No abstract

Gene targeting in embryonic stem (ES) cells is a powerful tool for generating mice with null alleles. Current methods of gene inactivation in ES cells introduce a neomycin gene (neo) cassette both as a mutagen and a selection marker for transfected cells. Although null alleles are valuable, changes at the nucleotide level of a gene are very important for functional analysis. One gene family in which subtle mutations would be particularly valuable are the clusters of Hox homeobox genes. Inactivation of gene in a cluster with a neo cassette that includes promoter/enhancer elements may deregulate transcription of neighbouring genes and generate a phenotype which is difficult to interpret. We describe here a highly efficient gene targeting method, termed the 'hit and run' procedure. This generates ES cells with subtle site-specific mutations with no selectable marker and may be useful for most genes. We have developed this procedure at the hypoxanthine phosphoribosyltransferase (hprt) locus and subsequently isolated ES cells with a premature stop codon in the homeobox of Hox-2.6 (ref. 14).

The Hox gene products are transcription factors involved in specifying regional identity along the anteroposterior body axis. In the mouse, several single mutants for Hox genes show variably penetrant, partial homeotic transformations of vertebrae at their anterior limits of expression, suggesting that compound Hox mutants might show more complete transformations with greater penetrance than the single Hox mutants. Compound mutants for the paralogous group 3 genes, hoxa-3 and hoxd-3, show deletion of a cervical vertebrae, which is not readily interpretable in terms of an alteration in regional identity. Here, we report the skeletal phenotypes of compound mutants in the group 4 Hox genes, hoxa-4, hoxb-4, and hoxd-4. Mice mutant for each of these genes were intercrossed to generate the three possible double mutant combinations and the triple mutant. In contrast to the hoxa-3, hoxd-3 double mutants, group 4 Hox compound mutants displayed clear alterations in regional identity, including a nearly complete transformation of the second cervical vertebrae toward the morphology of the first cervical vertebra in one double mutant combination. In comparing the types of homeotic transformations observed, different double mutant combinations showed different degrees of synergism. These results suggest a certain degree of functional redundancy among paralogous genes in specifying regional identity. Furthermore, there was a remarkable dose-dependent increase in the number of vertebrae transformed to a first cervical vertebra identity, including the second through the fifth cervical vertebrae in the triple mutant. Thus, these genes are required in a larger anteroposterior domain than is revealed by the single mutant phenotypes alone, such that multiple mutations in these genes result in transformations of vertebrae that are not at their anterior limit of expression. The specification of regional identity along the anteroposterior body axis of many organisms culminates in the generation of a diversity of structures along this axis. The proper specification of regional structures along this axis is dependent on the action of an evolutionarily conserved set of genes, which were first identified in Drosophila melanogaster for their ability to effect homeotic transformations, reidentification of a body segment

The family of WD40-repeat (WDR) proteins is one of the largest in eukaryotes, but little is known about their function in brain development. Among 26 WDR genes assessed, we found 7 displaying a major impact in neuronal morphology when inactivated in mice. Remarkably, all seven genes showed corpus callosum defects, including thicker (Atg16l1, Coro1c, Dmxl2, and Herc1), thinner (Kif21b and Wdr89), or absent corpus callosum (Wdr47), revealing a common role for WDR genes in brain connectivity. We focused on the poorly studied WDR47 protein sharing structural homology with LIS1, which causes lissencephaly. In a dosage-dependent manner, mice lacking Wdr47 showed lethality, extensive fiber defects, microcephaly, thinner cortices, and sensory motor gating abnormalities. We showed that WDR47 shares functional characteristics with LIS1 and participates in key microtubule-mediated processes, including neural stem cell proliferation, radial migration, and growth cone dynamics. In absence of WDR47, the exhaustion of late cortical progenitors and the consequent decrease of neurogenesis together with the impaired survival of late-born neurons are likely yielding to the worsening of the microcephaly phenotype postnatally. Interestingly, the WDR47-specific C-terminal to LisH (CTLH) domain was associated with functions in autophagy described in mammals. Silencing WDR47 in hypothalamic GT1-7 neuronal cells and yeast models independently recapitulated these findings, showing conserved mechanisms. Finally, our data identified superior cervical ganglion-10 (SCG10) as an interacting partner of WDR47. Taken together, these results provide a starting point for studying the implications of WDR proteins in neuronal regulation of microtubules and autophagy.WD40-repeat proteins | corpus callosum agenesis | microcephaly | neurogenesis | autophagy T he function of WD40-repeat (WDR)-containing proteins, one of the largest eukaryotic protein families, is largely unknown. Their importance is, however, evident based on their highly conserved repeating units from bacteria to mammals (1), commonly made of seven repetitive blades of 40 amino acids that end with a tryptophan-aspartic acid dipeptide at the C terminus.As shown by crystallography studies, including the crystal structure of the beta gamma dimer of the G-protein transducin (2), a classical WDR protein, all WDR proteins are predicted to fold into a circularized beta-propeller structure, serving as a rigid platform (or scaffold) for protein-protein interactions by providing many stable and symmetrical surfaces (3, 4). One reason why WDR domains may have been less studied than other common domains, such as kinases or PDZ or SH3 domains (3), is that no WDR domain has yet been found with catalytic activity (3), but this does not mean that the scaffold domains are less important. To the contrary, their serving as a platform for multiple enzymatic reactions and signaling events is highly significant (5).In recent years, human genetic studies have also begun to recognize the importance of WDR gen...