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An individual retina descends from a restricted and invariant group of nine animal blastomeres at the 32-cell stage. We tested which molecular signaling pathways are responsible for the competence of animal blastomeres to contribute to the retina. Inactivation of activin/Vg1 or fibroblast growth factor (FGF) signaling by expression of dominant-negative receptors does not prevent an animal blastomere from contributing to the retina. However, increasing bone morphogenetic protein (BMP) signaling in the retina-producing blastomeres significantly reduces their contribution. Conversely, reducing BMP signaling by expression of a dominant-negative BMP receptor or Noggin allows other animal blastomeres to contribute to the retina. Thus, the initial step in the retinal lineage is regulated by position within the BMP/Noggin field of epidermal versus neural induction. Vegetal tier blastomeres, in contrast, cannot contribute to the retina even when given access to the appropriate position and signaling fields by transplantation to the dorsal animal pole. We tested whether expression of molecules within the mesoderm inducing (activin, FGF), mesoderm-modifying (Wnt), or neural-inducing (BMP, Noggin) pathways impart a retinal fate on vegetal cell descendants. None of these, several of which induce secondary head structures, caused vegetal cells to contribute to retina. This was true even if the injected blastomeres were transplanted to the dorsal animal pole. Two pathways that specifically induce head tissues also were investigated. The simultaneous blockade of Wnt and BMP signaling, which results in the formation of a complete secondary axis with head and eyes, did not cause the vegetal clone to give rise to retina. However, Cerberus, a secreted protein that also induces an ectopic head with eyes, redirected vegetal progeny into the retina. These experiments indicate that vegetal blastomere incompetence to express a retinal fate is not due to a lack of components of known signaling pathways, but relies on a specific pathway of head induction.
FIG. 1. Thirty-two-cell-stage Xenopus embryo. Left: Animal pole view illustrating the five ipsilateral (right) and four contralateral (left)
blastomeres that contribute descendants to one retina. Center: Animal pole view illustrating the percentage of cells in one retina that
descend from each blastomere (data from Huang and Moody, 1993). Right: Lateral view of the right side showing the position of selected
ventral midline blastomeres used in this study. D1.1.1 is the major retinal progenitor and is the site for V2.1.1 transplantation. D, dorsal;
V, ventral; An, animal pole; Vg, vegetal pole. The nomenclature used is that assigned by Jacobson and Hirose (1981). D1.1.1 is equivalent
to A1; D1.2.1 to A2; V1.2.1 to A3; V1.1.1 to A4; D1.1.2 to B1; D1.2.2 to B2; V2.1.2 to D3; and V2.1.1 to D4 according to the nomenclature
of Nakamura et al. (1978).
FIG. 2. Volumes of control and experimental retinas were measured to indicate whether mRNA injections altered retinal size. Retinal
volume was significantly smaller (P , 0.05) in embryos that were injected with tAR mRNA at the 8-cell stage (D1), but not at the 32-cell
stage (D1.1.1). These results indicate that the effect at the 8-cell stage is due to interference with the early mesoderm inductive signaling.
FIG. 3. Transverse sections through stage 35/36 retinas showing
presence of GFP-labeled clones. (A) GFP mRNA was injected into
blastomere D1.1.1 as a control. GFP cells are distributed throughout
the retina, in a pattern similar to that observed with fluorescent
dextran lineage tracer (Huang and Moody, 1993). (B) Dominantnegative
activin receptor (tAR) and GFP mRNAs were coinjected
into D1.1.1. The affected D1.1.1 progeny populate the retina in a
pattern identical to that in controls. (C) BMP4 and GFP mRNAs
were coinjected into D1.1.1. This photo was overexposed to demonstrate
that the green-labeled clone is completely absent from the
retina. (D) Noggin and GFP mRNAs were coinjected into V1.1.1.
The V1.1.1 progeny are now present in the retina. L, lens vesicle.
Bar 5 50 mm.
FIG. 4. Population fate map comparing the D1.2.2 blastomereâs contribution to tissues in control GFP-injected (gray bars) and
BMPR-injected (black bars) embryos. The percentage of embryos containing D1.2.2 progeny in a particular tissue is plotted. A total of 10
GFP- and 17 BMPR-injected embryos were analyzed. In the BMPR-injected embryos there is a decrease in contribution to anterior and CNS
tissues and an increase in posterior and endodermal derivatives.
FIG. 5. Transverse sections through stage 37 heads in which vegetal blastomeres were injected with tAR. (A) DIC image at level of the
retinas (R) of embryo in which 500 pg tAR mRNA was injected into V2.1.1. The blastomere subsequently was transplanted to the position
of D1.1.1. Arrow points to ventral border of one retina. (B) DICâepifluorescence combined exposure of area in (A) marked by arrow. The
GFP-labeled clone derived from transplanted V2.1.1 (green cells) occupies the branchial arch mesoderm (Ba) just ventral to the retina (R).
(C) tAR mRNA (600 pg) was injected into the vegetal pole of the 8-cell vegetal blastomere (V2). Labeled cells are in the branchial arch
mesoderm (arrows), but not retina (R). (D) tAR mRNA (1 ng) was injected into the ventral vegetal 32-cell blastomere (V2.1.1). Occasionally
labeled cells are in the head mesoderm adjacent to the retina (arrow), but never populate the neural retina (R). (E) tAR mRNA (600 pg) was
injected into the equatorial region of the 8-cell vegetal blastomere (V2). Labeled cells frequently populated the branchial arch mesoderm (Ba)
and ectoderm. (F) tAR mRNA (600 pg) was injected into the equatorial, vegetal cell (V2.1.2) of the 32-cell embryo. Labeled cells frequently
populated the branchial arch mesoderm (Ba) and occasionally populated the retina (R, arrows).
FIG. 6. Population fate map comparing the V2.1.1 blastomereâs contribution to tissues in control GFP-injected (gray bars) and
cerberus-injected (black bars) embryos. The percentage of embryos containing V2.1.1 progeny in a particular tissue is plotted. A total of 42
GFP- and 35 cerberus-injected embryos were analyzed. In the cerberus-injected embryos there is an increase in contribution to anterior
tissues including retina and other CNS derivatives. All but the most anterior tissues (i.e., cement gland, olfactory placode) contain some
V2.1.1 progeny.
FIG. 7. (A) Stage 38/39 embryos showing examples of small secondary axes produced when low levels of Cerberus are expressed by the
V2.1.1 descendents. (B) Transverse section through the lower embryo in (A) at the level of the secondary axis showing an ectopic retina and
lens (L). V2.1.1 descendants populate both the retina and lens. (C) Transverse section through the head of an embryo in which Cerberus
mRNA was injected into V2.1.1, but no secondary axis formed. The V2.1.1 progeny form a small clone in the retina of the primary axis.
Bar 5 50 mm.
