Steel factor is an essential survival and proliferation factor for primordial germ cells (PGCs) during their migration in the early mouse embryo. PGCs arise during gastrulation, and migrate into the posterior endoderm that becomes the hindgut. Previous reports have suggested that PGCs become dependent on Steel factor when they colonize the hindgut. However, in the absence of a good marker for living PGCs, their behavior before hindgut colonization has not been previously studied. We report here the normal behavior of PGCs in live embryos before hindgut colonization, and the roles of Steel factor, using a reporter line in which GFP is driven by the promoter of the Stella gene, whose activation accompanies the initial specification of PGCs. We show first that PGCs are surrounded by Steel factor-expressing cells from their first appearance in the allantois to the time they enter the genital ridges. Second, fewer PGCs are found in the allantois in Steel-null embryos, but this is not due to a failure of PGC specification. Third, the analysis of cultured Steel-null early embryos shows that Steel factor is required for normal PGC motility, both in the allantois and in the hindgut. Germ cells migrate actively in the allantois, and move directionally from the allantois into the proximal epiblast. In the absence of Steel factor, caused by either null mutation or antibody blockade, PGC motility is dramatically decreased, but directionality is maintained, demonstrating a primary role for Steel factor in PGC motility. This was found both before and after colonization of the hindgut. These data,together with previously published data, show that PGCs are Steel factor dependent from their initial specification until they colonize the genital ridges, and suggest the existence of a `spatio-temporal niche' that travels with this important pluripotential cell population in the embryo.

Members of the bone morphogenetic protein (BMP) family play diverse roles in multiple developmental processes. However, in the mouse, mutations in many BMPs, BMP receptors and signaling components result in early embryonic lethality making it difficult to analyze the role of these factors during organogenesis or tissue homeostasis in the adult. To bypass this early lethality, we used an organ culture system to study the role of BMPs during primordial germ cell (PGC) migration. PGCs are the embryonic precursors of the sperm and eggs. BMPs induce formation of primordial germ cells within the proximal epiblast of embryonic day 7.5 (E7.5) mouse embryos. PGCs then migrate via the gut to arrive at the developing gonads by E10.5. Addition of BMP4 or the BMP-antagonist Noggin to transverse slices dissected from E9.5 embryos elevated PGC numbers or reduced PGC numbers, respectively. Noggin treatment also slowed and randomized PGC movements, resulting in a failure of PGCs to colonize the urogenital ridges (UGRs). Based on p-Smad1/5/8 staining, migratory PGCs do not respond to endogenous BMPs. Instead, the somatic cells of the urogenital ridges exhibit elevated p-Smad1/5/8 staining revealing active BMP signaling within the UGRs. Noggin treatment abrogated p-Smad staining within the UGRs and blocked localized expression of Kitl, a cytokine known to regulate the survival and motility of PGCs and Id1, a transcription factor expressed within the UGRs. We propose that BMP signaling regulates PGC migration by controlling gene expression within the somatic cells along the migration route and within the genital ridges.

ligand for FGFR2-IIIb, affects germ cell numbers. Loss of function, using a specific inhibitor of FGF signaling, causes increased apoptosis and inhibition of cell shape change in the migrating germ cells. Lastly, we confirm in vivo the effects seen in slice cultures in vitro, by examining germ cell positions and numbers in embryos carrying a loss-offunction allele of FGFR2-IIIb. In FGFR2-IIIb -/-embryos, germ cell migration is unaffected, but the numbers of germ cells are significantly reduced. These data show that a major role of FGF signaling through FGFR2-IIIb is to control germ cell numbers. The data do not discriminate between direct and indirect effects of FGF signaling on germ cells, and both may be involved.

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