Callosal Projection Neurons Research Articles

Early cortical GABAergic interneurons determine the projection patterns of L4 excitatory neurons.

During cerebral cortex development, excitatory pyramidal neurons (PNs) establish specific projection patterns while receiving inputs from GABAergic inhibitory interneurons (INs). Whether these inhibitory inputs can shape PNs' projection patterns is, however, unknown. While layer 4 (L4) PNs of the primary somatosensory (S1) cortex are all born as long-range callosal projection neurons (CPNs), most of them acquire local connectivity upon activity-dependent elimination of their interhemispheric axons during postnatal development. Here, we demonstrate that precise developmental regulation of inhibition is key for the retraction of S1L4 PNs' callosal projections. Ablation of somatostatin INs leads to premature inhibition from parvalbumin INs onto S1L4 PNs and prevents them from acquiring their barrel-restricted local connectivity pattern. As a result, adult S1L4 PNs retain interhemispheric projections responding to tactile stimuli, and the mice lose whisker-based texture discrimination. Overall, we show that temporally ordered IN activity during development is key to shaping local ipsilateral S1L4 PNs' projection pattern, which is required for fine somatosensory processing.

ARID1B controls transcriptional programs of axon projection in an organoid model of the human corpus callosum

Mutations in ARID1B, a member of the mSWI/SNF complex, cause severe neurodevelopmental phenotypes with elusive mechanisms in humans. The most common structural abnormality in the brain of ARID1B patients is agenesis of the corpus callosum (ACC), characterized by the absence of an interhemispheric white matter tract that connects distant cortical regions. Here, we find that neurons expressing SATB2, a determinant of callosal projection neuron (CPN) identity, show impaired maturation in ARID1B+/- neural organoids. Molecularly, a reduction in chromatin accessibility of genomic regions targeted by TCF-like, NFI-like, and ARID-like transcription factors drives the differential expression of genes required for corpus callosum (CC) development. Through an invitro model of the CC tract, we demonstrate that this transcriptional dysregulation impairs the formation of long-range axonal projections, causing structural underconnectivity. Our study uncovers new functions of the mSWI/SNF during human corticogenesis, identifying cell-autonomous axonogenesis defects in SATB2+ neurons as a cause of ACC in ARID1B patients.

Microtubule-binding protein MAP1B regulates interstitial axon branching of cortical neurons via the tubulin tyrosination cycle

Regulation of directed axon guidance and branching during development is essential for the generation of neuronal networks. However, the molecular mechanisms that underlie interstitial (or collateral) axon branching in the mammalian brain remain unresolved. Here, we investigate interstitial axon branching in vivo using an approach for precise labeling of layer 2/3 callosal projection neurons (CPNs). This method allows for quantitative analysis of axonal morphology at high acuity and also manipulation of gene expression in well-defined temporal windows. We find that the GSK3β serine/threonine kinase promotes interstitial axon branching in layer 2/3 CPNs by releasing MAP1B-mediated inhibition of axon branching. Further, we find that the tubulin tyrosination cycle is a key downstream component of GSK3β/MAP1B signaling. These data suggest a cell-autonomous molecular regulation of cortical neuron axon morphology, in which GSK3β can release a MAP1B-mediated brake on interstitial axon branching upstream of the posttranslational tubulin code.

Life after transcription: control of the fate of neurons

The neocortex is a complex structure responsible for higher-order cognitive abilities in mammals. It consists of six cell layers, each comprised of various subtypes of excitatory and inhibitory neurons. These neurons project their axons to specific targets within each layer. All neocortical projection neurons are generated by neural progenitor cells located in the proliferative ventricular zone. In recent decades, significant strides have been made in comprehending the transcriptional programs governing neuronal cell fate specification. Nonetheless, posttranscriptional mechanisms involved in this process remain largely unexplored. Recently, we found that TrkC-T1, an isoform of the TrkC receptor lacking the kinase domain, determines the fate of corticofugal projection neurons (CFuPN). Our study reveals that the balance between TrkC-T1 and TrkC-TK+, a more widely recognized isoform containing the kinase domain, depends on the type of cell within the developing cortex. Additionally, we demonstrate that two RNA-binding proteins, Srsf1 and Elavl1, work in opposition to establish this balance. Additionally, our data suggests that Srsf1 stimulates the CFuPN fate while Elavl1 stimulates the callosal projection neuron (CPN) fate in vivo by regulating the different TrkC-T1 to TrkC-TK+ ratios. In this study, we identified a protein translation-dependent mechanism that governs the cell fate switch in the developing neocortex. Our results demonstrate that Ire1α, the Inositol-Requiring Enzyme 1α, regulates global translation rates in the developing neocortex through its dynamic interaction with the ribosome, as well as the regulation of expression of translation elongation factors eIF4A1 and eEF-2. Inactivation of Ire1α leads to decreased protein synthesis rates that are associated with stalled ribosomes and a reduced number of sites for translation initiation. We demonstrate the distinctive sensitivity of neurons determined for the upper layer to translation rates. While eEF-2 is necessary for cortical lamination, eIF4A1 regulates the attainment of upper layer fate in a mechanism of translational control that is dependent on structural elements embedded in the 5’UTR of genes responsible for determining fate downstream of Ire1α. Our findings reveal the developmental control of ribosome dynamics as post-transcriptional mechanisms that coordinate the establishment of neuronal diversity and the assembly of cortical layers.

Srsf1 and Elavl1 act antagonistically on neuronal fate choice in the developing neocortex by controlling TrkC receptor isoform expression.

The seat of higher-order cognitive abilities in mammals, the neocortex, is a complex structure, organized in several layers. The different subtypes of principal neurons are distributed in precise ratios and at specific positions in these layers and are generated by the same neural progenitor cells (NPCs), steered by a spatially and temporally specified combination of molecular cues that are incompletely understood. Recently, we discovered that an alternatively spliced isoform of the TrkC receptor lacking the kinase domain, TrkC-T1, is a determinant of the corticofugal projection neuron (CFuPN) fate. Here, we show that the finely tuned balance between TrkC-T1 and the better known, kinase domain-containing isoform, TrkC-TK+, is cell type-specific in the developing cortex and established through the antagonistic actions of two RNA-binding proteins, Srsf1 and Elavl1. Moreover, our data show that Srsf1 promotes the CFuPN fate and Elavl1 promotes the callosal projection neuron (CPN) fate in vivo via regulating the distinct ratios of TrkC-T1 to TrkC-TK+. Taken together, we connect spatio-temporal expression of Srsf1 and Elavl1 in the developing neocortex with the regulation of TrkC alternative splicing and transcript stability and neuronal fate choice, thus adding to the mechanistic and functional understanding of alternative splicing in vivo.

A frontal transcallosal inhibition loop mediates interhemispheric balance in visuospatial processing

Interhemispheric communication through the corpus callosum is required for both sensory and cognitive processes. Impaired transcallosal inhibition causing interhemispheric imbalance is believed to underlie visuospatial bias after frontoparietal cortical damage, but the synaptic circuits involved remain largely unknown. Here, we show that lesions in the mouse anterior cingulate area (ACA) cause severe visuospatial bias mediated by a transcallosal inhibition loop. In a visual-change-detection task, ACA callosal-projection neurons (CPNs) were more active with contralateral visual field changes than with ipsilateral changes. Unilateral CPN inactivation impaired contralateral change detection but improved ipsilateral detection by altering interhemispheric interaction through callosal projections. CPNs strongly activated contralateral parvalbumin-positive (PV+) neurons, and callosal-input-driven PV+ neurons preferentially inhibited ipsilateral CPNs, thus mediating transcallosal inhibition. Unilateral PV+ neuron activation caused a similar behavioral bias to contralateral CPN activation and ipsilateral CPN inactivation, and bilateral PV+ neuron activation eliminated this bias. Notably, restoring interhemispheric balance by activating contralesional PV+ neurons significantly improved contralesional detection in ACA-lesioned animals. Thus, a frontal transcallosal inhibition loop comprising CPNs and callosal-input-driven PV+ neurons mediates interhemispheric balance in visuospatial processing, and enhancing contralesional transcallosal inhibition restores interhemispheric balance while also reversing lesion-induced bias.

Development of nanoRibo-seq enables study of regulated translation by cortical neuron subtypes, showing uORF translation in synaptic-axonal genes

Investigation of translation in rare cell types or subcellular contexts is challenging due to large input requirements for standard approaches. Here, we present "nanoRibo-seq" an optimized approach using 102- to 103-fold less input material than bulk approaches. nanoRibo-seq exhibits rigorous quality control features consistent with quantification of ribosome protected fragments with as few as 1,000 cells. We compare translatomes of two closely related cortical neuron subtypes, callosal projection neurons (CPN) and subcerebral projection neurons (SCPN), during their early postnatal development. We find that, while translational efficiency is highly correlated between CPN and SCPN, several dozen mRNAs are differentially translated. We further examine upstream open reading frame (uORF) translation and identify that mRNAs involved in synapse organization and axon development are highly enriched for uORF translation in both subtypes. nanoRibo-seq enables investigation of translational regulation of rare cell types invivo and offers a flexible approach for globally quantifying translation from limited input material.

Optogenetic stimulation shapes dendritic trees of infragranular cortical pyramidal cells.

Spontaneous or experimentally evoked activity can lead to changes in length and/or branching of neocortical pyramidal cell dendrites. For instance, an early postnatal overexpression of certain AMPA or kainate glutamate receptor subunits leads to larger amplitudes of depolarizing events driven by spontaneous activity, and this increases apical dendritic complexity. Whether stimulation frequency has a role is less clear. In this study, we report that the expression of channelrhodopsin2-eYFP was followed by a 5-day optogenetic stimulation from DIV 5-10 or 11-15 in organotypic cultures of rat visual cortex-evoked dendritic remodeling. Stimulation at 0.05 Hz, at a frequency range of spontaneous calcium oscillations known to occur in the early postnatal neocortex in vivo until eye opening, had no effect. Stimulation with 0.5 Hz, a frequency at which the cortex in vivo adopts after eye opening, unexpectedly caused shorter and somewhat less branched apical dendrites of infragranular pyramidal neurons. The outcome resembles the remodeling of corticothalamic and callosal projection neurons of layers VI and V, which in the adult have apical dendrites no longer terminating in layer I. Exposure to 2.5 Hz, a frequency not occurring naturally during the time windows, evoked dendritic damage. The results suggested that optogenetic stimulation at a biologically meaningful frequency for the selected developmental stage can influence dendrite growth, but contrary to expectation, the optogenetic stimulation decreased dendritic growth.

Remote neuronal activity drives glioma progression through SEMA4F.

The tumour microenvironment plays an essential role in malignancy, and neurons have emerged as a key component of the tumour microenvironment that promotes tumourigenesis across a host of cancers1,2. Recent studies on glioblastoma (GBM) highlight bidirectional signalling between tumours and neurons that propagates a vicious cycle of proliferation, synaptic integration and brain hyperactivity3-8; however, the identity of neuronal subtypes and tumour subpopulations driving this phenomenon is incompletely understood. Here we show that callosal projection neurons located in the hemisphere contralateral to primary GBM tumours promote progression and widespread infiltration. Using this platform to examine GBM infiltration, we identified an activity-dependent infiltrating population present at the leading edge of mouse and human tumours that is enriched for axon guidance genes. High-throughput, in vivo screening of these genes identified SEMA4F as a key regulator of tumourigenesis and activity-dependent progression. Furthermore, SEMA4F promotes the activity-dependent infiltrating population and propagates bidirectional signalling with neurons by remodelling tumour-adjacent synapses towards brain network hyperactivity. Collectively our studies demonstrate that subsets of neurons in locations remote to primary GBM promote malignant progression, and also show new mechanisms of glioma progression that are regulated by neuronal activity.

CDKL5 sculpts functional callosal connectivity to promote cognitive flexibility

Functional and structural connectivity alterations in short- and long-range projections have been reported across neurodevelopmental disorders (NDD). Interhemispheric callosal projection neurons (CPN) represent one of the major long-range projections in the brain, which are particularly important for higher-order cognitive function and flexibility. However, whether a causal relationship exists between interhemispheric connectivity alterations and cognitive deficits in NDD remains elusive. Here, we focused on CDKL5 Deficiency Disorder (CDD), a severe neurodevelopmental disorder caused by mutations in the X-linked Cyclin-dependent kinase-like 5 (CDKL5) gene. We found an increase in homotopic interhemispheric connectivity and functional hyperconnectivity across higher cognitive areas in adult male and female CDKL5-deficient mice by resting-state functional MRI (rs-fMRI) analysis. This was accompanied by an increase in the number of callosal synaptic inputs but decrease in local synaptic connectivity in the cingulate cortex of juvenile CDKL5-deficient mice, suggesting an impairment in excitatory synapse development and a differential role of CDKL5 across excitatory neuron subtypes. These deficits were associated with significant cognitive impairments in CDKL5 KO mice. Selective deletion of CDKL5 in the largest subtype of CPN likewise resulted in an increase of functional callosal inputs, without however significantly altering intracortical cingulate networks. Notably, such callosal-specific changes were sufficient to cause cognitive deficits. Finally, when CDKL5 was selectively re-expressed only in this CPN subtype, in otherwise CDKL5-deficient mice, it was sufficient to prevent the cognitive impairments of CDKL5 mutants. Together, these results reveal a novel role of CDKL5 by demonstrating that it is both necessary and sufficient for proper CPN connectivity and cognitive function and flexibility, and further validates a causal relationship between CPN dysfunction and cognitive impairment in a model of NDD.

MiR-409-3p represses Cited2 to refine neocortical layer V projection neuron identity.

The evolutionary emergence of the corticospinal tract and corpus callosum are thought to underpin the expansion of complex motor and cognitive abilities in mammals. Molecular mechanisms regulating development of the neurons whose axons comprise these tracts, the corticospinal and callosal projection neurons, remain incompletely understood. Our previous work identified a genomic cluster of microRNAs (miRNAs), Mirg/12qF1, that is unique to placental mammals and specifically expressed by corticospinal neurons, and excluded from callosal projection neurons, during development. We found that one of these, miR-409-3p, can convert layer V callosal into corticospinal projection neurons, acting in part through repression of the transcriptional regulator Lmo4. Here we show that miR-409-3p also directly represses the transcriptional co-regulator Cited2, which is highly expressed by callosal projection neurons from the earliest stages of neurogenesis. Cited2 is highly expressed by intermediate progenitor cells (IPCs) in the embryonic neocortex while Mirg, which encodes miR-409-3p, is excluded from these progenitors. miR-409-3p gain-of-function (GOF) in IPCs results in a phenocopy of established Cited2 loss-of-function (LOF). At later developmental stages, both miR-409-3p GOF and Cited2 LOF promote the expression of corticospinal at the expense of callosal projection neuron markers in layer V. Taken together, this work identifies previously undescribed roles for miR-409-3p in controlling IPC numbers and for Cited2 in controlling callosal fate. Thus, miR-409-3p, possibly in cooperation with other Mirg/12qF1 miRNAs, represses Cited2 as part of the multifaceted regulation of the refinement of neuronal cell fate within layer V, combining molecular regulation at multiple levels in both progenitors and post-mitotic neurons.

Unilateral cerebral ischemia induces morphological changes in the layer V projection neurons of the contralateral hemisphere

Decreased blood flow to the brain causes stroke and damage to neuronal networks. Neuronal damage occurs not only in the infarct core but also in areas away from the infarcts. This study was aimed to assess alterations of the cortical projection neurons that were distantly connected with the infarcts. Unilateral cortical ischemia was generated by middle cerebral artery occlusion in the right somatosensory cortex. Pre-labeled thalamocortical neurons disappeared, whereas contralateral callosal projection neurons survived 48 h post-ischemia. The unilateral ischemia increased the total length, segment length and the spine volume of dendrites from layer V callosal neurons in the homotopic cortex of the contralateral hemisphere. The morphological remolding of the contralateral cortical neurons cannot be reproduced by the spinal cord hemisection that cuts axons of corticospinal projection neurons of layer V. The data suggest that the retrograde degeneration of axons may not account for the early morphological changes in the contralateral cortex. We hypothesize that the loss of innervations from the ischemic cortex may bring in adaptive changes to the connected neurons, and adult cortical neurons can adjust their morphology to meet the reduction of synaptic inputs. This study may improve our understanding of the re-organization of cortical circuits following focal cerebral ischemia and help the development of new treatments designed to minimize the disability associated with stroke.

FOXG1 sequentially orchestrates subtype specification of postmitotic cortical projection neurons.

The mammalian neocortex is a highly organized six-layered structure with four major cortical neuron subtypes: corticothalamic projection neurons (CThPNs), subcerebral projection neurons (SCPNs), deep callosal projection neurons (CPNs), and superficial CPNs. Here, careful examination of multiple conditional knockout model mouse lines showed that the transcription factor FOXG1 functions as a master regulator of postmitotic cortical neuron specification and found that mice lacking functional FOXG1 exhibited projection deficits. Before embryonic day 14.5 (E14.5), FOXG1 enforces deep CPN identity in postmitotic neurons by activating Satb2 but repressing Bcl11b and Tbr1. After E14.5, FOXG1 exerts specification functions in distinct layers via differential regulation of Bcl11b and Tbr1, including specification of superficial versus deep CPNs and enforcement of CThPN identity. FOXG1 controls CThPN versus SCPN fate by fine-tuning Fezf2 levels through diverse interactions with multiple SOX family proteins. Thus, our study supports a developmental model to explain the postmitotic specification of four cortical projection neuron subtypes and sheds light on neuropathogenesis.

Differential gene expression between callosal and ipsilateral projection neurons in the monkey dorsolateral prefrontal and posterior parietal cortices.

Reciprocal connections between primate dorsolateral prefrontal (DLPFC) and posterior parietal (PPC) cortices, furnished by subsets of layer 3 pyramidal neurons (PNs), contribute to cognitive processes including working memory (WM). A different subset of layer 3 PNs in each region projects to the homotopic region of the contralateral hemisphere. These ipsilateral (IP) and callosal (CP) projections, respectively, appear to be essential for the maintenance and transfer of information during WM. To determine if IP and CP layer 3 PNs in each region differ in their transcriptomes, fluorescent retrograde tracers were used to label IP and CP layer 3 PNs in the DLPFC and PPC from macaque monkeys. Retrogradely-labeled PNs were captured by laser microdissection and analyzed by RNAseq. Numerous differentially expressed genes (DEGs) were detected between IP and CP neurons in each region and the functional pathways containing many of these DEGs were shared across regions. However, DLPFC and PPC displayed opposite patterns of DEG enrichment between IP and CP neurons. Cross-region analyses indicated that the cortical area targeted by IP or CP layer 3 PNs was a strong correlate of their transcriptome profile. These findings suggest that the transcriptomes of layer 3 PNs reflect regional, projection type and target region specificity.

Mllt11 Regulates Migration and Neurite Outgrowth of Cortical Projection Neurons during Development.

The formation of connections within the mammalian neocortex is highly regulated by both extracellular guidance mechanisms and intrinsic gene expression programs. There are two types of cortical projection neurons (CPNs): those that project locally and interhemispherically and those that project to subcerebral structures such as the thalamus, hindbrain, and spinal cord. The regulation of cortical projection morphologies is not yet fully understood at the molecular level. Here, we report a role for Mllt11 (Myeloid/lymphoid or mixed-lineage leukemia; translocated to chromosome 11/All1 Fused Gene From Chromosome 1q) in the migration and neurite outgrowth of callosal projection neurons during mouse brain formation. We show that Mllt11 expression is exclusive to developing neurons and is enriched in the developing cortical plate (CP) during the formation of the superficial cortical layers. In cultured primary cortical neurons, Mllt11 is detected in varicosities and growth cones as well as the soma. Using conditional loss-of-function and gain-of-function analysis we show that Mllt11 is required for neuritogenesis and proper migration of upper layer CPNs. Loss of Mllt11 in the superficial cortex of male and female neonates leads to a severe reduction in fibers crossing the corpus callosum (CC), a progressive loss in the maintenance of upper layer projection neuron gene expression, and reduced complexity of dendritic arborization. Proteomic analysis revealed that Mllt11 associates with stabilized microtubules, and Mllt11 loss affected microtubule staining in callosal axons. Taken together, our findings support a role for Mllt11 in promoting the formation of mature upper-layer neuron morphologies and connectivity in the cerebral cortex.SIGNIFICANCE STATEMENT The regulation of cortical projection neuron (CPN) morphologies is an area of active investigation since the time of Cajal. Yet the molecular mechanisms of how the complex dendritic and axonal morphologies of projection neurons are formed remains incompletely understood. Although conditional mutagenesis analysis in the mouse, coupled with overexpression assays in the developing fetal brain, we show that a novel protein called Mllt11 is sufficient and necessary to regulate the dendritic and axonal characteristics of callosal projection neurons in the developing mammalian neocortex. Furthermore, we show that Mllt11 interacts with microtubules, likely accounting for its role in neuritogenesis.

The Role of Zinc in Axon Formation via the mTORC1 Pathway.

Zinc is an essential micronutrient required for proper function during neuronal development because it can modulate neuronal function and structure. A fully functional description of zinc in axonal processing in the central nervous system remains elusive. Here, we define the role of intracellular zinc in axon formation and elongation, involving the mammalian target of rapamycin complex 1 (mTORC1). To investigate the involvement of zinc in axon growth, we performed an ex vivo culture of mouse hippocampal neurons and administrated ZnCl2 as a media supplement. At 2 days in vitro, the administration of zinc induced the formation of multiple and elongated axons in the ex vivo culture system. A similar outcome was witnessed in callosal projection neurons in a developing mouse brain. Treatment with extracellular zinc activated the mTORC1 signaling pathway in mouse hippocampal neuronal cultures. The zinc-dependent enhancement of neuronal processing was inhibited either by the deactivation of mTORC1 with RAPTOR shRNA or by mTOR-insensitive 4EBP1 mutants. Additionally, zinc-dependent mTORC1 activation enhanced the axonal translation of TC10 and Par3 may be responsible for axonal growth. We identified a promising role of zinc in controlling axonogenesis in the developing brain, which, in turn, may indicate a novel structural role of zinc in the cytoskeleton and developing neurons.

Studying the Role of P2X7 Receptor in Axonal Growth Using In Utero Electroporation Technique.

The nervous system is formed by a complex network of neuronal connections. During development, neurons elongate their axons through highly stereotyped anatomical pathways to form precise connections. Defects in these mechanisms are related with neurological disorders. Previous studies have reported that inhibition of the P2X7 receptor, an ionotropic purinergic receptor, promotes axonal growth and branching in cultured neurons. However, little is known about the in vivo mechanism of axonal elongation regulated by P2X7. Here, we detailed a step-by-step method to perform in utero cortical electroporation and quantified the electroporated axons employing accessible and open-source image processing software. This effective surgical procedure manipulates in vivo the gene expression in a discrete population of callosal projection neuron. Thus, a better understanding of the involvement of P2X7 in the in vivo establishment of neuronal circuits might help to clarify the basic biology of several neurodevelopmental disorders and axonal regenerative processes.

Synthetic modified Fezf2 mRNA (modRNA) with concurrent small molecule SIRT1 inhibition enhances refinement of cortical subcerebral/corticospinal neuron identity from mouse embryonic stem cells.

During late embryonic development of the cerebral cortex, the major class of cortical output neurons termed subcerebral projection neurons (SCPN; including the predominant population of corticospinal neurons, CSN) and the class of interhemispheric callosal projection neurons (CPN) initially express overlapping molecular controls that later undergo subtype-specific refinements. Such molecular refinements are largely absent in heterogeneous, maturation-stalled, neocortical-like neurons (termed “cortical” here) spontaneously generated by established embryonic stem cell (ES) and induced pluripotent stem cell (iPSC) differentiation. Building on recently identified central molecular controls over SCPN development, we used a combination of synthetic modified mRNA (modRNA) for Fezf2, the central transcription factor controlling SCPN specification, and small molecule screening to investigate whether distinct chromatin modifiers might complement Fezf2 functions to promote SCPN-specific differentiation by mouse ES (mES)-derived cortical-like neurons. We find that the inhibition of a specific histone deacetylase, Sirtuin 1 (SIRT1), enhances refinement of SCPN subtype molecular identity by both mES-derived cortical-like neurons and primary dissociated E12.5 mouse cortical neurons. In vivo, we identify that SIRT1 is specifically expressed by CPN, but not SCPN, during late embryonic and postnatal differentiation. Together, these data indicate that SIRT1 has neuronal subtype-specific expression in the mouse cortex in vivo, and that its inhibition enhances subtype-specific differentiation of highly clinically relevant SCPN / CSN cortical neurons in vitro.

ZBTB20 is crucial for the specification of a subset of callosal projection neurons and astrocytes in the mammalian neocortex.

Neocortical progenitor cells generate subtypes of excitatory projection neurons in sequential order followed by the generation of astrocytes. The transcription factor zinc finger and BTB domain-containing protein 20 (ZBTB20) has been implicated in regulation of cell specification during neocortical development. Here, we show that ZBTB20 instructs the generation of a subset of callosal projections neurons in cortical layers II/III in mouse. Conditional deletion of Zbtb20 in cortical progenitors, and to a lesser degree in differentiating neurons, leads to an increase in the number of layer IV neurons at the expense of layer II/III neurons. Astrogliogenesis is also affected in the mutants with an increase in the number of a specific subset of astrocytes expressing GFAP. Astrogliogenesis is more severely disrupted by a ZBTB20 protein containing dominant mutations linked to Primrose syndrome, suggesting that ZBTB20 acts in concert with other ZBTB proteins that were also affected by the dominant-negative protein to instruct astrogliogenesis. Overall, our data suggest that ZBTB20 acts both in progenitors and in postmitotic cells to regulate cell fate specification in the mammalian neocortex.

A Distinct Population of L6 Neurons in Mouse V1 Mediate Cross-Callosal Communication.

Through the corpus callosum, interhemispheric communication is mediated by callosal projection (CP) neurons. Using retrograde labeling, we identified a population of layer 6 (L6) excitatory neurons as the main conveyer of transcallosal information in the monocular zone of the mouse primary visual cortex (V1). Distinct from L6 corticothalamic (CT) population, V1 L6 CP neurons contribute to an extensive reciprocal network across multiple sensory cortices over two hemispheres. Receiving both local and long-range cortical inputs, they encode orientation, direction, and receptive field information, while are also highly spontaneous active. The spontaneous activity of L6 CP neurons exhibits complex relationships with brain states and stimulus presentation, distinct from the spontaneous activity patterns of the CT population. The anatomical and functional properties of these L6 CP neurons enable them to broadcast visual and nonvisual information across two hemispheres, and thus may play a role in regulating and coordinating brain-wide activity events.