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.

Repression of Irs2 by let-7 miRNAs is essential for homeostasis of the telencephalic neuroepithelium.

Structural integrity and cellular homeostasis of the embryonic stem cell niche are critical for normal tissue development. In the telencephalic neuroepithelium, this is controlled in part by cell adhesion molecules and regulators of progenitor cell lineage, but the specific orchestration of these processes remains unknown. Here, we studied the role of microRNAs in the embryonic telencephalon as key regulators of gene expression. By using the early recombiner Rx-Cre mouse, we identify novel and critical roles of miRNAs in early brain development, demonstrating they are essential to preserve the cellular homeostasis and structural integrity of the telencephalic neuroepithelium. We show that Rx-Cre;DicerF/F mouse embryos have a severe disruption of the telencephalic apical junction belt, followed by invagination of the ventricular surface and formation of hyperproliferative rosettes. Transcriptome analyses and functional experiments in vivo show that these defects result from upregulation of Irs2 upon loss of let-7 miRNAs in an apoptosis-independent manner. Our results reveal an unprecedented relevance of miRNAs in early forebrain development, with potential mechanistic implications in pediatric brain cancer.

Discrete domains of gene expression in germinal layers distinguish the development of gyrencephaly.

Gyrencephalic species develop folds in the cerebral cortex in a stereotypic manner, but the genetic mechanisms underlying this patterning process are unknown. We present a large-scale transcriptomic analysis of individual germinal layers in the developing cortex of the gyrencephalic ferret, comparing between regions prospective of fold and fissure. We find unique transcriptional signatures in each germinal compartment, where thousands of genes are differentially expressed between regions, including ~80% of genes mutated in human cortical malformations. These regional differences emerge from the existence of discrete domains of gene expression, which occur at multiple locations across the developing cortex of ferret and human, but not the lissencephalic mouse. Complex expression patterns emerge late during development and map the eventual location of folds or fissures. Protomaps of gene expression within germinal layers may contribute to define cortical folds or functional areas, but our findings demonstrate that they distinguish the development of gyrencephalic cortices.

Transient Cell-intrinsic Activity Regulates the Migration and Laminar Positioning of Cortical Projection Neurons.

Neocortical microcircuits are built during development and require the coordinated assembly of excitatory glutamatergic projection neurons (PNs) into functional networks. Neuronal migration is an essential step in this process. In addition to cell-intrinsic mechanisms, external cues including neurotransmitters regulate cortical neuron migration, suggesting that early activity could influence this process. Here, we aimed to investigate the role of cell-intrinsic activity in migrating PNs in vivo using a designer receptor exclusively activated by a designer drug (DREADD) chemogenetic approach. In utero electroporation was used to specifically express the human M3 muscarinic cholinergic Gq-coupled receptor (hM3Dq) in PNs and calcium activity, migratory dynamics, gene expression, and laminar positioning of PNs were assessed following embryonic DREADD activation. We found that transient embryonic DREADD activation induced premature branching and transcriptional changes in migrating PNs leading to a persistent laminar mispositioning of superficial layer PNs into deep cortical layers without affecting expression of layer-specific molecular identity markers. In addition, live imaging approaches indicated that embryonic DREADD activation increased calcium transients in migrating PNs and altered their migratory dynamics by increasing their pausing time. Taken together, these results support the idea that increased cell-intrinsic activity during migration acts as a stop signal for migrating cortical PNs.

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.

Secondary loss of miR-3607 reduced cortical progenitor amplification during rodent evolution.

The evolutionary expansion and folding of the mammalian cerebral cortex resulted from amplification of progenitor cells during embryonic development. This process was reversed in the rodent lineage after splitting from primates, leading to smaller and smooth brains. Genetic mechanisms underlying this secondary loss in rodent evolution remain unknown. We show that microRNA miR-3607 is expressed embryonically in the large cortex of primates and ferret, distant from the primate-rodent lineage, but not in mouse. Experimental expression of miR-3607 in embryonic mouse cortex led to increased Wnt/ß-catenin signaling, amplification of radial glia cells (RGCs), and expansion of the ventricular zone (VZ), via blocking the ß-catenin inhibitor APC (adenomatous polyposis coli). Accordingly, loss of endogenous miR-3607 in ferret reduced RGC proliferation, while overexpression in human cerebral organoids promoted VZ expansion. Our results identify a gene selected for secondary loss during mammalian evolution to limit RGC amplification and, potentially, cortex size in rodents.

Local Sustained GM-CSF Delivery by Genetically Engineered Encapsulated Cells Enhanced Both Cellular and Humoral SARS-CoV-2 Spike-Specific Immune Response in an Experimental Murine Spike DNA Vaccination Model.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic with recurrences. Therefore, finding a vaccine for this virus became a priority for the scientific community. The SARS-CoV-2 spike protein has been described as the keystone for viral entry into cells and effective immune protection against SARS-CoV-2 is elicited by this protein. Consequently, many commercialized vaccines focus on the spike protein and require the use of an optimal adjuvant during vaccination. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has demonstrated a powerful enhancement of acquired immunity against many pathogens when delivered in a sustained and local manner. In this context, we developed an encapsulated cell-based technology consisting of a biocompatible, semipermeable capsule for secretion of GM-CSF. In this study, we investigated whether murine GM-CSF (muGM-CSF) represents a suitable adjuvant for SARS-CoV-2 immunization, and which delivery strategy for muGM-CSF could be most beneficial. To test this, different groups of mice were immunized with intra-dermal (i.d.) electroporated spike DNA in the absence or presence of recombinant or secreted muGM-CSF. Results demonstrated that adjuvanting a spike DNA vaccine with secreted muGM-CSF resulted in enhancement of specific cellular and humoral immune responses against SARS-CoV-2. Our data also highlighted the importance of delivery strategies to the induction of cellular and humoral-mediated responses.

Transcriptomic and anatomic parcellation of 5-HT3AR expressing cortical interneuron subtypes revealed by single-cell RNA sequencing.

Cortical GABAergic interneurons constitute a highly diverse population of inhibitory neurons that are key regulators of cortical microcircuit function. An important and heterogeneous group of cortical interneurons specifically expresses the serotonin receptor 3A (5-HT3AR) but how this diversity emerges during development is poorly understood. Here we use single-cell transcriptomics to identify gene expression patterns operating in Htr3a-GFP+ interneurons during early steps of cortical circuit assembly. We identify three main molecular types of Htr3a-GFP+ interneurons, each displaying distinct developmental dynamics of gene expression. The transcription factor Meis2 is specifically enriched in a type of Htr3a-GFP+ interneurons largely confined to the cortical white matter. These MEIS2-expressing interneurons appear to originate from a restricted region located at the embryonic pallial-subpallial boundary. Overall, this study identifies MEIS2 as a subclass-specific marker for 5-HT3AR-containing interstitial interneurons and demonstrates that the transcriptional and anatomical parcellation of cortical interneurons is developmentally coupled.