Figure 1.
Schematic Map of the per3-luc Construct
The top graphic shows the exon-intron structures of per3 and the flanking gene. The BAC clone 8M06 screened for the first coding exon of per3 is approximately 72 kb long, and extends from about 26 kb upstream of exon 1 to intron 19 of per3. The bottom graphic shows the magnified view of the first coding exon in the BAC 8M06 and the modified BAC construct. The white and black boxes represent noncoding and coding sequences, respectively, of the first coding exon of per3. The coding sequence of this exon was replaced with an approximately 3-kb fragment containing luc and Kmr. Arrows under the construct represent primers used for the screening of transgenic lines.
Figure 2.
Bioluminescence in Embryos That Experienced Different Numbers of LD Cycles during Development
Embryos hemizygous for per3-luc were collected and monitored for bioluminescence while exposed to different numbers of 14:10 LD cycles starting on day 1 postfertilization followed by DD. (A) One LD, (B) one LD, (C) two LD, (D) three LD, (E) four LD, (F) five LD, (G) six LD, and (H) six LD. In (I), embryos were exposed to two LDs, one on day 1 and the other on day 6 of development. Black and white bars on top of each plot represent the times when the lights were off and on, respectively. Since overall bioluminescence levels can vary among clutches and experiments, normalized bioluminescence was averaged and plotted in each graph. Number of animals that were averaged is given at top right corner of each plot. Error bars represent standard error of the mean (SEM) For the one-LD and six-LD groups, plots for two experiments are shown here. These experiments showed small differences in developmental profiles, possibly due to differences in room temperature (about 1 °C), therefore could not be pooled. For the two- to five-LD groups, data from two experiments were pooled. The small but abrupt increase of luminescence that occurred only during the light period of LD cycles is considered an artifact made visible by low bioluminescence counts during the first several days of development, because the same level of fluctuation was observed in empty wells under LD condition.
Table 1.
Rhythmicity and Periods of Larval Zebrafish: Varying Number of LD Cycles
Table 2.
Rhythmicity and Periods of Larval Zebrafish: Varying LD and Temperature
Figure 3.
Temporal Expression of luc mRNA Is Similar to That of per3 during Development
Embryos hemizygous for per3-luc were collected from naturally breeding parents and kept in 14:10 LD cycles (lights on at 8 A.M. CST) at 22 °C for 6 d. Larval fish were shifted to DD at the end of the light phase on day 6. Total RNA was extracted from 2- to 3-d-old embryos and 7- to 8-d-old larval fish, and was subjected to real-time PCR for per3 and luc mRNA levels.
(A) Expression of per3 mRNA per embryo on day 3 was determined every 6 h starting at 10 A.M. (2 h after lights-on).
(B) The per3 mRNA per animal on day 8 was measured every 4 h starting at 10 A.M.
(C) Levels of luc mRNA on day 3 were determined as in (A). White and black bars at the bottom represent light and dark phases, respectively, for (A) and (C).
(D) Cycling of luc mRNA on day 8 was determined as in (B). The gray and black bars represent the time when the light would have been on and off, respectively, had the LD cycles continued.
For each of per3 and luc, mRNA levels were normalized to the peak level on day 8 (10 A.M. time point). The y-axis scales were set at 120% maximum for all plots to allow direct comparison of mRNA extracted on days 3 and 8. The x-axis scales are given in both hours and days postfertilization to facilitate the comparison with Figure 2. In order to show more detailed temporal profiles of mRNAs on day 3, plots with smaller y-axis scales were shown in the insets at top right corners of (A) and (C). Each plot is the average of three identical experiments, and error bars represent SEM. An identical experiment was also done at 24 °C with essentially the same results (unpublished data).
Figure 4.
Effects of Temperature on Development of per3-luc Expression
Transgenic embryos were entrained by two, four, or six LD cycles at either 22 °C or 28.5 °C, and monitored for bioluminescence in DD at 21–24 °C. (A) Two LDs at 22 °C, (B) two LDs at 28.5 °C, (C) four LDs at 22 °C, (D) four LDs at 28.5 °C, (E) six LDs at 22 °C, and (F) six LDs at 28.5 °C. The insets in (E) and (F) show the last 3 d of the record with magnified y-axis scales. Black and white bars on top of each plot represent the times when the lights were off and on, respectively. Actual amount of bioluminescence in cps is averaged and plotted in each graph. Number of animals that were averaged is given at top right corner of each plot. Error bars represent SEM. Results of one of two identical experiments with similar results are shown here.
Figure 5.
Levels of per3 and luc mRNA Are Elevated by High Temperatures during Development
(A) A schematic diagram showing how embryos were entrained and collected for RNA extraction. Embryos were entrained in 14:10 LD cycles (lights on at 8 A.M.; lights off at 10 P.M. CST) at two different temperatures, 22 °C and 28.5 °C. On day 6, half of the animals were sacrificed for RNA at 10 A.M. (2 h after lights-on) and at 10 P.M. (at lights-off). The rest of the animals were transferred to DD at 22 °C at 10 P.M. on that day, and sacrificed for RNA on day 10 at 10 A.M. and 10 P.M. The white and black bars represent day and night, respectively, and the gray bars the time at which lights would have been on had the LD cycles continued. The arrowheads indicate the time at which the animals were sacrificed for RNA extraction.
(B) Relative mRNA level per animal for per3 on days 6 and 10 of the experiment quantified by real-time qPCR. The levels were normalized to the value of the 10 A.M. time point on day 6 at 28.5 °C.
(C) Relative RNA level per animal for luc measured from the same samples used in (B). For both (B) and (C), averages of three experiments are shown. Error bars represent SEM.
Figure 6.
Bioluminescence Rhythms Mediated by per3-luc in DD and LD Measured for Six Days
(A) Average plot of bioluminescence rhythms in DD. Animals were entrained in 14:10 LD cycles for 6 d at 22 °C and tested for approximately 6.5 d in DD.
(B) Representative plot of bioluminescence rhythm in DD for an individual.
(C) Average plot of bioluminescence rhythms in 14:10 LD cycles. Animals were entrained in 6 LD cycles at 22 °C prior to the monitoring.
(D) Individual plot of bioluminescence rhythm in LD cycles. For each of DD and LD experiments, two experiments have been performed with essentially the same results. Only one of two experiments is shown for each of DD and LD. The first 12 h of data were deleted from each plot. Black and white bars on top of each plot represent the time when lights were off and on, respectively. Numbers of animals that were averaged is given at top right corner of (A) and (C). Error bars represent SEM in (A) and (C).