Axonogenesis and Morphogenesis in the Embryonic Zebrafish Brain
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The Journal of Neuroscience, February 1992, 72(2): 467-462 Axonogenesis and Morphogenesis in the Embryonic Zebrafish Brain Linda S. Ross, Timothy Parrett, and Stephen S. Easter, Jr. Biology Department, University of Michigan, Ann Arbor, Michigan 48109-1048 We have examined early neuronal differentiation and ax- A hallmark of the vertebrate brain is the complex yet precise onogenesis in the fore- and midbrain of zebrafish embryos set of interconnections between neurons; understandingthe de- to address general issues of early vertebrate brain devel- velopmental basisof this complex circuitry hasbeen a challenge opment. AChE expression and HNK-1 antibody immuno- to embryologistsfor decades.Most contemporary studies,such reactivity were used as markers for differentiated neurons as those of the retinotectal pathway, have examined relatively and axons, respectively. late phasesof ontogeny, long after early circuits have been es- The pattern of neuronal differentiation followed a stereo- tablished by the early outgrowth of pioneer fibers (Easter and typed sequence. AChE-positive cells first appeared between Taylor, 1989). The development of the earliest tracts used to 14 and 16 hr in three small, isolated, bilaterally symmetrical be a major concern of developmental neurobiologists,especially clusters on the surface of the brain. The three clusters-the Coghill (19 13, 1929), Tello (1923), Windle (1932) and Henick dorsorostral, ventrorostral, and ventrocaudal clusters- (1938). However, the techniquesavailable to them were limited, proved to be the progenitors of the telencephalon, ventral and eventually the publications regarding early brain develop- diencephalon, and mesencephalic tegmentum, respective- ment diminished. More recently, other techniqueshave become ly. With further development, more cells were added to these available, including electron microscopy, tract-tracing methods, three clusters, and new clusters appeared in the anlage of and immunocytochemistry; theseimproved techniquespromise the epiphysis (16 hr) and in the pituitary and dorsal mes- to reveal information that the older methodscould not. In fact, encephalon (by 24 hr). Subsequently, as more neurons dif- these improved methods have been exploited to great effect, ferentiated, the gaps of unlabeled cells were reduced; by 46 initially in the invertebrates (e.g., Bate, 1976; Goodman et al., hr, the cluster boundaries were indistinguishable. 1982; Bentley and Caudy, 1983) and more recently in the ver- Axonogenesis also followed a stereotyped sequence. The tebrates (Kimmel et al., 1982; Roberts and Clarke, 1982; Ja- first HNK-l-labeled processes arose from the first three cobson and Huang, 1985; Stallcup et al., 1985; Kuwada, 1986; clusters of AChE-positive cells and connected the clusters. Puelleset al., 1987a,b; Eaglesonand Harris, 1990). The earliest axonal growth cones appeared at 16 hr, directed While most of the vertebrate work has concentrated on the caudally from two to three neurons of the ventrocaudal clus- hindbrain and spinal cord, we have recently turned our attention ter and pioneering the ventral longitudinal tract. By 16 hr, to portions of the rostra1neural tube that give rise to the fore- the tract of the postoptic commissure was initiated by growth and midbrain. We have found that this region of the embryonic cones directed caudally from the ventrorostral cluster to- brain of the zebrafish is reasonably simple by 24 hr of devel- ward the ventrocaudal cluster. By 20 hr, axons from the dor- opment; the axonal tracts are confined to a simple scaffold of sorostral cluster projected ventrally to form the supraoptic five bilaterally symmetrical tracts and four commissures(Chit- tract. The other dorsoventral tracts (the dorsoventral dien- nis and Kuwada, 1990; Wilson et al., 1990). cephalic tract and the tract of the posterior commissure) Here, we examine the origin of the axonal scaffold by tracing became evident between 20 and 24 hr. the development of the first neuronsand their axonal tracts. We These observations provide a continuous record of the have used two anatomical markers for differentiated neurons: topological distortions involved in the conversion of the tu- AChE activity and HNK- 1 immunoreactivity. The spatiotem- bular embryonic brain into the contorted adult form. The poral expression of AChE has been used as an indicator of telencephalon, ventral diencephalon, and hypothalamus development in several vertebrates (chick: Layer, 1983; Layer originate from the same rostrocaudal level of the neural tube. and Spoms, 1987; Puelles et al., 1987a; Layer et al., 1988; The pattern of differentiation demonstrated that the early Weikert et al., 1990; frog: Moody and Stein, 1988; zebrafish: development of the rostra1 neural tube occurs simultaneous- Hannemanet al., 1988; Hanneman and Westerfield, 1989; Wil- ly in several independent centers, similar to the overtly seg- son et al., 1990). Newly generated neurons migrate from the mental development of the hindbrain. periventricular matrix zone out to the mantle layer where they begin to differentiate (Fujita, 1962, 1964), and transiently ex- press AChE (Miki et al., 198la,b; Miki and Mizoguti, 1982; Received June 27, 1991; revised Sept. 6, 1991; accepted Sept. 13, 1991. Layer, 1983; Mizoguti and Miki, 1985). This early embryonic This work was supported by NIH Research Grant EY-00168 to S.S.E. and NIH expressionof AChE is probably not linked to neurotransmitter Traineeships HD-07274 and EY-07022 to L.S.R. We thank Celeste Malinoski for her expert technical assistance, and Drs. Kathryn Tosney, Stephen Wilson, and catabolism, but it may be important to the early morphogenesis John Zook, Mr. John Burrill, and Ms. Riva Marcus for their useful comments on of the nervous system, becausein Drosophila, a mutant for the manuscript. Correspondence should be addressed to Dr. Linda S. Ross, Department of AChE shows abnormal neuronal development (Greenspan et Zoological and Biomedical Sciences, Ohio University, Athens, OH 45701. al., 1980; Hall et al., 1980). The HNK-1 antibody labels an Copyright 0 1992 Society for Neuroscience 0270-6474/92/120467-16$05.00/O epitope that is found on a variety of cell adhesion molecules 466 Ross et al. - Zebrafish Brain Development 12 hr Figure 1. Lateral views (camera lucida drawings) of brains (upper)and embryos (lower)of the ages discussed in this article. The optic recess (or) and otocyst (ot) are indicated as landmarks. bfm, boundary between fore- and midbrains; bmh, boundary between mid- and hindbrains; cb, cerebellum; dc, diencephalon; ep, epiphysis; ey, eye; hy, hypothalamus; op, olfactory placode; 1, tectum; tc, telencephalon; vf; ventral flexure. Scale bars, 50 pm. (Kruse et al., 1984), and this immunoreactivity has proven use- hindbrain segmentation is strong. However, the case for seg- ful in visualizing early axons (Nordlander, 1989; Metcalfe et al., mentation in rostra1 parts of the brain is much weaker. Earlier 1990; Wilson et al., 1990). workers (e.g., Bergquist, 1952)had described“neuromeres” there, The description of early tract formation not only provides but these descriptions lacked the elegant simplicity of the hind- information previously unavailable, but enablesus to address brain and spinal cord segments,at least partly becausethe brains two fundamental issuesin the development of the vertebrate described by Bergquist and his contemporaries were so non- CNS: the topology underlying the transformation of the rostra1 tubular. Wilson et al. (1990) usedAChE histochemistry to eval- neural tube into the brain and the segmentation of the rostra1 uate possible segmentationin the fore- and midbrains of 24 hr neural tube. zebrafish embryos, but the complexity of the brain at 24 hr may The early neural tube in vertebrates is quite cylindrical, but have obscured the possiblesubdivisions. In this article, we pre- as it develops, its rostra1 aspect becomesso distorted that the sent evidence for these subdivisions. original rostrocaudal axis is not evident. Others have used a variety of methods to identify this axis (Puelles et al., 1987b, Materials and Methods and publications cited therein). Here, we use markers for dif- Zebrafish embryos were obtained from daily spawnings of a colony in ferentiated neurons and axons to identify the earliest ones in our laboratory. Embryosat the four or eight cell stagewere grouped the -astral neural tube. As these axons and their cells of origin into separate Petri dishes and placed in an incubator at 28.5”C. Time are %lowed during the subsequentbrain flexures and distor- of fertilization was taken as 53 min (four cells) or 71 min (eight cells) prior to staging (Kimmel and Law, 1985); this estimate of age is accurate tions, it is possible to specify the direction of distortion and to within 18 min. Ages of embryos are expressed as hours postfertili- thus identify the original rostrocaudal axis. We show that the zation. rostrocaudalaxis parallelsthe tract of the postoptic commissure. These sameobservations are the basis for identifying the pre- Tissuepreparation cursors of the major subdivisions of the fore- and midbrain. Dechorionated embryos were anesthetized in a 0.03% aqueous solution Segmentationof the vertebrate CNS was s!atematically stud- of 3-aminobenzoic acid ethyl ester (Sigma), rinsed in 0.1 M maleate ied until the early 195Os,but was then largely ignored for thirty buffer (for AChE histochemistry)or 0.1 M phosphatebuffer (for all other years until interest was revived by more modem approaches procedures), and fixed