Axons of early generated neurons in cingulate cortex pioneer the corpus callosum

The internal capsule and corpus callosum are the two major efferent axonal pathways of the mammalian neocortex. Previous studies have shown that the first cortical axons to grow through the internal capsule, the pathway from cortex to its subcortical targets, are extended by subplate neurons, which are the earliest generated neurons in the neocortex. Here, we characterize the origin of the first axons to project through the other major efferent pathway of the cortex, the midline corpus callosum, which connects the two cortical hemispheres. Using anterograde Dil tracing, we show that cortical axons first cross the midline through the nascent corpus callosum at E17. Retrograde Dil labeling from medial cortex at E18 reveals that these axons originate from a discrete group of neurons in medial (presumptive cingulate) cortex. These early callosal cells have complex morphologies with highly branched dendrites and later appear to take on a pyramidal form characteristic of callosal neurons in deep layers of cingulate cortex. 3H-thymidine birthdating demonstrates that these cells are predominantly generated on E14, making them among the earliest generated neurons in this cortical region. Injections of retrograde tracers in one cortical hemisphere at late embryonic or early postnatal ages result in substantial numbers of neurons labeled in the ipsilateral subplate, but only a few neurons labeled in the contralateral subplate. Thus, subplate neurons do not pioneer or ever project in significant numbers through the corpus callosum. We conclude that the two major efferent pathways from cortex, the corpus callosum and the internal capsule, are pioneered by developmentally and spatially distinct populations of early generated cortical neurons.