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. 2012 Nov;139(22):4191-201.
doi: 10.1242/dev.083428.

Intercellular calcium signaling in a gap junction-coupled cell network establishes asymmetric neuronal fates in C. elegans

Jennifer A Schumacher  1 , Yi-Wen Hsieh, Shiuhwei Chen, Jennifer K Pirri, Mark J Alkema, Wen-Hong Li, Chieh Chang, Chiou-Fen Chuang
Affiliations

Intercellular calcium signaling in a gap junction-coupled cell network establishes asymmetric neuronal fates in C. elegans

Jennifer A Schumacher et al. Development. 2012 Nov.

Abstract

The C. elegans left and right AWC olfactory neurons specify asymmetric subtypes, one default AWC(OFF) and one induced AWC(ON), through a stochastic, coordinated cell signaling event. Intercellular communication between AWCs and non-AWC neurons via a NSY-5 gap junction network coordinates AWC asymmetry. However, the nature of intercellular signaling across the network and how individual non-AWC cells in the network influence AWC asymmetry is not known. Here, we demonstrate that intercellular calcium signaling through the NSY-5 gap junction neural network coordinates a precise 1AWC(ON)/1AWC(OFF) decision. We show that NSY-5 gap junctions in C. elegans cells mediate small molecule passage. We expressed vertebrate calcium-buffer proteins in groups of cells in the network to reduce intracellular calcium levels, thereby disrupting intercellular communication. We find that calcium in non-AWC cells of the network promotes the AWC(ON) fate, in contrast to the autonomous role of calcium in AWCs to promote the AWC(OFF) fate. In addition, calcium in specific non-AWCs promotes AWC(ON) side biases through NSY-5 gap junctions. Our results suggest a novel model in which calcium has dual roles within the NSY-5 network: autonomously promoting AWC(OFF) and non-autonomously promoting AWC(ON).

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Figures

Fig. 1.
Fig. 1.
NSY-5 gap junctions mediate small dye transfer between C. elegans embryonic neurons. (A) Schematic of the dye transfer technique. Black open circles, NPE-HCCC2; blue dots, HCCC2; red rectangles, gap junctions. (B-G) One pair of nsy-5p::mCherry cells from wild-type embryos. (B) Differential interference contrast (DIC) and (C) mCherry images. (D-G) Images of fluorescent HCCC2 in the coupled cells prior to UV (360 nm) uncaging (D), immediately after the first (E) and second (F) localized uncaging of one cell (donor), and when the transfer of fluorescent HCCC2 from donor cell to recipient cell reached equilibrium (G). (H) Time course of fluorescence intensities of HCCC2 (FHCCC2) in the wild-type coupled cells. D, E, F and G indicate the time points at which the images in D, E, F and G, respectively, were acquired. (I-N) One pair of nsy-5p::mCherry-expressing cells from nsy-5(ky634lf) embryos. (I) DIC and (J) mCherry images. (K-N) Images of fluorescent HCCC2 before localized uncaging of cell 1 (K), immediately after uncaging (L) and 40 seconds (M) and 380 seconds (N) after uncaging. (O) Time course of fluorescence intensities of HCCC2 (FHCCC2) in the nsy-5(lf) cell pair. K, L, M and N indicate the time points at which the images in K, L, M and N, respectively, were acquired. Scale bar: 10 μm.
Fig. 2.
Fig. 2.
Calcium buffer expression in the NSY-5 network disrupts AWC asymmetric gene expression and function. (A-D) Expression of the reporter gene str-2p::GFP in wild type (A), nsy-5(ky634lf) (B), unc-36(e251lf) (C) and odr-3p::calbindin D28K transgenic worms (D). Anterior is left; ventral is down. Arrowheads indicate AWC cell bodies. Scale bar: 10 μm. (E) AWC phenotypes of wild type, mutants and calcium buffer-expressing animals. (F) Mean relative chemotaxis indices. Wild-type animals are non-transgenic siblings from odr-3p::calbindin D28K extrachromosomal arrays. bu, 1:1000 butanone; pd, 1:10,000 2,3-pentanedione. Significance was calculated using t-test. Error bars indicate s.e.m.
Fig. 3.
Fig. 3.
Calbindin D28K acts as an intracellular calcium buffer in NSY-5 network embryonic neurons. (A) AWC phenotypes of calcium buffer (odr-3p::calbindin D28K) and calcium sensor (odr-3p::cmd-1) expressing animals alone or in the unc-2(zf35gf) background. (B) Representative traces of calcium dynamics in cultured C. elegans embryonic neurons during addition of the stimulus (ionomycin) followed by calcium chelator (EGTA). Horizontal lines indicate the duration of ionomycin and EGTA application. ΔF, the change in Fluo-4 fluorescence; F0, the resting Fluo-4 fluorescence prior to ionomycin stimulation. (C) Average maximum change in Fluo-4 intensity following ionomycin application. Significance was calculated using t-test. Error bars indicate s.e.m.
Fig. 4.
Fig. 4.
Calcium buffers antagonize the unc-2/unc-36 calcium signaling pathway. (A) AWC phenotypes of animals expressing calcium buffers alone or in mutants defective in AWC asymmetry. cbn, calbindin D28K; parv, parvalbumin. (B) The AWC asymmetry determination pathway.
Fig. 5.
Fig. 5.
Calcium acts autonomously in AWCs to promote AWCOFF and non-autonomously in non-AWCs to promote AWCON. (A-D) Expression of an integrated str-2p::GFP transgene and an unstable transgenic array bearing odr-3p::calbindin D28K or nsy-5p::calbindin D28K and the mosaic marker odr-1p::DsRed in non-transgenic (A), non-mosaic (B) and representative mosaic (C,D) worms. AWC neurons that express both GFP and DsRed appear yellow. (C) Mosaic animal that retains odr-3p::calbindin D28K in AWCL. (D) Mosaic animal that lost nsy-5p::calbindin D28K in both AWCs but retained the transgene in both AWBs. Arrowheads indicate AWC cell bodies; arrows indicate AWB cell bodies. Anterior is left; ventral is down. Scale bar: 10 μm.
Fig. 6.
Fig. 6.
Model of calcium function in left-right AWC asymmetry. (A) Calcium functions cell-autonomously within AWCs to promote the AWCOFF fate. Both AWC cells, before cell-cell communication, have calcium influx through voltage-gated calcium channels to maintain the default AWCOFF fate. (B) Calcium mediates intercellular signaling between non-AWCs and AWCs within the NSY-5 network to induce one AWCON and thus has a nsy-5-dependent non-cell-autonomous role in AWC asymmetry. The relative calcium level in the two AWC cells determines asymmetric AWC subtypes: the AWC with a lower calcium level becomes the induced AWCON, whereas the contralateral AWC with a higher level of calcium remains as the default AWCOFF. AWB cells express nsy-5, but do not directly contact AWC. AWBs may communicate with AWCs through other cells in the NSY-5 network. Gray rectangles represent gap junctions identified from EM reconstructions of adult (White et al., 1986). Blue cylinders represent NSY-5 gap junction channels.
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