14-3-3ε Is Required for Germ Cell Migration in Drosophila

Although 14-3-3 proteins participate in multiple biological processes, isoform-specific specialized functions, as well as functional redundancy are emerging with tissue and developmental stage-specificity. Accordingly, the two 14-3-3ε proteins in Drosophila exhibit functional specificity and redundancy. Homozygotes for loss of function alleles of D14-3-3ε contain significantly fewer germ line cells (pole cells) in their gonads, a phenotype not shared by mutants in the other 14-3-3 gene leo. We show that although D14-3-3ε is enriched within pole cells it is required in mesodermal somatic gonad precursor cells which guide pole cells in their migration through the mesoderm and coalesce with them to form the embryonic gonad. Loss of D14-3-3ε results in defective pole cell migration, reduced pole cell number. We present evidence that D14-3-3ε loss results in reduction or loss of the transcription factor Zfh-1, one of the main regulatory molecules of the pole cell migration, from the somatic gonad precursor cells

14-3-3ζ-Leo over-expression does not rescue pole cell number in <i>D14-3-3ε^ex4</i>homozygotes.

<p>Expression of the two ubiquitous Leo isoforms, LeoI and LeoII <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036702#pone.0036702-Messaritou1" target="_blank">[12]</a> in mutant homozygotes under Tub-Gal4, Act-Gal4 and NosVp16-Gal4 (light gray bars) failed to change the reduced number of pole cells in <i>D14-3-3ε^ex4</i> homozygotes. The dark bar indicates the number of pole cells per gonad in <i>D14-3-3ε^ex4</i> homozygotes. The number of pole cells in embryos expressing transgenes (light gray bars) was compared to that of the homozygotes (dark gray bar) and of genotype-matched heterozygotes (medium gray bars) carrying each UAS transgene on the same chromosome as that bearing the <i>D14-3-3ε^ex4</i> mutation. Full rescue was attained only when Tub-Gal4 drove the UAS<i>epsilon</i> (UAS<i>eps</i>) transgene. The line is drawn to aid comparisons with the number of pole cells in the mutant homozygotes.</p

Cell type limited transgenic rescue and phenocopy of the pole cell deficit.

<p>A. Equivalent expression of two independent <i>UASepsilon</i> transgenes in different chromosomal locations expressing either low (L) or high (H) levels of transgenic protein under either Tub-Gal4 or Act-Gal4. B. Transgenic rescue of the scattered pole cell phenotype of <i>D14-3-3ε^ex4</i> homozygous and heterozygous embryos under the Tub-Gal4 or Act-Gal4 drivers. The dark bar indicates the number of pole cells per gonad in <i>D14-3-3ε^ex4</i> homozygotes carrying a silent (no Gal4 driver) <i>UASepsilon</i> transgene. Pole cell number was significantly (p<0.0001, Dunnett's test) higher in mutant heterozygotes (medium gray bars) than in <i>D14-3-3ε^ex4</i> homozygotes. Importantly, expression of the transgene under Tub-Gal4 (middle light gray bar) resulted in a significant increase (p<0.0001, Dunnett's test) of pole cells per gonad over those in mutant homozygotes (dark bar). In contrast, pole cell number remained similar to that of the homozygous mutants under Act-Gal4 (rightmost light gray bar). C. Expression pattern of Tub-Gal4 and Act-Gal4 drivers. Expression of the transgenic protein b-Tau (red) using the drivers Tub-Gal4 (1–4) and Act-Gal4 (5–8) and in the embryonic stages 11 (1, 2, 5 and 6) and 14 (5, 6, 7 and 8). Pole cells are labeled with anti-vasa (green) and the arrows indicate their location in late with respect to cells expressing the Tub-Gal4 (3) and Act-Gal4 (7). Arrowheads in 2 and 4 indicate Tub-Gal4 driver expression in mesodermal cells surrounding the migrating pole cells and the of transgenic protein expression under Act-Gal4 in these cells (6, 8). D. RNAi-mediated attenuation of endogenous D14-3-3ε protein upon ubiquitous expression under Tub-Gal4 in embryos. The western blot probed with the chicken anti-D14-3-3ε antibody is also probed with anti-β-tubulin which serves as a loading control and demonstrates drastic reduction of D14-3-3ε to levels nearly as low as those in homozygous mutant embryos. E. RNA-interference(RNAi)-mediated phenocopy in wild type embryos of the pole cell deficit in <i>D14-3-3ε^ex4</i>homozygotes. Driving the RNAi-mediated transgene with Tub-Gal4 reduced pole cell number in the gonads of stage 12–13 embryos nearly to that observed in mutant homozygotes (dark gray bar). Partial, but statistically significant (p<0.001, Dunnett's test) reduction compared to controls (open bar) was attained under NosVp16-Gal4, but no deficit was precipitated under the mesodermal driver How24B-Gal4.</p

The Zfh-1 transcription factor interacts with and is regulated by D14-3-3ε.

<p>A. The amino acid sequence of Zfh-1 containing the two 14-3-3 binding sites (red) revealed by <i>in silico</i> analysis. The second site containing the amino acids RSTSSP represents a perfect fit to the typical consensus 14-3-3 binding. B. The Zfh-1 distribution in control (1, 5), <i>D14-3-3ε^ex4</i> (2) and <i>D14-3-3ε^J2B10</i> (6) homozygous mutant embryos is shown in green, while pole cells are stained for Vasa (red). In the independent experiment shown in panel 3 (control) and panel 4 (homozygous <i>D14-3-3ε^ex4</i> mutant), the Zfh-1 distribution is shown in blue and pole cells marked with anti-Vasa in green. The inserts are magnifications of one of the gonads to better reveal the severe reduction or absence (arrowheads in 2, 4, 6) of Zfh-1 in mesodermally derived gonadal cells (arrows in 1, 3, 5). In 5 and 6 the embryos are oriented and images were captured such as to reveal the distribution of Zfh-1 in the ventral nerve chord and other mesodermal tissues. Note the scattered pole cells in panel 6. C. Phenocopy of the reduction or loss (arrowheads) of Zfh-1 in mesodermally derived gonadal cells upon RNAi-mediated D14-3-3ε abrogation with Tub-Gal4 (4) and to a lesser degree with NosVp16-Gal4 (6) in comparison with the distribution of these cells in control (1), <i>D14-3-3ε^ex4</i> heterozygous (2) and homozygous (3) mutant embryos.</p

<i>D14-3-3ε</i> mutants are sterile.

<p>The number of single crosses that yielded larvae (% Fertile) over the total number of animals crossed (# crossed) per genotype is reported. D14<i>-3-3ε^ex5</i> are used as controls since they have the same genetic background as the mutants and express normal amounts of the protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036702#pone.0036702-Acevedo1" target="_blank">[13]</a>.</p

Mesodermally derived gonadal cells are present in <i>D14-3-3ε</i> mutant embryos.

<p><i>In situ</i> hybridization for transcripts of the 412 retrotransposon as a marker of the mesodermal component of the gonad (blue) and immunohistochemical labeling of the D14-3-3ε protein (brown) shows that the somatic cells of the gonad are present in controls, heterozygous and homozygous D14-3-3ε mutant embryos as indicated at stage 9 (1, 4, 7), stage 11 (2, 5, 8) and stage 14 (3, 6, 9).</p

Reduction in the pole cell number in <i>D14-3-3ε</i> mutant embryos.

<p>A. Reduced fecundity of homozygous mutant females reflected in the number of eggs laid per single female per day. Homozygous <i>D14-3-3ε^ex4</i> and <i>D14-3-3ε^J2B10</i> lay very few eggs (1 or 2 per day), while <i>D14-3-3ε^ex4</i> heterozygotes also exhibit significantly reduced fecundity compared to controls. B. Wild type embryos of stage 5 (1–3) and stage 11–12 (4–6) stained with anti-ε (green) and a-vasa (red). D14-3-3ε is expressed inside and outside of the pole cells. C. Quantification of the number of pole cells that reach each embryonic gonad estimated from at least 12 different embryos. The numberof pole cells per gonad of heterozygous (light gray bars) and homozygous (dark gray bars) mutant embryos is significantly different (p<0.001, Dunnett's test) from that in controls (open bars). D. Pole cell distribution in 16–18 hr homozygous mutants and <i>D14-3-3ε^ex4</i>/TM3GFP heterozygotes. anti-GFP staining (green) was used to distinguish heterozygotes from homozygous mutant embryos. Unlike in similarly aged control embryos, pole cells in heterozygous and homozygous mutant embryos appear dispersed all over the embryo and result in the formation of gonads with fewer pole cells. C. Quantification of the total number of pole cells per gonad in control and homozygous mutant embryos (n≥12 each) carefully staged according to morphological criteria. Although consistently somewhat reduced compared to controls at stage 5, pole cell number in mutant embryos became highly significantly different (p<0.001, Dunnett's test) from that in controls at stage 8 and 11–12.</p