Hypothalamic sonic hedgehog is required for cell specification and proliferation of LHX3/LHX4 pituitary embryonic precursors.

Sonic hedgehog (SHH) is an essential morphogenetic signal that dictates cell fate decisions in several developing organs in mammals. In vitro data suggest that SHH is required to specify LHX3+/LHX4+ Rathke's pouch (RP) progenitor identity. However, in vivo studies have failed to reveal such a function, supporting instead a crucial role for SHH in promoting proliferation of these RP progenitors and for differentiation of pituitary cell types. Here, we have used a genetic approach to demonstrate that activation of the SHH pathway is necessary to induce LHX3+/LHX4+ RP identity in mouse embryos. First, we show that conditional deletion of Shh in the anterior hypothalamus results in a fully penetrant phenotype characterised by a complete arrest of RP development, with lack of Lhx3/Lhx4 expression in RP epithelium at 9.0 days post coitum (dpc) and total loss of pituitary tissue by 12.5 dpc. Conversely, overactivation of the SHH pathway by conditional deletion of Ptch1 in RP progenitors leads to severe hyperplasia and enlargement of the Sox2+ stem cell compartment by the end of gestation.

Epithelial inactivation of Yy1 abrogates lung branching morphogenesis.

Yin Yang 1 (YY1) is a multifunctional zinc-finger-containing transcription factor that plays crucial roles in numerous biological processes by selectively activating or repressing transcription, depending upon promoter contextual differences and specific protein interactions. In mice, Yy1 null mutants die early in gestation whereas Yy1 hypomorphs die at birth from lung defects. We studied how the epithelial-specific inactivation of Yy1 impacts on lung development. The Yy1 mutation in lung epithelium resulted in neonatal death due to respiratory failure. It impaired tracheal cartilage formation, altered cell differentiation, abrogated lung branching and caused airway dilation similar to that seen in human congenital cystic lung diseases. The cystic lung phenotype in Yy1 mutants can be partly explained by the reduced expression of Shh, a transcriptional target of YY1, in lung endoderm, and the subsequent derepression of mesenchymal Fgf10 expression. Accordingly, SHH supplementation partially rescued the lung phenotype in vitro. Analysis of human lung tissues revealed decreased YY1 expression in children with pleuropulmonary blastoma (PPB), a rare pediatric lung tumor arising during fetal development and associated with DICER1 mutations. No evidence for a potential genetic interplay between murine Dicer and Yy1 genes during lung morphogenesis was observed. However, the cystic lung phenotype resulting from the epithelial inactivation of Dicer function mimics the Yy1 lung malformations with similar changes in Shh and Fgf10 expression. Together, our data demonstrate the crucial requirement for YY1 in lung morphogenesis and identify Yy1 mutant mice as a potential model for studying the genetic basis of PPB.

Gestational morphogenesis of the uterine epithelium of the gummy shark (Mustelus antarcticus).

Developing embryos of the non-placental, viviparous gummy shark (Mustelus antarcticus) are supplied with yolk from external and internal yolk sacs throughout the initial stages of gestation. Yolk supplies are exhausted by the 7th month of an 11-12 month gestation. During embryonic development, there is an approximate 800% gain in dry mass. These factors suggest nutrients are transferred from the mother to the young. The results of the present study provide some insights into how this is occurring. The uteri are paired and both are functional. Using both light and transmission electron microscopy, regions of the uterus were examined throughout maturation and gestation. The layers of the uterine wall throughout the entire length are similar to the uteri of other chondrichthyans previously examined. The uterine epithelium of the body of the uterus is smooth contoured, does not form villi, and undergoes cyclical and secretory changes throughout maturity and gestation. In immature uteri, the epithelium is simple columnar with minimal periodic acid-Schiff-positive and Alcian blue-positive secretory vesicles. In mature uteri, the epithelium is highly stratified with cuboidal cells containing numerous Alcian blue-positive and periodic acid-Schiff-positive vesicles. With pregnancy, prominent changes include a reduction in the number of cell layers, a reduction in cell size, a reduction in the connective tissue intervening between epithelium and blood vessel endothelium, and an increase in blood vessel number and size, so that at term, the uterine compartment consists of a single layer of squamous cells immediately underlain by sinusoidal-like blood vessels. These features along with a small number of secretory vesicles, dilated intercellular spaces, and clear transport vesicles suggest the transepithelial transfer of water and minerals from the maternal to embryonic environment, supplemented by minimal uterine secretions. This is defined as minimal histotrophy and this article represents the first detailed description of this reproductive mode.

Annexins as cell-type-specific markers in the developing chicken chorionallantoic membrane.

Between day E8 and E12 of embryonic development, the chicken chorioallantoic membrane (CAM) undergoes massive structural rearrangement enabling calcium-uptake from the eggshell to supply the growing embryo. However, the contribution of the various cell types of the chorionic epithelium including the capillary covering (CC) cells, villus cavity (VC) cells, endothelial-like cells, and basal cells to this developmental program is largely unknown. In order to obtain markers for the different cell types in the chorionic epithelium, we determined the expression patterns of various calcium-binding annexins in the developing chicken CAM. By reverse transcription/polymerase chain reaction with primers deduced from nucleotide sequences available in various databases, the presence of annexin (anx)-1, anx-2, anx-5, and anx-6 was demonstrated at days E8 and E12. Quantitative immunoblotting with novel antibodies raised against the recombinant proteins revealed that anx-1 and anx-5 were significantly up-regulated at day E12, whereas anx-2 and anx-6 expression remained almost unchanged in comparison to levels at day E8. Immunohistochemistry of paraffin-embedded sections of E12 CAM revealed anx-1 in CC cells and VC cells. Anx-2 was localized in capillaries in the chorionic epithelium and in basal cells of the allantoic epithelium, whereas anx-6 was detected in basal cells or endothelial-like cells of the chorionic epithelium and in the media of larger vessels in the mesenchyme. A 2-day exposure of the CAM to a tumor cell spheroid resulted in strong proliferation of anx-1-expressing CC cells suggesting that these cells participate in the embryonic response to experimental intervention. Thus, annexins exhibit complementary expression patterns and represent appropriate cell markers for the further characterization of CAM development and the interpretation of results obtained when using CAM as an experimental model.

Members of the Plag gene family are expressed in complementary and overlapping regions in the developing murine nervous system.

In the developing nervous system, cell fate specification and proliferation are tightly coupled events, ensuring the coordinated generation of the appropriate numbers and correct types of neuronal and glial cells. While it has become clear that tumor suppressor genes and oncogenes are key regulators of cell division in tumor cells, their role in normal cellular and developmental processes is less well understood. Here we present a comparative analysis of the expression profiles of the three members of the pleiomorphic adenoma gene (Plag) family, which encode zinc finger transcription factors previously characterized as tumor suppressors (Zac1) or oncogenes (Plag1, Plag-l2). We focused our analysis on the developing nervous system of mouse where we found that the Plag genes were expressed in both unique and overlapping patterns in the central and peripheral nervous systems, and in olfactory and neuroendocrine lineages. Based on their patterns of expression, we suggest that members of the Plag gene family might control cell fate and proliferation decisions in the developing nervous system and propose that deciphering these functions will help to explain why their inappropriate inactivation/activation leads to tumor formation.

Hmx2 and Hmx3 homeobox genes direct development of the murine inner ear and hypothalamus and can be functionally replaced by Drosophila Hmx.

The Hmx homeobox gene family appears to play a conserved role in CNS development in all animal species examined, and in higher vertebrates has an additional role in sensory organ development. Here, we show that murine Hmx2 and Hmx3 have both overlapping and distinct functions in the development of the inner ear's vestibular system, whereas their functions in the hypothalamic/pituitary axis of the CNS appear to be interchangeable. As in analogous knockin studies of Otx and En function, Drosophila Hmx can rescue conserved functions in the murine CNS. However, in contrast to Otx and En, Drosophila Hmx also rescues significant vertebrate-specific functions outside the CNS. Our work suggests that the evolution of the vertebrate inner ear may have involved (1) the redeployment of ancient Hmx activities to regulate the cell proliferation of structural components and (2) the acquisition of additional, vertebrate-specific Hmx activities to regulate the sensory epithelia.

Expression patterns of nectins and afadin during epithelial remodeling in the mouse embryo.

Cell-cell adhesion plays key roles in tissue morphogenesis and organogenesis. Nectins are Ca2+-independent immunoglobulin-like cell adhesion molecules connected to the actin cytoskeleton through afadin. Nectins play roles in a variety of cell-cell junctions in cooperation with or independently of cadherins. Here, we examined the cellular localization of nectins and afadin throughout primitive streak, neural plate, and early organogenesis stages of mouse development. Nectin and afadin localization coincided with a honeycomb-shaped meshwork of actin filaments at adherens junctions of polarized epithelia, including neuroepithelium, epithelial somites, and facial primordia. As organogenesis progressed, nectin-2 expression was maintained in general columnar epithelia, whereas nectin-1 and -3 became highly concentrated at sites of neural morphogenesis. Moreover, nectin-1 was highly expressed in keratinocytes of the skin, developing hair follicles, and epithelium of developing teeth. These results suggest that nectins and afadin are involved in dynamic epithelial remodeling during mouse development.

Identification of a novel glial cell line-derived neurotrophic factor-inducible gene required for renal branching morphogenesis.

In the developing kidney, activation of the rearrangement during transfection tyrosine kinase by glial cell line-derived neurotrophic factor (GDNF) is required for normal branching of the ureteric bud epithelium [corrected]. By differential display analysis we identified a novel GDNF-inducible gene (named GZF1) with a BTB/POZ (broad complex, tramtrack, and bric-a-brac)/(poxvirus and zinc finger) domain and 10 tandemly repeated zinc finger motifs. The up-regulation of the GZF1 gene showed two peaks at 1 h and 24-48 h after GDNF stimulation by Northern blotting. The late induction was also found at protein levels by Western blotting with anti-GZF1 antibody. As observed for other proteins with the BTB/POZ domain, the GZF1 protein had strong transcriptional repressive activity. Intriguingly, its expression was detected at high levels in branching ureteric buds and collecting ducts of mouse metanephric kidney in which RET was also expressed. Antisense phosphorothioated oligodeoxynucleotides of the GZF1 gene markedly impaired the ureteric bud branching in the metanephric organ culture, suggesting that the induction of GZF1 expression via the GDNF/RET signaling system is required for renal branching morphogenesis.

A search for tyrosine kinase receptors expressed in the rat embryonic pancreas.

It is known that during embryonic life, interactions between the pancreatic epithelium and its surrounding mesenchyme are important for proper development of the pancreas. These interactions are thought to be mediated by soluble factors, which could be, as in other tissues, the ligands of tyrosine kinase receptors. In this study, we screened for tyrosine kinase receptors expressed in pancreata of 13-day-old embryonic rats. Using a polymerase chain reaction-based approach that exploits sequence similarities within the catalytic kinase domains of these receptors, we identified 30 different tyrosine kinase receptors. The same approach was then used on cDNA prepared from fractions enriched in epithelium or in mesenchyme. Receptors for factors such as platelet derived growth factors were found to be expressed both in the epithelial and the mesenchymal fractions. Receptors for stem cell factor, for epidermal growth factor family members were mainly found in the epithelial fraction. The profile of expression of receptor tyrosine kinases in the embryonic pancreas was finally compared to the one found in other tissues and cell types, such as kidney, brain or INS-1 cells. Platelet derived growth factor receptors and ErbB2 were found to be enriched in the embryonic pancreas when compared with other tissues. It will now be possible to test the effects of the ligands of the different receptors we have cloned, on the differentiation and growth of the pancreas.

A novel mammalian wnt gene, WNT8B, shows brain-restricted expression in early development, with sharply delimited expression boundaries in the developing forebrain.

Our current knowledge of mammalian forebrain development is meagre. The comparatively few relevant anatomical landmarks are, however, being supplemented by gene expression studies which are able to identify subsets of anatomical structures. We previously described cloning, subchromosomal localization and preliminary structural characterization of the human WNT8B gene, the first mammalian Wnt8b gene to be reported. Wnt genes encode intercellular signalling molecules which play a variety of critical roles in early development, including, in several cases, a presumed role in brain development. In the current report we present the full-length cDNA sequence and genomic organization of the human Wnt8b gene and report studies of expression of the Wnt8b gene in human and mouse embryos. The human and mouse expression patterns appeared identical and were restricted to the developing brain, with the great majority of expression being found in the developing forebrain. In the latter case expression was confined to the germinative neuroepithelium of three sharply delimited regions: the dorsomedial wall of the telencephalic ventricles (which includes the developing hippocampus), a discrete region of the dorsal thalamus and the mammillary and retromammillary regions of the posterior hypothalamus. Expression in the developing hippocampus may suggest a role for WNT8B in patterning of this region and subchromosomal localization of the human gene to 10q24 may suggest it as a candidate gene for partial epilepsy in families where the disease has been linked to markers in this region.

Mash1 and neurogenin1 expression patterns define complementary domains of neuroepithelium in the developing CNS and are correlated with regions expressing notch ligands.

Genetic studies in Drosophila and in vertebrates have implicated basic helix-loop-helix (bHLH) genes in neuronal fate determination and cell type specification. We have compared directly the expression of Mash1 and neurogenin1 (ngn1), two bHLH genes that are expressed specifically at early stages of neurogenesis. In the PNS these genes are expressed in complementary autonomic and sensory lineages. In the CNS in situ hybridization to serial sections and double-labeling experiments indicate that Mash1 and ngn1 are expressed in adjacent and nonoverlapping regions of the neuroepithelium that correspond to future functionally distinct areas of the brain. We also showed that in the PNS several other bHLH genes exhibit similar lineal restriction, as do ngn1 and Mash1, suggesting that complementary cascades of bHLH factors are involved in PNS development. Finally, we found that there is a close association between expression of ngn1 and Mash1 and that of two Notch ligands. These observations suggest a basic plan for vertebrate neurogenesis whereby regionalization of the neuroepithelium is followed by activation of a relatively small number of bHLH genes, which are used repeatedly in complementary domains to promote neural determination and differentiation.

Bronchial ligation enhances murine fetal lung development in whole-organ culture.

Evidence exists from both congenital anomalies and animal models that normal fetal lung development is dependent on maintenance of fluid pressure within the developing "airways." Fetal tracheostomy, allowing free egress of airway fluids, results in lung hypoplasia, indicating that some airway distending pressure is required for normal lung development to occur. In contrast, fetal tracheal ligation, which increases fetal airway pressure, reverses lung hypoplasia in animal models. The authors' experiments test the hypothesis that large airway obstruction accelerates the development of murine lungs in vitro in whole-organ culture. Fetuses from time-dated pregnant CD-1 mice at day 14 of gestation were removed (term, 20 days), and the lungs were excised. The left bronchus of each lung was ligated (n = 26), after which the left lung was isolated and cultured at 37 degrees C (95% air, 5% CO2) in BGJb media supplemented with vitamin C and antibiotics. Some fetal lungs were cultured under similar conditions without bronchial ligation (n = 11). After 7 days in culture, the lungs were taken for various analyses. The lungs were fixed in either formaldehyde and processed for paraffin embedding for light microscopic evaluation and morphometric data collection, or were freshly minced and aliquots taken for total protein and DNA content. Several more ligated and unligated lungs were processed for ultrastructural analysis. Morphometric analysis on transverse sections of lungs showed significant differences in the lung tissue size, thickness, epithelial cell height, luminal areas, perimeters, and total number of airspaces (airway + primordial alveolar airspaces). It was evident that bronchial ligation promoted lung development. The ligated lungs displayed thinning of the primordial alveolar walls with cuboidal epithelial cells. The total number of airspaces per field was lower for better developed ligated lungs because of the increased area of airspaces compared with that of the unligated lungs. The dorsoventral tissue thickness (in micrometers) of the ligated lungs was significantly greater than that of the unligated lungs (124.1 +/- 7.0 v 89.6 +/- 8.0); the average outer perimeter of the primordial alveolar airspaces was greater for ligated lungs (404.56 +/- 19.0 microns v 256.85 +/- 17.0 microns). Similarly, the luminal diameter of the spaces of ligated lungs was almost double that of the unligated lungs (38.0 +/- 2.0 microns v 20.3 +/- 2.0 microns), as was the luminal surface area. The morphometric data, which suggest enhanced maturation of the ligated lungs, are supported by results of ultrastructural studies. Ligated lungs had significantly more lamellar bodies. Although total protein and DNA content were greater among the ligated lungs, the protein/DNA ratios did not differ among the groups. The intraluminal pressure (airway pressure) of ligated lungs was 2.9 mm Hg and 3.1 mm Hg at 2 and 4 days in organ culture; the respective pressures for unligated lungs were 1.0 mm Hg and 0.8 mm Hg. These data support the hypothesis that mechanical distending pressure resulting from airway obstruction not only improves pulmonary architecture but also accelerates lung development in vitro. Although these effects have been seen in in vivo models, this is the first proposed in vitro organ culture model. This model may prove to be a powerful tool for the study of molecular mechanisms of mammalian lung development with respect to mechanical and chemical (cytokines, hormones) stimuli.

Effect of hyperthermia on rat fetus palate epithelium / Efeito da hipertemia no epitélio palatino fetal do rato

Maternal hyperthermia during gestation induces delayed cellular growth and differentation in fetal rat palate epithelium, with increased nuclear, cytoplasmic and cellular volumes, increased epithelial thickness, decreased keratin and cellular numerical density

Development of sensory innervation in chick skin: comparison of nerve fibre and chondroitin sulphate distributions in vivo and in vitro.

In bird skin, nerve fibres develop in the dermis but do not enter the epidermis. In co-cultures of 7-day-old chick embryo dorsal root ganglia and epidermis, the neurites also avoid the epidermis. Previous studies have shown that chondroitin sulphate proteoglycans may be involved. Chondroitin sulphate has therefore been visualized by immunocytochemistry, using the monoclonal antibody CS-56, both in vivo and in vitro using light and electron microscopy. Its distribution was compared to those of 2 other chondroitin sulphate epitopes and to that of the growing nerve fibres. In cultures of epidermis from 7-day-old embryonic chicks, immunoreactivity is found uniformly around the epidermal cells while at 7.5 days the distribution in dermis is heterogeneous, and particularly marked in feather buds. In vivo, chondroitin sulphate immunoreactivity is detected in the epidermis, on the basal lamina, on the surfaces of fibroblasts and along collagen fibrils. This localization is complementary to the distribution of cutaneous nerves. Chondroitin sulphate in the basal lamina could prevent innervation of the epidermis and the dermal heterogeneities could partly explain the nerve fibres surrounding the base of the feathers. Chondroitin sulphate could therefore be important for neural guidance in developing chick skin.

Serum-free culture and characterization of renal epithelial cells isolated from human fetal kidneys of varying gestational age.

Cell cultures were initiated from seven human fetal kidneys that varied in gestational age from 90 days to newborn. The growth medium utilized was a 1:1 mixture of Dulbecco's modified Eagle's and Ham's F12 supplemented with selenium (5 ng/ml), insulin (5 micrograms/ml), transferrin (5 micrograms/ml), hydrocortisone (36 ng/ml), triiodothyronine (4 pg/ml), and epidermal growth factor (10 ng/ml). For all the kidney isolates, initial cell attachment occurred within 12 h through multicell spheroids, and by 24 h a rapidly growing population of cells was obtained. Confluency was reached within 3 to 6 days. A combination of light microscopy, immunohistochemistry, and ultrastructural evaluation was utilized to characterize the resulting cultures as epithelial and homogeneous within each isolate and among the isolates. That is, regardless of gestational age of the fetal kidney used as starting material, an identical or highly similar population of cells was obtained. By light microscopy, the cultures were noted to form very few domes, the number being an indication of transport activity. However, ultrastructural examination revealed that the cells were noted to form domes composed of only a few cells or "micro-domes" that would not be visible by light microscopy. Within the micro-domes as well as other areas of the monolayer an apparent absence of tight junctions was noted by routine transmission electron microscopy. However, by freeze fracture analysis cells were shown to possess sealing strands, the structural component of tight junctions. It is postulated that the tight junctions of fetal epithelial cells are structurally altered as compared to tight junctions in adult renal epithelial cell cultures.

Transplantation of facial processes for repair of induced branchial arch syndromes.

A technique has been developed by which facial processes with adequately migrated neural crest tissue can be cultured and transplanted to embryos with induced craniofacial malformations. Culturing was carried out in the anterior eye chambers of adult rats or as whole embryo cultures in glass vials (n = 71). Facial processes transplanted to the anterior eye chamber differentiated to cartilage, bone, and mesenchymal tissues. It was possible to keep the embryos alive in whole tissue culture for up to 24 hours. The addition of epidermal growth factor to the culture medium resulted in accelerated growth of epithelium on the surface of the facial processes. Facial processes were accepted when transplanted to either normal or etretinate treated embryos. Epithelium covered the transplanted facial processes, neural crest tissue was seen in the centre, and capillaries were in close contact to the base of each one.

Secretory differentiation and cell type identification of a human fetal bronchial epithelial cell line (HFBE).

A human fetal bronchial epithelial cell line (HFBE) grew in an undifferentiated pattern under conventional culture conditions. Despite a somewhat fibroblastic shape the cells maintained immunoreactivity to cytokeratin, carcinoembryonic antigen and epithelial membrane antigen. When grown on a collagen gel in a growth-hormone-supplemented medium, their spindle shape became more conspicuous. With an additional supplement of vitamin A (6 micrograms/ml), most of the cells underwent differentiation by producing many bright inclusion bodies which proved to be strongly positive with periodic acid-Schiff and weakly positive with alcian blue staining. Electron microscopy revealed a well-developed rough endoplasmic reticulum, an enlarged Golgi apparatus and many highly electron-dense secretory granules resembling those of Clara cells. Biochemical analysis demonstrated that HFBE cells cultured on collagen gel with vitamin A secreted hyaluronic acid and neutral glycoproteins containing mainly N-linked glycoproteins whose glycans were of a complex type. A monoclonal antibody (SEC-41) generated against the neutral glycoproteins detected a glycoprotein of approximately 52 kDa in the spent culture medium of differentiated HFBE cells. This antibody also reacted with the intracytoplasmic secretory granules in these cells. When tested on frozen sections of lung tissue, the immunohistochemical reactivity of the SEC-41 antibody was confined to Clara cells, some type II pneumocytes in the adult lung, and respiratory epithelial cells in the fetal lung. Moreover, this antibody could detect secretory glycoprotein in broncho-alveolar lavages from two patients. This paper clearly demonstrates that cells derived from human fetal bronchial epithelium can be cultivated in an undifferentiated precursor state and, under appropriate culture conditions, can be stimulated to undergo differentiation into a Clara cell type.

Induction and characterization of secretory differentiation in human fetal bronchial epithelial cell line (HFBE) cultured on collagen gel in growth hormone and vitamin A-supplemented medium.

A type of secretory differentiation was induced and characterized in a human fetal bronchial epithelial cell line (HFBE), which was grown on a collagen substratum in a basal differentiative medium (BDM) containing growth hormones and with supplementation of various concentrations of vitamin A (VA). HFBE cells grown on a collagen gel in BDM with or without VA assumed a spindle shape with thick cytoplasm arranged in strands running parallel to each other. Under a phase-contrast microscope, cells cultured in the absence of VA possessed a small number of bright inclusion bodies, which proved to be positive to PAS and almost negative to alcian-blue (AB) staining. Electron microscopy revealed well-developed rough endoplasmic reticulum (rER), enlarged Golgi apparatus and a small number of high-density granules resembling serous or Clara cell granules. HFBE cells treated with VA at levels higher than 6 mu/ml showed a remarkable increase of the secretory granules and contained amorphous material in the rER. Addition of a low concentration of VA (6 ng/ml) only stimulated the growth of HFBE cells. In contrast, higher concentrations of VA significantly inhibited the growth and 3H-thymidine incorporation into DNA in a dose-dependent manner. HFBE cells cultured on collagen gel with VA secreted products with 2 different molecular weights into the medium. A high molecular weight-product, consisting of void volume fractions from a Bio-gel A 15-m column, was identified as hyaluronic acid based on the results obtained from the DEAE-ion exchange chromatography and specific enzymatic digestion. A low molecular weight-product fractionated on the A 15-m was tentatively identified as mainly neutral glycoproteins containing N-linked glycans. While the secretion of hyaluronic acid was inhibited by VA in a dose-dependent manner, the secretion of the neural glycoproteins was most enhanced by VA in the range from the physiological concentration of 600 ng/ml to 6 micrograms/ml. These biochemical data on the secretory products, together with the morphological findings, demonstrate that the HFBE cell line serves as a new model for investigating the cellular differentiation of human lung epithelium.

Secretory differentiation of human fetal bronchial epithelial cells in culture. A study by histochemistry and electron microscopy.

Human fetal bronchial epithelial (HFBE) cells at 6-8 passages were cultivated on a collagen gel for 10 days. A basal differentiative medium (BDM), consisting of RPMI 1640 supplemented with hormones and growth factors, was employed. Histochemistry, scanning electron microscopy and transmission electron microscopy revealed that HFBE cells developed secretory granules when cultivated on collagen gel in BDM. They were electron-dense and stained positive for PAS but negative for alcian blue. On additional treatment with 8 micrograms/ml vitamin A (VA), the number of secretory granules was increased. Moreover, the HFBE cells lost their surface microvilli, and dilation of rough endoplasmic reticulum was more marked than in culture without VA.