Paper 6: HBL-1 patterns synaptic remodeling in C. elegans

TitleHBL-1 patterns synaptic remodeling in C. elegans

Year: 2012

Summary: C. elegans have five classes of motor neurons in the ventral nerve cord - A, B, VC, D, and AS. Within A,B, and D, there are both ventral and dorsal subgroups (e.g. D can be divided into VD and DD). However, at birth, only the DA, DB and DD neurons are present.

In this paper, they investigate a striking phenomenon, in which DD neurons, which initially form synapses with the ventral muscles, eliminate those synapses and instead form synapses onto the dorsal muscles (in order to be consistent with their name!). They mostly focus on the formation of new neuromuscular junctions, but note that the initial ventral axon converts to a dendritic fate (and thus presumably forms many new synapses which were not investigated in this paper), and that this rewiring process does not involve extension/retraction of neurites.

In contrast to the DD neurons, the VD neurons, which are created later in development, do not undergo this rewiring process - they initially innervate the ventral muscles, and they retain their neuromuscular junctions onto the ventral muscles.

How do DD neurons rewire? Why don't VD neurons rewire? These are the questions they want to address in this paper.

They start with a hypothesis from previous work, that a mutation in a protein called UNC-55 is responsible for the difference between VD and DD neurons. UNC-55 is highly expressed in VD, but not DD neurons. In unc-55 mutants, the VD neurons appear to rewire sometime after L2 from the ventral muscles to the dorsal muscles (as measured by UNC-57::GFP puncta, an endophilin marker for synapses, and also by recording IPSCs in the muscle, which more or less cease in the ventral muscle of the unc-55 mutant.). This means that both VD and DD target the dorsal muscles, and no other GABAergic neurons target the ventral muscles. Phenotypically, this leads to a ventral-coiling behavior, specifically during backwards movement (no idea why this only happens during backwards movement though. Perhaps VD doesn't need to drive muscle relaxation for forwards movement?)

In Drosophila, an ortholog of unc-55 represses expression of a zinc-finger transcription factor hunchback. They wondered if unc-55 repressed C. elegans hunchback ortholog, hbl-1. Driving GFP expression from an hbl-1 promoter, they find that hbl-1 promoter activity is lower in VD than DD, consistent with the hypothesis that unc-55 expression in VD is repressing the hbl-1 promoter. In unc-55 mutants, the promoter activity is much closer to, but still not quite equal. They thus conclude that hbl-1 must be repressed by unc-55 in VD, but there may be other factors that drive differential hbl-1 transcription too.

They then ask whether downregulating hbl-1 activity can suppress VD's rewiring. hbl-1 null mutations aren't viable, but there are alleles with reduced loss of function. Mutating hbl-1 in unc-55 worms partially suppresses VD rewiring. The use of the word partially here is interesting - it actually sometimes suppresses VD rewiring - for some VD neurons in some animals, they rewire to the dorsal side, or they don't. There doesn't seem to be any intermediate, as validated by looking both at microscopy images of endophilin puncta, and also looking at IPSCs in muscle. Either they get a near-normal IPSC rate, or they get no IPSCs whatsoever.

Based on this incomplete rescue effect of hbl-1 knockdown, they suggest "hbl-1 is unlikely to be the only UNC-55 target involved in D neuron remodeling." However, it seems to me equally possible that this is simply due to unc-55's repression of hbl-1 is not exactly the same as mutating hbl-1 and inducing some loss of function. What if unc-55 represses different amounts over time? Or if it represses hbl-1's function more or less than mutating hbl-1? Perhaps I'm missing something.

They then ask what the effect of hbl-1 mutations are on the DD neurons (where it is presumably active because unc-55 doesn't repress it).  They show that DD neurons switch from ventral to dorsal synapses temporally precisely, at 12-19 hr posthatching (late L1, early L2). In hbl-1 mutants. DD rewiring starts at around the same point, but by 23 hours posthatching, hasn't completed, retaining 'patches' of dorsal muscle with seemingly no NMJs present. This delayed DD rewiring doesn't seem to have to do with overall developmental slowing - other developmental processes seem to occur at the normal times.

What other than unc modulates hbl-1 expression? Driving GFP off a hbl-1 promoter and with a hbl-1 3'UTR, they find that in a mir-84 (a microRNA) mutant, GFP expression is increased nearly 8-fold. Changing the 3' UTR to a myosin 3'UTR abolished this effect. This suggests that hbl-1 is strongly post-transcriptionally regulated by mir-84. Consistent with this result, they showed that mir-84 mutants also remodel earlier.

Is activity necessary for rewiring? They first address this using GABA-related mutants which either lack a vesicular transporter VGAT or glutamic acid decarboxylase (GAD, the enzyme that makes GABA). Neither shows a change in the dynamics of DD remodeling.

What about general "network" activity (as opposed to just GABA signaling)? Is that necessary? This is a pretty loosely-defined question, but they throw some mutants at it anyhow, specifically:

  • unc-13 and unc-18 - mutants have problems with synaptic vesicle priming and docking, very low overall synaptic transmission
  • tom-1 and slo-1 - mutants that inactivate these BK channels have exaggerated synaptic transmission. tom-1 has an increased concentration of primed vesicles, and slo-1 has delayed repolarization of nerve terminals, leading to extended durations of transmission.

To cut to the chase, these mutants seem to impact hbl-1 expression, and change the developmental timing of rewiring. Whether they change the pattern of rewiring is seemingly not addressed (to be fair, I don't know how I'd address it).

Questions remaining:

This paper emphasized timing of rewiring, but what specific rewiring was happening wasn't always clear, and certainly whether certain mutations systematically changed the resulting wiring seems like it's still a very open question.

This paper was all about how neuromuscular junctions. But at the beginning of the paper, they note that the original ventral axon is converted into dendrites, and therefore must also form new synapses with upstream interneurons. How do these form?


Leftover thoughts: How do you make this kind of work quantitative? All these papers are starting to feel like laundry lists or parts lists, and I'm not very good at keeping track of parts, so I worry this isnt the approach I ought to be taking.