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The gallbladder excretes cytotoxic bile acids to the duodenum through the cystic duct and common bile duct system. Sox17 haploinsufficiency causes the biliary atresia-like phenotypes and hepatitis in late organogenesis mouse embryos. However, the molecular and cellular mechanisms in the Sox17-haploinsufficient gallbladder and liver in the etiology of biliary atresia remain unclear. In this study, transcriptomic analyses revealed the early onset of cholecystitis in the Sox17+/- embryos, together with the appearance of ectopic cystic duct-like epithelia in their gallbladders. The embryonic hepatitis showed positive correlations with the severities of cholecystitis in individual Sox17+/- embryos, in addition that the embryonic hepatitis could be induced by conditional deletion of Sox17 in the primordial gallbladder epithelia, but not in the fetal liver hepatoblasts. The Sox17+/- gallbladder also showed the drastic reduction in Sonic hedgehog expression, leading to aberrant smooth muscle formation and defective contraction of the fetal gallbladder. The defective gallbladder contraction positively correlated with the severity of embryonic hepatitis in Sox17+/- embryos, suggesting the potential contribution of embryonic cholecystitis and fetal gallbladder contraction in the early pathogenesis of congenital biliary atresia.

Congenital biliary atresia is an incurable disease of newborn infants, of unknown genetic causes, that results in congenital deformation of the gallbladder and biliary duct system. Here, we show that during mouse organogenesis, insufficient SOX17 expression in the gallbladder and bile duct epithelia results in congenital biliary atresia and subsequent acute ‘embryonic hepatitis’, leading to perinatal death in ~95% of the Sox17 heterozygote neonates in C57BL/6 (B6) background mice. During gallbladder and bile duct development, Sox17 was expressed at the distal edge of the gallbladder primordium. In the Sox17+/− B6 embryos, gallbladder epithelia were hypoplastic, and some were detached from the luminal wall, leading to bile duct stenosis or atresia. The shredding of the gallbladder epithelia is probably caused by cell-autonomous defects in proliferation and maintenance of the Sox17+/− gallbladder/bile duct epithelia. Our results suggest that Sox17 plays a dosage-dependent function in the morphogenesis and maturation of gallbladder and bile duct epithelia during the late-organogenic stages, highlighting a novel entry point to the understanding of the etiology and pathogenesis of human congenital biliary atresia.

In mouse embryogenesis, Sry is transiently activated in a center-to-pole wavelike manner along the anteroposterior (AP) axis of developing XY gonads. However, the mechanism and significance of the center-to-pole expansion of testis initiation pathways downstream of Sry expression remain unclear. Here we demonstrate that FGF9 can act as a diffusible conductor for a poleward expansion of tubulogenic programs at early phases of testis differentiation. In XY genital ridge cultures of anterior, middle and posterior segments at 11.0-11.25 days post-coitum, male-specific activation of Sry and its target gene, Sox9, was still observed in both anterior and posterior pole segments despite their isolation from the central domain. However, high-level Sox9 expression was not maintained, resulting in the failure of testis cord organization in most pole segments. A reconstruction experiment using ROSA:lacZ middle segments showed rescue of the tubulogenic defect in the poles without any appreciable contribution of lacZ-positive gonadal parenchyma cells. A partition culture assay also showed a possible contribution of soluble/diffusible factors secreted from the gonadal center domain to proper tubulogenesis in the poles. Among various signaling factors, Fgf9 expression was significantly lower in both anterior and posterior pole segments than in the central domain. The supportive role of the central domain could be substituted by exogenous FGF9 supply, whereas reduction of Wnt4 activity did not rescue the tubulogenesis defect in the pole segments. These observations imply that center-to-pole FGF9 diffusion directs a poleward expansion of testiculogenic programs along the AP axis of developing XY gonads.

In mammals, the Y-linked sex-determining gene Srycell-autonomously promotes Sertoli cell differentiation from bipotential supporting cell precursors through SRY-box containing gene 9 (Sox9),leading to testis formation. Without Sry action, the supporting cells differentiate into granulosa cells, resulting in ovarian development. However,how Sry acts spatiotemporally to switch supporting cells from the female to the male pathway is poorly understood. We created a novel transgenic mouse line bearing an inducible Sry transgene under the control of the Hsp70.3 promoter. Analysis of these mice demonstrated that the ability of Sry to induce testis development is limited to approximately 11.0-11.25 dpc, corresponding to a time window of only 6 hours after the normal onset of Sry expression in XY gonads. If Sry was activated after 11.3 dpc, Sox9 activation was not maintained, resulting in ovarian development. This time window is delimited by the ability to engage the high-FGF9/low-WNT4 signaling states required for Sertoli cell establishment and cord organization. Our results indicate the overarching importance of Sry action in the initial 6-hour phase for the female-to-male switching of FGF9/WNT4 signaling patterns.