Testing models of pharyngeal segmentation using the sea lamprey Petromyzon marinus and the frog Xenopus laevis

 描述(申请人提供)：颌骨、面部、颈部和内耳的骨骼和软骨由称为神经脊细胞(NCC)的迁移前体细胞发育而来。在胚胎头部的腹侧，NCC分布在称为咽弓的结构中。咽弓控制着NCC发育的多个方面，包括它们的迁移、图案化和分化。与此一致的是，参与咽弓形成的基因突变是DiGeorge(TBX1)和Bamforth-Lazarus(Foxe1)综合征头面部缺陷的基础。类似的骨骼异常也与子宫内维甲酸(RA)信号的中断有关，维甲酸是咽弓形成的重要调节因素。了解咽弓发育背后的复杂遗传和分子相互作用是确定遗传和环境诱导的头骨发育中断的原因和治疗的第一步。在形态上，咽弓的发育始于内胚层的外袋，称为咽囊。小袋的侧面突起，以及它们与覆盖的外胚层的融合，将壁分隔开 咽部，形成咽弓。一些驱动咽袋起始和生长的基因已经被发现，包括转录因子TBX1/10和Pax1/9。这些因子与其他几个转录调节因子和信号分子在咽内胚层中共表达。这些基因在进化上保守的表达和功能表明它们是眼袋形成的基因调控网络(GRN)的一部分。然而，这些假定的袋状GRN成分之间的确切调控关系以及它们的节段性表达是如何首先在咽内胚层建立的，目前尚不清楚。拟议的工作将使用一种创新的比较方法来解决我们知识中的这些空白，其中包括海鳗、海鳗和非洲爪蛙。七鳃鳗是最基本的脊椎动物，适合于发育操作。由于其发育缓慢，咽部器官形态简单，以及大量类似的咽袋，因此七鳃鳗特别适合研究动态的、重复的遗传交互作用驱动袋的形成。此外，技术的进步使该物种的遗传操作司空见惯。对七鳃鳗和四足脊椎动物莱维氏X.laevis之间的基因表达和功能进行比较，将突出咽袋GRN的核心、保守方面，可能在包括人类在内的所有脊椎动物中运行。这项工作的具体目的是解决这4个问题：1)SIX1、Eya和Foxq1在咽袋GRN中的位置是什么？2)来自邻近组织的节段性Wnt11r和FGF8信号是否激活了咽内胚层中的GRN组分的表达？3)RA信号梯度是否形成了调节GRN组分的重复激活的分化‘波前’？4)振荡基因表达的“时钟”是否建立了眼袋GRN组件的节段性表达？

 DESCRIPTION (provided by applicant): The bones and cartilages of the jaws, face, neck, and inner ear develop from migratory precursors called neural crest cells (NCCs). In the ventral portion of the embryonic head, NCCs populate structures called the pharyngeal arches. The pharyngeal arches control multiple aspects of NCC development, including their migration, patterning, and differentiation. Consistent with this, mutations in genes involved in pharyngeal arch formation underlie the craniofacial defects of DiGeorge (Tbx1) and Bamforth-Lazarus (Foxe1) syndromes. Similar skeletal abnormalities have been linked to in utero disruption of retinoic acid (RA) signaling, an essential regulator of pharyngeal arch formation. Understanding the complex genetic and molecular interactions underlying pharyngeal arch development is the first step toward identifying the causes of, and treatments for, genetic and environmentally-induced disruptions of head skeleton development. Morphologically, pharyngeal arch development begins with out-pocketings of endoderm called the pharyngeal pouches. The lateral outgrowth of the pouches, and their fusion with overlying ectoderm, partitions the walls of the pharynx, creating the pharyngeal arches. A few of the genes driving pharyngeal pouch initiation and outgrowth have been identified, including the transcription factors Tbx1/10 and Pax1/9. These factors are coexpressed with several other transcriptional regulators and signaling molecules in the pharyngeal endoderm. The evolutionarily conserved expression and function of these genes suggest they are part of a gene regulatory network (GRN) for pouch formation. However, the precise regulatory relationships between these putative pouch GRN components and how their segmental expression is first established in the pharyngeal endoderm, is unknown. The proposed work will address these gaps in our knowledge using an innovative comparative approach incorporating the sea lamprey Petromyzon marinus and the frog Xenopus laevis. The sea lamprey is the most basal vertebrate amenable to developmental manipulations. Due to its slow development, morphologically simple pharyngeal apparatus, and large number of similar pharyngeal pouches, the sea lamprey is particularly well-suited to studying the dynamic, iterated genetic interacts driving pouch formation. Furthermore, technical advances have made genetic manipulations in this species routine. Comparisons of gene expression and function between lamprey and X. laevis, a tetrapod vertebrate, will highlight the core, conserved aspects of the pharyngeal pouch GRN likely operating in all vertebrates, including humans. The specific aims of the work are to address these 4 questions: 1) what are the positions of Six1, Eya, and Foxq1 in the pharyngeal pouch GRN?; 2) do segmental Wnt11r and FGF8 signals from adjacent tissues activate expression of pouch GRN components in the pharyngeal endoderm?; 3) does a RA signaling gradient form a differentiation `wavefront' that regulates iterated activation of pouch GRN components? and 4) does a `clock' of oscillating gene expression establish segmental expression of pouch GRN components?