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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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Truncated CLKJrk protein accumulates to low levels. (A) Western blot containing 100 μg of head extract from ClkJrk or wild-type flies collected at ZT1 and probed with CLK GP50 antiserum. Full length CLK and truncated CLKJrk proteins are denoted in short (left) or long (right) exposures of the blot. (B) Brains from ClkJrk L3 larvae were dissected and fixed at ZT1, immunostained with a lower dilution of CLK GP50 antibody (diluted 1:1000 instead of 1:3000), and imaged by confocal microscopy. Images show a 28 μm Z-series projection of a right brain hemisphere, where dorsal is at the top. CLK GP50 immunostaining is detected in DN1*, DN2 and LN cells. DN1* and DN2 cells are as defined in the legend for Fig. 2, and LN cells include s-LNvs, l-LNvs and LNds. All images are representative of three or more independent experiments.
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Figure 6: Truncated CLKJrk protein accumulates to low levels. (A) Western blot containing 100 μg of head extract from ClkJrk or wild-type flies collected at ZT1 and probed with CLK GP50 antiserum. Full length CLK and truncated CLKJrk proteins are denoted in short (left) or long (right) exposures of the blot. (B) Brains from ClkJrk L3 larvae were dissected and fixed at ZT1, immunostained with a lower dilution of CLK GP50 antibody (diluted 1:1000 instead of 1:3000), and imaged by confocal microscopy. Images show a 28 μm Z-series projection of a right brain hemisphere, where dorsal is at the top. CLK GP50 immunostaining is detected in DN1*, DN2 and LN cells. DN1* and DN2 cells are as defined in the legend for Fig. 2, and LN cells include s-LNvs, l-LNvs and LNds. All images are representative of three or more independent experiments.

Mentions: Additional CLK antisera that we had generated were tested for cross-reactivity to DAC on western blots, and CLK antiserum GP50 showed no cross-reactivity to DAC (Fig. 4A). Based on the lack of CLK IR in non-oscillator cells from dac03 mutant larvae and L3 CNSs blocked with DAC antigen, we expected GP50 to detect CLK IR only in oscillator cells. Indeed, CLK and PER co-immunostained several clusters of cells including sLNvs, DN2s and DN1s in L3 larvae (Fig. 4B), but do not detect cells elsewhere in the CNS (compare CLK GP50 immunostaining with GP47 immunostaining in Fig. 1B, E), showing that CLK GP50 antiserum specifically detects oscillator cells in L3 brains. Four additional clusters of brain oscillator neurons are present in adult brains: LNds, l-LNvs, LPNs and DN3s [34,35]. Brains from adults collected at ZT23 were co-immunostained with GP50 and PER to determine if CLK expression is limited to oscillator neurons. CLK IR is also detected exclusively in oscillator cells from adult brains and is reduced or eliminated in brains from ClkJrk adults (Fig. 5), which express very low levels of truncated CLK protein (Fig. 6). These results demonstrate that GP50 specifically detects CLK, and that CLK is expressed exclusively in circadian oscillator cells in wild-type adult brains. Given that CLK is detected specifically in brain oscillator neurons in adults and L3 larvae, we used CLK GP50 antiserum to determine when brain oscillator neurons first appear during development.


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

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

Truncated CLKJrk protein accumulates to low levels. (A) Western blot containing 100 μg of head extract from ClkJrk or wild-type flies collected at ZT1 and probed with CLK GP50 antiserum. Full length CLK and truncated CLKJrk proteins are denoted in short (left) or long (right) exposures of the blot. (B) Brains from ClkJrk L3 larvae were dissected and fixed at ZT1, immunostained with a lower dilution of CLK GP50 antibody (diluted 1:1000 instead of 1:3000), and imaged by confocal microscopy. Images show a 28 μm Z-series projection of a right brain hemisphere, where dorsal is at the top. CLK GP50 immunostaining is detected in DN1*, DN2 and LN cells. DN1* and DN2 cells are as defined in the legend for Fig. 2, and LN cells include s-LNvs, l-LNvs and LNds. All images are representative of three or more independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: Truncated CLKJrk protein accumulates to low levels. (A) Western blot containing 100 μg of head extract from ClkJrk or wild-type flies collected at ZT1 and probed with CLK GP50 antiserum. Full length CLK and truncated CLKJrk proteins are denoted in short (left) or long (right) exposures of the blot. (B) Brains from ClkJrk L3 larvae were dissected and fixed at ZT1, immunostained with a lower dilution of CLK GP50 antibody (diluted 1:1000 instead of 1:3000), and imaged by confocal microscopy. Images show a 28 μm Z-series projection of a right brain hemisphere, where dorsal is at the top. CLK GP50 immunostaining is detected in DN1*, DN2 and LN cells. DN1* and DN2 cells are as defined in the legend for Fig. 2, and LN cells include s-LNvs, l-LNvs and LNds. All images are representative of three or more independent experiments.
Mentions: Additional CLK antisera that we had generated were tested for cross-reactivity to DAC on western blots, and CLK antiserum GP50 showed no cross-reactivity to DAC (Fig. 4A). Based on the lack of CLK IR in non-oscillator cells from dac03 mutant larvae and L3 CNSs blocked with DAC antigen, we expected GP50 to detect CLK IR only in oscillator cells. Indeed, CLK and PER co-immunostained several clusters of cells including sLNvs, DN2s and DN1s in L3 larvae (Fig. 4B), but do not detect cells elsewhere in the CNS (compare CLK GP50 immunostaining with GP47 immunostaining in Fig. 1B, E), showing that CLK GP50 antiserum specifically detects oscillator cells in L3 brains. Four additional clusters of brain oscillator neurons are present in adult brains: LNds, l-LNvs, LPNs and DN3s [34,35]. Brains from adults collected at ZT23 were co-immunostained with GP50 and PER to determine if CLK expression is limited to oscillator neurons. CLK IR is also detected exclusively in oscillator cells from adult brains and is reduced or eliminated in brains from ClkJrk adults (Fig. 5), which express very low levels of truncated CLK protein (Fig. 6). These results demonstrate that GP50 specifically detects CLK, and that CLK is expressed exclusively in circadian oscillator cells in wild-type adult brains. Given that CLK is detected specifically in brain oscillator neurons in adults and L3 larvae, we used CLK GP50 antiserum to determine when brain oscillator neurons first appear during development.

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
Related in: MedlinePlus