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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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PER and CLK expression in ES 12 – ES 15 embryos. 0 h – 24 h wild-type (W.T.), per01, or ClkJrk embryos were collected at CT33, immunostained with CLK and PER antisera, and imaged by confocal microscopy. (A, B) 12 μm Z-series projection of PER (A) or CLK + PER (B) IR in the VNC of a W.T. embryo during ES 12. (C, D) 20 μm Z-series projection of PER (C) or CLK + PER (D) IR in the VNC of a W.T. embryo during ES 13. (E, F) 18 μm Z-series projection of PER (E) or CLK + PER (F) IR in the brain (box) and VNC of a W.T. embryo during ES 14. (G, H) 30 μm Z-series projection of PER (G) or CLK + PER (H) IR in the brain (box) and VNC of a W.T. embryo during ES 15. (I) 42 μm Z-series projection of PER (E) or CLK + PER (F) IR of a per01 embryo during ES 15. (J) 22 μm Z-series projection of PER (E) or CLK + PER (F) IR in the brain (box) and VNC of a ClkJrk embryo during ES 15. Co-localization of CLK (red) and PER (green) is shown as yellow. All images are representative of three or more independent experiments.
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Figure 7: PER and CLK expression in ES 12 – ES 15 embryos. 0 h – 24 h wild-type (W.T.), per01, or ClkJrk embryos were collected at CT33, immunostained with CLK and PER antisera, and imaged by confocal microscopy. (A, B) 12 μm Z-series projection of PER (A) or CLK + PER (B) IR in the VNC of a W.T. embryo during ES 12. (C, D) 20 μm Z-series projection of PER (C) or CLK + PER (D) IR in the VNC of a W.T. embryo during ES 13. (E, F) 18 μm Z-series projection of PER (E) or CLK + PER (F) IR in the brain (box) and VNC of a W.T. embryo during ES 14. (G, H) 30 μm Z-series projection of PER (G) or CLK + PER (H) IR in the brain (box) and VNC of a W.T. embryo during ES 15. (I) 42 μm Z-series projection of PER (E) or CLK + PER (F) IR of a per01 embryo during ES 15. (J) 22 μm Z-series projection of PER (E) or CLK + PER (F) IR in the brain (box) and VNC of a ClkJrk embryo during ES 15. Co-localization of CLK (red) and PER (green) is shown as yellow. All images are representative of three or more independent experiments.

Mentions: To determine whether CLK is expressed in presumptive brain oscillator cells, embryos between 0 h and 24 h old were collected at CT33 and co-immunostained with PER and CLK. PER IR is detected in a segmented pattern along VNC as early as ES 12 (Fig. 7A, B), consistent with previous in situ hybridization results in embryos [24,25]. PER in the VNC encompasses more cells, increases in intensity, and expands into the brain by ES 15 (Fig. 7C–H). Loss of PER IR in per01 mutant confirms that this IR represents true PER expression (Fig. 7I). Surprisingly, no CLK IR is detected in PER-expressing cells, indicating that per is not activated by CLK-CYC during these early developmental stages. The CLK-CYC independent activation of per is similar to the situation in ovaries, where PER expression is not associated with circadian oscillator function [36]. To ensure that CLK expression below detectable levels does not activate PER at ES 12–15, ClkJrk embryos were immunostained for PER. These ClkJrk embryos show PER staining in the brain and VNC identical to that in wild-type embryos at ES 15 (Fig. 7J).


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

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

PER and CLK expression in ES 12 – ES 15 embryos. 0 h – 24 h wild-type (W.T.), per01, or ClkJrk embryos were collected at CT33, immunostained with CLK and PER antisera, and imaged by confocal microscopy. (A, B) 12 μm Z-series projection of PER (A) or CLK + PER (B) IR in the VNC of a W.T. embryo during ES 12. (C, D) 20 μm Z-series projection of PER (C) or CLK + PER (D) IR in the VNC of a W.T. embryo during ES 13. (E, F) 18 μm Z-series projection of PER (E) or CLK + PER (F) IR in the brain (box) and VNC of a W.T. embryo during ES 14. (G, H) 30 μm Z-series projection of PER (G) or CLK + PER (H) IR in the brain (box) and VNC of a W.T. embryo during ES 15. (I) 42 μm Z-series projection of PER (E) or CLK + PER (F) IR of a per01 embryo during ES 15. (J) 22 μm Z-series projection of PER (E) or CLK + PER (F) IR in the brain (box) and VNC of a ClkJrk embryo during ES 15. Co-localization of CLK (red) and PER (green) is shown as yellow. All images are representative of three or more independent experiments.
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Figure 7: PER and CLK expression in ES 12 – ES 15 embryos. 0 h – 24 h wild-type (W.T.), per01, or ClkJrk embryos were collected at CT33, immunostained with CLK and PER antisera, and imaged by confocal microscopy. (A, B) 12 μm Z-series projection of PER (A) or CLK + PER (B) IR in the VNC of a W.T. embryo during ES 12. (C, D) 20 μm Z-series projection of PER (C) or CLK + PER (D) IR in the VNC of a W.T. embryo during ES 13. (E, F) 18 μm Z-series projection of PER (E) or CLK + PER (F) IR in the brain (box) and VNC of a W.T. embryo during ES 14. (G, H) 30 μm Z-series projection of PER (G) or CLK + PER (H) IR in the brain (box) and VNC of a W.T. embryo during ES 15. (I) 42 μm Z-series projection of PER (E) or CLK + PER (F) IR of a per01 embryo during ES 15. (J) 22 μm Z-series projection of PER (E) or CLK + PER (F) IR in the brain (box) and VNC of a ClkJrk embryo during ES 15. Co-localization of CLK (red) and PER (green) is shown as yellow. All images are representative of three or more independent experiments.
Mentions: To determine whether CLK is expressed in presumptive brain oscillator cells, embryos between 0 h and 24 h old were collected at CT33 and co-immunostained with PER and CLK. PER IR is detected in a segmented pattern along VNC as early as ES 12 (Fig. 7A, B), consistent with previous in situ hybridization results in embryos [24,25]. PER in the VNC encompasses more cells, increases in intensity, and expands into the brain by ES 15 (Fig. 7C–H). Loss of PER IR in per01 mutant confirms that this IR represents true PER expression (Fig. 7I). Surprisingly, no CLK IR is detected in PER-expressing cells, indicating that per is not activated by CLK-CYC during these early developmental stages. The CLK-CYC independent activation of per is similar to the situation in ovaries, where PER expression is not associated with circadian oscillator function [36]. To ensure that CLK expression below detectable levels does not activate PER at ES 12–15, ClkJrk embryos were immunostained for PER. These ClkJrk embryos show PER staining in the brain and VNC identical to that in wild-type embryos at ES 15 (Fig. 7J).

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