May 1, 2007;
The competence of Xenopus blastomeres to produce neural and retinal progeny is repressed by two endo-mesoderm promoting pathways.
Only a subset of cleavage stage
blastomeres in the Xenopus embryo
is competent to contribute cells to the retina
vegetal blastomeres do not form retina
even when provided with neuralizing factors or transplanted to the most retinogenic position of the embryo
. These results suggest that endogenous maternal factors in the vegetal region repress the ability of blastomeres to form retina
. Herein we provide three lines of evidence that two vegetal-enriched maternal factors (VegT
), which are known to promote endo-mesodermal fates, negatively regulate which cells are competent to express anterior
neural and retinal fates. First, both molecules can repress the ability of dorsal-animal retinogenic blastomeres to form retina
, converting the lineage from neural/retinal to non-neural ectodermal and endo-mesodermal fates. Second, reducing the endogenous levels of either factor in dorsal-animal retinogenic blastomeres expands expression of neural/retinal genes and enlarges the retina
. The dorsal-animal repression of neural/retinal fates by VegT
is likely mediated by Sox17alpha
but not by XNr1
likely exert their effects on neural/retinal fates through at least partially independent pathways because Notch1
can reverse the effects of VegT
but not those of Vg1
. Third, reduction of endogenous VegT
vegetal blastomeres can induce a neural fate, but only allows expression of a retinal fate when both BMP and Wnt signaling pathways are concomitantly repressed.
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References [+] :
Fig. 5. Reduction of VegT and Vg1 in the D1.1 lineage increases the size of the neural ectoderm. (A) Reduction of VegT by morpholino injection (VegTMO) and of Vg1 by expression of a dominant-negative construct (dnVg1) enlarged the retina (r) and forebrain (fb). Note the abundant D1.1 progeny (green) in both structures (cf. Fig. 2E). Reduction of both factors (VegTMO/dnVg1) resulted in a similar phenotype. (B) The mean volumes of retinas in embryos in which control morpholinos (cMO, cVegTMO) were injected are not different from GFP controls (Fig. 2B). Those from VegTMO-injected embryos are significantly larger on both sides, and those from dnVg1-injected embryos are significantly larger on the injected side. Those from embryos injected with both constructs (VegTMO/dnVg1) are larger than for dnVg1 alone and similar to those from VegTMO alone. * Indicates p < 0.01 compared to GFP controls. Numbers in parentheses indicate size of sample. (C) The expression domains of pan-neural plate (sox3, notch1; white bars indicate measurement of width of domain) and retinal (rx1; arrows) genes are expanded on the side (right) injected with VegTMO or dnVg1. The expansion of neural plate markers is somewhat enhanced in embryos co-injected with VegTMO and dnVg1 (see also Table 1). (D) The expression domains of mesodermal (Xbra) and endodermal (sox17α, edd) genes after injection into D1.1 of the constructs indicated on the left. The only notable effect is repression of Xbra by dnVg1 (arrow). (E) The expansion of rx1 expression (arrow) by VegTMO is not altered by co-expression of Vg1, whereas the expansion caused by dnVg1 is reversed by co-expression of VegT. (F) The small retinal volumes displayed by Vg1-injected embryos were significantly increased by co-injection of VegTMO (* indicates p < 0.01). The small retinal volumes displayed by VegTMO-injected embryos were not altered by co-injection of dnVg1 (p > 0.05).
Fig. 6. The combination of sox17α and derriere phenocopies the VegT effect on the D1.1 lineage. (A) The retinas (r) in sox17α and Xnr1 mRNA injected embryos are normal in size, whereas derriere dramatically reduced both retinas. However, D1.1 progeny (green) were not located within the retina in sox17α, derriere or Xnr1 injected embryos. Co-expression of notch1 reversed the derriere phenotype, resulting in large retinas populated by abundant D1.1 progeny (green), but did not alter the Xnr1 phenotype. The expression of a dominant-negative derriere construct (Cm-derriere) resulted in a larger retina populated by large numbers of D1.1 progeny. (B) The mean volume of retinas in embryos injected with mRNAs for sox17α, derriere (Der), derriere plus Notch1 (Der/N), dominant-negative derriere (Cm-D), Xnr1 and Xnr1 plus notch1 (Xnr1/N). sox17α- and Xnr1-injected embryos do not significantly differ from GFP controls (Fig. 2B), whereas those from derriere embryos are significantly reduced on both sides. Co-expression of Notch1 partially rescues the derriere effect but has no effect on Xnr1 retinas (*, p < 0.01 compared to GFP controls; **, p < 0.05 compared to derriere). Cm-D significantly increased the size of both retinas (*, p < 0.01 compared to GFP controls). (C) Percentage of embryos in which D1.1 progeny populate the retina is dramatically reduced in embryos injected with sox17α, derriere (Der) or Xnr1 mRNAs. The derriere phenotype is partially rescued by co-expression of notch1 (Der/N), whereas the Xnr1 phenotype is not (Xnr1/N). All Cm-D expressing embryos have D1.1 progeny in the retinas, identical to GFP controls (Fig. 2C). (D) Domains of pan-neural plate (sox3, notch1), retinal (rx1), mesodermal (Xbra), and endodermal (sox17α, edd) genes after injection of one D1.1 blastomere with constructs listed on the left. The injected side of the embryo is on the right and the uninjected side is on the left. The sites of effects are indicated either by arrows, or in the neural plate by white bars indicating the width of the expression domains. Expression of sox17α does not affect neural/retinal genes, but inhibits Xbra and expands edd, consistent with its role in converting cells to an endodermal fate. Expression of derriere represses neural and retinal genes, and these effects are rescued by notch1. Cm-Derriere expanded neural and retinal markers and repressed Xbra. Xnr1 either represses (sox3, top rx1 panel) or expands (notch1; bottom rx1 panel) neural/retinal genes, and endo-mesoderm markers are dorsally expanded. No Xnr1 phenotype is rescued by notch1. (E) The expansion of neural (sox3, notch1) and retinal (rx1) expression domains by injection of VegTMO (Fig. 2C) is reversed by co-injection of derriere. Reduction of these markers by VegT (Fig. 2B) is reversed by cm-derriere (see also Table 1).
Fig. 7. In order to convert the V2.1.1 lineage to a retinal fate endo-mesodermal factors must be blocked in combination with suppression of BMP and Wnt signaling. (A) Expression domains of mesodermal (Xbra) and endodermal (sox17α, edd) genes are reduced by VegTMO injection into the vegetal pole, whereas controls (β-gal, cMO, cVegTMO) have no effects. (B) The extent to which the hindgut (g) is populated by V2.1.1 progeny (green) is strikingly reduced in VegTMO embryos (*) compared to controls (GFP). a, archenteron; s, somite. (C) Ectopic expression of rx1 (blue, arrows) in the vegetal pole was monitored by in situ hybridization after the V2.1.1 blastomere was injected with the indicated constructs. Red cells indicate the V2.1.1 progeny expressing those constructs. Reduction of VegT alone (VegTMO) or in combination with Vg1 (VegTMO/dnVg1) was not sufficient to induce ectopic rx1 expression. The long form of Cerberus (Cer-L), which can inhibit BMP, Wnt and Nodal signaling, was effective whereas the short version of Cerberus (Cer-S), which can inhibit only Nodal signaling, was not. Although reduction of both BMP signaling (by expression of noggin [Nog] and a dominant-negative BMP receptor [tBR]) and Wnt signaling (by expression of a dominant-negative Wnt8 construct [dnWnt]) causes ectopic rx1 expression, the cells expressing rx1 are not derived from the V2.1.1 clone (red cells), demonstrating an indirect consequence of the induction of a secondary head (as reported in Moore and Moody, 1999). However, combining the reduction of VegT or VegT plus Vg1 with the inhibition of BMP and Wnt signaling caused the V2.1.1 clone to ectopically express rx1, equivalent to the Cer-L phenotype.
Fig. 3. VegT and Vg1 alter cell fates at gastrulation stages and these changes affect later neural patterning. (A) The expression domains of markers of the germ layers (top labels) during gastrulation after injection of one D1.1 blastomere with mRNAs indicated on the left. Early neural ectoderm is identified by foxD5 and otx2, non-neural ectoderm by keratin, mesoderm by Xbra and endoderm by sox17α and edd. Red cells indicate the D1.1 progeny expressing the injected mRNA, and arrows indicate regions of gene repression or ectopic expression. Frequencies of phenotypes are presented in Table 1. (B) The expression domains of pan-neural (sox3, notch1), eye field (rx1), forebrain (otx2), midbrain (en2) and hindbrain (krox20) genes during neural plate stages. White bars indicate the width of the expression domains on the injected (right) versus uninjected (left) side of the neural plate. Arrows are as above. Frequencies of phenotypes are presented in Table 1.
Agius, Endodermal Nodal-related signals and mesoderm induction in Xenopus. 2000, Pubmed