Pmp22 super-enhancer deletion causes tomacula formation and conduction block in peripheral nerves.

Copy number variation of the peripheral nerve myelin gene Peripheral Myelin Protein 22 (PMP22) causes multiple forms of inherited peripheral neuropathy. The duplication of a 1.4 Mb segment surrounding this gene in chromosome 17p12 (c17p12) causes the most common form of Charcot-Marie-Tooth disease type 1A, whereas the reciprocal deletion of this gene causes a separate neuropathy termed hereditary neuropathy with liability to pressure palsies (HNPP). PMP22 is robustly induced in Schwann cells in early postnatal development, and several transcription factors and their cognate regulatory elements have been implicated in coordinating the gene's proper expression. We previously found that a distal super-enhancer domain was important for Pmp22 expression in vitro, with particular impact on a Schwann cell-specific alternative promoter. Here, we investigate the consequences of deleting this super-enhancer in vivo. We find that loss of the super-enhancer in mice reduces Pmp22 expression throughout development and into adulthood, with greater impact on the Schwann cell-specific promoter. Additionally, these mice display tomacula formed by excessive myelin folding, a pathological hallmark of HNPP, as have been previously observed in heterozygous Pmp22 mice as well as sural biopsies from patients with HNPP. Our findings demonstrate a mechanism by which smaller copy number variations, not including the Pmp22 gene, are sufficient to reduce gene expression and phenocopy a peripheral neuropathy caused by the HNPP-associated deletion encompassing PMP22.

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling.

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg's release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin's increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.

A requirement for the neuromodulators octopamine and tyramine in Drosophila melanogaster female sperm storage.

Female sperm storage is common among organisms with internal fertilization. It is important for extended fertility and, in cases of multiple mating, for sperm competition. The physiological mechanisms by which females store and manage stored sperm are poorly understood. Here, we report that the biogenic amines tyramine (TA) and octopamine (OA) in Drosophila melanogaster females play essential roles in sperm storage. D. melanogaster females store sperm in two types of organs, a single seminal receptacle and a pair of spermathecae. We examined sperm storage parameters in females mutant in enzymes required for the biochemical synthesis of tyrosine to TA and TA to OA, respectively. Postmating uterine conformational changes, which are associated with sperm entry and accumulation into storage, were unaffected by the absence of either TA or OA. However, sperm release from storage requires both TA and OA; sperm were retained in storage in both types of mutant females at significantly higher levels than in control flies. Absence of OA inhibited sperm depletion only from the seminal receptacle, whereas absence of both OA and TA perturbed sperm depletion from both storage organ types. We find innervation of the seminal receptacle and spermathecae by octopaminergic-tyraminergic neurons. These findings identify a distinct role for TA and OA in reproduction, regulating the release of sperm from storage, and suggest a mechanism by which Drosophila females actively regulate the release of stored sperm.

Single-cell RNA sequencing of neurofibromas reveals a tumor microenvironment favorable for neural regeneration and immune suppression in a neurofibromatosis type 1 porcine model.

Neurofibromatosis Type 1 (NF1) is one of the most common genetically inherited disorders that affects 1 in 3000 children annually. Clinical manifestations vary widely but nearly always include the development of cutaneous, plexiform and diffuse neurofibromas that are managed over many years. Recent single-cell transcriptomics profiling efforts of neurofibromas have begun to reveal cell signaling processes. However, the cell signaling networks in mature, non-cutaneous neurofibromas remain unexplored. Here, we present insights into the cellular composition and signaling within mature neurofibromas, contrasting with normal adjacent tissue, in a porcine model of NF1 using single-cell RNA sequencing (scRNA-seq) analysis and histopathological characterization. These neurofibromas exhibited classic diffuse-type histologic morphology and expected patterns of S100, SOX10, GFAP, and CD34 immunohistochemistry. The porcine mature neurofibromas closely resemble human neurofibromas histologically and contain all known cellular components of their human counterparts. The scRNA-seq confirmed the presence of all expected cell types within these neurofibromas and identified novel populations of fibroblasts and immune cells, which may contribute to the tumor microenvironment by suppressing inflammation, promoting M2 macrophage polarization, increasing fibrosis, and driving the proliferation of Schwann cells. Notably, we identified tumor-associated IDO1 +/CD274+ (PD-L1) + dendritic cells, which represent the first such observation in any NF1 animal model and suggest the role of the upregulation of immune checkpoints in mature neurofibromas. Finally, we observed that cell types in the tumor microenvironment are poised to promote immune evasion, extracellular matrix reconstruction, and nerve regeneration.

Insect seminal fluid proteins: identification and function.

Seminal fluid proteins (SFPs) produced in reproductive tract tissues of male insects and transferred to females during mating induce numerous physiological and behavioral postmating changes in females. These changes include decreasing receptivity to remating; affecting sperm storage parameters; increasing egg production; and modulating sperm competition, feeding behaviors, and mating plug formation. In addition, SFPs also have antimicrobial functions and induce expression of antimicrobial peptides in at least some insects. Here, we review recent identification of insect SFPs and discuss the multiple roles these proteins play in the postmating processes of female insects.

A non-canonical, interferon-independent signaling activity of cGAMP triggers DNA damage response signaling.

Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), produced by cyclic GMP-AMP synthase (cGAS), stimulates the production of type I interferons (IFN). Here we show that cGAMP activates DNA damage response (DDR) signaling independently of its canonical IFN pathways. Loss of cGAS dampens DDR signaling induced by genotoxic insults. Mechanistically, cGAS activates DDR in a STING-TBK1-dependent manner, wherein TBK1 stimulates the autophosphorylation of the DDR kinase ATM, with the consequent activation of the CHK2-p53-p21 signal transduction pathway and the induction of G1 cell cycle arrest. Despite its stimulatory activity on ATM, cGAMP suppresses homology-directed repair (HDR) through the inhibition of polyADP-ribosylation (PARylation), in which cGAMP reduces cellular levels of NAD+; meanwhile, restoring NAD+ levels abrogates cGAMP-mediated suppression of PARylation and HDR. Finally, we show that cGAMP also activates DDR signaling in invertebrate species lacking IFN (Crassostrea virginica and Nematostella vectensis), suggesting that the genome surveillance mechanism of cGAS predates metazoan interferon-based immunity.

17ß-Estradiol and estrogen receptor α protect right ventricular function in pulmonary hypertension via BMPR2 and apelin.

Women with pulmonary arterial hypertension (PAH) exhibit better right ventricular (RV) function and survival than men; however, the underlying mechanisms are unknown. We hypothesized that 17ß-estradiol (E2), through estrogen receptor α (ER-α), attenuates PAH-induced RV failure (RVF) by upregulating the procontractile and prosurvival peptide apelin via a BMPR2-dependent mechanism. We found that ER-α and apelin expression were decreased in RV homogenates from patients with RVF and from rats with maladaptive (but not adaptive) RV remodeling. RV cardiomyocyte apelin abundance increased in vivo or in vitro after treatment with E2 or ER-α agonist. Studies employing ER-α-null or ER-ß-null mice, ER-α loss-of-function mutant rats, or siRNA demonstrated that ER-α is necessary for E2 to upregulate RV apelin. E2 and ER-α increased BMPR2 in pulmonary hypertension RVs and in isolated RV cardiomyocytes, associated with ER-α binding to the Bmpr2 promoter. BMPR2 is required for E2-mediated increases in apelin abundance, and both BMPR2 and apelin are necessary for E2 to exert RV-protective effects. E2 or ER-α agonist rescued monocrotaline pulmonary hypertension and restored RV apelin and BMPR2. We identified what we believe to be a novel cardioprotective E2/ER-α/BMPR2/apelin axis in the RV. Harnessing this axis may lead to novel RV-targeted therapies for PAH patients of either sex.

Genetic feminization of the thoracic nervous system disrupts courtship song in male Drosophila melanogaster.

Despite the growing research investigating the sex-specific organization of courtship behavior in Drosophila melanogaster, much remains to be understood about the sex-specific organization of the motor circuit that drives this behavior. To investigate the sex-specification of a tightly patterned component of courtship behavior, courtship song, the authors used the GAL4/UAS targeted gene expression system to feminize the ventral ganglia in male Drosophila and analyzed the acoustic properties of courtship song. More specifically, the authors used the thoracic-specifying teashirt promoter (tsh(GAL4)) to express feminizing transgenes specifically in the ventral ganglia. When tsh(GAL4) drove expression of transformer (tra), males were unable to produce prolonged wing extensions. Transgenic expression of an RNAi construct directed against male-specific fruitless (fru(M)) transcripts resulted in normal wing extension, but highly defective courtship song, with 58% of males failing to generate detectable courtship song. Of those that did sing, widths of individual pulses were significantly broader than controls, suggesting thoracic fru(M) function serves to mediate proprioceptive-dependent wing vibration damping during pulse song. However, the most critical signal in the song, the interpulse interval, remained intact. The inability to phenocopy this effect by reducing fru(M) expression in motor neurons and proprioceptive neurons suggests thoracic interneurons require fru(M) for proper pulse song execution and patterning of pulse structure, but not for pulse timing. This provides evidence that genes establishing sex-specific activation of complex behaviors may also be used in establishing pattern-generating motor networks underlying these sex-specific behaviors.

Sex-specific deficits in neurite density and white matter integrity are associated with targeted disruption of exon 2 of the Disc1 gene in the rat.

Diffusion tensor imaging (DTI) has provided remarkable insight into our understanding of white matter microstructure and brain connectivity across a broad spectrum of psychiatric disease. While DTI and other diffusion weighted magnetic resonance imaging (MRI) methods have clarified the axonal contribution to the disconnectivity seen in numerous psychiatric diseases, absent from these studies are quantitative indices of neurite density and orientation that are especially important features in regions of high synaptic density that would capture the synaptic contribution to the psychiatric disease state. Here we report the application of neurite orientation dispersion and density imaging (NODDI), an emerging microstructure imaging technique, to a novel Disc1 svΔ2 rat model of psychiatric illness and demonstrate the complementary and more specific indices of tissue microstructure found in NODDI than those reported by DTI. Our results demonstrate global and sex-specific changes in white matter microstructural integrity and deficits in neurite density as a consequence of the Disc1 svΔ2 genetic variation and highlight the application of NODDI and quantitative measures of neurite density and neurite dispersion in psychiatric disease.

Complete B Cell Deficiency Reduces Allograft Inflammation and Intragraft Macrophages in a Rat Kidney Transplant Model.

BACKGROUND: Increasingly, it is being appreciated that B cells have broad roles beyond the humoral response and are able to contribute to and regulate inflammation. The specific role of B cells in the pathogenesis of early allograft inflammation remains unclear. METHODS: To address this question, we generated B cell-deficient (B) Lewis rats via clustered regularly interspaced short palindromic repeats (CRISPR) technology. In a full mismatch transplant model, kidneys from Brown Norway donors were transplanted into B Lewis recipients or wild type Lewis recipients. T cell-mediated rejection was attenuated with cyclosporine. RESULTS: Renal inflammation was reduced at 1 week after transplant (Banff scores for interstitial inflammation, microvascular inflammation, glomerulitis, and C4d) in allografts from B recipients. The reduction in interstitial inflammation was predominantly due to a decline in graft infiltrating macrophages. Intragraft T-cell numbers remained unchanged. In addition, B-cell deficiency was associated with increased T regulatory cells and reduced splenic T follicular helper cells at baseline; and significantly increased intragraft and splenic IL-10 mRNA levels after transplant. In vitro, B and wild type splenic T cells produced similar levels of IFN-γ in response to T cell-specific activation. CONCLUSIONS: B-cell deficiency in this model produced an anti-inflammatory phenotype with a shift toward regulatory T-cell populations, production of anti-inflammatory cytokines (IL-10), and a reduction in allograft inflammation. These findings define a role for B cells to influence the cell populations and mediators involved in the pathogenesis of early allograft inflammation.

Mouse Tmem135 mutation reveals a mechanism involving mitochondrial dynamics that leads to age-dependent retinal pathologies.

While the aging process is central to the pathogenesis of age-dependent diseases, it is poorly understood at the molecular level. We identified a mouse mutant with accelerated aging in the retina as well as pathologies observed in age-dependent retinal diseases, suggesting that the responsible gene regulates retinal aging, and its impairment results in age-dependent disease. We determined that a mutation in the transmembrane 135 (Tmem135) is responsible for these phenotypes. We observed localization of TMEM135 on mitochondria, and imbalance of mitochondrial fission and fusion in mutant Tmem135 as well as Tmem135 overexpressing cells, indicating that TMEM135 is involved in the regulation of mitochondrial dynamics. Additionally, mutant retina showed higher sensitivity to oxidative stress. These results suggest that the regulation of mitochondrial dynamics through TMEM135 is critical for protection from environmental stress and controlling the progression of retinal aging. Our study identified TMEM135 as a critical link between aging and age-dependent diseases.

CRISPR-Cas9 Genome Editing in Drosophila.

The CRISPR-Cas9 system has transformed genome engineering of model organisms from possible to practical. CRISPR-Cas9 can be readily programmed to generate sequence-specific double-strand breaks that disrupt targeted loci when repaired by error-prone non-homologous end joining (NHEJ) or to catalyze precise genome modification through homology-directed repair (HDR). Here we describe a streamlined approach for rapid and highly efficient engineering of the Drosophila genome via CRISPR-Cas9-mediated HDR. In this approach, transgenic flies expressing Cas9 are injected with plasmids to express guide RNAs (gRNAs) and positively marked donor templates. We detail target-site selection; gRNA plasmid generation; donor template design and construction; and the generation, identification, and molecular confirmation of engineered lines. We also present alternative approaches and highlight key considerations for experimental design. The approach outlined here can be used to rapidly and reliably generate a variety of engineered modifications, including genomic deletions and replacements, precise sequence edits, and incorporation of protein tags.

Reproductive hacking. A male seminal protein acts through intact reproductive pathways in female Drosophila.

Seminal proteins are critical for reproductive success in all animals that have been studied. Although seminal proteins have been identified in many taxa, and female reproductive responses to receipt of these proteins have been documented in several, little is understood about the mechanisms by which seminal proteins affect female reproductive physiology. To explore this topic, we investigated how a Drosophila seminal protein, ovulin, increases ovulation rate in mated females. Ovulation is a relatively simple physiological process, with known female regulators: previous studies have shown that ovulation rate is promoted by the neuromodulator octopamine (OA) in D. melanogaster and other insects. We found that ovulin stimulates ovulation by increasing OA signaling in the female. This finding supports a model in which a male seminal protein acts through "hacking" a well-conserved, regulatory system females use to adjust reproductive output, rather than acting downstream of female mechanisms of control or in parallel pathways altogether. We also discuss similarities between 2 forms of intersexual control of behavior through chemical communication: seminal proteins and pheromones.

Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila.

We and others recently demonstrated that the readily programmable CRISPR/Cas9 system can be used to edit the Drosophila genome. However, most applications to date have relied on aberrant DNA repair to stochastically generate frameshifting indels and adoption has been limited by a lack of tools for efficient identification of targeted events. Here we report optimized tools and techniques for expanded application of the CRISPR/Cas9 system in Drosophila through homology-directed repair (HDR) with double-stranded DNA (dsDNA) donor templates that facilitate complex genome engineering through the precise incorporation of large DNA sequences, including screenable markers. Using these donors, we demonstrate the replacement of a gene with exogenous sequences and the generation of a conditional allele. To optimize efficiency and specificity, we generated transgenic flies that express Cas9 in the germline and directly compared HDR and off-target cleavage rates of different approaches for delivering CRISPR components. We also investigated HDR efficiency in a mutant background previously demonstrated to bias DNA repair toward HDR. Finally, we developed a web-based tool that identifies CRISPR target sites and evaluates their potential for off-target cleavage using empirically rooted rules. Overall, we have found that injection of a dsDNA donor and guide RNA-encoding plasmids into vasa-Cas9 flies yields the highest efficiency HDR and that target sites can be selected to avoid off-target mutations. Efficient and specific CRISPR/Cas9-mediated HDR opens the door to a broad array of complex genome modifications and greatly expands the utility of CRISPR technology for Drosophila research.

Molecular social interactions: Drosophila melanogaster seminal fluid proteins as a case study.

Studies of social behavior generally focus on interactions between two or more individual animals. However, these interactions are not simply between whole animals, but also occur between molecules that were produced by the interacting individuals. Such "molecular social interactions" can both influence and be influenced by the organismal-level social interactions. We illustrate this by reviewing the roles played by seminal fluid proteins (Sfps) in molecular social interactions between males and females of the fruit fly Drosophila melanogaster. Sfps, which are produced by males and transferred to females during mating, are involved in inherently social interactions with female-derived molecules, and they influence social interactions between males and females and between a female's past and potential future mates. Here, we explore four examples of molecular social interactions involving D. melanogaster Sfps: processes that influence mating, sperm storage, ovulation, and ejaculate transfer. We consider the molecular and organismal players involved in each interaction and the consequences of their interplay for the reproductive success of both sexes. We conclude with a discussion of the ways in which Sfps can both shape and be shaped by (in an evolutionary sense) the molecular social interactions in which they are involved.