Transcriptional linkage analysis with in vivo AAV-Perturb-seq.

The ever-growing compendium of genetic variants associated with human pathologies demands new methods to study genotype-phenotype relationships in complex tissues in a high-throughput manner1,2. Here we introduce adeno-associated virus (AAV)-mediated direct in vivo single-cell CRISPR screening, termed AAV-Perturb-seq, a tuneable and broadly applicable method for transcriptional linkage analysis as well as high-throughput and high-resolution phenotyping of genetic perturbations in vivo. We applied AAV-Perturb-seq using gene editing and transcriptional inhibition to systematically dissect the phenotypic landscape underlying 22q11.2 deletion syndrome3,4 genes in the adult mouse brain prefrontal cortex. We identified three 22q11.2-linked genes involved in known and previously undescribed pathways orchestrating neuronal functions in vivo that explain approximately 40% of the transcriptional changes observed in a 22q11.2-deletion mouse model. Our findings suggest that the 22q11.2-deletion syndrome transcriptional phenotype found in mature neurons may in part be due to the broad dysregulation of a class of genes associated with disease susceptibility that are important for dysfunctional RNA processing and synaptic function. Our study establishes a flexible and scalable direct in vivo method to facilitate causal understanding of biological and disease mechanisms with potential applications to identify genetic interventions and therapeutic targets for treating disease.

Temporal controls over cortical projection neuron fate diversity.

During neocortex development, cortical projection neurons (PN) are sequentially produced and assemble into circuits underlying our interactions with the environment. Cortical PN are heterogeneous in terms of birthdate, layer position, molecular identity, connectivity, and function. This diversity increases in evolutionarily most recent species, but when and how it emerges during corticogenesis is still debated. While time-locked expression of determinant genes and early stochasticity allow the production of different types of PN, temporal differences in unfolding similar transcriptional programs, rather than fundamental differences in these programs, further account for anatomical variability between PN subtypes and across species. Altogether, these mechanisms, which will be discussed here, participate in increasing cortical PN diversity.

Heterogeneous fates of simultaneously-born neurons in the cortical ventricular zone.

Neocortical excitatory neurons belong to diverse cell types, which can be distinguished by their dates of birth, laminar location, connectivity, and molecular identities. During embryogenesis, apical progenitors (APs) located in the ventricular zone first give birth to deep-layer neurons, and next to superficial-layer neurons. While the overall sequential construction of neocortical layers is well-established, whether APs produce multiple neuron types at single time points of corticogenesis is unknown. To address this question, here we used FlashTag to fate-map simultaneously-born (i.e. isochronic) cohorts of AP daughter neurons at successive stages of corticogenesis. We reveal that early in corticogenesis, isochronic neurons differentiate into heterogeneous laminar, hodological and molecular cell types. Later on, instead, simultaneously-born neurons have more homogeneous fates. Using single-cell gene expression analyses, we identify an early postmitotic surge in the molecular heterogeneity of nascent neurons during which some early-born neurons initiate and partially execute late-born neuron transcriptional programs. Together, these findings suggest that as corticogenesis unfolds, mechanisms allowing increased homogeneity in neuronal output are progressively implemented, resulting in progressively more predictable neuronal identities.

miR-137 and miR-122, two outer subventricular zone non-coding RNAs, regulate basal progenitor expansion and neuronal differentiation.

Cortical expansion in primate brains relies on enlargement of germinal zones during a prolonged developmental period. Although most mammals have two cortical germinal zones, the ventricular zone (VZ) and subventricular zone (SVZ), gyrencephalic species display an additional germinal zone, the outer subventricular zone (oSVZ), which increases the number and diversity of neurons generated during corticogenesis. How the oSVZ emerged during evolution is poorly understood, but recent studies suggest a role for non-coding RNAs, which allow tight genetic program regulation during development. Here, using in vivo functional genetics, single-cell RNA sequencing, live imaging, and electrophysiology to assess progenitor and neuronal properties in mice, we identify two oSVZ-expressed microRNAs (miRNAs), miR-137 and miR-122, which regulate key cellular features of cortical expansion. miR-137 promotes basal progenitor self-replication and superficial layer neuron fate, whereas miR-122 decreases the pace of neuronal differentiation. These findings support a cell-type-specific role of miRNA-mediated gene expression in cortical expansion.

Temporal controls over inter-areal cortical projection neuron fate diversity.

Interconnectivity between neocortical areas is critical for sensory integration and sensorimotor transformations1-6. These functions are mediated by heterogeneous inter-areal cortical projection neurons (ICPN), which send axon branches across cortical areas as well as to subcortical targets7-9. Although ICPN are anatomically diverse10-14, they are molecularly homogeneous15, and how the diversity of their anatomical and functional features emerge during development remains largely unknown. Here we address this question by linking the connectome and transcriptome in developing single ICPN of the mouse neocortex using a combination of multiplexed analysis of projections by sequencing16,17 (MAPseq, to identify single-neuron axonal projections) and single-cell RNA sequencing (to identify corresponding gene expression). Focusing on neurons of the primary somatosensory cortex (S1), we reveal a protracted unfolding of the molecular and functional differentiation of motor cortex-projecting ([Formula: see text]) ICPN compared with secondary somatosensory cortex-projecting ([Formula: see text]) ICPN. We identify SOX11 as a temporally differentially expressed transcription factor in [Formula: see text] versus [Formula: see text] ICPN. Postnatal manipulation of SOX11 expression in S1 impaired sensorimotor connectivity and disrupted selective exploratory behaviours in mice. Together, our results reveal that within a single cortical area, different subtypes of ICPN have distinct postnatal paces of molecular differentiation, which are subsequently reflected in distinct circuit connectivities and functions. Dynamic differences in the expression levels of a largely generic set of genes, rather than fundamental differences in the identity of developmental genetic programs, may thus account for the emergence of intra-type diversity in cortical neurons.

Mapping the molecular and cellular complexity of cortical malformations.

The cerebral cortex is an intricate structure that controls human features such as language and cognition. Cortical functions rely on specialized neurons that emerge during development from complex molecular and cellular interactions. Neurodevelopmental disorders occur when one or several of these steps is incorrectly executed. Although a number of causal genes and disease phenotypes have been identified, the sequence of events linking molecular disruption to clinical expression mostly remains obscure. Here, focusing on human malformations of cortical development, we illustrate how complex interactions at the genetic, cellular, and circuit levels together contribute to diversity and variability in disease phenotypes. Using specific examples and an online resource, we propose that a multilevel assessment of disease processes is key to identifying points of vulnerability and developing new therapeutic strategies.

Do progenitors play dice?

The wide range of cell types produced by single progenitors in the neocortex of mice may result from stochastic rather than deterministic processes.

A Translaminar Genetic Logic for the Circuit Identity of Intracortically Projecting Neurons.

Neurons of the neocortex are organized into six radial layers, which have appeared at different times during evolution, with the superficial layers representing a more recent acquisition. Input to the neocortex predominantly reaches superficial layers (SL, i.e., layers (L) 2-4), while output is generated in deep layers (DL, i.e., L5-6) [1]. Intracortical connections, which bridge input and output pathways, are key components of cortical circuits because they allow the propagation and processing of information within the neocortex. Two main types of intracortically projecting neurons (ICPN) can be distinguished by their axonal features: L4 spiny stellate neurons (SSN) with short axons projecting locally within cortical columns [2-5], and SL and DL long-range projection neurons, including callosally projecting neurons (CPNSL and CPNDL) [5, 6]. Here, we investigate the molecular hallmarks that distinguish SSN, CPNSL, and CPNDL and relate their transcriptional signatures with their output connectivity. Specifically, taking advantage of the presence of CPN in both SL and DL, we identify lamina-independent genetic hallmarks of a constant projection motif (i.e., interhemispheric projection). By performing unbiased transcriptomic comparisons between CPNSL, CPNDL and SSN, we provide specific molecular profiles for each of these populations and show that target identity supersedes laminar position in defining ICPN transcriptional diversity. Together, these findings reveal a projection-based organization of transcriptional programs across cortical layers, which we propose reflects conserved strategy to protect canonical circuit structure (and hence function) across a diverse range of neuroanatomies.

Progenitor Hyperpolarization Regulates the Sequential Generation of Neuronal Subtypes in the Developing Neocortex.

During corticogenesis, ventricular zone progenitors sequentially generate distinct subtypes of neurons, accounting for the diversity of neocortical cells and the circuits they form. While activity-dependent processes are critical for the differentiation and circuit assembly of postmitotic neurons, how bioelectrical processes affect nonexcitable cells, such as progenitors, remains largely unknown. Here, we reveal that, in the developing mouse neocortex, ventricular zone progenitors become more hyperpolarized as they generate successive subtypes of neurons. Experimental in vivo hyperpolarization shifted the transcriptional programs and division modes of these progenitors to a later developmental status, with precocious generation of intermediate progenitors and a forward shift in the laminar, molecular, morphological, and circuit features of their neuronal progeny. These effects occurred through inhibition of the Wnt-beta-catenin signaling pathway by hyperpolarization. Thus, during corticogenesis, bioelectric membrane properties are permissive for specific molecular pathways to coordinate the temporal progression of progenitor developmental programs and thus neocortical neuron diversity.

Development and Organization of the Evolutionarily Conserved Three-Layered Olfactory Cortex.

The olfactory cortex is part of the mammalian cerebral cortex together with the neocortex and the hippocampus. It receives direct input from the olfactory bulbs and participates in odor discrimination, association, and learning (Bekkers and Suzuki, 2013). It is thought to be an evolutionarily conserved paleocortex, which shares common characteristics with the three-layered general cortex of reptiles (Aboitiz et al., 2002). The olfactory cortex has been studied as a "simple model" to address sensory processing, though little is known about its precise cell origin, diversity, and identity. While the development and the cellular diversity of the six-layered neocortex are increasingly understood, the olfactory cortex remains poorly documented in these aspects. Here is a review of current knowledge of the development and organization of the olfactory cortex, keeping the analogy with those of the neocortex. The comparison of olfactory cortex and neocortex will allow the opening of evolutionary perspectives on cortical development.

Schwannomin-interacting Protein 1 Isoform IQCJ-SCHIP1 Is a Multipartner Ankyrin- and Spectrin-binding Protein Involved in the Organization of Nodes of Ranvier.

The nodes of Ranvier are essential regions for action potential conduction in myelinated fibers. They are enriched in multimolecular complexes composed of voltage-gated Nav and Kv7 channels associated with cell adhesion molecules. Cytoskeletal proteins ankyrin-G (AnkG) and ßIV-spectrin control the organization of these complexes and provide mechanical support to the plasma membrane. IQCJ-SCHIP1 is a cytoplasmic protein present in axon initial segments and nodes of Ranvier. It interacts with AnkG and is absent from nodes and axon initial segments of ßIV-spectrin and AnkG mutant mice. Here, we show that IQCJ-SCHIP1 also interacts with ßIV-spectrin and Kv7.2/3 channels and self-associates, suggesting a scaffolding role in organizing nodal proteins. IQCJ-SCHIP1 binding requires a ßIV-spectrin-specific domain and Kv7 channel 1-5-10 calmodulin-binding motifs. We then investigate the role of IQCJ-SCHIP1 in vivo by studying peripheral myelinated fibers in Schip1 knock-out mutant mice. The major nodal proteins are normally enriched at nodes in these mice, indicating that IQCJ-SCHIP1 is not required for their nodal accumulation. However, morphometric and ultrastructural analyses show an altered shape of nodes similar to that observed in ßIV-spectrin mutant mice, revealing that IQCJ-SCHIP1 contributes to nodal membrane-associated cytoskeleton organization, likely through its interactions with the AnkG/ßIV-spectrin network. Our work reveals that IQCJ-SCHIP1 interacts with several major nodal proteins, and we suggest that it contributes to a higher organizational level of the AnkG/ßIV-spectrin network critical for node integrity.

The cytoskeleton-associated protein SCHIP1 is involved in axon guidance, and is required for piriform cortex and anterior commissure development.

SCHIP1 is a cytoplasmic partner of cortical cytoskeleton ankyrins. The IQCJ-SCHIP1 isoform is a component of axon initial segments and nodes of Ranvier of mature axons in peripheral and central nervous systems, where it associates with membrane complexes comprising cell adhesion molecules. SCHIP1 is also expressed in the mouse developing central nervous system during embryonic stages of active axonogenesis. Here, we identify a new and early role for SCHIP1 during axon development and establishment of the anterior commissure (AC). The AC is composed of axons from the piriform cortex, the anterior olfactory nucleus and the amygdala. Schip1 mutant mice displayed early defects in AC development that might result from impaired axon growth and guidance. In addition, mutant mice presented a reduced thickness of the piriform cortex, which affected projection neurons in layers 2/3 and was likely to result from cell death rather than from impairment of neuron generation or migration. Piriform cortex neurons from E14.5 mutant embryos displayed axon initiation/outgrowth delay and guidance defects in vitro. The sensitivity of growth cones to semaphorin 3F and Eph receptor B2, two repulsive guidance cues crucial for AC development, was increased, providing a possible basis for certain fiber tract alterations. Thus, our results reveal new evidence for the involvement of cortical cytoskeleton-associated proteins in the regulation of axon development and their importance for the formation of neuronal circuits.

CK2-regulated schwannomin-interacting protein IQCJ-SCHIP-1 association with AnkG contributes to the maintenance of the axon initial segment.

The axon initial segment (AIS) plays a central role in electrogenesis and in the maintenance of neuronal polarity. Its molecular organization is dependent on the scaffolding protein ankyrin (Ank) G and is regulated by kinases. For example, the phosphorylation of voltage-gated sodium channels by the protein kinase CK2 regulates their interaction with AnkG and, consequently, their accumulation at the AIS. We previously showed that IQ motif containing J-Schwannomin-Interacting Protein 1 (IQCJ-SCHIP-1), an isoform of the SCHIP-1, accumulated at the AIS in vivo. Here, we analyzed the molecular mechanisms involved in IQCJ-SCHIP-1-specific axonal location. We showed that IQCJ-SCHIP-1 accumulation in the AIS of cultured hippocampal neurons depended on AnkG expression. Pull-down assays and surface plasmon resonance analysis demonstrated that AnkG binds to CK2-phosphorylated IQCJ-SCHIP-1 but not to the non-phosphorylated protein. Surface plasmon resonance approaches using IQCJ-SCHIP-1, SCHIP-1a, another SCHIP-1 isoform, and their C-terminus tail mutants revealed that a segment including multiple CK2-phosphorylatable sites was directly involved in the interaction with AnkG. Pharmacological inhibition of CK2 diminished both IQCJ-SCHIP-1 and AnkG accumulation in the AIS. Silencing SCHIP-1 expression reduced AnkG cluster at the AIS. Finally, over-expression of IQCJ-SCHIP-1 decreased AnkG concentration at the AIS, whereas a mutant deleted of the CK2-regulated AnkG interaction site did not. Our study reveals that CK2-regulated IQJC-SCHIP-1 association with AnkG contributes to AIS maintenance. The axon initial segment (AIS) organization depends on ankyrin (Ank) G and kinases. Here we showed that AnkG binds to CK2-phosphorylated IQCJ-SCHIP-1, in a segment including 12 CK2-phosphorylatable sites. In cultured neurons, either pharmacological inhibition of CK2 or IQCJ-SCHIP-1 silencing reduced AnkG clustering. Overexpressed IQCJ-SCHIP-1 decreased AnkG concentration at the AIS whereas a mutant deleted of the CK2-regulated AnkG interaction site did not. Thus, CK2-regulated IQJC-SCHIP-1 association with AnkG contributes to AIS maintenance.