Quantification of gene expression patterns to reveal the origins of abnormal morphogenesis
- The Barcelona Institute for Science and Technology, Spain
- Universitat Pompeu Fabra, Spain
- European Molecular Biology Laboratory, Spain
- Universitat de Barcelona, Spain
- Universitat de les Illes Balears (UIB), Spain
- Pennsylvania State University, United States
- Institució Catalana de Recerca i Estudis Avançats, Spain
Figures
Figure 1
Quantitative size and shape comparison of forelimb bones in Fgfr2+/P253R newborn mice (P0) and unaffected littermates.
(A) Mouse skeleton at P0. 3D isosurface reconstruction of the skeleton of an unaffected littermate obtained from a high-resolution µCT scan. (B–F) Anatomical landmarks recorded on microCT scans of …
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Figure 1—source data 1
Source files for humerus shape data.
This zip archive contains the i) raw landmark coordinates registered to capture the shape of the humerus, as defined in Figure 1D; ii) the estimated centroid size and long-centroid size; iii) the Procrustes coordinates after Generalized Procrustes analysis (GPA); and iv) the PC scores resulting from the Principal Component Analysis (PCA) displayed in Figure 1H.
- https://doi.org/10.7554/eLife.36405.004
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Figure 1—source data 2
Source files for scapula shape data.
This zip archive contains the (i) raw landmark coordinates registered to capture the shape of the scapula, as defined in Figure 1E; (ii) the estimated centroid size and long-centroid size; iii) the Procrustes coordinates after Generalized Procrustes Analysis (GPA); and iv) the PC scores resulting from the Principal Component Analysis (PCA) displayed in Figure 1I.
- https://doi.org/10.7554/eLife.36405.005
Figure 2
Qualitative visualization of Dusp6 gene expression in unaffected and Fgfr2+/P253R mouse embryos at E10.5 and E11.5.
OPT scans of embryos WMISH stained for Dusp6 revealed the anatomical location of Dusp6 gene expression (shown in yellow). For each stage, the main expression domains are highlighted on the left for …
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Figure 2—source data 1
Original 2D images of the Dusp6 WMISH experiments.
This zip archive contains pictures, taken using a Leica MX16F microscope, of the right and left sides of the mouse embryos that underwent Dusp6 WMISH. Folders are organized by developmental stage and genotype.
- https://doi.org/10.7554/eLife.36405.008
Figure 3
New quantitative analysis method for 3D gene expression data, based on Geometric Morphometrics.
Mouse embryos between E10.5 and E11.5 were analyzed with WMISH to reveal the expression of Dusp6 (1), and then cleared with BABB, and OPT scanned using both fluorescence and transmission light (2). …
Figure 4
Developmental variation within litters of the Apert syndrome mouse model.
All of the limbs were individually staged using our publicly available web-based staging system (https://limbstaging.embl.es). The stage of the limb bud was estimated after alignment and shape …
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Figure 4—source data 1
Staging results.
Table containing the individual scores obtained from the staging system and used to produce the graph in Figure 4.
- https://doi.org/10.7554/eLife.36405.012
Figure 5 with 3 supplements
Tracing of limb phenotypes (anatomical and molecular) back through developmental time to the earliest moment of appearance.
Principal Component Analysis based on the Procrustes-based semi-landmark analysis was used to assess the shape of the limbs and the corresponding Dusp6 expression domains for each developmental …
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Figure 5—source data 1
Source files for limb shape data.
This zip archive contains the (i) raw landmark coordinates registered to capture the shape of the limbs and the associated Dusp6 expression domains, as defined in Figure 5—figure supplement 1; (ii) the estimated centroid size and long-centroid size of each structure; (iii) the Procrustes coordinates after Generalized Procrustes Analysis (GPA); and iv) the PC scores resulting from each of the Principal Component Analyses (PCA) displayed in Figure 5.
- https://doi.org/10.7554/eLife.36405.020
Figure 5—figure supplement 1
Anatomical landmarks recorded on the 3D reconstructions of the limbs and the Dusp6 expression domains obtained from OPT scans of Apert syndrome embryos at E10.5–E11.5.
In the limb, we manually recorded the 3D coordinates of five anatomical landmarks (A): L1–L2, the most medial points on the anterior and the posterior sides of wrist, collected along the curve that …
Figure 5—figure supplement 2
Principal Component analyses based on the Procrustes-based semi-landmark analysis of the shape of the hindlimbs and the corresponding Dusp6 expression domains for each developmental period.
Scatterplots of PC1 and PC2 axes with the corresponding percentage of total morphological variation explained are displayed for each analysis, along with morphings associated with the negative, mid …
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Figure 5—figure supplement 2—source data 1
Source files for hindlimb shape data.
This zip archive contains the i) raw landmark coordinates registered to capture the shape of the hindlimbs and the associated Dusp6 expression domains, as defined in Figure 5—figure supplement 1; ii) the estimated centroid size and long-centroid size of each structure; iii) the Procrustes coordinates after Generalized Procrustes Analysis (GPA); and iv) the PC scores resulting from each of the Principal Component Analyses (PCA) displayed in Figure 5—figure supplement 2.
- https://doi.org/10.7554/eLife.36405.017
Figure 5—figure supplement 3
Principal Component analyses based on the Procrustes-based semi-landmark analysis of the shape of the forelimbs and the corresponding Dusp6 expression domains for each developmental period.
Scatterplots of PC1 and PC2 axes with the corresponding percentage of total morphological variation explained are displayed for each analysis, along with morphings associated with the negative, mid …
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Figure 5—figure supplement 3—source data 1
Source files for forelimb shape data.
This zip archive contains the i) raw landmark coordinates registered to capture the shape of the forelimbs and the associates Dusp6 expression domains, as defined in Figure 5—figure supplement 1; ii) the estimated centroid size and long-centroid size of each structure; iii) the Procrustes coordinates after Generalized Procrustes Analysis (GPA); and iv) the PC scores resulting from each of the Principal Component Analyses (PCA) displayed in Figure 5—figure supplement 3.
- https://doi.org/10.7554/eLife.36405.019
Figure 6 with 2 supplements
Quantitative correlation between the size and shape of the limbs and the Dusp6 expression pattern.
(A–D) Comparison of limb bud size and Dusp6 volume in unaffected and Fgfr2+/P253R mutant littermates across development (Table 3). Limb size was measured as limb centroid size (A), whereas the size …
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Figure 6—source data 1
Source files for limb size and gene expression volume.
This Excel file contains tables with the individual scores for limb centroid size (µm) and Dusp6 volume (µm3) used to produce the graphs in (i) Figure 6A and Figure 6B; (ii) Figure 6C and Figure 6—figure supplement 1B,D; and (iii) Figure 6D and Figure 6—figure supplement 1A,C.
- https://doi.org/10.7554/eLife.36405.025
Figure 6—figure supplement 1
Quantitative size analyses of limb bud and Dusp6 volume in unaffected and Fgfr2+/P253R mutant littermates throughout development.
Comparison in hindlimbs (A, C) and forelimbs (B, D) at each developmental period, as defined in Table 2 and Figure 4. Limb size was measured in terms of limb centroid size (μm), whereas the size of …
Figure 6—figure supplement 2
Morphological integration between the shapes of the limb bud and the Dusp6 gene expression domain, as measured by partial least square (PLS) analysis.
Results display the morphings associated with the negative, mid and positive values of PLS1 for hindlimbs (A) and forelimbs (B), which accounted for more than 95% of the covariation between the limb …
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Figure 6—figure supplement 2—source data 1
Results of Ppartial Least Squares (PLS) analyses.
This zip archive contains the PLS scores resulting from each of the analyses displayed in Figure 6—figure supplement 2.
- https://doi.org/10.7554/eLife.36405.024
Videos
Video 1
Morphometric approach for the analysis of 3D gene expression data obtained by Optical Projection Tomography.
In this video, we show a superimposed view of a fluorescent and a transmission OPT scan of the same E11.5 mouse embryo, which was analyzed with WMISH to reveal the gene expression of Dusp6, a …
Tables
Table 1
Quantitative comparison of mean bone lengths and volumes.
Means were computed as the average lengths or volumes of the right and the left bones for Fgfr2+/P253R mutant (N = 12) and Fgfr2+/+ unaffected (N = 10) mice. Data for proximal phalanx I, middle …
| Mean bone length (mm ± SD) | Mean bone volume (mm3 ± SD) | ||||||
|---|---|---|---|---|---|---|---|
| Bone | Fgfr2+/+ | Fgfr2+/P253R | P-value | Fgfr2+/+ | Fgfr2+/P253R | P-value | |
| Autopod | Distal phalanx I | 0.11 ± 0.03 | 0.13 ± 0.02 | 0.004** | 0.0015 ± 0.0008 | 0.0015 ± 0.0008 | 0.86 |
| Distal phalanx II | 0.22 ± 0.03 | 0.21 ± 0.03 | 0.128 | 0.0021 ± 0.0007 | 0.0022 ± 0.0008 | 0.659 | |
| Middle phalanx II | 0.004 ± 0.003 | 0.01 ± 0.004 | 0.336 | 0.00002 ± 0.00002 | 0.00003 ± 0.00002 | 0.759 | |
| Proximal phalanx II | 0.15 ± 0.01 | 0.14 ± 0.01 | 0.057 | 0.0054 ± 0.0011 | 0.0053 ± 0.0014 | 0.82 | |
| Distal phalanx III | 0.26 ± 0.006 | 0.24 ± 0.01 | 0.068 | 0.0034 ± 0.0008 | 0.0033 ± 0.0017 | 0.796 | |
| Middle phalanx III | 0.10 ± 0.006 | 0.10 ± 0.006 | 0.243 | 0.0019 ± 0.0009 | 0.0022 ± 0.0013 | 0.436 | |
| Proximal phalanx III | 0.204 ± 0.02 | 0.18 ± 0.03 | 0.001** | 0.0087 ± 0.0003 | 0.0073 ± 0.0004 | 0.016* | |
| Distal phalanx IV | 0.24 ± 0.006 | 0.19 ± 0.02 | 0.061 | 0.0024 ± 0.0009 | 0.0024 ± 0.0014 | 0.923 | |
| Middle phalanx IV | 0.06 ± 0.009 | 0.07 ± 0.01 | 0.338 | 0.0009 ± 0.0002 | 0.0014 ± 0.0002 | 0.24 | |
| Proximal phalanx IV | 0.20 ± 0.01 | 0.20 ± 0.02 | 0.412 | 0.0080 ± 0.0016 | 0.0075 ± 0.0021 | 0.358 | |
| Distal phalanx V | 0.09 ± 0.01 | 0.08 ± 0.01 | 0.832 | 0.0005 ± 0.00007 | 0.0006 ± 0.0001 | 0.769 | |
| Proximal phalanx V | 0.14 ± 0.01 | 0.15 ± 0.02 | 0.027* | 0.0031 ± 0.0007 | 0.0036 ± 0.0012 | 0.089 | |
| Metacarpal II | 0.39 ± 0.02 | 0.41 ± 0.02 | 0.034* | 0.0270 ± 0.0028 | 0.0265 ± 0.0034 | 0.586 | |
| Metacarpal III | 0.49 ± 0.03 | 0.52 ± 0.03 | 0.004* | 0.0398 ± 0.0043 | 0.0382 ± 0.0062 | 0.342 | |
| Metacarpal IV | 0.43 ± 0.02 | 0.45 ± 0.03 | 0.011* | 0.0312 ± 0.0033 | 0.0291 ± 0.0048 | 0.114 | |
| Metacarpal V | 0.22 ± 0.01 | 0.22 ± 0.02 | 0.571 | 0.0120 ± 0.0019 | 0.0124 ± 0.0023 | 0.458 | |
| Zeugopod | Radius | 2.29 ± 0.02 | 2.22 ± 0.01 | 0.003* | 0.3187 ± 0.0292 | 0.3558 ± 0.0334 | 0.001** |
| Ulna | 2.76 ± 0.08 | 2.64 ± 0.07 | 0.000** | 0.4999 ± 0.0417 | 0.5366 ± 0.0766 | 0.010* | |
| Stylopod | Humerus | 1.65 ± 0.01 | 1.62 ± 0.007 | 0.007* | 0.9950 ± 0.0764 | 1.1444 ± 0.0649 | 0.001** |
| Not derived from limb bud | Scapula | 2.69 ± 0.08 | 2.75 ± 0.06 | 0.013* | 1.0757 ± 0.0733 | 1.2184 ± 0.0777 | 0.001** |
| Clavicle | 2.47 ± 0.06 | 2.34 ± 0.09 | 0.001** | 0.2214 ± 0.0147 | 0.2811 ± 0.0232 | 0.001** | |
Table 2
Sample composition by genotype and period.
Fgfr2+/+: unaffected littermates; Fgfr2+/P253R: Apert syndrome mutant littermates. Developmental periods were defined according to limb staging, as shown in Figure 4. Hindlimbs and forelimbs were …
| Genotype | Period | N limb | N gene | LIMB | |||
|---|---|---|---|---|---|---|---|
| Hindlimb (N = 34) | Forelimb (N = 46) | ||||||
| Early (~E10) | |||||||
| Limb | Gene | Limb | Gene | ||||
| Fgfr2+/+ | EARLY: E10-E10.5 | 30 | 24 | 13 | 9 | 17 | 15 |
| Fgfr2+/P253R | 16 | 15 | 11 | 9 | 5 | 6 | |
| Subtotal | 46 | 39 | 24 | 18 | 22 | 21 | |
| Mid early (~E10.5) | Mid late (~E11) | ||||||
| Limb | Gene | Limb | Gene | ||||
| Fgfr2+/+ | MID: E10.5-E11 | 15 | 10 | 7 | 3 | 8 | 7 |
| Fgfr2+/P253R | 16 | 9 | 8 | 3 | 8 | 6 | |
| Subtotal | 31 | 19 | 15 | 6 | 16 | 13 | |
| Late (~E11.5) | |||||||
| Limb | Gene | Limb | Gene | ||||
| Fgfr2+/+ | LATE: E11-E11.5 | 8 | 8 | 4 | 4 | 4 | 4 |
| Fgfr2+/P253R | 15 | 14 | 7 | 6 | 8 | 8 | |
| Subtotal | 23 | 22 | 11 | 10 | 12 | 12 | |
| Total | 100 | 80 | |||||
Table 3
Quantitative comparison of limb size (μm) and Dusp6 volume (μm3) in Fgfr2+/P253R mutant and Fgfr2+/+ unaffected mice.
Results from two-sided t-tests are provided separately for forelimbs and hindlimbs and for each developmental group, as defined in Table 2 and Figure 4. Statistically significant differences are …
| EARLY | MID | LATE | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Fgfr2+/+ | Fgfr2+/P253R | t | df | P-value | Fgfr2+/+ | Fgfr2+/P253R | t | df | P-value | Fgfr2+/+ | Fgfr2+/P253R | t | df | P-value | ||
| Forelimb | Limb size | 2041.63 | 2274.15 | 6.99 | 18.98 | <0.0001 | 2728.24 | 2579.69 | −1.70 | 7.74 | 0.13 | 3311.66 | 3097.72 | −3.57 | 5.39 | 0.01 |
| Dusp6 volume | 18160720 | 18387239 | 0.13 | 13.03 | 0.89 | 23002206 | 27432909 | 1.78 | 9.64 | 0.11 | 24792568 | 29875554 | 2.47 | 9.72 | 0.03 | |
| EARLY | MID | LATE | ||||||||||||||
| Fgfr2+/+ | Fgfr2+/P253R | t | df | P-value | Fgfr2+/+ | Fgfr2+/P253R | t | df | P-value | Fgfr2+/+ | Fgfr2+/P253R | t | df | P-value | ||
| Hindlimb | Limb size | 2280.36 | 2489.62 | 2.68 | 15.76 | 0.02 | 2769.51 | 2589.95 | −1.74 | 3.03 | 0.18 | 3191.12 | 2955.91 | −3.44 | 6.73 | 0.01 |
| Dusp6 volume | 16294103 | 16277958 | −0.01 | 15.36 | 0.99 | 23920390 | 23131303 | −0.19 | 2.45 | 0.86 | 41802827 | 45608477 | 0.52 | 4.14 | 0.63 | |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (M. musculus) | Fgfr2+/P253R | Wang et al., 2010; doi: 10.1186/1471 -213X-10–22 | Laboratory of Dr. Richtsmeier (Pennsylvania State University); inbred mouse model on a C57BL/6J background | |
| Chemical compound, drug | PBST | Sigma-Aldrich | P3563 | Phosphate-buffered saline, 0.1% tween 20 |
| Chemical compound, drug | paraformaldehyde | Sigma-Aldrich | P6148 | 4% in PBS |
| Chemical compound, drug | methanol | Sigma-Aldrich | 494437–2L-D | Methanol for protein sequencing, bioReagent,99.93% |
| Chemical compound, drug | digoxigenin-UTP | Sigma-Aldrich | 11277073910 | DIG RNA Labeling Mix (Roche) |
| Sequence -based reagent | Dusp6 | Dickinson et al. (2002) doi:10.1016/S0925 -4773 (02)00024–2 | m Mkp3-pCVM.sport6 | |
| Antibody | anti-DIG-AP (sheep polyclonal) | Sigma-Aldrich | 11093274910 | (1:2000) |
| Other | NBT | Sigma-Aldrich | 11585029001 | 4-nitro blue tetrazolium chloride, crystals (Roche) |
| Other | BCIP | Sigma-Aldrich | 11383221001 | 4-toluidine salt (Roche) |
| Chemical compound, drug | BABB | Sigma-Aldrich | 402834; W213802 | (one benzyl alcohol:two benzyl benzoate) |
| Chemical compound, drug | agarose | Sigma-Aldrich | A9414 | Agarose low gelling temperature |
| Software, algorithm | Matlab | The MathWorks, Inc | RRID:SCR_001622 | https://es.mathworks.com/products/matlab.html |
| Software, algorithm | R, CRAN | R Development Core Team, 2014 | RRID:SCR_003005 | http://www.R-project.org/ |
| Software, algorithm | Amira 6.3 | FEI | RRID:SCR_014305 | https://www.fei.com/software/amira-3d-for-life-sciences/ |
| Software, algorithm | Viewbox | dHAL software, Kifissia, Greece | RRID:SCR_016481 | http://www.dhal.com/ |
| Software, algorithm | Geomorph | Adams and Otárola-Castillo, 2013; doi: 10.1111/2041-210X.12035 | RRID:SCR_016482 | https://cran.r-project.org/web/packages/geomorph/index.html |
| Software, algorithm | MorphoJ | Klingenberg (2011): doi: 10.1111/j.1755 –0998.2010.02924.x | RRID:SCR_016483 | http://www.flywings.org.uk/morphoj_page.htm |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.36405.027
