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doi: 10.1242/10.1242/dev.00275

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Genetic rescue of cell number in a mouse model of microphthalmia: interactions between Chx10 and G1-phase cell cycle regulators

¹ Department of Ophthalmology and Visual Sciences, John A. Moran Eye Center, University of Utah, Salt Lake City, UT 84132, USA
² Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, UT 84132, USA

View larger version (24K): [in a new window]   Fig. 1. Kip1 is deregulated in Chx10-null retinas. (A,B) More P0 Chx10-null cells than wild-type cells stain positively with antibodies to Kip1 (red). All cells are stained with DAPI (blue). Scale bar: 40 µm. (C) Averaged counts from three animals for each genotype (>500 cells/animal) show a significant increase in Kip1 staining among Chx10-null cells (P<0.01).

View larger version (127K): [in a new window]   Fig. 2. The size and lamination defects of Chx10-null retinas are markedly rescued in Chx10, Kip1 double null retinas. The different eye sizes of adult wild type (A), Chx10-null (B), Chx10, Kip1 double null (C) and Kip1-null mice (D) are readily observable. (E-H) Retinas from postnatal day 19 for each genotype, photographed after dissection and removal of the lens. The pigmented tissue at the margins of the Chx10-null and (to a lesser extent) double null retinas was adherent to the retinal tissue. (I-L) Radial cross-sections through the retinas pictured in A-D, showing that the Chx10, Kip1 double null retina has normal lamination, in contrast to the Chx10-null retina. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar: 1 mm for E-H; 150 µm for I-L.

View larger version (97K): [in a new window]   Fig. 3. Müller glia and all of the major neuronal classes except bipolar cells (see Fig. 4) are present in the Chx10, Kip1 double null retinas at P19. Each cell type in the double null retina was found to have normal gross morphology and to be located in the appropriate layers, in comparison with wild type. By contrast, although all of these cell types are present in Chx10-null retinas, they lack normal morphology and organization. (A-D) Recoverin staining of photoreceptors in the outer nuclear layer. (E-H) Calbindin staining of horizontal and amacrine cells in the INL, and displaced amacrine and ganglion cells in the GCL.(I-L) CRALBP staining of Müller glia, which span the retina vertically. (M-P) Calretinin staining of AII amacrine cells in the INL and displaced AII amacrines in the GCL. (Q-T) ChAT staining of starburst amacrine cells in the INL and displaced starburst amacrines in the GCL. Scale bar: 50 µm.

View larger version (96K): [in a new window]   Fig. 4. Bipolar cells are absent in the otherwise morphologically normal Chx10, Kip1 double null retina. In a cross-section from P19 wild-type retina (A), both bipolar cell bodies (arrows) and the synaptic termini (arrowheads) that they make with ganglion cells are clearly visible with PKC- staining. In the double null (B), only amacrine (INL) and displaced amacrine cell (GCL) immunoreactivity is visible. ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar: 20 µm.

View larger version (50K): [in a new window]   Fig. 5. Deletion of Kip1 restores proliferation in Chx10-null retinas. (A-C) DAPI stained cross-sections show that Chx10, Kip1 double null retinas are morphologically rescued compared with Chx10 single null retinas at P0. Arrows indicate the neural retina. L, lens. (D) Counts of dissociated cells show that all three genotypes are significantly different from each other in cell number at P0 (***P<0.0003). (E-G) TUNEL-positive cells on sections from all genotypes were observed with similar spatial distributions, demonstrating that cell death is not significantly different in wild type, Chx10-null and Chx10, Kip1 double null retinas at P0. Note that because the eye is still growing, the Chx10-null retina is not yet as thin at this age as it will be by P19. Some positive cells are indicated with arrows. (H) In each genotype, fewer than 1% of cells were TUNEL positive, with no significant differences between genotypes. (I) A marker of proliferation (PCNA), and three markers of differentiated cells (recoverin, ß-tubulin and neurofilament), are all present in similar percentages of cells in wild type, Chx10-null and Chx10, Kip1 double null retinas. No marker showed significant differences between genotypes. Cell counts were performed at P0. Scale bar: 400 µm in A-C; 50 µm in E-G.

View larger version (47K): [in a new window]   Fig. 6. Progenitor cell populations are proportionally maintained across all genotypes. PCNA staining in (A) wild type, (B) Chx10-null and (C) Chx10, Kip1 double null retinas, shows that in each genotype the progenitor population size is commensurate with total retinal size at P0. Enlargements of each retina are shown in the insets. L, lens. Scale bar: 500 µm; 125 µm in inset.

View larger version (85K): [in a new window]   Fig. 7. In wild-type P0 retinas, the Chx10 and Kip1 proteins are present in almost entirely exclusive sets of cells, but cells in the neuroblast layer express both species of mRNA. Chx10 protein is present in the central neuroblast layer and in mitotic cells at the ventricular surface of the retina (A), while Kip1 protein is present in cells in the inner and outer thirds of the retina (B). (C) A merged image showing the mutually exclusive cellular staining pattern of these proteins. In situ hybridization shows that mRNA expression of both the Chx10 (D) and Kip1 (E) genes in wild-type retina at P0 is in the same set of cells, in the central neuroblast layer. (F-H) Double in situ of Chx10 and Kip1 mRNA demonstrates that individual RPCs in the neuroblast layer express both of these genes. (I) Northern blot hybridization of total RNA from P0 retinas shows that the Kip1 mRNA level is the same in wild-type and Chx10-null mice (by contrast, the CycD1 mRNA level does change; see Fig. 9C). Ethidium bromide-stained 28s rRNA bands are shown as a loading control. (J) Kip1 protein accumulates in progenitor cells as they exit the cell cycle and begin to differentiate. It is then downregulated in fully differentiated cells (except Müller glia). Arrowheads indicate cells that are positive for both mRNA probes. Scale bars: in A, 50 µm for A-C; in D, 50 µm doe D,E; in F, 10 µm for F-H.

View larger version (94K): [in a new window]   Fig. 8. Double-label immunofluorescence of retinal cross-sections reveals the intricate relationships between Chx10, CycD1 and Kip1 proteins. (A-C) Chx10 and CycD1 are present in the same cells in wild-type retinas. (D-F) CycD1, like Chx10, is in a complementary set of cells relative to Kip1. (G-I) In Chx10-null retinas, CycD1 is largely able to maintain a complementary expression pattern with Kip1. (J-L) However, in CycD1-null retinas, Chx10 fails to maintain complementarity with Kip1. (M-N) High magnification confocal images of Chx10 (red) and Kip1 (green) staining show more clearly that Kip1 and Chx10 are present in the same RPCs in the CycD1-null retina, but not in the wild-type retina. Both images are within the neuroblast layer. Scale bars: in A, 50 µm for A-L; in M, 10 µm for M,N.

View larger version (17K): [in a new window]   Fig. 9. Quantification of protein expression in CycD1 and Chx10-null mice. (A) In CycD1-null retinas, Kip1 expression is present in more cells than normal. The increased percentage of cells positive for both Chx10 and Kip1 indicates that RPCs cannot maintain their normal low levels of Kip1 protein in the absence of CycD1. (B) In Chx10-null retinas, the percentages of cells expressing both CycD1 and Kip1 are abnormal. (C) Northern blot hybridization of total RNA from P0 retinas shows that the CycD1 mRNA level decreases in the Chx10-null compared with wild type. Ethidium bromide-stained 28s rRNA bands are shown as loading controls. *A statistically significant difference (P<0.05); **P<0.01; ***P<0.0003.

View larger version (16K): [in a new window]   Fig. 10. Chx10 is necessary for proper regulation of CycD1. The percentage of cells positive for CycD1 is abnormally low in Chx10, Kip1 double null retinas, just as it is in Chx10 single null retinas. Chx10 is therefore necessary for proper CycD1 regulation even when proliferation is significantly restored. **P<0.01.