Abstract
Background: Potentially novel regulators of early human germline development have been identified recently, including SOX15 and SOX17, both of which show specific expression in human primordial germ cells. SOX17 is now known to be a critical specifier of human germ cell identity. There have been suggestions, as yet without evidence, that SOX15 might also play a prominent role. The early human germline is inaccessible for direct study, but an in vitro model of human primordial germ cell-like cell (hPGCLC) specification from human embryonic stem cells (hESCs) has been developed. This enables mechanistic study of human germ cell specification using genetic tools to manipulate the levels of SOX15 and SOX17 proteins to explore their roles in hPGCLC specification. Methods: SOX15 and SOX17 proteins were depleted during hPGCLC specification from hESCs using the auxin-inducible degron system, combined with a fluorescent reporter for tracking protein levels. Additionally, SOX15 protein was overexpressed using the ProteoTuner system. Protein-level expression changes were confirmed by immunofluorescence. The impact on hPGCLC specification efficiency was determined by flow cytometry at various time points. qPCR experiments were performed to determine some transcriptional effects of SOX15 perturbations. Results: We observed specific SOX15 expression in hPGCLCs by using immunofluorescence and flow cytometry analysis. Depletion of SOX15 had no significant effect on hPGCLC specification efficiency on day 4 after induction, but there was a significant and progressive decrease in hPGCLCs on days 6 and 8. By contrast, depletion of SOX17 completely abrogated hPGCLC specification. Furthermore, SOX15 overexpression resulted in a significant increase in hPGCLC fraction on day 8. qPCR analysis revealed a possible role for the germ cell and pluripotency regulator PRDM14 in compensating for changes to SOX15 protein levels. Conclusions: SOX17 is essential for hPGCLC specification, yet SOX15 is dispensable. However, SOX15 may have a role in maintaining germ cell identity.
Highlights
Novel regulators of early human germline development have been identified recently, including SOX15 and SOX17, both of which show specific expression in human primordial germ cells
Depletion of SOX15 during human primordial germ cell-like cell (hPGCLC) specification using auxin-inducible degron (AID) To determine the effects of SOX15 depletion on hPGCLC specification, a homozygous knock-in cell line with a C-terminal AID-Venus tag on SOX15 was generated by a strategy similar to one previously used for PRDM148
The neighboring somatic lineages were almost completely SOX15-negative: there were a small number of SOX15-positive, OCT4/BLIMP1 negative cells on days 2–4, but by day 5, SOX15 expression was completely confined to hPGCLCs
Summary
Novel regulators of early human germline development have been identified recently, including SOX15 and SOX17, both of which show specific expression in human primordial germ cells. The early human germline is inaccessible for direct study, but an in vitro model of human primordial germ cell-like cell (hPGCLC) specification from human embryonic stem cells (hESCs) has been developed. This enables mechanistic study of human germ cell specification using genetic tools to manipulate the levels of SOX15 and SOX17 proteins to explore their roles in hPGCLC specification. Results: We observed specific SOX15 expression in hPGCLCs by using immunofluorescence and flow cytometry analysis. QPCR analysis revealed a possible role for the germ cell and pluripotency regulator PRDM14 in compensating for changes to SOX15 protein levels. SOX15 may have a role in maintaining germ cell identity
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