kette affects the migration of the midline glial cells. Frontal views of dissected embryonic nerve cords stained for the overall axon pattern using the mAb BP102 and subsequent HRP immunohistochemistry. (B–C) On the basis of their mutant phenotypes, the different kette alleles were placed in the following allelic series: kette^C3–020 = kette^J4–048 > kette^P168 > kette^G1–037 > kette^J1–070. The genotype is indicated; not all genotypes are shown. The midline glial cells (E–H) are labeled in blue using the enhancer trap insertion AA142. Anterior is up. (A) In a wild-type embryo, most CNS axons are organized in a ladder-like pattern consisting of two longitudinal connectives (lc) and two commissures in each neuromere. Anterior commissure (ac) and posterior commissure (pc) are well separated. (B) Amorphic kette mutants show fused commissures and disrupted longitudinal connectives. (C) The hypomorphic mutation J1–70 results in a subtle commissural and connective phenotype. (D) The excision mutation Δ14–2 shows the amorphic kette phenotype. (E) In stage 12/13 wild-type embryos, the midline glial cells have migrated toward the commissures and start to intercalate between anterior and posterior commissures (arrowhead). (F) In stage 16 wild-type embryos, two midline glial cells have migrated between anterior and posterior commissure. (G) In mutant kette^C3–20 embryos, the midline glial cells migrate in an abnormal path and are found on the entire width of the commissure (arrowheads). (H) In stage 16 mutant kette embryos, the midline glial cells fail to intercalate between the segmental commissure and a fused commissure phenotype develops.

Phenotypic analysis of kette. Flat preparations of stage 16 CNS (A,B,E,D) or PNS (C,F) stained for the presence of different neural markers; (A,D) mAb 1D4, (B,E) mAb BP102 and anti-REPO antibodies, (C,F) mAb 22C10. (G,H) Whole mount embryos carrying a MHC–lacZ reporter, stained for β-Gal expression to label all somatic muscle cells. (A) In wild-type embryos, the FASCICLIN II protein is expressed in discrete fascicles. (D) In kette embryos, individual fascicles cannot be recognized. Often breaks are observed in the connectives. (B) Lateral glial cells are labeled in blue. They are found in a regular pattern in wild-type embryos. (E) In mutant kette embryos, lateral glial cells appear in their normal number. However, they do not migrate along the connectives and are found above the commissures. (C) A wild-type embryo stained for the presence of the 22C10 antigen, which labels the sensory neurons as well as their axonal projections. Sensory neurons are found in four discrete clusters, the dorsal cluster (d), the lateral cluster (l), the ventro-lateral (v′), and the ventral cluster (v). (F) In homozygous kette mutant embryos, reductions in the number of sensory neurons are found in all clusters. Axonal projections into the CNS appear unaffected. (G) In wild-type embryos, a regular pattern of 30 somatic muscle fibers is seen. (H) kette mutant embryos show reduced numbers of muscle fibers. In addition, the remaining muscle fibers appear smaller than in wild-type embryos.

Expression of kette during embryogenesis. (A,B) The P-element insertion into the kette gene carries a lacZ gene and was used to monitor kette transcription with anti-β-Gal antibodies. First β-Gal expression is detected in the midline neurons of stage 11 embryos and persists until stage 14. (C–F) Show whole mount in situ hybridizations of kette cDNA in wild-type embryos. (C–E) kette mRNA is broadly expressed until stage 14. (D) From stage 13 onward, kette expression can be seen in some CNS midline cells (arrow). (F) Lateral view of a stage 16 embryo. kette expression becomes confined to the CNS.

kette affects the cytoskeleton. GFP–MOESIN was used to monitor the organization of the F-actin bundles. (A–D) Confocal images obtained from whole mount stage 16 embryos, anterior is to the left. (A) Distinct F-actin expression can be detected in the CNS axons, the ectoderm and the somatic musculature (arrow). (B) In mutant kette embryos, the CNS axon tracts are disorganized as seen in Fig. 1. The organization of F-actin fibers in the lateral body wall is abnormal. No clear concentration of staining can be found at the apical surface of the embryo. In addition, a granular appearance of F-actin staining can be observed in the nervous system as well as in the lateral body wall (arrowheads). (C) Tangential view of the dorsal epidermis. F-actin bundles line up the cell boundaries. Some cells project fine hairs (arrowhead). (D) Similar view of a kette mutant embryo. The cell borders are not staining as clear as in wild type. In addition, long hair-like structures are visible that are not found in the wild type (arrow).

kette functions in midline neurons. Frontal views (A,B,E–G) of dissected embryonic CNS preparations; anterior is up. C and D are whole mount stainings of stage 16 embryos; anterior is to the left. CNS axon tracts are labeled in brown using mAb 22C10. The midline neurons (C,D) are labeled in blue using the enhancer trap insertion X55. (A) In stage 14 wild-type embryos, the VUM axons originate in the midline, project through the posterior commissure to the anterior commissure, where they bifurcate and turn to finally leave the CNS (arrows). (B,B′) In kette mutant embryos, the VUM axons project abnormally. In every neuromere, variable defects in the projection pattern can be observed (arrows, cf. with A). (C) Lateral view of a wild-type stage 16 embryonic CNS. (D) Lateral view of a kette mutant CNS stage 16 embryo. The number and position of the midline neurons has been determined using the marker X55. No changes in number and only slight changes in position were found. (E) In a stage 16 kette^J4–48 mutant embryo, commissures are fused (arrow) and the two longitudinal connectives (lc) are closer toward the midline. (F) In a stage 16 UAS-kette; simGAL4 mutant kette^J4–48 embryo, we observe a partial rescue of the kette commissure phenotype (arrow). A space appears between anterior and posterior commissure, and the two longitudinal connectives are located in further distance from the midline. The arrowhead indicates a bifurcating VUM axon. (G) Stage 13 wild-type embryo expressing elevated levels of kette in all midline cells (UAS-kette/simGAL4). Defects in the projection pattern of the VUM neurons can be observed (arrowheads, cf. with A).

The kette locus. The extent of the kette gene is shown, transcription is from left to right, the exon-intron structure, the P-element l(3)03335 integration site, and the extend of the deletion Δ14–2 are indicated. The deduced KETTE protein and the location of the kette mutations are shown. Shaded boxes indicate deduced transmembrane domains (Baumgartner et al. 1995). The deduced KETTE protein (1126 aa), the human HEM-2 protein (1134 amino acids, accession no. 3043698), and a deduced C. elegans HEM-2 protein (1141 amino acids, accession no. 3875597) show ∼60% identity throughout the entire ORF. Amino acid changes in different kette alleles are C3-20 (W → stop) change at position 256; G1-37 (H → L) change at position 334; J4-48 (W → stop) change at position 490; J1-70 (V → A) change at position 927. All mutations affect conserved amino acids.

kette genetically interacts with dock and Drac1. (A) In wild-type larvae, the 22C10 antigen labels the photoreceptor axons as they grow from the eye disc (ed) through the optic stalk (os) into the brain. Here they terminate in the lamina or the medulla. (B) In homozygous mutant dock^P2 larvae and (C) in homozygous kette^Δ2–6 larvae, the growth of retinula axons into the brain is disrupted. (D) Following further reduction of kette function in kette^Δ2–6/kette^C3–20 larvae, this axonal phenotype becomes more pronounced. Following removal of one copy of dock in a homozygous kette^Δ2–6 larvae (E) or one copy of kette in homozygous mutant dock^P2 larvae (F), we observed a much more severe axonal projection phenotype. (G–L) Frontal views of dissected embryonic CNS preparations. Anterior is up. (G) We used mAb BP102 to visualize the overall CNS axon pattern in mutant dock embryos. The zygotic dock phenotype is characterized by a reduction in the longitudinal connectives (arrow) and a slight fusion of the segmental commissures. (H) To determine the morphology of the VUM neurons, we used the mAb 22C10.As in mutant kette embryos, variable projection defects of the VUM axons were found in mutant dock embryos (arrows). (I) Stage 14 embryo of the genotype UAS–Drac1V12; simGAL4 expressing activated DRAC1 in all midline cells. Frequent defects in the projection pattern of the VUM neurons are observed (arrowheads). (K) In stage 16 embryos, a fused commissure phenotype can be seen (arrows). (L) Expression of Drac1 in all CNS midline cells of mutant kette embryos partially rescued the mutant CNS phenotype. The longitudinal connectives (lc) appeared further apart and the commissures appear separated. In addition, we often observed VUM axons projecting normally at the midline (arrow).