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First published online 16 December 2004
doi: 10.1242/dev.01560

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Dlx1 and Dlx2 function is necessary for terminal differentiation and survival of late-born retinal ganglion cells in the developing mouse retina

¹ Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Manitoba, R3E 3J7, Canada
² Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, Manitoba, R3A 1S1, Canada
³ Manitoba Institute of Cell Biology, Cancer Care Manitoba, Winnipeg, Manitoba, R3E 0V9, Canada
⁴ Department of Psychiatry, University of California, San Francisco, CA 94143, USA
⁵ Department of Ophthalmology, University of Manitoba, Winnipeg, Manitoba, R3E 0V9, Canada

View larger version (91K): [in a new window]   Fig. 3. Reduced optic nerve thickness in Dlx1/Dlx2 mutant eyes. (A,B) Wild-type (A) and Dlx1/Dlx2 mutant (B) optic nerves. Three measurements were made of the optic nerve as it exits the retina (optic nerve head) using the boundary between the GCL and NBL (A, top arrow), the outer retinal limit (A, bottom arrow) and at a point equidistant between these markers (A, middle arrow). There was a significant 23% decrease in the thickness of the mutant optic nerve (asterisk). Scale bars: 125 µm.

View larger version (100K): [in a new window]   Fig. 1. Histological characterization of the Dlx1/Dlx2 mutant retina. (A,B) Cresyl Violet staining of Dlx1/Dlx2 mutant and wild-type retinas at E18.5. The mutant displays a reduced GCL (B, arrows) but the remainder of the retina appears spared. (C-F) Expression of BRN3a (C,D) and BRN3b (E,F) in mutant and wild-type retina. Decreased numbers of BRN3a and BRN3b immunoreactive cells (C,E, arrows) indicate that fewer RGCs are present in the mutants. (G-J) Expression of SIX3 and PAX6 in mutant and wild-type retina. SIX3 and PAX6 are expressed in fewer cells in the mutant GCL than in the wild type (G,I, arrows). Expression of SIX3 and PAX6 in the neuroblastic layer appears unaffected in mutants (G,H,I,J, asterisks). (K-N) Expression of amacrine cell markers in the mutant and wild-type retina. (K,L) Syntaxin expression appears unchanged. (M,N) Calretinin immunoreactive cells are expressed in the mutant retina in numbers similar to those of wild type. (O-R) Horizontal cell markers are expressed in normal number and position in the Dlx1/Dlx2 mutants. Expression of NF165 (O,P, arrows) and PROX1 (Q,R, asterisks) are unaffected in the mutant. PROX1 expression in the GCL (Q,R, arrows) supports normal AII amacrine cell development. Scale bars: 250 µm in B; 50 µm in I,J,Q,R.

View larger version (38K): [in a new window]   Fig. 2. Quantification of early-born cells in the E18.5 mutant and wild-type retinas and late-born cells in retinal explants cultured for 7 days. (A) Mutant retinas featured a significant 33.76% reduction in the number of BRN3b-expressing RGCs in the mutant retinas compared with wild-type retinas (asterisk). No significant differences in the number of PAX6-expressing amacrine or NF165-expressing horizontal cells were identified. (B) Late-born retinal cell classes not identifiable in E18.5 retinas were quantified in 7-day-old explant cultures of paired mutant and wild-type retinas collected at E18.5. No significant differences in the number of rods, cones, Müller glia or bipolar interneurons as identified by Rho4D2, peanut agglutinin, glial fibrillary acidic protein (GFAP), and CHX10 immunoreactivity, respectively, were found (rods t=1.03, P>0.05, n=3; cones t=1.26, P>0.05, n=3; Müller glia t=0.64, P>0.05, n=3; bipolar cells t=0.79, P>0.51, n=3). PNA, peanut agglutinin.

View larger version (76K): [in a new window]   Fig. 4. (A) Quantification of cell death in the Dlx1/Dlx2 mutant. At E13.5, there is a significant (asterisk) 3-fold increase in activated caspase-3-expressing cells in the mutant. This amount of apoptosis is less marked at E16.5 (cross), when there is a significant 66% increase in the number of apoptotic cells. There is no statistical difference in cell death by E18.5. (B-G) Co-localization of activated caspase-3 and BRN3b indicates that cell death is occurring among RGCs (D, box, insert) at E13.5 and at E16.5 (G, box, insert). Scale bars: 100 µm in D; 50 µm in G. Inserts in D and G represent 2-fold enlargements.

View larger version (76K): [in a new window]   Fig. 5. Loss of function of Dlx1/Dlx2 results in a loss of late-born RGCs in the developing retina. (A,B) Expression of BRN3b in 7-day-old explant cultures of wild-type and Dlx1/Dlx2 mutant retinas collected at E18.5. BRN3b immunoreactivity is detected in wild type (A, box, insert) but not in mutant explant cultures. (C-T) BrdU birthdating assays identify retinal cells generated at the time of pulsing. (C-E,L-N) BrdU pulses at E12.5 label RGCs as identified by BRN3b protein expression in mutant (C, arrows) and wild-type (L, arrows) retinas collected at E18.5. (F-H,O-Q) E13.5 BrdU pulses also readily identify RGCs in both mutant (F, arrows) and wild-type (O, arrows) retinas. (I-K,R-T) BrdU pulses at E16.5 in mutant and wild-type retinas. Co-expression with BRN3b can be seen only in the wild-type retina (R, arrow). (U) Quantification of BRN3b-expressing RGCs born at E12.5, 13.5 and 16.5. RGCs born at E12.5 (asterisk) represent 10% of RGCs labeled in E18.5 wild-type retinas, but 16% of the RGCs in Dlx1/Dlx2 mutants yielding a significantly larger proportion of the population at E18.5 (t=8.8, P<0.05, n=5). RGCs born at E13.5 (cross) form a significantly larger proportion of the population of RGCs in wild-type retinas compared with mutants (15 versus 13.3%; t=3.32, P<0.05, n=5). RGCs born at E16.5 (#) form a significantly larger proportion (3 fold) of the population of wild-type retinas than mutant retinas (1.5 versus 0.6% of E18.5 population; t=4.85, P<0.05, n=5).

View larger version (98K): [in a new window]   Fig. 6. Expression of DLX1, DLX2 and BRN3b in the ocular retardation (orJ/orJ) mouse. (A-C) OrJ/orJ mutants (Chx10 null) lack expression of BRN3b (A), DLX1 (B) and DLX2 (C) at E13.5, indicating absent RGCs. (D-F)Wild-type controls exhibit normal patterns of expression of these markers. By E16.5, there are BRN3b-expressing cells in orJ/orJ mutants (G). BRN3b expression coincides with the onset of DLX1 and DLX2 expression (H, DLX1; I, DLX2). (J-L) Expression in controls (J, BRN3b; K, DLX1; L, DLX2). By E18.5, BRN3b expression is localized in the central inner retina in both orJ/orJ mutants and controls (M, mutant; P, wild type). DLX1 expression is not detected (N, mutant; Q, wild type) at E18.5. DLX2 expression is found in the inner and outer retina in both mutants and wild types (O, mutant; R, wild-type). The expression pattern of DLX2 is considerably disorganized in the orJ/orJ mutant compared with wild-type controls at this stage (O,R). Scale bars: 100 µm in F; 50 µm in L; 50 µm in R.

View larger version (78K): [in a new window]   Fig. 7. Co-localization of DLX2 with BRN3b in the ocular retardation (orJ/orJ) mutant. (A-C) Expression of BRN3b (A) and DLX2 (B) in the E16.5 retina with merged image (C). Co-expression is extensive between DLX2 and BRN3b at this time point (C, arrows), without BRN3b single-positive cells detected at this stage. (D-F) Expression of BRN3b (D) and DLX2 (E) in the E18.5 retina with merged image (F). Similar to E16.5, extensive co-localization is evident (F, arrows) and BRN3b single-positive cells are not detected. Scale bars: 33 µm.

View larger version (129K): [in a new window]   Fig. 8. Altered Chx10 and Crx homeobox gene expression in the E18.5 Dlx1/Dlx2 mutant retina. (A) Combined CHX10 immunohistochemistry with Crx digoxigenin in-situ hybridization in wild-type retina. CHX10 (brown stain) is localized primarily in the outer retina throughout the NBL. Crx RNA expression (blue stain) can be found in the extreme outer retina. (B) In the Dlx1/Dlx2 mutant there is increased Crx expression in the outer retina as well as ectopic expression in the central retina (arrows) and GCL (box, insert). Note the reduced GCL (double-headed arrows). (C) Crx in-situ hybridization of E18.5 wild-type retina. (D) Crx in-situ hybridization of E18.5 Dlx1/Dlx2 null retina. Ectopic Crx expression is clearly identified in the GCL of the mutant (D, box). Scale bars: 40 µm. Inserts in B,D represent a 3-fold enlargement.

View larger version (21K): [in a new window]   Fig. 9. Model for the generation of RGCs through a Dlx1/Dlx2 dependent pathway (A). Dlx1/Dlx2-expressing cells downregulate Chx10. These cells may then establish Brn3b expression and terminally differentiate into RGCs. The majority of RGCs are derived from Math5/Brn3b co-expressing populations of retinal progenitors (B). We suggest that Dlx1/Dlx2-specified RGCs might represent a distinct late-born RGC population in the murine retina.