FOXP transcription factors are an evolutionarily ancient protein subfamily coordinating the development of several organ systems in the vertebrate body. Association of their genes with neurodevelopmental disorders has sparked particular interest in their expression patterns and functions in the brain. Here, FOXP1, FOXP2, and FOXP4 are expressed in distinct cell type‐specific spatiotemporal patterns in multiple regions, including the cortex, hippocampus, amygdala, basal ganglia, thalamus, and cerebellum. These varied sites and timepoints of expression have complicated efforts to link FOXP1 and FOXP2 mutations to their respective developmental disorders, the former affecting global neural functions and the latter specifically affecting speech and language. However, the use of animal models, particularly those with brain region‐ and cell type‐specific manipulations, has greatly advanced our understanding of how FOXP expression patterns could underlie disorder‐related phenotypes. While many questions remain regarding FOXP expression and function in the brain, studies to date have illuminated the roles of these transcription factors in vertebrate brain development and have greatly informed our understanding of human development and disorders. This article is categorized under: Nervous System Development > Vertebrates: General Principles Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics Nervous System Development > Vertebrates: Regional Development

The molecular mechanisms driving brain development at risk in autism spectrum disorders (ASDs) remain mostly unknown. Previous studies have implicated the transcription factor FOXP1 in both brain development and ASD pathophysiology. However, the specific molecular pathways both upstream of and downstream from FOXP1 are not fully understood. To elucidate the contribution of FOXP1-mediated signaling to brain development and, in particular, neocortical development, we generated forebrain-specific conditional knockout mice. We show that deletion of in the developing forebrain leads to impairments in neonatal vocalizations as well as neocortical cytoarchitectonic alterations via neuronal positioning and migration. Using a genomics approach, we identified the transcriptional networks regulated by Foxp1 in the developing neocortex and found that such networks are enriched for downstream targets involved in neurogenesis and neuronal migration. We also uncovered mechanistic insight into Foxp1 function by demonstrating that sumoylation of Foxp1 during embryonic brain development is necessary for mediating proper interactions between Foxp1 and the NuRD complex. Furthermore, we demonstrated that sumoylation of Foxp1 affects neuronal differentiation and migration in the developing neocortex. Together, these data provide critical mechanistic insights into the function of FOXP1 in the developing neocortex and may reveal molecular pathways at risk in ASD.

Background Mutations in the gene encoding the transcription factor forkhead box P2, FOXP2, result in brain developmental abnormalities including reduced gray matter in both human patients and rodent models, and speech and language deficits. However, neither the region-specific function of FOXP2 in the brain, in particular the cerebellum, nor the effects of any post-translational modifications of FOXP2 in the brain and disorders have been explored. Methods We characterized sumoylation of FOXP2 biochemically, and analyzed the region-specific function and sumoylation of FOXP2 in the developing mouse cerebellum. Using in utero electroporation to manipulate the sumoylation-state of Foxp2 as well as Foxp2 expression levels in Purkinje cells (PCs) of the cerebellum in vivo, we reduced Foxp2 expression approximately 40% in the mouse cerebellum. Such a reduction approximates the haploinsufficiency observed in human patients who demonstrate speech and language impairments. Results We identified sumoylation of FOXP2 at K674 (K673 in mouse) in the cerebellum of neonates. In vitro co-immunoprecipitation and in vivo colocalization experiments suggest that PIAS3 acts as the SUMO E3 ligase for FOXP2 sumoylation. This sumoylation modifies transcriptional regulation by FOXP2. We demonstrate that Foxp2 sumoylation is required for regulation of cerebellar motor function and vocal communication, likely through dendritic outgrowth and arborization of PCs in the mouse cerebellum. Conclusions Sumoylation of Foxp2 in neonatal mouse cerebellum regulates PC development as well as motor functions and vocal communication, demonstrating evidence for sumoylation in regulating mammalian behaviors.

Genetic studies have associated FOXP2 variation with speech and language disorders and other neurodevelopmental disorders (NDDs) involving pathology of the cortex. In this brain region, FoxP2 is expressed from development into adulthood, but little is known about its downstream molecular and behavioral functions. Here, we characterized cortex-specific Foxp2 conditional knockout mice and found a major deficit in reversal learning, a form of behavioral flexibility. In contrast, they showed normal activity levels, anxiety, and vocalizations, save for a slight decrease in neonatal call loudness. These behavioral phenotypes were accompanied by decreased cortical dopamine D1 receptor (D1R) expression at neonatal and adult stages, while general cortical development remained unaffected. Finally, using single-cell transcriptomics, we identified at least five excitatory and three inhibitory D1R-expressing cell types in neonatal frontal cortex, and we found changes in D1R cell type composition and gene expression upon cortical Foxp2 deletion. Strikingly, these alterations included non-cell-autonomous changes in upper layer neurons and interneurons. Together, these data support a role for Foxp2 in the development of dopamine-modulated cortical circuits and behaviors relevant to NDDs.

Disruption of the transcription factor FoxP2, which is enriched in the basal ganglia, impairs vocal development in humans and songbirds. The basal ganglia are important for the selection and sequencing of motor actions, but the circuit mechanisms governing accurate sequencing of learned vocalizations are unknown. Here, we show that expression of FoxP2 in the basal ganglia is vital for the fluent initiation and termination of birdsong, as well as the maintenance of song syllable sequencing in adulthood. Knockdown of FoxP2 imbalances dopamine receptor expression across striatal direct-like and indirect-like pathways, suggesting a role of dopaminergic signaling in regulating vocal motor sequencing. Confirming this prediction, we show that phasic dopamine activation, and not inhibition, during singing drives repetition of song syllables, thus also impairing fluent initiation and termination of birdsong. These findings demonstrate discrete circuit origins for the dysfluent repetition of vocal elements in songbirds, with implications for speech disorders.