Sex-biased gene expression during neural differentiation of human embryonic stem cells.

Sex differences in the developing human brain are primarily attributed to hormonal influence. Recently however, genetic differences and their impact on the developing nervous system have attracted increased attention. To understand genetically driven sexual dimorphisms in neurodevelopment, we investigated genome-wide gene expression in an in vitro differentiation model of male and female human embryonic stem cell lines (hESC), independent of the effects of human sex hormones. Four male and four female-derived hESC lines were differentiated into a population of mixed neurons over 37 days. Differential gene expression and gene set enrichment analyses were conducted on bulk RNA sequencing data. While similar differentiation tendencies in all cell lines demonstrated the robustness and reproducibility of our differentiation protocol, we found sex-biased gene expression already in undifferentiated ESCs at day 0, but most profoundly after 37 days of differentiation. Male and female cell lines exhibited sex-biased expression of genes involved in neurodevelopment, suggesting that sex influences the differentiation trajectory. Interestingly, the highest contribution to sex differences was found to arise from the male transcriptome, involving both Y chromosome and autosomal genes. We propose 13 sex-biased candidate genes (10 upregulated in male cell lines and 3 in female lines) that are likely to affect neuronal development. Additionally, we confirmed gene dosage compensation of X/Y homologs escaping X chromosome inactivation through their Y homologs and identified a significant overexpression of the Y-linked demethylase UTY and KDM5D in male hESC during neuron development, confirming previous results in neural stem cells. Our results suggest that genetic sex differences affect neuronal differentiation trajectories, which could ultimately contribute to sex biases during human brain development.

Proline catabolism is a key factor facilitating Candida albicans pathogenicity.

Candida albicans, the primary etiology of human mycoses, is well-adapted to catabolize proline to obtain energy to initiate morphological switching (yeast to hyphal) and for growth. We report that put1-/- and put2-/- strains, carrying defective Proline UTilization genes, display remarkable proline sensitivity with put2-/- mutants being hypersensitive due to the accumulation of the toxic intermediate pyrroline-5-carboxylate (P5C), which inhibits mitochondrial respiration. The put1-/- and put2-/- mutations attenuate virulence in Drosophila and murine candidemia models and decrease survival in human neutrophils and whole blood. Using intravital 2-photon microscopy and label-free non-linear imaging, we visualized the initial stages of C. albicans cells infecting a kidney in real-time, directly deep in the tissue of a living mouse, and observed morphological switching of wildtype but not of put2-/- cells. Multiple members of the Candida species complex, including C. auris, are capable of using proline as a sole energy source. Our results indicate that a tailored proline metabolic network tuned to the mammalian host environment is a key feature of opportunistic fungal pathogens.

FRET-FISH probes chromatin compaction at individual genomic loci in single cells.

Chromatin compaction is a key biophysical property that influences multiple DNA transactions. Lack of chromatin accessibility is frequently used as proxy for chromatin compaction. However, we currently lack tools for directly probing chromatin compaction at individual genomic loci. To fill this gap, here we present FRET-FISH, a method combining fluorescence resonance energy transfer (FRET) with DNA fluorescence in situ hybridization (FISH) to probe chromatin compaction at select loci in single cells. We first validate FRET-FISH by comparing it with ATAC-seq, demonstrating that local compaction and accessibility are strongly correlated. FRET-FISH also detects expected differences in compaction upon treatment with drugs perturbing global chromatin condensation. We then leverage FRET-FISH to study local chromatin compaction on the active and inactive X chromosome, along the nuclear radius, in different cell cycle phases, and during increasing passage number. FRET-FISH is a robust tool for probing local chromatin compaction in single cells.

Blood-brain barrier-restricted translocation of Toxoplasma gondii from cortical capillaries.

The cellular barriers of the central nervous system proficiently protect the brain parenchyma from infectious insults. Yet, the single-celled parasite Toxoplasma gondii commonly causes latent cerebral infection in humans and other vertebrates. Here, we addressed the role of the cerebral vasculature in the passage of T. gondii to the brain parenchyma. Shortly after inoculation in mice, parasites mainly localized to cortical capillaries, in preference over post-capillary venules, cortical arterioles or meningeal and choroidal vessels. Early invasion to the parenchyma (days 1-5) occurred in absence of a measurable increase in blood-brain barrier (BBB) permeability, perivascular leukocyte cuffs or hemorrhage. However, sparse focalized permeability elevations were detected adjacently to replicative parasite foci. Further, T. gondii triggered inflammatory responses in cortical microvessels and endothelium. Pro- and anti-inflammatory treatments of mice with LPS and hydrocortisone, respectively, impacted BBB permeability and parasite loads in the brain parenchyma. Finally, pharmacological inhibition or Cre/loxP conditional knockout of endothelial focal adhesion kinase (FAK), a BBB intercellular junction regulator, facilitated parasite translocation to the brain parenchyma. The data reveal that the initial passage of T. gondii to the central nervous system occurs principally across cortical capillaries. The integrity of the microvascular BBB restricts parasite transit, which conversely is exacerbated by the inflammatory response.

Increased Expression of Y-Encoded Demethylases During Differentiation of Human Male Neural Stem Cells.

Human neural stem cells (hNSCs) have long been used as an in vitro model to study neurogenesis and as candidates for nervous system therapy. Many parameters have been considered when evaluating the success of transplantation, but sex of donor and recipients is often not discussed. We investigated two commercial NSC lines, the female hNSC-H9 and male hNSC-H14, and we observed faster growth rates in the male cells. At 4 days of differentiation, male cells presented a significant increase in expression of DCX, an immature neuronal marker, while female cells showed a significant increase in RMST, a long noncoding RNA, which is indispensable during neurogenesis. In addition, expression of neural markers MAP2, PSD95, SYP, DCX, and TUJ1 at day 14 of differentiation suggested a similar differentiation potential in both lines. The most significant differences at day 14 of differentiation were the expression levels of RELN, with almost 100-fold difference between the sexes, and MASH1, with more than 1,000-fold increase in male cells. To evaluate whether some of the observed differences may be sex related, we measured the expression of gametologous genes located on the X- and Y-chromosome. Most noticeable was the increase of Y-encoded demethylases KDM6C (UTY) and KDM5D during differentiation of male cells. Our results indicate that attention should be paid to sex when planning neurogenesis and transplantation experiments.

Novel Y-Chromosome Long Non-Coding RNAs Expressed in Human Male CNS During Early Development.

Global microarray gene expression analyses previously demonstrated differences in female and male embryos during neurodevelopment. In particular, before sexual maturation of the gonads, the differences seem to concentrate on the expression of genes encoded on the X- and Y-chromosomes. To investigate genome-wide differences in expression during this early developmental window, we combined high-resolution RNA sequencing with qPCR to analyze brain samples from human embryos during the first trimester of development. Our analysis was tailored for maximum sensitivity to discover Y-chromosome gene expression, but at the same time, it was underpowered to detect X-inactivation escapees. Using this approach, we found that 5 out of 13 expressed gametolog pairs showed unbalanced gene dosage, and as a consequence, a male-biased expression. In addition, we found six novel non-annotated long non-coding RNAs on the Y-chromosome with conserved expression patterns in newborn chimpanzee. The tissue specific and time-restricted expression of these long non-coding RNAs strongly suggests important functions during central nervous system development in human males.

EphA4-ADAM10 Interplay Patterns the Cochlear Sensory Epithelium through Local Disruption of Adherens Junctions.

The cochlear sensory epithelium contains a functionally important triangular fluid-filled space between adjacent pillar cells referred to as the tunnel of Corti. However, the molecular mechanisms leading to local cell-cell separation during development remain elusive. Here we show that EphA4 associates with ADAM10 to promote the destruction of E-cadherin-based adhesions between adjacent pillar cells. These cells fail to separate from each other, and E-cadherin abnormally persists at the pillar cell junction in EphA4 forward-signaling-deficient mice, as well as in the presence of ADAM10 inhibitor. Using immunolabeling and an in situ proximity ligation assay, we found that EphA4 forms a complex with E-cadherin and its sheddase ADAM10, which could be activated by ephrin-B2 across the pillar cell junction to trigger the cleavage of E-cadherin. Altogether, our findings provide a new molecular insight into the regulation of adherens junctions, which might be extended to a variety of physiological or pathological processes.

Author Correction: Essential role of mitochondrial Stat3 in p38MAPK mediated apoptosis under oxidative stress.

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Essential role of mitochondrial Stat3 in p38MAPK mediated apoptosis under oxidative stress.

Stat3 is an oncogene, frequently associated with malignant transformation. A body of evidence implicates that phospho-Stat3Y705 contributes to its nucleic translocation, while phospho-Stat3S727 leads to the accumulation in mitochondria. Both are of importance for tumor cell proliferation. In comparison to well-characterized signaling pathways interplaying with Stat3Y705, little is known about Stat3S727. In this work, we studied the influence of Stat3 deficiency on the viability of cells exposed to H2O2 or hypoxia using siRNA and CRISPR/Cas9 genome-editing. We found dysregulation of mitochondrial activity, which was associated with excessive ROS formation and reduced mitochondrial membrane potential, and observed a synergistic effect for oxidative stress-mediated apoptosis in Stat3-KD cells or cells carrying Stat3Y705F, but not Stat3S727D, suggesting the importance of functional mitochondrial Stat3 in this context. We also found that ROS-mediated activation of ASK1/p38MAPK was involved and adding antioxidants, p38MAPK inhibitor, or genetic repression of ASK1 could easily rescue the cellular damage. Our finding reveals a new role of mitochondrial Stat3 in preventing ASK1/p38MAPK-mediated apoptosis, wich further support the notion that selective inhibition mitochondrial Stat3 could provide a primsing target for chemotherapy.

Dissection and Culture of Mouse Embryonic Kidney.

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant. One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

SLC10A4 regulates IgE-mediated mast cell degranulation in vitro and mast cell-mediated reactions in vivo.

Mast cells act as sensors in innate immunity and as effector cells in adaptive immune reactions. Here we demonstrate that SLC10A4, also referred to as the vesicular aminergic-associated transporter, VAAT, modifies mast cell degranulation. Strikingly, Slc10a4 -/- bone marrow-derived mast cells (BMMCs) had a significant reduction in the release of granule-associated mediators in response to IgE/antigen-mediated activation, whereas the in vitro development of mast cells, the storage of the granule-associated enzyme mouse mast cell protease 6 (mMCP-6), and the release of prostaglandin D2 and IL-6 were normal. Slc10a4-deficient mice had a strongly reduced passive cutaneous anaphylaxis reaction and a less intense itching behaviour in response to the mast cell degranulator 48/80. Live imaging of the IgE/antigen-mediated activation showed decreased degranulation and that ATP was retained to a higher degree in mast cell granules lacking SLC10A4. Furthermore, ATP was reduced by two thirds in Slc10a4 -/- BMMCs supernatants in response to IgE/antigen. We speculate that SLC10A4 affects the amount of granule-associated ATP upon IgE/antigen-induced mast cell activation, which affect the release of granule-associated mast cell mediators. In summary, SLC10A4 acts as a regulator of degranulation in vitro and of mast cell-related reactions in vivo.

Multimodal Eph/Ephrin signaling controls several phases of urogenital development.

A substantial portion of the human population is affected by urogenital birth defects resulting from a failure in ureter development. Although recent research suggests roles for several genes in facilitating the ureter/bladder connection, the underlying molecular mechanisms remain poorly understood. Signaling via Eph receptor tyrosine kinases is involved in several developmental processes. Here we report that impaired Eph/Ephrin signaling in genetically modified mice results in severe hydronephrosis caused by defective ureteric bud induction, ureter maturation, and translocation. Our data imply that ureter translocation requires apoptosis in the urogenital sinus and inhibition of proliferation in the common nephric duct. These processes were disturbed in EphA4/EphB2 compound knockout mice and were accompanied by decreased ERK-2 phosphorylation. Using a set of Eph, Ephrin, and signaling-deficient mutants, we found that during urogenital development, different modes of Eph/Ephrin signaling occur at several sites with EphrinB2 and EphrinA5 acting in concert. Thus, Eph/Ephrin signaling should be considered in the etiology of congenital kidney and urinary tract anomalies.

Gene Expression of Quaking in Sporadic Alzheimer's Disease Patients is Both Upregulated and Related to Expression Levels of Genes Involved in Amyloid Plaque and Neurofibrillary Tangle Formation.

Quaking (QKI) is a gene exclusively expressed within glial cells. QKI has previously been implicated in various neurological disorders and diseases, including Alzheimer's disease (AD), a condition for which increasing evidence suggests a central role of glia cells. The objective of the present study was to investigate the expression levels of QKI and three QKI isoforms (QKI5, QKI6, and QKI7) in AD. Genes that have previously been related to the ontogeny and progression of AD, specifically APP, PSEN1, PSEN2, and MAPT, were also investigated. A real-time PCR assay of 123 samples from human postmortem sporadic AD patients and control brains was performed. The expression values were analyzed with an analysis of covariance model and subsequent multiple regressions to explore the possibility of related expression values between QKI, QKI isoforms, and AD-related genes. Further, the sequences of AD-related genes were analyzed for the presence of QKI binding domains. QKI and all measured QKI isoforms were found to be significantly upregulated in AD samples, relative to control samples. However, APP, PSEN1, PSEN2, and MAPT were not found to be significantly different. QKI and QKI isoforms were found to be predictive for the variance of APP, PSEN1, PSEN2, and MAPT, and putative QKI binding sites suggests an interaction with QKI. Overall, these results implicate a possible role of QKI in AD, although the exact mechanism by which this occurs remains to be uncovered.

Characterization and Expression of the Zebrafish qki Paralogs.

Quaking (QKI) is an RNA-binding protein involved in post-transcriptional mRNA processing. This gene is found to be associated with several human neurological disorders. Early expression of QKI proteins in the developing mouse neuroepithelium, together with neural tube defects in Qk mouse mutants, suggest the functional requirement of Qk for the establishment of the nervous system. As a knockout of Qk is embryonic lethal in mice, other model systems like the zebrafish could serve as a tool to study the developmental functions of qki. In the present study we sought to characterize the evolutionary relationship and spatiotemporal expression of qkia, qki2, and qkib; zebrafish homologs of human QKI. We found that qkia is an ancestral paralog of the single tetrapod Qk gene that was likely lost during the fin-to-limb transition. Conversely, qkib and qki2 are orthologs, emerging at the root of the vertebrate and teleost lineage, respectively. Both qki2 and qkib, but not qkia, were expressed in the progenitor domains of the central nervous system, similar to expression of the single gene in mice. Despite having partially overlapping expression domains, each gene has a unique expression pattern, suggesting that these genes have undergone subfunctionalization following duplication. Therefore, we suggest the zebrafish could be used to study the separate functions of qki genes during embryonic development.

SLC10A4 is a vesicular amine-associated transporter modulating dopamine homeostasis.

BACKGROUND: The neuromodulatory transmitters, biogenic amines, have profound effects on multiple neurons and are essential for normal behavior and mental health. Here we report that the orphan transporter SLC10A4, which in the brain is exclusively expressed in presynaptic vesicles of monoaminergic and cholinergic neurons, has a regulatory role in dopamine homeostasis. METHODS: We used a combination of molecular and behavioral analyses, pharmacology, and in vivo amperometry to assess the role of SLC10A4 in dopamine-regulated behaviors. RESULTS: We show that SLC10A4 is localized on the same synaptic vesicles as either vesicular acetylcholine transporter or vesicular monoamine transporter 2. We did not find evidence for direct transport of dopamine by SLC10A4; however, synaptic vesicle preparations lacking SLC10A4 showed decreased dopamine vesicular uptake efficiency. Furthermore, we observed an increased acidification in synaptic vesicles isolated from mice overexpressing SLC10A4. Loss of SLC10A4 in mice resulted in reduced striatal serotonin, noradrenaline, and dopamine concentrations and a significantly higher dopamine turnover ratio. Absence of SLC10A4 led to slower dopamine clearance rates in vivo, which resulted in accumulation of extracellular dopamine. Finally, whereas SLC10A4 null mutant mice were slightly hypoactive, they displayed hypersensitivity to administration of amphetamine and tranylcypromine. CONCLUSIONS: Our results demonstrate that SLC10A4 is a vesicular monoaminergic and cholinergic associated transporter that is important for dopamine homeostasis and neuromodulation in vivo. The discovery of SLC10A4 and its role in dopaminergic signaling reveals a novel mechanism for neuromodulation and represents an unexplored target for the treatment of neurological and mental disorders.

Impaired EphA4 signaling leads to congenital hydronephrosis, renal injury, and hypertension.

Experimental hydronephrosis induced by partial ureteral obstruction at 3 wk of age causes hypertension and renal impairment in adult rats and mice. Signaling by Ephrin receptors (Eph) and their ligands (ephrins) importantly regulates embryonic development. Genetically modified mice, where the cytoplasmic domain of the EphA4 receptor has been substituted by enhanced green fluorescent protein (EphA4gf/gf), develop spontaneous hydronephrosis and provide a model for further studies of the disorder. The present study aimed to determine if animals with congenital hydronephrosis develop hypertension and renal injuries, similar to that of experimental hydronephrosis. Ultrasound and Doppler techniques were used to visualize renal impairment in the adult mice. Telemetric blood pressure measurements were performed in EphA4gf/gf mice and littermate controls (EphA4+/+) during normal (0.7% NaCl)- and high (4% NaCl)-sodium conditions. Renal excretion, renal plasma flow, and glomerular filtration were studied, and histology and morphology of the kidneys and ureters were performed. EphA4gf/gf mice developed variable degrees of hydronephrosis that correlated with their blood pressure level. In contrast to EphA4+/+, the EphA4gf/gf mice displayed salt-sensitive hypertension, reduced urine concentrating ability, reduced renal plasma flow, and lower glomerular filtration rate. Kidneys from EphA4gf/gf mice showed increased renal injuries, as evidenced by fibrosis, inflammation, and glomerular and tubular changes. In conclusion, congenital hydronephrosis causes hypertension and renal damage, similar to that observed in experimentally induced hydronephrosis. This study further reinforces the supposed causal link between hydronephrosis and later development of hypertension in humans.

Ephrin-A5/EphA4 signalling controls specific afferent targeting to cochlear hair cells.

Hearing requires an optimal afferent innervation of sensory hair cells by spiral ganglion neurons in the cochlea. Here we report that complementary expression of ephrin-A5 in hair cells and EphA4 receptor among spiral ganglion neuron populations controls the targeting of type I and type II afferent fibres to inner and outer hair cells, respectively. In the absence of ephrin-A5 or EphA4 forward signalling, a subset of type I projections aberrantly overshoot the inner hair cell layer and invade the outer hair cell area. Lack of type I afferent synapses impairs neurotransmission from inner hair cells to the auditory nerve. By contrast, radial shift of type I projections coincides with a gain of presynaptic ribbons that could enhance the afferent signalling from outer hair cells. Ephexin-1, cofilin and myosin light chain kinase act downstream of EphA4 to induce type I spiral ganglion neuron growth cone collapse. Our findings constitute the first identification of an Eph/ephrin-mediated mutual repulsion mechanism responsible for specific sorting of auditory projections in the cochlea.

Adaptive changes in gene expression patterns in the somatosensory cortex after deletion of ephrinA5.

The role of wiring molecules in circuit assembly is tested directly in genetically engineered animals, in which the corresponding gene has been selectively mutated. Minor alterations in neuronal circuits in these mutant animals are explained by redundancy and/or adaptive changes of other genes relevant for brain development. There is very little known, however, about the extent and nature of the compensatory molecular mechanisms. Using gene microarrays, we compared gene expression patterns in the somatosensory cortex of wild type and ephrinA5 deficient mice, which exhibit subtle, but highly reproducible alterations of thalamocortical projections and intrinsic cortical circuits. We found that between 2.2%-5.7% of all transcripts (140-373 targets) detected in the somatosensory cortex are differentially expressed in comparing wild type and ephrinA5 mutants. A gene group analysis of the annotated transcripts revealed that a high proportion of the dysregulated genes encode proteins relevant for circuit development. Finer grain analysis by in situ hybridization and quantitative RT-PCR revealed that 20% of the Eph/ephrin family genes expressed in the somatosensory cortex are up-regulated in the mutant. One of these genes, EphB6, was up-regulated in all cortical layers, where it is normally expressed. However, the ephrinA2 and EphA5 were up-regulated only in selected layers in the cortex of the mutant; expression levels in other layers did not change. These findings indicate that there is specificity of adaptive and compensative changes in gene expression after the mutation of a single gene relevant for cortical development. Our results also point to the complexity of interpreting phenotypes of gene knock-out animals.

Connecting thalamus and cortex: the role of ephrins.

The complex task of wiring up the brain during embryonic development is achieved by a multitude of guidance signals acting in complex combinations to drive growing axons to their proper targets. The somatosensory system provides an extensively studied model system featuring many universal mechanisms of neural development. In rodents, it constitutes an important model to study how precise topographic connections are achieved. Recent evidence suggests that the Eph/ephrin family of guidance molecules is of pivotal importance for the development of the somatosensory system. Members of Eph/ephrin family are thought to be involved in the global presorting of thalamic axons projecting to the cortex, in labeling specific cortical areas for innervation, in providing topographic cues within the target area, and in distinguishing cortical layers for intracortical wiring. The Eph/ephrin system also seems to contribute to the formation of specific corticothalamic feedback projections. So far, the functions of only a few members of the Eph/ephrin family have been examined, but expression analysis indicates complex combinatorial effects of these signaling molecules. Understanding the Eph/ephrin wiring code is expected to yield new insights into the development and plasticity of brain circuits involved in higher functions.

Multiple roles of ephrins during the formation of thalamocortical projections: maps and more.

The functional architecture of the cerebral cortex is based on intrinsic connections that precisely link neurons from distinct cortical laminae as well as layer-specific afferent and efferent projections. Experimental strategies using in vitro assays originally developed by Friedrich Bonhoeffer have suggested that positional cues confined to individual layers regulate the assembly of local cortical circuits and the formation of thalamocortical projections. One of these wiring molecules is ephrinA5, a ligand for Eph receptor tyrosine kinases. EphrinA5 and Eph receptors exhibit highly dynamic expression patterns in distinct regions of the cortex and thalamus during early and late stages of thalamocortical and cortical circuit formation. In vitro assays suggest that ephrinA5 is a multifunctional wiring molecule for different populations of cortical and thalamic axons. Additionally, the expression patterns of ephrinA5 during cortical development are consistent with this molecule regulating, in alternative ways, specific components of thalamic and cortical connectivity. To test this directly, the organization of thalamocortical projections was examined in mice lacking ephrinA5 gene expression. The anatomical studies in ephrinA5 knockout animals revealed a miswiring of limbic thalamic projections and changes in neocortical circuits that were predicted from the expression pattern and the in vitro analysis of ephrinA5 function.