Loss of lmx1ba delays cartilage growth and maturation. (A) Confocal images of z-stack projections of the lower jaw at 3 dpf and 5 dpf in wild type and lmx1ba mutants immunostained for Col2a1 (white). Scale bars: 50 μm. Schematics indicating where measurements were made for (B) length of lower jaw, (C) length of Meckel's cartilage and (D) width of Meckel's cartilage (left), with quantification of results (right). N=7 for wild type; 8 for lmx1ba mutants. Student's unpaired t-test performed for all where ****P<0.0001. Confocal z-stack projections of the (E) lower jaw and (F) lower jaw joint at 5 dpf in wild type and lmx1ba mutants, immunostained for EdU (cyan) and Collagen Type II (Col2a1; red) following 24-h treatment with EdU Click-iT. Scale bars: 50 μm for E and 10 μm for F. Number of EdU-Col2a1-positive cells quantified for (G) the whole lower jaw and (H) jaw joint. N=6 for wild type; 8 for lmx1ba mutants. Student's unpaired t-test performed for all where *P=0.0362. (I-L) Electron microscopy of ethmoid plate in wild type and lmx1ba mutants at 5 dpf. (I,K) Pink arrowheads highlight areas of non-uniformity and non-intercalating chondrocytes. Scale bars: 5 µm. (M) Number of chondrocytes on periphery of cartilage that are not aligning down central line of stack. Calculated as percentage of total cell number along ethmoid plate in one section per fish. (N) Number of vesicles present per chondrocyte. Mann–Whitney unpaired t-test performed where, ****P=0.0001. N=30 chondrocytes total from three larvae per genotype.
|
