Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Selective knockout of NeuroD1 in the retina and pineal gland

ABSTRACT: NeuroD1 encodes a basic helix-loop-helix transcription factor involved in the development of neural and endocrine structures. NeuroD1 mRNA is highly abundant in the adult mammalian pineal gland and exhibits a developmental expression pattern similar to the retina. This is consistent with the common evolutionary origin of pinealocytes and retinal photoreceptors. Pinealocytes and retinal photoreceptors express a shared set of phototransduction genes and submammalian pinealocytes are photosensitive. In contrast to the retina, the pineal gland is a relatively homogeneous structure, composed 95% of pinealocytes. This makes the pineal gland a particularly useful model for understanding photoreceptor cell biology. The loss of NeuroD1 in the retina results in progressive photoreceptor degeneration and the molecular mechanisms underlying this retinal degeneration phenotype remain unknown. Similarly, the role that NeuroD1 plays in the pineal gland is unknown. To determine the function of NeuroD1 in the pineal gland and retina, a Cre/loxP recombination strategy was used to selectively target a NeuroD1 floxed allele and generate NeuroD1 conditional knockout (cKO) mice. Tissue specificity was conferred using Cre recombinase expressed under the control of the promoter of Crx, a transcription factor selectively expressed in the pineal gland and retina. Pineal and retinal tissues from two-month-old NeuroD1 cKO and control animals were used in microarray studies to identify candidate genes responsible for the photoreceptor degeneration phenotype. The Cre/loxP recombination strategy was used to target a NeuroD1 floxed allele and generate NeuroD1 conditional knockout mice (NeuroD1floxed::Crx-Cre+/-). NeuroD1 floxed mice (NeuroD1floxed::Crx-Cre-/-) served as the controls. Pineal glands and retinas from two-month-old control and conditional knockout mice were collected at ZT6 and ZT20. 3 pools of 6 pineal glands per genotype and respective time of day were collected for each sample. Similarly, 3 pools of 6 retinas each were also collected. RNA from each pool was extracted and hybridized on the Affymetrix Mouse 430 2.0 array.

ORGANISM(S): Mus musculus

SUBMITTER: Margaret Ochocinska

PROVIDER: E-GEOD-35396 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

NeuroD1 is required for survival of photoreceptors but not pinealocytes: results from targeted gene deletion studies.

Ochocinska Margaret J MJ Muñoz Estela M EM Veleri Shobi S Weller Joan L JL Coon Steven L SL Pozdeyev Nikita N Iuvone P Michael PM Goebbels Sandra S Furukawa Takahisa T Klein David C DC

Journal of neurochemistry 20120816 1

NeuroD1 encodes a basic helix-loop-helix transcription factor involved in the development of neural and endocrine structures, including the retina and pineal gland. To determine the effect of NeuroD1 knockout in these tissues, a Cre/loxP recombination strategy was used to target a NeuroD1 floxed gene and generate NeuroD1 conditional knockout (cKO) mice. Tissue specificity was conferred using Cre recombinase expressed under the control of the promoter of Crx, which is selectively expressed in the ...[more]

PMID: 22784109

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Project description:The cone-rod homeobox gene (Crx) encodes Crx, a transcription factor selectively expressed in two cell types, retinal photoreceptors and the melatonin secreting pinealocytes of the pineal gland. In this report the role of Crx in regulating gene expression in the mammalian pineal gland was extended using Affymetrix GeneChip technology. Deletion of Crx results in broad modulation of the mouse pineal transcriptome, including a >2-fold downregulation of 543 genes and a >2-fold upregulation of 745 genes. In addition to Crx, there was a >10-fold downregulation of 13 other genes. Of special interest was the discovery of a link between Crx and the homeobox gene Hoxc4, which was upregulated ~20-fold in the Crx-/- pineal gland. Analysis of night and day expression of genes indicated that a set of 51 genes exhibited differential expression in control animals. Of these genes, only eight were also differentially expressed in Crx-/- animals. This group included Aanat, which encodes the enzyme that controls the daily rhythm in melatonin synthesis in the vertebrate pineal gland. Accordingly, Crx appears to be essential for the 24-hour rhythmic component of expression of some genes in the pineal gland. In the Crx-/- mouse pineal gland, 41 genes exhibited differential night/day expression that was not seen in control animals, suggesting that Crx may function to modulate rhythmic expression of these genes as well. Together, the results of this investigation indicate that Crx broadly modulates the pineal transcriptome, perhaps in part through suppressive effects on expression of the homeobox gene Hoxc4. Pineal glands from control (129sv) mice and Crx-/- mice were collected at ZT6 and ZT20 for RNA extraction and hybridization on Affymetrix mouse 430_2 chip. Each condition were performed as triplicats. SUPPLEMENTARY FILES: The GCOS signal intensity data were analyzed using ChipInspector (Genomatix) version 2.1. FDR= 0, p-value <0.05, cut off=1, region size = 300 bp and 4 and 5 significant probes. Log(2) fold change independently of time of day. Up regulated genes: genes which are up regulated in Crx-/- compared to the control; down regulated genes: genes which are down regulated in Crx-/- compared to the control.

Project description:Pineal function follows a 24-hour schedule, dedicated to the conversion of night and day into a hormonal signal, melatonin. In mammals, 24-hour changes in pineal activity are controlled by a neural pathway that includes the central circadian oscillator in the suprachiasmatic nucleus and the superior cervical ganglia (SCG), which innervate the pineal gland. In this study, we have generated the first next-generation RNA sequencing evidence of neural control of the daily changes in the pineal transcriptome. We found over 3000 pineal transcripts that are differentially expressed (p <0.001) on a night/day basis (70% of these genes increase at night, 376 with fold change >4 or <1/4), the majority of which had not been previously identified as such. Nearly all night/day differences were eliminated by neonatal removal or decentralization of the SCG, confirming the importance of neural input for differential night/day changes in transcript abundance. In contrast, very few non-rhythmic genes showed evidence of changes in expression due to the surgical procedure itself, which is consistent with the hypothesis that post neonatal neural stimulation is not required for cell fate determination and maintenance of phenotype. Many of the transcripts that exhibit marked differential night/day expression exhibited similar changes in response to in vitro treatment with norepinephrine, the SCG neurotransmitter which mediates pineal regulation. Similar changes were also seen following treatment with an analog of the norepinephrine second messenger, cyclic AMP. For the in vivo data, there were 8 biological conditions: day and night time points for each of four surgical groups: Control (Ctrl) Sham-surgery (Sham), Decentralized (DCN), and Ganglionectomized (SCGX). Samples were pooled into three biological replicates for each biological condition. For the in vitro data there were 3 biological conditions: Untreated control (CN), DBcAMP-treated (DB), and Norepinephrine-treated (NE). For the pineal enrichment comparison, three samples (i.e. no biological replicates) were used: pineal-day, pineal-night and mixed-tissue. For the mixed tissues sample, the following tissues from three rats sacrificed at ZT7 were used: cortex, cerebellum, midbrain, hypothalamus, hindbrain, spinal cord, retina, pituitary, heart, liver, lung, kidney, skeletal muscle, small intestine, adrenal gland. Total RNA was extracted from each tissue, and then equal amounts of each of the 15 tissues were combined for the final pooled sample.

Dataset Information

Selective knockout of NeuroD1 in the retina and pineal gland

Publications

NeuroD1 is required for survival of photoreceptors but not pinealocytes: results from targeted gene deletion studies.

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