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.

Netrin-DCC signaling regulates corpus callosum formation through attraction of pioneering axons and by modulating Slit2-mediated repulsion.

The left and right sides of the nervous system communicate via commissural axons that cross the midline during development using evolutionarily conserved molecules. These guidance cues have been particularly well studied in the mammalian spinal cord, but it remains unclear whether these guidance mechanisms for commissural axons are similar in the developing forebrain, in particular for the corpus callosum, the largest and most important commissure for cortical function. Here, we show that Netrin1 initially attracts callosal pioneering axons derived from the cingulate cortex, but surprisingly is not attractive for the neocortical callosal axons that make up the bulk of the projection. Instead, we show that Netrin-deleted in colorectal cancer signaling acts in a fundamentally different manner, to prevent the Slit2-mediated repulsion of precrossing axons thereby allowing them to approach and cross the midline. These results provide the first evidence for how callosal axons integrate multiple guidance cues to navigate the midline.

Multiple Slits regulate the development of midline glial populations and the corpus callosum.

The Slit molecules are chemorepulsive ligands that regulate axon guidance at the midline of both vertebrates and invertebrates. In mammals, there are three Slit genes, but only Slit2 has been studied in any detail with regard to mammalian brain commissure formation. Here, we sought to understand the relative contributions that Slit proteins make to the formation of the largest brain commissure, the corpus callosum. Slit ligands bind Robo receptors, and previous studies have shown that Robo1(-/-) mice have defects in corpus callosum development. However, whether the Slit genes signal exclusively through Robo1 during callosal formation is unclear. To investigate this, we compared the development of the corpus callosum in both Slit2(-/-) and Robo1(-/-) mice using diffusion magnetic resonance imaging. This analysis demonstrated similarities in the phenotypes of these mice, but crucially also highlighted subtle differences, particularly with regard to the guidance of post-crossing axons. Analysis of single mutations in Slit family members revealed corpus callosum defects (but not complete agenesis) in 100% of Slit2(-/-) mice and 30% of Slit3(-/-) mice, whereas 100% of Slit1(-/-); Slit2(-/-) mice displayed complete agenesis of the corpus callosum. These results revealed a role for Slit1 in corpus callosum development, and demonstrated that Slit2 was necessary but not sufficient for midline crossing in vivo. However, co-culture experiments utilising Robo1(-/-) tissue versus Slit2 expressing cell blocks demonstrated that Slit2 was sufficient for the guidance activity mediated by Robo1 in pre-crossing neocortical axons. This suggested that Slit1 and Slit3 might also be involved in regulating other mechanisms that allow the corpus callosum to form, such as the establishment of midline glial populations. Investigation of this revealed defects in the development and dorso-ventral positioning of the indusium griseum glia in multiple Slit mutants. These findings indicate that Slits regulate callosal development via both classical chemorepulsive mechanisms, and via a novel role in mediating the correct positioning of midline glial populations. Finally, our data also indicate that some of the roles of Slit proteins at the midline may be independent of Robo signalling, suggestive of additional receptors regulating Slit signalling during development.

DCC regulates astroglial development essential for telencephalic morphogenesis and corpus callosum formation.

The forebrain hemispheres are predominantly separated during embryogenesis by the interhemispheric fissure (IHF). Radial astroglia remodel the IHF to form a continuous substrate between the hemispheres for midline crossing of the corpus callosum (CC) and hippocampal commissure (HC). Deleted in colorectal carcinoma (DCC) and netrin 1 (NTN1) are molecules that have an evolutionarily conserved function in commissural axon guidance. The CC and HC are absent in Dcc and Ntn1 knockout mice, while other commissures are only partially affected, suggesting an additional aetiology in forebrain commissure formation. Here, we find that these molecules play a critical role in regulating astroglial development and IHF remodelling during CC and HC formation. Human subjects with DCC mutations display disrupted IHF remodelling associated with CC and HC malformations. Thus, axon guidance molecules such as DCC and NTN1 first regulate the formation of a midline substrate for dorsal commissures prior to their role in regulating axonal growth and guidance across it.

Understanding the mechanisms of callosal development through the use of transgenic mouse models.

The cerebral cortex is the area of the brain where higher-order cognitive processing occurs. The 2 hemispheres of the cerebral cortex communicate through one of the largest fiber tracts in the brain, the corpus callosum. Malformation of the corpus callosum in human beings occurs in 1 in 4000 live births, and those afflicted experience an extensive range of neurologic disorders, from relatively mild to severe cognitive deficits. Understanding the molecular and cellular processes involved in these disorders would therefore assist in the development of prognostic tools and therapies. During the past 3 decades, mouse models have been used extensively to determine which molecules play a role in the complex regulation of corpus callosum development. This review provides an update on these studies, as well as highlights the value of using mouse models with the goal of developing therapies for human acallosal syndromes.