spondo Is a Dominant, Gain-of-Function Mutation in cmn (A) The spondo mutant phenotype is due to an early, nonsynonymous mutation in the calymmin gene (M10R). pd1211 mutation is a 2 bp deletion upstream of spondo that results in an early stop codon. (B) Expression of cmn during early zebrafish development is confined to the notochord (Figure S3). (C) Cross-section of 12 dpf larvae depicting the restriction of cmn expression to the notochord sheath cells. The spondo mutation causes disruption of the notochordal sheath (Figure S3). (D and D′) At later developmental stages, cmn expression becomes segmented and downregulated in cells that form the chordacentra. The expression is most highly upregulated in the col9a2 domain and in cells that are actively differentiating into mineralizing cells and express both col9a2 and entpd5a (arrows). (E) cmn is a highly derived paralog of an elastin-like extracellular protein. The locus containing cmn is duplicated in zebrafish, retaining synteny with neighboring genes. The gene altered in spondo (asterisk) is highly differentiated from its other paralog (“a”) as well as orthologs found in sarcopterygian lineages and is generally conserved among teleost fishes (Figure S2). (F and G) Overexpression of cmn containing the spondo mutation inhibits sheath cell differentiation into mineralizing cells. DNA constructs containing either QUAS:GFPCaax or QUAS:spod-p2A-EGFP sequences were mosaically overexpressed in a col9a2:QF2 transgenic line. Gaps in entpd5a:pkred expression within mineralizing domains were quantified (arrows). In control conditions, 14.1% of GFP+ sheath cells did not express entpd5a. In the experimental group of fish overexpressing the spondo mutation, 42.2% of GFP+ sheath cells were entpd5a negative. Two tailed p value = 0.0006. Overexpression of wild-type cmn does not have any effect (Figure S4). (H–K′) Reversion of spondo phenotype by creation of disruptive alleles in cis and trans to cmnspondo. Pooled guides targeting cmn injected into wild-type or cmnspod/+ embryos lead to mosaic deletions within the cmn locus. Targeting deletions to cmn reverted the spondo phenotype and partially restored notochord segmentation in 11/11 cmnspod/+ fish analyzed. Phenotype of injected fish closely resembles individuals containing the loss of function allele, pd1211 (Figure S5). See also Table S1.
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