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First published online 23 April 2008
doi: 10.1242/dev.010751

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ISL1 and BRN3B co-regulate the differentiation of murine retinal ganglion cells

¹ Department of Ophthalmology, University of Rochester, Rochester, NY 14642, USA.
² Center for Neural Development and Disease, University of Rochester, Rochester, NY 14642, USA.
³ Department of Neurobiology and Anatomy, University of Rochester, Rochester, NY 14642, USA.

View larger version (84K): [in this window] [in a new window]   Fig. 1. Expression of ISL1 in developing mouse retina. (A-F) The expression of ISL1 (red) is largely co-localized with that of BRN3B (green) in both migrating RGCs in the NBL and post-migrated RGCs in the GCL (arrows). Open and filled arrowheads indicate the occasional RGCs expressing BRN3B or ISL1 alone, respectively. (G-L) Most of ISL1+ cells (green) are not co-labeled with S- and M-phase markers (red), anti-BrdU (G-I) and anti-pH3 (J-L). (M-R) Expression of Isl1 is regulated by Math5 but not by Brn3b. (M,N,P,Q) Reduced expression of ISL1 (red) and BRN3B (green) in E13.5 Math5-null retinas. (O,R) The comparable Isl1 expression in wild-type (O) and Brn3b-null (R) retinas by in situ hybridization at E14.5. Insets show selected regions at higher magnification. R, retina; L, lens; NBL, neuroblast layer; GCL, ganglion cell layer. Scale bars: 100 µm.

View larger version (42K): [in this window] [in a new window]   Fig. 2. Generation of Isl1 conditional knockout and Isl1-lacZ knock-in alleles. (A) Generation of Isl1 conditional allele (Isl1CKO). Isl1 genomic structure and restriction enzyme map is shown at the top. White boxes are the non-coding exon sequences and black boxes the coding sequences. Thick bars are the sequences used to generate the homologous arms in the targeting vector. (B) Generation of Isl1lacZ knock-in allele. Neo, PGK-neomycin resistance gene; DTA, diphtheria toxin gene for negative selection of embryonic stem cells; lacZ, β-galactosidase-encoding gene; FRT, flipase recognition sequence; loxP, Cre recombinase recognition sequence; B, BamHI; RI, EcoRI; RV, EcoRV; K, KpnI; N, NotI. (C) Left panel, Southern genotyping confirmation of Isl1lacZ and Isl1lox mice using 3' probe and BamHI digestion. Right panel, PCR genotyping using primers indicated in A&B can distinguish Isl1lox, Isl1lacZ and wild-type allele. (D) Anti-ISL1 immunolabeling of E13.5 retina sections confirms the deletion of ISL1 in the retina of Isl1loxP/lacZ; Six3-Cre mice. The enlarged views of the corresponding boxed regions are showed on the right. Arrows indicate residual ISL1 expression. R, retina; L, lens. Scale bars: 50 µm.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Targeted disruption of Isl1 results in the developmental loss of RGCs. (A-H) Immunostaining of retina sections with anti-BRN3B reveals a significant loss of BRN3B+ RGCs in Isl1 nulls at E17.5 (C,G) and E18.5 (D,H). Insets show the enlarged views of the central retina. (I,J) Anti-activated caspase3 immunostaining shows an increase in apoptotic cells (arrows) in the GCL of Isl1-null retina. (K) The difference of apoptotic cell numbers in wild-type and Isl1-null retina is insignificant at E13.5-15.5. However, in Isl1 nulls, the apoptotic cells are significantly increased from E16.5. Each histogram represents the mean+s.d. n=3 or 4 for each genotype per stage. *P<0.05, **P<0.01; t-test. R, retina; L, lens; NBL, neuroblast layer; GCL, ganglion cell layer. Scale bars: 100 µm in A-D; 25 µm in I.

View larger version (96K): [in this window] [in a new window]   Fig. 4. Loss of RGCs in adult Isl1-null retina. (A-G) Immunostaining of whole-mount retina with anti-BRN3A (A,B), BRN3B (C,D) and SMI32 (F,G) antibodies reveals the reduction of RGCs in Isl1-null retina. (E) Quantification of BRN3A+ and BRN3B+ cells in the central retinal region of the whole mounts (n=4 for each genotype) reveals a loss of 66% RGCs in Isl1-null retina. (H,I) Ventral views of brain show the thinner optic nerves (arrows) in Isl1 nulls. (J) Quantification of optic nerve size by measuring the cross area of H&E stained optic nerve transverse sections at the level indicated by arrows. Mean size of optic nerve in Isl1-null mice is reduced to 31% of that in wild type (n=6 pairs). Each histogram represents the mean+s.d. ***P<0.001 (t-test). OC, optic chiasm; OT, optic tract. Scale bars: 50 µm in A; 100 µm in F; 1 mm in H.

View larger version (82K): [in this window] [in a new window]   Fig. 5. ISL1 and BRN3B regulate the expression of a common set of RGC-specific genes. (A-H) Compared with controls (left panels), in situ hybridization shows that at E14.5, the expression of RGC-specific genes Brn3a, Isl2, Olf1, Irx4, Ablim1, Gap43, L1cam and Shh decreases in the GCL (bracket) of both Isl1-null (middle panels) and Brn3b-null retinas (right panels). T, temporal; N, nasal. Scale bar: 100 µm.

View larger version (56K): [in this window] [in a new window]   Fig. 6. Axon growth defects in mice deficient for Isl1 or Brn3b. (A-F) Optic pathways at the ventral diencephalons after unilateral DiI labeling. In Isl1 nulls and Brn3b nulls, the majority of RGC axons fails to reach the midline at E13.5 (A-C). At E15.5, although the chiasms are formed in the null mice (D-F), the optic nerves are noticeably thinner and axons are de-fasciculated in the optic tract (arrowheads). ON, optic nerve; Ipsi-OT, ipsilateral optic tract; Contra-OT, contralateral optic tract. Scale bars: 100 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 7. More severe RGC loss in Isl1 and Brn3b compound null mice. (A-H) Immunostaining of adult whole-mount retinas with anti-BRN3A (A-D) and SMI32 (E-H). A more severe loss of BRN3A+ RGCs (red) and SMI32+ axon bundles (green) is observed in Isl1/Brn3b-null retina. (I-L) Ventral views of brains reveal optic nerves (arrows). Compared with the controls (I), optic nerve size is reduced in Isl1 nulls (J) and Brn3b nulls (K). The optic nerves are barely detectable in Isl1/Brn3b-null mice (L). (M-O) X-Gal staining of Brn3b-lacZ-expressing RGCs in E13.5 retina sections shows that the genesis and migration of RGCs are undisturbed in Brn3b-null (N) and Isl1/Brn3b-null (O) retinas. L, lens; GCL, ganglion cell layer. Scale bars: 50 µm in D and H; 800 µm in L and 100 µm in O.

View larger version (20K): [in this window] [in a new window]   Fig. 8. Functional mechanism of ISL1 and BRN3B in the development of RGCs. (A) Concurrent binding of ISL1 and BRN3B to RGC-specific promoters. Anti-BRN3B and anti-ISL1 antibodies but not IgG co-precipitate with the promoters of Brn3b, Shh, Brn3a and Isl2. Both antibodies do not precipitate with control Brn3b ORF. (B) Functional synergy of ISL1 and BRN3B on Brn3a luciferase reporter gene expression. Cells transfected with empty pcDNA expression vector and reporter are used as controls. Luciferase activity is determined by normalizing firefly activity with renilla activity. Then fold activation is calculated by dividing the luciferase activity of experimental groups with that of control. Each histogram represents the mean+s.d. (n=4). (C) The Math5-Brn3b/Isl1 pathway in the development of RGCs. Solid lines, direct regulation identified; broken lines, indirect or proposed regulation.

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