DEVELOPMENTAL DYNAMICS 2032553’10 (1995) Stages of Embryonic Development of the Zebrafish CHARLES B. KIMMEL, WILLIAM W. BALLARD, SETH R. KIMMEL, BONNIE ULLMANN, AND THOMAS F. SCHILLING Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403-1254 (C.B.K., S.R.K., B.U., T.F.S.); Department of Biology, Dartmouth College, Hanover, NH 03755 (W.W.B.) ABSTRACT We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad peri- ods of embryogenesis-the zygote, cleavage, blas- tula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental pro- cesses that occur during the first 3 days after fer- tilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages sub- divide the periods. Stages are named, not num- bered as in most other series, providing for flexi- bility and continued evolution of the staging series as we learn more about development in this spe- cies. The stages, and their names, are based on morphological features, generally readily identi- fied by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicro- graphs and composite camera lucida line draw- ings characterize the stages pictorially. Other figures chart the development of distinctive acters used as staging aid signposts. 0 1995 Wiley-Liss, Inc. Key words: Danio rerio, Morphogenesis, bryogenesis, Zygote, Cleavage, tula, Gastrula, Segmentation, ryngula, Hatching CONTENTS Introduction Organization Procedures Temperature and Standard Developmental Time Zygote Period (0-% h) Cleavage Period (3/4-21/4 h) Blastula Period (21/451/4 h) Gastrula Period (5%-10 h) 0 1995 WILEY-LISS, INC. char- Em- Blas- Pha- 253 254 254 260 260 261 263 268 Segmentation Period (10-24 h) 274 Pharyngula Period (24-48 h) 285 Hatching Period (48-72 h) 298 Early Larval Period 303 Acknowledgments 303 Glossary 303 References 309 INTRODUCTION A staging series is a tool that provides accuracy in developmental studies. This is because different em- bryos, even together within a single clutch, develop at slightly different rates. We have seen asynchrony ap- pearing in the development of zebrafish, Danio (Brachydanio) rerio, embryos fertilized simultaneously in vitro (C. Walker and G. Streisinger, in Westerfield, 1994) and incubated at an optimal temperature with- out crowding (28.S0C, 5-10 embryos/ml). Asynchrony arises at the earliest stages, and it becomes more pro- nounced as time passes. Comparisons reveal more of this variability among embryos from different clutches than from within a single clutch. Genetic uniformity may alleviate but does not eliminate this problem; even embryos of a clonal strain of zebrafish (Streis- inger et al., 1981) develop asynchronously. As for other kinds of embryos, staging by morpholog- ical criteria partly resolves this problem. For example, primary trigeminal sensory neurons in the head (Met- calfe et al., 1990) and primary motoneurons in the trunk (Eisen et al., 1986) both initiate axogenesis dur- ing stages when somites appear successively along the body axis. Staging by somite number more accurately predicts where these neurons will be in their develop- ment than does staging by elapsed time after fertiliza- tion (personal communications from W.K. Metcalfe and J.S. Eisen). Recording one’s experiments with refer- ence to a staging series provides a good way to ensure reproducibility and to allow subsequent incorporation Received August 22, 1994; accepted January 4, 1995. Address reprint requestsicorrespondence to Charles B. Kimmel, In- stitute of Neuroscience, 1254 University of Oregon, Eugene, OR 97403-1254. Thomas F. Schilling is now at the Department of Molecular Em- bryology, Imperial Cancer Research Fund, Lincoln’s Inn Fields, Lon- don WCZA 3PX, UK.
