SOX9 is a key factor for the postnatal maturation of the intrahepatic bile duct network.

It is widely recognized that sex-determining region Y-box 9 (SOX9) plays a critical role in the development of intrahepatic bile ducts (IHBDs) during the embryonic stage. IHBDs are composed of two distinct networks: a hierarchical network and a homogeneous network. The hierarchical network consists of large bile ducts that run along the portal veins (PVs) and branch into smaller bile ducts, forming a reticular structure around the PVs. By contrast, the homogeneous network is an early tubular structure composed of bile ductules surrounding the PVs and serves as a precursor to the mature hierarchical network. This study examined the role of SOX9 in the postnatal development of these networks using three-dimensional imaging analysis and a mouse model in which SOX9 deficiency predominates after birth. Our findings indicate that SOX9 is essential for the maturation and structural organization of the IHBD, particularly in terms of facilitating the proper connection between bile ductules and bile canaliculi. Furthermore, transcriptional changes in Sox9 conditional knockout mice activated compensatory pathways involved in bile acid transport and metabolism, while downregulating intercellular adhesion pathways, thereby impairing cholangiocyte adhesion and structural integrity. These results indicate that SOX9 plays a more pivotal role in the development of the bile duct network than previously thought. C57BL/6J mice (Japan SLC, Shizuoka, Japan), Sox9flox/flox mice (strain #013106, The Jackson Laboratory, Bar Harbor, ME) and Albumin (Alb)-Cre mice (strain #003574, The Jackson Laboratory) were used in this study. To delete Sox9 in liver epithelial cells, Sox9flox/flox mice were crossed with Alb-Cre mice to give Alb-Cre+/+;Sox9 flox/flox (Sox9 cKO) mice. For RNA-seq analysis of mouse liver samples (CT n=3, SOX9 cKO n=3), total RNA was prepared using Trizol extraction (Thermo Fisher, Waltham, MA, USA), and the quality of the total RNA was confirmed using a BioAnalyzer 2100 (RNA integrity number>9). DNA libraries were prepared according to the Illumina TruSeq protocol using a TruSeq Stranded mRNA LT sample prep kit (Illumina, San Diego, CA, USA) and sequenced using an Illumina NextSeq 500 (Illumina) with a NextSeq 500/550 High Output v2 kit (Illumina) to obtain single-end, 75-nt reads. RNA-seq data were aligned to the human genome (UCSC mm10) using STAR ver. 2.6.0a after trimming to remove adapter sequences and removal of low-quality ends using Trim Galore! v0.5.0 (cutadapt v1.16). Gene expression levels are reported as transcripts per million, as determined using RSEM v1.3.1.众所周知，性别决定区Y框蛋白9（SOX9）在胚胎时期的肝内胆管（intrahepatic bile ducts，IHBDs）发育过程中发挥关键作用。肝内胆管由两种截然不同的网络结构组成：层级化网络与均质化网络。层级化网络由沿门静脉（portal veins，PVs）走行的大胆管构成，其逐级分支为细小胆管，在门静脉周围形成网状结构。与之相反，均质化网络是一种早期管状结构，由环绕门静脉的胆管小叶构成，是成熟层级化网络的前体。 本研究借助三维成像分析与出生后SOX9特异性缺失的小鼠模型，探究了SOX9在上述两种网络出生后发育过程中的作用。研究结果表明，SOX9对肝内胆管的成熟与结构构建至关重要，尤其是在促进胆管小叶与胆小管（bile canaliculi）的正确连接方面。此外，Sox9条件性敲除（conditional knockout，cKO）小鼠的转录组变化激活了参与胆汁酸转运与代谢的代偿通路，同时下调了细胞间黏附通路，进而损伤胆管上皮细胞（cholangiocyte）的黏附能力与结构完整性。上述结果表明，SOX9在胆管网络发育过程中的作用比此前认知的更为关键。 本研究使用了C57BL/6J小鼠（购自日本静冈县日本SLC公司）、Sox9flox/flox小鼠（品系编号#013106，美国杰克逊实验室，巴尔港，缅因州）以及白蛋白（Alb）-Cre小鼠（品系编号#003574，美国杰克逊实验室）。为在肝上皮细胞中敲除Sox9，将Sox9flox/flox小鼠与Alb-Cre小鼠杂交，获得Alb-Cre+/+;Sox9 flox/flox（Sox9条件性敲除，Sox9 cKO）小鼠。 针对小鼠肝组织样本的RNA测序（RNA-seq）分析（对照组n=3，SOX9 cKO组n=3）中，采用Trizol法（Trizol extraction，Thermo Fisher，美国马萨诸塞州沃尔瑟姆市）提取总RNA，并通过BioAnalyzer 2100（生物分析仪2100）验证总RNA质量（RNA完整性数>9）。采用TruSeq Stranded mRNA LT样本制备试剂盒（Illumina，美国加利福尼亚州圣迭戈市），按照Illumina TruSeq流程构建DNA文库，并使用搭载NextSeq 500/550 High Output v2试剂盒的Illumina NextSeq 500测序仪（Illumina）进行测序，获取单端75核苷酸读长的测序数据。使用Trim Galore! v0.5.0（附带cutadapt v1.16）对测序数据进行接头修剪与低质量末端切除后，采用STAR v2.6.0a版本将RNA-seq数据比对至UCSC mm10人类基因组。采用RSEM v1.3.1软件计算基因表达量，以转录本每百万（transcripts per million，TPM）值进行报告。