BIOLOGICAL OVERVIEW

anterior open, more often referred to as yan, is a transcription factor that serves to inhibit neural and other types of differentiation. When yan is inactivated, differentiation proceeds. yan is modulated by the effects of Sevenless/Ras/MAPK, a major cytoplasmic signal transduction pathway. This pathway is a lengthy and complicated cascade of events leading to the activation of Pointed, and the inactivation of Yan. A look at Sevenless should prove instructive.

Sevenless is one of at least four Drosophila receptors that utilize the Ras/MAPK pathway to tranduce extracellular signals from outside the cell to the nucleus; the others are EGF-R or Torpedo, the homolog of the vertebrate epidermal growth factor receptor; Torso, the receptor responsible for terminal signaling; Breathless, the Drosophila FGF receptor homolog, and finally, Sevenless, the receptor for BOSS (Bride of Sevenless). Sevenless regulates the determination of cell fate of the R7 photoreceptor in the eye. R7 is the last to develop of the eight photoreceptors in each ommatidium (see The Drosophila Adult Ommatidium: Illustration and explanation with Quicktime animation).

To understand how the cascade functions physically, one must look at the proteins implicated in Sevenless signaling, and the signals of other receptor tyrosine kinases, including Ras1 and the mitogen actived protein kinase (MAPK) family of serine/threonine kinases. These include Raf (MAPKKK), DSor (MAPKK) and rolled (MAPK). These three kinases each add phosphate residues to the next protein in the signaling cascade as the activation signal is passed from molecule to molecule. Ultimately, the cascade reaches its targets; the transcription factors Yan and Pointed. While Pointed is a positive regulator of genes, activated by the cascade, Yan is negatively regulated by Sevenless signaling. Yan is an antagonist of the Sevenless/Ras1 proneural signal, meaning when the signal to differentiate is turned on, Yan is turned off.

Yan is a repressor. It is expressed in basally located nuclei of undifferentiated cells in the larval eye imaginal disc. As cells differentiate and their nuclei migrate to an apical position, yan expression is suddenly shut down, or downregulated. Phosphorylation by MAPK regulates the stability and subcellular localization of Yan. A mutant form of Yan, deficient in phosphorylation sites, results in cell death, possibly due to prolonged Yan-mediated inhibition of differentiation (Rebay, 1995).

Although yan is expressed in the embryonic ectoderm and mesoderm, it is absent in the developing central nervous system. Ectopic expression of yan in the CNS inhibits embryonic development. Overexpression of yan in the mesoderm, where it is normally expressed but later downregulated, strongly reduces twist expression. twist is vital to the differentiation of cells that will eventually become mesoderm. Such results indicated that yan is an inhibitor of differentiation, and does not just affect neural cells, but is also relevant in other sites. Yan is not a universal inhibitor of differentiation, since other ectodermal markers such as engrailed show no effects from yan overexpression. Notch function is similar to Yan. It too inhibits differentiation, and provides another example of the importance of negative regulation in the differentiation process (Rebay, 1995).

A key step in development is the establishment of cell type diversity across a cellular field. Segmental patterning within the Drosophila embryonic epidermis is one paradigm for this process. At each parasegment boundary, cells expressing the Wnt family member Wingless confront cells expressing the homeoprotein Engrailed. The Engrailed-expressing cells normally differentiate as one of two alternative cell types. In investigating the generation of this cell type diversity among the 2-cell-wide Engrailed stripe, it has been shown that Wingless, expressed just anterior to the Engrailed cells, is essential for the specification of anterior Engrailed cell fate. In a screen for additional mutations affecting Engrailed cell fate, anterior open (aop) (also known as yan) was identified, a gene encoding an inhibitory ETS-domain transcription factor that is negatively regulated by the Ras-MAP kinase signaling cascade. Anterior open must be inactivated for posterior Engrailed cells to adopt their correct fate. This is achieved by the EGF receptor (Egfr), which is required autonomously in the Engrailed cells to trigger the Ras1-MAP kinase pathway. Localized activation of Egfr is accomplished by restricted processing of the activating ligand, Spitz. Processing is confined to the cell row posterior to the Engrailed domain by the restricted expression of Rhomboid. These cells also express the inhibitory ligand Argos, which attenuates the activation of Egfr in cell rows more distant from the ligand source. Thus, distinct signals flank each border of the Engrailed domain, since Wingless is produced anteriorly and Spitz posteriorly. Since En cells have the capacity to respond to either Wingless or Spitz, these cells must choose their fate depending on the relative level of activation of the two pathways (O'Keefe, 1997).

The larval cuticle comprises a repeated array of precisely patterned denticle belts interspersed with smooth cuticle. In abdominal segments, each of these belts is made up of 6 rows of denticles, where each row is of a characteristic size and orientation reflecting fate decisions made by the underlying cells. Using a lacZ reporter gene expressed in the En cells, it has been demonstrated that the anterior En cells normally produce smooth cuticle, while the posterior En cells produce denticles and, thereby, form the first row of each belt. Thus, cells in the En domain adopt either a smooth or denticle fate depending on their position. To identify genes involved in specifying En cell fates, existing collections of mutants were screened for those in which anterior En cells inappropriately produce denticles. Ectopic denticles are observed immediately anterior to the denticle belts in aop mutants. The extra denticles are located at the lateral edges of denticle belts, and are more commonly observed in the posterior segments. To determine whether the En cell fates were altered in these mutants, the En cells were visualized with a lacZ reporter construct. Anterior En cells produce denticles instead of the normal smooth cuticle. Thus, aop function is required for some anterior En cells to adopt the smooth cell fate (O'Keefe, 1997).

Since aop activity is required for anterior En cells to adopt the smooth cell fate, Aop activity was tested to see if it was sufficient to force posterior En cells to produce smooth cuticle instead of first row denticles. A constitutively active form of Aop was examined, where all eight MAP kinase consensus phosphorylation sites were mutated, and its expression was driven in the En cells using the UAS/GAL4 system. While En-GAL4 embryos carrying UAS-Aop WT exhibit normal denticle pattern, such embryos carrying UAS-Aop Act are missing the normal first denticle row of each belt. Thus, if posterior En cells express a form of Aop that can not be inhibited by MAP kinase, then these cells adopt the smooth fate. This suggests that, normally, Aop must be inactivated in the posterior En cells for them to adopt denticle fates. Given that the Ras1-MAP kinase cascade is responsible for inhibiting Aop function in other tissues, it became a good candidate for inactivating Aop in the posterior En cells. If this pathway is indeed involved, then inappropriate activation of the pathway should mimic the aop mutant phenotype and allow anterior En cells to incorrectly produce denticles. To test this, embryos expressing a constitutively active form of Ras (UAS-Ras1 val-12 ) in the En cells were examined. These embryos have an ectopic row of denticles anterior to the normal first row, corresponding to the location of the anterior En cells. Thus, the anterior En cells are mis-specified by ectopic Ras1-MAP kinase activity, similar to the effects of loss of aop function. This suggests that Ras1-MAP kinase activity may normally be responsible for inactivating Aop in the posterior En cells, allowing them to adopt the denticle fate (O'Keefe, 1997).

Since the Ras1-MAP kinase cascade is activated by receptor tyrosine kinases, a test was performed to see whether such a receptor could be involved in specifying En cell fate. For several reasons, the best candidate was the Drosophila EGF Receptor (Egfr). (1) In the eye, an allele of aop was isolated as an enhancer of mutations in Ellipse, a gain-of-function allele of Egfr. (2) Egfr is ubiquitously expressed epidermis throughout embryogenesis and is required early for ventral-to-lateral patterning, as is Aop. Finally, at later stages, Egfr is required for cells to adopt denticle fates (O'Keefe, 1997 and references).

To address whether Egfr function is required for posterior En cells to adopt their correct fate, a dominant negative form of Egfr was expressed specifically in En cells. These embryos lack the first denticle row, corresponding to the position of the posterior En cells. Therefore, Egfr is autonomously required for the posterior En cells to adopt a denticle fate. It was next determined whether there is a source of Egfr ligand positioned appropriately to signal to the En cells. The Spitz source is posteriorly adjacent to En cells. It seemed likely that Egfr would be activated by Spi, its ligand in many other contexts. Spi is ubiquitously expressed as an inactive membrane-bound molecule with homology to TGF-alpha. A processing event, which requires Rhomboid (Rho) activity, releases active ligand. Thus, the spatially regulated expression of Rho marks cells that are the source for active, secreted Spi. These cells can trigger activation of Egfr in adjacent cells. The expression of Rhomboid suggests that there is a novel source of active Spi ligand at the appropriate time and place to influence En cell fate (O'Keefe, 1997).

To test directly whether the Egfr pathway is activated in these transverse stripes, the spatial distribution of activated MAP kinase was examined, using an antibody that is specific to the di-phosphorylated (active) form of MAP kinase (dp-ERK). In late stage embryos (9.5 hours AEL), a stripe of activated MAP kinase is detected just posterior to the En cells. This stripe is dependent on Egfr, since it is selectively removed in flb mutant embryos. In wild type, active MAP kinase is detectable within the En cells themselves, although at low levels. Thus, it appears that Egfr activation indeed spreads into the En cells. It could not be determined whether there is a difference between the anterior and posterior En cells. Activation of the Egfr pathway was confirmed by testing for the induction of a Egfr target gene, argos, the expression of which is closely correlated with regions of maximal Egfr activation. For instance, during earlier ventral-to-lateral patterning, argos is expressed in the ventralmost 1- to 2-cell rows, the point of highest Egfr activation. However, at later stages Argos mRNA is expressed in a stripe of cells posterior to the En cells, coincident with the expression of Rho and the highest levels of activated MAP kinase. Taken together, these data demonstrate that a secreted Egfr ligand (probably secreted Spitz), produced by cells just posterior to En cells, activates Egfr. Furthermore, it appears that the activation of Egfr is graded; highest posterior to En cells and at lower levels within the En cells. This signaling corresponds to the time when fates of the En cells are being determined, which is consistent with a role for Egfr in determining the fates of En cells. Experiments were carried out that revealed that anterior En cells can, in fact respond to Spitz (O'Keefe, 1997).

Spitz and Wingless signaling have been shown to have competing affects on En cell fate. Anterior En cells assume a denticle fate when wg function is eliminated at 8 hours AEL. Wg is expressed just anterior to the En domain, in a region of smooth cuticle. Thus, while Wg input instructs cells to adopt the smooth fate, activation of Egfr instructs cells to adopt denticle fates. The opposite response of En cells to these two signals raises the question of what fate these cells would adopt in the absence of both signals. To determine this, Egfr signaling was blocked by expressing Aop Act in En cells while concomitantly removing wg function using a conditional allele. When wg ts embryos carrying both En-GAL4 and UAS-Aop Act are shifted to non-permissive temperature at 8 hours AEL, the En cells adopt smooth fates. This suggests that smooth cuticle is the default cell fate. Wg signaling in this context is required primarily for antagonizing the effect of DER signaling in anterior En cells (O'Keefe, 1997).

The posterior En cells, which adopt a denticle fate, either cannot respond to Wg due to the absence of key signal transducers, or they do not see effective concentrations of Wg. In fact, it appears that the posterior En cell does not receive Wg input. The presence of downstream signal transducers was tested in posterior En cells. Cells expressing either an activated form of Armadillo or higher levels of wild-type Disheveled respond as if they have received the Wg signal. In embryos carrying both En-GAL4 and UAS-Arm S10, the expression of activated Armadillo causes the posterior En cells to inappropriately adopt the smooth cell fate. Identical results were obtained expressing Disheveled. Thus, Wg signal transducers downstream of Disheveled are present in posterior En cells. During normal patterning, these cells are probably not exposed to sufficient Wg levels to antagonize the effects of Egfr in these cells (O'Keefe, 1997).

A model is presented for the cooperation between Wingless and Spitz in specifying cell fate in Engrailed expressing cells. The En-expressing cells are flanked anteriorly by a cell row producing Wg and posteriorly by a cell row expressing Rhomboid, which produces secreted Spitz. The En cell nearest the Spi source receives a higher concentration of Spi, and thus activates the Egfr pathway sufficiently to specify a denticle fate. Reciprocally, the En cell nearest the Wg source receives a higher concentration of Wg and adopts a smooth fate. Spi also activates the Egfr pathway in the Rho-expressing cell, which therefore produces and secretes Argos. Argos can inhibit Spi activation of the Egfr pathway at a distance. As a consequence, the Egfr pathway is not sufficiently activated in the anterior En cell to out compete Wg signaling in this cell, and it adopts a smooth fate. In fact, the specific targets of Egfr signaling responsible for conferring the denticle fate are unknown (O'Keefe, 1997).

GENE STRUCTURE

Exons - two, separated by 16 kb

Bases in 3' UTR - 1683

PROTEIN STRUCTURE

Amino Acids - 732

Structural Domains

Yan has an ETS domain, bracketed by glutamate rich regions, two preceding it, and one following (Lai, 1992).

The members of the ets gene family of transcription factors are characterized by a conserved 85-residue DNA-binding region, termed the ETS domain, that lacks sequence homology to structurally characterized DNA-binding motifs. The ETS domain is composed of three alpha-helices (H) and four beta-strands (S) arranged in the order H1-S1-S2-H2-H3-S3-S4. The four-stranded antiparallel beta-sheet is the scaffold for a putative helix-turn-helix DNA recognition motif formed by helices 2 and 3. The 25 residues extending beyond the ETS domain to the native C-terminus of the truncated Ets-1 also contain a helical segment. On the basis of the similarity of this topology with that of catabolite activator protein (CAP), heat shock factor (HSF), and hepatocyte nuclear factor (HNF-3 gamma), it is proposed that ets proteins are members of the superfamily of winged helix-turn-helix DNA-binding proteins (Donaldson, 1994).

There are eight MAPK phosphorylation consensus repeats on Yan as well as six PEST sequences conferring rapid turnover (Rebay, 1995).

date revised: 12 Dec 96 

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.