The RH and whole (normal) embryos in which RD or RV was tracer-labeled were designated as W-RD, W-RV, RH-RD and RH-RV, respectively (Fig. 1c,d). The gastrula/ neurula embryos (Fig. 2) or tailbud larvae (DAI 5, Kao & Elinson 1988; Fig. 3a–d) bearing a normal appearance were investigated. Without any tracer injection, these DAI5 larvae developed at 60% or sometimes >90%, and the other morphological traits were for the most part a small head (DAI 2–4) or incomplete invagination, while larvae exhibiting a large head (DAI > 5) or no head (DAI 0, 1) were comparatively rare.
Each of the right blastomere clones populates the ectoderm on both sides, with similar dorsoventral and anteroposterior patterns to normal embryos
In the ectoderm of regulated late gastrula/early neurula embryos, the RD clone was distributed in the dorsal embryonic regions, which comprised the head epidermis and a longitudinal tissue region in the neural plate, most of which are positioned on the right side, but which also extend to the left side, with wider areas on the anterior side (Fig. 2a,c). Complementarily, the RV clone occupied the ventral/lateral regions on both sides, which are composed of the entire epidermis, excluding the head area, and also the neural plate regions along the left and right margins, with wider areas on the left and posterior sides (Fig. 2e,g). These results indicate that during the early cleavage stages, the prospectively most ventral RV descendants had been juxtaposed on the left side of the prospectively most dorsal RD descendants.
In normal embryos, most of the RD and RV clones were confined to the right side. The RD clone occupied the dorsal regions, the head epidermis and the entire neural plate excluding the outer margin in the tail, on the right side, with wider areas on the anterior side (Fig. 2b,d). The RV clone was distributed in the ventral/lateral regions on the right side, which are complementary to the RD clone regions, and also in a narrow left area of the ventral epidermis (Fig. 2f,h).
Thus, in regulated embryos, both the RD and RV clones were distributed on both sides, differing from normal embryos, with wider areas on the right and left sides, respectively. However, both clones exhibited distribution patterns similar to normal embryos along the dorsoventral and anteroposterior axes: the RD clone populated wider areas on the dorsal and anterior sides, while the RV clone occupied wider areas on the ventral and posterior sides. The observed distribution of the clones does not fit either of the two predictable most extreme blastomere fate shift modes, i.e. that the RD progeny contribute to the entire dorsal half of the embryo while the RV progeny form the ventral half if the dorsal endomesodermal tissue formation is controlled by the cell-autonomous action of dorsal and vegetally localized determinants, or that RD becomes the right half of the embryo while RV gives rise to the left half if the dorsal endomesoderm on the left is induced by the Nieuwkoop center on the right (Nieuwkoop 1969a,b; Nieuwkoop & Ubbels 1972; Gimlich & Gerhart 1984).
The anterior part of the dorsal endomesoderm exclusively originates from the dorsal blastomere
The distribution of the clones in the transverse histological sections of the tailbud stage larvae (Figs 3, 4) was quantitatively measured and reconstructed three-dimensionally (Fig. 5). This enabled comparison along the dorsoventral, anteroposterior and left-right axes between the larva types. To make it easier to follow the results, we provide only an overview of the clonal distribution in the larvae in the text, and the detailed distribution patterns in each tissue are summarized in Table S1, with figures representing the tissue and the examined sample number. The blastomere fate in regulated larvae was reproducible, but had a slightly larger variability than in normal larvae, as indicated by the standard errors for the measured progeny percentages in the grids (Table S2).
In regulated larvae, it is noteworthy that the RD progeny exclusively populated only the endoderm anterior to the first visceral pouch (Figs 4a, 5b), the most ventral endoderm region at the first visceral pouch level (Fig. 5c), the liver (Figs 4c, 5e) and the anterior tip of the notochord (Fig. 5c), which are AE derivatives (see Hausen & Riebesell 1991). This was the most anterior appearance of the distribution pattern of the RD clone: most of the RD progeny were distributed in tissue areas along the dorsal midline in the three germ layers, with wider areas and higher percentages on the anterior compared to the posterior side (Figs 4a–f, 5a–k and Table S1). This was closely similar to the distribution pattern in normal larvae along the dorsoventral and anteroposterior axes. They were located on both sides, with wider areas and higher percentages on the right side than the left, differing from normal larvae, where they were confined to the right side or midline tissues. The above distribution pattern was observed in the endoderm, somites, lateral plate, central nervous system (CNS) and epidermis. In the notochord and hypochord, this distribution pattern was observed only anteroposteriorly. Bilateral labeling throughout the tissues along the anteroposterior axis was seen in the notochord, hypochord, the areas in both somites adjoining the notochord and the ventral part of the CNS, but in the notochord and hypochord, difference of the labeling percentages was not detected between both sides at most of the levels. These tissues are the major parts of the dorsal axial structures, and the first three contain derivatives of the GRP, i.e. the ventral part of the notochord, the hypochord, and the medial ventral cells of the somites (Shook et al. 2004; see the legend for Fig. 7e,f). The labeled areas in both somites adjoining the notochord were broader in the dorsal direction than the region into which superficial cells had ingressed (Fig. 4b–f, Fig. 4 in Shook et al. 2004). Although the left area was narrower and had a smaller cell mass than the right area at each level, the labeled areas were found in all nine larvae examined throughout or almost throughout the tissue. Labeling throughout the tissues was also seen on the right side, in the dorsal endoderm underlying the hypochord, the dorsal part of the CNS and the dorsal epidermis. Bilateral labeling was detected also in the head mesenchyme and the ventral part of the body, which consisted of the head and trunk endoderm, the anterior portion of the lateral plate and the heart anlage.
In normal larvae, high RD-percentages were detected on the right side in all the AE derivatives described above, which are the endoderm areas anterior to the first visceral pouch (Fig. 5b′), the most ventral endoderm region at the first visceral pouch level (Fig. 5c′), the liver (Figs 4o, 5e′) and the anterior notochord tip (Fig. 5c′). The RD progeny were also present to a certain extent on the left. Most of the RD descendants were distributed on the right side along the dorsal midline in the three germ layers (Figs 4m–r, 5a′–k′ and Table S1). The further along the anterior side we observed, the wider the RD progeny distribution and the higher the percentages. Furthermore, bilateral labeling was observed throughout the notochord and hypochord (Figs 4m–r, 5c′–k′), as well as the ventral areas of the forebrain and midbrain, and in both retinas. Labeling throughout the tissues was seen on the right side in the dorsal region of the endoderm, the area in the somite flanking the notochord (Figs 4n–r, 5e′–k′) and the ventral region of the CNS. The breadth of the labeled right somite area was similar to the right counterpart area in regulated larvae. Thus, for the GRP derivatives, bilateral labeling was seen in the hypochord and the ventral region of the notochord, while high percentages of right side labeling and almost no left side labeling were seen in the medial regions of the somites. Relatively high percentages of right side labeling were observed in the head mesenchyme, and the ventral part of the larvae which consisted of the liver, anterior endoderm, anterior lateral plate and heart anlage.
The ventral blastomere progeny populate most of the ventral and posterior tissues derived from all of the germ layers
In regulated larvae, no RV progeny were observed in the endoderm anterior to the first visceral pouch (Figs 4g, 5m), the most ventral endoderm region at the first visceral pouch level (Fig. 5n), the liver (Figs 4i, 5p) or the anterior notochord tip (Fig. 5n). The RV clonal distribution was complementary to that of RD in each tissue. Most of the RV progeny were distributed in areas other than those along the dorsal midline, with wider areas and higher percentages in the posterior compared to the anterior regions, as well as in normal larvae, on the left side than the right, differing from normal larvae (Figs 4g–l, 5l–v and Table S1). This distribution pattern was seen in the somites, CNS and epidermis, in areas in which labeling on both sides throughout the tissues was observed, as well as in the endoderm and lateral plate, in which the labeling throughout the tissues was not observed. Considerable percentages of the RV progeny were detected throughout the tissues at the dorsal midline, consisting of the notochord excluding the anterior tip, and the hypochord (Figs 5o–u, 6a–c), which contain derivatives from the medial region of the GRP (Shook et al. 2004), unlike normal larvae. In the medial regions of the somites adjoining the notochord, to the ventral regions of which the GRP-derived cells contribute (Shook et al. 2004), the RV percentages throughout the tissue were higher on the left side of regulated larvae than on the right side of both regulated and normal larvae. In the head mesenchyme, some RV progeny were observed (Fig. 5l–o), and these were the RV progeny that had been derived from the margins of the neural plate (Fig. 2e,g).
Figure 6. Distribution of the right ventral blastomere progeny in the notochord and hypochord of regulated and normal larvae. (a–d) The labeled progeny distribution in RH-RV. In the notochord, the RV descendants are distributed in the ventral (a, level 9), right (b, level 6), center and left (c, level 6) regions in three independent larvae, but are not seen in another larva (d, level 6). In the hypochord, a descendant cell is seen in (a) and (b), but not in (c) and (d). (e, f) The labeled progeny distribution in an identical W-RV. In the notochord, no RV descendent is seen in either section. In the hypochord, a descendant cell is seen at level 8 (e), but not at level 7 (f). The abbreviations and orientations of the larvae are the same as in Figures 1, 2, and 4, respectively.
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In normal larvae, it is noteworthy that no RV progeny were detected in the endoderm at the level of the first visceral pouch (Fig. 5n′) and further to the anterior (Figs 4s, 5m′), the liver (Figs 4u, 5p′) or the notochord (Figs 4t–x, 5n′–u′, 6e,f). The RV clone for the most part occupied the major regions of the ventral tissues and also the posterior regions of some of the dorsal tissues derived from all the germ layers on the right side (Figs 4s–x, 5l′–v′ and Table S1). These regions consist of the tissue areas other than the RD progeny-rich areas located along the dorsal midline. In tissues derived from all three germ layers, the further along the posterior side we observed, the wider the distribution areas and the higher the clone percentages. This distribution pattern was seen not only in the somite, pronephric anlage, lateral plate, CNS and epidermis, in which labeling throughout the tissues was observed, but also in the endoderm, in which this labeling was not observed. In the hypochord at the trunk levels, the RV cells were observed at very low percentages (Figs 5q′,s′,t′, 6e). Thus, the GRP derivatives had certain percentages of RV progeny in the medial region of the right somite, but with very low to no percentages in the hypochord, the ventral part of the notochord and the medial region of the left somite.
The shift in the clonal distribution patterns in the tailbud larvae coincided well with that in the late gastrula/early neurula embryos, suggesting that most of the regulation had been achieved by the late gastrula stage.