Katarzyna Koltowska

Regulation of alternative polyadenylation by genomic imprinting

Maternally and paternally derived alleles can utilize different promoters, but allele-specific differences in cotranscriptional processes have not been reported. We show that alternative polyadenylation sites at a novel murine imprinted gene (H13) are utilized in an allele-specific manner. A differentially methylated CpG island separates polyA sites utilized on maternal and paternal alleles, and contains an internal promoter. Two genetic systems show that alleles lacking methylation generate truncated H13 transcripts that undergo internal polyadenylation. On methylated alleles, the internal promoter is inactive and elongation proceeds to downstream polyadenylation sites. This demonstrates that epigenetic modifications can influence utilization of alternative polyadenylation sites.

Getting out and about: the emergence and morphogenesis of the vertebrate lymphatic vasculature

The lymphatic vascular system develops from the pre-existing blood vasculature of the vertebrate embryo. New insights into lymphatic vascular development have recently been achieved with the use of alternative model systems, new molecular tools, novel imaging technologies and growing interest in the role of lymphatic vessels in human disorders. The signals and cellular mechanisms that facilitate the emergence of lymphatic endothelial cells from veins, guide migration through the embryonic environment, mediate interactions with neighbouring tissues and control vessel maturation are beginning to emerge. Here, we review the most recent advances in lymphatic vascular development, with a major focus on mouse and zebrafish model systems.

Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis

The VEGFC/VEGFR3 signaling pathway is essential for lymphangiogenesis (the formation of lymphatic vessels from pre-existing vasculature) during embryonic development, tissue regeneration and tumor progression. The recently identified secreted protein CCBE1 is indispensible for lymphangiogenesis during development. The role of CCBE1 orthologs is highly conserved in zebrafish, mice and humans with mutations in CCBE1 causing generalized lymphatic dysplasia and lymphedema (Hennekam syndrome). To date, the mechanism by which CCBE1 acts remains unknown. Here, we find that ccbe1 genetically interacts with both vegfc and vegfr3 in zebrafish. In the embryo, phenotypes driven by increased Vegfc are suppressed in the absence of Ccbe1, and Vegfc-driven sprouting is enhanced by local Ccbe1 overexpression. Moreover, Vegfc- and Vegfr3-dependent Erk signaling is impaired in the absence of Ccbe1. Finally, CCBE1 is capable of upregulating the levels of fully processed, mature VEGFC in vitro and the overexpression of mature VEGFC rescues ccbe1 loss-of-function phenotypes in zebrafish. Taken together, these data identify Ccbe1 as a crucial component of the Vegfc/Vegfr3 pathway in the embryo.

VEGFD regulates blood vascular development by modulating SOX18 activity.

Vascular endothelial growth factor-D (VEGFD) is a potent pro-lymphangiogenic molecule during tumor growth and is considered a key therapeutic target to modulate metastasis. Despite roles in pathological neo-lymphangiogenesis, the characterization of an endogenous role for VEGFD in vascular development has remained elusive. Here, we used zebrafish to assay for genetic interactions between the Vegf/Vegf-receptor pathway and SoxF transcription factors and identified a specific interaction between Vegfd and Sox18. Double knockdown zebrafish embryos for Sox18/Vegfd and Sox7/Vegfd exhibit defects in arteriovenous differentiation. Supporting this observation, we found that Sox18/Vegfd double but not single knockout mice displayed dramatic vascular development defects. We find that VEGFD-mitogen-activated protein kinase kinase-extracellular signal-regulated kinase signaling modulates SOX18-mediated transcription, functioning at least in part by enhancing nuclear concentration and transcriptional activity in vascular endothelial cells. This work suggests that VEGFD-mediated pathologies include or involve an underlying dysregulation of SOXF-mediated transcriptional networks.

Vegfc Regulates Bipotential Precursor Division and Prox1 Expression to Promote Lymphatic Identity in Zebrafish

Lymphatic vessels arise chiefly from preexisting embryonic veins. Genetic regulators of lymphatic fate are known, but how dynamic cellular changes contribute during the acquisition of lymphatic identity is not understood. We report the visualization of zebrafish lymphatic precursor cell dynamics during fate restriction. In the cardinal vein, cellular commitment is linked with the division of bipotential Prox1-positive precursor cells, which occurs immediately prior to sprouting angiogenesis. Following precursor division, identities are established asymmetrically in daughter cells; one daughter cell becomes lymphatic and progressively upregulates Prox1, and the other downregulates Prox1 and remains in the vein. Vegfc drives cell division and Prox1 expression in lymphatic daughter cells, coupling signaling dynamics with daughter cell fate restriction and precursor division.

mafba is a downstream transcriptional effector of Vegfc signaling essential for embryonic lymphangiogenesis in zebrafish.

The lymphatic vasculature plays roles in tissue fluid balance, immune cell trafficking, fatty acid absorption, cancer metastasis, and cardiovascular disease. Lymphatic vessels form by lymphangiogenesis, the sprouting of new lymphatics from pre-existing vessels, in both development and disease contexts. The apical signaling pathway in lymphangiogenesis is the VEGFC/VEGFR3 pathway, yet how signaling controls cellular transcriptional output remains unknown. We used a forward genetic screen in zebrafish to identify the transcription factor mafba as essential for lymphatic vessel development. We found that mafba is required for the migration of lymphatic precursors after their initial sprouting from the posterior cardinal vein. mafba expression is enriched in sprouts emerging from veins, and we show that mafba functions cell-autonomously during lymphatic vessel development. Mechanistically, Vegfc signaling increases mafba expression to control downstream transcription, and this regulatory relationship is dependent on the activity of SoxF transcription factors, which are essential for mafba expression in venous endothelium. Here we identify an indispensable Vegfc-SoxF-Mafba pathway in lymphatic development.

Arap3 is dysregulated in a mouse model of hypotrichosis-lymphedema-telangiectasia and regulates lymphatic vascular development

Mutations in SOX18, VEGFC and Vascular Endothelial Growth Factor 3 underlie the hereditary lymphatic disorders hypotrichosis-lymphedema-telangiectasia (HLT), Milroy-like lymphedema and Milroy disease, respectively. Genes responsible for hereditary lymphedema are key regulators of lymphatic vascular development in the embryo. To identify novel modulators of lymphangiogenesis, we used a mouse model of HLT (Ragged Opossum) and performed gene expression profiling of aberrant dermal lymphatic vessels. Expression studies and functional analysis in zebrafish and mice revealed one candidate, ArfGAP with RhoGAP domain, Ankyrin repeat and PH domain 3 (ARAP3), which is down-regulated in HLT mouse lymphatic vessels and necessary for lymphatic vascular development in mice and zebrafish. We position this known regulator of cell behaviour during migration as a mediator of the cellular response to Vegfc signalling in lymphatic endothelial cells in vitro and in vivo. Our data refine common mechanisms that are likely to contribute during both development and the pathogenesis of lymphatic vascular disorders.

Mural lymphatic endothelial cells regulate meningeal angiogenesis in the zebrafish

Mural cells of the vertebrate brain maintain vascular integrity and function, play roles in stroke and are involved in maintenance of neural stem cells. However, the origins, diversity and roles of mural cells remain to be fully understood. Using transgenic zebrafish, we identified a population of isolated mural lymphatic endothelial cells surrounding meningeal blood vessels. These meningeal mural lymphatic endothelial cells (muLECs) express lymphatic endothelial cell markers and form by sprouting from blood vessels. In larvae, muLECs develop from a lymphatic endothelial loop in the midbrain into a dispersed, nonlumenized mural lineage. muLEC development requires normal signaling through the Vegfc–Vegfd–Ccbe1–Vegfr3 pathway. Mature muLECs produce vascular growth factors and accumulate low-density lipoproteins from the bloodstream. We find that muLECs are essential for normal meningeal vascularization. Together, these data identify an unexpected lymphatic lineage and developmental mechanism necessary for establishing normal meningeal blood vasculature.

Ssrp1a controls organogenesis by promoting cell cycle progression and RNA synthesis.

Tightly controlled DNA replication and RNA transcription are essential for differentiation and tissue growth in multicellular organisms. Histone chaperones, including the FACT (facilitates chromatin transcription) complex, are central for these processes and act by mediating DNA access through nucleosome reorganisation. However, their roles in vertebrate organogenesis are poorly understood. Here, we report the identification of zebrafish mutants for the gene encoding Structure specific recognition protein 1a (Ssrp1a), which, together with Spt16, forms the FACT heterodimer. Focussing on the liver and eye, we show that zygotic Ssrp1a is essential for proliferation and differentiation during organogenesis. Specifically, gene expression indicative of progressive organ differentiation is disrupted and RNA transcription is globally reduced. Ssrp1a-deficient embryos exhibit DNA synthesis defects and prolonged S phase, uncovering a role distinct from that of Spt16, which promotes G1 phase progression. Gene deletion/replacement experiments in Drosophila show that Ssrp1b, Ssrp1a and N-terminal Ssrp1a, equivalent to the yeast homologue Pob3, can substitute Drosophila Ssrp function. These data suggest that (1) Ssrp1b does not compensate for Ssrp1a loss in the zebrafish embryo, probably owing to insufficient expression levels, and (2) despite fundamental structural differences, the mechanisms mediating DNA accessibility by FACT are conserved between yeast and metazoans. We propose that the essential functions of Ssrp1a in DNA replication and gene transcription, together with its dynamic spatiotemporal expression, ensure organ-specific differentiation and proportional growth, which are crucial for the forming embryo.

09-P069 The clamped s819 gene promotes liver differentiation and growth

The liver forms as part of digestive system. Despite the essential functions of the liver, little is known about its early embryonic development. The clamped s819 mutant was identified in a forward genetic screen for mutations causing defects in endodermal organogenesis in zebrafish. At 44 h post-fertilisation (hpf), clamped s819 mutant embryos display liver hypoplasia and defects in extrahepatic duct morphology. Examination of gene expression indicative of liver specification and differentiation in mutant embryos revealed that specification occurs normally while subsequent differentiation and growth are altered. Similar defects were observed in the developing eyes and fins. Interestingly, in clamped s819 mutants cell death was detected from 40 hpf onwards in the liver abutting mesoderm, and from 48 hpf onwards within the liver and extrahepatic duct. Concomitantly, cell proliferation in the liver of clamped s819 mutants was severely impaired from 44 hpf onwards. Therefore, we hypothesise that Clamped plays a role in promoting hepatic differentiation. Furthermore, Clamped may be required for cross-talk between the mesoderm and endoderm at this stage of liver organogenesis. Mosaic analysis will be carried out to test the tissue autonomous function of Clamped within this process. Positional cloning has mapped the molecular lesion underlying the clamped s819 mutant phenotype to a 240 kb interval on linkage group 14, containing three candidate genes. None of the candidates have so far been implicated in liver organogenesis, thus investigating their function promises to further our understanding of liver differentiation and growth.