Alterations of heart development in Xenopus laevis by galactoside-binding lectin or its sugar hapten inhibitor

The early heart anlagen of Xenopus laevis embryos were exposed to purified embryonic galactoside-binding lectin or its potent hapten inhibitor thiodigalactoside (TDG). Heart development was then studied using a variety of microscopical techniques. Conotruncal morphology and positioning with respect to the ventricle are altered in treated animals. In 34% of animals treated with lectin and 35% treated with TDG, the conotruncus leaves the ventricle from an abnormal location. Lectin or TDG treatments are also correlated with altered conotruncal shape, with the conotruncal regions showing greater radii of curvature compared to controls. Conotruncal myocyte differentiation is altered by the test treatments, with lack of development of organized myofibrillar arrays. Conotruncal cushion development is also affected. Changes occur in the shape and size of the primary conotruncal cushion, and alterations of outflow tract septation develop. Less maturation of ventricular myocytes is also observed in test animals. The results suggest that galactose-lectin interactions are important in heart development.     

Cerebellar connections in Xenopus laevis

Summary In the present study the cerebellar afferents in the clawed toad Xenopus laevis have been analysed with the horseradish peroxidase (HRP) technique. In addition, data on the efferent connections of the cerebellum could be gathered, based on the phenomenon of anterograde transport of HRP.Cerebellar afferents in Xenopus laevis appear to arise mainly in the vestibular nuclear complex, in a primordial inferior olive and in the spinal cord. Both primary (arising in the ipsilateral vestibular ganglion) and secondary vestibulocerebellar projections were found. A distinct crossed olivocerebellar projection to the molecular layer of the cerebellum was found. Two spinocerebellar pathways are present in Xenopus laevis, as in other anurans, viz. an ipsilateral dorsal spinocerebellar tract, presumably arising in dorsal root ganglion cells, and a larger ventral pathway, bilaterally arising in the spinal gray matter. The latter tract mainly originates in the ventrolateral and ventromedial spinal fields. Furthermore, a secondary trigeminocerebellar projection arising in the descending trigeminal nucleus, a cerebellar projection arising in the dorsal column nucleus, a small projection arising in a possible primordium of the mammalian nucleus prepositus hypoglossi, a raphecerebellar projection, and a small cerebellar projection originating in the ipsilateral mesencephalic tegmentum were demonstrated.Cerebellar efferents in Xenopus laevis are mainly aimed at the vestibular nuclear complex. A distinct ipsilateral cerebellovestibular projection present throughout the vestibular nuclear complex presumably arises in Purkyn cells, a smaller contralateral projection in the cerebellar nucleus. In addition, a small primordial brachium conjunctivum, projecting to the red nucleus, was noted.The basic pattern of cerebellar connections as suggested for terrestrial vertebrates (ten Donkelaar and Bangma 1984) is also found in the permanently aquatic anuran Xenopus laevis.     

Ontogeny of vasotocinergic and mesotocinergic systems in the brain of the South African clawed frog Xenopus laevis

For a better understanding of the development of neurotransmitter systems and of their putative functional significance during ontogenesis, the development of the vasotocin (AVT) and mesotocin (MST) systems in the brain of Xenopus laevis was studied by means of immunohistochemical techniques. Weakly immunoreactive fibers were already present at late embryonic stage 38 in the caudoventral part of the telencephalon and in the ventral part of the diencephalon. The earliest immunodetectable AVT and MST immunoreactive cell bodies were found in the developing preoptic area at late embryonic stage 43. At the end of the embryonic period (stage 45), AVT immunoreactive fibers have reached the future medial amygdala, the midbrain tegmentum, the median eminence and the neural lobe of the pituitary. When compared with AVT immunoreactive fibers, the development of MST fibers shows some temporal delay. During the premetamorphosis (stages 45–52), AVT immunoreactive cell bodies appear in the medial part of the suprachiasmatic nucleus, the dorsal infundibular region, and the midbrain tegmentum, whereas fibers can now be traced to the nucleus accumbens, the septum and the medial amygdala in the forebrain, to the midbrain tegmentum, the reticular formation, the raphe nuclei, and the solitary tract nucleus in the brainstem, and to the spinal cord. Further maturation of the AVT system during prometamorphosis (stages 53–58) includes the appearance of immunoreactive cell bodies in the lateral part of the suprachiasmatic nucleus, the ventral preoptic area, and the dorsal infundibular region. By the end of the metamorphosis (stage 65), the maturation of the AVT/MST systems reaches an almost adult-like pattern. It should be noted that in amphibians, in contrast to mammals, the early appearance of the AVT/MST systems, including their extensive extrahypothalamic component, suggests that the two neuropeptidergic systems may play a significant role during development.     

Co-localization of serotonin and GABA in neurons of the Xenopus laevis retina

Serotonin-synthesizing neurons in the retina of Xenopus laevis have been identified using anti-phenylalanine hydroxylase (PH) antibody which recognizes tryptophan 5-hydroxylase, the rate-limiting enzyme for serotonin synthesis. Double-labelling experiments, using anti-PH antibody and anti-serotonin antibody/5,7-dihydroxytryptamine (5,7-DHT) uptake, have shown that some serotonin-like immunoreactive/5,7-DHT-labelled neurons exhibit PH-like immunoreactivity (PH-LI) (serotonin-synthesizing neurons), but the others do not (serotonin-accumulating neurons). In the present study, triple-labelling experiments were performed using 5,7-DHT uptake and antibodies raised against GABA and PH, to determine the possible co-localization of -aminobutyric acid (GABA) in serotonin-synthesizing and/or -accumulating neurons in the Xenopus retina. All 5,7-DHT-labelled bipolar cells lacked PH-LI; all of them were immunoreactive to GABA. In contrast, all 5,7-DHT-labelled large amacrine cells exhibited PH-LI, but none of them expressed GABA-LI. Small amacrine cells labelled with 5,7-DHT but not PH-LI exhibited GABA-LI, whilst the small amacrine cells with PH-LI lacked GABA-LI. These observations indicate that GABA is co-localized in serotonin-accumulating amacrine and bipolar cells, whereas serotonin-synthesizing large and small amacrine cells do not contain GABA-LI.     

Microglia in tadpoles ofXenopus laevis: Normal distribution and the response to optic nerve injury

Summary We have studied the distribution of microglia in normalXenopus tadpoles and after an optic nerve lesion, using a monoclonal antibody (5F4) raised againstXenopus retinas of which the optic nerves had been cut 10 days previously. The antibody 5F4 selectively recognizes macrophages and microglia inXenopus. In normal animals microglia are sparsely but widely distributed throughout the retina, optic nerve, diencephalon and mesencephalon (other regions were not examined). After crush or cut of an optic nerve, or eye removal, there occurs an extensive microglial response along the affected optic pathway. Within 18 h an increase in the number of microglial cells in the optic tract and tectum can be detected. This response increases to peak at around 5 days after the lesion. At this time the nerve distal to the lesion contains many microglial cells; the entire optic tract is outlined by microglia, extended along the degenerating fibres; and the affected tectum shows a heavy concentration of microglia. This microglial response thereafter decreases and has mostly gone by 34 days. We conclude that the microglial response to optic nerve injury inXenopus tadpoles starts early, peaks just before the regenerating optic nerve axons enter the brain, and is much diminished by the time the retinotectal projection is re-established. The timing is such that the microglial response could play a major role in facilitating regeneration.     

The thymus and skin wound healing in Xenopus laevis adults

The capacity to heal wounds without scars is generally lost during the development in vertebrates. To explore the involvement of cells of the adaptive immune system in a scar-like tissue based repair, we studied the thymus in 15-month-old Xenopus after skin incisional wounding. After injury, the organ size significantly increased and marked changes in structure and TNF-α immunoreactivity were detected in the medullary microenvironment when the granulation tissue was present in the repair area. Most of the lymphocytes present in this wound connective tissue were found to be immunoreactive to specific T cell markers. Thymic mucocyte-like cells and epithelial cysts increased in number, the myoid cells acquired a faster turnover and associated in large clusters, blood vessels were dilated and corpuscles similar to mammalian Hassall's bodies were formed in medulla. A higher number of stronger medullary TNF-α immunoreactive cells, i.e., dendritic, epithelial, granular basophilic and myoid cells were also induced after wounding. With progression of healing the thymus gradually returned to histochemical patterns of controls. Our results suggest that during the scar-based skin repair of Xenopus adults the activity of the thymus may be stimulated and associated with the T lymphocyte infiltration observed into injured granulation tissue.     

Topographical relationship between neuronal nitric oxide synthase immunoreactivity and cyclic 3′,5′-guanosine monophosphate accumulation in the brain of the adult Xenopus laevis

Previous immunohistochemical staining procedures of the brain and pituitary in Xenopus laevis, using an antiserum against neuronal nitric oxide (NO) synthase (nNOS) and nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry, have revealed NOS activity in neurons and fibers in a number of brain areas, as well as in fibers in the pituitary. In the present study we have localized the target structures of the NOergic system in the Xenopus brain by visualizing the sites of NO-sensitive cyclic 3′,5′-guanosine monophosphate (cGMP) accumulation, according to a method for cGMP visualization in rat brain slices. Brain slices of unfixed Xenopus are incubated in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine and the NO donor sodium nitroprusside, followed by fixation and cryosectioning. Sections were then processed for immunohistochemistry using rabbit and sheep antisera against cGMP and a sheep antiserum against nNOS. Visualization of single and double labeling of cGMP immunoreactive and/or nNOS immunoreactive structures was performed with combined CY3/fluorescein isothiocyanate fluorescence microscopy. Following this procedure, we provide immunohistochemical evidence for the distribution of cGMP-accumulating neurons in the brain of adult Xenopus. In most brain areas, the distribution of nNOS and cGMP immunoreactive structures (neuron somata and fibers) is distinct and separate, for instance in the dorsal pallium, the lateral thalamic nuclei, the optic tectum, the locus coeruleus and the reticular formation. However, nNOS and cGMP immunoreactive structures are often found in the vicinity of each other, and in the optic tectum even in adjacent neuron fibers and somata. The present observations are in line with the presence of an NO-dependent soluble guanylate cyclase in distinct brain areas of Xenopus laevis, corroborating similar data in the mammalian brain. Further, our observations may add to the understanding of the anatomical connectivity pattern and functional relevance of the NOergic system in the amphibian brain.     

Retinal axons in Xenopus show different behaviour patterns on various glial substrates in vitro

Summary Several lines of evidence suggest that glial cells have major effects on neuronal pathfinding. We have examined in vitro whether the outgrowth pattern of Xenopus retinal fibres is influenced by the glial cells encountered as they grow to the optic tectum. Strips of retina were cultured on monolayers of glial cells from the diencephalon and from the rostral and caudal ends of the optic tectum. On glia from the caudal end of the tectum the growth of fibres from the nasal and temporal ends of the strips was different: temporal fibres were shorter and more fasciculated than nasal fibres. This difference was still discernible on glia isolated from the rostral end of the tectum, but to a lesser extent. On glia from the diencephalon there was no difference between nasal and temporal fibres.     

Application of lectin and B-lymphocyte-specific monoclonal antibodies for the demonstration of human microglia in formalin-fixed,paraffin-embedded brain tissue

Summary To evaluate the usefulness of microglial markers for routine neuropathological material, we studied formalin-fixed, paraffin-embedded human brain tissue with the immunoperoxidase method using the lectinRicinus communis agglutinin (RCA-1) and four monoclonal antibodies (LN-1, LN-2, LN-3, anti-HLA-DR/ alpha). RCA-1 stained resting microglia, but the staining intensity was mostly weak. LN-1 also stained resting microglia in paraffin sections first treated with protease. In contrast to LN-1, RCA-1 stained blood vessels heavily. LN-1 stained resting microglia more markedly than RCA-1 in brains fixed for a prolonged period of time. However, LN-1 recognized a small number of astrocytes in routine paraffin sections. LN-3 reactivity was detected on a few resting microglia, but was intensely expressed on large numbers of reactive microglia in many neurological diseases. Both LN-2 and anti-HLA-DR/alpha labelled microglia, but the reactions were inconsistent. This study suggests that the monoclonal antibodies LN-1 and LN-3 are useful for the demonstration of microglia in paraffin sections, and a combination of these antibodies and the antibody to glial fibrillary acidic protein is recommended in attempting to identify microglia.     

Two mannose-binding lectin homologues and an MBL-associated serine protease are expressed in the gut epithelia of the urochordate species Ciona intestinalis

The lectin complement pathway has important functions in vertebrate host defence and accumulating evidence of primordial complement components trace its emergence to invertebrate phyla. We introduce two putative mannose-binding lectin homologues (CioMBLs) from the urochordate species Ciona intestinalis. The CioMBLs display similarities with vertebrate MBLs and comprise a collagen-like region, α-helical coiled-coils and a carbohydrate recognition domain (CRD) with conserved residues involved in calcium and carbohydrate binding. Structural analysis revealed an oligomerization through interchain disulphide bridges between N-terminal cysteine residues and cysteines located between the neck region and the CRD. RT-PCR showed a tissue specific expression of CioMBL in the gut and by immunohistochemistry analysis we also demonstrated that CioMBL co-localize with an MBL-associated serine protease in the epithelia cells lining the stomach and intestine.In conclusion we present two urochordate MBLs and identify an associated serine protease, which support the concept of an evolutionary ancient origin of the lectin complement pathway.     

Expression of the human sodium/proton exchanger NHE-1 in Xenopus laevis oocytes enhances sodium/proton exchange activity and establishes sodium/lithium countertransport

We investigated whether the human sodium/proton (Na⁺/H⁺) exchanger isoform 1 (NHE-1) can mediate sodium/lithium (Na⁺/Li⁺) coutertransport. Using the Xenopus laevis oocyte expression system we determined amiloride-sensitive Li⁺ uptake, a measure of Na⁺/H⁺ exchange, in oocytes injected with water or NHE-1 cRNA. Amiloride-sensitive Li⁺ uptake was three-to tenfold enhanced over control in NHE-1 cRNA-injected cells and was selectively inhibited by 0.01 M HOE 694 [i.e. (3-methylsulphonyl-4-piperidinobenzoyl) guanidine methanesulphonate]. The endogenously present Na⁺/H⁺ exchanger was insensitive to HOE 694. After acidification of oocytes from pH 7.7 to 6.8, amiloride-sensitive Li⁺ uptake was four-to tenfold higher in NHE-1 cRNA-injected cells than in controls. Li⁺ efflux from control oocytes was independent of extracellular Na⁺, indicating that these cells expressed no measurable Na⁺/Li⁺ countertransport activity. In NHE-1 cRNA-injected oocytes, Li⁺ efflux was distinctly enhanced by extracellular Na⁺ ions. This Na⁺-dependent Li⁺ efflux was inhibited by ethylisopropylamiloride, phloretin and by cytosolic acidification. The data show that expression of the NHE-1 in X. laevis oocytes induces the expression of Na⁺/Li⁺ countertransport. The data confirm that Na⁺/H⁺ exchange and Na⁺/Li⁺ countertransport are mediated by the same transport system.     

Subunit stoichiometry of heterologously expressed G-protein activated inwardly rectifying potassium channels analysed by fluorescence intensity ratio measurement

The Xenopus laevis oocyte expression system offers the unique opportunity to heterologously express many proteins simultaneously and to control the expression level for every protein individually. By using the expression of fusion constructs of variants of the green fluorescence protein (eCFP, eGFP and eYFP) with GIRK1 and GIRK4 subunits and measuring the respective fluorescence intensity ratios (FIRs) of the expressed proteins by confocal laser scan microscopy, we were able to measure the amount of each of the individual subunits expressed. At equal amounts of injected RNAs encoding GIRK1 and GIRK4, we found that approximately 2.2 GIRK4 subunits per 1 GIRK1 subunit appeared at the surface of the oocyte, suggesting the coexistence of homooligomeric GIRK4 complexes with heterooligomeric GIRK1/GIRK4 complexes. Interestingly, when the ratio of injected RNA is increased in favour of GIRK1, the subunit stoichiometry changes accordingly until, at a RNA ratio of 25:1 (GIRK1/GIRK4), the subunit stoichiometry is shifted towards a protein complex with 3:1 stoichiometry (GIRK1/GIRK4). In parallel, the amount of GIRK1 protein appearing at the surface gets greatly reduced, supporting previous studies that showed that the GIRK1 subunit needs assembly with GIRK4 for surface localization. By using a genetically encoded marker for the endoplasmic reticulum (ER), we were able to show that the subunit stoichiometry in regions of the ER, which are located directly below the plasma membrane, closely resembles that observed directly at the surface. Generally, our study reveals that the subunit stoichiometry of GIRK1/GIRK4 channels in the Xenopus laevis oocyte expression system depends to a great extent on the molar ratio of the different RNAs injected.     

Characterization of lineage restricted forms of a Xenopus CD45 homologue

The leukocyte common antigen, also known as CD45, is a structurally heterogenous molecule ranging in molecular weight from 180 to 220 kDa. CD45 belongs to a family of high molecular weight, cell surface glycoproteins expressed on all hematopoietic lineages with the exception of mature erythrocytes. In higher vertebrates, the highly conserved cytoplasmic domain of CD45 exhibits protein tyrosine phosphatase activity and has been implicated in lymphocyte activation through dephosphorylation of critical tyrosine residues on substrates associated with signal transduction pathways. The monoclonal antibody CL21 recognizes a high molecular weight determinant expressed on the surface of Xenopus leukocytes which was postulated to be a CD45 homologue. In order to determine if lymphocyte subpopulations expressed different molecular weight variants, splenic B cells were identified and isolated on the basis of surface IgM and the CL21 determinant expressed by these cells was compared to the determinant expressed by thymocytes. Immunoprecipitation revealed that IgM+B cells expressed a 220 kDa molecular weight variant whereas thymocytes and IgM-cells expressed a 180 kDa variant. Bone marrow myeloid cells, isolated on the basis of light scatter properties, expressed a determinant which ranged from 150 to 160 kDa. Dephosphorylation experiments utilizing p-nitrophenyl phosphate, ³²P-labeIed Raytide [tyr(P)], or Kemptide [ser(P)] as substrates demonstrated that immunoprecipitated CL21 antigen exhibited tyrosine specific phosphatase activity which was inhibited by sodium orthovanadate. Thus, data based on the presence of enzymatic activity and lineage restricted molecular weight variants support the hypothesis that the CL21 determinant is the amphibian homologue of mammalian CD45, and suggest that both structural and functional elements of CD45 have been conserved during vertebrate evolution.     

Detection of chloroplast DNA by using fluorescent monoclonal anti-bromodeoxyuridine antibody and analysis of its fate during zygote formation in Chlamydomonas reinhardtii

Summary A monoclonal anti-bromodeoxyuridine antibody conjugated to fluorescein was used to detect the chloroplast nucleoids after specific incorporation of bromodeoxyuridine (BUdR) into the chloroplast DNA of Chlamydomonas reinhardtii. The incorporation of BUdR was enhanced by simultaneous treatment with fluorodeoxyuridine (FUdR). The method was applied to analyze the fate of chloroplast DNA in zygotes resulting from mating between BUdR-treated gametes (mt ⁺ or mt ^-) and untreated gametes of opposite mating-type. In crosses between wild-type strains, the nucleoids of mt ⁺ origin remained in the large majority of zygotes whereas those of mt ^- origin most often disappeared within the first hours following copulation. In crosses of the type mat-3 mt ⁺xwild-type mt ^- (the mat-3 mutation permits a high transmission of chloroplast genes from the mt ^- parent), the nucleoids of mt ^- origin were generally not eliminated which indicates that the mat-3 mutation prevents the selective destruction of paternal chloroplast DNA in the zygote.     

Multiple activation currents can be evoked inXenopus laevis eggs when cortical granule exocytosis is inhibited by weak bases

The fertilization potential inXenopus eggs under normal circumstances is considered to be a unique event. It is associated with a concomitantly occurring cortical granule exocytosis. If eggs were exposed to weak bases, exocytosis was inhibited but the fertilization potential could still be evoked. After recovery from this first transient increase in membrane conductance, a second could be elicited by a further stimulus. A fertilization potential could be triggered either before or after the egg had undergone an electrically induced activation potential. This suggests that sperm receptors and sperm activated ionic channels in the egg membrane remain functional following the conductance change, at least when the exocytotic event was prevented.A transient conductance increase could only be induced by NH ₄ ⁺ (pH 9.0) in unactivated eggs that had not undergone cortical granule exocytosis.Tremendous variation was noticed between successive activation currents elicited in the same egg. Under voltageclamp at 0 mV holding potential, the current often changed from inward to outward. Although cortical granule exocytosis may only play a minor role in the transient conductance change triggered at fertilization, it may well be involved in subsequent modifications of membrane conductance.     

A stereological approach to the study of neural organogenesis in Xenopus laevis

Summary Stereological methods are applied to the study of structural changes undergone, during neurulation, by the neuroepithelium of early embryos of Xenopus laevis. In light microscopy, we evaluate: the section area of the neuroepithelium, the nucleocytoplasmic ratio, the volume fraction of the intercellular spaces, of the nuclei and of the cytoplasm. In electron microscopy, the volumetric density and the surface ratio of mitochondria as well as the surface density of the endoplasmic reticulum are measured. Occasionally, the data found for Xenopus are compared to those found earlier in a similar study of the chicken embryo.     

Development of the mauthner cell in Xenopus laevis: A light and electron microscopic study of the perikaryon

Summary The Mauthner cell can first be recognized in Xenopus embryos at stage 31⁺–32 by its position and by the size of its nucleus and nucleolus. At early stages the perikaryon is dominated by a large ellipsoidal nucleus containing a single relatively compact nucleolus. The cytoplasm is characterized by numerous free ribosomes, poorly developed membrane systems, yolk platelets and globules, and by the presence of dense bodies containing what appear to be remnants of yolk breakdown.During subsequent development, membranes of both the endoplasmic reticulum and the Golgi apparatus become increasingly numerous. Long sinuous tubules of the endoplasmic reticulum are replaced by shorter repeatedly branching cisterns, and incidents of fusion between these elements and the nuclear envelope are less frequent. Large intramitochondrial granules and yolk elements are seldom encountered. The number of multivesicular bodies, alveolate vesicles and small secondary lysosomes increases. The originally nearly homogeneous cytoplasm is rearranged into areas rich in ribosomes and membranous organelles separated by channels containing predominantly neurofilaments and microtubules. At intermediate stages of development a cytoplasmic inclusion consisting of short arcs of parallel beaded strands, each 130–150 Å in diameter, is often present. The relationship of these morphological changes to the overall synthetic pattern of the cell is discussed. It is suggested that they signal the end of seeking growth by the cell.Supported by U.S. Public Health Service Training Grants GM 406 and NS 05591.     

Origin and distribution of enteric neurones in Xenopus.

Summary In Xenopus, we investigated the origin of enteric neurones and their distribution in relation to the extracellular matrix (ECM) components, fibronectin (FN) and tenascin (TN). Enteric neurone precursor cells originate from the anterior trunk neural crest (NC). They migrate along the ventromedial NC pathway (between somites and neural tube/notochord) into the primitive gut (via the dorsal mesentery/lateral plate mesoderm) where they differentiate into enteric neurones. NC cells were identified during their migration and in the gut using the X. laevis — X. borealis nuclear marker system. The neuronal character of NC cells in the gut could be demonstrated immunohistochemically with a monoclonal antibody against the HNK-1 epitope. This antibody is superior to N-CAM and neurofilament antibodies which proved insufficient in Xenopus.In early tadpoles (stage 45), enteric neurones occurred frequently in the mesenchymal lining of the oesophagus, either singly or in groups of two to three cells. In more distal portions of the digestive tract, enteric neurones were rarely found. In metamorphosing tadpoles (stage 62/63), enteric neurones were scattered singly beneath the mucosa, or formed small aggregates between the inner and outer muscle layer throughout the length of the digestive tract. The neurones occurred in positions corresponding to the myenteric and submucosal plexus of higher vertebrates.The distribution of enteric neurones was studied in relation to fibronectin (FN) and tenascin (TN), glycoproteins of the ECM, which support (FN) and inhibit (TN) amphibian NC cell migration. Using immunohisto-chemistry, FN was found during NC cell migration in ECM spaces along the ventromedial pathway, and in the gut between the mucosa and the muscle layers, where it would be able to support adhesion and migration of NC cells. TN, in contrast, appeared much later than FN, both in the dorsal trunk and also ventrally, in the gut. In older tadpoles, TN was present in the mesenchyme and muscle layers of the digestive tract, where it might have an inhibiting influence on the migration of enteric neurones within the gut wall.     

The development of the Merkel cells in the tentacles of Xenopus laevis larvae

Summary The Merkel cells in the larval tentacles of Xenopus laevis were examined by TEM. Different forms of Merkel cells were found, depending on the age of the larvae or the location in the tentacles. These forms have the appearance of intermediate states between Merkel cells and superficial epidermal cells; thus an epidermal origin for the Merkel cells seems more likely than an immigration from the neural crest. The forms differ in (1) their location in the epidermis, (2) their shape, (3) the number and extension of their desmosomes, (4) the content and distribution of dense-core granules, and (5) the outgrowth of their finger-like processes. Also the relation to a nerve ending is different. By marking Merkel cells with quinacrine, fluorescence spots were observed between the superficial and basal epidermal cells or, in the very tip, within the superficial epidermal cells. These latter spots represent immature Merkel cells, as confirmed by TEM. This indicates a development of Merkel cells from superficial epidermal cells and migration towards the basal layer. Dermal Merkel cells were nerver observed.Supported by the Deutsche Forschungsgemeinschaft (SFB 4/G1)