Distal limb development and specification of digit identities in tetrapods are under the control of a mesenchymal organizer called the polarizing region. Sonic Hedgehog (SHH) is the morphogenetic signal produced by the polarizing region in the posterior limb bud. Ectopic anterior SHH signaling induces digit duplications and has been suspected as a major cause underlying congenital malformations that result in digit polydactyly. Here, we report that the polydactyly of Gli3-deficient mice arises independently of SHH signaling. Disruption of one or both Gli3 alleles in mouse embryos lacking Shh progressively restores limb distal development and digit formation. Our genetic analysis indicates that SHH signaling counteracts GLI3-mediated repression of key regulator genes, cell survival, and distal progression of limb bud development.

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The bHLH transcription factor dHAND is required for establishment of SHH signaling by the limb bud organizer in posterior mesenchyme, a step crucial to development of vertebrate paired appendages. We show that the transcriptional repressor GLI3 restricts dHAND expression to posterior mesenchyme prior to activation of SHH signaling in mouse limb buds. dHAND, in turn, excludes anterior genes such as Gli3 and Alx4 from posterior mesenchyme. Furthermore, genetic interaction of GLI3 and dHAND directs establishment of the SHH/ FGF signaling feedback loop by restricting the BMP antagonist GREMLIN posteriorly. These interactions polarize the nascent limb bud mesenchyme prior to SHH signaling.

SUMMARY Wolf-Hirschhorn syndrome (WHS) is caused by deletions in the short arm of chromosome 4 (4p) and occurs in about one per 20,000 births. Patients with WHS display a set of highly variable characteristics including craniofacial dysgenesis, mental retardation, speech problems, congenital heart defects, short stature and a variety of skeletal anomalies. Analysis of patients with 4p deletions has identified two WHS critical regions (WHSCRs); however, deletions targeting mouse WHSCRs do not recapitulate the classical WHS defects, and the genes contributing to WHS have not been conclusively established. Recently, the human FGFRL1 gene, encoding a putative fibroblast growth factor (FGF) decoy receptor, has been implicated in the craniofacial phenotype of a WHS patient. Here, we report that targeted deletion of the mouse Fgfrl1 gene recapitulates a broad array of WHS phenotypes, including abnormal craniofacial development, axial and appendicular skeletal anomalies, and congenital heart defects. Fgfrl1 null mutants also display a transient foetal anaemia and a fully penetrant diaphragm defect, causing prenatal and perinatal lethality. Together, these data support a wider role for Fgfrl1 in development, implicate FGFRL1 insufficiency in WHS, and provide a novel animal model to dissect the complex aetiology of this human disease.

The orthodenticle/ otx and orthopedia/ otp classes of homeobox gene families have been identified in all three major classes of bilaterians: deuterostomes, lophotrochozoans, and ecdysozoans. Otx genes have been studied extensively and play a role in the development of anterior neural structures. Otp genes have been found to be involved in nervous system development in mouse and Drosophila. To date, no members of these genes are known in molluscs. We cloned orthologs of orthodenticle/ otx and orthopedia/ otpfrom the gastropod Patella vulgata, and designated them Pv-otx and Pv-otprespectively. Our analysis of the spatio-temporal expression pattern of otx and otp orthologs during P. vulgata embryogenesis leads to the following conclusions. First, Pv-otx is expressed in and around the stomodaeum and our analysis thus supports the previously suggested conservation of the protostome and deuterostome larval mouth regions. Second, we find that Pv-otp is involved in the development of the larval apical sensory organ, suggesting a conserved role for this gene family in nervous system development. A similar conserved role in nervous system development has been proposed for orthodenticle/otx genes and we suggest that part of the cells expressing Pv-otx are involved in the development of the anterior nervous system. Last, we postulate that otx genes were ancestrally involved in the development of ciliary bands in bilaterians.

Dynamic expressions of Homeobox (Hox) genes along the anterior-posterior axis of the neural tube (NT) are crucial in body patterning and neural crest (NC) development. At E12.5, Hoxb5 expression extends from the hindbrain throughout the entire NT with dorsally-restricted expression. By replacing the transactivation domain of Hoxb5 with a transcription repressor domain of the Drosophila engrailed (en) protein, we generate a chimeric protein enb5 that binds and represses the expression instead of inducing transcription of target genes. We have shown that Cre-mediated expression of enb5 in vagal NC caused defective NC migration and abnormal enteric nervous system (ENS) development.To further investigate the functions of Hoxb5 in nervous system development, we crossed enb5 mice to Wnt1-Cre mice.Wnt1-Cre/enb5 transgenic mice display hydrocephalus, abnormal skin pigmentation, and ENS defects.Hydrocephalus was firstly observed in P0 Wnt1-Cre/enb5 mice.In embryos, Hoxb5 was expressed by the brain structures responsible for the production and circulation of cerebrospinal fluid (CSF) namely the choroid plexus and the ependymal cell lining the brain ventricles. Therefore, hydrocephalus could be attributable to the defective flow and/or over-production of CSF in Wnt1-Cre/enb5 embryonic mice. Melanoblasts were found residing in the dermis and also penetrating into the epidermis in wildtype E12.5 embryos. In contrast, melanoblasts were undetectable and NC progenitors were drastically reduced in Wnt1-Cre/enb5 embryos, suggesting that NC induction and/or maintenance were defective in these embryos. We are investigating the molecular mechanisms by which perturbation of Hoxb5 signaling in the developing nervous system cause these anomalies in Wnt1-Cre/ enb5 mice.Hox genes encode a highly conserved family of transcription factors that play fundamental roles during embryonic development. In humans, mutations in HOXD13 cause the rare dominantly inherited limb synpolydactyly. Both polyalanine tract expansions and frameshift deletions in HOXD13 have been described to underlie this condition. We identified a new missense mutation substituting Alanine for Glycine at position 11 in the N-terminal part of HOXD13. We compared biochemical and biological properties of the HOXD13(G11A) mutant protein to wild-type HOXD13. We demonstrate, using invitro approaches, that the G11A mutation significantly impairs the stability of HOXD13. Furthermore, we observed a mild impairment of the DNA binding capacity of HOXD13(G11A) on TTAT, but not on TTAC target sequences. The subcellular localization of the protein is not affected by the mutation, suggesting distinct properties of the G11A mutant as compared to polyalanine tract expansions or the recently described HOXD13(G220V) mutant. To investigate biological activity of HOXD13(G11A), we are currently misexpressing it in the developing chick limb using RCAS system. Injection of RCAS expressing wild-type HOXD13 at stage HH10 of chick development induces a shortening of limb cartilages especially at the le...