Unrestricted Ketogenic Diet Feeding Enhances Epithelial Ovarian Cancer Growth In Vivo.

The ketogenic diet (KD) is hypothesized to impact tumor progression by altering tumor metabolism. In this study, we assessed the impact of an unrestricted KD on epithelial ovarian cancer (EOC) tumor growth, gene expression, and metabolite concentration in a mouse model. ID8 EOC cells, which were syngeneic with C57Bl/6J mouse strain and transfected with luciferase (ID8-luc), were injectedand monitored for tumor development. Female mice were fed either a strict KD, a high fat/low carbohydrate (HF/LC) diet, or a low fat/high carbohydrate (LF/HC) diet (n = 10 mice per group) ad libitum. EOC tumor growth was monitored weekly, and tumor burden was determined based on luciferase fluorescence (photons/second). At the endpoint (42 days), tumors were collected and processed for RNA sequencing. Plasma and tumor metabolites were evaluated using LC-MS. The KD-fed mice exhibited a statistically significant increase in tumor progression in comparison to the HF/LC- and LF/HC-fed groups (9.1 vs. 2.0 vs. 3.1-fold, respectively, p < 0.001). The EOC tumors of the KD-fed mice exhibited significant enrichment of the peroxisome proliferator-activated receptor (PPAR) signaling and fatty acid metabolism pathways based on the RNA sequencing analysis when compared to the LF/HC- and HF/LC-fed mice. Thus, unrestricted KD diet enhanced tumor progression in our mouse EOC model. KD was associated with the upregulation of fatty acid metabolism and regulation pathways, as well as enrichment of fatty acid and glutamine metabolites.

Transcriptional Behavior of Regulatory T Cells Predicts IBD Patient Responses to Vedolizumab Therapy.

BACKGROUND: Inflammatory bowel disease (IBD) involves chronic T cell-mediated inflammatory responses. Vedolizumab (VDZ), a monoclonal antibody against α4ß7 integrin, inhibits lymphocyte extravasation into intestinal mucosae and is effective in ulcerative colitis (UC) and Crohn's disease (CD). AIM: We sought to identify immune cell phenotypic and gene expression signatures that related to response to VDZ. METHODS: Peripheral blood (PBMC) and lamina propria mononuclear cells (LPMCs) were analyzed by flow cytometry and Cytofkit. Sorted CD4 + memory (Tmem) or regulatory T (Treg) cells from PBMC and LPMC were analyzed by RNA sequencing (RNA-seq). Clinical response (≥2-point drop in partial Mayo scores [UC] or Harvey-Bradshaw index [CD]) was assessed 14 to 22 weeks after VDZ initiation. Machine-learning models were used to infer combinatorial traits that predicted response to VDZ. RESULTS: Seventy-one patients were enrolled: 37 received VDZ and 21 patients remained on VDZ >2 years. Fourteen of 37 patients (38%; 8 UC, 6 CD) responded to VDZ. Immune cell phenotypes and CD4 + Tmem and Treg transcriptional behaviors were most divergent between the ileum and colon, irrespective of IBD subtype or inflammation status. Vedolizumab treatment had the greatest impact on Treg metabolic pathways, and response was associated with increased expression of genes involved in oxidative phosphorylation. The strongest clinical predictor of VDZ efficacy was concurrent use of thiopurines. Mucosal tissues offered the greatest number of response-predictive biomarkers, whereas PBMC Treg-expressed genes were the best predictors in combinatorial models of response. CONCLUSIONS: Mucosal and peripheral blood immune cell phenotypes and transcriptional profiles can inform VDZ efficacy and inform new opportunities for combination therapies.

Vedolizumab (VDZ) is effective in the treatment of IBD. Immunophenotyping and RNAseq of T cells were used to inform its mechanism of action. Changes in T regulatory cells in the periphery and mucosa have the greatest relationship to VDZ response.

Notch Inhibition Promotes Regeneration and Immunosuppression Supports Cone Survival in a Zebrafish Model of Inherited Retinal Dystrophy.

Photoreceptor degeneration leads to irreversible vision loss in humans with retinal dystrophies such as retinitis pigmentosa. Whereas photoreceptor loss is permanent in mammals, zebrafish possesses the ability to regenerate retinal neurons and restore visual function. Following acute damage, Müller glia (MG) re-enter the cell cycle and produce multipotent progenitors whose progeny differentiate into mature neurons. Both MG reprogramming and proliferation of retinal progenitor cells require reactive microglia and associated inflammatory signaling. Paradoxically, in zebrafish models of retinal degeneration, photoreceptor death does not induce the MG to reprogram and regenerate lost cells. Here, we used male and female zebrafish cep290 mutants to demonstrate that progressive cone degeneration generates an immune response but does not stimulate MG proliferation. Acute light damage triggered photoreceptor regeneration in cep290 mutants but cones were only restored to prelesion densities. Using irf8 mutant zebrafish, we found that the chronic absence of microglia reduced inflammation and rescued cone degeneration in cep290 mutants. Finally, single-cell RNA-sequencing revealed sustained expression of notch3 in MG of cep290 mutants and inhibition of Notch signaling induced MG to re-enter the cell cycle. Our findings provide new insights on the requirements for MG to proliferate and the potential for immunosuppression to prolong photoreceptor survival.SIGNIFICANCE STATEMENT Inherited retinal degenerations (IRDs) are genetic diseases that lead to the progressive loss of photoreceptors and the permanent loss of vision. Zebrafish can regenerate photoreceptors after acute injury by reprogramming Müller glia (MG) into stem-like cells that produce retinal progenitors, but this regenerative process fails to occur in zebrafish models of IRDs. Here, we show that Notch pathway inhibition can promote photoreceptor regeneration in models of progressive degeneration and that immunosuppression can prevent photoreceptor loss. These results offer insight into the pathways that promote MG-dependent regeneration and the role of inflammation in photoreceptor degeneration.

Endogenous Syngap1 alpha splice forms promote cognitive function and seizure protection.

Loss-of-function variants in SYNGAP1 cause a developmental encephalopathy defined by cognitive impairment, autistic features, and epilepsy. SYNGAP1 splicing leads to expression of distinct functional protein isoforms. Splicing imparts multiple cellular functions of SynGAP proteins through coding of distinct C-terminal motifs. However, it remains unknown how these different splice sequences function in vivo to regulate neuronal function and behavior. Reduced expression of SynGAP-α1/2 C-terminal splice variants in mice caused severe phenotypes, including reduced survival, impaired learning, and reduced seizure latency. In contrast, upregulation of α1/2 expression improved learning and increased seizure latency. Mice expressing α1-specific mutations, which disrupted SynGAP cellular functions without altering protein expression, promoted seizure, disrupted synapse plasticity, and impaired learning. These findings demonstrate that endogenous SynGAP isoforms with α1/2 spliced sequences promote cognitive function and impart seizure protection. Regulation of SynGAP-αexpression or function may be a viable therapeutic strategy to broadly improve cognitive function and mitigate seizure.

Mesenchymal stem cell-derived extracellular vesicles reduce senescence and extend health span in mouse models of aging.

Aging drives progressive loss of the ability of tissues to recover from stress, partly through loss of somatic stem cell function and increased senescent burden. We demonstrate that bone marrow-derived mesenchymal stem cells (BM-MSCs) rapidly senescence and become dysfunctional in culture. Injection of BM-MSCs from young mice prolonged life span and health span, and conditioned media (CM) from young BM-MSCs rescued the function of aged stem cells and senescent fibroblasts. Extracellular vesicles (EVs) from young BM-MSC CM extended life span of Ercc1-/- mice similarly to injection of young BM-MSCs. Finally, treatment with EVs from MSCs generated from human ES cells reduced senescence in culture and in vivo, and improved health span. Thus, MSC EVs represent an effective and safe approach for conferring the therapeutic effects of adult stem cells, avoiding the risks of tumor development and donor cell rejection. These results demonstrate that MSC-derived EVs are highly effective senotherapeutics, slowing the progression of aging, and diseases driven by cellular senescence.

Nonsense-mediated decay controls the reactivation of the oncogenic herpesviruses EBV and KSHV.

The oncogenic human herpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are the causative agents of multiple malignancies. A hallmark of herpesviruses is their biphasic life cycle consisting of latent and lytic infection. In this study, we identified that cellular nonsense-mediated decay (NMD), an evolutionarily conserved RNA degradation pathway, critically regulates the latent-to-lytic switch of EBV and KSHV infection. The NMD machinery suppresses EBV and KSHV Rta transactivator expression and promotes maintenance of viral latency by targeting the viral polycistronic transactivator transcripts for degradation through the recognition of features in their 3' UTRs. Treatment with a small-molecule NMD inhibitor potently induced reactivation in a variety of EBV- and KSHV-infected cell types. In conclusion, our results identify NMD as an important host process that controls oncogenic herpesvirus reactivation, which may be targeted for the therapeutic induction of lytic reactivation and the eradication of tumor cells.

SYNGAP1 Controls the Maturation of Dendrites, Synaptic Function, and Network Activity in Developing Human Neurons.

SYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. De novo loss-of-function variants in this gene cause a neurodevelopmental disorder defined by cognitive impairment, social-communication disorder, and early-onset seizures. Cell biological studies in mouse and rat neurons have shown that Syngap1 regulates developing excitatory synapse structure and function, with loss-of-function variants driving formation of larger dendritic spines and stronger glutamatergic transmission. However, studies to date have been limited to mouse and rat neurons. Therefore, it remains unknown how SYNGAP1 loss of function impacts the development and function of human neurons. To address this, we used CRISPR/Cas9 technology to ablate SYNGAP1 protein expression in neurons derived from a commercially available induced pluripotent stem cell line (hiPSC) obtained from a human female donor. Reducing SynGAP protein expression in developing hiPSC-derived neurons enhanced dendritic morphogenesis, leading to larger neurons compared with those derived from isogenic controls. Consistent with larger dendritic fields, we also observed a greater number of morphologically defined excitatory synapses in cultures containing these neurons. Moreover, neurons with reduced SynGAP protein had stronger excitatory synapses and expressed synaptic activity earlier in development. Finally, distributed network spiking activity appeared earlier, was substantially elevated, and exhibited greater bursting behavior in SYNGAP1 null neurons. We conclude that SYNGAP1 regulates the postmitotic maturation of human neurons made from hiPSCs, which influences how activity develops within nascent neural networks. Alterations to this fundamental neurodevelopmental process may contribute to the etiology of SYNGAP1-related disorders.SIGNIFICANCE STATEMENTSYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. While this gene is well studied in rodent neurons, its function in human neurons remains unknown. We used CRISPR/Cas9 technology to disrupt SYNGAP1 protein expression in neurons derived from an induced pluripotent stem cell line. We found that induced neurons lacking SynGAP expression exhibited accelerated dendritic morphogenesis, increased accumulation of postsynaptic markers, early expression of synapse activity, enhanced excitatory synaptic strength, and early onset of neural network activity. We conclude that SYNGAP1 regulates the postmitotic differentiation rate of developing human neurons and disrupting this process impacts the function of nascent neural networks. These altered developmental processes may contribute to the etiology of SYNGAP1 disorders.

Genomic insights of body plan transitions from bilateral to pentameral symmetry in Echinoderms.

Echinoderms are an exceptional group of bilaterians that develop pentameral adult symmetry from a bilaterally symmetric larva. However, the genetic basis in evolution and development of this unique transformation remains to be clarified. Here we report newly sequenced genomes, developmental transcriptomes, and proteomes of diverse echinoderms including the green sea urchin (L. variegatus), a sea cucumber (A. japonicus), and with particular emphasis on a sister group of the earliest-diverged echinoderms, the feather star (A. japonica). We learned that the last common ancestor of echinoderms retained a well-organized Hox cluster reminiscent of the hemichordate, and had gene sets involved in endoskeleton development. Further, unlike in other animal groups, the most conserved developmental stages were not at the body plan establishing phase, and genes normally involved in bilaterality appear to function in pentameric axis development. These results enhance our understanding of the divergence of protostomes and deuterostomes almost 500 Mya.

Comparative Analysis of Cleavage Specificities of Immobilized Porcine Pepsin and Nepenthesin II under Hydrogen/Deuterium Exchange Conditions.

Hydrogen/Deuterium Exchange (HDX) coupled with Mass Spectrometry (HDX-MS) is a sensitive and robust method to probe protein conformational changes and protein-ligand interactions. HDX-MS relies on successful proteolytic digestion of target proteins under acidic conditions to localize perturbations in exchange behavior to protein structure. The ability of the protease to produce small peptides and overlapping fragments and provide sufficient coverage of the protein sequence is essential for localizing regions of interest. While the acid protease pepsin has been the enzyme of choice for HDX-MS studies, recently, it was shown that aspartic proteases from carnivorous pitcher plants of the genus Nepenthes are active under low-pH conditions and cleave at basic residues that are "forbidden" in peptic digests. In this report, we describe the utility of one of these enzymes, Nepenthesin II (NepII), in a HDX-MS workflow. A systematic and statistical analysis of data from 11 proteins (6391 amino acid residues) digested with immobilized porcine pepsin or NepII under conditions compatible with HDX-MS was performed to examine protease cleavage specificities. The cleavage of pepsin was most influenced by the amino acid residue at position P1. Phe, Leu, and Met are favored residues, each with a cleavage probability of greater than 40%. His, Lys, Arg, or Pro residues prohibit cleavage when found at the P1 position. In contrast, NepII offers advantageous cleavage to all basic residues and produces shortened peptides that could improve the spatial resolution in HDX-MS studies.

Expression of SARS-CoV-2 Entry Molecules ACE2 and TMPRSS2 in the Gut of Patients With IBD.

BACKGROUND: Patients with inflammatory bowel disease (IBD) have intestinal inflammation and are treated with immune-modulating medications. In the face of the coronavirus disease-19 pandemic, we do not know whether patients with IBD will be more susceptible to infection or disease. We hypothesized that the viral entry molecules angiotensin I converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) are expressed in the intestine. We further hypothesized that their expression could be affected by inflammation or medication usage. METHODS: We examined the expression of Ace2 and Tmprss2 by quantitative polymerase chain reacion in animal models of IBD. Publicly available data from organoids and mucosal biopsies from patients with IBD were examined for expression of ACE2 and TMPRSS2. We conducted RNA sequencing for CD11b-enriched cells and peripheral and lamina propria T-cells from well-annotated patient samples. RESULTS: ACE2 and TMPRSS2 were abundantly expressed in the ileum and colon and had high expression in intestinal epithelial cells. In animal models, inflammation led to downregulation of epithelial Ace2. Expression of ACE2 and TMPRSS2 was not increased in samples from patients with compared with those of control patients. In CD11b-enriched cells but not T-cells, the level of expression of ACE2 and TMPRSS2 in the mucosa was comparable to other functional mucosal genes and was not affected by inflammation. Anti-tumor necrosis factor drugs, vedolizumab, ustekinumab, and steroids were linked to significantly lower expression of ACE2 in CD11b-enriched cells. CONCLUSIONS: The viral entry molecules ACE2 and TMPRSS2 are expressed in the ileum and colon. Patients with IBD do not have higher expression during inflammation; medical therapy is associated with lower levels of ACE2. These data provide reassurance for patients with IBD.

Synapse Formation Activates a Transcriptional Program for Persistent Enhancement in the Bi-directional Transport of Mitochondria.

Mechanisms that regulate the bi-directional transport of mitochondria in neurons for maintaining functional synaptic connections are poorly understood. Here, we show that in the pre-synaptic sensory neurons of the Aplysia gill withdrawal reflex, the formation of functional synapses leads to persistent enhancement in the flux of bi-directional mitochondrial transport. In the absence of a functional synapse, activation of cAMP signaling is sufficient to enhance bi-directional transport in sensory neurons. Furthermore, persistent enhancement in transport does not depend on NMDA and AMPA receptor signaling nor signaling from the post-synaptic neuronal cell body, but it is dependent on transcription and protein synthesis in the pre-synaptic neuron. We identified ∼4,000 differentially enriched transcripts in pre-synaptic neurons, suggesting a long-term change in the transcriptional program produced by synapse formation. These results provide insights into the regulation of bi-directional mitochondrial transport for synapse maintenance.

Long noncoding RNA GM12371 acts as a transcriptional regulator of synapse function.

Despite the growing evidence suggesting that long noncoding RNAs (lncRNAs) are critical regulators of several biological processes, their functions in the nervous system remain elusive. We have identified an lncRNA, GM12371, in hippocampal neurons that is enriched in the nucleus and necessary for synaptic communication, synapse density, synapse morphology, and dendritic tree complexity. Mechanistically, GM12371 regulates the expression of several genes involved in neuronal development and differentiation, as well as expression of specific lncRNAs and their cognate mRNA targets. Furthermore, we find that cAMP-PKA signaling up-regulates the expression of GM12371 and that its expression is essential for the activity-dependent changes in synaptic transmission in hippocampal neurons. Taken together, our data establish a key role for GM12371 in regulating synapse function.

Diverse pathways of escape from all well-characterized VRC01-class broadly neutralizing HIV-1 antibodies.

Many broadly neutralizing antibodies (bNAbs) against human immunodeficiency virus type 1 (HIV-1) were shown effective in animal models, and are currently evaluated in clinical trials. However, use of these antibodies in humans is hampered by the rapid emergence of resistant viruses. Here we show that soft-randomization can be used to accelerate the parallel identification of viral escape pathways. As a proof of principle, we soft-randomized the epitope regions of VRC01-class bNAbs in replication-competent HIV-1 and selected for resistant variants. After only a few passages, a surprisingly diverse population of antibody-resistant viruses emerged, bearing both novel and previously described escape mutations. We observed that the escape variants resistant to some VRC01-class bNAbs are resistant to most other bNAbs in the same class, and that a subset of variants was completely resistant to every well characterized VRC01-class bNAB, including VRC01, NIH45-46, 3BNC117, VRC07, N6, VRC-CH31, and VRC-PG04. Thus, our data demonstrate that soft randomization is a suitable approach for accelerated detection of viral escape, and highlight the challenges inherent in administering or attempting to elicit VRC01-class antibodies.

Protein kinase A activity leads to the extension of the acrosomal process in starfish sperm.

Acrosomal vesicles (AVs) of sperm undergo exocytosis during the acrosome reaction, which is immediately followed by the actin polymerization-dependent extension of an acrosomal process (AP) in echinoderm sperm. In the starfish Asterias amurensis, a large proteoglycan, acrosome reaction-inducing substance (ARIS), together with asteroidal sperm-activating peptide (asterosap) and/or cofactor for ARIS, induces the acrosome reaction. Asterosap induces a transient elevation of intracellular cGMP and Ca2+ levels, and, together with ARIS, causes a sustained increase in intracellular cAMP and Ca2+ . Yet, the contribution of signaling molecules downstream of cAMP and Ca2+ in inducing AV exocytosis and AP extension remain unknown. A modified acrosome reaction assay was used here to differentiate between AV exocytosis and AP extension in starfish sperm, leading to the discovery that Protein kinase A (PKA) inhibitors block AP extension but not AV exocytosis. Additionally, PKA-mediated phosphorylation of target proteins occurs, and these substrates localize at the base of the AP, demonstrating that PKA activation regulates an AP extension step during the acrosome reaction. The major PKA substrate was further identified, from A. amurensis and Asterias forbesi sperm, as a novel protein containing six PKA phosphorylation motifs. This protein, referred to as PKAS1, likely plays a key role in AP actin polymerization during the acrosome reaction.

Phylogenomic analyses of Echinodermata support the sister groups of Asterozoa and Echinozoa.

Echinoderms (sea urchins, sea stars, brittle stars, sea lilies and sea cucumbers) are a group of diverse organisms, second in number within deuterostome species to only the chordates. Echinoderms serve as excellent model systems for developmental biology due to their diverse developmental mechanisms, tractable laboratory use, and close phylogenetic distance to chordates. In addition, echinoderms are very well represented in the fossil record, including some larval features, making echinoderms a valuable system for studying evolutionary development. The internal relationships of Echinodermata have not been consistently supported across phylogenetic analyses, however, and this has hindered the study of other aspects of their biology. In order to test echinoderm phylogenetic relationships, we sequenced 23 de novo transcriptomes from all five clades of echinoderms. Using multiple phylogenetic methods at a variety of sampling depths we have constructed a well-supported phylogenetic tree of Echinodermata, including support for the sister groups of Asterozoa (sea stars and brittle stars) and Echinozoa (sea urchins and sea cucumbers). These results will help inform developmental and evolutionary studies specifically in echinoderms and deuterostomes in general.

Two-pore channels function in calcium regulation in sea star oocytes and embryos.

Egg activation at fertilization is an excellent process for studying calcium regulation. Nicotinic acid adenine dinucleotide-phosphate (NAADP), a potent calcium messenger, is able to trigger calcium release, likely through two-pore channels (TPCs). Concomitantly, a family of ectocellular enzymes, the ADP-ribosyl cyclases (ARCs), has emerged as being able to change their enzymatic mode from one of nucleotide cyclization in formation of cADPR to a base-exchange reaction in the generation of NAADP. Using sea star oocytes we gain insights into the functions of endogenously expressed TPCs and ARCs in the context of the global calcium signals at fertilization. Three TPCs and one ARC were found in the sea star (Patiria miniata) that were localized in the cortex of the oocytes and eggs. PmTPCs were localized in specialized secretory organelles called cortical granules, and PmARCs accumulated in a different, unknown, set of vesicles, closely apposed to the cortical granules in the egg cortex. Using morpholino knockdown of PmTPCs and PmARC in the oocytes, we found that both calcium regulators are essential for early embryo development, and that knockdown of PmTPCs leads to aberrant construction of the fertilization envelope at fertilization and changes in cortical granule pH. The calcium signals at fertilization are not significantly altered when individual PmTPCs are silenced, but the timing and shape of the cortical flash and calcium wave are slightly changed when the expression of all three PmTPCs is perturbed concomitantly, suggesting a cooperative activity among TPC isoforms in eliciting calcium signals that may influence localized physiological activities.

Deadenylase depletion protects inherited mRNAs in primordial germ cells.

A crucial event in animal development is the specification of primordial germ cells (PGCs), which become the stem cells that create sperm and eggs. How PGCs are created provides a valuable paradigm for understanding stem cells in general. We find that the PGCs of the sea urchin Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of inherited mRNAs. Enrichment of several germline determinants in the PGCs requires the RNA-binding protein Nanos to target the transcript that encodes CNOT6, a deadenylase, for degradation in the PGCs, thereby creating a stable environment for RNA. Misexpression of CNOT6 in the PGCs results in their failure to retain Seawi transcripts and Vasa protein. Conversely, broad knockdown of CNOT6 expands the domain of Seawi RNA as well as exogenous reporters. Thus, Nanos-dependent spatially restricted CNOT6 differential expression is used to selectively localize germline RNAs to the PGCs. Our findings support a 'time capsule' model of germline determination, whereby the PGCs are insulated from differentiation by retaining the molecular characteristics of the totipotent egg and early embryo.

The biology of the germ line in echinoderms.

The formation of the germ line in an embryo marks a fresh round of reproductive potential. The developmental stage and location within the embryo where the primordial germ cells (PGCs) form, however, differs markedly among species. In many animals, the germ line is formed by an inherited mechanism, in which molecules made and selectively partitioned within the oocyte drive the early development of cells that acquire this material to a germ-line fate. In contrast, the germ line of other animals is fated by an inductive mechanism that involves signaling between cells that directs this specialized fate. In this review, we explore the mechanisms of germ-line determination in echinoderms, an early-branching sister group to the chordates. One member of the phylum, sea urchins, appears to use an inherited mechanism of germ-line formation, whereas their relatives, the sea stars, appear to use an inductive mechanism. We first integrate the experimental results currently available for germ-line determination in the sea urchin, for which considerable new information is available, and then broaden the investigation to the lesser-known mechanisms in sea stars and other echinoderms. Even with this limited insight, it appears that sea stars, and perhaps the majority of the echinoderm taxon, rely on inductive mechanisms for germ-line fate determination. This enables a strongly contrasted picture for germ-line determination in this phylum, but one for which transitions between different modes of germ-line determination might now be experimentally addressed.

PIWI proteins and PIWI-interacting RNAs function in Hydra somatic stem cells.

PIWI proteins and their bound PIWI-interacting RNAs (piRNAs) are found in animal germlines and are essential for fertility, but their functions outside of the gonad are not well understood. The cnidarian Hydra is a simple metazoan with well-characterized stem/progenitor cells that provides a unique model for analysis of PIWI function. Here we report that Hydra has two PIWI proteins, Hydra PIWI (Hywi) and Hydra PIWI-like (Hyli), both of which are expressed in all Hydra stem/progenitor cells, but not in terminally differentiated cells. We identified ∼15 million piRNAs associated with Hywi and/or Hyli and found that they exhibit the ping-pong signature of piRNA biogenesis. Hydra PIWI proteins are strictly cytoplasmic and thus likely act as posttranscriptional regulators. To explore this function, we generated a Hydra transcriptome for piRNA mapping. piRNAs map to transposons with a 25- to 35-fold enrichment compared with the abundance of transposon transcripts. By sequencing the small RNAs specific to the interstitial, ectodermal, and endodermal lineages, we found that the targeting of transposons appears to be largely restricted to the interstitial lineage. We also identified putative nontransposon targets of the pathway unique to each lineage. Finally we demonstrate that hywi function is essential in the somatic epithelial lineages. This comprehensive analysis of the PIWI-piRNA pathway in the somatic stem/progenitor cells of a nonbilaterian animal suggests that this pathway originated with broader stem cell functionality.