The role of capsulin in the morphogenesis and differentiation of fetal rat gastric mucosa.

The signals that guide the morphogenesis and differentiation of rat fetal gastric mucosa remain largely unknown. We have investigated the role of capsulin in pit/gland formation and epithelial cell differentiation in cultured stomach tissue. Embryonic day 16.5 (E 16.5) stomach tissue cultured for three days in the presence of 1 microM hydrocortisone underwent dramatic transformation, from undifferentiated, stratified cells to differentiated epithelia composed of polarised columnar cells with mucous cells and pit/glands. In the presence of capsulin antisense oligonucleotides directed against capsulin mRNA, tissues do not undergo further development. Significantly, both mucous granules and pit/gland formation were inhibited compared to capsulin sense/scrambled oligonucleotide treated controls. However, in tissues treated with specific anti-rat HGF-antiserum to neutralise secreted HGF, pit/gland formation was inhibited, but the number of mucous granules remained unchanged compared to controls treated with non-specific antiserum (mouse monoclonal cytokeratin 8 antiserum). This data suggests that capsulin may have a role in the morphogenesis of pit/glands and mucin granule formation in the developing rat gastric mucosa. We discuss the possibility that this role of capsulin may be partly mediated through the actions of HGF.     

Changes with Development in the Expression of Cathepsin E in the Fetal Rat Stomach

The changes with development in the expression of cathepsin E in the fetal rat stomach were examined immunochemically and immunohistochemically. The activity of acid proteinase in fetal gastric extracts increased dramatically during late gestational stages, rising from 0.017 units per mg of protein on day 15 of gestation to 0.591 units per mg of protein on day 21 of gestation. Electrophoretic analysis, combined with immunological tests, showed that the increase was due exclusively to increases in the activity of the monomeric and dimeric forms of cathepsin E, while SDS-PAGE-immunoblot analysis revealed that both forms are present as a 43-kDa proenzyme. Immunohistochemically, cathepsin E was localized in the cytoplasm of all proliferating epithelial cells of pars glandularis on day 16 of gestation or later. As revealed by conventional histological methods, surface mucous cells and parietal cells appeared for the first time in specimens on day 19 of gestation, and all of these cells were immunopositive for cathepsin E. The present study further indicated that cathepsin E is the predominant aspartic proteinase in the stomach of young rats, until pepsinogen C appears. Based on these results, possible roles of gastric cathepsin E are discussed.     

Disorganization of stroma alters epithelial differentiation of the glandular stomach in adult mice

Summary The response of adult epithelium in contact with heterologous mesenchymes/stromas was studied in three digestive organs (forestomach, glandular stomach, and duodenum). After various tissues were implanted beneath the epithelial layer of adult mice, the epithelial differentiation was examined after sacrifice of animals at intervals up to 24 weeks. In the forestomach and duodenum, the epithelial differentiation was not affected at all by the tissue implantation. In the glandular stomach, in contrast, epithelial cells exhibited altered differentiation in which chief and parietal cells disappeared and were replaced by columnar epithelial cells with PAS-positive granules. These epithelial cells often formed immature villi. Such differentiation-altered columnar epithelium (DACE) was induced by implanting any type of tissue and even by sham operation, indicating that it was induced by disorganization of the tissue-implanted stroma. The size of DACE was significantly influenced by the stage of implanted tissue; 14.5-day fetal mesenchyme induced the largest DACE, and was followed by 16.5-day fetal mesenchyme, adult stroma, and sham operation. These results suggest the importance of stromal organization in maintaining epithelial differentiation in the glandular stomach.     

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.     

Analysis of Epithelial Protein Profiles of Prostatic Glands Induced Heterotypically in the Bladder Epithelium of the Rat

When urinary bladder epithelia of rats were grown in association with fetal urogenital sinus mesenchyme, prostatic morphogenesis was induced. The epithelial proteins were examined by HPLC fractionation followed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). More than 500 bands of silver-stained epithelial proteins were analyzed. The glandular epithelia induced from both adult and fetal bladder epithelia lost all of the 7 bladder-specific bands (BE 1–7) in most recombinants and expressed a number of prostate-specific bands. Among the 18 bands commonly found in all prostatic lobes, 13 (PE 4, 7–18) were constantly and 3 (PE 1–3) were sporadically detected, while the other 2 (PE 5 and 6) bands were not detected when the adult epithelium was used in recombination. Among the 7 prostatic lobe-specific bands (vPE 14, dPE 1–3), most of them were detected when the fetal epithelium was used, while few of them when the adult epithelium was used. These results demonstrate that prostatic morphogenesis induced in the bladder epithelium was associated with most of biochemical features of prostate. In addition to the biochemical study, histological examination revealed that the prostatic differentiation was more complete in the fetal bladder epithelium than the adult one.     

Ontogenetic expression of chromogranin A and its derived peptides, WE-14 and pancreastatin, in the rat neuroendocrine system

 The ontogenetic expression of chromogranin A (CgA) and its derived peptides, WE-14 and pancreastatin (PST), was studied in the rat neuroendocrine system employing immunohistochemical analysis of fetal and neonatal specimens from 12.5-day embryos (E12.5), to 42-day postnatal (P42) rats. CgA immunostaining was first detected in endocrine cells of the pancreas, stomach, intestine, adrenal gland and thyroid at E13.5, E14.5, E15.5, E15.5 and E18.5, respectively. PST-like immunoreactivity was detected in endocrine cells of the pancreas at E13.5, stomach, intestine at E15.5, adrenal gland at E17.5 and thyroid at E18.5. WE-14 immunoreactivity was first observed in the immature pancreas at E15.5, mucosal cells of the stomach at E15.5, scattered chromaffin cells in the immature adrenal gland and mucosal cells of the intestine at E17.5 and thyroid parafollicular cells at E18.5. These data confirm that the translation of the CgA gene is regulated differentially in various neuroendocrine tissues and, moreover, suggests that the posttranslational processing of the molecule is developmentally controlled. Accepted: 18 October 1996     

Pepsinogen Induction in Chick Stomach Epithelia by Reaggregated Proventricular Mesenchymal Cells In Vitro

In vitro organ culture system which permits embryonic chick proventriculus (glandular stomach) to synthesize pepsinogen de novo was developed. Explants of the proventricular rudiment were cultured on Millipore filters in Medium 199 with Earle's salts supplemented with 50% 12-day embryo extract at 38°C in 95% air and 5% CO₂.
In these culture conditions, pepsinogen, a functional marker protein of proventriculus, was first detected after 3 days of cultivation of 6-day chick proventricular rudiment. When recombined and cultured with 6-day proventricular mesenchyme, 6-day oesophageal, proventricular or gizzard (muscular stomach) epithelium expressed pepsinogen while small intestinal epithelium did not. These results were consistent with the previous results obtained by chorioallantoic membrane (CAM) grafting, and showed that the culture conditions are permissive for pepsinogen expression.
When recombined and cultured with reaggregated mesenchymal cells isolated from 6-day proventricular mesenchymal fragments, both 6-day proventricular and gizzard epithelia formed glandular structure and expressed pepsinogen. This indicates that the proventricular mesenchymal cells retain the ability to induce morphogenesis and cytodifferentiation of the proventricular epithelium even if the normal organization of proventricular mesenchyme is once destroyed.     

Gizzard epithelium of chick embryos can express embryonic pepsinogen antigen,a marker protein of proventriculus

Summary The avian stomach is composed of two distinct organs, the proventriculus and the gizzard. Pepsinogen expression in the proventricular and gizzard epithelia of chick embryos was investigated immunohistochemically with anti-embryonic chick pepsinogen (anti-ECPg) antiserum. In normal development, the ECPg antigen was expressed only in the glandular epithelial cells of the embryonic proventriculus from the 8th day of incubation onwards. However, both proventricular and gizzard epithelia of 6-day embryos expressed the ECPg antigen when recombined and cultured with the proventricular mesenchyme. Chronological studies revealed that the ECPg antigen was first detected in a few epithelial cells at 3 days of cultivation. The percentage of ECPg-positive cells among the total epithelial cells in each recombinant increased with the length of the culture period and all the glandular epithelial cells were positive at 9 days. During this process, the percentage of ECPg-positive cells in each cultured recombinant was similar in proventricular and gizzard epithelia. Moreover, both epithelia could express the ECPg antigen when recombined and cultured with the oesophageal or small-intestine mesenchyme for 9 days, though the percentage of ECPg-positive cells in each cultured recombinant was much lower than that in the cultured recombinant with the proventricular mesenchyme. These results indicate that the gizzard epithelium of 6-day chick embryos possesses a similar potential for pepsinogen expression as the proventricular epithelium of the same age.     

Analysis In Vitro of Capacity of Fetal Fat Pad to Support Mammary Gland Embryogenesis

Fourteen-day fetal mammary fat pad precursor tissue (FP) has the capacity to support various fetal epithelia allowing them to accomplish their characteristic development in vivo , without their own mesenchyme (1). This capacity decreases with age of fetal fat pad and is lost postnatally. To analyse the molecular mechanism of such interaction, a method for in vitro duplication of organogenesis is necessary. In the present paper, a co-culture system of fetal epithelium with prospective mammary fat pad is described. The explanted mammary epithelium started budding, then grew out forming branched mammary ducts with end buds. Ultrastructurally, the developing ductal structures exhibited the typical mammary gland morphogenesis.
³H-Thymidine incorportion assessed by autoradiography showed that the mammary gland morphogenesis in vitro was due to the proliferation of epithelial cells, not merely to a change of the shape of the epithelium. This supportive capacity of 14-day FP also decreased with aging; explanted mammary epithelium did not grow into 17-day FP. When insoluble, non-living biomatrix was used in place of living FP the epithelium grew into the matrix but the resulting structures lacked characteristic morphology of epithelium on living fetal FP. The difference of capacity between 14-day and 17-day tissues was also lost.     

Mouse oocytes derived from fetal germ cells are competent to support the development of embryos by in vitro fertilization

Mouse oocyte development in vitro has been studied in the past several years, but no evidence showed that the fertilizable oocytes could be obtained from the fetal mouse germ cells before the formation of the primordial follicles. In this study, an efficient and simple method has been established to obtain the mature oocytes from the fetal mouse germ cells at 16.5 days post-coitum (dpc). For the initial of follicular formation, fetal mouse 16.5 dpc ovaries were transplanted to the recipient under the kidney capsule, and the ovaries were recovered after 14 days. Subsequently, the growing preantral follicles in the ovarian grafts were isolated and cultured in vitro for 12 days. Practically, the mature oocytes ovulated from the antral follicles were able to be fertilized in vitro and support the embryonic development. The results demonstrate that the fetal mouse 16.5 dpc germ cells are able to form primordial follicles with the ovarian pregranulosa cells during the period of transplantation in the ectopic site, and the oocytes within the growing follicles are able to mature in vitro, then are able to support the embryonic development.     

Morphologic and functional development of whole human fetal stomachs grafted into nude mice

To study in vivo the cellular differentiation and secretion of human developing fetal stomach, ethically and technically impossible to perform in utero, 256 fetal stomachs were xenografted. Human stomachs from 6- to 10-week-old fetuses were grafted for 1-273 days into nude mice. Biopsies for immunohistochemistry, hybridization and electron microscopy were taken and a catheter introduced into the human stomach. Macroscopic growth was fast and cells in S phase were numerous during the first 9 weeks, then the stomach size was stable and the gastric mucosa, of adult type, remained normal. In situ hybridization detected only a minute mouse mesenchymal chimerism in the graft. Chromogranin A, intrinsic factor and H+/K+ adenosine triphosphatase were immunohistolocally detected in epithelial cells 20 days after grafting, gastrin was detected after 30 days and pepsinogen after 60 days. The pH in gastric juice, which was at 8.0 +/- 0.1 from days 10-25, dropped from 4.39 +/- 1.80 at 30 days to 1.58 +/- 0.29 at 90 days. Intrinsic factor was stable and pepsin ranged from 6.8 +/- 7.8 to 134 +/- 51 units at 90 days. The differentiation of the epithelial cells in xenografts was very accelerated in comparison to that in utero.     

Cell-specific hypomethylation of the pepsinogen gene in pepsinogen-producing cells

The pepsinogen gene is hypomethylated in the stomach, in which it is expressed. For demonstration that this hypomethylation of the pepsinogen gene in the stomach reflects pepsinogen-producing cells, we analyzed fractions of dispersed mucosal cells with various contents of pepsinogen-producing cells prepared from guinea pig stomach by centrifugal elutriation. mRNA expression and the extent of hypomethylation of the pepsinogen gene in each fraction was closely correlated with the content of pepsinogen-producing cells. These results suggested hypomethylation of the pepsinogen gene in pepsinogen-producing cells and differential pepsinogen gene methylation in cell subpopulations in the stomach.     

Angiogenesis and morphogenesis of murine fetal distal lung in an allograft model

Neovascularization is crucial to lung morphogenesis; however, factors determining vessel growth and formation are poorly understood. The goal of our study was to develop an allograft model that would include maturation of the distal lung, thereby ultimately allowing us to study alveolar development, including microvascular formation. We transplanted 14-day gestational age embryonic mouse lung primordia subcutaneously into the back of nude mice for 3.5-14 days. Lung morphogenesis and neovascularization were evaluated by light microscopy, in situ hybridization, and immunohistochemical techniques. Embryonic 14-day gestational age control lungs had immature structural features consistent with pseudoglandular stage of lung development. In contrast, 14 days after subcutaneous transplantation of a 14-day gestational age lung, the allograft underwent significant structural morphogenesis and neovascularization. This was demonstrated by continued neovascularization and cellular differentiation, resulting in mature alveoli similar to those noted in the 2-day postnatal neonatal lung. Confirmation of maturation of the allograft was provided by progressive type II epithelial cell differentiation as evidenced by enhanced local expression of mRNA for surfactant protein C and a threefold (P < 0.008) increase in vessel formation as determined by immunocytochemical detection of platelet endothelial cell adhesion molecule-1 expression. Using the tyrosine kinase Flk-1 receptor (flk-1) LacZ transgene embryos, we determined that the neovascularization within the allograft was from the committed embryonic lung endothelium. Therefore, we have developed a defined murine allograft model that can be used to study distal lung development, including neovascularization. The model may be useful when used in conjunction with an altered genetic background (knockout or knock in) of the allograft and has the further decided advantage of bypassing placental barriers for introduction of pharmacological agents or DNA directly into the lung itself.     

EGF-R and erbB-2 in murine tooth development after ethanol exposure

BACKGROUND: Alcohol consumption during pregnancy can frequently lead to a congenital disorder known as fetal alcohol syndrome (FAS); however, not all children born to alcoholic women develop FAS. Alcohol consumption may affect diverse organs and systems during embryonic development, including craniofacial structures. Small teeth, enamel alterations, and delayed eruption have been observed after ethanol exposure. Epidermal growth factor receptors (EGF-Rs) participate in dental proliferation and differentiation, and changes in these receptors were considered here to be a likely mechanism associated to the dental anomalies observed in this syndrome. Epidermal growth factor receptor type 1 (EGF-R) and epidermal growth factor receptor type 2 (erbB-2) immunoexpression during the lower first molar morphogenesis was investigated in mouse fetuses exposed to ethanol during gestation. METHODS: Pregnant female mice were divided into groups, consuming either 5, 10, 15, 20, or 25% ethanol solutions, or water (control group). Heads were obtained from 16.5- and 18.5-day fetuses. Immunohistochemistry was applied to EGF-R and erbB-2. RESULTS: At days 16.5 and 18.5, fetuses from 15%, 20%, and 25% ethanol groups showed delayed differentiation, degenerative changes in dental epithelial tissues and reduced dental size; additionally, they displayed an enhanced immunoreactivity to EGF-R and erbB-2. CONCLUSIONS: Our results suggest that ethanol consumption during pregnancy affects the expression of EGF receptors and induces a delay in murine fetal dental morphogenesis. Dental development is a process that involves a number of growth factors; hence we consider that further research is required to show whether the changes in glycosylation and growth-factor signaling pathways observed in other cells are also involved in the alterations observed in this study.     

Persistence of responsiveness of adult mouse mammary gland to induction by embryonic mesenchyme

Persistence of the capacity for embryogenic morphogenesis in adult mammary epithelium was demonstrated by allowing it to interact with grafted embryonic mesenchyme in vivo. When 14-day embryonic mammary or salivary mesenchyme was transplanted in the mammary gland of syngeneic young adult virgin mice, organogenetic development of the mammary epithelial cells occurred responding to closely attached mesenchyme. An early change, within 2–4 days, that was observed equally in both types of the mesenchymes was proliferation of mammary epithelial cells in multiple layers resembling rudimental architecture. Subsequently, ductal branching occurred from the rudimental architecture by mesenchyme-dependent branching pattern, of mammary gland type with mammary mesenchyme and of salivary gland-like type with salivary mesenchyme. This developmental response did not require hormones secreted from ovaries since it was observed similarly in ovariectomized mice. The mammary epithelium at the lactating stage did not show such a potential to the transplanted salivary mesenchyme.     

Immunolocalization of sonic hedgehog (Shh) in developing mouse lung.

Expression of sonic hedgehog (Shh) is required for normal development of the lung during embryogenesis. Loss of Shh expression in mice results in tracheoesophageal fistula, lung hypoplasia, and abnormal lung lobulation. To determine whether Shh may play a role later in lung morphogenesis, immunostaining for Shh was performed in mouse lung from embryonic day (E) 10.5 to postnatal day (PD) 24. Shh was detected in the distal epithelium of the developing mouse lung from E10.5 to E16.5. From E16.5 until PD15, Shh was present in epithelial cells in both the peripheral and conducting airways. Although all cells of the developing epithelium uniformly expressed Shh at E10.5, Shh expression was restricted to subsets of epithelial cells by E16.5. Between E16.5 and PD15, non-uniform Shh staining of epithelial cells was observed in the conducting airways in a pattern consistent with the distribution of non-ciliated bronchiolar cells (i.e., Clara cells) and the Clara cell marker CCSP. Shh did not co-localize with hepatocyte nuclear factor/forkhead homologue-4 (HFH-4), beta-tubulin, or with the presence of cilia. These results support the concept that Shh plays a distinct regulatory role in the lung later in morphogenesis, when it may influence formation or cytodifferentiation of the conducting airways.     

An immunocytochemical study on distinct intracellular localization of cathepsin E and cathepsin D in human gastric cells and various rat cells.

Immunocytochemical localization of two distinct intracellular aspartic proteinases, cathepsins E and D, in human gastric mucosal cells and various rat cells was investigated by immunogold technique using discriminative antibodies specific for each enzyme. Cathepsin D was exclusively confined to primary or secondary lysosomes in almost all the cell types tested, whereas cathepsin E was not detected in the lysosomal system. The localization of cathepsin E varied with different cell types. Microvillous localization of cathepsin E was found in the intracellular canaliculi of human and rat gastric parietal cells, rat renal proximal tubule cells, and the bile canaliculi of rat hepatic cells. The immunolocalization of each enzyme in gastric cells were essentially the same in humans and rats. In the gastric feveolar epithelial cells and parietal cells, definite immunolabeling for cathepsin E was observed in the cytoplasmic matrix, the cisternae of the rough endoplasmic reticulum, and the dilated perinuclear envelope. In rat kidney, cathepsin E was detected only in the proximal tubule cells, while cathepsin D was found mainly in the lysosomes of the distal tubule cells but not in those of the proximal tubule cells. These results clearly indicate the distinct intracytoplasmic localization of cathepsins E and D and suggest the possible involvement of cathepsin E in extralysosomal proteolysis that is related to specialized functions of each cell type.     

Development and characterization of conditionally immortalized gastric epithelial cell lines from transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene

Conditionally immortalized gastric epithelial cell lines were established from transgenic rats harboring temperature-sensitive simian virus 40 (tsSV40) large T-antigen gene. Gastric mucosal cells and epithelial tissues isolated from the stomach of the transgenic rats were cultured at permissive temperature (33 degrees C), and proliferative cells were cloned by colony formation. Six cell lines (designated as RGE1-01, RGE1-02, RGE1-03, RGE1-21, RGE1-22 and RGE2-01) showing epithelial-like morphology have been established. All cells grew at 33 degrees C, but did not at nonpermissive temperature (39 degrees C). High expression level of large T-antigen in the nuclei was observed at 33 degrees C, whereas the expression level was gradually decreased in a time-dependent manner at 39 degrees C. These results suggest that the temperature-sensitive growth characteristics arise as a result of a function of the tsSV40 large T-antigen. None of the cell lines were transformed as judged by anchorage-independent growth assay. Immunocytochemical findings indicated that all cells expressed epithelial cell markers including cytoskeletal (cytokeratin and actin), basement membrane (laminin and collagen type IV) and junctional complex (ZO-1 and desmoplakin I+II) proteins at 33 degrees C. All cells expressed mRNA of cathepsin E, a pit cell marker. Moreover, transepithelial resistance was observed between apical and basolateral sides in the cells. RGE1-22 cells produced prostaglandin E(2). Levels of mRNA for cathepsin E, transepithelial resistance and prostaglandin E(2) were influenced by the nonpermissive temperature. Thus, these conditionally immortalized gastric cell lines which preserve some epithelial cell characteristics will provide a useful in vitro model of gastric epithelium.     

In utero bone marrow transplantation of fetal baboons with mismatched adult baboon marrow.

In utero bone marrow transplantation to fetuses offers the potential advantage of ameliorating the effects of genetic disorders by transplanting allogeneic hematopoietic stem cells into recipients who are immunoincompetent and require no preparative regimen. Therefore, we undertook studies to examine the feasibility of in utero bone marrow transplantation of unrelated allogeneic adult bone marrow into fetal baboons. Thirty-one baboon fetuses were transplanted between the ages of 60 and 160 days gestation (normal gestation, 182 days) with unrelated allogeneic adult bone marrow containing a different isozyme of glucose-phosphate isomerase (GPI). Approximately one third of the 80-day fetuses demonstrated engraftment 1 month after transplantation. Three of three of the initial chimeras died in utero 45 to 80 days after transplantation and the remaining chimeras lost their graft. Furthermore, 80-day fetal baboons were able to recognize donor cells, maternal cells, and other adult baboon peripheral blood cells in a mixed lymphocyte culture (MLC) reaction but still could engraft with allogeneic bone marrow. In contrast all nonchimeric animals survived to term. These data suggest that fetal transplantation of primates is feasible using techniques employed in these studies and that transplantation of younger fetuses who are immunocompetent should be attempted.