Fig. 2

Fig. 2 Lmx1ba and lmx1bb regulate HA and Versican synthesis genes for morphogenesis. (A) The workflow illustrates screening and validation of F0 gRNA/Cas9-injected embryos, followed by a multiplex in situ hybridization experiment. gRNA/Cas9-injected embryos were screened at 50 hpf using their inner ear morphology. A small piece from the tail was clipped to assess genome editing using a T7 endonuclease assay. The remaining embryo body was fixed with PFA. Individually validated embryos from controls and lm1ba and lmx1bb knockdown were pooled in a single tube to perform HCR-FISH. dpf, days post-fertilization; SCC, semicircular canal. Created in BioRender. Munjal, A. (2024) https://BioRender.com/y43g100. (B-I) Effect of lmx1b double knockdown on ECM-associated gene expression in OV. Maximum intensity projections of OVs and quantification of probe fluorescence intensity of has3 (B,C), vcana (D,E), vcanb (F,G), and chsy1 (H,I) probes. col2a1a probe and the other ECM-associated gene probes are shown as white and green, respectively. The z-volume of each condition is individually set to capture all buds and show the OV morphology. Heatmaps represent fluorescence intensity with the same contrast for each probe of embryos injected with tyrosinase or lmx1b double gRNA. Representative images from two independent experiments are shown. Scale bar: 50 μm. Data are mean±s.e.m. n denotes the number of buds from individual embryos measured per condition from two independent experiments. P-values as labeled (unpaired, two-tailed Student's t-test).