Recent developments in targets for ischemic foot disease.

Diabetes is a key risk factor for ischaemic foot disease, which causes pain, tissue loss, hospital admission, and major amputation. Currently, treatment focuses on revascularisation, but many patients are unsuitable for surgery and revascularisation is frequently unsuccessful. The authors describe recent research in animal models and clinical trials investigating novel medical targets for ischaemia, including theories about impaired wound healing, animal models for limb ischaemia and recent randomised controlled trials testing novel medical therapies. Novel targets identified in animal models included stimulating mobilisation of CD34+ progenitor cells through upregulating oncostatin M or microRNA-181, downregulating tumour necrosis factor superfamily member 14, or activating the Wingless pathway. Within the ischaemic limb vasculature, upregulation of apolipoprotein L domain containing 1, microRNA-130b or long noncoding RNA that enhances endothelial nitric oxide synthase expression promoted limb blood supply recovery, angiogenesis, and arteriogenesis. Similarly, administration of soluble guanylate cyclase stimulators riociguat or praliciguat or 3-ketoacyl-CoA thiolase inhibitor trimetazidine promoted blood flow recovery. Translating pre-clinical findings to patients has been challenging, mainly due to limitations in clinically translatable animal models of human disease. Promising results have been reported for administering plasmids encoding hepatocyte growth factor or intra-arterial injection of bone marrow derived cells in small clinical trials. It remains to be seen whether these high resource therapies can be developed to be widely applicable. In conclusion, an ever-expanding list of potential targets for medical revascularisation is being identified. It is hoped that through ongoing research and further larger clinical trials, these will translate into new broadly applicable therapies to improve outcomes.

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.

35th International Mammalian Genome Conference: meeting overview.

The 35th International Mammalian Genome Conference (IMGC) was held on July 17-20, 2022 in Vancouver, British Columbia; this conference marked the first time the International Mammalian Genome Society (IMGS) hosted a meeting in Canada. Scientists from around the world participated to share advances in genetics and genomics research across mammalian species. A diverse attendance of pre-doctoral and post-doctoral trainees, young investigators, established researchers, clinicians, bioinformaticians, and computational biologists enjoyed a rich scientific program selected from 88 abstracts in the fields of cancer, conservation genetics, developmental biology, epigenetics, human disease modeling, immunology, infectious diseases, systems genetics, translational biology, and technological advances.

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.

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.

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.

Production of Digoxigenin-Labeled Riboprobes for In Situ Hybridization Experiments.

Experiments that visualize gene expression in intact tissues or organisms are fundamental to studies of gene function. These experiments, called in situ hybridization, require the production of a riboprobe, which is a labeled antisense RNA corresponding to a particular gene. The most commonly used system for visualizing gene expression via in situ hybridization is the incorporation of a digoxigenin label into an in vitro-transcribed RNA probe. After hybridization of the riboprobe to a target mRNA, its location can be detected via a high-affinity α-digoxigenin antibody conjugated to an alkaline-phosphatase enzyme. The article describes the design and production of digoxigenin-labeled riboprobes transcribed in vitro from template DNA (either plasmid or PCR amplicon). These riboprobes are suitable for use in tissue and whole-mount in situ hybridization protocols. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Plasmid-derived riboprobes Alternate Protocol: PCR-derived riboprobes Basic Protocol 2: Riboprobe synthesis with DIG label.

Whole-Mount In Situ Hybridization in Post-Implantation Staged Mouse Embryos.

Understanding RNA expression in space and time is a key initial step in dissecting gene function. The ability to visualize gene expression in whole-tissue or whole-specimen preparations, called in situ hybridization (ISH), was first developed 50 years ago. Two decades later, these protocols were adapted to establish robust methods for whole-mount ISH to murine embryos. The precise protocols vary somewhat between early-gestation and mid-gestation mouse embryos; the protocol presented here is optimal for use with post-implantation stage mouse embryos (stages 5.5-9.5 dpc). Routine uses of whole-mount ISH include documenting the wild-type expression pattern of individual genes and comparison of the expression pattern of signature genes (i.e., those that identify particular cells and tissues within an embryo) between wild-type and mutant embryos as part of a phenotyping experiment. This technique remains a mainstay of developmental biology studies and complements the massively parallel assessment of gene expression from dissociated tissues and cells via RNA-sequencing techniques. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Dissection of post-implantation (5.5-9.5 dpc) murine embryos Basic Protocol 2: Whole-mount in situ hybridization in post-implantation embryos Basic Protocol 3: Visualization of post-WMISH embryos Support Protocol 1: Creation of siliconized glass pipettes Support Protocol 2: Creation of embryo powder.

A murine Zic3 transcript with a premature termination codon evades nonsense-mediated decay during axis formation.

The ZIC transcription factors are key mediators of embryonic development and ZIC3 is the gene most commonly associated with situs defects (heterotaxy) in humans. Half of patient ZIC3 mutations introduce a premature termination codon (PTC). In vivo, PTC-containing transcripts might be targeted for nonsense-mediated decay (NMD). NMD efficiency is known to vary greatly between transcripts, tissues and individuals and it is possible that differences in survival of PTC-containing transcripts partially explain the striking phenotypic variability that characterizes ZIC3-associated congenital defects. For example, the PTC-containing transcripts might encode a C-terminally truncated protein that retains partial function or that dominantly interferes with other ZIC family members. Here we describe the katun (Ka) mouse mutant, which harbours a mutation in the Zic3 gene that results in a PTC. At the time of axis formation there is no discernible decrease in this PTC-containing transcript in vivo, indicating that the mammalian Zic3 transcript is relatively insensitive to NMD, prompting the need to re-examine the molecular function of the truncated proteins predicted from human studies and to determine whether the N-terminal portion of ZIC3 possesses dominant-negative capabilities. A combination of in vitro studies and analysis of the Ka phenotype indicate that it is a null allele of Zic3 and that the N-terminal portion of ZIC3 does not encode a dominant-negative molecule. Heterotaxy in patients with PTC-containing ZIC3 transcripts probably arises due to loss of ZIC3 function alone.