MLT-10 defines a family of DUF644 and proline-rich repeat proteins involved in the molting cycle of Caenorhabditis elegans

Abstract

The molting cycle of nematodes involves the periodic synthesis and removal of a collagen-rich exoskeleton, but the underlying molecular mechanisms are not well understood. Here, we describe the mlt-10 gene of Caenorhabditis elegans, which emerged from a genetic screen for molting-defective mutants sensitized by low cholesterol. MLT-10 defines a large family of nematode-specific proteins comprised of DUF644 and tandem P-X(2)-L-(S/T)-P repeats. Conserved nuclear hormone receptors promote expression of the mlt-10 gene in the hypodermis whenever the exoskeleton is remade. Further, a MLT-10::mCherry fusion protein is released from the hypodermis to the surrounding matrices and fluids during molting. The fusion protein is also detected in strands near the surface of animals. Both loss-of-function and gain-of-function mutations of mlt-10 impede the removal of old cuticles. However, the substitution mutation mlt-10(mg364), which disrupts the proline-rich repeats, causes the most severe phenotype. Mutations of mlt-10 are also associated with abnormalities in the exoskeleton and improper development of the epidermis. Thus, mlt-10 encodes a secreted protein involved in three distinct but interconnected aspects of the molting cycle. We propose that the molting cycle of C. elegans involves the dynamic assembly and disassembly of MLT-10 and possibly the paralogs of MLT-10.

Figure 1.

Genetic screen for mutants more sensitive to low cholesterol. (A) Representative Nomarski micrographs of C. elegans grown on LCNGM for one or two generations. Left, an adult; right, a larva trapped in partly shed cuticle (arrowhead). (B) Design of the screen. (C) Growth of L1 larvae to the adult stage on LCNGM (gray) or NGM (black) after cultivation for 5 d at 20°C. Parents of the observed animals were cultured on NGM.

Figure 2.

Mutations of mlt-10 hinder the removal of larval cuticles. (A) Representative Nomarski micrographs of mlt-10(mg364) mutants. Top, a midstage larva; bottom, an adult encased in the L4-stage cuticle (arrow). (B) Animals of each genotype were collected as embryos, cultured on NGM for 3–5 d at 20°C, and inspected using a dissecting microscope. Graph shows the percent of animals trapped in a larval cuticle. Not all genotypes were tested concurrently. (C and D) Representative Nomarski micrographs show animals of the indicated genotypes. (C) Animals were collected as embryos, cultured on NGM for 45 h at 20°C, and inspected using a compound microscope. Arrows indicate the buccal caps. (D) Hatchlings were collected and cultured on LCNGM for 3 d at 20°C. Arrows indicate unshed cuticles from the L4 larval stage. Scale bars, 10 μm.

Figure 3.

MLT-10 defines a large family of nematode-specific proteins. (A) Diagram of the C. elegans mlt-10 gene and flanking sequences. Black boxes represent exons, and gray boxes represent untranslated sequences. Nucleotide positions correspond to cosmid C09E8 (Accession no. gb AF077529). Table S3 further describes these mutations of mlt-10. (B) Diagram of the predicted MLT-10 protein (Accession no. gi 17531703) showing the signal sequence (gray), potential acceptor sites for N-linked glycans (●), conserved cysteine residues; DUF644 (red), and the proline-rich region (black). (C) Sequence alignment of the predicted paralogs and selected orthologues of MLT-10. Amino acid positions correspond to Ce MLT-10. Acidic and basic residues are shaded blue and red, respectively. Prolines are shaded black and hydroxy amino acids are shaded gray. The H590 residue affected by mg364 is boxed. Accession numbers for these sequences are Ce, ref NM_061354; Cbr, gi 187040316; Cre, gi 183180662; and Bm1_2748, gi 170584318. Table S4 provides additional information about the mltn genes of C. elegans.

Figure 4.

Spatial and temporal expression pattern of mlt-10. (A) Representative fluorescence and Nomarski micrographs show expression of the mlt-10p::gfp-pest fusion gene. GFP was detected in the major body hypodermal syncytium (arrow), the dorsal and ventral ridges of the hypodermis (asterisk), and the anterior hypodermis (arrowhead) of the late L4 stage larva. All fluorescence images were acquired with an exposure time of 187 ms. (B) Detection of mlt-10 transcripts by RT-PCR. Larvae were cultured on bacteria that expressed the indicated dsRNAs for 40 h at 25°C. Animals were harvested at the typical time of the L4-to-adult molt. Detection of ama-1 transcripts controlled for the quality of RNA samples and RT-PCR reactions.

Figure 5.

Localization of the MLT-10::mCherry and GFP::MLT-10 fusion proteins. (A–D) Representative fluorescence and Nomarski micrographs show expression of the fusion proteins. (A) MLT-10::mCherry detected in vesicle-like objects (arrow) in the lateral epidermis of a late L4 larva molting to the adult stage. Alae are visible on the underlying cuticle (arrowhead). (B) GFP::MLT-10 detected in vesicles (arrow) in the lateral epidermis of a molting larva. A double cuticle covers the mouth (arrowhead). (C) Cherry detected in a coelomocyte (arrow) of a young adult. (D) MLT-10::mCherry found in strands (arrows) near the surface of a young adult. Scale bars, 10 μm.

Figure 6.

Increased permeability of the cuticle in mlt-10 mutants. (A–D) Representative fluorescence micrographs of larvae stained with Hoechst 33258. All images were acquired with an exposure time of 50 ms. Scale bar, 10 μm. (E) The fraction of larvae with nuclei stained by Hoechst 33258. Values represent the average of two independent experiments; error bars, SEM. Asterisks indicate a significant difference from wild-type animals (p ≤ 0.05). (F) Early L1 stage larvae of the indicated genotypes were collected and cultured on high-salt or standard NGM plates for 3 d at 20°C. Values represent the average of two independent experiments; error bars, SEM.

Figure 7.

Disorganization of a cuticle collagen in mlt-10 mutants. (A–D) Representative confocal fluorescence micrographs show COL-19::GFP in the adult exoskeleton. Arrows point to disorganized assemblies of COL-19::GFP flanking the longitudinal alae. Scale bar, 10 μm.

Figure 8.

Malformation of the adult-specific alae in mlt-10 mutants. (A–F) Representative Nomarski micrographs show the adult exoskeleton. Arrow, abnormalities in the alae including gaps, branches, and regions with four ridges. Arrowhead, an atypical structure in the cuticle. Each region of interest was digitally magnified 2.5-fold for display in the inset. Scale bar, 10 μm.

Figure 9.

Abnormal development of the epidermis in mlt-10 mutants. (A–F) Fluorescence micrographs show expression of AJM-1::GFP and the nuclear scm::gfp reporter in the lateral hypodermis. The anterior end of each worm is to the right, and the ventral side is down. All images were acquired with an exposure time of 500 ms. Scale bars, 10 μm. (A–D) Hatchlings were cultured on NGM for 16 h at 25°C before imaging. Arrows, irregularly shaped seam cells in the late L1 larvae. (E and F) Larvae were imaged late in the L4 stage. Arrows, an irregularly shaped region of the syncytial seam with extra nuclei. (G and H) Representative fluorescence and Nomarski micrographs show adults. Arrows, examples of GFP detected outside of the syncytial seam. Ectopic expression of GFP was observed in 80% (n = 54) of mlt-10(tm3331) mutants, 73% (n = 44) of mlt-10(ok2581) animals, and 81% (n = 114) of mlt-10(mg416m364) adults, but was not observed in wild-type animals (n = 36).