Generation and characterization of Ptf1a antiserum and localization of Ptf1a in relation to Nkx6.1 and Pdx1 during the earliest stages of mouse pancreas development
Generation and characterization of Ptf1a antiserum and localization of Ptf1a in relation to Nkx6.1 and Pdx1 during the earliest stages of mouse pancreas development
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DOI：
10.1369/jhc.2008.950675
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发表时间：
2008-06-01
影响因子：
3.2
通讯作者：
Madsen, Ole D.
中科院分区：
文献类型：
--
作者：
Hald, Jacob;Sprinkel, Anne Ejrnaes;Madsen, Ole D.
Ptf1a 和 Pdx1 是早期胰腺发育的关键转录因子，功能丧失研究表明，单独缺乏每个基因会导致几乎完全的胰腺发育不全。 Ptf1a 特别有趣，因为它与最近报道的与多能病症相关的特征基因表达谱有关。很少有有用的抗体试剂可用于对早期发育的胰腺中 Ptf1a 蛋白表达进行一致且可靠的免疫组织化学可视化，其中这种关键调节因子的产生水平似乎非常低。我们描述了一种针对小鼠 Ptf1a 蛋白 C 末端部分的新型兔抗体，并首次报告了早在胚胎日 (e) 8.5-e8.75 小鼠胰腺背侧和腹侧芽以及 e10.0 神经管中的免疫检测。详细的共聚焦分析确定了丰富的三阳性（Ptf1a（+）/Nkx6.1（+）/Pdx1（+））假定的早期多能胰腺祖细胞，标记了e9.5背侧胰腺和e10.5腹侧胰腺。此外，Nkx6.1 与 Ptf1a 的表达模式随后在分支形态发生过程中分离（树干与尖端），最终在 e12.5 处标记出两个不同的祖细胞群。从 e15.5（小鼠）到成人胰腺（小鼠、大鼠和人），Ptf1a 抗体仅标记腺泡细胞核，正如预期的那样，其随后在使细胞处于这种分化状态中发挥/维持作用。总之，该抗体是进一步表征胰腺分化的重要早期步骤的新工具。本手稿包含 http://www.jhc.org 上的在线补充材料。请在线访问本文以查看这些材料。
Ptf1a and Pdx1 are critical transcription factors of early pancreatic development, as shown by loss of function studies where lack of each gene alone causes almost complete pancreas agenesis. Ptf1a is particularly interesting because it is linked to a recently reported signature gene expression profile associated with the multipotent condition. Few useful antibody reagents have been available for consistent and reliable immunohistochemical visualization of Ptf1a protein expression in the early developing pancreas in which the level of production of this critical regulator seems to be very low. We describe a novel rabbit antibody raised against the c-terminal portion of the mouse Ptf1a protein and report immunodetection, for the first time, as early as embryonic day (e) 8.5-e8.75 in the dorsal and ventral buds of the mouse pancreas as well as in the neural tube at e10.0. Detailed confocal analysis identifies an abundant triple-positive (Ptf1a(+)/Nkx6.1(+)/Pdx1(+)) putative early multipotent pancreatic progenitor cell that marks the e9.5 dorsal pancreas and e10.5 ventral pancreas. Furthermore, expression patterns of Nkx6.1 vs Ptf1a subsequently segregate during branching morphogenesis (trunk vs tip), ending up marking two distinct cell populations of progenitors at e12.5. From e15.5 (mouse) and in adult pancreas (mouse, rat, and human), the Ptf1a antibody marks only acinar cell nuclei, as expected for its subsequent role in committing/maintaining cells in this differentiated state. In summary, this antibody is a novel tool to further characterize important early steps of pancreas differentiation. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.