Cis-regulatory elements of the mitotic regulator, string/Cdc25.

Mitosis in most Drosophila cells is triggered by brief bursts of transcription of string (stg), a Cdc25-type phosphatase that activates the mitotic kinase, Cdk1 (Cdc2). To understand how string transcription is regulated, we analyzed the expression of string-lacZ reporter genes covering approximately 40 kb of the string locus. We also tested protein coding fragments of the string locus of 6 kb to 31.6 kb for their ability to complement loss of string function in embryos and imaginal discs. A plethora of cis-acting elements spread over >30 kb control string transcription in different cells and tissue types. Regulatory elements specific to subsets of epidermal cells, mesoderm, trachea and nurse cells were identified, but the majority of the string locus appears to be devoted to controlling cell proliferation during neurogenesis. Consistent with this, compact promotor-proximal sequences are sufficient for string function during imaginal disc growth, but additional distal elements are required for the development of neural structures in the eye, wing, leg and notum. We suggest that, during evolution, cell-type-specific control elements were acquired by a simple growth-regulated promoter as a means of coordinating cell division with developmental processes, particularly neurogenesis.

Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms.

In newly hatched Drosophila larvae, quiescent cells reenter the cell cycle in response to dietary amino acids. To understand this process, we varied larval nutrition and monitored effects on cell cycle initiation and maintenance in the mitotic neuroblasts and imaginal disc cells, as well as the endoreplicating cells in other larval tissues. After cell cycle activation, mitotic and endoreplicating cells respond differently to the withdrawal of nutrition: mitotic cells continue to proliferate in a nutrition-independent manner, while most endoreplicating cells reenter a quiescent state. We also show that ectopic expression of Drosophila Cyclin E or the E2F transcription factor can drive quiescent endoreplicating cells, but not quiescent imaginal neuroblasts, into S-phase. Conversely, we demonstrate that quiescent imaginal neuroblasts, but not quiescent endoreplicating cells, can be induced to enter the cell cycle when co-cultured with larval fat body in vitro. These results demonstrate a fundamental difference in the control of cell cycle activation and maintenance in these two cell types, and imply the existence of a novel mitogen generated by the larval fat body in response to nutrition.

Pointed, an ETS domain transcription factor, negatively regulates the EGF receptor pathway in Drosophila oogenesis.

Spatially regulated activation of the Drosophila epidermal growth factor (EGF) receptor by its ligand, Gurken, is required for establishment of the dorsal/ventral axis of the oocyte and embryo. During mid-oogenesis, Gurken is concentrated at the dorsal-anterior of the oocyte and is thought to activate the EGF receptor pathway in adjacent follicle cells. In response to this signal, dorsal follicle cell fate is determined. These cells further differentiate into either appendage-producing or midline cells, resulting in patterning in the dorsal follicle cell layer. We show here that Pointed, an ETS transcription factor, is required in dorsal follicle cells for this patterning. Loss of pointed results in the loss of midline cells and an excess of appendage-forming cells, a phenotype associated with overactivation of the EGF receptor pathway in the dorsal region. Overexpression of pointed leads to a phenotype similar to that generated by loss of the EGF receptor pathway. This suggests that Pointed normally down-regulates EGF receptor signaling in the midline to generate patterning in the dorsal region. Interestingly, pointed expression is induced by the EGF receptor pathway. These data indicate a novel antagonistic function for Pointed in oogenesis; in response to activation of the EGF receptor, pointed is expressed and negatively regulates the EGF receptor pathway, possibly by integrating information from a second pathway.

Mutations in an essential U2 small nuclear RNA structure cause cold-sensitive U2 small nuclear ribonucleoprotein function by favoring competing alternative U2 RNA structures.

Mutations in stem-loop IIa of yeast U2 RNA cause cold-sensitive growth and cold-sensitive U2 small nuclear ribonucleoprotein function in vitro. Cold-sensitive U2 small nuclear RNA adopts an alternative conformation that occludes the loop and disrupts the stem but does so at both restrictive and permissive temperatures. To determine whether alternative U2 RNA structure causes the defects, we tested second-site mutations in U2 predicted to disrupt the alternative conformation. We find that such mutations efficiently suppress the cold-sensitive phenotypes and partially restore correct U2 RNA folding. A genetic search for additional suppressors of cold sensitivity revealed two unexpected mutations in the base of an adjacent stem-loop. Direct probing of RNA structure in vivo indicates that the suppressors of cold sensitivity act to improve the stability of the essential stem relative to competing alternative structures by disrupting the alternative structures. We suggest that many of the numerous cold-sensitive mutations in a variety of RNAs and RNA-binding proteins could be a result of changes in the stability of a functional RNA conformation relative to a competing structure. The presence of an evolutionarily conserved U2 sequence positioned to form an alternative structure argues that this region of U2 is dynamic during the assembly or function of the U2 small nuclear ribonucleoprotein.