|
Figure 4. Developmental expression of XlNLRR-6. A: Reverse transcriptase-polymerase chain reaction (RT-PCR) of XlNLRR-6 reveals expression beginning at neurula stages 14-15, and continuing through the early larval period. Expected product size for the PCR was approximately 2.1 kb, as indicated by the bands shown. The portion of the gel below 1.5 kb has been cropped (and contained no extraneous bands). B-F: In situ hybridization of XlNLRR-6 transcript. B, lateral view of tail bud stage 26. C: Stage 29 lateral view. D: Stage 35 lateral view. E: Stage 37 dorsal view of head. F: Stage 39 lateral view of head. ey, eye; fb, forebrain; hb, hindbrain; mb, midbrain; oc, optic cup; ov, otic vesicle; re, retina; sc, spinal cord. Scale bars = 500 mu m in B-G.
|
|
Figure 9. Immunohistochemical analysis of Xenopus laevis neuronal leucine-rich repeat proteins (XlNLRR) -6 morpholino oligonucleotide (MO) -treated donor presumptive lens ectoderm (PLE) -derived lenses for gamma-crystallin expression at stage 35. A: Control MO-treated lens illustrating normal morphology and gamma-crystallin expression, indicated by the brown stain (see the Experimental Procedures section). Note the single, thin anterior epithelium and prominent fiber cell masses. B,C: Lenses from XlNLRR-6 MO-treated PLE donor specimens transplanted into control host embryos showing late differentiation defects. Multilayered lens epithelium and fiber cell disorganization are indicated. Fiber cells, although morphologically deranged, illustrate expression of gamma-crystallin protein markers. co, cornea; ld, lens defect in fiber cell differentiation; le, lens epithelium; ma, margin of lens equatorial zone; me, abnormal multilayered lens epithelium; pf, primary lens fiber cells; sf, secondary lens fiber cells. Scale bar = 20 mu m in C.
|
|
Figure 5. Examination of eye tissues at stage 25 after Xenopus laevis neuronal leucine-rich repeat proteins (XlNLRR) -6 knockdown. Example embryo injected with 9 ng of XlNLRR-6 morpholino oligonucleotide (MO) in a single cell at the two-cell stage. A: The left side of the embryo did not receive MO treatment. B: The right side of the embryo (asterisk in E) incorporated the MO. C,D: Red fluorescence images showing absence of the MO lissamine marker on the left (C) and presence on the right (D). E: A 10-μm section through the developing forebrain of the embryo shown in A–D demonstrates that both sides of the neural tube and both optic vesicles appear normal. This timing is approximately one stage before the morphological appearance of the lens vesicle (Schaefer et al.,1999). cg, cement gland; nt, neural tube lumen; ov, optic vesicles. Scale bar = 100 μm in E.
|
|
Figure 6. Effects of Xenopus laevis neuronal leucine-rich repeat proteins (XlNLRR) -6 knockdown on lens and retina development. All specimens are shown at stages 35–38, after retinal pigment epithelium has formed in control embryos. Fluorescent images in the second row are the identical specimens to those in the first row; these show the distribution of the lissamine-tagged morpholino oligonucleotide (MO). White arrowheads show position of the eye, when present. A,G: Uninjected control specimen. B,H: A total of 9 ng of control MO were injected at the two-cell stage, leading to normal eye morphology. C–F,I–L: A total of 9 ng of XlNLRR-6 MO were injected at the two-cell stage. Typical eye defects from this treatment are shown in order of increasing severity. C,D,I,J: Small eye with progressive ventral developmental defects. E,K: Eye dysgenesis. F,L: Missing eye. Note that head size decreases as eye phenotype severity increases in C–F. M–R: Ten-micrometer sections at the level of the dashed line in A (mid-eye), stained with hematoxylin. The dorsal–ventral axis is from top-to-bottom, and the proximal–distal axis is from right-to-left in each section. M: Normal eye with visible lens differentiation and formation of distinct retinal layers. N–R: Eye malformations, with poor or absent lens differentiation and disorganized, diminutive retinal development after treatment with 9 ng of XlNLRR-6 MO, injected at the two-cell stage. oc, outer cornea; le, lens epithelium; lf, lens fiber cells; gc, retinal ganglion cell layer; bc, bipolar cell layer; pc, photoreceptor cell layer (note that at this stage the rod and cone photoreceptors have not yet differentiated); pe, retinal pigment epithelium. Scale bar = 400 μm in L (applies to A–L); 100 μm in R (applies to M–R).
|
|
Reciprocal presumptive lens ectoderm (PLE) transplants. A: Schematic illustrating the surgical strategy. See the Experimental Procedures section for details. PLE transplants were conducted at neurula stage 14. B: Contralateral (i.e., unoperated) eye from a Xenopus laevis neuronal leucine-rich repeat proteins (XlNLRR) -6 morpholino oligonucleotide (MO) host/dextran-labeled donor PLE transplant specimen showing normal morphology. C: Operated eye from specimen shown in B, illustrating a dysgenic defect. D,E: Red and green (dextran) channel images of the specimen shown in C, showing the successful positioning of the dextran-labeled transplant at the position of the eye (arrowhead indicates green-labeled lens in E). F: Contralateral (i.e., unoperated) eye from a dextran-labeled host/XlNLRR-6 MO-treated donor PLE transplant specimen. G: Operated eye from the specimen shown in F. H,I: Red and green channel images of the specimen from G, showing the successful positioning of the red MO-labeled transplant at the position of the eye (arrowhead indicates red-labeled lens in H). JâO: Ten-micrometer sections of eyes resulting from transplant experiments at the same developmental stages shown in BâI. J: Normal retina from a control MO-treated host/dextran-labeled PLE donor transplant specimen. Proper differentiation of neuron and pigmented cell layers is indicated. K: Mild ventral defects in an XlNLRR-6 MO host/dextran-labeled PLE donor transplant specimen. Disorganized ventral neurons and absent ventral pigment epithelium are characteristic of this defect category (compare with J). L: Severe retinal defects in an XlNLRR-6 MO host/dextran-labeled PLE donor transplant specimen. Neural disorganization is evident throughout the retina, which is also diminished in size versus control (compare with J). This specimen's defect is also accompanied by lens disorganization. M: Normal lens from a control MO transplant specimen. The single-layer lens epithelium, equatorial margin of fiber cell formation, and differentiated fiber cell mass are indicated. N: Lens from dextran-labeled host/XlNLRR-6 MO-treated PLE donor specimen showing differentiation defect. Multilayered lens epithelium and fiber cell disorganization are indicated (compare with M). O: Whole eye section from a dextran-labeled host/XlNLRR-6 MO-treated PLE donor specimen showing lens and minor retinal differentiation defects (see text). bc, bipolar cell layer; co, cornea; dl, disorganized retinal cell layers; gc, ganglion cell layer; ip, inner plexiform layer; ld, lens defect in fiber cell differentiation; le, lens epithelium; lf, lens fiber cells; ma, margin of lens equatorial zone; me, abnormal multilayered lens epithelium; on, optic nerve; op, outer plexiform layer; pc, photoreceptor cell layer; pe, retinal pigment epithelium; vd, ventral disorganization. Scale bar = 400 μm in I (applies to BâI); 50 μm in O (applies to JâL,O); 20 μm in N (applies to MâN).
|
