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First published online 19 January 2005
doi: 10.1242/dev.01638

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ceh-16/engrailed patterns the embryonic epidermis of Caenorhabditis elegans

¹ ABI/Molecular Neurogenetics, LMU Munich, 80336 Munich, Germany
² IFOM (Firc Institute of Molecular Oncology Foundation), 20139 Milan, Italy
³ Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel
⁴ BioIII/Bioinformatics and Molecular Genetics, University of Freiburg, Schaenzlestrasse 1, D-79104 Freiburg, Germany

View larger version (51K): [in a new window]   Fig. 1. ceh-16 structure, mutants and expression pattern. (A) Exon/intron structure of ceh-16 (C13G5.1) on chromosome III. The ceh-16 gene is organized in five exons, the start methionine being in the second exon and the stop codon in the fifth exon. Structure of the deletion mutants. Bottom: structure of translational gfp fusion constructs used in this study. In both constructs ceh-16 expression is driven by the endogenous promoter region and contain the unc-54 3' UTR of the plasmid pPD95.75. The construct on top rescued the ceh-16 phenotype mutant. (B) Amino acid sequence of the proposed ceh-16 gene product (187 aa), underlined: epitope for Mab 4D9 (Patel et al., 1989). (C,D) Expression pattern of the rescuing gfp construct is most robust from 250 minutes after the first cleavage (C) throughout embryonic development (D) (1.5-fold stage of elongation) until early 3-fold stage (not shown). Expression was observed in the nuclei of hyp5, H0-H2, V1-V6, T. Some of these cells are not in the focal plane of the pictures. (E,F) Antibody staining of embryos at the same developmental stages as shown in C,D. All nuclei that expressed ceh-16::gfp were also stained with the monoclonal antibody 4D9 (Patel et al., 1989). (G,H) Schematic representation of the position of ceh-16 expressing cells [based on Sulston et al. (Sulston et al., 1983)]. Scale bar: 10 µm.

View larger version (76K): [in a new window]   Fig. 2. Phenotype of ceh-16(lg16) mutants and mosaic animals. (A,C,E,G,I,K) Fluorescence micrographs of living animals [1.5-fold stage, except for E,F (bean stage) and K,L (L1 larvae)] expressing the adherens junction marker ajm-1::gfp in order to visualize cell boundaries in the epithelia. (B,D,F,H,J,L) Corresponding Nomarski pictures. (A,B) Seam cells in wild-type embryos form one line of ten cells (C,D) ceh-16(lg16) embryo (approx. same stage) with disorganized epidermal structure. These animals normally do not elongate and do not hatch. The position of seam cell nuclei and the shape of the embryo are sketched. The initiation of ectopic cell fusion events can also be observed. (E,F) Mosaic analysis: Bean-stage ceh-16(lg16) embryo rescued with ceh-16::gfp. Seam cells that do not express nuclear ceh-16::gfp are misshapen and the boundaries intermingle with dorsal and ventral cells (E, arrows). At this stage the dorsal hypodermis has not yet fused to form a syncytium. (G,H) 1.5-fold ceh-16(lg16) embryo mosaic for ceh-16::gfp. Loss of ceh-16 results in fusion with the dorsal and ventral hypodermis (arrows). (I,J) Same genotype, one cell fusion event is shown (arrow in I). Leakage of internal cells through the epidermis is shown (arrow in J). (K,L) Mosaic (semi-rescued) L1 larva: fusions are shown (arrows in K). At the same position the larva has a lump (arrow in L). Scale bars: 10 µm for embryos; 20 µm for L1 larvae.

View larger version (71K): [in a new window]   Fig. 3. eff-1 expression is repressed by ceh-16 in the embryonic seam cells. (A) ceh-16(lg16) mosaic for the rescue marker ceh-16::gfp shows seam cell fusions (arrows) and cell migrations (asterisk) (compare Fig. 2). (B) Mosaic animal as in A crossed into eff-1(hy21); suppression of fusion (arrows). (C) eff-1(hy21) animal transgenic for an eff-1::gfp transcriptional construct that is expressed in cells committed to fuse. (D) as in C, but in addition this animal is ceh-16(RNAi). Ectopic eff-1::gfp expression (de-repression) and ectopic migration in the seam cells (arrows) as a result of ceh-16 inactivation. (E-G) ceh-16(RNAi) embryo transgenic for eff-1::gfp taken at three time points: eff-1 expression (eff-1p::gfp) in seam cells is de-repressed, resulting in fusion (arrows). All embryos are transgenic for ajm-1::gfp. All animals are at 1.5-fold stage except for E (bean stage). Scale bars: 10 µm.

View larger version (75K): [in a new window]   Fig. 4. ceh-16 regulates early seam cell markers. (A,C,E) Wild-type expression by means of integrated gfp constructs [elt-5 (Koh and Rothman, 2001)] or extrachromosomal arrays [nhr-73 and nhr-74 (Miyabayashi et al., 1999)]. (B,D,F) corresponding Nomarski micrographs. (G-L) Corresponding gfp strains in which ceh-16 was knocked down by ceh-16(RNAi). (K,L) Dorsal view. All the markers were downregulated and all animals show the phenotypic hallmarks of ceh-16(—), although to a lesser extent due to lower penetrance in RNAi experiments (Table 1). (M-P) ceh-16 ectopically induces elt-5::gfp expression. Ectopic expression of ceh-16 is achieved by a heatshock-inducible promoter (see Materials and methods). (M) Effects of ceh-16 misexpression in the epidermis; upper arrow points to irregularities at the tip of the tail, which are reminiscent of failed fusion events [similar as in eff-1 mutants (Mohler et al., 2002)]. (N) Ectopic expression of elt-5::gfp (arrows). (O) Similar to M (upper rightmost arrow points to the anus). (P) Lower arrow (also entire lower margin of the larva) points to ectopic dorsal expression of elt-5::gfp. Scale bars: 10 µm for all embryos; 20 µm for all L1 larvae.

View larger version (86K): [in a new window]   Fig. 5. ceh-16 is required for the differentiation of seam cells. (A) fused seam cell in a ceh-16 mosaic animal (bracket; see also Fig. 1). (B) Nomarski micrograph of the same animal, showing the lack of alae formation at the position where the seam cell fuse (bracket). (C) Seam cell that neither fused nor expressed ceh-16::gfp during embryogenesis (bracket); arrow shows a seam cell that moved ventrally and is not elongated. (D) The seam cell did not express ceh-16::gfp embryonically or generated alae (bracket). Both animals are L1. Scale bar: 20 µm.

View larger version (79K): [in a new window]   Fig. 6. ceh-16 is required for correct segregation of the seam cells into a straight row of cells. (A) Wild-type animal with straight compartment delineations (arrows). (B,C) Seam cells in mosaic animals (see also Fig. 1) that lack ceh-16::gfp expression (arrows) migrate and/or lose their shape. (D-G) Examples of mosaic embryos that did not show any ceh-16::gfp expression. Less penetrant phenotype is shown in C; more severe seam cell movements are shown in D-G (arrows point at extreme situations). Scale bar: 10 µm.

View larger version (31K): [in a new window]   Fig. 7. Model for the function of ceh-16/engrailed in C. elegans. (A) ceh-16/engrailed keeps the seam cells in a linear organization (green), due to its repression of seam cell fusion and cell migrations. (B) During elongation, hyp7 (dorsal) start to fuse; ceh-16/engrailed acts as a fusion repressor and as a regulator of seam-cell differentiation in order to maintain a straight boundary. At this stage genes important for `seam-cell-fate' are turned on/regulated by ceh-16/engrailed. (C) Transcriptional cascade regulating seam cell fate. (D) Summary of genetic regulatory cascades in the three rows of epidermal cells preceding and during the 1.5-fold stage: Dorsal: eff-1 is expressed and required for the fusion of hyp7 into a syncytium. It is not known which genes regulate eff-1 expression in the dorsal epidermis. Medial or lateral: in the seam cells ceh-16/engrailed represses the expression of eff-1 (directly or indirectly). This is necessary to allow ceh-16/engrailed to activate genes required for correct positioning of the seam cells (cell adhesion molecules) and for the differentiation of the seam cells (elt-5 etc.). Genes that regulate ceh-16/engrailed in C. elegans are unknown. Ventral: analogous to ceh-16/engrailed in the seam cells, lin-39/HoxD4/Dfd and ceh-20/Exd are required for the regulation of elt-5 and for the repression of eff-1 to allow vulva formation (Koh et al., 2002; Shemer et al., 2004). During postembryonic development, the seam cells act as a belt with two straight boundaries that are lost in the absence of ceh-16/engrailed.