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ARTICLE
Drosophila FGF cleavage is required for efficient
intracellular sorting and intercellular dispersal
Alex Sohr
1
,LijuanDu
1
, Ruofan Wang
1
,LiLin
2
,andSougataRoy
1
How morphogenetic signals are prepared for intercellular dispersal and signaling is fundamental to the understanding of
tissue morphogenesis. We discovered an intracellular mechanism that prepares Drosophila melanogaster FGF Branchless (Bnl)
for cytoneme-mediated intercellular dispersal during the development of the larval Air-Sac-Primordium (ASP). Wing-disc
cells expressBnl as a proprotein that is cleaved by Furin1 in the Golgi. Truncated Bnl sorts asymmetrically to the basal surface,
where it is received bycytonemes that extend from the recipient ASP cells. Uncleavable mutant Bnl has signaling activity but is
mistargeted tothe apical side, reducing its bioavailability. Since Bnl signaling levels feedback control cytoneme production in
the ASP, the reduced availability of mutant Bnl on the source basal surface decreases ASP cytoneme numbers, leading to a
reduced range of signal/signaling gradient and impaired ASP growth. Thus, enzymatic cleavage ensures polarized intracellular
sorting and availability of Bnl to its signaling site, thereby determining its tissue-specific intercellular dispersal and
signaling range.
Introduction
Intercellular communication mediated by signaling proteins is
essential for coordinating cellular functions during tissue mor-
phogenesis. Owing to decades of research, the core pathways of
developmental signaling and theirrolesandmodesofactionin
diverse morphogenetic contexts are well characterized. We now
know that a small set of conserved paracrine signals is universally
required for most developing tissues and organs. These signals are
produced in a restricted group of cells and disperse away from the
source to convey inductive information through their gradient
distribution (Christian, 2012;Akiyama and Gibson, 2015). It is
evident that to elicit a coordinated response, cells in a receptive
tissue field interpret at least three different parameters of the
gradient: the signal concentration, the timing, and the direction
from where they receive the signal (Briscoe and Small, 2015;
Kornberg, 2016). Therefore, understanding how different cellular
and molecular mechanisms in signal-producing cells prepare and
release the signals at the correct time and location and at an ap-
propriate level is fundamental to understanding tissue morpho-
genesis. It is also critical to know how these processes in source
cells spatiotemporally coordinate and integrate with cellular
mechanisms in the recipient cells to precisely shape signal gra-
dients and tissue patterns.
To address these questions, we focused on interorgan com-
munication ofa canonical FGF family protein, Bnl, that regulates
branching morphogenesis of tracheal airway epithelial tubes in
Drosophila melanogaster (Sutherland et al., 1996). Migration and
morphogenesis of each developing tracheal branch in embryo
and larvae is guided by a dynamically changing Bnl source
(Sutherland et al., 1996;Jarecki et al., 1999;Sato and Kornberg,
2002;Ochoa-Espinosa and Affolter, 2012;Du et al., 2017). For
instance, in third instar larva, Bnl produced by a restricted
group of columnar epithelial cells in the wing imaginal disc
activates its receptor Breathless (Btl) in tracheoblast cells in the
transverse connective (TC), a disc-associated tracheal branch
(Sato and Kornberg, 2002). Bnl signaling induces migration and
remodeling of the tracheoblasts to form a new tubular branch,
the Air-Sac-Primordium (ASP), an adult air-sac precursor and
vertebrate lung analogue (Fig. 1 A). Such dynamic and local
branch-specific signaling suggests a mechanism for precise
spatiotemporal regulation of Bnl release and dispersal in coor-
dination with the signaling response.
A critical role of regulated Bnl release can also be predicted
from the way cells exchange Bnl to sculpt recipient branch-
specificgradientshapes(Du et al., 2018a). Bnl is produced in
the wing disc cells, but it forms a long-range concentration
gradient only within the recipient ASP. Bnl gradient formation
depends on signaling through actin-based signaling filopodia
named cytonemes (Ram´
ırez-Weber and Kornberg, 1999;Sato
.............................................................................................................................................................................
1
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD;
2
Cardiovascular Research Institute, University of California, San Francisco,
San Francisco, CA.
Correspondence to Sougata Roy: sougata@umd.edu.
© 2019 Sohr et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the
publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0
International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).
Rockefeller University Press https://doi.org/10.1083/jcb.201810138 1653
J. Cell Biol. 2019 Vol. 218 No. 5 1653–1669
and Kornberg, 2002;Roy et al., 2011,2014;Du et al., 2018a). ASP
cells extend Btl-containing cytonemes to contact the basal sur-
face of the wing disc source and directly receive Bnl (Fig. 1 B).
Bnl reception through a graded number of cytonemes that are
formed along the distal-proximal (D-P) axis of the ASP epithe-
lium sculpts the Bnl gradient within the ASP. In the distal ASP
cells, high to medium levels of Bnl reception through cytonemes
induces an ETS transcription factor Pointed-P1 (PntP1), which
elicits positive feedback on Btl synthesis and cytoneme pro-
duction (Ohshiro et al., 2002;Du et al., 2018a). Gradually lower
levels of Bnl reception further away from the source induce
a homeobox transcription factor Cut, which suppresses Btl
synthesis and cytoneme production (Du et al., 2018a). Cut and
PntP1, expressed from the opposite poles of the ASP, reciprocally
antagonize each other’s expression. Consequently, zones of high
to low numbers of cytonemes are formed that can sculpt the Bnl
gradient in coordination with recipient ASP growth. Initiation of
this self-regulatory and tissue-specific gradient might require
limited signal release from the source, probably only at cyto-
neme contact sites.
The intracellular mechanisms in the source cells that prepare
Bnl for cytoneme-mediated exchange and branch-specific sig-
naling are uncharacterized. In this study, while analyzing vari-
ous functional forms of GFP-tagged Bnl, we uncovered a
Figure 1. Separate GFP fusion sites in Bnl result in different distribution patterns. (A) Drawing depicting the organization of the ASP andbnl-expressing
wing disc cells from third instar larva. DB, dorsal branch; TC, transverse connective. (B) Drawing of a sagittal view showing the tubular ASP epithelium, upper-
lower Z-axis, ASP cytonemes that contact the disc bnl-source (green nuclei), and the spatial domains of pntP1 and cut induced by high to low Bnl levels (green;
Du et al., 2018a). (C) Schematic map of the Bnl protein backbone showing its conserved FGF domain, signal peptide (SP), and four different GFP insertion sites.
(D–H9)Representative images of maximum-intensity projection of lower (wing disc source) and upper (ASP) Z-sections of third instar larval wing-discs ex-
pressing CD8-GFP, Bnl:GFP
1
,Bnl:GFP
2
,Bnl:GFP
3
,orBnl:GFP
4
under bnl-Gal4 as indicated. Red, αDlg staining marking cell outlines. (I–K) Representative ASP
images showing MAPK signaling (αdpERK, red) zones when Bnl:GFP
3endo
was expressed under native cis-regulatory elements (I), and when bnl-Gal4 over-
expressed Bnl:GFP
3
(J) or Bnl:GFP
1
(K). In D–K, white dashed line, ASP; white arrow, disc bnl-source; dashed arrow, Bnl:GFP puncta in the ASP; arrowhead, ASP
without Bnl:GFP
1
puncta. Scale bars: 30 µm.
Sohr et al. Journal of Cell Biology 1654
Cleavage prepares Drosophila FGF for signaling https://doi.org/10.1083/jcb.201810138
posttranslational endoproteolytic modification of Bnl. We show
that Bnl cleavage determines its polarized intracellular traf-
ficking to the basal surface of the source cells from whence ASP
cytonemes can receive the signal. This process limits Bnl avail-
ability to the ASP cytonemes and determines the range of Bnl
gradient dispersal and tissue morphogenesis. Given the con-
servation of fundamental developmental signaling mechanisms,
our demonstration of how a signal is endowed with information
for its target-specific intercellular distribution has fundamental
implications for understanding tissue morphogenesis.
Results
Bnl:GFP chimeras with different tag sites show different
dispersion patterns
To identify various functional forms of GFP-tagged Bnl proteins,
we generated four different Bnl:GFP variants and examined
their signaling activities (Fig. 1, A–C; Materials and methods; and
Tables S1 and S2). The Bnl protein is 770 amino acids long, with
an N-terminal 31-residue signal peptide and a conserved FGF
domain spanning from amino acids 243 to 379 (Fig. 1 C). Each of
the four variants contained a GFP tag at a single internal site: at
the 87th (Bnl:GFP
1
), 206th (Bnl:GFP
2
), 432nd (Bnl:GFP
3
), and
701st (Bnl:GFP
4
) amino acid residue. Transgenic Drosophila lines
harboring these constructs were crossed to bnl-Gal4 flies and
analyzed for activity in third instar larvae. In 3D confocal stacks
of wing discs, the lower Z sections revealed the Bnl-expressing
cells in the wing disc columnar epithelium, and the upper Z
sections (close to the objective) showed the associated ASP
(Fig. 1, B, D, and D9; and Video 1).
When the Bnl:GFP variants were expressed under bnl-Gal4
control, all ofthe variants were detectedin the disc Bnl source as
bright fluorescent puncta (Fig. 1, E–H). Overexpression of all
four Bnl:GFP variants led to ASP overgrowth (Fig. 1, E–H9),
which phenocopied a Bnl overexpression condition (Sato and
Kornberg, 2002). Thus, all of the Bnl:GFP variants could signal
nonautonomously. Unlike a membrane-tethered CD8:GFP pro-
tein, the fluorescent puncta comprising Bnl:GFP
2
,Bnl:GFP
3
,and
Bnl:GFP
4
were detected in the recipient ASP, suggesting that the
signals moved from the source to the ASP (Fig. 1, D–H9;and
Video 2). Surprisingly, although Bnl:GFP
1
puncta were visible in
the source cells and its overexpression induced ASP overgrowth,
the fluorescent puncta were absent from the recipient ASP
(Fig. 1, E–E9;Fig.S1,A–B9; and Video 3). Generally, as shownwith
an ASP derived from a genome-edited bnl:GFP
3endo
larva that
expressed the Bnl:GFP
3
at physiological levels (Du et al., 2018a),
only the distal ASP cells with high to moderate levels of Bnl
induce MAPK signaling (Fig. 1 I). In contrast, overexpression of
Bnl:GFP
3
or Bnl:GFP
1
in the source activated MAPK signaling in
all of the ASP cells (Fig. 1, J and K). Thus, Bnl:GFP
1
, like Bnl:GFP
3
,
is an active signal, but GFP fluorescence was undetectable in the
recipient ASP.
Bnl is cleaved before its transport to the recipient ASP
One possibility for Bnl:GFP
1
being functional yet undetectable in
the ASP could be that the protein was cleaved downstream of
tagging site 1 before the interorgan transport of its untagged
C-terminal fragment (Fig. 1 C). To test this possibility, we gen-
erated a double-tagged Bnl chimera with HA inserted at site
1 and GFP inserted at site 3 (Fig. 2 A). We performed Western
blot analyses on total protein lysates of cultured S2 cells that
were transfected with the bnl:GFP
1
,bnl:GFP
3
,orbnl:HA
1
GFP
3
constructs. An αGFP antibody recognized a common 150-kD
band, which likely represented the full-length protein (Fig. 2
B). Although the molecular weight of full-length Bnl:GFP was
predicted to be ∼113 kD, a larger band size could be due to
posttranslational modifications. Similar observations were re-
ported earlier for two Drosophila FGFs, Pyramus and Thisbe
(Tulin and Stathopoulos, 2010). Bnl:HA
1
GFP
3
and Bnl:GFP
3
had
similar band profiles, but Bnl:GFP
1
and Bnl:GFP
3
had multiple
variant-specificbands(Fig. 2 B). The detection of unique smaller
bands (∼37 and 60 kD) for N-terminally tagged Bnl:GFP
1
and
unique larger bands (>100 kD) for C-terminally tagged Bnl:GFP
3
or Bnl:HA
1
GFP
3
was consistent with a cleavage near tagging site
1. An αHA antibody recognized a weak ∼20-kD band (Fig. S2, A
and B) from lysates containing Bnl:HA
1
GFP
3
and Bnl:HA
1
(HA-
tag at position 1), but not from Bnl:HA
3
(HA-tag at site 3).
Therefore, the ∼20-kD band represented the N-terminal cleaved
product. These biochemical analyses suggested a cleavage in the
Bnl backbone, but it was difficult to estimate the actual molec-
ular size of the cleaved bands. Furthermore, the intracellular and
intercellular fates of the cleaved products cannot be directly
visualized in tissues using biochemical assays.
Therefore, we used a fluorescence microscopy–based assay to
simultaneously visualize both the HA- and GFP-tagged parts of
Bnl in cells. Immunostaining with αHA in S2 cells harboring
uncleaved Bnl:HA
1
GFP
3
molecules was expected to show both
HA
1
and GFP
3
localizing together. In contrast, a cleavage in the
molecules would separate the HA
1
tag from GFP
3
. Indeed, in
transfected S2 cells, Bnl:HA
1
GFP
3
was present in two distinct
spatially separated forms (Fig. 2, C–F). An internal perinuclear
zone showed colocalized GFP and HA signal, suggesting that the
zone contained uncleaved Bnl. In addition, there were a number
of exclusively GFP-positive puncta that localized more toward
the periphery of the S2 cells. Cells that were cultured and al-
lowed to adhere to a coverslip contained peripheral lamellipodial
and filopodial projections at the adherent surface. These pe-
ripheral lamellipodial/filopodial projections contained only a
truncated Bnl:GFP portion (Fig. 2, D–F; and Videos 4 and 5).
Spatial separation of the C-terminal GFP-tagged portion from
the rest of the Bnl:HA
1
GFP
3
molecule suggested Bnl cleavage.
To further test the peripheral distribution of the truncated
C-terminal fragment, we generated two different constructs:
bnl:HA
1
GFP
4
and bnl:GFP
1
HA
4
,wheretheHAandGFPtagswere
interchanged between sites 1 and 4 (Materials and methods).
Although the tag positions were changed in these constructs,
irrespective of the tags and tagging sites the cleaved N- and
C-terminal Bnl fragments showed consistent subcellular lo-
calization patterns (Fig. S2, C and D). These results showed
that Bnl is cleaved and a truncated C-terminal portion is
trafficked toward the cell periphery, probably for release. To
test interorgan dispersion of cleaved/uncleaved forms of Bnl,
we generated transgenic Drosophila lines harboring the bnl:
HA
1
GFP
3
construct. When bnl-Gal4 overexpressed Bnl:HA
1
GFP
3
Sohr et al. Journal of Cell Biology 1655
Cleavage prepares Drosophila FGF for signaling https://doi.org/10.1083/jcb.201810138
in the wing disc source, the N-terminal HA-tagged portion of
Bnl remained in the signal-producing cells, and a truncated
GFP-tagged C-terminal portion of Bnl (Bnl:GFP
3
)localized
only in the recipient ASP cells (Fig. 2, G–I;andFig.S2,E–G0).
These results strongly suggested that Bnl is cleaved in the
source and only a truncated Bnl derivative is received by
the ASP.
Bnl is cleaved at a single endoproteolytic site in the
Golgi network
Evolutionarily conserved serine proteases, namely the propro-
tein convertases (PCs) that include Furins, cleave many growth
factors and hormones that are synthesized in the form of pro-
ligands (Thomas, 2002). With an artificial neural network-based
in silico PC site prediction tool (Duckert et al., 2004), we iden-
tified three putative PC sites (PCS1–3) in the Bnl backbone.
Among them, PCS1 was Furin-specific with a core R–X–[R/K]–R
domain (Fig. 3, A–A9). Coincidentally, the four selected tagging
sites in the Bnl backbone were perfectly structured for testing
the putative cleavage sites (Fig. 3 A). To test for cleavage at PCS3
(Fig. 3 A), we generated a chimeric Bnl:GFP
3
HA
4
construct in
which the GFP and HA tags were inserted at sites 3 and 4, re-
spectively. Immunostaining with an αHA antibody on S2 cells
transfected with bnl:GFP
3
HA
4
showed colocalization of GFP
3
and
HA
4
(Fig. 3 B). Based on this cell biological assay, PCS3 is an
unlikely cleavage site. However, we did not investigate the
possibility of potential PCS3 cleaved products remaining closely
associated during their intracellular trafficking. In contrast, a
cleavage at either PCS1 or PCS2 could explain the observed
differential distribution of the N and C portions of Bnl:HA
1
GFP
3
(Fig. 2, C–H).
To test PCS1 and PCS2, we replaced their arginine (R) resi-
dues with glycine (G) and generated bnl:HA
1
GFP
3
-M1 (henceforth
referred as M1), a construct with mutations in PCS1 ((R/G)
161
TE
(R/G)
164
^SI(R/G)
166
), and bnl:HA
1
GFP
3
-M2 (henceforth referred
as M2) with mutations in PCS2 ((R/G)
233
NE(R/G)
236
^; Fig. 3 A).
R-to-G substitutions in PC sites were shown to successfully block
PC cleavage (Künnapuu et al., 2009). In transfected S2 cells,
Figure 2. Bnl is cleaved in producing cells
before its transport to the recipient ASP. (A)
Schematic map of a dual-tagged Bnl:HA
1
GFP
3
construct containing an HA-tag at site 1 and a
GFP-tag at site 3. (B) An αGFP Western blot
showing differential bands (*) obtained from S2
cell lysates containing Bnl:HA
1
GFP
3
,Bnl:GFP
1
,
and Bnl:GFP
3
. Mock, lysates from untransfected
cells. (C–F) Representative images of αHA-
immunostained (red) S2 cells expressing Bnl:
HA
1
GFP
3
; examples from adherent cells grown
on coverslip (D–F): XZY section (E and E9)and
XYZ section near coverslip (F); merged bright
field and fluorescent (C), merged fluorescent (C9,
E, and F), and split channels shown (C0,C90,and
E9). (G) A schematic drawing showing the ex-
pected localization pattern of the uncleaved Bnl:
HA
1
GFP
3
(yellow) and truncated Bnl:GFP
3
de-
rivative (green) in the αHA-stained (red) discs/
ASP. (H and I) A representative image of an
αHA-stained (red) wing disc and ASP (dashed
line) when bnl-Gal4 expressed Bnl:HA
1
GFP
3
(UAS-bnl:HA
1
GFP
3
). White, αDlg; split channels
(H9and H0); a graph (I) comparing the fractions
of GFP and HA (αHA) signal in the recipient ASP
relative to that of the wing disc source (n=14)
under this condition. In C–H0, arrowhead, un-
cleaved Bnl:HA
1
GFP
3
; arrow, truncated Bnl:GFP
3
derivative of Bnl:HA
1
GFP
3
. Scale bars: 10 µm
(C–F); 30 µm (H–H0).
Sohr et al. Journal of Cell Biology 1656
Cleavage prepares Drosophila FGF for signaling https://doi.org/10.1083/jcb.201810138
PCS1 mutation rendered the M1 molecules uncleavable, as HA
and GFP colocalized in the intracellular compartments (Fig. 3,
C–E; and Video 6). However, M2 molecules were cleaved like WT
proteins (Fig. 3 F). To compare the cleavage efficiency among the
Bnl mutants, we estimated the fraction (index of correlation
[I
corr
]) of colocalized pixels of HA and GFP channels from 3D
Figure 3. Bnl is cleaved at a single endoproteolytic site. (A) Location ofputative PCS1-3 in the Bnl backbone. #1–4: GFP insertion sites; *, point mutations
generated at PCS1 (M1) and PCS2 (M2). (A9)In silico predictions of PC sites. Upper panel, Furin-specific; lower panel, for general PC; green line, SP cleavage site;
red line, a set threshold above which the sequence is predicted to be a PC site. (B–G) Examples of αHA immunostained (red) S2 cells expressing Bnl:GFP
3
HA
4
(B) and Bnl:HA
1
GFP
3
mutants as indicated (C–G); XYZ section near coverslip (D) and XZY section (E) of M1-expressing adherent cells. (H) Graphs comparing
colocalization index (I
corr
) of the HA- and GFP-tagged parts of Bnl:HA
1
GFP
3
(WT), M1, M2, and M1M2 in αHA-stained (red) S2 cells. n= 15 (WT), 13 (M1), and 9
(M2 and M1M2); P values: ANOVA followed by Tukey HSD. In B–G, S2 cells were cotransfected with act-Gal4 and UAS-X, X= constructs as indicated.
(I–M) Maximum-intensity projections of the wing-disc source (I90,I00, K, and M) expressing Bnl:HA
1
GFP
3
mutants as indicated (bnl-Gal4 xUAS-X,X=M1,
M2, M1M2) and the recipient ASPs (I–I0,J–J0, and L–L0). Blue, αDlg; white dashed line, ASP. In B–M, arrow, truncated Bnl:GFP
3
derivative; arrowhead,
uncleaved Bnl:HA
1
GFP
3
. Scale bars: 10 µm (B–G); 30 µm (I–M).
Sohr et al. Journal of Cell Biology 1657
Cleavage prepares Drosophila FGF for signaling https://doi.org/10.1083/jcb.201810138
images (Jaskolski et al., 2005). The average I
corr
value was sig-
nificantly higher for M1 and M1M2 than either the control Bnl:
HA
1
GFP
3
or M2 cells, suggesting that the PCS1 mutation in-
hibited cleavage (Fig. 3 H). We also generated transgenic flies
harboring the M1, M2, or M1M2 constructs and analyzed their
distribution in the disc and ASP. When the M1 and M1M2 mu-
tants were expressed in the wing disc source, the recipient ASPs
received the colocalized HA-GFP puncta comprising the un-
cleaved full-length molecules (Fig. 3, I–I00 and L–M; and Video 7).
In contrast, only the GFP-tagged C-terminal part of M2 was
distributed within the ASP (Fig. 3, J and K). Collectively, these
results suggest that Bnl:HA
1
GFP
3
molecules are cleaved at PCS1
before their delivery from the disc source to the ASP.
Bnl cleavage could be intracellular or, alternatively, could
occur on the surface of the source cell plasma membrane where
the signal is delivered to the recipient ASP cytonemes (Fig. 1 B).
To test this possibility, we employed a detergent-free αGFP-
based immunostaining protocol (henceforth referred to as
αGFP
ex
), which was previously used to detect surface-exposed
Bnl:GFP (Du et al., 2018a). The αGFP
ex
assay detected only Bnl:
GFP
3
on the expressing source cell surface, but not Bnl:GFP
1
(Fig. 4, A and B). Thus, Bnl cleavage is intracellular, and only the
truncated C-terminal Bnl portion is displayed on the basal sur-
face of the source cells. To determine the subcellular location of
Bnl cleavage, we performed standard immunostaining with
αGM130 antibody, a cis-Golgi probe, on discs expressing Bnl:
GFP
1
,Bnl:HA
1
GFP
3
, or Bnl:HA
1
GFP
3
-M1. In the wing disc source,
100% of either Bnl:GFP
1
or uncleaved Bnl:HA
1
GFP
3
puncta were
localized in the GM130-marked cis-Golgi (Fig. 4, C–D9), whereas
the truncated Bnl:GFP
3
derivative (GFP-only puncta) localized in
many small uncharacterized intracellular vesicles, some of
which were enriched with Syntaxin16, a target-SNAP receptor
for intra/trans-Golgi sorting (Charng et al., 2014;Fig. 4, D, D9,F,
and F9). On the other hand, uncleaved M1 puncta were seen in all
of the vesicular compartments, indicating their routing through
the secretory pathway (Fig. 4, E, E9,G,andG9). Similar intra-
cellular distribution profiles of the cleaved and uncleaved por-
tions of Bnl were observed in cultured S2 cells (Fig. 4, H–J9).
Collectively, these results showed that Bnl is cleaved during its
trafficking through the Golgi network.
Bnl is cleaved by Furin1 in the wing disc bnl source
Intracellular Bnl cleavage at PCS1, which is a Furin-specificsite,
indicated that Bnl is likely cleaved by a Furin. To identify the
specific protease, we performed RNAi-mediated knockdown of
two Drosophila furin genes, Dfurin1 (fur1)andDfurin2 (fur2), in cell
culture assay. We did not investigate the role of amontillado
(amon), a mammalian PC2 orthologue, since it is expressed only
in neurons and neuroendocrine cells (Roebroek et al., 1992,1993;
Künnapuu et al., 2009). In S2 cells, RNAi treatment of fur1,fur2,
or both significantly reduced Bnl:HA
1
GFP
3
cleavage in compar-
ison to a nonspecific control RNAi (Fig. 5, A–E). Thus, Bnl
cleavage is Fur1and Fur2 dependent. However, invivo, only fur1
knockdown in the wing disc bnl source resulted in a stunted ASP
development, which phenocopied the bnl knockdown condition
(Fig. 5, F–I;Fig.S3,A–D; and Table S3). Measurement of the
allometric ratio of the recipient ASP length along its major D-P
axis to the width of the wing disc confirmed that the growth
abnormality was ASP specific and was not due to a systemic
developmental delay (Fig. 5, J and J9). Lack of a fur2 knockdown
phenotype in the ASP is likely due to the absence of fur2 ex-
pression in the bnl source, as expression analyses of fur1 and fur2
showed only fur1 expression in the bnl source (Fig. S3, E–K).
Thus, although both Fur1 and Fur2 could cleave Bnl in S2 cells,
their substrate specificity might depend on their tissue-specific
expression.
The RNAi analyses provided correlative evidence of Furin’s
role in Bnl cleavage. For direct evidence, we ex vivo cultured
larval wing discs expressing Bnl:HA
1
GFP
3
in the bnl source in
either the presence or absence of Furin inhibitors. In spite of the
prolonged (up to 16 h) ex vivo culture conditions, Bnl:HA
1
GFP
3
was cleaved in the absence of inhibitors, and the truncated Bnl:
GFP
3
moved to the growing ASPs (Fig. 6, A–C0). In the presence
of inhibitors (Fig. 6, D–F9), Bnl cleavage in the disc source was
blocked, and the amount of uncleaved puncta received by the
ASP gradually increased with the increase in incubation time
(Fig. 6 G). The time-dependent inhibition of Bnl cleavage by
Furin inhibitors confirmed Furin-dependent Bnl cleavage. Im-
portantly, these results, together with the M1 mutant analyses
(Fig. 2 I), showed that when Bnl cleavage is blocked the un-
cleaved signals can still move from the disc to the ASP. These
results indicated that cleavage might not be essential for mo-
lecular activation of the Bnl protein and led us to examine the
physiological roles of Bnl cleavage.
Uncleaved Bnl can signal and is dispersed by cytonemes, but
only within a narrow range
To examine M1 distribution and activity at its physiological
levels of expression, we modified a previously reported bnl:
GFP
3endo
allele into bnl:HA
1
GFP
3endo
(henceforth referred as wt
endo
)
and corresponding bnl:HA
1
GFP
3
-M1
endo
mutant alleles (hence-
forth referred as m1
endo
) by using genome editing (Materials and
methods; Fig. 7 A). Consistent with earlier observations for bnl:
GFP
3endo
(Du et al., 2018a), wt
endo
flies were homozygous viable
and had normal tissue morphology (Table S4).Although bnl is an
essential gene, m1
endo
mutant flies were homozygous viable, in-
dicating that the PCS1 mutation was nonlethal. As expected, the
endogenous Bnl:HA
1
GFP
3endo
(WT
endo
) molecules were cleaved
and ASPs received only the truncated Bnl:GFP
3
portion (hence-
forth referred as t-WT
endo
;Figs. 7 B and S4 A). The m1
endo
ASPs
also received uncleaved Bnl:HA
1
GFP
3-
M1
endo
(henceforth re-
ferred as M1
endo
) puncta containing both HA and GFP (Figs. 7 C
and S4 B). Furthermore, ex vivo cultured wt
endo
wing discs
grown in the presence of Furin inhibitors had uncleaved Bnl:
HA
1
GFP
3endo
puncta in the ASP (Fig. 7, D and E). Thus, in the
absence of cleavage, uncleaved Bnl could move to the ASP and
sustain tracheal growth.
When we genetically combined either wt
endo
or m1
endo
with a
btl:cherry
endo
allele, which expressed endo-tagged Btl:Cherry (Du
et al., 2018a), both t-WT
endo
and M1
endo
puncta colocalized with
the receptors in the ASPs (Fig. 7, F–G9). As reported earlier (Du
et al., 2018a), the distal ASP tip, which is closest to the disc bnl
source, had a high concentration of the receptor-colocalized
t-WT
endo
or M1
endo
puncta. With increasing distance from the
Sohr et al. Journal of Cell Biology 1658
Cleavage prepares Drosophila FGF for signaling https://doi.org/10.1083/jcb.201810138
source, their concentration gradually decreased. Bnl is known to
be transported by cytonemes to form a receptor-associated
gradient (Du et al., 2018a). To examine cytoneme-mediated
transport, we live imaged CD8:Cherry-marked ASPs in the ho-
mozygous wt
endo
or m1
endo
larvae (>30 discs/genotype). In both
conditions, ASPs extended long (>15-µm) polarized cytonemes
toward the source cells and received GFP-tagged fluorescent
puncta comprising either t-WT
endo
or M1
endo
(Fig. 7, H–I9;and
Fig. S4, C and D). Surface αGFP
ex
immunostaining showed that
both M1
endo
and t-WT
endo
colocalized with Btl:Cherry
endo
on the
recipient cytoneme surfaces before their endocytosis (Fig. 7, J
and J9; and Fig. S4, E–F9). Therefore, the pattern of tissue-specific
dispersion of M1
endo
was comparable to that of t-WT
endo
.
However, thorough scrutiny revealed that the m1
endo
allele
produced hypermorphic phenotypes due to a reduced signaling
range. The distal tip area of m1
endo
ASPs had significantly fewer
long (>15-µm) signal-receiving cytonemes than the wt
endo
ASPs
(Fig. 7 K). All of the cells (∼6–7 cells in Z-projected images)
within a 60-µm periphery surrounding the tip of wt
endo
ASPs
extended long signaling cytonemes. In contrast, only one to two
distal tip cells in the comparable region of the m1
endo
ASPs
extended M1
endo
-receiving cytonemes. A restricted zone of
M1
endo
-receiving cytonemes is reflected in the narrow gradient
range and attenuated m1
endo
ASP growth (Fig. 8, A–E). While
t-WT
endo
formed a long-range gradient along the ∼10–12-
cell–long ASP D-P axis, M1
endo
formed a narrow, steeper gradi-
ent along the ∼5–6-cell–long D-P axis (Fig. 8, D and E). Ac-
cordingly, the m1
endo
ASPs had a reduced zone of nuclear dpERK
in comparison to the wt
endo
ASPs (Fig. 8, G–I). Thus, M1
endo
had a
narrow distribution and signaling range compared with
t-WT
endo
(Fig. 8, G–I; and Fig. S4, G and H). Nevertheless, nor-
malization of either the signal concentration or the signaling
zone with recipient ASP length showed comparable scaling of
the t-WT
endo
and M1
endo
gradients and signaling zones in rela-
tion to the recipient ASP size (Fig. 8, F and I). Previously, our
work suggested that the Bnl gradient adopts recipient ASP-
specific shapes due to two counteracting Bnl signaling feed-
backs on cytonemes (Du et al., 2018a). Thus, scaling of the M1
endo
gradient to the recipient-specific shape indicated normal M1
endo
signaling, but within a limited range.
Ectopic expression in the salivary gland, a nontracheated
organ that does not normally express bnl (Jarecki et al., 1999),
Figure 4. Bnl is cleaved in the Golgi network
of Bnl-producing cells. (A and B) Projection
images of lower Z-stacks of the disc bnl source
showing detergent-free αGFP immunostaining
(αGFP
ex
;red)whenbnl-Gal4 expressed Bnl:GFP
1
(A) and Bnl:GFP
3
(B). White, phalloidin-Alexa
Fluor 647 to mark actin-rich cell outlines; ar-
rowhead, intracellular Bnl:GFP (only green);
arrow, surface-localized Bnl:GFP (green+red);
dashed line, ASP in the upper Z-stacks (not
shown). (C and C9)αGM130-stained (red) optical
sections of wing disc bnl-source expressing Bnl:
GFP
1
.(D–J9)Single optical sections of αHA-
immunostained (red) disc bnl-source (D–G9)and
S2 cells (H–J9)expressingeitherBnl:HA
1
GFP
3
or
M1 and marked with α-Stx-16 or αGM130 (blue)
as indicated. Arrow, truncated Bnl:GFP
3
deriva-
tive; arrowhead, uncleaved Bnl:HA
1
GFP
3
or M1
mutant; merged (D–J) and split (D9–J9)blue
channels shown. Scale bars: 20 µm (A–C); 5 µm
(C9–G9); 10 µm (H–J9).
Sohr et al. Journal of Cell Biology 1659
Cleavage prepares Drosophila FGF for signaling https://doi.org/10.1083/jcb.201810138
also showed a limited spatial distribution and signaling of M1.
Since Bnl expression is known to induce tracheal invasion to-
ward source cells, active Bnl expression in the salivary gland was
expected to induce easily scorable tracheal invasion. We took
advantage of a nonspecificexpressionofbnl-Gal4 (Du et al., 2017)
in the salivary gland to express the Bnl mutants. Except for a
CD8:GFP control, equivalent levels of expression of Bnl:HA
1
GFP
3
(WT), M1, M2, or M1M2 all induced tracheal invasion into the
salivary gland, confirming their nonautonomous signaling ir-
respective of cleavage (Fig. 8, J–N; and Fig. S5 A). Thus, M1 is an
active signal. However, the salivary glands expressing WT and
M2 had a significantly higher number of terminal branches
ramifying throughout the gland surface. In contrast, glands ex-
pressing M1 or M1M2 showed poor terminal branching fre-
quencies and surface coverage (Fig. 8, K–O). Thus, M1 induced a
spatially restricted response on the source cell surface. Since Bnl
distribution pattern on a producing cell surface determines the
spatial coverage of terminal branching on it (Peterson and
Krasnow, 2015), attenuated terminal branching on the M1-
expressing salivary glands suggested a reduced availability of
M1 on the exposed basal cell surface of the salivary gland.
Bnl cleavage ensures its trafficking to the basal cell surface
To examine this possibility, we performed the surface αGFP
ex
assay on salivary glands expressing the M1 or WT constructs. As
expected, a significantly lower fraction of total M1 molecules
were externalized on the basal surface of the salivary gland cells
in comparison to WT (Fig. 9, A–D). Strikingly, while the WT
protein covered the entire basal surface of the giant-sized sali-
vary gland cells, most of the externalized M1 molecules were
restricted to the cell junctions (Fig. 9, B and B9). Such abnor-
mality in spatial distribution might suggest mispolarized M1
Figure 5. Knockdown of furin expression affects Bnl cleavage. (A–D9)Images of αHA-immunostained (red) S2 cells cotransfected with act-Gal4,UAS-bnl:
HA
1
GFP
3
, and the synthesized RNAi as indicated. Control-i,nonspecificdsRNA;XYZ(A–D) and XZY (A9–D9) views; arrow, truncated Bnl:GFP
3
derivative; ar-
rowhead, uncleaved Bnl:HA
1
GFP
3
.(E) Graph comparing Bnl:HA
1
GFP
3
cleavage under various furin knockdown conditions in S2 cells. I
corr
, index of HA and GFP
colocalization, with lower values indicating cleavageand color separation; n=13(control),11(fur1-i), 12 (fur2-i), and 14 (fur1-i fur2-i); P values (ANOVA followed
by Tukey HSD): fur1-i versus fur1-i fur2-i,P=0.347;allothergroups,P<0.001.(F–I) αDlg-immunostained (white) wing disc and ASP (white dashed line) from
larvae where bnl-Gal4 expressed furin RNAi as indicated. Control, bnl-Gal4 xw
−
.(J and J9)Drawing depicting the scheme (J) of allometric measurement of ASP
length (L) relative to the corresponding wing disc (WD); graph (J9) comparing the length (L) ratio of ASP to wing-disc (WD) under conditions indicated. n=48
(control), 95 (fur1-i), 86 (fur2-i), 102 (fur1-i,fur2-i); P values (ANOVA followedby Tukey HSD): all groups versus fur1-i, P < 0.001; all groups versus fur1-i fur2-i,P<
0.001. Scale bars: 10 µm (A–D); 30 µm (F–I).
Sohr et al. Journal of Cell Biology 1660
Cleavage prepares Drosophila FGF for signaling https://doi.org/10.1083/jcb.201810138
trafficking, reducing its availability at the basal surface. Indeed,
confocal sections through the salivary glands showed that most
M1 signals were selectively enriched at the apical luminal sides
of the cells that were inaccessible to the external trachea (Fig. 9,
E–H). Notably, although salivary gland cells do not express Bnl,
they contain the Bnl cleavage machinery. Bnl:HA
1
GFP
3
(WT)
driven by bnl-Gal4 was cleaved leading to clear spatial separation
of the HA- and GFP-tagged fragments (Fig. S5, B and B9).
Therefore, these results suggested that Bnl cleavage promotes
efficient polarized trafficking to the basal signaling surface from
whence tracheal cells can receive the signal.
To confirm polarized Bnl sorting in the wing disc source, we
acquired XZY sections of the disc-ASP tissue complex along the
ASP D-P axis (Fig. 9, I–M). In the CD8:Cherry-marked disc bnl
source, overexpressed M1 molecules preferentially populated
the apical luminal and lateral sides of the columnar epithelial
cells. In contrast, the truncated WT molecules had relatively
higher density toward the basal side of the source cells (Fig. 9,
J–L). In αHA-immunostained discs that expressed the Bnl:
HA
1
GFP
3
construct under bnl-Gal4, the truncated Bnl:GFP
3
signal
was clearly polarized toward the basal surface of the columnar
epithelial cells facing the overlying ASP (Fig. 9 M). A surface
αGFP
ex
assay confirmed a higher percentage of basal external-
ization of Bnl:GFP
3
compared with M1 (Fig. S5 C). Similarly,
when examining the genome-edited wt
endo
and m1
endo
larvae, we
found that the basal surface of the disc source and recipient ASP
Figure 6. Furin-dependent Bnl cleavage in the wing
disc. (A–F9)The αHA-stained (red) wing disc that ex-
pressed Bnl:HA
1
GFP
3
under bnl-Gal4 and were ex vivo
cultured for 0 (pretreat) to 16 h in the absence and 1–5h
in the presence of Furin inhibitors as indicated. Arrow,
truncated Bnl:GFP
3
derivative; arrowhead, uncleaved
Bnl:HA
1
GFP
3
; blue, phalloidin-Alexa Fluor 647 marking
cell outlines; merged (A–D) and either split green, red
(A9–C0)oronlyred(D9–F9) channels are shown. (G)
Graphs comparing average levels of colocalized HA and
GFP in the ASP grown in presence and absence of Furin
inhibitors; samples were harvested at different time
points from the continuous culture. n= 11 (0 h), 11 (1 h),
10 (2.5 h), 9 (5 h control), 12 (5 h test), 5 (16 h); P values
(ANOVA followed by Tukey HSD): P = 0.0001 for 5 h
versus either 0 h, 1 h, or 2.5 h of Furin inhibition. Scale
bars: 30 µm.
Sohr et al. Journal of Cell Biology 1661
Cleavage prepares Drosophila FGF for signaling https://doi.org/10.1083/jcb.201810138
had significantly higher t-WT
endo
density in comparison to
M1
endo
(Fig. 9, N–P). Thus, Bnl cleavage in the source cells directs
efficient polarized sorting of the signal to the basal signaling
surface, thereby affecting intercellular signaling range and tis-
sue morphogenesis.
Discussion
This study showed that the FGF family protein Bnl is synthesized
as a proprotein and then endoproteolytically cleaved at a single
site by Furin1 in the Golgi network. The cleavage ensures effi-
cient polarized intracellular sorting of a truncated C-terminal
fragment containing the FGF domain to the signaling site,
where the signal is received by the ASP cytonemes for inter-
cellular dispersal and signaling.
Limited proteolysis is one of the versatile posttranslational
mechanisms that activates most, if not all, developmental signals
(LeMosy, 2006). Signals including Hedgehog (Hh); Dispatched;
EGF; Trunk; the TGF-β/BMP family proteins Decapentaplegic
(Dpp), Screw, and Glass bottom boat (Gbb); two Drosophila FGFs,
Pyr and Ths; and human FGF7 were all shown to be cleaved (Lee
et al., 1994;Schweitzer et al., 1995;Porter et al., 1996;Künnapuu
et al., 2009,2014;Wharton and Serpe, 2013;Constam, 2014;
Johnson et al., 2015;Anderson and Wharton, 2017;Stewart et al.,
2018). Although most signal cleavage is considered to activate
the signal and affect the range of signaling response (Künnapuu
et al., 2009,2014;Wharton and Serpe, 2013), full-length un-
cleaved signals were also found to activate receptors and were
shown to be secreted when expressed in cultured cells
(Künnapuu et al., 2009;Sopory et al., 2010;Tokhunts et al.,
2010;Tulin and Stathopoulos, 2010;Constam, 2014). There-
fore, why are signals synthesized as proproteins and subse-
quently cleaved for their activity or dispersion?
We showed that Bnl cleavage acts as a catalytic switch that
ensures its efficient polarized sorting to the basal signaling
surface from where it can be taken up by the recipient cyto-
nemes (Fig. 10). The uncleavable mutant Bnl can activate re-
ceptors but is presented on the basal surface at low levels (Fig. 9,
A–M). The reduced basal presentation of uncleavable Bnl is due
to its mistargeting to a far apical domain of the source cells,
Figure 7. Comparison of activities of endog-
enously expressed cleaved and uncleaved
Bnl. (A) Schematic map of the genomic bnl:
gfp
endo
locus and the products of its subsequent
CRISPR/Cas9-based editing; orange box, coding
exon; gray box, noncoding exon; line, introns; red
star, M1 mutation. (B and C) Representative
images of αHA-stained (red) ASP and wing disc
from homozygous wt
endo
(n= 85) and m1
endo
(n=
79) larvae. (D and E) Representative images of
αHA-immunostained (red) ASP and wing disc
from wt
endo
larvae after 5 h of ex vivo culture in
the absence (control; n=18)andpresence(n=
29) of Furin inhibitors. In B–E, white dashed line,
ASP; blue, phalloidin-Alexa Fluor 647; arrow,
t-WT
endo
; arrowhead, uncleaved WT
endo
or
M1
endo
.(F–G9)Receptor-colocalized t-WT
endo
and M1
endo
puncta (arrow) in trans-heterozygous
btl:Cherry
endo
/wt
endo
(F and F9)andbtl:Cherry
endo
/
m1
endo
(G and G9) ASP; split red channels (F9and
G9). (H–I9)Live images of CD8:Cherry-marked
ASPs showing the long (>15 µm) oriented ASP
cytonemes (arrows) containing t-WT
endo
(H and
H9)andM1
endo
(I and I9)puncta(arrowheads).(J
and J9)Surface αGFP
ex
immunostaining (white)
detecting M1
endo
on the ASP cytoneme surfaces
of btl:cherry
endo
/m1
endo
larvae; arrow and arrow-
head, receptor-colocalized intracellular (bright
green) and surface M1
endo
,respectively.(K)
Graph comparing the number of cytonemes
(>15 µm long) counted from a 60-µm perimeter
centering the ASP tip (Materials and methods) in
wt
endo
(n= 28) and m1
endo
(n= 38). Scale bars:
20 µm (B–G9,J,andJ9); 10 µm (H–I9).
Sohr et al. Journal of Cell Biology 1662
Cleavage prepares Drosophila FGF for signaling https://doi.org/10.1083/jcb.201810138
which the trachea cannot access. Therefore, we predict that a
pro-Bnl cleavage activates a delivery barcode for efficient target-
specific intercellular dispersal. Conceptually, the cleavage en-
sures a signaling polarity that is relayed from within the source
cells to the recipient ASP through cytonemes. Such signal bar-
coding for determining intercellular destination might be con-
served for all signals. Consistent with this view, a similar
cleavage-dependent polarized sorting mechanism was reported
for Hh in Drosophila retinal photoreceptor neurons (Huang and
Kunes, 1996;Chu et al., 2006;Daniele et al., 2017). A complex
choreography of apical and basal localization followed by the
basal cytoneme-dependent dispersion of Hh was also described
in Drosophila wing imaginal disc cells (Kornberg, 2011;Guerrero
and Kornberg, 2014).
Interestingly, the efficiency of intracellular and intercellular
Bnl trafficking depends on the enzymatic activity of Fur1
(Fig. 5 G and Fig. S3, A–D). Although Bnl expression is spatially
restricted in tissues, the molecular machinery that cleaves Bnl
Figure 8. Bnl cleavage determines the range of gradient distribution and signaling. (A–C) Images of αDlg immunostained (white) ASPs (white outline)
and wing discs from homozygous wt
endo
(n= 52) and m1
endo
(n= 64) larvae (A and B); a graphical comparison (C) of their ASP length relative to the wing disc
size. (D and E) Average intensity profiles of t-WT
endo
(D, n=3)andM1
endo
(E, n= 5) along the ASP D-P axis; lower panels, examples of signal distribution along
the ASP D-P axis. Red line, exponential fit trend line; C
max
, maximum average intensity; C
1/2
,1/2C
max
; slope for the trend line between C
max
and C
1/2
.(F)
Average intensity profiles of t-WT
endo
(n= 9) and M1
endo
(n= 12) normalized with recipient ASP length (D-P axes; Materials and methods). (G–I) Images of
αdpERK-stained (red) ASPs from homozygous wt
endo
(n= 16) and m1
endo
(n= 20) larvae (G and H) and graphicalcomparison(I) of their nuclear dpERK-positive
zones along the D-P axes; lower chart: average ratio (± SD) of number of dpERK-positive cells along the D-P axis to the total number of cells in the D-P axis.
(J–N) Larval salivary glands expressing CD8:GFP, Bnl:HA
1
GFP
3
(18), M1 (11), M2 (20), and M1M2 (18) under bnl-Gal4 as indicated. Red arrow, central branch
point. (O) A quantitative assessment of the frequency of terminal branching on salivary gland determined by Sholl analysis under the conditions indicated.
Scale bars: 30 µm (A, B, G, and H); 100 µm (J–N).
Sohr et al. Journal of Cell Biology 1663
Cleavage prepares Drosophila FGF for signaling https://doi.org/10.1083/jcb.201810138
exists even in salivary glands that do not normally express Bnl.
This might reflect the broad range of Fur1 expression, as re-
ported in several studies (Roebroek et al., 1992,1993;Künnapuu
et al., 2009;Nichols and Weinmaster, 2010;Johnson et al., 2015).
Alternatively, different types of cells might express different
furin/PC genes that can act redundantly. Furins are known to be
regulated enzymes that autoactivate in a Ca
2+
-dependent man-
ner during their intracellular trafficking (Thomas, 2002). How
and when the Furin activation pathway might intersect with the
pro-Bnl sorting itinerary is unknown. We also do not know why
a truncated Bnl is targeted only to the basal cell surface. Re-
cently, the trans-Golgi cargo receptor AP-1γ, a component of the
Clathrin AP-1 complex, was shown to be necessary for Bnl
trafficking to the basolateral membranes of bnl-expressing flight
muscle cells (Peterson and Krasnow, 2015). It is possible that Bnl
cleavage unmasks the cargo-receptor binding site. The current
Figure 9. Cleavage ensures polarized Bnl sorting to the basal cell surface for signaling. (A–C) High-magnification (40×) images of the exposed basal
surfaces (arrowhead) of salivary glands expressing WT or M1 under bnl-Gal4 from an area schematically shown in C. Red, surface αGFP
ex
immunostaining;
arrow, cell junction. (D) Graph comparing fractions (red surface stain/total GFP) of overexpressed WT (n=12)andM1(n= 10) that got externalized on the
salivary gland surface. (E–H) Images of sagittal sections of salivary glands expressing WT and M1 under bnl-Gal4.Arrow,apicallumen.(I) Drawings depicting
the ASP D-P axis (dashed line; upper panel) and an XZY section along the D-P axis (lowerpanel) showing the tubular ASP and disc epithelia as shown in J–M. (J
and K) Sagittal sections of αDlg immunostained (blue, sub-apical marker) wing disc and ASP when the disc bnl source coexpressed CD8:Cherry with either the
WT or M1 construct under bnl-Gal4. Arrow, basal side; arrowhead, apical side. (L) Graph comparing apical and basal percentage of WT and M1 relative to the
total amount in the disc source. n= 24 (WT) and 32 (M1). (M) Maximum projections of mid- and para-sagittal sections within ∼3 µm of mid-Y of an αDlg (blue)
and αHA (red) stained wing-disc/ASP, where bnl-Gal4 expressed Bnl:HA
1
GFP
3
. Arrow, truncated Bnl:GFP
3
; white dashed line, ASP and wing disc; arrowhead,
apical lumen of wing disc. (N–P) Comparison (graph in P) of levels of t-WT
endo
(n= 17) and M1
endo
(n= 33) on the surface of the disc source and ASP (dashed
line). Red and arrowhead, detergent-free αGFP-staining; arrow, intracellular puncta; white staining, phalloidin-Alexa Fluor 647. Scale bars: 50 µm (E–H); 20 µm
(all other panels).
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Cleavage prepares Drosophila FGF for signaling https://doi.org/10.1083/jcb.201810138
knowledge of intracellular Bnl/FGF targeting is rudimentary and
needs to be elucidated in the future.
Our findings revealed that although Bnl cleavage is intra-
cellular, it plays an important role in determining the range of
cytoneme-mediated intercellular Bnl dispersal. Insights on how
this intracellular event might influence the range of cytoneme-
dependent dispersal came from our earlier study (Du et al.,
2018a). As illustrated in Fig. 10, high to low levels of Bnl sig-
naling activate two counteracting feedback loops operating from
the opposite poles of the ASP, which help to establish the zones
of corresponding high to low number of Bnl-receiving cyto-
nemes along the ASP epithelium. The consequence is a systemic
self-regulatory process, where the number of Bnl-receiving cy-
tonemes produced by ASP cells is determined by the amount of
Bnl received by the cells through cytonemes, giving rise to the
recipient ASP-specific Bnl gradient shapes. Therefore, the in-
tracellular cleavage and polarized sorting pathway that modu-
late Bnl availability on the basal surface of source cells can
determine the spatial range of cytoneme formation, signal dis-
persion, and signaling. These results suggest an intricate coor-
dination of the intracellular events in the source and recipient
cells with the intercellular cytoneme-mediated dispersal, which
together can precisely shape signal gradients and tissue
patterns.
Materials and methods
Drosophila strains and genetic crosses
All crosses were incubated at 25°C. The following strains were
used in this study: UAS-bnlRNAi (34572), fur1-LacZ (10341), UAS-
fur1RNAi (25837), UAS-fur1RNAi (42481), UAS-fur1RNAi (41914),
UAS-fur2RNAi (51743), UAS-fur2RNAi (42577), UAS-fur1 (63077)
(from Bloomington Stock Center); UAS
attB
-Bnl:GFP
1
,UAS
attB
-Bnl:
GFP
2
,UAS
attB
-Bnl:GFP
3
,UAS
attB
-Bnl:GFP
4
,UAS
attB
-Bnl:HA
1
,UA-
S
attB
-Bnl:HA
2
,UAS
attB
-Bnl:HA
3
,UAS
attB
-Bnl:HA
4
(gifts from
Kornberg lab); fur2-Gal4 (NP 4074) (from Kyoto DGGR); UAS-
CD8:GFP,UAS-CD8:Cherry,btl-Gal4,bnl-Gal4 (Roy et al., 2014);
bnl:gfp
endo
,btl:cherry
endo
(Du et al., 2018a); and UAS-Bnl:GFP
1
,
UAS-Bnl:HA
1
,UAS-Bnl:GFP
2
,UAS-Bnl:GFP
3
,UAS-Bnl:HA
3
,UAS-Bnl:
GFP
4
,UAS-Bnl:HA
1
GFP
3
,UAS-Bnl:HA
1
GFP
3
-M1,UAS-Bnl:HA
1
GFP
3
-
M2,UAS-Bnl:HA
1
GFP
3
-M1M2,bnl:HA
1
GFP
3endo
,bnl:HA
1
GFP
3
-M1
endo
(this study).
Generation of transgenic Drosophila lines
UAS-bnl:GFP and UAS-bnl:HA variants
Each of the four Bnl:GFP variants contained an HA-tag upstream
to a GFP tag at a single internal site. Bnl:GFP
1
contained both HA
and GFP tags in tandem inserted between amino acids
RSSLVPSAVS
87
and E
88
RSVNQPT. Bnl:GFP
2
contained the tags
inserted between amino acids SNLDRNERST
206
and
V
207
PQSHLAWTS. Bnl:GFP
3
contained the tags inserted between
amino acids KAPPHCSSNT
432
and S
433
GSSSSISSS. Bnl:GFP
4
contained the tags between amino acids MSSGEEQDQDN
701
and
D
702
QDQEQSDPGE. Previously, transgenic Drosophila lines har-
boring the Bnl:GFP
3
construct at various attP loci in the second
and fourth chromosomes did not show any detectable Bnl:GFP
3
expression when driven by bnl-Gal4. Therefore, we subcloned
the Bnl:GFP constructs into the pUAST vector from the orig-
inal pUAST-attB constructs and resorted to the random
P-element–based transgenesis to avoid any positional effects
on Bnl:GFP expression. A summary of characterization of
different transgenic lines is presented in Table S2.
UAS-bnl:HA
1
GFP
3
UAS-bnl:HA
1
GFP
3
contained an HA-tag at site 1 (between 87 and
88 amino acid residues of the original protein) and a superfolder
Figure 10. Proposed model for the role of Bnl
cleavage in determining signaling range. Pro-
Bnl is cleaved by Furin1 in the Golgi into a
truncated-Bnl, which, through an unknown
process, is asymmetrically sorted to the basal
surface of the source cells. In the absence of
cleavage, mutant Bnl-M1 molecules trafficran-
domly and are mostly sequestered at a distant
apical domain, reducing their basal availability.
The ASP, which is present on the basal side of
the source, extends cytonemes to directly re-
ceive Bnl from the basal surface of the source
cells. High Bnl levels/signaling in the ASP induce
PntP1, which induces Bnl-receiving cytoneme
formation. Lower Bnl uptake in cells further from
the source induces Cut, which suppresses Bnl-
receiving cytoneme formation. Cut and PntP1
feedback inhibit each other’s expression, thereby
generating a Bnl gradient that adopts recipient
ASP-specificshapes(Du et al., 2018a). Conse-
quently, reduced Bnl-M1 availability results in
only a few ASP cells extending Bnl-receiving
cytonemes, leading to a restricted range of sig-
nal distribution and stunted ASP growth.
Sohr et al. Journal of Cell Biology 1665
Cleavage prepares Drosophila FGF for signaling https://doi.org/10.1083/jcb.201810138
GFP (P´
edelacq et al., 2006) at site 3 (between 432 and 433 res-
idues of the original protein). The construct was generated by
overlap extension PCR of three fragments using primers (Table
S1): the N-terminal HA-tagged part, the C-terminal Bnl coding
region (amplified from the pUAST-attB-Bnl:HA
1
), and the middle
sfGFP region from a sfGFP-containing construct (Addgene). The
final 3,060-bp PCR product was cloned into the pCR-Blunt II-
TOPO vector. The fully sequence-verified insert was subcloned
into the pUAST vector at the BglII and XbaI sites. UAS-bnl:HA
1
GFP
3
was used for analysis in S2 cells and for P-element–mediated
germline transformation and transgenesis.
UAS-bnl:HA
1
GFP
3
mutants
The M1 and M2 variants of Bnl:HA
1
GFP
3
contained the following
cleavage site mutations: M1, (R/G)TE(R/G)SI(R/G); M2, (R/G)NE(R/
G). These mutant constructs were created using overlap extension
PCR with the Bnl:HA
1
GFP
3
construct as a template. The primers
used are shown in Table S1. The final assembled PCR product was
cloned into the pCR-Blunt II-TOPO vector. The sequence-verified
constructs were subcloned into the BglII and XbaI sites of the
pUAST vector for either analysis in S2 cell culture or for
P-element–mediated germline transformation and transgenesis.
UAS-bnl:HA
1
GFP
4
and UAS-bnl:GFP
1
HA
4
UAS-bnl:HA
1
GFP
4
was cloned using overlap extension PCR to
insert a GFP tag at site 4 of Bnl:HA
1
.Similarly,UAS-bnl:GFP
1
HA
4
was cloned using overlap extension PCR to insert a GFP at site
1ofBnl:HA
4
. The primers used are listed in Table S1. These
constructs were verified and used in S2 cell culture analyses.
P-element based transgenesis was performed as described ear-
lier (Du et al., 2017). Various transgenic lines generated are
described in Table S2.
CRISPR/Cas9-based genome editing
The bnl:HA
1
GFP
3endo
and bnl:HA
1
GFP
3
-M1
endo
mutant alleles were
generated by in-frame insertion of an HA-tag into the first
coding exon of a previously characterized bnl:sfGFP
3endo
allele (Du
et al., 2018a) using CRISPR/Cas9-based genome editing following
previously described protocols (Du et al., 2017,2018b). The bnl:
HA
1
GFP
3
-M1 mutant allele includes the HA
1
tagaswellasmuta-
tions of three arginines (R) to glycines (G) at PCS1 that starts 82
amino acids upstream of the conserved FGF domain. For targeting
Cas9-based double-stranded break near tag site 1, a guide RNA
(BnlHA1gRNA, 59-CTACGTTCACTCACTGCGCTCGG-39; underlined
bases represent the PAM site) with zero off targets in the fly
genome was cloned by ligating two annealed complimentary
oligonucleotides into the pCFD3 vector (Table S1).
The replacement donors, pDonor-bnl:HA
1
GFP
3
and pDonor-bnl:
HA
1
GFP
3
-M1, were designed and generated following Du et al.
(2017). These constructs contained either HA
1
or the HA
1
-M1
mutations flanked by ∼1-kb long 59and 39arms that are ho-
mologous to the genomic sequence flanking tag site 1. Both 59
and 39homology arms were PCR-amplified from genomic DNA
from the nos-Cas9;;bnl:GFP
3endo
parent fly, sequence verified,
and assembled together into the pUC19 vector using Gibson
Assembly (primers in Table S1). To prevent retargeting of the
gRNA/Cas9 to the edited genome, a synonymous mutation was
introduced into the replacement cassette near the PAM se-
quence via the primers used for amplification (Table S1). The
constructs were fully sequenced before germline injection.
The gRNA-expressing vector and the respective replacement
donor vector were coinjectedinto the germline cells of nos-Cas9;;
bnl:sfGFP
3endo
embryos. For each genome-editing experiment, a
stepwise crossing strategy (Du et al., 2018b) was followed to
obtain G0-F2 progenies and establish individual fly lines for
screening. The desired “ends-out”homologous directed repair
(HDR) was screened for by a three-step PCR-based strategy (see
primers in Table S1), followed by sequencing and analyses of
tissue-specific expression patterns of the tagged genes under a
confocal microscope. The efficiency of genome editing–based
generation of the two different genotypes and their phenotypes
were summarized in Table S4. During generation of bnl:HA
1
GFP
3
-
M1
endo
several lines were obtained that had only the HA
1
inser-
tion without the M1 mutation. We predicted that the HDR had
taken place somewhere between the HA
1
tag site and M1 mu-
tation sites (219 bases apart). These lines were fully sequence
verified and found to have normal tissue expression. Therefore,
these lines were considered as bnl:HA
1
GFP
3endo
lines (see Table
S4). For subsequent analyses, we used a wt
endo
and an m1
endo
line
derived from the same genome-editing experiment. The wt
endo
F4–14 line and m1
endo
F4–9 line used in this study were fully
sequence verified and established after outcrossing as previ-
ously described (Du et al., 2018a).
Synthesis of double-stranded RNA (dsRNA) for gene
knockdown in S2 cells
dsRNA was synthesized by PCR from genomic DNA isolated
from S2 cells following a previously described protocol
(Künnapuu et al., 2009). The following PCR primers were used
to synthesize the T7 transcription template carrying the T7
promoter sequence at their 59ends: fur1:forward,59-TAATAC
GACTCACTATAGGGACGCAAAGATCCTCTGTGGCA-39; reverse,
59-TAATACGACTCACTATAGGGACATTGCTCCCGGAACTGC-39;
fur2:forward,59-TAATACGACTCACTATAGGGACGCTAGAGG
CCAATCCGGAA-39; reverse, 59-TAATACGACTCACTATAGGGA
CCCTTCTCGCCCCAAAAGTG-39. dsRNA against fur1 or fur2 were
synthesized using the MEGAscript RNAi Kit (Thermo Fisher
Scientific).
FISH
To probe for fur1 and fur2 mRNA, the desired probe regions of
540 and 552 bp were PCR amplified with primers (Table S1)
from respective cDNAs and cloned using Gibson assembly into
pSPT18 vector. The vector was linearized, and the RNA probe
was prepared using the DIG-RNA Labeling Kit (Roche) according
to the manufacturer’s protocol. RNA in situ hybridization on
third-instar larval tissues was performed as previously de-
scribed (Du et al., 2017). Hybridized probes were detected using
α-Dig antibody followed by immunofluorescence with Alexa
Fluor 647–conjugated secondary antibody.
Cell culture assay
S2 cells were cultured in 25-cm
2
flasks using Shields and Sang
M3 insect media (Sigma-Aldrich). For transfection, when cells
Sohr et al. Journal of Cell Biology 1666
Cleavage prepares Drosophila FGF for signaling https://doi.org/10.1083/jcb.201810138
were ∼90% confluent, the medium was removed and 6 ml of
fresh M3 medium was added to the flask. Cells were gently re-
suspended by pipetting and added to a 12-well plate with 1 ml of
cells per well. After 2 h, once the cells had adhered to the bottom
of the well, theM3 media was replaced with 1 ml serum-free M3
medium, and the cells were transfected with 1 µg of each DNA
using Lipofectamine 2000 following the manufacturer’s proto-
col. After 16 h, the serum-free medium was replaced with 1 ml
M3 medium containing serum. For experiments with furin RNAi,
5 µg of dsRNA was used for transfection. Under all conditions,
transient expression was examined 2–3daftertransfection.
Ex vivo organ culture and Furin inhibitor assay
Ex vivo culturing of wing discs was performed in WM1 medium as
described in Du et al. (2017). The discs were removed from a single
pool of culture after 0, 5, and 16 h of incubation at 25°C, followed
by fixation and αHA immunostaining of the tissues. For the Furin
inhibition assay, late third instar larval tissues were ex vivo cul-
tured in 2 ml of WM1 medium in the presence or absence of a
cocktail of Furin inhibitor I and II (50 µM final concentration
each; Calbiochem; 344930 and 344931) following recommended
concentrations in Johnson et al. (2015). The live tissues were in-
cubated for 1, 2.5, or 5 h. Following incubation, the carcasses were
transferred to a centrifuge tube, rinsed three times with 1× PBS,
and fixed in 4% PFA before αHA immunostaining.
Protein analyses
S2 cells were harvested 3 d posttransfection, and the cell pellets
were washed several times in 1× PBS. The pellet was re-
suspended in 70 µl RIPA cell lysis buffer (Sigma-Aldrich) in the
presence of a cocktail of protease inhibitors (Roche) and kept for
15 min at 4°C. An equal volume of lysed cells was combined with
2× Sample Buffer, heated at 95°C for 5 min, and loaded onto a
10% SDS-PAGE minigel. The gel was run at 50 V for 10 min for
stacking and then at 200 V until the desired amount of sepa-
ration occurred. Proteins were transferred from the gel to a
PVDF membrane using Transblot Turbo (Bio-Rad). A standard
protocol was followed to perform Western blot analyses using
primary antibodies: αGFP (1:1,000) or αHA (1:1,000) and HRP-
conjugated secondary antibody. The HRP activity was detected
with ECL substrate (GE) and imaged (Fuji LAS3000).
Immunostaining
Standard and detergent-free immunostaining protocols were as
previously described (Du et al., 2017). The following antibodies
were used in this study: α-Discs large (1:100; DSHB); α-HA
(1:1,000); α-dpERK (1:100; Cell Signaling); α-GFP (1:3,000 ex-
tracellular; Abcam); and α-PH3 (1:2,000; Cell Signaling). Alexa
Fluor–conjugated secondary antibodies (1:1,000; Molecular
Probes) were used for immunofluorescence detection. Phalloidin-
conjugated Alexa Fluor 647 was often used for marking cell
outlines.
Microscopic imaging
For live imaging, wing imaginal discs and their associated tra-
chea were prepared following Roy et al. (2014).Imageswere
obtained as previously described (Du et al., 2018a)usingaLeica
SP5X with HyD detector or an CSUX1 Yokogawa spinning disc
confocal equipped with an Andor iXon897 EMCCD camera. The
images were processed and analyzed with Fiji. Maximum-
intensity projections of sections were shown for most images.
All images were obtained using 40× objective in the micro-
scopes, except for Fig. 8 (J–N), which used a 20× objective. All
XZY images were obtained using the Leica SP5X with a 40×
objective for S2 cells and 20× objective for wing discs.
Analysis of ASP size
ASP length was measured from the TC along their longest
(major) D-P axisto the ASP tip. The disc size was determined by
measuring from the TC, along the ASP major axis to the edge of
the disc. A ratio of the ASP:disc size was used to compare dif-
ferent genotypes and conditions (Figs. 5 J9and 8C).
Sholl analysis for terminal branching
Salivary glands were gently dissected out from fixed larval tis-
sues overexpressing different cleaved and uncleaved variants of
Bnl and imaged under transmitted light to visualize tracheal
invasion. In WT overexpressing tissue, the terminal tracheal
branches ramified radially from a preexisting central branch
point. Due to its morphological resemblance with neuronal
dendritic arbors, we employed Sholl analysis (Binley et al., 2014)
using Fiji to measure the frequency of terminal branching. The
analysis created 20 concentric circles in increments of 5-µm
radius from the point of origin up to 100 µm and counted the
number of times any tracheal branch crossed these circles.
These values were averaged across several samples and com-
pared between the different Bnl variants expressed in the
salivary gland.
Quantitative analyses of fluorescence intensities
For Bnl levels, all fluorescent intensity measurements were
background corrected. 3D image stacks representing only either
the wing disc sections or the ASP were transformed into 2D by
maximum-intensity projections. The density of fluorescence
intensity was measured from a selected region of interest (ROI)
of the 2D images, outlining the either Bnl source cells or the
recipient ASP (Figs. 2 I,6G, and S5 C). For the recipient ASP, the
ROI encompassed the distal tip of the ASP (a region with ∼3–4-
cell diameter that received the maximum Bnl from the source).
Likewise, the density of the surface-localized Bnl:GFP variants,
probed by αGFP immunostaining (Fig. 9 P), was measured from
selected ROIs on the maximum-intensity projections of the rel-
evant optical sections encompassing either the ASP or wing disc
source. For Fig. 9 D, the ROIs represented each salivary gland cell
including the cell junctions, and the density of the redand green
channel intensities were measured from the maximum-
intensity projections of optical sections within the 5-µm
Z-stack from the most basal surface. The ratio of surface GFP
(red) to total GFP (green) was expected to be less than one.
However, some average ratios were slightly greater than one,
probably due to the immunofluorescent signal amplification of
the surface-exposed proteins obtained through αGFP im-
munostaining. Second, as reported earlier (Du et al., 2018a), the
surface exposed GFP was rapidly quenched, reducing its levels of
Sohr et al. Journal of Cell Biology 1667
Cleavage prepares Drosophila FGF for signaling https://doi.org/10.1083/jcb.201810138
detection on the cell surface. For Fig. 9 (J–L), ROIs representing
the basal or apical part of the source cells were selected from
maximum-intensity projections of the XZY sections. GFP in-
tensities measured from the ROIs were normalized to the total
intensity from the total source cell area.
For colocalization analyses in S2 cells (Figs. 3 H and 5E),
maximum-intensity projections of approximately four to five
stacks around the center of the cell were produced. An I
corr
value
was obtained using the Colocalization Colormap plugin of Fiji to
determine the degree of colocalization of two selected channels
(HA immunostain and GFP).
Gradients of intensities of Bnl:GFP variants in the ASP (Fig. 8,
D–F) were obtained along the ASP D-P axes as reported earlier
(Du et al., 2018a). For Fig. 8 F, gradients were measured from
homozygous wt
endo
(n=9)orm1
endo
(n= 12) ASPs. Each position
(x) within an ASP was normalized by length of the ASP (L)to
obtain x/L, the x-axis of the plot. Similarly, GFP intensity was
normalized by dividing each intensity value in a single sample
by the highest intensity value from that sample for the y-axis.
Normalized intensity values from each sample were taken at
0.05 x/Lincrements from 0 to 1 (i.e., 21 data points from each
sample). The normalized intensity values from each group (WT
or M1) were averaged together and plotted along the x/Laxis.
Cytoneme analysis
ASP cytoneme number was quantitated microscopically as previ-
ously described (Roy et al., 2011,2014). In brief, cytonemes >15 µm
in length that extended from a 60-µm total perimeter region
(30 µm from the tip of the ASP in both directions) were counted.
Statistical analyses
Statistical significance was determined with two-tailed ttests or
a one-way ANOVA followed by Tukey’shonestlysignificant
different (HSD) tests. All P values in the legends were obtained
using a ttest, unless otherwise stated.
Online supplemental material
Fig. S1 shows additional images comparing Bnl:GFP
1
and Bnl:
GFP
3
expression and dispersion. Fig. S2 shows additional bio-
chemical and cell-biological evidence of Bnl cleavage. Fig. S3
shows ASP phenotypes due to bnl and fur knockdown in the disc
bnl source and expression analyses of fur genes. Fig. S4 shows
additional examples of localization and activity of WT
endo
and
M1
endo
in the ASP. Fig. S5 shows cleavage of Bnl:HA
1
GFP
3
in the
salivary gland, a quantitative analysis performed to identify
lines of Bnl:HA
1
:GFP
3
variants that expressed at equivalent lev-
els, and a graph comparing the percentage of WT and M1 pro-
teins on the surface of the wing disc cells that overexpressedthe
proteins. Video 1 shows the organization of the ASP and wing
disc bnl-source. Video 2 shows the Bnl:GFP
3
distribution in a 3D
ASP section. Video 3 shows the lack of Bnl:GFP
1
in ASP 3D im-
ages. Videos 4 and 5 show the spatial distribution of cleaved Bnl
portions in XYZ and XZY S2 cell sections. Video 6 shows the
spatial distribution of the uncleaved M1 mutant Bnl in a S2 cell.
Video 7 shows the spatial distribution of the uncleaved M1
mutant Bnl in the wing disc-ASP. Table S1 lists primers used in
this study. Table S2 lists transgenic Drosophila lines created for
this study and their analyses. Table S3 lists phenotypic analyses
of fur expression knockdown by various RNAi lines. Table
S4 shows the efficiency of generation of bnl:HA
1
sfGFP
3
and
bnl:HA
1
sfGFP
3
-M1 mutant Drosophila lines using CRISPR/Cas9.
Acknowledgments
We thank Dr. T.B. Kornberg for the Bnl:GFP constructs and all
his mentorship and support; the Bloomington Stock Center for
Drosophila lines; Developmental Studies Hybridoma Bank for
antibodies; Drs. N.W. Andrews and T.B. Kornberg for comments
on the manuscript; and A.E. Beaven for assistance in the CBMG
Imaging Core.
This study was funded by the National Institutes of Health:
grants K99/R00HL114867 and R35GM124878 to S. Roy. A. Sohr
received a one-year fellowship from a Cell and Molecular Biol-
ogy Training Grant (T32-GM080201).
The authors declare no competing financial interests.
Author contributions: L. Lin generated the Bnl:GFP1-4 con-
structs; A. Sohr, L. Du, R. Wang, and S. Roy conducted the ex-
periments; S. Roy designed the project and supervised the work;
S. Roy, A. Sohr, R. Wang, and L. Du wrote the paper.
Submitted: 25 October 2018
Revised: 30 January 2019
Accepted: 5 February 2019
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