Lymnaea EGF and gigantoxin I, novel invertebrate members of the epidermal growth factor family.

In this review, we compare the sequence and structural relationships of two epidermal growth factor (EGF) family related proteins that have recently been discovered in invertebrate species. The first is L-EGF, a secreted growth factor from the gastropod mollusk Lymnaea stagnalis. The second is a peptide toxin (Gigantoxin I), isolated from the sea anenome Stichodactyla giganteus, which can paralyze crabs. L-EGF and Gigantoxin I share striking sequence similarity with mammalian erbB1 receptor ligands, including most of the essential receptor binding sites. Intriguingly, L-EGF's tertiary structure resembles more the structure of the EGF-like domain of coagulation factors. That is, the secondary and tertiary structure of L-EGF indicates the presence of a double-stranded beta-sheet but also suggests that this protein, in contrast to all other erbB1 ligands, contains a calcium-binding domain. One of the most remarkable features of L-EGF and Gigantoxin I however, is the indication that these protein are synthesized as non-membrane bound secreted peptides. This feature sets L-EGF and Gigantoxin I apart from all other members of the EGF family or EGF-like proteins identified thus far. We discuss sequence similarities and dissimilarities in the light of indications that, despite the more than 600 million years of phylogenetic distance separating both these invertebrates from mammals, Gigantoxin I and L-EGF retain some affinity for the mammalian erbB-family of receptors. Considering that mammalian EGF and its family members are frequently implicated in neoplastic diseases, the increasing number of identified and characterized invertebrate EGF family members may provide valuable leads in the design of erbB receptor antagonists.

Lymnaea epidermal growth factor promotes axonal regeneration in CNS organ culture.

Members of the epidermal growth factor (EGF) family are frequently implicated in the injury response of the mammalian nervous system. Although this implication is supported by extensive molecular evidence, it is not underpinned by conclusive functional data. Recently, we found that expression of an EGF homolog from the pond snail Lymnaea stagnalis (L-EGF) is upregulated after axotomy in the adult CNS, suggesting a role for this molecule in the injury response of the CNS. In the present study we asked whether L-EGF can promote axonal regeneration of three types of identified neurons in organ-cultured CNS. Treatment with purified L-EGF substantially enhanced axonal regeneration of all three types of neurons, an effect inhibited by submicromolar doses of PD153035, a specific EGF receptor (EGFR) tyrosine kinase inhibitor. In addition, PD153035 and K252a, a nonspecific kinase inhibitor, also reduced the degree of axonal regeneration that occurs without L-EGF supplementation, indicating that L-EGF or other EGFR ligands synthesized in the CNS participate in the regenerative response. An intriguing aspect of these results is that axonal regeneration of different, intrinsically L-EGF responsive and unresponsive neurons occurred in a coordinated manner. This observation suggests that indirect in addition to direct actions contribute to the beneficial effect of L-EGF. In conclusion, we provide functional evidence that an EGF homolog can promote axonal regeneration, substantiating existing molecular evidence implicating the EGF family in peripheral nerve regeneration and emphasizes the therapeutic potential of these molecules.

Neurotrophic actions of a novel molluscan epidermal growth factor.

The mammalian epidermal growth factor (EGF) is expressed in the developing and adult CNS, and it has been implicated in the control of cell proliferation, differentiation, and neurotrophic events. Despite extensive evolutionary conservation of the EGF motif in a range of different types of proteins, secreted EGF homologs with neurotrophic actions have not been reported in invertebrates. In this study, we present a novel member of the family of EGF-like growth factors, an EGF homolog from the mollusc Lymnaea stagnalis (L-EGF), and we demonstrate that this protein has neurotrophic activity. Purified L-EGF is a 43-residue peptide and retains the typical structural characteristics of the EGF motif. The L-EGF cDNA reveals a unique precursor organization. In contrast to the multidomain mammalian EGFs, it consists of only two domains, a signal peptide and a single EGF motif. Conspicuously, the L-EGF precursor lacks a transmembrane domain, setting it apart from all other members of the EGF-family. L-EGF mRNA is expressed throughout embryonic development, in the juvenile CNS, but not in the normal adult CNS. However, expression in the adult CNS is upregulated after injury, suggesting a role of L-EGF in repair functions. This notion is supported by the observation that L-EGF evokes neurite outgrowth in specific adult Lymnaea neurons in vitro, which could be inhibited by an EGF receptor tyrosine kinase inhibitor. In conclusion, our findings further substantiate the notion that the EGF family has an early phylogenetic origin, and our data support a neurotrophic role for L-EGF during development and injury repair.

Stamen development in the Ericaceae. I. Anther wall, microsporogenesis, inversion, and appendages.

Development of the introrse, tetrasporangiate, and normally dorsifixed and poricidal stamens has been studied at the gross morphological and cellular level in ten species of Ericaceae. Microsporogenesis, followed in four species, is normal, with cytokinesis simultaneous, forming tetrahedral tetrads. The tricolp(or)ate pollen is shed as permanent tetrads with each segment two-celled except in Enkianthus in which pollen grains are three-celled monads. Anther-wall development is similar in all four species initially, but no regular pattern of wall development could be recognized thereafter. The tapetum, of parietal origin, is binucleate, glandular, and mainly uniseriate. Viscin threads occur with the tetrads in the three rhododendroid species. A well-developed endothecium appears only in Enkianthus.Soon after stamen initiation, anthers of nine species invert at the eventual filament-anther junction to become introrse; in Enkianthus inversion occurs close to anthesis. Microsporogenesis starts during early inversion; greater cell elongation on the abaxial side of the young anther completes inversion by the late sporogenous-tissue stage. In Erica and, to a lesser extent Calluna, inversion results from greater abaxial than adaxial increase in cell number and length just above the filament-anther junction. The single vascular strand reflects the degree of inversion. Stamens of six species are appendaged; three have only awns, two only spurs, while one has both. Appendages arise from residual meristems after inversion is completed (or almost so) in all except Enkianthus. Awns develop at what will be the apex at maturity of each anther half. Their length and orientation vary among species. Only in Vaccinium do the awns become hollow (tubules). Spurs, varying in length, shape, and size, arise on the abaxial side from the filament, connective, or thecae.

Functional recovery of respiratory behavior during axonal regeneration in snails (Lymnaea stagnalis) is experience dependent.

This study investigated the role of experience in recovery of pulmonary respiration during axonal regeneration in Lymnaea stagnalis. Pulmonary respiration occurs when snails break the water surface and open the lung orifice, the pneumostome. It was shown that axotomy of all the axons innervating the pneumostome and surrounding area prevents the occurrence of lung respiration in 69% of snails. In the remaining 31%, lung respiration persisted, indicating that peripheral components alone are capable of initiating pneumostome openings and closures. Five weeks postsurgery, all snails with previous nerve crushes showed opening of the pneumostome with normal latency after breaking the water surface. However, prevention of pulmonary respiration during the recovery period dramatically changed the recovered behavior. Thus, experience in pulmonary respiration during axonal regeneration plays a role in the recovery of this behavior.

The lymnaea cardioexcitatory peptide (LyCEP) receptor: a G-protein-coupled receptor for a novel member of the RFamide neuropeptide family.

A novel G-protein-coupled receptor (GRL106) resembling neuropeptide Y and tachykinin receptors was cloned from the mollusc Lymnaea stagnalis. Application of a peptide extract from the Lymnaea brain to Xenopus oocytes expressing GRL106 activated a calcium-dependent chloride channel. Using this response as a bioassay, we purified the ligand for GRL106, Lymnaea cardioexcitatory peptide (LyCEP), an RFamide-type decapeptide (TPHWRPQGRF-NH2) displaying significant similarity to the Achatina cardioexcitatory peptide (ACEP-1) as well as to the recently identified family of mammalian prolactin-releasing peptides. In the Lymnaea brain, the cells that produce egg-laying hormone are the predominant site of GRL106 gene expression and appear to be innervated by LyCEP-containing fibers. Indeed, LyCEP application transiently hyperpolarizes isolated egg-laying hormone cells. In the Lymnaea pericardium, LyCEP-containing fibers end blindly at the pericardial lumen, and the heart is stimulated by LyCEP in vitro. These data confirm that LyCEP is an RFamide ligand for GRL106.

Developmental plasticity of respiratory behavior in Lymnaea.

The authors investigated the contribution of experience to development and maintenance of pulmonary respiration in Lymnaea stagnalis. Respiration in L. stagnalis is bimodal via both the skin and the lung. Rearing snails from eggs to adulthood while preventing lung respiration (differentially reared snails) showed that L. stagnalis can develop and survive without pulmonary respiration. These snails were able to open and close their pneumostome when given the opportunity as adults. However, quantitative aspects of their respiratory behavior were significantly altered. Prevention of pulmonary respiration in adult, normally reared snails also induced behavioral changes. Comparison of these changes with those in differentially reared snails revealed specific developmental effects, which were reversible. Thus, this is a suitable model system for studying questions related to behavioral plasticity.

Neurite outgrowth, RGD-dependent, and RGD-independent adhesion of identified molluscan motoneurons on selected substrates.

The extracellular matrix (ECM) provides structural support to cells and tissues and is involved in the regulation of various essential physiological processes, including neurite outgrowth. Most of the adhesive interactions between cells and ECM proteins are mediated by integrins. Integrins typically recognize short linear amino acid sequences in ECM proteins, one of the most common being Arginine-Glycine-Aspartate (RGD). The present study investigated neurite outgrowth and adhesion of identified molluscan neurons on a selection of substrates in vitro. Involvement of RGD binding sites in adhesion to the different substrates was investigated using soluble synthetic RGD peptides. The cells adhered to native (i.e., nondenatured) laminin and type IV collagen, but not to native plasma fibronectin. Denaturation of fibronectin dramatically enhanced cell adhesion. Only the adhesion to denatured fibronectin was inhibited by RGD peptides, indicating that denaturation uncovers a RGD binding site in the protein. Laminin as well as denatured fibronectin, but not type IV collagen, induced neurite outgrowth from a percentage of the RPA neurons. These results demonstrate that molluscan neurons can attach to various substrates using both RGD-dependent and RGD-independent adhesion mechanisms. This suggests that at least two different cell adhesion receptors, possibly belonging to the integrin family, are expressed in these neurons. Moreover, the results show that vertebrate ECM proteins can induce outgrowth from these neurons, suggesting that the mechanisms involved in adhesion as well as outgrowth promoting are evolutionarily well conserved.

Role of neuropeptides encoded on CDCH-1 gene in the organization of egg-laying behavior in the pond snail, Lymnaea stagnalis.

Egg laying in the pond snail Lymnaea stagnalis is triggered by a discharge of the neuroendocrine caudodorsal cells (CDCs). The CDCs expresses three different caudorsal cell hormone (CDCH) genes. This gene family expresses, in total, 11 different peptides among which is the ovulation hormone. Besides the CDCs, the CDCH gene family is expressed in other central and peripheral neurons. In this study, we investigated the roles the different CDCH peptides play in the organization of egg-laying behavior. Egg-laying behavior is a sequence of stereotyped movements in which three phases can be distinguished: resting, turning, and oviposition. We have used the excitation of right pedal N (RPeN) motor neurons as a simple analogue of shell-turning behavior, one of the elements of egg-laying behavior. RPeN motor neurons were inhibited during the resting phase of egg laying but were subsequently excited at the onset of and during the turning phase. The excitatory effect could be evoked by application of beta3-CDCP on RPeN motor neurons in the CNS as well as in isolation but not by the ovulation hormone, alpha-CDCP or Calfluxin, the other CDCH-1 peptides tested. The ovulation hormone itself caused inhibition of RPeN motor neurons. Anti-CDCH-1 positive fiber tracts were found close to the cell bodies and axons of the RPeN motor neurons. Electrical stimulation of a nerve that contains these fibers resulted in excitation of the RPeN motor neurons. The effects of injection of CDCH-1 peptides into intact animals correlated well with the effects of these peptides on RPeN motor neurons. Injection of beta3-CDCP or alpha-CDCP into intact animals resulted in immediate turning behavior in the absence of egg laying itself. The ovulation hormone and Calfluxin had no immediate effect on the behavior. Furthermore, our data indicate that the individual CDCH-1 peptides act on different targets.

Pronase acutely modifies high voltage-activated calcium currents and cell properties of Lymnaea neurons.

Pronase E ('pronase') is one of the proteolytic enzymes that are used in preparative procedures such as cell isolation and to soften the sheath of invertebrate ganglia. Although several effects of proteolytic enzymes on the physiology of non-neuronal tissues have been described, the effects of these enzymes on central neurons have received little attention. We examined the effects of bath-applied pronase on neurons in the Lymnaea central nervous system and in vitro. Pronase caused action potential broadening in neurons that exhibit a shoulder on the repolarization phase of their action potentials. This effect of pronase was accompanied by, although unrelated to, a depolarization and decrease in action potential interval. Some, but not all, effects of pronase in the central nervous system were reversible. For example, the decreases in membrane potential and action potential interval were both reversed after approximately 1 h of washing with saline. However, the effect of pronase on the action potential duration was not reversed after a period of 90 min. The modulation of action potential width prompted us to examine Ca2+ currents. Exposure to pronase resulted in an increase in both peak and late high voltage-activated Ca2+ currents in isolated neurons. Pronase neither changed the inactivation rate nor caused a shift in the current-voltage relationship of the current. The changes in action potential duration could be prevented by application of 0.1 mM Cd2+, indicating that the action potential broadening caused by pronase depends on Ca2+ influx. This is the first systematic study of the acute and direct actions of pronase on Ca2+ currents and cell properties both in the CNS and in vitro.

The two-oscillator circadian system of tree shrews (Tupaia belangeri) and its response to light and dark pulses.

The wheel-running activity rhythm of tree shrews (tupaias; Tupaia belangeri) housed in constant darkness (DD) phase-advanced following a 3-hr light pulse at circadian time (CT) 21. Dark pulses of 3 hr presented to tupaias in bright constant light (LL) did not induce significant phase shifts of the free-running activity rhythm, irrespective of the CT. In dim LL, tupaias showed simultaneous splitting of their circadian rhythm of wheel-running activity, nest-box activity, and feeding behavior. Light pulses of 6 hr and 2300 lux were presented to 13 tupaias with split wheel-running activity rhythms. These light pulses induced immediate phase shifts in the two components of the split rhythm in opposite directions. No differences were observed between the light-pulse phase response curves of the two components. Equally large immediate phase advances were induced in both components by light pulses of 230 lux, but not by 23 lux. The final phase shifts were small at all CTs. In two tupaias, activity rhythms transiently split and re-fused. Analysis of the relative position of the components in one of these indicates asymmetry in the coupling between the components.