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Supplementary Materials for
Construction of a Vertebrate Embryo from Two Opposing Morphogen
Gradients
Peng-Fei Xu, Nathalie Houssin, Karine F. Ferri-Lagneau, Bernard Thisse, Christine
Thisse*
*Corresponding author. E-mail: [email protected]
Published 4 April 2014, Science 344, 87 (2014)
DOI: 10.1126/science.1248252
This PDF file includes:
Materials and Methods
Figs. S1 to S10
Captions for Movies S1 to S4
Full Reference List
Other Supplementary Material for this manuscript includes the following:
(available at www.sciencemag.org/content/344/6179/87/suppl/DC1)
Movies S1 to S4
Materials and Methods
Engineering of opposing BMP and Nodal morphogen gradients
Gradients of BMP and Nodal were engineered at the animal pole of blastula embryos by
injecting, as previously described (3, 4), their corresponding mRNAs into two different
animal pole blastomeres at the 128-cell stage. By diffusing from their secreting source
and by stimulating their cognate receptor, BMP and Nodal form gradients of morphogen
activity, maximal in proximity to the secreting clones and progressively decreasing
further away from their source. The Nodal signaling gradient pathway has been
generated by injecting 10 pg of Nodal-related 2 (Ndr2, cyclops) mRNA. Ndr2 has been
described to act at short range but, when stimulating animal pole cells, it activates
transcription, after the midblastula transition, of nodal-related 1 (Ndr1, squint), known
to act at long range (12). Therefore the final Nodal activity gradient results from the
activity of both Ndr2, synthesized from the injected mRNA, and Ndr1, induced as a
direct response of cells stimulated by Ndr2. We therefore use “Nodal” as a generic term
to describe the sum of Ndr1 and Ndr2 activities that stimulate animal pole cells.
The establishment of a gradient of Nodal activity at the animal pole of the
blastula/gastrula by the clone of cells derived from the blastomere that had been injected
with Nodal mRNA is demonstrated by the concentric expression pattern of Nodal
downstream target genes, with those known to require the highest level of stimulation
(endoderm and prechordal plate markers) expressed close to the secreting clone while
those induced by a lower amount (dorsal and dorso-lateral mesoderm) observed to be
expressed in more distant cells.
Stimulation of the BMP pathway was performed by injecting 100 pg of bmp2b mRNA
or 20 pg of a 1:1 mix of bmp2b and bmp7 mRNAs. The functionally active BMP
morphogen has been shown to be a bmp2b/bmp7 heterodimer (28, 29). Therefore, after
injection of the bmp2b mRNA, the morphogen forms by heterodimerization between the
BMP2b synthesized from the injected mRNA and the BMP7 translated from
endogenous mRNAs transcribed after the MBT in the cells derived from the injected
blastomere.
The optimal distance between the injected blastomeres has been experimentally
determined and found to correspond to injection into two blastomeres of the animal pole
blastula at the 128-cell stage separated by one non-injected blastomere. This condition
allows induction of complete secondary embryonic axes in more that 50% of injected
embryos. Injection in adjacent cells results in Nodal and BMP clones overlapping
extensively, resulting into the formation of partial secondary axes lacking dorsal
mesendodermal structures. Injection into blastomeres separated by two or more
uninjected blastomeres strongly reduces the frequency of complete secondary embryonic
axes that have been observed.
All experiments involving generation of Nodal and/or BMP gradients by injection of
animal pole blastomeres have been performed at least in triplicate on 150 embryos each
time. After optimal conditions have been defined, every in situ marker used has been
tested on 50 embryos for each condition and stage (reproduced 3 times).
Labeling of injected blastomeres
To track cells derived from injected blastomeres, 50 pg of mRNA coding for Green
2
Fluorescent Protein (GFP) or Red Fluorescent Protein (RFP) have been coinjected
together with Nodal and BMP mRNA. To visualize BMP secreting cells in experiments
where injected embryos were analyzed by in situ hybridization, cells were labeled by
coinjection of 50 pg of LacZ mRNA or 50 pg of GFP mRNA. Localization of βgalactosidase activity was performed as previously described (30). GFP was localized by
HRP immunohistochemistry using an anti-GFP polyclonal primary antibody made in
rabbits (ab290 from abcam) used at a dilution of 1/100,000 and an HRP Goat AntiRabbit secondary antibody used at a dilution of 1/2000 followed by detection of HRP
using the DAB peroxidase substrate kit (Vector Laboratories, Inc).
In situ hybridization
Single and double color in situ hybridization have been performed as previously
described (31). The liv1 (slc39a6) probe contains the sequence of the second exon of the
slc39a6 gene cloned into the pCR-Topo vector. All other probes have been generated
from cDNA clones inserted into the pBS vector; antisense mRNA was synthesized using
the T7 RNA polymerase. The expression pattern during embryonic development for all
molecular markers used is available at http://zfin.org.
Labeling of blastula/early gastrula embryonic margin
To differentially label the animal pole territory and the embryonic margin, 50 pg of
mRNA coding for the photoactivable fluorescent Kaede protein was injected at the onecell stage. This was followed, at the 128-cell stage, by injection of BMP and Nodal
mRNAs into two different animal pole blastomeres. At the late blastula stage, the
fluorescence emission spectrum of the Kaede protein was irreversibly converted, as
previously described (32), from green to red by irradiation of the embryonic margin with
UV light. Embryos were examined immediately after photoconversion to verify that the
fluorescence of the Kaede protein was photoconverted from green to red at the
embryonic margin. Embryos were further analyzed at 24hpf for localization of the green
and red fluorescent signals looking for the presence or absence of red labeled cells in the
secondary embryonic axis.
Explantation of animal pole and in vitro culture of explants
Using a syringe needle (27G1/2 Becton Dickinson) as a tool, the animal pole region of
early embryos injected with Nodal and/or BMP at the 128-cell stage or of uninjected
controls was cut from the blastula at the 512-cell stage in a Petri dish coated with 1.5%
agarose filled with Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12
(DMEM/F-12) Media with 25 mM Hepes, 2.5 mM L-Glutamine and supplemented with
7 mM CaCl2, 1 mM sodium pyruvate, 50 µg/ml gentamycin, 1x antibiotic-antimycotic
(Gibco) and 3% heat inactivated fetal bovine serum (FBS). Animal pole explants
corresponding roughly to 1/3 of the blastula were allowed to form a sphere in this dish
for a few minutes and then placed in another Petri dish containing fresh culture medium
and incubated at 28.5°C.
Fusion of two animal pole explants is obtained by apposing the two freshly cut surfaces
(as shown in fig. S10A). After a few minutes, the two pieces of tissue form a single mass
of cells which is placed in another Petri dish with fresh culture medium and incubated at
28.5°C.
3
Time-lapse imaging
For time-lapse imaging, live embryos or embryoids were placed in a glass bottom
culture dish (MatTek corp.) properly oriented in one drop of 0.1% agarose /Danieau
buffer 0.3x, then covered with a solution of Agarose 0.1% (supplemented with
DMEM/F12 – 3% FBS and 7 mM CaCl2 for embryoids). A coverslip was used to seal
the preparation. Using a Leica TCS LSI confocal macroscope and the LAS AF (Leica
Application Suite Advanced Fluorescence) software, time-lapse movies were made of zstack captured every 5 minutes in bright field, green channel (for GFP) and red channel
(for RFP). Z-slices were acquired with a z-interval of 10 µm.
Supplementary references
28
29
30
31
32
B. Schmid et al. Equivalent genetic roles for bmp7/snailhouse and bmp2b/swirl in
dorsoventral pattern formation. Development 127, 957-967 (2000).
S. C. Little, M. C. Mullins. Bone morphogenetic protein heterodimers assemble
heteromeric type I receptor complexes to pattern the dorsoventral axis Nat Cell
Biol 11, 637-643 (2009).
M. Fürthauer, C. Thisse, B. Thisse. A role for FGF-8 in the dorsoventral patterning
of the zebrafish gastrula. Development 124, 4253-4264 (1997).
C. Thisse, B. Thisse. High resolution in situ hybridization on whole-mount
zebrafish embryo. Nat Protoc 3, 59-69 (2008).
K. Hatta, H. Tsujii, T. Omura. Cell tracking using a photoconvertible fluorescent
protein. Nat Protoc 1, 960-967 (2006).
4
Fig. S1
Organizing a vertebrate embryo with gradients of BMP and Nodal activity (A)
Spemann-Mangold grafting experiment (1), illustrated in the middle panel for a
zebrafish embryo, or gain of function of ventral morphogen antagonists at the ventral
margin results in the formation of a mirror image duplication (right panel) of the
gradient of ventralizing factors present in wild-type embryo (left panel). (B) The
BMP/Nodal ratio of activity varies continuously from ventral to dorsal along the
embryonic margin in the wild-type embryo (left panel). As previously reported (8),
stimulation of animal pole cells with a given ratio of BMP and Nodal or a graft onto the
animal pole of a corresponding domain of the embryonic margin induces different parts
of the embryonic axis. A high ratio or a graft of ventral margin organizes a tail; a low
ratio or graft of the dorso-lateral margin organizes a posterior head while a moderate
ratio or a graft of lateral margin organizes trunk structures. Stimulation of the animal
pole by Nodal or a graft of the dorsal margin induces formation of axial mesendodermal
tissues. (C) Engineering two opposing gradients of BMP and Nodal at the animal pole
generated by clones secreting these factors recapitulates, at the animal pole, the
continuous variation of BMP/Nodal activity ratios present at the wild-type embryonic
margin and generates conditions sufficient for the organization of a complete secondary
embryonic axis.
5
Fig. S2
The induced secondary embryonic axis is built from animal pole cells.
(A) Embryo injected at the one cell-stage with Kaede mRNA and at the 128-cell stage
with BMP and Nodal mRNAs into two different blastomeres and subsequently observed
at late blastula. ap: animal pole, m: embryonic margin. (B, C) At late blastula, Kaede
fluorescence is converted from green to red by UV irradiation of the embryonic margin.
(B) Red channel, (C) Merge of red and green channels. (D to G) Same embryo at 24 hpf
showing (D) primary (i) and secondary (ii) embryonic axes observed for fluorescence of
(E) unmodified Kaede protein (green channel) or (F) photoconverted Kaede protein (red
channel). (G) Merge of green and red channels. The secondary embryonic axis is devoid
of red-labeled cells and is built only with green-labeled cells originating from the animal
pole.
6
Fig. S3
BMP, by itself, does not induce formation of ectopic embryonic structures at the
animal pole.
(A, B) Clones derived from one animal pole blastomere injected with both BMP2b and
GFP mRNAs (A) spread in the ectoderm at gastrulation, (B) populate the epidermis at
24hpf without affecting embryo morphology or (C) stay at the animal pole at
gastrulation, (D) lead to dorsal expansion of the epidermis and decrease of anterior
neural tissues at 24hpf. (E, F) wild-type embryos at (E) gastrulation and (F) 24 hpf.
Embryos are shown in lateral view.
7
Fig. S4
Expression of Nodal stimulates generation of an attracting center at the animal
pole of the blastula/gastrula.
Left panels: wild-type embryos at blastula (40% epiboly) or gastrula (shield, 60%
epiboly) stages displaying two fluorescent clones of cells derived from two blastomeres
injected at the 128-cell stage with RFP or GFP mRNA that spreads within the ectoderm
during the gastrula stage. Right panels: embryos at the same developmental stages
injected at the 128-cell stage with GFP mRNA in two blastomeres (green cells) on both
sides of a central blastomere injected with a combination of Nodal and RFP mRNAs
(red cells). Stimulation by Nodal induces an attracting center, toward which surrounding
cells converge.
8
Fig. S5
Stimulation of the animal pole by Nodal induces expression of dorsal and dorsolateral marginal genes.
Stimulation of the animal pole by Nodal (+ Nodal) induces expression of dorsal specific
genes such as (A) goosecoid (gsc) and (B) chordin (chd) at the dome stage. At the
beginning of gastrulation, the expression patterns of genes induced by Nodal are
organized in concentric circles. Expression of the panmesodermal marker notail (ntl)
forms a ring (C). Double color in situ hybridizations reveals that genes requiring the
highest level of Nodal activity such as sox32 (endodermal marker, D) and frzb
(prechordal plate marker, E), are expressed in the center of the ntl expressing ring
(labelled in red). Expression of the notochordal marker sonic hedgehog a (shha) is
induced in the inner part of the ntl expressing ring (F) while wnt8a, which is excluded
from dorsal but is present in dorso-lateral marginal cells is induced in the outer part of
the ntl expression domain (G). Nodal does not induce expression of lateral and ventral
genes such as eve1 (H). At late gastrulation, the ectoderm present around the growing
notochord expressing ntl (red) is of dorsal identity and expresses the hindbrain marker
gbx1 (I). The embryos in the upper panels have been injected with Nodal RNA in one
animal pole blastomere and embryos in the lower panels correspond to the expression of
the genes indicated in the low left corner in wild-type (WT) embryos. Developmental
stages are indicated at the bottom. The red dashed line indicates the domain of the
dorso/dorso-lateral embryonic margin, which is induced when Nodal stimulates the
animal pole. Embryos are shown in animal pole view, dorsal to the right. Scale bar in
(A) 100 µm except for upper panels (C to G) 35 µm.
9
Fig. S6
Stimulation of the animal pole by Nodal and BMP induces lateral and ventral cell
identity.
(A, B) Expression of the paraxial mesoderm marker myf5 demonstrates the presence of
dorso-lateral mesoderm in the structure induced by stimulation of animal pole cells by
Nodal and BMP. (A) Animal pole view. BMP secreting cells are visualized using LacZ
staining (light blue). (B) Optical cross-section of the structure growing at the animal
pole of the embryo shown in (A). (C) Expression of myf5 in wild-type (WT) embryo.
Embryo is in vegetal pole view, dorsal to the right. pm: paraxial mesoderm. (D)
Expression of the epidermal (ventral ectoderm) marker foxi1 at late gastrula stage in an
embryo injected with Nodal and BMP and for which, at the onset of gastrulation, the
BMP secreting clone was located on the dorsal part of the animal pole. ep(i): epidermis
of the primary embryo, ep(ii): epidermis of the secondary embryo. (E, F) Double color
in situ hybridization at late gastrula stage (80% epiboly) with ntl (red, notochord) and
gbx1 (black, hindbrain) in an embryo injected with Nodal and BMP, with the BMP
secreting clone in the ventral part of the animal pole at the onset of gastrulation. The
interruption of the expression of gbx1 (between arrowheads in F) indicates that ventral
ectodermal identity has been induced by BMP. (F) is a high magnification of the animal
pole of the embryo shown in (E). Scale bar in (D): 200 µm (C to E), 60µm (A, B, F).
10
Fig. S7
Antero-Posterior polarity and elongation of the secondary embryonic axes induced
by Nodal or by Nodal and BMP
(A, B) Double color in situ hybridization revealing the anterior (a) - posterior (p)
polarity (gbx1, hindbrain marker being anterior and ntl, mesodermal marker being
posterior) of the primary (green arrows) and secondary (red arrows) axes induced by
Nodal (A) or by Nodal and BMP (B). (C, D) Expression of wnt8a and (E, F) of fgf8a at
gastrula stage visualized by in situ hybridization in embryos in which the animal pole
has been stimulated by Nodal. Triangles illustrate the action on the antero (a)-posterior
(p) polarity of Wnt8a and FGF8a, which act as morphogens, from the embryonic margin
(green) and from the blastopore lip induced by Nodal (red). (G) Stimulated solely by
Nodal, the induced radially symmetrical structure extends out of the animal pole (arrow)
parallel to the animal-vegetal axis of the egg (doted line). (H) In the presence of Nodal
and BMP signaling centers, at late gastula stage, the prechordal plate (pp) expressing
frzb and the notochord (n) expressing ntl migrate away from the BMP secreting cells.
ppi: prechordal plate of the primary axis. This results in a rotation of the a-p axis of the
extending structure of about 45° relative to the animal-vegetal axis (blue arrow).
Consequently, when the induced embryonic axis elongates posteriorly (arrow), it
extends in the direction of the BMP secreting center, which becomes located in its
ventral-posterior position. Scale bar in (G): 250 µm (G, H), 200 µm (A to F).
11
Fig. S8
Correlation between the relative position of the BMP and Nodal secreting clones
and the orientation of the secondary embryonic axis induced at the animal pole.
(A, B) When (A) the orientation of the vector between the Nodal and the BMP secreting
clones (yellow arrow) is parallel to the D-V axis (white arrow) of the embryonic margin,
where Nodal is strong dorsally and BMP strong ventrally, (B) the primary (i, blue arrow)
and the secondary (ii, red arrow) axes are parallel. (C, D) When (C) the Nodal-BMP
vector is perpendicular to the D-V axis, the secondary embryonic axis grows (D)
perpendicular to the primary axis. (E, F), When (E) the Nodal-BMP vector is
antiparallel to the D-V axis, primary and secondary axes grow (F) in opposite directions.
sh: embryonic shield, (A, C, E) animal pole views at the shield stage, dorsal to the right,
(B, D, F) lateral views at 30hpf.
12
Fig. S9
Characterization of animal pole explants of early zebrafish blastulae. Gene
expression analysis by in situ hybridization at gastrula stage in uninjected animal pole
explants for the Nodal (ndr2, lft1); FGF (fgf8, spry4), Wnt (wnt8a, sp5l), retinoic acid,
(aldh1a2), non canonical Wnt (wnt11) and BMP (bmp2b, bmp4, bmp7a, ved, szl, bambi)
signalling pathways, as well as the dorsal BMP superfamily member admp, an epidermal
marker (foxi1), neural plate markers (zic1, pax2a), an endoderm marker (sox32), a
notochord marker (flh), a muscle marker (msgn1), a ventral marginal marker (eve1), a
dorsal marginal marker (gsc) and a prechordal plate marker (hgg1). Altogether, explants
do not express ligands or downstream targets of the Nodal, Wnt or FGF pathways,
which are activated at the early embryonic margin. Similarly, expression of aldh1a2, an
enzyme involved in retinoic acid biosynthesis, as well as expression of molecular
markers of endoderm, mesoderm and dorsal ectoderm are absent. Expression of genes
coding for BMP as well as expression of BMP target genes is observed in all cells of the
explants, which have a ventral ectodermal identity and express epidermal markers such
as foxi1. Explants have been fixed for analysis by in situ hybridization when their
siblings have reached the midgastrula stage. Scale bars: 200 µm.
13
Fig. S10
Formation of embryoids from two fused animal pole explants. (A) Fusion of two
animal pole explants derived from an embryo injected at the 1-cell stage with
fluorescein dextran (green) and an embryo injected at the 1-cell stage with rhodamine
dextran (red) and with BMP and Nodal mRNAs into two separate blastomeres of the
animal pole (ap) at the 128-cell stage prior to isolation and fusion of the two animal
explants. This results in the formation of a single mass of cells containing two animal
pole regions for which the animal (a) – vegetal (v) axes (arrowheads) are oriented in
opposite directions. At the gastrula stage a blastopore forms in that part of the fused
explant derived from the explant injected with BMP and Nodal mRNAs (red) while a
cavity (blastocoele) is apparent in the part (green) derived from the fluorescein dextran
injected embryo. At 24 hpf, the mesendoderm of the resulting embryoid is labelled in
red and derives from the explant obtained from the embryo injected with BMP and
Nodal while the anterior central nervous system and the head epidermis is mainly
formed from cells derived from the explant obtained from the fluorescein dextran
injected embryo (green). (B) Volume of single and double explants compared to the
volume of a whole embryo at the sphere stage that was removed from the yolk. About
6,000 cells are present in a complete embryo at the sphere stage. Single explants contain
roughly 2,000 cells and double explants contain roughly 4,000 cells. (C) Size
comparison of embryoids derived from single explants or from double explants with the
size of a wild-type embryo at 24 hpf. ap: animal pole, bl: blastopore, br: brain, cns:
central nervous system e: eye, ep: epidermis, n: notochord. Scale bars: 200 µm.
14
Movie S1
Secondary embryonic axis induced by the combination of Nodal and BMP activity
gradients. Observed at 30 hpf, the secondary embryonic axis (bottom) includes a
complete head, a beating heart and shows spontaneous muscle contractions indicative of
a functional nervous system independent of muscle contractions of the primary axis (on
top). Remarkably, the antero-posterior orientation of this induced secondary axis is the
reverse of the antero-posterior orientation of the primary axis.
Movie S2
Time-lapse imaging of ectopic gastrulation induced by Nodal at the animal pole.
Embryos injected with 10 pg of Nodal mRNA and 50 pg of red fluorescent protein
(RFP) mRNA in one animal pole blastomere at the 128-cell stage and observed under
Leica TCS/LSI confocal macroscope during 4 hrs from germ ring to late gastrulation.
Nodal secreting cells (red) allow visualization of the internalization of animal pole cells
and formation of the blastopore and blastopore lip. Cells that do not internalize and
remain superficial at the animal pole are cells derived from the injected blastomere that
participate in the formation of the enveloping layer (EVL) at the early blastula stage.
Movie S3
Time-lapse imaging of ectopic gastrulation induced by Nodal and BMP at the animal
pole. Embryos injected with 10 pg of Nodal mRNA and 50 pg of RFP mRNA in one
blastomere and with 100 pg of BMP2b mRNA and 50 pg of GFP mRNA in another
blastomere of the animal pole at the 128-cell stage were observed under a Leica
TCS/LSI confocal macroscope for 3 hrs from germ ring to mid-gastrula stage. Red
labeling: Nodal secreting cells; green labeling: BMP secreting cells. Green cells close to
the Nodal signaling center are attracted by and converge toward the Nodal secreting
cells, while more distant cells are displaced vegetally and disperse in the ectoderm.
Movie S4
Time-lapse imaging of the gastrulation induced by Nodal in animal pole explants
cultured in vitro. Animal pole injected with 10 pg of Nodal mRNA and 50 pg of GFP
mRNA in one blastomere at the 128-cell stage, was explanted, placed in culture medium
and observed under a Leica TCS/LSI confocal macroscope for 4 hrs, from germ ring to
late gastrula stage.
15
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