Early Events in Pharyngeal Pouch Patterning

咽部是胚胎的一个复杂区域，因为它是由所有三个生殖层以及与相邻的、迁移的神经脊相互作用形成的。咽部形成前肠，但也负责一些内胚层器官的形成，包括甲状腺、甲状旁腺和胸腺。咽在前后(AP)轴和背腹(DV)轴上形成一个两极明显的空腔。沿着AP轴形成模式需要几个信号线索，包括Sonic Hedgehog(Shh)、BMP、Fgf和Wnt信号。这些途径汇聚在一起，定义了定义每个片段的转录网络。DV通路的定义较少，但我们发现它对咽部的正确构型至关重要。当内胚层从咽腹内胚层形成时，将形成的缝隙只出现在咽部的外侧。我们推测，不同的信号通路对于咽部AP和DV模式的形成是必要的，这些信号通路对于咽部鳃裂隙和咽部来源的内胚层器官的形成至关重要。我们的目标是使用特定的抑制剂、反义吗啉和Crispr技术的组合来发现定义咽部区域的关键信号通路。研究的特定通路包括Shh通路、成纤维细胞生长因子通路和典型的Wnt信号。我们预测，成纤维细胞生长因子通路是咽部对局部线索作出反应的整体能力所必需的。AP和DV模式都需要Shh通路，尽管每个轴对信号有不同的时间要求。WNT信号是建立腹侧咽所必需的，也是咽内胚层产生的所有器官形成所必需的。前咽的形成需要BMP信号。除了信号通路，我们还将定义特定转录因子在定义咽部区域中的作用。包括Hox家族成员在内的许多转录因子在定义咽部的片段方面都得到了很好的证实。我们建议测试其中几个基因在咽部模式中的作用，包括Hand1，Gcm2，Satb2和Tbx3。我们将使用反义吗啡和Crispr技术来降低这些转录因子的活性，并检查操纵胚胎的咽部是否存在分化和咽部内的图案缺陷。*目标3：我们将测试用于形成口腔的机制是否与用于打开咽鳃缝的机制相同。最近的研究表明，口腔的形成需要抑制Wnt信号和活跃的Shh信号。鉴于两者都需要相似的组织相互作用，包括内胚层和外胚层之间的密切相互作用，我们假设颧缝的形成将使用相同的潜在分子机制。**

The pharynx is a complex region of the embryo because it is formed from all three germ layers in addition to interactions with the adjacent, migrating neural crest. The pharynx forms the foregut but is also responsible for the formation of some endodermal organs including the thyroid, parathyroid and thymus. The pharynx forms a cavity with distinct polarity both in the anterior-posterior (AP) and dorsal-ventral (DV) axis. Several signaling cues are necessary for patterning along the AP axis including sonic hedgehog (Shh), Bmp, Fgf, and Wnt signaling. These pathways converge to define transcriptional networks that define each segment. The DV pathway is less defined but we have found it to be critical in proper patterning of the pharynx. The slits that will form only occur at the lateral sides of the pharynx while the endodermal organs form from the ventral pharyngeal endoderm. We hypothesize that distinct signaling pathways are necessary for AP and DV patterning of the pharynx and these are critical for formation of the pharyngeal gill slits and the endodermal organs derived from the pharynx. Our goal is to discover the key signaling pathways that define regions of the pharynx using a combination of specific inhibitors, antisense morpholinos, and Crispr technology. Specific pathways that investigated include the Shh pathway, Fgf pathway, and canonical Wnt signaling. We predict that that the Fgf pathway is necessary for the overall competency of the pharynx to respond to regional cues. The Shh pathway is required for both AP and DV patterning although each axis has distinct temporal requirements for the signal. Wnt signaling will be required for establishing the ventral pharynx and is necessary for formation of all organs arising from the pharyngeal endoderm. Bmp signaling will be required for formation of the anterior pharynx. In addition to the signaling pathways, we will define roles for specific transcription factors in defining the regions of the pharynx. A number of transcription factors are well established in defining the segments of the pharynx, including members of the Hox family. We propose to test the role of several of these in pharyngeal patterning including Hand1, Gcm2, Satb2, and Tbx3. We will use antisense morpholino and Crispr technology to reduce the activity of these transcription factors and examine the pharynx of manipulated embryos for differentiation and patterning defects within the pharynx.***Objective 3: We will test whether the mechanisms used for formation of the mouth are the same as those used for opening of the pharyngeal gill slits. Recent work has shown the formation of the mouth requires inhibition of Wnt signaling coupled with active Shh signaling. Given that similar tissue interactions, including close interactions between the endoderm and ectoderm, are required for both, we hypothesize that formation of the gill slits will use the same underlying molecular mechanism. **