Developing Regeneration Resources for a Model Amphibian

为模型两栖动物开发再生资源

• 批准号: 9272459
• 负责人: Jon Scott Thorson
• 金额: $ 57.33万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9272459
• 关键词: Accelerometer; Amaze; Ambystoma; Ambystoma mexicanum; Amphibia; Amputation; Animal Model; Biological; Biological Assay; Biological Process; Biology; CRISPR/Cas technology; Candidate Disease Gene; Cell Lineage; Cell Proliferation; Cells; Chemical Models; Chemicals; Chimeric Proteins; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Competence; Couples; Data; Development; Embryo; Event; FDA approved; Future; Gene Expression; Gene Targeting; Gene Transfer Techniques; Genes; Genetic; Genetic Models; Genetic Screening; Genetic Transcription; Guide RNA; Histology; Hormone use; Injectable; Insertional Mutagenesis; Investigation; Knock-out; Libraries; Mediating; Methods; Modeling; Molecular; Molecular Profiling; Monitor; Mosaicism; Muscle satellite cell; Mutagenesis; Mutation; Natural Products; Natural regeneration; Neuroglia; Organism; Pathway interactions; Pharmacology; Phenotype; Population; Process; Production; Reagent; Recruitment Activity; Regenerative Medicine; Regenerative response; Reporter; Reporting; Research; Research Infrastructure; Resources; Salamander; Signal Pathway; Signal Transduction; Site; Somatic Cell; Stem Cell Research; Stem cells; Tail; Testing; Thyroid Hormones; Time; Tissues; Transforming Growth Factor beta; Transgenic Organisms; Tretinoin; Vertebrate Biology; blastema; chemical genetics; comparative efficacy; design; dosage; feeding; in vivo; inhibitor/antagonist; mutant; nerve stem cell; network models; notch protein; novel; progenitor; promoter; public health relevance; regenerative; screening; stem cell biology; tool; web site; wound; wound epidermis

 DESCRIPTION (provided by applicant): This Multi-PD/PI application seeks to develop resources that will better enable studies of a highly regenerative amphibian, the Mexican axolotl (Ambystoma mexicanum). There is need to probe axolotl regeneration more deeply, with the same powerful approaches that have proven so effective in genetic model organisms. Specific Aim 1 will accomplish the first chemical genetic screen of axolotl regeneration using an embryo tail regeneration assay. Using pre-feeding axolotl embryos that are efficiently reared in micro-titer plates, up to 10,000 soluble chemicals from commercial, clinical-stage, and novel natural products libraries will be tested for impact on tail regeneration. Preliminary data show that the chemical screen and tail regeneration assay are likely to identify new molecules that impact regeneration. Positive hits from this screen, including previously identified inhibitors of Wnt, Tgfβ, and Fgf signaling, will be reported to the community and investigated further under Specific Aim 2, using cellular and transcriptional approaches. In particular, assays will be developed to assess chemical effects on formation of the wound epidermis, which acts as an early signaling center in the recruitment of progenitor/stem cells. Also, assays will be developed to assess cellular proliferation and de- differentiation, two processes that are associated with endogenous regeneration. Genes found to be expressed differently between control and chemically treated embryos will be prioritized for knock out using the CRISPR/Cas9 method. For each gene target, two gRNA pairs will be designed and injected into single cell axolotl embryos. Embryos will be reared to assess viability, and then administered tail amputations to confirm CRISPR gene editing and test for regeneration competence. Embryos associated with CRISPRs that block or cause abnormal regeneration, will be prioritized for founding stable lines. Specific Aim 3 will generate fluorescent reporter lines to assay signaling activity through major pathways known to function in regeneration and will also compare efficacies of two methods for developing gene-specific reporter lines, using as tester loci genes already known to mark cell populations critical for regenerative responses (neural stem cells, glia, muscle satellite cells). The chemical and genetic hits, and biological information arising from this model will be shared through a community website (Sal-Site). The proposed transgenics will be distributed by the Ambystoma Genetic Stock Center. Overall, this project integrates expertise across chemical screening, pharmacology, histology, transcription, transgenesis, and vertebrate biology to discover reagents and develop tools that are needed to enhance the axolotl for stem cell and regenerative biology.