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First published online December 22, 2004
doi: 10.1242/10.1242/jcs.01609

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Nuclear receptor NHR-25 is required for cell-shape dynamics during epidermal differentiation in Caenorhabditis elegans

Marie ilhánková^1,2, Marek Jindra^1,3 and Masako Asahina^2,*

¹ Department of Molecular Biology, Faculty of Biological Sciences, University of South Bohemia, CZ – 370 05, Czech Republic
² Institute of Parasitology ASCR, eské Budjovice, 37005 Czech Republic
³ Institute of Entomology ASCR, eské Budjovice, 37005 Czech Republic

View larger version (21K): [in a new window]   Fig. 1. A schematic drawing of seam-cell lineages. Ten seam cells in a freshly hatched worm (top) will produce 16 seam cells of the adult. The lineage of the tail seam cell T is not shown. Blue circles represent anterior daughters that fuse with the hyp7 syncytium and green diamonds denote stem seam cells. Pd indicates descendants of the V5.pa cell forming the postdeirid. Anterior is to the left. Based on Sulston and Horvitz (Sulston and Horvitz, 1977).

View larger version (112K): [in a new window]   Fig. 2. Seam cells fail to restore contacts in nhr-25(RNAi) L1 worms. Confocal sections are shown with adherens junctions visualized by using the anti-DLG-1 antibody (Bossinger et al., 2001). The images were taken approximately 3 hours (A), 5 hours (B), 7 hours (C) and 9 hours (D) after hatching. Examples of V and P cells are shown. Anterior seam-cell daughters fusing with the hyp7 syncytium are indicated with arrows. Arrowheads mark seam-cell elongation and the restoration of their mutual contacts. Anterior is to the left, dorsal up. Bars, 10 µm; bar in A applies to all panels except I.

View larger version (117K): [in a new window]   Fig. 3. The lack of cell-cell contacts in nhr-25(RNAi) L2 worms affects the fate of the seam-cell anterior daughters but not the ability of V5.p to produce a postdeirid neuron. Adherens junctions were stained with the anti-DLG-1 antibody. For simplicity, V cells are labeled with the names of their L1-stage ancestors. (A,B) In wild-type L2 worms, all seam cells remain in contact before and throughout their doubling division. V5.p produces an anterior neuroblast that is in the process of cell divisions, forming the future postdeirid (Pd). Contacts with descendants of V4.p and V6.p are shown by arrows. (C) In nhr-25(RNAi) animals, dividing seam cells remain round and isolated except for H1, whose contact with H0 is not interrupted because H1 had produced its hyp7 daughter towards the posterior. (D,E) The V5.pa daughters (brackets) divide and migrate dorsally in a postdeirid-like fashion regardless of the lack of contacts (arrowheads) with either V4.p (D) or V6.p (E) descendants in L2 nhr-25(RNAi) larvae. (F,G) Simultaneous propidium iodide and DLG-1 staining at the early L3 stage reveals the nuclei of the four postdeirid cells (Pd) and the normal adherens junctions (arrows) between V cells (F). The postdeirid structure differentiates normally in the absence of both anterior and posterior cell contacts of V5.p descendants (G, arrowheads) in rrf-3(pk1426); nhr-25(RNAi) worms. (H,I) Superfluous seam cells in nhr-25(RNAi) L2 worms express the scm::gfp marker (nuclear signal in H) and might form clusters (I). In all images, anterior is to the left and dorsal up. Bars, 10 µm; bar in A applies to all panels except C.

View larger version (46K): [in a new window]   Fig. 4. A mab-5::lacZ reporter gene is ectopically expressed in seam cells anterior to V6.p in nhr-25(RNAi) L2 larvae. Bracket in (B) shows ectopic ß-galactosidase activity in seam cells and their daughters. V6.p cells are indicated by arrows, arrowheads in (C) mark other seam cells on lateral sides. Additional staining comes from ventral P cells and neuroblasts. Anterior is to the left and dorsal up in (A,B); the animal in (C) is viewed from the ventral side. Tails of the nhr-25(RNAi) larvae show typical malformations. Bar, 20 µm.

View larger version (72K): [in a new window]   Fig. 5. nhr-25 RNAi affects development of L4-adult seam epidermis and expression of a cadherin cdh-3::gfp reporter. (A) Direct GFP fluorescence of cdh-3::gfp reveals continuity of the seam and proper formation of the vulva in control L4 larvae. (B,C) Large gaps separate seam cells in worms subjected to nhr-25 RNAi by feeding since hatching (B) or starting at 14 hours after hatching (C). Arrowhead points to a seam cell that failed to elongate in the anteroposterior direction, asterisks mark the positions where the vulva would normally develop. (D,E) Confocal images acquired under identical settings show that cdh-3::gfp expression is reduced or nearly undetectable in nhr-25 dsRNA-fed L4 animals (E) compared with controls (D). (F) Fusion of incorrectly aligned seam cells in late L4 nhr-25(RNAi) larvae (obtained by dsRNA injection of their parents) causes bifurcations and loops in the syncytium, as revealed by cdh-3::gfp. Arrows point to three seam nuclei. (G,H) L4 progeny of adults injected with nhr-25 dsRNA contain superfluous, irregularly spaced seam-cell nuclei marked by scm::gfp (H, arrows) compared with controls (G).

View larger version (53K): [in a new window]   Fig. 6. NHR-25 is expressed in the seam cells throughout development. (A,A') An antibody against NHR-25 stains nuclei of seam cells, marked with scm::gfp and outlined by MH27 antibody staining of adherens junctions in the same L1 larva. (B,C) Expression of an nhr-25::gfp construct in seam cells of L2 (B, arrows) and L4 animals. In all panels, anterior is to the left. Arrowheads point to hyp7 daughters of seam cells.

View larger version (163K): [in a new window]   Fig. 7. nhr-25 RNAi disrupts formation of the adult alae and the lateral epidermis. (A,B) Transmission electron microscopy sections reveal aberrant cuticle with shallow alae (arrows) in nhr-25 RNAi adults (B). The lateral hyp7 syncytium (h) has an abnormal structure, with much of the dense membrane system missing. The dark material (B) is yolk not taken up by the gonad. (C,D) Scanning electron micrographs of wild-type (C) and nhr-25(RNAi) (D) adults viewed from the lateral side. Alae are shown with arrows, bracket (D) indicates a lateral region of sparse annuli. (E) A gap in the alae (delimited by arrowheads) caused by nhr-25 RNAi. (F,G) Branching of the adult alae caused by a dominant mutation of the cuticle gene rol-6 in the SU93 strain (F) is enhanced by nhr-25 RNAi (G); a severe case is shown, corresponding to `>1 small loops' in Table 1. The underlying seam adherens junctions, marked by expression of the ajm-1::gfp reporter (F') mirrors the shape of the alae. Arrowheads indicate the branching points. Bars, 2 µm (A,B), 10 µm (C,D), 20 µm (E-G).

View larger version (105K): [in a new window]   Fig. 8. nhr-25 RNAi disrupts the ultrastructure of the epidermis and molting. (A,B) Compared with a control (A), the ventrolateral hyp7 epidermis is extremely thick in nhr-25(RNAi) adult (B, double arrow), thus separating muscles from the cuticle. The fibrous organelles that normally attach muscles to the cuticle (arrows) are not apparent (B). Likewise the struts between the cuticle layers (arrowheads) are missing; instead, abnormal vesicles of unknown content are present in nhr-25(RNAi) animals (B). Bar, 2 µm. (C,D) Wild-type worms fed on nhr-25 dsRNA-expressing E. coli from the time of hatching display molting defects. Examples show unshed L4 cuticle, attached to the head (C) or causing constrictions (D) in affected adults.

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