Dataset Information

The Mlc1 Promoter Directs Muller Cell-specific Gene Expression in the Retina.

ABSTRACT:

Purpose

Because the importance of glia in regulating brain functions has been demonstrated, genetic technologies that manipulate glial cell-specific gene expression in the brain have become essential and have made great progress. However, it is unknown whether the same strategy that is used in the brain can be applied to the retina because retinal glia differs from glia in the brain. Here, we aimed to find a method for selective gene expression in Müller cells (characteristic glial cells in the retina) and identified Mlc1 as a specific promoter of Müller cells.

Methods

Mlc1-tTA::Yellow-Cameleon-NanotetO/tetO (YC-Nano) mice were used as a reporter line. YC-Nano, a fluorescent protein, was ectopically expressed in the cell type controlled by the Mlc1 promotor. Immunofluorescence staining was used to identify the cell type expressing YC-Nano protein.

Results

YC-Nano-positive (+) signals were observed as vertical stalks in the sliced retina and spanned from the nerve fiber layer through the outer nuclear layer. The density of YC-Nano+ cells was higher around the optic nerve head and lower in the peripheral retina. The YC-Nano+ signals colocalized with vimentin, a marker of Müller cells, but not with the cell markers for blood vessels, microglia, neurons, or astrocytes.

Conclusions

The Mlc1 promoter allows us to manipulate gene expression in Müller cells without affecting astrocytes in the retina.

Translational relevance

Gene manipulation under control of Mlc1 promoter offers novel technique to investigate the role of Müller cells.

SUBMITTER: Danjo Y

PROVIDER: S-EPMC8764212 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Cell-specific expression of the gene that encodes brain-derived neurotrophic factor (BDNF) is required for the normal development of peripheral sensory neurons and efficient synaptic transmission in the mature central and peripheral nervous system. The control of BDNF gene expression involves multiple tissue and cell-specific promoters that are differentially regulated. The molecular mechanisms that are responsible for tissue and cell-specific expression of these promoters are still incompletely understood. RESULTS: The cloning and analysis of three additional zebrafish (Danio rerio) BDNF gene exons and two associated promoters, is reported. Among them are two exons that generate a novel tripartite mature transcript. The exons were located on the transcription unit, whose overall organization was determined by cloning, Southern blot hybridization and sequence analysis, and compared with the pufferfish (Fugu rubripes) and mammalian BDNF loci, revealing a conserved but more compact organization. Structural and functional analysis of the exons, their adjacent promoters and 5' flanks, showed that they are expressed cell-specifically. The promoter associated with the 5' exon of the tripartite transcript is GC-rich, TATA-less and the 5' flank adjacent to it contains multiple Sp1, Mef2, and AP1 elements. A fusion gene containing the promoter and 1.5 KB of 5' flank is directed exclusively to skeletal muscle of transiently transfected embryos. The second promoter, whose associated 5' exon contains a 25-nucleotide segment of identity with a mammalian BDNF gene exon, was transiently expressed in yolk of the early embryo. RT-PCR analysis of total RNA from whole juvenile fish and adult female skeletal muscle revealed tissue-specific expression of the 5' exons but the novel exon could not be detected even after two rounds of nested PCR. CONCLUSION: The zebrafish BDNF gene is as complex as the mammalian gene yet much more compact. Its exons are expressed in an independently regulated and cell-specific fashion. An initial structural and functional analysis has shown that the regions controlling zebrafish BDNF gene expression have been cloned and identified. They can now be subjected to detailed molecular and genetic analyses to identify the cellular mechanisms by which the transcription factors that act on these regions control BDNF gene expression.