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 Segmentation Period (10-24 h) 274 development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad peri- Pharyngula Period (24-48 h) 285 ods of embryogenesis-the zygote, cleavage, blas- Hatching Period (48-72 h) 298 tula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the Early Larval Period 303 changing spectrum of major developmental pro- Acknowledgments 303 cesses that occur during the first 3 days after fer- tilization, and we review some of what is known Glossary 303 about morphogenesis and other significant events that occur during each of the periods. Stages sub- References 309 divide the periods. Stages are named, not num- INTRODUCTION bered as in most other series, providing for flexi- A staging series is a tool that provides accuracy in bility and continued evolution of the staging series developmental studies. This is because different em- as we learn more about development in this spe- bryos, even together within a single clutch, develop at cies. The stages, and their names, are based on slightly different rates. We have seen asynchrony ap- morphological features, generally readily identi- pearing in the development of zebrafish, Danio fied by examination of the live embryo with the (Brachydanio) rerio, embryos fertilized simultaneously dissecting stereomicroscope. The descriptions in vitro (C. Walker and G. Streisinger, in Westerfield, also fully utilize the optical transparancy of the 1994) and incubated at an optimal temperature with- live embryo, which provides for visibility of even out crowding (28.S0C,5-10 embryos/ml). Asynchrony very deep structures when the embryo is examined arises at the earliest stages, and it becomes more pro- with the compound microscope and Nomarski nounced as time passes. Comparisons reveal more of interference contrast illumination. Photomicro- this variability among embryos from different clutches graphs and composite camera lucida line draw- than from within a single clutch. Genetic uniformity ings characterize the stages pictorially. Other may alleviate but does not eliminate this problem; figures chart the development of distinctive char- even embryos of a clonal strain of zebrafish (Streis- acters used as staging aid signposts. inger et al., 1981) develop asynchronously. 0 1995 Wiley-Liss, Inc. As for other kinds of embryos, staging by morpholog- ical criteria partly resolves this problem. For example, Key words: Danio rerio, Morphogenesis, Em- primary trigeminal sensory neurons in the head (Met- bryogenesis, Zygote, Cleavage, Blas- calfe et al., 1990) and primary motoneurons in the tula, Gastrula, Segmentation, Pha- trunk (Eisen et al., 1986) both initiate axogenesis dur- ryngula, Hatching ing stages when somites appear successively along the CONTENTS body axis. Staging by somite number more accurately predicts where these neurons will be in their develop- Introduction 253 ment than does staging by elapsed time after fertiliza- Organization 254 tion (personal communications from W.K. Metcalfe and J.S. Eisen). Recording one’s experiments with refer- Procedures 254 ence to a staging series provides a good way to ensure Temperature and Standard reproducibility and to allow subsequent incorporation Developmental Time 260 Zygote Period (0-% h) 260 Received August 22, 1994; accepted January 4, 1995. Address reprint requestsicorrespondenceto Charles B. Kimmel, In- Cleavage Period (3/4-21/4 h) 261 stitute of Neuroscience, 1254 University of Oregon, Eugene, OR 97403-1254. Blastula Period (21/451/4 h) 263 Thomas F. Schilling is now at the Department of Molecular Em- bryology, Imperial Cancer Research Fund, Lincoln’s Inn Fields, Lon- Gastrula Period (5%-10 h) 268 don WCZA 3PX, UK. 0 1995 WILEY-LISS, INC. 254 KIMMEL ET AL. of new observations and details. A series based on TABLE 1. Periods of Early Development morphology can also facilitate communication; the de- scriptor “18-somite embryo” has more meaning than Period h DescriDtion “18-hour-old embryo,” particularly in cross-species Zygote 0 The newly fertilized egg through the completion of the first zygotic cell comparisons. cycle An earlier staging series for zebrafish, although less Cleavage 3/4 Cell cycles 2 through 7 occur rapidly complete than the present one, fairly accurately por- and synchronously trays the first third (or 1st day) of embryonic develop- Blastula 2 1/4 Rapid, metasynchronous cell cycles (8, 9) give way to lengthened, ment, and includes useful sets of photographs (Hisaoka asynchronous ones at the and Battle, 1958; Hisaoka and Firlit, 1960). Warga and midblastula transition; epiboly Kimmel (1990) briefly described stages of the blastula then begins and gastrula. Preliminary versions of this series were Gastrula 5 v4 Morphogenetic movements of circulated and published in The Zebrafish Book (Wester- involution, convergence, and extension form the epiblast, field, 1994). The present version substantially revises, hypoblast, and embryonic axis; corrects, and expands the earlier ones. An up-to-date through the end of epiboly version is available in electronic form on the Internet Segmentation 10 Somites, pharyngeal arch primordia, World Wide Web server: “http:/zfish.uoregon.edu. and neuromeres develop; primary organogenesis; earliest movements; ORGANIZATION the tail appears Pharyngula 24 Phylotypic-stage embryo; body axis We have named the stages rather than numbering straightens from its early curvature them as in most other series, because named stages are about the yolk sac; circulation, more flexible and easier to remember or recognize than pigmentation, and tins begin development numbered ones. A stage defines more than an instant Hatching 48 Completion of rapid morphogenesis of in time, it is merely a device for approximately locating primary organ systems; cartilage a part of the continuum of development. With named development in head and pectoral stages one can easily add detail as it is learned, and tin; hatching occurs asynchronously Early larva 72 Swim bladder inflates; food-seeking intercalate new stages into the series as necessary for and active avoidance behaviors a particular study without resorting to cumbersome devices like decimal numbers, minuses, and pluses. For example, here we describe a five-somite stage, and next stage it, either manually with #5 watchmaker’s for- a 14-somite stage, but a particular study might use ceps, or by treatment with a proteolytic enzyme (Wes- eight stages in between (six-somite, seven-somite, and terfield, 1994). Late in the pharyngula period the em- so on), and one will immediately understand their bryo, if removed from the chorion, swims away in meaning. We emphasize that we do not mean to ex- response to touch; this can be prevented by anesthesia clude these extra stages, wherever they are in the se- in 0.003% tricaine (3-amino benzoic acid ethyl ester; ries, by ignoring them. Sigma Chemical Co.) at pH 7 (Westerfield, 1994). Re- As an aid to communicating a broader perspective peated anesthesia and rinsing appears to slightly but toward development, we group the stages into larger significantly retard subsequent development (see leg- time-blocks called periods (Table 11, and summarize in end to Fig. 2). the text the principal events during these periods. Spe- cialized terms are defined in the glossary, and boldface Nomarski Optics type in the text highlights terms important for staging. A compound microscope equipped with Nomarski dif- Table 2 collects brief descriptions of all the stages, and ferential interference contrast optics and objectives in- Figure 1 shows corresponding sketches. One can use the range of 25 x to 40 x reveals details otherwise not these two sources to approximately locate a stage of visible in the live preparation and permits the most interest, and then find additional detail as necessary in critical staging. For instance, Nomarski optics permits the text and other figures. the most accurate counts of cells in the blastula and of somites during segmentation. Moreover, “prim” stages PROCEDURES refer to the position of a structure, the primordium of Staging the posterior lateral line, that one must use Nomarski One can determine the approximate developmental optics to see for the sharpest determination of stages stage of a living embryo by examining it with a dis- during much of the pharyngula period. The embryo is secting stereo-microscope, generally with transmitted anesthetized, removed from the chorion, and mounted (not epi- or incident) illumination and high magnifica- between bridged coverslips (Westerfield, 1994), for ob- tion (about 50 x ). During the segmentation period the servation and later recovery. tail elongates, and if the embryo is left within its It is always better to stage embryos while they are chorion, the tail eventually curves over the trunk and alive rather than after killing and fixation. For exam- head so as to obscure the view. When this has hap- ple, staging during the straightening and hatching pe- pened, one must remove the embryo from its chorion to riods calls for a comparison of sizes of the head and the TABLE 2. Stages of Embryonic Developmenta