A. External srn phenotypes at 48 hpf include a bent tail (80% dorsal (534 embryos, 8 carrier pairs)) and aberrant hindbrain formation (brackets). Scale bar = 100 μm. B. Genetic and physical map of the srn locus (red arrow), including SSLP markers, number of recombinants, BAC clones and megabase positions from Ensembl Zv7. C, D. In srn, Gmds mutation is G to T (C, red box) resulting in a Glycine to Valine conversion (D, red box). GMDS amino acid sequence is highly conserved. E. Schematic of srn mutation in the short-chain dehydrogenase/reductase (SDR) domain of GMDS. F. Two splice variants exist in gmds mRNA, with (gmds-L, 377 aa) or without (gmds-S, 370 aa) exon 4. Gmds alternative splicing is not altered in srn mutants. G. Injection of gmds mRNA rescues srn mutants. Compared to uninjected embryos, 28.6±1.2% of embryos were mutant when scored by external phenotypes (3413 embryos, 27 carrier pairs). In embryos injected with WT gmds-gfp mRNA, the percentage of mutants scored by external phenotypes was significantly decreased, to ca. 5% (gmds-wtL-gfp 5.4±2.5%, 401 embryos, 3 carrier pairs; gmds-wtS-gfp 5.1±0.6%, 587 embryos, 4 carrier pairs; one-way ANOVA, followed by Dunn′s pairwise comparison, p<0.05). The percentage of embryos with mutant external phenotypes was unchanged in embryos injected with mutant gmds-gfp mRNA (gmds-srnL-gfp 30.2±0.9%, 387 embryos, 3 carrier pairs; gmds-wtS-gfp 25.1±1.9%, 516 embryos, 4 carrier pairs). This mRNA rescue experiment confirms that gmds is the gene responsible for srn mutation.
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