Distinct molecular profile and function of pioneer and follower neurons - Project Summary During development, axon growth is initiated by specialized types of neurons called “pioneer neurons”. As their name implies, they are the first to explore the dynamic environment of a developing tissue. After pioneer neurons extend their axons toward a particular target, their axon tracts act as a scaffold for the subsequent axon extension of other neurons, termed “followers”. Pioneer neurons have been well documented in the central and peripheral nervous systems of both vertebrates and invertebrates, but whether their molecular make up is distinct from that of followers is not known. We have recently shown that the neurotrophic factor receptor ret is specifically enriched in sensory pioneer neurons of the zebrafish, suggesting pioneer and follower neurons have different transcriptional profiles. To test this hypothesis, we conducted single-cell-RNA-sequencing (scRNA-seq) of these sensory neurons. Our scRNA-seq demonstrated that ret+ neurons indeed have a distinct transcriptional profile from ret- neurons. Expression analysis revealed 101 differentially expressed (DE) genes between these two populations. Furthermore, single molecule in situ hybridization of DE genes confirmed the presence of two distinct neuronal populations in vivo. To demonstrate that the presumptive pioneer-specific cluster indeed marks pioneer neurons, we knocked in a red fluorescent protein into rpz5 locus (rpz5 is almost exclusively expressed in the ret+ cell population). The reporter labeled pioneer axon terminals, confirming the ret+ population indeed represents pioneer neurons. We also discovered that a pioneer neuron “gene signature” is enriched in both developing peripheral and central nervous systems. Thus, the pioneer cell state we discovered represents a common feature in the nervous system. Based on this and additional preliminary data, we propose three specific aims that will determine: 1) the lineage relationship between pioneers and followers and molecular signals that specify pioneers; 2) the role of DE signaling pathways in differentiation of pioneers and followers; and 3) the molecular basis of distinct physiological properties of pioneers and followers. The successful completion of these aims will identify the mechanisms by which pioneer neurons arise and how they are specified. The core set of cellular functions in pioneers is characteristic of many types of neurons, so the knowledge gleaned from these studies will be relevant to pioneer neurons in other systems. Finally, identifying main factors regulating pioneer neuron fate commitment will create a list of novel therapeutic targets for studies involving nerve injury and regeneration.
