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@gu.se

Published 24 October 2017, Sci. Signal. 10, eaan0804 (2017)

DOI: 10.1126/scisignal.aan0804

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.

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

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

(A) Schematic representation of the cnk locus including genome coordinates and depicting

regions encoding for Cnk domains in different colors. SAM=sterile alpha motif in orange,

CRIC=conserved region in Cnk in light green, PDZ=post synaptic density protein (PSD95),

Drosophila discs large tumor suppressor (Dlg1), and zonula occludens-1 protein (zo-1) in

magenta, PH=pleckstrin homology domain in blue, RIM=Raf interacting motif in yellow,

IS=inhibitory sequence in brown, pYELI=phosphorylation site for Src42A in dark green, minimal

Alk interacting region (AIR) in red. UTRs appear narrower and in grey, introns as dashed lines.

The molecular lesions identified in different cnk alleles and the predicted changes in the resulting

Cnk proteins are indicated. (B-F) Absence of the visceral muscle in germ line clone-derived late

stage embryos carrying the HandC-GFP reporter and different cnk mutations “in trans” to the cnk

deficiency BSC161. Embryos were stained for GFP reporter gene expression (green) and FasIII

(red). N > 500 embryos for cnk63F and cnksag alleles, N ≥ 60 embryos for cnk14C and cnk116C. Scale

bar = 500 bp (A), 50 µm (B).

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

mutations.

(A-C) Eyes of control (A), homozygous cnk63F (B), and transheterozygous cnksag13L/cnksag32-3 (C)

female flies carrying the fTRG1248 insertion (Cnk.SGFP). Although Cnk.GFP rescues lethality

caused by cnk mutations, flies exhibit a weak rough eye phenotype which is slightly stronger in

the background of the proposed Raf-repressor alleles cnksag13L/cnksag32-3. N ≥ 200 flies for each

genotype. Scale bar = 50 µm.

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

Triple-selective (-Leu –Trp –His) media plate containing yeast colonies double-transformed with

the AlkICD-bait and a Cnk-prey construct (left) that comprises the range of all Alk-interacting Cnk

clones (His1182-Asn1538) with exception of the 42 amino acid CnkAIR (CnkY2H/AIR). AlkICD-bait

and an empty prey vector were used as a negative control (center). Human ALKICD (hALKICD)

and the growth factor receptor bound protein 2 (GRB2) adaptor protein (right) were employed as

a positive control.

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

Eyes of control female flies (A), and female flies carrying cnkAIR (B), cnkY2H (C), cnkCC9-110B “in

trans” to the cnk63F null allele. cnkAIR/cnk63Fanimals display wild-type eye morphology whereas

cnkY2H/cnk63F and cnkCC9-110B/cnk63F flies exhibit a rough-eye phenotype. N ≥ 100 flies for each

genotype. Scale bar = 50 µm.

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

(A) pERK antibody staining (green) of stage 5 control embryo to reveal terminal activity of the

RTK Torso. (B) Decreased pERK signals in a cnk63F (m-) embryo. N ≥ 30 embryos for A and B.

(C) Torso-dependent tll mRNA expression in a stage 5 control embryo revealed by in situ

hybridization. (D) The anterior tll expression domain is shifted and the posterior domain is

reduced in a cnk63F (m-) embryo. (E) Torso-dependent hkb mRNA expression in a stage 5 control

embryo. (F) Reduced hkb expression in a cnk63F (m-) embryo. Expression domains are indicated

as percentage of total egg length. N ≥ 15 embryos for C-F. (G-I) Stage 15-16 embryos stained

with antibodies against Cut protein (CT, red) and Drosophila epithelial cadherin (DE-Cad, green)

to analyze tll-dependent and hkb-dependent organ development of the alimentary tract and

balancer-associated β-Galactosidase (β-Gal, blue), respectively. Control embryo exhibiting

properly developed gut and Malpighian tubules (Mt, arrows point to the tip) (G). In accordance

with the reduced tll and hkb expression domains, Mts (arrows) and hindgut are shortened in

cnk63F(m-/z-)/BSC161 embryos (H). Mts are elongated but often misplaced (arrow points to a Mt

tip) in cnkY2H(m-/z-)/BSC161 embryos; fore- and hindgut appear normal (I). Notably, midgut

cells (arrowheads) are present and have spread in cnk- germ line clones, suggesting that Hkb

function is not grossly affected. (J-L) Stage 16-17 embryos stained for β3-Tubulin (β3-Tub, red)

and Hand-GFP (GFP, green) to reveal defects caused by impaired Heartless (FGFR)-signaling.

Control embryo (J). Strong defects in the heart and dorsal somatic muscles of a cnk63F(m-/z-

)/BSC161 embryo (K) but only mild defects in the case of cnkY2H(m-/z-)/BSC161 (L). (M-O) In

agreement with this observation, Even-skipped (EVE 3C10, red)-positive pericardial cells at stage

16 observed in control embryos (M) are absent in cnk63F(m-/z-)/BSC161- (N) but not in

cnkY2H(m-/z-)/BSC161 (O) embryos. (P-R) Heartless-dependent visceral longitudinal muscle

(LVM) development revealed by bHLH-lacZ reporter expression (β-Gal, green) and FasIII (red)

antibody staining in stage 13 embryos. In control embryos (P), LVMs migrate towards the

anterior pole. Impaired LVM development (arrows mark LVMs that exhibit the morphology of

dying cells) observed in a cnk63F(m-/z-)/BSC161 embryo (Q). cnkY2H(m-/z-)/BSC161 embryos

exhibit only mild LVM migration defects (R, arrows). (S-U) Breathless (FGFR)- and EGFR-

dependent tracheal development revealed by anti-GASP antibody staining (2A12, red) in stage

16-17 embryos. Control embryo (S). Phenotypic variation of the tracheal defects in cnksag32-3(m-

/z-)/BSC161 and cnk63F(m-/z-)/BSC161 embryos (T). Trachea development is only weakly

affected in cnkY2H(m-/z-)/BSC161 embryos (U). (V-X) EGFR-dependent development of muscle

attachment sites revealed by antibody staining against βPS-Integrin (βPS, cyan) at stage 16/17.

Control embryo (V). Abnormal muscle attachment sites in cnk63F(m-/z-)/BSC161 embryos (W)

and only mild defects in case of cnkY2H(m-/z-)/BSC161 (X). N ≥ 40 embryos for G to X. Scale

bars = 50 µm.

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

(A) Schematic representation of Ksr. The position of the proposed Raf-Ksr dimerization surface

is indicated by a green line, and the positions of the point mutations in krsS-627 and ksrS-638 alleles

and the predicted consequences for the Ksr-protein are indicated. CA = conserved area, KD =

protein kinase domain. (B) Antibody staining against GFP (green) and FasIII (red) reveals

impaired VM stretching and terminal defects in a late stage HandC-GFP; ksrS-638(m-/z-)/ksrS-627

embryo. N ≥ 40 embryos. (C) Late stage 11 zygotic mutant ksrS-627 embryo with slightly irregular

pERK staining (arrow indicates the visceral FC row). N ≥ 60 embryos. (D) Further reduction of

pERK signals in the VM (arrows) and severe irregularities in the pERK expression pattern of a

ksrS-627 germ line clone. N = 8 embryos. Scale bars = 100 amino acids (A), 50 µm (B, C).

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

mutants.

cnk allele (cnk-) cnk-/cnk-; fTRG1248/+ cnk-/Df(2R)BSC161;

fTRG1248/+

Df(2R)BSC161 (negative control)

no rescue no rescue

cnkk16314 rescued to viable and fertile flies n.d.

cnk63F rescued to viable and fertile flies n.d.

cnk14C some rescued to viable flies,

most die at pupal stage

rescued to viable and fertile flies

cnk116C rescued to viable and fertile flies n.d.

cnksag32-3 no rescue rescued to viable and fertile flies

cnksag13L no rescue rescued to viable and fertile flies

Table S2: Complementation tests for cnk alleles.

Alleles

employed for

crosses

Alk1 Alk10 Df(2R)BSC199

(Df jeb)

jebweli jebk05644 sag32-3 sag13L

Transheterozygous flies obtained in F1 (total progeny count is indicated in case

of non-complementation)

cnk63F Yes Yes Yes Yes Yes n.d. n.d.

cnkk16314 Yes Yes Yes Yes Yes No (618) No (653)

cnk14C Yes Yes Yes Yes Yes n.d. n.d.

cnk116C Yes Yes Yes Yes Yes n.d. n.d.

Df(2R)BSC161

(Df cnk)

n.d. n.d. n.d. n.d. n.d. No (260) No (181)

Table S3: Complementation tests for aveCC9 alleles.

Alleles employed for

crosses

aveCC9-36B aveCC9-20A

Transheterozygous flies obtained in F1 (total progeny count is

indicated in case of non-complementation)

Rpn20F Yes Yes

RpnMB09493 Yes Yes

ave108V No (325) No (306)