Localization of epidermal growth factor precursor in tooth and lung during embryonic mouse development

The murine epidermal growth factor (EGF) precursor is a 1217 amino acid protein which contains mature EGF (amino acid residues 977-1029) as well as eight EGF-like repeats. Although the highest levels of EGF are found in the adult male mouse submandibular gland, the results of in situ hybridization studies and mRNA analyses suggest that EGF precursor mRNA is synthesized in several adult mouse tissues including the lung and the incisor. To determine if EGF precursor gene expression is intrinsic to the developmental program for either embryonic tooth or lung organogenesis, sense and antisense oligodeoxyribonucleotide probes corresponding to amino acids 1070-1081 of the precursor were used to localize cellular sites of synthesis of EGF precursor mRNA by in situ hybridization. Antibodies directed against amino acid residues 348-691 of the precursor were used in immunodetection techniques to identify either EGF precursor protein or processed derivatives. In contrast to earlier reports indicating that embryonic mouse tissues do not synthesize EGF precursor mRNA, we found that EGF precursor mRNA is present in clusters of ectoderm-, mesoderm-, and ectomesenchyme-derived cells associated with embryonic teeth and lung organs. Moreover, epitopes common to the EGF precursor were immunolocalized in both the epithelial and mesenchymal tissues of embryonic mouse tooth and lung organs. These results suggest that the EGF precursor and/or motifs contained within the precursor molecule, including mature EGF, may play an instructive or permissive role in epithelial-mesenchymal interactions pursuant to organogenesis.     

Sonic hedgehog regulates growth and morphogenesis of the tooth

During mammalian tooth development, the oral ectoderm and mesenchyme coordinate their growth and differentiation to give rise to organs with precise shapes, sizes and functions. The initial ingrowth of the dental epithelium and its associated dental mesenchyme gives rise to the tooth bud. Next, the epithelial component folds to give the tooth its shape. Coincident with this process, adjacent epithelial and mesenchymal cells differentiate into enamel-secreting ameloblasts and dentin-secreting odontoblasts, respectively. Growth, morphogenesis and differentiation of the epithelium and mesenchyme are coordinated by secreted signaling proteins. Sonic hedgehog (Shh) encodes a signaling peptide which is present in the oral epithelium prior to invagination and in the tooth epithelium throughout its development. We have addressed the role of Shh in the developing tooth in mouse by using a conditional allele to remove Shh activity shortly after ingrowth of the dental epithelium. Reduction and then loss of Shh function results in a cap stage tooth rudiment in which the morphology is severely disrupted. The overall size of the tooth is reduced and both the lingual epithelial invagination and the dental cord are absent. However, the enamel knot, a putative organizer of crown formation, is present and expresses Fgf4, Wnt10b, Bmp2 and Lef1, as in the wild type. At birth, the size and the shape of the teeth are severely affected and the polarity and organization of the ameloblast and odontoblast layers is disrupted. However, both dentin- and enamel-specific markers are expressed and a large amount of tooth-specific extracellular matrix is produced. This observation was confirmed by grafting studies in which tooth rudiments were cultured for several days under kidney capsules. Under these conditions, both enamel and dentin were deposited even though the enamel and dentin layers remained disorganized. These studies demonstrate that Shh regulates growth and determines the shape of the tooth. However, Shh signaling is not essential for differentiation of ameloblasts or odontoblasts.     

Epidermal growth factor regulates gene expression of both epithelial and mesenchymal cells in mouse molar tooth organs in culture

Epidermal growth factor and cis-hydroxyproline specifically inhibited synthesis of type 1 collagen, a major gene product of the differentiated dental mesenchymal cells (odontoblasts). In tandem, synthesis of enamel proteins, specific gene products of differentiated dental epithelial cells (ameloblasts), was also inhibited. Under these culture conditions, total protein synthesis in tooth organs was not inhibited but rather increased. Inhibition curves of the gene products specific for epithelial and mesenchymal phenotypes were quite similar, indicating coordinate and intimately associated regulation of gene expression under conditions that perturb cytodifferentiation.     

Epidermal growth factor inhibits morphogenesis and cell differentiation in cultured mouse embryonic teeth

Although local epithelial-mesenchymal tissue interactions which are presumably mediated by extracellular matrix molecules are important regulators of tooth morphogenesis and differentiation, our studies have indicated that these developmental processes also depend on circulating molecules. The iron-carrying serum protein transferrin is necessary for the early morphogenesis of mouse tooth in organ culture (A-M. Partanen, I. Thesleff, and P. Ekblom, 1984, Differentiation 27, 59-66). In the present study we have examined the effects of other growth factors on mouse tooth germs grown in a chemically defined medium containing transferrin. Fibroblast growth factor and platelet derived growth factor had no detectable effects but epidermal growth factor (EGF) inhibited dramatically the morphogenesis of teeth, and prevented odontoblast and ameloblast cell differentiation. EGF stimulated cell proliferation in the explants measured as [3H]thymidine incorporation in DNA. However, when the distribution of dividing cells was visualized in autoradiographs, it was observed that cell proliferation was stimulated in the dental epithelium but was inhibited in the dental mesenchyme. The inhibition of cell proliferation in the dental mesenchyme apparently caused the inhibition of morphogenesis. We do not know whether the dental epithelium or mesenchyme was the primary target for the action of EGF in the inhibition of morphogenesis. It is, however, apparent that the response of the dental mesenchymal cells to EGF (inhibition of proliferation) is regulated by their local environment, since EGF enhanced proliferation when these cells were disaggregated and cultured as monolayers. This indicates that the organ culture system where the various embryonic cell lineages are maintained in their original environment corresponds better to the in vivo situation when the roles of exogenous growth factors during development are examined.     

Inhibition of mouse mammary ductal morphogenesis and down-regulation of the EGF receptor by epidermal growth factor

EGF, initially demonstrated to be a potent mitogen for a variety of cell types, has more recently been shown to inhibit proliferation of several cell lines. Few studies, however, have addressed the effects of EGF on growth and morphogenesis of tissues in vivo, particularly with regard to EGF as a possible inhibitor. We now demonstrate that EGF treatment of vigorously growing mammary ducts, administered directly to the glands by slow release plastic implants, inhibited normal ductal growth. Inhibition was restricted to the region around the implant and untreated glands in the same animal were normal, indicating direct effects of EGF. EGF-treated end buds were smaller and demonstrated reduced levels of DNA synthesis, although remnants of a stem (cap) cell layer persisted. Full inhibition of growth occurred within 3 days of implantation and required extended exposure to EGF, since treatment of 5 hr or less had no effect on ductal growth. At the lower inhibitory doses tested, growth resumed within 8 days, indicating reversibility of inhibition. No lobuloalveolar or hyperplastic response was seen. 125I-EGF autoradiography revealed that ductal growth inhibition was preceded by the disappearance of EGF receptors located in the cap cell layer of the end bud epithelium and in stromal cells adjacent to the buds. These results, in conjunction with our previous evidence demonstrating the growth-stimulatory effect by EGF on nonproliferating mammary ducts, suggest a growth regulatory role for EGF in mouse mammary ductal morphogenesis.     

Modulation of the epidermal growth factor receptor of mouse embryonic palatal mesenchyme cells in vitro by growth factors.

A single class of high-affinity receptors for EGF were detected on mouse embryonic palatal mesenchyme (MEPM) cells cultured in vitro. The degree of confluence of the cultured cells did not affect the number or affinity of the binding sites. Culture of MEPM cells in the presence of bFGF, IGF-II or TGF-beta 1 induced changes in 125I-EGF binding. TGF-beta 1 caused a marked reduction in binding to 40% of control levels. This reduction was achieved after 2 h and persisted for 24 h after addition of the growth factor. IGF-II induced a similar reduction but this effect was transitory; after a 12 h pretreatment with IGF-II, binding was restored to control levels. The effects of bFGF were biphasic. Initially, a short pre-treatment period (3-5 h) with bFGF caused a small reduction in 125I-EGF binding; longer periods of pre-incubation (24 h) resulted in a large increase in receptor number. Pre-incubation in medium containing both bFGF and TGF-beta 1 resulted in a decrease in EGF binding. Thus, TGF-beta 1 negated the large increase in receptor number induced by bFGF alone. Changes in receptor number were usually, but not always, directly related to changes in the biological activity of EGF, as assessed by a thymidine incorporation assay. This study highlights the possible interactive role of growth factors known to be present in the developing palate.     

Platelet-derived growth factor receptor regulates salivary gland morphogenesis via fibroblast growth factor expression

A coordinated reciprocal interaction between epithelium and mesenchyme is involved in salivary gland morphogenesis. The submandibular glands (SMGs) of Wnt1-Cre/R26R mice have been shown positive for mesenchyme, whereas the epithelium is beta-galactosidase-negative, indicating that most mesenchymal cells are derived from cranial neural crest cells. Platelet-derived growth factor (PDGF) receptor alpha is one of the markers of neural crest-derived cells. In this study, we analyzed the roles of PDGFs and their receptors in the morphogenesis of mouse SMGs. PDGF-A was shown to be expressed in SMG epithelium, whereas PDGF-B, PDGFRalpha, and PDGFRbeta were expressed in mesenchyme. Exogenous PDGF-AA and -BB in SMG organ cultures demonstrated increased levels of branching and epithelial proliferation, although their receptors were found to be expressed in mesenchyme. In contrast, short interfering RNA for Pdgfa and -b as well as neutralizing antibodies for PDGF-AB and -BB showed decreased branching. PDGF-AA induced the expression of the fibroblast growth factor genes Fgf3 and -7, and PDGF-BB induced the expression of Fgf1, -3, -7, and -10, whereas short interfering RNA for Pdgfa and Pdgfb inhibited the expression of Fgf3, -7, and -10, indicating that PDGFs regulate Fgf gene expression in SMG mesenchyme. The PDGF receptor inhibitor AG-17 inhibited PDGF-induced branching, whereas exogenous FGF7 and -10 fully recovered. Together, these results indicate that fibroblast growth factors function downstream of PDGF signaling, which regulates Fgf expression in neural crest-derived mesenchymal cells and SMG branching morphogenesis. Thus, PDGF signaling is a possible mechanism involved in the interaction between epithelial and neural crest-derived mesenchyme.     

RME-8 regulates trafficking of the epidermal growth factor receptor

We recently identified receptor-mediated endocytosis 8 (RME-8), a DnaJ domain protein localized to endosomes. We now demonstrate that RME-8 depletion leads to decreased levels of epidermal growth factor receptor (EGFR) without influencing receptors that primarily recycle to the plasma membrane. Decreases in EGFR are detected at both surface and intracellular pools and result from increased rates of EGFR degradation. Interestingly, RME-8 depletion also decreases EGFR levels in breast cancer cell lines in which overexpression of the EGFR family member ErbB2 has been shown to protect EGFR from degradation. These data implicate RME-8 in sorting decisions influencing EGFR at the level of endosomes and point to RME-8 as a potential regulatory target in ErbB2-positive breast cancers.     

Binding of epidermal growth factor from man, rat and mouse to the human epidermal growth factor receptor

Human, rat and mouse epidermal growth factors (EGF) bind to the same receptor on human placenta, but the binding characteristics differ. The apparent affinity constant (KA) for human EGF is higher (15 X 10(9) l/mol) than KA for rat EGF (10 X 10(9) l/mol). Mouse EGF binds with the lowest KA (5 X 10(9) l/mol). The pH optimum differs so that human and rat EGF bind with a pH optimum of 8.0, whereas mouse EGF binds with an optimum of pH 7.4. Half maximal dissociation is 130, 50 and 25 min for human, rat and mouse EGF, respectively. The structures of human, rat and mouse EGF differ somewhat. At least 11 of the first 24 residues differ. The N-terminal sequence of rat EGF is: Ala/Ser-Gly-X-Pro-Pro-Ser-Tyr-Asp-Gly-Tyr-X-Lys-Asp-Gly-Gly-Val-X-Met-Ty r-Val -Glu.     

Transferrin and tooth morphogenesis: Retention of transferrin by mouse embryonic teeth in organ culture

Transferrin is the only serum protein that is required for the early morphogenesis of mouse embryonic teeth in organ culture. Transferrin is able to support tooth morphogenesis and dental cell differentiation by stimulating cell proliferation. Its role in this process is restricted exclusively to iron transport, which takes place by receptor-mediated endocytosis of iron-loaded transferrin. A lipophilic iron chelator, pyridoxal isonicotinoyl hydrazone (PIH), can replace transferrin and support tooth morphogenesis in organ culture. We studied the effects of these two iron transporters on cell proliferation in tooth germs during culture. We found that Fe-PIH and transferrin stimulate proliferation to a similar extent in early cap-stage teeth of 14-day mouse embryos, but have no effect on cell proliferation in bell-stage teeth of 16-day mouse embryos. Day-16 teeth undergo morphogenesis in unsupplemented chemically defined medium, whereas transferrin or Fe-PIH is needed for the morphogenesis of day-14 teeth. Although the need for exogenous iron-transport molecules is lost with advancing development, the level of mitotic activity is still fairly high in bell-stage teeth. The abundant binding of transferrin in areas of active cell proliferation in bell-stage teeth also suggests that transferrin is still needed and used for the transport of iron into proliferating cells. Transferrin is not degraded by the process of receptor-mediated endocytosis. After releasing iron into a cell, transferrin is returned to the extracellular space and is reused. We therefore studied whether the transferrin needed by bell-stage teeth could be adequately supplied by endogenous transferrin synthesized or stored in tissue explants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization and quantitation of 125I-epidermal growth factor binding in mouse embryonic tooth and other embryonic tissues at different developmental stages

We have shown earlier that epidermal growth factor (EGF) inhibits morphogenesis and cell differentiation in mouse embryonic teeth in organ culture. This inhibition depends on the stage of tooth development so that only teeth at early developmental stages respond to EGF (A-M. Partanen, P. Ekblom, and I. Thesleff (1985) Dev. Biol. 111, 84-94). We have now studied the quantity and pattern of EGF binding in teeth at various stages of development by incubating the dissected tooth germs with 125I-labeled EGF. Although the quantity of 125I-EGF binding per microgram DNA stays at the same level, localization of 125I-EGF binding by autoradiography reveals that the distribution of binding sites changes dramatically. In bud stage the epithelial tooth bud that is intruding into the underlying mesenchyme has binding sites for EGF, but the condensation of dental mesenchymal cells around the bud does not bind EGF. At the cap stage of development the dental mesenchyme binds EGF, but the dental epithelium shows no binding. This indicates that the dental mesenchyme is the primary target tissue for the inhibitory effect of EGF on tooth morphogenesis during early cap stage. During advanced morphogenesis the binding sites of EGF disappear also from the dental papilla mesenchyme, but the dental follicle which consists of condensed mesenchymal cells surrounding the tooth germ, binds EGF abundantly. We have also studied EGF binding during the development of other embryonic organs, kidney, salivary gland, lung, and skin, which are all formed by mesenchymal and epithelial components. The patterns of EGF binding in various tissues suggest that EGF may have a role in the organogenesis of epitheliomesenchymal organs as a stimulator of epithelial proliferation during initial epithelial bud formation and branching morphogenesis. The results of this study indicate that EGF stimulates or maintains proliferation of undifferentiated cells during embryonic development and that the expression of EGF receptors in different organs is not related to the age of the embryo, but is specific to the developmental stage of each organ.     

Effects of hepatocyte growth factor anti-sense oligodeoxynucleotides or met D/D genotype on mouse molar crown morphogenesis

Hepatocyte growth factor (HGF) is considered to be one of the mediators of epithelio-mesenchymal interactions during early organogenesis and to be also involved in the development of murine molars. In the developing tooth, HGF is expressed in the cells of the dental papillae, and c-Met, its receptor, in the cells of dental epithelia. In order to study the functional role played by HGF in tooth development, we tested the effects of HGF translation arrest by anti-sense phosphorothioate oligodeoxynucleotides on E-14 molars cultured in vitro. We also analyzed the histo-morphogenesis and crown cytodifferentiation of transgenic met E-14 molars cultured in vitro. 3D reconstructions revealed perturbations of the cusp pattern. However, histo-morphogenesis and crown cytodifferentiation were normal at the histological level.     

Substitution for mesenchyme by basement-membrane-like substratum and epidermal growth factor in inducing branching morphogenesis of mouse salivary epithelium

Mouse salivary epithelium cannot undergo branching morphogenesis in the absence of the surrounding mesenchyme. To clarify the nature of the mesenchymal influence on the epithelium, we have investigated the culture conditions in which the epithelium could normally branch in the absence of mesenchymal cells. Combination of basement-membrane-like substratum (Matrigel) and epidermal growth factor (EGF) could substitute for the mesenchyme, the epithelium showing typical branching morphogenesis. Transforming growth factor alpha had the same effect as EGF. Matrigel plus basic fibroblast growth factor or transforming growth factor beta 1 and collagen gel plus EGF were not sufficient to support the branching of the epithelium. These results clearly reveal that the role of mesenchyme in salivary morphogenesis is both to provide the epithelium with an appropriate substratum and to accelerate growth of the epithelium.     

Signaling pathways activated by epidermal growth factor receptor or fibroblast growth factor receptor differentially regulate branching morphogenesis in fetal mouse submandibular glands

Although growth factor signaling is required for embryonic development of organs, individual signaling mechanisms regulating these organotypic processes are just beginning to be defined. We compared signaling activated in fetal mouse submandibular glands (SMGs) by three growth factors, epidermal growth factor (EGF), fibroblast growth factor (FGF) 7, or FGF10, and correlated it with specific events of branching morphogenesis. Immunoblotting showed that EGF strongly stimulated phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2) and weakly stimulated phosphorylation of phospholipase C γ 1 (PLC γ 1) and phosphatidylinositol-3 kinase (PI3K) in cultured E14 SMG. However, FGF7 and FGF10 stimulated phosphorylation of both PLC γ 1 and PI3K, but elicited only minimal phosphorylation of ERK-1/2. Morphological study of mesenchyme-free SMG epithelium cultured in Matrigel revealed that EGF induced cleft formation of endpieces, that FGF7 stimulated both cleft formation and stalk elongation, but that FGF10 induced only stalk elongation. In mesenchyme-free SMG epithelium cultured with EGF, FGF7 and FGF10, U0126 (MEK inhibitor) completely blocked cleft formation, whereas U73122 (PLC γ 1 inhibitor) suppressed stalk elongation. These finding suggest that EGF stimulates cleft formation and drives branch formation via ERK-1/2, and that FGF7 stimulates both cleft formation and stalk elongation via PLC γ 1 and partly via ERK-1/2, but that FGF10 stimulates stalk elongation mainly via PLC γ 1.     

Retinoids and epidermal growth factor alter embryonic mouse palatal epithelial and mesenchymal cell differentiation in organ culture

The mechanism by which retinoids (RA) induce cleft palate is not known. During normal palatogenesis, the medial epithelia of opposing palatal shelves cease DNA synthesis, come into contact, adhere, and undergo programmed cell death (PCD). In organ cultures of day 12 embryonic mouse palatal shelves, epidermal growth factor (EGF) blocks PCD, and DNA synthesis continues. In the present study, the effects of trans-RA, 13-cis-RA, EGF, and combinations of EGF and RA on surface morphology, DNA synthesis, and cellular ultrastructure are determined for CD-1 embryonic mouse palatal shelves cultured on day 12 of gestation. DNA synthesis in the medial cells was sustained and PCD was blocked by EGF, trans-RA, and 13-cis-RA. Exposure to trans-RA, but not to 1-cis-RA, induced the medial epithelia to undergo hyperplasia, and addition of EGF enhanced the effect. In the presence of RA, particularly trans-RA, medial epithelial cells acquired nasal cell characteristics, and EGF enhanced this effect. Expansion of the mesenchymal extracellular spaces was blocked by trans-RA and to a lesser degree by 13-cis-RA. The RA-induced alterations in normal epithelial and mesenchymal cell differentiation may be relevant to the etiology of RA-induced cleft palate in vivo.