Animals display complex behaviors. This functional complexity is mirrored by the high diversity of neuronal cell types of their nervous system. During development, the correct number of neurons of each neuronal cell type has to be produced so that the nervous system can function properly. How such a level of precision is reached in a robust manner remains poorly understood. The type-specific identity of a neuron is defined by the particular set of terminal selector transcription factors that it expresses (Deneris and Hobert, 2014; Hobert, 2011; Hobert and Kratsios, 2019). Terminal selectors are expressed throughout the life of the neuron and directly regulate the expression of large batteries of type-specific effector genes responsible for the functions of the neuron. During development, terminal selectors have to be activated in very precise sets of postmitotic neurons, at the right time and correct level. However, how this is achieved in a highly accurate manner is poorly characterized.

In animals, a conserved group of transcription factors, the neural bHLH factors, play key roles in early steps of nervous system development (Baker and Brown, 2018; Bertrand et al., 2002; Hartenstein and Stollewerk, 2015). They are involved in the choice between a neural and epidermal fate (classic proneural function), as well as in the specification of distinct neuronal-type identities. Neural bHLH factors include not only classic proneural bHLH factors but also bHLH factors involved in the establishment of the type-specific identity of the neuron. Neural bHLH factors are grouped in different families based on their sequences: Achaete-Scute, Neurogenin, Olig, Atonal and NeuroD. They act as heterodimers with another group of bHLH factors, the E-proteins, which are broadly expressed. Heterodimers of neural bHLHs with E-proteins bind to a specific DNA sequence, the E-box, to activate expression of their target genes. In this study, we analyzed how neural bHLH factors contribute to the precision in the initiation of terminal selector expression.

To characterize the mechanisms that ensure the robustness of neuronal specification, we used Caenorhabditis elegans as a model organism. Each C. elegans adult hermaphrodite has exactly 302 neurons divided in 118 distinct classes (Hobert et al., 2016; White et al., 1986). It is therefore a good system to analyze how a nervous system is built with high precision. We used the AIY cholinergic interneuron as a test lineage, because its specification network is well characterized (Barriere and Bertrand, 2020). There are two AIY neurons, one on the left side of the head and one on the right. The specific identity of the AIY neuron is regulated by a complex of two terminal selector transcription factors, the homeodomain transcription factors TTX-3 (ortholog of LHX2/9) and CEH-10 (ortholog of VSX1/2) (Fig. 1A). In the postmitotic AIY neuron, TTX-3 and CEH-10 directly activate and maintain the expression of a large battery of type-specific effector genes such as the acetylcholine vesicular transporter unc-17 or the neurotransmitter receptors ser-2 and mod-1 (Wenick and Hobert, 2004). In the embryo, the AIY neuron is produced during neurulation (epidermal enclosure) by an asymmetric division oriented along the anteroposterior axis (Sulston et al., 1983). This division generates the AIY neuron posteriorly and the SMDD motor neuron anteriorly. ttx-3 expression is initiated in the SMDD/AIY mother cell by direct binding of several bHLH transcription factors to its cis-regulatory regions: the neural bHLH factors HLH-3 (Achaete-Scute family) and HLH-16 (Olig family), and their cofactor HLH-2 (E-protein) (Bertrand et al., 2011; Bertrand and Hobert, 2009; Murgan et al., 2015). The Zic transcription factor REF-2 also contributes to the initiation of ttx-3 expression (Bertrand and Hobert, 2009). Following asymmetric division, the TTX-3 protein is inherited by both daughter cells. In the posterior daughter cell (AIY), the Wnt pathway is active leading to the formation of a TCF/β-catenin activator complex (POP-1/SYS-1) that acts with TTX-3 to directly activate ceh-10 expression by binding to its cis-regulatory regions (Bertrand and Hobert, 2009). In the anterior daughter cell (SMDD), the Wnt pathway is inactive, β-catenin is degraded, and ceh-10 expression is not activated. In the AIY neuron, following transcriptional activation, the CEH-10 protein forms a complex with TTX-3 that directly activates the expression of the AIY-specific effector gene battery (Wenick and Hobert, 2004). TTX-3 and CEH-10 also directly maintain their expression in the AIY neuron via a positive feedback loop throughout the life of the animal, locking in the type-specific identity of the neuron (Bertrand and Hobert, 2009; Wenick and Hobert, 2004).

In this study, we identify a third neural bHLH factor involved in AIY neuron specification, NGN-1, the only member of the Neurogenin family in C. elegans. By quantifying the initiation of the terminal selectors ttx-3 and ceh-10, using CRISPR-engineered lines, we show that the three neural bHLH factors, NGN-1, HLH-3 and HLH-16 act together to set the correct level of terminal selector expression, ensuring robust, 100% efficient, neuronal specification. The neural bHLHs directly act on the terminal selector cis-regulatory regions via E-boxes. In addition, we show that HLH-3 and NGN-1 act in an antagonistic manner on the expression of another target, egl-1, a proapoptotic gene of the BH3-only family: HLH-3 promotes its expression while NGN-1 represses it. This antagonism blocks egl-1 expression in the AIY neuron, preventing deleterious effects on AIY specification. Our study suggests that the use of several different neural bHLHs in a single neuron specification event allows the correct level of terminal selector expression to be reached, while simultaneously avoiding the ectopic activation of unwanted genes.