Human-specific paralogs of SRGAP2 induce neotenic features of microglia structural and functional maturation.

Microglia play key roles in shaping synaptic connectivity during neural circuits development. Whether microglia display human-specific features of structural and functional maturation is currently unknown. We show that the ancestral gene SRGAP2A and its human-specific (HS) paralogs SRGAP2B/C are not only expressed in cortical neurons but are the only HS gene duplications expressed in human microglia. Here, using combination of xenotransplantation of human induced pluripotent stem cell (hiPSC)-derived microglia and mouse genetic models, we demonstrate that (1) HS SRGAP2B/C are necessary and sufficient to induce neotenic features of microglia structural and functional maturation in a cell-autonomous manner, and (2) induction of SRGAP2-dependent neotenic features of microglia maturation non-cell autonomously impacts synaptic development in cortical pyramidal neurons. Our results reveal that, during human brain evolution, human-specific genes SRGAP2B/C coordinated the emergence of neotenic features of synaptic development by acting as genetic modifiers of both neurons and microglia.

Glioblastoma Neurovascular Progenitor Orchestrates Tumor Cell Type Diversity.

Glioblastoma (GBM) is the deadliest form of primary brain tumor with limited treatment options. Recent studies have profiled GBM tumor heterogeneity, revealing numerous axes of variation that explain the molecular and spatial features of the tumor. Here, we seek to bridge descriptive characterization of GBM cell type heterogeneity with the functional role of individual populations within the tumor. Our lens leverages a gene program-centric meta-atlas of published transcriptomic studies to identify commonalities between diverse tumors and cell types in order to decipher the mechanisms that drive them. This approach led to the discovery of a tumor-derived stem cell population with mixed vascular and neural stem cell features, termed a neurovascular progenitor (NVP). Following in situ validation and molecular characterization of NVP cells in GBM patient samples, we characterized their function in vivo. Genetic depletion of NVP cells resulted in altered tumor cell composition, fewer cycling cells, and extended survival, underscoring their critical functional role. Clonal analysis of primary patient tumors in a human organoid tumor transplantation system demonstrated that the NVP has dual potency, generating both neuronal and vascular tumor cells. Although NVP cells comprise a small fraction of the tumor, these clonal analyses demonstrated that they strongly contribute to the total number of cycling cells in the tumor and generate a defined subset of the whole tumor. This study represents a paradigm by which cell type-specific interrogation of tumor populations can be used to study functional heterogeneity and therapeutically targetable vulnerabilities of GBM.

A Meta-Atlas of the Developing Human Cortex Identifies Modules Driving Cell Subtype Specification.

Human brain development requires the generation of hundreds of diverse cell types, a process targeted by recent single-cell transcriptomic profiling efforts. Through a meta-analysis of seven of these published datasets, we have generated 225 meta-modules - gene co-expression networks that can describe mechanisms underlying cortical development. Several meta-modules have potential roles in both establishing and refining cortical cell type identities, and we validated their spatiotemporal expression in primary human cortical tissues. These include meta-module 20, associated with FEZF2+ deep layer neurons. Half of meta-module 20 genes are putative FEZF2 targets, including TSHZ3, a transcription factor associated with neurodevelopmental disorders. Human cortical organoid experiments validated that both factors are necessary for deep layer neuron specification. Importantly, subtle manipulations of these factors drive slight changes in meta-module activity that cascade into strong differences in cell fate - demonstrating how of our meta-atlas can engender further mechanistic analyses of cortical fate specification.

Common molecular features of H3K27M DMGs and PFA ependymomas map to hindbrain developmental pathways.

Globally decreased histone 3, lysine 27 tri-methylation (H3K27me3) is a hallmark of H3K27-altered diffuse midline gliomas (DMGs) and group-A posterior fossa ependymomas (PFAs). H3K27-altered DMGs are largely characterized by lysine-to-methionine mutations in histone 3 at position 27 (H3K27M). Most PFAs overexpress EZH inhibitory protein (EZHIP), which possesses a region of similarity to the mutant H3K27M. Both H3K27M and EZHIP inhibit the function of the polycomb repressive complex 2 (PRC2) responsible for H3K27me3 deposition. These tumors often arise in neighboring regions of the brainstem and posterior fossa. In rare cases PFAs harbor H3K27M mutations, and DMGs overexpress EZHIP. These findings together raise the possibility that certain cell populations in the developing hindbrain/posterior fossa are especially sensitive to modulation of H3K27me3 states. We identified shared molecular features by comparing genomic, bulk transcriptomic, chromatin-based profiles, and single-cell RNA-sequencing (scRNA-seq) data from the two tumor classes. Our approach demonstrated that 1q gain, a key biomarker in PFAs, is prognostic in H3.1K27M, but not H3.3K27M gliomas. Conversely, Activin A Receptor Type 1 (ACVR1), which is associated with mutations in H3.1K27M gliomas, is overexpressed in a subset of PFAs with poor outcome. Despite diffuse H3K27me3 reduction, previous work shows that both tumors maintain genomic H3K27me3 deposition at select sites. We demonstrate heterogeneity in shared patterns of residual H3K27me3 for both tumors that largely segregated with inferred anatomic tumor origins and progenitor populations of tumor cells. In contrast, analysis of genes linked to H3K27 acetylation (H3K27ac)-marked enhancers showed higher expression in astrocytic-like tumor cells. Finally, common H3K27me3-marked genes mapped closely to expression patterns in the human developing hindbrain. Overall, our data demonstrate developmentally relevant molecular similarities between PFAs and H3K27M DMGs and support the overall hypothesis that deregulated mechanisms of hindbrain development are central to the biology of both tumors.

Evaluation of advances in cortical development using model systems.

Compared with that of even the closest primates, the human cortex displays a high degree of specialization and expansion that largely emerges developmentally. Although decades of research in the mouse and other model systems has revealed core tenets of cortical development that are well preserved across mammalian species, small deviations in transcription factor expression, novel cell types in primates and/or humans, and unique cortical architecture distinguish the human cortex. Importantly, many of the genes and signaling pathways thought to drive human-specific cortical expansion also leave the brain vulnerable to disease, as the misregulation of these factors is highly correlated with neurodevelopmental and neuropsychiatric disorders. However, creating a comprehensive understanding of human-specific cognition and disease remains challenging. Here, we review key stages of cortical development and highlight known or possible differences between model systems and the developing human brain. By identifying the developmental trajectories that may facilitate uniquely human traits, we highlight open questions in need of approaches to examine these processes in a human context and reveal translatable insights into human developmental disorders.

Mounting evidence suggests human adult neurogenesis is unlikely.

In this issue of Neuron, Franjic et al. (2022) use a single-nuclei RNA sequencing approach that identified signatures of adult neurogenesis in mouse, pig, and macaque dentate gyrus, but not in humans, adding to a growing body of evidence that this process is likely lost in humans.

Cortical Cartography: Mapping Arealization Using Single-Cell Omics Technology.

The cerebral cortex derives its cognitive power from a modular network of specialized areas processing a multitude of information. The assembly and organization of these regions is vital for human behavior and perception, as evidenced by the prevalence of area-specific phenotypes that manifest in neurodevelopmental and psychiatric disorders. Generations of scientists have examined the architecture of the human cortex, but efforts to capture the gene networks which drive arealization have been hampered by the lack of tractable models of human neurodevelopment. Advancements in "omics" technologies, imaging, and computational power have enabled exciting breakthroughs into the molecular and structural characteristics of cortical areas, including transcriptomic, epigenomic, metabolomic, and proteomic profiles of mammalian models. Here we review the single-omics atlases that have shaped our current understanding of cortical areas, and their potential to fuel a new era of multi-omic single-cell endeavors to interrogate both the developing and adult human cortex.

Structure-activity mapping of ARHGAP36 reveals regulatory roles for its GAP homology and C-terminal domains.

ARHGAP36 is an atypical Rho GTPase-activating protein (GAP) family member that drives both spinal cord development and tumorigenesis, acting in part through an N-terminal motif that suppresses protein kinase A and activates Gli transcription factors. ARHGAP36 also contains isoform-specific N-terminal sequences, a central GAP-like module, and a unique C-terminal domain, and the functions of these regions remain unknown. Here we have mapped the ARHGAP36 structure-activity landscape using a deep sequencing-based mutagenesis screen and truncation mutant analyses. Using this approach, we have discovered several residues in the GAP homology domain that are essential for Gli activation and a role for the C-terminal domain in counteracting an N-terminal autoinhibitory motif that is present in certain ARHGAP36 isoforms. In addition, each of these sites modulates ARHGAP36 recruitment to the plasma membrane or primary cilium. Through comparative proteomics, we also have identified proteins that preferentially interact with active ARHGAP36, and we demonstrate that one binding partner, prolyl oligopeptidase-like protein, is a novel ARHGAP36 antagonist. Our work reveals multiple modes of ARHGAP36 regulation and establishes an experimental framework that can be applied towards other signaling proteins.