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Projection neurons underly cortical computation and are necessary for proper cortical function. Understanding how these cell types arise is a central question in neuroscience and has wide-ranging implications for treating brain diseases. Genes encoding for transcription factors or chromatin remodeling proteins have been identified that are necessary for generating projection neuron cell types in the cerebral cortex. Exactly how they function is unknown. Using genetically engineered mouse models combined with immunohistochemistry, bulk and single-cell RNA sequencing, chromatin profiling, circuit tracing, and electrophysiology, this work uncovers the molecular mechanisms by which the transcription factors Fezf2, Satb2, and the transcriptional corepressor Tle4, specify cell fate in the developing cerebral cortex. Fezf2 specifies cell fate by functioning as a selective repressor. It regulates subtype-specific identities of corticothalamic and subcerebral neurons by selectively repressing the expression of genes inappropriate for each neuronal subtype. Tle4 works collaboratively with Fezf2 in layer 6 to inhibit layer 5 subcerebral neuronal genes. On the other hand, Satb2 is involved in gene activation and repression; these functions are likely mediated by interactions with ATP-dependent chromatin remodeling complexes. This work also demonstrates that Satb2 dosage affects cortical development, shedding light on the etiology of patients with a mutant SATB2 allele.Fezf2, Tle4, and Satb2 all function to specify subtype identity in postmitotic neurons. These results indicate that a cortical glutamatergic identity is specified by progenitor cells, but subtype-specific identity is achieved through postmitotic fate refinement.

Neural stem cells (NSCs) in the embryonic neocortex sequentially generate different subtypes of glutamatergic projection neurons. Following that, NSCs undergo a major switch in their progenitor properties and produce g-aminobutyric acid (GABAergic) interneurons for the olfactory bulb (OB), cortical oligodendrocytes, and astrocytes. Herein, I provide evidence for the molecular mechanism that underlies this switch in the neocortical NSCs. At around E16.5, mouse neocortical NSCs start to generate Gsx2-expressing (Gsx2+) intermediate progenitor cells (IPCs). In vivo lineage tracing study revealed that Gsx2+ IPC population gives rise not only to OB interneurons (OB-INs) but also to cortical oligodendrocytes and astrocytes, suggesting that they are a multipotent population. I found that Sonic Hedgehog signaling is both necessary and sufficient for the generation of Gsx2+ IPCs by reducing Gli3R protein levels. Using single-cell RNA sequencing, I identified the transcriptional profile of Gsx2+ IPCs and the process of the lineage switch of cortical NSCs. The Gli3 ChIP-seq revealed 4 binding sites in Olig2/Olig1 locus and two of them are also bound by Pax6 shown by the Pax6 ChIP-seq data. Chromatin at these 4 binding sites show increased accessibility in the ATAC-seq assay. CUT&RUN analysis using histone marks indicates that they are potential enhancers. I generated the enhancer knock-out mice and found that these binding sites are essential for Olig2/Olig1 expression in NSCs and IPCs. The circularized chromosome conformation capture (4C) experiments revealed physical interactions between these binding sites and Olig2/Olig1 promoters. More importantly, I found that OB lineage cells and Olig2 positive cells are dramatically increased in the hGFAP-Cre; Gli3F/F; Pax6F/F (Gli3 Pax6 dcko) which indicates that Gli3 and Pax6 are involved in a common regulatory mechanism that regulate NSCs lineage switch.

The precise generation of distinct subtypes of neocortical projection neurons and their proper migration into appropriate laminar positions underlies our most advanced cognitive and perceptual abilities. Recent work suggests that a transcription factor network, including the genes Sox5, Satb2, Fezf2, and Bcl11b, is responsible for directing the fate specification of subcerebral projection neurons (SCPN) in layer Vb of the cerebral cortex, which is immediately adjacent to laver Va, which is comprised of callosal projection neurons (CPN). Because we observed the absence of SCPN in Sox5 null cortices, we hypothesized that Sox5 may specify subcerebral versus callosal neuronal fates in layer V of the cortex. Here, via a series of in situ hybridization and immunohistochemistry, and retrograde tracing experiments, we show that in Sox5 null cortices, layer V neurons do not take on the molecular identity of layer Vb SCPN and instead, they appear to take on the identity of layer Va CPN. I also demonstrate via birthdating experiments that there is a migration defect Sox5 null cortices, and layer V cells are generated prematurely. Overall, the work presented here provides an in-depth phenotypical characterization of layer V in Sox5 null cortices.

Projection neuron subtype identities in the cerebral cortex are established through the expression of pan-cortical and subtype-specific effector genes, which execute terminal differentiation programs that bestow neurons with a glutamatergic neuron phenotype and subtype-specific morphology, physiology, and axonal projections. Whether pan-cortical glutamatergic and subtype-specific characteristics are regulated by the same genes or controlled by distinct programs remains largely unknown. Here, I show that the transcriptional corepressor, TLE4, is expressed specifically in postmitotic corticothalamic projection neurons, where it functions to regulate the molecular, dendritic, and electrophysiological characteristics unique to corticothalamic neurons. I also demonstrate that TLE4 directly interacts with the forebrain embryonic zinc finger protein, FEZF2, within corticothalamic projection neurons to facilitate the transcriptional repression of subcerebral projection neuron identity. Through the utilization of our novel Fezf2-Bac-EnR transgenic mouse line, I was then able rescue the molecular defects of corticothalamic neurons in the cortex of Tle4 knockout mice and restore the dendritic and electrophysiological characteristics of theses neurons. Overall, the work presented provides an in-depth investigation into the transcriptional regulation of subtype-specific cortical projection neuron identity by Tle4 and Fezf2, thereby contributing novel insight into our current understanding of mammalian cortical development.

Radial glial cells (RGCs) are multipotent progenitors that give rise to excitatory projection neurons, OB interneurons, oligodendrocytes and astrocytes in a temporarily and spatially regulated manner. The transcription factor FEZF2 is necessary for cortical deep layer neuron specification in postmitotic neurons. However, the function of FEZF2 in regulating proliferation and differentiation of RGCs during cortical development and into adulthood is poorly understood. Temporally regulated lineage tracing of RGC clones in the Fezf2 KO and WT shows that an absence of FEZF2 did not substantially impact the generation of glia. Similarly, when Fezf2 is conditionally knocked out in RGCs at E13.5, neither glia nor OB interneuron generation is affected. Despite Fezf2’s negligent influence on differentiation, our quit fraction and intermediate progenitor analysis of the cKO in the V/SVZ suggests that FEZF2 reduces the proliferative lifespan of RGCs and leads to preferential differentiation of RGCs into neural intermediate progenitors.

The highly complex and specialized functions of the mammalian cortex rely on precise temporal development and organization of the diverse neuronal cell types found within by multiple cellular processes. A complete understanding of the molecular mechanisms regulating these events remains elusive. Given that many genes and cellular processes are conserved from mouse to human, I utilize the mammalian cerebral cortex in Mus musculus, as a model system for studying cortical development. Here, I present results from a detailed characterization of the transcription factor Myocyte Enhancing Factor 2C (Mef2c) during postnatal mouse cortical development. Mef2c, considered an upper layer gene, is expressed in all 6 layers of the cerebral cortex including a subpopulation of corticofugal projection neurons. Given its expression pattern in the cortex and previously identified functions in myocyte and CNS development, we hypothesized that Mef2c was a key regulator of neocortical development. Cell type specific and temporal expression analysis revealed that MEF2C is expressed in subcerebral projection neurons throughout corticogenesis. Using a conditional loss-of-function approach, we compared cortical development of control (Mef2ccontrol) and conditional knock out (Mef2ccKO) mice to identify the role of Mef2c. Results revealed that MEF2C expression pattern is similar to that of SATB2 and is in part positively regulated by Satb2. We showed that MEF2C is expressed in subcerebral neurons and is partially required to promote and maintain the identity of these neurons into early postnatal ages. Furthermore, we showed that MEF2C is required for axonal outgrowth of subcerebral projection neurons, such that Mef2ccKO brains displayed a significant loss of axon projections to subcerebral targets compared to Mef2ccontrol. Additionally, callosal neuron distribution analysis suggested that MEF2C acts to inhibit a callosal cell identity in deep layer neurons. Together, these data indicate the b

Transcriptional regulation at multiple steps of cortical development in Mus musculusByJennifer A. Betancourt The highly specialized functions of the mammalian cerebral cortex rely on the temporally precise development and organization of diverse cell types by various cellular processes. However, a complete understanding of the molecular mechanisms that regulate these events is still lacking. Here, I implement Mus musculus as a model system for studying neocortical development, given that many processes and genes involved in brain development are conserved from mouse to human. In this thesis, I primarily present results from a detailed characterization of transcription factor, Nuclear Factor One B (NfiB), during mouse neocortical development. Additionally I have included a short discussion regarding my contributions toward understanding how another transcription factor, Fezf2, functions as a key fate determinant of subcerebral neuron identity. NFIB is expressed in the cortical proliferative zone and in a subpopulation of cortical projection neurons. Given its expression pattern within the cerebral cortex and previously identified functions in CNS development, we hypothesized that NfiB was a key regulator of neocortical development. Cell–type specific and temporal expression analysis revealed that NFIB is primarily expressed in the radial glial subclass of neural stem/progenitors and in corticofugal projection neurons throughout corticogenesis. Using a loss of function approach, we compared neocortical development of wildtype (NfiB&plus/&plus) and NfiB–deficient (NfiB&minus/&minus) mice to identify the role of NfiB. Results revealed that NfiB is required for proper generation of outer radial glia and basal progenitors during late corticogenesis. Loss of these neural progenitor populations led to defects in late-stage neurogenesis and cortical lamination. Specifically, there was an increase in corticofugal–like neurons generated after the expect

Understanding how neuronal diversity is achieved within the cerebral cortex remains a major challenge in neuroscience. The surge of human embryonic stem cells (hESCs) as a novel model system provides a unique opportunity to study human corticogenesis in vitro and€ identify the mechanisms that promote neuronal differentiation to achieve neuronal diversity in the human brain. Here, we demonstrate the derivation of corticofugal neurons from a genetically engineered Fezf2-YFP hESC reporter line, and uncover two distinct Fezf2 subpopulations that are reminiscent of the 2 Fezf2-expressing neuronal subtypes in the developing mouse brain. Fezf2 is a transcription factor that is both necessary and sufficient for the specification of subcerebral projection neurons in mouse. Its role in human corticogenesis is still unknown. However, the high conservation of FEZF2 protein between mouse and human suggest that Fezf2 is a specific marker of human cerebral neurons as well. Two hypotheses drove our research: 1) Extrinsic factors that modulate in vivo developmental signaling pathways play a critical role in the differentiation of hESCs to a corticofugal fate in vitro; 2) Human embryonic stem cell-derived neurons can survive in vivo and extend axonal projections to specific targets in the rodent brain. Our research shows that hESCs-derived corticofugal neurons are an effective model system to investigate the molecular pathways that regulate human cortical differentiation, axon extension and survival, an endeavor that was until now proscribed due to the manipulation of human embryos.

The profound diversity of cell types within the nervous system has been appreciated for greater than a century. However, the mechanisms by which these different cell types are generated during development remain elusive. Here I focus on the roles of two closely related zinc-finger transcription factors, Fezf1 and Fezf2, during development of the olfactory system and cerebral cortex. I show that Fezf1 functions to promote the development and cell fate specification of sensory neurons within the main olfactory epithelium. Conversely, Fezf2 is required for developmental maintenance of the vomeronasal organ. Further, I show that during development of the cerebral cortex, Fezf2 expression identifies a multipotent progenitor for neocortical projection neurons and glia. Finally, I demonstrate that transcription of Fezf2 is regulated by multiple non-coding DNA elements that promote its expression across distinct domains of the cerebral cortex. Collectively, these studies illustrate the critical functions of Fez family transcription factors during development of the mammalian nervous system.

Dye Sensitized solar cells (DSSCs) are a promising candidate for next generation photovoltaic panels due to their low cost, easy fabrication process, and relative high efficiency. Despite considerable effort on the advancement of DSSCs, the efficiency has been stalled for nearly a decade due to the complex interplay among various DSSC components. DSSCs consist of a photoanode on a conducting substrate, infiltrated dye for light absorption and electron injection, and an electrolyte to regenerate the dye. On the photoanode is a high band-gap semiconducting material, primarily of a nanostructure morphology of titanium (II) dioxide (TiO2), dye molecules whose molar absorption is typically in the visible spectrum, are adsorbed onto the surface of TiO2. To improve the current DSSCs, there are many parameters that can be investigated.In a conventional DSSC, a thick semiconducting layer such as the nanoparticle TiO2 layer induces charge separation efficiently while concurrently increasing the charge transport distance, leading the cell to suffer from more charge recombination and deterioration in charge collection efficiency. To improve on this limitation, TiO2 nanowires (NW) and nanotubes (NT) are explored to replace the nanoparticle photoanode. One-dimensional nanostructures are known for the excellent electron transport properties as well as maintaining a relatively high surface area. Hence one of the focuses of this thesis explores at using different morphologies and composition of TiO2 nanostructures to enhance electron collection efficiency.Another challenge in conventional DSSCs is the limit in light absorption of solar irradiation. Dyes are limited to absorption only in the visible range, and have a low molar absorption coefficient in the near infrared (NIR). Tuning dyes is extremely complicated and may have more disadvantages than simply by extending light harvesting. Therefore our strategy is to incorporate quantum dots to replace the dye, as well as prepare a cel