Transcriptional Controls over Neocortical Projection Neuron Identity and Connectivity

The complex and sophisticated circuitry of the neocortex is assembled from an extraordinarily diverse repertoire of neuronal subtypes that reside in distinct functional areas. In recent years, a number of key regulators over neocortical projection neuron subtype and area specification have been identified. It is becoming increasingly clear that these regulators function within a highly-interconnected network, acting in parallel, synergistically, and cross-repressively to orchestrate cortical development. Moreover, an emerging understanding of cortical development has revealed that subtype and area identity are intimately interrelated, and that specification occurs based on several sequential molecular decision points. Although great strides have been made in recent years toward understanding molecular controls over neocortical projection neuron development, many important controls remain to be discovered, and mechanisms by which recently-identified regulators act to delineate subtype and area identity are not well understood. In this dissertation, I characterize functions of two zinc finger transcription factors, Ctip2 and Ctip1, in postmitotic projection neuron subtype and area identity acquisition, using in vivo gain- and loss-of-function approaches in the mouse. I find that Ctip2, known for several years as a central functional control over corticospinal motor neuron (CSMN) terminal differentiation and connectivity, is required both cell-autonomously (within CSMN) and non-cell-autonomously (within striatal medium-sized spiny neurons that surround CSMN axons traveling in the internal capsule) for CSMN to achieve proper connectivity with the spinal cord. In addition, I find that Ctip1, a transcription factor not previously functionally investigated in neocortical development, is a novel control over 1) corticothalamic and callosal projection neuron development and projection neuron migration; and 2) postmitotic area identity acquisition and the formation of sensory maps. Taken together, these results reveal previously unknown functions of Ctip1 in neocortical development, and novel sites of action for Ctip2 control over CSMN connectivity. Ctip1 and Ctip2 are transcriptional controls over the postmitotic specification of neocortical projection neuron subtype and area identity, and over projection neuron connectivity with distant targets

SnapShot: Cortical Development

Stem Cell and Regenerative Biolog

Molecular logic of neocortical projection neuron specification, development and diversity

The sophisticated circuitry of the neocortex is assembled from a diverse repertoire of neuronal subtypes generated during development under precise molecular regulation. In recent years, several key controls over the specification and differentiation of neocortical projection neurons have been identified. This work provides substantial insight into the 'molecular logic' underlying cortical development and increasingly supports a model in which individual progenitor-stage and postmitotic regulators are embedded within highly interconnected networks that gate sequential developmental decisions. Here, we provide an integrative account of the molecular controls that direct the progressive development and delineation of subtype and area identity of neocortical projection neurons.Stem Cell and Regenerative Biolog

Ctip1 Controls Acquisition of Sensory Area Identity and Establishment of Sensory Input Fields in the Developing Neocortex

While several transcriptional controls over the size and relative position of cortical areas have been identified, less is known about regulators that direct acquisition of area-specific characteristics. Here, we report that the transcription factor Ctip1 functions in primary sensory areas to repress motor and activate sensory gene expression programs, enabling establishment of sharp molecular boundaries defining functional areas. In Ctip1 mutants, abnormal gene expression leads to aberrantly motorized corticocortical and corticofugal output connectivity. Ctip1 critically regulates differentiation of layer IV neurons, and selective loss of Ctip1 in cortex deprives thalamocortical axons of their receptive “sensory field” in layer IV, which normally provides a tangentially and radially defined compartment of dedicated synaptic territory. Therefore, although thalamocortical axons invade appropriate cortical regions, they are unable to organize into properly configured sensory maps. Together, these data identify Ctip1 as a critical control over sensory area development.Stem Cell and Regenerative Biolog

Ctip1 Regulates the Balance between Specification of Distinct Projection Neuron Subtypes in Deep Cortical Layers

The molecular linkage between neocortical projection neuron subtype and area development, which enables the establishment of functional areas by projection neuron populations appropriate for specific sensory and motor functions, is poorly understood. Here, we report that Ctip1 controls precision of neocortical development by regulating subtype identity in deep-layer projection neurons. Ctip1 is expressed by postmitotic callosal and corticothalamic projection neurons, but is excluded over embryonic development from corticospinal motor neurons, which instead express its close relative, Ctip2. Loss of Ctip1 function results in a striking bias in favor of subcerebral projection neuron development in sensory cortex at the expense of corticothalamic and deep-layer callosal development, while misexpression of Ctip1 in vivo represses subcerebral gene expression and projections. As we report in a paired paper, Ctip1 also controls acquisition of sensory area identity. Therefore, Ctip1 couples subtype and area specification, enabling specific functional areas to organize precise ratios of appropriate output projections.Stem Cell and Regenerative Biolog

Somatic mutation in single human neurons tracks developmental and transcriptional history

Neurons live for decades in a postmitotic state, their genomes susceptible to DNA damage. Here we survey the landscape of somatic single-nucleotide variants (SNVs) in the human brain. We identified thousands of somatic SNVs by single-cell sequencing of 36 neurons from the cerebral cortex of three normal individuals. Unlike germline and cancer SNVs, which are often caused by errors in DNA replication, neuronal mutations appear to reflect damage during active transcription. Somatic mutations create nested lineage trees, allowing them to be dated relative to developmental landmarks and revealing a polyclonal architecture of the human cerebral cortex. Thus, somatic mutations in the brain represent a durable and ongoing record of neuronal life history, from development through postmitotic functionclose

Somatic Mutations Activating the mTOR Pathway in Dorsal Telencephalic Progenitors Cause a Continuum of Cortical Dysplasias

Focal cortical dysplasia (FCD) and hemimegalencephaly (HME) are epileptogenic neurodevelopmental malformations caused by mutations in mTOR pathway genes. Deep sequencing of these genes in FCD/HME brain tissue identified an etiology in 27 of 66 cases (41%). Radiographically indistinguishable lesions are caused by somatic activating mutations in AKT3, MTOR, and PIK3CA and germline loss-of-function mutations in DEPDC5, NPRL2, and TSC1/2, including TSC2 mutations in isolated HME demonstrating a “two-hit” model. Mutations in the same gene cause a disease continuum from FCD to HME to bilateral brain overgrowth, reflecting the progenitor cell and developmental time when the mutation occurred. Single-cell sequencing demonstrated mTOR activation in neurons in all lesions. Conditional Pik3ca activation in the mouse cortex showed that mTOR activation in excitatory neurons and glia, but not interneurons, is sufficient for abnormal cortical overgrowth. These data suggest that mTOR activation in dorsal telencephalic progenitors, in some cases specifically the excitatory neuron lineage, causes cortical dysplasia