From the transcription of genes involved in ectodermal dysplasias to the understanding of associated dental anomalies.

Orodental anomalies are one aspect of rare diseases and are increasingly identified as diagnostic and predictive traits. To understand the rationale behind gene expression during tooth or other ectodermal derivative development and the disruption of odontogenesis or hair and salivary gland formation in human syndromes we analyzed the expression patterns of a set of genes (Irf6, Nfkbia, Ercc3, Evc2, Map2k1) involved in human ectodermal dysplasias in mouse by in situ hybridization. The expression patterns of Nfkbia, Ercc3 and Evc2 during odontogenesis had never been reported previously. All genes were indeed transcribed in different tissues/organs of ectodermal origin. However, for Nfkbia, Ercc3, Evc2, and Map2k1, signals were also present in the ectomesenchymal components of the tooth germs. These expression patterns were consistent in timing and localization with the known dental anomalies (tooth agenesis, microdontia, conical shape, enamel hypoplasia) encountered in syndromes resulting from mutations in those genes. They could also explain the similar orodental anomalies encountered in some of the corresponding mutant mouse models. Translational approaches in development and medicine are relevant to gain understanding of the molecular events underlying clinical manifestations.

Embryonic retinoic acid synthesis is essential for heart morphogenesis in the mouse.

Retinoic acid (RA), the active derivative of vitamin A, has been implicated in various steps of cardiovascular development, but its contribution to early heart morphogenesis has not been clearly established in a mammalian system. To block endogenous RA synthesis, we have disrupted the gene encoding RALDH2, the first retinaldehyde dehydrogenase whose expression has been detected during early mouse post-implantation development. We describe here the heart abnormalities of the RA-deficient Raldh2 mutants that die in utero at gestational day 10.5. The embryonic heart tube forms properly, but fails to undergo rightward looping and, instead, forms a medial distended cavity. Expression of early heart determination factors is not altered in mutants, and the defect in heart looping does not appear to involve the Nodal/Lefty/Pitx2 pathway. Histological and molecular analysis reveal distinct anteroposterior components in the mutant heart tube, although posterior chamber (atria and sinus venosus) development is severely impaired. Instead of forming trabeculae, the developing ventricular myocardium consists of a thick layer of loosely attached cells. Ultrastructural analysis shows that most of the ventricular wall consists of prematurely differentiated cardiomyocytes, whereas undifferentiated cells remain clustered rostrally. We conclude that embryonic RA synthesis is required for realization of heart looping, development of posterior chambers and proper differentiation of ventricular cardiomyocytes. Nevertheless, the precise location of this synthesis may not be crucial, as these defects can mostly be rescued by systemic (maternal) RA administration. However, cardiac neural crest cells cannot be properly rescued in Raldh2(-/- )embryos, leading to outflow tract septation defects.

Retinoic acid synthesis and hindbrain patterning in the mouse embryo.

Targeted disruption of the murine retinaldehyde dehydrogenase 2 (Raldh2) gene precludes embryonic retinoic acid (RA) synthesis, leading to midgestational lethality (Niederreither, K., Subbarayan, V., Dolle, P. and Chambon, P. (1999). Nature Genet. 21, 444-448). We describe here the effects of this RA deficiency on the development of the hindbrain and associated neural crest. Morphological segmentation is impaired throughout the hindbrain of Raldh2-/- embryos, but its caudal portion becomes preferentially reduced in size during development. Specification of the midbrain region and of the rostralmost rhombomeres is apparently normal in the absence of RA synthesis. In contrast, marked alterations are seen throughout the caudal hindbrain of mutant embryos. Instead of being expressed in two alternate rhombomeres (r3 and r5), Krox20 is expressed in a single broad domain, correlating with an abnormal expansion of the r2-r3 marker Meis2. Instead of forming a defined r4, Hoxb1- and Wnt8A-expressing cells are scattered throughout the caudal hindbrain, whereas r5/r8 markers such as kreisler or group 3/4 Hox genes are undetectable or markedly downregulated. Lack of alternate Eph receptor gene expression could explain the failure to establish rhombomere boundaries. Increased apoptosis and altered migratory pathways of the posterior rhombencephalic neural crest cells are associated with impaired branchial arch morphogenesis in mutant embryos. We conclude that RA produced by the embryo is required to generate posterior cell fates in the developing mouse hindbrain, its absence leading to an abnormal r3 (and, to a lesser extent, r4) identity of the caudal hindbrain cells.

Restricted expression of the ron gene encoding the macrophage stimulating protein receptor during mouse development.

The human ron gene codes for a transmembrane protein tyrosine kinase which is a receptor for the macrophage stimulating protein. The ron receptor, together with the hepatocyte growth factor/scatter factor receptor encoded by the proto-oncogene met, and the product of the c-sea proto-oncogene, make up a family of structurally related receptors. We have cloned murine ron cDNA sequences and used them as probes for in situ hybridization and Northern blot experiments. We show that ron gene expression occurs relatively late in development, and is much more restricted than that of the met gene. ron gene expression is detected in specific areas of the central and the peripheric nervous system, as well as in discrete cells in developing bones, and in the glandular epithelia along the digestive tract.

Mouse Lbx1 and human LBX1 define a novel mammalian homeobox gene family related to the Drosophila lady bird genes.

We have cloned two novel homeobox genes which are the mouse (Lbx1) and human (LBX1) homologs of the Drosophila lady bird genes. They are highly related not only within the coding region but also in 5' and 3' untranslated regions. Several amino acid residues inside and around the homeodomain, have been conserved between the mammalian Lbx genes and their Drosophila counterparts. The mouse Lbx1 gene is located on chromosome 19 (region D) and the human LBX1 gene maps to the related q24 region of chromosome 10, known as a breakpoint region in translocations t(7;10) and t(10;14) involved in T-cell leukemias. Thus, LBX1 and the protooncogene HOX11 map to a common chromosomal region, as do their Drosophila counterparts, the lady bird and 93Bal genes. The mouse Lbx1 gene is specifically expressed during embryogenesis. From 10.5 days of gestation, Lbx1 expression is detected in the central nervous system and some developing muscles. In the CNS, Lbx1 transcripts are expressed in the dorsal part of the mantle layer of the spinal cord and hindbrain, up to a sharp boundary within the developing metencephalon. Thus, Lbx1 may be inolved in spinal cord and hindbrain differentiation and/or patterning, and its restricted expression pattern could depend upon evolutionarily conserved inductive signals involving some mammalian Wnt and Pax genes, as is the case for Drosophila lady bird genes and wingless or gooseberry.

Efficient cloning of cDNAs of retinoic acid-responsive genes in P19 embryonal carcinoma cells and characterization of a novel mouse gene, Stra1 (mouse LERK-2/Eplg2).

Pluripotent mouse P19 embryonal carcinoma (EC) cells have been extensively used as a developmental model system because they can differentiate in the presence of retinoic acid (RA) into derivatives of all three germ layers depending on RA dosage and culture conditions. The expression of several genes has been shown to be induced in RA-treated P19 EC cells and, interestingly, some of these genes may play important roles during mouse embryogenesis. In view of the increasing evidence that RA is a crucial signaling molecule during vertebrate development, we have initiated a study aimed at the systematic isolation of genes whose expression is induced in P19 cells at various times after exposure to RA. We describe here an efficient differential subtractive hybridization cloning strategy which was used to identify additional RA-responsive genes in P19 cells. Fifty different cDNA fragments corresponding to RA-induced genes were isolated. Ten cDNAs represent known genes, 4 of which have already been described as RA-inducible, while the remaining 40 correspond to novel genes. Many of these cDNA sequences represent low-abundance mRNAs. Kinetic analysis of mRNA accumulation following RA treatment allowed us to characterize four classes of RA-responsive genes. We also report the sequence and expression pattern in mouse embryos and adult tissues of one of these novel RA-inducible genes, Stra1, and show that it corresponds to the mouse ligand for the Cek5 receptor protein-tyrosine kinase.

Cloning a pseudogene and cDNA encoding a 17-kDa ribosomal protein from mouse: structure and regulation of expression.

An rp lambda 5 cDNA encoding a ribosomal protein (r-protein) and a pseudogenic form of the corresponding gene (rp lambda 7) have been cloned from mouse. This cDNA codes for a highly basic protein of 160 amino acids (aa) with a deduced M(r) of 17,601, and most likely represents the species homolog of a recently cloned rat cDNA, which has been proposed to encode a homolog of the yeast r-protein, YL43. The entire rp lambda 5 gene encompasses less than 1.5 kb of genomic DNA and apparently is composed of only two exons, as deduced from sequence comparison with its very similar pseudogenic variant, rp lambda 7. Southern analysis, using the rp lambda 5 cDNA as a probe, indicates the existence of a great number of highly related sequences in the mouse genome. The mRNA for rp lambda 5 is approximately 800 nucleotides (nt) long and is found to be ubiquitously expressed at high levels in embryonic and adult mouse tissues, as shown by Northern and in situ analyses. Retinoic acid (RA) seems to have a moderate down-regulatory effect on this mRNA in differentiating P19 embryonal carcinoma cells. Several degenerate/nondegenerate RA-response element (RARE) motifs are found within 560 bp upstream from the degenerate start codon in rp lambda 7. However, it is unknown whether this RA effect is exerted at the transcriptional and/or posttranscriptional levels.

Disruption of the Hoxd-13 gene induces localized heterochrony leading to mice with neotenic limbs.

Vertebrate Hoxd genes are sequentially activated during the morphogenesis and pattern formation of the limb. Using the approach of gene disruption via homologous recombination in embryonic stem cells, we have assessed the function of the last gene of the complex, Hoxd-13. Mutant mice displayed skeletal alterations along all body axes suggesting the existence of a general multiaxial patterning system. In limbs, abnormalities such as a reduction in the length of some bony elements, loss of phalanges, bone fusions, and the presence of an extra element were observed. We propose that the mutation induces local heterochrony, as illustrated by an important retardation in limb morphogenesis. The relevance of these observations to our understanding of the development and evolution of the tetrapod limb is discussed.

MHC-linked protection from diabetes dissociated from clonal deletion of T cells.

The I-E molecule of the major histocompatibility complex (MHC) can prevent the spontaneous development of diabetes in nonobese diabetic (NOD) mice. The mechanism of this protection has been investigated by breeding wild-type and promoter-mutated E kappa alpha transgenes onto the NOD genetic background. Animals carrying the various mutated transgenes expressed I-E on different subsets of immunocompetent cells, and thus cells important for the I-E protective effect could be identified. Although the wild-type transgene prevented the infiltration of lymphocytes into pancreatic islets, none of the mutants did. However, all of the transgenes could mediate the intrathymic elimination of T cells bearing antigen receptors with variable regions that recognize I-E. Thus, the I-E molecule does not protect NOD mice from diabetes simply by inducing the deletion of self-reactive T cells.