Exploring the mechanisms regulating neural progenitor cell fate decisions during Xenopus tropicalis spinal cord regeneration

Abstract

After injury, the mammalian spinal cord shows extensive damage, loss of neurons and scarring with very little functional regeneration. By contrast, non-mammalian species such as Xenopus tropicalis tadpoles can regenerate their spinal cord following injury. Across pro-regenerative species, spinal regeneration is characterised by the proliferation of neural progenitor cells (NPCs) from the injured stump, which are required for successful regeneration. The generation of new neurons is also a hallmark of successful regeneration in anamniotes, however the intrinsic mechanisms governing the balance between proliferation and differentiation are not fully understood. Here, I aimed to assess how cell fate decisions are regulated during regenerative outgrowth of the Xenopus tropicalis spinal cord. Our laboratory had previously identified a putative regulator of spinal cord regeneration, the transcription factor Forkhead Box M1 (Foxm1). Loss of foxm1 expression by CRISPR/Cas9-mediated knockout impairs regeneration of the tadpole tail. Using single cell transcriptomics, we identified a regeneration-specific population of neural progenitor cells (NPCs) in the spinal cord, which is characterised by the expression of Foxm1. However, although Foxm1 is canonically a regulator of proliferation, I established that loss of foxm1 expression does not affect the proliferation rate of NPCs, or the total length of their cell cycle. Instead, we found that Foxm1 is required to balance de novo neurogenesis in the regenerating spinal cord. Subsequently, I used discovery- and hypothesis-driven approaches to assess putative mechanisms underlying the cell fate decisions of NPCs during spinal cord regeneration. I showed that in NPCs, loss of Foxm1 impairs S phase progression at the expense of G1 phase, a phenotype which correlates to their impaired differentiation to neurons. I also showed that tissue oxidation is impaired in the regenerate in the absence of foxm1 expression. Lastly, to identify novel regulators of cell fate decisions, I developed an optimised protocol to isolate and multiplex label single cells from the regenerating spinal cord. Using this protocol, I generated a novel multiplex single cell RNA sequencing dataset, extending our single cell characterisation of the regenerating spinal cord up to 5 days post-amputation. Together, this work identifies a novel role for Foxm1 in regulating cell cycle dynamics and cell fate in NPCs during regeneration and extends our current knowledge of the mechanisms underlying epimorphic regeneration of the Xenopus tropicalis tadpole tail.

Date of Award	5 May 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Karel Dorey (Main Supervisor), Natalie Gardiner (Co Supervisor) & Enrique Amaya (Co Supervisor)