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.
Salamanders display an impressive ability to regenerate lost body parts, including complete restoration of lost limbs. Central to this process is the formation of a blastema; a mass of cells that develop into the new limb. I will use a combination of genome sequencing, single-cell transcriptomics and genome editing to discover the genes that are active specifically in the blastema and to develop transgenic animals where only blastema cells are florescent. These results will offer a quantum leap in our understanding of salamander limb regeneration and the tools available for additional studies, which will help pave the road to making human limb regeneration a clinical reality.
| Elewa, Ahmed; Wang, Heng; Talavera-López, Carlos et al. (2017) Reading and editing the Pleurodeles waltl genome reveals novel features of tetrapod regeneration. Nat Commun 8:2286 |
