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Plants alternate between diploid sporophyte and haploid gametophyte generations 1 . In mosses, which retain features of ancestral land plants, the gametophyte is dominant and has an independent existence. However, in flowering plants the gametophyte has undergone evolutionary reduction to just a few cells enclosed within the sporophyte. The gametophyte is thought to retain genetic control of its development even after reduction 2 . Here we show that male gametophyte development in Arabidopsis , long considered to be autonomous, is also under genetic control of the sporophyte via a repressive mechanism that includes large-scale regulation of protein turnover. We identify an Arabidopsis gene SHUKR as an inhibitor of male gametic gene expression. SHUKR is unrelated to proteins of known function and acts sporophytically in meiosis to control gametophyte development by negatively regulating expression of a large set of genes specific to postmeiotic gametogenesis. This control emerged late in evolution as SHUKR homologues are found only in eudicots. We show that SHUKR is rapidly evolving under positive selection, suggesting that variation in control of protein turnover during male gametogenesis has played an important role in evolution within eudicots. Ancestral land plants had a free-living gametophyte, but in flowering plants the gametophyte develops within the sporophyte. This study shows that male gametophyte development in Arabidopsis is directed by the sporophyte through repression of gametogenesis genes.

Aside from inheriting half of the genome of each of our parents, we are born with a small number of novel mutations that occurred during gametogenesis and postzygotically. Recent genome and exome sequencing studies of parent–offspring trios have provided the first insights into the number and distribution of these de novo mutations in health and disease, pointing to risk factors that increase their number in the offspring. De novo mutations have been shown to be a major cause of severe early-onset genetic disorders such as intellectual disability, autism spectrum disorder, and other developmental diseases. In fact, the occurrence of novel mutations in each generation explains why these reproductively lethal disorders continue to occur in our population. Recent studies have also shown that de novo mutations are predominantly of paternal origin and that their number increases with advanced paternal age. Here, we review the recent literature on de novo mutations, covering their detection, biological characterization, and medical impact.

Key Points Recent studies have shown that RNA-directed DNA methylation (RdDM) in Arabidopsis thaliana not only requires the production of 24-nucleotide small interfering RNAs (siRNAs) and the de novo DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) but also requires the production of RNA polymerase V (Pol V)-dependent intergenic non-coding (IGN) transcripts. Two crucial RdDM components, ARGONAUTE 4 (AGO4) and SUPPRESSOR OF TY INSERTION 5-LIKE (SPT5L), interact with Pol V-dependent transcripts, which suggests that they serve as a scaffold for the recruitment of the RdDM machinery. This process ultimately leads to DNA methylation and silencing at loci that produce siRNAs and IGN transcripts. Analyses of transposon expression and DNA methylation patterns in pollen grains and in embryo and endosperm tissues, respectively, suggest that genome-wide decreases in DNA methylation occur during male and female gametogenesis in A. thaliana , which might facilitate enhanced RdDM and transposon silencing in the sperm and egg cells by an unknown mechanism. Biochemical purification of DNA methyltransferase 3-like (DNMT3L) revealed an interaction between DNMT3L and unmethylated histone 3 lysine 4 (H3K4) tails. As DNMT3L also interacts with the DNMT3A de novo methyltransferase and because H3K4 methylation is anticorrelated with DNA methylation, a model has been proposed in which DNMT3L interaction with unmethylated H3K4 tails targets de novo methylation. Several recent findings suggest that Piwi-interacting RNAs (piRNAs) target de novo DNA methylation at transposons and other repetitive elements of the genome during male gametogenesis in mammals. piRNA populations isolated early in development were found to be enriched for such sequence elements, and mutations in MILI — a Piwi-clade Ago protein that binds piRNAs — showed DNA methylation defects at the stage of development at which de novo methylation is observed. Characterization of the Piwi clade of Ago proteins revealed the presence of symmetrical dimethylarginine modifications on several family members from Xenopus laevis , Drosophila melanogaster and mice. Tudor domains are known to interact with this modification, and Tudor domain-containing 1 (TDRD1), a protein with several Tudor domains, interacts with MILI and is required for DNA methylation and transposon silencing. In mammals, DNA methylation is maintained during DNA replication through the activity of DNMT1, which catalyses the methylation of hemimethylated CG sites in newly synthesized DNA. This activity depends heavily on the presence of ubiquitin-like plant homeodomain and RING finger domain 1 (UHRF1), a protein that specifically recognizes hemimethylated DNA and is proposed to recruit DNMT1 to chromatin. In A. thaliana , 5-methylcytosine DNA glycosylases and the base excision repair pathway catalyse active DNA demethylation during female gametogenesis and in vegetative plant tissues. Demethylation during gametogenesis is required for imprinting, whereas demethylation in vegetative tissues is proposed to combat robust DNA methylation by the RdDM pathway. In zebrafish there is evidence for an active DNA demethylation pathway that also involves DNA glycosylase activity and the base excision repair pathway. However, unlike in A. thaliana , in which methylated cytosines are directly recognized and removed, in zebrafish the methylated cytosine is first deaminated by the activation-induced cytosine deaminase (Aid)/apolipoprotein B mRNA-editing enzyme (Apobec) family of proteins, generating a G/T mismatch. This base is then removed by a thymine DNA glycosylase in what seems to be a tightly coupled manner. In mammals, mechanisms for active DNA demethylation remain unclear. However, an early model proposed a mechanism similar to that recently demonstrated in zebrafish, and a recent study showing that AID is necessary for the reduced levels of DNA methylation normally observed in primordial germ cells also supports this hypothesis. The discovery of the 5-hydroxymethylcytosine modification in certain mammalian cell types led to speculation that this modification could be a substrate for active DNA methylation. Recent studies have increased our understanding of how DNA methylation is accurately targeted, maintained and modified. Mechanistic similarities between plants and animals have emerged, including key roles for small RNAs, proteins with domains that bind methylated DNA and DNA glycosylases. Cytosine DNA methylation is a stable epigenetic mark that is crucial for diverse biological processes, including gene and transposon silencing, imprinting and X chromosome inactivation. Recent findings in plants and animals have greatly increased our understanding of the pathways used to accurately target, maintain and modify patterns of DNA methylation and have revealed unanticipated mechanistic similarities between these organisms. Key roles have emerged for small RNAs, proteins with domains that bind methylated DNA and DNA glycosylases in these processes. Drawing on insights from both plants and animals should deepen our understanding of the regulation and biological significance of DNA methylation.

Clonal animals do not sequester a germ line during embryogenesis. Instead, they have adult stem cells that contribute to somatic tissues or gametes. How germ fate is induced in these animals, and whether this process is related to bilaterian embryonic germline induction, is unknown. We show that transcription factor AP2 (Tfap2), a regulator of mammalian germ lines, acts to commit adult stem cells, known as i-cells, to the germ cell fate in the clonal cnidarian Hydractinia symbiolongicarpus. Tfap2 mutants lacked germ cells and gonads. Transplanted wild-type cells rescued gonad development but not germ cell induction in Tfap2 mutants. Forced expression of Tfap2 in i-cells converted them to germ cells. Therefore, Tfap2 is a regulator of germ cell commitment across germ line–sequestering and germ line–nonsequestering animals.

Malaria-causing parasites ( Plasmodium ) have complex life histories in the tissues of humans. For the most part, the parasites focus their efforts on replication within the human host cells. However, occasionally, some replicating cells release gametes into the bloodstream, which are picked up by biting mosquitoes. Filarsky et al. discovered that the Plasmodium parasite keeps the production of gametes under tight epigenetic control using heterochromatin protein 1 (HP1). Plasmodium gametocytogenesis is initiated when HP1 is evicted from upstream of gamete-specific genes by gametocyte development 1 (GDV1) protein. GDV1 is in turn regulated by its antisense RNA. What triggers GDV1 expression remains unclear. Elucidating this pathway could provide a target for interrupting malaria transmission. Science , this issue p. 1259 Plasmodium replication is interrupted for gamete production by eviction of a heterochromatin binding protein upstream of the relevant genes. Malaria is caused by Plasmodium parasites that proliferate in the bloodstream. During each replication cycle, some parasites differentiate into gametocytes, the only forms able to infect the mosquito vector and transmit malaria. Sexual commitment is triggered by activation of AP2-G, the master transcriptional regulator of gametocytogenesis. Heterochromatin protein 1 (HP1)–dependent silencing of ap2-g prevents sexual conversion in proliferating parasites. In this study, we identified Plasmodium falciparum gametocyte development 1 (GDV1) as an upstream activator of sexual commitment. We found that GDV1 targeted heterochromatin and triggered HP1 eviction, thus derepressing ap2-g . Expression of GDV1 was responsive to environmental triggers of sexual conversion and controlled via a gdv1 antisense RNA. Hence, GDV1 appears to act as an effector protein that induces sexual differentiation by antagonizing HP1-dependent gene silencing.

Protozoan parasites of the genus Plasmodium cause malaria and involve infection of multiple hosts and cell types during the life cycle. Producing sexually fit gametocytes is essential for transmitting the Plasmodium parasite into an anopheline mosquito vector. After the uptake of malaria parasites, male gametocytes undergo three rounds of DNA replication to produce eight nucleated flagellar gametes. Here, we report that the actin-like proteins Alp5a and Alp5b are involved in DNA segregation during male gametogenesis. The Plasmodium -specific Alp5a and Alp5b can be superimposed on human Arp2 and Arp3, localize to the nucleus, and interact with each other. Alp5a and Alp5b are individually dispensable for the development of P. berghei blood stages, but are simultaneously indispensable for parasite viability. Consistent with genetic studies, the inhibitory activity of the Arp2/3 complex inhibitor in Plasmodium supports an essential role for this complex during the blood stage. Deletion of Alp5a or Alp5b had no impact on actin nucleation, parasite growth, or gametocytemia during the blood stage. The knockout parasites were able to invade the mosquito midgut and form oocysts; however, these oocysts were significantly smaller in size and failed to mature, ultimately leading to their death. Genetic crosses revealed defects in male gamete integrity. We found that the reduced oocyst development was due to impaired DNA segregation during male gametogenesis. Our study provides molecular insights into the fundamental requirements of the Alps in Plasmodium , which are essential for malaria transmission.

Whole genome sequencing (WGS) studies have estimated the human germline mutation rate per basepair per generation (~1.2 × 10 −8 ) to be higher than in mice (3.5–5.4 × 10 −9 ). In humans, most germline mutations are paternal in origin and numbers of mutations per offspring increase with paternal and maternal age. Here we estimate germline mutation rates and spectra in six multi-sibling mouse pedigrees and compare to three multi-sibling human pedigrees. In both species we observe a paternal mutation bias, a parental age effect, and a highly mutagenic first cell division contributing to the embryo. We also observe differences between species in mutation spectra, in mutation rates per cell division, and in the parental bias of mutations in early embryogenesis. These differences between species likely result from both species-specific differences in cellular genealogies of the germline, as well as biological differences within the same stage of embryogenesis or gametogenesis. Estimates of mutation rates differ between species. Here, Lindsay et al. perform side-by-side analyses of germline mutation rates using multi-sibling mouse and human pedigrees and find different mutation rates between species, also stratified by sex and temporal stage of mutation acquisition.

Key Points Malaria is responsible for almost 600,000 deaths each year; a major challenge in eradicating this disease has been the lack of drugs targeting malaria parasites at a transmissible stage of the life cycle. Conversion of asexual blood-stage malaria parasites to sexual gametocytes is essential for transmission from the human host to the mosquito vector. This process occurs in only a small proportion of cells. Although several studies have investigated the transcriptional changes that occur during sexual development and attempted to identify genes that may be involved, the molecular basis of sexual conversion has been elusive. Recent work has identified an apicomplexan-specific transcription factor as a key regulator of commitment to gametocyte development. Epigenetic and post-transcriptional mechanisms are also crucial in regulating sexual development. The molecular events that integrate the signals that trigger commitment remain unknown and deserve further investigation. For transmission from mammalian host to mosquito vector, blood-stage malaria parasites must convert from an asexual form to the sexual gametocyte through a process known as gametocytogenesis. In this Review, Josling and Llinás discuss recent studies that have begun to elucidate the molecular basis of this process, in particular the factors involved in commitment to gametocytogenesis. Malaria is a devastating infectious disease that is caused by blood-borne apicomplexan parasites of the genus Plasmodium . These pathogens have a complex lifecycle, which includes development in the anopheline mosquito vector and in the liver and red blood cells of mammalian hosts, a process which takes days to weeks, depending on the Plasmodium species. Productive transmission between the mammalian host and the mosquito requires transitioning between asexual and sexual forms of the parasite. Blood- stage parasites replicate cyclically and are mostly asexual, although a small fraction of these convert into male and female sexual forms (gametocytes) in each reproductive cycle. Despite many years of investigation, the molecular processes that elicit sexual differentiation have remained largely unknown. In this Review, we highlight several important recent discoveries that have identified epigenetic factors and specific transcriptional regulators of gametocyte commitment and development, providing crucial insights into this obligate cellular differentiation process.

Cytoplasmic polyadenylation plays a vital role in gametogenesis; however, the participating enzymes and substrates in mammals remain unclear. Using knockout and knock-in mouse models, we describe the essential role of four TENT5 poly(A) polymerases in mouse fertility and gametogenesis. TENT5B and TENT5C play crucial yet redundant roles in oogenesis, with the double knockout of both genes leading to oocyte degeneration. Additionally, TENT5B-GFP knock-in females display a gain-of-function infertility effect, with multiple chromosomal aberrations in ovulated oocytes. TENT5C and TENT5D both regulate different stages of spermatogenesis, as shown by the sterility in males following the knockout of either gene. Finally, Tent5a knockout substantially lowers fertility, although the underlying mechanism is not directly related to gametogenesis. Through direct RNA sequencing, we discovered that TENT5s polyadenylate mRNAs encoding endoplasmic reticulum-targeted proteins essential for gametogenesis. Sequence motif analysis and reporter mRNA assays reveal that the presence of an endoplasmic reticulum-leader sequence represents the primary determinant of TENT5-mediated regulation. Cytoplasmic polyadenylation is vital for gametogenesis, yet key enzymes involved in mammals remain unclear. Here, authors describe how TENT5-mediated polyadenylation of mRNAs encoding secreted proteins is essential for gametogenesis in mice.

Transmission of the malaria parasite to the mosquito vector is critical for the completion of the sexual stage of the parasite life cycle and is dependent on the release of male gametes from the gametocyte body inside the mosquito midgut. In the present study, we demonstrate that PfCDPK4 is critical for male gametogenesis and is involved in phosphorylation of proteins essential for male gamete emergence. Gametocytes of the malaria parasite Plasmodium are taken up by the mosquito vector with an infectious blood meal, representing a critical stage for parasite transmission. Calcium-independent protein kinases (CDPKs) play key roles in calcium-mediated signaling across the complex life cycle of the parasite. We sought to understand their role in human parasite transmission from the host to the mosquito vector and thus investigated the role of the human-infective parasite Plasmodium falciparum CDPK4 in the parasite life cycle. P. falciparum cdpk4 − parasites created by targeted gene deletion showed no effect in blood stage development or gametocyte development. However, cdpk4 − parasites showed a severe defect in male gametogenesis and the emergence of flagellated male gametes. To understand the molecular underpinnings of this defect, we performed mass spectrometry-based phosphoproteomic analyses of wild-type and Plasmodium falciparum cdpk4 − late gametocyte stages to identify key CDPK4-mediated phosphorylation events that may be important for the regulation of male gametogenesis. We further employed in vitro assays to identify these putative substrates of Plasmodium falciparum CDPK4. This indicated that CDPK4 regulates male gametogenesis by directly or indirectly controlling key essential events, such as DNA replication, mRNA translation, and cell motility. Taken together, our work demonstrates that PfCDPK4 is a central kinase that regulates exflagellation and thereby is critical for parasite transmission to the mosquito vector. IMPORTANCE Transmission of the malaria parasite to the mosquito vector is critical for the completion of the sexual stage of the parasite life cycle and is dependent on the release of male gametes from the gametocyte body inside the mosquito midgut. In the present study, we demonstrate that PfCDPK4 is critical for male gametogenesis and is involved in phosphorylation of proteins essential for male gamete emergence. Targeting PfCDPK4 and its substrates may provide insights into achieving effective malaria transmission-blocking strategies.