Discovering signature genes and developing expression markers for blastema cells

这个为期三年的博士后培训计划的目标是赋予申请者 在美国建立研究再生的独立实验室所需的技能和网络 在蝾螈(火蜥蜴)中。蝾螈是再生研究版图的关键组成部分，因为它们 能够在失去后更换复杂的身体部位。肢体再生是这方面的一个重要模型。中环 纽特肢体再生指的是胚泡的形成；一群异质的细胞发育成 新的四肢。尽管它是中心性的，但我们对胚泡的了解很少，缺乏任何具体的 表达式标记。申请者将确定调控胚泡的基因和顺式调控元件。 在伊比利亚鳗中形成并开发荧光表达标记以识别伊比利亚鳗中的胚泡细胞类型 活着。这将通过以下研究计划来实现。第一，功能相关的基因组区域 将通过全基因组测序和选择性测序相结合的方式进行测序和组装 开放染色质(目标1)。第二，使用单细胞转录组技术，对胚泡有贡献的细胞类型 将被分类并识别标记基因。此外，通过整合这种单细胞转录 获得的基因组序列的信息，顺式调控元件在 将确定胚泡标记基因(目标2)。第三，使用CRISPR/Cas9基因组编辑，转基因 将开发表达荧光胚基标记的(目标3)。已识别的基因，顺式调控 元素和转基因蝾螈将提供前所未有的知识和工具来进一步了解 管理再生，从而使申请人能够开始一个有前途的独立职业生涯 再生研究。

The objective of this three-year postdoctoral training program is to empower the applicant with the necessary skills and network to establish an independent laboratory in the United States studying regeneration in newts (salamanders). Newts are critical components of the regenerative research landscape due to their ability to replace complex body parts after loss. Limb regeneration is an important model in this regard. Central to newt limb regeneration is the formation of a blastema; a heterogeneous mass of cells that develop into the new limb. Despite its centrality, we have a minimal understanding of the blastema and lack any specific expression markers. The applicant will identify the genes and cis-regulatory elements that regulate blastema formation in the Iberian newt and develop fluorescent expression markers to identify blastema cell types in vivo. This will be accomplished through the following research plan. First, functionally relevant genomic regions will be sequenced and assembled by a combination of whole genome sequencing and selectively sequencing open chromatin (Aim 1). Second, using single-cell transcriptomics, the cell types contributing to the blastema at will be classified and marker genes identified. Additionally, by integrating this single-cell transcriptomic information with obtained genomic sequences, cis-regulatory elements that are over- or under- represented in blastema markers genes will be identified (Aim 2). Third, using CRISPR/Cas9 genome editing, transgenic newts expressing fluorescent blastema markers will be developed (Aim 3). The identified genes, cis-regulatory elements and transgenic newts will offer unprecedented knowledge and tools to further understand the regulation of regeneration, thereby enabling the applicant to begin a promising independent career in regeneration research.