Biology subjects

Siekmann, A. F.

Publications and source records attributed to Siekmann, A. F..

5 recordsLinked to original sources

bioRxiv2023

Alk1/Endoglin dependent increases in vein endothelial cell sizes precipitate arteriovenous malformations

Aberrations in blood vessel diameters can disrupt the hierarchical patterning of the vasculature and cause congenital vascular anomalies, such as arteriovenous malformations (AVMs). Despite the identification of the Bone Morphogenetic Protein (BMP) pathway as a major driver in AVM pathology, we still lack an understanding of the early embryonic events regulating vessel hierarchy and arteriovenous shunt formation in vivo. We therefore studied blood vessel diameter control of the dorsal aorta (DA) and posterior cardinal vein (PCV) in zebrafish embryos. Our findings reveal that increases in blood flow during embryonic development result in increases in arterial endothelial cell (EC) sizes, ultimately enlarging DA diameters. By contrast, anterior regions of the PCV did not respond to changes in blood flow, while caudal regions, close to the artery-vein junction, responded to changes in flow like the DA, but to a lesser extent. To unravel the mechanisms underlying the reduced response of PCV ECs to flow, we studied zebrafish embryos mutant for the BMP pathway components endoglin and alk1. Through the generation of genetic mosaics, we discovered that both Endoglin and Alk1 were required cell autonomously in PCV cells to restrict EC sizes and thereby limit venous diameter increases in response to flow. We further revealed that initial increases in the diameter of the caudal PCV secondarily led to increased DA diameters and cell sizes. Therefore, Alk1/Endoglin signaling prevents vein ECs from behaving like arterial ECs. This differential response of arterial and venous EC cells to increases in flow is necessary to prevent the development of AVMs. This study thus offers insights into the spatiotemporal regulation of vessel hierarchy during early development and identifies changes in EC shapes as an important contributor in determining blood vessel diameters. Failure in this mechanism underlies the vein-specific initiation of AVMs in vertebrate models of HHT.

developmental biology↗

bioRxiv2022

Temporally and regionally distinct morphogenetic processes govern zebrafish tail fin blood vessel network expansion

Blood vessels form elaborate networks depending on tissue-specific signalling pathways and anatomical structures to guide their growth. However, it is not clear which morphogenetic principles organize the stepwise assembly of the vasculature. We thus performed a longitudinal analysis of zebrafish tail fin vascular assembly, revealing the existence of temporally and spatially distinct morphogenetic processes. Initially, vein-derived endothelial cells (ECs) generated arteries in a reiterative process requiring Vascular Endothelial Growth Factor (VEGF), Notch and cxcr4a signalling. Subsequently, veins produced veins in more proximal fin regions, transforming pre-existing artery-vein loops into a three-vessel pattern consisting of an artery and two veins. A distinct set of vascular plexuses formed at the base of the fin. They differed by virtue of diameter, flow magnitude and marker gene expression. At later stages, intussusceptive angiogenesis occurred from veins in distal fin regions. In proximal fin regions, we observed new vein sprouts crossing the inter-ray tissue through sprouting angiogenesis. Together, our results reveal a surprising diversity among the mechanisms generating the mature fin vasculature and suggest that these might be driven by separate local cues.

developmental biology↗

bioRxiv2021

DNA motif analysis of shear stress responsive endothelial enhancers reveals differential association of KLF and ETV/ETS binding sites with gained and lost enhancers

Endothelial cells (EC) lining blood vessels are exposed to mechanical forces, such as shear stress exerted by the flowing blood. These forces control many aspects of EC biology, including vascular tone, cell migration and proliferation in addition to cell size and shape. Despite a good understanding of the genes and signaling pathways responding to shear stress, our insights into the transcriptional regulation of these responses is much more limited. In particular, we do not know the different sets of regulatory elements (enhancers) that might control increases or decreases in gene expression. Here, we set out to study changes in the chromatin landscape of human umbilical vein endothelial cells (HUVEC) exposed to laminar shear stress. To do so, we performed ChIP-Seq for H3K27 acetylation, indicative of active enhancer elements and ATAC-Seq to mark regions of open chromatin in addition to RNA-Seq on HUVEC exposed to 6 hours of laminar shear stress. Our results show a correlation of gained and lost enhancers with up- and downregulated genes, respectively. DNA motif analysis revealed an over-representation of KLF transcription factor (TF) binding sites in gained enhancers, while lost enhancers contained more ETV/ETS motifs. We validated a subset of flow responsive enhancers using luciferase-based reporter constructs and CRISPR-Cas9 mediated genome editing. Lastly, we characterized shear stress responsive genes in ECs of zebrafish embryos using RNA-Seq. Together, our results reveal the presence of shear stress responsive DNA regulatory elements and lay the groundwork for the future exploration of these elements and the TFs binding to them in controlling EC biology.

genomics↗

bioRxiv2021

Regenerating vascular mural cells in zebrafish fin blood vessels are not derived from pre-existing ones and differentially require pdgfrb signaling for their development

Vascular networks are comprised of endothelial cells and mural cells, which include pericytes and smooth muscle cells. It is well established that new endothelial cells are derived from pre-existing ones during the angiogenic phase of blood vessel growth. By contrast, mural cell ontogeny is less clear with an ongoing debate whether mural cells possess mesenchymal stem cell properties. To elucidate the mechanisms controlling mural cell recruitment during development and tissue regeneration, we studied the formation of zebrafish caudal fin arteries. Mural cells showed morphological heterogeneity: cells colonizing arteries proximal to the body wrapped around them, while those in more distal regions extended protrusions along the proximo-distal vascular axis. Despite these differences, both cell populations expressed platelet-derived growth factor receptor beta (Pdgfrb) and the smooth muscle cell marker myosin heavy chain 11a (Myh11a). Loss of Pdgfrb signalling during development or tissue regeneration resulted in a substantial decrease in mural cells at the vascular front, while those proximal to the body were less affected. Using lineage tracing, we demonstrate that precursor cells located in periarterial regions of the caudal fin and expressing Pgdfrb can give rise to mural cells, while in regeneration newly formed mural cells were not derived from pre-existing ones. Together, our findings reveal conserved roles for pdgfrb signalling in development and regeneration, while at the same time illustrating a limited capacity of mural cells to self-renew or contribute to other cell types during tissue regeneration.

developmental biology↗

bioRxiv2020

Distinct Vegfa isoforms control endothelial cell proliferation through PI3 kinase signalling mediated regulation of cdkn1a/p21

The formation of appropriately patterned blood vessel networks requires endothelial cell migration and proliferation. Signaling through the Vascular Endothelial Growth Factor A (VEGFA) pathway is instrumental in coordinating these processes. mRNA splicing generates short (diffusible) and long (extracellular matrix bound) Vegfa isoforms. The differences between these isoforms in controlling cellular functions are not understood. In zebrafish, vegfaa generates short and long isoforms, while vegfab only generates long isoforms. We found that mutations in vegfaa affected endothelial cell migration and proliferation. Surprisingly, mutations in vegfab specifically reduced endothelial cell proliferation. Analysis of downstream signaling revealed no change in MAPK (ERK) activation, while inhibiting PI3 kinase signaling phenocopied vegfab mutants. The cell cycle inhibitor cdkn1a/p21 was upregulated in vegfab deficient embryos. Accordingly, reducing cdkn1a/p21 restored endothelial cell proliferation. Together, these results suggest that extracellular matrix bound Vegfa acts through PI3K signaling to specifically control endothelial cell proliferation during angiogenesis independently of MAPK (ERK) regulation.

developmental biology↗