hopscotch
Expression of HOP mRNA is ubiquitous throughout the embryo and not spatially activated (Binari, 1994).
The Drosophila egg develops through closely coordinated activities of associated
germline and somatic cells. An essential aspect of egg development is the differentiation of the somatic follicle cells into several distinct subpopulations with specific functions. The graded activity of the Janus kinase (JAK) pathway, stimulated by the Unpaired ligand, patterns the anterior-posterior axis of the follicular epithelium. Different levels of JAK activity instruct adoption of distinct anterior cell fates. Further, the coordinated activities of the JAK/STAT and epidermal growth factor receptor (EGFR) pathways are required to specify the posterior terminal cell fate. It is proposed that Upd secreted from the polar cells may act as a morphogen to stimulate A/P-derived follicular fates through JAK pathway activation (Xi, 2003).
The Drosophila egg is an intricately patterned structure with distinct specializations and polarities. These features are critical to subsequent embryonic development because the polarities of the egg are transmitted to the embryo, establishing the initial pattern in a developing zygote. The pattern of the mature egg is established by complex cellular interactions among and between both somatic follicle cells and germline cells. Each egg begins as a 16-cell germline cyst, from which one cell will become the oocyte and the remainder will become the supporting nurse cells. In the germarium, the anterior structure in which oogenesis is initiated, the germline cyst, is surrounded by a monolayer of somatic follicle cell precursors. As the encapsulated cyst exits from the germarium, approximately 10-14 of the somatic cells cease proliferation and differentiate. This group of cells forms two distinct populations: two polar cells at the anterior and posterior poles of each chamber and approximately seven stalk cells that form a bridge between the consecutive cysts. As the cyst exits the germarium, the other somatic cells covering each chamber, the epithelial follicle cells, remain undifferentiated (Xi, 2003 and references therein).
After pinching off from the germarium, each germline cyst grows, while the epithelial follicle cells proliferate. During this time, the anterior-posterior polarity that will ultimately determine all of the epithelial follicular fates is established. Elegant experiments have shown that the underlying prepattern of the follicular epithelium displays mirror image symmetry at the termini in the anterior-posterior (A/P) axis. Cells adopt one of three anterior terminal fates [border, stretched, and centripetal cells (terminal to central)], depending on proximity to the poles. In the intervening region between the terminal domains, cells will adopt a default 'main body' identity, and the posterior terminal cells form nearest the posterior pole. The symmetry of the A/P pattern is broken by EGFR signaling at the posterior. Secreted Grk from the posteriorly localized oocyte activates EGFR on the overlying follicle cells, establishing posterior terminal fate. In the absence of EGFR signaling, the anterior pattern is repeated at the posterior (Xi, 2003 and references therein).
By stage 7, the epithelial follicle cells cease proliferation and enter an endocycle. Afterward, these cells begin to show morphological and molecular signs of differentiation into the five epithelial fates: border, stretched, centripetal, posterior, and main body cells. Each of these subpopulations of follicle cells has a specific function with respect to the production of a mature egg, such that the correct number and position of each type is critical to ultimate egg morphology. These functions influence the production of structures that are essential to the egg, such as the dorsal respiratory appendages and the micropyle. These functions are also critical for proper anterior-posterior organization of the oocyte and, therefore, also for the resulting embryo (Xi, 2003 and references therein).
In early follicular differentiation, JAK activity is required for the production of stalk cells and the repression of polar cell fates. Later, JAK signaling is important for the proper recruitment and migration of border cells, a subpopulation of the follicular epithelium. Between these events, loss of JAK signaling in the follicular epithelium leads to persistent expression of Fas III, a marker for immature follicle cells (Xi, 2003 and references therein). The failure of these epithelial follicle cells to mature in hop mutant clones, as well as the persistent expression of upd in the polar cells, suggests that JAK signaling may have a role in differentiation of the entire follicular epithelium (Xi, 2003).
To address whether JAK signaling may play a role in distinguishing the terminal and main body domains, expression of mirror-lacZ (mirr-lacZ) was examined in egg chambers with aberrant JAK signaling. At ovarian stages 6-8, mirr-lacZ is strongly expressed in the main body follicle cells, with graded reduction toward the termini. In clones of hop mutant cells in the terminal regions, expression of mirr-lacZ is strongly induced, even prior to differentiation of those cells. It is concluded that JAK pathway activity distinguishes the terminal from the main body domains, at least as marked by the mirr-lacZ reporter. Specifically, JAK signaling is required to establish terminal identity and/or repress main body fate (Xi, 2003).
JAK activation is essential for specification of terminal fates, but is it also sufficient for terminal identity? To test this possibility, hop and upd were expressed in clones within the follicular epithelium, since either can activate JAK signaling. JAK pathway activation represses mirr-lacZ cell autonomously for Hop, but nonautonomously for the secreted Upd. This suggests that JAK pathway activity directly establishes the terminal domain. Furthermore, Upd expression causes graded repression of mirr-lacZ in the neighboring cells. Those cells closest to the Upd source have no expression of mirr-lacZ, but the amount of reporter in the neighboring cells increases with the distance from the Upd-expressing cells. It is concluded that JAK signaling must be activated in a graded fashion around the source of the Upd ligand, presumably because of extracellular diffusion of Upd. Moreover, the range over which the ectopic Upd can suppress mirr-lacZ is greater near the poles than in the center of the egg. Because endogenous Upd is secreted by the polar cells, it is easy to imagine that levels of JAK activity within the follicular epithelium would be greater near the poles. Consequently, the sum of the endogenous and ectopic Upd activities would be higher near the termini and could better repress mirr-lacZ. An alternative explanation is that the level of JAK signaling or another required signaling pathway is limited near the central region of the epithelium. It is concluded that JAK activation is necessary and sufficient for terminal identity in the follicular epithelium (Xi, 2003).
These results do not distinguish whether JAK signaling induces general terminal cell identity or is instructive for specific terminal fates. JAK activity could make the termini competent to respond to another signal that determines specific terminal fates or, alternatively, JAK activity could directly specify terminal fates, perhaps through varying levels of activation. To determine whether JAK signaling is essential for determining specific fates within the terminal domains, previously characterized markers for anterior subpopulations were examined. Work by others has already shown that JAK signaling can recruit the most terminal anterior fate, border cells. Here it is demonstrated that high levels of JAK activity are necessary and sufficient for border cell identity, at least within the anterior terminal domain. Consistent with the previous studies, the loss of JAK activity in clones of presumptive border cells invariably leads to failure of those cells to differentiate as border cells. However, this does not address whether JAK signaling is instructive for specific anterior fates. To address this question, the JAK pathway was activated to high levels in cells that are not at the terminus. Ectopic expression of either hop or upd stimulates additional cells to adopt border cell fate, again, in a cell-autonomous manner for hop and in a nonautonomous manner for upd. The location of the ectopic border cells suggests that they would normally have become stretched or centripetal cells. The ability of increased JAK signaling to alter fate within the anterior domain supports the hypothesis that levels of JAK signaling instruct specific fates within that domain. Increased JAK activity in the posterior terminal domain failed to induce ectopic border cells, presumably because of EGFR-mediated specification of posterior identity within this domain that would preclude expression of any anterior markers (Xi, 2003).
Involvement of JAK signaling in the specification of the more central anterior fates was examined with dpp-lacZ and MA33, which mark both stretched and centripetal cells, and BB127, which is specific for centripetal cells. JAK activity is essential for both fates. Loss of JAK signaling in either population results in a failure to express the population-specific markers. In addition to loss of the stretched cell marker, hop mutant cells also fail to migrate, spread out, and adopt the squamous morphology distinctive for stretched cells. On the basis of the expression of mirr-lacZ in JAK mutant clones above, it is presumed that these cells adopt a default main body fate. Moreover, effects of a weak general reduction of JAK signaling in the egg can be examined in ovaries of females transheterozygous for one severe and one weak allele of upd. In such eggs, the number of cells expressing a border cell marker is reduced. But, in addition, a marker for the stretched/centripetal fates is prominently expressed in the remaining border cells. Comparable expression of stretched cell markers in border cells is never observed in wild-type. Furthermore, the defective 'border cells' of upd mutant chambers also show aberrant migration. It is concluded that high levels of JAK activity are required for border cell fate, while lower levels direct stretched cell fate. Consistent with this, the border cells of upd mutant chambers did not express the centripetal cell-specific marker. Moreover, aberrant border cell specification in the Upd mutants indicates that Upd must normally activate JAK signaling during this process (Xi, 2003).
As with loss-of-function, clones of JAK-activated cells fail to express stretched and centripetal markers appropriate for their positions within the egg. On the basis of the morphology of the misexpressing cells and the complementary evidence with border cell markers, the presumptive stretched or centripetal cells with increased JAK activation are converted to the terminal-most border cell fate. Furthermore, JAK activation in the presumptive main body can induce the adoption of centripetal cell fate. However, it is somewhat surprising that JAK activation is unable to induce the most terminal (border cell) fate. One possible reason is that the endogenous JAK activity is likely highest near the poles and lowest in the central region, making it easier to convert cells closer to the terminus to border cell fate. This model assumes that the levels of ectopic activity must be lower than the highest levels of endogenous JAK activity, though evidence below suggests that this may not be true. A second possibility suggests that downstream components or cofactors required for high-level activation of JAK or for another required pathway may be in limited supply in the central region. Despite limited response in the central region, all the epithelial follicle cells are responsive to changes in levels of JAK activation. This indicates that the JAK pathway plays an active role, not a permissive role, in assigning specific terminal fates within the follicular epithelium (Xi, 2003).
The transformation of hop mutant cells in the posterior terminal domain into main body cells, as marked by mirr-lacZ, suggests that JAK signaling is essential for posterior terminal identity, as well as anterior fates. To address this hypothesis, two enhancer trap markers for posterior cells were analyzed in hop mutant clones. The pnt-lacZ marker is normally expressed strongly at the posterior, with a graded reduction in posterior cells farther from the pole. Cells mutant for hop show complete loss of pnt-lacZ expression in a cell-autonomous fashion. Moreover, because the cells at the posterior do not undergo the dramatic migrations seen at the anterior, it is possible to analyze the mutant and wild-type cells in their relative positions to one another. Significantly, it can be seen that wild-type cells express normal levels of pnt-lacZ, even when mutant cells intervene between them and the polar cells. Similar results were seen for a second posterior marker, blot01658. This suggests that the wild-type cells are receiving a signal directly from the polar cells and not via a local signal relay or 'bucket brigade' mechanism from neighboring cells (Xi, 2003).
To confirm that JAK activity influences posterior terminal fate, the function of those terminal cells was examined. At approximately stage 7, the posterior terminal cells send a signal to the underlying oocyte that stimulates microtubule reorganization in the oocyte. This microtubule reorganization is important for the migration of the oocyte nucleus to the dorsal anterior and for the proper sequestration of A/P determinants that direct development of the resulting embryo. After reorganization, one of these determinants, Staufen, is tightly associated with the posterior end of the oocyte. However, in egg chambers that lack JAK activity at the posterior terminus, Staufen fails to localize and is dispersed in the cytoplasm. Furthermore, in eggs with JAK mutant clones that cover only a portion of the presumptive posterior terminal cells, Staufen becomes localized directly underneath the wild-type cells at the posterior. This suggests that high JAK activity in the posterior follicle cells very precisely stimulates aggregation of a Staufen-bound complex in the underlying membrane. Despite the consistent mislocalization of Staufen in eggs with hop mutant clones at the posterior, the oocyte nucleus rarely fails to migrate to the dorsal anterior. This may indicate either that global microtubule reorganization is separable from Staufen localization or that Staufen is more sensitive to perturbations in microtubule reorganization (Xi, 2003).
Conversely, cell clones that express either hop or upd are able to activate pnt-lacZ, but only near the posterior of the chamber. Once again, the activation is autonomous for hop and nonautonomous for upd, supporting a direct role for JAK signaling in determining cell fates. Moreover, as with the mirr-lacZ reporter, the nonautonomous activity of Upd results in a graded response in the marker, such that the level of pnt-lacZ decreases as the distance from the ectopic Upd source increases. Again, this points to a gradient of JAK activity being established around the upd-expressing cells. Interestingly, activation of the posterior marker in cells neighboring upd-expressing clones was stronger on the posterior side of the clone. Again, this may be due to additive influences of the endogenous Upd signal coming from the polar cells and the ectopically expressing cells (Xi, 2003).
The initial A/P pattern in the follicular epithelium has a mirror image symmetry, such that cells at either end that are equidistant from the polar cells have equivalent identities. Subsequently, Grk from the oocyte, which always lies at the posterior of the egg, breaks the symmetry by stimulating EGFR in the follicle cells, inducing posterior terminal fate. Loss of EGFR activation in the posterior cells causes adoption of the underlying anterior fates. The requirement for both EGFR and JAK activation explains the failure of ectopic JAK activation to induce posterior identity at the anterior. But clones of cells that express activated EGFR can induce pnt-lacZ at the anterior. However, as in the posterior, induction of the marker at the anterior is graded, with highest levels closest to the pole. Furthermore, in the main body, activated EGFR is unable to induce posterior fate. This suggests that another factor essential for posterior identity is normally present, but limiting, in the anterior region. These domains that are competent to respond to activated EGFR coincide with the JAK activation. So, is EGFR limited in specifying posterior fate by the underlying activity of the JAK pathway? Consistent with this supposition, the coexpression of activated EGFR and JAK is capable of inducing posterior fate in all follicular epithelial cells. Thus, the coordinated activities of the two pathways are necessary and sufficient for induction of posterior identity (Xi, 2003).
Graded response of the mirr-lacZ marker and the ability of altered levels of JAK activity to change anterior fates are consistent with a model in which graded levels of JAK activity specify different follicular fates along the A/P axis. This model predicts that an overall increase or decrease of JAK activity would alter the number of cells adopting fates for each of the anterior subpopulations. Specifically, an overall reduction of JAK activity should reduce the number of border cells while shifting and/or reducing the number of cells adopting the more central stretched and centripetal fates and expanding the main body domain. To test this hypothesis, egg chambers from reduced function mutants of upd and hop were examined for the number and distribution of cells within each of the anterior subpopulations. Reduction of JAK activity dramatically reduces the number of border cells. Combination of one weak and one strong mutant allele of upd reduces the number of border cells by nearly half. Furthermore, combination of two weak hop alleles completely eliminates all border cells, despite producing morphologically normal eggs. Moreover, stretched cells are somewhat reduced in the hop mutant, while centripetal cells are only slightly affected. Similar results were seen at the posterior, where reduced hop activity results in marker expression that is only detectable to about four cell diameters from the posterior, rather than the normal eight cell diameters. However, graded marker expression is maintained, just shifted toward the posterior. A more substantial reduction of the most terminal fates strongly supports existence of graded JAK activity that is highest at the termini (Xi, 2003).
A model is presented for anterior-posterior follicular patterning. Patterning of the follicular epithelium requires the coordination of several signaling pathways. In the A/P axis, prominent roles for the EGFR and Notch pathways have been established. By incorporating the functions of the JAK pathway, an integrated model of A/P patterning in the follicular epithelium is proposed. The activation of the JAK pathway in the follicular epithelium is graded, with highest levels at the anterior and posterior poles. This is consistent with the production of the secreted ligand, Upd, from the polar cells, which is then received by cells of the follicular epithelium. The expression of Upd in the polar cells begins even within the germarium, so it is established as a potential graded signal from the earliest stages of follicular epithelial development. The polar cells have an organizer function in the establishment of A/P pattern. This organizer activity is consistent with the functions and behaviors described for JAK signaling in the surrounding epithelium. It is proposed that the gradient of JAK activity from both termini determines the presumptive border, stretched, and centripetal cells, on the basis of thresholds of JAK activity that define each fate, establishing a symmetrical prepattern. However, JAK signaling may not be the only patterning element in this process. Ectopic JAK activation in the main body domain is insufficient to induce the most terminal fate, the border cells. Though this could arise because of an inherent limitation to JAK signaling in the main body, the induction of Stat92E and Dome to high levels in similar activation clones argues against this. Alternatively, the main body may have low levels of some downstream coactivator for JAK signaling or of an independent patterning element, perhaps another signaling pathway. With the exception of this reduced response in the main body, adoption of each epithelial follicular fate can be simply ascribed to varying thresholds of JAK pathway activity (Xi, 2003).
The symmetrical prepattern established by JAK signaling is broken by EGFR activation in the posterior follicle cells stimulated by the secreted Grk ligand from the oocyte. The combined activation of JAK and EGFR signaling at the posterior defines posterior terminal follicle cell identity, overriding the default anterior fates specified by JAK activity alone. By the end of stage 6, when proliferation ceases, the cell fates of the follicular epithelium must already be determined. At that time, Notch pathway activation in all epithelial follicle cells triggers the transition from active division to an endocycle. By stage 9, the epithelial cells express markers for the various fates, begin migrations toward the posterior, and undergo morphological changes appropriate for ultimate function of that fate. Thus, the combined and sequential functions of the JAK, EGFR, and Notch pathways establish a series of anterior and posterior fates in the follicular epithelium (Xi, 2003).
The essential nature of morphogens, signals that have the ability to induce cell fates on the basis of levels of activity, is a central theme in animal development. Yet, despite this centrality, very few proteins have been demonstrated to have morphogenic function. Interestingly, most of the known morphogens have retained that activity throughout animal evolution. In both vertebrates and invertebrates, well-known signaling proteins of the Wnt, Hedgehog, and TGF-ß families act as morphogens. Though not all of the criteria have been explored, it is suggested that the properties of Upd and its stimulation of the JAK pathway in follicular epithelial cells are consistent with function as a morphogen. While the JAK intracellular cascade is highly conserved from flies to man, no proteins with significant homology to the Upd ligand have been found in other organisms. Therefore, Upd may be an unusual example of a morphogen that has rapidly diverged evolutionarily (Xi, 2003).
Morphogens are generally regarded to have four defining characteristics. (1) They are released from a localized source. In the ovary, Upd is secreted by the polar cells. (2) Morphogens form a concentration gradient from nearby to distant cells that respond directly to the signal, not through a relay mechanism. Although a gradient of Upd has not been directly visualized, the underlying gradient of JAK activation is apparent. Moreover, the response of cells to Upd activity requires downstream components of the JAK pathway in a cell-autonomous manner, demonstrating that the response to Upd is direct and not relayed. (3) Cells within the region of the gradient must show at least two different responses in addition to the default. In the follicular epithelium, the region that corresponds to the presumed JAK gradient gives rise to the border cells, stretched cells, and centripetal cells, in addition to the default main body cells. (4) Over- and underexpression should change cell fates in opposite directions. Clonal analysis clearly demonstrates that the anterior terminal and main body cell fates can be influenced by gain or loss of JAK pathway activity in an opposite and predictable manner. Thus, despite no direct visualization of a Upd gradient, the characteristics of the JAK pathway are consistent with a system that transduces a morphogenic signal (Xi, 2003).
The basement membrane (BM) represents a barrier to cell migration, which has to be degraded to promote invasion. However, the role and behaviour of the BM during the development of pre-invasive cells is only poorly understood. Drosophila border cells (BCs) provide an attractive genetic model in which to study the cellular mechanisms underlying the migration of mixed cohorts of epithelial cells. BCs are made of two different epithelial cell types appearing sequentially during oogenesis: the polar cells and the outer BCs. The pre-invasive polar cells undergo an unusual and asymmetrical apical capping with major basement membrane proteins, including the two Drosophila Collagen IV alpha chains, Laminin A and Perlecan. Capping of polar cells proceeds through a novel, basal-to-apical transcytosis mechanism that involves the small GTPase Rab5. Apical capping is transient and is followed by rapid shedding prior to the initiation of BC migration, suggesting that the apical cap blocks migration. Consistently, non-migratory polar cells remain capped. JAK/STAT signalling and recruitment of outer BCs are required for correct shedding and migration. The dynamics of the BM represent a marker of migratory BC, revealing a novel developmentally regulated behaviour of BM coupled to epithelial cell invasiveness (Medioni, 2005).
The migration of cohorts of cells is an alternative to single-cell migration, which is used by normal and cancer cells to invade tissues. One advantage for mixed clusters is to transport tumorigenic (for example, apoptotic resistant) cells with no migratory abilities to a distant destination that they could not reach on their own. In this case, migration is executed by migratory capable cells within the cluster. Clusters illustrate how separate functions (tumorigenesis and migration) can be merged through collaboration between two cell populations. It is thus important to understand how migrating cell clusters are assembled and organized. The BC cluster is made of two distinct populations of cells, i.e. the polar cells and the outer BCs, making it a good model with which to determine the cellular mechanisms underlying the recruitment and migration of mixed cohorts of cells. Three novel steps in the formation of BCs have been identified. (1) It was shown that a developmentally regulated basal to apical transport of BM material takes place in the polar cells, the first population of cells to form in the cluster. The apical cap is the earliest known marker of anterior polar cells. (2) The asymmetrical positioning of the apical cap suggests that despite an apparent identity, the two polar cells are different and might play distinct roles. (3) The data indicate that a two-way interaction takes place between the two differentiated subpopulations of invasive cells before they migrate. A first signal, activating the JAK/STAT pathway is sent by the polar cells to recruit the outer BCs. In a second step, the outer BCs are essential for shedding the apical cap of polar cells (Medioni, 2005).
Outer BCs are not required for apical cap formation. Similarly, outer BCs form normally in the absence of a cap, indicating that apical capping is not a pre-requisite for outer BCs to be recruited and the cluster to be assembled. Interestingly, it was found that immotile polar cells remain capped. Thus, a possible role for apical capping is to block the migration of immature clusters, a finding that could explain the long standing observation that isolated polar cells cannot migrate on their own. Indeed, the coordination between apical cap degradation and the recruitment of outer BCs indicates that degradation of the apical cap could serve as a check point or quality control ensuring that only finalized clusters can start migration. It is important to note that degradation of the ECM at the leading edge of migrating clusters is essential for tumour progression, and examples of cancer cells showing a reduction or absence of some basement membrane markers, including Collagen IV, have been reported. In particular, human alpha3/alpha4 type IV Collagen is found at the apical surface in normal colon tissue, but is absent in colorectal neoplastic cells, making the differential distribution of type IV collagens potential diagnostic markers for the invasiveness of cancer cells. The BC model will be central for future studies aimed at understanding BM dynamics and function in invasive clusters (Medioni, 2005).
The lethal
phase of null alleles of hop occurs at the larval-pupal interface associated with a small imaginal disc
phenotype. hop is required maternally because embryos derived from female hop mutants die with specific
defects. Embryos produced from homozygous hop mutants show segment specific defects.
The extent of these defects depends upon both the strength of the allele and the paternal
contribution. In the most extreme case embryos exhibit defects associated with five segments T2, T3,
A4, A5, and A8 [Images]. In the less extreme phenotype defects are only associated with A5. Thus, activity of
hop is required both for the maintenance and continued cell division of diploid imaginal
precursors and for the establishment of the full array of segments (Perrimon, 1986).
The Drosophila Tumorous-lethal (Tum-l) mutation acts as an activated oncogene, causing
hematopoietic neoplasms, overproliferation, and premature differentiation. Tum-l is a dominant
mutation in the hopscotch locus, which is required for cell division and for proper embryonic
segmentation. The Tum-l temperature-sensitive period for melanotic tumor formation includes most of
larval and pupal development (Hanratty, 1993).
A single amino acid change in HOP is associated with the Tum-l mutation.
Overexpression of either wild-type hop or Tum-l in the larval lymph glands causes melanotic
tumors and lymph gland hypertrophy. In
addition, overexpression of Hop in other larval tissues leads to pattern defects in the adult or to
lethality. Overexpression of either hop or Tum-l in Drosophila cell culture results in
tyrosine phosphorylation of HOP protein (Harrison, 1995).
A true revertant of the hop/Tum-l mutation has been generated in which both
the molecular lesion and the mutant hematopoietic phenotype revert back to wild type (Luo, 1995).
A dominant negative mutation, which results in a truncated Marelle protein, exhibits patterning defects similar to those seen in mutants of the epidermal growth factor pathway. Specifically, adults exhibit partial ectopic wing vein formation in the posterior wing compartment. Abormalities in embryonic and adult segmentation and in tracheal development are also observed. hopscotch and dominant negative marelle mutations can partially compensate for each other genetically, and hop overexpression can increase marelle transcriptional activity in vitro, indicating that the gene products act in a common regulatory pathway (Yan, 1996b).
The Jak (Janus) family of nonreceptor tyrosine kinases plays a critical role in cytokine
signal transduction pathways. In Drosophila, the dominant hopTum-l
mutation in the Hop Jak kinase causes leukemia-like and other developmental defects. The HopTum-l protein might be a hyperactive
kinase. The new dominant mutation hopT42, causes
abnormalities that are similar to but more extreme than those caused by hopTum-l. HopT42 contains a glutamic acid-to-lysine substitution at amino acid
residue 695 (E695K). This residue occurs in the JH2 (kinase-like) domain and is
conserved among all Jak family members. HopTum-l and HopT42
both hyperphosphorylate and hyperactivate D-Stat when overexpressed in
Drosophila cells. The hopT42 phenotype is partially
rescued by a reduction of wild-type D-stat activity. Generation of the
corresponding E695K mutation in murine Jak2 results in increased
autophosphorylation and increased activation of Stat5 in COS cells. These results
demonstrate that the mutant Hop proteins do indeed have increased tyrosine kinase
activity, that the mutations hyperactivate the Hop-D-Stat pathway, and that
Drosophila is a relevant system for the functional dissection of mammalian Jak-Stat
pathways. A model is presented for the role of the Hop-D-Stat pathway in
Drosophila hematopoiesis (Luo, 1997).
Gamma interferon (IFN-gamma) induces both the tyrosine and serine phosphorylation of Stat1. Stat1
serine phosphorylation is required for maximal transcriptional activity of Stat1. Stat1 tyrosine phosphorylation is not a prerequisite for Stat1 serine
phosphorylation, although an active Jak2 kinase is required for both phosphorylation events. Stat1
serine phosphorylation occurs with a more delayed time course than tyrosine phosphorylation. The
occurrence of serine phosphorylation without tyrosine phosphorylation suggests that serine
phosphorylation takes place in the cytoplasm. Experiments performed with cells expressing either
dominant-negative or constitutively active Ras protein indicate that the Ras-mitogen-activated protein
kinase pathway is probably not involved in IFN-gamma-induced Stat1 serine phosphorylation. A
kinase capable of correct Stat1 serine phosphorylation is detected in partially purified cytoplasmic
extracts from both IFN-gamma-treated and untreated cells (Zhu, 1997).
Loss of zygotic outstretched activity causes segmentation defects in the Drosophila embryo that resemble the
phenotype of hopscotch and stat92E mutant embryos. These defects
always include loss of the fifth abdominal denticle band and the posterior mid-ventral portion of the
fourth band. Defects in other segments are variable, but often include reduction of the second thoracic
and eighth abdominal denticle bands and fusion of the sixth and seventh bands. In contrast to hop or
stat92E, zygotic os activity is essential
but maternal activity is not, as evidenced by the lack of a maternal effect phenotype for os mutants The similarity between embryos that lack zygotic os and those that lack
maternal hop or stat92E suggests that os is a component of the JAK signaling pathway. This
hypothesis is further supported by genetic interactions between these genes. It has been observed
previously that certain allelic combinations of hop are viable, but have adult defects. The partial loss of hop activity in such animals causes reduced viability,
held-down wings, reduced production of mature eggs, and/or defects in eggs produced. Each of the
heteroallelic combinations results in a consistent and predictable degree of severity with respect to
these phenotypes. To test whether the hop and os genes interact genetically, one copy of os was
removed from animals carrying allelic combinations of hop. Altering the dose of os activity
exacerbates the defects observed for these hop mutant combinations. Such
enhancement is likely to occur if the two gene products are active in the same pathway (Harrison, 1998 and references).
Strong alleles of unpaired are embryonic lethal, but weaker alleles show an
outstretched (os) phenotype, resulting in adult flies with wings held out away from the body. Allelism
of upd and os is based on the failure of zygotic lethal upd alleles to complement the wing phenotype of
os alleles. For example, combination of the embryonic lethal
allele updYC43 with the viable allele oso results in viable adult flies with outstretched wings (Harrison, 1998 and references).
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, 1999a 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, 1999a 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, 1999a).
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, 1999a 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, 1999a).
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, 1999a).
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, 1999a).
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, 1999a).
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, 1999a).
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, 1999a).
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, 1999a).
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, 1999a).
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, 1999a).
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, 1999a).
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, 1999a).
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, 1999a).
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, 1999a).
Jak kinases are critical signaling components in hematopoiesis. While a large number of studies have been conducted on the roles of Jak kinases in the hematopoietic
cells, much less is known about the requirements for these tyrosine kinases in other tissues. Loss of function mutations in the Drosophila Jak kinase
Hopscotch (Hop) were used to determine the role of Hop in eye development. Hop is required for cell proliferation/survival in the eye imaginal disc, for the
differentiation of photoreceptor cells, and for the establishment of the equator and of ommatidial polarity. These results indicate that hop activity is required for
multiple developmental processes in the eye, both cell-autonomously and nonautonomously (Luo, 1999).
In the most extreme cases, eyes of homozygous or hemizygous hopmsv1 mutant flies are completely missing with a concomitant duplication of the antenna. A milder and also slightly more frequent abnormality is the small eye phenotype. Compared with the eye in wild-type, the small eye is reduced in size in the D/V axis, has fewer ommatidia, and exhibits irregular ommatidial arrays with duplicated bristles between ommatidia. Most frequently, the transheterozygotes show roughness along the equatorial region in which ommatidial fusion and duplicated bristles are seen. Both misrotations and changes of chirality of ommatidia are observed. For example, some of the ommatidia rotated either fewer or more than 180 degrees and display chirality defects. Finally, some of the ommatidia are fused (Luo, 1999).
The ommatidia within hop clones are generally normal, with occasional missing photoreceptor cells or polarity defects. However, many hop clones (in particular the rare larger clones) show a dramatic effect on the polarity of neighboring ommatidia. Strikingly, these clones generate an ectopic equator at the polar margin of the clone. Ommatidia at the polar margin of homozygous hop clones reverse their polarity such that the ommatidia point away from rather than toward the equator, thus creating an ectopic equator. Moreover, when a clone is observed very close to the D/V midline, the equator is deflected away and forms along the border of the clone. Most of the ommatidia with reversed polarity are composed entirely of wild-type photoreceptor cells. Thus, the requirement for hop in establishing ommatidial polarity is not cell autonomous. Since clones in both the dorsal and the ventral halves show the same phenotypes, Hop is required throughout the eye disc (Luo, 1999).
The lacZ insertion associated with a stat allele can be used as a reporter of Hop kinase activity. In wild-type eye discs, a gradient of lacZ activity is observed, with the highest and the lowest activity being observed at the poles and the equator, respectively. These studies show that Stat 92E expression is downregulated in the equatorial region of the eye disc and that Hop activity is graded, with its peak at the equator fading toward the poles. This phenomenon may suggest a negative feedback mechanism for Stat regulation (Luo, 1999).
Mutations in both wingless signaling components and hop affect equator formation nonautonomously, suggesting that a secondary diffusible signal exists downstream of them. However, their phenotypes are different in two ways: (1) mutants in wingless pathway components affect polarity on the equatorial side of the clone, whereas in hop mutant clones the polarity is reversed on the polar side, and (2) an ectopic equator forms in the center of a wingless pathway mutant clone, whereas such an ectopic equator forms outside a hop clone. The wg-signaling data suggest that (in addition to its indirect role on fng expression and thus Notch activation) Wg signaling controls a diffusible factor as a secondary signal that is either up- or down-regulated
(depending on whether it is a positive or negative factor. The nonautonomy of the hop mutant clones also suggests that a secondary diffusible signal acts downstream of Hop. However, it might act in the opposite direction from the one regulated by Wg due to the opposite influence on polarity by hop- and wg-signaling components, respectively. The full understanding of the nature of the Wg- and Hop-associated phenotypes, and of their potential interactions and secondary signals activated, will only be possible when the presumptive secondary signals are identified (Luo, 1999)
Metazoans use diverse and rapidly evolving mechanisms to determine sex.
In Drosophila an X-chromosome-counting mechanism
determines the sex of an individual by regulating the master switch gene,
Sex-lethal (Sxl). The X-chromosome dose is communicated
to Sxl by a set of X-linked signal elements (XSEs), which activate
transcription of Sxl through its 'establishment' promoter,
SxlPe. A new XSE called sisterlessC
(sisC) is described whose mode of action differs from that of previously characterized
XSEs, all of which encode transcription factors that activate Sxl
Pe directly. In contrast, sisC encodes a secreted ligand
for the Drosophila Janus kinase (JAK) and 'signal transducer
and activator of transcription' (STAT) signal transduction pathway and
is allelic to outstretched (os, also called unpaired). sisC works indirectly on Sxl through this signaling
pathway because mutations in sisC or in the genes encoding Drosophila
JAK or STAT reduce expression of SxlPe similarly.
The involvement of os in sex determination confirms that secreted ligands
can function in cell-autonomous processes. Unlike sex signals for other organisms,
sisC has acquired its sex-specific function while maintaining non-sex-specific
roles in development, a characteristic that it shares with all other Drosophila
XSEs (Sefton, 2000).
If os acts on SxlPe indirectly through effects
on Drosophila JAK (encoded by hopscotch [hop]) and on
Drosophila STAT (encoded by Stat92E), then the effect on Sxl
Pe of eliminating either hop or Stat92E should
be the same as eliminating os. This prediction was confirmed. Because
only maternal rather than zygotic hop and Stat92E are likely
to be relevant at the very early embryonic stage when SxlPe
is activated, the maternal contribution
of these two genes was eliminated by inducing homozygous mutant germline clones in mothers
heterozygous for null alleles. Expression of SxlPe:lacZ
in these experimentals was compared with that for control embryos derived
from hop-/+ and Stat92E-/+ germ
cells. Loss of maternal hop+ does not eliminate Sxl
Pe expression, but expression is substantially reduced: although
49% of the experimental embryos expressed SxlPe:lacZ
, essentially identical to the 50% figure for the controls, 32% of the experimental embryos were in the intermediate
staining class compared with only 6% for the controls. The reduction was generally
more uniform across the embryos than in the os experiment. Similar results were seen for Stat92E. Sixteen per cent of controls stained in the intermediate range, compared with
45% for the experimentals; thus, SxlPe expression was clearly
reduced. Curiously, the fraction of experimental embryos staining above background
is greater than 50%, suggesting that although loss of maternal Stat92E
decreases SxlPe expression in females, it might also
increase SxlPe expression in males. Alternatively, this
increase might be due to effects on the lacZ enhancer trap present
in Stat92E6346. The
observation that Drosophila STAT is a regulator of SxlPe
is consistent with the finding of STAT binding sites (TTCNNNGAA)
253, 393 and 428 bp upstream of the SxlPe transcription
start site. The tandem arrangement of these sites in Sxl would facilitate
the kind of cooperative binding of STAT dimers shown to be important in some
systems (Sefton, 2000).
With the discovery of sisC, the collection of fly XSEs may be nearly
complete. The impression given by this collection is that
Drosophila relies on biochemically diverse proteins to assess X-chromosome
dose, but they all act on Sxl at the level of transcription. In contrast,
the XSEs of Caenorhabditis elegans include both transcriptional and
post-transcriptional regulators of their target, xol-1. Characterization of sisC reveals that both
C. elegans and Drosophila XSEs seem to include proteins that work
extracellularly (Sefton, 2000).
Transcriptional activation by (and therefore the physiologic impact of) activated tyrosine-phosphorylated STATs (signal transducers and
activators of transcription) may be negatively regulated by proteins termed PIAS (protein inhibitors of activated stats), as shown by
experiments with mammalian cells in culture. By using the genetic modifications in Drosophila, in
vivo functional interaction of the Drosophila homologs stat92E and a Drosophila PIAS gene (dpias) have been demonstrated. A loss-of-function
allele was used and dpias was conditionally overexpressed in JAK-STAT pathway mutant backgrounds. It is concluded that the correct dpias/stat92E
ratio is crucial for blood cell and eye development (Betz, 2001).
Because the dpias03697 allele is a homozygous lethal, genetic interaction crosses were designed in which flies heterozygous for the recessive dpias03697 allele were scored for the possible enhancement or suppression of known phenotypes in JAK-STAT pathway mutants. hopTum-l is a dominant hyperactive allele (increased HOP activity at elevated temperature) that causes tumor formation. This tumor formation, which is suppressed by stat92E LOF mutants, results from excessive proliferation of blood cells (plasmatocytes) that form melanotic abdominal tumors in larvae and pupae that can be scored in adults. At 25°C, 37% of heterozygous hopTum-l adult females had at least one abdominal tumor. Reduction of a negative activating regulator of this pathway should cause an increase in tumors. The percentage of flies with at least one tumor more than doubled in the hopTum-l/+;dpias03697/+ genotype compared with the progeny with two WT dpias alleles. Experiments on tumor frequency support the conclusion that dPIAS interacts negatively with the JAK-STAT pathway made overactive by hopTum-l: this leads to tumor formation. It is concluded that dPIAS decreases the transcriptional impact of the overactive STAT92E (Betz, 2001).
The role of dpias in eye development was examined because hypomorphic mutants of hop and os have small eyes. Two different lines, GMR-Gal4 and ey-Gal4, in which dpias overexpression depends on Gal4 activation at different times during eye development, were used. When the GMR-Gal4 line was used to drive UAS-dpias(537), no obvious effect on eye size or texture was observed. When UAS-dpias(537) was activated with the ey-Gal4 driver, eye size was severely reduced and the remaining small eye had a rough texture. A doubling of the transgene dosage further aggravated this phenotype and resulted in complete loss of the eyes in most of the surviving progeny. Because ey-Gal4 is active very early in eye development (before cellular differentiation) and GMR-Gal4 at later stages (during cellular differentiation), it is concluded that overexpression of dpias(537) has an effect primarily on cells in the early proliferating eye disc (Betz, 2001).
Whether this occurs because of a decreased activity of the JAK-STAT pathway was investigated. To this end Small-eyed UAS-dpias(537)/CyO;ey-Gal4 flies were crossed to a stock carrying a heat shock-inducible stat92E gene (hs-stat92E) and the progeny were raised under mild heat-shock conditions. A significant rescue of eye size and texture was observed only in progeny that carried the hs-stat92E transgene but not in genotypes without the hs-stat92E transgene segregating from the same cross. Moreover, a similar eye-size rescue effect was achieved by crossing the hopTum-l stock with small-eyed UAS-dpias(537)/CyO;ey-Gal4 flies, further bolstering the notion that activated STAT92E is required for eye development and that dPIAS counteracts the activated STAT92E (Betz, 2001).
The posterior spiracle defects of the domeless/mom mutation have led
to an examination of functions of the Hop/Stat92E pathway in tracheal formation.
Trachea form from 10 tracheal pits, 1 per hemisegment. The
trachealess gene selects the tracheal primordia in the
embryonic ectoderm and drives the conversion of these planar epithelial regions into tracheal pits. The tracheal pits then sprout successively finer branches and fuse together, forming
the tracheal network. The trachea is further connected to the posterior
spiracle, forming a functional tracheal system. Tracheal formation was examined in mom, hop, and STAT92E mutants by using an enhancer trap line in the trachealess gene (1-eve-1) and an antibody [(mAb)2A12] that stains tracheal branches and trunks. In hop null
embryos, trachealess expression is completely abolished and
tracheal formation is completely blocked. In paternally rescued embryos, a defective tracheal system forms, generally with several disruptions in the main trunk and several branches. Because all of the mom and
STAT92E mutants examined were enhancer trap lines, trachealess gene expression could not be examined by directly using the 1-eve-1
enhancer trap line. However, in the paternally rescued STAT92E
and mom mutant embryos, similar to the hop mutant embryos, a
defective tracheal system formed,
generally with several disruptions in the main trunk and several
branches. These data suggest that Mom and the Hop/Stat92E
signal transduction pathway play an indispensable role in tracheal formation (Chen, 2002).
The JAK/STAT signal transduction pathway regulates many developmental processes in Drosophila. However, the functional mechanism of this pathway is poorly understood. The Drosophila cyclin-dependent kinase 4 (Cdk4) exhibits embryonic mutant phenotypes identical to those in the Hopscotch/JAK kinase and stat92E/STAT mutations. Specific genetic interactions between Cdk4 and hop mutations suggest that Cdk4 functions downstream of the HOP tyrosine kinase. Cyclin D-Cdk4 (as well as Cyclin E-Cdk2) binds and regulates STAT92E protein stability. STAT92E regulates gene expression for various biological processes, including the endocycle S phase. These data suggest that Cyclin D-Cdk4 and Cyclin E-Cdk2 play more versatile roles in Drosophila development (Chen, 2003).
In a large screen for autosomal P element-induced zygotic lethal mutations associated with specific maternal effect lethal phenotypes, a mutation, l(2)sh0671, located at 53C, was identified that showed a maternal effect segmentation phenotype. The phenotype is similar to the effect of loss of hop and stat92E gene activity during oogenesis. The P element, l(2)sh0671, was inserted into the second intron of the Cdk4 gene before the ATG translation initiation code (Chen, 2003).
The similarity of the Cdk4 mutant phenotype to that of hop and stat92E suggests that these latter genes are involved in the same developmental process. A prediction of this hypothesis is that mutations in Cdk4 would affect the expression of segmentation genes in the same manner as hop and stat92E. The removal of either hop or stat92E activity is known to result in the stripe-specific loss of expression of several pair-rule genes. 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 gene construct containing a 5.2 kb eve promoter element driving lacZ shows expression of lacZ in eve stripes 2, 3, and 7. Removal of maternal activity of either hop or stat92E results in the loss of the third stripe from the reporter construct. Similarly, removal of maternal activity of Cdk4 also causes the specific loss of the third stripe, without affecting the second or seventh stripes (Chen, 2003).
The HOP/STAT92E pathway regulates tracheal formation through regulating trachealess (trh) gene expression in the embryo. It was reasoned that Cdk4 might also regulate tracheal formation. Tracheal formation was examined in wild-type, hop, and Cdk4 embryos by using an antibody [(mAb)2A12] that stains tracheal branches and trunks. In paternally rescued hop and cdk4 embryos, a similar defective tracheal system was formed that generally had several disruptions in the main trunk and several branches. These data suggest that Cdk4 regulates tracheal formation in a manner similar to the HOP/STAT92E signal transduction pathway (Chen, 2003).
To determine whether hop and Cdk4 genetically interact, a test was performed to see whether a reduction in the amount of maternal Cdk4 gene activity could enhance the maternal effect associated with a partial loss of function in the hop mutation. Embryos that are derived from mothers that carry GLCs of the hopmsv1 hypomorphic allele show weak segmentation defects, and many of them hatch. However, when these embryos are derived from females that also carry a single copy of Cdk43, they exhibit segmentation defects that are similar to embryos derived from females that lack all maternal hop activity, and none of them hatch. This result suggests that hop and Cdk4 act in concert to regulate embryonic segmentation (Chen, 2003).
Whether Cdk4 operates upstream or downstream of HOP was examined by testing whether the effect of a hyperactive hop allele could be negated by a reduction in the amount of Cdk4 gene activity. If Cdk4 is required for transducing the HOP signal, then reduction of Cdk4 should suppress a hop gain-of-function phenotype. The dominant temperature-sensitive hop allele, hopTum-l, was used for this experiment. When grown above 29°C, flies heterozygous for hopTum-l have reduced viability and the emerging adults develop melanotic tumors. Viability and formation of melanotic tumors at 29°C were compared in females heterozygous for hopTum-l and Cdk4 with females heterozygous only for hopTum-l. An improved survival rate was obtained by removing a single copy of Cdk4 in hopTum-l heterozygous females. However, the formation of melanotic tumors is affected less by removing a single copy of Cdk4 in hopTum-l heterozygous females (Chen, 2003).
To further examine the function of Cdk4 in the HOP/STAT92E signal transduction pathway, the genetic interactions of Cdk4 with hop and stat92E were tested in embryos. The hop (hopC111) and stat92E (stat92E6346) null embryos show a consistent deletion of the fifth abdominal segment and the posterior mid-ventral portion of the fourth abdominal segment, and none of them hatch. When HS-Cdk4 is ubiquitously expressed in hopC111 embryos, most embryos have complete fourth and fifth abdominal segments, and many of them hatch. Ubiquitous expression of Cdk4 has no effect in stat92E mutant embryos (Chen, 2003).
In mammals and Drosophila, Cdk4 forms a protein complex that regulates the cell cycle progression. The Cyclin D and Cdk4 complex (CycD-Cdk4) phosphorylates and releases RB from RB/E2F; free E2F then activates gene expression, including Cyclin E (CycE). Cyclin E and Cdk2 form a complex (CycE-Cdk2) and regulate the cell cycle at the G1-S transition point. To further examine relations between the HOP/STAT92E signal transduction pathway and cell cycle regulation, the genetic interaction of hop with CycE was tested. Like HS-Cdk4, HS-CycE rescues hopC111 embryo segmentation defects but has no effect on stat92E mutant embryos (Chen, 2003).
The viability and formation of melanotic tumors at 29°C were compared in females heterozygous for hopTum-l and CycE with females heterozygous only for hopTum-l. An improved survival rate was observed by removing a single copy of CycE in hopTum-l heterozygous females. As in the case of Cdk4, the formation of melanotic tumors is less affected by removing a single copy of CycE in hopTum-l heterozygous females. These results suggest that CycD-Cdk4 and CycE-Cdk2 complexes are members of the HOP/STAT92E signal transduction pathway and function downstream of the HOP tyrosine kinase and either upstream of or parallel to the STAT92E transcription factor (Chen, 2003).
Thus Cdk4 functions in the HOP/STAT92E pathway and regulates embryonic segmentation, tracheal formation, eye development, and melanotic tumor formation. Specific genetic interactions between Cdk4 and hop or stat92E mutations suggest that Cdk4 functions upstream of STAT and parallel to or downstream of the HOP tyrosine kinase. Furthermore, CycD-Cdk4 and CycE-Cdk2 bind and regulate STAT92E protein stability. These data demonstrate that, besides their role in regulating the cell cycle, CycD-Cdk4 and CycE-Cdk2 have a role in regulating cell fate determination and proliferation via STAT signaling (Chen, 2003).
STAT92E binds directly to the promoter of pair-rule genes and regulates their expression for segmentation. This occurs during the first 13 embryonic cell cycles, which are nearly synchronous and lack G1 and G2 gap phases. Obviously, the function of CycD-Cdk4 and CycE-Cdk2 is not to regulate the cell cycle during this period. The CycD-Cdk4 and CycE-Cdk2 complexes may regulate pair-rule gene expression through stabilizing STAT92E protein and increasing its transcription activity (Chen, 2003).
The duplication of genes and genomes is believed to be a major force in the evolution of eukaryotic organisms. However, different models have been presented about how duplicated genes are preserved from elimination by purifying selection. Preservation of one of the gene copies due to rare mutational events that result in a new gene function (neo-functionalization) necessitates that the other gene copy retain its ancestral function. Alternatively, preservation of both gene copies due to rapid divergence of coding and non-coding regions such that neither retains the complete function of the ancestral gene (sub-functionalization) may result in a requirement for both gene copies for organismal survival. The duplication and divergence of the tandemly arrayed homeotic clusters have been studied in considerable detail and have provided evidence in support of the sub-functionalization model. However, the vast majority of duplicated genes are not clustered tandemly, but instead are dispersed in syntenic regions on different chromosomes, most likely as a result of genome-wide duplications and rearrangements. The Myb oncogene family provides an interesting opportunity to study a dispersed multigene family because invertebrates possess a single Myb gene, whereas all vertebrate genomes examined thus far contain three different Myb genes (A-Myb, B-Myb and c-Myb). A-Myb and c-Myb appear to have arisen by a second round of gene duplication, which was preceded by the acquisition of a transcriptional activation domain in the ancestral A-Myb/c-Myb gene generated from the initial duplication of an ancestral B-Myb-like gene. B-Myb appears to be essential in all dividing cells, whereas A-Myb and c-Myb display tissue-specific requirements during spermatogenesis and hematopoiesis, respectively. The absence of Drosophila Myb (Dm-Myb) causes a failure of larval hemocyte proliferation and lymph gland development, while Dm-Myb(-/-) hemocytes from mosaic larvae reveal a phagocytosis defect. Vertebrate B-Myb, but neither vertebrate A-Myb nor c-Myb, can complement these hemocyte proliferation defects in Drosophila. Indeed, vertebrate A-Myb and c-Myb cause lethality in the presence or absence of endogenous Dm-Myb. These results are consistent with a neomorphic origin of an ancestral A-Myb/c-Myb gene from a duplicated B-Myb-like gene. In addition, these results suggest that B-Myb and Dm-Myb share essential conserved functions that are required for cell proliferation. Finally, these experiments demonstrate the utility of genetic complementation in Drosophila to explore the functional evolution of duplicated genes in vertebrates (Davidson, 2004).
To establish whether Dm-Myb is generally required for proliferation
of hemocytes, epistasis experiments were conducted using a Dm-Myb null mutation and
dominant substitution mutations of the Toll receptor (Tl10b) and the Jak kinase, hopscotch (hopTuml); dominant gain-of-function mutations in these genes result in hyperactivation of their respective pathways leading to hemocyte overproliferation and abnormal lamellocyte differentiation. To determine whether Dm-Myb is required for the dysregulated overproliferation and differentiation phenotypes of Tl10b and
hopTuml mutants, double mutant larvae lacking Dm-Myb in conjunction with these dominant alleles of Toll and hopscotch were generated.
It was found that, in addition to an overproliferation of plasmatocytes in the primary
lymph gland lobes, the secondary lymph gland lobe hemocytes aberrantly differentiate
into lamellocytes in hopTuml mutants. It is thought that the normally smaller
secondary lymph gland lobes serve as a reservoir of undifferentiated prohemocytes, however, in hopTuml larvae the secondary lobes enlarge with
concomitant abnormal differentiation of lamellocytes. While hemocytes in the secondary lymph gland lobes of hopTuml,
Dm-Myb-/- double mutants show an increased expression of the lamellocyte enhancer-trap marker, these ß-gal positive cells fail to overproliferate and do not adopt the flattened shape characteristic of differentiated lamellocytes. In summary, an activated JAK/STAT pathway cannot drive the proliferation of hemocytes in the absence of Dm-Myb. In addition, an activated Toll pathway cannot drive the proliferation of hemocytes in the absence of Dm-Myb (Davidson, 2004).
It has been proposed that germ-line-encoded pattern recognition
receptors bind microbial cell wall determinants (such as lipopolysaccharides, mannans, and peptidoglycans) and initiate an immune response, either by activating
associated proteases in circulation or by directly triggering intracellular signaling pathways in immune responsive cells. To date, no
pattern recognition receptor has been firmly identified in Drosophila and shown to activate an immune response. A primitive
complement-like system, evocative of the alternative or the lectin pathways of complement, could be involved in the activation of some of the Drosophila host
defense mechanisms. This hypothesis was made attractive by the recent reports that invertebrates such as sea urchins and tunicates have a complement-like system,
and produce proteins with structural similarities to vertebrate complement C3 proteins, containing an intrachain thiolester bond. Similar proteins have also been
described in the horseshoe crab, a member of the class of arthropods to which Drosophila also belongs (Lagueux, 2000 and references therein).
Drosophila expresses four genes encoding proteins with significant similarities with the thiolester-containing proteins of
the complement C3/alpha2-macroglobulin superfamily. The genes are transcribed at a low level during all stages of development,
and their expression is markedly up-regulated after an immune challenge. For one of these genes, which is predominantly expressed in
the larval fat body, a constitutive expression was observed in gain-of-function mutants of the Janus kinase (JAK) hop and a reduced
inducibility in loss-of-function hop mutants. A constitutive expression was observed in gain-of-function Toll mutants. These novel
complement-like proteins are likely to play roles in the Drosophila host defense (Lagueux, 2000).
In higher vertebrates, the complement system consists of about 30 serum
and cell surface proteins and mediates inflammatory reactions,
opsonization of microorganisms for phagocytosis, and direct killing of
some pathogens. Activation can occur via the classical
antibody-dependent pathway, the alternative pathway, and the lectin
pathway, which all converge on the central complement C3 protein. The presence in
Drosophila of several proteins with basic structural characteristics similar to complement C3 makes attractive the working hypothesis that an ancient equivalent of the alternative pathway and/or the lectin pathway exists in this species. In vertebrates, the
activation of the alternative pathway is initiated by spontaneous hydrolysis of the thiolester bond of complement C3, resulting, through
association with other proteins of the complement system, in an active
C3 convertase that is normally inactivated by regulatory proteins
present on self tissue, but absent from non-self, providing for a
relative primitive mode of discriminating self from non-self. In turn, active C3
convertase activates complement C3 and, through an
amplification loop, triggers the conventional effector mechanisms of
complement. Activation of the lectin pathway is initiated when various
sugars present on the surface of microorganisms bind to a collectin,
the mannan-binding lectin (MBL), thereby inducing proteolytic cascades
that activate complement C3 and the downstream events common to all
three activation pathways (Lagueux, 2000).
In the mid-nineties, it became apparent that the complement system is
not a unique property of the host defense armatarium of vertebrates.
ESTs from cDNA libraries of sea urchin coelomocytes were found to
encode a protein with structural similarities to vertebrate complement
C3, including an intrachain thiolester motif, plus a homolog of
vertebrate factor B, which participates in the activation of complement
C3 through the alternative pathway. More recently, an ascidian
species was reported to possess homologs of complement C3 and of two
mannose-binding lectin-associated proteases (MASPs), plus a homolog
of factor B, raising the possibility that equivalents of both the
lectin and the alternative activation pathways are present in these
deuterostome invertebrates. Experiments with ascidian coelomocytes
further indicate that the complement C3-like molecules act as opsonic
factors and are activated through a complement-like cascade (Lagueux, 2000 and references therein).
TEPs are also structurally close to
alpha2-macroglobulins, which are evolutionary
ancient protease inhibitors, from which complement C3 has been proposed
to have arisen by gene duplication. Protease
inhibitors related to alpha2-macroglobulin have
been described in several invertebrates and have been particularly well
studied in the horseshoe crab Limulus. Indeed, Limulus alpha2-macroglobulin has been
proposed to function as a protease inhibitor, particularly of proteases
released by tissue damage caused by injury or pathogens and of soluble
or surface bound proteases produced by invading microorganisms.
It has been suggested that the first opsonic system could have
required no specific recognition or activation mechanism other than the presence of exogenous proteases causing
alpha2-macroglobulin to bind directly to the
protease-producing organism (Lagueux, 2000 and references therein).
Because Drosophila has four expressed genes encoding
proteins with structural similarities to the superfamily of complement C3/alpha2-macroglobulin,
significant functional versatilities may be expected, all the more so, because one of
the Tep genes, Tep2, gives rise to five different
transcripts. Interestingly, the Tep2 transcripts are
identical except for a short region of 30 aa. This region is encoded by
alternatively spliced exons corresponding to the hypervariable region
of the TEPs; it is located in a relative position similar to the bait
domain in alpha2-macroglobulins or the anaphylatoxins in complement C3. Alternative splicing has not been
reported in vertebrates for members of the complement
C3/alpha2-macroglobulin superfamily. By
increasing the number of putative recognition motifs for microorganisms
or proteases, it may contribute to the fine-tuning of recognition of
noxious structural patterns in the absence of the large repertoire of
receptors of the adaptive immune response in vertebrates (Lagueux, 2000 and references therein).
The Drosophila plasmatocytes are macrophage-like blood cells
that readily engulf bacteria or fungal spores, as well as various cellular debris resulting from injury or apoptosis. Nothing is known about possible opsonization in this model, and a tempting working
hypothesis is that the complement-like proteins described here
precisely fulfill such a role in the host defense. Future efforts
will be directed toward experimentally testing this hypothesis, and it is
anticipated that the generation of mutants of the various Tep
genes, which all map to the left arm of the second chromosome, will be
invaluable in this endeavor (Lagueux, 2000).
TEP1 is produced mainly in the fat body, and its expression is
up-regulated by immune challenge. It is hypothesized that, as is the case
for immune-induction of antimicrobial peptides in this tissue, the
up-regulation would be dependent on either the Toll or the
imd pathway. This
control is strongly dependent on the JAK hopscotch.
Hopscotch is the only JAK identified in Drosophila, and in
the gain-of-function mutant hopTum-l,
Tep1 is constitutively expressed, whereas its
immune-inducibility is dramatically reduced in the loss-of-function
mutant hopM38. Gain-of-function
mutations of hop have remarkable effects on hemopoiesis in
Drosophila and result in overproliferation of blood cells,
increased differentiation of lamellocytes, and aggregation of blood
cells into masses, which tend to become melanized, a process referred
to as melanotic tumor formation. The data thus show that these events
are concomitant with an increased transcription of the Tep1
gene. Whether they are causally related remains an open question, but
it will be worthwhile investigating whether the TEP1 protein can affect
the aggregation of blood cells and the localized induction of melanization (Lagueux, 2000).
The mechanisms by which an organism becomes immune competent during its development are largely unknown. When
infected by eggs of parasitic wasps, Drosophila larvae mount a complex cellular immune reaction in which specialized host
blood cells, lamellocytes and crystal cells, are activated and recruited to build a capsule around the parasite egg to block its
development. Parasitization by the wasp Leptopilina boulardi leads to a dramatic increase in the number of both lamellocytes and crystal cells in the Drosophila larval lymph gland. Furthermore, a limited burst of mitosis follows shortly after infection, suggesting that both cell division and differentiation of lymph gland hemocytes are required
for encapsulation. These changes, observed in the lymph glands of third-instar, but never of second-instar hosts, are almost
always accompanied by dispersal of the anterior lobes themselves. To confirm a link between host development and immune competence, mutant hosts in which development is blocked during larval or late larval stages were infected. In genetic backgrounds where ecdysone levels are low (ecdysoneless) or ecdysone signaling is blocked (nonpupariating allele of the transcription factor broad), the encapsulation response is severely compromised. In the third-instar ecdysoneless hosts, postinfection mitotic amplification in the lymph glands is absent and there is a reduction in crystal cell maturation and postinfection circulating lamellocyte concentration. These results suggest that an
ecdysone-activated pathway potentiates precursors of effector cell types to respond to parasitization by proliferation and
differentiation. It is proposed that, by affecting a specific pool of hematopoietic precursors, this pathway thus confers immune
capacity to third-instar larvae (Sorrentino, 2002).
To confirm the correlation between ecdysone deficiency
and reduction in encapsulation capacity, the effect of ecd1 on encapsulation was studied in a background in which
lamellocytes are produced in lymph glands without parasitization.
The temperature-sensitive semidominant lethal
mutation hopTum-l causes an overproliferation of circulating hemocytes, the appearance of lamellocytes in large numbers,
and the encapsulation of self tissue.
The temperature-sensitive period of hopTum-l, like that of ecd1, begins in the second larval instar. The size and
number of melanotic capsules was analyzed in third-instar larvae at 18,
25, and 29°C, and adults at 25°C. At all three
temperatures, 100% of hopTum-l/Y larvae exhibit multiple
capsules. Lowering of ecdysone levels in hopTum-l/Y; ecd1/ecd1 animals results in a mild, but significant, suppression of the hopTum-l melanotic capsule phenotype. Thus, at 18 and 25°C, over 10% of double mutants were completely clear of melanotic capsules, and at 29°C, nearly 20% were devoid of capsules. Furthermore, most double-mutant larvae
that did score positively for capsules exhibited fewer and
smaller capsules. Tumor penetrance in surviving double-mutant adults at 25°C (86.2%) is consistent with the corresponding larval value. Thus,
ecd1 is able to partially suppress the hopTum-l-induced melanotic capsule phenotype (Sorrentino, 2002).
A model is proposed in which lymph gland
development and specific steps in hematopoiesis required
for the encapsulation response are both tied to a signal
transduction pathway triggered by ecdysone, possibly at the
L2-L3 transition. This pathway regulates the capacity of
lamellocyte and crystal cell precursors to respond to infection.
While the precise role of the ecdysone pathway in
these steps is not clear, it is possible that ecdysone induces
division of hematopoietic progenitors in order to maintain a
critical basal population of immature immune effector
cells. In addition, ecdysone may trigger differentiation in
lamellocyte and/or crystal cell precursors. If so, third-instar
ecd mutant lymph glands would have fewer lamellocyte
and crystal cell precursors, most or all of which in an
immature or unpotentiated state that prevents them from
responding to parasitization; the aggregate effect of this
would be an unresponsive lymph gland. Availability and application of molecular markers for progenitors versus differentiated lamellocytes and crystal cells will allow validation of this model and facilitate
examination of whether or not lymph gland overgrowth
mutants, such as hopTum-l, affect cell populations and bypass the requirement for ecdysone. Such studies can then reveal
whether suppression of the hopTum-l phenotype in hopTum-l/Y;ecd1
/ecd1 double mutants (with fewer and smaller melanotic
tumors than hopTum-l/Y larvae) is due to changes in these progenitor cells or unrelated effects 'downstream' in the
encapsulation process (Sorrentino, 2002).
Innate immunity is essential for metazoans to fight microbial infections. Genome-wide expression profiling was used to analyze the outcome of impairing specific signaling pathways after microbial challenge. These transcriptional patterns can be dissected into distinct groups. In addition to signaling through the Toll/NFkappaB or Imd/Relish pathways, signaling through the JNK and JAK/STAT pathways controls distinct subsets of targets induced by microbial agents. Each pathway shows a specific temporal pattern of activation and targets different functional groups, suggesting that innate immune responses are modular and recruit distinct physiological programs. In particular, the results may imply a close link between the control of tissue repair and antimicrobial processes (Boutros, 2002).
Lipopolysaccharides (LPS) are the principal cell wall components of gram-negative bacteria. In mammals, exposure to LPS causes septic shock through a Toll-like receptor TLR4-dependent signaling pathway. LPS treatment of Drosophila SL2 cells leads to rapid expression of antimicrobial peptides, such as Cecropins (Cec). SL2 cells resemble embryonic hemocytes and have also been used as a model system to study JNK and other signaling pathways. LPS-responsive induction of the antimicrobial peptides AttacinA (AttA), Diptericin (Dipt), and Cec relies on IKK and Relish. In order to obtain a broad overview on the transcriptional response to LPS in Drosophila, genome-wide expression profiles of SL2 cells were generated at different time points following LPS treatment. Altered expression of 238 genes was detected (Boutros, 2002).
In time-course experiments, a complex pattern of gene expression was observed that can be separated into different temporal clusters. A first group, with peak expression at 60 min after LPS, primarily consists of cytoskeletal regulators, signaling, and proapoptotic factors. This group includes cytoskeletal and cell adhesion modulators such as Matrix metalloprotease-1, WASp, Myosin, and Ninjurin, proapoptotic factors such as Reaper, and signaling proteins such as Puckered and VEGF-2. A second group, with peak expression at 120 min, includes many known defense and immunity genes, such as Cec, Mtk, and AttA, but not the gram-positive-induced peptide Drs. Interestingly, this cluster also includes PGRP-SA, which is a gram-positive pattern recognition receptor in vivo, suggesting possible crossregulation between gram-positive- and gram-negative-induced factors. A third group is transiently downregulated upon LPS stimulation. This cluster includes genes that play a role in cell cycle control, such as String and Rca1. Altogether, these results show that, in response to LPS, a defined gram-negative stimulus, cells elicit a complex transcriptional response (Boutros, 2002).
Clustering revealed a noncanonical group with small proteins that are expressed late and transiently with peak expression at 6 hr after septic injury. One of the clustered transcripts, CG11501, encodes a small Cys-rich protein that is 115 amino acids long and is strongly induced after septic injury. By RT-PCR, it was confirmed that CG11501 is upregulated after septic injury. In order to characterize how CG11501 is controlled after microbial challenge, a candidate pathway approach was undertaken. In an independent study, it was found that totM gene induction, which is part of the same cluster, is dependent on a JAK/STAT signaling pathway. Whether CG11501 induction requires JAK/STAT signaling was examined. Mutations in JAK/STAT pathways in Drosophila have been implicated in various processes during embryonic and larval development. In Anopheles, STAT is activated in response to bacterial infection. Similarly, gain-of-function STAT has been implicated in the transcriptional control of thiolester proteins. Mutant alleles of hopscotch (hop), the Drosophila homolog of JAK were examined. Quantitative PCR shows that CG11501 induction after septic injury is diminished in hop loss-of-function mutants, whereas the expression of Toll and Imd targets drs, and cec is not affected (Boutros, 2002).
This study shows that in addition to known innate immune cascades, JNK and JAK/STAT are required for the transcriptional response during microbial challenge. One transcriptional signature of small secreted peptides can be traced to JAK/STAT signaling. Additionally, JNK signaling controls cytoskeletal genes after an LPS stimulus and after septic injury in vivo. Both in cells and in vivo, JNK pathways are connected to the same upstream signaling cassette that induces NFkappaB targets. Altogether, these results suggest that innate immune signaling pathways closely link cytoskeletal remodeling, as required for tissue repair, and direct antimicrobial actions. The data also provide insights into the connection of temporal patterns and the activation of distinct signaling pathways (Boutros, 2002).
NFkappaB pathways play a central role for innate and adaptive immune response in mammals. In innate immune responses, TLRs on dendritic cells recognize microbial agents and activate NFkappaB, leading to the expression of proinflammatory cytokines and other costimulatory factors required to initiate an adaptive immune response. Additionally, other signaling pathways have been implicated at later stages during immune responses in mammals, but their physiological role in innate immunity remains rather poorly understood. For example, several cytokines, such as IL-6 and IL-11, signal through a JAK/STAT pathway to induce the expression of acute phase proteins. Similarly, JNK pathways are activated in response to TNF and IL-1, may lead to the expression of immune modulators, and are required for T cell differentiation. In Drosophila, studies have investigated two distinct NFkappaB-pathways --Toll and Imd/Rel -- that have been shown to mediate gram-positive/fungal and gram-negative responses. Both pathways induce specific antimicrobial peptides and thereby focus the response on the invading microbial agent. Genetic analysis has shown that functions of the NFkappaB-pathways are separable; flies that are mutant for only one of these pathways are susceptible to subgroups of pathogens. Could the contribution of NFkappaB-dependent and, possibly, other signaling pathways be identified by examining global expression profiles? The obtained data set demonstrates that NFkappaB-independent signaling pathways contribute to the transcriptional patterns observed after microbial infection. Both in cells and in vivo, JNK-dependent targets precede the peak expression of antimicrobial peptides that require NFkappaB. JAK/STAT targets are induced with a distinct temporal pattern that shows late, but only transient, expression characteristics. The stereotyped pathway patterns after microbial challenge suggest that the correct temporal execution of signaling events, similar to signaling during development, may play an important role in the regulation of homeostasis (Boutros, 2002).
To characterize the features of JAK/STAT signaling in Drosophila immune response, totA was identified as a gene that is regulated by the JAK/STAT pathway in response to septic injury. Septic injury triggers the hemocyte-specific expression of upd3, a gene encoding a novel Upd-like cytokine that is necessary for the JAK/STAT-dependent activation of totA in the Drosophila counterpart of the mammalian liver, the fat body. In addition, totA activation is shown to require the NF-KB-like Relish pathway, indicating that fat body cells integrate the activity of NF-KB and JAK/STAT signaling pathways upon immune response. This study reveals that, in addition to the pattern recognition receptor-mediated NF-kappaB-dependent immune response, Drosophila undergoes a complex systemic response that is mediated by the production of cytokines in blood cells, a process that is similar to the acute phase response in mammals (Agaisse, 2003).
In order to identify genes that are regulated by the JAK/STAT pathway in response to septic injury in adult flies, a screen was performed for candidates that display an inducible expression upon immune challenge and that are constitutively expressed in flies carrying a gain-of-function mutation in the JAK/STAT pathway. To this end, custom-made cDNA microarrays were used to compare gene expression profiles of nonchallenged wild-type flies to gene expression profiles of challenged wild-type flies and to gene expression profiles of nonchallenged TumL flies displaying a gain-of-function mutation in the Drosophila JAK kinase Hopscotch. MP1 was identified as a gene that fulfilled both criteria for induction upon challenge and constitutive expression in a JAK/STAT gain-of-function mutation. MP1 expression was not induced in challenged flies displaying loss-of-function mutation in hop (hopM38/hopmsv1), confirming the involvement of Drosophila JAK in MP1 expression (Agaisse, 2003).
Drosophila larvae defend themselves against parasitoid wasps by completely surrounding the egg with layers of specialized hemocytes called lamellocytes. Similar capsules of lamellocytes, called melanotic capsules, are also formed around 'self' tissues in larvae carrying gain-of-function mutations in Toll and hopscotch. Constitutive differentiation of lamellocytes in larvae carrying these mutations is accompanied by high concentrations of plasmatocytes, the major hemocyte class in uninfected control larvae. The relative contributions of hemocyte concentration vs. lamellocyte differentiation to wasp egg encapsulation are not known. To address this question, Leptopilina boulardi was used to infect more than a dozen strains of host larvae harboring a wide range of hemocyte densities. A significant correlation exists between hemocyte concentration and encapsulation capacity among wild-type larvae and larvae heterozygous for mutations in the Hopscotch-Stat92E and Toll-Dorsal pathways. Larvae carrying loss-of-function mutations in Hopscotch, Stat92E, or dorsal group genes exhibit significant reduction in encapsulation capacity. Larvae carrying loss-of-function mutations in dorsal group genes (including Toll and tube) have reduced hemocyte concentrations, whereas larvae deficient in Hopscotch-Stat92E signaling do not. Surprisingly, unlike hopscotch mutants, Toll and tube mutants are not compromised in their ability to generate lamellocytes. These results suggest that circulating hemocyte concentration and lamellocyte differentiation constitute two distinct physiological requirements of wasp egg encapsulation and Toll and Hopscotch proteins serve distinct roles in this process (Sorrentino, 2004).
These results suggest that the suppression of encapsulation capacity by loss of function of hop, Tl, or tub is likely to be due to distinct requirements of these genes. The suppression of lymph gland response to parasitization in the hopM4 background is consistent with the observed reduction in hopM4/Y encapsulation capacity and suggests that the Hopscotch protein is necessary for a parasite-induced signal for lamellocyte differentiation. This signal for lamellocyte differentiation is most likely mediated by the transcription factor Stat92E: Loss of function of one copy of Stat92E suppresses the penetrance of the hopTum-l-induced melanotic tumor phenotype and Stat92E is constitutively activated in Drosophila cell cultures that overexpress HopTum-l. These results are consistent with the proposed Stat92E-dependent lamellocyte signal: stat92E larvae are immune compromised and are unable to mount an efficient egg encapsulation response despite exhibiting control circulating hemocyte concentration levels. Additionally, mean circulating lamellocyte percentage in hopTum-l/Y; stat92EHJ/stat92EHJ larvae that are tumor-free is ~1%, which is indistinguishable from the control value (Sorrentino, 2004).
In contrast to Hop and Stat92E, Toll and Tube appear not to play a role in lamellocyte differentiation; rather, loss-of-function mutations in Toll or tube probably suppress encapsulation via other mechanisms. Since Toll and tube larvae have very few circulating hemocytes, reduction in encapsulation in Tl and tub mutants might be due to defects in wasp egg recognition or a reduction in hemocyte proliferation that normally follows parasitization. The effect of these mutations on crystal cells is unclear. While the possibility that these mutations reduce encapsulation capacity by reducing the crystal cell population cannot be ruled out, this is unlikely, since Black cells mutant larvae without functional crystal cells are immune competent and can still successfully encapsulate wasp eggs. The fact that lymph glands of loss-of-function Tl and tub mutant larvae can support lamellocyte differentiation suggests that the low circulating hemocyte concentration in Tl and tub larvae in itself does not hinder lamellocyte differentiation or the ability of the lymph gland to disperse after the wasp egg is introduced into the hemocoel. Given that gain-of-function Tl alleles induce lamellocyte differentiation, the lack of effect of Tl- on lamellocyte differentiation is somewhat unexpected, and it is possible that lamellocyte differentiation is in some way secondarily activated in the Tl10b background. Thus, the wasp egg encapsulation assay is a useful tool for evaluating the genetic requirements for lamellocyte differentiation (Sorrentino, 2004).
In conclusion, this study shows that while there is substantial variation in hemocyte concentration in control larvae, this variation is consistent with a log-normal distribution. Such a distribution could be a result of the inherently logarithmic process of cell division. Using this quantitative method of circulating hemocyte concentration data analysis, it was found that previously reported circulating hemocyte concentration values for mutant larvae that exhibit reduced or increased hemocyte densities are also log-normally distributed and that approximately half of each of these mutant distributions lie beyond the limits of the control distribution, allowing ranges of circulating hemocyte concentration values to be defined as being low, control, and high. In addition, encapsulation capacity in control and DV mutant larvae correlates with circulating hemocyte concentration. Evidence for biological significance of this correlation also comes from observations that D. melanogaster larvae selected for higher resistance against A. tabida have twice as many circulating hemocytes as compared to control larvae. These observations support the notion that circulating hemocytes, possibly plasmatocytes, contribute to the efficiency of the egg encapsulation response. However, high circulating hemocyte concentration alone is insufficient to trigger encapsulation; lamellocytes must be present. For example, massive 20- to 300-fold increases in circulating hemocyte concentration involving plasmatocytes and crystal cells, but not lamellocytes, are insufficient to trigger constitutive encapsulation of self tissue in the larva. The combined use of genetic and immune approaches used in this study demonstrates that different developmental signals independently contribute to the maintenance of the steady-state hemocyte concentration in circulation and the ability to differentiate lamellocytes. Together, these physiological parameters enable larval hosts to efficiently defend themselves against wasp infections (Sorrentino, 2004).
The JAK/STAT signaling pathway, renowned for its effects on cell proliferation and survival, is constitutively active in various human cancers, including ovarian. JAK and STAT are required to convert the border cells in the Drosophila ovary from stationary, epithelial cells to migratory, invasive cells. The ligand for this pathway, Unpaired (Upd), is expressed by two central cells within the migratory cell cluster. Mutations in upd or jak cause defects in migration and a reduction in the number of cells recruited to the cluster. Ectopic expression of either Upd or JAK is sufficient to induce extra epithelial cells to migrate. Thus, a localized signal activates the JAK/STAT pathway in neighboring epithelial cells, causing them to become invasive (Silver, 2001).
Polar cells emit a short-range signal that causes adjacent follicle cells to surround them and acquire the ability to migrate through the nurse cells. The results reported here suggest that Upd is the major signal secreted by the polar cells that both recruits adjacent follicle cells into the cluster and causes them to become migratory. Both of these functions are carried out by activation of JAK and STAT in the neighboring follicle cells. Signaling through this pathway is necessary, both for recruitment of border cells to the cluster and for motility once the cells are recruited. This is based on the observations that in the majority of mutant egg chambers, border cell clusters contain fewer than the normal number of cells, and that even clusters with normal numbers of cells fail to migrate normally (Silver, 2001).
It is worth noting that while some migration is observed in JAK and STAT border cell mutants, the loss of Upd in the polar cells completely prevents migration. This may reflect greater perdurance of JAK and STAT proteins in the mosaic clones, compared to Upd, if Upd is normally present at lower levels and/or is more labile. Alternatively, these differences may imply that in addition to its activation of JAK and STAT, Upd can activate other signaling pathways (Silver, 2001).
Activation of the JAK/STAT pathway is not only necessary but is also sufficient to convert epithelial follicle cells to become migratory. Numerous extra border cells were observed following overexpression of upd, hop, or hopTum, many of which invaded the nurse cell cluster. These extra cells did not result from excess proliferation because follicle cells cease dividing at stage 6, at least 12 hr prior to border cell differentiation. Furthermore, no difference in phospho-histone H3 antibody labeling was observed in cells overexpressing upd or in cells lacking stat, ehrn compared to wild-type. Moreover, it was possible to obtain large clones lacking upd, hop, or stat activity, indicating that homozygous mutant cells retain the ability to divide numerous times. Thus, activation of the JAK/STAT pathway leads to border cell specification and migration, without effects on proliferation. In addition, while extra follicle cells could become migratory as a secondary consequence of ectopic polar cell formation, activation of the JAK/STAT pathway results in the appearance of additional migratory cells in the absence of extra polar cells (Silver, 2001).
The question of whether signaling through this pathway might be sufficient to cause epithelial cells to become invasive was addressed ectopically expressing Upd, Hopscotch (Hop), or the constitutively active form of Hop, HopTum1, using the GAL4/UAS expression system. In this method, the yeast transcriptional activator GAL4 is expressed under the control of a cell type-specific enhancer, in this case slbo-GAL4 and c306-GAL4. In stage 9 egg chambers, slbo-GAL4 induces expression of genes that are under the control of the yeast upstream activating sequence (UAS) in approximately 20 anterior follicle cells, a subset of which normally become the border cells. This is nearly identical to the ß-gal expression from an enhancer trap insertion into the slow border cells (slbo) locus, even though Slbo protein expression is normally restricted to the border cells at stage 9. C306-GAL4 drives expression in a larger number of anterior, as well as posterior, follicle cells, compared to slbo-GAL4. C306-GAL4 also begins expressing earlier in oogenesis than slbo-GAL4 (Silver, 2001).
Egg chambers from c306-GAL4; UAS-hop females exhibit a dramatic increase in the number of border cells compared to wild-type. Up to 90 slbo expressing cells are produced, about 60 of which invade the nurse cell cluster and 20 of which have completed migration by early stage 10. Similar, though less dramatic, phenotypes are observed when the constitutively activated kinase is expressed with either slbo-GAL4 or c306-GAL4. Likewise, slbo-GAL4;UAS-upd and c306-GAL4;UAS-upd females contain numerous extra slbo-expressing cells compared to wild-type, in the absence of extra polar cells. This is in marked contrast to the effect of excessive Hedgehog pathway signaling, which causes ectopic border cells to form as a secondary consequence of ectopic polar cell specification. Overexpression of upd does not appear to cause excess cell proliferation, sinces no difference was detected in phospho-histone H3 antibody labeling, which marks mitotic cells, as compared to wild-type (Silver, 2001).
Some of the extra border cells migrate as single cells, whereas others form multiple small clusters, and still others form one large cluster. The ability of the cells to migrate varies according to which protein is being expressed as well as with the timing and level of expression. High levels of ectopic Upd result in egg chambers in which both normal and extra border cells frequently fail to migrate, whereas high levels of wild-type Hop produce the most migratory cells. Thus, ectopic activation of the JAK/STAT pathway is sufficient to cause extra epithelial follicle cells to invade the nurse cell cluster (Silver, 2001).
Janus kinase (JAK) plays several signaling roles in Drosophila oogenesis. The gene for a JAK pathway ligand, unpaired, is expressed specifically in the polar follicle cells, two pairs of somatic cells at the anterior and posterior poles of the developing egg chamber. Consistent with unpaired expression, reduced JAK pathway activity results in the fusion of developing egg chambers. A primary defect of these chambers is the expansion of the polar cell population and concomitant loss of interfollicular stalk cells. These phenotypes are enhanced by reduction of unpaired activity, suggesting that Unpaired is a necessary ligand for the JAK pathway in oogenesis. Mosaic analysis of both JAK
pathway transducers, hopscotch and Stat92E, reveals that JAK signaling is specifically required in the somatic follicle cells. Moreover, JAK activity is also necessary for the initial commitment of epithelial follicle cells. Many of these roles are in common with, but distinct from, the known functions of Notch signaling in oogenesis. Consistent with these data is a model in which Notch signaling determines a pool of cells to be competent to adopt stalk or polar fate, while JAK signaling assigns specific identity within that competent pool (McGregor, 2002).
The somatic cells of the ovary consist of multiple subpopulations, each with its
own function(s) in the developing egg. While the germline cyst is dividing and developing within the germarium, a monolayer of somatic cells surrounds the cyst as it
moves posteriorly through the germarium. As the cyst becomes enveloped by the somatic cells, the egg chamber pinches off from the germarium,
entering the vitellarium. At that time, approximately 5-8 somatic cells differentiate into stalk. These flattened, disc-shaped cells are stacked together to form the spacer between successive cysts. Stalk cells connect the anterior end of a more mature egg chamber to the posterior end of the next younger chamber. Also at that time, molecular markers can distinguish the stalk cells from the polar cells, which arise from the same precursors. The polar cells are arranged as two pairs of follicle cells, one pair at either end of each chamber near the stalk cells. While the stalk cells and polar cells
cease proliferation at the end of the germarium, the remaining follicle cells, which are referred to as epithelial follicle cells, divide approximately five times to expand the pool of follicle cells. Those epithelial cells later differentiate into various subpopulations with specific functions
in the vitellarium. Those subpopulations are pre-patterned with mirror image symmetry along the anterior-posterior axis of the egg. Imposed on that pre-pattern, signaling from the oocyte by the TGFalpha molecule Gurken stimulates the induction of posterior polarity on the somatic cells at the posterior end. The result is an egg with coordinated polarities of the somatic and germline cells. This coordination is essential for the proper localization of maternal determinants that pattern the resulting embryo (McGregor, 2002).
Strikingly, unpaired is expressed very specifically within the
ovary. After egg chambers pinch off from the germarium, upd is restricted to the two pairs of polar cells found at the anterior and posterior tips of the egg. In the germarium, upd is expressed in a cluster of somatic cells at the posterior of region 3. Presumably these are the cells that give rise to the stalk and polar cells. Expression in the polar and border cells persists until egg maturation. In situ hybridization to Stat92E RNA reveals that Drosophila STAT is expressed in both the germarium and the vitellarium. Expression in the germarium occurs in all follicle cells in region 2a and 2b; it then begins to be restricted to terminal follicle cells in region 3. In the vitellarium, Stat92E is expressed weakly at the termini of the egg chamber, but in a broader domain than only the two polar cells. After stage 9, Stat92E is strongly expressed in the nurse cells, consistent with the maternal role of Stat92E in the segmentation of the early embryo. Moreover, weak ubiquitous expression of hop is detectable in the follicular epithelium. These data are consistent with a potential role for JAK signaling in oogenesis (McGregor, 2002).
The loss of JAK pathway function in the somatic cells of the Drosophila ovary results in the fusion of adjacent cysts and/or the mislocalization of the oocyte within a cyst. Based on molecular markers for cell identity, mutations in hop or Stat92E cause the loss of stalk cells and an increase in the number of polar cells. This shift in cell fates correlates with the fusion of adjacent cysts. An allelic series of hop mutant combinations shows a range of phenotypic severity, from occasional fusion of two adjacent chambers to complete fusion of all cysts with no morphological distinction between germarium and vitellarium. The severity of the visible phenotypes is reflective of the severity of the follicle cell fate transformations. Effects on fate range from frequent appearance of one extra polar cell in the weakest mutation to consistent appearance of a dozen or more extra polar cells in more severe alleles. Phenotypes seen in mutant clones of hop and Stat92E ovaries are similar to those seen in the heteroallelic combinations of hop mutations. By using the directed mosaic technique, clone production is limited specifically to the somatic cells, thereby demonstrating that the activity of the JAK pathway is required in the follicle cells. Mosaic analysis also demonstrates that the adoption of proper epithelial cell fates requires JAK activity (McGregor, 2002).
All follicle cell subpopulations in an egg are derived from approximately three stem cells in the germarium of each ovariole. While still in the germarium, a common pool of distinct stalk and polar cell precursors is set aside from the epithelial follicle cells. Those precursors then differentiate into either stalk or polar cells. The remaining epithelial cells are pre-patterned with mirror image symmetry along the anteroposterior axis, with three distinct subpopulations at each end. The symmetry is broken at stage 6 when Gurken in the oocyte stimulates EGF receptor in the posterior terminal cells to determine posterior polarity of the egg. The three anterior terminal cell populations then become border cells, stretched (nurse cell-associated) cells, and centripetal cells. The posterior terminal cells are essential for the reorganization of the cytoskeleton in the oocyte. Those cells send an unknown signal to the germline that stimulates the reversal of microtubular polarity in the egg which is necessary for the migration of the oocyte nucleus to the anterior and for the correct localization of polarity determinants in the egg (McGregor, 2002).
Loss of JAK pathway signaling clearly influences the terminal fate of the stalk/polar cell precursors. In heteroallelic mutant combinations of hop, the number of polar cells increases while the number of stalk cells decreases. However, the sum of stalk cells plus polar cells remains approximately the same as in wild type, indicating that loss of JAK signaling is not influencing proliferation of the precursor pool, nor is it causing recruitment of epithelial follicle cells to a polar fate. This suggests a model in which the normal function of the JAK pathway is to promote the adoption of stalk cell fate in a subset of the stalk/polar cell precursor pool. JAK pathway activation may either instruct the adoption of stalk cell fates or prevent the adoption of polar cell fate. Current data do not distinguish between these alternatives (McGregor, 2002).
A second role for JAK signaling in the follicle cells was highlighted by analysis of mosaics. In chambers of the vitellarium, the immature cell marker Fas III is rapidly downregulated in all but the polar cells. However, the epithelial follicle cells do not begin to express markers of terminal differentiation until stage 7. Indeed, these cells continue to proliferate through stage 6. Nonetheless, the loss of Fas III in the epithelial cells beginning around stage 2 suggests that the identity of these cells has already begun to change. Presumably they become preliminarily committed to an epithelial follicle cell fate. In hop or Stat92E mutant clones, younger chambers retain high levels of Fas III in all the mutant cells. In more mature egg chambers (stage 7 or later) there is a consistent lack of Fas III expansion in mutant cells. The transient nature of the increase in Fas III expression suggests that the mutant cells remain in an immature state until later stages. In this model, JAK pathway activity would be necessary for the preliminary commitment step in epithelial cell differentiation that occurs after the egg chamber pinches off from the germarium. At approximately stage 7, the normal stage for terminal differentiation, the Fas III-positive JAK mutant cells lose Fas III expression, presumably because they are cued to differentiate by another signal. The consequence of loss of JAK signaling on terminal epithelial cell fates remains to be investigated (McGregor, 2002).
Several signaling pathways have been implicated in the patterning of the follicular epithelium. The best characterized are the Notch, EGFR and Hedgehog pathways. In the earliest of these activities, strong expression of hh in the terminal filament and cap cells at the anterior tip of the germarium stimulates the proliferation of the somatic stem cells. Loss of Hh signaling results in reduced follicle cell number and consequent failure to properly encapsulate the germline cyst. Recent work has demonstrated that the normal role of Hh in the ovaries is as a somatic stem cell factor and that it is necessary for the proliferation of somatic stem cells (McGregor, 2002).
After Hh activity promotes the production of a pool of follicular precursors, the stalk/polar cell precursor pool is set aside from the epithelial cell pool. The stalk/polar cell precursor pool is distinct from the epithelial pool because it ceases to proliferate as the cyst reaches the posterior end of the germarium. The method by which the stalk/polar cell precursors are determined is not known, but it has been suggested that Notch signaling, enhanced by localized Fringe activity, may be involved in the process. Similar to JAK mutants, the loss of Notch activity causes chamber fusions that are apparently the result of a failure to produce stalk cells. But unlike JAK mutants, N pathway mutants also fail to produce polar cells. Therefore, N signaling is required for the differentiation of both polar and stalk cell fates (McGregor, 2002).
So what distinguishes stalk and polar cells from one another? JAK signaling induces the adoption of stalk cell fates in a subset of the stalk/polar cell precursors. Loss of JAK pathway activity expands polar cells at the expense of stalk cells, while ectopic activation of the pathway causes a reduction of polar cells. Therefore, it is proposed that JAK pathway activity determines the terminal fate of stalk and polar cells. However, JAK activity is limited in assigning stalk cell fates to only competent cells, that is, the stalk/polar cell precursor pool. Thus, another activity, perhaps N signaling, is necessary to induce competence for stalk and polar fates. Alternatively, N signaling may be primarily responsible for the assignment of polar cell fates. One could imagine a mechanism of lateral inhibition, already linked to N signaling in various tissues, in which all the cells of the precursor pool have N activity, but that the signal becomes limited to and maintained only in the polar cells. It may be the activity of the N pathway that then drives stable expression of upd and allows the induction of stalk cell fates in neighboring cells (McGregor, 2002).
While polar and stalk cell fates are adopted as chambers exit the germarium, differentiation of the epithelial follicle cell fates is not obvious until later. At approximately stage 7, epithelial follicle cells express markers for each of the terminal identities with a clear anterior-posterior orientation. But in the absence of Grk/EGFR signaling at the posterior, a symmetrical mirror image pattern of three terminal populations of epithelial fates at each end is revealed. In wild-type ovaries, up to approximately stage 6, the oocyte signals to the overlying posterior follicle cells through Gurken. The terminal follicle cells that receive the Grk signal are induced to become posterior follicle cells. The resulting posterior follicle cells then signal to the oocyte to stimulate a cytoskeletal rearrangement. The resulting microtubular polarity drives the migration of the oocyte nucleus from the posterior to the anterior and establishes the AP axis that allows the sequestration of anterior and posterior maternal products to their respective poles. The signal from the soma for polarization of the oocyte microtubules is not yet known (McGregor, 2002).
When the developing cyst exits the germarium, there is a distinct change in the epithelial cell precursors. The level of Fas III, a marker for immature follicle cells, is rapidly reduced in all epithelial cell precursors. However, these cells do not begin to express markers for new cell identities until around stage 7. Therefore, it seems that the epithelial cells become committed to a fate early in the vitellarium, but do not terminally differentiate until later. This is consistent with the fact that the epithelial follicle cells continue to divide until stage 6. Furthermore, Grk/EGFR signaling does not impose posterior identity on epithelial cells until stage 6. So the loss of Fas III in epithelial cell precursors in the early vitellarium marks an intermediate step in specific epithelial identities. JAK signaling is involved in this step, because clones of JAK pathway mutations cause the persistence of Fas III in epithelial cell precursors in the early vitellarium. The normal loss of Fas III expression in epithelial precursors of the early vitellarium may indicate the establishment of a pre-pattern of epithelial identities determined by JAK signaling. It is attractive to speculate such a role because the secreted JAK pathway ligand Upd is expressed symmetrically at the termini of the chamber. It is easy to envision a scheme in which the strength of the Upd signal received by the epithelial cell precursors determines the ultimate epithelial identity. However, these epithelial cells would remain in a proliferative, undifferentiated program until stage 7. The event that allows terminal differentiation is unclear, but could also be a N signal, as suggested above for competence of stalk and polar cells. This is consistent with the report of a pulse of Delta protein, a N ligand, that occurs at stages 5-7. Additional work will determine whether JAK signaling is instructive for specific epithelial fates (McGregor, 2002).
The anterior-posterior axis of Drosophila becomes polarized early in oogenesis, when the oocyte moves to the posterior of the germline cyst because it preferentially adheres to posterior follicle cells. The source of this asymmetry is unclear, however, since anterior and posterior follicle cells are equivalent until midoogenesis, when Gurken signaling from the oocyte induces posterior fate. Asymmetry is shown to arise because each cyst polarizes the next cyst through a series of posterior to anterior inductions. Delta signaling from the older cyst induces the anterior polar follicle cells, the anterior polar cells signal through the JAK/STAT pathway to induce the formation of the stalk between adjacent cysts, and the stalk polarizes the younger anterior cyst by inducing the shape change and preferential adhesion that positions the oocyte at the posterior. The anterior-posterior axis is therefore established by a relay mechanism, which propagates polarity from one cyst to the next (Torres, 2003).
The follicle stem cells reside in region 2b of the germarium and give rise to two distinct lineages: the epithelial follicle cell precursors, which proliferate until stage 6 to generate most of the cells that surround each cyst, and the polar/stalk precursors. The latter exit mitosis at stage 1 to 2 of oogenesis and give rise to the symmetric pairs of polar cells at the anterior and posterior poles of the cyst and to the stalk that separates each cyst from the adjacent one. Delta mutant germline clones and Notch follicle cell clones fail to form polar cells, indicating that Delta signals from the germline to activate the Notch receptor in the polar/stalk precursors to induce them to adopt the polar cell fate. This induction requires fringe, which is upregulated in the polar/stalk precursors and renders these precursors competent to respond to the Delta signal. Once the polar cells are specified, they express Unpaired, the ligand for the JAK/STAT pathway, and the resultant activation of JAK/STAT signaling plays two key roles in patterning the rest of the follicle cells. (1) The polar cells induce uncommitted polar/stalk cell precursors to become stalk cells. Overexpression of Unpaired causes all polar/stalk cell precursors to differentiate as stalk, whereas loss-of-function mutations in hopscotch (JAK) or STAT92E cause a loss of the stalk. (2) Unpaired signaling from the polar cells induces the adjacent epithelial follicle cells at each pole of the egg chamber to adopt a terminal fate. This induction is essential for axis formation because only the terminal cells are competent to respond to Gurken by becoming posterior. Unpaired also acts as a morphogen to specify three distinct terminal cell types at the anterior: the border cells, the stretched follicle cells, and the centripetal cells. In the absence of Gurken signaling, all three cell types also form at the posterior of the egg chamber, indicating that the graded activity of JAK/STAT pathway creates a symmetric prepattern at both poles (Torres, 2003 and references therein).
X-linked signal elements (XSEs) communicate the dose of X chromosomes to the regulatory-switch gene Sex-lethal (Sxl) during Drosophila sex determination. Unequal XSE expression in precellular XX and XY nuclei ensures that only XX embryos will activate the establishment promoter, SxlPe, to produce a pulse of the RNA-binding protein, SXL. Once XSE protein concentrations have been assessed, SxlPe is inactivated and the maintenance promoter, SxlPm, is turned on in both sexes; however, only in females is SXL present to direct the SxlPm-derived transcripts to be spliced into functional mRNA. Thereafter, Sxl is maintained in the on state by positive autoregulatory RNA splicing. Once set in the stable on (female) or off (male) state, Sxl controls somatic sexual development through control of downstream effectors of sexual differentiation and dosage compensation. Most XSEs encode transcription factors that bind SxlPe, but the XSE unpaired (upd) encodes a secreted ligand for the JAK/STAT pathway. Although STAT directly regulates SxlPe, it is dispensable for promoter activation. Instead, JAK/STAT is needed to maintain high-level SxlPe expression in order to ensure Sxl autoregulation in XX embryos. Thus, upd is a unique XSE that augments, rather than defines, the initial sex-determination signal (Avila, 2007).
The question of how embryos differentiate between precise 2-fold differences in X-linked signal element (XSE) doses is central to understanding how genetic constitution defines sexual fate. Current X-chromosome-counting models posit that the female fate is set when XSE proteins exceed a threshold concentration and activate SxlPe. The XSE threshold is set by interactions between the XSEs and other proteins in the embryo. Some XSEs interact with maternally supplied proteins to form dose-sensitive transcription factors, such as Scute/Daughterless, that bind SxlPe, but XSE doses are also assessed with reference to maternally and zygotically expressed repressors. Three XSE proteins, SisA, Scute, and Runt, are viewed as acting similarly by binding directly to and activating SxlPe. The fourth XSE, unpaired (upd, also called outstretched or sisC), encodes a secreted ligand that signals through the JAK kinase (hopscotch) to activate the Stat92E transcription factor. Although upd meets the criteria of an XSE, its effects on sex determination are weaker than those of sisA, scute, and runt, and changes in its gene dose have only moderate effects on Sxl. To understand how this comparatively dose-insensitive XSE regulates sex, when and where upd, JAK, and STAT act on the Sxl switch was examined (Avila, 2007).
Using in situ hybridization, the early embryonic expression pattern of upd was defined. No evidence was found for maternally supplied transcripts and it was observed that upd mRNA was first detectable in nuclear cycle 13. The fact that the first upd transcripts are present throughout the embryo, including at the poles, is consistent with the distribution of phosphorylated Stat92E. As cellularization progresses past early cycle 14, the upd pattern resolves into indistinct stripes that developed into a 14 stripe pattern during gastrulation. These results show that upd expression begins later than that of the other XSEs (sisA in cycle 8; scute in cycle 9) and also, paradoxically, that it begins after the onset of transcription of its target, Sxl, in cycle 12 (Avila, 2007).
To understand how upd functions in Sxl activation and how it differs from other XSEs, upd mutations were examined for their effects on SxlPe by using in situ hybridization and on Sxl protein levels by using immunostaining with SXL antibody. Significantly, the RNA probes detected nascent Sxl transcripts, allowing monitoring of both the spatial and temporal responses of SxlPe on a cell-cycle by cell-cycle basis (Avila, 2007).
updsisC1, a loss-of-function mutation that appears to specifically affect sex determination was examined, because it has no observable effect on later upd functions. Consistent with the fact that upd has a modest effect on SxlPe, it was found that two-thirds of homozygous updsisC embryos expressed SxlPe in a manner indistinguishable from that of the wild-type. A small proportion of embryos, 15%, had within their middle sections several clusters of 5-15 nuclei that did not express SxlPe, whereas the remaining 18% had severe defects, with SxlPe expression being absent from most of the central regions of the embryos. Despite early aberrations in SxlPe activity, immunostaining revealed no lasting defect in the expression of SXL, because updsisC1 embryos that reached germband extension stained in a 1:1 male:female ratio. To determine the effects of a complete loss of zygotic upd activity, updYC43, a probable null mutation, and the deficiency Df(1)ue69, which deletes upd and the upd-like gene, upd3, were examined. With respect to SxlPe, it was found that upd-null-mutant females were more severely affected than were updsisC1 embryos. At cellularization, the defects ranged from embryos containing large clusters of nuclei that did not express SxlPe in the central part of embryo to those in which the entire central region failed to express the promoter. The poles, however, expressed SxlPe normally. Immunostainings of updYC43 and Df(1)ue69 embryo collections revealed that these alleles had strong but incompletely penetrant effects on the later distribution of SXL. The fact that an estimated 40% of mutant female embryos stage 6 and older failed to express SXL in their central regions is consistent with the observed defects in SxlPe activity. The remainder eventually expressed normal levels of SXL in all their tissues, indicating that most upd mutant females were able to compensate for reduced SxlPe activity and ultimately engaged autoregulatory Sxl mRNA splicing. Two upd-like genes, upd2 and upd3, map adjacent to upd. Loss of zygotic upd2 had no effect on SxlPe, and the effects of Df(1)ue69 (upd3-,upd-) appeared identical to those of updYC43 when analyzed in a common genetic background. This shows that XSE activity in this region of the X is due to upd alone (Avila, 2007).
Except for the ligands, each component of the JAK/STAT pathway is maternally deposited into the embryo. To eliminate JAK/STAT activity completely, the dominant female-sterile technique was used to generate females lacking maternal hopscotch (hop) or Stat92E, which encode the only JAK kinase and STAT in Drosophila. It was expected that by removing maternal hop, STAT would remain unphosphoryated, allowing a determination of the effects of the loss of the entire pathway on SxlPe (Avila, 2007).
When Sxl expression was examined in cycle 14 embryos derived from hopC111 germline clones, it was found that SxlPe was active in the anterior and posterior regions of the embryos but almost completely inactive in the central region of the embryos. In contrast to the results with upd mutants and deficiencies, all of which exhibited considerable embryo-to-embryo variation, loss of maternal hop had nearly identical effects on SxlPe in every embryo. This more potent effect of maternal hopC111 as compared to upd mutants suggests that zygotic Upd might not be the only activator of JAK in the precellular embryo (Avila, 2007).
The findings with hopC111 were confirmed by using the Stat92E06346 mutation. Cycle 14 embryos derived from Stat92E06346 germline clones also lacked nearly all SxlPe expression in their central regions, but they were even more strongly affected than hopC111 females because SxlPe activity was also reduced in the termini. These findings are contrary to predictions of a linear JAK/STAT pathway going from zygotic Upd through receptor and kinase to activated STAT. Instead, the progressive weakening of SxlPe by removal of upd and Stat92E suggests that there is hop-independent control of Stat92E function in sex determination. The possibility of cross-talk between signaling systems is supported by the finding that the torso receptor-tyrosine-kinase pathway activates STAT92E in the embryo termini (Avila, 2007).
Although the hopC111 and Stat92E06346 mutations had large effects on SxlPe during cycle 14, the period of maximum SxlPe expression, it was found that these mutations had little effect on SxlPe at earlier stages. In wild-type females, SxlPe is first activated in cycle 12. Expression increases throughout cycle 13 and reaches a peak in the first minutes of cycle 14. In embryos from hopC111 mothers, SxlPe was expressed as in the wild-type during cycles 12 and 13. However, upon entry into cycle 14, SxlPe activity ceased in the middle sections of the embryos. A similar phenomenon was observed in embryos carrying strong upd mutants and in those derived from Stat92E06346 germline clones. These results show that JAK/STAT, and thus upd XSE function, is not needed for the initial activation of SxlPe. Instead, upd must function as a different kind of XSE: one dispensable for the initial assessment of X-chromosome dose, but needed to maintain SxlPe activity in the final stage of the X-counting process (Avila, 2007).
When the progeny of hopC111 mutant mothers were examined for Sxl protein, it was found that defects in SxlPe expression led to a permanent failure to express SXL in the central regions in 35% of female embryos. This suggests that the loss of SxlPe activity in cycle 14 can reduce the level of early Sxl to below the threshold normally required to activate autoregulatory mRNA splicing. Although 35% of female embryos were defective for later Sxl expression, most females that completed gastrulation expressed Sxl uniformly. This striking discordance between the effects of hop (and upd and Stat92E) mutants on SxlPe activity and ultimate Sxl levels suggests that stable Sxl autoregulation can be established even when SxlPe function has been seriously compromised. Although some rescuing Sxl mRNA or protein may have diffused from the poles, an alternative explanation is that expression of SxlPe during cycles 12 and 13 might often have provided sufficient Sxl to trigger autoregulation once the maintenance promoter, SxlPm, had been activated (Avila, 2007).
SxlPe is thought to have two main functional elements: a proximal 390 bp X-counting region responsible for sex-specific activation, and a more distal (to -1.4 kb) element that elevates Sxl transcription. Three predicted STAT-binding sites are located in these elements at positions -253, -393, and -428 bp. To test their roles, consensus TTC sequences were changed to TTT because such changes block binding by STAT92E and the mammalian homologs STATs 5 and 5a. In situ hybridizations revealed that the mutation in the proximal STAT site, S1, greatly reduced the number of nuclei expressing SxlPe-lacZ, creating a patchy staining pattern and lower overall mRNA levels. Mutations in S1 and S2, or in all three sites together, caused a strong but variable loss of SxlPe-lacZ expression in most nuclei, resulting in dramatically reduced accumulation of lacZ mRNA. Although the S1, S2, S3 mutant appeared to have a slightly stronger effect than the double mutant, both transgenes exhibited phenotypes reminiscent of those seen in embryos derived from Stat92E06346 germline clones. These results show that STAT92E acts through the consensus binding sites at SxlPe (Avila, 2007).
SxlPe is remarkable for both its rapid response and exquisite sensitivity to X-chromosome dose. In male embryos, it is always off. In female embryos, SxlPe is strongly expressed, but only during a 35-40 min period from mid cycle 12 until about 10-15 min into cycle 14. Given these time constraints, many have assumed that all XSEs would function to establish the initial on or off state of SxlPe. However, it was found that upd behaved very differently than sisA and scute, both of which are required for SxlPe activation and expression. Loss of upd or the JAK/STAT pathway had little or no effect on SxlPe during cycles 12 or 13. Instead, JAK/STAT mutations blocked SxlPe expression late in the process, during cycle 14. This observation is interpreted as revealing that SxlPe is regulated in two mechanistically distinct phases: the first controlling the initial response to X-chromosome dose, and the second acting to maintain or reinforce the initial decision (Avila, 2007).
The relatively late actions of upd and hop offer explanations for several puzzling aspects of upd's function in sex determination. First, upd is considered a weak XSE. This is both because Sxl is comparatively insensitive to upd dose and because loss of upd or JAK/STAT function doesn't eliminate Sxl expression. Both effects are consistent with expectations of a two-step, initiation and maintenance, model for SxlPe function. JAK/STAT mutations would not be expected to eliminate all Sxl function in a two-step model because the STAT-independent initiation step would produce Sxl mRNA and protein. The exact gene dose of upd would not be particularly important for sex because excess active STAT could not induce SxlPe without the prior actions of the initiating XSEs and because even a single dose of upd+ could provide enough active STAT to augment an earlier decision to become female. Thus, the proposed STAT maintenance function explains both the failure of the constitutively active hoptum-l allele to induce ectopic SxlPe expression in males and the ability of hoptum-l to further stimulate SxlPe activity in females. Likewise, the requirement for STAT site S2, located just distal to the 390 bp X-counting region of SxlPe, and the finding that upd is first expressed after Sxl can be explained if STAT's role is to bolster transcription from SxlPe in embryos that already have counted two Xs. Although neither essential for SxlPe expression nor highly dose sensitive, upd, hop, and Stat92E nonetheless play important roles at SxlPe. In their absence, the period of SxlPe activity is cut short, reducing the concentration of Sxl and preventing a large fraction of embryos from engaging the maintenance mode of Sxl expression (Avila, 2007).
How might STAT92E function in a two-step model? One possibility is that STAT might antagonize the late-acting repressor Dpn. Alternatively, the STAT transcription factor might augment, stabilize, or replace earlier-acting XSE activator complexes as their concentrations diminish in cycle 14. BAP60, a core component of the Brahma chromatin-remodeling complex, has been shown to interact with two components of the sex-determination signal. If STAT92E also interacts with the Brahma complex, it might maintain SxlPe chromatin in an active state, facilitating the restoration of transcription after the 13th mitosis (Avila, 2007).
Understanding the commonalities and unique mechanisms STATs employ in their multitude of roles is a fundamental goal of research on this ubiquitous signaling pathway. It is also essential for understanding why the pathway has so often been co-opted into new roles during evolution. STATS seem primarily permissive rather than instructive. They are rarely the primary signals defining cell fate. In these respects, comparison of the even-skipped (eve) stripe 3 enhancer and SxlPe reveals interesting parallels. Both SxlPe and eve stripe 3 are regulated by the balance between several activators and repressors. The responses of both elements to JAK/STAT signaling are extremely rapid, occurring within the dynamic environment of the precellular embryo. Stat92E is important for each, but its roles augment the actions of other factors, rather than being responsible for defining the initiating signals (Avila, 2007).
With respect to the evolution of the sex signal, it has been proposed that a diffusible JAK/STAT signal might have been recruited to allow non linear signal amplification or, alternatively, that a diffusible ligand might render SxlPe less sensitive to random fluctuations in cell-autonomous XSE protein concentrations. Although the weak dose dependence of upd argues against signal amplification, a buffering function is consistent with existing data. These findings suggest another possibility. STAT proteins respond rapidly to a range of regulatory signals; it may be this ability to act within a matter of minutes that brought JAK/STAT into the temporally dynamic X-chromosome-counting process (Avila, 2007).
Rearrangement of cells constrained within an epithelium is a key process that contributes to tubular morphogenesis. Activation in a gradient of the highly conserved JAK/STAT pathway is essential for orienting the cell rearrangement that drives elongation of a genetically tractable model. Using loss-of-function and gain-of-function experiments, it has been shown that the components of the pathway from ligand to the activated transcriptional regulator STAT are required for cell rearrangement in the Drosophila embryonic hindgut. The difference in effect between localized expression of ligand (Unpaired) and dominant active JAK (Hopscotch) demonstrates that the ligand plays a cell non-autonomous role in hindgut cell rearrangement. Taken together with the appearance of STAT92E in a gradient in the hindgut epithelium, these results support a model in which an anteroposterior gradient of ligand results in a gradient of activated STAT. These results provide the first example in which JAK/STAT signaling plays a required role in orienting cell rearrangement that elongates an epithelium (Johansen, 2003).
upd, encoding the ligand for the Drosophila JAK/STAT
pathway, is expressed only in the small intestine and is regulated by
genes controlling hindgut cell rearrangement. In drm and
bowl mutants, expression of upd is missing from the small
intestine, while in lin mutants, upd expression is expanded throughout much of the hindgut. These
results raise the possibility that localized Upd might provide an orienting
cue for rearranging hindgut cells (Johansen, 2003).
If it plays a role in hindgut cell rearrangement, upd must be
expressed before and during the period of major hindgut elongation, i.e.
between stages 11 and 16; genes encoding the other known components of the
Drosophila JAK/STAT signaling pathway should also be
expressed at the same stages, both within and adjacent to
upd-expressing cells. In situ hybridization was used to characterize
the expression of upd, dome, hop and Stat92E during stages
just prior to and during hindgut elongation (Johansen, 2003).
Expression of upd in the hindgut is first detected at stage 9 in a
narrow ring of cells that will become the small intestine. Expression in the
prospective small intestine is maintained during stages 10 and 11, where it can be seen
just posterior to the everting renal tubules (note that in the hindgut at
these germband-extended stages, 'posterior' is toward the head). During stages
12-14, when the hindgut undergoes a major part of its elongation, upd
expression is seen throughout the now distinct small intestine. Expression of
upd is maintained throughout the small intestine during the remainder
of embryogenesis (Johansen, 2003).
The Janus kinase hop is expressed uniformly throughout the embryo,
including the hindgut as it elongates. Expression of both the receptor-encoding gene dome and Stat92E is detected weakly at
the anterior of the hindgut beginning at stage 9; it becomes significantly
stronger by stage 11, and is maintained through stage 14. For both the
receptor- and STAT-encoding genes, expression domains in the hindgut
epithelium overlap with and extend beyond the narrow domain of upd
expression.
Most significantly, expression of dome and Stat92E extends
to a more posterior position in the hindgut epithelium than does expression of
upd. Thus, the mRNA expression of the ligand, receptor and STAT
components in the hindgut prior to and during its elongation is consistent
with a role for JAK/STAT signaling in hindgut cell rearrangement (Johansen, 2003).
Elongation of the Drosophila hindgut by cell
rearrangement requires the Upd ligand and the JAK/STAT pathway components Dome
(receptor), Hop (JAK) and Stat92E. Since elongation does not occur when
expression of ligand or activation of the pathway is uniform, but only when
the source of ligand is localized to the hindgut anterior, the requirement for
localized JAK/STAT signaling in hindgut elongation can be characterized as
instructive, rather than permissive. Since patterning is normal in hindguts both
lacking and uniformly expressing upd, the required role of JAK/STAT
signaling in hindgut morphogenesis is likely via direct effects on cell
movement (Johansen, 2003).
The rescue of the upd phenotype by anteriorly localized expression
in the hindgut of upd, but not of activated JAK (Hopscotch),
demonstrates that there is a requirement for upd function that is not
cell autonomous. In other words, upd is required in cells (those of
the large intestine that undergo the greatest rearrangement) that are different from
cells that produce it (those of the small intestine). A number of examples
have been described in which localized expression of a signaling molecule
(including Upd) is required non-autonomously for cell rearrangement,
morphogenesis or motility. In the Drosophila eye imaginal disc,
expression of Upd at the midline is required to establish a dorsoventral
polarity that orients ommatidial rotation. In
both Drosophila tracheae and the vertebrate lung, branching
morphogenesis of the epithelium depends on localized expression of FGF in
adjacent mesenchyme (Johansen, 2003).
Localized activation of JAK/STAT signaling has been shown to play a role in
cell motility in a number of contexts. In Drosophila, localized
expression of Upd in the anterior polar cells of the egg chamber acts to
coordinate the migration of the adjacent border cells. In
mammals, cytokines expressed in target tissues act to attract both migrating
lymphocytes and tumor. The finding that localized (only in the small intestine)
expression of upd is both necessary and sufficient for rearrangement
of cells in the large intestine indicates that Upd must have an
organizational, action-at-a-distance function in controlling cell
rearrangement during tubular morphogenesis (Johansen, 2003).
Rescue experiments establish that there is a cell
non-autonomous requirement for upd in hindgut elongation. Consistent
with this, there is evidence that Upd is present and required in an
anteroposterior gradient in the hindgut. Prior to and during hindgut
elongation, both Stat92E mRNA and Stat92E protein are detected not
only in the small intestine epithelium (and the visceral mesoderm surrounding
the small intestine), but also in the epithelium posterior to the small
intestine; this expression of Stat92E appears to be in a gradient. In the
Drosophila eye imaginal disc, a gradient of Upd is required to orient
the rotation of ommatidial cell clusters; in addition, there is evidence for a gradient of Upd and Stat92E in patterning of the follicular epithelium of the Drosophila egg chamber. Since expression of Stat92E depends on upd, it is likely that Upd protein is present in the hindgut epithelium as an anteroposterior gradient, with its highest level in the upd-expressing cells of the small intestine, and lowest level in posterior, upd non-expressing cells of the large intestine. Expression of SOCS36E (suppressor of cytokine signaling at 36E), which is regulated by upd, overlaps with and extends significantly beyond the domain of upd expression,
further supporting the idea that there is a gradient of Upd in the hindgut (Johansen, 2003).
In the Drosophila eye imaginal disc, anti-Upd staining and the
behavior of clones of mutant cells that have lost components of the JAK/STAT
pathway indicate that Upd is present in a gradient that extends at least 50
µm beyond its midline mRNA expression domain. In the
Drosophila hindgut, Stat92E is a reliable reporter
for the presence of Upd. Two to four hours after upd is first
expressed at the anterior of the hindgut (stage 9), Stat92E can be detected at
least 30-40 µm from the site of upd expression (stages 11 and 12).
These time and distance parameters are similar to those observed during
generation of the Upd gradient in the eye, and the Dpp and Wg gradients in
wing imaginal discs, which form over distances of roughly 40-80 µm in 1-8
hours. Thus, it is reasonable to imagine that a gradient of Upd is established in the
developing hindgut in a short enough time frame to affect cell rearrangement (Johansen, 2003).
The essential consequence of JAK/STAT signaling is activation of the STAT
protein, which leads to altered transcriptional programs. STAT has been
shown in a number of contexts to be required for cell motility, and
therefore probably regulates expression of genes controlling cytoskeletal
assembly and cell adhesion. In these contexts, however, activation of STAT
does not appear to be required to orient cell movement, but rather to
facilitate or promote it. As Stat92E is required for hindgut elongation, and
its protein product appears to be present in a gradient along the
anteroposterior axis, this raises the intriguing question of how a gradient of
a transcription factor might orient cell rearrangement (Johansen, 2003).
Blood cells play a crucial role in both morphogenetic and immunological processes in Drosophila, yet the factors regulating their proliferation remain largely unknown. In order to address this question, antibodies were raised against a tumorous blood cell line and an antigenic determinant was identified that marks the surface of prohemocytes and also circulating plasmatocytes in larvae. This antigen was identified as PDGF- and VEGF-receptor related, a Drosophila homolog of the mammalian receptor for platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF). The Drosophila receptor controls cell proliferation in vitro. By overexpressing in vivo one of its putative ligands, PVF2, a dramatic increase was induced in circulating hemocytes. These results identify the PDGF/VEGF receptor homolog and one of its ligands as important players in Drosophila hematopoiesis (Munier, 2002).
The distribution of the antigenic determinant on the different Drosophila hemocyte types was examined by immunocytochemistry. Wild-type and hopTum-l mutants were used for this purpose. hopTum-l is a Janus kinase (JAK) gain-of-function mutation resulting in an overproliferation of circulating blood cells, of which a large number are lamellocytes. Plasmatocytes were stained and lamellocytes were not. In hopTum-l mutants, the most strongly reacting cells were small rounded cells that correspond to circulating progenitor blood cells (prohemocytes). In wild-type larvae, the presence of prohemocytes is mostly restricted to lymph glands. Strong staining was observed on the prohemocytes of wild-type lymph glands. In hop-Tum-l lymph glands, the small rounded prohemocytes were also strongly stained, but other cells that have been described as lamellocytes but did not react with the antibody. Stained circulating crystal cells could not be observed due to their fragility. No staining was ever observed on larval tissues other than hemocytes, namely on fat body, muscles, imaginal discs, epidermal cells, brain or trachea (Munier, 2002).
In Drosophila gain-of-function alleles in the JAK Hopscotch (e.g. hopTum-l) cause overproliferation of hemocytes. This report raises the obvious question as to what kind of interconnection exists between pathway(s) activated by PVR and the JAK/STAT pathway itself. Some cross-talk could occur, such as phosphorylation of STAT by PVF2-induced PVR activation. In mammals, for instance, PDGFR can directly activate some STATs. Conversely, evidence exists that JAK can activate the D-raf/D-MEK/MAP kinase pathway, one that is frequently activated by receptor tyrosine kinases. As is the case for PVF2, however, neither JAK nor STAT seem absolutely required for blood cell proliferation. Indeed, in loss-of-function mutants of hop or stat that permit larval development, blood cell counts are normal. This leaves open the possibility that upstream components of the JAK/STAT pathway, e.g., the receptor Domeless (DOME) and its ligand Unpaired, could act in synergy with the PVF2/PVR pathway. Both DOME and Upd are implicated in embryonic pair-rule gene expression, but their role in hematopoeisis awaits investigation (Munier, 2002).
In summary, the data indicate that PVR integrates two functions shared by mammalian receptors of the same subfamily. Like its mammalian VEGFR homologs (Flt1, KDR and Flt4), it regulates cell migration; and like c-Kit, Flt-3, c-Fms and most PDGFRs, it is implicated in the control of blood cell proliferation. In the light of the importance of hemocytes in development and in the innate immune response, it would be highly relevant to investigate further the interaction between PVFs, PVR, the JAK/STAT pathway and the downstream mitogenic factors that they induce (Munier, 2002).
The nucleosome remodeling factor (NURF) is one of several ISWI-containing protein complexes that catalyze ATP-dependent nucleosome sliding and facilitate transcription of chromatin in vitro. To establish the physiological requirements of NURF, and to distinguish NURF genetically from other ISWI-containing complexes, mutations were isolated in the gene encoding the large NURF subunit, nurf301. NURF is shown to be required for transcription activation in vivo. In animals lacking NURF301, heat-shock transcription factor binding to and transcription of the hsp70 and hsp26 genes are impaired. Additionally, NURF is shown to be required for homeotic gene expression. Consistent with this, nurf301 mutants recapitulate the phenotypes of Enhancer of bithorax, a positive regulator of the Bithorax-Complex previously localized to the same genetic interval. Finally, mutants in NURF subunits exhibit neoplastic transformation of larval blood cells that causes melanotic tumors to form (Badenhorst, 2002).
During the course of this analysis it was noticed that nurf301
mutant animals display a high incidence of melanotic tumors. Melanotic tumors have previously been reported in a number of mutant backgrounds and are generally caused by neoplastic transformation of the larval blood cells. The circulating cells (hemocytes) of the larval blood or
hemolymph provide one tier of the innate immune system of insects by
encapsulating or engulfing pathogens. A number of mutations have been shown to trigger the overproliferation and premature differentiation of hemocytes. Tumors form when these cells aggregate, or invade and encapsulate normal larval tissues Badenhorst, 2002).
Melanotic tumors are observed both in EMS-induced nurf301
mutants that truncate NURF301, the P-element induced mutation that reduces nurf301 transcript levels, and allelic combinations of these mutants. Tumor penetrance is extremely high (100% for nurf3012 at 25°C). Consistent with tumor development, circulating hemocyte cell number was increased dramatically in hemolymph isolated from nurf301
mutant animals. A large percentage of animals lacking ISWI,
the catalytic subunit of NURF, also displayed melanotic tumors
confirming that disrupted NURF function induces tumor formation. In iswi mutant animals the number of circulating hemocytes is also increased. In both nurf301 and
iswi mutant hemolymph, small aggregates of hemocytes are often
observed. All hemocyte cell types are present, from small round cells
(prohemocytes) to crystal cells and lamellocytes (Badenhorst, 2002).
In Drosophila, larval blood cell transformation and melanotic
tumor formation can be induced by inappropriate activation of either of
two distinct signaling cascades: the Toll or the JAK/STAT pathway.
Inappropriate activation and nuclear-localization of the Drosophila NF-kappaB homolog Dorsal, caused either by
constitutive activation of the Toll receptor or removal of the
inhibitor, the Drosophila IkappaB Cactus, leads to melanotic tumors in third instar larvae. In the second pathway, gain-of-function mutations in Hopscotch (Hop), the Drosophila Janus Kinase (JAK), induce melanotic tumors. Hop gain-of-function mutants cause tumor development by triggering constitutive activation and DNA-binding by the Drosophila STAT transcription factor, STAT92E (Badenhorst, 2002).
To resolve whether the melanotic tumors seen in the nurf301
mutants were caused by misregulation of either the TOLL or HOP/STAT92E pathways, whether nurf301 mutants enhance tumor
phenotypes seen in constitutively active Toll or Hop mutant lines was tested. Tumor incidence in animals carrying one copy of a
gain-of-function Hop mutation -- hopTum-1
-- is increased by simultaneous reduction in NURF301
levels. In contrast, removal of
one copy of NURF301 fails to enhance the Toll gain-of-function allele
Tl10b. The results suggest that
NURF acts as a negative regulator within the Drosophila
JAK/STAT signaling pathway (Badenhorst, 2002).
Molecular signatures of both JAK and Toll activation have been defined.
It is known that Hop gain-of-function mutants induce expression of a
complement-like protein TEP1. Overactivation of
the Toll pathway also induces TEP1 synthesis but primarily induces
expression of antimicrobial peptides, including Drosomycin (Drs) and
Diptericin (Dpt). Loss of nurf301 induces
tep1 but fails to induce drs or dpt, demonstrating that NURF301 principally affects the Hop/STAT92E
pathway. Whether nurf301 interacts genetically with
other known components of the Hop/STAT92E pathway was tested. Certain mutations in unpaired (upd, also known as outstretched),
which encodes a ligand for the Hop receptor, display a characteristic
wings-out phenotype, due to decreased activation of Hop and
consequently decreased STAT92E function. When NURF301 levels are
simultaneously decreased in these mutant backgrounds, animals are
mostly restored to the wild-type. These genetic interactions
confirm that NURF301 acts as a negative regulator of the Hop/STAT92E
pathway, at a point downstream of Hop. Hence, disruption of NURF could
affect either STAT92E or the targets of STAT92E. In
nurf301 mutants, levels of the STAT92E transcription factor
are not elevated, suggesting that NURF acts to repress the activity of STAT92E or the expression of some STAT92E target genes (Badenhorst, 2002).
Striking similarities continue to emerge between the mammalian and Drosophila JAK/STAT signaling pathway. However, until now there has not been the ability to monitor global pathway activity during development. A transgenic animal was generated with a JAK/STAT responsive reporter gene that can be used to monitor pathway activation in whole Drosophila embryos. Expression of the lacZ reporter regulated by STAT92E binding sites can be detected throughout embryogenesis, and is responsive to the Janus Kinase hopscotch and the ligand Upd. The system has enabled identification of the effect of a predicted gene related to upd, designated upd2, whose expression initiates during germ band extension. The stimulatory effect of upd2 on the JAK/STAT reporter can also be demonstrated in Drosophila tissue culture cells. This reporter system will benefit future investigations of JAK/STAT signaling modulators both in whole animals and tissue culture (Gilbert, 2005; full text of article).
To visualize global JAK/STAT pathway activation in the whole animal a transgenic Drosophila line containing the lacZ gene regulated by STAT DNA binding sequences. This construct included three STAT target sites (GAS) upstream of a minimal Drosophila heat shock promoter in the pCaSpeR.hsp.bas vector. This reporter gene and the Drosophila line is referred to as (GAS)3-lacZ. Expression during embryogenesis is strikingly dynamic (Gilbert, 2005).
To evaluate expression of the reporter gene, in situ hybridization was performed to detect the lacZ mRNA transcript in homozygous (GAS)3-lacZ embryos during various stages of development. Expression of the lacZ gene is not detectable in syncytial blastoderm embryos. However, at the onset of cellularization, lacZ mRNA is detected throughout the embryo with strongest expression in the ventral region. Just prior to gastrulation, expression becomes more spatially restricted. At the onset of gastrulation, an intense lacZ signal is detected in a broad region anterior to the presumptive cephalic furrow and invaginating presumptive mesoderm. As germ band extension proceeds, expression is reduced, but reappears by early stage 9 in the head region and as a weak 14 stripe pattern. By stage 10 this pattern resolves into strong expression in 14 parasegments. The lacZ signal then recedes and is detected in small clusters of segmentally repeated cells. These data indicate that STAT binding sites within a promoter context can drive expression of a reporter gene in the Drosophila embryo. Homozygous Drosophila were also generated that contain a lacZ transgene regulated by a single GAS element. The (GAS)1-lacZ embryos exhibited a similar pattern of gene expression but with significantly weaker intensity (Gilbert, 2005).
To ensure that expression of the reporter system was in fact responsive to JAK/STAT activity, (GAS)3-lacZ expression was evaluated in embryos lacking maternal hop. These hop embryos have also been shown to exhibit reduced STAT92E protein levels. Maternal hop was removed using the FLP-DFS technique to generate females with hopC111 homozygous germ cells. These females were crossed to (GAS)3-lacZ males to generate embryos that lack maternal hop and carry a single copy of the (GAS)3-lacZ transgene. Embryos were evaluated for expression of lacZ transcript by in situ hybridization. Expression was found to be dramatically reduced in all stages examined. During early cellularization only a weak anterior signal is detected with little or no signal in the remainder of the embryo. At the onset of gastrulation, the embryos exhibited reduced expression throughout, particularly in the area corresponding to mesoderm. During germ-band extension, the expression in 14 parasegments was reduced with residual signals in small clusters of cells at the midline of the original 14 stripes. The residual activity detected may be hop independent. stat92E null alleles were also tested and showed residual activity. These results indicate that the (GAS)3-lacZ reporter system is responsive to a reduction of in vivo levels of JAK/STAT pathway components, and it was confirmed that early JAK/STAT signaling is established in cellularizing embryos in a ubiquitous manner (Gilbert, 2005).
The result suggests that the presence of Upd ligand alone is not sufficient to activate the pathway. In addition, when Upd was ectopically expressed with the paired-Gal4 driver, (GAS)3-lacZ was hyperactivated, but only in a subset of cells. These data support the finding of the inability of ectopic ligand to induce Domeless dimerization (Brown, 2003), and hence pathway activation in early embryos (Gilbert, 2005).
The removal of maternal hop by the production of germline clones had a clear inhibitory effect on the establishment of (GAS)3-lacZ expression in early cellularized embryos, gastrulation, and the maintenance of reporter expression during germ-band extension. Since loss of maternal hop has been shown to negatively regulate STAT92E protein levels, it was expected that this loss of function allele would cause the strongest loss of (GAS)3-lacZ expression. However, all three stages displayed some residual expression. Given that the hopC111 mutation in the germ line clone corresponds to a small internal deletion, it is possible that the residual reporter expression is due to low activity of a mutant Hop protein. The possiblility that residual expression is due to the activity of an unknown DNA binding factor that can interact with the reporter gene cannot be ruled out. It is also possible that there is minimal but constitutive activity of the promoter used in the construction of the (GAS)3-lacZ gene. The presence of a CAAT box and a TATA box could facilitate a low level of expression by the basal transcriptional machinery (Gilbert, 2005).
The contribution of upd, upd3 and upd2 to JAK/STAT signaling during embryo development was also evaluated. The removal of upd and upd3 or upd, upd3 and upd2 significantly decreased pathway activity. The effect was similar to the removal of maternal hop during the establishment of (GAS)3-lacZ expression in cellularized embryos. The earliest developmental stage examined for both deficiencies is slightly later than that examined for hop embryos. Given the slight difference in staging, the residual expression is similar, consisting of a weak head stripe and 2 weak stripes in the trunk. The maintenance of (GAS)3-lacZ expression in germ-band extended embryos was also analyzed. The removal of both upd, upd3 and upd2 had a more severe effect on reporter gene expression than removal of upd and upd3 alone. This increase in severity was manifest as a reduction in the number of expressing cells within the segmentally repeated cell clusters (Gilbert, 2005).
These studies performed both in vivo and in Drosophila tissue culture cells provide evidence that Upd2 can act to stimulate activation of the JAK/STAT pathway. Since the expression pattern of both upd and upd2 is similar during germ-band extension, it is possible that they serve certain biologically redundant functions similar to the manner in which IL-6 cytokines function in mammalian systems. These are pleiotropic cytokines that share structural similarity and functional redundancies in part due to the fact that they share a common receptor subunit. Alternatively, signaling by Upd and Upd2 may serve specific functions either in the embryo or during other stages of larval, pupal, or adult development. In monitoring upd2 expression by Western blot analysis, multiple isoforms were detected that may indicate post-translational modifications. The nature of the Upd2 proteins remains to be characterized and could provide insight on additional levels of signaling specificity and receptor binding. Upd2 may not be associated with the extracellular matrix like Upd and thereby able to act at a greater distance from its production to influence gene expression. The in vitro studies in Drosophila S2 cells clearly demonstrated the ability of Upd2 to stimulate specific expression of (GAS)3-lacZ (Gilbert, 2005).
This report provides evidence that JAK/STAT signaling can be monitored in vivo using the lacZ reporter gene regulated by STAT DNA binding elements. A complementary assay has recently been developed to monitor the pattern of STAT92E phosphorylation in the embryo, however this method of detecting lacZ by in situ hybridization provides a highly sensitive assay with little background to detect pathway activation from the cell surface receptor to gene expression in the nucleus. In addition, dynamic expression of the reporter can be visualized by a simple X-gal staining of whole embryos and remains sensitive enough to allow detection of changes in reporter activity in response to the removal and ectopic expression of pathway components. This capability could facilitate a genetic screen for enhancers or suppressors of JAK/STAT pathway activity during specific developmental stages. In addition, since (GAS)3-lacZ can be used to monitor JAK/STAT activation in tissue culture cells, this could facilitate a screen in tissue culture cells as well as providing a method of verifying screen-based genetic interactions. The reporter line should also be useful to characterize JAK/STAT function during later developmental stages. Preliminary experiments with X-gal staining of (GAS)3-lacZ third instar larval structures revealed β-galactosidase activity in a subset of structures known to require or possess competence for JAK/STAT signaling (Gilbert, 2005).
Until now direct monitoring of JAK/STAT pathway activation has only been possible in tissue culture cells. The establishment of an in vivo monitor of JAK/STAT pathway activation will provide an indispensable tool for the discovery of interacting proteins and tissue-specific requirements during Drosophila development. This assay can be used to visualize pathway activation and identify novel regulators of JAK/STAT signaling during embryogenesis and the isolation of a novel gene, upd2, which bears sequence homology to upd and encodes a functional ligand of the JAK/STAT pathway is specifically described. These data add to the mounting evidence that suggests the Drosophila JAK/STAT pathway is not simple, but contains multiple ligands that may act to elicit tissue and gene-specific responses (Gilbert, 2005).
The JAK/STAT pathway was first identified in mammals as a signaling mechanism central to hematopoiesis and has since been shown to exert a wide range of pleiotropic effects on multiple developmental processes. Its inappropriate activation is also implicated in the development of numerous human malignancies, especially those derived from hematopoietic lineages. The JAK/STAT signaling cascade has been conserved through evolution and although the pathway identified in Drosophila has been closely examined, the full complement of genes required to correctly transduce signaling in vivo remains to be identified. A dosage-sensitive dominant eye overgrowth phenotype caused by ectopic activation of the JAK/STAT pathway was used to screen 2267 independent, newly generated mutagenic P-element insertions. After multiple rounds of retesting, 23 interacting loci that represent genes not previously known to interact with JAK/STAT signaling have been identified. Analysis of these genes has identified three signal transduction pathways, seven potential components of the pathway itself, and six putative downstream pathway target genes. The use of forward genetics to identify loci and reverse genetic approaches to characterize them has allowed us to assemble a collection of genes whose products represent novel components and regulators of this important signal transduction cascade (Mukherjee, 2006).
Cell cycle proteins: The screen identified genes responsible for the modification of the overgrown eye phenotype associated with P{w+, GMR-updδ3'}.
The eye overgrowth induced by P{w+, GMR-updδ3'} results from additional rounds of mitosis in eye-imaginal disc cells anterior to the morphogenetic furrow. Despite the ectopic JAK/STAT pathway activation caused by the misexpression of upd, these cells are patterned essentially normally and go on to form an increased number of ommatidia in the P{w+, GMR-updδ3'} eye disc. Despite this proliferation-dependent phenotype, core cell cycle regulatory proteins failed to show consistent interactions when assayed as part of a candidate approach. While unexpected, this result suggests that the core cell cycle regulatory proteins do not represent components that become rate limiting in the proliferative environment tested (Mukherjee, 2006).
Despite the lack of interaction with core cell cycle components, alleles of did, trbls, and Mob1 were identified as modifiers of the overgrown eye phenotype. Indeed, homozygous did mutants have been described as having small imaginal discs, and a phenotype similar to that is observed in hopM13 mutant third instar larval discs. While not central to cell cycle progression, these loci appear to be involved in its regulation and may imply that the interaction between JAK/STAT signaling and cellular proliferation is indirect (Mukherjee, 2006).
Of particular interest are the inconsistent interactions observed between Cdk4 alleles. Although cdk4 represents the only Drosophila component of the cell cycle machinery proposed to interact with the JAK/STAT pathway, the assay identified only one of the three alleles tested as a weak suppressor of the eye overgrowth phenotype. Previous studies did not utilize loss-of-function experiments but rather utilized the converse approach. When misexpressed by a P{w+, GMR-Gal4} driver, the coexpression of P{w+, UAS-CycD}, P{w+, UAS-Cdk4}, and P{w+, UAS-upd} dramatically enhanced the eye overgrowth phenotype over that mediated by P{w+, UAS-upd} or P{w+, UAS-CycD} and P{w+, UAS-Cdk4} alone. Although it is possible that loss of a single copy of the cdk4 locus does not reduce protein levels below a rate-limiting threshold, the inconsistency of interactions produced by multiple cdk4 alleles is puzzling and true existence or nature of any potential interaction between JAK/STAT signaling and endogenous Cdk4 remains to be established (Mukherjee, 2006).
Transcription factors and coregulators: A number of transcription factors were identified as interacting loci in the screen. One of these is the Drosophila homolog of the nuclear factor of activated T-cells (NFAT), a locus originally identified as an inducer of cytokine gene expression. Intriguingly, it has been shown that human NFAT, in conjunction with NF-kappaB, AP-1, and STATs, represents factors involved in mediating cytokine and T-cell-receptor-induced interferon-γ signaling. Intriguingly, activation of these transcription factors results in the production of numerous intrinsic antiviral factors in the vertebrate system, a role that has also been shown to depend on JAK/STAT signaling within Drosophila fat-body cells. Although further analysis of this interaction is required, this is the first report that suggests an evolutionarily conserved link between NFAT and JAK/STAT signaling in Drosophila (Mukherjee, 2006).
C-terminal binding protein (CtBP), a transcriptional corepressor previously characterized as an enhancer of the Drosophila JAK/STAT pathway, was also identified in the screen. While not all alleles of CtBP show consistent interaction with P{w+, GMR-updδ3'}, cell culture assays utilizing dsRNA-mediated knockdown imply that CtBP is a component of the JAK/STAT pathway, which acts as a positive regulator of signaling. In addition, an independent genomewide RNAi-based screen for JAK/STAT pathway interactors also identified dsRNAs targeting CtBP as a suppressor of pathway signaling. Finally, an upregulation of CtBP transcript is observed in P{w+, GMR-updδ3'} eye discs compared to wild-type eyes. Given the results from cell-based assays and in situ analysis, it appears most likely that CtBP does indeed represent a positive regulator of JAK/STAT pathway activity. This finding is particularly surprising, given the previously identified role for CtBP as a transcriptional repressor, which, in combination with the Groucho corepressor, is involved in repressing Su(H)-mediating expression of Notch pathway target genes. The significance of this result, however, remains to be determined and it is conceivable that the observed interaction with the eye overgrowth phenotype represents an indirect effect, possibly via interaction with Notch pathway signaling activity (Mukherjee, 2006).
Extracellular proteins: One aspect of the screen undertaken is the paracrine mode of Upd signaling required for cellular overproliferation. In the P{w+, GMR-updδ3'} eye, the region of upd expression is spatially separate from the domain in which increased levels of cellular proliferation are observed and the ligand must therefore be able to move to and activate the pathway in neighboring cells. Although it has been shown that Unpaired represents a secreted extracellular signaling molecule that is both post-translationally glycosylated and able to associate with the extracellular matrix (ECM), very little is known regarding the mechanisms regulating these processes (Mukherjee, 2006).
One class of molecules previously shown to be involved in the extracellular trapping and movement of signaling ligands is the heparan sulfate proteoglycans (HSPGs) Dally, Dally-like, Perlecan, and Syndecan. These molecules, and their extensive post-translational modifications, not only play important roles in providing shape and biomechanical strength to organs and tissues, but also have been shown to be required for the transduction of signaling by the Wingless, Hedgehog, and the FGF-like ligands Heartless and Breathless. Despite the significance of HSPGs for the transduction of these ligands, mutations in the HSPGs themselves, as well as mutations in the HSPG-modifying enzymes sugarless and sulphateless, do not appear to interact with the eye overgrowth phenotypes associated with P{w+, GMR-updδ3'} and suggest that Upd is likely to interact with the ECM via different mechanisms. One potential component of this alternative mechanism identified in the screen is Tenascin-major (Ten-m). Ten-M, also known as odd Oz, encodes an extracellular adhesion molecule that was also classified as a component of the JAK/STAT pathway in the tissue-culture-based paracrine signaling assay. Although the tissue culture results imply a direct function of the molecule in pathway signaling, further analysis of the role of Ten-m in controlling the secretion and/or movement of Upd remains to be determined in vivo (Mukherjee, 2006).
Signaling pathways: The Drosophila eye is dispensable in a laboratory environment and sensitized genetic screens that compromise its function have proven to be powerful tools for the identification of signal transduction pathway components. Drosophila eye development is, however, a complex process involving multiple signal transduction pathways including EGFR, Hh, Notch, Dpp, and Wingless. A number of examples of interactions between these pathways and JAK/STAT signaling have been described. For example, a gradient of four-jointed in the developing eye disc is determined by the coordinated activities of Notch, Wingless, and JAK/STAT pathways. Also, at the posterior dorso/ventral border of the eye, Notch and eye gone (eyg) have been shown to cooperatively induce expression of upd, which then acts to promote cell proliferation. Consistent with these complex interactions, the screen identified Bunched (bun), a member of the Dpp signal transduction pathway, and Bearded (brd), a member of the Notch signaling pathway. bunched is a transcription factor that genetically interacts with dpp. Strikingly, Dpp pathway components have previously been reported as modulators of the P{w+, GMR-updδ3'} eye phenotype, with hypomorphic alleles of dpp and Mothers against dpp (Mad) representing strong suppressors of eye overgrowth. Similar interactions in mammalian systems have identified the synergistic activity of STAT3 and Smad1 in the differentiation of astrocytes from their progenitor cells. These proteins, however, do not physically interact, but bind to p300/CBP to promote the transactivation of target genes (Mukherjee, 2006).
The screen also identified mth-like8, a seven-pass trans-membrane protein with predicted G-protein-coupled receptor activity. Although expression of mth-like8 changes in response to JAK/STAT pathway activation, an in-depth analysis of its interaction remains to be undertaken (Mukherjee, 2006).
Drosophila has emerged as an important model system to discover and analyze genes controlling hematopoiesis. One regulatory network known to control hemocyte differentiation is the Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signal-transduction pathway. A constitutive activation mutation of the Janus kinase Hopscotch (hopscotchTumorous-lethal; hopTum-l) results in a leukemia-like over-proliferation of hemocytes and copious differentiation of lamellocytes during larval stages. Friend of GATA (FOG) protein U-shaped (Ush) is expressed in circulating and lymph gland hemocytes, where it plays a critical role in controlling blood cell proliferation and differentiation. These findings demonstrate that a reduction in ush function results in hematopoietic phenotypes strikingly similar to those observed in hopTum-l animals. These include lymph gland hypertrophy, increased circulating hemocyte concentration, and abundant production of lamellocytes. Forced expression of N-terminal truncated versions of Ush likewise leads to larvae with severe hematopoietic anomalies. In contrast, expression of wild-type Ush results in a strong suppression of hopTum-l phenotypes. Taken together, these findings demonstrate that U-shaped acts to control larval hemocyte proliferation and suppress lamellocyte differentiation, likely regulating hematopoietic events downstream of Hop kinase activity. Such functions appear to be facilitated through Ush interaction with the hematopoietic GATA factor Serpent (Srp) (Sorrentino, 2007).
In wild-type lymph glands, Ush is not detectable in the second instar larva (L2) but is expressed in L3, beginning in the cortical zone and eventually spreading to the entire lymph gland. It stands to reason that during the normal dispersal of the hematopoietic organs in late L3, those lymph gland hemocytes in the cortical zone will be the first to enter circulation. In such a model, Ush-expressing lymph gland hemocytes enter the circulating hemocyte population, which (with the exception of crystal cells) already express Ush. Thus Ush can be viewed as a hemocyte maturation marker. This begs the question of why Ush is expressed in what are apparently the most mature hemocytes. The current observations strongly implicate Ush as being present in order to suppress proliferation and lamellocyte differentiation among mature plasmatocytes (Sorrentino, 2007).
The strongest evidence for a mechanism in which Ush suppresses hemocyte proliferation and lamellocyte differentiation comes from analyses of ush mutants. Reducing Ush function causes lymph gland hypertrophy, which is a direct result of an increase in the number of lymph gland hemocytes. Furthermore, in a manner similar in quality (but somewhat less in intensity) to those of hopTum-l larvae, ush mutant lymph glands disperse precociously, and cortical zone hemocytes appear to be morphologically consistent with lamellocytes. Additionally, total circulating hemocyte concentration (CHC) of ush mutants is over four-fold greater than that of wild-type larvae, and two different alleles of ush when heterozygous, induce a less severe but nonetheless significant hematopoietic phenotype. High CHC is consistent with the mechanism of a large number of hemocytes, including lamellocytes, leaving the lymph gland and entering circulation. The possibility cannot be ruled out that circulating hemocytes, which are of a different embryonic origin than lymph gland hemocytes, can also over-proliferate and/or differentiate into lamellocytes in ush mutants. Since cortical zone hemocytes (predominantly plasmatocytes) express Ush and can differentiate into lamellocytes, the fact that circulating plasmatocytes also express Ush would support the notion of circulating plasmatocytes also being able to differentiate into lamellocytes (Sorrentino, 2007).
Importantly, it was observed that wild-type L2 lymph glands do not express Ush, while L3 organs do (in the cortical zone first, then throughout the lymph gland). Clearly, a mechanism that represses a developmental decision is not necessary unless a cell has the potential to actually make the choice. Thus the existence of a Ush-regulated mechanism for suppression of hemocyte proliferation and lamellocyte differentiation in L3 cortical zone hemocytes is interpreted as supportive of the hypothesis that Ush+ cells are in a different genetic state in which they can, given the proper cues, hyperproliferate and differentiate into lamellocytes. It follows that wild-type L2 lymph gland hemocytes cannot hyperproliferate and become lamellocytes. Such a putative mechanism is consistent with previous findings; Jung (2005) observed that L3 lymph gland hemocyte proliferation takes place primarily within the cortical zone, while Sorrentino (2002) observed that, in larvae parasitized by the wasp Leptopilina boulardi, L2 lymph glands are immune-unresponsive (as indicated by mitotic index, crystal cell population size, and a lamellocyte marker) whereas L3 lymph glands do respond (Sorrentino, 2007).
Additional strong evidence for the role of Ush is provided by transgene expression data. Expression of wild-type Ush in hopTum-l/Y larvae produced an effect opposite in quality to that of ushVX22/ushr24, that being a significant 90% reduction in the hopTum-l-induced circulating lamellocyte population. The CgGAL4 driver is active in hopTum-l/Y L2 hemocytes, thus transgenic Ush has an opportunity to act on hemocytes prior to lymph gland dispersal. The significant reduction could be explained by the suppression of lamellocyte differentiation and/or the suppression of the proliferation of pro-lamellocytes. An apoptotic mechanism may also be partially involved. The reason for the observation that the hopTum-l non-lamellocyte population was not significantly affected by transgenic Ush is unknown, but one explanation would be a dosage-dependent mechanism in which experimental expression of just one copy of a UASush transgene is insufficient to suppress hopTum-l over-proliferation. It is also possible that hemocyte over-proliferation is a secondary effect of lamellocyte differentiation, and thus not under the direct control of Ush (Sorrentino, 2007).
If Ush normally suppresses crystal cell differentiation why do ush mutants not exhibit a severe overabundance of crystal cells? Using the strong amorphic ush1 background, an approximately 30% increase in mean crystal cell counts has been observed in stage-16 embryos. However, since the wild-type mean number of crystal cells in stage-16 embryos is about 24-25 per embryo, a 30% increase amounts to about 8 additional crystal cells. Since there are hundreds of plasmatocytes in an embryo, the overwhelming majority of plasmatocytes do not become crystal cells in the absence of Ush (Sorrentino, 2007).
Such findings can be explained by a model in which Ush suppresses crystal cell differentiation in a small subset of hemocytes, with the primary role of Ush in hematopoiesis being to control lamellocyte differentiation and hemocyte proliferation. In this model, the down-regulation of ush expression in embryonic crystal cells occurs because hemocytes committed to the crystal cell lineage cannot become lamellocytes, and thus require no mechanism to suppress lamellocyte differentiation (Sorrentino, 2007).
Srp is expressed in all hopTum-l/Y hemocytes, both lamellocytes and non-lamellocytes, in circulation and in the lymph gland. Thus, all Drosophila hemocyte classes studied thus far express this hematopoietic GATA factor, and the role of Srp in the differentiation of all hemocyte types is worthy of investigation. The fact that the lamellocyte population observed in ushVX22/+ larvae is strongly reduced by srpneo45/+ and completely reduced to wild-type levels by srp3/+ suggests Srp plays an active role in lamellocyte differentiation. In humans, GATA-3 determines the differentiation of Th2 cells, which like lamellocytes are the primary effectors in a cellular immune response against metazoan endoparasites. FOG-1 inhibits Th2 differentiation by inhibiting GATA-3 activity via physical interaction. However, if Srp is indeed necessary for lamellocyte differentiation, the finding that Srp is expressed in all hemocytes likely means it is not the sole determinant of lamellocyte differentiation. Thus it is considered likely that an additional transcriptional regulator, either another GATA factor (e.g., Grain, dGATAd, dGATAe) or a non-GATA factor, works in conjunction with Srp to specify the lamellocyte differentiation program (Sorrentino, 2007).
Ush232-1191, Ush302-1191, and Ush365-1191 driven by CgGAL4 are dominant inducers of hematopoietic tumor phenotypes, measurably stronger than that of the ushVX22/ushr24 loss-of-function condition. This finding is not interpreted as coincidental. In an important parallel all three of these constructs, when activated by the mesodermal twiGAL4 driver, exhibit a failure to suppress the expression of a cardiac-active Dmef2-lacZ reporter gene. Additionally, it was observed that Ush232-1191, though missing zinc finger 1, is still able to bind to Srp in vitro just as it is able to bind to Pnr. Such observations are consistent with the possibility that endogenous and transgenic Ush may compete in their binding to Srp. In such a situation, transgenic Ush232-1191, even if bound to Srp, might fail to suppress Srp-induced hemocyte proliferation and lamellocyte differentiation. Alternative explanations include: (1) Ush232-1191 bound to Srp may actually enhance normal Srp activity; (2) the Srp:Ush232-1191 complex may behave neomorphically; (3) Ush may normally dimerize while not bound to Srp, if so a Ush:Ush232-1191 complex may not be able to separate into active Ush monomers. The transgenic Ush proteins are assumed to be sufficiently stable and functional as to validate these observations, since the UASush constructs used have been shown to generate stable proteins in other cell types and also to induce measurable phenotypes (Sorrentino, 2007).
There is significantly more Ush present in the nuclei of hopTum-l/Y hemocytes than in nuclei of wild-type blood cells. Interestingly, Ush appears to be exclusively nuclear in hopTum-l/Y hemocytes, whereas there appears to be some cytoplasmic anti-Ush staining in wild-type hemocytes. It is possible that Ush function is in part determined by its cytoplasmic/nuclear ratio. Perhaps the qualitatively higher Ush concentration in hopTum-l hemocytes is the result of nuclear translocation of all cellular Ush. Based on work with human 293T and mouse erythroleukemia cell cultures, Garriga-Canut (2004) proposed a model in which TACC-3 and GATA-1 compete in binding to FOG-1, with FOG-1 bound to TACC-3 retained in the cytoplasm. Such a mechanism may also be at work in Drosophila hemocytes and the possibility of a Ush cytoplasmic sequestration phenomenon remains to be investigated (Sorrentino, 2007).
This study also found that there exist high concentrations of exclusively nuclear Ush in other tumorous backgrounds, those being in Tl10b/+ and CgGAL4>UAScol animals. Larvae carrying the dominant Tl10b allele exhibit a hematopoietic tumor phenotype similar to that of hopTum-l/Y larvae. In addition to srpDGAL4>UAScol, CgGAL4>UAScol is sufficient to induce lamellocyte differentiation (although it also induces L2 developmental arrest). All hemocytes, including lamellocytes, in both of these backgrounds also exhibit high concentrations of nuclear Ush. These observations are consistent with a model in which three different signaling pathways (Hop-Stat, Toll-Dorsal, and the early B-cell related factor Collier) all make use of a single common downstream lamellocyte induction program that involves Ush. Examination of hemocytes from additional tumorous backgrounds will reveal whether such a model is truly universal. An important question remains as to how Ush suppresses hemocyte proliferation and lamellocyte differentiation, yet is expressed so strongly in tumorous hemocytes. Taken together, the findings are supportive of Ush having an early function in repressing lamellocyte differentiation. Up-regulation of the protein in lamellocytes would be suggestive of a second, separate function for Ush within this differentiated hemocyte. Comparable multi-functional properties have been reported for FOG-1 in vertebrate hematopoiesis (Sorrentino, 2007).
Therefore, reduction of Ush function results in a classic hematopoietic tumor phenotype: lymph gland hypertrophy and early dispersal, a significant increase in total circulating hemocyte concentration, large-scale lamellocyte differentiation, and melanotic tumors. These anomalies can be partially induced by the loss-of-function of a single copy of ush. The identification of this FOG class protein as a tumor suppressor raises questions about the roles of other FOG proteins in mammalian leukemias. While mutations in murine fog1 have been associated with hematopoietic dysfunction such as the failure of megakaryopoiesis and the arrest of erythropoiesis, FOG proteins have not been implicated as a causal factor in any human leukemia. While there is no guarantee that the observations in Drosophila will directly translate to specific human hematopoietic pathologies, it may now be worthwhile to examine the state of fog gene expression and function in human leukemias (Sorrentino, 2007).
Neuroblasts (NBs) generate a variety of neuronal and glial cells in the central nervous system of the Drosophila embryo. These NBs, few in number, are selected from a field of neuroepithelial (NE) cells. In the optic lobe of the third instar larva, all NE cells of the outer optic anlage (OOA) develop into either NBs that generate the medulla neurons or lamina neuron precursors of the adult visual system. The number of lamina and medulla neurons must be precisely regulated because photoreceptor neurons project their axons directly to corresponding lamina or medulla neurons. This study shows that expression of the proneural protein Lethal of scute [L(1)sc] signals the transition of NE cells to NBs in the OOA. L(1)sc expression is transient, progressing in a synchronized and ordered 'proneural wave' that sweeps toward more lateral NEs. l(1)sc expression is sufficient to induce NBs and is necessary for timely onset of NB differentiation. Thus, proneural wave precedes and induces transition of NE cells to NBs. Unpaired (Upd), the ligand for the JAK/STAT signaling pathway, is expressed in the most lateral NE cells. JAK/STAT signaling negatively regulates proneural wave progression and controls the number of NBs in the optic lobe. These findings suggest that NBs might be balanced with the number of lamina neurons by JAK/STAT regulation of proneural wave progression, thereby providing the developmental basis for the formation of a precise topographic map in the visual center (Yasugi, 2008).
NE cells are programmed to differentiate into NBs from the medial edge of
the developing optic lobe. The wave of differentiation progresses
synchronously in a row of cells from medial to lateral optic lobe sweeping
across the entire NE sheet; it is preceded by the transient expression of the
proneural gene l(1)sc. As the NBs at the medial edge are oldest and
the more lateral ones are youngest, developmental process of medulla neurons
can be viewed as an array of progressively aged cells across optic lobe
mediolaterally. This contrasts with NB formation in the embryonic CNS in which
a small number of cells are selected from NE cells to become NBs, leaving the
majority of NE cells to develop into non-neural cells. The optic lobe
proneural wave is reminiscent of the morphogenetic furrow that moves across
the developing eye imaginal disc. The morphogenetic furrow is the site where
differentiation from neuroepithelium to photoreceptor neurons is initiated. The
progression is driven by the secreted Hh expressed in the differentiated
photoreceptor cells. By contrast, the proneural wave still progresses even when
NB differentiation is impaired, suggesting that its progression is not driven
by a factor emanating from differentiated NBs. No
progression-defective phenotypes were observed when Hh or Decapentaplegic (Dpp) signaling was reduced. The model is favored that the proneural wave
progression is driven by an intrinsic mechanism such as a segmentation clock
and is negatively regulated by JAK/STAT pathway. As the JAK/STAT
ligand Upd is expressed only by the most lateral NE cells, proliferation of
the NE cells moves the source of ligand laterally and as a consequence
releases more medial NE cells from negative regulation and allows the
proneural wave to progress laterally. Alternatively, distribution of the Upd
ligand and/or the response to Upd changes as the NE cells age as graded
10xSTAT-GFP activities are more prominent in the early stage. Non-autonomous
action of JAK/STAT signal indicates that it does not directly regulate L(1)sc
expression and there are second signal(s) that regulate the expression of
L(1)sc under the control of JAK/STAT signal (Yasugi, 2008).
Three out of the four AS-C genes [sc, l(1)sc and
ase] are expressed during medulla neurogenesis. l(1)sc is
expressed in NE cells and ase in NBs, while sc is expressed
both in NE cells and NBs. Deleting all AS-C genes causes as significant
delay as da in NB formation but does not completely eliminate NB
formation, suggesting that Da-dependent proneural gene activities are required
for timely onset of NB formation. Mutation for sc or ase
alone does not affect NB formation, but the simultaneous deletion of
sc and l(1)sc causes the delay in NB formation and the
additional deletion of ase further delays NB formation. ase
expression is not altered in the absence of l(1)sc and
l(1)sc is not altered in the absence of ase, indicating that
l(1)sc and ase both contribute to the differentiation from
NE cells to NBs. Although the contribution of Sc cannot be formally excluded,
the highly specific expression pattern led to the inference that L(1)sc plays a
major role in the proneural wave (Yasugi, 2008).
JAK/STAT signaling is known to regulate stem cell maintenance in the adult
germline of Drosophila. In the male testis, germline stem cells (GSCs)
attach to a cluster of somatic support cells at the tip (hub) of the testis.
When a GSC divides, the daughter retaining contact with the hub maintains
self-renewing GSC identity, while the other daughter differentiates into
gonialblast. Upd is specifically expressed in the hub cells and activates
JAK/STAT signal in the GSCs to maintain stem cell state. In
the female ovary, JAK/STAT signaling is required in the somatic escort stem
cells whose daughters encase developing cysts.
This study shows that in the optic lobe development, JAK/STAT signaling maintains
NE cells in an undifferentiated state. It is suggested that a common mechanism
operates in both these developmental systems. Loss of Hop or Stat92E function
decreases number of stem cells and ectopic expression of Upd results in over
proliferation of undifferentiated cells. The cell fate may be determined by
the distance of the cells from the source of ligand; the cells farther from
the source commence to differentiate (Yasugi, 2008).
In the vertebrate CNS, NE cells first proliferate by symmetric cell
divisions and differentiate into neurons and glia in later developmental
stages. JAK/STAT
signaling has been implicated in maintenance of neural precursor cells, but
there is no clear evidence that those cells are in the same developmental
stage as described in this study for Drosophila. Further study of JAK/STAT
signaling will reveal whether a common mechanism underlies stem cell
development in both Drosophila and vertebrates, and should give new
insights into vertebrate CNS neurogenesis (Yasugi, 2008).
Development of a precise topographic map (retinotopic map) in
Drosophila is known to involve regulation of lamina neuron
development with respect to the incoming R axons.
The lateral NE sheet is continuous with a groove called the lamina furrow
where NE cells are arrested at G1/S phase. The
arriving R axons deliver Hh and liberate the arrested NE cells to proliferate
and develop into lamina neuron precursors. And,
thus, R axons can induce the development of their synaptic partners in their
vicinity to balance the number of R axonal termini and lamina neurons.
However, medulla development does not depend on inputs from the R axons in the
early phase. This study shows that both lamina and medulla neurons are
derived from the continuous NE sheet. Large clones of cells mutant for the
JAK/STAT signaling cause immature proliferation of medulla NBs at the expense
of lamina neurons, suggesting that the number of NE cells serves as the
limiting factor to generate precursors for lamina and medulla neurons. Thus,
the number of medulla neurons is roughly regulated at the level of NBs whose
generation might be balanced indirectly with the number of lamina neurons
through regulating proneural wave progression by JAK/STAT signaling. JAK/STAT
signaling therefore plays an important role in the formation of a precise
retinotopic map in the visual center (Yasugi, 2008).
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