Molecular dissection of the migrating posterior lateral line primordium during early development in zebrafish

<p>Abstract</p> <p>Background</p> <p>Development of the posterior lateral line (PLL) system in zebrafish involves cell migration, proliferation and differentiation of mechanosensory cells. The PLL forms when cranial placodal cells delaminate and become a coherent, migratory primordium that traverses the length of the fish to form this sensory system. As it migrates, the primordium deposits groups of cells called neuromasts, the specialized organs that contain the mechanosensory hair cells. Therefore the primordium provides both a model for studying collective directional cell migration and the differentiation of sensory cells from multipotent progenitor cells.</p> <p>Results</p> <p>Through the combined use of transgenic fish, Fluorescence Activated Cell Sorting and microarray analysis we identified a repertoire of key genes expressed in the migrating primordium and in differentiated neuromasts. We validated the specific expression in the primordium of a subset of the identified sequences by quantitative RT-PCR, and by <it>in situ </it>hybridization. We also show that interfering with the function of two genes, <it>f11r </it>and <it>cd9b</it>, defects in primordium migration are induced. Finally, pathway construction revealed functional relationships among the genes enriched in the migrating cell population.</p> <p>Conclusions</p> <p>Our results demonstrate that this is a robust approach to globally analyze tissue-specific expression and we predict that many of the genes identified in this study will show critical functions in developmental events involving collective cell migration and possibly in pathological situations such as tumor metastasis.</p

The tip-link antigen, a protein associated with the transduction complex of sensory hair cells, is protocadherin-15

Sound and acceleration are detected by hair bundles, mechanosensory structures located at the apical pole of hair cells in the inner ear. The different elements of the hair bundle, the stereocilia and a kinocilium, are interconnected by a variety of link types. One of these links, the tip link, connects the top of a shorter stereocilium with the lateral membrane of an adjacent taller stereocilium and may gate the mechanotransducer channel of the hair cell. Mass spectrometric and Western blot analyses identify the tip-link antigen, a hitherto unidentified antigen specifically associated with the tip and kinocilial links of sensory hair bundles in the inner ear and the ciliary calyx of photoreceptors in the eye, as an avian ortholog of human protocadherin-15, a product of the gene for the deaf/blindness Usher syndrome type 1F/DFNB23 locus. Multiple protocadherin-15 transcripts are shown to be expressed in the mouse inner ear, and these define four major isoform classes, two with entirely novel, previously unidentified cytoplasmic domains. Antibodies to the three cytoplasmic domain-containing isoform classes reveal that each has a different spatiotemporal expression pattern in the developing and mature inner ear. Two isoforms are distributed in a manner compatible for association with the tip-link complex. An isoform located at the tips of stereocilia is sensitive to calcium chelation and proteolysis with subtilisin and reappears at the tips of stereocilia as transduction recovers after the removal of calcium chelators. Protocadherin-15 is therefore associated with the tip-link complex and may be an integral component of this structure and/or required for its formatio

Transcriptional signature of accessory cells in the lateral line, using the Tnk1bp1:EGFP transgenic zebrafish line

<p>Abstract</p> <p>Background</p> <p>Because of the structural and molecular similarities between the two systems, the lateral line, a fish and amphibian specific sensory organ, has been widely used in zebrafish as a model to study the development/biology of neuroepithelia of the inner ear. Both organs have hair cells, which are the mechanoreceptor cells, and supporting cells providing other functions to the epithelium. In most vertebrates (excluding mammals), supporting cells comprise a pool of progenitors that replace damaged or dead hair cells. However, the lack of regenerative capacity in mammals is the single leading cause for acquired hearing disorders in humans.</p> <p>Results</p> <p>In an effort to understand the regenerative process of hair cells in fish, we characterized and cloned an <it>egfp </it>transgenic stable fish line that trapped <it>tnks1bp1</it>, a highly conserved gene that has been implicated in the maintenance of telomeres' length. We then used this Tg(<it>tnks1bp1</it>:EGFP) line in a FACsorting strategy combined with microarrays to identify new molecular markers for supporting cells.</p> <p>Conclusions</p> <p>We present a Tg(<it>tnks1bp1</it>:EGFP) stable transgenic line, which we used to establish a transcriptional profile of supporting cells in the zebrafish lateral line. Therefore we are providing a new set of markers specific for supporting cells as well as candidates for functional analysis of this important cell type. This will prove to be a valuable tool for the study of regeneration in the lateral line of zebrafish in particular and for regeneration of neuroepithelia in general.</p

Phoenix Is Required for Mechanosensory Hair Cell Regeneration in the Zebrafish Lateral Line

In humans, the absence or irreversible loss of hair cells, the sensory mechanoreceptors in the cochlea, accounts for a large majority of acquired and congenital hearing disorders. In the auditory and vestibular neuroepithelia of the inner ear, hair cells are accompanied by another cell type called supporting cells. This second cell population has been described as having stem cell-like properties, allowing efficient hair cell replacement during embryonic and larval/fetal development of all vertebrates. However, mammals lose their regenerative capacity in most inner ear neuroepithelia in postnatal life. Remarkably, reptiles, birds, amphibians, and fish are different in that they can regenerate hair cells throughout their lifespan. The lateral line in amphibians and in fish is an additional sensory organ, which is used to detect water movements and is comprised of neuroepithelial patches, called neuromasts. These are similar in ultra-structure to the inner ear's neuroepithelia and they share the expression of various molecular markers. We examined the regeneration process in hair cells of the lateral line of zebrafish larvae carrying a retroviral integration in a previously uncharacterized gene, phoenix (pho). Phoenix mutant larvae develop normally and display a morphologically intact lateral line. However, after ablation of hair cells with copper or neomycin, their regeneration in pho mutants is severely impaired. We show that proliferation in the supporting cells is strongly decreased after damage to hair cells and correlates with the reduction of newly formed hair cells in the regenerating phoenix mutant neuromasts. The retroviral integration linked to the phenotype is in a novel gene with no known homologs showing high expression in neuromast supporting cells. Whereas its role during early development of the lateral line remains to be addressed, in later larval stages phoenix defines a new class of proteins implicated in hair cell regeneration

Criblage d'une mutagenèse chimique dans les embryons de poisson-zèbre axé sur la mobilité

Mon travail de thèse a porté sur un criblage de mutagenèse chimique dans des embryons de poisson-zèbre induisant des mutations ponctuelles. Les phénotypes retenus correspondent à deux critères, la mobilité déficiente des embryons et la présence de nécrose dans le tube neural, structure à l'origine des tissus nerveux de l'adulte. Le but était de muter des gènes de la mise en place des tissus nerveux et musculaires. Dans la première partie de ma thèse j'ai analysé et caractérisé ces lignées. En collaboration avec une équipe INRA, nous les avons criblé, pour trouver des mutants de l'acétylcholinestérase, l'enzyme chargé de la dégradation de l'acétylcholine, le neurotransmetteur neuromusculaire. Le mutant que nous avons trouvé s'appelle acetylcholinesterase (ache). Nous avons analysé le phénotype en détail. En faisant un double mutant, avec le mutant nicotinic receptors (nic), totalement dépourvu de récepteurs nicotiniques de l'acétylcholine, nous avons pu montrer clairement, que le phénotype musculaire dans ache se fait à travers ce récepteur. La plupart des insecticides et des médicaments palliatifs contre l'Alzheimer ou la myasthénis gravis, fonctionnent par blocage de cet enzyme. Le mutant ache est le premier modèle vertébré, pour tester leurs effets secondaires. L'ensemble de la démarche est donc validé par ce mutant, qui fourni un modèle pour résoudre des questions ne pouvant être adressées avec la souris " Knock out ". Ce travail fournit aussi un ensemble de mutants, dont la nature moléculaire reste à élucider, mais qui pourra contribuer à la compréhension de mécanismes fondamentaux. Ces connaissances pourraient aussi être rapidement applicables à l'Homme, tout particulièrement pour établir des thérapies pour certaines maladies génétiques humaines, comme les myopathies, dont les symptômes sont souvent très proches, des phénotypes de nos lignées mutantes.During my PhD, I did a screen of a chemical mutagenesis in zebrafish embryos, inducing point mutations. We used two screening criteria's, the altered mobility of embryos and the presence of necroses in the neural tube, the structure giving rise to most of the adult nervous system. The goal was to find mutated genes implicated in the layout of a functional loco motor apparatus. In the first part of my thesis I analysed and characterized the mutant lines. In collaboration with an INRA group, we screened the lines for mutants in the acetylcholinesterase gene, coding for the enzyme (AChE) degrading the neuromuscular neurotransmitter acetylcholine (Ach). We called the mutant acetylcholinesterase (ache). We analysed the phenotype and by crossing it with the mutant nicotinic receptors (nic), lacking functional nicotinic receptors for Ach, we could show that the muscular phenotype is receptor dependant. Most of the insecticides and drugs against Alzheimer disease and myasthenis gravis are inhibitors of the AChE. So the mutant ache provides the first vertebrate model to test side effects of theses drugs. This mutant validates our screen and provides a model to tackle questions which can not be addressed in the mouse knock out. This work also provide a set of neuromuscular mutants, in which the molecular nature still remains to be determine, but which could improve our knowledge in the setting up of neuronal and muscular tissues. They might be of particular relevance in genetic diseases, like myopathies as some symptoms are highly reminiscent of some of the phenotypes of our lines.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Effect of water composition on aluminium, calcium and organic carbon extraction in tea infusions

Tea infusion is the second world wide consumed beverage. In this study, total aluminium and calcium concentrations, total organic carbon and total polyphenol content were determined and compared with respect to the origin of tea leaves, their particle size (broken or whole leaves) and also the mineral composition of four waters. It appeared that the higher the mineral content, the lower the extraction yield of aluminium, total organic carbon and total polyphenols. This could be due to calcium uptake by leaves. Calcium present in mineral waters could be complexed with pectins present in cell walls thus leading to a decrease in the element extraction