Methods
Generation Targeting and Breeding Schemes
Apert 252 Mice - 252ApertFgfr2(+/S252W)
We used a 6.28 kb genomic DNA fragment containing exons IIIa, IIIb and IIIc (exons 7, 8 and 9) of the murine Fgfr2 gene cloned into pBluescript SK– (Stratagene) to make a construct (Fig. 1A). A 755_756CA>GG substitution, resulting in a Ser252Trp mutation at the residue homologous to human FGFR2 amino acid 252, was introduced into exon IIIa using site-directed mutagenesis. We inserted a thymidine kinase (TK) gene cassette at the 5′ XhoI site upstream of the targeting DNA fragment and a neo cassette with flanking loxPrecognition sites into intron IIIa, oriented in an opposite transcriptional direction to the targetFgfr2. The final targeting vector of 13.6 kb was confirmed by sequencing, linearized by NotI digestion, and introduced into R1 ES cells by electroporation (carried out by The Jackson Laboratory).
Text from: Wang, Y., Xiao, R., Yang, F., Karim, B. O., Iacovelli, A. J., Cai, J., Lerner, C. P., Richtsmeier, J. T., Leszl, J. M., Hill, C. A., et al. (2005). Abnormalities in cartilage and bone development in the Apert syndrome FGFR2(+/S252W) mouse. Development 132, 3537–3548.
Fgfr2+/S252W Apert syndrome mouse models were bred on C57BL/6J genetic background for 20 generations to minimize phenotypic variation and were generated, euthanized, fixed and imaged in compliance with animal welfare guidelines approved by the Johns Hopkins University, the Mount Sinai School of Medicine and the Pennsylvania State University Animal Care and Use Committees.
Text From: Martínez-Abadías,N, G Holmes, T Pankratz,Y Wang,X Zhou, EW Jabs, JT Richtsmeier 2013 From shape to cells: mouse models reveal mechanisms altering palate development in Apert syndrome. Disease models and mechanisms, in press doi:10.1242/dmm.010397.
Images of Apert 252 Mice seen on this website were used in the following articles:
Martínez-Abadías,N, G Holmes, T Pankratz,Y Wang,X Zhou, EW Jabs, JT Richtsmeier 2013 From shape to cells: mouse models reveal mechanisms altering palate development in Apert syndrome. Disease models and mechanisms, in press doi:10.1242/dmm.010397
Richtsmeier, JT, K Flaherty 2013 Hand in glove: brain and skull in development and dysmorphogenesis. Acta Neuropathol, 125(4), doi 10.1007/s00401-013-1104-y
Apert 253 Mice- 253ApertFgfr2(+/P253R)
Targeting construct created with a TK cassette, neo cassette with flanking loxP sequences, and a portion of the wild-type Fgfr2 gene (including exons IIIa, IIIb, and IIIc, but not exon 10) introduced by Hind III (H) digestion; mutant allele produced by homologous recombination and neo deletion mediated by Cre. The 758C>G, P253R mutation (*) was introduced into exon IIIa. Probes (-) and restriction enzyme Sty I (St) or Sac I (S) used for Southern blot analysis and PCR primers (F, Fn, R arrows) for genotyping are shown. B) Identification of mutant and wild-type alleles in ES cell clones using Southern-blot analysis with 5'- and 3'-probes. The mutant allele shows 4.1 kb and 8.3 kb bands with the 5'-and 3'-probes, respectively. C) Genotyping results from PCR of tail DNAs. Lane 1: 100 bp DNA ladder; lane 2: wild-type +/+, 290 bp; lane 3: heterozygote with +/P253Rneo [wild-type allele and P253R allele with neo casette, 400 bp]; and lane 4: heterozygote mutant +/P253R [wild-type allele and mutant allele after neo deletion with only one remaining loxP sequence, 350 bp].
Southern blot hybridization, PCR, and sequencing confirmed the predicted mutant allele. An inbred line was generated by mating our heterozygote Fgfr2+/P253Rneo mice with C57BL/6J mice for more than ten generations. The inserted neo cassette was found by RT-PCR to decrease dramatically the expression of the mutant allele. We then excised the floxed neo cassette by mating heterozygote Fgfr2+/P253Rneo mice with EIIA-Cre transgenic mice that were also generated on a B6 genetic background. Heterozygous Fgfr2+/P253R mice were born at the expected frequency of 50%, and their mutant and wild-type alleles resulted in similar Fgfr2 mRNA and protein expression levels (see additional file 2). The Fgfr2 P253R allele was expressed in multiple organs including the skull, limb, and other tissues (i.e., brain, lung, skin, heart, liver, stomach, intestine, and spleen).
Text From: Wang, Y., Sun, M., Uhlhorn, V. L., Zhou, X., Peter, I., Martínez-Abadias, N., Hill, C. A., Percival, C. J., Richtsmeier, J. T., Huso, D. L., et al. (2010). Activation of p38 MAPK pathway in the skull abnormalities of Apert syndrome Fgfr2(+P253R) mice. BMC Dev. Biol. 10, 22
Fgfr2+/P253R Apert syndrome mouse models were bred on C57BL/6J genetic background for 20 generations to minimize phenotypic variation and were generated, euthanized, fixed and imaged in compliance with animal welfare guidelines approved by the Johns Hopkins University, the Mount Sinai School of Medicine and the Pennsylvania State University Animal Care and Use Committees.
Text From: Martínez-Abadías,N, G Holmes, T Pankratz,Y Wang,X Zhou, EW Jabs, JT Richtsmeier 2013 From shape to cells: mouse models reveal mechanisms altering palate development in Apert syndrome. Disease models and mechanisms, in press doi:10.1242/dmm.010397.
Images of Apert 253 Mice seen on this website were used in the following articles:
Martínez-Abadías,N, G Holmes, T Pankratz,Y Wang,X Zhou, EW Jabs, JT Richtsmeier 2013 From shape to cells: mouse models reveal mechanisms altering palate development in Apert syndrome. Disease models and mechanisms, in press doi:10.1242/dmm.010397
Richtsmeier, JT, K Flaherty 2013 Hand in glove: brain and skull in development and dysmorphogenesis. Acta Neuropathol, 125(4), doi 10.1007/s00401-013-1104-y
Targeting construct created with a TK cassette, neo cassette with flanking loxP sequences, and a portion of the wild-type Fgfr2 gene (including exons IIIa, IIIb, and IIIc, but not exon 10) introduced by Hind III (H) digestion; mutant allele produced by homologous recombination and neo deletion mediated by Cre. The 758C>G, P253R mutation (*) was introduced into exon IIIa. Probes (-) and restriction enzyme Sty I (St) or Sac I (S) used for Southern blot analysis and PCR primers (F, Fn, R arrows) for genotyping are shown. B) Identification of mutant and wild-type alleles in ES cell clones using Southern-blot analysis with 5'- and 3'-probes. The mutant allele shows 4.1 kb and 8.3 kb bands with the 5'-and 3'-probes, respectively. C) Genotyping results from PCR of tail DNAs. Lane 1: 100 bp DNA ladder; lane 2: wild-type +/+, 290 bp; lane 3: heterozygote with +/P253Rneo [wild-type allele and P253R allele with neo casette, 400 bp]; and lane 4: heterozygote mutant +/P253R [wild-type allele and mutant allele after neo deletion with only one remaining loxP sequence, 350 bp].
Southern blot hybridization, PCR, and sequencing confirmed the predicted mutant allele. An inbred line was generated by mating our heterozygote Fgfr2+/P253Rneo mice with C57BL/6J mice for more than ten generations. The inserted neo cassette was found by RT-PCR to decrease dramatically the expression of the mutant allele. We then excised the floxed neo cassette by mating heterozygote Fgfr2+/P253Rneo mice with EIIA-Cre transgenic mice that were also generated on a B6 genetic background. Heterozygous Fgfr2+/P253R mice were born at the expected frequency of 50%, and their mutant and wild-type alleles resulted in similar Fgfr2 mRNA and protein expression levels (see additional file 2). The Fgfr2 P253R allele was expressed in multiple organs including the skull, limb, and other tissues (i.e., brain, lung, skin, heart, liver, stomach, intestine, and spleen).
Text From: Wang, Y., Sun, M., Uhlhorn, V. L., Zhou, X., Peter, I., Martínez-Abadias, N., Hill, C. A., Percival, C. J., Richtsmeier, J. T., Huso, D. L., et al. (2010). Activation of p38 MAPK pathway in the skull abnormalities of Apert syndrome Fgfr2(+P253R) mice. BMC Dev. Biol. 10, 22
Fgfr2+/P253R Apert syndrome mouse models were bred on C57BL/6J genetic background for 20 generations to minimize phenotypic variation and were generated, euthanized, fixed and imaged in compliance with animal welfare guidelines approved by the Johns Hopkins University, the Mount Sinai School of Medicine and the Pennsylvania State University Animal Care and Use Committees.
Text From: Martínez-Abadías,N, G Holmes, T Pankratz,Y Wang,X Zhou, EW Jabs, JT Richtsmeier 2013 From shape to cells: mouse models reveal mechanisms altering palate development in Apert syndrome. Disease models and mechanisms, in press doi:10.1242/dmm.010397.
Images of Apert 253 Mice seen on this website were used in the following articles:
Martínez-Abadías,N, G Holmes, T Pankratz,Y Wang,X Zhou, EW Jabs, JT Richtsmeier 2013 From shape to cells: mouse models reveal mechanisms altering palate development in Apert syndrome. Disease models and mechanisms, in press doi:10.1242/dmm.010397
Richtsmeier, JT, K Flaherty 2013 Hand in glove: brain and skull in development and dysmorphogenesis. Acta Neuropathol, 125(4), doi 10.1007/s00401-013-1104-y
Beare Stevenson Mice- Fgfr2+/Y394C
(Text Refers to Image Within Cited Article Below)
Generation of Fgfr2+/Y394C mice. (A) The Fgfr2 targeting construct contains exons IIIa, IIIb, IIIc, and exon 10, which codes for the transmembrane domain (TM) of the gene, a neo cassette flanked by loxP sequences (open arrows), and the 1181A>G (based on nucleotide sequence NM_010207.2 and results in Y394C protein mutation) (*) in exon 10. Screening for recombinant clones was done by Southern blot analysis using restriction enzymes HindIII (H) or DrdI (D) and probes indicated (-). Other restriction enzyme sites noted are SacI (S), BsaHI (B), XcmI (X), and EcoNI (E). PCR primers (F, Fn, R; arrows) used for genotyping are shown. (B) (Upper panel) Genotyping results from PCR of tail DNAs. Lane 1: 100 bp DNA ladder; lane 2: wild-type +/+, 300 bp; lane 3: heterozygote +/Y394C [wild-type allele 300 bp and mutant allele after neo deletion with only one remaining loxP sequence, 350 bp]; and lane 4: heterozygote +/Y394Cneo [wild-type allele 300 bp and Y394C allele with neo cassette, 200 bp]. (Lower panel) RT-PCR results from PpuM1/Xho1 restriction enzyme-digest of Fgfr2 alleles expressed in calvaria (lanes 2 and 3) and skin (lanes 4 and 5). Lane 1: 100 bp ladder; lane 2: wild-type +/+, 460 bp and 244 bp digestion products; lane 3: heterozygote +/Y394C [wild-type allele 460 bp, wild- type & Y394 allele 244 bp and Y394 allele 196 bp digestion products]; lane 4: wild-type +/+; lane 5: heterozygote +/Y394C. (C and D) Gross appearance of Fgfr2+/Y394C mice. (C) Note no significant difference in the body size (P0, +/Y394C [n=12] versus +/+ [n=9]: 1.402 ± 0.081 g versus 1.374 ± 0.122 g, P=0.5470) and limb length between the wild-type and mutant at P0. The mutant has a dome-shaped skull (arrow). (D) Note significant difference in the body size between the wild-type and mutant at P24. (E and F) Weight and survival curves of Fgfr2+/Y394C (red) and wild-type (green) mice. (E) Weights of mice with age show growth retardation in the mutant (e.g., P5, +/Y394C [n=8] versus +/+ [n=4]: 2.160 ± 0.2613g versus 3.400 ± 0.342 g, P<0.0001). (F) Survival curves show that almost half of the Fgfr2+/Y394C mutants died within 24-36 hours after birth; most died within 2 weeks.
Text From: Wang Y, Zhou X, Oberoi K, et al. (2012) p38 inhibition ameliorates skin and skull abnormalities in Fgfr2 Beare-Stevenson mice. J Clini Investig 122, 2153–2164.
Our sample was composed of litters of Beare-Stevenson Fgfr2+/Y394C heterozygote mice and unaffected littermates. Litters of mice were euthanized at postnatal day zero (P0) and postnatal day eight (P8) with inhalation anesthetics and fixed in 4% paraformaldehyde. Care and use of mice for this study were in compliance with relevant animal welfare guidelines approved by Mount Sinai School of Medicine and Pennsylvania State University Animal Care and Use Committees.
Text From: Percival, CJ, Y Wang, X Zhou, EW Jabs, JT Richtsmeier 2012 The effect of a Beare-Stevenson syndrome Fgfr2 Y394C mutation on early craniofacial bone volume and relative bone mineral density in mice. J Anat., 221:434-442.
Images of Beare Stevenson Mice seen on this website were used in the following articles:
Percival, CJ, Y Wang, X Zhou, EW Jabs, JT Richtsmeier 2012 The effect of a Beare-Stevenson syndrome Fgfr2 Y394C mutation on early craniofacial bone volume and relative bone mineral density in mice. J Anat., 221:434-442.
Wang, Y, X Zhou, K Oberoi, R Phelps, R Couwenhoven, M Sun, A Rezza, G Holmes, CJ Percival, J Friedenthal, P Krejci, JT Richtsmeier, DL Huso, M Rendl, EW Jabs 2012. p38 Inhibition ameliorates skin and skull abnormalities in Fgfr2 Beare-Stevenson mice. J Clinical Investigation, 122(6):2153-2164..
Generation of Fgfr2+/Y394C mice. (A) The Fgfr2 targeting construct contains exons IIIa, IIIb, IIIc, and exon 10, which codes for the transmembrane domain (TM) of the gene, a neo cassette flanked by loxP sequences (open arrows), and the 1181A>G (based on nucleotide sequence NM_010207.2 and results in Y394C protein mutation) (*) in exon 10. Screening for recombinant clones was done by Southern blot analysis using restriction enzymes HindIII (H) or DrdI (D) and probes indicated (-). Other restriction enzyme sites noted are SacI (S), BsaHI (B), XcmI (X), and EcoNI (E). PCR primers (F, Fn, R; arrows) used for genotyping are shown. (B) (Upper panel) Genotyping results from PCR of tail DNAs. Lane 1: 100 bp DNA ladder; lane 2: wild-type +/+, 300 bp; lane 3: heterozygote +/Y394C [wild-type allele 300 bp and mutant allele after neo deletion with only one remaining loxP sequence, 350 bp]; and lane 4: heterozygote +/Y394Cneo [wild-type allele 300 bp and Y394C allele with neo cassette, 200 bp]. (Lower panel) RT-PCR results from PpuM1/Xho1 restriction enzyme-digest of Fgfr2 alleles expressed in calvaria (lanes 2 and 3) and skin (lanes 4 and 5). Lane 1: 100 bp ladder; lane 2: wild-type +/+, 460 bp and 244 bp digestion products; lane 3: heterozygote +/Y394C [wild-type allele 460 bp, wild- type & Y394 allele 244 bp and Y394 allele 196 bp digestion products]; lane 4: wild-type +/+; lane 5: heterozygote +/Y394C. (C and D) Gross appearance of Fgfr2+/Y394C mice. (C) Note no significant difference in the body size (P0, +/Y394C [n=12] versus +/+ [n=9]: 1.402 ± 0.081 g versus 1.374 ± 0.122 g, P=0.5470) and limb length between the wild-type and mutant at P0. The mutant has a dome-shaped skull (arrow). (D) Note significant difference in the body size between the wild-type and mutant at P24. (E and F) Weight and survival curves of Fgfr2+/Y394C (red) and wild-type (green) mice. (E) Weights of mice with age show growth retardation in the mutant (e.g., P5, +/Y394C [n=8] versus +/+ [n=4]: 2.160 ± 0.2613g versus 3.400 ± 0.342 g, P<0.0001). (F) Survival curves show that almost half of the Fgfr2+/Y394C mutants died within 24-36 hours after birth; most died within 2 weeks.
Text From: Wang Y, Zhou X, Oberoi K, et al. (2012) p38 inhibition ameliorates skin and skull abnormalities in Fgfr2 Beare-Stevenson mice. J Clini Investig 122, 2153–2164.
Our sample was composed of litters of Beare-Stevenson Fgfr2+/Y394C heterozygote mice and unaffected littermates. Litters of mice were euthanized at postnatal day zero (P0) and postnatal day eight (P8) with inhalation anesthetics and fixed in 4% paraformaldehyde. Care and use of mice for this study were in compliance with relevant animal welfare guidelines approved by Mount Sinai School of Medicine and Pennsylvania State University Animal Care and Use Committees.
Text From: Percival, CJ, Y Wang, X Zhou, EW Jabs, JT Richtsmeier 2012 The effect of a Beare-Stevenson syndrome Fgfr2 Y394C mutation on early craniofacial bone volume and relative bone mineral density in mice. J Anat., 221:434-442.
Images of Beare Stevenson Mice seen on this website were used in the following articles:
Percival, CJ, Y Wang, X Zhou, EW Jabs, JT Richtsmeier 2012 The effect of a Beare-Stevenson syndrome Fgfr2 Y394C mutation on early craniofacial bone volume and relative bone mineral density in mice. J Anat., 221:434-442.
Wang, Y, X Zhou, K Oberoi, R Phelps, R Couwenhoven, M Sun, A Rezza, G Holmes, CJ Percival, J Friedenthal, P Krejci, JT Richtsmeier, DL Huso, M Rendl, EW Jabs 2012. p38 Inhibition ameliorates skin and skull abnormalities in Fgfr2 Beare-Stevenson mice. J Clinical Investigation, 122(6):2153-2164..
Crouzon Mice- 342Fgfr2c(C342Y/+)
Fgfr2C342Y/+ mice (1) were maintained on a CD1 genetic background and intercrossed to generate litters with wild-type (WT), Fgfr2C342Y/+, and Fgfr2C342Y/C342Y embryos. Embryonic dates were determined by observing a vaginal plug in female mice; noon on the day of plug visualization was considered to be E0.5. Embryos were harvested via caesarian section after CO2 asphyxiation of the pregnant mothers during the time of palatogenesis (E13.5–E15.5). Embryonic tail clippings were used to obtain DNA for genotyping with genomic PCR analysis.
Text From: Alison K. Snyder-Warwick, Chad A. Perlyn, Jing Pan, Kai Yu, Lijuan Zhang, David M. Ornitz. Analysis of a gain-of-function FGFR2 Crouzon mutation provides evidence of loss of function activity in the etiology of cleft palate.Proc Natl Acad Sci U S A. 2010 February 9; 107(6): 2515–2520. Published online 2010 February 1. doi: 10.1073/pnas.0913985107
Images of Crouzon Mice seen on this website were used in the following articles:
Martínez-Abadías,N, Motch, S, T Pankratz,Y Wang, K Aldridge, EW Jabs, JT Richtsmeier 2013 Tissue specific responses to aberrant FGF signaling in complex head phenotypes. Dev Dyn, 242(1):80-94. doi: 10.1002/ dvdy.23903. PMID: 2317272
Richtsmeier, JT, K Flaherty 2013 Hand in glove: brain and skull in development and dysmorphogenesis. Acta Neuropathol, 125(4), doi 10.1007/s00401-013-1104-y
Text From: Alison K. Snyder-Warwick, Chad A. Perlyn, Jing Pan, Kai Yu, Lijuan Zhang, David M. Ornitz. Analysis of a gain-of-function FGFR2 Crouzon mutation provides evidence of loss of function activity in the etiology of cleft palate.Proc Natl Acad Sci U S A. 2010 February 9; 107(6): 2515–2520. Published online 2010 February 1. doi: 10.1073/pnas.0913985107
Images of Crouzon Mice seen on this website were used in the following articles:
Martínez-Abadías,N, Motch, S, T Pankratz,Y Wang, K Aldridge, EW Jabs, JT Richtsmeier 2013 Tissue specific responses to aberrant FGF signaling in complex head phenotypes. Dev Dyn, 242(1):80-94. doi: 10.1002/ dvdy.23903. PMID: 2317272
Richtsmeier, JT, K Flaherty 2013 Hand in glove: brain and skull in development and dysmorphogenesis. Acta Neuropathol, 125(4), doi 10.1007/s00401-013-1104-y
Meuke Syndrome Mice (Fgfr3) - 244Fgfr3(P244R/P244R) and 244Fgfr3(P244R/+)
Muenke Syndrome Mouse models were bred by Mimi Jabs using the procedure laid out in the following article:
Twigg, S. R., Healy, C., Babbs, C., Sharpe, J. A., Wood, W. G., Sharpe, P. T., Morriss-Kay, G. M., and Wilkie, A. O. (2009) Skeletal analysis of the Fgfr3(P244R) mouse, a genetic model for the Muenke craniosynostosis syndrome. Dev Dyn 238, 331-342
Twigg, S. R., Healy, C., Babbs, C., Sharpe, J. A., Wood, W. G., Sharpe, P. T., Morriss-Kay, G. M., and Wilkie, A. O. (2009) Skeletal analysis of the Fgfr3(P244R) mouse, a genetic model for the Muenke craniosynostosis syndrome. Dev Dyn 238, 331-342
Cre Mice
All methods regarding Cre mice can be found on the Jackson Laboratories website (jaxmice.jax.org). The strain numbers for all models included in this website are as follows:
-Wnt1 (mutation only expressed in Nueral Tube, etc.): ?????
-Mesp1 (mutation expressed in mesoderm derived cells):?????
-TEK (mutation expressed only in endothelial cells): 008863
-Col1a2 (mutation expressed only in chondrogenic cells): 016237
-Osx (mutation expressed only in Osteoblasts): 006361
-Wnt1 (mutation only expressed in Nueral Tube, etc.): ?????
-Mesp1 (mutation expressed in mesoderm derived cells):?????
-TEK (mutation expressed only in endothelial cells): 008863
-Col1a2 (mutation expressed only in chondrogenic cells): 016237
-Osx (mutation expressed only in Osteoblasts): 006361
Imaging Protocol
MRM and μ-CT- The fixed
animals were immersed in a 2% Magnevist (Bayer Health Care, Wayne, NJ)
phosphor-buffered solution for 10 days to reduce the T1 and T2 relaxation
times. The achieved short T1 (32 ms) and T2 (8 ms) times allowed for fast
imaging with a high contrast-to-noise ratio. To prevent the animals from drying
out and to minimize magnetic susceptibility artifacts during scanning the
specimens were surrounded by a flourinert liquid FD-43 (3M, St. Paul, MN). All
experiments were conducted on a vertical 14.1 Tesla Varian (Varian Inc., Palo
Alto, CA) imaging system with direct drive technology. A home-built loop gap
resonator with a diameter of 2.0 cm was used to acquire standard
three-dimensional spin echo images of the head of the animal. Images up to an
isotropic resolution of 40 μm were acquired. A standard imaging experiment with
an isotropic resolution of 80 μm comprised a field of view of 15.4 × 14 × 11
mm3 and a matrix size of 192 × 132 (75% partial Fourier: 176) × 137. With eight
averages and a repetition time of 75 ms (echo time 25 ms) the total scan time
was three hours. Matlab (The MathWorks, Inc., Natick, MA) was used for
postprocessing. By zero-filling each direction by a factor of two the pixel
resolution of the standard imaging experiment was 40 μm3.
μ-CT images were acquired at the Center for Quantitative Imaging at the Pennsylvania State University (www.cqi.psu.edu) using the HD-600 OMNI-X high-resolution X-ray computed tomography system (Bio-Imaging Research Inc, Lincolnshire, IL) following already established protocols (Parsons et al., 2007; Hill et al., 2007) with pixel size of 0.15–0.02 mm and 0.15–0.025 mm slice thickness.
Text From: Aldridge, K, CA Hill, JR Austin, C Perival, N Martinez-Abadias, T Neuberger, Y Wang, EW Jabs, JT Richtsmeier. 2010. Brain phenotypes in two FGFR2 mouse models for Apert syndrome. Devel Dyn. 239: 987-997.
μ-CT images were acquired at the Center for Quantitative Imaging at the Pennsylvania State University (www.cqi.psu.edu) using the HD-600 OMNI-X high-resolution X-ray computed tomography system (Bio-Imaging Research Inc, Lincolnshire, IL) following already established protocols (Parsons et al., 2007; Hill et al., 2007) with pixel size of 0.15–0.02 mm and 0.15–0.025 mm slice thickness.
Text From: Aldridge, K, CA Hill, JR Austin, C Perival, N Martinez-Abadias, T Neuberger, Y Wang, EW Jabs, JT Richtsmeier. 2010. Brain phenotypes in two FGFR2 mouse models for Apert syndrome. Devel Dyn. 239: 987-997.
Clearing and Staining Protocol
Specimens were histologically prepared and imaged by Kazz Kawazaki at The Pennsylvania State University using standard protocol for Alcian Blue/Alizarin red staining of bone and cartilage.
Standard protocol is outlined in: Hogan B, Beddington R, Constantini F, Lacy E: Staining embryos for cartilage and bone. In Manipulating the Mouse Embryo: A Laboratory Manual. 2nd Edition. Plainview, New York: Cold Spring Harbor Laboratory Press; 1994: 379.
and can also be read here: http://cshprotocols.cshlp.org/content/2009/3/pdb.prot5170.full
Standard protocol is outlined in: Hogan B, Beddington R, Constantini F, Lacy E: Staining embryos for cartilage and bone. In Manipulating the Mouse Embryo: A Laboratory Manual. 2nd Edition. Plainview, New York: Cold Spring Harbor Laboratory Press; 1994: 379.
and can also be read here: http://cshprotocols.cshlp.org/content/2009/3/pdb.prot5170.full