recovery-of-adult-zebrafish-hearts-for-high-throughput-applications

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

University of California San Francisco

Max-Planck Institute for Heart and Lung Research

Changes in tissue homeostasis, acquisition of invasive cell characteristics, and tumor formation can often be linked to the loss of epithelial cell polarity. In carcinogenesis, the grade of neoplasia correlates with impaired cell polarity. In Drosophila, lethal giant larvae (lgl), discs large (dlg), and scribble, which are components of the epithelial apico-basal cell polarity machinery, act as tumor suppressors, and orthologs of this evolutionary conserved pathway are lost in human carcinoma with high frequency. However, a mechanistic link between neoplasia and vertebrate orthologs of these tumor-suppressor genes remains to be fully explored at the organismal level. Here, we show that the pen/lgl2 mutant phenotype shares two key cellular and molecular features of mammalian malignancy: cell autonomous epidermal neoplasia and epithelial-to-mesenchymal-transition (EMT) of basal epidermal cells including the differential expression of several regulators of EMT. Further, we found that epidermal neoplasia and EMT in pen/lgl2 mutant epidermal cells is promoted by ErbB signalling, a pathway of high significance in human carcinomas. Intriguingly, EMT in the pen/lgl2 mutant is facilitated specifically by ErbB2 mediated E-cadherin mislocalization and not via canonical snail-dependent down-regulation of E-cadherin expression. Our data reveal that pen/lgl2 functions as a tumor suppressor gene in vertebrates, establishing zebrafish pen/lgl2 mutants as a valuable cancer model.

Lgl2 executes its function as a tumor suppressor by regulating ErbB signaling in the zebrafish epidermis.

The heart's continuous motion makes it difficult to capture high-resolution images of this organ in vivo. We developed tools based on high-speed selective plane illumination microscopy (SPIM), offering pristine views into the beating zebrafish heart. We captured three-dimensional cardiac dynamics with postacquisition synchronization of multiview movie stacks, obtained static high-resolution reconstructions by briefly stopping the heart with optogenetics and resolved nonperiodic phenomena by high-speed volume scanning with a liquid lens.

High-resolution reconstruction of the beating zebrafish heart.

Dilated cardiomyopathy is a leading cause of congestive heart failure and a debilitating complication of antineoplastic therapies. Despite disparate causes for dilated cardiomyopathy, maladaptive cardiac remodeling and decreased systolic function are common clinical consequences, begging an investigation of in vivo contractile dynamics in development and disease, one that has been impossible to date.

Actin Binding GFP Allows 4D In Vivo Imaging of Myofilament Dynamics in the Zebrafish Heart and the Identification of Erbb2 Signaling as a Remodeling Factor of Myofibril Architecture.

Heterogeneity within a population of cells of the same type is a common theme in metazoan biology. Dissecting complex developmental and physiological processes crucially relies on our ability to probe the expression profile of these cell subpopulations. Current strategies rely on cell enrichment based on sequential or simultaneous use of multiple intersecting markers starting from a heterogeneous cell suspension. The extensive tissue manipulations required to generate single-cell suspensions, as well as the complexity of the required equipment, inherently complicate these approaches. Here, we propose an alternative methodology based on a genetically encoded system in the model organism Danio rerio (zebrafish). In transgenic fish, we take advantage of the combinatorial biotin transfer system, where polysome-associated mRNAs are selectively recovered from cells expressing both a tagged ribosomal subunit, Rpl10a, and the bacterial biotin ligase BirA. We have applied this technique to skeletal muscle development and identified new genes with interesting temporal expression patterns. Through this work we have thus developed additional tools for highly specific gene expression profiling.

Article	Year
Lgl2 executes its function as a tumor suppressor by regulating ErbB signaling in the zebrafish epidermis. PLoS genetics\| PubMed ID: 19911055	2009
High-resolution reconstruction of the beating zebrafish heart. Nature methods\| PubMed ID: 25042787	2014
Actin Binding GFP Allows 4D In Vivo Imaging of Myofilament Dynamics in the Zebrafish Heart and the Identification of Erbb2 Signaling as a Remodeling Factor of Myofibril Architecture. Circulation research\| PubMed ID: 25228389	2014
Translational profiling through biotinylation of tagged ribosomes in zebrafish. Development (Cambridge, England)\| PubMed ID: 25231762	2014

Sven Reischauer

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