Function of the dead end gene during zebrafish primordial germ cell formation

Weiyi Liu, Purdue University

Abstract

Primordial germ cells (PGCs) are the progenitors of the germ cell lineage able to develop into either spermatogonia in the male or oogonia in the female. Zebrafish dead end (dnd) mRNA is specifically restricted to PGCs and is required for PGC polarization, motility and survival. Previous studies have shown that zebrafish Dnd functions by protecting the 3’UTRs of nanos and TDRD7 from miR-430b-mediated RNA decay. In this work, we demonstrate that zebrafish Dnd protein possesses ATPase activity that resides in the last 91 amino acids of the Dnd C terminus. Rescue experiments conducted by injecting mRNAs encoding the Dnd ATPase mutants into embryos in which the endogenous dnd expression was inhibited demonstrated that the ATPase activity is required for normal zebrafish PGC survival. The expression of PGC markers nanos and TDRD7 but not vasa were down-regulated when dnd mutant proteins lacking ATPase were expressed in the rescued embryos, indicating that the Dnd ATPase is involved in protecting nanos and TDRD7 transcripts. To further examine the proposed function of dnd and to investigate how it affects nanos expression in the PGC, we used an in vitro luciferase assay with the zebrafish ZSSJ cell line to evaluate dnd’s effect on nanos-3’UTR stability and expression. Surprisingly, transfection of a construct containing the luciferase gene linked to nanos-3’UTR along with a plasmid encoding Dnd significantly repressed luciferase activity at the translational level. The repression of nanos-3’UTR by Dnd was confirmed by in vivo studies using intact zebrafish embryos. The repression was miR-430 independent and it required the Dnd RNA recognition motif but not the ATPase domain. Interestingly, co-expression of Apobec3, a mouse Dnd interacting RNA editing enzyme, along with zebrafish Dnd in ZSSJ cells increased the translational repression of nanos 3’UTR, indicating a coordinated role of Apobec3 and Dnd. The results from this study suggest a novel scenario of Dnd function in which Dnd may inhibit nanos expression at the translational level to maintain primordial germ cell integrity. Finally, to identify Dnd associated molecules, protein pull-down and RNA pull-down were performed and the candidate partners were investigated for their potential roles in PGC migration, proliferation and survival.

Degree

Ph.D.

Advisors

Collodi, Purdue University.

Subject Area

Genetics|Cellular biology|Developmental biology

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