Equitable Expanded Carrier Screening Needs Indigenous Clinical and Population Genomic Data.

Expanded carrier screening (ECS) for recessive monogenic diseases requires prior knowledge of genomic variation, including DNA variants that cause disease. The composition of pathogenic variants differs greatly among human populations, but historically, research about monogenic diseases has focused mainly on people with European ancestry. By comparison, less is known about pathogenic DNA variants in people from other parts of the world. Consequently, inclusion of currently underrepresented Indigenous and other minority population groups in genomic research is essential to enable equitable outcomes in ECS and other areas of genomic medicine. Here, we discuss this issue in relation to the implementation of ECS in Australia, which is currently being evaluated as part of the national Government's Genomics Health Futures Mission. We argue that significant effort is required to build an evidence base and genomic reference data so that ECS can bring significant clinical benefit for many Aboriginal and/or Torres Strait Islander Australians. These efforts are essential steps to achieving the Australian Government's objectives and its commitment "to leveraging the benefits of genomics in the health system for all Australians." They require culturally safe, community-led research and community involvement embedded within national health and medical genomics programs to ensure that new knowledge is integrated into medicine and health services in ways that address the specific and articulated cultural and health needs of Indigenous people. Until this occurs, people who do not have European ancestry are at risk of being, in relative terms, further disadvantaged.

WNT-responsive SUMOylation of ZIC5 promotes murine neural crest cell development, having multiple effects on transcription.

Zinc finger of the cerebellum (Zic) proteins act as classic transcription factors to promote transcription of the Foxd3 gene during neural crest cell specification. Additionally, they can act as co-factors that bind proteins from the T-cell factor/lymphoid enhancing factor (TCF/LEF) family (TCFs) to repress WNT-ß-catenin-dependent transcription without contacting DNA. Here, we show that ZIC activity at the neural plate border is influenced by WNT-dependent SUMOylation. In the presence of high canonical WNT activity, a lysine residue within the highly conserved zinc finger N-terminally conserved (ZF-NC) domain of ZIC5 is SUMOylated, which reduces formation of the ZIC-TCF co-repressor complex and shifts the balance towards transcription factor function. The modification is crucial in vivo, as a ZIC5 SUMO-incompetent mouse strain exhibits neural crest specification defects. This work reveals the function of the ZF-NC domain within ZIC, provides in vivo validation of target protein SUMOylation and demonstrates that WNT-ß-catenin signalling directs transcription at non-TCF DNA-binding sites. Furthermore, it can explain how WNT signals convert a broad region of Zic ectodermal expression into a restricted region of neural crest cell specification.

SUMOylation Potentiates ZIC Protein Activity to Influence Murine Neural Crest Cell Specification.

The mechanisms of neural crest cell induction and specification are highly conserved among vertebrate model organisms, but how similar these mechanisms are in mammalian neural crest cell formation remains open to question. The zinc finger of the cerebellum 1 (ZIC1) transcription factor is considered a core component of the vertebrate gene regulatory network that specifies neural crest fate at the neural plate border. In mouse embryos, however, Zic1 mutation does not cause neural crest defects. Instead, we and others have shown that murine Zic2 and Zic5 mutate to give a neural crest phenotype. Here, we extend this knowledge by demonstrating that murine Zic3 is also required for, and co-operates with, Zic2 and Zic5 during mammalian neural crest specification. At the murine neural plate border (a region of high canonical WNT activity) ZIC2, ZIC3, and ZIC5 function as transcription factors to jointly activate the Foxd3 specifier gene. This function is promoted by SUMOylation of the ZIC proteins at a conserved lysine immediately N-terminal of the ZIC zinc finger domain. In contrast, in the lateral regions of the neurectoderm (a region of low canonical WNT activity) basal ZIC proteins act as co-repressors of WNT/TCF-mediated transcription. Our work provides a mechanism by which mammalian neural crest specification is restricted to the neural plate border. Furthermore, given that WNT signaling and SUMOylation are also features of non-mammalian neural crest specification, it suggests that mammalian neural crest induction shares broad conservation, but altered molecular detail, with chicken, zebrafish, and Xenopus neural crest induction.

Zic2 mutation causes holoprosencephaly via disruption of NODAL signalling.

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.

Identification of reference genes suitable for RT-qPCR studies of murine gastrulation and patterning.

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.

ZIC2 in Holoprosencephaly.

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.

Overview of Rodent Zic Genes.

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.

Mutation of the diamond-blackfan anemia gene Rps7 in mouse results in morphological and neuroanatomical phenotypes.

The ribosome is an evolutionarily conserved organelle essential for cellular function. Ribosome construction requires assembly of approximately 80 different ribosomal proteins (RPs) and four different species of rRNA. As RPs co-assemble into one multi-subunit complex, mutation of the genes that encode RPs might be expected to give rise to phenocopies, in which the same phenotype is associated with loss-of-function of each individual gene. However, a more complex picture is emerging in which, in addition to a group of shared phenotypes, diverse RP gene-specific phenotypes are observed. Here we report the first two mouse mutations (Rps7(Mtu) and Rps7(Zma)) of ribosomal protein S7 (Rps7), a gene that has been implicated in Diamond-Blackfan anemia. Rps7 disruption results in decreased body size, abnormal skeletal morphology, mid-ventral white spotting, and eye malformations. These phenotypes are reported in other murine RP mutants and, as demonstrated for some other RP mutations, are ameliorated by Trp53 deficiency. Interestingly, Rps7 mutants have additional overt malformations of the developing central nervous system and deficits in working memory, phenotypes that are not reported in murine or human RP gene mutants. Conversely, Rps7 mouse mutants show no anemia or hyperpigmentation, phenotypes associated with mutation of human RPS7 and other murine RPs, respectively. We provide two novel RP mouse models and expand the repertoire of potential phenotypes that should be examined in RP mutants to further explore the concept of RP gene-specific phenotypes.

Fibroblast-specific upregulation of Flightless I impairs wound healing.

The cytoskeletal protein Flightless (Flii) is a negative regulator of wound healing. Upregulation of Flii is associated with impaired migration, proliferation and adhesion of both fibroblasts and keratinocytes. Importantly, Flii translocates from the cytoplasm to the nucleus in response to wounding in fibroblasts but not keratinocytes. This cell-specific nuclear translocation of Flii suggests that Flii may directly regulate gene expression in fibroblasts, providing one potential mechanism of action for Flii in the wound healing response. To determine whether the tissue-specific upregulation of Flii in fibroblasts was important for the observed inhibitory effects of Flii on wound healing, an inducible fibroblast-specific Flii overexpressing mouse model was generated. The inducible ROSA26 system allowed the overexpression of Flii in a temporal and tissue-specific manner in response to tamoxifen treatment. Wound healing in the inducible mice was impaired, with wounds at day 7 postwounding significantly larger than those from non-inducible controls. There was also reduced collagen maturation, increased myofibroblast infiltration and elevated inflammation. The impaired healing response was similar in magnitude to that observed in mice with non-tissue-specific upregulation of Flii suggesting that fibroblast-derived Flii may have an important role in the wound healing response.

Flightless I over-expression impairs skin barrier development, function and recovery following skin blistering.

Development of an intact epidermis is critical for maintaining the integrity of the skin. Patients with epidermolysis bullosa (EB) experience multiple erosions, which breach the epidermal barrier and lead to increased microbial colocalization of wounds, infections and sepsis. The cytoskeletal protein Flightless I (Flii) is a known regulator of both development and wound healing. Using Flii(+/-), WT and Flii(Tg/Tg) mice, we investigated the effect of altering Flii levels in embryos and adult mice on the development of the epidermal barrier and, consequently, how this affects the integrity of the skin in EB. Flii over-expression resulted in delayed formation of the epidermal barrier in embryos and decreased expression of tight junction (TJ) proteins Claudin-1 and ZO-2. Increased intercellular space and transepidermal water loss was observed in Flii(Tg)(/Tg) adult mouse skin, while Flii(Tg/Tg) keratinocytes showed altered TJ protein localization and reduced transepithelial resistance. Flii is increased in the blistered skin of patients with EB, and over-expression of Flii in experimental EBA showed impaired Claudin-1 and -4 TJ protein expression and delayed recovery of functional barrier post-blistering. Immunoprecipitation confirmed Flii associated with TJ proteins and in vivo actin assays showed that the effect of Flii on actin polymerization underpinned the impaired barrier function observed in Flii(Tg/Tg) mice. These results therefore demonstrate an important role for Flii in the development and regulation of the epidermal barrier, which may contribute to the impaired healing and skin fragility of EB patients.

The role of Zic genes in inner ear development in the mouse: Exploring mutant mouse phenotypes.

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.

The Zic2 gene directs the formation and function of node cilia to control cardiac situs.

The first molecular herald of organ asymmetry during murine embryogenesis is found at the periphery of the node in early-somite stage embryos. Asymmetric gene expression and calcium accumulation at the node occurs in response to a left-ward flow of extracellular fluid across the node, generated by motile cilia within the pit of the node and likely sensed by immotile cilia in the periphery of the node. The ciliation of node cells is controlled by a cascade of node-restricted transcription factor activity during mid-late gastrulation. Mutation of the murine Zic2 transcription factor is associated with random cardiac situs and a loss of asymmetric gene expression at the early-somite node and in the lateral plate. Zic2 is not expressed in these regions but is transiently expressed in the mid-late gastrula node at the time of ciliogenesis. The cilia of the node are overtly abnormal in Zic2 mutant embryos being dysmorphic and short relative to wild-type littermates. The expression of the Noto, Rfx3, and Foxj1 transcription factors known to regulate ciliogenesis is greatly depleted in the mid-gastrula node of mutants, as is the expression of the Pkd1l1 gene required for cilia function. Zic2 appears to be a component of the gene regulatory network that drives ciliation of node cells during gastrulation.

Attenuation of flightless I improves wound healing and enhances angiogenesis in a murine model of type 1 diabetes.

AIMS/HYPOTHESIS: Skin lesions and ulcerations are severe complications of diabetes that often result in leg amputations. In this study we investigated the function of the cytoskeletal protein flightless I (FLII) in diabetic wound healing. We hypothesised that overexpression of FLII would have a negative effect on diabetic wound closure and modulation of this protein using specific FLII-neutralising antibodies (FnAb) would enhance cellular proliferation, migration and angiogenesis within the diabetic wound. METHODS: Using a streptozotocin-induced model of diabetes we investigated the effect of altered FLII levels through Flii genetic knockdown, overexpression or treatment with FnAb on wound healing. Diabetic wounds were assessed using histology, immunohistochemistry and biochemical analysis. In vitro and in vivo assays of angiogenesis were used to assess the angiogenic response. RESULTS: FLII levels were elevated in the wounds of both diabetic mice and humans. Reduction in the level of FLII improved healing of murine diabetic wounds and promoted a robust pro-angiogenic response with significantly elevated von Willebrand factor (vWF) and vascular endothelial growth factor (VEGF)-positive endothelial cell infiltration. Diabetic mouse wounds treated intradermally with FnAb showed improved healing and a significantly increased rate of re-epithelialisation. FnAb improved the angiogenic response through enhanced formation of capillary tubes and functional neovasculature. Reducing the level of FLII led to increased numbers of mature blood vessels, increased recruitment of smooth muscle actin-α-positive cells and improved tight junction formation. CONCLUSIONS/INTERPRETATION: Reducing the level of FLII in a wound may be a potential therapeutic approach for the treatment of diabetic foot ulcers.

Overexpression of the Flii gene increases dermal-epidermal blistering in an autoimmune ColVII mouse model of epidermolysis bullosa acquisita.

Epidermolysis bullosa (EB) is a severe genetic skin fragility syndrome characterized by blister formation. The molecular basis of EB is still largely unknown and wound healing in patients suffering from EB remains a major challenge to their survival. Our previous studies have identified the actin remodelling protein Flightless I (Flii) as an important mediator of wound repair. Here we identify Flii as a novel target involved in skin blistering. Flii expression was significantly elevated in 30 patients with EB, most prominently in patients with recessive dystrophic EB (RDEB) who have defects in production of type VII collagen (ColVII). Using an autoimmune ColVII murine model of EB acquisita (EBA) and an immunocompetent-ColVII-hypomorphic genetic mouse model of RDEB together with murine Flii alleles, we investigated the contribution of Flii to EB. Overexpression of Flii produced severe blistering post-induction of EBA, while decreased Flii reduced blister severity, elevated integrin expression, and improved ColVII production. Flii(+/-) blistered skin showed reduced α-SMA, TGF-ß1, and Smad 2/3 expression, suggesting that decreasing Flii may affect fibrosis. In support of this, Flii-deficient fibroblasts from EBA mice were less able to contract collagen gels in vitro; however, addition of TGF-ß1 restored collagen contraction, suggesting an interplay between Flii and TGF-ß1. Elevated Flii gene and protein expression was further observed in the blisters of ColVII hypomorphic mice, a murine model of RDEB, suggesting that reducing Flii in blistered skin could be a potential new approach for treating patients with EB.

Patterning of the antero-ventral mammalian brain: Lessons from holoprosencephaly comparative biology in man and mouse.

Adult form and function are dependent upon the activity of specialized signaling centers that act early in development at the embryonic midline. These centers instruct the surrounding cells to adopt a positional fate and to form the patterned structures of the phylotypic embryo. Abnormalities in these processes have devastating consequences for the individual, as exemplified by holoprosencephaly in which anterior midline development fails, leading to structural defects of the brain and/or face. In the 25 years since the first association between human holoprosencephaly and the sonic hedgehog gene, a combination of human and animal genetic studies have enhanced our understanding of the genetic and embryonic causation of this congenital defect. Comparative biology has extended the holoprosencephaly network via the inclusion of gene mutations from multiple signaling pathways known to be required for anterior midline formation. It has also clarified aspects of holoprosencephaly causation, showing that it arises when a deleterious variant is present within a permissive genome, and that environmental factors, as well as embryonic stochasticity, influence the phenotypic outcome of the variant. More than two decades of research can now be distilled into a framework of embryonic and genetic causation. This framework means we are poised to move beyond our current understanding of variants in signaling pathway molecules. The challenges now at the forefront of holoprosencephaly research include deciphering how the mutation of genes involved in basic cell processes can also cause holoprosencephaly, determining the important constituents of the holoprosencephaly permissive genome, and identifying environmental compounds that promote holoprosencephaly. This article is categorized under: Congenital Diseases > Stem Cells and Development Congenital Diseases > Genetics/Genomics/Epigenetics Congenital Diseases > Molecular and Cellular Physiology Congenital Diseases > Environmental Factors.

Mouse strains for the ubiquitous or conditional overexpression of the Flii gene.

The gelsolin related actin binding protein, Flii, is able to regulate wound healing; mice with decreased Flii expression show improved wound healing whereas mice with elevated Flii expression exhibit impaired wound healing. In both mice and humans Flii expression increases with age and amelioration of FLII activity represents a possible therapeutic strategy for improved wound healing in humans. Despite analysis of Flii function in a variety of organisms we know little of the molecular mechanisms underlying Flii action. Two new murine alleles of Flii have been produced to drive constitutive or tissue-specific expression of Flii. Each strain is able to rescue the embryonic lethality associated with a Flii null allele and to impair wound healing. These strains provide valuable resources for ongoing investigation of Flii function in a variety of biological processes.

Decreased expression of Flightless I, a gelsolin family member and developmental regulator, in early-gestation fetal wounds improves healing.

Up until late in the third trimester of gestation and through to adulthood, the healing response acts more to regenerate than to repair a wound. The mechanisms underlying this "scar-free" healing remain unknown although the actin cytoskeleton has a major role. Flightless I (Flii), an actin-remodelling protein and essential developmental regulator, negatively affects wound repair but its effect on scar-free fetal healing is unknown. Using fetal skin explants from E17 (regenerate) and E19 (repair) rats, the function of Flii in fetal wound repair was determined. Expression of Flii increased between E17 and E19 days of gestation and wounding transiently increased Flii expression in E17 but not E19 wounds. However, both confocal and immunofluorescent analysis showed E17 keratinocytes immediately adjacent to the wounds downregulated Flii. As a nuclear coactivator and inhibitor of proliferation and migration, the absence of Flii in cells at the edge of the wound could be instrumental in allowing these cells to proliferate and migrate into the wound deficit. In contrast, Flii was strongly expressed within the cytoplasm and nucleus of keratinocytes within epidermal cells at the leading edge of E19 wounded fetal skin explants. This increase in Flii expression in E19 wounds could affect the way these cells migrate into the wound space and contribute to impaired wound healing. Neutralising Flii protein improved healing of early- but not late-gestation wounds. Flii did not colocalise with actin cables formed around E17 wounds suggesting an independent mechanism of action distinct from its actin-binding function in scar-free wound repair.

Disruption of entire Cables2 locus leads to embryonic lethality by diminished Rps21 gene expression and enhanced p53 pathway.

In vivo function of CDK5 and Abl enzyme substrate 2 (Cables2), belonging to the Cables protein family, is unknown. Here, we found that targeted disruption of the entire Cables2 locus (Cables2d) caused growth retardation and enhanced apoptosis at the gastrulation stage and then induced embryonic lethality in mice. Comparative transcriptome analysis revealed disruption of Cables2, 50% down-regulation of Rps21 abutting on the Cables2 locus, and up-regulation of p53-target genes in Cables2d gastrulas. We further revealed the lethality phenotype in Rps21-deleted mice and unexpectedly, the exon 1-deleted Cables2 mice survived. Interestingly, chimeric mice derived from Cables2d ESCs carrying exogenous Cables2 and tetraploid wild-type embryo overcame gastrulation. These results suggest that the diminished expression of Rps21 and the completed lack of Cables2 expression are intricately involved in the embryonic lethality via the p53 pathway. This study sheds light on the importance of Cables2 locus in mouse embryonic development.

Zic2-associated holoprosencephaly is caused by a transient defect in the organizer region during gastrulation.

The putative transcription factor ZIC2 is associated with a defect of forebrain development, known as Holoprosencephaly (HPE), in humans and mouse, yet the mechanism by which aberrant ZIC2 function causes classical HPE is unexplained. The zinc finger domain of all mammalian Zic genes is highly homologous with that of the Gli genes, which are transcriptional mediators of Shh signalling. Mutations in Shh and many other Hh pathway members cause HPE and it has been proposed that Zic2 acts within the Shh pathway to cause HPE. We have investigated the embryological cause of Zic2-associated HPE and the relationship between Zic2 and the Shh pathway using mouse genetics. We show that Zic2 does not interact with Shh to produce HPE. Moreover, molecular defects that are able to account for the HPE phenotype are present in Zic2 mutants before the onset of Shh signalling. Mutation of Zic2 causes HPE via a transient defect in the function of the organizer region at mid-gastrulation which causes an arrest in the development of the prechordal plate (PCP), a structure required for forebrain midline morphogenesis. The analysis provides genetic evidence that Zic2 functions during organizer formation and that the PCP develops via a multi-step process.