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Identification of molecular markers of bipolar cells in the murine retina.

Kim DS, Ross SE, Trimarchi JM, Aach J, Greenberg ME, Cepko CL - J. Comp. Neurol. (2008)

Bottom Line: Additionally, the expression of bipolar cell genes was analyzed in Bhlhb4 knockout retinas, in which rod bipolar cells degenerate postnatally, to delineate further the identity of bipolar cells in which novel markers are found.From the analysis of Bhlhb4 mutant retinas, cone bipolar cell gene expression appears to be relatively unaffected by the degeneration of rod bipolar cells.Identification of molecular markers for the various subtypes of bipolar cells will lead to greater insights into the development and function of these diverse interneurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.

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Mutagenesis of the mouse Bhlhb4 gene. A: Gene targeting strategy showing partial restriction map of WT Bhlhb4 allele, the targeting vector, the targeted ES cell allele, and the Bhlhb4  allele. The Bhlhb4 gene (which is a single exon gene), the PGK-neomycin cassette, and the PGK-diptheria toxin cassette are represented by rectangles; the arrows represent open reading frames, and the triangles represent loxP sites. Thin lines show the positions of 5′ and 3′ probes used in Southern blotting analysis. BsmI restriction sites (B), used for screening for integration by homologous recombination from the 5′ side of the gene, and NheI restriction sites (N), used for screening from the 3′ side, are indicated. B: Southern blot analysis of ES cells. Genomic DNA was digested with either BsmI or NheI, and Southern blots were analyzed by using either the 5′ or the 3′ probe, respectively. Fragment sizes for WT (+/+) and targeted (−/+) DNA are indicated. C: PCR genotyping from mouse tail DNA from WT (+/+), heterozygous (−/+), and Bhlhb4- (−/−) animals. WT allele, 216 bp; Bhlhb4- allele, 299 bp. D: RNA in situ hybridization for Bhlhb4 RNA. Retinal sections from P21 WT (+/+) and Bhlhb4- (−/−) mice are shown. Scale bar = 100 μm in D.
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fig04: Mutagenesis of the mouse Bhlhb4 gene. A: Gene targeting strategy showing partial restriction map of WT Bhlhb4 allele, the targeting vector, the targeted ES cell allele, and the Bhlhb4 allele. The Bhlhb4 gene (which is a single exon gene), the PGK-neomycin cassette, and the PGK-diptheria toxin cassette are represented by rectangles; the arrows represent open reading frames, and the triangles represent loxP sites. Thin lines show the positions of 5′ and 3′ probes used in Southern blotting analysis. BsmI restriction sites (B), used for screening for integration by homologous recombination from the 5′ side of the gene, and NheI restriction sites (N), used for screening from the 3′ side, are indicated. B: Southern blot analysis of ES cells. Genomic DNA was digested with either BsmI or NheI, and Southern blots were analyzed by using either the 5′ or the 3′ probe, respectively. Fragment sizes for WT (+/+) and targeted (−/+) DNA are indicated. C: PCR genotyping from mouse tail DNA from WT (+/+), heterozygous (−/+), and Bhlhb4- (−/−) animals. WT allele, 216 bp; Bhlhb4- allele, 299 bp. D: RNA in situ hybridization for Bhlhb4 RNA. Retinal sections from P21 WT (+/+) and Bhlhb4- (−/−) mice are shown. Scale bar = 100 μm in D.

Mentions: To assess further the identity of bipolar cells in which the novel markers are expressed, in situ hybridization studies were conducted in retinas from P21 WT and Bhlhb4-deficient mice in which rod bipolar cells have previously been shown to die from P8 to P12 (Bramblett et al., 2004). A mutation at the Bhlhb4 gene locus was introduced by gene targeting and Cre/loxP-mediated recombination in ES cells, as shown in Figure 4. Bhlhb4-positive cells were completely absent in retinas of homozygous mice as assessed by in situ hybridization (Fig. 4D). The specific loss of rod bipolar cells in Bhlhb4 mutants was confirmed by examining Prkca expression, a known rod bipolar cell gene (Greferath et al., 1990). Whereas WT retinas displayed robust expression of Prkca in bipolar cell bodies closely apposed to the OPL and in a subset of amacrine cells, bipolar cells expressing Prkca were completely absent from the Bhlhb4- retina; amacrine cell expression was maintained, however (Fig. 5A,B).Fig. 4


Identification of molecular markers of bipolar cells in the murine retina.

Kim DS, Ross SE, Trimarchi JM, Aach J, Greenberg ME, Cepko CL - J. Comp. Neurol. (2008)

Mutagenesis of the mouse Bhlhb4 gene. A: Gene targeting strategy showing partial restriction map of WT Bhlhb4 allele, the targeting vector, the targeted ES cell allele, and the Bhlhb4  allele. The Bhlhb4 gene (which is a single exon gene), the PGK-neomycin cassette, and the PGK-diptheria toxin cassette are represented by rectangles; the arrows represent open reading frames, and the triangles represent loxP sites. Thin lines show the positions of 5′ and 3′ probes used in Southern blotting analysis. BsmI restriction sites (B), used for screening for integration by homologous recombination from the 5′ side of the gene, and NheI restriction sites (N), used for screening from the 3′ side, are indicated. B: Southern blot analysis of ES cells. Genomic DNA was digested with either BsmI or NheI, and Southern blots were analyzed by using either the 5′ or the 3′ probe, respectively. Fragment sizes for WT (+/+) and targeted (−/+) DNA are indicated. C: PCR genotyping from mouse tail DNA from WT (+/+), heterozygous (−/+), and Bhlhb4- (−/−) animals. WT allele, 216 bp; Bhlhb4- allele, 299 bp. D: RNA in situ hybridization for Bhlhb4 RNA. Retinal sections from P21 WT (+/+) and Bhlhb4- (−/−) mice are shown. Scale bar = 100 μm in D.
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fig04: Mutagenesis of the mouse Bhlhb4 gene. A: Gene targeting strategy showing partial restriction map of WT Bhlhb4 allele, the targeting vector, the targeted ES cell allele, and the Bhlhb4 allele. The Bhlhb4 gene (which is a single exon gene), the PGK-neomycin cassette, and the PGK-diptheria toxin cassette are represented by rectangles; the arrows represent open reading frames, and the triangles represent loxP sites. Thin lines show the positions of 5′ and 3′ probes used in Southern blotting analysis. BsmI restriction sites (B), used for screening for integration by homologous recombination from the 5′ side of the gene, and NheI restriction sites (N), used for screening from the 3′ side, are indicated. B: Southern blot analysis of ES cells. Genomic DNA was digested with either BsmI or NheI, and Southern blots were analyzed by using either the 5′ or the 3′ probe, respectively. Fragment sizes for WT (+/+) and targeted (−/+) DNA are indicated. C: PCR genotyping from mouse tail DNA from WT (+/+), heterozygous (−/+), and Bhlhb4- (−/−) animals. WT allele, 216 bp; Bhlhb4- allele, 299 bp. D: RNA in situ hybridization for Bhlhb4 RNA. Retinal sections from P21 WT (+/+) and Bhlhb4- (−/−) mice are shown. Scale bar = 100 μm in D.
Mentions: To assess further the identity of bipolar cells in which the novel markers are expressed, in situ hybridization studies were conducted in retinas from P21 WT and Bhlhb4-deficient mice in which rod bipolar cells have previously been shown to die from P8 to P12 (Bramblett et al., 2004). A mutation at the Bhlhb4 gene locus was introduced by gene targeting and Cre/loxP-mediated recombination in ES cells, as shown in Figure 4. Bhlhb4-positive cells were completely absent in retinas of homozygous mice as assessed by in situ hybridization (Fig. 4D). The specific loss of rod bipolar cells in Bhlhb4 mutants was confirmed by examining Prkca expression, a known rod bipolar cell gene (Greferath et al., 1990). Whereas WT retinas displayed robust expression of Prkca in bipolar cell bodies closely apposed to the OPL and in a subset of amacrine cells, bipolar cells expressing Prkca were completely absent from the Bhlhb4- retina; amacrine cell expression was maintained, however (Fig. 5A,B).Fig. 4

Bottom Line: Additionally, the expression of bipolar cell genes was analyzed in Bhlhb4 knockout retinas, in which rod bipolar cells degenerate postnatally, to delineate further the identity of bipolar cells in which novel markers are found.From the analysis of Bhlhb4 mutant retinas, cone bipolar cell gene expression appears to be relatively unaffected by the degeneration of rod bipolar cells.Identification of molecular markers for the various subtypes of bipolar cells will lead to greater insights into the development and function of these diverse interneurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.

Show MeSH
Related in: MedlinePlus