Anne-Gaëlle Borycki

Differentiation of Avian Craniofacial Muscles: I. Patterns of Early Regulatory Gene Expression and Myosin Heavy Chain Synthesis

Myogenic populations of the avian head arise within both epithelial (somitic) and mesenchymal (unsegmented) mesodermal populations. The former, which gives rise to neck, tongue, laryngeal, and diaphragmatic muscles, show many similarities to trunk axial, body wall, and appendicular muscles. However, muscle progenitors originating within unsegmented head mesoderm exhibit several distinct features, including multiple ancestries, the absence of several somite lineage‐determining regulatory gene products, diverse locations relative to neuraxial and pharyngeal tissues, and a prolonged and necessary interaction with neural crest cells. The object of this study has been to characterize the spatial and temporal patterns of early muscle regulatory gene expression and subsequent myosin heavy chain isoform appearance in avian mesenchyme‐derived extraocular and branchial muscles, and compare these with expression patterns in myotome‐derived neck and tongue muscles. Myf5 and myoD transcripts are detected in the dorsomedial (epaxial) region of the occipital somites before stage 12, but are not evident in the ventrolateral domain until stage 14. Within unsegmented head mesoderm, myf5 expression begins at stage 13.5 in the second branchial arch, followed within a few hours in the lateral rectus and first branchial arch myoblasts, then other eye and branchial arch muscles. Expression of myoD is detected initially in the first branchial arch beginning at stage 14.5, followed quickly by its appearance in other arches and eye muscles. Multiple foci of myoblasts expressing these transcripts are evident during the early stages of myogenesis in the first and third branchial arches and the lateral rectus‐pyramidalis/quadratus complex, suggesting an early patterned segregation of muscle precursors within head mesoderm. Myf5‐positive myoblasts forming the hypoglossal cord emerge from the lateral borders of somites 4 and 5 by stage 15 and move ventrally as a cohort. Myosin heavy chain (MyHC) is first immunologically detectable in several eye and branchial arch myofibers between stages 21 and 22, although many tongue and laryngeal muscles do not initiate myosin production until stage 24 or later. Detectable synthesis of the MyHC‐S3 isoform, which characterizes myofibers as having “slow” contraction properties, occurs within 1–2 stages of the onset of MyHC synthesis in most head muscles, with tongue and laryngeal muscles being substantially delayed. Such a prolonged, 2‐ to 3‐day period of regulatory gene expression preceding the onset of myosin production contrasts with the interval seen in muscles developing in axial (approximately 18 hr) and wing (approximately 1–1.5 days) locations, and is unique to head muscles. This finding suggests that ongoing interactions between head myoblasts and their surroundings, most likely neural crest cells, delay myoblast withdrawal from the mitotic pool. These descriptions define a spatiotemporal pattern of muscle regulatory gene and myosin heavy chain expression unique to head muscles. This pattern is independent of origin (somitic vs. unsegmented paraxial vs. prechordal mesoderm), position (extraocular vs. branchial vs. subpharyngeal), and fiber type (fast vs. slow) and is shared among all muscles whose precursors interact with cephalic neural crest populations. Dev Dyn 1999;216:96–112. ©1999 Wiley‐Liss, Inc.

Gli2 and Gli3 have redundant and context-dependent function in skeletal muscle formation.

The Gli family of zinc finger transcription factors are mediators of Shh signalling in vertebrates. In previous studies, we showed that Shh signalling, via an essential Gli -binding site in the Myf5 epaxial somite (ES) enhancer, is required for the specification of epaxial muscle progenitor cells. Shh signalling is also required for the normal mediolateral patterning of myogenic cells within the somite. In this study, we investigate the role and the transcriptional activities of Gli proteins during somite myogenesis in the mouse embryo. We report that Gli genes are differentially expressed in the mouse somite. Gli2 and Gli3 are essential for Gli1 expression in somites, establishing Gli2 and Gli3 as primary mediators and Gli1 as a secondary mediator of Shh signalling. Combining genetic studies with the use of a transgenic mouse line expressing a reporter gene under the control of the Myf5 epaxial somite enhancer, we show that Gli2 or Gli3 is required for Myf5 activation in the epaxial muscle progenitor cells. Furthermore, Gli3, but not Gli2 represses Myf5 transcription in a dose-dependent manner in the absence of Shh. Finally, we provide evidence that hypaxial and myotomal gene expression is mispatterned in Gli2–/–Gli3–/– and Gli3–/–Shh–/– somites. Together, our data demonstrate both positive and negative regulatory functions for Gli2 and Gli3 in the control of Myf5 activation in the epaxial muscle progenitor cells and in dorsoventral and mediolateral patterning of the somite.

5 Multiple Tissue Interactions and Signal Transduction Pathways Control Somite Myogenesis

Publisher Summary This chapter reviews embryological and molecular studies that have established a central role for developmental signaling mechanisms in the establishment of myogenic cells in the somites of vertebrate embryos, and current models of embryonic signaling are also evaluated in the control of skeletal myogenic determination. In the context of the avian and mammalian studies, the chapter also discusses recent genetic studies of myogenesis in the zebrafish and embryological studies in Xenopus. Current evidence provides a general view that somite myogenesis is controlled by complex tissue interactions that expose somitic cells to multiple, independently functioning, and partially redundant signals that promote and inhibit myogenesis. These complex tissue interactions and signal transduction processes apparently have a common genetic outcome, which is the activation of myogenic regulatory factor (MRF) genes that commit somitic cells to a myogenic fate. This complexity of interactions and signaling processes, therefore, can be viewed as an adaptive mechanism to exploit the diversity of signaling mechanisms available throughout the embryo to activate a common MRF genetic regulatory pathway for muscle differentiation at widely dispersed anatomical sites in the vertebrate body.

Muscle determination: Another key player in myogenesis?

The steps that commit multipotential somite cells to muscle differentiation are being elucidated. Recent results show that pax3 is an upstream regulator of myoD, one of the key genes in myogenic lineage determination.

FatJ acts via the Hippo mediator Yap1 to restrict the size of neural progenitor cell pools

The size, composition and functioning of the spinal cord is likely to depend on appropriate numbers of progenitor and differentiated cells of a particular class, but little is known about how cell numbers are controlled in specific cell cohorts along the dorsoventral axis of the neural tube. Here, we show that FatJ cadherin, identified in a large-scale RNA interference (RNAi) screen of cadherin genes expressed in the neural tube, is localised to progenitors in intermediate regions of the neural tube. Loss of function of FatJ promotes an increase in dp4-vp1 progenitors and a concomitant increase in differentiated Lim1+/Lim2+ neurons. Our studies reveal that FatJ mediates its action via the Hippo pathway mediator Yap1: loss of downstream Hippo components can rescue the defect caused by loss of FatJ. Together, our data demonstrate that RNAi screens are feasible in the chick embryonic neural tube, and show that FatJ acts through the Hippo pathway to regulate cell numbers in specific subsets of neural progenitor pools and their differentiated progeny.

Sonic hedgehog acts cell-autonomously on muscle precursor cells to generate limb muscle diversity

How muscle diversity is generated in the vertebrate body is poorly understood. In the limb, dorsal and ventral muscle masses constitute the first myogenic diversification, as each gives rise to distinct muscles. Myogenesis initiates after muscle precursor cells (MPCs) have migrated from the somites to the limb bud and populated the prospective muscle masses. Here, we show that Sonic hedgehog (Shh) from the zone of polarizing activity (ZPA) drives myogenesis specifically within the ventral muscle mass. Shh directly induces ventral MPCs to initiate Myf5 transcription and myogenesis through essential Gli-binding sites located in the Myf5 limb enhancer. In the absence of Shh signaling, myogenesis is delayed, MPCs fail to migrate distally, and ventral paw muscles fail to form. Thus, Shh production in the limb ZPA is essential for the spatiotemporal control of myogenesis and coordinates muscle and skeletal development by acting directly to regulate the formation of specific ventral muscles.

Sonic hedgehog-dependent synthesis of laminin α1 controls basement membrane assembly in the myotome

Basement membranes have essential structural and signalling roles in tissue morphogenesis during embryonic development, but the mechanisms that control their formation are still poorly understood. Laminins are key components of basement membranes and are thought to be essential for initiation of basement membrane assembly. Here, we report that muscle progenitor cells populating the myotome migrate aberrantly in the ventral somite in the absence of sonic hedgehog (Shh) signalling, and we show that this defect is due to the failure to form a myotomal basement membrane. We reveal that expression of Lama1, which encodes laminin α1, a subunit of laminin-111, is not activated in Shh-/- embryos. Recovery of Lama1 expression or addition of exogenous laminin-111 to Shh-/-;Gli3-/- embryos restores the myotomal basement membrane, demonstrating that laminin-111 is necessary and sufficient to initiate assembly of the myotomal basement membrane. This study uncovers an essential role for Shh signalling in the control of laminin-111 synthesis and in the initiation of basement membrane assembly in the myotome. Furthermore, our data indicate that laminin-111 function cannot be compensated by laminin-511.

Dystroglycan Protein Distribution Coincides With Basement Membranes and Muscle Differentiation During Mouse Embryogenesis

Using immunohistochemistry, we have examined β‐Dystroglycan protein distribution in the mouse embryo at embryonic stages E9.5 to E11.5. Our data show that Dystroglycan expression correlates with basement membranes in many tissues, such as the notochord, neural tube, promesonephros, and myotome. In the myotome, we describe the timing of Dystroglycan protein re‐distribution at the surface of myogenic precursor cells as basement membrane assembles into a continuous sheet. We also report on non‐basement‐membrane‐associated Dystroglycan expression in the floor plate and the myocardium. This distribution often corresponds to sites of expression previously reported in adults, suggesting that Dystroglycan is continuously produced during development. Developmental Dynamics 236:2627–2635, 2007.

Expression of avian C-terminal binding proteins (Ctbp1 and Ctbp2) during embryonic development

C‐terminal binding proteins (CtBPs) are transcriptional corepressors of mediators of Notch, Wnt, and other signalling pathways. Thus, they are potential players in the control of several developmentally important processes, including segmentation, somitogenesis, and neural tube and limb patterning. We have cloned the avian orthologues of Ctbp1 and Ctbp2 and examined their expression pattern by whole‐mount in situ hybridization between Hamburger and Hamilton (HH) stages 3 and 24. Both Ctbp genes show similar expression patterns during embryonic development, and both are detected from HH stage 3 in the developing central nervous system, by HH stage 7 in the paraxial mesoderm and later in the limb bud. In most places, Ctbp1 and Ctbp2 are expressed in overlapping domains. However, there are interesting domains and/or temporal expression patterns that are specific to each Ctbp gene. For instance, Ctbp1 is predominantly expressed in the epiblast, whereas Ctbp2 is in the primitive streak at HH stage 3. However, by HH stage 4, both genes are found in the primitive streak and in the ectoderm. Similarly, although both genes display similar expression patterns in early somitogenesis, in mature somites, Ctbp1 transcripts are located in myotomal cells, whereas Ctbp2 transcripts are observed in dermomyotomal cells. Finally, we found that emigrating neural crest cells express Ctbp2, whereas dorsal root ganglia express Ctbp1. These data suggest that Ctbp1 and Ctbp2 may be functionally redundant in some tissues and have unique functions in other tissues. Developmental Dynamics 235:490–495, 2005.

Isolation and characterization of a cDNA clone encoding for rat CSF-1 gene. Post-transcriptional repression occurs in myogenic differentiation.

A major CSF-1 (Colony-Stimulating Factor 1) mRNA 4.0 kb long was expressed during the proliferation of the L6 alpha 1 rat myogenic cells and was down-regulated after their differentiation into myotubes. A complete cDNA encoding the rat CSF-1 gene (rmCSF-1) was isolated from a cDNA library of L6 alpha 1 myoblasts and sequenced. The overall deduced amino acid sequence was 100% and 68% identical to the mouse and human CSF-1, respectively. While the previously reported mechanisms about the regulation of CSF-1 expression in TPA-treated-monocytes (Horiguchi, J., Sariban, E. and Kufe, D. (1988) Mol. Cell. Biol. 8, 3951-3954) and in fibroblasts (Falkenburg, J.H.F., Harrington, M.A., De Paus, R.A., Walsh, M.K., Daub, R., Landegent, J.E. and Broxmeyer, H.E. (1991) Blood 78, 658-665) involved a control at the transcriptional level, in contrast, the CSF-1 mRNA (half-life approximately 3 h in L6 alpha 1 myoblasts) was post-transcriptionally down-regulated during myogenesis. Inhibition of protein synthesis with cycloheximide (CHX) increased differentially the half-life of CSF-1 mRNA in L6 alpha 1 myotubes compared to L6 alpha 1 myoblasts. Finally, L6 alpha 1 myoblasts were shown to synthesize a 140 kDa homodimeric form of CSF-1. Thus, these findings, together with other results, indicate that CSF-1 gene products may play a role in the normal and neoplastic proliferation of muscular cells.