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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
maximal outliers. Student T-test, with * = p<0.05; ** = p<0.01; *** = p<0.0001. (D-F)
Automatic cell tracking in posterior mandible with use of membrane-targeted GFP
(R26mT/mG). (D) Automatically recognized cells and tracks. (E) Color coded tracks. (F) Color
coded tracks merged with displacement vectors. (G-W) Effect of blebbistatin treatment on
tooth morphogenesis. (G) Cartoon showing region studied. (H, J) K14-GFP visualization of
oral epithelium in control mandible culture (H) and blebbistatin treated mandible (J).
Morphology of dental epithelium was 3D reconstructed from optical confocal sections (I, K).
Blebbistatin treated mandible (K) shows mislocalised shallow circular invagination instead of
dental lamina formation as in control (I). (L-W) Dysregulation of expression of genes after
blebbistatin treatment: Pitx2 in dental epithelium (L, R); Msx1 in dental mesenchyme (M, S);
Shh in enamel knot (N, T); Eda in dental epithelium (O, U); Wnt10b in enamel knot (P, V);
Bmp4 in enamel knot and dental mesenchyme (Q, W). Scale bars: 100 µm.
Supplemental Figure 4: Directed cell migration occurs specifically in progeny of Fgf8-
expressing cells from the rosette (related to Figure 2). (A) The mandible was segmented
into four parts (blue – rosette, green – incisor, purple – labial, red – posterior areas). All
descendants of Fgf8-expressing cells were automatically tracked (B) and average values of
cell migration parameters (track length and displacement) were plotted (C). (D-G)
Visualization of track length and displacement for cells in each part of mandible (D) in
rosette, (E) in incisor, (F) in labial, (G) in posterior. (H-J) The specificity of cell migration
properties was tested with additional cre drivers to determine if the origin of cells is important
for the migratory behavior. Sox2creER was used to label an additional epithelial population
within the embryonic mandible, and cells were automatically tracked (I). Tracks of Sox2-
derived cells showed significantly lower migration track length, displacement and straightness
in movement than Fgf8 derived cells (J). (K-M) Live imaging of mesenchymal cells adjacent
to dental epithelium. Cells show only limited movement (L), which is in a circular pattern in
the presumptive rosette area (M represents white rectangle in L). (N-P) Quantification of cell
migration parameters: (N) track length, (O) displacement, (P) straightness among cells from
different segments of the mandible (color coded), as well as Sox2 progeny and mesenchymal
cells labeled with Wnt1cre. 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
maximal outliers. Student T-test, with * = p<0.05; ** = p<0.01; *** = p<0.0001. (Q) Track
translation analysis to compare the general direction of movement of descendants of Fgf8-
expressing cells from rosette with Sox2 progeny (see also Supplemental Video 3).
Supplemental Figure 5: Descendants of Fgf8-expressing cells have features of collective
migration, can form supernumerary tooth in the diastema, and are attracted by SHH
(related to Figures 2-5). (A-D) Descendants of Fgf8-expressing cells maintain E-cadherin
expression; quantification of fluorescence intensity between neighboring cells (D) suggests
that the cell contacts between descendants of Fgf8 expressing population are enriched in E-
cadherin. X-axis in D represents fluorescence units. (E-J) Whole mandible view showing
lower magnification versions of images in Figure 3D-H, as well as of Wnt10b. (K-U)
Proliferation and apoptosis assay on cyclopamine and SU5402 treated organ cultures. (K-M)
control, (O-Q) cyclopamine, (Q-S) SU5402. (T) Quantification showing no significant
differences in proliferation in prospective dental epithelium. (U) Quantification of apoptosis
shows no significant differences in cell death within cultured dental epithelia. (V-Y) Spry4
null embryonic molars at E14.5. Compared to controls (V), in Spry4 null mice destined for a
supernumerary tooth phenotype (W), the descendants of Fgf8-expressing cells expanded
anteriorly and were localized in the supernumerary tooth primordium. (X) In Spry4 null
embryos without a supernumerary cap, descendants of Fgf8-expressing cells lineage were not
evident in the diastema. S – supernumerary cap, M1 – first molar primordium. (Y)
Quantification of the expansion of descendants from Fgf8-expressing cells along the antero-
posterior length of the dental epithelium. (Z-Zd) Descendants of Fgf8-expressing cells are
more attracted to SHH-soaked beads than control beads. Standard deviation was used for error
estimates, Student T-test * = p<0.05; ** = p<0.01; *** = p<0.0001.
Supplemental Table:
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
al., 2005), Fgf8ires-cre (MGI: 4839641) (Toyoda et al., 2010), R26RLacZ (MGI: 1861932)
(Soriano, 1999), R26mT/mG (MGI: 3716464) (Muzumdar et al., 2007), R26RConfetti (MGI:
4835542) (Snippert et al., 2010), R26RDTA (MGI: 3618991) (Wu et al., 2006), K14EGFP/Actb
(K14-GFP-Actin) (MGI: 4421514) (Vaezi et al., 2002), Ptc1LacZ (MGI: 1857447) (Goodrich
et al., 1997), Gli1LacZ (MGI: 2449767) (Bai et al., 2002), Spry4– (MGI: 3701941) (Klein et al.,
2006), R26RRFP (MGI: 3809524) (Madisen et al., 2010), ShhEGFP/cre (MGI: 3053959) (Harfe et
al., 2004), Smoflox (MGI: 2176256) (Long et al., 2001), R26RSmoM2 (MGI: 3576373) (Jeong et
al., 2004), Fgf8flox (MGI: 2150347) (Meyers et al., 1998), EcadCFP (MGI: 4838590) (Snippert
et al., 2010), Fgf8creER (Hoch et al., 2015) Wnt1cre (Danielian et al., 1998) (MGI: 2386570)
Sox2creER (Arnold et al., 2011) (MGI: 5295990), LifeAct (Riedl et al., 2010) (MGI: 4831038).
Tamoxifen was administrated to pregnant females intraperitoneally at a dose of 9 mg per 40 g
of mouse weight. All embryos were staged by embryonic day and wet body weight to
optimize comparison between litters. Representative body weights for individual stages were
as follows: E11.5 – 50 mg, E12.5 – 90-100 mg, E14.5 – 230-280 mg.
Mating and embryo harvest schemes
male female embryos harvested
Fgf8LacZ B6 wild-type (WT) Fgf8LacZ
Fgf8ires-cre R26RLacZ Fgf8ires-cre;R26RLacZ
Fgf8ires-cre R26RConfetti Fgf8ires-cre ;R26RConfetti
Fgf8ires-cre R26mT/mG Fgf8ires-cre;R26mT/mG
ShhEGFP/cre R26RConfetti ShhEGFP/cre;R26RConfetti
Fgf8creER;K14EGFP/Actb R26RDTA Fgf8creER;K14EGFP/Actb;R26RDTA Fgf8creER;K14EGFP/Actb
Fgf8creER R26RLacZ Fgf8creER;R26RLacZ
Fgf8creER R26mT/mG Fgf8creER;R26mT/mG
Fgf8LacZ;ShhEGFP/cre B6 WT Fgf8LacZ;ShhEGFP/cre
Fgf8creER R26RRFP;EcadCFP Fgf8creER;R26RRFP;EcadCFP
LifeAct B6 WT LifeAct
Fgf8creER;K14EGFP/Actb Fgf8flox Fgf8creER/flox;K14EGFP/Actb Fgf8creER;K14EGFP/Actb Fgf8creER;K14EGFP/Actb; Smoflox/+ Smoflox/flox Fgf8creER;K14EGFP/Actb;Smoflox/flox Fgf8creER ;K14EGFP/Actb;
Smoflox/+
Fgf8creER;K14EGFP/Actb R26RSmoM2 Fgf8creER;K14EGFP/Actb;R26RSmoM2 Fgf8creER;K14EGFP/Actb Spry4+/-; Fgf8creER;R26mT/mG Spry4+/-;R26mT/mG Spry4-/-;Fgf8creER;R26mT/mG Spry4+/-;
Fgf8creER;R26mT/mG Spry4+/+;Fgf8creER; R26mT/mG
Gli1LacZ B6 WT Gli1LacZ
Ptc1LacZ B6 WT Ptc1LacZ
Sox2creER R26mT/mG Sox2creER;R26mT/mG
Wnt1cre R26RConfetti Wnt1cre;R26RConfetti
Histology and in situ hybridization
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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