Dazl functions in maintenance of pluripotency and genetic and epigenetic programs of differentiation in mouse primordial germ cells in vivo and in vitro

Abstract

Background: Mammalian germ cells progress through a unique developmental program that encompasses proliferation and migration of the nascent primordial germ cell (PGC) population, reprogramming of nuclear DNA to reset imprinted gene expression, and differentiation of mature gametes. Little is known of the genes that regulate quantitative and qualitative aspects of early mammalian germ cell development both in vivo, and during differentiation of germ cells from mouse embryonic stem cells (mESCs) in vitro.

Methodology and principal findings: We used a transgenic mouse system that enabled isolation of small numbers of Oct4DeltaPE:GFP-positive germ cells in vivo, and following differentiation from mESCs in vitro, to uncover quantitate and qualitative phenotypes associated with the disruption of a single translational regulator, Dazl. We demonstrate that disruption of Dazl results in a post-migratory, pre-meiotic reduction in PGC number accompanied by aberrant expression of pluripotency genes and failure to erase and re-establish genomic imprints in isolated male and female PGCs, as well as subsequent defect in progression through meiosis. Moreover, the phenotypes observed in vivo were mirrored by those in vitro, with inability of isolated mutant PGCs to establish pluripotent EG (embryonic germ) cell lines and few residual Oct-4-expressing cells remaining after somatic differentiation of mESCs carrying a Dazl null mutation. Finally, we observed that even within undifferentiated mESCs, a nascent germ cell subpopulation exists that was effectively eliminated with ablation of Dazl.

Conclusions and significance: This report establishes the translational regulator Dazl as a component of pluripotency, genetic, and epigenetic programs at multiple time points of germ cell development in vivo and in vitro, and validates use of the ESC system to model and explore germ cell biology.

Figure 1. Requirement for Dazl in Embryonic Germ Cells.

(A) Total germ cell counts in E12.5 testis. (B) Total germ cell counts in E12.5 ovary. (C) Total germ cell counts in E14.5 testis. (D) Total germ cell counts in E14.5 ovary. (E) Total germ cell counts in E16.5 testis. (F) Total germ cell counts in E16.5 ovary. Number of germ cells is on the y-axis. ‘*’ denotes a statistically significant difference in germ cell count with the Dazl−/− mutant (p<0.05). ‘**’ denotes a statistically significant difference in germ cell count with the Dazl−/− mutant (p<0.001).

Figure 2. Aberrant Germ Cell Gene Expression in the Germ Cells of Dazl Mutants.

Expression profiles are shown in isolated germ cells at E12.5, E13.5, E14.5, and E15.5. Males are displayed in green boxes (A), (C), and (E). Females are displayed in purple boxes (B), (D), and (F). cKit, Oct4, Pum2 (Pumilio2), Stella, Nanos3, and Nanos2 represent genes, which are expressed beginning in migrating PGCs, while Vasa, Sycp3 (Synaptonemal complex protein 3), and Sycp1 (Synaptonemal complex protein 1) represent meiotic markers. Expression levels on the y-axis are normalized to the GAPDH housekeeping gene. Each bar on the graph represents average expression of sorted germ cells from the following number of embryos. Males: E12.5 +/+ (N = 3), +/− (N = 13), −/− (N = 5); E13.5 +/+ (N = 4), +/− (N = 8), −/− (N = 3); E14.5 +/+ (N = 2), +/− (N = 17), −/− (N = 3); E15.5 +/+ (N = 7), +/− (N = 10), −/− (N = 3). Females: E12.5 +/+ (N = 5), +/− (N = 7), −/− (N = 6); E13.5 +/+ (N = 3), +/− (N = 5), −/− (N = 4); E14.5 +/+ (N = 8), +/− (N = 15), −/− (N = 4); E15.5 +/+ (N = 7), +/− (N = 7), −/− (N = 4). Note the Y-axis on all graphs display different scales.

Figure 3. Methylation Marks at the H19 Imprinted Locus are disrupted in Germ Cells of Dazl Mutants.

(A) Schematic of expected methylation status of 15 CpG islands in region 1304–1726 of the H19 differentially methylated region (DMR) (GenBank acc. No. U19619) at different time points in the lifecycle of male germ cell development. Open circles denote an unmethylated status of a CpG, while filled circles denote a methylated CpG. (B) Results of bisulphite sequencing displaying an average percent methylation status (y-axis) of 60 clones per datapoint (20 clones per individual sample, 3 replicates for each genotype/age) for both E13.5 and post E16.5 GFP-positive germ cells isolated by FACS. Clones that were hemi-methylated are not shown. ‘***’ denotes a very significant difference (P<0.001) in unmethylated status relative to somatic. ‘*’ denotes a statistically significant difference (P<0.05) in methylated status relative to somatic. ‘‡ ‡ ‡’ denotes an extremely significant difference (P<0.001) in methylated status with E13.5 Dazl+/+. ‘†††’ denotes an extremely significant difference (P<0.001) in methylated status with E13.5 Dazl+/−.

Figure 4. Dazl is required for Derivation of Embryonic Germ Cells Lines from E12.5 PGCs.

Brightfield (BF) and GFP fluorescent images of Oct4ΔPE:GFP-positive PGCs during EG derivation on STO feeders from male and female E12.5 PGCs. (A–D) BF and (E–H) GFP expression of day 2 post-FACS wildtype and Dazl null PGCs. (I–L) BF of flattened, cobblestone like colonies at day 9 with no GFP expression (M–P). (Q–T) BF and (U–X) GFP expression in day 16 post-FACS cells, with only wildtype wells displaying EG colony formation (Q, S) and GFP expression (U, W).

Figure 5. Dazl is required for Meiotic Progression, but Not for Entry into Meiosis.

Results of analysis of 100 cells/genotype FACS isolated E17.5 Oct4ΔPE:GFP-positive cells, immunostained for SYCP3 (red) to indicate pachytene stage chromosomal alignment of Prophase I, and DAPI (blue) to label the nucleus. (A) Wildtype germ cell in pachytene. (B) Dazl−/− mutant germ cell in late zygotene/early pachytene. (C) Dazl−/− mutant germ cell displaying non-aligned SYCP3 staining. (D) Dazl−/− mutant germ cell displaying no SYCP3 staining. (E) Bar graph displaying percentage of cells per 100 cells/genotype showing appropriate SYCP3 alignment (A, B, red bars), and percentage of cells determined to be ambiguous or not showing SYPC3 alignment (yellow bars).

Figure 6. Dazl is required for in vitro Germ Cell Development.

Counts of Oct4ΔPE:GFP positive cells in wildtype (+/+), Dazl heterozygotes (+/−), and Dazl-null (−/−) mutant cell lines during a 35-day embryoid body differentiation. Percent GFP positive of all live cells counted is on the y-axis. ‘***’ denotes an extremely significant difference (P<0.001) in percent GFP positive cells relative to wildtype. ‘**’ denotes a very significant difference (P<0.01) in percent GFP positive cells relative to wildtype. ‘*’ denotes a significant difference (P<0.05) in percent GFP positive cells relative to wildtype.

Figure 7. Dazl Mutant mESCs Display Aberrant Gene Expression of Pluripotency and Germ Cell Markers During Differentiation.

Gene expression profiles are shown from GFP-positive cells, isolated by FACs, from day 0, 3, 7, 14, 21, 28, and 35 of differentiation. Expression is represented by heat map generated on DChip displaying a continuum with high expression depicted by red to low expression, in blue. The numeric values are from the formula 2∧^(−ΔCt), with GAPDH as the housekeeping gene, on a log scale. Genes are classified based by functional group as follows. Group A consisted of the pluripotency related genes Oct4, Sox2, Nanog, and TNAP (Tissue-non-specific alkaline phosphatase). Group B contains epigenetic regulators from the DNA methyltransferase family, Dnmt1, Dnmt3a, and Dnmt3b. Group C consists of the primordial germ cell specific markers Blimp1, Fragilis, Stella, cKit, Pumilio1 (Pum1), Pumilio2 (Pum2), and Nanos3; Group D contains the gonocyte markers Vasa, Stra8, Sycp3 (Synaptonemal Complex Component 3), GCNF, and GDF9. Group E contains the late germ cell markers Tdrd1 (Tudor domain containing 1), Tekt1 (tektin1), and Acrosin; Group F contains pro-apoptotic markers Casp6 (Caspase6) and Bax (BCL2-associated X), as well as the anti-apoptotic marker Bcl-2; Group G contains markers of autophagy including Atg5 (autophagy related 5) and Beclin1.

Figure 8. Dazl is required for Erasure and Re-establishment of H19 DMR Imprints.

Results of bisulphite sequencing on GFP-positive germ cells isolated by FACS, displaying an average percent methylation status (y-axis) and days of differentiation on the x-axis. Average values are from 40 clones per datapoint (20 clones per individual sample, 2 replicates for each genotype/timepoint).

Figure 9. Proposed Model Denotes Dazl as Link Between Pluripotency, Reprogramming and Differentiation in vivo and in vitro.

Green signifies Oct4ΔPE:GFP expression during the lifecycle of germ cell development. (A) mESCs derived from Dazl−/− blastocysts display a reduction in pluripotency and germ cell markers, and do not differentiate towards the germ cell lineage as seen in wildtype and Dazl heterozygous mESC lines. (B) Gonocytes in vivo and putative germ cell in vitro show a reduction in Oct4ΔPE:GFP -positive cells, and do not display stage specific genetic markers of germ cell development, or erasure of imprinting methylation marks at the H19 locus in males. (C) Gonia isolated from Dazl−/− mutant gonads show a significant reduction in Oct4ΔPE:GFP -positive cells and aberrant gene expression, indicating a loss of pluripotency, self-renewal, and germ cell potential. (D) Early stage oocytes and spermatocytes from Dazl−/− embryos display a significant loss of Oct4ΔPE:GFP -positive cells, aberrant gene expression, failure to re-establish imprinting methylation marks at the H19 locus in males, and failure progress though meiosis.