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

The essential roles played by Nodal and Bmp signalling during early mouse development have been extensively documented. Here we used 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 discovered 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. Importantly we found that beside Blimp1, down-regulated phosphoSmad159 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.


Introduction
Primordial germ cells (PGCs), the precursors of sperm and eggs, are initially detectable in the early mouse embryo at around embryonic day (e) 6.25, prior to the onset of gastrulation 1 . Early fate mapping experiments revealed that the proximal posterior epiblast (PPE) gives rise to both the extra-embryonic mesoderm (ExM) and PGC cell populations 2 . The regulatory signals governing these cell fate decisions remain ill-defined. The PR domain containing zinc finger transcription factor Blimp1 (encoded by Prdm1) plays an essential role during PGC specification and expression at e6.25 identifies precursor PGCs (pre-PGCs) 3,4 . Commitment to the PGC lineage becomes evident slightly later between e6.75-e7.5 when pre-PGCs activate expression of germ cell markers such as Stella (Dppa3) and Ap2g (Tfap2c), concomitantly reactivate expression of pluripotency genes including Sox2 and repress somatic gene expression 1,5 . By e8.5, specified PGCs have migrated from the base of the allantois into the overlying endoderm, and subsequently migrate along the dorsal hindgut endoderm before homing to and colonising the genital ridges from e10.5 1 . Extensive chromatin remodelling and genome-wide epigenetic reprogramming occurs during migration 6  Smad9 (Smad159) respectively, that associate with the co-Smad Smad4, translocate to the nucleus and activate cell-type specific target gene expression 11 . Nodal signalling in the early epiblast is required for correct patterning of the VE, formation of the anterior-posterior (A-P) axis and also promotes development of the ExE 12 .
Together with Bmp signals from the ExE, Nodal initiates mesoderm induction and primitive streak formation (PS) within the PPE 12,13 . During gastrulation, graded Nodal signals pattern the PS 10 . Thus, highest levels of Nodal signalling are required for specification of anterior PS derivatives. Lowering Nodal expression levels in the PS, or depleting Smad23 in the epiblast results in failure to correctly specify the anterior definitive endoderm (DE) and the embryonic midline 14 .
In contrast Bmp/Smad159 signals promote the formation of ExM. Loss of Bmp4 from the ExE disrupts gastrulation and is associated with truncation of posterior structures including the allantois and yolk sac 13 . Both Smad1 and Smad5 null embryos also display ExM tissue defects 15,16 . Mutant embryos lacking Bmp4 or Bmp8b expression in the ExE or those lacking Bmp2 in the VE display compromised PGC development in the epiblast [17][18][19] . Bmp4 null embryos entirely lack mature PGCs, while Bmp4 heterozygous embryos contains reduced numbers of PGCs. The observation of reduced numbers of PCGs in embryos lacking either Smad1 or Smad5 15,16 , as well as in Smad1/5 double heterozygous embryos 20 , provides convincing evidence that dose-dependent Bmp signalling governs PGC specification.
The Wnt signalling pathway also regulates PGC development 21 . Wnt3 is normally induced in the posterior epiblast in response to Nodal/Bmp signalling 22 . Wnt3 mutants fail to gastrulate 23 and lack a detectable pre-PGC population 21 . Collectively, these findings demonstrate that patterning during gastrulation and PGC formation are co-ordinately regulated by dynamic signalling events. However, given the complex morphological disturbances observed in loss of function mutant embryos, it has proven difficult to further dissect the crosstalk between the embryonic and extraembryonic tissues. In particular since Nodal null embryos arrest prior to gastrulation 24 , any possible role of Nodal signalling during PGC specification remains to be explored.
Here we exploited tissue specific conditional deletion strategies to investigate functional contributions made by Nodal and Bmp signalling within the embryonic versus extra-embryonic tissues. We have directly assessed the distinct roles played by various signalling pathway components in the VE and the epiblast during formation of the PGC lineage. Collectively our experiments provide new insights into the signalling cues that cooperatively regulate the size of the founding pre-PGC population and govern the PGC developmental programme during induction, specification and migration at early post-implantation stages of mouse development.

Results
Smad2 is required in the VE to restrict Bmp signalling to the proximal region In Smad2 null embryos, lacking the anterior visceral endoderm (AVE) signalling centre, the epiblast adopts an exclusively ExM fate 12,25 . These mis-patterned embryos arrest at around e9.5. Here, to examine cell type specific Smad2 functional contributions within the VE, we crossed animals carrying a conditional Smad2 allele (Smad2CA) with heterozygous Smad2 +/mice carrying the Ttr-Cre transgene 26 (Supplemental Fig. S1A). As shown below, we found that the Smad2DVE embryos phenocopy the Smad2 -/embryos, strengthening the idea that the dramatic tissue disturbances observed in the null embryos predominantly reflect the loss of Smad2 signalling in the AVE.
Whole-mount in situ hybridisation experiments demonstrate that Bmp4 expression remains unchanged, whereas Smad2DVE embryos lack Bmp2 transcripts (Supplemental Fig. 1B). Thus, higher levels of p-Smad159 cannot simply be explained due to increased expression of Bmp ligands.
Antagonistic Bmp and Nodal signalling cues govern VE specification 9 .
However, the regulatory mechanisms that normally restrict p-Smad159 signalling to the proximal VE have yet to be fully characterised. The TGFb antagonist Gdf3, expressed in the epiblast and distal VE, directly antagonises Bmp4 activity 27,28 .
Moreover, selective mesoderm expansion in double homozygous embryos lacking both Gdf3 and the closely related ligand Gdf1 has been documented 29 . Here we observe in Smad2DVE embryos that Gdf3 expression is absent in the VE and reduced in the epiblast (Fig. 1B). Thus, up-regulated p-Smad159 activity in Smad2DVE embryos potentially reflects decreased Gdf3 expression levels.
Conditional inactivation of Smad2 in the visceral endoderm results in expansion of the PGC niche and increased number of PGCs Alkaline phosphatase (AP) positive PGC clusters were previously identified in e8.5 Smad2 -/embryos 15 . To evaluate PGC formation in Smad2DVE embryos we examined PGC marker gene expression. Nanog, normally reactivated in the early proximal epiblast 30 , is also strongly expressed in developing PGCs 31 . As expected in control embryos, we detected cells co-expressing Nanog and the pluripotency marker Oct4 in the PPE (Fig. 1C). Similarly, Smad2DVE e6.5 embryos contain Nanog/Oct4 double positive cells adjacent to the ExE, but these were located in a central position in the epiblast (Fig. 1C).
Next, to assess whether these cells correspond to pre-PGCs, we used the Blimp1-Venus (BV) BAC transgene that faithfully recapitulates Blimp1 expression in both the VE and the developing PGCs 32 . In Smad2DVE mutants expressing the BV transgene we detected BV-positive (BV + ) pre-PGCs that also co-express E-Cadherin (Supplemental Fig. 1C). As judged by immunohistochemistry these cells strongly express endogenous Blimp1 protein (Supplemental Fig. 1D). Interestingly in Smad2DVE embryos BV + pre-PGCs are initially detectable within the epiblast at e5.5, 12-18 hours before their appearance in wild type embryos (Fig. 1A). Slightly later at e6.5, the Smad2DVE proximal epiblast contains increased numbers of BV + cells as compared to control wild type embryos ( Fig. 1D & 1E). BV + cells in the proximal epiblast at e6.5 normally express Brachyury and retain E-cadherin expression, whereas the adjacent mesodermal cells also strongly express Brachyury but down-regulate E-cadherin expression (Fig. 1D, Supplemental Overall in Smad2DVE embryos we observe increased numbers of BV, Oct4, Nanog and Stella co-expressing PGCs surrounded by Brachyury-positive, E-Cadherinnegative mesodermal cells. Thus, Smad2 expression in the VE normally restricts expansion of the PGC niche so that only a small subset of posterior epiblast cells become allocated to a PGC fate. Next, to examine Smad2 functional contributions within the epiblast we made use of the Sox2-Cre deleter strain 33 . Stella + PGCs are readily detectable at e8.5 in Smad2DEpi embryos, suggesting that Smad2 is dispensable for PGC specification (Supplemental Fig. 2A). However, it seems likely that the closely related Smad3 effector, known to be robustly expressed in the epiblast 14 Fig. 2D). Interestingly, Nodal -/embryos display precocious induction of BV + epiblast cells (Figure 2A), which only weakly express Nanog ( Fig. 2A). As for Smad2DVE embryos, e5.5 Nodal -/mutants similarly exhibit increased levels of p-Smad159 staining in the distal VE (Fig. 2B). Moreover p-Smad159 is also detectable throughout the epiblast (Fig. 2B), strongly suggesting that Nodal functions in vivo to promote optimal levels of Nanog during pre-PGC development and also to spatially restrict the PGC niche.
The T-box transcription factor Eomes, acting downstream of Nodal signalling, plays essential roles during gastrulation 36,37 . Conditional deletion in the epiblast (EomesDEpi) results in defective epithelial to mesenchymal transition (EMT) and the failure of nascent mesoderm cells to down-regulate E-cadherin and exit the PS 36 .
Eomes is expressed in early pre-PGCs but becomes down-regulated by late streak stages (Supplemental Fig. 1E, Supplemental Fig. 2E). Interestingly, EomesDEpi e7.5 embryos contain an expanded epithelial-like BV + cell population (Fig. 2C) that also express endogenous Blimp1 protein (Supplemental Fig. 2F). Nanog expression is reactivated within the epiblast, however the BV + cell population in EomesDEpi embryos is largely Nanog negative (Fig. 2D). Moreover, at e7.5 BV + cells fail to activate the PGC markers Ap2g, Stella and Sox2 ( Eomes is also required to pattern the VE 38 . As shown in Supplemental Fig.   2F, EomesDVE embryos contain Blimp1 + epiblast cells. Thus, Eomes activity in the VE is non-essential for Blimp1 induction in the epiblast. Smad1 is required for specification whereas Smad4 controls the maintenance and migration of PGCs Smad4 in association with the receptor Smads, Smad159 activates Bmp target gene expression. As judged by AP staining, both Smad1 null and Smad4DEpi embryos lack PGCs at e8.5 15,39 . However, cooperative or possibly unique functional roles at distinct developmental stages during PGC lineage specification have yet to be examined. Initially to explore Smad4 functional requirements in the VE, we generated Smad4DVE embryos. We found at e7.5 that Blimp1-expressing PGCs are formed appropriately at the base of the allantois (Supplemental Fig. 3A). Similarly, Smad4 activity in the epiblast is non-essential for PGC specification. BV/Stella coexpressing cells are detectable on the posterior side of Smad4DEpi embryos at e7.5 (Supplemental Fig. 3B). However, at e8.5 these Stella/ BV + PGCs remain at the base of the allantois and fail to migrate towards the gut endoderm (Fig. 3A).
Additionally, we used a Blimp1-Cre deleter strain 40 to selectively eliminate Smad4 expression within the pre-PGC cell population (Smad4DPGC). Relatively few Stella expressing cells were present in e8.5 Smad4DPGC embryos (Supplemental Fig.   3C). Thus, Smad4 is dispensable for initial PGC specification but is required for PGC maintenance and/or migration.  5A). In contrast in e5.5 Smad2DVE embryos, p-Smad159 is detectable throughout the entire VE (Fig. 1A), but slightly later at e6.5 VE expression is downregulated (Fig. 5A). The mesodermal cell population surrounding BV + cells display strong p-Smad159 immunoreactivity at e6.5. However, the BV + cells themselves are devoid of p-Smad159 staining (Fig. 5A). Likewise, the BV high expressing cells in control e7.5 embryos, as well as the expanded numbers of BV + cells present in e7.5 Smad2DVE embryos, lack p-Smad159 reactivity (Fig. 5B).
These results demonstrate that down-regulated p-Smad159 levels distinguishes specified PGCs from their somatic neighbours and therefore specified PGCs do not respond to high levels of Bmp signalling. Similarly, EomesDEpi embryos display a high proportion of BV + epiblast cells lacking p-Smad159 activity (Supplemental Fig.   5A). Decreased p-Smad159 staining was also observed within the BV + cells generated during in vitro PGCLC differentiation (Fig. 5C). Thus, decreased responsiveness to Bmp signalling is a characteristic feature of PGC cell populations, both in in vitro and in vivo settings.
To directly assess whether Blimp1 expression influences Smad159 phosphorylation levels, we generated Blimp1 -/embryos carrying the BV-transgene.
BV + epiblast cells were visible at the base of the allantoic bud at e7.5 in Blimp1 -/embryos (Fig. 5D), strengthening the notion that Blimp1 functional activity is not required at early stages during pre-PGC formation. Interestingly we found that the BV high cell population partially retains p-Smad159 reactivity (Fig. 5D). To test whether loss of p-Smad159 within the maturing PGCs potentially reflects reduced levels of transcription, next we examined RNA-seq data sets 42,43  Here we describe a novel cellular mechanism that allows PGCs to insulate themselves and become refractory to Bmp signals. Thus, we found at e7.5 that BV + cells display reduced Smad159 phosphorylation levels. Moreover within d6 PGCLC aggregates p-Smad159 activity and BV expression are mutually exclusive. Imaging studies demonstrate that PGCs actively migrate as individual cells from the base of the allantois towards the overlying endoderm 54 . However, the molecular pathways guiding this initial directional migration remain ill-defined.
Besides intrinsic regulators of cell motility the posterior endoderm also plays a critical role guiding PGC migration. For example, in Sox17 mutant embryos that display defective endoderm formation, PGCs are formed, mature and initiate migration towards the endoderm but lack the ability to appropriately integrate to the endoderm 55 . Similarly we found here in Smad2DVE and Smad4DEpi embryos that PGCs are appropriately specified but fail to migrate towards the endoderm. PGCs normally extend long filopodia-like structures at e7.5 54 . In contrast in Smad2DVE embryos endoderm formation is compromised, filopodia-like structures are never observed and PGCs remain as clusters of epithelial-like cells. Likewise, Smad4DEpi embryos fail to form DE 39 . Migration defects could potentially reflect the intrinsic loss of Smad4-dependent PGC functional activities, or alternatively, defective endoderm formation potentially results in the failure to produce chemo-attractants. The extracellular matrix (ECM) also plays a crucial role in guiding PGC migration 56,57 . We previously reported that Smad4 controls ECM deposition in early developmental stages 58 . Further studies on the trophic signals emitted from the posterior endoderm and the potential role of Smad4-dependent signalling in ECM composition will be required to further define Bmp/Nodal Smad requirements during PGC migration.
An antagonistic relationship between Nodal and Bmp pathways has been described in a variety of developmental contexts including within the VE for establishing initial proximal-distal polarity, patterning of the PS, morphogenesis of the amnion and correct establishment of the left-right body plan 9,10,13,59,60 . The present experiments further demonstrate that these regulatory cues govern cell fate decisions in the posterior epiblast causing a discrete subpopulation to adopt a germ cell versus somatic cell fate. Importantly, we found that Blimp1 induction within the PGCs is associated with down-regulated pSmad159 levels and thus provide a cell intrinsic regulatory mechanism that allows this subpopulation to become nonresponsive to local Bmp signalling cues. Future experiments will be needed to further define dynamic cellular events controlling this developmental switch.

PGCLC cultures
EpiLCs and PGCLCs were induced as previously described 63 Table S3. RT-qPCR 250ng RNA was reverse transcribed to cDNA using Superscript III First Strand Synthesis System (Life Technologies, Cat#18080-051) and diluted to 100μl final volume in H2O (2.5ng/ul). 2μl (5ng) cDNA were used per RT-qPCR reaction in duplicate using SYBR-green kit (Qiagen, Cat#204143). Relative gene expression was normalised to Gapdh expression and calculated as 2 ΔΔ Ct. Average expression levels were calculated for two technical replicates from two independent cell lines per genotype. RT-qPCR primer sequences are listed in Table S4.

In situ hybridisation and immunohistochemistry
Whole-mount in situ hybridisation analysis was performed as before 50  Haematoxylin and eosin staining was performed as previously described 36 .