Axonogenesis is coordinated by alternative splicing programming and splicing regulator PTBP2

How a neuron acquires an axon is a fundamental question. Piecemeal identification of many axonogenesis-related genes has been done, but coordinated regulation is unknown. Through unbiased transcriptome profiling of immature primary cortical neurons during early axon formation, we discovered an association between axonogenesis and neuron-specific alternative splicing. Known axonogenesis genes exhibit little expression alternation but widespread splicing changes. Axonogenesis-associated splicing is governed by RNA binding protein PTBP2, which is enriched in neurons and peaks around axonogenesis in the brain. Cortical depletion of PTBP2 prematurely induces axonogenesis-associated splicing, causes imbalanced expression of axonogenesis-associated isoforms, and specifically affects axon formation in vitro and in vivo. PTBP2-controlled axongeneisis-associated Shtn1 splicing determines SHTN1's capacity to regulate actin interaction, polymerization, and axon growth. Precocious Shtn1 isoform switch contributes to disorganized axon formation of Ptbp2-/- neurons. We conclude that PTBP2-orchestrated alternative splicing programming is required for robust generation of a single axon in mammals. Overall design: Immature cortical neurons were cultured from embryonic day 14 mouse neocortices at the density of 1 million cells per 35 mm petri dish. At 1 day in vitro (1 DIV), these neurons extend multiple indistinguishable processes in a seemingly random manner, just like pre-axonogenesis neurons in vivo. Around 3 DIV, one of the neurites accelerates its growth to become the axon. We performed unbiased deep RNA sequencing (RNA-Seq) of wildytpe DIV 1, wildtype DIV 3, and Ptbp2-/- DIV1 neurons. There are two biological replicates per sample (specific gentoype and age).