BACKGROUND
a considerable percentage of cardiac birth defects is caused by a failure in normal migration, differentiation or patterning of the cardiac neural crest .
this subset of pluripotent neural crest stem cells forms in the dorsal aspect of the neural tube at the level of the mid-otic placode to the third somite  <cit> .
subsequently cardiac neural crest cells  delaminate, undergo a phenotypic transformation from an epithelial to mesenchymal cell type, and migrate latero-ventrally into the 3rd, 4th and 6th pharyngeal arch arteries , where they contribute to the formation of the smooth muscle cell layer of endothelial structures derived from the aortic arch arteries  <cit> .
a subset of cnccs continues to migrate deeper into the aortic sac to form the aortico-pulmonary septum; a vital structure, which separates the pulmonary trunk from the aorta  <cit> .
an indispensable role of cnccs in the development of the cardiac outflow tract was originally demonstrated by pioneering studies of kirby and coworkers  <cit> , who showed that ablation of the cnc in the chick led to severe outflow tract  defects including persistent truncus arteriosus , mispatterning of the great vessels and outflow tract mal-alignments  <cit> .
early migratory cnccs have been shown to retain a considerable degree of plasticity and their fate is largely controlled by instructional signals from local environments into which nccs migrate  <cit> .
several recent studies have indicated that members of the tgf-β superfamily, i.e., tgf-βs and bmps are likely candidates to provide some of these signals.
mice deficient in tgf-β <dig> display fourth aortic arch artery defects  <cit> , while neural crest cell specific abrogation of tgf-β type ii receptor  results in interruption of the aortic arch and pta  <cit> .
bmps  <dig> and - <dig> are required for proper formation of the outflow tract cushions  <cit> , while bmp type ii receptor is needed for proper development of the conotruncal ridges  <cit> .
moreover, neural crest-specific deletion of the bmp type i receptors alk <dig> and alk <dig> has been shown to lead to defective aortico-pulmonary septation, among other cardiac defects  <cit> .
tgf-β subfamily ligands signal via a receptor complex composed of two type ii receptors and two type i receptors  <cit> .
ligand binding leads to phosphorylation and activation of type i receptors, which, in turn, phosphorylate and activate a specific set of downstream signaling molecules called smads.
in general terms, tgf-βs bind to the tgf-β type ii receptor  and tgf-β type i receptor  activating tgf-β smads , while bmps bind to the bmp type ii receptor and type i receptors alk <dig>  - <dig>  or  <dig>  activating bmp smads .
however, it is likely that these signaling interactions are more complex in vivo, possibly allowing formation of heterotetrameric complexes composed of different type ii and type i receptors  <cit> .
in addition, recent studies have identified novel tgf-β-related ligands, which can bind to entire different combinations of receptors.
for instance, growth and differentiation factors   <dig> and  <dig> can bind to activin type ii receptor and alk <dig> to activate tgf-β smads  <cit> .
therefore, we hypothesized that deletion of alk <dig> in a specific cell lineage should reveal phenotypes which cannot be seen in comparable mutants lacking tgfbr <dig>  indeed, we recently showed that neural crest cell specific alk <dig> mutants display a unique spectrum of craniofacial developmental defects, e.g., cleft snout and severe mandibular hypoplasia  <cit> ; these phenotypes were not seen in corresponding tgfbr <dig> mutants  <cit> .
to determine, whether alk <dig> would also mediate unique non-redundant signaling events in cardiac neural crest cells, we focused on cardiac and pharyngeal phenotypes of mouse embryos lacking alk <dig> specifically in neural crest cells.
we discovered that in alk5/wnt1-cre mutants, pharyngeal organs  fail to migrate appropriately.
moreover, the mutant embryos display severe aortic sac and pharyngeal arch artery defects, and failed aortico-pulmonary septation leading to pta.
our data further suggest that at least some of these abnormal detected phenotypes result from a dramatic increase in apoptosis of postmigratory cardiac neural crest cells.
these phenotypes differ remarkably from those seen in corresponding tgfbr <dig> mutants, suggesting that alk <dig> mediates a wider spectrum of signaling events than its classical binding partner tgfβrii in cardiac neural crest cells during cardiac and pharyngeal development.
RESULTS
persistent truncus arteriosus and abnormal large vessels in mice lacking alk <dig> in cardiac nccs
to inactivate alk <dig> in cardiac nccs, mice homozygous for the floxed alk <dig> allele   <cit>  were crossed with transgenic wnt1-cre mice  <cit> , which were also heterozygous for the alk <dig> knockout allele  allele.
the resulting mice heterozygous for the alk5flox and alk5ko alleles, which also carried the wnt1-cre transgene, had the alk <dig> gene specifically inactivated in nccs , while the littermates with remaining allelic combinations were phenotypically normal and served as controls .
when embryos were harvested during the last day of gestation, an expected number  of alk5/wnt1-cre mutants were recovered.
however, all mutant offspring died either during the birth or during the first post-natal hours.
to determine, if alk5-mediated tgf-β-signaling had a role in development of the oft and large vessels of the aortic arch, we performed casting dye experiments on e <dig> embryos .
in wild-type embryos , the aorta was clearly separated from the pulmonary trunk, and the right brachiocephalic, left carotid and left subclavian arteries branched directly off the aortic arch.
in contrast, alk5/wnt1cre mutant embryos consistently displayed a single prominent arterial trunk , while corresponding tgfbr <dig> mutant embryos  displayed interrupted aortic arch, as reported earlier  <cit> .
approximately 40% of the alk <dig> mutants had a right-sided outflow tract, with the retroesophageal arch connecting to the descending aorta and to the left subclavian artery.
the carotid arteries originated either from a common bud located in the ventral side of the ascending arch, or from separate adjacent sites, as verified by serial sectioning .
the remaining mutants displayed a left-sided aortic arch, where the right carotid arteries originated from the right lateral aspect of the ascending trunk, while the left carotid arteries budded from the ventral or right ventral aspects of the trunk .
both right and left subclavian arteries consistently originated from the descending part of the aortic arch.
similarly, in all mutants both left and right pulmonary arteries always branched out from the common arterial trunk.
to conclude, alk5/wnt1-cre mutants consistently displayed pta, which differed significantly from the characteristic interrupted aortic arch phenotype seen in tgfbr2/wnt1-cre mutants  <cit> .
abnormal patterning of the pharyngeal arch arteries and aortic sac in alk5/wnt1cre mutants
during cardiovascular development, the paas undergo a complex set of sequential asymmetric remodeling steps resulting in the left-sided aortic arch.
to determine, whether alk5-mediated signaling was involved in remodeling of paas, we performed intracardiac india ink injections at different developmental stages.
while at e <dig>  alk5/wnt1-cre mutants did not show obvious differences in the paas, abnormal remodeling became obvious in mutants a day later at e <dig> .
the controls displayed the well-formed 3rd, 4th and 6th paas.
moreover, the carotid duct  was already regressing as demonstrated by the reduced size .
in alk5/wnt1-cre mutants, the 3rd and 4th pairs of paas were bilaterally hypoplastic, whereas the 6th pair of paas was notably hyperplastic .
furthermore, the carotid duct was remarkably large, when compared to controls.
while the controls displayed an interruption of the carotid duct at e <dig> and e <dig> as expected , the mutants demonstrated an uncharacteristic break of the dorsal aorta between the 4th and 6th pairs of paas .
around e <dig> , the aortic sac normally forms a distinctive t-shaped structure, as seen in frontal sections of the control sample in fig.
<dig>  subsequently, the right horn of this structure transforms into the prospective brachiocephalic artery, while the left horn together with the left 4th paa gives rise to the definitive aortic arch  <cit> .
in alk5/wnt1-cre mutants, the t-shaped aortic sac failed to form .
instead, the truncus bifurcated to a left and right arm, which further branched to the paas, particularly to the predominant pair of 6th paas .
the observed phenotype is consistent with the absence or severe hypoplasia of structures derived from the aortic sac in late stage embryos , e.g., the missing brachiocephalic artery and severe shortening of the ascending truncus as shown in the figure  <dig>
cardiac nccs deficient in alk <dig> can populate the outflow tract
next we used the r26r lineage-tracing assay to determine whether cnccs could appropriately populate the outflow tract region.
briefly, alk5flox/flox mice were crossed with the rosa <dig> cre reporter mice, and subsequently alk5flox/flox;r26r females were crossed with alk5ko/wt;wnt1-cre males.
the resulting embryos had the nc-lineage permanently labeled with β-galactosidase expression, and displayed identical phenotypes to those obtained without the r26r reporter.
staining of embryos for β-galactosidase at e8-e <dig> did not reveal detectable differences in ncc migration between mutants and controls .
similarly, serial transverse sectioning of whole mount embryos  and subsequent analysis of positively stained cells in the oft region demonstrated that cnccs deficient in alk <dig> were capable of populating the paas, aortic sac and conotruncal ridges at a level comparable to that of controls .
to conclude, the observed phenotypes in alk5/wnt1-cre mutants were certainly not due to defective migration of cnccs to the pharyngeal and outflow tract regions.
aortic sac and aortico-pulmonary septal defects in alk5/wnt1cre mutant embryos
septation of the outflow tract lumen begins in a cranial-to-caudal direction, starting distally in the aortic sac and proceeding toward the heart  <cit> .
initially, the condensed mesenchyme derived from the nc forms in the base of the aortic sac between the origins of 4th and 6th paas.
subsequently, two prongs of the developing aortico-pulmonary  septum extend into the truncal cushions and the aortico-pulmonary septation complex crosses the aortic sac cranially.
in ink-injected control embryos at e <dig> , a characteristic conotruncal transition separating the truncus and conus could be seen as a twisted configuration, resulting from a change in orientation of the truncal and conal cushions .
in contrast, in alk5/wnt1-cre mutants the outflow tract appeared unusually straight, failing to demonstrate the distinct conotruncal transition .
this assay also clearly showed a dramatic reduction in the size of the aortic sac.
histological analysis of control samples displayed the characteristic rotation of the aortic sac and truncal oft at the level where the ap septation takes place and verified the presence of the distinctive condensed ap-septal mesenchyme, which gradually divided the oft to the aorta and the pulmonary trunk .
r26r lineage tracing showed that this tissue is derived from the nc, while immunostaining for α-sma showed differentiation into smooth muscle .
in alk5/wnt1-cre mutants the characteristic rotation of the aortic sac and truncal oft fails to take place , and a properly formed ap-septum was not detectable .
r26r lineage tracing demonstrated that the defects were not due to failure of nccs to reach the oft region.
nc-derived cells around the abnomally bifurcated aortic sac, the abnormally large sixth paas and the truncus demonstrated strong αsma staining .
recently, we showed that the nc-specific mutants of the related type i receptor, alk <dig>  display pta as well  <cit> .
in alk2/wnt1-cre mutants, the rotation of the aortic sac and truncal oft failed to occur  as seen in alk5/wnt1-cre mutants.
however, in alk <dig> mutants the 6th pair of the paas was grossly hypoplastic, and while the alk2/wnt1-cre mutants displayed a noticeable amount of septal tissue between the 4th and 6th paas , the condensed septal mesenchyme lacking alk <dig> failed to extend the prongs into the truncal cushions and to form the ap septum.
concurrently, the 6th paas were losing their patency, which may have further contributed to the failed ap septation .
while cnccs managed to migrate to the aortic sac and the truncal cushion level , immunostaining for αsma appeared much weaker when compared to controls and alk <dig> mutants, implying that alk2-mediated signaling is involved in smooth muscle cell differentiation as previously suggested  <cit> .
to conclude, while both alk <dig> and alk <dig> mutants demonstrate a failure in both the rotation of the aortic sac and the truncal oft, and in the formation of the ap septum, the pathogenetic mechanisms behind these defects appear remarkably different.
alk5/wnt1-cre mutants display increased apoptosis of post-migratory neural crest cells
as described above, alk5/wnt1-cre mutants displayed an inadequate amount of ap-septal tissue in the base of the aortic sac between the origins of 4th and 6th paas.
to analyze whether this phenotype resulted either from defective cncc proliferation or inappropriate apoptosis, we used brdu and tunel staining, respectively.
while cncc proliferation was not affected in alk <dig> mutants , we could detect a dramatic increase in the number of tunel positive cells in tissues surrounding the aortic sac including the site where the ap-septum forms .
dual staining for lacz and tunel positive cells demonstrated that these cells were postmigratory cnccs; this phenotype was already clearly detectable at e <dig> .
these results were confirmed by using immunostaining for cleaved caspase- <dig>  another marker for apoptosis .
in the chick, apoptotic neural crest-derived cells have also been found at the sites, where the prongs of the ap septum penetrate into the oft cushion mesenchyme  <cit> .
thus, we compared apoptosis patterns also on the more proximal level, but found no detectable differences at e <dig>  between alk5/wnt1-cre mutants and controls .
unlike in alk5/wnt1-cre mutant embryos, increased apoptosis of nc-derived cells is not responsible for the observed defects in the oft septation in corresponding alk <dig> mutants .
to conclude, our results suggest that in alk5/wnt1-cre mutants a noticeable increase in apoptosis coincides with the abnormal patterning of the paas and the aortic sac, and with the failed ap-septation.
these data support a specific role for alk <dig> signaling, either directly or indirectly, in cncc survival, since a similar apoptosis of nc-derived cells is not seen in tgfbr2/wnt1-cre mutants  <cit> .
pharyngeal organs fail to migrate in alk5/wnt1-cre mutants
in addition to the cardiac oft, development of pharyngeal organs, i.e., the parathyroid glands and the thymus was also abnormal in alk5/wnt1-cre mutants .
normally the thymus develops from the third pharyngeal pouch endoderm and migrates caudally to its final location in the superior mediastinum as seen in controls at e <dig> .
in contrast, the thymic primordia of the alk <dig> mutant littermates failed to descend caudally, and were located bilaterally in the neck region, where they were surrounded by neural crest-derived mesenchyme .
the fate determination assay demonstrated that the thymic primordia were equally populated by nccs both in controls and alk <dig> mutants .
likewise the parathyroid glands failed to migrate normally in alk5/wnt1-cre mutants.
during normal development, the parathyroids first migrate in association with the thymic primordia, until they reach the thyroids in the neck region as seen in fig 8c.
in alk5/wnt1-cre mutants, the parathyroids remained associated with the thymic primordia, and despite this abnormal rostral location, expression of parathyroid hormone was indistinguishable between alk <dig> mutants and controls at e <dig> .
to conclude, the observed pharyngeal organ phenotypes were also in striking contrast to those seen in tgfbr2/wnt1-cre mutants  <cit> .
discussion
during the last few years the wnt1-cre transgenic driver line has proven to be a powerful tool for tissue-specific gene deletion in nccs  <cit> .
using this approach, several studies have independently shown that the nc-specific deletion of the tgfbr <dig> gene leads to a distinct set of calvarial, facial and cardiac defects  <cit> .
interestingly, these defects appear quite different both in the craniofacial and pharyngeal regions, including the heart, when compared to the corresponding mutants of alk <dig>  which encodes the tgf-β type i receptor, a prototypical binding partner of tgf-βrii  <cit>  and the present study).
while tgfbr2/wnt1-cre mutants as well as mice deficient in tgf-β <dig> display the pta type a <dig> , alk5/wnt1-cre mutants reported here demonstrate earlier patterning defects of the paas, which is particularly obvious in the 3rd pair of the paas.
moreover, the alk5/wnt1-cre mutants display an abnormal patterning of the aortic sac and defective ap septation leading to pta, reminiscent of type a <dig> .
however, our results also demonstrate that significant hypoplasia of the aortic sac leads to a severe shortening of the ascending truncal arch, which masks possible defects in derivatives of the 4th paas, i.e., interruption of the aortic arch.
these observed differences are likely due to substantial apoptosis of alk5-deficient post-migratory neural crest cells, which is clearly detectable at e <dig> , whereas similar intense cell death has not been reported in tgfbr2/wnt1-cre mutants <cit> .
our present results suggest that while tgf-β signaling in cardiac nccs is predominantly mediated via the alk5/tgf-βrii receptor complex, alk <dig> also mediates signaling of other related ligands, which are either directly or indirectly required for appropriate ncc survival.
in fact, it has been shown that, besides tgf-βrii, alk <dig> can also form a complex with the activin type iib receptor to activate downstream smads 2/ <dig>  <cit> .
furthermore, a subset of tgf-β-related growth and differentiation factors , e.g., gdf <dig>  gdf <dig>  gdf <dig> and gdf <dig> could induce these events  <cit> .
although relevant gdfs  <dig>   <dig>  <dig> and  <dig> are not expressed in the developing heart, nor do the mice deficient in these gdfs display developmental cardiac defects, we cannot exclude the possibility that circulating gdfs, perhaps in concert with tgf-βs may contribute to ncc survival during cardiac and pharyngeal morphogenesis.
we specifically studied apoptosis at the level of the aortic sac, where the ap-septum forms between the origins of 4th and 6th paas.
already at e <dig> , we could see intense apoptosis among the postmigratory nccs in the mesenchyme surrounding the aortic sac at the site where the prospective ap septum forms, i.e., this cell death precedes the ap septal defect seen in mutants.
although some nc-derived cells appeared to be differentiating to smooth muscle cells in the oft , we could never detect the ap septum forming in alk5/wnt1-cre mutants between e <dig>  and e <dig> .
these findings suggest that the pool of cells forming the ap septum is severely affected by the cell death.
moreover, it is likely that these cells forming the ap-septum die the before majority of them can differentiate to smooth muscle cells.
several in vitro studies have suggested an indispensable role for tgf-β-signaling in differentiation of nccs into smooth muscle cells.
moreover, a recent in vivo study suggested that mice lacking tgfbr <dig> in cnccs display defective ncc differentiation into αsma-positive cells in the ap septum  <cit> , although this result was later disputed by another study  <cit> .
our immunohistochemical staining of αsma in the oft unequivocally demonstrated that signaling via alk <dig> is not required for smooth muscle differentiation in vivo.
moreover, it has been suggested that deletion of tgfbr <dig> in nccs leads to other phenotypic features reminiscent of those seen in the velocardiofacial/digeorge syndrome   <cit>  caused by a deletion of the so called digeorge critical region  on chromosome 22q <dig>  <cit> .
our present results suggest that although many of the observed phenotypes seen in alk5/wnt1-cre mutants superficially resemble those seen in vcf/dgs, a detailed examination shows that the nc-specific abrogation of alk <dig> does not lead to vcf/dgs-like phenotypes.
firstly, while the pharyngeal organ migration fails in alk5/wnt1-cre mutants, perhaps as a result of increased mesenchymal cell death in the pharyngeal region, both the thymus, thyroid and parathyroid seem to develop relatively normally on the histological level in these mutants.
secondly, the ncc death seen in alk <dig> mutants affects a predominantly postmigratory population of nccs, while genes located in the dgcr, i.e., tbx <dig> and crkl, control ncc survival earlier at e <dig> -e <dig> by regulating proliferation of the secondary heart field , and endoderm expansion, which in turn provides survival signal for nccs allowing them to populate the pharyngeal region  <cit> .
ncc ablation in the chick has been shown to lead to pta and to a failure of addition of myocardium from the secondary heart field  <cit> .
it was suggested that the defective migration of cells from the secondary heart field to the oft in turn resulted in shortening and inappropriate rotation of the oft, leading to mal-alignment of the arterial pole with the ventricles  <cit> .
while the detected oft phenotype in alk5/wnt1-cre mutants shared many similarities with that seen in the chick nc ablation models, e.g., pta and the hypoplastic aortic sac, our current results suggest that the secondary heart field is not severely affected in alk <dig> mutants .
since we could not detect appropriate rotation of the oft in neural crest-specific mutants of alk <dig> or alk <dig>  it appears that cells derived from the nc, as well as those from the shf, are mutually required for proper oft rotation in mice.
however, it appears that these two tgf-β/bmp type i receptors contribute to oft rotation through different mechanisms.
in alk <dig> mutants there is very little, if any, detectable septal mesenchyme present, and thus it could be argued that in these mutants oft rotation fails due to a lack of penetration of septal prongs into the cushion mesenchyme.
in contrast, in alk2/wnt1-cre mutants a sizeable septal mesenchyme could be seen, still without any obvious looping of the aortic sac and truncal oft, suggesting that the mere presence of the septal mesenchyme, without correct smooth muscle cell differentiation, is not sufficient for oft rotation.
CONCLUSIONS
in this study, we have deleted the tgf-β type i receptor  gene specifically in the mouse neural crest  cell lineage.
our data suggest that alk <dig> is required cell autonomously in the nc to mediate non-redundant signaling events that are essential for appropriate patterning of the pharyngeal organs and cardiac oft.
the cardiac and pharyngeal defects observed in the nc-specific alk <dig> mutants differ significantly from those seen in corresponding mutants lacking the tgf-β type ii receptor, suggesting that signaling mediated by alk <dig> is not limited to the classical tgf-β ligands during cardiac/pharyngeal development.

 a considerable percentage of cardiac birth defects is caused by a failure in normal migration, differentiation or patterning of the cardiac neural crest. this subset of pluripotent neural crest stem cells forms in the dorsal aspect of the neural tube at the level of the mid-otic placode to the third somite. subsequently cardiac neural crest cells delaminate, undergo a phenotypic transformation from an epithelial to mesenchymal cell type, and migrate latero-ventrally into the 3rd, 4th and 6th pharyngeal arch arteries, where they contribute to the formation of the smooth muscle cell layer of endothelial structures derived from the aortic arch arteries. a subset of cnccs continues to migrate deeper into the aortic sac to form the aortico-pulmonary septum; a vital structure, which separates the pulmonary trunk from the aorta. an indispensable role of cnccs in the development of the cardiac outflow tract was originally demonstrated by pioneering studies of kirby and coworkers, who showed that ablation of the cnc in the chick led to severe outflow tract defects including persistent truncus arteriosus, mispatterning of the great vessels and outflow tract mal-alignments. early migratory cnccs have been shown to retain a considerable degree of plasticity and their fate is largely controlled by instructional signals from local environments into which nccs migrate. several recent studies have indicated that members of the tgf-β superfamily, i.e., tgf-βs and bmps are likely candidates to provide some of these signals. mice deficient in tgf-β display fourth aortic arch artery defects, while neural crest cell specific abrogation of tgf-β type ii receptor results in interruption of the aortic arch and pta. bmps and - are required for proper formation of the outflow tract cushions, while bmp type ii receptor is needed for proper development of the conotruncal ridges. moreover, neural crest-specific deletion of the bmp type i receptors alk and alk has been shown to lead to defective aortico-pulmonary septation, among other cardiac defects. tgf-β subfamily ligands signal via a receptor complex composed of two type ii receptors and two type i receptors. ligand binding leads to phosphorylation and activation of type i receptors, which, in turn, phosphorylate and activate a specific set of downstream signaling molecules called smads. in general terms, tgf-βs bind to the tgf-β type ii receptor and tgf-β type i receptor activating tgf-β smads, while bmps bind to the bmp type ii receptor and type i receptors alk - or activating bmp smads. however, it is likely that these signaling interactions are more complex in vivo, possibly allowing formation of heterotetrameric complexes composed of different type ii and type i receptors. in addition, recent studies have identified novel tgf-β-related ligands, which can bind to entire different combinations of receptors. for instance, growth and differentiation factors and can bind to activin type ii receptor and alk to activate tgf-β smads. therefore, we hypothesized that deletion of alk in a specific cell lineage should reveal phenotypes which cannot be seen in comparable mutants lacking tgfbr indeed, we recently showed that neural crest cell specific alk mutants display a unique spectrum of craniofacial developmental defects, e.g., cleft snout and severe mandibular hypoplasia; these phenotypes were not seen in corresponding tgfbr mutants. to determine, whether alk would also mediate unique non-redundant signaling events in cardiac neural crest cells, we focused on cardiac and pharyngeal phenotypes of mouse embryos lacking alk specifically in neural crest cells. we discovered that in alk5/wnt1-cre mutants, pharyngeal organs fail to migrate appropriately. moreover, the mutant embryos display severe aortic sac and pharyngeal arch artery defects, and failed aortico-pulmonary septation leading to pta. our data further suggest that at least some of these abnormal detected phenotypes result from a dramatic increase in apoptosis of postmigratory cardiac neural crest cells. these phenotypes differ remarkably from those seen in corresponding tgfbr mutants, suggesting that alk mediates a wider spectrum of signaling events than its classical binding partner tgfβrii in cardiac neural crest cells during cardiac and pharyngeal development.  persistent truncus arteriosus and abnormal large vessels in mice lacking alk in cardiac nccs to inactivate alk in cardiac nccs, mice homozygous for the floxed alk allele were crossed with transgenic wnt1-cre mice, which were also heterozygous for the alk knockout allele allele. the resulting mice heterozygous for the alk5flox and alk5ko alleles, which also carried the wnt1-cre transgene, had the alk gene specifically inactivated in nccs, while the littermates with remaining allelic combinations were phenotypically normal and served as controls. when embryos were harvested during the last day of gestation, an expected number of alk5/wnt1-cre mutants were recovered. however, all mutant offspring died either during the birth or during the first post-natal hours. to determine, if alk5-mediated tgf-β-signaling had a role in development of the oft and large vessels of the aortic arch, we performed casting dye experiments on e embryos. in wild-type embryos, the aorta was clearly separated from the pulmonary trunk, and the right brachiocephalic, left carotid and left subclavian arteries branched directly off the aortic arch. in contrast, alk5/wnt1cre mutant embryos consistently displayed a single prominent arterial trunk, while corresponding tgfbr mutant embryos displayed interrupted aortic arch, as reported earlier. approximately 40% of the alk mutants had a right-sided outflow tract, with the retroesophageal arch connecting to the descending aorta and to the left subclavian artery. the carotid arteries originated either from a common bud located in the ventral side of the ascending arch, or from separate adjacent sites, as verified by serial sectioning. the remaining mutants displayed a left-sided aortic arch, where the right carotid arteries originated from the right lateral aspect of the ascending trunk, while the left carotid arteries budded from the ventral or right ventral aspects of the trunk. both right and left subclavian arteries consistently originated from the descending part of the aortic arch. similarly, in all mutants both left and right pulmonary arteries always branched out from the common arterial trunk. to conclude, alk5/wnt1-cre mutants consistently displayed pta, which differed significantly from the characteristic interrupted aortic arch phenotype seen in tgfbr2/wnt1-cre mutants. abnormal patterning of the pharyngeal arch arteries and aortic sac in alk5/wnt1cre mutants during cardiovascular development, the paas undergo a complex set of sequential asymmetric remodeling steps resulting in the left-sided aortic arch. to determine, whether alk5-mediated signaling was involved in remodeling of paas, we performed intracardiac india ink injections at different developmental stages. while at e alk5/wnt1-cre mutants did not show obvious differences in the paas, abnormal remodeling became obvious in mutants a day later at e. the controls displayed the well-formed 3rd, 4th and 6th paas. moreover, the carotid duct was already regressing as demonstrated by the reduced size. in alk5/wnt1-cre mutants, the 3rd and 4th pairs of paas were bilaterally hypoplastic, whereas the 6th pair of paas was notably hyperplastic. furthermore, the carotid duct was remarkably large, when compared to controls. while the controls displayed an interruption of the carotid duct at e and e as expected, the mutants demonstrated an uncharacteristic break of the dorsal aorta between the 4th and 6th pairs of paas. around e, the aortic sac normally forms a distinctive t-shaped structure, as seen in frontal sections of the control sample in fig. subsequently, the right horn of this structure transforms into the prospective brachiocephalic artery, while the left horn together with the left 4th paa gives rise to the definitive aortic arch. in alk5/wnt1-cre mutants, the t-shaped aortic sac failed to form. instead, the truncus bifurcated to a left and right arm, which further branched to the paas, particularly to the predominant pair of 6th paas. the observed phenotype is consistent with the absence or severe hypoplasia of structures derived from the aortic sac in late stage embryos, e.g., the missing brachiocephalic artery and severe shortening of the ascending truncus as shown in the figure cardiac nccs deficient in alk can populate the outflow tract next we used the r26r lineage-tracing assay to determine whether cnccs could appropriately populate the outflow tract region. briefly, alk5flox/flox mice were crossed with the rosa cre reporter mice, and subsequently alk5flox/flox;r26r females were crossed with alk5ko/wt;wnt1-cre males. the resulting embryos had the nc-lineage permanently labeled with β-galactosidase expression, and displayed identical phenotypes to those obtained without the r26r reporter. staining of embryos for β-galactosidase at e8-e did not reveal detectable differences in ncc migration between mutants and controls. similarly, serial transverse sectioning of whole mount embryos and subsequent analysis of positively stained cells in the oft region demonstrated that cnccs deficient in alk were capable of populating the paas, aortic sac and conotruncal ridges at a level comparable to that of controls. to conclude, the observed phenotypes in alk5/wnt1-cre mutants were certainly not due to defective migration of cnccs to the pharyngeal and outflow tract regions. aortic sac and aortico-pulmonary septal defects in alk5/wnt1cre mutant embryos septation of the outflow tract lumen begins in a cranial-to-caudal direction, starting distally in the aortic sac and proceeding toward the heart. initially, the condensed mesenchyme derived from the nc forms in the base of the aortic sac between the origins of 4th and 6th paas. subsequently, two prongs of the developing aortico-pulmonary septum extend into the truncal cushions and the aortico-pulmonary septation complex crosses the aortic sac cranially. in ink-injected control embryos at e, a characteristic conotruncal transition separating the truncus and conus could be seen as a twisted configuration, resulting from a change in orientation of the truncal and conal cushions. in contrast, in alk5/wnt1-cre mutants the outflow tract appeared unusually straight, failing to demonstrate the distinct conotruncal transition. this assay also clearly showed a dramatic reduction in the size of the aortic sac. histological analysis of control samples displayed the characteristic rotation of the aortic sac and truncal oft at the level where the ap septation takes place and verified the presence of the distinctive condensed ap-septal mesenchyme, which gradually divided the oft to the aorta and the pulmonary trunk. r26r lineage tracing showed that this tissue is derived from the nc, while immunostaining for α-sma showed differentiation into smooth muscle. in alk5/wnt1-cre mutants the characteristic rotation of the aortic sac and truncal oft fails to take place, and a properly formed ap-septum was not detectable. r26r lineage tracing demonstrated that the defects were not due to failure of nccs to reach the oft region. nc-derived cells around the abnomally bifurcated aortic sac, the abnormally large sixth paas and the truncus demonstrated strong αsma staining. recently, we showed that the nc-specific mutants of the related type i receptor, alk display pta as well. in alk2/wnt1-cre mutants, the rotation of the aortic sac and truncal oft failed to occur as seen in alk5/wnt1-cre mutants. however, in alk mutants the 6th pair of the paas was grossly hypoplastic, and while the alk2/wnt1-cre mutants displayed a noticeable amount of septal tissue between the 4th and 6th paas, the condensed septal mesenchyme lacking alk failed to extend the prongs into the truncal cushions and to form the ap septum. concurrently, the 6th paas were losing their patency, which may have further contributed to the failed ap septation. while cnccs managed to migrate to the aortic sac and the truncal cushion level, immunostaining for αsma appeared much weaker when compared to controls and alk mutants, implying that alk2-mediated signaling is involved in smooth muscle cell differentiation as previously suggested. to conclude, while both alk and alk mutants demonstrate a failure in both the rotation of the aortic sac and the truncal oft, and in the formation of the ap septum, the pathogenetic mechanisms behind these defects appear remarkably different. alk5/wnt1-cre mutants display increased apoptosis of post-migratory neural crest cells as described above, alk5/wnt1-cre mutants displayed an inadequate amount of ap-septal tissue in the base of the aortic sac between the origins of 4th and 6th paas. to analyze whether this phenotype resulted either from defective cncc proliferation or inappropriate apoptosis, we used brdu and tunel staining, respectively. while cncc proliferation was not affected in alk mutants, we could detect a dramatic increase in the number of tunel positive cells in tissues surrounding the aortic sac including the site where the ap-septum forms. dual staining for lacz and tunel positive cells demonstrated that these cells were postmigratory cnccs; this phenotype was already clearly detectable at e. these results were confirmed by using immunostaining for cleaved caspase- another marker for apoptosis. in the chick, apoptotic neural crest-derived cells have also been found at the sites, where the prongs of the ap septum penetrate into the oft cushion mesenchyme. thus, we compared apoptosis patterns also on the more proximal level, but found no detectable differences at e between alk5/wnt1-cre mutants and controls. unlike in alk5/wnt1-cre mutant embryos, increased apoptosis of nc-derived cells is not responsible for the observed defects in the oft septation in corresponding alk mutants. to conclude, our results suggest that in alk5/wnt1-cre mutants a noticeable increase in apoptosis coincides with the abnormal patterning of the paas and the aortic sac, and with the failed ap-septation. these data support a specific role for alk signaling, either directly or indirectly, in cncc survival, since a similar apoptosis of nc-derived cells is not seen in tgfbr2/wnt1-cre mutants. pharyngeal organs fail to migrate in alk5/wnt1-cre mutants in addition to the cardiac oft, development of pharyngeal organs, i.e., the parathyroid glands and the thymus was also abnormal in alk5/wnt1-cre mutants. normally the thymus develops from the third pharyngeal pouch endoderm and migrates caudally to its final location in the superior mediastinum as seen in controls at e. in contrast, the thymic primordia of the alk mutant littermates failed to descend caudally, and were located bilaterally in the neck region, where they were surrounded by neural crest-derived mesenchyme. the fate determination assay demonstrated that the thymic primordia were equally populated by nccs both in controls and alk mutants. likewise the parathyroid glands failed to migrate normally in alk5/wnt1-cre mutants. during normal development, the parathyroids first migrate in association with the thymic primordia, until they reach the thyroids in the neck region as seen in fig 8c. in alk5/wnt1-cre mutants, the parathyroids remained associated with the thymic primordia, and despite this abnormal rostral location, expression of parathyroid hormone was indistinguishable between alk mutants and controls at e. to conclude, the observed pharyngeal organ phenotypes were also in striking contrast to those seen in tgfbr2/wnt1-cre mutants. discussion during the last few years the wnt1-cre transgenic driver line has proven to be a powerful tool for tissue-specific gene deletion in nccs. using this approach, several studies have independently shown that the nc-specific deletion of the tgfbr gene leads to a distinct set of calvarial, facial and cardiac defects. interestingly, these defects appear quite different both in the craniofacial and pharyngeal regions, including the heart, when compared to the corresponding mutants of alk which encodes the tgf-β type i receptor, a prototypical binding partner of tgf-βrii and the present study). while tgfbr2/wnt1-cre mutants as well as mice deficient in tgf-β display the pta type a, alk5/wnt1-cre mutants reported here demonstrate earlier patterning defects of the paas, which is particularly obvious in the 3rd pair of the paas. moreover, the alk5/wnt1-cre mutants display an abnormal patterning of the aortic sac and defective ap septation leading to pta, reminiscent of type a. however, our results also demonstrate that significant hypoplasia of the aortic sac leads to a severe shortening of the ascending truncal arch, which masks possible defects in derivatives of the 4th paas, i.e., interruption of the aortic arch. these observed differences are likely due to substantial apoptosis of alk5-deficient post-migratory neural crest cells, which is clearly detectable at e, whereas similar intense cell death has not been reported in tgfbr2/wnt1-cre mutants. our present results suggest that while tgf-β signaling in cardiac nccs is predominantly mediated via the alk5/tgf-βrii receptor complex, alk also mediates signaling of other related ligands, which are either directly or indirectly required for appropriate ncc survival. in fact, it has been shown that, besides tgf-βrii, alk can also form a complex with the activin type iib receptor to activate downstream smads 2/. furthermore, a subset of tgf-β-related growth and differentiation factors, e.g., gdf gdf gdf and gdf could induce these events. although relevant gdfs and are not expressed in the developing heart, nor do the mice deficient in these gdfs display developmental cardiac defects, we cannot exclude the possibility that circulating gdfs, perhaps in concert with tgf-βs may contribute to ncc survival during cardiac and pharyngeal morphogenesis. we specifically studied apoptosis at the level of the aortic sac, where the ap-septum forms between the origins of 4th and 6th paas. already at e, we could see intense apoptosis among the postmigratory nccs in the mesenchyme surrounding the aortic sac at the site where the prospective ap septum forms, i.e., this cell death precedes the ap septal defect seen in mutants. although some nc-derived cells appeared to be differentiating to smooth muscle cells in the oft, we could never detect the ap septum forming in alk5/wnt1-cre mutants between e and e. these findings suggest that the pool of cells forming the ap septum is severely affected by the cell death. moreover, it is likely that these cells forming the ap-septum die the before majority of them can differentiate to smooth muscle cells. several in vitro studies have suggested an indispensable role for tgf-β-signaling in differentiation of nccs into smooth muscle cells. moreover, a recent in vivo study suggested that mice lacking tgfbr in cnccs display defective ncc differentiation into αsma-positive cells in the ap septum, although this result was later disputed by another study. our immunohistochemical staining of αsma in the oft unequivocally demonstrated that signaling via alk is not required for smooth muscle differentiation in vivo. moreover, it has been suggested that deletion of tgfbr in nccs leads to other phenotypic features reminiscent of those seen in the velocardiofacial/digeorge syndrome caused by a deletion of the so called digeorge critical region on chromosome 22q. our present results suggest that although many of the observed phenotypes seen in alk5/wnt1-cre mutants superficially resemble those seen in vcf/dgs, a detailed examination shows that the nc-specific abrogation of alk does not lead to vcf/dgs-like phenotypes. firstly, while the pharyngeal organ migration fails in alk5/wnt1-cre mutants, perhaps as a result of increased mesenchymal cell death in the pharyngeal region, both the thymus, thyroid and parathyroid seem to develop relatively normally on the histological level in these mutants. secondly, the ncc death seen in alk mutants affects a predominantly postmigratory population of nccs, while genes located in the dgcr, i.e., tbx and crkl, control ncc survival earlier at e -e by regulating proliferation of the secondary heart field, and endoderm expansion, which in turn provides survival signal for nccs allowing them to populate the pharyngeal region. ncc ablation in the chick has been shown to lead to pta and to a failure of addition of myocardium from the secondary heart field. it was suggested that the defective migration of cells from the secondary heart field to the oft in turn resulted in shortening and inappropriate rotation of the oft, leading to mal-alignment of the arterial pole with the ventricles. while the detected oft phenotype in alk5/wnt1-cre mutants shared many similarities with that seen in the chick nc ablation models, e.g., pta and the hypoplastic aortic sac, our current results suggest that the secondary heart field is not severely affected in alk mutants. since we could not detect appropriate rotation of the oft in neural crest-specific mutants of alk or alk it appears that cells derived from the nc, as well as those from the shf, are mutually required for proper oft rotation in mice. however, it appears that these two tgf-β/bmp type i receptors contribute to oft rotation through different mechanisms. in alk mutants there is very little, if any, detectable septal mesenchyme present, and thus it could be argued that in these mutants oft rotation fails due to a lack of penetration of septal prongs into the cushion mesenchyme. in contrast, in alk2/wnt1-cre mutants a sizeable septal mesenchyme could be seen, still without any obvious looping of the aortic sac and truncal oft, suggesting that the mere presence of the septal mesenchyme, without correct smooth muscle cell differentiation, is not sufficient for oft rotation.  in this study, we have deleted the tgf-β type i receptor gene specifically in the mouse neural crest cell lineage. our data suggest that alk is required cell autonomously in the nc to mediate non-redundant signaling events that are essential for appropriate patterning of the pharyngeal organs and cardiac oft. the cardiac and pharyngeal defects observed in the nc-specific alk mutants differ significantly from those seen in corresponding mutants lacking the tgf-β type ii receptor, suggesting that signaling mediated by alk is not limited to the classical tgf-β ligands during cardiac/pharyngeal development. 
failure in signaling via alk leads to severe cardiovascular and pharyngeal defects, including inappropriate remodeling of pharyngeal arch arteries, abnormal aortic sac development, failure in pharyngeal organ migration and persistent truncus arteriosus. while alk is not required for neural crest cell migration, our results demonstrate that it plays an important role in the survival of post-migratory cardiac neural crest cells. our results demonstrate that alk5-mediated signaling in neural crest cells plays an essential cell-autonomous role in the pharyngeal and cardiac outflow tract development. we deleted the tgf-β type i receptor alk specifically in the mouse neural crest cell lineage.
 congenital cardiovascular diseases are the most common form of birth defects in humans. a substantial portion of these defects has been associated with inappropriate induction, migration, differentiation and patterning of pluripotent cardiac neural crest stem cells. while tgf-β-superfamily signaling has been strongly implicated in neural crest cell development, the detailed molecular signaling mechanisms in vivo are still poorly understood.  we deleted the tgf-β type i receptor alk specifically in the mouse neural crest cell lineage. failure in signaling via alk leads to severe cardiovascular and pharyngeal defects, including inappropriate remodeling of pharyngeal arch arteries, abnormal aortic sac development, failure in pharyngeal organ migration and persistent truncus arteriosus. while alk is not required for neural crest cell migration, our results demonstrate that it plays an important role in the survival of post-migratory cardiac neural crest cells.  our results demonstrate that alk5-mediated signaling in neural crest cells plays an essential cell-autonomous role in the pharyngeal and cardiac outflow tract development. 