In vivo imaging and quantitative analysis of zebrafish embryos by digital holographic microscopy.

Digital holographic microscopy (DHM) has been applied extensively to in vitro studies of different living cells. In this paper, we present a novel application of an off-axis DHM system to in vivo study of the development of zebrafish embryos. Even with low magnification microscope objectives, the morphological structures and individual cell types inside developing zebrafish embryos can be clearly observed from reconstructed amplitude images. We further study the dynamic process of blood flow in zebrafish embryos. A calibration routine and post-processing procedures are developed to quantify physiological parameters at different developmental stages. We measure quantitatively the blood flow as well as the heart rate to study the effects of elevated D-glucose (abnormal condition) on circulatory and cardiovascular systems of zebrafish embryos. To enhance our ability to use DHM as a quantitative tool for potential high throughput screening application, the calibration and post-processing algorithms are incorporated into an automated processing software. Our results show that DHM is an excellent non-invasive imaging technique for visualizing the cellular dynamics of organogenesis of zebrafish embryos in vivo.

Zebrafish eve1 regulates the lateral and ventral fates of mesodermal progenitor cells at the onset of gastrulation.

Zebrafish eve1, a member of the even-skipped related gene family, is expressed initially in the animal pole of late blastula embryo and subsequently restricted to the ventral mesoderm of the gastrula embryo under the signaling control of bone morphogenetic protein (Bmp). Overexpression of eve1 in embryos results in similar ventralized phenotypes to that seen in embryos overexpressing Bmp, suggesting that Eve1 acts downstream of the Bmp signaling pathway to regulate the fate of mesodermal progenitor cells (MPCs). How eve1 functions in the normal development of MPCs is unknown. Using overexpression of a chimeric protein of Eve1 fused to the Gal4 activation domain and gene-knockdown approaches, we investigated the role of eve1 in MPC development of zebrafish embryos at early gastrulation. We find that Eve1 functions as a transcriptional repressor and is required for normal MPC development. The role of eve1 in MPCs requires the redundant and cooperative functions of Bmp-activated downstream homeobox genes, ved, vent and vox. Inhibition of eve1, ved, vent and vox in double and triple combinations results in dorsalized phenotypes. Furthermore, specific inhibition of eve1 and ved causes the expression of an ectopic patch of the brachyury ortholog no tail and leads to the formation of an ectopic tail. Our data show that Eve1 functions together with Ved, Vent and Vox in a transcriptional network to prevent the spread of anti-Bmp gene activity from the dorsal side, leading to the establishment of the Bmp gradient activity along the dorsoventral axis to induce distinct transcriptional outputs in MPCs to maintain the lateral and ventral MPC fates during gastrulation.

Biocompatibility and biodistribution of surface-enhanced Raman scattering nanoprobes in zebrafish embryos: in vivo and multiplex imaging.

Nanoparticles are increasingly being used to investigate biological processes in various animal models due to their versatile chemical, unique optical, and multifunctional properties. In this report we address the biocompatibility and biodistribution of nanoparticle sensors used for Raman chemical imaging in live zebrafish (Danio rerio) embryos. Surface-enhanced Raman scattering (SERS) nanoprobes (NPs) comprising gold nanoparticles (AuNPs) as enhancing substrate and nonfluorescent Raman labels were synthesized and microinjected into zebrafish embryos at the one-cell stage. Raman mapping was performed to assess their distribution in various cell-types and tissues of developing embryo at five different stages between 6 and 96 hpf (hours post-fertilization). Biocompatibility and toxicity studies indicate that the NPs are not toxic and the embryos were found to exhibit normal morphological and gene expression in addition to the proper form and function of major organs such as the heart and vasculature (of 7 day old NPs injected zebrafish embryos). A multiplex in vivo detection protocol was developed by SERS imaging to demonstrate that multiple labels can be detected by Raman mapping in undifferentiated cells as they develop into distinct cell- and tissue-types. The present work is the first to report on multiplex Raman imaging of zebrafish embryos with potential implications in tracking tissue development and biological processes at single molecule sensitivity using appropriate target molecules in vivo.

Disruption of zebrafish cyclin G-associated kinase (GAK) function impairs the expression of Notch-dependent genes during neurogenesis and causes defects in neuronal development.

BACKGROUND: The J-domain-containing protein auxilin, a critical regulator in clathrin-mediated transport, has been implicated in Drosophila Notch signaling. To ask if this role of auxilin is conserved and whether auxilin has additional roles in development, we have investigated the functions of auxilin orthologs in zebrafish. RESULTS: Like mammals, zebrafish has two distinct auxilin-like molecules, auxilin and cyclin G-associated kinase (GAK), differing in their domain structures and expression patterns. Both zebrafish auxilin and GAK can functionally substitute for the Drosophila auxilin, suggesting that they have overlapping molecular functions. Still, they are not completely redundant, as morpholino-mediated knockdown of the ubiquitously expressed GAK alone can increase the specification of neuronal cells, a known Notch-dependent process, and decrease the expression of Her4, a Notch target gene. Furthermore, inhibition of GAK function caused an elevated level of apoptosis in neural tissues, resulting in severe degeneration of neural structures. CONCLUSION: In support of the notion that endocytosis plays important roles in Notch signaling, inhibition of zebrafish GAK function affects embryonic neuronal cell specification and Her4 expression. In addition, our analysis suggests that zebrafish GAK has at least two functions during the development of neural tissues: an early Notch-dependent role in neuronal patterning and a late role in maintaining the survival of neural cells.

The regulation of mesodermal progenitor cell commitment to somitogenesis subdivides the zebrafish body musculature into distinct domains.

The vertebrate musculature is produced from a visually uniform population of mesodermal progenitor cells (MPCs) that progressively bud off somites populating the trunk and tail. How the MPCs are regulated to continuously release cells into the presomitic mesoderm throughout somitogenesis is not understood. Using a genetic approach to study the MPCs, we show that a subset of MPCs are set aside very early in zebrafish development, and programmed to cell-autonomously enter the tail domain beginning with the 16th somite. Moreover, we show that the trunk is subdivided into two domains, and that entry into the anterior trunk, posterior trunk, and tail is regulated by interactions between the Nodal and bone morphogenetic protein (Bmp) pathways. Finally, we show that the tail MPCs are held in a state we previously called the Maturation Zone as they wait for the signal to begin entering somitogenesis.

Visualizing morphogenesis in transgenic zebrafish embryos using BODIPY TR methyl ester dye as a vital counterstain for GFP.

Green fluorescent protein (GFP) technology is rapidly advancing the study of morphogenesis, by allowing researchers to specifically focus on a subset of labeled cells within the living embryo. However, when imaging GFP-labeled cells using confocal microscopy, it is often essential to simultaneously visualize all of the cells in the embryo using dual-channel fluorescence to provide an embryological context for the cells expressing GFP. Although various counterstains are available, part of their fluorescence overlaps with the GFP emission spectra, making it difficult to clearly identify the cells expressing GFP. In this study, we report that a new fluorophore, BODIPY TR methyl ester dye, serves as a versatile vital counterstain for visualizing the cellular dynamics of morphogenesis within living GFP transgenic zebrafish embryos. The fluorescence of this photostable synthetic dye is spectrally separate from GFP fluorescence, allowing dual-channel, three-dimensional (3D) and four-dimensional (4D) confocal image data sets of living specimens to be easily acquired. These image data sets can be rendered subsequently into uniquely informative 3D and 4D visualizations using computer-assisted visualization software. We discuss a variety of immediate and potential applications of BODIPY TR methyl ester dye as a vital visualization counterstain for GFP in transgenic zebrafish embryos.

Combinatorial gene regulation by Bmp and Wnt in zebrafish posterior mesoderm formation.

Combinatorial signaling is an important mechanism that allows the embryo to utilize overlapping signaling pathways to specify different territories. In zebrafish, the Wnt and Bmp pathways interact to regulate the formation of the posterior body. In order to understand how this works mechanistically, we have identified tbx6 as a posterior mesodermal gene activated by both of these signaling pathways. We isolated a genomic fragment from the tbx6 gene that recapitulates the endogenous tbx6 expression, and used this to ask how the Bmp and Wnt signaling pathways combine to regulate gene expression. We find that the tbx6 promoter utilizes distinct domains to integrate the signaling inputs from each pathway, including multiple Tcf/LEF sites and a novel Bmp-response element. Surprisingly, we found that overexpression of either signaling pathway can activate the tbx6 promoter and the endogenous gene, whereas inputs from both pathways are required for the normal pattern of expression. These results demonstrate that both Bmp and Wnt are present at submaximal levels, which allows the pathways to function combinatorially. We present a model in which overlapping Wnt and Bmp signals in the ventrolateral region activate the expression of tbx6 and other posterior mesodermal genes, leading to the formation of posterior structures.

HrT is required for cardiovascular development in zebrafish.

The recently identified zebrafish T-box gene hrT is expressed in the developing heart and in the endothelial cells forming the dorsal aorta. Orthologs of hrT are expressed in cardiovascular cells from Drosophila to mouse, suggesting that the function of hrT is evolutionarily conserved. The role of hrT in cardiovascular development, however, has not thus far been determined in any animal model. Using morpholino antisense oligonucleotides, we show that zebrafish embryos lacking hrT function have dysmorphic hearts and an absence of blood circulation. Although the early events in heart formation were normal in hrT morphant embryos, subsequently the hearts failed to undergo looping, and late onset defects in chamber morphology and gene expression were observed. In particular, we found that the loss of hrT function led to a dramatic upregulation of tbx5, a gene required for normal heart morphogenesis. Conversely, we show that overexpression of hrT causes a significant downregulation of tbx5, indicating that one key role of hrT is to regulate the levels of tbx5. Secondly, we found that HrT is required to inhibit the expression of the blood lineage markers gata1 and gata2 in the most posterior lateral plate mesoderm. Finally, we show that HrT is required for vasculogenesis in the trunk, leading to similar vascular defects to those observed in midline mutants such as floating head. hrT expression in the vascular progenitors depends upon midline mesoderm, indicating that this expression is one important component of the response to a midline-derived signal during vascular morphogenesis.