Lateral interactions account for the pattern of the hair cell array in the chick basilar papilla.

It has been suggested that lateral interactions set up the array of hair cells and supporting cells in the chick basilar papilla. The presence of a hair cell would inhibit adjacent cells from becoming hair cells, and promote the formation of supporting cells. Models of cell specification were tested, starting with a closely packed array of multipotent progenitor cells. Lateral interactions, in which emerging hair cells promoted a supporting cell phenotype in adjacent cells, and in which emerging supporting cells promoted a hair cell phenotype in adjacent cells, produced an array of cells similar to that observed experimentally in the distal and central parts of the basilar papilla. In these areas, the ratio of supporting cells to hair cells is very close to 2:1, each hair cell on average being surrounded by six supporting cells, and each supporting cell being surrounded by three hair cells and three supporting cells. Identical patterns of hair and supporting cells could be produced by models in which either of the lateral inhibitory factors was replaced by a diffusive factor, i.e. a factor which acts on all cells in the model irrespective of position. The agreement of the model with observed cell ratios supports the view that the fate of both hair cells and supporting cells in the chick basilar papilla is a product of cell interactions within the papilla. It is possible that one factor, that provides contact lateral inhibition and promotes the formation of supporting cells, is the Notch/Delta system. It is possible that the other factor is retinoic acid, a diffusive factor that promotes the formation of hair cells.

Expression of EphA4 in developing inner ears of the mouse and guinea pig.

The expression of EphA4, an Eph-class receptor tyrosine kinase, was determined by immunohistochemistry in developing inner ears of the mouse and the guinea pig. In the mouse, EphA4 expression was visible in the fibroblasts of the spiral ligament and in the structures that were to become the osseous spiral lamina. Cochlear nerve ganglion cells expressed ephrin-B2, and the modiolus expressed mRNA coding for ephrin-B3, both transmembrane ligands for EphA4. In contrast, in the guinea pig, cells of the cochlear nerve ganglion expressed EphA4, as did supporting cells of the organ of Corti (Hensen's cells and inner pillar cells). There was also some expression in fibroblasts of the spiral ligament but none in the structures that were to become the osseous spiral lamina. It is suggested that in the mouse, EphA4 may help direct the cochlear innervation towards the organ of Corti by a repulsive interaction, but that this is highly species dependent.

A tyrosine kinase screen of mouse vestibular maculae.

Receptor tyrosine kinases allow extracellular signals to influence intracellular events, while other tyrosine kinases are involved in intracellular signalling. They may therefore be involved in the development, maintenance and repair of the sensory epithelia of the inner ear, since these are believed to be affected by inter- and intracellular signalling. In order to analyse possible tyrosine kinases expressed in sensory areas of the inner ear, a reverse transcription polymerase chain reaction screen of microdissected sensory epithelia was undertaken, using primers targeted at conserved sequences in tyrosine kinase domains. Tissue was taken from the maculae of the mouse vestibular organs, and consisted mainly of hair cells and their supporting cells. Of 80 clones sequenced, 49 coded for tyrosine kinases, and 11 for other known molecules. Further analysis of one of the sequences, for FGF receptor 4, showed a novel variant, expressed in the inner ear and elsewhere, with a variation in the intracellular domain which suggests differential activation of known signalling pathways. Other clones coded for tyrosine kinases expected to be involved in cell surface and intracellular signalling. The technique forms a powerful tool for analysing a range of the tyrosine kinases expressed, and provides a starting point for the analysis of cell-cell signalling in the inner ear.

Fibroblast growth factor receptor-4 splice variants cause deletion of a critical tyrosine.

We have identified two novel isoforms of fibroblast growth factor receptor-4 (FGFR4). They result from alternative splicing of intron 17. Two transcripts, both slightly larger than the one coding for the known mouse FGFR4, are generated. The shortest (FGFR4-17a) includes the 31-most 3'-nucleotides of intron 17; the longest (FGFR4-17b) includes all 114 nucleotides of intron 17. Translation of the FGFR4-17a and FGFR4-17b splice variants predicts that both novel putative FGFR4 isoforms have a truncated C-terminal intracellular tail. The first amino acid residue affected by the insertions in both novel isoforms is Tyr-760, a residue that may play a crucial role in intracellular signaling through stimulation of the phosphatidylinositol-biphosphate pathway.

Expression and genetic variation of the Aplysia egg-laying hormone gene family in the atrial gland.

We have screened an Aplysia atrial gland cDNA library using an egg-laying hormone (ELH) precursor probe and have isolated and characterized five different clones, four of which are full-length and approximately 0.8 kb in size. The characterization of these cDNA clones firmly established the genetic variation of the ELH-related precursors expressed in the atrial gland and provided a rational basis for their revised nomenclature proposed herein. The five precursor ELH-related cDNA sequences obtained predicted the following genetically distinct polypeptide precursors designated as: A, [Asp143]A, [Glu94,Gln139]A, [Pro25]B, and [Phe96,Asp107]BT. The [Phe96,Asp107]Br cDNA sequence predicted a truncated form of a B-type precursor. Northern blot analysis of atrial gland RNA identified two transcripts of about equal intensity of 0.9 kb and 1.1 kb. Polymerase chain reaction of genomic DNA, together with DNA sequence analysis, resolved previously reported discrepancies between genomic and cDNA sequences of the ELH-related precursors. Taken together the results obtained identified the expression of five ELH-related precursor genes in the atrial gland of Aplysia from at least two genetic loci per haploid genome.

The expression of messenger RNAs coding for growth factors, their receptors, and eph-class receptor tyrosine kinases in normal and ototoxically damaged chick cochleae.

Messenger RNAs coding for growth factors and receptor tyrosine kinases were measured by quantitative competitive and by semi-quantitative reverse-transcription polymerase chain reaction in whole and dissected chick inner ears. The fibroblast growth factor (FGF) receptor 1 chick embryonic kinase (CEK) 1 was expressed in all structures examined (otocyst, hatchling whole cochlea, cochlear nerve ganglion, and cochlear and vestibular sensory epithelia), although slightly more heavily in the otocyst. The related fibroblast growth factor receptors CEK 2 and 3 were preferentially expressed in the nerve ganglion and in the vestibular sensory epithelium, respectively. FGF1 mRNA was low in early development, increasing to mature levels at around embryonic age 11 days, while FGF2 mRNA was expressed at constant levels at all ages. In response to ototoxic damage, FGF1 mRNA levels were increased in the early damaged cochlear sensory epithelium. Immunohistochemistry for CEK1 showed that normal hair cells expressed the receptor heavily on the hair cell stereocilia, while with early damage, CEK1 came to be expressed heavily on the apical surfaces of the supporting cells. In normal chicks, the CEK4 and CEK8 eph-class receptor tyrosine kinases were expressed relatively heavily by the cochlear nerve ganglion, and CEK10 was expressed relatively heavily by the cochlear hair cell sensory epithelium. The results suggest that the FGF system may be involved in the response of the cochlear epithelium to ototoxic damage. The eph-class receptor tyrosine kinase CEK10 may be involved in cell interactions in the cochlear sensory epithelium, while CEK4 and CEK8 may play a role in the cochlear innervation.

Sequence and tissue distribution of chicken cellular nucleic acid binding protein cDNA.

We sequenced cDNAs coding for chicken cellular nucleic acid binding protein (CNBP). Two slightly different variations of the open reading frame were found, each of which translates into a protein with seven zinc finger domains. The longest transcript contains an in-frame insert of 3 bp. The sequence conservation between chick CNBP cDNAs with human, rat and mouse CNBP cDNAs is extreme, especially in the coding region, where the deduced amino acid sequence identity with human, rat and mouse CNBP is 99%. CNBP-like transcripts were also found in various tissues from insect, shrimp, fish and lizard. Regions with remarkable nucleotide conservation were also found in the 3' untranslated region, indicating important functions for these regions. Quantitative reverse transcription polymerase chain reaction (RT-PCR) indicated that in the chick, CNBP in present in all tissues examined in approximately equal ratios to total RNA. RT-PCR of total RNA isolated from different phyla indicate CNBP-like proteins are widespread throughout the animal kingdom. The extraordinary level of conservation suggests an important physiological role for CNBP.

Molecular cloning and cellular localization of a furin-like prohormone convertase from the atrial gland of Aplysia.

Prohormone convertases (PCs) are Ca(2+)-dependent subtilisin-related endoproteases that have been implicated in the post-translational processing of prohormones and other proproteins. Furin is an ubiquitously expressed PC that has been shown to hydrolyze a wide variety of precursor proteins in secretory pathways. We have screened an Aplysia atrial gland cDNA library using a furin probe prepared by polymerase chain reaction (PCR) and have isolated an Aplysia furin-related 6.7-kb cDNA partial clone that was truncated on the 5' end. The remaining 5' atrial gland furin nucleotide sequence was obtained by two stages of reverse transcription PCR. The final composite nucleotide sequence of the atrial gland furin cDNA was 7,837 bp in length. This sequence encoded a putative preproendoprotease (Afurin2) of 824 amino acid residues that was related to other eukaryotic furins, and showed a high sequence identity with a recently reported Aplysia nervous system furin-like sequence. In situ hybridization demonstrated extensive expression of Afurin2 in atrial gland secretory cells. The cDNA clone contained a relatively long 3' untranslated region of 5,230 nucleotides that included a microsatellite repeat region (TG)n. The characterized Aplysia Afurin2 is a candidate PC that may play an important role in the processing of egg-laying hormone (ELH)-related precursors in the secretory cells of the atrial gland. In addition, comparative structural studies of Afurin2, together with previously reported localization studies, argue for the occurrence of a furin-like convertase within secretory granules.

Molecular cloning, cDNA sequence, and localization of a prohormone convertase (PC2) from the Aplysia atrial gland.

Neuropeptides and peptide hormones are synthesized as part of larger precursor proteins that are processed post-translationally by subtilisin-related calcium-dependent prohormone convertases (PCs), frequently at multiple basic sites, to generate biologically active peptides. The atrial gland of Aplysia californica produces large quantities of egg-laying hormone (ELH)-related peptides, providing a unique opportunity to study prohormone processing. We have screened an Aplysia atrial gland cDNA library using a Lymnaea stagnalis PC2 probe and have isolated an Aplysia PC2-related 4.6-kb cDNA partial clone that was truncated on the 5' end. The remaining 5' atrial gland PC2 nucleotide sequence was obtained by reverse transcription/polymerase chain reaction (RT-PCR). The composite cDNA structure (5.6 kb) was deduced from sequence analysis of the RT-PCR product combined with the sequence obtained from the cDNA clone. The deduced cDNA of Aplysia atrial gland PC2 encoded a putative preproendoprotease of 653 amino acids that was evolutionarily related to other eukaryotic PC2s, and showed the strongest sequence identity with recently reported Aplysia nervous tissue PC2 sequences. In situ hybridization demonstrated extensive expression of PC2 in atrial gland secretory cells. The cDNA clone contained a relatively long 3'untranslated region (3'-UTR) of 3,632 nucleotides. Strikingly, the 3'-UTR also contained several major nucleotide repeat sequences including the microsatellite repeats, (CA)n and (TG)n, and a TA-rich region comprised largely of the triplet repeat (TTA)n. The characterized Aplysia PC2 is a candidate endoprotease that may play an important role in the processing of ELH-related precursors in the atrial gland and represents the first example of PC2 expression in exocrine tissue.

Ultrastructural localization of egg-laying prohormone-related peptides in the atrial gland of Aplysia californica.

The atrial gland is an exocrine organ that secretes into the oviduct of Aplysia californica and expresses three homologous genes belonging to the egg-laying hormone gene family. Although post-translational processing of the egg-laying hormone precursor in the neuroendocrine bag cells has been examined in detail, relatively little is known about the post-translational processing of egg-laying hormone-related gene products in the atrial gland. A combination of morphologic techniques that included light-microscopic histology and immunocytochemistry, transmission electron microscopy, and immuno-electron microscopy were used to localize egg-laying hormone-related peptides in the atrial gland and to evaluate the characteristic morphology of their secretory cells. Results of these studies showed that there were at least three major types of secretory cells in the atrial gland (types 1-3). Significantly, of these three cell types, only type 1 was immunoreactive to antisera against egg-laying hormone-related precursor peptides. The immunoreactivity studies established that all three egg-laying hormone-related precursor genes are expressed in type-1 cells and indicated that the processing of these precursors also occurs within the secretory granules of this cell type. Evidence was also obtained that proteolytic processing of the egg-laying hormone-related precursors differed significantly from that observed in the bag cells. In contrast to the bag cells, the NH2-terminal and COOH-terminal products of the egg-laying hormone-related precursors of the atrial gland were not sorted into different types of vesicles.

The changing microtubule arrangements in developing hair cells of the chick cochlea.

It has been suggested that microtubules in auditory hair cells might be involved in directing the morphological and hence functional polarisation of the sensory hair bundles. The distribution of microtubules was studied in hair cells of the chick cochlea, during the developmental stages when the stereocilia and cuticular plate were being formed. Cochleae were immunofluorescently labelled with antibodies to tubulin at specific stages in development, and hair cell ultrastructure was observed by electron microscopy. We found that the microtubule array changed from a simple symmetrical apical plate with a central kinocilium before the cuticular plate forms, to a ring with the kinocilium to one side when the cuticular plate begins to form, through to a cup-like arrangement below the cuticular plate once the plate has formed. In the earliest stages, no asymmetries were observed in the distribution of the microtubules, suggesting that structures other than the microtubules set up the functional polarisation of the stereociliary bundle.

Peptide processing and release by the neuroendocrine caudodorsal cells of Lymnaea stagnalis during an egg-laying cycle.

The freshwater mollusc Lymnaea stagnalis exhibits an egg-laying cycle that takes about 24 h. The cycle is under control of the cerebral neuroendocrine Caudodorsal Cells (CDC), which release various peptides, one of which is the ovulation-inducing hormone, CDCH. During the egg-laying cycle the CDC reveal three consecutive states of electrical activity, the resting, the active and the inhibited state. Processing and release of CDCH have been studied in relation to the states of electrical (and secretory) activity of the CDC, with quantitative immuno-electron microscopy and a newly raised CDCH antiserum. In somata of CDC in the resting state 1 (just before activation of the CDC), in the active state (30 min after activation) and in the inhibited state (3 h after activation) more secretory granules are immunolabelled, and to a higher degree, than in somata of CDC in the resting state 2 (10 h after activation). Axon terminals of CDC in resting states 1 and 2 are equally immunoreactive, whereas in terminals in the active state more granules are labelled, and to a higher degree, than in the resting states. Secretory granules in terminals in the inhibited state are intermediate in both respects. Immuno-electron microscopy combined with the tannic acid method for the demonstration of exocytosis showed that in terminals in the active state, the percentage of immunolabelled exocytosed granule contents is much higher than in the other states. The same holds for the degree of immunopositivity of these contents.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of Aplysia atrial gland secretory granules containing egg-laying prohormone-related peptides.

A purification scheme is described for the isolation of secretory granules containing egg-laying prohormone-related peptides from the atrial gland of Aplysia californica, an exocrine organ in the reproductive tract. Granules were purified by differential centrifugation of atrial gland homogenates followed by centrifugation on continuous Percoll-sucrose gradients. Quantitative enzyme assays in conjunction with electron microscopic analyses demonstrated that secretory granules thus isolated were significantly purified with respect to other subcellular organelles such as mitochondria and lysosomes. Immunoelectron microscopy demonstrated that the majority (approximately 85%) of the purified secretory granules were immunoreactive for A-NTP (N-terminal peptide), a cleavage product of the egg-laying prohormone-related A and A' precursors (residues 22-34). The purified granules represented an enriched source of peptides that were readily resolved by reversed-phase high performance liquid chromatography.

Occurrence of a furin-like prohormone processing enzyme in Aplysia neuroendocrine bag cells.

1. Strong evidence is accumulating that the endoproteases which process prohormones at dibasic residue cleavage sites are members of a subtilisin-related class of proteases. 2. Using the polymerase chain reaction (PCR), we have isolated and characterized an Aplysia californica neuroendocrine bag cell cDNA product (270 base pairs) that encodes a sequence which is highly homologous to the subtilisin-related class of processing proteases that includes yeast Kex2, human/mouse/Drosophila furin, human PC2, and mouse PC1/PC3 and PC2. 3. The characterized cDNA PCR product showed the highest degree of residue identity with the furin-related group of proteins (human/mouse furin 71%; Drosophila furin 63%). 4. These results establish that Aplysia contain a subtilisin-like gene and suggest that the expression of this gene may play a role in processing Aplysia precursor proteins in the bag cells and likely also in the exocrine atrial gland. 5. Furthermore, the Aplysia nucleotide sequence results, together with available sequence information from human, mouse, and Drosophila furins, provide reasonable evidence that the furin-like enzymes may represent a separate subclass of the subtilisin-like processing enzymes.

Light and electron microscopic immunocytochemical demonstration of synthesis, storage, and release sites of the neuropeptide calfluxin in Lymnaea stagnalis.

The cerebral caudodorsal cells (CDC) of the pulmonate snail Lymnaea stagnalis control egg-laying and associated behaviors. They produce various peptides derived from two precursor molecules, proCDCH-I and II, one of which is calfuxin (CaFl). CaFL is involved in the control of the activity of a female accessory sex gland, the albumen gland. At the light microscope level, using an antibody raised against synthetic CaFl, immunoreactivity was demonstrated in all CDC somata as well as in the neurohemal CDC terminals in the periphery of the cerebral commissure and in the CDC axon collaterals in the inner region of the commissure. A group of small neurons in each cerebral ganglion was also immunopositive. At the ultrastructural level, secretory granules (SG) and large electron-dense granules (LG), formed by the Golgi apparatus and thought to be involved in intracellular degradation of secretory material, were clearly immunolabeled. The density of immunolabeling of LG was 3.3 times greater than that of SG, indicating that CaFl is preferentially packed into LG. In the LG, the density of immunolabeling with anti-alpha CDCP (alpha CDCP is also a peptide derived from proCDCH-I and II) was 10 times greater than in SG, suggesting that CaFl and alpha CDCP are processed and sorted in (quantitatively) different ways. In the neurohemal terminals SG release their CaFl-immunopositive contents into the hemolymph by the process of exocytosis, whereas collaterals release such contents into the intracellular space of the intercerebral commissure. It is proposed that neurohemally released CaFl acts upon the albumen gland, whereas CaFl released from the collaterals may influence the activity of central neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Light- and electron-microscopic immunocytochemistry of a molluscan insulin-related peptide in the central nervous system of Planorbarius corneus.

Two groups of cerebral dorsal cells of the pulmonate snail Planorbarius corneus stain positively with antisera raised against synthetic fragments of the B- and C-chain of the molluscan pro-insulin-related prohormone, proMIP-I, of another pulmonate snail, Lymnaea stagnalis. At the light-microscopic level the somata of the dorsal cells and their axons and neurohemal axon terminals in the periphery of the paired median lip nerves are immunoreactive with both antisera. Furthermore, the canopy cells in the lateral lobes of the cerebral ganglia are positive. In addition, MIPB-immunoreactive neurons are found in most other ganglia of the central nervous system. At the ultrastructural level, pale and dark secretory granules are found in somata and axon terminals of the dorsal cells. Dark granules are about 4 times as immunoreactive to both antisera as pale granules. Release of anti-MIPB- and anti-MIPC-immunopositive contents of the secretory granules by exocytosis is apparent in material treated according to the tannic acid method. It is concluded that the dorsal and canopy cells synthesize a molluscan insulin-related peptide that is packed in the cell body into secretory granules and that is subsequently transported to the neurohemal axon terminals and released into the hemolymph by exocytosis. Thus, MIP seems to act as a neurhormone on peripheral targets. On the basis of the analogy between the dorsal cells and the MIP-producing cells in L. stagnalis, it is proposed that the dorsal cells of P. corneus are involved in the control of body growth and associated processes.

Immuno-electron microscopy of sorting and release of neuropeptides in Lymnaea stagnalis.

The cerebral caudodorsal cells of the pulmonate snail Lymnaea stagnalis control egg laying and egg laying behavior by releasing various peptides derived from two precursors. The biosynthesis, storage, intracellular breakdown and release of three caudodorsal cell peptides were studied by means of immuno-electron microscopy using antisera raised to fragments of these peptides: (1) Caudodorsal Cell Hormone-I (CDCH-I; derived from precursor I), (2) Caudodorsal Cell Hormone-II (CDCH-II; from precursor II), and (3) alpha-Caudodorsal Cell Peptide (alpha CDCP; from both precursors). After affinity purification of the antisera, the specificity of the sera was confirmed with dotting immunobinding assays. From the ultrastructural immunocytochemical data it has been concluded that the precursor molecules are cleaved at the level of the Golgi apparatus after which the C-terminal parts (containing alpha CDCP) and N-terminal parts (containing DCDH-I or CDCH-II) are sorted and preferentially packaged into large electron-dense granules (MD 150 nm), respectively. Very probably, the content of the large electron-dense granules is degraded within the cell body. The immunoreactivity of the secretory granules increases during discharge from the Golgi apparatus, indicating further processing. At least a portion of the secretory granules contains all three peptides, as shown by double and triple immunopositive stainings whereas other granules appear to contain only one or two of these peptides. The caudodorsal cells release multiple peptides via exocytosis from neurohemal axon terminals into the hemolymph and from blindly ending axon collaterals into the intercellular space of the cerebral commissure (nonsynaptic release).

Ultrastructural evidence for synthesis, storage and release of insulin-related peptides in the central nervous system of Lymnaea stagnalis.

The cerebral neuroendocrine Light Green Cells of the pulmonate snail Lymnaea stagnalis, which control body growth and associated processes, stain positively with an affinity-purified antiserum raised to a large part of the C-chain of pro-molluscan insulin-related peptides. At the ultrastructural level, the rough endoplasmic reticulum is immunonegative, the Golgi apparatus is slightly positive and secretory granules in the process of budding from the Golgi apparatus are strongly positive. These observations indicate that the Light Green Cells synthesize molluscan insulin-related peptides, which are processed before packing by the Golgi apparatus into secretory granules. The two morphologically distinct secretory granule types, i.e. with pale and dark contents, respectively, are equally immunoreactive with antiserum raised to the C-chain of molluscan insulin-related peptides. Secretory granules within lysosomal structures reveal various degrees of immunoreactivity, indicating their graded breakdown. The Light Green Cells release secretory material by the process of exocytosis into the haemolymph from neurohaemal axon terminals located in the periphery of the median lip nerve. The electron-dense (tannic acid method) released contents are clearly immunopositive. The same holds for secretory granule contents released from Light Green Cells axon profiles in the centre of the lip nerve. Some immunoreactivity is also present in the intercellular space between these axon profiles. It is concluded that molluscan insulin-related peptides may act in two ways, namely (1) as neurohormones via the haemolymph at peripheral targets and (2) in a non-synaptic (paracrine) fashion at targets within the central nervous system.