Discovering signature genes and developing expression markers for blastema cells

 描述(由申请者提供)：这个为期三年的博士后培训计划的目标是赋予申请者必要的技能和网络，以便在美国建立一个独立的实验室，研究蝾螈(火蜥蜴)的再生。蝾螈是再生研究领域的重要组成部分，因为它们有能力在丢失后替换复杂的身体部位。肢体再生是这方面的一个重要模型。纽特肢体再生的核心是胚泡的形成，即发育成新肢体的异质细胞团。尽管它是中心性的，但我们对胚泡的了解很少，缺乏任何特定的表达标记。申请者将识别调节伊比利亚鳗胚芽形成的基因和顺式调控元件，并开发荧光表达标记来鉴定体内的胚泡细胞类型。这将通过以下研究计划来实现。首先，功能相关的基因组区域将通过全基因组测序和选择性开放染色质测序相结合的方式进行测序和组装(目标1)。其次，利用单细胞转录组学技术，将有助于胚泡形成的细胞类型进行分类并识别标记基因。此外，通过将这种单细胞转录信息与获得的基因组序列相结合，将识别在胚泡标记基因中表达过多或表达不足的顺式调控元件(目标2)。第三，利用CRISPR/Cas9基因组编辑，将开发表达荧光胚芽标记的转基因蝾螈(目标3)。已确定的基因、顺式调控元件和转基因蝾螈将提供前所未有的知识和工具，以进一步了解再生的调节，从而使申请者能够在再生研究中开始一段有前途的独立职业生涯。

 DESCRIPTION (provided by applicant): 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.