SAT013 characterization of CRISPR/Cas9 induced RXR isotype mutants n Xenopus Laevis

Abstract

Retinoid-X receptors (RXRs) occupy a unique position in the nuclear receptor (NR) superfamily. While some actions of the highly conserved RXRs may be independent, RXRs most often are heterodimer partners for several NRs that regulate development, reproduction and metabolism. As such, environmental chemicals interfering with the activity of RXRs and their partners may have significant deleterious effects on human health, often in developmental window, cell-type, and gene selective ways. The frog Xenopus has long served as an excellent model for studying vertebrate development including thyroid hormone action, and recent advances in genome editing have greatly enhanced its utility for investigating basic cell and organismal biology as well as human disease processes. To determine isotype specific roles in response to natural and synthetic ligands as well as candidate environmental chemicals interacting with RXRs in vivo, we created targeted germline mutations in the Xenopus tropicalis RXRα (rxra), RXRβ (rxrb), and RXRγ (rxrg) genes. For CRISPR/Cas9 mediated genome editing, guide RNAs were designed to target sequences just prior to or within the first RXR DNA binding domains. We raised all three RXR homozygous null mutants up to and through metamorphosis. The rxrb and rxrg homozygous null mutants completed metamorphosis and allowed us to establish adult null lines, but the rxra homozygous null mutants die at metamorphic climax, reminiscent of the thyroid hormone receptor (thra/thrb) double mutants. Some rxra mutant froglets also lack forearms or have forelimb defects, implying a role for rxra in RA mediated forelimb specification. The X. tropicalis rxrg homozygous null mutants show a trend toward an increased rate of metamorphosis versus matched wild-type controls, with significantly elevated tshb gene expression as newly metamorphosed froglets suggesting impaired negative feedback by TH. Significantly altered opsin gene expression in the froglet eye was also observed in rxrg mutant retinas: elevated short wave opsins opn1sw1 and opn1sw2 and sharply reduced long wave opsin opn1lw. This elevated tshb and opn1sw1 expression is highly similar to what is found in mouse rxrg null mutants; thus RXRγ action in the eye and pituitary appears to be highly conserved between mammals and amphibians. The establishment of these new RXR isotype mutant specific lines will support the expanded use of Xenopus to study basic biology and environmental toxicology involving TR-RXR and other NR-RXR partnerships.