Abstract Cortical inhibitory neurons (CIN) populate the neocortex and hippocampus by extensive tangential migration. This process is highly vulnerable to genetic and environmental disturbances and is linked to neuropsychiatric disorders. However, the mechanisms by which transient migratory abnormalities translate into persistent functional deficits remain poorly understood. Here, we utilized a conditional Cxcr4 knockout to investigate the consequences of disrupted migratory guidance - a feature of genetic schizophrenia models. This demonstrated that despite migrating in ectopic cortical layers, CIN quantitatively colonized the neocortex until birth, whereas limbic regions developed a permanent deficit in CIN numbers. Furthermore, CIN failed to populate the neocortical marginal zone, a transient reservoir for late-born CIN destined for superficial cortical layers. Consequently, the layering and molecular identities of CIN, as well as the synaptic connectivity and spontaneous activity of the neuronal network, were significantly altered in the early postnatal neocortex. Although abnormal CIN layering was gradually compensated before maturity, functional differences persisted, as evidenced by facilitated propagation of sensory stimuli between cortical areas. These results demonstrate that CIN migration is an instructive process critical for activity-driven early postnatal network maturation and CIN identity, thus providing a mechanistic link between migratory guidance and the integrity of mature circuits.

Mammalian hematopoietic cells arise from mesodermal progenitors in a close developmental relationship with endothelium and along three distinct cell lineages known as primitive, prodefinitive, and definitive hematopoiesis. However, the developmental hierarchies between early mesodermal progenitors, endothelium, and blood cell lineages are incompletely understood. Here, fate-mapping, cloning, and genetic experiments identified a population of KDR+CXCR4+ prodefinitive angio-hematopoietic progenitors (PDAPs) in murine primitive streak stage mesoderm and human induced pluripotent stem cell (hiPSC) cultures. PDAPs gave rise to yolk sac (YS) and rostral (cephalic) endothelial cells and RUNX1-dependent prodefinitive hematopoietic cells, including erythro-myeloid progenitors (EMPs) and tissue macrophages, via NOTCH1-independent hemogenic endothelial cells. Notably, PDAPs did not give rise to primitive erythropoiesis or to caudal endothelium and definitive hematopoiesis. These results identify a previously unrecognized layer of lineage segregation in early mammalian mesoderm that characterizes the prodefinitive angio-hematopoietic lineage and the origin of tissue macrophages and rostral blood vessels that may be of interest for pathophysiology and cell-based therapy efforts.

In central nervous system (CNS) diseases characterized by late-onset neurodegeneration, the interplay between innate and adaptive immune responses remains poorly understood. This knowledge gap is exacerbated by the prolonged protracted disease course as it complicates the delineation of brain-resident and infiltrating cells. Here, we conducted comprehensive profiling of innate and adaptive immune cells in a murine model of spastic paraplegia 15 (SPG15), a complicated form of hereditary spastic paraplegia. Using fate-mapping of bone marrow-derived cells, we identified microgliosis accompanied by infiltration and local expansion of T cells in the CNS of Spg15-/- mice. Single-cell analysis revealed an expansion of disease-associated microglia (DAM) and effector CD8+ T cells prior to neuronal loss. Analysis of potential cell-cell communication pathways suggested bidirectional interactions between DAM and effector CD8+ T cells, potentially contributing to disease progression in Spg15-/- mice. In summary, we identified a shift in microglial phenotypes associated with the recruitment and expansion of T cells as a new characteristic of Spg15-driven neuropathology.

In central nervous system (CNS) diseases characterized by late-onset neurodegeneration, the interplay between innate and adaptive immune responses remains poorly understood. This knowledge gap is amplified by the prolonged nature of these diseases, complicating the delineation of brain-resident and infiltrating cells. Here, we conducted a comprehensive profiling of innate and adaptive immune cells across various CNS regions in a murine model of spastic paraplegia 15 (SPG15), a complicated form of hereditary spastic paraplegia (HSP). Using fate-mapping of bone marrow-derived cells via genetic labeling, we identified microgliosis and microglial MHC-II upregulation accompanied by infiltration and local expansion of T cells in the CNS of Spg15-/- mice. Single-cell analysis revealed an increase of disease-associated microglia (DAM) and clonal expansion of effector CD8+ T cells across CNS regions occurring prior to neuronal loss. Analysis of potential cell-cell communication pathways suggested bidirectional interactions between DAM and effector CD8+ T cells potentially contributing to disease progression in Spg15-/- mice. In summary, we identified a shift in microglial phenotypes associated with recruitment and clonal expansion of T cells as a new characteristic of Spg15-driven neuropathology. Targeting activated microglia, CD8+ T cells and their communication represent promising avenues to prevent the loss of neuronal function in HSP . ### Competing Interest Statement The authors have declared no competing interest.

Binocular vision requires the segregation of retinal ganglion cell (RGC) axons extending from the retina into the ipsilateral and contralateral optic tracts. RGC axon segregation occurs at the optic chiasm, which forms at the ventral diencephalon midline. Using expression analyses, retinal explants and genetically modified mice, we demonstrate that CXCL12 (SDF1) is required for axon segregation at the optic chiasm. CXCL12 is expressed by the meninges bordering the optic pathway, and CXCR4 by both ipsilaterally and contralaterally projecting RGCs. CXCL12 or ventral diencephalon meninges potently promoted axon outgrowth from both ipsilaterally and contralaterally projecting RGCs. Further, a higher proportion of axons projected ipsilaterally in mice lacking CXCL12 or its receptor CXCR4 compared with wild-type mice as a result of misrouting of presumptive contralaterally specified RGC axons. Although RGCs also expressed the alternative CXCL12 receptor ACKR3, the optic chiasm developed normally in mice lacking ACKR3. Our data support a model whereby meningeal-derived CXCL12 helps drive axon growth from CXCR4-expressing RGCs towards the diencephalon midline, enabling contralateral axon growth. These findings further our understanding of the molecular and cellular mechanisms controlling optic pathway development.

Mammalian hematopoietic cells arise from mesodermal progenitors in a close developmental relationship with endothelium, and along three distinct cell lineages known as primitive, pro-definitive, and definitive hematopoiesis. However, the developmental hierarchy between mesodermal progenitors, endothelium, and blood cell lineages is incompletely understood. We report here the identification in mouse gastrula and human (h)iPSC cultures of a population of CXCR4+ primitive streak stage mesodermal progenitors that give rise to yolk sac endothelium, blood islands, yolk sac hematopoiesis, and resident macrophages, corresponding to the pro-definitive lineage. Strikingy, this progenitor does not give rise to primitive erythropoiesis or to caudal endothelium and definitive hematopoiesis. Interestingly however, the pro-definitive progenitor population also gives rise to rostral endothelium that persists in adults. Finally, pro-definitive progenitor- derived endothelium is the first and main source of macrophages in embryo and hiPSC cultures, both directly and via previously described multipotent Erythro-Myeloid Progenitors. The identification and isolation of this mesodermal progenitor defines a revised pro-definitive angio-hematopoietic lineage and provides a framework for resident macrophage and endothelial differentiation relevant to disease pathophysiology and their manipulation for therapeutic purposes. ### Competing Interest Statement The authors have declared no competing interest.

Supplementary Data from Overexpression of Stromal Cell–Derived Factor 1 and Its Receptor CXCR4 Induces Autocrine/Paracrine Cell Proliferation in Human Pituitary Adenomas

Olfaction depends on lifelong production of sensory neurons from CXCR4 expressing neurogenic stem cells. Signaling by CXCR4 depends on the concentration of CXCL12, CXCR4's principal ligand. Here, we use several genetic models to investigate how regulation of CXCL12 in the olfactory stem cell niche adjusts neurogenesis. We identify subepithelial tissue and sustentacular cells, the olfactory glia, as main CXCL12 sources. Lamina propria-derived CXCL12 accumulates on quiescent gliogenic stem cells via heparan sulfate. Additionally, CXCL12 is secreted within the olfactory epithelium by sustentacular cells. Both sustentacular-cell-derived and lamina propria-derived CXCL12 are required for CXCR4 activation. ACKR3, a high-affinity CXCL12 scavenger, is expressed by mature glial cells and titrates CXCL12. The accurate adjustment of CXCL12 by ACKR3 is critical for CXCR4-dependent proliferation of neuronal stem cells and for proper lineage progression. Overall, these findings establish precise regulation of CXCL12 by glia cells as a prerequisite for CXCR4-dependent neurogenesis and identify ACKR3 as a scavenger influencing tissue homeostasis beyond embryonic development.

Abstract Background The innate lymphoid cell (ILC) family consists of NK cells, ILC type 1, 2, 3 and lymphoid tissue inducer cells. They have been shown to play important roles in homeostasis and immune responses and are generally considered tissue resident. Not much is known about the presence of ILC members within the central nervous system and whether they are tissue resident in this organ too. Therefore, we studied the presence of all ILC members within the central nervous system and after ischemic brain insult. Methods We used the photothrombotic ischemic lesion method to induce ischemic lesions within the mouse brain. Using whole-mount immunofluorescence imaging, we established that the ILCs were present at the rim of the lesion. We quantified the increase of all ILC members at different time-points after the ischemic lesion induction by flow cytometry. Their migration route via chemokine CXCL12 was studied by using different genetic mouse models, in which we induced deletion of Cxcl12 within the blood–brain barrier endothelium, or its receptor, Cxcr4, in the ILCs. The functional role of the ILCs was subsequently established using the beam-walk sensorimotor test. Results Here, we report that ILCs are not resident within the mouse brain parenchyma during steady-state conditions, but are attracted towards the ischemic stroke. Specifically, we identify NK cells, ILC1s, ILC2s and ILC3s within the lesion, the highest influx being observed for NK cells and ILC1s. We further show that CXCL12 expressed at the blood–brain barrier is essential for NK cells and NKp46+ ILC3s to migrate toward the lesion. Complementary, Cxcr4-deficiency in NK cells prevents NK cells from entering the infarct area. Lack of NK cell migration results in a higher neurological deficit in the beam-walk sensorimotor test. Conclusions This study establishes the lack of ILCs in the mouse central nervous system at steady-state and their migration towards an ischemic brain lesion. Our data show a role for blood–brain barrier-derived CXCL12 in attracting protective NK cells to ischemic brain lesions and identifies a new CXCL12/CXCR4-mediated component of the innate immune response to stroke.

Platelet activation plays a critical role in thrombosis. Inhibition of platelet activation is a cornerstone in treatment of acute organ ischemia. Platelet ACKR3 surface expression is independently associated with all-cause mortality in CAD patients. In a novel genetic mouse strain, we show that megakaryocyte/platelet-specific deletion of ACKR3 results in enhanced platelet activation and thrombosis in vitro and in vivo. Further, we performed ischemia/reperfusion experiments (transient LAD-ligation and tMCAO) in mice to assess the impact of genetic ACKR3 deficiency in platelets on tissue injury in ischemic myocardium and brain. Loss of platelet ACKR3 enhances tissue injury in ischemic myocardium and brain and aggravates tissue inflammation. Activation of platelet-ACKR3 via specific ACKR3 agonists inhibits platelet activation and thrombus formation and attenuates tissue injury in ischemic myocardium and brain. Here we demonstrate that ACKR3 is a critical regulator of platelet activation, thrombus formation and organ injury following ischemia/reperfusion.

The function of the cerebral cortex depends on various types of interneurons (cortical interneurons [cINs]) and their appropriate allocation to the cortical layers. Caudal ganglionic eminence-derived cINs (cGE-cINs) are enriched in superficial layers. Developmental mechanisms directing cGE-cINs toward superficial layers remain poorly understood. We examine how developmental and final positioning of cGE-cINs are influenced by the Cxcl12, Cxcr4, Ackr3 module, the chief attractant system guiding medial ganglionic eminence-derived cINs (mGE-cINs). We find that Cxcl12 attracts cGE-cINs through Cxcr4 and supports their layer-specific positioning in the developing cortex. This requires the prevention of excessive Cxcr4 stimulation by Ackr3-mediated Cxcl12 sequestration. Postnatally, Ackr3 confines Cxcl12 action to the marginal zone. Unlike mGE-cINs, cGE-cINs continue to express Cxcr4 at early postnatal stages, which permits cGE-cINs to become positioned in the forming layer 1. Thus, chemoattraction by Cxcl12 guides cGE-cINs and holds them in superficial cortical layers.

Inflammation triggers secondary brain damage after stroke. The meninges and other CNS border compartments serve as invasion sites for leukocyte influx into the brain thus promoting tissue damage after stroke. However, the post-ischemic immune response of border compartments compared to brain parenchyma remains poorly characterized. Here, we deeply characterize tissue-resident leukocytes in meninges and brain parenchyma and discover that leukocytes respond differently to stroke depending on their site of residence. We thereby discover a unique phenotype of myeloid cells exclusive to the brain after stroke. These stroke-associated myeloid cells partially resemble neurodegenerative disease-associated microglia. They are mainly of resident microglial origin, partially conserved in humans and exhibit a lipid-phagocytosing phenotype. Blocking markers specific for these cells partially ameliorates stroke outcome thus providing a potential therapeutic target. The injury-response of myeloid cells in the CNS is thus compartmentalized, adjusted to the type of injury and may represent a therapeutic target.

Midbrain dopaminergic (mDA) neurons are generated from a ventral midbrain progenitor zone over a time span of several days [embryonic day 10.0 (E10.0) to E14.5 in mouse]. Within this neurogenic period, a progressively changing fate potential of mDA progenitors could contribute to the generation of diverse mDA neuronal subpopulations. To test this idea, we combined inducible genetic fate mapping and intersectional labeling approaches to trace the lineage of cells expressing the chemokine receptor CXCR4. The Cxcr4 transcript is expressed in mDA progenitors and precursors, but not in differentiated mDA neurons. Cxcr4-expressing mDA progenitors/precursors labeled at E11.5 develop into a broad range of mDA neurons, whereas labeling of the Cxcr4 lineage at later time points (E12.5-E15.5) results in an increasingly restricted contribution to mDA neurons proceeding from lateral to medial in the substantia nigra and from dorsal to ventral in the ventral tegmental area. In parallel, the innervation of dopaminergic projection targets by mDA neurons derived from Cxcr4-expressing cells is becoming more restricted: the late-generated mDA neurons innervate only the medial-rostral regions in the dorsal striatum and only the medial shell in the nucleus accumbens. Our results suggest that mDA progenitor cells become increasingly restricted in their cell fate potential over time.

Perivascular macrophages (pvMs) are associated with cerebral vasculature and mediate brain drainage and immune regulation. Here, using reporter mouse models, whole brain and section immunofluorescence, flow cytometry, and single cell RNA sequencing, besides the Lyve1+F4/80+CD206+CX3CR1+ pvMs, we identify a CX3CR1- pvM population that shares phagocytic functions and location. Furthermore, the brain parenchyma vasculature mostly hosts Lyve1+MHCII- pvMs with low to intermediate CD45 expression. Using the double Cx3cr1GFP x Cx3cr1-Cre;RosatdT reporter mice for finer mapping of the lineages, we establish that CD45lowCX3CR1- pvMs are derived from CX3CR1+ precursors and require PU.1 during their ontogeny. In parallel, results from the Cxcr4-CreErt2;Rosa26tdT lineage tracing model support a bone marrow-independent replenishment of all Lyve1+ pvMs in the adult mouse brain. Lastly, flow cytometry and 3D immunofluorescence analysis uncover increased percentage of pvMs following photothrombotic induced stroke. Our results thus show that the parenchymal pvM population is more heterogenous than previously described, and includes a CD45low and CX3CR1- pvM population.

Among the five somatostatin receptors (SST1–SST5), SST4 is the least characterized, which is in part due to the lack of specific monoclonal antibodies. We generated a knockin mouse model that expresses a carboxyl-terminal SST4-eGFP fusion protein. In addition, we extensively characterized the novel rabbit monoclonal anti-human SST4 antibody 7H49L61 using transfected cells and receptor-expressing tissues. 7H49L61 was then subjected to immunohistochemical staining of a series of formalin-fixed, paraffin-embedded normal and neoplastic human tissues. Characterization of SST4-eGFP mice revealed prominent SST4 expression in cortical pyramidal cells and trigeminal ganglion cells. In the human cortex, 7H49L61 disclosed a virtually identical staining pattern. Specificity of 7H49L61 was demonstrated by detection of a broad band migrating at 50–60 kDa in immunoblots. Tissue immunostaining was abolished by preadsorption of 7H49L61 with its immunizing peptide. In the subsequent immunohistochemical study, 7H49L61 yielded a predominant plasma membrane staining in adrenal cortex, exocrine pancreas, and placenta. SST4 was also found in glioblastomas, parathyroid adenomas, gastric and pancreatic adenocarcinomas, pheochromocytomas, and lymphomas. Altogether, we provide the first unequivocal localization of SST4 in normal and neoplastic human tissues. The monoclonal antibody 7H49L61 may also prove of great value for identifying SST4-expressing tumors during routine histopathological examinations.

Biallelic mutations inDONSON, an essential gene encoding for a replication fork protection factor, were linked to skeletal abnormalities and microcephaly. To better understand DONSON function in corticogenesis, we characterizedDonsonexpression and consequences of conditionalDonsondeletion in the mouse telencephalon.Donsonwas widely expressed in the proliferation and differentiation zones of the embryonic dorsal and ventral telencephalon, which was followed by a postnatal expression decrease.Emx1-Cre-mediatedDonsondeletion in progenitors of cortical glutamatergic neurons caused extensive apoptosis in the early dorsomedial neuroepithelium, thus preventing formation of the neocortex and hippocampus. At the place of the missing lateral neocortex, these mutants exhibited a dorsal extension of an early-generated paleocortex. Targeting cortical neurons at the intermediate progenitor stage usingTbr2-Cre evoked no apparent malformations, whereasNkx2.1-Cre-mediatedDonsondeletion in subpallial progenitors ablated 75% ofNkx2.1-derived cortical GABAergic neurons. Thus, the early telencephalic neuroepithelium depends critically on Donson function. Our findings help explain why the neocortex is most severely affected in individuals withDONSONmutations and suggest that DONSON-dependent microcephaly might be associated with so far unrecognized defects in cortical GABAergic neurons. TargetingDonsonusing an appropriate recombinase is proposed as a feasible strategy to ablate proliferating and nascent cells in experimental research.

Abstract Perivascular macrophages (pvM) are closely associated with cerebral vasculature and play an essential role in drainage of the brain and regulation of the immune response. Here, using reporter mouse models and immunofluorescence on sections and whole brain, flow cytometry and single cell sequencing, we identify a Lyve1+ brain perivascular population lacking classical macrophage markers such as CD45 and Cx3cr1. We named the new non-conventional CD45 negative perivascular macrophages pvM2. These cells have a similar location, morphology and phagocytic function as conventional pvM. The pvM2 are not derived from hematopoietic stem cells, as they are negative in the VavtdT lineage tracing model. They increase in number after photothrombotic induced stroke established by flow cytometry and 3D immunofluorescence analysis. Since CD45 negative cells were typically excluded from macrophage studies, the presence of pvM2 has been previously missed and their role is of importance to assess in the brain disease models.

The olfactory epithelium (OE) possesses unique lifelong neuroregenerative capacities and undergoes constitutive neurogenesis throughout mammalian lifespan. Two populations of stem cells, frequently dividing globose basal cells (GBCs) and quiescent horizontal basal cells (HBCs), readily replace olfactory neurons throughout lifetime. Although lineage commitment and neuronal differentiation of stem cells has already been described in terms of transcription factor expression, little is known about external factors balancing between differentiation and self-renewal. We show here that expression of the CXC-motif chemokine receptor 4 (CXCR4) distinguishes both types of stem cells. Extensive colocalization analysis revealed exclusive expression of CXCR4 in proliferating GBCs and their neuronal progenies. Moreover, only neuronal lineage cells were derived from CXCR4-CreER-tdTomato reporter mice in the OE. Furthermore, Cre-tdTomato mice specific for HBCs (Nestin+ and Cytokeratin14+) did not reduce CXCR4 expression when bred to mice bearing floxed CXCR4 alleles, and did not show labeling of the neuronal cells. CXCR4 and its ligand CXCL12 were markedly upregulated upon induction of GBC proliferation during injury-induced regeneration. in vivo overexpression of CXCL12 did downregulate CXCR4 levels, which results in reduced GBC maintenance and neuronal differentiation. We proved that these effects were caused by CXCR4 downregulation rather than over-activation by showing that the phenotypes of CXCL12-overexpressing mice were highly similar to the phenotypes of CXCR4 knockout mice. Our results demonstrate functional CXCR4 signaling in GBCs regulates cell cycle exit and neural differentiation. We propose that CXCR4/CXCL12 signaling is an essential regulator of olfactory neurogenesis and provide new insights into the dynamics of neurogenesis in the OE.

Conventional dendritic cells (cDC) are key activators of naive T cells, and can be targeted in adults to induce adaptive immunity, but in early life are considered under-developed or functionally immature. Here we show that, in early life, when the immune system develops, cDC2 exhibit a dual hematopoietic origin and, like other myeloid and lymphoid cells, develop in waves. Developmentally distinct cDC2 in early life, despite being distinguishable by fate mapping, are transcriptionally and functionally similar. cDC2 in early and adult life, however, are exposed to distinct cytokine environments that shape their transcriptional profile and alter their ability to sense pathogens, secrete cytokines and polarize T cells. We further show that cDC2 in early life, despite being distinct from cDC2 in adult life, are functionally competent and can induce T cell responses. Our results thus highlight the potential of harnessing cDC2 for boosting immunity in early life.