Implementing new genomic tools to study ventral midbrain development and injury response in the newt Pleurodeles waltl

Abstract

Mammals have a poor ability to regenerate organs or tissues. In particular, brain neurons do not regenerate after damage. A good example of this limitation is the irreversible degeneration of midbrain dopaminergic neurons in Parkinson’s disease (PD). Several strategies are being developed to treat PD. The proposed treatments include transplantations and local reprogramming of brain cells. Transplantation approaches involve different sources of dopaminergic progenitors or neurons which can be sourced either in vivo from fetal ventral midbrain tissues, or in vitro from either embryonic stem cells or induced pluripotent stem cells. Reprogramming approaches try to directly convert local glial cells into dopaminergic neurons in vivo. While clinical progress has been made in trials, several issues need to be addressed, such as the safety of the techniques, and ethical considerations.

In this thesis I explored how dopaminergic neurons could be generated locally in the ventral midbrain from the endogenous neural stem cells. I used newts as the animal model, which have an extraordinary ability to regenerate organs and tissues including limb, tail, heart, lens, spinal cord, and the brain. Previous studies showed that newts functionally recovered after mesencephalic and diencephalic ablation of dopaminergic neurons by a process which is fueled by regenerative neurogenesis. However, the cellular and molecular mechanisms after dopaminergic lesion have remained largely unrevealed, mostly because of the paucity of available molecular tools. In addition to the regeneration studies, studying newts broadens our understanding of the function and the shaping of the midbrain from a developmental and evolutionary perspective. The work in this thesis made use of recent technological advances, such as a newly assembled genomic resource, single-cell RNAseq (scRNAseq) and single-nucleus RNAseq (snRNAseq) methods, and genomic modification techniques. I implemented and refined those tools in the following projects.

In Project I, I made use of new genomic and transcriptomic resources for the Iberian ribbed newt, Pleurodeles waltl. I identified gene orthologues of key dopaminergic determinants and other genes expressed in vertebrate ventral midbrain. I found examples of both evolutionary conservation and divergence. I designed and produced plasmids and gRNAs which were used to generate transgenic and mutant newts. I predicted reactivities of multiple commercially available antibodies that I tested in Pleurodeles waltl tissues. I also designed probes for single and multiplexed in situ hybridizations.

In Project II, I studied the development of the Pleurodeles waltl ventral midbrain. Using scRNAseq/snRNAseq analyses in combination with immunohistochemical and functional analyses I focused on two aspects: (i) cellular heterogeneity, and (ii) transition of neural stem/progenitor cells from active proliferation to quiescence. Based on marker gene expression, I found high degree of conservations of major cell types between Pleurodeles waltl and mammals. I then studied in detail the development of the ventral midbrain with emphasis on the dopaminergic system. I uncovered that the cellular diversity observed in the adult ventral midbrain is established at the late larval stage, though the proportions of cell types change as development proceeds. I used the snRNAseq data to detect changes in gene expression from development to adulthood. The in silico data were confirmed by validating protein expression in the midbrain, by which I could observe defined subpopulations in their spatial distribution. Furthermore, I found that overexpression of the transcription factor NFI in vivo promotes the natural transition of stem/progenitor cells into quiescence.

In Project III, I explored the cellular and molecular responses to dopaminergic injury in the Pleurodeles waltl ventral midbrain. I found, in accordance with the results of Project II, a downregulation of NFI correlating with the reactivation of ependymolial cells, which are neural stem cells in the newt brain. Overexpression of NFI after dopaminergic ablation inhibited the activation of ependymoglia. Bulk RNAseq analyses revealed sets of up- and downregulated genes which I explored further in the snRNAseq datasets. Furthermore, using the transgenic line mpeg1:GFP and PLX3397-mediated depletion, I revealed a role for microglia/macrophages in the reactivation of ependymolia after the ablation of dopaminergic neurons.

In sum, the work provides new resources and entry points in how to consider the development and evolution of the newt ventral midbrain, and its ability to recover following loss of dopaminergic neurons.