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The toxic effect of di(2-ethylhexyl) phthalate (DEHP) on prepubertal testes was examined in this study. We treated 3-week-old male mice with 4.8 mg/kg/day (milligram/kilogram/day) (no observed adverse effect level), 30 mg/kg/day (high exposure dose relative to humans), 100 mg/kg/day (level causing a reproductive system disorder), and 500 mg/kg/day (dose causing a multigenerational reproductive system disorder) of DEHP via gavage. Obvious abnormalities in the testicular organ coefficient, spermatogenic epithelium, and testosterone levels occurred in the 500 mg/kg DEHP group. Ribonucleic acid sequencing (RNA-seq) showed that differentially expressed genes (DEGs) in each group could enrich reproduction and reproductive process terms according to the gene ontology (GO) results, and coenrichment of metabolism pathway was observed by the Reactome pathway analysis. Through the analysis of common genes in the metabolism pathway, we discovered that DEHP exposure at 4.8 to 500 mg/kg or 100 mg/kg caused the same damages to the prepubertal testis. In general, we identified two key transcriptional biomarkers (fatty acid binding protein 3 (Fabp3) and carboxylesterase (Ces) 1d), which provided new insight into the gene regulatory mechanism associated with DEHP exposure and will contribute to the prediction and diagnosis of prepuberty testis injury caused by DEHP.

Significance Low availability of nitrogen (N), mainly nitrate in aerobic soils, is a primary limiting factor for crop production. Most terrestrial plants live in symbiosis with arbuscular mycorrhizal (AM) fungi to increase nutrient uptake, including N, from soil. Research on the AM symbiosis field has focused almost exclusively on ammonium as the form of N transferred to the plants, and there has been no direct evidence of N transfer as nitrate thus far. Here, we report that mycorrhizal rice could receive more than 40% of its N via the mycorrhizal pathway and that the AM-specific nitrate transporter OsNPF4.5 accounted for approximately 45% of the mycorrhizal nitrate uptake. Our work suggests the presence of a mycorrhizal route for nitrate uptake in plants., Low availability of nitrogen (N) is often a major limiting factor to crop yield in most nutrient-poor soils. Arbuscular mycorrhizal (AM) fungi are beneficial symbionts of most land plants that enhance plant nutrient uptake, particularly of phosphate. A growing number of reports point to the substantially increased N accumulation in many mycorrhizal plants; however, the contribution of AM symbiosis to plant N nutrition and the mechanisms underlying the AM-mediated N acquisition are still in the early stages of being understood. Here, we report that inoculation with AM fungus Rhizophagus irregularis remarkably promoted rice (Oryza sativa) growth and N acquisition, and about 42% of the overall N acquired by rice roots could be delivered via the symbiotic route under N-NO3− supply condition. Mycorrhizal colonization strongly induced expression of the putative nitrate transporter gene OsNPF4.5 in rice roots, and its orthologs ZmNPF4.5 in Zea mays and SbNPF4.5 in Sorghum bicolor. OsNPF4.5 is exclusively expressed in the cells containing arbuscules and displayed a low-affinity NO3− transport activity when expressed in Xenopus laevis oocytes. Moreover, knockout of OsNPF4.5 resulted in a 45% decrease in symbiotic N uptake and a significant reduction in arbuscule incidence when NO3− was supplied as an N source. Based on our results, we propose that the NPF4.5 plays a key role in mycorrhizal NO3− acquisition, a symbiotic N uptake route that might be highly conserved in gramineous species.

Most land plants can establish symbiosis with arbuscular mycorrhizal (AM) fungi to increase fitness to environmental challenges. The development of AM symbiosis is controlled by intricate procedures involving all phytohormones. However, the mechanisms underlying the auxin-mediated regulation of AM symbiosis remains largely unknown. Here, we report that AM colonisation promotes auxin response and indole-3-acetic acid (IAA) accumulation, but downregulates IAA biosynthesis genes in tomato (Solanum lycopersicum). External IAA application modulates the AM symbiosis by promoting arbuscule formation at low concentrations but repressing it at high concentrations. An AM-induced GH3 gene, SlGH3.4, encoding a putative IAA-amido synthetase, negatively regulates mycorrhization via maintaining cellular auxin homoeostasis. Loss of SlGH3.4 function increased free IAA content and arbuscule incidence, while constitutively overexpressing SlGH3.4 in either tomato or rice resulted in decreased IAA content, total colonisation level and arbuscule abundance in mycorrhizal roots. Several auxin-inducible expansin genes involved in AM formation or resistance to pathogen infection were upregulated in slgh3.4 mycorrhizal roots but downregulated in the SlGH3.4-overexpressing plants. Taken together, our results highlight a positive correlation between the endogenous IAA content and mycorrhization level, particularly arbuscule incidence, and suggest that the SlGH3.4-mediated auxin homoeostasis and regulation of expansin genes is involved in finely tuning the AM development.

Most land plants can form symbiosis with arbuscular mycorrhizal (AM) fungi to enhance uptake of mineral nutrients, particularly phosphate (Pi) and nitrogen (N), from the soil. It is established that transport of Pi from interfacial apoplast into plant cells depends on the H+ gradient generated by the H+ -ATPase located on the periarbuscular membrane (PAM); however, little evidence regarding the potential link between mycorrhizal N transport and H+ -ATPase activity is available to date. Here, we report that a PAM-localized tomato H+ -ATPase, SlHA8, is indispensable for arbuscule development and mycorrhizal P and N uptake. Knockout of SlHA8 resulted in truncated arbuscule morphology, reduced shoot P and N accumulation, and decreased H+ -ATPase activity and acidification of apoplastic spaces in arbusculated cells. Overexpression of SlHA8 in tomato promoted both P and N uptake, and increased total colonization level, but did not affect arbuscule morphology. Heterogeneous expression of SlHA8 in the rice osha1 mutant could fully complement its defects in arbuscule development and mycorrhizal P and N uptake. Our results propose a pivotal role of the SlHA8 in energizing both the symbiotic P and N transport, and highlight the evolutionary conservation of the AM-specific H+ -ATPase orthologs in maintaining AM symbiosis across different mycorrhizal plant species.

Flight, an active fear response to imminent threat, is dependent on the rapid risk assessment of sensory information processed by the cortex. The thalamic reticular nucleus (TRN) filters information between the cortex and the thalamus, but whether it participates in the regulation of flight behavior remains largely unknown. Here, we report that activation of parvalbumin-expressing neurons in the limbic TRN, but not those in the sensory TRN, mediates flight. Glutamatergic inputs from the cingulate cortex (Cg) selectively activate the limbic TRN, which in turn inhibits the intermediodorsal thalamic nucleus (IMD). Activation of this Cg→limbic TRN→IMD circuit results in inhibition of the IMD and produces flight behavior. Conversely, removal of inhibition onto the IMD results in more freezing and less flight, suggesting that the IMD may function as a pro-freeze center. Overall, these findings reveal a novel corticothalamic circuit through the TRN that controls the flight response.

Di-(2-ethylhexyl) phthalate (DEHP) is an environmental endocrine disruptor that accumulates in organisms in various ways and induces male reproductive system disorders. In this study, we established a testicular injury model by gavage with different concentrations of DEHP. The testes were then collected for RNA sequencing (RNA-seq), and the results were analyzed by bioinformatics and verified by experiments. Our research results show that different concentrations of DEHP interfere with testicular development differently. Weighted gene coexpression network analysis (WGCNA) generated sixteen modules and identified the turquoise module as key. Then, estrogen receptor 1 (ESR1), filamin A (Flna) and Furin were identified as hub genes. qPCR and immunohistochemistry results revealed that all three hub genes were upregulated. We detected the locations of these genes by immunohistochemistry. ESR1 was mainly located in Leydig cells; Flna immunostaining is observed in the Leydig and some germ cells and Furin staining was seen in almost all types of testicular cells. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed enrichment mainly in MAPK signaling pathways, p53 signaling pathways, HIF-1 signaling pathways, protein processing in the endoplasmic reticulum, apoptosis, the cell cycle, RNA degradation, etc. This is the first study using WGCNA to investigate the mechanism of DEHP-induced injury in the prepubertal testis, providing new research angles to further understand the mechanism of DEHP-induced injury in the prepubertal testis.

N6-methyladenosine (m6A) modification, the most prevalent form of RNA methylation, modulates gene expression post-transcriptionally. Di-(2-ethylhexyl) phthalate (DEHP) is a common environmental endocrine disrupting chemical that induces testicular injury due to the inhibition of the demethylase fat mass and obesity-associated protein (FTO) and increases the m6A modification. How FTO-mediated m6A modification in testicular Leydig cell injury induced by DEHP remains unclear. Here, the TM3 Leydig cell line was treated with mono-(2-ethylhexyl) phthalate (MEHP), the main metabolite of DEHP in the body, as well as FB23-2, an inhibitor of FTO. Decreased levels of testosterone in the culture supernatant, significantly increased apoptosis, and a remarkable upregulation of global m6A modification were found in both TM3 cells treated with MEHP and FB23-2. Transcriptome sequencing showed that both treatments significantly induced apoptosis-associated gene expression. Methylated RNA immunoprecipitation sequencing showed that the Leydig cell injury induced by upregulated m6A modification could be associated with multiple physiological disorders, including histone acetylation, reactive oxygen species biosynthesis, MAPK signaling pathway, hormone secretion regulation, autophagy regulation, and male gonadal development. Overall, the inhibition of FTO-mediated up-regulation of m6A could be involved in MEHP-induced Leydig cell apoptosis.

Di-(2-ethylhexyl) phthalate (DEHP) is the most common phthalate that can affect the male reproductive system. DEHP exposure at the prepubertal stage could lead to the injury of immature testes, but the mechanism has not been fully clarified. In the present study, we elucidated the possible underlying mechanism of DEHP-induced prepubertal testicular injury through stereological analysis and transcriptome profiling. Compared with the control group, the DEHP-treated rats had lower body weight gain and decreased testicular weight and organ coefficient. Moreover, lower serum levels of testosterone and LH were observed in the DEHP group, in contrast to the increased FSH level. Additionally, the serum level of estradiol had no significant difference after DEHP exposure. Stereological analysis showed significant reduction in volumes of most testicular structures, especially in the seminiferous tubule and seminiferous epithelium, along with a vast decrease of spermatogenic cells and obvious structural damages with substantial pathological signs (germ cracks, cytoplasmic vacuolization, sloughing, multinucleated giant cell formation, chromatolysis desquamation and dissolution, pyknosis of nuclei) in the seminiferous tubule upon DEHP exposure at the prepubertal stage. Furthermore, transcriptome profiling identified 5548 differentially expressed genes (DEGs) upon DEHP exposure. Pathway enrichment analysis revealed several crucial signaling pathways related to retinol metabolism, oxidative phosphorylation, steroid hormone biosynthesis, and cell adhesion molecules (CAMs). In addition, seven DEGs selected from RNA-seq data were validated by quantitative real-time polymerase chain reaction (qRT-PCR), and the results showed the same trends as the RNA-seq results. In conclusion, the above findings provide basic morphological data and lay a foundation for systematic research on transcriptome profiling in prepubertal testicular injury induced by DEHP.

There are a large number of abandoned mining areas in China, where external-soil spray seeding is a common technique used to assist with the restoration of these areas. However, the soil component of external-soil spray seeding is deficient, and they are prone to collapse, which complicates ecological restoration. In this study, we added a mineral-solubilizing microbial strain to an external-soil spray seeding substrate in Robinia pseudoacacia and Lespedeza bicolor pots, which were monitored from December 2018 to November 2019. We investigated their root growth and root tensile properties, as well as root-reinforced soil shear strength. The results revealed that the addition of the microbial strain in the substrate improved root growth of Robinia pseudoacacia. The root-reinforced soil shear strength, tensile force and strength were also strengthened by the added microbial strain. Although the growth rate of Robinia pseudoacacia was faster than that of Lespedeza bicolor, the shear strength of the root-reinforced Robinia pseudoacacia soil was lower than that of the Lespedeza bicolor root-reinforced soil of the same diameter. Finally, compared with the cohesion, the change in the friction angle is relatively small, and differences in cohesion resulted in shear strength changes under the same treatment. Our results suggested that the addition of a mineral-solubilizing microbial strain to the external-soil spray seeding substrate could help plants strengthen the soil and positively enhance its effects. These results might also enrich the existing data on the effects of mineral-solubilizing microbial strains on plant roots, while guiding further studies toward improving the efficacy of external-soil spray seeding technologies.

Summary Pain is a source of substantial discomfort. Abnormal activity in both the zona incerta (ZI) and posterior complex of the thalamus (Po) are implicated in neuropathic pain, but their exact roles remain unclear. In particular, the precise cell types and molecular mechanisms of the ZI-Po circuit that regulate nociception are largely uncharacterized. Here, we found that parvalbumin (PV)-positive neuronal projections from the ventral ZI (ZIv) to the Po (ZIv-Po) are critical for promoting nocifensive behaviors, whereas selectively inhibiting ZIv-Po activity reduces nocifensive withdrawal responses. Furthermore, cannabinoid type 1 receptors (CB1Rs) are expressed specifically at ZIv-Po axon terminals in this circuit, and cannabinoids attenuate nocifensive responses through presynaptic inhibition. Selective inhibition of the ZIv-Po circuit or administration of cannabinoids into the Po are sufficient to ameliorate pathological pain. These findings identify the critical role of the ZIv-Po circuit and its modulation by endocannabinoids in controlling nocifensive behaviors.

In the developing human neocortex, progenitor cells generate diverse cell types prenatally. Progenitor cells and newborn neurons respond to signaling cues, including neurotransmitters. While single-cell RNA sequencing has revealed cellular diversity, physiological heterogeneity has yet to be mapped onto these developing and diverse cell types. By combining measurements of intracellular Ca2+ elevations in response to neurotransmitter receptor agonists and RNA sequencing of the same single cells, we show that Ca2+ responses are cell-type-specific and change dynamically with lineage progression. Physiological response properties predict molecular cell identity and additionally reveal diversitynot captured by single-cell transcriptomics. We find that the serotonin receptor HTR2A selectively activates radial glia cells in the developing human, butnot mouse, neocortex, and inhibiting HTR2A receptorsin human radial glia disrupts the radial glial scaffold. We show highly specific neurotransmittersignaling during neurogenesis in the developing human neocortex and highlight evolutionarilydivergent mechanisms of physiological signaling.

Neuropathic pain and bladder dysfunction represent significant quality-of-life issues for many spinal cord injury patients. Loss of GABAergic tone in the injured spinal cord may contribute to the emergence of these symptoms. Previous studies have shown that transplantation of rodent inhibitory interneuron precursors from the medial ganglionic eminence (MGE) enhances GABAergic signaling in the brain and spinal cord. Here we look at whether transplanted MGE-like cells derived from human embryonic stem cells (hESC-MGEs) can mitigate the pathological effects of spinal cord injury. We find that 6 months after transplantation into injured mouse spinal cords, hESC-MGEs differentiate into GABAergic neuron subtypes and receive synaptic inputs, suggesting functional integration into host spinal cord. Moreover, the transplanted animals show improved bladder function and mitigation of pain-related symptoms. Our results therefore suggest that this approach may be a valuable strategy for ameliorating the adverse effects of spinal cord injury.

Many terrestrial plants can form root symbiosis with beneficial microorganisms for enhancing uptake of mineral nutrients or increasing fitness to adverse environmental challenges. Arbuscular mycorrhizal (AM) symbiosis that is formed by AM fungi and the roots of vascular flowering plants is the most widespread mutualistic associations in nature. As a typical endosymbiosis, AM interactions involves the differentiation of both symbionts to create novel symbiotic interfaces within the root cells, and requires a continuous nutrient exchange between the two partners. AM plants have two pathways for nutrient uptake, either direct uptake via the root hairs and root epidermis at the plant-soil interface, or indirectly through the AM fungal hyphae at the plant-fungus interface. Over the last few years, great progress has been made in deciphering the mechanisms underlying the AM-mediated modulation of nutrient uptake processes, and an increasing number of plant and fungal genes responsible for transporting nutrients from the soil or across the intraradical symbiotic interfaces have been identified and functionally characterized. Here, we summarize the recent advances in the nitrogen uptake, assimilation and translocation in the AM symbiosis, and also explore the current understanding of how the N status and interplay with C and P in modulating the development of AM associations.

WRKY transcription factors play important roles in various stress responses in diverse plant species. In cotton, this family has not been well studied, especially in relation to fiber development. Here, the genomes and transcriptomes of Gossypium raimondii and Gossypium arboreum were investigated to identify fiber development related WRKY genes. This represents the first comprehensive comparative study of WRKY transcription factors in both diploid A and D cotton species. In total, 112 G. raimondii and 109 G. arboreum WRKY genes were identified. No significant gene structure or domain alterations were detected between the two species, but many SNPs distributed unequally in exon and intron regions. Physical mapping revealed that the WRKY genes in G. arboreum were not located in the corresponding chromosomes of G. raimondii, suggesting great chromosome rearrangement in the diploid cotton genomes. The cotton WRKY genes, especially subgroups I and II, have expanded through multiple whole genome duplications and tandem duplications compared with other plant species. Sequence comparison showed many functionally divergent sites between WRKY subgroups, while the genes within each group are under strong purifying selection. Transcriptome analysis suggested that many WRKY genes participate in specific fiber development processes such as fiber initiation, elongation and maturation with different expression patterns between species. Complex WRKY gene expression such as differential Dt and At allelic gene expression in G. hirsutum and alternative splicing events were also observed in both diploid and tetraploid cottons during fiber development process. In conclusion, this study provides important information on the evolution and function of WRKY gene family in cotton species.

Heterosynaptic long-term depression (hLTD) at untetanized synapses accompanying the induction of long-term potentiation (LTP) spatially sharpens the activity-induced synaptic potentiation; however, the underlying mechanism remains unclear. We found that hLTD in the hippocampal CA1 region is caused by stimulation-induced ATP release from astrocytes that suppresses transmitter release from untetanized synaptic terminals via activation of P2Y receptors. Selective stimulation of astrocytes expressing channelrhodopsin-2, a light-gated cation channel permeable to Ca2+, resulted in LTD of synapses on neighboring neurons. This synaptic modification required Ca2+ elevation in astrocytes and activation of P2Y receptors, but not N-methyl-D-aspartate receptors. Furthermore, blocking P2Y receptors or buffering astrocyte intracellular Ca2+ at a low level prevented hLTD without affecting LTP induced by SC stimulation. Thus, astrocyte activation is both necessary and sufficient for mediating hLTD accompanying LTP induction, strongly supporting the notion that astrocytes actively participate in activity-dependent synaptic plasticity of neural circuits. (c) 2012 Wiley Periodicals, Inc.

Circuits for brain building The brain is made up of circuits. Chen and Kriegstein analyzed how one particular circuit functions differently in developing and mature mouse brains (see the Perspective by Spitzer). This circuit connects the brain's diencephalon to the cortex. Early in development, the circuit is excitatory and affects how the cortical neurons it touches build their dendrites and synapses. Later on, after the fundamentals of brain construction are in place, the circuit switches to inhibitory functions, helping the brain resist epileptiform activity. Science , this issue p. 554 ; see also p. 510

Non-neurogenic cell types, such as cortical astroglia and fibroblasts, can be directly converted into neurons by the overexpression of defined transcription factors. Normally, the cellular phenotype of such differentiated cells is remarkably stable and resists direct cell transdifferentiation. Here we show that theInk4a/Arf(also known asCdkn2a) locus is a developmental barrier to direct neuronal transdifferentiation induced by transcription factor overexpression. With serial passagein vitro, wild-type postnatal cortical astroglia become progressively resistant toDlx2-induced neuronal transdifferentiation. In contrast, the neurogenic competence ofInk4a/Arf-deficient astroglia is both greatly increased and does not diminish through serial cell culture passage. Electrophysiological analysis further demonstrates the neuronal identity of cells induced fromInk4a/Arf-null astroglia, and short hairpin RNA-mediated acute knockdown of p16Ink4a and p19Arf p16Ink4aand p19Arfindicates that these gene products function postnatally as a barrier to cellular transdifferentiation. Finally, we found that mouse fibroblasts deficient forInk4a/Arfalso exhibit greatly enhanced transcription factor-induced neuronal induction. These data indicate thatInk4a/Arfis a potent barrier to direct neuronal transdifferentiation and further suggest that this locus functions normally in the progressive developmental restriction of postnatal astrocytes.

SummaryThe ventricular-subventricular zone (V-SVZ) is an extensive germinal niche containing neural stem cells (NSCs) in the walls of the lateral ventricles of the adult brain. How the adult brain’s neural activity influences the behavior of adult NSCs remains largely unknown. We show that serotonergic (5HT) axons originating from a small group of neurons in the raphe form an extensive plexus on most of the ventricular walls. Electron microscopy revealed intimate contacts between 5HT axons and NSCs (B1) or ependymal cells (E1) and these cells were labeled by a transsynaptic viral tracer injected into the raphe. B1 cells express the 5HT receptors 2C and 5A. Electrophysiology showed that activation of these receptors in B1 cells induced small inward currents. Intraventricular infusion of 5HT2C agonist or antagonist increased or decreased V-SVZ proliferation, respectively. These results indicate that supraependymal 5HT axons directly interact with NSCs to regulate neurogenesis via 5HT2C.

Directed differentiation from human pluripotent stem cells (hPSCs) has seen significant progress in recent years. However, most differentiated populations exhibit immature properties of an early embryonic stage, raising concerns about their ability to model and treat disease. Here, we report the directed differentiation of hPSCs into medial ganglionic eminence (MGE)-like progenitors and their maturation into forebrain type interneurons. We find that early-stage progenitors progress via a radial glial-like stem cell enriched in the human fetal brain. Both invitro and posttransplantation into the rodent cortex, the MGE-like cells develop into GABAergic interneuron subtypes with mature physiological properties along a prolonged intrinsic timeline of up to 7months, mimicking endogenous human neural development. MGE-derived cortical interneuron deficiencies are implicated in a broad range of neurodevelopmental and degenerative disorders, highlighting the importance of these results for modeling human neural development and disease.