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. Overall design: 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.

学界普遍认为，性别决定区Y框蛋白9（sex-determining region Y-box 9, SOX9）在胚胎期肝内胆管（intrahepatic bile ducts, IHBDs）发育过程中发挥关键调控作用。肝内胆管可分为两类截然不同的网络结构：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，后者逐级分支为细小胆管，在门静脉周围形成网状结构。与之相对，均质化网络是一类早期管状结构，由环绕门静脉的胆管小泡构成，是成熟层级化网络的发育前体。本研究借助三维成像分析技术，以及出生后特异性敲除SOX9的小鼠模型，探究了SOX9在上述两类胆管网络出生后发育过程中的作用。本研究结果显示，SOX9对于肝内胆管的成熟与结构构建至关重要，尤其在促进胆管小泡与胆小管的正确连接方面发挥关键作用。此外，SOX9条件性敲除小鼠的转录组发生显著变化：其胆汁酸转运与代谢相关的代偿通路被激活，而细胞间黏附通路则被下调，进而损伤胆管上皮细胞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核心作用远超此前认知。实验整体设计：本研究使用了三类实验动物：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（Albumin, Alb）-Cre小鼠（品系编号#003574，美国杰克逊实验室）。为在肝上皮细胞中特异性敲除Sox9，本研究将Sox9flox/flox小鼠与Alb-Cre小鼠交配，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）分析中，我们设置对照组（Control, CT，n=3）与SOX9 cKO组（n=3），采用TRIzol试剂（Trizol，购自美国马萨诸塞州沃尔瑟姆市赛默飞世尔科技公司）提取总RNA，并通过生物分析仪2100（BioAnalyzer 2100）验证总RNA质量，其RNA完整性指数≥9。采用TruSeq链特异性mRNA LT样本制备试剂盒（购自美国加利福尼亚州圣地亚哥市伊卢米纳（Illumina）公司），按照伊卢米纳TruSeq实验流程构建DNA文库；随后使用搭载NextSeq 500/550 High Output v2试剂盒的伊卢米纳NextSeq 500测序平台进行测序，获取单端75碱基（75-nt）读段。测序数据需先经过预处理：使用Trim Galore! v0.5.0（搭载cutadapt v1.16）去除接头序列并修剪低质量末端，随后采用STAR v2.6.0a软件将RNA-seq数据比对至UCSC mm10参考基因组。采用RSEM v1.3.1软件计算基因表达量，以每百万转录本数（transcripts per million, TPM）作为表达量单位。