Neuronal subtypes are specified by the level of neurod expression in the zebrafish lateral line.

During neural development, even in seemingly homogeneous cell populations, individual neurons acquire diverse morphology and behavior, and even adjacent neurons can establish synaptic connections with distinct targets. Although diversity among individual neurons is essential for a functional nervous system, the underlying molecular mechanism of establishing heterogeneity in a population of neuronal precursors has been poorly addressed at the single-cell level. We focused on the development of the zebrafish posterior lateral line (PLL) and revealed a molecular mechanism that differentiates a homogenous neuronal population in the ganglion into the two types of neurons, leaders and followers. We developed a method to analyze gene expression levels in leaders and followers at the single-cell resolution, and found that leaders expressed significantly higher levels of neurod compared with followers. Furthermore, neurod expression was found to be heterogeneous among neurons before the onset of phenotypic differentiation of leaders and followers, and neurod overexpression in single PLL neurons promoted differentiation into leaders. These results suggest that the quantity, rather than quality (i.e., the ON/OFF states), of neurod expression directly or indirectly determines the two subtypes of PLL neurons.