Larval endocycles regulation at GS by cell growth and nutrition

Drosophila larvae are built for eating and growth. Larvae increase their mass ~ 200fold in just four days as they prepare for metamorphosis. Almost all of this growth is due to increases in cell size in differentiated larval-specific tissues that progress through a modified cell cycle — the endocycle—consisting of successive rounds of DNA synthesis without intervening mitoses. Endoreplicative tissues (ERTs) include the larval gut, fat body, salivary glands, malpighian tubules, trachea, muscles and epidermis. The switch from mitotic proliferation to endocycles occurs in late embryogenesis, and appears to be accomplished by loss of String and the mitotic cyclins A and B, whilst continuing periodic expression of the S phase-promoter, cyclin E. Studies of mutants defective in DNA replication and using replication inhibitors show that the increased DNA ploidy resulting from endocycling is absolutely required for larval growth (Royzman et al 1997, B. Edgar, J. Britton, A. de la Cruz, L. Johnston, D. Lehman, C. Martin-Castellanos & D. Prober, unpublished results).

Our studies of the ERT cell cycles show that they are regulated by nutrition (Britton & Edgar 1998). If the newly hatched larva is starved for dietary amino acids, DNA replication in most ERTs is not initiated. Under starvation conditions these tissues express low levels of cyclin E and E2F, the transcription factor which is probably responsible for cyclin E expression. If either E2F or cyclin E is induced in starved larvae, DNA replication in the ERTs is activated, and thus expression of these genes appears to limit the ERT cell cycle. When nutrient-deprived larvae are fed, expression of E2F and cyclin E mRNAs increases approximately sixfold, and DNA replication is initiated in most ERT cells. If the animal is first fed and then starved, the ERT cell cycle is activated and then inactivated quite rapidly. These experiments all indicate that the ERT cell cycle is nutrition-responsive, rather than controlled by a rigid developmental program.

This mode of regulation seems appropriate to the ERTs since their cells are already terminally differentiated, and their primary function is to grow and provide a nutrient rich 'incubator' for the undifferentiated neuroblasts and imaginal cells that eventually produce the reproductive adult. The response of these undifferentiated progenitor cells to food withdrawal is quite unlike that of the ERTs. Larval neuroblasts and imaginal disc cells continue to proliferate for many days after a larva is starved, and seem to complete their normal proliferation programs. In this instance the ERTs lose mass, presumably as they transfer stored nutrients to the developing nervous system and the discs.

These experiments suggest a mechanism in which E2F and cyclin E, which drive G/S transitions in the ERTs, respond directly to nutritional inputs (Fig. 2B). Our working hypothesis is that the link between nutrition and endoreplication in the ERTs is simply cell growth. This is supported by several observations. First, ectopic expression of genes that drive cell growth can promote extra DNA replication in ERTs in fed animals, and can trigger unscheduled DNA replication in the ERTs in starved animals. These genes include the transcription factor d-Myc (Johnston et al 1999), phosphatidylinositol-3-kinase (PI3K) (Weinkove et al 1999) and cyclin D/Cdk4 (unpublished results). Second, we've found that levels of cyclin E protein are post-transcriptionally responsive to increased rates of cellular growth in imaginal discs (see below; Prober & Edgar 2000). Assuming the same mechanism operates in ERTs, we suggest that ERT cells simply grow when nutritional conditions permit, and that one cellular response is increased cyclin E activity and DNA endoreplication.

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