QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online January 31, 2007
Journal of Experimental Biology 210, 676-684 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02694

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Yu, C.-J. Articles by Li, L. Search for Related Content PubMed PubMed Citation Articles by Yu, C.-J. Articles by Li, L.

Synchronizing multiphasic circadian rhythms of rhodopsin promoter expression in rod photoreceptor cells

Chuan-Jiang Yu, Yan Gao, Ping Li and Lei Li^*

Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA

View larger version (4K): [in this window] [in a new window]   Fig. 1. Circadian rhythms of rhodopsin mRNA expression in isolated zebrafish retinas in constant darkness (DD). Between the subjective day and night, the expression of rhodopsin mRNA fluctuated. The expression increased steadily during the day, peaked in the late afternoon and then decreased at night. Horizontal bar: black indicates night; gray, subjective day without light. Values are means ± s.e.m. (N=8 at each time point).

View larger version (19K): [in this window] [in a new window]   Fig. 2. Construction and expression of rhodopsin::GFP. (A) A diagram of the DNA fragment that contained 1.2 kb of zebrafish rhodopsin promoter and 0.7 kb of pd2EGFP cDNA with a short poly(A) (pA) tail. (B) Rhodopsin promoter-driven GFP expression in rod photoreceptor cells in the ventral patch of the retina (arrows) at 4 days post-fertilization. Scale bar, 40 µm. (C) Germline transmission of the transgene determined by PCR. Lane 1, molecular markers. Lane 2, positive control (plasmid DNA that contained 0.7 kb of pd2EGFP cDNA). Lanes 3–11, PCR of genomic DNA from nine embryos that were selected from a cross between a transgenic and a wild-type fish. Lanes 3–6, non-transgenic siblings. Lanes 7–11, transgenic siblings. Each lane represents PCR from an individual embryo. Wnt (internal control) was detected in every embryo.

View larger version (57K): [in this window] [in a new window]   Fig. 3. A cryostat section across the outer retina of a transgenic fish showing GFP (left), anti-rhodopsin immunoreactivity (middle), and the co-localization of GFP and rhodopsin (right). GFP was found in the soma (arrowheads) and inner segment (arrow with broken line) of rod photoreceptor cells. The antibody labeled rhodopsin in the inner (arrow with broken line) and outer (arrow with solid line) segments. The merged image shows the co-localization of GFP and rhodopsin. Scale bar, 40 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Circadian rhythms of rhodopsin promoter-driven GFP expression in individual rod photoreceptor cells. (A) GFP intensity in individual rod cells during the first (top row) and second cycles (bottom row) in constant darkness (DD). Note that the times of peak expression (normalized to 1.0) varied in different cells. (B) Time-lapse images of rhodopsin promoter-driven GFP expression in two rod cells from the same slice preparation in 24 h of DD. The highest expression was detected at 13:00 h and 07:00 h, respectively, in cell 1 and cell 2. Black bars, night; gray bars, subjective day without light.

View larger version (44K): [in this window] [in a new window]   Fig. 5. Synchronization of rhodopsin promoter expression in individual rod photoreceptor cells. (A) Rhodopsin promoter-driven GFP expression in 24 h in constant darkness (DD), except at 22:00 h, when a 30-min light pulse was applied. Exposure to light (indicated by the arrow) synchronized the expression in all rod cells (N=10). After light treatment, the expression began to increase. (B) Light exposure (N=21) or the application of dopamine (N=16) or dopamine D2 receptor agonist quinpirole (N=14) decreased rhodopsin promoter expression. In the presence of the dopamine D2 receptor antagonist sulpiride, light produced no effect on rhodopsin promoter expression (N=19). Each line represents time-lapse imaging data from one rod cell. Horizontal lines represent the duration of treatment.

View larger version (28K): [in this window] [in a new window]   Fig. 6. Cytoplasmic free Ca2+ concentrations in individual rod cells measured before, during and after exposure to light (A; N=16), quinpirole (B; N=17) or 8-pCPT-cGMP and dopamine treatment (C; N=17). (A,B) Light or quinpirole treatment decreased cytoplasmic Ca2+ concentrations. Horizontal lines represent the duration of light or drug treatment. (C) The application of 8-pCPT-cGMP (a membrane-permeable cGMP analog) increased Ca2+ influx. However, when dopamine was added to the medium, cytoplasmic Ca2+ concentrations decreased. Horizontal solid and broken lines represent 8-pCPT-cGMP and dopamine treatments, respectively. Each line represents time-lapse imaging data from one rod cell. (D) Decreases in cytoplasmic Ca2+ concentrations after 8-pCPT-cGMP and dopamine treatments in the absence (N=17; open bar) or presence of Co2+ (N=8; hatched bar). Values are the means ± s.e.m.; ns, no significant difference.