Humans and other mammals have extremely limited postnatal regenerative abilities, and these limitations pose a significant challenge to health and quality of life. In contrast to humans, axolotl salamanders regenerate many organs and appendages, such as limbs, with astonishing success. Axolotl limbs are very similar anatomically to human limbs, so they offer an ideal opportunity for discovering regenerative mechanisms that might lead to the development of future therapeutics. In my new laboratory, we are investigating the molecular mechanisms of limb regeneration in axolotls so that we can later apply this knowledge to understanding why humans cannot regenerate limbs. An outstanding question is why highly-regenerative organisms use a structure called a blastema, where internal progenitor cells accumulate, to drive regeneration. Blastema cells are heterogeneous in their lineage and likely their potentials, but very little is known about how these attributes are controlled or even how progenitor cells are cued to become activated and join the blastema. To understand these questions, we have initiated a large RNA-seq based approach, and we are coupling this approach to powerful new tools for examining gene function in these organisms. In our first analysis, we have profiled the transcriptomes of individual cells from two key tissues, at one time point. We also generated a tissue-coded de novo transcriptome to use as a reference for gene assignment and for differential gene expression analysis. The initial individual cells sequenced were fully-formed blastema cells and wound epidermis cells, which overly the blastema and are thought to control key aspects of regeneration. We chose this time point, 23 days post- amputation, as the first sampling point because at this time the blastema population is at its height for numbers of cells but there are not yet any overt signs of differentiation. We have thus far discovered many transcripts that are specifically upregulated in individual cells in these important tissues, and we have performed functional analyses with two of the genes. In this proposal, we aim to use this powerful strategy to identify the gene expression changes that support the transition from intact tissue to activated progenitor cells during the creation of the blastema. We will profile the transcriptomes of more individual cells, but now we will query cells harvested from time points between amputation and the full blastema. In parallel, we will further examine genes uncovered in the first analysis, specifically those that show binary expression patterns and may therefore distinguish subtypes of blastema cells or wound epidermis cells. We will use recently-developed loss-of-function and gain-of-function technologies to interrogate specific genes in vivo, in regenerating limbs. This work is innovative because it takes a completely a priori approach to discovering mechanisms of limb regeneration, it does so at the single-cell level, and it capitalizes on powerful new techniques for examining gene function.

Public Health Relevance

Humans have extremely limited limb regenerative abilities, but axolotl salamanders can regenerate an entire limb even in adulthood, though the molecular mechanisms enabling limb regeneration are very poorly understood. We have established an analytical pipeline for discovering, and assessing the function of, genes that facilitate essential behaviors of specific cells during this complex process. We aim to use these technologies to interrogate the role of specific cellular processes in specific populations of progenitor cells in he regenerating limb.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
NIH Director’s New Innovator Awards (DP2)
Project #
1DP2HD087953-01
Application #
8953925
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Mukhopadhyay, Mahua
Project Start
2015-09-30
Project End
2020-06-30
Budget Start
2015-09-30
Budget End
2020-06-30
Support Year
1
Fiscal Year
2015
Total Cost
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
Bryant, Donald M; Sousounis, Konstantinos; Farkas, Johanna E et al. (2017) Repeated removal of developing limb buds permanently reduces appendage size in the highly-regenerative axolotl. Dev Biol 424:1-9
Bryant, Donald M; Sousounis, Konstantinos; Payzin-Dogru, Duygu et al. (2017) Identification of regenerative roadblocks via repeat deployment of limb regeneration in axolotls. NPJ Regen Med 2:30
Bryant, Donald M; Johnson, Kimberly; DiTommaso, Tia et al. (2017) A Tissue-Mapped Axolotl De Novo Transcriptome Enables Identification of Limb Regeneration Factors. Cell Rep 18:762-776
Haas, Brian J; Whited, Jessica L (2017) Advances in Decoding Axolotl Limb Regeneration. Trends Genet 33:553-565