Open Access Highly Accessed Research article

Circadian rhythms in the pineal organ persist in zebrafish larvae that lack ventral brain

Ramil R Noche25, Po-Nien Lu12, Lauren Goldstein-Kral3, Eric Glasgow4 and Jennifer O Liang1*

Author Affiliations

1 Department of Biology, University of Minnesota-Duluth, 1035 Kirby Drive, Duluth, Minnesota, 55812 USA

2 Department of Biology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio, 44106 USA

3 Hathaway Brown High School, 19600 North Park Boulevard, Shaker Heights, Ohio, 44122 USA

4 Department of Oncology, Georgetown University Medical Center, 4000 Reservoir Road NW Washington, DC, 20057 USA

5 Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts, 02115 USA

For all author emails, please log on.

BMC Neuroscience 2011, 12:7  doi:10.1186/1471-2202-12-7

Published: 13 January 2011

Additional files

Additional File 1:

Exorh protein is expressed without a significant rhythm (A, B) Embryos were injected with (A) control or (B) exorh atg MO, fixed at 64 hpf, and processed for fluorescent whole mount immunostaining with the anti-bovine Rhodopsin antibody 4D2. (A) Control embryos have robust fluorescent signal in the pineal organ that is (B) severely reduced in Exorh depleted embryos. (C-C') Embryos injected with beta-galactosidase mRNA have undetectable levels of immunoreactivity with the 4D2 antibody at 8 hpf. (D-D') In contrast, embryos injected with exorh mRNA show strong antibody staining at 8 hpf. (E, F) Embryos were fixed in a circadian time course and then processed for 4D2 antibody staining. One-way Analysis of Variance (ANOVA) and Tukey's analysis revealed no significant changes in pineal Exorh protein levels that followed a daily rhythm (n≥9 embryos per time point). However, a few time points were significantly different (p ≤ 0.05) from each other including 72 and 108 hpf, 72 and 116 hpf, 76 and 116 hpf, and 80 and 116 hpf. (A-B, and E) are dorsal views, anterior to the top and (C-D') are lateral views. (C') and (D') are higher magnification images of the regions boxed in (C) and (D), respectively. Scale bars = 20 μm for (A-B, C', D', E) and 100 μm for (C, D).

Format: PDF Size: 737KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional File 2:

Two days in a L/D cycle is not sufficient to initiate robust circadian cycling of aanat2 expression. Embryos were raised in a 14:10 h L/D cycle. At 47.5 hpf, ZT 23.5, a set of embryos was transferred to constant dark, constant temperature conditions. Embryos were fixed at the indicated stages and ZT and (A) processed for WISH for expression of aanat2 and (B) the WISH signal was quantified. Note the time of peak (closed arrowheads) expression is similar between the cyc embryos and their WT siblings. All images are dorsal views, anterior to the top. For the samples in a L/D cycle, position within the photoperiod is indicated by ZT and light conditions by the white (light period) and black (dark period) bars. For the constant dark samples, the original L/D cycle is indicated by the ZT and the black (original dark period) and dark grey (original light period) bars. Experiment was repeated two times with similar results, and representative images from one of the experiments are shown. Scale bars = 20 μm.

Format: PDF Size: 975KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional File 3:

Comparison between larva processed for WISH with antisense and sense probes reveals low background staining. Embryos were raised in a 14:10 h L/D cycle and fixed and processed for WISH using aanat2 antisense or sense probe. Note that the sense probe produces no detectable signal, as it would recognize antisense mRNA, which should not be present. The brown regions are melanocytes in the skin, which have a natural pigment. All images are dorsal views, anterior to the top, with the pineal indicated (closed arrowheads). Position within the photoperiod is indicated by ZT and light conditions by the white (light period) and black (dark period) bars. Representative images are shown. Scale bar = 30 μm.

Format: PDF Size: 463KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data