Biochemical interactions of Off-track and Off-track2.

<p>(A, B) Otk and Otk2 form homooligomers and heterooligomers. Otk-Myc or Otk2-Myc and Otk-GFP or Otk2-GFP expression vectors were transfected as indicated in <i>Drosophila</i> S2r+ cells. Relevant bands corresponding to tagged Otk and Otk2 are marked by an asterisk in the bottom right panel of (A). (C) Homo- and heterodimerization of Otk and Otk2 requires the transmembrane domain. Otk-Myc or Myc-tagged Otk deletion contructs and Otk-GFP or Otk2-GFP expression vectors were transfected as indicated in <i>Drosophila</i> S2r+ cells. OtkDeltaCy lacks the cytoplasmic domain (aa 776–1033) and OtkDeltaEx lacks the extracellular domain (aa 2–474). (D) Off-track and Off-track2 interact with Frizzled1 and Frizzled2. Otk-GFP or Otk2-GFP and Fz1-Myc or Fz2-Myc expression vectors were transfected as indicated in <i>Drosophila</i> S2r+ cells. Cell lysates were immunoprecipitated and analyzed by Western Blot with the indicated antibodies. IP, Immunoprecipitation; WB, Western Blot.</p

The PTK7-Related Transmembrane Proteins Off-track and Off-track 2 Are Co-receptors for <i>Drosophila</i> Wnt2 Required for Male Fertility

<div><p>Wnt proteins regulate many developmental processes and are required for tissue homeostasis in adult animals. The cellular responses to Wnts are manifold and are determined by the respective Wnt ligand and its specific receptor complex in the plasma membrane. Wnt receptor complexes contain a member of the Frizzled family of serpentine receptors and a co-receptor, which commonly is a single-pass transmembrane protein. Vertebrate protein tyrosine kinase 7 (PTK7) was identified as a Wnt co-receptor required for control of planar cell polarity (PCP) in frogs and mice. We found that flies homozygous for a complete knock-out of the <i>Drosophila</i> PTK7 homolog <i>off track</i> (<i>otk</i>) are viable and fertile and do not show PCP phenotypes. We discovered an <i>otk</i> paralog (<i>otk2</i>, <i>CG8964</i>), which is co-expressed with <i>otk</i> throughout embryonic and larval development. Otk and Otk2 bind to each other and form complexes with Frizzled, Frizzled2 and Wnt2, pointing to a function as Wnt co-receptors. Flies lacking both <i>otk</i> and <i>otk2</i> are viable but male sterile due to defective morphogenesis of the ejaculatory duct. Overexpression of Otk causes female sterility due to malformation of the oviduct, indicating that Otk and Otk2 are specifically involved in the sexually dimorphic development of the genital tract.</p></div

<i>otk, otk2^D72</i> homozygous mutant males are sterile.

<p><i>otk, otk2^D72</i> homozygous mutant males are sterile.</p

<i>otk, otk2</i> loss of function causes malformation and obstruction of the ejaculatory duct.

<p>(A) Overview of the reproductive tract of a male heterozygous for <i>otk, otk2^D72</i> and carrying a Protamin B-eGFP transgene. (A″) and (A‴) show higher magnifications of the insets highlighted in (A′). (B) Overview of the reproductive tract of a male homozygous for <i>otk, otk2^D72</i> and carrying a Protamin B-eGFP transgene. Note that the ejaculatory duct is severely shortened and thickened compared to (A). Also note that sperm marked by Protamine B-eGFP (B′) accumulates in the ejaculatory duct of the homozygous mutant male. (B″) and (B‴) show higher magnifications of the insets highlighted in (B′). (C) Disorganization of the muscle sheath of the ejaculatory duct in <i>otk, otk2</i> homozygous mutant males. (C″) Enlarged view of the boxed area in (C′). (D, E) The muscle sheath of the anterior (D) and posterior (E) ejaculatory duct from heterozygous control males. (D″, E″) Enlarged views of the boxed areas in (D′, E′). Fluorescent Phalloidin was used to stain F-actin. aED, anterior ejaculatory duct; pED, posterior ejaculatory duct; PG, paragonium (accessory gland); SP, sperm pump; SV, seminal vesicle; TE, testis. Scale bars: A, B = 500 µm, A″, B″ = 100 µm, A‴, B‴ = 50 µm, C–E = 100 µm, C″–E″ = 50 µm.</p

Generation of <i>otk</i> and <i>otk2</i> null alleles.

<p>(A) Overview of the genomic region of <i>otk</i>, <i>otk2</i> and the neighboring gene <i>Mppe</i> located on chromosome 2R. Insertion positions of the three P-element transposons utilized are shown. Null alleles for <i>otk</i> and <i>otk2</i> alone as well as for both <i>otk</i> and <i>otk2</i> were generated via FLP/FRT mediated recombination between FRT sites contained in P(XP)d01360 and PBac(RB)e03992 (<i>otk^A1</i>), PBac(PB)c01790 and P(XP)d01360 (<i>otk2^C26</i>) or PBac(PB)c01790 and PBac(RB)e03992 (<i>otk, otk2^D72</i>). (B–F) Verification of the three generated alleles by whole mount immunofluorescent stainings. Homozygous mutant embryos of the indicated genotypes were stained with antibodies against Otk, Otk2, GFP and the CNS axon marker Bp102 as control. (B–D) Wild type embryos expressing CD8-GFP under control of <i>engrailed::GAL4</i> were mixed with <i>otk^A1</i> or <i>otk2^C26</i> homozygous mutant embryos prior to fixation and staining. The gain of the confocal microscope was adjusted to the staining intensity of Otk and Otk2 in the wild type embryos and subsequently images of the mutant embryos were taken at exactly the same settings. (B–B‴) In <i>otk^A1</i> homozygous mutant embryos no Otk protein (B) can be detected, but the staining for Otk2 (B′) is normal. (C–C‴) In <i>otk2^C26</i> homozygous mutant embryos no Otk2 protein (C′) can be detected, but the staining for Otk (C) is normal. (D–D‴) wild type control showing normal levels of Otk (D) and Otk2 (D′) expression. The GFP staining of the respective embryos is shown in panels (B″–D″) and the DAPI staining in (B‴–D‴). (E–E‴) In <i>otk, otk2^D72</i> homozygous mutant embryos neither Otk (E) nor Otk2 (E′) protein can be detected. (F–F‴) Heterozygous <i>otk, otk2^D72</i>/+ embryos are shown as control. BP102 staining to label the nervous system is shown in (E″, F″) and DAPI staining in (E‴, F‴). Anterior is to the left. Scale bar = 100 µm.</p

Sterility <i>otk, otk2^D72</i> homozygous mutant males can be rescued by an Otk transgene.

<p>Sterility <i>otk, otk2^D72</i> homozygous mutant males can be rescued by an Otk transgene.</p

Protein expression of Off-track and Off-track2 in embryos.

<p>(A–D) <i>white</i>⁻ embryos were stained with an antibody against the extracellular domain of Otk. (E–H) <i>white</i>⁻ embryos were stained with a peptide antibody raised against Otk2. Stages of embryonic development are indicated. (I–I‴) Detail of the hindgut and Malpighian tubules of a wild type embryo at stage 14 stained for DNA (DAPI, I), Otk (I′) and Baz (I″), merged image in (I‴). Otk is strongly expressed in the visceral mesoderm (vm) surrounding the hindgut and is also expressed in the hindgut epithelium (hg) and in the Malpighian tubules (mt). Note that Otk is localized to the whole plasma membrane and does not show enrichment at the zonula adherens marked by the Baz protein. (J–J‴) Otk is localized to axons and to perikarya of CNS neurons. The image shows a detail from the ventral side of a stage 15 embryo stained for DNA (DAPI, J) Otk (J′) and the neuronal nuclear marker Elav (J″), merged image in (J‴). Note that Otk is strongly enriched on axons (ax) but also stains the plasma membrane of neuronal perikarya (np). In all panels, anterior is to the left. Scale bars in (A–H) = 100 µm, scale bars in (I, J) = 20 µm.</p

Otk and Otk2 bind to Wnt2.

<p>(A) Otk and Otk2 co-precipitate <i>Drosophila</i> Wnt2. Otk-GFP or Otk2-GFP and Wnt2-Myc expression vectors were transfected as indicated in Drosophila S2r+ cells. Cell lysates were immunoprecipitated and analyzed by Western Blot with the indicated antibodies. IP, Immunoprecipitation; WB, Western Blot. (B–D) Wnt2 protein binds to S2 cells transfected with Otk-GFP or Otk2-GFP. S2 cells transfected with Otk-GFP (B), Otk2-GFP (C) and DE-Cadherin-GFP (D) were incubated with conditioned medium from S2 cells producing Wnt2-Myc and subsequently stained with anti-Myc antibody. GFP signals are shown in (B–D), Myc signal is shown in (B′–D′), DAPI staining is shown in (B″–D″) and the merged images in (B‴–D‴). Scale bar: 20 µm.</p

Off-track (Otk) and Off-track2 (CG8964, Otk2) are paralogs evolved by gene duplication.

<p>(A) Phylogenetic tree representation of an alignment of sequences from different species homologous to <i>Drosophila</i> Otk. Blast-P of Dm-Otk in <i>Drosophila</i> returned Dm-CG8964 (Otk2) as the best hit; Dm-Ror and Dm-Heartless-A were the second and third hit, repectively. Blast-P of Dm-Otk in Mouse returned Mm-PTK7 as the best hit, while Mm-FGF receptor3 was the second best hit. Blast-P of Dm-Otk in Human returned Hs-PTK7 as the best hit. ClustalW alignment of these sequences and Neighbor-Joining tree confirms that the PTK7 branch is separated from the other proteins by a bootstrap value of 100. In this branch, two <i>Drosophila</i> genes, but only one in the other species can be found. (B) Protein structures of Otk and Otk2. Domains were predicted using the SMART sequence analysis tool. Dm, <i>Drosophila melanogaster</i>; Mm, <i>Mus musculus</i>; Hs, <i>Homo sapiens</i>.</p