Background: How do existing motor neurons acquire new traits?

A study in PNAS reports that existing nematode motor neurons can acquire a serotonin reuptake trait while retaining their cholinergic identity, altering their egg-laying response to external serotonin. The researchers examined how epigenetic regulation that suppresses gene expression and enhancers, sequences that regulate gene expression, contribute, respectively, to environment-dependent and persistent expression of this trait.

A key question in nervous system evolution is how stable cell functions coexist with the acquisition of new traits. In the nematode Caenorhabditis elegans, VC4 and VC5 are cholinergic motor neurons in the egg-laying circuit that use acetylcholine as a neurotransmitter. The researchers compared the conditions under which these cells, which normally show weak serotonin staining that varies among individuals, accumulate serotonin, and how the same trait appears in related species.

Key findings: A reuptake trait revealed by repression mechanisms and enhancer replacement

In C. elegans, the histone H3 lysine 9 methylation (H3K9me) pathway was found to suppress the serotonin reuptake trait. Mutations in some components of this pathway strengthened serotonin staining in VC4 and VC5, and restoring expression of the normal genes in those neurons suppressed this change. Eliminating the function of the serotonin transporter gene mod-5/Sert abolished the staining. Removing HSN, the neighboring serotonin-synthesizing neurons, also reduced staining, supporting the explanation that the cells take up serotonin from their surroundings rather than synthesize it themselves. However, the researchers did not directly detect mod-5 expression using an endogenous marker in H3K9me pathway mutants. Low expression levels or indirect changes in transporter activity remain possible explanations.

A comparison of 26 species found strong serotonin staining in VC4 and VC5 in five species of the Angaria lineage. The corresponding neurons in C. angaria also retained cholinergic markers. This is consistent with the interpretation that they gained a reuptake trait rather than abandoning their existing identity and becoming a different cell type.

The key causal experiment involved replacing a regulatory sequence. Replacing the first intron of C. elegans mod-5 with the C. angaria sequence produced strong serotonin staining in VC4 and VC5. The interpretation is that an enhancer in this region functions by becoming incorporated into the existing gene-regulatory network. The loss of enhancer-reporter activity following loss of the transcription factor UNC-4 or mutation of predicted binding sites also supports this interpretation.

Behavioral experiments measured how egg-laying induction changed after two hours of exposure to external serotonin. C. elegans expressing mod-5 in VC4 and VC5 showed a smaller increase in egg laying than controls. Conversely, eliminating mod-5 in C. angaria abolished its ability to buffer egg-laying induction. C. elegans raised for one generation in an environment with a high concentration of serotonin also showed increased serotonin staining and buffering of the egg-laying response. This buffering disappeared with loss of mod-5 and was reduced with loss of UNC-4.

Meaning and outlook: Connecting environmental responses with evolutionary fixation

These findings show that a trait added to existing motor neurons can vary with the environment or persist because of differences in regulatory sequences. The changes demonstrated in this study concern the reuptake trait of existing egg-laying circuit neurons and their behavioral response.

The intermediate mechanism connecting environmental serotonin with H3K9me regulation and mod-5 expression remains unknown. It is also unresolved whether this trait benefits survival or reproduction in natural environments, or whether an environment-dependent state preceded the persistent trait in the Angaria lineage. The possibility that a similar principle applies to serotonin transporter regulation in mammals remains a hypothesis requiring further testing.

The study is available in the public full text of the PNAS paper.