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First published online 12 January 2005
doi: 10.1242/dev.01646

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Functional equivalence of Brn3 POU-domain transcription factors in mouse retinal neurogenesis

Ling Pan^1,*, Zhiyong Yang^1,*, Liang Feng¹ and Lin Gan^1,2,3,

¹ Center for Aging and Developmental Biology, University of Rochester, Rochester, NY 14642, USA
² Department of Ophthalmology, University of Rochester, Rochester, NY 14642, USA
³ Department of Neurobiology and Anatomy, University of Rochester, Rochester, NY 14642, USA

View larger version (70K): [in a new window]   Fig. 1. Spatiotemporal expression profiles of Brn3a and Brn3b in developing mouse retinas. (A-L) The expression of Brn3a and Brn3b in RGCs. (A) The onset of Brn3b expression (green) starts at E11.5 in the central retina. (D,G,J) At E12.5 to E15.5, Brn3b expression expands towards the peripheral retina and is detected in cells in the NBL and in post-migration RGCs in the GCL. (B) Brn3a expression (red) is not detected in the retina at E11.5. (E,H,K) The appearance of Brn3a begins at E12.5 and is limited to RGCs in the GCL throughout the development. (C,F,I,L) Overlaid images of Brn3a and Brn3b expression. (M-R) Postmitotic expression of Brn3b in the developing retina. (M,P) Anti-Brn3b labeling shows Brn3b in nuclei of cells in the GCL and the NBL. (N) Anti-BrdU (red) labels the nuclei of proliferating cells at S phase. (O) Merged image of M,N. (Q) Anti-PH3 (green) reveals the nuclei of proliferating cells at M phase. (R) Merged image of P,Q. Inserts showed the enlarged view of the corresponding boxed regions. NBL, neuroblast layer; GCL, ganglion cell layer; l, lens. Scale bar: 100 µm.

View larger version (91K): [in a new window]   Fig. 2. Downregulation of Brn3a expression in Brn3b-null retinas and co-localization of Brn3a and Brn3b in heterozygous Brn3b3a/+ retinas. (A-F) In situ hybridization using Brn3b, Brn3a 3'UTR and lacZ probes to show the expression patterns of Brn3b, Brn3a and Brn3aki mRNA in E11.5-15.5 retinas. (A) Expression of endogenous Brn3a in wild-type mice. (B) Brn3b expression in wild-type mice. (C) Reduced expression of Brn3a in Brn3b-null retinas. (D) Absence of Brn3b mRNA in Brn3b-null retinas. (E) Expression of endogenous Brn3a in Brn3b3a/+ retinas. (F) Onset of Brn3aki expression at E11.5 and the expression of Brn3aki mRNA in the GCL and the NBL of Brn3b3a/+ retinas. (G-I) Immunolabeling of Brn3b3a/+ retinas at E11.5-15.5 with anti-Brn3a (red) and anti-Brn3b (green). (G) Detection of Brn3a protein in Brn3b3a/+ retinas in the NBL and the GCL. (H) Brn3b expression in Brn3b3a/+ retinas in the NBL and the GCL. (I) Complete overlap of Brn3a and Brn3b expression in cells in the NBL and the GCL. Insets show the enlarged view of the corresponding boxed regions and revealed overlap of Brn3b and Brn3a. NBL, neuroblast layer; GCL, ganglion cell layer; l, lens. Scale bar: 100 µm.

View larger version (67K): [in a new window]   Fig. 3. Rescue of retinal defects in adult Brn3b-null mice by Brn3a knock-in in Brn3b locus. Top, middle and bottom panels are representative of wild-type (Brn3blacZ/+), Brn3blacZ/AP mutant and Brn3b3a/lacZ mice, respectively. (A-C) Hematoxylin and Eosin staining of the retinal structure. (D-F) X-Gal staining of Brn3b-lacZ-positive RGCs. (G-I) SMI32 immunostaining of neurofilament. (J-L) Whole-mount optic nerves and tracks. (M-O) Hematoxylin and Eosin staining of optic nerve cross-sections cut at the broken lines in J-L. Arrowheads indicate optic nerves. RPE, retinal pigmented epithelium; OS, outer segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars: 100 µm in A-I; 300 µm in J-O.

View larger version (101K): [in a new window]   Fig. 4. Absence of programmed cell death of Brn3b-null RGCs in Brn3aki knock-in mice during embryonic development. Cryosections of retinas at E13.5 to P0 were stained with X-Gal to reveal the RGCs by the expression of Brn3b-lacZ reporter gene at Brn3b locus. (A,D,G,J) Control heterozygous Brn3blacZ/+ mice. During retinal development, RGCs are generated and remained in the GCL. (B,E,H,K) Brn3blacZ/AP mice. The lacZ-expressing RGCs were initially formed normally in the GCL (E) but a majority of them degenerated before birth (E,H,K). (C,F,I,L) Brn3b3a/lacZ mice. Replacement of Brn3b with Brn3aki prevents the apoptosis of Brn3b-null RGCs marked by the strong X-Gal staining in the GCL. RPE, retinal pigmented epithelium; NBL, neuroblast layer; GCL, ganglion cell layer; l, lens. Scale bar: 100 µm.

View larger version (64K): [in a new window]   Fig. 5. Normal retinal axon projections in Brn3b3a/3a knock-in mice. Lipophilic dye tracings of the proximal visual pathway in E17.5 mice. (A) Schematic view of the visual system at the ventral surface of the brain. Anterior is towards the top, posterior towards the bottom. Contralateral pathway is shown in red and ipsilateral pathway in blue. The area within the broken lines is imaged with a Nikon fluorescent dissecting microscope. (B) Wild-type mouse. The majority of axons cross the optic chiasm towards the contralateral pathway. Certain axons from ventrotemporal quadrant of the retina do not cross the optic chiasm and form the ipsilateral projection pathway. (C) Brn3b-null mouse. Abnormally high percentage of axon fibers travel towards the ipsilateral optic tract and alongside the left optic nerve to the left eye. (D) Brn3b3a/3a knock-in mouse. Normal axon projection pattern is restored by Brn3aki expression in the absence of Brn3b. ON, optic nerve; OT, optic tract; Ipsi, ipsilateral projection; Contra, contralateral projection. Asterisk indicates the optic chiasm. Scale bar: 200 µm.

View larger version (139K): [in a new window]   Fig. 6. Restoration of Brn3b downstream target genes in retinas at E14.5 by Brn3aki expression. For each in situ hybridization set of retinal sections at E14.5, left, middle and right panels represent wild-type control, Brn3bAP/lacZ mutant and Brn3b3a/3a knock-in, respectively. (A) Control Brn3b probe shows the absence of Brn3b expression in Brn3bAP/lacZ and Brn3b3a/3a retinas. (B) Brn3a ORF probe detects the endogenous and ectopic Brn3a expression. Expression of the following genes is downregulated in Brn3bAP/lacZ mice but is restored by Brn3aki expression: (C) endogenous Brn3a, (D) Ablim, (G) Gfi1, (H) Gli1, (I) Isl2, (J) Irx4, (K) Irx6, (L) Olf1, (M) Gap43, (N) L1, (O) Shh and (P) Hermes. Conversely, Dlx1 (E) and Dlx2 (F) expression is upregulated in the absence of Brn3b but restored to near-normal levels in Brn3b3a/3a knock-in retina. Scale bar: 100 µm.

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