© 2008 Nature Publishing Group http://www.nature.com/naturemethods
ARTICLES
Imaging dynamic cell-cell junctional coupling in vivo
using Trojan-LAMP
Yan-Ming Guo1,3, Shiuhwei Chen1,3, Premnath Shetty2, Genhua Zheng1, Rueyling Lin2 & Wen-hong Li1
To study the physiological regulation and function of
cell-cell gap junction communication in vivo, we developed
a bioconjugate of caged dye, named dextran-CANPE-HCC, for
imaging cell coupling in small model organisms. In vitro, the
compound was photolyzed efficiently with robust fluorescence
enhancement. Dextran-CANPE-HCC delivered into Caenorhabditis
elegans oocytes was retained in cells throughout development.
Using local uncaging, we photolyzed dextran-CANPE-HCC to
release the small HCC dye and imaged the dynamics of
intercellular dye transfer through gap junction channels, a
technique we named Trojan–local activation of molecular
fluorescent probes (LAMP). Early during embryonic development,
the pattern of cell coupling undergoes dramatic remodeling
and imaging revealed that the germ cell precursors, P2, P3
and P4, were isolated from the somatic cell communication
compartment. As dextran-CANPE-HCC is chemically and
metabolically stable, labeled worms showed very bright signal
upon photoactivation after hatching, which allowed us to
examine cell coupling in living worms noninvasively.
Intercellular communication through gap junction channels is
ubiquitous in multicellular organisms and is essential for many
vital physiological processes including heart beating, labor, secretion and neuronal communication1,2. There have been extensive
studies of the regulation of junctional coupling in cultured cells, in
heterologous expression systems such as Xenopus laevis oocytes and
in isolated or reconstituted membranes3,4. Although these studies
have generated biochemical, biophysical and cellular-biological
insights into gap junction channels, relatively few studies are
carried out in living organisms. The paucity of in vivo data is
due, at least in part, to the fact that we do not yet have a noninvasive
and sensitive assay to examine the distribution, strength and
dynamics of cell coupling in living organisms.
To better examine gap junction coupling in intact living cells or
in dissected tissues, we recently developed photouncaging and
fluorescence imaging techniques, LAMP and infrared-LAMP5,6,
which involve loading cells with a caged, membrane-permeant
and acetoxymethyl (AM) ester–containing coumarin dye called
NPE-HCCC2/AM (ref. 7). There are several challenges in adapting
LAMP to study gap junction coupling in vivo. First, cellular uptake
of dyes containing AM esters is generally poor in living organisms,
in particular for loading cells that are far away from the body
surface. Second, small organic molecules tend to slowly leak out of
cells or become compartmentalized in cellular organelles, making it
difficult to image them for a long time. Third, these probes must be
chemically and metabolically stable, and should not be toxic to the
labeled animals.
To develop a noninvasive imaging assay to study cell coupling
in vivo, we used C. elegans, which is transparent at all stages of
development and is thus ideal for optical imaging. The relatively
small number of cells (B560 cells in the first larval stage and
B1,000 in the adult), the invariant cell lineage and the capacity to
image the entire cellular architecture by serial section electron
microscopy8 are advantageous for tracking cell-cell communication
networks at all stages of this nematode’s development.
As invertebrates, C. elegans express innexins rather than
connexins9, and 25 innexin genes were identified in the C. elegans
genome sequencing project. More recently, innexin-like genes
named pannexins have been discovered in mammals10. Although
innexins or pannexins share no substantial sequence similarity
with connexins, the overall topologies of these different classes
of proteins are remarkably similar11, and these proteins have
been suggested to be important in mediating cell-cell communication. Currently, little is known about the functions of pannexins
in vivo.
We report here the development of a new class of bioconjugates
of photoactivatable dyes, dextran-CANPE-HCC, for imaging cell
coupling in living worms. After injection into the ovary of
C. elegans adult hermaphrodites, dextran-CANPE-HCC was
retained in cells throughout embryonic development and in
hatched worms, with no observable toxic effects on development.
This dye remained stable in cells and fluoresced brightly upon
photolysis. We characterized the pattern of junctional cell coupling
in developing C. elegans embryos, revealing a dramatic remodeling
of cell coupling among early blastomeres even before the 4-cell
stage, with the germ-line blastomere quickly becoming poorly
coupled to somatic cells. We could also uncage the probe in specific
cells in hatched larvae to generate bright fluorescence. This allows
us to study cell-cell communication in live worms noninvasively
with high spatiotemporal resolution.
1Departments
of Cell Biology and of Biochemistry, and 2Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd.,
Dallas, Texas 75390, USA. 3These authors contributed equally to this work. Correspondence should be addressed to W.-H.L. ([email protected]).
RECEIVED 16 APRIL; ACCEPTED 4 JULY; PUBLISHED ONLINE 1 AUGUST 2008; DOI:10.1038/NMETH.1238
NATURE METHODS | VOL.5 NO.9 | SEPTEMBER 2008 | 835
ARTICLES
worms with the dye premixed with bacteria
(Supplementary Fig. 1 online).
Photolysis
MacroWe therefore designed a new class of
MacroCage
Cage
Coumarin
+ Coumarin
molecule
molecule
bioconjugates of caged probes in which a
macromolecule such as dextran served as a
carrier for the caged probe and, notably, the
Dextran
Dextran
carrier was linked to a fluorophore indirO
ectly through the caging group (type 2
O
O
–O
O
O O
O O
Photolysis
C NH
C NH
H
NO
H
bioconjugate; Fig. 1). This design is distinct
N
+
N
O2N
N
N
COO–
COO–
from conventional bioconjugates of caged
O
O
dyes, in which a fluorophore is permanently
Dextran-HCC-NPE
Dextran-HCC
linked to a macromolecule12 (type 1 bioconjugate; Fig. 1a). In dextran-CANPEb
HCC, a coumarin dye, 7-hydroxy coumarin
Photolysis
MacroMacroCoumarin
Cage
Coumarin
Cage
+
3-carboxamide (HCC) is linked to dextran
molecule
molecule
through a bifunctional photolabile protecting group (cage), 1-(4-carbamoyl-2-nitroO
phenyl)ethyl (CANPE). Upon photolysis,
O O
O
COO–
H
–
ON
O O
O
O2N
N
the coumarin fluorophore is released from
N
–
Photolysis
COO
H
COO–
+
O
N
N
the dextran-cage conjugate, becomes fluo–
COO
O
rescent and freely diffusible, and passes
O N
O N
H
Dextran
Dextran
H
through gap junction channels. We named
this technique Trojan-LAMP.
Dextran-CANPE-HCC
HCC
We synthesized dextran-CANPE-HCC in
12 steps (Fig. 2a). Dextran-CANPE-HCC
Figure 1 | Bioconjugates of caged probes. (a,b) Schematic showing photolysis of conventional bioconjugates (type 1; a) and a new type of bioconjugate (type 2, in which coumarin is released from the
has negligible fluorescence. After complete
carrier upon photolysis; b) of caged probes (top). Examples for each type of bioconjugates used in this
photolysis at 365 nm, the fluorescence
study and their photolyzed products are also shown (bottom).
intensity increased over 260-fold with
410-nm excitation (Fig. 2b). Previously, we
RESULTS
have shown that the uncaging efficiency of NPE-caged coumarins is
A new class of bioconjugates of photoactivatable probes
extraordinarily high, about two orders of magnitude higher than
We initially applied the LAMP technique to living worms that of caged fluorescein or rhodamine7, and dextran-CANPEusing NPE-HCCC2/AM (refs. 5,7). However, we detected no HCC retains almost as high a photolytic efficiency (Fig. 2c,d). We
calculated its UV light–mediated uncaging cross-section (product
cellular loading after incubating worms with the caged dye, and
of the uncaging quantum yield (Qu) and extinction coefficient (e)
only intestinal cells and lumen were loaded when we fed the
b
a
1. HNO3, –15 °C, 95%
2. 2-Trimethylsilylethanol,
COOH EDC/HCI, pyridine, 67%
O2N
O
O
COOCH2CH2TMS
COOCH2CH2TMS
2
3
O
COO Bu
H
N
O2N
1. DIEA, CH3CN, 51%
t
N
O
HO
O
O
HO
4 steps
O
2. Tetrabutylammonium
fluoride, THF, 66%
O
H
N
OH
COOH
5
t
N(CH2COO Bu)2
O
O
O
O
O2N
O
2. TFA, CH2Cl2, 100%
O
O
O
H
N
O2N
H
N
N
9
6
Before
photolysis
3
0
420
460
440
480
500
c
4
1. TFP, EDC/HCI, DMAP, 89%
After complete
photolysis
12
Wavelength (nm)
O
O
t
COO Bu
COOH Dextran amine (40 kDa), DMSO,
triethylamine; then dialysis, 60%
COOH
O
O
N
H
Conversion (%)
O2N
Emission intensity
(a.u.)
Br
Benzoyl peroxide,
NBS, 83%
–
COO
N
COO
–
Dextran
OTFP
6
100
80
60
NPE-HCC
Dextran-CANPE-HCC
40
20
0
Dextran-CANPE-HCC
0
100
200
300
400
500
Time (s)
d
100
Figure 2 | Synthesis and characterization of dextran-CANPE-HCC. (a) (2-Trimethylsilyl)ethyl-4-ethyl-3-nitrobenzoate
(2), prepared from 4-ethylbenzoic acid, was converted to (2-trimethylsilyl)ethyl-4-(1-bromo)ethyl-3-nitrobenzoate
80
(3), which was then coupled with 7-hydroxycoumarin-3-carboxamide (4, from 7-hydroxycoumarin-3-carboxylate) to
NPE-HCC
60
Dextran-CANPE-HCC
give caged coumarin-3-carboxamide (5). Subsequent transformations provided 7-{1-[4-(2,3,5,6-tetrafluoro40
phenoxy)carbonyl-2-nitrophenyl]ethoxy}-coumarin-3-carboxamide (6), which yielded dextran-CANPE-HCC upon
0
10
20
30
conjugation. EDC, N-(3-dimethylaminopropyl)-N¢-ethylcarbodiimide; DIEA, N,N-diisopropylethylamine; THF,
Time (min)
tetrahydrofuran; DMAP, 4-(dimethylamino)pyridine; NBS, N-bromosuccinimide; TFP, 2,3,5,6-tetrafluorophenol; TMS,
trimethylsilyl. (b) Fluorescence emission spectra (excitation, 410 nm) of dextran-CANPE-HCC before and after complete photolysis. (c,d) Time course of
photoconversion of dextran-CANPE-HCC and NPE-HCC (reference compound) photolyzed at 365 nm (c) or at 740 nm by two-photon excitation (d).
Caged HCC
remaining (%)
© 2008 Nature Publishing Group http://www.nature.com/naturemethods
a
836 | VOL.5 NO.9 | SEPTEMBER 2008 | NATURE METHODS
40
ARTICLES
a
b
c2
FHCC (a.u.)
0
Ex
P1
AB
d
UV
e
P1
f
P1
1
Ex
AB
ABp
0
0
40
80
120
160
ABa
Time (s)
g
UV
FHCC (a.u.)
ABp
3
ABa
Figure 3 | Dye transfer in early embryos. (a–c) Differential interference contrast (DIC) (a) and coumarin fluorescence
2
P1
(b) images immediately after UV-light uncaging of the P1 cell in a 2-cell embryo. Time course (c) of the average HCC
1
fluorescence (FHCC) in three regions of interest (outlined in a in colors that correspond to those in c) in P1, AB and an
0
extracellular area (Ex) within the eggshell. (d–g) DIC (d), GFP–histone H2B fluorescence (e) and coumarin fluorescence (f)
80
120
0
40
images immediately after uncaging ABp in a 3-cell embryo. Dashed lines in the HCC fluorescence time course (g) are the slopes
Time (s)
indicating the initial dye intensity increase in recipient cells, representing the initial rate of dye transfer from ABp. In this
experiment, HCC fluorescence of ABp decreased little owing to saturation of the detector. Dashed yellow outlines indicate the uncaging area. Double-sided arrows
indicate the detected coumarin signal from light scattering immediately after uncaging, and single-sided arrows mark the UV-light uncaging (c,g). Scale bars, 10 mm.
at 365 nm) to be 5,000 M–1 cm–1 and its two-photon-uncaging
cross-section to be 0.5 Goeppert-Mayer (GM; 1 GM ¼
1050 cm4 s/photon) at 740 nm. (It took longer for two-photon
uncaging to be detected to an appreciable degree because we
measured the accumulated photolysis of the bulk solution, whereas
two-photon excitation only occurs at the focal point of a laser
beam.) The high uncaging efficiency makes it possible to use much
lower doses of light for photoactivation, thus minimizing photodamage. This is an important consideration for live-cell imaging,
especially in experiments involving developing embryos that are
more sensitive to phototoxicity13.
Patterns of cell coupling in early developing embryos
To introduce dextran-CANPE-HCC into C. elegans, we injected the
probe into the distal end of gonads of young hermaphrodites. We
also injected rhodamine-dextran as a fluorescent marker. The
injected dye gradually filled the gonad syncytium and was detected
in fertilized embryos in a few hours (Supplementary Fig. 2 online).
Throughout development, labeled embryos showed little coumarin
fluorescence signal, yet they displayed robust coumarin fluorescence after uncaging, confirming that dextran-CANPE-HCC is
chemically and metabolically stable in vivo.
We collected embryos from injected worms and studied the
pattern of cell coupling during early development14 (see Supplementary Fig. 3 online for a schematic of C. elegans cell lineage).
a
b
P2
c
d
e
FHCC (a.u.)
Figure 4 | Dye transfer in early 4-cell embryos.
(a–j) Analysis of embryos after local uncaging of
EMS (a–e) or ABp (f–j). DIC images (a,f; cell label
colors correspond to those of traces in e,j; scale
bars, 10 mm), GFP–histone H2B fluorescence
images (b,g) and coumarin fluorescence
images immediately after (c,h) or B30 s after
(d,i) uncaging. Dashed yellow outlines indicate
the uncaging areas. Time courses of the average
HCC fluorescence intensity of the bulk cytoplasm
(e,j). Regions of interest for each cell cover the
cytoplasm in the center of the cell in each case.
The boundary of each region was typically at least
2 mm away from the cell-cell contact. The pattern
of cell coupling is representative of five embryos
assayed in the early 4-cell stage.
In 1-cell embryos, local uncaging at one end of an embryo
generated a sudden increase in coumarin fluorescence near the
uncaging area (Supplementary Fig. 4 online). The released HCC
rapidly diffused across the entire cell and equilibrated in the cytosol
in approximately 6 s, suggesting that HCC fluorophore does not
bind strongly to cytoplasmic proteins or granules. This is an
important requirement for tracers used to track cell-cell communication, so that the rate-limiting step of cell-cell dye diffusion is
through gap junction channels.
In 2-cell stage embryos, we locally photolyzed dextran-CANPEHCC in one cell (P1, the donor cell) by directing a narrow
beam of UV light to areas away from the cell-cell interface, to
minimize UV-light exposure of neighboring cells. After
uncaging, we detected a fluorescent signal immediately in the
neighboring cell (the AB cell; Fig. 3a–c). This was also seen in
the extracellular space and is mainly caused by scattering of
coumarin fluorescence from the donor cell to the neighboring
areas. We also observed this phenomenon in embryos at other
developmental stages (Fig. 3d–g) and in cultured cells at high
confluence (data not shown), but it is more pronounced in
developing C. elegans embryos, most likely because of extensive
cell-cell contact in three dimensions and because of the presence of
yolk granules.
Apart from the scattering effect, in the majority of experiments
done at the 2-cell stage, we did not detect obvious dye transfer
ABp
UV
2
EMS
1
P2
ABp
ABa
EMS
0
0
ABa
f
g
P2
ABp
EMS
h
i
j
FHCC (a.u.)
© 2008 Nature Publishing Group http://www.nature.com/naturemethods
4
60
20
40
Time (s)
UV
ABp
2
ABa
1
P2
EMS
0
ABa
0
30
60
90
Time (s)
NATURE METHODS | VOL.5 NO.9 | SEPTEMBER 2008 | 837
ARTICLES
leakage as well as of potential indirect dye
transfer from a donor to a recipient through
ABa
an intermediate cell. This approach allowed
ABa
1
EMS
us to compare relative coupling efficiency
P2
ABp
among blastomeres and also reduced or
0
P2
120
0
40
80
eliminated variations in absolute fluoresTime (s)
cence intensities from experiment to experig
f
e ABa ABp
h
UV
ment, thus allowing comparisons between
EMS
embryos. Using this approach, the coupling
1
ABa
ABp
P2
between AB cells was 10.2 ± 3.5 times (n ¼ 6
P2
0
EMS
embryos, mean ± s.d.) stronger than the
0
20
40 60
80
coupling between ABp and P1. Even taking
Time (s)
P2
i
j
k
l
into account the cell volume difference
UV
between ABp and P1, these data still suggest
P2
1
ABp
that AB cells are selectively coupled at much
ABp
greater strength. The coupling between ABa
EMS
EMS
0
and P1 was also very weak compared with
0
40
80
120
ABa
that between AB cells (data not shown).
Time (s)
p
This selective coupling between AB cells
m
UV
n
o
ABa
persisted at the early 4-cell stage. Shortly
ABp
ABa
1
ABp
after the cytokinesis of P1, we uncaged one
P2
EMS
of its daughter cells, EMS. Coupling between
0
EMS
EMS and P2, the other daughter of P1, was
80
120
0
40
Time (s)
strong, and there was little dye transfer to
q
either ABa or ABp (Fig. 4a–e and SuppleFigure 5 | Patterns of cell coupling in late 4-cell embryos.
1
mentary
Movie 1 online). Uncaging ABp
(a–p) Dye transfer after locally uncaging ABp (a–d), EMS (e–h),
resulted in faster dye transfer to ABa
P2 (i–l) or ABa (m–p). DIC images (a,e,i,m; cell labels were
0.5
(Fig. 4f–j) than to EMS or P2.
colored to correspond to cell traces in d,h,l,p; scale bars,
10 mm), coumarin fluorescence images immediately after
As development progresses, the pattern of
uncaging (b,f,j,n; dashed yellow outlines indicate uncaging
gap
junction coupling among these 4 cells
0
areas) and B30 s after uncaging (c,g,k,o), and time courses of
underwent a dynamic reorganization.
the average HCC fluorescence intensity of the bulk cytoplasm
Toward the mid 4-cell stage, the coupling
(d,h,i,l,p). Regions of interest for each cell cover the center
between AB cells was still strong, and there
cytoplasm of the cell, with the boundary of each region typically at least 2 mm away from the cell-cell
was little dye transfer from ABp to P2 (data
contact. (q) Normalized relative coupling strengths (n ¼ 5, mean ± s.d.) between neighboring cells
not shown). However, EMS starts to estabcalculated from the initial dye transfer data after uncaging ABp or EMS. The relative coupling strength
between the AB cells was arbitrarily set to 1.
lish communication with ABp and ABa. At
the same time, the coupling between P2 and
between AB and P1 cells (Fig. 3c). Occasionally, however, there was EMS progressively weakened as embryos develop. By the late 4-cell
weak coupling between these two cells in late–2-cell embryos. In stage (characterized by the elongation of EMS, which projects
addition, there was also a gradual leakage of HCC out of cells, as cellular processes around ABa), P2 is nearly uncoupled from
indicated by the decay of P1 fluorescence and the increase in fluoreboth EMS and ABp. Uncaging either ABp or EMS (Fig. 5a–h and
scence intensity in the extracellular space (Fig. 3c), possibly mediated Supplementary Movie 2 online) resulted in very slow dye transfer
through hemichannels or nonspecific anion transporters15.
to P2, but communication between ABp and EMS, ABp and ABa,
The next round of cell division starts from the AB cell, followed and EMS and ABa was still strong. Consistent with these data,
by the P1 cell. To define the stage of cell cycle more reliably, we
uncaging P2 showed little dye transfer to ABp or EMS (Fig. 5i–l),
monitored chromosome structure by fluorescence imaging using
and uncaging ABa revealed the reciprocal strong coupling between
GFP-tagged histone H2B as the marker (strain RW10006 (ref. 16)).
AB cells and between ABa and EMS (Fig. 5m–p). We quantified the
When the AB cell finishes mitosis, the division of P1 is approaching
relative coupling strengths among these cells based on the initial
telophase (Fig. 3e). When the cytokinesis of AB cells was complete
rate of coumarin intensity increase in recipient cells (Fig. 5q).
(see Supplementary Methods online for criteria used), we waited
When the P2 cell divides, it generates the C and P3 cells, which
for at least another 30 s before uncaging one of its daughter cells,
like P2 are located at the posterior end of the developing embryo.
ABp. Subsequent imaging revealed that ABa and ABp were strongly
After C and P3 formed, we uncaged dextran-CANPE-HCC at the
coupled, and that dye transfer reached equilibrium within approxianterior end of an embryo. HCC dye diffused rapidly across the
mately 60 s. However, dye transfer between ABp and the dividing
embryo. However, when it reached the P3 cell either at interphase
P1 cell was much slower (Fig. 3g).
or undergoing mitosis, dye diffusion into P3 cell was restricted,
To compare cell coupling strength, we calculated the ratio of the suggesting that the P3 cell was weakly coupled to neighboring
initial rates of coumarin intensity increase in recipient cells somatic cells throughout its life (Supplementary Movies 3 and 4
(Fig. 3g). We designed this quantification procedure to minimize online). Notably, dye transfer to the C cell appeared to be the same
the effects of the initial light scattering, of the subsequent dye
as transfer to other somatic cells. The division of P3 generates 2
c
d
FHCC (a.u.)
b
UV
ABp
2
FHCC (a.u.)
FHCC (a.u.)
FHCC (a.u.)
EMS
838 | VOL.5 NO.9 | SEPTEMBER 2008 | NATURE METHODS
pEM
AB S
pP
EM 2
SP2
S
EM
a-
AB
AB
AB
a-
AB
p
Relative coupling
strength
© 2008 Nature Publishing Group http://www.nature.com/naturemethods
a
ARTICLES
a
b
P2
ABp
c
d
FHCC (a.u.)
cells, P4 and D, at the posterior end in a 28cell stage embryo. Photolysis of dextranCANPE-HCC at the anterior end released
HCC dye, which barely diffused into P4 and
D cells (Supplementary Movie 5 online).
UV
1
ABp
ABa
EMS
P2
EMS
Fdextran-HCC (a.u.)
© 2008 Nature Publishing Group http://www.nature.com/naturemethods
0
40
80
120
160
0
Blastomere gap junction coupling
ABa
Time (s)
mediates dye transfer
g
h 4 UV
P2
To confirm that the observed cell-cell dye
ABp
e
f
ABp
transfer is through gap junction channels
3
ABa
rather than by other mechanisms such
EMS
ABp
2
P2
as cytoplasmic bridges (which allow macroP2
1
EMS
EMS
ABa
molecules over tens of kilodaltons to
0
pass17,18), we carried out control experiABa
160
0
40
80
120
ments using 18a-glycyrrhetinic acid (aTime (s)
19
GA) , a compound that has been used to
block gap junction transmission. We did Figure 6 | Cell-cell dye transfer in developing C. elegans embryos is through gap junction channels.
not observe dye transfer in the presence of (a–d) DIC image (a) and coumarin fluorescence image immediately after (b) or B30 s after (c) uncaging
a-GA in a cell that is normally coupled to dextran-CANPE-HCC (type 2) in ABp of a late 4-cell embryo that has been treated with bleach, chitinase
and a-GA. Time course of the average HCC fluorescence intensity is plotted in d. (e–h) DIC image (e) and
neighboring blastomeres (Fig. 6a–d and
coumarin fluorescence immediately after (f) or B80 s after (g) UV-light uncaging of dextran-HCC-NPE
Supplementary Methods). In another con- (type 1) in ABp of a late 4-cell embryo. Time course of average dextran-HCC fluorescence intensity of the
trol experiment, we photolyzed dextran- bulk cytoplasm is shown in h. Dashed yellow outlines (b,f) indicate the local uncaging area. Regions of
HCC-NPE (type-1 bioconjugate) to gener- interest for each cell cover the center cytoplasm of the cell, with the boundary of each region typically at
ate dextran-HCC (10 kDa dextran; Fig. 1a least 2 mm away from the cell-cell contact. The results are representative of four uncaging experiments for
and Supplementary Fig. 5 online). We did each condition. Scale bars, 10 mm.
not expect this dextran-dye to pass through
gap junction channels because its molecular weight is above the by B11 s, in the metacarpus by B30 s and it nearly filled the
typical molecular exclusion limit (B1,500 Da) of connexin or entire pharynx in B100 s (Supplementary Fig. 6). During the
innexin channels3,20. Indeed, we did not observe any movement of experiment, the worm was alive and showed some minor movedextran-HCC to neighboring cells after a local uncaging (Fig. 6e–h), ments sporadically (Supplementary Movie 7 online).
further supporting that cell-cell transfer of free HCC in
C. elegans embryos is mediated through gap junction channels.
Combining Trojan-LAMP with two-photon excitation
To assess potential UV-light damage to living embryos during
As dextran-CANPE-HCC has high two-photon uncaging efficiency
uncaging, we exposed the whole labeled embryos to UV light (Fig. 2d), and because HCC is a good fluorophore for two-photon
for the same duration used for local uncaging. These embryos imaging6, we also applied the technique of two-photon excitation
developed normally into adults and showed no observable to monitor cell coupling in C. elegans. We photolyzed dextranCANPE-HCC near the posterior terminal bulb of the pharynx by
behavioral defects (n ¼ 10 embryos). Moreover, even when we
excitation at 740 nm. Subsequent two-photon imaging of released
increased the UV light dose tenfold, we still did not observe
any abnormality in the embryos or in the adults (n ¼ 10 embryos). HCC by 820-nm excitation showed similar dye diffusion as
UV-light uncaging experiments (Supplementary Fig. 7 online).
The treated worms showed usual morphology, moved and fed
normally, and laid many embryos. This suggests that the Integrating two-photon uncaging and imaging techniques would
be most useful in examining cell coupling in three dimensions
amount of UV light required for uncaging dextran-CANPE-HCC
is well below the UV light dose that may harm cells or perturb when UV-light uncaging alone does not provide sufficient spatial
selectivity to mark cells of interest.
embryonic development.
Imaging cell coupling in living worm larvae
In both L1 and L2 stage larvae labeled with dextran-CANPE-HCC,
global uncaging of the probe generated intense coumarin signal
throughout the worm (Supplementary Movie 6 online). To
examine dye transfer in live worms, we performed local photouncaging experiments in the pharynx, which consists of eight
groups of muscle cells, pharyngeal muscles 1–8. Neighboring
pharyngeal muscle cells are connected by gap junction channels21.
To uncage dextran-CANPE-HCC, we focused a UV light laser
(365 nm) at the posterior terminal bulb, approximately where
group 7 pharyngeal muscles reside (Supplementary Fig. 6a
online). Upon delivering laser pulses, HCC fluorescence immediately rose in the irradiated area and the dye rapidly spread toward
the anterior of pharynx. We observed HCC throughout the isthmus
DISCUSSION
To study the regulation and physiological function of cell-cell
communication in vivo, major technological advancements are
needed in at least two areas: new methods to follow the dynamics
of cell coupling in living animals and new reagents to specifically
modulate cellular junctional coupling strength in physiological
preparations. To establish functional correlation between cell coupling and physiology or behavior in living animals, these techniques need to be minimally invasive, yet capable of tracking or
altering the dynamics of gap junction coupling with high spatiotemporal resolution over the course of a physiological measurement or a behavioral task.
To address the first technical gap, we developed a new class of
bioconjugates of photoactivatable dyes, the first member of which
NATURE METHODS | VOL.5 NO.9 | SEPTEMBER 2008 | 839
ARTICLES
strength as part of their developmental
program. At 20 1C, the entire 4-cell stage
P2
only lasts for about 10 min, meaning that
P1
EMS
EMS
EMS and P2 cells maintain gap junction
EMS
coupling only for minutes in the early 4-cell
2-cell
3-cell
Early 4-cell
Mid 4-cell
Late 4-cell
stage. Transient coupling behavior has been
Figure 7 | Dynamic gap junction communication network in early developing embryos. Strong cell-cell
observed in other organisms, such as in the
communication or fast dye transfer is represented by solid arrows; weak coupling or slow dye transfer is
developing nervous systems of Daphnia25 or
represented by dashed arrows.
the leech embryo26, yet with coupling durations ranging from hours to a day. Such
is dextran-CANPE-HCC. By comparison with the traditional type I
temporary junctional communication has been proposed to have a
bioconjugates of caged fluorophores, carrier molecules of type II role in controlling neurogenesis and in the formation of neural
bioconjugates can be heavily labeled with caged dyes without
circuits27. Future application of the Trojan-LAMP assay is likely to
reveal other transient cell couplings during development. In addishowing self-quenching, thus maximizing the efficiency of probe
tion, by using multicolor imaging and two-photon uncaging and
delivery for a given amount of carrier molecules.
Cell-cell communication via gap junction channels is important imaging techniques, we can correlate the dynamics of gap junction
in the orderly development of multicellular organisms22. To study coupling with other biochemical or cellular processes in three
the developmental importance of gap junction coupling, it would dimensions, which may offer additional insights into the role of
be desirable to construct a high-resolution communication map cell coupling in development.
which marks the occurrence, the strength and the dynamics of
We were able to image uncaged dextran-CANPE-HCC in L1 and
junctional coupling with cellular resolution over the course of 2 stage larvae, likely because of its long cellular retention and its
development. Because of its limited number of cells, C. elegans is an outstanding stability in vivo. By this time, the development of several
attractive system to achieve this goal. Using dextran-CANPE-HCC
tissue systems is finished or nearly complete, so coupling dynamics
and our new Trojan-LAMP technique, we characterized the can be characterized in many functional systems in wild-type or
dynamics of dye transfer at different stages in the C. elegans embryo. mutant worms, including ones with defects in innexin expresOur experiments revealed a dynamic pattern of selective cell
sion28,29. Besides C. elegans, other small organisms such as zebrafish,
flies and several marine species are also appropriate models for
coupling during early development (Fig. 7). Strong cell coupling
is first seen between AB daughter cells and then between P1 studying gap junction coupling. As embryos of these animals only
daughter cells, so an early 4-cell embryo has two communication modestly increase in size during development, the Trojan-LAMP
compartments: one contains ABa and ABp, and the other contains technique may also be applicable to these systems. Also, in mamEMS and P2. By the late 4-cell stage, ABa, ABp and EMS form a malian systems, local electroporation30 may serve as an alternative
approach to deliver dextran-dye conjugates to cells, possibly allowstrong communication compartment that is poorly connected with
the P2 cell, the germline precursor. The weak communication ing the study of junctional cell coupling in living mammals.
In addition to photoactivatable fluorophores, the concept of type
between P2 and other somatic cells persisted during subsequent
2 bioconjugates of caged probes can be extended to cage other
cell divisions. When the embryo developed to the 28-cell stage, D
molecules such as second messengers, agonists or antagonists of
and P4 were the only two cells separated from the somatic
cellular receptors or ion channels. Macromolecular conjugates of
communication compartment.
these bioactive molecules linked through appropriate caging groups
This high-resolution map of cell-cell coupling offers much more
dynamic and detailed information than what was known previously would likewise have prolonged cellular retention time. This would
about cell communication during the early embryogenesis of allow us to noninvasively perturb cell signaling with light in living
C. elegans. In contrast to our results, another group examined animals over time. Such probes will offer new opportunities to
dye transfer in C. elegans embryos by microinjection and ionto- investigate the organization, timing, interaction and function of
various biochemical events in vivo.
phoresis of Lucifer yellow dye, and concluded that from the 4-cell
stage, all blastomeres are well coupled and that restricted dye
METHODS
diffusion does not start until P4 and D cells are born23. The failure
to identify the transient coupling domains in early embryos could Photo-uncaging and imaging dye transfer in C. elegans. Dexbe due to technical limitations of microinjection, including low tran-CANPE-HCC (3–4.5 mM of caged HCC, kept in the dark
temporal resolution or cell damage, poor quantification of dye
and protected from light during use; measured by its absortransfer kinetics or coupling strength and difficulty of reliably bance at 350 nm, using e350 nm ¼ 25,000 M–1 cm–1) and
injecting the dye into a cell at precise moments of development 4–5 mg/ml dextran-rhodamine (40 kDa; Sigma) were injected into
gonads of young hermaphrodites. Three to four hours later, we
or cell cycle. In addition, Lucifer yellow binds strongly to yolk
granules in early C. elegans embryos23,24. This complicates the collected early embryos from injected worms by cutting the worms
interpretation of dye diffusion data and diminishes the accuracy near the uterus. Labeled embryos were transferred onto a 2% agar
and sensitivity of measuring the rate of cell-cell dye transfer.
pad, covered with a coverslip and mounted on an inverted
The early separation of germ-line precursor cells from the fluorescence microscope (Axiovert 200M; Carl Zeiss). During
somatic cells may suggest a unique cytoplasmic characteristic
uncaging and imaging, we used motorized filter wheels (Ludl
important for maintaining germ-line potential. Moreover, the Electronics Products) to select excitation and emission wavelengths
dramatic remodeling of the coupling pattern during early develby passing light from a xenon lamp (75 W) through bandpass
opment suggests that embryos may rapidly adjust cellular coupling
filters (Chroma Technology). A neutral density filter was also
© 2008 Nature Publishing Group http://www.nature.com/naturemethods
AB
P1
ABp
ABp
ABa
ABa
ABp
P2
840 | VOL.5 NO.9 | SEPTEMBER 2008 | NATURE METHODS
ABa
ABp
P2
ABa
© 2008 Nature Publishing Group http://www.nature.com/naturemethods
ARTICLES
added to the light path to reduce the excitation light intensity by
B70%. The typical UV-light uncaging duration is B2 s. Bandpass
filters were chosen for UV-light uncaging (360 ± 20 nm), coumarin
imaging (excitation 425 ± 5 nm, emission 460 ± 15 nm), GFP
imaging (excitation 488 ± 7.5 nm, emission 530 ± 20 nm) and
rhodamine imaging (excitation 560 ± 20 nm, emission 615 ± 30
nm). A customized multiple-path dichroic mirror was used for the
UV-light uncaging and three-color imaging. Epifluorescence was
collected with a cooled charge-coupled device (CCD) camera
(ORCA-ER; Hamamatsu) under the control of OpenLab software
(Improvision). The coupling patterns at different stages of early
developing embryos were representative of at least 5 uncaging
experiments. Coupling between newly formed sister cells was
assayed at least 30 s after the completion of cytokinesis as described
in Supplementary Methods.
Single-cell uncaging of dextran-CANPE-HCC in living worms
was carried out with a MicroPoint laser system (Photonic Instruments) installed on the Axiovert 200M microscope, using a
nitrogen UV-light laser (NL100; Stanford Research Systems) and
a 2-(4-biphenylyl)-5-(4-t-butylphenyl)-1,3,4-oxadiazol (BPBD)
dye cell to provide laser output at 364 nm for uncaging. When
imaging worms, 1-phenoxy-2-propanol (0.1% in water) was
added to the agar pad to reduce worm movement.
Two-photon uncaging and imaging were performed on a LSM
510 imaging system (Carl Zeiss) equipped with a Chameleon-XR
laser (Coherent)6. We typically set the laser power below 20 mW at
the entrance of the scanning head as measured by a power meter
(FieldMate; PM10 sensor; Coherent). The incident laser power at
the specimen, estimated by placing the power meter just above the
objective, was about half of the value measured at the entrance of
the scan head. To uncage dextran-CANPE-HCC, we rasterscanned an area of the posterior terminal bulb at 740 nm briefly.
Then, we switched the excitation wavelength to 820 nm to image
HCC fluorescence by two-photon excitation.
Additional methods. Details of synthesis, purification and characterization of the two types of dextran conjugates, developmental
staging and blocking of dye transfer in early embryos, and
quantification of the relative cell coupling strength are available
in Supplementary Methods.
Note: Supplementary information is available on the Nature Methods website.
ACKNOWLEDGMENTS
Imaging experiments involving two-photon excitation were performed at the Live
Cell Imaging Core Facility of University of Texas Southwestern Medical Center and
directed by K. Luby-Phelps, who also provided critical comments on the
manuscript. We thank the Caenorhabditis Genetics Center for providing worm
strains and X. Wang (University of Texas Southwestern) for providing the
microinjection apparatus. We thank the Welch Foundation (I-1510) and the US
National Institutes of Health for financial support.
Published online at http://www.nature.com/naturemethods/
Reprints and permissions information is available online at
http://npg.nature.com/reprintsandpermissions/
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