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doi: 10.1242/10.1242/dev.00551

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The Caenorhabditis elegans ems class homeobox gene ceh-2 is required for M3 pharynx motoneuron function

Gudrun Aspöck^1,*, Gary Ruvkun² and Thomas R. Bürglin^1,3,

¹ Division of Cell Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
² Department of Molecular Biology, Massachusetts General Hospital and Department of Genetics, Harvard Medical School, Wellman 8, 50 Blossom Street, Boston, MA 02114, USA
³ Department of Biosciences at Novum, and Center for Genomics and Bioinformatics, Karolinska Institutet, Södertörns Högskola, Alfred Nobels Allé 7, SE-141 89 Huddinge, Sweden

View larger version (43K): [in a new window]   Fig. 1. Genomic structure of ceh-2 and sequence comparisons. (A) Alignment of homeodomains and hexapeptides of ems class genes from various species. Dots designate identical amino acids, vertical lines indicate intron positions and dashes denote artificial gaps that are introduced for alignment purposes. Abbreviations and sources of sequence data: Bf, Branchiostoma floridiae (amphioxus) (AF261146) (Williams and Holland, 2000); Dm, Drosophila melanogaster (X51653, X66270) (Dalton et al., 1989; Walldorf and Gehring, 1992); Dr, zebrafish (D32214, D32215) (Morita et al., 1995); Hs, human (X68879, X68880) (Simeone et al., 1992); Hys, Hydractinia symbiolongicarpus (cnidarian) (Mokady et al., 1998); Nv, Notophthalmus viridescens (newt) (Beauchemin et al., 1998); Mm, mouse (X68881, X68882) (Simeone et al., 1992); Xl, Xenopus laevis (Pannese et al., 1998). (B) Genomic structure of the ceh-2 gene and flanking ORFs on cosmid C17A12. The extent of the ch4 deletion is indicated underneath the gene. Rescue subclones (pTRB203, pTRB204) and reporter constructs (pTRB201, pTRB202), as well as the homeodomain (HD), hexapeptide (HP) and the position of the peptide antigen are shown. Grey boxes indicate acidic regions.

View larger version (79K): [in a new window]   Fig. 2. Expression pattern of ceh-2 in the anterior pharynx, determined by anti-peptide antibody staining (A,C) and gfp reporter constructs (D-F). Animals are oriented with anterior towards the left; cells are labeled. (A,B,E) Right subdorsal views to show all cell types; (D) dorsal view; (F) ventral view showing both pairs of M3 and NSM neurons. (A,C) An antibody against a CEH-2 peptide stains 14 nuclei in the anterior pharynx of larvae (A) and five or six cells in gastrulating embryos (C). (B) Pharynx muscle corresponding to A, stained with 3NB12 mouse monoclonal antibody (Miller et al., 1983) against a pharyngeal myosin. (D-F) ceh-2::gfp under the control of 1.6 kb promoter sequence (pTRB 201) in wild-type (D,E) and ceh-2(ch4) animals (F) is expressed in the embryonic pharynx primordium (D) and neurons of the pharynx (E,F). ceh-2::gfp expression levels and cell morphology in ceh-2(ch4) mutants are unchanged (F).

View larger version (22K): [in a new window]   Fig. 3. Homozygous ceh-2(ch4) mutants are retarded in larval development. (A) Duration of larval development in wild type (N2) and ceh-2(ch4). (B) Rescue of the slow growth defect by a ceh-2 transgene. Most ceh-2(ch4) mutants take more than 51 hours to develop into adults. Worms carrying the genomic rescue transgene pTRB203 develop faster. The error bar represents the s.d. of four independent transgenic strains; less than wild-type rescue of ceh-2(ch4) is probably due to a slight growth disadvantage of the rol-6D co-injection marker used.

View larger version (23K): [in a new window]   Fig. 4. Most electropharyngeograms (EPGs) from ceh-2(ch4) mutants lack M3 transients. Each EPG corresponds to one pharynx pump cycle (contraction and relaxation). (A-C) Typical EPGs from wild type N2 (A) and ceh-2(ch4) (B). M3 transients are marked with asterisks. Note the longer pump duration in mutants (B). EPGs of ceh-2(ch4) worms carrying a rescue transgene have normal M3 transients (C). (D) Percentage of animals with wild-type EPGs. Recordings of 10-20 EPGs from each of 9-24 animals were classified according to the presence of M3 transients in the EPGs: all EPGs from one animal lack M3 transients (white), at least one EPG from that animal has M3 transients (hatched), or all EPGs have normal (wild type) M3 transients (black). Two independent strains carrying the rescue transgene pTRB203 show rescue. The non-rescue control strain was transgenic with pTRB204.

View larger version (13K): [in a new window]   Fig. 5. ceh-2(ch4) animals display locomotion behaviors that require functional NSM neurons. Locomotion rates on agar plates of wild-type (black columns) or ceh-2(ch4) animals (white columns) were recorded in the presence (+) or absence (-) of food, with or without prior food deprivation for 30 minutes (starved/satiated) on plates containing either no fluoxetine or 75 µM fluoxetine (+). Wild type, black; ceh-2(ch4), white. Error bars indicate s.e.m.

View larger version (56K): [in a new window]   Fig. 6. hs-ceh-2 is able to partially rescue the fly ems phenotype in the brain. Laser confocal images of stage 15 embryonic Drosophila brains, lateral views. Anterior is towards the left, dorsal is upwards. ceh-2 cDNA was expressed under the control of a heat-shock promoter at embryonic stage 11 in offspring of ems9H83/TM3 sb Ubx-lacZ heterozygotes. Embryos were fixed at stage 15 and stained with anti-horseradish peroxidase (green) and anti-ß-galactosidase antibodies (not shown). Homozygous ems mutant embryos were recognized by the absence of anti-ß-galactosidase staining. (A) ems/+ heterozygous mutants have wild-type brain morphology. Head involution movements cause a backward bending of the brain towards the ventral nerve cord. Arrow in A indicates connectives at the level of neuromere b3. (B) ems/ems mutant embryos have no neurons or connectives between neuromere b1 and the ventral nerve cord (arrow) (Hirth et al., 1995). The frontal connectives that usually project to b3 (arrowhead in A) project ectopically into b1(arrowhead in B). (C) hs-ceh-2 is able to restore the missing neuromeres (arrow) and correct the projection of the frontal nerve (arrowhead) in homozygous ems mutants. Head involution movements do not occur in ems homozygous mutants (Jürgens et al., 1984) and are not restored by hs-ceh-2 expression (nor by hs-ems; data not shown). Therefore the angle between brain and ventral nerve cord is larger in rescued animals (C) compared with heterozygotes (A).