Supplemental Material for Vargas-Velazquez, Besnard, and Félix, 2019

Figure S1. Dissecting microscope pictures of wild-type and mutant O. tipulae adult hermaphrodites.

WT: wild-type. Oti-lin-3(mf86): fully-penetrant egg-laying defective, forming a bag of worms. Oti-mom-5(sy493): also partially egg-laying defective and protruding vulva. Oti-plx-1(mf78): protruding vulva. All the images are set to the same scale. Scale bar: 100 micrometers.

Figure S2. Example of mapping-by-sequencing in O. tipulae.

Graphs showing the frequency of JU170 calls for single-nucleotide polymorphisms between CEW1 (reference wild isolate, on which the mutagenesis was conducted) and JU170 (alternative wild isolate used for mapping) along scaffold 10 of genome assembly nOt.2.0, for two alleles of the cov-4 locus, called sy465 and sy493. The location of Oti-mom-5 is marked by a grey line. See Besnard et al. 2017 for further details.

Figure S3. Single-molecule FISH of Wnt genes.

A) Examples of the localization of WNT mRNAs as revealed by fluorescence and DAPI signal. The size of the bar is 10 micrometers. B) Average number of mRNAs of WNT genes in the L3 stage along an antero-posterior axis defined from the beginning of the gut to the last nucleus seen in the tail by DAPI.

Figure S4. Phylogenetic relationship between Wnt genes inside and outside the Caenorhabditis clade.

The cladograms were inferred using the Neighbor-Joining method with 1000 replicates for boostrapping. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. Evolutionary analyses were conducted in MEGA X. Abbreviations: Cbr (C. briggsae), Cel (C. elegans), Cjp (C. japonica), Dmel (Drosophila melagonaster), Oti (O. tipulae), Ovo (Onchocerca volvulus).

Figure S5. Identification and phylogenetic relationships of Delta/Serrate/Lag-2 (DSL) proteins in O. tipulae.

A) Cladogram inferred using the Neighbor-Joining method with 1000 replicates for boostrapping. Abbreviations: Cbr (C. briggsae), Cel (C. elegans), Cjp (C. japonica), Dm (Drosophila melagonaster), Oti (O. tipulae), Ppa (Pristionchus pacificus).

B) Alignment of the delta motif used to calculate the molecular distances between DSL proteins.

Figure S6. Expression profile of Cel-lin-44 revealed by smFISH.

A) Expression of lin-44 (red) in the sex myoblast precursors (identified with the hlh-8::GFP transgene, in blue) before P6.p division.

B) Expression of lin-44 (red) in the sex myoblast precursors (blue) and in P6.p daughters. The anchor cell is labeled with probes against lag-2 (green).

The scale bar measures 10 micrometers and is the same for both images.

Figure S7. Cis-regulation of Cel-lin-3 and Oti-lin-3 expression.

A) Diagram representing binding sites (rectangles) known to be recurrent in the upstream region of Caenorhabditis species lin-3 (Barkoulas et al. 2016). The orientation of the binding sequence is defined by its position with respect to the line (i.e., above for the forward strand and below for the reverse strand).

B) Oti-lin-3 expression patterns revealed by smFISH in wild-type and Oti-lin-3(mf86) animals. The animals are shown in ventral view, thus showing the developing gonad primordium on either side of the anchor cell. The arrows indicate the position of the anchor cell as revealed by the presence of Oti-dsl mRNA. The scale bar measures 10 micrometers and is the same for both images.

Table S1. List of strains used in this study.

Table S2. Sequences of DNA primers used in this study.

Sequencing primers to verify by Sanger sequencing the mutations identified by the mapping by sequencing approach, and to identify the molecular lesion in additional alleles.

Table S3. Sequences of smFISH probes used in this study.

The fluorophore coupled to each probe is noted at the end of the set name.

Table S4. smFISH quantifications, distance measurements and vulval cell fates used in this study.