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CLOCK expression identifies developing circadian oscillator neurons in the brains of Drosophila embryos.

Houl JH, Ng F, Taylor P, Hardin PE - BMC Neurosci (2008)

Bottom Line: Although PER expression in CLK-negative cells continues in ClkJrk embryos, PER expression in cells that co-express PER and CLK is eliminated.These data demonstrate that brain oscillator neurons begin development during embryogenesis, that PER expression in non-oscillator cells is CLK-independent, and that oscillator phase is an intrinsic characteristic of brain oscillator neurons.These results define the temporal and spatial coordinates of factors that initiate Clk expression, imply that circadian photoreceptors are not activated until the end of embryogenesis, and suggest that PER functions in a different capacity before oscillator cell development is initiated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Research on Biological Clocks, Department of Biology, Texas A&M University, 3258 TAMU, College Station, TX 77843, USA. jhoul@mail.bio.tamu.edu

ABSTRACT

Background: The Drosophila circadian oscillator is composed of transcriptional feedback loops in which CLOCK-CYCLE (CLK-CYC) heterodimers activate their feedback regulators period (per) and timeless (tim) via E-box mediated transcription. These feedback loop oscillators are present in distinct clusters of dorsal and lateral neurons in the adult brain, but how this pattern of expression is established during development is not known. Since CLK is required to initiate feedback loop function, defining the pattern of CLK expression in embryos and larvae will shed light on oscillator neuron development.

Results: A novel CLK antiserum is used to show that CLK expression in the larval CNS and adult brain is limited to circadian oscillator cells. CLK is initially expressed in presumptive small ventral lateral neurons (s-LNvs), dorsal neurons 2 s (DN2s), and dorsal neuron 1 s (DN1s) at embryonic stage (ES) 16, and this CLK expression pattern persists through larval development. PER then accumulates in all CLK-expressing cells except presumptive DN2s during late ES 16 and ES 17, consistent with the delayed accumulation of PER in adult oscillator neurons and antiphase cycling of PER in larval DN2s. PER is also expressed in non-CLK-expressing cells in the embryonic CNS starting at ES 12. Although PER expression in CLK-negative cells continues in ClkJrk embryos, PER expression in cells that co-express PER and CLK is eliminated.

Conclusion: These data demonstrate that brain oscillator neurons begin development during embryogenesis, that PER expression in non-oscillator cells is CLK-independent, and that oscillator phase is an intrinsic characteristic of brain oscillator neurons. These results define the temporal and spatial coordinates of factors that initiate Clk expression, imply that circadian photoreceptors are not activated until the end of embryogenesis, and suggest that PER functions in a different capacity before oscillator cell development is initiated.

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DAC is co-expressed with CLK in the CNS of L3 larvae. L3 larval brains were dissected and fixed at ZT1, immunostained with CLK and DAC antibodies, and imaged by confocal microscopy. (A-C) Projected Z-series images of the CNS, where dorsal is at the top. (A) DAC expression in the CNS. Arrows denote DAC immunoreactivity in Kenyon cells (KC) and the optic lobe (OL). (B) CLK expression in the same brain as panel A. Arrows denote CLK immunoreactivity in KCs and the OL. (C) Superimposed dual laser image of DAC and CLK immunostaining in the same larval CNS as panels A and B. Co-localization of DAC (green) and CLK (red) is seen as yellow. Arrowheads, CLK positive/DAC negative cells. (D-F) Magnified 1 μm optical section through the left hemisphere of an L3 larval brain. OL, optic lobe. (D) DAC immunostaining. (E) CLK immunostaining. (F) Superimposed dual laser image of DAC and CLK immunostaining. Arrowheads, CLK positive/DAC negative cells. Images are representative of three independent experiments. Six or more larval CNSs were examined in each experiment. Z-series images are projections over 32 optical sections at 2.5 μm per optical section.
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Figure 1: DAC is co-expressed with CLK in the CNS of L3 larvae. L3 larval brains were dissected and fixed at ZT1, immunostained with CLK and DAC antibodies, and imaged by confocal microscopy. (A-C) Projected Z-series images of the CNS, where dorsal is at the top. (A) DAC expression in the CNS. Arrows denote DAC immunoreactivity in Kenyon cells (KC) and the optic lobe (OL). (B) CLK expression in the same brain as panel A. Arrows denote CLK immunoreactivity in KCs and the OL. (C) Superimposed dual laser image of DAC and CLK immunostaining in the same larval CNS as panels A and B. Co-localization of DAC (green) and CLK (red) is seen as yellow. Arrowheads, CLK positive/DAC negative cells. (D-F) Magnified 1 μm optical section through the left hemisphere of an L3 larval brain. OL, optic lobe. (D) DAC immunostaining. (E) CLK immunostaining. (F) Superimposed dual laser image of DAC and CLK immunostaining. Arrowheads, CLK positive/DAC negative cells. Images are representative of three independent experiments. Six or more larval CNSs were examined in each experiment. Z-series images are projections over 32 optical sections at 2.5 μm per optical section.

Mentions: We previously demonstrated CLK immunostaining in all circadian oscillator cells and some non-oscillator cells in adults [26]. One group of non-oscillator cells that showed CLK immunostaining was Kenyon Cells (KCs), which are involved in olfactory learning and memory [27]. To characterize CLK immunostaining during development, we co-stained L3 larval CNSs with CLK, oscillator cell marker PER, and the KC cell marker DAC [23,28-30]. As expected, we observed CLK staining in every PER-expressing cell, but also detected CLK in every DAC-expressing cell (Fig. 1). This surprising correspondence between CLK and DAC expression in non-oscillator cells suggests a relationship between Clk and dac activation: Clk and dac are activated by the same activator, Clk activates dac, or dac activates Clk. Alternately, CLK and DAC co-immunostaining could also result from cross-reactivity of CLK antiserum to DAC, though little, if any, sequence identity is evident between these two proteins (data not shown). Because CLK and DAC run at a similar apparent molecular weight of ~130 kDa on western blots [13,31], we tested whether our CLK antiserum (GP47) cross-reacted with in vitro translated DAC. As expected, GP47 detected CLK on western blots, but this CLK antiserum also detected DAC (Fig. 2A). Competition with purified DAC protein blocked GP47 detection of DAC on western blots (Fig. 2B), confirming DAC cross-reactivity. Likewise, incubating L3 CNSs with purified DAC specifically blocks GP47 immunostaining in DAC expressing non-oscillator cells (Fig. 2C). GP47 antiserum detects more than the canonical 4 to 5 LNvs, 2 DN2s, and 2 DN1s in L3 brains blocked with purified DAC. Although these additional cells could be due to incomplete blocking by DAC, they may also represent DN3s, LNds, and/or l-LNvs, which have been detected previously in L3 brains [23,32]. CLK immunostaining in non-oscillator cells is also eliminated in the CNS of dac03 mutant larvae (Fig. 3), which lack DAC protein [33]. Taken together, these results demonstrate that CLK immunoreactivity (IR) in non-oscillator cells is due to cross-reactivity with DAC, which implies that CLK expression is limited to oscillator cells.


CLOCK expression identifies developing circadian oscillator neurons in the brains of Drosophila embryos.

Houl JH, Ng F, Taylor P, Hardin PE - BMC Neurosci (2008)

DAC is co-expressed with CLK in the CNS of L3 larvae. L3 larval brains were dissected and fixed at ZT1, immunostained with CLK and DAC antibodies, and imaged by confocal microscopy. (A-C) Projected Z-series images of the CNS, where dorsal is at the top. (A) DAC expression in the CNS. Arrows denote DAC immunoreactivity in Kenyon cells (KC) and the optic lobe (OL). (B) CLK expression in the same brain as panel A. Arrows denote CLK immunoreactivity in KCs and the OL. (C) Superimposed dual laser image of DAC and CLK immunostaining in the same larval CNS as panels A and B. Co-localization of DAC (green) and CLK (red) is seen as yellow. Arrowheads, CLK positive/DAC negative cells. (D-F) Magnified 1 μm optical section through the left hemisphere of an L3 larval brain. OL, optic lobe. (D) DAC immunostaining. (E) CLK immunostaining. (F) Superimposed dual laser image of DAC and CLK immunostaining. Arrowheads, CLK positive/DAC negative cells. Images are representative of three independent experiments. Six or more larval CNSs were examined in each experiment. Z-series images are projections over 32 optical sections at 2.5 μm per optical section.
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Figure 1: DAC is co-expressed with CLK in the CNS of L3 larvae. L3 larval brains were dissected and fixed at ZT1, immunostained with CLK and DAC antibodies, and imaged by confocal microscopy. (A-C) Projected Z-series images of the CNS, where dorsal is at the top. (A) DAC expression in the CNS. Arrows denote DAC immunoreactivity in Kenyon cells (KC) and the optic lobe (OL). (B) CLK expression in the same brain as panel A. Arrows denote CLK immunoreactivity in KCs and the OL. (C) Superimposed dual laser image of DAC and CLK immunostaining in the same larval CNS as panels A and B. Co-localization of DAC (green) and CLK (red) is seen as yellow. Arrowheads, CLK positive/DAC negative cells. (D-F) Magnified 1 μm optical section through the left hemisphere of an L3 larval brain. OL, optic lobe. (D) DAC immunostaining. (E) CLK immunostaining. (F) Superimposed dual laser image of DAC and CLK immunostaining. Arrowheads, CLK positive/DAC negative cells. Images are representative of three independent experiments. Six or more larval CNSs were examined in each experiment. Z-series images are projections over 32 optical sections at 2.5 μm per optical section.
Mentions: We previously demonstrated CLK immunostaining in all circadian oscillator cells and some non-oscillator cells in adults [26]. One group of non-oscillator cells that showed CLK immunostaining was Kenyon Cells (KCs), which are involved in olfactory learning and memory [27]. To characterize CLK immunostaining during development, we co-stained L3 larval CNSs with CLK, oscillator cell marker PER, and the KC cell marker DAC [23,28-30]. As expected, we observed CLK staining in every PER-expressing cell, but also detected CLK in every DAC-expressing cell (Fig. 1). This surprising correspondence between CLK and DAC expression in non-oscillator cells suggests a relationship between Clk and dac activation: Clk and dac are activated by the same activator, Clk activates dac, or dac activates Clk. Alternately, CLK and DAC co-immunostaining could also result from cross-reactivity of CLK antiserum to DAC, though little, if any, sequence identity is evident between these two proteins (data not shown). Because CLK and DAC run at a similar apparent molecular weight of ~130 kDa on western blots [13,31], we tested whether our CLK antiserum (GP47) cross-reacted with in vitro translated DAC. As expected, GP47 detected CLK on western blots, but this CLK antiserum also detected DAC (Fig. 2A). Competition with purified DAC protein blocked GP47 detection of DAC on western blots (Fig. 2B), confirming DAC cross-reactivity. Likewise, incubating L3 CNSs with purified DAC specifically blocks GP47 immunostaining in DAC expressing non-oscillator cells (Fig. 2C). GP47 antiserum detects more than the canonical 4 to 5 LNvs, 2 DN2s, and 2 DN1s in L3 brains blocked with purified DAC. Although these additional cells could be due to incomplete blocking by DAC, they may also represent DN3s, LNds, and/or l-LNvs, which have been detected previously in L3 brains [23,32]. CLK immunostaining in non-oscillator cells is also eliminated in the CNS of dac03 mutant larvae (Fig. 3), which lack DAC protein [33]. Taken together, these results demonstrate that CLK immunoreactivity (IR) in non-oscillator cells is due to cross-reactivity with DAC, which implies that CLK expression is limited to oscillator cells.

Bottom Line: Although PER expression in CLK-negative cells continues in ClkJrk embryos, PER expression in cells that co-express PER and CLK is eliminated.These data demonstrate that brain oscillator neurons begin development during embryogenesis, that PER expression in non-oscillator cells is CLK-independent, and that oscillator phase is an intrinsic characteristic of brain oscillator neurons.These results define the temporal and spatial coordinates of factors that initiate Clk expression, imply that circadian photoreceptors are not activated until the end of embryogenesis, and suggest that PER functions in a different capacity before oscillator cell development is initiated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Research on Biological Clocks, Department of Biology, Texas A&M University, 3258 TAMU, College Station, TX 77843, USA. jhoul@mail.bio.tamu.edu

ABSTRACT

Background: The Drosophila circadian oscillator is composed of transcriptional feedback loops in which CLOCK-CYCLE (CLK-CYC) heterodimers activate their feedback regulators period (per) and timeless (tim) via E-box mediated transcription. These feedback loop oscillators are present in distinct clusters of dorsal and lateral neurons in the adult brain, but how this pattern of expression is established during development is not known. Since CLK is required to initiate feedback loop function, defining the pattern of CLK expression in embryos and larvae will shed light on oscillator neuron development.

Results: A novel CLK antiserum is used to show that CLK expression in the larval CNS and adult brain is limited to circadian oscillator cells. CLK is initially expressed in presumptive small ventral lateral neurons (s-LNvs), dorsal neurons 2 s (DN2s), and dorsal neuron 1 s (DN1s) at embryonic stage (ES) 16, and this CLK expression pattern persists through larval development. PER then accumulates in all CLK-expressing cells except presumptive DN2s during late ES 16 and ES 17, consistent with the delayed accumulation of PER in adult oscillator neurons and antiphase cycling of PER in larval DN2s. PER is also expressed in non-CLK-expressing cells in the embryonic CNS starting at ES 12. Although PER expression in CLK-negative cells continues in ClkJrk embryos, PER expression in cells that co-express PER and CLK is eliminated.

Conclusion: These data demonstrate that brain oscillator neurons begin development during embryogenesis, that PER expression in non-oscillator cells is CLK-independent, and that oscillator phase is an intrinsic characteristic of brain oscillator neurons. These results define the temporal and spatial coordinates of factors that initiate Clk expression, imply that circadian photoreceptors are not activated until the end of embryogenesis, and suggest that PER functions in a different capacity before oscillator cell development is initiated.

Show MeSH