pointed
Two chromosomal domains have been identified with opposite regulatory effects on the transcriptional activity of the pointed P2 promoter: one trans-activates and the other trans-represses Pointed P2 expression. By deletion mapping these control regions were found to be within the 5' region of the Pointed P2 transcript (Scholz, 1993).
Tracheal expression of pointed and Serum response factor (also known as pruned, blistered and DSRF) are targeted by branchless, acting through breathless. To define the role of bnl in later branching events, expression of secondary (pointed) and terminal (Srf) branch genes in bnl loss-of-function mutants were assayed. pnt and Srf fail to be expressed in the tracheal system of bnl mutants. In contrast, in embryos that ectopically express bnl, both markers are activated throughout the tracheal system, and the expressing cells later give rise to secondary and terminal barnches. These results support the hypothesis that bnl expression near the ends of the primary branches not only guides primary branch outgrowth, but also activates the program of secondary and terminal branching in cells at these positions (Sutherland, 1996 and Samakovlis, 1996).
In the differentiation of photoreceptors in eye imaginal discs, activated Ras1 up-regulates the transcriptional activity of P2, but not the P1 form of Pointed. Pointed P2 may be a direct target of a Drosophila MAPK called ERKA, encoded by the rolled locus at the same time that Ras 1 and ERKA negatively regulate the ability of yan to repress transcription. (O'Neill, 1994). Pointed P2 is phosphorylated by MAPK at a single site that is required for its in vivo function as a transcriptional activator. This site is located within the so-called 'pointed' or RII domain which is shared by a subset of ETS proteins (Brunner, 1994).
Two classes of glial cells are found in the embryonic Drosophila CNS: midline glial
cells and lateral glial cells. Midline glial development is triggered by EGF-receptor
signaling, whereas lateral glial development is controlled by the glial cells missing (gcm) gene. Subsequent
glial cell differentiation depends partly on pointed . tramtrack (ttk) is required for all CNS glia development. Mutant ttk embryos are characterized by an embryonic CNS axon pattern phenotype of fused segmental commissures, indicating a requirement of ttk during midline glial development. In ttk embryos, longitudinal axon tract formation is impaired and the connnectives appear thinner. This phenotype is indicative of a defect in the longitudinal glia (Giesen, 1997).
tramtrack encodes two
zinc-finger proteins, one of which, ttkp69, is expressed in all non-neuronal CNS cells. ttk expression in the ventral cord is restricted to lateral and midline glial cells. All cells that express the glial marker Repo also express ttkp69. The transverse nerve exit glial cells (or DM cells) express ttkp69. In the CNS of stage 16 ttk mutants, there are about 20% less lateral glial cells than a wild-type CNS. In mutants, although the midline glial cells are initially present in normal number and position, they fail to perform their normal migration. Therefore ttk is required for normal glial development. The exit glial cells in mutant ttk embryos are slightly enlarged, but they are still able to ensheath both the segmental and intersegmental axon bundles. Like ttk, pointed is expressed in glial cells. However, unlike ttk, pointed is required for glial cell development. Ectopic ttkp69 expression in the neuroectoderm leads to a partial block of neuronal development as indicated by substantially reduced expression of the neuronal Elav antigen as well as other neuronal markers examined (Giesen, 1997).
Both Ttkp69 and pointed are downstream of gcm. gcm, however, is not expressed in midline glia, and ttkp69 as well as pointed expression in midline cells is normal in gcm mutants. pointed and ttkp69 are both expressed under the control of gcm in lateral glial cells; the expression of these genes appears to be independent of one another. Thus the two targets of gcm appear to act in parallel. Glial cell differentiation may depend on a dual process, requiring the activation of glial
differentiation by pointed and the concomitant repression of neuronal development by
tramtrack (Giesen, 1997).
During normal tracheal development, secondary and terminal branching genes are induced at the ends
of growing primary branches by
localized expression of Branchless. Because the
ectopic branches in sprouty mutants are formed by the prestalk cells located near the cells that are
normally induced to branch, the extra branches could arise from overactivity of the Bnl pathway. To
test whether sty functions by limiting the Bnl pathway or by preventing branching in some other way, an examination was made of downstream effectors in the Bnl pathway that regulate the later branching events (Hacohen, 1998).
One such effector is pointed (pnt), a downstream target of several receptor tyrosine kinase pathways. pnt expression is induced by Bnl at the ends of
primary branches and promotes secondary and terminal branching. Similarly, the DSRF gene and three other
marker genes (Terminal -2,-3, and -4) are induced at the ends of growing primary branches; all
promote terminal branching. In sty mutants, all five downstream effectors
are expressed in expanded domains that include the prestalk cells, which later form ectopic branches. The DSRF marker is activated at the same time as in the normal branching cells (Hacohen, 1998).
The transcriptional repressor Yan is another critical target of Bnl signaling. As in other RTK pathways, activation of the Btl receptor leads to MAPK-dependent phosphorylation and degradation of Yan, which is necessary to
activate the later programs of tracheal branching. Normally, Yan is
degraded only in the tip cells of the outgrowing primary branches. In sty
mutants, Yan is degraded in an expanded domain that coincides with the expanded domains of pnt
and DSRF expression. A yan-lacZ transcriptional reporter continues to be
expressed normally, implying that down-regulation of Yan occurs posttranscriptionally as in other RTK
pathways. The results show that sty loss of function mutations enhance all known downstream effects in this Bnl pathway. An engineered gain of function condition, in which the sty gene
product is overexpressed during embryonic stages 10-12, severely blocks induction of downstream
effectors and branching by Bnl. The reciprocal is also true: overexpression
of Bnl can overcome the opposition of sty and induce secondary and terminal branching throughout the tracheal system. Thus, sty behaves genetically as a competitive
inhibitor of the Bnl pathway (Hacohen, 1998).
Hox genes have large expression domains, yet these genes control the formation of fine pattern elements at specific locations. The mechanism underlying subdivision of the abdominal-A (abdA) Hox domain in the visceral mesoderm has been examined. AbdA directs formation of an embryonic midgut constriction at a precise location within the broad and uniform abdA expression domain. The constriction divides the abdA domain of the midgut into two chambers, the anterior one producing the Pointed (Pnt) ETS transcription factors and the posterior one the Odd-paired (Opa) zinc finger protein. Transcription of both pnt and opa is activated by abdA. Near the anterior limit of the abdA domain, two signals, Decapentaplegic and Wingless, are produced, in adjacent non-overlapping patterns, under Hox control in mesoderm cells.
AbdA is proposed to activate three targets, in distinct subsets of its broad domain of expression: wg at the anterior boundary of Connectin (Con) patch 7; pnt from anterior Con patch 7 to anterior Con patch 8, and opa, from anterior Con patch 8 through Con patch 11. Dpp signaling plays a central role in setting these distinct expression domains. The initial activation of wg by AbdA requires dpp. opa is activated in all abdA-expressing cells that do not receive a Dpp signal, defining the site of the posterior constriction. wg, in collaboration with abdA, activates pnt to generate the appropriate number of cells in the third midgut chamber, positioning the posterior constriction at the proper distance from the central constriction and partitioning the posterior midgut appropriately. Fine patterning of the posterior midgut is achieved by the activity of diffusible signals emanating from the central midgut, a remarkably long-range organizing effect (Bilder, 1998).
Intercellular signaling through the EGF receptor (EGFR) patterns the Drosophila egg. The TGF alpha-like ligand Gurken signals from the oocyte to the
receptor in the overlying somatic follicle cells. In the dorsal follicle cells, this initial paracrine signaling event triggers an autocrine amplification
by two other EGFR ligands: Spitz and Vein. Spitz becomes an effective ligand only in the presence of the multitransmembrane domain protein Rhomboid.
Consequent high-level EGFR activation leads to localized expression of the diffusible inhibitor Argos, which alters the profile of signaling. This sequential
activation, amplification, and local inhibition of the EGFR forms an autoregulatory cascade that leads to the splitting in two of an initial single peak of signaling, thereby patterning the egg (Wasserman, 1998).
Egfr signaling specifies the dorsoventral axis and patterns the eggshell. It is suggested that these
two functions are controlled by temporally separate phases of Egfr activation. When amplification
and splitting of Egfr signaling do not occur, eggs have only a single, fused appendage. Surprisingly,
larvae emerge from these eggs at the frequency predicted by Mendelian principles, and those that
emerge have no apparent dorsoventral defects. When follicle cell clones of a spitz null are induced, the hatching rate of eggs with fused appendages os 82% of the predicted number. Similarly, all of the predicted number of eggs with a single fused appendage hatch from mutant females. The same is true of
eggs with fused appendages caused by follicle cell clones of argos null mutations.
Therefore, disruption of the amplifying and splitting process does not perturb dorsoventral axis
specification, implying that the initial Gurken signal to the Egfr is sufficient to specify the axis. The subsequent cascade of amplification and splitting then patterns the eggshell (Wasserman, 1998).
Cell proliferation in the developing renal tubules of Drosophila is strikingly patterned, occurring in two phases to
generate a consistent number of tubule cells. The later phase of cell division is promoted by EGF receptor signaling
from a specialized subset of tubule cells, the tip cells, which express the protease Rhomboid and are thus able to
secrete the EGF ligand, Spitz. The response to EGF signaling, and in consequence cell division, is patterned by the specification of a second cell type in the tubules. These cells are primed to respond to EGF signaling
by the transcription of two pathway effectors, PointedP2, which is phosphorylated on pathway activation, and Seven up. While expression of
pointedP2 is induced by Wingless signaling, seven up is initiated in a subset of the PointedP2 cells through the activity of the proneural genes. Both signaling and responsive cells are set aside in each tubule primordium from a proneural gene-expressing cluster of cells, in a
two-step process: (1) a proneural cluster develops within the domain of Wingless-activated, pointedP2-expressing cells to initiate the co-expression
of seven up; (2) lateral inhibition, mediated by the neurogenic genes, acts within this cluster of cells to segregate the tip cell precursor, in which
proneural gene expression strengthens to initiate rhomboid expression. As a consequence, when the precursor cell divides, both daughters secrete
Spitz and become signaling cells. Establishing domains of cells competent to transduce the EGF signal and divide ensures a rapid and reliable
response to mitogenic signaling in the tubules and also imposes a limit on the extent of cell division, thus preventing tubule hyperplasia (Sudarsan, 2002).
The ETS domain protein PointedP2 (PntP2) functions downstream of Egfr/Ras signaling. This protein contains a single MAPK phosphorylation site and upon phosphorylation, competes with the ETS domain transcriptional repressor, Yan, to activate the expression of target genes. In the absence of pnt function, cell proliferation in the tubules is reduced in a manner similar to svp mutants (Sudarsan, 2002).
It was therefore asked whether early expression of pnt as well as svp is required to prime the mitogenic response in tubule cells. pntP2 is initiated in the posterior side of each tubule during stage 10. This domain is characterized by high levels of wg expression, which are required for the normal development of AS-C expression in the PNC, during the time it develops within this domain. The domain of wg and pntP2 expression is slightly wider than the PNC and pntP2 expression is initiated well before Egfr activity is required for tubule cell divisions. The expression of pntP2 persists in this posterior domain when the tip mother cell is specified. In wgCX4 mutant embryos, tubule expression of pntP2 is completely abolished, showing that Wg signaling is required to initiate its expression. Conversely, the overexpression of wg, using a hs-wg construct, results in expansion of pntP2 expression to the anterior side of the tubule primordium and elevation of expression to high levels. Thus, Wg is necessary and sufficient to activate the expression of pntP2 in the tubules (Sudarsan, 2002).
Hox proteins play fundamental roles in generating pattern diversity during development and evolution, acting in broad domains but controlling localized cell diversification and pattern. Much remains to be learned about how Hox selector proteins generate cell-type diversity. In this study, regulatory specificity was investigated by dissecting the genetic and molecular requirements that allow the Hox protein Abdominal A to activate wingless in only a few cells of its broad expression domain in the Drosophila visceral mesoderm. The Dpp/Tgfß signal controls Abdominal A function, and Hox protein and signal-activated regulators converge on a wingless enhancer. The signal, acting through Mad and Creb, provides spatial information that subdivides the domain of Abdominal A function through direct combinatorial action, conferring specificity and diversity upon Abdominal A activity (Grienenberger, 2003).
This study provides a conceptual framework for understanding the molecular basis of regional Hox protein transcriptional activity. Dpp and Wg signaling subdivide the AbdA Hox domain, allowing activation of pointed (pnt) and opa target genes in the third and fourth midgut chambers, respectively. Based upon the data presented here, it is suspected that the localized activation of pnt and opa by AbdA also relies on direct enhancer integration of Hox and signaling inputs. Accordingly, a Hox/signaling combinatorial code functionally subdivides the domain where a single Hox protein is made, giving rise to discrete patterns of target gene activation. The structures of relevant cis-regulatory regions of AbdA target genes are instrumental for determining which signal is required to allow activation by AbdA. The pnt midgut enhancer would contain AbdA and Wg response elements and would be activated by AbdA specifically in the third midgut chamber through the combinatorial action of AbdA and the Drosophila Tcf/Arm transcriptional effector of Wg signaling. Similarly, the opa midgut enhancer would contain AbdA and Dpp response elements and would be activated only in the fourth gut chamber by AbdA, in this case because of an inhibitory effect of the Dpp-regulated transcription factor on AbdA activity (Grienenberger, 2003).
Members of the Eyes absent (Eya) protein family play important roles in tissue specification and patterning by serving as both transcriptional activators and protein tyrosine phosphatases. These activities are often carried out in the context of complexes containing members of the Six and/or Dach families of DNA binding proteins. eyes absent, the founding member of the Eya family is expressed dynamically within several embryonic, larval, and adult tissues of Drosophila. Loss-of-function mutations are known to result in disruptions of the embryonic head and central nervous system as well as the adult brain and visual system, including the compound eyes. In an effort to understand how eya is regulated during development, a genetic screen was carried out designed to identify genes that lie upstream of eya and govern its expression. This study identified a large number of putative regulators, including members of several signaling pathways. Of particular interest is the identification of both yan/anterior open and pointed, two members of the EGF Receptor (EGFR) signaling cascade. The EGFR pathway is known to regulate the activity of Eya through phosphorylation via MAPK. These findings suggest that this pathway is also used to influence eya transcriptional levels. Together these mechanisms provide a route for greater precision in regulating a factor that is critical for the formation of a wide range of diverse tissues (Salzer, 2010).
This report describes a genetic screen that identified factors that direct the expression of the retinal determination gene eyes absent to the developing embryonic head and eye imaginal disc. Putative regulators were identified by the loss or expansion of Eya protein distribution within the embryonic head of stage 9 loss-of-function mutants. The findings indicate multiple signaling cascades including Notch, Hedgehog, TGFβ, and the EGFR regulate eya expression. These results are consistent with previous studies identifying Hedgehog, Ras, and TGFβ as regulators of eya function in eye development. No mutations were recovered in any of known Wingless pathway members. This was slightly unexpected as Wnt signaling and eya are known to reciprocally regulate each other. This result could imply, however, that eya is regulated differently in diverse tissues (Salzer, 2010).
A screen similar to the one described in this study successfully identified the TGFβ pathway as an important upstream regulator of another retinal determination gene, dachshund. Of interest is the observation that the loss of TGFβ signaling has differential effects on eya and dac expression. In TGFβ mutant embryos ectopic dac expression was observed in cells of the visual primordium. However, eya expression remains unaffected in this tissue and is instead lost in the subsets of cells that give rise to the protocerebrum. These differential effects are interesting as eya and dac interact genetically within the retinal determination network. Therefore it seems that these regulatory relationships vary among different tissues. It also appears that the number of distinct signaling pathways that regulate eya expression outnumbers that of dac. This is unsurprising as the expression pattern of eya, when compared to dac, is considerably more dynamic, at least within the embryonic head (Salzer, 2010).
It was of particular interested to finding that mutations in spitz, argos, anterior open/yan and pointed, all members of the EGFR signaling pathway, altered the transcriptional pattern of eya. Previous work has demonstrated that the EGFR pathway post-translationally regulates Eya activity in the developing eye through phosphorylation via Ras/MAPK at two sites within the transactivation domain. Experiments in both flies and in insect cell culture indicate that phosphorylation augments, but is not absolutely essential, for either the transcriptional activation potential of Eya or for the induction of ectopic eyes in forced expression assays (Salzer, 2010).
These findings suggest that the EGFR pathway is also required to regulate eya transcription. This is consistent with findings that eya expression is lost in mago- clones, which reduce Ras signaling (Firth, 2009). Indeed, loss of aop/yan behind the morphogenetic furrow results in the higher levels of Eya and its facultative partner So. Both proteins are required for photoreceptor cell fate specification and maintenance. Elevated levels of Eya and So proteins in yan mutant clones are consistent with roles for Yan in suppressing photoreceptor cell fate during normal development. In yan clones, Eya protein levels are activated to significantly higher levels than that of So. One possible explanation for these results is that EGFR signaling may in fact regulate eya expression but not that of so. As EGFR signaling also regulates Eya activity, in a yan clone there may be a feedback loop that ultimately results in lowered levels of Eya phosphorylation. Reduced levels of the Eya phospho-protein, while still able to stimulate so transcription, may do so at a less efficient rate thereby leading to lower levels of ectopic So protein (Salzer, 2010).
Unexpectedly, it was found that dac, a putative downstream target of the So-Eya complex, is not up regulated in yan clones. Rather, dac expression is down-regulated when yan is removed. As So-Eya is thought to positively regulate dac expression this result is somewhat puzzling. The result does suggest that dac is regulated not only by the Eya-So complex but also by other mechanisms, possible through EGFR signaling and an intermediate repressor. The So-Eya-Dac subcircuit is under complex regulatory control. This study suggests that still greater complexity exists in the form of differential regulation by signal transduction cascades both at transcriptional and post-translational levels (Salzer, 2010).
The transcriptional repressor Yan prevents inappropriate responses to receptor tyrosine kinase signaling by outcompeting Pointed for access to target gene promoters. The molecular mechanism underlying downregulation of Yan involves CRM1-mediated nuclear export. A novel role in this context is defined for MAE, a co-factor previously implicated in facilitating MAPK phosphorylation of Yan. In addition to promoting Yan downregulation, MAE also participates in an inhibitory feedback loop that attenuates Pointed-P2 activation. Thus, it is proposed that MAE plays multiple independent roles in fine-tuning the levels of Pointed and Yan activity in accordance with changing RTK signaling conditions (Tootle, 2002).
MAPK-mediated recognition and phosphorylation of Yan at Serine 127 is thought to be facilitated by a protein called Modulator of the Activity of ETS (MAE). Mechanistically, MAE binds to Yan via a protein-protein interaction motif found at the N terminus of Yan and the C terminus of MAE, referred to as the Pointed Domain (PD). Interestingly, it has been suggested that MAE binds to the PD of PNT-P2, and enhances the transcriptional activation of PNT-P2; this has led to the proposal that MAE promotes PNT-P2 phosphorylation by MAPK. Thus, it has been speculated that by promoting phosphorylation events that simultaneously downregulate Yan and upregulate PNT-P2, MAE facilitates downstream responses to RTK signaling (Tootle, 2002).
In addition to promoting homotypic Yan-Yan interactions, PD-mediated
binding to heterologous proteins may also influence Yan localization and
activity. MAE, the only protein known to interact with the PD of Yan, appears to serve such a function. Co-immunoprecipitation experiments have confirmed that MAE can bind to Yan in the absence of signaling, and show that the complex is destabilized in the presence of RAS/MAPK activation. However, because MAE
inhibits Yan-mediated transcriptional repression, it is expected that, in the
absence of signaling, not all Yan will be bound to MAE. The finding that MAE
can also be co-immunoprecipitated with PNT-P2, suggests a mechanism for
sequestering MAE away from Yan to allow efficient repression and prevent
inappropriate differentiation in the absence of signaling (Tootle, 2002).
Upon activation of the RAS/MAPK cascade, dual phosphorylated MAPK enters
the nucleus and phosphorylates Yan, triggering a cascade of events that
ultimately leads to the removal of transcriptional repression. MAE is needed for MAPK-mediated phosphorylation of Yan at Serine 127 in vitro, the same site previously shown
to be critical for initiating Yan downregulation both in cell culture and in
vivo. This study sheds new light on the sequence of steps in this process. CRM1-mediated nuclear export is a necessary step in
downregulation of Yan. How is this achieved? A model is supported
whereby in response to pathway stimulation, the PNT-P2-MAE complex is
phosphorylated, releasing PNT-P2 to activate transcription and MAE to interact
with Yan. Binding to MAE inhibits the transcriptional repression of Yan, and may facilitate phosphorylation of serine 127 by activated MAPK, although
the order in which these two events happen remains to be determined. These data
suggest MAE then plays a third role in presenting Yan to CRM1, thereby
promoting nuclear export (Tootle, 2002).
The ultimate outcome of this complex series of events is abrogation of
Yan-mediated repression of target genes and freeing the promoters for
interaction with Pointed. In unstimulated cells, unphosphorylated PNT-P2
localizes to the nucleus in a complex with MAE, but is effectively out
competed for binding to target gene promoters by Yan. Upon
activation of the RAS/MAPK cascade, phosphorylation of PNT-P2 transforms it
into a potent transcriptional activator.
In vitro experiments show that MAE binding to PNT-P2 leads to activation
of transcription, and this is assumed to occur via MAE promoting MAPK
phosphorylation, and hence activation, of PNT-P2. It has been shown that PNT-P2 contains a MAPK binding site, suggesting PNT-P2
interacts directly with MAPK without requiring a facilitator protein.
Consistent with this second scenario, it has been found that MAE inhibits PNT-P2
transcriptional activation. However, it is formally possible that MAE could
have dual and antagonistic roles with respect to PNT-P2 regulation, first
stimulating its activity by promoting MAPK phosphorylation and later limiting
its ability to activate transcription. Definitive validation of either model
will require in vivo analysis of the role of MAE with respect to PNT-P2
regulation (Tootle, 2002).
Superficially, this proposed role in antagonizing PNT-P2 function seems to
disagree with the finding that loss of mae function suppresses the
rough eye phenotype of Sev-RASV12. However, in the absence of MAE,
Yan cannot be downregulated. Thus, the effect of loss of mae function
on PNT-P2 regulation is irrelevant in this context, since the target sites will
still be occupied by Yan. However, the dual function of MAE as both a positive
and a negative regulator of RTK signaling may explain the relatively weak
suppression of Sev-RASV12 and the fact that it has not been
isolated in any of the numerous RTK pathway-based genetic modifier
screens (Tootle, 2002).
In summary, these data lead to a model in which, in
unstimulated cells, Yan binds with high affinity to the DNA and blocks PNT-P2
from contacting and activating the promoters of downstream target genes. Upon stimulation by RAS, MAPK phosphorylation of Yan and PNT-P2 allows CRM1 to interact with and export Yan, in a process that disrupts Yan and MAE binding and disrupts the
PNT-P2-MAE complex, allowing PNT-P2 to bind to the DNA and activate
transcription. Free
MAE could then interact again with PNT-P2, resulting either in its removal
from the DNA, inhibition of transcriptional activation or interaction with a
phosphatase that returns it to an inactive state. Thus, a negative
feedback loop would be created to prevent runaway signaling by PNT-P2. An
alternative, and not necessarily mutually exclusive, mechanism with respect to
PNT-P2, is that the interaction of MAE with PNT-P2 might prevent efficient
phosphorylation by MAPK, thereby limiting the pool of activated PNT-P2 and
keeping the signaling response in check. It is likely that additional
co-factors that bind MAE, Yan and/or PNT-P2 will be required for fine-tuning
activation and downregulation in response to changing RTK signaling
conditions (Tootle, 2002).
In the context of TEL downregulation, it is interesting to note that no
mammalian orthologs of mae have been identified yet. However, a
second mammalian TEL-like gene, referred to as TEL2 or TELB, has been isolated. TEL2
also functions as a transcriptional repressor, is capable of oligomerizing
with itself and with TEL, and may thus serve as a regulator of TEL. Of
particular interest with respect to this work defining the role of MAE, TEL2
encodes six splice variants, one of which, TEL2a, yields a protein with just
the PD. TEL2a closely resembles the structure of MAE, and BLAST results show that the
PD of MAE is most closely related to the PD of TEL2, with 39% identity and 51%
similarity. Thus, it seems likely that TEL2a may regulate TEL activity by a
mechanism similar to that used by MAE for regulating Yan. With respect to the
interactions that have been demonstrated between PNT-P2 and MAE, it will be
interesting to investigate whether TEL2a also interacts with and regulates
other PD containing ETS family transcriptional activators, such as ETS1, the
mammalian ortholog of PNT-P2 (Tootle, 2002).
Inductive patterning mechanisms often use negative regulators to coordinate the effects and efficiency of induction. During Spitz EGF-mediated neuronal induction in the Drosophila compound eye and chordotonal organs, Spitz causes activation of Ras signaling in the induced cells, resulting in the activation of Ets transcription factor Pointed P2. Developmental roles are described for a novel negative regulator of Ras signaling, EDL/MAE (Modulator of the activity of Ets), a protein with an Ets-specific Pointed domain but not an ETS DNA-binding domain. The loss of EDL/MAE function results in a reduced number of photoreceptor neurons and chordotonal organs, suggesting a positive role in the induction by Spitz EGF. However, EDL/MAE functions as an antagonist of Pointed P2, by binding to its Pointed domain and abolishing its transcriptional activation function. Furthermore, edl/mae appears to be specifically expressed in cells with inducing ability. This suggests that inducing cells, which can respond to Spitz they themselves produce, must somehow prevent activation of Pointed P2. Indeed hyperactivation of Pointed P2 in inducing cells interferes with their inducing ability, resulting in the reduction in inducing ability. It is proposed that EDL/MAE blocks autocrine activation of Pointed P2 so that inducing cells remain induction-competent. Inhibition of inducing ability by Pointed probably represents a novel negative feedback system that can prevent uncontrolled spread of induction of similar cell fates (Yamada, 2003).
The edl/mae gene
was identified through enhancer trap lines that harbor P-element insertions at 55E. mae encodes a 177 amino acid polypeptide that contains
a region similar to the Pointed domain found in many Ets proteins. In contrast to all
other proteins that contain the Pointed domain, EDL/MAE lacks the conserved
DNA-binding domain, the ETS domain. Because of the potential function of
EDL/MAE in Ras/MAPK signaling, the expression pattern of
mae was examined in two tissues where Ets proteins function as downstream targets of Ras/MAPK signaling. In the eye imaginal disc mae mRNA is
expressed in clusters of cells in two rows in the morphogenetic furrow. Expression is seen
in a small number of cells in each cluster, with a spacing roughly
corresponding to that of the ommatidial clusters. To examine mae
expression at the cellular level, an mae enhancer trap line
maeJS was used that expresses lacZ in the eye imaginal disc. Expression of this mae-lacZ reporter
initiates in R8 cells within the morphogenetic furrow, corresponding to the
stage in which R8 induces R2 and R5. Subsequently, R2/R5, which act as the secondary source of
induction, also initiate edl/mae-lacZ expression at lower levels. During the development of the embryonic chordotonal organs, mae mRNA is present in chordotonal organ precursor (COP) C1-C5, but was undetectable in C6-C8. As in the eye
imaginal disc, mae expression is transient and disappears from the
COPs before they started dividing. Thus, in both the ommatidium and the
chordotonal organ, mae expression is detectable only in cells with
inducing ability (Yamada, 2003).
To address the role of mae in inducing cells, loss-of-function mutations were identified
in mae. The
maeJV line contains a P-element insertion in the
vicinity of the presumptive transcription initiation site of mae
and has viability of
5% in trans to a deletion of the 55E/F region, Df(2R)P34. Since this effect on viability is reverted upon excision of the P-element and is completely suppressed by a transgene containing the entire mae coding
region, maeJV represents a reduction of function
allele of mae. In addition, a lethal allele, maeL19, was generated that removes the entire mae gene. Both maeL19 homozygotes and
maeL19/Df(2R)P34 animals die as late
embryos or early larvae (Yamada, 2003).
Analysis of mae mutants reveals that in both the eye and
chordotonal organ, the loss of mae reduces the efficiency of
Spitz-mediated induction. In retinal sections of
maeJV/Df(2R)P34 and
maeJV/L19 animals, about 3% of ommatidia show
loss of photoreceptor cells, of the R1-R6 and R7 photoreceptor subtype. The R8 cell, which most strongly expresses mae expression within the ommatidium, is always present, even in ommatidia where other photoreceptor cells are missing. A similar
phenotype is seen in maeL19 mutant clones, which
entirely lack mae function. This phenotype is almost completely
rescued by an mae+ transgene. The requirement of
mae is more pronounced when the level of the inducing signal is
compromised. Star is a dosage-sensitive component of Spitz-mediated
induction in the eye, and is required for the transport of Spitz EGF to the
Golgi apparatus. In Star-/+ animals, 30% of ommatidia
show a reduction in the number of photoreceptor neurons, with the average
number of R1-R7 cells reduced per ommatidium of 0.39. When
maeJV/L19 mutation
is placed in the Star-/+ background, 65% of ommatidia
lacked at least one neuron, with 1.71 photoreceptor cells missing per
ommatidium on average. Similarly, the mae mutation enhances the
reduction in the number of photoreceptor neurons in a hypomorphic allele of
spitz. These synergistic effects of mae and Star/spitz suggest that mae participates in the induction of R1-R7 by Spitz EGF (Yamada, 2003).
Although the role of the
Pointed domain as the target of the MAPK phosphorylation is well established, the Pointed domain of EDL/MAE does not contain the consensus phosphorylation site and thus is unlikely to be regulated by the upstream signal. Emerging evidence indicates that this domain is also the site of protein-protein interaction, mediating homo- or hetero-oligomerization among Pointed domain-containing proteins. The in vivo significance of such oligomerization, however, has not been
demonstrated. EDL/MAE binding to Yan is required for
MAPK-mediated phosphorylation of Yan, leading to inactivation of Yan function as a repressor of Ets target genes. Since EDL/MAE has activities in the absence of Yan, EDL/MAE
must have targets other than YAN. The results of this study show that the binding of EDL/MAE
to the Pointed domain of PntP2 causes a profound effect on the activity of
Pnt; expression of EDL/MAE abrogates the activity of PntP2 as a transcription activator in culture cell transfection assays. This effect is
supported by misexpression studies in vivo, which show that EDL/MAE
misexpression causes phenotypes that mimic the loss of PntP2 function. Phenotypes of mae loss of function are also similar to the consequences of Pnt hyperactivation. It is proposed that EDL/MAE
acts by antagonizing PntP2 protein in photoreceptor neuronal differentiation
and chordotonal organ development (Yamada, 2003).
The EDL/MAE misexpression experiments in vivo support the idea
that EDL/MAE antagonizes, rather than promotes, PntP2 activity. The effects of EDL/MAE misexpression cannot be explained by the promotion of phosphorylation of Yan, because phosphorylation causes the inactivation of Yan, and
loss of yan produces effects that are the opposite of what has been
observed by EDL/MAE misexpression. The
opposite effects of EDL/MAE on PntP2-mediated transcription may be due to the
difference in the cell lines employed in the transfection assays. It is also
possible that EDL/MAE activity is used differently in diverse tissues; for
example, the effect seen on the ventral denticle belts in the embryonic
cuticle may be due to the promotion of Yan inactivation within the ventral
neuroectoderm, allowing PntP1 to function in the specification of medial fates (Yamada, 2003).
Within the developmental contexts examined in this study, mae
expression appears to be confined to cells with the ability to induce other
cells using Spitz EGF. This suggests that Mae may have a role in regulating
induction by Spitz. Secreted Spitz acts not only on the induced cells, but is
also received by the inducing cells themselves. Although the molecular events
leading to the activation of Pnt within the induced cells is well established,
whether the same regulatory cascade operates within the inducing cells had not
been studied. Hyperactivation of Pnt in inducing cells was found to have a
deleterious effect on induction; in the embryo, COP C3 loses expression of
rhomboid, a factor that is essential for the production of Spitz EGF.
Although inducing cells are positioned so that they receive highest levels of
Spitz EGF that they produce, they may possess a mechanism to prevent
hyperactivation of Pnt. The phenotypes of the mae loss-of-function
mutants and the effect of Pnt hyperactivation are similar in both ommatidial
and chordotonal organ development. Mae is thus likely to
be a part of the machinery that antagonizes PntP2 to prevent the negative
effect of Pnt on induction in the inducing cells (Yamada, 2003).
A major challenge to the proposal that Mae acts in inducing cells by
antagonizing Pnt is that the loss of Mae function produces a rather mild
effect on induction; most ommatidia are constructed normally in the
mae null clone, and the loss of scolopidia is observed in only 25% of
hemisegments in mae- embryos. Since this phenotype is weaker
than that which can be achieved by an artificial activation of pnt
using the GAL4/UAS-mediated overexpression, it can be argued that the
role that Mae plays in repressing Pnt function might be minor. For
example, inducing cells may possess multiple mechanisms to inhibit Pnt
activation, and deleting Mae alone may not lead to full activation. However,
it is likely that the overexpression paradigm results in a high level of
Pnt activation that cannot be achieved under physiological conditions. It is
also possible that Mae does not completely block Pnt activation in inducing
cells, but just needs to keep the level from reaching the state that results in interference of induction (Yamada, 2003).
This raises the question when and where Pnt uses the activity to curb
induction. During both ommatidial assembly and the development of the
chordotonal system, Pnt promotes neuronal development in the induced cells. It is
suggested that Pnt may also suppress inducing ability in such cells. This would
create a negative feedback loop so that the cell, once induced, does not itself
acquire inducing ability. Although such a mechanism would be effective in
preventing uncontrolled spread of homeogenetic induction, the need for such
regulatory system arises only if induced cells also have the opportunity to
acquire inducing ability. This is indeed the case for R2/R5; these cells form
via induction by R8, and then express rhomboid and become a secondary
source of Spitz EGF. Other cells, such as R3/R4 could also potentially become
inducers, because they reside within the proneural cluster
prior to the onset of induction and have probably experience Atonal expression, which
promotes rhomboid expression. The repressive effect of Pnt on rhomboid would thus be a mechanism to safeguard against the potential activation of rhomboid by Atonal within the proneural cluster. Pnt may cause this repression via activating expression of a repressor or by acting as a repressor itself (Yamada, 2003).
The inhibition of rhomboid expression is not the only way that Pnt
negatively regulates induction. In the eye, a rhomboid paralog
roughoid plays a critical role in generating mature Spitz EGF. It is
possible that roughoid may also be regulated by Pnt to control
induction. Furthermore, upon activation of Ras signaling, induced cells produce negative regulators of the Ras pathway, such as Sprouty, Argos and Kekkon, generating negative feedback loops. Because Argos is a secreted
antagonist of Spitz EGF, its production by inducing cells could be detrimental for induction. The inhibition of Pnt function by Mae may also serve to reduce Argos production in the inducing cells, allowing efficient
induction (Yamada, 2003).
Although induction in Drosophila eye and the chordotonal organ
is 'homeogenetic' in the sense that both the inducing cell and
the induced cell are of the same cell type (photoreceptor neurons or COPs),
they differ in genetic and molecular properties. Although neuronal
specification of founder cells R8 and C1-C5 requires atonal function
but not pnt, induced cells R1-R7 and C6-C8 depend on Pnt activation
and need atonal only indirectly. In addition, the induction itself
generates a dichotomy between cells with inducing ability and those without,
because induced cells acquire a different character (lack of inducing ability) from the inducing cell. Inducing cells, however, are prevented from expressing these characteristics through the repression of Pnt function by Mae. Other
instances of homeogenetic induction may also possess such properties, in order
to generate cellular diversity, rather than equivalence. During the
development of muscle progenitors in Drosophila, the size of the
inductive field is defined by a group of cells similar to the proneural
cluster; a small number of founder muscles are selected based on the activity
of the bHLH transcription factor lethal of scute and
EGF-mediated induction. Because mae is also expressed in a subset of
muscle progenitors, it may act in founder muscle selection in
a similar way as it does in the eye and chordotonal organs (Yamada, 2003).
Previous phylogenetic analysis reveals that the Ets protein family
originated early during metazoan evolution and most of the functional
diversity was already established prior to the separation of protostomes and
deuterostomes. Although it is likely that such an ancestral Ets protein
already contained a Pointed domain, the Pointed domain of Mae could not be
classified as similar to any of the previously known Ets protein subclasses. This suggests that a
Mae-like protein may have already existed before the divergence of Ets
proteins. It is tempting to speculate that Mae or Mae-like proteins
may regulate inductive processes in other developmental processes in
Drosophila and vertebrates (Yamada, 2003).
During Drosophila melanogaster eye development, signaling through receptor tyrosine kinases (RTKs) leads to activation of Rolled, a mitogen activated protein tyrosine kinase. Key nuclear targets of Rolled are two
antagonistic transcription factors: Yan, a repressor, and Pointed-P2 (Pnt-P2),
an activator. A critical regulator of this process, Mae, can interact with both
Yan and Pnt-P2 through their SAM domains. Although earlier work showed that Mae
derepresses Yan-regulated transcription by depolymerizing the Yan polymer, the
mechanism of Pnt-P2 regulation by Mae remained undefined. This study finds
that efficient
phosphorylation and consequent activation of Pnt-P2 requires a three-dimensional
docking surface on its SAM domain for the MAP kinase, Rolled. Mae binding to
Pnt-P2 occludes this docking surface, thereby acting to downregulate Pnt-P2
activity. Docking site blocking provides a new mechanism whereby the cell can
precisely modulate kinase signaling at specific targets, providing another layer
of regulation beyond the more global changes effected by alterations in the
activity of the kinase itself (Qiao, 2005).
The findings in this work, combined with prior results, suggest how Yan, Pnt-P2 and Mae work together to determine cell fate in response to the activation of the
RTK pathways. In the absence of MAPK activation, unphosphorylated Yan polymers
outcompete Pnt-P2 for access to ETS-binding sites, creating a repressed state of
the target genes. Upon RTK activation, activated phospho-Rolled MAPK
enters the nucleus and phosphorylates a small amount
of the monomeric Yan. By binding to Yan and blocking polymer interactions, basal
levels of Mae likely help to maintain an appropriate concentration of free Yan
in the nucleus. Phosphorylation of Yan triggers its
cytoplasmic export with the help of CRM1. The decrease in
free Yan then drives the equilibrium away from the DNA-bound polymer. Meanwhile,
the antagonist of Yan, Pnt-P2, becomes activated by Rolled MAPK phosphorylation,
presumably through enhanced binding to transcriptional coactivators CREB binding
protein (CBP) and p300, which act to bridge the DNA-bound transcription factors
and the basal transcription complex. Since Mae is regulated by
Yan and Pnt-P2, inactivation of Yan and activation of
Pnt-P2 leads to increasing amounts of Mae and further removal of Yan repression.
These processes could easily lead to runaway expression of differentiation
genes. As revealed in this study, however, another job of Mae is to block the
MAPK/Rolled docking site on Pnt-P2, inhibiting Pnt-P2 phosphorylation, which in
turn attenuates transcriptional activity. This negative feedback loop ensures a
level of transcription appropriate for normal development (Qiao, 2005).
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