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First published online 8 November 2006
doi: 10.1242/dev.02664

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Requirement for Bhlhb5 in the specification of amacrine and cone bipolar subtypes in mouse retina

¹ Center for Aging and Developmental Biology, University of Rochester, Rochester, NY 14642, USA.
² Department of Biology, University of Victoria, Victoria, BC V8W 3N5, Canada.
³ Department of Ophthalmology, University of Rochester, Rochester, NY 14642, USA.
⁴ Department of Neurobiology and Anatomy, University of Rochester, Rochester, NY 14642, USA.

View larger version (79K): [in this window] [in a new window]   Fig. 1. Expression profile of Bhlhb5 in retinogenesis. Retinal sections from the indicated developmental stages were immunolabeled with anti-Bhlhb5 (green) and the nuclei counter-stained with Propidium Iodide (PI, red) (A-I) or probed with a Bhlhb5 in situ probe (J-O). The onset of Bhlhb5 expression starts at E11.5 in the central retina (A). At E12.5 to E15.5, Bhlhb5 expression expands toward the peripheral retina and is mostly detected in cells in the NBL (B-D,J-M). At E17.5 to P0, Bhlhb5 expression becomes localized in the inner boundary of the NBL and the GCL (E,F,N,O). Inserts in E and F show the enlarged view of the corresponding boxed regions. At P7 to P28, two rows of Bhlhb5 expression are seen in the INL and one in the GCL (arrowheads, G-I). (P-U) Bhlhb5 expression is mostly observed in post-mitotic cells of the developing retina. Anti-Bhlhb5 (green) labeling of E12.5 retina shows Bhlhb5 in nuclei of cells in the NBL (P) and anti-BrdU (red) labels the nuclei of proliferating cells at S-phase (Q). (R) Overlay image of P and Q. (S-U) Anti-Bhlhb5 (S, red) and anti-phosphorylated histone H3 (PH3) (T, green) show that Bhlhb5+ cells are mostly negative for PH3 labeling. Abbreviations for this and other figures: L, lens; NBL, neuroblast layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Scale bars: 100 µm.

View larger version (58K): [in this window] [in a new window]   Fig. 2. Expression of Bhlhb5 in GABAergic amacrine and OFF-cone bipolar subtypes. Sections from P28 mouse retinas were doubleimmunolabeled with anti-Bhlhb5 (red) and subtype-specific markers (green) as indicated. (A-C) The Bhlhb5+ cells in the GCL and the ACL are Pax6+ amacrine cells. (D-F) Bhlhb5 is expressed in GAD65+ GABAergic amacrine cells. (G-I) Bhlhb5 is co-expressed with Prox1 in displaced amacrine cells (arrows) in the GCL and bipolar cells (arrowheads) in the INL. (J-L) Bhlhb5 is co-expressed with Chx10 in bipolar cells (arrowheads). Inserts show the double-labeling of bipolar cells (asterisks) at high magnification. (M-O) All Bhlhb5+ bipolar cells are Vsx1+ cone bipolar cells (white arrowheads). (P-R) All Bhlhb5+ bipolar cells (white arrowheads) are recoverin+ Type 2 OFF-bipolar cells; red arrowheads indicate the OFF-bipolar cells immunoreactive to recoverin only. Scale bar: 50 µm.

View larger version (39K): [in this window] [in a new window]   Fig. 3. Developmental abnormality of Bhlhb5-null retinas. (A-J) Retinal sections from Bhlhb5-/- and wild-type control retinas at indicated developmental stages were stained with Haemotoxylin and Eosin. Compared with the control retina (A,C,E), no overt change in retinal thickness and laminar organization is seen in the mutant (B,D,F) from E15.5-P0. At P14 to P28, the INL of Bhlhb5-null retinas is thinner and the number of cells in the INL is reduced by approximately 40% (G-J). (K) Quantitation of cells in the INL and the GCL per 250 µm length of retinal section at E17.5 to P28. Each bar represents the mean±s.d. for three or more retinas. Scale bars: 50 µm.

View larger version (93K): [in this window] [in a new window]   Fig. 4. Selective loss of retinal cell subtypes in Bhlhb5-null retinas. (A-X) Sections from P21 mouse retinas were immunolabeled with subtype-specific markers (green) and nuclear-counterstained with PI (red). Loss of Bhlhb5 leads to a severe loss of amacrine cells immunoreactive for Pax6 (A,G) and GAD65 (B,H) and to an absence of TH+ (C,I) and Prox1+ (D,J) amacrine cells. There is no overt change in the number of amacrine cells immunoreactive to ChAT (E,K), calretinin (F,L) and Isl1 (M,S). A significant loss of Vsx1+ CB cells was observed in Bhlhb5-null retina (N,T). However, no discernible change is seen in the number of PKC+ RB (O,U) and Go+ ON-bipolar (P,V) cells, and Brn3b+ (Q,W) and Brn3a+ (R,X) ganglion cells. Scale bar: 100 µm.

View larger version (111K): [in this window] [in a new window]   Fig. 5. Decreased genesis of Type 2 cone bipolar, GABAergic and displaced amacrine cells. (A-H) Immunolabeling of retinal sections at P6 with bipolar subtype-specific markers reveals a dramatic drop in the genesis of CB cells immunoreactive for Vsx1 (A,E), recoverin (B,F, white arrowheads) and NK3R (C,G, white arrowheads) in Bhlhb5-null mice, whereas the total number of bipolar cells labeled by Chx10 is unchanged (D,H). (I,J,M,N) Anti-Prox1 labeling of retinal sections at P0 (I,M) and P6 (J,N) demonstrates the absence of displaced amacrine genesis in the GCL (white arrowheads) in Bhlhb5-null mice, whereas the Prox1+ horizontal cells (red arrowheads) in Bhlhb5-null mice are formed normally. (K,L,O,P) Anti-Pax6 (K,O) and anti-GAD65 (L,P) labeling also demonstrate a significant decrease in the genesis of amacrine cells in the INL. Scale bar: 100 µm.

View larger version (72K): [in this window] [in a new window]   Fig. 6. Normal expression of retinogenic bHLH factors in Bhlhb5-null retinas. (A-F) Immunolabeling shows a largely overlapping expression of Bhlhb5 (green) and NeuroD (red) in E13 wild-type retina. B, D and F show the enlarged view of the corresponding boxed regions in A, C and E, respectively. (G,L) Anti-NeuroD labeling reveals no change in NeuroD expression in Bhlhb5-null retinas at E13. (H-K,M-P) Similarly, the expression of Math3 (H,M), Ngn2 (I,N), Math5 (J,O) and Mash1 (K,P) is unaffected in Bhlhb5-null retina at E14.5 as assessed by in situ hybridization. Scale bars: 100 µm.

View larger version (98K): [in this window] [in a new window]   Fig. 7. Upregulation of Bhlhb5 and NeuroD expression in the Math5-LacZ cell lineage in Math5-null retina. (A-H) Retinal sections immunolabeled with anti-Bhlhb5 (green) and nuclei counterstained with PI (red) show that loss of Math5 results in a large increase in Bhlhb5+ displaced amacrine cells and a significant decrease in Bhlhb5+ bipolar cells. (A,E) Low magnification of retinas at P21. (B,F) Enlarged view of the corresponding boxed regions in A and E, respectively. (C,G) Confocal sections of Bhlhb5+ displaced amacrine cells in the GCL. (D,H) Confocal sections of Bhlhb5+ cells in the INL. (I-P) Co-immunolabeling of Bhlhb5 (red) and LacZ (green) indicates that loss of Math5 results in a significant increase in the number of cells expressing Bhlhb5 in E13 retina and that a majority of these Bhlhb5+ cells express Math5-LacZ. The boxed areas in K and O are shown at high magnification in L and P. (Q-X) Similarly, an increase in retinal cells expressing Math5-LacZ and NeuroD is observed in E13.5 Math5-null mice. The boxed areas of S and W are shown at high magnification in T and X. The Math5-GFP fluorescence is undetectable under the fixation and detection conditions used and all green signals are derived from either LacZ or anti-Bhlhb5 staining. Scale bars: in A, 200 µm for A and E; in all other panels, 100 µm.

View larger version (27K): [in this window] [in a new window]   Fig. 8. A model for the role of Bhlhb5 in the generation of GABAergic amacrine and Type 2 CB cells. The retinal progenitors exit the cell cycle and are divided into the Math5+ and Math5- precursor pools based on the expression of Math5. Precursors with the transient activation of Math5 are RGC-competent. Some of these precursors choose the RGC differentiation pathway and generate nearly all RGCs. The remaining precursors lose RGC-competence when Math5 expression ceases, express other retinogenic bHLH factors and generate horizontal cells or, along with precursors from the Math5- pool, produce amacrine and cone cells. Together with NeuroD and Math3, Bhlhb5 determines the genesis GABAergic amacrine cells. The rod, bipolar and Müller cells are derived from the Math5- pool of precursors and Bhlhb5 expression provides the precursors with competence to differentiate into Type 2 bipolar cells.