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A Circadian Clock Gene, Cry, Affects Heart Morphogenesis and Function in Drosophila as Revealed by Optical Coherence Microscopy.

Alex A, Li A, Zeng X, Tate RE, McKee ML, Capen DE, Zhang Z, Tanzi RE, Zhou C - PLoS ONE (2015)

Bottom Line: Notably, heart rate (HR) and cardiac activity period (CAP) of Drosophila showed significant variations during the pupa stage, when heart remodeling took place.Silencing of dCry resulted in slower HR, reduced CAP, smaller heart chamber size, pupal lethality and disrupted posterior segmentation that was related to increased expression of a posterior compartment protein, wingless.Collectively, our studies provided novel evidence that the circadian clock gene, dCry, plays an essential role in heart morphogenesis and function.

View Article: PubMed Central - PubMed

Affiliation: Department of Electrical and Computer Engineering, Lehigh University, Bethlehem, PA, United States of America, 18015; Center for Photonics and Nanoelectronics, Lehigh University, Bethlehem, PA, United States of America, 18015.

ABSTRACT
Circadian rhythms are endogenous, entrainable oscillations of physical, mental and behavioural processes in response to local environmental cues such as daylight, which are present in the living beings, including humans. Circadian rhythms have been related to cardiovascular function and pathology. However, the role that circadian clock genes play in heart development and function in a whole animal in vivo are poorly understood. The Drosophila cryptochrome (dCry) is a circadian clock gene that encodes a major component of the circadian clock negative feedback loop. Compared to the embryonic stage, the relative expression levels of dCry showed a significant increase (>100-fold) in Drosophila during the pupa and adult stages. In this study, we utilized an ultrahigh resolution optical coherence microscopy (OCM) system to perform non-invasive and longitudinal analysis of functional and morphological changes in the Drosophila heart throughout its post-embryonic lifecycle for the first time. The Drosophila heart exhibited major morphological and functional alterations during its development. Notably, heart rate (HR) and cardiac activity period (CAP) of Drosophila showed significant variations during the pupa stage, when heart remodeling took place. From the M-mode (2D + time) OCM images, cardiac structural and functional parameters of Drosophila at different developmental stages were quantitatively determined. In order to study the functional role of dCry on Drosophila heart development, we silenced dCry by RNAi in the Drosophila heart and mesoderm, and quantitatively measured heart morphology and function in those flies throughout its development. Silencing of dCry resulted in slower HR, reduced CAP, smaller heart chamber size, pupal lethality and disrupted posterior segmentation that was related to increased expression of a posterior compartment protein, wingless. Collectively, our studies provided novel evidence that the circadian clock gene, dCry, plays an essential role in heart morphogenesis and function.

No MeSH data available.


Related in: MedlinePlus

Validation of the OCM observations indicating cardiac dysfunction and structural defects in UAS-dCry-RNAi; 24B-GAL4 flies through whole heart and cardiac ultrastructure analysis.a) En face OCM projection of an adult UAS-dCry-RNAi; 24B-GAL4 fly. The red dotted line shows the imaging location corresponding to the axial OCM image shown in (c). (b, c) Representative axial OCM images of adult heart: (b) the cardiac tube of a control fly (24B-GAL4/+) appeared normal; however, (c) the cardiac tube of a UAS-dCry-RNAi; 24B-GAL4 fly on AD1 appeared slightly deformed with under-developed conical chamber (dotted circle) and folds along the dorsal surface (arrow). (d, e) Micrographs of whole adult heart by F-actin immuno-fluorescent staining: (d) Control fly (24B-GAL4/+) showed a normal cardiac tube. (e) Silencing of dCry in heart (UAS-dCry-RNAi; 24B-GAL4) resulted in a smaller, underdeveloped and irregular cardiac tube. Yellow dotted arrows point towards the cardiac tube. (f, g) Ultrastructure of adult heart longitudinal sections between A1 to A3 segments by TEM: (f) Control fly showed normal myofibril structure. (g) Silencing of dCry in heart resulted in immature and discontinuous Z discs, immature and irregular myofilament arrays and degenerated mitochondria. Scale bars: 200 μm in (a), (b) and (c), 100 μm in (d) and (e), and 500 nm in (f) and (g).
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pone.0137236.g003: Validation of the OCM observations indicating cardiac dysfunction and structural defects in UAS-dCry-RNAi; 24B-GAL4 flies through whole heart and cardiac ultrastructure analysis.a) En face OCM projection of an adult UAS-dCry-RNAi; 24B-GAL4 fly. The red dotted line shows the imaging location corresponding to the axial OCM image shown in (c). (b, c) Representative axial OCM images of adult heart: (b) the cardiac tube of a control fly (24B-GAL4/+) appeared normal; however, (c) the cardiac tube of a UAS-dCry-RNAi; 24B-GAL4 fly on AD1 appeared slightly deformed with under-developed conical chamber (dotted circle) and folds along the dorsal surface (arrow). (d, e) Micrographs of whole adult heart by F-actin immuno-fluorescent staining: (d) Control fly (24B-GAL4/+) showed a normal cardiac tube. (e) Silencing of dCry in heart (UAS-dCry-RNAi; 24B-GAL4) resulted in a smaller, underdeveloped and irregular cardiac tube. Yellow dotted arrows point towards the cardiac tube. (f, g) Ultrastructure of adult heart longitudinal sections between A1 to A3 segments by TEM: (f) Control fly showed normal myofibril structure. (g) Silencing of dCry in heart resulted in immature and discontinuous Z discs, immature and irregular myofilament arrays and degenerated mitochondria. Scale bars: 200 μm in (a), (b) and (c), 100 μm in (d) and (e), and 500 nm in (f) and (g).

Mentions: The cardiac structural and functional parameters of the few dCry-RNAi flies that emerged as adult flies (n = 23) were compared to those of age-matched control flies (n = 25) on AD1. EDA and ESA were significantly smaller (p < 0.01) in dCry-RNAi flies (Fig 2f and 2g; see S3 Table for comparison of other parameters). Underdeveloped conical chamber and folds along the dorsal abdomen were clearly observed in dCry-RNAi flies at AD1 compared to controls (Fig 3b and 3c). Fluorescent labelling of the adult heart on AD1 with F-actin immunostaining confirmed that silencing of dCry led to an underdeveloped, irregular and smaller cardiac tube compared to controls (Fig 3d and 3e). Transmission electron microscopy (TEM) imaging showed that adult hearts of control flies exhibited normal myofibril structure, regular myofilament arrays with continuous Z discs, normal mitochondria (Fig 3f). In contrast, hearts from the dCry-RNAi flies revealed immature and discontinuous Z discs, immature and irregular myofilament arrays and degenerated mitochondria (Fig 3g). This TEM ultrastructural analysis demonstrated that many cellular structural elements needed for normal cardiac functioning were not properly developed in the dCry-RNAi flies, even in the ones that managed to emerge as adult flies and indicates the critical role played by dCry in the structural development of cardiac tissue at a cellular/sub-cellular level.


A Circadian Clock Gene, Cry, Affects Heart Morphogenesis and Function in Drosophila as Revealed by Optical Coherence Microscopy.

Alex A, Li A, Zeng X, Tate RE, McKee ML, Capen DE, Zhang Z, Tanzi RE, Zhou C - PLoS ONE (2015)

Validation of the OCM observations indicating cardiac dysfunction and structural defects in UAS-dCry-RNAi; 24B-GAL4 flies through whole heart and cardiac ultrastructure analysis.a) En face OCM projection of an adult UAS-dCry-RNAi; 24B-GAL4 fly. The red dotted line shows the imaging location corresponding to the axial OCM image shown in (c). (b, c) Representative axial OCM images of adult heart: (b) the cardiac tube of a control fly (24B-GAL4/+) appeared normal; however, (c) the cardiac tube of a UAS-dCry-RNAi; 24B-GAL4 fly on AD1 appeared slightly deformed with under-developed conical chamber (dotted circle) and folds along the dorsal surface (arrow). (d, e) Micrographs of whole adult heart by F-actin immuno-fluorescent staining: (d) Control fly (24B-GAL4/+) showed a normal cardiac tube. (e) Silencing of dCry in heart (UAS-dCry-RNAi; 24B-GAL4) resulted in a smaller, underdeveloped and irregular cardiac tube. Yellow dotted arrows point towards the cardiac tube. (f, g) Ultrastructure of adult heart longitudinal sections between A1 to A3 segments by TEM: (f) Control fly showed normal myofibril structure. (g) Silencing of dCry in heart resulted in immature and discontinuous Z discs, immature and irregular myofilament arrays and degenerated mitochondria. Scale bars: 200 μm in (a), (b) and (c), 100 μm in (d) and (e), and 500 nm in (f) and (g).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4565115&req=5

pone.0137236.g003: Validation of the OCM observations indicating cardiac dysfunction and structural defects in UAS-dCry-RNAi; 24B-GAL4 flies through whole heart and cardiac ultrastructure analysis.a) En face OCM projection of an adult UAS-dCry-RNAi; 24B-GAL4 fly. The red dotted line shows the imaging location corresponding to the axial OCM image shown in (c). (b, c) Representative axial OCM images of adult heart: (b) the cardiac tube of a control fly (24B-GAL4/+) appeared normal; however, (c) the cardiac tube of a UAS-dCry-RNAi; 24B-GAL4 fly on AD1 appeared slightly deformed with under-developed conical chamber (dotted circle) and folds along the dorsal surface (arrow). (d, e) Micrographs of whole adult heart by F-actin immuno-fluorescent staining: (d) Control fly (24B-GAL4/+) showed a normal cardiac tube. (e) Silencing of dCry in heart (UAS-dCry-RNAi; 24B-GAL4) resulted in a smaller, underdeveloped and irregular cardiac tube. Yellow dotted arrows point towards the cardiac tube. (f, g) Ultrastructure of adult heart longitudinal sections between A1 to A3 segments by TEM: (f) Control fly showed normal myofibril structure. (g) Silencing of dCry in heart resulted in immature and discontinuous Z discs, immature and irregular myofilament arrays and degenerated mitochondria. Scale bars: 200 μm in (a), (b) and (c), 100 μm in (d) and (e), and 500 nm in (f) and (g).
Mentions: The cardiac structural and functional parameters of the few dCry-RNAi flies that emerged as adult flies (n = 23) were compared to those of age-matched control flies (n = 25) on AD1. EDA and ESA were significantly smaller (p < 0.01) in dCry-RNAi flies (Fig 2f and 2g; see S3 Table for comparison of other parameters). Underdeveloped conical chamber and folds along the dorsal abdomen were clearly observed in dCry-RNAi flies at AD1 compared to controls (Fig 3b and 3c). Fluorescent labelling of the adult heart on AD1 with F-actin immunostaining confirmed that silencing of dCry led to an underdeveloped, irregular and smaller cardiac tube compared to controls (Fig 3d and 3e). Transmission electron microscopy (TEM) imaging showed that adult hearts of control flies exhibited normal myofibril structure, regular myofilament arrays with continuous Z discs, normal mitochondria (Fig 3f). In contrast, hearts from the dCry-RNAi flies revealed immature and discontinuous Z discs, immature and irregular myofilament arrays and degenerated mitochondria (Fig 3g). This TEM ultrastructural analysis demonstrated that many cellular structural elements needed for normal cardiac functioning were not properly developed in the dCry-RNAi flies, even in the ones that managed to emerge as adult flies and indicates the critical role played by dCry in the structural development of cardiac tissue at a cellular/sub-cellular level.

Bottom Line: Notably, heart rate (HR) and cardiac activity period (CAP) of Drosophila showed significant variations during the pupa stage, when heart remodeling took place.Silencing of dCry resulted in slower HR, reduced CAP, smaller heart chamber size, pupal lethality and disrupted posterior segmentation that was related to increased expression of a posterior compartment protein, wingless.Collectively, our studies provided novel evidence that the circadian clock gene, dCry, plays an essential role in heart morphogenesis and function.

View Article: PubMed Central - PubMed

Affiliation: Department of Electrical and Computer Engineering, Lehigh University, Bethlehem, PA, United States of America, 18015; Center for Photonics and Nanoelectronics, Lehigh University, Bethlehem, PA, United States of America, 18015.

ABSTRACT
Circadian rhythms are endogenous, entrainable oscillations of physical, mental and behavioural processes in response to local environmental cues such as daylight, which are present in the living beings, including humans. Circadian rhythms have been related to cardiovascular function and pathology. However, the role that circadian clock genes play in heart development and function in a whole animal in vivo are poorly understood. The Drosophila cryptochrome (dCry) is a circadian clock gene that encodes a major component of the circadian clock negative feedback loop. Compared to the embryonic stage, the relative expression levels of dCry showed a significant increase (>100-fold) in Drosophila during the pupa and adult stages. In this study, we utilized an ultrahigh resolution optical coherence microscopy (OCM) system to perform non-invasive and longitudinal analysis of functional and morphological changes in the Drosophila heart throughout its post-embryonic lifecycle for the first time. The Drosophila heart exhibited major morphological and functional alterations during its development. Notably, heart rate (HR) and cardiac activity period (CAP) of Drosophila showed significant variations during the pupa stage, when heart remodeling took place. From the M-mode (2D + time) OCM images, cardiac structural and functional parameters of Drosophila at different developmental stages were quantitatively determined. In order to study the functional role of dCry on Drosophila heart development, we silenced dCry by RNAi in the Drosophila heart and mesoderm, and quantitatively measured heart morphology and function in those flies throughout its development. Silencing of dCry resulted in slower HR, reduced CAP, smaller heart chamber size, pupal lethality and disrupted posterior segmentation that was related to increased expression of a posterior compartment protein, wingless. Collectively, our studies provided novel evidence that the circadian clock gene, dCry, plays an essential role in heart morphogenesis and function.

No MeSH data available.


Related in: MedlinePlus