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Original Studies |
Division of Endocrinology and Metabolism (J.J.W., W.M.P., P.D., J.-P.Z.), Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8020; INSERM U-426 et Institut Fédératif de Recherche 02 (S.C., C.S.), Faculté de Médecine Xavier Bichat, 75018, Paris, France; Service d’Histo-Embryologie et Cytogénétique (J.R.), Hôpital Saint Antoine, Debré AP-HP, 75012, Paris, France; and Service de Biologie du Développement (A.-L.D.), Hôpital Robert Debré, 75935 Paris Cedex 19, France
Address all correspondence and requests for reprints to: Caroline Silve, M.D., Ph.D., INSERM U-426 et Institut Fédératif de Recherche 02, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France. E-mail: silve{at}bichat.inserm.fr
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In recent years, increasing attention has been given to the functions of PTHrP and its receptor during development. Both PTHrP and the PTHR1 are widely expressed during fetal life, beginning as early as the morula stage of embryogenesis (4, 5). Experiments using a variety of genetically-altered mice have suggested two principal roles for PTHrP/PTHR1 signaling in mammalian development. Initially, it was found that the disruption of either the PTHrP or PTHR1 genes by homologous recombination causes skeletal abnormalities that lead to the neonatal death of PTHrP or PTHR1-knockout animals (6, 7). These experiments demonstrated the importance of PTHrP/PTHR1 signaling in the regulation of chondrocyte proliferation and differentiation during endochondral bone formation. Subsequently, the genetic rescue of the skeletal defects in PTHrP-knockout mice, using cartilage-specific PTHrP or constitutively-active PTHR1 transgenes, led to the realization that PTHrP/PTHR1 signaling also regulates epithelial-mesenchymal interactions during the organogenesis of epithelial organs such as the skin, mammary glands, and teeth (8, 9, 10, 11). In the absence of PTHrP or the PTHR1 in mice, mammary glands do not form, and tooth eruption fails.
Little is known about the role of the PTHrP/PTHR1 signaling pathway in human development. Although mutations in the PTHrP gene have not been recognized as a cause of human disease, inherited mutations in the PTHR1 have. Gain-of-function mutations in the PTHR1 gene have been shown to cause Jansen’s type metaphyseal chondrodysplasia, a form of dwarfism caused by a delay in the differentiation of chondrocytes in developing bones (12, 13). Interestingly, perhaps reflecting the involvement of PTHrP/PTHR1 signaling in the physiology of the breast and skin, one female patient with Jansen’s type metaphyseal chondrodysplasia was reported to be unable to breast-feed and to have (similar to her affected daughter) dry and scaly skin (13). Recently, another form of short-limbed dwarfism, Blomstrand chondrodysplasia, has been recognized to result from loss-of-function mutations in the PTHR1 gene (14, 15, 16, 17). This disorder, first described in 1985, is characterized by accelerated skeletal maturation, inappropriate ossification of cartilage, craniofacial malformations, coarctation of the aorta, and fetal death (14, 15, 16, 17, 18). Histologically, there seems to be an acceleration of chondrocyte differentiation and primary ossification within the growth plates of endochondral bones. Thus, both activating and inactivating mutations of the PTHR1 result in abnormalities in human endochondral bone formation. In this report, we present evidence that fetuses with Blomstrand chondrodysplasia have striking abnormalities in breast and tooth development, demonstrating that PTHrP/PTHR1 signaling is critical to the development of these organs in humans.
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Subjects and Methods |
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Case 1 (fetus B1) was a male fetus that was diagnosed as suffering from Blomstrand chondrodysplasia at 26 weeks gestation, by routine ultrasonography. The ultrasound exam revealed short limbs and hydropic features. The mother was 26 yr old at the time and was gravida 2 para 1; her first pregnancy resulted in a healthy baby. The parents were not related, and there was no prior history of dwarfism in either family. The pregnancy was terminated, and the diagnosis of Blomstrand chondrodysplasia was confirmed by radiographic and histological examination. Case 2 (fetus B2) was a sister of the index case and was stillborn at 33 weeks gestation. She was the product of the mother’s fourth pregnancy, and the diagnosis of Blomstrand chondrodysplasia was made by postmortem examination. Both siblings displayed the typical features of Blomstrand chondrodysplasia, including very short limbs, extremely advanced skeletal maturation, ossification of the thyroid and hyoid cartilages, hypoplastic mandibles with protruding tongues, generalized edema, and preductal coarctation of the aorta. Detailed descriptions of these cases have been published (18). Both were found to be compound heterozygotes for the PTHR1 gene, having one allele that was not expressed and one that coded for a nonfunctional receptor (14).
Methods
Tissue procurement and histology. The Blomstrand fetuses were preserved in 10% formalin at the time of their original necropsy. Tissue for the present studies was obtained from these samples after the informed consent of the parents and according to the French Ethical Committee recommendations. Control tissue from fetuses showing no evidence of skeletal abnormalities was obtained from spontaneously or voluntarily terminated pregnancies and came from two sources. Some samples were obtained after the informed consent of the parents and according to the French Ethical Committee recommendations. Other samples were obtained from the Critical Technologies Program of the Department of Pathology at the Yale University School of Medicine under protocols approved by the Yale University Human Investigation Committee.
For routine histological analysis, the tissue was embedded in paraffin, sectioned, and stained with hematoxylin and eosin using standard procedures. Histological analysis of tooth development was performed on the maxillary bones, which were decalcified in 2% EDTA for 3–4 weeks before embedding. Three horizontal sections were taken through the length of the maxillary bone blocks from the 26-week-GA (gestational age) Blomstrand fetus (fetus B1); and seven sagittal (vertical) sections, from those from the 33-week-GA Blomstrand fetus (fetus B2). Five-micrometer-thick serial sections were then performed at each of these levels. Vertical sections were obtained from a 30-week age-matched control. Histological analysis was performed on serial sections stained with hematoxylin/eosin/safran.
Computed tomography (CT) scan analysis. CT scan analysis of the superior and inferior maxillary bones, from the two fetuses with Blomstrand chondrodysplasia and a control (31-week GA), were performed, in parallel, using an Elscint CT twin apparatus.
Immunohistochemistry. Immunohistochemistry was performed on paraffin-embedded, formalin-fixed sections. Primary incubations were performed using a rabbit antirat PTHR1 antibody, which was the kind gift of Dr. Robert Nissesson (San Francisco, CA). This antibody was incubated with tissue sections, for 8–12 h, at a concentration of 2–4 µg/mL, at 4 C. Specificity was determined by incubations with nonspecific IgG and by performing competition studies using a synthetic peptide containing the antibody epitopes (also kindly supplied by Dr. Nissenson). Primary antibody staining was detected using the Vector Elite avidin-biotin kit and 3,3'diaminobenzidine as a chromogen (Vector Laboratories, Inc., Burlingame, CA).
In situ hybridization. In situ hybridization was performed on sections of human breast buds as previously described (8). Sections of mandibular bone were hybridized with probes generated from a 308-bp complementary DNA fragment of the human PTHrP gene and from a 312-bp complementary DNA fragment of the human PTH/PTHrP receptor gene (19), using protocols previously reported (20).
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Results |
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Although readily identifiable in control fetuses of similar GA, no
nipples were identified on examination of the thoracic regions of the
patients described above (Fig. 1
, A and
B). To ascertain whether the lack of nipples also meant that no breast
ducts were present, portions of the thorax that should have contained
the breast tissue (skin and sc tissues extending from the sternum to
the axilla, see Fig. 1B) were removed from cases B1 and B2, serially
sectioned at intervals of 50 or 100 microns, and examined
histologically. As shown in Fig. 1C, the epidermis and other skin
appendages (hair follicles, sebaceous glands, and sweat glands) seemed
to be normal. However, no breast structures were identified on either
side of either fetus. Therefore, in human fetuses, the lack of
functional receptors for PTHrP leads to a failure of breast and nipple
development.
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To examine tooth development, we first performed CT scans of
the maxillas of the affected fetuses and compared them with scans of a
control fetus of comparable GA. As seen in Fig. 2, A and B, developing teeth were easily
identified in the control; but in the Blomstrand fetuses, only the two
upper lateral incisors were clearly seen. On examination of serial
scans, one could also detect a faint outline of the central incisors,
but the molars were not visualized (data not shown). Although the
lateral incisors were easily detected, their orientation was highly
abnormal. The long axis of each lateral incisor appeared to be rotated
by approximately 90 degrees, relative to that of normal incisors; and,
on serial sections, it was clear that these teeth were oriented in a
plane parallel to the oral cavity, not in the normal perpendicular
plane. Because of these abnormalities, the lateral incisors were
encroaching into the alveolar space of the central incisors.
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, C and D). However, tooth development was
uniformly abnormal. First, confirming the findings made on CT scan
analysis, the orientation of the developing teeth was strikingly
altered, on histological analysis. As opposed to normal teeth, which
grew along parallel axes, the teeth from patients with Blomstrand
chrondodysplasia showed markedly divergent axes of growth (Fig. 2, C
and D). Furthermore, sections made parallel to the oral cavity did not
cut the teeth from patients with Blomstrand chondrodysplasia in a
transverse plane, but nearly parallel to their long axis (compare C and
D in Fig. 2). Second, severe impaction was observed. Normally, the
developing tooth is found in a regularly shaped crypt that is free of
alveolar bone (Fig. 2C). In contrast, in the fetuses with Blomstrand
chondrodysplasia, alveolar bone was found within the crypts, and all
the teeth were encased in bone (see Fig. 2, D and E). The alveolar bone
bordering the crypts was distorted and irregular, and fingers of bone
appeared to extend into the enamel organ and to distort the tooth germ
architecture (Fig. 2E). In addition, ectopic areas of dentin were
located on the lateral aspect of the teeth, perpendicular to the normal
layers located at the developing crown (Fig. 2C). In spite of the
impaction, the amelogenesis and dentinogenesis of the tooth buds
appeared normal. Each tooth had the proper arrangement of ameloblast
and odontoblast layers, as well as normal formation of dentin and
enamel matrix (Fig. 2F). PTHrP and PTHR1 expression during human fetal breast and tooth development
To document further the role of PTHrP/PTHR1 signaling during human
breast and tooth development, we analyzed the patterns of expression of
PTHrP and the PTHR1 in these organs in normal human fetuses. As seen in
Fig. 3A, the human breast begins as a
bud-like growth of the epidermis into the underlying mesenchyme. Using
in situ hybridization, it appeared that the PTHrP gene is
expressed in epithelial cells of the 16-week breast bud (Fig. 3, B and
C) but not within the surrounding mesenchyme. Similar in
situ hybridization experiments to detect PTHR1 messenger RNA
(mRNA) were not successful. However, by immunohistochemical techniques,
it appeared that the PTHR1 is found on mesenchymal cells surrounding
the breast bud. In addition, at this stage, staining for the receptor
is also present on epithelial cells within the bud (see Fig. 3, D and
E).
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. PTHrP
mRNA is found within enamel epithelial cells, with the highest levels
of expression observed in the dental lamina, although lower (but clear)
expression was also identified in the enamel organ and in the
vestibular lamina (Fig. 4, A–C). PTHR1 mRNA is found principally
within osteoblasts lining mandibular and maxillary bone (Fig. 4, D–F).
However, there is also a low level of expression seen within the dental
mesenchyme, a loose connective tissue located within the tooth crypt
itself (data not shown).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The lack of any breast or nipple tissue in fetuses suffering from Blomstrand chondrodysplasia demonstrates that an intact PTHrP/PTHR1 signaling pathway is a prerequisite for breast development in human beings. This is also the case in mice. Disruption of either the murine PTHrP or PTHR1 genes, by homologous recombination, leads to a failure of embryonic mammary gland development (8). In these knockout mice, a bud-shaped embryonic mammary rudiment forms; but instead of growing out into the mammary fat pad and initiating ductal branching morphogenesis, the mammary bud degenerates, and the mammary epithelial cells die. A series of experiments has demonstrated that, in mice, PTHrP serves as a critical message from the epithelial component of the mammary bud to the mesenchymal component (8, 21, 22). In response to PTHrP, immature mesenchymal cells surrounding the mammary epithelial bud commit to a mammary stromal cell fate, a decision that is necessary for the mesenchymal cells to acquire the ability to support the survival and subsequent morphogenesis of the epithelial cells (8, 21). Given the rarity of this condition and the difficulty in procuring tissue from affected fetuses, it is impossible to determine whether the defects in breast development in Blomstrand chondrodysplasia are exactly identical to the defects in mammary development seen in PTHrP and PTHR1 knockout mice. However, similar to the pattern seen in mice, we have found that, in early human breast development, PTHrP is produced by mammary epithelial cells, and the PTHR1 is located on stromal cells surrounding the mammary bud. This observation and the similar absence of breast tissue in late-term Blomstrand fetuses and in neonatal PTHrP and PTHR1 knockout mice represent strong evidence that, in fact, the defects are analogous.
As illustrated by the finding of a distorted tooth architecture and orientation in our patients, tooth development is also abnormal in Blomstrand chondrodysplasia. The defects seen in our patients did not seem to be attributable to abnormal tooth bud formation, for all the teeth were present, and each tooth had the appropriate histological layers. Instead, the abnormalities seemed to be primarily the result of progressive impaction of the developing teeth by the surrounding bone. These abnormalities in Blomstrand chondrodysplasia are highly reminiscent of those seen in patients with osteopetrosis (23), a series of diseases characterized by a lack of osteoclastic bone resorption (24). They are also similar to those observed in PTHrP knockout mice that have been rescued from their neonatal deaths by the expression of a chondrocyte-specific PTHrP transgene (9). As in our Blomstrand patients, in patients with osteopetrosis and in the rescued knockout mice, tooth buds form normally, and amelogenesis and dentinogenesis are normal. However, as the teeth grow and develop, they become increasingly distorted and impacted; and in both the rescued knockout mice and the osteopetrotic patients, they fail to erupt. Studies in murine models have shown that the PTHrP gene is expressed in the enamel epithelium during early tooth formation and that its expression is markedly up-regulated in stellate reticulum cells located beneath the roof of the tooth crypt, just preceding tooth eruption (9, 25). It seems that during murine tooth development, PTHrP functions to activate bone resorption around the tooth crypt and within the eruption pathway. This allows the tooth initially to grow within the bone and subsequently to erupt. As with breast development, the patterns of PTHrP and PTHR1 expression during tooth development are similar in mice and humans. Therefore, it is likely that PTHrP functions in a similar fashion during human tooth development and that the abnormalities in the teeth of human fetuses with Blomstrand chondrodysplasia result from a defect in alveolar bone resorption.
Whether the defect in alveolar bone resorption observed in Blomstrand chondrodysplasia reflects a generalized abnormality in osteoclast function, as in osteopetrosis, or a defect restricted to the microenvironment of the tooth, as described in the rescued knockout mice, is unclear. In favor of the former possibility, abnormalities in bone remodeling were detected in the two cases with Blomstrand chondrodysplasia studied in this report (18).
Recently, it has been suggested that PTHrP, signaling through the PTHR1, may participate in the regulation of vascular tone, cardiac inotrophy and chronotrophy, and heart development (26, 27, 28, 29). Therefore, it is interesting that these two fetuses with Blomstrand chondrodysplasia suffered from preductal coartation of the aorta and hydrops fetalis. It is possible that these cardiovascular abnormalities might have contributed to the death of fetus B2 in utero, a situation perhaps analogous to the premature cardiovascular deaths of some PTHR1 knockout mouse embryos (29). However, other than these cardiovascular abnormalities, other internal organs in these fetuses seemed to develop normally (18), a situation also analogous to the PTHrP and PTHR1 knockout mice (6, 7).
In summary, in addition to the known abnormalities described in bone, impairment of the PTHrP/PTHR1 signaling pathway in humans with Blomstrand chondrodysplasia is associated with severe abnormalities in tooth and breast development. Thus, in addition to regulating chondrocyte proliferation and differentiation during the process of endochondral bone formation in humans, this signaling pathway seems to regulate epithelial-mesenchymal interactions necessary for the formation of the human breast and local bone resorption necessary for human tooth development and eruption. Further investigation into the role of the PTHrP/PTHR1 signaling pathway in development may provide new clues to the pathogenesis of diseases as diverse as osteoporosis, ectodermal dysplasias, and breast cancer.
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Acknowledgments |
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Footnotes |
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Received September 9, 2000.
Revised December 28, 2000.
Accepted January 4, 2001.
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