Highlights － Regulation of expression of the vesicular glutamate transporter is highly modular － Homeodomain proteins initiate and maintain VGLUT expression in distinct neuron types － Distinct terminal neuronal features including cotransmitter identity are coregulated － Homeodomain regulation of glutamatergic identity is phylogenetically conserved
Summary The choice of using one of many possible neurotransmitter systems is a critical step in defining the identity of an individual neuron type. We show here that the key defining feature of glutamatergic neurons, the vesicular glutamate transporter EAT-4/VGLUT, is expressed in 38 of the 118 anatomically defined neuron classes of the C. elegans nervous system. We show that distinct cis-regulatory modules drive expression of eat-4/VGLUT in distinct glutamatergic neuron classes. We identify 13 different transcription factors, 11 of them homeodomain proteins, that act in distinct combinations in 25 different glutamatergic neuron classes to initiate and maintain eat-4/VGLUT expression. We show that the adoption of a glutamatergic phenotype is linked to the adoption of other terminal identity features of a neuron, including cotransmitter phenotypes. Examination of mouse orthologs of these homeodomain proteins resulted in the identification of mouse LHX1 as a regulator of glutamatergic neurons in the brainstem.