Cystathionine β-Synthase Is Necessary for Axis Development in Vivo

Abstract

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.

A hypomorphic cystathionine ß-synthase gene contributes to cavefish eye loss by disrupting optic vasculature

“…The Astyanax cbsa and cbsb genes do not appear to be co-expressed in the eyes, which is the likely reason that hypomorphic cbsa effects are restricted to these organs and do not engender a lethal response. We also observed cbsa expression in the SF optic tectum, as also reported for cbs genes in zebrafish 19 and mouse 36 , and cbsa is downregulated in this region of the CF brain. The optic tecta are regressed coordinately with the eyes during CF evolution 37,38 .…”

“…2o, p). The pattern of cbsa and cbsb expression in Astyanax SF resembled that reported in zebrafish 19 . The results suggest that cbsa and cbsb show mostly overlapping expression during SF and PA-CF development, except for the PA-CF eyes, brain, and myotomes in which neither of the cbs paralogs was strongly expressed.…”

Cystathione β-synthase regulates HIF-1α stability through persulfidation of PHD2

“…Hypoxic stress manifested by decreased EC proliferation was also rescued by H 2 S supplementation, all of which support a cytoprotective role for CBS and H 2 S (41). Supporting our in vitro findings, CBS-silenced zebrafish embryos exhibited the same phenotypes observed during hypoxic signaling, including bent axis and abnormal ISVs (25); as expected, these phenotypes were reversed by supplementation with H 2 S.…”

“…All zebrafish studies performed under the Institutional Animal Care and Use Committee (IACUC) protocol 320, which is approved by the Medical College of Wisconsin (MCW) Institutional Animal Care and Use Committee. Zebrafish maintenance and embryo stages were performed as per the protocols published before (25). The generation and characterization of the cbsb MO studies and cbsb CRISPR fish have already been described before by us (25).…”

Zebrafish, a Novel Model System to Study Uremic Toxins: The Case for the Sulfur Amino Acid Lanthionine

“…As for CBS in particular, it is known that two CBS paralogues are present in zebrafish, as CBSa and CBSb . CBSa has been proven to be redundant [ 29 ] while in adult animals CBSb is expressed primarily in the kidney and muscle, with lower levels in the brain, gill, and heart [ 30 ] and its loss of function during embryogenesis, leads to increased levels of homocysteine [ 29 ]. In this work we only focused our qPCR analysis on the transcript encoding for the CBSb , while the anti-CBS antibody, used for WB was not paralog-specific.…”