Loss of Lmx1b affects muscle formation in the trunk but not in the head. Confocal z-stack projections of wt and dKO mutants at 5 dpf immunostained with myosin, showing the whole body, scale bars: 250 μm, (A) and zoomed in regions of trunk muscle where defects in muscle formation increase in severity from no defect in wild type (B) to increasing number of defects in dKOs (C-E). Scale bars: 50 μm. (C,D) Pink asterisks indicate large gaps between myofibrils; (D) white arrowheads indicate abnormal fibre branching. (F) Percentage of larvae showing muscle defects at different severities quantified for wt and dKO larvae at 5 dpf. Differences in muscle formation between wt and dKO larvae at 3 dpf and 5 dpf quantified as (G) somite area and (H) the percentage coverage of muscle (myosin) staining per somite across all genotypes. Each data point is an average of measurements taken from three separate somites per fish. Schematics (right) show how measurements were made. N=11 for wild type and dKO mutants. Welch's t-test performed for G where ***P=.0001. Student's unpaired t-tests performed for H where *P=.01 and **P=.0092. (I) Confocal maximum projections of wt and dKO mutant larvae at 5 dpf immunostained for muscle (green) and co-stained with DAPI (blue). Scale bars: 100 μM. (J) Confocal z-stack projections of the lower jaw of wild type and dKO mutants immunostained for the tendon marker Thbs4 (cyan) and Collagen type II (Col2a1; red) at 5 dpf. Scale bars: 50 μM. (K) Schematic showing location and names of key muscles in the lower jaw of zebrafish. Muscles measured here are filled in in green. (L) Graphs showing quantification of muscle fibre length and width in the intermandibularis posterior (imp) and interhyal (ih) muscles at 5 dpf. N=5 for wild type and 7 for dKOs. Two datapoints plotted per fish, per age as measurements taken from right and left side of the lower jaw. Student's unpaired t-test performed at each age for all graphs.
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