Transfection of Mv1Lu mink lung type II alveolar cells with ␤1-6-N-acetylglucosaminyl transferase V is associated with the expression of large lysosomal vacuoles, which are immunofluorescently labeled for the lysosomal glycoprotein lysosomal-associated membrane protein-2 and the ␤1-6-branched N-glycan-specific lectin phaseolis vulgaris leucoagglutinin. By electron microscopy, the vacuoles present the morphology of multilamellar bodies (MLBs). Treatment of the cells with the lysosomal protease inhibitor leupeptin results in the progressive transformation of the MLBs into electron-dense autophagic vacuoles and eventual disappearance of MLBs after 4 d of treatment. Heterologous structures containing both membrane lamellae and peripheral electron-dense regions appear 15 h after leupeptin addition and are indicative of ongoing lysosome-MLB fusion. Leupeptin washout is associated with the formation after 24 and 48 h of single or multiple foci of lamellae within the autophagic vacuoles, which give rise to MLBs after 72 h. Treatment with 3-methyladenine, an inhibitor of autophagic sequestration, results in the significantly reduced expression of multilamellar bodies and the accumulation of inclusion bodies resembling nascent or immature autophagic vacuoles. Scrape-loaded cytoplasmic FITC-dextran is incorporated into lysosomal-associated membrane protein-2-positive MLBs, and this process is inhibited by 3-methyladenine, demonstrating that active autophagy is involved in MLB formation. Our results indicate that selective resistance to lysosomal degradation within the autophagic vacuole results in the formation of a microenvironment propicious for the formation of membrane lamella. INTRODUCTIONMultilamellar bodies (MLBs) are membrane-bound cellular organelles, which vary in size from 100-2400 nm, are composed of concentric membrane layers, and frequently exhibit an electron-dense core. MLBs are found in numerous cell types where they function in lipid storage and secretion (Schmitz and Mü ller, 1991). In lung type II alveolar cells, MLBs function as secretory granules whose exocytosis results in the deposition of the tubular myelin forms of surfactant on the surface of the alveolae (Hatasa and Nakamura, 1965;Ryan et al., 1975;Williams, 1977). The surfactant film over the alveolar epithelium regulates the surface tension at the air-cell interface and protects the alveola from collapse during respiration (Haagman and van Golde, 1991).Although the secretory function of MLBs in type II alveolar cells is well established, the precise mechanism of MLB biogenesis remains unclear. Autoradiographic studies of murine type II alveolar cells of mouse lungs showed that although phospholipids labeled with [ 3 H]choline are delivered directly from the Golgi to the MLB, proteins metabolically labeled with [ 3 H]leucine are visualized within multivesicular bodies before delivery to MLBs (Chevalier and Collet, 1972). Surfactant proteins A, B, and C are delivered via multivesicular bodies to MLBs, and multivesicular bodies are proposed as th...

Development of the fetal mouse esophageal epithelium was followed using light microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and radioautography. At 15 days of gestation in the cervical (C), mediastinal (M), and abdominal (A) segments of the esophagus, the epithelium was two or three cells thick, and only cells located in the basal (germinal) layer incorporated tritiated thymidine. Ciliated cells were sparse in all three segments. At 17 days of gestation, longitudinal mesenchymal ridges became more differentiated in the distal segment. Labeling indices were lower than at preceding stages in each segment. Ciliated cells had increased in number and appeared to be evenly distributed along the whole esophagus. In periodic acid-Schiff (PAS)-stained sections, an increasing proximodistal distribution of glycogen stores was observed, with greatest concentrations found in segment A. At 18 days of gestation, labeling indices were comparable in segments C and M (11.7% +/- 2.9% and 12.8% +/- 1.9%, respectively) but remained higher in segment A (17.9% +/- 2.0%). Ciliated cells were still present. At this stage, transverse circular furrows and ridges started to appear. They increased in number at 4 days after birth and were very closely distributed in the adult. In longitudinal sections, these ridges corresponded to projections of stratum granulosum and of the overlying stratum corneum. After birth, ciliated cells desquamated rapidly but some patches were still present at 4 days. At 8 days, the esophageal epithelium was not yet keratinized.

We have previously shown that monoclonal antibodies (MAb) prepared against the duodenal mucosa of 4-day-old mice disclosed the presence of two antigens associated with the formation of intestinal crypts. One of these, MIM-1/39, was found in the apical cytoplasm of undifferentiated epithelial crypt cells of the duodenum and colon. We report here the immunolocalization of MIM-1/39 in different glands associated with the gastrointestinal tract, using a polyclonal antibody produced against antigen MIM-1/39. By indirect immunofluorescence on 1-micron thick Lowicryl K4M sections, MIM-1/39 was detected in secretory granules of serous cells in lingual (von Ebner's gland), sublingual, submandibular and parotid glands, and in pancreas; it was also found in epithelial cells of the gallbladder and in the secretory granules of chief cells of gastric glands. Liver, kidney, and Brunner's glands were not immunoreactive. Immunocytochemistry revealed the presence of antigen MIM-1/39 in small secretory granules of the gallbladder, duodenum, colon, and in the large secretory granules in serous cells of lingual and parotid glands, in pancreas, and in gastric chief cells. In Western blotting, the MIM-1/39 MAb revealed two bands (330 and 350 KD) in adult mouse duodenal mucosa, gallbladder and stomach, whereas only one (330 KD) was disclosed in pancreatic juice. However, two bands (330 and 350 KD) were detected in pancreatic juice with the polyclonal antibody. The distribution of MIM-1/39 was different from that reported for IgA, bound and free secretory components, cryptdin, and Tamm-Horsfall protein. Therefore, MIM-1/39 appears to be a unique protein. Its exact role remains to be elucidated.

Two monoclonal antibodies were prepared against the duodenal mucosa of fourday‐old mice (MIM‐1/39 and MIM 1/130). The expression of the antigens was associated with the crypts of the small and large intestine in the fetus and adult. MIM‐1/39 was present in epithelial cells of the intervillous areas in the small intestine at 17 and 18 days of gestation; afterwards its expression was detected only in crypt cells from birth to adulthood. Transition from the mouth of the crypts to intestinal villi was abrupt. Expression of MIM‐1/39 was first detected at time of birth in the colon: In the adult, only crypt cells expressed the antigen and goblet cells were negative. Antigen MIM‐1/130 was detected from 16 to 18 days of gestation in the small intestine, in the mesenchymal matrix lying under the intervillous epithelium. After birth, it was present in the pericryptal mesenchymal matrix. This antigen was also expressed at birth in the colon and remained in the pericryptal matrix in the adult. In vivo, multiple injections of an organic extract of rat amniotic fluid to mothers, starting at 14 days of gestation, induced a profound modulation in the pattern of expression of both antigens at 17 days of gestation: The pattern of expression was comparable to that observed at least 5 days after birth in untreated animals. The expression of both antigens before crypt appearance may reflect some molecular differentiation in preparation for the formation of crypts, while their association with differentiated crypts may indicate that they have a role in the maintenance of crypt functional and/or morphological integrity. Finally, the fact that their expression can be modulated experimentally may prove to be a breakthrough for the study of crypt formation. © 1992 Wiley‐Liss, Inc.

These observations indicate that the extracellular matrix associated with the epithelium of pyloric glands, of intestinal and colonic crypts, and of gallbladder contains a new antigen whose function remains to be determined. The neonatal mouse hence constitutes a good model to study the role of extracellular matrix components in determining organ differentiation in vivo.