DEVEL
OPMENT
AL BIOL
OGY
Recons
titution
of morphogen
shuttling
circuits
Ronghui
Zhu
1
†
, Leah
A. Santa
t
1
†
, Joseph
S. Markson
1
, Nagar
ajan Nandagopal
2
, Jan Gregrowicz
1
,
Michael
B. Elowitz
1
*
Developing
tissues
form
spatial patterns
by establishing
concentr
ation gradients
of diffusible
signaling
proteins
called
morphogens.
The bone
morphogenetic
protein
(BMP)
morphogen
pathway uses
a family
of extracellular
modula
tors to reshape
signaling
gradients
by actively
“
shuttling
”
ligands
to differ
ent locations.
It has remained
unclear
what circuits
are sufficient
to enable
shuttling,
what other
patterns
they can gener
ate, and
whether
shuttling
is evolutionarily
conserv
ed. Here, using
a synthetic,
bottom-up
appr
oach, we compar
ed the spatiotem-
poral dynamics
of differ
ent extracellular
circuits.
Three proteins
—
Chordin,
Twsg,
and the BMP-1
protease
—
suc-
cessfully
displa
ced gradients
by shuttling
ligands
away from the site of production.
A mathema
tical
model
explained
the differ
ent spatial dynamics
of this and other
circuits.
Last, combining
mammalian
and
Drosophila
components
in the same
system sugges
ts that shuttling
is a conserv
ed capability
. Together,
these
results
reveal
principles
through
which
extracellular
circuits
contr
ol the spatiotempor
al dynamics
of morphogen
signaling.
Copyright
© 2023 The
Authors,
some
rights
reserved;
exclusive licensee
American
Associa
tion
for the Advancement
of Science.
No claim to
original
U.S. Government
Works. Distributed
under
a Creative
Commons
Attribution
License
4.0 (CC BY).
INTRODUCTION
In
multicellular
organisms,
long-r
ange
signaling
molecules
called
morphogens
establish
spa
tial
concentr
ation
gradients
to pattern
de-
veloping
tissues
(
1
–
4
).
The
bone
morphogenetic
protein
(BMP)
sig-
naling
pathw
ay is
broadly
conserv
ed
across
metazoans
and
functions
in nearly
every
tissue
conte
xt.
BMP
ligands
form
gradi-
ents
tha
t contr
ol the
patterning
of
early
embry
os
(
5
,
6
),
neur
al
tube
specifica
tion
(
7
),
and
limb
bud
forma
tion
(
8
),
among
many
other
processes.
BMP
gradients
need
to be
precisely
contr
olled
to
ensur
e
normal
morphogenesis.
For
example,
during
early
Xenopus
embry
o dev
elopment,
dorsal-v
entr
al BMP
gradients
scale
with
embry
o size
to ensur
e a normal
body
plan,
even
when
the
embry
o is cut
in half
(
9
–
11
).
Similarly
, in
Drosophila
,
perturba
tions
tha
t affect
the
sharpness
of orthologous
decapentaplegic
(Dpp)
morphogen
gradients
alter
dev
elopmental
outcomes
(
12
).
BMP
gradients
are shaped
by
a set
of inter
acting
extracellular
components.
Among
them,
the
inhibitor
Chordin,
a cofa
ctor
termed
Twis
ted
gas
trula
tion
(Twsg1),
and
the
BMP-1
protease
(Fig.
1A)
pla
y key roles
in shaping
BMP
ligand
distributions
(
13
,
14
).
Chordin
binds
to BMP
, preventing
it from
activa
ting
its
recep-
tors
(
15
),
and
this
inter
action
can
be
strengthened
by
Twsg1
(
16
–
19
).
Thus,
Chordin
is
mos
tly
kno
wn
as
a BMP
antagonis
t.
Ho
wever,
Chordin
has
been
pos
tula
ted
to
activ
ely
redis
tribute
BMP
ligands
across
dev
eloping
tissues,
thr
ough
a phenomenon
termed
“
shuttling
”
(
20
,
21
).
In
this
process,
Chordin
binds
to
BMP
ligands,
protecting
them
from
receptor-media
ted
internaliza-
tion
and
degr
ada
tion
(
22
).
These
Chordin-BMP
comple
xes
can
diffuse
extracellularly
. Las
t, clea
vage
of Chordin
by
BMP-1
(
23
)
can
“
r elease
”
BMP
ligands
at new sites.
Depending
on
their
expr
es-
sion
domains,
these
components
can
gener
ate shuttling
processes
tha
t either
displa
ce
signaling
gradients
away from
the
zone
in
which
the
ligand
is produced
(
10
,
24
–
29
)
or compr
ess
signaling
gra-
dients
into
regions
narr
ower than
those
in which
the
ligands
are se-
creted
(Fig.
1B)
(
20
,
22
,
30
–
32
).
Despite
much
work
char
acterizing
the
inter
actions
among
BMP
ligands
and
modula
tors,
key ques
tions
about
how the
y work
togeth-
er as a system
to shape
signaling
in spa
ce and
time
have remained
unclear.
Firs
t, are Chordin,
Twsg,
and
BMP-1
sufficient
for
ligand
shuttling?
These
components
are all
present
during
early
zebr
afish
dev
elopment.
Ho
wever,
two
recent
studies
argued
agains
t a role
for
shuttling
(
33
,
34
).
In
this
case,
it is unclear
whether
other
compo-
nents
(
26
,
27
)
or feedba
ck loops
are requir
ed
for
shuttling,
whether
shuttling
is activ
ely
suppr
essed,
or whether
the
system
is simply
not
oper
ating
in the
appr
opria
te par
ameter
regime
to enable
shuttling.
These
possibilities
are difficult
to disentangle
in the
embry
onic
conte
xt,
wher
e one
cannot
readily
isola
te the
effects
of just a few
components
among
the
larger
inter
connected
system.
Second,
wha
t is the
full
repertoir
e of beha
viors
tha
t a circuit
consis
ting
of
Chordin,
Twsg1,
and
BMP-1
can
gener
ate?
While
individual
com-
ponents
have been
perturbed
in
dev
eloping
embry
os,
it has
re-
mained
challenging
to
systema
tically
and
quantita
tively
tune
multiple
components
in combina
tions.
Third,
is shuttling
an
evolu-
tionarily
conserv
ed
fea
tur
e of
the
BMP
pathw
ay?
Biochemical
studies
indica
te tha
t protein
inter
actions
among
Chordin,
Twsg1,
and
BMP-1
are largely
conserv
ed
(
16
,
35
–
38
),
but
it remains
unclear
whether
this
leads
to functional
conserva
tion
of shuttling.
Ideally
, one
would
like to test conserva
tion
by
combining
compo-
nents
from
differ
ent
species,
something
tha
t is difficult
to do
sys-
tema
tically
in embry
os.
An
ideal
way to addr
ess
these
ques
tions
is by recons
tituting
mor-
phogen
gradients
outside
of embry
os (
39
,
40
).
In this
appr
oach,
en-
gineer
ed
cells
tha
t produce
circuit
components
or
report
on
morphogen
signaling
can
be
arr
anged
spa
tially
in a cell
cultur
e
system,
allo
wing
time-lapse
imaging
of spa
tiotempor
al gradient
dy-
namics.
This
bottom-up
appr
oach enables
one
to study
pattern
for-
ma
tion
circuits
in an
isola
ted,
quantita
tive manner
and
ther
eby
test
the
sufficiency
of specific
component
combina
tions
for
patterning
beha
viors,
rationally
engineer
novel patterns,
and
analyze
how evo-
lutionary
changes
in circuit
components
affect
patterning.
Her
e, we created
a bottom-up
gradient
recons
titution
system
to
study
the
BMP
gradient
forma
tion
process,
analyze
it in spa
ce and
time,
and
perturb
it thr
ough
systema
tic,
quantita
tive contr
ol of key
modula
tors
—
Chordin,
Twsg1,
and
BMP-1.
We found
tha
t these
1
Howard Hughes
Medical
Institute and Division
of Biology
and Biological
Engi-
neering,
California
Institute
of Technology
, Pasadena,
CA, USA.
2
Department
of
Systems Biology
, Harvard
Medical
School,
Boston, MA, USA.
*Corresponding
author.
Email:
melowitz@caltech.edu
†
These
authors
contributed
equally
to this work.
SCIENCE
ADVANCES
|
RESEARCH
ARTICLE
Zhu
et al.
,
Sci. Adv.
9
, eadf9336
(2023)
12 July 2023
1 of 14
Downloaded from https://www.science.org at California Institute of Technology on October 18, 2023
Fig. 1. Synthetic
gradient
recons
titution
enables
systematic analy
sis of BMP gradient
modula
tion.
(
A
) Chordin
canbindtoBMPligands,
andTwsg1strengthens
this
interaction. BMP-1
can cleave Chordin
(dashed
lines represent protein cleavage) in its free form orcomplex
forms and release
BMP for binding
to receptor
and signaling.
(
B
) Schema
tic data. Left: In
Drosophila
early embry
o, Dpp (
Dr osophila
BMP ortholog),
Tsg, and Tld are expressed
dorsally
. Ventrally expressed
Sog (
Dr osophila
Chordin
ortholog)
has been sugges
ted to sharpen
the BMP distribution
to a narrower region.
Tsg,
Drosophila
Twsg1 ortholog;
Tld,
Drosophila
BMP-1
ortholog.
Right:
Chordin
has
been shown to push signaling
away from the dorsal
mesoderm
after BMP4 injection
into early
Xenopus
embry
os (
10
). xTsg,
Xenopus
Twsg1 ortholog;
Xld,
Xenopus
Twsg1
ortholog.
(
C
) We reconstituted
BMP gradients
into
“
blank
slate
”
NMuMG
cellsthat express minimal
levels of most morphogen
pathway components
aside from receptors
and intracellular
signal
transducers
(table S1) and exhibit minimal
transcriptional
changes
in response
to morphogen
signaling
(
41
–
43
).
Synthetic
pathway components
were engineer
ed into these cells to generate morphogen
or modula
tor sender
and receiver cells (left). Engineer
ed senders
and receivers were cocultur
ed in different
configur
ations (middle
and right) to reconst
itute BMP gradients.
(
D
) We constructed
BMP4
and Chordin
senders
and BMP4
receivers with inducible
BMP-1
expression,
such that circuit components
BMP4,
Chordin,
and BMP-1
can be orthogonally
controlled by 4-OHT, Dox, and ABA, respectiv
ely. Note that Sender-B
and Sender-C
also
constitutively express Citrine
for initial sender
region labeling.
mChe,
mCherry;
mTurq, mTurquoise2;
Cit, Citrine.
BMPR
denotes
BMP receptors,
which
are endogenously
expressed
in NMuMG
cells (table
S1). Details
of these cell lines, and the constructs
used to build them,
are available
in tables
S2 and S3.
SCIENCE
ADVANCES
|
RESEARCH
ARTICLE
Zhu
et al.
,
Sci. Adv.
9
, eadf9336
(2023)
12 July 2023
2 of 14
Downloaded from https://www.science.org at California Institute of Technology on October 18, 2023