A genome-wide screen for modifiers of transgene variegation identifies genes with critical roles in development
© Ashe et al.; licensee BioMed Central Ltd. 2008
Received: 21 June 2008
Accepted: 19 December 2008
Published: 19 December 2008
Some years ago we established an N-ethyl-N-nitrosourea screen for modifiers of transgene variegation in the mouse and a preliminary description of the first six mutant lines, named MommeD1-D6, has been published. We have reported the underlying genes in three cases: MommeD1 is a mutation in SMC hinge domain containing 1 (Smchd1), a novel modifier of epigenetic gene silencing; MommeD2 is a mutation in DNA methyltransferase 1 (Dnmt1); and MommeD4 is a mutation in Smarca 5 (Snf2h), a known chromatin remodeler. The identification of Dnmt1 and Smarca5 attest to the effectiveness of the screen design.
We have now extended the screen and have identified four new modifiers, MommeD7-D10. Here we show that all ten MommeDs link to unique sites in the genome, that homozygosity for the mutations is associated with severe developmental abnormalities and that heterozygosity results in phenotypic abnormalities and reduced reproductive fitness in some cases. In addition, we have now identified the underlying genes for MommeD5 and MommeD10. MommeD5 is a mutation in Hdac1, which encodes histone deacetylase 1, and MommeD10 is a mutation in Baz1b (also known as Williams syndrome transcription factor), which encodes a transcription factor containing a PHD-type zinc finger and a bromodomain. We show that reduction in the level of Baz1b in the mouse results in craniofacial features reminiscent of Williams syndrome.
These results demonstrate the importance of dosage-dependent epigenetic reprogramming in the development of the embryo and the power of the screen to provide mouse models to study this process.
Random mutagenesis screens for modifiers of position effect variegation were initiated in Drosophila in the 1960s [1, 2]. The screens used a fly strain, called w v , that displays variegated expression of the white (w) locus resulting in red and white patches in the eye. Variegation refers to the 'salt and pepper' expression of some genes due to the stochastic establishment of an epigenetic state at their promoters. The best characterized example of variegation in mammals is the coat color of mice carrying the A vy allele [3, 4]. In this case there is a correlation between DNA methylation at the promoter and silencing of the gene [5, 6]. Alleles of this type provide us with an opportunity to study epigenetic gene silencing at a molecular level in a whole organism.
The extent of the variegation at the w v locus, that is, the ratio of red to white patches in the eye, was known to be sensitive to strain background, suggesting the existence of genetic modifiers. Offspring of mutagenized flies were screened for changes in the degree of variegation. These screens have been continued to saturation and the results suggest that there are between 100 and 150 such genes [7, 8]. Approximately one-third of these have been identified and, as expected, most turn out to play critical roles in epigenetic gene silencing [1, 9]. These include genes encoding proteins involved in heterochromatin formation, for example, HP1 and histone methyltransferases .
Recently, we established a similar screen in the mouse using a transgenic line that expresses green fluorescent protein (GFP) in a variegated manner in erythrocytes . We anticipated that the screen would produce mutant lines that would help clarify the role of epigenetic gene silencing in mammals. Offspring of N-ethyl-N-nitrosourea (ENU) treated males were screened for changes in the percentage of erythrocytes expressing GFP (measured by flow cytometry). In those individuals in which the percentage of expressing cells was higher or lower than the wild-type mean by more than two standard deviations, heritability was tested. A preliminary description of the first six heritable mutations, which we refer to as Modifiers of murine metastable epialleles or Mommes, following the screening of 608 G1 offspring, has been published . We reported that all six were dosage-effect genes and five of the six were homozygous lethal, with loss of homozygotes apparent at weaning, but no knowledge of when death occurred. At the time of publication in 2005, none of the underlying genes had been identified. Since then we have identified the underlying mutation in three cases, MommeD1, MommeD2 and MommeD4. MommeD1 is a mutation in SMC hinge domain containing 1 (SmcHD1), encoding a previously uncharacterized protein containing a domain normally found in SMC proteins and we have gone on to show that this protein has a critical role in X inactivation . MommeD2 is a mutation in DNA methyltransferase 1, Dnmt1, encoding a DNA methyltransferase, and MommeD4 is a mutation in Smarca5, encoding Snf2h, a chromatin-remodeling enzyme . The finding of Dnmt1 and Smarca5, both known to be involved in epigenetic reprogramming, validates the screen. Here we report an update of the screen, adding four new MommeDs, identifying the underlying point mutation in two more cases, and further characterizing the phenotypes associated with hetero- and homozygosity.
Results and discussion
Integration site of the GFP transgene
We have previously reported that the GFP transgene used in this screen has integrated as an approximately 11 copy array on chromosome 1 . We were keen to further characterize the integration site. PCR using primers specific to the 3' end of the transgene construct in combination with degenerate random tagging primers (genome walking) revealed that the transgene had integrated into chromosome 1 at 47,747,486 bp (UCSC web browser, July 2007 assembly). This site of integration is neither centromeric nor telomeric, and so presumably the silencing is related to the multicopy nature of the transgene array [13, 14]. The integration site does not disrupt any annotated genes, and is approximately 1 Mb from the closest annotated transcript.
The identification of MommeD7-D10
Quantitative analysis of GFP expression following heterozygous intercrosses
Percentage expressing cells
55 ± 7 (7)
80 ± 3* (13)
286 ± 43 (7)
700 ± 52* (13)
54 ± 4 (40)
41 ± 4* (55)
15 ± 4* (15)
287 ± 25 (40)
244 ± 28* (55)
161 ± 27* (15)
59 ± 4 (8)
39 ± 6* (13)
326 ± 37 (8)
228 ± 27* (13)
55 ± 4 (46)
44 ± 3* (87)
22 ± 2* (6)
295 ± 33 (46)
269 ± 26* (87)
243 ± 19* (6)
For each MommeD, the heritability of the mutation has been tested and confirmed over at least 5 generations by using at least 30 litters. During the breeding of each mutant line, the expression profiles remained constant, consistent with the idea that we were following a single mutation in each case. The frequency with which we found these mutations, 1 in 100 G1 offspring, was similar to our previous results .
Genotype ratios of offspring at weaning following heterozygous intercrosses
Expected if semidominant, homozygous viable
Expected if semidominant, homozygous lethal
Homozygous lethality occurs at different stages of development
Litter size reductions following heterozygous intercrosses have already been reported for MommeD1-D6 at weaning , but the timing of the losses has only been reported for MommeD1, D2 and D4. MommeD1 is homozygous lethal in females only, with death occurring around mid-gestation [10, 11]. MommeD2 and MommeD4 are homozygous lethal at 8.5 days post-coitus (dpc) and 17.5 dpc, respectively [10, 12]. Here we describe the timing of the losses for MommeD3, D5, D6, D7, D8, D9 and D10.
Embryo dissections to determine time of death of homozygotes
Abnormal or resorbed
Abnormal or resorbed
Abnormal or resorbed
28 (26%)* †
12 (26%)* ‡
We hypothesized that this increase in reticulocytes was responsible for the larger than expected increase in the average fluorescence level of the GFP transgene in expressing cells observed in this line (Table 1). We performed FACS analysis on whole blood after staining for reticulocytes with propidium iodide. As expected, MommeD7-/+ mice had a threefold increase in the percentage of reticulocytes compared to MommeD7+/+ mice (Figure 3d), and the percentage of GFP fluorescence in both MommeD7-/+ and MommeD7+/+ was higher in reticulocytes than mature red cells (Figure 3e). Although this is only significant for MommeD7-/+ (p < 0.005), the trend is there for MommeD7+/+ mice (p = 0.07). This is consistent with the idea that a change in the erythroid cell populations contributes to the dramatic increase in the average fluorescence level of the GFP transgene in MommeD7-/+ mice.
Some MommeD8-/- mice (classified by their GFP expression profile and progeny testing) were viable at weaning but they were rare (Figure 1, Table 2). Following MommeD8-/+ intercrosses, dissections at 14.5 dpc showed no increase in the number of abnormal or resorbed embryos (Table 3). Litter size at birth was not significantly different from that seen in wild-type litters (data not shown), suggesting that the death of most MommeD8-/- individuals occurred after birth and before weaning. The only obvious phenotypic abnormality seen in MommeD8-/- mice that survived to weaning was reduced size. MommeD8 homozygotes were significantly smaller (6.60 g ± 0.25 standard error of the mean (SEM)) than their wild-type (8.54 g ± 0.33 SEM, p < 0.001) and heterozygous (8.65 g ± 0.29 SEM, p < 0.0001) littermates.
Dissections following MommeD9-/+ (determined by GFP fluorescence and progeny testing) intercrosses revealed a pattern similar to that seen for MommeD5 and MommeD6, suggesting MommeD9-/- embryos arrest before 9.5 dpc (Table 3). In the case of MommeD10 the data suggest that death of homozygotes occurred after birth (Table 3), and preliminary data collected at P7 indicated death in the first week of life (data not shown). Some MommeD10-/- individuals survived to weaning but they were extremely rare. This was confirmed by genotyping once the point mutation was identified.
Summary of MommeD screen for epigenetic modifiers
Suppressor or enhancer of variegation
Mutated gene, or number of genes in interval
Chr 17 (2.4)
Females only E10
Chr 9 (1.4)
Chr 11 (6)
Chr 8 (2.5)
Chr 4 (1.4)
Chr 14 (2.5)
Chr 7 (0.25)
Chr 4 (4)
Chr 7 (4)
Chr 5 (4)
Abnormal phenotypes associated with heterozygosity for MommeD7-D10
We have mapped the mutations in all ten cases to relatively small regions of the genome (Table 4). The mapping of MommeD1-D6 has been documented . Here we report the mapping of MommeD7-10. MommeD7 maps to a 0.25 Mb region on chromosome 7 between markers D7Mit220 and rs13479441 (using 134 phenotypically mutant and 135 phenotypically wild-type mice). This region contains 10 genes. MommeD8 maps to a 4 Mb region on chromosome 4 between markers rs6337795 and D4Mit279 (using 234 phenotypically mutant and 177 phenotypically wild-type mice). This region contains 54 genes. MommeD9 maps to a 3 Mb region on chromosome 7 between markers rs31712695 and rs6193818 (using 103 phenotypically mutant and 127 phenotypically wild-type mice). This region contains 80 genes. MommeD10 maps to a 4 Mb region between markers D5Mit165 and rs13478547 on chromosome 5. Twenty-four phenotypically homozygous and 312 phenotypically non-homozygous (heterozygous and wild-type mice combined) were used (see Materials and methods). These data show that each of the ten MommeD mutations maps to a unique region of the genome.
MommeD5 has a mutation in Histone deacetylase 1
The mutation produces a frameshift, resulting in changes to the last 12 amino acids, and an additional 25 amino acids. Protein modeling predictions based on human HDAC8, for which the crystal structure has been solved [18, 19], suggest that the carboxyl terminus of Hdac1 is relatively unstructured and so the mutation is unlikely to affect the stability of the protein (J Matthews, personal communication). An antibody that recognizes the carboxyl terminus of Hdac1 showed a 50% reduction of binding in 10.5 dpc MommeD5-/+ embryos, and negligible binding in MommeD5-/- embryos (Figure 5b), confirming that this region of the protein has been altered in the MommeD5 line. An antibody that recognizes the amino terminus of Hdac1 showed that the levels of the protein are not altered between mutant and wild-type mice (Figure 5b). Lysine 476 at the carboxyl terminus has been shown to be sumoylated and important for enzymatic function of the wild-type protein  and the absence of this amino acid in the MommeD5 mutant protein is likely to impair function. A knockout of Hdac1 has been reported and the homozygous embryos died around 9.5 dpc , similar to the time of death observed in MommeD5-/- embryos (Figure 5c). Together, these results argue that the mutation in Hdac1 is causative of the MommeD5 phenotype. Consistent with this, the level of Hdac2 increased in both MommeD5-/+ and MommeD5-/- embryos, as predicted from the reports of compensatory upregulation of Hdac2 in embryonic stem cells null for Hdac1 . Indeed, this upregulation may explain why MommeD5 was identified as an enhancer, rather than a suppressor, of variegation. Loss of histone deacetylase function is generally associated with transcriptional activation, but exceptions to this have been reported and the upregulation of Hdac2 could explain these results .
MommeD10 has a mutation in Baz1b
Effects of MommeD5 and MommeD10on DNA methylation at the transgene locus
Craniofacial analysis of MommeD10mice
Twenty cranial landmarks and nine mandibular landmarks were located on each skull using approximately 70 micron resolution datasets and inter-landmark measurements were compared (Figure 8d and Additional data file 1). Statistical analyses were carried out using the data collected from males only. Homozygote skulls were significantly different to wild type (Student's t-test, length:height ratio, p < 0.01; width:height ratio, p < 0.01; length:width ratio, p < 0.05), confirming the bulbous appearance of the skulls on the reconstructed images. Much of this difference could be attributed to reduction of the parietal and nasal bones (both > 12.5% shorter in homozygotes compared to an overall mean length and width reduction of approximately 9%). The reduced parietal bone length and the reduction and upward angulation of the nasal bones in these mice (Figure 8c, d) are reminiscent of the decrease in the volume of the parieto-occipital region and the appearance of the nose in WBS patients [34, 35]. Heterozygotes also had a decreased cranium width:height ratio (Student's t-test, p < 0.05) and decreased length:height ratio (Student's t-test, p < 0.05) compared to wild-type skulls. Of note, heterozygotes showed a reduction in palatine bone width of similar magnitude to that seen in homozygotes, suggesting a greater sensitivity of some parts of the skull to decreased Baz1b protein levels. Measurements of the lower jaw revealed relative mandibular hypoplasia in homozygotes that was most pronounced in the posterior region (approximately 20% reduction), encompassing the condyle, angle and coronoid processes (Figure 8d and Additional data file 1). The posterior aspects of the mandibles of heterozygotes were also reduced in size when compared to wild-type mandibles, albeit to a lesser degree than in the homozygotes.
Expression of Baz1bduring mouse embryogenesis
In the facial primordia, Baz1b is expressed in branchial arch 1 as it first emerges (Figure 9g), and continues later in development in both the maxillary and mandibular components of branchial arch 1 and branchial arch 2 (Figure 9c-e, h, i). Expression in the branchial arches is primarily mesenchymal, and is enriched in the rostral distal margin of the mandible, and the caudal distal margin of branchial arch 2 (Figure 9c-e, h). Baz1b is also expressed in the frontonasal process (Figure 9b) and later in the mesenchyme of both the medial and lateral nasal prominences as they elevate to surround the nasal pits (Figure 9c, d, h, i). Baz1b is expressed strongly in all the major facial primordia from early in embryogenesis.
A possible role for Baz1b in Williams syndrome
Overall, the skull shape in mutant animals is reminiscent of the head shape seen in WBS, including a small upturned nose with flat nasal bridge, micrognathia (or mandibular hypoplasia), malocclusion, bi-temporal narrowing and prominent forehead . WBS is known to be associated with a hemizygous deletion of approximately 28 genes in humans, but which of these genes are responsible for the craniofacial phenotype remains controversial. People with atypical deletions, and varying degrees of craniofacial abnormalities, implicate both proximal and distal ends of the deletion, suggesting that more than one gene is responsible [36–41]. Tassabehji and colleagues  reported craniofacial defects in a transgenic (c-myc; Tgf-α) mouse line that harbored a chromosomal translocation fortuitously disrupting the Gtf2ird1 gene, the human orthologue of which resides in the WBS critical interval . Mice homozygous for this transgene produced little Gtf2ird1 mRNA and exhibited craniofacial dysmorphism, suggesting a role for Gtf2ird1 in the craniofacial phenotype. Mice hemizygous for the transgene were indistinguishable from wild-type animals. Disruption of Gtf2ird1 in this mouse was associated with a 40 kb deletion at the site of integration, the addition of 5-10 tandem copies of a c-myc transgene, and translocation of the entire segment to chromosome 6 , providing opportunity for disruption to the expression of other genes, such as Baz1b, in the region. A targeted knockout of Gtf2ird1, produced by others, failed to find craniofacial dysmorphology or dental abnormalities . We checked the sequence and expression of Gtf2ird1 in MommeD10 mutants and found no abnormalities (data not shown). The chance of a second point mutation occurring in a coding region in this linked interval is extremely small (p = 0.0008, based on a mutation rate of 1 in 1.82 Mb) [15–17].
Our studies show that the chromatin remodeler Baz1b is expressed strongly in cranial neural crest-derived mesenchyme that drives facial morphogenesis and that homozygosity for a missense mutation in Baz1b produces an array of craniofacial features that are similar to those that characterize the typical WBS face. Significantly, some craniofacial features are also apparent in heterozygotes. These results suggest that reduction in Baz1b protein levels contributes to the elfin facies characteristic of WBS and that WBS is, at least in part, a chromatin-remodeling factor disease.
Extension of the screen has produced four new MommeDs, MommeD7-D10, all of which behave in a semidominant fashion, as do the six previously reported . We have now identified the underlying genes in five of the ten cases, two of which, Hdac1 and Baz1b, we report here. Both are already known to be involved in epigenetic processes, further validating the screen design. In the case of Baz1b this is the first report of a mouse carrying a disrupted allele at this locus and we have shown a role for the protein in craniofacial development.
The screen, designed primarily to identify genes involved in the epigenetic gene silencing of foreign DNA, such as transgenes, has revealed the critical role that such genes play in embryonic and fetal development. It is interesting that at least half of the MommeDs are important during gastrulation. Furthermore, the identification of heterozygous effects suggests that a reduction in the amount of protein product of less than 50% has effects on the health of the mouse. One of the hallmarks of epigenetic gene silencing across all multicellular organisms from plants to humans is the stochastic nature by which they operate  and these studies re-emphasize the importance of probabilistic events during embryogenesis. We believe that this screen will provide new tools with which to study these processes.
Materials and methods
Wild-type inbred C57BL/6J and FVB/NJ mice were purchased from ARC Perth. Procedures were approved by the Animal Ethics Committee of the University of Sydney and the Animal Ethics Committee of the Queensland Institute of Medical Research. The ENU screen was carried out in the FVB/NJ inbred transgenic line as described previously . All MommeD lines were maintained in this strain unless stated otherwise. Most crosses between MommeD individuals were performed on individuals three generations or more removed from the MommeD progenitor. A total of 1,000 G1 offspring were screened at 3 weeks of age, from which ten heritable dominant mutations were identified.
The Dnmt3b null mice were a kind gift from En Li. They have been subsequently backcrossed for more than ten generations to the C57BL/6J strain. GFP fluorescence in Dnmt3b+/- mice was analyzed in the F1 offspring of crosses between Dnmt3b+/- mice and the FVB/NJ transgenic line, and as such each mouse was hemizygous for the transgene.
GFP fluorescence in erythrocytes was analyzed by flow cytometry at weaning. A drop of blood was collected in Osmosol buffer (Lab Aids Pty Ltd, Narrabeen, NSW, Australia) and analyzed on a FACScan (Becton Dickinson, Franklin Lakes, NJ, USA) with excitation at 488 and 550 nm. The 488 nm channel predominantly measures GFP fluorescence and the 550 nm channel measures autofluoresence. The data were analyzed using CELL QUEST software with a GFP-positive gate set to exclude 99.9% of wild-type erythrocytes. Mean fluorescence was calculated using cells within the positive gate. Histograms depict only the GFP fluorescence channel.
Genome walking was performed using the APAgene™ GOLD Genome Walking Kit (Bio S&T Inc., Montreal, Quebec, Canada) following the manufacturer's instructions. Gene specific primers used were (5'-3'): WalkA CCATATTTTCACCATACACGACA; WalkB GAGACTTTCTCATCCCCAAAACT; WalkC CCCCAAAACTTGTACCCAAA.
For MommeD7, D8 and D9, FVB/C57 F1 heterozygous individuals were backcrossed to C57, and linkage analysis performed on their offspring. Phenotype was assigned based on GFP fluorescence profile. A panel of microsatellite markers that differ in size between FVB and C57 were used to localize the mutation to a small area of the genome. Mice wild type for the mutation should only have C57 chromosomes at the linked interval, while mice heterozygous for the mutation should carry both FVB and C57 chromosomes.
Linkage analysis in MommeD10 was carried out using an FVB/C57 F1 MommeD10-/+ intercross to produce 337 F2 individuals. MommeD10-/- mice were distinguished from their littermates by their dramatically reduced size at weaning and their reduced GFP expression profile. Recombination events allowed the linked region to be localized to a small genomic interval. MommeD10-/- mice should only carry FVB chromosomes at the MommeD10 linked region, while the remaining mice should be either FVB/C57 or C57/C57.
Blood smears were made from blood taken from the tail tip of MommeD7-/+ and MommeD7+/+ mice, and stained with New methylene blue. Full blood analyses were done on the automated haematology analyzer Sysmex Xe-2100 (Sysmex Corporation, Woodlands Spectrum, Singapore).
Reticulocyte analysis by FACS
Nucleated cells and reticulocytes were separated from mature erythrocytes based on propidium iodide fluorescence levels. An RNase control was performed and the presumptive reticulocyte cell population could no longer be detected. Mean GFP fluorescence was determined for reticulocyte and mature erythrocyte cell populations. This was done essentially as described in . Three adult MommeD7-/+ and three wild-type littermate controls were used. Approximately 25 μl of whole blood was collected from the tail in cold Osmosol buffer (Lab Aids Pty Ltd). Cells were fixed for 1 h at 4°C in 1% paraformaldehyde in Osmosol and then washed once in cold Osmosol. Cells were then permeabilized by adding -20°C ethanol to a cell pellet whilst vortexing, and incubated with rotation for 2.5 h at 4°C. A 40 μg/ml solution of propidium iodide was added to pelleted cells and the cells were incubated at 37°C for 30 minutes. Analysis was performed on a FACScan (Becton Dickinson). The data were analyzed using CELL QUEST software.
Following identification of the MommeD10 point mutation, genotyping was carried out by AciI digestion of a PCR product of exon 7 of Baz1b (primers available on request). The AciI site is created by the MommeD10 point mutation. Following identification of the MommeD5 mutation, genotyping was carried out by PCR amplification across the deleted region, and gel electrophoresis using Ultra low-range agarose (Bio-Rad, Hercules, CA, USA).
We prepared whole-cell lysates of 10.5 dpc embryos (MommeD5) and 14.5 dpc heads and adult spleen (MommeD10) in 2-3× volume lysis buffer (0.05 M Tris, 7 M urea, 150 mM NaCl). Samples were incubated on ice for 20 minutes, sonicated, centrifuged, and the supernatant collected. Approximately 5 μg (MommeD5) or 10 μg (MommeD10) of protein was separated by SDS-PAGE on a 4-12% gradient gel (Invitrogen, Carlsbad, CA, USA) and was analyzed with antibodies to the Hdac1 amino terminus (sc-6299, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Hdac1 carboxyl terminus (05-100, Millipore, Billerica, MA, USA), Hdac2 (05-814, Millipore), Baz1B (BL2067, Bethyl Laboratories, Montgomery, TX, USA) or γ-tubulin (T5192, Sigma-Aldrich, St Louis, MO, USA). Blots were quantified by normalizing the levels of Hdac1, Hdac2 or Baz1B in each lane to that of γ-tubulin. The normalized levels were then averaged across genotypes and the ratio to wild-type levels calculated. A peptide blocking experiment was carried out for the Hdac1 amino terminus using sc-6299P (Santa Cruz Biotechnology).
RNA preparation and quantitative RT-PCR
Total RNA was extracted from 14.5 dpc embryo heads using TRI reagent (Sigma-Aldrich). cDNA was synthesized from total RNA using SuperScriptII reverse transcriptase (Invitrogen) and random hexamer primers. Quantitative RT-PCR was performed with the Platinum SYBR Green qPCR SuperMix-UDG with primers designed to span exon/intron boundaries (available on request). All reactions were performed in triplicate and normalized to both GAPDH and ribosomal highly basic 23-kDa protein (Rpl13A) . The reaction was run on a Corbett Research Rotor-Gene (Qiagen, Doncaster, Vic, Australia).
Bisulfite sequencing of the transgene HS-40 enhancer
Bisulfite sequencing was carried out as described in . Oligonucleotides to the bisulfite converted HS-40 enhancer were as follows (5'-3'): GFPbisF1 AAAATAAAATTTTTGGATTGTTATTATTATAA; GFPbisF2 ATATTTGTAATTTTAGTATTTTGGGAGGTT; and GFPbisR AATCTCTACTCACTACAAACTCCATCTC. Cycling conditions were as follows: 94°C for 2 minutes for 1 cycle; 94°C for 30 s, 60°C for 30 s, 72°C for 45 s for 35 cycles; and 72°C for 6 minutes for 1 cycle.
Three heads of 4-week-old male mice of each genotype, and one head of female mice of each genotype (MommeD10-/-, MommeD10-/+ and MommeD10+/+) were scanned at 8.7 micron resolution using a SkyScan 1076 micro-computed tomography unit and the data set reduced to approximately 17 microns for three-dimensional reconstruction of the serial slices (CT Analyser software V.184.108.40.206; SkyScan, Kontich, Belgium). Twenty cranial landmarks and nine mandibular landmarks (based on those of Richtsmeier ) were located on each of nine skulls and inter-landmark measurements recorded using DataViewer software (V.220.127.116.11; SkyScan). To verify accuracy of the measurements, any landmarks showing marked differences between genotype groups were re-located on a separate day and the measurement repeated. The mean value of each measurement was compared between homozygotes, heterozygotes and wild-type animals.
RNA probes and whole-mount in situhybridization
DIG-labeled RNA probes were transcribed from linearized DNA templates and used in whole mount in situ hybridization analysis of wild-type FVB/NJ mouse embryos at a range of gestational stages. The probe was directed to 1.1 kb of the 3' untranslated region of Baz1b. A sense probe was used in earlier experiments to confirm specificity of the antisense probe. Whole mount in situ hybridization was performed as previously described . Embryo images were captured digitally using an Olympus SZX12 microscope with DP Controller software (Olympus Corporation).
Additional data files
The following additional data file is available with the online version of this paper. Additional data file 1 shows more craniofacial measurements between MommeD10+/+, MommeD10-/+ and MommeD10-/- mice.
fluorescence activated cell sorting
green fluorescent protein
Modifier of murine metastable epialle
standard error of the mean
Williams Beuren syndrome.
The authors would like to thank Belinda Washbourne and Dr Peter Self at Adelaide Microscopy for assistance with the microcomputed tomography scans and logistics, and Dr J Matthews at The University of Sydney for assistance with protein modeling. This study was supported by NHMRC grants to EW. AA, DKM, NCW, TB and NCB were supported by Australian Postgraduate Awards. NV was supported by an International Postgraduate Award (University of Sydney). CW and GJA are NHMRC Senior Research Fellows. EW was supported by a fellowship from the Queensland Institute of Medical Research.
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