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 Drosophila transcription factor Tramtrack (Ttk) is involved in a wide range of developmental decisions, ranging from early embryonic patterning to differentiation processes in organogenesis. Given the wide spectrum of functions and pleiotropic effects that hinder a comprehensive characterisation, many of the tissue specific functions of this transcription factor are only poorly understood. Multiple roles of Ttk have been discovered in the development of the tracheal system on the morphogenetic level. This study sought to identify some of the underlying genetic components that are responsible for the tracheal phenotypes of Ttk mutants. Gene expression changes were profiled after Ttk loss- and gain-of-function in whole embryos and cell populations enriched for tracheal cells. The analysis of the transcriptomes revealed widespread changes in gene expression. Interestingly, one of the most prominent gene classes that showed significant opposing responses to loss- and gain-of-function was annotated with functions in chitin metabolism, along with additional genes that are linked to cellular responses, which are impaired in ttk mutants. The expression changes of these genes were validated by quantitative real-time PCR and further functional analysis of these candidate genes and other genes also expected to control tracheal tube size revealed at least a partial explanation of Ttk's role in tube size regulation. The computational analysis of tissue-specific gene expression data highlighted the sensitivity of the approach and revealed an interesting set of novel putatively tracheal genes (Rotstein, 2011).
The microarray results confirm previous observations and provide new data for the different Ttk tracheal requirements. For instance, the transcription factor Esg, which plays a pivotal role in fusion cell identity specification is lost when Ttk is over-expressed, but still present in Ttk loss-of-function conditions. The microarray data confirm this regulation, and in addition identifies other genes already shown to directly or indirectly modulate fusion fate as Ttk targets, like hdc, CG15252, or pnt. Similarly, polychaetoid (pyd), which has been identified as a Ttk target in in situ hybridisation analysis, is differentially expressed in the microarray conditions (it should be noted however that pyd is not formally a candidate due to inconsistencies between microarray replicates; in fact only splice variant pyd-RE shows a response), explaining in part the requirement of Ttk in tracheal cell intercalation. In addition, it is tempting to speculate about other candidate targets to mediate this function of Ttk in intercalation, like canoe for instance, which has been recently shown to act with pyd during embryogenesis (Rotstein, 2011).
The microarray analysis pointed to a regulation of the Notch signalling pathway or its activity by Ttk, likely acting as a negative regulator. In contrast, it has previously been observed that Ttk acts as a downstream effector of N activity in the specification of different tracheal identitites. Indeed, it was shown that Ttk levels depend on N activity in such a way that when N is active, Ttk levels are high, whereas when N is not active, Ttk levels are low. Thus, lower levels of Ttk were observed in tracheal fusion cells due to the inactivity of N there. Therefore, Ttk acts as a target of N in fusion cell determination. Now, the results of the microarray add an extra level of complexity to the Ttk-N interaction. The observation that in turn Ttk also transcriptionally regulates several N pathway components suggests that Ttk is involved in a feedback mechanism that could play a pivotal role in biasing or amplifying N signalling outcome (Rotstein, 2011).
Interactions between Ttk and N have been observed in different developmental contexts, emphasising the importance of such regulations. Several examples illustrate the regulation, either positive or negative, of Ttk expression by N activity. In addition, a recent report provides evidence of a regulation of N activity by Ttk and proposes a mutually repressive relationship between N and Ttk which would also involve Ecdysone signalling. The results are consistent with many of these observations, indicating that they could represent general molecular mechanisms of morphogenesis. Thus, tracheal cell specification could serve as an ideal scenario to investigate the intricate, and often contradictory, interactions between N and Ttk and the complexity of N signaling (Rotstein, 2011).
A dynamic balance between stem cell maintenance and differentiation paces generation of post-mitotic progeny during normal development and maintenance of homeostasis. Recent studies show that Notch plays a key role in regulating the identity of neuroepithelial stem cells, which generate terminally differentiated neurons that populate the adult optic lobe via the intermediate progenitor cell type called neuroblast. Thus, understanding how Notch controls neuroepithelial cell maintenance and neuroblast formation will provide critical insight into the intricate regulation of stem cell function during tissue morphogenesis. This study shows that a low level of Notch signaling functions to maintain the neuroepithelial cell identity by suppressing the expression of pointedP1 gene through the transcriptional repressor Anterior open. Increased Notch signaling, which coincides with transient cell cycle arrest but precedes the expression of PointedP1 in cells near the medial edge of neuroepithelia, defines transitioning neuroepithelial cells that are in the process of acquiring the neuroblast identity. Transient up-regulation of Notch signaling in transitioning neuroepithelial cells decreases their sensitivity to PointedP1 and prevents them from becoming converted into neuroblasts prematurely. Down-regulation of Notch signaling combined with a high level of PointedP1 trigger a synchronous conversion from transitioning neuroepithelial cells to immature neuroblasts at the medial edge of neuroepithelia. Thus, changes in Notch signaling orchestrate a dynamic balance between maintenance and conversion of neuroepithelial cells during optic lobe neurogenesis (Weng, 2012).
A deregulated conversion of neuroepithelial cells into neuroblasts perturbs formation of the neuronal network and will almost certainly lead to visual impairment of the adult fly. Thus, a dynamic balance between neuroepithelial cell maintenance and differentiation plays a pivotal role during morphogenesis of the optic lobe. This study provides evidence that changes in Notch signaling regulate the dynamic balance between maintenance of neuroepithelial cells and formation of neuroblasts. A low level of Notch signaling maintains the neuroepithelial cell identity by triggering Aop-dependent repression of the pntP1 gene. Transient up-regulation of Notch signaling in transitioning neuroepithelial cells raises their threshold of response to PntP1 preventing them from precociously converting into immature neuroblasts. Finally, abrupt down-regulation of Notch signaling together with a high level of PntP1 trigger the conversion from transitioning neuroepithelial cells into immature neuroblasts at the medial edge of neuroepithelia. Thus, interplay between changes in Notch signaling and transient up-regulation of pntP1 orchestrates synchronous and progressive formation of neuroblasts in a medial-to-lateral orientation across the entire neuroepithelial swath (Weng, 2012).
Lack of Notch reporter transgene expression throughout neuroepithelia located laterally from transitioning neuroepithelial cells has been perplexing in light of recent studies reporting that Notch signaling is necessary for maintenance of their identity. One possibility might be that these Notch reporter transgenes including E(spl)mγ-GFP might not contain all necessary regulatory response elements to respond to Notch signaling in most neuroepithelial cells. Alternatively, the level of Notch signaling might simply be too low to activate the expression of the Notch reporter transgene. The second hypothesis is favored for the following reasons. Since over-expression of Notchintra is sufficient to trigger robust cell autonomous expression of E(spl)mγ-GFP in neuroepithelia located laterally from transitioning neuroepithelial cells, this transgene does contain all necessary regulatory elements to respond to a high level of Notch signaling. Furthermore, the Notch ligand Delta is expressed in a low level throughout neuroepithelia located laterally from transitioning neuroepithelial cells and Delta likely functions to trans-activate Notch signaling in these cells. In the context of trans-activation of Notch signaling by Delta, the level of the ligand correlates with the level of signaling output. Taken together, it is concluded that maintenance of the neuroepithelial cell identity requires a low level of Notch signaling (Weng, 2012).
It is proposed that Notch maintains the identity of neuroepithelial cells by activating Aop-dependent repression of the pntP1 gene. The Suppressor of Hairless protein, which is necessary for activating transcription of Notch targets genes, directly binds to the promoter of the aop gene. Furthermore, removing the Notch or aop function triggered premature conversion of neuroepithelia into neuroblasts whereas over-expressing Notch or aop prevented conversion of neuroepithelial cells into neuroblasts. Most importantly, over-expression of aop suppressed premature differentiation of Notch mutant neuroepithelial cells. Finally, heterozygosity of the pntP1 gene completely suppressed premature conversion of neuroepithelial cells into neuroblasts in a hypomorphic aop mutant genetic background. These data lead to the conclusion that Notch signaling maintains the identity of neuroepithelial cells by activating an Aop-dependent repression of pntP1. In the future, analyses of Notch and pntP1 double mutants will be necessary to confirm this regulatory mechanism (Weng, 2012).
Down-regulation of Notch signaling is necessary for formation of neuroblasts, so transient up-regulation of Notch signaling in transitioning neuroepithelial cells appears rather counterproductive. One possibility might be that up-regulation of Notch signaling paces the conversion from transitioning neuroepithelial cells into neuroblasts by increasing their threshold of response to PntP1. Consistent with this hypothesis, constitutively activated Notch signaling prevented transitioning neuroepithelial cells from becoming converted into neuroblasts despite expressing PntP1. This hypothesis was further supported by co-expression of pntP1 overcoming the blockade by constitutively activated Notch signaling and restoring conversion of transitioning neuroepithelial cells into neuroblasts. Thus, it is proposed that up-regulation of Notch signaling in transitioning neuroepithelial cells raises their threshold of response to PntP1 and functions to prevent them from becoming converted into immature neuroblasts precociously. Such an elaborated mechanism only permits transitioning neuroepithelial cells expressing the highest level of PntP1 to convert into immature neuroblasts. This mechanism is consistent with a recent study reporting that the EGF ligand is processed and secreted by cells near the medial edge of the optic lobe neuroepithelia. As a result of simple diffusion, transitioning neuroepithelial cells at the medial edge of neuroepithelia will be exposed to the highest level of the EGF ligand and will express the highest level of PntP1. As such, EGF signaling likely creates a vector field establishing the directionality of conversion from neuroepithelial cells into neuroblasts whereas Notch signaling refines the functional output of EGF signaling by raising the threshold response to PntP1 (Weng, 2012).
Many important questions arise from this highly plausible mechanism by which the interplay between Notch and EGF signaling paces synchronous conversion of neuroepithelial cells into neuroblasts one row at a time. This model will predict that immature neuroblasts immediately adjacent to transitioning neuroepithelial cells should secrete the processed EGF ligand. However, the antibody specific for the Rhomboid (Rho) protease required for proteolytic activation of the EGF protein is currently unavailable and a genomic fragment encompassing the rho-1 locus tagged with YFP did not show detectable expression in the larval optic lobe. Alternatively, a recent study shows that pntP1 is a direct target of Notch in vivo. Thus, up-regulation of Notch signaling might directly activate transcription of the pntP1 gene in transitioning neuroepithelial cells. Since Notch signaling becomes abruptly down-regulated at the medial edge of neuroepithelia, it is highly possible that the threshold of response to PntP1 also becomes lowered in the same cells. Thus, the pre-existing level of PntP1 protein will likely be sufficient to trigger the conversion from transitioning neuroepithelial cells into immature neuroblasts. More experiments including identification of the cell type from which the processed EGF ligand is released and a direct test to confirm the role of EGF signaling during conversion of neuroepithelia into neuroblasts will be key to distinguish these two possible mechanisms (Weng, 2012).
The fat-hippo signaling mechanism controls tissue growth by regulating proliferation and cell death and promotes timely differentiation of optic lobe neuroepithelial cells). While inactivation of fat-hippo signaling delays conversion of neuroepithelia into neuroblasts, removal of the downstream effecter yorkie only accelerates the conversion near the medial edge of the optic lobe neuroepithelia. Thus, fat-hippo signaling likely functions as a gatekeeper to prevent over-growth of optic lobe neuroepithelia by triggering transient cell cycle arrest. Intriguingly, transient cell cycle arrest precedes increased Notch signaling in transitioning neuroepithelial cells. Detailed studies in the future will be necessary to determine whether activation of the fat-hippo signaling might contribute to increased Notch signaling in transitioning neuroepithelial cells (Weng, 2012).
beta amyloid protein precursor-like (Appl), the ortholog of human APP, which is a key factor in the pathogenesis of Alzheimer's disease, was found in a genome-wide expression profile search for genes required for Drosophila R7 photoreceptor development. Appl expression was found in the eye imaginal disc and it is highly accumulated in R7 photoreceptor cells. The R7 photoreceptor is responsible for UV light detection. To explore the link between high expression of Appl and R7 function, Appl null mutants were analyzed and reduced preference for UV light was found, probably because of mistargeted R7 axons. Moreover, axon mistargeting and inappropriate light discrimination are enhanced in combination with neurotactin mutants. R7 differentiation is triggered by the inductive interaction between R8 and R7 precursors, which results in a burst of Ras1/MAPK, activated by the tyrosine kinase receptor Sevenless. Therefore, whether Ras1/MAPK is responsible for the high Appl expression was examined. Inhibition of Ras1 signaling leads to reduced Appl expression, whereas constitutive activation drives ectopic Appl expression. Appl was shown to be directly regulated by the Ras/MAPK pathway through a mechanism mediated by PntP2, an ETS transcription factor that specifically binds ETS sites in the Appl regulatory region. Zebrafish appb expression increased after ectopic fgfr activation in the neural tube of zebrafish embryos, suggesting a conserved regulatory mechanism (Mora, 2013).
Two main conclusions can be drawn from this work. First, Drosophila Appl is involved in R7 axonal targeting. Moreover, the finding that the Appl loss-of-function defects are enhanced when combined with Nrt heterozygous mutant suggest that Appl acts at the membrane of R7, where it interacts with other proteins such as Nrt. Second, Appl activation downstream of the RTK/Ras1 is independent of neural specification, occurs in vivo, and is mediated by direct binding of PntP2 to ETS sequences in the Appl regulatory region (Mora, 2013).
Together, these findings may provide insights into the pathogenesis of neurological disorders such as Alzheimer's disease. The β-amyloid peptides, which accumulate in the amyloid plaques found in the brain of Alzheimer's disease patients, are produced after APP proteolysis. However, Alzheimer's disease has not only been associated to the production of the primary component Aβ by proteolysis of APP, but also by transcriptional regulation. Increased APP transcription underlies the phenotype in some cases of familial Alzheimer's disease. In addition, overexpression of APP appears to be responsible for the early onset of Alzheimer's disease in individuals with Down syndrome. Thus, the current results open the possibility to explore whether in some cases of Alzheimer's disease a burst of RTK/Ras1/MAPK occurs and whether this signaling activity ends with high APP accumulation (Mora, 2013).
Amyloid β peptides are known to be involved in vision dysfunction caused by age-related retinal degeneration in mouse models. Thus, the current in vivo observations could be the basis for further research in mammalian models for neurodegenerative retinal disorders that share several pathological features with Alzheimer's disease (Mora, 2013).
Epithelial renewal in the Drosophila intestine is orchestrated by Intestinal Stem Cells (ISCs). Following damage or stress the intestinal epithelium produces ligands that activate the epidermal growth factor receptor (EGFR) in ISCs. This promotes their growth and division and, thereby, epithelial regeneration. This study demonstrates that the HMG-box transcriptional repressor, Capicua (Cic), mediates these functions of EGFR signaling. Depleting Cic in ISCs activated them for division, whereas overexpressed Cic inhibited ISC proliferation and midgut regeneration. Epistasis tests showed that Cic acted as an essential downstream effector of EGFR/Ras signaling, and immunofluorescence showed that Cic's nuclear localization was regulated by EGFR signaling. ISC-specific mRNA expression profiling and DNA binding mapping using DamID indicated that Cic represses cell proliferation via direct targets including string (Cdc25), Cyclin E, and the ETS domain transcription factors Ets21C and Pointed (pnt). pnt was required for ISC over-proliferation following Cic depletion, and ectopic pnt restored ISC proliferation even in the presence of overexpressed dominant-active Cic. These studies identify Cic, Pnt, and Ets21C as critical downstream effectors of EGFR signaling in Drosophila ISCs (Jin 2015).
It is well established that EGFR signaling is essential to drive ISC growth and division in the fly midgut. However, the precise mechanism via which this signal transduction pathway activates ISCs has remained a matter of inference from experiments with other cell types. Moreover, despite a vast literature on the pathway and ubiquitous coverage in molecular biology textbooks, the mechanisms of action of the pathway downstream of the MAPK are not well understood for any cell type. From this study, a model is proposed (see Model for Cic control of Drosophila ISC proliferation). Multiple EGFR ligands and Rhomboid proteases are induced in the midgut upon epithelial damage, which results in the activation of the EGFR, RAS, RAF, MEK, and MAPK in ISCs. MAPK phosphorylates Cic in the nucleus, which causes it to dissociate from regulatory sites on its target genes and also translocate to the cytoplasm. This allows the de-repression of target genes, which may then be activated for transcription by factors already present in the ISCs. The critical Cic target genes identified in this study include the cell cycle regulators stg (Cdc25) and Cyclin E, which in combination are sufficient to drive dormant ISCs through S and M phases, and pnt and Ets21C, ETS-type transcriptional activators that are required and sufficient for ISC activation (Jin 2015).
Upon damage, activated EGFR signaling mediates activation of ERK, which phosphorylates Cic, and relocates it to the cytoplasm. As a result, stg, CycE, Ets21C and pnt transcription are relieved from Cic repression, and induce ISC proliferation (Jin 2015).
Although this study found more than 2000 Cic binding sites in the ISC genome, not all of the genes associated with these sites were significantly upregulated, as assayed by RNA-Seq, upon Cic depletion. One possible explanation for this is that Cic binding sites from DamID-Seq were also associated with other types of transcription units (miRNAs, snRNAs, tRNAs, rRNAs, lncRNAs) that were not scored for activation by the RNA-Seq analysis. Indeed a survey of the Cic binding site distributions suggests this. This might classify some binding sites as non-mRNA-associated. However, it is also possible that many Cic target genes may require activating transcription factors that are not expressed in ISCs. Such genes might not be strongly de-repressed in the gut upon Cic depletion (Jin 2015).
In other Drosophila cells MAPK phosphorylation is thought to directly inactivate the ETS domain repressor Yan, and to directly activate the ETS domain transcriptional activator Pointed P2 (PNTP2). In fact Pnt and Yan have been shown to compete for common DNA binding sites on their target genes. Thus, previous studies proposed a model of transcriptional control by MAPK based solely on post-translational control of the activity of these ETS factors. However, this study found that Pnt and Ets21C were transcriptionally induced by MAPK signaling, and could activate ISCs if overexpressed, and that depleting yan or pntP2 had no detectable proliferation phenotype. In addition, overexpression of PNTP2 was sufficient to trigger ISC proliferation, suggesting either that basal MAPK activity is sufficient for its post-translational activation, or that PNTP2 phosphorylation is not obligatory for activity. Moreover, pntP2 loss of function mutant ISC clones had no deficiency in growth even after inducing proliferation by P.e. infection, which increases MAPK signaling. These observations indicate that the direct MAPKāPNTP2 phospho-activation pathway is not uniquely or specifically required for ISC proliferation. These results suggest instead that transcriptional activation of pnt and Ets21c via MAPK-dependent loss of Cic-mediated repression is the predominant mode of downstream regulation by MAPK in midgut ISCs (Jin 2015).
In addition to activating ISCs for division, EGFR signaling activates them for growth. Previous studies showed loss of EGFR signaling prevented ISC growth and division, and that ectopic RasV12 expression could accelerate the growth not only of ISCs but also post-mitotic enteroblasts. Similarly, this study shows that loss of cic caused ISC clones to grow faster than controls, by increasing cell number as well as cell size. For instance, increased size of GFP+ ISCs and EBs was observed when cic-RNAi was induced by the esgts or esgtsF/O systems. Therefore, in a search for Cic target genes probable growth regulatory genes such as Myc, Cyclin D, the Insulin/TOR components InR, PI3K, S6K and Rheb, Hpo pathway components, and the loci encoding rRNA, tRNAs and snRNAs were specifically checked. It was found that Cic bound to the InR, Akt1, Rheb, Src42A and Yki loci. However, of these only InR mRNA was significantly upregulated in Cic-depleted progenitor cells. In surveying the non-protein coding genome, it was found that Cic had binding sites in many loci encoding tRNA, snRNA, snoRNA and other non-coding RNAs, though not in the 28S rRNA or 5S rRNA genes. Due to the method used for RNA-Seq library production, RNA expression profiling experiments could not detect expression of these loci, and so it remains to be tested whether Cic may regulate some of those non-coding RNA's transcription to control cell growth. It is also possible that Cic controls cell growth regulatory target genes indirectly, for instance via its targets Ets21C and Pnt, which are also strong growth promoters in the midgut. But given that no conclusive model can be drawn from the data regarding how Cic restrains growth, one must consider the possibility that ERK signaling stimulates cell growth via non-transcriptional mechanisms, as proposed by several studies (Jin 2015).
The critical role of Cic as a negative regulator of cell proliferation in the fly midgut is consistent with its tumor suppressor function in mammalian cancer development. Also consistent with the current findings are the observations that the ETS transcription factors ETV1 and ETV5 are upregulated in sarcomas that express CIC-DUX, an oncogenic fusion protein that functions as a transcriptional activator, and that knockdown of CIC induces the transcription of ETV1, ETV4 and ETV5 in melanoma cells. Moreover the transcriptional regulation by ETS transcription factors is important in human cancer development. Their expression is induced in many tumors and cancer cell lines. For example, ERG, ETV1, and ETV4 can be upregulated in prostrate cancers, and ETV1 is upregulated in post gastrointestinal stromal tumors and in more than 40% of melanomas. In addition, the mRNA expression of these ETS genes was correlated with ERK activity in melanoma and colon cancer cell lines with activating mutations in BRAF (V600E), such that their expression decreased upon MEK inhibitor treatment. Furthermore, overexpression of the oncogenic ETS proteins ERG or ETV1 in normal prostate cells can activate a Ras/MAPK-dependent gene expression program in the absence of ERK activation. These cancer studies imply that there is an unknown factor that links Ras/Mapk activity to the expression of ETS factors, and that some of the human ETS factors might act without MAPK phosphorylation, as does Drosophila PntP1. Combining the knowledge of Cic with what was previously known about CIC in tumor development, it is proposed that CIC is the unknown factor that regulates ETS transcription factors in Ras/MAKP-activated human tumors (Jin 2015).
In summary, this study has elucidated a mechanism wherein Cic controls the expression of the cell cycle regulators stg (Cdc25) and Cyclin E, along with the Ets transcription factor Pnt, and perhaps also Ets21C, by directly binding to regulatory sites in their promoters and introns. Using genetic tests it was shown that these interactions are meaningful for regulating stem cell proliferation. Therefore, it is suggested that human CIC may also lead to the transcriptional induction of cell cycle genes and ETS transcription factors in RAS/MAPK activated- or loss-of-function-CIC tumors such as brain or colorectal cancers (Jin 2015).
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).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
pointed : Biological Overview
| Evolutionary Homologs
| Targets of Activity
| Developmental Biology
| Effects of Mutation
| References
Society for Developmental Biology's Web server.