Paper #12 - Food and metabolic signalling defects in a Caenorhabditis elegans serotonin-synthesis mutant

Title: Food and metabolic signalling defects in a Caenorhabditis elegans serotonin-synthesis mutant

Year: 2000

Summary: Here they set out to characterize the overall role of serotonin in C. elegans  by constructing a mutant that cannot synthesize serotonin.

First, they note that the C. elegans genome contains only one ortholog to mammalian tryptophan hydroxylase, an enzyme that makes 5-hydroxytryptophan, which is then decarboxylated to form 5-hydroxytryptamine. In C. elegans, this gene is called tph-1, and shares about 60% amino acid identity in its catalytic domain with mammalian tryptophan hydroxylase (and admittedly, 53% and 51% with mammalian phenylalanine and tyrosine hydroxylases, respectively). This, combined with the fact a tph-1::GFP fusion is expressed in known serotonergic neurons (HSN, NSM, ADF, CP, and rarely AIM and RIH) and a tph-1 null mutant doesn't have detectable levels of serotonin by immunostaining, suggests that tph-1 is in fact C. elegans sole tryptophan hydroxylase. They also note that dopamine appears unaffected in the tph-1 mutant, as indicated by 'formaldehyde-induced fluorescence staining.' How they know this is dopamine and not any other kind of amine neurotransmitter (tyramine, octopamine) I don't know. Perhaps they didn't know those were used in C. elegans at the time? (Structurally they're super similar so I imagine they'd all fluoresce when fixed - tyramine is dopamine minus one aromatic hydroxyl group, and octopamine is tyramine with a hydroxyl added to the ethyl linker between the amine and the aromatic ring).

How messed up are the tph-1 mutants? Morphologically, the serotonergic neurons look okay when visualized via tph-1::GFP fusion (the tph-1 in this fusion is presumably dead, since it only goes from the 5' upstream region to exon 4).

Behaviorally, the tph-1 mutants are definitely abnormal. 10-15% enter dauer, another 10-15% form 'partial dauer' (I am not sure what this means). If the plates are supplemented with serotonin, ~1% enter dauer. In the tph-1 background, daf-3 or daf-16 mutants also do not enter dauer.  daf-16 is a transcription factor that drives entry to dauer, and is negatively regulated by daf-2 (the initial receptor of the insulin-like signaling cascade). daf-3 is also a transcription factor that drives entry into dauer, and is negatively regulated by daf-7 (a TGF-beta ortholog).  So, one more time, daf-3 and daf-16 mutants really don't enter dauer, but daf-7 and daf-2 mutants have presumably overactive daf-3 and daf-16, and mostly produce dauers.

tph-1 mutants also have rates of pumping (~2 Hz) that are not affected by the presence of food, which are comparable to starved WT (~2.5-3Hz) but not fed WT (3.5-4 Hz). They also have lower rates of egg-laying. Both egg-laying and pumping behaviors had previously been identified as modulated by serotonin), and are also both behaviors seen typically in starved animals.  During L2 and L3, they also accumulate larger quantities of fat, although its not clear to me whether these fat stores disappear as adults or not.

The authors conclude the tph-1 behavioral patterns are probably not due to signaling of reduced food intake due to slower pharyngeal pumping, as eat mutants with similarly reduced pumping don't arrest at dauer.

Questions that I still have:

  • The article states "There is a serotonergic input to the DAF-7 pathway at the level of production of daf-7 TGF-beta neuroendocrine signal." Where does this happen? ASI?
  • ... I should probably have more questions but I worked so damn hard reading this thing that I just need to let it sink in for a while. Hopefully will come back and update.