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Correspondence to: Juha Kere, Dept. of Medical Genetics, Haartman Institute, PO Box 21 (Haartmaninkatu 3), 00014 University of Helsinki, Finland.
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Summary |
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Anhidrotic ectodermal dysplasia (EDA) is characterized by defects in the development of teeth, hair, and sweat glands. To study the expression of the human gene defective in EDA in human fetal development (Weeks 623 of gestational age) and in adult tissues, in situ hybridization and immunohistochemistry were used. First signs of expression were detected at Week 8 in epidermis and in neuroectodermal cells. Starting at Week 12, osteoblasts and thymus were positive for EDA mRNA. Hair follicles expressed EDA mRNA from 18 weeks. The presence of the EDA protein coincided with mRNA expression in the tissues examined. The expression pattern of the EDA gene is consistent with typical involvement of the skin in the syndrome. However, the expression is not limited to the ectodermal tissues and many sites of expression are not obviously reflected in the clinical features of the syndrome. (J Histochem Cytochem 46:281289, 1998)
Key Words: skin, syndrome, in situ hybridization, immunohistochemistry, mRNA, protein
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Introduction |
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X-LINKED ANHIDROTIC (HYPOHIDROTIC) ECTODERMAL DYSPLASIA (EDA; MIM #305100) (
Recently, we isolated an X chromosomal gene with mutations in EDA patients (
To gain knowledge of the role of the EDA gene in the developing human fetus, we have studied its expression patterns by mRNA in situ hybridization and immunohistochemistry. We report that the expression of the EDA gene is not restricted to tissues affected in the syndrome. Its expression starts as early as Week 8 of gestation in some tissues and continues through fetal life to some adult cell types.
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Materials and Methods |
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Tissues
Formalin-fixed, paraffin-embedded archival specimens were obtained from the Departments of Pathology of the Universities of Oulu and Helsinki, Finland. The study was approved by the ethics committees of the Departments of Medical Genetics and Dermatology. The following subgroups of histological sections were examined: (a) complete embryos and fetuses at gestational ages of 6, 7, 8, 9, 10, and 12 weeks. All material originated from medical abortions. Fetal age was estimated by menstrual age and morphological criteria; (b) selected tissues from older fetuses: skin biopsies from fetal scalp and trunk at 15, 16, 18, 21, and 23 weeks of gestation; kidney, testis, and gastric and colon mucosa at Weeks 15, 19, and 20; liver at Weeks 15 and 20; pancreas at Weeks 15 and 19; and esophagus at Weeks 19 and 20; and (c) histologically normal adult organ specimens: liver (n = 3), kidney (n = 3), pancreas (n = 5), prostate gland (n = 3), adrenal gland (n = 3), bronchi (n = 3), colon mucosa (n = 5), endometrium (n = 3), hypothalamus (n = 2), spinal ganglia (n = 3), sympathetic ganglia (n = 3), mammary gland (n = 3), and incisor tooth (n = 1). The tissues were chosen on the basis of our previous Northern analyses (
In Situ Hybridization
The sequence and specificity of the 551-BP EDA anti-sense cRNA probe have been described (
Immunohistochemistry
Polyclonal antibodies against the EDA protein expressed in E. coli were raised in rabbits and purified by affinity columns (
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Results |
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A summary of tissues showing EDA mRNA expression by in situ hybridization is shown in Table 1. More detailed descriptions of different tissues and timing of expression are described below according to organ groups.
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Expression of EDA mRNA in Epidermis and Its Appendages
The first signs of expression were detected in Week 8 of gestation, when mRNA for EDA was seen in epidermis covering the lower jaw. At Week 9, epidermis of the developing limb was positive and by Week 10, epidermis of fingers, upper and lower jaw, acoustic canal, and scalp displayed EDA mRNA (Figure 1AC, Figure 1G, Figure 1H). No expression was seen in a tooth germ at an early stage of development at Week 10. The epidermis of the trunk was consistently positive beginning at Week 16, whereas the condensed mesenchymal cells in the hair papilla were negative (Figure 1DF). EDA mRNA was consistently expressed in the outer root sheath of scalp hair follicles from Week 18 of gestation (Figure 1I and Figure 1J). In contrast, appendages in their early stages of development, including developing hair pegs, were consistently negative. In addition to hair follicles, eccrine sweat glands and sebaceous glands were positive in adult skin (Figure 1KN).
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Expression in Neuroectodermal Cells
From Week 8, neuroectoderm surrounding the cerebral vesicles and the spinal canal was positive for EDA mRNA (Figure 2AH). The expression continued consistently in all brain sections that were studied (Table 1). From Week 10, EDA mRNA was detected in dorsal root ganglia and anterior horn cells of the spinal cord (Figure 2I and Figure 2J). EDA-positive neurons were also seen in adult sympathetic and spinal ganglia as well as hypothalamus (Figure 2KM).
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Osteoblasts, but not Chondrocytes, Express EDA mRNA
At Week 12 of gestation, EDA mRNA was detected in osteoblasts of ribs (Figure 3A and Figure 3B). In contrast, chondrocytes and hypertrophic chondrocytes of developing bones showed no signal (Figure 3A). Osteoblasts lining the newly deposited bone matrix also showed intense expression in the calvarial bones at Week 16 (Figure 3C and Figure 3D).
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EDA mRNA Expression in Internal Organs
Mesenchymal cells in most organs were negative for EDA mRNA. Bronchial epithelium and lungs were negative in two specimens studied (Figure 3G). Scattered cells in the developing heart showed abundant signal for EDA mRNA at Week 8, and the developing thymus showed strong signal for EDA at Week 12 (Figure 3E and Figure 3F). The esophageal surface epithelium was positive at least at Weeks 12 and 19 (Figure 3J and Figure 3K). At Week 20, the renal collecting tubules and pelvic epithelium were positive (Figure 3L and Figure 3M).
Patterns of Expression in Adult Tissues Other than Skin
EDA mRNA was detected in the duct epithelium of adult prostate (Figure 3O and Figure 3P), mammary gland, kidney, sympathetic ganglia, and hypothalamus (Figure 2LN). Pancreas, liver, colon mucosa, endometrium, and bronchial epithelium showed no signal. In the one adult tooth section available for study, mesenchymal cells around the tooth were positive for EDA mRNA (Figure 3H and Figure 3I).
EDA Protein Co-localizes with Sites of mRNA Expression
To study whether EDA protein was translated at sites of mRNA expression, immunohistochemistry using a polyclonal antibody was performed on several of the specimens. EDA protein co-localized with mRNA in tissues examined, including the neuroectoderm of brain vesicles (Figure 2H), the dorsal root ganglia (Figure 2K), thymus (Figure 3G), kidney tubules (Figure 3N), esophageal epithelium, and developing bone.
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Discussion |
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Cloning of the EDA gene revealed a predicted transmembrane protein with no homologies to known proteins that would have suggested a functional role (
A more detailed study of the expression of the EDA gene using both in situ hybridization and immunohistochemistry is motivated by several points. First, expression in a wide range of fetal and adult tissues (as suggested by Northern analyses) impelled further study and the identification of cell types expressing EDA mRNA. Second, data on the timing of expression could aid in defining the role of the EDA gene in the chain of morphogenetic events. Third, information on the expression pattern in various organs and cell types might offer clues to the phenotypic features of EDA. Finally, it is useful to know whether transcription of the gene in various tissues and cell types coincides with translation and the presence of the protein product.
Expression of EDA in Fetal Tissues
Northern analyses had suggested that many fetal and adult organs consisting of different cell types express EDA mRNA (
The strong expression of EDA mRNA in osteoblasts contrasts with the absence of EDA mRNA in chondrocytes, fibroblasts, and dermal papillae in the skin and in mesenchymal cells in many other organs. These results indicate that EDA expression is not restricted to epithelial cells and suggest the presence of specific regulatory mechanisms for controlling EDA gene expression.
Timing of Expression Suggests Multiple Roles
Further support for the specificity of regulation came from the staged timing pattern of EDA mRNA expression during fetal development. Expression was first detected in the epithelial and neuroectodermal cells at Week 8. Within the next few weeks, expression spread to other tissues, and by Week 18 was observed in mature appendages throughout the skin. In general, some tissues showed early expression that was later turned off, whereas in other tissues mRNA first appeared later (Table 1). These observations suggest a double role for the EDA gene: first, a developmental role that is necessary for morphogenesis to be completed and, second, a maintenance role, e.g., in adult skin, supported by continued expression.
Phenotypic Correlations
Expression of EDA mRNA in the developing and adult skin, including consistent expression in hair follicles, is in good agreement with hypotrichosis, one of the prominent phenotypic features in EDA. Hypodontia is another major feature, but teeth other than those in early germ stages in fetal sections from Week 10 and one adult section were not available for study. The absence of expression from the early developmental stages in hair and around tooth buds, but expression in mesenchymal cells around one adult tooth, warrants further study.
On the basis of the symptoms in EDA patients, it is conceivable that the function of the EDA protein is associated with the regulation of early morphogenetic events during the development of hair follicles, sweat glands, and teeth. These organs share striking morphological similarities during the initiation of development and during early morphogenesis. Our observation of the lack of EDA expression during early hair follicle morphogenesis and in tooth buds was unexpected but is not incompatible with a developmental role, because expression was seen even earlier (Week 8) throughout the epidermis. A possible interpretation is that an intact EDA gene product may be needed for normal morphogenesis very early in the epithelium, and again in more mature forms of hair follicles, sweat and sebaceous glands, and mesenchyme around teeth.
Consistent symptoms and phenotypic features in EDA include mild dysmorphic features of the skull and facial bones and an increased tendency to infections, such as bronchitis (
The airway symptoms might have suggested that the EDA gene was involved in bronchial gland development. Surprisingly, we did not observe EDA signal in bronchial epithelium in fetal development (two samples) or in the adult. The absence of signal may be artifactual and these negative results should be viewed with caution. If the EDA gene is actually not involved in the development of the bronchial epithelium, then bronchial oversensitivity to infections in EDA might result from an indirect mechanism, possibly involving the immune system. Our observation that the fetal thymus specifically expressed EDA provides evidence that the gene product may be associated with the maturation or function of the immune system.
Translation of EDA Protein
The presence of EDA mRNA in a cell type does not necessarily imply translation and expression of the protein. To study whether the corresponding protein antigens could be correlated to mRNA expression, adjacent tissue sections were stained by a specific antibody. We conclude that the sites of transcription and occurrence of protein were in good agreement, suggesting that mRNA in situ hybridization predicts sites of protein expression.
Clues to Molecular Interactions
The overall spatiotemporal pattern of EDA expression in embryonic and adult tissues bears similarities to, but to our knowledge is different from, that of any other known molecule studied thus far in hair and teeth (
The expression of EDA in some epithelia resembles EGFR expression (
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Acknowledgments |
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Supported by the Sigrid Juselius Foundation, Finska Läkaresällskapet, Academy of Finland, and Folkhälsan Institute of Genetics.
We thank Johanna Pispa for discussions and Alli Tallqvist and Liisa Sund for excellent technical assistance.
Received for publication April 4, 1997; accepted September 3, 1997.
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