APOE4 impairs the microglial response in Alzheimer's disease by inducing TGFß-mediated checkpoints.

The APOE4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease (AD). The contribution of microglial APOE4 to AD pathogenesis is unknown, although APOE has the most enriched gene expression in neurodegenerative microglia (MGnD). Here, we show in mice and humans a negative role of microglial APOE4 in the induction of the MGnD response to neurodegeneration. Deletion of microglial APOE4 restores the MGnD phenotype associated with neuroprotection in P301S tau transgenic mice and decreases pathology in APP/PS1 mice. MGnD-astrocyte cross-talk associated with ß-amyloid (Aß) plaque encapsulation and clearance are mediated via LGALS3 signaling following microglial APOE4 deletion. In the brains of AD donors carrying the APOE4 allele, we found a sex-dependent reciprocal induction of AD risk factors associated with suppression of MGnD genes in females, including LGALS3, compared to individuals homozygous for the APOE3 allele. Mechanistically, APOE4-mediated induction of ITGB8-transforming growth factor-ß (TGFß) signaling impairs the MGnD response via upregulation of microglial homeostatic checkpoints, including Inpp5d, in mice. Deletion of Inpp5d in microglia restores MGnD-astrocyte cross-talk and facilitates plaque clearance in APP/PS1 mice. We identify the microglial APOE4-ITGB8-TGFß pathway as a negative regulator of microglial response to AD pathology, and restoring the MGnD phenotype via blocking ITGB8-TGFß signaling provides a promising therapeutic intervention for AD.

A high-resolution enhancer atlas of the developing telencephalon.

The mammalian telencephalon plays critical roles in cognition, motor function, and emotion. Though many of the genes required for its development have been identified, the distant-acting regulatory sequences orchestrating their in vivo expression are mostly unknown. Here, we describe a digital atlas of in vivo enhancers active in subregions of the developing telencephalon. We identified more than 4,600 candidate embryonic forebrain enhancers and studied the in vivo activity of 329 of these sequences in transgenic mouse embryos. We generated serial sets of histological brain sections for 145 reproducible forebrain enhancers, resulting in a publicly accessible web-based data collection comprising more than 32,000 sections. We also used epigenomic analysis of human and mouse cortex tissue to directly compare the genome-wide enhancer architecture in these species. These data provide a primary resource for investigating gene regulatory mechanisms of telencephalon development and enable studies of the role of distant-acting enhancers in neurodevelopmental disorders.

Single cell enhancer activity distinguishes GABAergic and cholinergic lineages in embryonic mouse basal ganglia.

Enhancers integrate transcription factor signaling pathways that drive cell fate specification in the developing brain. We paired enhancer labeling and single-cell RNA-sequencing (scRNA-seq) to delineate and distinguish specification of neuronal lineages in mouse medial, lateral, and caudal ganglionic eminences (MGE, LGE, and CGE) at embryonic day (E)11.5. We show that scRNA-seq clustering using transcription factors improves resolution of regional and developmental populations, and that enhancer activities identify specific and overlapping GE-derived neuronal populations. First, we mapped the activities of seven evolutionarily conserved brain enhancers at single-cell resolution in vivo, finding that the selected enhancers had diverse activities in specific progenitor and neuronal populations across the GEs. We then applied enhancer-based labeling, scRNA-seq, and analysis of in situ hybridization data to distinguish transcriptionally distinct and spatially defined subtypes of MGE-derived GABAergic and cholinergic projection neurons and interneurons. Our results map developmental origins and specification paths underlying neurogenesis in the embryonic basal ganglia and showcase the power of scRNA-seq combined with enhancer-based labeling to resolve the complex paths of neuronal specification underlying mouse brain development.

Disruptions in a cluster of computationally identified enhancers near FOXC1 and GMDS may influence brain development.

Regulatory elements are more evolutionarily conserved and provide a larger mutational target than coding regions of the human genome, suggesting that mutations in non-coding regions contribute significantly to development and disease. Using a computational approach to predict gene regulatory enhancers, we found that many known and predicted embryonic enhancers cluster in genomic loci harboring development-associated genes. One of the densest clusters of predicted enhancers in the human genome is near the genes GMDS and FOXC1. GMDS encodes a short-chain mannose dehydrogenase enzyme involved in the regulation of hindbrain neural migration, and FOXC1 encodes a developmental transcription factor required for brain, heart, and eye development. We experimentally validate four novel enhancers in this locus and demonstrate that these enhancers show consistent activity during embryonic development in domains that overlap with the expression of FOXC1 and GMDS. These four enhancers contain binding motifs for several transcription factors, including the ZIC family of transcription factors. Removal of the ZIC binding sites significantly alters enhancer activity in three of these enhancers, reducing expression in the eye, hindbrain, and limb, suggesting a mechanism whereby ZIC family members may transcriptionally regulate FOXC1 and/or GMDS expression. Our findings uncover novel enhancer regions that may control transcription in a topological domain important for embryonic development.

A comparative evaluation of the strengths and potential caveats of the microglial inducible CreER mouse models.

The recent proliferation of new Cre and CreER recombinase lines provides researchers with a diverse toolkit to study microglial gene function. To determine how best to apply these lines in studies of microglial gene function, a thorough and detailed comparison of their properties is needed. Here, we examined four different microglial CreER lines (Cx3cr1YFP-CreER(Litt), Cx3cr1CreER(Jung), P2ry12CreER, and Tmem119CreER), focusing on (1) recombination specificity, (2) leakiness (the degree of tamoxifen-independent recombination in microglia and other cells), (3) the efficiency of tamoxifen-induced recombination, (4) extraneural recombination (the degree of recombination in cells outside of the CNS, particularly myelo/monocyte lineages), and (5) off-target effects in the context of neonatal brain development. We identify important caveats and strengths for these lines, which will provide broad significance for researchers interested in performing conditional gene deletion in microglia. We also provide data emphasizing the potential of these lines for injury models that result in the recruitment of splenic immune cells.

Radial glia promote microglial development through integrin αVß8 -TGFß1 signaling.

Microglia diversity emerges from interactions between intrinsic genetic programs and environment-derived signals, but how these processes unfold and interact in the developing brain remains unclear. Here, we show that radial glia-expressed integrin beta 8 (ITGB8) expressed in radial glia progenitors activates microglia-expressed TGFß1, permitting microglial development. Domain-restricted deletion of Itgb8 in these progenitors establishes complementary regions with developmentally arrested "dysmature" microglia that persist into adulthood. In the absence of autocrine TGFß1 signaling, we find that microglia adopt a similar dysmature phenotype, leading to neuromotor symptoms almost identical to Itgb8 mutant mice. In contrast, microglia lacking the TGFß signal transducers Smad2 and Smad3 have a less polarized dysmature phenotype and correspondingly less severe neuromotor dysfunction. Finally, we show that non-canonical (Smad-independent) signaling partially suppresses disease and development associated gene expression, providing compelling evidence for the adoption of microglial developmental signaling pathways in the context of injury or disease.

Generation of Cre-transgenic mice using Dlx1/Dlx2 enhancers and their characterization in GABAergic interneurons.

DLX1 and DLX2 transcription factors are necessary for forebrain GABAergic neuron differentiation, migration, and survival. We generated transgenic mice that express Cre-recombinase under the control of two ultra-conserved DNA elements near the Dlx1 and 2 locus termed I12b and URE2. We show that Cre-recombinase is active in a "Dlx-pattern" in the embryonic forebrain of transgenic mice. I12b-Cre is more active than URE2-Cre in the medial ganglionic eminences and its derivatives. Fate-mapping of EGFP+ cells in adult Cre;Z/EG animals demonstrated that GABAergic neurons, but not glia, are labeled. Most NPY+, nNOS+, parvalbumin+, and somatostatin+ cells are marked by I12b-Cre in the cortex and hippocampus, while 25-40% of these interneuron subtypes are labeled by URE2-Cre. Labeling of neurons generated between E12.5 to E15.5 indicated differences in birth-dates of EGFP+ cells that populate the olfactory bulb, hippocampus, and cortex. Finally, we provide the first in vivo evidence that both I12b and URE2 are direct targets of DLX2 and require Dlx1 and Dlx2 expression for proper activity.

Lack of Flvcr2 impairs brain angiogenesis without affecting the blood-brain barrier.

Fowler syndrome is a rare autosomal recessive brain vascular disorder caused by mutation in FLVCR2 in humans. The disease occurs during a critical period of brain vascular development, is characterized by glomeruloid vasculopathy and hydrocephalus, and is almost invariably prenatally fatal. Here, we sought to gain insights into the process of brain vascularization and the pathogenesis of Fowler syndrome by inactivating Flvcr2 in mice. We showed that Flvcr2 was necessary for angiogenic sprouting in the brain, but surprisingly dispensable for maintaining the blood-brain barrier. Endothelial cells lacking Flvcr2 had altered expression of angiogenic factors, failed to adopt tip cell properties, and displayed reduced sprouting, leading to vascular malformations similar to those seen in humans with Fowler syndrome. Brain hypovascularization was associated with hypoxia and tissue infarction, ultimately causing hydrocephalus and death of mutant animals. Strikingly, despite severe vascular anomalies and brain tissue infarction, the blood-brain barrier was maintained in Flvcr2 mutant mice. Our Fowler syndrome model therefore defined the pathobiology of this disease and provided new insights into brain angiogenesis by showing uncoupling of vessel morphogenesis and blood-brain barrier formation.

A new genetic strategy for targeting microglia in development and disease.

As the resident macrophages of the brain and spinal cord, microglia are crucial for the phagocytosis of infectious agents, apoptotic cells and synapses. During brain injury or infection, bone-marrow derived macrophages invade neural tissue, making it difficult to distinguish between invading macrophages and resident microglia. In addition to circulation-derived monocytes, other non-microglial central nervous system (CNS) macrophage subtypes include border-associated meningeal, perivascular and choroid plexus macrophages. Using immunofluorescent labeling, flow cytometry and Cre-dependent ribosomal immunoprecipitations, we describe P2ry12-CreER, a new tool for the genetic targeting of microglia. We use this new tool to track microglia during embryonic development and in the context of ischemic injury and neuroinflammation. Because of the specificity and robustness of microglial recombination with P2ry12-CreER, we believe that this new mouse line will be particularly useful for future studies of microglial function in development and disease.

Functionally Distinct Connectivity of Developmentally Targeted Striosome Neurons.

One long-standing model of striatal function divides the striatum into compartments called striosome and matrix. While some anatomical evidence suggests that these populations represent distinct striatal pathways with differing inputs and outputs, functional investigation has been limited by the methods for identifying and manipulating these populations. Here, we utilize hs599CreER mice as a new tool for targeting striosome projection neurons and testing their functional connectivity. Extending anatomical work, we demonstrate that striosome neurons receive greater synaptic input from prelimbic cortex, whereas matrix neurons receive greater input from primary motor cortex. We also identify functional differences in how striosome and matrix neurons process excitatory input, providing the first electrophysiological method for delineating striatal output neuron subtypes. Lastly, we provide the first functional demonstration that striosome neurons are the predominant striatal output to substantia nigra pars compacta dopamine neurons. These results identify striosome and matrix as functionally distinct striatal pathways.

Genomic Resolution of DLX-Orchestrated Transcriptional Circuits Driving Development of Forebrain GABAergic Neurons.

DLX transcription factors (TFs) are master regulators of the developing vertebrate brain, driving forebrain GABAergic neuronal differentiation. Ablation of Dlx1&2 alters expression of genes that are critical for forebrain GABAergic development. We integrated epigenomic and transcriptomic analyses, complemented with in situ hybridization (ISH), and in vivo and in vitro studies of regulatory element (RE) function. This revealed the DLX-organized gene regulatory network at genomic, cellular, and spatial levels in mouse embryonic basal ganglia. DLX TFs perform dual activating and repressing functions; the consequences of their binding were determined by the sequence and genomic context of target loci. Our results reveal and, in part, explain the paradox of widespread DLX binding contrasted with a limited subset of target loci that are sensitive at the epigenomic and transcriptomic level to Dlx1&2 ablation. The regulatory properties identified here for DLX TFs suggest general mechanisms by which TFs orchestrate dynamic expression programs underlying neurodevelopment.

Mafb and c-Maf Have Prenatal Compensatory and Postnatal Antagonistic Roles in Cortical Interneuron Fate and Function.

Mafb and c-Maf transcription factor (TF) expression is enriched in medial ganglionic eminence (MGE) lineages, beginning in late-secondary progenitors and continuing into mature parvalbumin (PV+) and somatostatin (SST+) interneurons. However, the functions of Maf TFs in MGE development remain to be elucidated. Herein, Mafb and c-Maf were conditionally deleted, alone and together, in the MGE and its lineages. Analyses of Maf mutant mice revealed redundant functions of Mafb and c-Maf in secondary MGE progenitors, where they repress the generation of SST+ cortical and hippocampal interneurons. By contrast, Mafb and c-Maf have distinct roles in postnatal cortical interneuron (CIN) morphological maturation, synaptogenesis, and cortical circuit integration. Thus, Mafb and c-Maf have redundant and opposing functions at different steps in CIN development.

Subpallial Enhancer Transgenic Lines: a Data and Tool Resource to Study Transcriptional Regulation of GABAergic Cell Fate.

Elucidating the transcriptional circuitry controlling forebrain development requires an understanding of enhancer activity and regulation. We generated stable transgenic mouse lines that express CreERT2 and GFP from ten different enhancer elements with activity in distinct domains within the embryonic basal ganglia. We used these unique tools to generate a comprehensive regional fate map of the mouse subpallium, including sources for specific subtypes of amygdala neurons. We then focused on deciphering transcriptional mechanisms that control enhancer activity. Using machine-learning computations, in vivo chromosomal occupancy of 13 transcription factors that regulate subpallial patterning and differentiation and analysis of enhancer activity in Dlx1/2 and Lhx6 mutants, we elucidated novel molecular mechanisms that regulate region-specific enhancer activity in the developing brain. Thus, these subpallial enhancer transgenic lines are data and tool resources to study transcriptional regulation of GABAergic cell fate.

Olig1 function is required to repress dlx1/2 and interneuron production in Mammalian brain.

Abnormal GABAergic interneuron density, and imbalance of excitatory versus inhibitory tone, is thought to result in epilepsy, neurodevelopmental disorders, and psychiatric disease. Recent studies indicate that interneuron cortical density is determined primarily by the size of the precursor pool in the embryonic telencephalon. However, factors essential for regulating interneuron allocation from telencephalic multipotent precursors are poorly understood. Here we report that Olig1 represses production of GABAergic interneurons throughout the mouse brain. Olig1 deletion in mutant mice results in ectopic expression and upregulation of Dlx1/2 genes in the ventral medial ganglionic eminences and adjacent regions of the septum, resulting in an ∼30% increase in adult cortical interneuron numbers. We show that Olig1 directly represses the Dlx1/2 I12b intergenic enhancer and that Dlx1/2 functions genetically downstream of Olig1. These findings establish Olig1 as an essential repressor of Dlx1/2 and interneuron production in developing mammalian brain.

Dlx1&2-dependent expression of Zfhx1b (Sip1, Zeb2) regulates the fate switch between cortical and striatal interneurons.

Mammalian pallial (cortical and hippocampal) and striatal interneurons are both generated in the embryonic subpallium, including the medial ganglionic eminence (MGE). Herein we demonstrate that the Zfhx1b (Sip1, Zeb2) zinc finger homeobox gene is required in the MGE, directly downstream of Dlx1&2, to generate cortical interneurons that express Cxcr7, MafB, and cMaf. In its absence, Nkx2-1 expression is not repressed, and cells that ordinarily would become cortical interneurons appear to transform toward a subtype of GABAergic striatal interneurons. These results show that Zfhx1b is required to generate cortical interneurons, and suggest a mechanism for the epilepsy observed in humans with Zfhx1b mutations (Mowat-Wilson syndrome).

Fezf2 expression identifies a multipotent progenitor for neocortical projection neurons, astrocytes, and oligodendrocytes.

Progenitor cells in the cerebral cortex sequentially generate distinct classes of projection neurons. Recent work suggests the cortex may contain intrinsically fate-restricted progenitors marked by expression of Cux2. However, the heterogeneity of the neocortical ventricular zone as well as the contribution of lineage-restricted progenitors to the overall cortical neurogenic program remains unclear. Here, we utilize in vivo genetic fate mapping to demonstrate that Fezf2-expressing radial glial cells (RGCs) exist throughout cortical development and sequentially generate all major projection neuron subtypes and glia. Moreover, we show that the vast majority of CUX2⁺ cells in the VZ and SVZ are migrating interneurons derived from the subcortical telencephalon. Examination of the embryonic cortical progenitor population demonstrates that Cux2⁺ RGCs generate both deep- and upper-layer projection neurons. These results identify Fezf2⁺ radial glial cells as a multipotent neocortical progenitor and suggest that the existence, and molecular identity, of laminar-fate-restricted RGCs awaits further investigation.