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Basal cell carcinomas (BCCs) of the skin exhibit a wide range of histological growth patterns as well as a highly variable rate of invasiveness. A large body of experimental and clinical studies supports a role for the CD44 glycoprotein family in the latter process. In the present study, we explored the distribution and the level of expression of pan-CD44, CD44v3, CD44v5 and CD44v6 in BCCs. The use of paraffin sections, combined with an antigen retrieval procedure, yielded far more detailed data than would have been possible with frozen sections. On average, the level of expression of the four CD44 isoforms studied appeared to differ relatively little. However, tumours or tumour areas consisting of thin tumour cell strands showed a significantly stronger expression of all four isoforms than those consisting of solid tumour cell groups. Furthermore, the highest CD44 expression was frequently observed in the smallest tumour cell strands in the tumour periphery. In these strands, the label seemed to be located not only at the tumour cell-tumour cell interface, as in other tumour areas, but also on the tumour cell surfaces facing the stroma. We are presently assessing the exact localization of CD44 at the cellular level by immunoelectron microscopy. In most cases, different growth patterns with significantly different levels of CD44 expression were found side by side within individual tumours. CD44 expression is therefore not a static tumour cell characteristic but is correlated with tumour architecture and tumour-stroma interaction.

The VD1 and RPD2 neurons of Lymnaea stagnalis innervate other central neurons, certain skin areas, the pneumostome area, and the auricle of the heart. Recently, a set of four (λ, ω, α, β) neuropeptides produced by these giant neurons and by certain other central neurons has been characterized. Although alternative splicing of the preprohormone of these neurons yields at least 10 different α neuropeptides, an affinity-purified antiserum directed against a domain common to all α neuropeptides has previously been shown to be highly selective in staining VD1, RPD2 and other neurons that produce the preprohormone. Since the gene encoding the neuropeptides is structurally similar to that expressed in R15 of the marine opisthobranch Aplysia californica, we have used the affinity purified antiserum as a marker for VD1/RPD2-related systems in other molluscs. Immunopositive neurons and fibers are observed in the central nervous systems of all species studied (Achatina fulica, Anodonta sp., Aplysia brasiliana, A. californica, Bulinus truncatus, Cepea sp., Eobania vermiculata, Helix aspersa, H. pomatia, Limax maximus, Mytilus edulis, Nassarius reticulatus, Viviparus viviparus). Several medium-sized and small neurons and 1–4 giant neurons are found in the pulmonates and opisthobranchs. The giant neurons in pulmonates have locations in the subesophageal ganglion, axonal branching patterns, and terminal arborizations in the auricle of the heart; all these characteristics are similar to those of VD1 and RPD2. Double-labelling (Lucifer yellow injection, immunocytochemistry) confirms that the two giant neurons in Helix pomatia are Br and Br. The immunoreactive cells in A. fulica appear to include the VIN and PON neurons. The antiserum also stains cells that appear to be the R15 neurons in two Aplysia species. The small and medium-sized neurons are distributed widely over the central ganglia of opisthobranchs and pulmonates. In prosobranchs and bivalves, small neurons are found in the cerebral and abdominal ganglia. No evidence has been found for innervation of the heart in these latter groups but in M. edulis, immunoreactive terminals can be observed in the gill. The results suggest the evolutionary conservation of immunoreactive peptides and the neurons that produce them, and thus support and extend previous hypotheses regarding the homology of certain giant neurons across molluscan species.

In the pond snail Lymnaea stagnalis, the growth regulating system consists of (1) about 200 neuroendocrine light green cells, located in four clusters in the cerebral ganglia, and (2) the paired canopy cells, located in the lateral lobes. These cells express genes encoding the molluscan insulin-related peptides (MIPs). Six MIP genes have previously been identified. Four of these (I, II, III and V) are expressed in the light green cells and the canopy cells. The MIP-VI gene is a pseudogene. In the present in situ hybridization study, using oligonucleotide probes specific to the transcripts of MIP-I, -II, -III, -IV and -V, no signal was obtained with the MIP-IV probe, indicating that gene IV is also a pseudogene. With the other four probes, two types of light green cells were distinguished. Type-A cells express all four MIP genes, whereas type-B cells do not (or only faintly) express the MIP-I gene. Gene III is relatively strongly expressed in type-B cells. Genes II and V are moderately expressed in both cell types. Type-A cells are mainly located in the periphery of the clusters, whereas type-B cells are present in the center. The canopy cell resembles type-A light green cells. The expression levels of the MIP-II and MIP-V genes are low in the canopy cell. The expression pattern of the MIP genes correlates with the staining pattern of the anti-MIP-C antibody, which has been raised to a synthetic C-fragment shared by MIP-I, -II and -V. Type-A cells stain more intensely with the antibody than type-B cells.

The functional morphology of the neuroendocrine system producing sodium influx-stimulating (SIS) peptide in the pond snail, Lymnaea stagnalis, was studied by in situ hybridization and immunocytochemistry. The SIS-peptide, which is 76 amino acids long, stimulates sodium uptake from the ambient medium. Two synthetic DNA probes were used for in situ hybridization. The nucleotide sequences were chosen from the cDNA structure; they encode amino acids 8–17 and 64–73, respectively. SIS-peptide sequences 10–20 and 67–76 were synthesized and antibodies were raised to them and affinity-purified. In addition to these antibodies, a monoclonal antibody raised to a bioactive, high-pressure liquid chromatography (HPLC)-purified brain extract was used for immunocytochemistry. Paraffin sections of central nervous systems and of whole snails were studied. The SIS-peptide system could be identified as the previously described yellow cell (YC) system by comparing alternate sections treated with the DNA probes, stained with the antibodies, or stained with alcian blue-alcian yellow. SIS-peptide neurons (∼45) occur in the ganglia of the visceral ring and in the proximal parts of visceral nerves. Axons run in the nerves of these and in several nerves of other ganglia. Numerous axon branches penetrate the perineurium forming a vast central neurohemal area. The SIS-peptide system innervates the pericardium, the nephridial gland, the reno-pericardial canal, the ureter, the spermoviduct and gonadal acini, the anterior aorta, the ventral buccal artery, and the penis protractor muscle. The morphology of the system is discussed in relation to the process of sodium ion uptake from the ambient medium and from pro-urine, and to that of regulating blood pressure. In the central nervous system and other organs, neurons and axons not labeled with the DNA probes, but immunoreactive to one or two of the antibodies, were observed. It seems unlikely that these elements are functionally related to the SIS-peptide system.

Of gastropod molluscs, only in the Aplysiidae and the Lymnaeidae have the genes encoding the respective egg-laying hormones been cloned and the neurons controlling egg laying and egg-laying behavior been identified. Immunocytochemistry, using antibodies raised against alpha-CDCP (one of the neuropeptides encoded on the egg-laying hormone gene of Lymnaea stagnalis), identified neurons in various species of gastropods. In the basommatophoran snail, Biomphalaria glabrata, large and small neurons were observed in areas of the central nervous system similar to where immunoreactive cells exist in L. stagnalis, i.e., in the cerebral and pleural ganglia. In the stylommatophoran snail (Helix aspersa) and the slug (Limax maximus), large immunopositive neurons occur in the visceral and right parietal ganglia. In L. maximus, small immunoreactive neurons were found in the cerebral ganglia while in H. aspersa similar cells were observed intermingled with the large cells in the visceral and right parietal ganglia. Similar to the situation in L. stagnalis, in the female part of the reproductive tract of B. glabrata, L. maximus, and A. californica, but not in H. aspersa, neurons and/or fiber tracts are present. The results indicate that egg-laying hormone precursor molecules of gastropod molluscs are phylogenetically closely related. The alpha-CDCP antiserum may allow the identification of hitherto unknown egg-laying regulating systems of gastropod molluscs.

VD1 and RPD2 are two giant neuropeptidergic neurons located respectively in the visceral and right parietal ganglion of the central nervous system (CNS) of the pond snail Lymnaea stagnalis. They are the most prominent representatives of a system of neurons expressing a gene that is similar to the gene expressed in R15 of Aplysia californica. Both neuronal systems are involved in the regulation of cardiorespiratory phenomena. In the present study the axonal branches of VD1 and RPD2 were mapped using immunocytochemical and tracer studies. To this end the α1-antiserum (directed to one of the VD1/RPD2 neuropeptides) was used in combination with Lucifer yellow (LY) and Ni-lys tracers. In whole mount preparations of the CNS, immunostained axons of VD1 and RPD2 were observed to run to the pleural, cerebral and pedal ganglia and in several nerves. Upon LY injection of VD1 thin axon branches were observed in the internal right parietal nerve. These run to the skin in the mantle area near the pneumostome and osphradium. The skin of the lips appeared to receive a similar innervation via the lip nerves. Thick LY filled axons of VD1 and RPD2 were observed in the intestinal nerve. They could be traced to the heart region. The pericardial branch of the intestinal nerve innervates the pericardium and heart (Ni-lys tracing). Immunocytochemically, using the α1-antiserum, it was demonstrated that this nerve branch carries the axons of VD1 and RPD2 to the venous side of the auricle, where they enter the pericardial cavity and ramify in the auricle (but not in the ventricle). Many immunopositive varicosities were observed on the auricular muscle fibers. Comparison of the stainings in the auricle with those obtained with antisera to the cardioactive neuropeptides FMRFamide and SCP showed no colocalization. The functional significance of the axonal distribution patterns of VD1 and RPD2 and the morphological and functional homology of the neurons to R15 of Aplysia are discussed.

VD1 and RPD2 are two giant neuropeptidergic neurons in the central nervous system (CNS) of the pond snail Lymnaea stagnalis. We wished to determine whether other central neurons in the CNS of L. stagnalis express the VD1/RPD2 gene. To this end, in situ hybridization with the cDNA probe of the VD1/RPD2 gene and immunocytochemistry with antisera specific to VD1 and RPD2 (the alpha 1-antiserum, Mab4H5 and ALMA 6) and to R15 (the alpha 1 and 16-mer antisera) were performed on alternate tissue sections. A VD1/RPD2 neuronal system comprising three classes of neurons (A1-A3) was found. All neurons of the system express the gene. Division into classes is based on immunocytochemical characteristics. Class A1 neurons (VD1 and RPD2) immunoreact with the alpha 1-antiserum, Mab4H5 and ALMA 6. Class A2 neurons (1-5 small and 1-5 medium sized neurons in the visceral and right parietal ganglion, and two clusters of small neurons and 5 medium-sized neurons in the cerebral ganglia) immunoreact with the alpha 1-antiserum and Mab4H5, but not with ALMA 6. Class A3 neurons (3-4 medium-sized neurons and a cluster of 4-5 small neurons located in the pedal ganglion) immunoreact with the alpha 1-antiserum only. All neurons of the system are immunonegative to the R15 antisera. The observations suggest that the neurons of the VD1/RPD2 system produce different sets of neuropeptides. A group of approximately 15 neurons (class B), scattered in the ganglia, immunostained with one or more of the antisera, but did not react with the cDNA probe in in situ hybridization.