leg1a^zju3/zju3leg1b^zju1/zju1 double mutant exhibits a smaller liver and exocrine pancreas and thinner intestinal tube phenotype.

a, b Representative WISH images using the fabp10a probe (liver marker) at 3.5 dpf (a) and statistical analysis of the liver size (b). leg1a^zju3/zju3leg1b^zju1/zju1 double homozygous mutants (leg1_dm) exhibited a small liver phenotype when compared with WT, leg1a^zju1/zju1 (leg1a_mu) single and leg1b^zju1/zju1 (leg1b_mu) single homozygous mutants. a Top: the date of experiments performed; bottom right: number of embryos showing the phenotype over total embryos examined. b The y axis shows the positive signal area in each embryo stained with the fabp10a probe. c–f Representative WISH images using the combination of fabp10a (liver) and trypsin (exocrine pancreas) (c) and of fabp10a (liver) and fabp2 (intestine marker) (e) probes on WT, leg1a_mu single, leg1b_mu single and leg1_dm double mutant embryos at 3.5 dpf. c, e Top: the date of experiments performed; bottom right: number of embryos showing the phenotype over total embryos examined. L, liver (outlined by a yellow line in c, e); P, exocrine pancreas (outlined by a magenta line in c); I, intestinal tube (outlined by a magenta line in e). Statistical analysis of the sizes of exocrine pancreas (left) and liver (middle) (d) and of the intestine (left) and liver (middle) (f) was performed, respectively. The ratios of liver vs exocrine pancreas (right panel in d) and of liver vs intestine (right panel in f) for each genotype were also shown. ***P < 0.001; ns, no significance.

RNA-seq analysis revealed activation of GCR in both leg1a^zju1/zju1 single and leg1b^zju1/zju1 single mutants.

a–d Genome browser view (a, c) and statistical analysis (b, d) of leg1a and leg1b transcript counts (FPKM) together with actb2 and bhmt two control genes in RNA-seq samples obtained from WT, leg1a_mu single, leg1b_mu single and leg1_dm double mutant embryos at 3 dpf (a, b) and at 5 dpf (c, d), respectively. ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001.

Knockdown of leg1b by MO in leg1a^zju1/zju1 caused a small liver phenotype.

a Statistical analysis of the ratios of the precursor and total leg1b transcripts in WT and leg1a^zju1/zju1 at 3 dpf and 5 dpf, respectively. Nine RNA-seq datasets each for WT and leg1a^zju1/zju1 at 3 dpf and 5 dpf were used for this analysis (refer to Supplementary Tables S1, S2, S7, S8, S13, S14). b Western blot of Leg1 from embryos at 3.5 dpf after injection of leg1b-MO in different genotypes as shown. Tubulin, loading control. c, d Representative images showing the WISH result using the fabp10a probe (c) and statistical analysis of the liver size (d) in different genotypes injected with leg1b-MO or control mismatch morpholino (mismatch-MO) as indicated. c Top: the date of experiments performed; bottom right: number of embryos showing the phenotype over total embryos examined. d The y axis shows the positive signal area in each embryo stained with the fabp10a probe.

upf3a^−/−leg1a^zju1/zju1 double mutant exhibits a smaller liver phenotype.

a Statistical analysis of transcript counts (FPKM) of the genes encoding components of the NMD pathway and COMPAS complex in RNA-seq samples obtained from WT, leg1a_mu single and leg1b_mu single mutant embryos at 3 dpf and at 5 dpf, respectively. b, c Representative images showing the WISH result using the fabp10a probe (b) and the statistical analysis of liver sizes (c) in the WT, leg1a_mu, upf3a_mu, upf3a;leg1a_dm and leg1_dm embryos at 3.5 dpf. d Western blot of Leg1 protein in different genotypes at 3.5 dpf and 5 dpf, respectively. Tubulin, loading control. e, f Representative images showing the WISH result using the fabp10a probe (e) and the statistical analysis of liver sizes (f) in the WT, leg1a_mu, upf1_mu, and upf1;leg1a_dm embryos at 3.5dpf. b, e Top: the date of experiments performed; bottom right: number of embryos showing the phenotype over total embryos examined. ns, no significance; **P < 0.01; ***P < 0.001.

RNA-seq analysis showed that the upregulated leg1b transcript level in leg1a^zju1/zju1 was downregulated by Upf3a depletion but was unaffected by Upf1 depletion.

a–d Showing genome browser view (a, c) and statistical analysis (b, d) of leg1a and leg1b transcript counts (FPKM) together with actb2 and bhmt two control genes in RNA-seq samples obtained from WT, upf3a^−/− (upf3a_mu) single, leg1a^zju1/zju1 (leg1a_mu) single and upf3a^−/−leg1a^zju1/zju1 (upf3a;leg1a_dm) double mutant embryos at 3 dpf (a, b) and at 5 dpf (c, d), respectively. e–h Genome browser view (e, g) and statistical analysis (f, h) of leg1a and leg1b transcript counts (FPKM) together with actb2 and bhmt two control genes in RNA-seq samples obtained from WT, upf1^−/− (upf1_mu) single, leg1a^zju1/zju1 (leg1a_mu) single and upf1^−/−leg1a^zju1/zju1 (upf1;leg1a_dm) double mutant embryos at 3 dpf (e, f) and at 5 dpf (g, h), respectively. ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001.

Depletion of Upf3a but not of Upf1 obviously alters the DEGs in the leg1a^zju1/zju1 mutant.

a, b DEGs between WT and leg1a^zju1/zju1 (leg1a_mu) at 3 dpf (a) and 5 dpf (b) were identified by analyzing the RNA-seq data, respectively. The heat-map shows the hierarchical clustering of these DEGs in the RNA-seq samples obtained from upf3a^−/− (upf3a_mu) single and upf3a^−/−leg1a^zju1/zju1 (upf3a;leg1a_dm) double mutant embryos at 3 dpf (a) and 5 dpf (b), respectively. c, d DEGs between WT and leg1a^zju1/zju1 (leg1a_mu) at 3 dpf (a) and 5dpf (b) were identified by analyzing the RNA-seq data, respectively. The heat-map shows the hierarchical clustering of these DEGs in the RNA-seq samples obtained from upf1^−/− (upf1_mu) single and upf1^−/−leg1a^zju1/zju1 (upf1;leg1a_dm) double mutant embryos at 3 dpf (c) and 5 dpf (d), respectively. e The qPCR analysis of leg1a and leg1b transcripts in WT and leg1b^zju1/zju1 at 1.5 dpf. The embryos were injected with st-MO, upf1-MO, or upf3a-MO at the one-cell stage.

Upregulation of leg1b in leg1a^zju1/zju1 is not coupled with an enrichment of H3K4me3 around its TSS.

a Graph showing the normalized reads distribution profiles of the genome-wide H3K4me3 enrichment (RPKM) in the liver dissected from WT, leg1a_mu, upf3a_mu and upf3a;leg1a_dm embryos at 5 dpf. TSS, transcription start site. b–e Genome browser view of the H3K4me3 enrichment in the genomic region of representative liver-specific (b), muscle-enriched (c), intestine-enriched genes (d) and leg1a and leg1b (e) genes. The y axis shows the ULI-NChIP counts across the genomic region shown.

Cross-comparison of H3K4me3 enrichment in the genomic regions of 70 liver-enriched genes between WT adult and embryonic liver.

a–d Genome browser view of the genomic region of representative genes in the category of TSS specific-enrichment of H3K4me3 in both adult and embryonic liver (a), TSS-specific enrichment of H3K4me3 in the adult liver but not the embryonic liver (b), H3K4me3 enrichment in the TTS together with gene body region in both adult and embryonic liver (c) and different patterns of H3K4me3 enrichment in the TSS together with gene body region between adult and embryonic liver (d). The y axis shows the ChIP-seq counts across the genomic region shown. The adult liver ChIP-seq data were extracted from the database (SRA: SRX6422954 and SRX6422955) and the embryonic liver data were obtained in this work.