Germline specific dgcr8 deletion has no functional influence on gonad development. (A) A basic schematic diagram of generating dgcr8 germline conditional knockout zebrafish. (B) Real-time analysis of the expression of dgcr8 mRNA and primary miRNAs in ovary of dgcr8 kop-cKO fish. (C) Real-time PCR analysis of the expression of mature miRNAs in ovary of dgcr8 kop-cKO fish. (D) Expression of germline markers in the ovary of dgcr8 kop-cKO. (E) Histology of ovaries in the wild-type and dgcr8 kop-cKO female zebrafish. *p < 0.05, **p < 0.01, and ****p < 0.0001.

Rescue of Mdgcr8 and MZdgcr8 using miR-430 duplex. (A) A schematic diagram of generating Mdgcr8 mutant zebrafish by out-crossing with wild-type male. (B) A schematic diagram of generating MZdgcr8 mutant zebrafish by in-crossing. (C) The expression of dgcr8 was analyzed by in situ hybridization in early embryonic stage. (D) Mdgcr8 and MZdgcr8 exhibit gastrulation defect, brain malformations, body curvature, and heart developmental defects compared with wild-type. miR-430 duplex was used to rescue the defects. (E) Epiboly defects in Mdgcr8 and MZdgcr8. At 14 hpf, the phenotype of yolk excision could be observed in MZdgcr8 mutant (5 X), but not in Mdgcr8. (F) Mdgcr8 embryonic survival rate become normal after rescue. (G) MZdgcr8 mutant embryos only survived up to 6–10 dpf after rescue. (H) Mdgcr8 mutant embryos after rescue could develop to adults and exhibit proper sex ratio. Scale bar 1 cm.

Maternal Dgcr8 regulates mitosis during early embryonic development in Mdgcr8 and MZdgcr8. (A) Representative images of 25 hpf WT, Mdgcr8 and MZdgcr8 embryos showing the distribution of PH3 immunolabeling cells (B) and quantification of positive cells in the hindbrain before the otic vesicle in the dashed box region (N = 3). (C) Mitotic centrosomes were detected using γ-tubulin immunostaining. ***p < 0.001 and ****p < 0.0001.

Heart defects was successfully rescued in Mdgcr8, but not in MZdgcr8. (A) The cardiac progenitor cells failed to migrate to the middle-line at prim-16 stage (32 hpf) in Mdgcr8 and MZdgcr8 (red arrows). (B) After miR430 rescue, cardiogenesis appeared normal including structure of atrium and ventricle. (C) MZdgcr8 failed to be rescued by mir430, exhibiting hydropericardium phenotype. (D,E) Heart-rate results indicated that the heart defects were completely rescued in Mdgcr8, but only partially in MZdgcr8, and MZdgcr8 heart rate gradually decreased at 4 days. **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Maternal mutants exhibit reduced erythropoiesis and could be rescued in Mdgcr8, but not in MZdgcr8. Whole embryo O-dianisidine staining assay was used for assessing functional hemoglobin in mature primitive erythrocytes in wild-type, Mdgcr8, MZdgcr8, and miR-430 rescued groups at 50 (A) and 74 hpf (B). Hemoglobinized cells accumulate in the yolk sac in wild-type but mainly accumulate in the caudal vein (red arrows) in MZdgcr8 and MZdgcr8 mutants. 10–15 embryos per groups. Scale bar: 200 μm. (C) The touch response was categorized as strong, weak, and no response based on the analysis of the video frames. (D) At 60 hpf, the rescue rate was determined in wild-type and maternal mutant embryos. (E) At 48 hpf, the hatching rate was detected in wild-type and maternal mutant embryos. ****p < 0.0001.

Expression value of miRNAs in wild-type and MZdgcr8 embryos and qPCR validations. (A) 80 miRNAs for values greater than 30 were obtained in wild-type embryos. (B) 16 miRNAs for values greater than 30 were obtained in MZdgcr8 embryos. (C) miRNAs with higher values in wild-type and MZdgcr8. (D) Total miRNAs read count and miR-430 family read count in wild-type and MZdgcr8. (E) Percentage of miR-430 family in total miRNAs read count in wild-type. (F) Percentage of miR-430 family in total miRNAs read count in MZdgcr8. (G) Validation of some mature miRNAs by real-time PCR. **p < 0.01 and ****p < 0.0001.

mRNA expression variation in wild-type and MZdgcr8 embryos during 30% epiboly stage. (A) Top 30 downregulated genes from RNA-seq data. (B) Top 30 upregulated genes from RNA-seq data. (C) Expression count of miRNAs biogenesis related genes.