Fig 2: Ercc2/Xpd-deficient phenotypes are independent of an activated p53 response(A) qPCR analysis of expressions of genes associated with tp53 activation in ercc2/xpd mutants and siblings at 5 and 7 dpf. Data are presented as mean ± SD from three independent biological repeats. Student’s t test, ∗, p < 0.05, ∗∗, p < 0.01, NS, non-significant.(B) Whole-mount in situ hybridization showed a large up-regulation of tp53 in digestive organs (white arrowheads), retina (white arrow) and arches (black arrowheads) of ercc2/xpd mutants at 5 dpf. Scale bars, 100 μm.(C) Whole-mount in situ hybridization revealed that tp53 mutation did not affect ercc2/xpd mutant phenotypes in digestive organs or hematopoiesis. Scale bars, 100 μm.(D) Schematic diagram showing the possible mechanism of how Ercc2/Xpd deficiency leads to failure of digestive organ growth. See also Figure S9.

Fig 3: Perturbed rRNA synthesis results in nucleolar stress(A) Transmission electron microscopy images of intestinal endothelial cell (IEC) nuclei in ercc2/xpd mutants and siblings at 5 dpf. Arrowheads point to enlarged nucleoli. Scale bars, 1 μm.(B–D) DAPI staining and immunostaining of the nucleolar markers fibrillarin and nucleolin in IECs in ercc2/xpd mutants and siblings at 5 dpf. Areas of dashed boxes are magnified. Arrowheads point to enlarged nucleoli, arrows indicate the translocation of nucleolar proteins to the nucleoplasm. Scale bars, 20 μm. See also Figure S7.(E) Schematic diagram showing stepwise processing of the pre-rRNA transcript. a-c corresponds to the rRNA intermediates. 5′ETS and ITS1 probes are indicated with green and red lines, respectively. ETS, external transcribed spacer; ITS, internal transcribed spacer.(F) Northern blot using 5′ETS and ITS1 probes to detect precursor forms of rRNA in ercc2/xpd mutants and siblings at 5 dpf. Methylene blue staining was used as a loading control.(G and H) Representative E-Bioanalyzer analysis and measurement of the 28S/18S rRNA ratio in ercc2/xpd mutants (M) and siblings (S) at 5 and 7 dpf. Data are presented as mean ± SD, Student’s t test, NS, non-significant.

Fig 4: Mitochondrial abnormalities, autophagy, and inflammation are highly induced(A) Transmission electron microscope images of intestinal endothelial cells (IECs) in ercc2/xpd mutants and siblings at 5 dpf. Arrows point to swollen and disarrayed mitochondria, red arrowheads point to autophagosome-like vesicles. m, mitochondrion. Scale bars, 1 μm. See also Figures S4B and S6A.(B) Immunostaining of the mitochondrial marker MT-CO1 in IECs in ercc2/xpd mutants and siblings at 5 dpf. Areas of dashed boxes are magnified. Scale bars, 20 μm.(C) Immunostaining of the autophagosomal marker LC3BII in IECs in ercc2/xpd mutants and siblings at 5 dpf. Areas of dashed boxes are magnified. Scale bars, 10 μm.(D) Transcriptomic analysis revealed increased expression of genes related to inflammation in ercc2/xpd mutants compared to siblings at 5 dpf.(E) qPCR verification of inflammation-related gene expression in ercc2/xpd mutants and siblings at 5 and 7 dpf. Data are presented as mean ± SD from three independent biological repeats. Student’s t test, ∗, p < 0.05, ∗∗, p < 0.01, NS, non-significant. See also Figure S6.

Fig 5: Ercc2/Xpd-deficient intestinal endothelial cells exhibit loss of cell polarity and impaired proliferation(A) Tg(Nkx2.2a:EGFP) marked intestinal endocrine cells in ercc2/xpd mutants and siblings at 5 dpf. Scale bars, 100 μm.(B) Alcian blue staining showed intestinal goblet cells (left) and quantification of their numbers (right) in ercc2/xpd mutants and siblings at 5 dpf. Scale bars, 100 μm. Data are presented as mean ± SD. Student’s t test, ∗∗∗∗, p < 0.0001.(C) EdU incorporation assay and immunostaining of the mitotic marker phospho-histone H3 revealed reduced the proliferation of intestinal endothelial cells (IECs) in ercc2/xpd mutants compared to siblings at 5 dpf. Scale bars, 100 μm.(D) Quantification of percentages of EdU-positive IECs or pH3-positive cells in different regions of the intestine. i.b., intestinal bulb; m.i., mid-intestine; p.i., posterior intestine. Data are presented as mean ± SD, Student’s t test, ∗∗∗∗, p < 0.0001, ∗∗∗, p < 0.001. See also Figure S4D.(E) The TUNEL assay showed no detectable apoptotic signals in IECs in ercc2/xpd mutants and siblings compared to the DNase-I-treated positive control at 7 dpf. Scale bars, 100 μm. See also Figure S5A.(F) Immunostaining of γ-H2A.X revealed no increased apoptosis or DNA instability in IECs in ercc2/xpd mutants compared to siblings at 7 dpf. Scale bars, 100 μm.