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First published online 3 March 2004
doi: 10.1242/dev.01071

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Role of the Barhl2 homeobox gene in the specification of glycinergic amacrine cells

Center for Advanced Biotechnology and Medicine and Department of Pediatrics, UMDNJ-Robert Wood Johnson Medical School, 679 Hoes Lane, Piscataway, NJ 08854, USA

View larger version (114K): [in a new window]   Fig. 1. Expression of Barhl2 during CNS development. (A-P) The Barhl2 transcript was detected by in situ hybridization in whole-mount embryos (A,B) and in sagittal (C) and coronal (D-P) sections from embryonic and postnatal mice at the indicated developmental stages. Barhl2 expression is initiated at E9.5 in two symmetric stripes within the diencephalon (A). At later stages, its expression is found in select regions of the telencephalon, diencephalon, mesencephalon, cerebellum, medulla and spinal cord (B-P), as well as in the retina and pineal gland (E,F). aes, anterior extramural migratory stream; AP, alar plate; BP, basal plate; Cb, cerebellum; D, diencephalon; D1, D1 interneuron; DB, diagonal band; EGL, external granule layer; ET, epithalamic neuroepithelium; IC, inferior colliculus; ION, inferior olivary nucleus; IT, intermediate thalamic neuroepithelium; LRL, lower rhombic lip; M, mesencephalon; Me, medulla; MM, mammillary region; NR, neural retina; NT, neural tube; PA, periaqueductal gray; PC, pyriform cortex; PG, pineal gland; PGN, pontine gray nucleus; PO, preoptic area; PV, paraventricular nucleus; SC, superior colliculus; SpC, spinal cord; ST, spinal trigeminal nucleus; T, telencephalon; URL, upper rhombic lip; VT, ventral thalamus. Scale bar: 25 µm in I; 50 µm in C,O; 100 µm in D,P; 250 µm in A,E-N; 333 µm in B.

View larger version (82K): [in a new window]   Fig. 2. Spatial and temporal expression patterns of Barhl genes during mouse retinogenesis. (A,B) In situ hybridization analysis of E14.5 and P1 retinal sections using a Barhl2 antisense probe showed prominent signals in the inner retina. (C-F) Retinal sections from the indicated developmental stages were immunostained with an anti-Barhl2 antibody. In embryonic, postnatal and adult retinas, the anti-Barhl2 antibody labeled many cells in the inner nuclear and ganglion cell layers (C-F), as well as some migratory cells within the outer neuroblastic layer (C,D). Arrows indicate representative differentiating and mature horizontal cells. (G) In situ hybridization analysis of E16.5 retinal sections using a Barhl1 antisense probe detected no signals in the retina. (H) X-gal staining of retinal sections from E16.5 Barhl1lacZ/+ embryos counterstained with Fast Red. No ß-galactosidase activity was detected in the retina. GCL, ganglion cell layer; inbl, inner neuroblastic layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segment; onbl, outer neuroblastic layer; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment. Scale bar: 25 µm.

View larger version (86K): [in a new window]   Fig. 3. Localization of Barhl2 protein in postmitotic amacrine, horizontal and ganglion cells. (A) Retinal sections from BrdU-labeled E17.5 embryos were double-immunostained with antibodies against Barhl2 (red) and BrdU (green). Barhl2-immunoreactive cells, even those within the outer neuroblastic layer (indicated by arrowheads), do not overlap the S-phase cells immunoreactive for BrdU. (B-I) Double-immunolabeling of adult retinal sections using an anti-Barhl2 antibody and antibodies against several indicated cell type-specific marker proteins. Barhl2 appears to be expressed in all amacrine cells immunoreactive for syntaxin (B), glycine transporter 1 (GLYT1) (C), GABA receptor GABAB (D), calretinin (E) and calbindin D-28k (F); and in all horizontal cells immunoreactive for calbindin (F), but only in a subset of ganglion cells immunoreactive for Brn3a (G). By contrast, Barhl2 is absent from rod bipolar and Müller cells immunoreactive for protein kinase C (PKC) and glutamine synthase (GS), respectively (H,I). Arrows indicate representative colocalized cells. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; onbl, outer neuroblastic layer; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bar: 10 µm in A-F,H,I; 16.7 µm in G.

View larger version (45K): [in a new window]   Fig. 4. Effect of forced Barhl2 expression on retinal inner nuclear cells. (A) Schematic of control-GFP and Barhl2-GFP retroviral constructs. In Barhl2-GFP, a bicistronic transcript containing an internal ribosomal entry site (IRES) is produced from the viral LTR promoter, which allows efficient expression of both Barhl2 and the GFP marker protein. (B,C) Virus-transduced GFP+ (green) cells were visualized in P30 retinal sections that were weakly counterstained with nuclear DAPI. Infection with Barhl2-GFP viruses caused a significant increase of cells situated within the inner region of the inner nuclear layer. Arrows in C indicate two such cells. (D) Percentages of virus-transduced cells located in different cellular layers of the retina (means±s.d.). More than 600 GFP+ cells in three independent retinas were scored for either control-GFP or Barhl2-GFP virus. The inner nuclear layer was divided into the outer and inner halves for quantitation. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bar: 10 µm.

View larger version (109K): [in a new window]   Fig. 5. Effect of misexpressed Barhl2 on the formation of different retinal cell types. (A-P) Sections from P30 retinas infected with control-GFP (A,C,E,G,I,K,M,O) or Barhl2-GFP (B,D,F,H,J,L,N,P) viruses were double-immunostained with an anti-GFP antibody and antibodies against syntaxin (A,B), Pax6 (C,D), glycine transporter 1 (GLYT1) (E,F), calbindin (G,H), GABA (I,J), PKC (K,L), glutamine synthase (GS) (M,N) or Brn3a (O,P). All sections were weakly counterstained with nuclear DAPI. Forced Barhl2 expression resulted in a significant increase in the number of amacrine cells immunoreactive for syntaxin (A,B), Pax6 (C,D), GLYT1 (C,D) and calbindin (E,F), whereas it had no effect on amacrine cells immunoreactive for GABA (I,J) and ganglion cells immunoreactive for Brn3a (O,P). Moreover, it greatly suppressed the formation of rod bipolar cells immunoreactive for PKC (K,L) and Müller cells immunoreactive for GS (M,N). Arrows indicate representative colocalized cells. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer. Scale bar: 25 µm.

View larger version (27K): [in a new window]   Fig. 6. Quantitation of virus-transduced retinal cells that became immunoreactive for a series of cell type-specific markers. (A) P0 retinas infected in vivo. (B) E17.5 retinal explants infected in vitro. Each histogram represents the mean±s.d. for three or four retinas. More than 530 GFP+ cells were scored in each retina. ChAT, choline acetyltransferase; GAT-1, GABA transporter 1; GLYT1, glycine transporter 1; GS, glutamine synthase; TH, tyrosine hydroxylase.

View larger version (73K): [in a new window]   Fig. 7. Effect of a dominant-negative form of Barhl2 on the formation of different retinal cell types. (A) Schematic of control-GFP and Barhl2-EnR-GFP retroviral constructs. (B) Percentages of virus-transduced cells located in different cellular layers of the retina (means±s.d.). More than 500 GFP+ cells in three or four independent retinas were scored for either control-GFP or Barhl2-EnR-GFP virus. (C) Percentages of virus-transduced cells that became immunoreactive for a series of cell type-specific markers. Each histogram represents the mean±s.d. for three to five retinas. More than 500 GFP+ cells were scored in each retina. (D,E) Virus-transduced GFP+ cells were visualized in P30 retinal sections that were weakly counterstained with DAPI. Infection with Barhl2-EnR-GFP viruses resulted in a significant decrease of INL cells. (F-K) Sections from P30 retinas infected with control-GFP or Barhl2-EnR-GFP viruses were double-immunostained with an anti-GFP antibody and antibodies against Pax6 (F,G), GLYT1 (H,I) or GABA (J,K). All sections were weakly counterstained with DAPI. Forced Barhl2-EnR expression reduced the number of amacrine cells immunoreactive for Pax6 (F,G) and GLYT1 (H,I) but had no effect on amacrine cells immunoreactive for GABA (J,K). Arrows indicate representative colocalized cells. EnR, engrailed repressor domain; GCL, ganglion cell layer; GS, glutamine synthase; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bar: 25 µm.

View larger version (16K): [in a new window]   Fig. 8. Schematic depicting genetic relationships between Barhl2 and other transcription factors involved in the development of amacrine cells. Retinal progenitors may acquire the competence to produce amacrine cells by the activation of an unknown factor(s). Competent progenitor cells are then specified by Math3 and Neurod1 to become amacrine cells. Some of the newly generated amacrine cells may differentiate into glycinergic cells by the action of Barhl2 and Pax6. Alternatively, Math3 and Neurod1 may also be able to specify Pax6+ progenitors into amacrine cells (broken arrow). It is known that Pax6+ progenitor cells have the potential to differentiate into rod, cone, horizontal, bipolar, ganglion and Müller cells.