Segmental pattern of development of the hindbrain and spinal cord of the zebrafish embryo

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1 Development (1988) Printed in Great Britain The Company of Biologists Limited Segmental pattern of development of the hindbrain and spinal cord of the zebrafish embryo ERIC HANNEMAN*, BILL TREVARROW, WALTER K. METCALFE, CHARLES B. KIMMEL and MONTE WESTERFIELD Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA Current address: Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, OR 97201, USA Summary In the ventral hindbrain and spinal cord of zebrafish embryos, the first neurones that can be identified appear as single cells or small clusters of cells, distributed periodically at intervals equal to the length of a somite. In the hindbrain, a series of neuromeres of corresponding length is present, and the earliest neurones are located in the centres of each neuromere. Young neurones within both the hindbrain and spinal cord were identified in live embryos using Nomarski optics, and histochemically by labelling for acetylcholinesterase activity and expression of an antigen recognized by the monoclonal antibody zn-1. Among them are individually identified hindbrain reticulospinal neurones and spinal motoneurones. These obervations suggest that early development in these regions of the CNS reflects a common segmental pattern. Subsequently, as more neurones differentiate, the initially similar patterning of the cells in these two regions diverges. A continuous longitudinal column of developing neurones appears in the spinal cord, whereas an alternating series of large and small clusters of neurones is present in the hindbrain. Key words: segmentation, neuromere, zebrafish, acetylcholinesterase, pattern formation, hindbrain, spinal cord. Introduction A segment is usually taken to mean a set of structures that is iterated serially along the body axis (discussed in Stent, 1985), and studies in diverse organisms indicate that segmental patterns of organization are common in nature. Serially repeated arrangements of neurones and their development have been studied in the grasshopper (Wilson & Hoyle, 1978; Doe & Goodman, 1985), Drosophila (Ghysen & Jan, 1985; Hartenstein & Campos-Ortega, 1984), the leech (Zipser, 1982; Kramer & Weisblat, 1985; Weisblat & Shankland, 1985) and the nematode (White et al. 1976; Sulston et al. 1983). In contrast, much less is known about segmental neurones or their development in vertebrates (review: Keynes & Stern, 1985). In particular, the extent of segmentation of the vertebrate brain is controversial (Jarvik, 1980; Gans & Northcutt, 1983). Recent studies of the central nervous system of zebrafish have suggested that several types of identified neurones develop segmentally (review: Kimmel & Westerfield, 1988). In the spinal cord, individually identifiable primary motoneurones that repeat in register with the segmental series of myotomes have been characterized (Westerfield et al. 1986; Myers et al. 1986). The primary motoneurones on each side of a segment contribute their axons to a single ventral root and innervate muscle fibres restricted to one overlying myotome. In the hindbrain, iterated series of identified interneurones, including local reticular neurones (Kimmel et al. 1985) and reticulospinal neurones have been described (Metcalfe et al. 1986). The morphology of the reticulospinal neurones suggested that they were present as several families of segmental homologues (Metcalfe et al. 1986). Developmental studies have shown that the primary motoneurones (Eisen et al. 1986; Myers et al. 1986), and a subset of the reticulospinal neurones (Mendelson, 1986a,b) are generated and develop axons during the first day after fertilization, in a pattern that looks overtly segmental.

2 50 E. Hanneman and others Although these data suggest that discrete types of neurones in the CNS are arranged and develop segmentally, it was unknown whether the apparent segmental pattern is a special feature of these particular cells or it reflects a more general developmental plan. Furthermore, classical evidence for CNS segmentation included the identification of a series of swellings, or neuromeres, along the surface of the hindbrain in a variety of vertebrate species (reviewed in Jacobson & Meier, 1984). Neuromeres have not been described in zebrafish, thus it has not been possible to ask how the serially repeating sets of neurones are positioned within neuromeres. Here we report studies in which we have identified the earliest neurones to arise in the ventral regions of the zebrafish spinal cord and hindbrain. We have observed the young neurones with Nomarski optics in live embryos, distinguishing their somata by their large size and characteristic positions (Eisen et al. 1986). In addition, we have used two labelling techniques to reveal developing neurones; expression of the enzyme acetylcholinesterase (AChE; Hanneman & Westerfield, 1988) and of a neuronal antigen that is recognized by the monoclonal antibody zn-1 (Trevarrow & Hanneman, 1985; Myers et al. 1986; Trevarrow, 1988). We found that the first neurones that can be identified by all three methods in the ventral hindbrain and spinal cord of zebrafish embryos are present as single cells or small clusters of cells that repeat with the same periodicity in both locations. We identified hindbrain neuromeres and found that single segmental sets of hindbrain neurones are located within single neuromeres, whose length corresponds to the length of a trunk somite. We describe this early pattern and the subsequent changes that occur during the next few hours of development. Materials and methods Embryos Embryos were collected from spontaneous spawnings and staged when 4-8 cells were present in order to estimate the time of fertilization within about 15 min (fertilization occurs 1 h before the 4-cell stage and 1-3 h before the 8-cell stage). The embryos were incubated at 28-5 C. Prior to live observations and histochemistry, their chorions were removed with forceps and the embryos were anaesthetized in 008% tricaine methane sulphonate ("Finquel", Ayerst Laboratories). Observations of live embryos Embryos at h (h: hours postfertilization at 28-5 C) were mounted between coverslips to permit oblique side views of the hindbrain and rostral spinal cord, and examined using Nomarski interference contrast optics (Zeiss Universal Microscope, x25 and x40 Plan objectives). In some cases, the cells were photographed and, in other cases, drawings were made with the aid of a camera lucida. Distances were measured either on photographic prints or the drawings; equivalent measurements were obtained with both methods. Distances were not corrected for foreshortening due to the curvature of the neuraxis in young embryos, which produces some underestimate of the actual lengths. A cetylcholinesterase The techniques for fixation and staining of AChE activity were based on the method of Karnovsky & Roots (1964) as previously described (Hanneman & Westerfield, 1988). Briefly, anaesthetized embryos were fixed in cold 4 % buffered formaldehyde for 4-12 h, rinsed in 0-3M-sucrose and embedded in gelatin. Sections 30^m thick were cut on a vibratome and dried onto gelatin-coated slides. Activity of AChE was developed for 4-12 h at 4 C. The reaction product could be intensified by immersing the reacted tissue in a solution containing diaminobenzidine (Sigma, DAB) in 0-1 M-phosphate buffer at 4 C for ]0min. The activity has previously been shown to be specifically due to AChE (Hanneman & Westerfield, 1988). Measurements made from the sections were corrected for shrinkage due to fixation, estimated to be 20% from comparisons with unfixed material (unpublished observations). Lengths are reported as the mean ± the standard deviation. zn-1 antibody Details of the production of the zn-1 monoclonal antibody, and the types of cells it recognizes, are described elsewhere (Trevarrow, 1988). Labelling of cells with zn-1 was performed by the indirect peroxidase-antiperoxidase (PAP) method (Sternberger, 1979). Vibratome sections, mounted on slides, were preincubated with 1 % bovine serum albumin in phosphate-buffered saline (0-13M-NaCl, M- KC1, 0-05M-sodium phosphate, ph ), and then overnight in media from hybridoma cultures. After several washes, goat anti-mouse IgG (1:100) was applied as a bridge, followed by another rinse. Monoclonal PAP (1:100) was then applied, and subsequently the slides were rinsed before being incubated in 0-05mgml" 1 DAB and 0003% H 2 O 2. Following histochemistry, sections were dehydrated in increasing concentrations of ethanol followed by xylene, and a coverslip applied with Permount. Scanning electron microscopy Embryos at 17 h were fixed overnight in a solution of 1 % paraformaldehyde, 1-25% glutaraldehyde, and 0-1 M- sodium phosphate buffer (ph 7-4). They were then washed in 0-1 M-phosphate buffer, rinsed in distilled water, dehydrated through a series of ethanols and critical-point dried. The dried embryos were then mounted on SEM stubs with a small drop of rubber cement and the skin over the hindbrain was removed by touching the skin with a fine needle covered with a small amount of rubber cement. The animals were sputter-coated with gold-paladium and viewed with an AMR-1000A scanning electron microscope. Shrinkage due to specimen preparation was approximately 40%.

3 Segmental CNS development Results Segmental arrangement of neurones in the embryonic spinal cord Primary motoneurones in the spinal cord are segmentally arranged, as demonstrated with three different methods; AChE staining, Nomarski observation of live embryos and staining with the zn-l monoclonal antibody. Spinal segments are in register with the myotomes (Myers, 1985), of which about 30 develop, 15 in the trunk and 15 in the tail. The first neurones to express AChE activity in the zebrafish were described previously (Hanneman & Westerfield, 1988). They are visible before 15 h as bilateral pairs of cells repeating once per segment in the spinal cord and are thought to be young CaP motoneurones, expressing this marker before their axons develop. CaP motoneurones are one of three types of primary motoneurones that are present as single cells on each side of each spinal segment (Westerfield etal. 1986; Myers etal. 1986). Using Nomarski differential interference contrast optics, CaP motoneurones can also be identified by their large size in live embryos before their axons develop and were shown to be the first motoneurones to grow axons, at about 18 h in trunk segments (Eisen et al. 1986). What appear to be the same neurones can also be identified at about the time of axogenesis by their expression of the antigen recognized by the monoclonal antibody zn-l. In the later embryo, zn-l stains a wide variety of, and perhaps all, the neurones in the CNS (Trevarrow, 1988), but at h only about one bilateral pair of ventrally located cells is labelled per segment (Fig. 1). Within a few hours, cells now well stained by this method at the same locations can be positively identified as CaP motoneurones from the distribution of their axons within the myotomes 51 (shown in Myers et al. 1986). The putative CaP motoneurones are the most intensely staining of a very small set of zn-l positive cells that repeat at segmental intervals. Among the other stained cells at 24 h are the other two types of primary motoneurones (MiP and RoP; see Myers et al. 1986). These three assays, large cell size, expression of AChE and staining by zn-l, demonstrate that the first neurones to appear in the ventral spinal cord are arranged segmentally. We compare this simple pattern with that of the hindbrain. Hindbrain neuromeres contain segmental sets of neurones The segmental structure of the hindbrain is apparent by comparing the sculpturing of its surface and the positions of the earliest neurones to develop. Fig. 2, drawn from a side view of a live embryo, summarizes these relationships which we now consider in detail. Regions along the neuraxis proposed to represent neural segments are assigned names, following convention used previously (legend to Fig. 2). Inspection of live h embryos using Nomarski optics revealed a series of swellings of the hindbrain surface (Figs 2, 3A) that appear to correspond to the neuromeres that have been described previously in embryos of other fishes (Harrison, 1895; Goodrich, 1930). The same pattern was visible in fixed animals viewed by SEM (Fig. 3B). Both methods showed that the presence and locations of neuromeres were consistent features of all embryos, although the prominence of the neuromeres varied considerably among embryos. The segmental structure of the hindbrain was also apparent at the cellular level. By examining live h embryos, we could usually locate large and v Fig. 1. The first neurones to express zn-l antigen in the zebrafish spinal cord are located at segmental intervals. Horizontal section through rostral tail segments at 21 h. A repeat pattern is evident; the intervals correspond to the lengths of the myotomes, which are not clearly shown here. The locations of the cells suggest they are CaP motoneurones, which are known to be labelled by zn-l at early times in their development (Myers et al. 1986). Bar, 50 /tm.

4 52 E. Hanneman and others Fig. 2. Segmental patterning of the hindbrain. Camera-lucida drawing showing the positions of neuromeres, young neurones and other landmark features distinguishable in a selected living embryo viewed with Nomarski interference contrast optics at 20-5 h. Left side, with dorsal to the top. The neuronal somata were identified by their sizes and ventrolateral locations (see Fig. 3C). Single cells or small clusters of cells were visible in each hindbrain segment. The Rol neuromere was not prominent in this animal. Identification was also more uncertain in the caudal (Ca) hindbrain. Neuromeres (or segments) are named following Metcalfe et al. (1986). Rol-3, rostral hindbrain segments; Mil-3, middle hindbrain segments; Cal-3, caudal hindbrain segments; Spl-3, spinal segments; Mth, Mauthner reticulospinal neurone; MiD2-3, Middle Dorsal reticulospinal neurones (see Metcalfe et al. 1986); CaPl, Caudal Primary motoneurone in Spl; myol-3, the first three myotomes; oto, otic vesicle. Bar, 50fim. prominent cells, present as individuals or as small clusters of two or three cells on each side of the midline of the brain and placed ventrolaterally at the middle of each neuromere. These cells looked similar in soma morphology to the primary motoneurones of the spinal cord, and probably were postmitotic young neurones in early stages of their differentiation (see below). In particular, as illustrated in Fig. 3C, we could nearly always specifically identify a distinctive large cell (arrow) in the neuromere at the level of the rostral margin of the otic capsule. The position of this cell corresponded to that of the Mauthner neurone, a well-known reticulospinal neurone. A single Mauthner neurone is present on each side of the midline of the brain. It is located in the centre of the Mil neuromere (Metcalfe et al. 1986) and can be uniquely identified, even at early stages (Mendelson, 19866) because, among reticulospinal neurones in this neuromere, its axon is the only one to take a crossed pathway to the spinal cord. Staining with appropriate monoclonal antibodies reveals its axon to be present by 18 h (Kuwada et al. 1987; W. K. Metcalfe, unpublished observations). Developing neurones in the hindbrain were studied by AChE histochemistry and zn-1 immunocytochemistry. The positions of the first cells labelled with either method are the same, and both methods appear to label the large neurones identified in live embryos. For example, the Mauthner neurone was shown to express both AChE (Hanneman & Westerfield, 1988) and zn-1 (Trevarrow, 1988). Initially only one or a few cells per segment were labelled (Fig. 4) and, as in the spinal cord, AChE activity precedes zn- 1 staining by about 3h. At the earliest times, we occasionally observed labelled cells at one half segment intervals with either staining method, suggesting that cells located at the neuromere borders were beginning to differentiate (see below). We measured the intervals between the cell clusters observed in live embryos, and the intervals between cells stained with AChE, to obtain estimates of hindbrain segment length. The data, shown in Table 1, reveal that at h the hindbrain neuromeres and rostral myotomes in the trunk are both about 55 fim long. Similar lengths were obtained by both methods. The hindbrain contains about nine neuromeres The most-distinctive neuromeres, shown in Fig. 3A, contained the most-prominent cells or cell clusters by the Nomarski observations. These are the segments Ro2 through Mi3. These five segments were previously shown to contain the earliest hindbrain reticulospinal neurones to develop (Mendelson, 19866). There are reproducible differences in their sizes; Figs 2 and 3A show that the Ro3 and Mi2 neuromeres are larger than their neighbours. The Rol segment typically was less distinctive by the Nomarski assay. Distinctive large cells were not observed at this stage of development within the primordium of the cerebellum, located at the rostral border of Rol.

5 Segmental CNS development 53 Fig. 3. Neuromeres and young neurones of the hindbrain. (A) Hindbrain neuromeres in a live embryo at 18h, photographed using Nomarski optics. Left side, with dorsal to the top. Three rostral-level (Rol-3), three middle-level (Mil-3), and the first caudal-level (Cal) neuromeres are shown (see Fig. 2 for the complete set). The borders of one neuromere, Mil, are indicated for comparison with panel C. Its neighbouring neuromeres are the two most prominent ones; Ro3 rostrally, and Mi2 caudally at the level of the otic vesicle (oto). (B) Scanning electron micrograph of the dorsal surface of the hindbrain of an embryo fixed at 17 h. The skin overlying the hindbrain has been removed, revealing the three neuromeres of middle (Mi) hindbrain, and the otic vesicle (oto). The fourth ventricle has not yet opened at this stage of development. (C) The Mil neuromere, photographed using Nomarski optics from the left side of a live embryo at 17 h. The neuromere bulges dorsally (top) and laterally; its borders are indicated by arrowheads. A developing neurone (arrow), positioned ventrolaterally near the centre of the neuromere, is larger than surrounding cells. It is probably the young Mauthner neurone, a reticulospinal cell known to develop very early and to be present at this location (at the rostral end of the overlying otic vesicle). Bars, A, 50^m; B and C, 10/im. In addition to three Ro and three Mi segments, we have tentatively identified three caudal-level or Ca segments between the level of the otic vesicle and the first myotome, based upon the distribution of large cells (Fig. 2). Assuming this assignment is correct (see Discussion), there would be a total of nine

6 54 E. Hanneman and others hindbrain segments. The next neural segment caudally, which we call the first spinal segment (Spl; Fig. 2), contains the CaP motoneurone that innervates muscles of the first myotome. This cell, CaPl, and more frequently CaP2 (in Sp2) could be identified by their axonal growth cones entering the myotome as described by Eisen et al. (1986). Divergence in pattern between the spinal cord and hindbrain Our observations have revealed a marked similarity in the early development of the segmental pattern in the spinal cord and hindbrain. On the other hand, some differences are present from the times of our earliest observations (16h). For example, the width of the hindbrain is considerably greater than the spinal cord (Table 2, and compare Figs 1 and 4), indicating that a hindbrain segment must contain considerably more cells than a spinal segment, since at this stage their other dimensions are similar. The differences in dimensions between these two regions of the CNS become more pronounced with continued development. As reported above, at h both the hindbrain and spinal cord segments are about 55 fim long. By 120 h (5 days postfertilization), the length of a spinal segment has increased to about 100 //m (Myers, 1985; determined from the positions of ventral roots), whereas hindbrain segment length decreases to about 35 ^m (Metcalfe et al. 1986; determined from the positions of segmentally iterated sets of reticulospinal neurones). We observed that between 16 and 120 h the width of the hindbrain increases about threefold, whereas the width of the spinal cord only increases about 1-4-fold (Table 2). Divergence between the spinal cord and hindbrain is also observed in the pattern of early neuronal development. In both regions, the number of differentiating cells increases rapidly after the first ones are observed, as shown in Fig. 5 for hindbrain AChE expression from 16 20h. However, the distribution of these additional cells differs: in the spinal cord, clusters of smaller neurones appear between the Table 1. Lengths of hindbrain neuromeres and trunk myotomes Method Live* AChEt Neuromere 57 ±11 53 ±6 Length (nm ±s.d.) n 36 8 Myotome 52 ±4 56±4 * Measurements using Nomarski optics in five embryos at h. t Measurements in fixed and stained sections from three embryos at 16h. The data are corrected for 20% shrinkage. n 17 8 Table 2. Width of the hindbrain and spinal cord Age (h) * Widths t Widths 20- were were Width (>m±s.d.) Hindbrain* 151 ± ±22 measured at the measured at the Spinal cordf 57 ± O±17 Mil segment. Sp5 segment. Rol Ro2 Ro3 Mil Mi2 Mi3 Cal Ca2 Ca3 Neuromere Fig. 5. More cells in the hindbrain express AChE as development proceeds. The numbers of cells stained for AChE activity were counted in sections from sets of 10 embryos fixed at 2h intervals. Closed circles, 16 h; open squares, 18 h; closed squares, 20 h. labelled larger cells that were observed initially and, by 28 h, the increased numbers of these cells produce continuous labelling along the longitudinal axis of the spinal cord as shown in Fig. 6. In contrast, in the Ro and Mi regions of the hindbrain the addition of new labelled cells produces a discontinuous pattern, an alternating series of small and large clusters that was evident with both staining methods (Fig. 7). On each side of the midline a single large cluster is present at the centre of each neuromere and a single small cluster is present at the border between adjacent Fig. 4. The first neurones to express AChE (in A) and the zn-1 antigen (B) occur at segmental intervals in the hindbrain. The panels compare horizontal sections from 18 h embryos, with rostral to the left, and photographed with Nomarski optics. The bilateral nature of the pattern is particularly evident in B. Labelled young neurones on one side of the midline within the Mi2, Mi3, Cal and Ca2 neuromeres, are indicated by arrowheads, oto, otic vesicle. Bar, n 7 5 5

7 4A 4

8 Segmental CNS development 55 Fig. 6. Cells stained for AChE activity form a continuous column on each side of the midline of the spinal cord at 28 h. The Nomarski photograph is of a horizontal section in the region of Sp2-5. Rostral is to the left. Large cells located near the outside borders of the spinal cord are labelled, while smaller cells deep in the spinal cord are not. In the periphery, the transverse myosepta are also AChE-positive, and a pair bordering a single myotome is evident as oblique lines in the lower left of the figure, giving a measure of segment length. Bar, 50 fan. neuromeres. Thus single bilateral pairs of large and small clusters comprise a single segmental equivalent. It was clear from the positions of the large clusters of stained neurones in the centres of the neuromeres at h that they were present exactly where the first developing neurones were observed at the earlier stages (e.g. 16 h). Moreover, in experiments reported elsewhere (Hanneman & Westerfield, 1988) the Mauthner, MiD2, and MiD3 reticulospinal neurones were located within the large clusters stained by AChE by doubly labelling them with AChE and retrogradely transported HRP. We also were able to locate the same cells within the large clusters as revealed by the zn-1 antibody because their axons are stained by this antibody, permitting unambiguous identification of the cells. Thus, the large clusters identified by zn-1 and AChE indeed correspond, and contain the Mauthner, MiD2, MiD3 serial family (see Metcalfe et al. 1986) of identified reticulospinal neurones. The small clusters, developing at neuromere borders where we only occasionally saw singlecell staining at earlier times, would contain other neurones, including MiR reticulospinal neurones, that are present a half-segment away from the ones described above (Metcalfe et al. 1986). At 28 h, the Ca2 and Ca3 segments exhibit a more continuous column of labelled neurones; in this respect, they are more like segments of the spinal cord in their organization at this stage. Discussion The earliest neurones to arise in the ventral hindbrain and spinal cord of the zebrafish appear at periodic intervals, as demonstrated by direct observations of live zebrafish embryos and two independent markers of neuronal differentiation; AChE activity and the antigen recognized by the monoclonal antibody zn-j. The periodicity is identical initially in both of these regions and corresponds in length to that of the trunk somites. Moreover, we observed in the hindbrain a corresponding set of swellings, which we termed neuromeres (see Jacobson & Meier, 1984, for a recent discussion of neuromeres in vertebrate embryos). Although the locations of spinal roots has long been known to be segmental in vertebrates, a segmental patterning of neurones (including motoneurones) within the CNS is not commonly observed, even in embryos (but see Bennet & DiLulio, 1985). For example, the embryonic rat spinal cord initially contains a continuous column of young neurones (Altman & Bayer, 1984), such as we observed in older zebrafish embryos. The difference may be due to the presence of fewer neurones in zebrafish embryos than in other species that have been studied, for as shown here even in the zebrafish the segmental pattern becomes obscured as many other neurones develop. Our observations suggest that nine hindbrain segments are present between the cerebellum and the level of the first myotome. Previous evidence for segmentation of the zebrafish hindbrain was based on

9 56 E. Hanneman and others Fig. 7. Alternating large and small clusters of cells are segmentally arranged, one each per segment, in the hindbrain. Nomarski photographs of parasagittal sections of the left side (dorsal up), stained for AChE (A) and zn-1 antigen (B) at 30h. As is particularly clear in the middle of panel A, the large clusters are in the centres of the hindbrain neuromeres and the small clusters are at the neuromere borders. Bar, 50fim. the patterning and development of identified reticular interneurones (Kimmel et al. 1985; Metcalfe et al. 1986; Mendelson, 1986a,b). The present observations reveal that the neurones identified earlier are a part of a larger population of cells that develop at segmental intervals. The Ro and Mi neuromeres, described here, each contain a single segmental set of reticulospinal cells. Furthermore, the segments that contain the reticulospinal cells that develop earliest (Mendelson, 1986i>) form the most prominent neuromeres. These are the Ro2, Ro3 and the three Mi segments. The designation of the two caudalmost hindbrain segments, Ca2 and Ca3, is tentative, since the neuromeres of this region are not prominent and the early neurones in this region have not been individually identified. The Ca2 and Ca3 segments are transitional between the brain and the spinal cord and have some distinguishing features that are described elsewhere (Trevarrow, 1988). The similarity in the early development of the spinal cord and hindbrain suggests a common plan of segmental patterning in both of these regions of the

10 Segmental CNS development 57 CNS. It has been proposed that the hindbrain and spinal cord have a common evolutionary origin (e.g. see Goodrich, 1930; Jarvik, 1980), and if this were true it would help to explain the similar patterning of early development of these two regions. Previous evidence for the homology of hindbrain and spinal segments was based on the presence of neuromeres, and on morphological considerations in older animals, e.g. the arrangements of cranial nerve nuclei and roots (Ariens Kappers et al. 1936; Jarvik, 1980). More recently, an alternative view has appeared that the brain and spinal cord are entirely separate in their evolution (Gans & Northcutt, 1983; Northcutt & Gans, 1983). Our observations, in so far as they go, could be construed as developmental evidence in support of spinal cord and hindbrain homology, rather than their origins being independent. What is clear from our work, however, is that it is important to examine early embryonic stages to look for common patterning features, since we observed marked divergence between the hindbrain and spinal cord within only a few hours after their first neurones appear. Whereas differences in morphology from one spinal segment to another have not been identified, hindbrain segments vary considerably from one another as well as from spinal segments. Individual hindbrain neuromeres are distinctive in size even at early times, and they develop distinctive sets of interneurones (Kimmel et al. 1985; Metcalfe et al. 1986). In Drosophila, genes that control segmentspecific differences have been identified (Lewis, 1978) and their expression during early development has been studied (Hafen etal. 1984; Carroll etal. 1986). 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