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Human Molecular Genetics Advance Access originally published online on July 6, 2006
Human Molecular Genetics 2006 15(16):2457-2467; doi:10.1093/hmg/ddl168
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Cardiac malformations and midline skeletal defects in mice lacking filamin A

Alan W. Hart1, Joanne E. Morgan1, Jürgen Schneider2, Katrine West1, Lisa McKie1, Shoumo Bhattacharya2, Ian J. Jackson1 and Sally H. Cross1,*

1 Comparative and Developmental Genetics Section, MRC Human Genetics Unit, Edinburgh EH4 2XU, UK and 2 Department of Cardiovascular Medicine, University of Oxford, Wellcome Trust Centre for Human Genetics, Oxford OX3 7BN, UK

* To whom correspondence should be addressed. Tel: +44 1313322471; Fax: +44 1314678456; Email: sally.cross{at}hgu.mrc.ac.uk

Received May 1, 2006; Revised June 9, 2006; Accepted July 1, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The X-linked gene filamin A (Flna) encodes a widely expressed actin-binding protein that crosslinks actin into orthogonal networks and interacts with a variety of other proteins including membrane proteins, integrins, transmembrane receptor complexes and second messengers, thus forming an important intracellular signalling scaffold. Heterozygous loss of function of human FLNA causes periventricular nodular heterotopia in females and is generally lethal (cause unknown) in hemizygous males. Missense FLNA mutations underlie a spectrum of disorders affecting both sexes that feature skeletal dysplasia accompanied by a variety of other abnormalities. Dilp2 is an X-linked male-lethal mouse mutation that was induced by N-ethyl-N-nitrosourea. We report here that Dilp2 is caused by a T-to-A transversion that converts a tyrosine codon to a stop codon in the Flna gene (Y2388X), leading to absence of the Flna protein and male lethality because of incomplete septation of the outflow tract of the heart, which produces common arterial trunk. A proportion of both male and female mutant mice have other cardiac defects including ventricular septal defect. In addition, mutant males have midline fusion defects manifesting as sternum and palate abnormalities. Carrier females exhibit milder sternum and palate defects and misshapen pupils. These results define crucial roles for Flna in development, demonstrate that X-linked male lethal mutations can be recovered from ENU mutagenesis screens and suggest possible explanations for lethality of human males hemizygous for null alleles of FLNA.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Filamin A, encoded by the X-linked gene FLNA, is a large, widely expressed protein of 280 kDa with an actin-binding domain at its N-terminus followed by 24 tandem immunoglobulin-like repeat units of about 96 amino acids that are thought to fold into antiparallel beta-pleated sheet domains forming a rod-like structure (1). These repeats are interrupted by two flexible loops forming hinges, one between repeat units 15 and 16 and the other between repeat units 23 and 24. Filamin A dimerizes through the final repeat unit at the C-terminal end to form a flexible V-shaped homodimer, and this dimerization is essential for function. Filamin A crosslinks actin filaments into orthogonal networks and it is involved in the anchoring of membrane proteins to the actin cytoskeleton (1,2). It also regulates reorganization of the actin cytoskeleton by interacting with integrins, transmembrane receptor complexes, ion channels and second messengers (3). Remodelling of the cytoskeleton is key to the modulation of cell shape and migration; in melanoma cell lines deficient for filamin A, the plasma membrane displays blebbing and cell migration is impaired (4). The interaction of filamin A with both actin and membrane receptors for cell signalling molecules suggests that it may function as an important interface that connects and coordinates a large variety of cellular processes to the dynamic regulation of actin cytoskeleton (5).

In humans, a range of mutations of the X-linked FLNA gene have been identified: nonsense, missense and splice variants that give rise to various conditions (reviewed in 6). X-linked periventricular nodular heterotopia (PVNH) is associated with nonsense, splice and frameshift mutations that presumably lead to loss of function (7). In this disorder, neurons fail to migrate into the cerebral cortex and remain as nodules in the subependymal or periventricular region and female patients may suffer from epileptic seizures and other defects, including patent ductus arteriosus (8,9). Although most hemizygous males harbouring FLNA mutations that cause PVNH in females die early during embryogenesis, some cases have been reported where affected males have died post-natally owing to haemorrhaging (9,10). However, a few males with PVNH, some with other complications, have been identified who have mutations that are thought to retain residual function and/or who are mosaics of normal and mutation-bearing cells (11–13). Missense mutations and in-frame deletions thought to confer gain of function disrupt developmental processes involved in craniofacial, skeletal and other organ systems, resulting in a spectrum of disorders: otopalatodigital syndrome types 1 and 2 (OPD1 and OPD2), Melnick–Needles syndrome (MNS) and frontometaphyseal dysplasia (FMD) (14). Missense and loss-of-function mutations have also been found underlying a syndrome incorporating PVNH and Ehlers-Danlos syndrome (15). Compound phenotypes have been found where single base substitutions lead to the production of two types of transcript. In one case of a female patient with a combined PVNH/FMD phenotype, a missense mutation is incorporated into one transcript and the other contains a deletion (16). In a male patient with PVNH, constipation and facial dysmorphism resembling cerbro-fronto-facial syndrome, a normal transcript and a truncated transcript owing to a frameshift, have been found (17).

We report here the identification of a loss-of-function mutation in the mouse Flna gene that causes mild skeletal abnormalities in female carrier mice and results in male lethality because of incomplete septation of the heart during gestation accompanied by other cardiac, skeletal and palate defects. This Flna mouse mutant provides a model for the study of filamin A function and will be useful for understanding how mutations in this gene lead to human disease.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Dilp2 is caused by a point mutation in the Flna gene
We identified the X-linked, male-lethal, N-ethyl-N-nitrosourea (ENU)-induced mouse mutation Dilp2 in a screen for dominant eye mutations as a founder female with misshapen pupils (18). The ENU-mutagenized parental strain was Balb/c, and this was crossed to C3H/HeN for screening. We backcrossed Dilp2 to C57BL/6, and 0/114 of the male offspring carried the mutant X-chromosome. In addition, the number of carrier females (65/159) was smaller than expected ({chi}21d.o.f.=5.29, P=<0.05). This ~30% reduction in the number of carrier females is probably caused by the random nature of X chromosome inactivation such that those females with the mutant X chromosome active in a higher proportion of cells do not survive. The misshapen pupil phenotype was incompletely penetrant; most obligate carriers had only one eye affected or were unaffected. We therefore mapped the Dilp2 mutation using the fully penetrant male embryonic lethal phenotype. We had previously mapped Dilp2 on the X chromosome between DXMit81 and DXMit38 (18). To refine the map position, we analysed the C57BL/6 backcross progeny and reduced the genetic interval to 386 kb between the single nucleotide polymorphism (SNP) rs3157210 located in the third intron of the L1cam gene and SNP rs29035820 located in the third intron of the Emd gene. This interval contains all or part of 13 predicted genes (NCBI Mouse Build 35, http://www.ensembl.org) (Supplementary Material, Table S1). We prioritized candidate genes on the basis of expression patterns, likely function and known human or mouse mutant phenotypes and sequenced the exons and splice junctions of five candidates from the region, a total of 88 protein-coding exons, comprising about 19.5 kb of protein-coding sequence and splice sites. We found a single nucleotide change, a T-to-A transversion in exon 44 of the Flna gene at position 7164, which converts a tyrosine codon to a nonsense codon at position 2388 in the protein (Fig. 1A). On the basis of abnormal skeletal phenotype of affected male embryos and female carriers (described in the Results section entitled "The Dilp2 mutation causes skeletal defects"), we considered this to be a particularly good candidate for the Dilp2 mutation, as mutations of the human orthologue can cause skeletal abnormalities (14). In our screen, we found 25 inherited ENU-induced mutations that cause eye defects (18). Two have not yet been mapped to high resolution. For the others, we have attempted to isolate the underlying causative lesion and for all 23 we have found a nucleotide base change that affects either the coding region sequence or splice site of a gene from within each candidate interval (18–22, unpublished data and this work). Similar observations have been made for ENU-induced mutations affecting the formation of T lymphocyte subsets, leading to the suggestion that most mutations in non-protein-coding genomic sequence do not have profound effects on phenotype (23). Taken together, these findings indicate that ENU mutations occurring in functional DNA outside coding exons are extremely unlikely to be detected in screens designed to detect major phenotypic changes. As the frequency of functional base changes in DNA using the mutagenesis regime employed is about 1 in 1.82 Mb (24), it is highly likely that the Dilp2 phenotype can be attributed solely to the mutation found in the Flna gene and that no nearby mutation is responsible for or contributes to the mutant phenotype.


Figure 1681
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  		Figure 1. Flna is mutated in Dilp2 mice. (A) Genomic DNA sequence traces from Flna exon 44 from wild-type, a carrier female (Dilp2/+) and hemizygous Dilp2 male (Dilp2/–). (B) Sequence analysis of Flna RT–PCR products from wild type, three carrier females (Dilp2/+) and hemizygous Dilp2 male (Dilp2/–) E14.5 embryos. The arrowhead points to the position of the T-to-A transversion in the Dilp2 allele in (A) and (B). (C) Immunoblotting of protein extracts from E15.5 embryos and MEFs of wild type (WT), heterozygous female (H) and hemizygous mutant male (M) genotypes with anti-Flna antibody BL877 (upper part) and anti-tubulin antibody as a loading control (lower part).

 
What is the effect of the T7164A transversion on Flna expression?
Translation of the Dilp2 mutant transcript would produce a truncated protein terminating in the immunoglobulin-like repeat unit 22 lacking the final 260 amino acids of the protein, including the dimerization domain and several of the domains known to be necessary for interactions with binding partners. To determine whether the defects we observe in mutant mice are associated with a truncated Flna protein, or whether the protein is absent, we first sequenced Flna RT–PCR products. RNA from affected male embryos yielded products containing only the Dilp2 mutation, as expected, and the weak sequence trace implied low yield of PCR product and suggested that very low levels of transcript were present (Fig. 1B). In samples from three heterozygous female embryos, only the wild-type sequence was evident (Fig. 1B). These results suggest one of the two alternatives; either there is skewing of X chromosome inactivation in favour of the X chromosome carrying the wild-type allele in the heterozygous females or the mutant transcript is subject to nonsense-mediated decay (NMD). We tested the first possibility by sequencing RT–PCR products generated from a second X-linked gene (Maged1) containing a coding SNP (rs13459181). Transcripts from both X chromosomes were present in samples from three heterozygous female embryos and thus inactivation was not grossly skewed (data not shown). The presence of a termination codon more than 50 nucleotides upstream of the final exon–exon junction (measured after splicing) leads to NMD (25). The termination codon introduced by the Dilp2 mutation in the Flna transcript is 590 nucleotides before the final exon–exon junction between exons 46 and 47. It seems likely that NMD is responsible for the low level of mutant transcript observed in the mutant male and the absence of mutant transcript observed in heterozygous females (Fig. 1B). Immunoblotting of protein extracts from embryos and mouse embryonic fibroblasts (MEFs) using an anti-Flna antibody showed that Flna protein was undetectable in mutant males (Fig. 1C), indicating that the amount of protein translated from the mutant allele is below detection levels, or that any truncated protein produced from residual mutant transcript is rapidly degraded.

Functional consequences of Flna mutation
To examine the effects of the mutation on cell morphology, we stained MEFs prepared from wild-type, mutant male and carrier female embryos with anti-Flna antibody and for actin (Fig. 2). Flna staining is not detectable in the Dilp2 male cells (Fig. 2I) and there is a mixed population in the carrier female cells, as a consequence of X chromosome inactivation (Fig. 2E). Although filamin A binds to actin filaments, there is no observable change in the distribution of the actin network in mutant cells (Fig. 2B, C, F, G, J and K). It has been reported that FLNA-deficient human melanoma cells have blebbing and impaired locomotion (4). However, there is no apparent difference in cell morphology of mutant versus wild-type cells, in particular, there is no blebbing, indicating that Flna deficiency does not affect fibroblast morphology (Fig. 2D, H and L). In addition, Flna is thought to play a role in cell migration (4). We used a wound-healing assay to test cell migration in culture and found no difference between wild-type and mutant MEFs in their ability to migrate (Fig. 3). Furthermore, lack of Flna has no adverse effect on cell growth rates (data not shown).


Figure 1682
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  		Figure 2. Flna protein is absent from Dilp2 mutant cells. MEFs were prepared from wild-type (A–D), Dilp2 carrier female (E–H) and Dilp2 mutant male (I–L) E13.5 embryos. Flna protein (red) is present in all wild-type cells (A) and some of the Dilp2 female carrier cells (E) and is absent from the Dilp2 male mutant cells (I). Actin (green) is present in all cells of all three genotypes (B, F and J). (C, G and K) Merged images. (A–C, E–G, I–K) DAPI staining is shown in blue. Absence of Flna protein does not affect cell morphology: compare the DIC images for wild-type (D), mixed population of cells from the female Dilp2 carrier (H) and the cells from the Dilp2 male mutant (L). Bar=20 µm.

 


Figure 1683
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  		Figure 3. Cell migration is unaffected in Dilp2 mutant MEFs. Wild-type (A–D) and Dilp2 mutant (E–H) MEFs were plated on tissue culture dishes coated with fibronectin. The cells were wounded by scoring with a pipette tip (edges of wound are shown by white dotted lines in A, E, C and G) and images were captured at 0 h (A and E) and 6 h (B and F) or 0 h (C and G) and 12 h (D and H). Representative images are shown from at least three independent experiments. The absence of the Flna protein does not appear to effect cell migration (compare B with F and D with H). Bar=100 µm.

 
Dilp2 mutant mice die during mid-gestation and have cardiac defects
In humans, loss-of-function mutations of FLNA are associated with male embryonic lethality, but the cause of lethality is unknown (8,9). To investigate the embryonic lethality of the mutant male mice, we examined embryonic day 14.5 (E14.5) and E15.5 embryos. At a gross level, mutant male embryos were oedematous, exhibited haemorrhaging and presented with delayed resorption of their umbilical hernia (Fig. 4B). After E15.5, male embryos died and were not present at later stages of development. Magnetic resonance imaging (MRI) revealed major heart defects. All the affected males examined by MRI (n=7 from four litters) had common arterial trunk in which there is no separate aorta and pulmonary artery, but a single vessel emerges from the right ventricle (Fig. 5B). In addition to common arterial trunk, other heart abnormalities were observed: ventricular septal defects (VSDs) (Fig. 5F), thickening of the mitral valve (dysplasia) and atrial septal defect primum. Two of the eight heterozygous females examined by MRI also had VSDs; one also had double outlet right ventricle, in accord with the ~30% lethality of female carriers. The defects were confirmed by haematoxylin and eosin staining of serial sections from MRI-scanned embryos and also embryos from additional litters (Fig. 5D and H). A common arterial trunk would indicate that developmental processes had gone awry at mid-gestation. To investigate this, we examined the developing heart at E10.5 and E11.5. Separation of the major arteries requires migration of cells of neural crest origin into the heart (26) as well as primary heart cells in situ. By in situ hybridization for Erbb3, we examined cardiac neural crest cells in mutant and heterozygous embryos at E10.5. No differences could be detected in the spatial distribution of these cells (Fig. 6A and B). Thus it appears that the neural crest cells required for outflow tract development are recruited normally to the heart in the Dilp2 mutant. However, in situ analysis of the expression of the transcription factor Tbx20, a marker for primary heart cells, at E11.5 demonstrates subtle differences in the mutant heart (Fig. 6C–H). Examination of the gross structure reveals that in the mutant heart, the outflow tract seems to be more centrally located and the right ventricle appears smaller compared with the wild type and heterozygous female (Fig. 6C–E). Using optical projection tomography (OPT), we scanned these embryos and made 3D reconstructions of the expression pattern in the developing heart (Fig. 6F–H). The Tbx20 expression shows the outflow tract separating in the wild type and heterozygous female but failing to separate in the mutant (Fig. 6F–H). These findings are similar to those observed in the Tbx20 knockdown model where similar right ventricle hypoplasia and outflow tract abnormalities have been described (27).


Figure 1684
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  		Figure 4. Gross phenotype of Dilp2 mutant embryos. (A) Normal wild-type E15.5 embryo. (B) Hemizygous Dilp2 male E15.5 embryo demonstrating oedema, haemorrhaging and an umbilical hernia (asterisk).

 


Figure 1685
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  		Figure 5. Cardiac malformations in Dilp2 male embryos. (A, C, E, Gand I) Transverse sections and 3D reconstruction (left-ventral oblique view) of hearts from wild-type embryos at E15.5. Corresponding images from Dilp2 hemizygous male embryos (B, D, F, H and J). Arrow heads in (A) and (C) indicate the correct location of the aorta and pulmonary artery in wild-type embryos. In contrast, the single arrowhead in (B) and (D) reveals a common arterial trunk in the Dilp2 male embryos. In addition, arrows in (F) and (H) show a VSD present in the Dilp2 male embryos. (A, B, E and F) Analysis using MRI. (C, D, G and H) Haematoxylin and eosin staining of 7 µm thick sections. (I and J) 3D reconstructions where the grey areas show the ventricular muscle rendered semi-transparent. Bar=500 µm (A, B, E, F, I and J); 400 µm (C, D, G and H).

 


Figure 1686
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  		Figure 6. Origins of the cardiac defect in Dilp2 mutant embryos. In situ hybridization of E10.5 embryos with Erbb3 to detect migrating cardiac neural crest cells (A and B). Hybridization reveals no difference between heterozygous female (A) and hemizygous mutant male (B) embryos. Whole-mount in situ hybridization analysis of E11.5 embryos showing Tbx20 expression in cardiac development (C–H). The outflow tract loops leftwards in the wild type and Dilp2 heterozygous female (C and D), whereas it has a central trajectory in the Dilp2 male (E). The edges of the outflow tract are highlighted with dashed white lines (C–E). The right ventricle appears smaller in the Dilp2 male (compare E with C and D). OPT of the in situ hybridization shows the outflow tract separating in wild type and heterozygous female embryos (white arrows) (F and G) but failing to divide in male mutants (H). CNCC3, CNCC4, CNCC6, cardiac neural crest cells in aortic arches 3, 4 and 6. OFT, outflow tract; LV, left ventricle; RV, right ventricle; RA, right atrium; LA, left atrium; OV, otic vesicle.

 
The Dilp2 mutation causes skeletal defects
Skeletal analysis of Dilp2 males revealed sternum defects with a failure to fuse at E15.5 (Fig. 7F). This defect could underlie the poor resorption of the umbilical hernia observed in Dilp2 males (Fig. 4B), as the growing visceral organs are allowed to spill out of the abdominal cavity. In all carrier females examined, we observed incomplete fusion of the sternum at E15.5 (n=8), although this was less severe than in Dilp2 males (Fig. 7E). By E18.5 (n=3), sternal fusion in females is still not complete (Fig. 7H) and although fusion occurs by the adult stage, the sternum appears compressed and misshapen and the xiphoid process is abnormal (Fig. 7J). In contrast to the incomplete penetrance of the dilated pupils phenotype, the sternum defect of female carriers is highly penetrant.


Figure 1687
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  		Figure 7. The Dilp2 mutation causes palate and skeletal abnormalities. (A–C) Ten micrometre thick coronal sections of palates from E15.5 embryos (A) Wild type (B) Dilp2 carrier female and (C) Dilp2 hemizygous male. In the wild type, the palatal shelves have elevated and fused (A); in the female carrier, there is still a small gap (B) and in the Dilp2 male mutant, the palatal shelves have failed to elevate (C). Skeletal preparations from (D–F) E15.5 embryos, (G and H) E18.5 embryos and (I and J) adult mice. The sternum has fused in the wild-type animals at all ages (D, G and I). There is no fusion in the Dilp2 male mutant embryo (F) and fusion is incomplete in the Dilp2 carrier female embryos (E and H); in the adult Dilp2 carrier female, although fusion has occurred, the sternum is thicker and shorter than wild-type, the xiphoid process is misshapen and the second to fourth sternebrae are all fused together (square bracket) (J). Bar=500 µm (A–C), 800 µm (D–F), 1600 µm (G and H).

 
Histopathology revealed further skeletal defects in mutant males and some carrier females (Fig. 7A–C). At E15.5 in mutant males, the palatal shelves fail to elevate (Fig. 7C), resulting in a cleft palate. In many carrier females, the palatal shelves, although elevated correctly, remain incompletely fused, probably representing a delay in development. The extent of the defect in carrier females presumably depends on the proportion of cells in which the mutant X chromosome is active (Fig. 7B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Loss of function of Flna does not affect fibroblast cell morphology or migration
Dilp2 is a nonsense mutation of Flna leading to NMD of mRNA and absence of Flna protein. The finding that Dilp2 mutant fibroblasts have normal morphology and migrate in a fashion indistinguishable from wild-type cells (Figs 3 and 4) is surprising in the light of previous work. Human melanoma cells deficient for FLNA display abnormal cell morphology and cell migration defects that can be rescued by the introduction of exogenous FLNA (4). The different cell types studied may account for the dissimilar phenotypes. Melanoma cells have a greater migratory capacity than many other cell types and FLNA may be partly responsible for their enhanced migration. Furthermore, their transformed nature may also lead melanoma cells to have altered response to environmental cues, possibly mediated through signalling systems interacting with FLNA.

Dilp2 mutant mice as models for human FLNA diseases
Loss of Flna in Dilp2 males is lethal and affected embryos die at E15.5. They display oedema, have an umbilical hernia and have common arterial trunk accompanied by other cardiac defects (Figs 4 and 5). In addition, Dilp2 causes the sternum to remain unfused and the elevation of the palatal shelves to fail (Fig. 7). All the defects are found in the midline and involve structures where cell movement and fusion events are crucial in their formation. In humans, the consequences of different types of mutations in the FLNA gene fall into two classes. Loss-of-function mutations underlie PVNH in female patients and cause lethality in males (7,9,10). Although most males with truncating mutations die early during embryogenesis, some die as neonates owing to haemorrhaging (9,10). In one case, an affected male died at 1 week of age and was found to have cardiovascular malformations including both atrial and ventricular septal defects along with other abnormalities (12). Our finding that male Dilp2 mice have common arterial trunk may indicate the basis of embryonic lethality in human males with loss-of-function mutations in FLNA.

Localized missense mutations in FLNA, thought to confer gain of function, have been found in disorders that all involve skeletal dysplasia accompanied by a spectrum of additional developmental abnormalities (14). Clustering of the mutations in particular regions of the gene indicates that specific interactions of the FLNA with one or more of its partners is being disrupted or altered. It is striking that the skeletal and palate defects we find in mice deficient for Flna have similarities to those of the missense disorders in humans. Both OPD1 and OPD2 syndromes feature cleft palate, and some affected OPD2 males have been reported with omphalocele, persistence of the umbilical hernia (28). Omphalocele and cleft palate have also been found in males with MNS (6,29). Both these anomalies were found in affected Dilp2 males (Figs 4B and 7C). Although sternal defects are not a major component of FLNA disorders, they have been noted in association with MNS (30). One of the cardiac abnormalities that we observe in Dilp2 mutant males is mitral valve dysplasia. In humans, missense FLNA mutations have been reported to cause X-linked myxomatous plurivalvular dystrophy where the mitral valve is affected (31). In contrast to the human loss-of-function mutations, we find no evidence of abnormal neuronal migration as a consequence of the Dilp2 mutation. We examined transverse brain sections of five carrier female for signs of PVNH but did not observe nodules (data not shown). This suggests that there may be compensatory factors responsible for the successful migration of neurons in the absence of Flna in mice. One possible candidate is filamin B, which shares ~70% homology with Flna and is highly expressed in the ventricular and subventricular zones in the mouse (32).

Dilated pupil phenotype
Why does the Dilp2 mutation cause the pupil to be misshapen? This phenotype is clearly modulated by background effects as the incidence of abnormal pupils decreased with repeated backcrossing onto C57BL/6. Flna is thought to be involved in sequestering of the transcription factor Foxc1 to a heterochromatin-rich inactive partition of the nucleus (33). Absence of Flna may thus result in an increase in available Foxc1. In humans, both deletion and duplication of the chromosomal region containing FOXC1 can cause anterior segment defects including iris hypoplasia (34–36). In the mouse, although the consequences of elevated levels of Foxc1 are unknown, haploinsufficiency results, as in humans, in eye defects, including abnormal pupils, in a strain-dependent manner (37).

Functional consequences of loss of function of Flna in the mouse
Although Flna deficiency does not appear to have any major effect on fibroblast cells, the defects in both Dilp2 hemizygous male mice and female carriers all involve fusion defects. The heart defects caused by Flna deficiency are particularly striking. Outflow tract abnormalities account for about one-third of congenital heart defects in humans. The separation of the outflow tract into a separate aorta and pulmonary artery is crucially dependent on a number of tissues and cells: outflow myocardium and endocardium, secondary heart field, neural crest cells and primary heart field. We investigated expression patterns for cells marked by Erbb3 (migrating cardiac neural crest cells) and Tbx20 (primary heart field) (Fig. 6). Neural crest cells appear to be grossly normal with respect to spatial pattern, which suggests that the septation defect observed may lie elsewhere. Indeed, in situ hybridization analysis of Tbx20 at E11.5 suggests that the spatial expression pattern of this gene may be altered. An in-depth analysis of this phenotype with other markers may reveal other proteins and/or pathways affected by loss of Flna. It is possible that the palatal and sternal defects are due to localized migratory defects or to a particular change in cell shape or polarity. This possibility is supported by work investigating the mechanistic role that FLNA plays in the genesis of PVNH where expression of mutant FLNA resulted in cells being unable to adopt consistent polarity and their motility was affected (38). The range of abnormalities seen in hemizygous and heterozygous embryos and cells are not consistent with profound defects in cell migration; rather, Flna appears to be required for cellular interactions and tissue fusion in the formation of the outflow tract and midline structures. Dilp2 provides a mouse model for further studies on Flna function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Animals
The animal studies described in this paper were carried out under the guidance issued by the Medical Research Council in Responsibility in the Use of Animals for Medical Research (July 1993) and Home Office Project Licence nos. PPL60/2242 and PPL60/3124. The Dilp2 strain has been submitted to the European Mouse Mutant Archive (http://www.emmanet.org/) strain number EM:00387.

Linkage analysis
PCR products containing informative SNPs were amplified from genomic DNA extracted using standard procedures from BALB/c (the ENU-mutagenized strain), C57BL/6 (the strain used for backcrossing) and backcross progeny and sequenced as described in the following section.

Mutational analysis of candidate genes
Exons and the immediate flanking sequences of candidate genes were amplified from Dilp2, BALB/c, C3H and C57BL/6 genomic DNA using intronic primers that were also used for subsequent sequence analysis. PCR products were purified using Millipore Multi-screen PCR 96-well filtration system on a Biomek 2000 robotic platform and sequenced directly using Big DyeTM terminator cycle sequencing. Sequences were analysed using the SequencherTM program.

Reverse transcription–polymerase chain reaction
Total RNA was isolated from E14.5 embryos using Tri-ReagentTM (Sigma). First strand cDNA was synthesized using a first Strand cDNA Synthesis Kit for RT–PCR (AMV) (Roche) using an oligo-(dT) primer. An aliquot was then amplified by the PCR using primers Flnaex42-44F (5'-TACCTGATAGCCCCTTCGTG-3') and Flnaex42-44R (5'-GGATGAAACGCACAGCATAC-3') using BIO-X-ACT (Bioline). PCR products were purified and sequenced as described in the previous section.

Immunoblotting
Protein extracts from wild-type, heterozygous and Dilp2 mutant E15.5 embryos and wild-type and Dilp2 mutant MEFs were prepared using standard protocols. Samples were separated on 10% SDS–PAGE gel, transferred to Hybond-P (Amersham Biosciences) and probed with anti-Flna antibody BL877 (Bethyl Laboratories, Inc.) diluted 1:1000 using standard protocols and visualized with horseradish-peroxidase secondary antibody and ECL detection (Amersham Biosciences) following the manufacturer's instructions. The epitope recognized by BL877 maps to a region between residues 400 and 450 of human Flna, which would be present in truncated protein produced from the FlnaDilp2 allele. We used immunoblotting with anti-tubulin antibody (gift of Dr Richard R. Meehan) as a loading control.

MEF preparation and immunohistochemistry
MEFs were prepared from E13.5 embryos after the removal of the head and red organs by mincing the torso, digesting with 1.5% trypsin and seeding onto 10 cm cell culture dishes before splitting once at 1:4 and freezing. Red organs were used to genotype the embryos. For fluorescent immunohistochemistry, cells were plated at 105 cells/ml (250 µl/well) on eight well chamber slides. After 24 h culture, the wells of slides were removed and treated for 10 min with 0.1% Triton X-100 in PBS. After washing twice in PBS for 5 min, they were blocked for 1 h in 90% FCS and 10% PBS at room temperature. Cells were labelled with Oregon Green Phalloidin (Molecular Probes) and anti-Flna antibody pab228 (39) diluted 1:40 and 1:500, respectively, in PBS + 10% FCS and incubated for 1 h at room temperature. After washing in PBS, slides were incubated with TXR-conjugated donkey anti-rabbit IgG (Jackson Immunoresearch) diluted 1:400 in PBS+10% FCS. Finally, slides were incubated for 5 min at room temperature with 0.05 µg/µl DAPI (Sigma)+0.01% Triton X-100 and then mounted in Vectashield (Vector). The pab228 antibody was raised against the C-terminal end of Flna, only 31 amino acids of which would be present in the putative truncated protein produced from the Dilp2 allele. Lack of staining in this experiment cannot be taken as evidence of lack of expression, but serves to identify mutant cells. The BL877 antibody used for immunoblotting did not work in immunohistochemistry. Cells were visualized using a Zeiss Axioplan II fluorescence microscope fitted with Chroma #83000 filter set (Chroma Technology Corp, Rockingham, VT, USA) and Coolsnap HQ CCD camera (Roper Scientific, Tucson, Arizona, USA). Fluorescent and differential interference contrast (DIC) image capture was controlled by in-house scripting of IPLab Spectrum (Scanalytics, Fairfax, VA, USA) and processed using Adobe Photoshop 7.0 (Adobe, Inc.).

Wound-healing assay
Wound-healing assays were carried out essentially as described (40). Lines were etched using a razor blade on the underside of 24-well tissue culture dishes and used as reference marks during image acquisition. The plates were incubated with 10 µg/ml fibronectin (Sigma) diluted in PBS overnight at 4°C. This was replaced with 2 mg/ml BSA (Sigma) diluted in PBS for 1 h at 37°C. Wells were then washed once with PBS, and 105 MEF cells were plated/well in culture medium to create a confluent monolayer. After 4 h incubation under standard conditions to allow the cells to adhere and spread, wounds were created by scoring with a sterile P200 pipette tip. After washing the wells once with culture medium and replacing with fresh medium, cells were incubated under standard conditions for up to 12 h and images were captured at appropriate time points. The imaging system comprises a Hamamatsu Orca ER CCD camera [Hamamatsu Photonics (UK) Ltd, Welwyn Garden City, UK] and Zeiss Axiovert 100 inverted microscope (Carl Zeiss, Welwyn Garden City, UK). Image capture was performed using in-house scripts written for IPLab Spectrum (Scanalytics).

Magnetic resonance imaging
Pregnant mice were sacrificed by cervical dislocation at E14.5 or E15.5. Embryos were dissected and fixed for 7 days in 4% paraformaldehyde and 2 mM Gd-DTPA (Magnevist, Schering) in PBS at 4°C. Multiembryo MRI was performed as described (41) and the image files analysed using Amira software (TGS Europe, Merignas, France).

Embryo histology and skeletal analysis
After dissection, embryos were photographed and fixed overnight in 4% paraformaldehyde in PBS at 4°C. A small part of the tail was used for genotyping. After washing in PBS, they were dehydrated by immersion in a series of increasing concentrations of alcohol, embedded in paraffin wax and sectioned and stained with haematoxylin and eosin. Skeletal samples were prepared and stained with Alcian Blue and Alizarin Red as described (http://www.eumorphia.org/EMPReSS). Slides and embryonic skeletal samples were viewed on a Leica MZFLIII fluorescence stereo microscope fitted with a Coolsnap colour camera (Roper Scientific). Image capture was controlled by in-house scripting of IPLab Spectrum (Scanalytics).

Whole-mount RNA in situ and OPT
Digoxygenin-labelled riboprobes for Tbx20 and Erbb3 were generated and whole-mount in situ hybridization was performed essentially as described (42). OPT was carried out essentially as described (43). Analysis and visualization of OPT data were performed by BIOPTONICS (http://www.bioptonics.com/).


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We would like to thank Nick Hastie for critical reading of the manuscript. We thank Bo van Deurs and Richard Meehan for the gift of antibodies, Paul Perry for help with microscopy, Nelly da Silva for help with MEF culture, Gwen Cranston for help with OPT and Sandy Bruce for assistance in preparing the figures. This work was funded by the Medical Research Council, UK, a grant from the European Commission under FP5, EUMORPHIA project (QLG2-CT-2002-00930), the Wellcome Trust and the British Heart Foundation. The original ENU mutagenesis program was supported by funding from GlaxoSmithKline.

Conflict of Interest statement. The authors declare that they have no conflicting interests.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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