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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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CLK and PER expression in embryos during ES 16 and ES 17. 0 h – 24 h wild-type (W.T.) embryos were collected at CT33, immunostained with CLK and PER antisera, and imaged by confocal microscopy. Anterior is to the left and dorsal is on the top. (A-C) 58 μm Z-series projection of PER (A), CLK (B) or CLK + PER (C) IR in the brain (box) and VNC during early ES 16. (A'-C') Magnified view of the boxed brain regions in panels A-C, respectively. (D-F) 18 μm Z-series projection of PER (D), CLK (E) or CLK + PER (F) IR in the brain (box) and VNC during late ES 16. (D'-F') Magnified view of the boxed brain regions in panels D-F, respectively. (G-I) 20 μm Z-series projection of PER (G), CLK (H) or CLK + PER (I) IR in the brain (box) and VNC during ES 17. (G'-I') Magnified view of the boxed brain regions in panels G-I, respectively. 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 8: CLK and PER expression in embryos during ES 16 and ES 17. 0 h – 24 h wild-type (W.T.) embryos were collected at CT33, immunostained with CLK and PER antisera, and imaged by confocal microscopy. Anterior is to the left and dorsal is on the top. (A-C) 58 μm Z-series projection of PER (A), CLK (B) or CLK + PER (C) IR in the brain (box) and VNC during early ES 16. (A'-C') Magnified view of the boxed brain regions in panels A-C, respectively. (D-F) 18 μm Z-series projection of PER (D), CLK (E) or CLK + PER (F) IR in the brain (box) and VNC during late ES 16. (D'-F') Magnified view of the boxed brain regions in panels D-F, respectively. (G-I) 20 μm Z-series projection of PER (G), CLK (H) or CLK + PER (I) IR in the brain (box) and VNC during ES 17. (G'-I') Magnified view of the boxed brain regions in panels G-I, respectively. Co-localization of CLK (red) and PER (green) is shown as yellow. All images are representative of three or more independent experiments.

Mentions: Early in ES 16, weak CLK immunoreactivity is first detected in brain cells that do not express PER (Fig. 8A–C). During mid to late ES 16, CLK expression becomes stronger and expands to additional cells in the dorsal brain (Fig. 8D–F). In addition, PER starts to be detected in CLK positive cells in the ventral portion of the brain (Fig. 8F). By ES 17, CLK positive cells form three distinct clusters in each brain hemisphere, with two dorsal cells, four ventral cells, and two cells between and slightly posterior to these dorsal and ventral clusters (Fig. 8G–I). The positions of these CLK positive cells are reminiscent of oscillator cells in larvae; a dorsal cluster of DN1s, a ventral cluster of s-LNvs, and a medial cluster of DN2s [23,37].


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

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

CLK and PER expression in embryos during ES 16 and ES 17. 0 h – 24 h wild-type (W.T.) embryos were collected at CT33, immunostained with CLK and PER antisera, and imaged by confocal microscopy. Anterior is to the left and dorsal is on the top. (A-C) 58 μm Z-series projection of PER (A), CLK (B) or CLK + PER (C) IR in the brain (box) and VNC during early ES 16. (A'-C') Magnified view of the boxed brain regions in panels A-C, respectively. (D-F) 18 μm Z-series projection of PER (D), CLK (E) or CLK + PER (F) IR in the brain (box) and VNC during late ES 16. (D'-F') Magnified view of the boxed brain regions in panels D-F, respectively. (G-I) 20 μm Z-series projection of PER (G), CLK (H) or CLK + PER (I) IR in the brain (box) and VNC during ES 17. (G'-I') Magnified view of the boxed brain regions in panels G-I, respectively. Co-localization of CLK (red) and PER (green) is shown as yellow. All images are representative of three or more independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 8: CLK and PER expression in embryos during ES 16 and ES 17. 0 h – 24 h wild-type (W.T.) embryos were collected at CT33, immunostained with CLK and PER antisera, and imaged by confocal microscopy. Anterior is to the left and dorsal is on the top. (A-C) 58 μm Z-series projection of PER (A), CLK (B) or CLK + PER (C) IR in the brain (box) and VNC during early ES 16. (A'-C') Magnified view of the boxed brain regions in panels A-C, respectively. (D-F) 18 μm Z-series projection of PER (D), CLK (E) or CLK + PER (F) IR in the brain (box) and VNC during late ES 16. (D'-F') Magnified view of the boxed brain regions in panels D-F, respectively. (G-I) 20 μm Z-series projection of PER (G), CLK (H) or CLK + PER (I) IR in the brain (box) and VNC during ES 17. (G'-I') Magnified view of the boxed brain regions in panels G-I, respectively. Co-localization of CLK (red) and PER (green) is shown as yellow. All images are representative of three or more independent experiments.
Mentions: Early in ES 16, weak CLK immunoreactivity is first detected in brain cells that do not express PER (Fig. 8A–C). During mid to late ES 16, CLK expression becomes stronger and expands to additional cells in the dorsal brain (Fig. 8D–F). In addition, PER starts to be detected in CLK positive cells in the ventral portion of the brain (Fig. 8F). By ES 17, CLK positive cells form three distinct clusters in each brain hemisphere, with two dorsal cells, four ventral cells, and two cells between and slightly posterior to these dorsal and ventral clusters (Fig. 8G–I). The positions of these CLK positive cells are reminiscent of oscillator cells in larvae; a dorsal cluster of DN1s, a ventral cluster of s-LNvs, and a medial cluster of DN2s [23,37].

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