Department of Integrated Biosciences, Graduate School of Frontier Science, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba Prefecture 277-8562, Japan
*Author for correspondence (e-mail: shonan{at}biol.k.u-tokyo.ac.jp)
Accepted September 28, 2001
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SUMMARY |
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Key words: Sea urchin, Adult rudiment, Left-right asymmetry, Half embryo, Handedness
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INTRODUCTION |
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The adult rudiment comprises the left coelomic sac and the vestibular ectoderm, which are initially located remote from each other within the left side of the larva. During the initial stage of development of the adult rudiment, the hydropore canal and the hydrocoel develop in the left coelomic sac, and a small epithelial invagination begins to develop on the left side of the oral ectoderm as the primary vestibule (Runnström, 1917; Gustafson and Wolpert, 1963; Czihak, 1965; Hörstadius, 1973; Pehrson and Cohen, 1986). The corresponding parts on the right side of the larva do not develop these organs, so that the hydropore canal, the hydrocoel and the vestibule are left-side-specific traits in normal development. Although the exact timing of development of these organs differs between different species, these traits characterize the earliest LR asymmetry in the morphology of the larva generally among indirect-developing species (Okazaki, 1975; Raff, 1987).
Previous studies of indirect-developing sea urchins have shown that the polarity for differential LR development can be re-established in half embryos dissected meridionally to the AV axis. The first and second cleavages are meridional to the AV axis, and the halves separated along these planes in early development up to the blastula stage have been shown to develop into the pluteus (Hörstadius and Wolsky, 1936; Hörstadius, 1973), and further into sexually mature sea urchins (Marcus, 1979; Cameron et al., 1996). The ability to re-establish LR polarity in the meridional twins of Lytechinus variegatus was studied by McCain and McClay (McCain and McClay, 1994). Their study demonstrated that the meridional halves of the cleavage stage were able to coordinate the polarity between the OA and the LR axes to re-establish the normal LR development of the hydropore canal. The meridional dissection was performed as late as the mesenchyme blastula stage. Although 22% of the meridional halves showed abnormalities, such as a right pore canal or bilateral pore canals, many of the mesenchyme blastula halves (78%) re-established normal LR polarity. The study indicated that the meridional halves re-establish normal LR polarity during cleavage, and the ability of the halves to re-establish normal LR polarity was not completely lost as late as the mesenchyme blastula stage (McCain and McClay, 1994).
Development of the adult rudiment is accelerated in a direct-developing sea urchin, Heliocidaris erythrogramma, whose vestibule develops soon after the gastrula stage (Williams and Anderson, 1975; Wray and Raff, 1990). Although the orientation of the first cleavage to the LR axis is variable in some sea urchin species (Kominami, 1988; Henry et al., 1992; Ruffins and Ettensohn, 1996; Summers et al., 1996), the first cleavage invariably occurs in the mid-sagittal plane in H. erythrogramma (Wray and Raff, 1990; Henry and Raff, 1990; Emlet 1995). The developmental potential of the left and right halves to re-establish the LR polarity for the vestibule was investigated in H. erythrogramma (Henry and Raff, 1990; Henry and Raff, 1994), by taking advantage of the first cleavage, which predicts the future left and right sides of the larvae. Both the left and right halves directed the formation of a vestibule throughout the early blastula stage, although the regulatory ability declined in the left halves as development proceeded. Normal LR polarity was demonstrated to be retained in the left halves, whereas the polarity was sometimes reversed in the right halves (Henry and Raff, 1990; Henry and Raff, 1994). The potential of the left and the right halves to re-establish LR polarity was also investigated in two starfish species (Hörstadius, 1928; Hörstadius, 1973). The LR development of the hydrocoel in a direct developer, Asterina gibbosa, and of the hydopore canal in an indirect developer, Astropecten aranciacus remained normal in the left halves dissected at the gastrula stage, but was impaired in an unpredictable manner in the right halves, with the hydrocoel or the hydropore canal being formed on the left, on the right, on both the left and the right, or on neither side (Hörstadius, 1928; Hörstadius, 1973). Thus, the results of the dissection experiments for H. erythrogramma and for the two starfish species all indicated that the normal LR polarity was fixed in the left halves, whereas the LR polarity was unstable in the right halves.
There have been no experiments to examine the potential of indirect-developing sea urchins to re-establish the LR polarity in half embryos that were clearly identified as having originated from the left or the right half of the embryo. The finding that the meridional halves of indirect developing species re-established normal LR polarity indicates that determination of LR polarity is mediated by cell interactions (McCain and McClay, 1994), but it is not known when or which cell interactions determine the normal LR position of the adult rudiment during development. As a first step in investigating the process that determines the LR placement of the adult rudiment in spatial and temporal contexts, we investigated whether excisions on the left and right side of the embryos have a different effect on LR placement of the adult rudiment in two indirect-developing sea urchins, Hemicentrotus pulcherrimus and Scaphechinus mirabilis. We excised whole or part of the tissue on the left or the right side of embryos during the stages that ranged from the early gastrula stage to the two-armed pluteus stage. The operated larvae were cultured and examined for the LR position of the adult rudiment, and of components of the adult rudiment that develop morphological LR differences in the larva, such as the hydropore canal, the hydrocoel and the primary vestibule. In contrast to the previous results found in the direct-developing sea urchin H. erythrogramma and the two starfish species, the indirect-developing sea urchins examined in the present study showed abnormal LR patterning in the left halves but not in the right halves. Furthermore, regional excisions in the mid-late gastrula stage revealed that a part of right side tissue was indispensable for the formation of an adult rudiment on the left side of the larva, whereas that on the left side was not. Thus, the left-right differential effects of excisions in the present study indicate that LR polarity in formation of the adult rudiment is directed by the right side, in larvae of the indirect-developing sea urchins.
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MATERIALS AND METHODS |
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Microsurgery
Microsurgical operations were performed under a dissecting microscope (WILD MZ8: Leica) with a glass needle produced by manually pulling over a tiny gas flame. After the operation, the larvae were examined through an optical microscope (OPTIPHOTO or BIOPHOTO: Nikon) to confirm that the excision had been made as planned.
Vital staining
Vital staining was performed with 1% Nile Blue Sulfate (Schmid Gmbh) in a solution containing 10% agarose. A glass capillary whose opening was about the same diameter of an egg was filled with the dye and used to apply it to the embryos. The stained embryos were immediately transferred to a new dish and operated on. The position of the dye marks in the operated larvae was checked with an optical microscope.
Measurements of arm length
The length of the larval arms was measured with a micrometer under an optical microscope with a x20 or x10 power objective.
Statistics
The statistical significance of consistent inversion (in the right-EME cut) was tested against the null hypothesis that the probability of forming the adult rudiment on the left side or the right side of an individual was equal. A formation of a left adult rudiment and a right adult rudiment in the outcomes were scored as 1 point for the left side and for the right side, respectively. Outcomes such as bilateral adult rudiments or no adult rudiment on either side were given a score of 0.5 point each on the left and right side. Significance was tested by cumulating one tail of binomial distributions (Sokal and Rohlf, 1995).
The significance of differences of the mean and the variations in the length of arms among different groups of the left and right halves was tested by analysis of variance (ANOVA) for a single classification (Sokal and Rohlf, 1995). An Fs value of less than 1 means that variations between groups are smaller than the variation within each group, indicating that the difference between groups is not significant. Probability was tested only when Fs was more than 1.
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RESULTS |
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First, we investigated whether dissection of the early gastrulae along the midplane evoked reversal of the OA polarity, as reversal of the OA polarity had previously been observed in embryos dissected meridionally during cleavage (Hörstadius and Wolsky, 1936; McCain and McClay, 1994) and in the blastula stage (Hörstadius and Wolsky, 1936). A medium concentration of Nile Blue Sulfate was used to stain the embryos, because it had been demonstrated to have no biasing effect on the polarity of the OA or the LR axis (Lindhal, 1932; Hörstadius, 1973; Henry et al., 1992; Henry and Raff, 1994), as confirmed by the following findings. The left or right side of an embryo was stained with Nile Blue Sulfate at the earliest stage of gastrulation. The embryo was immediately bisected along the midplane (Fig. 1A), and the location of the staining in the each half was investigated in the two-armed pluteus stage. However, as no staining was ever detected in the opposite half, the left halves were examined after staining the left side, and the right halves were examined after staining the right side. In the 30 left halves examined, staining was always found on the left side (n=24/30, Fig. 1B), the only exceptions being some specimens in which the staining was so weak that its exact position could not be identified (n=6/30). Similarly, right staining was found exclusively on the right side (n=27/31) of right-half larvae, and the only exceptions were specimens in which no staining could be identified (n=4/31). These results indicate that left and right halves obtained by midplane dissection at the earliest gastrula stage do not reverse the polarity of the OA axis. Therefore, the left side of the left halves was always derived from the left side of the unseparated whole embryos, and the right side of the right halves from the right side.
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We first examined the unoperated side of the left and the right halves of S. mirabilis dissected during the early to late gastrula stage for differences in arm length by measuring the length of postoral-arm-rod plus body-rod (po-length), and the length of the anterolateral-arm-rod plus body-rod (al-length) (see Fig. 3A for the arm notation). The results showed no differences in po- or al-length between the left and right halves at the two-armed pluteus stage (n=22, Fs=0.0017 and Fs=1.33 for po- and al-length, respectively, single classification ANOVA for two samples, see Materials and Methods), or in po-length (n=20, Fs=0.073), al-length (n=19, Fs=0.13), or in length of the posterodorsal rod (pd-length) (n=17, Fs=0.59) between the left and right halves at the six-armed stage. These results indicate that midplane dissection has no differential effect on arm growth on the unoperated side in left and right halves dissected during the gastrula stage. The same test was performed on larvae dissected in the two-armed pluteus stage, and no significant differences were found between the left and right halves (data not shown).
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To summarize, arm length on the cut side and on the unoperated side did not significantly differ either between the left and right halves, or between larvae with a left adult rudiment and larvae with a right adult rudiment in the left halves. These results indicated that even though LR patterning was defective in the left halves alone, there was no difference in damage to arm growth between the left and the right halves. Reversal of the LR position of the adult rudiment was not linked with the severe damage evaluated on the basis of arm length.
LR positioning of the hydropore canal in H. pulcherrimus half larvae
We next investigated the LR position of the hydropore canal in the left and the right halves of H. pulcherrimus as the earliest morphological LR marker in this species. The hydropore canal is a tubular organ that develops from the left coelomic sac and connects the coelomic cavity of the adult rudiment to the dorsal opening. It normally develops only on the left side. In several species of sea urchins, including H. pulcherrimus, the left hydropore canal forms during the late two-armed or early four-armed pluteus stage (Gustafson and Wolpert, 1963; Pehrson and Cohen, 1986; McCain and McClay, 1994), and in other species, including S. mirabilis, it develops later, during the six-armed stage. Although formation of the hydropore canal usually results in eventual development of the adult rudiment, degeneration of the pore canal sometimes occurs under experimental conditions. For example, two types of abnormal LR patterning of the hydropore canal and adult rudiment have been reported for the meridional halves of Lytechinus variegatus: a right pore canal and bilateral pore canals (McCain and McClay, 1994), and in one of the larvae with bilateral pore canals in that study the left canal degenerated and the adult rudiment developed on the right side alone. In the present study, the majority of the left halves dissected in the mid-late gastrula stage formed a right adult rudiment, and no larvae with bilateral adult rudiments were found (Table 2A). The present results raise two possibilities: (1) that the hydropore canals develop on both sides in (some of) the left halves but the left one degenerates subsequently; or (2) that the hydropore canal develops on only the right side of the left halves. To determine which actually occurs, we examined the LR position of the hydropore canal in the half larvae of H. pulcherrimus dissected in the mid-late gastrula stage (Table 2B, Fig. 4).
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The position of the pore canal in the right halves was examined next. Of the 22 right halves examined in the four-armed stage, 19 had a left pore canal and the other three had no identifiable pore canal. All three larvae with no pore canal were confirmed to eventually develop a left adult rudiment during subsequent development, indicating that the right halves consistently develop a left pore canal.
To summarize, the pore canal formed only on the right side in the majority of the left halves, indicating that development of the pore canal was generally not initiated on the left side of the left halves. In the right halves, the pore canal generally formed on the left side. In both the left and right halves, the LR position of the hydropore canal at the four-armed stage generally coincided with the LR placement of the adult rudiment, although the pore canal degenerated in a minority of the left half larvae.
Regional operations in the mid- to late-gastrula stage
Excision of tissue containing a portion of the archenteron
The results of the midplane dissection in the early gastrula and in the mid-late gastrula stage indicated frequent occurrence of reversal of LR polarity during formation of the adult rudiment in the left halves, but that reversal seldom occurred in the right halves. One possible interpretation of the different outcomes is that removal of tissue on the right side and left side has different effects on LR positioning during formation of the adult rudiment. Next, we examined the effect of regional excisions on LR placement of the adult rudiment (Fig. 5). The regional excisions were performed in the mid-late gastrula stage to take advantage of the fact that the percentage of polarity reversals in the left halves was highest at this stage so that the left and the right halves would be most distinctly different with regard to LR placement of the adult rudiment.
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The right EME cut resulted in reversal of the LR placement of the adult rudiment in most of the operated larvae (Table 3A, Fig. 6A). Among the H. pulcherrimus specimens, seven out of the nine larvae had a right adult rudiment, and only two had a left adult rudiment. Among the S. mirabilis specimens, 12 out of the 17 larvae that survived formed a right adult rudiment, four had no adult rudiment on either side, and only one had a left adult rudiment. Statistical analysis confirmed that the right-EME cut, rather than randomizing the direction of adult rudiment formation, consistently biased it toward the right side (H. pulcherrimus P<0.09; S. mirabilis, P<0.007, exact probability). The results indicate that the LR placement of the adult rudiment was basically reversed in the larvae subjected to the right EME cut.
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The left-EME cut was performed in H. pulcherrimus embryos (Table 3A), and the adult rudiment was formed on the normal left side of all 10 larvae that survived. The results of the EME cut indicate that the effect of the left- and the right-animal side excisions of the embryo on the LR placement of the adult rudiment is basically the same as the effect of excision of the whole left side and the whole right side in the midplane dissection, respectively.
We then examined whether the excision of the right vegetal side affects LR placement of the adult rudiment. The tissue on the right vegetal side including the archenteron, the ectodermal epithelium and the mesenchyme cells was excised (Fig. 5D,E). All H. pulcherrimus and S. mirabilis larvae formed the adult rudiment on the left side after this operation (Table 3A, Fig. 6D), indicating that excision of the right vegetal side of the embryo had no effect on the normal LR placement of the adult rudiment. The effect of the sham operation along the midline of the embryos was examined next (Fig. 5F). The sham operation consisted of cutting the embryos almost completely in half along the midplane by stopping the cut slightly before the left and right halves were completely separated. The only connection between the left and right halves was the extracellular matrix (hyaline layer) in the plane of dissection. The embryos eventually developed into apparently normal plutei, and all formed a left adult rudiment (Table 3A). These findings indicate that the normal LR placement of the adult rudiment was not disrupted by the sham operation.
The above results of the regional operations indicated that removal of the right animal side generally resulted in reversal of the LR position of the primary vestibule and the adult rudiment, and that removal of the left animal side or the right vegetal side had no effect on the normal LR placement of the adult rudiment.
Excision of tissues beside the midplane region
The series of EME cut experiments revealed that removal of the right animal side was effective in disrupting the LR position of the adult rudiment, whereas the excisions that avoided this region had no effect on the LR position of the adult rudiment. We next wondered whether removal of only the epithelium on the right animal side, avoiding the archenteron, would affect LR placement (Fig. 7A,B). However, in reality it was impossible to leave the archenteron completely intact in the mid-late gastrula stage, because filopodial tracts from part of the secondary mesenchyme cells (SMCs) attached the inner wall of the ectodermal epithelium (Hardin and McClay, 1990). Besides, among four types of the SMCs, part of pigment and blastocoelar cells migrated out of the archenteron at the early gastrula stage (Okazaki, 1975; Ruffins and Ettensohn, 1996; Kominami, 2000), and as a result the cells that had already left the archenteron were removed from embryos by this cut. We refer to this operation the EM cut, as the ectodermal and mesodermal (EM) tissue was excised from the embryo. The EM cut did not directly damage the endoderm or the SMCs that evaginate from the archenteron in a later stage, such as the coelomic sac and pharyngeal muscle cells, because these cells do not leave the archenteron until the late prism stage (Gustafson and Wolpert, 1963; Okazaki, 1975; Ishimoda-Takagi, 1984; Ettensohn and Ruffins, 1993). The morphology of the tip of the archenteron at the two-armed stage appeared normal in the EM-cut larvae (Fig. 7C), whereas the coelomic sacs of the EME-cut larvae were clearly smaller on the cut side (Fig. 5C).
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To summarize, removal of all of the tissue on the right side did not affect the formation of the left adult rudiment from at the prism stage of H. pulcherrimus onwards. In addition, removal of the whole right side in the prism stage and two-armed pluteus stage elicited formation of the adult rudiment on the right side. The fact that the left halves did not fail to form an adult rudiment on the left side suggests that a critical change involved in the process of formation of the adult rudiment occurs on the left side of the embryo during the period between the gastrula stage and the prism stage. In addition, removal of the whole left side in the two-armed pluteus stage was shown not to affect formation of an adult rudiment on this side in larvae of H. pulcherrimus.
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DISCUSSION |
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Regional dissection of sea urchin embryos and larvae at different stages between the early gastrula and the two-armed pluteus in this study revealed that the LR placement of the adult rudiment in larvae was basically impaired only in the left halves and not in the right halves. The present findings and previous reports suggest that the three mechanisms described below are involved in determination of LR placement of the adult rudiment.
First, the present findings suggest that in the mid- to late-gastrula (mid-late gastrula) stage the left side of the embryo is not yet specified to autonomously form the adult rudiment. The left halves dissected in the mid-late gastrula stage and the larvae subjected to the right EME cut generally failed to develop any components of the adult rudiment, such as the hydropore canal, the hydrocoel or the primary vestibule, on their left side (Fig. 4A, Fig. 6C, Table 2, Table 3B). The left side of the left halves and the right EME cut-larvae was derived from the left side of the unseparated whole embryo, as demonstrated by the vital staining experiments for the left and right halves (Fig. 1). Because the vestibule region, which is located between the anterolateral and postoral arms (Czihak, 1965; Okazaki, 1975) (Fig. 6B), is remote from the midplane, it is reasonable to conclude that the tissue adjacent to the vestibule was not displaced by the operation and shifted into the cut side of the operated larvae. Thus, the position of the vestibule region in relation to the adjacent tissue may not have been significantly changed by the operation. Indeed, the fact that many (50%) of the left halves dissected in the early gastrula stage formed a left adult rudiment indicates that the presumptive vestibule region was able to differentiate a vestibule (Fig. 2C). Therefore, the finding that the primary vestibule invagination did not occur cannot be attributed to a direct effect of the operation. The fact that an excision far from the vestibule region altered the fate of this region suggests that the initial cues that determine the formation of the vestibule in intact whole embryos originate either in the midplane region or the right side. The fact that formation of the vestibule is a prerequisite for the formation of the adult rudiment suggests that formation of the adult rudiment cannot be initiated autonomously by the left side.
Second, the findings suggest that normal LR polarity for the direction of formation of the adult rudiment is directed by the right side. It has been shown previously that meridional halves dissected during cleavage stages are capable of coordinating LR polarity with the polarity of the oral-aboral (OA) axis to develop a left hydrocoel (McCain and McClay, 1994). The present study showed that the establishment of LR polarity was disrupted in the left halves dissected during the period between the early gastrula and the two-armed pluteus stages (Fig. 9A-C), whereas normal LR polarity was correctly re-established in the right halves (Fig. 9D-F). This finding strongly suggests that LR polarity is coordinated with OA polarity by the right side during normal development, and therefore that the right side controls the process of polarity establishment in left side tissue. The hypothetical effect of the right side on the left side appears to proceed during the period between the mid-late gastrula stage and the prism stage, because the left halves dissected in the prism stage and the pluteus stage never failed to develop the adult rudiment on their left side (Fig. 8A, Fig. 9C, Table 5), indicating that contact with the right side of the intact whole embryos before the prism stage was sufficient to initiate the formation of the left adult rudiment and that no further influence from the normal right side was needed. This suggests that some induction from the right side to the left side that takes place between the mid-late gastrula and the prism stages is involved as one of the mechanisms that establishes normal LR polarity in the direction of formation of the adult rudiment.
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Taken together, the above findings suggest that the left side is not committed to form the adult rudiment autonomously in the mid-late gastrula stage, and that some induction from the right side to the left side that takes place between the mid-late gastrula stage and the prism-pluteus stages is involved in the determination of normal LR polarity in the direction of formation of the adult rudiment. These findings suggest that one of crucial changes during the process of determining the LR placement of the adult rudiment occurs between the gastrula stage and the prism stage. This period coincides with the period when some evidence of LR asymmetry first arises at the tip of the archenteron, as previously shown by asymmetric expression of transcripts such as the snail-related gene (Hardin, 1995), ß-catenin (Miller and McClay, 1997a), and cadherins (Miller and McClay, 1997b), or by asymmetric allocation of descendants of small micromeres into the coelomic sacs (Pehrson and Cohen, 1986; Ettensohn and Ruffins, 1993) (M. Aihara and S. A., unpublished). It has also been suggested previously and here in this study that the definitive decision to turn on or turn off morphogenesis of the adult rudiment is not yet complete in the two-armed pluteus stage.
Modes of re-establishing LR polarity in the left and right halves of different animal species
Previous studies of separated or conjoined half embryos of echinoderms (Hörstadius, 1928; Hörstadius, 1973; Henry and Raff, 1990; Henry and Raff, 1994) and vertebrates (Spemann and Falkenberg, 1919; Levin et al., 1996; Nascone and Mercola, 1997) showed that LR patterning defects occurred in the right halves but not in the left halves. LR polarity was either randomly re-established among different individuals (Henry and Raff, 1994) or was randomized among different organs within the same individual (Hörstadius, 1928; Hörstadius, 1973; Spemann and Falkenberg, 1919; Levin et al., 1996; Nascone and Mercola, 1997), and thus there was no consistency in the type of LR patterning defects that occurred in the right halves.
In comparison with the above results in previous studies, the results obtained for the dissected left and right halves of indirect-developing sea urchin larvae in this study showed unique features. First, LR patterning defects were generally found only in the left halves, not in the right halves (Fig. 9), providing the first examples of LR patterning defects being elicited in left halves but not in right halves. Besides the two species investigated in this study, we confirmed that LR defects in five other indirect-developing sea urchins belonging to three different orders are also confined to occurring only in the left halves after dissection in the gastrula stage (M. A. and S. A., unpublished), suggesting that the confinement of LR defects to the left halves is common to indirect-developing sea urchins. A second unique feature of our findings was uniformity in the manner of occurrence of LR defects between different individuals that was observed in the two stages: (1) LR polarity in the placement of the adult rudiment was reversed in almost all of the left halves dissected in the mid-late gastrula stage (Table 2), indicating that the reversal of polarity in the larvae was not random, which was further confirmed by the right EME cut in the same stage (Table 3); and (2) the left halves of H. pulcherrimus larvae dissected in the prism stage and pluteus stage consistently formed bilateral adult rudiments (Table 5). The consistent occurrence of one specific type of LR patterning defect being induced in every individual has never been found for LR halves in other animal species. Thus, the unique features found for indirect developing sea urchins in regard to bias in the occurrence of defects between the LR halves and to uniformity in the occurrence of defects provided evidence that the confinement of defects to the right halves or the randomness in the occurrence of defects, which have been generally found in the previous studies (Spemann and Falkenberg, 1919; Wilhelmi, 1921; Hörstadius, 1928; Hörstadius, 1973; Henry and Raff, 1990; Henry and Raff, 1994; Nascone and Mercola, 1997; Brown and Wolpert, 1990; Levin, 1997; Ramsdell and Yost, 1998), are not equally conserved among animal species.
Despite the differences described above, the mode of occurrence of LR defects in all of the previous and the present experiments to produce left and right halves, including both separated and conjoined twins, is universal in that the left and right halves have shown different susceptibility, with LR defects consistently biased to occur in only one of the halves. The finding that patterning in only one side of the halves sustained the damage of the disruptions of LR interaction suggests that cell fate determination of the LR patterning of only one side is influenced by the opposite side in the normal whole embryos. Thus, these different animals appear to share a common process in which determination of cell fate on the left or right side is influenced by the other side as a general framework for the mechanism that establishes LR patterning.
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ACKNOWLEDGMENTS |
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REFERENCES |
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