Transposons as tools for enhancer trap screens in vertebrates

DNA transposons are efficient tools in transgenesis and have therefore become popular in the analysis of the regulatory genome in vertebrates via enhancer trap screens. Here, I discuss recent progress in this field of research, with a focus on the application of one of these transposons, namely the medaka fish derived Tol2, to enhancer trapping in zebrafish, and how this approach compares with others that have a similar objective

The role of vasculature and blood circulation in zebrafish swimbladder development

<p>Abstract</p> <p>Background</p> <p>Recently we have performed a detailed analysis of early development of zebrafish swimbladder, a homologous organ of tetrapod lung; however, the events of swimbladder development are still poorly characterized. Many studies have implicated the role of vascular system in development of many organs in vertebrates. As the swimbladder is lined with an intricate network of blood capillaries, it is of interest to investigate the role of the vascular system during early development of swimbladder.</p> <p>Results</p> <p>To investigate the role of endothelial cells (ECs) and blood circulation during development of the swimbladder, phenotypes of swimbladder were analysed at three different stages (~2, 3 and 5 dpf [day postfertilization]) in <it>cloche </it>(<it>clo</it>) mutant and Tnnt2 morphants, in the background of transgenic lines <it>Et(krt4:EGFP)</it>^{<it>sq33-2 </it>}and <it>Et(krt4:EGFP)</it>^{<it>sqet3 </it>}which express EGFP in the swimbladder epithelium and outer mesothelium respectively. Analyses of the three tissue layers of the swimbladder were performed using molecular markers <it>hb9</it>, <it>fgf10a</it>, <it>acta2</it>, and <it>anxa5 </it>to distinguish epithelium, mesenchyme, and outer mesothelium. We showed that the budding stage was independent of ECs and blood flow, while early epithelial growth, mesenchymal organization and its differentiation into smooth muscle, as well as outer mesothelial organization, were dependent on ECs. Blood circulation contributed to later stage of epithelial growth, smooth muscle differentiation, and organization of the outer mesothelium. Inflation of the swimbladder was also affected as a result of absence of ECs and blood flow.</p> <p>Conclusion</p> <p>Our data demonstrated that the vascular system, though not essential in swimbladder budding, plays an important role in the development of the swimbladder starting from the early growth stage, including mesenchyme organization and smooth muscle differentiation, and outer mesothelial organization, which in turn may be essential for the function of the swimbladder as reflected in its eventual inflation.</p

Localized rbp4 expression in the yolk syncytial layer plays a role in yolk cell extension and early liver development

<p>Abstract</p> <p>Background</p> <p>The number of genes characterized in liver development is steadily increasing, but the origin of liver precursor cells and the molecular control of liver formation remain poorly understood. Existing theories about formation of zebrafish visceral organs emphasize either their budding from the endodermal rod or formation of independent anlage followed by their later fusion, but none of these is completely satisfactory in explaining liver organogenesis in zebrafish.</p> <p>Results</p> <p>Expression of a gene encoding the retinol binding protein 4 (Rbp4) was analyzed in zebrafish. <it>rbp4</it>, which is expressed mainly in the liver in adults, was shown to be expressed in the yolk syncytial layer (YSL) during early embryogenesis. At 12–16 hpf <it>rbp4 </it>expression was restricted to the ventro-lateral YSL and later expanded to cover the posterior YSL. We demonstrated that <it>rbp4 </it>expression was negatively regulated by Nodal and Hedgehog (Hh) signalling and positively controlled by retinoic acid (RA). Knockdown of Rbp4 in the YSL resulted in shortened yolk extension as well as the formation of two liver buds, which could be due to impaired migration of liver progenitor cells. <it>rbp4 </it>appears also to regulate the extracellular matrix protein Fibronectin1 (Fn1) specifically in the ventro-lateral yolk, indicating a role of Fn1 in liver progenitor migration. Since exocrine pancreas, endocrine pancreas, intestine and heart developed normally in Rbp4 morphants, we suggest that <it>rbp4 </it>expression in the YSL is required only for liver development.</p> <p>Conclusion</p> <p>The characteristic expression pattern of <it>rbp4 </it>suggests that the YSL is patterned despite its syncytial nature. YSL-expressed Rbp4 plays a role in formation of both yolk extension and liver bud, the latter may also require migration of liver progenitor cells.</p

The chemokine Sdf-1 and its receptor Cxcr4 are required for formation of muscle in zebrafish

<p>Abstract</p> <p>Background</p> <p>During development cell migration takes place prior to differentiation of many cell types. The chemokine receptor Cxcr4 and its ligand Sdf1 are implicated in migration of several cell lineages, including appendicular muscles.</p> <p>Results</p> <p>We dissected the role of <it>sdf1</it>-<it>cxcr4 </it>during skeletal myogenesis. We demonstrated that the receptor <it>cxcr4a </it>is expressed in the medial-anterior part of somites, suggesting that chemokine signaling plays a role in this region of the somite. Previous reports emphasized co-operation of Sdf1a and Cxcr4b. We found that during early myogenesis Sdf1a co-operates with the second Cxcr4 of zebrafish – Cxcr4a resulting in the commitment of myoblast to form fast muscle. Disrupting this chemokine signal caused a reduction in <it>myoD </it>and <it>myf5 </it>expression and fast fiber formation. In addition, we showed that a dimerization partner of MyoD and Myf5, E12, positively regulates transcription of <it>cxcr4a </it>and <it>sdf1a </it>in contrast to that of Sonic hedgehog, which inhibited these genes through induction of expression of <it>id2</it>.</p> <p>Conclusion</p> <p>We revealed a regulatory feedback mechanism between <it>cxcr4a</it>-<it>sdf1a </it>and genes encoding myogenic regulatory factors, which is involved in differentiation of fast myofibers. This demonstrated a role of chemokine signaling during development of skeletal muscles.</p

The prolyl isomerase Pin1 stabilizes NeuroD during differentiation of mechanoreceptors

The peptidyl prolyl cis-trans isomerase Pin1 plays vital roles in diverse cellular processes and pathological conditions. NeuroD is a differentiation and survival factor for a subset of neurons and pancreatic endocrine cells. Although multiple phosphorylation events are known to be crucial for NeuroD function, their mechanisms remain elusive. In this study, we demonstrate that zebrafish embryos deficient in Pin1 displayed phenotypes resembling those associated with NeuroD depletion, characterized by defects in formation of mechanosensory hair cells. Furthermore, zebrafish Pin1 interacts with NeuroD in a phosphorylation-dependent manner. In Pin1-deficient cell lines, NeuroD is rapidly degraded. However, the protein stability of NeuroD is restored upon overexpression of Pin1. These findings suggest that Pin1 functionally regulates NeuroD protein levels by post-phosphorylation cis-trans isomerization during neuronal specification

Zebrafish transgenic Enhancer TRAP line database (ZETRAP)

BACKGROUND: The zebrafish, Danio rerio, is used as a model organism to study vertebrate genetics and development. An effective enhancer trap (ET) in zebrafish using the Tol2 transposon has been demonstrated. This approach could be used to study embryogenesis of a vertebrate species in real time and with high resolution. DESCRIPTION: The information gathered during the course of systematic investigation of many ET transgenic lines have been collected and compiled in the form of an online database – the Zebrafish Enhancer TRAP lines database (ZETRAP). CONCLUSION: ZETRAP is a web-based system that provides data and information to the scientific community about the developmental, genetic and genomic aspects of transgenic zebrafish lines obtained using Tol2 transposon-mediated transgenesis. The current version (version 1.0) contains description of 27 ET lines that express EGFP in various organs and tissues, for example, heart, brain, notochord, gut, etc. It also includes information on insertion sites of the Tol2 transposon in these lines

Il-verbi f'sekwenza fil-Malti

F’dan l-artiklu l-awtriċi tistħarreġ in-natura tal-katina verbali fil-Malti, li għalissa nistgħu niddeskrivuha bħala sekwenza ta’ verbi li jokkorru wara xulxin mingħajr l-ebda ndħil ta’ xi element ieħor.peer-reviewe

Optogenetic in vivo cell manipulation in KillerRed-expressing zebrafish transgenics

<p>Abstract</p> <p>Background</p> <p>KillerRed (KR) is a novel photosensitizer that efficiently generates reactive oxygen species (ROS) in KR-expressing cells upon intense green or white light illumination <it>in vitro</it>, resulting in damage to their plasma membrane and cell death.</p> <p>Results</p> <p>We report an <it>in vivo </it>modification of this technique using a fluorescent microscope and membrane-tagged KR (mem-KR)-expressing transgenic zebrafish. We generated several stable zebrafish <it>Tol2 </it>transposon-mediated enhancer-trap (ET) transgenic lines expressing mem-KR (SqKR series), and mapped the transposon insertion sites. As mem-KR accumulates on the cell membrane and/or Golgi, it highlights cell bodies and extensions, and reveals details of cellular morphology. The photodynamic property of KR made it possible to damage cells expressing this protein in a dose-dependent manner. As a proof-of-principle, two zebrafish transgenic lines were used to affect cell viability and function: SqKR2 expresses mem-KR in the hindbrain rhombomeres 3 and 5, and elsewhere; SqKR15 expresses mem-KR in the heart and elsewhere. Photobleaching of KR by intense light in the heart of SqKR15 embryos at lower levels caused a reduction in pumping efficiency of the heart and pericardial edema and at higher levels - in cell death in the hindbrain of SqKR2 and in the heart of SqKR15 embryos.</p> <p>Conclusions</p> <p>An intense illumination of tissues expressing mem-KR affects cell viability and function in living zebrafish embryos. Hence, the zebrafish transgenics expressing mem-KR in a tissue-specific manner are useful tools for studying the biological effects of ROS.</p

miR-7 Controls the Dopaminergic/Oligodendroglial Fate through Wnt/\u3b2-catenin Signaling Regulation

During the development of the central nervous system, the proliferation of neural progenitors and differentiation of neurons and glia are tightly regulated by different transcription factors and signaling cascades, such as the Wnt and Shh pathways. This process takes place in cooperation with several microRNAs, some of which evolutionarily conserved in vertebrates, from teleosts to mammals. We focused our attention on miR-7, as its role in the regulation of cell signaling during neural development is still unclear. Specifically, we used human stem cell cultures and whole zebrafish embryos to study, in vitro and in vivo, the role of miR-7 in the development of dopaminergic (DA) neurons, a cell type primarily affected in Parkinson's disease. We demonstrated that the zebrafish homologue of miR-7 (miR-7a) is expressed in the forebrain during the development of DA neurons. Moreover, we identified 143 target genes downregulated by miR-7, including the neural fate markers TCF4 and TCF12, as well as the Wnt pathway effector TCF7L2. We then demonstrated that miR-7 negatively regulates the proliferation of DA-progenitors by inhibiting Wnt/\u3b2-catenin signaling in zebrafish embryos. In parallel, miR-7 positively regulates Shh signaling, thus controlling the balance between oligodendroglial and DA neuronal cell fates. In summary, this study identifies a new molecular cross-talk between Wnt and Shh signaling pathways during the development of DA-neurons. Being mediated by a microRNA, this mechanism represents a promising target in cell differentiation therapies for Parkinson's disease