The chick embryo: a leading model in somitogenesis studies

Mechanisms of Development 121 (2004) 1069–1079 www.elsevier.com/locate/modo

Review

The chick embryo: a leading model in somitogenesis studies Olivier Pourquie´* Stowers Institute for Medical Research, 1000E 50th Street, Kansas City, MO 64110, USA Received 26 April 2004; received in revised form 3 May 2004; accepted 4 May 2004

Abstract The vertebrate body is built on a metameric organization which consists of a repetition of functionally equivalent units, each comprising a vertebra, its associated muscles, peripheral nerves and blood vessels. This periodic pattern is established during embryogenesis by the somitogenesis process. Somites are generated in a rhythmic fashion from the presomitic mesoderm and they subsequently differentiate to give rise to the vertebrae and skeletal muscles of the body. Somitogenesis has been very actively studied in the chick embryo since the 19th century and many of the landmark experiments that led to our current understanding of the vertebrate segmentation process have been performed in this organism. Somite formation involves an oscillator, the segmentation clock whose periodic signal is converted into the periodic array of somite boundaries by a spacing mechanism relying on a traveling threshold of FGF signaling regressing in concert with body axis extension. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Somite; Presomitic mesoderm; FGF; Segmentation; Clock; Notch; Retinoic acid

1. Introduction The somitic lineage forms with the head mesoderm the so-called paraxial mesoderm. The somitic series starts anteriorly immediately caudal to the otic vesicle and runs posteriorly on both sides of the neural tube and notochord to the caudal tip of the embryo. In the chick embryo, somites appear as paired epithelial blocks of cells forming sequentially from the rostral extremity of the mesenchymal presomitic mesoderm (PSM). The rhythm of somite production is characteristic of the species at a given temperature. In the chick embryo, a pair of somite forms every 90 min at 37 8C and a total of 52 somites pairs are formed during the somitogenesis process which lasts from day 1 to day 5 of development. Somitogenesis can be subdivided into three major phases. First a growth phase during which new paraxial mesoderm cells are produced by a growth zone (epiblast and then primitive streak and later on tail bud) and become organized as two rods of mesenchymal tissue, forming the PSM. As we will see, cells entering the caudal PSM are already endowed with some segmental information. Second a patterning phase occurring in the PSM, during which this segmental information is translated into a segmental pre-pattern in * Tel.: þ1-816-926-4442; fax: þ 1-816-926-2095. E-mail address: [email protected] (O. Pourquie´). 0925-4773/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mod.2004.05.002

the rostral PSM. Third, the rostro-caudal (RC) identity of the somites is established in the rostral-most PSM soon followed by the formation of the somitic boundary. During their maturation in the PSM, all cells of the paraxial mesoderm go successively through these phases, which are tightly regulated at the spatio-temporal level. In this review, I will discuss essentially the origin of the somitic territory and the mechanisms involved in the metamery generation in the presomitic mesoderm. Our understanding of the later stages of paraxial mesoderm differentiation such as boundary formation (Kulesa and Fraser, 2002; Sato et al., 2002) and somite differentiation (Hirsinger et al., 2000; Brent and Tabin, 2002) as well as of the regional patterning of somitic derivatives along the antero-posterior (AP) axis also largely rely on work performed in the chick embryo (Kieny et al., 1972; Nowicki and Burke, 2000). These topics have been reviewed in detail elsewhere and will not be discussed here (Hirsinger et al., 2000; Saga and Takeda, 2001; Brent and Tabin, 2002).

2. Mapping the origin of somites in the chick: stem cells and pre-pattern Following the pioneering work of Vogt in the 1920s, several workers attempted to produce fate maps of

1070

O. Pourquie´ / Mechanisms of Development 121 (2004) 1069–1079

the gastrulating chick embryo (Vogt, 1929). Using techniques similar to that developed in amphibians such as colored marks placed on the epiblast before gastrulation, the territory giving rise to somites was mapped to the posterior epiblast and shown to ingress at the level of the rostral primitive streak (Wetzel, 1929; Pasteels, 1937; Spratt, 1955). These early studies led to two different views of the function of the primitive streak in the generation of the paraxial mesoderm. On one hand, authors like Wetzel or Spratt proposed that the primitive streak acts as a blastema generating the different germ layers of the embryo during its regression (Wetzel, 1929; Spratt and Haas, 1965). In this view, the streak was composed of indifferent precursors, which acquire their fate as they become positioned in their definitive location by the streak regression movements (Fig. 1A). On the other hand authors like Pasteels or Waddington viewed the primitive streak like the amphibian blastopore, i.e. a transit area for the cells of the superficial epiblast destined to form the future mesoderm (Fig. 1B) (Pasteels, 1937; Waddington, 1952). These two conceptions imply different mechanisms for the specification of future somites along the AP axis. In the first view, it is the timing of the production of the cells by the primitive streak, which define their position in the somitic series (Fig. 1A). In contrast, the second view was associated with the notion of well-defined presumptive somites arrayed in their definitive sequence on the surface of the epiblast, as originally proposed in amphibians (Fig. 1B) (Vogt, 1929). During primitive streak regression, these territories would progressively ingress in an orderly fashion and thus yield the definitive somitic series (Fig. 1B). Such a preformationist conception of somitogenesis implies the existence of a segmental prepattern in the epiblast and does not provide any explanation for the generation of metamery. Both conceptions shared the view that the AP distribution of the territories in the streak reflects their future medio-lateral distribution in the embryo, in that they both position the somitic territory rostrally and the lateral plate caudally. Recent reinvestigation of these questions using more modern fate mapping techniques such as DiI labeling or quail-chick chimeras have not yet completely solved this controversy. It is now clear that the primitive streak is not only a transit place for cells on their way to the presomitic mesoderm, but also contains resident precursor cells which generate the paraxial mesoderm during its regression (Selleck and Stern, 1991; Schoenwolf et al., 1992; Hatada and Stern, 1994; Psychoyos and Stern, 1996) (Fig. 1C). DiI labeling experiments demonstrated the existence of such resident stem cells in the streak, a finding that was further supported by different fate mapping strategies in the mouse (Stern et al., 1992; Nicolas et al., 1996; Wilson and Beddington, 1996). These observations led to the idea that the rostral streak contains a pool of stem cells responsible for the generation of the whole paraxial mesoderm. These stem cells would become resident in the primitive streak after ingression of the epiblastic presumptive territory of

the paraxial mesoderm. This stem cell zone was further proposed to show some organization along the AP axis with the future medial cells of the somites lying more anteriorly in the streak and Hensen’s node, while the cells fated to contribute to the lateral somite would be located more caudally (Selleck and Stern, 1991; Eloy-Trinquet and Nicolas, 2002a). This distribution is consistent with the overall organization of the primitive streak mentioned above whereby its AP axis reflects the future medio-lateral organization of the tissues. The growth pattern in this stem cell model is therefore similar to that proposed for the limb bud, in which cells exiting early the precursor area called the progress zone contribute to proximal elements while cells exiting the area late will form distal structures (Summerbell et al., 1973). For the paraxial mesoderm, the position of the cells along the AP axis in the somitic series would be strictly defined by the timing of their exit from the stem cell zone. Patterning mechanisms such as those proposed for the limb bud based on the progress zone model would therefore perfectly apply to the paraxial mesoderm. These include the possibility of the cells of the stem cell zone to somehow count cell divisions or oscillations of the segmentation clock (see below) to convert these into positional information such as Hox gene expression (Duboule, 1994; Dale and Pourquie, 2000; Dubrulle et al., 2001; Eloy-Trinquet and Nicolas, 2002a). This stem cell model does not, however, easily explain a series of experimental findings. For instance, when different AP levels of the chick embryo primitive streak are labeled either by colored marks or by orthotopic quail cell grafts, cells from more anterior levels usually contribute to a territory extending more anteriorly than cells located more posteriorly (Pasteels, 1937; Schoenwolf et al., 1992). This, therefore, suggests that there is also a form of AP prepattern in the streak as originally proposed by several authors.

3. The somite center conundrum The beginning of the experimental approach of somitogenesis in the chick embryo can be traced back to the work of Spratt in the early 1950s. He first performed a series of fate mapping experiments using charcoal, carmine and colored marks to precisely define the position of somitic precursors at primitive streak stages (Spratt, 1955). Spratt observed that a significant portion of the anterior primitive streak contains somite precursors (Fig. 2, in blue). Then he realized a series of blastoderm transection experiments in primitive streak stage embryos, and observed the development of somites from the embryonic parts cultured independently (Fig. 2). Expectedly, the caudal portion of embryos transected at the level of the rostral streak and thus including the prospective somite territory was able to give rise to a tail-like extension containing somites (Fig. 2A). However, there was a level of the streak below which

O. Pourquie´ / Mechanisms of Development 121 (2004) 1069–1079

1071

Fig. 1. Origin of the somitic territory. The paraxial mesoderm is shown in light blue and its presumptive territory in the epiblast and in the primitive streak is in darker blue. The lateral plate and its presumptive territory are shown in grey. (A) Dynamics of the somitic presumptive territory during gastrulation in the chick embryo according to Wetzel (1929). Initially located as a large posterior stripe in the epiblast (left and middle panels, the presumptive territory of the somites stretches along the AP axis and becomes localized in the primitive streak (right panel) where it acts as a blastema and generates the paraxial mesoderm during streak regression (arrows). (B) Dynamics of the somitic presumptive territory according to Pasteels (1937). Somites are already arrayed in their definitive order on the surface of the epiblast (left and middle panels). Invagination of this material occurs at the primitive streak resulting in their positioning according to their final distribution early on in caudal-most part embryo (right panel). Primitive streak regression results in the progressive unfolding of this prespecified somitic series (right panel). In the middle and right panels, the left side corresponds to the superficial epiblastic layer, and the right side to the invaginated material. (C) Stem cell model for somitogenesis based on DiI labeling analysis and mouse restrospective lineage studies (Stern et al., 1992; Nicolas et al., 1996). The presumptive territory of the paraxial mesoderm initially located in the caudal epiblast ingresses at the level of the rostral primitive streak where it constitutes a population of resident stem cells, which generate the presomitic mesoderm during primitive streak regression (arrows). This territory is organized along the AP axis such that cells located anteriorly produce cells of the medial PSM while those located more posteriorly produce lateral PSM cells. Medial and lateral paraxial mesoderm are separated by a hatched line in the right panel.

1072

O. Pourquie´ / Mechanisms of Development 121 (2004) 1069–1079

Fig. 2. (A, B) Spratt’s and Bellairs’ (C) transection experiments. The presumptive territory of the somites in the primitive streak as mapped by Spratt (1955) is indicated in blue. Stage 4 HH (Hamburger and Hamilton, 1951) embryos (left panels) were sectioned at the indicated level (hatched line) and the anterior (grey) and posterior parts were allowed to develop 24 h in culture to observe somite formation (right panels). In (A), the caudal fragment includes the entire presumptive territory of the somites in the primitive streak and generates somites in culture. In (B), the caudal fragment contains the caudal presumptive somitic territory of the streak but does not generate somites. This led Spratt (1955) to propose the existence of somite centers shown in (C) (dotted circles) located adjacent to the rostral primitive streak. These centers are transit areas where paraxial mesoderm precursors entering the caudal PSM acquire their segmental information. (D) shows Bellairs’ (1963) experiment in which the same caudal fragment as in B plus the rostral primitive streak can give rise to somites, arguing against the existence of the somite centers and indicating that the rostral primitive streak is required for somite formation.

the caudal part became unable to generate somites (Fig. 2B). Surprisingly, this level did not correspond to the caudal limit of the somite territory in the streak defined by his mapping experiments. It, therefore, suggested that somite formation from more caudal portions of the streak required the presence of the rostral territory. Based on these findings he proposed the existence of somite centers, which are bilateral areas adjacent to the Hensen’s node required for

the formation of somites (Fig. 2C). In his view, these centers were following the regression movement of the node and were continuously traversed by a constantly changing population of somite precursor cells on which they imprint the segmental pattern. This work was subsequently challenged by experiments of Bellairs who performed similar transections except that she left in place the rostral primitive streak attached to

O. Pourquie´ / Mechanisms of Development 121 (2004) 1069–1079

the caudal fragment that was unable to generate somites in Spratt’s experiments (Bellairs, 1963) (Fig. 2D). In these conditions, she observed that the caudal portion of the blastoderm was able to generate somites, indicating (i) that no somite centers adjacent to the node are required for somite production and (ii) that the rostral primitive streak is required for somite generation. Hensen’s node ablations performed by several workers independently argued against a major role of the node in somite formation from the caudal territory, suggesting that in Bellairs experiments it was the rostral streak and not the Hensen’s node which was able to rescue the ability to form somites of the caudal most portion of the streak somitic territory (Wolff, 1936; Joubin and Stern, 1999) and references therein). To account for these results, Bellairs proposed a model invoking a dual origin of somite cells (Bellairs, 1985). She postulated the existence of pre-somite clusters corresponding to a specific cell population set-aside early in development and located adjacent to the Hensen’s node. These cells, which would be endowed with some segmental information, would be released as a trail during Hensen’s node regression and associate with naı¨ve cells deriving from the primitive streak. Thus, these presomite clusters would provide a frame for segmentation to the naive primitive streak derived cells. This model is consistent with the observation that orthotopic grafts of the rostral-most portion, but not of more caudal segments of the anterior primitive streak can form independent segmental series in the host (Nicolet, 1971; Schoenwolf et al., 1992; Selleck and Stern, 1992). Whereas both types of grafts contribute to somite formation, only the cells of the rostral-most streak territory appear committed to their segmental pattern. Also, surgical ablation of the medial PSM, which derives from the rostral-most primitive streak, prevents segmentation of the lateral PSM while ablation of the lateral PSM has no effect on medial PSM segmentation (Freitas et al., 2001). Thus only the derivatives of the rostral streak, which form the medial PSM, are able to segment autonomously. The lateral PSM derivatives which derive from more caudal areas of the streak require a signal from the medial cells to segment. Altogether, these results suggest that somites are formed from two kinds of precursors which differ in their segmentation ability. A set of precursors corresponding to Bellairs’ somite clusters, which are endowed with some segmental information, derive from the rostral-most primitive streak and will form the medial PSM and somites. These cells will associate with naı¨ve precursors derived from more caudal portions of the primitive streak which will form the lateral PSM and somites. This early segregation is consistent with the clonal coherence of medial and lateral myotomal lineages reported in the mouse (Eloy-Trinquet and Nicolas, 2002a,b). Interestingly, this early subdivision is reflected in the later lineages derived from these primitive streak populations. Medial somitic cells give rise to the vast majority of the body segmented structures, including the vertebral column and its associated muscles, whereas cells

1073

derived from the lateral somite will yield the majority of non segmented somite-derived structures including limb muscles and dermis (Selleck and Stern, 1991; Ordahl and Le Douarin, 1992; Olivera-Martinez et al., 2000). The idea that a segmental prepattern is already set in the PSM is consistent with important experiments performed in the chick embryo in the 1970s by Menkes and Sandor and by Bodo Christ (Christ et al., 1974; Menkes and Sandor, 1977). These authors realized a series of embryonic manipulations in culture and in ovo, in which they inverted the orientation of the segmental plate along the AP axis (Fig. 3). In all these experiments, the inverted PSM fragment kept its endogenous segmentation schedule and formed somites in a posterior to anterior direction. Further experiments in which discontinuities were introduced in the segmental plate by killing cells by UV irradiation, or in which endoderm and ectoderm were removed from the adjacent segmental plate did not succeed in altering the endogenous segmentation schedule of the PSM (Menkes et al., 1968; Sandor and Amels, 1971; Sandor and Fazakas-Todea, 1980). Together, these findings argued in favor of a surprisingly high degree of autonomy of the PSM with respect to the segmentation program. The experiments described above contributed to propagate the idea that the PSM is already segmentally prepatterned and that segmentation is established earlier on in the epiblast or in the primitive streak. Evidence for such a pre-pattern was first reported in the chick embryo by Meier who described condensations prefigurating the somites in the PSM (Meier, 1979). These structures, which he termed somitomeres, could only be observed by stereo-scanning electron microscopy (Jacobson, 1988). Together with Packard, they showed that when isolated, the avian segmental plate was able to generate between 10 and 12 somites which corresponded to the number of somitomeres they could detect in the PSM (Packard and Jacobson, 1976; Packard and Meier, 1983). However, since this work, no further evidence in support of the existence of the somitomeres was provided. In particular, no molecular evidence for the existence of such a segmental prepattern all along the PSM was ever obtained. As we discuss below, the first genes expressed in a striped fashion are found in the rostral PSM and the high degree of autonomy of the PSM can be explained without inferring the existence of a somite prepattern in the PSM.

4. The clock and wavefront In a series of experiments aimed at testing the mechanisms involved in the control of somite number in the amphibian embryo, Cooke showed in the 1970s that reducing the number of cells in the blastula leads to the development of smaller embryos with a normal number of somites (Cooke, 1975). To explain these results, together with Zeeman they proposed a theoretical model for

1074

O. Pourquie´ / Mechanisms of Development 121 (2004) 1069–1079

Fig. 3. Menkes and Sandor (1977) experiment in which they inverted along the AP axis a segment of the posterior embryo including part of the PSM. In the inverted fragment, segmentation proceeds from posterior to anterior according to its endogenous schedule.

somitogenesis called the ‘clock and wavefront’ (Cooke and Zeeman, 1976). This model posits that cells of the PSM possess an intrinsic oscillator, the clock, which controls their somite forming ability. Neighboring cells of the PSM oscillate in phase between a permissive and a nonpermissive state for somite formation. This model comprises a second component called the wavefront, which corresponds to a maturation front slowly moving posteriorly in concert with the AP differentiation gradient of the embryo. PSM cells form a somite when they are hit by the wavefront when in the permissive phase of the clock. First indirect evidence for this model came from heatshock experiments performed in the frog embryo (Elsdale et al., 1976). This treatment resulted in localized segmentation defects seen in cells that were in the PSM at the time of heat-shock. They provided evidence for some coordination among PSM cells and were taken as an argument for the existence of the wavefront. The clock and wavefront model was subsequently tested in the avian embryo (Bellairs and Veini, 1984). Early observations of Lutz had shown that several embryos could be generated out of a single avian blastoderm when transected very early on (Lutz, 1948). Veini and Bellairs used this experimental strategy to produce quail embryos with a reduced number of cells in which they observed no alterations of the somite count (Bellairs and Veini, 1984). Also, heat-shock experiments on chick embryos by Stern and Colleagues led to discrete segmentation defects like in frog (Primmett et al., 1988, 1989). Altogether, these experiments did not provide strong support in favor of the model, but they could be interpreted within its frame. The clock and wavefront was the first of a series of somitogenesis models postulating the existence of an oscillator acting in PSM cells (Keynes and Stern, 1988; Dale and Pourquie, 2000; Stern and Vasiliauskas, 2000) for reviews of the different somitogenesis models).

Evidence for an oscillator associated to the segmentation process was first recognized in the chick embryo as rhythmic waves of expression in PSM cells of the mRNA coding for the basic helix loop helix (b-HLH) transcription factor c-hairy1, a vertebrate homologue of the protein encoded by the fly pair rule gene hairy (Palmeirim et al., 1997). This oscillator, which was termed segmentation clock, controls the periodic transcription of a group of genes called cyclic genes, which are related to the Notch and to the Wnt signaling pathways (reviewed in Pourquie, 2003a). This rhythmic expression begins during gastrulation in the paraxial mesoderm precursors and their descendants and is maintained throughout somitogenesis (Jouve et al., 2002). This early onset of the segmentation clock prior to entering the presomitic mesoderm is consistent with the notion described earlier that some segmental information is already acquired in cells of the rostral primitive streak. This further implies that the number of oscillations experienced by PSM cells is directly correlated to their position along the AP axis (Jouve et al., 2002). Little is known about the mechanism underlying the oscillations. Clock elements localized in the promoter of the cyclic genes can recapitulate their periodic expression in transgenic mice suggesting that oscillations are regulated at the transcriptional level (Cole et al., 2002; Morales et al., 2002). The clockwork of the oscillator appears to involve a series of Notch-based negative feedback loops involving lunatic fringe and hes genes acting downstream of a Wnt-based negative feedback loop involving axin2 (Aulehla et al., 2003; Bessho et al., 2003; Dale et al., 2003). The role of the segmentation clock in the somitogenesis process is still unclear. One of the output of the oscillator is the periodic Notch activation in the PSM, which could act as a signal periodically initiating the process of somite boundary specification (Jiang et al., 1998; Pourquie, 1999).

O. Pourquie´ / Mechanisms of Development 121 (2004) 1069–1079

Whereas the segmentation clock is believed to set the rhythm of somitogenesis it does not specify the positioning of the boundaries. In the clock and wavefront model, the interval between two consecutive somites corresponds to the distance roamed by the wavefront during one period of the clock. Recent experiments in chick and fish identified the wavefront as a traveling threshold of FGF signaling generated by a gradient of fgf8 mRNA in the PSM (Dubrulle et al., 2001; Sawada et al., 2001). By performing microsurgical inversions of small PSM fragments, the PSM was subdivided into two domains: a caudal domain in which the inversion results in a normal segmentation pattern in the operated embryos, indicating that cells retain some plasticity, and a rostral domain in which the inverted PSM fragment shows an aberrant segmentation pattern, indicating that the segmental program is already activated (Dubrulle et al., 2001). The boundary between the two regions was located at the level of somite-IV/-V in the PSM and called the determination front. While this boundary had not been previously recognized in the PSM, it corresponds to the level at which PSM cells change from a loose mesenchymal organization to a more compact one, with cell nuclei becoming aligned beneath the ectoderm and above the endoderm (Duband et al., 1987; Dubrulle et al., 2001; Pourquie, 2003b). These results might be surprising in the light of the inversion experiments of Menkes and Sandor or Christ described earlier. However, plasticity of the PSM fragments was only observed when small territories of approximately one somite in length were inverted (Dubrulle et al., 2001). No such plasticity was seen when larger PSM fragments were inverted. As discussed above, some form of segmental programming is already found at the primitive streak level, and is therefore likely to be maintained in the caudal PSM. This programming is consistent with the result of the larger inversions, which segment according to

1075

their endogenous schedule. However, it is clearly not a definitive commitment as it can be challenged in the small PSM inversions. fgf8 mRNA is expressed in the caudal undetermined region of the PSM according to a caudo-rostral gradient and its anterior expression limit maps to the determination front level (Fig. 4, in grey) (Dubrulle et al., 2001). FGF8 protein is also expressed in a graded fashion in the caudal embryo as well as the phosphorylated form of AKT, a kinase acting downstream of the PI3 kinase pathway activated by FGF signaling (Dubrulle and Pourquie, 2004). Therefore, the fgf8 mRNA gradient is translated into a gradient of FGF signaling across the PSM. Disrupting the FGF signaling gradient by massive overexpression of fgf8 in the PSM by in ovo electroporation completely blocks segmentation and maintains a caudal PSM character in paraxial mesoderm cells (Dubrulle et al., 2001). This suggests that PSM cells are maintained immature by high FGF signaling posteriorly and that they activate their segmentation program only when they reach lower levels of FGF signaling in the rostral PSM. Altogether, this indicates that the determination front identified by the microsurgical experiments described above corresponds to a threshold of FGF signaling which is initially set by an fgf8 mRNA gradient (Fig. 4). Grafting beads soaked in FGF8 results in a rostral displacement of the determination front leading to smaller somite formation, whereas blocking FGF signaling shifts the front caudally and results in the formation of larger somites (Dubrulle et al., 2001; Sawada et al., 2001). In these experiments, only the position of the boundary is affected indicating that somite boundary positioning occurs at the determination front level. In the clock and wavefront model, the wavefront corresponds to a smooth maturation wave slowly progressing caudally, which freezes the clock pattern in PSM cells, and thus positions the somitic boundaries. Although in

Fig. 4. Model for segment formation in the chick embryo. On the left side of the embryos are shown the antagonistic gradients of FGF signaling (in grey) and of retinoic acid (in blue), which define the position of the determination front (thick black line). The periodic signal of the segmentation clock is shown in yellow as the phase I expression of Notch-related cyclic genes (Pourquie and Tam, 2001). Cells reaching the determination front which are exposed to this periodic signal activate the expression of segmental genes such as Mesp (in black) in a segment-wide domain thus establishing the segmental pattern. Subsequently, rostro-caudal identity of the prospective somite is established and ultimately somite boundaries are formed.

1076

O. Pourquie´ / Mechanisms of Development 121 (2004) 1069–1079

the original model, the wavefront was positioned at the limit between the PSM and the somitic region, the heat-shock experiments argued for a more caudal position in the PSM (Elsdale et al., 1976). The determination front therefore fulfills all the criteria of the wavefront in that it corresponds to a maturation front traveling caudally as the embryonic axis extends which defines the level where somitic boundaries are first specified. The definition of the position of the determination front was recently shown to be further refined by a gradient of retinoic acid (RA) antagonizing the FGF signaling gradient (Diez del Corral et al., 2003; Moreno and Kintner, 2004). This RA gradient (Fig. 4, in blue) is established in opposite orientation to the FGF gradient, between the rostral-most PSM and somites where the retinaldehyde dehydrogenase enzyme, raldh2 required for retinoic acid synthesis is expressed, and the tail-bud and posterior PSM region where the Cyp26 enzyme involved in the catabolism of RA is produced. Establishment of the RA gradient is controlled by FGF signaling which represses raldh2 in the paraxial mesoderm and activates cyp26 expression in the tail bud. Exposure of the PSM to RA results in a similar phenotype to that seen when FGF signaling is blocked using inhibitors such as SU5402: fgf8 mRNA is downregulated and the determination front is shifted caudally resulting in the formation of larger somites. Conversely, absence of RA signaling in the retinoid deficient VAD quail embryos or RA pathway inhibition by using chemical antagonists or receptor dominant-negative constructs result in an upregulation of fgf8 expression and in a phenotype similar to an excess FGF signaling with a rostral shift of the determination front and the formation of smaller somites (Diez del Corral et al., 2003; Moreno and Kintner, 2004). Therefore, RA signaling limits the rostral extension of the FGF gradient and controls the positioning of the determination front (Fig. 4). Recent experiments in the frog indicate that RA further acts as a transcriptional activator for genes involved in segmentation and maturation of the PSM such as the Mesp genes (Moreno and Kintner, 2004). Therefore, an important role of the FGF signaling gradient is not only to maintain PSM cells immature by repressing the activation of PSM maturation genes, but it is also to position the level at which RA can begin to act as a transcriptional activator for these genes. Intriguingly, as will be discussed below, fgf8 mRNA is not actively transcribed in the PSM suggesting that the RA gradient controls the stability of fgf8 mRNA. The first genes to be expressed with a strict segmental pattern are the bHLH transcription factors of the mesp/meso/thylacine family (Saga et al., 1997; Buchberger et al., 1998; Sparrow et al., 1998; Sawada et al., 2000; Saga and Takeda, 2001). These genes act upstream of a genetic cascade involving the Notch pathway, which ultimately results in boundary positioning and formation of rostral and caudal somitic compartments. Mesp genes are periodically activated in a segment-wide domain at the determination

front level of the PSM (Fig. 4, in black) (Buchberger et al., 1998). Mesp genes are repressed by high levels of FGF signaling and become activated at the front level in response to RA signaling (Moreno and Kintner, 2004). In frog and mouse, their activation was shown to require periodic Notch signaling, which likely places them downstream of the segmentation clock (Jen et al., 1999; Takahashi et al., 2000). This therefore supports the idea that segments are first specified at the level of determination front in response to the segmentation clock (Fig. 4). These data indicate that the pace of somitogenesis along the AP axis is controlled by the regression of the determination front in the PSM. Examination of the transcription of fgf8 mRNA reveals that it is constantly transcribed in the precursor cell area of the tail bud during axis extension and that fgf8 mRNA transcription stops when cells enter the PSM (Dubrulle and Pourquie, 2004). The progressive decay of the fgf8 mRNA in the PSM results in the formation of the mRNA gradient which positions the determination front. Due to axis elongation which results in the addition of new cells expressing high levels of fgf8 mRNA selectively in the posterior PSM and to the decay of fgf8 mRNA in the PSM, the gradient is dynamic and becomes constantly displaced caudally (Fig. 4). This displacement is responsible for the caudal movement of the determination front, and thus the pace of somitogenesis directly depends on the degradation rate of fgf8 mRNA. From the 10 to 25-somite stage in the chick embryo the size of the PSM is maintained by the constant addition of cells caudally, compensating for the somites formed from the rostral PSM. Thus, at these stages, the speed of axis elongation and the speed of determination front regression must be identical to maintain the constant length of the PSM. Later in development the PSM appears to progressively shorten along the AP axis and to contain less presumptive somites (Millet and Pourquie´, unpublished observations), suggesting that the speed of axis elongation has becomes slower than the caudal progression of the determination front. Ultimately, this imbalance between the two processes will result in the exhaustment of the PSM, leading the determination front to reach the tail bud level. This could explain the almost synchronous arrest of somitogenesis and axis extension which is observed to occur in chick after a total number of 52 somites is reached (Bellairs, 1986). This RNA decay system can also act as an hourglass type of time counting device in the PSM allowing the cells to know precisely when they must activate their segmentation program autonomously. This system thus explains the surprising degree of autonomy of segmentation observed in the inversion experiments or in cultures in which isolated PSM were found to segment autonomously (Christ et al., 1974; Menkes and Sandor, 1977; Palmeirim et al., 1998). It, therefore, provides a valuable alternative to the PSM prepatterning hypothesis previously described. A gradient of Wnt signaling has also been implicated in the control of the oscillations along the PSM (Aulehla et al.,

O. Pourquie´ / Mechanisms of Development 121 (2004) 1069–1079

2003). In this model, the oscillations, which depend on Wnt signaling would cease at the determination front level once Wnt concentration drops below a given threshold. Accordingly the wavefront would be defined by a threshold of Wnt signaling. However, because Wnt3a acts upstream of fgf8in the posterior embryo, it could also be that these Wnt effects are indirect acting though FGF signaling.

5. Specification of rostral and caudal somitic compartments in the PSM Somites can be subdivided into a rostral and a caudal compartment which exhibit different properties with respect to neural crest cell and motoneuron axon migration (Keynes and Stern, 1984, 1988). This RC subdivision of the somites provides the frame for peripheral nervous system segmentation (Bronner-Fraser, 2000). The importance of this subdivision was noticed early in the chick embryo by Remak who realized that vertebrae form from the fusion of the posterior part of a somite to the anterior part of the consecutive one during a process termed Resegmentation (Remak, 1850). Acquisition of these rostral and caudal identities by anterior PSM cells is seen by the striped expression of several genes which are later expressed either in the rostral or in the caudal compartments of the formed somites (reviewed in Pourquie, 2001; Saga and Takeda, 2001). PSM inversions result in an inversion of the somite RC polarity indicating that it is acquired prior to somite formation (Keynes and Stern, 1984; Aoyama and Asamoto, 1988; Stern et al., 1991). In fact, uncoupling segmentation from acquisition of RC identities was never achieved experimentally in the chick embryo. This led to the idea that acquisition of RC fates was required for segment formation (reviewed in Pourquie, 2001). Such a requirement was formalized in Meinhardt’s somitogenesis model, in which somite formation results from the juxtaposition of rostral and caudal somitic identities. In this hypothesis, cells of the PSM oscillate between a rostral and a caudal cell state (Meinhardt, 1986). When the definitive rostral or caudal identity of the anterior PSM cells becomes stabilized, they sort out into the prospective rostral and caudal somitic compartments due to their inability to mix. Boundaries would be formed as a result of the juxtaposition of these two cell states. This was supported by the observation that in addition to the intersomitic boundaries, there is an intrasomitic boundary called the Von Ebner’s fissure which forms in the sclerotome at the interface between rostral and caudal somite compartments (Keynes and Stern, 1988). Furthermore, when juxtaposed by grafting, somitic cells of similar identity would mix whereas cells of different identity would segregate (Stern and Keynes, 1987). However, this model does not account for the distinction between intersomitic and intrasomitic boundaries. This led Meinhardt to postulate the existence of a third cell identity associated with intersomitic boundary formation. However,

1077

examination of the process of intersomitic boundary formation in the zebrafish trilobite or knypek single and double mutants which are defective in the convergenceextension process demonstrates that in these mutants, somites containing only two rows of cells form and RC identity of the cells is properly specified (Henry et al., 2000). Thus, in these somites only two cell types, either rostral or caudal, are seen, arguing against the existence of a third specific cell type devoted to boundary formation. This hypothesis is also in conflict with other types of somitogenesis models such as the ‘clock and wavefront’, the ‘cell cycle’ or the ‘clock and trail’, which proposed that somites become determined in the PSM as a cohort of cells fated to segment together whose number is gated by the clock (Stern et al., 1988; Cooke, 1998; Kerszberg and Wolpert, 2000). According to these models, acquisition of rostral and caudal identities is subsequent to the earlier segmentation event. The process of acquisition of RC identity begins to be understood at the genetic level and largely relies on Notch signaling. It now appears based on studies in mouse and frog embryos that specification of the RC identities is subsequent to segment definition. In the mouse, Mesp2 was shown to play a critical role in the acquisition of somite rostral identity (Saga et al., 1997). Mesp2 is initially activated in a one segment-wide domain at the determination front level and becomes subsequently downregulated in the future caudal somite half in a Notch dependent fashion (Buchberger et al., 1998; Takahashi et al., 2000). Lowering the dose of Mesp2 activity in a mouse allelic series results in the formation of segments deprived of RC identities (NomuraKitabayashi, 2002). Segmentation and acquisition of RC identities are nevertheless tightly linked and in the frog, generation of larger or smaller somites by modifying the wavefront position by interfering with FGF or RA signaling results in a corresponding increase or decrease of the size of the RC compartments, respectively (Moreno and Kintner, 2004).

6. Conclusion The process of animal segmentation has long fascinated biologists and the conservation of the segmental mechanisms across animal species has been debated for more than two centuries. Because somitogenesis corresponds to a developmental window easily accessible to manipulations in ovo, the chicken embryo has constituted a major model for studies of vertebrate segmentation. In the last century, major breakthroughs that have shaped our current understanding of this process have been performed in this organism. There is no doubt that with the release of the chicken genome and the recent development of approaches to perform gain or loss of function in the embryo, the chicken embryo model will continue to dominate the field of vertebrate segmentation.

1078

O. Pourquie´ / Mechanisms of Development 121 (2004) 1069–1079

Acknowledgements I thank Silvia Esteban for artwork. Work in the Pourquie´ lab is supported by the Stowers Institute for Medical Research and an NIH Grant #1 R01 HD043158-01 to O. Pourquie´.

References Aoyama, H., Asamoto, K., 1988. Determination of somite cells: independence of cell differentiation and morphogenesis. Development 104, 15–28. Aulehla, A., Wehrle, C., Brand-Saberi, B., Kemler, R., Gossler, A., Kanzler, B., Herrmann, B.G., 2003. Wnt3a plays a major role in the segmentation clock controlling somitogenesis. Dev. Cell 4, 395 –406. Bellairs, R., 1963. The development of somites in the chick embryo. J. Embryol. Exp. Morphol. 11, 697 –714. Bellairs, R., 1985. A new theory about somite formation in the chick. Prog. Clin. Biol. Res. 171, 25 –44. Bellairs, R., 1986. The tail bud and cessation of segmentation in the chick embryo. In: Bellairs, E., Lash, J.W. (Eds.), Somitogenesis in Developing Embryos, Plenum Press, New York, pp. 161 –178. Bellairs, R., Veini, M., 1984. Experimental analysis of control mechanisms in somite segmentation in avian embryos. II. Reduction of material in the gastrula stages of the chick. J. Embryol. Exp. Morphol. 79, 183– 200. Bessho, Y., Hirata, H., Masamizu, Y., Kageyama, R., 2003. Periodic repression by the bHLH factor Hes7 is an essential mechanism for the somite segmentation clock. Genes Dev. 17, 1451–1456. Brent, A.E., Tabin, C.J., 2002. Developmental regulation of somite derivatives: muscle, cartilage and tendon. Curr. Opin. Genet. Dev. 12, 548– 557. Bronner-Fraser, M., 2000. Rostrocaudal differences within the somites confer segmental pattern to trunk neural crest migration. Curr. Top Dev. Biol. 47, 279– 296. Buchberger, A., Seidl, K., Klein, C., Eberhardt, H., Arnold, H.H., 1998. cMeso-1, a novel bHLH transcription factor, is involved in somite formation in chicken embryos. Dev. Biol. 199, 201–215. Christ, B., Jacob, H.J., Jacob, M., 1974. Somitogenesis in the chick embryo. Determination of the segmentation direction. Verh. Anat. Ges. 68, 573– 579. Cole, S.E., Levorse, J.M., Tilghman, S.M., Vogt, T.F., 2002. Clock regulatory elements control cyclic expression of lunatic fringe during somitogenesis. Dev. Cell 3, 75–84. Cooke, J., 1975. Control of somite number during morphogenesis of a vertebrate, Xenopus laevis. Nature 254, 196– 199. Cooke, J., 1998. A gene that resuscitates a theory-somitogenesis and a molecular oscillator. Trends Genet. 14, 85–88. Cooke, J., Zeeman, E.C., 1976. A clock and wavefront model for control of the number of repeated structures during animal morphogenesis. J. Theor. Biol. 58, 455 –476. Dale, K.J., Pourquie, O., 2000. A clock-work somite. Bioessays 22, 72– 83. Dale, J.K., Maroto, M., Dequeant, M.L., Malapert, P., McGrew, M., Pourquie, O., 2003. Periodic Notch inhibition by Lunatic Fringe underlies the chick segmentation clock. Nature 421, 275–278. Diez del Corral, R., Olivera-Martinez, I., Goriely, A., Gale, E., Maden, M., Storey, K., 2003. Opposing FGF and retinoid pathways control ventral neural pattern, neuronal differentiation, and segmentation during body axis extension. Neuron 40, 65–79. Duband, J.L., Dufour, S., Hatta, K., Takeichi, M., Edelman, G.M., Thiery, J.P., 1987. Adhesion molecules during somitogenesis in the avian embryo. J. Cell Biol. 104, 1361–1374.

Duboule, D., 1994. Temporal colinearity and the phylotypic progression: a basis for the stability of a vertebrate Bauplan and the evolution of morphologies through heterochrony. Dev. Suppl., 135–142. Dubrulle, J., Pourquie, O., 2004. fgf8 mRNA decay establishes a gradient that couples axial elongation to patterning in the vertebrate embryo. Nature 427, 419–422. Dubrulle, J., McGrew, M.J., Pourquie, O., 2001. FGF signaling controls somite boundary position and regulates segmentation clock control of spatiotemporal Hox gene activation. Cell 106, 219 –232. Eloy-Trinquet, S., Nicolas, J.F., 2002a. Cell coherence during production of the presomitic mesoderm and somitogenesis in the mouse embryo. Development 129, 3609–3619. Eloy-Trinquet, S., Nicolas, J.F., 2002b. Clonal separation and regionalisation during formation of the medial and lateral myotomes in the mouse embryo. Development 129, 111 –122. Elsdale, T., Pearson, M., Whitehead, M., 1976. Abnormalities in somite segmentation following heat shock to Xenopus embryos. J. Embryol. Exp. Morphol. 35, 625– 635. Freitas, C., Rodrigues, S., Charrier, J.B., Teillet, M.A., Palmeirim, I., 2001. Evidence for medial/lateral specification and positional information within the presomitic mesoderm. Development 128, 5139– 5147. Hamburger, V., Hamilton, H.L., 1951. A series of normal stages in the development of the chick embryo (1951). Dev. Dyn. 195, 231–272. Hatada, Y., Stern, C.D., 1994. A fate map of the epiblast of the early chick embryo. Development 120, 2879–2889. Henry, C.A., Hall, L.A., Burr Hille, M., Solnica-Krezel, L., Cooper, M.S., 2000. Somites in zebrafish doubly mutant for knypek and trilobite form without internal mesenchymal cells or compaction. Curr. Biol. 10, 1063–1066. Hirsinger, E., Jouve, C., Dubrulle, J., Pourquie, O., 2000. Somite formation and patterning. Int. Rev. Cytol. 198, 1–65. Jacobson, A.G., 1988. Somitomeres: mesodermal segments of vertebrate embryos. Development 104(Suppl), 209–220. Jen, W.C., Gawantka, V., Pollet, N., Niehrs, C., Kintner, C., 1999. Periodic repression of Notch pathway genes governs the segmentation of Xenopus embryos. Genes Dev. 13, 1486–1499. Jiang, Y.J., Smithers, L., Lewis, J., 1998. The clock is linked to notch signalling. Curr. Biol. 8, R868– R871. Joubin, K., Stern, C.D., 1999. Molecular interactions continuously define the organizer during the cell movements of gastrulation. Cell 98, 559 –571. Jouve, C., Iimura, T., Pourquie, O., 2002. Onset of the segmentation clock in the chick embryo: evidence for oscillations in the somite precursors in the primitive streak. Development 129, 1107–1117. Kerszberg, M., Wolpert, L., 2000. A clock and trail model for somite formation, specialization and polarization. J. Theor. Biol. 205, 505 –510. Keynes, R.J., Stern, C.D., 1984. Segmentation in the vertebrate nervous system. Nature 310, 786–789. Keynes, R.J., Stern, C.D., 1988. Mechanisms of vertebrate segmentation. Development 103, 413–429. Kieny, M., Mauger, A., Sengel, P., 1972. Early regionalization of somitic mesoderm as studied by the development of axial skeleton of the chick embryo. Dev. Biol. 28, 142–161. Kulesa, P.M., Fraser, S.E., 2002. Cell dynamics during somite boundary formation revealed by time-lapse analysis. Science 298, 991 –995. Lutz, H., 1948. Sur la polyembryonnie experimentale resultant de fissuration en croix du blastoderme non incube de cane. C.R. Acad. Sci. 226, 841. Meier, S., 1979. Development of the chick embryo mesoblast. Formation of the embryonic axis and establishment of the metameric pattern. Dev. Biol. 73, 24 –45. Meinhardt, H., 1986. Models of segmentation. In: Bellairs, R., Ede, D.A., Lash, J.W. (Eds.), Somites in Developing Embryos, Plenum press, New York, pp. 179–191. Menkes, B., Sandor, S., 1977. Somitogenesis, regulation potencies, sequence determination and primordial interactions. In: Ede, D.A.,

O. Pourquie´ / Mechanisms of Development 121 (2004) 1069–1079 (Ed.), Vertebrate Limb and Somite Morphogenesis, British Society Developmental Biology Symposium 3, Cambridge University Press, Cambridge, pp. 405 –419. Menkes, B., Sandor, S., Elias, S., 1968. Researches on the formation of axial organs of the chick embryo. IV. Rev. Roum. Embryol. Cytol. 5, 131–137. Morales, A.V., Yasuda, Y., Ish-Horowicz, D., 2002. Periodic lunatic fringe expression is controlled during segmentation by a cyclic transcriptional enhancer responsive to notch signaling. Dev. Cell 3, 63–74. Moreno, T.A., Kintner, C., 2004. Regulation of segmental patterning by retinoic acid signaling during Xenopus somitogenesis. Dev. Cell 6, 205–218. Nicolas, J.F., Mathis, L., Bonnerot, C., Saurin, W., 1996. Evidence in the mouse for self-renewing stem cells in the formation of a segmented longitudinal structure, the myotome. Development 122, 2933–2946. Nicolet, G., 1971. Avian gastrulation. Adv. Morphog. 9, 231 –262. Nomura-Kitabayashi, A., Takahashi, Y., Kitajima, S., Inoue, T., Takeda, H., Saga, Y., 2002. Hypomorphic Mesp allele distinguishes establishment of rostrocaudal polarity and segment border formation in somitogenesis. Development 129(10), 2473– 2481. Nowicki, J.L., Burke, A.C., 2000. Hox genes and morphological identity: axial versus lateral patterning in the vertebrate mesoderm. Development 127, 4265–4275. Olivera-Martinez, I., Coltey, M., Dhouailly, D., Pourquie, O., 2000. Mediolateral somitic origin of ribs and dermis determined by quailchick chimeras. Development 127, 4611–4617. Ordahl, C.P., Le Douarin, N.M., 1992. Two myogenic lineages within the developing somite. Development 114, 339– 353. Packard, D.S.J., Jacobson, A., 1976. The influence of axial structures on chick somite formation. Dev. Biol. 53, 36–48. Packard, D.S.J., Meier, S., 1983. An experimental study of the somitomeric organization of the avian segmental plate. Dev. Biol. 97, 191 –202. Palmeirim, I., Henrique, D., Ish-Horowicz, D., Pourquie´, O., 1997. Avian hairy gene expression identifies a molecular clock linked to vertebrate segmentation and somitogenesiss. Cell 91, 639 –648. Palmeirim, I., Dubrulle, J., Henrique, D., Ish-Horowicz, D., Pourquie´, O., 1998. Uncoupling segmentation and somitogenesis in the chick presomitic mesoderm. Dev. Genet. 23, 77 –85. Pasteels, J., 1937. Etudes sur la gastrulation des Verte´bre´s me´roblastiques. III. Oiseaux. IV. Conclusions ge´ne´rales. Arch. Biol. 48, 381 –488. Pourquie, O., 1999. Notch around the clock. Curr. Opin. Genet. Dev. 9, 559–565. Pourquie, O., 2001. Vertebrate somitogenesis. Annu. Rev. Cell Dev. Biol. 17, 311 –350. Pourquie, O., 2003a. The segmentation clock: converting embryonic time into spatial pattern. Science 301, 328–330. Pourquie, O., 2003b. Vertebrate somitogenesis: a novel paradigm for animal segmentation? Int. J. Dev. Biol. 47, 597 –603. Pourquie, O., Tam, P.P., 2001. A nomenclature for prospective somites and phases of cyclic gene expression in the presomitic mesoderm. Dev. Cell. 1, 619–620. Primmett, D.R., Stern, C.D., Keynes, R.J., 1988. Heat shock causes repeated segmental anomalies in the chick embryo. Development 104, 331–339. Primmett, D.R., Norris, W.E., Carlson, G.J., Keynes, R.J., Stern, C.D., 1989. Periodic segmental anomalies induced by heat shock in the chick embryo are associated with the cell cycle. Development 105, 119–130. Psychoyos, D., Stern, C.D., 1996. Fates and migratory routes of primitive streak cells in the chick embryo. Development 122, 1523–1534. Remak, R., 1850. Untersuchungen u¨ber die entwicklung der Wirbeltiere, Reimer, Berlin. Saga, Y., Takeda, H., 2001. The making of the somite: molecular events in vertebrate segmentation. Nat. Rev. Genet. 2, 835–845. Saga, Y., Hata, N., Koseki, H., Taketo, M.M., 1997. Mesp2: a novel mouse gene expressed in the presegmented mesoderm and essential for segmentation initiation. Genes Dev. 11, 1827–1839.

1079

Sandor, S., Amels, D., 1971. Researches in the formation of axial organs in the chick embryo. VII. Rev. Roum. Embryol. 8, 37 –41. Sandor, S., Fazakas-Todea, I., 1980. Researches on the formation of axial organs in the chick embryo. X. Further investigations on the role of ecto- and endoderm in somitogenesis. Morphol. Embryol. (Bucur) 26, 29– 32. Sato, Y., Yasuda, K., Takahashi, Y., 2002. Morphogical boundary forms by a novel inductive enevt mediated by Lunatic fringe and Notch during somitic segmentation. Development 129, 3633–3644. Sawada, A., Fritz, A., Jiang, Y., Yamamoto, A., Yamasu, K., Kuroiwa, A., Saga, Y., Takeda, H., 2000. Zebrafish Mesp family genes, mesp-a and mesp-b are segmentally expressed in the presomitic mesoderm, and Mesp-b confers the anterior identity to the developing somites. Development 127, 1691–1702. Sawada, A., Shinya, M., Jiang, Y.J., Kawakami, A., Kuroiwa, A., Takeda, H., 2001. Fgf/MAPK signalling is a crucial positional cue in somite boundary formation. Development 128, 4873– 4880. Schoenwolf, G.C., Garcia-Martinez, V., Dias, M.S., 1992. Mesoderm movement and fate during avian gastrulation and neurulation. Dev. Dyn. 193, 235–248. Selleck, M.A., Stern, C.D., 1991. Fate mapping and cell lineage analysis of Hensen’s node in the chick embryo. Development 112, 615 –626. Selleck, M.A., Stern, C.D., 1992. Commitment of mesoderm cells in Hensen’s node of the chick embryo to notocord and somite. Development 114, 403–415. Sparrow, D.B., Jen, W.C., Kotecha, S., Towers, N., Kintner, C., Mohun, T.J., 1998. Thylacine 1 is expressed segmentally within the paraxial mesoderm of the Xenopus embryo and interacts with the Notch pathway. Development 125, 2041–2051. Spratt, N.T., 1955. Analysis of the organizer center in the early chick embryo. I Localisation of prospective notochord and somite cells. J. Exp. Zool. 128, 121 –162. Spratt, N.T. Jr, Haas, H., 1965. Germ layer formation and the role of the primitive streak in the chick. I. Basic Architecture and Morphogenetic Tissue Movements. J. Exp. Zool. 158, 9– 38. Stern, C.D., Keynes, R.J., 1987. Interactions between somite cells: the formation and maintenance of segment boundaries in the chick embryo. Development 99, 261–272. Stern, C.D., Vasiliauskas, D., 2000. Segmentation: a view from the border. Curr. Top Dev. Biol. 47, 107–129. Stern, C.D., Fraser, S.E., Keynes, R.J., Primmett, D.R., 1988. A cell lineage analysis of segmentation in the chick embryo. Development 104, 231– 244. Stern, C.D., Artinger, K.B., Bronner-Fraser, M., 1991. Tissue interactions affecting the migration and differentiation of neural crest cells in the chick embryo. Development 113, 207–216. Stern, C.D., Hatada, Y., Selleck, M.A., Storey, K.G., 1992. Relationships between mesoderm induction and the embryonic axes in chick and frog embryos. Dev. Suppl., 151–156. Summerbell, D., Lewis, J.H., Wolpert, L., 1973. Positional information in chick limb morphogenesis. Nature 244, 492 –496. Takahashi, Y., Koizumi, K., Takagi, A., Kitajima, S., Inoue, T., Koseki, H., Saga, Y., 2000. Mesp2 initiates somite segmentation through the Notch signalling pathway. Nat. Genet. 25, 390– 396. Vogt, W., 1929. Gestaltanalyse ma Amphibienkein mit o¨rtlicher Vitalfa¨rbung. II. Teil. Gastrulation und Mesodermbildung bei Urodelen und Anuren. Wilhelm Roux’ Arch. Entwicklungsmech. Org. 120, 384–706. Waddington, C.H., 1952. The Epigenetics of Birds, Cambridge University Press, Cambridge, London. Wetzel, R., 1929. Untersuchung am Hunchen, Berlin. Wilson, V., Beddington, R.S., 1996. Cell fate and morphogenetic movement in the late mouse primitive streak. Mech. Dev. 55, 79–89. Wolff, E., 1936. Les bases de la teratogenese experimentale des vertebres amniotes, d’apres les resultats de methodes directes. Arch. Anat., Strasbourg, 22.