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Brn3b/Brn3c double knockout mice reveal an unsuspected role for Brn3c in retinal ganglion cell axon outgrowth

¹ Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA
² Center for Aging and Developmental Biology and Department of Neurology, University of Rochester School of Medicine, Rochester, NY 14642, USA
³ Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA

View larger version (61K): [in a new window]   Fig. 1. Expression of Brn3a, Brn3b and Brn3c in developing retinas of wild-type and Brn3b–/– mice. Brn3a, Brn3b, Brn3c and ß-actin transcripts were detected by RT-PCR. Total RNA was collected from wild-type (+/+) and Brn3b–/– retinas at E14.5 and E16.5. PCR products were visualized by ethidium bromide staining.

View larger version (51K): [in a new window]   Fig. 2. Construction of the Brn3c-AP allele and genotype of Brn3b/Brn3c double knockout mice. (A) The 5' and 3' homology arms of Brn3c were inserted into the multiple cloning site of the AP-ki targeting vector (Gan et al., 1999). The targeting vector contains TK, neo and AP (hPLAP) cassettes as depicted. The position and exon/intron organization of the Brn3c gene with a 1.5 kb Sac1- BamH1 fragment used for genotyping are shown below the wild-type gene. B, BamHI; E, EcoRI; H, HindIII; S, SacI; V, EcoRV; X, XbaI. (B) Genotypes from a Brn3b–/–:Brn3c (+/–) intercross using Southern hybridization. Genomic DNA was digested with HindIII and BamHI. Genotyping for Brn3b was performed as detailed by Gan et al. (Gan et al., 1999), and a band at 6-kb representing the targeted allele is observed in all lanes. Genotyping for Brn3c was performed using the 3' probe shown in A, which resulted in a wild-type band (7.1 kb) and a mutant band (5.5 kb). The three right hand lanes represent genotypes of Brn3b/Brn3c double mutant mice.

View larger version (99K): [in a new window]   Fig. 3. Axons projecting from Brn3c-expressing cells in retinal explants. (A) Weak AP activity detected in axons projecting from a Brn3c (AP/+) retinal explant. (B) AP activity was twice as intense as in A in axons projecting from a Brn3c (AP/AP) retinal explant. (C,D) DIC images of A and B, respectively, showing abundant axons emanating from both of the retinal explants. (E) AP expression in neurites and migrating cells from a Brn3b (AP/AP) retinal explant. (F) Lack of AP expression in neurites and migrating cells from a Brn3b (GFP/GFP):Brn3c (AP/AP) double mutant retinal explant. Arrows point to nonexpressing neurites. (G) AP expression in neurites (arrows) within the tissue body of Brn3b (GFP/GFP):Brn3c (AP/+) retinal explants. (H) AP expression in Brn3c-expressing cells (arrowheads) within the tissue body of a Brn3b (GFP/GFP):Brn3c (AP/AP) double mutant retinal explant.

View larger version (161K): [in a new window]   Fig. 4. Retinal flat-mounts showing RGCs and optic fiber layers from E16.5 retinas. (A) Low magnification of a wild-type retina labeled with anti-NFL (axons, green) and propidium iodide (nuclei, red). Axons were well fasciculated and were growing counter-radially into the optic disk (OD). (B) An enlarged view of the retina from the area highlighted in A. (C) Same image as B with the green channel (axons) removed. Note numerous nuclei in the ganglion cell layer. (D) Low magnification of Brn3b–/– retina labeled with the same antibodies as in A. (E) An enlarged view of the retina from the area highlighted in D. The axon number was greatly reduced when compared with that of a wild-type retina. Axons were not fasciculated and many did not enter the OD. (F) Same image as E with the green channel (axons) removed. The density of nuclei was not reduced when compared with that of the wild type. (G) Low magnification of a Brn3b/Brn3c double mutant retina labeled with the same antibodies as in A. Axons were barely detectable (arrows). (H) An enlarged view from the highlighted area shown in G. The axon number was further reduced when compared with E, but misrouted axons were not observed. The thick green-labeled tubule structures were capillaries that label non-specifically. (I) Same image as that in H with green channel removed. A slightly reduced nuclei number was detected in the vacant areas, but the reduction was not significant when compared with Brn3b–/– retinas.

View larger version (109K): [in a new window]   Fig. 5. RGC and axon distribution in 3-week-old retinas. (A-C) Wild-type (A), Brn3b–/– (B) and Brn3b/Brn3c double mutant (C) retinas from the dorsal-nasal region. Retinas were labeled with anti-ß3-tubulin (RGCs and axons, green) and propidium iodide (nuclei, red). The number of axons in the Brn3b–/– retina (B) was greatly reduced when compared with that of the wild type (A). The number of axons deceased only slightly in the double-mutant retina (C). The thick branching green-labeled structures are blood vessels and capillaries nonspecifically labeled. (D-F) Enlarged views of wild-type (D), Brn3b–/– (E) and Brn3b/Brn3c double-mutant (F) retinas from the ventral-temporal region. Optic disks are located at the bottom out of frame. Retinas were labeled with anti-ß3-tubulin (RGCs and axons, green), ChAT (displaced amacrine cells, blue) and propidium iodide (nuclei, red). The number of axons was greatly reduced in the Brn3b/Brn3c double mutant retina (F) when compared with a Brn3b–/– retina (E). The thick branching green-labeled structures (arrows) are blood vessels and capillaries that were nonspecifically labeled with anti-mouse IgG secondary antibody. Note that excess capillaries were embedded in the RGC layer. Some large nuclei are surrounded by ChAT-positive signals in F. It is possible that these signals resulted from the processes of the overproduced amacrine cells. (G-I) The same images as D-F with the green and blue channels removed. The smaller condensed nuclei (blue arrows) correspond to ChAT-positive amacrine cells, while the large diffuse nuclei (green arrows) correspond to RGCs. (J-L) Low-magnification views of cross sections collected from wild-type (J), Brn3b–/– (K) and Brn3b/Brn3c double mutant (L) retinas. Sections were labeled as in D-F. Note the optic fiber layer in green at the bottom of the Brn3b–/– retina (K) is much thinner than that of the wild-type retina (J), and the optic fiber in the double mutant retina (L) is barely detectable. (M-O) Enlarged views of cross sections collected from wild-type (M), Brn3b–/– (N) and Brn3b/Brn3c double mutant (O) retinas. Labeling was the same as in D-F. RGCs are indicated by arrowheads.

View larger version (23K): [in a new window]   Fig. 6. Altered cell population in the ganglion cell layer of Brn3b–/– and Brn3b/Brn3c double mutant retinas. Six 200 x 200 µm images collected from three different retinas from each indicated genotype were scored for small (amacrine, blue) and large (RGC, green) nuclei. Ambiguous nuclei were counted and assigned equally to each category.

View larger version (91K): [in a new window]   Fig. 7. Anterograde-labeled optic chiasms from wild-type, Brn3b–/– and Brn3b/Brn3c-double mutant mice. At P2, DiI (red) was injected into the vitreous space of right eyes and DiASP (green) into left eyes. Images were collected with a confocal microscope at P3 through the visible thickness of the optic chiasms at 5 µm intervals. (A) Wild-type optic chiasm showing representative decussation. (B) Brn3b–/– optic chiasm showing highly abnormal crossing patterns. Optic nerves were thinner than those from wild-type and many axons were observed sprouting from the optic nerve and projecting to the ventral hypothalamus. (C) Brn3b/Brn3c double-mutant optic chiasm showing partially restored decussation. (D-F) Axons from the left eye of those shown in A-C to visualize the small portion of ipsilaterally projecting axons. Note the abnormally abundant ipsilateral axons in E and the absence of misrouted and ipsilateral axons in F.

View larger version (72K): [in a new window]   Fig. 8. Overexpression of HSV-GFPBrn3c in retinal explants. (A) HSV-GFP-Brn3c amplicon. The coding region of Brn3c was inserted between the XbaI and BamHI sites of the amplicon vector. GFP was driven by the CMV promoter, and Brn3c by the HSV IE4/5 promoter. The vector without the Brn3b insert served as a control. (B) Low-magnification view of a Brn3b–/– retinal explant infected with the HSV-GFP control amplicon. (C) An enlarged view of the peripheral region of the same sample as in B, showing a small number of abnormal neurites emanating from the tissue body. (D) Low-magnification view of a Brn3b–/– retinal explant infected with the HSV-GFP-Brn3c amplicon. (E) An enlarged view of the peripheral region of the same sample as in D showing a large number of neurites emanating from the tissue body. Inset shows branched structures (arrows).