Student Theses and Dissertations

Alina Rashid

Date of Award

2022

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Shaham Laboratory

Abstract

During animal development, neurons and neuron-like cells are generated from progenitor cells that often line tubes. For example, in the developing spinal cord, basal processes of radial glial stem cells line the fluid-filled spinal canal. Notch/Delta signaling is implicated in the differentiation of these cells into motoneurons through control of their proliferative state. Down-regulation of the Notch-dependent Hes1 and Hes5 transcriptional inhibitors in differentiating cells induces expression of the proneural bHLH transcription factors Olig2 and Ngn2, leading to adherens junction loss, delamination from the epithelium, cell migration, and neuronal maturation. A similar sequence of events characterizes formation of pancreatic insulin-secreting β-cells, which are innervated and express genes also active in motoneurons. Here, epithelial progenitor cells lining pancreatic ducts express high levels of Notch, which activates Hes1 repressor, in turn blocking Ngn3 expression. Down-regulation of Notch drives delamination, cell migration, and subsequent differentiation by lifting inhibition of Ngn3 and by allowing Notch to directly activate Ngn3 expression. The Olig family bHLH factor bHLHb4 is expressed in delaminating/migrating cells, and may be involved in β-cell differentiation. The dynamics of gene expression and the regulatory interactions among genes driving motoneuron formation are not fully understood. Furthermore, distinguishing whether genes governing this differentiation event control cell division or cell fate acquisition can be challenging. To address these issues, I have studied a similar transformation process in the genetically amenable nematode, Caenorhabditis elegans. The C. elegans Y cell is one of six epithelial cells that line the rectal tube in first-larval-stage (L1) animals. In L2 animals, Y loses apical junctions with neighboring cells, migrates anterodorsally, and transforms into the PDA motoneuron, which innervates the intestinal and anal depressor muscles. Remarkably, this transformation takes place without cell division. Forward and reverse genetic studies identified a number of genes required for the Y-to-PDA transition, including lin-12/Notch, sem-4/Sall, egl-5/Hox, and several chromatin remodeling genes. Thus, the Y-to-PDA transition is an excellent setting in which to identify regulators that specifically affect motoneuron progenitor-cell differentiation, and not cell division. Here, I present my identification of novel regulators of Y-to-PDA transformation and explore the interactions among them and previously identified genes. I find that loss of lin-12/Notch blocks PDA neuron formation, and gain of lin-12/Notch function generates a precocious PDA neuron. Thus, lin-12/Notch acts as a timing rheostat for motoneuron generation in a cell-division-independent capacity. lin-12/Notch functions, at least in part, by regulating ngn-1/Ngn, through control of the bHLH gene hlh-16/Olig. ngn-1/Ngn basal expression levels are set early on by sem-4/Sall, egl-5/Hox, and hlh-16/Olig. Coincident with the onset of Y-cell morphological changes and migration, I find an increase in ngn-1/Ngn gene expression, governed by sem-4/Sall and egl-5/Hox, but not hlh-16/Olig. Y-cell migration is accompanied by retrograde extension of a process that remains anchored at the rectal slit. The tip of this process serves as a growth point for the PDA axon. Following axonal growth, I identify the axonal cytoskeleton genes unc-119, unc-44/Ank, and unc-33/Crmp as targets of hlh-16/Olig and ngn-1/Ngn, and demonstrate a previously unappreciated role for these genes in regulating the expression of motoneuron terminal differentiation genes. My results highlight intriguing similarities with spinal-cord motoneuron and pancreatic islet formation, suggesting a conserved module for the differentiation of tube-lining cells into motoneuron/motoneuron-like progeny.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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