Induction of alveolar type II cell differentiation in embryonic tracheal epithelium in mesenchyme-free culture

We have previously shown that fetal lung mesenchyme can reprogram embryonic rat tracheal epithelium to express a distal lung phenotype. We have also demonstrated that embryonic rat lung epithelium can be induced to proliferate and differentiate in the absence of lung mesenchyme. In the present study we used a complex growth medium to induce proliferation and distal lung epithelial differentiation in embryonic tracheal epithelium. Day-13 embryonic rat tracheal epithelium was separated from its mesenchyme, enrobed in growth factor-reduced Matrigel, and cultured for up to 7 days in medium containing charcoal-stripped serum, insulin, epidermal growth factor, hepatocyte growth factor, cholera toxin, fibroblast growth factor 1 (FGF1), and keratinocyte growth factor (FGF7). The tracheal epithelial cells proliferated extensively in this medium, forming lobulated structures within the extracellular matrix. Many of the cells differentiated to express a type II epithelial cell phenotype, as evidenced by expression of SP-C and osmiophilic lamellar bodies. Deletion studies showed that serum, insulin, cholera toxin, and FGF7 were necessary for maximum growth. While no single deletion abrogated expression of SP-C, deleting both FGF7 and FGF1 inhibited growth and prevented SP-C expression. FGF7 or FGF1 as single additions to the medium, however, were unable to induce SP-C expression, which required the additional presence of serum or cholera toxin. FGF10, which binds the same receptor as FGF7, did not support transdifferentiation when used in place of FGF7. These data indicate that FGF7 is necessary, but not sufficient by itself, to induce the distal rat lung epithelial phenotype, and that FGF7 and FGF10 play distinct roles in lung development.     

Early Development of Mouse Anterior Pituitary: Role of Mesenchyme

Epithelial-mesenchymal interaction in the early development of the anterior pituitary gland was examined by chronological observations on fetal pituitary epithelium grafted in vivo with and without its own mesenchyme. At 8.5 days of gestation, the RATHKE'S pouch began to evaginate toward the diencephalon. The mesenchymal tissue around the pouch was at first very sparsely scattered, but then condensed, on day 10 becoming visible under a dissecting microscope. When RATHKE'S pouch epithelia from 10- and 12-day fetuses were transplanted alone under the kidney capsule, they proliferated slightly to form cysts, the cells of which differentiated into ACTH-producing cells, but not into prolactin-producing cells. Pituitary morphogenesis did not occur. When these epithelia were recombined with homotypic mesenchyme and transplanted, the epithelia proliferated remarkably on one side of the wall of the pouch, resulting in formation of a pars distalis that contained both ACTH-producing cells and prolactin-producing cells. Heterotypic mesenchyme, such as lung, dermis and mammary gland mesenchyme, could induce 12-day epithelium, but not 10-day epithelium to develop into pars distalis. Thus, fetal pituitary epithelium has the capacity of autodifferentiation into ACTH-producing cells, not into prolactin-producing cells, and requires mesenchymal support for development of the pars distalis.     

Molecular analysis of the determination of developmental fate in the small intestinal epithelium in the chicken embryo

Determination of the developmental fate in the small intestinal epithelium of the chicken embryo has not been fully analyzed up to the present. This study was carried out to analyze the determination time of the developmental fate of the small intestinal epithelium under the influence of other mesenchymes. The small intestinal epithelium reassociated and cultivated with the proventricular or gizzard mesenchyme or the dermis expressed chicken intestinal fatty acid binding protein, sucrase and CdxA as occurs during the normal development of the small intestinal epithelium. The presumptive intestinal endoderm taken from an earlier stage embryo and associated and cultivated with the proventricular or gizzard mesenchyme, showed gene expression patterns which were the same as those found in normal development. However, when the dermis was associated, the epithelium expressed sonic hedgehog, but never expressed intestinal epithelial- or stomach epithelial-markers. These results indicate that the determination of the developmental fate in the small intestinal epithelium and acquisition of autodifferentiation potency occur at the early stage of the gut development. Moreover the presumptive intestinal endoderm needs the supportive influence of the gut mesenchyme in order to differentiate fully into the intestinal epithelium.     

Differentiation of proventricular epithelium in xenoplastic associations with mesenchymal or fibroblastic cells

Summary Proventricular epithelium (PV epithelium) from 6-day chicken embryos was associated with cultured cells, derived from fetal rat small intestine, or with fetal rat or human skin fibroblasts. The cytodifferentiation of PV epithelium was investigated using antibodies to chicken pepsinogen, a marker protein of PV epithelium, and to chicken sucrase, a marker enzyme of the small-intestinal brush-border membrane. PV epithelium formed complex glands and produced pepsinogen in association with cultured gut mesenchymal cells and skin fibroblasts. Its development was comparable to that achieved under the influence of PV mesenchyme. PV epithelial development was severely inhibited, however, under the influence of intact chicken or rat intestinal mesenchyme. The data are consistent with the idea that during the first step of epithelial-mesenchymal interactions, the epithelium and not the mesenchyme may be responsible for the determination of the developmental fate.     

The concentric structure of the developing gut is regulated by Sonic hedgehog derived from endodermal epithelium

The embryonic gut of vertebrates consists of endodermal epithelium, surrounding mesenchyme derived from splanchnic mesoderm and enteric neuronal components derived from neural crest cells. During gut organogenesis, the mesenchyme differentiates into distinct concentric layers around the endodermal epithelium forming the lamina propria, muscularis mucosae, submucosa and lamina muscularis (the smooth muscle layer). The smooth muscle layer and enteric plexus are formed at the outermost part of the gut, always some distance away from the epithelium. How this topographical organization of gut mesenchyme is established is largely unknown. Here we show the following: (1) Endodermal epithelium inhibits differentiation of smooth muscle and enteric neurons in adjacent mesenchyme. (2) Endodermal epithelium activates expression of patched and BMP4 in adjacent non-smooth muscle mesenchyme, which later differentiates into the lamina propria and submucosa. (3) Sonic hedgehog (Shh) is expressed in endodermal epithelium and disruption of Shh-signaling by cyclopamine induces differentiation of smooth muscle and a large number of neurons even in the area adjacent to epithelium. (4) Shh can mimic the effect of endodermal epithelium on the concentric stratification of the gut. Taken together, these data suggest that endoderm-derived Shh is responsible for the patterning across the radial axis of the gut through induction of inner components and inhibition of outer components, such as smooth muscle and enteric neurons.     

Neutral morphogenetic activity of epithelium in heterologous tissue recombinations

To assess the existence of specific and nonspecific epithelial instructions for mesenchymal cell differentiation we compared homospecific and heterospecific mouse and quail tissue recombinations. In heterospecific recombinants between trypsin-dissociated mouse molar mesenchyme and quail epithelia neither odontoblasts nor chondrocytes differentiated. Cartilage appeared if the quail epithelium was contaminated with homologous limb mesenchyme and odontoblasts differentiated if the mouse dental epithelium was contaminated with dental papilla cells.     

Role of uterine epithelium in the development of myometrial smooth muscle cells

To study the role of epithelial-mesenchymal interactions in myometrial development, uteri from neonatal Balb/c mice 1 to 60 days postpartum were utilized. Intact (untrypsinized) uteri, trypsinized but unseparated uteri, homotypic uterine tissue recombinants (separated-recombined), or uterine mesenchyme alone were grafted beneath the renal capsule of syngeneic female hosts and grown for 1 mo. Uterine mesenchyme from 1-day mice grafted alone produced small amounts of smooth muscle, most of which was associated with vasculature, whereas uterine mesenchyme from older donors possessing a rudimentary myometrium at the time of grafting formed intermediate amounts of myometrium (actin-positive smooth muscle bundles). In contrast, all specimens containing epithelium (intact, trypsinized, and separated-recombined) developed large amounts of myometrium. Uterine epithelia from neonatal through adult stages were equally effective in permissively inducing myometrial development in 1-day uterine mesenchyme. From these data, it is apparent that uterine epithelium plays an important promotional role in the differentiation and possibly the spatial organization of the myometrium.     

Epithelial and mesenchymal cell differentiation in the fetal rat genital ducts: changes in the expression of cytokeratin and vimentin type of intermediate filaments and desmosomal plaque proteins

Mesonephric and paramesonephric ducts develop in different ways in male and female fetuses. We have analyzed the changes in the expression of cytokeratin and vimentin type of intermediate filaments and desmosomal plaque proteins in progressing and regressing genital ducts of rat fetuses. The concomitant changes in the basement membranes were detected by laminin antibody. Epithelial cells of the indifferent (Day 15) male and female mesonephric and paramesonephric ducts contained faint vimentin positivity which, however, later disappeared. Indifferent mesonephric duct epithelium stained strongly for cytokeratin, whereas in the corresponding paramesonephric duct only a weak and spotty positivity was seen. Immunocytochemical localization of cytokeratin filaments and desmosomal plaque proteins correlated with the ultrastructural differences in the apical junctional complexes of the mesonephric and paramesonephric ducts. Regardless of the ongoing regression of the male paramesonephric duct, cytokeratin positivity increased in the disorganizing epithelium; the most weak and a granular immunoreaction was seen in the cells found in the intensively vimentin-positive periductal mesenchyme. In the regressing female mesonephric duct cytokeratin positivity was lost before the final dissolution of the basement membrane. Immunoblotting analysis of cytokeratin and vimentin polypeptides of the individual genital ducts were in agreement with the immunocytochemical results obtained in 15- and 16-day-old fetuses. The results suggest that the expression of vimentin type intermediate filaments is an indication of the mesothelial origin of the genital ducts. The increase in cytokeratin positivity of the regressing paramesonephric duct epithelium suggests that the degenerative changes are initiated by the mesenchyme. Cytokeratin-positive cells found in the periductal mesenchyme of the male paramesonephric duct may be epithelial cells transforming into mesenchyme. The results emphasize a close relationship between the changes of the intermediate filament system and extracellular matrix upon differentiation of the fetal genital ducts.     

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.     

Chondrogenesis of mandibular mesenchyme from the embryonic chick is inhibited by mandibular epithelium and by epidermal growth factor

This study documents the role of mandibular epithelium and epidermal growth factor (EGF) in the initiation, maturation and maintenance of Meckel's cartilage using percent 3H-thymidine-labelled cells as an index of proliferative activity and distribution of labelled cells, chondrocyte size and relative amount of extracellular matrix as indices of chondrogenesis. Mandibular mesenchyme from embryos of H.H. stages 18, 22, 25 was cultured for 2 to 10 days (a) unseparated from mandibular epithelium, (b) in isolation, or (c) after recombination with mandibular epithelium in the presence or absence of 5-40 ng/ml EGF. Epithelium delayed both initiation of chondrogenesis and maturation of already formed cartilage. The 3H-thymidine-labelling index was reduced in cartilage that differentiated in the presence of mandibular epithelium. Epithelium influenced the timing of mesenchymal differentiation (a) by delaying cytodifferentiation through prolonging high levels of proliferation, and (b) by directly affecting differentiation itself. EGF, especially at 10-20 ng/ml, affected both proliferation of mesenchyme and chondrogenesis in mesenchyme cultured with or without epithelium. All observed effects of epithelium on intact tissues could be duplicated by exposing isolated mesenchyme to EGF at 10 ng/ml, i.e. a role for EGF in chondrogenesis is suggested.     

Independent regulation of Dlx2 expression in the epithelium and mesenchyme of the first branchial arch

Dlx2, a member of the distal-less gene family, is expressed in the first branchial arch, prior to the initiation of tooth development, in distinct, non-overlapping domains in the mesenchyme and the epithelium. In the mesenchyme Dlx2 is expressed proximally, whereas in oral epithelium it is expressed distally. Dlx2 has been shown to be involved in the patterning of the murine dentition, since loss of function of Dlx1 and Dlx2 results in early failure of development of upper molar teeth. We have investigated the regulation of Dlx2 expression to determine how the early epithelial and mesenchymal expression boundaries are maintained, to help to understand the role of these distinct expression domains in patterning of the dentition. Transgenic mice produced with a lacZ reporter construct, containing 3.8 kb upstream sequence of Dlx2, led to the mapping of regulatory regions driving epithelial but not mesenchymal expression in the first branchial arch. We show that the epithelial expression of Dlx2 is regulated by planar signalling by BMP4, which is coexpressed in distal oral epithelium. Mesenchymal expression is regulated by a different mechanism involving FGF8, which is expressed in the overlying epithelium. FGF8 also inhibits expression of Dlx2 in the epithelium by a signalling pathway that requires the mesenchyme. Thus, the signalling molecules BMP4 and FGF8 provide the mechanism for maintaining the strict epithelial and mesenchymal expression domains of Dlx2 in the first arch.     

Induction of functional cytodifferentiation in the epithelium of tissue recombinants. I. Homotypic seminal vesicle recombinants

Functional cytodifferentiation of seminal vesicle epithelium was investigated in tissue recombinants. Neonatal rat and mouse seminal vesicles were separated into epithelium and mesenchyme using trypsin. Epithelium and mesenchyme were then recombined in vitro to form interspecific rat/mouse homotypic recombinants. Growth as renal grafts in adult male athymic mice resulted in seminal vesicle morphogenesis in 70% of the recombinants (the remaining 30% failed to grow). Functional cytodifferentiation was judged by the expression of the major androgen-dependent secretory proteins characteristic of the seminal vesicles of adult rats and mice. Antibodies specific for each of these proteins were used to screen tissue sections by immunocytochemistry and to probe protein extracts by immunoblotting techniques. The heterospecific recombinants synthesized the full range of seminal vesicle secretory proteins that typifies the species providing the epithelium of the recombinant, not the mesenchyme. There was little functional variation between individual recombinants. The time course of development corresponded to that of intact neonatal seminal vesicles grown under the same conditions. Morphogenesis and functional cytodifferentiation were not evident after one week, but were well advanced after two weeks. Seminal vesicle recombinants grown for three weeks were indistinguishable morphologically and functionally from normal adult seminal vesicles. In addition, the ability of adult seminal vesicle epithelium to be induced to proliferate was examined. In association with neonatal seminal vesicle mesenchyme, the epithelium of the adult seminal vesicle proliferated and retained its normal functional activity. Thus, seminal vesicle functional cytodifferentiation can be faithfully reproduced in homotypic tissue recombinants. The methods used in this study will be used to investigate seminal vesicle development in instructive inductions of heterotypic epithelia.     

Neutral morphogenetic activity of epithelium in heterologous tissue recombinations

Abstract. To assess the existence of specific and nonspecific epithelial instructions for mesenchymal cell differentiation we compared homospecific and heterospecific mouse and quail tissue recombinations. In heterospecific recombinants between trypsin-dissociated mouse molar mesenchyme and quail epithelia neither odontoblasts nor chondrocytes differentiated. Cartilage appeared if the quail epithelium was contaminated with homologous limb mesenchyme and odontoblasts differentiated if the mouse dental epithelium was contaminated with dental papilla cells.     

FGF10 is required for cell proliferation and gland formation in the stomach epithelium of the chicken embryo

The development of digestive organs in vertebrates involves active epithelial-mesenchymal interactions. In the chicken proventriculus (glandular stomach), the morphogenesis and cytodifferentiation of the epithelium are controlled by the inductive signaling factors that are secreted from the underlying mesenchyme. Previous studies have shown that Fgf10 is expressed in the developing chicken proventricular mesenchyme, whereas its receptors are present in the epithelium. In our present study, we show that FGF10 is an early mesenchymal signal that is critically associated with the developmental processes in the proventricular epithelium. Furthermore, virus-mediated Fgf10 overexpression in ovo results in a hypermorphic epithelial structure and an increase in epithelial cell number. In contrast, the overexpression of a secreted FGFR2b (sFGFR2b), an FGF10 antagonist, blocks cell proliferation and gland formation in the proventricular epithelium in ovo. This downregulation of proliferative activity was subsequently found to retard gland formation and also to delay differentiation of the epithelium. These results demonstrate that FGF10 signaling, mediated by FGFR1b and/or FGFR2b, is required for proliferation and gland formation in the epithelium in the developing chick embryo.     

Branching morphogenesis of mouse salivary epithelium in basement membrane-like substratum separated from mesenchyme by the membrane filter

Branching morphogenesis of mouse salivary gland has been studied with organ-culture system. We developed a novel transfilter culture system for analyzing branching morphogenesis of the salivary epithelium. The submandibular salivary epithelium from early 13-day mouse fetus, clotted with Matrigel and separated from the mesenchyme by membrane filter, showed extensive growth and branching morphogenesis, morphological differentiation of lobules and stalks, and a typical cleft shape. The epithelium showed little growth and no branching without Matrigel clot or without the mesenchyme. This branching morphogenesis was induced even when the pore size of the filter was reduced to 0.05 microns. Use of type I collagen gel instead of Matrigel mostly induced incomplete morphogenesis with various histological abnormalities. These results suggest that the salivary epithelium can undergo branching morphogenesis in the absence of the mechanical action of mesenchymal cells although it needs an appropriate extracellular matrix and some mesenchymal factors transmitted through the filter.     

Partial removal of the mesenchyma disrupts the growth and differentiation of the epithelium in respiratory tract explants from strain A and C57BL mouse embryos

Differentiation of respiratory epithelium (E) of 17-day old embryos of A and C57BL mice after partial removal of mesenchyme (M) was studied in organ culture. In control explants, tissue-specific growth and differentiation of epithelium were observed during long-term culturing. Ih both mouse strains, partial removal of mesenchyme prevented the development of alveolar-like structures in explants of distal part of respiratory tract. In most explants of proximal part of respiratory tract (65.1% in A mice and 85.7% in C57BL mice) with partially removed mesenchyme, we found atypical epithelial structures and foci of poorly differentiated cells with high proliferation rate.     

Morphogenesis of Mouse Mammary Epithelium In Vivo in Response to Biomatrix Prepared from a Stimulatory Fetal Mesenchyme

Insoluble "biomatrix" of mesenchyme is a stimulator of mammary cell differentiation in vitro , but its effect in the morphogenesis is unknown. Fetal salivary mesenchyme induces intense local duct formation when implanted into adult mammary gland. We have therefore tested whether biomatrix prepared from fetal salivary mesenchyme retains this abillity to stimulate duct formation in vivo . Salivary mesenchyme isolated from mouse fetuses at 13.5–14.0 days of gestation, extracted sequentially with water and with 1 M NaCl, then digested with DNAse and RNAse was implanted into mammary glands of female mice and left for periods of 1–35 days. In approximately 40% of recipients, the local epithelium either formed cyst like structures, or else "spikes" of mammary epithelium penetrated the matrix forming a simplified ductwork inside it. Similar responses were elicited by salivary mesenchyme killed by freezing and also by biomatrix prepared from fetal mammary fat pad precursor tissue, mesenchyme of fetal lung, and fetal heart, liver, and brain. However when mesenchyme was either fixed with glutaraldehyde or sonicated and embedded in polymer blocks before implantation, no epithelial response was noted. These observations suggest that the biomatrix provides a passive scaffolding that contributes to morphogenesis of mammary ducts, is insufficient to support normal morphogenesis.