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First published online June 22, 2006
doi: 10.1242/10.1242/dev.02411
Review |
,
Department of Molecular Developmental Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Author for correspondence (e-mail:
m.zeidler{at}sheffield.ac.uk)
Accepted 19 April 2006
SUMMARY
High levels of interspecies conservation characterise all signal transduction cascades and demonstrate the significance of these pathways over evolutionary time. Here, we review advances in the field of JAK/STAT signalling, focusing on recent developments in Drosophila. In particular, recent results from genetic and genome-wide RNAi screens, as well as studies into the developmental roles played by this pathway, highlight striking levels of physical and functional conservation in processes such as cellular proliferation, immune responses and stem cell maintenance. These insights underscore the value of model organisms for improving our understanding of this human disease-relevant pathway.
Introduction
The transduction of information from the outside of a cell to produce a specific response is an essential prerequisite for development, homeostasis and cellular survival, and is mediated by a small number of signal transduction cascades. As a result, the study of these pathways from a developmental viewpoint has been a focus of research for many years and have shown that signalling pathways are frequently reused in multiple tissues for diverse developmental processes. Here, we focus on one such cascade - the JAK/STAT pathway - the misregulation of which is associated with a wide range of human malignancies, including diverse haematopoietically derived cancers.
The human genome encodes multiple isoforms of all major JAK/STAT pathway
components, many of which are co-expressed within the same cells and can form
homo- and heterodimers to determine a variety of specific effects in response
to a diverse range of ligands. By contrast, Drosophila contains a
simpler `streamlined' pathway that is nonetheless sufficient to mediate a
multitude of different processes (reviewed by
Bach and Perrimon, 2003
;
Castelli-Gair Hombría and Brown,
2002; Hou et al.,
2002). However, despite the relative simplicity of the
Drosophila pathway, molecular and functional data clearly indicate
that a high level of conservation exists between the structural components of
the insect and mammalian pathways. As a result, the low levels of redundancy
in JAK/STAT pathway components, together with the availability of gain- and
loss-of-function mutations, advanced genetic and molecular tools, and a
multitude of markers, make Drosophila an excellent model for
investigations into the JAK/STAT pathway.
In this review, we focus on two aspects of JAK/STAT pathway biology. First,
we examine recent advances in the identification and characterisation of
pathway components and regulators. Second, we review results that highlight
the evolutionary conservation of pathway function in vivo - with an emphasis
on developmental aspects. In particular, we summarise information about
conserved roles of the JAK/STAT pathway in the control of proliferation during
larval development, and in immune responses and germ/stem cell development.
Although the immune response is not central to the field of developmental
biology, and has been reviewed in greater depth elsewhere
(Agaisse and Perrimon, 2004;
Tzou et al., 2002), these
pathway functions serve to illustrate the functional conservation between
flies and humans.
Given the levels of conservation in this pathway that are now becoming evident, it is hoped that the newly identified regulators and developmental roles being discovered in the Drosophila model system will advance our understanding of this important pathway and its roles in human disease.
The canonical JAK/STAT signalling pathway
Since the initial identification of a novel class of interferon-activated
transcription factors over 14 years ago
(Fu et al., 1992;
Schindler et al., 1992),
extensive studies have characterised the core components of the JAK/STAT
signal transduction pathway, which include a wide and diverse range of
extracellular ligands and transmembrane receptors, four Janus kinases (JAKs)
and seven genes coding for signal transducers and activators of transcription
(STATs) (Kisseleva et al.,
2002). In addition to the identification of the pathway components
themselves, a model of pathway activation has also been established
(Fig. 1). In general, this
entails the binding of an extracellular ligand to a transmembrane receptor,
which results in the activation of the receptor-associated JAKs. These
tyrosine kinases then phosphorylate themselves and their associated receptors
to generate docking sites for the SH2 domains of STATs. According to the
established models, STATs are normally present in the cytoplasm as inactive
monomers before recruitment to the receptor/JAK complex. However, it has also
been shown that STATs constitutively shuttle between the cytoplasm and nucleus
before being retained in the nucleus following activation (reviewed by
Vinkemeier, 2004). Once bound
to the receptor/JAK complex, STAT molecules are themselves phosphorylated and
dimerise. These dimers, stabilised by the interaction between the SH2 domain
of one molecule and the phospho-Tyr of the other molecule, translocate to the
nucleus where they bind to a palindromic DNA sequence in the promoters of
pathway target genes to activate transcription (as shown in
Fig. 1). Finally, although the
dimerisation of STATs via an N-terminal domain interaction can occur prior to
pathway stimulation, only complexes activated by Tyr-phosphorylation appear to
induce target gene expression (Braunstein
et al., 2003).
Over the past decade, numerous studies have determined a wealth of
information about the functions and activities of these core JAK/STAT
signalling components (reviewed by Hou et
al., 2002; Kisseleva et al.,
2002). Studies in model systems, such as the fruitfly
Drosophila melanogaster, the fish Danio rerio and the
nematode Caenorhabditis elegans, have also identified conserved
pathway components in these lower organisms
(Hou et al., 2002). In
particular, genetic analysis in Drosophila has identified all the
components of the canonical JAK/STAT signalling cascade, and over recent
years, this genetically tractable model organism has become an important focus
of research in the field. In Drosophila, the core components of the
JAK/STAT pathway consist of: three ligands [Unpaired (UPD; OS - FlyBase), UPD2
and UPD3 (Fig. 2A)
(Agaisse et al., 2003;
Castelli-Gair Hombría et al.,
2005; Gilbert et al.,
2005; Harrison et al.,
1998)]; a transmembrane receptor called Domeless (DOME)
(Fig. 2B)
(Brown et al., 2001;
Chen et al., 2002); a JAK
kinase known as Hopscotch (HOP) (Fig.
2C) (Binari and Perrimon,
1994); and a transcription factor called STAT92E
(Fig. 2D)
(Hou et al., 1996;
Yan et al., 1996). Although
the core pathway components have been known for some time (reviewed by
Bach and Perrimon, 2003;
Castelli-Gair Hombría and Brown,
2002; Hou et al.,
2002), these molecules still represent an area of active research
that aims to characterise their biochemical and functional activity in vivo
(Brown et al., 2003;
Henriksen et al., 2002;
Karsten et al., 2005).
However, it is the identification and characterisation of additional
components and regulators of the core pathway that will be discussed here.
|
Given the multitude of roles played by signal transduction pathways and the potential developmental consequences of their inappropriate activity, it is not surprising that multiple regulatory mechanisms exist to control them. These regulators can be broadly divided into two classes. Those whose activity is required for the transduction of pathway signalling, referred to as positive regulators, and those that act to reduce the strength of signalling - negative regulators.
Both positively and negatively acting factors that modulate JAK/STAT
pathway signalling have been identified and include not only new ligands
(Castelli-Gair Hombría et al.,
2005), but also other interacting signal transduction cascades and
other less well characterised molecules
(Bach et al., 2003;
Baeg et al., 2005;
Mukherjee et al., 2006;
Müller et al., 2005).
These are discussed below.
Positive regulators
Pathway ligands
While the activation of JAK/STAT pathway signalling in mammalian systems is
triggered by a wide range of interleukins, interferons and growth factors
(Langer et al., 2004;
Subramaniam et al., 2001),
only a single closely related group of Unpaired-like pathway ligands have been
identified in invertebrates (Agaisse et
al., 2003; Castelli-Gair
Hombría et al., 2005;
Gilbert et al., 2005;
Harrison et al., 1998). In
addition, although retrospective alignments of Type II cytokines suggest that
UPD may be related to the Leptin family of mammalian pathway ligands
(Langer et al., 2004), no
obvious UPD-like proteins can be detected beyond the Drosophilidae
family of flies (Castelli-Gair
Hombría and Brown, 2002;
Castelli-Gair Hombría et al.,
2005). In silico searches have, nonetheless, identified the three
related upd-like genes (Fig.
2A) in the Drosophilids, for which genome sequence is
available (Castelli-Gair Hombría et
al., 2005), and characterisation of upd mutations in
Drosophila melanogaster suggest that the canonical requirements for
JAK/STAT pathway activity are likely to be mediated exclusively by the ligands
UPD, UPD2 and UPD3 (Agaisse et al.,
2003; Castelli-Gair
Hombría et al., 2005;
Harrison et al., 1998).
Mutations in the upd locus were first identified over 75 years ago
(Müller, 1930), and the
upd gene itself has since been shown to encode a secreted
glycoprotein that activates the pathway via DOME
(Brown et al., 2001;
Harrison et al., 1998). When
ectopically expressed, UPD can function over at least 20 cell diameters in
vivo (Tsai and Sun, 2004) and
non-autonomous signalling induced by endogenous levels of UPD expression has
also been demonstrated (Bach et al.,
2003; Karsten et al.,
2002).
Although null mutations of upd show strong segmentation and
posterior spiracle phenotypes (Brown et
al., 2001; Harrison et al.,
1998), these are generally less severe than those exhibited by
loss of more downstream components, such as hop or stat92E,
indicating that other ligands may partly compensate for the loss of
upd (Castelli-Gair Hombría
et al., 2005). The recent characterisation of the upd2
locus has confirmed that UPD2 represents a second pathway activator that
functions redundantly to UPD
(Castelli-Gair Hombría et al.,
2005; Gilbert et al.,
2005). Like UPD, UPD2 can activate JAK/STAT pathway reporters both
in tissue culture and in vivo. However, in contrast to UPD, UPD2 does not
appear to attach to the extracellular matrix, but rather is secreted as a
freely diffusible ligand (Castelli-Gair
Hombría et al., 2005). The third UPD-like protein, UPD3,
has been less extensively characterised. However, it is expressed in the
developing gonads (Castelli-Gair
Hombría et al., 2005), the larval lymph gland
(Jung et al., 2005) and
circulating haemocytes following septic injury
(Agaisse et al., 2003).
|
).
Mutations in BRWD3 also genetically interact with the JAK/STAT
pathway to decrease the frequency of the melanotic tumour phenotype associated
with the gain-of-function mutation hopTum-l
(Müller et al.,
2005).
Of particular significance for the analysis of BRWD3 is the recent
discovery that the human homologue of BRWD3 is disrupted in a large
proportion of individuals with B-cell chronic lymphocytic leukaemia (B-CLL)
(Kalla et al., 2005). Although
a link between JAK/STAT signalling and the development of other haematopoietic
malignancies has been demonstrated
(Calò et al., 2003),
the precise role of BRWD3 with regard to JAK/STAT signalling and the
development of B-CLL has yet to be investigated.
Other signalling pathways
Recent investigations have identified that interactions occur between the
JAK/STAT pathway and several other signal transduction cascades. For example,
the Notch signalling cascade activates the JAK/STAT pathway in developing eye
imaginal discs, where it induces expression of upd
(Chao et al., 2004;
Moberg et al., 2005;
Reynolds-Kenneally and Mlodzik,
2005), and both pathways co-operate during foregut morphogenesis
(Josten et al., 2004). In
addition, the Notch cascade is known to affect JAK/STAT signalling via
activation of JAK2 in the mouse (Kamakura
et al., 2004), and strong genetic interactions between JAK/STAT
pathway signalling and mutations in Notch pathway components, such as
Notch, Delta and bearded, have been demonstrated in
Drosophila (Mukherjee et al.,
2006). In addition, a requirement for STAT92E in mediating the eye
overgrowth phenotype caused by ectopic activation of SRC has been reported
(Read et al., 2004), and
genetic screens have also identified interactions with components of the
DPP/BMP pathway (such as dpp, mothers against dpp, thickveins,
bunched) and the Hedgehog (hh) signalling pathway
(Bach et al., 2003;
Mukherjee et al., 2006).
Overall, it is becoming clear that no single pathway functions in isolation in a developmental context and although care must be taken in interpreting genetic interaction data, it is likely that an analysis of inter-pathway crosstalk will represent a key step towards our understanding of JAK/STAT signalling in vivo.
Other factors
Recent forward and reverse genetic screens
(Box 1)
(Bach et al., 2003;
Baeg et al., 2005;
Mukherjee et al., 2005;
Müller et al., 2005) have
identified multiple novel loci, the knockdown of which is sufficient to
modulate the strength of JAK/STAT pathway activity. Strikingly, a comparison
of the genes identified in these different screens reveals a relatively small
overlap in the candidates identified. In the case of the genetic interaction
screens (Bach et al., 2003;
Mukherjee et al., 2005),
genetic interaction can occur at many levels and may result from effects
downstream of the pathway itself. In addition, as the genetic screens
undertaken are not saturating, a limited overlap between the loci identified
by genetic and RNAi screens is not necessarily surprising. By contrast, the
results of two published RNAi screens (Baeg
et al., 2005; Müller et
al., 2005) are more striking and show a maximum overlap of only
20%. As both RNAi screens identified known pathway components, it is unlikely
that false negatives have lead to the exclusion of many true interactors.
Rather, it seems more likely that differences in the transcriptomes of the two
cell lines used, in conjunction with alternative strategies of pathway
stimulation and post-screen in silico analysis, are responsible for most of
the differences in the results of these two screens. Given that false positive
hits are also likely to be present, the in vivo validation of candidate
pathway-regulating loci is still required.
| Box 1. Screens for JAK/STAT pathway regulators
Whole-genome RNAi screens can now be routinely performed in
Drosophila using libraries of double stranded (ds) RNA molecules,
each
More-traditional `forward' genetic screens have also been employed to
identify mutations that interact with JAK/STAT signalling, using the principal
of a `sensitised system'. In this approach, candidate pathway modulators are
identified on the basis of a genetic interaction with a dominant phenotype. In
the case of the recently completed JAK/STAT pathway screens, ectopic pathway
activation in the developing fly eye leads to a massive increase in cellular
proliferation and an overgrown adult eye. Importantly, this large eye
phenotype is sensitive to the strength of the endogenous pathway and loss of
one copy of a pathway component is sufficient to produce a readily
recognisable decrease in eye size (Bach et
al., 2003
|
| Box 2. Feedback loops and JAK/STAT pathway signalling Several
feedback loops consisting of putative pathway target genes function in the
JAK/STAT pathway to modulate its signalling. In such feedback loops, pathway
activation leads to target gene expression, so increasing the influence of
that gene product. Where a target gene normally negatively regulates JAK/STAT
signalling a negative-feedback loop is formed that acts to decrease the
strength and duration of pathway activity. Where the target gene is a
positively acting pathway component, a positive feedback loop acts to increase
the strength of signalling.
One of the first feedback loops identified in JAK/STAT signalling is that
formed by the SOCS protein family, the pathway-dependent expression and
negative regulation of the JAK/receptor complex of which acts as a potent
negative feedback loop in both mammals and Drosophila
(Kile and Alexander, 2001
By contrast, positive feedback loops appear to be involved in regulating
JAK/STAT pathway activity. For example, upregulation of STAT3 is often
observed in mammalian cells upon interleukin 6 (IL6) stimulation, a response
elicited by IL6-response elements found in the STAT3 promoter
(Ichiba et al., 1998 Although yet to be demonstrated molecularly, the interplay of positive feedback loops generated by the upregulation of pathway components, and of negative feedback loops generated by SOCS and PTP61F, is likely to represent an important aspect of the differing levels of pathway sensitivity and perdurance in vivo.
|
Negative regulators
SOCS
The SOCS (suppressors of cytokine signalling) genes, originally
characterised in vertebrates, are the best characterised of the JAK/STAT
pathway negative regulators. In vertebrates, SOCS proteins have been shown to
suppress JAK/STAT pathway signalling via several distinct mechanisms
(Kile and Alexander, 2001;
Krebs and Hilton, 2001). They
are themselves target genes of this pathway, which are expressed in response
to STAT activation, so forming negative-feedback loops to downregulate pathway
activity (Box 2). On the basis
of sequence homology, three SOCS-like genes have been identified in the
Drosophila genome (Fig.
3A). Of these, socs36E functions as a potent pathway
repressor, which, when ectopically expressed, mimics the outstretched wing
phenotype of upd mutants and the venation defects associated with the
stat92EHJ allele (Baeg
et al., 2005; Rawlings et al.,
2004). socs36E is also a transcriptional target of
STAT92E (Baeg et al., 2005;
Callus and Mathey-Prevot,
2002; Karsten et al.,
2002; Rawlings et al.,
2004). Although socs44A does not show JAK/STAT-dependent
expression, it can inhibit pathway activity
(Rawlings et al., 2004).
socs16D is as yet uncharacterised.
|
;
Ungureanu et al., 2003;
Wormald and Hilton, 2004). A
single Drosophila PIAS-like protein, ZIMP
(Fig. 3B), also physically
interacts with STAT92E and can suppress the formation of haematopoietic
tumours caused by ectopic overactivation of the pathway
(Betz et al., 2001).
NSTAT92E
Although the Drosophila genome contains a single STAT-encoding
gene (Yan et al., 1996), a
negatively acting truncated form of STAT92E has recently been characterised
(Fig. 3C)
(Henriksen et al., 2002). In
contrast to the C-terminal truncated ß-forms of mammalian STATs, which
lack a transcriptional activation domain, Drosophila NSTAT92E
lacks an N-terminal region, which has been shown in mammalian STATs to promote
tetramerisation and nuclear re-export to the cytoplasm
(Johnson et al., 1999;
Levy and Darnell, 2002;
Meyer and Vinkemeier, 2004;
Vinkemeier et al., 1998).
When ectopically expressed, NSTAT92E exerts a dominant-negative effect
on the expression of the JAK/STAT pathway target genes even-skipped
and trachealess (Henriksen et
al., 2002; Karsten et al.,
2005). The ratio of full-length to NSTAT92E expression also
varies throughout development (Henriksen
et al., 2002; Mukherjee et
al., 2005) and it will be intriguing to determine the mechanisms
by which the truncated:full-length STAT92E ratio is regulated in vivo.
Phosphatases
Given the central role of the JAK tyrosine kinase in pathway signalling,
phosphatase activity is likely to represent an important regulatory mechanism.
Recently, two independent RNAi screens have identified Drosophila
PTP61F (Fig. 3D), a homologue
of human PTPB1 (phospho-Tyr phosphatase B1), as a suppressor of
STAT92E-dependent transcription (Baeg et
al., 2005; Müller et al.,
2005). Ptp61f is expressed in a pattern that mirrors that
of upd and that appears to be JAK/STAT pathway-dependent (see also
Box 2). As expected, reducing
PTP61F activity in cultured Kc167 and S2-NP cells by RNAi leads to
dramatic increases in both the activity of STAT-responsive reporters and the
levels of Tyr-phosphorylated HOP and STAT92E protein
(Baeg et al., 2005). However,
the exact mechanism by which PTP61F can downregulate pathway signalling is not
entirely clear. Only a nuclear localised splice form of PTP61F, termed
PTP61FC, can affect pathway activity in vivo but RNAi knockdown of both splice
forms affects the pathway epistatically downstream of HOP
(Müller et al., 2005).
PTP61FC therefore probably acts at the level of STAT92E, a conclusion that is
also supported by in silico modelling, which shows that pathway activity is
most strongly influenced by altering the level of nuclear STAT-interacting
phosphatases (Zi et al.,
2005).
Ken & Barbie/BCL6
A role for Ken & Barbie (KEN) in the selective regulation of STAT92E
activity has also recently been demonstrated, a finding that could advance our
understanding of how a single pathway can mediate diverse developmental
processes (Arbouzova et al.,
2006). KEN is the Drosophila homologue of human BCL6
(B-cell lymphoma 6) and belongs to the family of BTB/POZ domain-containing
transcriptional repressors (Fig.
3E). In vitro selection experiments show that KEN recognises a DNA
sequence that partially overlaps that of STAT92E in vitro
(Fig. 4A), while tissue culture
assays indicate that it can specifically downregulate JAK/STAT pathway
reporters only when they contain the consensus KEN DNA-binding site
(Fig. 4B). In vivo, both
ectopically expressed and endogenous KEN downregulate only a subset of likely
STAT92E target genes.
The human BCL6 locus is itself the focus of considerable interest because
of its mutation in a large proportion of diffuse large B-cell non-Hodgkins
lymphomas (Pasqualucci et al.,
2003). Although no direct link has been made between the
requirement for BCL6 during B-cell development and the JAK/STAT pathway in
vivo, BCL6 has also been shown to repress STAT6-dependent transcription in
cell culture (Harris et al.,
1999; Harris et al.,
2005; Hartatik et al.,
2001). Further analysis of the interplay between KEN/BCL6 and
JAK/STAT signalling promises to be an interesting area for future
research.
Conserved roles for JAK/STAT signalling during development
In addition to the identification of new factors that regulate JAK/STAT
signalling, significant progress has also been made in our understanding of
the roles played by the pathway during development (reviewed by
Bach and Perrimon, 2003;
Castelli-Gair Hombría and Brown,
2002; Hou et al.,
2002). Several recent studies of the role of the
Drosophila JAK/STAT pathway have identified examples in which both
the mechanisms and functions of the pathway are conserved between vertebrates
and Drosophila. Here, we review these findings with a particular
focus on cellular proliferation, the innate immune response and stem/germ cell
development.
|
)].
Second, chronic myeloproliferative diseases (MPDs; a term for diseases in
which myeloid cell types undergo abnormal proliferation and/or survival) are
often associated with the activation of tyrosine kinases, many of which have
the potential to activate STATs (De
Keersmaecker and Cools, 2005). In particular, a recent analysis of
polycythaemia vera, a MPD characterised by the overproliferation of
erythrocytes, has revealed that over 80% of affected individuals carry a V617F
mutation in the regulatory pseudokinase domain of JAK2. This mutation results
in constitutive kinase activity and, presumably, in ectopic pathway activation
(James et al., 2005;
Staerk et al., 2005).
Similar gain-of-function mutations in the Drosophila JAK homologue
HOP have also been identified. The hopT42 allele, for
example, also contains a pseudo-kinase domain mutation, which leads to the
constitutive activity of Drosophila HOP, while the corresponding
mutation generated in mouse JAK2 also results in pathway overactivation
(Luo et al., 1997). In
vivo, hopT42 induces the overproliferation and premature
differentiation of larval blood cells, which then form melanotic tumours
(Fig. 5B)
(Luo et al., 1997), a
phenotype essentially identical to that observed in the
hopTum-l gain-of-function mutant
(Hanratty and Dearolf, 1993;
Harrison et al., 1995;
Luo et al., 1995). Thus, it
appears that in addition to the conservation of the JAKs themselves, both the
potential to generate activating mutations and their in vivo haematopoietic
overproliferation phenotypes have been conserved through evolution. A role for
the pathway during normal haematopoietic development is also implied by the
observation that upd3 and dome are expressed in the main
Drosophila larval haematopoietic organ - the lymph gland
(Jung et al., 2005). Although
parallels between Drosophila and vertebrate haematopoiesis have been
drawn in the past (Evans et al.,
2003), it will be intriguing to determine exactly how far the
functional conservation at the level of individual signalling pathways, such
as the JAK/STAT cascade, has been maintained.
As described above, analyses of gain-of-function alleles have shown that
ectopic JAK/STAT pathway activation is sufficient to induce overproliferation
of at least some cell types. Another tissue in which this occurs is the
developing eye imaginal disc. During normal eye development, a physical
indentation, termed the morphogenetic furrow (MF), moves from the posterior to
the anterior of a field of undetermined retinal cells. The MF marks the
beginning of cellular determination and is associated with the first and
second mitotic `waves' of cell division that occur ahead of and behind the MF,
respectively (Fig. 5C)
(reviewed by Voas and Rebay,
2004). This pattern of proliferation is enhanced by the ectopic
stimulation of the JAK/STAT pathway, which results in a large increase in the
number of mitotic cells within the first mitotic wave
(Fig. 5D)
(Bach et al., 2003;
Tsai and Sun, 2004). However,
despite the overproliferation of pluripotent cells ahead of the MF, cells
already determined to generate the adult eye posterior to the furrow do not
respond proliferatively and differentiate essentially normally
(Fig. 5D)
(Bach et al., 2003). As a
result of the increase in proliferation, more cells are available to be
recruited into ommatidia, giving rise to an enlarged and overgrown adult eye
(compare Fig. 5E with
5F). This phenotype is
sensitive to the dose of downstream pathway genes and has been used as the
basis of genetic interaction screens (see
Box1)
(Bach et al., 2003;
Mukherjee et al., 2006).
The analyses of loss-of-function mutations also indicate that the JAK/STAT
pathway is required for normal cellular proliferation. The epithelial cells
that make up the imaginal discs destined to form the adult fly are diploid
cells, have a normal G1-S-G2-M cell cycle, and so represent a good model for
analysing cellular proliferation in vivo (reviewed by
Baker, 2001). Loss of
hop leads to the under-proliferation of these imaginal disc cells,
resulting in small discs (Mukherjee et
al., 2005; Perrimon and
Mahowald, 1986). In addition, several hypomorphic upd
alleles display small eye phenotypes
(Müller, 1930) - an
effect that is also sensitive to the dose of downstream pathway genes
(Mukherjee et al., 2006;
Tsai and Sun, 2004).
More recently, a detailed analysis of the requirement for stat92E
in the cellular proliferation of the wing imaginal disc has revealed an
unexpected and more complex situation. By inducing mitotic recombination,
heterozygous stat92E mutant cells can be forced to divide to generate
a pair of daughter cells: one of which is homozygous mutant for
stat92E, while the other is wild type. Given that the two daughter
cells are born at the same time and in the same local environment, differences
in the subsequent proliferation of these two cells can be directly compared.
By analysing the growth of such clonally related cell populations at different
time points during larval development, a major (although not essential) role
for stat92E in the promotion of cell proliferation has been
demonstrated during early larval development. Surprisingly, the converse
occurs at later larval stages with stat92E mutant cells proliferating
significantly more rapidly than wild type
(Mukherjee et al., 2005). This
changing proliferative phenotype implies that endogenous STAT92E present in
wing disc cells at later stages normally functions to reduce the rate of
proliferation. Even more surprising is that this endogenous anti-proliferative
activity is a result of non-canonical (UPD- and HOP-independent) STAT92E
activation - although ectopically induced canonical pathway stimulation is
also sufficient to produce the same effect
(Mukherjee et al., 2005).
|
; Silva et al.,
2003). Similarly in Drosophila, cellular
overproliferation caused by ectopic SRC activation in the adult eye is a
consequence of STAT92E stimulation (Read
et al., 2004), while STAT92E activity in embryonic primordial germ
cells (PGCs), also termed pole cells, is a consequence of stimulation by the
receptor tyrosine kinase TORSO (Li et al.,
2003). Second, although it is as yet unclear how STAT92E elicits
both proliferative and anti-proliferative effects, this finding also has
parallels in mammalian systems. STAT1 is known to promote cell cycle arrest
and apoptosis (Stephanou and Latchman,
2005), while the activities of STAT3 and STAT5 promote
pro-proliferative cellular responses
(Bowman et al., 2000;
Stephanou and Latchman, 2005).
By contrast, the single Drosophila STAT-like gene can fulfil both of
these functions. Whether this is a consequence of an ancestral activity
subsequently assigned to distinct vertebrate STAT proteins or an example of
genome `simplification' in Drosophilids
(Raible et al., 2005) is as
yet unknown.
Although the activation of JAK/STAT signalling appears to be both necessary
and sufficient to modulate cellular proliferation in multiple tissues, the
exact mechanism by which the pathway regulates cell division is not clear. In
humans, the STAT-activated expression of cyclin D1, which encodes a
regulatory subunit of CYCD/CDK4 complex that promotes G1/S transition, and of
c-myc (dm - FlyBase), which encodes a transcriptional regulator of
cell cycle progression, can account for the proliferative effect of the
JAK/STAT pathway (Bowman et al.,
2000; Calò et al.,
2003). Similarly, Drosophila CYCD/CDK4 and CYCE/CDK2
complexes have been reported to interact with STAT92E
(Chen et al., 2003). However,
these and recent observations describing JAK/STAT induced upregulation of
cycD in the eye imaginal disc
(Tsai and Sun, 2004) do not
represent a complete explanation of the overproliferation phenotype. Rather,
the Drosophila homologues of the CYCD/CDK4 complex is known to
promote cellular growth with only an indirect effect on proliferation
(Datar et al., 2000;
Meyer et al., 2000;
Tsai and Sun, 2004).
Furthermore, loss of a single copy of cycD does not reduce the eye
overgrowth phenotype caused by ectopic activation of the JAK/STAT pathway
(Mukherjee et al., 2006).
Thus, although links have been demonstrated, the exact mechanisms that connect
the Drosophila JAK/STAT pathway to the cell cycle remain to be
elucidated.
Immune responses
The JAK/STAT cascade was originally discovered as a cytokine-induced
signalling pathway required by myeloid and lymphoid cell lineages (reviewed by
Ihle, 1995). It has
subsequently been shown to play a central role in orchestrating mammalian
immune responses (reviewed by Boehm et
al., 1997; Hanlon et al.,
2002; Trinchieri,
2003; Watford et al.,
2003). Consequently, lack of pathway activity leads to defects in
B- and T-cell functions, and results in severe immunodeficiencies, such as
SCID (severe combined immunodeficiency) (reviewed by
Schindler, 2002). An
increasing amount of evidence also indicates that the pathway is required to
control the innate immune response and the haematopoietic development of the
fruitfly. One of the first demonstrations of this was the melanised
haematopoietic tumour phenotype that results from the overactivation of the
pathway (Fig. 5A,B)
(Hanratty and Dearolf, 1993;
Harrison et al., 1995;
Luo et al., 1997). In this
case, tumourigenesis is caused by gain-of-function hop alleles, the
constitutive activation of which results in the overproliferation of
lamellocytes, one of the three lineages of the Drosophila haemocytes
(Sorrentino et al., 2004).
Lamellocyte differentiation and proliferation, similar to that observed in
hop gain-of-function mutants, is also part of the normal immune
response to wounding and infection (Tzou
et al., 2002). Given this similarity, a role for the endogenous
JAK/STAT pathway in immune responses has also been suggested
(Lanot et al., 2001;
Markus et al., 2005). Indeed,
while the number of circulating haemocytes is not affected, the ability to
activate immune responses upon infection is largely suppressed in hop
and stat92E partial loss-of-function mutants
(Sorrentino et al., 2004).
Consistent with this are recent studies that show STAT92E is activated in fat
body cells upon immune challenge. The Drosophila fat body is an
essential organ of the fly immune response that is the functional analogue of
the mammalian liver. STAT92E activation in the fat body results in the
expression of the antimicrobal peptides TotA and TotM, as well as of the
D-raf (phl - FlyBase) proto-oncogene. In the case of TotA, this
response can also be blocked by a dominant-negative form of DOME
(Agaisse et al., 2003;
Boutros et al., 2002;
Dostert et al., 2005;
Kwon et al., 2000).
Interestingly, this fat body specific activation of JAK/STAT signalling
requires upd3, but not upd or upd2. upd3 is
expressed by circulating haemocytes at the site of septic injury, with the
cytokine functioning non-autonomously to activate the pathway in fat body
cells throughout the larva (Agaisse et al.,
2003). Further evidence also suggests a role of the
Drosophila JAK/STAT pathway in mediating an innate antiviral
response. Transcription of vir-1 (virus-induced RNA 1), an as yet
uncharacterised protein that is expressed upon viral challenge, may be STAT92E
dependent, as STAT92E binds to the vir-1 promoter in vitro. Moreover,
hop mutants are hyper-sensitive to viral infection
(Dostert et al., 2005).
Consistent with this, studies in the mosquito Anopheles gambiae (Ag)
also implicate the JAK/STAT pathway in the immune response, with the
activation of Ag-STAT occurring in cell culture and in cell lysates
obtained from immune-challenged adult individuals
(Barillas-Mury et al.,
1999).
Taken together, it appears that key roles for the JAK/STAT signal transduction cascade in the development of the immune system, and the signalling required to activate it, have been conserved during evolution. Although the details and cell types involved in Drosophila and mammals differ, analysis of the promoters of the putative STAT92E target genes represents a future direction for studies into the immune functions of JAK/STAT signalling.
Primordial germ cells, stem cells and JAK/STAT signalling
The identification, maintenance and potential use of pluripotent stem cells
is a field of considerable potential significance for human health. As such,
research into stem cell biology has attracted considerable interest over
recent years. In particular, a thorough understanding of the cellular
environment (the so-called stem cell niche) in which stem cells are maintained
in vivo represents an essential prerequisite for such efforts and, as a
result, the niches required by stem cells in vivo are increasingly being
studied in Drosophila. Although several signalling pathways,
including the Wnt and BMP pathways, have been shown to be repeatedly required
to define stem cell niches in both mammals and flies (reviewed by
Li and Xie, 2005), the role of
JAK/STAT pathway signalling in this process is especially pertinent for this
review (Rao, 2004;
Varga and Wrana, 2005). One
of the first indications for such a requirement is derived from experience in
cell culture, which has shown that stimulation of mouse STAT3 by LIF
(leukaemia inhibitory factor) and other interleukin 6-family cytokines is
required to maintain the undifferentiated status of murine embryonic stem (ES)
cells ex vivo (Boeuf et al.,
1997; Hao et al.,
2006; Humphrey et al.,
2004; Niwa et al.,
1998). Although stimulation by STAT3 does not appear to be
sufficient to support the self-renewal of human ES cells
(Daheron et al., 2004;
Sato et al., 2004), it is
possible that the activity of other STATs may be required by human ES
cells.
The JAK/STAT pathway is also essential for stem cell maintenance in
Drosophila. At early stages of Drosophila embryogenesis, the
zygotic nuclei that migrate to the posterior pole of the embryo give rise to
PGCs, termed pole cells, which require JAK/STAT signalling at multiple stages.
During the early blastoderm stage, pole cell proliferation requires the
activation of STAT92E, which is mediated by the receptor tyrosine kinase TORSO
(Li et al., 2003). Later, 6-7
hours after egg laying, STAT92E activity is necessary for the pole cells to
migrate through the wall of the hindgut and towards the embryonic gonads
(Li et al., 2003). After
12 hours, the pole cells use guidance cues, probably supplied by the
localised expression of JAK/STAT pathway ligands, to coalesce and form the
embryonic gonads (Brown et al.,
2006; Li et al.,
2003).
Although it is not entirely clear whether the proliferation and migration
of PGCs requires JAK/STAT pathway activity in both sexes, a specific role for
JAK/STAT pathway signalling in embryonic male gonads has also been
established. In particular, a recent report has shown that upd is
expressed in somatic cells of the embryonic testis, where it induces the
phosphorylation of STAT92E specifically in the male germ cells
(Wawersik et al., 2005). This
pathway activation results in the expression of the male-specific markers
mgm-1, dpa and mcm5. Blocking the activity of the pathway by
removing zygotic STAT92E in the male germ cells results in the loss of marker
expression, while activation of the pathway in female germ cells induces
STAT92E activity and masculinisation
(Wawersik et al., 2005).
A key role of JAK/STAT signalling for the maintenance and proliferation of
the stem cells within the gonads of both sexes has also been demonstrated
during adult life. In adult Drosophila males, self-renewing germline
stem cells (GSCs) are arranged around a small group of upd-expressing
somatic cells at the apical tip of each testis, called the hub
(Fig. 6A)
(Fuller, 1998). The polarised
division of stem cells produces one self-renewed stem cell proximal to the hub
and one distal gonialblast committed to differentiation
(Fig. 6A).
It has been proposed that JAK/STAT signalling in GSCs is required to maintain stem cell state and/or proliferation, a suggestion strongly supported by the expression of UPD in hub cells (Fig. 6B). In this model, the more distal daughter cell arising from the division of GSCs is further away from the source of pathway ligand (Fig. 6A) and so is no longer exposed to sufficiently high levels of stimulation.
Consistent with this, analysis of hop and stat92E mutants
reveals that mutant testes lose their GSCs during larval development, although
mutant spermatogonia are able to differentiate normally into sperm
(Brawley and Matunis, 2004;
Kiger et al., 2001;
Tulina and Matunis, 2001).
Conversely, ectopic expression of upd results in the expansion of
GSCs at the expense of differentiating spermatogonia
(Kiger et al., 2001;
Tulina and Matunis, 2001).
Strikingly, one report has indicated that spermatogonia, which have initiated
differentiation following a decrease in JAK/STAT signalling, can repopulate
the niche after restoration of appropriate signalling, implying that pathway
activity may also be sufficient to `de-differentiate' cells in this particular
environment (Brawley and Matunis,
2004).
|
;
Ghiglione et al., 2002;
McGregor et al., 2002;
Silver and Montell, 2001).
More specifically related to stem cell maintenance is a recent description of
somatic `escort stem cells' and germline stem cells present at the very
earliest stages of oocyte development
(Decotto and Spradling, 2005).
The maintenance of the escort stem cells and the development of the oocyte
have also been shown to depend on the activity of the JAK/STAT pathway, and
although the precise interactions between escort and germline stem cells
remains to be determined, it appears that the pathway is required for the
maintenance of at least a subset of male and female stem cells in vivo
(Decotto and Spradling,
2005).
Conclusions and perspectives
Comparative analysis of data published over recent years shows that the
JAK/STAT pathway has been conserved during evolution, not only at the level of
its structural components but also functionally. However, despite the relative
simplicity of the Drosophila JAK/STAT pathway, it still fulfils many
of the same functions observed in vertebrates.
Given this degree of inter-species conservation, the systematic exploitation of the genetic strengths inherent in the Drosophila system represents a particularly valuable approach for the future. Indeed, genetic and genome-wide RNAi screens that have already been undertaken (Box 1) have almost certainly identified pathway components, regulators and interacting genes whose human homologues fulfil similar roles in vivo. Indeed, an essential task for the near future will be to identify these human orthologues from amongst the genetic `noise' inherent in any screen.
After the identification of the orthologous genes, one approach that will
undoubtedly prove to be helpful for the further characterisation of both human
and Drosophila pathway regulators is the analysis of common roles in
the conserved pathway functions of stem cell maintenance, immunity and
cellular proliferation. For example, the JAK2 mutations recently identified as
being responsible for human polycythema vera have been known to produce
leukaemia-like haemocyte overproliferation in Drosophila for almost
10 years (Luo et al., 1995;
James et al., 2005).
In the longer term, it is to be hoped that both the Drosophila and mammalian JAK/STAT fields can recognise and exploit their inter-species synergies to use the strengths of both systems to determine the identify and functions of the key regulators of this medically important signal transduction pathway.
ACKNOWLEDGMENTS
The authors thank Herbert Jäckle, Patrick Müller and Michael Boutros for valuable comments on the manuscript, and are especially grateful to Iris Plischke, Rebecca Stearns and Erika Bach for supplying data used in figures. Funding to the Zeidler laboratory is provided by an Emmy Noether fellowship of the D.F.G. and the Max Planck Society.
Footnotes
* Present address: Samuel Lunenfeld Research Institute, 600 University
Avenue, Room 884, Toronto, ON, M5G 1X5, Canada 
Present address: Deparment of Biomedical Science, The University of
Sheffield, Western Bank, Sheffield S10 2TN, UK
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