Project 1: Neural Regeneration and Deciding When to Stop Nerve Regrowth
Above: Control (on left) eye regeneration. Regeneration of excess of eyes (on right) following inhibition of PCP.
One of the largest black boxes in regeneration is how an organism decides when patterning is complete. A mechanism to stop regeneration must exist, since planarian do not keeping regenerating new tissue indefinitely. But how this happens is unknown. My data have revealed that for one tissue, specially the nervous system, the Planar Cell Polarity (PCP) pathway is required for the planarian nervous system to stop regenerating. Without PCP the entire nervous system continues to grow for months after regeneration normally ends! Strikingly, the excess visual neurons produced results in regenerates that continually produce new eyes.
We found that the restriction of neural growth by PCP is conserved in vertebrate regeneration and development, suggesting this is an ancestral role. Together the data reveal PCP inhibition may be a novel target for therapies aimed at initiating nerve regrowth in humans, where damage to the central nervous system is usually permanent. The overall goal of this project is to determine how PCP functions to restrict nerve growth. We are investigating which cells require PCP, how the PCP pathway interacts with stem cells to regulate neural progenitors, and how neural termination is integrated with the mechanisms that control the overall regeneration of shape. The ultimate aim is to identify therapeutic reagents for the initiation of neural regeneration.
The planarian nervous system (above, in green), consists of a true brain, two ventral nerve cords that run the length of the body, and an intricate network of commissural and peripheral nerves. When PCP is inhibited, the regenerating worm regrows too many commissural neurons (yellow arrows).
Project 2: Bioelectrical Signaling as a Master Regulator of Regenerative Shape
The restoration of shape during planarian regeneration requires very sophisticated coordination between new and old tissues, in order to align pre-existing organs with the newly proliferating ones. My data has revealed that membrane de-polarization (mediated by the H,K-ATPase ion transporter) is upstream of both head formation and the anterior identity of new tissues (which drives new tissue shape), as well as apoptotic-mediated tissue remodeling in pre-existing tissues.
Thus a knockout of H,K-ATPase activity by chemical inhibition results in trunk fragments (which need to regenerate both a head and tail) that regenerate without heads and fail to reshape their old tissues:
Left: Control (top row) – new tissues in the anterior are depolarized (red arrow), resulting in head regeneration. H,K-ATPase Inhibited (bottom row) – without H,K-ATPase activity, new tissues are hyperpolarized (open arrow) producing regenerates lacking heads and tissue remodeling.
Above: H,K-ATPase inhibited regenerates.
If H,K-ATPase activity is only partially knocked-out, regenerates are able to form heads, but lack the ability to shape pre-existing tissues, failing to maintain overall body shape (image to the right). Thus RNA interference (RNAi) to a single H,K-ATPase alpha subunit (on bottom) results in worms with tiny, shrunken heads and anteriorly shifted, abnormally large pharynges (green brackets). The overall goal of this project is to determine how changes in voltage at the plasma membrane can coordinate overall animal shape in both new and old tissues. We are doing this through a systems approach investigating how the genes regulating planarian shape are/are not linked, as well as studying the role of calcium signaling downstream of membrane voltage changes and upstream of head gene transcription and tissue apoptosis.