Cis-regulatory mechanisms of left/right asymmetric neuron-subtype specification in C. elegans.

Anatomically and functionally defined neuron types are sometimes further classified into individual subtypes based on unique functional or molecular properties. To better understand how developmental programs controlling neuron type specification are mechanistically linked to programs controlling neuronal subtype specification, we have analyzed a neuronal subtype specification program that occurs across the left/right axis in the nervous system of the nematode C. elegans. A terminal selector transcription factor, CHE-1, is required for the specification of the ASE neuron class, and a gene regulatory feedback loop of transcription factors and miRNAs is required to diversify the two ASE neurons into an asymmetric left and right subtype (ASEL and ASER). However, the link between the CHE-1-dependent ASE neuron class specification and the ensuing left-right subtype specification program is poorly understood. We show here that CHE-1 has genetically separable functions in controlling bilaterally symmetric ASE neuron class specification and the ensuing left-right subtype specification program. Both neuron class specification and asymmetric subclass specification depend on CHE-1-binding sites (;ASE motifs') in symmetrically and asymmetrically expressed target genes, but in the case of asymmetrically expressed target genes, the activity of the ASE motif is modulated through a diverse set of additional cis-regulatory elements. Depending on the target gene, these cis-regulatory elements either promote or inhibit the activity of CHE-1. The activity of these L/R asymmetric cis-regulatory elements is indirectly controlled by che-1 itself, revealing a feed-forward loop configuration in which che-1 restricts its own activity. Relative binding affinity of CHE-1 to ASE motifs also depends on whether a gene is expressed bilaterally or in a left/right asymmetric manner. Our analysis provides insights into the molecular mechanisms of neuronal subtype specification, demonstrating that the activity of a neuron type-specific selector gene is modulated by a variety of distinct means to diversify individual neuron classes into specific subclasses. It also suggests that feed-forward loop motifs may be a prominent feature of neuronal diversification events.