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First published online October 27, 2004
doi: 10.1242/10.1242/dev.01408

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NCR-1 and NCR-2, the C. elegans homologs of the human Niemann-Pick type C1 disease protein, function upstream of DAF-9 in the dauer formation pathways

Jie Li, Gemma Brown, Michael Ailion^*, Samuel Lee and James H. Thomas

Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA

View larger version (142K): [in a new window]   Fig. 1. Pleiotropic phenotypes of ncr-2; ncr-1 mutants. (A-C) Characteristic morphology of ncr-2; ncr-1 dauer-like larvae. (A) ncr-2(nr2023); ncr-1(nr2022) dauer-like larva. (B) daf-9(m540) dauer-like larva. (C) daf-7(e1372) dauer larva. Left and right panels show the pharynx and the alae, respectively. In the left panels, the black and white arrowheads indicate the isthmus and the terminal bulb of the pharynx. Compared with the fully constricted pharynx of a daf-7 dauer, which is similar to normal dauers, those of the ncr-2; ncr-1 and daf-9 mutants are only partially constricted. In the right panels, the black arrowheads indicate the dauer lateral alae consisting of morphologically distinct longitudinal ridges. (D-G) Pleiotropic developmental phenotypes of ncr-2; ncr-1 adults. (D) In a young wild-type adult, normal alae consist of three or four parallel longitudinal ridges. (E) Broken alae in an ncr-2; ncr-1 adult. White arrowheads indicate the remaining faint alae up to the point where they are broken. (F) Normal vulval morphology of a wild-type adult. The vulva is a slightly protruding structure, forming the opening through which fertilized eggs are expelled from the uterus. (G) Abnormal vulval morphology of a ncr-2; ncr-1 mutant. The white arrow indicates the vulval protrusion and black arrowheads indicate late stage embryos about to hatch inside the parent. Scale bars: 20 µm.

View larger version (29K): [in a new window]   Fig. 2. Cholesterol is at limiting concentration in ncr-2; ncr-1 mutants. Higher cholesterol levels suppress the Daf-c phenotype of ncr-2; ncr-1, but not the other Daf-c mutants, including daf-2(e1370ts), daf-7(e1372ts) and daf-9(m540). The dauer formation of Gen 2 worms is presented here (see Materials and methods). The error bars indicate the s.d. values of binomial populations. (B) The brood size of ncr-2; ncr-1 mutant is enhanced by higher cholesterol levels. *P<0.0001, Kruskal-Wallis nonparametric ANOVA test. (C) The average life span (±s.d.) of recovered ncr-2; ncr-1 dauers on plates containing 5 µg/ml and 20 µg/ml cholesterol is 7.7±5.1 days and 12.9±6.2 days respectively, P<0.0001, Student's t-test. The experiments in B,C were conducted at 20°C. n2; n1 is ncr-2(nr2023); ncr-1(nr2022) and n2; d12 n1 is ncr-2(nr2023); daf-12(m20) ncr-1(nr2022).

View larger version (84K): [in a new window]   Fig. 3. Progesterone treatment of wild-type worms mimics phenotypes caused by cholesterol deprivation. (A,C) Normaski micrographs of wild-type worms grown on normal plates. (B,D) Normaski micrographs of representative wild-type worms cultured on low-sterol NGM plates containing 20 µg/ml progesterone. The phenotypes of Gen 1 adults are presented (see Materials and methods). (A) Normal gonadal cell migration. (B) Abnormal gonadal cell migration in animals cultured on progesterone plates. The distal tip cell migrated on the ventral side all the way up to the pharynx, failing to make either of the programmed turns. The black arrow indicates the direction of dtc migration. (C) Under normal conditions, the sperm (white arrowheads) are enclosed within spermatheca (sp). (D) Loose sperm in N2 animals cultured on progesterone plates. White arrowheads indicate sperm found within pseudocoelomic space, probably owing to rupture of the basement membrane surrounding the somatic gonad. Scale bars: 20 µm.

View larger version (29K): [in a new window]   Fig. 4. ncr-1 and ncr-2; ncr-1 mutants are hypersensitive to progesterone. Synchronized populations of N2, ncr-1, ncr-2, or ncr-2; ncr-1 strains (Gen 1, see Materials and methods) were cultured on NGM plates with (+) or without (–) 5 µg/ml cholesterol and 20 µg/ml progesterone. The development of wild-type and mutant worms were scored after 72 hours at 20°C. The results from duplicate experiments were pooled together and expressed as a percent value (n>50).

View larger version (38K): [in a new window]   Fig. 5. Gene structures of the ncr-1 and ncr-2 loci and reporter constructs. (A) Structure of ncr-1A and ncr-1B cDNA isoforms. Black boxes represent coding and white boxes represent non-coding exon sequences. The genomic subclone represented by ncr-1A rescued the ncr-2; ncr-1 Daf-c phenotype. Also shown are corresponding positions of the original nr2022 deletion allele and additional mutations isolated from our noncomplementation screen. (B) Structure of ncr-1 reporter constructs and corresponding expression patterns. (C) Structure of ncr-2-coding region and position of nr2023 deletion allele. The corresponding genomic fragment rescued the ncr-2; ncr-1 Daf-c phenotype. (D) Structure of the ncr-2 reporter construct and its expression pattern.

View larger version (64K): [in a new window]   Fig. 6. Expression patterns of ncr-1 and ncr-2. (A-G) Expression pattern of the ncr-1Ap(l)::gfp construct. (A) Pharynx (pha), excretory cell (exc), and intestine (int) strongly express GFP, in an L1/L2 larva. (B) Enlarged view of the head region in A. Two out of the six IL2 neurons are in focus, indicated by arrowheads. The other head neurons are out of focus or obscured by strong fluorescence from the pharynx. (C) Expression in the excretory canal (ec, arrow) and rectal epithelial cells (arrowhead). (D) GFP is strongly expressed in the spermatheca (sp) of an adult worm, and also weakly expressed in the gonadal sheath and uterus wall. (E-G) Dynamic hypodermal expression of ncr-1Ap(l)::gfp during development. (E) GFP expression is first observed in the lateral hypodermal seam cells during late L1/early L2 stages. Individual seam cells are indicated by white arrowheads. (F) By the L4 stage, the entire hypodermis, consisting of seam cells and the lateral hypodermis, expresses GFP. White arrowheads indicate the edge of the lateral hypodermis. (G) After the L4/adult molt, the hypodermal expression diminishes. (H-J) Expression pattern of ncr-1Ap(s)::gfp fusion. (H) Expression in identified head neurons, including three out of the six IL2 neurons (the other three are out of focus), ASGL, pharyngeal neuron I6 and XXXL cell. (I,J) Starvation induces strong GFP expression in lateral hypodermis, but not in seam cells. Shown here are L2 stage larvae from normal growth conditions (I) and from a plate that has been starved for 12-24 hours (J). (K) The ncr-1Bp::gfp fusion is expressed in about 10 pairs of head neurons, in an L1 stage larva. (L,M) Expression pattern of ncr-2p::gfp. (L) In an adult animal, ncr-2p::gfp is strongly expressed in proximal gonadal sheath cells and weakly expressed in spermatheca. (M) ncr-2p::dsRed2 expression (left) colocalizes with daf-9::gfp (middle) in the XXX cells. ncr-2p::dsRed2 is expressed diffusely throughout the cells, compared with the perinuclear pattern of daf-9::gfp expression. In the merged view (right), daf-9::gfp is pseudocolored yellow to reveal the red color better. Scale bars: 20 µm.

View larger version (37K): [in a new window]   Fig. 7. Morphological abnormality of the ASER neuron in the ncr-2; ncr-1 mutant. (A) GFP labeled wild-type and mutant ASER neurons. The wild-type ASER neuron (top panel) consists of a cell body (c), a dendritic process (d) to the amphid sensory organ in the nose, and an axon process (a) to the nerve ring. The bottom panel shows the morphology of an ASER neuron of the ncr-2; ncr-1 mutant at the dauer stage. The mutant neuron sends out an ectopic posterior process (epp), often terminating in one or more swellings. (B) The morphological defect of the ASER neuron in ncr-2; ncr-1 mutant is transient and associated with the dauer stage. The percent value of each morphological class is reported.