Development of automated imaging and analysis for zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos

Impaired embryonic motility in dusp27 mutants reveals a developmental defect in myofibril structure

An essential step in muscle fiber maturation is the assembly of highly ordered myofibrils that are required for contraction. Much remains unknown about the molecular mechanisms governing the formation of the contractile apparatus. We identified an early embryonic motility mutant in zebrafish caused by integration of a transgene into the pseudophosphatase dual specificity phosphatase 27 (dusp27) gene. dusp27 mutants exhibit near complete paralysis at embryonic and larval stages, producing extremely low levels of spontaneous coiling movements and a greatly diminished touch response. Loss of dusp27 does not prevent somitogenesis but results in severe disorganization of the contractile apparatus in muscle fibers. Sarcomeric structures in mutants are almost entirely absent and only rare triads are observed. These findings are the first to implicate a functional role of dusp27 as a gene required for myofiber maturation and provide an animal model for analyzing the mechanisms governing myofibril assembly

Brd4 and P300 Confer Transcriptional Competency during Zygotic Genome Activation

The awakening of the genome after fertilization is a cornerstone of animal development. However, the mechanisms that activate the silent genome after fertilization are poorly understood. Here, we show that transcriptional competency is regulated by Brd4- and P300-dependent histone acetylation in zebrafish. Live imaging of transcription revealed that genome activation, beginning at the miR-430 locus, is gradual and stochastic. We show that genome activation does not require slowdown of the cell cycle and is regulated through the translation of maternally inherited mRNAs. Among these, the enhancer regulators P300 and Brd4 can prematurely activate transcription and restore transcriptional competency when maternal mRNA translation is blocked, whereas inhibition of histone acetylation blocks genome activation. We conclude that P300 and Brd4 are sufficient to trigger genome-wide transcriptional competency by regulating histone acetylation on the first zygotic genes in zebrafish. This mechanism is critical for initiating zygotic development and developmental reprogramming.This research was supported by the Fonds de Recherche du Québec Santé (postdoctoral fellowship to J.-D.B.), the Surdna Foundation and the Yale Genetics Venture Fund (postdoctoral fellowship to L.M.), and the NIH (grants R01 HD074078, GM103789, GM102251, GM101108, GM081602, R35 GM122580, and 4DNucleome program to A.J.G.). M.A.M.-M. was supported by Programa de Movilidad en Areas de Investigación priorizadas por la Consejería de Igualdad, Salud y Políticas Sociales de la Junta de Andalucía and is currently supported by Ramon y Cajal program from Ministerio de Ciencia, Innovación y Universidades of the Spanish government (RYC-2017-23041). The Giraldez lab is supported by the Howard Hughes Medical Institute Faculty Scholar program and was supported by the Pew Scholars Program in the Biomedical Sciences, March of Dimes 1-FY12-230, the Yale Scholars Program, and Whitman fellowship funds provided by E.E. Just, Lucy B. Lemann, Evelyn and Melvin Spiegel, The Haldan Keffer Hartline and Edward F. MacNichol, Jr., of the Marine Biological Laboratory in Woods Hole, Massachusetts to A.J.G