disc proliferation abnormal


DEVELOPMENTAL BIOLOGY

Embryonic

The high levels of transcript in 0-3 hour embryos indicate a maternally supplied message. Expression levels increase in 3-6 h embryos and reach a maximum level at 6-9 hours. The transcript level is reduced by 9-12h and only very low levels are detected after 12 hours (Feger, 1995).

The expression pattern of dpa is dynamic during gastrulation and germband extension. Most tissues express the transcript at high levels but some areas have very little or no expression. The epidermal expression remains high until late stage 11, when it is extinguished. Expression is then found in the delaminated neuroblasts of the CNS and the sensory organ precursors of the PNS. The dividing sensory organ precursors of the PNS continue to express the gene until stage 13/14 when expression ceases, concomitantly with the end of cell division. Most or all cells in the ventral nerve cord and brain show high transcript levels from stages 10 through 12, whereas only a subset of CNS cells express the transcript from stages 13 onwards. The gene is also expressed in endoreplicating tissues, starting in the anterior and posterior midgut at stage 12 followed by hindgut expression at stage 13 and, shortly afterwards, expression in the Malpighian tubules (Feger, 1995).

Larval

Expression of dpa is detected in cells of the third instar larvae in eye imaginal disc cells ahead of the morphogenetic furrow, and in a tight band behind it, but not in the furrow itself, coinciding with the first and second mitotic waves of proliferating cells. In the CNS of third instar larvae, expression is evident in the mitotically active optic lobes and in those neuroblasts that produce ganglion mother cells (Feger, 1995).

Minichromosome maintenance (MCM) proteins are essential eukaryotic DNA replication factors. The binding of MCMs to chromatin oscillates in conjunction with progress through the mitotic cell cycle. This oscillation is thought to play an important role in coupling DNA replication to mitosis and limiting chromosome duplication to once per cell cycle. The coupling of DNA replication to mitosis is absent in Drosophila endoreplication cycles (endocycles), during which discrete rounds of chromosome duplication occur without intervening mitoses. The behavior of MCM proteins was examined in endoreplicating larval salivary glands, to determine whether oscillation of MCM-chromosome localization occurs in conjunction with passage through an endocycle S phase. MCMs in polytene nuclei exist in two states: either associated with or dissociated from chromosomes. DmMCM2, DmMCM4, and DmMCM5 are detected as nuclear proteins in polytene nuclei of salivary glands during the three larval instars. In a majority of polytene nuclei from second- and third- instar larvae (>80%), most of the nuclear MCM stain is excluded from the region occupied by DNA and the nucleolus. It is inferred that most of the nuclear MCMs are not asociated with chromosomes in these nuclei and this pattern is referred to as nucleoplasmic. In contrast, in a small fraction of nuclei (~10%), nuclear MCM staining is coincident with the DNA. This state is interpreted as indicating a chromosomal association of MCMs. Cyclin E is expressed in transient pulses, each of which overlaps the beginning of each endocycle S phase. cyclin E mutants fail to undergo endoreplication. Heat induction of cyclin E produces a synchronous burst of DNA synthesis in polytene cells lasting between 3 and 6 hours. 40% of nuclei display chromosomal DmMCM2 after heat shock, and this association rapidly diminishes. Thus heat induction of Cyclin E drives chromosome association of DmMCM2. Subsequently, DNA synthesis erases this association. To test whether DNA synthesis is required for the dissociation of DmMCM2, DNA synthesis was blocked with an inhibitor, aphidicolin. In the presence of aphidicolin, chromosome-associated DmMCM2 is retained for up to 3 hours after induction of S phase by cyclin E, apparently stabilizing the association of DmMCM2 with chromosomes. It is concluded that DNA replication is required for dissociation of DmMCM2 from chromosomes. Thus, mitosis is not required for oscillations in chromosome binding of MCMs and it is proposed that cycles of MCM-chromosome association normally occur in endocycles. These results demonstrate that the cycle of MCM-chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles (Su, 1998).

Adult

There is maternal expression of dpa throughout oogenesis. The transcript accumulates in late oogenesis in the nurse cells before it is transferred to the oocyte (Feger, 1995).

Effects of Mutation or Deletion

Homozygous mutant animals develop into third instar larvae of normal size but pupariation is slightly delayed. Mutant animals die during pupal development. No imaginal discs are found in larval animals and the CNS of mutant larvae is reduced in size. Presence of endoreplicating tissues such as the gut, fat body and polytene chromosomes suggests that dpa is required during mitotic but not during endoreplicating cell cycles. Disc primordia are normal, as revealed by an escargot mRNA hybridization probe and antibodies specific for the Snail protein (Feger, 1995).

More cells are labeled with BrdU in stage 16 embryos than are normally found. Despite the ectopic labeling, mutant larvae show fewer ganglion mother cells surrounding putative neuroblasts. This suggests that the cell cycle is prolonged in mutant flies, and that dpa function is required for the regulation of DNA replication in neuroblasts (Feger, 1995).

Visualization of replication initiation and elongation in Drosophila

Chorion gene amplification in the ovaries of Drosophila is a powerful system for the study of metazoan DNA replication in vivo. Using a combination of high-resolution confocal and deconvolution microscopy and quantitative realtime PCR, it was found that initiation and elongation occur during separate developmental stages, thus permitting analysis of these two phases of replication in vivo. Bromodeoxyuridine, origin recognition complex, and the elongation factors minichromosome maintenance proteins (MCM)2-7 and proliferating cell nuclear antigen were precisely localized, and the DNA copy number along the third chromosome chorion amplicon was quantified during multiple developmental stages. These studies revealed that initiation takes place during stages 10B and 11 of egg chamber development, whereas only elongation of existing replication forks occurs during egg chamber stages 12 and 13. The ability to distinguish initiation from elongation makes this an outstanding model to decipher the roles of various replication factors during metazoan DNA replication. This system was used to demonstrate that the pre-replication complex component, Double-parked protein/Cdt1, is not only necessary for proper MCM2-7 localization, but, unexpectedly, is present during elongation (Claycomb, 2002).

Three independent lines of evidence are presented that initiation and the bulk of elongation at a chorion amplicon occur during two separate developmental periods. (1) Deconvolution microscopy shows that ORC and BrdU initially colocalize at origins and then diverge, since ORC is lost in stage 11 and BrdU resolves into a double bar structure. (2) Elongation factors PCNA and MCM2-7 follow the same pattern as BrdU, resolving from foci early in amplification to a double bar structure by stage 12 to 13. (3) Quantitative realtime PCR shows a peak increase in DNA copy number at the origins by stage 11, with increases in flanking sequences becoming substantial in stages 12 and 13. Thus initiation ends by stage 11, and during stages 12 and 13 only the existing forks progress outward. Furthermore, these observations led to the unanticipated conclusion that DUP/Cdt1 travels with replication forks (Claycomb, 2002).

The realtime PCR and immunofluorescence data are remarkably consistent. (1) Both methods restrict initiation to stages 10B and 11 of oogenesis, and elongation to stages 12 and 13. Between stages 10B and 11, the maximum fold amplification was detected at amplification control element (ACE) on third chromosome (ACE3) by realtime PCR, ORC localized to origins, and the deconvolution showed a maximum increase in bar length. During stages 12 and 13, increases in fold amplification were detected only proximal and distal to ACE3, and ORC no longer localized to origins, whereas BrdU incorporation resolved into the double bar structure. (2) The distances of fork movement are consistent. Deconvolution measurements predicted that forks were maximally 30 +/- 3 kb apart in stage 10B, and this correlates with the 40-kb span of peak copy number detected by realtime PCR. In stage 11, forks were measured to have progressed across a 55 +/- 13-kb region by deconvolution and across a 45-kb region by realtime PCR. By stage 13, deconvolution showed that replication forks were maximally separated by 74 +/- 7 kb, whereas realtime PCR measured a 75-kb span (Claycomb, 2002).

The quantitative analysis of the amplification gradient provides insight into mechanisms affecting fork movement and termination and suggests that an onionskin structure impedes fork movement. The maximal rate of fork movement during amplification has been calculated to be 90 bp/min on average. In comparison, replication forks in the polytene larval salivary glands travel at ~300 bp/min (Steinemann, 1981), whereas rates of fork movement in both diploid Drosophila cell culture and embryo syncytial divisions are ~2.6 kb/min. From these rates, it seems that polyteny hinders replication fork movement, an effect even more pronounced in amplification, given that the chorion cluster has a rate of fork movement three times less than polytene salivary glands. The fact that by stage 13 there is a gradient of copy number, and not a plateau, further demonstrates the inefficiency of fork movement along the chorion cluster (Claycomb, 2002).

There do not seem to be specific termination sites to stop forks either along or at the ends of the chorion region, but fork movement may display some sequence or chromatin preference. The gradient of decreasing copy number implies that forks stop at a range of sites, because the presence of specific termination points along the region would be expected to cause steep drops in copy number. Despite this lack of specific termination sites, during stages 12 and 13 a greater increase is seen in copy number to one side of ACE3, and it was often observe by immunofluorescence that one of the two bars is shorter. This suggests that the sequence or chromatin structure to the other side of ACE3 hinders fork movement, and as fewer forks move out, less BrdU incorporation occurs and a shorter bar results (Claycomb, 2002).

These studies highlight the complex regulation of chorion gene amplification. How are the number of origin firings restricted to the proper developmental time? It is known that the number of rounds of origin firing at the chorion amplicons is limited by the action of Rb, E2F1, and DP. Perhaps Dup and MCM2-7 are also a part of this regulation, with origins firing only when MCM2-7 are properly loaded. It will also be interesting to decipher the regulation of Dup/Cdt1 during amplification. Recent studies have demonstrated that a Drosophila homologue of the metazoan re-replication inhibitor, Geminin, exists and interacts biochemically and genetically with Dup/Cdt1. Female-sterile mutations in geminin result in increased BrdU incorporation during amplification, raising the possibility that Geminin acts on DUP/Cdt1 at the chorion loci to limit origin firing. In addition to permitting the delineation of the regulatory circuitry controlling origin firing, the ability to developmentally distinguish initiation from elongation provides a powerful tool for the analysis of the properties of metazoan replication factors in vivo (Claycomb, 2002).


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disc proliferation abnormal: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology

date revised: 10 November 2012

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