Fig 2: Phenotypes of maternal cecal microbiota in maternal hepatocyte-specific Ascl1 ablated mice. Maternal cecal samples were collected from GD18 Ascl1fl/fl and hep-Ascl1-/- mice. (A) Cecal microbiota analysis (n = 5). (B) Hepatic Hamp2 mRNA levels were measured using qRT-PCR and presented as the mean fold changes relative to nonpregnant (NP) controls ± SD (n = 4–5). ∗∗∗∗P < .0001.

Fig 3: Maternal pancreas phenotypes in maternal hepatocyte-specific Ascl1 ablated mice. Maternal pancreases were collected and weighed from nonpregnant (NP) and GD18 or GD19 Ascl1fl/fl and hep-Ascl1-/- mice. (A) Morphology of the pancreas. (B) Maternal pancreas-to-total-body-weight ratios are presented as means ± SD (n = 4–5). (C) Maternal pancreatic sections were subjected to insulin immunostaining. (D) The quantification of the insulin-positive islets to the pancreatic section area are presented as means ± SD (n = 3–4). (E) β-cell mass is presented as the means ± SD (n = 3–4). Serums were collected from NP and GD15 and GD18 Ascl1fl/fl and hep-Ascl1-/- mice. Serum (F) insulin and (G) glucose are presented as the means ± SD (n = 3–6). ∗∗P < .01, and ∗∗∗P < .001. Glu, glucose.

Fig 4: Ascl1 activation in maternal livers of pregnant mice and human beings. Timed pregnancies were generated in 3-month-old C57BL/6 female mice. Livers were collected from nonpregnant (NP) mice and maternal livers from pregnant mice at the indicated GDs. (A–C) Ascl1 expression in maternal livers. (A) Hepatic Ascl1 mRNA levels were measured using qRT-PCR and presented as the mean fold changes relative to NP controls ± SD (n = 3). ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001, compared with NP controls. (B) Western blot was performed using liver nuclear lysates with antibodies against ASCL1. Lamin B1 was used as a loading control. (C) Liver sections were subjected to Ascl1 in situ hybridization. Ascl1 mRNA is stained dark brown. (D) Liver sections were prepared from archived paraffin blocks of human liver tissues. Ascl1 mRNA was visualized on liver sections. Representative results are shown with liver sections from a NP woman, a 6 months’ pregnant mother, a hepatocellular carcinoma (HCC) patient, a hepatocellular adenoma (HCA) patient, and a 4.5-month-old child with hepatitis.

Fig 5: Placental phenotypes in maternal hepatocyte-specific Ascl1 ablated mice. Placentas were collected and weighed from GD15 and GD18 Ascl1fl/fl and hep-Ascl1-/- mice. (A) Placental weights are presented as means ± SD (n = 4–9 dams). (B) Placental sections were stained with H&E. (C) Frozen placental sections underwent periodic acid–Schiff (PAS) staining. Glycogen is stained red to purple. (D) Frozen placental sections were subjected to PL-I and PL-II in situ hybridization staining using the RNAscope 2.5 HD Assay-BROWN kit. The PL-I and PL-II mRNAs are stained dark brown. (E) Western blot was performed using placental lysates prepared from 1 placenta per dam with antibodies against the proteins indicated. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Serums were collected from nonpregnant (NP) and GD15 and GD18 Ascl1fl/fl and hep-Ascl1-/- mice. (F) The concentrations of circulating alkaline phosphatase (ALP) are presented as means ± SD (n = 4–6). ∗∗P < .01 and ∗∗∗∗P < .0001.