DNA replication events during larval silk gland development in the silkworm, Bombyx mori

DNA replication events during larval silk gland development in the silkworm,Bombyx mori

Chun-Dong Zhang 1,Fang-Fang Li 1,Xiang-Yun Chen,Mao-Hua Huang,Jun Zhang,Hongjuan Cui,Min-Hui Pan ?,Cheng Lu ?

State Key Laboratory of Silkworm Genome Biology,Institute of Sericulture and Systems Biology,Southwest University,Chongqing 400715,PR China

a r t i c l e i n f o Article history:

Received 29February 2012

Received in revised form 28April 2012Accepted 30April 2012

Available online 16May 2012Keywords:

DNA replication Endomitosis Silkworm Silk gland

a b s t r a c t

The silk gland is an important organ in silkworm as it synthesizes silk proteins and is critical to spinning.The genomic DNA content of silk gland cells dramatically increases 200–400thousand times for the larval life span through the process of https://www.360docs.net/doc/d93510380.html,ing in vitro culture,DNA synthesis was measured using BrdU labeling during the larval molt and intermolt periods.We found that the cell cycle of endomitosis was activated during the intermolt and was inhibited during the molt phase.The anterior silk gland,mid-dle silk gland,and posterior silk gland cells asynchronously exit the endomitotic cycle after day 6in 5th instar larvae,which correlated with the reduced expression of the cell cycle-related cdt1,pcna,cyclin E,cdk2and cdk1mRNAs in the wandering phase.Additional starvation had no effect on the initiation of silk gland DNA synthesis of the freshly ecdysed larvae.

1.Introduction

The silkworm is economically important due to their produc-tion of silk.Silk is produced by a pair of silk glands that are divided into three morphologically and physiologically distinct regions:the anterior silk gland (ASG)containing approximately 200cells,the middle silk gland (MSG)containing approximately 250cells and the posterior silk gland (PSG),which contains approximately 525cells (Perdrix-Gillot,1979).The PSG synthesizes the silk struc-tural proteins,the ?broin L and H chains and ?brohexamerin (for-merly known as p25)(Couble et al.,1985;Kikuchi et al.,1992;Zhou et al.,2000),the MSG synthesizes the three major compo-nents of the glue proteins,sericin 1,2and 3(Takasu et al.,2002),and the ASG serves as the ducts to carry the silk proteins to the silk spinning apparatus.The development of the silk gland occurs be-tween stages 17–18and 26–27of embryonic development,after which the silk glands that contain approximately 975cells are completely developed (Dhawan and Gopinathan,2003;Matsunami et al.,1999).

The silk gland continues to enlarge in size during larval devel-opment;however,the number of cells remains unchanged as the cells undergo the endomitotic cell cycle (Dhawan and Gopinathan,2003;Perdrix-Gillot,1979).During endomitosis,the G1and S cell cycle phases alternate without an intervening mitotic division (Zimmet and Ravid,2000),leading to an increase in DNA content and DNA replication machinery proteins (Niranjanakumari and Gopinathan,1991,1992,1993).The endomitotic cell cycle begins during embryogenesis when the ASG,MSG,and PSG are formed (Dhawan and Gopinathan,2003).During the larval stage,17–19rounds of endomitotic DNA replication occur in the MSG and PSG,resulting in a 217–219-fold increase in DNA content,with each cell containing approximately 400,000times the haploid genomic DNA content (Gage,1974).Although morphogenesis of the silk gland nuclei can be observed microscopically in larvae from the ?rst instar to the wandering phase,DNA synthesis in the ASG,MSG,and PSG during each instar have not been characterized well to date.

There are two distinct physiological processes during silkworm larval ontogeny:the molt and intermolt,during which the silk gland cells undergo multiple endomitotic cell cycles.Previous re-search has demonstrated that cyclin E protein is highly expressed during the intermolt phase,and is signi?cantly reduced during the molt (Dhawan and Gopinathan,2003).Cyclin E-CDK2is known to play an important role in the initiation of DNA synthesis (Ekholm and Reed,2000;Grana and Reddy,1995;Hengstschlager et al.,1999).In Drosophila ,salivary gland DNA synthesis is regu-lated by oscillations in cyclin E expression (Weiss et al.,1998).It is not known whether altered cyclin E expression affects DNA syn-thesis in the silkworm larvae silk gland cells during the molt.The role of feeding during the intermolt on the initiation of DNA syn-

Corresponding authors.Address:State Key Laboratory of Silkworm Genome

Biology,Southwest University 216Tiansheng Rd.,Beibei District,Chongqing 400716,PR China.Tel.:+862368250793;fax:+862368251128(M-.H.Pan).

E-mail addresses:pmh0476@https://www.360docs.net/doc/d93510380.html, (M.-H.Pan),lucheng@https://www.360docs.net/doc/d93510380.html, (C.Lu).1

These two authors contributed equally to this work.

thesis has not been investigated yet.In order to determine the DNA replication events in the silk worm,Bombyx mori,we analyzed DNA synthesis in the larval silk gland during different developmental stages in vitro,and investigated the effects of starvation on silk gland cell DNA synthesis in the freshly ecdysed larvae.

2.Materials and methods

2.1.Insects and silk gland culture

Larvae of the B.mori strain09-030were obtained from the Silk-worm Gene Bank of Southwestern University,Chong Qing,China and were reared on fresh mulberry leaves at25°C with a12h light/12h dark photoperiod.The larvae were surface sterilized in 70%ethanol for2–3min,rinsed in sterile water and the silk glands were dissected and cultured in Grace insect medium supple-mented with10%fetal bovine serum at27°C.

2.2.BrdU incorporation and immunostaining

BrdU incorporation was performed using the In Situ Cell Prolif-eration Kit(Roche,Basel,Switzerland).Brie?y,the silk glands were dissected from the larvae,washed extensively in Grace medium and incubated in BrdU in Grace medium supplemented with10% fetal bovine serum for30min at27°C.The samples were washed extensively in PBS and incubated with a nuclease to generate sin-gle-stranded DNA fragments to which the BrdU antibody could bind.The samples were mounted in50%glycerol in PBS and visu-alized using the Olympus BX21microscope(Olympus,Tokyo,CTATTA-30and reverse50-GACGGGCTCTTCCATTTC-30),cyclin A (forward50-GCCCTCGGAGACCTCAGTCA-30and reverse50-TGTCGG TCTGTGCAGTGGTG-30);cdk2(forward50-CAGCTATTCCGCGTATTC CG-30and reverse50-TTTAAGGATGTAGAGGAGGCGGCAC-30),and cdk1(forward50-CAACTCTTCCGCATTTTCAG-30and reverse50-CCTA TACACTTTGAACAGAATCCGT-30).PCR reactions were performed at 95°C for5min followed by25cycles at95°C for60s,52°C for 60s and72°C for60s with a?nal extension at72°C for5min.

B.mori ribosomal protein L3(rpl3)primers(forward50-GTCGTCA TCGTGGTAAGGTC-30and reverse50-GGTCTCAATGTATCCAA-CAAC-30)were included as the internal controls.

3.Results

3.1.Low levels of endomitotic DNA replication in larval silk glands during the molt phase

Larval silk glands are generally considered to be quiescent dur-ing the molt.BrdU was employed to label endomitotic DNA syn-thesis in the silk glands of2nd,3rd and4th advanced apolysis larvae.Fig.1a presents the typical results obtained from the silk gland of the2nd advanced apolysis larvae;similar results were ob-tained from the silk gland of the3rd and4th advanced apolysis lar-vae.Several BrdU-positive cells were observed in the MSG and PSG. The positive cells were not equally distributed in the MSG and PSG. However,no BrdU-positive cells were observed in the ASG.Fig.2a shows the quanti?cation of BrdU-positive cells in the MSG and PSG during the2nd,3rd and the4th advanced apolysis larvae.

labeling of silk gland cells during the molting and intermolt larvae.Silk glands were labeled with BrdU in vitro for30min.(a)Silk glands of2nd instar (advanced apolysis).(b and c)Third instar larvae fed with mulberry leaves for6h and24h after ecdysis,respectively.(d and e)Fourth instar larvae

after ecdysis,respectively.At least?ve silk glands were used in each group.White arrowheads mark weak labeling.Scale bars represent100l m.

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D.Zhang et al./Journal of Insect Physiology58(2012)974–978975

MSG and PSG,which was similar to silk glands24h after feeding (data not shown).Fig.2b showed the quantitation of BrdU-positive cells in the ASG,MSG and PSG during the2nd instar larvae6h,24h and48h after feeding.

In order to investigate the effect of feeding on endomitosis,the 4th instar larvae were starved for6h,24h and48h after molting and were labeled using BrdU.After6h starving,BrdU-positive cells

ASG,MSG and PSG(Fig.1d),which was sim-

larvae that had been fed for6h(Fig.1b).In

had been starved for24h,no BrdU-positive 3.3.Endomitotic cell cycle arrest occurs in the silk glands of late5th instar larvae

Silk gland enlargement occurs rapidly during the5th instar.The silk gland developmental process in the5th instar is poorly under-stood,and therefore we analyzed endomitosis in the silk gland during the5th instar using BrdU labeling.A large number of BrdU-positive cells were observed in the MSG

day1(Fig.3a)and day5(Fig.3b)of the5th

time the silk gland cells rapidly expanded in

BrdU labeled cells in the silk gland.Silk glands labeled with BrdU in vitro for30min.Ten silk glands were used and treated groups.(a)The silk glands of2nd,3rd and4th instar larvae18h after ecdysis(advanced apolysis).(b)The silk glands h,24h and48h,respectively.

silk gland cells during the5th instar and wandering phase larvae.Silk glands were labeled with BrdU in vitro for1h.(a–c)The leaves for24h,day5and day6,respectively.(d)The silk glands of the wandering phase at12h.At least?ve silk glands were labeling.Scale bars indicate100l m.

glands were dramatically enlarged during the5th instar.DNA granular processes appeared on the surface of nuclei from day1 of the5th instar larvae,and as the larvae development goes,the processes became increasingly obvious.These results indicated that PSG cells exit from the endomitotic cell cycle before MSG cells during the5th instar.

3.4.Cell cycle-related genes are expressed in5th instar larvae

BrdU labeling demonstrated that endomitosis continued in the 5th instar larvae to day5in the PSG and day6in the MSG;there-fore,we investigated the expression of cell cycle-related genes using RT-PCR during the5th instar.The mRNA expression of?ve cell cycle-related genes,cdt1,pcna,cycE,cdk1and cdk2,decreased and was almost undetectable in the wandering phase at6h and after24h spinning compared with during the4th molt and from day1to day6of the5th instar(Fig.4).

4.Discussion

The silk glands are the silk protein synthesizing organ in the silkworm.Normal growth and development of the silk gland is essential to spinning.In this study,we investigated the develop-ment of the silk gland in larvae during the2nd–5th instar.This is the?rst systematic analysis on silk gland development during the2nd–5th instar by immuno?uorescence staining.B.mori silk-worm variety09–030was used for these experiments.This has a similar ontogeny cycle to the Da Zao variety and contains a trans-lucent silk gland that can be easily studied by immuno?uorescence staining.

Our study demonstrates that DNA synthesis is not quiescent in the MSG and PSG in the molt phase,during which DNA synthesis does not occur in ASG cells.Low levels of cyclin E are detected in the silkworm silk glands during the molt(Dhawan and Gopinathan, 2003),and our results indicate that endomitosis occurs at low levels during this stage of development.This suggests that silk gland endomitosis is inhibited during the molt and gradually activated in the ASG during the intermolt.After molting,ASG,MSG and PSG cells undergo endomitosis,with the highest levels of endomitosis observed in the MSG and PSG.These results suggest that the MSG and PSG endomitotic cell cycle is short during the eating phase. Conversely,only small numbers of BrdU-positive cells were observed in the ASG,which indicates that the endomitotic cell cycle is longer in the ASG during the eating phase.ASG cells may have a longer endomitotic cell cycle,as they would consequently undergo fewer cycles of DNA replication and be smaller than the MSG and PSG cells.

This study demonstrates that larval silk glands expand rapidly as a result of DNA replication between day1and5in5th instar lar-vae.Furthermore,the silk gland cells undergoing endomitosis ar-rest asynchronously;PSG cells begin to arrest on day5of the5th instar,and exit endomitosis during the wandering phase,while ASG cells continue endomitosis in the wandering phase.

In Drosophila,DNA synthesis in endomitotic cells is affected by nutrition and feeding,and DNA synthesis is arrested in the endo-mitotic cells,but not in the mitotic cells of starved larvae(Britton and Edgar,1998).The starvation of4th instar silkworm larvae can inhibit the growth of the silk gland.We detected DNA synthesis in the ASG,MSG and PSG of starved larvae6h after molting.It ap-pears that DNA synthesis is activated by a self-regulating process after molting and does not depend on feeding,which suggests that the initiation of DNA synthesis is a complex process that may be regulated by many different factors,including nutrition and envi-ronmental conditions.However,BrdU-positive cells were only ob-served in the ASG after24h starvation,which suggests that feeding is required for endomitosis in the MSG and PSG to continue.

The5th instar larvae of the silkworm B.mori(09-030)takes mulberry leaf from day1to day6,but day7is the wandering phase. RT-PCR showed that the expression of genes involved in the cell cycle began to reduce on day6in5th instar larvae,when MSG and PSG cells were observed to exit endomitosis.The expression of Cdt1,a component of the DNA replication initiation complex that is essential for the initiation of DNA replication(Rialland et al., 2002),pcna,a critical component of the DNA polymerase complex (Bowman et al.,2004;Zhang et al.,1999),Cdk2,which forms a kinase complex with cyclin E required for cells to enter S phase, and Cdk1were reduced and almost undetectable in the wandering and spinning phases.The expression pattern of cell cycle regulatory genes and the results of the BrdU labeling experiments demonstrate that PSG and MSG silk gland cells start to exit endomitosis on day6 of the5th instar,which indicates that silk gland development has been completed.

Acknowledgments

This work is supported by grants from the National Basic Research Program of China(No.2012cb114603)and the National Natural Science Foundation of China(Grant Nos.30972146and 31172269).

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