RESUMEN
In our previous work, two-dimensional gel electrophoretic analysis of the translational products from mRNA of lens from rats maintained on 50% galactose up to 45 days has suggested that synthesis of mRNA was not arrested by the disease process, but it decreased significantly relative to the control. The loss in mRNA number was due mainly to loss in cell population. Specifically, the gamma-crystallin mRNA product decreased to very low levels at onset of the disease. However, this mRNA was resynthesized in the surviving cells as the cataracts matured. Therefore, it became of interest to explore whether reversal or inhibition of cataracts would lead to some measurable changes in mRNA population in the experimental lens. The results show that the percentage (relative to the total mRNA population) of the in vitro [35S]-labeled translational products from alpha-, beta- and gamma-crystallin mRNAs combined, remained unchanged irrespective of the state of the lens. The severity of the cataracts was examined by indirect immunofluorescence with polyclonal MP26 antibody. Reversal of cataracts led to partial recovery of normal fiber cell morphology. Treatment with sorbinil in combination with galactose led to inhibition of cataracts with indication of appearance of vacuoles at longer periods of exposure to the drug. The translational products profile reflected the expected variation in non-crystallin and crystallin mRNA synthesis. It is concluded that there appears to be a combined fixed level of synthesis for the crystallins, such that inhibition in synthesis of gamma-crystallin mRNAs appears to lead to an increase in synthesis of alpha-crystallin mRNAs, while synthesis of beta-crystallin mRNAs showed insignificant fluctuation. A similar conclusion may also be drawn relative to the combined synthesis for the non-crystallin mRNAs.
Asunto(s)
Catarata/metabolismo , Galactosemias/metabolismo , Cristalino/análisis , Cristalino/metabolismo , ARN Mensajero/análisis , HumanosRESUMEN
Using C57/10Sn (B10, H-2b) and B10.A/SgSn (B10.A,H-2a) congenic mice, we measured 1) the level of endogenous pulmonary corticosterone during mouse development; 2) the degree of lung morphological maturation on gestation day 17, with or without corticosteroid treatment; and 3) the maternal influence on normal lung development and fetal response to corticosteroids. The results of our study indicate that there was a progressive increase in the level of endogenous hormone with time in fetal B10 (H-2b) and B10.A (H-2a) mice; throughout mid- to late gestation, the detectable amount of hormone was almost identical in lungs of both strains. Evaluating the degree of lung maturation by morphometry, B10.A mouse lungs were found to be less mature than B10 mouse lungs. Following corticosteroid treatment on day 12 of gestation, H-2a lungs were equal to or more mature than H-2b lungs. We also compared heterozygous mouse lungs from reciprocal crosses (B10.B10.A, b/a and B10.A.B10, a/b). Mice with a maternally derived H-2a haplotype had less mature lungs than those with a maternally derived H-2b haplotype, suggesting a maternal effect. When exogenous hormone was administered, all heterozygous mouse lungs increased in maturity regardless of the origin of the H-2a haplotype. The treated a/b or b/a lungs were more mature than homozygous b/b and less mature than homozygous a/a lungs. We conclude that progressive lung maturation is associated with a gene(s) at or near the H-2 complex, as is the ability to respond to corticosteroids.
Asunto(s)
Pulmón/embriología , Complejo Mayor de Histocompatibilidad , Corticoesteroides/análisis , Corticoesteroides/farmacología , Animales , Femenino , Feto , Pulmón/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos , EmbarazoRESUMEN
Epithelial differentiation during lung development appears to be influenced by mesenchyme-derived instructions coupled with hormonal regulations. The basal lamina which is associated with progenitor and differentiating epithelia during mouse embryogenesis (Theiler-stages 16-28) was examined by transmission electron microscopy and indirect-immunofluorescence microscopy. During the embryonic phase of lung development, progenitor epithelia for the pulmonary acinus projected microvilli or cytoplasmic "feet" through the basal lamina, which resulted in discontinuities and a close approximation of the adjacent mesenchymal-cell processes. These changes were also associated with the transitory polarization of mesenchymal cells perpendicular to the plane of the basal lamina, which resulted in a sheet of cuboidal mesenchymal cells adjacent to the developing acinar-tubule epithelium. During the embryonic phase of lung development, these specific interstitial or mesenchymal cells stained for heparan-sulfate proteoglycans; no other cell types were immunostained. By Theiler-stage 25, the acinar-tubule epithelia had differentiated into type-II pneumonocytes which contained lamellar bodies and significant amounts of glycogen. Fibronectin, laminin, and heparan-sulfate proteoglycan were localized in the basement membranes during the embryonic, canalicular, and terminal sac phases of lung morphogenesis. A diffuse localization of fibronectin of the interstitial cell surfaces was observed. These observations indicate that major changes in the structure and composition of basal lamina occur during the embryonic and fetal phases of pulmonary-acinus epithelial-cell differentiation and the production of pulmonary surfactant. The major changes in the basal lamina may be partly mediated by mesenchyme-derived instructions for type-II epithelial-cell differentiation.
Asunto(s)
Pulmón/embriología , Animales , Membrana Basal/citología , Membrana Basal/ultraestructura , Femenino , Fibronectinas/análisis , Técnica del Anticuerpo Fluorescente , Laminina/análisis , Pulmón/ultraestructura , Ratones , Ratones Endogámicos , Microscopía Electrónica , Morfogénesis , Embarazo , Proteoglicanos/análisisRESUMEN
The development of the middle ear and tympanum of Gallus gallus has been studied in embryos Hamilton-Hamburger stages 20-46. Particular attention was paid to the pattern of expansion of the pharyngeal pouch forming the tympanic cavity, and the histogenesis of the tissues of the region of the vestibular window. It is concluded that pouch expansion is brought about by simple epithelial growth into regions devoid of mesenchyme. The mesenchyme does not show significant cell death, but differentiates as connective tissue, macrophages, and sinus vascular spaces. The epithelium of the mature cavity is of endodermal origin, and there is no indication of celomic contributions. We provide a detailed morphological analysis of the development of the footplate from cells of both the second visceral arch and the otic capsule. These detailed observations on the patterns of chondrification of columella and capsular tissues permit the presentation of a model for cellular interactions leading to the differentiation of the annular ligament.
Asunto(s)
Embrión de Pollo/crecimiento & desarrollo , Oído Medio/embriología , Animales , Diferenciación Celular , Células Epiteliales , Estratos Germinativos/citología , Faringe/embriologíaRESUMEN
Single doses of N-formyl hydroxyaminoacetic acid (hadacidin) were injected into chick embryos HH stages 5-30. Embryos surviving for 8+ days total incubation time were recovered and examined for gross and microscopically observable defects. Particular attention was paid to the development of the columella. Survival was only 20%, but a high incidence of cranio-facial and hind limb defects was observed in most age groups. While three categories of columella defect were observed, reflecting some degree of age dependency, this chondrogenic tissue is less susceptible to teratogenic action than other skeletal tissues. The types of columella defect observed are interpreted in the light of a model of cellular interactions in fenestra vestibularis differentiation presented previously. It is concluded that hadacidin produces abnormalities of skeletal differentiation by disrupting morphogenetic, rather than the initiative phases of development of particular elements.
Asunto(s)
Huesos/anomalías , Oído Medio/anomalías , Glicina/análogos & derivados , Teratógenos , Anomalías Inducidas por Medicamentos/clasificación , Animales , Huesos/efectos de los fármacos , Embrión de Pollo , Relación Dosis-Respuesta a Droga , Oído Medio/efectos de los fármacos , Oído Medio/patología , Formaldehído/análogos & derivados , Formaldehído/farmacología , Glicina/farmacología , Factores de TiempoRESUMEN
The timing, position and mechanism(s) for determining type II cytodifferentiation during mammalian lung development are not known. To approach this problem, we have cultured Theiler stage 16 embryonic B10.A strain mouse lung primordia (12-days gestation, E12) in serumless, chemically defined medium in the presence or absence of dexamethasone (DEX) for periods up to 27 days in vitro. Morphogenesis and cytodifferentiation were evaluated by light and transmission electron microscopy and immunochemical techniques. Pulmonary surfactant-associated apoproteins (PSAP) were initially expressed by type II cells at 16.5-day gestation in vivo. DEX-supplementation to the culture medium resulted in the accelerated expression of PSAP; the apoprotein isoforms (A1, A2, and A3) produced in vitro were comparable to those synthesized during fetal and postnatal in vivo development by high resolution, two-dimensional gel electrophoresis coupled with immunoblot staining. Cultures without DEX produced PSAP A2 and A3 isoforms, but did not produce A1 (26-31 kDa, pI 5.2-5.3). DEX-treated cultures produced more lamellar bodies within type II cells than non-treated controls. The results demonstrate that long-term cultures of embryonic lung primordia express morphogenesis, cytodifferentiation and the synthesis and secretion of PSAP in the absence of exogenous hormones or growth factors. The data set further supports the hypothesis that morphogenesis and type II cytodifferentiation are regulated by autocrine and paracrine factors intrinsic to the embryonic lung developmental program and independent of exogenous hormone controls.
Asunto(s)
Pulmón/embriología , Corticoesteroides/análisis , Animales , Diferenciación Celular , Células Cultivadas , Medios de Cultivo , Electroforesis en Gel de Poliacrilamida , Epitelio/análisis , Epitelio/ultraestructura , Técnica del Anticuerpo Fluorescente , Pulmón/análisis , Pulmón/citología , Ratones , Ratones Endogámicos , Morfogénesis , Organoides/ultraestructura , Proteolípidos/análisis , Proteolípidos/biosíntesis , Proteínas Asociadas a Surfactante Pulmonar , Surfactantes Pulmonares/análisis , Surfactantes Pulmonares/biosíntesisRESUMEN
Previous work from this laboratory has shown that in cataractous lens from rats fed a 50% galactose diet up to 32 days, synthesis of crystallin mRNAs was reduced while synthesis of mRNAs for the non-crystallin proteins appeared to be maintained at equivalent or higher levels than found in the controls. In this study, we find that the population of mRNA from the 45-day cataractous lens included all of the crystallin mRNAs and substantial amounts of mRNAs for the non-crystallin proteins, in particular those proteins of molecular weights ranging from 45,000 to 92,000, and pIs from 5.5 to 7.0. At the 45-day cataractous stage, the observed mRNA products totaled between 100 and 120, while for the control they numbered between 40 and 50. In addition, hybrid-select measurements with a lambda gt11 MP26 clone established that MP26 mRNA persisted in the fiber cells of 11-, 20-, 32- and 45-day galactosemic lens. The data, therefore, suggest that continuous exposure of the lens to galactose apparently leads to significant fluctuations in mRNA synthesis and survival. Indirect immunofluorescence (with a monospecific polyclonal rabbit anti-MP26 antibody) and light-microscopy studies demonstrated that although the cortical fiber cells were swollen and structurally disoriented, they retained their nuclei, while the enucleated fiber cells were absent. The microscopy data also suggest that differentiation of the epithelial cells to fiber cells continued in the cataractous lens. Fiber-cell migration was backward as compared with the control, and the surviving cells were localized within the cortex and appeared to be surrounded by damaged cells or cell debris. It is suggested that persistent fiber-cell survival, and continued synthesis of various classes of mRNA by the nucleated cells in the cataractous lens could explain why reversal of cataracts results in recovery in both cell morphology and transparency in the non-nuclear portion of the lens.
Asunto(s)
Catarata/metabolismo , Cristalino/metabolismo , ARN Mensajero/biosíntesis , Animales , Catarata/inducido químicamente , Catarata/patología , Supervivencia Celular , Cristalinas/biosíntesis , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Galactosa , Cristalino/patología , Peso Molecular , Ratas , Factores de TiempoRESUMEN
During lung development type II alveolar epithelial cells produce extracellular pulmonary surfactant. Polyclonal antibodies were produced against nonserum proteins associated with human surfactant. The present studies were designed (i) to determine if mouse surfactant proteins were antigenically cross-reactive with polyclonal antibodies directed against human surfactant proteins; and (ii) to determine surfactant protein localization during fetal, neonatal, and adult mouse lung development. Two-dimensional gel electrophoresis studies in conjunction with immunologic techniques provided evidence that mouse and human surfactant proteins shared antigenic determinants. The major monomeric form of mouse surfactant protein in a glycoprotein of approximately Mr 35,000 under reducing conditions. A less abundant form was identified as a Mr 45,000 polypeptide. Immunohistochemical localization showed that type II cells contain surfactant protein at Theiler stage 26. A gradient of immunostaining was localized within alveolar surfaces. The antigen was not detected in heart, blood vessels, or pulmonary interstitial cells. Surfactant protein was detected lining alveolar surfaces in mature adult lung. The distribution of this protein during fetal and neonatal lung morphogenesis suggests that this extracellular constituent of pulmonary surfactant may be extremely useful as a phenotypic marker with which to evaluate normal and abnormal lung development.
Asunto(s)
Pulmón/crecimiento & desarrollo , Proteolípidos/análisis , Surfactantes Pulmonares/análisis , Animales , Electroforesis en Gel de Poliacrilamida , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Ratones , Peso Molecular , Fenotipo , Embarazo , Proteolípidos/inmunología , Proteínas Asociadas a Surfactante Pulmonar , Surfactantes Pulmonares/inmunologíaRESUMEN
One of the major problems in developmental biology concerns how differential gene activity is regionally controlled. One approach to this problem is the use of mesenchyme specification of epithelial-specific gene expression, such as, during tooth morphogenesis or lung morphogenesis. In the example of tooth morphogenesis, dental papilla ectomesenchyme induces de novo gene expression as assayed by detection of amelogenin transcripts, or immunodetection of amelogenin polypeptides within ameloblast cells. This process does not require serum supplementation or exogenous factors during epithelial-mesenchymal interactions in vitro. In contrast, lung morphogenesis requires hormones to mediate mesenchyme-derived influences upon type II epithelial cell differentiation and the production of pulmonary surfactant (eg, neutral and phospholipids, surfactant proteins). Glucocorticoids are required to stimulate the release of fetal pneumonocyte factor (FPF) from fibroblasts which, in turn, enhance the production of pulmonary surfactant. Thyroxin appears to regulate the relative responsiveness of progenitor type II cells to steroid-stimulated release of FPF. This review will highlight key concepts associated with these developing organ systems and emphasize the problem of regional controls which regulate epithelial cell-specific gene activity.