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BMP-gated cell cycle progression drives anoikis during
mesenchymal collective migration
Frank Macabenta
1
,
Hsuan-Te Sun
1
,
Angelike Stathopoulos
1,2,*
1
California Institute of Technology, Division of Biology and Biological Engineering 1200 East
California Boulevard, Pasadena, CA 91125 USA
2
Lead contact
SUMMARY
Tissue homeostasis involves the elimination of abnormal cells to avoid compromised patterning
and function. While quality control through cell competition is well-studied in epithelial tissues, it
is unknown if and how homeostasis is regulated in mesenchymal collectives. Here we demonstrate
that collectively migrating
Drosophila
muscle precursors utilize both Fibroblast growth factor
(FGF) and Bone morphogenetic protein (BMP) signaling to promote homeostasis via anoikis,
a form of cell death in response to substrate de-adhesion. Cell cycle-regulated expression of
cell death gene
head involution defective
is responsible for caudal visceral mesoderm (CVM)
anoikis. Secreted BMP ligand drives cell cycle progression via a visceral mesoderm-specific
cdc25/string
enhancer to synchronize collective proliferation, as well as apoptosis of cells that
have lost access to substrate-derived FGF. Perturbation of BMP-dependent cell cycle progression
is sufficient to confer anoikis resistance to mismigrating cells, facilitating invasion of other tissues.
This BMP-gated cell cycle checkpoint defines a quality control mechanism during mesenchymal
collective migration.
Graphical Abstract
*
Corresponding author: angelike@caltech.edu.
AUTHOR CONTRIBUTIONS:
A.S. and F.M. conceived the project. F.M. planned the experimental approach. A.S. directed the
project. F.M. and H.S. performed all experiments and analyzed the data with input from A.S. The manuscript was written by F.M. and
A.S. with input from H.S.
DECLARATION OF INTERESTS:
The authors declare no competing interests.
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Author manuscript
Dev Cell
. Author manuscript; available in PMC 2023 July 25.
Published in final edited form as:
Dev Cell
. 2022 July 25; 57(14): 1683–1693.e3. doi:10.1016/j.devcel.2022.05.017.
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eTOC Blurb
Quality control during collective cell migration is essential for proper organ assembly. Macabenta
et al
. show that
Drosophila
embryonic muscle precursors utilize secreted BMP ligand to
coordinate mitosis along with concomitant expression of the cell death gene Hid, which eliminates
cells that lose access to substrate-derived FGF ligand during migration.
Keywords
cell migration; caudal visceral mesoderm; FGF signaling; BMP signaling; cell cycle; anoikis; cell
death; Decapentaplegic; Tolkin
INTRODUCTION
The coordinated directional movement of groups of cells, which is known as collective cell
migration, is primarily responsible for the highly stereotyped assembly of specific organs
and involves an incredible diversity of strategies and heterotypic interactions depending on
the organ or tissue type (
Macabenta and Stathopoulos, 2019a
;
Scarpa and Mayor, 2016
).
While originally used to describe only epithelial or epithelial-like groups of cells that
have stable adhesions, the definition of collective cell migration has since expanded to
accommodate more transient, dynamic adhesive contacts within groups of mesenchymal
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cells (
Theveneau and Mayor, 2012
,
2013
). Maintaining homeostasis via the elimination of
abnormal or hyperplastic cells in migrating collectives is essential for normal development,
as dysregulated collective migration is a hallmark of metastatic cancer and chronic
inflammation. While cell competition has been identified as an evolutionarily-conserved
strategy for eliminating abnormal cells in epithelial tissues, it is unknown if, and how,
mesenchymal collectives exert quality control via the removal of abnormal or mismigrating
cells.
In this study, we use the
Drosophila
embryonic caudal visceral mesoderm (CVM) as a model
for understanding how a collectively-migrating cohort of mesenchymal cells maintains
integrity via removal of lost cells prior to organ assembly. CVM migration is a multistep
process that involves the synchronous bilateral migration of cell cohorts along the trunk
visceral mesoderm (TVM), which serves as a substrate track (Figure 1A). Additionally,
CVM cells form heterotypic interactions with co-migrating primordial germ cells (PGCs)
(
Stepanik et al., 2016
), which is similar to the chase-and-run mechanism described in
collectively-migrating
Xenopus
cranial neural crest and placodal cells (
Theveneau et al.,
2013
). Our lab and others have previously characterized an essential role for FGF signaling
in supporting CVM cell migration and survival (
Kadam et al., 2012
;
Reim et al., 2012
).
The
Drosophila
FGFs Pyramus (Pyr) and Thisbe (Ths) are expressed in the TVM, where
they interact with the FGF receptor Heartless (Htl) expressed by the migrating CVM
cohorts to promote proper pathfinding and integrin-dependent adhesion (
Kadam et al., 2012
;
Macabenta and Stathopoulos, 2019b
;
Sun and Stathopoulos, 2018
). Furthermore, unlike
other examples of FGF-dependent collective cell migration in
Drosophila
[e.g. mesoderm
spreading during gastrulation (
McMahon et al., 2010
;
Sun and Stathopoulos, 2018
)], CVM
cells undergo apoptosis when FGF signaling is ablated. In wildtype embryos, posterior CVM
cells that have lost contact with the TVM undergo apoptosis (
Kadam et al., 2012
;
Reim
et al., 2012
), while mutants for
biniou
(
bin
) and
bagpipe
(
bap
) result in more widespread
CVM cell death due to complete loss of the TVM (
Reim et al., 2012
). This makes the
CVM an ideal system for studying anoikis, which is a form of apoptosis induced by the
detachment of cells from the extracellular matrix, therefore preventing adhesion-independent
growth. Acquiring anoikis resistance is a prerequisite to cancer progression and metastatic
colonization of other tissues (
Paoli et al., 2013
). Furthermore, we have uncovered the
unprecedented ability of CVM cells to influence the morphogenesis of their tissue substrate
(
Macabenta and Stathopoulos, 2019b
). During normal development, migrating CVM cells
appear to reorient cells in the TVM as they pass over, such that a complete absence of CVM
results in abnormal TVM morphology. In FGF mutants, an even more dramatic phenotype
is observed: CVM cells mismigrate and alter the directional growth of the TVM, forming
contralateral ‘bridges’ (
Macabenta and Stathopoulos, 2019b
). There is clearly a significant
morphogenetic consequence to allowing mismigrating CVM cells to survive; therefore, one
can extrapolate that a mechanism for inducing apoptosis in cells that have lost access to FGF
signaling (such as cells that have moved off-track) is critical to establishing normal midgut
musculature.
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RESULTS
Temporally regulated cell death in CVM cells requires
hid
To investigate programmed cell death over the course of CVM migration, we immunostained
stage 11-13 embryos with antibodies to Teyrha-meyrha (Tey) to visualize the CVM and
Fasciclin III (FasIII) to visualize the TVM (Figure 1A,B,B’). Tey nuclear staining is
completely lost in cells that undergo apoptosis, which is apparent when used in conjunction
with the CVM-specific
HLH54F
-Gap-Venus (HGV) reporter to visualize the cytoplasm and
cell membranes (
Stepanik et al., 2016
); Tey signal is absent in cellular debris and blebs
visualized by HGV, presumably due to nuclear envelope breakdown in dying cells (Figure
1B, arrowheads). Consistent with previously reported findings (
Kadam et al., 2012
;
Mandal
et al., 2004
), CVM cells in
htl
mutant embryos are almost completely lost to apoptosis
by stage 13 as indicated by visualization of terminal deoxynucleotidyl transferase-mediated
dUTP nick-end labeling (TUNEL)-positive cells and a near-total absence of Tey staining
(Figures S1A,B and 1C,D,G). However, apoptosis only occurs during the later stages of
migration; CVM cells in
htl
mutants remain alive through stages 10-12, albeit with highly
abnormal migration (
Kadam et al., 2012
;
Reim et al., 2012
). Apoptosis is also observed
in WT embryos, with mismigrating cells and posterior CVM cells that have lost contact
with the TVM going off-track (Movie S1). We therefore wanted to investigate how this
temporally-restricted cell death is achieved in migratory CVM.
The gene
head involution defective
(
hid
), which is an ortholog of mammalian
Smac
/
DIABLO (
Verhagen and Vaux, 2002
), is well-known for its function in promoting
programmed cell death (
Grether et al., 1995
;
Verhagen and Vaux, 2002
). Hid protein
targets the cell survival factor
Drosophila
Inhibitor of Apoptosis 1 (DIAP1) for proteasomal
degradation, initiating the downstream signaling cascade that culminates in Caspase-3
cleavage and subsequent apoptosis (
Goyal et al., 2000
;
Holley et al., 2002
;
Ryoo et al.,
2002
;
Wang et al., 1999
;
Yoo et al., 2002
). Expression of
hid
was observed in the CVM and
verified via fluorescence
in situ
hybridization (FISH) with
hid
riboprobes (Figure 1K). To
test whether CVM cell apoptosis relates to
hid
expression, double mutants of
htl
and
hid
were generated and CVM cell survival was quantified with anti-Tey staining (Figure 1E,G).
We found that
hid
loss of function attenuated apoptosis of CVM observed in
htl
single
mutants, suggesting that
hid
is essential for the cell death observed in later stages (Figure
1E, compare with C,D). Intriguingly, this rescue of cell survival also resulted in “undead”
htl hid
mutant CVM cells occasionally invading other tissues, including the embryonic
central nervous system (CNS) (Figure 1H-J), demonstrating that Hid initiates anoikis in
aberrantly migrating CVM cells. Consistent with previously published literature, the cell
death phenotype was also attenuated by expressing constitutively active Ras (Ras1a) in the
CVM, which works downstream of FGF and is known to antagonize
hid
[Figure S1C-E’;
(
Bergmann et al., 1998
;
Gisselbrecht et al., 1996
;
Kurada and White, 1998
;
Vincent et al.,
1998
)].
hid
expression in CVM cells is restricted to the later stages of migration, starting at stage
12 (Figure 1K), which coincides with CVM cell division [as detected by phosphorylated
histone H3 (PH3) immunostaining to label mitotic cells] at the posterior turn before
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rearranging into discrete linear cohorts along the TVM prior to myoblast fusion (Figure
1M;
Shibata et al., 1990
;
Su et al., 1998
). We investigated whether there is a correlation
between timed CVM cell division and the onset of
hid
expression by first looking at mutants
for
string
(
stg
), which encodes a Cdc25 phosphatase that promotes expression of the mitotic
kinase Cdk1 (Cdc2;
Edgar and O’Farrell, 1990
). Expression of
stg
is therefore essential for
the G2-M transition, such that loss of function mutants have fewer cells due to delayed
mitosis. If
hid
expression in the CVM is regulated by the cell cycle,
stg
mutants would
likely present a delay or absence of
hid
mRNA expression. Indeed, FISH assays indicate a
highly reduced and delayed onset of
hid
expression in
stg
single mutants, with detectable
expression starting only at stage 13, well after the CVM have completed their migration
(Figure 1N,N’,O,O’, compare with M,M’). Furthermore, double mutants for
htl
and
stg
showed that a loss of
stg
function largely attenuated apoptosis caused by a loss of FGF
signaling (Figure 1F,G). By counting the numbers of CVM nuclei at stage 11 prior to cell
division, we found that the numbers of CVM cells in WT,
htl
, and
htl hid
mutants was
not significantly different, with only
htl stg
mutants presenting a reduced number of cells
(Figure S1F-H, K). This suggests that temporal activation of
hid
in the CVM is regulated by
the cell cycle and correlated with mitosis. Additionally, we examined the effect of the
E2f1
transcription factor on
hid
expression dynamics in the CVM.
E2f1
plays a central role in
cell cycle regulation and has been shown to regulate
hid
expression in a context-dependent
manner (
Bilak and Su, 2009
;
Davidson and Duronio, 2012
;
Tanaka-Matakatsu et al., 2009
).
We found that in
E2f1
mutant embryos,
hid
expression is delayed in a manner similar to
what is observed in mitosis-defective
stg
mutants, suggesting that cell cycle regulation as the
cells reach the posterior turn at stage 12 plays an important role in
hid
expression dynamics
in the CVM (Figure S1I,J).
A BMP-responsive enhancer controls
stg
expression in the CVM
To investigate how the timing of cell division is controlled in migratory CVM, we used
publicly-available ChIP-seq data for the transcription factors Biniou (Bin) and Myocyte
Enhancer Factor 2 (Mef2), which are known to support enhancer activity in the visceral
mesoderm (i.e. CVM and TVM) (
Junion et al., 2012
;
Zinzen et al., 2009
) in conjunction
with published enhancer data (
Kvon et al., 2014
) to identify candidate
stg
visceral mesoderm
enhancers. Published enhancer constructs VT49281 and VT49282 (
Kvon et al., 2014
)
contain an overlapping region that shows strong occupancy by both Bin and Mef2 from
6-8hr ChIP-seq data, which allowed us to identify a 1.5-kb enhancer that supports expression
of
stg
in both the CVM and TVM (Figure 2A,B). Additionally, phosphorylated Mothers
Against Decapentaplegic (pMAD) ChIP-seq data from 6-8hr embryos (
Junion et al., 2012
)
showed strong occupancy at this enhancer, suggesting a direct requirement for BMP
signaling in controlling expression (Figure 2A). We verified that this enhancer is expressed
in the CVM by checking colocalization of the
lacZ
reporter mRNA signal with Tey antibody
to label CVM and Bin antibody to label both CVM and TVM (Figure S2A-C’). To test
whether this
stg
visceral mesoderm (
stgVM
) enhancer is BMP-responsive, we crossed the
stgVM-lacZ
reporter into a background that is mutant for the BMP type I receptor encoded
by
thickveins
(
tkv
) (
Brummel et al., 1994
;
Penton et al., 1994
;
Terracol and Lengyel,
1994
). Crossing the
stgVM
reporter construct into a
tkv
mutant background resulted in a
loss of
lacZ
expression as visualized by FISH (Figure 2D, compare with C, S2F), which
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was also observed when a dsRNA construct targeting
tkv
was expressed specifically in the
CVM (Figure S2D,F); conversely, overexpressing constitutively-active Tkv specifically in
the CVM resulted in increased
lacZ
mRNA signal (Figure S2E,F), demonstrating that this
enhancer is BMP-responsive. Identification of the
stgVM
enhancer complements previous
studies that described a role for BMP signaling in influencing context-dependent
stg
expression dynamics throughout development, including in the early embryo and the trachea
(
Djabrayan and Casanova, 2016
;
Edgar et al., 1994
).
We therefore investigated how BMP signaling is transduced in the CVM. The secreted
BMP2/4-related ligand Decapentaplegic (Dpp) is essential for coordinating growth and
proliferation in wing discs, where Dpp is expressed in a thin stripe and laterally diffuses as
a gradient (
Akiyama and Gibson, 2015
). In addition to strong lateral ectoderm and dorsal
epidermis expression in the embryo, Dpp is expressed in the TVM, where it eventually
refines into a discretely localized stripe of expression at parasegments 3 and 7 (
Jackson
and Hoffmann, 1994
). Immunostaining with pMAD antibody to visualize active BMP
signaling demonstrated a correlation between expression of Dpp and pMAD expression
domains in the embryo, including PS7 of the TVM (Figures 2F, S3A-A”). Additionally,
we verified the expression of
tkv
mRNA in the CVM (Figure 2G-H’), and found that
ectopic expression of constitutively active Tkv using the CVM-specific G447-GAL4 driver
was sufficient to promote precocious cell division (Movie S2). Discrete Dpp expression
in the TVM, particularly in segments PS3 and PS7, requires inputs from the homeotic
genes
abdominal-A
(
abd-A
) and
Ultrabithorax
(
Ubx
) (
Capovilla et al., 1994
;
Rauskolb and
Wieschaus, 1994
;
Reuter et al., 1990
;
Thüringer and Bienz, 1993
) and is required for normal
midgut morphogenesis (
Galeone et al., 2017
;
Panganiban et al., 1990
). Additionally, the
Drosophila Tbx1
ortholog
optomotor-blind-related-gene-1
(
org-1
) is required for expression
of
Ubx
and
dpp
in the TVM, while not affecting specification of the midgut musculature
(
Schaub and Frasch, 2013
). Over the course of migration, CVM cells directly contact and
pass over PS7 (Figures 2E,F,S3). As Dpp is expressed by a large number of tissues in the
embryo in addition to PS7-specific expression in the TVM (Figure 2E,I) over the course
of stages 11-13, and due to the difficulty of separating earlier functions of Dpp from
potential later functions in the lateral ectoderm and dorsal epidermis expression domains, we
investigated whether PS7-derived Dpp serves as a more specific source of ligand for BMP
signaling in the CVM. To this end, we used
org-1
OJ487
(referred to as
org-1
mutant in this
study), an allele that results in highly-reduced PS7-specific
dpp
expression and complete
loss of PS3-specific expression [(
Schaub and Frasch, 2013
); Figure 2J,M,Q compare with
I,L,P)]. While CVM cell number between WT and
org-1
mutants was not significantly
different at stage 11 (Figure 2O), we observed a reduced number of CVM cells in
org-1
mutant embryos at the conclusion of stage 13, suggesting that TVM-derived Dpp helps
coordinate CVM proliferation (Figure 2S). However, ectopic expression of Dpp throughout
the TVM was not sufficient to induce precocious or otherwise dysregulated proliferation
of CVM cells (Figure 2K,N,O,R,S), suggesting another layer of regulation limits BMP
signaling capacity during migration. Furthermore,
tkv
mutant embryos exhibit wide-ranging
defects due to multiple roles of BMP signaling in supporting earlier patterning, including
TVM specification and morphogenesis (
Bradley et al., 2003
;
Frasch, 1995
). In order to
investigate a more CVM-specific role for BMP signaling and its apparent regulation during
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collective migration, we used published RNAseq data to identify BMP signaling effectors
that are enriched in the CVM (
Bae et al., 2017
).
Secreted Tok metalloprotease regulates BMP signal transduction in the CVM
The secreted metalloprotease encoded by the gene
tolkin
(
tok
), a known effector of BMP
signaling, is expressed specifically in the CVM throughout migration (
Bae et al., 2017
;
Finelli et al., 1995
) (Figure 3A-B’). Like its closely-linked gene,
tolloid
(
tld
), the product of
tok
is an ortholog of mammalian BMP-1 and participates in BMP signaling by cleaving the
inhibitor Short gastrulation (Sog), freeing BMP ligands, including Dpp (
Serpe et al., 2005
).
We hypothesized that CVM-derived Tok works cell-autonomously to process Dpp ligand
secreted from tissue substrates, transducing BMP signaling over the course of migration and
influencing the timing of proliferation in migratory CVM. At stage 12, in both WT and
htl
mutants, the CVM contains a subset of cells that are pMAD-positive as visualized by
immunostaining (Figure 3C-D’). However, in
tok
loss of function mutants, we observed an
absence of pMAD staining in CVM cells at this same stage, suggesting that Tok functions as
a BMP effector in migratory CVM (Figure 3E-E’, S3B-B”).
Furthermore, CVM cell number in
tok
mutants was significantly reduced compared to WT
in a stage-specific manner as quantified via Tey antibody staining (Figure 3H,N,J,P). At
stage 11, a similar number of CVM cells is present in
tok
mutants compared to wildtype
(Figure 3T); however at stage 13, subsequent to cell division, wildtype cell number has
increased but the increase observed in
tok
mutants is significantly smaller (Figure 3U).
Surprisingly, double mutants for
tok
and
htl
presented significantly improved CVM cell
survival (Figure 3I,O, compare with K,Q; T,U). This was also observed in double mutants
for
htl
and
tkv
as well as in
htl
mutants expressing a dsRNA hairpin targeting
tkv
specifically in the CVM (Figure 3L,R,M,S,T,U). Based on our data showing how BMP
signaling regulates
stg
expression in the visceral mesoderm, we believe that the reduction
in cell number in the
tok
mutant background compared to WT is due to a disruption in
stg
-mediated proliferation due to loss of BMP signaling activity. Therefore, the BMP-FGF
double mutants phenocopy the
htl stg
mutant phenotype, suggesting that the role of BMP
signaling in coordinating proliferation directly impacts the timing of cell death during CVM
migration. Collectively, these results suggest that in this context, 1) BMP signaling promotes
timely anoikis via its role in cell cycle progression to facilitate
hid
expression, and that
2) activating BMP signaling to promote cell cycle progression requires CVM-specific Tok
metalloprotease, which in turn allows CVM collectives to transduce BMP signaling in the
presence of secreted Dpp ligand in the extracellular milieu.
We further investigated whether spatial constraints relate to CVM cell death. TUNEL
staining at stage 13 supports the view that cells not contacting the TVM undergo apoptosis
(Figure 3F). Using a reporter of
hid
expression,
hid5’F-GFP
(
Tanaka-Matakatsu et al.,
2009
), to assay CVM cells at stage 13, we show that even in wildtype embryos a few CVM
cells are associated with elevated
hid
expression but no Tey staining, which indicates cell
death. Furthermore,
hid5’F-GFP
signal is elevated only in cells located at a distance from
the TVM and reduced in the majority of cells that directly contact the TVM (Figure 3G).
Increased
hid5’F-GFP
expression in CVM cells that appear at a distance from the TVM
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may relate to loss of FGF signaling and subsequent de-adhesion of the CVM, as the TVM
expresses Pyramus (Pyr), a membrane-associated FGF ligand (
Kadam et al., 2012
;
Reim et
al., 2012
;
Stepanik et al., 2020
).
The G2-M transition temporally positions CVM anoikis during migration
To gain further insight into the mechanism by which BMP signaling promotes both
cell proliferation and apoptosis in the CVM, we used the fly Fluorescent Ubiquitin-
associated Cell Cycle Indicator (Fly-FUCCI) system (Figure 4A-C). Fly-FUCCI consists
of a bicistronic construct under the control of a UAS promoter that allows for expression
of fluorophore-conjugated peptides of the cell cycle genes E2f1 and CycB to present a
readout of cell cycle stage; the RFP-conjugated CycB degron is expressed through DNA
synthesis (S) and growth phase 2 (G2) before being degraded, while the GFP-conjugated
E2f1 degron is expressed beginning in G2 before being degraded prior to S phase (
Zielke et
al., 2014
). CVM-specific expression of Fly-FUCCI reveals the transition from G2 through
mitosis followed by a transient G1 at the posterior turn at stage 12 in both WT and
htl
mutant backgrounds (Figure 4A,B); however, in a
tok htl
double mutant background, CVM
cells are arrested at G2 at stages 12-13 (Figure 4C). Furthermore, direct manipulation of
cell cycle effectors can recapitulate survival or cell death phenotypes in the CVM in a cell
cycle-dependent manner (Figure 4D-M); CVM-specific expression of a
stg
dsRNA construct
using the GAL4 system (
G447>stgRNAi
) in a
htl
mutant background results in prolonged
survival past stage 12 (Figure 4E,I, compare with D,H), while ectopic expression of
stg
using this same expression system (
G447>stg)
has the opposite effect. Expression of
stg
in a
tok htl
double mutant ‘un-rescues’ the survival associated with loss of both FGF and
BMP signaling components (Figure 4G,K,M compare with Figure 3K,Q,U), suggesting
that cell proliferation is sufficient for temporally positioning cell death in migratory CVM.
Additionally, precocious cell death was observed when a dsRNA construct targeting
tribbles
(
trbl
), which encodes a negative regulator of Stg (
Mata et al., 2000
), was expressed
specifically in the CVM in a
htl
mutant background (
G447>trblRNAi
; Figure 4F,J,L,M),
further supporting a model in which cell cycle progression positions a quality control
checkpoint for the collective (Figure 4N,O).
DISCUSSION
In this study, we described a mechanism for correcting errors in mesenchymal collective cell
migration prior to organ assembly. First, we described how initiation of CVM cell death due
to loss of FGF signaling requires cell cycle-regulated expression of
hid
(e.g. Figure 1I,K).
Next, we demonstrated how BMP signaling transduced by cell-autonomous Tok activity
coordinates the timing of cell cycle progression of CVM cells over the course of migration
by promoting BMP signaling (e.g. Figure 3C-E and 4C). Third, we showed how coordinated
cell cycle progression ensures timely apoptosis of cells that have gone off track, ensuring
that cells that remain on-track undergo myoblast fusion (e.g. Figure 3F,G). In this system,
fitness is conferred by proximity and ability to transduce both FGF and BMP signals by
members of the collective. Although both FGF and BMP receptors are expressed in the
CVM, FGF ligand expression is restricted to the TVM track, while Dpp is expressed broadly
throughout the embryo, including in a subpopulation of TVM cells anterior to migratory
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CVM (Figures 2E and 4N). As such, Tok metalloprotease secreted by CVM cells is critical
to ‘sensing’ a given source of Dpp, triggering either growth or anoikis (Figure 4O).
Cell proliferation and negative regulation of Hid-mediated apoptosis proceeds when CVM
cells are both adhered to an FGF ligand-presenting substrate as well as close to a source
of secreted Dpp, which is normally bound by Sog -- necessitating cleavage by Tok to
free Dpp ligand for activation of BMP signaling in the CVM (Figure 4O). In cases of
CVM de-adhesion or mismigration to a substrate that expresses Dpp but not FGF, secreted
Tok processes Sog and frees Dpp for signaling and subsequent cell cycle progression as
normal; however, the absence of FGF-dependent Hid antagonism results in rapid apoptosis
of the lost cell (Figure S4A). Finally, when both FGF and BMP signaling components (i.e.
either Tok ligand-activating protease or Tkv receptor) are lost, CVM cells are rendered
refractory to secreted Dpp, resulting in cell cycle arrest and survival in a senescence-like
state (Figure S4B). This is a striking example of a developmental context in which Dpp can
promote apoptosis, as opposed to its more well-known role in promoting cellular growth,
thus complementing studies in epithelial tissues describing a requirement for boundary Dpp
in positioning cell death for leg morphogenesis (
Manjón et al., 2007
) and the extrusion of
BMP mutant cells from epithelial wing primordia (
Gibson and Perrimon, 2005
).
Additionally, Dpp’s function as a cell cycle regulator is sufficient for timing apoptosis in
migratory CVM, as direct manipulation of cell cycle effectors can either recapitulate or
ablate the survival phenotype presented by double mutants for FGF and BMP signaling
components (Figure 4D-M). Positioning of the G2-M transition by Stg is a critical step of
the regulatory cascade leading to apoptosis, but likely requires the concomitant activity of
the E2f1 transcription factor, which has a known role in promoting apoptosis via regulation
of
hid
(
Davidson and Duronio, 2012
;
Moon et al., 2005
). Surprisingly, surviving CVM cells
in
tok htl
double mutants that arrest at G2 in stage 13 (Figure 4C) re-enter the cell cycle
by stage 15 (Figure S4C,D); this suggests that the absence of both FGF and BMP signaling
first induces an arrested senescence-like state, whereby survival after a prolonged G2 results
in cell cycle re-entry, perhaps through the activation of additional signaling pathways. This
is reminiscent of the cell cycle stalling induced by JNK signaling in the fly wing disc in
response to cellular damage (
Cosolo et al., 2019
;
Ohsawa et al., 2012
). Therefore, our study
has tantalizing implications regarding the existence of additional cell types in
Drosophila
that undergo a similar senescence-like state. Furthermore, the role for BMP in promoting
apoptosis has potential parallels in higher organisms, as reduced apoptosis in pulmonary
artery smooth muscle cells (PASMCs) is associated with BMP signaling, and can lead to
hypertrophy of the pulmonary vasculature in primary pulmonary hypertension (PPH) (
Zhang
et al., 2003
).
We have shown that
hid
is required for eliminating CVM cells that have gone off-track;
in
hid
single mutants, we observed cells that have migrated ventrally that are absent in
WT embryos (Figure S4G, compare with E). However, it is also likely that additional
pro-apoptotic genes like
grim
and
reaper
(
rpr
) are involved, as the
htl hid
double mutant
background resulted in a significantly lower number of surviving CVM cells when compared
to WT (Figure 1G). Indeed, when we compared
hid
single mutants with a deficiency in
which the three pro-apoptotic genes
grim
,
rpr
, and
hid
are deleted [
Df(3L)H99
, referred to
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as
DfH99
in this study (
Chen et al., 1996
;
Grether et al., 1995
;
Mackay and Bewley, 1989
;
White et al., 1996
;
Zhou et al., 1997
)], we not only found a significantly higher number of
CVM cells in
DfH99
embryos when compared to both WT and
hid
single mutants (Figure
S4F), but also observed differences in cell morphology between the different genotypes;
strikingly, a continuous ‘bridge’ of CVM cells was observed in the
DfH99
embryo (Figure
S4E-H’). This may hint at additional components of the quality control system that not
only support apoptosis of different populations of CVM cells, but may potentially regulate
non-apoptotic functions, such as directional myoblast fusion.
We expect that this study will open new avenues for understanding how a continuum of
cell interactions can contribute to development and cancer. While it is generally accepted
that the acquisition of a mesenchymal phenotype is required for metastasis in most cancers,
the extent to which cooperative interactions are required between migratory mesenchymal
cancer cells and other tissue types has yet to be determined. Furthermore, the invasion of
other tissues by CVM that have lost the cell death program resembles pathologies associated
with Tuberous Sclerosis Complex (TSC), a developmental disorder that can result in the
formation of smooth muscle cell-derived benign tumors in other tissues (
Lesma et al., 2005
).
The developmental basis for how these tumors arise and the role that collective migration
may play remain to be investigated, and is a promising avenue for identifying broadly
conserved regulatory mechanisms governing tissue homeostasis.
Limitations of the study
Although we focused on the canonical role for Tok metalloprotease in mediating BMP
signaling via cleavage of the Dpp inhibitor Sog for this study, we observed migration
defects in the
tok
and BMP mutant backgrounds that were apparent at stage 11, prior to
the cells reaching the posterior turn. This suggests that there may be an additional role
for BMP signaling in ensuring proper CVM migration, and perhaps additional targets for
Tok-dependent cleavage that might contribute to this system, which will require further
study. Further work will also be needed to identify which signaling pathways might be
responsible for preferential migration of FGF mutant CVM towards the CNS in the
htl hid
double mutant background.
STAR METHODS
RESOURCE AVAILABILITY
Lead contact—
Further information and requests for resources and reagents should
be directed to and will be fulfilled by the lead contact, Angelike Stathopoulos
(angelike@caltech.edu).
Materials availability—
Drosophila
strains and other reagents generated in this study will
be available upon request from the lead contact, or the commercial sources listed in the key
resources table.
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Data and code availability
No large-scale datasets have been generated in this study. The raw microscopy
data that support the findings of this study are available from the lead contact
upon reasonable request.
This study did not generate any software and code. Any additional information
required to reanalyze the data shown in this paper is available from the lead
contact upon request.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Fly stocks and husbandry—
All fly stocks were kept at 22-25°C in standard medium.
The CVM reporter
HLH54F-gap-Venus
was described previously (
Kadam et al., 2012
;
Stepanik et al., 2016
) and combined with
htl
AB42
mutant and GAL4/UAS lines via standard
genetic crosses. Wild type (WT) refers to
yw
unless otherwise noted. For experiments, flies
were kept in collection cages with apple juice agar plates supplemented with yeast paste
and allowed to lay for at least eight hours before egg collection and dechorionation in 100%
bleach.
METHOD DETAILS
Whole-mount
In situ
hybridization, immunofluorescence staining—
Embryos
were collected and fixed using previously described methods (
Frasch, 1995
;
Jiang et al.,
1991
). The antisense Digoxigenin (DIG) labeled RNA probe targeting
hid
was generated
with linearized DGRC CDNA clone AT13267 (Stock #11756), and the
lacZ
RNA probe
was generated from linearized plasmid. For alkaline phosphatase
in situ
hybridization
(AP ISH), an anti-DIG antibody conjugated to alkaline phosphatase (1:500, Sigma,
11093274910) was used in conjunction with NBT/BCIP (Roche) to visualize signal. For
fluorescent
in situ
hybridization (FISH), a sheep anti-DIG polyclonal antibody (1:200,
Thermo Fisher, PA1-85378) was used in conjunction with Alexa Fluor secondary antibody
(1:500, Molecular Probes).
Antibody stainings were performed as previously described. The primary antibodies and
dilutions used in this study were Rabbit anti-Tey (
Macabenta and Stathopoulos, 2019b
),
Mouse anti-FasIII (1:200, DSHB, 7G10), Goat anti-GFP (1:5000, Rockland, 600-101-215),
Rabbit anti-RFP (1:500, MBL, PM005), Rabbi anti-Beta-Galactosidase (1:500, MP,
559761), Rabbit anti-Phospho-SMAD1/5 (1:50, Cell Signaling, 9516S), Rabbit anti-PH3
(1:500, EMD, 06-570), Sheep anti-DIG (1:200, Thermo Fisher, PA1-85378), Anti-DIG
AP (1:200, Sigma, 11093274910), and Rabbit anti-Biniou (1:500,
Jakobsen et al., 2007
).
Immunofluorescence staining was performed using Alexa Fluor 488, 555 and 647 secondary
antibodies (1:500, Molecular Probes). TUNEL was performed by using the
In Situ
Cell
Death Detection Kit (Roche) according to manufacturer protocols. Embryos were mounted
in Permount (Fisher Scientific) for whole-mount AP ISH experiments or in 70% glycerol
in 1× PBS buffer for whole-mount immunofluorescence-stained experiments. Imaging was
performed using an Axio Imager Z2 (Zeiss) for AP ISH embryos, and an LSM 800 (Zeiss)
for immunofluorescence-stained embryos. Zen blue edition (Zeiss) was used to process
images for subsequent analysis and figures.
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Quantification of CVM cell number—
Confocal images of embryos immunostained
with Tey antibody were obtained for stage 11 (dorsal view and 13 (lateral view) using an
LSM800 with 20X objective. Embryos were staged by assessing both FasIII antibody signal
intensity and lateral amnioserosa morphology over the course of germband retraction. Tey-
positive nuclei were counted manually in Fiji/ImageJ (
Schindelin et al., 2012
) by planing
through the z stacks and using the Cell Counter plug-in. For lateral views, ventral CNS and
lateral M12 muscle-specific expression of Tey was excluded from counts. Experiments were
repeated 2-3 times and power analysis to determine sample size was performed using the
G*power tool (
Faul et al., 2009
). Graphs and statistical analyses were generated using Prism
9 (GraphPad).
Hybridization chain reaction—
Hybridization chain reaction (HCR) was performed
as described in (
Slaidina et al., 2021
) with modifications to initial steps to account for
embryonic as opposed to ovarian tissue. The probes used were
dpp
,
tkv
, and
HLH54F
.
Cloning of reporter construct and generation of transgenic flies—
The
stgVM
sequence was PCR amplified using Phusion High Fidelity polymerase and ligated into the
eve2promoter-lacZ
vector using standard techniques. Site-directed transgenesis was carried
out using a
D. melanogaster
stock containing an attP insertion site at positions ZH-51C
(Bloomington stock #24482) and ZH-86Fb (Bloomington stock #23648). The primers used
were:
Stg
VM
_F: GATCCGGGAATTGGGAATTCTATCGAGAAATATAT
Stg
VM
_R: GCAGATCTGTTAACGAATTCCGTGTGCATTTGCCA
Quantification of
lacZ
mRNA signal—
Embryos were collected and FISH was
performed using
lacZ
riboprobes in conjunction with Tey antibody staining. Individual
embryos were subsequently staged and imaged using an LSM 800 confocal microscope.
Three replicates were used for each genotype, with 23-25 total CVM measurements across
three samples. Raw .czi files were processed using ImageJ software, with single planes used
for quantification. Regions of interest (ROIs) were demarcated by using Tey-positive CVM
nuclei as a guide, and mean fluorescence intensity values for
lacZ
riboprobe signal within
each ROI were obtained via the Analyze
Measure
Mean Value function. Corrected
mean values were obtained by measuring background signal/noise in a separate ROI and
subtracting the resulting fluorescence intensity value from each CVM-associated
lacZ
mean
fluorescence intensity measurement. One-way ANOVA was applied to assess statistical
significance.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
ACKNOWLEDGMENTS:
We thank Norbert Perrimon for sharing fly stocks, and Eileen Furlong for sharing the Biniou antibody. We are
also grateful to the Stathopoulos lab members, in particular Heather Curtis, for helpful discussions and technical
Macabenta et al.
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support. This study was supported by funding from National Institutes of Health grant R01HD10018 to A.S. and
Baxter Postdoctoral Fellowship to F.M. Model figures were created with
BioRender.com
.
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Highlights
Cell death in migrating caudal visceral mesoderm (CVM) cells requires Hid
Hid levels are correlated with onset of CVM cell division
A BMP-responsive
stg
enhancer controls timing of cell division during CVM
migration
Cell cycle-coupled Hid expression eliminates cells that have lost access to
FGF
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Figure 1. CVM cell death due to loss of FGF signaling is temporally regulated and requires
hid
.
(A) Schematic of CVM cells (red) migrating bilaterally along the TVM track (cyan). (B-B’)
WT embryos expressing HLH54F-Gap-Venus (HGV) reporter immunostained with GFP
antibody to mark CVM cell membranes (green), Tey antibody (red) to mark CVM nuclei,
and FasIII antibody to mark the TVM (cyan). Loss of Tey nuclear stain was observed in
GFP-positive regions that undergo apoptosis (yellow arrowhead). (C-F) Stage 13 embryos
immunostained with Tey antibody (red) and Fas-III antibody (cyan). Tey-positive nuclei
for each genotype were counted and quantified as a metric for cell survival by stage 13
(G, n=10 per genotype). (H) Schematic demonstrating relative positions of CVM (red),
TVM (cyan), and central nervous system (CNS, dark blue) in a stage 13 embryo. (I, J)
Stage 13 embryos immunostained with Tey antibody to visualize CVM and ventral muscles
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(red), GFP to visualize the CVM-specific reporter (green), and BP102 to visualize the CNS
(dark blue). In
htl hid
double mutants, CVM cells occasionally invade the CNS (J). (K)
FISH reveals temporal activation of
hid
expression (green) in the CVM marked with anti-
Tey (purple). (L) Temporal dynamics of cell division over the course of CVM migration.
Embryos expressing the HLH54F-Gap-Venus (HGV) reporter were immunostained with
GFP antibody to visualize cell membranes (red) and PH3 antibody to visualize actively
dividing cells (white). (M-O’) In
stg
mutant embryos,
hid
expression is highly delayed (N,
N’ O, O’), compared to a
stg
heterozygote (M, M’). Insets show magnified views. p<0.05,
Scale bars = 20μm. See also Figure S1.
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Figure 2. A BMP-responsive
stg
enhancer controls timing of cell division in the CVM in response
to destination-derived Dpp and cell-autonomous Tok activity.
(A) Location of ~1.5kb
stgVM
enhancer relative to
stg
locus and previously published Stark
Lab constructs (
Kvon et al., 2014
) and ChIP-seq data for Bin, Mef2, and pMAD (
Junion
et al., 2012
;
Zinzen et al., 2009
). (B) Alkaline phosphatase ISH with
lacZ
riboprobe in
stage 12 and stage 13 embryos expressing the
stgVM-lacZ
reporter construct. (C, D) FISH
with lacZ probe (green) and anti-Tey antibody staining (purple) in stage 12 WT (C) and
tkv
mutant (D) embryos. Yellow arrowheads denote CVM-specific signal. (E) Hybridization
chain reaction (HCR) staining with
dpp
riboprobes (white) and immunostaining with Tey
antibody (purple) in sequentially-staged embryos. Lateral ectoderm expression is denoted by
green arrowheads and dorsal epidermis expression is denoted by light blue arrowheads. (F)
Embryos expressing HGV reporter immunostained with anti-GFP (red), anti-FasIII (green),
and anti-pMAD (blue) antibodies. PS7 expression domain is indicated by orange arrowhead
in E and F. (G-H’) HCR staining with
tkv
(green) and
HLH54F
(purple) riboprobes in
WT stage 11 sagittal (G) and transverse (H) views, with magnified view (H’) showing
tkv
mRNA expression in the CVM. (I-J) Embryos stained with pMAD antibody, with
WT (I) showing normal PS3 (blue border) and PS7 (yellow border) expression domains.
In
org-1
OJ487
(J) embryos, PS7 expression domain is highly reduced and PS3 expression
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is missing. In
bap>dpp
(K) embryos, pMAD expression domain is expanded along the
length of the TVM. (L-N) Dorsal-view stage 11 WT,
org-1
OJ487
, and
bap-GAL4>dpp
(
bap>dpp
) embryos immunostained with anti-Tey antibody (white). (O) Quantification of
stage 11 dorsal view CVM cell number using Tey antibody. The number of Tey-positive
nuclei in both left and right sides was counted and tabulated for each genotype (n=8-9
per genotype). (P-R) Lateral-view stage 13 WT,
org-1
OJ487
, and
bap-GAL4>dpp
(
bap>dpp
)
embryos immunostained with anti-Tey antibody (white). (S) Quantification of CVM cell
number in stage 13 WT,
org-1
OJ487
, and
bap>dpp
embryos (n=10 per genotype). p<0.05,
Scale bars 20μm. See also Figure S2.
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