GSBS Dissertations and Theses

Approval Date

3-16-2015

Document Type

Doctoral Dissertation

Academic Program

Neuroscience

Department

Department of Neurobiology; Tzumin Lee Lab

First Thesis Advisor

Tzumin Lee, MD, PhD

Keywords

Drosophila melanogaster, Cerebrum, Neural Stem Cells, Body Patterning, Gene Targeting, Cell Lineage, CRISPR-Cas Systems

Subjects

Dissertations, UMMS; Drosophila melanogaster; Cerebrum; Neural Stem Cells; Body Patterning; Gene Targeting; Cell Lineage; CRISPR-Cas Systems

Abstract

Understanding the complex mechanisms to assemble a functional brain demands sophisticated experimental designs. Drosophila melanogaster, a model organism equipped with powerful genetic tools and evolutionarily conserved developmental programs, is ideal for such mechanistic studies. Valuable insights were learned from research in Drosophila ventral nerve cord, such as spatial patterning, temporal coding, and lineage diversification. However, the blueprint of Drosophila cerebrum development remains largely unknown.

Neural progenitor cells, called neuroblasts (NBs), serially and stereotypically produce neurons and glia in the Drosophila cerebrum. Neuroblasts inherit specific sets of early patterning genes, which likely determine their individual identities when neuroblasts delaminate from neuroectoderm. Unique neuroblasts may hence acquire the abilities to differentially interpret the temporal codes and deposit characteristic progeny lineages. We believe resolving this age-old speculation requires a tracing system that links patterning genes to neuroblasts and corresponding lineages, and further allows specific manipulations.

Using modern transgenic systems, one can immortalize transient NB gene expressions into continual labeling of their offspring. Having a collection of knockin drivers that capture endogenous gene expression patterns would open the door for tracing specific NBs and their progenies based on the combinatorial expression of various early patterning genes. Anticipating the need for a high throughput gene targeting system, we created Golic+ (gene targeting during oogenesis with lethality inhibitor and CRISPR/Cas “plus”), which features efficient homologous recombination in cystoblasts and a lethality selection for easy targeting candidate recovery. Using Golic+, we successfully generated T2AGal4 knock-ins for 6 representative early patterning genes, including lab, unpg, hkb, vnd, ind, and msh. They faithfully recapitulated the expression patterns of the targeted genes. After preserving initial NB expressions by triggering irreversible genetic labeling, we revealed the lineages founded by the NBs expressing a particular early patterning gene.

Identifying the neuroblasts and lineages that express a particular early patterning gene should elucidate the genetic origin of neuroblast diversity. We believe such an effort will lead to a deeper understanding of brain development and evolution.

DOI

10.13028/M2401R

Rights and Permissions

Copyright is held by the author, with all rights reserved.

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