Molekulare Untersuchungen zur Innenohrmorphogenese bei der Maus (mus musculus).

Die Expressionsmuster der Homeoboxgene Nkx5-1, Nkx5-2 und des 'paired box' Gens Pax2 sowie des Tyrosinkinase Rezeptorgens sek wurden zu unterschiedlichen Zeitpunkten der Innenohrmorphogenese, während der normalen Entwicklung und unter veränderten in vivo und in vitro Situationen, untersucht. Die Expressionsmuster im sich entwickelnden Innenohr von Wildtyp Embryonen stellen die Basis für vergleichende Analysen in verschiedenen Innenohrmutanten der Maus dar. Die fidget Maus, eine Gleichgewicht-Mutante, die mit der Transkriptverteilung des Nkx5-1 Gens im vestibulären Apparat korrelierende Innenohrdefekte aufweist, wurde untersucht. Um Hinweise auf externe, die Innenohrentwicklung steuernde Signale, die insbesondere die Aktivität des Nkx5-1 Gens regulieren, zu erhalten, wurden auch Mutanten, deren Innenohrdefekte auf Hinterhirnmißbildungen zurückzuführen sind (splotch, Hoxa-1), analysiert. Es wurde keine Veränderung in der Expression der untersuchten Gene detektiert. Mittels des Expressionsnachweises des Melanoblasten-spezifischen Markergens Trp2 konnte gezeigt werden, daß in splotch Mausmutanten keine Melanoblasten aus der Neuralleiste in das Innenohrepithel einwandern. In einem hier optimierten Innenohr in vitro Kultursystem wurden einzelne Schritte der Innenohrmorphogenese nachgestellt und demonstriert, daß die in vivo charakterisierten Genexpressionsmuster unter in vitro Bedingungen in Organ- und Organ-ähnlichen Kulturen nachvollziehbar sind. Verschiedene, modifizierende Bedingungen und funktionelle Anwendungen dieses in vitro Modellsystems wurden untersucht und diskutiert.Differential gene expression during mouse inner ear development was analysed under normal and altered in vivo and in vitro conditions. The expression pattern of the homeoboxgenes Nkx5-1 and Nkx5-2, the paired box gene Pax-2 and the tyrosinkinase receptor genesek was analysed in detail in structures of the developing inner ear. These genes play regulatory roles in transcription or in cell-cell signalling respectively (sek) and may be important for embryonal patterning and/or cell type specification within the inner ear. To investigate signals which regulate especially the activity of the Nkx5-1 gene, the wildtype expression of this gene was compared with those of inner ear mutant embryos. Mouse mutants with primary inner ear defects (fidget) and those with inner ear defects resulting from hindbrain malformations (splotch, Hoxa-1) were analysed. The mouse mutants were also analysed for Nkx5-2, Msx-1, Pax-2 and FGFR2 gene expression. There was no altered gene expression detectable. Further more it could be shown that in splotch mutant embryos migrating melanoblasts from neural crest which populate the inner ear epithelium in wildtype embryos are absent. An inner ear in vitro culture system was established, here the different steps of inner ear morphogenesis such as gene expression pattern could be performed. Modification and functional application of this system were investigated and discussed

Retroviral enhancer detection insertions in zebrafish combined with comparative genomics reveal genomic regulatory blocks - a fundamental feature of vertebrate genomes

A large-scale enhancer detection screen was performed in the zebrafish using a retroviral vector carrying a basal promoter and a fluorescent protein reporter cassette. Analysis of insertional hotspots uncovered areas around developmental regulatory genes in which an insertion results in the same global expression pattern, irrespective of exact position. These areas coincide with vertebrate chromosomal segments containing identical gene order; a phenomenon known as conserved synteny and thought to be a vestige of evolution. Genomic comparative studies have found large numbers of highly conserved noncoding elements (HCNEs) spanning these and other loci. HCNEs are thought to act as transcriptional enhancers based on the finding that many of those that have been tested direct tissue specific expression in transient or transgenic assays. Although gene order in hox and other gene clusters has long been known to be conserved because of shared regulatory sequences or overlapping transcriptional units, the chromosomal areas found through insertional hotspots contain only one or a few developmental regulatory genes as well as phylogenetically unrelated genes. We have termed these regions genomic regulatory blocks (GRBs), and show that they underlie the phenomenon of conserved synteny through all sequenced vertebrate genomes. After teleost whole genome duplication, a subset of GRBs were retained in two copies, underwent degenerative changes compared with tetrapod loci that exist as single copy, and that therefore can be viewed as representing the ancestral form. We discuss these findings in light of evolution of vertebrate chromosomal architecture and the identification of human disease mutations

Mosaic hoxb4a Neuronal Pleiotropism in Zebrafish Caudal Hindbrain

To better understand how individual genes and experience influence behavior, the role of a single homeotic unit, hoxb4a, was comprehensively analyzed in vivo by clonal and retrograde fluorescent labeling of caudal hindbrain neurons in a zebrafish enhancer-trap YFP line. A quantitative spatiotemporal neuronal atlas showed hoxb4a activity to be highly variable and mosaic in rhombomere 7–8 reticular, motoneuronal and precerebellar nuclei with expression decreasing differentially in all subgroups through juvenile stages. The extensive Hox mosaicism and widespread pleiotropism demonstrate that the same transcriptional protein plays a role in the development of circuits that drive behaviors from autonomic through motor function including cerebellar regulation. We propose that the continuous presence of hoxb4a positive neurons may provide a developmental plasticity for behavior-specific circuits to accommodate experience- and growth-related changes. Hence, the ubiquitous hoxb4a pleitropism and modularity likely offer an adaptable transcriptional element for circuit modification during both growth and evolution

Activity of etv5a and etv5b genes in the hypothalamus of fasted zebrafish is influenced by serotonin

Serotonin has been implicated in the inhibition of food intake in vertebrates. However, the mechanisms through which serotonin acts has yet to be elucidated. Recently, ETV5 (ets variant gene 5) has been asso- ciated with obesity and food intake control mechanisms in mammals. We have analyzed a putative phys- iological function of the two etv5 paralogous genes (etv5a and etv5b) in neuronal food intake control in adult zebrafish that have been exposed to different nutritional conditions. A feeding assay was estab- lished and fluoxetine, a selective serotonin re-uptake inhibitor (SSRI), was applied. Gene expression changes in the hypothalamus were determined using real-time PCR. Fasting induced an up-regulation of etv5a and etv5b in the hypothalamus, whereas increased serotonin levels in the fasted fish counter- acted the increase in expression. To investigate potential mechanisms the expression of further food intake control genes was determined. The results show that an increase of serotonin in fasting fish causes a reduction in the activity of genes stimulating food intake. This is in line with a previously demonstrated anorexigenic function of serotonin. Our results suggest that obesity-associated ETV5 has a food intake stimulating function and that this function is modulated through serotonin.Fil: Mechaly, Alejandro. University of Sydey. Sydney Medical School; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Richardson, Ebony. University of Sydey. Sydney Medical School; AustraliaFil: Rinkwitz, Silke. University of Sydney. Sydney Medical School; Australi

Effective heritable gene knockdown in zebrafish using synthetic microRNAs

Although zebrafish is used to model human diseases through mutational and morpholino-based knockdown approaches, there are currently no robust transgenic knockdown tools. Here we investigate the knockdown efficiency of three synthetic miRNA-expressing backbones and show that these constructs can downregulate a sensor transgene with different degrees of potency. Using this approach, we reproduce spinal muscular atrophy (SMA) in zebrafish by targeting the smn1 gene. We also generate different transgenic lines, with severity and age of onset correlated to the level of smn1 inhibition, recapitulating for the first time the different forms of SMA in zebrafish. These lines are proof-of-concept that miRNA-based approaches can be used to generate potent heritable gene knockdown in zebrafish

A Simple and efficient protocol for the treatment of zebrafish colonies infected with parasitic nematodes

Our zebrafish colony experienced a period of increased mortality rate of 6.5 times more deaths per month in a colony of over 13,000 zebrafish (Danio rerio), which developed over 3 months. We observed that before death, affected fish appeared emaciated, often with an abdominal bulge. We performed dissection on 18 fish that had this appearance and found in 15 that their gut was infected with a nematode that closely resembled Pseudocapillaria tomentosa. We devised a treatment protocol for this nematode infection, which involved addition of fenbendazole, a drug used to treat nematode infections in cattle and sheep, to the fish feed. Fenbendazole produced no severe side effects in the fish and several treatments have effectively eradicated the parasite from our colony. The mortality rate of our fish has decreased to a value of 0.7%/month (p<0.001, equal to that before the infection). We propose this protocol as an inexpensive alternative to having to rederive an entire colony from bleached eggs, and as a prophylactic measure used in quarantine facilities on a regular basis. A correction exists for this article, and may be found at doi:10.1089/zeb.2013.0868.cxn.4 page(s

Tissue-specific models of spinal muscular atrophy confirm a critical role of SMN in motor neurons from embryonic to adult stages

Spinal muscular atrophy (SMA) is an autosomal recessive disease linked to survival motor neuron (SMN) protein deficiency. While SMN protein is expressed ubiquitously, its deficiency triggers tissue-specific hallmarks, including motor neuron death and muscle atrophy, leading to impaired motor functions and premature death. Here, using stable miR-mediated knockdown technology in zebrafish, we developed the first vertebrate system allowing transgenic spatio-temporal control of the smn1 gene. Using this new model it is now possible to investigate normal and pathogenic SMN function(s) in specific cell types, independently or in synergy with other cell populations. We took advantage of this new system to first test the effect of motor neuron or muscle-specific smn1 silencing. Anti-smn1 miRNA expression in motor neurons, but not in muscles, reproduced SMA hallmarks, including abnormal motor neuron development, poor motor function and premature death. Interestingly, smn1 knockdown in motor neurons also induced severe late-onset phenotypes including scoliosis-like body deformities, weight loss, muscle atrophy and, seen for the first time in zebrafish, reduction in the number of motor neurons, indicating motor neuron degeneration. Taken together, we have developed a new transgenic system allowing spatio-temporal control of smn1 expression in zebrafish, and using this model, we have demonstrated that smn1 silencing in motor neurons alone is sufficient to reproduce SMA hallmarks in zebrafish. It is noteworthy that this research is going beyond SMA as this versatile gene-silencing transgenic system can be used to knockdown any genes of interest, filling the gap in the zebrafish genetic toolbox and opening new avenues to study gene functions in this organism

Motor neuron-expressed microRNAs 218 and their enhancers are nested within introns of Slit2/3 genes

miR218-1 and miR218-2 are embedded in introns of SLIT2 and SLIT3, respectively, an arrangement conserved throughout vertebrate genomes. Both miR218 genes are predicted to be transcribed in the same orientation as their host genes and were assumed to be spliced from Slit2/3 primary transcripts. In zebrafish miR218 is active in cranial nerve motor nuclei and spinal cord motor neurons, while slit2 and slit3 are expressed predominantly in the midline. This differential expression pattern suggested independent regulation of miR218 genes by distinct enhancers. We tested conserved noncoding elements for regulatory activity by reporter gene transgenesis in zebrafish. Two human enhancers, 76 kb and 130 kb distant from miR218-2, were identified that drove GFP expression in zebrafish in an almost complete miR218 expression pattern. In the zebrafish slit3 locus, two enhancers with identical activity were discovered. In human SLIT2 one enhancer 52 kb upstream of miR218-1 drove an expression pattern very similar to the enhancers of miR218-2. This establishes that miR218-1/-2 regulatory units are nested within SLIT2/3 and that they are duplicates of an ancestral single locus. Due to the strong activity of the enhancers, unique transgenic lines were created that facilitate morphological and gene functional genetic experiments in motor neurons