Transcriptional repression by FEZF2 restricts alternative identities of cortical projection neurons.

Projection neuron subtype identities in the cerebral cortex are established by expressing pan-cortical and subtype-specific effector genes that execute terminal differentiation programs bestowing 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, we show that FEZF2 functions as a transcriptional repressor, and it regulates subtype-specific identities of both corticothalamic and subcerebral neurons by selectively repressing expression of genes inappropriate for each neuronal subtype. We report that TLE4, specifically expressed in layer 6 corticothalamic neurons, is recruited by FEZF2 to inhibit layer 5 subcerebral neuronal genes. Together with previous studies, our results indicate that a cortical glutamatergic identity is specified by multiple parallel pathways active in progenitor cells, whereas projection neuron subtype-specific identity is achieved through selectively repressing genes associated with alternate identities in differentiating neurons.

Neonatal Tbr1 Dosage Controls Cortical Layer 6 Connectivity.

An understanding of how heterozygous loss-of-function mutations in autism spectrum disorder (ASD) risk genes, such as TBR1, contribute to ASD remains elusive. Conditional Tbr1 deletion during late mouse gestation in cortical layer 6 neurons (Tbr1layer6 mutants) provides novel insights into its function, including dendritic patterning, synaptogenesis, and cell-intrinsic physiology. These phenotypes occur in heterozygotes, providing insights into mechanisms that may underlie ASD pathophysiology. Restoring expression of Wnt7b largely rescues the synaptic deficit in Tbr1layer6 mutant neurons. Furthermore, Tbr1layer6 heterozygotes have increased anxiety-like behavior, a phenotype seen ASD. Integrating TBR1 chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) data from layer 6 neurons and activity of TBR1-bound candidate enhancers provides evidence for how TBR1 regulates layer 6 properties. Moreover, several putative TBR1 targets are ASD risk genes, placing TBR1 in a central position both for ASD risk and for regulating transcriptional circuits that control multiple steps in layer 6 development essential for the assembly of neural circuits.

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.