HMGCR Pathway Mediates Cerebral-Vascular Stability and Angiogenesis in Developing Zebrafish

Intracerebral hemorrhage (ICH) is a severe form of stroke, with a high mortality rate and often resulting in irreversible neurological deterioration. Although animal studies have provided insight into the etiology of the disease, many of the causative genes and mechanisms implicated in cerebral-vascular malformations are unknown. Treatment options remain ineffective. With the present models, the pathophysiological consequences of ICH can only be assessed in situ and after histological analysis. Furthermore, common deficiencies of the current models include the heterogeneity, low expression and low reproducibility of the desired phenotype. Hence, there is a requirement for novel approaches to model ICH pathogenesis. Zebrafish (Danio rerio) has gained recognition as a vertebrate model for stroke research. Through a combination of pharmacological blockers, metabolite rescue, genetic approaches, and confocal imaging analysis, I demonstrate a requirement for the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway in regulating developmental cerebral-vascular stabilization. A transient loss in HMGCR function induces ICH, characterised by progressive dilation of blood vessels, vascular permeability and vessel rupture. These effects are likely due to reduced prenylation of Rho GTPases, evidenced by morpholino-mediated blocking of the prenylation pathway and in vivo assessment of endothelial-specific localization of cdc42, a Rho GTPase family protein. These results are in conformity with recent clinical and experimental evidence. I have further shown that this model consistently replicates common pathoghysiological processes associated with ICH. The hemorrhages are associated with the disruption of the blood-brain barrier, vessel disintegration, hematoma expansion and edema into the adjacent brain regions. Also, enhanced apoptosis, activation of inflammatory mediators in the periphery of the hematoma, enriched heme oxygenase 1 (HO-1) expression and localised thrombosis were observed in these embryos. I show that the patterning and distribution of catecholaminergic neurons, response to sensory stimulus and swimming speed were impaired as a consequence of ICH. These results suggest that HMGCR contributes to cerebral-vascular stabilisation through Rho GTPase mediated-signalling and that zebrafish can serve as a powerful paradigm for the systemic analysis of the etiological and pathophysiological underpinnings of ICH and can help establish the basis for future studies into screening for putative therapeutics and elucidating mechanisms aiding functional recovery

Signaling Molecules Governing Pluripotency and Early Lineage Commitments in Human Pluripotent Stem Cells

Signaling in pluripotent stem cells is a complex and dynamic process involving multiple mediators, finely tuned to balancing pluripotency and differentiation states. Characterizing and modifying the necessary signaling pathways to attain desired cell types is required for stem-cell applications in various fields of regenerative medicine. These signals may help enhance the differentiation potential of pluripotent cells towards each of the embryonic lineages and enable us to achieve pure in vitro cultures of various cell types. This review provides a timely synthesis of recent advances into how maintenance of pluripotency in hPSCs is regulated by extrinsic cues, such as the fibroblast growth factor (FGF) and ACTIVIN signaling pathways, their interplay with other signaling pathways, namely, wingless- type MMTV integration site family (WNT) and mammalian target of rapamycin (mTOR), and the pathways governing the determination of multiple lineages

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.The publication of this article was covered with a generous support from BARZAN HOLDINGS. RR is partly supported by R61HL154254 and funds from Department of Pediatrics and Children’s Hospital

Disruption of pdgfra alters endocardial and myocardial fusion during zebrafish cardiac assembly

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

Characterization of Endothelial Cilia Distribution During Cerebral-Vascular Development in Zebrafish (Danio rerio)

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 because 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, because of 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. Conclusions—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.S. Eisa-Beygi is supported by funds from Kelleigh’s Cause Foundation. R. Ramchandran is also supported by 1R01HL123338,and partly supported by funds from Women’s Health Research Program at the Medical College of Wisconsin. S. Prabhudesai is supported by Department of Pediatrics, and Children’s Research Institute funds to R. Ramchandran. H.C. Yalcin is supported by QNRF (Qatar National Research Fund), National Priority Research Program NPRP 10-0123-170222, and Qatar University internal funds QUUGBRC-2017-3 and QUST-BRC-SPR\2017-1

Image1_Cystathionine β-Synthase Is Necessary for Axis Development in Vivo.PDF

<p>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.</p