Osteocalcin (OCN), the most abundant noncollagenous protein in the bone matrix, is reported to be a bone-derived endocrine hormone with wide-ranging effects on many aspects of physiology, including glucose metabolism and male fertility. Many of these observations were made using an OCN-deficient mouse allele (Osc-) in which the 2 OCN-encoding genes in mice, Bglap and Bglap2, were deleted in ES cells by homologous recombination. Here we describe mice with a new Bglap and Bglap2 double-knockout (dko) allele (Bglap/2p.Pro25fs17Ter) that was generated by CRISPR/Cas9-mediated gene editing. Mice homozygous for this new allele do not express full-length Bglap or Bglap2 mRNA and have no immunodetectable OCN in their serum. FTIR imaging of cortical bone in these homozygous knockout animals finds alterations in the collagen maturity and carbonate to phosphate ratio in the cortical bone, compared with wild-type littermates. However, μCT and 3-point bending tests do not find differences from wild-type littermates with respect to bone mass and strength. In contrast to the previously reported OCN-deficient mice with the Osc-allele, serum glucose levels and male fertility in the OCN-deficient mice with the Bglap/2pPro25fs17Ter allele did not have significant differences from wild-type littermates. We cannot explain the absence of endocrine effects in mice with this new knockout allele. Possible explanations include the effects of each mutated allele on the transcription of neighboring genes, or differences in genetic background and environment. So that our findings can be confirmed and extended by other interested investigators, we are donating this new Bglap and Bglap2 double-knockout strain to the Jackson Laboratories for academic distribution.

In mammals, parathyroid hormone (PTH) is a key regulator of extracellular calcium and inorganic phosphorus homeostasis. Although the parathyroid glands were thought to be the only source of PTH, extra-parathyroid PTH production in the thymus, which shares a common origin with parathyroids during organogenesis, has been proposed to provide an auxiliary source of PTH, resulting in a higher than expected survival rate for aparathyroid Gcm2⁻/⁻ mutants. However, the developmental ontogeny and cellular identity of these "thymic" PTH-expressing cells is unknown. We found that the lethality of aparathyroid Gcm2⁻/⁻ mutants was affected by genetic background without relation to serum PTH levels, suggesting a need to reconsider the physiological function of thymic PTH. We identified two sources of extra-parathyroid PTH in wild-type mice. Incomplete separation of the parathyroid and thymus organs during organogenesis resulted in misplaced, isolated parathyroid cells that were often attached to the thymus; this was the major source of thymic PTH in normal mice. Analysis of thymus and parathyroid organogenesis in human embryos showed a broadly similar result, indicating that these results may provide insight into human parathyroid development. In addition, medullary thymic epithelial cells (mTECs) express PTH in a Gcm2-independent manner that requires TEC differentiation and is consistent with expression as a self-antigen for negative selection. Genetic or surgical removal of the thymus indicated that thymus-derived PTH in Gcm2⁻/⁻ mutants did not provide auxiliary endocrine function. Our data show conclusively that the thymus does not serve as an auxiliary source of either serum PTH or parathyroid function. We further show that the normal process of parathyroid organogenesis in both mice and humans leads to the generation of multiple small parathyroid clusters in addition to the main parathyroid glands, that are the likely source of physiologically relevant "thymic PTH."

Mammary epithelial progenitors are the normal cell-of-origin of breast cancer. We previously defined a population of p27+ quiescent hormone-responsive progenitor cells in the normal human breast whose frequency associates with breast cancer risk. Here, we describe that deletion of the Cdkn1b gene encoding the p27 cyclin-dependent kinase inhibitor in the estrogen-induced mammary tumor-susceptible ACI rat strain leads to a decrease in the relative frequencies of Cd49b+ mammary luminal epithelial progenitors and pregnancy-related differentiation. We show by comprehensive gene expression profiling of purified progenitor and differentiated mammary epithelial cell populations that p27 deletion has the most pronounced effects on luminal progenitors. Cdkn1b-/- females have decreased fertility, but rats that are able to get pregnant had normal litter size and were able to nurse their pups implying that loss of p27 in ACI rats does not completely abrogate ovarian function and lactation. Reciprocal mammary gland transplantation experiments indicate that the p27-loss-induced changes in mammary epithelial cells are not only caused by alterations in their intrinsic properties, but are likely due to altered hormonal signaling triggered by the perturbed systemic endocrine environment observed in Cdkn1b-/- females. We also observed a decrease in the frequency of mammary epithelial cells positive for progesterone receptor (Pr) and FoxA1, known direct transcriptional targets of the estrogen receptor (Erα), and an increase in phospho-Stat5 positive cells commonly induced by prolactin (Prl). Characterization of genome-wide Pr chromatin binding revealed distinct binding patterns in mammary epithelial cells of Cdkn1b+/+ and Cdkn1b-/- females and enrichment in genes with known roles in Notch, ErbB, leptin, and Erα signaling and regulation of G1-S transition. Our data support a role for p27 in regulating the pool size of hormone-responsive luminal progenitors that could impact breast cancer risk.

Specialized endocrine cells produce and release steroid hormones that govern development, metabolism and reproduction. In order to synthesize steroids, all the genes in the biosynthetic pathway must be coordinately turned on in steroidogenic cells. In Drosophila, the steroid producing endocrine cells are located in the prothoracic gland (PG) that releases the steroid hormone ecdysone. The transcriptional regulatory network that specifies the unique PG specific expression pattern of the ecdysone biosynthetic genes remains unknown. Here, we show that two transcription factors, the POU-domain Ventral veins lacking (Vvl) and the nuclear receptor Knirps (Kni), have essential roles in the PG during larval development. Vvl is highly expressed in the PG during embryogenesis and is enriched in the gland during larval development, suggesting that Vvl might function as a master transcriptional regulator in this tissue. Vvl and Kni bind to PG specific cis-regulatory elements that are required for expression of the ecdysone biosynthetic genes. Knock down of either vvl or kni in the PG results in a larval developmental arrest due to failure in ecdysone production. Furthermore, Vvl and Kni are also required for maintenance of TOR/S6K and prothoracicotropic hormone (PTTH) signaling in the PG, two major pathways that control ecdysone biosynthesis and PG cell growth. We also show that the transcriptional regulator, Molting defective (Mld), controls early biosynthetic pathway steps. Our data show that Vvl and Kni directly regulate ecdysone biosynthesis by transcriptional control of biosynthetic gene expression and indirectly by affecting PTTH and TOR/S6K signaling. This provides new insight into the regulatory network of transcription factors involved in the coordinated regulation of steroidogenic cell specific transcription, and identifies a new function of Vvl and Knirps in endocrine cells during post-embryonic development.

The conserved TOR kinase signaling network links nutrient availability to cell, tissue and body growth in animals. One important growth-regulatory target of TOR signaling is ribosome biogenesis. Studies in yeast and mammalian cell culture have described how TOR controls rRNA synthesis-a limiting step in ribosome biogenesis-via the RNA Polymerase I transcription factor TIF-IA. However, the contribution of TOR-dependent ribosome synthesis to tissue and body growth in animals is less clear. Here we show in Drosophila larvae that ribosome synthesis in muscle is required non-autonomously to maintain normal body growth and development. We find that amino acid starvation and TOR inhibition lead to reduced levels of TIF-IA, and decreased rRNA synthesis in larval muscle. When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth. This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2-a secreted factor that binds and inhibits dILP activity-from muscle. We also observed that maintaining TIF-IA levels in muscle could partially reverse the starvation-mediated suppression of systemic insulin signaling. Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function. These data suggest that muscle ribosome synthesis functions as a nutrient-dependent checkpoint for overall body growth: in nutrient rich conditions, TOR is required to maintain levels of TIF-IA and ribosome synthesis to promote high levels of systemic insulin, but under conditions of starvation stress, reduced muscle ribosome synthesis triggers an endocrine response that limits systemic insulin signaling to restrict growth and maintain homeostasis.

It is now established that the central nervous system plays an important role in regulating whole body metabolism and energy balance. However, the extent to which sensory systems relay environmental information to modulate metabolic events in peripheral tissues has remained poorly understood. In addition, it has been challenging to map the molecular mechanisms underlying discrete sensory modalities with respect to their role in lipid metabolism. In previous work our lab has identified instructive roles for serotonin signaling as a surrogate for food availability, as well as oxygen sensing, in the control of whole body metabolism. In this study, we now identify a role for a pair of pheromone-sensing neurons in regulating fat metabolism in C. elegans, which has emerged as a tractable and highly informative model to study the neurobiology of metabolism. A genetic screen revealed that GPA-3, a member of the Gα family of G proteins, regulates body fat content in the intestine, the major metabolic organ for C. elegans. Genetic and reconstitution studies revealed that the potent body fat phenotype of gpa-3 null mutants is controlled from a pair of neurons called ADL(L/R). We show that cAMP functions as the second messenger in the ADL neurons, and regulates body fat stores via the neurotransmitter acetylcholine, from downstream neurons. We find that the pheromone ascr#3, which is detected by the ADL neurons, regulates body fat stores in a GPA-3-dependent manner. We define here a third sensory modality, pheromone sensing, as a major regulator of body fat metabolism. The pheromone ascr#3 is an indicator of population density, thus we hypothesize that pheromone sensing provides a salient 'denominator' to evaluate the amount of food available within a population and to accordingly adjust metabolic rate and body fat levels.

In order to identify genetic factors related to thyroid cancer susceptibility, we adopted a candidate gene approach. We studied tag- and putative functional SNPs in genes involved in thyroid cell differentiation and proliferation, and in genes found to be differentially expressed in thyroid carcinoma. A total of 768 SNPs in 97 genes were genotyped in a Spanish series of 615 cases and 525 controls, the former comprising the largest collection of patients with this pathology from a single population studied to date. SNPs in an LD block spanning the entire FOXE1 gene showed the strongest evidence of association with papillary thyroid carcinoma susceptibility. This association was validated in a second stage of the study that included an independent Italian series of 482 patients and 532 controls. The strongest association results were observed for rs1867277 (OR[per-allele] = 1.49; 95%CI = 1.30-1.70; P = 5.9x10(-9)). Functional assays of rs1867277 (NM_004473.3:c.-283G>A) within the FOXE1 5' UTR suggested that this variant affects FOXE1 transcription. DNA-binding assays demonstrated that, exclusively, the sequence containing the A allele recruited the USF1/USF2 transcription factors, while both alleles formed a complex in which DREAM/CREB/alphaCREM participated. Transfection studies showed an allele-dependent transcriptional regulation of FOXE1. We propose a FOXE1 regulation model dependent on the rs1867277 genotype, indicating that this SNP is a causal variant in thyroid cancer susceptibility. Our results constitute the first functional explanation for an association identified by a GWAS and thereby elucidate a mechanism of thyroid cancer susceptibility. They also attest to the efficacy of candidate gene approaches in the GWAS era.

Acquired resistance to endocrine therapy is responsible for half of the therapeutic failures in the treatment of breast cancer. Recent findings have implicated increased expression of the ETS transcription factor ELF5 as a potential modulator of estrogen action and driver of endocrine resistance, and here we provide the first insight into the mechanisms by which ELF5 modulates estrogen sensitivity. Using chromatin immunoprecipitation sequencing we found that ELF5 binding overlapped with FOXA1 and ER at super enhancers, enhancers and promoters, and when elevated, caused FOXA1 and ER to bind to new regions of the genome, in a pattern that replicated the alterations to the ER/FOXA1 cistrome caused by the acquisition of resistance to endocrine therapy. RNA sequencing demonstrated that these changes altered estrogen-driven patterns of gene expression, the expression of ER transcription-complex members, and 6 genes known to be involved in driving the acquisition of endocrine resistance. Using rapid immunoprecipitation mass spectrometry of endogenous proteins, and proximity ligation assays, we found that ELF5 interacted physically with members of the ER transcription complex, such as DNA-PKcs. We found 2 cases of endocrine-resistant brain metastases where ELF5 levels were greatly increased and ELF5 patterns of gene expression were enriched, compared to the matched primary tumour. Thus ELF5 alters ER-driven gene expression by modulating the ER/FOXA1 cistrome, by interacting with it, and by modulating the expression of members of the ER transcriptional complex, providing multiple mechanisms by which ELF5 can drive endocrine resistance.

As model organism-based research shifts from forward to reverse genetics approaches, largely due to the ease of genome editing technology, a low frequency of abnormal phenotypes is being observed in lines with mutations predicted to lead to deleterious effects on the encoded protein. In zebrafish, this low frequency is in part explained by compensation by genes of redundant or similar function, often resulting from the additional round of teleost-specific whole genome duplication within vertebrates. Here we offer additional explanations for the low frequency of mutant phenotypes. We analyzed mRNA processing in seven zebrafish lines with mutations expected to disrupt gene function, generated by CRISPR/Cas9 or ENU mutagenesis methods. Five of the seven lines showed evidence of altered mRNA processing: one through a skipped exon that did not lead to a frame shift, one through nonsense-associated splicing that did not lead to a frame shift, and three through the use of cryptic splice sites. These results highlight the need for a methodical analysis of the mRNA produced in mutant lines before making conclusions or embarking on studies that assume loss of function as a result of a given genomic change. Furthermore, recognition of the types of adaptations that can occur may inform the strategies of mutant generation.

Honey bees (Apis mellifera) provide a system for studying social and food-related behavior. A caste of workers performs age-related tasks: young bees (nurses) usually feed the brood and other adult bees inside the nest, while older bees (foragers) forage outside for pollen, a protein/lipid source, or nectar, a carbohydrate source. The workers' transition from nursing to foraging and their foraging preferences correlate with differences in gustatory perception, metabolic gene expression, and endocrine physiology including the endocrine factors vitellogenin (Vg) and juvenile hormone (JH). However, the understanding of connections among social behavior, energy metabolism, and endocrine factors is incomplete. We used RNA interference (RNAi) to perturb the gene network of Vg and JH to learn more about these connections through effects on gustation, gene transcripts, and physiology. The RNAi perturbation was achieved by single and double knockdown of the genes ultraspiracle (usp) and vg, which encode a putative JH receptor and Vg, respectively. The double knockdown enhanced gustatory perception and elevated hemolymph glucose, trehalose, and JH. We also observed transcriptional responses in insulin like peptide 1 (ilp1), the adipokinetic hormone receptor (AKHR), and cGMP-dependent protein kinase (PKG, or "foraging gene" Amfor). Our study demonstrates that the Vg-JH regulatory module controls changes in carbohydrate metabolism, but not lipid metabolism, when worker bees shift from nursing to foraging. The module is also placed upstream of ilp1, AKHR, and PKG for the first time. As insulin, adipokinetic hormone (AKH), and PKG pathways influence metabolism and gustation in many animals, we propose that honey bees have conserved pathways in carbohydrate metabolism and conserved connections between energy metabolism and gustatory perception. Thus, perhaps the bee can make general contributions to the understanding of food-related behavior and metabolic disorders.

Diapause, a programmed developmental arrest primarily induced by seasonal environmental changes, is very common in the animal kingdom, and found in vertebrates and invertebrates alike. Diapause provides an adaptive advantage to animals, as it increases the odds of surviving adverse conditions. In insects, individuals perceive photoperiodic cues and modify endocrine signaling to direct reproductive diapause traits, such as ovary arrest and increased fat accumulation. However, it remains unclear as to which endocrine factors are involved in this process and how they regulate the onset of reproductive diapause. Here, we found that the long day-mediated drop in the concentration of the steroid hormone ecdysone is essential for the preparation of photoperiodic reproductive diapause in Colaphellus bowringi, an economically important cabbage beetle. The diapause-inducing long-day condition reduced the expression of ecdysone biosynthetic genes, explaining the drop in the titer of 20-hydroxyecdysone (20E, the active form of ecdysone) in female adults. Application of exogenous 20E induced vitellogenesis and ovarian development but reduced fat accumulation in the diapause-destined females. Knocking down the ecdysone receptor (EcR) in females destined for reproduction blocked reproductive development and induced diapause traits. RNA-seq and hormone measurements indicated that 20E stimulates the production of juvenile hormone (JH), a key endocrine factor in reproductive diapause. To verify this, we depleted three ecdysone biosynthetic enzymes via RNAi, which confirmed that 20E is critical for JH biosynthesis and reproductive diapause. Importantly, impairing Met function, a component of the JH intracellular receptor, partially blocked the 20E-regulated reproductive diapause preparation, indicating that 20E regulates reproductive diapause in both JH-dependent and -independent manners. Finally, we found that 20E deficiency decreased ecdysis-triggering hormone signaling and reduced JH production, thereby inducing diapause. Together, these results suggest that 20E signaling is a pivotal regulator that coordinates reproductive plasticity in response to environmental inputs.

The feeling of hunger or satiety results from integration of the sensory nervous system with other physiological and metabolic cues. This regulates food intake, maintains homeostasis and prevents disease. In C. elegans, chemosensory neurons sense food and relay information to the rest of the animal via hormones to control food-related behaviour and physiology. Here we identify a new component of this system, SKN-1B which acts as a central food-responsive node, ultimately controlling satiety and metabolic homeostasis. SKN-1B, an ortholog of mammalian NF-E2 related transcription factors (Nrfs), has previously been implicated with metabolism, respiration and the increased lifespan incurred by dietary restriction. Here we show that SKN-1B acts in two hypothalamus-like ASI neurons to sense food, communicate nutritional status to the organism, and control satiety and exploratory behaviours. This is achieved by SKN-1B modulating endocrine signalling pathways (IIS and TGF-β), and by promoting a robust mitochondrial network. Our data suggest a food-sensing and satiety role for mammalian Nrf proteins.

Chemicals that are highly prevalent in our environment, such as phthalates and pesticides, have been linked to problems associated with reproductive health. However, rapid assessment of their impact on reproductive health and understanding how they cause such deleterious effects, remain challenging due to their fast-growing numbers and the limitations of various current toxicity assessment model systems. Here, we performed a high-throughput screen in C. elegans to identify chemicals inducing aneuploidy as a result of impaired germline function. We screened 46 chemicals that are widely present in our environment, but for which effects in the germline remain poorly understood. These included pesticides, phthalates, and chemicals used in hydraulic fracturing and crude oil processing. Of the 46 chemicals tested, 41% exhibited levels of aneuploidy higher than those detected for bisphenol A (BPA), an endocrine disruptor shown to affect meiosis, at concentrations correlating well with mammalian reproductive endpoints. We further examined three candidates eliciting aneuploidy: dibutyl phthalate (DBP), a likely endocrine disruptor and frequently used plasticizer, and the pesticides 2-(thiocyanomethylthio) benzothiazole (TCMTB) and permethrin. Exposure to these chemicals resulted in increased embryonic lethality, elevated DNA double-strand break (DSB) formation, activation of p53/CEP-1-dependent germ cell apoptosis, chromosomal abnormalities in oocytes at diakinesis, impaired chromosome segregation during early embryogenesis, and germline-specific alterations in gene expression. This study indicates that this high-throughput screening system is highly reliable for the identification of environmental chemicals inducing aneuploidy, and provides new insights into the impact of exposure to three widely used chemicals on meiosis and germline function.

Appropriate nutrient response is essential for growth and reproduction. Under favorable nutrient conditions, the C. elegans nuclear receptor DAF-12 is activated by dafachronic acids, hormones that commit larvae to reproductive growth. Here, we report that in addition to its well-studied role in controlling developmental gene expression, the DAF-12 endocrine system governs expression of a gene network that stimulates the aerobic catabolism of fatty acids. Thus, activation of the DAF-12 transcriptome coordinately mobilizes energy stores to permit reproductive growth. DAF-12 regulation of this metabolic gene network is conserved in the human parasite, Strongyloides stercoralis, and inhibition of specific steps in this network blocks reproductive growth in both of the nematodes. Our study provides a molecular understanding for metabolic adaptation of nematodes to their environment, and suggests a new therapeutic strategy for treating parasitic diseases.

The visual system converts the distribution and wavelengths of photons entering the eye into patterns of neuronal activity, which then drive motor and endocrine behavioral responses. The gene products important for visual processing by a living and behaving vertebrate animal have not been identified in an unbiased fashion. Likewise, the genes that affect development of the nervous system to shape visual function later in life are largely unknown. Here we have set out to close this gap in our understanding by using a forward genetic approach in zebrafish. Moving stimuli evoke two innate reflexes in zebrafish larvae, the optomotor and the optokinetic response, providing two rapid and quantitative tests to assess visual function in wild-type (WT) and mutant animals. These behavioral assays were used in a high-throughput screen, encompassing over half a million fish. In almost 2,000 F2 families mutagenized with ethylnitrosourea, we discovered 53 recessive mutations in 41 genes. These new mutations have generated a broad spectrum of phenotypes, which vary in specificity and severity, but can be placed into only a handful of classes. Developmental phenotypes include complete absence or abnormal morphogenesis of photoreceptors, and deficits in ganglion cell differentiation or axon targeting. Other mutations evidently leave neuronal circuits intact, but disrupt phototransduction, light adaptation, or behavior-specific responses. Almost all of the mutants are morphologically indistinguishable from WT, and many survive to adulthood. Genetic linkage mapping and initial molecular analyses show that our approach was effective in identifying genes with functions specific to the visual system. This collection of zebrafish behavioral mutants provides a novel resource for the study of normal vision and its genetic disorders.

XLalphas and ALEX are structurally unrelated mammalian proteins translated from alternative overlapping reading frames of a single transcript. Not only are they encoded by the same locus, but a specific XLalphas/ALEX interaction is essential for G-protein signaling in neuroendocrine cells. A disruption of this interaction leads to abnormal human phenotypes, including mental retardation and growth deficiency. The region of overlap between the two reading frames evolves at a remarkable speed: the divergence between human and mouse ALEX polypeptides makes them virtually unalignable. To trace the evolution of this puzzling locus, we sequenced it in apes, Old World monkeys, and a New World monkey. We show that the overlap between the two reading frames and the physical interaction between the two proteins force the locus to evolve in an unprecedented way. Namely, to maintain two overlapping protein-coding regions the locus is forced to have high GC content, which significantly elevates its intrinsic evolutionary rate. However, the two encoded proteins cannot afford to change too quickly relative to each other as this may impair their interaction and lead to severe physiological consequences. As a result XLalphas and ALEX evolve in an oscillating fashion constantly balancing the rates of amino acid replacements. This is the first example of a rapidly evolving locus encoding interacting proteins via overlapping reading frames, with a possible link to the origin of species-specific neurological differences.

Exposure to endocrine disruptors is associated with developmental defects. One compound of concern, to which humans are widely exposed, is bisphenol A (BPA). In model organisms, BPA exposure is linked to metabolic disorders, infertility, cancer, and behavior anomalies. Recently, BPA exposure has been linked to DNA methylation changes, indicating that epigenetic mechanisms may be relevant. We investigated effects of exposure on genomic imprinting in the mouse as imprinted genes are regulated by differential DNA methylation and aberrant imprinting disrupts fetal, placental, and postnatal development. Through allele-specific and quantitative real-time PCR analysis, we demonstrated that maternal BPA exposure during late stages of oocyte development and early stages of embryonic development significantly disrupted imprinted gene expression in embryonic day (E) 9.5 and 12.5 embryos and placentas. The affected genes included Snrpn, Ube3a, Igf2, Kcnq1ot1, Cdkn1c, and Ascl2; mutations and aberrant regulation of these genes are associated with imprinting disorders in humans. Furthermore, the majority of affected genes were expressed abnormally in the placenta. DNA methylation studies showed that BPA exposure significantly altered the methylation levels of differentially methylated regions (DMRs) including the Snrpn imprinting control region (ICR) and Igf2 DMR1. Moreover, exposure significantly reduced genome-wide methylation levels in the placenta, but not the embryo. Histological and immunohistochemical examinations revealed that these epigenetic defects were associated with abnormal placental development. In contrast to this early exposure paradigm, exposure outside of the epigenetic reprogramming window did not cause significant imprinting perturbations. Our data suggest that early exposure to common environmental compounds has the potential to disrupt fetal and postnatal health through epigenetic changes in the embryo and abnormal development of the placenta.

Saliva plays important roles in insect feeding, but its roles in insect reproduction were rarely reported. Here we reported that the knockdown of a salivary gland-specific gene NlG14 disrupted the reproduction through inhibiting the ovulation of the brown planthopper (BPH), Nilaparvata lugens (Stål), one of the most devastating rice pests in Asia. NlG14 knockdown caused the displacement of the lateral oviduct secreted components (LOSC), leading to the ovulation disorder and the accumulation of mature eggs in the ovary. The RNAi-treated females laid much less eggs than their control counterparts, though they had the similar oviposition behavior on rice stems as controls. NlG14 protein was not secreted into the hemolymph, indicating an indirect effect of NlG14 knockdown on BPH reproduction. NlG14 knockdown caused the malformation of A-follicle of the principal gland and affected the underlying endocrine mechanism of salivary glands. NlG14 reduction might promote the secretion of insulin-like peptides NlILP1 and NlILP3 from the brain, which up-regulated the expression of Nllaminin gene and then caused the abnormal contraction of lateral oviduct muscle. Another explanation was NlG14 reduction disrupted the ecdysone biosynthesis and action through the insulin-PI3K-Akt signaling in ovary. Altogether, this study indicated that the salivary gland specific protein NlG14 indirectly mediated BPH ovulation process, which established a connexon in function between insect salivary gland and ovary.

Mutant dwarf and calorie-restricted mice benefit from healthy aging and unusually long lifespan. In contrast, mouse models for DNA repair-deficient progeroid syndromes age and die prematurely. To identify mechanisms that regulate mammalian longevity, we quantified the parallels between the genome-wide liver expression profiles of mice with those two extremes of lifespan. Contrary to expectation, we find significant, genome-wide expression associations between the progeroid and long-lived mice. Subsequent analysis of significantly over-represented biological processes revealed suppression of the endocrine and energy pathways with increased stress responses in both delayed and premature aging. To test the relevance of these processes in natural aging, we compared the transcriptomes of liver, lung, kidney, and spleen over the entire murine adult lifespan and subsequently confirmed these findings on an independent aging cohort. The majority of genes showed similar expression changes in all four organs, indicating a systemic transcriptional response with aging. This systemic response included the same biological processes that are triggered in progeroid and long-lived mice. However, on a genome-wide scale, transcriptomes of naturally aged mice showed a strong association to progeroid but not to long-lived mice. Thus, endocrine and metabolic changes are indicative of "survival" responses to genotoxic stress or starvation, whereas genome-wide associations in gene expression with natural aging are indicative of biological age, which may thus delineate pro- and anti-aging effects of treatments aimed at health-span extension.

WNK (With no Lysine [K]) kinases have critical roles in the maintenance of ion homeostasis and the regulation of cell volume. Their overactivation leads to pseudohypoaldosteronism type II (Gordon syndrome) characterized by hyperkalemia and high blood pressure. More recently, WNK family members have been shown to be required for the development of the nervous system in mice, zebrafish, and flies, and the cardiovascular system of mice and fish. Furthermore, human WNK2 and Drosophila Wnk modulate canonical Wnt signaling. In addition to a well-conserved kinase domain, animal WNKs have a large, poorly conserved C-terminal domain whose function has been largely mysterious. In most but not all cases, WNKs bind and activate downstream kinases OSR1/SPAK, which in turn regulate the activity of various ion transporters and channels. Here, we show that Drosophila Wnk regulates Wnt signaling and cell size during the development of the wing in a manner dependent on Fray, the fly homolog of OSR1/SPAK. We show that the only canonical RF(X)V/I motif of Wnk, thought to be essential for WNK interactions with OSR1/SPAK, is required to interact with Fray in vitro. However, this motif is unexpectedly dispensable for Fray-dependent Wnk functions in vivo during fly development and fluid secretion in the Malpighian (renal) tubules. In contrast, a structure function analysis of Wnk revealed that the less-conserved C-terminus of Wnk, that recently has been shown to promote phase transitions in cell culture, is required for viability in vivo. Our data thus provide novel insights into unexpected in vivo roles of specific WNK domains.