Functional Analysis of Signaling Pathways Controlling Limb Regeneration

University Of Florida, Gainesville FL

Investigators

Abstract

Urodele amphibians (newts and salamanders) are unique throughout the animal kingdom in their ability to perfectly regenerate whole organs such as limbs, brains and the heart. For example, if the limb is amputated anywhere along its length a perfect replacement will regenerate in about 1-2 months and this regenerate will contain the bones, cartilage, joints, muscles, nerves, blood vessels and skin that were originally there and all in the exactly the same relationship to each other so that the limb will work again. The goal of this project is to determine how growth factors and signaling molecules drive this process. It has long been known that the nerves supply a factor that stimulates the proliferation of the regenerating cells in the limb, and several candidates have been identified recently. This project functionally tests each of these candidates in a novel assay based on gene expression in the wound epidermis. The identification of a factor that stimulates proliferation would have a huge impact on mammalian systems where the lack of proliferation is the major impediment to tissue regeneration. In addition to the nerve factor, two additional signaling pathways are utilized to guide the regenerative process. These will also be examined using the latest gene knockout techniques to determine how these pathways interact with other known signals. This project will impact several scientific communities, and the public at large because regeneration has huge public appeal. Examples of this interest include that fact that these recent axolotl regeneration studies were highlighted in TV programs and other public media, and received public acclaim. Additionally, this project includes specifically-designed mini-research projects that involve 4-6 undergraduates per year, and promotes active involvement of under-represented minorities and women. The first part of this project concentrates on the function of the apical epidermis that forms after limb amputation as a crucial regulator of regeneration. Transcriptomic analysis has identified the limb-specific genes expressed in this apical epidermis, including Wnt, BMP and FGF - as well as two novel pathways: IGF and RA. These new pathways will be examined by inhibition using antisense morpholinos, pharmacological inhibition and Crispr/Cas9 targeted mutagenesis to identify interactions with other pathways by qPCR and in situ hybridization. The transcriptional network downstream of RA signaling will also be examined by RNA-Seq to identify targets of this signaling molecule. The second objective is to discover the molecular basis of the neurotrophic control of limb regeneration. A novel approach will be used to screen 12 potential neurotrophic factors for their effect on gene expression in the apical epidermis in vitro since the apical epidermis is the primary target of the nerve. Included in this screen are 7 novel neurotrophic factors that were identified in a microarray for secreted factors from sensory nerves. Successful candidates will be taken further using in vivo analyses including expression patterns during limb regeneration and over-expression in transgenic animals.