Fig. 2.

Fig. 2.

The suppression of early lethality of the msmo1^nu81 mutation yields adult fish with kol^nu7-like phenotype. (A) Survival of larvae from msmo1^nu81/+ in-crosses, from 7 dpf to 70 dpf. Most msmo1^nu81 mutants die by 9 dpf. [7 dpf wild type (wt; black) n=19, heterozygotes (het; pale gray) n=49, knockout mutants (KO; dark gray) n=16, P=0.02794; 8 dpf wt n=21, het n=39, KO n=17, P=0.8065; 9 dpf wt n=12, het n=40, KO n=3, ***P=0.0008; 10 dpf wt n=37, het n=44, KO n=3, ****P<0.0001; 15 dpf wt n=26, het n=51, KO n=3, ****P<0.0001; 70 dpf wt n=18, het n=35, KO n=0, ***P=0.001.] (B) Overexpression of msmo1 driven by daily heat shock of the transgenic line Tg(hsp70l:msmo1:IRESnlsGFP)^nu99 rescued the lethality of msmo1^nu8 mutants. Transgenic screening based on cardiac GFP. (Control non-transgenic siblings 14 dpf wt n=14, het n=32, KO n=2, ***P=0.0035; transgenic siblings 14 dpf wt n=18, het n=44, KO n=28, P=0.3214.) cont, control; hs, heat shock. (C) Dietary cholesterol supplementation does not improve the survivability of msmo1^nu81 mutants. Clutches from msmo1^nu81/+ in-crosses were fed either a high-cholesterol diet (hcd) or a control standardized diet (cont) beginning at 5 dpf until collection at 10 dpf. (HCD wt n=26, het n=50, KO n=9, P=0.0089; control diet wt n=22, het n=66, KO n=5, P<0.0001.) All two-tailed P-values were calculated using chi-squared test. (D-F′) Whole-mount in situ hybridization during the first 5 days of development shows msmo1 expression predominately in the yolk syncytial layer (YSL) and liver. Expression is first detected during early somitogenesis in the YSL (D) and continues there at 3 dpf (E). At 4 dpf, strong expression is observed in the differentiated liver (F,F′). (G-I) Generation of msmo1^nu81/wild-type chimeras using endoderm replacement rescues early lethality of msmo1^nu81 mutants and reveals a strong kol^nu7-like phenotype. (G) Schematic of the procedure. Wild-type Tg(ubi:Zebrabow-M)^a131 donor embryos were injected with sox32 RNA to force an endodermal fate. At high stage (∼3 hpf), cells were transplanted from donor to host embryos collected from msmo1^nu81/+ in-crosses. (H) Surviving msmo1^nu81 mutants display a strong kol^nu7-like phenotype. msmo1^nu81 n=4. (I) The majority of organs of endodermal origin displayed a high enrichment in transplanted Tg(ubi:Zebrabow-M)^a131 cells (red). msmo1^nu81 n=4. (J-L) Liver-specific msmo1 expression in msmo1^nu81 mutants rescues early lethality and produces juvenile msmo1^nu81 mutants with strong kol^nu7-like phenotype. (J) Liver-specific regulatory element fabp10a was used to drive msmo1 expression in msmo1^nu81 mutants. (K) Adult Tg(fabp10a:msmo1:pA)^nu100;msmo1^nu81 mutants phenocopy kol^nu7 mutant. Tg(fabp10:msmo1:pA)^nu100 n=50, Tg(fabp10a:msmo1:pA)^nu100;msmo1^nu81 n=50. (L) Whole-mount skeletal preparations reveal gross malformations throughout Tg(fabp10a:msmo1:pA)^nu100;msmo1^nu81 craniofacial and axial skeleton, similar to those observed in kol^nu7. Tg(fabp10:msmo1:pA)^nu100 n=3, Tg(fabp10a:msmo1:pA)^nu100;msmo1^nu81 n=7.