Publication Date

2012-06-05

Availability

Open access

Embargo Period

2012-06-05

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2012-05-24

First Committee Member

Mary Lou King

Second Committee Member

Zafar Nawaz

Third Committee Member

Ralf Landgraf

Fourth Committee Member

Athula Wikramanayake

Fifth Committee Member

Pantelis Tsoulfas

Sixth Committee Member

Lidia Kos

Abstract

Nanos is expressed in multipotent cells, stem cells, and primordial germ cells (PGCs) of organisms as diverse as jellyfish and humans. It functions together with Pumilio to translationally repress targeted mRNAs. Here we show by loss-of-function experiments that Xenopus Nanos1 is required to preserve PGC fate. Morpholino knockdown of maternal Nanos1 resulted in a striking decrease in PGCs and loss of germ cells from the gonads. Lineage tracing and TUNEL staining reveals that Nanos1 deficient PGCs fail to migrate out of the endoderm. They appear to undergo apoptosis rather than convert to normal endoderm. Whereas normal PGCs do not become transcriptionally active until neurula, Nanos1 depleted PGCs prematurely express a hyperphosphorylated RNA Pol II-CTD at the mid-blastula transition. Furthermore, they now inappropriately express somatic genes characteristic of endoderm regulated by maternal VegT including Xsox17-alpha, Bix4, Mixer, GATA4, and Edd. We further demonstrate that Pumilio specifically binds VegT RNA in vitro and represses, along with Nanos1, VegT translation within PGCs. Repressed VegT RNA in wild type PGCs is significantly less stable than VegT in Nanos1 depleted PGCs. Our data indicate maternal VegT RNA is an authentic target of Nanos1/Pumilio translational repression. We propose that Nanos1 functions to translationally repress RNAs that normally specify endoderm and promote apoptosis, thus preserving the germline.

Keywords

Xenopus; PGCs; Nanos/Xcat2; VegT; germline determination; endoderm; translational repression

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