Falix Farah A, Weeda Víola B, Labruyere Wilhelmina T, Poncy Alexis, de Waart Dirk R, Hakvoort Theodorus B M, Lemaigre Frédéric, Gaemers Ingrid C, Aronson Daniël C, Lamers Wouter H
Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Amsterdam, The Netherlands; Emma Children's Hospital AMC and Pediatric Surgical Center of Amsterdam, Academic Medical Center, Amsterdam, The Netherlands.
Tytgat Institute for Liver and Intestinal Research, Academic Medical Center, Amsterdam, The Netherlands; Emma Children's Hospital AMC and Pediatric Surgical Center of Amsterdam, Academic Medical Center, Amsterdam, The Netherlands.
Dev Biol. 2014 Dec 15;396(2):201-13. doi: 10.1016/j.ydbio.2014.10.002. Epub 2014 Oct 18.
Notch signaling plays an acknowledged role in bile-duct development, but its involvement in cholangiocyte-fate determination remains incompletely understood. We investigated the effects of early Notch2 deletion in Notch2(fl/fl)/Alfp-Cre(tg/-) ("Notch2-cKO") and Notch2(fl/fl)/Alfp-Cre(-/-) ("control") mice. Fetal and neonatal Notch2-cKO livers were devoid of cytokeratin19 (CK19)-, Dolichos-biflorus agglutinin (DBA)-, and SOX9-positive ductal structures, demonstrating absence of prenatal cholangiocyte differentiation. Despite extensive cholestatic hepatocyte necrosis and growth retardation, mortality was only ~15%. Unexpectedly, a slow process of secondary cholangiocyte differentiation and bile-duct formation was initiated around weaning that histologically resembled the ductular reaction. Newly formed ducts varied from rare and non-connected, to multiple, disorganized tubular structures that connected to the extrahepatic bile ducts. Jaundice had disappeared in ~30% of Notch2-cKO mice by 6 months. The absence of NOTCH2 protein in postnatally differentiating cholangiocyte nuclei of Notch2-cKO mice showed that these cells had not originated from non-recombined precursor cells. Notch2 and Hnf6 mRNA levels were permanently decreased in Notch2-cKO livers. Perinatally, Foxa1, Foxa2, Hhex, Hnf1β, Cebpα and Sox9 mRNA levels were all significantly lower in Notch2-cKO than control mice, but all except Foxa2 returned to normal or increased levels after weaning, coincident with the observed secondary bile-duct formation. Interestingly, Hhex and Sox9 mRNA levels remained elevated in icteric 6 months old Notch2-cKOs, but decreased to control levels in non-icteric Notch2-cKOs, implying a key role in secondary bile-duct formation.
Cholangiocyte differentiation becomes progressively less dependent on NOTCH2 signaling with age, suggesting that ductal-plate formation is dependent on NOTCH2, but subsequent cholangiocyte differentiation is not.
Notch信号通路在胆管发育中发挥着公认的作用，但其在胆管细胞命运决定中的参与情况仍未完全了解。我们研究了Notch2(fl/fl)/Alfp-Cre(tg/-)（“Notch2-cKO”）和Notch2(fl/fl)/Alfp-Cre(-/-)（“对照”）小鼠中早期Notch2缺失的影响。胎儿和新生Notch2-cKO肝脏缺乏细胞角蛋白19（CK19）、双花扁豆凝集素（DBA）和SOX9阳性的导管结构，表明产前胆管细胞分化缺失。尽管存在广泛的胆汁淤积性肝细胞坏死和生长迟缓，但死亡率仅约为15%。出乎意料的是，在断奶前后启动了一个缓慢的继发性胆管细胞分化和胆管形成过程，组织学上类似于小胆管反应。新形成的导管从罕见且不相连到多个杂乱的管状结构不等，这些结构与肝外胆管相连。到6个月时，约30%的Notch2-cKO小鼠黄疸消失。Notch2-cKO小鼠出生后分化的胆管细胞核中不存在NOTCH2蛋白，表明这些细胞并非起源于未重组的前体细胞。Notch2-cKO肝脏中Notch2和Hnf6 mRNA水平永久降低。围产期，Notch2-cKO小鼠中Foxa1、Foxa2、Hhex、Hnf1β、Cebpα和Sox9 mRNA水平均显著低于对照小鼠，但除Foxa2外，所有这些水平在断奶后均恢复正常或升高，这与观察到的继发性胆管形成一致。有趣的是，6个月大黄疸型Notch2-cKO小鼠中Hhex和Sox9 mRNA水平仍然升高，但在无黄疸的Notch2-cKO小鼠中降至对照水平，这意味着它们在继发性胆管形成中起关键作用。
胆管细胞分化随年龄增长对NOTCH2信号的依赖性逐渐降低，这表明胆管板形成依赖于NOTCH2，但随后的胆管细胞分化并非如此。
