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The Kallmann syndrome gene homolog in C. elegans is involved in epidermal morphogenesis and neurite branching

Elena I. Rugarli^1,*,, Elia Di Schiavi^2,*, Massimo A. Hilliard², Salvatore Arbucci², Cristina Ghezzi¹, Anna Facciolli², Giuseppe Coppola², Andrea Ballabio^1,3 and Paolo Bazzicalupo^2,

¹ Telethon Institute of Genetics and Medicine (TIGEM), via P. Castellino III, 80131 Naples, ltaly
² International Institute of Genetics and Biophysics (IIGB), via P. Castellino III, 80131 Naples, ltaly
³ Faculty of Medicine, II University of Naples, Naples, ltaly
^* These two authors contributed equally to this work

View larger version (67K): [in a new window]   Fig. 1. Evolutionary conservation of KAL proteins. (A) Amino acid sequence alignment of known KAL homologs. KAL, human KAL protein; KALc, chicken KAL protein; zKAL1.1 and zKAL1.2, zebrafish KAL proteins (Ardouin et al., 2000); and CeKAL, C. elegans KAL protein. Stars highlight conserved cysteines. The WAP domain is underlined. In the most conserved part of the protein, containing the cysteine-rich region, the WAP domain and the first FNIII repeat, identity and similarity of residues between KAL and CeKAL are approximately 30% and 50%, respectively. (B) The domain topology of KAL proteins. The order and the relative distance of all domains are conserved. Abbreviations: SP, signal peptide; C-rich, cysteine-rich domain; WAP, WAP domain; FNIII, fibronectin type III repeat; GPI, glycosyl-phosphatidyl-inositol anchoring site.

View larger version (18K): [in a new window]   Fig. 2. Structure of the kal-1 locus and of the constructs used in this study. (A) Structure of the kal-1 locus on cosmid K03D10. Introns 1, 2, 3, 4 and 5 (not drawn to scale) are 7025, 1968, 949, 471, 888 nucleotides long, respectively. There is a putative transplicing site 3 bp upstream of the ATG. The 3' untranslated region is 211 nucleotides long. kal-1 is at one extremity of linkage group 1, near the gene unc-54 (map coordinates 22,88). The regions corresponding to the conserved protein domains are indicated. The approximate position of the gb503 deletion is indicated. (B) Reporter expression constructs and KAL proteins overexpression constructs used in this work. The kal-1 regulatory region, present in all constructs, corresponds to 4.3 kb of genomic sequences upstream and including the ATG of C. elegans kal-1. It drives expression of an E. coli lacZ gene (GB105), a gfp gene (GB102), the kal-1 cDNA, including the signal peptide (CeKAL), or the human KAL cDNA (HuKAL, see Materials and Methods). Abbreviations: SP, signal peptide; C-rich, cysteine-rich domain; WAP, WAP domain; FNIII, fibronectin type III repeat; NLS, nuclear localization signal.

View larger version (136K): [in a new window]   Fig. 3. kal-1 mutants show embryonic lethality and L1 larvae morphological abnormalities. (A-D) DIC photomicrographs of embryos. (A) Control embryo at one and a half-fold stage; (B-D) mutant embryos in which ventral enclosure has failed; cells protrude ventrally outside of the embryonic mass (white arrows). These embryos will not hatch and they will eventually die. Confocal images of embryos stained with rhodaminated phalloidin (E,F) or AbMH27 (G,H). (E) Wild-type comma stage embryo showing the ordered pattern of cell boundaries. (F) This pattern is disrupted in a mutant, where cells establish ectopic contacts and are abnormally oriented (white arrow), clumping together and generating star-like shapes (white arrowhead). (G) In later wild-type embryos (threefold stage), each epithelial cell contacts only one anterior and one posterior partner. In mutant embryos of comparable stage (H), epithelial cells clump together (white arrow) and the normal pattern cannot be recognized. (I-L) DIC photomicrographs of L1 larvae. (I) Control L1 larva. (J-L) Mutant L1 larvae with abnormal body shape consisting in enlargements and bulges (white arrows), most often present in the head and tail regions. (M,N) Confocal images of larvae stained with AbMH27. In M, the line of lateral epithelial cells in a newly hatched larva is disorganized and the shape of the individual cells is altered. The white arrow points to a three partners boundary. In N, a group of epithelial cells has detached from the main lateral line and has organized a separate islet of epithelial cells. The outline of the animals (broken white lines) is drawn from parallel visible light micrographs (G, M and N).

View larger version (121K): [in a new window]   Fig. 4. kal-1 mutants show altered male tails. (A-F) DIC photomicrographs; ventral views. Control tail (A): the nine rays on each side are indicated. (B-F) kal-1 mutants. White arrows indicate loss or strong reduction of rays; black arrows indicate abnormal shape, extra rays or fusion of rays. In D, combination of DIC and epifluorescence image of a kal-1(gb503) mutant worm, which is also transgenic for evIs82a [unc-129 ns::GFP; dpy-20(+)] (Colavita and Culotti, 1998). This transgene specifically expresses GFP in one of ray 5 sensory neurons (white arrowhead), allowing to establish that ray 5 maintains its identity, but is posterior to ray 6 (black arrowhead). (G) Schematic representation of the outline of epithelial cells at three different times during tail formation in wild-type L4 males [modified from Baird et al. (Baird et al., 1991)]. During early L4, the tail seam cells (indicated by stars), which are next to the clusters of ray precursors (numbered 1 to 9), are still separated from each other. At mid L4, tail seam cells have partially fused together to form the SET (seam tail) cell, which maintains its connection with the most posterior body seam cell and with the ray clusters. At late L4, the fusion of tail seam is complete and the flanking hyp7 cell has engulfed the ray clusters. The SET cell maintains its contact with body seam throughout the process. (H,I) Confocal images of developing male tails at the L4 stage stained with AbMH27: wild type in H; kal-1(gb503) in I. Numbers from 1 to 9 indicate the clusters of precursors of sensory rays. The typical triangular arrangement of precursors to rays 4, 5 and 6 of the control tail (H) is changed to an almost straight line in the mutant tail (white arrow in I). This arrangement of epithelial cells in L4 will result, in the adult tail, in the inversion between the position of ray 5 and 6 (D) or in their fusion. In I, the shape of the SET cell, which does not contact the posterior body seam cell, is also abnormal.

View larger version (88K): [in a new window]   Fig. 5. kal-1 mutants show neuronal growth defects. Epifluorescence images of neurons of adult worms, harboring the reporter plasmid GB102 on an extrachromosomal transgenic array. In all panels, anterior is towards the left. (A,D) control; (B,C,E) kal-1 mutants. (A-C) The EF3 male-specific neuron is visible in these worms because of GFP expression from the transgene. In mutant males, the EF3 dendrite presents an extra-branching with the formation of a second dendrite running in the same direction and parallel to the normal one. (D-F) A neuron of the head, RIC, is visible in these worms because of GFP expression from the transgene. In mutant worms overexpressing CeKAL the axon presents an extra-branching with the formation of a spike running parallel but in the opposite direction to the normal path. In F, the trace of the neuron is drawn from micrograph D, and the broken line represents the extrabranching visible in E. Arrows indicate extra-branching in the mutants.

View larger version (126K): [in a new window]   Fig. 6. Expression of kal-1 reporter constructs in transgenic worms (anterior is towards the left). In all cases, except in D, worms harboring plasmid GB102 (no nuclear localization signal) are shown and GFP expression was detected by epifluorescence; in D, the plasmid was GB105, (containing a NLS) and ß-galactosidase expression was detected histochemically. (A,B) Embryonic expression begins in a group of ventral neuroblasts in 120- to 200-cell stage embryos, ventral view, superficial focal planes; (C) ventral view, intermediate focal plane of an embryo between 310 and 360 minutes after fertilization; the expressing neuroblasts have split in an anterior and a posterior group and have been covered by the epithelial cells that have joined at the ventral midline. (D) ß-gal staining of an L3 larva showing three groups of neurons expressing the transgene after hatching, see text. (E) Neurons of the anterior ganglia express GFP; arrow points to their axonal projections in the nerve ring. (F) Arrows point to the dendrites of sensory neurons, in the head of an adult male. (G) Ventral view of the mid section of an L3 larva; the canal associated neurons, CAN cells, express the construct. (H) Lateral view of the mid section of an L4 hermaphrodite; the cell body of an HSN neuron is visible, partially out of focus; arrows indicate its anteriorly directed process. (I,J) Tail region of L4 hermaphrodites. In I, the PDB cell body is partially out of focus and the arrow points to the characteristic process reaching the tail tip before turning anteriorly. In J, a tail interneuron, possibly PVW can be seen.