A Prox1 enhancer represses haematopoiesis in the lymphatic vasculature.

Transcriptional enhancer elements are responsible for orchestrating the temporal and spatial control over gene expression that is crucial for programming cell identity during development1-3. Here we describe a novel enhancer element that is important for regulating the expression of Prox1 in lymphatic endothelial cells. This evolutionarily conserved enhancer is bound by key lymphatic transcriptional regulators including GATA2, FOXC2, NFATC1 and PROX1. Genome editing of the enhancer to remove five nucleotides encompassing the GATA2-binding site resulted in perinatal death of homozygous mutant mice due to profound lymphatic vascular defects. Lymphatic endothelial cells in enhancer mutant mice exhibited reduced expression of genes characteristic of lymphatic endothelial cell identity and increased expression of genes characteristic of haemogenic endothelium, and acquired the capacity to generate haematopoietic cells. These data not only reveal a transcriptional enhancer element important for regulating Prox1 expression and lymphatic endothelial cell identity but also demonstrate that the lymphatic endothelium has haemogenic capacity, ordinarily repressed by Prox1.

Single-cell analysis of lymphatic endothelial cell fate specification and differentiation during zebrafish development.

During development, the lymphatic vasculature forms as a second network derived chiefly from blood vessels. The transdifferentiation of embryonic venous endothelial cells (VECs) into lymphatic endothelial cells (LECs) is a key step in this process. Specification, differentiation and maintenance of LEC fate are all driven by the transcription factor Prox1, yet the downstream mechanisms remain to be elucidated. We here present a single-cell transcriptomic atlas of lymphangiogenesis in zebrafish, revealing new markers and hallmarks of LEC differentiation over four developmental stages. We further profile single-cell transcriptomic and chromatin accessibility changes in zygotic prox1a mutants that are undergoing a LEC-VEC fate shift. Using maternal and zygotic prox1a/prox1b mutants, we determine the earliest transcriptomic changes directed by Prox1 during LEC specification. This work altogether reveals new downstream targets and regulatory regions of the genome controlled by Prox1 and presents evidence that Prox1 specifies LEC fate primarily by limiting blood vascular and haematopoietic fate. This extensive single-cell resource provides new mechanistic insights into the enigmatic role of Prox1 and the control of LEC differentiation in development.

Multiple cis-regulatory elements control prox1a expression in distinct lymphatic vascular beds.

During embryonic development, lymphatic endothelial cell (LEC) precursors are distinguished from blood endothelial cells by the expression of Prospero-related homeobox 1 (Prox1), which is essential for lymphatic vasculature formation in mouse and zebrafish. Prox1 expression initiation precedes LEC sprouting and migration, serving as the marker of specified LECs. Despite its crucial role in lymphatic development, Prox1 upstream regulation in LECs remains to be uncovered. SOX18 and COUP-TFII are thought to regulate Prox1 in mice by binding its promoter region. However, the specific regulation of Prox1 expression in LECs remains to be studied in detail. Here, we used evolutionary conservation and chromatin accessibility to identify enhancers located in the proximity of zebrafish prox1a active in developing LECs. We confirmed the functional role of the identified sequences through CRISPR/Cas9 mutagenesis of a lymphatic valve enhancer. The deletion of this region results in impaired valve morphology and function. Overall, our results reveal an intricate control of prox1a expression through a collection of enhancers. Ray-finned fish-specific distal enhancers drive pan-lymphatic expression, whereas vertebrate-conserved proximal enhancers refine expression in functionally distinct subsets of lymphatic endothelium.

Phylogenetic analysis and embryonic expression of panarthropod Dmrt genes.

BACKGROUND: One set of the developmentally important Doublesex and Male-abnormal-3 Related Transcription factors (Dmrt) is subject of intense research, because of their role in sex-determination and sexual differentiation. This likely non-monophyletic group of Dmrt genes is represented by the Drosophila melanogaster gene Doublesex (Dsx), the Caenorhabditis elegans Male-abnormal-3 (Mab-3) gene, and vertebrate Dmrt1 genes. However, other members of the Dmrt family are much less well studied, and in arthropods, including the model organism Drosophila melanogaster, data on these genes are virtually absent with respect to their embryonic expression and function. RESULTS: Here we investigate the complete set of Dmrt genes in members of all main groups of Arthropoda and a member of Onychophora, extending our data to Panarthropoda as a whole. We confirm the presence of at least four families of Dmrt genes (including Dsx-like genes) in Panarthropoda and study their expression profiles during embryogenesis. Our work shows that the expression patterns of Dmrt11E, Dmrt93B, and Dmrt99B orthologs are highly conserved among panarthropods. Embryonic expression of Dsx-like genes, however, is more derived, likely as a result of neo-functionalization after duplication. CONCLUSIONS: Our data suggest deep homology of most of the panarthropod Dmrt genes with respect to their function that likely dates back to their last common ancestor. The function of Dsx and Dsx-like genes which are critical for sexual differentiation in animals, however, appears to be much less conserved.

Embryonic expression patterns and phylogenetic analysis of panarthropod sox genes: insight into nervous system development, segmentation and gonadogenesis.

BACKGROUND: Sox (Sry-related high-mobility-group box) genes represent important factors in animal development. Relatively little, however, is known about the embryonic expression patterns and thus possible function(s) of Sox genes during ontogenesis in panarthropods (Arthropoda+Tardigrada+Onychophora). To date, studies have been restricted exclusively to higher insects, including the model system Drosophila melanogaster, with no comprehensive data available for any other arthropod group, or any tardigrade or onychophoran. RESULTS: This study provides a phylogenetic analysis of panarthropod Sox genes and presents the first comprehensive analysis of embryonic expression patterns in the flour beetle Tribolium castaneum (Hexapoda), the pill millipede Glomeris marginata (Myriapoda), and the velvet worm, Euperipatoides kanangrensis (Onychophora). 24 Sox genes were identified and investigated: 7 in Euperipatoides, 8 in Glomeris, and 9 in Tribolium. Each species possesses at least one ortholog of each of the five expected Sox gene families, B, C, D, E, and F, many of which are differentially expressed during ontogenesis. CONCLUSION: Sox gene expression (and potentially function) is highly conserved in arthropods and their closest relatives, the onychophorans. Sox B, C and D class genes appear to be crucial for nervous system development, while the Sox B genes Dichaete (D) and Sox21b likely play an additional conserved role in panarthropod segmentation. The Sox B gene Sox21a likely has a conserved function in foregut and Malpighian tubule development, at least in Hexapoda. The data further suggest that Sox D and E genes are involved in mesoderm differentiation, and that Sox E genes are involved in gonadal development. The new data expand our knowledge about the expression and implied function of Sox genes to Mandibulata (Myriapoda+Pancrustacea) and Panarthropoda (Arthropoda+Onychophora).

The relationship between the secondary vascular system and the lymphatic vascular system in fish.

New technologies have resulted in a better understanding of blood and lymphatic vascular heterogeneity at the cellular and molecular levels. However, we still need to learn more about the heterogeneity of the cardiovascular and lymphatic systems among different species at the anatomical and functional levels. Even the deceptively simple question of the functions of fish lymphatic vessels has yet to be conclusively answered. The most common interpretation assumes a similar dual setup of the vasculature in zebrafish and mammals: a cardiovascular circulatory system, and a lymphatic vascular system (LVS), in which the unidirectional flow is derived from surplus interstitial fluid and returned into the cardiovascular system. A competing interpretation questions the identity of the lymphatic vessels in fish as at least some of them receive their flow from arteries via specialised anastomoses, neither requiring an interstitial source for the lymphatic flow nor stipulating unidirectionality. In this alternative view, the 'fish lymphatics' are a specialised subcompartment of the cardiovascular system, called the secondary vascular system (SVS). Many of the contradictions found in the literature appear to stem from the fact that the SVS develops in part or completely from an embryonic LVS by transdifferentiation. Future research needs to establish the extent of embryonic transdifferentiation of lymphatics into SVS blood vessels. Similarly, more insight is needed into the molecular regulation of vascular development in fish. Most fish possess more than the five vascular endothelial growth factor (VEGF) genes and three VEGF receptor genes that we know from mice or humans, and the relative tolerance of fish to whole-genome and gene duplications could underlie the evolutionary diversification of the vasculature. This review discusses the key elements of the fish lymphatics versus the SVS and attempts to draw a picture coherent with the existing data, including phylogenetic knowledge.

Epigenetic Regulation of Endothelial Cell Lineages During Zebrafish Development-New Insights From Technical Advances.

Epigenetic regulation is integral in orchestrating the spatiotemporal regulation of gene expression which underlies tissue development. The emergence of new tools to assess genome-wide epigenetic modifications has enabled significant advances in the field of vascular biology in zebrafish. Zebrafish represents a powerful model to investigate the activity of cis-regulatory elements in vivo by combining technologies such as ATAC-seq, ChIP-seq and CUT&Tag with the generation of transgenic lines and live imaging to validate the activity of these regulatory elements. Recently, this approach led to the identification and characterization of key enhancers of important vascular genes, such as gata2a, notch1b and dll4. In this review we will discuss how the latest technologies in epigenetics are being used in the zebrafish to determine chromatin states and assess the function of the cis-regulatory sequences that shape the zebrafish vascular network.

mafba and mafbb differentially regulate lymphatic endothelial cell migration in topographically distinct manners.

Lymphangiogenesis, formation of lymphatic vessels from pre-existing vessels, is a dynamic process that requires cell migration. Regardless of location, migrating lymphatic endothelial cell (LEC) progenitors probe their surroundings to form the lymphatic network. Lymphatic-development regulation requires the transcription factor MAFB in different species. Zebrafish Mafba, expressed in LEC progenitors, is essential for their migration in the trunk. However, the transcriptional mechanism that orchestrates LEC migration in different lymphatic endothelial beds remains elusive. Here, we uncover topographically different requirements of the two paralogs, Mafba and Mafbb, for LEC migration. Both mafba and mafbb are necessary for facial lymphatic development, but mafbb is dispensable for trunk lymphatic development. On the molecular level, we demonstrate a regulatory network where Vegfc-Vegfd-SoxF-Mafba-Mafbb is essential in facial lymphangiogenesis. We identify that mafba and mafbb tune the directionality of LEC migration and vessel morphogenesis that is ultimately necessary for lymphatic function.