Neuronal diversity critically relies on precise spatiotemporal regulation of gene expression in the nervous system. The cholinergic motor neurons (MNs) of the C. elegans nerve cord provide a prime model to investigate the gene regulatory mechanisms underlying neuronal diversity. These MNs are grouped into six classes (SAB, DA, DB, VA, VB, AS) based on anatomical and molecular criteria. A remarkable wealth of MN class-specific markers provides a unique entry point into the problem of neuronal diversity. Such markers report expression of terminal identity genes (e.g., genes coding for ion channels, neurotransmitter receptors, signaling molecules, neuropeptides etc.), whose continuous expression is essential for MN function. It has been previously reported that the phylogenetically conserved Collier/Olf/Ebf (COE)-type transcription factor (TF) UNC-3 is essential for cholinergic MN diversity by directly activating a large battery of MN class-specific terminal identity genes. However, an important mechanistic question remains: since UNC-3 is expressed in all cholinergic MN classes, what are the mechanisms that prevent UNC-3 from activating class-specific genes more broadly, i.e., in all MN classes? A recent study identified seven MN class-specific TFs that act as transcriptional repressors to counteract UNC-3's ability to activate MN class-specific genes. However, the underlying mechanisms are poorly understood. Here, we focus on one repressor, the AT-rich Interaction Domain (ARID) protein CFI-1. CFI-1 is expressed in head muscles, interneurons and DA, DB, VA, VB cholinergic MNs. Through automated worm tracking, we found that
cfi-1 (-) mutants exhibit severe locomotion defects, suggesting a critical role for CFI-1 in MNs. To uncover the downstream targets of CFI-1 in MNs, we are currently employing RNA-seq and ChIP-seq, as only one target of CFI-1 (glutamate receptor subunit
glr-4) has been described to date. In wildtype animals,
glr-4 is activated by UNC-3 in SAB neurons but not in DA or DB MNs, due to repression by CFI-1. Overexpression of CFI-1 in SAB neurons leads to downregulation of
glr-4, suggesting that CFI-1 is sufficient to silence
glr-4. Furthermore, post-developmental depletion of CFI-1 using the auxin-inducible degradation system uncovered a continuous requirement for CFI-1 in
glr-4 repression. Lastly, we investigated the mechanisms that induce and maintain
cfi-1 expression in MNs. We found that the midbody HOX genes
lin-39 and
mab-5 are required for
cfi-1 induction during early development, while UNC-3 appears to be indispensable for
cfi-1 maintenance in adulthood. Together, our experiments aim to shed light into the mechanisms upstream and downstream of CFI-1, a critical regulator of cholinergic MN diversity.