Research ArticleDevelopmental Biology

The scaffolding protein Cnk binds to the receptor tyrosine kinase Alk to promote visceral founder cell specification in Drosophila

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Science Signaling  24 Oct 2017:
Vol. 10, Issue 502, eaan0804
DOI: 10.1126/scisignal.aan0804
  • Fig. 1 Alk and Cnk interact through an AIR.

    (A) Schematic of the cnk locus indicating the Y2H AlkICD-interacting Cnk prey constructs. PDZ, postsynaptic density protein (PSD95), Drosophila discs-large tumor suppressor (Dlg1), and zonula occludens–1 protein (zo-1); RIM, Raf-interacting motif; IS, inhibitory sequence; pYELI, phosphorylation site for Src42A. Untranslated regions are noted in gray. The minimal AIR (Ala1384-Ser1425) is shown in red. (B) Yeast growth on double-selective transformation-mating control (−Leu, −Trp) and triple-selective (−Leu, −Trp, −His) media plates. Each streak corresponds to diploid yeast colonies coexpressing the indicated Gal4_AD Cnk fusion proteins and either Gal4_BD-AlkICD or Gal4_BD (empty vector control). (C) Late-stage female Drosophila pupae expressing pan-neuronal UAS-jeb under the control of elavC155-Gal4 in control, cnkk16314/+, cnk63F/+, and Df(2R)rl10a/+ genetic backgrounds. Pupae carrying no UAS transgene were used as a negative control. Quantification of the Cnk-dependent modification of body size (right). Outliers from the 2.5 to 97.5 percentile (whiskers) appear as circles, ***P < 0,001; n.s., not significant. One-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test were used. n = 559 pupae, >100 pupae for each genotype; values were pooled from four experimental replicates. (D to I) In situ hybridization showing cnk expression in cnk63F(m/z)/BSC161 embryos (D) and in embryos expressing cnk under control of the en2.4-Gal4 driver (E). Maternal cnk transcripts in a stage 2 w1118 embryo (F), ubiquitous zygotic cnk expression in cnk63F(m/z)/+ embryos at stages 5 (G) and 10 (H). Cnk expression in the brain (br), ventral nerve chord (vnc), and peripheral nervous system (arrowheads) at the end of embryogenesis (I). n ≥ 100 embryos for each genotype. (J and K) Cnk and Alk protein localization in stage 10/11 fTRG1248 (cnk.SGFP) control (J) and Alk1/Exel7144; cnk.SGFP transheterozygous (K) embryos. Dotted yellow lines indicate VM clusters; n ≥ 100 embryos for each genotype. Scale bars, 500 bp (A), 500 μm (C), 50 μm (D to I), and 10 μm (J and K).

  • Fig. 2 Ectopic expression of CnkAIR inhibits Alk signaling.

    (A to E) Heads of adult female flies expressing hemagglutinin (HA)–tagged 5xCnkAIR (cnkAIR.HA) (A), HA-tagged 5xMyr-CnkAIR (Myr::cnkAIR.HA) (B), UAS-Alk alone (C), and UAS-Alk in combination with either HA-tagged 5xCnkAIR (D) or HA-tagged 5xMyr-CnkAIR (E) under the control of the sev-Gal4 driver. n ≥ 400 flies for each genotype. (F to H) Alk antibody staining (blue) in twist.2xPE (2xPE) driver control (F), 2xPE>cnkAIR.HA (G), and 2xPE>Myr::cnkAIR.HA (H) embryos at stage 11/12. rP298-LacZ [β-galactosidase (β-Gal), red] reporter gene expression in visceral FCs and somatic FCs (arrows in G and H). Construct expression is revealed by HA tag antibody staining (HA, green). (I to K) Stage 16 embryos of the indicated genotype stained for HandC-GFP reporter gene expression (GFP, green), FasIII (red), and HA (blue). Asterisks in (J) and (K) indicate somatic muscles labeled by HA staining (blue). n ≥ 200 embryos for each genotype and staining. Scale bars, 50 μm.

  • Fig. 3 Loss of maternal and zygotic cnk genocopies Alk and jeb mutants.

    (A, C, E, G, I, and K) VM specification in stage 11/12 Drosophila embryos with the indicated genotypes revealed by rP298-lacZ (β-Gal, red) and Alk (green) expression. In (I), embryos are stained for FasIII (green) instead of Alk. (B, D, F, H, J, and L) Stage 16 embryos carrying the HandC-GFP reporter stained for FasIII and GFP to reveal the presence of visceral muscles. GFP expression in garland cells (gc) confirms the presence of the HandC-GFP reporter in (F), (H), and (J). n ≥ 600 embryos for (A) to (J); n = 50 embryos for (K) and (L). Scale bars, 50 μm.

  • Fig. 4 Cnk functions between Ras and Raf in the Ras-ERK pathway.

    Stage 11/12 embryos were stained for pERK (blue), HandC-GFP reporter gene expression (GFP, green), and Alk (red). Dorsal views are shown. (A) bap3-Gal4 (bap>) driver control. Arrow marks pERK- and GFP-positive visceral FCs. (B to D) pERK staining and HandC-GFP reporter expression in HandC-GFP, bap3>UAS-jeb (B), HandC-GFP bap3>UAS-Ras85DV12 (C), and HandC-GFP bap3>UAS-Raf.gof (D) embryos. (E) Maternal and zygotic HandC-GFP; cnksag13L(m/z)/BSC161 mutant. (F to H) pERK and GFP staining in cnksag13L(m/z)/BSC161 embryos that carry the HandC-GFP reporter and express either UAS-jeb (F), UAS-Ras85DV12 (G), or UAS-Raf.gof (H) under the control of the bap3-Gal4 driver. n ≥ 40 embryos for each genotype. Scale bar, 50 μm.

  • Fig. 5 CnkAIR is required for robust activation of ERK in visceral FCs.

    (A) Schematic of the cnk locus indicating deletions in cnkΔAIR and cnkΔY2H and the cnkCC9-110B allele. cnkΔAIR flies harbor a 126-bp in-frame deletion in the endogenous cnk locus, which removes the region encoding the 42–amino acid AIR (Ala1384-Ser1425) of the Cnk protein. cnkΔY2H flies harbor a 357–amino acid deletion (His1182-Asn1538). (B to C) Stage 11/12 balanced siblings (B) and cnkΔAIR(m/z)/BSC161 (C) embryos expressing the rP298-LacZ FC reporter were stained for β-Gal (red) and Alk (green). (D and E) Stage 16 embryos stained for HandC-GFP reporter gene expression (green) and FasIII (red). β-Gal (blue) staining reveals balancer-associated LacZ expression in a sibling embryo (D). n ≥ 100 embryos for (B) to (E). (F to H) pERK (black) in visceral FCs (arrows) and tracheal pits (arrowheads) of ventrally orientated stage 11 control (F), cnkΔAIR(m/z) (G), and AlkKO (H) embryos. The bottom panel in (F), (G), and (H) depicts a [rainbow RGB color lookup table (LUT)] heat map representation of close-ups from the embryo in the upper half. (I) Quantification of (background-)corrected total fluorescence intensities (CTF) in the VM relative to pERK CTFs of the adjacent tracheal pits. One-way ANOVA with Dunett’s multiple comparisons test was used to reveal statistical significance (***P < 0.001; n = 40 measurements from three different staining replicates for each genotype). (J) VM development in a stage 11/12 cnkΔY2H(m/z)/BSC161 embryo, expressing the rP298-LacZ FC reporter stained for β-Gal (red) and Alk (green). (K) Stage 16 HandC-GFP and cnkΔY2H(m/z)/BSC161 embryo stained for GFP (green) and FasIII (red). n ≥ 50 embryos for (J) and (K). (L and M) pERK (black) in visceral FCs (arrows) and tracheal pits (arrowheads) of ventrally orientated stage 11 cnkΔY2H(m/z) (L) and cnk63F(m/z)/BSC161 (M) embryos. n ≥ 30 embryos. Bottom panels in (L) and (M) depict a (rainbow RGB color LUT) heat map representation of close-ups from the embryo in the top panels. Scale bars, 50 μm.

  • Fig. 6 Ave, but not Ksr, is required for Cnk-mediated Alk signaling in the VM.

    (A) Table summarizing the binding partners and numbers of interacting preys obtained by Y2H screening using full-length Cnk as bait. (B) Schematic representation of the ave locus. The first exon of the minus-orientated Rpn6 gene is shown below; CRISPR/Cas9-induced molecular lesions of the aveCC9-20A and aveCC9-36A null alleles are indicated. (C to K) Antibody staining against HandC-GFP reporter gene expression (green), FasIII (red), and balancer-associated β-Gal expression [blue in (C) to (E)] in stage 11/12 (C, F, and I), stage 13/14 (D, G, and J), and stage 16 (E, H, and K) embryos of the indicated genotypes. n ≥ 100 embryos for (C) to (H); n ≥ 40 embryos for (I) to (K). Scale bars, 50 μm.

  • Fig. 7 Model incorporating a role for Cnk and Ave in Alk signaling during visceral FC specification.

    Alk activation at the membrane of a prospective visceral FC by the ligand Jeb, which is secreted from the neighboring somatic mesoderm, induces the Raf/MAPK/ERK signaling cascade, eventually leading to the transcriptional activation of downstream targets including kirre, org-1, and Hand. Cnk and Ave are core components of the Alk signaling pathway that are required downstream of the receptor and upstream of Raf to mediate visceral FC specification. Although not essential for Jeb-Alk signaling, Ksr appears to be required for full activation of ERK.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/502/eaan0804/DC1

    Fig. S1. Characterization of the cnk alleles used in this study.

    Fig. S2. The fTRG library line fTRG1248 (Cnk.SGFP) rescues the lethality of cnk mutations.

    Fig. S3. CnkY2H/ΔAIR binds to the AlkICD in Y2H.

    Fig. S4. Eye morphology of mutants with CRISPR/Cas9-modified cnk alleles.

    Fig. S5. Morphological analysis of cnk germline clone–derived embryos.

    Fig. S6. Visceral phenotypes caused by mutations in ksr.

    Table S1. The fTRG library line fTRG1248 (Cnk.SGFP) rescues the lethality of cnk mutants.

    Table S2. Complementation tests for cnk alleles.

    Table S3. Complementation tests for aveCC9 alleles.

  • Supplementary Materials for:

    The scaffolding protein Cnk binds to the receptor tyrosine kinase Alk to promote visceral founder cell specification in Drosophila

    Georg Wolfstetter, Kathrin Pfeifer, Jesper R. van Dijk, Fredrik Hugosson, Xiangyi Lu, Ruth H. Palmer*

    *Corresponding author. Email: ruth.palmer{at}gu.se

    This PDF file includes:

    • Fig. S1. Characterization of the cnk alleles used in this study.
    • Fig. S2. The fTRG library line fTRG1248 (Cnk.SGFP) rescues the lethality of cnk mutations.
    • Fig. S3. CnkY2H/ΔAIR binds to the AlkICD in Y2H.
    • Fig. S4. Eye morphology of mutants with CRISPR/Cas9-modified cnk alleles.
    • Fig. S5. Morphological analysis of cnk germline clone–derived embryos.
    • Fig. S6. Visceral phenotypes caused by mutations in ksr.
    • Table S1. The fTRG library line fTRG1248 (Cnk.SGFP) rescues the lethality of cnk mutants.
    • Table S2. Complementation tests for cnk alleles.
    • Table S3. Complementation tests for aveCC9 alleles.

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    Citation: G. Wolfstetter, K. Pfeifer, J. R. van Dijk, F. Hugosson, X. Lu, R. H. Palmer, The scaffolding protein Cnk binds to the receptor tyrosine kinase Alk to promote visceral founder cell specification in Drosophila. Sci. Signal. 10, eaan0804 (2017).

    © 2017 American Association for the Advancement of Science

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