Hyperplastic Conotruncal Endocardial Cushions and Transposition of Great Arteries in Perlecan-Null Mice
Perlecan is a heparan-sulfate proteoglycan abundantly expressed in pericellular matrices and basement membranes during development. Inactivation of the perlecan gene in mice is lethal at two developmental stages: around E10 and around birth. We report a high incidence of malformations of the cardiac outflow tract in perlecan-deficient embryos. Complete transposition of great arteries was diagnosed in 11 out of 15 late embryos studied (73%). Three of these 11 embryos also showed malformations of semilunar valves. Mesenchymal cells in the outflow tract were abnormally abundant in mutant embryos by E9.5, when the endocardial-mesenchymal transformation starts in wild-type embryos. At E10.5, mutant embryos lacked well-defined spiral endocardial ridges, and the excess of mesenchymal cells obstructed sometimes the outflow tract lumen. Most of this anomalous mesenchyme expressed the smooth muscle cell-specific α-actin isoform, a marker of the neural crest in the outflow tract of the mouse. In wild-type embryos, perlecan is present in the basal surface of myocardium and endocardium, as well as surrounding presumptive neural crest cells. We suggest that the excess of mesenchyme at the earlier stages of conotruncal development precludes the formation of the spiral ridges and the rotation of the septation complex in order to achieve a concordant ventriculoarterial connection. The observed mesenchymal overpopulation might be due to an uncontrolled migration of neural crest cells, which would arrive prematurely to the heart. Thus, perlecan is involved in the control of the outflow tract mesenchymal population size, underscoring the importance of the extracellular matrix in cardiac morphogenesis.
Perlecan is a multidomain heparan-sulfate proteoglycan with a wide distribution in extracellular matrices.1,2⇓ Perlecan is an important element for assembly and function of basement membranes, and its expression during embryonic development suggests critical functions in other extracellular matrices as well. Its highest expression in the mammalian embryo coincides with the development of the blood vessels, heart, and skeletal muscle. Furthermore, perlecan is later expressed during organogenesis of most visceral organs and cartilage.3 Diverse interactions have been reported between perlecan and other extracellular matrix proteins,4 cell adhesion molecules,5 and growth factors.6–8⇓⇓
Recently, we and others have shown that perlecan plays a critical role in the development of the heart, cartilage, and nervous system.9,10⇓ Perlecan-null embryos show pericardial hemorrhage, chondrodysplasia, and exencephaly. The mutation is lethal, with embryonic death showing two frequency peaks, between E10 and E12, and perinatally. Death was apparently due to myocardial defects in the earlier embryos and a respiratory failure caused by the severe osteochondrodysplasia in the newborns. We described discontinuities in the myocardial wall of embryos before E12, which probably explained the intense coelomic hemorrhages observed by these stages.9 However, the myocardium, although thinned, was continuous in late embryos, which did not show pericardial hemorrhages.
We have further investigated the heart development in perlecan-deficient embryos, and we have observed a high frequency of malformations in the outflow tract (OFT), resulting, in most cases, in complete transposition of great arteries (TGA).
TGA is a malformation characterized by the aorta arising from the anatomically right ventricle and the pulmonary trunk emerging from the anatomically left ventricle.11 The aortic root is located to the right and slightly anterior to the pulmonary trunk, and there is no fibrous mitroaortic continuity. This malformation, which keeps pulmonary and systemic circulation in parallel instead of in series, is lethal in newborns due to generalized hypoxia, unless venous and oxygenated blood can partially mix through atrial and/or ventricular septal defects. Even in this case, the malformation is life-threatening and requires urgent surgical intervention.11 The pathogenesis of TGA is controversial, due to the lack of appropriate animal models. TGA has been previously related only with genetic deficiencies involved in control of the left-right asymmetry, such as cryptic12 and the type IIB activin receptor gene.13 TGA can also be induced in all-trans retinoic acid–treated embryos.14,15⇓
Our present results represent the identification of a genetic defect that leads to complete TGA without further alterations of the lateral asymmetry, and point to an essential role of perlecan during conotruncal septation.
Materials and Methods
Generation of Perlecan-Deficient Mice
Perlecan-deficient mice were generated by gene targeting, as previously described.9 Genotyping of the embryos was performed by Southern blot analysis of EcoRI-digested genomic DNA obtained from tail fragments. Thirty-eight homozygous embryos and 30 heterozygous/wild-type control littermates were studied. All the embryos studied were alive and showed beating hearts at the moment of their collection. The perlecan-deficient mice were originally generated in the University of Lund (Sweden), as described in Reference 9, and breeding was performed in the University of Valencia (animal house registration No. 46005). The animals were cared for under the guideline Council Directive 86/609/EEC. The mice were killed in accordance with the European Commission DGXI (Laboratory Animals: Recommendation for Euthanasia of Experimental Animals).
Histology and Immunohistochemistry
For histology and immunohistochemistry, the embryos were fixed in 4% buffered paraformaldehyde in PBS or in methanol-acetone-water (2:2:1), dehydrated, and paraffin-embedded. Sections (10 μm) were stained with hematoxylin/eosin or immunostained. For immunoperoxidase staining, endogenous peroxidase and endogenous biotin were blocked and nonspecific binding sites were saturated with 16% sheep serum, 1% bovine serum albumin, and 0.5% Triton X-100 in Tris-PBS (SBT). The slides were then incubated overnight in the primary antibody diluted in SBT, washed, incubated for 1 hour in biotin-conjugated secondary antibody diluted 1:100 in SBT, washed again, and incubated for 1 hour in avidin-peroxidase complex diluted 1:150 in TPBS. Peroxidase activity was developed with Sigma Fast DAB tablets.
For double-labeling, we used a mixture of primary polyclonal and monoclonal antibodies followed by incubation with TRITC-conjugated anti-mouse IgG and biotin-conjugated anti-rabbit IgG (1:100). Finally, sections were incubated with FITC-conjugated avidin (1:150).
The rabbit polyclonal antibody against mouse perlecan domain II had been described earlier.16 It was used at a 1:1000 dilution for immunoperoxidase and 1:100 for immunofluorescence. The monoclonal anti–smooth muscle cell (SMC) α-actin (clone 1A4, Sigma) was used at a 1:2000 and 1:100 dilution in immunoperoxidase and immunofluorescence, respectively. The rabbit polyclonal L53, against myosin heavy chain of striated muscle (a gift of Dr Franco, University of Jaén, Spain), was used at a 1:25 dilution.
Cardiac Phenotype in Late Perlecan Mutant Embryos
In the heterozygous and wild-type embryos from E13.5 until E17.5, the heart showed normal septation of the conus and concordant connections between great arteries and ventricles. However, in 11 out of the 15 perlecan-null embryos studied at these stages, a complete TGA was observed (Figures 1A through 1D). The remaining mutant embryos showed ventriculoarterial concordance, although the aortic root was slightly dextropositioned when compared with the normal embryos.
Ventricular septation was complete in the perlecan-deficient embryos (Figure 1D), except for one embryo that showed a wide interventricular communication and a pulmonary ostium located at the right side of the interventricular septum, resulting in a double outlet right ventricle (Figure 1E). Other malformations were found in the semilunar valves of three late perlecan-null embryos bearing TGA. These malformations, two affecting the pulmonary valve and one affecting the aortic valve, basically consisted of asymmetrical and abnormal shapes of cushions, which were sometimes obstructing the vascular lumen (Figures 1F through 1H).
Perlecan-null embryos showed no anomalies related with the left-right asymmetry. Lungs, liver, stomach, and spleen were arranged as in the wild-type embryos. The vascular connections of right and left atria and the morphology of the atrioventricular valves were also normal (Figure 1I).
Excess of Mesenchymal Cells in the OFT of Early Perlecan Mutant Embryos
The main difference between the hearts of the early perlecan-deficient embryos and their heterozygous/wild-type littermates was found in the amount of mesenchymal cells populating the cardiac jelly of the OFT. In the heterozygous and wild-type embryos, only a few mesenchymal cells were detected by E9.5 in the distal half of the conotruncus (Figure 2A). In striking contrast, the same areas of perlecan mutant embryos were abundantly populated with mesenchymal cells. In some embryos, these cells formed relatively compact cell clusters that reduced the conal lumen (Figure 2B).
By E10.5, clear differences were also found at the level of the OFT between the homozygous mutants and heterozygous/wild-type embryos (Figures 2C, 2D, and 2G through 2L). In the latter, mesenchymal cells of the endocardial cushions formed two main ridges, the septal and the parietal (Figure 2C). These ridges showed a distinct spiral arrangement and a wide acellular area between the mesenchymal cells and the myocardium (Figures 2C and 2G through 2I). At the distal part of the conotruncus, two well-defined masses of condensed cells were observed in the dorsal and ventral wall (Figure 2G). These prongs of cells were continuous distally with the condensed mesenchyme of the aortic sac septum and proximally with the endocardial ridges.
In E10.5 perlecan-null embryos, the abundant mesenchymal cells of the OFT filled most of the space between endocardium and myocardium (Figures 2D and 2K). Compact clusters of mesenchymal cells were sometimes found. Defined, spirally arranged endocardial ridges were never observed. The lumen of the OFT was irregular, circular, or triangular instead of elliptic (compare Figure 2K with 2H). In some embryos, the conal lumen appeared severely narrowed, with the hepatic sinusoids and the posterior cardinal veins abnormally enlarged, probably due to a hypertensive venous return. Signs of myocardial damage, including ruptures, were sometimes observed in the proximal conus and conoventricular junction. No significant differences were observed in the most distal (truncal) part of the OFT and in the aortic sac. The septum of the aortic sac and the prongs of condensed mesenchymal cells formed normally in the perlecan-deficient embryos (Figure 2J). On the other hand, no significant differences were found in the atrioventricular endocardial cushions (Figures 2E and 2F).
Anomalous Conotruncal Septation in Perlecan Mutant Embryos
Although the conotruncal septation began at E11.5 in both homozygous and heterozygous/wild-type embryos, clear differences were observed in the organization of the mesenchyme of the endocardial ridges (Figure 3). In all embryos, the aorticopulmonary septum was observed between the aortic and pulmonary roots. However, only in wild-type and heterozygous embryos the endocardial ridges showed well-defined contours and a spiral arrangement. The conal lumen was narrowed in the medial part, giving rise to the aortic and pulmonary pathways. The aortic pathway, distally located at the right part, turns proximally to the dorsal side and opens to the ventricular lumen in the proximity of the interventricular foramen (Figure 3A).
In the perlecan-deficient embryos, the aortic and pulmonary trunks and the distal truncus appeared normally arranged. However, in the proximal conus, most of the mesenchyme was irregularly dispersed throughout the extracellular matrix, and defined endocardial ridges were not recognizable (Figure 3B and 3C). The conal lumen was misplaced from the center of the conus and showed a very mild curvature along the conus wall. Thus, the opening of the aortic pathway into the presumptive right ventricle is located far away of the interventricular foramen.
Septation of the OFT was virtually complete by E12.5. The result of this septation was different between perlecan-null embryos and their heterozygous or wild-type littermates. One of the three perlecan-null embryos studied at this stage showed TGA with a conus septum located in a sagittal plane at the level of the valvular primordium. The other two homozygous embryos showed concordant ventriculoarterial connection but the aorta was misplaced to the right of its normal position.
Immunohistochemical Characterization of the OFT Mesenchyme
At E10.5, perlecan immunoreactivity was very strong in the mesenchyme of the OFT and showed a distinct distoproximal gradient in wild-type/heterozygous embryos. The staining was more intense on the surface of the mesenchyme in the distal (truncal) area and decreased along the conus (Figure 4A). The most proximal mesenchyme, located at the conoventricular junction, and the mesenchyme of the atrioventricular endocardial cushions were faintly immunoreactive (Figure 4A and 4K). The basal surface of the myocardial, endocardial, and epicardial cells was also immunoreactive (Figures 4A, 4J, and 4K).
Because the distal OFT is being populated by migrating neural crest cells by 10.5 dpc, we attempted to identify presumptive neural crest cells with a monoclonal antibody against the SMC α-actin isoform. Perlecan immunoreactivity was especially marked around a number of SMC α-actin–positive cells in the distal OFT (Figure 4J).
SMC α-actin immunoreactivity revealed clear differences between wild-type and perlecan-deficient embryos. Positive cells were very scarce in the OFT of E9.5 heterozygous and wild-type embryos (Figure 4B). In normal E10.5 embryos, immunoreactive cells were detected around the aortic sac and in the truncus area, as well as scattered in the mesenchyme of the endocardial ridges of the conus (Figures 4D, 4F, and 4J). The immunoreactivity of these cells ranged from faint to moderately intense, but was always lower than the immunoreactivity of the conal myocardial cells.
In contrast to heterozygous and wild-type embryos, E9.5 and E10.5 perlecan-null embryos contained abundant SMC α-actin immunoreactive cells in the OFT, cells that sometimes extended along the whole conus (Figures 4C, 4E, 4G, and 4L). These cells reached even the trabeculate portion of the right ventricle (Figure 4E). The immunoreactivity of these cells was stronger than in the control embryos and similar to the labeling of the myocardial cells. However, they were not stained with antibodies against myocardial markers (Figure 4H).
In a previous article,9 we showed that disruption of the perlecan gene in mice results in embryonic lethality, either around E10 or around birth. To determine the cause of death, we have now performed a thorough analysis of the heart of perlecan-null embryos. This analysis revealed major abnormalities of cardiac development. In particular, complete TGA was observed in 73% of the embryos surviving until E17.5. TGA was sometimes associated with abnormalities in the semilunar valve leaflets. Other mutant embryos without TGA showed dextroposition of the aortic root.
Mutations in a number of genes in mouse have been correlated with OFT malformations, but among them, TGA is a rare event. TGA has been observed in mice after treatment of pregnant animals with all-trans retinoic acid,14,15⇓ and as a consequence of mutations of genes involved in establishing left-right asymmetry.12,13⇓ In the retinoic acid–treated mice, hypoplastic conal swellings and the lack of well-developed proximal endocardial ridges prevent the proximal rotation of the aortic and pulmonary orifices. On the other hand, TGA resulting from a deficiency in genes such as cryptic, a member of the EGF-CFC family of membrane-associated receptors,12 or the type IIB activin receptor13 is always associated with ventricular and atrial septal defects and laterality anomalies, such as randomized heart position or pulmonary isomerism. In contrast, TGA in the perlecan-null embryos was usually accompanied by complete ventricular septation, and we did not observe anomalies in laterality. Therefore, perlecan deficiency results in a model alike to the human TGA with situs solitus.
The first phenotypic manifestation related to the absence of perlecan is the abnormal abundance of cells within the OFT at the earliest stages of formation of the endocardial cushions (E9.5). However, the atrioventricular cushions of the mutant embryos were indistinguishable from those from the wild type. By E10.5, lack of defined endocardial ridges, together with an anomalous excess of mesenchymal cells in the OFT, were evident in the perlecan-null embryos. As a consequence, the lumen of the conus became rounded or irregular, and the mesenchyme filled the whole cardiac jelly, invading areas close to the myocardium that are normally devoid of mesenchymal cells. The conal lumen became dramatically obstructed in some embryos, and this observation, together with the evidence of hypertension in the venous return, myocardial damage, and ruptures, might explain the massive hemorrhages and the embryonic death that peaks at this stage.9
Several mutations that result in overgrowth of endocardial cushions are known to produce cardiac malformations. Absence of glypican-3, an heparan sulfate proteoglycan implicated in the Simpson-Golabi-Behmel syndrome, produces a general overgrowth during development,17 although cardiac malformations have only been observed in humans,18 with highest incidence of ventricular and atrial septal defects. On the other hand, unregulated Ras activity leads to hyperproliferation of mesenchymal cells, as shown in neurofibromin (Nf1) deficient mice,19 which develop overabundant endocardial cushions. Cell proliferation in Nf1-null embryos is observed after E12.5 and affects both the outflow tract and the atrioventricular endocardial cushions. Besides, double-outlet right ventricle was invariably observed in Nf1−/− embryos. The malformations observed in perlecan-null embryos clearly differ from these overgrowth syndromes, because the abnormal overpopulation of the OFT endocardial cushions is limited to the early stages.
Besides perlecan, other components of the cardiac jelly, such as versican and hyaluronan, are essential to form the endocardial cushions. Mice bearing targeted deletion of hyaluronan synthase-2 (Has2) do not express hyaluronan in the endocardial cushions, resulting in compaction of the extracellular space and absence of endocardial-mesenchyme transformation. These mice lack of endocardial cushions and trabeculae in the heart.20 Similarly, the initial swellings of the endocardial cushions are absent in both the conotruncal and the atrioventricular segments of the heart of hdf homozygous mice, an embryonic lethal mutation due to disruption of the versican (Cspg2) gene.21 Unlike hyaluronan and versican, which are located throughout the acellular cardiac jelly, perlecan is only detectable in the neighborhood of the cushion mesenchymal cells, as well as in the basal laminae of the endocardium and myocardium. Significantly, perlecan-null embryos showed normal swelling of the endocardial cushions and endocardial-mesenchyme transformation.
Perlecan was abundantly expressed in the distal endocardial ridges of E10.5 embryos. However, the endocardial-derived mesenchymal cells of the proximal OFT are only stained in the subendocardial layer. This staining pattern was identical to that found in the AV cushions and suggests that perlecan expression declines rapidly in the endocardial-derived cells. On the other hand, the strongest perlecan immunoreactivity was found, in the distal OFT, on mesenchymal cells expressing SMC α-actin, a reliable marker of the migrating neural crest cells in the mouse OFT.22,23⇓
The normal localization of perlecan should be correlated to the anomalous amount of OFT mesenchyme found in the perlecan-deficient embryos. This excess of mesenchyme might be explained, at least in part, by a premature migration of neural crest cells into the OFT. In E9.5 wild-type embryos, only scattered neural crest cells have arrived to the most distal OFT.23,24⇓ However, our E9.5 mutant embryos already showed an abnormal amount of mesenchymal cells in the OFT, and many of them were SMC α-actin+. Furthermore, α-actin immunoreactive cells were found at unusually proximal levels by E10.5 in the perlecan-deficient embryos, sometimes reaching the trabeculated portion of the right ventricle.
It seems plausible that an uncontrolled migration of the neural crest accounts for the mesenchymal excess reported in the OFT of perlecan-deficient embryos. However, we cannot discard that part of the exceeding mesenchymal cells have an endocardial origin. Both neural crest and endocardial-derived cells are exposed to potent mitogens in the OFT, mainly FGFs as well as insulin-like growth factors.25,26⇓ Perlecan specifically binds FGF2 and promotes receptor activation27 and mitogenesis.6 Binding sites for FGFs have been recently described in the core protein.28 In addition, several growth factors and growth factor–regulator molecules bind to heparan sulfate moieties. Perlecan could thus bind and sequester these mitogens, in line with its ability to potently inhibit smooth muscle cell proliferation.29 Perlecan covers the surface of presumptive neural crest cells and the endocardium, the sources of the OFT mesenchyme, as well as the myocardium, a tissue that secretes mitogenic growth factors. Therefore, it is conceivable that the lack of perlecan increases the availability of mitogenic signals into the OFT and leads to an increased proliferation of the OFT mesenchyme, independently of its origin. However, it is significant that the atrioventricular endocardial cushions, which are populated by endocardial-derived cells but not by neural crest cells, were of normal size in perlecan-deficient embryos. This observation strengthens the hypothesis that perlecan deficiency affects mainly to neural crest cells.
Neural crest plays an essential role in OFT septation.30 Cardiac neural crest ablation and diverse mouse gene mutations that affect the cardiac neural crest migration have demonstrated that the neural crest is required for formation of the conotruncal septum. Disruption of genes such as Pax3/Splotch,31 or the endothelin-A receptor,32 predominantly result in abnormalities of the aorticopulmonary septation that are consistent with a severe cardiac neural crest deficiency. In contrast, an excess of neural crest cells explains the ventricular hypertrophy and OFT obstructions in CMV43 mice, a transgenic strain that overexpresses the gap junction gene Cx43 driven by the CMV-promotor.33 This observation suggests that in perlecan-null embryos TGA cannot be explained only from overabundance of cardiac neural crest, but rather from changes in the differentiation and organization of all the OFT mesenchymal population. This is also suggested by the phenotype of embryos deficient in the transcription factor Sox4 that show lack of fusion of the linearly arranged OFT ridges.34 Although Sox4 is expressed in the endocardially-derived tissue of both the OFT and AV cushions, malformations are restricted to the OFT development, suggesting again a defect in the interaction between endocardial-derived and neural crest–derived mesenchymal cells.34
In conclusion, we suggest that a disorganization of the spiral endocardial ridges due to an excess of mesenchymal cells is the main cause of the abnormal septation in perlecan-deficient embryos. It is remarkable that the ultimate result, a malposition of the great arteries, is similar in our model, in the retinoic acid-treated embryos, and in the Nf1-deficient embryos, which show hypoplasia and hyperplasia of the endocardial cushions, respectively. This illustrates the need of well-defined spiral endocardial ridges for the rotation of the aorticopulmonary septum and the correct ventriculoarterial alignment. It is important to remark that most hypotheses about the origin of TGA have hitherto focused on much later events, such as an arrested rotation of the OFT,35 or a differential conal development,36,37⇓ whereas virtually no attention has been paid to the involvement of neural crest cells in this pathology. It would be a matter of further research to test whether the present model of TGA relates to the development of congenital anomalies of the OFT in humans.
This work was supported by grants PM98-0219, PM99-0179, and 1FD97-0693 (DGESIC, Spain). We thank Gerardo Atencia for their valuable technical help, Diego Franco for their kind gift of the L53 antibody, and Amalia Capilla and Mónica Gomez-Lor for genotyping mice and embryos.
Original received December 21, 2001; revision received June 3, 2002; accepted June 4, 2002.
- ↵Murdoch AD, Liu B, Schwarting R, Tuan RS, Iozzo RV. Widespread expression of perlecan proteoglycan in basement membranes and extracellular matrices of human tissues as detected by a novel monoclonal antibody against domain III and by in situ hybridization. J Histochem Cytochem. 1994; 42: 239–249.
- ↵SundarRaj N, Fite D, Ledbetter S, Chakravarti S, Hassell JR. Perlecan is a component of cartilage matrix and promotes chondrocyte attachment. J Cell Sci. 1995; 108: 2663–2672.
- ↵Mongiat M, Taylor K, Otto J, Aho S, Uitto J, Whitelock JM, Iozzo RV. The protein core of the proteoglycan perlecan binds specifically to fibroblast growth factor-7. J Biol Chem. 2000; 275: 7095–7100.
- ↵Costell M, Gustafsson E, Aszodi A, Mörgelin M, Bloch W, Hunziker E, Addicks K, Timpl R, Fässler R. Perlecan maintains the integrity of cartilage and some basement membranes. J Cell Biol. 1999; 147: 1109–1122.
- ↵Thiene G, Frescura C. Cardiopatie congenite.In: Cali A, Fiore-Donati L, eds. Anatomia Patologica Generale e Applicata. Firenze: Uses; 1988: 795–837.
- ↵Oh SP, Li E. The signaling pathway mediated by the type IIB activin receptor controls axial patterning and lateral asymmetry in the mouse. Genes Dev. 1997; 11: 1812–1826.
- ↵Yasui H, Nakazawa M, Morishima M, Miyagawa-Tomita S, Momma K. Morphological observations on the pathogenetic process of transposition of the great arteries induced by retinoic acid in mice. Circulation. 1995; 91: 2478–2486.
- ↵Nakajima Y, Hiruma T, Nakazawa M, Morishima M. Hypoplasia of cushion ridges in the proximal OFT elicits formation of a right ventricle-to-aortic route in retinoic acid-induced complete transposition of the great arteries in the mouse: scanning electron microscopic observations of corrosion cast models. Anat Rec. 1996; 245: 76–82.
- ↵Cano-Gauci DF, Song HH, Yang H, McKerlie C, Cho B, Shi W, Pullano R, Piscione T, Grisaru S, Soon S, Sedlackova L, Tanswell AK, Mak TW, Yeger H, Lockwood GA, Rosenblum ND, Filmus J. Glypican-3-deficient mice exhibit developmental overgrowth and some of the abnormalities typical of Simpson-Golabi-Behmel syndrome. J Cell Biol. 1999; 146: 255–264.
- ↵Lakkis MM, Epstein JA. Neurofibromin modulation of ras activity is required for normal endocardial-mesenchymal transformation in the developing heart. Development. 1998; 125: 4359–4367.
- ↵Camenish TD, Spicer AP, Brehm-Gibson T, Biesterfeldt J, Augustine ML, Calabro A Jr, Kubalak S, Klewer SE, McDonald JA. Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme. J Clin Invest. 2000; 106: 349–360.
- ↵Ya J, van den Hoff MJ, de-Boer PA, Tesink-Taekema S, Franco D, Moorman AF, Lamers WH. Normal development of the OFT in the rat. Circ Res. 1998; 82: 464–472.
- ↵Mjaatvedt CH, Yamamura H, Wessels A, Ramsdell A, Turner D, Markwald RR. Mechanisms of segmentation, septation and remodeling of the tubular heart: endocardial cushion fate and cardiac looping.In: Harvey RP, Rosenthal N, eds. Heart Development. San Diego, Calif: Academic Press; 1999: 159–177.
- ↵Joseph SJ, Ford MD, Barth C, Portbury S, Bartlett PF, Nurcombe V, Greferath U. A proteoglycan that activates fibroblast growth factors during early neuronal development is a perlecan variant. Development. 1996; 122: 3443–3452.
- ↵Mongiat M, Otto J, Oldershaw R, Ferrer FJ, Sato D, Iozzo RV. Fibroblast growth factor-binding protein is a novel partner for perlecan protein core. J Biol Chem. 2001; 276: 10263–10271.
- ↵Conway SJ, Henderson DJ, Copp AJ. Pax3 is required for cardiac neural crest migration in the mouse: evidence from the splotch (Sp2H) mutant. Development. 1997; 124: 505–514.
- ↵Clouthier DE, Hosoda K, Richardson JA, Williams SC, Yanagisawa H, Kuwaki T, Kumada M, Hammer RE, Yanagisawa M. Cranial and cardiac neural crest defects in endothelin-A receptor-deficient mice. Development. 1998; 125: 813–824.
- ↵Ewart JL, Cohen MF, Meyer RA, Huang GY, Wessels A, Gourdie RG, Chin AJ, Park SM, Lazatin BO, Villabon S, Lo CW. Heart and neural tube defects in transgenic mice overexpressing the Cx43 gap junction gene. Development. 1997; 124: 1281–1292.
- ↵Ya J, Schilham MW, de-Boer PA, Moorman AF, Clevers H, Lamers WH. Sox4-deficiency syndrome in mice is an animal model for common trunk. Circ Res. 1998; 83: 986–994.
- ↵Anderson RH, Wilkinson JL, Arnold R, Becker AE, Lubkiewicz L. Morphogenesis of bulboventricular malformations, II: observations on malformed hearts. Br Heart J. 1979; 36: 948–970.