Neuronal and Progenitor/Stem Cell Function During Salivary Gland Development

唾液腺发育过程中的神经元和祖细胞/干细胞功能

• 批准号: 8148648
• 负责人: Matthew Hoffman
• 金额: $ 104.97万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8148648
• 关键词: Cell physiology; Salivary Glands; Stem cells; gland development; nerve stem cell

Identifying factors that maintain epithelial stem/progenitor cells and drive their cell fate decisions within a developing organ is essential to understand development and has implications for organ regeneration. This project has two approaches, one investigating the bidirectional communication between parasympathetic nerve development and SMG epithelial morphogenesis, and the second identifying embryonic mouse salivary gland stem/progenitor cell populations within the gland. We have made advances in understanding the bidirectional communication between parasympathetic nerve development and SMG epithelial morphogenesis. Surprisingly, parasympathetic nerve function also influences the stem/progenitor cells within the epithelium. The maintenance of a progenitor cell population as a reservoir of undifferentiated cells is required for organ development and regeneration. However, the mechanisms by which epithelial progenitor cells are maintained during salivary organogenesis are poorly understood. Both parasympathetic and sympathetic branches of the autonomic nervous system innervate the adult SMG, but it is the parasympathetic axons that extend from the parasympathetic submandibular ganglion (PSG) and innervate the embryonic SMG. We reported that removal of the parasympathetic ganglion in mouse explant organ culture decreased the number of keratin 5-positive epithelial progenitor cells which decreased epithelial morphogenesis. These effects were rescued with an acetylcholine analog. In addition, chemical inhibition of acetylcholine release, chemical antagonism of muscarinic receptor activity, and gene knockdown of muscarinic receptors in the epithelium all have a similar effect on growth as removing the PSG. We demonstrate that acetylcholine signaling, via the muscarinic M1 receptor and EGFR, increased epithelial morphogenesis and proliferation of the keratin 5-positive progenitor cells. Our analysis of the keratin-5 positive cells in salivary gland development involved genetic lineage tracing experiments to prove that the keratin-5 positve cells were epithelial progenitor cells. Therefore, the parasympathetic innervation maintained the epithelial keratin 5-positive progenitor cell population in an undifferentiated state, which was required for organogenesis. This mechanism for epithelial progenitor cell maintenance may be targeted for organ repair or regeneration. Early cell fate decisions are coordinated by transcription factors (TFs), which control expression of genes involved in self-renewal and differentiation. We have characterized the expression of known stem/progenitor cell-related genes during embryonic SMG development. TFs involved in stem cell self-renewal (Sox2, Nanog, Oct3/4, cMyc, and the Etv family), progenitor differentiation (Sox family), basal progenitor cell markers (cytokeratins), and proliferation markers for transit amplifying cells, were evaluated by Agilent microarray and qPCR. Embryonic stem (ES) cells are maintained by four important TFs (Sox2, Nanog, Oct3/4, cMyc) whose expression is influenced by different growth factors. The TFs Nanog, Sox2, and Klf4 were detectable in E13 SMGs. However, we were unable to detect Oct3/4 levels in the SMG at the beginning of development by qPCR, suggesting the SMG cells were already committed along a lineage pathway and had made at least one cell fate decision with the loss of Oct3/4 expression. Primary salivary epithelia were treated with growth factors (2 hours) to detect direct transcriptional changes and to investigate the molecular basis of cell fate specification. Growth factors previously shown to influence SMG development were used and the expression of stem/progenitor cell markers were evaluated by qPCR. Nanog, Sox2, and Klf4 expression decreased after FGF10 treatment, suggesting FGF10 drives the cells along a more differentiated end bud fate. FGF10 upregulated the expression of cMyc, Sox9, and the TFs Etv4 and Etv5. None of the growth factors tested influenced Sox10 or delta-Np63 expression, suggesting they were downstream of the signaling pathway or controlled by other factors. Taken together, our data suggest that early E13 SMGs contain two distinct major epithelial cell compartments, the end bud and duct, in which different TFs and stem/progenitor cell markers influence the maintenance, differentiation and/or proliferation of these cell populations. Understanding the cell lineage of progenitor cells within the salivary glands will be important from the clinical perspective where progenitor cells of specific lineages may be more appropriate than pluripotent stem cells for clinical transplantation to regenerate irradiation-damaged salivary glands.

识别维持上皮茎/祖细胞并在发育器官中推动其细胞命运决策的因素对于了解发育至关重要，并且对器官再生具有影响。该项目有两种方法，一种研究了副交感神经发育与SMG上皮形态发生之间的双向通信，以及第二个识别腺体内的胚胎小鼠唾液腺茎/祖细胞群体。我们在理解副交感神经发展与SMG上皮形态发生之间的双向交流方面取得了进步。令人惊讶的是，副交感神经功能还影响上皮内的茎/祖细胞。 器官发育和再生需要维持祖细胞种群作为未分化细胞的储层。但是，在唾液器官发生过程中保持上皮祖细胞的机制知之甚少。自主神经系统的副交感神经和交感神经分支都支配了成年SMG，但是副交感神经轴突从副交感神经下皮神经节（PSG）延伸并支配胚胎SMG。我们报道说，小鼠外植体培养中副交感神经节的去除减少了角蛋白5阳性上皮祖细胞的数量，从而降低了上皮形态发生。 这些作用是用乙酰胆碱类似物救出的。此外，上皮细胞中乙酰胆碱释放的化学抑制作用，毒蕈碱受体活性的化学拮抗作用以及毒蕈碱受体的基因敲低均对生长的作用与去除PSG相似。 我们证明，通过毒蕈碱M1受体和EGFR，乙酰胆碱信号传导增加了角蛋白5阳性祖细胞的上皮形态发生和增殖。我们对唾液腺发育中角蛋白-5阳性细胞的分析涉及遗传谱系追踪实验，以证明角蛋白-5阳性细胞是上皮祖细胞。因此，副交感神经维持在未分化的状态下，维持上皮角蛋白5阳性祖细胞群，这是器官发生所必需的。这种上皮祖细胞维持的机制可以用于器官修复或再生。 早期的细胞命运决策由转录因子（TFS）协调，该因子控制着与自我更新和分化有关的基因表达。我们已经表征了胚胎SMG发育过程中已知的茎/祖细胞相关基因的表达。与干细胞自我更新有关的TF（SOX2，Nanog，Oct3/4，CMYC和ETV家族），祖细胞分化（SOX家族），基底祖细胞标记（细胞角蛋白）以及用于过境扩增细胞的增殖标记物，由敏捷的微型递增细胞评估。胚胎干细胞（ES）细胞由四个重要的TF（SOX2，Nanog，Oct3/4，CMYC）维持，其表达受不同生长因子的影响。在E13 SMG中可以检测到TFS Nanog，Sox2和KLF4。 但是，我们无法检测到QPCR开发开始时SMG中的Oct3/4水平，这表明SMG细胞已经沿着谱系途径进行了，并且至少做出了一个细胞命运的决定，而OCT3/4表达的丧失。用生长因子（2小时）处理原发性唾液上皮，以检测直接的转录变化并研究细胞命运规范的分子基础。先前显示出影响SMG发育的生长因子，并通过QPCR评估了茎/祖细胞标记的表达。 FGF10处理后Nanog，Sox2和KLF4表达降低，表明FGF10沿着更分化的末端芽驱动细胞。 FGF10上调了CMYC，SOX9和TFS ETV4和ETV5的表达。测试的生长因子均未影响Sox10或Delta-NP63表达，这表明它们是信号通路的下游或其他因素控制的。综上所述，我们的数据表明，E13早期SMG包含两个不同的主要上皮细胞室，即末端芽和管道，其中不同的TFS和茎/祖细胞标记物会影响这些细胞群体的维持，分化和/或增殖。从临床的角度来看，了解唾液腺内祖细胞的细胞谱系将很重要，因为特定谱系的祖细胞可能比多能干细胞更合适，以便对临床移植以再生受辐照受损的唾液腺。