Genetic dissection of Nodal and Bmp signalling requirements during primordial germ cell development in mouse

Abstract

The essential roles played by Nodal and Bmp signalling during early mouse development have been extensively documented. Here we use conditional deletion strategies to investigate functional contributions made by Nodal, Bmp and Smad downstream effectors during primordial germ cell (PGC) development. We demonstrate that Nodal and its target gene Eomes provide early instructions during formation of the PGC lineage. We discover that Smad2 inactivation in the visceral endoderm results in increased numbers of PGCs due to an expansion of the PGC niche. Smad1 is required for specification, whereas in contrast Smad4 controls the maintenance and migration of PGCs. Additionally we find that beside Blimp1, down-regulated phospho-Smad159 levels also distinguishes PGCs from their somatic neighbours so that emerging PGCs become refractory to Bmp signalling that otherwise promotes mesodermal development in the posterior epiblast. Thus balanced Nodal/Bmp signalling cues regulate germ cell versus somatic cell fate decisions in the early posterior epiblast.

Fig. 1

Imbalanced Bmp/Nodal signalling and expansion of the PGC niche caused by Smad2 inactivation in the VE. a Representative images of p-Smad159 immunofluorescence (IF) staining of e5.5 embryos carrying the Blimp1-mVenus (BV) transgene, counter stained with DAPI. Dashed line indicates extent of proximal p-Smad159 staining in control embryos. The arrow indicates expanded p-Smad159 in the distal VE of Smad2ΔV embryos. b Whole-mount in situ hybridisation analysis of Gdf3 expression in control and Smad2ΔVE embryos at e5.5 and e6.0. c Nanog and Oct4 co-staining in e6.5 control and Smad2ΔVE embryos. d Brachyury IF in e6.5 control and Smad2ΔVE BV-expressing embryos. e Otx2 staining and BV expression at e6.5. All IF staining images were counter stained with DAPI. Scale bars = 100 μm

Fig. 2

Nodal and its downstream target Eomes regulate early stages of PGC development. a Reduced levels of Nanog expression in e5.5 Nodal null BV⁺ embryos. b Nodal null BV⁺ embryos display an expansion of p-Smad159 staining in the distal VE, as indicated by arrows. Arrowhead indicates lowered p-Smad159 staining within BV⁺ cells. c Expansion of the BV⁺ cell population in EomesΔEpi embryos at e7.5. d Nuclear Nanog staining in EomesΔEpi and control BV⁺ embryos at e7.5. e Analysis of Ap2γ, Sox2 and Stella in e7.5 control and EomesΔEpi BV⁺ cells. f Brachyury staining in control and EomesΔEpi BV⁺ e7.5 embryos. All IF images are counter stained with DAPI. Scale bars = 100 μm

Fig. 3

Smad1 is required for PGC specification whereas Smad4 controls PGC maintenance and migration. a Stella staining at e8.5 shows PGCs migrating along the hindgut endoderm in BV⁺ control but not Smad4ΔEpi embryos. b Oct4 staining in Smad1 null and control e7.5 BV⁺ embryos. c Single optical sections (2D) and z-stack projections (3D) showing Oct4 staining in the posterior side of Smad1ΔEpi and control BV⁺ embryos. Arrows indicate Oct4 and BV co-expressing cells. All IF images are counter stained with DAPI. Scale bars = 100 μm

Fig. 4

Abilities of mutant ES cells lacking Nodal/Bmp/Smad pathway components to undergo PGCLC differentiation. a Time-line and culture protocol used for PGCLC differentiation. b Stella IF staining of day 2 and day 4 BV⁺ PGCLC aggregates derived from the indicated genotypes, counter stained with DAPI. Scale bars = 100 μm. c Heatmap showing the log2 fold changes (log2FC) of Prdm1, Tfap2c and Hoxb1 expression, as judged by RT-qPCR, at day 2, 4 and 6 of PGCLC differentiation in control and mutant cells of the indicated genotypes, relative to EpiLCs

Fig. 5

PGCs down-regulate p-Smad159 to become refractory to the high-Bmp signalling environment. a p-Smad159 staining of BV⁺ cell populations. Arrowhead confirms e6.5 Smad2ΔVE embryos lack p-Smad159 in the VE, while arrows indicates weak activity in BV-expressing epiblast cells. b p-Smad159 staining in control and Smad2ΔVE BV⁺ embryos at e7.5. Arrows indicate loss of p-Smad159 staining in BV high-expressing cells. c p-Smad159 staining in BV⁺ wild-type d6 PGCLC aggregates. Arrows indicate BV⁺ cell populations lacking p-Smad159 reactivity. d p-Smad159 staining in control and Blimp1^−/− BV⁺ embryos at e7.5. Arrows indicate reduction of p-Smad159 reactivity in BV high-expressing cells in expanded panels. Arrowheads indicate retained p-Smad159 activity in BV⁺ cells in the Blimp1^−/− mutant embryo. All IF images are counter stained with DAPI. Scale bars = 100 μm. e Genome browser track view of GFP-ChIP wiggle plots from GFP-Blimp1 expressing PGCLC cultures at day (d)2 (blue) and d6 (red) showing enrichment of GFP-Blimp1 density at a Smad1 intronic region. The sequence below the GFP-Blimp1 ChIP site contains a Blimp1 binding motif indicated in red. The consensus Blimp1 binding motif previously identified by genome-wide ChIP-seq data sets is also indicated