STAT/marelle: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Signal-transducer and activator of

transcription protein at 92E

Synonyms - D-STAT, marelle (mrl)

Cytological map position - 92E2

Function - transcription factor, signal transduction molecule

Keyword(s) - JAK/STAT pathway, regulation of pair rule genes, tumor suppressor, germband extension

Symbol - Stat92E

FlyBase ID:FBgn0015512

Genetic map position -

Classification - STAT homolog - src homology 2 domain

Cellular location - cytoplasmic and nuclear



NCBI links: Entrez Gene | HomoloGene | UniGene
BIOLOGICAL OVERVIEW

The JAK/STAT pathway pathway is involved in communicating extracellular events on an intracellular level: signals must be carried across the cell membrane, through the cytoplasm and finally into the nucleus. Receptors that function through Janus kinase (JAK) proteins do not themselves have kinase activity, but rely on the kinase activity of the JAK family to transduce their signals into the cell.

Two laboratories have simultaneously described STAT, referred to here as marelle, the Drosophila homolog for mammalian STAT (signal transducer and activator of transcription) proteins. One characterized a maternal lethal effect that exhibited a segmentation phenotype similar to the effect of the loss of hopscotch. hopscotch is the Drosophila homolog of the mammalian JAK family kinases. The second lab had been searching for a Drosophila homolog for the mammalian STAT gene (Hou, 1996 and Yan, 1996a). Discovery of marelle completes the identification of the JAK-STAT pathway in the fly.

The mammalian janus family tyrosine kinases (JAK kinases) associate with the intracellular domains of particular cytokine receptors, and become activated following ligand-induced assembly of receptor subunits at the cell surface. The STATs are a family of src homology 2 proteins that are cytoplasmic transcription factors that serve to transduce signals from the activated JAK kinases to the nucleus. The src homology 2 domain serves to dock STAT proteins to phosphorylated substrates. STAT proteins are phosphorylated in turn. When activated by tyrosine phosphorylation, STAT proteins undergo dimerization. They translocate to the nucleus and promote transcriptional activation of cytokine inducible genes (Schindler, 1995). The identity of possible Drosophila cytokines, if there are any in this system, is not known.

Drosophila STAT, named marelle (the French term for "hopscotch"), is present in the egg at the time of fertilization and is expressed early in development. It is transcribed initially in a pair-rule striped pattern and later in a 14 striped segment polarity pattern. MRL binds to and regulates the even-skipped stripe 3 promoter, and regulates the pair rule gene runt (Hou, 1996 and Yan, 1996).

Polarity of the Drosophila compound eye is established at the level of repeating multicellular units (known as ommatidia), which are organized into a precise hexagonal array (see The Drosophila Adult Ommatidium: Illustration and explanation with Quicktime animation). The adult eye is composed of ~800 ommatidia, each of which forms one facet. Sections through the eye reveal that each ommatidium contains eight photoreceptor cells in a stereotypic trapezoidal arrangement that has two mirror-symmetric forms: a dorsal form present above the dorsoventral (DV) midline, and a ventral form below. An axis of mirror-image symmetry runs along the DV midline and is known as the equator. By analogy to the terrestrial equator, the extreme dorsal and ventral points of the eye are referred to as the poles. Differentiation of ommatidia begins during the third instar larval stage when a furrow moves from posterior to anterior over the epithelium of the eye imaginal disc. Each ommatidial unit is born as a bilaterally symmetrical cluster of photoreceptor precursors, that is polarized on its anteroposterior axis. The clusters then become polarized on the DV (or equatorial-polar) axis, by the process of proto-ommatidium rotation via two 45° steps away from the DV midline, forming the equator. It has been suggested that the direction of this rotation is a consequence of a gradient of positional information emanating from either the midline or the polar regions of the disc (Zeidler, 1999 and references).

A number of recent studies have shed light on some of the mechanisms involved in the positioning of the equator on the DV midline of the eye imaginal disc. It is now clear that a critical step is the activation of Notch activity in a line of cells along the midline, and that this localized activation of Notch is a consequence of the restricted expression of the fringe (fng) gene product in the ventral half of the disc and the homeodomain transcription factor Mirror (Mirr) in the dorsal half of the disc. Furthermore, an important role for Wingless (Wg) in polarity determination on the DV axis has been demonstrated. Wg is a secreted protein (and the founder member of the Wnt family of morphogens) that is expressed at the poles of the eye disc. Wg has been shown to act as an activator of mirr expression; increasing the levels of Wg expression in the eye disc shifts mirr expression and the position of the equator ventrally, whereas reduction of wg function shifts mirr expression dorsally. Additionally, it has been shown convincingly that a gradient of Wg signaling across the DV axis of the eye disc regulates ommatidial polarity such that the lowest point of Wg signaling coincides with the equator (Zeidler, 1999 and references).

The JAK/STAT pathway is central to the establishment of planar polarity during Drosophila eye development. A localized source of the pathway ligand, Unpaired/Outstretched, present at the midline of the developing eye, is capable of activating the JAK/STAT pathway over long distances. A gradient of JAK/STAT activity across the DV axis of the eye regulates ommatidial polarity via an unidentified second signal. Additionally, localized Unpaired influences the position of the equator via repression of mirror (Zeidler, 1999).

The data points to a model in which Upd and Wg first act to define the equator via restriction of mirr expression to the dorsal hemisphere and localized activation of Notch along the DV midline. Definition of the equator is known to occur early in development, while the disc is still small, and divides the disc into two hemispheres separated by a straight boundary that will form the future equator. Such boundaries evidently serve as a source of a second signal that can polarize ommatidia, since fng loss of function clones that induce ectopic regions of activated Notch result in changes in ommatidial polarity. Subsequently in development, it is surmised that gradients of JAK/STAT and Wg-pathway activity across the DV axis of the eye disc are responsible for setting up a gradient(s) of one or more second signals (most likely detected by the receptor Frizzled) that can determine ommatidial polarity. These signals might be responsible for maintaining longer range polarization of ommatidia away from the equator and the localized Notch-dependent polarizing signal (Zeidler, 1999 and references).

Loss of function (LOF) clones for mutations in the Drosophila JAK and STAT homologs were generated by the FLP/FRT system. Tangential sections through LOF clones of both hop and stat alleles show a regular array of ommatidia containing a wild-type complement of correctly differentiated and correctly positioned photoreceptor cells. Thus, the JAK/STAT pathway is not absolutely required for imaginal disc cell proliferation, cell fate specification, or differentiation. Mutant clones are, however, associated with stereotyped defects in ommatidial polarity (Zeidler, 1999).

A large proportion of hop LOF clones result in polarity defects in which ommatidia straddling the polar boundary of the clone exhibit inverted DV polarity. The phenotype is strongest in larger clones and in clones in which the polar boundary runs parallel to the equator. Typically, one or two ommatidial rows are inverted, with the strongest phenotype observed showing about five inverted rows. Mutant ommatidia in the center of the clone and on the equatorial margin of the clone show a normal orientation. Both totally mutant ommatidia adjacent to the polar boundary and chimeric ommatidia comprising both wild-type and mutant cells on the clonal border can assume an inverted fate. Occasional inversions are observed in clusters immediately outside the clone in which all of the photoreceptors are wild type. LOF hop clones examined in third instar imaginal discs show the same phenotype (Zeidler, 1999).

The downstream pathway component STAT was also tested by inducing clones of stat92E alleles. These give qualitatively identical phenotypes to hop clones, but at a lower penetrance. The frequency with which inversions are recovered is increased in a genetic background heterozygous for hop, demonstrating that removal of a single copy of hop can sensitize the pathway to loss of stat92E. The weak nature of the stat92E phenotype would appear to indicate that the stat92E gene product is only partially required to transduce the hop-mediated signal. Although unexpected, this finding is consistent with previous evidence that more than one STAT homolog exists in flies, and suggests that they act semiredundantly in ommatidial polarity determination. Thus, the juxtaposition of wild-type cells and cells unable to transduce the JAK/STAT signal can generate ectopic axes of ommatidial mirror-image symmetry that resemble the normal equator (Zeidler, 1999).

As LOF JAK/STAT clones result in ectopic axes of ommatidial symmetry, the effects of ectopic activation of the pathway were examined by misexpression of the pathway ligand Upd/Outstretched. GOF Upd clones were generated by a combination of the FLP/FRT cassette, such that Upd is expressed in discrete groups of marked cells in the developing eye. This results in inversion of ommatidial polarity in the wild-type tissue on the equatorial side of the clone, with the greatest effect observed in clones closer to the poles of the disc. Taken together, these LOF and GOF results indicate that JAK/STAT function across the DV axis of the eye disc is necessary for the normal establishment of a single axis of ommatidial mirror-image symmetry along the DV midline, and is sufficient to define ectopic axes of mirror-image symmetry (Zeidler, 1999).

An interesting aspect of the original P-element-mediated insertional mutation in the stat92E locus (stat92E06346) is the lacZ expression pattern produced by this enhancer detector. Eye discs from larvae carrying this insertion (subsequently referred to as stat92E-lacZ) show a gradient of lacZ activity that is highest at the poles and decreases to a low point at the DV midline. Increased expression is also seen in the ocellar spot region, and, independently, in many of the macrophage-like blood cells often associated with the eye imaginal disc complex. However, in situ hybridization experiments undertaken with probes specific for the stat92E transcript show ubiquitous expression of stat92E mRNA in third instar eye discs, suggesting that this enhancer detector might only report a subset of stat92E transcript expression (Zeidler, 1999).

An intriguing possibility was that stat92E-lacZ expression might be related to JAK/STAT pathway activity. stat92E-lacZ staining was therefore examined in larvae carrying the constitutively active hopTuml allele of Drosophila JAK. In hopTuml eye discs with uniformly increased JAK/STAT activity, the overall level of lacZ activity is consistently lower than in discs from wild-type siblings stained in parallel. Additional experiments show that the level of stat92E-lacZ expression is inversely proportional to the level of JAK/STAT pathway activation: High activation produced by Upd expression abolishes stat92E-lacZ activity; moderate activation produced by the hopTuml allele gives reduced activity, whereas cells in which there is no JAK/STAT signaling (such as hop clones) show maximal levels of stat92E-lacZ activity. Comparing the results of these experiments with the endogenous pattern of stat92E-lacZ staining in the eye disc, it is concluded that JAK/STAT activity must be highest at the DV midline (where stat92E-lacZ activity is lowest) and low at the poles (where stat92E-lacZ activity is upregulated to levels similar to those seen in hop clones) with a gradient of JAK/STAT activity present between these extremes (Zeidler, 1999).

Given the role of Upd in restricting mirr expression, one possible mechanism by which JAK/STAT LOF clones might induce ectopic axes of mirror-image symmetry would be through the generation of ectopic boundaries of mirr expression. The expression of mirr-lacZ was examined in hop clones. Many clones lying both dorsally and ventrally were examined in eye discs, and in no case was an alteration in mirr-lacZ expression observed. Additionally, hundreds of adults carrying mirr-lacZ were examined, in which hop clones had been induced, and, again, in no case was a change in mirr-regulated white+ expression observed (Zeidler, 1999).

Thus, ommatidial polarity inversions generated by hop clones are mirr independent. It is therefore concluded that the process of midline equator definition by dorsally restricted mirr expression and the regulation of ommatidial polarity by the JAK/STAT pathway are separable processes. It is also noted that these results suggest that Upd might act independently of Hop to regulate mirr expression (Zeidler, 1999).

The ommatidial polarity phenotype produced by removal of JAK activity in mosaic clones has a number of important features: (1) the phenotype observed is an inversion of ommatidial polarity in which either the dorsal rotational form is seen in the ventral hemisphere of the eye or vice versa; (2) the phenotype is only observed on the polar boundary of the mosaic tissue; (3) the strength of the phenotype (in terms of the number of inverted ommatidia seen) is dependent on the size and shape of the clone; (4) the phenotype is cell nonautonomous as either fully mutant, fully wild-type, or as mosaic clusters that can manifest the phenotype (Zeidler, 1999).

From these characteristics, the following can be deduced: the nonautonomy of the phenotype produced by removal of the autonomously acting pathway component JAK, and its dependence on clone size and shape, suggests that JAK/STAT affects ommatidial polarity via a secreted downstream signal (which subsequently will be referred to as a second signal, most likely detected by Frizzled). The direction of the nonautonomy (only in a polar direction) and the strict DV nature of the polarity inversions indicates that this second signal must be graded in its activity along the DV axis, with a change in direction of the gradient at the equator. The direction of this gradient would then be the instructive cue to which ommatidia respond when rotating to establish their mature polarity (Zeidler, 1999).

The simplest model would be that there is a single second signal secreted from the equator, which is downstream of mirr/fng/Notch, and that Wg and Upd/JAK/STAT feed into this pathway upstream of Notch. This is consistent with the roles of Wg and Upd as regulators of mirr expression and, thus, in positioning the endogenous equator. However, it is not consistent with the observed ommatidial polarity inversions produced in the eye field both dorsally and ventrally by Wg-pathway and JAK/STAT-pathway LOF and GOF clones. These phenotypes indicate that second-signal concentration is dependent on Wg pathway and JAK/STAT pathway activity across the whole of the eye field, and thus the second signal cannot be only secreted from the DV midline as a consequence of localized Notch activation. It is conceivable that Notch is activated on the polar boundary of JAK/STAT LOF clones, but in this context the only known mechanism of Notch activation is via mirr/fng interactions, and this possibility has been ruled out (Zeidler, 1999).

Instead, the data points to a model in which Upd and Wg first act to define the equator via restriction of mirr expression to the dorsal hemisphere and localize activation of Notch along the DV midline. Definition of the equator is known to occur early in development, while the disc is still small, and divides the disc into two hemispheres separated by a straight boundary that will form the future equator. Such boundaries evidently serve as a source of a second signal that can polarize ommatidia, becausefng LOF clones that induce ectopic regions of activated Notch result in changes in ommatidial polarity (Zeidler, 1999).

Subsequently in development, it is surmised that gradients of JAK/STAT and Wg-pathway activity across the DV axis of the eye disc are responsible for setting up a gradient(s) of one or more second signals that can determine ommatidial polarity. These signals might be responsible for maintaining longer range polarization of ommatidia away from the equator and the localized Notch-dependent polarizing signal. A number of observations provide a great deal of support for such a model. (1) It is consistent with the known timing of the events involved. The requirement for fng function has been shown to lie between late first instar and mid second instar, which coincides with the first appearance of high levels of Upd expression at the optic stalk. However, the ommatidia are not formed (and thus do not respond to the polarity signal) until the start of the third instar, a stage when localized Upd expression still persists. Furthermore, extracellular Upd protein can be seen in a concentration gradient many cell diameters from the optic stalk at the early third instar stage, consistent with Upd being at least partly responsible for setting up the long-range gradient of JAK/STAT activity across the DV axis of the eye disc that is revealed by the stat92E-lacZ reporter. (2) This model does not require that a single source of second signal secreted by a narrow band of cells at the equator should be capable of determining ommatidial polarity across the whole of the DV axis of the disc during the third instar stage of development. Instead, the band of activated Notch at the equator would serve to draw a straight line between the fields of dorsally and ventrally polarized ommatidia, and need only secrete a localized source of second signal to polarize ommatidia in this region. Further from the equator, the opposing gradients of Upd and Wg signaling would provide a robust mechanism for maintenance of correct ommatidial polarity across the DV axis. Conversely, without the mirr/fng/Notch mechanism to draw a straight line, it would be impossible to imagine how Upd at the posterior margin and Wg at the poles alone could provide the perfectly straight equator that is ultimately formed. (3) The phenotypes that are observed are consistent with multiple competing mechanisms responsible for determining ommatidial polarity. When inversions of ommatidial polarity are induced by generating hop clones or ectopically expressing Upd, straight equators are not produced, such that two cleanly abutting fields of dorsal and ventral ommatidia are produced. Instead, there is usually some confusion of ommatidial identities as if they might be receiving conflicting signals. Additionally, when upd activity is removed from the optic stalk, an equator still forms (albeit at the ventral edge of the disc), but some ommatidia dorsal to the equator still adopt a ventral fate as if the determination of ommatidial polarity is less robust in the absence of Upd (Zeidler, 1999).


GENE STRUCTURE

The mrl transcript is 4.0 kb, composed of seven exons. The first exon is 200 bp consisting of 5' untranslated DNA and separated from the remaining exons by approximately 7 kb (Hou, 1996).

PROTEIN STRUCTURE

Amino Acids - 761

Structural Domains

MRL contains a src homology2-like domain and a DNA binding motif (Hou, 1996 and Yan, 1996a).


STAT/marelle: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 15 NOV 97

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