Objective
Endothelial cells (ECs) sense and respond to flow-induced mechanical stress, in part, via microtubule-based projections called primary cilia. However, many critical steps during vascular morphogenesis occur independent of flow. The involvement of cilia in regulating these stages of cranial vascular morphogenesis is poorly understood, as cilia have not been visualized in primary head vessels. The objective of this study was to investigate involvement of cilia in regulating the early stages of cranial vascular morphogenesis.
Approach and Results
Using high-resolution imaging of the Tg(kdrl:mCherry-CAAX)
y171;(bactin::Arl13b:GFP) zebrafish line, we showed that cilia are enriched in the earliest formed cranial vessels that assemble via vasculogenesis and in angiogenic hindbrain capillaries. Cilia were more prevalent around the boundaries of putative intravascular spaces in primary and angiogenic vessels. Loss of cardiac contractility and blood flow, due to knockdown of cardiac troponin T type 2a (tnnt2a) expression, did not affect the distribution of cilia in primary head vasculature. In later stages of development, cilia were detected in retinal vasculature, areas of high curvature, vessel bifurcation points, and during vessel anastomosis. Loss of genes crucial for cilia biogenesis (ift172 and ift81) induced intracerebral hemorrhages in an EC-autonomous manner. Exposure to high shear stress induced premature cilia disassembly in brain ECs and was associated with intracerebral hemorrhages.
Conclusion
Our study suggests a functional role for cilia in brain ECs, which is associated with the emergence and remodeling of the primary cranial vasculature. This cilia function is flow-independent, and cilia in ECs are required for cerebral vascular stability.
Spontaneous intracranial hemorrhage is a debilitating form of stroke, often leading to death or permanent cognitive impairment. Many of the causative genes and the underlying mechanisms implicated in developmental cerebral-vascular malformations are unknown. Recent in vitro and in vivo studies in mice have shown inhibition of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway to be effective in stabilizing cranial vessels. Using a combination of pharmacological and genetic approaches to specifically inhibit the HMGCR pathway in zebrafish (Danio rerio), we demonstrate a requirement for this metabolic pathway in developmental vascular stability. Here we report that inhibition of HMGCR function perturbs cerebral-vascular stability, resulting in progressive dilation of blood vessels, followed by vessel rupture, mimicking cerebral cavernous malformation (CCM)-like lesions in humans and murine models. The hemorrhages in the brain are rescued by prior exogenous supplementation with geranylgeranyl pyrophosphate (GGPP), a 20-carbon metabolite of the HMGCR pathway, required for the membrane localization and activation of Rho GTPases. Consistent with this observation, morpholino-induced depletion of the β-subunit of geranylgeranyltransferase I (GGTase I), an enzyme that facilitates the post-translational transfer of the GGPP moiety to the C-terminus of Rho family of GTPases, mimics the cerebral hemorrhaging induced by the pharmacological and genetic ablation of HMGCR. In embryos with cerebral hemorrhage, the endothelial-specific expression of cdc42, a Rho GTPase involved in the regulation of vascular permeability, was significantly reduced. Taken together, our data reveal a metabolic contribution to the stabilization of nascent cranial vessels, requiring protein geranylgeranylation acting downstream of the HMGCR pathway.
Objective
Tyrosine kinase containing immunoglobulin and epidermal growth factor homology1 (Tie1), an endothelial and hematopoietic cell-specific receptor tyrosine kinase, is an important regulator of angiogenesis and critical for maintaining vascular integrity. The post-transcriptional regulation of tie1 mRNA expression is not understood, but it might partly explain Tie1’s differential expression pattern in endothelium. Following up on our previous work that identified natural antisense transcripts from the tie1 locus—tie1 antisense (tie1AS), which regulates tie1 mRNA levels in zebrafish—we attempted to identify the mechanism of this regulation.
Approach and Results
Through in vitro and in vivo ribonucleoprotein binding studies, we demonstrated that tie1AS lncRNA interacts with an RNA binding protein—embryonic lethal and abnormal vision Drosophila-like1 (Elavl1)—that regulates tie1 mRNA levels. When we disrupted the interaction between tie1AS and Elavl1 by using constitutively active anti-sense morpholino oligonucleotides (MOs) or photoactivatable MOs, tie1 mRNA levels increased between 26 and 31 hours post fertilization (hpf), particularly in the head. This increase correlated with dilation of primordial midbrain channels, smaller eyes, and reduced ventricular space. We also observed these phenotypes when we used CRISPRi to knock down tie1AS. Treatment of the MO-injected embryos with a small molecule that decreased tie1 mRNA levels rescued all three abnormal phenotypes.
Conclusions
We identified a novel mode of temporal and spatial post-transcriptional regulation of tie1 mRNA. It involves lncRNA, tie1AS, and Elavl1 (an interactor of tie1AS).
Cardiac development in vertebrates is a finely tuned process regulated by a set
of conserved signaling pathways. Perturbations of these processes are often
associated with congenital cardiac malformations. Platelet-derived growth factor
receptor α (PDGFRα) is a highly conserved tyrosine kinase
receptor, which is essential for development and organogenesis. Disruption of
Pdgfrα function in murine models is embryonic lethal
due to severe cardiovascular defects, suggesting a role in cardiac development,
thus necessitating the use of alternative models to explore its precise
function. In this study, we generated a zebrafish pdgfra mutant
line by gene trapping, in which the Pdgfra protein is truncated and fused with
mRFP (Pdgfra-mRFP). Our results demonstrate that pdgfra mutants
have defects in cardiac morphology as a result of abnormal fusion of myocardial
precursors. Expression analysis of the developing heart at later stages
suggested that Pdgfra-mRFP is expressed in the endocardium. Further examination
of the endocardium in pdgfra mutants revealed defective
endocardial migration to the midline, where cardiac fusion eventually occurs.
Together, our data suggests that pdgfra is required for proper
medial migration of both endocardial and myocardial precursors, an essential
step required for cardiac assembly and development.
The cystathionine ß-synthase (CBS) is a critical enzyme in the transsulfuration pathway and is responsible for the synthesis of cystathionine from serine and homocysteine. Cystathionine is a precursor to amino acid cysteine. CBS is also responsible for generation of hydrogen sulfide (H2S) from cysteine. Mutation in CBS enzyme causes homocysteine levels to rise, and gives rise to a condition called hyperhomocysteinuria. To date, numerous mouse knockout models for CBS enzyme has been generated, which show panoply of defects, reflecting the importance of this enzyme in development. In zebrafish, we and others have identified two orthologs of cbs, which we call cbsa and cbsb. Previous gene knockdown studies in zebrafish have reported a function for cbsb ortholog in maintaining ion homeostasis in developing embryos. However, its role in maintaining H2S homeostasis in embryos is unknown. Here, we have performed RNA analysis in whole zebrafish embryos that showed a wide expression pattern for cbsa and cbsb primarily along the embryonic axis of the developing embryo. Loss-of-function analysis using a combination of approaches which include splice morpholinos and CRISPR/Cas9 genomic engineering show evidence that cbsb ortholog is responsible for anterior-posterior axis development, and cbsa function is redundant. Cbsb loss of function fish embryos show shortened and bent axis, along with less H2S and more homocysteine, effects resulting from loss of Cbsb. Using a chemical biology approach, we rescued the axis defects with betaine, a compound known to reduce homocysteine levels in plasma, and GYY4137, a long term H2S donor. These results collectively argue that cells along the axis of a developing embryo are sensitive to changes in homocysteine and H2S levels, pathways that are controlled by Cbsb, and thus is essential for development.
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