Dual mode of paraxial mesoderm formation during chick gastrulation

Results

To study the establishment of AP identity in the paraxial mesoderm, we first set out to test whether cells produced at the same time by the anterior and posterior parts of the paraxial mesoderm territory in the primitive streak end up in the same somites. We labeled primitive streak cells with both 3,3′-dilinoleyloxacarbocyanine perchlorate (DiO) at the 90% level (see Materials and Methods) and DiI at the 70% level (Fig. 1 a and b) and monitored the position of the descendants of the labeled cells along the AP axis. Descendants of the DiO-labeled cells were found in the medial part of somites, whereas DiI-labeled cells were found in the lateral part of somites (Fig. 1c). Strikingly, however, the anteriormost descendants of DiO-labeled cells were always observed to be located up to two to five somites anterior to DiI-labeled cells (Fig. 1c, n = 12). Such labeled embryos were also analyzed in time-lapse movies [supporting information (SI) Movies 1 and 2]. The analysis of these movies suggests that the difference in the final positioning of labeled cells in the medial and lateral somites essentially reflects the initial AP difference in the primitive streak, and that the path of posterior cells to reach the lateral paraxial mesoderm is longer than that of the anterior streak cells to reach the medial column. To confirm this observation, we used an alternative labeling technique, in which we replaced a fragment from the 90% level of the primitive streak with an EGFP-labeled equivalent piece from a stage-matched donor embryo and performed in the same embryo a second homotopic graft of a DsRed-labeled streak fragment from the 70% to the 80% level (Fig. 1d). This electroporation-based labeling technique made it possible to track the fate of the labeled cells after longer incubation periods than the vital dye-based labeling, in which labeling, especially in the caudal part of an embryo, largely faded out because of rapid dilution of the dye 2 days after incubation. In these embryos, descendants of the EGFP-labeled cells were observed mostly in the medial somites, whereas the descendants of the DsRed-labeled cells were observed in the lateral somites (Fig. 1e). As observed in the DiI- and DiO-labeling experiments, the anteriormost medial derivatives were always found to lie two to five somites anterior to the lateral cells (Fig. 1e, n = 4). The same result was observed when DsRed was used to label anterior streak cells and EGFP to label the posterior streak graft (Fig. 1g, n = 4). Thus, these experiments are consistent with previous fate maps (ref. 12 and refs. therein) and demonstrate that the medial and the lateral parts of a single somite are produced at distinct times by the primitive streak.

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Fig. 1.

Medial and lateral somites are produced at different times by the anterior primitive streak. (a) Schematic representation of the DiI-/DiO-labeling procedure. (b) Stage 4+HH chicken embryo immediately after DiI/DiO labeling showing the position of labeled cells along the primitive streak. (c) Same embryo as in b after 12 h of reincubation. (d) Double-homotopic and -homochronic grafts of different primitive streak levels labeled by electroporation with EGFP and DsRed. (e and f) Embryo grafted at the 90% streak level with an EGFP-labeled fragment and at the 70% streak level with a DsRed-labeled fragment 24 h (e) and 48 h (f) after reincubation. (g and h) Embryo grafted at the 90% streak level with a DsRed-labeled fragment and at the 70% streak level with an EGFP-labeled fragment 24 h (g) and 48 h (h) after reincubation. (i and l) Homotopic and homochronic graft of a fragment of EGFP-electroporated anterior primitive streak at 90% level (i) and at 70% level (l). (j and k) EGFP-labeled descendants of the primitive streak fragment in an embryo grafted at the 90% level of the streak after reincubation times of 24 h (j) and 48 h (k). (m and n) EGFP-labeled descendants of the primitive streak fragment in an embryo grafted at the 70% level of the streak after reincubation times of 24 h (m) and 48 h (n). Ventral views, anterior to the top.

We have investigated further the contribution of different regions of the primitive streak to the different axial levels of the paraxial mesoderm. In double- (Fig. 1d) or single-graft (Fig. 1 i and l) experiments analyzed after 24 h, cells from the 90% level of the streak mostly gave rise to cells in medial somites from the anterior level of the somitic region to the tail bud (SI Table 1; Fig. 1 e, g, and j). In contrast, grafts of more posterior regions of the primitive streak (70–80% level) essentially produced descendants in the lateral part of the somites extending over more posterior levels (SI Table 1; Fig. 1 e, g, and m). As reported (12), however, labeled cells were not always entirely restricted either to the medial or to the lateral somitic compartments, suggesting some intermingling between the two populations. When grafted embryos were incubated for 48 h, derivatives of the 90% streak level continued to contribute to more posterior paraxial mesoderm and tail bud (SI Table 1; Fig. 1 f, h, and k). In contrast, derivatives of grafts from more posterior regions of the streak were not seen to contribute to the posterior part of the embryo (Fig. 1 f, h, and n). Therefore, these experiments support the existence of stem cells contributing to the medial somites located in the anteriormost part of the primitive streak, caudal to the notochord precursors in Hensen's node (6). These stem cells contribute to all axial levels of the paraxial mesoderm. These observations also demonstrate that lateral somite precursors in the anterior streak do not behave as stem cells, indicating that at these stages, the precursors of the posterior lateral somites are not located in the primitive streak.

These experiments raised the question of the origin of cells contributing to the lateral somites in more posterior regions of the AP axis. Available fate maps indicate that until stage 4 Hamburger and Hamilton (HH), the paraxial mesoderm territory is located in the epiblast lateral to the primitive streak and that between stages 4HH and 7HH, somitic cells arise from a region corresponding to the 70–90% level of the primitive streak (12–15). After stage 4HH, ingression of the epiblast territory adjacent to the anterior streak was proposed to be completed (16). Thus, at the beginning of primitive streak regression, the contribution of the epiblast adjacent to the anterior streak was essentially thought to be restricted to the neural plate (17). However, this view is in contrast to studies in the chicken embryo that indicate paraxial mesoderm progenitors can be found in the epiblast adjacent to the anterior streak even after the beginning of streak regression (18, 19). Because the fate of the epiblast adjacent to the streak between stages 4HH and 7HH has been poorly characterized, we have undertaken an extensive fate mapping of this region. To that end, we performed a series of homotopic grafts of EGFP-labeled fragments of different levels of this epiblast (SI Table 2; Fig. 2 a–c and g). Our fate map of the epiblast adjacent to the primitive streak indicates that after the beginning of Hensen's node regression, the anteriormost epiblast that lies adjacent to the node and the anterior primitive streak (90% level) essentially gives rise to neural and ectodermal derivatives (Fig. 2g, dark blue). More posteriorly, extending from the 90–60% level of the primitive streak, a territory essentially contributing to the lateral somites is found (Fig. 2g, red). Posterior to this territory, the epiblast contributes to the lateral plate and extraembryonic mesoderm (Fig. 2g, blue and pink, respectively). To ensure that the results were not biased by our grafting method, we used an alternative labeling strategy in which a small DiI crystal was transiently applied on the epiblast in ovo to label only cells in the superficial layer of the embryo (SI Table 3; Fig. 2 d–f). A similar contribution to the somites was observed from the DiI-labeled epiblast (Fig. 2 e and f). Using both labeling procedures, the contribution of the epiblast to the posterior somites was not exclusively restricted to the lateral compartment and included some medial derivatives, which must, therefore, intermix with derivatives of the anterior primitive streak. Thus, these data indicate that the paraxial mesoderm prof the lateral epiblast mostly contribute to the lateral somites.

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Fig. 2.

Fate mapping of the epiblast. (a–c) Fate mapping of the epiblast using EGFP labeling followed by grafting. Epiblast cells adjacent to the anterior streak were labeled by electroporation with EGFP at stage 5HH (a), grafted into an unlabeled host, and reincubated for 12 h (b). (c) Transverse section at anterior 70% level of the primitive streak level of the embryo shown in a. PS, primitive streak. (d–f) Epiblast fate mapping using DiI labeling in ovo. Epiblast cells adjacent to the anterior 70% of the streak were labeled with a DiI crystal at stage 5HH (d) and reincubated for 20 h (e). (f) Transverse sections at the posterior PSM level of the embryo shown in e. (g) Summary of the results of the epiblast fate mapping. Each square indicates the level of individual epiblast graft, and the colors indicate the fate of the grafted tissue. Dark blue, neural tube; red, paraxial mesoderm; blue, lateral plate mesoderm; pink, extraembryonic mesoderm; green, surface ectoderm.

We then analyzed in more detail the fate of cells of the epiblastic paraxial mesoderm territory in terms of its contribution along the AP body axis. We noticed that grafts of more anterior regions of the somitic domain of the epiblast gave rise to descendants contributing to more anterior regions of the AP axis than grafts of more posterior regions (SI Table 2; compare Fig. 3 a–c and d–f). Therefore, the presumptive territory of the somites in the epiblast is regionalized along the AP axis.

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Fig. 3.

Regionalization of PSM precursor cells in the epiblast. (a–c) Embryo grafted at the 80% streak level before reincubation (a), after 24 h (b), and after 48 h (c). (d–f) Embryo grafted at 60% streak level before reincubation (d), after 24 h (e), and after 48 h (f). Ventral views, anterior to the top.