M-Cadherin Is a PAX3 Target During Myotome Patterning.

PAX3 belongs to the paired-homeobox family of transcription factors and plays a key role as an upstream regulator of muscle progenitor cells during embryonic development. Pax3-mutant embryos display impaired somite development, yet the consequences for myotome formation have not been characterized. The early myotome is formed by PAX3-expressing myogenic cells that delaminate from the dermomyotomal lips and migrate between the dermomyotome and sclerotome where they terminally differentiate. Here we show that in Pax3-mutant embryos, myotome formation is impaired, displays a defective basal lamina and the regionalization of the structural protein Desmin is lost. In addition, this phenotype is more severe in embryos combining Pax3-null and Pax3 dominant-negative alleles. We identify the adhesion molecule M-Cadherin as a PAX3 target gene, the expression of which is modulated in the myotome according to Pax3 gain- and loss-of-function alleles analyzed. Taken together, we identify M-Cadherin as a PAX3-target linked to the formation of the myotome.

A tensile ring drives tissue flows to shape the gastrulating amniote embryo.

Tissue morphogenesis is driven by local cellular deformations that are powered by contractile actomyosin networks. How localized forces are transmitted across tissues to shape them at a mesoscopic scale is still unclear. Analyzing gastrulation in entire avian embryos, we show that it is driven by the graded contraction of a large-scale supracellular actomyosin ring at the margin between the embryonic and extraembryonic territories. The propagation of these forces is enabled by a fluid-like response of the epithelial embryonic disk, which depends on cell division. A simple model of fluid motion entrained by a tensile ring quantitatively captures the vortex-like "polonaise" movements that accompany the formation of the primitive streak. The geometry of the early embryo thus arises from the transmission of active forces generated along its boundary.

PAX3 Confers Functional Heterogeneity in Skeletal Muscle Stem Cell Responses to Environmental Stress.

Muscle satellite cells (MuSCs) are the quiescent muscle stem cells required for adult skeletal muscle repair. The impact of environmental stress such as pollution on MuSC behavior remains unexplored. We evaluated the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure, a ubiquitous and highly toxic pollutant, on MuSCs by combining in vivo mouse molecular genetic models with ex vivo studies. While all MuSCs express the transcription factor PAX7, we show that a subset also express PAX3 and exhibit resistance to environmental stress. Upon systemic TCDD treatment, PAX3-negative MuSCs display impaired survival, atypical activation, and sporadic differentiation through xenobiotic aryl hydrocarbon receptor signaling. We further show that PAX3-positive MuSCs become sensitized to environmental stress when PAX3 function is impaired and that PAX3-mediated induction of mTORC1 is required for protection. Our study, therefore, identifies a functional heterogeneity of MuSCs in response to environmental stress controlled by PAX3.

Timed Collinear Activation of Hox Genes during Gastrulation Controls the Avian Forelimb Position.

Limb position along the body is highly consistent within one species but very variable among vertebrates. Despite major advances in our understanding of limb patterning in three dimensions, how limbs reproducibly form along the antero-posterior axis remains largely unknown. Hox genes have long been suspected to control limb position; however, supporting evidences are mostly correlative and their role in this process is unclear. Here, we show that limb position is determined early in development through the action of Hox genes. Dynamic lineage analysis revealed that, during gastrulation, the forelimb, interlimb, and hindlimb fields are progressively generated and concomitantly patterned by the collinear activation of Hox genes in a two-step process. First, the sequential activation of Hoxb genes controls the relative position of their own collinear domains of expression in the forming lateral plate mesoderm, as demonstrated by functional perturbations during gastrulation. Then, within these collinear domains, we show that Hoxb4 anteriorly and Hox9 genes posteriorly, respectively, activate and repress the expression of the forelimb initiation gene Tbx5 and instruct the definitive position of the forelimb. Furthermore, by comparing the dynamics of Hoxb genes activation during zebra finch, chicken, and ostrich gastrulation, we provide evidences that changes in the timing of collinear Hox gene activation might underlie natural variation in forelimb position between different birds. Altogether, our results that characterize the cellular and molecular mechanisms underlying the regulation and natural variation of forelimb positioning in avians show a direct and early role for Hox genes in this process.

Cell Division Drives Epithelial Cell Rearrangements during Gastrulation in Chick.

During early embryonic development, cells are organized as cohesive epithelial sheets that are continuously growing and remodeled without losing their integrity, giving rise to a wide array of tissue shapes. Here, using live imaging in chick embryo, we investigate how epithelial cells rearrange during gastrulation. We find that cell division is a major rearrangement driver that powers dramatic epithelial cell intercalation events. We show that these cell division-mediated intercalations, which represent the majority of epithelial rearrangements within the early embryo, are absolutely necessary for the spatial patterning of gastrulation movements. Furthermore, we demonstrate that these intercalation events result from overall low cortical actomyosin accumulation within the epithelial cells of the embryo, which enables dividing cells to remodel junctions in their vicinity. These findings uncover a role for cell division as coordinator of epithelial growth and remodeling that might underlie various developmental, homeostatic, or pathological processes in amniotes.

Notch regulation of myogenic versus endothelial fates of cells that migrate from the somite to the limb.

Multipotent Pax3-positive (Pax3(+)) cells in the somites give rise to skeletal muscle and to cells of the vasculature. We had previously proposed that this cell-fate choice depends on the equilibrium between Pax3 and Foxc2 expression. In this study, we report that the Notch pathway promotes vascular versus skeletal muscle cell fates. Overactivating the Notch pathway specifically in Pax3(+) progenitors, via a conditional Pax3(NICD) allele, results in an increase of the number of smooth muscle and endothelial cells contributing to the aorta. At limb level, Pax3(+) cells in the somite give rise to skeletal muscles and to a subpopulation of endothelial cells in blood vessels of the limb. We now demonstrate that in addition to the inhibitory role of Notch signaling on skeletal muscle cell differentiation, the Notch pathway affects the Pax3:Foxc2 balance and promotes the endothelial versus myogenic cell fate, before migration to the limb, in multipotent Pax3(+) cells in the somite of the mouse embryo.

Six homeoproteins directly activate Myod expression in the gene regulatory networks that control early myogenesis.

In mammals, several genetic pathways have been characterized that govern engagement of multipotent embryonic progenitors into the myogenic program through the control of the key myogenic regulatory gene Myod. Here we demonstrate the involvement of Six homeoproteins. We first targeted into a Pax3 allele a sequence encoding a negative form of Six4 that binds DNA but cannot interact with essential Eya co-factors. The resulting embryos present hypoplasic skeletal muscles and impaired Myod activation in the trunk in the absence of Myf5/Mrf4. At the axial level, we further show that Myod is still expressed in compound Six1/Six4:Pax3 but not in Six1/Six4:Myf5 triple mutant embryos, demonstrating that Six1/4 participates in the Pax3-Myod genetic pathway. Myod expression and head myogenesis is preserved in Six1/Six4:Myf5 triple mutant embryos, illustrating that upstream regulators of Myod in different embryonic territories are distinct. We show that Myod regulatory regions are directly controlled by Six proteins and that, in the absence of Six1 and Six4, Six2 can compensate.

Itm2a is a Pax3 target gene, expressed at sites of skeletal muscle formation in vivo.

The paired-box homeodomain transcription factor Pax3 is a key regulator of the nervous system, neural crest and skeletal muscle development. Despite the important role of this transcription factor, very few direct target genes have been characterized. We show that Itm2a, which encodes a type 2 transmembrane protein, is a direct Pax3 target in vivo, by combining genetic approaches and in vivo chromatin immunoprecipitation assays. We have generated a conditional mutant allele for Itm2a, which is an imprinted gene, by flanking exons 2-4 with loxP sites and inserting an IRESnLacZ reporter in the 3' UTR of the gene. The LacZ reporter reproduces the expression profile of Itm2a, and allowed us to further characterize its expression at sites of myogenesis, in the dermomyotome and myotome of somites, and in limb buds, in the mouse embryo. We further show that Itm2a is not only expressed in adult muscle fibres but also in the satellite cells responsible for regeneration. Itm2a mutant mice are viable and fertile with no overt phenotype during skeletal muscle formation or regeneration. Potential compensatory mechanisms are discussed.

Lack of in vivo functional compensation between Pax family groups II and III in rodents.

Pax genes encode evolutionarily conserved transcription factors that play critical roles in embryonic development and organogenesis. Pax proteins are subdivided into four subfamilies: group I (Pax1and 9), II (Pax2, 5, and 8), III (Pax3 and 7), and IV (Pax4 and 6), based on the presence of a paired domain, an octapeptide motif and part or all of the homeodomain. Studies of the evolution of this gene family are incomplete. Nevertheless, it is known that each family evolved via duplication from four corresponding ancestral genes. Pax gene functions have been shown to be conserved within subgroups. It remains unclear, however, whether any (early) conserved function is shared between subgroups. To investigate conserved functions between subfamily II and III, we replaced an allele of Pax3 with a Pax8-coding sequence via gene targeting in the mouse. Homozygote Pax3(Pax8/Pax8) embryos display phenotypes indistinguishable from Pax3-deficient mutant embryos, with neural tube closure defects, a deficit in neural crest cells in the trunk, and skeletal muscle defects including absence of long-range migratory myogenic progenitors and impaired somite development. Interestingly, despite Pax8 expression in the neural tube in a domain ventral to that of Pax3, Pax8 cannot replace Pax3 function in the dorsal neural tube. Altogether, our results demonstrate that expression of Pax8 fails to compensate for Pax3 deficiency, demonstrating the absence of functional compensation between one subfamily of Pax genes and another in the mouse embryo. Our result suggests that Pax3/7 and Pax2/5/8 functions evolved independently after duplication of the ancestral progenitor Pax genes.

A Pax3/Dmrt2/Myf5 regulatory cascade functions at the onset of myogenesis.

All skeletal muscle progenitor cells in the body derive from the dermomyotome, the dorsal epithelial domain of developing somites. These multipotent stem cells express Pax3, and this expression is maintained in the myogenic lineage where Pax3 plays an important role. Identification of Pax3 targets is therefore important for understanding the mechanisms that underlie the onset of myogenesis. In a microarray screen of Pax3-GFP sorted cells, with analysis on Pax3 gain and loss of function genetic backgrounds, we identify Dmrt2, expressed in the dermomyotome, as a Pax3 target. In vitro gel shift analysis and chromatin immunoprecipitation with in vivo extracts show that Pax3 binds to a conserved 286 bp sequence, situated at -18 kb from Dmrt2. This sequence directs reporter transgene expression to the somite, and this is severely affected when the Pax3 site is mutated in the context of the locus. In Dmrt2 mutant embryos, somite maturation is perturbed and the skeletal muscle of the myotome is abnormal. We now report that the onset of myogenesis is also affected. This depends on activation, in the epaxial dermomyotome, of the myogenic determination gene, Myf5, through its early epaxial enhancer. This sequence contains sites that bind Dmrt2, which belongs to the DM class of DNA-binding proteins. Mutation of these sites compromises activity of the enhancer in transgenic embryos where the reporter transgene is under the control of the Myf5 epaxial enhancer. Transactivation of this site by Dmrt2 is demonstrated in vitro, and conditional overexpression of Dmrt2 in Pax3 expressing cells in the somite confirms the role of this factor in the activation of Myf5. These results reveal a novel genetic network, comprising a Pax3/Dmrt2/Myf5 regulatory cascade that operates in stem cells of the epaxial dermomyotome to initiate skeletal muscle formation.

Muscle stem cell behavior is modified by microRNA-27 regulation of Pax3 expression.

Skeletal muscle stem cells are regulated by Pax3/7. During development, Pax3 is required for the maintenance of these cells in the somite and their migration to sites of myogenesis; high levels of Pax3 interfere with muscle cell differentiation, both in the embryo and in the adult. Quantitative fine-tuning of Pax3 is critical, and microRNAs provide a potential mechanism. We identify microRNA-27b (miR-27b), which directly targets the 3'-UTR of Pax3 mRNA, as such a regulator. miR-27b is expressed in the differentiating skeletal muscle of the embryonic myotome and in activated satellite cells of adult muscle. In vivo overexpression of a miR-27b transgene in Pax3-positive cells in the embryo leads to down-regulation of Pax3, resulting in interference with progenitor cell migration and in premature differentiation. In a complementary experiment, miR-27b inhibitors were transfected into cultures of adult muscle satellite cells that normally express miR-27b at the onset of differentiation, when Pax3 protein levels undergo rapid down-regulation. Interference with miR-27b function results in continuing Pax3 expression leading to more proliferation and a delay in the onset of differentiation. Pax7 levels are not affected. Introduction of miR-27b antagomirs at a site of muscle injury in vivo also affects Pax3 expression and regeneration in vivo. We therefore conclude that miR-27b regulates Pax3 protein levels and this down-regulation ensures rapid and robust entry into the myogenic differentiation program.

The regulatory mechanisms that underlie inappropriate transcription of the myogenic determination gene Myf5 in the central nervous system.

Myf5 is a key myogenic determination factor, specifically present at sites of myogenesis. Surprisingly, during mouse development, this gene is also transcribed in restricted areas of the central nervous system, although the Myf5 protein is not detectable. We have investigated the regulation of Myf5 expression in the central nervous system. Using both in ovo electroporation in the chick embryo and transgenesis in the mouse, we show that regulatory sequences that direct neuronal Myf5 transcription are present in a distal element located between -55 and -54.3 Kb from the Myf5 gene. An Oct6/Tst1 binding site is required for embryonic brain expression, and in the Oct6 mutant mouse embryo, Myf5 transcripts are no longer detectable in the brain. The Wnt-beta catenin signalling pathway is also implicated. Finally we show that post-transcriptional regulation of Myf5 gene expression involves miRNA repression acting through the Myf5-3'UTR.

Pax3 regulation of FGF signaling affects the progression of embryonic progenitor cells into the myogenic program.

Pax3/7-dependent stem cells play an essential role in skeletal muscle development. We now show that 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.

Myogenic progenitor cells in the mouse embryo are marked by the expression of Pax3/7 genes that regulate their survival and myogenic potential.

The transcription factors Pax3 and Pax7 are important regulators of myogenic cell fate, as demonstrated by genetic manipulations in the mouse embryo. Pax3 lies genetically upstream of MyoD and has also been shown recently to directly control Myf5 transcription in derivatives of the hypaxial somite, where it also plays an important role in ensuring cell survival. Both Pax3 and Pax7 are expressed in myogenic progenitor cells derived from the central dermomyotome that make a major contribution to skeletal muscle growth. In Pax3/Pax7 double mutants, the myogenic determination genes, Myf5 and MyoD, are not activated in these cells which become incorporated into other tissues or die. This again demonstrates the dual function of Pax factors in regulating the entry of progenitor cells into the myogenic programme and in ensuring their survival. Pax3 expression marks cells in the dermomyotome that either become myogenic or downregulate Pax3 and assume another cell fate. The latter include the smooth muscle cells of the dorsal aorta that share a common clonal origin with the skeletal muscle of the myotome, thus illustrating the initial multipotency of Pax3 expressing cells.

A novel genetic hierarchy functions during hypaxial myogenesis: Pax3 directly activates Myf5 in muscle progenitor cells in the limb.

We address the molecular control of myogenesis in progenitor cells derived from the hypaxial somite. Null mutations in Pax3, a key regulator of skeletal muscle formation, lead to cell death in this domain. We have developed a novel allele of Pax3 encoding a Pax3-engrailed fusion protein that acts as a transcriptional repressor. Heterozygote mouse embryos have an attenuated mutant phenotype, with partial conservation of the hypaxial somite and its myogenic derivatives, including some hindlimb muscles. At these sites, expression of Myf5 is compromised, showing that Pax3 acts genetically upstream of this myogenic determination gene. We have characterized a 145-base-pair (bp) regulatory element, at -57.5 kb from Myf5, that directs transgene expression to the mature somite, notably to myogenic cells of the hypaxial domain that form ventral trunk and limb muscles. A Pax3 consensus site in this sequence binds Pax3 in vitro and in vivo. Multimers of the 145-bp sequence direct transgene expression to sites of Pax3 function, and an assay of its activity in the chick embryo shows Pax3 dependence. Mutation of the Pax3 site abolishes all expression controlled by the 145-bp sequence in transgenic mouse embryos. We conclude that Pax3 directly regulates Myf5 in the hypaxial somite and its derivatives.

Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells.

The growth and repair of skeletal muscle after birth depends on satellite cells that are characterized by the expression of Pax7. We show that Pax3, the paralogue of Pax7, is also present in both quiescent and activated satellite cells in many skeletal muscles. Dominant-negative forms of both Pax3 and -7 repress MyoD, but do not interfere with the expression of the other myogenic determination factor, Myf5, which, together with Pax3/7, regulates the myogenic differentiation of these cells. In Pax7 mutants, satellite cells are progressively lost in both Pax3-expressing and -nonexpressing muscles. We show that this is caused by satellite cell death, with effects on the cell cycle. Manipulation of the dominant-negative forms of these factors in satellite cell cultures demonstrates that Pax3 cannot replace the antiapoptotic function of Pax7. These findings underline the importance of cell survival in controlling the stem cell populations of adult tissues and demonstrate a role for upstream factors in this context.

A Pax3/Pax7-dependent population of skeletal muscle progenitor cells.

During vertebrate development, successive phases of embryonic and fetal myogenesis lead to the formation and growth of skeletal muscles. Although the origin and molecular regulation of the earliest embryonic muscle cells is well understood, less is known about later stages of myogenesis. We have identified a new cell population that expresses the transcription factors Pax3 and Pax7 (paired box proteins 3 and 7) but no skeletal-muscle-specific markers. These cells are maintained as a proliferating population in embryonic and fetal muscles of the trunk and limbs throughout development. Using a stable green fluorescent protein (GFP) reporter targeted to Pax3, we demonstrate that they constitute resident muscle progenitor cells that subsequently become myogenic and form skeletal muscle. Late in fetal development, these cells adopt a satellite cell position characteristic of progenitor cells in postnatal muscle. In the absence of both Pax3 and Pax7, further muscle development is arrested and only the early embryonic muscle of the myotome forms. Cells failing to express Pax3 or Pax7 die or assume a non-myogenic fate. We conclude that this resident Pax3/Pax7-dependent progenitor cell population constitutes a source of myogenic cells of prime importance for skeletal muscle formation, a finding also of potential value in the context of cell therapy for muscle disease.

Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice.

In vertebrates, skeletal muscle is a model for the acquisition of cell fate from stem cells. Two determination factors of the basic helix-loop-helix myogenic regulatory factor (MRF) family, Myf5 and Myod, are thought to direct this transition because double-mutant mice totally lack skeletal muscle fibres and myoblasts. In the absence of these factors, progenitor cells remain multipotent and can change their fate. Gene targeting studies have revealed hierarchical relationships between these and the other MRF genes, Mrf4 and myogenin, where the latter are regarded as differentiation genes. Here we show, using an allelic series of three Myf5 mutants that differentially affect the expression of the genetically linked Mrf4 gene, that skeletal muscle is present in the new Myf5:Myod double-null mice only when Mrf4 expression is not compromised. This finding contradicts the widely held view that myogenic identity is conferred solely by Myf5 and Myod, and identifies Mrf4 as a determination gene. We revise the epistatic relationship of the MRFs, in which both Myf5 and Mrf4 act upstream of Myod to direct embryonic multipotent cells into the myogenic lineage.

Divergent functions of murine Pax3 and Pax7 in limb muscle development.

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, we replaced Pax3 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. We show that 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. We conclude 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.