TGFβ-related growth factor signaling is reduced in chondrocytes of galnsm/m mutant larvae. (A) Live confocal images of Meckel's cartilage fli1a:EGFP;BRE:dmKO-positive galns+/+ (wild-type) and galnsm/m (mutant) larvae show reduced levels of BMP signaling in mutant chondrocytes and in perichondrial cells (yellow arrows). Right column shows that signaling is also decreased in mutant nasal placodes. M, Meckel's cartilage. Scale bars: 10 µm. (B) Graphs show that the percentage of cells with positive dmKO signal and fluorescence intensity are reduced in mutant cells. n=19 larvae from three independent matings. Error bars=s.e.m. Two-tailed paired Student's t-test, **P<0.01. (C) Live confocal images of the Meckel's and ceratohyal cartilages in fli1a:EGFP; SBE:nmCherry (nuclear)-positive galns+/+ (wild-type) and galnsm/m (mutants) larvae show that TGFβ signaling is also reduced in mutant cells. Yellow arrows highlight regions with mCherry signal, in chondrocytes and perichondrium. In right panels, blue arrows highlight reduced signal in perichondrium. Scale bar: 10 µm. (D,E) Graphs show that the percentage of cells with positive nuclear mCherry signal and fluorescence intensity are reduced in the Meckel's (D) and ceratohyal (E) cartilages in mutant larvae. n=13 larvae from three independent matings. Error bars=s.e.m. Two-tailed paired Student's t-test. *P<0.05, **P<0.01. (F,G) qRT-PCR indicated that the transcript expression levels of the BMP-regulated col10a1a (F) and TGFβ-regulated col2a1a (G) are reduced in galnsm/m mutant larvae at 4 and 8 dpf. n=5 biological replicates of samples containing 20 embryos per genotype per timepoint. Error bars=s.e.m. Two-tailed paired Student's t-test, **P<0.01, ***P<0.001, ****P<0.0001. (H) Live confocal images of fli1a:EGFP;BRE:dmKO-positive galns+/+ (wild-type) and galnsm/m (mutants) larvae treated at 3-4 dpf with either 0.025% DMSO or 25 µM E64d suggest that limited cathepsin inhibition does increase BMP signaling. Scale bar: 10 µm. (I) Graphs showing the percent of cells in larvae of each genotype and treatment condition with dmKO signal or fluorescence intensity in cartilage. n=17 larvae per genotype per treatment condition. Error bars=s.e.m. One-way ANOVA with Dunnett's multi-comparison correction, *P<0.05, **P<0.01, ***P<0.001. (J,K) Graphs show that the cell shape (J) and the percentage of cells intercalated (K) both improve in galnsm/m mutant larvae treated with E64d. Data from wild-type larvae are presented in Fig. S3. n=17 larvae per genotype per treatment condition from three independent matings. Error bars=s.e.m. One-way ANOVA with Dunnett’s multi-comparison correction, ****P<0.001. (L) Live confocal images of fli1a:EGFP-positive larvae show that E64d treatment does not improve vacuolation in galnsm/m mutants. Yellow arrows highlight vacuoles. (M) Graph shows that E64d treatment does not improve vacuolation. Error bars=s.e.m. Two-tailed paired Student's t-test, *P<0.05. Data from wild-type larvae are presented in Fig. S3.
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