Molecular mechanisms underlying interstitial axon branching in the mammalian brain

The cerebral cortex is a six-layered brain structure that is essential for sensory processing, motor skills, learning and memory and thought. The establishment of functional cortical connectivity requires newly born neurons to migrate into appropriate layers during embryogenesis and then elaborate complex and unique axon and dendrite branching patterns. This results in laminar-specific organization of interstitial axon branches and also the elaboration of select intra- and inter-areal cortical connections. Given the complexity of axon trajectories, precise regulation of collateral axon branch formation is vital for the generation of a functional brain connectome. However, the intracellular signaling pathways, receptors, cell surface molecules and extracellular ligands that regulate laminar-specific interstitial axon branching in the cortex are poorly understood. Thus, a central remaining question is how laminar-specific innervation is established in the cerebral cortex. In this proposal, we investigate interstitial axon branching in vivo using novel approaches for precise labeling of layer 2/3 callosal projection neurons (CPNs), allowing for quantitative analysis of axonal morphology at high acuity and also manipulation of gene expression in well-defined temporal windows. Our recent work identifies an intracellular signaling pathway that regulates interstitial axon branching in vivo in layer 2/3 CPNs – we show that GSK3β activates MAP1B to promote interstitial axon branching via increased tyrosination of tubulin. In the Aim 1 of the current proposal, we will investigate the role of this pathway in other classes of cortical excitatory projection neurons, and we will investigate the molecular mechanisms underlying how MAP1B and tubulin tyrosination lead to the formation of interstitial axon branches. Next, our preliminary analysis of single cell RNA sequencing (scRNA seq) data acquired from the developing somatosensory cortex identifies multiple cell surface molecules enriched in layer 2/3 CPNs, and a follow-up genetic survey of these molecules show that an adhesion synaptic protein LRRTM4 has strong potential to act as an interstitial axon branch-restricting factor. In the Aim 2, we will investigate the function of LRRTM4 and identify its ligands in cortical layer 4. In the Aim 3, we will broaden our analysis on the Auditory cortex and will also address if GSK3β/MAP1B signaling and LRRTM4 adhesion molecule regulate interstitial axon branching in the contralateral axons. In summary, our work will lead to the identification of molecular principles governing the development of cortical connectivity and will provide a robust foundation for future independent research program.