BACKGROUND
congenital malformations are a major cause of death in childhood, and are typically characterized by lesions that do not compromise fetal survival.
for instance, congenital heart disease  typically consists of lesions such as ventricular and atrial septal defects, which are compatible with fetal hemodynamics  <cit> .
although human genetic studies have identified some genes that cause congenital cardiac malformations, the molecular and developmental mechanisms underlying most of these defects remain largely unknown.
despite the high incidence of chd , only a handful of genes have been identified that when mutated, result in congenital heart disease  <cit> .
the mouse is a particularly good model for studying mechanisms of cardiac diseases as its anatomy and development resembles that of the human more closely than any other genetically tractable organism.
importantly, the mouse is amenable to genotype-driven approaches such as transgenic knockouts of defined candidate genes  <cit> , genome wide mutagenesis approaches using gene-trap  <cit>  or transposon insertion screens  <cit> , and high-throughput phenotype-driven screens that rely, for instance, on n-ethyl-n-nitrosourea  mutagenesis  <cit> .
however, high-throughput cardiovascular genomic approaches in the mouse have been hampered by the paucity of phenotyping tools that allow efficient identification of complex cardiac malformations.
as mouse embryos are opaque, late developmental defects are particularly difficult to identify.
for instance, cardiac septal defects or outflow tract abnormalities can only be confidently identified after  <dig>  days post coitum  when cardiac and outflow tract septation are completed in normal embryos.
the identification of malformations in late gestation embryos typically relies on serial histological sectioning, which is extremely labor intensive.
furthermore, this often results in the irretrievable loss of 3d information, which is essential for the interpretation of complex cardiac malformations.
in addition, standard pathological analysis is not amenable to high-throughput phenotype screening protocols that are required for any mutagenesis screen aiming at the functional dissection of the developmental biology of cardiac diseases.
therefore, new technological approaches must be harnessed that allow an efficient phenotyping of heart defects and also of subtle cardiac abnormalities that are at danger of being overseen in traditional histopathology screens.
this is even more important in the light of upcoming new endeavors in functional mouse genome annotation  <cit> .
currently, new large-scale mouse mutagenesis screens are being set up in the us and in europe that aim to produce heritable mutations in every gene in the mouse genome  <cit> .
to make full use of these new mouse mutant resources more precise and efficient phenotyping methods are urgently needed  <cit> .
the success of genome wide saturation mutagenesis screens depends therefore on improved phenotyping, and new high-resolution imaging approaches for mouse mutants are one of the most important which need to be established.
we previously reported the development of fast gradient-echo mri of single mouse embryos  <cit> .
this resulted in the acquisition of a 3d dataset in under  <dig> hours, with an experimental image resolution of  <dig> ×  <dig> ×  <dig> μm/voxel.
we showed that mri is capable of accurately identifying normal embryonal structures, and cardiac and adrenal malformations in knockout mouse embryos, and we have validated this technique by performing in depth histological examinations of imaged embryos  <cit> .
these experiments showed that single-embryo mri could correctly identify all cardiac lesions  except those under  <dig> μm – which is below the resolution of the mri technique.
as this method images single embryos in overnight runs, it still lacks the throughput required for phenotype-driven mutagenesis screens.
for instance, in a typical recessive enu mutagenesis screen, to screen  <dig> enu mutant lines using a 3-generation breeding scheme would require the analysis of ~ <dig> embryos  <cit> .
we now report the development of a method of imaging up to  <dig> embryos simultaneously in a single unattended overnight run, at high spatial resolution.
allowing ~ <dig> minutes per embryo, the analysis of  <dig> embryos would take  <dig> working days for a single trained individual.
we show that this high-throughput multi-embryo mri technique can be used to rapidly identify unsuspected embryonal cardiac and visceral malformations.
using this technique we could identify a novel, and hitherto unsuspected role for the phosphatidylserine receptor  in controlling ventricular septal, outflow tract and pulmonary artery development.
in addition, we found thymus hypoplasia in ptdsr-deficient embryos.
these findings suggest that a novel ptdsr-mediated pathway is required for cardiac and thymus development.
RESULTS
multi-embryo imaging
we modified our previously described fast gradient echo magnetic resonance imaging technique  <cit>  to image embryos embedded in four to eight layers  in  <dig> mm nuclear magnetic resonance tubes using a single quadrature driven birdcage coil .
preparation and embedding of embryos typically took less than an hour.
in initial experiments we imaged up to  <dig> embryos simultaneously in overnight runs of < <dig> hours, with an experimental resolution of  <dig> ×  <dig> ×  <dig> μm.
subsequently, we used a custom made optimized probe with an increased sensitivity range to enhance imaging throughput.
this allowed us to image  <dig> embryos simultaneously , but with a larger matrix size and an increased field-of-view in the long axis of the tube.
for these experiments we imaged embryos for ~ <dig> hours, and achieved an improved experimental resolution of  <dig> ×  <dig> ×  <dig> μm.
the optimized coil used for  <dig> embryos mri  has a sensitivity range in z-direction of close to  <dig> mm.
the artefacts seen at both ends in the longitudinal image  are caused by b1-inhomogeneities at the end-rings of the coil.
however, accurate image analysis for the entire data set remains possible if the height of the embryo stack does not exceed approximately  <dig> mm as demonstrated in the corresponding axial views of the top and the bottom layer .
the resolution achieved with the multi-embryo mri technique allowed us to visualize the heart, cardiac septa, central nervous system, and visceral organs in fine detail , in embryos taken from each layer.
the data are permanently archived on dvds, for subsequent analysis.
construction of 3d reconstructions of the heart typically takes ~ <dig> hours, but was not necessary for the identification of cardiovascular defects.
sensitivity and specificity of multi-embryo imaging
to assess the sensitivity and specificity of multi-embryo imaging in comparison to single-embryo imaging, we used a model of cited <dig> deficiency  <cit> .
embryos lacking cited <dig>  have diverse cardiac malformations, including atrial and ventricular septal defects, outflow tract and aortic arch malformations, and adrenal agenesis  <cit> .
as the trp53-repressor gene cdkn2ap19arf is a target of cited <dig>  <cit> , we also examined embryos lacking both cited <dig> and trp <dig> to determine if this would rescue the heart and adrenal defects in cited2-/- mice.
we imaged  <dig> embryos using the multi-embryo technique in the 16-embryo mode.
embryonal genotypes included  <dig> wild-type,  <dig> cited2+/-,  <dig> cited2-/-, three cited2-/-: trp53-/-, four cited2-/-: trp53+/-, two trp53+/-, and two trp53-/-.
we analyzed the data from each embryo for cardiac malformations without knowledge of the genotype.
this typically took a maximum of  <dig> minutes per embryo.
we scored each embryo for atrial and ventricular septal defects , outflow tract , and aortic arch malformations , and for adrenal agenesis.
each embryo was then re-imaged singly at high resolution, and the data re-analyzed as before.
in this group we identified  <dig> embryos with asd,  <dig> with vsd,  <dig> with outflow tract defects,  <dig> with aortic arch defects, and  <dig> with bilateral adrenal agenesis, using high-resolution single embryo imaging.
in comparison to single embryo imaging, the overall sensitivity and specificity of multi-embryo imaging for cardiac malformations was 88% and 92% respectively.
for asd  the sensitivity and specificity was 85% and 95%; for vsd 94% and 94%; for outflow tract malformations 94% and 100%; and for aortic arch malformations 91% and 100% respectively .
for bilateral adrenal agenesis, the sensitivity was 100% and specificity was 95% .
embryos lacking both cited <dig> and trp <dig> had cardiovascular defects and adrenal agenesis, indicating that trp <dig> does not play a major role in the genesis of these defects in mice lacking cited <dig>  these results indicate that multi-embryo mri is a potentially powerful high-throughput tool for efficiently characterizing cardiovascular malformations and identifying other defects in organogenesis.
cardiac malformations in mice lacking ptdsr
we next evaluated the role of multi-embryo mri in analyzing unexplained lethality in embryos generated in collaborating laboratories.
recently, we have generated mice lacking the phosphatidylserine receptor  on a c57bl/6j background, by gene targeting in embryonic stem cells  <cit> .
ptdsr is a nuclear protein of unknown function, which is essential for the development and differentiation of multiple organs during embryogenesis  <cit> .
ablation of ptdsr function in knockout mice causes perinatal lethality, growth retardation  <cit>  and a delay in terminal differentiation of the kidney, intestine, liver and lungs during embryogenesis  <cit> .
in addition, ptdsr-/- embryos develop complex ocular lesions  <cit>  as well as haematopoietic defects  <cit> .
however, as many malformations have been described in ptdsr mutants, none of those detected could explain the observed perinatal lethality of ptdsr-/- mice.
in the process of phenotypical characterization of our ptdsr-deficient mouse line, we frequently observed subcutaneous edema of varying sizes in ptdsr-/- embryos by gross inspection .
as the development of edema in various mouse mutants is frequently associated with cardiovascular defects  <cit>  we started to investigate if this also holds true for ptdsr-deficient mice.
we examined  <dig> embryos lacking ptdsr, and  <dig> littermate wild-type or heterozygous controls using multi-embryo mri.
we found that  <dig> of  <dig> ptdsr-/- embryos had cardiac malformations, which included ventricular septal defects, double outlet right ventricle, and pulmonary artery hypoplasia .
none of the wild-type or ptdsr+/- embryos had cardiac malformations.
these findings were confirmed on single embryo imaging .
furthermore, to verify the identified cardiac defects in the ptdsr-/- mice we performed serial transverse sectioning of all analyzed embryos.
in all cases, we could recognize again the same heart defects that were identified before using the multi-embryo mri technique .
in addition, we analyzed by multi-embryo mri a second ptdsr-knockout mouse line , which is identical to the initially analyzed ptdsr mutant except that the loxp-flanked neomycin selection cassette was removed by breeding the original ptdsrtm <dig> gbf knockout line  <cit>  to a cmv-cre deleter mouse line  <cit> .
from this ptdsrtm <dig>  gbf knockout mouse line we analyzed  <dig> ptdsr-/- embryos, and as littermate controls,  <dig> ptdsr+/+ and  <dig> ptdsr+/- embryos.
we found ventricular septal defects in five out of the eight ptdsr-/- embryos .
again we found no evidence for cardiac malformations in wild-type or heterozygous littermate control embryos.
we also observed a modest degree of thymic hypoplasia in ptdsr-/- embryos .
to confirm this, thymus and embryo volumes were measured in  <dig> ptdsr-/- embryos and  <dig> wild-type or heterozygous control embryos at  <dig>  dpc.
embryo volume measured from the mri datasets correlated very strongly with embryo weight , and was modestly reduced in ptdsr-/- embryos .
the volume of the thymus was significantly reduced in ptdsr-/- embryos, even after correction for embryo volume, to 58% of the control value .
to correlate the identified cardiopulmonary malformations in ptdsr-/- embryos with expression of the ptdsr gene during heart development, we made use of our ptdsr gene-trap reporter mouse line  <cit> .
using x-gal staining in heterozygous embryos staged between  <dig>  dpc and  <dig>  dpc we found specific ptdsr expression in the heart starting at  <dig>  dpc and getting more defined to the compact zone and the trabeculi from  <dig>  dpc onwards .
furthermore, when we analyzed ptdsr-/- hearts by histopathology at  <dig>  dpc we observed a severe differentiation defect in the compact zone as well as in the trabeculi , thus demonstrating that ptdsr is in addition required for heart muscle differentiation at later stages of development.
taken together these results indicate that ptdsr plays a hitherto unsuspected role in cardiovascular development as well as in cardiac muscle differentiation.
discussion
utility of mri in identifying mouse models of human malformations
our results show that it is possible to efficiently identify and quantitate relatively subtle cardiac and visceral malformations in late gestation mouse embryos using multi-embryo mri.
we have deliberately optimized our technique at late gestation in order to identify those congenital defects that allow survival through most of gestation.
importantly, these defects resemble human congenital malformations, and would provide mouse models for the study of these diseases.
our method represents a simpler alternative to the multi-coil approach published recently  <cit>  in which up to eight fixed mouse embryos were imaged simultaneously, with a resolution of  <dig> μm.
in comparison, the multi-embryo method described here has a higher experimental resolution of  <dig> μm.
notably, it requires substantial developmental and financial effort to equip an experimental mr-system with multiple coil and receive capability.
role of mri in investigating murine embryonal or perinatal lethality
many mouse gene knockouts display late gestational lethality, but incomplete analysis and loss of 3d information consequent to histological sectioning, results in major developmental malformations being frequently missed.
as shown here, ptdsr-/- embryos on a c57bl/6j background develop heart defects.
this was not noted in recent reports  <cit> , and emphasizes the need not only for completely examining several mutant embryos, but also repeating these examinations in different genetic backgrounds.
a major advantage of mri is the ability to easily ship fixed embryos from referring laboratories to the laboratory that performs the mri analysis.
this minimizes the expense of animal relocation, re-derivation, and breeding required to generate the embryos, and significantly reduces animal experimentation.
role of mri in high-throughput phenotype driven screens
mri screens performed at late gestation would be expected to identify genes that affect later aspects of development, and identify hypomorphic and haploinsufficient alleles of genes that affect earlier steps of development.
published data from genome-wide enu mutagenesis screens in the mouse indicate that ~30% progeny carry a heritable recessive phenotype, making 3-generation recessive screens the method of choice for identifying developmental malformations  <cit> .
at least  <dig> 3rd generation progeny per 1st generation mutant are typically screened, resulting in a >78% probability of identifying at least one fully penetrant recessive homozygous mutant.
a typical recessive screen  would require the analysis of ~ <dig> –  <dig> embryos per year.
our results show that multi-embryo mri is eminently suitable for such throughput.
although, screening of  <dig> embryos overnight requires typically about  <dig> hours of data analysis, multi-embryo mri mutagenesis screens can be easily performed at a reasonable scale if multiple operators analyze the data in parallel.
as the data are permanently stored on dvds, and can be analyzed easily by commercially available software, they can be without difficulty disseminated to specialists for further and more detailed analysis.
another powerful application of multi-embryo mri will likely be the investigation and screening of potentially teratogenic drugs.
functional role of ptdsr in heart development
our results presented here indicate a new, and hitherto unsuspected role for the phosphatidylserine receptor in controlling ventricular septal, outflow tract, pulmonary artery, and thymus development.
this finding suggests that a novel ptdsr-mediated pathway is required for cardiac and thymus development.
recently, we have demonstrated that in contrast to previously reported hypothetical ptdsr functions, the ptdsr protein is not required for the clearance of apoptotic cells  <cit> .
moreover, detailed analysis of apoptosis induction and apoptotic cell clearance in ptdsr+/+ and ptdsr-/- embryos during heart development did not reveal any difference in the number and location of apoptotic cells between the genotypes .
this further excludes that ptdsr has any function in apoptotic cell clearance and points to other developmental mechanisms that are affected by ptdsr ablation.
the neural crest plays an important role in the development of the cardiac outflow tract, aortic arches, and the thymus  <cit> .
as ptdsr-deficient embryos lack intestinal ganglia  <cit>  which are also derived from the neural crest, these results suggest that ptdsr-/- mice may have an underlying neural crest defect.
importantly, dysfunction of these ptdsr-mediated pathways during development could also potentially result in heart defects in humans.
CONCLUSIONS
our results validate the utility of multi-embryo mri for high-throughput identification of murine models for human congenital cardiac malformations, and using this technique we have shown that ptdsr is essential for normal cardiac development.
further experiments are needed to define exactly in which pathways ptdsr is involved during heart development.
we expect that multi-embryo mri will be an important technology for future phenotype-driven mouse mutagenesis screens.
the technology can be easily implemented at standard mri imaging centers, thus allowing by collaboration with individual researchers or mouse mutagenesis centers, a high-throughput functional genetic dissection of mechanisms underlying cardiac development and congenital heart diseases.

 congenital malformations are a major cause of death in childhood, and are typically characterized by lesions that do not compromise fetal survival. for instance, congenital heart disease typically consists of lesions such as ventricular and atrial septal defects, which are compatible with fetal hemodynamics. although human genetic studies have identified some genes that cause congenital cardiac malformations, the molecular and developmental mechanisms underlying most of these defects remain largely unknown. despite the high incidence of chd, only a handful of genes have been identified that when mutated, result in congenital heart disease. the mouse is a particularly good model for studying mechanisms of cardiac diseases as its anatomy and development resembles that of the human more closely than any other genetically tractable organism. importantly, the mouse is amenable to genotype-driven approaches such as transgenic knockouts of defined candidate genes, genome wide mutagenesis approaches using gene-trap or transposon insertion screens, and high-throughput phenotype-driven screens that rely, for instance, on n-ethyl-n-nitrosourea mutagenesis. however, high-throughput cardiovascular genomic approaches in the mouse have been hampered by the paucity of phenotyping tools that allow efficient identification of complex cardiac malformations. as mouse embryos are opaque, late developmental defects are particularly difficult to identify. for instance, cardiac septal defects or outflow tract abnormalities can only be confidently identified after days post coitum when cardiac and outflow tract septation are completed in normal embryos. the identification of malformations in late gestation embryos typically relies on serial histological sectioning, which is extremely labor intensive. furthermore, this often results in the irretrievable loss of 3d information, which is essential for the interpretation of complex cardiac malformations. in addition, standard pathological analysis is not amenable to high-throughput phenotype screening protocols that are required for any mutagenesis screen aiming at the functional dissection of the developmental biology of cardiac diseases. therefore, new technological approaches must be harnessed that allow an efficient phenotyping of heart defects and also of subtle cardiac abnormalities that are at danger of being overseen in traditional histopathology screens. this is even more important in the light of upcoming new endeavors in functional mouse genome annotation. currently, new large-scale mouse mutagenesis screens are being set up in the us and in europe that aim to produce heritable mutations in every gene in the mouse genome. to make full use of these new mouse mutant resources more precise and efficient phenotyping methods are urgently needed. the success of genome wide saturation mutagenesis screens depends therefore on improved phenotyping, and new high-resolution imaging approaches for mouse mutants are one of the most important which need to be established. we previously reported the development of fast gradient-echo mri of single mouse embryos. this resulted in the acquisition of a 3d dataset in under hours, with an experimental image resolution of × × μm/voxel. we showed that mri is capable of accurately identifying normal embryonal structures, and cardiac and adrenal malformations in knockout mouse embryos, and we have validated this technique by performing in depth histological examinations of imaged embryos. these experiments showed that single-embryo mri could correctly identify all cardiac lesions except those under μm – which is below the resolution of the mri technique. as this method images single embryos in overnight runs, it still lacks the throughput required for phenotype-driven mutagenesis screens. for instance, in a typical recessive enu mutagenesis screen, to screen enu mutant lines using a 3-generation breeding scheme would require the analysis of ~ embryos. we now report the development of a method of imaging up to embryos simultaneously in a single unattended overnight run, at high spatial resolution. allowing ~ minutes per embryo, the analysis of embryos would take working days for a single trained individual. we show that this high-throughput multi-embryo mri technique can be used to rapidly identify unsuspected embryonal cardiac and visceral malformations. using this technique we could identify a novel, and hitherto unsuspected role for the phosphatidylserine receptor in controlling ventricular septal, outflow tract and pulmonary artery development. in addition, we found thymus hypoplasia in ptdsr-deficient embryos. these findings suggest that a novel ptdsr-mediated pathway is required for cardiac and thymus development.  multi-embryo imaging we modified our previously described fast gradient echo magnetic resonance imaging technique to image embryos embedded in four to eight layers in mm nuclear magnetic resonance tubes using a single quadrature driven birdcage coil. preparation and embedding of embryos typically took less than an hour. in initial experiments we imaged up to embryos simultaneously in overnight runs of < hours, with an experimental resolution of × × μm. subsequently, we used a custom made optimized probe with an increased sensitivity range to enhance imaging throughput. this allowed us to image embryos simultaneously, but with a larger matrix size and an increased field-of-view in the long axis of the tube. for these experiments we imaged embryos for ~ hours, and achieved an improved experimental resolution of × × μm. the optimized coil used for embryos mri has a sensitivity range in z-direction of close to mm. the artefacts seen at both ends in the longitudinal image are caused by b1-inhomogeneities at the end-rings of the coil. however, accurate image analysis for the entire data set remains possible if the height of the embryo stack does not exceed approximately mm as demonstrated in the corresponding axial views of the top and the bottom layer. the resolution achieved with the multi-embryo mri technique allowed us to visualize the heart, cardiac septa, central nervous system, and visceral organs in fine detail, in embryos taken from each layer. the data are permanently archived on dvds, for subsequent analysis. construction of 3d reconstructions of the heart typically takes ~ hours, but was not necessary for the identification of cardiovascular defects. sensitivity and specificity of multi-embryo imaging to assess the sensitivity and specificity of multi-embryo imaging in comparison to single-embryo imaging, we used a model of cited deficiency. embryos lacking cited have diverse cardiac malformations, including atrial and ventricular septal defects, outflow tract and aortic arch malformations, and adrenal agenesis. as the trp53-repressor gene cdkn2ap19arf is a target of cited, we also examined embryos lacking both cited and trp to determine if this would rescue the heart and adrenal defects in cited2-/- mice. we imaged embryos using the multi-embryo technique in the 16-embryo mode. embryonal genotypes included wild-type, cited2+/-, cited2-/-, three cited2-/-: trp53-/-, four cited2-/-: trp53+/-, two trp53+/-, and two trp53-/-. we analyzed the data from each embryo for cardiac malformations without knowledge of the genotype. this typically took a maximum of minutes per embryo. we scored each embryo for atrial and ventricular septal defects, outflow tract, and aortic arch malformations, and for adrenal agenesis. each embryo was then re-imaged singly at high resolution, and the data re-analyzed as before. in this group we identified embryos with asd, with vsd, with outflow tract defects, with aortic arch defects, and with bilateral adrenal agenesis, using high-resolution single embryo imaging. in comparison to single embryo imaging, the overall sensitivity and specificity of multi-embryo imaging for cardiac malformations was 88% and 92% respectively. for asd the sensitivity and specificity was 85% and 95%; for vsd 94% and 94%; for outflow tract malformations 94% and 100%; and for aortic arch malformations 91% and 100% respectively. for bilateral adrenal agenesis, the sensitivity was 100% and specificity was 95%. embryos lacking both cited and trp had cardiovascular defects and adrenal agenesis, indicating that trp does not play a major role in the genesis of these defects in mice lacking cited these results indicate that multi-embryo mri is a potentially powerful high-throughput tool for efficiently characterizing cardiovascular malformations and identifying other defects in organogenesis. cardiac malformations in mice lacking ptdsr we next evaluated the role of multi-embryo mri in analyzing unexplained lethality in embryos generated in collaborating laboratories. recently, we have generated mice lacking the phosphatidylserine receptor on a c57bl/6j background, by gene targeting in embryonic stem cells. ptdsr is a nuclear protein of unknown function, which is essential for the development and differentiation of multiple organs during embryogenesis. ablation of ptdsr function in knockout mice causes perinatal lethality, growth retardation and a delay in terminal differentiation of the kidney, intestine, liver and lungs during embryogenesis. in addition, ptdsr-/- embryos develop complex ocular lesions as well as haematopoietic defects. however, as many malformations have been described in ptdsr mutants, none of those detected could explain the observed perinatal lethality of ptdsr-/- mice. in the process of phenotypical characterization of our ptdsr-deficient mouse line, we frequently observed subcutaneous edema of varying sizes in ptdsr-/- embryos by gross inspection. as the development of edema in various mouse mutants is frequently associated with cardiovascular defects we started to investigate if this also holds true for ptdsr-deficient mice. we examined embryos lacking ptdsr, and littermate wild-type or heterozygous controls using multi-embryo mri. we found that of ptdsr-/- embryos had cardiac malformations, which included ventricular septal defects, double outlet right ventricle, and pulmonary artery hypoplasia. none of the wild-type or ptdsr+/- embryos had cardiac malformations. these findings were confirmed on single embryo imaging. furthermore, to verify the identified cardiac defects in the ptdsr-/- mice we performed serial transverse sectioning of all analyzed embryos. in all cases, we could recognize again the same heart defects that were identified before using the multi-embryo mri technique. in addition, we analyzed by multi-embryo mri a second ptdsr-knockout mouse line, which is identical to the initially analyzed ptdsr mutant except that the loxp-flanked neomycin selection cassette was removed by breeding the original ptdsrtm gbf knockout line to a cmv-cre deleter mouse line. from this ptdsrtm gbf knockout mouse line we analyzed ptdsr-/- embryos, and as littermate controls, ptdsr+/+ and ptdsr+/- embryos. we found ventricular septal defects in five out of the eight ptdsr-/- embryos. again we found no evidence for cardiac malformations in wild-type or heterozygous littermate control embryos. we also observed a modest degree of thymic hypoplasia in ptdsr-/- embryos. to confirm this, thymus and embryo volumes were measured in ptdsr-/- embryos and wild-type or heterozygous control embryos at dpc. embryo volume measured from the mri datasets correlated very strongly with embryo weight, and was modestly reduced in ptdsr-/- embryos. the volume of the thymus was significantly reduced in ptdsr-/- embryos, even after correction for embryo volume, to 58% of the control value. to correlate the identified cardiopulmonary malformations in ptdsr-/- embryos with expression of the ptdsr gene during heart development, we made use of our ptdsr gene-trap reporter mouse line. using x-gal staining in heterozygous embryos staged between dpc and dpc we found specific ptdsr expression in the heart starting at dpc and getting more defined to the compact zone and the trabeculi from dpc onwards. furthermore, when we analyzed ptdsr-/- hearts by histopathology at dpc we observed a severe differentiation defect in the compact zone as well as in the trabeculi, thus demonstrating that ptdsr is in addition required for heart muscle differentiation at later stages of development. taken together these results indicate that ptdsr plays a hitherto unsuspected role in cardiovascular development as well as in cardiac muscle differentiation. discussion utility of mri in identifying mouse models of human malformations our results show that it is possible to efficiently identify and quantitate relatively subtle cardiac and visceral malformations in late gestation mouse embryos using multi-embryo mri. we have deliberately optimized our technique at late gestation in order to identify those congenital defects that allow survival through most of gestation. importantly, these defects resemble human congenital malformations, and would provide mouse models for the study of these diseases. our method represents a simpler alternative to the multi-coil approach published recently in which up to eight fixed mouse embryos were imaged simultaneously, with a resolution of μm. in comparison, the multi-embryo method described here has a higher experimental resolution of μm. notably, it requires substantial developmental and financial effort to equip an experimental mr-system with multiple coil and receive capability. role of mri in investigating murine embryonal or perinatal lethality many mouse gene knockouts display late gestational lethality, but incomplete analysis and loss of 3d information consequent to histological sectioning, results in major developmental malformations being frequently missed. as shown here, ptdsr-/- embryos on a c57bl/6j background develop heart defects. this was not noted in recent reports, and emphasizes the need not only for completely examining several mutant embryos, but also repeating these examinations in different genetic backgrounds. a major advantage of mri is the ability to easily ship fixed embryos from referring laboratories to the laboratory that performs the mri analysis. this minimizes the expense of animal relocation, re-derivation, and breeding required to generate the embryos, and significantly reduces animal experimentation. role of mri in high-throughput phenotype driven screens mri screens performed at late gestation would be expected to identify genes that affect later aspects of development, and identify hypomorphic and haploinsufficient alleles of genes that affect earlier steps of development. published data from genome-wide enu mutagenesis screens in the mouse indicate that ~30% progeny carry a heritable recessive phenotype, making 3-generation recessive screens the method of choice for identifying developmental malformations. at least 3rd generation progeny per 1st generation mutant are typically screened, resulting in a >78% probability of identifying at least one fully penetrant recessive homozygous mutant. a typical recessive screen would require the analysis of ~ – embryos per year. our results show that multi-embryo mri is eminently suitable for such throughput. although, screening of embryos overnight requires typically about hours of data analysis, multi-embryo mri mutagenesis screens can be easily performed at a reasonable scale if multiple operators analyze the data in parallel. as the data are permanently stored on dvds, and can be analyzed easily by commercially available software, they can be without difficulty disseminated to specialists for further and more detailed analysis. another powerful application of multi-embryo mri will likely be the investigation and screening of potentially teratogenic drugs. functional role of ptdsr in heart development our results presented here indicate a new, and hitherto unsuspected role for the phosphatidylserine receptor in controlling ventricular septal, outflow tract, pulmonary artery, and thymus development. this finding suggests that a novel ptdsr-mediated pathway is required for cardiac and thymus development. recently, we have demonstrated that in contrast to previously reported hypothetical ptdsr functions, the ptdsr protein is not required for the clearance of apoptotic cells. moreover, detailed analysis of apoptosis induction and apoptotic cell clearance in ptdsr+/+ and ptdsr-/- embryos during heart development did not reveal any difference in the number and location of apoptotic cells between the genotypes. this further excludes that ptdsr has any function in apoptotic cell clearance and points to other developmental mechanisms that are affected by ptdsr ablation. the neural crest plays an important role in the development of the cardiac outflow tract, aortic arches, and the thymus. as ptdsr-deficient embryos lack intestinal ganglia which are also derived from the neural crest, these results suggest that ptdsr-/- mice may have an underlying neural crest defect. importantly, dysfunction of these ptdsr-mediated pathways during development could also potentially result in heart defects in humans.  our results validate the utility of multi-embryo mri for high-throughput identification of murine models for human congenital cardiac malformations, and using this technique we have shown that ptdsr is essential for normal cardiac development. further experiments are needed to define exactly in which pathways ptdsr is involved during heart development. we expect that multi-embryo mri will be an important technology for future phenotype-driven mouse mutagenesis screens. the technology can be easily implemented at standard mri imaging centers, thus allowing by collaboration with individual researchers or mouse mutagenesis centers, a high-throughput functional genetic dissection of mechanisms underlying cardiac development and congenital heart diseases. 
genetic studies in the mouse have been crucial to uncover new genes and signaling pathways associated with heart development and congenital heart disease. the technique can be easily adapted for mouse mutagenesis screens and, thus provides an important new tool for identifying new mouse models for human congenital heart diseases. our novel multi-embryo mri technique enables high-throughput identification of murine models for human congenital cardiopulmonary malformations at high spatial resolution. the identification of murine models of congenital cardiac malformations in high-throughput mutagenesis screens and in gene-targeted models is hindered by the opacity of the mouse embryo. using this approach we identified cardiac malformations in phosphatidylserine receptor deficient embryos. these results indicate that ptdsr plays a key role in cardiac development. these included ventricular septal defects, double-outlet right ventricle, and hypoplasia of the pulmonary artery and thymus. we developed and optimized a novel method for high-throughput multi-embryo magnetic resonance imaging.
 congenital heart defects are the leading non-infectious cause of death in children. genetic studies in the mouse have been crucial to uncover new genes and signaling pathways associated with heart development and congenital heart disease. the identification of murine models of congenital cardiac malformations in high-throughput mutagenesis screens and in gene-targeted models is hindered by the opacity of the mouse embryo.  we developed and optimized a novel method for high-throughput multi-embryo magnetic resonance imaging. using this approach we identified cardiac malformations in phosphatidylserine receptor deficient embryos. these included ventricular septal defects, double-outlet right ventricle, and hypoplasia of the pulmonary artery and thymus. these results indicate that ptdsr plays a key role in cardiac development.  our novel multi-embryo mri technique enables high-throughput identification of murine models for human congenital cardiopulmonary malformations at high spatial resolution. the technique can be easily adapted for mouse mutagenesis screens and, thus provides an important new tool for identifying new mouse models for human congenital heart diseases. 