Domingo C and Keller R (2000), Cells remain competent to respond to mesoderm-i...

XB-ART-10419

Dev Biol 2000 Sep 01;2251:226-40. doi: 10.1006/dbio.2000.9769.

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Cells remain competent to respond to mesoderm-inducing signals present during gastrulation in Xenopus laevis.

Domingo C , Keller R .

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During gastrulation, the vertebrate embryo is patterned and shaped by complex signaling pathways and morphogenetic movements. One of the first regions defined during gastrulation is the prospective notochord, which exhibits specific cell behaviors that drive the extension of the embryonic axis. To examine the signals involved in notochord formation in Xenopus laevis and the competence of cells to respond to these signals, we performed cell transplantation experiments during gastrulation. Labeled cells from the prospective notochord, somitic mesoderm, ventrolateral mesoderm, neural ectoderm, and epidermis, between stages 9 (pregastrulation) and 12 (late gastrulation), were grafted into the prospective notochord region of the early gastrula. We show that cells from each region are competent to respond to notochord-inducing signals and differentiate into notochordal tissue. Cells from the prospective neural ectoderm are the most responsive to notochord-inducing signals, whereas cells from the ventrolateral and epidermal regions are the least responsive. We show that at the end of gastrulation, while transplanted cells lose their competence to form notochord, they remain competent to form somites. These results demonstrate that at the end of gastrulation cell fates are not restricted within germ layers. To determine whether notochord-inducing signals are present throughout gastrulation, grafts were made into progressively older host embryos. We found that regardless of the age of the host, grafted cells from each region give rise to notochordal tissue. This indicates that notochord-inducing signals are present throughout gastrulation and that these signals overlap with somite-inducing signals at the end of gastrulation. We conclude that it is the change of competence that restricts cells to specific tissues rather than the regulation of the inducing signals.

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	FIG. 1. Fate maps of the interior show the prospective tissues for the pregastrula (stage 9), early gastrula (stage 10), mid gastrula (stage 11), and late gastrula (stage 12). These fate maps and nomenclature are based on Keller (1975, 1976, 1991). The prospective areas include EP, epidermis; NE, neural ectoderm; N, notochord; SM, somitic mesoderm; VLM, ventrolateral mesoderm; DMZ dorsal marginal zone; and LEM, leading edge mesoderm.
	FIG. 2. Experimental design. (A) Embryos were injected with either fluorescein or rhodamine dextran at the single cell stage. At stages 9 through 12, approximately 30 cells were grafted from the deep cell layers of the N, SM, VLM, NE, and EP to the prospective notochord (N) region of unlabeled, stage 10 embryos. (B) Approximately 30 cells were grafted from fluorescently labeled embryos at stage 10 to the prospective notochord region of embryos at stages 10 through 12. (C) A piece of tissue was dissected from the SM, VLM, NE, and EP regions of rhodaminelabeled gastrulae (stage 10). Small clumps of about 30 cells were then grafted either to the same region they came from (homotopic graft) or to the prospective notochord region (heterotopic graft) of host gastrulae. Five homotopic and five heterotopic grafts were performed using the same piece of donor tissue. In all cases, the grafted embryos developed to tailbud (stages 22 to 28) and the fate of the grafted cells was determined.
	FIG. 3. Distribution of labeled cells grafted from the prospective notochord. Cells were grafted from a stage 10 donor into a stage 10 host. (A) A cleared whole-mount embryo (stage 27) with labeled cells scattered throughout the notochord. (B) A longitudinal section of a grafted embryo, showing that the grafted cells differentiated into notochordal tissue as indicated by the bipolar, elongated cell shapes and the formation of vacuoles (arrow). Scale bars, 100 mm.
	FIG. 4. Distribution of labeled cells grafted from the somitic mesoderm. (A) Cells were grafted from a stage 10 donor into a stage 10 host. A cleared whole-mount embryo shows that the grafted cells differentiated into notochord (arrow) and somitic (arrowhead) tissues. (B) A longitudinal section of a grafted embryo from the same experiment, showing that the grafted cells differentiated into notochordal tissue as shown by the presence of vacuoles (arrow). A couple of labeled cells are also found within the notochordalâsomitic boundary (arrowhead). (C) Cells were grafted from a stage 11 donor to a stage 10 host. A cleared whole-mount embryo shows that the labeled cells have differentiated into notochordal (arrow) and somitic (arrowhead) tissues in about equal proportions. (D) Cells were grafted from a stage 10 donor to a stage 11 host. A cleared whole-mount embryo shows labeled cells that have differentiated into notochordal tissue (arrow) while other labeled cells are positioned ventrally to the notochord, within the notochordalâsomitic boundary (arrowhead). (E) Cells were grafted from a stage 10 donor to the somitic region of a stage 10 host. A cleared whole-mount embryo shows that labeled cells have differentiated into somitic tissue. Scale bars, 100 mm.
	FIG. 5. Distribution of labeled cells grafted from the ventrolateral mesoderm. (A) Cells were grafted from a stage 10 donor to a stage 10 host. A cleared whole-mount embryo shows labeled cells both in the notochord (arrow) and ventral to the notochord (arrowhead). (B) This embryo was longitudinally sectioned and the labeled cells were found in the notochord showing the typical bipolar elongated cell shape (arrow). Cells excluded from the notochord did not show the bipolar cell shape (arrowhead). (C) Cells were grafted from a stage 10 donor to a stage 11 host. A transverse section shows the labeled cells within the notochord (arrow) and also a labeled cell ventral to the notochord (arrowhead). (D) Cells were grafted from a stage 10 donor to the VLM region of a stage 10 host. A cleared whole-mount embryo shows groups of labeled cells that are likely blood islands.
	FIG. 6. Distribution of labeled cells grafted from the neural ectoderm. (A) Cells were grafted from a stage 10 donor to a stage 10 host. A cleared whole-mount embryo shows labeled cells in the notochord with vacuoles clearly visible (arrow). In addition, labeled cells are present both dorsal and ventral to the notochord (arrowheads). (B) A sectioned embryo from this experiment shows labeled cells that are elongated and vacuolated in the notochord (arrow). Ventral to the notochord are labeled cells that are more rounded and lack vacuoles (arrowhead). (C) Cells were grafted from a stage 12 donor to a stage 10 host. A cleared whole-mount embryo shows labeled cells in the floor plate of the neural tube. (D) A transverse section shows that the labeled cells are located to the floor plate of the neural tube (arrow). (E) Cells were grafted from a stage 10 donor to the neural ectoderm of a stage 10 host. A cleared whole-mount embryo shows labeled cells in the neural tube. The arrow points at the axonal projections extending from the labeled cells in the neural tube.
	FIG. 7. The distribution of labeled cells grafted from the prospective epidermis. (A) Cells were grafted from a stage 10 donor to a stage 10 host. A cleared whole-mount embryo shows labeled cells within the notochord showing the stack of coins array (arrow). Some labeled cells are also present ventral to the notochord and dorsal to the notochord (arrowheads). (B) Transverse section through an embryo from this experiment shows labeled cells both in the notochord (arrow) and in the floor plate of the neural tube (arrowhead). (C) Cells were grafted from a stage 11 donor to a stage 10 host. A cleared whole-mount embryo shows a few labeled cells present in the notochord (arrow) and several cells ventral to the notochord. (D) A transverse section shows labeled cells positioned ventral to the notochord within the matrix-filled region surrounding the notochord and in the hypochord. (E) Cells were grafted from a stage 10 donor to the prospective epidermal region of a stage 10 host. A cleared whole-mount embryo shows labeled cells in the epidermis.
	FIG. 8. Stage-dependent competence to form notochord and somites. (A) A line graph shows the percentage of cases in which grafted cells gave rise to notochord cells. The y axis represents the percentage of cases in which grafted cells differentiated into notochord cells. The x axis represents the age of the donor embryos. (B) A line graph shows the percentage of cases in which grafted cells gave rise to somitic cells. The y axis represents the percentage of cases in which grafted cells differentiated into somitic cells. The x axis represents the age of the donor embryos. EP, epidermis; NE, neural ectoderm; N, notochord; SM, somitic mesoderm; VLM, ventrolateral mesoderm.
	FIG. 9. Notochord-inducing signals persist through gastrula stages and overlap with somite-inducing signals throughout gastrulation. (A) A line graph shows the percentage of cases in which grafted cells gave rise to notochord cells. The y axis represents the percentage of cases in which grafted cells differentiated into notochord cells. The x axis represents the age of the host embryos. (B) A line graph shows the percentage of cases in which grafted cells gave rise to somitic cells. The y axis represents the age of the host embryos. EP, epidermis; NE, neural ectoderm; N, notochord; SM, somitic mesoderm; VLM, ventrolateral mesoderm.