Gene Dosage Effects at the Imprinted Gnas Cluster

Genomic imprinting results in parent-of-origin-dependent monoallelic gene expression. Early work showed that distal mouse chromosome 2 is imprinted, as maternal and paternal duplications of the region (with corresponding paternal and maternal deficiencies) give rise to different anomalous phenotypes with early postnatal lethalities. Newborns with maternal duplication (MatDp(dist2)) are long, thin and hypoactive whereas those with paternal duplication (PatDp(dist2)) are chunky, oedematous, and hyperactive. Here we focus on PatDp(dist2). Loss of expression of the maternally expressed Gnas transcript at the Gnas cluster has been thought to account for the PatDp(dist2) phenotype. But PatDp(dist2) also have two expressed doses of the paternally expressed Gnasxl transcript. Through the use of targeted mutations, we have generated PatDp(dist2) mice predicted to have 1 or 2 expressed doses of Gnasxl, and 0, 1 or 2 expressed doses of Gnas. We confirm that oedema is due to lack of expression of imprinted Gnas alone. We show that it is the combination of a double dose of Gnasxl, with no dose of imprinted Gnas, that gives rise to the characteristic hyperactive, chunky, oedematous, lethal PatDp(dist2) phenotype, which is also hypoglycaemic. However PatDp(dist2) mice in which the dosage of the Gnasxl and Gnas is balanced (either 2∶2 or 1∶1) are neither dysmorphic nor hyperactive, have normal glucose levels, and are fully viable. But PatDp(dist2) with biallelic expression of both Gnasxl and Gnas show a marked postnatal growth retardation. Our results show that most of the PatDp(dist2) phenotype is due to overexpression of Gnasxl combined with loss of expression of Gnas, and suggest that Gnasxl and Gnas may act antagonistically in a number of tissues and to cause a wide range of phenotypic effects. It can be concluded that monoallelic expression of both Gnasxl and Gnas is a requirement for normal postnatal growth and development

Zic2 is required for neural crest formation and hindbrain patterning during mouse development

The Zic genes are the vertebrate homologues of the Drosophila pair rule gene odd-paired. It has been proposed that Zic genes play several roles during neural development including mediolateral segmentation of the neural plate, neural crest induction, an

Zic2 is required for neural crest formation and hindbrain patterning during mouse development

AbstractThe Zic genes are the vertebrate homologues of the Drosophila pair rule gene odd-paired. It has been proposed that Zic genes play several roles during neural development including mediolateral segmentation of the neural plate, neural crest induction, and inhibition of neurogenesis. Initially during mouse neural development Zic2 is expressed throughout the neural plate while later on expression in the neurectoderm becomes restricted to the lateral region of the neural plate. A hypomorphic allele of Zic2 has demonstrated that in the mouse Zic2 is required for the timing of neurulation. We have isolated a new allele of Zic2 that behaves as a loss of function allele. Analysis of this mutant reveals two further functions for Zic2 during early neural development. Mutation of Zic2 results in a delay of neural crest production and a decrease in the number of neural crest cells that are produced. These defects are independent of mediolateral segmentation of the neurectoderm and of dorsal neurectoderm proliferation, both of which occur normally in the mutant embryos. Additionally Zic2 is required during hindbrain patterning for the normal development of rhombomeres 3 and 5. This work provides the first genetic evidence that the Zic genes are involved in neural crest production and the first demonstration that Zic2 functions during hindbrain patterning

KATNAL1 regulation of sertoli cell microtubule dynamics is essential for spermiogenesis and male fertility

Spermatogenesis is a complex process reliant upon interactions between germ cells (GC) and supporting somatic cells. Testicular Sertoli cells (SC) support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1). We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC) from 15.5 days post-coitum (dpc) and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives

Incidence and Survival of PatDp(dist2).

*<p>23 PatDp altogether but 2 were PatDp(dist2)2∶2 and 3 were PatDp(dist2)2∶0.</p>∼<p>36 PatDp altogether but 3 were PatDp(dist2)0∶2 and 2 were PatDp(dist2)2∶0.</p>∧<p>10 PatDp altogether but 3 were PatDp(dist2)2∶1 and one was not classified for <i>ΔEx1A.</i></p><p>Incidence No shows the number of PatDps born/total number of mice born.</p><p>Survival No shows number of PatDps/total number of PatDps scored.</p><p>For comparison of PatDp(dist2)2∶0 with other PatDp classes:</p>1<p>P<0.05,</p>2<p>P<0.01,</p>3<p>P<0.0001 (Fisher’s exact test, 2 tailed).</p><p>For comparison of PatDp(dist2)2∶1 with other PatDp classes except PatDp(dist2)2∶0:</p>4<p>P<0.002,</p>5<p>P<0.0001 (Fisher’s exact test, 2 tailed).</p

KATNAL1 is a microtubule severing protein.

<p>Hek293 cells were stably transfected with constructs encoding either KATANIN p60 or KATNAL1 protein. Separate wells were treated +/− tetracycline or vehicle for a period of 12 hours, fixed then stained for α-tubulin as a marker of microtubules. This demonstrated that, like KATANIN p60, KATNAL1 functions as a microtubule severing protein. Scale bars = 20 µm.</p

Katnal1 co-localises with Sertoli cell microtubules, but is restricted to basal regions in mutant testes.

<p>At d35, immunohistochemical localisation of the Sertoli cell-specific isoform of beta-tubulin TUBB3 reveals an apparent disruption to the microtubule network in <i>Katnal1^1H/1H</i> testes, (a, b). In Wild-Type animals, KATNAL1 localisation (arrows) tracks the SC microtubule network from basal to apical regions (c, e). Conversely, the mutant KATNAL1 protein is restricted to the basal region of Sertoli cells in <i>Katnal1^1H/1H</i> animals (d, f). Images representative of stage VI of the spermatogenic cycle. Bars = 20 µm.</p

Sertoli Cell and Germ Cell composition of the testes.

<p>Comparison of Sertoli and Germ cell composition of the testes from Wild-type (+/+) and homozygous <i>Katnal1^1H/1H</i> (1H/1H) mutant mice at d22, d35 and d70. (n = 5 per group; Mean ± SEM).</p

Crosses to generate PatDp(dist2).

<p>Crosses to generate PatDp(dist2).</p

Growth retardation.

<p>(<b>A</b>) Growth curve of +/<i>ΔEx1A</i> and wild-type littermates from 1 day to 12 weeks. The weights of wild-type littermates have been normalised to 1 at each timepoint and the weights of +/<i>ΔEx1A</i> mice have been taken as a percentage of wild-type weights. (n = 5–26). (<b>B</b>) Growth curve of PatDp(dist2)2∶2 and +/<i>ΔEx1A</i>. The weights of +/<i>ΔEx1A</i> littermates have been normalised to 1 at each timepoint and the weights of PatDp(dist2)2∶2 mice have been taken as a percentage of +/<i>Ex1A</i> weights (n = 9–12). Error bars show standard errors of the means.</p