Human primordial germ cells (hPGCs), the precursors of sperm and eggs, originate during week 2-3 of early postimplantation development1. Using in vitro models of hPGC induction2–4, recent studies suggest striking mechanistic differences in specification of human and mouse PGCs5. This may partly be due to the divergence in their pluripotency networks, and early postimplantation development6–8. Since early human embryos are inaccessible for direct studies, we considered alternatives, including porcine embryos that, as in humans, develop as bilaminar embryonic discs. Here we show that porcine PGCs (pPGCs) originate from the posterior pre-primitive streak competent epiblast by sequential upregulation of SOX17 and BLIMP1 in response to WNT and BMP signalling. Together with human and monkey in vitro models simulating peri-gastrulation development, we show conserved principles for epiblast development for competency for PGC fate, followed by initiation of the epigenetic programme9–11, regulated by a balanced SOX17–BLIMP1 gene dosage. Our combinatorial approach using human, porcine and monkey in vivo and in vitro models, provides synthetic insights on early human development.

PRDM14 is a crucial regulator of mouse primordial germ cells (mPGCs), epigenetic reprogramming and pluripotency, but its role in the evolutionarily divergent regulatory network of human PGCs (hPGCs) remains unclear. Besides, a previous knockdown study indicated that PRDM14 might be dispensable for human germ cell fate. Here, we decided to use inducible degrons for a more rapid and comprehensive PRDM14 depletion. We show that PRDM14 loss results in significantly reduced specification efficiency and an aberrant transcriptome of hPGC-like cells (hPGCLCs) obtained in vitro from human embryonic stem cells (hESCs). Chromatin immunoprecipitation and transcriptomic analyses suggest that PRDM14 cooperates with TFAP2C and BLIMP1 to upregulate germ cell and pluripotency genes, while repressing WNT signalling and somatic markers. Notably, PRDM14 targets are not conserved between mouse and human, emphasising the divergent molecular mechanisms of PGC specification. The effectiveness of degrons for acute protein depletion is widely applicable in various developmental contexts.

Loss-of-function mutations of cyclic-AMP response element binding protein, binding protein (CREBBP) are prevalent in lymphoid malignancies. However, the tumour suppressor functions of CREBBP remain unclear. We demonstrate that loss of Crebbp in murine haematopoietic stem and progenitor cells (HSPC) leads to increased development of B-cell lymphomas. This is preceded by accumulation of hyperproliferative lymphoid progenitors with a defective DNA damage response (DDR) due to a failure to acetylate p53. We identify a pre-malignant lymphoma stem cell population with decreased H3K27ac, which undergoes transcriptional and genetic evolution due to the altered DDR, resulting in lymphomagenesis. Importantly, when Crebbp is lost later in lymphopoiesis, cellular abnormalities are lost and tumour generation attenuated. We also document that CREBBP mutations may occur in HSPC from patients with CREBBP-mutated lymphoma. These data suggest that earlier loss of Crebbp is advantageous for lymphoid-transformation and inform the cellular origins and subsequent evolution of lymphoid malignancies.

PRDM14 is a crucial regulator of mouse primordial germ cells (mPGC), epigenetic reprogramming and pluripotency, but its role in the evolutionarily divergent regulatory network of human PGCs (hPGCs) remains unclear. Besides, a previous knockdown study indicated that PRDM14 might be dispensable for human germ cell fate. Here, we decided to use inducible degrons for a more rapid and comprehensive PRDM14 depletion.We show that PRDM14 loss results in significantly reduced specification efficiency and an aberrant transcriptome of human PGC-like cells (hPGCLCs) obtained in vitro from human embryonic stem cells (hESCs). Chromatin immunoprecipitation and transcriptomic analyses suggest that PRDM14 cooperates with TFAP2C and BLIMP1 to upregulate germ cell and pluripotency genes, while repressing WNT signalling and somatic markers. Notably, PRDM14 targets are not conserved between mouse and human, emphasising the divergent molecular mechanisms of PGC specification. The effectiveness of degrons for acute protein depletion is widely applicable in various developmental contexts. MainGametes develop from primordial germ cells (PGCs), the embryonic precursors, which are apparently specified in ~week 2-3 (Wk2-3) human embryos 1, 2 . While the requirement for BMP and WNT signalling for PGC specification is conserved between mouse and human 3-5 , the gene regulatory network for hPGC fate has diverged 6 . Mouse PGCs (mPGCs) are specified by three core transcription factors (TFs): Prdm1 (encoding BLIMP1), Prdm14 and Tfap2c (encoding AP2γ) 7, 8 , among which PRDM14 plays a central role; loss of Prdm14 abrogates mPGC specification 9 , while its overexpression is sufficient to induce mPGC fate in vitro 8 .During mPGC specification, PRDM14 induces upregulation of germline-specific genes, assists BLIMP1mediated repression of somatic transcripts and initiates global epigenetic reprogramming 7, 8, 10, 11 . PRDM14 also has a significant role in preimplantaion development 12 , as well as pluripotency induction and maintenance in both mouse and human [13][14][15][16] . Indeed, PRDM14 knockdown in hESCs led to a decrease in OCT4 levels and elevated expression of lineage markers 13, 17, 18 .Despite its critical function in mPGC specification, the role of PRDM14 in hPGC development remains uncertain, due to its low and potentially cytoplasmic expression in gonadal hPGCs 3 . Furthermore, a partial PRDM14 knockdown suggested it might not be important for hPGC specification in vitro 19 , within the TF network for hPGC specification that has diverged significantly from mouse 1, 6, 20 . In particular, SOX17 is a key determinant of hPGC fate, acting upstream of BLIMP1 and TFAP2C 3 , but it is dispensable for mPGC 2 development 21, 22 . Understanding whether PRDM14 has a role in hPGC specification is critical towards gaining insights on the molecular divergence between mouse and human PGCs.An inducible system for PRDM14 loss of function during hPGCLC specification from hESCs is critical, since PRDM14 is also vital for hESC pluripotency 13 . According...

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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.

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