Person:

Fame, Ryan

The mammalian neocortex is an exquisite, highly organized brain structure composed of hundreds of subpopulations of neurons and glia, precisely connected to enable motor control, sensory perception, information integration, and planning. Unique molecular, structural, and anatomical neuronal properties underlie diverse functionality, endowing much of the neocortex’s complex processing power. Neocortical size correlates with information processing capacity, suggesting that increased neuronal number and diversity begets increased sophistication. One excitatory projection neuron type, callosal projection neurons (CPN), has disproportionately expanded with cortical size increase. CPN directly connect homotypic regions of the two neocortical hemispheres by sending axons via the largest white matter fiber tract in the brain, the corpus callosum (CC), allowing quick relay, integration, and comparison of information. In humans, the CC contains over 300,000 axons, CPN have been centrally implicated in autism spectrum disorders, and absence or surgical disruption of CPN connectivity in humans is associated with defects in abstract reasoning, problem solving, and generalization. Therefore, CPN are critical to complex brain functions, and their diversity likely contributes to these roles. Work presented in this dissertation addresses molecular controls over CPN development, specifically genes that are expressed by, and function in, particular subpopulations of CPN. While much progress has been made in identifying molecular controls over neocortical arealization, lamination, and broad subtype specification, CPN diversity has remained largely unaddressed. Therefore, this work begins by identifying genes more highly expressed in CPN than other closely related projection neuron populations, and uncovers molecular diversity within CPN. From this molecular diversity, functional analysis of three candidate molecular controls over CPN subtype diversity follows. Cited2 acts broadly in neocortical progenitor development and postnatally in refining somatosensory CPN identity. Caveolin1 identifies a population of CPN with dual axonal projections. Tmtc4 is mutated in human CC disease and can function in CPN axonal development. These analyses of CPN molecular diversity in mouse then expand to an investigation of which molecular subpopulations are conserved, expanded, or uncommon between rodent and primate, allowing both for comparative evolutionary theories of CPN function, and indicating which CPN populations critical for human brain function can be best studied in rodent models.

The neocortex contains hundreds to thousands of distinct subtypes of precisely connected neurons, allowing it to perform remarkably complex tasks of high-level cognition. Callosal projection neurons (CPN) connect the cerebral hemispheres via the corpus callosum, integrating cortical information and playing key roles in associative cognition. CPN are a strikingly diverse set of neuronal subpopulations, and development of this diversity requires precise control by a complex, interactive set of molecular effectors. We have found that the transcriptional coregulator Cited2 regulates and refines two stages of CPN development. Cited2 is expressed broadly by progenitors in the embryonic day 15.5 subventricular zone, during the peak of superficial layer CPN birth, with a progressive postmitotic refinement in expression, becoming restricted to CPN of the somatosensory cortex postnatally. We generated progenitor-stage and postmitotic forebrain-specific Cited2 conditional knock-out mice, using the Emx1-Cre and NEX-Cre mouse lines, respectively. We demonstrate that Cited2 functions in progenitors, but is not necessary postmitotically, to regulate both (1) broad generation of layer II/III CPN and (2) acquisition of precise area-specific molecular identity and axonal/dendritic connectivity of somatosensory CPN. This novel CPN subtype-specific and area-specific control from progenitor action of Cited2 adds yet another layer of complexity to the multistage developmental regulation of neocortical development.

Little is known about the molecular development and heterogeneity of callosal projection neurons (CPN), cortical commissural neurons that connect homotopic regions of the two cerebral hemispheres via the corpus callosum and that are critical for bilateral integration of cortical information. Here we report on the identification of a series of genes that individually and in combination define CPN and novel CPN subpopulations during embryonic and postnatal development. We used in situ hybridization analysis, immunocytochemistry, and retrograde labeling to define the layer-specific and neuron-type-specific distribution of these newly identified CPN genes across different stages of maturation. We demonstrate that a subset of these genes (e.g., Hspb3 and Lpl) appear specific to all CPN (in layers II/III and V–VI), whereas others (e.g., Nectin-3, Plexin-D1, and Dkk3) discriminate between CPN of the deep layers and those of the upper layers. Furthermore, the data show that several genes finely subdivide CPN within individual layers and appear to label CPN subpopulations that have not been described previously using anatomical or morphological criteria. The genes identified here likely reflect the existence of distinct programs of gene expression governing the development, maturation, and function of the newly identified subpopulations of CPN. Together, these data define the first set of genes that identify and molecularly subcategorize distinct populations of callosal projection neurons, often located in distinct subdivisions of the canonical cortical laminae.