Supplemental Material for Sorkaç et al., 2018

Raw Data for Table 1 in the article entitled:<div><br></div><div>"LIN-12/Notch regulates GABA signaling at the <em>C. elegans</em>neuromuscular junction"</div

OSM-11 and C. elegans DSL Ligands Interact with LIN-12 Notch Extracellular Domain EGF Repeats in the Two-Hybrid System

<p>DSL-1, OSM-11, LAG-2 extracellular domain (LAG-2Ex), EGL-17, or LIN-3 was fused to the GAL4 DNA binding domain (DB); the first six LIN-12 EGF repeats were fused to the GAL4 activation domain (AD). Pairwise interactions were tested with the yeast two-hybrid assay; positive interactions are indicated by blue staining. Both Notch DSL ligands and OSM-11 interacted with LIN-12 EGF repeats, whereas no interaction of LIN-3 EGF or EGL-17 FGF with LIN-12 Notch receptor EGF repeats was detected. LIN-12::DB fusion proteins exhibited strong self-activation (unpublished data); therefore, reciprocal fusions were not tested. Interaction controls are: (1) empty vectors; (2) DB-pRb and AD-E2F; (3) DB-Fos and AD-Jun; (4) Gal4p and pPC86; and (5) DB-DP1 and AD-E2F1.</p

OSM-11 Is Required for Normal Development

<p>(A) Thirty-one percent of <i>osm-11(lf)</i> adult animals had overtly normal vulva and did not retain eggs resembling control animals. (B) Fifty-seven percent of <i>osm-11(lf)</i> animals inappropriately retained eggs and/or had a single misshapen or protruding vulva (15% and 42%, respectively). (C) Twelve percent of <i>osm-11(lf)</i> animals had an extra protrusion near the normally positioned vulva. (D) <i>osm-11(lf)</i> animals had defects in head morphology at low frequency (arrowhead). (E) Two thirds of <i>osm-11(lf)</i> animals had a ventral protrusion behind the anus (arrowhead). <i>n</i> > 100 animals were scored.</p

<i>osm-11</i> Is Expressed in VPCs and Other Tissues

<div><p>(A) OSM-11 expression in seam cells of L1 larvae detected using α-OSM-11 antisera. The seam cells on the right side of an L1 animal are in focus; the seam cells on the left side are visible and slightly out of focus. OSM-11 was not expressed in seam cells or hypoderm at other larval stages.</p> <p>(B) OSM-11 expression in the developing uterus of L4 larvae. Left, α-OSM-11 antisera staining; right, visible light image.</p> <p>(C) OSM-11 expression in vulval precursor cells (VPCs; arrowheads) in L3 larvae. The top panels show α-OSM-11 antisera staining of VPCs prior (top left) and immediately after (top right) cell fate specification as assessed by <i>lip-1p::gfp</i> expression. An overlay of α-OSM-11 staining and <i>lip-1p::gfp</i> expression shows that OSM-11 is concentrated on the apical surface of the VPCs (bottom right); this was confirmed using an <i>ajm-1::gfp</i> fusion (unpublished data).</p> <p>(D) OSM-11 expression in seam cells and spermatheca in adult animals. An <i>osm-11p::gfp</i> reporter gene containing <i>unc-54</i> 3′ UTR sequences is expressed in adult seam cells (left); α-OSM-11 antisera was used to confirm seam cell and spermatheca expression (right). No OSM-11 was detected in neurons of larvae or adult animals (unpublished data); embryonic expression was not characterized.</p> <p>In (A–D), the scale bar represents 10 μm.</p></div

<i>osm-11</i> Normally Increases Notch Signaling during Vulval Development

<div><p>(A and B) <i>lin-12(lf)</i> is epistatic to <i>osm-11(lf)</i>. <i>lin-12(lf)</i> is the null allele <i>n941</i>; animals carrying this allele have a protruding vulva (pVul; [A]) that is distinct from the defective vulva seen in <i>osm-11(lf)</i> animals (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0060196#pbio-0060196-g001" target="_blank">Figure 1</a>). <i>lin-12(lf);osm-11(lf)</i> animals were indistinguishable from <i>lin-12(lf)</i> animals (B).</p> <p>(C and D) <i>osm-11(lf)</i> suppresses <i>lin-12(csgf)</i> at 15 °C. <i>lin-12(csgf)</i> is <i>n137n460</i>, a recessive cold-sensitive gain-of-function allele; animals carrying this mutation have multiple pseudovulvae (Muv; [C]). <i>lin-12(csgf);osm-11(lf)</i> animals were significantly less Muv (nonMuv) than <i>lin-12(csgf)</i> animals ([D]; <i>p</i> < 0.05).</p> <p>(E and F) <i>osm-11(lf)</i> does not suppress <i>lin-12(gf)</i>. <i>lin-12(gf)</i> is <i>n137</i>, a dominant gain-of-function allele that is ligand independent; animals carrying this mutation are Muv (E). <i>lin-12(gf);osm-11(lf)</i> animals were indistinguishable from <i>lin-12(gf)</i> animals (F). <i>n</i> > 50 animals were scored for each genotype.</p></div

<i>osm-11</i> Encodes a Secreted Protein Required for Vulval Development

<div><p>(A) Western blot of conditioned media from <i>Drosophila</i> S2 cells containing an OSM-11 cDNA expression construct or empty vector. OSM-11 was not detected in cell lysates (unpublished data). The molecular weight of mature OSM-11 was predicted at 18.9 kDa; the detected protein migrated at 20.7 kDa (arrowhead). OSM-11 may be O-linked glycosylated (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0060196#pbio-0060196-g002" target="_blank">Figure 2</a>).</p> <p>(B) Transgenic rescue of <i>osm-11(lf)</i> vulval defects. <i>osm-11(lf)</i> animals harboring transgenes with empty expression vectors were indistinguishable from nontransgenic <i>osm-11(lf)</i> animals (<i>n</i> = 129 animals, 5 transgenic lines) and were used as controls. Multiple transgenic lines were scored for all rescue experiments; data are reported as mean ± standard error of the mean (S.E.M.) In addition to a genomic <i>osm-11</i> construct, expression of the <i>osm-11</i> cDNA using the following promoters also significantly rescued <i>osm-11(lf)</i> vulval defects: <i>osm-11p</i>, <i>hsp-16p</i> (ubiquitous expression; 79% normal vulval; unpublished data), <i>wrt-6p</i> (hypodermal), <i>osm-10p</i> (sensory neurons), and <i>glr-1p</i> (nonoverlapping set of neurons vs. <i>osm-10p</i>). In addition, heterologous expression of mammalian DLK1 driven by the <i>hsp-16</i> promoter also significantly rescued <i>osm-11(lf)</i> vulval phenotypes. <i>n</i> > 52 animals for each transgene, <i>p</i> < 0.05 by χ².</p></div

OSM-11 Loss Results in Cell Fate Specification Defects

<div><p>(A) A simplified diagram of cell fate GFP marker expression in P5.p, P6.p, and P7.p. GFP expression is schematically shown in green. Note that equivalence group members P3.p, P4.p, and P8.p are not shown. In wild-type animals, primary (1°) cell fate markers are expressed in P6.p (top left), whereas secondary (2°) cell fate markers are normally expressed in P5.p and P7.p (top right). The first Pn.p division (by P5.p, P6.p, and P7.p) in mid-L3 larvae gives rise to Pn.px cells; the next divisions give rise to Pn.pxx cells in late-L3 larvae. Loss of Notch signaling does not stop 1° cell fate assumption by P6.p, but results in inappropriate adoption of 1° cell fates by P5.p, P7.p, and their descendents. Loss of EGF/Ras signaling results in adoption of the tertiary fate by P5.p and P7.p, and in some cases, P6.p, depending on the severity of the defect [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0060196#pbio-0060196-b096" target="_blank">96</a>].</p> <p>(B) Quantification of data from (C–E). <i>p</i> < 0.05 based on χ² for each marker.</p> <p>(C) Ten percent of L3 <i>osm-11(lf)</i> animals (right) ectopically express the 1° cell fate marker <i>egl-17p::gfp</i> in P5.p or P7.p, which normally adapt the 2° fate (left).</p> <p>(D) Sixty-seven percent of L3 <i>osm-11(lf)</i> animals lack expression of the 2° cell fate marker <i>lin-11p::gfp</i> in descendants of P5.p and/or P7.p.</p> <p>(E) Thirty-five percent of L3 <i>osm-11(lf)</i> animals do not up-regulate expression of the 2° fate marker <i>lip-1p::gfp</i> in P5.p and/or P7.p. <i>p</i> < 0.05 based on χ² for each marker. These data suggest <i>osm-11(lf)</i> animals have a loss of 2° cell fate specification consistent with loss of LIN-12 Notch signaling.</p> <p>In (C–E), arrowheads indicate the positions of P5.p, P6.p, and P7.p.</p></div