Date of Award

December 2015

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

Advisor(s)

Kate E. Lewis

Keywords

development, GABA, neurons, specification, spinal cord, zebrafish

Subject Categories

Life Sciences

Abstract

Spinal cord contains different types of neurons. A vast majority of these neurons are interneurons. Therefore, a better understanding of how interneurons in the spinal cord develop is particularly important, as it will hopefully enable researchers to establish better treatments for spinal cord injury patients. In this thesis, I used zebrafish to study development of two classes of spinal cord neurons - KA cells (which correspond to cerebrospinal fluid-contacting neurons, CSF-cNs) and V2b cells. Both KA and V2b neurons are functionally important in vertebrate locomotor circuitry.

Development of functional neurons involves progression of genetic cascades that lead to correct cell specification. Some of the most important genes expressed during cell development encode for transcription factors, which are regulatory proteins that can either activate or repress expression of downstream genes. Both V2b and KA neurons in zebrafish express a common set of transcription factors - Tal1, Gata2a, and Gata3. These proteins are not present in any other spinal cord cell type. Previous work used a knockdown approach (morpholinos) to show that gata2a and gata3 have different functions in specification of KA cell types - KA" cells require gata2a and KA' cells require gata3 to develop correctly. In this thesis, I test whether the same phenotypes occur in null mutants. Also, I investigate the role of tal1 in specification of KA cells, and the role of all three (gata2a, gata3, tal1) genes in V2b specification. To do this, I used tal1, gata2a and gata3 zebrafish mutants.

KA and V2b neurons also share another characteristic - all these cells are GABAergic. Therefore, I investigate whether tal1, gata2a and gata3 genes are required for correct specification of V2b and/or KA global cell fate and/or GABAergic phenotypes of these cells. In addition, I identify a subset of genes expressed by either KA", KA' and/or V2b cells and analyze the expression of some of these in these mutants.

My results show that tal1, gata2a, and gata3 have distinct functions in each neuron type. In KA" cells, gata2a is required for correct expression of the majority of KA" markers and the GABAergic phenotype of these cells. Interestingly, both tal1 and gata3 are not required for correct specification of KA" cells. In KA' cells, the situation seems to be reversed - tal1 and gata3 are required for correct expression of all KA' markers, but gata2a is required for only some aspects of the KA' cell fate in a subset of KA' cells. In V2b cells, the phenotypic effects of these mutations are more complicated - neither tal1, gata2a nor gata3 are required for correct expression of all V2b genes. However, tal1 is required for expression of a subset of these genes, and for the GABAergic phenotype of V2b cells.

This thesis contributes to better understanding of KA", KA' and V2b neuron specification. Also, the presented results have broader implications, as they underlie the importance of cell type specificity of genetic cascades that lead to correct development of spinal cord neurons.

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