Absence of sympathetic innervation hampers the generation of tertiary lymphoid structures upon acute lung inflammation.

Tertiary lymphoid structures (TLS) are lymphoid organs present in inflammatory non-lymphoid tissues. Studies have linked TLS to favorable outcomes for patients with cancers or infectious diseases, but the mechanisms underlying their formation are not fully understood. In particular, secondary lymphoid organs innervation raises the question of sympathetic nerve fibers involvement in TLS organogenesis. We established a model of pulmonary inflammation based on 5 daily intranasal instillations of lipopolysaccharide (LPS) in immunocompetent mice. In this setting, lung lymphoid aggregates formed transiently, evolving toward mature TLS and disappearing when inflammation resolved. Sympathetic nerve fibers were then depleted using 6-hydroxydopamine. TLS quantification by immunohistochemistry showed a decrease in LPS-induced TLS number and surface in denervated mouse lungs. Although a reduction in alveolar space was observed, it did not impair overall pulmonary content of transcripts encoding TNF-α, IL-1ß and IFN-γ inflammation molecules whose expression was induced by LPS instillations. Immunofluorescence analysis of immune infiltrates in lungs of LPS-treated mice showed a drop in the proportion of CD23+ naive cells among CD19+ B220+ B cells in denervated mice whereas the proportion of other cell subsets remained unchanged. These data support the existence of neuroimmune crosstalk impacting lung TLS neogenesis and local naive B cell pool.

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.

Heterogeneity and developmental dynamics of LYVE-1 perivascular macrophages distribution in the mouse brain.

Brain perivascular macrophages (PVMs) are border-associated macrophages situated along blood vessels in the Virchow-Robin space and are thus found at a unique anatomical position between the endothelium and the parenchyma. Owing to their location and phagocytic capabilities, PVMs are regarded as important components that regulate various aspects of brain physiology in health and pathophysiological states. Here, we used LYVE-1 to identify PVMs in the mouse brain using brain-tissue sections and cleared whole-brains to learn about how they are distributed within the brain and across different developmental postnatal stages. We find that LYVE-1+ PVMs associate with the vasculature in different patterns and proportions depending on vessel diameter or arterio-venous differentiation. LYVE-1+ PVMs relate to blood vessels in a brain-region-dependent manner. We show that their postnatal distribution is developmentally dynamic and peaks at P10-P20 depending on the brain region. We further demonstrate that their density is reduced in the APP/PS1 mouse model of Alzheimer's Disease proportionally to beta-amyloid deposits. In conclusion, our results reveal unexpected heterogeneity and dynamics of LYVE-1+ PVMs, with selective coverage of brain vasculature, compatible with potential unexplored roles for this population of PVMs in postnatal development, and in regulating brain functions in steady-state and disease conditions.

Myelinating Schwann cells and Netrin-1 control intra-nervous vascularization of the developing mouse sciatic nerve.

Peripheral nerves are vascularized by a dense network of blood vessels to guarantee their complex function. Despite the crucial role of vascularization to ensure nerve homeostasis and regeneration, the mechanisms governing nerve invasion by blood vessels remain poorly understood. We found, in mice, that the sciatic nerve invasion by blood vessels begins around embryonic day 16 and continues until birth. Interestingly, intra-nervous blood vessel density significantly decreases during post-natal period, starting from P10. We show that, while the axon guidance molecule Netrin-1 promotes nerve invasion by blood vessels via the endothelial receptor UNC5B during embryogenesis, myelinated Schwann cells negatively control intra-nervous vascularization during post-natal period.

Megalencephalic leukoencephalopathy with subcortical cysts is a developmental disorder of the gliovascular unit.

Absence of the astrocyte-specific membrane protein MLC1 is responsible for megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare type of leukodystrophy characterized by early-onset macrocephaly and progressive white matter vacuolation that lead to ataxia, spasticity, and cognitive decline. During postnatal development (from P5 to P15 in the mouse), MLC1 forms a membrane complex with GlialCAM (another astrocytic transmembrane protein) at the junctions between perivascular astrocytic processes. Perivascular astrocytic processes along with blood vessels form the gliovascular unit. It was not previously known how MLC1 influences the physiology of the gliovascular unit. Here, using the Mlc1 knock-out mouse model of MLC, we demonstrated that MLC1 controls the postnatal development and organization of perivascular astrocytic processes, vascular smooth muscle cell contractility, neurovascular coupling, and intraparenchymal interstitial fluid clearance. Our data suggest that MLC is a developmental disorder of the gliovascular unit, and perivascular astrocytic processes and vascular smooth muscle cell maturation defects are primary events in the pathogenesis of MLC and therapeutic targets for this disease.

[The functions of arterial sympathetic innervation: from development to pathology]. / Les fonctions de l'innervation sympathique artérielle - Du développement à la pathologie.

Arterial sympathetic innervation (ASI) is a complex biological process requiring a fine axonal guidance by arteries. Its physiological impact has remained unknown for decades but recently started to be better understood and recognized. ASI is a key element of the adaptive response of the cardiovascular system to challenging situations (exposure to cold, exercise…) as ASI controls the diameter of resistance arteries, thus blood supply to organs and systemic arterial blood pressure via arterial tone modulation. Defaults in ASI can lead to diseases, acting as a main cause or as an aggravating factor. Its impact is actively studied in cardiovascular diseases representing major public health issues, like hypertension, but ASI could also play a role in aging and many more pathological processes including cancer.

TITLE: Les fonctions de l'innervation sympathique artérielle - Du développement à la pathologie. ABSTRACT: L'innervation sympathique artérielle (ISA) est un processus biologique complexe nécessitant un guidage fin des axones des neurones sympathiques par les artères. L'ISA est un élément clé de l'adaptation du système cardiovasculaire aux différentes contraintes (exposition au froid, exercice, etc.) : elle contrôle le diamètre des artères de résistance, donc le flux sanguin parvenant aux organes et la pression artérielle systémique via la modulation du tonus artériel. Son importance lors du vieillissement et dans de nombreux contextes pathologiques est de mieux en mieux reconnue et comprise. Son intégration à la prise en charge de nombreuses maladies (hypertension, cancer, etc.) permettrait d'en améliorer traitements et pronostic.

Sympathetic Innervation Promotes Arterial Fate by Enhancing Endothelial ERK Activity.

RATIONALE: Arterial endothelial cells are morphologically, functionally, and molecularly distinct from those found in veins and lymphatic vessels. How arterial fate is acquired during development and maintained in adult vessels is incompletely understood. OBJECTIVE: We set out to identify factors that promote arterial endothelial cell fate in vivo. METHODS AND RESULTS: We developed a functional assay, allowing us to monitor and manipulate arterial fate in vivo, using arteries isolated from quails that are grafted into the coelom of chick embryos. Endothelial cells migrate out from the grafted artery, and their colonization of host arteries and veins is quantified. Here we show that sympathetic innervation promotes arterial endothelial cell fate in vivo. Removal of sympathetic nerves decreases arterial fate and leads to colonization of veins, whereas exposure to sympathetic nerves or norepinephrine imposes arterial fate. Mechanistically, sympathetic nerves increase endothelial ERK (extracellular signal-regulated kinase) activity via adrenergic α1 and α2 receptors. CONCLUSIONS: These findings show that sympathetic innervation promotes arterial endothelial fate and may lead to novel approaches to improve arterialization in human disease.

Listeners Exploit Syntactic Structure On-Line to Restrict Their Lexical Search to a Subclass of Verbs.

Many experiments have shown that listeners actively build expectations about up-coming words, rather than simply waiting for information to accumulate. The online construction of a syntactic structure is one of the cues that listeners may use to construct strong expectations about the possible words they will be exposed to. For example, speakers of verb-final languages use pre-verbal arguments to predict on-line the kind of arguments that are likely to occur next (e.g., Kamide, 2008, for a review). Although in SVO languages information about a verb's arguments typically follows the verb, some languages use pre-verbal object pronouns, potentially allowing listeners to build on-line expectations about the nature of the upcoming verb. For instance, if a pre-verbal direct object pronoun is heard, then the following verb has to be able to enter a transitive structure, thus excluding intransitive verbs. To test this, we used French, in which object pronouns have to appear pre-verbally, to investigate whether listeners use this cue to predict the occurrence of a transitive verb. In a word detection task, we measured the number of false alarms to sentences that contained a transitive verb whose first syllable was homophonous to the target monosyllabic verb (e.g., target "dort" /dɔʁ/ to sleep and false alarm verb "dorlote" /dɔʁlɔt/ to cuddle). The crucial comparison involved two sentence types, one without a pre-verbal object clitic, for which an intransitive verb was temporarily a plausible option (e.g., "Il dorlote" / He cuddles) and the other with a pre-verbal object clitic, that made the appearance of an intransitive verb impossible ("Il le dorlote" / He cuddles it). Results showed a lower rate of false alarms for sentences with a pre-verbal object pronoun (3%) compared to locally ambiguous sentences (about 20%). Participants rapidly incorporate information about a verb's argument structure to constrain lexical access to verbs that match the expected subcategorization frame.

NG2 glia are required for vessel network formation during embryonic development.

The NG2(+) glia, also known as polydendrocytes or oligodendrocyte precursor cells, represent a new entity among glial cell populations in the central nervous system. However, the complete repertoire of their roles is not yet identified. The embryonic NG2(+) glia originate from the Nkx2.1(+) progenitors of the ventral telencephalon. Our analysis unravels that, beginning from E12.5 until E16.5, the NG2(+) glia populate the entire dorsal telencephalon. Interestingly, their appearance temporally coincides with the establishment of blood vessel network in the embryonic brain. NG2(+) glia are closely apposed to developing cerebral vessels by being either positioned at the sprouting tip cells or tethered along the vessel walls. Absence of NG2(+) glia drastically affects the vascular development leading to severe reduction of ramifications and connections by E18.5. By revealing a novel and fundamental role for NG2(+) glia, our study brings new perspectives to mechanisms underlying proper vessels network formation in embryonic brains.

Vascular Mural Cells Promote Noradrenergic Differentiation of Embryonic Sympathetic Neurons.

The sympathetic nervous system controls smooth muscle tone and heart rate in the cardiovascular system. Postganglionic sympathetic neurons (SNs) develop in close proximity to the dorsal aorta (DA) and innervate visceral smooth muscle targets. Here, we use the zebrafish embryo to ask whether the DA is required for SN development. We show that noradrenergic (NA) differentiation of SN precursors temporally coincides with vascular mural cell (VMC) recruitment to the DA and vascular maturation. Blocking vascular maturation inhibits VMC recruitment and blocks NA differentiation of SN precursors. Inhibition of platelet-derived growth factor receptor (PDGFR) signaling prevents VMC differentiation and also blocks NA differentiation of SN precursors. NA differentiation is normal in cloche mutants that are devoid of endothelial cells but have VMCs. Thus, PDGFR-mediated mural cell recruitment mediates neurovascular interactions between the aorta and sympathetic precursors and promotes their noradrenergic differentiation.

Arterial innervation in development and disease.

Innervation of arteries by sympathetic nerves is well known to control blood supply to organs. Recent studies have elucidated the mechanisms that regulate the development of arterial innervation and show that in addition to vascular tone, sympathetic nerves may also influence arterial maturation and growth. Understanding sympathetic arterial innervation may lead to new approaches to treat peripheral arterial disease and hypertension.

Netrin-1 controls sympathetic arterial innervation.

Autonomic sympathetic nerves innervate peripheral resistance arteries, thereby regulating vascular tone and controlling blood supply to organs. Despite the fundamental importance of blood flow control, how sympathetic arterial innervation develops remains largely unknown. Here, we identified the axon guidance cue netrin-1 as an essential factor required for development of arterial innervation in mice. Netrin-1 was produced by arterial smooth muscle cells (SMCs) at the onset of innervation, and arterial innervation required the interaction of netrin-1 with its receptor, deleted in colorectal cancer (DCC), on sympathetic growth cones. Function-blocking approaches, including cell type-specific deletion of the genes encoding Ntn1 in SMCs and Dcc in sympathetic neurons, led to severe and selective reduction of sympathetic innervation and to defective vasoconstriction in resistance arteries. These findings indicate that netrin-1 and DCC are critical for the control of arterial innervation and blood flow regulation in peripheral organs.

Friend or foe? Early social evaluation of human interactions.

We report evidence that 29-month-old toddlers and 10-month-old preverbal infants discriminate between two agents: a pro-social agent, who performs a positive (comforting) action on a human patient and a negative (harmful) action on an inanimate object, and an anti-social agent, who does the converse. The evidence shows that they prefer the former to the latter even though the agents perform the same bodily movements. Given that humans can cause physical harm to their conspecifics, we discuss this finding in light of the likely adaptive value of the ability to detect harmful human agents.

Olfactory receptor responding to gut microbiota-derived signals plays a role in renin secretion and blood pressure regulation.

Olfactory receptors are G protein-coupled receptors that mediate olfactory chemosensation and serve as chemosensors in other tissues. We find that Olfr78, an olfactory receptor expressed in the kidney, responds to short chain fatty acids (SCFAs). Olfr78 is expressed in the renal juxtaglomerular apparatus, where it mediates renin secretion in response to SCFAs. In addition, both Olfr78 and G protein-coupled receptor 41 (Gpr41), another SCFA receptor, are expressed in smooth muscle cells of small resistance vessels. Propionate, a SCFA shown to induce vasodilation ex vivo, produces an acute hypotensive response in wild-type mice. This effect is differentially modulated by disruption of Olfr78 and Gpr41 expression. SCFAs are end products of fermentation by the gut microbiota and are absorbed into the circulation. Antibiotic treatment reduces the biomass of the gut microbiota and elevates blood pressure in Olfr78 knockout mice. We conclude that SCFAs produced by the gut microbiota modulate blood pressure via Olfr78 and Gpr41.

Semaphorin3A, Neuropilin-1, and PlexinA1 are required for lymphatic valve formation.

RATIONALE: The lymphatic vasculature plays a major role in fluid homeostasis, absorption of dietary lipids, and immune surveillance. Fluid transport depends on the presence of intraluminal valves within lymphatic collectors. Defective formation of lymphatic valves leads to lymphedema, a progressive and debilitating condition for which curative treatments are currently unavailable. How lymphatic valve formation is regulated remains largely unknown. OBJECTIVE: We investigated if the repulsive axon guidance molecule Semaphorin3A (Sema3A) plays a role in lymphatic valve formation. METHODS AND RESULTS: We show that Sema3A mRNA is expressed in lymphatic vessels and that Sema3A protein binds to lymphatic valves expressing the Neuropilin-1 (Nrp1) and PlexinA1 receptors. Using mouse knockout models, we show that Sema3A is selectively required for lymphatic valve formation, via interaction with Nrp1 and PlexinA1. Sema3a(-/-) mice exhibit defects in lymphatic valve formation, which are not due to abnormal lymphatic patterning or sprouting, and mice carrying a mutation in the Sema3A binding site of Nrp1, or deficient for Plxna1, develop lymphatic valve defects similar to those seen in Sema3a(-/-) mice. CONCLUSIONS: Our data demonstrate an essential direct function of Sema3A-Nrp1-PlexinA1 signaling in lymphatic valve formation.

Extracellular Engrailed participates in the topographic guidance of retinal axons in vivo.

Engrailed transcription factors regulate the expression of guidance cues that pattern retinal axon terminals in the dorsal midbrain. They also act directly to guide axon growth in vitro. We show here that an extracellular En gradient exists in the tectum along the anterior-posterior axis. Neutralizing extracellular Engrailed in vivo with antibodies expressed in the tectum causes temporal axons to map aberrantly to the posterior tectum in chick and Xenopus. Furthermore, posterior membranes from wild-type tecta incubated with anti-Engrailed antibodies or posterior membranes from Engrailed-1 knockout mice exhibit diminished repulsive activity for temporal axons. Since EphrinAs play a major role in anterior-posterior mapping, we tested whether Engrailed cooperates with EphrinA5 in vitro. We find that Engrailed restores full repulsion to axons given subthreshold doses of EphrinA5. Collectively, our results indicate that extracellular Engrailed contributes to retinotectal mapping in vivo by modulating the sensitivity of growth cones to EphrinA.

Autophosphorylation-independent and -dependent functions of focal adhesion kinase during development.

Focal adhesion kinase (FAK) regulates numerous cellular functions and is critical for processes ranging from embryo development to cancer progression. Although autophosphorylation on Tyr-397 appears required for FAK functions in vitro, its role in vivo has not been established. We addressed this question using a mutant mouse (fakDelta) deleted of exon 15, which encodes Tyr-397. The resulting mutant protein FAKDelta is an active kinase expressed at normal levels. Our results demonstrate that the requirement for FAK autophosphorylation varies during development. FAK(Delta/Delta) embryos developed normally up to embryonic day (E) 12.5, contrasting with the lethality at E8.5 of FAK-null embryos. Thus, autophosphorylation on Tyr-397 is not required for FAK to achieve its functions until late mid-gestation. However, FAK(Delta/Delta) embryos displayed hemorrhages, edema, delayed artery formation, vascular remodeling defects, multiple organ abnormalities, and overall developmental retardation at E13.5-14.5, and died thereafter demonstrating that FAK autophosphorylation is also necessary for normal development. Fibroblasts derived from mutant embryos had a normal stellate morphology and expression of focal adhesion proteins, Src family members, p53, and Pyk2. In contrast, in FAK(Delta/Delta) fibroblasts and endothelial cells, spreading and lamellipodia formation were altered with an increased size and number of focal adhesions, enriched in FAKDelta. FAK mutation also decreased fibroblast proliferation. These results show that the physiological functions of FAK in vivo are achieved through both autophosphorylation-independent and autophosphorylation-dependent mechanisms.

Guidance of vascular development: lessons from the nervous system.

The vascular system of vertebrates consists of an organized, branched network of arteries, veins, and capillaries that penetrates all the tissues of the body. One of the most striking features of the vascular system is that its branching pattern is highly stereotyped, with major and secondary branches forming at specific sites and developing highly conserved organ-specific vascular patterns. The factors controlling vascular patterning are not yet completely understood. Recent studies have highlighted the anatomic and structural similarities between blood vessels and nerves. The 2 networks are often aligned, with nerve fibers and blood vessels following parallel routes. Furthermore, both systems require precise control over their guidance and growth. Several molecules with attractive and repulsive properties have been found to modulate the proper guidance of both nerves and blood vessels. These include the Semaphorins, the Slits, and the Netrins and their receptors. In this review, we describe the molecular mechanisms by which blood vessels and axons achieve proper path finding and the molecular cues that are involved in their guidance.

Silencing of choline acetyltransferase expression by lentivirus-mediated RNA interference in cultured cells and in the adult rodent brain.

RNA interference (RNAi) is a potent mechanism for local silencing of gene expression and can be used to study loss-of-function phenotypes in mammalian cells. We used RNAi to knockdown specifically the expression of choline acetyltransferase (ChAT), the enzyme of acetylcholine biosynthesis, both in cultured cells and in the adult brain. We first identified a 19-nucleotide sequence in the coding region of rat and mouse ChAT transcripts that constitutes a target for potent silencing of ChAT expression by RNAi. We generated a lentiviral vector that produces both a small hairpin RNA (shRNA) targeting ChAT mRNAs and the enhanced green fluorescent protein (EGFP) reporter protein to facilitate identification of transduced cells. In the cholinergic cell line NG108-15, there was at least 90% less of the ChAT protein, as measured by assaying its enzymatic activity, 3 days postinfection with this vector than in cells infected with a control vector. The vector was used to transduce cholinergic neurons in vivo and reduced ChAT expression strongly and specifically in the cholinergic neurons of the medial septum in adult rats, without affecting the expression of the vesicular acetylcholine transporter. This lentiviral vector is thus a powerful tool for specific inactivation of cholinergic neurotransmission and can therefore be used to study the role of cholinergic nuclei in the brain. This lentiviral-mediated RNAi approach will also allow the development of new animal models of diseases in which cholinergic neurotransmission is specifically altered.