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. 2011 Aug;9(8):e1001121.
doi: 10.1371/journal.pbio.1001121. Epub 2011 Aug 9.

Opposing activities of LIT-1/NLK and DAF-6/patched-related direct sensory compartment morphogenesis in C. elegans

Grigorios Oikonomou  1 , Elliot A Perens, Yun Lu, Shigeki Watanabe, Erik M Jorgensen, Shai Shaham
Affiliations

Opposing activities of LIT-1/NLK and DAF-6/patched-related direct sensory compartment morphogenesis in C. elegans

Grigorios Oikonomou et al. PLoS Biol. 2011 Aug.

Abstract

Glial cells surround neuronal endings to create enclosed compartments required for neuronal function. This architecture is seen at excitatory synapses and at sensory neuron receptive endings. Despite the prevalence and importance of these compartments, how they form is not known. We used the main sensory organ of C. elegans, the amphid, to investigate this issue. daf-6/Patched-related is a glia-expressed gene previously implicated in amphid sensory compartment morphogenesis. By comparing time series of electron-microscopy (EM) reconstructions of wild-type and daf-6 mutant embryos, we show that daf-6 acts to restrict compartment size. From a genetic screen, we found that mutations in the gene lit-1/Nemo-like kinase (NLK) suppress daf-6. EM and genetic studies demonstrate that lit-1 acts within glia, in counterbalance to daf-6, to promote sensory compartment expansion. Although LIT-1 has been shown to regulate Wnt signaling, our genetic studies demonstrate a novel, Wnt-independent role for LIT-1 in sensory compartment size control. The LIT-1 activator MOM-4/TAK1 is also important for compartment morphogenesis and both proteins line the glial sensory compartment. LIT-1 compartment localization is important for its function and requires neuronal signals. Furthermore, the conserved LIT-1 C-terminus is necessary and sufficient for this localization. Two-hybrid and co-immunoprecipitation studies demonstrate that the LIT-1 C-terminus binds both actin and the Wiskott-Aldrich syndrome protein (WASP), an actin regulator. We use fluorescence light microscopy and fluorescence EM methodology to show that actin is highly enriched around the amphid sensory compartment. Finally, our genetic studies demonstrate that WASP is important for compartment expansion and functions in the same pathway as LIT-1. The studies presented here uncover a novel, Wnt-independent role for the conserved Nemo-like kinase LIT-1 in controlling cell morphogenesis in conjunction with the actin cytoskeleton. Our results suggest that the opposing daf-6 and lit-1 glial pathways act together to control sensory compartment size.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. daf-6 restricts amphid sensory compartment size.
In longitudinal sections and diagrams (A, B, D, F, and H) anterior is left. White scale bars, 10 µm. Black scale bars, 1 µm. (A) Schematic of the C. elegans amphid. Top: Each amphid consists of 12 neurons (only one is depicted here) and two glial cells, the sheath and the socket. Bottom: Detail of the anterior tip of the amphid. Matrix is secreted by the Golgi apparatus. tj, tight junction. Adapted from . (B, D) The ASER neuron and the amphid sheath glia visualized, respectively, with mCherry (red; driven by the gcy-5 promoter) and GFP (green; driven by the T02B11.3 amphid sheath promoter in a wild-type (B) or daf-6(e1377) (D) animal (transgenes nsEx2766 and nsEx2752, respectively)). The ASER neuron extends a single cilium through the length of the amphid channel in the wild type (arrow). In the mutant, the cilium is bent and not exposed to the environment, and the amphid pocket is bloated (asterisk). (C, E) Electron micrograph of a cross-section through the anterior portion of the amphid sheath glia channel in an adult wild-type animal (C) or a daf-6(e1377) adult mutant (E). Arrow in (C), sensory cilium. Red arrowheads indicate subcortical electron dense material. Arrow in (E), bent cilium. Asterisk, bloated sheath glia channel. Note difference in magnification between (C) and (E). (F, H) Longitudinal section through the amphid primordium of a wild-type (F) or daf-6(e1377) (H) embryo at approximately 400 min of development. Asterisk, filaments. Arrow, basal body. (G, I) Cross-section through the amphid primordium of a wild-type (G) or daf-6(e1377) (I) embryo at approximately 420 min of development. Arrow, basal body. Asterisk, bloated channel. Note difference in magnification between (G) and (I). See also Figure S1.
Figure 2
Figure 2. Loss of lit-1 suppresses the loss of daf-6.
(A) Dye-filling assay for indicated genotypes (n≥90). The lit-1(t1512) strain also contained the unc-32(e189) mutation. unc-32(e189) does not affect dye filling (unpublished data). Error bars, standard error of the mean (SEM). (B) The ASER neuron and the amphid sheath glia, visualized with mCherry (red) and GFP (green), respectively, in a lit-1(ns132); daf-6(e1377) animal (transgene nsEx2761). Arrow, ASER cilium. Left is anterior. Scale bar, 10 µm. (C) Electron micrograph of a cross-section through the amphid sheath channel of a lit-1(ns132); daf-6(e1377) adult animal. Arrow, cilium. Scale bar, 1 µm. (D) Top: Schematic of the LIT-1 protein. Light blue, non-conserved N-terminal domain. Red, conserved kinase domain. Dark blue, conserved C-terminal domain. Bottom: Alignment of the region truncated in lit-1(ns132) from different species. See also Figure S2.
Figure 3
Figure 3. Suppression of daf-6 mutations requires loss of lit-1 in glia.
(A) Image of an amphid sheath glial cell body expressing lit-1p::NLS-RFP (red; in nucleus) and vap-1p::GFP (green) (transgene nsEx2308). Yellow, overlapping expression. Left is anterior. Scale bar, 10 µm. (B) Image of an adult (head) expressing lit-1p::GFP (green) and ptr-10p::NLS-RFP (red) (transgene nsEx2159). Arrows, cells with overlapping expression. Left is anterior. Scale bar, 10 µm. (C) Dye-filling assay for indicated genotypes (n≥90). None of the transgenes had an effect on the dye filling of wild type animals (n>100, unpublished data). Error bars, SEM. p value calculated using Chi-squared test.
Figure 4
Figure 4. LIT-1 is required for amphid sensory compartment morphogenesis.
(A, B) Dye filling in animals carrying the indicated mutations (n≥100). Error bars, SEM. In (B) a sensitized dye-filling assay was used (see Experimental Procedures). (C) Left: Schematic of the arrangement of the cilia (red) and the sheath glial channel (green) in a wild-type adult animal. Not all cilia are depicted. Right: electron micrograph of cross-sections of the amphid channel. Section outlined in yellow is just below the socket-sheath junction; blue outlined section is approximately one micron posterior. Scale bars, 1 µm. (D) Same as in (C), but for a dye-filling defective lit-1(t1512) adult animal. The panel arrangement is a reflection of the one in (C). Arrowheads, tight ensheathment of individual cilia by the sheath glia. Scale bars, 1 µm.
Figure 5
Figure 5. mom-4/TAK1 mutations suppress the loss of daf-6.
(A) Dye filling in animals of the indicated genotypes (n≥90). The alleles used are: daf-6(n1543), lit-1(ns132), mom-4(ne1539). daf-6 is marked with unc-3(e151) in all strains except for mom-4; daf-6. unc-3(e151) does not affect dye filling (unpublished data). Error bars, SEM. p value calculated using Chi-squared test. (B) Schematic of Wnt signaling during endoderm specification in C. elegans. In contrast to the LIT-1 MAPK module (red), Wnt signaling does not appear to be involved in amphid sheath channel formation (see text and Table S1).
Figure 6
Figure 6. LIT-1 and MOM-4 localize to the amphid sensory compartment.
(A–F) Images of adult animals expressing the indicated GFP fusion proteins. Animals are otherwise wild-type except in (C). daf-19(m86) animals also carried the daf-16(mu86) allele to prevent dauer entry. The T02B11.3 amphid sheath promoter was used to drive all constructs. Transgenes depicted: nsEx2606 (A), nsEx2840 (B), nsEx2829 (C), nsEx2609 (D), nsEx2747 (E), and nsEx2626 (F). Anterior is to the left. Scale bars, 10 µm. (G) Quantification of channel localization of indicated LIT-1 protein fusions (n≥100). See also Figure S3.
Figure 7
Figure 7. The actin cytoskeleton is involved in amphid sensory compartment morphogenesis.
(A) Growth assay (left) and quantitative β-galactosidase enzymatic activity assay (right) demonstrating the interaction between LexA fused to the LIT-1 carboxy-terminal domain and GAD fused to fragments of ACT-4 or WSP-1. Error bars, standard deviation. f, fragment. −WL, medium without Tryptophan and Leucine. –WLH, medium without Tryptophan, Leucine, and Histidine. 3AT, 3-amino-1,2,4-triazole. A.U., arbitrary units. (B) Amphid channel localization of GFP::ACT-4 (transgene nsEx2876). Anterior is to the left. Scale bar, 10 µm. (C, D) fEM (see Experimental Procedures) of a cross-section through the amphid channel (blue trace) just below the socket-sheath junction (C) or 2 µm posterior (D). White puncta indicate mEos::ACT-4 localization. Transgene used nsEx2970. Asterisks, cilia. Scale bars, 1 µm. (E–G) Co-localization of GFP::WSP-1 and mCherry::LIT-1 at the amphid sensory compartment (transgene nsEx3245). The T02B11.3 amphid sheath promoter was used to drive all constructs. Anterior is to the left. Scale bars, 10 µm. (H) The carboxy-terminal domain of LIT-1 co-immunoprecipitates with WSP-1. Drosophila S2 cells were transfected with HA::eGFP::LIT-1Ct and with or without MYC::WSP-1. Cell lysates were immunoprecipitated using anti-MYC-conjugated agarose beads and analyzed by anti-HA immunoblot. (I) Dye filling in animals of the indicated genotypes (n≥90). The alleles used are: daf-6(n1543), lit-1(ns132), wsp-1(gm324). daf-6 is marked with unc-3(e151) in all strains. unc-3(e151) does not affect dye filling (unpublished data). Error bars, SEM. See also Table S2.
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