Venous endothelial cells are molecularly and functionally distinct from their arterial counterparts. Although veins are often considered the default endothelial state, genetic manipulations can ...modulate both acquisition and loss of venous fate, suggesting that venous identity is the result of active transcriptional regulation. However, little is known about this process. Here we show that BMP signalling controls venous identity via the ALK3/BMPR1A receptor and SMAD1/SMAD5. Perturbations to TGF-β and BMP signalling in mice and zebrafish result in aberrant vein formation and loss of expression of the venous-specific gene Ephb4, with no effect on arterial identity. Analysis of a venous endothelium-specific enhancer for Ephb4 shows enriched binding of SMAD1/5 and a requirement for SMAD binding motifs. Further, our results demonstrate that BMP/SMAD-mediated Ephb4 expression requires the venous-enriched BMP type I receptor ALK3/BMPR1A. Together, our analysis demonstrates a requirement for BMP signalling in the establishment of Ephb4 expression and the venous vasculature.
Insights into oncogenesis derived from cancer susceptibility loci (SNP) hold the potential to facilitate better cancer management and treatment through precision oncology. However, therapeutic ...insights have thus far been limited by our current lack of understanding regarding both interactions of these loci with somatic cancer driver mutations and their influence on tumorigenesis. For example, although both germline and somatic genetic variation to the p53 tumor suppressor pathway are known to promote tumorigenesis, little is known about the extent to which such variants cooperate to alter pathway activity. Here we hypothesize that cancer risk-associated germline variants interact with somatic
mutational status to modify cancer risk, progression, and response to therapy. Focusing on a cancer risk SNP (rs78378222) with a well-documented ability to directly influence p53 activity as well as integration of germline datasets relating to cancer susceptibility with tumor data capturing somatically-acquired genetic variation provided supportive evidence for this hypothesis. Integration of germline and somatic genetic data enabled identification of a novel entry point for therapeutic manipulation of p53 activities. A cluster of cancer risk SNPs resulted in increased expression of prosurvival p53 target gene
and attenuation of p53-mediated responses to genotoxic therapies, which were reversed by pharmacologic inhibition of the prosurvival c-KIT signal. Together, our results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and identify novel combinatorial therapies. SIGNIFICANCE: These results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and present novel therapeutic targets.
Angiogenesis, the fundamental process by which new blood vessels form from existing ones, depends on precise spatial and temporal gene expression within specific compartments of the endothelium. ...However, the molecular links between proangiogenic signals and downstream gene expression remain unclear. During sprouting angiogenesis, the specification of endothelial cells into the tip cells that lead new blood vessel sprouts is coordinated by vascular endothelial growth factor A (VEGFA) and Delta-like ligand 4 (Dll4)/Notch signaling and requires high levels of Notch ligand DLL4. Here, we identify MEF2 transcription factors as crucial regulators of sprouting angiogenesis directly downstream from VEGFA. Through the characterization of a Dll4 enhancer directing expression to endothelial cells at the angiogenic front, we found that MEF2 factors directly transcriptionally activate the expression of Dll4 and many other key genes up-regulated during sprouting angiogenesis in both physiological and tumor vascularization. Unlike ETS-mediated regulation, MEF2-binding motifs are not ubiquitous to all endothelial gene enhancers and promoters but are instead overrepresented around genes associated with sprouting angiogenesis. MEF2 target gene activation is directly linked to VEGFA-induced release of repressive histone deacetylases and concurrent recruitment of the histone acetyltransferase EP300 to MEF2 target gene regulatory elements, thus establishing MEF2 factors as the transcriptional effectors of VEGFA signaling during angiogenesis.
We describe the creation of a transgenic zebrafish expressing GFP driven by a 7.5 kb promoter region of the tbx16 gene. This promoter segment is sufficient to recapitulate early embryonic expression ...of endogenous tbx16 in the presomitic mesoderm, the polster and, subsequently, in the hatching gland. Expression of GFP in the transgenic lines later in development diverges to some extent from endogenous tbx16 expression with the serendipitous result that one line expresses GFP specifically in commissural primary ascending (CoPA) interneurons of the developing spinal cord. Using this line we demonstrate that the gene mafba (valentino) is expressed in CoPA interneurons.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Zebrafish aplnra functions in epiboly Nornes, Svanhild; Tucker, Ben; Lardelli, Michael
BMC research notes,
11/2009, Letnik:
2, Številka:
1
Journal Article
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The zebrafish, Danio rerio, possesses the paralogous genes aplnra and aplnrb that are duplicates of an ancestral orthologue of the human APLNR gene encoding a G-protein coupled receptor that binds ...the peptide ligand APELIN and is required for normal cardiovascular function. aplnrb is required for migration of cells contributing to heart development in zebrafish embryos. aplnra is transcribed in a complex pattern during early development but its function in embryogenesis is largely unknown.
Blockage of translation of aplnra mRNA in zebrafish embryos results in retarded or failed epiboly with the blastoderm apparently disconnected from the nuclei of the yolk syncytial layer. Gastrulation is also defective. Failure of correct tail extension is observed with ectopic structures resembling somites positioned dorsal to the spinal cord.
aplnra, unlike its duplicate aplnrb, is essential for normal epiboly, although this function appears to be independent of signalling activated by zebrafish Apelin. The defects in epiboly caused by loss of aplnra activity appear, at least partially, to be due to a requirement for aplnra activity in the yolk syncytial layer.
Correct vascular differentiation requires distinct patterns of gene expression in different subtypes of endothelial cells. Members of the ETS transcription factor family are essential for the ...transcriptional activation of arterial and angiogenesis-specific gene regulatory elements, leading to the hypothesis that they play lineage-defining roles in arterial and angiogenic differentiation directly downstream of VEGFA signalling. However, an alternative explanation is that ETS binding at enhancers and promoters is a general requirement for activation of many endothelial genes regardless of expression pattern, with subtype-specificity provided by additional factors. Here we use analysis of Ephb4 and Coup-TFII (Nr2f2) vein-specific enhancers to demonstrate that ETS factors are equally essential for vein, arterial and angiogenic-specific enhancer activity patterns. Further, we show that ETS factor binding at these vein-specific enhancers is enriched by VEGFA signalling, similar to that seen at arterial and angiogenic enhancers. However, while arterial and angiogenic enhancers can be activated by VEGFA in vivo, the Ephb4 and Coup-TFII venous enhancers are not, suggesting that the specificity of VEGFA-induced arterial and angiogenic enhancer activity occurs via non-ETS transcription factors. These results support a model in which ETS factors are not the primary regulators of specific patterns of gene expression in different endothelial subtypes.
•Vein-specific enhancers can contain essential ETS motifs.•VEGFA induced an increase in ETS binding at vein, arterial and angiogenic enhancers.•VEGFA stimulation cannot induce vein-specific enhancer activity.
OBJECTIVE—The vascular endothelial growth factor (VEGF) receptor Flk1 is essential for vascular development, but the signaling and transcriptional pathways by which its expression is regulated in ...endothelial cells remain unclear. Although previous studies have identified 2 Flk1 regulatory enhancers, these are dispensable for Flk1 expression, indicating that additional enhancers contribute to Flk1 regulation in endothelial cells. In the present study, we sought to identify Flk1 enhancers contributing to expression in endothelial cells.
APPROACH AND RESULTS—A region of the 10th intron of the Flk1 gene (Flk1in10) was identified as a putative enhancer and tested in mouse and zebrafish transgenic models. This region robustly directed reporter gene expression in arterial endothelial cells. Using a combination of targeted mutagenesis of transcription factor–binding sites and gene silencing of transcription factors, we found that Gata and Ets factors are required for Flk1in10 enhancer activity in all endothelial cells. Furthermore, we showed that activity of the Flk1in10 enhancer is restricted to arteries through repression of gene expression in venous endothelial cells by the Notch pathway transcriptional regulator Rbpj.
CONCLUSIONS—This study demonstrates a novel mechanism of arterial–venous identity acquisition, indicates a direct link between the Notch and VEGF signaling pathways, and illustrates how cis-regulatory diversity permits differential expression outcomes from a limited repertoire of transcriptional regulators.
We describe the isolation of cDNA clones for zebrafish Pax9. Pax9 expression was initiated at the end of the segmentation period in mesenchymal sclerotome cells on both sides of the notochord
...similarly to the corresponding mouse and chick genes. Two transcripts, Pax9a and - b , are generated by alternative splicing. The gene contains 4 exons with exon 3 being included in the Pax9a transcript and spliced out in the Pax9b transcript. The Pax9a and -b proteins are identical for 212 amino acids from the N terminus but contain distinct C-terminal
regions of 131 and 58 amino acids, respectively. The paired domain of Pax9 displayed a binding-site specificity distinct from
Pax6 but similar to Pax1 and â2. Both Pax9a and -b activated a promoter containing a paired domain binding site. However,
this activation was observed when low amounts of Pax9 expression vectors were used. Higher amounts led to a sharp decrease
in the activation and even turned into repression. Both the distinct C-terminal regions of Pax9a and -b harbored transcriptional
activating domains of different potency not revealed in the context of the full-length proteins due to a negative influence
of the N-terminal region including the paired domain.
Presenilin1 (PSEN1) and presenilin2 (PSEN2) are involved in the processing of type-1 transmembrane proteins including the amyloid precursor protein (APP), Notch and several others. PSEN1 has been ...shown to be crucial for proteolytic cleavage of Notch in developing animal embryos. Mouse embryos lacking Psen1 function show disturbed neurogenesis and somite formation, resembling Notch pathway mutants. However, loss of Psen2 activity reveals only a minor phenotype. Zebrafish embryos are a valuable tool for analysis of the molecular genetic control of cell differentiation since endogenous gene expression can be modulated in subtle and complex ways to give a phenotypic readout. Using injection of morpholino antisense oligonucleotides to inhibit protein translation in zebrafish embryos, we show that reduced Psen2 activity decreases the number of melanocytes in the trunk but not in the cranial area at 2 days post fertilisation (dpf). Reduced Psen2 activity apparently reduces Notch signalling resulting in perturbed spinal
neurogenin1 (
neurog1) expression, neurogenesis and trunk and tail neural crest development. Similar effects are seen for reduced Psen1 activity. These results suggest that Psen2 plays a more prominent role in Notch signalling and embryo development in zebrafish than in mammals. Intriguingly, decreased Psen2 activity increases the number of Dorsal Longitudinal Ascending (DoLA) interneurons in the spinal cord while decreased Psen1 activity has no effect. However, the effect on DoLAs of reduced Psen2 can be ameliorated by Psen1 loss. The effects of changes in Psen2 activity on DoLA interneurons and other cells in zebrafish embryos provide bioassays for more detailed dissection of Psen2 function.