Ventral Epithelium Research Articles

Purine and pyrimidine nucleotide-sensitive phosphoinositidase C in ampulla from frog semicircular canal.

A microassay was developed to screen the abilities of ATP analogues to stimulate phosphoinositidase C in single ventral regions (including dark cells and sensory cells) of ampullas microdissected from posterior vertical semicircular canals of Rana ridibundo and labeled with myo-[3H]inositol. ATP induced a dose-dependent and saturable increase of total [3H]linositol phosphate production accompanied by an equivalent decrease in the [3H]phosphoinositide pool. The rank order of analogues revealing agonistic potencies for phosphoinositidase C activation was as follows: uridine 5'-triphosphate > or = adenosine 5'-O-[3-thiophosphate] tetralithium > adenosine 5'-O-[2-thiodiphosphate] trilithium > or = ATP > or = ADP = inosine 5'-triphosphate > or = guanosine 5'-triphosphate > or = 2-methylthio-adenosine 5'-triphosphate tetrasodium > or = 2'-desoxy-thymidine 5'-triphosphate > or = cytidine 5'-triphosphate = (alpha, beta)-methyl ATP > AMP, whereas adenosine 3',5'-cyclic monophosphate and adenosine were almost devoid of activity. For antagonists, 1,4'-diisothiocyanostilbene-2, 2'-disulfonic acid was far more active than suramin for competitive inhibition of ATP-induced enzyme stimulation, whereas reactive blue 2 acted as a noncompetitive inhibitor. Results indicate that the putative P2 receptors triggering phosphoinositidase C activation in ventral ampullary epithelium from frog semicircular canal exhibit mainly the functional properties of P2Y and P2U receptors.

Distinct spatial and temporal functions for PS integrins during Drosophila wing morphogenesis.

At the onset of pupariation in the Drosophila wing, the PS1 and PS2 integrins are expressed preferentially on the dorsal and ventral wing epithelia, respectively. Clonal analysis experiments have indicated that integrins are required to maintain the tight association of the wing surfaces. Surprisingly, we find that even in clones of cells lacking integrins the wing layers become apposed early in metamorphosis. However, following the normal period of wing separation, large integrin mutant clones do not become re-apposed in the pupa, and integrins are not organized in basal plaques in cells opposite a mutant clone. Paradoxically, our experiments indicate that at least one integrin function requires different integrins on the dorsal and ventral wing surfaces, however in some cases both alphaPS subunits can function to some degree on each wing surface. Finally, overexpression of an alphaPS subunit throughout the wing leads to a dominant wing blister phenotype, and the critical period for this phenotype is the beginning of pupariation. These data indicate that integrin requirements in wing morphogenesis can be separated into early (prepupal) and late (pupal) functions. The late function seems to reflect the traditional view of integrins as cell-matrix adhesion proteins. The early requirement, which probably requires dorsoventral segregation of PS1 and PS2, suggests functions for PS1 and PS2 in signaling events that regulate morphogenesis.

Role of the PS integrins in Drosophila development.

The PS1 and PS2 integrins of Drosophila are heterodimers of alphaPS1betaPS and alphaPS2betaPS subunits, respectively, with very strong structural similarity to vertebrate integrins. Cell transfection experiments indicate that the PS integrins are receptors for extracellular matrix components and are functionally analogous to vertebrate integrins. Matrix ligands from Drosophila tissues have been identified for PS1 and PS2 integrins, using transformed cells and a cell-spreading assay. Mutations in all three subunit genes have been identified, and the phenotypes of mutants indicate that PS integrins are required for the proper morphogenesis of a number of embryonic tissues. Using methods to produce genetic mosaics and transformation of integrin transgenes into whole animals, integrin functions in adult morphogenesis also have been examined. In the pupal wing, integrins are critically required to maintain the connection between dorsal and ventral epithelia, and recent results suggest that in early pupal development, the integrins are acting as specific receptors, as opposed to a non-specific cell-matrix glue.

Cell Type- and Differentiation Stage-Dependent Expression of PML Domains in Rat, Detected by Monoclonal Antibody HIS55

Using mouse monoclonal antibody (mAb) HIS55, we identified a nuclear antigen (ag) that exhibited a staining pattern of discrete foci. Such foci could be detected in cells of many mammalian species. These nuclear foci were not associated with nuclear membrane, nucleoli, or mitotic chromosomes. In isolated rat liver nuclei, HeLa cells, and normal human and rat lymph nodes, staining of HIS55 colocalized with that of 5E10. 5E10 recognizes PML nuclear domains, multimolecular complexes of unknown function containing the product of PML gene and at least two other components. HIS55 foci were expressed widely in many tissues but the expression level varied in a cell type-specific manner, with the number of HIS55 nuclear foci ranging from 0 (as in neurons) to over 100 (as in megakaryocytes) and the size ranging from fine (as in cortical thymocytes) to very large (as in urethra epithelium). HIS55 ag expression level also varied among cells of the same lineage, as observed in embryonic development of rat and in the hemopoietic system of adult rat. The expression level of HIS55 loci roughly correlated with the overall rate of protein synthesis of cells, supporting a role of PML domains as transcription regulatory units. The expression of HIS55 foci, however, did not correlate with the growth index of cell populations. Our observations on normal tissues agreed with the hypothesis that PML domain expression is regulated by external, possibly site-dependent factors. We further supported this by demonstrating that PML domains in rat ventral prostate epithelia were upregulated upon castration.

Postembryonic development of the ovary in the penicillate diplopod, Eudigraphis nigricans (Miyosi) (Diplopoda, Penicillata)

Postembryonic development of the ovary through the larval stages was studied in a penicillate diplopod, Eudigraphis nigricans. In the first instar larva a single young cell cluster, consisting of about 20 spherical gonial cells and some smaller interstitial cells, exists beneath the alimentary canal in the third body segment. The gonadal epithelium encompasses the upper surface of this young cell cluster by the end of the first instar. The epithelium then extends forward and backward to form a single long sac-like gonad, leaving the young cell cluster on the center of the gonadal floor as a mound-shaped germarium. In an early second-instar larva, very early previtellogenic oocytes accompanied by some interstitial cells appear in the front and rear surfaces of the ovarian germarium. During the period from the third through the seventh (the last) larval instar, some cell clusters containing several previtellogenic oocytes and interstitial cells successively separate forward and backward from the germarium to form a series of paired patch-shaped vitellarial areas on the extending ventral ovarian epithelium. In each vitellarial area, some of the interstitial cells surround the oocytes to form the follicles. In the seventh instar, the ovarian lumen is extremely expanded, and the late previtellogenic oocytes in the vitellarial areas encroach upward into the ovarian lumen. These oocytes floating in the ovarian lumen are still connected with their own vitellarial areas by partial extensions of their follicles. Some phylogenetic implications of the basic characteristics in structure and postembryonic development of the ovary are discussed. © 1995 Wiley-Liss, Inc.

Spatiotemporal Immunolocalization of Stage-Spacific Embryonic Antigen-1 and Related Antigens in the Developing Trachea and Lung of Fetal Hamsters.

We examined the immunolocalization of stage-specific embryonic antigen-1 (SSEA-1) and related antigens in the developing trachea and lungs of fetal hamsters both in vitro and in vitro. On gestational days 10-16 (the day ot birth), fetal tracheas and lungs were fixed in phosphate-buffered paraform-aldehyde and frozen. Frozen sections were incubated with monoclonal antibodies against SSEA-1 (Lex hapten), sialyl SSEA-1 carrying i antigen and fucosyl SSEA-1 (LeYhapten). Some sections were treated with FITC-labeled secondary antibody for observation by confocal laser microscopy, and others were treated with colloidal gold-labeled secondary antibody for ultrastructural observation. In the developing tracheas, preferential staining in the ventral and lateral tracheal epithelium appeared for all of the antibodies by day 15, and ultrastructural study demonstrated that the regional difference in staining occurred regardless of cell type. In the developing lungs, positive sialyl SSEA-1-i antigen immunostaining was seen in the terminal portion of the epithelium by day 14, and the staining disappeared during the postnatal period. An in vitro study using explant organ culture of gestational day-11 lung with trachea revealed similar immuno-localization patterns. In the cultured trachea, the regional difference in immuno-staining was also seen with development of cartilage and smooth muscle, and in the cultured lung, the staining intensity became stronger in the terminal epithelium than in larger airway epithelium.

Phylogenetic implications of structure of adult ovary and oogenesis in the penicillate diplopod, Eudigraphis nigricians (miyosi) (diplopoda: Myriapoda).

We describe some significant structures of the adult ovary in a Japanese penicillate diplopod, Eudigraphis nigricans, with respect to phylogenetic implications. The ovary is a long, saclike organ lying between the alimentary canal and the ventral nerve cord from the fourth through the ninth body segment. The ovarian wall consists of a thin ovarian epithelium and a sparse muscle covering. There are two types of oogenetic sites: a single, mound-shaped germarium sitting on the center of the ventral ovarian epithelium, and ∼ 10 pairs of patchlike vitellarial areas metamerically arranged anterior and posterior to the germarium. The germarium consists of oogonia, early previtellogenic oocytes, and some somatic interstitial cells. In contrast, the vitellarial areas are composed of more advanced oocytes, follicle cells surrounding the oocytes, and some interstitial cells, but no oogonia. A few larger previtellogenic oocytes rise up from each vitellarial area into the ovarian lumen. Each of these oocytes is still connected with its own vitellarial area by a partial extension of its follicle. Vitellogenesis takes place in these oocytes rising in the ovarian lumen. The ripe primary oocytes leave their follicles to be transported forward into the oviducts. Some phylogenetic implications of the basic characteristics in ovarian structure and oogenesis of E. nigricans are discussed. © 1994 Wiley-Liss, Inc.

Blistered: a gene required for vein/intervein formation in wings of Drosophila.

We have characterized the blistered (bs) locus phenotypically, genetically and developmentally using a set of new bs alleles. Mutant defects range from wings with ectopic veins and intervein blisters to completely ballooned wings where the distinction between vein and intervein is lost. Mosaic analyses show that severe bs alleles behave largely autonomously; homozygous patches having vein-like properties. Developmental analyses were undertaken using light and electron microscopy of wild-type and bs wings as well as confocal microscopy of phalloidin- and laminin-stained preparations. bs defects were first seen early in the prepupal period with the failure of apposition of dorsal and ventral wing epithelia. Correspondingly, during definitive vein/intervein differentiation in the pupal period (18-36 hours after puparium formation), the extent of dorsal/ventral reapposition is reduced in bs wings. Regions of the wing that fail to become apposed differentiate properties of vein cells; i.e. become constricted apically and acquire a laminin-containing matrix basally. To further understand bs function, we examined genetic interactions between various bs alleles and mutants of two genes whose products have known functions in wing development. (i) rhomboid, a component of the EGF-R signalling pathway, is expressed in vein cells and is required for specification of vein cell fate. rhove mutations (lacking rhomboid in wings) suppress the excess vein formation and associated with bs. Conversely, rho expression in prepupal and pupal bs wings is expanded in the regions of increased vein formation. (ii) The integrin genes, inflated and myospheroid, are expressed in intervein cells and are required for adhesion between the dorsal and ventral wing surfaces. Loss of integrin function results in intervein blisters. Integrin mutants interact with bs mutants to increase the frequency of intervein blisters but do not typically enhance vein defects. Both developmental and genetic analyses suggest that the bs product is required during metamorphosis for the initiation of intervein development and the concomitant inhibition of vein development.

Towards a standard method to demonstrate adenylate cyclase activity at the electron microscopical level

The ventral epidermis of the frog Rana fuscigula is a typical tight epithelium which acts as a functional syncytium in the active transepithelial transport of sodium ions. Transport across this epithelium is regulated by cyclic adenosine monophosphate (cAMP). This study was undertaken to formulate an optimal protocol for the localization, within this epithelium, of adenylate cyclase; the enzyme involved in cAMP synthesis. The ventral epithelium of R. fuscigula was collagenase treated and processed using five different fixation/incubation protocols. The components of a basal incubating medium were modified by changing the localizing agent, adding adenylate cyclase stimulators and inhibitors of other enzymes. Control incubations undertaken included a) leaving the substrate out, b) prior heat inactivation of the enzyme, c) specific blockers and d) incubation for alkaline phosphatase as an alternative enzyme. The samples were then processed for electron microscopy. Localization of adenylate cyclase was best obtained, when fixing the tissue after incubation for 30 min at 37 degrees C. The medium that gave the best and most consistent localization contained magnesium chloride; as a required ion, theophylline, dithiothreitol, ouabain, levamisole; as enzyme inhibitors, forskolin; as a stimulator of adenylate cyclase, lead nitrate; as the capture agent and column purified adenylyl imidodiphosphate; as the substrate.

Androgen regulation of cell adhesion molecule gene expression in rat prostate during organ degeneration. C-CAM belongs to a class of androgen-repressed genes associated with enriched stem/amplifying cell population after prolonged castration.

Androgen is required for maintaining the differentiated state of prostatic epithelium, and that cell adhesion molecules play active roles in controlling the development of epithelial cells. However, little is known about the regulation of cell adhesion molecules during prostate development. In this study, we demonstrated that the expression of an epithelial cell adhesion molecule, C-CAM, in rat ventral prostatic epithelium are repressed by androgen. A similar regulatory pattern was also observed in the seminal vesicle but not in other androgen-dependent organs (the coagulating gland and the dorsolateral prostate), or other organs (the liver and kidney). These observations suggest that regulation of C-CAM expression by androgen is tissue-specific. Unlike the other androgen-repressed genes associated with apoptosis, which have transient expression patterns, the elevated level of C-CAM in the ventral prostate persisted for more than 30 days postcastration, suggesting that C-CAM belongs to a class of androgen-repressed genes which are not associated with apoptosis. Immunohistochemical study detected C-CAM on the apical surface of secretory epithelium in control rats; castration resulted in an altered localization of C-CAM expression from columnar epithelium to cuboidal basal epithelium. Taken together, the up-regulation and altered expression pattern of C-CAM in ventral prostatic epithelium by androgen deprivation may be associated with the enriched epithelial stem/amplifying cell population in degenerated ventral prostate. These observations suggest that C-CAM may play an active role in the process of prostatic epithelial differentiation.

Postimplantation expression patterns indicate a role for the mouse forkhead/HNF-3 α, β and γ genes in determination of the definitive endoderm, chordamesoderm and neuroectoderm

The HNF-3 alpha, beta and gamma genes constitute a family of transcription factors that are required for hepatocyte-specific gene expression of a number of genes, e.g. transthyretin, alpha-1 antitrypsin and tyrosine aminotransferase. These genes share a highly conserved DNA-binding domain first found in the Drosophila gene, forkhead, which is required for the normal patterning of the developing gut and central nervous system in Drosophila. In adult mouse tissues, transcripts from HNF-3 alpha and beta have been localised to the liver, intestine and lung, whereas HNF-3 gamma is found in the liver, intestine and testis. In light of the early developmental significance of forkhead in Drosophila, we have compared the patterns of expression of HNF-3 alpha, beta and gamma mRNAs during murine embryogenesis. We find that these genes are sequentially activated during development in the definitive endoderm. HNF-3 beta mRNA is expressed in the node at the anterior end of the primitive streak in all three germ layers and is the first gene of this family to be activated. Subsequently, HNF-3 alpha is transcribed in the primitive endoderm in the region of the invaginating foregut and HNF-3 gamma appears upon hindgut differentiation. These genes have different anterior boundaries of mRNA expression in the developing endoderm and transcripts are found in all endoderm-derived structures that differentiate posterior to this boundary. Therefore, we propose that these genes define regionalization within the definitive endoderm. Furthermore, differential mRNA expression of HNF-3 alpha and beta is detected in cells of the ventral neural epithelium, chordamesoderm and notochord. In the neural epithelium, expression of HNF-3 alpha and beta mRNA becomes localised to cells of the floor plate. We propose that, in addition to their characterised requirement for liver-specific gene expression, HNF-3 alpha and beta are required for mesoderm and neural axis formation. We also conclude that HNF-3 beta is the true orthologue of the Drosophila forkhead gene.

The distribution of PS integrins, laminin A and F-actin during key stages in Drosophila wing development.

We first summarize wing development during metamorphosis of Drosophila and identify four critical steps in the conversion of a folded single layered wing disc to a flat bilayered wing. Each step occurs twice, once during the 12 hour prepupal period and again during the 84 hour pupal period. (1) Apposition in which basal surfaces of dorsal and ventral epithelia come close together. (2) Adhesion in which basal junctions form between the apposed basal surfaces. (3) Expansion in which wing area increases as a result of cells flattening. (4) Separation in which dorsal and ventral epithelia are separated by a bulky extracellular matrix but remain connected by slender cytoplasmic processes containing the microtubules and microfilaments of the transalar cytoskeleton. Disc ultrastructure is correlated with the distribution of the beta chain of integrin, laminin A, and filamentous actin for each key stage of pupal development. Integrin and laminin exhibit a mutually exclusive distribution from the adhesion stage onwards. Integrin is present on the basal surface of intervein cells but not on vein cells whereas laminin A is absent from the basal surfaces of intervein cells but is present on vein cells. We conclude that laminin is not a ligand for integrin in this context. During apposition and adhesion stages integrin is broadly distributed over the basal and lateral surfaces of intervein cells but subsequently becomes localized to small basal foci. These foci correspond to basal contact zones between transalar processes. The distribution of filamentous actin is dynamic, changing from an apical distribution during hair morphogenesis to a basal distribution as the transalar cytoskeleton develops. Basal adherens-type junctions are first evident during the adhesion stage and become closely associated with the transalar cytoskeleton during the separation stage. Thus, basal junction formation occurs in two discrete steps; intercellular connections are established first and junction/cytoskeletal connections are formed about 20 hours later. These observations provide a basis for future investigations of integrin mediated adhesion in vivo.

Relationship of saccular ultrastructure to otolith growth in the teleost Oreochromis niloticus.

The sacculus of Oreochromis niloticus is anatomically separated from the utriculus and semicircular canals. The saccular wall is composed of the sensory epithelium, transitional epithelia, and squamous epithelium. Cellular granules are abundant in the sensory and transitional epithelia but scarce in the squamous epithelium. Over the dorsal side of the dorsal transitional epithelium there exists an oval patch of cells with distinctive microvilli. New finding is a shallow groove which extends from the anterior end of the sensory epithelium approximately halfway down along the ventral perimacular transitional epithelium. Small vesicles, which appear "empty" under transmission electron microscopy (TEM), are aggregated in the posterior region of the groove. These small vesicles are also present in both the sensory and transitional epithelia. A second kind of vesicle is comparatively large and appears filled with stainable contents. These vesicles are restricted to the sensory region. Both kinds of vesicles appear to be involved in apical secretion and possibly provide the otolithic membrane with fibers. The otolithic membrane is composed of a gelatinuous layer and subcupular meshwork. The meshwork appears to contribute to the formation of the otolith. The small empty vesicles appear to originate in sensory and transitional epithelial cells and may form the subcupular meshwork. The larger filled vesicles are derived predominantly from sensory cells in the sensory epithelium and appear to contribute to the gelatinuous layer of otoliths. © 1992 Wiley-Liss, Inc.

Mitotic activity and cell deletion in ventral prostate epithelium of intact and castrated oxytocin-treated rats.

Previous study has shown that oxytocin (OT) attenuated the postcastration regression of ventral prostate epithelium in rats. To decide if this effect is associated with stimulation of cell division or improvement of cell survival, metaphase index of secretory cells and frequency of apoptotic bodies in the epithelium, combined with stereological parameters, were determined in the present study. OT was injected subcutaneously in a dose of 0.25 IU/100 g/d during 5 postorchiectomal days and the same treatment was applied to intact rats. Only the prostate of castrated animals responded to OT. They had greater total volume of the epithelium, total number and metaphase index of secretory cells, but lower frequency of apoptotic bodies. These findings demonstrate the ability of OT to stimulate mitotic activity and to diminish mortality of secretory cells caused by orchiectomy.

Salinity tolerance and structure of external and internal gills in tadpoles of the crab-eating frog, Rana cancrivora

Salinity tolerance and histology of gills were studied in Rana cancrivora larvae. The tadpoles at the external gill stages (W stages 21-22) were able to survive in media containing up to 40% seawater, but died in water of higher salinity. Their external gills appear to have no critical role in adaptation to seawater. However, advanced tadpoles with internal gills (T-K stages I-XVIII) were able to tolerate 50% or higher seawater. In the internal gills, there are numerous mitochondria-rich cells (MR cells) scattered on the ventral and lateral epithelia of the gill arches and the gill tufts in both freshwater- and seawater-acclimated tadpoles. In freshwater-acclimated tadpoles there are three types of MR cell: (1) microplicated, (2) microvillous, and (3) apically vacuolated. In tadpoles acclimated to dilute seawater, the ratio of type-1 to type-2 cells is lower, although all three types of MR cell are present. In 60%-seawater-acclimated tadpoles, a few MR cells with a lumen and concave cytoplasm at the apical membrane (type 4) are present. The changes in MR cell morphology under ambient conditions of low or high salinity may reflect alterations in the physiological roles of the gills with regard to transport of ions.

Use of Horseradish Peroxidase Conjugated Lectins for Detection of Glycoconjugate Changes in Developing Lingual Epithelium of the Chick Embryo: (lectins//glycoconjugates/epithelium/tongue/chick embryo).

A battery of six different horseradish peroxidase-labelled lectins (SBA, DBA, PNA, WGA, ConA and LTA) was used to study the distribution of carbohydrate residues in glycoconjugates during the development of the chick embryo tongue anlage (7th-21st day of incubation) and in the tongue of 3 day-old chicken, at the dorsal and ventral epithelium. From the 7th to the 16th day β-D-galactose, D-galactose-(β1 → 3)-N-acetyl-D-galactosamine, D-mannose and β-N-acetyl-D-glucosamine show the same epithelial distribution at two lingual aspects. From the 17th day to hatching the epithelium of the dorsal and ventral surfaces is characterized by a different distribution of sugar residues. The functional adaptation of the epithelium, as far as the saccharidic moletie distribution is concerned, seems to be completed at the 21st day of incubation.

Characterization of cytokeratin patterns in the developing human tongue.

The characterization of cytokeratin (CK) in adult oral mucosa and developing teeth have been well documented in human. Cytokeratin distribution in developing oral mucosa has not yet been described. The aim of this study was to identify the expression of CK in human fetal tongue (week 10 to week 23) and to correlate the results with morphological maturation. Simple epithelial CK are expressed in all cell layers during the early stages, essentially in peridermal cells. From the 14th week, CK 18 is present only in the taste buds, making this polypeptide a reliable marker for this sensory organ. CK 4 and 13 are expressed from the 10th to the 23rd week by both ventral and dorsal lingual epithelia. Terminal differentiation keratins (CK 1, 2 and 10-11) can only be detected immunohistochemically at the 14th week in some cells on the external surface of some papillae. The number of these papillae and positive cells increase at the 19th and 23rd weeks. The terminal differentiation markers are expressed several weeks earlier than the formation of a well-distinguished keratinized layer.

Alternative modes of asexual reproduction inTrichoplax adhaerens (Placozoa)

Hollow swarmers are budded off at the dorsal surface ofTrichoplax and are covered by dorsal epithelium. Their inner cavity is lined with the flagellated cells of the ventral epithelium. There is no indication that the fiber cells included between the epithelia take any part either in morphologenesis or the separation of the bud from the mother animal. The early primordium forms in the interspace. A single layer of cells derived from both epithelia surrounds a cavity filled with granular matter that stains like proteins. The latter is used up during the floating phase of the swarmers that may last for a week. After settling at the bottom, the hollow sphere opens at one point. The concave ventral epithelium gradually flattens as more cells become incorporated in it. The latter form new flagella and flagellar pits. More frequently found than swarmers are small spherical forms that are unable to float and possess a distinct polarity. Their upper half is covered by dorsal epithelium and their lower half by ventral epithelium. Large fiber cells are in the center. Their site and mode of formation is unknown. Rarely observed are dorsal stolons whose bulbous end flattens upon touching the substrate. Since they are totally covered by the flat cells of the dorsal epithelium, they may have to undergo a transformation, like the hollow swarmers, to bring the ventral epithelium into contact with the substrate.

Ganglion formation from the otic placode and the otic crest in the chick embryo: Mitosis, migration, and the basal lamina

We have studied the morphogenesis of the cochleo-vestibular (CVG) and distal cranial ganglia in the early chick embryo (White Leghorn embryos). Light microscopy and immunocytochemical staining for fibronectin and laminin were used to trace the cellular contributions to these ganglia from the otic placode and otocyst. Serial semi-thin plastic sections (3-5 microns) stained with toluidine blue at each Hamburger-Hamilton stage (St.) from 10 to 21 were used. We were able to trace individual groups of cells derived from these epithelial structures into the anlagen of the CVG and the distal parts of cranial n. ganglia VII, IX, and X. For immunostaining, antisera were used to visualize the basal lamina in 15-microns cryostat sections from St. 14 to 21 embryos. Described here for the first time is the otic crest, a ridge of epithelium surrounding the placode. Cells migrate from the otic crest (St. 11 to 14) during the period when the otocyst is forming. These cells become continuous spatially with those derived from the epibranchial placodes and the presumptive ganglia of cranial nerves VII, IX, and X. Furthermore, rostral otic crest cells merge with neural crest cells, which appose the myelencephalon, and they join with the newly formed neuroblasts of the CVG, which migrate from the ventral epithelium of the otocyst at St. 14 to 21. This region of the epithelium forms the bulk of the CVG; it also has many more mitotic figures than the rest of the otocyst. Cells in the rostralmost CVG (vestibular part) are the first to complete their migration and send axons into both the medulla and incipient crista ampullaris. Immuno-staining for fibronectin and laminin shows that these two basal-lamina-associated glycoproteins appear in a continuous layer beneath the otic epithelium just prior to CVG migration. Thus there is no evidence that the migration is launched by a prior decomposition of the basal lamina. The cells migrating from the epithelium bridge the basal lamina with their leading processes while the trailing processes are withdrawing from the epithelium. These trailing processes must withdraw after the neuroblast migrates, since most of the neuroblasts undergo mitosis in subsequent stages. The migrating cells appear to push out of the epithelium by displacing immunostained fragments of the basal lamina ahead of their leading processes. This suggests that the exodus of cells is accompanied by forces within the epithelium itself. Whether this is generated by the migratory neuroblasts themselves or by other sources remains to be determined.

Organization of projections from olfactory epithelium to olfactory bulb in the frog, Rana pipiens

One hypothesis for the coding of olfactory quality is that regions of the olfactory epithelium are differentially sensitive to particular odor qualities and that this regional sensitivity is conveyed to the olfactory bulb in a topographic manner by the olfactory nerve. A corollary to this hypothesis is that there is a sufficiently orderly connection between the epithelium and the olfactory bulb to convey this topographical coding. Thus we examined topography in the projection from epithelium to bulb in the frog, which has been the subject of numerous electrophysiological studies but has not yet been examined using modern neuroanatomical techniques. The tracer WGA-HRP was applied to the ventral or to the dorsal olfactory epithelium, or both. Anterograde transport of label to the olfactory bulb was seen after as few as 2 days; label was still present in the bulb as long as 21 days postinjection. In cases where WGA-HRP was applied to the entire epithelium, there was dense anterograde labelling of the ipsilateral olfactory bulb. In addition, a small medial portion of the contralateral bulb was labelled. Injections limited to either the ventral or dorsal epithelium produced patterns of anterograde labelling in the glomerular layer of the olfactory bulb, which varied with the size and location of the injection. With very large injections in either the dorsal or ventral epithelium, label appeared to be evenly distributed in the glomerular layer. With smaller injections in the ventral epithelium, there was heavier labelling in the lateral than in the medial portions of the glomerular layer, although light labelling was found in all regions of the glomerular layer. In contrast, injection sites restricted to the dorsal epithelium produced more anterograde labelling in the medial than lateral portions of the glomerular layer. These patterns extended throughout the dorsal-ventral extent of the bulb. Within the limits of the anterograde tracing technique used, we were unable to detect any systematic relationship between the pattern of labelling in the glomerular layer and the medial-lateral or rostral-caudal location of the injection site in either the ventral or dorsal epithelium. We conclude that in the frog, as in other amphibia, there is only a limited degree of topographic order between the epithelium and the olfactory bulb.