Fig. 1

Genes encoding the transcription factor Lmx1b, lmx1ba and lmx1bb, are differentially patterned and necessary for semicircular canal morphogenesis. (A) Illustrations show typical morphological features of OVs at relevant stages of semicircular canal morphogenesis. Images are maximum intensity projections of OVs at select time points stained with multiplex in situ probes against lmx1bb (green), lmx1ba (magenta), and col2a1a (white). The z-volume and the contrast of each time point are individually set to capture all buds and achieve better visualization. Yellow lines indicate buds or pillars. Scale bar: 50 ?m. (B) Maximum intensity projections of OVs of membrane-NeonGreen-expressing embryos at 50 hpf injected with control or jag1b MO stained with multiplex in situ probes against lmx1bb (green) and lmx1ba (magenta). 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 control and jag1b MO. Crista and macula are marked by yellow and black asterisks, respectively. Scale bar: 50 ?m. (C) 3D-rendered OVs at 50 or 72 hpf from membrane-NeonGreen-expressing embryos injected with control or jag1b MO. Scale bars: 50 ?m. (D) Illustrations showing the difference in gene expression of lmx1ba and lmx1bb at 50 hpf (left) or semicircular canal morphology at 72 hpf (right) between embryos injected with control and jag1b MO. (E) 3D-rendered OVs at 50 or 72 hpf from membrane-mCherry-expressing embryos injected with tyrosinase or lmx1b double gRNA. Scale bars: 50 ?m. (F,F?) Quantification of bud length (F) and lateral bud width (F?) at 50 hpf in embryos injected with tyrosinase or lmx1b double gRNA. Data are mean±s.e.m. In the absence of buds, bud lengths correspond to cell lengths. 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). a, anterior; al, anterior-lateral; hc, hair cells; p, posterior; pl, posterior-lateral; v, ventral. Representative images and data from two independent experiments are shown.