouse BMP4- or human BMP2-producing cells grafted dorsally to the neural tube at E2 or E3 increase considerably the amount of Msx-expressing mesenchymal cells which are normally recruited from the somite to form the spinous process of the vertebra. Later on, the dorsal part of the vertebra is enlarged, resulting in vertebral fusion and, in some cases (e.g. grafts made at E3), in the formation of a 'giant' spinous process-like structure dorsally. In strong contrast, BMP-producing cells grafted laterally to the neural tube at E2 exerted a negative effect on the expression of Pax1 and Pax3 genes in the somitic mesenchyme, which then turned on Msx genes. Moreover, sclerotomal cell growth and differentiation into cartilage were then inhibited. Dorsalization of the neural tube, manifested by expression of Msx and Pax3 genes in the basal plate contacting the BMP-producing cells, was also observed. In conclusion, this study demonstrates that differentiation of the ventrolateral and dorsal parts of the vertebral cartilage is controlled by different molecular mechanisms. The former develops under the influence of signals arising from the floor plate-notochord complex. These signals inhibit the development of dorsal subcutaneous cartilage forming the spinous process, which requires the influence of BMP4 to differentiate (Monsoro-Burg, 1996).

Neural crest cells originating in the occipital region of the avian embryo are known to play a vital role in formation of the septum of the cardiac outflow tract and to contribute cells to the aortic arches, thymus, thyroid and parathyroids. This 'cardiac' neural crest sub-population is assumed to exist in mammals. Pax3 expression can serve as a marker of cardiac neural crest cells in the mouse embryo. Cells of this lineage were traced from the occipital neural tube, via branchial arches 3, 4 and 6, into the aortic sac and aorto-pulmonary outflow tract. Confirmation that these Pax3-positive cells are indeed cardiac neural crest is provided by experiments in which hearts were deprived of a source of colonizing neural crest, by organ culture in vitro, with consequent lack of up-regulation of Pax3. Occipital neural crest cell outgrowths in vitro were also shown to express Pax3. Mutation of Pax3, as occurs in the splotch (Sp2H) mouse, results in development of conotruncal heart defects including persistent truncus arteriosus. Homozygotes also exhibit defects of the aortic arches, thymus, thyroid and parathyroids. Pax3-positive neural crest cells are found to emigrate from the occipital neural tube of Sp2H/Sp2H embryos in a relatively normal fashion, but there is a marked deficiency or absence of neural crest cells traversing branchial arches 3, 4 and 6, and entering the cardiac outflow tract. This decreased expression of Pax3 in Sp2H/Sp2H embryos was not due to down-regulation of Pax3 in neural crest cells, as use of independent neural crest markers, Hoxa-3, CrabpI, Prx1, Prx2 and c-met also reveals a deficiency of migrating cardiac neural crest cells in homozygous embryos. This work demonstrates the essential role of the cardiac neural crest in formation of the heart and great vessels in the mouse; furthermore, it shows that Pax3 function is required for the cardiac neural crest to complete its migration to the developing heart (Conway, 1997).

The paired-related homeobox gene, prx-1, is expressed in the postmigratory cranial mesenchyme of all facial prominences and is required for the formation of proximal first arch derivatives. lacZ was introduced into the prx-1 locus to study the developmental fate of cells destined to express prx-1 in the prx-1 mutant background. lacZ is normally expressed in prx-1(neo);prx-1(lacZ) mutant craniofacial mesenchyme up until 11.5 d.p.c. At later time points, lacZ expression is lost from structures that are defective in the prx-1(neo) mutant mice. A related gene, prx-2, demonstrates overlapping expression with prx-1. To test the idea that prx-1 and prx-2 perform redundant functions, prx-1(neo;)prx-2 compound mutant mice were generated. Double mutant mice have novel phenotypes in which the rostral aspect of the mandible is defective; the mandibular incisor arrested as a single, bud-stage tooth germ, and Meckel's cartilage is absent. Expression of two markers for tooth development, pax9 and patched, are downregulated. Using a transgene that marks a subset of prx-1-expressing cells in the craniofacial mesenchyme, it has been shown that cells within the hyoid arch take on the properties of the first branchial arch. These data suggest that prx-1 and prx-2 coordinately regulate gene expression in cells that contribute to the distal aspects of the mandibular arch mesenchyme and that prx-1 and prx-2 play a role in the maintenance of cell fate within the craniofacial mesenchyme (Lu, 1999b).

The mechanisms by which pluripotent embryonic cells generate unipotent tissue progenitor cells during development are unknown. Molecular/genetic experiments in cultured cells have led to the hypothesis that the product of a single member of the MyoD gene family (MDF) is necessary and sufficient to establish the positive aspects of the determined state of myogenic precursor cells: i.e., the ability to initiate and maintain the differentiated state. Embryonic cell type determination also involves negative regulation, such as the restriction of developmental potential for alternative cell types, that is not directly addressed by the MDF model. In the experiments reported here, phenotypic restriction in myogenic precursor cells is assayed by an in vivo 'notochord challenge' to evaluate their potential to 'choose' between two alternative cell fate endpoints: cartilage and muscle. The notochord challenge assay used here to demonstrate the loss of cartilage potential in precursor cells of the limb muscles is fundamentally different from previous assays of limb muscle precursor cell specification in vitro: (1) the assay is designed to challenge muscle phenotype commitment with cartilage-inducing signals in the native embryonic environment, using embryonic signals that are known to affect somite cell specification; (2) two differentiated cell type endpoints ('choices'), muscle and cartilage, are scored simultaneously; (3) the assay is well-defined anatomically, allowing detailed analysis of the morphogenetic potential of the implanted cells (Williams, 2000).

Two separate myogenic precursor cell populations were found to be phenotypically restricted while expressing the Pax3 gene and prior to MDF gene activation. Therefore, while MDF family members act positively during myogenic differentiation, the process of phenotypic restriction (the negative aspect of cell specification) requires cellular and molecular events and interactions that precede MDF expression in myogenic precursor cells. The qualities of muscle formed by the determined myogenic precursor cells in these experiments further indicate that their developmental potential is intermediate between that of myoblastic stem cells taken from fetal or adult tissue (which lack mitotic and morphogenetic potential when tested in vivo) and embryonic stem cells (which are multipotent). It is hypothesized that such embryonic myogenic progenitor cells represent a distinct class of determined embryonic cell, one that is responsible for both tissue growth and tissue morphogenesis (Williams, 2000).

Muscle satellite cells represent a distinct lineage of myogenic progenitors responsible for the postnatal growth, repair, and maintenance of skeletal muscle. At birth, satellite cells account for ~30% of sublaminar muscle nuclei in mice followed by a decrease to <5% in a 2-month-old adult. This decline in satellite cell nuclei reflects the fusion of satellite cells during the postnatal growth of skeletal muscle. Satellite cells were originally defined on the basis of their unique position in mature skeletal muscle and are closely juxtaposed to the surface of myofibers such that the basal lamina surrounding the satellite cell and its associated myofiber is continuous (Seale, 2000).

In mice <2 months of age, satellite cells in resting skeletal muscle are mitotically quiescent and are activated in response to diverse stimuli, including stretching, exercise, injury, and electrical stimulation. The descendents of activated satellite cells, called myogenic precursor cells, undergo multiple rounds of cell division before fusion with new or existing myofibers. The total number of quiescent satellite cells in adult muscle remains constant over repeated cycles of degeneration and regeneration, suggesting that the steady-state satellite cell population is maintained by self-renewal. Therefore, satellite cells have been suggested to form a population of monopotential stem cells that are distinct from their daughter myogenic precursor cells as defined by biological and biochemical criteria (Seale, 2000 and references therein).

Satellite cells clearly represent the progenitors of the myogenic cells that give rise to the majority of the nuclei within adult skeletal muscle. However, recent studies have identified a population of pluripotential stem cells, also called side-population (SP) cells, in adult skeletal muscle. Muscle-derived SP cells are readily isolated by fluorescence-activated cell sorting (FACS) on the basis of Hoechst dye exclusion. Purified SP cells derived from muscle exhibit the capacity to differentiate into all major blood lineages after tail vein injection into lethally irradiated mice. Of particular significance is the observation that transplanted SP cells isolated from bone marrow or muscle actively participate in myogenic regeneration. However, only muscle-derived SP cells appear to give rise to myogenic satellite cells. In addition, SP cells convert to desmin-expressing myoblasts after exposure to appropriate cell culture conditions. However, it remains unclear whether SP cells are equivalent to satellite cells, are progenitors for satellite cells, or represent an entirely independent cell population (Seale, 2000).

The gene expression profile of quiescent satellite cells and their activated progeny is largely unknown. Quiescent satellite cells express the c-Met receptor (receptor for hepatocyte growth factor) and M-cadherin protein. Activated satellite cells upregulate MyoD or Myf5 before entering S-phase. Proliferating myogenic precursor cells, the daughter cells of satellite cells, express desmin, Myf5, MyoD, and other myoblast specific markers. Nevertheless, the paucity of cell-lineage specific markers has been a significant impediment to understanding the relationship between satellite cells and their progeny (Seale, 2000).

The paired box transcription factor Pax7 was isolated by representational difference analysis as a gene specifically expressed in cultured satellite cell-derived myoblasts. In situ hybridization reveals that Pax7 was also expressed in satellite cells residing in adult muscle. Cell culture and electron microscopic analysis reveals a complete absence of satellite cells in Pax7-/- skeletal muscle. Surprisingly, fluorescence-activated cell sorting analysis indicatesthat the proportion of muscle-derived stem cells is unaffected. Importantly, stem cells from Pax7-/- muscle display almost a 10-fold increase in their ability to form hematopoietic colonies. These results demonstrate that satellite cells and muscle-derived stem cells represent distinct cell populations. Together these studies suggest that induction of Pax7 in muscle-derived stem cells induces satellite cell specification by restricting alternate developmental programs (Seale, 2000).

Pax3 is a key transcription factor implicated in development and human disease. To dissect the role of Pax3 in myogenesis and establish whether it is a repressor or activator, loss- and gain-of-function alleles were generated by targeting an nLacZ reporter and a sequence encoding the oncogenic fusion protein PAX3-FKHR into the Pax3 locus. Rescue of the Pax3 mutant phenotypes by PAX3-FKHR suggests that Pax3 acts as a transcriptional activator during embryogenesis, since the C-terminal sequence of FKHR, present in the fusion protein, contains a potent transcriptional activation domain, and PAX3-FKHR has been shown to act as an efficient transactivator on Pax3-binding sites in vitro. The ability of Pox3 to activate transcription was confirmed by a Pax reporter mouse. However, mice expressing PAX3-FKHR display developmental defects, including ectopic delamination and inappropriate migration of muscle precursor cells. These events result from overexpression of c-met, leading to constitutive activation of Met signaling, despite the absence of the ligand SF/HGF. Haploinsufficiency of c-met rescues this phenotype, confirming the direct genetic link with Pax3. The gain-of-function phenotype is also characterized by overactivation of MyoD. The consequences of PAX3-FKHR myogenic activity in the limbs and cervical and thoracic regions point to differential regulation of muscle growth and patterning. This gain-of-function allele provides a new approach to the molecular and cellular analysis of the role of Pax3 and of its target genes in vivo (Relaix, 2003).

Satellite cells are myogenic precursors responsible for skeletal muscle regeneration. Satellite cells are absent in the Pax-7-/- mouse, suggesting that this transcription factor is crucial for satellite cell specification. Analysis of Pax-7 expression in activated satellite cells unexpectedly revealed substantial heterogeneity within individual clones. Further analyses show that Pax-7 and myogenin expression are mutually exclusive during differentiation, where Pax-7 appears to be up-regulated in cells that escape differentiation and exit the cell cycle, suggesting a regulatory relationship between these two transcription factors. Indeed, overexpression of Pax-7 down-regulates MyoD, prevents myogenin induction, and blocks MyoD-induced myogenic conversion of 10T1/2 cells. Overexpression of Pax-7 also promotes cell cycle exit even in proliferation conditions. Together, these results suggest that Pax-7 may play a crucial role in allowing activated satellite cells to reacquire a quiescent, undifferentiated state. These data support the concept that satellite cell self-renewal may be a primary mechanism for replenishment of the satellite cell compartment during skeletal muscle regeneration (Olguin, 2004).

A working model is presented for the role of Pax-7 in satellite cell physiology. Mitotically quiescent satellite cells express a subset of characteristic proteins including the markers syndecan-3, syndecan-4, and c-met but are heterogeneous for Pax-7 protein. Upon activation, satellite cells proliferate and up-regulate MyoD. Proliferating myoblasts that are positive for both Pax-7 and MyoD behave as a heterogeneous population where a small fraction of cells are prone to precocious differentiation, inducing myogenin and losing Pax-7 expression, while a small number retain precursor characteristics. Co-expression of Pax-7 and MyoD might be required to retain myoblasts in a proliferative state and prevent premature differentiation. As the myogenic program proceeds, MyoD family transcription factors are up-regulated and Pax-7 is down-regulated in cells committed to differentiation. A small number of precursor cells up-regulate Pax-7 and down-regulate MyoD, exit the cell cycle, and form a new satellite cell pool. Additional events might be involved to evade apoptosis and acquire the final satellite cell position beneath the basal lamina of regenerated muscle fibers (Olguin, 2004).

In mammals, Six5, Six4 and Six1 genes are co-expressed during mouse myogenesis. Six4 and Six5 single knockout (KO) mice have no developmental defects, while Six1 KO mice die at birth and show multiple organ developmental defects. Six1Six4 double KO mice were generated and an aggravation of the phenotype previously reported for the single Six1 KO was demonstrated. Six1Six4 double KO mice are characterized by severe craniofacial and rib defects, and general muscle hypoplasia. At the limb bud level, Six1 and Six4 homeogenes control early steps of myogenic cell delamination and migration from the somite through the control of Pax3 gene expression. Impaired in their migratory pathway, cells of the somitic ventrolateral dermomyotome are rerouted, lose their identity and die by apoptosis. At the interlimb level, epaxial Met expression is abolished, while it is preserved in Pax3-deficient embryos. Within the myotome, absence of Six1 and Six4 impairs the expression of the myogenic regulatory factors myogenin and Myod1, and Mrf4 expression becomes undetectable. Myf5 expression is correctly initiated but becomes restricted to the caudal region of each somite. Early syndetomal expression of scleraxis is reduced in the Six1Six4 embryo, while the myotomal expression of Fgfr4 and Fgf8 but not Fgf4 and Fgf6 is maintained. These results highlight the different roles played by Six proteins during skeletal myogenesis (Grifone, 2005).

Skeletal muscle serves as a paradigm for the acquisition of cell fate, yet the relationship between primitive cell populations and emerging myoblasts has remained elusive. A novel population of resident Pax3+/Pax7+, muscle marker-negative cells, has been identifed that is present throughout development. Using mouse mutants that uncouple myogenic progression, this study shows that these Pax+ cells give rise to muscle progenitors. In the absence of skeletal muscle, they apoptose after down-regulation of Pax7. Furthermore, they mark the emergence of satellite cells during fetal development, and do not require Pax3 function (Kassar-Duchossoy, 2005).

The classification of different cell states in muscle, notably the identification of a novel uncommitted Pax3/Pax7+ population, provides a framework for refining the cell order in this lineage, and characterizing the skeletal muscle niche. Moreover, it is proposed that differentiated skeletal muscle or precursor cells are a functional component of this niche since Pax7+ stem cells, as well as MPCs, require the downstream myogenic program for their self-renewal, but surprisingly, not for their birth. In future studies it would important to identify factors secreted by skeletal muscle that will mediate the self-renewal of engrafted cells, and thereby enhance their regenerative potential (Kassar-Duchossoy, 2005).

Postnatal growth and regeneration of skeletal muscle requires a population of resident myogenic precursors named satellite cells. The transcription factor Pax7 is critical for satellite cell biogenesis and survival and has been also implicated in satellite cell self-renewal; however, the underlying molecular mechanisms remain unclear. Pax7 overexpression in adult primary myoblasts has been shown to down-regulate MyoD and prevent myogenin induction, inhibiting myogenesis. This study shows that Pax7 prevents muscle differentiation independently of its transcriptional activity, affecting MyoD function. Conversely, myogenin directly affects Pax7 expression and may be critical for Pax7 down-regulation in differentiating cells. These results provide evidence for a cross-inhibitory interaction between Pax7 and members of the muscle regulatory factor family. This could represent an additional mechanism for the control of satellite cell fate decisions resulting in proliferation, differentiation, and self-renewal, necessary for skeletal muscle maintenance and repair (Olguin, 2007).

Pax3 and Pax7 play distinct but overlapping roles in developmental and postnatal myogenesis. The mechanisms involved in the differential regulation of these highly homologous proteins are unknown. Evidence is presented that Pax3, but not Pax7, is regulated by ubiquitination and proteasomal degradation during adult muscle stem cell activation. Intriguingly, only monoubiquitinated forms of Pax3 could be detected. Mutation of two specific lysine residues in the C-terminal region of Pax3 reduced the extent of its monoubiquitination and susceptibility to proteasomal degradation, whereas introduction of a key lysine into the C-terminal region of Pax7 rendered that protein susceptible to monoubiquitination and proteasomal degradation. Monoubiquitinated Pax3 was shuttled to the intrinsic proteasomal protein S5a by interacting specifically with the ubiquitin-binding protein Rad23B. Functionally, sustained expression of Pax3 proteins inhibited myogenic differentiation, demonstrating that Pax3 degradation is an essential step for the progression of the myogenic program. These results reveal an important mechanism of Pax3 regulation in muscle progenitors and an unrecognized role of protein monoubiquitination in mediating proteasomal degradation (Boutet, 2007).

Pax3/7-dependent stem cells play an essential role in skeletal muscle development. Fgfr4 lies genetically downstream from Pax3 and is a direct target. In chromatin immunoprecipitation (ChIP)-on-chip experiments, Pax3 binds to a sequence 3' of the Fgfr4 gene that directs Pax3-dependent expression at sites of myogenesis in transgenic mouse embryos. The activity of this regulatory element is also partially dependent on E-boxes, targets of the myogenic regulatory factors, which are expressed as progenitor cells enter the myogenic program. Other FGF signaling components, notably Sprouty1, are also regulated by Pax3. In vivo manipulation of Sprouty expression reveals that FGF signaling affects the balance between Pax-positive progenitor cells and committed myoblasts. These results provide new insight into the Pax-initiated regulatory network that modulates stem cell maintenance versus tissue differentiation (Lagha, 2008).

Although FoxO and Pax proteins represent two important families of transcription factors in determining cell fate, they had not been functionally or physically linked together in mediating regulation of a common target gene during normal cellular transcription programs. This study identified MyoD, a key regulator of myogenesis, as a direct target of FoxO3 and Pax3/7 in myoblasts. Cell-based assays and in vitro studies reveal a tight codependent partnership between FoxO3 and Pax3/7 to coordinately recruit RNA polymerase II and form a preinitiation complex (PIC) to activate MyoD transcription in myoblasts. The role of FoxO3 in regulating muscle differentiation is confirmed in vivo by observed defects in muscle regeneration caused by MyoD downregulation in FoxO3 null mice. These data establish a mutual interdependence and functional link between two families of transcription activators serving as potential signaling sensors and regulators of cell fate commitment in directing tissue specific MyoD transcription (Hu, 2008).

Sequence analysis revealed four potential FRE sites in a 6 kb region upstream of the myod transcription initiation site, which are between the DRR and PRR elements important for MyoD regulation during embryonic muscle development. ChIP experiments were performed to determine promoter occupancy of FoxOs at the myod gene using antibodies specifically directed against each of the three FoxO proteins. FoxO3 was efficiently detected at the myod promoter regions overlapping putative FRE -940 and -1598, but not at -1928 and -2351. In marked contrast to FoxO3, neither FoxO1 nor FoxO4 was detected significantly above background at the four potential FREs in C2C12 cells. As a control, the same FoxO1 and FoxO4 antibodies were used to successfully detect their occupancy at the p21 promoter, which is known to be regulated by these FoxOs. Indeed, it appears that FoxO3 selectively binds two of the putative FREs at the myod promoter in myoblasts, suggesting that FoxO3 binding may correspond to one of the important steps regulating MyoD expression. RNA polymerase II was also found at the myod promoter by ChIP, further confirming the correlation between FoxO3 promoter occupancy and transcription activation. Although FoxO4 appeared to have low transcriptional activity, little, if any, FoxO4 was detected at the myod promoter by ChIP in myoblasts. Taken together, these results suggest that at least FoxO3 is likely part of the active transcription machinery directly recruited to the myod promoter in myoblasts (Hu, 2008).

Since FoxO3 binds to putative FRE -940 and FRE -1598 of the myod promoter in C2C12 cells, whether FoxO3 is also able to recognize and bind these putative FREs in vitro was tested by electrophoretic mobility shift assays (EMSA). Purified recombinant FoxO3 efficiently bound to both the FRE -940 and FRE -1598 probes, but not to mutant probes, confirming that FoxO3 can bind to both FRE sequences specifically in vitro. Consistent with ChIP results, in luciferase reporter assays, mutations in the FREs at -940 or -1598 significantly debilitated myod transcription, while mutations in FRE -1928 and FRE -2351 had little or no effect on transcription activity. These results suggest that FRE -940 and -1598 bound by FoxO3 are critical for myod transcription activation. Taken together, these assays indicate that FoxO3 can specifically bind two FREs in the myod promoter and activate transcription (Hu, 2008).

Although FoxOs have previously been implicated functioning in muscle differentiation, skeletal muscle-specific genes directly targeted by FoxOs had not been identified. The current findings indicate that FoxO3 (but not FoxO1 or FoxO4) binds a subset of FREs in the myod promoter to work in concert with Pax3/7 in regulating cell type-specific transcription activation in myoblasts. The contribution of FoxO3 in directing myod transcription activation in vivo was further confirmed by the observed muscle regeneration defects in FoxO3 null mice (Hu, 2008).

Identification of FoxO3 as an important myod transcription activator may provide a handle to explore the potential signaling pathways governing muscle regeneration. Interestingly, FoxO1 was found to negatively regulate MyoD expression indirectly through the Delta-Notch pathway. The potential repression of MyoD by FoxO1 together with the results of direct activation of MyoD by FoxO3 suggests an intriguing mechanism to fine tune MyoD expression. Muscle differentiation may therefore utilize selected FoxOs in partnership with Pax3/7 to integrate inputs from multiple signaling pathways (Hu, 2008).

The apparent Kd of FoxO3 and Pax3/7 binding individually to the DNA elements in the myod promoter is on the order of 10−7M in vitro, which is rather modest compared to a typical DNA binding protein, such as GAL4 (apparent Kd, ~10−11M). This is consistent with the finding that neither FoxO3 nor Pax3/7 alone binds promoter DNA efficiently to form a stable DNA-activator complex capable of recruiting a functional PIC. It appears that to efficiently assemble an active PIC via FoxO3 and Pax3/7 at the myod promoter both protein-DNA and protein-protein interactions mediated by this hitherto unknown activator partnership must take place to trigger transcription activation synergistically. This may therefore represent a useful and efficient combinatorial mechanism to direct cell type-specific transcription while utilizing two activators shared by many cell types. This codependence is reminiscent of the mechanism utilized by Sox2 and Pax6 to drive lens-specific transcription of δ-crystallin gene (Hu, 2008).

Although under normal conditions FoxO3 is predominantly detected occupying the FRE at -1598 of the myod promoter, curiously, it was found that some FoxO4 can be detected at this site in myoblasts when Pax3/7 is depleted. It is known that Pax3/7 is not expressed in myotubes, but MyoD expression persists in myotubes. It will be interesting to survey the identity of FoxOs binding to the myod promoter in myotubes versus myoblasts and explore additional mechanisms involved in cell type-specific transcription activation during later stages of myogenesis. Intriguingly, there is a switching of the core transcription machinery from the canonical holo-TFIID to a TRF3/TAF3 complex during myoblasts differentiation to myotubes (Deato, 2007). While FoxO3 and Pax3/7 appear to work in concert with TFIID at the MyoD promoter in myoblasts, it will be important to identify the key activator(s) that function together with the TRF3/TAF3 complex in myotubes (Hu, 2008).

Maintenance of multipotency and how cells exit this state to adopt a specific fate are central questions in stem cell biology. During vertebrate development, multipotent cells of the dorsal somite, the dermomyotome, give rise to different lineages such as vascular smooth and skeletal muscle, regulated by the transcription factors Foxc2 and Pax3, respectively. Reciprocal inhibition was found between Pax3 and Foxc2 in the mouse embryo. Using both genetic approaches and manipulation of external signals in somite explants, it was demonstrated that the Pax3:Foxc2 ratio modulates myogenic versus vascular cell fates. This provides insight into how cell fate choices are orchestrated by these lineage genes in the dermomyotome (Lagha, 2009).

Pax3 and cell adhesion

Pax3 is expressed in the developing somites, dorsal spinal cord, mesencephalon and neural crest derivatives. Several loss-of-function mutations are correlated with the Splotch phenotype in mice and Waardenburg syndrome in humans. Malformations include a lack of muscle in the limb, a failure of neural tube closure and dysgenesis of numerous neural crest derivatives. In this study embryonic stem (ES) cells were used to generate a lacZ knock-in into the Pax3 locus. The Pax3 knock-in Splotch allele (Sp2G) was used to generate Pax3-deficient ES cells in order to investigate whether, in chimeric embryos, Pax3 is acting cell autonomously in the somites and the neural tube. While Pax3 function is essential for the neuroepithelium and somites, a wild-type environment rescues mutant neural crest cells. In the two affected embryonic tissues, mutant and wild-type cells undergo segregation and do not intermingle. The contribution of mutant cells to the neural tube and the somites displays temporal differences. All chimeric embryos show a remarkable contribution of blue cells to the neural tube at all stages analyzed, indicating that the Pax3-deficient cells are not excluded from the neural epithelium while development proceeds. In contrast, this is not true for the paraxial mesoderm. The somite contribution of Pax3-/- ES cells becomes less frequent in older embryos as compared to controls with Pax3+/- ES cells. It is proposed that although Pax3 function is related to cell surface properties, its role may differ in various tissues. In fact, apoptosis was found in Pax3-deficient cells of the lateral dermomyotome but not in the neural tube (Mansouri, 2001).

The chimeric embryos provide a system in which Pax3-deficient cells are allowed to mix and interact with surrounding wild-type cells, enabling them to be rescued in the affected tissues. A failure to rescue mutant cells would suggest that Pax3 acts cell autonomously in that specific tissue. Somite patterning is under the control of different signals provided by the neural tube and other tissues such as the surface ectoderm, and the axial and the lateral mesoderm. The multiple roles of Pax3 in the paraxial mesoderm and the neural tube do not disclose which tissue requires Pax3 function. In chimeric embryos, where only the neural tube exhibits a high proportion of mutant cells, the dermomyotome develops normally. Thus, Pax3 function in the neural tube is not related to signals exerted on the somites. However, the contribution of mutant cells to the dermomyotome leads to disorganized somites, as has been reported for Splotch embryos. In addition, Pax3 mutant cells in the lateral dermomyotome are not able to migrate into the limb. Accordingly, Pax3 acts cell autonomously in the lateral dermomyotome. Furthermore, mutant cells in the neural tube are not able to interact with wild-type cells, resulting in an open neuroepithelium and suggesting a cell autonomous role for Pax3 in this tissue (Mansouri, 2001).

The analysis of chimeric embryos further reveals that in the neural tube, somites and olfactory epithelium, mutant and wild-type cells do not intermingle. Pax3-/- and wild-type cells fail to mix and clear segregation is readily detectable in the affected tissues in whole-mount X-gal-stained embryos. The affected tissues consist exclusively of patches of blue cells (mutant) or white cells (wild type) revealing a distinct boundary. However, this abnormal participation of mutant cells is not observed before E10 of gestation. In addition, the neural tube alone displays segregation of heterozygous mutant and wild-type cells in some chimeras generated by the aggregation of Pax3+/- ES cells with wild-type embryos. This is in close correlation with the often observed failure of neural tube closure at the posterior neuropore (spina bifida) in heterozygous Splotch embryos. The failure of mutant cells to mix and interact normally with surrounding wild-type cells suggests that Pax3 may control cell surface properties. Similar observations have been made in chimeras generated with mutant Pax6 Sey cells. Differences in cell-cell adhesion documented by the expression of various cadherins may confer segregation behavior and thus define cell identity. In fact, it has been suggested that some Pax genes act on some or all of the following: cell surface molecules (R-, N-cadherin); members of the immunoglobulin superfamily (N-CAM, L1), and/or integrins. Alternatively, molecules involved in modulating cell surface properties may also act downstream of Pax genes. Such a protein may be the c-met tyrosine kinase, the receptor for HGF/SF (hepatocyte growth factor/scatter factor), which has been proposed to act downstream of Pax3 and initiate the migration of myoblasts from the lateral dermomyotome. Accordingly, the tendency of Pax-deficient cells to segregate from wild-type cells in the affected tissues indicates a common mechanism, reflecting a similar role for Pax genes in various organs (Mansouri, 2001).

In addition, the contributions of mutant cells to the neural tube and the somites display temporal differences. All chimeric embryos show a remarkable contribution of blue cells to the neural tube at all stages analyzed, indicating that the Pax3-deficient cells are not excluded from the neural epithelium while development proceeds. In contrast, this is not true for the paraxial mesoderm. Somite contribution of Pax3-/- ES cells becomes less frequent in older embryos, when compared with controls with Pax3+/- ES cells. This suggests that although in the neural tube and somites cell surface properties are related to Pax3 function, Pax3 may play different roles in both tissues. In the somites, Pax3 may be necessary for cell proliferation and/or survival. Although natural cell death was described in the somites, it is thought that in the lateral dermomyotome of Sp2H embryos, observed apoptosis is significant. There results support the idea that Pax3 function in the dermomyotome is related to cell survival. Furthermore, the findings suggest that Pax3 is also required for maintenance of the integrity of the dermomyotome, where it may drive a proper epithelial-to-mesenchymal transition process that is necessary for the formation of the myotome. This is in close correlation with earlier observations in Splotch embryos, where after E9.5 the lateral dermomyotome becomes truncated, leading to a loss of epithelial morphology. The lack of Fgf8 expression in the dermomyotome of Splotch embryos provides further evidence for the role of Pax3 in the cytoarchitecture of this structure. In the absence of Fgf8, cell survival is affected in the first branchial arch. During gastrulation, the lack of Fgf8 causes a failure of cell migration. Fgf8 may therefore be one of the factors that mediates cell proliferation and/or migration in the dermomyotome. In addition, the expression of Fgf8 in the rostral and caudal dermomyotomal lips points to a role in the epithelial-to-mesenchymal transition during the differentiation of the dermomyotome. In the absence of the Fgf8 receptor, Fgfr1, it was proposed that the primary defect is a deficiency in the ability of cells to make the transition from an epithelial-to-mesenchymal morphology. Altogether, these results suggest an important role for Pax3 in the morphogenesis of the epithelium of the lateral dermomyotome. Fgf8 acts downstream of Pax3 to achieve this function. Pax7 possibly restores the integrity of the medial dermomyotome, because in Pax3/Pax7 double mutants the whole epithelium is truncated (Mansouri, 2001).

In the neural tube, Pax3 has been also suggested to be necessary for the migration of neural crest cells. However, grafting of dorsal neural tube tissue from Splotch mice into chick host embryo results in normal neural crest migration. These studies clearly provide further evidence that Pax3-deficient neural crest cells are able to migrate from the neural tube and that wild-type cells always rescue neural crest derivatives (DRG and SG). The role of Pax3 in neural crest cells may be related to the maintenance of other properties required post migration, such as proliferation and/or survival. Similar findings have recently been described for cardiac neural crest cells. Alternatively, neural crest migration may require interaction with neighboring tissues. It is conceivable that these neighboring cues are also related to cell surface molecules, such as extracellular matrix proteins. Strikingly, overexpression of the extracellular matrix protein versican has been described and associated with defective neural crest migration in Splotch embryos. The results however, do not correlate with earlier studies that suggest a cell autonomous role for Pax3 in neural crest cells. The analysis of ES cell grafts into the chick neural tube supports the idea that Pax3 does not act cell autonomously in neural crest migration. In addition, these results indicate that Pax3 is not necessary for ES cells to migrate in chicken embryo via the DV and DL pathways. A slight difference in the behavior of the cells that lack Pax3 should be noticed. By an as yet unexplained mechanism, the absence of Pax3 seems to favor slightly the DL migration (Mansouri, 2001).

The possibility that Pax3 function in neural crest is related to intrinsic properties, which are required post migration, cannot be excluded. The difference in the migration potential of mutant neural crest cells between rostral and caudal neural tube of Splotch embryos is most likely related to a redundant function of Pax7 in the hindbrain (Mansouri, 2001).

In summary, the chimeric analysis using Pax3-deficient ES cells reveals a cell-autonomous function of Pax3 in the somites and neural tube. A common denominator of Pax3 function may be the modulation of cell surface properties, although distinct roles are enacted in various tissues (Mansouri, 2001).

Table of contents

paired continued: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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