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. 2012 Jan 17:12:4.
doi: 10.1186/1471-213X-12-4.

Fine scale analysis of gene expression in Drosophila melanogaster gonads reveals Programmed cell death 4 promotes the differentiation of female germline stem cells

Amy C Cash  1 , Justen Andrews
Affiliations

Fine scale analysis of gene expression in Drosophila melanogaster gonads reveals Programmed cell death 4 promotes the differentiation of female germline stem cells

Amy C Cash et al. BMC Dev Biol. .

Abstract

Background: Germline stem cells (GSCs) are present in the gonads of Drosophila females and males, and their proper maintenance, as well as their correct differentiation, is essential for fertility and fecundity. The molecular characterization of factors involved in maintenance and differentiation is a major goal both in Drosophila and stem cell research. While genetic studies have identified many of these key factors, the use of genome-wide expression studies holds the potential to greatly increase our knowledge of these pathways.

Results: Here we report a genome-wide expression study that uses laser cutting microdissection to isolate germline stem cells, somatic niche cells, and early differentiating germ cells from female and male gonads. Analysis of this data, in association with two previously published genome-wide GSC data sets, revealed sets of candidate genes as putatively expressed in specific cell populations. Investigation of one of these genes, CG10990 the Drosophila ortholog of mammalian Programmed cell death 4 (Pdcd4), reveals expression in female and male germline stem cells and early differentiating daughter cells. Functional analysis demonstrates that while it is not essential for oogenesis or spermatogenesis, it does function to promote the differentiation of GSCs in females. Furthermore, in females, Pdcd4 genetically interacts with the key differentiation gene bag of marbles (bam) and the stem cell renewal factor eIF4A, suggesting a possible pathway for its function in differentiation.

Conclusions: We propose that Pdcd4 promotes the differentiation of GSC daughter cells by relieving the eIF4A-mediated inhibition of Bam.

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Figures

Figure 1
Figure 1
Gene expression in the germarium and apex of the testis. (A-D) Laser cutting microdissection. Photomicrographs show fixed and dehydrated ovariole (A, C) and testis (B, D), before (A, B) and after (C, D) the tissue was captured. Scale bars are 50 μm. The captured tissue was used for RNA isolation and subsequent expression profiling. (E) Differential gene expression in the germarium compared to the testis apex. Volcano plot shows M value (log2(testis apex/germarium) on the X-axis and statistical significance (-log10 adjusted p-values) on the Y-axis.
Figure 2
Figure 2
mRNA expression patterns of sex-biased candidate genes in ovaries and testes. (A-D) Light micrographs showing in situ hybridization to CG7777 (A), CG4404 (B), CG9975 (C) and Pp1-13C (D) in ovary (left panel) and testis (right panel) tissue. Scale bars are 50 μm.
Figure 3
Figure 3
Expression patterns of candidate genes in ovaries and testes. (A-B) Light micrographs showing in situ hybridization to CG9925 (A) and CG10990 (B) in ovary (left panel) and testis (right panel) tissue. Scale bars are 50 μm. (C-G) Immunofluorescence images of GFP protein traps [8,28] reporting expression of CG10990 (C), Tropomyosin I (Tm1; D), CG1600 (E), nervana 2 (nrv2; F) and GlcAT-S (G) in ovary (left panel) and testis (right panel) tissue. Immunostained proteins are listed at the bottom right of each panel. Immunofluorescence images are either confocal projections (C-E) or single Z-stack images (F-G). Scale bars are 25 μm.
Figure 4
Figure 4
Sequence similarity and genetic organization of Drosophila Programmed cell death 4 (Pdcd4, formally called CG10990). (A) Alignment of Pdcd4 protein sequences from H. sapiens (human) and D. melanogaster. The alignment of identical residues and chemically similar residues are indicated by asterisks and paired dots respectively. The extent of the two MA-3 protein domains are indicated by blue (N-terminal) and red (C-terminal) shading. Yellow shading indicates the extent of highly similar sequences outside the MA-3 domains. (B) Organization of the Pdcd4 locus. Exons are indicated by rectangles, the transcription start site is indicated by an arrow, open reading frame is shaded yellow, and sequences encoding the two MA-3 domains are shaded blue and red (corresponding to the shading in A). The locations of the transgene insertions are indicated by triangles. The CG10990-GFP protein trap line G00093 is referred to as Pdcd4G93. The Pdcd41 allele was induced by FRT mediated recombination between the insertions PBac{WH}CG10990f06108 and P{XP}CG10990d04016 and the region deleted in this mutation is indicated by a dashed line.
Figure 5
Figure 5
Loss-of-function Pdcd4 mutations result in an increase in the number of cells with a spherical spectrosome. (A-F) Immunofluorescence images showing single projections of confocal Z-stacks of germaria from 5-day-old females. Genotypes are listed in the bottom right corner of each panel. Immunostaining with anti-Hts (green channel in A-C and red channel in D-F) decorated the spectrosomes (ss) and fusomes (fs). Anti-Vasa (red in A-C) decorated germ cells, and the GFP protein trap Pdcd4G93 decorated the apical tips of the germaria. Scale bars are 10 μm. (A) Representative wild-type germarium showing the typical four cells with spherical spectrosomes. (B-F) Representative germaria from Pdcd4 mutants showing an increased number of cells with spherical spectrosomes. (G) Frequency histogram showing the mean number of cells containing spherical spectrosomes ± standard error for the indicated genotypes. Cells numbers were scored by scanning up and down through confocal Z-stacks. A minimum of 38 germaria from at least 19 females were scored for each genotype. ** indicates a p-value less than or equal to 1 × 10-16 for a comparison between the number of spectrosome-containing cells in the respective mutants vs wild-type.
Figure 6
Figure 6
Loss-of-function Pdcd4 mutations result in an increase in the number of pre-cystoblasts. (A-C) Immunofluorescence images showing germaria from 5-day-old females of the indicated genotypes. Immunostaining with pMad (red channel) was used as a marker for GSCs. Anti-Hts (green in A&B) decorated spectrosomes and fusomes and Pdcd4-GFP (the GFP protein trap Pdcd4G93, green channel in C) reported the expression of Pdcd4. Arrowheads indicate examples of high nuclear pMad expression. (D-E) Immunofluorescence images showing single projections of confocal Z-stacks of germaria from 5-day-old females of the indicated genotypes. Immunostaining for bam-GFP (green channel) was used as a marker for CBs, and immunostaining with anti-Hts (red channel) decorated spectrosomes and fusomes. Asterisks indicate the locations of apical tips of the germaria. Scale bars are 10 μm. (F) A schematic depicting the relationship between spectrosome/fusome morphology, pMad and Bam expression, and cell identity within the germarium. Pre-cystoblasts are defined as cells containing spherical spectrosomes that do not express pMad or Bam.
Figure 7
Figure 7
Pdcd4 interacts genetically with eIF4A and bam. (A-D, F-I) Immunofluorescence images showing single projections of confocal Z-stacks of germaria from 5-day-old females. Genotypes are listed in the bottom right corner of each panel. Hts is labeled in green and Vasa is labeled in red. Asterisks indicate the locations of apical tips of the germaria. Scale bars are 10 μm. (E, J) Frequency histograms showing the mean number of cells containing spherical spectrosomes ± standard error for the indicated genotypes. (E) The p-value shown corresponds to the comparison of the number of spectrosome-containing cells in Pdcd41/Pdcd41compared to Pdcd41/Pdcd41; eIF4A1013/+. A minimum of 34 germaria from at least 17 females were scored for each genotype. (J) The p-values shown correspond to the comparison of the number of spectrosome-containing cells in Pdcd41/Pdcd41; bamΔ86/+ compared to the control genotypes bamΔ86/+ and Pdcd41/Pdcd41. A minimum of 37 germaria from at least 18 females were scored for each genotype.
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