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.  ...

Medical College of Wisconsin

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 (HS) from cysteine. Mutation in  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 , which we call  and . Previous gene knockdown studies in zebrafish have reported a function for  ortholog in maintaining ion homeostasis in developing embryos. However, its role in maintaining HS homeostasis in embryos is unknown. Here, we have performed RNA analysis in whole zebrafish embryos that showed a wide expression pattern for  and  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  ortholog is responsible for anterior-posterior axis development, and  function is redundant.  loss of function fish embryos show shortened and bent axis, along with less HS 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 HS donor. These results collectively argue that cells along the axis of a developing embryo are sensitive to changes in homocysteine and HS levels, pathways that are controlled by Cbsb, and thus is essential for development.

Cystathionine β-Synthase Is Necessary for Axis Development .

Tie1 (tyrosine kinase containing immunoglobulin and epidermal growth factor homology 1), 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  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  locus- (), which regulates  mRNA levels in zebrafish-we attempted to identify the mechanism of this regulation.

Temporal and Spatial Post-Transcriptional Regulation of Zebrafish  mRNA by Long Noncoding RNA During Brain Vascular Assembly.

[This corrects the article DOI: 10.3389/fcell.2018.00014.].

Corrigendum: Cystathionine β-Synthase Is Necessary for Axis Development .

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)  ;(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.

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

Skeletal muscle injuries are one of the most common problems in the worldwide which impose a substantial financial burden to the health care system. Accordingly, it widely accepted that muscle regeneration is a promising approach that can be used to treat muscle injury patients. However, the underlying mechanisms of muscle regeneration have yet to be elucidated. The muscle structure and muscle-related gene expression are highly conserved between human and zebrafish. Therefore, the zebrafish can be considered as an ideal animal model in muscle regeneration studies. In this study, Tol2 transposase was applied to produce Tg(mylpfa: cfp-nfsB) zebrafish model that express a fusion protein composed of cyan fluorescent protein (CFP) and nitrorudactase (NTR) under control of mylpfa promoter. The results showed that MTZ (Metronidazole) treatment of Tg(mylpfa:cfp-nfsB) zebrafish larvae can lead to muscle injury by selective ablation of muscle cells. And also, results confirmed the muscle regeneration ability of the transgenic larvae after withdrawal of Mtz for three days. Overall, The results of this study suggest that the Tg(mylpfa:cfp-nfsB) zebrafish model can be used in muscle regeneration study in order to elucidate the mechanisms of this process.

Establishing a new animal model for muscle regeneration studies.

Capillary malformation-arteriovenous malformation (CM-AVM) syndrome is a class of capillary anomalies that are associated with arteriovenous malformations and arteriovenous fistulas, which carry a risk of hemorrhages. There are no broadly effective pharmacological therapies currently available. Most CM-AVMs are associated with a loss of RASA1, resulting in constitutive activation of RAS signaling. However, protein interaction analysis revealed that RASA1 forms a complex with Rho GTPase-activating protein (RhoGAP), a negative regulator of RhoA signaling. Herein, we propose that loss of RASA1 function results in constitutive activation of RhoA signaling in endothelial cells, resulting in enhanced vascular permeability. Therefore, strategies aimed at curtailing RhoA activity should be tested as an adjunctive therapeutic approach in cell culture studies and animal models of RASA1 deficiency.

Shahram Eisa-Beygi

Department of Cell Biology,
Neurobiology and Anatomy,
Department of Cell Biology, Neurobiology and Anatomy

Medical College of Wisconsin

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

Shahram Eisa-Beygi

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cell Biology,Neurobiology and Anatomy,Department of Cell Biology, Neurobiology and Anatomy

Medical College of Wisconsin

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

Department of Cell Biology,
Neurobiology and Anatomy,
Department of Cell Biology, Neurobiology and Anatomy