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Order and Coherence in the Fate Map of the Zebrafish Nervous System

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Order and Coherence in the Fate Map of the Zebrafish Nervous System Development 121, 2595-2609 (1995) 2595 Printed in Great Britain © The Company of Biologists Limited 1995 Order and coherence in the fate map of the zebrafish nervous system Katherine Woo and Scott E. Fraser Division of Biology, Beckman Institute (139-74), California Institute of Technology, Pasadena, Ca. 91125, USA SUMMARY The zebrafish is an excellent vertebrate model for the study embryo. Time-lapse video microscopy shows that the of the cellular interactions underlying the patterning and rearrangement of blastoderm cells during gastrulation is the morphogenesis of the nervous system. Here, we report highly ordered. Cells near the dorsal midline at 6 hours, regional fate maps of the zebrafish anterior nervous system primarily forebrain progenitors, display anterior-directed at two key stages of neural development: the beginning (6 migration. Cells more laterally positioned, corresponding hours) and the end (10 hours) of gastrulation. Early in gas- to midbrain and hindbrain progenitors, converge at the trulation, we find that the presumptive neurectoderm midline prior to anteriorward migration. These results displays a predictable organization that reflects the future demonstrate a predictable order in the presumptive anteroposterior and dorsoventral order of the central neurectoderm, suggesting that patterning interactions may nervous system. The precursors of the major brain subdi- be well underway by early gastrulation. The fate maps visions (forebrain, midbrain, hindbrain, neural retina) provide the basis for further analyses of the specification, occupy discernible, though overlapping, domains within induction and patterning of the anterior nervous system, as the dorsal blastoderm at 6 hours. As gastrulation proceeds, well as for the interpretation of mutant phenotypes and these domains are rearranged such that the basic order of gene-expression patterns. the neural tube is evident at 10 hours. Furthermore, the anteroposterior and dorsoventral order of the progenitors Key words: Danio rerio, fate map, neural patterning, morphogenetic is refined and becomes aligned with the primary axes of the movements, retina, dye microinjection, gastrulation INTRODUCTION The zebrafish (Danio rerio; formerly known as Brachydanio rerio) provides several unique characteristics suitable for the The vertebrate central nervous system is the product of study of vertebrate neural-induction and -patterning mecha- inductive interactions between the ectoderm and mesoderm. nisms. Its optical transparency permits direct observations of The patterning of the neurectoderm is thought to be a pro- tissue movements, cell behaviors, and cell interactions during gressive process. At the end of gastrulation, anteroposterior neurogenesis (Warga and Kimmel, 1990; Kimmel et al., 1994). and dorsoventral axes of the neurectoderm are organized in Its amenability to genetic analysis has already resulted in the broad domains that can be defined morphologically and mole- isolation of a variety of mutants (Mullins et al., 1994; Driever cularly. These are further refined as neurulation proceeds, as et al., 1994) that will be powerful tools for examining devel- evident, for example, in the formation of rhombomeres opmental mechanisms. At present, the results of experimental (Keynes and Lumsden, 1990), and the acquisition of dorsal and studies and developmental mutants must be interpreted with ventral neural tube fates (Ruiz i Altaba and Jessel, 1993). Any reference to Xenopus, in which most modern studies of neural detailed investigation of the regional patterning of the neural induction and axis formation have been performed (reviewed plate requires knowledge of the positions of various primordia in Kessel and Melton, 1994; Donaich, 1993; Ruiz-i-Altaba, of the CNS before, during and after the process. Such infor- 1993; Slack and Tannahill, 1992). While the expression mation is contained in fate maps. These depict, at a specific patterns of a variety of gene homologues suggest similarities developmental stage, what the different regions of an embryo in the components of the neural-patterning machinery of will become during normal development (Slack, 1991). Fate Xenopus, zebrafish, and other vertebrates, it is unclear if the maps of different vertebrates have been invaluable in aiding analogy can be extended to the workings of this machinery. the design of grafting and explant experiments, as well as the For example, there are clear differences in the early cellular interpretation of molecular markers (for example, chick: Couly dynamics between species: in Xenopus, blastomeres change and Le Douarin, 1988; mouse: Lawson et al., 1991; Xenopus: neighbors slowly during cleavage and blastula stages (Wetts Eagleson and Harris, 1990; fish: Oppenheimer, 1947; Ballard, and Fraser, 1989); in zebrafish, cell mixing has an earlier onset 1973; Kimmel et al., 1990). A series of fate maps for different and is more dramatic (Kimmel and Law, 1985; Warga and stages may also provide information about the cell and tissue Kimmel, 1990; Helde et al., 1994). Such differences might rearrangements of gastrulation and neurulation (Keller, 1975; require the operation of distinct signaling mechanisms to Keller, 1976; Lawson et al., 1991; Selleck and Stern, 1991). pattern the embryo. Furthermore, significant differences exist 2596 K. Woo and S. E. Fraser 3 in tissue distribution within their respective organizer regions. Single cells were injected iontophoretically with either 3×10 Mr 3 For example, in Xenopus, the organizer (dorsal lip) contains fluorescein dextran or 3×10 Mr rhodamine dextran (Molecular Probes precursors of only mesodermal (notochord, somite) and endo- D-3306 and D-3308). To reduce toxicity, these dyes were cleaned of × 3 dermal tissues (Keller, 1975, 1976); in zebrafish, recent impurities with a 3 10 Mr size-exclusion HPLC column. Ion- evidence suggests that the organizer (shield) encompasses an tophoretic injections were performed essentially as described previ- intermixed group of mesodermal, endodermal and neural pre- ously (Wetts and Fraser, 1988). Micropipettes, pulled from AlSi thin- walled capillaries (A-M systems), were filled at their tips with a cursors (Shih and Fraser, 1995). Thus, the results of experi- fluorescent dextran solution (about 100 mg/ml) and then back-filled mental studies and the phenotypes of mutants cannot be inter- with 1.2 M LiCl. Injection was accomplished by passing a positive preted solely by analogy with data from Xenopus; instead, a current of 4-5 nA for 3-10 seconds. Each injection was immediately detailed description of the zebrafish neural fate map is verified by viewing the embryo under epi-fluorescence optics. required. The dye injections were made into the cells that the pipette first In this study, we present detailed regional neural fate maps encountered in the blastoderm, as judged by the membrane potential of the zebrafish at the beginning and end of gastrulation (6 recorded through the pipette. Thus, even though the deep cell layer of hours and 10 hours of development, respectively, both key the 6h embryo is between 3-4 cells thick at the animal pole (5-6 cells stages in zebrafish neurogenesis), and use video time-lapse thick close to the germ ring), most injections were within its outermost microscopy to observe directly the cell rearrangements that 1-2 cells. The injections were intended to label single cells; however, occasionally 2 or 3 closely placed cells were labeled after a single transform one fate map to the other. Gastrulation begins at 50% injection, presumably due to cytoplasmic bridges among cells. At the epiboly (about 5.2 hours); by 6 hours, the embryonic shield has time of initial scoring, animals containing florescent debris or labeled become morphologically pronounced, enabling unambiguous periderm were discarded. Those containing 1-3 distinctly labeled cells identification of ‘dorsal’ (staging table in Westerfield, 1994). were kept for later analysis. At 10 hours, the epiblast of the zebrafish embryo completely The 10h fate map data were collected using two different covers the yolk, marking the end of gastrulation (Westerfield, approaches: in the first, we targeted our injections into particular 1994). In the period between 6 and 10 hours of development, regions at 6h, scored the positions of labeled cells immediately, and several genes implicated in embryonic patterning and then re-scored the same embryo at 10h. This enabled us to compare signaling, such as goosecoid (Stachel et al., 1993; Schulte- the relative positions of a labeled cell at 6h and its progeny at 10h. In Merker et al., 1994), axial (Strahle et al., 1993), hedgehog the second, we labeled cells at 10h and scored cell position immedi- ately. These two approaches gave essentially identical fate maps, so (Krauss et al., 1993) and the rtk genes (Xu et al., 1994), initiate they were combined in the final map presented here. their expression in the dorsal side of the embryo. Although the Since injections were performed on either the left or right sides of presumptive neurectoderm appears outwardly homogeneous at the embryos, we examined the assumption of left-right symmetry in these stages, genes believed to play roles in anteroposterior the 6h embryo. We calculated the percentage of each tissue-fate patterning including pax6 (Krauss et al., 1991; Puschel et al., obtained from injections to either the left or the right side. The two 1992), engrailed (Hatta et al., 1991a), and Krox20 (Oxtoby and sides displayed nearly identical tissue-fate distributions. (Data not Jowett, 1993)
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