Benoît Robert

Insertional mutation of the mouse Msx1 homeobox gene by an nlacZ reporter gene

We have generated a null allele of the mouse Msx1 homeobox gene by insertion of an nlacZ reporter gene into its homeobox. The sensitivity of beta-galactosidase detection permitted us to reveal novel aspects of Msx1 gene expression in heterozygous embryos, in particular in ectoderm and mesoderm during gastrulation, and in migrating neural crest cells. Homozygous mutant mice die at birth with facial defects (see Satokata, I. and Maas, R. (1994) Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development. Nat. Genet. 6, 348-356). To investigate the reason for this limited phenotype, we compared the pattern of Msx1 expression with that of the closely related Msx2 gene in wild type embryos and in Msx1-/- mutants. Notably, whereas the expression of Msx1 and Msx2 overlap in the developing limb, this is not the case in the facial regions most affected in the mutant.

Analysis of Msx1; Msx2 double mutants reveals multiple roles for Msx genes in limb development

The homeobox-containing genes Msx1 and Msx2 are highly expressed in the limb field from the earliest stages of limb formation and, subsequently, in both the apical ectodermal ridge and underlying mesenchyme. However, mice homozygous for a null mutation in either Msx1 or Msx2 do not display abnormalities in limb development. By contrast, Msx1; Msx2 double mutants exhibit a severe limb phenotype. Our analysis indicates that these genes play a role in crucial processes during limb morphogenesis along all three axes. Double mutant limbs are shorter and lack anterior skeletal elements (radius/tibia, thumb/hallux). Gene expression analysis confirms that there is no formation of regions with anterior identity. This correlates with the absence of dorsoventral boundary specification in the anterior ectoderm, which precludes apical ectodermal ridge formation anteriorly. As a result, anterior mesenchyme is not maintained, leading to oligodactyly. Paradoxically, polydactyly is also frequent and appears to be associated with extended Fgf activity in the apical ectodermal ridge, which is maintained up to 14.5 dpc. This results in a major outgrowth of the mesenchyme anteriorly, which nevertheless maintains a posterior identity, and leads to formation of extra digits. These defects are interpreted in the context of an impairment of Bmp signalling.

The mouse homeobox gene, S8, is expressed during embryogenesis predominantly in mesenchyme

The murine S8 gene, originally identified by Kongsuwan et al. [EMBO J. 7(1988)2131-2138] encodes a homeodomain which resembles those of the paired family. We studied the expression pattern during mid-gestation embryogenesis of S8 by in situ hybridization. Expression was detected locally in craniofacial mesenchyme, in the limb, the heart and the somites and sclerotomes all along the axis, and was absent from the central and peripheral nervous system, splanchnopleure, and endodermal derivatives. This pattern differs considerably from that of most previously described homeobox containing genes. By genetic analysis, the gene was located on chromosome 2, about 20 cM from the HOX-4 cluster.

Msx1 is required for dorsal diencephalon patterning

The dorsal midline of the neural tube has recently emerged as a major signaling center for dorsoventral patterning. Msx genes are expressed at the dorsal midline, although their function at this site remains unknown. Using Msx1nlacZ mutant mice, we show that the normal expression domain of Msx1 is interrupted in the pretectum of mutant embryos. Morphological and gene expression data further indicate that a functional midline is not maintained along the whole prosomere 1 in Msx1 mutant mice. This results in the downregulation of genes expressed laterally to the midline in prosomere 1, confirming the importance of the midline as a signaling center. Wnt1 is essential for dorsoventral patterning of the neural tube. In the Msx1 mutant, Wnt1 is downregulated before the midline disappears, suggesting that its expression depends on Msx1. Furthermore, electroporation in the chick embryo demonstrates that Msx1 can induce Wnt1 expression in the diencephalon neuroepithelium and in the lateral ectoderm. In double Msx1/Msx2 mutants, Wnt1 expression is completely abolished at the dorsal midline of the diencephalon and rostral mesencephalon. This indicates that Msx genes may regulate Wnt1 expression at the dorsal midline of the neural tube. Based on these results, we propose a model in which Msx genes are intermediary between Bmp and Wnt at this site.

The human homeobox gene HOX7 maps to chromosome 4p16.1 and may be implicated in Wolf-Hirschhorn syndrome

SummaryA cosmid containing the human sequence (HOX7) homologous to the mouse homeogene Hox-7 was isolated from a genomic cosmid library. There is only one highly conserved homologous gene in the human genome. The C-terminal two-thirds of the HOX7 homeobox DNA sequence has been determined; there are no predicted amino acid changes from the mouse sequence. Data from mouse/human hybrid cell lines show that HOX7 maps to human chromosome 4p16.1, a region that is syntenic with part of mouse chromosome 5, the site of the murine Hox-7 gene. Analysis of chromosomes from two patients with Wolf-Hirschhorn syndrome, which is characterised by profound dysmorphologies, indicates that the HOX7 locus is deleted. Although not all Wolf-Hirschhorn syndrome patients analysed were deleted for HOX7, the combination of positional data and functional correlation with mouse expression implicates HOX7 as a candidate gene for this syndrome.

Msx1 and Dlx5 act independently in development of craniofacial skeleton, but converge on the regulation of Bmp signaling in palate formation.

Msx and Dlx homeoproteins control the morphogenesis and organization of craniofacial skeletal structures, specifically those derived from the pharyngeal arches. In vitro Msx and Dlx proteins have opposing transcriptional properties and form heterodimeric complexes via their homeodomain with reciprocal functional repression. In this report we examine the skeletal phenotype of Msx1; Dlx5 double knock-out (DKO) mice in relationship with their expression territories during craniofacial development. Co-expression of Dlx5 and Msx1 is only observed in embryonic tissues in which these genes have independent functions, and thus direct protein interactions are unlikely to control morphogenesis of the cranium. The DKO craniofacial phenotypes indicate a complex interplay between these genes, acting independently (mandible and middle ear), synergistically (deposition of bone tissue) or converging on the same morphogenetic process (palate growth and closure). In the latter case, the absence of Dlx5 rescues in part the Msx1-dependent defects in palate growth and elevation. At the basis of this effect, our data implicate the Bmp (Bmp7, Bmp4)/Bmp antagonist (Follistatin) signal: in the Dlx5(-/-) palate changes in the expression level of Bmp7 and Follistatin counteract the reduced Bmp4 expression. These results highlight the importance of precise spatial and temporal regulation of the Bmp/Bmp antagonist system during palate closure.

Anteroposterior patterning in the limb and digit specification: Contribution of mouse genetics

The limb has been a privileged object of investigation and reflection for scientists over the past two centuries and continues to provide a heuristic framework to analyze vertebrate development. Recently, accumulation of new data has significantly changed our view on the mechanisms of limb patterning, in particular along the anterior‐posterior axis. These data have led us to revisit the mode of action of the zone of polarizing activity. They shed light on the molecular and cellular mechanisms of patterning linked to the Shh‐Gli3 signaling pathway and give insights into the mechanism of activation of these cardinal factors, as well as the consequences of their activity. These new data are in good part the result of systematic Application of tools used in contemporary mouse molecular genetics. These have extended the power of mouse genetics by introducing mutational strategies that allow fine‐tuned modulation of gene expression, interchromosomal deletions and duplication. They have even made the mouse embryo amenable to cell lineage analysis that used to be the realm of chick embryos. In this review, we focus on the data acquired over the last five years from the analysis of mouse limb development and discuss new perspectives opened by these results. Developmental Dynamics 235:2337–2352, 2006.

The same myosin alkali light chain gene is expressed in adult cardiac atria and in fetal skeletal muscle

SummaryWe have isolated from a cDNA library constructed using mouse cardiac mRNA sequences, a clone (pC6) homologous to part of the mRNA encoding the myosin alkali light chain MLC1A from adult mouse atria. This sequence also hybridizes to mRNA encoding the fetal light chain form MLC1emb expressed in both fused myotubes in culture and in 18 day fetal skeletal muscle. These mRNA sequences are indistinguishable from the MLC1A messenger both on the basis of size and of their thermal stability of hybridization.In vitro translation of mRNA selected by hybridization with pC6 results in a protein that comigrates with the fetal MLC1emb isoform, and two-dimensional gel electrophoresis of adult atrial and fetal skeletal muscle proteins shows MLC1A and MLC1emb to be indistinguishable in the mouse. Southern blot hybridization of clone pC6 to mouse genomic DNA and the analysis of restriction fragment length polymorphisms between different mouse species demonstrates the presence of a single hybridizing locus in the mouse genome. These data provide strong evidence that the atrial MLC1A and fetal skeletal MLC1emb isoform are encoded by the same gene and by the same mRNA and are thus identical proteins.

Msx1 is a regulator of bone formation during development and postnatal growth: in vivo investigations in a transgenic mouse model.

The present study is devoted to Msx1 distribution and function from birth to 15 months, events and periods still unexplored in vivo using Msx1 knock in transgenic mice. The study is focused on the mandible, as an exemplary model system for Msx1-dependent neural crest-derived skeletal unit. The transgenic line enabled study of morphological abnormalities in Msx1 null mutation mice and Msx1 protein expression in Msx1+/ m heterozygous mice. In Msx1 null mutation, the most striking feature was an inhibition of the mandibular basal convexity, the absence of teeth and alveolar bone processes, and absence of endochondral ossification in the mandibular condyle. At birth, in Msx1+/ m heterozygous animals, we identified for the first time a double Msx1 aboral-oral and disto-proximal gradient field developmental pattern located in the low border of the mandibular bone in relation with this bone segment modeling. Msx1 expression involved both osteoblast and osteoclast cells. A distinct pattern characterized bone surfaces: Periosteum osteoblast differentiation was related to Msx1 downregulation, while in the endosteum both differentiated osteoblasts and osteoclasts expressed the homeoprotein. In postnatal stages, Msx1 expression was maintained in the alveolar bone processes and dento-alveolar cells in relation with tooth function. Our data suggest that Msx1 play a role in a site-specific manner not only in early patterning but also in skeletal growth and modeling by acting on heterogenous bone cell populations.

Changes in gene expression during myogenic differentiation. I. Regulation of messenger RNA sequences expressed during myotube formation.

Abstract The patterns of gene expression at three different stages of cellular differentiation have been compared by examining polysomal and nuclear RNA base sequence complexities. The changes in nuclear RNA and polysomal poly(A)-containing messenger RNA (poly(A)+ mRNA) sequences in passing from a multipotential mouse embryonal carcinoma cell, to a committed (teratocarcinoma-derived) myoblast cell and finally to terminally differentiated myotube structures, have been followed by saturation hybridization of highly labelled single-copy mouse DNA and by RNA excess hybridization with polysomal complementary DNA. The saturation hybridization of total and fractionated sc-DNA § shows that there is a 30% increase in nRNA complexity in passing from the EC to the Mb and Mt stages, and that the EC stage nRNA sequences remain a subset of Mb and Mt stage nRNA. At the polysomal level, with each stage of differentiation a new group of mRNA sequences enters the polysomes. Mt cDNA probes enriched in the new sequences which appear at the Mb and Mt stages were prepared. From hybridization experiments between these cDNA probes and total nRNA from the three stages of differentiation, it would appear that the expression of qualitatively new sequences at the polysomal level is controlled transcriptionally and not post-transcriptionally. Further, support for this conclusion was furnished by experiments where chromatin from EC and Mb cells was partially digested with DNase I. These experiments show that gene sequences specific to the Mt stage of differentiation are resistant to attack by DNase I in the chromatin of EC and Mb cells, whereas gene sequences of mRNAs expressed at all three stages are readily digested. Some sequences present in Mb cells increase in concentration after cell fusion, possibly as a consequence of quantitative post-transcriptional modulation.