William L. McKenna

Tbr1 and Fezf2 Regulate Alternate Corticofugal Neuronal Identities during Neocortical Development

The molecular mechanisms regulating fate divergence of closely related, but distinct, layer 6 corticothalamic and layer 5 subcerebral projection neurons are largely unknown. We present evidence for central transcriptional mechanisms that regulate fate specification of corticothalamic (layer 6) and subcerebral (layer 5) projection neurons. We found that TBR1 promotes the identity of corticothalamic neurons and represses subcerebral fates through reducing expression of Fezf2 and CTIP2. These conclusions are based on the following: (1) In Tbr1−/− mice, the number of cells expressing layer 6 markers was reduced, and the number of cells expressing layer 5 markers was increased. Early-born (birthdated on E11.5) neurons ectopically expressed subcerebral neuronal markers, and extended their axons into subcerebral targets. (2) Ectopic Tbr1 expression in layer 5 neurons prevented them from extending axons into the brainstem and the spinal cord. (3) Chromatin immunoprecipitation analysis using TBR1 antibodies showed that TBR1 bound to a conserved region in the Fezf2 gene. (4) Analysis of Fezf2 mutants and Tbr1−/−; Fezf2−/− compound mutants provided evidence that Fezf2 blocks corticothalamic fate in layer 5 by reducing Tbr1 expression in subcerebral neurons. All neocortical regions appear to use this core transcriptional program to specify corticothalamic (layer 6) and subcerebral (layer 5) projection neurons.

Fezf2 expression identifies a multipotent progenitor for neocortical projection neurons, astrocytes and oligodendrocytes

Progenitor cells in the cerebral cortex sequentially generate distinct classes of projection neurons. Recent work suggests the cortex may contain intrinsically fate-restricted progenitors marked by expression of Cux2. However, the heterogeneity of the neocortical ventricular zone as well as the contribution of lineage-restricted progenitors to the overall cortical neurogenic program remains unclear. Here, we utilize in vivo genetic fate mapping to demonstrate that Fezf2-expressing radial glial cells (RGCs) exist throughout cortical development and sequentially generate all major projection neuron subtypes and glia. Moreover, we show that the vast majority of CUX2⁺ cells in the VZ and SVZ are migrating interneurons derived from the subcortical telencephalon. Examination of the embryonic cortical progenitor population demonstrates that Cux2⁺ RGCs generate both deep- and upper-layer projection neurons. These results identify Fezf2⁺ radial glial cells as a multipotent neocortical progenitor and suggest that the existence, and molecular identity, of laminar-fate-restricted RGCs awaits further investigation.

Protein Interacting with C-Kinase 1/Protein Kinase Cα-Mediated Endocytosis Converts Netrin-1-Mediated Repulsion to Attraction

In vertebrates, the receptor families deleted in colorectal cancer (DCC) and UNC5 mediate responses to the bifunctional guidance cue netrin-1. DCC mediates attraction, whereas a complex of DCC and UNC5 mediates repulsion. Thus, a primary determinant of the responsiveness of an axon to netrin-1 is the presence or absence of UNC5 family members on the cell surface. Currently, little is known about the role of receptor trafficking in regulating neuronal responses to netrin-1. We show that protein interacting with C-kinase 1 (PICK1) recruits activated protein kinase Cα (PKCα) to MycUNC5A at the plasma membrane, stimulating its endocytosis. We identify two PKCα phosphorylation sites at serines 408 and 587, as well as dileucine internalization motifs, which are required for this endocytosis. We find that PKCα-stimulated internalization of UNC5A alters the functional response of developing hippocampal axons to netrin-1, preventing UNC5A-mediated growth cone collapse and converting netrin-1-stimulated chemorepulsion to attraction. To address whether this conversion in axonal response occurs in neurons expressing endogenous levels of UNC5, we show that mouse cerebellar granule axons exhibit chemorepulsion in a netrin-1 gradient and that this chemorepulsion is converted to chemoattraction after PKCα activation. We demonstrate that this repulsion depends on UNC5A because Unc5a−/− axons are not repelled and show this conversion depends on PICK1 because PICK1−/− axons are not converted to chemoattraction after PKCα activation. Together, these data provide a potential mechanism to explain how developing neurons alter their responsiveness to netrin-1 at intermediate choice points as they navigate to their targets.

UNC5A promotes neuronal apoptosis during spinal cord development independent of netrin-1

In addition to their role as chemorepellent netrin-1 receptors, UNC5 proteins may mediate cell death because they induce apoptosis in cultured cells. To test this in vivo, we generated Unc5a (formerly Unc5h1) knockout mice and found that this deletion decreased apoptosis and increased the number of neurons in the spinal cord. In contrast, loss of netrin-1 (Ntn1) did not affect the amount of apoptosis, suggesting that NTN1 is not required for neuronal apoptosis in vivo.

Foxg1 Coordinates the Switch from Nonradially to Radially Migrating Glutamatergic Subtypes in the Neocortex through Spatiotemporal Repression

The specification of neuronal subtypes in the cerebral cortex proceeds in a temporal manner; however, the regulation of the transitions between the sequentially generated subtypes is poorly understood. Here, we report that the forkhead box transcription factor Foxg1 coordinates the production of neocortical projection neurons through the global repression of a default gene program. The delayed activation of Foxg1 was necessary and sufficient to induce deep-layer neurogenesis, followed by a sequential wave of upper-layer neurogenesis. A genome-wide analysis revealed that Foxg1 binds to mammalian-specific noncoding sequences to repress over 12 transcription factors expressed in early progenitors, including Ebf2/3, Dmrt3, Dmrta1, and Eya2. These findings reveal an unexpected prolonged competence of progenitors to initiate corticogenesis at a progressed stage during development and identify Foxg1 as a critical initiator of neocorticogenesis through spatiotemporal repression, a system that balances the production of nonradially and radially migrating glutamatergic subtypes during mammalian cortical expansion.

Fezf1 and Fezf2 Are Required for Olfactory Development and Sensory Neuron Identity

The murine olfactory system consists of main and accessory systems that perform distinct and overlapping functions. The main olfactory epithelium (MOE) is primarily involved in the detection of volatile odorants, while neurons in the vomeronasal organ (VNO), part of the accessory olfactory system, are important for pheromone detection. During development, the MOE and VNO both originate from the olfactory pit; however, the mechanisms regulating development of these anatomically distinct organs from a common olfactory primordium are unknown. Here we report that two closely related zinc‐finger transcription factors, FEZF1 and FEZF2, regulate the identity of MOE sensory neurons and are essential for the survival of VNO neurons respectively. Fezf1 is predominantly expressed in the MOE while Fezf2 expression is restricted to the VNO. In Fezf1‐deficient mice, olfactory neurons fail to mature and also express markers of functional VNO neurons. In Fezf2‐deficient mice, VNO neurons degenerate prior to birth. These results identify Fezf1 and Fezf2 as important regulators of olfactory system development and sensory neuron identity. J. Comp. Neurol. 519:1829–1846, 2011.

Netrin‐1‐independent adenosine A2b receptor activation regulates the response of axons to netrin‐1 by controlling cell surface levels of UNC5A receptors

Growth cone response to the bifunctional guidance cue netrin‐1 is regulated by the activity of intracellular signaling intermediates such as protein kinase C‐alpha (PKCα) and adenylyl cyclase. Among the diverse cellular events these enzymes regulate is receptor trafficking. Netrin‐1, itself, may govern the activity of these signaling intermediates, thereby regulating axonal responses to itself. Alternatively, other ligands, such as activators of G protein‐coupled receptors, may regulate responses to netrin‐1 by governing these signaling intermediates. Here, we investigate the mechanisms controlling activation of PKCα and the subsequent downstream regulation of cell surface UNC5A receptors. We report that activation of adenosine receptors by adenosine analogs, or activation of the putative netrin‐1 receptor, the G protein‐coupled receptor adenosine A2b receptor (A2bR) results in PKCα‐dependent removal of UNC5A from the cell surface. This decrease in cell surface UNC5A reduces the number of growth cones that collapse in response to netrin‐1 and converts repulsion to attraction. We show these A2bR‐mediated alterations in axonal response are not because of netrin‐1 because netrin‐1 neither binds A2bR, as assayed by protein overlay, nor stimulates PKCα‐dependent UNC5A surface loss. Our results demonstrate that netrin‐1‐independent A2bR signaling governs the responsiveness of a neuron to netrin‐1 by regulating the levels of cell surface UNC5A receptor.

TBR1 regulates autism risk genes in the developing neocortex

Exome sequencing studies have identified multiple genes harboring de novo loss-of-function (LoF) variants in individuals with autism spectrum disorders (ASD), including TBR1, a master regulator of cortical development. We performed ChIP-seq for TBR1 during mouse cortical neurogenesis and show that TBR1-bound regions are enriched adjacent to ASD genes. ASD genes were also enriched among genes that are differentially expressed in Tbr1 knockouts, which together with the ChIP-seq data, suggests direct transcriptional regulation. Of the nine ASD genes examined, seven were misexpressed in the cortices of Tbr1 knockout mice, including six with increased expression in the deep cortical layers. ASD genes with adjacent cortical TBR1 ChIP-seq peaks also showed unusually low levels of LoF mutations in a reference human population and among Icelanders. We then leveraged TBR1 binding to identify an appealing subset of candidate ASD genes. Our findings highlight a TBR1-regulated network of ASD genes in the developing neocortex that are relatively intolerant to LoF mutations, indicating that these genes may play critical roles in normal cortical development.

Mutual regulation between Satb2 and Fezf2 promotes subcerebral projection neuron identity in the developing cerebral cortex

Significance Mutations in special AT-rich sequence-binding protein 2 (Satb2) cause severe intellectual deficiency in humans. However, its function in brain development is not completely understood. Our study focuses on the function of Satb2 in specifying cortical projection neuron fates. We find that, although Satb2 activates the expression of some subcerebral neuronal genes, it also inhibits the expression of other genes that are expressed in subcerebral neurons. We report that Satb2 promotes Fezf2 and Sox5 expression in subcerebral neurons, and that Fezf2 in turn inhibits high-level Satb2 expression. We show that the mutual regulation between Satb2 and Fezf2 is essential for Satb2 to promote subcerebral neuron fate. Generation of distinct cortical projection neuron subtypes during development relies in part on repression of alternative neuron identities. It was reported that the special AT-rich sequence-binding protein 2 (Satb2) is required for proper development of callosal neuron identity and represses expression of genes that are essential for subcerebral axon development. Surprisingly, Satb2 has recently been shown to be necessary for subcerebral axon development. Here, we unravel a previously unidentified mechanism underlying this paradox. We show that SATB2 directly activates transcription of forebrain embryonic zinc finger 2 (Fezf2) and SRY-box 5 (Sox5), genes essential for subcerebral neuron development. We find that the mutual regulation between Satb2 and Fezf2 enables Satb2 to promote subcerebral neuron identity in layer 5 neurons, and to repress subcerebral characters in callosal neurons. Thus, Satb2 promotes the development of callosal and subcerebral neurons in a cell context-dependent manner.

Multiple conserved regulatory domains promote Fezf2 expression in the developing cerebral cortex

BackgroundThe genetic programs required for development of the cerebral cortex are under intense investigation. However, non-coding DNA elements that control the expression of developmentally important genes remain poorly defined. Here we investigate the regulation of Fezf2, a transcription factor that is necessary for the generation of deep-layer cortical projection neurons.ResultsUsing a combination of chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) we mapped the binding of four deep-layer-enriched transcription factors previously shown to be important for cortical development. Building upon this we characterized the activity of three regulatory regions around the Fezf2 locus at multiple stages throughout corticogenesis. We identified a promoter that was sufficient for expression in the cerebral cortex, and enhancers that drove reporter gene expression in distinct forebrain domains, including progenitor cells and cortical projection neurons.ConclusionsThese results provide insight into the regulatory logic controlling Fezf2 expression and further the understanding of how multiple non-coding regulatory domains can collaborate to control gene expression in vivo.