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ATRX is an X-encoded member of the SNF2 family of ATPase/helicase proteins thought to regulate gene expression by modifying chromatin at target loci. Mutations in ATRX provided the first example of a human genetic disease associated with defects in such proteins. To better understand the role of ATRX in development and the associated abnormalities in the ATR-X (alpha thalassemia mental retardation, X-linked) syndrome, we conditionally inactivated the homolog in mice, Atrx, at the 8- to 16-cell stage of development. The protein, Atrx, was ubiquitously expressed, and male embryos null for Atrx implanted and gastrulated normally but did not survive beyond 9.5 days postcoitus due to a defect in formation of the extraembryonic trophoblast, one of the first terminally differentiated lineages in the developing embryo. Carrier female mice that inherit a maternal null allele should be affected, since the paternal X chromosome is normally inactivated in extraembryonic tissues. Surprisingly, however, some carrier females established a normal placenta and appeared to escape the usual pattern of imprinted X-inactivation in these tissues. Together these findings demonstrate an unexpected, specific, and essential role for Atrx in the development of the murine trophoblast and present an example of escape from imprinted X chromosome inactivation. [ABSTRACT FROM AUTHOR]

ENU mutagenesis is a powerful tool for the identification of novel models of human disease. Haploinsufficiency of the insulin receptor (IR) and associated mild insulin resistance has been utilised to sensitise an ENU screen to identify novel mutations resulting in impaired glucose tolerance and diabetes. An intraperitoneal glucose tolerance test (IPGTT) was utilised in a phenotype driven screen to identify F1 individuals with impaired glucose tolerance. Animals deviating more than 2 SDs from an unmutagenised control population entered inheritance testing. Here we describe two dominant mutations in the highly conserved HMG box of the transcription factor Sox4, one identified in the phenotype driven screen and the other subsequently obtained from an embryonic screen. Sox4 has previously been associated with pancreas development and, as expected, homozygotes for either mutation or compound heterozygotes were embryonic lethal, suggesting both mutations affect gene function and are nulls. Heterozygous Sox4 adult mice had mildly impaired glucose tolerance, and insulin secretion assays in both whole animals and in isolated islets indicated a defect in insulin secretion. In association with IR+/- induced insulin resistance, this insulin secretory defect causes a more severe impaired glucose tolerance and represents a novel polygenic model of type 2 diabetes. Knockdown of Sox4 in INS1 cells with two different siRNAs, targeted to separate regions of the gene, abolished glucose-stimulated insulin release. Interestingly, we also observed a significant decrease in insulin release in response to tolbutamide in Sox4 siRNA treated cells. Tolbutamide stimulates insulin secretion in pancreatic beta cells by closing KATP channels in the plasma membrane, leading to membrane depolarisation and activation of voltage-gated calcium channels. This results in calcium influx and a rise in intracellular calcium ([Ca2+]i) that triggers insulin release. Changes in [Ca2+]i evoked by glucose in Sox4-siRNA treated cells, however, were normal, indicating that KATP channels close normally. Taken together with the impaired secretory response to tolbutamide, this suggests the insulin secretory defect produced by Sox4 knockdown lies downstream of calcium signalling, perhaps at the level of exocytosis. [ABSTRACT FROM AUTHOR]

Presents a study which examined the reappearance of ...-sarcomeric actin transcripts and protein in mouse striated muscles. Details on the profile characteristic of adult skeletal muscle; Methodology used to conduct the study; Results of the study.

The ZIC proteins are a family of transcription regulators with a well-defined zinc finger DNA-binding domain and there is evidence that they elicit functional DNA binding at a ZIC DNA binding site. Little is known, however, regarding domains within ZIC proteins that confer trans-activation or -repression. To address this question, a new cell-based trans-activation assay system suitable for ZIC proteins in HEK293T cells was constructed. This identified two previously unannotated evolutionarily conserved regions of ZIC3 that are necessary for trans-activation. These domains are found in all Subclass A ZIC proteins, but not in the Subclass B proteins. Additionally, the Subclass B proteins fail to elicit functional binding at a multimerised ZIC DNA binding site. All ZIC proteins, however, exhibit functional binding when the ZIC DNA binding site is embedded in a multiple transcription factor locus derived from ZIC target genes in the mouse genome. This ability is due to several domains, some of which are found in all ZIC proteins, that exhibit context dependent trans-activation or -repression activity. This knowledge is valuable for assessing the likely pathogenicity of variant ZIC proteins associated with human disorders and for determining factors that influence functional transcription factor binding.

Quantitative reverse transcriptase PCR (RT-qPCR), a powerful and efficient means of rapidly comparing gene expression between experimental conditions, is routinely used as a phenotyping tool in developmental biology. The accurate comparison of gene expression across multiple embryonic stages requires normalisation to reference genes that have stable expression across the time points to be examined. As the embryo and its constituent tissues undergo rapid growth and differentiation during development, reference genes known to be stable across some time points cannot be assumed to be stable across all developmental stages. The immediate post-implantation events of gastrulation and patterning are characterised by a rapid expansion in cell number and increasing specialisation of cells. The optimal reference genes for comparative gene expression studies at these specific stages have not been experimentally identified. In this study, the expression of five commonly used reference genes (H2afz, Ubc, Actb, Tbp and Gapdh) was measured across murine gastrulation and patterning (6.5-9.5 dpc) and analysed with the normalisation tools geNorm, Bestkeeper and Normfinder. The results, validated by RT-qPCR analysis of two genes with well-documented expression patterns across these stages, indicated the best strategy for RT-qPCR studies spanning murine gastrulation and patterning utilises the concurrent reference genes H2afz and Ubc.

The ZIC2 transcription factor is one of the most commonly mutated genes in Holoprosencephaly (HPE) probands. HPE is a severe congenital defect of forebrain development which occurs when the cerebral hemispheres fail to separate during the early stages of organogenesis and is typically associated with mispatterning of the embryonic midline. Recent study of genotype-phenotype correlations in HPE cases has defined distinctive features of ZIC2-associated HPE presentation and genetics, revealing that ZIC2 mutation does not produce the craniofacial abnormalities generally thought to characterise HPE but leads to a range of non-forebrain phenotypes. Furthermore, the studies confirm the extent of ZIC2 allelic heterogeneity and that pathogenic variants of ZIC2 are associated with both classic and middle interhemispheric variant (MIHV) HPE which arise from defective ventral and dorsal forebrain patterning, respectively. An allelic series of mouse mutants has helped to delineate the cellular and molecular mechanisms by which one gene leads to defects in these related but distinct embryological processes.

The five murine Zic genes encode multifunctional transcriptional regulator proteins important for a large number of processes during embryonic development. The genes and proteins are highly conserved with respect to the orthologous human genes, an attribute evidently mirrored by functional conservation, since the murine and human genes mutate to give the same phenotypes. Each ZIC protein contains a zinc finger domain that participates in both protein-DNA and protein-protein interactions. The ZIC proteins are capable of interacting with the key transcriptional mediators of the SHH, WNT and NODAL signalling pathways as well as with components of the transcriptional machinery and chromatin-modifying complexes. It is possible that this diverse range of protein partners underlies characteristics uncovered by mutagenesis and phenotyping of the murine Zic genes. These features include redundant and unique roles for ZIC proteins, regulatory interdependencies amongst family members and pleiotropic Zic gene function. Future investigations into the complex nature of the Zic gene family activity should be facilitated by recent advances in genome engineering and functional genomics.

The ZIC2 transcription factor is one of the genes most commonly mutated in Holoprosencephaly (HPE) probands. Studies in cultured cell lines and mice have shown a loss of ZIC2 function is the pathogenic mechanism but the molecular details of this ZIC2 requirement remain elusive. HPE arises when signals that direct morphological and fate changes in the developing brain and facial primordia are not sent or received. One critical signal is sent from the prechordal plate (PrCP) which develops beneath the ventral forebrain. An intact NODAL signal transduction pathway and functional ZIC2 are both required for PrCP establishment. We now show that ZIC2 acts downstream of the NODAL signal during PrCP development. ZIC2 physically interacts with SMAD2 and SMAD3, the receptor activated proteins that control transcription in a NODAL dependent manner. Together SMAD3 and ZIC2 regulate FOXA2 transcription in cultured cells and Zic2 also controls the foxA2 expression during Xenopus development. Variant forms of the ZIC2 protein, associated with HPE in man or mouse, are deficient in their ability to influence SMAD-dependent transcription. These findings reveal a new mechanism of NODAL signal transduction in the mammalian node and provide the first molecular explanation of how ZIC2 loss-of-function precipitates HPE., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Murine Zic genes (Zic1-5) are expressed in the dorsal hindbrain and in periotic mesenchyme (POM) adjacent to the developing inner ear. Zic genes are involved in developmental signaling pathways in many organ systems, including the ear, although their exact roles haven't been fully elucidated. This report examines the role of Zic1, Zic2, and Zic4 during inner ear development in mouse mutants in which these Zic genes are affected., Results: Zic1/Zic4 double mutants don't exhibit any apparent defects in inner ear morphology. By contrast, inner ears from Zic2(kd/kd) and Zic2(Ku/Ku) mutants have severe but variable morphological defects in endolymphatic duct/sac and semicircular canal formation and in cochlear extension in the inner ear. Analysis of otocyst patterning in the Zic2(Ku/Ku) mutants by in situ hybridization showed changes in the expression patterns of Gbx2 and Pax2., Conclusions: The experiments provide the first genetic evidence that the Zic genes are required for morphogenesis of the inner ear. Zic2 loss-of-function doesn't prevent initial otocyst patterning but leads to molecular abnormalities concomitant with morphogenesis of the endolymphatic duct. Functional hearing deficits often accompany inner ear dysmorphologies, making Zic2 a novel candidate gene for ongoing efforts to identify the genetic basis of human hearing loss., (© 2014 Wiley Periodicals, Inc.)

Embryonic development and adult homeostasis are dependent upon the coordinated action of signal transduction pathways such as the Wnt signalling pathway which is used iteratively during these processes. In the early post-implantation mouse embryo, Wnt/β-catenin signalling activity plays a critical role in the formation of the primitive streak, progression of gastrulation and tissue patterning in the anterior-posterior axis. The net output of the signalling pathway is influenced by the delivery and post-translational modification of the ligands, the counteracting activities of the activating components and the negative modulators, and the molecular interaction of β-catenin, TCF and other factors regulating the transcription of downstream target genes., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The Zic genes encode zinc finger containing proteins that can bind proteins and DNA. The understanding of Zic molecular networks has been hampered by functional redundancy amongst family members, and because their loss-of-function phenotypes are indicative of a role in many signalling pathways. Recently molecular evidence has emerged confirming the pleiotropic nature of these proteins: they act both as classical transcription factors and as co-factors to directly and indirectly influence gene expression. It has long been known that germ-line mutation of the Zic genes in human and mouse causes a range of congenital disorders. Recently connections between Zic proteins and stem cell function have also emerged suggesting a role in adult onset diseases. The immediate challenge is to determine when and where these proteins act as transcription factors/co-factors during development and disease and how the switch between these roles is controlled., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Flightless I (Flii) is an actin-remodeling protein that influences diverse processes including cell migration and gene transcription and links signal transduction with cytoskeletal regulation. Here, we show that Flii modulation of focal adhesions and filamentous actin stress fibers is Rac1-dependent. Using primary skin fibroblasts from Flii overexpressing (Flii(Tg/Tg)), wild-type, and Flii deficient (Flii(+/-)) mice, we show that elevated expression of Flii increases stress fiber formation by impaired focal adhesion turnover and enhanced formation of fibrillar adhesions. Conversely, Flii knockdown increases the percentage of focal complex positive cells. We further show that a functional effect of Flii at both the cellular level and in in vivo mouse wounds is through inhibiting paxillin tyrosine phosphorylation and suppression of signaling proteins Src and p130Cas, both of which regulate adhesion signaling pathways. Flii is upregulated in response to wounding, and overexpression of Flii inhibits paxillin activity and reduces adhesion signaling by modulating the activity of the Rho family GTPases. Overexpression of constitutively active Rac1 GTPase restores the spreading ability of Flii(Tg/Tg) fibroblasts and may explain the reduced adhesion, migration, and proliferation observed in Flii(Tg/Tg) mice and their impaired wound healing, a process dependent on effective cellular motility and adhesion.

Monosomy of the human chromosome 6p terminal region results in a variety of congenital malformations that include brain, craniofacial, and organogenesis abnormalities. To examine the genetic basis of these phenotypes, we have carried out an unbiased functional analysis of the syntenic region of the mouse genome (proximal Mmu13). A genetic screen for recessive mutations in this region recovered thirteen lines with phenotypes relevant to a variety of clinical conditions. These include two loci that cause holoprosencephaly, two that underlie anophthalmia, one of which also contributes to other craniofacial abnormalities such as microcephaly, agnathia, and palatogenesis defects, and one locus responsible for developmental heart and kidney defects. Analysis of heterozygous carriers of these mutations shows that a high proportion of these loci manifest with behavioral activity and sensorimotor deficits in the heterozygous state. This finding argues for the systematic, reciprocal phenotypic assessment of dominant and recessive mouse mutants. In addition to providing a resource of single gene mutants that model 6p-associated disorders, the work reveals unsuspected genetic complexity at this region. In particular, many of the phenotypes associated with 6p deletions can be elicited by mutation in one of a number of genes. This finding implies that phenotypes associated with contiguous gene deletion syndromes can result not only from dosage sensitivity of one gene in the region but also from the combined effect of monosomy for multiple genes that function within the same biological process.

We have developed a sensitized screen to identify genes involved in gene silencing, using random N-ethyl-N-nitrosourea mutagenesis on mice carrying a variegating GFP transgene. The dominant screen has produced six mutant lines, including both suppressors and enhancers of variegation. All are semidominant and five of the six are homozygous embryonic lethal. In one case, the homozygous lethality depends on sex: homozygous females die at midgestation and display abnormal DNA methylation of the X chromosome, whereas homozygous males are viable. Linkage analysis reveals that the mutations map to unique chromosomal locations. We have studied the effect of five of the mutations on expression of an endogenous allele known to be sensitive to epigenetic state, agouti viable yellow. In all cases, there is an effect on penetrance, and in most cases, parent of origin and sex-specific effects are detected. This screen has identified genes that are involved in epigenetic reprogramming of the genome, and the behavior of the mutant lines suggests a common mechanism between X inactivation and transgene and retrotransposon silencing. Our findings raise the possibility that the presence or absence of the X chromosome in mammals affects the establishment of the epigenetic state at autosomal loci by acting as a sink for proteins involved in gene silencing. The study demonstrates the power of sensitized screens in the mouse not only for the discovery of novel genes involved in a particular process but also for the elucidation of the biology of that process.