EvoprintHD of os

BIOLOGICAL OVERVIEW

According to FlyBase, the small eye (sy) mutation was described by Bridges on July 3, 1919. Originally considered two separate loci, outstretched (od) and small eye, Muller proposed a single gene based on the recovery of a mutant with both phenotypes, which was not resolvable into two components by recombination, as well as on the failure to recover recombinants between os and sy in transheterozygotes (Verderosa, 1954). The original os mutation was described by Abrahamson in 1953 (Verderosa, 1954). The os mutation gave a phenotype of wings held virtually at right angles to body and small and rounded eyes. The unpaired term for this gene originated with Carroll (1986) in a description of zygotically active genes that affect the spatial expression of the fushi tarazu during early Drosophila embryogenesis. Referred to as sisterless C (Cline, 1993), the gene is considered as an X:A numerator element in sex determination. Finally cloned and described in 1998, outstretched, termed unpaired by Harrison, 1998, has been found to be a secreted protein, associated with the extracellular matrix, that activates the JAK pathway. This essay will conform to the FlyBase convention and refer to this much encountered protein is Outstretched.

Our current understanding of the JAK/STAT pathway is deduced primarily from studies in mammals. JAKs are a novel class of nonreceptor tyrosine kinases, of which four mammalian members have been characterized. The unique feature of these molecules is the presence of two tandem kinase-homologous domains. The carboxy-terminal domain of these proteins has been shown to catalyze tyrosine phosphorylation, whereas the more amino-terminal domain is apparently catalytically inactive. The major substrates for JAK tyrosine phosphorylation are the STATs. This family of molecules, with seven identified members in mammals, is able to bind to specific DNA sequences and activate transcription. Together, the JAKs and STATs comprise a simple intracellular signal relay that can be activated by a number of extracellular factors. In vertebrates, the cytokines are the largest class of molecules to utilize the cascade. Although the cytokines have significantly different amino acid sequences, they are divided into two categories on the basis of their structures. The larger class, type I cytokines, have a characteristic up-up-down-down helical structure, referring to the arrangement of alpha helices in the protein. The receptors for these molecules are heteromultimeric subunits, with many cytokines using a common subunit in the receptor complex. Whereas the type II cytokines have different structure than the type I class, they also bind to heteromultimeric receptor complexes (Harrison, 1998 and references).

Mechanistically, the JAK/STAT pathway provides a direct means to respond to extracellular signals. The first step in activation is binding of the extracellular ligand to the appropriate transmembrane receptor. Ligand binding typically induces receptor dimerization or multimerization. Current models hold that receptor dimerization brings their associated JAK molecules into close proximity to each other, presumably facilitating trans-phosphorylation of the JAKs. Signals are then transmitted directly to the nucleus through the activation of appropriate STATs (The term STAT stands for signal transducer and activator of transcription). Inactive STATs are found in the cytoplasm and, upon JAK activation, are recruited to the inner surface of the cell membrane. Here, they are tyrosine phosphorylated by the JAKs, then translocate to the nucleus and bind specific DNA sequences to regulate transcription. In this manner, direct response to signals is achieved via transcriptional activation of downstream target genes by activated STATs (Harrison, 1998 and references).

Recently, a Drosophila paradigm for the JAK-STAT pathway has emerged in which a single JAK, termed hopscotch (hop) and a STAT, termed stat92E or marelle, have been identified. Drosophila hop was originally identified as a gene involved in embryonic pattern formation. Embryos derived from females that do not express the hop gene product in their germ lines show novel embryonic defects characterized by the loss and/or fusion of a specific subset of body segments. This phenotype is unusual because it does not resemble any of the stereotypical classes of segmentation genes (gap, pair-rule, or segment polarity genes). This same embryonic phenotype is also seen in animals derived from females that lack stat92E gene activity. A third gene, outstretched or unpaired (upd), with the distinctive embryonic mutant phenotype of hop and stat92E, has now been cloned. It is suggested that this gene encodes a ligand for the JAK/STAT pathway during Drosophila segmentation. The conceptual translation product does not resemble that of any of the cytokines (Harrison, 1998). It is similar to only one other identified protein, the Om(1E) protein of Drosophila ananassae, a very closely related species (Juni, 1996).

The similarity of the os mutant phenotype with that of hop and stat92E, along with the genetic interactions that exist between them, suggest that all three genes are involved in the same developmental process. A prediction of this hypothesis is that mutations in os would affect the expression of segmentation genes in the same manner as hop and stat92E. Analysis of the expression of pair-rule genes in os mutant embryos confirms this prediction. The removal of os results in the stripe-specific loss of expression of several pair-rule genes. Specifically, in os mutants, the fifth stripes of expression of the genes even-skipped (eve), fushi tarazu (ftz), and runt are reduced or absent. Additionally, the third stripes of eve and ftz, and the second stripe of runt are variably reduced. These stripe-specific effects are identical to those described for maternal loss of hop and stat92E activities (Binari, 1994 and Hou, 1996).

The enhancer elements responsible for control of the third stripe of eve expression have been mapped to a 500-bp element upstream of the eve transcriptional start site. A reporter construct carrying 5.2 kb of eve upstream sequence, including this enhancer region, fused to the lacZ gene drives expression of lacZ in the second, third, and seventh stripes of eve. Previous work has shown that this fragment contains sequences that bind Stat92E protein in vitro. Removal of maternal activity of either hop or stat92E results in the loss of the third stripe from the reporter construct. Similarly, zygotic mutation of os also causes the specific loss of the third stripe, without affecting the second or seventh stripes (Harrison, 1998 and references).

The lack of sequence similarity between Os and any known vertebrate activators is intriguing. But like cytokines, the predicted secondary structure for Os includes several stretches of alpha-helical regions. Whereas Os has no sequence homology with cytokines, perhaps the alpha-helices fold into a structure reminiscent of cytokines. There is precedent among mammalian ligands for structural similarities in molecules that do not have sequence homology with type I cytokines. These molecules also utilize JAKs for signal transduction. Although there may not have been strong evolutionary conservation of specific sequences, there may be conservation of general structure in ligands because of functional requirement for binding to the appropriate receptor. On the other hand, some aspects of Os structure are less consistent with a cytokine-type molecule. First, the Os protein is extremely basic, with a predicted pI of nearly 12. Second, in contrast with many cytokines, Os is associated with the ECM, which may limit the range of activity of the ligand. These characteristics suggest that Os may be representative of a new family of ligands that stimulates the JAK pathway. Determination of the relationship between Os and other JAK activating ligands must wait for the identification of Os homologs in other species. Finally, characterization of the Os receptor will reveal whether Os signals through a conventional receptor molecule or whether it defines a novel mechanism by which the JAK/STAT pathway becomes activated (Harrison, 1998).

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. 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, known as the midline, runs along the DV midline (see Specification of the eye disc primordium and establishment of dorsal/ventral asymmetry). 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 45ƒ 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).

GENE STRUCTURE

os untranslated regions (UTRs) contain multiple copies of two sequence elements that have been shown to decrease mRNA stability. In the 3' UTR there are four dispersed ATTTA motifs, which are found in a number of cytokines, lymphokines, and proto-oncogenes. In addition, a A/TTTGTA motif that was identified in the pair-rule gene ftz is present in two copies in the 3' UTR and one copy in the 5' UTR of os. These elements could contribute to the rapidly changing pattern of os mRNA accumulation found in the early embryo (Harrison, 1998 and references).

Bases in 5' UTR - 226

Exons - 3

Bases in 3' UTR - 734

PROTEIN STRUCTURE

Amino Acids - 413

Structural Domains

Sequencing of the largest recovered os cDNA has identified an ORF that could encode a peptide of 46.8 kD. The conceptual translation product is similar to only one other identified protein, the Om(1E) protein of Drosophila ananassae, a very closely related species (Juni, 1996). The proteins from the two species are ~90% identical over two-thirds of their length but diverge at the termini. Little is known about the developmental function of the Om(1E) gene, except that overexpression of the gene causes defects in the adult fly. In the developing eye imaginal disc, expression of Om(1E) causes overproliferation of cells. Also, overexpression in the developing wing imaginal disc causes ectopic bristles, outstretched wings, and deformations of the notum. Interestingly, these phenotypes are identical to defects caused by overexpression of the hop gene in Drosophila melanogaster (Harrison, 1995), thus lending further support to the hypothesis that Os is a component of JAK signaling in flies. The only striking structural features of the Om(1E) protein are a potential signal sequence at the amino terminus and several sites for possible amino-linked glycosylation, both of which suggest that the protein is extracellular. These features are also present in D. melanogaster Os. A simple model for the role of such a protein in JAK signaling is that it codes for a ligand that activates the JAK pathway (Harrison, 1998).