DNA interference: DNA-induced gene silencing in the appendicularian Oikopleura dioica.

RNA interference is widely employed as a gene-silencing system in eukaryotes for host defence against invading nucleic acids. In response to invading double-stranded RNA (dsRNA), mRNA is degraded in sequence-specific manner. So far, however, DNA interference (DNAi) has been reported only in plants, ciliates and archaea, and has not been explored in Metazoa. Here, we demonstrate that linear double-stranded DNA promotes both sequence-specific transcription blocking and mRNA degradation in developing embryos of the appendicularian Oikopleura dioica. Introduced polymerase chain reaction (PCR) products or linearized plasmids encoding Brachyury induced tail malformation and mRNA degradation. This malformation was also promoted by DNA fragments of the putative 5'-flanking region and intron without the coding region. PCR products encoding Zic-like1 and acetylcholine esterase also induced loss of sensory organ and muscle acetylcholinesterase activity, respectively. Co-injection of mRNA encoding EGFP and mCherry, and PCR products encoding these fluorescent proteins, induced sequence-specific decrease in the green or red fluorescence, respectively. These results suggest that O. dioica possesses a defence system against exogenous DNA and RNA, and that DNA fragment-induced gene silencing would be mediated through transcription blocking as well as mRNA degradation. This is the first report of DNAi in Metazoa.

Atypical guanylyl cyclase from the pond snail Lymnaea stagnalis: cloning, sequence analysis and characterization of expression.

Soluble guanylyl cyclases (sGCs) are traditionally recognized as the main molecular receptor for nitric oxide (NO), a gaseous transmitter involved in many functions of the nervous system. Some sGCs are however insensitive to NO and therefore are known as atypical. Although atypical sGCs have been shown to exist in both vertebrate and invertebrate nervous systems, our understanding of their functional role is incomplete. Here we report on the cloning, sequencing and localization of an atypical sGC named Lym-sGCbeta3 from the snail Lymnaea stagnalis. We found that Lym-sGCbeta3 shares a number of structural characteristics with some previously characterized atypical sGCs including the presence of Tyr140 in the regulatory domain. This residue is thought to be of a critical importance in determining sensitivity of atypical sGCs to oxygen. These findings raise the possibility that Lym-sGCbeta3 is an oxygen receptor. The results of our in situ hybridization and RT-PCR experiments support this idea further by showing that Lym-sGCbeta3 is expressed in the osphradium, a peripheral sense organ in which oxygen-sensing neurons are located. Also of interest are our observations that many neurons in Lymnaea CNS co-express conventional and atypical sGC subunits. These data are consistent with a possible dominant negative regulatory role of atypical sGC subunits through the formation of heterodimers exhibiting low enzymatic activity.

Dynamic expression of the retinoic acid-synthesizing enzyme retinol dehydrogenase 10 (rdh10) in the developing mouse brain and sensory organs.

Organs develop through many tissue interactions during embryogenesis, involving numerous signaling cascades and gene products. One of these signaling molecules is retinoic acid (RA), an active vitamin A derivative, which in mammalian embryos is synthesized from maternal retinol by two oxidative reactions involving alcohol/retinol dehydrogenases (ADH/RDHs) and retinaldehyde dehydrogenases (RALDHs), respectively. The activity of RALDHs is known to be crucial for RA synthesis; however, recently a retinol dehydrogenase (RDH10) has been shown to represent a new limiting factor in this synthesis. We investigated the spatiotemporal distribution of Rdh10 gene transcripts by in situ hybridization and quantitative polymerase chain reaction (PCR) during development of the brain and sensory organs. Although Rdh10 relative mRNA levels decline throughout brain development, we show a strong and lasting expression in the meninges and choroid plexuses. Rdh10 expression is also specifically seen in the striatum, a known site of retinoid signaling. In the eye, regional expression is observed both in the prospective pigmented epithelium and neural retina. In the inner ear Rdh10 expression is specific to the endolymphatic system and later the stria vascularis, both organs being involved in endolymph homeostasis. Furthermore, in the peripheral olfactory system and the vibrissae follicles, expression is present from early stages in regions where sensory receptors appear and mesenchymal/epithelial interactions take place. The distribution of Rdh10 transcripts during brain and sensory organ development is consistent with a role of this enzyme in generating region-specific pools of retinaldehyde that will be used by the various RALDHs to refine the patterns of RA synthesis.

Gene identification and evidence for expression of G protein alpha subunits, phospholipase C, and an inositol 1,4,5-trisphosphate receptor in Aplysia californica rhinophore.

In the marine mollusk Aplysia californica, waterborne protein pheromones that are released during egg laying act in concert to stimulate mate attraction. However, molecular information concerning the cellular receptors and signaling mechanisms that may be involved in waterborne peptide and protein pheromonal communication is lacking. As a first step toward examining whether members of the G protein family and phosphoinositide signaling pathway are present in the primary peripheral chemosensory organs (i.e., rhinophores), we isolated five full-length cDNA clones from an A. californica central nervous system cDNA library. These clones encoded (1) the G protein alpha subunits of the Gq, Gi, and Go families, (2) a protein with homology to phospholipase C (PLC) isoforms, and (3) an inositol 1,4,5-trisphosphate receptor (IP3R). The expression of these genes was examined using laser capture microdissection/reverse transcription-polymerase chain reaction and in situ hybridization. All of them are expressed in the rhinophore sensory epithelium, suggesting that Galphaq, Galphai, Galphao, PLC-like protein, and IP3R may be involved in waterborne protein pheromone detection in Aplysia-possibly via a phosphoinositide signaling mechanism.

Cholinergic neurons mediate CaMKII-dependent enhancement of courtship suppression.

In Drosophila, calcium/calmodulin-dependent protein kinase II (CaMKII) activity is crucial in associative courtship conditioning for both memory formation and suppression of courtship during training with a mated female. We have previously shown that increasing levels of constitutively active CaMKII, but not calcium-dependent CaMKII, in a subset of neurons can decrease the initial level of courtship and enhance the rate of suppression of courtship in response to a mated female. In this study, we demonstrate that a subpopulation of noncholinergic, nondopaminergic, non-GABAergic neurons can cause CaMKII-dependent reductions in initial courtship, but only cholinergic neurons enhance training-dependent suppression. These data suggest that processing of pheromonal signals in two subpopulations of neurons, likely antennal lobe projection neurons, is critical for behavioral plasticity.

Receptor protein tyrosine phosphatase gamma is a marker for pyramidal cells and sensory neurons in the nervous system and is not necessary for normal development.

In order to gain insight into the biological role of receptor protein tyrosine phosphatase gamma (RPTPgamma), we have generated RPTPgamma-null mice. RPTPgamma was disrupted by insertion of the beta-galactosidase gene under the control of the RPTPgamma promoter. As the RPTPgamma-null mice did not exhibit any obvious phenotype, we made use of these mice to study RPTPgamma expression and thus shed light on potential biological functions of this phosphatase. Inspection of mouse embryos shows that RPTPgamma is expressed in a variety of tissues during embryogenesis. RPTPgamma is expressed in both embryonic and adult brains. Specifically, we detected RPTPgamma expression in cortical layers II and V and in the stratum pyramidale of the hippocampus, indicating that RPTPgamma is a marker for pyramidal neurons. Mixed primary culture of glial cells showed a lack of expression of RPTPgamma in astrocytes and a low expression of RPTPgamma in oligodendrocytes and in microglia. Interestingly, RPTPgamma expression was detected in all sensory organs, including the ear, nose, tongue, eye, and vibrissa follicles, suggesting a potential role of RPTPgamma in sensory neurons. An initial behavioral analysis showed minor changes in the RPTPgamma-null mice.

Synaptic ultrastructure of Drosophila Johnston's organ axon terminals as revealed by an enhancer trap.

The role of auditory circuitry is to decipher relevant information from acoustic signals. Acoustic parameters used by different insect species vary widely. All these auditory systems, however, share a common transducer: tympanal organs as well as the Drosophila flagellar ears use chordotonal organs as the auditory mechanoreceptors. We here describe the central neural projections of the Drosophila Johnston's organ (JO). These neurons, which represent the antennal auditory organ, terminate in the antennomechanosensory center. To ensure correct identification of these terminals we made use of a beta-galactosidase-expressing transgene that labels JO neurons specifically. Analysis of these projection pathways shows that parallel JO fibers display extensive contacts, including putative gap junctions. We find that the synaptic boutons show both chemical synaptic structures as well as putative gap junctions, indicating mixed synapses, and belong largely to the divergent type, with multiple small postsynaptic processes. The ultrastructure of JO fibers and synapses may indicate an ability to process temporally discretized acoustic information.

A tissue specific cytochrome P450 required for the structure and function of Drosophila sensory organs.

Cytochrome P450s have generally been acknowledged as broadly tuned detoxifying enzymes. However, emerging evidence argues P450s have an integral role in cell signaling and developmental processes, via their metabolism of retinoic acid, arachidonic acid, steroids, and other cellular ligands. To study the morphogenesis of Drosophila sensory organs, we examined mutants with impaired mechanosensation and discovered one, nompH, encodes the cytochrome P450 CYP303a1. We now report the characterization of nompH, a mutant defective in the function of peripheral chemo- and mechanoreceptor cells, and demonstrate CYP303a1 is essential for the development and structure of external sensory organs which mediate the reception of vital mechanosensory and chemosensory stimuli. Notably this P450 is expressed only in sensory bristles, localizing in the apical region of the socket cell. The wide diversity of the P450 family and the growing number of P450s with developmental phenotypes suggests the exquisite tissue and subcellular specificity of CYP303a1 illustrates an important aspect of P450 function; namely, a strategy to process critical developmental signals in a tissue- and cell-specific manner.

Developmental expression pattern of monoamine oxidases in sensory organs and neural crest derivatives.

Serotonin (5-HT) has been shown to act as a morphogen in craniofacial and heart development and in the migration of neural crest derivatives. Some of these structures are capable of capturing 5-HT during development, but nothing is known about the localization of the main monoamine degradation enzymes, monoamine oxidase (MAO) A and B, in these developing tissues. We generated a highly specific antibody to MAOB; immunoreactivity is entirely abolished in brain extracts or brain sections of mice lacking MAOB. From the use of this antibody and specific riboprobes, we report that MAOB is expressed early in a variety of neural crest derivatives, in facial sensory organs, and in the heart. From E11.5 to P0, MAOB was found to be strongly expressed in the following neural crest derivatives: the aorta, cranial mesenchyme (developing bones, sensory neurons of the cranial ganglia, cartilages, thyroid, and striate muscles), dental mesenchyme, several soft palate derivatives, and boundary cap cells (E11.5-P4). Boundary cap cells contribute to the formation of nerve exit-entry points between the central and the peripheral nervous systems. Several facial sensory organs also contained MAOB mRNA, protein, and activity. High MAOB expression was noted in the olfactory placode, the dorsal part of the olfactory epithelium, the olfactory nerve layer (probably the ensheathing glia), the cochlear ganglionic cells, the taste buds, and the Merkel cells in the vibrissae follicles. Finally, we found that MAOB is massively expressed in the pharyngeal organ, heart, liver, and mast cells. In contrast, MAOA expression was restricted to the sympathetic ganglia and to the meningeal and capillary blood vessels. The pattern of MAOB expression generally matched the previously reported patterns of expression of the plasma 5-HT transporter expression or of the histamine biosynthetic enzyme L-histidine decarboxylase, suggesting a role for MAOB in fine regulation of the levels of 5-HT and histamine in the developing embryo.

New fluorinated derivatives as esterase inhibitors. Synthesis, hydration and crossed specificity studies.

A variety of new fluorinated chemicals have been prepared for the first time and tested as inhibitors of esterases, one of the main enzymes involved in pheromone catabolism, in two economically important pests, the Egyptian armyworm Spodoptera littoralis (SL) and the Mediterranean corn borer Sesamia nonagrioides (SN). Using the respective major component of the pheromone as substrate, the K(m) and V(max) of the antennal esterase of both insects resulted to be 5.66 x 10(-4) M and 8.47 x 10(-6) Mmin(-1) for SL and 1.61 x 10(-7) M and 1.25 x 10(-7) Mmin(-1) for SN, pointing out that SN esterase has a higher affinity for its corresponding substrate than SL. In general, the trifluoromethyl ketones (TFMKs) exhibited higher inhibitory potency than the corresponding difluoromethyl ketones (DFMKs) or difluoroaldehydes (DFAs). The compounds appeared to hydrate differently in aqueous solution, the extent of hydration following the order: alpha,alpha-DFMKs

Two Na,K-ATPase beta 2 subunit isoforms are differentially expressed within the central nervous system and sensory organs during zebrafish embryogenesis.

We have identified cDNAs encoding a second zebrafish ortholog of the human Na,K-ATPase beta 2 subunit. The beta 2b cDNA encodes a 292 amino acid-long polypeptide with 74% identity to the previously characterized zebrafish beta 2a subunit. By using a zebrafish meiotic mapping panel, we determined that the beta 2b gene (atp1b2b) was tightly linked to markers on linkage group 5, whereas the beta 2a gene was located on linkage group 23. In situ hybridization analysis shows that in developing zebrafish embryos, atp1b2a and atp1b2b are predominantly expressed in the nervous system. beta 2a transcripts were abundantly expressed throughout brain as well as spinal cord neurons and lateral line ganglia. In contrast, beta 2b mRNA expression was primarily detected in sensory organs, including retina, otic vesicles, and lateral line neuromast cells. These results suggest that the beta 2a and beta 2b genes play distinct roles in developing brain and sensory organs, and raise the possibility that the functions encoded by the single mammalian beta 2 gene may be partitioned between the two zebrafish beta 2 orthologs.

A CUB-serine protease in the olfactory organ of the spiny lobster Panulirus argus.

csp, a gene encoding a protein with high sequence identity to trypsinlike serine protease and CUB domains, was identified from a cDNA library from the olfactory organ (antennular lateral flagellum) of the spiny lobster Panulirus argus. The full-length cDNA sequence of csp is 1801 bp, encoding a protein of 50.25 kD, with three domains: signal peptide, trypsinlike serine protease, and CUB (named for a class of compounds including Complement subcomponents Clr/Cls, Uegf, and Bone morphogenic protein-1). RT-PCR, Northern blots, and immunoblots showed that csp is predominantly expressed in the lateral flagellum and eyestalk. Immunocytochemistry showed that Csp is present in olfactory (aesthetasc) sensilla around auxiliary cells (glia that surround the inner dendrites of olfactory receptor neurons, ORNs) and ORN outer dendrites. We propose that Csp is expressed and secreted by auxiliary cells, associates with ORN cell membranes or extracellular matrix via the CUB domain, and has trypsinlike activity. In the eyestalk, Csp is associated with cells surrounding axons between neuropils of the eyestalk ganglia. Possible functions in the olfactory organ and eyestalk are discussed. To our knowledge, this is the first report from any olfactory system of a gene encoding a protein with serine protease and CUB domains.

Preferential expression of biotransformation enzymes in the olfactory organs of Drosophila melanogaster, the antennae.

Biotransformation enzymes have been found in the olfactory epithelium of vertebrates. We now show that in Drosophila melanogaster, a UDP-glycosyltransferase (UGT), as well as a short chain dehydrogenase/reductase and a cytochrome P450 are expressed specifically or preferentially in the olfactory organs, the antennae. The evolutionarily conserved expression of biotransformation enzymes in olfactory organs suggests that they play an important role in olfaction. In addition, we describe five Drosophila UGTs belonging to two families. All five UGTs contain a putative transmembrane domain at their C terminus as is the case for vertebrate UGTs where it is required for enzymatic activity. The primary sequence of the C terminus, including part of the transmembrane domain, differs between the two families but is highly conserved not only within each Drosophila family, but also between the members of one of the Drosophila families and vertebrate UGTs. The partial overlap of the conserved primary sequence with the transmembrane domain suggests that this part of the protein is involved in specific interactions occurring at the membrane surface. The presence of different C termini in the two Drosophila families suggests that they interact with different targets, one of which is conserved between Drosophila and vertebrates.

Glutamine synthetase is a glial-specific marker in the olfactory regions of the lobster (Panulirus argus) nervous system.

Glutamine synthetase (GS) has been qualified as a very specific marker of astroglial-type neuroglia in vertebrate neural tissues. In this paper we have begun to examine the possibility that glial localization of GS could be a ubiquitous characteristic of complex nervous systems. To this end we have used immunohistochemistry to localize GS-like immunoreactivity in the olfactory regions of the complex nervous system of the arthropod, the spiny lobster Panulirus argus. We describe a novel method for affinity isolation of antibodies from crude serum. Using this approach we purified GS-specific antibodies to chick retina GS and used these to analyze the lobster brain and the primary olfactory organ. Western blots showed that the lobster brain contains an immunoreactive peptide with nearly the same molecular mass as that of chick retina GS. Northern blot analyses of mRNA and enzymatic activity assays also confirm that the lobster brain produces GS. Immunohistochemical staining of sectioned lobster olfactory lobes and sensory sensilla showed strong reactivity in specific cells. Comparison of the GS immunostaining pattern with that for FMRFamide, a well characterized marker of neurons in invertebrate neural tissues, it became clear that GS is indeed glial-specific in lobster neural tissues as it is in vertebrates. These results suggest that the compartmentalization of GS in non-neuronal cells is either an early step in neural evolution or is an obligate and fundamental characteristic of complex neural systems composed of both neurons and neuroglia.

Cellular events during development of the olfactory sense organs in Drosophila melanogaster.

The olfactory sensilla on the antenna of adult Drosophila melanogaster develop during the first 36 hr after pupariation, from their anlagen in the cephalic disc. We have used tissue-specific beta-galactosidase expression in the enhancer trap strain A101.IF3 and the monoclonal antibody 22C10 as sensory cell markers, as well as the lineage tracer 5-bromo-2'-deoxyuridine (BrdU), to describe this process. The development of an olfactory sensillum begins with the selection of a "founder cell" (FC). These cells are distinct in that they possess large apically located nuclei revealed by beta-galactosidase expression in A101.IF3. In the following 6 hr, a few cells neighboring the FC also start expressing beta-galactosidase and together comprise a group. Cells of this group, denoted a "presensillum-cluster" (PSC), undergo at least one round of replication and give rise to all of the cells of a sensillum. A subset of the cells within each PSC and, later, all the sensory neurons are recognized by MAb22C10. The antennae of the mutant lozenge3 (lz3) lack all basiconic and some trichoid sensilla. The mutation apparently affects early steps in sensillum development and many of the FCs fail to form. Those that are present, however, proceed to form mature olfactory sensilla. Therefore, we conclude that the selection of an FC is the first step in olfactory sense organ development. Our study reveals novel aspects of sensory development in Drosophila.

Choline acetyltransferase immunoreactive neurons innervating labyrinthine and lateral line sense organs in amphibians.

The goal of the present study was to investigate aspects of the central organization of the neurons belonging to the octavolateralis efferent system of amphibians. The perikarya of three genera, Pleurodeles, Xenopus, and Discoglossus, were located in the brainstem by applying retrograde tracers to the appropriate cranial nerves and choline acetyltransferase immunohistochemistry was used to identify cholinergic neurons. The efferent neurons supplying lateral line (Pleurodeles, Xenopus) and labyrinthine (Pleurodeles, Xenopus, and Discoglossus) end organs were found to intermingle in a single octavolateralis efferent nucleus. The neurons lie bilateral to the labelled nerves in Pleurodeles and ipsilateral in Xenopus and Discoglossus. Separate labelling of the anterior and posterior octavus rami provided no evidence for distinct groupings of efferent neurons that could be associated with auditory and vestibular end organs. In all three species many if not all octavolateral efferent neurons displayed immunoreactivity for choline acetyltransferase. They could be distinguished from the cholinergic facial motoneurons, with which they sometimes intermingle, on the basis of either their distinctive size and shape (Pleurodeles, Xenopus) or their location (Discoglossus). Double labelling in Xenopus confirmed the cholinergic nature of the efferent neurons.

High-affinity cAMP phosphodiesterase and adenosine localized in sensory organs.

Odorant-stimulated formation of cAMP in olfactory receptor neurons may mediate olfactory signal transduction. The response is short and desensitization occurs rapidly, possibly by induction of cyclic nucleotide phosphodiesterase (PDE) activity. Previously, we showed that two low Km PDEs regulate hydrolysis of cAMP in olfactory cilia. One PDE is Ca2+/calmodulin-dependent and non-selective for both cAMP-PDE and cGMP; the other is Ca2+/calmodulin-independent, sensitive to rolipram and selective for cAMP. We have localized cAMP-selective PDE in olfactory, gustatory and retinal sensory systems by autoradiography with the selective inhibitor [3H]rolipram. We observe dense binding over olfactory neurons, particularly over olfactory nerve bundles and olfactory cilia. In the tongue apical regions of taste buds of the circumvallate papillae are strongly labeled as well as portions of the glossopharyngeal nerve. Retinal binding is most dense over the inner plexiform layer, ganglion cells and the optic nerve but is also substantial over the inner nuclear layer. The pattern of [3H]rolipram-binding in retina is reminiscent of adenosine localization. Accordingly, adenosine was immunohistochemically localized in olfactory, gustatory and retinal tissues. Adenosine immunoreactivity is observed in olfactory neurons, in the basal regions of taste buds and in retinal ganglion cells.