The development of a functional vascular system is an essential process during vertebrate embryogenesis. The blood vasculature, composed of a hierarchical branched network of arteries, veins and capillaries, provides efficient supply of oxygen and nutrients to reach all organs and tissues. Vascular endothelial cells, which form the inner layer of blood vessels, not only crucially control the formation and maintenance of a functional circulatory system (Trimm and Red-Horse, 2023), but also secrete specific factors, named angiocrines, that regulate the functions of individual organs (Rafii et al., 2016). Achieving specificity in the induction of mesodermal progenitors to become endothelial cell fated, the subsequent acquisition of arteriovenous or organotypic endothelial cell identity, as well as the correct formation of vascular structure and function is a formidable feat. Although there has been significant progress in understanding these processes in recent decades (Hogan and Schulte-Merker, 2017), the control mechanisms governing them remain incompletely understood.

The coordinated regulation of gene expression is crucial, not only for the induction of endothelial cells, but also for the subsequent development of a functional vascular system. Gene expression involves transcription and translation, as well as the turnover of mRNAs and proteins. In recent decades, numerous gene regulatory mechanisms involving a plethora of factors have been shown to play a role in endothelial cell specification and vascular development (De Val and Black, 2009; Minami and Aird, 2005). A number of recent preprint articles provide novel insights into gene regulatory factors and mechanisms, which not only control endothelial cell fate induction (Chen et al., 2024 preprint; Van Wauwe et al., 2024 preprint), but also the development of vascular structures (Beckröge et al., 2024 preprint; Patel et al., 2024 preprint).

In vertebrates, endothelial cells differentiate from mesodermal progenitor cells (Minasi et al., 2002), in a process that depends on the ETS pioneer factor ETV2 (Lee et al., 2008; Ferdous et al., 2009). Using a focused CRISPR screen, Chen and colleagues studied factors that cooperate with ETV2 to drive the specification of human endothelial cells from mesoderm progenitor cells (Chen et al., 2024 preprint). They identified that GABPA, a transcriptional activator, and REST, a transcriptional repressor, act in concert with ETV2 as functional regulators of endothelial cell commitment. By subsequently employing the chromatin profiling strategy, CUT&RUN, during in vitro endothelial cell differentiation, they determined that GABPA or REST each co-occupied a substantial proportion of ETV2-bound genomic regions, suggesting cooperation between these factors and ETV2. However, only a few regions that were solely occupied by GABPA or REST were found to be within de novo accessible chromatin regions, indicating that these factors bind to open chromatin regions but have little pioneering activity themselves (Chen et al., 2024 preprint). Bulk RNA sequencing (RNA-seq) on GABPA knockout (KO) cells in which ETV2 was overexpressed, determined that genes involved in angiogenesis, including FLT1, HHEX and ECM1, were downregulated. Conversely, REST KO following ETV2 overexpression resulted in an upregulation of genes known to play a role in heart development, including GATA4, GATA5, SMAD6 and WNT5A. This indicates that REST is recruited to ETV2-induced open chromatin to repress the expression of non-endothelial cell lineage genes, in particular those involved in cardiac development. These findings are in line with previous work demonstrating that ETV2 inactivation results in the expansion of other mesodermal fates, including the cardiac lineage (Liu et al., 2012; Rasmussen et al., 2011).

Following specification, endothelial cells proliferate rapidly and assemble vascular tubes, forming the first axial blood vessels in vertebrates. These are already composed of endothelial cells that are distinguishable from one another as the result of a process known as arteriovenous endothelial cell differentiation. Although a number of key factors, including VEGF and Notch, are crucial for arteriovenous endothelial cell differentiation (Trimm and Red-Horse, 2023), the molecular mechanisms governing the induction of arterial and venous fate remain incompletely understood. In their new study, Van Wauwe and colleagues found that prdm16, a zinc-finger transcription factor best known for controlling adipocyte and cardiac muscle cell fates (Cibi et al., 2020; Wang et al., 2019; Wu et al., 2022), is expressed in arteries, but not veins, of zebrafish and mammalian embryos (Van Wauwe et al., 2024 preprint). Transcriptomic analysis of human endothelial progenitor cells overexpressing PRDM16 revealed that this transcription factor predominantly induces the expression of Notch signalling pathway components, such as DLL4, HEY1, HEY2 and EFNB2, which are genes known to play roles in arterial endothelial cell lineage commitment (Phng and Gerhardt, 2009). Furthermore, by knocking down prdm16 expression in zebrafish embryos using morpholinos, and then performing RNA-seq on FACS-isolated endothelial cells, numerous genes previously implemented in venous endothelial cell development (including flt4, mrc1a, stab1, stab2 and nr2f2) were found to be upregulated in prdm16 knockdown (KD) embryos (Van Wauwe et al., 2024 preprint). Interestingly, Notch signalling pathway genes did not significantly differ in expression between prdm16 KD and control embryos, suggesting that the primary in vivo role of Prdm16 is to suppress the venous transcriptional programme, at least in zebrafish. This may also be the case in mammals, as elevated levels of the venous marker endomucin were observed in arteries of Prdm16 KO mouse embryos when compared with littermate controls (Van Wauwe et al., 2024 preprint), corroborating data from a previous study in postnatal mice (Thompson et al., 2023). Finally, the authors observed that the KD of prdm16 led to arteriovenous malformations in zebrafish embryos in which canonical Notch signalling was partially inhibited. This may result from a physical interaction between PRDM16 and HEY, which is also reported in their study.

Transcriptional co-factors do not bind to DNA directly, but instead interact with DNA-binding transcriptional regulators. In their recent preprint, Patel and colleagues investigate the role(s) of the transcriptional co-factor, Zmiz1, in vascular development (Patel et al., 2024 preprint). Zmiz1-null mice die during embryogenesis (Beliakoff et al., 2008), so Patel and colleagues generated constitutive, endothelial cell-specific Zmiz1 KO mice. Using this system, they investigated vasculogenesis and angiogenesis during development. Endothelial-specific Zmiz1 KO embryos exhibited a number of vascular phenotypes including defective patterning of cranial and trunk vasculature at embryonic day (E) 12.5, an absence of blood-filled yolk sac vessels at E13.5, and a failure to form large caliber vessels. Homozygous null endothelial cell-specific Zmiz1−/− embryos were found to be non-viable (Patel et al., 2024 preprint), suggesting that defective angiogenesis contributes to embryonic lethality in Zmiz1−/− mice. Using inducible endothelial-specific Zmiz1 KO mice, along with a well-established model of angiogenesis, the mouse retina (Uemura et al., 2006), the authors identified a defect in sprouting angiogenesis in the absence of Zmiz1. Specifically, a significant decrease in the number of vascular sprouts formed in endothelial cell-specific Zmiz1 KO retinas relative to controls at postnatal day 7, correlating with a reduction in the expression of the tip cell-associated genes Angpt2, Apln, Esm1 and Cxcr4 in Zmiz1 mutant retinas (Patel et al., 2024 preprint). Identifying transcription factor partners of Zmiz1 will be an exciting avenue to explore in future studies.

Not only have pioneer factors, transcription factors and transcriptional co-factors been implicated in endothelial cell biology, but numerous studies have identified roles for RNA-binding proteins in the induction of endothelial cell fate and function (Volkers et al., 2024). One recent example of this is Trim71, an RNA-binding protein that controls the quantity of target mRNAs by binding their 3′UTR (Kumari et al., 2018; Torres-Fernandez et al., 2019), resulting in target mRNA degradation (Loedige et al., 2013). When performing single-cell RNA-sequencing on Trim71 KO and control mouse embryos during gastrulation (E7.5), the Kolanus lab identified elevated expression of Eomes (Beckröge et al., 2024 preprint), a transcription factor expressed in the early mesoderm, which antagonizes brachyury function during mesoderm specification (Schule et al., 2023). Direct binding of Trim71 to Eomes mRNA was confirmed by cross-linking RNA precipitation. Trim71 KO embryos display a number of vascular developmental phenotypes, including the loss of the large vitelline vessels, decreased vascular branching and a reduction in the endothelial extensions in the yolk sac microvasculature. Given that the loss of Eomes expression results in impaired mesoderm formation (Russ et al., 2000), the authors speculate that a delicate balance of Eomes expression is required for proper formation of the vasculature. Therefore, elevation in the expression of Eomes in Trim71 KO embryos may, at least in part, explain the vascular phenotypes observed in these mice.

Together, the four preprints highlighted in this article demonstrate the diversity in gene regulatory factors and mechanisms controlling endothelial cell induction, their differentiation, and the development of a functional vascular system. As our understanding of molecular players involved in these processes grows, it is becoming increasingly clear that, as in so many other instances during development, precise coordination in gene expression, both spatially and temporally, is required in order to prevent endothelial dysfunction and pathologies related to this. Evolutionary tinkering has generated molecular control mechanisms on a number of levels, which each delicately contribute to the generation of correct gene expression programmes. As such, endothelial cell specification and vascular development can serve as models not only for studies investigating topics such as fate induction, morphogenesis and vascular pathologies, but also for those that aim to dissect the relevance of gene expression control mechanisms on a variety of different levels in developmental biology.

We are grateful to the preprint authors for fact-checking this article.

Funding

This work was supported by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation; grants CRC1444, CRC1470, and CRC1588 to H.G.).

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Competing interests

The authors declare no competing or financial interests.