Integrative and Comparative Biology Advance Access originally published online on July 26, 2007
Integrative and Comparative Biology 2007 47(3):360-372; doi:10.1093/icb/icm064
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A revised fate map for amphioxus and the evolution of axial patterning in chordates
Scripps Institution of Oceanography, University of California San Diego, La Jolla CA 92093, USA
Correspondence: 1E-mail: lzholland{at}ucsd.edu
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Synopsis
IntroductionThe chordates include vertebrates plus two groups of invertebrates (the cephalochordates and tunicates). Previous embryonic fate maps of the cephalochordate amphioxus (Branchiostoma) were influenced by preconceptions that early development in amphioxus and ascidian tunicates should be fundamentally the same and that the early amphioxus embryo, like that of amphibians, should have ventral mesoderm. Although detailed cell lineage tracing in amphioxus has not been done because of limited availability of the embryos and because cleavage is radial and holoblastic with the blastomeres nearly equal in size and not tightly adherent until the mid-blastula stage, a compilation of data from gene expression and function, blastomere isolation and dye labeling allows a more realistic fate map to be drawn. The revised fate map is substantially different from that of ascidians. It shows (1) that the anterior pole of the amphioxus embryo is offset dorsally from the animal pole only by about 20°, (2) that the ectoderm/mesendoderm boundary (the future rim of the blastopore) is at the equator of the blastula, which approximately coincides with the 3rd cleavage plane, and (3) that there is no ventral mesoderm during the gastrula stage. Involution or ingression of cells over the blastopore lip is negligible, and the blastopore, which is posterior, closes centripetally as if by a purse string. During the gastrula stage, the animal pole shifts ventrally, coming to lie about 20° ventral to the anterior tip of the late gastrula/early neurula. Comparisons of the embryos of amphioxus and vertebrates indicate that in spite of large differences in the mechanics of cleavage and gastrulation, anterior/posterior and dorsal/ventral patterning occur by homologous genetic mechanisms. Therefore, the small, nonyolky embryo of amphioxus is probably a reasonable approximation of the basal chordate embryo before the evolution of determinate cleavage in the tunicates and the evolution large amounts of yolk in basal vertebrates.
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Introduction |
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Recent phylogenetic analyses have reversed the positions of amphioxus and tunicates within the chordates and placed amphioxus basal to a tunicate plus vertebrate clade (Blair and Hedges 2005
; Philippe et al. 2005; Bourlat et al. 2006; Delsuc et al. 2006). Because of this rearrangement, the amphioxus embryo (with indeterminant cleavage and regulative development) rather than the tunicate embryo (with determinant cleavage and early decision of cell fates) can be regarded as the likely starting point for the striking variations of early development in vertebrates. Indeed, comparisons of the expression and function of early axial patterning genes between amphioxus and vertebrates indicate that, in spite of major morphological differences in gastrulation between them, they share common genetic mechanisms mediating early embryonic patterning (Yu et al. 2007).
The morphological diversity of chordate embryos preceding the more uniform neurula/tailbud ("phylotypic") stage has resulted in considerable confusion about two related aspects of early development—fate mapping and correlating the egg (animal/vegetal) axis with the later embryonic axes. Within the chordates, embryonic fate maps have been accurately described for tunicates (from precise cell lineage analysis) and vertebrates (although not at the single-cell level); in contrast, existing fate maps for amphioxus are based on only a few studies, and those have not agreed. Relating the animal–vegetal (A/V) axis of early embryos to the anterior–posterior (A/P) and dorsal–ventral (D/V) axes of later life-history stages is even more problematic. In tunicates and vertebrates, extensive tissue movements during early development make it difficult to correlate axes between early and later embryonic stages (Nishida 2005). These uncertainties continue to engender fundamental differences of opinion about axis orientation, even for such extensively studied embryos as amphibians (Kumano and Smith 2002).
The previous literature contains widely divergent opinions about virtually every aspect of amphioxus embryology (Figs. 1 and 2; Table 1). In the present review, we show how new data have settled at least some of these old controversies and discuss the broader subject of the evolution of axial patterning in chordates in light of recent advances in amphioxus embryology. Importantly, our revised amphioxus fate map (Fig. 6) is inconsistent with previous claims that early development is very similar in amphioxus and tunicates. Moreover, our results suggest that the early amphioxus embryo is probably a reasonable approximation of the embryo of the ancestral vertebrate.
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Establishment of the embryonic axes |
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Because of wide variations in the early development of vertebrates, the common denominators of axial patterning in vertebrate embryos have been sought in the invertebrate chordates (amphioxus and tunicates), both of which typically have small, relatively nonyolky eggs. Initial attempts to understand the commonalities of axial patterning in chordates were based on the assumption (Conklin 1905
) that because amphioxus and tunicates are phylogenetically close, their development must be fundamentally the same. However, it is now recognized that tunicate development is highly derived compared to that of other chordates (Holland and Gibson-Brown 2003; Seo et al. 2004). In ascidians, the most widely studied tunicate class, the myoplasm, which gives rise to most of the larval tail musculature, becomes localized soon after fertilization to the future posterior pole of the embryo, which is to one side of the A/V axis just vegetal to the equator. The myoplasm is bisected by the first cleavage, and the A/P axis of the ascidian embryo is typically considered to be almost at right angles to the A/V axis (Fig. 1A). Consequently, early discussion of amphioxus development centered on the orientation of the embryonic axes and whether or not amphioxus segregates the myoplasm as in ascidians. These discussions were shaped by the authors’ conceptions of (1) whether amphioxus has myoplasm marking the posterior pole, (2) whether the amphioxus blastopore is oriented as in ascidians, (3) whether there is disproportionate elongation of one side of the gastrula, (4) whether cells involute over the blastopore lip, and (5) whether the blastopore closes from one side or uniformly from all sides at once, as if by a purse-string (Fig. 1; Table 1).
The author with the most pervasive influence has been Conklin (1905, 1932, 1933). In his 1905 monograph, Conklin postulated that early development in amphioxus is fundamentally the same as that in ascidians, although he had not yet seen an amphioxus embryo and earlier views on amphioxus development were widely divergent concerning such important topics as the orientation of the early embryonic axes, which side was dorsal, how the blastopore closed, whether there was ingression over the rim of the blastopore, and where the mesoderm first appeared (Figs. 1 and 2; Table 1). For ascidians, Conklin (1905) concluded that the blastopore was not oriented precisely dorsally but was inclined slightly posteriorly (Fig. 1K), but agreed with earlier embryologists that the blastopore closed by the anterior (or antero-dorsal) lip growing posteriorly and that the mesoderm arose from cells just posterior and lateral to the blastopore. With this view of ascidian development in mind, Conklin sided with studies of amphioxus development that painted a similar picture, for example, that of Klaatsch (1897) who thought that mesoderm formation in amphioxus resembled that in ascidians: namely that presumptive mesoderm cells on the rim of the amphioxus blastopore were destined to ingress over the blastoporal lip to become the definitive mesoderm. Conversely, Conklin (1905) was highly critical of previous suggestions that there might be substantial differences between amphioxus and ascidian embryology. For example, he firmly disagreed with (Samassa 1898), who correctly recognized that amphioxus and ascidian embryos only resembled one another once the neurula stage (the phylotypic stage) was reached and, therefore, considered development of tunicates to be highly shortened and modified.
When Conklin had the opportunity to observe amphioxus embryos directly, he did not substantially alter his views on the fundamental similarity of amphioxus and ascidian embryos, although he proposed that the amphioxus blastopore was oriented more posteriorly than he had originally thought and that the anterior pole is offset from the animal pole by about 45°, not by about 70° as he had previously proposed (compare Fig. 1L with 1N). Most importantly, he did not change his ideas about the origin of the amphioxus mesoderm. Indeed, he pushed the origin of the mesoderm much earlier in development (Fig. 4A) and proposed that in amphioxus, as in tunicates, the myoplasm segregates to the future posterior pole soon after fertilization (Conklin 1932, 1933).
Conklin (1932, 1933) was not required to consider any information counter to his preconceptions (discussed subsequently) because, by his own admission, he never had an opportunity to observe amphioxus embryos at any stage between fertilization and first cleavage. He thus imagined that in amphioxus, as in ascidians, cytoplasmic movements occurring soon after fertilization set aside the myoplasm at the posterior part of the egg and that the first cleavage furrow, which is along the A/P axis, starts at the posterior pole and bisects the myoplasm. In turn, Conklin claimed that the myoplasm gave rise to small, round, rapidly dividing cells at the ventrolateral edge of the vegetal plate, and that later, at the mid-gastrula stage, pouches of mesoderm protruded into the archenteron (Fig. 2F). Figure 4A shows Conklin's fate map reflecting these views (Conklin, 1933), which influenced conclusions from more recent experimental work (Tung et al. 1958, 1960, 1962) (Fig. 4B) and has been widely copied in textbooks and review articles (McEwen 1957; Browder et al. 1991; Lane and Sheets 2006). However, to further confuse an already complicated and largely incorrect picture, Balinsky (1981) depicted not only a dorsolateral migration of supposed ventral mesoderm but also a posterior shift of notochord cells, which he attributed to Conklin (1932), who was far more vague concerning a dorso-lateral shift of the mesoderm and did not mention any migration of the notochord. To confuse matters even worse, recent reviews by Chea et al. (2005) and Lane and Sheets (2006) have placed the amphioxus A/P axis at 90° to the A/V axis (Fig. 1O and P, which is something that Conklin (1932) himself never proposed.
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A corrected fate map of amphioxus |
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The basis of Conklin's fate map of amphioxus (Fig. 4A) is the assumption that in amphioxus development, as in ascidians, the myoplasm is set aside at the posterior pole of the embryo shortly after fertilization and that, as in amphibians, the amphioxus gastrula has both ventral and dorsal mesoderm. Our re-examination of the experiments of Tung et al. (1958
, 1960), which involved isolation of blastomeres as well as labeling them with Nile blue sulphate, together with more recent studies on cell movements (Zhang et al. 1997) and gene expression in amphioxus embryos (Yu et al. 2007), shows that Conklin's basic assumptions were wrong and allows a more accurate fate map of amphioxus embryos to be constructed.
Tung and colleagues followed the fates of isolated blastomeres and of cells in intact embryos labeled with Nile blue sulphate (Tung et al. 1958, 1960, 1962). Their experiments with isolated blastomeres confirmed the results of Wilson (1893), who showed that unlike ascidian embryos, the amphioxus embryo is highly regulative with each of the first four blastomeres, when separated, capable of developing into an entire embryo. This result is in marked contrast to ascidian embryos in which not even the first two isolated blastomeres can give rise to a complete larva (Conklin 1911) and shows that myoplasm cannot be segregated to one side of the zygote shortly after fertilization. Importantly, contrary to Conklin's reports, Tung and his colleagues found that 
92.5% of the time, the first cleavage plane is very close to the A/V axis. Isolation of the animal and vegetal halves of 8-cell or 16-cell embryos showed that the 3rd cleavage plane divided the embryos into ectoderm and mesendoderm, with the animal halves typically developing into ciliated ectodermal vesicles, sometimes with a tail fin, and the vegetal halves typically developing mesendodermal derivatives. Therefore, the 3rd cleavage plane must approximately mark the future posterior pole of the embryo, and the D/V and right/left (R/L) axes must lie approximately in this plane, at right angles to the first two cleavage planes. Confirmation that the D/V and R/L axes approximately coincide with the 3rd cleavage plane came from separating the four tiers of blastomeres (a1, a2, v1, and v2) at the 32-cell stage. The a1 tier gave rise to an ectodermal vesicle, and the an2 layer to an ectodermal vesicle with sometimes some somites or endoderm. The veg1 layer typically gave rise to a mass of cells with little or no ectoderm containing mesendodermal derivatives. The veg2 layer typically gave rise to an endodermal vesicle, which sometimes gastrulated. Embryos lacking any one of the four layers usually developed into a complete larva although many lacking an a2 or veg1 layer were foreshortened and those lacking a veg2 layer had a reduced gut.
In addition, Tung et al. (1962) stained individual cells and groups of cells in intact embryos at the 2-cell to 32 cell stages. Staining of one of the first four blastomeres resulted in two major phenotypes at the early larval stage—one in which all tissues were stained except for the ventral ectoderm and the posteriormost endoderm and somites, and the second with the stain only in the nonneural (ventral and lateral) ectoderm and anteriormost somites and gut. These patterns indicate that the first cleavage divides the embryo into right and left halves and the second cleavage into dorsal (or dorso-anterior) and posterior (or posterio-ventral) tissues. At the 8-cell stage, staining of one of the four animal blastomeres resulted either in staining of ectoderm and neural tube or of ectoderm only, in agreement with the animal half of the embryo giving rise only to ectoderm. Staining of one vegetal blastomere resulted in two phenotypes. In one, most of the endoderm and the posterior 2/3 of the somites were stained. In the other, staining was in the posterior tip of the nerve cord, most of the notochord, the anterior third of the somites and the anteriormost endoderm. The authors concluded from these experiments that "the distribution of mesodermal material is quite different from that of ascidians." Even so, their fate map (Fig. 4B) differs only slightly from that of Fig. 1 in Conklin (1932) (Fig. 4).
The experiments of Tung et al. (1958, 1960, 1962) are remarkable technically, since amphioxus embryos are very small, about 140 µm in diameter, and they had no micromanipulators. Given the difficulty of the experiments and the lack of equipment for photomicroscopy, the possibility remains that in some labeling experiments, the dye may not have completely labeled some blastomeres, while in others, it may have also labeled a portion of the neighboring blastomeres. Moreover, since Tung et al. (1962) were unaware that ventral mesoderm derives at the neurula stage from outgrowths of the somites (Kozmik et al. 2001) (see subsequently), they may have misinterpreted some mesodermal staining as endodermal for the larval stages. Consequently, the cell fates inferred from the pattern of labeled cells at the early larval stage might not have been entirely accurate. An added difficulty is that since Nile blue sulphate does not survive fixation, all of the observations with dye-labeling were on living embryos, which are motile from the late gastrula stage. To hold the embryos down, in order to draw them with a camera lucida, embryos were trapped under the agar film the culture dishes were coated with and could not be easily manipulated to facilitate observations. For the blastomere isolation experiments, Tung et al. (1958, 1960) were able to fix and section the embryos; however, since the larvae are only about 450 µm long by 50 µm wide, the resolution that can be obtained with paraffin sections is suboptimal. Moreover, these authors pointed out that during cleavage stages, the arrangement of blastomeres is not always regular and that for the blastomere separation experiments, they had to laboriously select embryos with a regular arrangement. Such an irregular arrangement of blastomeres is often seen in Branchiostoma floridae as well, and yet such embryos subsequently develop normally, underscoring the regulative nature of early amphioxus development. Another impediment to cell lineage studies is that once the fertilization envelope is removed, the polar bodies are often lost, making it is difficult to distinguish animal from vegetal blastomeres, since they are nearly the same size (Tung et al. 1958). Even so, we have shown that it is possible to label small groups of cells at the gastrula stage (Zhang et al. 1997). Using modern methods to examine the cell-lineage of amphioxus embryos should be feasible and would help clarify the results of Tung et al. (1958).
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Early amphioxus embryos do not segregate myoplasm and they lack ventral mesoderm |
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Electron microscopy suggests that what Conklin (1932
, 1933) thought was basophilic myoplasm in amphioxus (Fig. A in the 1933 paper and Figs. 118 and 119 in the 1932 paper) is probably the ribosome-rich pole plasm that forms shortly after fertilization at the vegetal pole of the egg. As we have shown, soon after fertilization, the sperm is rapidly translocated to the vegetal pole of the egg where it decondenses (Holland and Holland 1992). Simultaneously, endoplasmic reticulum in the vegetal cortical cytoplasm detaches from the cortex and forms large, ribosome-rich whorls (the putative pole plasm) that remain at the vegetal pole and are segregated into a single blastomere at each cleavage until at least the late blastula (our unpublished data). This region of the cytoplasm is relatively yolk-free, as is the track that the sperm nucleus makes as it subsequently migrates to the animal hemisphere accompanied by a cloud of mitochondria. It may be that these areas of relatively yolk-free cytoplasm correspond to the presumed myoplasm of Conklin (1932, 1933), although they are much closer to the vegetal pole than he depicted. In light, the work of Coffman et al. (2004) indicating that in sea urchin embryos, the oral (ventral) side of the embryo is established on the side of the embryo with the highest concentration of mitochondria, it seems likely that in amphioxus, the concentration of mitochondria near the fusing pronuclei, which meet near the periphery of the zygote about 25° from the animal pole (Holland and Holland 1992), may influence the position of the D/V axis.
Semi-thin histological sections together with gene expression show that early amphioxus embryos completely lack ventral mesoderm, which forms during the neurula stage as ventral outgrowths from the somites. Most of the expression domains of genes that turn on by the early gastrula stage do not change their position throughout gastrulation (Yu et al. 2007). For example, the chordin domain marks the dorsal axial mesoderm (notochord) and overlying floor plate of the neuroectoderm throughout gastrulation. By the early- to mid-gastrula, a number of genes turn on in the paraxial mesoderm. These include Wnt8, involved in specification of somitic mesoderm and Pax3/7, upstream in the myogenic cascade. By the mid-gastrula, genes involved in segmentation of the somites and in myogenesis are also expressed in the paraxial mesoderm, and by the late gastrula, the somites start segmenting in an anterior to posterior pattern. During the gastrula stage, the ventral mesodermal marker Vent is expressed throughout the mesendoderm (Yu et al. 2007). However, by the late gastrula, it is substantially down-regulated ventrally but remains expressed dorsolaterally in the paraxial mesoderm and the edges of the neural plate. Then, at the mid-neurula stage, Vent-expressing ventral mesoderm forms as outgrowths from the somites, which extend ventrally around the gut and fuse in the ventral midline (Kozmik et al. 2001). Moreover, the scanning electron microscopic (SEM) study of Hirakow and Kajita (1991) and studies with semi-thin sections (Langeland et al. 2006) demonstrate that the pouch-like bulging of the ventral mesoderm at the early to mid-gastrula stage described by Conklin (1932) does not exist and was probably an artifact as was the pear-shape of the blastopore he described. Taken together, results from gene expression, dye labeling, and blastomeres separation show that previous fate maps of amphioxus (Fig. 4) are incorrect in depicting a zone of presumptive mesoderm in ventral blastomeres.
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DiI labeling and gene expression shows the near absence of involution during gastrulation amphioxus |
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In tunicates and vertebrates, cell rearrangements and movements during gastrulation make it very difficult to extrapolate the embryonic axes of the neurula back to early embryos (Nishida 2005
). Consequently, even today, the orientation of the axes in Xenopus is in dispute (Gerhart 2002; Kumano and Smith 2002; Lane and Sheets 2006). Moreover, Nishida (2005) noted that although the A/P axis of the tunicate embryo is by convention placed at right angles to the A/V axis, it does not correspond exactly to the A/P axis of the larva because of massive cellular movements. In contrast, in amphioxus, DiI labeling shows that cell and tissue movements during gastrulation are so minimal that territories marked by expression of particular marker genes at the early gastrula can be extrapolated back to the blastula stage (Zhang et al. 1997). This is in marked contrast to the situation in vertebrates, where extensive cell movements virtually preclude the use of gene expression domains in generating fate maps (Fraser and Harland 2000). The critical experiment for amphioxus involved placing spots of DiI at intervals around the lip of the blastopore at the early gastrula stage (Zhang et al. 1997). These spots stretch slightly during blastopore closure, but remain at the rim of the blastopore as it closes, demonstrating that gastrulation is by simple invagination with only very slight involution of cells over the blastopore lip. Because cell movements during gastrulation are so minimal, domains of gene expression at the early gastrula stage can be combined with data from the experiments of Tung et al. (1960, 1962) to infer the fates of territories in the late blastula. For example, at the late blastula stage, the blastopore marker brachyury turns on in a stripe around the equator (Zhang et al. 1997). In addition, Wnt8 turns on at the late blastula throughout the presumptive mesendoderm with the highest expression around the equator (Zhang et al. 1997; Yasui et al. 2001; Yu et al. 2007). Thus, given the near absence of involution plus continued expression of brachyury around the blastopore throughout gastrulation, it is likely that the brachyury-expressing cells at the late blastula are the same cells expressing brachyury a few minutes later at the early gastrula. Together, these data strongly indicate that the equator of the amphioxus blastula is the future rim of the blastopore and that the blastopore is posterior and closes centripetally as though by a purse string, as claimed by some early embryologists (Table 1). The results are consistent with the experiments of Tung et al. (1958) who showed that the mesendoderm/ectoderm boundary approximately coincides with the 3rd cleavage plane. |
Gene markers help define the embryonic axes |
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For construction of a fate map, it is important to know the orientation of the D/V axis. As noted previously, this is difficult to do even in material fixed before the neural plate flattens. In the 8-cell stage amphioxus embryo, the four animal blastomeres, destined to be ectoderm are slightly smaller than the four vegetal blastomeres, destined to be largely mesendoderm. However, there are no obvious morphological differences between the dorsal and ventral or right and left sides of the embryo before the early neurula stage. For example, relatively loosely associated or more rounded cells have sometimes been demonstrated in fixed amphioxus gastrulae in one sector of the blastopore lip: some authors considered these cells to be dorsal (Fig. 2A, D, and E), while others thought them ventral (Fig. 2B, C, and F). Conklin (1932
) thought they were ventral–posterior and constituted the mesoderm. In contrast, Hirakow and Kajita (1991), in their SEM study, could not decide on which side of the blastopore rim lay the more rounded cells. To answer the question of whether the more rounded cells are dorsal or ventral, we labeled the dorsal ectoderm (presumptive neural plate) by in situ hybridization for Sox1/2/3 and did SEM after photographing the critical point-dried embryos on the SEM stub (Fig. 5). We found that the cells at the dorsal blastopore lip are tightly adherent to one another while those at the ventral lip of the gastrula are rounder and more loosely packed (Fig. 5). According to our fate map these cells are presumptive endoderm. Finally, to identify the anterior pole of the embryo, we used DiI labeling and the expression of marker genes. Expression of the anterior ectodermal marker FoxQ2 shows that at the early to mid-gastrula stage, the anterior pole is offset dorsally about 20° from the animal pole (Fig. 3). This domain continues to mark the anterior tip of the late gastrula/early neurula as the blastopore closes (Yu et al. 2003) (Fig. 6). Similarly, the center of the domain of the anterior endodermal marker Hex underlies the animal pole throughout gastrulation (Yu et al. 2007) (Fig. 6). DiI labeling confirms that the anterior pole is about 20° dorsal to the animal pole; thus, a spot of DiI placed on the animal pole, comes to lie 20° ventral to the anterior tip of the neurula (Zhang and Holland, unpublished data).
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Other markers help refine the fate map. As noted earlier, since DiI labeling shows that there is virtually no involution or cell migration during gastrulation in amphioxus, gene expression patterns that mark large territories and do not change substantially during gastrulation can help in constructing a fate map at the late blastula. For example, throughout the gastrula and early neurula stages, Sox1/2/3 expression marks neuroectoderm (Holland et al. 2000
), while Distalless marks the remainder of the ectoderm (Holland et al. 1996) (Fig. 6). The anterior limit of the neuroectoderm as marked by the boundary of Sox1/2/3 and Dll expression is about 35–40° dorsal to the animal pole. The prospective mesoderm is marked by ADMP and goosecoid expression at the early gastrula stage (Yu et al. 2007) and occupies most of the dorsal side of the involuting mesendoderm. As noted above, there is no ventral mesoderm until the mid-neurula stage, when it forms as Vent-expressing, ventral outgrowths from the somites (Kozmik et al. 2001). Expression of Chordin marks the presumptive notochord (axial mesoderm) at the early gastrula stage, permitting the distinction of axial from paraxial mesoderm (Yu et al. 2007). Chordin also marks the center of the neural plate (presumptive floor plate). At the mid-gastrula, Wnt8 turns on throughout all but the anteriormost 10–15% of the paraxial mesoderm, which extends to the anterior tip of the embryo (Schubert et al. 2000a). Taken together with blastomere isolation and labeling with DiI and Nile blue sulphate, the expression of tissue markers allows the construction of an approximate fate map for the late blastula and gastrula stages (Fig. 3). This fate map shows that at the late blastula stage, the anterior pole is offset about 20° from the animal pole and that the future posterior pole is located around the equator. Contrary to previous fate maps, there is no ventral mesodermal territory per se, since ventral mesoderm derives from the somites. By the neurula stage, the anterior pole is at the anterior tip of the embryo, and the animal pole lies about 20° towards the ventral side.
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Evolutionary conservation of the genetic mechanisms for A/P and D/V patterning of amphioxus and vertebrate embryos |
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It has been difficult to determine the common denominators of early embryonic patterning in vertebrates, because of the wide variation in gastrulation movements among the classes. For example, in the zebrafish, the first sign of gastrulation is epiboly of the blastoderm over the yolk, while the frog embryo gastrulates by involution, which is much more pronounced at the dorsal blastopore lip. Chick and mouse embryos gastrulate by ingression of cells through the primitive streak. Even so, in all vertebrates, the gastrula organizer (Spemann's organizer in amphibians, the node in the chick and mammals) functions in axial patterning and head induction. Both experimental data and gene expression indicate that amphioxus embryos also have a gastrula organizer and that the molecular mechanisms for D/V and A/P patterning are fundamentally the same as in vertebrates (Tung et al. 1962
; Yu et al. 2007). For example, in amphioxus, homologs of the vertebrate organizer genes Nodal, Lefty, Lim-l, goosecoid, and the BMP-modulators ADMP, chordin, and Twisted gastrulation (Tsg) are all expressed in the dorsal mesendoderm during the gastrula stage (Neidert et al. 2000; Langeland et al. 2006; Yu et al. 2007) (Fig. 6). Both BMP5-8 and BMP2/4 are initially broadly expressed in the mesendoderm, but by the late gastrula stage, they both become downregulated in the dorsal axial mesoderm. As in vertebrates, exogenous BMP4 protein results in ventralized embryos lacking a notochord, neural plate and a head. In addition, as noted earlier, Hex is expressed in the anterior endoderm in amphioxus as in Xenopus. It is expressed in functionally comparable extraembryonic tissues in the chick (the hypoblast) and mouse (the anterior visceral endoderm), which are involved in neural induction.
Gene expression also indicates that in both amphioxus and vertebrates, Wnt signaling is high posteriorly and low anteriorly. All of the eight amphioxus Wnt genes described to date (Wnts 1, 3, 4, 5, 6, 7b, and 11) are expressed around the blastopore during the gastrula stage. Nuclear ß-catenin is localized to cells around the blastopore, and Wnt antagonists are expressed either anteriorly (Dkk3, sFRP2-like) or both anteriorly and posteriorly (Dkk1/2/4) (Holland and Holland 2000; Schubert et al. 2000a, 2000b; 2001; Holland et al. 2005; Yu et al. 2007).
Together, these results indicate the genetic mechanisms for patterning the D/V and A/P axes of amphioxus embryos are conserved with vertebrates. Thus, at least three important features were present before the split of cephalochordates and vertebrates: (1) a dorsal organizer using BMP antagonists and Nodal and its antagonist Lefty to mediate D/V patterning, (2) an anterior endodermal signaling center expressing Hex, and (3) a posterior Wnt-signaling center specifying posterior identity. Because phylogenetic analyses with large gene sets place amphioxus basally within the chordates and group tunicates with vertebrates, it is likely that the early embryonic patterning mechanisms in amphioxus, with separate signaling centers producing BMP- and Wnt-signaling gradients along the D/V and A/P axes, respectively, reflects the ancestral state of axial patterning for chordates. In contrast, tunicates, which have independently evolved early segregation of the myoplasm and early determination of cell fates, have lost the gastrular organizer. BMP and chordin, although expressed during cleavage stages, are not involved in dorso-ventral patterning (Darras and Nishida 2001), while Wnts are not expressed posteriorly at the gastrula stage.
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Conclusion |
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In sum, the hypothesis of Conklin and others that patterning of amphioxus and ascidian embryos is fundamentally the same was universally accepted during the first half of the 20th century. Then about 50 years ago, the experiments of Tung and his colleagues cast serious doubt on this notion. More recent work on the genetic basis of early embryonic patterning in amphioxus and tunicates has demonstrated that axial patterning in amphioxus, with separate signaling centers generating BMP and Wnt signaling gradients along the D/V and A/P axes, reflects the ancestral state of axial patterning in chordates. Although the amphioxus fate map in Fig. 3 may in the future be refined in light of additional studies, current data are sufficient to demonstrate its general overall accuracy. In amphioxus, the anterior pole is only offset from the animal pole by about 20°. The A/P axis is not at right angles to the animal/vegetal axis as depicted in recent reviews. There is no ventral mesoderm at the gastrula stage in amphioxus; it does not form until the mid-neurula. It is perhaps not surprising that major inaccuracies in the amphioxus fate map have persisted in the secondary literature even in light of increasing amounts of primary data that together draw a more accurate picture. Although a fate map done with modern labeling techniques is sorely needed, there is at present sufficient evidence to show that axial patterning in amphioxus and tunicates is fundamentally quite different from the one proposed by Conklin (1905
, 1932, 1933).
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Acknowledgments |
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We thank Frietson Galis and David Carrier for their invitation to participate in the SICB symposium and Evelyn York for her assistance with SEM. This work was supported by grants from the National Science Foundation to LZH and NDH (IOB-0416292) and from the March of Dimes Foundation (to LZH 1-FY05-108).
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Footnotes |
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From the symposium "Linking Genes and Morphology in Vertebrates" presented at the annual meeting of the Society for Integrative and Comparative Biology, January 3–7, 2007, at Phoenix, Arizona.
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