Gina E. Elsen

Tbr1 regulates regional and laminar identity of postmitotic neurons in developing neocortex

Areas and layers of the cerebral cortex are specified by genetic programs that are initiated in progenitor cells and then, implemented in postmitotic neurons. Here, we report that Tbr1, a transcription factor expressed in postmitotic projection neurons, exerts positive and negative control over both regional (areal) and laminar identity. Tbr1 null mice exhibited profound defects of frontal cortex and layer 6 differentiation, as indicated by down-regulation of gene-expression markers such as Bcl6 and Cdh9. Conversely, genes that implement caudal cortex and layer 5 identity, such as Bhlhb5 and Fezf2, were up-regulated in Tbr1 mutants. Tbr1 implements frontal identity in part by direct promoter binding and activation of Auts2, a frontal cortex gene implicated in autism. Tbr1 regulates laminar identity in part by downstream activation or maintenance of Sox5, an important transcription factor controlling neuronal migration and corticofugal axon projections. Similar to Sox5 mutants, Tbr1 mutants exhibit ectopic axon projections to the hypothalamus and cerebral peduncle. Together, our findings show that Tbr1 coordinately regulates regional and laminar identity of postmitotic cortical neurons.

The protomap is propagated to cortical plate neurons through an Eomes-dependent intermediate map

The cortical area map is initially patterned by transcription factor (TF) gradients in the neocortical primordium, which define a “protomap” in the embryonic ventricular zone (VZ). However, mechanisms that propagate regional identity from VZ progenitors to cortical plate (CP) neurons are unknown. Here we show that the VZ, subventricular zone (SVZ), and CP contain distinct molecular maps of regional identity, reflecting different gene expression gradients in radial glia progenitors, intermediate progenitors, and projection neurons, respectively. The “intermediate map” in the SVZ is modulated by Eomes (also known as Tbr2), a T-box TF. Eomes inactivation caused rostrocaudal shifts in SVZ and CP gene expression, with loss of corticospinal axons and gain of corticotectal projections. These findings suggest that cortical areas and connections are shaped by sequential maps of regional identity, propagated by the Pax6 → Eomes → Tbr1 TF cascade. In humans, PAX6, EOMES, and TBR1 have been linked to intellectual disability and autism.

Tbr2 Expression in Cajal-Retzius Cells and Intermediate Neuronal Progenitors Is Required for Morphogenesis of the Dentate Gyrus

The dentate gyrus (DG) is a unique cortical region whose protracted development spans the embryonic and early postnatal periods. DG development involves large-scale reorganization of progenitor cell populations, ultimately leading to the establishment of the subgranular zone neurogenic niche. In the developing DG, the T-box transcription factor Tbr2 is expressed in both Cajal-Retzius cells derived from the cortical hem that guide migration of progenitors and neurons to the DG, and intermediate neuronal progenitors born in the dentate neuroepithelium that give rise to granule neurons. Here we show that in mice Tbr2 is required for proper migration of Cajal-Retzius cells to the DG; and, in the absence of Tbr2, formation of the hippocampal fissure is abnormal, leading to aberrant development of the transhilar radial glial scaffold and impaired migration of progenitors and neuroblasts to the developing DG. Furthermore, loss of Tbr2 results in decreased expression of Cxcr4 in migrating cells, leading to a premature burst of granule neurogenesis during early embryonic development accompanied by increased cell death in mutant animals. Formation of the transient subpial neurogenic zone was abnormal in Tbr2 conditional knock-outs, and the stem cell population in the DG was depleted before proper establishment of the subgranular zone. These studies indicate that Tbr2 is explicitly required for morphogenesis of the DG and participates in multiple aspects of the intricate developmental process of this structure.

Intermediate Progenitor Cohorts Differentially Generate Cortical Layers and Require Tbr2 for Timely Acquisition of Neuronal Subtype Identity

Intermediate progenitors (IPs) amplify the production of pyramidal neurons, but their role in selective genesis of cortical layers or neuronal subtypes remains unclear. Using genetic lineage tracing in mice, we find that IPs destined to produce upper cortical layers first appear early in corticogenesis, by embryonic day 11.5. During later corticogenesis, IP laminar fates are progressively limited to upper layers. We examined the role of Tbr2, an IP-specific transcription factor, in laminar fate regulation using Tbr2 conditional mutant mice. Upon Tbr2 inactivation, fewer neurons were produced by immediate differentiation and laminar fates were shifted upward. Genesis of subventricular mitoses was, however, not reduced in the context of a Tbr2-null cortex. Instead, neuronal and laminar differentiation were disrupted and delayed. Our findings indicate that upper-layer genesis depends on IPs from many stages of corticogenesis and that Tbr2 regulates the tempo of laminar fate implementation for all cortical layers.

Neurog2 Simultaneously Activates and Represses Alternative Gene Expression Programs in the Developing Neocortex

Progenitor cells undergo a series of stable identity transitions on their way to becoming fully differentiated cells with unique identities. Each cellular transition requires that new sets of genes are expressed, while alternative genetic programs are concurrently repressed. Here, we investigated how the proneural gene Neurog2 simultaneously activates and represses alternative gene expression programs in the developing neocortex. By comparing the activities of transcriptional activator (Neurog2-VP16) and repressor (Neurog2-EnR) fusions to wild-type Neurog2, we first demonstrate that Neurog2 functions as an activator to both extinguish Pax6 expression in radial glial cells and initiate Tbr2 expression in intermediate neuronal progenitors. Similarly, we show that Neurog2 functions as an activator to promote the differentiation of neurons with a dorsal telencephalic (i.e., neocortical) identity and to block a ventral fate, identifying 2 Neurog2-regulated transcriptional programs involved in the latter. First, we show that the Neurog2-transcriptional target Tbr2 is a direct transcriptional repressor of the ventral gene Ebf1. Secondly, we demonstrate that Neurog2 indirectly turns off Etv1 expression, which in turn indirectly regulates the expression of the ventral proneural gene Ascl1. Neurog2 thus activates several genetic off-switches, each with distinct transcriptional targets, revealing an unappreciated level of specificity for how Neurog2 prevents inappropriate gene expression during neocortical development.

Stable Respiratory Activity Requires Both P/Q-Type and N-Type Voltage-Gated Calcium Channels

P/Q-type voltage-gated calcium channels (Cav2.1) play critical presynaptic and postsynaptic roles throughout the nervous system and have been implicated in a variety of neurological disorders. Here we report that mice with a genetic ablation of the Cav2.1 pore-forming α1A subunit (α1A−/−) encoded by CACNA1a (Jun et al., 1999) suffer during postnatal development from increasing breathing disturbances that lead ultimately to death. Breathing abnormalities include decreased minute ventilation and a specific loss of sighs, which was associated with lung atelectasis. Similar respiratory alterations were preserved in the isolated in vitro brainstem slice preparation containing the pre-Bötzinger complex. The loss of Cav2.1 was associated with an alteration in the functional dependency on N-type calcium channels (Cav2.2). Blocking N-type calcium channels with conotoxin GVIA had only minor effects on respiratory activity in slices from control (CT) littermates, but abolished respiratory activity in all slices from α1A−/− mice. The amplitude of evoked EPSPs was smaller in inspiratory neurons from α1A−/− mice compared with CTs. Conotoxin GVIA abolished all EPSPs in inspiratory neurons from α1A−/− mice, while the EPSP amplitude was reduced by only 30% in CT mice. Moreover, neuromodulation was significantly altered as muscarine abolished respiratory network activity in α1A−/− mice but not in CT mice. We conclude that excitatory synaptic transmission dependent on N-type and P/Q-type calcium channels is required for stable breathing and sighing. In the absence of P/Q-type calcium channels, breathing, sighing, and neuromodulation are severely compromised, leading to early mortality.

Conditional ablation of Tbr2 results in abnormal development of the olfactory bulbs and subventricular zone-rostral migratory stream

Development of the olfactory bulb (OB) is a complex process that requires contributions from several progenitor cell niches to generate neuronal diversity. Previous studies showed that Tbr2 is expressed during the generation of glutamatergic OB neurons in rodents. However, relatively little is known about the role of Tbr2 in the developing OB or in the subventricular zone‐rostral migratory stream (SVZ‐RMS) germinal niche that gives rise to many OB neurons. Results: Here, we use conditional gene ablation strategies to knockout Tbr2 during embryonic mouse olfactory bulb morphogenesis, as well as during perinatal and adult neurogenesis from the SVZ‐RMS niche, and describe the resulting phenotypes. We find that Tbr2 is important for the generation of mitral cells in the OB, and that the olfactory bulbs themselves are hypoplastic and disorganized in Tbr2 mutant mice. Furthermore, we show that the SVZ‐RMS niche is expanded and disordered following loss of Tbr2, which leads to ectopic accumulation of neuroblasts in the RMS. Lastly, we show that adult glutamatergic neurogenesis from the SVZ is impaired by loss of Tbr2. Conclusions: Tbr2 is essential for proper morphogenesis of the OB and SVZ‐RMS, and is important for the generation of multiple lineages of glutamatergic olfactory bulb neurons. Developmental Dynamics 243:440–450, 2014.

The Epigenetic Factor Landscape of Developing Neocortex Is Regulated by Transcription Factors Pax6→ Tbr2→ Tbr1

Epigenetic factors (EFs) regulate multiple aspects of cerebral cortex development, including proliferation, differentiation, laminar fate, and regional identity. The same neurodevelopmental processes are also regulated by transcription factors (TFs), notably the Pax6→ Tbr2→ Tbr1 cascade expressed sequentially in radial glial progenitors (RGPs), intermediate progenitors, and postmitotic projection neurons, respectively. Here, we studied the EF landscape and its regulation in embryonic mouse neocortex. Microarray and in situ hybridization assays revealed that many EF genes are expressed in specific cortical cell types, such as intermediate progenitors, or in rostrocaudal gradients. Furthermore, many EF genes are directly bound and transcriptionally regulated by Pax6, Tbr2, or Tbr1, as determined by chromatin immunoprecipitation-sequencing and gene expression analysis of TF mutant cortices. Our analysis demonstrated that Pax6, Tbr2, and Tbr1 form a direct feedforward genetic cascade, with direct feedback repression. Results also revealed that each TF regulates multiple EF genes that control DNA methylation, histone marks, chromatin remodeling, and non-coding RNA. For example, Tbr1 activates Rybp and Auts2 to promote the formation of non-canonical Polycomb repressive complex 1 (PRC1). Also, Pax6, Tbr2, and Tbr1 collectively drive massive changes in the subunit isoform composition of BAF chromatin remodeling complexes during differentiation: for example, a novel switch from Bcl7c (Baf40c) to Bcl7a (Baf40a), the latter directly activated by Tbr2. Of 11 subunits predominantly in neuronal BAF, 7 were transcriptionally activated by Pax6, Tbr2, or Tbr1. Using EFs, Pax6→ Tbr2→ Tbr1 effect persistent changes of gene expression in cell lineages, to propagate features such as regional and laminar identity from progenitors to neurons.

Transcriptional control of corticothalamic projection neuron identity by TBr1

individuals ranging in age from 6weeks to 49years (n = 68). Additionally we measured expression of doublecortin mRNA in the DLPFC of patients with schizophrenia (n = 37) and controls (n = 37). We found a striking decline (54% in mRNA, 76% in protein) in doublecortin expression in the first year of life between neonates (<3 months) and infants (3months-1year). Doublecortin expression continued to decrease in toddler/school aged individuals then plateaued. Doublecortin expression was detectable in adults at levels ∼7% of neonates. We saw no change in doublecortin mRNA expression in the DLPFC of patients with schizophrenia. Protracted expression of doublecortin in the first years of life is consistent with the hypothesis that new neurons arrive in the cortex in postnatal life. Expression of doublecortin is maintained in adulthood which may indicate continued arrival of neurons or a further constitutive function of doublecortin. The lack of change in doublecortin in patients with schizophrenia suggests that levels of adult neuronal migration may be similar in patients compared to controls; however, further work is needed to determine if other aspects of postnatal neurogenesis are intact.