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Dynamics of endoreplication during Drosophila posterior scutellar macrochaete development.

Kawamori A, Shimaji K, Yamaguchi M - PLoS ONE (2012)

Bottom Line: We also found that the timing of endoreplication differs, depending on the type of macrochaete.Moreover, endocycling in shaft cells of both the left and right sides of pSC bristle lineages occurs in the same pattern, indicating that the process is synchronized for specific types of macrochaete.Our findings suggest that endocycling in macrochaete cell lineages can be a model for understanding mechanisms of endoreplication at the single-cell level.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto, Japan.

ABSTRACT
Endoreplication is a variant type of DNA replication, consisting only of alternating G1 and S phases. Many types of Drosophila tissues undergo endoreplication. However, the timing and the extent to which a single endocycling macrochaete undergoes temporally programmed endoreplication during development are unclear. Here, we focused on the dynamics of endoreplication during posterior scutellar (pSC) macrochaete development. Quantitative analyses of C values in shaft cells and socket cells revealed a gradual rise from 8C and 4C at 8 hours after pupal formation (APF) to 72C and 24C at 29 hours APF, respectively. The validity of the values was further confirmed by the measurement of DNA content with a confocal laser microscope. BrdU incorporation assays demonstrated that shaft cells undergo four rounds of endoreplication from 18 to 29.5 hours APF. In contrast, socket cells undergo two rounds of endoreplication during the same period. Statistical analyses showed that the theoretical C values, based on BrdU assays, nearly coincide with the actually measured C values in socket cells, but not in shaft cells after 22 hours APF. These analyses suggest that socket cells undergo two rounds of endoreplication. However, the mechanism of endoreplication in the shaft cells may change from 22 hours APF, suggesting the possibility that shaft cells undergo two or four rounds of endoreplication during the periods. We also found that the timing of endoreplication differs, depending on the type of macrochaete. Moreover, endocycling in shaft cells of both the left and right sides of pSC bristle lineages occurs in the same pattern, indicating that the process is synchronized for specific types of macrochaete. Our findings suggest that endocycling in macrochaete cell lineages can be a model for understanding mechanisms of endoreplication at the single-cell level.

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The C values of pSC shaft and socket cells at 29 hours APF as measured with a confocal laser microscope.Wild type female pupae were grown till 29 hours APF and dissected thoraxes were stained with PI. (A) The white arrowhead indicates a shaft cell nucleus. Open arrowheads indicate epidermal cell nuclei. (B) The arrow indicates a socket cell nucleus. (C) Quantification of C values in shaft or socket cell nuclei at 29 hours APF. The Y axis indicates C values.
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pone-0038714-g003: The C values of pSC shaft and socket cells at 29 hours APF as measured with a confocal laser microscope.Wild type female pupae were grown till 29 hours APF and dissected thoraxes were stained with PI. (A) The white arrowhead indicates a shaft cell nucleus. Open arrowheads indicate epidermal cell nuclei. (B) The arrow indicates a socket cell nucleus. (C) Quantification of C values in shaft or socket cell nuclei at 29 hours APF. The Y axis indicates C values.

Mentions: Next, to determine to what extent the shaft and socket cells undergo endoreplication during development, we carried out DAPI-staining and measured the dynamics of chromatin values (C values) of shaft and socket cell nuclei in pSC bristle cell lineages from 8, 12, 18 hours APF and 20 to 29 hours APF at one hour intervals. The C values are defined as the relative genomic DNA contents of shaft or socket cell nuclei relative to mean values of those of surrounding epidermal cells (see Materials and Methods). The results showed that the C values in shaft cell nuclei were 8C at 8 hours APF and finally increased up to 72C at 29 hours APF (Fig. 2A), a nine-fold increase in genomic DNA content during 21 hours of development. In contrast, the C value in socket cell nuclei was about 4C at 8 hours APF and finally increased up to about 24C at 29 hours APF (Fig. 2B), a six-fold increase. However, relatively large variability was seen in shaft and socket cells at some developmental stages (compare DNA contents at 23 with 24 hours APF). This may be due to the asynchronous age of pupae, influenced by the growing conditions and/or inaccuracy of the imaging method used. The genomic DNA contents were measured by a conventional fluorescent microscope equipped with a cooled CCD camera. This may lead to overlap of genomic DNA with that of neighboring cells in some cases. To further confirm the validity of the measurements, we performed Propidium iodide (PI) staining, and determined the C values of shaft and socket cells by measuring the DNA content of shaft, socket and surrounding epidermal cells at 29 hours APF with a confocal laser microscope (see Materials and Methods). The results showed that the C values of both shaft and socket cells were about 65C and 28C, respectively (Fig. 3A–C). These C values are nearly equal with those measured with DAPI staining utilizing a conventional fluorescent microscope, confirming that the quantification of C values with the conventional microscope is appropriate. Taken together, these results suggest that two rounds of endoreplication occur for shaft and socket cell from 18 to 29 hours APF.


Dynamics of endoreplication during Drosophila posterior scutellar macrochaete development.

Kawamori A, Shimaji K, Yamaguchi M - PLoS ONE (2012)

The C values of pSC shaft and socket cells at 29 hours APF as measured with a confocal laser microscope.Wild type female pupae were grown till 29 hours APF and dissected thoraxes were stained with PI. (A) The white arrowhead indicates a shaft cell nucleus. Open arrowheads indicate epidermal cell nuclei. (B) The arrow indicates a socket cell nucleus. (C) Quantification of C values in shaft or socket cell nuclei at 29 hours APF. The Y axis indicates C values.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368872&req=5

pone-0038714-g003: The C values of pSC shaft and socket cells at 29 hours APF as measured with a confocal laser microscope.Wild type female pupae were grown till 29 hours APF and dissected thoraxes were stained with PI. (A) The white arrowhead indicates a shaft cell nucleus. Open arrowheads indicate epidermal cell nuclei. (B) The arrow indicates a socket cell nucleus. (C) Quantification of C values in shaft or socket cell nuclei at 29 hours APF. The Y axis indicates C values.
Mentions: Next, to determine to what extent the shaft and socket cells undergo endoreplication during development, we carried out DAPI-staining and measured the dynamics of chromatin values (C values) of shaft and socket cell nuclei in pSC bristle cell lineages from 8, 12, 18 hours APF and 20 to 29 hours APF at one hour intervals. The C values are defined as the relative genomic DNA contents of shaft or socket cell nuclei relative to mean values of those of surrounding epidermal cells (see Materials and Methods). The results showed that the C values in shaft cell nuclei were 8C at 8 hours APF and finally increased up to 72C at 29 hours APF (Fig. 2A), a nine-fold increase in genomic DNA content during 21 hours of development. In contrast, the C value in socket cell nuclei was about 4C at 8 hours APF and finally increased up to about 24C at 29 hours APF (Fig. 2B), a six-fold increase. However, relatively large variability was seen in shaft and socket cells at some developmental stages (compare DNA contents at 23 with 24 hours APF). This may be due to the asynchronous age of pupae, influenced by the growing conditions and/or inaccuracy of the imaging method used. The genomic DNA contents were measured by a conventional fluorescent microscope equipped with a cooled CCD camera. This may lead to overlap of genomic DNA with that of neighboring cells in some cases. To further confirm the validity of the measurements, we performed Propidium iodide (PI) staining, and determined the C values of shaft and socket cells by measuring the DNA content of shaft, socket and surrounding epidermal cells at 29 hours APF with a confocal laser microscope (see Materials and Methods). The results showed that the C values of both shaft and socket cells were about 65C and 28C, respectively (Fig. 3A–C). These C values are nearly equal with those measured with DAPI staining utilizing a conventional fluorescent microscope, confirming that the quantification of C values with the conventional microscope is appropriate. Taken together, these results suggest that two rounds of endoreplication occur for shaft and socket cell from 18 to 29 hours APF.

Bottom Line: We also found that the timing of endoreplication differs, depending on the type of macrochaete.Moreover, endocycling in shaft cells of both the left and right sides of pSC bristle lineages occurs in the same pattern, indicating that the process is synchronized for specific types of macrochaete.Our findings suggest that endocycling in macrochaete cell lineages can be a model for understanding mechanisms of endoreplication at the single-cell level.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biology, Kyoto Institute of Technology, Sakyo-ku, Kyoto, Japan.

ABSTRACT
Endoreplication is a variant type of DNA replication, consisting only of alternating G1 and S phases. Many types of Drosophila tissues undergo endoreplication. However, the timing and the extent to which a single endocycling macrochaete undergoes temporally programmed endoreplication during development are unclear. Here, we focused on the dynamics of endoreplication during posterior scutellar (pSC) macrochaete development. Quantitative analyses of C values in shaft cells and socket cells revealed a gradual rise from 8C and 4C at 8 hours after pupal formation (APF) to 72C and 24C at 29 hours APF, respectively. The validity of the values was further confirmed by the measurement of DNA content with a confocal laser microscope. BrdU incorporation assays demonstrated that shaft cells undergo four rounds of endoreplication from 18 to 29.5 hours APF. In contrast, socket cells undergo two rounds of endoreplication during the same period. Statistical analyses showed that the theoretical C values, based on BrdU assays, nearly coincide with the actually measured C values in socket cells, but not in shaft cells after 22 hours APF. These analyses suggest that socket cells undergo two rounds of endoreplication. However, the mechanism of endoreplication in the shaft cells may change from 22 hours APF, suggesting the possibility that shaft cells undergo two or four rounds of endoreplication during the periods. We also found that the timing of endoreplication differs, depending on the type of macrochaete. Moreover, endocycling in shaft cells of both the left and right sides of pSC bristle lineages occurs in the same pattern, indicating that the process is synchronized for specific types of macrochaete. Our findings suggest that endocycling in macrochaete cell lineages can be a model for understanding mechanisms of endoreplication at the single-cell level.

Show MeSH
Related in: MedlinePlus