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Synergistic induction of the clock protein PERIOD by insulin-like peptide and prothoracicotropic hormone in Rhodnius prolixus (Hemiptera): implications for convergence of hormone signaling pathways.

Vafopoulou X, Steel CG - Front Physiol (2014)

Bottom Line: PER is severely reduced in the nuclei of all clock cells in continuous light, but on transfer of tissues to darkness in vitro, it is rapidly induced.In the peripheral oscillators salivary gland (SG) and fat body cells, neither bombyxin nor PTTH nor darkness induced PER, but a combination of both bombyxin and PTTH induced PER.We infer clock cells are able to integrate light cycle information with internal signals from hormones.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, York University Toronto, ON, Canada.

ABSTRACT
We showed previously that release of the cerebral neurohormones, bombyxin (an insulin-like peptide, ILP) and prothoracicotropic hormone (PTTH) from the brain have strong circadian rhythms, driven by master clock cells in the brain. These neurohormone rhythms synchronize the photosensitive brain clock with the photosensitive peripheral clock in the cells of the prothoracic glands (PGs), in which both regulate steroidogenesis. Here, using immunohistochemistry and confocal laser scanning microscopy, we show these neurohormones likely act on clock cells in the brain and PGs by regulating expression of PERIOD (PER) protein. PER is severely reduced in the nuclei of all clock cells in continuous light, but on transfer of tissues to darkness in vitro, it is rapidly induced. A 4h pulse of either PTTH or ILPs to brain and PGs in vitro both rapidly and highly significantly induce PER in the nuclei of clock cells. Administration of both neurohormones together induces more PER than does either alone and even more than does transfer to darkness, at least in PG cells. These are clearly non-steroidogenic actions of these peptides. In the peripheral oscillators salivary gland (SG) and fat body cells, neither bombyxin nor PTTH nor darkness induced PER, but a combination of both bombyxin and PTTH induced PER. Thus, PTTH and ILPs exert synergistic actions on induction of PER in both clock cells and peripheral oscillators, implying their signaling pathways converge, but in different ways in different cell types. We infer clock cells are able to integrate light cycle information with internal signals from hormones.

No MeSH data available.


Related in: MedlinePlus

Images of control and treated brains 12 days after a blood meal. All images are a dorsal view of the right brain hemisphere. (A–C) show in vivo brains. Control scotophase brains from entrained animals in 12L:12D stained with anti-PER (A) or anti-PDF (B). Fluorescence is shown as yellow/white. Images show the lateral clock neurons (LNs) at the junction of protocerebrum (pc) and optic lobe (ol) and their axonal projections. (C) a brain from animals transferred to LL for 3 weeks and stained with anti-PDF; note absence of PDF fluorescence in LNs. LL brains similarly stained with anti-PER also showed no staining in the LNs (not shown). (D–I) show LL brains incubated in vitro.(D–E) show brains stained with anti-PER. (D) LL brain transferred to darkness. (E) brain challenged with bombyxin. Note induction of PER (compare with C). (F–I) show brains stained with anti-PDF. (F) brain transferred to darkness. (G) brain challenged with bombyxin. (H) brain challenged with anti-PTTH. (I) brain challenged with bombyxin plus PTTH. Scale bars = 10 μm.
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Figure 3: Images of control and treated brains 12 days after a blood meal. All images are a dorsal view of the right brain hemisphere. (A–C) show in vivo brains. Control scotophase brains from entrained animals in 12L:12D stained with anti-PER (A) or anti-PDF (B). Fluorescence is shown as yellow/white. Images show the lateral clock neurons (LNs) at the junction of protocerebrum (pc) and optic lobe (ol) and their axonal projections. (C) a brain from animals transferred to LL for 3 weeks and stained with anti-PDF; note absence of PDF fluorescence in LNs. LL brains similarly stained with anti-PER also showed no staining in the LNs (not shown). (D–I) show LL brains incubated in vitro.(D–E) show brains stained with anti-PER. (D) LL brain transferred to darkness. (E) brain challenged with bombyxin. Note induction of PER (compare with C). (F–I) show brains stained with anti-PDF. (F) brain transferred to darkness. (G) brain challenged with bombyxin. (H) brain challenged with anti-PTTH. (I) brain challenged with bombyxin plus PTTH. Scale bars = 10 μm.

Mentions: Vafopoulou et al. (2010) showed that PER fluorescence in Rhodnius brain clock cells showed circadian cycling in 12L:12D; the main group of clock cells (lateral clock neurons; LNs) were also filled with PDF, which enabled tracing of their axons. Figure 3A shows the location of LNs stained with anti-PER (Figure 3A) and anti-PDF (Figure 3B) on the border of the optic lobe and protocerebrum of a control scotophase animal entrained in 12L:12D at day 12 after a blood meal. We have shown previously (and discussed in depth) that PER fluorescence in LNs is exclusively cytoplasmic (Vafopoulou et al., 2010; Vafopoulou and Steel, 2012a,b). Here, we first examined the effect of transfer of 12 such animals to LL for 3 weeks on levels of PER and PDF in LNs in vivo. All brains were completely devoid of PER fluorescence and 10 of the 12 were also devoid of PDF fluorescence. Two of the 12 brains showed trace levels of PDF in their somata and none in their axons. Figure 3C shows an LL brain stained with anti-PDF in which PDF fluorescence in LNs is reduced to background levels. PER fluorescence in LNs is similarly reduced to background levels (not shown). Thus, chronic LL completely abolishes PER in brain clock cells and PDF is abolished or drastically reduced.


Synergistic induction of the clock protein PERIOD by insulin-like peptide and prothoracicotropic hormone in Rhodnius prolixus (Hemiptera): implications for convergence of hormone signaling pathways.

Vafopoulou X, Steel CG - Front Physiol (2014)

Images of control and treated brains 12 days after a blood meal. All images are a dorsal view of the right brain hemisphere. (A–C) show in vivo brains. Control scotophase brains from entrained animals in 12L:12D stained with anti-PER (A) or anti-PDF (B). Fluorescence is shown as yellow/white. Images show the lateral clock neurons (LNs) at the junction of protocerebrum (pc) and optic lobe (ol) and their axonal projections. (C) a brain from animals transferred to LL for 3 weeks and stained with anti-PDF; note absence of PDF fluorescence in LNs. LL brains similarly stained with anti-PER also showed no staining in the LNs (not shown). (D–I) show LL brains incubated in vitro.(D–E) show brains stained with anti-PER. (D) LL brain transferred to darkness. (E) brain challenged with bombyxin. Note induction of PER (compare with C). (F–I) show brains stained with anti-PDF. (F) brain transferred to darkness. (G) brain challenged with bombyxin. (H) brain challenged with anti-PTTH. (I) brain challenged with bombyxin plus PTTH. Scale bars = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Images of control and treated brains 12 days after a blood meal. All images are a dorsal view of the right brain hemisphere. (A–C) show in vivo brains. Control scotophase brains from entrained animals in 12L:12D stained with anti-PER (A) or anti-PDF (B). Fluorescence is shown as yellow/white. Images show the lateral clock neurons (LNs) at the junction of protocerebrum (pc) and optic lobe (ol) and their axonal projections. (C) a brain from animals transferred to LL for 3 weeks and stained with anti-PDF; note absence of PDF fluorescence in LNs. LL brains similarly stained with anti-PER also showed no staining in the LNs (not shown). (D–I) show LL brains incubated in vitro.(D–E) show brains stained with anti-PER. (D) LL brain transferred to darkness. (E) brain challenged with bombyxin. Note induction of PER (compare with C). (F–I) show brains stained with anti-PDF. (F) brain transferred to darkness. (G) brain challenged with bombyxin. (H) brain challenged with anti-PTTH. (I) brain challenged with bombyxin plus PTTH. Scale bars = 10 μm.
Mentions: Vafopoulou et al. (2010) showed that PER fluorescence in Rhodnius brain clock cells showed circadian cycling in 12L:12D; the main group of clock cells (lateral clock neurons; LNs) were also filled with PDF, which enabled tracing of their axons. Figure 3A shows the location of LNs stained with anti-PER (Figure 3A) and anti-PDF (Figure 3B) on the border of the optic lobe and protocerebrum of a control scotophase animal entrained in 12L:12D at day 12 after a blood meal. We have shown previously (and discussed in depth) that PER fluorescence in LNs is exclusively cytoplasmic (Vafopoulou et al., 2010; Vafopoulou and Steel, 2012a,b). Here, we first examined the effect of transfer of 12 such animals to LL for 3 weeks on levels of PER and PDF in LNs in vivo. All brains were completely devoid of PER fluorescence and 10 of the 12 were also devoid of PDF fluorescence. Two of the 12 brains showed trace levels of PDF in their somata and none in their axons. Figure 3C shows an LL brain stained with anti-PDF in which PDF fluorescence in LNs is reduced to background levels. PER fluorescence in LNs is similarly reduced to background levels (not shown). Thus, chronic LL completely abolishes PER in brain clock cells and PDF is abolished or drastically reduced.

Bottom Line: PER is severely reduced in the nuclei of all clock cells in continuous light, but on transfer of tissues to darkness in vitro, it is rapidly induced.In the peripheral oscillators salivary gland (SG) and fat body cells, neither bombyxin nor PTTH nor darkness induced PER, but a combination of both bombyxin and PTTH induced PER.We infer clock cells are able to integrate light cycle information with internal signals from hormones.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, York University Toronto, ON, Canada.

ABSTRACT
We showed previously that release of the cerebral neurohormones, bombyxin (an insulin-like peptide, ILP) and prothoracicotropic hormone (PTTH) from the brain have strong circadian rhythms, driven by master clock cells in the brain. These neurohormone rhythms synchronize the photosensitive brain clock with the photosensitive peripheral clock in the cells of the prothoracic glands (PGs), in which both regulate steroidogenesis. Here, using immunohistochemistry and confocal laser scanning microscopy, we show these neurohormones likely act on clock cells in the brain and PGs by regulating expression of PERIOD (PER) protein. PER is severely reduced in the nuclei of all clock cells in continuous light, but on transfer of tissues to darkness in vitro, it is rapidly induced. A 4h pulse of either PTTH or ILPs to brain and PGs in vitro both rapidly and highly significantly induce PER in the nuclei of clock cells. Administration of both neurohormones together induces more PER than does either alone and even more than does transfer to darkness, at least in PG cells. These are clearly non-steroidogenic actions of these peptides. In the peripheral oscillators salivary gland (SG) and fat body cells, neither bombyxin nor PTTH nor darkness induced PER, but a combination of both bombyxin and PTTH induced PER. Thus, PTTH and ILPs exert synergistic actions on induction of PER in both clock cells and peripheral oscillators, implying their signaling pathways converge, but in different ways in different cell types. We infer clock cells are able to integrate light cycle information with internal signals from hormones.

No MeSH data available.


Related in: MedlinePlus