XB-ART-8263J Neurobiol November 5, 2001; 49 (2): 129-41.
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Cellular competence plays a role in photoreceptor differentiation in the developing Xenopus retina.
Factors in the environment appear to be responsible for inducing many of the cell fates in the retina, including, for example, photoreceptors. Further, there is a conserved order of histogenesis in the vertebrate retina, suggesting that a temporal mechanism interacts in the control of cellular determination. The temporal mechanism involved could result from different inducing signals being released at different times. Alternatively, the inducing signals might be present at many stages, but an autonomous clock could regulate the competence of cells to respond to them. To differentiate between these mechanisms, cells from young embryonic retinas were dissociated and grown together with those from older embryos, and the timing of photoreceptor determination assayed. Young cells appeared uninfluenced by older cells, expressing photoreceptor markers on the same time schedule as when cultured alone. A similar result was obtained when the heterochronic mixing was done in vivo by grafting a small plug of optic vesicle from younger embryos into older hosts. Even the graft cells at the immediate margin of the transplant failed to express photoreceptor markers earlier than normal, despite their being in contact with older, strongly expressing host cells. We conclude that retinal progenitors intrinsically acquire the ability to respond to photoreceptor-inducing cues by a mechanism that runs on a cell autonomous schedule, and that the conserved order of histogenesis is based in part on this competence clock.
PubMed ID: 11598920
Article link: J Neurobiol
Species referenced: Xenopus laevis
Genes referenced: bag3 clock hspa5
Antibodies: Hspa5 Ab1 Photoreceptors Ab1
Article Images: [+] show captions
|Figure 1 Photomicrographs of dissociated, cultured retinal cells. The color scheme is as follows: blue: bis-benzamide, stains all cell nuclei; red: dextran-TRITC filled cells, marks the population of cells from the earlier injected embryo; green: indicates XAP antibody stained cells. Cells which are red and blue stained indicate one population of dissociated cells, either young or old (different for different cases), and those which are blue stained only make up the other. Panels (A) and (B) illustrate a case in which the optic vesicles from a Stage 21 embryo (blue only cells) and those from a dextran injected, Stage 24 embryo [red cell in (A)] were dissociated, mixed, plated, and 24 h later fixed and processed for XAP-1 immunoreactivity. The older cell is XAP-1 positive [green cell in (B)], while neither of the younger cells (blue only) expresses XAP-1, even the cell at the far left that is directly contacting an older cell. Panel (C) illustrates the results of an experiment in which optic vesicles from a dextran injected, Stage 26 embryo (red cells) were dissociated and mixed with cells (blue only) from a Stage 30 optic vesicle. Twenty-four hours later the culture was fixed and immunostained with anti-XAP-2 (green cell). Because, in this case, it is the younger cells which are dextran labeled, separate panels are not needed to illustrate the data. By this stage some of both the younger and older cells have elaborated a phenotype typical of cultured photoreceptors (arrowed cells). The older one of these (horizontal arrow, not red stained) is XAP-2 positive, while the younger one (vertical arrow, red cell) is not. Indeed, the younger photoreceptor failed to express XAP-2 despite being surrounded by several older cells, and contacted by at least one. Scale bars: (A,B) 5 10 mm; (C) 5 5 mm.|
|Figure 2 Histograms illustrating the proportion of cultured embryonic retinal cells that express antigens [XAP-1 (A) and XAP-2 (B)] at various developmental stages and when cultured alone (open bars) or when mixed with older cells (black bars). XAP-1 is expressed earlier than XAP-2. Both XAP-1 and -2 immunoreactivity are gradually up-regulated over approximately 6 h. Note that at the age at which the older cells of the heterochronic mix are taken, XAP-1 and -2 are expressed at high levels. Nevertheless, there is no significant difference in the proportion of younger cells in such a mix that express XAP-1 or -2 compared to the isochronic control.|
|Figure 3 Photomicrographs of sections through Xenopus retinas that received transplantation of presumptive retinal tissue from dextran-fluorescein (FITC) labeled embryos of the same age [isochronic (A,B)], or from an earlier age [heterochronic (C,D)]. Embryos were allowed to survive to the stages of initial expression of photoreceptor antigens XAP-1 (A,C) or XAP-2 (B,D). This is apparent because all cases demonstrated a spatial gradient of XAP antigen expression: strong in the central retina and gradually lost at the margins. Thus, at the age of sacrifice, XAP expression was at an early stage, and it was important that transplants were made in central retina. Note that the XAP-1 stained retinas are as yet undifferentiated with no obvious cytoarchitectural lamination. In addition, XAP-1 is expressed in the skin as well as the photoreceptors. By the time XAP-2 begins to be expressed the inner and outer plexiform layers of the retina are apparent. This antigen, specific to cone photoreceptors, exhibits a lower density of punctate staining. In isochronic transplants, XAP-1 and -2 staining extends across the grafted portion of retina. In contrast, the graft of a heterochronic transplant fails to express XAP-1 or -2 at any level in any region until the graft itself reaches the stage of normal antigen expression. Scale bar: 50 mm.|
|Figure 4 Photomicrographs of sections through two Stage 41 Xenopus retinas, each of which received a transplant of optic vesicle tissue from a younger, dextran-FITC labeled embryo. In both instances the host embryo was at Stage 28, and the donor at Stage 24 at the time of transplant. The host embryos were allowed to grow to a stage when the retina was mature, that is, when both graft and host tissue were at Stage 41. The sections are counterstained with bis-benzimide, which demonstrates that the retinal cytoarchitecture develops normally following the transplant surgery. The graft survives and is apparent as a wedge of green labeled cells well integrated into the host retina and with normal cytoarchitecture. Finally, the sections are immunostained with XAP-1 (A) and XAP-2 (B) antibodies (red), which are normally expressed, in both cases, in the host and the graft. Thus, the younger graft of a heterochronic transplant is seen to eventually express XAP-1 and -2, but to do so on its own schedule. Scale bar: 50 mm.|
|Figure 5 High power photomicrographs of sections through Xenopus retinas that received heterochronic transplants of presumptive retinal tissue from dextran-FITC labeled embryos. Embryos were allowed to survive to the stages of initial expression of photoreceptor antigens XAP-1 (A) or XAP-2 (B). Note that both XAP-1 and -2 immunoreactivity extends to the border of the transplant, but not at all into it. Even transplant cells that are in immediate contact with older host cells fail to express XAP-1 or -2 to any extent. Scale bar: 25 mm.|