ArticlePDF Available

Abstract and Figures

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 express Bnl as a proprotein that is cleaved by Furin1 in the Golgi. Truncated Bnl sorts asymmetrically to the basal surface, where it is received by cytonemes that extend from the recipient ASP cells. Uncleavable mutant Bnl has signaling activity but is mistargeted to the 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.
This content is subject to copyright.
Drosophila FGF cleavage is required for efcient
intracellular sorting and intercellular dispersal
Alex Sohr
, Ruofan Wang
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-specic intercellular dispersal and
signaling range.
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 eld 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-specic 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-
specicgradientshapes(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 lopodia
named cytonemes (Ram´
ırez-Weber and Kornberg, 1999;Sato
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD;
Cardiovascular Research Institute, University of California, San Francisco,
San Francisco, CA.
Correspondence to Sougata Roy:
© 2019 Sohr et al. This article is distributed under the terms of an AttributionNoncommercialShare AlikeNo Mirror Sites license for the rst six months after the
publication date (see After six months it is available under a Creative Commons License (AttributionNoncommercialShare Alike 4.0
International license, as described at
Rockefeller University Press 1653
J. Cell Biol. 2019 Vol. 218 No. 5 16531669
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 others 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-specic 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-specic 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.
(DH9)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
under bnl-Gal4 as indicated. Red, αDlg staining marking cell outlines. (IK) Representative ASP
images showing MAPK signaling (αdpERK, red) zones when Bnl:GFP
was expressed under native cis-regulatory elements (I), and when bnl-Gal4 over-
expressed Bnl:GFP
(J) or Bnl:GFP
(K). In DK, white dashed line, ASP; white arrow, disc bnl-source; dashed arrow, Bnl:GFP puncta in the ASP; arrowhead, ASP
without Bnl:GFP
puncta. Scale bars: 30 µm.
Sohr et al. Journal of Cell Biology 1654
Cleavage prepares Drosophila FGF for signaling
posttranslational endoproteolytic modication of Bnl. We show
that Bnl cleavage determines its polarized intracellular traf-
cking 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-specic intercellular distribution has fundamental
implications for understanding tissue morphogenesis.
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, AC; 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
), 206th (Bnl:GFP
), 432nd (Bnl:GFP
), and
701st (Bnl:GFP
) amino acid residue. Transgenic Drosophila lines
harboring these constructs were crossed to bnl-Gal4 ies 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 uorescent puncta (Fig. 1, EH). Overexpression of all
four Bnl:GFP variants led to ASP overgrowth (Fig. 1, EH9),
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 uorescent puncta comprising Bnl:GFP
were detected in the recipient ASP, suggesting that the
signals moved from the source to the ASP (Fig. 1, DH9;and
Video 2). Surprisingly, although Bnl:GFP
puncta were visible in
the source cells and its overexpression induced ASP overgrowth,
the uorescent puncta were absent from the recipient ASP
(Fig. 1, EE9;Fig.S1,AB9; and Video 3). Generally, as shownwith
an ASP derived from a genome-edited bnl:GFP
larva that
expressed the Bnl:GFP
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
or Bnl:GFP
in the source activated MAPK signaling in
all of the ASP cells (Fig. 1, J and K). Thus, Bnl:GFP
, like Bnl:GFP
is an active signal, but GFP uorescence was undetectable in the
recipient ASP.
Bnl is cleaved before its transport to the recipient ASP
One possibility for Bnl:GFP
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
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 modications. Similar observations were re-
ported earlier for two Drosophila FGFs, Pyramus and Thisbe
(Tulin and Stathopoulos, 2010). Bnl:HA
and Bnl:GFP
similar band proles, but Bnl:GFP
and Bnl:GFP
had multiple
variant-specicbands(Fig. 2 B). The detection of unique smaller
bands (37 and 60 kD) for N-terminally tagged Bnl:GFP
unique larger bands (>100 kD) for C-terminally tagged Bnl:GFP
or Bnl:HA
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
and Bnl:HA
tag at position 1), but not from Bnl:HA
(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 difcult 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 uorescence microscopybased 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
molecules was expected to show both
and GFP
localizing together. In contrast, a cleavage in the
molecules would separate the HA
tag from GFP
. Indeed, in
transfected S2 cells, Bnl:HA
was present in two distinct
spatially separated forms (Fig. 2, CF). 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 lopodial projections at the adherent surface. These pe-
ripheral lamellipodial/lopodial projections contained only a
truncated Bnl:GFP portion (Fig. 2, DF; and Videos 4 and 5).
Spatial separation of the C-terminal GFP-tagged portion from
the rest of the Bnl:HA
molecule suggested Bnl cleavage.
To further test the peripheral distribution of the truncated
C-terminal fragment, we generated two different constructs:
and bnl:GFP
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
trafcked 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:
construct. When bnl-Gal4 overexpressed Bnl:HA
Sohr et al. Journal of Cell Biology 1655
Cleavage prepares Drosophila FGF for signaling
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
only in the recipient ASP cells (Fig. 2, GI;andFig.S2,EG0).
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 articial neural network-based
in silico PC site prediction tool (Duckert et al., 2004), we iden-
tied three putative PC sites (PCS13) in the Bnl backbone.
Among them, PCS1 was Furin-specic with a core RX[R/K]R
domain (Fig. 3, AA9). 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
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
showed colocalization of GFP
(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 trafcking. In contrast, a
cleavage at either PCS1 or PCS2 could explain the observed
differential distribution of the N and C portions of Bnl:HA
(Fig. 2, CH).
To test PCS1 and PCS2, we replaced their arginine (R) resi-
dues with glycine (G) and generated bnl:HA
-M1 (henceforth
referred as M1), a construct with mutations in PCS1 ((R/G)
), and bnl:HA
-M2 (henceforth referred
as M2) with mutations in PCS2 ((R/G)
^; 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
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
and Bnl:GFP
. Mock, lysates from untransfected
cells. (CF) Representative images of αHA-
immunostained (red) S2 cells expressing Bnl:
; examples from adherent cells grown
on coverslip (DF): XZY section (E and E9)and
XYZ section near coverslip (F); merged bright
eld and uorescent (C), merged uorescent (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:
(yellow) and truncated Bnl:GFP
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
). 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 CH0, arrowhead, un-
cleaved Bnl:HA
; arrow, truncated Bnl:GFP
derivative of Bnl:HA
. Scale bars: 10 µm
(CF); 30 µm (HH0).
Sohr et al. Journal of Cell Biology 1656
Cleavage prepares Drosophila FGF for signaling
PCS1 mutation rendered the M1 molecules uncleavable, as HA
and GFP colocalized in the intracellular compartments (Fig. 3,
CE; and Video 6). However, M2 molecules were cleaved like WT
proteins (Fig. 3 F). To compare the cleavage efciency among the
Bnl mutants, we estimated the fraction (index of correlation
]) 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. #14: GFP insertion sites; *, point mutations
generated at PCS1 (M1) and PCS2 (M2). (A9)In silico predictions of PC sites. Upper panel, Furin-specic; 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. (BG) Examples of αHA immunostained (red) S2 cells expressing Bnl:GFP
(B) and Bnl:HA
mutants as indicated (CG); XYZ section near coverslip (D) and XZY section (E) of M1-expressing adherent cells. (H) Graphs comparing
colocalization index (I
) of the HA- and GFP-tagged parts of Bnl:HA
(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 BG, S2 cells were cotransfected with act-Gal4 and UAS-X, X= constructs as indicated.
(IM) Maximum-intensity projections of the wing-disc source (I90,I00, K, and M) expressing Bnl:HA
mutants as indicated (bnl-Gal4 xUAS-X,X=M1,
M2, M1M2) and the recipient ASPs (II0,JJ0, and LL0). Blue, αDlg; white dashed line, ASP. In BM, arrow, truncated Bnl:GFP
derivative; arrowhead,
uncleaved Bnl:HA
. Scale bars: 10 µm (BG); 30 µm (IM).
Sohr et al. Journal of Cell Biology 1657
Cleavage prepares Drosophila FGF for signaling
images (Jaskolski et al., 2005). The average I
value was sig-
nicantly higher for M1 and M1M2 than either the control Bnl:
or M2 cells, suggesting that the PCS1 mutation in-
hibited cleavage (Fig. 3 H). We also generated transgenic ies
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, II00 and LM; 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
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
), which was previously used to detect surface-exposed
Bnl:GFP (Du et al., 2018a). The αGFP
assay detected only Bnl:
on the expressing source cell surface, but not Bnl:GFP
(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:
, or Bnl:HA
-M1. In the wing disc source,
100% of either Bnl:GFP
or uncleaved Bnl:HA
puncta were
localized in the GM130-marked cis-Golgi (Fig. 4, CD9), whereas
the truncated Bnl:GFP
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 proles of the cleaved and uncleaved por-
tions of Bnl were observed in cultured S2 cells (Fig. 4, HJ9).
Collectively, these results showed that Bnl is cleaved during its
trafcking through the Golgi network.
Bnl is cleaved by Furin1 in the wing disc bnl source
Intracellular Bnl cleavage at PCS1, which is a Furin-specicsite,
indicated that Bnl is likely cleaved by a Furin. To identify the
specic 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 signicantly reduced Bnl:HA
cleavage in compar-
ison to a nonspecic control RNAi (Fig. 5, AE). 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, FI;Fig.S3,AD; 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 conrmed that the growth
abnormality was ASP specic 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, EK).
Thus, although both Fur1 and Fur2 could cleave Bnl in S2 cells,
their substrate specicity might depend on their tissue-specic
The RNAi analyses provided correlative evidence of Furins
role in Bnl cleavage. For direct evidence, we ex vivo cultured
larval wing discs expressing Bnl:HA
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
was cleaved in the absence of inhibitors, and the truncated Bnl:
moved to the growing ASPs (Fig. 6, AC0). In the presence
of inhibitors (Fig. 6, DF9), 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 conrmed 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 modied a previously reported bnl:
allele into bnl:HA
(henceforth referred as wt
and corresponding bnl:HA
mutant alleles (hence-
forth referred as m1
) by using genome editing (Materials and
methods; Fig. 7 A). Consistent with earlier observations for bnl:
(Du et al., 2018a), wt
ies were homozygous viable
and had normal tissue morphology (Table S4).Although bnl is an
essential gene, m1
mutant ies were homozygous viable, in-
dicating that the PCS1 mutation was nonlethal. As expected, the
endogenous Bnl:HA
) molecules were cleaved
and ASPs received only the truncated Bnl:GFP
portion (hence-
forth referred as t-WT
;Figs. 7 B and S4 A). The m1
also received uncleaved Bnl:HA
(henceforth re-
ferred as M1
) puncta containing both HA and GFP (Figs. 7 C
and S4 B). Furthermore, ex vivo cultured wt
wing discs
grown in the presence of Furin inhibitors had uncleaved Bnl:
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
or m1
with a
allele, which expressed endo-tagged Btl:Cherry (Du
et al., 2018a), both t-WT
and M1
puncta colocalized with
the receptors in the ASPs (Fig. 7, FG9). 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
or M1
puncta. With increasing distance from the
Sohr et al. Journal of Cell Biology 1658
Cleavage prepares Drosophila FGF for signaling
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
or m1
larvae (>30 discs/genotype). In both
conditions, ASPs extended long (>15-µm) polarized cytonemes
toward the source cells and received GFP-tagged uorescent
puncta comprising either t-WT
or M1
(Fig. 7, HI9;and
Fig. S4, C and D). Surface αGFP
immunostaining showed that
both M1
and t-WT
colocalized with Btl:Cherry
on the
recipient cytoneme surfaces before their endocytosis (Fig. 7, J
and J9; and Fig. S4, EF9). Therefore, the pattern of tissue-specic
dispersion of M1
was comparable to that of t-WT
However, thorough scrutiny revealed that the m1
produced hypermorphic phenotypes due to a reduced signaling
range. The distal tip area of m1
ASPs had signicantly fewer
long (>15-µm) signal-receiving cytonemes than the wt
(Fig. 7 K). All of the cells (67 cells in Z-projected images)
within a 60-µm periphery surrounding the tip of wt
extended long signaling cytonemes. In contrast, only one to two
distal tip cells in the comparable region of the m1
extended M1
-receiving cytonemes. A restricted zone of
-receiving cytonemes is reected in the narrow gradient
range and attenuated m1
ASP growth (Fig. 8, AE). While
formed a long-range gradient along the 1012-
celllong ASP D-P axis, M1
formed a narrow, steeper gradi-
ent along the 56-celllong D-P axis (Fig. 8, D and E). Ac-
cordingly, the m1
ASPs had a reduced zone of nuclear dpERK
in comparison to the wt
ASPs (Fig. 8, GI). Thus, M1
had a
narrow distribution and signaling range compared with
(Fig. 8, GI; 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
and M1
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-
specic shapes due to two counteracting Bnl signaling feed-
backs on cytonemes (Du et al., 2018a). Thus, scaling of the M1
gradient to the recipient-specic shape indicated normal M1
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
;red)whenbnl-Gal4 expressed Bnl:GFP
(A) and Bnl:GFP
(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:
.(DJ9)Single optical sections of αHA-
immunostained (red) disc bnl-source (DG9)and
S2 cells (HJ9)expressingeitherBnl:HA
M1 and marked with α-Stx-16 or αGM130 (blue)
as indicated. Arrow, truncated Bnl:GFP
tive; arrowhead, uncleaved Bnl:HA
or M1
mutant; merged (DJ) and split (D9J9)blue
channels shown. Scale bars: 20 µm (AC); 5 µm
(C9G9); 10 µm (HJ9).
Sohr et al. Journal of Cell Biology 1659
Cleavage prepares Drosophila FGF for signaling
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 nonspecicexpressionofbnl-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
(WT), M1, M2, or M1M2 all induced tracheal invasion into the
salivary gland, conrming their nonautonomous signaling ir-
respective of cleavage (Fig. 8, JN; and Fig. S5 A). Thus, M1 is an
active signal. However, the salivary glands expressing WT and
M2 had a signicantly 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, KO). 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 trafcking to the basal cell surface
To examine this possibility, we performed the surface αGFP
assay on salivary glands expressing the M1 or WT constructs. As
expected, a signicantly lower fraction of total M1 molecules
were externalized on the basal surface of the salivary gland cells
in comparison to WT (Fig. 9, AD). 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. (AD9)Images of αHA-immunostained (red) S2 cells cotransfected with act-Gal4,UAS-bnl:
, and the synthesized RNAi as indicated. Control-i,nonspecicdsRNA;XYZ(AD) and XZY (A9D9) views; arrow, truncated Bnl:GFP
derivative; ar-
rowhead, uncleaved Bnl:HA
.(E) Graph comparing Bnl:HA
cleavage under various furin knockdown conditions in S2 cells. I
, 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.(FI) 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 (AD); 30 µm (FI).
Sohr et al. Journal of Cell Biology 1660
Cleavage prepares Drosophila FGF for signaling
trafcking, 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,
EH). Notably, although salivary gland cells do not express Bnl,
they contain the Bnl cleavage machinery. Bnl:HA
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
efcient polarized trafcking to the basal signaling surface from
whence tracheal cells can receive the signal.
To conrm 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, IM). 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,
JL). In αHA-immunostained discs that expressed the Bnl:
construct under bnl-Gal4, the truncated Bnl:GFP
was clearly polarized toward the basal surface of the columnar
epithelial cells facing the overlying ASP (Fig. 9 M). A surface
assay conrmed a higher percentage of basal external-
ization of Bnl:GFP
compared with M1 (Fig. S5 C). Similarly,
when examining the genome-edited wt
and m1
larvae, we
found that the basal surface of the disc source and recipient ASP
Figure 6. Furin-dependent Bnl cleavage in the wing
disc. (AF9)The αHA-stained (red) wing disc that ex-
pressed Bnl:HA
under bnl-Gal4 and were ex vivo
cultured for 0 (pretreat) to 16 h in the absence and 15h
in the presence of Furin inhibitors as indicated. Arrow,
truncated Bnl:GFP
derivative; arrowhead, uncleaved
; blue, phalloidin-Alexa Fluor 647 marking
cell outlines; merged (AD) and either split green, red
(A9C0)oronlyred(D9F9) 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
had signicantly higher t-WT
density in comparison to
(Fig. 9, NP). Thus, Bnl cleavage in the source cells directs
efcient polarized sorting of the signal to the basal signaling
surface, thereby affecting intercellular signaling range and tis-
sue morphogenesis.
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 ef-
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 efcient 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,
AM). 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:
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
(n= 85) and m1
79) larvae. (D and E) Representative images of
αHA-immunostained (red) ASP and wing disc
from wt
larvae after 5 h of ex vivo culture in
the absence (control; n=18)andpresence(n=
29) of Furin inhibitors. In BE, white dashed line,
ASP; blue, phalloidin-Alexa Fluor 647; arrow,
; arrowhead, uncleaved WT
.(FG9)Receptor-colocalized t-WT
and M1
puncta (arrow) in trans-heterozygous
(F and F9)andbtl:Cherry
(G and G9) ASP; split red channels (F9and
G9). (HI9)Live images of CD8:Cherry-marked
ASPs showing the long (>15 µm) oriented ASP
cytonemes (arrows) containing t-WT
(H and
(I and I9)puncta(arrowheads).(J
and J9)Surface αGFP
immunostaining (white)
detecting M1
on the ASP cytoneme surfaces
of btl:cherry
larvae; arrow and arrow-
head, receptor-colocalized intracellular (bright
green) and surface M1
Graph comparing the number of cytonemes
(>15 µm long) counted from a 60-µm perimeter
centering the ASP tip (Materials and methods) in
(n= 28) and m1
(n= 38). Scale bars:
20 µm (BG9,J,andJ9); 10 µm (HI9).
Sohr et al. Journal of Cell Biology 1662
Cleavage prepares Drosophila FGF for signaling
which the trachea cannot access. Therefore, we predict that a
pro-Bnl cleavage activates a delivery barcode for efcient target-
specic 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 efciency of intracellular and intercellular
Bnl trafcking depends on the enzymatic activity of Fur1
(Fig. 5 G and Fig. S3, AD). 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. (AC) Images of αDlg immunostained (white) ASPs (white outline)
and wing discs from homozygous wt
(n= 52) and m1
(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 proles of t-WT
(D, n=3)andM1
(E, n= 5) along the ASP D-P axis; lower panels, examples of signal distribution along
the ASP D-P axis. Red line, exponential t trend line; C
, maximum average intensity; C
; slope for the trend line between C
and C
Average intensity proles of t-WT
(n= 9) and M1
(n= 12) normalized with recipient ASP length (D-P axes; Materials and methods). (GI) Images of
αdpERK-stained (red) ASPs from homozygous wt
(n= 16) and m1
(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.
(JN) Larval salivary glands expressing CD8:GFP, Bnl:HA
(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 (JN).
Sohr et al. Journal of Cell Biology 1663
Cleavage prepares Drosophila FGF for signaling
exists even in salivary glands that do not normally express Bnl.
This might reect 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
-dependent man-
ner during their intracellular trafcking (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
trafcking to the basolateral membranes of bnl-expressing ight
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. (AC) High-magnication (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
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. (EH) 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 JM. (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
. Arrow, truncated Bnl:GFP
; white dashed line, ASP and wing disc; arrowhead,
apical lumen of wing disc. (NP) Comparison (graph in P) of levels of t-WT
(n= 17) and M1
(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 (EH); 20 µm
(all other panels).
Sohr et al. Journal of Cell Biology 1664
Cleavage prepares Drosophila FGF for signaling
knowledge of intracellular Bnl/FGF targeting is rudimentary and
needs to be elucidated in the future.
Our ndings 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 inuence 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-specic 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
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
(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);
(Du et al., 2018a); and UAS-Bnl:GFP
(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
contained both HA
and GFP tags in tandem inserted between amino acids
and E
contained the tags
inserted between amino acids SNLDRNERST
contained the tags inserted between
amino acids KAPPHCSSNT
and S
contained the tags between amino acids MSSGEEQDQDN
QDQEQSDPGE. Previously, transgenic Drosophila lines har-
boring the Bnl:GFP
construct at various attP loci in the second
and fourth chromosomes did not show any detectable Bnl:GFP
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-elementbased transgenesis to avoid any positional effects
on Bnl:GFP expression. A summary of characterization of
different transgenic lines is presented in Table S2.
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 trafcran-
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 others expression, thereby
generating a Bnl gradient that adopts recipient
ASP-specicshapes(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
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 (amplied from the pUAST-attB-Bnl:HA
), and the middle
sfGFP region from a sfGFP-containing construct (Addgene). The
nal 3,060-bp PCR product was cloned into the pCR-Blunt II-
TOPO vector. The fully sequence-veried insert was subcloned
into the pUAST vector at the BglII and XbaI sites. UAS-bnl:HA
was used for analysis in S2 cells and for P-elementmediated
germline transformation and transgenesis.
The M1 and M2 variants of Bnl:HA
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
construct as a template. The primers
used are shown in Table S1. The nal assembled PCR product was
cloned into the pCR-Blunt II-TOPO vector. The sequence-veried
constructs were subcloned into the BglII and XbaI sites of the
pUAST vector for either analysis in S2 cell culture or for
P-elementmediated germline transformation and transgenesis.
and UAS-bnl:GFP
was cloned using overlap extension PCR to
insert a GFP tag at site 4 of Bnl:HA
was cloned using overlap extension PCR to insert a GFP at site
. The primers used are listed in Table S1. These
constructs were veried 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
and bnl:HA
mutant alleles were
generated by in-frame insertion of an HA-tag into the rst
coding exon of a previously characterized bnl:sfGFP
allele (Du
et al., 2018a) using CRISPR/Cas9-based genome editing following
previously described protocols (Du et al., 2017,2018b). The bnl:
-M1 mutant allele includes the HA
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
bases represent the PAM site) with zero off targets in the y
genome was cloned by ligating two annealed complimentary
oligonucleotides into the pCFD3 vector (Table S1).
The replacement donors, pDonor-bnl:HA
and pDonor-bnl:
-M1, were designed and generated following Du et al.
(2017). These constructs contained either HA
or the HA
mutations anked by 1-kb long 59and 39arms that are ho-
mologous to the genomic sequence anking tag site 1. Both 59
and 39homology arms were PCR-amplied from genomic DNA
from the nos-Cas9;;bnl:GFP
parent y, sequence veried,
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 amplication (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;;
embryos. For each genome-editing experiment, a
stepwise crossing strategy (Du et al., 2018b) was followed to
obtain G0-F2 progenies and establish individual y lines for
screening. The desired ends-outhomologous 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-specic expression patterns of the tagged genes under a
confocal microscope. The efciency of genome editingbased
generation of the two different genotypes and their phenotypes
were summarized in Table S4. During generation of bnl:HA
several lines were obtained that had only the HA
tion without the M1 mutation. We predicted that the HDR had
taken place somewhere between the HA
tag site and M1 mu-
tation sites (219 bases apart). These lines were fully sequence
veried and found to have normal tissue expression. Therefore,
these lines were considered as bnl:HA
lines (see Table
S4). For subsequent analyses, we used a wt
and an m1
derived from the same genome-editing experiment. The wt
F414 line and m1
F49 line used in this study were fully
sequence veried 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
CCCTTCTCGCCCCAAAAGTG-39. dsRNA against fur1 or fur2 were
synthesized using the MEGAscript RNAi Kit (Thermo Fisher
To probe for fur1 and fur2 mRNA, the desired probe regions of
540 and 552 bp were PCR amplied 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 manufacturers 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 immunouorescence with Alexa
Fluor 647conjugated secondary antibody.
Cell culture assay
S2 cells were cultured in 25-cm
asks 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
were 90% conuent, the medium was removed and 6 ml of
fresh M3 medium was added to the ask. 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 manufacturers 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 23daftertransfection.
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 xation 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 nal 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 xed 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).
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
Fluorconjugated secondary antibodies (1:1,000; Molecular
Probes) were used for immunouorescence detection. Phalloidin-
conjugated Alexa Fluor 647 was often used for marking cell
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 (JN), 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 xed 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 ramied 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 uorescence intensities
For Bnl levels, all uorescent 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 uorescence
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 34-
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 immunouorescent signal amplication 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
detection on the cell surface. For Fig. 9 (JL), 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 ve
stacks around the center of the cell were produced. An I
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,
DF) 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
(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 signicance was determined with two-tailed ttests or
a one-way ANOVA followed by Tukeyshonestlysignicant
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
and Bnl:
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
in the ASP. Fig. S5 shows cleavage of Bnl:HA
in the
salivary gland, a quantitative analysis performed to identify
lines of Bnl:HA
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
distribution in a 3D
ASP section. Video 3 shows the lack of Bnl:GFP
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 efciency of generation of bnl:HA
-M1 mutant Drosophila lines using CRISPR/Cas9.
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 nancial 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
Akiyama, T., and M.C. Gibson. 2015. Morphogen transport: theoretical and
experimental controversies. Wiley Interdiscip. Rev. Dev. Biol. 4:99112.
Anderson, E.N., and K.A. Wharton. 2017. Alternative cleavage of the bone
morphogenetic protein (BMP), Gbb, produces ligands with distinct
developmental functions and receptor preferences. J. Biol. Chem. 292:
Binley, K.E., W.S. Ng, J.R. Tribble, B. Song, and J.E. Morgan. 2014. Sholl analysis:
aquantitativecomparisonofsemi-automatedmethods.J. Neurosci.
Methods. 225:6570.
Briscoe, J., and S. Small. 2015. Morphogen rules: design principles of
gradient-mediated embryo patterning. Development. 142:39964009.
Charng, W.-L., S. Yamamoto, M. Jaiswal, V. Bayat, B. Xiong, K. Zhang, H.
Sandoval, G. David, S. Gibbs, H.-C. Lu, et al.2014. Drosophila Tempura,
a novel protein prenyltransferase αsubunit, regulates notch signaling
via Rab1 and Rab11. PLoS Biol. 12:e1001777.
Christian, J.L. 2012. Morphogen gradients in development: from form to
function. Wiley Interdiscip. Rev. Dev. Biol.1:315.
Hedgehog to axons, coordinating assembly of the Drosophila eye and
brain. Dev. Cell. 10:635646.
Constam, D.B. 2014. Regulation of TGFβand related signals by precursor
processing. Semin. Cell Dev. Biol. 32:8597.
Daniele, J.R., T. Chu, and S. Kunes. 2017. A novel proteolytic event controls
Hedgehog intracellular sorting and distribution to receptive elds. Biol.
Open. 6:540550.
Du, L., A. Zhou, A. Patel, M. Rao, K. Anderson, and S. Roy. 2017. Unique
patterns of organization and migration of FGF-expressing cells during
Drosophila morphogenesis. Dev. Biol. 427:3548.
Du, L., A. Sohr, G. Yan, and S. Roy. 2018a. Feedback regulation of cytoneme-
mediated transport shapes a tissue-specic FGF morphogen gradient.
eLife. 7:e38137.
Sohr et al. Journal of Cell Biology 1668
Cleavage prepares Drosophila FGF for signaling
Du,L.,A.Zhou,A.Sohr,andS.Roy.2018b.Anefcient strategy for generating
tissue-specic binary transcription systems in Drosophila by genome
editing. J. Vis. Exp. doi: 10.3791/58268.
Duckert, P., S. Brunak, and N. Blom. 2004. Prediction of proprotein con-
vertase cleavagesites. Protein Eng. Des. Sel. 17:107112.
Guerrero, I., and T.B. Kornberg. 2014. Hedgehog and its circuitous journey
from producing to target cells. Semin. Cell Dev. Biol. 33:5262. https://doi
Huang, Z., and S. Kunes. 1996. Hedgehog, transmitted along retinal axons,
triggers neurogenesis in the developing visual centers of the Drosophila
brain. Cell. 86:411422.
Jarecki, J., E. Johnson, and M.A. Krasnow. 1999. Oxygen regulation of airway
branching in Drosophila is mediated by branchless FGF. Cell. 99:
Jaskolski, F., C. Mulle, and O.J. Manzoni. 2005. An automated method to
quantify and visualize colocalized uorescent signals. J. Neurosci.
Methods. 146:4249.
Johnson, T.K., M.A. Henstridge, A. Herr, K.A. Moore, J.C. Whisstock, and C.G.
Warr. 2015. Torso-like mediates extracellular accumulation of Furin-
cleaved Trunk to pattern the Drosophila embryo termini. Nat. Commun.
Kornberg, T.B. 2011. Barcoding Hedgehog for intracellular transport. Sci.
Signal. 4:pe44.
Kornberg, T.B. 2016. A Path to Pattern. Curr. Top. Dev. Biol. 116:551567.
Künnapuu, J., I. Bj¨
orkgren, and O. Shimmi. 2009. The Drosophila DPP signal
is produced by cleavage of its proprotein at evolutionary diversied
furin-recognition sites. Proc. Natl. Acad. Sci. USA. 106:85018506.
Künnapuu, J., P.M. Tauscher, N. Tiusanen, M. Nguyen, A. L¨
oytynoja, K. Ar-
ora, and O. Shimmi. 2014. Cleavage of the Drosophila screw prodomain
is critical for a dynamic BMP morphogen gradient in embryogenesis.
Dev. Biol. 389:149159.
Lee, J.J., S.C. Ekker, D.P. von Kessler, J.A. Porter, B.I. Sun, and P.A. Beachy.
1994. Autoproteolysis in hedgehog protein biogenesis. Science. 266:
LeMosy, E.K. 2006. Proteolytic regulatory mechanisms in the formation of
extracellular morphogen gradients. Birth Defects Res. C Embryo Today.
Nichols, J.T., and G. Weinmaster. 2010. Proteolytic Activation of Notch Signal-
ing: Roles for Ligand Endocytosis and Mechanotransduction. Vol. 2. Second
edition. Elsevier Inc., New York; 7 pp.
Ochoa-Espinosa, A., and M. Affolter. 2012. Branching morphogenesis: from
cells to organs and back. Cold Spring Harb. Perspect. Biol. 4:a008243.
Ohshiro, T., Y. Emori, and K. Saigo. 2002. Ligand-dependent activation of
breathless FGF receptor gene in Drosophila developing trachea. Mech.
Dev. 114:311.
edelacq, J.-D., S. Cabantous, T. Tran, T.C. Terwilliger, and G.S. Waldo. 2006.
Engineering and characterization of a superfolder green uorescent
protein. Nat. Biotechnol. 24:7988.
Peterson, S.J., and M.A. Krasnow. 2015. Subcellular trafcking of FGF con-
trols tracheal invasion of Drosophila ight muscle. Cell. 160:313323.
Porter, J.A., K.E. Young, and P.A. Beachy. 1996. Cholesterol modication of
hedgehog signaling proteins in animal development. Science. 274:
ırez-Weber, F.A., and T.B. Kornberg. 1999. Cytonemes: cellular pro-
cesses that project to the principal signaling center in Drosophila
imaginal discs. Cell. 97:599607.
Roebroek, A.J., J.W. Creemers, I.G. Pauli, U. Kurzik-Dumke, M. Rentrop, E.A.
Gateff, J.A. Leunissen, and W.J. Van de Ven. 1992. Cloning and func-
tional expression of Dfurin2, a subtilisin-like proprotein processing
enzyme of Drosophila melanogaster with multiple repeats of a cysteine
motif. J. Biol. Chem. 267:1720817215.
Roebroek, A.J., J.W. Creemers, I.G. Pauli, T. Bogaert, and W.J. Van de Ven.
1993. Generation of structural and functional diversity in furin-like
proteins in Drosophila melanogaster by alternative splicing of the
Dfur1 gene. EMBO J. 12:18531870.
Roy, S., F. Hsiung, and T.B. Kornberg. 2011. Specicity of Drosophila cyto-
nemes for distinct signaling pathways. Science. 332:354358. https://doi
Roy, S., H. Huang, S. Liu, and T.B. Kornberg. 2014. Cytoneme-mediated
contact-dependent transport of the Drosophila decapentaplegic signal-
ing protein. Science. 343:1244624.
Sato, M., and T.B. Kornberg. 2002. FGF is an essential mitogen and chemo-
attractant for the air sacs of the drosophila tracheal system. Dev. Cell. 3:
Schweitzer, R., M. Shaharabany, R. Seger, and B.Z. Shilo. 1995.SecretedSpitz
triggers the DER signaling pathway and is a limiting component in
embryonic ventral ectoderm determination. Genes Dev. 9:15181529.
Sopory, S., S. Kwon, M. Wehrli, and J.L. Christian. 2010. Regulation of Dpp
activity by tissue-specic cleavage of an upstream site within the
prodomain. Dev. Biol. 346:102112.
Stewart, D.P., S. Marada, W.J. Bodeen, A. Truong, S.M. Sakurada, T. Pandit, S.
M. Pruett-Miller, and S.K. Ogden. 2018. Cleavage activates dispatched
for Sonic Hedgehog ligand release. eLife. 7:e31678.
Sutherland, D., C. Samakovlis, and M.A. Krasnow. 1996. branchless encodes a
Drosophila FGF homolog that controls tracheal cell migration and the
pattern of branching. Cell. 87:10911101.
Thomas, G. 2002. Furin at the cutting edge: from protein trafc to embryo-
genesis and disease. Nat. Rev. Mol.Cell Biol. 3:753766.
Tokhunts, R., S. Singh, T. Chu, G. DAngelo, V. Baubet, J.A. Goetz, Z. Huang, Z.
Yuan, M. Ascano, Y. Zavros, et al. 2010. The full-length unprocessed
hedgehog protein is an active signaling molecule. J. Biol. Chem. 285:
Tulin, S., and A. Stathopoulos. 2010. Analysis of Thisbe and Pyramus func-
tional domains reveals evidence for cleavage of Drosophila FGFs. BMC
Dev. Biol. 10:83100.
Wharton, K.A., and M. Serpe. 2013. Fine-tuned shuttles for bone morpho-
genetic proteins. Curr. Opin. Genet. Dev. 23:374384.
Sohr et al. Journal of Cell Biology 1669
Cleavage prepares Drosophila FGF for signaling
... However, to drive heterophilic CAM-like bidirectional recognition for synapse, Bnl needs to be tightly associated with the source cell membrane. The source surface localization of Bnl was known to be critical for its dispersion and functions 36,37 . Moreover, Bnl is likely to be a membrane-associated protein 38 , despite its ability to disperse over long range 9 . ...
... How might a secreted protein be associated exclusively on the source cell surface, and be both inhibited and activated for dispersal? A probable mechanism emerged while exploring post-translational Bnl modifications during its intracellular trafficking 36 . We knew that a small N-terminal portion (residue 1-164) upstream of the central 'FGF domain' of Bnl is cleaved off in the source cell Golgi by Furin1 to facilitate polarized trafficking of the remaining C-terminal signaling portion of Bnl to the basal side of the source cell ( Fig. 4a; ref. 36 ). ...
... A probable mechanism emerged while exploring post-translational Bnl modifications during its intracellular trafficking 36 . We knew that a small N-terminal portion (residue 1-164) upstream of the central 'FGF domain' of Bnl is cleaved off in the source cell Golgi by Furin1 to facilitate polarized trafficking of the remaining C-terminal signaling portion of Bnl to the basal side of the source cell ( Fig. 4a; ref. 36 ). When cells expressed a Furin-sensor HA 1 Bnl:GFP 3 construct with HA (site 1) and GFP (site 3) flanking the Furin cleavage site, the cleaved HA-tagged portion was retained in the Golgi, and the truncated Bnl:GFP 3 fragment was externalized for dispersal 36 . ...
Full-text available
How signaling proteins generate a multitude of information to organize tissue patterns is critical to understanding morphogenesis. In Drosophila, FGF produced in wing-disc cells regulates the development of the disc-associated air-sac-primordium (ASP). Here, we show that FGF is Glycosylphosphatidylinositol-anchored to the producing cell surface and that this modification both inhibits free FGF secretion and promotes target-specific cytoneme contacts and contact-dependent FGF release. FGF-source and ASP cells extend cytonemes that present FGF and FGFR on their surfaces and reciprocally recognize each other over distance by contacting through cell-adhesion-molecule (CAM)-like FGF-FGFR binding. Contact-mediated FGF-FGFR interactions induce bidirectional responses in ASP and source cells that, in turn, polarize FGF-sending and FGF-receiving cytonemes toward each other to reinforce signaling contacts. Subsequent un-anchoring of FGFR-bound-FGF from the source membrane dissociates cytoneme contacts and delivers FGF target-specifically to ASP cytonemes for paracrine functions. Thus, GPI-anchored FGF organizes both source and recipient cells and self-regulates its cytoneme-mediated tissue-specific dispersion.
... Bnl is expressed by a restricted group of wing disc cells and subsequently transported to the ASP to control its morphogenesis (Sato and Kornberg, 2002;Du et al., 2018). Bnl is cleaved by Furin-1 at the 164 th amino acid residue prior to the interorgan transport of only its C-terminal truncated portion that contains the receptor binding site (Sohr et al., 2019). ...
... The results from this assay together with other experiments showed that uncleaved Bnl is active and can be transported to the recipient cells to induce signaling. However, Bnl cleavage ensures its efficient polarized target-specific dispersion, thereby modulating its range and tissue-specific activity (Sohr et al., 2019). The ex vivo culture method with pharmacological Furin inhibition strategy is not limited to only the Drosophila wing imaginal disc and ASP tissue system. ...
Furin is an evolutionarily conserved proprotein convertase (PC) family enzyme with a broad range of substrates that are essential for developmental, homeostatic, and disease pathways. Classical genetic approaches and in vitro biochemical or cell biological assays identified that precursor forms of most growth factor family proteins are processed by Furin. To quantitatively assess the potential role of Furin in cleaving and modulating intercellular dispersion of a Drosophila signaling protein, we developed a simple assay by combining genetics, ex vivo organ culture, pharmacological treatment, and imaging analyses. The protocol herein describes how to ex vivo culture Drosophila wing imaginal discs expressing a fluorescently tagged Drosophila Fibroblast Growth Factor (FGF, Branchless/Bnl) over a long period of time in the presence of Furin inhibitors and monitor the cleavage and intercellular dispersion of the truncated Bnl parts using microscopy. Although the assay described here is for assessing the effect of Furin inhibition on Bnl cleavage in the Drosophila larval wing imaginal disc, the principle and methodology can easily be adopted for any other signals, tissue systems, or organisms. This strategy and protocol provide an assay for examining Furin activity on a specific substrate by directly visualizing the spatiotemporal distribution of its truncated parts in an ex vivo-cultured organ.
... Cytonemes containing specific receptors respond to different signals depending on the cell type. Examples include cytonemes that detect Decapentaplegic in eye discs, Delta-Notch in the air sac primordium, FGF in both air sac primordium and tracheal cells, and Wingless in wing imaginal discs [105][106][107]. Wing imaginal discs also release exosomes containing Hedgehog [108,109] and Wingless [110]. ...
Full-text available
During embryonic development, cells communicate with each other to determine cell fate, guide migration, and shape morphogenesis. While the relevant secreted factors and their downstream target genes have been characterized extensively, how these signals travel between embryonic cells is still emerging. Evidence is accumulating that extracellular vesicles (EVs), which are well defined in cell culture and cancer, offer a crucial means of communication in embryos. Moreover, the release and/or reception of EVs is often facilitated by fine cellular protrusions, which have a history of study in development. However, due in part to the complexities of identifying fragile nanometer-scale extracellular structures within the three-dimensional embryonic environment, the nomenclature of developmental EVs and protrusions can be ambiguous, confounding progress. In this review, we provide a robust guide to categorizing these structures in order to enable comparisons between developmental systems and stages. Then, we discuss existing evidence supporting a role for EVs and fine cellular protrusions throughout development.
... Regarding cytoneme formation, in the most studied tissue, the Drosophila wing disc, filopodial polarization is suggested to be controlled by the formation of the Rho GTPase Rac gradient (Couto et al., 2017;Georgiou and Baum, 2010). Vesicle sorting is also proposed to play an important role in the basolateral formation of cytonemes by transporting signaling ligands to the basolateral side (Bilioni et al., 2013;Callejo et al., 2011;Sohr et al., 2019). In zebrafish, local cytoneme nucleation is induced by the Wnt8a-dependent recruitment of CDC42-dependent assembly protein 1 (Ho et al., 2004;Stanganello et al., 2015). ...
Full-text available
Actin-based protrusions called cytonemes are reported to function in cell communication by supporting events such as morphogen gradient establishment and pattern formation. Despite the crucial roles of cytonemes in cell signaling, the molecular mechanism for cytoneme establishment remains elusive. In this study, we showed that the leukocyte common antigen-related (LAR) receptor protein tyrosine phosphatase plays an important role in cytoneme-like protrusion formation. Overexpression of LAR in HEK293T cells induced the formation of actin-based protrusions, some of which exceeded 200 µm in length and displayed a complex morphology with branches. Upon focusing on the regulation of LAR dimerization or clustering and the resulting regulatory effects on LAR phosphatase activity, we found that longer and more branched protrusions were formed when LAR dimerization was artificially induced and when heparan sulfate was applied. Interestingly, although the truncated form of LAR lacking phosphatase-related domains promoted protrusion formation, the phosphatase-inactive forms did not show clear changes, suggesting that LAR dimerization triggers the formation of cytoneme-like protrusions in a phosphatase-independent manner. Our results thus emphasize the importance of LAR and its dimerization in cell signaling. This article has an associated First Person interview with the first author of the paper.
... Around 300 cells of the system differentiate into TCs in the embryo in a process that involves cell growth and elongation driven by Fibroblast Growth Factor (FGF) signal, presented by neighboring tissues. Simultaneously, each TC elaborates a subcellular lumen de novo [9][10][11]. TCs are the functional respiratory units of the system and undergo multiple rounds of growth and branching through the larval stages to form highly ramified cells that reach out and attach to their target tissues. ...
Cells with subcellular lumens form some of the most miniature tubes in the tubular organs of animals. These are often crucial components of the system, executing functions at remote body locations. Unlike tubes formed by intercellular or autocellular junctions, the cells with junctionless subcellular lumens face unique challenges in modifying the cell shape and plasma membrane organization to incorporate a membrane-bound tube within, often associated with dramatic cellular growth and extensions. Results in the recent years have shown that membrane dynamics, including both the primary delivery and recycling, is crucial in providing the cell with the flexibility to face these challenges. A significant portion of this information has come from two in vivo invertebrate models; the Drosophila tracheal terminal cells and the C. elegans excretory cell. This review focuses on the data obtained from these systems in the recent past about how trafficking pathways influence subcellular tube and branching morphogenesis. Given that such tubes occur in vertebrate vasculature, these insights are relevant to human health, and we contrast our conclusions with the less understood subcellular tubes of angiogenesis.
... Tip cells hence follow Bnl-expressing cells and thereby elongate the branch. Imaging Bnl-GFP fusion proteins during imaginal disc development demonstrated that Btl-expressing tracheal cells directly contact Bnl-GFP-expressing source cells (Sohr et al., 2019). Due to adhesive intercellular junctions that connect tip cells and stalk cells, the entire elongating branch resembles somehow, metaphorically speaking, an ''axon-like'' structure. ...
Full-text available
Neurons have evolved specialized growth structures to reach and innervate their target cells. These growth cones express specific receptor molecules that sense environmental cues and transform them into steering decisions. Historically, various concepts of axon guidance have been developed to better understand how axons reach and identify their targets. The essence of these efforts seems to be that growth cones require solid substrates and that major guidance decisions are initiated by extracellular cues. These sometimes highly conserved ligands and receptors have been extensively characterized and mediate four major guidance forces: chemoattraction, chemorepulsion, contact attraction and contact repulsion. However, during development, cells, too, do migrate in order to reach molecularly-defined niches at target locations. In fact, axonal growth could be regarded as a special case of cellular migration, where only a highly polarized portion of the cell is elongating. Here, I combine several examples from genetically tractable model organisms, such as Drosophila or zebrafish, in which cells and axons are guided by attractive cues. Regardless, if these cues are secreted into the extracellular space or exposed on cellular surfaces, migrating cells and axons seem to keep close contact with these attractants and seem to detect them right at their source. Migration towards and along such substrate-derived attractants seem to be particularly robust, as genetic deletion induces obvious searching behaviors and permanent guidance errors. In addition, forced expression of these factors in ectopic tissues is highly distractive too, regardless of the pattern of other endogenous cues. Thus, guidance and migration towards and along attractive tissues is a powerful steering mechanism that exploits affinity differences to the surroundings and, in some instances, determines growth trajectories from source to target region.
... However, this probability depends on the relative number of cytonemes connecting to each target cell. Recent experimental studies of gradient formation of a FGF family protein, Branchless (Bnl), in Drosophila have shown that the number of cytonemes can be time-dependent [6,20]. In Drosophila, Bnl is the primary signal that guides the branching morphogenesis of tracheal epithelial tubes in the wing imaginal disc. ...
Morphogen protein gradients play a vital role in regulating spatial pattern formation during development. The most commonly accepted mechanism of protein gradient formation involves the diffusion and degradation of morphogens from a localized source. However, there is growing experimental evidence for a direct cell-to-cell signaling mechanism via thin actin-rich cellular extensions known as cytonemes. Recent modeling studies of cytoneme-based morphogenesis in invertebrates ignore the discrete nature of vesicular transport along cytonemes, focusing on deterministic continuum models. In this paper, we develop an impulsive signaling model of morphogen gradient formation in invertebrates, which takes into account the discrete and stochastic nature of vesicular transport along cytonemes. We begin by solving a first passage time problem with sticky boundaries to determine the expected time to deliver a vesicle to a target cell, assuming that there is a 'nucleation' time for injecting the vesicle into the cytoneme. We then use queuing theory to analyze the impulsive model of morphogen gradient formation in the case of multiple cytonemes and multiple targets. In particular, we determine the steady-state mean and variance of the morphogen distribution across a one-dimensional array of target cells. The mean distribution recovers the spatially decaying morphogen gradient of previous deterministic models. However, the burst-like nature of morphogen transport can lead to Fano factors greater than unity across the array of cells, resulting in significant fluctuations at more distant target sites.
Full-text available
Rhes (RASD2) is a thyroid hormone-induced gene that regulates striatal motor activity and promotes neurodegeneration in Huntington disease (HD) and tauopathy. Previously, we showed that Rhes moves between cultured striatal neurons and transports the HD protein, polyglutamine-expanded huntingtin (mHTT) via tunneling nanotube (TNT)-like membranous protrusions. However, similar intercellular Rhes transport has not yet been demonstrated in the intact brain. Here, we report that Rhes induces TNT-like protrusions in the striatal medium spiny neurons (MSNs) and transported between dopamine-1 receptor (D1R)-MSNs and D2R-MSNs of intact striatum and organotypic brain slices. Notably, mHTT is robustly transported within the striatum and from the striatum to the cortical areas in the brain, and Rhes deletion diminishes such transport. Moreover, we also found transport of Rhes to the cortical regions following restricted expression in the MSNs of the striatum. Thus, Rhes is a first striatum-enriched protein demonstrated to move and transport mHTT between neurons and brain regions, providing new insights on interneuronal protein transport in the brain.
Full-text available
How signaling proteins generate a multitude of information to organize tissue patterns is critical to understanding morphogenesis. In Drosophila, FGF produced in wing-disc cells regulates the development of the disc-associated air-sac-primordium/ASP. We discovered that FGF is GPI-anchored to the producing cell surface and that this modification both inhibits free FGF secretion and activates target-specific bidirectional FGF-FGFR signaling through cytonemes. Source and ASP cells extend cytonemes that present FGF and FGFR on their surfaces and reciprocally recognize each other over distance by contacting each other through CAM-like FGF-FGFR binding. Contact-mediated FGF-FGFR binding induces bidirectional signaling, which, in turn, promotes ASP and source cells to polarize cytonemes toward each other and reinforce signaling contacts. Subsequent un-anchoring of FGFR-bound-FGF from the source membrane dissociates cytoneme contacts and delivers FGF target-specifically to ASP cytonemes for paracrine functions. Thus, GPI-anchored FGF organizes both source and recipient cells and self-regulates its cytoneme-mediated tissue-specific dispersion and signaling.
Most fibroblast growth factors (FGFs) function as receptor ligands through their conserved FGF domain, but sequences outside this domain vary and are not well studied. This core domain of 120 amino acids (aa) is flanked in all FGFs by highly divergent amino-terminal and carboxy-terminal sequences of variable length. Drosophila has fewer FGF genes, with only three identified to date, pyramus (pyr), thisbe (ths), and branchless (bnl), and all three encoding relatively large FGF proteins (∼80 kDa). We hypothesized that the longer FGF proteins present in Drosophila and other organisms may relate to an ancestral form, in which multiple functions or regulatory properties are present within a single polypeptide. Here, we focused analysis on Pyr, finding that it harbors a transmembrane domain (TMD) and extended C-terminal intracellular domain containing a degron. The intracellular portion limits Pyr levels, whereas the TMD promotes spatial precision in the paracrine activation of Heartless FGF receptor. Additionally, degron deletion mutants that upregulate Pyr exhibit cell polarity defects that lead to invagination defects at gastrulation, demonstrating a previously uncharacterized cell-autonomous role. In summary, our data show that Pyr is the first demonstrated transmembrane FGF, that it has both extracellular and intracellular functions, and that spatial distribution and levels of this particular FGF protein are tightly regulated. Our results suggest that other FGFs may be membrane tethered or multifunctional like Pyr.