Cell Death & Differentiation (2021) 28:2296–2299 https://doi.org/10.1038/s41418-021-00812-0 COMMENT Natural selection at the cellular level: insights from male germ cell differentiation 1 1 Daniel H. Nguyen ● Diana J. Laird Received: 9 February 2021 / Revised: 20 May 2021 / Accepted: 20 May 2021 / Published online: 2 June 2021 © The Author(s) 2021. This article is published with open access Waddington’s concept of differentiation as an epigenetic The germline is a fascinating context for investigating landscape provides an enduring metaphor visualizing the the consequences of heterogeneity on differentiation and options faced by stem and progenitor cells. However, cell fate. As fetal germ cells establish the gametes, their increasing understanding of cellular heterogeneity poses population dynamics can greatly influence inheritance. The new questions about the identities and behaviors of the cells conflict between diversity and orderly differentiation looms beginning this process. We now recognize a greater diver- centrally over germline development. In mouse embryos, sity of initial states for individual progenitor cells, which germ cells undertake an epic journey, from specification may affect their trajectories and disrupt progress entirely. through sex differentiation, replete with opportunities for 1234567890();,: 1234567890();,: Here, we consider how developmental selection occurs heterogeneity to develop and be assessed. Notably, an when heterogeneity in differentiating progenitors produces excess of germ cells is produced and then pruned by pro- divergent cellular outcomes of survival versus elimination. grammed cell death [5]. This occurs across diverse species Heterogeneity is a fundamental property of biological regardless of sex, suggesting that differential fitness and systems. Individual cell properties like location or cell cycle elimination are critical. can yield vastly different behaviors, and single cell analysis Deciduous progenitor cells feature in many develop- offers deeper characterization of transcriptional and genetic mental settings, including gastrulation and neural develop- diversity. Heterogeneity in progenitors can wield lasting ment [6], and may be strategic for resolving heterogeneity impacts on the composition of lineages. Variable Myc during differentiation. In the germline, the basis for this expression in epiblast cells generates selection for Myc-high potential selection is obtuse, although recent studies illu- populations [1] and similar enrichment occurs based on minate how variation emerges and affects differentiation at differential Hippo signaling [2]. Heterogeneous expression a more granular population level. Here, we focus on the of P53 [3] or mTOR [4] forms the basis for clonal expan- male lineage in the fetal period, as developmental diversi- sion in hematopoietic stem cells and early mouse embryos. fication and elimination during fetal oogenesis is more In these examples, differentiation from heterogeneous pro- complex, involving meiotic entry and asymmetric nurse genitors involves cell competition and active elimination cell-oocyte cytoplasmic transfer [7]. based on fitness. Importantly, competitive states are heri- In mouse fetal testes, germ cells undergo a stereotypical table across cell division, evoking principles similar to period of apoptosis shortly after male differentiation. This Darwinian selection. Moreover, evidence of developmental event represents a developmentally-programmed selective failure and selection challenges the notion that differentia- barrier, which we examined by characterizing apoptotic tion follows a robust and stable trajectory, especially at the germ cells and germ cell diversity [8]. Multicolor lineage individual cell level. labeling revealed that dying germ cells were clonally related and shared a common cellular ancestry. This indicated that heritable factors determine distinct apoptotic fates among germ cell subpopulations. It should be noted that germ cells * Diana J. Laird at this stage are connected by intercellular bridges that [email protected] permit cytoplasmic exchange. These conserved germ cell structures are mitotically-produced and can reinforce clonal 1 Department of Obstetrics, Gynecology and Reproductive Science, behavior through sharing of cytoplasmic factors between Center for Reproductive Sciences, Eli and Edythe Broad Center Drosophila for Regeneration Medicine and Stem Cell Research, UCSF, daughter cells [9]. In testes, bridges can syn- San Francisco, CA, USA chronize apoptosis in response to DNA damage even when Natural selection at the cellular level: insights from male germ cell differentiation 2297 Fig. 1 Instability during differentiation provides an opportunity forming in the landscape. The adjacent presence of an apoptotic for assessment of individual cellular phenotypes present in a het- attractor state alongside a differentiation attractor state creates an erogeneous differentiating population. In the above figure, a pro- unstable equilibrium (box). Differentiating cells proceed, balancing genitor cell prior to differentiation is at a multipotent state (1). along this ridge until individual cellular phenotypes exert sufficient Acquisition of epimutations or other forms of heritable cellular attraction toward either state (4), causing trajectories to diverge. Het- diversity, as represented by dotted or dashed outlines (2), can alter the erogeneous cells are consequently sorted into differentiated or apop- propensities of descendent cells toward attractor states. At the onset of totic fates, with apoptotic cells terminating progress in an inescapable differentiation (3), attractor states emerge, represented by depressions well (5). only a subset of germ cells suffer insults [10]. While such What disrupts sex differentiation in certain germ cells to connectivity resembles clonal behavior, we distinguished produce clonal elimination? An interesting link emerges that clonal apoptosis in mouse fetal germ cells persists in from evidence for epigenetic control of sex differentiation. mutants incapable of forming bridges—thereby sub- Germ cell sex differentiation can be modulated by inter- stantiating that apoptotic fate is heritable and intrinsic. fering with DNA demethylation machinery, including To ascertain the basis of this apoptotic fate, we identified Dnmt1 and Tet1; these regulate the expression of a a male apoptosis-prone subpopulation characterized by restricted set of germline reprogramming-responsive (GRR) elevated expression of pro-apoptotic genes such as Bad and genes via promoter methylation [13]. Timely, efficient male p53. This transcriptional signature was associated with differentiation relies upon activation of GRR genes by DNA persistent expression of genes from a sex-undifferentiated demethylation and protection from aberrant methylation. state, whereas a reciprocal population was demarcated by We found that GRRs were hypermethylated and under- male-differentiated genes and downregulation of the apop- transcribed in apoptosis-prone germ cells, which likely totic signature. Together, these transcriptional profiles hinders male differentiation. What might cause aberrant suggest a dichotomy between germ cell death and methylation? Early germ cell development involves exten- differentiation. sive resetting of the epigenome for totipotency and germ Tethering apoptotic resistance to successful differentia- cell and sex-specific identities. Such intense epigenetic tion may function as a quality-control mechanism. Pros- remodeling in an acute period risks erroneous demethyla- permatogonial differentiation genes such as Nanos2 also tion. These “epimutations” are far more likely to arise in promote survival, as evidenced by elevated apoptosis in progenitors than de novo genetic mutations [14] and could Nanos2−/− [11]. Preceding male differentiation, apoptotic potentially provide a heritable, differentiation-deficient transcripts remain high in germ cells but diminish at the phenotype. onset of Nanos2 [8]. Thus, apoptosis can act as a differ- Epigenetic regulation of differentiation is commonplace in entiation fail-safe to eliminate aberrant or suboptimally developing tissues including blood, skin, and intestine [15]. differentiated cells. Coordinating apoptosis with differ- We predict that epigenetically-regulated gene sets analogous entiation in progenitors may prevent multipotent cells like to GRRs control differentiation in other contexts. These loci early germ cells from tumorogenesis due to inappropriate could therefore be epimutation hotspots to generate hetero- differentiation. Similarly tight control over differentiation geneous differentiation potential. Single-cell methylomics may direct removal of aneuploid progenitors in early promises exciting insights into the epigenetic diversity of embryos to prevent aberrant development and cancer [12]. subpopulations and their varied fates during differentiation. 2298 D. H. Nguyen, D. J. Laird With evidence that aberrant epigenetic regulation cells. How does cellular variation evolve over time? What underlies the choice between differentiation and apoptosis strategies guide selection? Clonal tracing and single cell in male-differentiating germ cells, how might this influence analysis techniques will be powerful tools for answering these germ cell diversity? The heritable nature of epimutations questions and examining tissue biology through a new lens of predicts that certain clones will predominate the pool of cellular population dynamics. prospermatogonia; indeed,