Modeling Cartilage-Hair Hypoplasia in zebrafish through modulation of the rmrp locus

Abstract

Cartilage-Hair Hypoplasia (CHH) is an autosomal recessive genetic disorder caused by mutations in RMRP (RNA Component of the mitochondrial RNA Processing Complex). Although extremely rare in the general population, CHH is highly prevalent in the Amish (~1:1,300 births) and Finnish (~1:20,000 births), and can manifest in dwarfism, bone dysplasia, hypotrichosis, gastrointestinal dysfunction, anemia, and predisposition to certain cancers. Currently, no animal models exist for CHH; dysfunctional RMRP is embryologically lethal in mice. The goal of this project was to develop a model of rmrp dysfunction in zebrafish, Danio rerio, to study the developmental mechanisms underlying CHH. We designed and injected four CRISPR-Cas9 guide RNAs targeting the zebrafish ortholog, rmrp, and isolated DNA from F0 mutants at two days post-fertilization (dpf). Three of these guide RNAs induced genome editing as evidenced by heteroduplexing on a PAGE (polyacrylamide gel electrophoresis) analysis. We quantified mosaicism through cloning and sequencing analysis of embryos corresponding to these three guides, which demonstrated an estimated 50.24%, 100%, 95.99% mosaicism respectively. Next, we characterized phenotypes relating to cartilage dysplasia, anemia, and lack of enteric neurons (Hirschsprung Disease). We used automated imaging of a collagen-fluorescent reporter line (-1.4col1a1:egfp) to examine cartilage dysplasia, using ceratohyal angle as a proxy. We determined erythrocyte count using a fluorescent transgenic line (gata1:dsRed) to test for anemia, and we used HuC/D immunostaining, which allowed us to quantify enteric neurons. The F0 rmrp mutants recapitulate cartilage dysplasia and gastrointestinal deficiency, as indicated by a significantly more obtuse ceratohyal angle in mutants compared to controls for guides 2 and 3 (ex., p= 7.5 x 10-11, p= 1.9 x 10-4, respectively) and a significant reduction of enteric neurons in guide 2 F0 mutants (p=0.016 vs controls). We did not observe an anemia phenotype. This model will provide us with a deeper understanding of the cellular and molecular mechanism of RMRP, guide novel therapeutic avenues, and will likely uncover broader insight into the treatment of other phenotypically overlapping ribosomopathies in the long term.