Abstract
The cell intrinsic programming that regulates mammalian primordial germ cell (PGC) development in the pre-gonadal stage is challenging to investigate. To overcome this we created a transgene-free method for generating PGCs in vitro (iPGCs) from mouse embryonic stem cells (ESCs). Using labeling for SSEA1 and cKit, two cell surface molecules used previously to isolate presumptive iPGCs, we show that not all SSEA1+/cKit+ double positive cells exhibit a PGC identity. Instead, we determined that selecting for cKitbright cells within the SSEA1+ fraction significantly enriches for the putative iPGC population. Single cell analysis comparing SSEA1+/cKitbright iPGCs to ESCs and embryonic PGCs demonstrates that 97% of single iPGCs co-express PGC signature genes Blimp1, Stella, Dnd1, Prdm14 and Dazl at similar levels to e9.5–10.5 PGCs, whereas 90% of single mouse ESC do not co-express PGC signature genes. For the 10% of ESCs that co-express PGC signature genes, the levels are significantly lower than iPGCs. Microarray analysis shows that iPGCs are transcriptionally distinct from ESCs and repress gene ontology groups associated with mesoderm and heart development. At the level of chromatin, iPGCs contain 5-methyl cytosine bases in their DNA at imprinted and non-imprinted loci, and are enriched in histone H3 lysine 27 trimethylation, yet do not have detectable levels of Mvh protein, consistent with a Blimp1-positive pre-gonadal PGC identity. In order to determine whether iPGC formation is dependent upon Blimp1, we generated Blimp1 null ESCs and found that loss of Blimp1 significantly depletes SSEA1/cKitbright iPGCs. Taken together, the generation of Blimp1-positive iPGCs from ESCs constitutes a robust model for examining cell-intrinsic regulation of PGCs during the Blimp1-positive stage of development.
Highlights
The molecular events that regulate cell fate decisions in postimplantation mammalian embryonic development are largely uncharacterized due to the challenge in identifying and isolating small populations of specific precursor cells that are developmentally transient in the early embryo
To generate a transgene-free method of in vitro PGCs (iPGCs) differentiation, we correlated expression of Oct4 protein in day 6 embryoid bodies (EBs) derived from V6.5 embryonic stem cells (ESCs) with the cell surface marker Stage Specific Embryonic Antigen 1 (SSEA1)
SSEA1 is highly expressed on B-lymphocyte induced maturation protein 1 (Blimp1)-positive stage primordial germ cell (PGC) and PGC precursors derived from epiblast stem cells [24,25]
Summary
The molecular events that regulate cell fate decisions in postimplantation mammalian embryonic development are largely uncharacterized due to the challenge in identifying and isolating small populations of specific precursor cells that are developmentally transient in the early embryo. The PGCs migrate out of the allantois and into the embryonic hindgut endoderm at e8.0–8.5 where they continue to proliferate and begin to accumulate nuclear histone H3 lysine 27 trimethylation (H3K27m3) [3]. By e10.5–e11.0 a single embryo has approximately 1,000–2,000 PGCs, which exit the hindgut and begin colonization of the indifferent gonad and express Mvh protein [3,4,5]. One of the most characterized regulators of PGC fate is the transcriptional repressor B-lymphocyte induced maturation protein 1 (Blimp1), the transcriptional product of the PRD1-BF1 and RIZ (PR) domain 1 (Prdm1) gene. Blimp expression is detected in epiblast-derived PGCs and persists until e11.5, when PGCs have colonized the gonad [2,6]. Direct binding of Blimp at the Hoxb locus in PGCs has not been demonstrated
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