Day–night cycles and the sleep-promoting factor,Sleepless, affect stem cell activity in the Drosophila testis

Adult stem cells maintain tissue integrity and function by renewing cellular content of the organism through regulated mitotic divisions. Previous studies showed that stem cell activity is affected by local, systemic, and environmental cues. Here, we explore a role of environmental day–night cycles in modulating cell cycle progression in populations of adult stem cells. Using a classic stem cell system, the Drosophila spermatogonial stem cell niche, we reveal daily rhythms in division frequencies of germ-line and somatic stem cells that act cooperatively to produce male gametes. We also examine whether behavioral sleep–wake cycles, which are driven by the environmental day–night cycles, regulate stem cell function. We find that flies lacking the sleep-promoting factor Sleepless, which maintains normal sleep in Drosophila, have increased germ-line stem cell (GSC) division rates, and this effect is mediated, in part, through a GABAergic signaling pathway. We suggest that alterations in sleep can influence the daily dynamics of GSC divisions.In a constantly changing environment, the transitions between day and night represent the sole nearly invariable component of our living habitat and have influenced the evolution of life since its origin (1). Environmental rhythms in light, either directly or through endogenous circadian clocks, coordinate many biological functions, including gene expression, enzyme activity, and various cellular and behavioral processes (2). One of the most pronounced 24-h cycles, the rhythm in sleep and wakefulness, reflects neuronal activity in the brain and also affects the organism’s systemic milieu, evoking physiological responses in many peripheral tissues (3). In this study, we address possible roles of environmental rhythms and the behavioral status of the organism in regulating cellular homeostasis in adult tissue.Cell renewal is essential for maintaining tissue function over the lifetime of the organism (4). To replace damaged and aging cellular content, comparatively small populations of adult stem cells reside within specialized microenvironments called stem cell niches, where they undergo repeated mitotic divisions, generating new stem and differentiated cells (5). Although the rate of stem cell division is sustained at a tissue and cell type-specific level, an abrupt decrease in the number of cells, caused by physical injury or massive cell death, can initiate a quick response within stem cell populations, leading to increased division activity and thus, enhanced cell production (6). Behavioral factors and environmental influences are also known to affect the frequency of cell divisions, adjusting tissue homeostasis to the changing conditions inside and outside the organism (7). Twenty-four–hour rhythms of division have been documented in several mammalian tissues, reflecting dynamic activity in complex populations of dividing cells consisting of stem cells and their differentiating progeny (8–15). Altered behavioral states, such as insufficient sleep, pregnancy, and physical exercise, induce mitotic activity in the brain and other stem cell-supported tissues (4, 7). However, in most of these studies, stem cells, in particular, were not singled out for analysis, leaving open the question of whether these master regulators are subject to environmental and behavioral influences. To assess this question, we take advantage of a classic Drosophila stem cell system, the spermatogonial stem cell niche, which supports two easily identifiable and thoroughly characterized populations of adult stem cells (16).Located in the apical part of the testis, this niche is formed by a tight cluster of somatic support cells, called the hub, surrounded by germ-line stem cells (GSCs) and cyst progenitor cells (CPCs) (Fig. 1A). Hub cells produce short-range signaling molecules that activate downstream signaling pathways specifically in adjacent stem cells but not in more remotely located cells, thereby confining the stem cell domain in the testis (17). Thus, in contrast to more complex stem cell niches that maintain mammalian tissues, both GSCs and CPCs can be unequivocally identified in vivo and in fixed whole-mount testes based on their position next to the hub and by using simple lineage-specific markers (17) (Fig. 1). GSCs and CPCs self-renew while also generating differentiating cell progeny: gonialblasts and somatic cyst cells, respectively. Gonialblasts enclosed by cyst cell pairs form cysts, the units of sperm development (Fig. 1A). Within cysts, early germ cells continue to amplify, producing 2–16 cell clusters called spermatogonial cells before undergoing meiosis and maturation (16). Molecular communication between the germ line and somatic cell partners ensures proper progression through spermatogenesis (18).Open in a separate windowFig. 1.The spermatogonial stem cell niche and early stages of spermatogenesis in the fly testis. (A) Schematic representation of the niche composed of somatic hub cells (light blue), 5–10 GSCs (peach), and approximately two times as many CPCs (blue). GSCs and CPCs produce differentiating gonialblasts (GBs; yellow) and cyst cells (gray) that form cysts. GBs undergo four additional mitotic divisions to become spermatogonial cells before proceeding into meiosis. (B and C) Single confocal sections show (B, arrowheads) duplicating GSCs and (C, arrow) CPCs. Hub cells are labeled with antibodies against protein Armadillo (membrane green; asterisks). GSCs are positive for a germ cell-specific marker Vasa (red) and positioned immediately next to the hub. CPCs contact hub cells and are Vasa-negative. Antibodies against the phosphorylated form of Histone H3 label mitotic chromatin (nuclear green). DNA counterstain is in blue.Here, we show that, in the presence of environmental day–night cycles, both stem cell populations occupying the Drosophila spermatogonial stem cell niche show daily rhythms in division frequencies that do not persist in constant darkness and thus, do not seem to constitute free-running circadian rhythms. Because sleep–wake rhythms can be driven by environmental cycles, we further address the effect of sleep duration on stem cell activity. Using a combination of genetic and pharmacological assays, we find that loss of the sleep-promoting factor Sleepless (SSS) stimulates GSC division rates in the testis. At least some of the effects of SSS on the GSCs are mediated by reduced GABA levels, which also contribute to the short sleep phenotype. Based on these results, we suggest that some sleep-regulating pathways influence the rate of stem cell division in the fly.     

Glycolipid GD3 and GD3 synthase are key drivers for glioblastoma stem cells and tumorigenicity

The cancer stem cells (CSCs) of glioblastoma multiforme (GBM), a grade IV astrocytoma, have been enriched by the expressed marker CD133. However, recent studies have shown that CD133⁻ cells also possess tumor-initiating potential. By analysis of gangliosides on various cells, we show that ganglioside D3 (GD3) is overexpressed on eight neurospheres and tumor cells; in combination with CD133, the sorted cells exhibit a higher expression of stemness genes and self-renewal potential; and as few as six cells will form neurospheres and 20–30 cells will grow tumor in mice. Furthermore, GD3 synthase (GD3S) is increased in neurospheres and human GBM tissues, but not in normal brain tissues, and suppression of GD3S results in decreased GBM stem cell (GSC)-associated properties. In addition, a GD3 antibody is shown to induce complement-dependent cytotoxicity against cells expressing GD3 and inhibition of GBM tumor growth in vivo. Our results demonstrate that GD3 and GD3S are highly expressed in GSCs, play a key role in glioblastoma tumorigenicity, and are potential therapeutic targets against GBM.Glioblastoma multiforme (GBM) is extremely infiltrative and difficult to treat, and most patients develop recurrence after therapy. Over the past decade, many studies have suggested that bulk GBM tumors harbor cancer stem cells (CSCs) (1, 2), a distinct subpopulation of cancer cells that are able to initiate new tumors efficiently, have long-term self-renewal capacity, and survive better against chemo- or radiotherapy (2–4). CD133 has become a widely used marker for the enrichment of GBM CSCs (GSCs) and other tumor types (5–10). However, recent studies have shown that CD133 is not specific for GSCs because CD133⁻ cells also possess tumor-initiating potential (11–13), indicating the need to identify more specific and exclusive markers for GSCs to facilitate our understanding of GSCs and therapeutic development against GBM. Several reports have proposed L1CAM, A2B5, integrin α6, MET, and CD15 as markers for GSCs (14–18). However, none of these protein markers could be used specifically to identify GSCs, and no study was reported with respect to glycans as potential markers, although glycan biosynthesis involves multiple genes and it is possible to create different structures in cancer progression. It is noted that ganglioside D2 (GD2) and ganglioside D3 (GD3) were found on the surface of neural stem cells (NSCs) and that stage-specific embryonic antigen 3 (SSEA3) and SSEA4 were found on embryonic stem cells and cancer cells (19–21), but there is no glycan marker found on the surface of GSCs.Gangliosides are sialic acid-containing glycosphingolipids (GSLs) that are most abundant in the nervous system (22). The expression levels and patterns of gangliosides during brain development shift from simple gangliosides, such as GM3 and GD3, to complex gangliosides, such as GM1, GD1a, GD1b, and GT1b (23, 24). Moreover, several unique ganglioside markers, including SSEA3, SSEA4, GD2, and GD3, have been identified in stem cells (19). GD3, a b-series ganglioside containing two sialic acids, is highly expressed in mouse and human embryonic NSCs (20, 25). In cancers, GD3 is highly accumulated in human primary melanoma tissues as well as in established melanoma cell lines (26), whereas human normal melanocytes express no or minimal levels of GD3 (27). Moreover, malignant gliomas contain higher levels of GD3, and its expression correlates with the degree of malignancy (28). GD3 is produced from the precursor GM3 by the activity of GD3 synthase (GD3S), which mediates the properties of CSCs through the c-MET signaling pathway and correlates with poor prognosis in triple-negative human breast tumors (29). These findings suggest that GD3 may play an important role in the transformation of normal cells into tumors, and imply that GD3 could be a cell surface marker for GSCs.This study was designed to identify glycan markers for the enrichment of GBM stem cells and then uses these enriched GBM stem cells to characterize tumorigenicity, their association with clinical GBM specimens, and their regulation in tumor progression. The results showed that GD2 and GD3 were positively stained on GBM neurospheres. We found that cells with high GD3 expression display functional characteristics of GSCs. Suppression of GD3S, a critical enzyme for GD3 synthesis, impeded neurosphere formation and tumor initiation. The expression of GD3S correlated with the grades of astrocytomas and mediated self-renewal through c-Met activation. Furthermore, a GD3 antibody was found to eliminate the GD3⁺ cells through complement-dependent cytotoxicity (CDC) in vitro and to suppress tumor growth in mice. These results suggest that GD3 could be a significant biomarker for GSCs, that CD3 could be combined with CD133 for the enrichment of GSCs, and that both GD3 and GD3S could be targets for the development of new therapies against GBM.     

Life-long in vivo cell-lineage tracing shows that no oogenesis originates from putative germline stem cells in adult mice

Whether or not oocyte regeneration occurs in adult life has been the subject of much debate. In this study, we have traced germ-cell lineages over the life spans of three genetically modified mouse models and provide direct evidence that oogenesis does not originate from any germline stem cells (GSCs) in adult mice. By selective ablation of all existing oocytes in a Gdf9-Cre;iDTR mouse model, we have demonstrated that no new germ cells were ever regenerated under pathological conditions. By in vivo tracing of oocytes and follicles in the Sohlh1-CreER^T2;R26R and Foxl2-CreER^T2;mT/mG mouse models, respectively, we have shown that the initial pool of oocytes is the only source of germ cells throughout the life span of the mice and that no adult oogenesis ever occurs under physiological conditions. Our findings clearly show that there are no GSCs that contribute to adult oogenesis in mice and that the initial pool of oocytes formed in early life is the only source of germ cells throughout the entire reproductive life span.Whether or not oogenesis occurs in the adult mammalian ovary has been a long-standing question in developmental biology. It has been generally accepted for more than half a century that in most mammalian species oocytes cannot renew themselves in postnatal or adult life (1), but studies in the past decade have raised the possibility of adult oogenesis in both mouse and human ovaries and have increased the intensity of the debate (2–5). These studies have proposed that oocytes can be regenerated from putative germline stem cells (GSCs) or oogonial stem cells (OSCs) in adult mouse and human ovaries (these are both referred to as GSCs in this paper) (2–5). By calculating the number of healthy follicles and atretic follicles at different ages, Johnson et al. proposed that 77 new oocytes could be regenerated from putative GSCs in the mouse ovary every day (2). Moreover, they proposed that a group of GSCs, which had originated from the epithelium of the ovarian surface, served as the source of the regenerated oocytes (2). One year later, in response to criticism from the field (6), Johnson et al. amended their previous result and reported that the GSCs had actually originated from the bone marrow and peripheral blood (3).More recently, isolation of mouse and human GSCs using the DEAD box polypeptide 4 (DDX4) antibody-based cell sorting was reported, and these GSCs were suggested to serve as the source of the oocytes that fueled the follicular replenishment (4, 5). Due to their potential implications for treating female infertility, these studies have attracted the attention of researchers as well as the popular press (7).In contrast to these reports, other recent reports have provided evidence that adult oogenesis and the so-called GSCs do not exist and have questioned the above-mentioned findings (8–12). For example, by tracing the proliferation of cultured Ddx4-positive cells in vitro, a recent study from our group reported that no mitotically active GSCs exist in the postnatal mouse ovary (10). More recently, Lei and Spradling provided evidence to support our findings by showing that no active GSCs could be detected in adult mouse ovaries (11).Although the existence of adult oogenesis has been debated for more than a century, most of the studies supporting or opposing the existence of adult oogenesis have been based on indirect approaches such as mathematical analysis of oocyte numbers and in vitro cell purification and cell cultures. There is a lack of functional in vivo evidence for or against the existence of adult oogenesis in mammals. Or, if adult oogenesis does exist, it is not known if such a process is physiologically relevant.In the current study, we have generated three genetically modified mouse models and performed in vivo cell-lineage tracing of oocytes—under both physiological and pathological conditions—over the entire life span of the animals. Our results clearly show that no oocyte neogenesis occurs from putative GSCs in adult mammalian ovaries and that the initial pool of oocytes formed in early life is the only source of germ cells throughout the entire reproductive life span.     

Female mice lack adult germ-line stem cells but sustain oogenesis using stable primordial follicles

Whether or not mammalian females generate new oocytes during adulthood from germ-line stem cells to sustain the ovarian follicle pool has recently generated controversy. We used a sensitive lineage-labeling system to determine whether stem cells are needed in female adult mice to compensate for follicular losses and to directly identify active germ-line stem cells. Primordial follicles generated during fetal life are highly stable, with a half-life during adulthood of 10 mo, and thus are sufficient to sustain adult oogenesis without a source of renewal. Moreover, in normal mice or following germ-cell depletion with Busulfan, only stable, single oocytes are lineage-labeled, rather than cell clusters indicative of new oocyte formation. Even one germ-line stem cell division per 2 wk would have been detected by our method, based on the kinetics of fetal follicle formation. Thus, adult female mice neither require nor contain active germ-line stem cells or produce new oocytes in vivo.Mammalian females ovulate periodically over their reproductive lifetimes, placing significant demands on their ovaries for oocyte production. Murine females produce up to 500 oocytes during 50 cycles, whereas human females release a similar number during 40 y of monthly cycles. Before birth, their ovaries contain thousands (mice) or millions (human) of prefollicular germ cells. A large “ovarian reserve” of primordial follicles is generated around the time of birth from prefollicular germ cells, and follicle numbers decline slowly (1). Because histological evidence of prefollicular germ cells also disappears at birth, it has been widely thought that the initial follicles are stable enough to sustain oogenesis throughout the normal reproductive lifespan (2).During the last decade, it has been claimed that primordial follicles in adult ovaries are highly unstable and that mouse and human females consequently require adult germ-line stem cells (GSCs) to maintain a pool of follicles and sustain ovulation. Active stem cells were placed in the ovarian surface epithelium (3) or in the bone marrow (4). However, others failed to reproduce these data and their predictions (5–10). Subsequently, evidence for ovarian GSCs came from transplantation assays. Selected ovarian cells explanted into culture gave rise to rare cells capable of forming oocytes following transplantation into a host ovary (11, 12). This work has also been challenged (13). Recently, patterns of somatic mutations in female germ cells during adulthood were interpreted to be consistent with the presence of adult stem cells (14). Normal adult female GSCs, should they exist, might prove useful for advancing reproductive health and for stem-cell-based therapies. Lineage tracing in vivo constitutes the definitive method for detecting stem cells (15). Here we show by single-cell lineage tracing that GSCs are not required to maintain the mouse primordial follicle pool and are undetectable in adult mouse ovaries.     

Detection of genomic variations and DNA polymorphisms and impact on analysis of meiotic recombination and genetic mapping

DNA polymorphisms are important markers in genetic analyses and are increasingly detected by using genome resequencing. However, the presence of repetitive sequences and structural variants can lead to false positives in the identification of polymorphic alleles. Here, we describe an analysis strategy that minimizes false positives in allelic detection and present analyses of recently published resequencing data from Arabidopsis meiotic products and individual humans. Our analysis enables the accurate detection of sequencing errors, small insertions and deletions (indels), and structural variants, including large reciprocal indels and copy number variants, from comparisons between the resequenced and reference genomes. We offer an alternative interpretation of the sequencing data of meiotic products, including the number and type of recombination events, to illustrate the potential for mistakes in single-nucleotide polymorphism calling. Using these examples, we propose that the detection of DNA polymorphisms using resequencing data needs to account for nonallelic homologous sequences.DNA polymorphisms are ubiquitous genetic variations among individuals and include single nucleotide polymorphisms (SNPs), insertions and deletions (indels), and other larger rearrangements (1–3) (Fig. 1 A and B). They can have phenotypic consequences and also serve as molecular markers for genetic analyses, facilitating linkage and association studies of genetic diseases, and other traits in humans (4–6), animals, plants, (7–10) and other organisms. Using DNA polymorphisms for modern genetic applications requires low-error, high-throughput analytical strategies. Here, we illustrate the use of short-read next-generation sequencing (NGS) data to detect DNA polymorphisms in the context of whole-genome analysis of meiotic products.Open in a separate windowFig. 1.(A) SNPs and small indels between two ecotype genomes. (B) Possible types of SVs. Col genotypes are marked in blue and Ler in red. Arrows indicate DNA segments involved in SVs between the two ecotypes. (C) Meiotic recombination events including a CO and a GC (NCO). Centromeres are denoted by yellow dots.There are many methods for detecting SNPs (11–14) and structural variants (SVs) (15–25), including NGS, which can capture nearly all DNA polymorphisms (26–28). This approach has been widely used to analyze markers in crop species such as rice (29), genes associated with diseases (6, 26), and meiotic recombination in yeast and plants (30, 31). However, accurate identification of DNA polymorphisms can be challenging, in part because short-read sequencing data have limited information for inferring chromosomal context.Genomes usually contain repetitive sequences that can differ in copy number between individuals (26–28, 31); therefore, resequencing analyses must account for chromosomal context to avoid mistaking highly similar paralogous sequences for polymorphisms. Here, we use recently published datasets to describe several DNA sequence features that can be mistaken as allelic (32, 33) and describe a strategy for differentiating between repetitive sequences and polymorphic alleles. We illustrate the effectiveness of these analyses by examining the reported polymorphisms from the published datasets.Meiotic recombination is initiated by DNA double-strand breaks (DSBs) catalyzed by the topoisomerase-like SPORULATION 11 (SPO11). DSBs are repaired as either crossovers (COs) between chromosomes (Fig. 1C), or noncrossovers (NCOs). Both COs and NCOs can be accompanied by gene conversion (GC) events, which are the nonreciprocal transfer of sequence information due to the repair of heteroduplex DNA during meiotic recombination. Understanding the control of frequency and distribution of CO and NCO (including GC) events has important implications for human health (including cancer and aneuploidy), crop breeding, and the potential for use in genome engineering. COs can be detected relatively easily by using polymorphic markers in the flanking sequences, but NCO products can only be detected if they are accompanied by a GC event. Because GCs associated with NCO result in allelic changes at polymorphic sites without exchange of flanking sequences, they are more difficult to detect. Recent advances in DNA sequencing have made the analysis of meiotic NCOs more feasible (30–32, 34); however, SVs present a challenge in these analyses. We recommend a set of guidelines for detection of DNA polymorphisms by using genomic resequencing short-read datasets. These measures improve the accuracy of a wide range of analyses by using genomic resequencing, including estimation of COs, NCOs, and GCs.     

The evolution of self-control

Cognition presents evolutionary research with one of its greatest challenges. Cognitive evolution has been explained at the proximate level by shifts in absolute and relative brain volume and at the ultimate level by differences in social and dietary complexity. However, no study has integrated the experimental and phylogenetic approach at the scale required to rigorously test these explanations. Instead, previous research has largely relied on various measures of brain size as proxies for cognitive abilities. We experimentally evaluated these major evolutionary explanations by quantitatively comparing the cognitive performance of 567 individuals representing 36 species on two problem-solving tasks measuring self-control. Phylogenetic analysis revealed that absolute brain volume best predicted performance across species and accounted for considerably more variance than brain volume controlling for body mass. This result corroborates recent advances in evolutionary neurobiology and illustrates the cognitive consequences of cortical reorganization through increases in brain volume. Within primates, dietary breadth but not social group size was a strong predictor of species differences in self-control. Our results implicate robust evolutionary relationships between dietary breadth, absolute brain volume, and self-control. These findings provide a significant first step toward quantifying the primate cognitive phenome and explaining the process of cognitive evolution.Since Darwin, understanding the evolution of cognition has been widely regarded as one of the greatest challenges for evolutionary research (1). Although researchers have identified surprising cognitive flexibility in a range of species (2–40) and potentially derived features of human psychology (41–61), we know much less about the major forces shaping cognitive evolution (62–71). With the notable exception of Bitterman’s landmark studies conducted several decades ago (63, 72–74), most research comparing cognition across species has been limited to small taxonomic samples (70, 75). With limited comparable experimental data on how cognition varies across species, previous research has largely relied on proxies for cognition (e.g., brain size) or metaanalyses when testing hypotheses about cognitive evolution (76–92). The lack of cognitive data collected with similar methods across large samples of species precludes meaningful species comparisons that can reveal the major forces shaping cognitive evolution across species, including humans (48, 70, 89, 93–98).To address these challenges we measured cognitive skills for self-control in 36 species of mammals and birds (Fig. 1 and Tables S1–S4) tested using the same experimental procedures, and evaluated the leading hypotheses for the neuroanatomical underpinnings and ecological drivers of variance in animal cognition. At the proximate level, both absolute (77, 99–107) and relative brain size (108–112) have been proposed as mechanisms supporting cognitive evolution. Evolutionary increases in brain size (both absolute and relative) and cortical reorganization are hallmarks of the human lineage and are believed to index commensurate changes in cognitive abilities (52, 105, 113–115). Further, given the high metabolic costs of brain tissue (116–121) and remarkable variance in brain size across species (108, 122), it is expected that the energetic costs of large brains are offset by the advantages of improved cognition. The cortical reorganization hypothesis suggests that selection for absolutely larger brains—and concomitant cortical reorganization—was the predominant mechanism supporting cognitive evolution (77, 91, 100–106, 120). In contrast, the encephalization hypothesis argues that an increase in brain volume relative to body size was of primary importance (108, 110, 111, 123). Both of these hypotheses have received support through analyses aggregating data from published studies of primate cognition and reports of “intelligent” behavior in nature—both of which correlate with measures of brain size (76, 77, 84, 92, 110, 124).Open in a separate windowFig. 1.A phylogeny of the species included in this study. Branch lengths are proportional to time except where long branches have been truncated by parallel diagonal lines (split between mammals and birds ∼292 Mya).With respect to selective pressures, both social and dietary complexities have been proposed as ultimate causes of cognitive evolution. The social intelligence hypothesis proposes that increased social complexity (frequently indexed by social group size) was the major selective pressure in primate cognitive evolution (6, 44, 48, 50, 87, 115, 120, 125–141). This hypothesis is supported by studies showing a positive correlation between a species’ typical group size and the neocortex ratio (80, 81, 85–87, 129, 142–145), cognitive differences between closely related species with different group sizes (130, 137, 146, 147), and evidence for cognitive convergence between highly social species (26, 31, 148–150). The foraging hypothesis posits that dietary complexity, indexed by field reports of dietary breadth and reliance on fruit (a spatiotemporally distributed resource), was the primary driver of primate cognitive evolution (151–154). This hypothesis is supported by studies linking diet quality and brain size in primates (79, 81, 86, 142, 155), and experimental studies documenting species differences in cognition that relate to feeding ecology (94, 156–166).Although each of these hypotheses has received empirical support, a comparison of the relative contributions of the different proximate and ultimate explanations requires (i) a cognitive dataset covering a large number of species tested using comparable experimental procedures; (ii) cognitive tasks that allow valid measurement across a range of species with differing morphology, perception, and temperament; (iii) a representative sample within each species to obtain accurate estimates of species-typical cognition; (iv) phylogenetic comparative methods appropriate for testing evolutionary hypotheses; and (v) unprecedented collaboration to collect these data from populations of animals around the world (70).Here, we present, to our knowledge, the first large-scale collaborative dataset and comparative analysis of this kind, focusing on the evolution of self-control. We chose to measure self-control—the ability to inhibit a prepotent but ultimately counterproductive behavior—because it is a crucial and well-studied component of executive function and is involved in diverse decision-making processes (167–169). For example, animals require self-control when avoiding feeding or mating in view of a higher-ranking individual, sharing food with kin, or searching for food in a new area rather than a previously rewarding foraging site. In humans, self-control has been linked to health, economic, social, and academic achievement, and is known to be heritable (170–172). In song sparrows, a study using one of the tasks reported here found a correlation between self-control and song repertoire size, a predictor of fitness in this species (173). In primates, performance on a series of nonsocial self-control control tasks was related to variability in social systems (174), illustrating the potential link between these skills and socioecology. Thus, tasks that quantify self-control are ideal for comparison across taxa given its robust behavioral correlates, heritable basis, and potential impact on reproductive success.In this study we tested subjects on two previously implemented self-control tasks. In the A-not-B task (27 species, n = 344), subjects were first familiarized with finding food in one location (container A) for three consecutive trials. In the test trial, subjects initially saw the food hidden in the same location (container A), but then moved to a new location (container B) before they were allowed to search (Movie S1). In the cylinder task (32 species, n = 439), subjects were first familiarized with finding a piece of food hidden inside an opaque cylinder. In the following 10 test trials, a transparent cylinder was substituted for the opaque cylinder. To successfully retrieve the food, subjects needed to inhibit the impulse to reach for the food directly (bumping into the cylinder) in favor of the detour response they had used during the familiarization phase (Movie S2).Thus, the test trials in both tasks required subjects to inhibit a prepotent motor response (searching in the previously rewarded location or reaching directly for the visible food), but the nature of the correct response varied between tasks. Specifically, in the A-not-B task subjects were required to inhibit the response that was previously successful (searching in location A) whereas in the cylinder task subjects were required to perform the same response as in familiarization trials (detour response), but in the context of novel task demands (visible food directly in front of the subject).     

Axin2 marks quiescent hair follicle bulge stem cells that are maintained by autocrine Wnt/β-catenin signaling

How stem cells maintain their identity and potency as tissues change during growth is not well understood. In mammalian hair, it is unclear how hair follicle stem cells can enter an extended period of quiescence during the resting phase but retain stem cell potential and be subsequently activated for growth. Here, we use lineage tracing and gene expression mapping to show that the Wnt target gene Axin2 is constantly expressed throughout the hair cycle quiescent phase in outer bulge stem cells that produce their own Wnt signals. Ablating Wnt signaling in the bulge cells causes them to lose their stem cell potency to contribute to hair growth and undergo premature differentiation instead. Bulge cells express secreted Wnt inhibitors, including Dickkopf (Dkk) and secreted frizzled-related protein 1 (Sfrp1). However, the Dickkopf 3 (Dkk3) protein becomes localized to the Wnt-inactive inner bulge that contains differentiated cells. We find that Axin2 expression remains confined to the outer bulge, whereas Dkk3 continues to be localized to the inner bulge during the hair cycle growth phase. Our data suggest that autocrine Wnt signaling in the outer bulge maintains stem cell potency throughout hair cycle quiescence and growth, whereas paracrine Wnt inhibition of inner bulge cells reinforces differentiation.The hair follicle is a complex miniorgan that repeatedly cycles through stages of rest (telogen), growth (anagen), and destruction (catagen) throughout life (1). During anagen, growing hair follicles emerge adjacent to the old telogen hair follicles that remain there throughout the cycle and create an epithelial protrusion known as the “bulge.” At the end of the hair cycle, in catagen, cells from the follicle migrate along the retracting epithelial strand and join the two epithelial layers of the telogen bulge—the inner and outer bulge layers—surrounding the club hair shaft (2).Several studies have established that stem cells residing in the outer bulge are the source of the regenerative capacity of the cycling hair follicle (3–5). During telogen, these stem cells are thought to be generally quiescent (6). In response to signals from their microenvironment during anagen, the stem cells divide and produce proliferative progeny that participate in the growth of the new follicle (7). Some of these activated stem cells and their progeny are believed to migrate away from the bulge, but are subsequently able to rejoin it after anagen is complete (2, 5). Cells that return to the outer bulge take on a follicular stem cell identity, ready to divide and participate in the next hair cycle (2, 8). Conversely, cells returning to the inner bulge do not divide and, instead, form an inner bulge niche of differentiated cells for the outer bulge cells (2). Stem cells remain quiescent during telogen for an extended period, and the identity of signals that maintain stem cell identity during this time are poorly understood.In the hair, Wnt/β-catenin signaling is required right from the earliest stages of development, for the initiation of hair placode formation (9). Wnt signals are needed later during postnatal homeostasis as well, for the initiation of anagen in postnatal hair (10). Therefore, in view of their well-established importance for stem cell maintenance in multiple adult tissues, including the skin (11), Wnts are candidate hair follicle stem cell (HFSC)-maintaining signals. However, Wnt signaling is generally believed to be inactive in the telogen bulge (8, 10, 12), which is thought to be quiescent. Wnt signaling becomes strongly elevated when bulge cells are “activated” to undergo the transition from telogen to anagen (13, 14). During anagen, Wnt signaling has been described to primarily specify differentiated cell fates in the anagen follicle (12, 15). As anagen proceeds and the follicle enters catagen and telogen again, the bulge is thought to revert to a Wnt-inhibited state (12, 13, 16, 17).Conversely, there is evidence for a functional requirement of Wnt/β-catenin signaling in the bulge other than initiating anagen and specifying differentiation during anagen. For instance, postnatal deletion of β-catenin in outer bulge cells results in the loss of label-retention and HFSC markers, suggesting that β-catenin is required for maintenance of HFSC identity (10).Here, beyond its role in hair differentiation and anagen initiation, we sought to determine whether Wnt/β-catenin signaling is also involved in HFSC maintenance during telogen. We found that Axin2 expression persists in HFSCs in the outer bulge throughout telogen and anagen, suggesting that active Wnt signaling is a consistent feature of bulge stem cells. Furthermore, these hair outer bulge stem cells produce autocrine Wnts and paracrine-acting Wnt inhibitors that may specify the positional identity of cells residing within the bulge niche.     

Human pluripotent stem cell-derived neural constructs for predicting neural toxicity

Human pluripotent stem cell-based in vitro models that reflect human physiology have the potential to reduce the number of drug failures in clinical trials and offer a cost-effective approach for assessing chemical safety. Here, human embryonic stem (ES) cell-derived neural progenitor cells, endothelial cells, mesenchymal stem cells, and microglia/macrophage precursors were combined on chemically defined polyethylene glycol hydrogels and cultured in serum-free medium to model cellular interactions within the developing brain. The precursors self-assembled into 3D neural constructs with diverse neuronal and glial populations, interconnected vascular networks, and ramified microglia. Replicate constructs were reproducible by RNA sequencing (RNA-Seq) and expressed neurogenesis, vasculature development, and microglia genes. Linear support vector machines were used to construct a predictive model from RNA-Seq data for 240 neural constructs treated with 34 toxic and 26 nontoxic chemicals. The predictive model was evaluated using two standard hold-out testing methods: a nearly unbiased leave-one-out cross-validation for the 60 training compounds and an unbiased blinded trial using a single hold-out set of 10 additional chemicals. The linear support vector produced an estimate for future data of 0.91 in the cross-validation experiment and correctly classified 9 of 10 chemicals in the blinded trial.There is a pressing need for improved methods to assess the safety of drugs and other compounds (1–5). Success rates for drug approval are declining despite higher research and development spending (6), and clinical trials often fail due to toxicities that were not identified through animal testing (7). In addition, most of the chemicals in commerce have not been rigorously assessed for safety despite growing concerns over the potential impact of industrial and environmental exposures on human health (2–5). Animal models are costly, time consuming, and fail to recapitulate many aspects of human physiology, which has motivated agencies such as the National Institutes of Health (NIH) and the US Environmental Protection Agency (EPA) to initiate programs that emphasize human cellular approaches for assessing the safety of drugs (1) and environmental chemicals (2, 3). In vitro cellular models that accurately reflect human physiology have the potential to improve the prediction of drug toxicity early in the development pipeline (1) and would provide a cost-effective approach for testing other sources of chemical exposure, including food additives, cosmetics, pesticides, and industrial chemicals (2–5).The human brain is particularly sensitive to toxic insults during development and early childhood (8), and there is growing concern that exposure to environmental chemicals may be linked to the rising incidence of neurodevelopmental disorders worldwide (4). Human brain development is mediated by highly coordinated cellular interactions between functionally distinct cell types that include neurons, glia, blood vessels, and microglia (9–15), each of which may be involved in neurotoxicity mechanisms (16–18). The cellular diversity of the developing brain complicates efforts to assess developmental neurotoxicity in vitro, because toxins might target numerous distinct cell types or cellular interactions and the underlying toxicity mechanisms are often unknown (3–5). Neurotoxicity has been evaluated using brain-derived cells in aggregate culture or coculture, neural stem cells, and other in vitro platforms, and these studies suggest that complex neurotoxic effects can be mimicked by incorporating cellular diversity into the model system (16, 18–20). However, many of these studies rely on animal cells that poorly reflect human physiology or primary human cells that are not scalable and introduce batch variability.Although in vitro human cellular models have historically been hampered by inadequate access to cellular components of the human brain, human embryonic stem (ES) cells (21) and induced pluripotent stem (iPS) cells (22, 23) now offer a scalable source for tissue-specific cell types. Here, reproducible 3D neural constructs that incorporated vascular and microglial components were fabricated for developmental neurotoxicity screening by culturing precursor cells derived from the H1 human ES cell line on synthetic hydrogels under defined conditions. Machine learning was used to build a predictive model from RNA sequencing (RNA-Seq) data for neural constructs exposed to a training set of 60 toxic and nontoxic chemicals and then to make predictions in a blinded trial using a set of 10 additional compounds.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Lagging adaptation to warming climate in Arabidopsis thaliana

If climate change outpaces the rate of adaptive evolution within a site, populations previously well adapted to local conditions may decline or disappear, and banked seeds from those populations will be unsuitable for restoring them. However, if such adaptational lag has occurred, immigrants from historically warmer climates will outperform natives and may provide genetic potential for evolutionary rescue. We tested for lagging adaptation to warming climate using banked seeds of the annual weed Arabidopsis thaliana in common garden experiments in four sites across the species’ native European range: Valencia, Spain; Norwich, United Kingdom; Halle, Germany; and Oulu, Finland. Genotypes originating from geographic regions near the planting site had high relative fitness in each site, direct evidence for broad-scale geographic adaptation in this model species. However, genotypes originating in sites historically warmer than the planting site had higher average relative fitness than local genotypes in every site, especially at the northern range limit in Finland. This result suggests that local adaptive optima have shifted rapidly with recent warming across the species’ native range. Climatic optima also differed among seasonal germination cohorts within the Norwich site, suggesting that populations occurring where summer germination is common may have greater evolutionary potential to persist under future warming. If adaptational lag has occurred over just a few decades in banked seeds of an annual species, it may be an important consideration for managing longer-lived species, as well as for attempts to conserve threatened populations through ex situ preservation.Rapid climate change has already caused species range shifts and local extinctions (1) and is predicted to have greater future impacts (2). As the suitable climate space for a species shifts poleward (3), populations previously well adapted to the historical climate in a particular region may experience strong selection to adapt to rapidly warming local temperatures (4–10). Rapid evolutionary response to climate change has already been observed (11, 12), but it remains unclear whether evolutionary response can keep pace with rapidly changing local adaptive optima (6, 8, 13–15). If local adaptation is slower than the rate of climate change, the average fitness of local populations may decline over time (7, 14, 16, 17), possibly resulting in local extinctions and range collapse at the warmer margin. Where such lag exists, we expect that local seeds banked for conservation may no longer be well adapted to their sites of origin (18). However, such adaptational lag may be mitigated by migration or gene flow from populations in historically warmer sites if those populations are better adapted to current conditions in a site than local populations (8, 13, 19, 20). Although adaptational lag has been predicted (4–6, 8, 14, 15, 19, 21, 22), the distinctive signature of mismatch between local population performance and current climate optima has not yet been explicitly demonstrated in nature.Despite evidence for local adaptation in many organisms (23), there have been few explicit tests for the role of specific climate factors in shaping local fitness optima (4, 9, 13). Such tests require growing many genotypes from populations spanning a range of climates in common gardens across a species’ range to decouple climate of origin from geographic variation in other selective factors (4, 6, 14). If adaptation to local climate has occurred, then genotypes from climates similar to each planting site are expected to have high fitness in that site relative to genotypes from dissimilar climates (6). However, if local adaptive optima have shifted with rapid warming trends over the last 50 y, we expect that banked genotypes from historically warmer climates will have higher fitness within a site than banked genotypes of local origin (6, 21, 22).We tested for lagging adaptation to climate using Arabidopsis thaliana, a naturally inbreeding annual species that inhabits a broad climate space across its native Eurasian range (24). A. thaliana exhibits strong circumstantial evidence of climate adaptation, including geographic clines in ecologically important life-history traits (25–28) and in candidate genes associated with these traits (29, 30), as well as genome-wide associations of single nucleotide polymorphisms with climatic factors (31–34). To test explicitly for local adaptation to climate we measured the lifetime fitness of more than 230 accessions from banked seeds originating from a broad range of climates in replicated field experiments in four sites across the species’ native climate range (Fig. 1). We observed that genotypes originating in historically warmer climates outperformed local genotypes, particularly at the northern range limit.Open in a separate windowFig. 1.Map of common garden sites and sites of origin of the 241 native A. thaliana accessions represented in our experiments.