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We have identified Xenopus MADM-like (xMADML), a Xenopus laevis gene related to the murine MADM and the human NRBP genes. xMADML is expressed throughout early development and is expressed most strongly in the developing lens and more weakly in the retina and other anterior tissues. We demonstrate that disruption of xMADML translation by means of morpholino injection results in impaired retina and lens development. Reciprocal transplantation of the presumptive lensectoderm between morpholino-injected embryos and those injected solely with a dextran lineage tracer demonstrates that xMADML is necessary in both the lens and the retina for correct development of these eye tissues. Analysis of gene expression after knockdown of xMADML revealed significant alterations in the expression of some genes, including Pax6, xSix3, Sox2, and Sox3, suggesting that xMADML plays a role in regulating gene expression during development of the eye. This investigation is the first in vivo study examining the developmental role of this novel gene and reveals an important role of xMADML in eyetissue development and differentiation.
Figure 2. Xenopus MADM-like (xMADML) expression. A-F: Embryonic expression of xMADML by in situ hybridization. Anterior is to the right in all cases. White arrowheads denote the rightlateral extent of anterior staining, and white arrows indicate eye and lens placode staining. A is a dorsal view, dorsal is up in B-F. A: Stage 14: faint xMADML expression visible in the anterior neural plate. The black arrowhead indicates the blastopore, and the dotted line indicates the dorsal midline of the embryo. B: Stage 19: faint xMADML expression visible in a region of the anterior neural plate and adjacent headectoderm. C-E: Stages 25, 32, and 35, respectively: revealing expression in the presumptive lensectoderm (PLE), lens, retina, and surrounding areas. F: Stage 35 sense control showing no staining. G: Section of eye of animal shown in E. H: RT-PCR analysis of xMADML expression in stages as noted. Lane 1 is the 1-kb DNA ladder, lane 2 is a negative control, and lane 3 is positive control plasmid. The white arrow indicates the expected 1,513-bp amplified product, based on the full-length cDNA coding sequence recovered. The white arrowheads indicate two alternative splice forms (see text). ce, corneal epithelium; le, lens; nr, neural retina. Scale bar = 400 mu m in A-F, 100 mu m in G.
Figure 4. Morpholino phenotype. Stage 35 animals developing from embryos injected unilaterally at the two-cell stage with 18 ng of indicated morpholino (MO). Each row shows one embryo in whole-mount in both white light and red morpholino fluorescence and the affected eye in section in transmitted light. For panels in the first two columns, anterior is to the right and dorsal is up. Arrows indicate the location of eye. For the panels in the third column, dorsal is to the right. A–C: Uninjected control animal. D–F:Xenopus MADM-like (xMADML) -MO injected animal. Mild phenotype showing reduced development of ventralretina and poorly differentiated, misorganized fiber cells in the lens. G–I: xMADML-MO injected animal. Severe phenotype showing retina and lens with severely impaired gross morphology. J–L: Control morpholino (CTL-MO) -injected animal exhibiting normal morphology. ce, corneal epithelium; le, lens; nr, neural retina. Scale bar = 400 μm in the first two columns, 100 μm in the third column.Download figure to PowerPoint
Figure 5. Specificity of Xenopus MADM-like (xMADML) morpholino (MO). Stage 35 animals developing from embryos injected unilaterally at the two-cell stage with 18 ng of indicated morpholino. A,B,D–H: Anterior is right and dorsal is up. Arrows indicate the location of eye. C,I: Dorsal is to the right and distal is up. A–C: Rescue phenotype by means of coinjection of 18 ng of xMADML-MO and 500 pg of ALT-xMADML mRNA. D–F: With 18 ng of green fluorescent xMADML-MO and 1 ng of xMADML-DsRED2-N1 fusion construct. D: Animal with mild defect after xMADML-MO injection. E: Lack of red fluorescence, showing that xMADML-MO has prevented the translation of the fusion protein. F: Green fluorescence showing the presence of xMADML-MO. G–I: Injection of 1 ng of xMADML-DsRED2-N1 fusion plasmid alone. G: Brightfield view. H: High degree of red fluorescence, indicating expression of the fusion construct. I: Section of fusion construct-injected eye, showing normal morphology. ce, corneal epithelium; le, lens; nr, neural retina. Scale bars = 400 μm in A,B,D–H, 100 μm in C–I.Download figure to PowerPoint
Figure 7. Reciprocal presumptive lensectoderm (PLE) transplants. Embryos were injected unilaterally at the two-cell stage with either morpholino or rhodamine green dextran, and PLEs were transplanted between injected animals at stage 14. The resulting stage 35 larvae are shown. For the panels in the first three columns, anterior is to the right and dorsal is up. Arrows indicate the locations of eyes. For the panels in the fourth column, dorsal is to the right. Each row shows, left to right, one embryo as whole-mount in transmitted light, red morpholino fluorescence, and green dextran fluorescence, and the affected eye in section, respectively. A–D: Rhodamine green dextran host with a red lissamine Xenopus MADM-like (xMADML) morpholino (MO) PLE transplant. E–H: Red lissamine xMADML-MO host with rhodamine green dextran PLE transplant. I–L: Rhodamine green dextran host with a red lissamine control morpholino (CTL-MO) PLE transplant. M–P: Red lissamine CTL-MO host with a rhodamine green dextran PLE transplant. ce, corneal epithelium; le, lens; nr, neural retina. Scale bars = 400 μm in the first three columns, 100 μm in the fourth column.Download figure to PowerPoint
Figure 8. In situ analysis of selected genes in Xenopus MADM-like (xMADML) morpholino (MO) animals. Embryos injected unilaterally with 18 ng of xMADML-MO at the two-cell stage and showing severely defective eye phenotypes at stage 35 were fixed and assayed for expression of specific genes as indicated below. Each row of panels show staining of one gene. The first column shows the control (non–xMADML-MO injected) side of animals showing the normal in situ expression pattern for each gene. Anterior is to the right and dorsal is up. The second column shows the xMADML-MO–injected side of animals, showing the in situ expression pattern in the knockdown phenotype. Anterior is to the left and dorsal is up. The third column shows a section of the control (uninjected) eye. Distal is up and dorsal is to the left. The fourth column shows a section of the xMADML-MO–injected eye. Distal is up and dorsal is to the right. A–D:Xotx2 staining. E–H:XL-maf staining. I–L: γ6-crystallin staining. M–P:Sox2 staining. Q–T:xSix3 staining. U–X:Sox3 staining. Y–BB:Pax6 staining. The white arrow indicates the eye, the black arrow indicates the lens. nr, neural retina; cns, neural tube. Scale bar = 400 μm in the first two columns, 100 μm in the last two columns.Download figure to PowerPoint
nrbp2 (nuclear receptor binding protein 2) expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 19, lateral view, anteriorright, dorsal up.
nrbp2 (nuclear receptor binding protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anteriorleft, dorsal up.
