Worm Breeder's Gazette 16(4): 28 (October 1, 2000)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

A Genetic Screen for Mutants Interacting with the C. elegans Orthologue of the Kallmann Syndrome Protein

Hannes E. Bülow, Katherine L. Berry, Jun Zhu, Oliver Hobert

Columbia University, College of Physicians and Surgeons, Dept. of Biochem. & Mol. Biophysics, New York, NY 10032, U.S.A.

Kallmann Syndrome is a human hereditary disease characterized by anosmia and infertility that occurs in both X-linked and autosomal forms. Only one causative gene, the gene responsible for the X-linked form, has been discovered. This gene, alternatively named anosmin-1 or kal-1 (1,2), codes for a secreted protein containing a four disulfide core domain and four fibronectin III repeats. Biochemical and histochemical studies suggest it to be involved in olfactory axon pathfinding and/or recognition of post-synaptic mitral cells (for review see 3).

However, the molecular mechanism by which anosmin-1 acts has remained elusive, as have its hypothesized receptor and downstream signalling molecules. In order to discover molecular partners of anosmin-1, we turned to its structurally conserved orthologue in C. elegans, Cekal-1. A transcriptional fusion between the Cekal-1 promoter and GFP showed expression in a subset of neurons in the head and the tail. We employed a two step approach of first generating a Cekal-1 specific gain-of-function phenotype and subsequently screening for modifier mutations of this phenotype.

Step 1: Cekal-1-Specific Gain-of-Function Phenotype

To generate a gain-of-function phenotype we expressed Cekal-1 cDNA under neural and cell-specific promoters and looked for defects in sets of neurons labelled with GFP. These studies show that, while mis/over-expression of Cekal-1 has no effect in most neurons, it strongly affects the integrity of certain neurons. For example, if the cDNA is overexpressed in AIY, an interneuron implicated in thermosensation, we observe ectopic neurites originating from the cell body and the axon. The penetrance of this defect is consistently 100% as demonstrated by three independent integrated overexpressing lines. Control experiments in which either a truncated CeKAL-1 protein or structurally related proteins were ectopically expressed in AIY yielded no effect on AIY interneuron morphology, strongly suggesting that the gain-of-function phenotype is specific to CeKAL-1 protein activity.

Step 2: Genetic Screen for Modifier Mutants

To determine whether mutants previously implicated in axon outgrowth and/or pathfinding could suppress our gain-of-function phenotype, we created doubles between such mutants and lines overexpressing Cekal-1 in AIY. We failed to detect significant suppresion of Cekal-1 induced neurites by the netrin system (unc-6, unc-5, and unc-40), Robo (sax-3), ephrin receptor (vab-1), FGFR (egl-15), and other genes involved in axon outgrowth/pathfinding (ina-1, unc-52, unc-73, unc-34, unc-61, unc-13, unc-51, and unc-119).

To isolate modifier mutations of the Cekal-1 specific gain-of-function phenotype in AIY we conducted a clonal F1 screen after EMS mutagenesis. We screened through 1356 haploid genomes and isolated seven mutants (o16-ot21, ot24). Six mutations suppress this neurite growth phenotype to various degrees between 24% and 90%. Additionally, one mutation (ot21) enhances this phenotype such that the observed neurites are considerably longer than in unmutagenized animals. The mutations fall into at least four complementation groups. Initial mapping experiments using STS markers place ot16 within three map units on LGIII, and ot17/ot19 (which are allelic) on LGX close to lon-2. For ot21 linkage to LGV could be established.

To assess the specificity of the modifier mutants, we constructed double mutants with ttx-3, which causes neurite outgrowth in AIY interneurons (4). While ot20 did not suppress the ttx-3 neurites, ot16 and ot18 showed a slight suppression of 11% and 17%, respectively. The enhancer ot21 did not enhance ttx-3 dependent neurites, suggesting CeKAL-1 specificity. In an additional specificity test, ot21 could not enhance sax-2(ot10) dependent ectopic neurites in AIY interneurons or sensory neurons (5). We plan to construct double mutants between sax-2(ot10) or ttx-3(ks-5) and the remaining suppressor mutants to corroborate their specific suppression of CeKAL-1 generated neurites.

Cloning of the Cekal-1 specific modifier genes should provide insight into the role of Cekal-1 and its partners in C. elegans. Additionally, the human orthologues of these interacting genes could account for autosomal forms of Kallmann Syndrome.


  1. Legouis R, Hardelin JP, et al. (1991) The candidate gene for the X-linked Kallmann Syndrome encodes a protein related to adhesion molecules. Cell, 67:423-435
  2. Franco B, Guiloli S, Pragliola A, et al. (1991) A gene deleted in Kallmann’s syndrome shares homology with neural cell adhesion and axonal pathfinding molecules. Nature, 353:529-536
  3. Rugarli E, Ballabio A (1993) Kallmann Syndrome. From Genetics to Neurobiology. JAMA, 270(22):2713-2716
  4. Hobert O, Mori I, Yamashita Y, Honda H, Ohshima Y, Liu Y, Ruvkun G. (1997) Regulation of interneuron function in the C. elegans thermoregulatory pathway by the ttx-3 LIM homeobox Neuron. 19:345-357
  5. Zallen JA, Kirch SA, Bargmann CI. (1999) Genes required for axon pathfinding and extension in the C. elegans nerve ring. Development. 126(16):3679-92.