| Human Molecular Genetics | Pages |
©1999 Oxford University Press |
The expression of Jagged1 in the developing mammalian heart correlates with cardiovascular disease in Alagille syndrome
Introduction
Results
JAG1 expression in the heart
JAG1 expression in the lung
JAG1 expression in the kidney, vertebral column, limb bud and eye
Discussion
Materials And Methods
Section in situ hybridization
Immunohistochemistry
Acknowledgements
References
The expression of Jagged1 in the developing mammalian heart correlates with cardiovascular disease in Alagille syndrome
Received July 21, 1999; Revised and Accepted September 14, 1999
The establishment of the cardiovascular system represents an early, critical event essential for normal embryonic development, and defects in cardiovascular development are a frequent cause of both in utero and neonatal demise. Congenital cardiovascular malformations, the most frequent birth defect, can occur as isolated events, but are frequently presented clinically within the context of a constellation of defects that involve multiple organs and that define a specific syndrome. In addition, defects can be a primary effect of gene mutations or result from secondary effects of altered cardiac physiology. Alagille syndrome (AGS) is an autosomal dominant disorder characterized by developmental abnormalities of the heart, liver, eye, skeleton and kidney. Congenital heart defects, the majority of which affect the right-sided or pulmonary circulation, contribute significantly to mortality in AGS patients. Recently, mutations in Jagged1 (JAG1), a conserved gene of the Notch intercellular signaling pathway, have been found to cause AGS. In order to begin to delineate the role of JAG1 in normal heart development we have studied the expression pattern of JAG1 in both the murine and human embryonic heart and vascular system. Here, we demonstrate that JAG1 is expressed in the developing heart and multiple associated vascular structures in a pattern that correlates with the congenital cardiovascular defects observed in AGS. These data are consistent with an important role for JAG1 and Notch signaling in early mammalian cardiac development.
INTRODUCTION
Alagille syndrome (AGS) is an autosomal dominant disorder characterized by developmental abnormalities of the heart, liver, eye, skeleton and kidney. Congenital heart defects, the majority of which are right-sided, contribute significantly to mortality in AGS patients. Overall, the incidence of AGS is at least 1:70 000 live births; however, the disease is under-diagnosed due to the variability in clinical presentation, even within the same family. JAG1, a ligand for the Notch receptors, was identified as the gene for AGS by positional cloning techniques in 1997 (1,2). (Throughout the text, the gene Jagged1 has been written as JAG1, whether it refers to mouse, human or rat; JAG1 is the human nomenclature and is used throughout for consistency.) AGS is the first congenital malformation syndrome caused by mutations in a gene in the Notch signaling pathway (1). With a long-term view to defining the role of JAG1 in normal heart development and thereby understanding the underlying basis for the heart defects seen in AGS, we have studied the expression pattern of JAG1 in the developing mammalian heart.
The Notch gene family encodes transmembrane receptors and ligands critical for cell fate decisions during development in both invertebrates and vertebrates (3). Studies on the role of the members of the Notch signaling pathway in human disease have only recently begun to emerge. The Notch genes themselves have been implicated in human disease in two instances. A T cell leukemia has been shown to be caused by a t(7;9) chromosomal translocation that interrupts the Notch1 (TAN1) locus at 9q34 (4). Germline mutations in Notch3 have been found in patients with a disorder termed CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy). CADASIL is a disease with onset during the fifth decade that is characterized by stroke and dementia and is associated with diffuse white matter abnormalities on neuro-imaging (5).
AGS, or arteriohepatic dysplasia, the disease caused by mutations in JAG1, is an autosomal dominant multisystem disorder that has been defined by paucity of the intrahepatic bile ducts associated with cholestasis, heart murmur, characteristic facial features, vertebral anomalies and eye abnormalities. Other recognized manifestations of the disorder include structural or functional renal disease and growth failure (6). Heart manifestations in AGS vary from minor to severe. Most (97%) patients have a heart murmur and, of these, the majority (67%) have peripheral pulmonic stenosis (6). Cardiovascular lesions associated with AGS include pulmonary artery stenosis, hypoplasia of the entire pulmonary vascular tree, pulmonary atresia, tetralogy of Fallot, truncus arteriosus, secundum atrial septal defect, patent ductus arteriosus and ventriculoseptal defect (7). The molecular pathways regulating development of these cardiac structures have not been well defined. The clinical features of AGS causing the most morbidity are congenital heart defects and chronic cholestatic liver disease.
Because mutations in JAG1 cause AGS (1,2), JAG1 and the Notch pathway are implicated in cardiovascular development. Whereas the patterns of JAG1 and Notch expression have been defined in the central nervous system, limb bud, tooth bud, eye and inner ear (8-12), no systematic study of JAG1 expression in the AGS target organs has been performed. The purpose of this study was to investigate the expression of JAG1 during critical periods of cardiovascular development in the mammalian embryo. Surveys of JAG1 expression in mouse and human development performed in our laboratory are presented here, focusing on heart and lung development. The mouse is an appropriate model for dissecting the role of this gene in mammalian heart development since multiple developmental time points can be obtained. Importantly, our mouse analyses are corroborated by data obtained using a 56 day human embryo sample.
RESULTS
JAG1 expression in the heart
Expression of the mouse JAG1 genehas been analyzed by RT-PCR and RNA in situ hybridization using sectioned tissues. Corresponding JAG1 expression in a human sectioned 56 day embryo has also been investigated by in situ hybridization. RT-PCRanalysis was used to detect expression in RNA obtained from whole mouse embryos at a variety of developmental stages ranging from 7.5 to 16.5 days post-coitum (d.p.c.). JAG1 was expressed in all stages that were tested (data not shown). JAG1 expression was also assayed in mouse 5 day post-natal liver, adult liver and adult heart, kidney, lung and eye and was present in all cases (data not shown).
To spatially localize JAG1 expression in the developing embryo, whole mount in situ hybridization was performed on 8.75-9.75 d.p.c. mouse embryos using a JAG1 antisense mRNA probe. Expression was detected in structures that include those destined to contribute to the cardiovascular system of the adult animal. In particular, JAG1 is expressed in the first pharyngeal arch at 8.5-8.75 d.p.c. and in pharyngeal arches 2, 3, 4 and 6 as well as in the heart, eye and intersomitic spaces by 9.5-9.75 d.p.c. (10, and data not shown). The sixth pharyngeal arch gives rise to the pulmonary artery. In all of the whole mount embryos, pharyngeal arch expression of JAG1 was bilaterally symmetrical. In addition, JAG1 expression was detected in the mesencephalon and rhombencephalon at 8.75 d.p.c. and strong expression was noted in the otic vesicle at 9.5 d.p.c. (data not shown). Based on these data, and the fact that JAG1 may be involved in heart development as observed by heart defects in AGS patients, we used section in situ hybridization to examine the expression in greater detail.
Sections of tissue were obtained from mouse embryos harvested at 8.75, 9.75, 10.5 and 12.5 d.p.c. and from a human 56 day embryo. JAG1 expression was detected in the 8.75 d.p.c. embryo with strong signal in the pharyngeal arches (10, and data not shown). At 9.75 d.p.c., expression was evident at multiple sites including the heart, the first pharyngeal arch, the descending aorta and the otic vesicle (data not shown). At mid-gestation in the mouse (10.5-12.5 d.p.c.) JAG1 expression was detected in multiple cardiovascular structures including the valve precursors of the heart. Transmural expression was detected in the atria but a much lower level of expression is present in the ventricles at 10.5 d.p.c. Robust JAG1 expression is detected in the descending aorta, the aortic arch arteries (AAA) and the neural tube (NT) (Fig. 1a). Figure 1b is a serial section probed with PECAM. To verify and compare the location of JAG1 expression, vascular structures were identified with PECAM-1 (CD31) (13), an endothelial cell marker. PECAM-1 is a member of the immunoglobulin superfamily expressed exclusively on early endothelial cells and is thought to be involved in endothelial cell-cell adhesion (14,15). We have utilized this marker to distinguish the pattern of JAG1 expression from that of endothelial cells in the vascular structures that were being studied here. Figure 1c is a JAG1 sense strand control on a 10.5 d.p.c. embryo section that confirms probe specificity. The next stage of development examined is 12.5 d.p.c.; at this time JAG1 is expressed in the pulmonary outflow tract (Fig. 1d and e) with a serial sense strand control shown in Figure 1f. Figure 1d also demonstrates JAG1 expression in the pharynx (Ph) and mandible (Man). JAG1 is also expressed in the aortic outflow tract (Fig. 1g); signal is detected in the intervertebral arteries and ductus arteriosus (Fig. 1g) and the endothelial cells of the endocardial cushions within the atrioventricular canal (Fig. 1g and h). These endocardial cells within the atrioventricular canal will undergo epithelial to mesenchymal transformation to initiate valve leaflet formation (16). JAG1 is also expressed at a high level in the pulmonary arteries (Fig. 1i) and the aorta (Fig. 1j). The combined studies with JAG1 and PECAM-1 show overlapping patterns of expression in the aortic arch arteries and descending aorta (Fig. 1a, b, j and k). However, in contrast to PECAM-1, JAG1 is not restricted to endothelial cells, but is also expressed in the perivascular cells and smooth muscle cells of developing vessels. Smooth muscle markers stain the muscle throughout the wall of the aorta. We examined the staining pattern of smooth muscle using an antibody in an immunohistochemistry experiment in the developing mouse aorta. A pattern of expression is seen for JAG1 mRNA (Fig. 1j) similar to the smooth muscle staining pattern in Figure 1l, i.e. throughout the vessel wall. Overall, JAG1 expression is particularly accentuated in the right-sided outflow tract, ductus arteriosus and pulmonary arteries, observations that correlate with the aforementioned right-sided abnormalities in the pulmonary circulation of AGS patients.
Figure 1. Section in situ hybridizations using JAG1 as probe. (a) A 10.5 d.p.c. mouse embryo probed with JAG1 shows expression in the atrium (At), aortic arch arteries (AAA), descending aorta (DAo) and neural tube. Vt, ventricle (b) A section through the 10.5 d.p.c. mouse heart (H) showing PECAM-1 expression in the endothelial cells of the aortic arch arteries (AAA, arrowheads). (c) A 10.5 d.p.c. mouse embryo section probed with a JAG1 sense probe to illustrate probe specificity. (d) The 12.5 d.p.c. mouse embryo expresses JAG1 in the pulmonary outflow tract (POFT), expression in the developing valve is limited to a thin layer of endothelium (arrow), whereas, outside the heart, expression is transmural (arrowhead). Expression is also seen in the pharynx and mandible. At, atrium; Vt, ventricle. (e) A higher magnification view of the pulmonary outflow tract (POFT), showing robust JAG1 expression. (f) A 12.5 d.p.c. mouse embryo probed with JAG1 sense control to illustrate probe specificity. At, atrium; POFT, pulmonary outflow tract; Vt, ventricle. (g) At 12.5 d.p.c. JAG1 expression is seen in the outflow tract (OFT), intervertebral arteries (IVA) and descending aorta and around the atrio-ventricular canal (AVC) and ductus arteriosus (DA). At, atrium; Vt, ventricle. (h) A higher magnification illustrating JAG1 expression around the atrio-ventricular canal between the endocardial cushions at 12.5 d.p.c. (i) JAG1 is expressed in the pulmonary arteries (PA's) but not obviously in the 12.5 d.p.c. mouse lung (Lg). H, heart. (j) JAG1 is strongly expressed in descending aorta (DAo) at 12.5 d.p.c. H, heart; Lv, liver. (k) A section through a 12.5 d.p.c. mouse embryo showing expression of PECAM-1 in the endothelial cells within the descending aorta (DAo) (the similarly colored staining at either end of the sectioned vessel represents red blood cells). (l) Immunohistochemical staining using a smooth muscle antibody on a section through the mouse aorta at 12.5 d.p.c. shows the pattern of smooth muscle staining throughout the walls of the vessel, the staining is seen as the dark brown lining of the aorta against the blue and white background. DAo, descending aorta. Scale bar [in (a)] for (a)-(d), (f), (g) and (i)-(k), 100 µm; scale bar [in (e)] for (e) and (h), 50 µm; scale bar for (l), 150 µm. The signal is a beige color and the background is blue.
In order to compare the patterns of JAG1 expression in the developing mouse heart with those of the human heart, sections of ahuman embryo at ~8 weeks of gestation (equivalent to ~13.5 d.p.c. in the mouse) were probed with JAG1. Expression of JAG1 throughout the myocardium was detected in the atria (Fig. 2a). However, expression in the ventricle was limited to the endocardium (the endothelial cells within the wall of the ventricle) (Fig. 2a). No expression was detected in the myocardial cell layers. Interestingly, expression was also detected in the coronary arteries (Fig. 2a) and epicardium (Fig. 2b), which have recently been defined as structures that differentiate from the liver primordium (17). From these human data, we propose that the mouse and human JAG1 expression patterns in the heart are comparable.
Figure 2. In situ hybridization using human embryos. (a) A section through a human 56 day embryo showing myocardial JAG1 expression in the atria (At) and in the coronary artery (CA), but expression in the ventricle (Vt) is limited to the endocardium. (b) A section through a 56 day human embryo showing rings of JAG1 expression around the airways (A) and blood vessels. Arrows indicate JAG1 expression in the epicardium. At, atrium; Vt, ventricle. (c) A section through a human kidney showing JAG1 expression in the kidney. (d) A section through a human 56 day embryo demonstrating JAG1 expression in the intervertebral spaces. Scale bar [in (d)] for (a)-(d), 100 µm. The signal is a beige color and the background is blue.
JAG1 expression in the lung
The timing of crucial events in the development of the airways and vascular system in human and mouse proceeds at different rates and the molecular events that control the development of the lungs in the mammal are not clearly defined. The adhesion molecules that play a large part in lung development have been described (15), but the role of signaling molecules such as JAG1 in this process are not well understood. Figure 2b shows JAG1 expression in the human 56 day embryonic lung with rings of expression around the airways and blood vessels. In contrast, in the mouse very little expression is detected in the corresponding 12-13 d.p.c. lung (Fig. 3a) except in the proximal pulmonary vasculature. In contrast, hybridization of a 12.5 d.p.c. mouse embryo with a probe for PECAM-1 (Fig. 3b) demonstrates robust expression in small vascular branches within the lung parenchyma, but spares the airways. Hybridization with a probe for JAG1 in 18.5 d.p.c. and newborn mouse lungs demonstrates expression in airways and large vessels but spares the small capillaries (Fig. 3c and data not shown). Thus, the timing of expression of JAG1 between these two species is different, but distinctive rings of JAG1 expression around the developing airways in both species point to a role for JAG1 and the Notch signaling pathway in lung development. PECAM-1 expression in a mouse 18.5 d.p.c. lung section shows ubiquitous expression in the endothelial cells except in the epithelium lining the airways (Fig. 3d).
Figure 3. Section in situ hybridization in the mouse lung. (a) JAG1 expression in a 12.5 d.p.c. mouse lung showing expression in the pulmonary arteries (PA) but little expression elsewhere. (b) PECAM-1 in situ hybridization in the 12.5 d.p.c. mouse lung showing expression in the small vascular branches within the lung parenchyma, but not around the airways themselves. (c) A section through an 18.5 d.p.c. mouse lung showing rings of JAG1 expression around the airways (A) and large blood vessels. (d) PECAM expression in the mouse 18.5 d.p.c. lung showing ubiquitous expression in the endothelial cells but no expression in the epithelial cells surrounding the airways. Scale bar for (a), 100 µm; scale bar [in (d)] for (b)-(d), 50 µm. The signal is a beige color and the background is blue.
JAG1 expression in the kidney, vertebral column, limb bud and eye
JAG1 expression was detectedin a variety of sites that correlate with disease in AGS patients. For example, robust, spatially unrestricted JAG1 expression is detected throughout the parenchyma of the human kidney (Fig. 2c); in a collection of 92 patients with AGS, 40% were documented to have structural or functional renal disease, the most common abnormality being renal tubular acidosis (6). JAG1 expression is detected in the vertebral column of a human 56 day embryo (Fig. 2d), an observation that correlates with 51% of patients exhibiting vertebral anomalies including butterfly vertebrae and shortened interpeduncular distances (6). JAG1 is also expressed in the developing mouse limb bud (10,18, and data not shown); some distal limb abnormalities have been associated with AGS. In several reports, detailed examination of the upper extremities revealed subtle abnormalities in 40-80% of AGS patients studied. The defects are clinically insignificant and include such findings as clinodactyly, shortened ulnae and distal phalanges and proximal placement of the thumbs (19). JAG1 expression is detected in the developing mouse eye at 9.5 and 10.5 d.p.c. (9, and data not shown). The most common ophthalmological abnormalities seen in AGS patients include anterior chamber anomalies such as posterior embryotoxon, Axenfeld's anomaly or Rieger anomaly. Pigmentary changes of the retina may be observed in some patients (19). Abnormalities of the optic disc have also been reported (6). The majority of the eye abnormalities in AGS do not impair vision. The liver, a major site of disease in AGS patients, has a highly restricted and well-defined pattern of JAG1 expression (data not shown). The sites of expression in the liver correlate with bile duct paucity, one of the major phenotypes in addition to heart disease in affected children.
DISCUSSION
The identification of the gene whose disruption is responsible for AGS in humans has prompted us to investigate the expression of JAG1 in heart structures affected in AGS patients and determine whether this correlates with disease. A histological analysis of a series of mouse and human embryos hybridized with JAG1 has revealed a strong correlation between affected structures and sites of expression. A role for JAG1 in heart development is implied by these expression data and the wealth of prior evidence that the Notch signaling pathway is known to be important in cell fate determinations in the developing embryo (3,20,21).
The expression of JAG1 has been studied previously in other systems, including the central nervous system, eye, limb bud and developing tooth (8-11,22). However, no systematic study has been made of JAG1 expression in the mammalian cardiovascular system. Targeted disruption of JAG1 in the mouse causes embryonic lethality at 10.5 d.p.c. in homozygous mutants. Mutant embryos have cranial hemorrhages and defects in remodeling of yolk sac vasculature (by angiogenesis). No structural heart defects are reported in the mutant embryos. Heterozygous mutant mice are normal except for an eye dysmorphology similar to that seen in Coloboma (Cm) mice (23). The results demonstrate that JAG1 has an important role in vascular development but early embryonic lethality in homozygous JAG1 null mice did not allow conclusions to be made about the contribution of JAG1 to development of the heart valves and lung vasculature.
Patients with AGS have specific cardiovascular defects which we have correlated with JAG1 mRNA expression in the developing human and mouse. In a recent study of 92 AGS patients, Emerick et al. (6) found that 24% of the population had structural intracardiac heart disease. Of these 22 patients, 77% were found to have a lesion involving the pulmonary valve or outflow tract (including tetralogy of Fallot or pulmonary stenosis or atresia in combination with other structural abnormalities). Twenty of the 22 patients had either atrial septal defect, ventriculoseptal defect or both. In this study, no patients had structural abnormalities of the mitral, tricuspid or aortic valves, but one patient was found to have coarctation of the aorta (6). In a review of the literature by Silberbach et al. (24) a total of three patients were reported to have aortic stenosis, one had a dysplastic atrio-ventricular valve and one had coarctation of the aorta. Thus, although right-sided cardiac abnormalities are clearly more common in AGS, defects in the left-sided circulation have been reported as well.
Strong JAG1 expression in the developing pulmonary valve and outflow tract correlate with the predominance of right-sided heart lesions seen in AGS patients, especially pulmonic stenosis. During formation of the valve leaflets, a population of endocardial cells from the region of the outflow tract undergo epithelial to mesenchymal transformation; JAG1 expression in the valve is restricted to this thin layer of cells (Fig. 1d, e, g and h). JAG1 is expressed throughout the endocardium, myocardium and epicardium of the atrium in both mouse and human, correlating with atrial septal defects (Figs 1a and 2a). In the ventricle, expression is more restricted to the endocardium and epicardium and is not present in the myocardium (Fig. 1b). Expression in the ductus arteriosus correlates with the finding of patent ductus arteriosus seen in some AGS patients (Fig. 1g). We detected JAG1 expression in the epicardium of the developing heart at 8 weeks gestation in the human (Fig. 2b). Although this epicardial expression does not correlate with a disease phenotype in AGS, it is interesting in the light of the fact that these epicardial cells undergo epithelial to mesenchymal transformation to give rise to coronary vascular smooth muscle, perivascular fibroblasts and intermyocardial fibroblasts (25).
JAG1 is also expressed in the aortic outflow tract (Fig. 1g) and in the descending aorta (Fig. 1j). Interestingly, aortic expression in both mouse and human is not restricted to endothelial cells but is also clearly evident in the peri-endothelial population of cells. Given the recent evidence to suggest an intimate and critical relationship between endothelial and smooth muscle differentiation during vascular development (26,27), these observations are particularly intriguing.
JAG1 expression is detected in the developing lung (Figs 2b and 3c) and in the blood vessels leading to the lungs (for example, the pulmonary arteries) (Figs 1i and 3a). In a study of 92 patients, 67% of those undergoing a formal cardiac evaluation were found to have stenosis at some level in the pulmonary vascular tree. This site of JAG1 expression correlates with this disease phenotype (peripheral pulmonic stenosis), a narrowing of the blood vessels that lead to the lungs, one of the most common heart lesions in AGS. The development of the pulmonary vasculature is known to involve both angiogenesis and vasculo-genesis (28). JAG1 has been shown previously to affect angiogenesis in vitro (29). In the human lung, JAG1 is expressed in the smooth muscle layer of the developing airways and in the large vessels. In the mouse lung, JAG1 expression is detected in the large but not in the smaller ramifications of the pulmonary vascular bed and in the airways. One explanation for the clinical manifestation of hypoplasia of the pulmonary vasculature in combination with JAG1 expression in the airways would be that Notch signaling is required for normal angiogenesis and vasculogenesis (involving the differentiation of blood vessels from mesenchymal cells) to occur surrounding the developing airways.
Levels of JAG1 expression do not always correlate with severity of the disease phenotype in AGS. For example, high levels of expression are detected in the developing kidney (Fig. 2c) but only ~40% of patients have renal involvement, which may range from a relatively inconsequential renal tubular acidosis to a more severe structural abnormality of the kidney or collecting system. Many studies have shown JAG1 to be expressed throughout the developing central nervous system (8,10,11,22); however, structural abnormalities of the brain or nervous system have never been documented in AGS. The incidence of mental retardation has ranged from 2 to 16% in previous studies, but developmental delay in some of these patients may have resulted from severe nutritional deficiencies (6). We have detected JAG1 expression in the otic pit at 9.5 d.p.c. in the mouse, but hearing loss has not been reported as a defining feature of AGS. Some patients with deletions of larger regions of chromosome 20p and AGS do have hearing loss, but this is thought to be the result of deletion of a different gene contiguous to JAG1 (N.B. Spinner, unpublished data). Similarly, JAG1 has been shown to be involved in the development of hematopoietic precursor cells, but patients with AGS do not have recognized abnormalities of bone marrow function (30). We hypothesize that in patients with a deletion or mutation of one copy of JAG1, haploinsufficiency of the gene product results in different clinical phenotypes depending on the tissue and other local factors influencing cell differentiation.
In summary, we have shown that JAG1 is expressed in the murine heart valves as well as the great vessels in a pattern similar to that seen in the developing human at a single time point. An abundance of JAG1 expression is documented in the outflow tracts (structures derived from the conotruncus), the pulmonary artery, ductus arteriosus and descending aorta. Many of the structures expressing JAG1 correlate with the disease phenotypes seen in patients with AGS. Taken together, our observations strongly implicate a role for JAG1 in early and late stages of mammalian cardiovascular development.
MATERIALS AND METHODS
Section in situ hybridization
Section in situ hybridization was performed on embryos harvested from timed matings from 8.5 to 14.5 d.p.c. and human 56 day embryos. The mouse embryos were dissected using a Nikon SMZ-U dissection microscope and fixed in 4% paraformaldehyde overnight. The human embryos were obtained from Alan Fantel (Director, Central Laboratory for Human Embryology at the University of Washington, Department of Pediatrics, Seattle, WA). Fixed embryos were embedded in paraffin wax using a Shandon Histocenter 2 embedder. Sections 7 µm thick were cut in ribbons onto glass slides using a microtome. Riboprobes were transcribed in the presence of [35S]UTP. Sense (control) and antisense (test) probes were generated for each region of the gene to be tested. Probes from the DSL (conserved delta-serrate-lag-2) region at the 5[prime]-end of the gene and the EGF repeat region of JAG1 were used in preliminary section in situ hybridization. The human JAG1 probe contained the DSL region and the EGF repeats cloned into the pCRII vector. The mouse probe was an expressed sequence tag (EST) containing 2.2 kb of mouse JAG1 cloned into pT7T3, including the poly(A) tail and 3[prime]-UTR, cytoplasmic domain and part of the EGF repeats. PECAM-1 in situ hybridi-zations used a mouse probe developed from an EST containing the 3[prime]-UTR and 2.6 kb of mouse PECAM-1 cloned into pT7T3. For both EST probes, the antisense strand was linearized with EcoRI and transcribed with T3 polymerase. The sense probe was linearized with NotI and transcribed with T7 polymerase.
The general protocol for section in situ hybridization and probe production was essentially as described previously (31,32) with some additional modifications. Essentially, slides containing the sections of interest were taken through a series of washes, including a de-waxing step with xylene and ethanol, acid treatment and treatment with proteinase K. The slides were then post-fixed in paraformaldehyde, washed in acetic anhydride and left to air dry. The slides were incubated in hybridization solution containing the probe in a humidified chamber overnight at 50°C. The sections were then washed and the signal was detected by dipping the slides in a photographic emulsion and exposing for 1-2 weeks. Slides were developed and counterstained and the signal was visualized under dark field or fluorescence microscopy.
Immunohistochemistry
Immunohistochemistry was performed essentially as described (33). Mouse anti-human [alpha]-smooth muscle actin antibody was obtained from Dako (Carpenteria, CA) and used at a dilution of 1:200. Paraffin sections were dewaxed, rehydrated and washed thoroughly in phosphate-buffered saline. The sections were incubated in 3% hydrogen peroxide in methanol for 30 min, washed in phosphate-buffered saline and then incubated with 10% goat serum for 1 h at room temperature. Diluted primary antibody was applied and the slides incubated at 4°C overnight. Following a phosphate-buffered saline wash, diluted goat anti-mouse biotin-conjugated secondary antibody was applied (Vector Laboratories, Burlingame, CA). Slides were washed and ABC reagent (Vector Laboratories), consisting of avidin conjugated to horseradish peroxidase, was applied. Detection was with diaminobenzene, as per the instructions from Vector Laboratories. The control slides were treated in the same way but with omission of the primary antibody.
ACKNOWLEDGEMENTS
We thank Dr J. Golden for his expert technical advice on whole mount in situ hybridization, Dr I. Krantz for his ongoing advice, Drs B.E. Emanuel and M.L. Budarf for human embryo sections, P.L. Jones for smooth muscle antibody, B. Sellinger for technical assistance and Dr M.H. Malim for critically reading this manuscript. This work was supported by Institutional start up funds (R.J.O.), NIH grant P50HL62177-01 (H.S.B. and R.J.O.) and NIH grant DK53104 (N.B.S.). K.M.L. is supported by training grants T32 DK07066 and 5T32GM08638.01.
REFERENCES
+To whom correspondence should be addressed. Tel: +1 215 590 3856; Fax: +1 215 590 3764; Email: oakey{at}mail.med.upenn.edu
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M. Noseda, G. McLean, K. Niessen, L. Chang, I. Pollet, R. Montpetit, R. Shahidi, K. Dorovini-Zis, L. Li, B. Beckstead, et al. Notch Activation Results in Phenotypic and Functional Changes Consistent With Endothelial-to-Mesenchymal Transformation Circ. Res., April 16, 2004; 94(7): 910 - 917. [Abstract] [Full Text] [PDF]
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M. T. Tetzlaff, W. Yu, M. Li, P. Zhang, M. Finegold, K. Mahon, J. W. Harper, R. J. Schwartz, and S. J. Elledge Inaugural Article, From The Cover: Defective cardiovascular development and elevated cyclin E and Notch proteins in mice lacking the Fbw7 F-box protein PNAS, March 9, 2004; 101(10): 3338 - 3345. [Abstract] [Full Text] [PDF]
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 T. Iso, Y. Hamamori, and L. Kedes Notch Signaling in Vascular Development Arterioscler Thromb Vasc Biol, April 1, 2003; 23(4): 543 - 553. [Abstract] [Full Text] [PDF]
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 B. McCright, J. Lozier, and T. Gridley A mouse model of Alagille syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency Development, March 4, 2003; 129(4): 1075 - 1082. [Abstract] [Full Text] [PDF]
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 T. Lebestky, S.-H. Jung, and U. Banerjee A Serrate-expressing signaling center controls Drosophila hematopoiesis Genes & Dev., February 1, 2003; 17(3): 348 - 353. [Abstract] [Full Text] [PDF]
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 V. Lindner, C. Booth, I. Prudovsky, D. Small, T. Maciag, and L. Liaw Members of the Jagged/Notch Gene Families Are Expressed in Injured Arteries and Regulate Cell Phenotype via Alterations in Cell Matrix and Cell-Cell Interaction Am. J. Pathol., September 1, 2001; 159(3): 875 - 883. [Abstract] [Full Text] [PDF]
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B McCright, X Gao, L Shen, J Lozier, Y Lan, M Maguire, D Herzlinger, G Weinmaster, R Jiang, and T Gridley Defects in development of the kidney, heart and eye vasculature in mice homozygous for a hypomorphic Notch2 mutation Development, January 2, 2001; 128(4): 491 - 502. [Abstract] [PDF]
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 Z. A. Eldadah, A. Hamosh, N. J. Biery, R. A. Montgomery, M. Duke, R. Elkins, and H. C. Dietz Familial Tetralogy of Fallot caused by mutation in the jagged1 gene Hum. Mol. Genet., January 1, 2001; 10(2): 163 - 169. [Abstract] [Full Text] [PDF]
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 D. A Cabezas, R. Slaugh, F. Abidi, J F. Arena, R. E Stevenson, C. E Schwartz, and H. A Lubs A new X linked mental retardation (XLMR) syndrome with short stature, small testes, muscle wasting, and tremor localises to Xq24-q25. J. Med. Genet., September 1, 2000; 37(9): 663 - 668. [Abstract] [Full Text]
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