galnsm/m mutant larvae have craniofacial cartilage and bone development. (A) Alcian Blue staining of galns wild-type (+/+) and mutant (m/m) larvae showing that mutant cartilages are shorter and slightly dysmorphic. Lines demarcating the edge of the eyes (black line), the tip of the nose (black dashed lines) and the tip of Meckel's cartilage show that wild-type cartilage (red dashed lines) extends much further past the eyes than the mutant cartilage. Red arrows highlight cartilages affected by Galns defiency. Scale bar: 20 µm. (B) Schematics highlighting the parameters measured, including the lengths of the Meckel's (M; blue) and ceratohyal (CH; green) cartilages, and angle between the ceratohyal structures (red, palatoquadrate). The distance Meckel's cartilage protrudes past the eyes and area of the head was also measured. With the exception of the ceratohyal angle, all measurements were reduced in mutant larvae. n=14-18 larvae per genotype per timepoint from three independent biological crosses analyzed. Error bars=s.e.m. Two-tailed paired Student's t-test, *P<0.05, **P<0.01, ****P<0.0001. (C) Confocal analyses of 7 dpf fli1a:EGFP-positive cells from the lateral and central portions of Meckel's cartilage identified alterations in the shape and organization of mutant chondrocytes. Scale bar: 10 µm. (D) Graphs show quantitative measurements of cell shape and organization in the central and lateral portions of Meckel's cartilage. n=20-25 larvae from three biological matings. Error bars=s.e.m. Two-tailed paired Student's t-test, ****P<0.0001. (E) Confocal images showing that mutant chondrocytes are highly vacuolated. Yellow arrows highlight vacuoles. Scale bar: 10 µm. (F) Analysis of vacuolation shows that nearly 100% of mutant chondrocytes contain vacuoles, whereas very few wild-type cells are vacuolated. n=23 larvae per genotype from three independent biological crosses were analyzed. Error bars=s.e.m. Two-tailed paired Student's t-test, ****P<0.0001. (G) Alizarin Red staining of larvae at 7 and 10 dpf identified multiple regions in galnsm/m mutants with decrease mineralization. The boxed area surrounding Meckel's cartilage represents region of interest (ROI)1, and the boxed area surrounding the ceratohyal represents ROI2. Scale bar: 20 µm. (H) The fluorescence intensity of Alizarin Red staining was measured in each of the ROIs. Data from n=12 larvae per genotype from three independent biological crosses were analyzed and graphed. Data from 7 dpf (left column) and 10 dpf (right column) are presented. Error bars=s.e.m. Two-tailed paired Student's t-test, *P<0.05, **P<0.01, ***P<0.001. (I) Alizarin Red staining of larval vertebrae also shows reduced mineralization in mutant vertebrae. Asterisks highlight regions of difference at 7 dpf. Bars highlight the reduction in the diameter of mineralized structures in 10 dpf mutants. (J-L) Graphs show quantitative measurements of the number of mineralized vertebrae in 7 dpf (left) and 10 dpf (right) larvae (J), the intensity of Alizarin Red (AR) fluorescence in 7 dpf larvae (K) and the diameter of Alizarin Red-positive vertebrae in 10 dpf larvae (L). n=21 larvae from three biological replicates. Error bars=s.e.m. Two-tailed paired Student's t-test, *P<0.05, ***P<0.001, ****P<0.01.
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