Jessica E. Treisman

The Drosophila Tuberous Sclerosis Complex Gene Homologs Restrict Cell Growth and Cell Proliferation

The inherited human disease tuberous sclerosis, characterized by hamartomatous tumors, results from mutations in either TSC1 or TSC2. We have characterized mutations in the Drosophila Tsc1 and Tsc2/gigas genes. Inactivating mutations in either gene cause an identical phenotype characterized by enhanced growth and increased cell size with no change in ploidy. Overall, mutant cells spend less time in G1. Coexpression of both Tsc1 and Tsc2 restricts tissue growth and reduces cell size and cell proliferation. This phenotype is modulated by manipulations in cyclin levels. In postmitotic mutant cells, levels of Cyclin E and Cyclin A are elevated. This correlates with a tendency for these cells to reenter the cell cycle inappropriately as is observed in the human lesions.

Sightless has homology to transmembrane acyltransferases and is required to generate active Hedgehog protein

Proteins of the Hedgehog (Hh) family act as important developmental signals in a variety of species [1]. Hh proteins are synthesized as full-length precursors that are autocatalytically cleaved by their C-terminal domains to release the signaling N-terminal domains [2]. The addition of a cholesterol molecule to the C terminus of the signaling domain is concomitant with cleavage [3]. Vertebrate Sonic hedgehog (Shh) proteins have also been shown to acquire a fatty acid chain on the N-terminal cysteine of this domain [4], which is required for a subset of their in vivo functions [5, 6]. A mutation of the corresponding cysteine in Drosophila Hh transforms it into a dominant-negative protein [6]. We have identified a novel gene, sightless (sit), which is required for the activity of Drosophila Hh in the eye and wing imaginal discs and in embryonic segmentation. sit acts in the cells that produce Hh, but does not affect hh transcription, Hh cleavage, or the accumulation of Hh protein. sit encodes a conserved transmembrane protein with homology to a family of membrane-bound acyltransferases. The Sit protein could act by acylating Hh or by promoting other modifications or trafficking events necessary for its function.

Osa associates with the Brahma chromatin remodeling complex and promotes the activation of some target genes.

The yeast SWI/SNF complex and its Drosophila and mammalian homologs are thought to control gene expression by altering chromatin structure, but the mechanism and specificity of this process are not fully understood. The Drosophila osa gene, like yeast SWI1, encodes an AT‐rich interaction (ARID) domain protein. We present genetic and biochemical evidence that Osa is a component of the Brahma complex, the Drosophila homolog of SWI/SNF. The ARID domain of Osa binds DNA without sequence specificity in vitro, but it is sufficient to direct transcriptional regulatory domains to specific target genes in vivo. Endogenous Osa appears to promote the activation of some of these genes. We show evidence that some Brahma‐containing complexes do not contain Osa and that Osa is not required to localize Brahma to chromatin. These data suggest that Osa modulates the function of the Brahma complex.

The ubiquitin ligase Hyperplastic discs negatively regulates hedgehog and decapentaplegic expression by independent mechanisms

Photoreceptor differentiation in the Drosophila eye disc progresses from posterior to anterior in a wave driven by the Hedgehog and Decapentaplegic signals. Cells mutant for the hyperplastic discs gene misexpress both of these signaling molecules in anterior regions of the disc, leading to premature photoreceptor differentiation and overgrowth of surrounding tissue. The two genes are independently regulated by hyperplastic discs; decapentaplegic can still be misexpressed in cells mutant for both hyperplastic discs and hedgehog, and a repressor form of the transcription factor Cubitus interruptus can block decapentaplegic misexpression but not hedgehog misexpression. Loss of hyperplastic discs causes the accumulation of full-length Cubitus interruptus protein, but not of Smoothened, in both the eye and wing discs. hyperplastic discs encodes a HECT domain E3 ubiquitin ligase that is likely to act by targeting Cubitus interruptus and an unknown activator of hedgehog expression for proteolysis.

Cell-autonomous and -nonautonomous functions of LAR in R7 photoreceptor axon targeting.

During Drosophila visual system development, photoreceptors R7 and R8 project axons to targets in distinct layers of the optic lobe. We show here that the LAR receptor tyrosine phosphatase is required in the eye for correct targeting of R7 axons. In LAR mutants, R7 axons initially project to their correct target layer, but then retract to the R8 target layer. This targeting defect can be fully rescued by transgenic expression of LAR in R7, and partially rescued by expression of LAR in R8. The phosphatase domains of LAR are required for its activity in R7, but not in R8. These data suggest that LAR can act both as a receptor in R7, and as a ligand provided by R8. Genetic interactions implicate both Enabled and Trio in LAR signal transduction.

Pygopus activates Wingless target gene transcription through the mediator complex subunits Med12 and Med13

Wnt target gene transcription is mediated by nuclear translocation of stabilized β-catenin, which binds to TCF and recruits Pygopus, a cofactor with an unknown mechanism of action. The mediator complex is essential for the transcription of RNA polymerase II-dependent genes; it associates with an accessory subcomplex consisting of the Med12, Med13, Cdk8, and Cyclin C subunits. We show here that the Med12 and Med13 subunits of the Drosophila mediator complex, encoded by kohtalo and skuld, are essential for the transcription of Wingless target genes. kohtalo and skuld act downstream of β-catenin stabilization both in vivo and in cell culture. They are required for transcriptional activation by the N-terminal domain of Pygopus, and their physical interaction with Pygopus depends on this domain. We propose that Pygopus promotes Wnt target gene transcription by recruiting the mediator complex through interactions with Med12 and Med13.

act up Controls Actin Polymerization to Alter Cell Shape and Restrict Hedgehog Signaling in the Drosophila Eye Disc

Cells in the morphogenetic furrow of the Drosophila eye disc undergo a striking shape change immediately prior to their neuronal differentiation. We have isolated mutations in a novel gene, act up (acu), that is required for this shape change. acu encodes a homolog of yeast cyclase-associated protein, which sequesters monomeric actin; we show that acu is required to prevent actin filament polymerization in the eye disc. In contrast, profilin promotes actin filament polymerization, acting epistatically to acu. However, both acu and profilin are required to prevent premature Hedgehog-induced photoreceptor differentiation ahead of the morphogenetic furrow. These findings suggest that dynamic changes in actin filaments alter cell shape to control the movement of signals that coordinate a wave of differentiation.

Pattern formation in the Drosophila eye disc

Differentiation of the Drosophila compound eye from the eye imaginal disc is a progressive process: columns of cells successively differentiate in a posterior to anterior sequence, clusters of cells form at regularly spaced intervals within each column, and individual photoreceptors differentiate in a defined order within each cluster. The progression of differentiation across the eye disc is driven by a positive autoregulatory loop of expression of the secreted molecule Hedgehog, which is temporally delayed by the intercalation of a second signal, Spitz. Hedgehog refines the spatial position at which each column initiates its differentiation by inducing secondary signals that act over different ranges to control the expression of positive and negative regulators. The position of clusters within each column is controlled by secreted inhibitory signals from clusters in the preceding column, and a single founder neuron, R8, is singled out within each cluster by Notch-mediated lateral inhibition. R8 then sequentially recruits surrounding cells to differentiate by producing a short-range signal, Spitz, which induces a secondary short-range signal, Delta. Intrinsic transcription factors act in combination with these two signals to produce cell-type diversity within the ommatidium. The Hedgehog and Spitz signals are transported along the photoreceptor axons and reused within the brain as long-range and local cues to trigger the differentiation and assembly of target neurons.

4 Eye Development in Drosophila: Formation of the Eye Field and Control of Differentiation

Publisher Summary This chapter discusses the early stages of eye development in Drosophila , focusing on the mechanisms that determine the global pattern rather than those involved in the establishment of specific cell fates. It discusses the specification of the eye disk to form an eye, the control of morphogenetic furrow initiation and propagation, and the coordination of growth with differentiation. Most adult Drosophila structures, including the eye, develop from imaginal disks. These are groups of cells set aside in the embryo that grow and differentiate inside the larva and evert to become functional during metamorphosis. Differentiation in the eye disk is progressive, moving across the disk in a wavelike manner from posterior to anterior. The front of the wave is marked by an indentation in the disk known as the morphogenetic furrow. Most cell division occurs in the unpatterned cells anterior to the furrow, while on the posterior side of the furrow the cells are organized into clusters that develop into the ommatidia. The Drosophila eye disk develops in a remarkably coordinated manner. Its differentiation begins at a precisely defined point and expands at a steady rate, laying down rows of evenly spaced ommatidial founder cells.

Two subunits of the Drosophila mediator complex act together to control cell affinity.

The organizing centers for Drosophila imaginal disc development are created at straight boundaries between compartments; these are maintained by differences in cell affinity controlled by selector genes and intercellular signals. skuld and kohtalo encode homologs of TRAP240 and TRAP230, the two largest subunits of the Drosophila mediator complex; mutations in either gene cause identical phenotypes. We show here that both genes are required to establish normal cell affinity differences at the anterior-posterior and dorsal-ventral compartment boundaries of the wing disc. Mutant cells cross from the anterior to the posterior compartment, and can distort the dorsal-ventral boundary in either the dorsal or ventral direction. The Skuld and Kohtalo proteins physically interact in vivo and have synergistic effects when overexpressed, consistent with a skuld kohtalo double-mutant phenotype that is indistinguishable from either single mutant. We suggest that these two subunits do not participate in all of the activities of the mediator complex, but form a submodule that is required to regulate specific target genes, including those that control cell affinity.