Phenotypes of elt-5(RNAi) embryos and larvae. (A,C,E,G) Wild-type; (B,D,F,H) elt-5(RNAi) animals. (A) Nomarski image of wild-type L1 larva. Arrow points to the buccal capsule, which is attached to the anterior end of the worm. (B) elt-5(RNAi) L1 larva showing lumps (arrowhead) and a detached buccal capsule (arrow) at the anterior end of the pharynx, which has contracted toward the posterior. (C-F) Embryos stained with mAb MH27 to visualize adherens junctions of epidermal cells. The asterisks mark the V1 seam cell. (C) Lateral view of a wild-type embryo at the ∼2.5-fold stage. The row of 10 lateral seam cells, all completely surrounded by adherens junctions, is prominently visible. (D) Lateral view of an elt-5(RNAi) embryo at the ∼2.5-fold stage. One of the seam cells, V1 (asterisk), does not show adherens junctions, indicating that it has fused with the neighboring hyp7 syncytium on the dorsal and ventral sides. (E) Lateral view of a wild-type embryo slightly past the comma stage. All ten seam cells are visible, although part of the most posterior seam cell, T, is out of focus. (F) Lateral view of an elt-5(RNAi) embryo slightly past the comma stage. One of the seam cells, V1 (asterisk), is ventrally misplaced, and its neighbors, H2 and V2, inappropriately contact each other. (G,H) L1-stage larvae expressing the seam cell marker SCM (nuclear signal) and JAM-1::GFP, a maker for adherens junctions. (G) Wild-type larva showing SCM expression in seam cells only. (H) elt-5(RNAi) larva showing SCM expression in nuclei of syncytial cells (arrowheads) near a fused seam cell (arrow).

Alae defects in elt-5(RNAi) larvae. (A,C,E) Wild-type; (B,D,F) elt-5(RNAi) larvae. (A,B) Nomarski images of L1 larvae. (A) Wild-type larva, showing normal alae, visible as two ridges along the length of the body (arrowheads). (B) elt-5(RNAi) larva in which alae are absent over most seam cells (short and long arrow) and malformed over two others (box). (C,D) Fluorescence images of the adherens junctions (revealed with JAM-1::GFP) of the larvae shown in A and B, respectively. (C) All seam cells show adherens junctions in the wild-type larva. (D) Some of the seam cells (short arrow) have fused to the epidermal syncytium in the elt-5(RNAi) larva. Some of the unfused seam cells (long arrow) do not have alae. (E,F) Details from A and B, respectively. Areas of detail are marked by the boxes in A,B.

Embryonic expression of seam and non-seam epidermal markers. Wild-type expression patterns are shown in the left panels, expression in elt-5(RNAi) embryos is shown on the right. (A-F) Approximately threefold stage embryos carrying NR reporters. (A,B) nhr-75::GFP is expressed in seam cells in wild-type (A) but not in elt-5(RNAi) (B) embryos. (C,D) nhr-73::GFP is expressed at high levels in both wild-type (C) and elt-5(RNAi) (D) embryos. (E,F) nhr-77 is expressed at high levels in seam cells in wild-type (E) and weakly and sporadically in elt-5(RNAi) (F) embryos. In the elt-5(RNAi) embryo, a single seam cell shows a barely detectable level of GFP expression (arrow). (G,H) elt-3::GFP expression in wild-type and elt-5(RNAi) embryos at the ∼1.5-fold stage. JAM-1::GFP expression in adherens junctions was included to help in identifying seam cells. The arrows indicates two seam cells, H2 and V3, in G,H. (G) elt-3::GFP is expressed in all non-seam major epidermal nuclei, and is excluded from all seam nuclei in a wild-type embryo. (H) elt-5(RNAi) embryo showing ectopic expression of elt-3::GFP in seam cells. Eight seam nuclei show expression, two of which are indicated by the arrows. A gap in the adherens junction of H0 (arrowhead) reveals that the cell is undergoing fusion.

Summary of elt-5 and -6 reporter construct expression. The exon-intron structures of the elt-5 and -6 genes and approximate distances in kb are shown at the top. Short and long oval shapes represent GFP and lacZ-coding regions, respectively. +/- indicates weak expression. See Materials and Methods for a more detailed description of the constructs. See text for a description of the AB+MS lineage, seam and nervous system expression.

Expression patterns of elt-5 and -6 reporters and endogenous ELT-5 and -6 proteins. (A,B) Expression of the elt-5 translational fusion, pKK52. (A) Ventral view of a 28-cell stage embryo. GFP is seen in the nuclei of all four MS granddaughters (arrowheads) and all 16 AB great great granddaughters. Only 14 AB descendants are visible in this focal plane. (B) Lateral view of a comma-stage embryo. Strong GFP expression is present in seam cells and several cells in the head, tentatively identified as neurons and/or neuronal support cells. Somewhat weaker expression is observed in many other cells, mostly in the head and tail regions; these are likely to be descendants of the AB and MS founder cells. (C,D) Embryos at the ∼1.5-fold (C) and ∼threefold (D) stages stained with anti-ELT-5 (red) and MH27 (green). High levels of ELT-5 are detected in all seam cells and in many other cells in the head and tail regions at these stages. (E,F) Expression patterns of the elt-6 transcriptional fusion reporter, pKK41. (E) Embryo at the ∼1.5-fold stage shows strong GFP expression in several cells in the head region, tentatively identified as neurons and/or support cells, and much weaker expression in seam cells. (F) Head region of an L1-stage larva showing long processes in GFP-expressing cells. These are likely to be neurons and/or support cells. GFP is also present in seam cells at this stage, but they are not visible in this focal plane. (G,H) Embryos stained with anti-ELT-6 (red) and MH27 (green) at the ∼1.5-fold (G) and ∼2.5-fold (H) stages. The staining pattern is similar to the expression pattern of the reporter construct shown in E. The arrowheads point to a seam cell (V2) in B-E,G,H, and the arrows point to cells in the nervous system.

Rescue by ELT-6 restores normal alae and uncovers post-embryonic defects in elt-5(RNAi) animals. (A,B) L1-stage elt-5(RNAi) larva in which the nhr-74::GFP::ELT-6 fusion (pKK49) was expressed in seam cells. (A) Interior view showing that the buccal capsule (arrow) is not attached. (B) Surface view showing that alae are restored in larvae expressing GFP::ELT-6 in seam cells (compare with Fig. 3B,F). (C,D) Fluorescence image of epidermal adherens junctions (JAM-1::GFP) in L2-stage larvae. (C) Wild-type larva showing that adherens junctions surround all seam cells. (D) elt-5(RNAi) larva carrying a construct (pKK25) that drives ELT-6::GFP expression in AB and MS descendants. All but one of the seam cells (arrow) lack visible adherens junctions and have therefore fused with the surrounding epidermal syncytium. Arrows point to the H0 seam cell in C,D. (E,F) Molting defects in elt-5(RNAi) larvae expressing GFP::ELT-6 in AB and MS descendants but not in seam cells (pKK25). (E) Larva arrested at the L2-L3 molt showing its inability to break through the old cuticle, resulting in a plugged mouth. (F) Larva arrested at the L3-L4 molt with cuticle in the tail region still attached. The space between the old and new cuticle has collected waste material (arrows) and the old cuticle has formed a constriction (arrowhead).