Alagille syndrome (AGS, MIM 118450) is an autosomal dominant disorder with a variable phenotype characterised by hepatic, eye, cardiac, and skeletal malformations and a characteristic facial appearance. Mutations within the geneJAGGED1 (JAG1), which encodes a ligand for NOTCH receptor(s), has been shown to cause Alagille syndrome. Interactions of NOTCH receptors and their ligands influence cell fate decisions in several developmental pathways. We report the tissue expression of JAG1 in human embryos.
We have performed tissue in situ hybridisation on human embryos aged 32-52 days using 35S labelled riboprobes forJAG1. JAG1 is expressed in the distal cardiac outflow tract and pulmonary artery, major arteries, portal vein, optic vesicle, otocyst, branchial arches, metanephros, pancreas, mesocardium, around the major bronchial branches, and in the neural tube. We conclude thatJAG1 is expressed in the structures affected in Alagille syndrome, such as the pulmonary artery, anterior chamber of the eye, and face.
- Alagille syndrome
- arteriohepatic dysplasia
- NOTCH signalling
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Alagille syndrome (AGS, MIM 118450) is an autosomal dominant disorder associated with abnormalities of the liver, heart, eye, skeleton, and a characteristic facial appearance described first in 1969.1 It is one of the most common paediatric causes of chronic liver disease and has an estimated incidence in a mixed white population of 1 in 100 000 live births with no gender differences when ascertained on the basis of neonatal liver disease.2 3 The diagnosis is based on the finding of paucity of the interlobular bile ducts associated with three to five major features: chronic cholestasis, cardiac disease, skeletal abnormalities, ocular abnormalities, and a characteristic facial phenotype (table 1). However, the phenotype is very variable and within the same family one patient may present with life threatening congenital heart disease, whereas others may have only mild cholestasis. As yet, no specific phenotype has been associated with a particular genotype.4 The clinical features of AGS are shown in table 1.
Interstitial deletions within chromosome 20p were initially identified in a small number of Alagille cases and subsequently mutations in JAG1 were identified, showing that this was the disease gene.9-11 JAG1 stretches over 36 kb of genomic DNA on chromosome 20p12 and comprises 26 exons varying in size from 28 to 2284 bp to produce a 5.9 kb mRNA transcript.9 TheJAG1 gene encodes a protein belonging to the family of NOTCH ligands. These contain conserved sequences: a DSL domain (named after Delta, Serrate, and Lag-2 ligands forC elegans NOTCH), a varying number of EGF-like repeats (16 in human JAG1), a cysteine rich NOTCH region, and a transmembrane domain. In a recent study by Crosnier et al,12mutational analysis was performed on samples from 109 unrelated patients; 63% had intragenic mutations including 14 nonsense mutations, 31 frameshifts, 11 splice site mutations, and 13 missense mutations. Mutations were de novo in 40 of 57 probands. All the mutations mapped to the extracellular domain of the protein and the majority are predicted to give rise to truncated proteins.
Transgenic mice homozygous for the Jag1 mutation die from haemorrhage during early embryogenesis, exhibiting defects in remodelling of the embryonic and yolk sac vasculature. Mice heterozygous for the Jag1 null allele exhibit an eye defect similar to that seen in AGS patients, but do not have a similar hepatic or cardiac phenotype.13 Therefore, there is a discrepancy between the mouse and the human phenotype.
NOTCH and its ligands are well conserved between species and are involved in the determination of cell fate.14 However, the differences in phenotype between JAG1 mutations in human and mouse suggest that their roles may not be identical or there is a greater degree of redundancy in mice. Northern blot analysis of human adult RNA indicates thatJAG1 is widely expressed and most abundant in ovary, prostate, pancreas, placenta, and heart. Lower levels are seen in colon, small intestine, spleen, and skeletal muscle and the lowest levels in testis, thymus, leucocytes, kidney, liver, and lung.9 Human JAG1 studies have shown that JAG1 is expressed in the developing ductal plate of the fetal liver at 14 weeks, but earlier embryonic material was not examined.15 A recent study by Loomes et al 16 has shown that the expression of JAG1 in the developing mouse heart correlates with the picture of cardiovascular disease seen in Alagille syndrome. This study also included examination of a 56 day old embryonic human heart. Unfortunately, this is at the end of the embryonic period and cardiogenesis will almost have been completed. We have investigated JAG1 in earlier human embryonic development in order to determine the gene expression pattern and correlate it with the defects seen in Alagille syndrome.
Collection and use of human embryonic tissue was carried out following ethical approval from the Newcastle Health Authority. Embryos were collected following medically (RU486) or surgically induced termination of pregnancy, staged immediately according to the Carnegie classification system by stereomicroscopy, fixed overnight in 4% paraformaldehyde in phosphate buffered saline, and embedded in paraffin wax.17-19
IN SITU HYBRIDISATION
A 550 bp fragment of exon 26 of the humanJAG1 gene was used as a template to make sense and antisense RNA probes (IMAGE clone 430227) and also a 500 bp fragment of a similar region of mouse Jag1 (IMAGE clone 1227823). RNA probes were labelled with 35S using RNA in vitro transcription.19 Paraffin embedded mouse embryos between 9 and 13 ed and human embryos between 32 and 52 days post ovulation (dpo) were sectioned at 5 μm intervals and tissue in situ hybridisations were performed as previously described.19
At 32 dpo expression is seen in the lens vesicle and the tip of the optic cup (fig 1B, D). By 52 dpo expression was confined to the circumference of the developing lens and the anterior part of the visual layer of the retina (fig 2B).
JAG1 is expressed strongly in the distal cardiac outflow tract, aortic sac, and dorsal aortae at 32 dpo (fig1H). A lower level of expression was seen in the atria, particularly the right, but no convincing expression has been seen in the cardiac ventricles (fig 1H). By 52 dpo expression is detected in the pulmonary outflow tract, pulmonary artery, and aorta (fig 2F), but at lower levels than 32 dpo.
JAG1 is expressed in the portal vein and hepatic artery by 41 dpo (fig 3B). It is not expressed in the hepatic parenchyma during the age range studied.
Embryos aged 32 dpo showed strong JAG1 expression in the left and right sinus horns, branchial arches (fig1F), rhombencephalon, otocyst (fig 1B), and neural tube (fig 1F and fig3B). There was also expression around the bronchi. A striking feature was the strong expression of JAG1 in all the major arteries, such as the aorta and iliac and vertebral arteries. By 52 dpo JAG1 was expressed strongly in the ependymal layer of the spinal cord and the omentum of the midgut. At 13 weeks there was also expression in the pancreas (results not shown).
Jag1 expression was also examined in tissue sections of mouse embryos (embryonic day 9-13) and the distribution of expression was similar to that seen in human embryos.
We have shown that JAG1 expression during human embryonic development predominately localises to the organs or tissues that are affected in Alagille syndrome. The expression in the outflow tract, particularly the developing pulmonary trunk, correlates with common cardiac manifestations; 67% of patients with AGS have either right outflow tract obstruction (for example, pulmonary valve stenosis) or peripheral pulmonary artery stenosis.5 It is interesting that althoughJAG1 is also expressed in the developing aorta, the majority of cardiac defects affect the right side of the heart.
The peripheral margin of the optic cup differentiates into the ciliary body and iris.20 JAG1 is expressed in the presumptive ciliary body region before it is morphologically distinguishable from the adjacent neural retina and iris. Therefore, it is not surprising that ophthalmological findings in patients with AGS predominately affect the anterior chamber. Posterior embryotoxon, although not entirely pathognomonic of AGS, occurs in 55% of patients. It is characterised by a prominent Schwalbe's line (a line of material in the anterior chamber at the junction of the cornea and the uveal trabecular network). Although usually asymptomatic, such anterior chamber malformations can result in glaucoma. By 52 dpoJAG1 is expressed in the circumference of the lens and in the anterior part of the visual layer of the retina. This may explain the findings reported by Emericket al 5 of retinal pigmentary changes and optic disc drusen.
JAG1 is expressed in the portal vein and hepatic artery by 41 dpo. The ductal plate forms around the branches of the portal vein at about 56 dpo and is subsequently remodelled to produce mature bile duct architecture. During development,JAG1 is expressed in the ductal plate and postnatally in the biliary epithelium.15 The classical histopathological lesion in AGS is bile duct paucity but this lesion is progressive and is often not evident in the newborn period.21 The initial formation of the bile ducts appears to be macroscopically normal and then there is a gradual loss of these bile ducts. Initial correct formation of the ductal plate may well be in keeping with the phenotype of AGS. However, a defect in the development of the ductal plate may lead to later regression of the bile ducts. This is in contrast to the structural lesions in the heart and eye, which are primary malformations. It is possible that the bile duct loss is not the result of abnormal bile duct formation, but rather a later insult that affects the integrity of the bile ducts. This may be because of haploinsufficiency of JAG1 in the bile duct epithelium or a vascular defect.
One of the striking findings of the expression pattern ofJAG1 was the strong expression in all the major arteries. This is interesting as diffuse vascular abnormalities are a minor feature of Alagille syndrome. Renal artery stenosis,22 middle aortic syndrome,23 and intracranial bleeding8 have all been associated with Alagille syndrome. Additionally, patients sometimes have ileal stenosis or atresia suggesting possible prenatal ischaemic injury. Thus, it is possible that defects in the NOTCH signalling cascade result in vascular anomalies leading to these manifestations in Alagille syndrome.
Mutations in the gene for NOTCH3 receptor cause adult onset CADSIL which is associated with intracranial bleeding.24 Patients with CADSIL have small, deep cerebral infarcts, leucoencephalopathy, and a non-atherosclerotic, non-amyloid angiopathy involving the media of small cerebral arteries. Histopathological analysis shows major lesions of vascular smooth muscle cells that eventually disappear. Thus, NOTCH signalling pathways have roles in adult vertebrate tissues and in mature cells. If these pathways are perturbed then serious disease may result. The mechanism of the vasculopathy in AGS may be similar to that seen in CADSIL.
Patients with AGS have clefting of their vertebrae known as butterfly vertebrae. Notch1 and 2 are expressed in the presomatic mesoderm and Notch1 mutant mice undergo disorganised somitogenesis.25 Thus, the vertebral anomalies may be the result of disrupted NOTCH signalling. However, asJAG1 is not expressed in the developing vertebrae but is expressed in the vertebral arteries it is possible that the clefting is the result of vascular compromise during development.
JAG1 is expressed in both the meso- and metanephros. Renal anomalies have been reported in 23-74% of patients in series that have examined renal function.6 In the study of Emerick et al,5 the commonest abnormality was renal tubular acidosis but a number of structural abnormalities were also noted. These included small, hyperechogenic kidney, uretopelvic obstruction, renal cysts, and infantile onset renal insufficiency, which may be considered either structural or functional. Therefore, it is possible that more than one mechanism of disease may play a part in the renal abnormalities.
JAG1 is also expressed at sites that do not appear to be affected in AGS, such as in the neural tube. Structural defects of the nervous system are not commonly seen in AGS. It is possible that there is functional redundancy in the NOTCH signalling system and other ligands may substitute forJAG1. For instance, in mice,Jag2 is also expressed in the spinal cord.26
In summary, the embryonic expression pattern ofJAG1 shows correlation with many of the tissues affected in Alagille syndrome and gives clues to the pathogenetic mechanisms operating in this disease.
This work was funded by the Wellcome Trust and the Knott Trust.
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Corresponding to: Dr Jones,