Nmo phosphorylates Pk.

<p>(<b>A</b>) Prickle is phosphorylated by Nmo kinase but not dROK or Hpo kinases. Band shift assay of <i>in vitro</i> translated Pk (common region), Pan/dTCF and Myosin binding subunit (Mbs). The open arrowhead denotes the size of the unphosphorylated form (compared to no kinase lane). Note the band shift of Pk. Pan and Mbs serve as positive and negative controls, respectively. (<b>B</b>) Schematic of Pk constructs used in this study. The Pk protein scheme is shown above with the Pk-Sple N terminus (blue), short Pk N terminus as black line, PET domain (green) and three LIM domains (yellow). Constructs as denoted here are indicated by thick black lines: common (sequence shared between Pk and Pk-Sple isoforms); C-terminus; N-Δ1; N-Δ2 (both C-terminal deletions as indicated); C1; C2; Dom (PET and LIM domains); Sple N-terminus (Pk-Sple specific N-terminal extension); PET domain; M (Middle sequence) (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007391#sec011" target="_blank">Methods</a> for sequence details). Full lines indicate fragments that are phosphorylated by Nmo, dashed lines indicate unphosphorylated fragments. (<b>C</b>) <i>In vitro</i> kinase assay gel using purified Nmo kinase and <i>in vitro</i> translated Pk fragments (from panel <b>B</b>). Upper panel; radiograph showing phosphorylation; autophosphorytion of Nmo is denoted by *N. Vang C-term is used as negative control (also [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007391#pgen.1007391.ref024" target="_blank">24</a>]). Common, C-term, N-Δ1, N-Δ2, and M fragments are phosphorylated by Nmo. Corresponding Coomassie-stained gel is shown below—full-length fragments of individual constructs are indicated by *.</p

<i>nmo</i> enhances the PCP defects of <i>pk</i>^<i>sple1</i> mutants.

<p>(<b>A—D</b>) <i>nmo</i> genetically interacts with <i>pk</i>^<i>sple1</i> in the eye. Tangential sections of adult eyes of the indicated genotype are shown with corresponding schematics below. Chiral ommatidia are depicted as black (dorsal) or red (ventral) flagged arrows and symmetrical clusters are green straight arrows (also Suppl. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007391#pgen.1007391.s002" target="_blank">S2 Fig</a>). The dorso-ventral midline (line of mirror symmetry) can be seen in <i>wt</i> (<b>A</b>) and <i>nmo</i>^<i>P</i> (<b>B</b>) mutants, whereas chiral forms are intermixed in homozygous <i>pk</i>^<i>sple1</i> mutants (<b>C</b>). In <i>pk</i>^<i>sple1</i>; <i>nmo</i>^<i>P</i> double mutants (<b>D</b>), the proportion of chirality defects significantly increases with many symmetrical clusters (quantified in <b>M</b>). (<b>E-F</b>) Detection of symmetrical clusters in larval eye discs. Immunofluorescence of an R4-specific molecular marker (<i>mδ-LacZ</i>, green) and a pan-neuronal marker (Elav, blue). Examples of <i>mδ-LacZ</i>-negative cells are marked in white (R3 or *) and <i>mδ-LacZ</i>-positive cells are marked in red (R4 or *). (<b>E</b>) Wild-type: Note regular arrangement of β-gal-positive cells with one cell located at the polar side/R4 of each cluster (equator is in center of panel, see Suppl. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007391#pgen.1007391.s002" target="_blank">S2 Fig</a> for detail of frame positioning). (<b>F</b>) R3/R4 fate is affected in <i>pk</i>^<i>sple1</i>; <i>nmo</i>^<i>P</i> animals. Note disorganization of β-gal-positive cell pattern, and the presence of clusters with either two or none <i>mδ-LacZ</i> positive cells (symmetrical clusters). (<b>G-J,N</b>) <i>nmo</i>^<i>P</i> enhances the <i>pk</i>^<i>sple1</i> PCP defects in legs. Tarsal segments of adult legs of the indicated genotype are shown. <i>wt</i> and <i>nmo</i>^<i>P</i> mutant legs have four tarsal joints (<b>G,H,N</b>) joints are marked with arrowheads in <b>G</b>). Extra joints form in <i>pk</i>^<i>sple1</i> mutants, and incomplete joint tissue can be seen within tarsal segments (<b>I,N</b>). The line shows a region of reversed polarity (bristles point proximally). The median joint number increases in <i>pk</i>^<i>sple1</i>; <i>nmo</i>^<i>P</i> double mutants (<b>J</b>) from five to seven (<b>N</b>), and the extent of polarity reversal is increased (lines in <b>J</b>). (<b>K-L</b>) Loss of <i>nmo</i> function enhances the <i>act-EGFP-Pk</i> gain of function leg phenotype. Extra joint tissue forms in <i>act-EGFP-Pk</i> tarsal segments (<b>K</b>), and joint number further increases in <i>act-EGFP-Pk/nmo</i>^<i>DB</i> animals (<b>L</b>; quantified in panel <b>N</b>: median joint number increases from five to six). (<b>M</b>) Quantification of chirality defects (**** <i>P</i>< 5E^-24, ** <i>P</i><0.005, Chi-squared test n>200). (<b>N</b>) Quantification of tarsal joints (**** <i>P</i> = 5E^-24, ** <i>P</i><0.0001, Mann Whitney U test n>54).</p

The Cul1/SkpA/Slmb SCF complex promotes Pk degradation.

<p>(<b>A-D</b>) Eye phenotypes of <i>sevGal4</i>, <i>UAS-Pk</i> (<i>sev>Pk</i>) in the indicated backgrounds of reducing levels of components of SCF-E3 ligase complex (Cul1, SkpA, and Slmb). Knockdown of <i>Cul1</i> (<b>B</b>) and <i>SkpA</i> (<b>C</b>) as well as <i>slmb</i>^<i>+/-</i> background (<b>D</b>) all enhance the Pk gain-of-function phenotype (<b>A</b>), quantified in (<b>E</b>) also quantified are <i>white</i> RNAi as a negative control and <i>slmb</i> knockdown (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007391#pgen.1007391.g007" target="_blank">Fig 7</a> and Suppl. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007391#pgen.1007391.s007" target="_blank">S7 Fig</a> for additional sections). In each genotype <i>n</i>>200 and <i>P</i><0.02, 0.001, and 0.0005 in *, ***, and ****, respectively). (<b>F</b>) Reduction of Slmb function increases Pk protein level in eye discs. Lysates from eye discs expressing <i>act</i>-EGFP-Pk in either a <i>w</i>^<i>1118</i> or <i>slmb</i>^<i>+/-</i> background were immunoblotted using GFP and γ-tubulin (control) antibodies. <i>GMR</i>><i>DNProsbeta6</i> is included for comparison.</p

Nmo acts with dTAK and Hipk to regulate Pk.

<p>(<b>A-B</b>) The phenotype of <i>sev</i>>Pk is enhanced in a <i>dTAK</i>^<i>179</i> heterozygous background (<b>A</b>), quantified in (<b>B</b>) (See also Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007391#pgen.1007391.g005" target="_blank">5</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007391#pgen.1007391.g006" target="_blank">6</a> for <i>sev</i>><i>Pk</i> section examples). (<b>C-E</b>) Knockdown of <i>Hipk</i> (<b>D</b>) enhances the <i>sev</i>><i>Pk</i>, <i>>wIR</i> phenotype (<b>E</b>), similarly to knockdown of <i>nmo</i> (<b>C</b>). (<b>F</b>) Quantification of eye phenotypes in <b>C-E</b> (in each genotype <i>n</i>>200 and <i>P</i><0.005, and <0.0005 for *** and ****, respectively). (<b>G</b>) A schematic of the Pk protein showing location of the consensus Hipk phosphorylation site S880, within the consensus HE<b>S</b>PSR, along with the two clusters of Nmo phosphorylation sites.</p

<i>nmo</i> is required in the R4 photoreceptor for correct chirality establishment.

<p>Clonal analysis of <i>nmo</i> function in a <i>pk</i>^<i>sple1</i> background, all cells are mutant for <i>pk</i>^<i>sple1</i>, and marked clones are also mutant for the <i>nmo</i> null allele, <i>nmo</i>^<i>DB</i> (marked by lack of pigment granules, small black dots adjacent to rhabdomeres—examples of <i>nmo</i>+ Rs are labelled with black arrowheads and <i>nmo</i>- Rs are labelled with yellow arrowheads in A). Only the genotype of the R3-R4 pair was scored in pairs in which R3 and R4 were of different genotypes. (<b>A</b>) Representative image of a tangential section of an adult mosaic eye; black arrow points to a correctly-oriented cluster in which R3 and R4 exhibit pigment granules and are thus <i>nmo</i>+ (or <i>wt</i> for <i>nmo</i>, marked by black arrowheads); yellow arrow points to a symmetrical cluster in with both R3 and R4 lack pigment granules and therefore are <i>nmo-</i> (marked by yellow arrowheads); green arrow points to a correctly-oriented cluster in which <i>nmo</i> function is only present in R4 (R3: <i>nmo</i>^-/R4: <i>nmo</i>⁺); red arrow points to an incorrectly-oriented cluster in which <i>nmo</i> function is only present in R3 (R3: <i>nmo</i>⁺/R4: <i>nmo</i>^-). (<b>B</b>) Schematic of mosaic analysis. Clusters in which <i>nmo</i> function is present in R3 only (pigment is represented by black circle, left column), or R4 only (right column), can develop in a wild-type orientation (below) or can exhibit PCP defects (above line). Red arrow points to an incorrectly-oriented cluster with <i>nmo</i> function in R3 only (as in real cluster in panel <b>A</b>) and green arrow points to a correctly-oriented cluster with <i>nmo</i> function in R4 (as in panel <b>A</b>). (<b>C</b>) Quantification of wt and PCP defects in clusters in which only R3 has <i>nmo</i> function (first column) compared to those with <i>nmo</i> function in R4 (second column). There is a significant increase in the proportion of WT clusters when <i>nmo</i> is present only in R4, compared to only in R3 (<i>P</i> = 0.0022, Fisher’s exact test n = 140 pairs) For comparison, the data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007391#pgen.1007391.g002" target="_blank">Fig 2M</a> are also shown; <i>pk</i>^<i>sple1</i> (third column) and <i>pk</i>^<i>sple1</i>; <i>nmo</i>^<i>P</i> double mutants (fourth column). There is no significant difference between <i>pk</i>^<i>sple1</i> mutants and loss of <i>nmo</i> function in R3 only, or between <i>pk</i>^<i>sple1</i>; <i>nmo</i>^<i>P</i> double mutants and loss of <i>nmo</i> function in R4 only (<i>P</i>>0.05). (<b>D</b>) (<b>D′</b>) In ommatidia that have Nmo function in R3 only (R3 <i>nmo</i>⁺/R4 <i>nmo</i>^-) in <i>pk</i>^<i>sple1</i> background, 42% ommatidia develop as wild-type clusters with correct chirality. (<b>D′′</b>) In ommatidia that have Nmo function in R4 (R3 <i>nmo</i>^-/R4 <i>nmo</i>⁺) in <i>pk</i>^<i>sple1</i> background, 68% develop as wild-type clusters with correct chirality, resembling the <i>pk</i>^<i>sple1</i> single mutant background. (<b>D′′′</b>) In <i>pk</i>^<i>sple1</i>; <i>nmo</i>^<i>P</i> double mutants with both R3 and R4 lacking Nmo function (R3 <i>nmo</i>^-/R4 <i>nmo</i>^-) 43% of clusters develop with correct chirality, comparable to genotype in panel (<b>D′</b>). (<b>D′′′′</b>) In <i>pk</i>^<i>sple1</i> single mutants, 60% of clusters develop with correct chirality. Compare panels <b>D′</b> and <b>D′′′</b>: when R4 lacks Nmo function, the proportion of wild-type clusters is similar, irrespective of the genotype of R3. This suggests that Nmo acts in R4 during chirality establishment/R3 vs R4 specification. Compare panels <b>D′′</b> and <b>D′′′′</b>: when R4 has Nmo function, the proportion of wild-type clusters is similar to the <i>pk</i>^<i>sple1</i> single mutant, irrespective of the genotype of R3. Together this suggests that <i>nmo</i> is only required in R4 for correct chirality. Green denotes Nmo function and magenta denotes lack of Nmo function. Note, in all cases described, all cells are mutant for <i>pk</i>^<i>sple1</i>.</p

Nmo limits Pk^Pk activity.

<p>(<b>A</b>) Identification of Nmo phosphorylation sites. <i>In vitro</i> kinase assay with increasing amounts of M fragment (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007391#pgen.1007391.g001" target="_blank">Fig 1</a>) in which the first or second cluster of MAPK consensus sites, (potential Nmo target sites), or both, are mutated. Positions of the Nmo S/T phosphorylation sites are indicated in above schematic of fragment with S and T, respectively (the S/T residues in Pk are for cluster 1: S515, S519, S595, and S599, and for cluster 2: T708, S725, T737, and S762). Upper panel; radiograph showing phosphorylated Pk fragments (arrowhead; * denotes Nmo autophosphorylation), lower panel; Coomassie-stained gel. Note that phosphorylation is significantly reduced only when both clusters are mutated. (<b>B-D</b>) Pk lacking both clusters of Nmo phosphorylation sites shows a stronger phenotype. <i>sevenless(sev)</i>-<i>Gal4</i> driven Pk overexpression (myc-Pk-WT; panel <b>B</b>) displays a gain of function phenotype with both flips and symmetrical clusters. The phenotype is more severe when a Pk construct in which both Nmo consensus site clusters have been mutated (myc-Pk-Mut; note increased symmetrical clusters, panel <b>C</b>) is expressed. Both transgenes are inserted in the same attP site and thus expressed at same levels; quantified in panel <b>D</b> (**** <i>P</i><0.00005 Chi-squared test, n>300). (<b>E-I</b>) Dose-dependent effect of Nmo on the <i>sev</i>-<i>Gal4</i> driven Pk gain-of-function phenotype. <i>sev</i>-driven Pk expression causes chirality defects (<b>E</b>). (<b>F-G</b>) Reduction of Nmo function through either one copy of the hypomorphic allele, <i>nmo</i>^<i>P/+</i> (<b>F</b>), or the null allele, <i>nmo</i>^<i>DB/+</i> (<b>G</b>) enhances these PCP defects, particularly number of symmetrical clusters (quantified in panel <b>I</b>); in contrast Nmo co-overexpression with Pk, (<i>sev>Pk</i>, <i>>Nmo</i>; panel <b>H</b>) suppresses the Pk-induced formation of symmetrical clusters (quantified in panel <b>I</b>; <i>P</i><0.0002, circle represents cluster with R-cell loss). (<b>I</b>) Quantification of chirality defects (****<i>P</i><0.0005, Chi-squared test n>300).</p

Nmo regulates Pk levels via proteasome-mediated degradation.

<p>(<b>A</b>) Loss of <i>nmo</i> phosphorylation sites increases the Pk protein level in eye discs. Lysates from myc-Pk-WT- or myc-Pk-Mut-expressing eye discs were immunoblotted using myc and γ-tubulin (control) antibodies. <i>w</i>^<i>1118</i> discs were used as a control. (<b>B</b>) Loss of <i>nmo</i> function increases Pk protein level in eye discs. The relative amount of EGFP-Pk protein in a <i>wt</i> or <i>nmo</i>^<i>DB/+</i> background was calculated and normalized to Arm levels. A representative blot is shown, data from four independent experiments are quantified in graph to the right. Reduction of <i>nmo</i> function increases the amount of EGFP-Pk protein, but not that of EGFP-Sple (see Suppl. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007391#pgen.1007391.s006" target="_blank">S6 Fig</a> for blot; paired t-test, * <i>P</i> = 0.038, ns <i>P</i>>0.05). (<b>C-F</b>) Eye phenotypes of <i>sevGal4</i>, <i>UAS-Pk</i> (<i>sev>Pk</i>) and <i>act-EGFP-Pk</i> co-expressing dominant negative (DN) proteasome components. The <i>sev</i>><i>Pk</i> phenotype (<b>C</b>) is enhanced by co expression of DNProsβ2 (<b>D</b>). The <i>act</i>-<i>EGFP-Pk</i> phenotype (<b>E</b>) is enhanced by <i>GMR>DNProsβ6</i> co-expression (<b>F</b>). Note the increase in symmetrical clusters. (<b>G</b>) Quantification of eye phenotypes (****<i>P</i><0.0001, Chi-squared test n>300). (<b>H</b>) Inhibition of proteasome function increases Pk protein level in eye discs. Lysates from eye discs expressing <i>act</i>-<i>EGFP-Pk</i> in either a <i>w</i>^<i>1118</i> or <i>GMR</i>> <i>DNProsβ6</i> background were immunoblotted using GFP and γ-tubulin (control) antibodies.</p

Normalized pathological angiogenesis in <i>Gpr116</i>^-/- retinas.

<p>A. Confocal images of post-OIR retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates at P12 (the images shown are representative of 5 mice per genotype). B. Confocal images of post-OIR retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates at P17 (the images shown are representative of 5 mice per genotype). C. Quantification of the avascular area on the post-OIR retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates at P12 (n = 5 mice at least per genotype). D. Quantification of the avascular area on the post-OIR retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates at P17 (n≥7 mice at least per genotype). E. Confocal images of post-OIR tufts (blue arrows) in <i>Gpr116</i> WT, heterozygous and knockout littermates at P17 (the images shown are representative of 5 mice per genotype)</p

Massive accumulation phenotype in lungs of aged <i>Gpr116</i>^-/- mice.

<p>A. Bright field image of the inflated lung from <i>Gpr116</i> WT, heterozygous and knockout littermates. B. Weights of whole lungs over total body weight from <i>Gpr116</i> WT, heterozygous and knockout littermates (n≥5 mice per genotype). C. Bright field images of heart from <i>Gpr116</i> WT, heterozygous and knockout littermates. D. Weights of the heart (left) over total body weight from <i>Gpr116</i> WT, heterozygous and knockout littermates (n≥5 mice per genotype). E. Bright field images of the spleen from <i>Gpr116</i> WT, heterozygous and knockout littermates. F. Weights of the spleen (left) over total body weight from <i>Gpr116</i> WT, heterozygous and knockout littermates (n≥5 mice per genotype). G. BALF collected from <i>Gpr116</i> WT, heterozygous and knockout littermates (The picture shown is representative of 3 mice for each genotype). H. Quantification of saturated phosphatydilcholine in BALF by ELISA (n = 3 mice per genotype). I. Quantification of protein content in BALF by BCA assay (n = 3 mice per genotype). J. Surfactant proteins detection in BALF by western blot. Molecular weights are indicated on the right. (n = 2 mice per genotype). K. Bright field images of the lung, after hematoxylin and eosin staining. The black arrowheads indicate alveolar macrophages (the image is representative of 4 mice for each genotype). L. Electron microscopy view of <i>Gpr116</i> wildtype and knockout lungs (n = 2 mice for each genotype). M. Confocal images of lung sections stained with ADRP (white) and nuclear stain (Hoechst, blue). Note that a red autofluorescent signal appears in knockout lungs. (the image shown is representative of 2 mice for each genotype). N. Confocal images of lung sections stained with nuclear marker Hoechst (blue) to show autofluorescent cells accumulated in the alveolar space, either in the green or red channel (the image is representative of 3 mice for each genotype). O. Autofluorescence emission spectrum of macrophages in the old knockout lung, upon 405 nm excitation (the image is representative of 2 mice). P. Detection of autofluorescent cells from <i>Gpr116</i> knockout lung by FACS (n = 2 mice per genotype)</p

Retinal vascular patterning in <i>Gpr116</i>^-/- mice.

<p>A. Vascular network in P4 retinas. Dashed line indicates the limits of the retina (the picture shown is representative of at least 5 mice for each genotype). B. Quantification of the retinal vascular outgrowth at P4 (n = 5 for WT, n = 12 for heterozygotes and n = 6 for knockout). C. Vascular patterning in P7 retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates. Isolectin (red), CD31 (green) and Erg (grey) were used to visualize endothelium, and NG2 (green) and ASMA (red) to detect mural cells (the images shown are representative of 3 mice for each genotype). D. Vascular patterning in P7 retinas from <i>Gpr116</i> ECKO and littermates controls. Isolectin (red) is used to visualize endothelium, and NG2 (green) and smooth muscle actin α (ASMA, blue) to detect mural cells (the images show are representative of 2 mice per genotype). E. Isolectin (red) and FITC-dextran (green) distribution in P21 retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates. CD31 (green) is used to stain the endothelium, and nuclei are stained with Hoechst (blue) (the images shown are representative of 3 mice per genotype). F. Monolayers formed by isolated endothelial cells from <i>Gpr116</i> WT, heterozygous and knockout brain. Endothelial cells (CD31) and nuclei (Hoechst) are indicated in green and blue, respectively (the pictures shown are representative of 3 mice for each genotype)</p