Cortical activity emerges in region-specific patterns during early brain development.

The development of precise neural circuits in the brain requires spontaneous patterns of neural activity prior to functional maturation. In the rodent cerebral cortex, patchwork and wave patterns of activity develop in somatosensory and visual regions, respectively, and are present at birth. However, whether such activity patterns occur in noneutherian mammals, as well as when and how they arise during development, remain open questions relevant for understanding brain formation in health and disease. Since the onset of patterned cortical activity is challenging to study prenatally in eutherians, here we offer an approach in a minimally invasive manner using marsupial dunnarts, whose cortex forms postnatally. We discovered similar patchwork and travelling waves in the dunnart somatosensory and visual cortices at stage 27 (equivalent to newborn mice) and examined earlier stages of development to determine the onset of these patterns and how they first emerge. We observed that these patterns of activity emerge in a region-specific and sequential manner, becoming evident as early as stage 24 in somatosensory and stage 25 in visual cortices (equivalent to embryonic day 16 and 17, respectively, in mice), as cortical layers establish and thalamic axons innervate the cortex. In addition to sculpting synaptic connections of existing circuits, evolutionarily conserved patterns of neural activity could therefore help regulate other early events in cortical development.

Divergent evolution of developmental timing in the neocortex revealed by marsupial and eutherian transcriptomes.

Only mammals evolved a neocortex, which integrates sensory-motor and cognitive functions. Significant diversifications in the cellular composition and connectivity of the neocortex occurred between the two main therian groups: marsupials and eutherians. However, the developmental mechanisms underlying these diversifications are largely unknown. Here, we compared the neocortical transcriptomes of Sminthopsis crassicaudata, a mouse-sized marsupial, with those of eutherian mice at two developmentally equivalent time points corresponding to deeper and upper layer neuron generation. Enrichment analyses revealed more mature gene networks in marsupials at the early stage, which reverted at the later stage, suggesting a more precocious but protracted neuronal maturation program relative to birth timing of cortical layers. We ranked genes expressed in different species and identified important differences in gene expression rankings between species. For example, genes known to be enriched in upper-layer cortical projection neuron subtypes, such as Cux1, Lhx2 and Satb2, likely relate to corpus callosum emergence in eutherians. These results show molecular heterochronies of neocortical development in Theria, and highlight changes in gene expression and cell type composition that may underlie neocortical evolution and diversification. This article has an associated 'The people behind the papers' interview.

Loss-of-function variants in ZEB1 cause dominant anomalies of the corpus callosum with favourable cognitive prognosis.

BACKGROUND: The neurodevelopmental prognosis of anomalies of the corpus callosum (ACC), one of the most frequent brain malformations, varies extremely, ranging from normal development to profound intellectual disability (ID). Numerous genes are known to cause syndromic ACC with ID, whereas the genetics of ACC without ID remains poorly deciphered. METHODS: Through a collaborative work, we describe here ZEB1, a gene previously involved in an ophthalmological condition called type 3 posterior polymorphous corneal dystrophy, as a new dominant gene of ACC. We report a series of nine individuals with ACC (including three fetuses terminated due to ACC) carrying a ZEB1 heterozygous loss-of-function (LoF) variant, identified by exome sequencing. RESULTS: In five cases, the variant was inherited from a parent with a normal corpus callosum, which illustrates the incomplete penetrance of ACC in individuals with an LoF in ZEB1. All patients reported normal schooling and none of them had ID. Neuropsychological assessment in six patients showed either normal functioning or heterogeneous cognition. Moreover, two patients had a bicornuate uterus, three had a cardiovascular anomaly and four had macrocephaly at birth, which suggests a larger spectrum of malformations related to ZEB1. CONCLUSION: This study shows ZEB1 LoF variants cause dominantly inherited ACC without ID and extends the extraocular phenotype related to this gene.

A diffusion MRI-based spatiotemporal continuum of the embryonic mouse brain for probing gene-neuroanatomy connections.

The embryonic mouse brain undergoes drastic changes in establishing basic anatomical compartments and laying out major axonal connections of the developing brain. Correlating anatomical changes with gene-expression patterns is an essential step toward understanding the mechanisms regulating brain development. Traditionally, this is done in a cross-sectional manner, but the dynamic nature of development calls for probing gene-neuroanatomy interactions in a combined spatiotemporal domain. Here, we present a four-dimensional (4D) spatiotemporal continuum of the embryonic mouse brain from E10.5 to E15.5 reconstructed from diffusion magnetic resonance microscopy (dMRM) data. This study achieved unprecedented high-definition dMRM at 30- to 35-µm isotropic resolution, and together with computational neuroanatomy techniques, we revealed both morphological and microscopic changes in the developing brain. We transformed selected gene-expression data to this continuum and correlated them with the dMRM-based neuroanatomical changes in embryonic brains. Within the continuum, we identified distinct developmental modes comprising regional clusters that shared developmental trajectories and similar gene-expression profiles. Our results demonstrate how this 4D continuum can be used to examine spatiotemporal gene-neuroanatomical interactions by connecting upstream genetic events with anatomical changes that emerge later in development. This approach would be useful for large-scale analysis of the cooperative roles of key genes in shaping the developing brain.

Functional and clinical studies reveal pathophysiological complexity of CLCN4-related neurodevelopmental condition.

Missense and truncating variants in the X-chromosome-linked CLCN4 gene, resulting in reduced or complete loss-of-function (LOF) of the encoded chloride/proton exchanger ClC-4, were recently demonstrated to cause a neurocognitive phenotype in both males and females. Through international clinical matchmaking and interrogation of public variant databases we assembled a database of 90 rare CLCN4 missense variants in 90 families: 41 unique and 18 recurrent variants in 49 families. For 43 families, including 22 males and 33 females, we collated detailed clinical and segregation data. To confirm causality of variants and to obtain insight into disease mechanisms, we investigated the effect on electrophysiological properties of 59 of the variants in Xenopus oocytes using extended voltage and pH ranges. Detailed analyses revealed new pathophysiological mechanisms: 25% (15/59) of variants demonstrated LOF, characterized by a "shift" of the voltage-dependent activation to more positive voltages, and nine variants resulted in a toxic gain-of-function, associated with a disrupted gate allowing inward transport at negative voltages. Functional results were not always in line with in silico pathogenicity scores, highlighting the complexity of pathogenicity assessment for accurate genetic counselling. The complex neurocognitive and psychiatric manifestations of this condition, and hitherto under-recognized impacts on growth, gastrointestinal function, and motor control are discussed. Including published cases, we summarize features in 122 individuals from 67 families with CLCN4-related neurodevelopmental condition and suggest future research directions with the aim of improving the integrated care for individuals with this diagnosis.

The evolution, formation and connectivity of the anterior commissure.

The anterior commissure is the most ancient of the forebrain interhemispheric connections among all vertebrates. Indeed, it is the predominant pallial commissure in all non-eutherian vertebrates, universally subserving basic functions related to olfaction and survival. A key feature of the anterior commissure is its ability to convey connections from diverse brain areas, such as most of the neocortex in non-eutherian mammals, thereby mediating the bilateral integration of diverse functions. Shared developmental mechanisms between the anterior commissure and more evolutionarily recent commissures, such as the corpus callosum in eutherians, have led to the hypothesis that the former may have been a precursor for additional expansion of commissural circuits. However, differences between the formation of the anterior commissure and other telencephalic commissures suggest that independent developmental mechanisms underlie the emergence of these connections in extant species. Here, we review the developmental mechanisms and connectivity of the anterior commissure across evolutionarily distant species, and highlight its potential functional importance in humans, both in the course of normal neurodevelopment, and as a site of plastic axonal rerouting in the absence or damage of other connections.

Differential timing of a conserved transcriptional network underlies divergent cortical projection routes across mammalian brain evolution.

A unique combination of transcription factor expression and projection neuron identity demarcates each layer of the cerebral cortex. During mouse and human cortical development, the transcription factor CTIP2 specifies neurons that project subcerebrally, while SATB2 specifies neuronal projections via the corpus callosum, a large axon tract connecting the two neocortical hemispheres that emerged exclusively in eutherian mammals. Marsupials comprise the sister taxon of eutherians but do not have a corpus callosum; their intercortical commissural neurons instead project via the anterior commissure, similar to egg-laying monotreme mammals. It remains unknown whether divergent transcriptional networks underlie these cortical wiring differences. Here, we combine birth-dating analysis, retrograde tracing, gene overexpression and knockdown, and axonal quantification to compare the functions of CTIP2 and SATB2 in neocortical development, between the eutherian mouse and the marsupial fat-tailed dunnart. We demonstrate a striking degree of structural and functional homology, whereby CTIP2 or SATB2 of either species is sufficient to promote a subcerebral or commissural fate, respectively. Remarkably, we reveal a substantial delay in the onset of developmental SATB2 expression in mice as compared to the equivalent stage in dunnarts, with premature SATB2 overexpression in mice to match that of dunnarts resulting in a marsupial-like projection fate via the anterior commissure. Our results suggest that small alterations in the timing of regulatory gene expression may underlie interspecies differences in neuronal projection fate specification.

NFIA and NFIB function as tumour suppressors in high-grade glioma in mice.

Nuclear factor one (NFI) transcription factors are implicated in both brain development and cancer in mice and humans and play an essential role in glial differentiation. NFI expression is reduced in human astrocytoma samples, particularly those of higher grade, whereas over-expression of NFI protein can induce the differentiation of glioblastoma cells within human tumour xenografts and in glioblastoma cell lines in vitro. These data indicate that NFI proteins may act as tumour suppressors in glioma. To test this hypothesis, we generated complex mouse genetic crosses involving six alleles to target gene deletion of known tumour suppressor genes that induce endogenous high-grade glioma in mice, and overlaid this with loss of function Nfi mutant alleles, Nfia and Nfib, a reporter transgene and an inducible Cre allele. Deletion of Nfi resulted in reduced survival time of the mice, increased tumour load and a more aggressive tumour phenotype than observed in glioma mice with normal expression of NFI. Together, these data indicate that NFI genes represent a credible target for both diagnostic analyses and therapeutic strategies to combat high-grade glioma.

NFIB Haploinsufficiency Is Associated with Intellectual Disability and Macrocephaly.

The nuclear factor I (NFI) family of transcription factors play an important role in normal development of multiple organs. Three NFI family members are highly expressed in the brain, and deletions or sequence variants in two of these, NFIA and NFIX, have been associated with intellectual disability (ID) and brain malformations. NFIB, however, has not previously been implicated in human disease. Here, we present a cohort of 18 individuals with mild ID and behavioral issues who are haploinsufficient for NFIB. Ten individuals harbored overlapping microdeletions of the chromosomal 9p23-p22.2 region, ranging in size from 225 kb to 4.3 Mb. Five additional subjects had point sequence variations creating a premature termination codon, and three subjects harbored single-nucleotide variations resulting in an inactive protein as determined using an in vitro reporter assay. All individuals presented with additional variable neurodevelopmental phenotypes, including muscular hypotonia, motor and speech delay, attention deficit disorder, autism spectrum disorder, and behavioral abnormalities. While structural brain anomalies, including dysgenesis of corpus callosum, were variable, individuals most frequently presented with macrocephaly. To determine whether macrocephaly could be a functional consequence of NFIB disruption, we analyzed a cortex-specific Nfib conditional knockout mouse model, which is postnatally viable. Utilizing magnetic resonance imaging and histology, we demonstrate that Nfib conditional knockout mice have enlargement of the cerebral cortex but preservation of overall brain structure and interhemispheric connectivity. Based on our findings, we propose that haploinsufficiency of NFIB causes ID with macrocephaly.

Verbal Adynamia and Conceptualization in Partial Rhombencephalosynapsis and Corpus Callosum Dysgenesis.

Verbal adynamia is characterized by markedly reduced spontaneous speech that is not attributable to a core language deficit such as impaired naming, reading, repetition, or comprehension. In some cases, verbal adynamia is severe enough to be considered dynamic aphasia. We report the case of a 40-year-old, left-handed, male native English speaker who presented with partial rhombencephalosynapsis, corpus callosum dysgenesis, and a language profile that is consistent with verbal adynamia, or subclinical dynamic aphasia, possibly underpinned by difficulties selecting and generating ideas for expression. This case is only the second investigation of dynamic aphasia in an individual with a congenital brain malformation. It is also the first detailed neuropsychological report of an adult with partial rhombencephalosynapsis and corpus callosum dysgenesis, and the only known case of superior intellectual abilities in this context.

A pan-mammalian map of interhemispheric brain connections predates the evolution of the corpus callosum.

The brain of mammals differs from that of all other vertebrates, in having a six-layered neocortex that is extensively interconnected within and between hemispheres. Interhemispheric connections are conveyed through the anterior commissure in egg-laying monotremes and marsupials, whereas eutherians evolved a separate commissural tract, the corpus callosum. Although the pattern of interhemispheric connectivity via the corpus callosum is broadly shared across eutherian species, it is not known whether this pattern arose as a consequence of callosal evolution or instead corresponds to a more ancient feature of mammalian brain organization. Here we show that, despite cortical axons using an ancestral commissural route, monotremes and marsupials share features of interhemispheric connectivity with eutherians that likely predate the origin of the corpus callosum. Based on ex vivo magnetic resonance imaging and tractography, we found that connections through the anterior commissure in both fat-tailed dunnarts (Marsupialia) and duck-billed platypus (Monotremata) are spatially segregated according to cortical area topography. Moreover, cell-resolution retrograde and anterograde interhemispheric circuit mapping in dunnarts revealed several features shared with callosal circuits of eutherians. These include the layered organization of commissural neurons and terminals, a broad map of connections between similar (homotopic) regions of each hemisphere, and regions connected to different areas (heterotopic), including hyperconnected hubs along the medial and lateral borders of the cortex, such as the cingulate/motor cortex and claustrum/insula. We therefore propose that an interhemispheric connectome originated in early mammalian ancestors, predating the evolution of the corpus callosum. Because these features have been conserved throughout mammalian evolution, they likely represent key aspects of neocortical organization.

Altered structural connectivity networks in a mouse model of complete and partial dysgenesis of the corpus callosum.

Corpus callosum dysgenesis (CCD) describes a collection of brain malformations in which the main fiber tract connecting the two hemispheres is either absent (complete CCD, or 'agenesis of the corpus callosum') or reduced in size (partial CCD). Humans with these neurodevelopmental disorders have a wide range of cognitive outcomes, including seemingly preserved features of interhemispheric communication in some cases. However, the structural substrates that could underlie this variability in outcome remain to be fully elucidated. Here, for the first time, we characterize the global brain connectivity of a mouse model of complete and partial CCD. We demonstrate features of structural brain connectivity that model those predicted in humans with CCD, including Probst bundles in complete CCD and heterotopic sigmoidal connections in partial CCD. Crucially, we also histologically validate the recently predicted ectopic sigmoid bundle present in humans with partial CCD, validating the utility of this mouse model for fine anatomical studies of this disorder. Taken together, this work describes a mouse model of altered structural connectivity in variable severity CCD and forms a foundation for future studies investigating the function and mechanisms of development of plastic tracts in developmental disorders of brain connectivity.

Long-distance aberrant heterotopic connectivity in a mouse strain with a high incidence of callosal anomalies.

Corpus callosum dysgenesis (CCD) is a developmental brain condition in which some white matter fibers fail to find their natural course across the midplane, reorganizing instead to form new aberrant pathways. This type of white matter reorganization is known as long-distance plasticity (LDP). The present work aimed to characterize the Balb/c mouse strain as a model of CCD. We employed high-resolution anatomical MRI in 81 Balb/c and 27 C57bl6 mice to show that the Balb/c mouse strain presents a variance in the size of the CC that is 3.9 times higher than the variance of normotypical C57bl6. We also performed high-resolution diffusion-weighted imaging (DWI) in 8 Balb/c and found that the Balb/c strain shows aberrant white matter bundles, such as the Probst (5/8 animals) and the Sigmoid bundles (7/8 animals), which are similar to those found in humans with CCD. Using a histological tracer technique, we confirmed the existence of these aberrant bundles in the Balb/c strain. Interestingly, we also identified sigmoid-like fibers in the C57bl6 strain, thought to a lesser degree. Next, we used a connectome approach and found widespread brain connectivity differences between Balb/c and C57bl6 strains. The Balb/c strain also exhibited increased variability of global connectivity. These findings suggest that the Balb/c strain presents local and global changes in brain structural connectivity. This strain often presents with callosal abnormalities, along with the Probst and the Sigmoid bundles, making it is an attractive animal model for CCD and LDP in general. Our results also show that even the C57bl6 strain, which typically serves as a normotypical control animal in a myriad of studies, presents sigmoid-fashion pattern fibers laid out in the brain. These results suggest that these aberrant fiber pathways may not necessarily be a pathological hallmark, but instead an alternative roadmap for misguided axons. Such findings offer new insights for interpreting the significance of CCD-associated LDP in humans.

Transcription factors NFIA and NFIB induce cellular differentiation in high-grade astrocytoma.

INTRODUCTION: Malignant astrocytomas are composed of heterogeneous cell populations. Compared to grade IV glioblastoma, low-grade astrocytomas have more differentiated cells and are associated with a better prognosis. Therefore, inducing cellular differentiation to alter the behaviour of high-grade astrocytomas may serve as a therapeutic strategy. The nuclear factor one (NFI) transcription factors are essential for normal astrocytic differentiation. Here, we investigate whether family members NFIA and NFIB act as effectors of cellular differentiation in glioblastoma. METHODS: We analysed expression of NFIA and NFIB in mRNA expression data of high-grade astrocytoma and with immunofluorescence co-staining. Furthermore, we induced NFI expression in patient-derived subcutaneous glioblastoma xenografts via in vivo electroporation. RESULTS: The expression of NFIA and NFIB is reduced in glioblastoma as compared to lower grade astrocytomas. At a cellular level, their expression is associated with differentiated and mature astrocyte-like tumour cells. In vivo analyses consistently demonstrate that expression of either NFIA or NFIB is sufficient to promote tumour cell differentiation in glioblastoma xenografts. CONCLUSION: Our findings indicate that both NFIA and NFIB may have an endogenous pro-differentiative function in astrocytomas, similar to their role in normal astrocyte differentiation. Overall, our study establishes a basis for further investigation of targeting NFI-mediated differentiation as a potential differentiation therapy.

Callosal agenesis and congenital mirror movements: outcomes associated with DCC mutations.

Pathogenic variants in the gene encoding deleted in colorectal cancer (DCC) are the first genetic cause of isolated agenesis of the corpus callosum (ACC). Here we present the detailed neurological, brain magnetic resonance imaging (MRI), and neuropsychological characteristics of 12 individuals from three families with pathogenic variants in DCC (aged 8-50y), who showed ACC and mirror movements (n=5), mirror movements only (n=2), ACC only (n=3), or neither ACC nor mirror movements (n=2). There was heterogeneity in the neurological and neuroimaging features on brain MRI, and performance across neuropsychological domains ranged from extremely low (impaired) to within normal limits (average). Our findings show that ACC and/or mirror movements are associated with low functioning in select neuropsychological domains and a DCC pathogenic variant alone is not sufficient to explain the disability. WHAT THIS PAPER ADDS: Neuropsychological impairment severity is related to presence of mirror movements and/or agenesis of the corpus callosum. A DCC pathogenic variant in isolation is associated with the best prognosis.

Variants in nuclear factor I genes influence growth and development.

The nuclear factor one (NFI) site-specific DNA-binding proteins represent a family of transcription factors that are important for the development of multiple organ systems, including the brain. During brain development in mice, the expression patterns of Nfia, Nfib, and Nfix overlap, and knockout mice for each of these exhibit overlapping brain defects, including megalencephaly, dysgenesis of the corpus callosum, and enlarged ventricles, which implies a common but not redundant function in brain development. In line with these models, human phenotypes caused by haploinsufficiency of NFIA, NFIB, and NFIX display significant overlap, sharing neurodevelopmental deficits, macrocephaly, brain anomalies, and variable somatic overgrowth. Other anomalies may be present depending on the NFI gene involved. The possibility of variants in NFI genes should therefore be considered in individuals with intellectual disability and brain overgrowth, with individual NFI-related conditions being differentiated from one another by additional signs and symptoms. The exception is provided by specific NFIX variants that act in a dominant negative manner, as these cause a recognizable entity with more severe cognitive impairment and marked bone dysplasia, Marshall-Smith syndrome. NFIX duplications are associated with a phenotype opposite to that of haploinsufficiency, characterized by short stature, small head circumference, and delayed bone age. The spectrum of NFI-related disorders will likely be further expanded, as larger cohorts are assessed.

Transcriptional regulation of intermediate progenitor cell generation during hippocampal development.

During forebrain development, radial glia generate neurons through the production of intermediate progenitor cells (IPCs). The production of IPCs is a central tenet underlying the generation of the appropriate number of cortical neurons, but the transcriptional logic underpinning this process remains poorly defined. Here, we examined IPC production using mice lacking the transcription factor nuclear factor I/X (Nfix). We show that Nfix deficiency delays IPC production and prolongs the neurogenic window, resulting in an increased number of neurons in the postnatal forebrain. Loss of additional Nfi alleles (Nfib) resulted in a severe delay in IPC generation while, conversely, overexpression of NFIX led to precocious IPC generation. Mechanistically, analyses of microarray and ChIP-seq datasets, coupled with the investigation of spindle orientation during radial glial cell division, revealed that NFIX promotes the generation of IPCs via the transcriptional upregulation of inscuteable (Insc). These data thereby provide novel insights into the mechanisms controlling the timely transition of radial glia into IPCs during forebrain development.

DCC mutation update: Congenital mirror movements, isolated agenesis of the corpus callosum, and developmental split brain syndrome.

The deleted in colorectal cancer (DCC) gene encodes the netrin-1 (NTN1) receptor DCC, a transmembrane protein required for the guidance of commissural axons. Germline DCC mutations disrupt the development of predominantly commissural tracts in the central nervous system (CNS) and cause a spectrum of neurological disorders. Monoallelic, missense, and predicted loss-of-function DCC mutations cause congenital mirror movements, isolated agenesis of the corpus callosum (ACC), or both. Biallelic, predicted loss-of-function DCC mutations cause developmental split brain syndrome (DSBS). Although the underlying molecular mechanisms leading to disease remain poorly understood, they are thought to stem from reduced or perturbed NTN1 signaling. Here, we review the 26 reported DCC mutations associated with abnormal CNS development in humans, including 14 missense and 12 predicted loss-of-function mutations, and discuss their associated clinical characteristics and diagnostic features. We provide an update on the observed genotype-phenotype relationships of congenital mirror movements, isolated ACC and DSBS, and correlate this to our current understanding of the biological function of DCC in the development of the CNS. All mutations and their associated phenotypes were deposited into a locus-specific LOVD (https://databases.lovd.nl/shared/genes/DCC).

Transcriptional regulation of Nfix by NFIB drives astrocytic maturation within the developing spinal cord.

During mouse spinal cord development, ventricular zone progenitor cells transition from producing neurons to producing glia at approximately embryonic day 11.5, a process known as the gliogenic switch. The transcription factors Nuclear Factor I (NFI) A and B initiate this developmental transition, but the contribution of a third NFI member, NFIX, remains unknown. Here, we reveal that ventricular zone progenitor cells within the spinal cord express NFIX after the onset of NFIA and NFIB expression, and after the gliogenic switch has occurred. Mice lacking NFIX exhibit normal neurogenesis within the spinal cord, and, while early astrocytic differentiation proceeds normally, aspects of terminal astrocytic differentiation are impaired. Finally, we report that, in the absence of Nfia or Nfib, there is a marked reduction in the spinal cord expression of NFIX, and that NFIB can transcriptionally activate Nfix expression in vitro. These data demonstrate that NFIX is part of the downstream transcriptional program through which NFIA and NFIB coordinate gliogenesis within the spinal cord. This hierarchical organisation of NFI protein expression and function during spinal cord gliogenesis reveals a previously unrecognised auto-regulatory mechanism within this gene family.

EMX1 regulates NRP1-mediated wiring of the mouse anterior cingulate cortex.

Transcription factors act during cortical development as master regulatory genes that specify cortical arealization and cellular identities. Although numerous transcription factors have been identified as being crucial for cortical development, little is known about their downstream targets and how they mediate the emergence of specific neuronal connections via selective axon guidance. The EMX transcription factors are essential for early patterning of the cerebral cortex, but whether EMX1 mediates interhemispheric connectivity by controlling corpus callosum formation remains unclear. Here, we demonstrate that in mice on the C57Bl/6 background EMX1 plays an essential role in the midline crossing of an axonal subpopulation of the corpus callosum derived from the anterior cingulate cortex. In the absence of EMX1, cingulate axons display reduced expression of the axon guidance receptor NRP1 and form aberrant axonal bundles within the rostral corpus callosum. EMX1 also functions as a transcriptional activator of Nrp1 expression in vitro, and overexpression of this protein in Emx1 knockout mice rescues the midline-crossing phenotype. These findings reveal a novel role for the EMX1 transcription factor in establishing cortical connectivity by regulating the interhemispheric wiring of a subpopulation of neurons within the mouse anterior cingulate cortex.