Developmental Cell Supplemental Information Migration of Founder Epithelial Cells Drives Proper Molar Tooth Positioning and Morphogenesis Jan Prochazka, Michaela Prochazkova, Wen Du, Frantisek Spoutil, Jolana Tureckova, Renee Hoch, Tomomi Shimogori, Radislav Sedlacek, John L. Rubenstein, Torsten Wittmann, and Ophir D. Klein
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Developmental Cell
Supplemental Information
Migration of Founder Epithelial Cells Drives
Proper Molar Tooth Positioning and Morphogenesis
Jan Prochazka, Michaela Prochazkova, Wen Du, Frantisek Spoutil, Jolana Tureckova,
Renee Hoch, Tomomi Shimogori, Radislav Sedlacek, John L. Rubenstein, Torsten
Wittmann, and Ophir D. Klein
Supplemental Figures:
Supplemental Figure 1: A narrow window of Fgf8 expression labels the future tooth
germ and is distinct from the site of Shh expression (related to Figure 1). (A-F) Lineage
tracing experiments using constitutive Fgf8ires-cre;R26RLacZ provided similar results to
experiments in which Fgf8creER was induced at approximately E11.5 (1 day after tamoxifen
injection) shown in Figure 1. The constitutive cre activity shown here provided higher
efficiency of recombination that was more suitable for live imaging experiments and
statistical analysis. (G-I) Induction of cre in Fgf8creER;R26mT/mG embryos by tamoxifen
injection at E10.75 showed exclusive labeling of the oral epithelium without any labeling in
adjacent mesenchyme at E11.5 (G), E12.5 (H) and E14.5 (G). (J-L) Induction of cre in
Fgf8creER;R26RLacZ embryos by tamoxifen injection at E11.5 (J) showed only a few labeled
cells in the E14.5 tooth germ. Injection of tamoxifen at E12.5 (K) or E13.5 (L) showed few to
no labeled cells. Asterisk labels the site where jaw joint was cut. (M-R) Comparison of clonal
growth of dental epithelium (N-P) and oral epithelium of tongue (Q). (R) Statistical plot of
clone probability density between tooth and tongue epithelia shows highly significant
difference in clonal behavior of dental and tongue epithelium. Mann-Whitney unpaired, non-
parametric test; p = 1.5x10-10. (S-Z) ShhEGFP;Fgf8LacZ co-localization during embryonic
development at E11.5 (T-V, 50 mg wet weight) and E12.5 (X-Z, 100 mg wet weight).
Supplemental Figure 2: The descendants of Fgf8-expressing cells are organized in a
transient posterior rosette structure that is sensitive to fixation (related to Figure 2). (A)
Quantification of cell shapes from Figure 2C, D shows that cells lost their elongated shape
after fixation in 4% PFA. (B, C) Images from Fgf8ires-cre;R26RConfetti embryos of large rosette
cells after fixation (B) and live (C). (D, E) Higher magnification view (400x) of central part of
large rosette shows enrichment of E-cadherin. (D) EcadCFP, (E) EcadCFP;R26RTomato. (F)
Similar view with use of LifeAct embryo showing actin enriched rosette structure
(arrowhead).
Supplemental Figure 3: The descendants of Fgf8-expressing cells are organized in a
transient posterior rosette structure that is released by intraepithelial migration during
development (related to Figure 2). (A-C) Automatic cell tracking in rosette. (A)
Segmentation of the posterior mandible into rosette population (blue) and surrounding cells
(yellow). (B) Tracks and displacement vectors for all cells. (C) Quantification of cell
migration parameters for both populations. Plots are presented with end of whiskers set at the
1.5x interquartile range above the third quartile and below the first quartile; open circles mark
Supplemental Table 1 related to quantification of data from Figure 1, Figure 3, Figure 4, Figure 5, Supplemental Figure 3, Supplemental Figure 4 and Supplemental Figure 5. Outline of table is provided below; actual table provided as .xls sheet.
Sheet name Refers to Description
Fig1_Supplemental data Figure 1w-z; Supplemental Figure 2n,o Statistical evaluation of clonal growth
Fig3_Supplemental data Figure 3p-r Formation of dental lamina after Shh and Fgf inhibition
Fig4a_Supplemental data Figure 4a-h, q Formation of dental lamina in conditional cell autonomous mutants
Fig4b_Supplemental data Figure 4i-l, r Epithelial cell migration in conditional cell autonomous mutants
Fig5_Supplemental data Figure 5 Epithelial cell migration after Shh annd Fgf inhibition
SFig3_Supplemental data Supplemental Figure 3c-e Automatic cell tracking data in rosette
SFig4a_Supplemental data Supplemental Figure 4a-g, n-p Automatic cell tracking data in rosette and other parts of mandible
SFig4b_Supplemental data Supplemental Figure 4h-j, n-p Automatic cell tracking of Sox2-positive cells
SFig4c_Supplemental data Supplemental Figure 4k-m, n-p Automatic cell tracking data in mesenchyme
SFig5a_Supplemental data Supplemental Figure 5d-g Length of dental lamina in Spry4 null embryos with and without supernumerary tooth germ
SFig5b_Supplemental data Supplemental Figure 5h-l Cell attraction to SHH soaked beads and EcadCFP intensity
Supplemental Video Legends:
Supplemental Video 1 related to Figure 1: 3D reconstruction generated in Imaris from Scale cleared K14-GFP-actin control and Fgf8creER;R26RDTA embryo.
Supplemental Video 2 related to Figure 2: (a) 14 hour time-lapse imaging of rosette
structure in Fgf8ires-cre;R26RConfetti embryonic mandible showing rosette release followed by
oriented cell movement. (b) 30 hour time-lapse imaging of control embryonic mandible in
Fgf8ires-cre;R26RConfetti embryo (YFP channel) showing cell migration within oral epithelium.
Most of the cell movement originates in the rosette region, and all cell movement is oriented
towards the dental lamina site. (c, d) Higher magnification time-lapse imaging of individual
cell clusters from Fgf8 expressing area show membrane dynamics and protrusion formation
during migration. (e) 48 hour time-lapse imaging of embryonic mandible in Fgf8ires-
cre;R26mT/mG embryo showing cell migration within oral epithelium. Most of the cell
movement originates in the rosette region, and all cell movement is oriented towards the
dental lamina site, where a strong contraction is visible. Asterisk indicates center of the
rosette.
Supplemental Video 3 related to Figure 2: 14 hour time-lapse imaging of embryonic
mandible in Sox2creER;R26mT/mG embryonic mandible showing non-directed, non-oriented cell
movement.
Supplemental Video 4 related to Figure 4 and Figure 5: 36 hour time-lapse imaging of
embryonic mandible showing cell migration within oral epithelium in (a) Fgf8creER;R26mT/mG
control embryo, (b) Fgf8creER/flox;R26mT/mG, (c) Fgf8creER;R26mT/mG;Smoflox/flox and (d)
Fgf8creER;R26mT/mG; R26RSmoM2 embryos. (e) 30 hour time-lapse imaging in YFP channel of
Fgf8ires-cre;R26RConfetti embryo after cyclopamine and SU5402 treatment. (f) Cells from
cyclopamine treated mandible show a disruption in oriented cell migration within the oral
epithelium. Cells from SU5402 treated mandible show a decrease of cell migration, with most
cells remaining in the rosette region. Asterisk indicates center of the rosette.
Supplemental Material and Methods:
Mouse lines
The following transgenic mouse strains were used: Fgf8LacZ (MGI: 3612999) (Grieshammer et
For histological analysis of LacZ expression, samples were fixed in Mirsky’s fixative
(National Diagnostics) overnight and subsequently stained in X-Gal solution. Stained samples
were post-fixed in 4% PFA and imaged on a Leica MZ 16F stereoscope with Spot 2.3.1
camera (Diagnostic Instruments). Samples were embedded in paraffin, cut in 10 µm sections,
and counter-stained with nuclear fast red (Sigma). Imaging of histological sections was done
with a Leica DM5000 microscope. Three embryos at each stage were analyzed. To analyze
3D morphology of dental epithelium, samples were fixed for 1 hour in 4% PFA and then
cleared in scaleA2 solution for two weeks as described in (Hama et al., 2011). Imaging was
done with an inverted Leica SP5 LSM, optical sections were generated every 4 µm, and 3D
reconstructions were obtained using Imaris software (Bitplane) A minimum of 4 samples was
processed for 3D analysis and quantification of each stage and genotype. In situ hybridization
was done according to standard protocols. To generate digoxigenin labeled riboprobes,
plasmids containing Shh, Fgfr2, Etv4, Bmp4, Wnt10b, Eda, Pitx2 and Msx1 sequences were
used for in vitro transcription. A minimum of 3 independent samples was processed for in situ
hybridization. Cell proliferation was analyzed by adding an EdU pulse into organ cultures (10
µM final concentration) (Life technologies) and co-staining with TUNEL kit (Roche)
according to manufacture protocol.
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