Regulated expression of FLRT genes implies a functional role in the regulation of FGF signalling during mouse development

Within the mammalian genome, there are many multimember gene families that encode membrane proteins with extracellular leucine rich repeats which are thought to act as cell adhesion or signalling molecules. We previously showed that the members of the NLRR gene family are expressed in a developmentally restricted manner in the mouse with NLRR-1 being expressed in the developing myotome. The FLRT gene family shows a similar genomic layout and predicted protein secondary structure to the NLRRs so we analysed expression of the three FLRT genes during mouse development. FLRTs are glycosylated membrane proteins expressed at the cell surface which localise in a homophilic manner to cell-cell contacts expressing the focal adhesion marker vinculin. Each member of the FLRT family has a distinct, highly regulated expression pattern, as was seen for the NLRR family. FLRT3 has a provocative expression pattern during somite development being expressed in regions of the somite where muscle precursor cells migrate from the dermomyotome and move into the myotome, and later in myotomal precursors destined to migrate towards their final destination, for example, those that form the ventral body wall. FLRT3 is also expressed at the midbrain/hindbrain boundary and in the apical ectodermal ridge, regions where FGF signalling is known to be important, suggesting that the role for FLRT3 in FGF signalling identified in Xenopus is conserved in mammals. FLRT1 is expressed at brain compartmental boundaries and FLRT2 is expressed in a subset of the sclerotome, adjacent to the region that forms the syndetome, suggesting that interaction with FGF signalling may be a general property of FLRT proteins. We confirmed this by showing that all FLRTs can interact with FGFR1 and FLRTs can be induced by the activation of FGF signalling by FGF-2. We conclude that FLRT proteins act as regulators of FGF signalling, being induced by the signal and then able to interact with the signalling receptor, in many tissues during mouse embryogenesis. This process may, in part, be dependent on homophilic intercellular interactions between FLRT molecules.     

Expression of the Lingo/LERN gene family during mouse embryogenesis

We have analysed the expression during mouse development of the four member Lingo/LERN gene family which encodes type 1 transmembrane proteins containing 12 extracellular leucine rich repeats, an immunoglobulin C2 domain and a short intracellular tail. Each family member has a distinct pattern of expression in the mouse embryo as is the case for the related NLRR, FLRT and LRRTM gene families. Lingo1/LERN1 is expressed in the developing trigeminal, facio-acoustic and dorsal root ganglia. An interesting expression pattern is also observed in the somites with expression localising to the inner surface of the dermomyotome in the ventro-caudal lip. Further expression is seen in lateral cells of the hindbrain and midbrain, lateral cells in the motor horn of the neural tube, the otic vesicle epithelium and epithelium associated with the developing gut. Lingo3/LERN2 is expressed in a broad but specific pattern in many tissues across the embryo. Lingo2/LERN3 is seen in a population of cells lying adjacent to the epithelial lining of the olfactory pit while Lingo4/LERN4 is expressed in the neural tube in a subset of progenitors adjacent to the motor neurons. Expression of all Lingo/LERN genes increases as the embryo develops but is low in the adult with only Lingo1/LERN1 and Lingo2/LERN3 being detectable in adult brain.     

Mox-1 and Mox-2 define a novel homeobox gene subfamily and are differentially expressed during early mesodermal patterning in mouse embryos.

We have isolated two mouse genes, Mox-1 and Mox-2 that, by sequence, genomic structure and expression pattern, define a novel homeobox gene family probably involved in mesodermal regionalization and somitic differentiation. Mox-1 is genetically linked to the keratin and Hox-2 genes of chromosome 11, while Mox-2 maps to chromosome 12. At primitive streak stages (approximately 7.0 days post coitum), Mox-1 is expressed in mesoderm lying posterior of the future primordial head and heart. It is not expressed in neural tissue, ectoderm, or endoderm. Mox-1 expression may therefore define an extensive 'posterior' domain of embryonic mesoderm before, or at the earliest stages of, patterning of the mesoderm and neuroectoderm by the Hox cluster genes. Between 7.5 and 9.5 days post coitum, Mox-1 is expressed in presomitic mesoderm, epithelial and differentiating somites (dermatome, myotome and sclerotome) and in lateral plate mesoderm. In the body of midgestation embryos, Mox-1 signal is restricted to loose undifferentiated mesenchyme. Mox-1 signal is also prominent over the mesenchyme of the heart cushions and truncus arteriosus, which arises from epithelial-mesenchymal transformation and over a limited number of craniofacial foci of neural crest-derived mesenchyme that are associated with muscle attachment sites. The expression profile of Mox-2 is similar to, but different from, that of Mox-1. For example, Mox-2 is apparently not expressed before somites form, is then expressed over the entire epithelial somite, but during somitic differentiation, Mox-2 signal rapidly becomes restricted to sclerotomal derivatives. The expression patterns of these genes suggest regulatory roles for Mox-1 and Mox-2 in the initial anterior-posterior regionalization of vertebrate embryonic mesoderm and, in addition, in somite specification and differentiation.     

Ventral axial organs regulate expression of myotomal Fgf-8 that influences rib development

Fgf-8 encodes a secreted signaling molecule mediating key roles in embryonic patterning. This study analyzes the expression pattern, regulation, and function of this growth factor in the paraxial mesoderm of the avian embryo. In the mature somite, expression of Fgf-8 is restricted to a subpopulation of myotome cells, comprising most, but not all, epaxial and hypaxial muscle precursors. Following ablation of the notochord and floor plate, Fgf-8 expression is not activated in the somites, in either the epaxial or the hypaxial domain, while ablation of the dorsal neural tube does not affect Fgf-8 expression in paraxial mesoderm. Contrary to the view that hypaxial muscle precursors are independent of regulatory influences from axial structures, these findings provide the first evidence for a regulatory influence of ventral, but not dorsal axial structures on the hypaxial muscle domain. Sonic hedgehog can substitute for the ventral neural tube and notochord in the initiation of Fgf-8 expression in the myotome. It is also shown that Fgf-8 protein leads to an increase in sclerotomal cell proliferation and enhances rib cartilage development in mature somites, whereas inhibition of Fgf signaling by SU 5402 causes deletions in developing ribs. These observations demonstrate: (1) a regulatory influence of the ventral axial organs on the hypaxial muscle compartment; (2) regulation of epaxial and hypaxial expression of Fgf-8 by Sonic hedgehog; and (3) independent regulation of Fgf-8 and MyoD in the hypaxial myotome by ventral axial organs. It is postulated that the notochord and ventral neural tube influence hypaxial expression of Fgf-8 in the myotome and that, in turn, Fgf-8 has a functional role in rib formation.     

Pax3 functions in cell survival and in pax7 regulation

In developing vertebrate embryos, Pax3 is expressed in the neural tube and in the paraxial mesoderm that gives rise to skeletal muscles. Pax3 mutants develop muscular and neural tube defects; furthermore, Pax3 is essential for the proper activation of the myogenic determination factor gene, MyoD, during early muscle development and PAX3 chromosomal translocations result in muscle tumors, providing evidence that Pax3 has diverse functions in myogenesis. To investigate the specific functions of Pax3 in development, we have examined cell survival and gene expression in presomitic mesoderm, somites and neural tube of developing wild-type and Pax3 mutant (Splotch) mouse embryos. Disruption of Pax3 expression by antisense oligonucleotides significantly impairs MyoD activation by signals from neural tube/notochord and surface ectoderm in cultured presomitic mesoderm (PSM), and is accompanied by a marked increase in programmed cell death. In Pax3 mutant (Splotch) embryos, MyoD is activated normally in the hypaxial somite, but MyoD-expressing cells are disorganized and apoptosis is prevalent in newly formed somites, but not in the neural tube or mature somites. In neural tube and somite regions where cell survival is maintained, the closely related Pax7 gene is upregulated, and its expression becomes expanded into the dorsal neural tube and somites, where Pax3 would normally be expressed. These results establish that Pax3 has complementary functions in MyoD activation and inhibition of apoptosis in the somitic mesoderm and in repression of Pax7 during neural tube and somite development.     

Developmentally regulated expression of the LRRTM gene family during mid-gestation mouse embryogenesis

We have analysed the expression during mid-gestation mouse development of the four member LRRTM gene family which encodes type 1 transmembrane proteins containing 10 extracellular leucine rich repeats and a short intracellular tail. Each family member has a developmentally regulated pattern of expression distinct from all other members. LRRTM1 is expressed in the neural tube, otic vesicle, apical ectodermal ridge, forebrain and midbrain up to a sharp central boundary. LRRTM2 is expressed in a subset of progenitors in the neural tube. LRRTM3 is expressed in a half somite wide stripe in the presomitic mesoderm adjacent to the boundary with the most recently formed somite. Additional expression is seen in the neural tube, forebrain and hindbrain. LRRTM4 is expressed in the limb mesenchyme, neural tube, caudal mesoderm and in three distinct regions of the head. Later expression occurs in a subset of the developing sclerotome. Each family member has a unique expression domain within the neural tube.     

Clonal separation and regionalisation during formation of the medial and lateral myotomes in the mouse embryo.

In vertebrates, muscles of the back (epaxial) and of the body wall and limbs (hypaxial) derive from precursor cells located in the dermomyotome of the somites. In this paper, we investigate the mediolateral regionalisation of epaxial and hypaxial muscle precursor cells during segmentation of the paraxial mesoderm and myotome formation, using mouse LaacZ/LacZ chimeras. We demonstrate that precursors of medial and lateral myotomes are clonally separated in the mouse somite, consistent with earlier studies in birds. This clonal separation occurs after segmentation of the paraxial mesoderm. We then show that myotome precursors are mediolaterally regionalised and that this regionalisation precedes clonal separation between medial and lateral precursors. Strikingly, the properties of myotome precursors are remarkably similar in the medial and lateral domains. Finally, detailed analysis of our clones demonstrates a direct spatial relationship between the myocytes in the myotome and their precursors in the dermomyotome, and earlier in the somite and presomitic mesoderm, refuting several models of myotome formation, based on permanent stem cell systems or extensive cell mingling. This progressive mediolateral regionalisation of the myotome at the cellular level correlates with progressive changes in gene expression in the dermomyotome and myotome.     

Somite subdomains, muscle cell origins, and the four muscle regulatory factor proteins

We show by immunohistology that distinct expression patterns of the four muscle regulatory factor (MRF) proteins identify subdomains of mouse somites. Myf-5 and MyoD are, at specific stages, each expressed in both myotome and dermatome cells. Myf-5 expression is initially restricted to dorsal cells in all somites, as is MyoD expression in neck somites. In trunk somites, however, MyoD is initially expressed in ventral cells. Myogenin and MRF4 are restricted to myotome cells, though the MRF4-expressing cells are initially less widely distributed than the myogenin-expressing cells, which are at all stages found throughout the myotome. All somitic myocytes express one or more MRFs. The transiently distinct expression patterns of the four MRF proteins identify dorsal and ventral subdomains of somites, and suggest that skeletal muscle cells in somites originate at multiple sites and via multiple molecular pathways.     

Alternatively spliced variants of protocadherin 8 exhibit distinct patterns of expression during mouse development

Protocadherins, a subgroup of the cadherin superfamily of calcium-dependent cell adhesion molecules, are considered to play important roles in the developing embryo particularly in the central nervous system. The Protocadherin 8 (Pcdh8) gene comprises three coding exons in both human and mouse, and the exon junctions are precisely conserved between these two species. Alternative splicing of Pcdh8 RNA leads to the formation of two isoforms that differ in the length of the cytoplasmic domains. We have investigated the expression of these short and long variants of Pcdh8 during early mouse development by RT/PCR and in situ hybridization. We found that both isoforms were predominantly expressed in the nervous system, and that their expression patterns appeared to be developmentally regulated. However, the short variant had a broader pattern of expression than the long variant and was found in some non-neuronal tissues, such as paraxial mesoderm, developing somites, and in limb interdigital mesenchyme where massive programmed cell death occurs. The differential expression of two alternative cytoplasmic domain variants suggests that Pcdh8 may regulate cell adhesion in a variety of developmental processes, and that this may involve different intracellular interactions.