Collagen IV differentially regulates planarian stem cell potency and lineage progression

The extracellular matrix (ECM) provides a precise physical and molecular environment for cell maintenance, self-renewal, and differentiation in the stem cell niche. However, the nature and organization of the ECM niche is not well understood. The adult freshwater planarian Schmidtea mediterranea maintains a large population of multipotent stem cells (neoblasts), presenting an ideal model to study the role of the ECM niche in stem cell regulation. Here we tested the function of 165 planarian homologs of ECM and ECM-related genes in neoblast regulation. We identified the collagen gene family as one with differential effects in promoting or suppressing proliferation of neoblasts. col4-1, encoding a type IV collagen α-chain, had the strongest effect. RNA interference (RNAi) of col4-1 impaired tissue maintenance and regeneration, causing tissue regression. Finally, we provide evidence for an interaction between type IV collagen, the discoidin domain receptor, and neuregulin-7 (NRG-7), which constitutes a mechanism to regulate the balance of symmetric and asymmetric division of neoblasts via the NRG-7/EGFR pathway.

Across the animal kingdom, stem cell function is regulated by the microenvironment in the surrounding niche (1), where the concentration of molecular signals for self-renewal and differentiation can be precisely regulated (2). The niche affects stem cell biology in many processes, such as aging and tissue regeneration, as well as pathological conditions such as cancer (3). Most studies have been done in tissues with large stem cell populations, such as the intestinal crypt (4) and the hair follicle (5) in mice. Elucidation of the role of the stem cell niche in tissue regeneration requires the study of animals with high regenerative potential, such as freshwater planarians (flatworms) (6). Dugesia japonica and Schmidtea mediterranea are two well-studied species that possess the ability to regenerate any missing body part (6, 7).Adult S. mediterranea maintain a high number of stem cells (neoblasts)—∼10 to 30% of all somatic cells in the adult worm—with varying potency, including pluripotent cells (8–14). Neoblasts are the only proliferating somatic cells: they are molecularly heterogeneous, but all express piwi-1 (15–18). Lineage-committed neoblasts are “progenitors” that transiently express both piwi-1 and tissue-specific genes (15, 19). Examples include early intestinal progenitors (γ neoblast, piwi-1⁺/hnf4⁺) (8, 10, 15, 19–21) and early epidermal progenitors (ζ neoblast, piwi-1⁺/zfp-1⁺) (8, 15). Other progenitor markers include collagen for muscles (22), ChAT for neurons (23), and cavII for protonephridia (24, 25). During tissue regeneration, neoblasts are recruited to the wound site, where they proliferate then differentiate to replace the missing cell types (16, 26). Some neoblasts express the pluripotency marker tgs-1, and are designated as clonogenic neoblasts (cNeoblasts) (10, 11). cNeoblasts are located in the parenchymal space adjacent to the gut (11).Neoblasts are sensitive to γ-irradiation and can be preferentially depleted in the adult planarian (27). After sublethal γ-irradiation, remaining cNeoblasts can repopulate the stem cell pool within their niche (10, 11). The close proximity of neoblasts to the gut suggests gut may be a part of neoblast niche (28, 29). When gut integrity was impaired by silencing gata4/5/6, the egfr-1/nrg-1 ligand-receptor pair, or wwp1, maintenance of non–γ-neoblasts were also disrupted (20, 30, 31), but whether that indicates the gut directly regulates neoblast remains unclear. There is evidence indicating the dorsal-ventral (D/V) transverse muscles surrounding the gut may promote neoblast proliferation and migration, with the involvement of matrix metalloproteinase mt-mmpB (32, 33). The central nervous system has also been implicated in influencing neoblast maintenance through the expression of EGF homolog neuregulin-7 (nrg-7), a ligand for EGFR-3, affecting the balance of neoblast self-renewal (symmetric or asymmetric division) (34).In other model systems, an important component of the stem-cell niche is the extracellular matrix (ECM) (35). Germline stem cells in Drosophila are anchored to niche supporting cells with ECM on one side, while the opposite side is exposed to differentiation signals, allowing asymmetric cell fate outcomes for self-renewal or differentiation following division (36–38). Few studies have addressed the ECM in planarians, largely due to the lack of genetic tools to manipulate the genome, the absence of antibodies to specific planarian ECM homologs, or the tools required to study cell fate changes. However, the genomes of D. japonica (39–41) and S. mediterranea (41–45), and single-cell RNA-sequencing (scRNA-seq) datasets for S. mediterranea are now available (11, 46–50). A recent study of the planarian matrisome demonstrated that muscle cells are the primary source of many ECM proteins (51), which, together with those produced by neoblasts and supporting parenchymal cells, may constitute components of the neoblast niche. For example, megf6 and hemicentin restrict neoblast’s localization within the parenchyma (51, 52). Functional studies also implicate ECM-modifiers, such as matrix metalloproteases (MMPs) in neoblast migration and regeneration. For example, reducing the activity of the ECM-degrading enzymes mt-mmpA (26, 33), mt-mmpB (53), or mmp-1 (33) impaired neoblast migration, proliferation, or overall tissue growth, respectively. Neoblasts are also likely to interact with ECM components of the niche via cell surface receptors, such as β1 integrin, inactivation of which impairs brain regeneration (54, 55).Here, we identified planarian ECM homologs in silico, followed by systematic functional assessment of 165 ECM and ECM-related genes by RNA interference (RNAi), to determine the effect on neoblast repopulation in planarians challenged by a sublethal dose of γ-irradiation (10). Surprisingly, multiple classes of collagens were shown to have the strongest effects. In particular, we show that the type IV collagens (COLIV) of basement membranes (BMs), were required to regulate the repopulation of neoblasts as well as lineage progression to progenitor cells. Furthermore, our data support an interaction between COLIV and the discoidin domain receptor (DDR) in neurons that activates signaling of NRG-7 in the neoblasts to regulate neoblast self-renewal versus differentiation. Together, these data demonstrate multifaceted regulation of planarian stem cells by ECM components.     

SLFN11 promotes CDT1 degradation by CUL4 in response to replicative DNA damage,while its absence leads to synthetic lethality with ATR/CHK1 inhibitors

Schlafen-11 (SLFN11) inactivation in ∼50% of cancer cells confers broad chemoresistance. To identify therapeutic targets and underlying molecular mechanisms for overcoming chemoresistance, we performed an unbiased genome-wide RNAi screen in SLFN11-WT and -knockout (KO) cells. We found that inactivation of Ataxia Telangiectasia- and Rad3-related (ATR), CHK1, BRCA2, and RPA1 overcome chemoresistance to camptothecin (CPT) in SLFN11-KO cells. Accordingly, we validate that clinical inhibitors of ATR (M4344 and M6620) and CHK1 (SRA737) resensitize SLFN11-KO cells to topotecan, indotecan, etoposide, cisplatin, and talazoparib. We uncover that ATR inhibition significantly increases mitotic defects along with increased CDT1 phosphorylation, which destabilizes kinetochore-microtubule attachments in SLFN11-KO cells. We also reveal a chemoresistance mechanism by which CDT1 degradation is retarded, eventually inducing replication reactivation under DNA damage in SLFN11-KO cells. In contrast, in SLFN11-expressing cells, SLFN11 promotes the degradation of CDT1 in response to CPT by binding to DDB1 of CUL4^CDT2 E3 ubiquitin ligase associated with replication forks. We show that the C terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation. Furthermore, we identify a therapy-relevant ATPase mutant (E669K) of the SLFN11 gene in human TCGA and show that the mutant contributes to chemoresistance and retarded CDT1 degradation. Taken together, our study reveals new chemotherapeutic insights on how targeting the ATR pathway overcomes chemoresistance of SLFN11-deficient cancers. It also demonstrates that SLFN11 irreversibly arrests replication by degrading CDT1 through the DDB1–CUL4^CDT2 ubiquitin ligase.

Schlafen-11 (SLFN11) is an emergent restriction factor against genomic instability acting by eliminating cells with replicative damage (1–6) and potentially acting as a tumor suppressor (6, 7). SLFN11-expressing cancer cells are consistently hypersensitive to a broad range of chemotherapeutic drugs targeting DNA replication, including topoisomerase inhibitors, alkylating agents, DNA synthesis, and poly(ADP-ribose) polymerase (PARP) inhibitors compared to SLFN11-deficient cancer cells, which are chemoresistant (1, 2, 4, 8–17). Profiling SLFN11 expression is being explored for patients to predict survival and guide therapeutic choice (8, 13, 18–24).The Cancer Genome Atlas (TCGA) and cancer cell databases demonstrate that SLFN11 mRNA expression is suppressed in a broad fraction of common cancer tissues and in ∼50% of all established cancer cell lines across multiple histologies (1, 2, 5, 8, 13, 25, 26). Silencing of the SLFN11 gene, like known tumor suppressor genes, is under epigenetic mechanisms through hypermethylation of its promoter region and activation of histone deacetylases (HDACs) (21, 23, 25, 26). A recent study in small-cell lung cancer patient-derived xenograft models also showed that SLFN11 gene silencing is caused by local chromatin condensation related to deposition of H3K27me3 in the gene body of SLFN11 by EZH2, a histone methyltransferase (11). Targeting epigenetic regulators is therefore an attractive combination strategy to overcome chemoresistance of SLFN11-deficient cancers (10, 25, 26). An alternative approach is to attack SLFN11-negative cancer cells by targeting the essential pathways that cells use to overcome replicative damage and replication stress. Along these lines, a prior study showed that inhibition of ATR (Ataxia Telangiectasia- and Rad3-related) kinase reverses the resistance of SLFN11-deficient cancer cells to PARP inhibitors (4). However, targeting the ATR pathway in SLFN11-deficient cells has not yet been fully explored.SLFN11 consists of two functional domains: A conserved nuclease motif in its N terminus and an ATPase motif (putative helicase) in its C terminus (2, 6). The N terminus nuclease has been implicated in the selective degradation of type II tRNAs (including those coding for ATR) and its nuclease structure can be derived from crystallographic analysis of SLFN13 whose N terminus domain is conserved with SLFN11 (27, 28). The C terminus is only present in the group III Schlafen family (24, 29). Its potential ATPase activity and relationship to chemosensitivity to DNA-damaging agents (3–5) imply that the ATPase/helicase of SLFN11 is involved specifically in DNA damage response (DDR) to replication stress. Indeed, inactivation of the Walker B motif of SLFN11 by the mutation E669Q suppresses SLFN11-mediated replication block (5, 30). In addition, SLFN11 contains a binding site for the single-stranded DNA binding protein RPA1 (replication protein A1) at its C terminus (3, 31) and is recruited to replication damage sites by RPA (3, 5). The putative ATPase activity of SLFN11 is not required for this recruitment (5) but is required for blocking the replication helicase complex (CMG-CDC45) and inducing chromatin accessibility at replication origins and promoter sites (5, 30). Based on these studies, our current model is that SLFN11 is recruited to “stressed” replication forks by RPA filaments formed on single-stranded DNA (ssDNA), and that the ATPase/helicase activity of SLFN11 is required for blocking replication progression and remodeling chromatin (5, 30). However, underlying mechanisms of how SLFN11 irreversibly blocks replication in DNA damage are still unclear.Increased RPA-coated ssDNA caused by DNA damage and replication fork stalling also triggers ATR kinase activation, promoting subsequent phosphorylation of CHK1, which transiently halts cell cycle progression and enables DNA repair (32). ATR inhibitors are currently in clinical development in combination with DNA replication damaging drugs (33, 34), such as topoisomerase I (TOP1) inhibitors, which are highly synergistic with ATR inhibitors in preclinical models (35). ATR inhibitors not only inhibit DNA repair, but also lead to unscheduled replication origin firing (36), which kills cancer cells (37, 38) by inducing genomic alterations due to faulty replication and mitotic catastrophe (33).The replication licensing factor CDT1 orchestrates the initiation of replication by assembling prereplication complexes (pre-RC) in G1-phase before cells enter S-phase (39). Once replication is started by loading and activation of the MCM helicase, CDT1 is degraded by the ubiquitin proteasomal pathway to prevent additional replication initiation and ensure precise genome duplication and the firing of each origin only once per cell cycle (39, 40). At the end of G2 and during mitosis, CDT1 levels rise again to control kinetochore-microtubule attachment for accurate chromosome segregation (41). Deregulated overexpression of CDT1 results in rereplication, genome instability, and tumorigenesis (42). The cellular CDT1 levels are tightly regulated by the damage-specific DNA binding protein 1 (DDB1)–CUL4^CDT2 E3 ubiquitin ligase complex in G1-phase (43) and in response to DNA damage (44, 45). How CDT1 is recognized by CUL4^CDT2 in response to DNA damage remains incompletely known.In the present study, starting with a human genome-wide RNAi screen, bioinformatics analyses, and mechanistic validations, we explored synthetic lethal interactions that overcome the chemoresistance of SLFN11-deficient cells to the TOP1 inhibitor camptothecin (CPT). The strongest synergistic interaction was between depletion of the ATR/CHK1-mediated DNA damage response pathways and DNA-damaging agents in SLFN11-deficient cells. We validated and expanded our molecular understanding of combinatorial strategies in SLFN11-deficient cells with the ATR (M4344 and M6620) and CHK1 (SRA737) inhibitors in clinical development (33, 46, 47) and found that ATR inhibition leads to CDT1 stabilization and hyperphosphorylation with mitotic catastrophe. Our study also establishes that SLFN11 promotes the degradation of CDT1 by binding to DDB1, an adaptor molecule of the CUL4^CDT2 E3 ubiquitin ligase complex, leading to an irreversible replication block in response to replicative DNA damage.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:The effect of parathyroid hormone on osteogenesis is mediated partly by osteolectin

We previously described a new osteogenic growth factor, osteolectin/Clec11a, which is required for the maintenance of skeletal bone mass during adulthood. Osteolectin binds to Integrin α11 (Itga11), promoting Wnt pathway activation and osteogenic differentiation by leptin receptor⁺ (LepR⁺) stromal cells in the bone marrow. Parathyroid hormone (PTH) and sclerostin inhibitor (SOSTi) are bone anabolic agents that are administered to patients with osteoporosis. Here we tested whether osteolectin mediates the effects of PTH or SOSTi on bone formation. We discovered that PTH promoted Osteolectin expression by bone marrow stromal cells within hours of administration and that PTH treatment increased serum osteolectin levels in mice and humans. Osteolectin deficiency in mice attenuated Wnt pathway activation by PTH in bone marrow stromal cells and reduced the osteogenic response to PTH in vitro and in vivo. In contrast, SOSTi did not affect serum osteolectin levels and osteolectin was not required for SOSTi-induced bone formation. Combined administration of osteolectin and PTH, but not osteolectin and SOSTi, additively increased bone volume. PTH thus promotes osteolectin expression and osteolectin mediates part of the effect of PTH on bone formation.

The maintenance and repair of the skeleton require the generation of new bone cells throughout adult life. Osteoblasts are relatively short-lived cells that are constantly regenerated, partly by skeletal stem cells within the bone marrow (1). The main source of new osteoblasts in adult bone marrow is leptin receptor-expressing (LepR⁺) stromal cells (2–4). These cells include the multipotent skeletal stem cells that give rise to the fibroblast colony-forming cells (CFU-Fs) in the bone marrow (2), as well as restricted osteogenic progenitors (5) and adipocyte progenitors (6–8). LepR⁺ cells are a major source of osteoblasts for fracture repair (2) and growth factors for hematopoietic stem cell maintenance (9–11).One growth factor synthesized by LepR⁺ cells, as well as osteoblasts and osteocytes, is osteolectin/Clec11a, a secreted glycoprotein of the C-type lectin domain superfamily (5, 12, 13). Osteolectin is an osteogenic factor that promotes the maintenance of the adult skeleton by promoting the differentiation of LepR⁺ cells into osteoblasts. Osteolectin acts by binding to integrin α11β1, which is selectively expressed by LepR⁺ cells and osteoblasts, activating the Wnt pathway (12). Deficiency for either Osteolectin or Itga11 (the gene that encodes integrin α11) reduces osteogenesis during adulthood and causes early-onset osteoporosis in mice (12, 13). Recombinant osteolectin promotes osteogenic differentiation by bone marrow stromal cells in culture and daily injection of mice with osteolectin systemically promotes bone formation.Osteoporosis is a progressive condition characterized by reduced bone mass and increased fracture risk (14). Several factors contribute to osteoporosis development, including aging, estrogen insufficiency, mechanical unloading, and prolonged glucocorticoid use (14). Existing therapies include antiresorptive agents that slow bone loss, such as bisphosphonates (15, 16) and estrogens (17), and anabolic agents that increase bone formation, such as parathyroid hormone (PTH) (18), PTH-related protein (19), and sclerostin inhibitor (SOSTi) (20). While these therapies increase bone mass and reduce fracture risk, they are not a cure.PTH promotes both anabolic and catabolic bone remodeling (21–24). PTH is synthesized by the parathyroid gland and regulates serum calcium levels, partly by regulating bone formation and bone resorption (23–25). PTH1R is a PTH receptor (26, 27) that is strongly expressed by LepR⁺ bone marrow stromal cells (8, 28–30). Recombinant human PTH (Teriparatide; amino acids 1 to 34) and synthetic PTH-related protein (Abaloparatide) are approved by the US Food and Drug Administration (FDA) for the treatment of osteoporosis (19, 31). Daily (intermittent) administration of PTH increases bone mass by promoting the differentiation of osteoblast progenitors, inhibiting osteoblast and osteocyte apoptosis, and reducing sclerostin levels (32–35). PTH promotes osteoblast differentiation by activating Wnt and BMP signaling in bone marrow stromal cells (28, 36, 37), although the mechanisms by which it regulates Wnt pathway activation are complex and uncertain (38).Sclerostin is a secreted glycoprotein that inhibits Wnt pathway activation by binding to LRP5/6, a widely expressed Wnt receptor (7, 8), reducing bone formation (39, 40). Sclerostin is secreted by osteocytes (8, 41), negatively regulating bone formation by inhibiting the differentiation of osteoblasts (41, 42). SOSTi (Romosozumab) is a humanized monoclonal antibody that binds sclerostin, preventing binding to LRP5/6 and increasing Wnt pathway activation and bone formation (43). It is FDA-approved for the treatment of osteoporosis (20, 44) and has activity in rodents in addition to humans (45, 46).The discovery that osteolectin is a bone-forming growth factor raises the question of whether it mediates the effects of PTH or SOSTi on osteogenesis.     

4E-BP2–dependent translation in parvalbumin neurons controls epileptic seizure threshold

The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) integrates multiple signals to regulate critical cellular processes such as mRNA translation, lipid biogenesis, and autophagy. Germline and somatic mutations in mTOR and genes upstream of mTORC1, such as PTEN, TSC1/2, AKT3, PIK3CA, and components of GATOR1 and KICSTOR complexes, are associated with various epileptic disorders. Increased mTORC1 activity is linked to the pathophysiology of epilepsy in both humans and animal models, and mTORC1 inhibition suppresses epileptogenesis in humans with tuberous sclerosis and animal models with elevated mTORC1 activity. However, the role of mTORC1-dependent translation and the neuronal cell types mediating the effect of enhanced mTORC1 activity in seizures remain unknown. The eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and 2 (4E-BP2) are translational repressors downstream of mTORC1. Here we show that the ablation of 4E-BP2, but not 4E-BP1, in mice increases the sensitivity to pentylenetetrazole (PTZ)- and kainic acid (KA)–induced seizures. We demonstrate that the deletion of 4E-BP2 in inhibitory, but not excitatory neurons, causes an increase in the susceptibility to PTZ-induced seizures. Moreover, mice lacking 4E-BP2 in parvalbumin, but not somatostatin or VIP inhibitory neurons exhibit a lowered threshold for seizure induction and reduced number of parvalbumin neurons. A mouse model harboring a human PIK3CA mutation that enhances the activity of the PI3K-AKT pathway (Pik3ca^H1047R-Pvalb) selectively in parvalbumin neurons shows susceptibility to PTZ-induced seizures. Our data identify 4E-BP2 as a regulator of epileptogenesis and highlight the central role of increased mTORC1-dependent translation in parvalbumin neurons in the pathophysiology of epilepsy.

Epilepsy is a prevalent (0.5 to 1% of the general population) (1) heterogeneous neurological disorder affecting all age groups and is characterized by seizures and associated psychological and social stigmas (2–4). Hyperactivation of the mechanistic/mammalian target of rapamycin (mTOR) pathway has been reported in brain lesions of epileptic patients with neurodevelopmental disorders (5, 6), and human genetic studies have shown that mutations in mTOR (7, 8) and other components of its pathway are linked to epileptogenesis (6, 9–13). mTOR is a highly conserved serine/threonine protein kinase that forms two distinct complexes: mTORC1 and mTORC2. mTORC1 integrates multiple environmental and intracellular signals to modulate brain functions by controlling key cellular processes such as mRNA translation, nucleotide, lipid and mitochondrial biogenesis, and autophagy (14, 15). Germline or somatic mutations, which result in enhanced mTORC1 activity, including in PIK3CA, PTEN, AKT3, TSC1/2, RHEB, and MTOR, are associated with neurodevelopmental disorders with epilepsy (16–23). Recent studies have also identified mutations in mTORC1 upstream amino acid–sensing GATOR1-KICSTOR-Rag GTPase pathways as a common cause of epilepsy (24), revealing that mutations in GATOR1 (DEPDC5, NPRL2, and NPRL3) (25, 26) and KICSTOR (ITFG2, KPTN, SZT2, and C12ORF66) (6, 7, 27) genes are often found in epileptic pathologies. The link between the mTORC1 and epilepsy has been recapitulated in animal models with enhanced mTORC1 activity (e.g., Pten^+/−, TSC1/2^+/−, and activating mutations in Pik3ca Nestin-Cre knockin (KI), Akt3 KI, MTOR, and Rheb KI) (18, 20, 23, 28–31) while inhibition of mTORC1 reversed epileptogenesis in TSC1^GFAP-Cre and Pten^+/− mice (20, 31). Notably, the mTORC1 rapalog, everolimus, has been approved by the US Food and Drug Administration (FDA) for the treatment of epilepsy in tuberous sclerosis complex (TSC) patients (32, 33).mTORC1 is a master regulator of mRNA translation. Upon activation, mTORC1 phosphorylates S6 protein kinases 1 and 2 (S6K1/2) and 4E-binding proteins (4E-BPs) (34, 35). In the hypophosphorylated form, 4E-BPs bind and prevent the association of the cap-binding protein eIF4E with the large scaffolding protein eIF4G, thereby inhibiting the formation of the eIF4F complex (composed of eIF4E, eIF4G, and an mRNA helicase eIF4A), which is essential for the initiation of cap-dependent translation. Phosphorylation of 4E-BPs by mTORC1 results in the release of eIF4E from 4E-BPs, allowing eIF4F complex formation and initiation of translation (36, 37). Among the three 4E-BP family members (4E-BP1, 4E-BP2, and 4E-BP3), 4E-BP2 is the most abundant paralog in the mammalian brain (38, 39). A recent study (5) has identified aberrant activation of eIF4E as a major mechanism for translational changes in focal malformations of cortical development (FMCD), a condition that is often caused by brain somatic activating mutations in MTOR and presents with intractable epilepsy in children, accompanied by developmental abnormalities and autism spectrum disorder (ASD) (5, 40–42). Increased eIF4E activity has a pathogenic role in inducing epileptic seizures in FMCD as eIF4E knockdown prevented spontaneous seizures in mTOR Cys1483Tyr and Leu2427Pro mutant mice (5), which show mTORC1 hyperactivation.Despite the progress in understanding the causal link between enhanced mTORC1 activity and epilepsy, the mTORC1-downstream molecular mechanisms promoting epileptogenesis and the cell types mediating the effect on seizure threshold and severity remain poorly understood. In this work, we investigated the role of two main mTORC1-downstream effectors, 4E-BP1 and 4E-BP2, in regulating seizure susceptibility and studied the neuronal cell types mediating epileptogenic effects. We report that mice with whole-body or parvalbumin neuron–specific deletion of 4E-BP2 exhibit reduced threshold and increased severity of epileptic seizures. Moreover, we show that Pik3ca^H1047R-Pvalb mutant mice harboring a conditional parvalbumin neuron–specific KI gain-of-function mutation (H1047R) in the PIK3CA kinase domain are prone to seizures. Collectively, these findings demonstrate a central role of 4E-BP2 and parvalbumin neurons in mediating mTORC1-dependent epileptogenesis, thus expanding our understanding of cell type–specific molecular mechanisms of translation dysregulation in epilepsy and other neurodevelopmental disorders.     

From the Cover: Evidence from South Africa for a protracted end-Permian extinction on land

Earth’s largest biotic crisis occurred during the Permo–Triassic Transition (PTT). On land, this event witnessed a turnover from synapsid- to archosauromorph-dominated assemblages and a restructuring of terrestrial ecosystems. However, understanding extinction patterns has been limited by a lack of high-precision fossil occurrence data to resolve events on submillion-year timescales. We analyzed a unique database of 588 fossil tetrapod specimens from South Africa’s Karoo Basin, spanning ∼4 My, and 13 stratigraphic bin intervals averaging 300,000 y each. Using sample-standardized methods, we characterized faunal assemblage dynamics during the PTT. High regional extinction rates occurred through a protracted interval of ∼1 Ma, initially co-occurring with low origination rates. This resulted in declining diversity up to the acme of extinction near the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary. Regional origination rates increased abruptly above this boundary, co-occurring with high extinction rates to drive rapid turnover and an assemblage of short-lived species symptomatic of ecosystem instability. The “disaster taxon” Lystrosaurus shows a long-term trend of increasing abundance initiated in the latest Permian. Lystrosaurus comprised 54% of all specimens by the onset of mass extinction and 70% in the extinction aftermath. This early Lystrosaurus abundance suggests its expansion was facilitated by environmental changes rather than by ecological opportunity following the extinctions of other species as commonly assumed for disaster taxa. Our findings conservatively place the Karoo extinction interval closer in time, but not coeval with, the more rapid marine event and reveal key differences between the PTT extinctions on land and in the oceans.

Mass extinctions are major perturbations of the biosphere resulting from a wide range of different causes including glaciations and sea level fall (1), large igneous provinces (2), and bolide impacts (3, 4). These events caused permanent changes to Earth’s ecosystems, altering the evolutionary trajectory of life (5). However, links between the broad causal factors of mass extinctions and the biological and ecological disturbances that lead to species extinctions have been difficult to characterize. This is because ecological disturbances unfold on timescales much shorter than the typical resolution of paleontological studies (6), particularly in the terrestrial record (6–8). Coarse-resolution studies have demonstrated key mass extinction phenomena including high extinction rates and lineage turnover (7, 9), changes in species richness (10), ecosystem instability (11), and the occurrence of disaster taxa (12). However, finer time resolutions are central to determining the association and relative timings of these effects, their potential causal factors, and their interrelationships. Achieving these goals represents a key advance in understanding the ecological mechanisms of mass extinctions.The end-Permian mass extinction (ca. 251.9 Ma) was Earth’s largest biotic crisis as measured by taxon last occurrences (13–15). Large outpourings from Siberian Trap volcanism (2) are the likely trigger of calamitous climatic changes, including a runaway greenhouse effect and ocean acidification, which had profound consequences for life on land and in the oceans (16–18). An estimated 81% of marine species (19) and 89% of tetrapod genera became extinct as established Permian ecosystems gave way to those of the Triassic. In the ocean, this included the complete extinction of reef-forming tabulate and rugose corals (20, 21) and significant losses in previously diverse ammonoid, brachiopod, and crinoid families (22). On land, many nonmammalian synapsids became extinct (16), and the glossopterid-dominated floras of Gondwana also disappeared (23). Stratigraphic sequences document a global “coral gap” and “coal gap” (24, 25), suggesting reef and forest ecosystems were rare or absent for up to 5 My after the event (26). Continuous fossil-bearing deposits documenting patterns of turnover across the Permian–Triassic transition (PTT) on land (27) and in the oceans (28) are geographically widespread (29, 30), including marine and continental successions that are known from China (31, 32) and India (33). Continental successions are known from Russia (34), Australia (35), Antarctica (36), and South Africa’s Karoo Basin (Fig. 1 and 37–40), the latter providing arguably the most densely sampled and taxonomically scrutinized (41–43) continental record of the PTT. The main extinction has been proposed to occur at the boundary between two biostratigraphic zones with distinctive faunal assemblages, the Daptocephalus and Lystrosaurus declivis assemblage zones (Fig. 1), which marks the traditional placement of the Permian–Triassic geologic boundary [(37) but see ref. 44]. Considerable research has attempted to understand the anatomy of the PTT in South Africa (38, 39, 45–52) and to place it in the context of biodiversity changes across southern Gondwana (53, 54) and globally (29, 31, 32, 44, 47, 55).Open in a separate windowFig. 1.Map of South Africa depicting the distribution of the four tetrapod fossil assemblage zones (Cistecephalus, Daptocephalus, Lystrosaurus declivis, Cynognathus) and our two study sites where fossils were collected in this study (sites A and B). Regional lithostratigraphy and biostratigraphy within the study interval are shown alongside isotope dilution–thermal ionization mass spectrometry dates retrieved by Rubidge et al., Botha et al., and Gastaldo et al. (37, 44, 80). The traditional (dashed red line) and associated PTB hypotheses for the Karoo Basin (37, 44) are also shown. Although traditionally associated with the PTB, the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary is defined by first appearances of co-occurring tetrapod assemblages, so its position relative to the three PTB hypotheses is unchanged. The Ripplemead member (*) has yet to be formalized by the South African Committee for Stratigraphy.Decades of research have demonstrated the richness of South Africa’s Karoo Basin fossil record, resulting in hundreds of stratigraphically well-documented tetrapod fossils across the PTT (37, 39, 56). This wealth of data has been used qualitatively to identify three extinction phases and an apparent early postextinction recovery phase (39, 45, 51). Furthermore, studies of Karoo community structure and function have elucidated the potential role of the extinction and subsequent recovery in breaking the incumbency of previously dominant clades, including synapsids (11, 57). Nevertheless, understanding patterns of faunal turnover and recovery during the PTT has been limited by the scarcity of quantitative investigations. Previous quantitative studies used coarsely sampled data (i.e., assemblage zone scale, 2 to 3 Ma time intervals) to identify low species richness immediately after the main extinction, potentially associated with multiple “boom and bust” cycles of primary productivity based on δ¹³C variation during the first 5 My of the Triassic (41, 58). However, many details of faunal dynamics in this interval remain unknown. Here, we investigate the dynamics of this major tetrapod extinction at an unprecedented time resolution (on the order of hundreds of thousands of years), using sample-standardized methods to quantify multiple aspects of regional change across the Cistecephalus, Daptocephalus, and Lystrosaurus declivis assemblage zones.     

Violet light suppresses lens-induced myopia via neuropsin (OPN5) in mice

Myopia has become a major public health concern, particularly across much of Asia. It has been shown in multiple studies that outdoor activity has a protective effect on myopia. Recent reports have shown that short-wavelength visible violet light is the component of sunlight that appears to play an important role in preventing myopia progression in mice, chicks, and humans. The mechanism underlying this effect has not been understood. Here, we show that violet light prevents lens defocus–induced myopia in mice. This violet light effect was dependent on both time of day and retinal expression of the violet light sensitive atypical opsin, neuropsin (OPN5). These findings identify Opn5-expressing retinal ganglion cells as crucial for emmetropization in mice and suggest a strategy for myopia prevention in humans.

Myopia (nearsightedness) in school-age children is generally axial myopia, which is the consequence of elongation of the eyeball along the visual axis. This shape change results in blurred vision but can also lead to severe complications including cataract, retinal detachment, myopic choroidal neovascularization, glaucoma, and even blindness (1–3). Despite the current worldwide pandemic of myopia, the mechanism of myopia onset is still not understood (4–8). One hypothesis that has earned a current consensus is the suggestion that a change in the lighting environment of modern society is the cause of myopia (9, 10). Consistent with this, outdoor activity has a protective effect on myopia development (9, 11, 12), though the main reason for this effect is still under debate (7, 12, 13). One explanation is that bright outdoor light can promote the synthesis and release of dopamine in the eye, a myopia-protective neuromodulator (14–16). Another suggestion is that the distinct wavelength composition of sunlight compared with fluorescent or LED (light-emitting diode) artificial lighting may influence myopia progression (9, 10). Animal studies have shown that different wavelengths of light can affect the development of myopia independent of intensity (17, 18). The effects appear to be distinct in different species: for chicks and guinea pigs, blue light showed a protective effect on experimentally induced myopia, while red light had the opposite effect (18–22). For tree shrews and rhesus monkeys, red light is protective, and blue light causes dysregulation of eye growth (23–25).It has been shown that visible violet light (VL) has a protective effect on myopia development in mice, in chick, and in human (10, 26, 27). According to Commission Internationale de l’Eclairage (International Commission on Illumination), VL has the shortest wavelength of visible light (360 to 400 nm). These wavelengths are abundant in outside sunlight but can only rarely be detected inside buildings. This is because the ultraviolet (UV)-protective coating on windows blocks all light below 400 nm and because almost no VL is emitted by artificial light sources (10). Thus, we hypothesized that the lack of VL in modern society is one reason for the myopia boom (9, 10, 26).In this study, we combine a newly developed lens-induced myopia (LIM) model with genetic manipulations to investigate myopia pathways in mice (28, 29). Our data confirm (10, 26) that visible VL is protective but further show that delivery of VL only in the evening is sufficient for the protective effect. In addition, we show that the protective effect of VL on myopia induction requires OPN5 (neuropsin) within the retina. The absence of retinal Opn5 prevents lens-induced, VL-dependent thickening of the choroid, a response thought to play a key role in adjusting the size of the eyeball in both human and animal myopia models (30–33). This report thus identifies a cell type, the Opn5 retinal ganglion cell (RGC), as playing a key role in emmetropization. The requirement for OPN5 also explains why VL has a protective effect on myopia development.     

Patterns of bacterial motility in microfluidics-confining environments

Understanding the motility behavior of bacteria in confining microenvironments, in which they search for available physical space and move in response to stimuli, is important for environmental, food industry, and biomedical applications. We studied the motility of five bacterial species with various sizes and flagellar architectures (Vibrio natriegens, Magnetococcus marinus, Pseudomonas putida, Vibrio fischeri, and Escherichia coli) in microfluidic environments presenting various levels of confinement and geometrical complexity, in the absence of external flow and concentration gradients. When the confinement is moderate, such as in quasi-open spaces with only one limiting wall, and in wide channels, the motility behavior of bacteria with complex flagellar architectures approximately follows the hydrodynamics-based predictions developed for simple monotrichous bacteria. Specifically, V. natriegens and V. fischeri moved parallel to the wall and P. putida and E. coli presented a stable movement parallel to the wall but with incidental wall escape events, while M. marinus exhibited frequent flipping between wall accumulator and wall escaper regimes. Conversely, in tighter confining environments, the motility is governed by the steric interactions between bacteria and the surrounding walls. In mesoscale regions, where the impacts of hydrodynamics and steric interactions overlap, these mechanisms can either push bacteria in the same directions in linear channels, leading to smooth bacterial movement, or they could be oppositional (e.g., in mesoscale-sized meandered channels), leading to chaotic movement and subsequent bacterial trapping. The study provides a methodological template for the design of microfluidic devices for single-cell genomic screening, bacterial entrapment for diagnostics, or biocomputation.

Many motile bacteria live in confining microenvironments (e.g., animal or plant tissue, soil, waste, granulated, and porous materials) and consequently are important to many applications like health [infectious diseases (1, 2), pharmaceuticals (3), and nutrition (4)], agriculture [veterinary (5) and crops (6)], environmental science [photosynthesis (7), biodegradation (8), and bioremediation (9)], and industrial activities [mining (10) and biofouling (11)]. Bacterial motility is essential in the search for available physical space as well as for enabling bacterial taxis in response to external stimuli, such as temperature (12), chemical gradients (13, 14), mechanical cues (15), or magnetic fields (16).To thrive in environments with diverse geometrical and physical characteristics, from open spaces to constraining environments, motile bacteria have evolved a multitude of propelling mechanisms (17), with flagellum-driven being the most common (18, 19). Flagellum-based machinery features various numbers of flagella (20) and designs: monotrichous, lophotrichous, amphitrichous, or peritrichous. The mechanics of this machinery, coupled with cell morphology (21) (e.g., coccus, rod-like, or curved) translates into several motility modes (e.g., turn angle, run-and-tumble, or run-and-flick) (22), and various motility behaviors (e.g., swimming, tumbling, and swarming) (17, 23). Environmental factors (24, 25) (e.g., chemical composition, viscosity, temperature, pH, and the chemistry and the roughness of adjacent surfaces) also influence bacterial motility.“Pure” bacterial motility, unbiased by chemotaxis or fluid flow, was reported near simple flat surfaces (26, 27) and in channels (28–30). Simulations of model bacteria in analogous conditions were also undertaken (31–37), but owing to the complexity of bacterial mechanics (38), modeling from first principles did not provide sufficient understanding to accurately predict movement patterns of different species in complex, confined environments. Consequently, studies of the effects of bacterial geometry in confined geometries were limited to models of simple, monotrichous bacteria with an assumed rigid flagellum (32, 39).Microfluidic devices (40, 41) are commonly used for the manipulation of individual or small populations of cells in micrometer-sized channels for medical diagnostics (42), drug screening (43), cell separation (44, 45), detection and sorting (46), and single-cell genomics (47). While microfluidic structures are used for the study of the motility of mammalian cells (48, 49), and microorganisms [e.g., fungi (50, 51), algae (52), or bacteria (29, 53–56)], these studies typically focus on a single species.To make progress toward a more general understanding of the motility of individual bacterial cells in confining microenvironments, as well as to assess the extent to which the behavior of bacteria with complex architectures can be assimilated with that of the more predictable monotrichous bacteria, the present work investigated the movement of five species (i.e., Vibrio natriegens, Magnetococcus marinus, Pseudomonas putida, Vibrio fischeri, and Escherichia coli) in microfluidic geometries with various levels of confinement and geometrical complexity.     

CYK-1/Formin activation in cortical RhoA signaling centers promotes organismal left–right symmetry breaking

Proper left–right symmetry breaking is essential for animal development, and in many cases, this process is actomyosin-dependent. In Caenorhabditis elegans embryos active torque generation in the actomyosin layer promotes left–right symmetry breaking by driving chiral counterrotating cortical flows. While both Formins and Myosins have been implicated in left–right symmetry breaking and both can rotate actin filaments in vitro, it remains unclear whether active torques in the actomyosin cortex are generated by Formins, Myosins, or both. We combined the strength of C. elegans genetics with quantitative imaging and thin film, chiral active fluid theory to show that, while Non-Muscle Myosin II activity drives cortical actomyosin flows, it is permissive for chiral counterrotation and dispensable for chiral symmetry breaking of cortical flows. Instead, we find that CYK-1/Formin activation in RhoA foci is instructive for chiral counterrotation and promotes in-plane, active torque generation in the actomyosin cortex. Notably, we observe that artificially generated large active RhoA patches undergo rotations with consistent handedness in a CYK-1/Formin–dependent manner. Altogether, we conclude that CYK-1/Formin–dependent active torque generation facilitates chiral symmetry breaking of actomyosin flows and drives organismal left–right symmetry breaking in the nematode worm.

The emergence of left–right asymmetry is essential for normal animal development and, in the majority of animal species, one type of handedness is dominant (1). The actin cytoskeleton plays an instrumental role in establishing the left–right asymmetric body plan of invertebrates like fruit flies (2–6), nematodes (7–11), and pond snails (12–15). Moreover, an increasing number of studies showed that vertebrate left–right patterning also depends on a functional actomyosin cytoskeleton (13, 16–22). Actomyosin-dependent chiral behavior has even been reported in isolated cells (23–28) and such cell-intrinsic chirality has been shown to promote left–right asymmetric morphogenesis of tissues (29, 30), organs (21, 31), and entire embryonic body plans (12, 13, 32, 33). Active force generation in the actin cytoskeleton is responsible for shaping cells and tissues during embryo morphogenesis. Torques are rotational forces with a given handedness and it has been proposed that in plane, active torque generation in the actin cytoskeleton drives chiral morphogenesis (7, 8, 34, 35).What could be the molecular origin of these active torques? The actomyosin cytoskeleton consists of actin filaments, actin-binding proteins, and Myosin motors. Actin filaments are polar polymers with a right-handed helical pitch and are therefore chiral themselves (36, 37). Due to the right-handed pitch of filamentous actin, Myosin motors can rotate actin filaments along their long axis while pulling on them (33, 38–42). Similarly, when physically constrained, members of the Formin family rotate actin filaments along their long axis while elongating them (43). In both cases the handedness of this rotation is determined by the helical nature of the actin polymer. From this it follows that both Formins and Myosins are a potential source of molecular torque generation that could drive cellular and organismal chirality. Indeed, chiral processes across different length scales, and across species, are dependent on Myosins (19), Formins (13–15, 26), or both (7, 8, 21, 44). It is, however, unclear how Formins and Myosins contribute to active torque generation and the emergence chiral processes in developing embryos.In our previous work we showed that the actomyosin cortex of some Caenorhabditis elegans embryonic blastomeres undergoes chiral counterrotations with consistent handedness (7, 35). These chiral actomyosin flows can be recapitulated using active chiral fluid theory that describes the actomyosin layer as a thin-film, active gel that generates active torques (7, 45, 46). Chiral counterrotating cortical flows reorient the cell division axis, which is essential for normal left–right symmetry breaking (7, 47). Moreover, cortical counterrotations with the same handedness have been observed in Xenopus one-cell embryos (32), suggesting that chiral counterrotations are conserved among distant species. Chiral counterrotating actomyosin flow in C. elegans blastomeres is driven by RhoA signaling and is dependent on Non-Muscle Myosin II motor proteins (7). Moreover, the Formin CYK-1 has been implicated in actomyosin flow chirality during early polarization of the zygote as well as during the first cytokinesis (48, 49). Despite having identified a role for Myosins and Formins, the underlying mechanism by which active torques are generated remains elusive.Here we show that the Diaphanous-like Formin, CYK-1/Formin, is a critical determinant for the emergence of actomyosin flow chirality, while Non-Muscle Myosin II (NMY-2) plays a permissive role. Our results show that cortical CYK-1/Formin is recruited by active RhoA signaling foci and promotes active torque generation, which in turn tends to locally rotate the actomyosin cortex clockwise. In the highly connected actomyosin meshwork, a gradient of these active torques drives the emergence of chiral counterrotating cortical flows with uniform handedness, which is essential for proper left–right symmetry breaking. Together, these results provide mechanistic insight into how Formin-dependent torque generation drives cellular and organismal left–right symmetry breaking.     

Conditional destabilization of the TPLATE complex impairs endocytic internalization

In plants, endocytosis is essential for many developmental and physiological processes, including regulation of growth and development, hormone perception, nutrient uptake, and defense against pathogens. Our toolbox to modulate this process is, however, rather limited. Here, we report a conditional tool to impair endocytosis. We generated a partially functional TPLATE allele by substituting the most conserved domain of the TPLATE subunit of the endocytic TPLATE complex (TPC). This substitution destabilizes TPC and dampens the efficiency of endocytosis. Short-term heat treatment increases TPC destabilization and reversibly delocalizes TPLATE from the plasma membrane to aggregates in the cytoplasm. This blocks FM uptake and causes accumulation of various known endocytic cargoes at the plasma membrane. Short-term heat treatment therefore transforms the partially functional TPLATE allele into an effective conditional tool to impair endocytosis. Next to their role in endocytosis, several TPC subunits are also implicated in actin-regulated autophagosomal degradation. Inactivating TPC via the WDX mutation, however, does not impair autophagy, thus enabling specific and reversible modulation of endocytosis in planta.

Endocytosis is an evolutionarily conserved eukaryotic pathway by which extracellular material and plasma membrane (PM) components are internalized via vesicles (1, 2). Clathrin-mediated endocytosis (CME), relying on the scaffolding protein clathrin, is the most prominent and the most studied endocytic pathway (3–5). As clathrin does not interact directly with the PM, nor does it recognize cargoes, adaptor proteins are required to act as essential links between the clathrin coat and the PM (6). In plant cells, material selected for CME is recognized by two adaptor complexes, the adaptor complex 2 (AP-2) and the TPLATE complex (TPC) (7–9). In contrast to TPC, single subunit mutants of AP-2 are viable (7, 8, 10–13) and AP-2 recruitment and dynamics appear to rely on TPC function (8, 14).TPC represents an ancestral adaptor complex, which is however absent in present-day metazoans and yeasts. It was experimentally identified as an octameric complex in Arabidopsis and as a hexametric complex in Dictyostelium (8, 15). Plants, however, are the only eukaryotic supergroup identified so far where TPC is essential for life (8, 15), as knockout or severe knockdown of single subunits of TPC in Arabidopsis leads to pollen or seedling lethality, respectively (8, 13). Two TPC subunits, AtEH1/Pan1 and AtEH2/Pan1, were not associated with the other TPC core components when the complex was forced into the cytoplasm by truncating the TML subunit and did not copurify with the other TSET components in Dictyostelium. This indicates that they may be auxiliary components to the core TPC (8, 15). These AtEH/Pan1 proteins were recently identified as important players in actin-regulated autophagy in plants. AtEH/Pan1 proteins recruit several components of the endocytic machinery to the autophagosomes, and are degraded together with them under stress conditions (16). However, whether this pathway serves to degrade specific cargoes or to regulate the endocytic machinery itself (17), and whether the whole TPC is required for this degradation pathway, remains unclear.Genetic and chemical tools to manipulate endocytosis have been extensively investigated via interfering with the functions of endocytic players, such as clathrin (18–22), adaptor proteins (7, 10–12, 14, 23–25), and dynamin-related proteins (26–30). The chemical inhibitors originally used to affect CME in plants have recently been described to possess undesirable side effects (31) or to affect proteins that are not only specific for endocytosis: for example, clathrin itself, as it is also involved in TGN trafficking (19, 22). The same is true for several genetic tools currently available to affect CME in plants (18, 21, 22, 30). Manipulation of TPC, functioning exclusively at the PM, represents a very good candidate to affect CME more specifically. So far however, there are no chemical tools to target TPC functions or dominant-negative mutants available. Inducible silencing works, but causes seedling lethality and takes several days to become effective (8). The only tools to manipulate TPC function in viable plants consist of knock-down mutants with very mild reduction of expression and consequently similar mild effects on CME (8, 14, 16, 32).     

The Widened Pipe Model of plant hydraulic evolution

Shaping global water and carbon cycles, plants lift water from roots to leaves through xylem conduits. The importance of xylem water conduction makes it crucial to understand how natural selection deploys conduit diameters within and across plants. Wider conduits transport more water but are likely more vulnerable to conduction-blocking gas embolisms and cost more for a plant to build, a tension necessarily shaping xylem conduit diameters along plant stems. We build on this expectation to present the Widened Pipe Model (WPM) of plant hydraulic evolution, testing it against a global dataset. The WPM predicts that xylem conduits should be narrowest at the stem tips, widening quickly before plateauing toward the stem base. This universal profile emerges from Pareto modeling of a trade-off between just two competing vectors of natural selection: one favoring rapid widening of conduits tip to base, minimizing hydraulic resistance, and another favoring slow widening of conduits, minimizing carbon cost and embolism risk. Our data spanning terrestrial plant orders, life forms, habitats, and sizes conform closely to WPM predictions. The WPM highlights carbon economy as a powerful vector of natural selection shaping plant function. It further implies that factors that cause resistance in plant conductive systems, such as conduit pit membrane resistance, should scale in exact harmony with tip-to-base conduit widening. Furthermore, the WPM implies that alterations in the environments of individual plants should lead to changes in plant height, for example, shedding terminal branches and resprouting at lower height under drier climates, thus achieving narrower and potentially more embolism-resistant conduits.

Water transport through plants is a key driver of the carbon and other biogeochemical cycles (1–3) and is a crucial link in plant adaptation to climate and vegetation response to climate change (4–9). The water conducting cells of plants, xylem conduits, widen with distance from the stem tip, and, therefore, taller plants have wider conduits (6, 10–12). Xylem conduits are of two main types: tracheids, found in most gymnosperms, and vessels, found in most flowering plants. Tracheids have intact cell membranes, so water must flow from cell to cell through these membranes. Vessels are made up of cells aligned vertically end to end, with the cell membranes dissolved between successive members, forming a tube. Whatever their differences in structure, wider conduits are beneficial because they conduct more water. Tip-to-base widening is expected to help maintain conductance per unit leaf area constant as an individual plant grows taller, counterbalancing the resistance that would otherwise accrue with increasing conductive path length the individual grows (2, 13). Wider conduits, however, are more vulnerable to embolisms caused by cold and likely drought (8, 14–18) and cost more in terms of carbon for a plant (ref. 1; cf. ref. 19). Embolisms in the xylem even affect transport of photosynthates in the phloem (8, 20). This means that as trees grow taller, conductance, embolism vulnerability, and carbon costs must interrelate in a delicate evolutionary balance.Because of the importance of this balance in plant hydraulic evolution and in forest reactions to climate change (3, 6, 21–23), an important goal of plant biology is to construct models that predict how and why plants deploy conduit diameters throughout their bodies (1, 2, 17, 24–26). Some models predict that conduits should be of uniform diameter (27, 28), while others predict that they should widen tip to base (1, 2, 13, 24, 29, 30). But even current models include untested assumptions and large numbers of parameters, making it difficult to identify the biological causes of the predictions they make. For example, some invoke Da Vinci’s rule, the largely untested assumption that the summed wood area of the twigs is the same as that at the base (24, 26). Other models depict plant conduits as branching as they do in mammalian circulatory systems, but whether this happens along the entire stem in plants is unclear (30–33). There is an expectation that conduit diameter D should widen with distance from the stem tip L following a power-law (D ∝ L^b), but there is no agreement on the value of b, the conduit widening exponent (1, 2). Furthermore, even though within-individual tip-to-base conduit widening has been confirmed in a handful of species (34–36), and the scaling of conduit diameter with plant size across species is consistent with it (6, 10–12, 34), the expectation that conduits should widen similarly within stems across terrestrial vascular plant lineages and habits has yet to be empirically confirmed. Here we present the Widened Pipe Model (WPM), which correctly predicts the form of tip-to-base conduit widening across the span of plant size, life form, and habitat across the terrestrial plant phylogeny.     

Ammonium transporter expression in sperm of the disease vector Aedes aegypti mosquito influences male fertility

The ammonium transporter (AMT)/methylammonium permease (MEP)/Rhesus glycoprotein (Rh) family of ammonia (NH₃/NH₄⁺) transporters has been identified in organisms from all domains of life. In animals, fundamental roles for AMT and Rh proteins in the specific transport of ammonia across biological membranes to mitigate ammonia toxicity and aid in osmoregulation, acid–base balance, and excretion have been well documented. Here, we observed enriched Amt (AeAmt1) mRNA levels within reproductive organs of the arboviral vector mosquito, Aedes aegypti, prompting us to explore the role of AMTs in reproduction. We show that AeAmt1 is localized to sperm flagella during all stages of spermiogenesis and spermatogenesis in male testes. AeAmt1 expression in sperm flagella persists in spermatozoa that navigate the female reproductive tract following insemination and are stored within the spermathecae, as well as throughout sperm migration along the spermathecal ducts during ovulation to fertilize the descending egg. We demonstrate that RNA interference (RNAi)-mediated AeAmt1 protein knockdown leads to significant reductions (∼40%) of spermatozoa stored in seminal vesicles of males, resulting in decreased egg viability when these males inseminate nonmated females. We suggest that AeAmt1 function in spermatozoa is to protect against ammonia toxicity based on our observations of high NH₄⁺ levels in the densely packed spermathecae of mated females. The presence of AMT proteins, in addition to Rh proteins, across insect taxa may indicate a conserved function for AMTs in sperm viability and reproduction in general.

Ammonium transporters (AMTs), methylammonium permeases (MEPs), and Rhesus glycoproteins (Rh proteins) comprise a protein family with three clades, and homologs from each have been identified in virtually all domains of life (1). AMT proteins were first identified in plants (2) with the simultaneous discovery of MEP proteins in fungi (3), followed by Rh proteins in humans (4). Ammonia (NH₃/NH₄⁺) is vital for growth in plants and microorganisms and is retained in some animals for use as an osmolyte (5, 6), for buoyancy (7, 8), and for those lacking sufficient dietary nitrogen (9). In the majority of animals, however, ammonia is the toxic by-product of amino acid and nucleic acid metabolism and, accordingly, requires efficient mechanisms for its regulation, transport, and excretion (10–13). AMT, MEP, and Rh proteins are responsible for the selective movement of ammonia (NH₃) or ammonium (NH₄⁺) across biological membranes, a process that all organisms require. Unlike their vertebrate, bacterial, and fungal counterparts which function as putative NH₃ gas channels (14–18), a myriad of evidence suggests that plant AMT proteins and closely related members in some animals are functionally distinct and facilitate electrogenic ammonium (NH₄⁺) transport (17, 19–22). In contrast to vertebrates which only possess Rh proteins (23), many invertebrates are unique in that they express both AMT and Rh proteins, sometimes in the same cell (24–28). Among insects, the presence of both AMT and Rh proteins has been described in Drosophila melanogaster (29, 30) and mosquitoes that vector disease-causing pathogens, Anopheles gambiae (22, 31) and Aedes aegypti (32, 33). It is unclear whether, in these instances, AMT and Rh proteins can functionally substitute for one another, but in the anal papillae of A. aegypti larvae, knockdown of either Amt or Rh proteins causes decreases in ammonia transport, suggesting that they do not (32–34). To date, studies on ammonia transporter (AMT and Rh) function in insects have focused on ammonia sensing and tasting in sensory structures (22, 30, 31, 35), ammonia detoxification and acid–base balance in muscle, digestive, and excretory organs (15, 36), and ammonia excretion in a variety of organs involved in ion and water homeostasis (9, 24, 32–34).A. aegypti is the primary vector for the transmission of the human arboviral diseases Zika, yellow fever, chikungunya, and dengue virus, which are of global health concern due to rapid increases in the geographical distribution of this species, presently at its highest ever (37, 38). In light of the well-documented evolution of insecticide resistance in mosquitoes (39–42), more recent methods to control disease transmission such as the sterile insect technique (43), transinfection and sterilization of mosquitoes with the bacterium Wolbachia (44), and targeted genome editing rendering adult males sterile (45) have proven effective. These methods take advantage of various aspects of mosquito reproductive biology; however, an understanding of male reproductive biology and the male contributions to female reproductive processes is still in its infancy (46). Here, we describe the expression of an A. aegypti ammonium transporter (AeAmt1) in the sperm during all stages of spermatogenesis, spermiogenesis, and egg fertilization, which is critical for fertility.     

The feeding system of Tiktaalik roseae: an intermediate between suction feeding and biting

Changes to feeding structures are a fundamental component of the vertebrate transition from water to land. Classically, this event has been characterized as a shift from an aquatic, suction-based mode of prey capture involving cranial kinesis to a biting-based feeding system utilizing a rigid skull capable of capturing prey on land. Here we show that a key intermediate, Tiktaalik roseae, was capable of cranial kinesis despite significant restructuring of the skull to facilitate biting and snapping. Lateral sliding joints between the cheek and dermal skull roof, as well as independent mobility between the hyomandibula and palatoquadrate, enable the suspensorium of T. roseae to expand laterally in a manner similar to modern alligator gars and polypterids. This movement can expand the spiracular and opercular cavities during feeding and respiration, which would direct fluid through the feeding apparatus. Detailed analysis of the sutural morphology of T. roseae suggests that the ability to laterally expand the cheek and palate was maintained during the fish-to-tetrapod transition, implying that limited cranial kinesis was plesiomorphic to the earliest limbed vertebrates. Furthermore, recent kinematic studies of feeding in gars demonstrate that prey capture with lateral snapping can synergistically combine both biting and suction, rather than trading off one for the other. A “gar-like” stage in early tetrapod evolution might have been an important intermediate step in the evolution of terrestrial feeding systems by maintaining suction-generation capabilities while simultaneously elaborating a mechanism for biting-based prey capture.

Although suction feeding is a primary mode of prey capture among aquatic vertebrates (1), it is physically impractical on land due to the lower viscosity of air as compared to water (2–4). Terrestrial-feeding vertebrates must resort to other means, such as biting or tongue capture, to procure food (2). Naturally, researchers seeking to understand shifts in feeding strategies in tetrapodomorph vertebrates during the water-to-land transition have focused primarily on whether feeding systems in fossil forms showed adaptations for either suction or biting (2, 5). Generally, plesiomorphic “fish-like” morphology is interpreted as a means to create suction during the feeding cycle, and derived “tetrapod-like” morphology is interpreted as suggestive of biting (5–8). Suction feeding in fish is typically associated with jointed, kinetic skulls that allow for large volumetric expansion to draw in food (1, 9). In contrast, many lineages of modern tetrapods have consolidated skulls, such as mammals, crocodilians, and amphibians, thought to strengthen the skull for biting (9–12). While there is evidence for kinetic joints in the palate and skull roof of multiple early stem tetrapods (6, 13–16), it is uncertain if they represent plesiomorphic holdovers of limited fish-like cranial kinesis (13, 17, 18) or were independently derived mechanisms to improve biting capabilities on land (17, 19).A central challenge of paleontology has been to understand how, and when, transitions in the feeding system of early terrestrial vertebrates occurred. Late Devonian finned tetrapodomorphs, typified by Eusthenopteron foordi, have expansive, kinetic skulls with open sutures, robust gill covers, large hyomandibulae, tall palatal elements, and a jointed neurocranium all thought to be features that play a role in suction feeding (5, 9, 20). In contrast, the Late Devonian limbed tetrapodomorph Acanthostega gunnari has a flat skull, interdigitating sutures between the bones of the skull roof, absent gill covers, reduced hyomandibulae, horizontal palatal elements, and a consolidated neurocranium that are hypothesized to be derived adaptations for biting (5, 6, 21, 22). Analyses of tetrapodomorph lower jaws have produced equivocal results, noting few differences between presumed aquatic and terrestrial forms (7, 8). These results suggest that either a fish-like suction-based feeding mechanism was maintained well into the Carboniferous (7, 8, 23) or that a biting-based feeding mechanism had evolved in water prior to the origin of terrestrial tetrapods (24).To understand how feeding modes shifted among tetrapodomorphs and assess the origin of novel feeding mechanisms in the tetrapod lineage, we use high-resolution microcomputed tomography (µCT) to analyze multiple specimens of a well-preserved elpistostegalian-grade tetrapodomorph, Tiktaalik roseae, and compare the anatomy resolved from those µCT scans to features of other extinct tetrapodomorphs and extant fishes with analogous features. T. roseae is a tetrapodomorph from the Upper Devonian (Frasnian, ∼375 Mya) of Arctic Canada (Ellesmere Island, Nunavut Territory) (25, 26) that, according to most-recent phylogenies (27, 28), is representative of the outgroup of limbed vertebrates (tetrapods). Although plesiomorphic in lower jaw morphology (7, 8, 29), elpistostegalian-grade tetrapodomorphs (a group also including Panderichthys rhombolepis and Elpistostege watsoni) represent a period of rapid cranial evolution that could nevertheless suggest shifts in feeding strategies (5, 26, 30, 31). µCT was performed on four specimens of T. roseae from the Nunavut Fossil Vertebrate Collection (NUFV) consisting of three-dimensionally (3D) preserved palatal material in articulation with the cranium, as well as individual bones from multiple disarticulated specimens (SI Appendix, Table S1). Sutural cross-sections were compared with homologous sutures reported for E. foordi (5, 20) and A. gunnari (5, 21). Cranial joints were compared with possible modern analogs, alligator gar (Atractosteus spatula) (32–34) and ornate bichir (Polypterus ornatipinnis) (5, 32, 35), which were selected on the basis of convergent feeding morphologies with T. roseae. Finally, joints between the palate, hyomandibula, and braincase were modeled with the same kinematic range of motion as reported in A. spatula for comparison purposes (34).