The gene reporter and rescue constructs were generated by PCR fusion as previously described [26]. The promotor regions of the clec genes (1.0–1.6 kb upstream of start codon) were amplified from genomic DNA by PCR with primer A and primer B of which the latter contains an overlap to the sequence of the gfp vector (S1B Table). The gfp or mCherry coding sequence plus the 3’-UTR of unc-54 was amplified from the Fire vector pPD95.75 with primer C (5’-agcttgcatgcctgcaggtcgact-3’) and D (5’-aagggcccgtacggccgactagtagg-3’). The transgenic constructs were finally synthesized using PCR fusion with primer A* (S1B Table) and D* (5’-ggaaacagttatgtttggtatattggg-3’) and directly injected at a concentration of 10 ng/μl. The plasmids carrying ttx-3p::RFP (40 ng/μl), which is expressed in the AIY interneuron pair of successfully transformed animals, or myo-2p::RFP (25 ng/μl), expressed in pharyngeal muscle, were used as co-injection markers in lines carrying the clec-4 promotor construct or in lines carrying the clec-41 or clec-43 promotor construct, respectively. The fusion constructs with the clec-41 and clec-42 promotors were injected into the unc-119(ed3) background together with plasmid pPK605 (gift from Patricia Kuwabara, Addgene plasmid # 38148) which served as rescue for the unc-119 phenotype (S1B Table). At least three lines were generated per reporter construct and microscopically evaluated. As the lines injected with the same fluorescent reporter construct showed similar expression patterns we focused on the ones listed in S1B Table for further analyses.

For microscopy worms were mounted on slides with a 2% agarose patch and immobilized with sodium azide. All pictures were taken with the confocal microscope LSM 700 or the Axio Observer Z.1 by Zeiss (Carl Zeiss AG, Jena, Germany).

Transgenic strains MY1121 and MY1122 for rescuing clec-4(ya1) were generated by injecting 50 ng/μl of a fusion construct with 0.5 kb intestinal promotor of mtl-2 [27] and the coding region of clec-4, or the complete coding region of clec-4 including 1.4 kb of the upstream promotor into the unc-119(ed3) background (S1C Table). The rescue for the unc-119 mutation and myo-2p::RFP served as co-injection markers as described above.

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