Src kinase function controls progenitor cell pools during regeneration and tumor onset
in the Drosophila intestine
Alexander Kohlmaier1, Christina Fassnacht1, Jin Yinhua1, Hanna Reuter1, Jamila Begum1,
Devanjali Dutta and Bruce A. Edgar1
1
DKFZ-ZMBH Alliance, Universität Heidelberg, Im Neuenheimer Feld 282, 69120
Heidelberg, Germany
SUPPLEMENTARY INFORMATION
SUPPLEMENTARY MATERIALS AND METHODS:
Antisera:
The following primary antibodies were used: rabbit anti-phospho 4E-BP (Cell Signaling)
1/500; mouse anti-Delta (DSHB), 1/100; rabbit anti-phospho-Histone H3 S10(Millipore)
1/1000; chicken anti-GFP (Invitrogen) 1/1000); mouse anti-pH3 S10(Cell Signaling) 1/300;
rabbit anti-β-galactosidase (Cappell), 1/2000; rabbit anti-Pdm1 (kind gift from W. Chia,
Temasek Laboratories, Singapore) 1/1500; mouse anti-Pros (DSHB) at 1/100; chicken antiGFP (Invitrogen (1/1000); Secondary Alexa coupled antibodies (Invitrogen) were used at
1/1000. mouse anti-dpERK (Sigma) 1/100; rabbit anti-pSrc (S. Hayashi) at 1/1000
RNAseq analysis:
Total RNA was directly isolated from 20-30 whole midgut tissues of different Src- or Ackexpressing intestines and their respective controls (esgts controls or esgFO control) after 1
week of transgene expression (Suppl. Table 1,2). RNA was isolated with Trizol (Invitrogen)
without prior tissue dissociation. For esgFO>Src, esgFO>Ack and esgts>Src expression 6x
multiplexed libraries were made and run on an Illumina HiSeq2000 sequencer with 50 bp read
length. For esgts and dlts mediated Ack expression, 4x multiplexed libraries were made with
the Illumina TruSeq preparation kit from normalized amounts of RNA and run on a GAIIX
sequencer with 72 bp read length. 8-14M reads were obtained and 4-8M unique reads mapped
and normalized using RPKM normalization. RPKM values for all experimental conditions are
presented in the form of log2 transformed normalized expression (N.E.) values in the Suppl.
Tables.
For esgts >Notch RNAi and the respective esgts control samples (Suppl. Table 1,2), midguts
were dissociated with 75μl of 50mg/ml Collagenase for 1 hour at 27°C. 2000 esgGFP+ cells
were sorted using a FACS Aria II sorter (BD Biosciences). Total RNA by extraction using the
PicoPure RNA isolation kit (Arcturus) resulted in approx. 2ng of RNA. RNA was
subsequently linearly amplified (5000x) using the RiboAmp HS PLUS RNA amplification kit
(Applied Biosciences). RNA sequencing was performed using an Illumina HiSeq2000
sequencer with 2x multiplexed libraries with 50 bp read length. 35-40M reads were obtained
and 15-20M unique reads mapped and normalized using RPM normalization. Samples were
analyzed in replicates of three at 95% confidence level.
For WT esg+, ISC and EB cell type-specific profiles (Suppl. Fig. S3A) 100 dissected guts
were dissociated and 2000 GFP+ cells each were sorted using esg>GFP (progenitors), GbeSu(H)ts>GFP (EBs) or Dlts>GFP (ISCs) cell-type specific Gal4 drivers and analyzed as
above. RNA sequencing was performed using an Illumina HiSeq2000 sequencer with 2x
multiplexed libraries with 50 bp read length. Data from 3 replicates per genotype were
analyzed using DNAStar’s QSeq software.
Reads were mapped to the Drosophila melanogaster genome (NCBI release 5.10). Data were
analyzed using DNAStar’s QSeq software. The gene set of functional cell cycle regulators
analyzed in Suppl. Table 1, 2 was previously assembled1 and imported into QSeq software.
No cutoff for minimal expression was applied to exclude weakly expressed genes, because
also Delta, the standard marker for stem cells, is weakly expressed in wildtype ISCs.
Fly strains:
w; MyoIAGal4; tubGal80ts UAS-GFP (myo1ats), yw; esgGal4; tubGal80ts UAS-GFP (esgts),
w;esgGal4, tubGal80ts, UAS-GFP; UAS-Flp, Act>CD2>Gal4 (esgFO) have been described
previously2. w; Gbe-Su(H)Gal4, UAS-CD8-GFP; tubGal80ts and w;tubGal80ts; UASGFP;DlGal4 were described3. UAS-Src42a.CA, UAS-Src64b, UAS-Mkp3, and the EgfrCO
mutant allele were obtained from Bloomington stock center (Bloomington, IN, USA).
(MAE)Src64b4 (Fig. 2A, A') was obtained via Leslie Saucedo (University of Puget Sound,
Tacoma, USA). UAS-Ack lines were described5 and a kind gift from N. Harden (Simon Fraser
University Burnaby, British Columbia, CA), Su(H)Gbe-LacZ was a kind gift from S. Bray
(University of Cambridge, UK), the DeltaGal4 and Gbe-Su(H)Gal4 lines were kind gifts
from S. Hou (NIH Frederick, Maryland, USA). The bantam microRNA GFP sensor lines was
a kind gift from M. Milan (IRB Barcelona, ESP), Pon-GFP line was a kind gift from Y.-N.
Jan (HHMI UCSF, USA). stg::GFP and cycB3::GFP trap lines were from the Yale Flytrap
consortium (USA), the STAT-GFP6 ([BAC CH321-73F24 P[acman] line was a gift from Sol
Sotillos (CABD, Sevilla, ESP). UAS-Src42a30-1, UAS-Src42a43-3, UAS-Src64bYF42(constitut.
act.), UAS-Src64b9-2 were all kind gifts from Tian Xu7 (Yale, School of Medicine, USA).
UAS-DomeCyt and UAS-DomeCyt8 were obtained via Parthive Patel from James
Castelli-Gair Hombría (CAPD, Sevilla, ESP). RNAi lines were obtained from Vienna
Drosophila VDRC RNAi or the TRIP RNAi Stock Centers: Src42a RNAi (VDRC GD10610
v26019), Src64b RNAi (VDRC GC12263 v35252), Notch RNAi (v27228), PS3/4 RNAi
(VDRC v4891) targeting CG8095 (PS3) and with 4/270 19-mer hits also CG16827 (PS4),
aPKC (VDRC v2907), UAS-stg, UAS-cycE, UAS-E2f1, UAS-Dp and UAS-Cdk2 and their
combinations were reported9, Egfr RNAi and Stat92E RNAi flies published2, 10. UAS-rca1
and UAS-fzr lines were reported11-13. Flies were maintained on standard media, only female
flies were analysed.
1
Guest ST, Yu J, Liu D, Hines JA, Kashat MA, Finley RL, Jr. A protein network-guided
screen for cell cycle regulators in Drosophila. BMC Syst Biol 2011; 5: 65.
2
Jiang H, Patel PH, Kohlmaier A, Grenley MO, McEwen DG, Edgar BA.
Cytokine/Jak/Stat signaling mediates regeneration and homeostasis in the
Drosophila midgut. Cell 2009; 137: 1343-1355.
3
Zeng X, Chauhan C, Hou SX. Characterization of midgut stem cell- and enteroblastspecific Gal4 lines in drosophila. Genesis 2010; 48: 607-611.
4
Pagarigan KT, Bunn BW, Goodchild J, Rahe TK, Weis JF, Saucedo LJ. Drosophila
PRL-1 is a growth inhibitor that counteracts the function of the Src oncogene.
PLoS One 2013; 8: e61084.
5
Zahedi B, Shen W, Xu X, Chen X, Mahey M, Harden N. Leading edge-secreted Dpp
cooperates with ACK-dependent signaling from the amnioserosa to regulate
myosin levels during dorsal closure. Dev Dyn 2008; 237: 2936-2946.
6
Sotillos S, Krahn M, Espinosa-Vazquez JM, Hombria JC. Src kinases mediate the
interaction of the apical determinant Bazooka/PAR3 with STAT92E and increase
signalling efficiency in Drosophila ectodermal cells. Development 2013; 140:
1507-1516.
7
Pedraza LG, Stewart RA, Li DM, Xu T. Drosophila Src-family kinases function with
Csk to regulate cell proliferation and apoptosis. Oncogene 2004; 23: 4754-4762.
8
Brown S, Hu N, Hombria JC. Identification of the first invertebrate interleukin
JAK/STAT receptor, the Drosophila gene domeless. Curr Biol 2001; 11: 17001705.
9
Buttitta LA, Katzaroff AJ, Perez CL, de la Cruz A, Edgar BA. A double-assurance
mechanism controls cell cycle exit upon terminal differentiation in Drosophila.
Dev Cell 2007; 12: 631-643.
10
Jiang H, Grenley MO, Bravo MJ, Blumhagen RZ, Edgar BA. EGFR/Ras/MAPK
signaling mediates adult midgut epithelial homeostasis and regeneration in
Drosophila. Cell Stem Cell 2011; 8: 84-95.
11
Zielke N, Querings S, Grosskortenhaus R, Reis T, Sprenger F. Molecular dissection
of the APC/C inhibitor Rca1 shows a novel F-box-dependent function. EMBO Rep
2006; 7: 1266-1272.
12
Sigrist SJ, Lehner CF. Drosophila fizzy-related down-regulates mitotic cyclins and
is required for cell proliferation arrest and entry into endocycles. Cell 1997; 90:
671-681.
13
Grosskortenhaus R, Sprenger F. Rca1 inhibits APC-Cdh1(Fzr) and is required to
prevent cyclin degradation in G2. Dev Cell 2002; 2: 29-40.
14
Buchon N, Broderick NA, Kuraishi T, Lemaitre B. Drosophila EGFR pathway
coordinates stem cell proliferation and gut remodeling following infection. BMC
Biol 2010; 8: 152.
15
Shindo M, Wada H, Kaido M, Tateno M, Aigaki T, Tsuda L et al. Dual function of Src
in the maintenance of adherens junctions during tracheal epithelial
morphogenesis. Development 2008; 135: 1355-1364.
16
Brennecke J, Hipfner DR, Stark A, Russell RB, Cohen SM. bantam encodes a
developmentally regulated microRNA that controls cell proliferation and
regulates the proapoptotic gene hid in Drosophila. Cell 2003; 113: 25-36.
SUPPLEMENTARY FIGURE LEGEND
Suppl. Figure S1 Src levels determine progenitor proliferation capacity
(A) Conditional Src kinase expression in esg+ progenitors for 7 days triggers progenitor
division and esg+ progenitor pool size increase. Note that wildtype stem cell nests contain 1-3
esg+ cells. 6-8 esg+ cells are observed per stem cell nest following Src64b expression.
Amplified esg+ cells following Src42a.CA expression cover the intestinal epithelium. Some
surplus esg+ cells following Src kinase expression are diploid, while some show ectopic
endoreplication. Zoom-in (right panels). (B) Overexpression of different transgenic alleles of
Src64b or Src42a in esg+ cells for 1 day, including the constitutively active mutant Src64bYF42
and wildtype Src64b9-2, wildtype Src42a30-1 and wildtype Src42a43-3 alleles. All these alleles
have been classified as exhibiting moderate (M) or strong (S) mutant tissue growth
phenotypes when expressed with GMR-GAL4 drivers in larval eye imaginal discs7.
Overexpression of moderate Src42a alleles stimulates ISC division more strongly than strong
or constitutively active Src64b alleles. (B') Real-time PCR analysis. mRNA abundance of
selected cycle regulators cyclin A, cyclin B, string, of escargot and of stress-related cytokines
rhomboid and spaetzle following 1 day pulse-expression of Src42a.CA, Src64b or Ack in esg+
cells. (B'') Src64b expression in ECs triggers EC delamination from the basement membrane
into the intestinal lumen more strongly than Src42a.CA overexpression. This was judged from
a decrease of midgut longitudinal and circumferential extensions, which is known to be due to
available EC numbers14. (C) esgFO-Gal4-mediated clonal labelling of esg+ progenitors and
their progeny for 2 days shows that expression of Src42a.CA or of Src64b causes progenitor
cell overproliferation (non-overlapping GFP+ clones marked by dotted line). Zoom-in of
boxed area in the right panels. pERK staining (extracellular signal-regulated kinase) marks
ERK MAPK signaling activity and distinguishes undifferentiated progenitors from
differentiated cells. Extra cells produced by Src kinases activation maintain pERK activity in
many accumulating cells.
Suppl. Figure S2 Src kinases are required for esg+ progenitor cell proliferation rate
(A) Co-depletion of Src42a and Src64b in esg+ cells for 14 days (at 29°C) causes a reduction
in the number of esg+ marked progenitor cells. Since ISCs (marked with Delta) are still
detectable, this reduction is due to impaired replenishment of differentiating EBs by
regenerative division of ISC division. (B) Src42a or Src64b inhibition by trangenic RNAi (IR)
for 7 days triggers reduction of DNA replication capacity in esg+ progenitors. FACS analysis
of DNA content. Note reduced numbers of progenitors in S-phase following Src42a depletion
during P.e. infection-mediated tissue regeneration (middle panel, red trace) compared to
infected wildtype controls (left panel, red trace). For reference, red dashed horizontal line
marks S-phase levels in P.e. infected wildtype. After Src42a depletion reduced numbers of
diploid nuclei and increased numbers of endoreplicative nuclei are observed in uninfected
esg+ cells (middle panel, black trace) compared to uninfected wildtype progenitors (left panel
black trace). (C) EsgFO-mediated Src42a depletion for 7 days does not lead to ISC
differentiation. Red arrows indicate remaining weak endocytic Delta foci marking ISCs,
which are display reduced p4E-BP levels, a Notch-signaling activity-dependent marker of
undifferentiated ISCs. (C') Prolonged Src42a depletion for 28 days does not trigger
differentiation of ISCs, as judged from absence of absence of Gbe-Su(H)-LacZ levels, a
transcriptional reporter for Notch signaling activity. (D) Levels and distribution of
phosphorylated active Src42a (pSrc42aY40015) in healthy wildtype and P.e. infected midguts.
Suppl. Figure S3 Asymmetric ISC division following core cell cycle stimulation
(A) pERK signaling strength per individual cell was not increased upon Src42a.CA or Src64b
activation compared to controls. Quantification of mean nuclear pERK intensity per
individual cell plotted in arbitrary units (a.u.) after normalization to the mean nuclear
escargot-GFP intensity within a cell. Each bar represents one cell, values were ranked from
low to high. (A') Endogenous STAT92E protein levels and distribution, as reported from a
STAT92E-GFP BAC transgene, in healthy wildtype or following Src42a.CA expression in
esg+ progenitors. White arrow mark diploid esg+ progenitors, and red arrows mark aberrant
endoreplicating esg+ cells. (B) String (Stg), Cyclin E (CycE), E2F1/Dp were expressed alone
or in combination with Cdk2, as indicated, in Delta+ ISCs or Gbe-Su(H)+ EBs. Mitotic rates
were measured. For E2F1/Dp expression in EBs the numbers of total mitoses per midgut and,
separately, of mitoses within GFP-marked EBs were plotted. Note that E2f1, Dp;CycE;Cdk2
coexpression triggered much higher levels of mitoses and that data were plotted on a different
scale. (C) Different classes of esg+ progenitor cell clusters form following co-expression of
Stg and CycE in esg+ cells. Gbe-Su(H)-LacZ levels report Notch activity in the history of
these cells: note rare clusters that are Notch-negative (outlined with white dotted line), as well
as clusters with weak Notch levels or containing several distinctly Notch positive cells
(orange) and frequent clusters containing mostly Notch positive cells (red). (C') Mitotic cell
cycle stimulation by E2f1 + Dp co-expression for 4 days in ISC by MARCM clonal analysis
reveals amplification of Notch-positive (Gbe-Su(H)+) EBs (red arrows) and maintenance of a
single Notch-negative bona fide ISCs (white arrow).
Suppl. Figure S4 Ack kinase expression triggers amplification of a specified progenitor
cell type
Ack overexpression leads to particularly strong amplification of Dl+ cells. We tested whether
these Ack-expressing Dl+ cells represented ISC-like cells by expression profiling. (A) We
established wildtype ‘ISC-selective’ and wildtype ‘EB-selective’ RNA-seq expression
signatures, by comparison of FACS-sorted wildtype ISCs with sorted wildtype EBs in
triplicates at 95% confidence. These profiles were compared to the profile of whole midgut
tissues consisting mostly of esg+ progenitors, many of which where Dl+, after 1 week of Ackoverexpression. The mRNA expression profile of midguts following Ack expression in esg+
progenitors for 7 days (cutoff=2x differential expression) is more similar to the expression
profile of FACS-sorted wildtype enteroblasts than to FACS-sorted wildtype ISCs. No cutoff
for minimal normalized expression level was applied to exclude any weakly expressed genes,
because also Delta, the standard marker for stem cells, is weakly expressed in uninfectd
wildtype progenitor cells.
EB-selective and ISC-selective expression profiles were determined by comparison of
wildtype EB with ISC profiles at 2-fold or 10-fold differential upregulation (top) or
downregulation (bottom). See Suppl. Materials and Methods for determination of ISC- and
EB selective profiles. (B) Bantam microRNA displays higher activity in ISCs (red arrow) and
reduced activity in differentiated ISC daughter cells (white dashed arrows, left panel) using a
transgenic Tub-GFP reporter controlled by two 31 nucleotide long bantam micro RNA target
sites integrated in the 3'UTR of the P element in the CaSpeR4 transgenesis vector16. Ack
expression for 7 days can amplify the number of cells with high bantam activity (red arrows).
Suppl. Figure S5 Molecular control of the mitotic-to-endocycle switch in the intestine
(A-C') APC/C activity levels control maintenance of diploid esg+ progenitor cell fate.
(A) Cyclin B3 is not degraded in differentiating ISC daughter cells residing in one stem cell
nest (preplicating ISCs are marked by EdU labelling, red arrow). (B) Rca1 overexpression
inhibits mitotic-to-endocycle switching in progenitors as well as impairs the maturation of
differentiating EBs to Pdm1+ ECs compared to esgFO controls of the same age. Note Pdm1
staining in mature EC derived from ISC division within 12 days in the wildtype (white
arrows). Nuclear Pdm1 staining is absent in arrested GFP+ cells following Rca1 expression
(yellow dashed arrows). (C) Fzr expression stimulates formation of high rates of
endoreplication in wildtype esg+ cells and reduces esg+ progenitor overproliferation triggered
by Notch depletion when Fzr is coexpressed with the Notch RNAi constructs. In this case, Fzr
decreases the number of ectopic EEs following Notch depletion, at least in part by causing
aberrant endoreplication in diploid Prospero-marked EEs. (C'). Zoom-in of C: Fzr
overexpression reduces EE cluster formation of Notch-depleted progenitors. Arrow marks
ectopic endoreplication in a Notch-depleted Prospero marked EE.
Suppl. Table 1 Src kinases expression levels in wildtype intestinal cell types after P.e.
infection
Numbers represent fold-change (up or down as indicated) in mRNA abundance of P.e.
infected adult intestinal cell types compared to healthy wildtype counterparts. For RNAseq
analysis, esgGFP-labeled progenitors, or Delta-GFP labeled ISCS, or Gbe-Su(H)-GFP labeled
EBs were FACS-sorted.
Suppl. Table 2 List of induced cell cycle regulators following Src42a or Ack expression or
Notch inhibition.
Ack or Src42a.Ca kinases were expressed using either the esgts flipout (esgFO) system2 for
permanently marking ISCs and their progeny or esg-Gal4 expression (esgts) for progenitorspecific gene expression affecting ISCs and their EB daughters. Numbers represent foldchange increase in mRNA abundance of functional cell cycle regulators compared to esgFO
or esgts controls, respectively. The list was ranked by highest upregulation following
esgFO>Src42a.CA expression. Transgenes were induced for 7 days. Asterisks indicate direct
E2F1 targets. The cut-off value was 2.0 fold increase of mRNA abundance. The genes in this
list were independently determined to be expressed in wildtype intestinal progenitor cell
types: following tissue dissociation and FACS sorting of wildtype esg-GFP+ cells, or
wildtype dl-GFP+ cells or wildtype Gbe-Su(H)-GFP+ cells, a log2 normalized expression
(N.E) value >1.000 in either sorted esg+ progenitors, or sorted Dl+ ISCs or sorted Notch+ EBs
was applied as cutoff for a factor to be included in this analysis. N.E values represent log2
transformed RPKM values (See Suppl. Materials and Methods for details).
Suppl. Table 3 Normalized expression values of cell cycle regulators listed in Suppl.
Table 1
The N.E. values (log2 transformed RPKM values) of cell cycle regulators found to be
commonly induced following Src42a.CA or Ack expression or Notch inhibition (Suppl. table
2) are listed. Different samples were prepared independently on different Illumina platforms
(Hiseq2000 or GAIIX as indicated), and should be quantitatively compared only to their
respective controls (boxed) and not between each other.