Loss of the Polycomb group protein Rnf2 results in derepression of tbx-transcription factors and defects in embryonic and cardiac development.

The Polycomb group (PcG) protein family is a well-known group of epigenetic modifiers. We used zebrafish to investigate the role of Rnf2, the enzymatic subunit of PRC1. We found a positive correlation between loss of Rnf2 and upregulation of genes, especially of those whose promoter is normally bound by Rnf2. The heart of rnf2 mutants shows a tubular shaped morphology and to further understand the underlying mechanism, we studied gene expression of single wildtype and rnf2 mutant hearts. We detected the most pronounced differences at 3 dpf, including upregulation of heart transcription factors, such as tbx2a, tbx2b, and tbx3a. These tbx genes were decorated by broad PcG domains in wildtype whole embryo lysates. Chamber specific genes such as vmhc, myh6, and nppa showed downregulation in rnf2 mutant hearts. The marker of the working myocard, nppa, is negatively regulated by Tbx2 and Tbx3. Based on our findings and literature we postulate that loss of Rnf2-mediated repression results in upregulation and ectopic expression of tbx2/3, whose expression is normally restricted to the cardiac conductive system. This could lead to repression of chamber specific gene expression, a misbalance in cardiac cell types, and thereby to cardiac defects observed in rnf2 mutants.

Continuous addition of progenitors forms the cardiac ventricle in zebrafish.

The vertebrate heart develops from several progenitor lineages. After early-differentiating first heart field (FHF) progenitors form the linear heart tube, late-differentiating second heart field (SHF) progenitors extend the atrium and ventricle, and form inflow and outflow tracts (IFT/OFT). However, the position and migration of late-differentiating progenitors during heart formation remains unclear. Here, we track zebrafish heart development using transgenics based on the cardiopharyngeal gene tbx1. Live imaging uncovers a tbx1 reporter-expressing cell sheath that continuously disseminates from the lateral plate mesoderm towards the forming heart tube. High-speed imaging and optogenetic lineage tracing corroborates that the zebrafish ventricle forms through continuous addition from the undifferentiated progenitor sheath followed by late-phase accrual of the bulbus arteriosus (BA). FGF inhibition during sheath migration reduces ventricle size and abolishes BA formation, refining the window of FGF action during OFT formation. Our findings consolidate previous end-point analyses and establish zebrafish ventricle formation as a continuous process.

Regulation of ß1 integrin-Klf2-mediated angiogenesis by CCM proteins.

Mechanotransduction pathways are activated in response to biophysical stimuli during the development or homeostasis of organs and tissues. In zebrafish, the blood-flow-sensitive transcription factor Klf2a promotes VEGF-dependent angiogenesis. However, the means by which the Klf2a mechanotransduction pathway is regulated to prevent continuous angiogenesis remain unknown. Here we report that the upregulation of klf2 mRNA causes enhanced egfl7 expression and angiogenesis signaling, which underlies cardiovascular defects associated with the loss of cerebral cavernous malformation (CCM) proteins in the zebrafish embryo. Using CCM-protein-depleted human umbilical vein endothelial cells, we show that the misexpression of KLF2 mRNA requires the extracellular matrix-binding receptor ß1 integrin and occurs in the absence of blood flow. Downregulation of ß1 integrin rescues ccm mutant cardiovascular malformations in zebrafish. Our work reveals a ß1 integrin-Klf2-Egfl7-signaling pathway that is tightly regulated by CCM proteins. This regulation prevents angiogenic overgrowth and ensures the quiescence of endothelial cells.

High-resolution reconstruction of the beating zebrafish heart.

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.

Light sheet microscopy.

This chapter introduces the concept of light sheet microscopy along with practical advice on how to design and build such an instrument. Selective plane illumination microscopy is presented as an alternative to confocal microscopy due to several superior features such as high-speed full-frame acquisition, minimal phototoxicity, and multiview sample rotation. Based on our experience over the last 10 years, we summarize the key concepts in light sheet microscopy, typical implementations, and successful applications. In particular, sample mounting for long time-lapse imaging and the resulting challenges in data processing are discussed in detail.

Multilayer mounting for long-term light sheet microscopy of zebrafish.

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

Phylogenetic analysis and expression of zebrafish transient receptor potential melastatin family genes.

BACKGROUND: The transient receptor potential melastatin (TRPM) gene family belongs to the superfamily of nonselective TRP ion channels. TRP channels are cellular sensors, detecting a multitude of inputs, including temperature, light, chemical, and mechanical stimuli. Recent studies revealed diverse roles during development, linking TRP channels to differentiation, proliferation, cell motility, cell death, and survival. A detailed description of this gene family in the zebrafish is still missing. RESULTS: Phylogenetic analysis revealed 11 trpm genes in the zebrafish genome. The zebrafish orthologs of mammalian TRPM1 and TRPM4 are duplicated and quadruplicated, respectively, and TRPM8, a cold sensitive channel has been lost in zebrafish. Whole-mount in situ hybridization experiments revealed dynamic expression pattern of trpm genes in the developing embryo and early larva. Transcripts were mainly found in neural cell clusters, but also in tissues involved in ion homeostasis. CONCLUSIONS: Our results suggest a role of TRPM channels in sensory information processing, including vision, olfaction, taste, and mechanosensation. An involvement in developmental processes is likely, as some trpm genes were found to be expressed in differentiating cells. Our data now provide a basis for functional analyses of this gene family of ion channels in the vertebrate model organism Danio rerio.

Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.

Light sheet microscopy techniques, such as selective plane illumination microscopy (SPIM), are ideally suited for time-lapse imaging of developmental processes lasting several hours to a few days. The success of this promising technology has mainly been limited by the lack of suitable techniques for mounting fragile samples. Embedding zebrafish embryos in agarose, which is common in conventional confocal microscopy, has resulted in severe growth defects and unreliable results. In this study, we systematically quantified the viability and mobility of zebrafish embryos mounted under more suitable conditions. We found that tubes made of fluorinated ethylene propylene (FEP) filled with low concentrations of agarose or methylcellulose provided an optimal balance between sufficient confinement of the living embryo in a physiological environment over 3 days and optical clarity suitable for fluorescence imaging. We also compared the effect of different concentrations of Tricaine on the development of zebrafish and provide guidelines for its optimal use depending on the application. Our results will make light sheet microscopy techniques applicable to more fields of developmental biology, in particular the multiview long-term imaging of zebrafish embryos and other small organisms. Furthermore, the refinement of sample preparation for in toto and in vivo imaging will promote other emerging optical imaging techniques, such as optical projection tomography (OPT).

Regulation of hub mRNA stability and translation by miR430 and the dead end protein promotes preferential expression in zebrafish primordial germ cells.

The Hu proteins are RNA-binding proteins known to be involved in various aspects of RNA metabolism, such as nucleo-cytoplasmic shuttling, translation, and stability. These proteins are predominantly expressed in neuronal tissues and are important for neuronal differentiation and plasticity. Here, we report on the regulation over hub mRNA stability and function in zebrafish embryos. Using reporters encoding for fluorescent proteins, we show that hub RNA is a target of global miRNA-mediated repression, while the RNA-binding protein Dead end (Dnd) contributes to maintenance of the expression in the primordial germ cells (PGCs).