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

Two regulatory genes, cNkx5-1 and cPax2, show different responses to local signals during otic placode and vesicle formation in the chick embryo

The early stages of otic placode development depend on signals from neighbouring tissues including the hindbrain. The identity of these signals and of the responding placodal genes, however, is not known. We have identified a chick homeobox gene cNkx5-1, which is expressed in the otic placode beginning at stage 10 and exhibits a dynamic expression pattern during formation and further differentiation of the otic vesicle. In a series of heterotopic transplantation experiments, we demonstrate that cNkx5-1 can be activated in ectopic positions. However, significant differences in otic development and cNkx5-1 gene activity were observed when placodes were transplanted into the more rostral positions within the head mesenchyme or into the wing buds of older hosts. These results indicate that only the rostral tissues were able to induce and/or maintain ear development. Ectopically induced cNkx5-1 expression always reproduced the endogenous pattern within the lateral wall of the otocyst that is destined to form vestibular structures. In contrast, cPax2 which is expressed in the medial wall of the early otic vesicle later forming the cochlea never resumed its correct expression pattern after transplantation. Our experiments illustrate that only some aspects of gene expression and presumably pattern formation during inner ear development can be established and maintained ectopically. In particular, the dorsal vestibular structures seem to be programmed earlier and differently from the ventral cochlear part

Using fish models to investigate the links between microbiome and social behaviour: the next step for translational microbiome research?

Recent research has revealed surprisingly important connections between animals’ microbiome and social behaviour. Social interactions can affect the composition and function of the microbiome; conversely, the microbiome affects social communication by influencing the hosts’ central nervous system and peripheral chemical communication. These discoveries set the stage for novel research focusing on the evolution and physiology of animal social behaviour in relation to microbial transmission strategies. Here, we discuss the emerging roles of teleost fish models and their potential for advancing research fields, linked to sociality and microbial regulation. We argue that fish models, such as the zebrafish (Danio rerio, Cyprinidae), sticklebacks (‎Gasterosteidae), guppies (Poeciliidae) and cleaner–client dyads (e.g., obligate cleaner fish from the Labridae and Gobiidae families and their visiting clientele), will provide valuable insights into the roles of microbiome in shaping social behaviour and vice versa, while also being of direct relevance to the food and ornamental fish trades. The diversity of fish behaviour warrants more interdisciplinary research, including microbiome studies, which should have a strong ecological (field‐derived) approach, together with laboratory‐based cognitive and neurobiological experimentation. The implications of such integrated approaches may be of translational relevance, opening new avenues for future investigation using fish models

Functional Assessment of Disease-Associated Regulatory Variants <i>In Vivo</i> Using a Versatile Dual Colour Transgenesis Strategy in Zebrafish

Disruption of gene regulation by sequence variation in non-coding regions of the genome is now recognised as a significant cause of human disease and disease susceptibility. Sequence variants in cis-regulatory elements (CREs), the primary determinants of spatio-temporal gene regulation, can alter transcription factor binding sites. While technological advances have led to easy identification of disease-associated CRE variants, robust methods for discerning functional CRE variants from background variation are lacking. Here we describe an efficient dual-colour reporter transgenesis approach in zebrafish, simultaneously allowing detailed in vivo comparison of spatio-temporal differences in regulatory activity between putative CRE variants and assessment of altered transcription factor binding potential of the variant. We validate the method on known disease-associated elements regulating SHH, PAX6 and IRF6 and subsequently characterise novel, ultra-long-range SOX9 enhancers implicated in the craniofacial abnormality Pierre Robin Sequence. The method provides a highly cost-effective, fast and robust approach for simultaneously unravelling in a single assay whether, where and when in embryonic development a disease-associated CRE-variant is affecting its regulatory function

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