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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). |
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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. |
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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). |
