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In a screen for human kinases that regulate Xenopus laevis embryogenesis, we identified Nagk and other components of the UDP-GlcNAc glycosylation salvage pathway as regulators of anteroposterior patterning and Wnt signaling. We find that the salvage pathway does not affect other major embryonic signaling pathways (Fgf, TGFβ, Notch, or Shh), thereby demonstrating specificity for Wnt signaling. We show that the role of the salvage pathway in Wnt signaling is evolutionarily conserved in zebrafish and Drosophila. Finally, we show that GlcNAc is essential for the growth of intestinal enteroids, which are highly dependent on Wnt signaling for growth and maintenance. We propose that the Wnt pathway is sensitive to alterations in the glycosylation state of a cell and acts as a nutritional sensor in order to couple growth/proliferation with its metabolic status. We also propose that the clinical manifestations observed in congenital disorders of glycosylation (CDG) in humans may be due, in part, to their effects on Wnt signaling during development.
Copyright © 2019 Elsevier B.V. All rights reserved.
SET domain-containing proteins play a vital role in regulating gene expression during development through modifications in chromatin structure. Here we show that SET domain-containing 5 (Setd5) is divergently transcribed with Gt(ROSA26)Sor, is necessary for mammalian development, and interacts with the PAF1 co-transcriptional complex and other proteins. Setd5-deficient mouse embryos exhibit severe defects in neural tube formation, somitogenesis and cardiac development, have aberrant vasculogenesis in embryos, yolk sacs and placentas, and die between embryonic day 10.5 and 11.5. Setd5-deficient embryonic stem cells have impaired cellular proliferation, increased apoptosis, defective cell cycle progression, a diminished ability to differentiate into cardiomyocytes and greatly perturbed gene expression. SETD5 co-immunoprecipitates with multiple components of the PAF1 and histone deacetylase-containing NCoR complexes and is not solely required for major histone lysine methylation marks. In the absence of Setd5, histone acetylation is increased at transcription start sites and near downstream regions. These findings suggest that SETD5 functions in a manner similar to yeast Set3p and Drosophila UpSET, and that it is essential for regulating histone acetylation during gene transcription.
© 2016. Published by The Company of Biologists Ltd.
The regulation of β-catenin turnover is the central mechanism governing activation of the Wnt signaling pathway. All components of the pathway are present in the early embryo of Xenopus laevis, and Xenopus egg extracts have been used to recapitulate complex biological reactions such as microtubule dynamics, DNA replication, chromatin assembly, and phases of the cell cycle. Herein, we describe a biochemical method for analyzing β-catenin degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extracts. We show that in such a biochemical system, cytoplasmic β-catenin degradation is regulated by soluble components of the Wnt pathway as well as small molecules.
During pregnancy, maternal β-cells undergo compensatory changes, including increased β-cell mass and enhanced glucose-stimulated insulin secretion. Failure of these adaptations to occur results in gestational diabetes mellitus. The secreted protein connective tissue growth factor (CTGF) is critical for normal β-cell development and promotes regeneration after partial β-cell ablation. During embryogenesis, CTGF is expressed in pancreatic ducts, vasculature, and β-cells. In adult pancreas, CTGF is expressed only in the vasculature. Here we show that pregnant mice with global Ctgf haploinsufficiency (Ctgf(LacZ/+)) have an impairment in maternal β-cell proliferation; no difference was observed in virgin Ctgf(LacZ/+) females. Using a conditional CTGF allele, we found that mice with a specific inactivation of CTGF in endocrine cells (Ctgf(ΔEndo)) develop gestational diabetes during pregnancy, but this is due to a reduction in glucose-stimulated insulin secretion rather than impaired maternal β-cell proliferation. Moreover, virgin Ctgf(ΔEndo) females also display impaired GSIS with glucose intolerance, indicating that underlying β-cell dysfunction precedes the development of gestational diabetes in this animal model. This is the first time a role for CTGF in β-cell function has been reported.
BACKGROUND - Dynamic activation and inactivation of gene regulatory DNA produce the expression changes that drive the differentiation of cellular lineages. Identifying regulatory regions active during developmental transitions is necessary to understand how the genome specifies complex developmental programs and how these processes are disrupted in disease. Gene regulatory dynamics are mediated by many factors, including the binding of transcription factors (TFs) and the methylation and acetylation of DNA and histones. Genome-wide maps of TF binding and DNA and histone modifications have been generated for many cellular contexts; however, given the diversity and complexity of animal development, these data cover only a small fraction of the cellular and developmental contexts of interest. Thus, there is a need for methods that use existing epigenetic and functional genomics data to analyze the thousands of contexts that remain uncharacterized.
RESULTS - To investigate the utility of histone modification data in the analysis of cellular contexts without such data, I evaluated how well genome-wide H3K27ac and H3K4me1 data collected in different developmental stages, tissues, and species were able to predict experimentally validated heart enhancers active at embryonic day 11.5 (E11.5) in mouse. Using a machine-learning approach to integrate the data from different contexts, I found that E11.5 heart enhancers can often be predicted accurately from data from other contexts, and I quantified the contribution of each data source to the predictions. The utility of each dataset correlated with nearness in developmental time and tissue to the target context: data from late developmental stages and adult heart tissues were most informative for predicting E11.5 enhancers, while marks from stem cells and early developmental stages were less informative. Predictions based on data collected in non-heart tissues and in human hearts were better than random, but worse than using data from mouse hearts.
CONCLUSIONS - The ability of these algorithms to accurately predict developmental enhancers based on data from related, but distinct, cellular contexts suggests that combining computational models with epigenetic data sampled from relevant contexts may be sufficient to enable functional characterization of many cellular contexts of interest.
Ror2 is a signaling receptor for Wnt ligands that is known to play important roles in limb development, but having no essential roles known in adult tissues. Recent evidence has implicated Ror2 in mediating both canonical and non-canonical signaling pathways. Ror2 was initially found to be highly expressed in osteosarcoma and renal cell carcinomas, and has recently been found in an increasingly long list of cancers currently including melanoma, colon cancer, melanoma, squamous cell carcinoma of the head and neck, and breast cancer. In the majority of these cancer types, Ror2 expression is associated with more aggressive disease states, consistent with a role mediating Wnt signaling regardless of the canonical or noncanonical signal. Because of the pattern of tissue distribution, the association with high-risk diseases, and the cell surface localization of this receptor, Ror2 has been identified as a potential high value target for therapeutic development. However, the recent discovery that Ror2 may function through non-kinase activities challenges this strategy and opens up opportunities to target this important molecule through alternative means.
Copyright © 2015 Elsevier Inc. All rights reserved.
The winged-helix transcription factor Foxh1 is an essential regulator of Nodal signaling during the key developmental processes of gastrulation, anterior-posterior (A-P) patterning, and the derivation of left-right (L-R) asymmetry. Current models have Foxh1 bound to phospho-Smad2/3 (pSmad2/3) as a central transcriptional activator for genes targeted by Nodal signaling including Nodal itself, the feedback inhibitor Lefty2, and the positive transcriptional effector Pitx2. However, the conserved Engrailed homology-1 (EH1) motif present in Foxh1 suggests that modulated interaction with Groucho (Grg) co-repressors would allow Foxh1 to function as a transcriptional switch, toggling between transcriptional on and off states via pSmad2-Grg protein-switching, to ensure the properly timed initiation and suppression, and/or amplitude, of expression of Nodal and its target genes. We minimally mutated the Foxh1 EH1 motif, creating a novel Foxh1(mEH1) allele to test directly the contribution of Foxh1-Grg-mediated repression on the transient, dynamic pattern of Nodal signaling in mice. All aspects of Nodal and its target gene expression in Foxh1(mEH1/mEH1) embryos were equivalent to wild type. A-P patterning and organ situs in homozygous embryos and adult mice were also unaffected. The finding that Foxh1-Grg-mediated repression is not essential for Nodal expression during mouse embryogenesis suggests that other regulators compensate for the loss of repressive regulatory input that is mediated by Grg interactions. We suggest that the pervasive inductive properties of Nodal signaling exist within the context of a strongly buffered regulatory system that contributes to resilience and accuracy of its dynamic expression pattern.
Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
Haematopoietic stem and progenitor cells are maintained by special microenvironments known as niches in bone marrow. Many studies have identified diverse candidate cells that constitute niches for haematopoietic stem cells in the marrow, including osteoblasts, endothelial cells, Schwann cells, α-smooth muscle actin-expressing macrophages and mesenchymal progenitors such as CXC chemokine ligand (CXCL)12-abundant reticular (CAR) cells, stem cell factor-expressing cells, nestin-expressing cells and platelet-derived growth factor receptor-α (PDGFR-α)(+)Sca-1(+)CD45(-)Ter119(-) (PαS) cells. However, the molecular basis of the formation of the niches remains unclear. Here we find that the transcription factor Foxc1 is preferentially expressed in the adipo-osteogenic progenitor CAR cells essential for haematopoietic stem and progenitor cell maintenance in vivo in the developing and adult bone marrow. When Foxc1 was deleted in all marrow mesenchymal cells or CAR cells, from embryogenesis onwards, osteoblasts appeared normal, but haematopoietic stem and progenitor cells were markedly reduced and marrow cavities were occupied by adipocytes (yellow adipose marrow) with reduced CAR cells. Inducible deletion of Foxc1 in adult mice depleted haematopoietic stem and progenitor cells and reduced CXCL12 and stem cell factor expression in CAR cells but did not induce a change to yellow marrow. These data suggest a role for Foxc1 in inhibiting adipogenic processes in CAR progenitors. Foxc1 might also promote CAR cell development, upregulating CXCL12 and stem cell factor expression. This study identifies Foxc1 as a specific transcriptional regulator essential for development and maintenance of the mesenchymal niches for haematopoietic stem and progenitor cells.
Over the past decades, studies using zebrafish have significantly advanced our understanding of the cellular basis for development and human diseases. Zebrafish have rapidly developing transparent embryos that allow comprehensive imaging of embryogenesis combined with powerful genetic approaches. However, forward genetic screens in zebrafish have generated unanticipated findings that are mirrored by human genetic studies: disruption of genes implicated in basic cellular processes, such as protein secretion or cytoskeletal dynamics, causes discrete developmental or disease phenotypes. This is surprising because many processes that were assumed to be fundamental to the function and survival of all cell types appear instead to be regulated by cell-specific mechanisms. Such discoveries are facilitated by experiments in whole animals, where zebrafish provides an ideal model for visualization and manipulation of organelles and cellular processes in a live vertebrate. Here, we review well-characterized mutants and newly developed tools that underscore this notion. We focus on the secretory pathway and microtubule-based trafficking as illustrative examples of how studying cell biology in vivo using zebrafish has broadened our understanding of the role fundamental cellular processes play in embryogenesis and disease.