Temporal and sequential transcriptional dynamics define lineage shifts in corticogenesis.

The cerebral cortex contains billions of neurons, and their disorganization or misspecification leads to neurodevelopmental disorders. Understanding how the plethora of projection neuron subtypes are generated by cortical neural stem cells (NSCs) is a major challenge. Here, we focused on elucidating the transcriptional landscape of murine embryonic NSCs, basal progenitors (BPs), and newborn neurons (NBNs) throughout cortical development. We uncover dynamic shifts in transcriptional space over time and heterogeneity within each progenitor population. We identified signature hallmarks of NSC, BP, and NBN clusters and predict active transcriptional nodes and networks that contribute to neural fate specification. We find that the expression of receptors, ligands, and downstream pathway components is highly dynamic over time and throughout the lineage implying differential responsiveness to signals. Thus, we provide an expansive compendium of gene expression during cortical development that will be an invaluable resource for studying neural developmental processes and neurodevelopmental disorders.

Transcriptional regulation of MGE progenitor proliferation by PRDM16 controls cortical GABAergic interneuron production.

The mammalian cortex is populated by neurons derived from neural progenitors located throughout the embryonic telencephalon. Excitatory neurons are derived from the dorsal telencephalon, whereas inhibitory interneurons are generated in its ventral portion. The transcriptional regulator PRDM16 is expressed by radial glia, neural progenitors present in both regions; however, its mechanisms of action are still not fully understood. It is unclear whether PRDM16 plays a similar role in neurogenesis in both dorsal and ventral progenitor lineages and, if so, whether it regulates common or unique networks of genes. Here, we show that Prdm16 expression in mouse medial ganglionic eminence (MGE) progenitors is required for maintaining their proliferative capacity and for the production of proper numbers of forebrain GABAergic interneurons. PRDM16 binds to cis-regulatory elements and represses the expression of region-specific neuronal differentiation genes, thereby controlling the timing of neuronal maturation. PRDM16 regulates convergent developmental gene expression programs in the cortex and MGE, which utilize both common and region-specific sets of genes to control the proliferative capacity of neural progenitors, ensuring the generation of correct numbers of cortical neurons.

The Eya1 Phosphatase Mediates Shh-Driven Symmetric Cell Division of Cerebellar Granule Cell Precursors.

During neural development, stem and precursor cells can divide either symmetrically or asymmetrically. The transition between symmetric and asymmetric cell divisions is a major determinant of precursor cell expansion and neural differentiation, but the underlying mechanisms that regulate this transition are not well understood. Here, we identify the Sonic hedgehog (Shh) pathway as a critical determinant regulating the mode of division of cerebellar granule cell precursors (GCPs). Using partial gain and loss of function mutations within the Shh pathway, we show that pathway activation determines spindle orientation of GCPs, and that mitotic spindle orientation correlates with the mode of division. Mechanistically, we show that the phosphatase Eya1 is essential for implementing Shh-dependent GCP spindle orientation. We identify atypical protein kinase C (aPKC) as a direct target of Eya1 activity and show that Eya1 dephosphorylates a critical threonine (T410) in the activation loop. Thus, Eya1 inactivates aPKC, resulting in reduced phosphorylation of Numb and other components that regulate the mode of division. This Eya1-dependent cascade is critical in linking spindle orientation, cell cycle exit and terminal differentiation. Together these findings demonstrate that a Shh-Eya1 regulatory axis selectively promotes symmetric cell divisions during cerebellar development by coordinating spindle orientation and cell fate determinants.

The Elegance of Sonic Hedgehog: Emerging Novel Functions for a Classic Morphogen.

Sonic Hedgehog (SHH) signaling has been most widely known for its role in specifying region and cell-type identity during embryonic morphogenesis. This mini-review accompanies a 2018 SFN mini-symposium that addresses an emerging body of research focused on understanding the diverse roles for Shh signaling in a wide range of contexts in neurodevelopment and, more recently, in the mature CNS. Such research shows that Shh affects the function of brain circuits, including the production and maintenance of diverse cell types and the establishment of wiring specificity. Here, we review these novel and unexpected functions and the unanswered questions regarding the role of SHH and its signaling pathway members in these cases.

PRDM16 co-operates with LHX2 to shape the human brain.

PRDM16 is a dynamic transcriptional regulator of various stem cell niches, including adipocytic, hematopoietic, cardiac progenitors, and neural stem cells. PRDM16 has been suggested to contribute to 1p36 deletion syndrome, one of the most prevalent subtelomeric microdeletion syndromes. We report a patient with a de novo nonsense mutation in the PRDM16 coding sequence, accompanied by lissencephaly and microcephaly features. Human stem cells were genetically modified to mimic this mutation, generating cortical organoids that exhibited altered cell cycle dynamics. RNA sequencing of cortical organoids at day 32 unveiled changes in cell adhesion and WNT-signaling pathways. ChIP-seq of PRDM16 identified binding sites in postmortem human fetal cortex, indicating the conservation of PRDM16 binding to developmental genes in mice and humans, potentially at enhancer sites. A shared motif between PRDM16 and LHX2 was identified and further examined through comparison with LHX2 ChIP-seq data from mice. These results suggested a collaborative partnership between PRDM16 and LHX2 in regulating a common set of genes and pathways in cortical radial glia cells, possibly via their synergistic involvement in cortical development.

Astrocytes as master modulators of neural networks: Synaptic functions and disease-associated dysfunction of astrocytes.

Astrocytes are the most abundant glial cell type in the central nervous system and are essential to the development, plasticity, and maintenance of neural circuits. Astrocytes are heterogeneous, with their diversity rooted in developmental programs modulated by the local brain environment. Astrocytes play integral roles in regulating and coordinating neural activity extending far beyond their metabolic support of neurons and other brain cell phenotypes. Both gray and white matter astrocytes occupy critical functional niches capable of modulating brain physiology on time scales slower than synaptic activity but faster than those adaptive responses requiring a structural change or adaptive myelination. Given their many associations and functional roles, it is not surprising that astrocytic dysfunction has been causally implicated in a broad set of neurodegenerative and neuropsychiatric disorders. In this review, we focus on recent discoveries concerning the contributions of astrocytes to the function of neural networks, with a dual focus on the contribution of astrocytes to synaptic development and maturation, and on their role in supporting myelin integrity, and hence conduction and its regulation. We then address the emerging roles of astrocytic dysfunction in disease pathogenesis and on potential strategies for targeting these cells for therapeutic purposes.

Developmental origin and local signals cooperate to determine septal astrocyte identity.

Astrocyte specification during development is influenced by both intrinsic and extrinsic factors, but the precise contribution of each remains poorly understood. Here we show that septal astrocytes from Nkx2.1 and Zic4 expressing progenitor zones are allocated into non-overlapping domains of the medial (MS) and lateral septal nuclei (LS) respectively. Astrocytes in these areas exhibit distinctive molecular and morphological features tailored to the unique cellular and synaptic circuit environment of each nucleus. Using single-nucleus (sn) RNA sequencing, we trace the developmental trajectories of cells in the septum and find that neurons and astrocytes undergo region and developmental stage-specific local cell-cell interactions. We show that expression of the classic morphogens Sonic hedgehog (Shh) and Fibroblast growth factors (Fgfs) by MS and LS neurons respectively, functions to promote the molecular specification of local astrocytes in each region. Finally, using heterotopic cell transplantation, we show that both morphological and molecular specifications of septal astrocytes are highly dependent on the local microenvironment, regardless of developmental origins. Our data highlights the complex interplay between intrinsic and extrinsic factors shaping astrocyte identities and illustrates the importance of the local environment in determining astrocyte functional specialization.

A developmentally defined population of neurons in the lateral septum controls responses to aversive stimuli.

When interacting with their environment, animals must balance exploratory and defensive behavior to evaluate and respond to potential threats. The lateral septum (LS) is a structure in the ventral forebrain that calibrates the magnitude of behavioral responses to stress-related external stimuli, including the regulation of threat avoidance. The complex connectivity between the LS and other parts of the brain, together with its largely unexplored neuronal diversity, makes it difficult to understand how defined LS circuits control specific behaviors. Here, we describe a mouse model in which a population of neurons with a common developmental origin (Nkx2.1-lineage neurons) are absent from the LS. Using a combination of circuit tracing and behavioral analyses, we found that these neurons receive inputs from the perifornical area of the anterior hypothalamus (PeFAH) and are specifically activated in stressful contexts. Mice lacking Nkx2.1-lineage LS neurons display increased exploratory behavior even under stressful conditions. Our study extends the current knowledge about how defined neuronal populations within the LS can evaluate contextual information to select appropriate behavioral responses. This is a necessary step towards understanding the crucial role that the LS plays in neuropsychiatric conditions where defensive behavior is dysregulated, such as anxiety and aggression disorders.

Astrocyte-neuron crosstalk through Hedgehog signaling mediates cortical synapse development.

Neuron-glia interactions play a critical role in the regulation of synapse formation and circuit assembly. Here we demonstrate that canonical Sonic hedgehog (Shh) pathway signaling in cortical astrocytes acts to coordinate layer-specific synaptic connectivity. We show that the Shh receptor Ptch1 is expressed by cortical astrocytes during development and that Shh signaling is necessary and sufficient to promote the expression of genes involved in regulating synaptic development and layer-enriched astrocyte molecular identity. Loss of Shh in layer V neurons reduces astrocyte complexity and coverage by astrocytic processes in tripartite synapses; conversely, cell-autonomous activation of Shh signaling in astrocytes promotes cortical excitatory synapse formation. Furthermore, Shh-dependent genes Lrig1 and Sparc distinctively contribute to astrocyte morphology and synapse formation. Together, these results suggest that Shh secreted from deep-layer cortical neurons acts to specialize the molecular and functional features of astrocytes during development to shape circuit assembly and function.

Transcriptional profiling of sequentially generated septal neuron fates.

The septum is a ventral forebrain structure known to regulate innate behaviors. During embryonic development, septal neurons are produced in multiple proliferative areas from neural progenitors following transcriptional programs that are still largely unknown. Here, we use a combination of single-cell RNA sequencing, histology, and genetic models to address how septal neuron diversity is established during neurogenesis. We find that the transcriptional profiles of septal progenitors change along neurogenesis, coinciding with the generation of distinct neuron types. We characterize the septal eminence, an anatomically distinct and transient proliferative zone composed of progenitors with distinctive molecular profiles, proliferative capacity, and fate potential compared to the rostral septal progenitor zone. We show that Nkx2.1-expressing septal eminence progenitors give rise to neurons belonging to at least three morphological classes, born in temporal cohorts that are distributed across different septal nuclei in a sequential fountain-like pattern. Our study provides insight into the molecular programs that control the sequential production of different neuronal types in the septum, a structure with important roles in regulating mood and motivation.

Epigenetic regulation of cortical neurogenesis; orchestrating fate switches at the right time and place.

Over the last several decades the field has made tremendous progress in understanding the proliferative behavior of cortical progenitors and the lineage relationships of their clonal progeny. The genetic and epigenetic mechanisms that control the dynamic patterns of gene expression during cortical development are only beginning to be characterized. In this review we highlight the most well characterized epigenetic modifications and their influence on progenitor proliferation and cortical neuron cell fate.

Cortical ChAT+ neurons co-transmit acetylcholine and GABA in a target- and brain-region-specific manner.

The mouse cerebral cortex contains neurons that express choline acetyltransferase (ChAT) and are a potential local source of acetylcholine. However, the neurotransmitters released by cortical ChAT+ neurons and their synaptic connectivity are unknown. We show that the nearly all cortical ChAT+ neurons in mice are specialized VIP+ interneurons that release GABA strongly onto other inhibitory interneurons and acetylcholine sparsely onto layer 1 interneurons and other VIP+/ChAT+ interneurons. This differential transmission of ACh and GABA based on the postsynaptic target neuron is reflected in VIP+/ChAT+ interneuron pre-synaptic terminals, as quantitative molecular analysis shows that only a subset of these are specialized to release acetylcholine. In addition, we identify a separate, sparse population of non-VIP ChAT+ neurons in the medial prefrontal cortex with a distinct developmental origin that robustly release acetylcholine in layer 1. These results demonstrate both cortex-region heterogeneity in cortical ChAT+ interneurons and target-specific co-release of acetylcholine and GABA.

Sonic hedgehog signaling in astrocytes mediates cell type-specific synaptic organization.

Astrocytes have emerged as integral partners with neurons in regulating synapse formation and function, but the mechanisms that mediate these interactions are not well understood. Here, we show that Sonic hedgehog (Shh) signaling in mature astrocytes is required for establishing structural organization and remodeling of cortical synapses in a cell type-specific manner. In the postnatal cortex, Shh signaling is active in a subpopulation of mature astrocytes localized primarily in deep cortical layers. Selective disruption of Shh signaling in astrocytes produces a dramatic increase in synapse number specifically on layer V apical dendrites that emerges during adolescence and persists into adulthood. Dynamic turnover of dendritic spines is impaired in mutant mice and is accompanied by an increase in neuronal excitability and a reduction of the glial-specific, inward-rectifying K+ channel Kir4.1. These data identify a critical role for Shh signaling in astrocyte-mediated modulation of neuronal activity required for sculpting synapses.

The Epigenetic State of PRDM16-Regulated Enhancers in Radial Glia Controls Cortical Neuron Position.

The epigenetic landscape is dynamically remodeled during neurogenesis. However, it is not understood how chromatin modifications in neural stem cells instruct the formation of complex structures in the brain. We report that the histone methyltransferase PRDM16 is required in radial glia to regulate lineage-autonomous and stage-specific gene expression programs that control number and position of upper layer cortical projection neurons. PRDM16 regulates the epigenetic state of transcriptional enhancers to activate genes involved in intermediate progenitor cell production and repress genes involved in cell migration. The histone methyltransferase domain of PRDM16 is necessary in radial glia to promote cortical neuron migration through transcriptional silencing. We show that repression of the gene encoding the E3 ubiquitin ligase PDZRN3 by PRDM16 determines the position of upper layer neurons. These findings provide insights into how epigenetic control of transcriptional enhancers in radial glial determines the organization of the mammalian cerebral cortex.

Radial glia in the ventral telencephalon.

The ventral telencephalon is the developmental origin of the basal ganglia and the source of neuronal and glial cells that integrate into developing circuits in other areas of the brain. Radial glia in the embryonic subpallium give rise to an enormous diversity of mature cell types, either directly or through other transit-amplifying progenitors. Here, we review current knowledge about these subpallial neural stem cells and their progeny, focusing on the period of neurogenesis. We describe their cell biological features and the extrinsic and intrinsic molecular codes that guide their fate specification in defined temporal and spatial sequences. We also discuss the role of clonal lineage in the organization and specification of mature neurons.

Lineage Relationships Do Not Drive MGE/PoA-Derived Interneuron Clustering in the Brain.

Neocortical excitatory and inhibitory neurons derive from distinct progenitor domains during embryonic development and migrate to their final positions, where they assemble into functional circuits. This process appears to be influenced by lineage relationships among locally born excitatory neurons, raising the intriguing possibility that this might be true for cortical interneurons. Two recent articles by the Fishell laboratory and our own used retrovirus-encoded DNA barcodes as unambiguous lineage-tracing tools to address this question, finding that clonally related inhibitory interneurons dispersed widely across the forebrain (Harwell et al., 2015; Mayer et al., 2015). This Matters Arising Response addresses the Sultan et al. (2016) Matters Arising paper, published concurrently in Neuron, where the authors reanalyze the datasets from both studies and propose a new interpretation, whereby clonally related interneurons would be considered clustered according to specific spatial constraints. After studying the report from Sultan et al. (2016) and carefully revisiting previously published studies, we find no evidence of lineage-dependent MGE/PoA-derived interneuron clustering in the forebrain.

Wide Dispersion and Diversity of Clonally Related Inhibitory Interneurons.

The mammalian neocortex is composed of two major neuronal cell types with distinct origins: excitatory pyramidal neurons and inhibitory interneurons, generated in dorsal and ventral progenitor zones of the embryonic telencephalon, respectively. Thus, inhibitory neurons migrate relatively long distances to reach their destination in the developing forebrain. The role of lineage in the organization and circuitry of interneurons is still not well understood. Utilizing a combination of genetics, retroviral fate mapping, and lineage-specific retroviral barcode labeling, we find that clonally related interneurons can be widely dispersed while unrelated interneurons can be closely clustered. These data suggest that migratory mechanisms related to the clustering of interneurons occur largely independent of their clonal origin.

Sonic hedgehog expression in corticofugal projection neurons directs cortical microcircuit formation.

VIDEO ABSTRACT: The precise connectivity of inputs and outputs is critical for cerebral cortex function; however, the cellular mechanisms that establish these connections are poorly understood. Here, we show that the secreted molecule Sonic Hedgehog (Shh) is involved in synapse formation of a specific cortical circuit. Shh is expressed in layer V corticofugal projection neurons and the Shh receptor, Brother of CDO (Boc), is expressed in local and callosal projection neurons of layer II/III that synapse onto the subcortical projection neurons. Layer V neurons of mice lacking functional Shh exhibit decreased synapses. Conversely, the loss of functional Boc leads to a reduction in the strength of synaptic connections onto layer Vb, but not layer II/III, pyramidal neurons. These results demonstrate that Shh is expressed in postsynaptic target cells while Boc is expressed in a complementary population of presynaptic input neurons, and they function to guide the formation of cortical microcircuitry.

Persistent sonic hedgehog signaling in adult brain determines neural stem cell positional identity.

Neural stem cells (NSCs) persist in the subventricular zone (SVZ) of the adult brain. Location within this germinal region determines the type of neuronal progeny NSCs generate, but the mechanism of adult NSC positional specification remains unknown. We show that sonic hedgehog (Shh) signaling, resulting in high gli1 levels, occurs in the ventral SVZ and is associated with the genesis of specific neuronal progeny. Shh is selectively produced by a small group of ventral forebrain neurons. Ablation of Shh decreases production of ventrally derived neuron types, while ectopic activation of this pathway in dorsal NSCs respecifies their progeny to deep granule interneurons and calbindin-positive periglomerular cells. These results show that Shh is necessary and sufficient for the specification of adult ventral NSCs.