Involvement of Calcium-Dependent Pathway and β Subunit-Interaction in Neuronal Migration and Callosal Projection Deficits Caused by the Cav1.2 I1166T Mutation in Developing Mouse Neocortex

Introduction: Gain-of-function mutations in the L-type Ca2+ channel Cav1.2 cause Timothy syndrome (TS), a multisystem disorder associated with neurologic symptoms, including autism spectrum disorder (ASD), seizures, and intellectual disability. Cav1.2 plays key roles in neural development, and its mutation can affect brain development and connectivity through Ca2+-dependent and -independent mechanisms. Recently, a gain-of-function mutation, I1166T, in Cav1.2 was identified in patients with TS-like disorder. Its channel properties have been analyzed in vitro but in vivo effects of this mutation on brain development remain unexplored.Methods:In utero electroporation was performed on ICR mice at embryonic day 15 to express GFP, wild-type, and mutant Cav1.2 channels into cortical layer 2/3 excitatory neurons in the primary somatosensory area. The brain was fixed at postnatal days 14–16, sliced, and scanned using confocal microscopy. Neuronal migration of electroporated neurons was examined in the cortex of the electroporated hemisphere, and callosal projection was examined in the white matter and contralateral hemisphere.Results: Expression of the I1166T mutant in layer 2/3 neurons caused migration deficits in approximately 20% of electroporated neurons and almost completely diminished axonal arborization in the contralateral hemisphere. Axonal projection in the white matter was not affected. We introduced second mutations onto Cav1.2 I1166T; L745P mutation blocks Ca2+ influx through Cav1.2 channels and inhibits the Ca2+-dependent pathway, and the W440A mutation blocks the interaction of the Cav1.2 α1 subunit to the β subunit. Both second mutations recovered migration and projection.Conclusion: This study demonstrated that the Cav1.2 I1166T mutation could affect two critical steps during cerebrocortical development, migration and axonal projection, in the mouse brain. This is mediated through Ca2+-dependent pathway downstream of Cav1.2 and β subunit-interaction.

A microRNA cluster downstream of the selector gene Fezf2 coordinates fate specification with dendritic branching in cortical neurons

In the cerebral cortex, cortical projection neurons comprise classes of neurons project to distant regions of the central nervous system. These neurons develop from the same progenitor pool, but they acquire strikingly different inputs and outputs to underpin strikingly different functions. The question of how corticospinal projection neurons - involved in motor function and implicated in paralysis - and callosal projection neurons - involved in cognitive function and implicated in autism - develop represents a fundamental and clinically important question in neurodevelopment. A network of transcription factors, including the selector gene Fezf2, is central to specifying cortical projection neuron fates. Gene regulation up- and down-stream of these transcription factors, however, is not well understood, particularly as it relates to the development of the major inputs to cortical projection neurons. Here we show that the miR-193b~365 microRNA cluster downstream of Fezf2 cooperatively represses the signaling molecule Mapk8, and impacts dendritic branching of cortical projection neurons.

miR-409-3p represses Cited2 at the evolutionary emergence of the callosal and corticospinal projections

Callosal projection neurons are a broad population of interhemispheric projection neurons that extend an axon across the corpus callosum to connect the two cerebral hemispheres. The corticospinal tract, comprised of the axons of corticospinal projection neurons, is unique to mammals, and its full extension to the lumbar segments that control walking is, like the corpus callosum, unique to placental mammals. The emergence of these two distinct axonal tracts is thought to underpin the evolutionary expansion of complex motor and cognitive abilities. The molecular mechanisms regulating the divergence of corticospinal and callosal projection neurons are incompletely understood. Our recent work identifies a genomic cluster of microRNAs (12qF1/Mirg) unique to placental mammals. These clustered miRNAs are specifically expressed by corticospinal vs. callosal projection neurons during the molecular refinement of corticospinal vs. callosal projection neuron fate (1). One of these, miR-409-3p, can convert layer V callosal into corticospinal projection neurons, acting in part through repression of the callosal-expressed transcriptional regulator Lmo4. This conversion is partial, however, suggesting that miR-409-3p represses multiple callosal projection neuron control genes in order to specify corticospinal projection neurons. One potential additional target of miR-409-3p repression is the callosal-expressed transcriptional co-activator Cited2. Cited2 interacts genetically with Lmo4, and Lmo4 can partially functionally compensate for Cited2 in thymus development(2). Further, Cited2 and Lmo4 function as opposing molecular controls over specific areal identity within superficial layer callosal projection neurons of the somatosensory and motor cortices, respectively (3). Cited2 is highly expressed by callosal, relative to corticospinal, projection neurons from the earliest stages of neurogenesis. Cited2 is necessary for the expansion of intermediate progenitor cells (IPCs) in the subventricular zone (SVZ), and the resulting generation of superficial layer callosal projection neurons. Here we show that miR-409-3p and Cited2 interact in IPCs and in corticospinal vs. deep layer callosal projection neuron development. miR-409-3p represses the Cited2 3UTR in luciferase assays. Mirg, which encodes miR-409-3p, and Cited2, are reciprocally expressed in IPCs at e15.5 by qPCR. Furthermore, miR-409-3p gain-of-function results in a phenocopy of established Cited2 loss-of-function in IPCs. Later on, miR-409-3p and Cited2 exert opposing effects on the adoption of corticospinal vs. callosal projection neuron subtype identity. Taken together, our work suggests that miR-409-3p, and possibly other 12qF1 miRNAs, represses Cited2 in IPCs to limit their proliferation, and in developing corticospinal and deep layer callosal projection neurons to favor corticospinal fate.

Human-specific enrichment of schizophrenia risk-genes in callosal neurons of the developing neocortex

SummaryHuman genetic studies have provided a wealth of information on genetic risk factors associated with neuropsychiatric diseases. However, whether different brain cell types are differentially affected in disease states and when in their development and maturation alterations occur is still poorly understood. Here we generated a longitudinal transcriptional map of excitatory projection neuron (PN) and inhibitory interneuron (IN) subtypes of the cerebral cortex, across a timeline of mouse embryonic and postnatal development, as well as fetal human cortex and human cortical organoids. We found that three types of gene signatures uniquely defined each cortical neuronal subtype: dynamic (developmental), adult (terminal), and constitutive (stable), with individual neuronal subtypes varying in the degree of similarity of their signatures between species. In particular, human callosal projection neurons (CPN) displayed the greatest species divergence, with molecular signatures highly enriched for non-coding, human-specific RNAs. Evaluating the association of neuronal class-specific signatures with neuropsychiatric disease risk genes using linkage disequilibrium score regression showed that schizophrenia risk genes were enriched in CPN identity signatures from human but not mouse cortex. Human cortical organoids confirmed the association with excitatory projection neurons. The data indicate that risk gene enrichment is both species- and cell type-specific. Our study reveals molecular determinants of cortical neuron diversification and identifies human callosal projection neurons as the most species-divergent population and a potentially vulnerable neuronal class in schizophrenia.

ZBTB20 is critical 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 regulating 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. 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 postmitotic cells to regulate cell-fate specification in the mammalian neocortex.

Laminar-specific interhemispheric connectivity mapping with bilateral line-scanning fMRI

ABSTRACTDespite extensive studies detecting blood-oxygen-level-dependent (BOLD) fMRI signals across two hemispheres to present cognitive processes in normal and diseased brains, the role of corpus callosum (CC) to mediate interhemispheric functional connectivity remains controversial. Several studies show maintaining low-frequency fluctuation of resting-state (rs)-fMRI signals in homotopic brain areas of acallosal humans and post-callosotomy animals, raising the question: how can we specify the circuit-specific rs-fMRI signal fluctuation from other sources? To address this question, we have developed a bilateral line-scanning fMRI (BiLS) method to detect bilateral laminar BOLD fMRI signals from symmetric cortical regions with high spatial (100 μm) and temporal (100 ms) resolution in rodents under anesthesia. In addition to ultra-slow oscillation (0.01-0.02 Hz) patterns across all cortical layers, a layer-specific bilateral coherence pattern was observed with a peak at Layer (L)2/3, where callosal projection neurons are primarily located and reciprocal transcallosal projections are received. In particular, the L2/3-specific coherence pattern showed a peak at 0.05 Hz based on the stimulation paradigm, depending on the interhemispheric CC activation. Meanwhile, the L2/3-specific rs-fMRI coherence was peaked at 0.08-0.1Hz which was independent of the varied ultra-slow oscillation patterns (0.01-0.02 Hz) presumably involved with global neuromodulation. This work provides a unique laminar fMRI mapping scheme to characterize the CC-mediated evoked fMRI and frequency-dependent rs-fMRI responses, presenting crucial evidence to distinguish the circuit-specific fMRI signal fluctuations across two hemispheres.Significance statementLaminar fMRI is a promising method to better understand neuronal circuit contribution to functional connectivity (FC) across cortical layers. Here, we developed a bilateral line-scanning fMRI method, allowing the detection of laminar-specific BOLD-fMRI signals from homologous cortical regions in rodents with high spatial and temporal resolution. Laminar coherence patterns of both evoked and rs-fMRI signals revealed that CC-dependent interhemispheric FC is significantly strong at Layer 2/3, where callosal projection neurons are primarily located. The Layer 2/3-specific rs-fMRI coherence is independent of ultra-slow oscillation based on global neuromodulation, distinguishing the circuit-specific rs-fMRI signal fluctuation from different regulatory sources.

New Molecular Players in the Development of Callosal Projections

Cortical development in humans is a long and ongoing process that continuously modifies the neural circuitry into adolescence. This is well represented by the dynamic maturation of the corpus callosum, the largest white matter tract in the brain. Callosal projection neurons whose long-range axons form the main component of the corpus callosum are evolved relatively recently with a substantial, disproportionate increase in numbers in humans. Though the anatomy of the corpus callosum and cellular processes in its development have been intensively studied by experts in a variety of fields over several decades, the whole picture of its development, in particular, the molecular controls over the development of callosal projections, still has many missing pieces. This review highlights the most recent progress on the understanding of corpus callosum formation with a special emphasis on the novel molecular players in the development of axonal projections in the corpus callosum.

Increased Callosal Connectivity in Reeler Mice Revealed by Brain-Wide Input Mapping of VIP Neurons in Barrel Cortex

The neocortex is composed of layers. Whether layers constitute an essential framework for the formation of functional circuits is not well understood. We investigated the brain-wide input connectivity of vasoactive intestinal polypeptide (VIP) expressing neurons in the reeler mouse. This mutant is characterized by a migration deficit of cortical neurons so that no layers are formed. Still, neurons retain their properties and reeler mice show little cognitive impairment. We focused on VIP neurons because they are known to receive strong long-range inputs and have a typical laminar bias toward upper layers. In reeler, these neurons are more dispersed across the cortex. We mapped the brain-wide inputs of VIP neurons in barrel cortex of wild-type and reeler mice with rabies virus tracing. Innervation by subcortical inputs was not altered in reeler, in contrast to the cortical circuitry. Numbers of long-range ipsilateral cortical inputs were reduced in reeler, while contralateral inputs were strongly increased. Reeler mice had more callosal projection neurons. Hence, the corpus callosum was larger in reeler as shown by structural imaging. We argue that, in the absence of cortical layers, circuits with subcortical structures are maintained but cortical neurons establish a different network that largely preserves cognitive functions.

An evolutionarily acquired microRNA shapes development of mammalian cortical projections

The corticospinal tract is unique to mammals and the corpus callosum is unique to placental mammals (eutherians). The emergence of these structures is thought to underpin the evolutionary acquisition of complex motor and cognitive skills. Corticospinal motor neurons (CSMN) and callosal projection neurons (CPN) are the archetypal projection neurons of the corticospinal tract and corpus callosum, respectively. Although a number of conserved transcriptional regulators of CSMN and CPN development have been identified in vertebrates, none are unique to mammals and most are co-expressed across multiple projection neuron subtypes. Here, we discover seventeen CSMN-enriched microRNAs (miRNAs), fifteen of which map to a single genomic cluster that is exclusive to eutherians. One of these, miR-409-3p, promotes CSMN subtype identity in part via repression of LMO4, a key transcriptional regulator of CPN development. In vivo, miR-409-3p is sufficient to convert deep-layer CPN into CSMN. This is the first demonstration of an evolutionarily acquired miRNA in eutherians that refines cortical projection neuron subtype development. Our findings implicate miRNAs in the eutherians’ increase in neuronal subtype and projection diversity, the anatomic underpinnings of their complex behavior.Significance StatementThe mammalian central nervous system contains unique projections from the cerebral cortex thought to underpin complex motor and cognitive skills, including the corticospinal tract and corpus callosum. The neurons giving rise to these projections - corticospinal and callosal projection neurons - develop from the same progenitors, but acquire strikingly different fates. The broad evolutionary conservation of known genes controlling cortical projection neuron fates raises the question of how the more narrowly conserved corticospinal and callosal projections evolved. We identify a microRNA cluster selectively expressed by corticospinal projection neurons and exclusive to placental mammals. One of these microRNAs promotes corticospinal fate via regulation of the callosal gene LMO4, suggesting a mechanism whereby microRNA regulation during development promotes evolution of neuronal diversity.

Neuron-class specific responses govern adaptive remodeling of myelination in the neocortex

Myelination plasticity plays a critical role in neurological function, including learning and memory. However, it is unknown whether this plasticity is enacted through uniform changes across all neuronal subtypes, or whether myelin dynamics vary between neuronal classes to enable fine-tuning of adaptive circuit responses. We performed in vivo two-photon imaging to investigate the dynamics of myelin sheaths along single axons of both excitatory callosal projection neurons and inhibitory parvalbumin+ interneurons in layer 2/3 of adult mouse visual cortex. We find that both neuron types show dynamic, homeostatic myelin remodeling under normal vision. However, monocular deprivation results in experience-dependent adaptive myelin remodeling only in parvalbumin+ interneurons, but not in callosal projection neurons. Monocular deprivation induces an initial increase in elongation events in myelin segments of parvalbumin+ interneurons, followed by a contraction phase affecting a separate cohort of segments. Sensory experience does not alter the generation rate of new myelinating oligodendrocytes, but can recruit pre-existing oligodendrocytes to generate new myelin sheaths. Parvalbumin+ interneurons also show a concomitant increase in axonal branch tip dynamics independent from myelination events. These findings suggest that adaptive myelination is part of a coordinated suite of circuit reconfiguration processes, and demonstrate that distinct classes of neocortical neurons individualize adaptive remodeling of their myelination profiles to diversify circuit tuning in response to sensory experience.