Fig. 2

(A) Linkage analysis of gemütlich revealed a region of homozygosity on chromosome 9 with a peak between 32 to 33 Mb. (B) The MOB family member 4 (mob4) gene was located within the linked region. (C) Genomic sequences show that the mutant gemütlich harboured a mob4 allele with a premature stop codon in exon 2 (Q41X). (D) Western blot analysis using antibodies against human MOB4 showed epitope loss in mob4^geh homozygotes. (E) Knockdown of mob4 by the morpholinos mob4_3D(+93–16) that targets the splice donor of exon 3 or (F) mob4_ATG(-9+16) that targets the translation start codon led to a reduction in birefringence. (G) Compared to control injected 3-dpf-old larvae (100 ± 1% and 100 ± 2%, respectively), administration of mob4_3D(+93–16) induced a reduction in birefringence to 71 ± 2% and mob4_ATG(-9+16) to 65 ± 6%. Crosses represent individual larvae (n = 6). (H) RT-PCR using primers targeting exons 1 and 5 of mob4 revealed altered splicing in mob4_3D(+93–16)-injected larvae. (I) The mob4^-13 allele harboured a genomic deletion of 13 bp from exon 1 (g.5_17del). (J) Compared to 3-dpf-old siblings (both 100 ± 1%), the birefringence of mob4^-13 homozygotes and mob4^-13/geh compound heterozygotes was significantly reduced to 63 ± 1% and 61.1 ± 0.8%, respectively. Crosses represent averaged birefringence of clutches with a minimum of 6 larvae per genotype (n = 5 clutches). Data are presented as mean ± SEM; *** P < 0.001 calculated by Student’s t-test.