Microglia maintain structural integrity during fetal brain morphogenesis.

Microglia (MG), the brain-resident macrophages, play major roles in health and disease via a diversity of cellular states. While embryonic MG display a large heterogeneity of cellular distribution and transcriptomic states, their functions remain poorly characterized. Here, we uncovered a role for MG in the maintenance of structural integrity at two fetal cortical boundaries. At these boundaries between structures that grow in distinct directions, embryonic MG accumulate, display a state resembling post-natal axon-tract-associated microglia (ATM) and prevent the progression of microcavities into large cavitary lesions, in part via a mechanism involving the ATM-factor Spp1. MG and Spp1 furthermore contribute to the rapid repair of lesions, collectively highlighting protective functions that preserve the fetal brain from physiological morphogenetic stress and injury. Our study thus highlights key major roles for embryonic MG and Spp1 in maintaining structural integrity during morphogenesis, with major implications for our understanding of MG functions and brain development.

Dual ontogeny of disease-associated microglia and disease inflammatory macrophages in aging and neurodegeneration.

Brain macrophage populations include parenchymal microglia, border-associated macrophages, and recruited monocyte-derived cells; together, they control brain development and homeostasis but are also implicated in aging pathogenesis and neurodegeneration. The phenotypes, localization, and functions of each population in different contexts have yet to be resolved. We generated a murine brain myeloid scRNA-seq integration to systematically delineate brain macrophage populations. We show that the previously identified disease-associated microglia (DAM) population detected in murine Alzheimer's disease models actually comprises two ontogenetically and functionally distinct cell lineages: embryonically derived triggering receptor expressed on myeloid cells 2 (TREM2)-dependent DAM expressing a neuroprotective signature and monocyte-derived TREM2-expressing disease inflammatory macrophages (DIMs) accumulating in the brain during aging. These two distinct populations appear to also be conserved in the human brain. Herein, we generate an ontogeny-resolved model of brain myeloid cell heterogeneity in development, homeostasis, and disease and identify cellular targets for the treatment of neurodegeneration.

Microglial ontogeny, diversity and neurodevelopmental functions.

Microglia are instrumental to the development, function, homeostasis and pathologies of the central nervous system. These brain-resident macrophages arise early in embryogenesis and seed the developing brain, where they differentiate in response to cues provided by their neural niche. Throughout life, microglia regulate the neural tissue through a variety of cellular functions influenced by intrinsic and extrinsic factors. Despite their importance, we are only starting to uncover how microglia colonize the brain, adopt distinct functional states during development and the long-term impact of early alteration of their functions. This review highlights the latest knowledge on the ontogeny of microglia, their developmental trajectory and emerging roles. Characterizing these processes will be critical for our understanding of both brain physiology and pathologies.

Early Fate Defines Microglia and Non-parenchymal Brain Macrophage Development.

Central nervous system (CNS) macrophages comprise microglia and border-associated macrophages (BAMs) residing in the meninges, the choroid plexus, and the perivascular spaces. Most CNS macrophages emerge during development, with the exception of choroid plexus and dural macrophages, which are replaced by monocytes in adulthood. Whether microglia and BAMs share a developmental program or arise from separate lineages remains unknown. Here, we identified two phenotypically, transcriptionally, and locally distinct brain macrophages throughout development, giving rise to either microglia or BAMs. Two macrophage populations were already present in the yolk sac suggesting an early segregation. Fate-mapping models revealed that BAMs mostly derived from early erythro-myeloid progenitors in the yolk sac. The development of microglia was dependent on TGF-ß, whereas the genesis of BAMs occurred independently of this cytokine. Collectively, our data show that developing parenchymal and non-parenchymal brain macrophages are separate entities in terms of ontogeny, gene signature, and requirement for TGF-ß.

Biphasic Impact of Prenatal Inflammation and Macrophage Depletion on the Wiring of Neocortical Inhibitory Circuits.

The etiology of neurodevelopmental disorders is linked to defects in parvalbumin (PV)-expressing cortical interneurons and to prenatal immune challenges. Mouse models of maternal immune activation (MIA) and microglia deficits increase the postnatal density of PV interneurons, raising the question of their functional integration. Here, we show that MIA and embryonic depletion of macrophages including microglia have a two-step impact on PV interneurons wiring onto their excitatory target neurons in the barrel cortex. In adults, both challenges reduced the inhibitory drive from PV interneurons, as reported in neurodevelopmental disorders. In juveniles, however, we found an increased density of PV neurons, an enhanced strength of unitary connections onto excitatory cells, and an aberrant horizontal inhibition with a reduced lateral propagation of sensory inputs in vivo. Our results provide a comprehensive framework for understanding the impact of prenatal immune challenges onto the developmental trajectory of inhibitory circuits that leads to pathological brain wiring.

Induced-Pluripotent-Stem-Cell-Derived Primitive Macrophages Provide a Platform for Modeling Tissue-Resident Macrophage Differentiation and Function.

Tissue macrophages arise during embryogenesis from yolk-sac (YS) progenitors that give rise to primitive YS macrophages. Until recently, it has been impossible to isolate or derive sufficient numbers of YS-derived macrophages for further study, but data now suggest that induced pluripotent stem cells (iPSCs) can be driven to undergo a process reminiscent of YS-hematopoiesis in vitro. We asked whether iPSC-derived primitive macrophages (iMacs) can terminally differentiate into specialized macrophages with the help of growth factors and organ-specific cues. Co-culturing human or murine iMacs with iPSC-derived neurons promoted differentiation into microglia-like cells in vitro. Furthermore, murine iMacs differentiated in vivo into microglia after injection into the brain and into functional alveolar macrophages after engraftment in the lung. Finally, iPSCs from a patient with familial Mediterranean fever differentiated into iMacs with pro-inflammatory characteristics, mimicking the disease phenotype. Altogether, iMacs constitute a source of tissue-resident macrophage precursors that can be used for biological, pathophysiological, and therapeutic studies.

Subrepellent doses of Slit1 promote Netrin-1 chemotactic responses in subsets of axons.

BACKGROUND: Axon pathfinding is controlled by guidance cues that elicit specific attractive or repulsive responses in growth cones. It has now become clear that some cues such as Netrin-1 can trigger either attraction or repulsion in a context-dependent manner. In particular, it was recently found that the repellent Slit1 enables the attractive response of rostral thalamic axons to Netrin-1. This finding raised the intriguing possibility that Netrin-1 and Slit1, two essential guidance cues, may act more generally in an unexpected combinatorial manner to orient specific axonal populations. To address this major issue, we have used an innovative microfluidic device compatible not only with dissociated neuronal cultures but also with explant cultures to systematically and quantitatively characterize the combinatorial activity of Slit1 and Netrin-1 on rostral thalamic axons as well as on hippocampal neurons. RESULTS: We found that on rostral thalamic axons, only a subthreshold concentration of the repellent Slit1 triggered an attractive response to a gradient of Netrin-1. On hippocampal neurons, we similarly found that Slit1 alone is repulsive and a subthreshold concentration of Slit1 triggered a potent attractive or repulsive behavioral response to a gradient of Netrin-1, depending on the nature of the substrate. CONCLUSIONS: Our study reveals that at subthreshold repulsive levels, Slit1 acts as a potent promoter of both Netrin-1 attractive and repulsive activities on distinct neuronal cell types, thereby opening novel perspectives on the role of combinations of cues in brain wiring.

Microglia modulate wiring of the embryonic forebrain.

Dysfunction of microglia, the tissue macrophages of the brain, has been associated with the etiology of several neuropsychiatric disorders. Consistently, microglia have been shown to regulate neurogenesis and synaptic maturation at perinatal and postnatal stages. However, microglia invade the brain during mid-embryogenesis and thus could play an earlier prenatal role. Here, we show that embryonic microglia, which display a transiently uneven distribution, regulate the wiring of forebrain circuits. Using multiple mouse models, including cell-depletion approaches and cx3cr1(-/-), CR3(-/-), and DAP12(-/-) mutants, we find that perturbing microglial activity affects the outgrowth of dopaminergic axons in the forebrain and the laminar positioning of subsets of neocortical interneurons. Since defects in both dopamine innervation and cortical networks have been linked to neuropsychiatric diseases, our study provides insights into how microglial dysfunction can impact forebrain connectivity and reveals roles for immune cells during normal assembly of brain circuits.

Pathfinding of corticothalamic axons relies on a rendezvous with thalamic projections.

Major outputs of the neocortex are conveyed by corticothalamic axons (CTAs), which form reciprocal connections with thalamocortical axons, and corticosubcerebral axons (CSAs) headed to more caudal parts of the nervous system. Previous findings establish that transcriptional programs define cortical neuron identity and suggest that CTAs and thalamic axons may guide each other, but the mechanisms governing CTA versus CSA pathfinding remain elusive. Here, we show that thalamocortical axons are required to guide pioneer CTAs away from a default CSA-like trajectory. This process relies on a hold in the progression of cortical axons, or waiting period, during which thalamic projections navigate toward cortical axons. At the molecular level, Sema3E/PlexinD1 signaling in pioneer cortical neurons mediates a "waiting signal" required to orchestrate the mandatory meeting with reciprocal thalamic axons. Our study reveals that temporal control of axonal progression contributes to spatial pathfinding of cortical projections and opens perspectives on brain wiring.

Emergent growth cone responses to combinations of Slit1 and Netrin 1 in thalamocortical axon topography.

How guidance cues are integrated during the formation of complex axonal tracts remains largely unknown. Thalamocortical axons (TCAs), which convey sensory and motor information to the neocortex, have a rostrocaudal topographic organization initially established within the ventral telencephalon [1-3]. Here, we show that this topography is set in a small hub, the corridor, which contains matching rostrocaudal gradients of Slit1 and Netrin 1. Using in vitro and in vivo experiments, we show that Slit1 is a rostral repellent that positions intermediate axons. For rostral axons, although Slit1 is also repulsive and Netrin 1 has no chemotactic activity, the two factors combined generate attraction. These results show that Slit1 has a dual context-dependent role in TCA pathfinding and furthermore reveal that a combination of cues produces an emergent activity that neither of them has alone. Our study thus provides a novel framework to explain how a limited set of guidance cues can generate a vast diversity of axonal responses necessary for proper wiring of the nervous system.

Role of Fgf8 signalling in the specification of rostral Cajal-Retzius cells.

Cajal-Retzius (CR) cells play a key role in the formation of the cerebral cortex. These pioneer neurons are distributed throughout the cortical marginal zone in distinct graded distributions. Fate mapping and cell lineage tracing studies have recently shown that CR cells arise from restricted domains of the pallial ventricular zone, which are associated with signalling centres involved in the early regionalisation of the telencephalic vesicles. In this study, we identified a subpopulation of CR cells in the rostral telencephalon that expresses Er81, a downstream target of Fgf8 signalling. We investigated the role of the rostral telencephalic patterning centre, which secretes FGF molecules, in the specification of these cells. Using pharmacological inhibitors and genetic inactivation of Fgf8, we showed that production of Fgf8 by the rostral telencephalic signalling centre is required for the specification of the Er81+ CR cell population. Moreover, the analysis of Fgf8 gain-of-function in cultivated mouse embryos and of Emx2 and Gli3 mutant embryos revealed that ectopic Fgf8 signalling promotes the generation of CR cells with a rostral phenotype from the dorsal pallium. These data showed that Fgf8 signalling is both required and sufficient to induce rostral CR cells. Together, our results shed light on the mechanisms specifying rostral CR cells and further emphasise the crucial role of telencephalic signalling centres in the generation of distinct CR cell populations.

Dlx-dependent and -independent regulation of olfactory bulb interneuron differentiation.

Olfactory bulb interneuron development is a complex multistep process that involves cell specification in the ventral telencephalon, tangential migration into the olfactory bulb, and local neuronal maturation. Although several transcription factors have been implicated in this process, how or when they act remains to be elucidated. Here we explore the mechanisms that result in olfactory bulb interneuron defects in Dlx1&2-/- (distal-less homeobox 1 and 2) and Mash1-/- (mammalian achaete-schute homolog 1) mutants. We provide evidence that Dlx1&2 and Mash1 regulate parallel molecular pathways that are required for the generation of these cells, thereby providing new insights into the mechanisms underlying olfactory bulb development. The analysis also defined distinct anatomical zones related to olfactory bulb development. Finally we show that Dlx1&2 are required for promoting tangential migration to the olfactory bulb, potentially via regulating the expression of ErbB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4), Robo2 (roundabout homolog 2), Slit1 (slit homolog 1), and PK2 (prokineticin 2), which have all been shown to play essential roles in this migration.

Tangential neuronal migration controls axon guidance: a role for neuregulin-1 in thalamocortical axon navigation.

Neuronal migration and axon guidance constitute fundamental processes in brain development that are generally studied independently. Although both share common mechanisms of cell biology and biochemistry, little is known about their coordinated integration in the formation of neural circuits. Here we show that the development of the thalamocortical projection, one of the most prominent tracts in the mammalian brain, depends on the early tangential migration of a population of neurons derived from the ventral telencephalon. This tangential migration contributes to the establishment of a permissive corridor that is essential for thalamocortical axon pathfinding. Our results also demonstrate that in this process two different products of the Neuregulin-1 gene, CRD-NRG1 and Ig-NRG1, mediate the guidance of thalamocortical axons. These results show that neuronal tangential migration constitutes a novel mechanism to control the timely arrangement of guidance cues required for axonal tract formation in the mammalian brain.

Patterning of the lateral ganglionic eminence by the Gsh1 and Gsh2 homeobox genes regulates striatal and olfactory bulb histogenesis and the growth of axons through the basal ganglia.

The function of the Gsh1 and Gsh2 homeobox transcription factors during development of the mouse telencephalon was studied using loss of function mutations. No telencephalic phenotype was observed in Gsh1 mutants, whereas Gsh2 and Gsh1/2 mutants showed progressively more severe defects in development of neurons derived from the lateral ganglionic eminence (LGE). These defects arise from abnormal dorsoventral specification of LGE progenitor cells. Mice lacking both Gsh1 and Gsh2 have severe hypoplasia of the striatum, olfactory tubercle, and interneurons that migrate from the dorsal LGE to the olfactory bulb. In addition, Gsh function is linked to the development of telencephalic dopaminergic neurons. These observations show that Gsh1 and Gsh2 have early roles in defining the identity of LGE progenitor cells. As a result of the basal ganglia defects in the Gsh1/2 mutants, there are pallial heterotopia near the cortical/subcortical limit and defects in the pathfinding of corticofugal and thalamocortical fibers. These findings highlight the developmental interdependence of adjacent telencephalic structures.

DLX5 regulates development of peripheral and central components of the olfactory system.

Induction, neurogenesis, and synaptogenesis of the olfactory bulb are thought to require interactions with the olfactory epithelium. The Dlx family of homeobox genes is expressed in both the olfactory bulb and olfactory epithelium. In particular, Dlx5 is expressed in the olfactory placode, olfactory epithelium, and local circuit neurons of the olfactory bulb. Here we analyzed mice lacking DLX5 function. The Dlx5-/- mutation reduces the size of the olfactory epithelium. Although some olfactory neurons are formed, they fail to generate olfactory axons that innervate the olfactory bulb. Despite the lack of innervation, the olfactory bulb forms, and neurogenesis of projection and local circuit neurons proceeds. However, the mutation has a cell-autonomous effect on the ability of neural progenitors to produce olfactory bulb local circuit neurons, with granule cells more severely affected than periglomerular cells. In addition, the mutation has a noncell-autonomous effect on the morphogenesis of mitral cells.