Institute of Neuroscience, 1254 University of Oregon, Eugene, OR 97403-1254, USA
* Present address: Department of Genetics, Campus Box 7264, University of North Carolina, Chapel Hill, NC, 27599-7264, USA
Author for correspondence (e-mail: eisen{at}uoneuro.uoregon.edu)
Accepted 17 May 2002
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SUMMARY |
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Key words: Anteroposterior, Compartments, Hox expression, Resegmentation, Sclerotome, Segmentation, Somite, Vertebral column, Zebrafish
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INTRODUCTION |
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The vertebral column and ribs that make up the post-cranial axial skeleton have a metameric organization. The functional segmental unit of the vertebral column, the vertebra, is composed, in the simplest terms, of the vertebral body or centrum that develops around the embryonic notochord, dorsally extending neural arches and spines, ventrally extending hemal arches, attachment sites for ribs, and various other decorations of these regions (Fig. 1A). These vertebral column elements are distributed in a region-specific manner along the AP axis. Based on such attributes, vertebrae can be grouped into common types, the relative number of each type providing the so-called vertebral or axial formula for a given vertebrate. For example, tetrapods generally have a characteristic number of cervical, thoracic, lumbar, sacral and caudal vertebrae; however, in other vertebrates the vertebral types are less easily categorized (see Romer, 1956).
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The post-cranial axial skeleton is derived from somitic mesoderm but the nature of the segmental relationship between these structures remains obscure. The vertebral column develops from sclerotome, a mesenchymal cell population derived from ventral somite. Sclerotome cells that will contribute to the vertebral column move to surround axial midline structures, condense and differentiate as chondrocytes, thus forming a cartilaginous skeletal framework that later is replaced by bone. The somites themselves are segmentally repeating units of paraxial mesoderm. The segmental register of the somite column and vertebral column are offset, a fact recognized from the early days of modern embryology (see Remak, 1855) (reviewed by Brand-Saberi and Christ, 2000
). Experimental studies of somite contribution to the vertebral column has been confined to avian embryos in which sclerotome comprises a major part of the somite. The overall picture that has emerged is that each somite contributes, essentially without significant AP dispersal, to adjacent body structures including sclerotome-derived vertebral components (Fig. 1B) (Beresford, 1983
; Aoyama and Asamoto, 1988
; Bagnall et al., 1988
; Lance-Jones, 1988
; Ewan and Everett, 1992
; Aoyama and Asamoto, 2000
; Huang et al., 2000a
). The original boundary between somites ultimately aligns near the midline of the adjacent vertebral segment, a phenomenon commonly referred to as resegmentation (reviewed by Brand-Saberi and Christ, 2000
; Christ et al., 2000
; Saga and Takeda, 2001
).
Recent work has begun to elucidate the events of paraxial mesoderm segmentation and somitogenesis (reviewed by Christ et al., 2000; Holley and Nüsslein-Volhard, 2000
; Stickney et al., 2000
; Stockdale et al., 2000
; Maroto and Pourquié, 2001
; Saga and Takeda, 2001
). It is now clear that anterior (A) and posterior (P) domains exist within each segmental unit and are established prior to somite epithelialization. Evidence of A and P domains within the somite and the offset segmental register of somites and their vertebral derivatives underlie comparisons between somite/vertebral development and segmentation in Drosophila melanogaster (Christ et al., 1998
; Christ et al., 2000
; Huang et al., 2000a
; Stern and Vasiliauskas, 2000
). In fact, the textbook resegmentation presentation of development from somites to vertebral column (Fig. 1B) coincides closely with the current model of segmental development in fly integument. A prominent feature of early metamerism in D. melanogaster is the repeating anterior and posterior subdivisions of the elemental segmental unit the parasegment (Martinez-Arias and Lawrence, 1985
). The register of parasegments is offset from that of the segments, which become morphologically evident later in development. These AP subdivisions are lineage-based ectodermal compartments (García-Bellido et al., 1973
), a feature that is thought to be important for formation and maintenance of developmentally relevant boundaries (reviewed by Dahmann and Basler, 1999
). In vertebrates, single cell labels in chick segmental plate, prior to somite formation, have ruled out lineage-restricted compartments for formation of somites or their AP domains (Stern et al., 1988
). Half-somite transplant experiments in avian embryos (Aoyama and Asamoto, 2000
), however, appear consistent with a strict resegmentation (Fig. 1B) and suggest anterior and posterior lineage-restricted compartments after somite formation.
In contrast to avian embryos, many other vertebrate embryos, including representatives in fish (Swaen and Brachet, 1899; Swaen and Brachet, 1901
; Sunier, 1911
; Morin-Kensicki and Eisen, 1997
) and frogs (reviewed by Keller, 2000
), develop sclerotome as a relatively minor somite component. How these minor-component sclerotome cells distribute along the AP axis has not been described, although it is clear that significant cell movement is associated with sclerotome development (Morin-Kensicki and Eisen, 1997
). Thus, the generality of restricted anterior and posterior sclerotome contribution to vertebrae needs to be explored in other vertebrate embryos, in addition to avians. For example, scant sclerotome production might support increased dispersal along the AP axis as schematized in Fig. 1C, which depicts a leaky resegmentation model in which sclerotome contribution to the vertebral column is not strictly dependent upon the anterior or posterior somite domain of origin.
Two outstanding issues motivated us to analyze vertebral column development in zebrafish. First, understanding the true developmental sequence leading from somites, in which patterning genes such as those of the Hox complex are expressed, to vertebrae, the structures in which we typically identify evidence of altered patterning and homeosis, provides insight into patterning along the AP body axis. Second, understanding how somite and vertebral numbers relate illuminates evolutionary variation of the vertebrate body plan. In this work, we explore the validity of current views on the segmental relationship of somites and vertebral column to zebrafish development and we perform the critical test of AP somite domain distribution at the single cell level. We find that while the distribution of sclerotome along the AP axis is consistent with a leaky resegmentation, the anterior and posterior sclerotome domains clearly are not lineage-restricted with respect to future vertebrae. We also describe a somites/vertebrae alignment that suggests the two anterior-most somites do not contribute to the zebrafish vertebral column. We characterize the development of the zebrafish vertebral column and relate the AP regional character of the axial skeleton to Hox gene expression patterns. These results support co-localization of anterior expression boundaries of hoxc6 homologs with the cervical/thoracic transition zone in zebrafish, as described for tetrapods. Finally, we find that patterned posterior vertebral types fall outside the domain of Hox family anterior expression boundaries, suggesting alternative patterning mechanisms for these regions.
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MATERIALS AND METHODS |
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Cell labeling
Embryos of 16-22 h had chorions removed, were anesthetized in dilute tricaine methanesulfonate (TMS, Sigma) in physiological saline (Westerfield et al., 1986) and mounted in 1.2% agar on a microslide (Eisen et al., 1989
). Individual sclerotome or presumptive myotome cells were injected with fluorescent dextrans (Molecular Probes) as described by Morin-Kensicki and Eisen (Morin-Kensicki and Eisen, 1997
). Because somite cells that will contribute to myotome remain within their somite of origin (DeVoto et al., 1996
; Morin-Kensicki and Eisen, 1997
), one or more cells of the myotome in a more anterior somite also were labeled to serve as reference points for following sclerotome migration. Data from somites 5-17 were included in this study. For analysis of the cell distribution from anterior and posterior sclerotome domains, only those labeled cells in anterior-most or posterior-most positions within the somite were considered. Early migration data were included from some of the labeled sclerotome cells by Morin-Kensicki and Eisen (Morin-Kensicki and Eisen, 1997
).
Sclerotome progenitors were labeled at 18 h and clone size assayed at 3 d. The fluorescent label often became faint and punctate, precluding a rigorous analysis of sclerotome cell proliferation, although clone size appeared to range between two to six cells. We recorded the distribution of labeled sclerotome cell progeny along the dorsoventral axis in 13 embryos. From a total of 55 cells counted at 3 d, 44 were located dorsal to the notochord or dorsal or lateral to the neural tube, nine were located ventral to the notochord and two were located lateral to the notochord.
Videomicroscopy
Embryos were mounted either in agar or between bridged coverslips (Myers et al., 1986) and examined using a Leitz 50x water immersion objective on a Zeiss Universal microscope. Low light level fluorescent images were obtained through a Dark Invader (Night Vision Systems) and Koyo camera. Images, contrast-enhanced and averaged to remove noise with an Argus 10 image processor (Hamamatsu), were stored on an optical disc recorder (Panasonic). Fluorescent cells were viewed first 5 to 20 minutes after labeling and then at 24 h and 48 h and again between 3-5 d. Zebrafish remain optically clear at these stages allowing three-dimensional localization of labeled cells in live fish by the z-series record at the light microscope. We collected positional data on every labeled cell of each clone in this manner (Morin-Kensicki and Eisen, 1997
). Somites were counted in 24 h embryos as described by Kimmel et al. (Kimmel et al., 1995
). In 4 d fish, the number of somites was inferred from the number of myotomes.
Visualizing vertebrae and myotomes
The early vertebral cartilaginous framework was visualized by in situ hybridization using an -coll2a1 riboprobe (a gift from Y.-L. Yan) (Yan et al., 1995
) as described by Thisse et al. (Thisse et al., 1993
) with the following modifications for larger tissue: fixation, soak and wash times were increased approx. threefold; proteinase K concentration was doubled; tissue was rinsed in 2 mg/ml sodium borohydride for 30 minutes prior to prehybridization; and prehybridization and hybridization buffer contained 1% SDS.
Sclerotome cells injected with fluorescein dextran were visualized in frozen sections at 10 d according to the methods of DeVoto et al. (DeVoto et al., 1996) with an alkaline phosphatase conjugated anti-fluorescein antibody according to manufacturers recommendations (Roche). To visualize vertebrae in these sections, tissue was rinsed twice for 10 minutes in PBS at pH 8.3 and incubated overnight at 4°C in Xylenol Orange (Rahn and Perren, 1971
) (Sigma, X0127) at 1 mg/ml in PBS at pH 8.3. The tissue was then rinsed five times for 10 minutes in PBS at pH 8.3, coverslipped and visualized on an Axioplan microscope (Zeiss) with a rhodamine filter set. Images were captured with a MicroMAX 1300Y5M digital camera (Princeton Instruments) using MetaView software (Universal Imaging Corporation).
Myotome cells injected with fluorescein dextran were visualized by antibody staining in 12-13 d whole-mount zebrafish after 24 hour/4°C fixation in 4% paraformaldehyde and careful removal of the skin. Fish were post-fixed and then washed extensively prior to staining in Alizarin Red (see below). Embryos were stored in a 0.5% solution of KOH in water.
Histological staining
To visualize developing vertebrae, Alcian Blue, 8GX (CI 74240), a common marker for non-mineralized cartilage matrix, and Alizarin Red S (CI 58005), a stain for mineralized cartilage and bone matrix were used. Zebrafish from 3-30 d were immobilized in ice water, anesthetized in dilute TMS, then fixed for 12-72 hours in buffered 4% paraformaldehyde. After washing, fish were placed in either 0.1 mg/ml Alcian Blue in 1:4 glacial acetic acid:95% ethanol (pH 4.5) for 12 hours, or 0.1 mg/ml alizarin red in 0.5% KOH for 2-5 hours. Alcian Blue-stained zebrafish were either transferred to Alizarin Red or dehydrated in 100% ethanol for 1 hour and cleared in methyl salicylate. Alizarin Red-stained zebrafish were dehydrated in 100% methanol, cleared in methyl salicylate and mounted between bridged coverslips in Permount (Fisher Scientific). Procedures for staining adult zebrafish were similar, but fixation, staining and dehydration times were each 1 week.
A modification of a Phloxine-Methylene Blue-Thomas Method staining procedure (Humason, 1972) was used to visualize boundaries between successive myotomes and successive vertebrae. After fixation for 12-72 hours in buffered 4% paraformaldehyde, 15 d zebrafish were immersed in a solution of Methylene Blue (CI 52015), Azure B (CI 52010) and borax, each at 1.25 mg/ml, in water for 2 hours. Fish were then destained in 0.2% aqueous acetic acid for 20 minutes followed by 30 minutes each in 95% then 100% ethanol, cleared in methyl salicylate and mounted between bridged coverslips in Permount.
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RESULTS |
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Relationship between somites and vertebrae
In zebrafish, mesenchymal cells derived from a ventromedial cell cluster of the somite migrate to positions adjacent to the notochord and neural tube (Morin-Kensicki and Eisen, 1997), as expected for sclerotome contribution to connective tissue and vertebral cartilages. To show these mesenchymal cells contribute to the developing vertebral column, we labeled cells of the ventromedial cluster with vital fluorescent dye and processed fish to visualize the label at stages when the developing vertebral column was apparent. We were able to visualize both labeled cells and vertebral components in sectioned material from eight fish. In each case, some of the labeled cells contributed to developing vertebral components that labeled with Xylenol Orange (Rahn and Perren, 1971
) (Fig. 3) verifying that they derived from sclerotome, and thus, that sclerotome makes the expected contributions in zebrafish.
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DISCUSSION |
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Sclerotome distribution pattern in zebrafish supports leaky resegmentation but not lineage-restricted compartments
Models describing development of the vertebral column from somites, usually referred to as resegmentation, have relied on partitioning of the somite into anterior and posterior domains. The shift in register observed between somites and vertebral column is posited to occur when the posterior sclerotome of one somite pair recombines with the anterior sclerotome of the next posterior somite pair. The compartmental version of resegmentation holds that the anterior and posterior domains are lineage-restricted compartments. This conception of resegmentation has a direct correlate in the anterior and posterior compartments that subdivide ectodermal parasegments in D. melanogaster. But does this model describe how the vertebral column is derived from somites in vertebrate embryos?
Compartmental resegmentation in vertebrates has only recently been tested experimentally (Aoyama and Asamoto, 2000). Previous work had shown by several methods that a single avian somite contributes to more than one vertebra (Beresford, 1983
; Aoyama and Asamoto, 1988
; Bagnall et al., 1988
; Lance-Jones, 1988
; Ewan and Everett, 1992
; Huang et al., 2000a
) but the relative contribution of individual anterior or posterior domains had not been assessed. Aoyama and Asamoto (Aoyama and Asamoto, 2000
) showed that in chick-quail chimeras, individual anterior or posterior half-somite transplants had progeny restricted to one half of one adjacent vertebra in a manner consistent with strict compartmental resegmentation (Fig. 1B). Here, we show that cells derived from individual anterior or posterior sclerotome cells in zebrafish do not distribute along the AP axis in a manner strictly dependent upon their anterior or posterior origin. In fact, clonal analysis (Table 2) indicates that individual cells can contribute progeny to both adjacent vertebral locations, a result that is incompatible with the idea of lineage-restricted compartments. Our results in zebrafish are thus more consistent with a leaky resegmentation model (Fig. 1C).
Recent genetic evidence indicates that AP subdivisions within paraxial mesoderm relate to morphogenesis of the vertebral column. Disruption of gene pathways important for establishing future somite AP subdivisions in presomitic mesoderm (reviewed by McGrew and Pourquié, 1998; Stickney et al., 2000
; Christ et al., 2000
; Pourquie, 2001
; Saga and Takeda, 2001
) results in perturbed somite and vertebral column segmentation (Evrard et al., 1998
; Sparrow et al., 1998
; Zhang and Gridley, 1998
; Schubert et al., 2001
) notably without disturbing differentiation of somite into sclerotome and dermamyotome or disrupting the broad pattern of anteroposterior regional identity (e.g. thoracic versus lumbar). Perhaps AP distinctions exist in sclerotome important for vertebral segmentation that are maintained via cell interactions rather than lineage restrictions. In D. melanogaster, the sharp boundary of engrailed expression stripes is maintained by cell interactions, despite movement of individual cells away from the boundary and their coincident loss of engrailed expression (Vincent and OFarrell, 1992
). In addition, distinct domains in the developing leg (Brook and Cohen, 1996
; Jiang and Struhl, 1996
; Johnston and Schubiger, 1996
; Penton and Hoffman, 1996
; Theisen et al., 1996
) and body wall (Diez del Corral et al., 1999
; Calleja et al., 2000
) are maintained by cell interactions and not lineage restriction. Moreover, cell-labeling experiments in chick embryo hindbrain indicate that while the majority of cells respect rhombomere boundaries (Fraser et al., 1990
), some cells cross those boundaries (Birgbauer and Fraser, 1994
) in a manner inconsistent with lineage-restricted compartments, as we have described here for zebrafish somite domains. Thus, while D. melanogaster may use lineage-restricted anterior and posterior compartments as a patterning mechanism in the segmented ectoderm, at a more general level both flies and vertebrates may rely on fundamental cell interactions for refining and maintaining boundary information in the establishment of distinct domains with subsequent patterning roles.
Alignment of somites and vertebral column in zebrafish reveals possible occipital somites and aberrant vertebrae
The question of the involvement of somites in head skeleton development has a long history (Jeffs and Keynes, 1990). Experimental manipulation in chick (Couly et al., 1993
; Huang et al., 2000b
) indicates that a specific number of somites contributes cells to formation of the posterior-most bones of the skull. In mouse, manipulating Hox gene expression via retinoic acid (Kessel and Gruss, 1991
), creation of transgenics (Kessel et al., 1990
) or mutation (Chisaka and Capecchi, 1991
; Lufkin et al., 1991
; Chisaka et al., 1992
) also supports this notion. In zebrafish the alignment of the myotome derived from somite 5 with the second and third vertebrae provides evidence that the first two somites and perhaps part of the third may not contribute to the vertebral column. Whether these anterior somites contribute sclerotome cells to formation of posterior skull bones as documented in chick (Couly et al., 1993
; Huang et al., 2000b
) is currently an unanswered question that can be addressed by future lineage-labeling studies.
One possible relationship between somites and vertebrae is shown in Fig. 6. However, our comparison of total somite number with total vertebral number indicates that ultimate resolution of somites and vertebrae during development remains obscure. We found that long fish often have an extra vertebra that lacks a hemal arch just before the more rigidly patterned tail fin set, while short fish have various patterning defects in the size, shape and presence, absence or duplication of various vertebral elements, primarily in posterior regions. Finding that anomalous vertebrae are more frequently associated with extremes of total vertebral number suggests that the mechanisms underlying patterning are not entirely able to compensate for extreme paucity or excess of material.
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The anterior expression boundaries of all known zebrafish Hox genes fit within the region encompassed by trunk somites [somites 1-17 as defined by Kimmel et al. (Kimmel et al., 1995)]. We show here that this region corresponds to somites forming the cervical and rib-bearing vertebrae (Fig. 6). Zebrafish hoxd12a with the posterior-most described anterior expression boundary at somite 17 (van der Hoeven et al., 1996
) may correspond to the posterior end of the trunk and the transition from rib-bearing to hemal arch bearing vertebrae. The anterior expression boundaries of zebrafish hoxc10a (somite mid-13) (Prince et al., 1998a
) and hoxd12a fall within the region of the zebrafish vertebral column that shows a variable distribution of vertebral types (Fig. 6). It would be interesting to learn if any variability exists in anterior expression boundaries of these Hox genes that might predict a specific vertebral formula outcome in individual fish.
The tail shows patterned vertebrae but no Hox code
The vertebrae of the zebrafish tail (with origins posterior to somite 17) showed regional distinctions. These included differences in neural arch, hemal arch and centrum size, presence of dorso- and ventroposterior extensions from the centrum and unique shapes of the tail set vertebrae. Such region-specific tail vertebrae morphologies may indicate a tail-specific AP regional patterning mechanism distinct from but similar to the Hox code of the trunk. Alternatively, these characteristic morphologies may reveal unique local patterning mechanisms. Using intracellular labeling techniques, Müller and colleagues (Müller et al., 1996), showed that the first 13-14 somites form during gastrulation in zebrafish but that somites more posterior to this arise from the tail bud. This mechanistic transition also coincides with the transition to somites contributing to the axial skeleton variable region. It is intriguing that the region of variability may coincide with a region of transition between distinct mechanisms of defining AP regional specificity, that is, trunk- and tail-specific patterning. While a similar distinction between somites formed by primary and secondary gastrulation noted by Christ and colleagues (Christ et al., 2000
) does not appear to correlate with Hox gene expression domains in chick (Burke et al., 1995
; Christ et al., 2000
), our results lend support to the concept that the tail region develops with mechanisms that are in some ways distinct from those employed for head and trunk patterning (Kanki and Ho, 1997
; Griffin et al., 1998
; Ahn and Gibson, 1999
; Kimelman and Griffin, 2000
). Thus, in zebrafish, AP patterning along the trunk vertebral column may be influenced by Hox gene expression patterns but the regional identity of tail vertebrae may be defined by different mechanisms.
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ACKNOWLEDGMENTS |
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