Early Events in Pharyngeal Pouch Patterning

咽是胚胎的一个复杂区域，因为它是由所有三个胚层形成的，并且与相邻的迁移神经嵴相互作用。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。咽形成一个在前后（AP）和背腹（DV）轴上具有明显极性的腔。在AP轴上形成图案需要几个信号信号，包括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途径是不太明确的，但我们发现它是关键的正确模式的咽。狭缝只在咽部外侧形成，而内胚层器官则在咽部腹侧内胚层形成。我们假设不同的信号通路对于咽的AP和DV模式是必要的，这些信号通路对于咽鳃缝和来自咽的内皮器官的形成至关重要。我们的目标是使用特异性抑制剂、反义morpholinos和Crispr技术的组合来发现定义咽部区域的关键信号通路。研究的具体途径包括Shh途径、Fgf途径和典型Wnt信号。我们预测Fgf通路对于咽的整体能力对区域信号的反应是必要的。Shh通路对于AP和DV模式都是必需的，尽管每个轴对信号有不同的时间要求。Wnt信号是腹咽部形成所必需的，也是所有源自咽部内胚层的器官形成所必需的。形成前咽需要Bmp信号。除了信号通路外，我们还将定义特定转录因子在定义咽部区域中的作用。许多转录因子在定义咽的片段，包括Hox家族的成员中得到了很好的确立。我们建议测试其中几个在咽部模式中的作用，包括Hand1， Gcm2， Satb2和Tbx3。我们将使用反义morpholino和Crispr技术来降低这些转录因子的活性，并检查操作胚胎的咽分化和咽内的模式缺陷。***目的3：我们将测试用于口腔形成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。鉴于两者都需要类似的组织相互作用，包括内胚层和外胚层之间的密切相互作用，我们假设鳃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. **