Loss of canonical WNT signaling in the AER after attenuation of FGFR2b-ligands signaling.

<p>(<b>A,B</b>) <i>Topgal</i>, a WNT signaling reporter shows strong expression in the AER of both fore- and hind-limbs of the control embryos at E11. (<b>C,F</b>) <i>Topgal</i> expression is mostly lost in the AER of DTG embryos 1 hour (C,D) and 4 hrs (E,F) after Dox-IP injection at E11. (<b>G–H</b>) IF for the activated form of β-catenin in control (G) and DTG one hour after Dox-IP at E11 (H). Note the strong reduction in β-catenin positive cells in the AER and in the adjacent mesenchyme confirming the <i>Topgal</i> results. (<b>I</b>) Quantification of G and H. Bars represent the mean ± s.e.m. of at least 5 independent samples of each. Mann-Whitney non-parametric test was performed; *p≤0.05. (<b>J,K</b>) WMISH for <i>Dkk1</i> indicating robust up-regulation of the WNT inhibitor 1 hour after Dox-IP (K) compared to the control limbs (J). Insets in J and K are corresponding vibratome cross sections through the limb focusing on the AER. (B,D,F are high magnification of A,C,E respectively). (<b>L–P</b>) Quantification by qRT-PCR of <i>Dkk1</i> (L), <i>Wnt3</i> (M), <i>Wnt3a</i> (N), <i>Axin2</i> (O) and <i>Fgf10</i> (P) at different time points after single Dox-IP injection at E11. h, hindimb; f, forelimb. Scale bar A,C: 170 µm; B,D,H,I: 50 µm; E,F: 50 µm.</p

Cell adhesion and cell death are reduced in the AER after attenuation of FGFR2b-ligands signaling.

<p>Histology (<b>A, D, G</b>) and TEM (<b>B, C, E, F, H, I</b>) images of the AER at E11, E11+ 1 hr and E11+ 2 hrs post Dox-IP. (D) Note that at 1 hr the AER is spreading and no longer a compact pseudostratified epithelium like in the control (A). SEM analysis does not indicate major changes except for irregularly shaped nuclei. (G–I) after 2 hrs Dox-IP, the AER seemingly reformed as a compact structure (G) but SEM analysis indicated that the most superficial layer, the periderm is missing. (<b>J–R</b>) Cell-cell adhesion was tested by IF for β1-integrin (J,M,P), P63 (K,N,Q) and E-Cadherin (L,O,R) expression. β1-integrin expression is reduced in DTG-AER 1 and 2 hours after Dox-IP (<b>M, P</b>) in comparison to the control AER at E11 (<b>J</b>). P63 expression is reduced in DTG-AER 1 hours after Dox-IP (<b>N</b>) compared to the control AER at E11 (<b>K</b>). No significant difference is observed at 2 hours after Dox-IP (<b>Q</b>) compared to the control AER. E-cadherin expression is reduced in DTG-AER 1 and 2 hours after Dox-IP (<b>O,R</b>) in comparison to the control AER at E11 (<b>L</b>). (<b>U–W</b>) TUNEL staining for control AER at E10.5 (<b>U</b>), DTG-AER 1 hour after Dox-IP (<b>V</b>) and 2 hours after Dox-IP (<b>W</b>) demonstrate reduction in cell death at 1 hour after Dox-IP in the DTG-AER. (<b>S and T</b>) Quantification by q-PCR of <i>β1-integrin</i> and <i>P63</i> expression. Scale bar A,D,G: 25 µm; B,E,H: 5 µm; C,F,I: 2 µm; J–R: 20 µm; U–W: 20 µm.</p

Signaling induced by FGFR2b-ligands interactionplays a critical function to control the amplification of the mesenchymal progenitors throughout limb development.

<p>(<b>A</b>) FGFR2b-ligands signaling plays a major function in the amplification of the mesenchymal cells that will give rise to the stylopod prior to limb bud induction. Transient inhibition of FGFR2b-ligands signaling at E8.5 is compatible with limb induction while inhibition at E9.5, 10.5 and E11.5 leads to the irreversible loss of the AER. (<b>B</b>) Consequences of Dox-IP at E8.5. In double heterozygous embryos, the mesenchymal progenitors that are specified to give rise to the stylopod are not amplified but the AER is induced at E9.5 allowing the formation and amplification of the mesenchymal progenitors for the <i>zeugopod</i> and autopod. Note that in our model the efficiency of the AER may not be the same as in WT as progenitors for Z and A are not amplified at the same rate leading to corresponding skeletal defects. In double homozygous embryos, the stylopod progenitors are not amplified and the AER is not functional (dotted line) leading to limb agenesis. (<b>C</b>) Consequences of Dox-IP at E10/E10.5. In double heterozygous embryos, the AER is lightly affected at E10.5 (dotted lined with big distances between gaps) the progenitors for the autopod are partially amplified leading to shorter digits. In <i>[R26^rtTA/+; Tg/Tg]</i> embryos, the AER is mildly affected at E10.5 (dotted line with smaller distance between gaps) the progenitors for the autopod are not amplified at all leading to absence of autopod. In double homozygous embryos, the AER is no longer functional at E10.5 (dotted line) and leads to absence of amplification of the mesenchymal progenitors for the zeugopod and autopod). (<b>C</b>) Schematic representation of the AER showing the superficial layer of periderm cells covering the compact AER cells expressing FGFR2b. FGFR2b ligand, FGF10 is expressed in the underlying mesenchyme. (<b>D</b>) FGFR2b signaling in the AER cells allows β-catenin stabilization. Such stabilization downstream of FGFR2b signaling, allows β-catenin function in cell adhesion and in signaling, <i>Fgf8</i>, <i>Dkk1</i> and <i>Wnt3</i> are known downstream targets.</p

Dynamics of soluble <i>Fgfr2b</i> expression and impact on AER maintenance after a single Dox-IP injection.

<p>(<b>A</b>) Embryos were collected at 0.5, 1,2,4,6,12 and 24 hrs after Dox IP at E11. (<b>B</b>) Schematic of the <i>soluble Fgfr2b</i> structure indicating the position of the specific primers P1 and P2 used to detect <i>sFgfr2b</i> expression. (<b>C</b>) Quantification of <i>soluble Fgfr2b</i> by qRT-PCR indicating a peak of expression at 6 hrs and a steep decrease at 24 hrs. (<b>D</b>) Analysis of the AER at these different time points showing a progressive diseappearance of the AER. (<b>E</b>) Western blot from whole E11 embryos exposed to Dox at different time points. Significant decrease in P-ERK levels is observed after 4 hrs. (<b>F</b>) Quantification of the P-ERK/Total ERK ratio at the different time points. (<b>G</b>) BEK (FGFR2) expression by IHC indicating that FGFR2 is still expressed in the rudimentary AER at 24 hrs post Dox-IP. (<b>H</b>) qRT-PCR for endogenous <i>Fgfr2b</i> expression supporting the IHC results. Scale bar D: 50 µm; G-upper panels: 50 µm; G-lower panels: 25 µm.</p

Attenuation of FGFR2b-ligands signaling leads to cellular disorganization of β-catenin.

<p>(<b>A</b>) Western blot of total β-catenin using protein extract from whole embryo isolated at different time-points after a single Dox-IP injection at E11. (<b>B</b>) Quantification of the western blot shown in A. (<b>C–E</b>) IF for β-catenin in the AER of E11 forelimbs at 0 hr, (C), 1 hr (D) and 6 hrs (E) after Dox-IP injection. (<b>F–H</b>) Schematic representation of the individual cells corresponding to the images shown in (C–E). (<b>I–K</b>) Quantification of total β-catenin in the AER (I), total β-catenin at the plasma membrane (J) and total β-catenin in the cytoplasmic compartment (K). Scale bars: A,C,E: 500 µm; B,D,F,F,J: 300 µm; C–E: 20 µm.</p

FGFR2b-ligands signaling controls cell proliferation in the mesenchyme of the limb bud.

<p>(<b>A–D</b>) Phosphohistone H3 and E-cadherin double IF staining in control E11.5 (<b>A,B</b>) and DTG [<i>R26^rtTA/rtTA; Tg/Tg</i>] (<b>C,D</b>) limb bud exposed to Dox-IP at E10.5 and analyzed at E11.5 demonstrate significant reduction in cell proliferation of both the AER and the adjacent mesenchyme. (<b>E–H</b>) Caspase 3 IF staining for cell death in control limb bud at E11.5 (<b>E,F</b>) and DTG (<b>G,H</b>) limb buds exposed to Dox-IP at E10.5 and analyzed at E11.5 display significant decrease in apoptosis of the rudimentary AER, there is no change in the apoptosis of the adjacent mesenchyme. (Adj mesenchyme = Adjacent mesenchyme). (<b>I</b>) Quantification of PHH3 positive cells. (<b>J</b>) Quantification of caspase 3 positive cells. Bars represent the mean ± s.e.m. of at least 5 independent samples of each. Mann-whitney non-parametric test was performed. *p≤0.05. Scale bar A–H: 50 µm.</p

Signaling induced by FGFR2b-ligands interactioncontrols progressive limb growth along the proximal-distal axis.

<p>Pregnant females carrying [<i>R26^rtTA/+;Tg/+</i>] double transgenic (DTG) embryos and single transgenic [<i>R26^rtTA/+ or Tg/+</i>] control embryos were treated continuously with Doxycycline food starting at different developmental stages; (<b>A,B</b>) Treatment at E8.5, before limb induction: loss of both hindlimbs and forelimbs in E13.5 DTG embryos. (<b>C,D</b>) Treatment at E10.5, after limb bud induction: Formation of rudimentary forelimbs and almost complete absence of hindlimbs in E14.5 DTG embryos. (<b>E–F</b>) Treatment at E11.5: Absence of autopod in both hindlimbs and forelimbs of E13.5 DTG embryos. (<b>G</b>) Dissected hindlimbs in DTG and controls shown in (E,F). (<b>H–I</b>) Treatment at E13.0: control (H) and DTG (I) embryos at E16. Note that the <i>Topgal</i> allele was introduced in DTG and control embryos to visualize the extent of mesenchymal condensation in the limb. (J,K) Dissected left hindlimbs from embryos shown in H and I displaying failure of separation of the digits in DTG hindlimb. (<b>L–O</b>) Treatment at E13.5: truncation of the digits in both forelimbs and hindlimbs. (<b>P–S</b>) Alcian blue/alizarin red staining indicates the reduction in the size of the P3 phalange in the forelimb and complete loss of the P3 phalange in the hindlimb of DTG embryos treated from E13.5 to E16.5. d, digits; p, phalanges.</p

Transient attenuation of FGFR2b-ligands signaling, before and after limb bud induction, affects different skeletal elements along the A/P axis.

<p>Alcian blue/alizarin red staining of embryos was used to visualize the bone versus cartilage respectively. Images of E18.5 embryos which were exposed to Dox at E8.5 (<b>A–D</b>), E9.5 (<b>E–F</b>), E10.5 (<b>G,H</b>) and E11.5 (<b>I–J</b>). Hindlimbs demonstrate more drastic defects than forelimbs. Also, homozygous vs. heterozygous embryos and right vs. left limbs are differently affected.</p

Fate of the mesenchymal progenitors upon FGFR2b-ligands inactivation.

<p>(<b>A–C</b>) Quantification by qRT-PCR of <i>Meis1</i>, <i>Hoxa11</i> and <i>Hoxa13</i> expression in the developing forelimb at different time-points after Dox injection at E11. (<b>D–U</b>) WMISH at 0 hr (D–F; J–L; P–R) and 2 hours Dox-IP (G–I; M–O; S–U) for <i>Meis1</i> (D–I), <i>Hoxa11</i> (J–O) and <i>Hoxa13</i> (P–U). Note the increase in the expression of proximal/stylopod progenitor marker <i>Meis1</i> at the expense of the distal/autopod marker <i>Hoxa13</i>. Scale bars: D,J,P,G,M,S: 500 µm; E,F,K,L,Q,R,H,I,N,O,T,U: 300 µm.</p

Expression of <i>Fgf8</i> in the AER following Dox-IP.

<p>(<b>A–F</b>) WMISH for <i>Fgf8</i> in control (<b>A,B</b>), <i>Rosa26^rtTA/rtTA; Tg/Tg</i> embryo after 1 hour Dox-IP injection (<b>C,D</b>) and 2 hours after Dox-IP injection (<b>E,F</b>). B,D,F are high magnification of A,C,E. (C,D) <i>Fgf8-AER</i> expression is decreased at 1 hour after Dox-IP injection. (inset in D) Close up examination of the limb shows that the AER is detaching. (E,F) <i>Fgf8-AER</i> is significantly expressed 2 hours after Dox-IP injection. However, note that the expression at 2 hours is still lower than the one observed in the control and still exhibits gaps in <i>Fgf8</i> expression indicating defective AER at this time point. (<b>B′–B′″,D′–D′″,F′–F′″</b>) SEM images of control (<b>B′–B″</b>), 1 hour Dox-IP (<b>D′–D″</b>) and 2 hours Dox-IP (<b>F′–F″</b>) embryos showing that the AER is still present at 1 and 2 hrs after Dox-IP. (<b>G,H</b>) <i>Fgf8</i> expression at 24 hrs post Dox-IP showing complete absence of <i>Fgf8</i> expression in the hindlimb and a residual expression in the forelimb. (K) Quantification of <i>Fgf8</i> expression by qRT-PCR in the forelimbs at different times post Dox-IP. Note that <i>Fgf8</i> expression is still present at 1 hr post Dox-IP supporting the hypothesis that the lack of <i>Fgf8</i> expression at 1 Dox-IP by WMISH is due to the physical loss of the AER. Scale bar A,C,E,G,I: 500 µm; B,D,F,H,J: 300 µm; B′,D′,F′: 100 µm; B″,D″,F″: 50 µm; B′″,D′″,F′″: 10 µm.</p