Limits...
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.

Show MeSH

Related in: MedlinePlus

Dynamics of BrdU incorporation in pSC shaft and socket cells from 17.5 to 29 hours APF.Pupae with the +/+ genotype were grown until particular times of development and BrdU incorporation assays were performed as described in the Materials and Methods. Images show pSC shaft and socket cells stained with DAPI and anti-BrdU antibodies (A–H). Arrows indicate socket cell nuclei. Arrowheads indicate shaft cell nuclei. Note the BrdU incorporation observed in A, C, F, G in shaft cells and in C, D, H in socket cells. (I) Pattern of BrdU incorporation in shaft cells from 17.5 to 29 hours APF. The Y axis indicates the % of BrdU incorporation in shaft and socket cells. Sample numbers and detailed data are described in Table S1.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3368872&req=5

pone-0038714-g004: Dynamics of BrdU incorporation in pSC shaft and socket cells from 17.5 to 29 hours APF.Pupae with the +/+ genotype were grown until particular times of development and BrdU incorporation assays were performed as described in the Materials and Methods. Images show pSC shaft and socket cells stained with DAPI and anti-BrdU antibodies (A–H). Arrows indicate socket cell nuclei. Arrowheads indicate shaft cell nuclei. Note the BrdU incorporation observed in A, C, F, G in shaft cells and in C, D, H in socket cells. (I) Pattern of BrdU incorporation in shaft cells from 17.5 to 29 hours APF. The Y axis indicates the % of BrdU incorporation in shaft and socket cells. Sample numbers and detailed data are described in Table S1.

Mentions: To confirm the validity of the results (Fig. 2), we examined the timing of S phase in pSC shaft and socket cells of a left side of a thorax by carrying out BrdU incorporation assays at 30 minute intervals from 17.5 to 29 hours APF (Note that N.D. at 28.5 hours APF) (see Materials and Methods). In this study, S phase is defined as the period where more than 50% of shaft or socket cells show BrdU incorporation. It is difficult to obtain 100% BrdU staining for a variety of reasons such as the asynchronous stage of the pupae as stated earlier. Based on 50% BrdU staining in shaft cells S phases were observed at 17.5, 21, 22.5, 26.5 and 28 hours APF (Figs. 4, Table S1) (in detail, 50% for 17.5, 91% for 21, 87% for 22.5, 57% for 26.5 and 67% for 28 hours APF). The timing of BrdU incorporation was generally consistent with times when statistically significant changes in the C value of shaft cells were observed (Fig. 2A, see brackets, an exception is the period of 18 to 20 hours APF). In contrast, in the case of socket cells, S phases were observed at 17.5, 22.5 and 29 hours APF (Figs. 4, Table S1). The timing again was largely consistent with times when statistically significant changes in the C value of socket cells were observed (Fig. 2B, see arrows, an exception is the period of 28 to 29 hours APF). However, slight BrdU incorporation signals were also observed between 21.5 and 22 hours APF and between 24 and 26 hours APF where no significant increase in the amount of DNA is seen (Fig. 2 and 4I). This again may be due to the asynchronous stage of the pupae, stated earlier. However, we cannot exclude the possibility that these signals reflect the length of individual S phases as S phase lengths in shaft and socket cells of microchaetes vary between 187 and 291 minutes at 30°C [23]. All these results suggest that S phase in pSC shaft cells may occur at around 17.5, 21, 22.5, around 26.5 and 28 hours APF in pSC shaft cells, whereas in pSC socket cells it may occur at 17.5, around 22.5 and 29 hours APF.


Dynamics of endoreplication during Drosophila posterior scutellar macrochaete development.

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

Dynamics of BrdU incorporation in pSC shaft and socket cells from 17.5 to 29 hours APF.Pupae with the +/+ genotype were grown until particular times of development and BrdU incorporation assays were performed as described in the Materials and Methods. Images show pSC shaft and socket cells stained with DAPI and anti-BrdU antibodies (A–H). Arrows indicate socket cell nuclei. Arrowheads indicate shaft cell nuclei. Note the BrdU incorporation observed in A, C, F, G in shaft cells and in C, D, H in socket cells. (I) Pattern of BrdU incorporation in shaft cells from 17.5 to 29 hours APF. The Y axis indicates the % of BrdU incorporation in shaft and socket cells. Sample numbers and detailed data are described in Table S1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038714-g004: Dynamics of BrdU incorporation in pSC shaft and socket cells from 17.5 to 29 hours APF.Pupae with the +/+ genotype were grown until particular times of development and BrdU incorporation assays were performed as described in the Materials and Methods. Images show pSC shaft and socket cells stained with DAPI and anti-BrdU antibodies (A–H). Arrows indicate socket cell nuclei. Arrowheads indicate shaft cell nuclei. Note the BrdU incorporation observed in A, C, F, G in shaft cells and in C, D, H in socket cells. (I) Pattern of BrdU incorporation in shaft cells from 17.5 to 29 hours APF. The Y axis indicates the % of BrdU incorporation in shaft and socket cells. Sample numbers and detailed data are described in Table S1.
Mentions: To confirm the validity of the results (Fig. 2), we examined the timing of S phase in pSC shaft and socket cells of a left side of a thorax by carrying out BrdU incorporation assays at 30 minute intervals from 17.5 to 29 hours APF (Note that N.D. at 28.5 hours APF) (see Materials and Methods). In this study, S phase is defined as the period where more than 50% of shaft or socket cells show BrdU incorporation. It is difficult to obtain 100% BrdU staining for a variety of reasons such as the asynchronous stage of the pupae as stated earlier. Based on 50% BrdU staining in shaft cells S phases were observed at 17.5, 21, 22.5, 26.5 and 28 hours APF (Figs. 4, Table S1) (in detail, 50% for 17.5, 91% for 21, 87% for 22.5, 57% for 26.5 and 67% for 28 hours APF). The timing of BrdU incorporation was generally consistent with times when statistically significant changes in the C value of shaft cells were observed (Fig. 2A, see brackets, an exception is the period of 18 to 20 hours APF). In contrast, in the case of socket cells, S phases were observed at 17.5, 22.5 and 29 hours APF (Figs. 4, Table S1). The timing again was largely consistent with times when statistically significant changes in the C value of socket cells were observed (Fig. 2B, see arrows, an exception is the period of 28 to 29 hours APF). However, slight BrdU incorporation signals were also observed between 21.5 and 22 hours APF and between 24 and 26 hours APF where no significant increase in the amount of DNA is seen (Fig. 2 and 4I). This again may be due to the asynchronous stage of the pupae, stated earlier. However, we cannot exclude the possibility that these signals reflect the length of individual S phases as S phase lengths in shaft and socket cells of microchaetes vary between 187 and 291 minutes at 30°C [23]. All these results suggest that S phase in pSC shaft cells may occur at around 17.5, 21, 22.5, around 26.5 and 28 hours APF in pSC shaft cells, whereas in pSC socket cells it may occur at 17.5, around 22.5 and 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