Student Theses and Dissertations

Peter Insley

Date of Award

2017

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Shaham Laboratory

Abstract

The positioning of neuronal cell bodies and neurites is critical for intact functioning of the nervous system. Mapping the positions of the soma and neurites in the brains of developing embryos as important central nervous system structures are being created may yield novel insight into the role of distinct cell groups in creating these structures. New developments in microscopy have made this an excellent time to study neural development in the C. elegans embryo. In the past decade, implementations of highly light efficient methods such as single plane illumination microscopy have rendered it possible to follow development of embryonic structures in 3D with excellent temporal resolution (Huisken et al., 2004) and low phototoxicity. Recent work has resulted in quantitative characterization of the outgrowth of a single neurite in the late, rapidly moving three-fold stage of the C. elegans embryo for the first time (Christensen et al., 2015). In this thesis, I first describe the construction and programming of a single plane illumination microscope (SPIM) based on a design from Hari Shroff's lab (Wu et al., 2011). The microscope is developed especially for use with C. elegans embryos and permits fast image acquisition without excessive photodamage, compared to other forms of microscopy. Second, I describe the use of the SPIM microscope to image the development of a subset of sublateral neurons, the earliest known entrants to the nerve ring (Rapti et al, in preparation), into which they grow in the 1.5-fold stage. I describe an algorithm for automatically aligning developing embryos onto one another until the beginning of the rapid embryonic movements known as twitching, which begin at the start of the twofold stage. I employ my algorithm to align a group of identically imaged embryos onto one another and deduce information about the positioning of the nerve ring in an approximately uniform coordinate system. I determine that nerve rings are precisely positioned in the embryo to within about a micrometer while the cell bodies that grow into the nerve ring are positioned over a much wider distance. My work suggests that the nerve ring grows out towards the ALA neuron as an anchor, and that twitching may begin when the developing nerve ring reaches the ALA. I additionally describe observation of new phenotypes related to the cam-1 mutation, which was previously identified as a regulator of anterior-posterior placement of the nerve ring (Kennerdell et al., 2009). Third, I describe an application of the SPIM microscope for imaging the death of the tail spike cell, a complex, multi-compartment differentiated cell which dies over a period of hours during the three-fold stage, when the animal is rapidly moving in its shell, and cannot be imaged otherwise than with a rapid, light efficient microscope such as the one described here. I determined the time course and confirmed the sequence of events of wild type tail spike cell death. Additionally, I report stronger phenotypes for some known tail spike cell death genes when imaged in the embryo, suggesting that eff-1 plays a stronger role than previously known in clearance of the distal part of the tail spike cell process, and additionally that ced-5 has a strong role in clearance of the same compartment (in addition to its known role in soma clearance). In an appendix I describe work beginning on an extension of the microscope, which will hopefully see the microscope used as a tool for selectively inducing fluorescence in individual cells and following the development of those cells in time. My results demonstrate the utility of single plane illumination microscopy for study of C. elegans embryogenesis and establish fundamental facts about the variability of the C. elegans central nervous system by making direct comparisons between animals. This work contributes to our understanding of the C. elegans nervous system by establishing fundamental bounds on the range of nerve ring positioning between individuals.

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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