Fig. 1.

Late-onset skeletal defects observed in the koliber^nu7 (kol^nu7) mutant are the result of downregulation of msmo1 expression. (A) Compared to wild-type (wt) siblings (top), adult kol^nu7 mutants display a reduced body length and small head size. wt n=300, kol^nu7 n=300. (B) Whole-mount skeletal preparations reveal gross malformations and hyperossification throughout the adult kol^nu7 craniofacial and axial skeleton after Alcian Blue (cartilage) and Alizarin Red (ossified bone and mineralized tissues) staining. wt n=100, kol^nu7 n=100. (C-F) Early larval mutants do not display patterning defects or premature ossification. Whole-mount Alizarin Red staining of 4.7 mm (∼8 dpf) wt (C,E) and kol^nu7. (D,F). Ventral view (C,D) and lateral view (E,F). wt n=3, kol^nu7 n=4. (G) Positional cloning reveals that the kol^nu7 locus is located to the ∼457 kb critical region flanked by polymorphic markers with one or two recombinants out of 1844 meioses, corresponding to a genetic distance of 0.16 cM. (H) Screen of gene expression using quantitative RT-PCR from RNA extracted from hypural complex of ∼18 mm SL kol^nu7 and wt siblings. wt n=2, kol^nu7 =2. Only msmo1 level was significantly different out of 11 tested genes, located in the ∼1.2 Mb region encompassing the kol^nu7 locus. Initial screen results: grhprb not detected (ND); uba6 mean difference 0.64, s.d. 0.36; abpp2 −1.62, s.d. 1.00; mettl14 1.16, s.d. 0.68; prss12 1.20, s.d. 0.72; ndst3 1.51, s.d. 1.50; ugt8 −1.03, s.d. 0.07; spock3 ND; tll1 0.67, s.d. 0.27; cpe 0.90, s.d. 0.42; msmo1 −10.07, s.d. 4.32. Confirmation test of msmo1 expression (msmo1 −11.47, s.d. 8.49; P=0.0012), wt n=3, kol^nu7 =5. Three technical replicates were included for all assays. Gene expression was normalized to the reference gene eefla1. Fold change was calculated using Livak method (Livak and Schmittgen, 2001). P-value calculated using unpaired Student's t-test on dCt values. (I) The msmo1^nu81 mutant allele is not able to complement the kol^nu7 mutation. Adult kol^nu7/+:msmol^nu81/+ transheterozygotes phenocopy the kol^nu7 mutant. wt n=100, kol^nu7/+:msmol^nu81/+ n=100. (J) Whole-mount skeletal preparations reveal gross malformations throughout the kol^nu7/+:msmol^nu81/+ craniofacial and axial skeleton, similar to those observed in kol^nu7. wt n=10, kol^nu7/+:msmo1^nu81/+ n=10. (K) The msmo1^nu81 allele is the result of a 37 bp insertion, allowing for allele-specific expression analysis between msmo1^nu81 and kol^nu7. PAM, protospacer adjacent motif (underlined in red). (L) Strong downregulation of the kol^nu7-linked allele (asterisks) in kol^nu7/+:msmo1^nu81/+ compared to the wt allele in msmo1^nu81/+ suggests that the kol^nu7 mutation is cis-acting. kol^nu7/+:msmo1^nu81/+ n=3, msmo1^nu81/+ n=3. The top band is a heterodimer of wt and mutant strands.