Annexins are a large family of calcium binding proteins that associate with cell membrane phospholipids and are involved in various cellular processes including endocytosis, exocytosis and ...membrane-cytoskeletal organization. Despite studies on numerous Annexin proteins, the function of Annexin A3 (Anxa3) is largely unknown. Our studies identify Anxa3 as a unique marker of the endothelial and myeloid cell lineages of Xenopus laevis during development. Anxa3 transcripts are also detected in endothelial cells (ECs) of zebrafish and mouse embryos, suggesting an important evolutionary function during formation of blood vessels. Indeed, Anxa3 loss-of-function experiments in frog embryos reveal its critical role during the morphogenesis of early blood vessels, as angioblasts in MO injected embryos fail to form vascular cords. Furthermore, in vitro experiments in mammalian cells identify a role for Anxa3 in EC migration. Our results are the first to reveal an in vivo function for Anxa3 during vascular development and represent a previously unexplored aspect of annexin biology.
Vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed by lymphatic endothelial cells and has been shown to stimulate lymphangiogenesis in adult mice. However, the role VEGFR2 ...serves in the development of the lymphatic vascular system has not been defined. Here we use the Cre-lox system to show that the proper development of the lymphatic vasculature requires VEGFR2 expression by lymphatic endothelium. We show that Lyve-1(wt/Cre);Vegfr2(flox/flox) mice possess significantly fewer dermal lymphatic vessels than Vegfr2(flox/flox) mice. Although Lyve-1(wt/Cre);Vegfr2(flox/flox) mice exhibit lymphatic hypoplasia, the lymphatic network is functional and contains all of the key features of a normal lymphatic network (initial lymphatic vessels and valved collecting vessels surrounded by smooth muscle cells (SMCs)). We also show that Lyve-1(Cre) mice display robust Cre activity in macrophages and in blood vessels in the yolk sac, liver and lung. This activity dramatically impairs the development of blood vessels in these tissues in Lyve-1(wt/Cre);Vegfr2(flox/flox) embryos, most of which die after embryonic day14.5. Lastly, we show that inactivation of Vegfr2 in the myeloid lineage does not affect the development of the lymphatic vasculature. Therefore, the abnormal lymphatic phenotype of Lyve-1(wt/Cre);Vegfr2(flox/flox) mice is due to the deletion of Vegfr2 in the lymphatic vasculature not macrophages. Together, this work demonstrates that VEGFR2 directly promotes the expansion of the lymphatic network and further defines the molecular mechanisms controlling the development of the lymphatic vascular system.
Vascular development begins when mesodermal cells differentiate into endothelial cells, which then form primitive vessels. It has been hypothesized that endothelial-specific gene expression may be ...regulated combinatorially, but the transcriptional mechanisms governing specificity in vascular gene expression remain incompletely understood. Here, we identify a 44 bp transcriptional enhancer that is sufficient to direct expression specifically and exclusively to the developing vascular endothelium. This enhancer is regulated by a composite
cis-acting element, the FOX:ETS motif, which is bound and synergistically activated by Forkhead and Ets transcription factors. We demonstrate that coexpression of the Forkhead protein FoxC2 and the Ets protein Etv2 induces ectopic expression of vascular genes in
Xenopus embryos, and that combinatorial knockdown of the orthologous genes in zebrafish embryos disrupts vascular development. Finally, we show that FOX:ETS motifs are present in many known endothelial-specific enhancers and that this motif is an efficient predictor of endothelial enhancers in the human genome.
Zinc Finger MIZ-Type Containing 1 (Zmiz1), also known as ZIMP10 or RAI17, is a transcription cofactor and member of the Protein Inhibitor of Activated STAT (PIAS) family of proteins. Zmiz1 is ...critical for a variety of biological processes including vascular development. However, its role in the lymphatic vasculature is unknown. In this study, we utilized human dermal lymphatic endothelial cells (HDLECs) and an inducible, lymphatic endothelial cell (LEC)-specific Zmiz1 knockout mouse model to investigate the role of Zmiz1 in LECs. Transcriptional profiling of ZMIZ1-deficient HDLECs revealed downregulation of genes crucial for lymphatic vessel development. Additionally, our findings demonstrated that loss of Zmiz1 results in reduced expression of proliferation and migration genes in HDLECs and reduced proliferation and migration in vitro. We also presented evidence that Zmiz1 regulates Prox1 expression in vitro and in vivo by modulating chromatin accessibility at Prox1 regulatory regions. Furthermore, we observed that loss of Zmiz1 in mesenteric lymphatic vessels significantly reduced valve density. Collectively, our results highlight a novel role of Zmiz1 in LECs and as a transcriptional regulator of Prox1, shedding light on a previously unknown regulatory factor in lymphatic vascular biology.
Hereditary hemorrhagic telangiectasia is an autosomal dominant vascular disorder caused by heterozygous, loss-of-function mutations in 4 transforming growth factor beta (TGFβ) pathway members, ...including the central transcriptional mediator of the TGFβ pathway, Smad4. Loss of Smad4 causes the formation of inappropriate, fragile connections between arteries and veins called arteriovenous malformations (AVMs), which can hemorrhage leading to stroke, aneurysm, or death. Unfortunately, the molecular mechanisms underlying AVM pathogenesis remain poorly understood, and the TGFβ downstream effectors responsible for hereditary hemorrhagic telangiectasia-associated AVM formation are currently unknown.
To identify potential biological targets of the TGFβ pathway involved in AVM formation, we performed RNA- and chromatin immunoprecipitation-sequencing experiments on BMP9 (bone morphogenetic protein 9)-stimulated endothelial cells (ECs) and isolated ECs from a Smad4-inducible, EC-specific knockout ( Smad4-iECKO) mouse model that develops retinal AVMs. These sequencing studies identified the angiopoietin-Tek signaling pathway as a downstream target of SMAD4. We used monoclonal blocking antibodies to target a specific component in this pathway and assess its effects on AVM development.
Sequencing studies uncovered 212 potential biological targets involved in AVM formation, including the EC surface receptor, TEK (TEK receptor tyrosine kinase) and its antagonistic ligand, ANGPT2 (angiopoietin-2). In Smad4-iECKO mice, Angpt2 expression is robustly increased, whereas Tek levels are decreased, resulting in an overall reduction in angiopoietin-Tek signaling. We provide evidence that SMAD4 directly represses Angpt2 transcription in ECs. Inhibition of ANGPT2 function in Smad4-deficient mice, either before or after AVMs form, prevents and alleviates AVM formation and normalizes vessel diameters. These rescue effects are attributed to a reversion in EC morphological changes, such as cell size and shape that are altered in the absence of Smad4.
Our studies provide a novel mechanism whereby the loss of Smad4 causes increased Angpt2 transcription in ECs leading to AVM formation, increased blood vessel calibers, and changes in EC morphology in the retina. Blockade of ANGPT2 function in an in vivo Smad4 model of hereditary hemorrhagic telangiectasia alleviated these vascular phenotypes, further implicating ANGPT2 as an important TGFβ downstream mediator of AVM formation. Therefore, alternative approaches that target ANGPT2 function may have therapeutic value for the alleviation of hereditary hemorrhagic telangiectasia symptoms, such as AVMs.
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder that leads to abnormal connections between arteries and veins termed arteriovenous malformations (AVM). ...Mutations in TGFβ pathway members
ALK1
,
ENG
and
SMAD4
lead to HHT. However, a
Smad4
mouse model of HHT does not currently exist. We aimed to create and characterize a
Smad4
endothelial cell (EC)-specific, inducible knockout mouse (
Smad4
f/f
;
Cdh5
-Cre
ERT2
) that could be used to study AVM development in HHT. We found that postnatal ablation of
Smad4
caused various vascular defects, including the formation of distinct AVMs in the neonate retina. Our analyses demonstrated that increased EC proliferation and size, altered mural cell coverage and distorted artery–vein gene expression are associated with
Smad4
deficiency in the vasculature. Furthermore, we show that depletion of
Smad4
leads to decreased
Vegfr2
expression, and concurrent loss of endothelial
Smad4
and
Vegfr2
in vivo leads to AVM enlargement. Our work provides a new model in which to study HHT-associated phenotypes and links the TGFβ and VEGF signaling pathways in AVM pathogenesis.
Wnt signaling is essential to many events during organogenesis, including the development of the mammalian lung. The Wnt family member Wnt4 has been shown to be required for the development of ...kidney, gonads, thymus, mammary and pituitary glands. Here, we show that Wnt4 is critical for proper morphogenesis and growth of the respiratory system. Using in situ hybridization in mouse embryos, we identify a previously uncharacterized site of Wnt4 expression in the anterior trunk mesoderm. This expression domain initiates as early as E8.25 in the mesoderm abutting the tracheoesophageal endoderm, between the fusing dorsal aortae and the heart. Analysis of Wnt4−/− embryos reveals severe lung hypoplasia and tracheal abnormalities; however, aortic fusion and esophageal development are unaffected. We find decreased cell proliferation in Wnt4−/− lung buds, particularly in tip domains. In addition, we observe reduction of the important lung growth factors Fgf9, Fgf10, Sox9 and Wnt2 in the lung bud during early stages of organogenesis, as well as decreased tracheal expression of the progenitor factor Sox9. Together, these data reveal a previously unknown role for the secreted protein Wnt4 in respiratory system development.
•Wnt4 is strongly and transiently expressed in a domain of the trunk mesoderm near the developing respiratory system.•Wnt4 null embryos exhibit defective tracheal and lung development.•Expression of the lung growth factors Fgf9, Fgf10, Sox9 and Wnt2 is decreased in the absence of Wnt4.•Cell proliferation in nascent lung buds is reduced in Wnt4 null embryos.
The Rho family of small GTPases has been shown to be required in endothelial cells (ECs) during blood vessel formation. However, the underlying cellular events controlled by different GTPases remain ...unclear. Here, we assess the cellular mechanisms by which Cdc42 regulates mammalian vascular morphogenesis and maintenance. In vivo deletion of Cdc42 in embryonic ECs (Cdc42(Tie2KO)) results in blocked lumen formation and endothelial tearing, leading to lethality of mutant embryos by E9-10 due to failed blood circulation. Similarly, inducible deletion of Cdc42 (Cdc42(Cad5KO)) at mid-gestation blocks angiogenic tubulogenesis. By contrast, deletion of Cdc42 in postnatal retinal vessels leads to aberrant vascular remodeling and sprouting, as well as markedly reduced filopodia formation. We find that Cdc42 is essential for organization of EC adhesion, as its loss results in disorganized cell-cell junctions and reduced focal adhesions. Endothelial polarity is also rapidly lost upon Cdc42 deletion, as seen by failed localization of apical podocalyxin (PODXL) and basal actin. We link observed failures to a defect in F-actin organization, both in vitro and in vivo, which secondarily impairs EC adhesion and polarity. We also identify Cdc42 effectors Pak2/4 and N-WASP, as well as the actomyosin machinery, to be crucial for EC actin organization. This work supports the notion of Cdc42 as a central regulator of the cellular machinery in ECs that drives blood vessel formation.
The vascular system is a complex, largely stereotyped network of interconnecting and branching vessels. How thousands of vessels form at precise locations is a key question regarding vascular ...morphogenesis. In order to achieve this defined architecture, the embryo integrates a multitude of attractive and repulsive cues to guide and shape the developing vasculature. This review summarizes recent studies investigating the interactions between endothelial cells and signals from surrounding tissues that pattern the initial blood vessel network.
Proper vascular formation is regulated by multiple signaling pathways. The vascular endothelial growth factor (VEGF) signaling promotes endothelial proliferation. Notch and its downstream targets act ...to lead endothelial cells toward an arterial fate through regulation of arterial gene expression. However, the mechanisms of how endothelial cells (ECs) in the artery maintain their arterial characteristics remain unclear. Here, we show that PRDM16 (positive regulatory domain-containing protein 16), a zinc finger transcription factor, is expressed in arterial ECs, but not venous ECs in developing embryos and neonatal retinas. Endothelial-specific deletion of
induced ectopic venous marker expression in the arterial ECs and reduced vascular smooth muscle cell (vSMC) recruitment around arteries. Whole-genome transcriptome analysis using isolated brain ECs show that the expression of
(encoding ANGIOPOIETIN2, which inhibits vSMC recruitment) is upregulated in the
knockout ECs. Conversely, forced expression of PRDM16 in venous ECs is sufficient to induce arterial gene expression and repress the ANGPT2 level. Together, these results reveal an arterial cell-autonomous function for PRDM16 in suppressing venous characteristics in arterial ECs.