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Supplementary Materials for
Vertebrate Limb Bud Formation Is Initiated by Localized Epithelial-toMesenchymal Transition
Jerome Gros and Clifford J. Tabin*
*Corresponding author. E-mail: [email protected]
Published 14 March 2014, Science 343, 1253 (2014)
DOI: 10.1126/science.1248228
This PDF file includes:
Materials and Methods
Figs. S1 to S6
References
Materials and Methods
Immunostaining
Embryos were embedded in 7.5% gelatin / 15% sucrose and sectioned using a Leica
CM3000 cryostat. Sections were then incubated with primary antibodies in (PBS/BSA
0.2%, Triton 0,1% / SDS 0,02%) overnight, washed 2x10min in PBS and incubated for
30min in (PBS/BSA 0.2%, Triton 0,1% / SDS 0,02%) with secondary antibodies.
Antibodies used were anti β−catenin (BD BioSciences), aPkc (Santa cruz), laminin
(Sigma), Brdu (BD BioSciences) and Phalloidin (Invitrogen), Vimentin (Abcam), Ncadherin (BD BioSciences). Apoptosis was detected using the click-it Tunel detection kit
from Invitrogen and Proliferation by incubation of embryos with a 10-2M BrdU solution.
Cell counting was performed using ImageJ and the plugins “Colocalization” and “ITCN”
written by Pierre Bourdoncle (Institut Jacques Monod, Paris) and Thomas Kuo (UCSB).
Electroporation and expression constructs
White leghorns chicken embryos were ordered form Charles River Laboratory.
Electroporation was performed as previously described (13). Briefly, a plasmidic solution
of DNA at 1µg. µl-1 was injected into the coelomic cavity of stage 13 embryos and 3
pulses of 5ms at 50V were applied using homemade electrodes. RhoA expression vector
was kindly provided by Dr. Sheng (4); Fgf10 was cloned from a limb cDNA library and
subcloned into a pCAG-ires-GFP construct.
Mouse strains
Fgf10 (11) and Tbx5 (9) mutant strains were used in this study, as previously
published.
Fig. S1. Expression of Ncadherin, Vimentin, β-catenin and aPKC throughout the
early stages of chick limb bud formation.
Transverse sections at the forelimb level of stage 13 (A, D), 15 (B, E) and 19 (C, F).
Sections were immunostained with Ncadherin (green), Vimentin (red) in (A to C) and
aPKC (green), β−catenin (red) in (D to F). nt: neural tube; no: notochord; en: endoderm;
so: somite; im: intermediate mesoderm; ec: ectoderm; spp: splanchnopleure; sp:
somatopleure.
Fig. S2. Timing of emergence of mesenchymal cells within the somatopleure.
(A to L) Transverse sections of stage 15, 16, 17 and 18 showing the emergence of
mesenchymal cells as observed by F-actin organization (Green) and nuclear morphology
(Dapi, white) at the forelimb (A to D), trunk (E to H) and hindlimb (I to L) level. Pictures
of the stage at which mesenchymal cells were first observed for each region are framed in
red.
Fig. S3. Cellular state of the mouse somatopleure across development.
(A to C) Transverse sections at the forelimb level of E8.75 (G), E9.0 (H) and E9.25 (I).
Sections were stained with Dapi (blue), β-catenin (red) and with anti laminin antibody
(green). (D to F) Higher magnification of region from (A to D), respectively, as indicated
by dashed boxes. nt: neural tube; no: notochord; en: endoderm; so: somite; im:
intermediate mesoderm; ec: ectoderm; spp: splanchnopleure; sp: somatopleure.
Fig. S4: Changes in subcellular localization of N-cadherin, Vimentin, β-catenin and
aPKC as electroporated cells of the somatopleure undergo EMT.
(A to C) Higher magnification of Fig.1,G, H and I, focusing on the GFP electroporated
epithelial somatopleure 3h, 12h and 24h after electroporation. (D to G) Transverse
sections at the forelimb level of stage 13 chick embryo electroporated with a GFP
reporter gene, re-incubated for 3 hours (D, E) or 24 hours (F, G) and stained for GFP,
vimentin (red) and N-cadherin (blue) antibody. (H to K) Higher magnification of regions
from (D to G), as indicated by dashed boxes. Note the basal localization of Vimentin and
apical localization of N-cadherin in GFP epithelial somatopleure cells (yellow
arrowheads and white arrows respectively) and relocalization of N-cadherin and
increased Vimentin staining in GFP positive cells that have left the somatopleure
epithelium (G to H, white arrowheads).(L to O) Transverse sections at the forelimb level
of stage 13 chick embryo electroporated with a GFP reporter gene, re-incubated for 3
hours (L, M) or 24 hours (N, O) and stained for GFP (green), β-catenin (red) and aPKC
(blue) antibody. (P to S) Higher magnification of region from (L to O), as indicated by
dashed boxes. Note the specific enrichment of aPKC and β-catenin staining at the apical
end of electroporated cells (white arrows) and relocalization of β-catenin and aPKC after
cells have left the epithelium (R, S, yellow arrowheads).
Fig. S5: Rhoa overexpressing cells maintain expression of epithelial determinants.
(A to C) Transverse sections at the forelimb level of stage 15 chick embryo
electroporated with RhoA and GFP, re-incubated for 24 hours and stained for GFP
(green), β-catenin (red) and aPKC (blue) antibody. (D to F) Higher magnification of the
electroporated region from (A to C). Note the increased levels of β-catenin and aPKC
specifically in electroporated cells as cells fail to leave the epithelial somatopleure (White
arrows). (G to I) Transverse sections at the forelimb level of stage 15 chick embryo
electroporated with RhoA and GFP, re-incubated for 24 hours and stained for GFP
(green), Vimentin (red) and N-cadherin (blue) antibody. (J to L) Higher magnification of
the electroporated region from (G to I).
Fig. S6: RhoA Overexpression does not induce dramatic reduction of proliferation
or increase in apoptosis.
(A to C) Transverse sections stained with BrdU (red) at the forelimb level of RhoA/GFP
(green) electroporated side (B) and control un-electroporated side (A). (C) Magnification
of the region shown in (B). Arrowheads in in C point at numerous proliferating cells. (D
to G) Transverse sections assayed for apoptosis using TUNEL (red) at the level of the
forelimb of (E, F) of RhoA/GFP (green) electroporated (D) and WT un-electroporated
limb buds and un-electroporated embryos at the level of the flank (G). (F) is a
Magnification of the region shown in (E). No apoptosis is seen in the RhoA-expressing
cells (green) indicating that their failure to transform into mesenchymal cells and to
migrate into the limb bud is not due to cell death. A slight increase in apopotosis can be
seen in the non-RhoA transfected mesenchyme of the electroporated limb region relative
to WT limb progenitors (compare E and F). This apoptosis does not relate spatially to the
RhoA/GFP electroporated cells and resembles the pattern of apoptosis seen in the
interlimb region where mesenchyme is produced by EMT, but (as in the RhoAelectroporated limb field) no limb bud emerges and an AER is not induced
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