Microbiome Influences Prenatal and Adult Microglia in a Sex-Specific Manner.

Microglia are embryonically seeded macrophages that contribute to brain development, homeostasis, and pathologies. It is thus essential to decipher how microglial properties are temporally regulated by intrinsic and extrinsic factors, such as sexual identity and the microbiome. Here, we found that microglia undergo differentiation phases, discernable by transcriptomic signatures and chromatin accessibility landscapes, which can diverge in adult males and females. Remarkably, the absence of microbiome in germ-free mice had a time and sexually dimorphic impact both prenatally and postnatally: microglia were more profoundly perturbed in male embryos and female adults. Antibiotic treatment of adult mice triggered sexually biased microglial responses revealing both acute and long-term effects of microbiota depletion. Finally, human fetal microglia exhibited significant overlap with the murine transcriptomic signature. Our study shows that microglia respond to environmental challenges in a sex- and time-dependent manner from prenatal stages, with major implications for our understanding of microglial contributions to health and disease.

Y-complex nucleoporins independently contribute to nuclear pore assembly and gene regulation in neuronal progenitors.

Besides assembling nuclear pore complexes, the conduits of nuclear transport, many nucleoporins also contribute to chromatin organization and gene expression, with critical roles in development and pathologies. We previously reported that Nup133 and Seh1, two components of the Y-complex subassembly of the nuclear pore scaffold, are dispensable for mouse embryonic stem cell viability but required for their survival during neuroectodermal differentiation. Here, a transcriptomic analysis revealed that Nup133 regulates a subset of genes at early stages of neuroectodermal differentiation, including Lhx1 and Nup210l, which encodes a newly validated nucleoporin. These genes are also misregulated in Nup133ΔMid neuronal progenitors, in which nuclear pore basket assembly is impaired. However, a four-fold reduction of Nup133 levels, despite also affecting basket assembly, is not sufficient to alter Nup210l and Lhx1 expression. Finally, these two genes are also misregulated in Seh1-deficient neural progenitors, which only show a mild reduction in nuclear pore density. Together these data reveal a shared function of Y-complex nucleoporins in gene regulation during neuroectodermal differentiation, apparently independent of nuclear pore basket integrity.

Krox20 defines a subpopulation of cardiac neural crest cells contributing to arterial valves and bicuspid aortic valve.

Although cardiac neural crest cells are required at early stages of arterial valve development, their contribution during valvular leaflet maturation remains poorly understood. Here, we show in mouse that neural crest cells from pre-otic and post-otic regions make distinct contributions to the arterial valve leaflets. Genetic fate-mapping analysis of Krox20-expressing neural crest cells shows a large contribution to the borders and the interleaflet triangles of the arterial valves. Loss of Krox20 function results in hyperplastic aortic valve and partially penetrant bicuspid aortic valve formation. Similar defects are observed in neural crest Krox20-deficient embryos. Genetic lineage tracing in Krox20-/- mutant mice shows that endothelial-derived cells are normal, whereas neural crest-derived cells are abnormally increased in number and misplaced in the valve leaflets. In contrast, genetic ablation of Krox20-expressing cells is not sufficient to cause an aortic valve defect, suggesting that adjacent cells can compensate this depletion. Our findings demonstrate a crucial role for Krox20 in arterial valve development and reveal that an excess of neural crest cells may be associated with bicuspid aortic valve.

Regulatory T Cells Expressing Tumor Necrosis Factor Receptor Type 2 Play a Major Role in CD4+ T-Cell Impairment During Sepsis.

Sepsis causes inflammation-induced immunosuppression with lymphopenia and alterations of CD4+ T-cell functions that renders the host prone to secondary infections. Whether and how regulatory T cells (Treg) are involved in this postseptic immunosuppression is unknown. We observed in vivo that early activation of Treg during Staphylococcus aureus sepsis induces CD4+ T-cell impairment and increases susceptibility to secondary pneumonia. The tumor necrosis factor receptor 2 positive (TNFR2pos) Treg subset endorsed the majority of effector immunosuppressive functions, and TNRF2 was particularly associated with activation of genes involved in cell cycle and replication in Treg, probably explaining their maintenance. Blocking or deleting TNFR2 during sepsis decreased the susceptibility to secondary infection. In humans, our data paralleled those in mice; the expression of CTLA-4 was dramatically increased in TNFR2pos Treg after culture in vitro with S. aureus. Our findings describe in vivo mechanisms underlying sepsis-induced immunosuppression and identify TNFR2pos Treg as targets for therapeutic intervention.

Krox20 hindbrain regulation incorporates multiple modes of cooperation between cis-acting elements.

Developmental genes can harbour multiple transcriptional enhancers that act simultaneously or in succession to achieve robust and precise spatiotemporal expression. However, the mechanisms underlying cooperation between cis-acting elements are poorly documented, notably in vertebrates. The mouse gene Krox20 encodes a transcription factor required for the specification of two segments (rhombomeres) of the developing hindbrain. In rhombomere 3, Krox20 is subject to direct positive feedback governed by an autoregulatory enhancer, element A. In contrast, a second enhancer, element C, distant by 70 kb, is active from the initiation of transcription independent of the presence of the KROX20 protein. Here, using both enhancer knock-outs and investigations of chromatin organisation, we show that element C possesses a dual activity: besides its classical enhancer function, it is also permanently required in cis to potentiate the autoregulatory activity of element A, by increasing its chromatin accessibility. This work uncovers a novel, asymmetrical, long-range mode of cooperation between cis-acting elements that might be essential to avoid promiscuous activation of positive autoregulatory elements.

Blood vessels guide Schwann cell migration in the adult demyelinated CNS through Eph/ephrin signaling.

Schwann cells (SC) enter the central nervous system (CNS) in pathophysiological conditions. However, how SC invade the CNS to remyelinate central axons remains undetermined. We studied SC migratory behavior ex vivo and in vivo after exogenous transplantation in the demyelinated spinal cord. The data highlight for the first time that SC migrate preferentially along blood vessels in perivascular extracellular matrix (ECM), avoiding CNS myelin. We demonstrate in vitro and in vivo that this migration route occurs by virtue of a dual mode of action of Eph/ephrin signaling. Indeed, EphrinB3, enriched in myelin, interacts with SC Eph receptors, to drive SC away from CNS myelin, and triggers their preferential adhesion to ECM components, such as fibronectin via integrinß1 interactions. This complex interplay enhances SC migration along the blood vessel network and together with lesion-induced vascular remodeling facilitates their timely invasion of the lesion site. These novel findings elucidate the mechanism by which SC invade and contribute to spinal cord repair.

Physiological adjustments and transcriptome reprogramming are involved in the acclimation to salinity gradients in diatoms.

Salinity regimes in estuaries and coastal areas vary with river discharge patterns, seawater evaporation, the morphology of the coastal waterways and the dynamics of marine water mixing. Therefore, microalgae have to respond to salinity variations at time scales ranging from daily to annual cycles. Microalgae may also have to adapt to physical alterations that induce the loss of connectivity between habitats and the enclosure of bodies of water. Here, we integrated physiological assays and measurements of morphological plasticity with a functional genomics approach to examine the regulatory changes that occur during the acclimation to salinity in the estuarine diatom Thalassiosira weissflogii. We found that cells exposed to different salinity regimes for a short or long period presented adjustments in their carbon fractions, silicon pools, pigment concentrations and/or photosynthetic parameters. Salinity-induced alterations in frustule symmetry were observed only in the long-term (LT) cultures. Whole transcriptome analyses revealed a down-regulation of nuclear and plastid encoded genes during the LT response and identified only a few regulated genes that were in common between the ST and LT responses. We propose that in diatoms, one strategy for acclimating to salinity gradients and maintaining optimal cellular fitness could be a reduction in the cost of transcription.

Metabolic intermediates selectively stimulate transcription factor interaction and modulate phosphate and purine pathways.

Cells use strategic metabolites to sense the metabolome and accordingly modulate gene expression. Here, we show that the purine and phosphate pathways are positively regulated by the metabolic intermediate AICAR (5'-phosphoribosyl-5-amino-4-imidazole carboxamide). The transcription factor Pho2p is required for up-regulation of all AICAR-responsive genes. Accordingly, the binding of Pho2p to purine and phosphate pathway gene promoters is enhanced upon AICAR accumulation. In vitro, AICAR binds both Pho2p and Pho4p transcription factors and stimulates the interaction between Pho2p and either Bas1p or Pho4p in vivo. In contrast, SAICAR (succinyl-AICAR) only affects Pho2p-Bas1p interaction and specifically up-regulates purine regulon genes. Together, our data show that Bas1p and Pho4p compete for Pho2p binding, hence leading to the concerted regulation of cellular nucleotide synthesis and phosphate consumption.

A long nuclear-retained non-coding RNA regulates synaptogenesis by modulating gene expression.

A growing number of long nuclear-retained non-coding RNAs (ncRNAs) have recently been described. However, few functions have been elucidated for these ncRNAs. Here, we have characterized the function of one such ncRNA, identified as metastasis-associated lung adenocarcinoma transcript 1 (Malat1). Malat1 RNA is expressed in numerous tissues and is highly abundant in neurons. It is enriched in nuclear speckles only when RNA polymerase II-dependent transcription is active. Knock-down studies revealed that Malat1 modulates the recruitment of SR family pre-mRNA-splicing factors to the transcription site of a transgene array. DNA microarray analysis in Malat1-depleted neuroblastoma cells indicates that Malat1 controls the expression of genes involved not only in nuclear processes, but also in synapse function. In cultured hippocampal neurons, knock-down of Malat1 decreases synaptic density, whereas its over-expression results in a cell-autonomous increase in synaptic density. Our results suggest that Malat1 regulates synapse formation by modulating the expression of genes involved in synapse formation and/or maintenance.

Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress.

Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages.

Boundary cap cells are peripheral nervous system stem cells that can be redirected into central nervous system lineages.

Boundary cap cells (BC), which express the transcription factor Krox20, participate in the formation of the boundary between the central nervous system and the peripheral nervous system. To study BC stemness, we developed a method to purify and amplify BC in vitro from Krox20(Cre/+), R26R(YFP/+) mouse embryos. We show that BC progeny are EGF/FGF2-responsive, form spheres, and express neural crest markers. Upon growth factor withdrawal, BC progeny gave rise to multiple neural crest and CNS lineages. Transplanted into the developing murine forebrain, they successfully survived, migrated, and integrated within the host environment. Surprisingly, BC progeny generated exclusively CNS cells, including neurons, astrocytes, and myelin-forming oligodendrocytes. In vitro experiments indicated that a sequential combination of ventralizing morphogens and glial growth factors was necessary to reprogram BC into oligodendrocytes. Thus, BC progeny are endowed with differentiation plasticity beyond the peripheral nervous system. The demonstration that CNS developmental cues can reprogram neural crest-derived stem cells into CNS derivatives suggests that BC could serve as a source of cell type-specific lineages, including oligodendrocytes, for cell-based therapies to treat CNS disorders.

Single nuclei transcriptomics reveal the differentiation trajectories of periosteal skeletal/stem progenitor cells in bone regeneration.

Bone regeneration is mediated by skeletal stem/progenitor cells (SSPCs) that are mainly recruited from the periosteum after bone injury. The composition of the periosteum and the steps of SSPC activation and differentiation remain poorly understood. Here, we generated a single-nuclei atlas of the periosteum at steady-state and of the fracture site during early stages of bone repair ( https://fracture-repair-atlas.cells.ucsc.edu ). We identified periosteal SSPCs expressing stemness markers ( Pi16 and Ly6a /SCA1) and responding to fracture by adopting an injury-induced fibrogenic cell (IIFC) fate, prior to undergoing osteogenesis or chondrogenesis. We identified distinct gene cores associated with IIFCs and their engagement into osteogenesis and chondrogenesis involving Notch, Wnt and the circadian clock signaling respectively. Finally, we show that IIFCs are the main source of paracrine signals in the fracture environment, suggesting a crucial paracrine role of this transient IIFC population during fracture healing. Overall, our study provides a complete temporal topography of the early stages of fracture healing and the dynamic response of periosteal SSPCs to injury, redefining our knowledge of bone regeneration.

The pelvic organs receive no parasympathetic innervation.

The pelvic organs (bladder, rectum, and sex organs) have been represented for a century as receiving autonomic innervation from two pathways - lumbar sympathetic and sacral parasympathetic - by way of a shared relay, the pelvic ganglion, conceived as an assemblage of sympathetic and parasympathetic neurons. Using single-cell RNA sequencing, we find that the mouse pelvic ganglion is made of four classes of neurons, distinct from both sympathetic and parasympathetic ones, albeit with a kinship to the former, but not the latter, through a complex genetic signature. We also show that spinal lumbar preganglionic neurons synapse in the pelvic ganglion onto equal numbers of noradrenergic and cholinergic cells, both of which therefore serve as sympathetic relays. Thus, the pelvic viscera receive no innervation from parasympathetic or typical sympathetic neurons, but instead from a divergent tail end of the sympathetic chains, in charge of its idiosyncratic functions.

Motor innervation directs the correct development of the mouse sympathetic nervous system.

The sympathetic nervous system controls bodily functions including vascular tone, cardiac rhythm, and the "fight-or-flight response". Sympathetic chain ganglia develop in parallel with preganglionic motor nerves extending from the neural tube, raising the question of whether axon targeting contributes to sympathetic chain formation. Using nerve-selective genetic ablations and lineage tracing in mouse, we reveal that motor nerve-associated Schwann cell precursors (SCPs) contribute sympathetic neurons and satellite glia after the initial seeding of sympathetic ganglia by neural crest. Motor nerve ablation causes mispositioning of SCP-derived sympathoblasts as well as sympathetic chain hypoplasia and fragmentation. Sympathetic neurons in motor-ablated embryos project precociously and abnormally towards dorsal root ganglia, eventually resulting in fusion of sympathetic and sensory ganglia. Cell interaction analysis identifies semaphorins as potential motor nerve-derived signaling molecules regulating sympathoblast positioning and outgrowth. Overall, central innervation functions both as infrastructure and regulatory niche to ensure the integrity of peripheral ganglia morphogenesis.

MEK-SHP2 inhibition prevents tibial pseudarthrosis caused by NF1 loss in Schwann cells and skeletal stem/progenitor cells.

Congenital pseudarthrosis of the tibia (CPT) is a severe pathology marked by spontaneous bone fractures that fail to heal, leading to fibrous nonunion. Half of patients with CPT are affected by the multisystemic genetic disorder neurofibromatosis type 1 (NF1) caused by mutations in the NF1 tumor suppressor gene, a negative regulator of RAS-mitogen-activated protein kinase (MAPK) signaling pathway. Here, we analyzed patients with CPT and Prss56-Nf1 knockout mice to elucidate the pathogenic mechanisms of CPT-related fibrous nonunion and explored a pharmacological approach to treat CPT. We identified NF1-deficient Schwann cells and skeletal stem/progenitor cells (SSPCs) in pathological periosteum as affected cell types driving fibrosis. Whereas NF1-deficient SSPCs adopted a fibrotic fate, NF1-deficient Schwann cells produced critical paracrine factors including transforming growth factor-ß and induced fibrotic differentiation of wild-type SSPCs. To counteract the elevated RAS-MAPK signaling in both NF1-deficient Schwann cells and SSPCs, we used MAPK kinase (MEK) and Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) inhibitors. Combined MEK-SHP2 inhibition in vivo prevented fibrous nonunion in the Prss56-Nf1 knockout mouse model, providing a promising therapeutic strategy for the treatment of fibrous nonunion in CPT.

Comparative transcriptomics reveals different strategies of Trichoderma mycoparasitism.

BACKGROUND: Trichoderma is a genus of mycotrophic filamentous fungi (teleomorph Hypocrea) which possess a bright variety of biotrophic and saprotrophic lifestyles. The ability to parasitize and/or kill other fungi (mycoparasitism) is used in plant protection against soil-borne fungal diseases (biological control, or biocontrol). To investigate mechanisms of mycoparasitism, we compared the transcriptional responses of cosmopolitan opportunistic species and powerful biocontrol agents Trichoderma atroviride and T. virens with tropical ecologically restricted species T. reesei during confrontations with a plant pathogenic fungus Rhizoctonia solani. RESULTS: The three Trichoderma spp. exhibited a strikingly different transcriptomic response already before physical contact with alien hyphae. T. atroviride expressed an array of genes involved in production of secondary metabolites, GH16 ß-glucanases, various proteases and small secreted cysteine rich proteins. T. virens, on the other hand, expressed mainly the genes for biosynthesis of gliotoxin, respective precursors and also glutathione, which is necessary for gliotoxin biosynthesis. In contrast, T. reesei increased the expression of genes encoding cellulases and hemicellulases, and of the genes involved in solute transport. The majority of differentially regulated genes were orthologues present in all three species or both in T. atroviride and T. virens, indicating that the regulation of expression of these genes is different in the three Trichoderma spp. The genes expressed in all three fungi exhibited a nonrandom genomic distribution, indicating a possibility for their regulation via chromatin modification. CONCLUSION: This genome-wide expression study demonstrates that the initial Trichoderma mycotrophy has differentiated into several alternative ecological strategies ranging from parasitism to predation and saprotrophy. It provides first insights into the mechanisms of interactions between Trichoderma and other fungi that may be exploited for further development of biofungicides.

Neural tube-associated boundary caps are a major source of mural cells in the skin.

In addition to their roles in protecting nerves and increasing conduction velocity, peripheral glia plays key functions in blood vessel development by secreting molecules governing arteries alignment and maturation with nerves. Here, we show in mice that a specific, nerve-attached cell population, derived from boundary caps (BCs), constitutes a major source of mural cells for the developing skin vasculature. Using Cre-based reporter cell tracing and single-cell transcriptomics, we show that BC derivatives migrate into the skin along the nerves, detach from them, and differentiate into pericytes and vascular smooth muscle cells. Genetic ablation of this population affects the organization of the skin vascular network. Our results reveal the heterogeneity and extended potential of the BC population in mice, which gives rise to mural cells, in addition to previously described neurons, Schwann cells, and melanocytes. Finally, our results suggest that mural specification of BC derivatives takes place before their migration along nerves to the mouse skin.

Topical delivery of mitogen-activated protein kinase inhibitor binimetinib prevents the development of cutaneous neurofibromas in neurofibromatosis type 1 mutant mice.

Cutaneous neurofibromas (cNFs) are a hallmark of patients with the neurofibromatosis type 1 (NF1) genetic disorder. These benign nerve sheath tumors, which can amount to thousands, develop from puberty onward, often cause pain and are considered by patients to be the primary burden of the disease. Mutations of NF1, encoding a negative regulator of the RAS signaling pathway, in the Schwann cell (SCs) lineage are considered to be at the origin of cNFs. The mechanisms governing cNFs development are poorly understood, and therapeutics to reduce cNFs are missing, mainly due to the lack of appropriate animal models. To address this, we designed the Nf1-KO mouse model that develops cNFs. Using this model, we found that cNFs development is a singular event and goes through 3 successive stages: initiation, progression, and stabilization characterized by changes in the proliferative and MAPK activities of tumor SCs. We found that skin trauma accelerated the development of cNFs and further used this model to explore the efficacy of the MEK inhibitor binimetinib to cure these tumors. We showed that while topically delivered binimetinib has a selective and minor effect on mature cNFs, the same drug prevents their development over long periods.

Filopodia-like protrusions of adjacent somatic cells shape the developmental potential of oocytes.

The oocyte must grow and mature before fertilization, thanks to a close dialogue with the somatic cells that surround it. Part of this communication is through filopodia-like protrusions, called transzonal projections (TZPs), sent by the somatic cells to the oocyte membrane. To investigate the contribution of TZPs to oocyte quality, we impaired their structure by generating a full knockout mouse of the TZP structural component myosin-X (MYO10). Using spinning disk and super-resolution microscopy combined with a machine-learning approach to phenotype oocyte morphology, we show that the lack of Myo10 decreases TZP density during oocyte growth. Reduction in TZPs does not prevent oocyte growth but impairs oocyte-matrix integrity. Importantly, we reveal by transcriptomic analysis that gene expression is altered in TZP-deprived oocytes and that oocyte maturation and subsequent early embryonic development are partially affected, effectively reducing mouse fertility. We propose that TZPs play a role in the structural integrity of the germline-somatic complex, which is essential for regulating gene expression in the oocyte and thus its developmental potential.

Thyroid-stimulating hormone receptor signaling restores skeletal muscle stem cell regeneration in rats with muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe and progressive myopathy leading to motor and cardiorespiratory impairment. We analyzed samples from patients with DMD and a preclinical rat model of severe DMD and determined that compromised repair capacity of muscle stem cells in DMD is associated with early and progressive muscle stem cell senescence. We also found that extraocular muscles (EOMs), which are spared by the disease in patients, contain muscle stem cells with long-lasting regenerative potential. Using single-cell transcriptomics analysis of muscles from a rat model of DMD, we identified the gene encoding thyroid-stimulating hormone receptor (Tshr) as highly expressed in EOM stem cells. Further, TSHR activity was involved in preventing senescence. Forskolin, which activates signaling downstream of TSHR, was found to reduce senescence of skeletal muscle stem cells, increase stem cell regenerative potential, and promote myogenesis, thereby improving muscle function in DMD rats. These findings indicate that stimulation of adenylyl cyclase leads to muscle repair in DMD, potentially providing a therapeutic approach for patients with the disease.