Esni Farzad, Ghosh Bidyut, Biankin Andrew V, Lin John W, Albert Megan A, Yu Xiaobing, MacDonald Raymond J, Civin Curt I, Real Francisco X, Pack Michael A, Ball Douglas W, Leach Steven D
Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.
Development. 2004 Sep;131(17):4213-24. doi: 10.1242/dev.01280. Epub 2004 Jul 27.
Notch signaling regulates cell fate decisions in a variety of adult and embryonic tissues, and represents a characteristic feature of exocrine pancreatic cancer. In developing mouse pancreas, targeted inactivation of Notch pathway components has defined a role for Notch in regulating early endocrine differentiation, but has been less informative with respect to a possible role for Notch in regulating subsequent exocrine differentiation events. Here, we show that activated Notch and Notch target genes actively repress completion of an acinar cell differentiation program in developing mouse and zebrafish pancreas. In developing mouse pancreas, the Notch target gene Hes1 is co-expressed with Ptf1-P48 in exocrine precursor cells, but not in differentiated amylase-positive acinar cells. Using lentiviral delivery systems to induce ectopic Notch pathway activation in explant cultures of E10.5 mouse dorsal pancreatic buds, we found that both Hes1 and Notch1-IC repress acinar cell differentiation, but not Ptf1-P48 expression, in a cell-autonomous manner. Ectopic Notch activation also delays acinar cell differentiation in developing zebrafish pancreas. Further evidence of a role for endogenous Notch in regulating exocrine pancreatic differentiation was provided by examination of zebrafish embryos with homozygous mindbomb mutations, in which Notch signaling is disrupted. mindbomb-deficient embryos display accelerated differentiation of exocrine pancreas relative to wild-type clutchmate controls. A similar phenotype was induced by expression of a dominant-negative Suppressor of Hairless [Su(H)] construct, confirming that Notch actively represses acinar cell differentiation during zebrafish pancreatic development. Using transient transfection assays involving a Ptf1-responsive reporter gene, we further demonstrate that Notch and Notch/Su(H) target genes directly inhibit Ptf1 activity, independent of changes in expression of Ptf1 component proteins. These results define a normal inhibitory role for Notch in the regulation of exocrine pancreatic differentiation.
Notch信号通路调控多种成年和胚胎组织中的细胞命运决定，是外分泌型胰腺癌的一个特征。在发育中的小鼠胰腺中，Notch信号通路成分的靶向失活已明确了Notch在调控早期内分泌分化中的作用，但对于Notch在调控后续外分泌分化事件中可能发挥的作用，提供的信息较少。在此，我们表明激活的Notch及其靶基因在发育中的小鼠和斑马鱼胰腺中积极抑制腺泡细胞分化程序的完成。在发育中的小鼠胰腺中，Notch靶基因Hes1在外分泌前体细胞中与Ptf1-P48共表达，但在分化的淀粉酶阳性腺泡细胞中不表达。利用慢病毒递送系统在E10.5小鼠背侧胰腺芽的外植体培养物中诱导异位Notch信号通路激活，我们发现Hes1和Notch1-IC均以细胞自主方式抑制腺泡细胞分化，但不抑制Ptf1-P48的表达。异位Notch激活也会延迟发育中的斑马鱼胰腺中腺泡细胞的分化。通过检查纯合mindbomb突变的斑马鱼胚胎，提供了内源性Notch在调控外分泌胰腺分化中作用的进一步证据，其中Notch信号传导被破坏。与野生型同窝对照相比，mindbomb缺陷胚胎的外分泌胰腺分化加速。表达显性负性无翅型MMTV整合位点家族成员抑制子[Su(H)]构建体诱导了类似的表型，证实Notch在斑马鱼胰腺发育过程中积极抑制腺泡细胞分化。使用涉及Ptf1反应性报告基因的瞬时转染实验，我们进一步证明Notch和Notch/Su(H)靶基因直接抑制Ptf1活性，而与Ptf1组成蛋白表达的变化无关。这些结果确定了Notch在调控外分泌胰腺分化中的正常抑制作用。
