Ror interacts with members of the Wnt pathways

3.5.1 Ror genetically interacts with the ligand Wnt5

In a first approach to identify the pathway or biological process in which Ror is involved, I have analyzed genetic interactions of Ror⁴ with alleles for fz, fz2, otk, otk2 and wnt5.

Through the analysis of genetic interactions it is possible to find functional relationships between genes and pathways. For instance, when the double mutant of two genes is showing a more severe phenotype than the two single mutants alone, functional redundancies can be identified. This indicates an in vivo relationship between the two proteins.

I have crossed Ror⁴ flies together with mutants for fz, fz2, otk, otk2 and wnt5 and analyzed their adult progeny for severity of phenotypes and viability. From each allelic combination I have analyzed flies heterozygous for both mutant alleles, flies homozygous for Ror⁴ and heterozygous for the second mutation, as well as flies homozygous for both. For fz and fz2 I have additionally analyzed Ror⁴ in transheterozygous flies carrying two different alleles for fz or fz2 above each other.

Homozygous fz^J22 flies are viable but display PCP defects in eyes, wings and body, the other two tested fz alleles are homozygous lethal. Transheterozygous flies for all three allele combinations are also viable with PCP defects. I did not detect any genetic interactions of Ror⁴ with fz (Table 9). Both fz2 alleles I used are homozygous lethal, transheterozygous animals are viable and display no PCP defects. However, they are male and female sterile. For fz2 I did not observe any genetic interactions either.

Flies homozygous mutant for Wnt5 are viable but display defects in the central nervous system (Fradkin et al., 2004). When additionally removing one copy of Ror, this phenotype is not enhanced and the flies are also viable. Only when both copies of Ror are missing it is lethal (Table 9). I have analyzed the CNS of late stage Wnt5⁴⁰⁰/Wnt5⁴⁰⁰; Ror⁴/Ror⁴ embryos for defects and did not detect a more severe phenotype than in Wnt5⁴⁰⁰/Wnt5⁴⁰⁰ embryos (data not shown). A lethality test confirmed that the lethality is not embryonic but occurs soon after hatching in the first larval stage (data not shown).

Flies homozygous mutant for the otk and otk2 single mutations are viable without any discernible phenotype, double mutants for otk and otk2 are male sterile

(Linnemannstöns et al., 2014). Double mutants for Ror and otk or Ror and otk2 are viable and fertile. Homozygous flies mutant for all three genes are naturally also male sterile but as demonstrated above, the weak CNS defect observed in Ror⁴ embryos cannot be observed (3.4.2). Interestingly, these flies lay an increased number of unfertilized eggs (3.4.1).

In conclusion, I have not observed any functional relationship between Ror and fz, fz2, otk or otk2. It seems however, that there is a synthetic genetic interaction between Ror and Wnt5, indicating that the two proteins possibly have a common function during larval or pupal development.

Due to the limited time frame of this study, genetic interactions of Ror with the Wnt ligands Wingless, Wnt2 and Wnt4, which are all located on the second chromosome as well, were not analyzed. The recombinations between the Ror⁴ allele and the single mutants otk^A1 and otk2^C26, respectively were performed by Dr. Karen Linnemannstöns.

Table 9: Ror genetically interacts with Wnt5. Genetic interactions of the Ror⁴ allele with three fz alleles, two fz2 alleles, single and double mutant alleles for otk and otk2 and one Wnt5 allele were tested.

Genotype Viability/Phenotype

Ror⁴/Ror⁴ viable

fz^J22/ fz^J22 viable with PCP defects

Ror⁴/CyO; fz^J22/TM6 viable

Ror⁴/Ror⁴; fz^J22/TM6 viable

Ror⁴/CyO; fz^J22/ fz^J22 viable with PCP defects Ror⁴/Ror⁴; fz^J22/ fz^J22 (zygotic) viable with PCP defects Ror⁴/Ror⁴; fz^J22/ fz^J22(maternal) viable with PCP defects

fz^R52/ fz^R52 lethal

Ror⁴/CyO; fz^R52/TM6 viable

Ror⁴/Ror⁴; fz^R52/TM6 viable

Ror⁴/CyO; fz^R52/ fz^R52 lethal Ror⁴/Ror⁴; fz^R52/ fz^R52 (zygotic) lethal Ror⁴/Ror⁴; fz^R52/ fz^R52(maternal) lethal

fz^P21/ fz^P21 lethal

Ror⁴/CyO; fz^P21/TM6 viable

Ror⁴/Ror⁴; fz^P21/TM6 viable

Ror⁴/CyO; fz^P21/ fz^P21 lethal Ror⁴/Ror⁴; fz^P21/ fz^P21 (zygotic) lethal Ror⁴/Ror⁴; fz^P21/ fz^P21(maternal) lethal

fz^J22/fz^P21 viable with PCP defects

Ror⁴/CyO; fz^J22/fz^P21 viable with PCP defects Ror⁴/Ror⁴; fz^J22/fz^P21(zygotic) viable with PCP defects Ror⁴/Ror⁴; fz^J22/fz^P21(maternal) viable with PCP defects

fz^J22/fz^R52 viable with PCP defects

Ror⁴/CyO; fz^J22/fz^R52 viable with PCP defects Ror⁴/Ror⁴; fz^J22/fz^R52(zygotic) viable with PCP defects Ror⁴/Ror⁴; fz^J22/fz^R52(maternal) viable with PCP defects

fz^P21/fz^R52 viable with PCP defects

Ror⁴/CyO; fz^P21/fz^R52 viable with PCP defects Ror⁴/Ror⁴; fz^P21/fz^R52(zygotic) viable with PCP defects Ror⁴/Ror⁴; fz^P21/fz^R52(maternal) viable with PCP defects

Dfz2^C2/ Dfz2^C2 lethal

Ror⁴/CyO; Dfz2^C2/TM6 viable

Ror⁴/CyO; Dfz2^C2/ Dfz2^C2 lethal Ror⁴/Ror⁴; Dfz2^C2/TM6 viable Ror⁴/Ror⁴; Dfz2^C2/ Dfz2^C2(zygotic) lethal

Df(3L)469-2/ Df(3L)469-2 lethal

Ror⁴/CyO; Df(3L)469-2/ TM6 viable Ror⁴/CyO; Df(3L)469-2/ Df(3L)469-2 lethal Ror⁴/Ror⁴; Df(3L)469-2/ TM6 viable Ror⁴/Ror⁴; Df(3L)469-2/ Df(3L)469-2 (zygotic) lethal

Dfz2^C2/ Df(3L)469-2 viable (sterile) Ror⁴/CyO; Dfz2^C2/ Df(3L)469-2 viable (sterile) Ror⁴/ Ror⁴; Dfz2^C2/ Df(3L)469-2 viable (sterile)

Wnt5⁴⁰⁰/Wnt5⁴⁰⁰ viable, CNS defects

Wnt5⁴⁰⁰/Wnt5⁴⁰⁰; Ror⁴/CyO viable, CNS defects Wnt5⁴⁰⁰/Wnt5⁴⁰⁰; Ror⁴/Ror⁴ lethal

otk^A1/ otk^A1 viable

otk2^C26/ otk2^C26 viable

Df(otk,otk2)D72/ Df(otk,otk2)D72 viable (male sterile) Ror⁴, otk^A1/Ror⁴, otk^A1 viable

Ror⁴, otk2^C26/ Ror⁴,otk2^C26 viable

Ror⁴,Df(otk,otk2)D72/ Ror⁴,Df(otk,otk2)D72 viable (male sterile)

3.5.2 Ror binds to the Wnt ligands Wg, Wnt2 and Wnt4

Vertebrate Ror proteins have been shown to bind to several Wnt ligands and also to Fz receptors (Oishi et al., 2003). In order to gain more insight into the function of Ror proteins it is important to identify the Wnt ligands binding to Ror in Drosophila. I have studied biochemical interactions of Ror via immunoprecipitation. To achieve this, I co-overexpressed GFP-tagged Ror with Myc-tagged Wg, Wnt2 and Wnt4 constructs under the control of the actin5C promoter in S2R+ cells. As negative control I co-transfected mCD8-GFP with the same Myc-tagged Wnts. The GFP-tagged proteins were immunoprecipitated from cell lysates using an anti-GFP antibody. Western blotting and detection with an anti-Myc antibody showed that Wg-Myc, Wnt2-Myc as well as Wnt4-Myc were co-immunoprecipitated with Ror-GFP, while none of them were pulled down together with mCD8-GFP (Figure 28). This indicates that all three Wnt ligands bind to Ror-GFP. Although in other experiments I was also able to pull down Wnt5-Myc with Ror-GFP as well, I could not reliably reproduce this result. This was due to a constantly low transfection efficiency of the construct. The transfections, lysate preparations, pull-down and western blotting in this experiment were performed by Julia Loth.

Figure 28: Ror-GFP binds to Myc-tagged Wg, Wnt2 and Wnt4. Indicated constructs were transfected into Drosophila S2R+ cells. Co-immunoprecipitation from cell lysates was performed using a rabbit- anti-GFP antibody followed by Western blotting using mouse anti-Myc and rabbit anti-GFP antibodies. In the GFP blot the denatured heavy chain of the antibody used in the IP is visible. IP: immunoprecipitation; WB:

Western Blot. Protein sizes are indicated in kDa.

3.5.3 Ror binds to the Wnt receptors Fz and Dfz2 and the Wnt co-receptors Otk and Otk2 In Drosophila, Fz and Fz2 constitute the core receptors for Wnt signaling (Bhanot et al., 1996, 1999). To receive some indication as to whether Ror acts as an independent Wnt receptor or as a receptor together with Fz or Fz2, I performed co-immunoprecipitations with GFP-tagged Ror and Myc-tagged Fz and Fz2 in S2R+ cells.

Same as above, I pulled down Ror-GFP with a GFP antibody and after Western blotting detected bound Myc-tagged Fz proteins with a Myc antibody. As negative control mCD8-GFP was used again. At the same time I also analyzed possible biochemical interactions of Ror-GFP with Myc-tagged Otk and Otk2.

All four receptors, Fz, Fz2, Otk and Otk2 co-immunoprecipitated with Ror-GFP (Figure 29). This indicates that Ror is able to bind to all of them and suggests that Drosophila Ror

Figure 29: Ror-GFP binds to Myc-tagged Fz, Fz2, Otk and Otk2. Indicated constructs were co-transfected into Drosophila S2R+ cells. Co-immunoprecipitation from cell lysates was performed using a rabbit anti-GFP antibody followed by Western blotting using mouse anti-Myc and rabbit anti-anti-GFP antibodies. IP:

immunoprecipitation; WB: Western Blot. Protein sizes are indicated in kDa.