pointed
Although there is a single EGF receptor in Drosophila, multiple ligands activate it. This
work explores the role of two ligands, Spitz and Vein, in the embryonic ventral ectoderm. Spitz is a
potent ligand, whereas Vein is an intrinsically weak activating ligand. Prior to gastrulation, vein is expressed in the
future neuroectoderm. This early phase of expression appears to cooperate with Spitz in the induction
of medial neuroblast cell fates. Following gastrulation at stage 9, vein expression in the
neuroectoderm becomes confined to three to four cell rows on each side of the midline. This pattern
gradually restricts, such that by stage 11 only one cell row on each side of the midline expresses vein. Secreted Spitz,
emanating from the midline, triggers expression of vein in the ventral-most cell rows, by inducing
expression of the ETS domain transcription factor Pointed P1. In the absence of Vein, lateral cell fates
are not induced when Spitz levels are compromised. The positive feedback loop of Vein generates a
robust mechanism for patterning the ventral ectoderm (Golembo, 1999).
Induction of gene expression by Egfr in the ventral-most rows of cells occurs
for the argos, orthodenticle, and tartan genes. Induction is obtained through inactivation
of Yan, an ETS domain transcriptional repressor, and induction of Pointed P1, an ETS domain
transcriptional activator. To test if induction of vein by Egfr is also mediated by Pointed P1, vein expression was examined in pointed mutant embryos. Traces of expression are observed in the
midline in stage 10 embryos only, whereas no expression is displayed by the ventral-most and lateral
ectodermal cell rows at stage 11, thus showing that vein induction is defective in pointed mutants.
Elimination of Pointed activity can also be obtained in the following manner: an activated form of Yan,
in which the inactivating MAP kinase phosphorylation sites have been mutated, blocks the activity of Pointed by competing for the same DNA-binding sites. Indeed, when activated, Yan is ectopically expressed in the Kruppel domain the endogenous
expression of vein is abolished in that region. To examine if Pointed P1 is sufficient for
induction, vein expression was examined in embryos in which Pointed P1 was expressed in the Kruppel
domain. Indeed, expression of vein in the same domain is observed. These results
demonstrate that under conditions of ectopic expression, Pointed P1 is necessary and sufficient for
vein expression (Golembo, 1999).
During oogenesis, dorsal follicle cells differentiate into either
appendage-producing or midline cells, resulting in patterning in the dorsal follicle cell layer. Pointed, an ETS transcription factor, is required in dorsal follicle cells for
this patterning. Loss of pointed results in the loss of midline cells and an excess of
appendage-forming cells, a phenotype associated with overactivation of the EGF receptor
pathway in the dorsal region. Overexpression of pointed leads to a phenotype similar to that
generated by loss of the EGF receptor pathway. This suggests that Pointed normally
down-regulates EGF receptor signaling in the midline to generate patterning in the dorsal
region. pointed P1 transcript expression, beginning at stage 6-7, is induced by the EGF receptor pathway. By stage 8, expression is restricted to posterior follicle cells. During stage 9-10, P1 is detected in dorsal-anterior and posterior follicle cells. During later stages, both these expression patterns refine into two areas: two dorsal patches and two posterior half-circles. P2 transcript expression is also detected during oogenesis, both in the germ line and follicle cells. Follicle cell expression is observed in stage 9-10 egg chambers in a pattern resembling the early P1 expression pattern in dorsal-anterior follicle cells. During later stages, P2 expression is restricted to two groups of anterior dorsolateral follicle cells that flank the oocyte nucleus. Whereas two dorsolateral dorsal appendages are detected in control eggs, pointed mutant appendages are four times wider than a single wild type appendage, suggesting that the phenotype does not result from two appendages fusing together, but rather fromcells in the middle region taking on an appendage-producing cell fate. These
data indicate a novel antagonistic function for Pointed in oogenesis; in response to activation
of the EGF receptor, pointed is expressed and negatively regulates the EGF receptor
pathway (Morimoto, 1996).
The specific tracheal branching defects observed in unplugged mutant
embryos prompted an examination of the relationship between unpg
and pointed (pnt); pnt is expressed in tracheal placodes
and in the developing tracheal branches. In the absence of pnt gene function, tracheal cells fail to migrate and branches do not extend to target tissues. In particular, stalling of the ganglionic branches at the ventral oblique musculature in hypomorphic pnt embryos is reminiscent
of the most extreme phenotype observed in unpg embryos.
To determine the regulatory relationship between pnt and unpg genes, pnt homozygous embryos were immunostained with Unplugged-specific antibody, in order to follow the fate of the ganglionic
branches. At stage 12.5, ganglionic branch precursor cells migrating toward the CNS are clearly visible in wild-type embryos. In pnt mutant embryos, no migratory cells
that are expressing Unpg protein can be detected; however, a few precursor cells accumulating low levels of Unpg protein occasionally can be identified. By stage 14, the ganglionic branches of normal embryos are well
developed, as is evident from a group of 8 to 9 unpg-expressing
cells along the ventrolateral region of each hemisegment. In pnt mutant embryos, only 3 to 4
unpg-expressing cells can be detected; these unpg-expressing
cells are clustered in a group suggesting that the precursor cells remain immobile and fail to extend from the
tracheal pits. These results suggest that the ganglionic branch phenotype in pointed embryos may be in part due to a loss or reduction of unpg gene expression and failure of unpg-expressing
cells to extend into the CNS (Chiang, 1995). It would be of interest to determine if unpg functions downstream of pnt in the ventral midline of the CNS.
adrift is expressed in the leading cells of growing tracheal
branches, near clusters of branchless FGF-expressing cells
and in a pattern very similar to that of several known
branchless-induced genes including pointed, DSRF/pruned and
sprouty. This suggested that adrift
expression might also be induced by the bnl signaling
pathway. Expression of an adrift lacZ reporter was examined
in embryos mutant for four components of the branchless
pathway: bnl, breathless, pnt and pruned. Initial expression of the adrift reporter in stage 11
tracheal cells is normal in all four mutants, but
subsequent expression in the leading cells of the branches is
absent in bnl, btl and pnt mutants. Expression in
pruned mutants is unaffected. In a
complementary experiment in which bnl was misexpressed
under the control of the hsp70 promoter, expression of the
adrift reporter expands to include additional cells in each
branch. Thus, the Branchless FGF pathway induces
adrift expression in the leading cells of tracheal branches, and
this induction requires the bnl FGF, the btl FGF receptor and
the pointed ETS domain transcription factor (Englund, 1999).
Although the target genes for Yan and Pointed in eye morphogenesis are not yet known, it is known that Pointed acts at the promoter level to activate transcription. Conversely, Yan acts at the same promoter sites to inhibit transcription (O'Neill, 1994). The targets of Pointed and Yan are identified as ETS-binding sites (EBS). ETS-binding sites are also found in several virus promoters (Nye, 1992).
pointed is a target of JUN and the ras pathway in eye morphogenesis. R7 photoreceptor fate in the Drosophila eye is induced by the activation of the Sevenless receptor
tyrosine kinase and the RAS/MAP kinase signal transduction pathway. Constituatively activated JUN is sufficient to induce R7 fate
independent of ras pathway signals. JUN interacts with Pointed to promote R7 formation. This
interaction is cooperative when both proteins are targeted to the same promoter and is antagonized
by YAN, a negative regulator of R7 development. Furthermore,
phyllopod, another target of RAS pathway activation,
behaves as a suppressor of activated JUN. These data suggest that JUN and
Pointed act on common target genes to promote neuronal differentiation in the Drosophila eye, and
that phyllopod might be such a common target (Treier, 1995).
prospero gene becomes transcriptionally activated at a low level in all Sevenless-competent cells prior to Sevenless signaling, and this requires the activities of Ras1 and two Ras1/MAP kinase-response ETS transcription factors, Yan and Pointed. Activation of pros transcription in all cells within the R7 equivalence group requires the down-regulation of Yan activity through phosphorylation by MAPK in R7 and cone cell precursors. Loss of pointed results in a reduction in the number of pros expressing cells. Two other nuclear factors, Seven in absentia (SINA) and Phyllopod are required for R7 determination, but are not absolutely for pros expression. However, the presence of phyl in cells is sufficient to induce them to express elevated levels of pros. phyl requires sina activity to stimulate pros expression. SINA protein can be shown to form a complex with PHYL (Kauffmann, 1996)
The tracheal cells that express sprouty are located very close to the small clusters of epidermal and
mesodermal cells that express Branchless, the sole known Drosophila FGF ligand; the sty expression pattern is very similar to that of pointed and other genes induced by Bnl. These observations suggest that sty might also be induced by the Bnl pathway. Consistent with this, in bnl and breathless mutants, sty is not expressed or is expressed only weakly. When Bnl is ubiquitously expressed, sty turns on at high levels throughout the tracheal system. The downstream effector pnt is also required for sty expression, and sty expression is activated outside its normal expression domain when pnt P1 protein is ubiquitously expressed. Consistent with this, pnt sty double mutants display the same tracheal phenotype as the pnt mutant alone. These results show that sty expression is induced by the Branchless activated signaling pathway that Sprouty inhibits. Sty limits induction of its own gene, just as it limits induction of other genes by Bnl, as shown by the broadened expression domain of a sty-lacZ marker in a sty- background (Hacohen, 1998).
Ras signaling elicits diverse outputs, yet how Ras specificity is generated remains incompletely understood. Wingless and
Decapentaplegic confer competence for receptor tyrosine kinase-mediated induction of a subset of Drosophila muscle and cardiac progenitors by acting
both upstream of and in parallel to Ras. In addition to regulating the expression of proximal Ras pathway components, Wg and Dpp coordinate the direct effects
of three signal-activated transcription factors (dTCF, Mad, and Pointed that function in the Wg, Dpp, and Ras/MAPK pathways, respectively) and two tissue-restricted transcription factors (Twist and
Tinman) on a progenitor identity gene enhancer. The integration of Pointed with the combinatorial effects of dTCF, Mad, Twist, and Tinman
determines inductive Ras signaling specificity in muscle and heart development (Halfon, 2000).
Cell fate specification in the somatic mesoderm of the Drosophila embryo has been examined as a model for dissecting the molecular basis of combinatorial
signaling involving receptor tyrosine kinases (RTKs). The somatic musculature and the cells that compose the heart develop from specialized cells called progenitors. Each progenitor divides asymmetrically to produce two founder cells that possess
information that specifies individual muscle fate and that seed the formation of multinucleate myofibers. The focus of this study has been a small subset of somatic mesodermal cells that express the transcription factor Even skipped. Eve is expressed in the progenitors and
founders of both the dorsal muscle fiber DA1 and a pair of heart accessory cells, the Eve pericardial cells or EPCs. Since eve is the earliest known marker for these cells and is required for their formation, eve is referred to here as a progenitor identity gene (Halfon, 2000).
Previous genetic experiments have defined multiple intercellular signaling events that govern the progressive determination of the Eve progenitors. Signaling from both the Wnt family member Wingless (Wg) and the TGF family member Decapentaplegic (Dpp) prepatterns the
mesoderm and renders cells competent to respond to Ras/MAPK activation. Localized Ras activation within the competence domain determined by the
intersection of Wg and Dpp expression occurs through the action of two RTKs: the Drosophila epidermal growth factor receptor (Egfr) and the Heartless (Htl)
fibroblast growth factor receptor. This RTK signaling induces two distinct equivalence groups, each of which expresses Eve. Lateral inhibition mediated by
Notch then selects a single progenitor from each equivalence group (Halfon, 2000).
The present study explores how the prepattern genes wg and dpp establish competence for mesodermal cells both to activate and to respond to the
Ras/MAPK cascade; how multiple intercellular signals are integrated to establish Eve progenitor fates, and how muscle- and cardiac-specific responses to Ras
signaling are generated. Wg provides competence for the generation of the Ras/MAPK inductive signal by regulating the expression of key
proximal components of the Egfr and Htl RTK pathways. Wg and Dpp then create competence for a specific response to the inductive signal both through their
own respective downstream transcriptional effectors, dTCF and Mothers against
dpp (Mad), and through their regulation of the mesoderm-specific transcription factors Tinman (Tin) and Twist (Twi). Specificity of the Ras/MAPK response is achieved though the
integration of these signal-activated and tissue-restricted transcription factors, along with the Ras/MAPK-activated Ets domain transcription factor PointedP2
(Pnt), at a single transcriptional enhancer. These results provide a direct link between the initial axis patterning processes in the early embryo and the subsequent
combinatorial signaling events that lead to the progressive determination of muscle and cardiac progenitors (Halfon, 2000).
The Eve progenitors in each mesodermal hemisegment arise during embryonic stage 11 in a dorsal region demarcated by the intersecting domains of Wg and Dpp expression. The cells exposed to both Wg and Dpp are competent to respond to localized Ras signaling, which induces the initial expression of Eve in two clusters of equipotent cells. In each of these equivalence groups, activity of the Notch pathway leads to the rapid refinement of Eve expression to a single muscle or cardiac progenitor. The two Eve equivalence groups arise sequentially. Cluster C2, from which progenitor P2 derives, is first to form. P2 divides asymmetrically, with one daughter maintaining Eve expression and becoming the founder of the two EPCs (F2EPC), and the other losing Eve expression and becoming the founder of muscle DO2. The second Eve-expressing cluster, C15, forms slightly later and produces the progenitor P15, which in turn divides to yield the founder of the Eve-expressing muscle, DA1, and an Eve-negative cell of as-yet-undetermined identity. Activation of the Ras/MAPK pathway in C15 depends on both the DER and Htl RTKs, but only Htl signaling is required for C2 formation (Halfon, 2000).
The progressive determination of Eve mesodermal progenitors requires that Wg prepattern the mesoderm, rendering cells competent to respond to inductive RTK/Ras signaling. To further investigate the basis of this competence, whether or not the Ras pathway is active in the absence of Wg signaling was examined by monitoring the expression of the activated, diphosphorylated form of MAPK in wg mutant embryos. Diphospho-MAPK is expressed in progenitor P2 in early stage 11 wild-type embryos. Not only is this progenitor missing from wg mutant embryos, but activation of MAPK in the C2 equivalence group, which is dependent on Htl, fails to occur. Similarly, Wg is essential both for P15 formation and for the DER- and Htl-dependent activation of MAPK in the equivalence group from which this progenitor is derived (Halfon, 2000).
Next to be determined was at what level in the RTK/Ras pathway Wg is required for MAPK activation. In wg mutant embryos, there is loss of (1) the P2-specific expression of Htl; (2) its specific downstream signaling component, Heartbroken (Hbr, also known as Dof and Stumps), and (3) Rhomboid (Rho), a protein involved in the presentation of the Egfr ligand Spitz. Conversely, constitutive Wg signaling, achieved by ectopic expression of Wg or an activated form of the downstream Wg pathway component Armadillo (Arm), induces Htl, Hbr, and Rho expression in more dorsal mesodermal cells than the single P2 progenitor found at a comparable developmental stage. This effect is less prominent for Rho than for Htl and Hbr, which may reflect different threshold responses to Wg. Alternatively, the effect on Htl and Hbr may be more pronounced because ectopic Wg signaling prolongs their earlier expression in the entire C2 cluster; Rho, in contrast, is normally expressed in P2 but not in C2, possibly making it more refractory to a prepattern factor such as Wg. Expanded expression of these RTK pathway components is associated with increased MAPK activation and Eve expression. However, these effects of Wg hyperactivation are transient, with a normal number of Eve progenitors eventually segregating. Moreover, activated Arm is able to fully rescue Htl, Hbr, Rho, diphospho-MAPK, and Eve expression in wg mutant embryos. Htl, Hbr, and Rho expression, as well as MAPK activation, are also Dpp dependent. In summary, Wg and Dpp regulate the production of several key proximal components of the DER and Htl signal transduction pathways (Halfon, 2000).
Given the involvement of Wg in the expression of Htl, Hbr, and Rho, it was reasoned that a constitutively activated form of Ras1 might bypass the requirement of Wg for MAPK activation. Constitutively activated Ras1, when targeted to the mesoderm of wild-type embryos, leads to an overproduction of Eve progenitors, as well as to the expected hyperactivation of MAPK in these cells. In the absence of Wg signaling, diphospho-MAPK expression is restored by activated Ras1. However, despite this recovery of MAPK activation, constitutive Ras1 does not rescue Eve progenitor formation in a wg mutant background. This is in marked contrast to the ability of activated Arm to fully rescue RTK signaling and Eve progenitor specification in a wg mutant. These results suggest that, in addition to enabling activation of Ras/MAPK signaling as a result of the induction of Htl, Hbr, and Rho expression, Wg signaling must contribute other factors that are essential for the specification of mesodermal Eve progenitors (Halfon, 2000).
Given the importance of Ras/MAPK signaling in Eve progenitor determination, a determination was made of whether Pnt, an Ets domain transcriptional activator that functions downstream of MAPK, is also involved in this process. In pnt mutant embryos, there is a severe reduction in the number of both Eve progenitors, although this loss is more pronounced for the P15 lineage. Since mesoderm migration is normal in pnt embryos, Pnt must only be required for the progenitor specification function of Htl. Consistent with this conclusion, an activated form of Pnt induces extra Eve progenitors (Halfon, 2000).
In embryos mutant for yan, which encodes a MAPK-regulated Ets-domain transcriptional repressor, there is an increase in the number of Eve progenitors and their differentiated derivatives. Conversely, a constitutively activated form of Yan inhibits Eve progenitor formation. Thus, two MAPK-regulated transcription factors are involved in the development of Eve progenitors (Halfon, 2000).
One mechanism that would ensure the convergence of the multiple regulatory inputs required for the formation of P2 and P15 is integration by a transcriptional enhancer. Since Eve expression is the feature that uniquely identifies these progenitors, an investigation was made of whether eve itself is a direct target for regulation by both signal-activated and tissue-specific transcription factors. Regulatory sequences responsible for mesodermal eve expression are located approximately 6 kb downstream of the transcription start site. Deletions of this region were generated and a 312 bp minimal enhancer was defined that has been termed the eve Muscle and Heart Enhancer (MHE). When fused to a nuclear-lacZ reporter gene, the MHE drives expression in a mesodermal pattern identical to that of the endogenous eve gene. Reporter expression initiates at early stage 11, coincident with the onset of Eve expression in the equivalence group C2. Following formation of P2, MHE activity is observed in P15 and in the P2 daughters, F2EPC and F2DO2, then in the EPCs and the F15 daughters of P15, and finally in muscle fiber DA1. Colocalization of MHE-driven ß-galactosidase expression with Runt, which marks the F2DO2 founder and muscle DO2, establishes that the reporter gene expression present in Eve-negative sibling cells is a result of ß-galactosidase perdurance. Of note, the MHE mimics endogenous Eve expression despite its lack of a consensus binding site for the transcription factor Zfh-1 that had previously been proposed to play a role in mesodermal eve regulation (Halfon, 2000).
Strikingly, the MHE is only active in a single nucleus of the mature DA1 and DO2 muscles. It is inferred that these are the original nuclei of the F15DA1 and F2DO2 founders based on prior reporter expression in those cells. Similar results were obtained when DNA flanking the MHE by several hundred base pairs on either side (+4.96 to +7.36 kb), including the previously described Zfh-1 site, was included in the reporter construct, or when the MHE was placed 3' to a reporter gene fused to the endogenous eve promoter. Thus, additional sequences are required for eve expression in non-founder myofiber nuclei. Of critical importance to the present study, the MHE fully recapitulates mesodermal Eve expression during the signal-dependent induction of progenitor and founder cells (Halfon, 2000).
Genetic manipulation of the Wg, Dpp, and RTK/Ras signaling pathways causes predictable alterations of endogenous mesodermal Eve expression. A determination was made of whether the isolated MHE responds appropriately to these signals. In all genetic backgrounds, reporter gene expression corresponds precisely to that of endogenous eve. For example, constitutively activated Arm transiently increases the expression of both genes. However, Wg hyperactivation does not have a stable effect on MHE function. In contrast, both endogenous eve and the MHE-driven reporter are induced throughout the initial competence domain by constitutively activated Pnt, and expression of both markers extends laterally in the presence of activated Arm plus Pnt. Ectopic Dpp leads to both endogenous Eve and MHE-driven reporter expression in the ventral mesoderm, while coexpression of Dpp and activated Ras1 induces expression of both genes in a dorsal-ventral stripe. These results demonstrate that the isolated MHE is responsive to all of the known signals that are essential for the specification of Eve progenitors (Halfon, 2000).
Given that the MHE recapitulates early mesodermal Eve expression, a determination was made of whether this enhancer contains binding sites for candidate signal-dependent and mesoderm-specific transcription factors. Focus was placed on two mesoderm-specific factors, Tin and Twi, as well as the nuclear factors that act downstream of Wg (dTCF), Dpp (Mad) and Ras (Pnt, Yan). A computer-based search of the MHE sequence has suggested the presence of potential binding sites for each of these transcription factors. Gel-shift assays confirm that these putative sites actually bind the relevant factors. This analysis establishes the existence of one binding site for dTCF, six for Mad, two for Twist, and four each for Tin and Pnt. Since Yan binds to each of the Pnt sites, these are referred to as Ets sites (Halfon, 2000).
To ascertain whether these in vitro binding sites have in vivo functional significance, the sites were mutated, both singly and in combination, within the context of the entire MHE. All mutagenesis was by base substitution so as not to affect the spacing between other potential cis-regulatory elements. The ability of the mutated MHEs to drive reporter gene expression was tested in transgenic embryos and this expression was compared to that of endogenous Eve. Of the six Mad sites, only Mad4, 5, and 6 are critical for MHE function when inactivated singly or in combination. Mutation of the single dTCF site or of individual binding sites for Twi, Tin, or the Ets factors also lead to loss of reporter gene expression in some, but not all, Eve-expressing cells, with some mutant sites associated with a more severe loss than others. Of note, both the EPC and DA1 lineages are affected equally by all of the mutations. In addition, the activity level in those Eve-expressing cells that do maintain reporter gene expression is on average lower than that seen with the wild-type MHE. In contrast to the single site mutants, mutation of the two Twi, all four Tin, or all four Ets sites completely eliminate MHE activity. It is concluded that binding sites for two tissue-specific and three signal-responsive transcription factors are required for full activity of the MHE in both the muscle and the heart lineages (Halfon, 2000).
The finding that the three Wg-dependent factors, dTCF, Twi, and Tin, that directly regulate eve could explain why activated Ras is incapable of bypassing Wg in the induction of Eve progenitors. Therefore attempts were made to rescue Eve expression in wg mutant embryos by ectopically expressing Twi and Tin together with activated Ras. However, Eve progenitors were not recovered by this manipulation, perhaps due to the direct requirement of dTCF for eve MHE activity. While activated Arm can supply the missing downstream Wg transcription factor in this rescue experiment, Arm alone is capable of fully rescuing not only the Eve progenitors but also all of the Wg-dependent factors that regulate the MHE, including Twi, Tin, and the RTK/Ras pathway components. Thus, the combined effects of the MHE transcription factors could not be further evaluated in the absence of Wg signaling. Nevertheless, the rescue and enhancer mutagenesis data strongly support the involvement of Wg as a mesodermal competence determinant both upstream of the Ras pathway and directly (via dTCF) as well as indirectly (via Twi and Tin) in the transcriptional response to inductive RTK signaling (Halfon, 2000).
Since mutation of any single transcription factor binding site in the MHE causes only a partial loss of enhancer activity, it was considered whether different sites might function together synergistically. To test this possibility, binding site mutations for two different activators were combined. Simultaneous mutation of the dTCF and Twi1 sites led to reporter gene expression in approximately 5-fold fewer cells than would be expected from the additive independent effects of each mutation. A similar, though slightly less robust, synergy was observed when the dTCF and Ets3 mutations were combined (Halfon, 2000).
An assessment was made of whether ectopic coexpression of individual transcription factors or upstream signals would lead to cooperative effects on endogenous Eve expression. As previously reported, ectopic Wg has no effect on Eve expression at late stage 11, activated Ras1 induces extra Eve progenitors, and ectopic Wg plus activated Ras1 cause a lateral expansion of the progenitor clusters. When Twi is expressed using a twi-Gal4 driver, a few Eve-positive cells develop at ectopic positions. The magnitude of this effect is increased by coexpression of Wg and Twi, and even more so by coexpression of Twi with activated Ras1. The latter effect strikingly resembles that of Wg plus activated Ras1. With the simultaneous ectopic expression of Wg, Twi, and activated Ras1, Eve progenitors form an almost continuous anteroposterior stripe confined to the dorsal mesoderm. These results demonstrate a synergistic induction of Eve progenitors by various combinations of Wg, Twi, and activated Ras1 that parallels the synergistic loss of MHE activity seen by mutating the dTCF, Twi, and Ets binding sites. Taken together, these loss- and gain-of-function findings suggest that dTCF, Twi, and Pnt cooperate at the MHE to synergistically regulate Eve transcription and, by extension, to induce the specification of Eve progenitor fates (Halfon, 2000).
It is concluded that Wg and Dpp coordinate a series of signal-activated (dTCF and Mad) and mesoderm-specific (Twi and Tin) transcription factors in a temporal and spatial pattern that facilitates cooperation with the Ras transcriptional effector Pnt. The synergistic integration of these five transcription factors by a single enhancer generates a specific developmental response to Ras/MAPK signaling. Moreover, Wg and Dpp exert proximal effects in this signaling network by enabling Ras/MAPK activation through the regulated localized expression of upstream components of the RTK signal transduction machinery. A model governing the acquisition of developmental competence, signal integration and response specificity in this system is presented. Wg and Dpp provide competence through the regulation of tissue-specific transcription factors (Tin and Twi), signal-responsive transcription factors (Mad and dTCF), and proximal components of the RTK/Ras pathways (Htl, Hbr, and Rho). The Ras signaling cascade leads to activation of the inductive transcription factor, Pnt, and inactivation of the Yan repressor. While a direct role for Mad in regulating Tin expression has been demonstrated, Wg regulation of Tin, Twi, Htl, Hbr, and Rho may be either direct or indirect. Dpp has additional effects on the proximal RTK factors. The five transcriptional activators assemble at and are integrated by the MHE, where they function synergistically to promote eve expression. Specificity of the response to inductive RTK/Ras signaling derives from the combinatorial effects of the tissue-restricted and signal-activated transcription factors that converge at the MHE. In the absence of inductive signaling, Yan would repress eve by binding to the Ets sites. Since eve is a muscle and heart identity gene, the regulatory mechanisms are inferred to have a more general function in determining progenitor fates. Additional complexity attendant upon the control of RTK activity in this system derives from positive feedback regulation of the Ras/MAPK cascade and from reciprocal regulatory interactions between the Ras and Notch pathways (Halfon, 2000).
Regulated transcription of the prospero gene in the Drosophila eye provides a model for how gene expression is specifically controlled by signals from receptor
tyrosine kinases. prospero is controlled by signals from the Egfr receptor and the Sevenless receptor. A direct link is established between
Egfr activation of a transcription enhancer in prospero and binding of two transcription factors that are targets of Egfr signaling. Binding of the cell-specific
Lozenge protein is also required for activation, and overlapping Lozenge protein distribution and Egfr signaling establishes expression in a subset of equivalent
cells competent to respond to Sevenless. Sevenless activates prospero independent of the enhancer and involves targeted degradation of
Tramtrack, a transcription repressor (Xu, 2000).
Thus, Egfr signaling is required to activate pros expression in the R7 equivalence group but is restricted from activating pros expression in other cells by the distribution of the transcription factor Lz. The transcriptional effectors of the Egfr pathway combinatorially interact with Lz at an eye-specific pros enhancer to restrict enhancer activity to the R7 equivalence group. It is suggested that this mechanism is a primary means by which pros transcription is restricted to the R7 equivalence group. This combinatorial mechanism supposes that Egfr signaling inactivates Yan and activates Pnt, but modification of these transcription factors is not sufficient to activate the enhancer. Lz is also required to activate the enhancer. The only cells that contain Lz, activated Pnt, and inactivated Yan are R1, R6, R7, and cone cells. Thus, the enhancer is activated in a subset of Egfr-responsive cells. A similar combinatorial mechanism regulates shaven expression in cone cells. shaven expression requires both Lz and Egfr-induced regulation of Yan and Pnt. However, Notch signaling through Su(H) is also required for shaven expression in cone cells. This third input may explain why shaven has a more restricted expression pattern than pros, given that cone cells receive a robust Notch signal (Flores, 2000). In muscle and cardiac cells, RTK signaling is similarly integrated with other signal inputs and tissue-restricted transcription factors to regulate enhancer activity of the even skipped gene (Halfon, 2000). Thus, differential expression of genes in response to an RTK/Ras signal appears to be controlled by each gene's capacity to bind and be regulated by different combinations of transcription factors (Xu, 2000).
A model is presented for the regulatory inputs into prospero. (1) In eye progenitor cells, the presence of Yan represses pros transcription through its binding to the enhancer and competitively excluding Pnt from binding to the same sites. (2) Lz begins to be produced in progenitor cells after the first wave of photoreceptor differentiation. However, Lz alone cannot activate the enhancer in progenitor cells that have not received a Spitz signal. (3) When a progenitor cell receives a Spitz signal, Egfr is activated. This inactivates Yan, allowing activated Pnt to bind to the enhancer. At the morphogenetic furrow, the enhancer is inactive despite Egfr-stimulated cells containing inactive Yan and active Pnt since progenitor cells in this region do not contain Lz, which is also required for enhancer activity. Hence, photoreceptors R2, R3, R4, R5, and R8 do not express pros. It is only in cells that receive a Spitz signal and contain Lz that the combination of Lz and Pnt bound to the enhancer activate the enhancer. (4) Ttk88 reduces the level of pros transcription through a mechanism independent of the eye enhancer. This repression may not be strong enough to block the eye enhancer in the R7 equivalence group but acts to limit its level of transcription. (5) When a progenitor cell receives both a Spitz and Boss signal, stronger or longer signal transduction induces Ttk88 inactivation. This Egf represses pros transcription and leads to a specific increase of Pros in R7 cells (Xu, 2000).
The ETS factors Yan and Pnt have been implicated as substrates for activated MAPK, whose activities are modified upon phosphorylation. Both Yan and Pnt bind to the same sites in the pros eye enhancer except for one site that is Yan-specific. Their effects on enhancer activity are antagonistic; Yan represses while Pnt activates. One model is that Yan represses transcription by outcompeting Pnt for their binding sites, thereby preventing Pnt from activating transcription. This model is attractive since it has been found that Yan has a 100-fold greater affinity than Pnt for ETS factor binding sites in vitro. If this difference between purified fusion proteins in vitro is extrapolated to the fly eye, it would explain how Yan can outcompete Pnt and repress transcription. Results from mutagenesis of the binding sites is also consistent with this model. Mutated binding sites cause the enhancer to be inactive, which is the result predicted if Yan merely prevents Pnt from interacting with those sites. If Yan were actively repressing transcription in a manner dependent upon binding, then mutated binding sites would cause derepression and ectopic expression. Although a model where the binding sites are obligatory for both active repression by Yan and activation by Pnt cannot be excluded, the competitive binding model is the simplest one consistent with these data (Xu, 2000).
From these data it is proposed that two RTKs, Egfr and Sev, regulate pros by activating the Ras1 intracellular pathway in R7 cells, but these RTKs regulate pros differentially. Egfr regulates pros by modifying Yan and Pnt, which act directly through the eye-specific enhancer. The Egfr signal in R7 cells appears to occur before Sev, and it sufficiently inactivates Yan and activates Pnt to switch on the enhancer before the Sev signal. This sufficiency is demonstrated in sev mutants where enhancer activity in R7 cells is no different from wild-type. In contrast, the Sev signal in R7 cells is not sufficient to switch on the enhancer in the absence of the Egfr signal since the enhancer is inactive in Egfr mutant R7 cells (Xu, 2000).
How do these RTKs selectively regulate particular transcription factors and thereby regulate different aspects of pros transcription? The most attractive model is that RTK selection reflects the timing or intensity of each signal. If it is timing, then there must be a time period of competence during which a factor is sensitive to any RTK signal, and the time period is different for each factor. Alternatively, the intensity of a signal may dictate which transcription factor activities are sensitive. For example, Yan and Pnt activities may be insensitive to signal strength that is less than or equal to the level achieved by Sev but not Egfr within R7 cells. Ttk88 activity may be insensitive to signal strength that is less than or equal to the level achieved by Sev or Egfr alone but not the combination of the two within R7 cells. Signal 'strength' may be determined by the level of Ras pathway activity or the length of time that the Ras pathway is active. Sensitivity of transcription factors might be set either by the affinities of these factors for binding sites in a gene such as pros, or by the ability of factors to be substrates for RTK-stimulated modification. Given that Yan and Pnt are modified by a very different mechanism from Ttk88, substrate sensitivity is a possible determinant. In summary, RTK signals may provide specificity to gene regulation based on quantitative variation in which threshold transcription responses are set by transcription factors that have different sensitivities to RTK signal strength (Xu, 2000).
How multifunctional signals combine to specify unique cell fates during pattern formation is not well understood. Together with the
transcription factor Lozenge, the nuclear effectors of the Egfr and Notch signaling pathways directly regulate D-Pax2 (shaven) transcription in cone cells of the
Drosophila eye disc. Moreover, the specificity of shaven expression can be altered upon genetic manipulation of these inputs. Thus, a relatively small number
of temporally and spatially controlled signals received by a set of pluripotent cells can create the unique combinations of activated transcription factors required
to regulate target genes and ultimately specify distinct cell fates within this group. It is expected that similar mechanisms may specify pattern formation in vertebrate
developmental systems that involve intercellular communication (Flores, 2000).
shaven is the Drosophila homolog of the vertebrate Pax2 gene. This locus is represented by at least two classes of mutant alleles: shaven (sv) and sparkling (spa). spa mutants show cone cell defects resulting from mutations in the fourth intron of the gene, which have led to the identification of a 926 bp SpeI fragment within this intron that includes the eye-specific enhancer (Flores, 2000).
In EGFRts third-instar larvae raised at 29°C for 36 hr prior to dissection, Shaven expression is lost in cone cell precursors. To restrict the loss of Egfr function to the undifferentiated cells posterior to the furrow and cells that acquire their fates during the second phase of morphogenesis, a lz-Gal4 driver was used to express a dominant-negative form of Egfr. In these discs, Shaven expression is lost from cone cell precursors, while neuronal patterning in the precluster is maintained. Shaven expression was further examined in mutants of genes encoding the nuclear components of the Egfr signaling pathway, the repressor Yan and the activator PntP2. Shaven expression is also lost in discs in which lz-Gal4 drives the expression of a nonphosphorylatable form of Yan refractory to the Egfr signal. Similarly, in the hypomorphic pnt1230 mutant, a modest reduction of Shaven expression occurs in cone cell precursors, while a stronger reduction is observed upon expression of a dominant-negative form of PntP2. These experiments together suggest that the Egfr signaling pathway activates shaven expression in cone cell precursors by relieving Yan-mediated repression and stimulating PntP2 activation (Flores, 2000).
The above genetic analysis does not address whether the effects of Egfr signaling on shaven transcription are direct or indirect. Therefore, in vitro mutagenesis was used to examine potential direct effects. Six ETS domain consensus binding sites were found in the SME. EMSAs show that two of these sites (1 and 6) are bound by both Yan and PntP2. Yan also binds to two additional sites (2 and 4). All six ETS sites were mutated to 5'-TTAA/T-3' in the context of SME-lacZ, and the resulting SMEmETSx6-lacZ construct was transformed into flies. In these transgenic flies, ß-galactosidase expression is lost from cone cell precursors. Since PntP2 was found to bind only to Ets sites 1 and 6, a SME-lacZ construct in which only these sites were mutated (SMEmETS(1,6)-lacZ) was transformed into flies. ß-galactosidase expression in cone cells is completely eliminated. These in vitro and in vivo results together demonstrate that PntP2 directly controls shaven expression in cone cell precursors by binding to ETS domain sites in the SME (Flores, 2000).
Mutating Su(H) and ETS binding
sites eliminates expression of the target gene in the cone cells, which demonstrates a direct role for these pathways in transcriptional activation of shaven. Clonal analysis was undertaken to establish the requirement of the Notch and Egfr pathways in shaven expression. Unfortunately, these pathways are necessary for proliferation and have many layers of function. Therefore a flip-out strategy was used to inhibit N and Egfr function in GFP-labeled single-cell clones. This was best achieved in clones induced by GMR-flp. The GMR enhancer is only active behind the furrow and only a single cell division takes place in this population of cells. As a result, the clone size is very small. In a wild-type background, single cells marked with GFP express Shaven. However, when these single cells also express EGFRDN or NECN, they do not express Shaven. Thus, cone cells need functional Notch and Egfr receptors in order to express Shaven (Flores, 2000).
The results described so far suggest that shaven expression is limited to cells which (1) express Lz; (2) receive a sufficiently strong Egfr signal to both alleviate Yan-imposed repression and stimulate PntP2 activation, and (3) receive a N signal able to stimulate Su(H) activation. The tripartite control of shaven expression in the cone cell precursors requires that they receive all three inputs at the proper time in their development. Lz expression in cone cell precursors has been documented. Consistent with their reception of the Egfr signal, activated MAPK is detected in cone cell precursors at the time when they initiate Shaven expression. Dl is expressed in developing photoreceptor clusters at the time when the cone cell precursors express Shaven. Thus, the neuronal clusters signal through an inductive Dl/N pathway to activate shaven expression in the neighboring cone cell precursors. These results suggest that, in addition to expressing Lz, the cone cell precursors receive the Egfr and N signals at the time of fate acquisition and Shaven expression. Presumably, at least one of these three activation mechanisms is lacking in cells that do not express shaven. This hypothesis was tested through genetic manipulation of the system (Flores, 2000).
Undifferentiated cells immediately posterior to the furrow receive the N signal and express Lz, but they do not express Shaven. It is hypothesized that the absence of Shaven expression in these cells is caused by a lack of the Egfr signal. This hypothesis is consistent with the observation that Egfr signaling causes these cells to differentiate. Indeed, Shaven is ectopically expressed in undifferentiated cells that express an activated form of Egfr. Loss-of-function yane2D/yanpokX8 discs also show ectopic expression of Shaven in undifferentiated cells. Similarly, in discs expressing SMEmETSx6-lacZ, in which the six ETS sites in the SME are mutated, ß-galactosidase is also expressed in undifferentiated cells. Presumably, relief of Yan repression is sufficient to activate some shaven in undifferentiated cells. In SMEmETS(1,6)-lacZ,where the Pnt binding sites are eliminated but two of the Yan binding sites are still intact, there is no expression of ß-galactosidase in the undifferentiated cells. These results suggest that while the undifferentiated cells posterior to the furrow express Lz and receive the N signal, they fail to express Shaven because they do not receive the Egfr signal and are therefore unable to relieve the Yan-imposed repression of shaven (Flores, 2000).
Enhancers integrate spatiotemporal information to generate precise patterns of gene expression. How complex is the regulatory logic of a typical developmental enhancer, and how important is its internal organization? This study examined in detail the structure and function of sparkling, a Notch- and EGFR/MAPK-regulated, cone cell-specific enhancer of the Drosophila Pax2 gene, in vivo. In addition to its 12 previously identified protein-binding sites, sparkling is densely populated with previously unmapped regulatory sequences, which interact in complex ways to control gene expression. One segment is essential for activation at a distance, yet dispensable for other activation functions and for cell type patterning. Unexpectedly, rearranging sparkling's regulatory sites converts it into a robust photoreceptor-specific enhancer. These results show that a single combination of regulatory inputs can encode multiple outputs, and suggest that the enhancer's organization determines the correct expression pattern by facilitating certain short-range regulatory interactions at the expense of others (Swanson, 2010).
The goal of this study was to use a well-characterized, signal-regulated developmental enhancer to examine, in fine detail, the regulatory interactions and structural rules governing transcriptional activation in vivo. This study used functional in vivo assays to test the power of the proposed combinatorial code of 'Notch/Su(H) + Lz + MAPK/Ets' to explain the activity and cell type specificity of the spa cone cell enhancer of dPax2. In the course of this work, several surprising properties of spa were discovered that are not accounted for in current models of enhancer function (Swanson, 2010).
The spa enhancer for fine-scale analysis because (1) the known direct regulators and their binding sites are well defined, (2) they could, in theory, constitute the sum total of the patterning information received by the enhancer, and (3) the enhancer, at 362 bp, is relatively small, simplifying mutational analyses. Surprisingly, a large proportion of the previously uncharacterized sequence within spa is vital for normal enhancer activity in vivo, and of that subset, a large proportion directly influences cell type specificity (Swanson, 2010).
In addition to necessary inputs from Lz, Pnt, and Su(H), three segments of spa were identified, regions 4, 5, and 6, that make essential contributions to gene expression in cone cells. In addition, region 2 makes a relatively minor contribution. (Region 1, another essential domain, will be discussed separately.) Fine-scale mutagenesis reveals that within regions 4, 5, and 6, very little DNA is dispensable for cone cell activation. The previously uncharacterized regulatory sites in spa are very likely bound by factors other than Lz/Pnt/Su(H), for the following reasons: no sequences resembling Lz/Pnt/Su(H)-binding sites reside in these regions; mutations in the newly mapped sites have different effects than removing the defined TFBSs or the proteins that bind them; doubling the known TFBSs fails to compensate for the loss of the newly mapped sequences; and, most importantly, mutating the newly mapped regulatory regions does not significantly affect binding of the known activators to nearby binding sites in vitro. It is not known whether the proposed novel regulators are cone cell-specific, eye-specific, or ubiquitous in their expression. It is known that the newly mapped sites are necessary both for normal cone cell expression and ectopic PR expression. Cut, Prospero, and Tramtrack are expressed in cone cells, but are thought to act as transcriptional repressors. The transcription factor Hindsight is required for dPax2 expression and cone cell induction, but acts indirectly, activating Delta in R1/R6 to induce Notch signaling in cone cells (Swanson, 2010).
Unsurprisingly, placing the enhancer closer to the promoter boosts expression of spa(wt), as well as some of the impaired mutants. The spa enhancer is located at +7 kb in its native locus, and nearly all mutational studies place the enhancer immediately upstream of the promoter. If the entire analysis had been performed at −121 bp, the functional significance of several critical regulatory sequences would have been underrated, and region 1 would have been dismissed as nonregulatory DNA. Other well-characterized enhancers, which have been analyzed in a promoter-proximal position only, may therefore contain more critical regulatory sites than is currently realized (Swanson, 2010).
Like many transcriptional activators, all three known direct activators of spa (or their orthologs) recruit p300/CBP histone acetyltransferase coactivator complexes. Doubling the number of binding sites for these transcription factors (to 6 Lz, 8 Ets, and 10 Su(H) sites) does not suffice to drive cone cell expression in the absence of the newly mapped regulatory regions. It may be, then, that factors recruited to the newly mapped regulatory sites within spa employ mechanisms that are distinct from those of the known activators. The remote activity of spa, mediated by region 1, appears to be an example of such a mechanism (Swanson, 2010).
It was possible to convert spa into a R1/R6-specific enhancer in three ways: (1) by moving the defined TFBSs to one side of the enhancer in a tight cluster; (2) by placing Lz and Ets sites next to regions 1, 4, and 6a; and (3) by mutating regions 2, 3, 5, and 6b within spa while maintaining the native spacing of all other sites. From these experiments, it is concluded that spa contains short-range repressor sites that prevent ectopic activation in PRs by Lz + Pnt + regions 4 + 6a. spa contains at least two redundant repressor sites, because both region 5 and regions 2, 3, and 6b must be mutated to attain ectopic R1/R6 expression (Swanson, 2010).
klumpfuss, which encodes a putative transcriptional repressor, is directly activated by Lz in R1/R6/R7, but is also present in cone cells, making it an unlikely repressor of spa. seven-up, another known transcriptional repressor, is expressed in R3/R4/R1/R6 and could therefore act to repress spa in PRs. However, no putative Seven-up-binding sites were identified within spa. Phyllopod, an E3 ubiquitin ligase component, represses dPax2 and the cone cell fate in R1/R6/R7, but the transcription factor mediating this effect is not yet known (Shi, 2009). Perhaps the best candidate for a PR-specific direct repressor of spa is Bar, which encodes the closely related and redundant homeodomain transcription factors BarH1 and BarH2. Bar expression is activated by Lz in R1/R6 and is required for R1/R6 cell fates. Furthermore, misexpression of BarH1 in presumptive cone cells can transform them into PRs. It is unclear whether Bar-family proteins act as repressors, activators, or both. BarH1/2 can bind sequences containing the homeodomain-binding core consensus TAAT, and region 5 of spa contains two TAAT motifs. Future studies will explore the possibility that Bar directly represses spa in PRs (Swanson, 2010).
The combinatorial code of spa, then, requires multiple inputs in addition to Lz, MAPK/Ets, and Notch/Su(H). Indeed, the data suggest that the known regulators can contribute to expression in multiple cell types, depending on context. The newly mapped control elements identified within spa are necessary not only to facilitate transcriptional activation, but also to steer the Lz + Ets + Su(H) code toward cone cell-specific gene expression (Swanson, 2010).
Enhancers are often located many kilobases from the promoters they regulate. Enhancer-promoter interactions over such distances are very likely to require active facilitation. Even so, few studies have focused specifically on transcriptional activation at a distance, and the majority of this work involves locus control regions (LCRs) and/or complex multigenic loci, which are not part of the regulatory environment of most genes and enhancers. Like spa, many developmental enhancers act at a distance in their normal genomic context, yet can autonomously drive a heterologous promoter in the proper expression pattern, without requiring an LCR or other large-scale genomic regulatory apparatus. However, in nearly all assays of enhancer function, the element to be studied is placed immediately upstream of the promoter. In such cases, regulatory sites specifically mediating remote interactions cannot be identified. Because the initial mutational analysis of spa was performed on enhancers placed at a moderate distance from the promoter (−846 bp), it was possible to screen for sequences required only at a distance, by moving crippled enhancers to a promoter-proximal position. Only one segment of spa, region 1, was absolutely essential at a distance but completely dispensable near the promoter. This region, which contains the only block of extended sequence conservation within spa, plays no apparent role in patterning, or in basic activation at close range. Therefore this segment of spa is termed a 'remote control' element (RCE) (Swanson, 2010).
The remote enhancer regulatory activity described in this study differs from previously reported long-range regulatory mechanisms in two important ways. First, the remote function of spa does not require any sequences in or near the dPax2 promoter. This functionally distinguishes spa from enhancers in the Drosophila Hox complexes that require promoter-proximal 'tethering elements' and/or function by overcoming insulators. This distal activation mechanism also likely differs from enhancer-promoter interactions mediated by proteins that bind at both the enhancer and the promoter, as occurs in looping mediated by ER, AR, and Sp1. Second, studies of distant enhancers of the cut and Ultrabithorax genes have revealed a role for the cohesin-associated factor Nipped-B, especially with respect to bypassing insulators, but it has not been demonstrated that Nipped-B, or any other enhancer-binding regulator, is required only when the enhancer is remote (Swanson, 2010).
The spa RCE is the first enhancer subelement demonstrated to be essential for enhancer-promoter interactions at a distance, but unnecessary for proximal enhancer function and cell type specificity. However, the present work contains only a limited examination of this activity, as part of a broader study of enhancer function. These functional studies, testing for potential promoter preferences and distance limitations, and the identities of factors binding to the RCE are being persued(Swanson, 2010).
As discussed above, it is fairly easy to switch spa from cone cell expression to R1/R6 expression (though, curiously, a construct that is active in both cell types has yet to be constructed). The results show that multiple regions of spa mediate a repression activity in R1/R6, but not in cone cells. It is further concluded that these spa-binding repressors act in a short-range manner; that is, they must be located very near to relevant activator-binding sites, because moving Lz and Pnt sites to one side of spa, without removing the repressor sites (KO+synthCS), abolishes repression. Despite this failure of repression, synergistic interactions among Lz and Ets sites and the newly mapped sites still occur in this reorganized enhancer -- at least in R1/R6 cells. Cone cell-specific expression is lost, however, revealing (along with other experiments) that transcriptional activation in cone cells is highly sensitive to the organization of regulatory sites within spa. Slightly wider spacing of regulatory sites (KO+synthNS) kills the enhancer altogether, suggesting that synergistic positive interactions within spa, though apparently longer in range than repressive interactions, are severely limited in their range. The structural organization of spa, then, appears to be constrained by a complex network of short-range positive and negative interactions. Activator sites must be spaced closely enough to trigger synergistic activation in cone cells; at the same time, repressor sites must be positioned to disrupt this synergy in noncone cells, preventing ectopic activation (Swanson, 2010).
Recent work has shown that changes to enhancer organization can 'fine-tune' the output of a combinatorial code, subtly changing the sensitivity of the enhancer to a morphogen. Given the importance of the structure of the spa enhancer for its proper function, it is proposed that any combinatorial code model, no matter how complex, is insufficient to describe the regulation of spa, because the same components can be rearranged to produce drastically different patterns (Swanson, 2010).
One might expect that the regulatory and organizational complexity of the spa enhancer, and its extreme sensitivity to mutation, would be reflected in strict evolutionary constraints upon enhancer sequence and structure. Yet, very poor conservation of spa sequence was observed, both in the known TFBSs and in most of the newly mapped essential regulatory elements. The reduced presence of Lz/Ets/Su(H) sites in D. pseudoobscura could potentially be attributed to redundancy of those sites in D. melanogaster, or to compensatory gain of binding sites for alternate factors in the D. pse enhancer. Perhaps more difficult to understand is the apparent loss of critical regulatory sequences in regions 4, 5, and 6a in D. pse; the experiments in D. mel suggest that the absence of those inputs would result in loss of cone cell expression and/or ectopic activation. It remains possible that many of these inputs are in fact conserved, but that conservation is not obvious due to binding site degeneracy and/or rearrangement of elements within the enhancer. Fine-scale comparative studies are ongoing (Swanson, 2010).
spa is by no means the first example of an enhancer that is functionally maintained despite a lack of sequence conservation. The most thoroughly characterized example of this phenomenon is the eve stripe 2 enhancer; its function is conserved despite changes in binding site composition and organization. Note, however, that spa has undergone much more rapid sequence divergence than eve stripe 2, with no apparent change in function. In general, the ability of an enhancer to maintain its function in the face of rapid sequence evolution suggests that enhancer structure must be quite flexible. These observations support the 'billboard' model of enhancer structure, which proposes that as long as individual regulatory units within an enhancer remain intact, the organization of those units within the enhancer is flexible. Yet, the findings concerning the importance of local interactions among densely clustered, precisely positioned transcription factors are more consistent with the tightly structured 'enhanceosome' model. Further structure-function analysis will be necessary to fully understand the players and rules governing this regulatory element (Swanson, 2010).
In addition to a post-translational regulation of Head involution defective (Hid), the Ras/MAPK pathway
promotes cell survival in Drosophila by downregulating the expression of hid.
Conversely, downregulation of the Ras/MAPK pathway induces cell death by upregulating hid
expression. hid transcript levels are downregulated in dominantly active Dras1- (Dras1Q13) expressing embryos when assayed 3 hr after heat shock. In wild-type embryos, total HID mRNA levels do not change dramatically between stage 11, when Ras expression was ectopically induced, and stage 14, when HID mRNA levels were assayed. This eliminates the concern that developmental arrest might account for the observed difference in HID mRNA levels. It was observed that hid levels return to normal in Dras1Q13 embryos by 5 hr after heat shock. Cell death also resumes in these embryos several hours later. This indicates that a transient increase in Ras activity leads to a transient suppression of hid expression, accompanied by a transient protection from naturally occurring cell death. HID mRNA levels were also assayed through an alternative procedure: whole mount in situ analysis. These results confirm that hid transcript levels decline in dominantly active Dras1- (Dras1Q13) expressing embryos. This is particularly apparent in the midline glia, which strongly express hid. The survival of midline glia is known to depend on the activity of the Epidermal growth factor receptor pathway. To confirm that Ras regulation of hid utilizes the Raf/MAPK pathway, the effect of a constitutively active form of Draf (phlF22) on hid expression has been investigated. In situ analyses were performed on embryos expressing activated Draf under the control of the heat shock promoter. Heat-induced expression of phlF22 results in downregulation of hid transcript levels, suggesting that Ras functions through the Raf/MAPK pathway to downregulate hid expression (Kurada, 1998).
Reduction in pointed (pnt) activity has been observed to enhance ectopic Hid induced cell death in the eye. The pointed transcription factor is a target of MAPK function and acts as a positive regulator in the R7 pathway. The pnt gene encodes two related proteins, pnt1 and pnt2. pnt2 operates downstream of the MAPK rolled in the Ras pathway. Therefore, the consequences of ectopic expression of pnt2 were examined. Embryos were generated that carry UAS-Pnt2 and a midline glia-specific Gal4 driver (52A-Gal4), resulting in the expression of pnt2 in the midline glia cells. Such embryos were tested for hid levels by whole-mount in situ analysis. Like embryos expressing activated Dras1 and activated Draf, pnt2-expressing embryos show decreased hid transcript levels, indicating that the Ras/MAPK pathway, acting through pnt, downregulates hid transcription (Kurada, 1998).
Since upregulation of the Ras/MAPK pathway promotes cell survival and downregulates hid expression, it was predicted that increased hid expression is the cause of the increased apoptosis observed when Ras activity is decreased. Ubiquitous expression of the negative regulator yan is able to induce massive embryonic apoptosis. In these same embryos HID mRNA levels are increased within 2 hr of yanAct induction and continue to rise for many more hours. Thus, downregulation of Ras activity in the embryo results in increased hid transcription and apoptosis, and this transcription is regulated either directly or indirectly by yan. These results imply that Ras activation of MAPK and inactivation of yan is an important cell survival pathway in embryos (Kurada, 1998).
Blocking Epidermal growth factor receptor activity in the developing eye also enhances apoptosis. If hid is a target of Egfr/Ras/MAPK activity in this tissue, then hid levels should increase when Egfr activity is blocked. Expression of a dominant negative Egfr in the developing eye results in a band of increased hid transcription in the eye disc. This band lies several rows posterior to the furrow and corresponds well with the first developmental defects seen in these eye discs. In sum, these data implicate the downregulation of hid transcription as an important component of Egfr antiapoptotic activity. The post-transcriptional modification of Hid appears to be equally important (Kurada, 1998).
Receptor tyrosine kinase (RTK) signaling plays an instructive role in cell fate decisions, whereas Notch signaling is often involved in restricting cellular competence for differentiation. Genetic interactions between these two
evolutionarily conserved pathways have been extensively documented. The underlying molecular mechanisms, however, are not well understood. Yan, an Ets transcriptional repressor that blocks cellular potential for specification and differentiation, is a target of Notch signaling during Drosophila eye
development. The Suppressor of Hairless (Su[H]) protein of the Notch pathway is required for activating yan
expression, and Su(H) binds directly to an eye-specific yan enhancer in vitro. In contrast, yan expression is repressed by Pointed (Pnt), which is a key component of the RTK pathway. Pnt binds specifically to the yan enhancer and competes with Su(H) for DNA binding. This competition illustrates a potential mechanism for RTK and Notch signals to oppose each other. Thus, yan serves as a common target of Notch/Su(H) and RTK/Pointed signaling pathways during cell fate specification (Rohrbaugh, 2002).
To investigate how yan expression is regulated, DNA fragments comprising a 20-kb genomic sequence surrounding the first exon of yan were tested for regulatory potential in corresponding transgenic flies. Through this approach, a 122-bp eye-specific enhancer located approximately 3.5 kb upstream of the first exon was identified. In eight out of nine transgenic lines, this enhancer activated expression of a bacterial lacZ reporter gene within posterior undifferentiated cells of eye discs. This recapitulates the endogenous yan gene expression in eye discs with the exception of the MF region. Three putative Su(H) binding sites were found in the yan enhancer. When tested through an in vitro electrophoretic mobility shift assay (EMSA), the Su(H) protein was shown to specifically bind to these sequences. Further, the yan enhancer became inactive in most of the posterior undifferentiated retinal cells when the Su(H) function was removed. All together, these loss-of-function and DNA binding analyses support the notion that Su(H) is required to promote yan transcription and that yan is a target gene of Su(H) in the eye (Rohrbaugh, 2002).
An Ets domain binding site (EBS, 5'-GGAA/T-3') was found within the S2 site. Since Yan is an Ets domain protein and a transcriptional repressor, whether Yan could be involved in autoregulation was examined. When a constitutively activated Yan (YanAct) was overproduced in eye discs, the reporter gene expression was strongly reduced. Since Yan is capable of negatively regulating yan transcription, this autoinhibitory mechanism might be used to prevent overproduction of Yan in undifferentiated cells. DNA binding data suggests that Yan can be directly involved in this negative regulation. However, this Yan-mediated autoinhibitory feedback appears to play a minor role in regulating yan expression, because the yan enhancer activity is apparently not affected in yan mutant clones produced in eye discs (Rohrbaugh, 2002).
A role for RTK signaling in regulating yan transcription was investigated. When the RTK pathway is constitutively activated by torD-DER or Ras1V12, the yan enhancer activity is greatly reduced. Thus, RTK signaling appears to negatively regulate yan transcription, in addition to its effect on Yan protein stability. Evidence supports a view that the inhibitory effect of RTK/Ras1 signaling on yan expression is mediated through the pointed (pnt) gene. Taken together, the results demonstrated that Pnt negatively regulates yan expression, and it is likely that Pnt is directly involved in repressing yan transcription. Although a role for Pnt as a transcriptional repressor has not been extensively investigated, pnt has been shown to negatively regulate hid transcription in embryos. Interestingly, a P-DLS motif is present in the Pnt protein (amino acids 356–360 in PntP1), which might mediate interaction with the transcriptional corepressor dCtBP. At this point, the data does not exclude the possibility that Pnt might also activate expression of a repressor, which in turn switches off yan transcription (Rohrbaugh, 2002).
The nesting of an Ets binding site within the S2 site suggests a possible mechanism whereby the binding of Pnt could interfere with Su(H)'s DNA binding activity. Indeed, increasing the amount of Pnt effectively prevents Su(H) from DNA binding. Such competition provides a mechanism by which RTK/Pnt signaling directly antagonizes Notch-mediated lateral inhibition at the transcriptional level. Since Ets binding sites are nested in many Su(H) binding sites, competitive occupancy of the common sequence could be a general mechanism for regulating expression of genes targeted by both Notch and RTK pathways (Rohrbaugh, 2002).
It is proposed that spatially restricted yan expression in the developing eye is coordinated by actions of multiple regulatory factors that include Su(H) and Pnt. Consequently, the yan enhancer provides an interface for Notch and RTK signals to oppose one another. The DNA binding analysis and mutagenesis of yan Su(H) binding sites provide evidence that supports a cell-autonomous role of Notch and RTK signaling in the regulation of yan expression. Interestingly, Yan expression is reduced not only in Su(H)D47 clones but also in some Su(H)+ cells that surround the mutant clones in eye discs. This result implies that loss-of-Su(H) function might also cause a cell-nonautonomous effect on yan expression, possibly due to upregulation of RTK signaling in those Su(H)+ cells. This upregulation may occur via an increase of a diffusible activator of the RTK pathway due to the loss of Su(H). The model presented here illustrates a mechanism that should help explain how progenitor cells are maintained in an undifferentiated state by Notch-mediated inhibitory signals and how they can be effectively induced for cellular differentiation by RTK-mediated inductive signals (Rohrbaugh, 2002).
Although glial cells are an important component in any
complex nervous system, not much is known about the
molecular mechanisms underlying glial development. In
Drosophila, a number of gene functions and mechanisms
required during glial development are emerging. Following
lineage specification, terminal differentiation of glial cells is
mediated by transcription factors encoded by repo and pointed.
The identification of genes activated by pointed in glial cells
should provide new insights in the molecular mechanisms
underlying glial differentiation. loco
, a regulator of G-protein signalling that functions as a GTPase-activating protein towards G-proteins) might represent such a pointed target gene.
Analysis of the loco promotor region reveals the presence of
GCM- and ETS-binding sites suggesting that loco might be a
direct target of gcm as well. loco
promotor-lacZ fusion constructs reveal a small promotor
fragment that is capable of directing lacZ expression in almost
all loco-expressing glial cells. This promotor fragment is
indeed dependent on pointed function and ectopic pointed
expression as well as ectopic gcm expression result in a
corresponding ectopic lacZ expression. Sequence analysis and
in vitro mutagenesis reveal These data, as well as the phenotypes observed in loco and
pointed mutant embryos, suggest that loco
is indeed a target of pointed (Granderath, 1999).
In Drosophila, lateral glial cell development is initiated by the transcription factor encoded by glial cells missing. gcm activates downstream transcription factors such as repo and pointed, which subsequently control terminal glial differentiation. The gene loco has been identified as a potential target gene of pointed and is involved in terminal glial differentiation. It encodes an RGS domain protein expressed specifically by the lateral glial cells in the developing embryonic CNS. The loco promoter and the control of the glial-specific transcription pattern has been analyzed. Using promoter-reporter gene fusions a 1.9 kb promoter element capable of directing the almost complete loco gene expression pattern has been identified. Sequence analysis suggests the presence of Gcm and Pointed DNA binding sites. Following in vitro mutagenesis of these sites their relevance in vivo has been demonstrated. The expression of loco is initially dependent on gcm. During subsequent stages of embryonic development Gcm and Pointed appear to activate loco transcription synergistically. In addition, at least two other factors appear to repress loco expression in the ectoderm and in the CNS midline cells (Granderath, 2000).
Two alternative modes are presented as to how loco transcription might
be regulated. In the simple model, a linear array of transcriptional regulators results in the correct expression of loco. gcm acts on top of this cascade
and activates pointed, which in turn leads to glial-specific
loco expression. Alternatively, loco gene activation might be biphasic. Initially gcm concomitantly activates both loco and pointed. In a second phase, gcm and pointed act synergistically on the loco promoter to mediate high levels of glial-specific loco expression. The
data favour the latter model (Granderath, 2000).
The 1.9 kb Rrk promoter element is capable of directing expression of a lacZ reporter in the complete loco expression
domain. The Rrk fragment itself appears to contain more
than one crucial regulatory element. The US1 construct, which overlaps the Rrk fragment, which harbors two gcm binding sites located in the 5'
part of the Rrk fragment, directs glial expression resembling
the expression of loco in a pointed mutant background. The
3' sequences of the Rrk fragment are found in the Nrk fragment. This promoter fragment, which harbours one gcm and
one pointed binding site, is not able to confer any glial
expression. Only the complete Rrk fragment is able to direct
the entire loco transcriptional profile, pointing to synergistic
effects of proteins binding to the 3' and 5' portions of the
Rrk element. This notion is supported by the observation
that pointed cannot activate the Rrk element when both
Gcm binding sites GBS1 and GBS3 are deleted. Ectopic expression of either gcm or pointed alone within the neuroectoderm leads to sporadic activation of the Rrk enhancer, suggesting the presence of both gcm and pointed
responsive elements. Coexpression of gcm and pointed in
the rhomboid expression pattern shows two interesting
results: (1) it is evident that cells within neuroectoderm
activate the Rrk reporter fragment very strongly, showing that the
two transcription factors act synergistically; (2) it is
important to note that although comparably high levels of
gcm and pointed are found in the CNS midline and the
mesodermal cells, they never activate the Rrk reporter (Granderath, 2000).
Coexpression of gcm and pointed can also direct expression
of the Nrk reporter. Within the Nrk fragment only one Gcm
and one Pointed binding site are found, 370 bp apart.
gcm is the master regulatory gene controlling lateral glial
cell development. The gene pointed is not expressed in
mutant gcm embryos suggesting that pointed expression depends on gcm. However, only coexpression of pointed and gcm leads to an efficient activation of the Rrk enhancer, indicating that gcm can not efficiently activate pointed transcription in the neuroectoderm.
Despite the fact that pointedP1 is thought to act as a
transcriptional activator it appears that cofactors such as
gcm are required to allow full activation. This observation parallels
results obtained in vertebrate systems, where it has been suggested that the binding of
cofactors is a mechanism to relieve auto-inhibition of ETS proteins. pointed is expressed in many tissues during
development and activates very different sets of genes
(e.g., depending on the cells in which pointedP1 is
expressed it activates tracheal, epidermal, neuronal or
glial development). Thus, interaction with different tissue-specific coactivators might be an important step in selecting the appropriate downstream target genes (Granderath, 2000). Direct coactivation of glial target genes by both gcm and pointedP1 is possibly not confined to loco; the analysis of a second
pointed-dependent enhancer element has revealed the presence
of putative binding sites for both gcm and pointed (Granderath, S. and Klambt, C., unpublished data cited in Granderath, 2000).
The synergistic activation of loco by Gcm and Pointed could suggest that Pointed might be able to recruit or stabilize Gcm at the regulatory regions of terminal differentiation genes. This would lead to an increased expression of the respective genes but concomitantly could also disrupt the positive auto-regulatory feedback loop found for the gcm gene. This would provide a possible mechanism as to how the positive auto-regulation of gcm is terminated. How loco expression is maintained in vivo remains to be addressed (Granderath, 2000).
Terminal differentiation of glial cells is controlled by
pointed. Two different isoforms are generated from the
pointed locus, PointedP1 and PointedP2. They share the
DNA binding domain and during embryonic CNS development they are expressed in the lateral glia (PointedP1) or the midline glia (PointedP2). Despite the common DNA binding activity, the two factors activate non-overlapping sets of
target genes in the different glial cell types. The mechanism by which the selection of glial PointedP1 and PointedP2 target genes occurs appears to be complex. A
simple model would be to postulate that specific, as yet
unidentified cofactors are expressed either in the neuroectoderm or the CNS midline cells. However, in the midline, PointedP2 function can be substituted by PointedP1. This might be explained by postulating that PointedP1 is able to interact with a pointedP2 coactivator. Besides Gcm, additional factors appear to be required to
specify PointedP1 target genes, because the coexpression of PointedP1 and Gcm in the CNS midline is not sufficient to evoke any Rrk reporter gene expression.
Alternatively, the discrimination of PointedP1 and PointedP2 target genes might be mediated by transcriptional repressors. Two such proteins are known to be expressed
in the CNS midline: Single minded and Abrupt. No potential Single
minded binding sites were found in the Rrk construct. One
potential Abrupt binding site (CTTAATTAA at position 1537-1547
of the Rrk fragment) was predicted by DNA sequence analysis. However, disruption of this site does not alter the reporter gene expression directed by the Rrk fragment in vivo. Thus, if Abrupt directly acts on the lococ1 promoter, it
must bind to a different site in the Rrk fragment. Abrupt
apparently represses Rrk-mediated expression (and possibly
expression of other gcm-dependent genes) only in the
apodemata, which might explain the muscle attachment
defects observed in abrupt mutant embryos. In the CNS midline, however, the function of abrupt is not required for the repression of loco. Thus, additional experiments are required to determine which mechanisms
are used in vivo to discriminate between lateral and midline
glial gene expression (Granderath, 2000).
At least two transcripts of loco are expressed in oogenesis: (1) loco-c2 is observed in the anterior-dorsal follicle cells and is downstream of the epidermal growth factor receptor signaling pathway, initiated in the oocyte; (2) loco-c3 is a new transcript of loco that is
expressed in the nurse cells from stage 6 onwards. Disrupting loco in
follicle cells results in ventralized eggs, while disrupting loco in nurse cells results in short eggs, due to defective dumping of the nurse cell cytoplasm into the oocyte. How does loco fit into the existing EGF pathway? loco is downstream of Egfr/torpedo in the follicle cells and appears to be activated both at high and moderate levels
of torpedo activation. loco is downstream of pointed, a target of Egf signaling. loco has been identified
in a screen for genes downstream of pointed in specific
subsets of cells in the central nervous system (CNS). pointed P1 and P2 are expressed in the anterior-dorsal follicle cells in oogenesis. The expression of pointed in oogenesis is dynamic,
expression first being observed in the germarium, then later
at stage 8, downstream of torpedo, in the posterior follicle
cells and again at stage 10, also downstream of torpedo, in
anterior-dorsal follicle cells. Since pointed is observed in anterior-dorsal follicle cells in a similar pattern to loco at stage 10, it is possible that loco is downstream of pointed at this particular stage of oogenesis. The relationship between loco and
pointed was analyzed in these cells. Using a pointed 1/UAS sense fly line, expression of pointed was driven in all the follicle
cells using a T155 GAL4 driver. In situ
hybridization to RNA in the GAL4/UAS-pointed ovaries,
using a pointed probe, clearly shows high levels of
pointed expression in all the follicle cells that cover the
oocyte at stage 10. The expression of
loco-c2 was examined in egg chambers where pointed was being ectopically expressed. A normal spatial distribution was seen in
anterior-dorsal follicle cells at stage 10, though levels of
expression were somewhat reduced. This reduction
in expression is not uniform over the anterior-dorsal
region, with the anterior-most follicle cells maintaining
their normal level of expression. At later stages the expression
pattern is slightly different from wild type, since there is a
patch of cells expressing loco in the dorsal position that
has not migrated as far anteriorly as would be expected at
this stage. This is due to overexpression of pointed in oogenesis
resulting in failure to make dorsal appendages. This
suggests that cells normally expressing loco do not migrate
as far as in wild type egg chambers. This experiment
shows that there is not a simple relationship between
loco and pointed. Ectopic pointed expression does disrupt the normal loco-c2
expression pattern at stage 10, with its level of expression
dropping in the anterior-dorsal follicle cells, except in the
anterior-most follicle cells. This indicates that loco-c2 is
downstream of pointed, although not directly (Pathirana, 2002).
Signaling by the Drosophila EGF receptor (Egfr) is modulated by four known EGF-like proteins: the agonists Vein (Vn), Spitz (Spi), and Gurken (Grk) and the
antagonist Argos (Aos). Egfr is broadly expressed and thus tissue-specific regulation of ligand expression and activity is an important mechanism for controlling
signaling. The tissue-specific regulation of Vn signaling was investigated by examining vn transcriptional control and Vn target gene activation in the embryo and the
wing. The results show a complex temporal and spatial regulation of vn transcription involving multiple signaling pathways and tissue-specific activation of Vn target
genes. In the embryo, vn is a target of Spi/Egfr signaling mediated by the ETS transcription factor PointedP1 (PntP1). This establishes a positive feedback loop in
addition to the negative feedback loop involving Aos. The simultaneous production of Vn provides a mechanism for dampening Aos inhibition and thus fine-tunes
signaling. In the larval wing pouch, vn is not a target of Spi/Egfr signaling but is expressed along the anterior-posterior boundary in response to Hedgehog (Hh)
signaling. Repression by Wingless (Wg) signaling further refines the vn expression pattern by causing a discontinuity at the dorsal-ventral boundary. The potential for
vn to activate Egfr target genes correlates with its roles in development: vn has a minor role in embryogenesis and does not induce Egfr target genes such as aos and
pntP1 in the embryo. Conversely, vn has a major role in wing development and Vn/Egfr signaling is a potent inducer of Egfr target genes in the wing disk. Spi also
has the potential to induce Egfr target genes in the wing disk. However, the ligands appear to evoke specific responses that result in different patterns of target gene
expression. Other factors modulate the potential of Vn so that induction of Vn/Egfr target genes in the wing pouch is cell specific (Wessells, 1999).
Differences between Vn and Spi are apparent in the patterns of target
gene induction resultant from the ectopic expression of Vn and soluble Spitz (sSpi) in the
wing pouch. Effects have been noted for three Egfr target genes:
aos, pntP1, and kekkon-1 (kek1). In each case, a different
response to the ligands is seen. Both ligands induce ectopic aos
expression but Vn does so in a broader domain than sSpi, however, neither induces aos in the L3/L4
intervein region. In the embryo, the Egfr target
gene pntP1 mediates aos induction by sSpi. Likewise in the wing, ectopic sSpi induces pntP1
expression in cells that also expressed aos. However, following ectopic Vn no detectable
change in pntP1 expression is seen using in situ hybridization and
only very weak induction of pntP1-lacZ is seen in a domain that
does not correspond fully with ectopic aos expression. This suggests either that another transcription
factor mediates the induction of aos in response to Vn or
that PntP1 is capable of inducing aos, even when changes in
its own expression level are too low to be detected by
current methods. Both Vn and sSpi induce ectopic expression
of kek1, but predominantly in posterior cells rather
than throughout the domain of their ectopic expression. Thus the action of both ligands appears
limited by the presence or absence of some other factor in
anterior cells. There is also a difference in the level of
induction: Vn, which functions to induce kek1
expression in normal development, is a potent
inducer of high levels of ectopic kek1 expression,
whereas sSpi induces low levels of ectopic kek1 expression
and appears to reduce expression of endogenous kek1 (Wessells, 1999).
blistered is expressed in the precursors of the terminal tracheal cells and in the future intervein
territories of the third instar wing imaginal disc. Dissection of the blistered regulatory region reveals that a single enhancer element, which is
under the control of the fibroblast growth factor (FGF)-receptor signaling pathway, is sufficient to induce blistered expression in the terminal
tracheal cells. In contrast, two separate enhancers direct expression in distinct intervein sectors of the wing imaginal disc. One element is
active in the central intervein sector and is induced by the Hedgehog signaling pathway. The other element is under the control of
Decapentaplegic and is active in two separate territories, which roughly correspond to the intervein sectors flanking the central sector.
Hence, each of the three characterized enhancers constitutes a molecular link between a specific territory induced by a morphogen signal and
the localized expression of a gene required for the final differentiation of this territory (Nussbaumer, 2000).
A 500 bp enhancer element (TCE) has been isolated whose activity reproduces the blistered expression pattern
in terminal tracheal cells, with respect to its temporal,
spatial and regulatory cues. The TCE is the first enhancer
identified in Drosophila that responds to FGF signaling. It
has been reported that the FGF signaling cascade activates
the MAP kinase pathway and that the Ets-domain containing
protein Pnt is a target for the ERK-MAP kinase. Therefore, the expression of the TCE is likely to be controlled by FGF signaling which, through the ERK-MAP kinase pathway, activates a Pnt-DNA-bound complex
in conjunction with other factors.
Further dissection of the enhancer and the identification of
the individual DNA sites and the relevant transcription
factors should help to elucidate how FGF triggers specific
nuclear responses. Interestingly, during early mesoderm
induction in Xenopus laevis, an Ets-SRF complex has
been implicated in transducing FGF signaling. Therefore, blistered might not only be a target gene whose transcription is activated in response to FGF
signaling, but it might also encode a protein that assembles into a
complex to integrate the FGF signal. However, the TCE
does not require Blistered itself for signal induction since it is
still active in a blistered loss-of-function mutant. Nevertheless, other
putative target genes induced via FGF signaling in the
terminal tracheal cells could require the activation of an
Ets-Blistered-DNA complex (Nussbaumer, 2000).
In the early Drosophila embryo, a system of coordinates is laid down by segmentation genes and dorsoventral patterning genes. Subsequently, these coordinates must be interpreted to define particular tissues and organs. To begin understanding this process for a single organ, a study has been carried out of how one of the first salivary gland genes, fork head (fkh), is turned on in the primordium of this organ, the salivary placode. A placode-specific fkh enhancer was identified 10 kb from the coding sequence. Dissection of this enhancer shows that the apparently homogeneous placode is actually composed of at least four overlapping domains. These domains appear to be developmentally important because they predict the order of salivary invagination, are evolutionarily conserved, and are regulated by patterning genes that are important for salivary development. Three dorsoventral domains are defined by Egf receptor (Egfr) signaling, while stripes located at the anterior and posterior edges of the placode depend on wingless signaling. Further analysis has identified sites in the enhancer that respond either positively to the primary activator of salivary gland genes, Sex combs reduced (Scr), or negatively to Egfr signaling. These results show that fkh integrates spatial pattern directly, without reference to other early salivary gland genes. In addition, a binding site for Fkh protein was identified that appears to act in fkh autoregulation, keeping the gene active after Scr has disappeared from the placode. This autoregulation may explain how the salivary gland maintains its identity after the organ is established. Although the fkh enhancer integrates information needed to define the salivary placode, and although fkh mutants have the most extreme effects on salivary gland development thus far described, it is argued that fkh is not a selector gene for salivary gland development and that there is no master, salivary gland selector gene. Instead, several genes independently sense spatial information and cooperate to define the salivary placode (Zhou, 2001).
Expression of fkh is limited at the dorsal edge of the placode by dpp and at the ventral edge of the placode by Egfr and the spitz group genes. Expression driven by the two halves of the enhancer has identified a new dorsoventral limit, the boundary between the dorsal and ventral parts of the placode. What genes establish this boundary? Expression of 1-506:lacZ in sim or pnt embryos shows that the Egfr pathway is involved. In these embryos, staining is as strong in the dorsal placode as it is in wild-type embryos, but now there is also weak staining that extends all the way to the ventral midline. Thus, expression of 1-506:lacZ must normally be limited by Egfr signaling. In contrast to this result, dpp mutations have no effect on this boundary (Zhou, 2001).
The Epidermal growth factor receptor (Egfr) pathway controls cell fate decisions throughout phylogeny. Typically, binding of secreted ligands to Egfr on the cell surface initiates a well-described cascade of events that ultimately invokes transcriptional changes in the nucleus. In contrast, the mechanisms by which autocrine effects are regulated in the ligand-producing cell are unclear. In the Drosophila eye, Egfr signaling, induced by the Spitz ligand, is required for differentiation of all photoreceptors except for R8, the primary source of Spitz. R8 differentiation is instead under the control of the transcription factor Senseless. High levels of Egfr activation are incompatible with R8 differentiation; the mechanism by which Egfr signaling is actively prevented in R8 is described. Specifically, Senseless does not affect cytoplasmic transduction of Egfr activation, but does block nuclear transduction of Egfr activation through transcriptional repression of pointed, which encodes the nuclear effector of the pathway. Thus, Senseless promotes normal R8 differentiation by preventing the effects of autocrine stimulation by Spitz. An analogous relationship exists between Senseless and Egfr pathway orthologs in T-lymphocytes, suggesting that this mode of repression of Egfr signaling is conserved (Frankfort, 2004).
In this analysis of sens function in R8 differentiation, it was found
that the extra R2/R5 cell that develops from the pre-R8 in sens
mutants expresses Ro, which is normally expressed in R2/R5 but not R8. Ro is
expressed downstream of Egfr pathway activation, and both ro function
and high levels of Egfr pathway activation are required for R2/R5
differentiation. Since the pre-R8 cell consistently expresses Ro and
differentiates as an R2/R5 cell in sens mutants, it was hypothesized that
this transformation occurs as a consequence of high levels of Egfr activation
in the pre-R8 cell (Frankfort, 2004).
This hypothesis was tested by simultaneously removing sens function
and blocking Egfr activation in the developing Drosophila eye. Egfr
activation was blocked by removing function of both rhomboid-1 (rho-1)
and rhomboid-3 (rho-3; FlyBase: roughoid, ru). Loss
of both rho-1 and rho-3 function prevents processing of
secreted Egfr ligands, including Spi, and results in the loss of all ERK (MAP
kinase) activation. Furthermore, loss of rho-1 and rho-3
phenocopies Egfr loss-of-function in that only R8 cells differentiate. Loss
of sens function results in pre-R8 differentiation as a founder R2/R5
cell which is sufficient to recruit a reduced number of photoreceptors. However, the absence of rho-1, rho-3 and sens together causes total photoreceptor loss, except for a few photoreceptors near the clonal boundary that are rescued non-autonomously by neighboring wild-type cells that produce and
process Spi appropriately. A similar phenotype is detected in tissue mutant for both spi and sens. This loss of photoreceptors seen in rho-1 rho-3 sens and spi sens mutants is not due to cell death because apoptosis was
prevented in these experiments by expression of GMR-p35. Furthermore, pre-R8 selection still occurs in both rho-1 rho-3 and rho-1 rho-3 sens mutant tissue, suggesting that a potential founding photoreceptor is present. Therefore, these results are interpreted to mean that, in the absence of sens function, pre-R8 differentiation as a founder R2/R5 photoreceptor requires activation of the Egfr signaling pathway via the Spi ligand. In other words, in sens mutants, the pre-R8 switches from a Spi/Egfr-independent R8
differentiation pathway to a Spi/Egfr-dependent R2/R5 differentiation
pathway (Frankfort, 2004).
This work suggests that Sens acts to ensure that the organizing center of
each ommatidium is refractory to the developmental signals it produces --
the R8 cell can secrete Spi and even activate Egfr on its own cell membrane,
yet remains protected from the deleterious effects of activation of Pnt and
other Egfr targets, such as Ro, in R8 (Frankfort, 2004).
The mechanism by which Sens regulates the discrepancy between levels of
Egfr activation at the receptor/cytoplasmic and nuclear levels in R8 is
probably through repression of pnt transcription. This is supported
by the observation that pnt transcription is not induced by
misexpression of an activated form of Egfr when sens is
co-misexpressed. Furthermore, expression of the pnt-P1 isoform in R8 disrupts R8 differentiation. Since
misexpression of pnt-P2 has no effect on R8 differentiation, this
suggests that Sens negatively regulates transcription of pnt-P1, but
not pnt-P2. This mode of regulation is consistent with established
models for transduction of the Egfr signal to the nucleus. Specifically, ERK
phosphorylates Pnt-P2, which is thought to be a transient positive regulator
of pnt-P1 transcription. In this model, transduction of Egfr activation occurs all
the way into the nucleus of R8, but Sens represses the pathway at the final
step -- positive regulation of pnt-P1 by Pnt-P2. When sens
function is removed, the block on pnt-P1 transcription is relieved,
and Pnt-P1 can exert its transcriptional effects on the nucleus, including
ro induction (Frankfort, 2004).
There is evidence that pnt-P1 transcription can be regulated by
Egfr signaling independently of pnt-P2 during Drosophila
embryogenesis. If this is the case during eye development, the model would
remain essentially the same -- Sens would still act as a negative
regulator of pnt-P1 in R8. However, this regulation would occur
independently of pnt-P2 rather than downstream of pnt-P2 (Frankfort, 2004).
Sens is also a potent negative regulator of ro and this
relationship appears to specifically affect the cell fate decision between R8
and R2/R5 differentiation. Several lines of evidence suggest that Sens-mediated
repression of ro is distinct from other effects of Sens in R8: (1)
loss of ro function does not rescue R8 differentiation in all
ommatidia in sens mutants; (2) even those R8 cells that do differentiate in sens ro double mutants require Spi/Egfr pathway activation; (3) misexpression
of ro in R8 causes a different phenotype than misexpression of
pnt-P1 in R8. Specifically, even though Egfr pathway activation is
necessary and sufficient for Ro expression, misexpression of pnt-P1
in R8 does not cause an obvious cell fate transformation from R8 to R2/R5,
while misexpression of ro in R8 does.
Indeed, R8 markers are still expressed when pnt-P1 is misexpressed in
R8. However, aberrant nuclear movements and the absence of small rhabdomeres
at the level of R8 in adults suggest that misexpression of pnt-P1
does perturb R8 differentiation. Together, these results suggest that Sens repression of pnt-P1 occurs independently of Sens function as a repressor of ro, and that Sens-mediated repression of pnt-P1 is probably required for normal R8 differentiation upstream or independently of cell fate determination (Frankfort, 2004).
Since Sens acts as a transcription factor and its mammalian homolog, Gfi-1,
binds directly to enhancer regions of Ets1 and Ets3, two
mammalian orthologs of pnt, it is possible that Sens repression of
pnt-P1 expression occurs directly.
Gfi-1 also interacts with nuclear matrix proteins to repress transcription. Thus,
it is possible that Sens represses transcription of Egfr nuclear effectors via
a similar mechanism. Future experiments are required to determine which of
these or other mechanisms are important during R8 differentiation. However, it
is likely that Sens does not act as a positive regulator of Edl/Mae, a
proposed cell-autonomous repressor of Egfr signaling, because edl/mae
function is not required for normal R8 differentiation. Finally, it is also unlikely that sens functions as an activator of yan, which encodes a nuclear repressor of the Egfr pathway, because yan loss-of-function mutations also do not impact R8 differentiation (Frankfort, 2004).
The positioning of Sens repression downstream of ERK activation may help
explain interactions observed between sens and Egfr pathway homologs
in T-lymphocytes. In Jurkat T-cells, activation induced cell death (AICD), a
process that is required to prevent non-specific activation of T-cells, is
dependent, in part, on ERK1/2 activation.
Intriguingly, high levels of Gfi-1 have been shown to inhibit AICD despite
high levels of ERK1/2 activation. The antagonistic relationship between Sens and the Egfr pathway in R8, in conjunction with the observation that Gfi-1 can bind to the enhancer regions of Ets1 and Ets3, suggest that this inhibition of AICD may occur via Gfi-1-mediated repression of ERK1/2 targets
(such as Ets/pnt) in T-cells. Thus, these results may establish R8 development as a powerful and novel system with which to study mechanisms of
lymphomagenesis, apoptosis and cancer (Frankfort, 2004 and references therein).
In Drosophila, commitment of a cell to a sense organ precursor (SOP) fate requires bHLH proneural transcription factor upregulation, a process that depends in most cases on the interplay of proneural gene autoregulation and inhibitory Notch signaling. A subset of SOPs are selected by a recruitment pathway involving EGFR signaling to ectodermal cells expressing the proneural gene atonal. EGFR signaling drives recruitment by directly facilitating atonal autoregulation. Pointed, the transcription factor that mediates EGFR signaling, and Atonal protein itself bind cooperatively to adjacent conserved binding sites in an atonal enhancer. Recruitment is therefore contingent on the combined presence of Atonal protein (providing competence) and EGFR signaling (triggering recruitment). Thus, autoregulation is the nodal control point targeted by signaling. This exemplifies a simple and general mechanism for regulating the transition from competence to cell fate commitment whereby a cell signal directly targets the autoregulation of a selector gene (zur Lage, 2004).
Paracrine signaling is a widespread trigger of cell fate determination during development. However, it is well known that the information that such signals impart depends on the context. Thus, signaling allows or prevents a target cell from committing to a fate for which it is already predisposed or competent. Sense organ precursor (SOP) determination in the developing Drosophila PNS provides an important model system for understanding the mechanisms underlying competence and commitment, and particularly how the transition from competence to commitment is controlled. In this case, competence and commitment requires the function of the bHLH proneural genes achaete and scute (ac/sc), atonal (ato), and amos, which can be viewed as selector genes of SOP fate. Much progress has been made in understanding this process during 'classical' SOP selection. Proneural genes are initially expressed in groups of ectodermal cells known as proneural clusters (PNCs). This initial expression provides cells with neural competence but does not necessarily lead to commitment. The key event in SOP commitment is the upregulation of proneural protein expression in specific PNC cells. For ac/sc, a complex network of cell interactions and signaling feedback loops determines whether a cell upregulates or downregulates ac/sc expression, thereby ensuring that a single SOP is selected. In this process, Notch signaling within proneural clusters inhibits ac/sc autoregulation by directly interacting with autoregulatory enhancers (zur Lage, 2004 and references).
In addition to this classical mode, there is a second mode of SOP formation that has been characterized for ato during chordotonal stretch receptor development: chordotonal SOPs can be recruited by EGFR signaling. In each embryonic abdominal segment, five chordotonal SOPs are selected from two ato-expressing PNCs in a conventional manner, involving the interplay of ato with Notch signaling. These 'primary' precursors express rhomboid, (rho) which activates secretion of the EGFR ligand Spitz, and the subsequent signaling to adjacent ectodermal cells leads to their recruitment as 'secondary' chordotonal SOPs. As a result, in each abdominal segment five primary precursors recruit three secondary precursors, which together differentiate as the eight chordotonal organs. As in all cases of EGFR signaling, the recruited cell's immediate response is the activation of the ETS family transcription factor, Pointed (Pnt). The pnt gene encodes two isoforms, Pnt-P1 and P2 (the combined activity of the two isoforms is referred to as Pnt). Pnt-P2 is activated by ERK MAPK phosphorylation upon EGFR stimulation, whereas Pnt-P1 is regulated by EGFR signaling at the transcriptional level. The favored model is that ERK phosphorylates Pnt-P2, which then activates the transcription of Pnt-P1. Apart from their regulation, it is thought that Pnt-P1 and P2 function similarly as transcription factors by binding to the same sites via their common ETS domains. Interestingly, signaling from the dorsal-most primary SOP triggers the recruitment of oenocyte precursors rather than chordotonal SOPs. Clearly, specificity of cellular response must depend on factors other than Pnt (zur Lage, 2004).
A similar process of recruitment occurs for the adult femoral chordotonal organ (FCO), but with key differences. As in the embryo, chordotonal SOPs recruit further SOPs from the ectoderm, but in this case recruitment is reiterative: newly recruited SOPs themselves express rho and in turn recruit further SOPs from the ato-expressing PNC. Thus, unlike in the embryo, the recruitment cycle is repeated many times as new SOPs become new signaling sources. As a result, some 80 SOPs are recruited over time. In the leg disc, SOP recruitment correlates with ato upregulation. To understand the basis of this, ato regulation during recruitment has been investigated. Analysis of key target gene enhancers has greatly increased understanding of the logic of cell fate determination. An enhancer upstream of ato is active specifically during recruitment in both the leg disc and the embryo. This enhancer is regulated directly by Pnt and Ato binding cooperatively to adjacent sites. The consequence of this is that the enhancer responds only to the combined input of both Pnt and Ato. Thus, Ato ensures the specificity of EGFR signaling in this context. Importantly, SOP recruitment depends on the direct manipulation of Ato autoregulation: such autoregulation is contingent on EGFR signaling. Thus, to promote the transition from competence to cell fate commitment, a cell signal directly targets the autoregulation of a selector gene (zur Lage, 2004).
Sun (1998) described the approximate location of several regulatory elements up- and downstream of the ato ORF. An enhancer supporting reporter gene expression in FCO precursors was inferred indirectly to exist between SmaI and BamHI restriction sites 3.6-5.5 kb upstream of the ato ORF. When this 1.9 kb region was cloned into a Gal4 P element vector, it indeed supported Gal4 expression in the FCO precursors. The fragment was subdivided and each subfragment was inserted into the GFP reporter vector, pHStinger. Using these transgenes, the FCO enhancer could be localized to a 367 bp fragment. GFP expression driven by this fragment was observed in the chordotonal SOPs (marked by Ato and the SOP protein, Senseless [Sens] but not in the overlying PNC (marked by Ato), suggesting that it is active during SOP commitment (zur Lage, 2004).
The ato FCO enhancer is also active during embryonic chordotonal recruitment. The enhancer drives GFP expression in embryonic sensory cells that derive from a subset of Ato-dependent SOPs. Owing to the delayed acquisition of GFP fluorescence, the onset of GFP expression appears shortly after Ato is downregulated in these SOPs. Nevertheless, examination of perduring GFP in older embryos relative to a sensory neuron differentiation marker, 22C10, revealed expression in two chordotonal sensilla of the five that make up the lateral chordotonal array (lch5). This correlates with the two recruited SOPs that contribute to lch5. The GFP-expressing sensilla are usually the most posterior of the lch5 cluster. Significantly, the anterior-most sensillum never expresses GFP, which is consistent with it deriving from a primary chordotonal precursor. Similarly, GFP expression is observed weakly in one of the two ventral chordotonal organs (vchB), which derives from a recruited SOP. In the head, there is notable expression in cells that give rise to the larval eye (Bolwig's organ). This is also an ato-specified sense organ that requires EGFR signaling. Overall, these patterns suggest that this enhancer is responsible not for general ato regulation in SOPs, but specifically in situations where it depends on EGFR signaling. Moreover, the enhancer appears to mediate the EGFR signaling response in a variety of developmental situations. This element is referred as the ato recruitment enhancer (ato-RE). Significantly, ato-RE is not expressed in oenocytes, even though these are recruited by EGFR signaling from a primary chordotonal SOP (zur Lage, 2004).
How ato-RE-GFP expression responds to ectopic expression of pnt-P1 was analyzed using 109-68-Gal4, a Gal4 driver line that is expressed in many proneural clusters and SOPs in the imaginal discs. However, misexpression of UAS-pnt-P1 induced very little change in the ato-RE-GFP expression pattern in leg or wing discs. It was reasoned that another tissue-restricted factor is required for the response of ato-RE to Pnt. Ato protein itself may be such a factor, since it is already expressed at a low level in the leg PNC cells that are recruited. UAS-ato misexpression (109-68-Gal4 UAS-ato) results in a modest change in expression of ato-RE-GFP, even though such misexpression induces significant (nonrecruitment) chordotonal SOP commitment. In contrast, co-misexpression of both Pnt-P1 and Ato results in a significant increase in ectopic ato-RE-GFP expression in the leg and wing. This finding suggests two things: (1) Pnt/EGFR activation of ato-RE is contingent on the presence of Ato. This restricts its function to Ato-expressing PNC cells. (2) ato-RE is an autoregulatory enhancer, but unlike other proneural autoregulatory enhancers, autoregulation is contingent on the cell receiving an EGFR/Pnt signal (zur Lage, 2004).
These results suggest that the ato-RE is activated by the simultaneous presence of Ato and Pnt in recruited SOPs. As expected from previous evidence, Ato and Pnt-P1 are indeed both expressed in leg FCO SOPs. In the embryo, too, in double labeling experiments, cells that coexpress Ato and Pnt-P1 are clearly present at the time and location expected of recruited SOPs. Consistent with this, a GFP reporter transgene that responds directly to Ato regulation is expressed in both primary and recruited chordotonal SOPs and is coexpressed with Pnt-P1 (zur Lage, 2004).
ato-RE was tested for its ability to bind Pnt and Ato proteins in vitro (the latter as a heterodimer with Daughterless [Da] protein). In gel mobility shift assays using purified proteins and the entire ato-RE as a probe, Pnt and Ato/Da proteins both bind in a manner that is consistent with a single binding site each. In general, Ato/Da binds to the bHLH A-class E-box core consensus. On this criterion, there are two potential Ato/Da binding sites in ato-RE (E1 and E2). Ato/Da binding could be competed strongly by an E1-containing competitor oligonucleotide, but not strongly by an E2-containing competitor nor by a competitor with a mutated version of the E1 site [E1(M): CAGGTG→CCTAGG]. This suggests that Ato/Da binds to ato-RE largely via E1. Mobility shifts with site-specific oligonucleotides as probes supported this. To assess the in vivo function of these sites, site-directed mutagenesis was carried out on ato-RE and the effect on reporter gene expression was assessed in transgenic flies. Mutation of E2 had no discernable effect on ato-RE-GFP expression, but mutation of E1 completely abolished expression in the embryo, leg imaginal disc, and also the eye disc. Thus, E1 is likely to be a binding site through which Ato regulates its own expression in recruited chordotonal precursors (zur Lage, 2004).
Pnt binds to a consensus sequence around a GGAA core that has been characterized for vertebrate ETS-1, and a number of functional Pnt binding sites have been characterized that conform to this consensus. In ato-RE there are two potential Pnt binding sites (ETS-A and ETS-B). Purified Pnt-P1 protein binds to ato-RE, and this binding is competed efficiently with a competitor oligonucleotide containing the ETS-A site but not one containing the ETS-B site. This suggests that Pnt-P1 binds ato-RE via the ETS-A site. Mutating ETS-A abolished GFP reporter gene expression in the embryo, leg FCO region, and eye. A few GFP-expressing cells remaining in the leg may correspond to a tibial chordotonal organ. Mutation of ETS-B had no apparent effect. Thus, the ETS-A site is likely to be a binding site through which Pnt regulates ato expression in the recruited chordotonal precursors (zur Lage, 2004).
In summary, virtually the entire activity of the ato-RE requires the E1 and ETS-A sites, most likely by binding Ato/Da and Pnt, respectively (zur Lage, 2004).
A remarkable feature of the E1 and ETS-A sites is their proximity, their core sequences being separated by 4 bp. This proximity is maintained precisely in the sequence upstream of ato in the genomes of D. pseudoobscura and virilis, where the sites are within an identical stretch of 58 bp. The proximity and its conservation suggest that protein interactions between these transcription factors may contribute to the mechanism by which they specifically regulate ato. Molecular modeling suggests that the Pnt ETS domain and the E1 bHLH domains of Da/Ato heterodimer can bind simultaneously to this DNA sequence and may make direct contact with each other. Although no structures are known for any of these proteins, the conformations of the domains are likely to be highly similar to other proteins of similar sequence for which structures are available. The bHLH domains in the Ato:Da heterodimer were modeled using MyoD homodimer in complex with DNA, and Pnt was modeled using a structure of PU1's ETS domain in complex with DNA. The DNA molecules in each complex were superimposed such that the sites were the correct number of base pairs apart to resemble the E1-ETS-A sequence. The resultant model shows no serious steric clashes between Ato and Pnt domains and, indeed, the two proteins are close enough to form direct contacts (zur Lage, 2004).
This possibility was explored by investigating the binding of Ato/Da and Pnt in gel mobility shift assays. In the presence of all three proteins, a slower migrating protein-DNA complex was observed that represents all three proteins bound to the DNA. In a supershift assay, this complex is lost if antibodies to Ato or Pnt-P1 are included. Moreover, it appears that the triple binding is synergistic. In particular, although Pnt binds relatively poorly to ETS-A alone, the presence of all three proteins appears to drive strong binding of the ternary complex. Interestingly, the ternary complex also formed (albeit less efficiently) when the Pnt site was mutated such that it no longer bound Pnt when added alone. Thus, although Pnt requires the ETS-A site in vivo, in vitro Ato/Da can pull Pnt into the DNA-protein complex even when Pnt cannot interact as efficiently with the DNA itself. Consistent with this, Pnt can also interact with Ato in a GST pull-down assay in the absence of DNA. These data suggest that protein-protein interactions stabilize the DNA-protein complex and that cooperative binding may be important for this enhancer's function and specificity in vivo (zur Lage, 2004).
An interesting question is whether the synergistic interaction between Pnt and Ato/Da allows the E1/ETS-A sites to function in vivo outside the context of the ato-RE enhancer. A construct was made with GFP driven by two tandem repeats of a 35 bp fragment from the conserved ato-RE region, including the E1 and ETS-A sites [(E1+ETS-A)2-GFP]. In the embryo, expression of (E1+ETS-A)2-GFP was strikingly similar to ato-RE-GFP. It is strongly expressed in the precursors of vchAB, v'td2, and two sensilla of lch5. In the head there is particularly strong expression in cells giving rise to Bolwig's organ, as well as other ato-dependent locations. This construct, however, does not support any expression in the femoral precursors of the leg disc, suggesting that additional ato-RE sequences are required here for correct regulation (zur Lage, 2004).
To ascertain the contribution of the ETS-A site, a reporter transgene driven by six copies of the E1 site alone [(E1)6-GFP] was generated. Unlike the (E1+ETS-A)2-GFP construct, (E1)6-GFP is expressed in all ato-dependent SOPs in the embryo. Thus, the E1 site is capable of supporting Ato/Da-dependent regulation in all SOPs, but regulation is normally restricted to recruited SOPs by the need for Ato/Da to interact with Pnt. Presumably, this requirement is subverted when the E1 site is highly multimerized. A second possibility is that as well as binding Pnt, the ETS-A site can also bind a repressor. A likely candidate is the ETS repressor Yan. Yan acts in opposition to Pnt, and its repressor activity is relieved upon EGFR signaling by phosphorylation by ERK. Indeed, Yan protein is expressed during embryonic chordotonal recruitment, and recruitment is more extensive in yan mutant embryos. Consistent with this, Yan protein is able to bind the ETS-A site in vitro, and (E1+ETS-A)2-GFP is expressed in more cells in yan mutant embryos. This suggests that the ETS-A site is bound by Yan repressor. This repression of ato-RE is relieved by EGFR-dependent phosphorylation and by displacement by Pnt proteins. Interestingly, there is no evidence that Yan functions in leg disc SOP recruitment since its expression is undetectable during FCO development. However, FCO recruitment is susceptible to Yan function, since expression of a UAS-yanAct construct strongly inhibits chordotonal SOP recruitment (zur Lage, 2004).
A summary of the regulation of ato during recruitment is presented. Initially, Ato is expressed at a low level in PNC cells as a result of regulation by an enhancer(s) that is distinct from ato-RE. Such PNC enhancers are known for both the leg and the embryo. Coexpression of E(spl) in response to Notch signaling inhibits SOP commitment, but the low level of Ato provides the competence for these cells to respond to EGFR signaling from other SOPs. If an Ato-expressing cell receives this signal, the combined action of Pnt and Ato/Da acts via the ato-RE element to upregulate Ato expression, leading to SOP commitment. Although this model describes a mechanism for SOP recruitment, it is likely that the firstborn chordotonal SOPs (including the primary SOPs in the embryo) are selected from the PNC by mechanisms involving an interplay of ato regulation with Notch/E(spl) signaling, as described for ac/sc. This conventional SOP selection route would function via enhancers other than the ato-RE (zur Lage, 2004).
The combined response to Pnt and Ato/Da is mediated at the molecular level by cooperative binding of the transcription factors to adjacent sites that are evolutionarily conserved. The juxtaposition of sites allows high affinity of protein complex binding in vitro, and hence high specificity of enhancer activity in vivo even when the two sites form a synthetic enhancer in isolation from the rest of the enhancer. Binding by Pnt-P1 alone is rather poor but is much stronger in the presence of Ato/Da. Thus, cooperativity increases specificity. As in the case of Pnt, direct cooperative interaction with a selector gene product has been found to underlie the specificity of mammalian ETS-1 proteins, including interaction with Runx and Pax5. Significantly, cooperative binding has been characterized between ETS-1 and the bHLH protein, USF. Preliminary molecular modeling of bHLH and ETS domains on the ato-RE shows the feasibility of contact between the Ato HLH and Pnt ETS domains. Among proneural proteins, this interaction may be very specific for Ato: bHLH residues available for interaction with Pnt are uniquely conserved in Ato and its vertebrate homologs compared with Sc and its homologs. It is suggested that Sc is unable to make appropriate interactions with Pnt. Consistent with this, when the Ato/Da E1 site is altered to conform to the binding consensus for Sc/Da, there is a dramatic loss of ato-RE enhancer activity. In this light, Pnt can be thought of as a specificity cofactor that ensures that Ato is the only proneural protein that can regulate the ato-RE (zur Lage, 2004).
Positive autoregulation is a common transcriptional control mechanism. It is suggested that commonly, if not universally, positive autoregulation is contingent on other conditions being fulfilled in addition to the factor itself being present. In consequence, promotion or inhibition of autoregulation provides a sensitive nodal point of regulation that can be modulated by extrinsic factors. In the case of ato, autoregulation provides the switch through which EGFR signaling can drive the transition from SOP competence to commitment. In a related way, autoregulation plays an important part in conventional SOP determination by Ac and Sc proneural proteins. In current models, Notch inhibits SOP selection by antagonizing the activity of Ac and Sc autoregulatory enhancers. Genetically, chordotonal precursor recruitment is also inhibited by Notch signaling. This does not appear to be by direct DNA binding, however, since there are no good candidate consensus sites for Su(H) or E(spl) in ato-RE. One possibility is that E(spl) proteins interact directly with Ato/Da and that this inhibitory interaction is displaced by interaction with Pnt on the ato-RE. Other possibilities include direct interaction between Notch and EGFR pathways farther upstream than Pnt and Ato and the activation of yan expression by Notch signaling (zur Lage, 2004).
Autoregulation of ac and sc is relatively simple in the sense that a single autoregulatory enhancer functions in many or all of the locations in which these genes are expressed. In contrast, by requiring Pnt, ato-RE activity is limited to the subset of areas of ato expression in which recruitment signaling occurs. Such spatial restriction of autoregulatory enhancer activity appears to be an important part of ato regulation, since in addition to the ato-RE, the gene is proposed to have a number of distinct autoregulatory enhancers, with different ones required in different locations. Presumably, the autoregulatory action of each of these enhancers is contingent on spatially restricted factor(s) equivalent to Pnt. It will be important to find out what these factors are and whether they too interact directly with Ato/Da proteins at their respective binding sites (zur Lage, 2004).
Ets transcription factors play crucial roles in regulating diverse cellular processes including cell proliferation, differentiation and survival. Coordinated regulation of the Drosophila Ets transcription factors Yan and Pointed is required for eliciting appropriate responses to Receptor Tyrosine Kinase (RTK) signaling. Yan, a transcriptional repressor, and Pointed, a transcriptional activator, compete for regulatory regions of common target genes, with the ultimate outcome likely influenced by context-specific interactions with binding partners such as Mae (FlyBase name: ETS-domain lacking). Previous work in cultured cells has led to a proposal that Mae attenuates the transcriptional activity of both Yan and Pointed, although its effects on Pointed remain controversial. A new layer of complexity to this regulatory hierarchy is provided whereby mae expression is itself directly regulated by the opposing action of Yan and Pointed. In addition, Mae can antagonize Pointed function during eye development; a finding that suggests Mae operates as a dual positive and negative regulator of RTK-mediated signaling in vivo. Together these results lead to a proposal that a combination of protein-protein and transcriptional interactions between Mae, Yan and Pointed establishes a complex regulatory circuit that ensures that both down-regulation and activation of the RTK pathway occur appropriately according to specific developmental context (Vivekanand, 2004).
Because mae expression
in wild-type embryos is reminiscent of the expression patterns of
genes such as argos (aos) and orthodenticle
(otd) that have been shown to be regulated by Epidermal growth
factor receptor (EGFR) signaling, whether mae expression might be similarly regulated by the downstream EGFR pathway effectors, Yan and Pnt, was investigated. Analysis of the genomic region around mae reveals two clusters of ETS DNA
binding consensus sites (EBS; defined as GGAA/T), one
upstream of the transcription start site (MaeEBS1) and the other in
the intron of mae (MaeEBS2),
further suggesting that Yan and Pnt might regulate mae
expression. To explore this possibility, in situ hybridization
experiments were performed to determine whether mae expression
is affected by altering the dosage of Yan and Pnt. As predicted,
based on the presence of EBS clusters in the mae genomic
region, mae expression is significantly increased in
yan mutant embryos, while it is lost in pnt mutant embryos. Conversely, ubiquitous overexpression of YanACT or Pnt results in down-regulation and up-regulation of mae expression, respectively. In
addition to regulating mae expression in the embryo, Yan and
Pnt also regulate mae expression in eye imaginal discs. Over-expression of PntP1 results in almost three-fold increase in mae levels, while
over-expression of YanACT results in a decrease. Taken
together, these results suggest that mae expression is
regulated by the Ets transcription factors Pnt and Yan in multiple
developmental contexts (Vivekanand, 2004).
To determine whether Pnt and Yan regulate mae levels
directly, the EBS clusters were cloned upstream of a minimal promoter
and luciferase cDNA to generate two different MaeEBS-luciferase
reporters, MaeEBS1-luciferase (upstream cluster) and MaeEBS2-luciferase (intronic cluster). This enabled an assessment of the effects of
Pnt and Yan on these putative regulatory elements by performing
transcription assays in Drosophila S2 cells. If Pnt and Yan
directly regulate mae transcription, then the prediction would
be that Pnt and Yan would bind to the EBSs and activate and repress
transcription of the reporter, respectively (Vivekanand, 2004).
Both the upstream and
the intronic EBS clusters behaved similarly in these luciferase
reporter assays. Addition of the constitutively activated form of
Pnt, PntP1, resulted in activation of the reporter, while co-transfection
of Yan with PntP1 resulted in two to three fold repression in
transcription. Similarly,
co-transfection of PntP2 and RASV12 resulted in
transcriptional activation of the reporter. The transcriptional modulation of the MaeEBS-luciferase reporters by Pnt and Yan supports the hypothesis that mae
expression is directly regulated by Pnt and Yan in vivo (Vivekanand, 2004).
MAE has been shown to antagonize Pnt function, putting
it in the unique position of being a dual positive and negative
regulator of EGFR-mediated signals. Intriguingly,
mae expression is itself regulated by Pnt and Yan, suggesting
a whole new layer of feedback loops that fine-tune and down-regulate
signaling (Vivekanand, 2004).
While overexpression of MAE
blocks Yan's repression capability (Tootle, 2003), this occurs without altering
Yan nuclear localization. Thus increased MAE expression appears to
interfere directly with Yan-mediated transcriptional repression. An
intriguing model to explain this finding originates from the
observation that homotypic interactions mediated by the Pointed
Domain (PD) of TEL, the mammalian ortholog of Yan, result in the
formation of TEL polymer that may facilitate transcriptional
repression by wrapping around the target DNA (Kim, 2001). Yan is similarly capable of
self-association and the residues that are required for TEL
polymerization have been conserved, suggesting Yan-Yan polymerization
might similarly be critical for repression (Jousset, 1997 and Qiao, 2004). In this context, perhaps
clusters of EBSs, similar to those described in mae,
by recruiting multiple Yan molecules to a common target site may
provide a scaffold for nucleating and promoting Yan
polymerization (Vivekanand, 2004).
Such a model requires a mechanism to limit the
extent of polymer formation, such that the cell can achieve efficient
but reversible repression of target genes. Considering its
multifaceted role in down-regulating Yan activity and its ability to
bind the PD of Yan, MAE is a prime candidate to fill such a role.
Consistent with this prediction, recent studies have found that
PD-mediated polymerization of Yan is required for transcriptional
repression and that MAE effectively 'caps' Yan
oligomerization by occluding the residues required for polymerization
(Qiao, 2004). Thus it is tempting to speculate that MAE's ability to abrogate
Yan-mediated repression may reflect a role in
'depolymerizing' Yan at the DNA, an intriguing model
that remains to be validated in vivo (Vivekanand, 2004).
In addition to antagonizing
Yan activity, this work suggests that MAE also negatively regulates
PntP2 function, thus positioning it uniquely within the RTK pathway
as both a positive and negative regulator. For example, the
phenotypes associated with misexpression of MAE in the
Drosophila visual system are completely suppressed by
co-expression of PntP2, arguing strongly that MAE can antagonize EGFR
signaling in the eye by interfering with the activity of PntP2. While
the photoreceptor loss and increased apoptosis phenotypes associated
with MAE overexpression resemble the consequences of blocking Yan
nuclear export and down-regulation, the reduced Yan expression
observed in MAE-expressing eye disc argues against such an
explanation. Furthermore, if MAE were inducing premature
down-regulation of Yan in these cells, one would expect to observe
ectopic photoreceptors, rather than the neuronal loss that actually
occurs. Thus, although a direct effect on Yan cannot be excluded, the interpretation is favored that the primary consequence of MAE
overexpression is reduction in activity of PntP2, and that the loss
of Yan expression is a secondary outcome. It is important to note
that both cell culture and in vivo experiments employ
overexpression strategies that are subject to the caveats inherent to
such analyses. Thus these experiments are viewed as an opportunity to
reveal new mechanistic hypotheses that will provide an important
foundation for future studies designed to unravel the complex
regulatory circuitries that exist between MAE, Yan and Pnt in vivo (Vivekanand, 2004).
Induction of both positive and negative feedback
loops by signal transduction pathways plays an important role in
regulating the response to pathway activation. Activation of PntP2 by EGFR/RAS/MAPK
results in the transcription of target genes including Argos
and Kekkon1, which have been shown to negatively regulate the
pathway. This study identifies another target of the Ets transcription
factors Pnt and Yan, mae, which performs the dual role of
promoting and inhibiting signaling by the EGFR/RAS/MAPK pathway.
Based on the effects on mae expression pattern observed in
pnt and yan mutants and in embryos and eye imaginal
discs overexpressing Pnt and Yan, it is proposed that Pnt activates while
Yan represses mae transcription (Vivekanand, 2004).
Based on MAE's ability to antagonize EGFR signaling output,
activation of mae transcription by PntP2 provides a negative
feedback loop that would prevent runaway pathway activation. While Kekkon-1 and the secreted antagonist Argos act at the level of the receptor to down-regulate signaling, the
induction of mae transcription would ensure the
down-regulation of the pathway by inhibiting the function of the
effector PntP2. This would result in cell autonomous inhibition of
the EGFR/RAS/MAPK pathway at the level of the transcription factor.
Moreover, while the previously identified inhibitors Argos, Sprouty and
Kekkon1 function solely as antagonists of RTK signaling, MAE is unusual in that it acts both as a positive and negative
regulator of the pathway by inhibiting both Yan and PntP2
function (Vivekanand, 2004).
Because MAE negatively regulates both Yan and PntP2
function, imposing constraints on MAE protein levels becomes
critical. This appears to be achieved by regulating mae
expression levels directly by Yan and Pnt. For example, because excess MAE could potentially break up
Yan-Yan polymer to such an extent that Yan would no longer
able to repress appropriate target genes (Qiao, 2004), the negative regulation of
mae expression by Yan sets up a situation whereby excessive
levels of MAE do not accumulate. Thus in the absence of RTK
signaling, repression of mae by Yan would ensure that only low
MAE levels are present in the nucleus, allowing Yan to repress
transcription. Emphasizing the importance of fine-tuning the
expression levels of these three nuclear RTK pathway regulators and
further complicating the circuitry, it has been suggested that Yan
and Pnt may also directly regulate each other's transcription, setting
up additional positive and/or negative regulatory loops. For
example, the finding that overexpression of PntP2 leads to
up-regulation of Yan in the eye disc is consistent with a feedback
loop whereby the activity of PntP2, a positive pathway effector,
attenuates its own activity by increasing expression of the Yan
repressor. A great deal of future work will be needed to unravel the
precise in vivo contexts in which these complex transcriptional
regulatory networks operate (Vivekanand, 2004).
In conclusion, MAE joins the panoply
of regulators of EGFR signaling that have been shown to play an
important role in modulating and restricting the strength, range and
duration of signaling events. By establishing negative feedback loops
that act at multiple levels within a signal transduction cascade, a
robust checkpoint is established to attenuate as well as prevent
constitutive signaling by the RTK pathway (Vivekanand, 2004).
Drosophila development depends on stable boundaries between cellular territories, such as the embryonic parasegment boundaries and the compartment boundaries in the imaginal discs. Patterning in the compound eye is fundamentally different: the boundary is not stable, but moves (the morphogenetic furrow). Paradoxically, Hedgehog signaling is essential to both: Hedgehog is expressed in the posterior compartments in the embryo and in imaginal discs, and posterior to the morphogenetic furrow in the eye. Therefore, uniquely in the eye, cells receiving a Hedgehog signal will eventually produce the same protein. The mechanism that underlies this difference is the special regulation of hedgehog (hh) transcription through the dual regulation of an eye specific enhancer. This enhancer requires the Egfr/Ras pathway transcription factor Pointed. Recently, others have shown that this same enhancer also requires the eye determining transcription factor Sine oculis (So). These data are discussed in terms of a model for a combinatorial code of furrow movement (Rogers, 2005).
There are two known eye-specific hedgehog (hh) mutations:
hhbar3 (also known as hh1) and
hhfurrow stops early (or hhfse). Both
are associated with deletions in the first intron.
hhbar3 is a homozygous viable allele with a strong
recessive eye phenotype resulting from arrest of the morphogenetic furrow.
hhfse is a gamma-induced viable allele with a weaker eye
phenotype. PCR and direct sequencing were used to determine the precise end-points of the deletions. The hhbar3 deletion
is 1885 bp and the hhfse deletion lies within the span of
hhbar3, but is shorter. Both
hhbar3 and hhfse are viable and can be
maintained as homozygous stocks, although they are not as vigorous as wild
type. This is probably not due to second-site recessive lethal mutations, since
lines were derived that are isogenic for the X and major autosomes and they are
no more vigorous. The cuticles and nervous system (by anti-Elav
and anti-Futsch stains) of the isogenic hhbar3 and
hhfse embryos were examined, and no detectable phenotypes were found (Rogers, 2005).
To determine if either of these two eye-specific alleles are null for
hedgehog function in the eye, all viable pair-wise
combinations of these alleles, wild-type and two zygotic lethal alleles
(hhAC and hh8), were derived.
hhAC is a single gene deletion that removes both the start
sites for transcription and translation.
hh8 (also known as hh13C) is a
chain-terminating mutation in the coding sequence. Both
alleles are zygotic lethal with strong cuticle phenotypes.
hhAC is thought to be a null because of the strength of
its phenotype and the nature of its lesion. On
phenotypic grounds and comparison with other alleles, other groups have also
reported hh8 to be functionally amorphic (Rogers, 2005).
These alleles form a series for adult eye phenotype. This
was quantified by counting eye facets in adult females; hhfse, hhbar3 and hh8
heterozygotes are not significantly different from wild type. However,
hhAC is slightly dominant, with an eye that is about 10%
smaller than wild type (although this difference is not statistically
significant) (Rogers, 2005).
By facet number, hhbar3 is a strong, eye-specific
hypomorph. It is fully recessive in trans to wild type, has a severely reduced
eye when homozygous (68% smaller than
hhbar3/hh+) and in trans to the null
hhAC it is smaller still (82% smaller than
hhbar3/hh+). This suggests that
hhbar3 is not an amorph for eye size by Muller's test: the
phenotype becomes stronger in trans to the null.
hhfse is similar to but weaker than
hhbar3: the hhfse homozygous eye is
only 32% smaller than hhfse/hh+ and in
trans the null (hhAC), it is further reduced to 78%. Thus,
by both measures (phenotype as a homozygote and in trans to a null),
hhbar3 is a strong hypomorphic allele and
hhfse is a weaker hypomorph. From the 95% confidence
limits, all these results are statistically significant (Rogers, 2005).
Probably hhbar3 and hhfse affect a
transcriptional enhancer and not the protein itself or the gene promoter,
because neither lesion directly affects the coding sequence. In sequencing 23
cDNAs from eye-imaginal discs, no alternative first exon or start
site was found in the region of the two mutations (Rogers, 2005).
These two eye-specific alleles of hedgehog were characterized and they were found to delete elements that are specifically necessary for expression
in the developing eye, posterior to the morphogenetic furrow. This
hedgehog eye enhancer drives expression in all of the developing
ommatidial cells except the R8. This element was reduced to a 203 bp minimal
fragment that is sufficient for reporter expression. The
hedgehog eye enhancer is regulated by pointed in vivo and
bound by Pointed in vitro. Since Egfr/Ras-driven Pointed activates reporters in
all the cells except the R8, it is suggested that the hedgehog expression
in the developing eye is driven by this enhancer and that Hedgehog is
expressed in the developing ommatidial cells excepting the R8 (Rogers, 2005).
It is proposed that hhbar3 is indeed null for
hedgehog expression in the developing eye, consistent with the loss
of detectable antigen. This appears to contradict facet count data, which
show that hhbar3 is not null for eye size. It is suggested that
hedgehog functions elsewhere (probably in the eye disc margin),
expressed at some lower level, and acts redundantly with Decapentaplegic to
drive the early phases of furrow progression. This is consistent with data
from others for an early role for hedgehog in the eye margin for
furrow initiation, and with a proposed redundancy between
hedgehog and dpp in the furrow.
The enhancement of the hhbar3 phenotype when it is placed
in trans to a null (hhAC) suggests that hhbar3 may reduce, but not eliminate this early function (Rogers, 2005).
Several examples of eye-specific transcriptional enhancers have been
characterized. A number of these are in genes that act early in retinal
determination (eyes absent, dachshund and sine oculis), and
are not directly involved in the morphogenetic furrow. Some
enhancers that function in and posterior to the morphogenetic furrow have also
been studied. One example is the atonal gene, which has been shown to
have two regulatory enhancers with specific and different activities in the
furrow.
Interestingly the atonal enhancers produce almost the reciprocal
expression pattern of the hedgehog eye enhancer described here:
hedgehog is expressed in all cells except the R8 and atonal
expression is in only the R8, posterior to the furrow. Furthermore,
atonal mutations can affect hedgehog signaling, although
this may be indirect, and indeed, hedgehog is also known to regulate
atonal. Other enhancers that act posterior to the furrow have been
characterized in the rough, sevenless and prospero genes,
but none of these appears to show the particular type of regulation
described in this study (Rogers, 2005).
A similar DNA fragment from the hhbar3 region confers post-furrow, eye-specific expression on a lacZ reporter (Pauli, 2005). The consensus binding site for another transcription factor was characterized: the retinal determination protein Sine oculis (So). Two So-binding sites were found in the hhbar3 region, and it was shown that these are necessary for the normal function of the hedgehog eye enhancer. A So site tetramer is
sufficient to drive reporter expression in the entire presumptive eye field in
the third instar disc. One of the two So sites lies
within the 203 bp minimal element (Rogers, 2005).
Taken together, both sets of data suggest that Pointed and So activation
at the minimal element are each necessary, but that neither is sufficient for
the specific activation of the hedgehog eye enhancer posterior to the
furrow. It is proposed that they act together to confer this dual regulation. This
is consistent with the following model: that special dual regulation of hedgehog is the mechanism which makes the morphogenetic furrow move, unlike the stable compartment boundaries. It is suggested that this dual regulation depends on one 'selector' signal that is eye specific (So), to differentiate the furrow from boundaries in other organs. The second component must act to close a loop such that cells which receive the furrow inducing signal will later send it, after a delay, to make the boundary move forward. This 'signal' component is Pointed, acting downstream of Egfr/Ras signaling in the assembling ommatidia. This may be a case of 'selector' and 'signal' transcriptional integration. Indeed, pointed itself has been shown to integrate 'selector' factors in muscle development. It is proposed that by this dual regulatory mechanism, a system that first evolved to divide the bauplan into metameric parasegments has been co-opted to drive a moving wave of differentiation in the developing eye (Rogers, 2005).
Roughest (Rst) is a cell adhesion molecule of the immunoglobulin superfamily that has multiple and diverse functions during the development of Drosophila melanogaster. The pleiotropic action of Rst is reflected by its complex and dynamic expression during the development of Drosophila. By an enhancer detection screen, several cis-regulatory modules have been identified that mediate specific expression of the roughest gene in Drosophila developmental processes. To identify trans-regulators of rst expression, the Gal4/UAS system was used to screen for factors that were sufficient to activate Rst expression when ectopically expressed. By this method the transcription factors Single-minded, Pointed.P1, and Su(H)-VP16 were identified. Furthermore, these factors and, in addition, Dmef2 are able to ectopically activate rst expression via the previously described rst cis-regulatory modules. This fact and the use of mutant analysis allocates the action of the transcription factors to specific developmental contexts. In the case of Sim, it could be shown to regulate rst expression in the embryonic midline, but not in the optic lobes. Mutagenesis of Sim consensus binding sites in the regulatory module required for rst expression in the embryonic midline, abolishes rst expression; indicating that the regulation of rst by Sim is direct (Apitz, 2005).
Rst has complex and multifaceted functions throughout the development of the fly, which include myogenesis, eye development, as well as axonal pathfinding in the optic lobes. To gain a better understanding of these functions at the levels of gene regulation and signal transduction, a number of tests were designed to identify both the transcriptional activators and their respective targets surrounding the rst locus. In a preceding study (Apitz, 2004), a number of DNA segments upstream of rst were characterized and regulatory regions were discovered that mediate gene expression in myoblasts, midline, and eyes, respectively. In the present
study these results were supplemented with an in vivo screen to identify regulators of rst expression using the Gal4/UAS system. Several factors were discovered that are able to induce ectopic Rst expression and to activate reporter gene expression via rst cis-regulatory sequences (Apitz, 2005).
The experimental route taken to identify protein factors involved in the regulation of the rst gene is based on the detection of their potential to induce ectopic Rst expression in vivo. The use of sca-Gal4 as a driver line in this experimental approach is based on the following criteria. sca-Gal4 mediates expression in neuroectodermal cells of the embryo. At embryonic stage 10, these cells can be examined for ectopic Rst expression because no endogenous Rst expression is found at this time in these cells; this allows operators to obtain clear-cut and unequivocal results. The use of alternative Gal4 driver lines did not prove suitable because of the dynamic expression of Rst during all developmental stages, and due to its subcellular localization. For example, when dll-Gal4 is used as a driver, it is difficult to distinguish between ectopic Rst expression induced in the apical tips of cells of the leg discs, and endogenous Rst expression in the overlaying ectodermal cells. Furthermore, the use of sca-Gal4 has the advantage that neuroectodermal cells are not fully differentiated cells. This may more closely resemble the developmental state of the cells in which Rst expression is normally induced endogenously, e.g., in undifferentiated cells of the developing eye disc. However, this approach generally fails to reveal transcription factors that need a coactivator for induction of rst expression, which is not present in the cells of the neuroectoderm at embryonic stage 10. This may explain the failure of Dmef2 to induce rst expression at this stage. It was shown, however, that Dmef2 is able to induce rst expression at later stages by the use of rst-lacZ constructs. The function of the different rst-lacZ constructs has been linked to specific developmental circumstances (Apitz, 2004) and their activation by corresponding factors is consistent with the known roles of these proteins in development (Apitz, 2005).
Ectopic expression of a constitutively active Pnt variant (Pnt.P1) mediates strong activation of Rst expression in neuroectodermal cells. Since Pnt.P1 recognizes the same target sequences as its splice variant Pnt.P2, the nuclear effector of the Ras-MAPK pathway, ectopic activation of Rst expression by Pnt.P1 is consistent with a regulation by the Ras-MAPK pathway. Similarly, the ectoptic activation of Rst expression in neuroectodermal cells by Su(H)-VP16 points to a regulation of rst by the Notch pathway (Apitz, 2005).
The Ras-MAPK and the Notch pathways display significant crosstalk during developmental processes in Drosophila, e.g., in cell fate specification of the eye disc. It is difficult to elucidate a possible regulation of a candidate gene by mutant analysis if it is activated by both pathways. In mutants, for one of the pathways, the activity of the other pathway will ensure residual expression of the candidate gene under scrutiny. rst expression is activated by both pathways and single mutant analysis did not reveal a significant loss of expression. Both pathways converge on regulatory elements contained within F6 and not in F5. Furthermore, a regulatory module is present in the nonoverlapping part of F6 that is activated in IOC before apoptotic decisions are made in these cells (Apitz, 2004). This module is located within an approximately 600-bp sequence and is active during several apoptotic decisions (Apitz, 2004). Consensus binding sites for Su(H) and Pnt in this module are conserved between D. melanogaster and D. pseudoobscura. Both the Ras-MAPK and Notch pathways are involved in apoptotic processes of IOC cells. Together, these data suggest that rst transcription is regulated by these pathways in the context of apoptotic decisions (Apitz, 2005).
The transactivation screen shows that Dmef2 acts on rst cis-regulatory sequences: the overlapping reporter gene constructs rstF5 -lacZ and rstF6 -lacZ are both ectopically activated by Dmef2 in the scabrous expression domain. Regions F5 and F6 contain partially overlapping sequences and it is likely that Dmef2 acts on a sequence interval bracketed by this overlap. This region contains a regulatory module for expression in the mesoderm (Apitz, 2004). This sequence contains a putative Dmef2 binding site that is also conserved between D. melanogaster and D. pseudoobscura. The sequence of this putative element exactly matches a Dmef2 binding site found in the enhancer of β3 tubulin. Therefore, it is argued that rst may be a direct target of Dmef2. This is consistent with similar mesodermal expression patterns and mutant phenotypes of Dmef2 and rst. In contrast, Rst is expressed in the mesoderm of Dmef2 loss-of-function mutants. An explanation for this result is provided by analysis of rst cis-regulatory sequences. rst is regulated in the mesoderm by at least two independent regulatory modules that mediate a differential expression pattern (Apitz, 2004). One is contained in F6p, the other in F5p. This points to a mesodermal regulation of rst by at least two factors, one of which is still active in Dmef2 mutants (Apitz, 2005).
In Drosophila, restricted expression of the Iroquois complex
(Iro-C) genes in the proximal region of the wing imaginal disc contributes to
its territorial subdivision, specifying first the development of the notum
versus the wing hinge, and subsequently, that of the lateral versus medial
notum. Iro-C expression is under the control of the EGFR and Dpp signalling
pathways. To analyze how both pathways cooperate in the regulation of Iro-C,
several wing disc-specific cis-regulatory elements of the complex were isolated.
One of these (IroRE2) integrates competing inputs of the EGFR and
Dpp pathways, mediated by the transcription factors Pointed (downstream of
EGFR pathway) and Pannier/U-shaped and Mothers against Dpp (Mad), in the case
of Dpp. By contrast, a second element (IroRE1) mediates activation
by both the EGFR and Dpp pathways, thus promoting expression of Iro-C in a
region of elevated levels of Dpp signalling, the prospective lateral notum
near the anterior-posterior compartment boundary. These results help define
the molecular mechanisms of the interplay between the EGFR and Dpp pathways in
the specification and patterning of the notum (Letizia, 2007).
The Iro-C genes ara and caup show similar patterns of
expression in the wing disc. In early second instar larvae, they are expressed
in the whole prospective mesothorax region.
Later, in the third instar, their expression is restricted to the lateral
notum. In addition, at this developmental stage, novel domains of expression appear in the prospective regions of the L1, L3 and L5 veins, tegula, dorsal radius, dorsal
and ventral pleura and alula.
The expression of mirr is slightly different, being absent from the
L3, L5 and tegula domains but present at the other domains. The Iro-C
harbours two additional transcription units, lincoyan
(linc), whose pattern of expression at the notum is identical to that
of ara and/or caup and quilapan (quil),
which is ubiquitously expressed. Previous genetic analysis suggested
the existence of enhancer-like REs that would drive the coincident expression
of ara and caup in the wing disc.
Thus, In(3L)iroDFM2, associated with a breakpoint within
the ara transcription unit, removes ara expression in the wing disc except in the L3 vein domain, in contrast to caup expression which is only lost
from that domain. This suggests the existence of vein L3-specific RE(s)
distal to the In(3L)iroDFM2 breakpoint and other RE(s),
specific for the remaining domains of Iro-C expression, located proximal to
such breakpoint. To identify notum-specific REs, the regulatory
potential of 31 different genomic fragments, spanning approximately 110 kb of
genomic Iro-C DNA was analyzed (Letizia, 2007).
Only five of those fragments drove lacZ expression at specific
regions of the imaginal wing disc. One of them, 3.3 kb in length and named Iro regulatory
element2. The IroRE2 was reduced to a
1.6 kb subfragment (sequence of the IroRE2-B fragment), which maintained
enhancer activity in the notum and was activated by EGFR and repressed by Dpp signalling.
Thus, IroRE2-lacZ was expressed in the proximal region of
early third instar wing discs (the presumptive notum region) and at the
presumptive lateral notum in third instar wing discs. Note,
however, that the pattern of IroRE2-mediated lacZ
expression does not exactly coincide with that of ara/caup. Thus,
ß-gal was not detected in a triangular area, located near the notum/hinge
border and centred around the AP compartment boundary, where expression of
ara/caup is enhanced. This is precisely the
region where expression of lacZ was driven by another Iro-C genomic
fragment of 3.9 kb, IroRE1. Accordingly, an IroRE1-IroRE2 composite RE was found to drive lacZ expression in a pattern very similar, albeit not identical, to that of the endogenous ara/caup
genes (Letizia, 2007).
Two other genomic fragments, IroRE3 and IroRE4 (3.4
and 3.7 kb), adjacent to each other, drove lacZ expression in a stripe of cells located at the proximal region of the presumptive lateral notum, which partially
overlapped with the caup expression domain. Finally,
IroRE5 (2.8 kb) drove expression mainly in the prospective alula
and peripodial membrane (Letizia, 2007).
A common theme in development is the convergence of different signalling
pathways to implement a given developmental program. For instance during
embryonic development, the antagonistic activity of the EGFR and Dpp pathways
sets the limits between the neuroectoderm and the dorsal ectoderm. A
similar situation applies to the specification of prospective body regions
within the wing imaginal disc. During the early second instar, EGFR and
Dpp pathways act antagonistically on the regulation of the Iro-C
restricting its expression to the prospective notum region where it specifies
notum development rather than hinge. Later, at the early third instar, again
the concomitant activity of EGFR and Dpp signals (the latter now also
emanating form the most proximal region of the wing disc) partition the
prospective notum into two different subdomains, the medial and the lateral
notum, the latter being specified by ara/caup expression.
Thus, to understand how regionalization of the adult fly body is achieved it
is important to elucidate the mechanisms responsible for the joint
interpretation of both signalling pathways (Letizia, 2007).
This study shows that the opposing effects of the EGFR and Dpp pathways on
Iro-C expression result from the convergence of both pathways on at least two
distinct Iro-C regulatory elements, IroRE1 and IroRE2.
These two REs drive gene expression in two complementary domains of the
prospective notum region of the wing disc, and appear to mediate most of the
regulation of the Iro-C genes by the Dpp and EGFR pathways in this region of the wing disc. Furthermore, IroRE1 provides a regulatory mechanism for the coexistence at the prospective lateral notum of Iro-C expression and Dpp pathway activity, notwithstanding the negative regulation of Iro-C by such pathway (Letizia, 2007).
The transcriptional regulation of the Iro-C genes is modular. Thus, the
non-coding Iro-C genomic DNA contains a series of five separate enhancers that
control the expression of a reporter gene in sub-domains within the realm of
Iro-C expression in the prospective notum region of the wing disc. None of the
identified fragments reproduces on its own the entire pattern of expression of
Iro-C in the prospective notum. However, IroRE1 and
IroRE2 promote expression in complementary domains that entirely
cover the territory of the presumptive lateral notum. Furthermore,
IroRE2-mediated transcription recapitulates expression of Iro-C at
the whole prospective notum at the second larval instar. It is hypothesized that
the combined activity of both REs would be responsible for a great part of the
regulation of Iro-C expression in the notum territory. Moreover, although
IroRE3, IroRE4 and IroRE5 mediate
lacZ expression in patterns only partly related to that of the Iro-C
genes, these REs probably contribute to the complex regulation of the Iro-C.
In addition, the possibility cannot be excluded of other RE(s) located outside
the tested region that would help to establish the final pattern of Iro-C
expression. Indeed, IroDFM3, a deficiency obtained by imprecise excision of the irorF209 P-lacZ element that extends up to the mirr promoter, maintains some lacZ expression in part of the central notum (Letizia, 2007).
The identified REs might act simultaneously on ara and
caup expression to give rise to their almost coincident patterns of
expression. Such coincidence cannot
be attributed to cross-regulation between ara and caup since
in irorF209 mutant discs (irorF209 is
an ara null allele expression of caup is unmodified. Regulation of
ara/caup would be, accordingly, similar to that of the
achaete-scute genes of the AS-C, which show identical patterns of
expression due to the use of shared enhancers.
Expression of the vertebrate Iroquois (Irx) genes appears to be
similarly regulated. Thus, the analysis of the regulatory potential of highly
and ultra conserved non-coding regions present in the intergenic regions of
the Irx clusters suggests these genes to be regulated by partially redundant
enhancers shared by the components of each cluster (Letizia, 2007).
Expression of mirr in the notum region of the wing disc largely
coincides with that of ara/caup and most likely is under the control
of the same REs. Thus, activity of the IroRE2 may account for the
unmodified expression of mirr in iro1 imaginal
discs (associated with an inversion breakpoint located within the
caup transcription unit). In addition, differences in the expression of
ara/caup and mirr might be due to the presence of repressor
RE(s) or insulator sequences that would prevent the action of the RE(s)
controlling ara/caup on the mirr promoter. This is
consistent with the previous observation of ectopic expression of mirr in Mob1 mutants, a regulatory mutation mapped within the Iro-C (Letizia, 2007).
The identification of REs present in the Iro-C has allowed unveiling of
some of the molecular mechanisms of its transcriptional regulation at the
level of DNA-protein interaction and analysis of the interplay of positive and
negative inputs from convergent signalling pathways (Letizia, 2007).
EGFR activation in the proximal region of the wing disc leads to expression
of Iro-C. This study demonstrates that both IroRE1 and IroRE2 mediate positive regulation by the EGFR pathway. It is shown that Pnt
mediates activation of IroRE2-lacZ by the EGFR pathway.
Furthermore, EGFR-dependent activation is cell context dependent. This suggests the
existence, in the cells receiving EGFR signalling, of presently unknown
factors that would contribute to ara/caup activation and/or the
presence of counteracting repressing mechanisms, which should prevent their
activation. Clearly, the Dpp pathway is so far the best candidate, since it
has been shown that it can repress Iro-C and
the IroRE2-lacZ transgene (Letizia, 2007).
The molecular mechanism of Dpp-dependent regulation of Iro-C expression
appears to be more complex. The Dpp pathway can repress or activate Iro-C
through different REs and different effector proteins. IroRE2
appears to mediate Dpp-dependent repression at the medial notum (most probably
through direct binding of the heterodimer Pnr/Ush and Mad) and at the hinge
and lateral notum (independently of Pnr, Ush and the GATA factor Grn in these
domains). Dpp-dependent repression of Iro-C may be mediated, in addition,
through a different RE, namely, through a brk silencer element (brkSE), shown to mediate Dpp-dependent repression of brk by binding of a Medea/Mad/Schnurri repressor complex, which is present at the Iro-C within IroRE5 (Letizia, 2007).
Despite the Dpp-mediated repression through IroRE2, a high level
of Iro-C proteins accumulates in the lateral region of the notum, near the
strong source of Dpp at the AP border. Furthermore, in this region of the wing disc Iro-C expression is refractory to Dpp-dependent repression. It
is noteworthy that, IroRE1 mediates lacZ expression
exclusively in that region of the wing disc and it appears to provide a
regulatory mechanism for the co-existence of Iro-C expression and Dpp pathway
activity, since the Dpp pathway does not repress but, on the contrary,
activates IroRE1-mediated lacZ expression. Activation is
restricted to the lateral notum, most likely because of the presence, in the
hinge and medial notum territories, of repressors [Muscle segment homeobox,
Msh; also known as Drop and Pnr/Ush, respectively] that would counteract
activation. Putative binding sites for both Msh (consensus sequence G/C
TTAATTG) and GATA proteins are indeed present in IroRE1. Thus, IroRE1 and IroRE2 represent two different REs in the same gene that respond in opposite ways to the same positional information, i.e. Dpp signalling. In addition a Dpp-independent mechanism based in the mutual repression between Iro-C and the homeoprotein Msh helps to maintain the distal border of
Iro-C expression. This repression could be mediated by direct binding of Msh to one putative Msh binding site present in the Iro-RE2-B sequence (Letizia, 2007).
Coding sequences are usually the most highly conserved sectors of DNA, but genomic regions controlling the expression pattern of certain genes can also be conserved across diverse species. In this study, five enhancers were identified capable of activating transcription in the midline glia of Drosophila melanogaster and each contains sequences conserved across at least 11 Drosophila species. In addition, the conserved sequences contain reiterated motifs for binding sites of the known midline transcriptional activators, Single-minded, Tango, Dichaete, and Pointed. To understand the molecular basis for the highly conserved genomic subregions within enhancers of the midline genes, the ability of various motifs to affect midline expression, both individually and in combination, were tested within synthetic reporter constructs. Multiple copies of the binding site for the midline regulators Single-minded and Tango can drive expression in midline cells; however, small changes to the sequences flanking this transcription factor binding site can inactivate expression in midline cells and activate expression in tracheal cells instead. For the midline genes described in this study, the highly conserved sequences appear to juxtapose positive and negative regulatory factors in a configuration that activates genes specifically in the midline glia, while maintaining them inactive in other tissues, including midline neurons and tracheal cells (Fulkerson, 2010).
The results described in this study indicate that the four genes expressed in the midline glia contain enhancers with subregions conserved in 11 or 12 of the sequenced Drosophila genomes. These conserved subregions contain one or more of the four motifs previously identified in the wrapper regulatory region, are highly A/T rich, and needed for robust expression in the midline. These results confirm the importance of several transcription factor-binding sites for midline glial activation. One of these sites, the CME, binds both Sim/Tgo and Trh/Tgo heterodimers and, when multimerized, can drive reporter gene expression in both midline and tracheal cells. Two lines of evidence indicate that the context of the CME determines whether or not it can be utilized to drive expression in these two tissues. (1) The sequences flanking the CMEs are highly conserved in the four genes discussed in this study, Glec, oatp26f, liprinγ and wrapper, suggesting that the location and sequence of other transcription factor-binding sites are constrained. (2) Changing the sequences flanking the CME in the synthetic multimers can eliminate expression in the midline, trachea, or both tissues (Fulkerson, 2010).
A multimerized CME in the context of the 4Toll:GFP reporter was expressed in both the midline and trachea and quite sensitive to slight modifications in flanking sequences. Changing 5-7 nucleotides on either side of the CME within this multimerized construct either substantially elevated expression in the trachea and eliminated it in the midline (T rich:GFP) or eliminated expression in both tissues (Sox:GFP). Additional combinations between the CME and one of the other midline glial motifs restricted expression to the midline (Pnt:GFP) or the trachea (POU:GFP). These results indicate that testing binding sites for two different factors next to one another can disrupt the endogenous ordering and spacing of the sites within the enhancers. Significantly, the Toll:GFP and Pnt:GFP reporters, unlike the intact enhancers described in this study, drive GFP expression in both midline neurons and glia. This midline expression pattern suggests that the synthetic multimers may lack repressor-binding sites that restrict expression to midline glia. Taken together, these results demonstrate the sensitivity of CME function to flanking sequences within the midline enhancers (Fulkerson, 2010).
Existing experimental evidence suggests that unlike most transcription factors, Sim/Tgo heterodimers (as well as Trh/Tgo heterodimers) preferentially binds one sequence over all others: ACGTG, the CME. Within the enhancers described in this study, sites flanking the CME have remained unchanged over evolutionary time due, in part, to similarities between binding sites for Sim and Trh and the molecular consequences of changing nucleotides adjacent to the CME. This conservation may ensure transcription is restricted to the midline glia and repressed in tracheal cells. In addition to the midline enhancers reported in this study, regions conserved among Drosophila species were found within the known midline enhancers. For instance, a 1.0-kb enhancer present in the first intron of slit drives expression in the midline glia and it contains a single CME and a 32-bp sequence conserved in 11 Drosophila species. It is important to note that the number of midline enhancers described in this study is limited and not all the midline glial enhancers are likely to exhibit such a high degree of conservation. For instance, a midline enhancer of the ectoderm3 gene, was identified that exhibits much less conservation among Drosophila species and presently, the basis for the observed variation among enhancers is unknown (Fulkerson, 2010).
The Ets transcriptional activator, pnt, a downstream effector of EGFR signaling, and Drifter, a POU domain protein, are expressed in both embryonic midline glia and tracheal cells. Previous studies have shown that deleting a POU domain-binding site within an enhancer of rhomboid eliminated expression in tracheal cells, but did not affect its midline glial expression. The results described in this study confirm and extend these results and suggest that the location of a POU domain-binding site relative to the CME can play a role in determining if a gene is expressed in the midline glia, the trachea or both. Moreover, swapping the PAS domains between Sim and Trh proteins indicated that additional, midline or tracheal specific cofactors bind to the PAS domains of the individual proteins and likely to determine which genes are expressed in the two different cell types. This may be the reason sequences adjacent to the CME play such a critical and sensitive role in determining which tissues express the various reporter genes described here. To activate the midline genes, Sim may interact with Drifter and Pnt and bind to sequences flanked by different binding sites compared with sequences bound by Trh, Drifter, and Pnt needed to activate tracheal genes. The simplicity of the multimers studied in this paper raise the possibility that different PAS heterodimers may specifically interact with other factors, such as Drifter and Pnt, in a manner that depends on the relative location and/or distance between each binding site, as has been described for nuclear hormone receptor complexes (Fulkerson, 2010).
The results confirm those of Swanson (2010), who found binding sites can be juxtaposed in different ways within enhancers to favor particular short-range interactions, and, in this way, various combinations of transcription factor binding sites (inputs) can result in more than one output. Similarly, the motifs described in this study can be combined in different ways that result in either midline or tracheal expression. The results indicate the proximity of the CME to activators, one another and/or to repressors could contribute to the level of expression observed in the trachea and midline. This study focused on activator sites, but repressor sites are also likely present and restrict expression to certain cell types. Previous studies in Drosophila embryos have revealed the complexity of the transcriptional regulatory 'grammar' and have shown that the transcriptional output from various genes can be determined by the stoichiometry, affinity, spacing, arrangement, and distance between activator and repressor sites (Fulkerson, 2010).
The high degree of conservation within the midline enhancer subregions examined in this study here belies known properties of transcription factors and their recognition sequences, as well as observations made for many early developmental regulators of Drosophila development. Most transcription factors can vary considerably in the sequences they recognize and tend to bind to related sites with different affinities. This property would suggest that enhancers need not be strictly conserved to function, in contrast to what is reported here. The pattern of conserved sequences within these identified enhancers suggests that the transcription factors that bind these regions do so in a conserved order and spacing pattern. These results suggest that Sim and Trh may interact with other proteins to form an 'enhanceosome'-like complex, similar to that observed in the regulation of the interferon-β gene, in which activators and HMG proteins interact to form a specific multiprotein complex, with a defined structure. This model contrasts with the 'information display/billboard' model of enhancer function. In that model, enhancers are bound by a group of independent factors or group of factors that work together to promote or repress transcription in particular cell types. An important distinction between the two models is the arrangement of binding sites within an enhancer. Within an enhanceosome, the arrangement of binding sites relative to each other is constrained, whereas within a billboard enhancer, the relative arrangement of binding sites is rather flexible as long as a sufficient number of binding sites work together, in many possible configurations, to recruit factors for transcriptional activation (Fulkerson, 2010).
Results obtained with the midline glial genes examined in this study suggest that midline enhancers may consist of a nucleating enhanceosome-like region that combines with an 'information display/billboard' constellation of additional binding sites. This is supported by results obtained with the 70-bp conserved region of wrapper. When tested alone, it only marginally drives midline expression, whereas in the context of the 166-bp enhancer, it works quite well. Moreover, the 166-bp region of virilis cannot function on its own, but drives high levels of expression in the midline glia of melanogaster in the context of the larger, 476 bp region. That the 166-bp region from virilis cannot work efficiently in the midline suggests the transcription complex that binds to this region may be slightly different in virilis compared with melanogaster. For each enhancer described in this study, the presence of the conserved region is required to obtain expression in the midline glia (Fulkerson, 2010).
After comparing vertebrate genomes and generating reporter constructs with highly conserved noncoding sequences, Bailey (2006) noticed that many of these direct expression to regions of the CNS. It is possible that enhancers of CNS genes are more conserved compared with other gene sets, such as early developmental regulators of Drosophila that have been studied in detail. This may be due to the highly conserved nature of the transcription factors that regulate gene expression in this tissue, many of which have analogous functions in flies and mammals). Sox-binding sites are present throughout conserved regions of CNS genes and one of the similarities between these conserved CNS genes, the extensively characterized interferon-β enhanceosome and midline glial genes is the importance of HMG proteins. These proteins may bend the DNA, facilitating binding to highly structured, multiprotein complexes. The enhancers described here likely bind PAS and Sox proteins together with other conserved CNS regulators and it may be this combination of transcription factors that contributes to the similarly conserved arrangement of binding sites (Fulkerson, 2010).
Numerous combinations of transcription factor binding sites can be used to drive expression in many tissue types. Despite the conservation found in this study, binding sites for transcription factors do vary considerably, making it, at times, difficult to identify enhancers based on sequence conservation. In certain cases, changes within enhancers can generate diverse phenotypes between Drosophila populations. The continuing challenge is to understand both the forces constraining the enhancer sequences between Drosophila species, as well as how changes in these regions lead to significant modifications in the expression pattern of a gene, which over the long term, leads to variation among Drosophila populations and eventually, Drosophila species. For the midline genes described in this study, selection has stabilized the constellation of binding sites found within enhancers, resulting in their conservation among Drosophila species over approximately 40 million years of evolution (Fulkerson, 2010).
Primary branching in the Drosophila trachea is regulated by the Trachealess (Trh) and Tango (Tgo) basic helix-loop-helix-PAS (bHLH-PAS) heterodimers, the POU protein Drifter (Dfr)/Ventral Veinless (Vvl), and the Pointed (Pnt) ETS transcription factor. The jing gene encodes a zinc finger protein also required for tracheal development. Three Trh/Tgo DNA-binding sites, known as CNS midline elements, in 1.5 kb of jing 5'cis-regulatory sequence (jing1.5) previously suggested a downstream role for jing in the pathway. This study shows that jing is a direct downstream target of Trh/Tgo and that Vvl and Pnt are also involved in jing tracheal activation. In vivo lacZ enhancer detection assays were used to identify cis-regulatory elements mediating embryonic expression patterns of jing. A 2.8-kb jing enhancer (jing2.8) drove lacZ expression in all tracheal cell lineages, the CNS midline and Engrailed-positive segmental stripes, mimicking endogenous jing expression. A 1.3-kb element within jing2.8 drove expression that was restricted to Engrailed-positive CNS midline cells and segmental ectodermal stripes. Surprisingly, jing1.5-lacZ expression was restricted to tracheal fusion cells despite the presence of consensus DNA-binding sites for bHLH-PAS, ETS, and POU domain transcription factors. Given the absence of Trh/Tgo DNA-binding sites in the jing1.3 enhancer, these results are consistent with previous observations suggesting a combinatorial basis to Trh-/Tgo-mediated transcriptional regulation in the trachea (Morozova, 2010). In the developing Drosophila trachea, transcriptional regulation must be precisely coordinated with growth factor signaling to induce the appropriate cellular response. Studies of downstream transcriptional response elements in the transforming growth factor β (TGF-β) signaling pathway show the importance of discrete sequence changes differentiating an activation versus repressive response. Furthermore, such an activating enhancer element in the knirps gene in this pathway requires a cooperative effect with Trh and Tgo to possibly direct tissue specificity in the trachea. Tracheal gene expression is also controlled combinatorially by Trh/Tgo and Dfr/Vvl or either alone. Similarly, this study shows that Trh/Tgo response elements in the jing gene require additional elements to specify embryonic tracheal expression (Morozova, 2010). Jing is implicated in transcriptional regulation in numerous biological processes, but its exact role is not known. This study extend previous observations of a role for jing in the trachea by establishing it as a direct downstream target of Trh/Tgo heterodimers. By analyzing jing 5' cis-regulatory regions, this study shows combinatorial basis to Trh/Tgo-mediated jing activation. A 2.8-kb jing enhancer recapitulates endogenous jing expression in the embryonic trachea, ectodermal stripes, and CNS midline. jing2.8 includes a distal 1.5-kb of genomic DNA that has three CMEs which are known for their involvement in combinatorial transcriptional regulation. The best evidence that Trh/Tgo complexes are able to directly activate the jing1.5 enhancer was gathered from Drosophila S2 cells by Luciferase reporter and ChIP assays. The CMEs in jing1.5-luc were required for activation by Trh/Tgo suggesting a protein-DNA interaction. Furthermore, Trh/Tgo heterodimers associated with and activated the jing1.5 enhancer. However, the combination of DNA-binding sites for bHLH-PAS, POU, and ETS transcription factors in jing1.5 is not capable of driving tracheal β-Gal expression in a pattern similar to that of endogenous jing. The jing1.3 enhancer cannot drive tracheal expression. Evidence is shown, in vitro and in vivo, that trh, pnt, and dfr/vvl regulate jing mRNA and even jing1.5-lacZ fusion cell expression. Given these results, along with the absence of additional CMEs and consensus POU domain-binding sites in jing1.3, it is proposed that trh and dfr/vvl regulate jing tracheal expression in combination with additional elements in jing1.3 (Morozova, 2010). jing1.5 specifies a fusion cell component of jing expression that may instead be regulated by the bHLH-PAS transcription factors, Dys/Tgo. This is consistent with the presence of preferred and less preferred Dys/Tgo DNA-binding sites in the jing 1.5-lacZ enhancer. Prior to embryonic stage 12, trh is required for dys expression and then Dys and Archipelago downregulate trh specifically in fusion cells during stage 12. Therefore, Trh cannot activate jing1.5-lacZ in fusion cells from stage 12 which is consistent with the presence of fusion cell lacZ expression in embryos carrying CME deletions in jing1.5. The reductions in jing1.5-lacZ expression in the fusion cells of trh mutants may therefore result from subsequent reductions in dys expression (Morozova, 2010). This study also characterized jing cis-regulatory elements controlling different aspects of jing expression in CNS glia and Engrailed-expressing midline neurons and segmental ectodermal cells. The midline expression of jing enhancers provided an opportunity to compare jing transcriptional regulation in two tissues. The data show that jing1.5 is sufficient to drive expression in MG and neurons where Jing is normally expressed. The CNS midline identity of jing1.5-lacZ-expressing cells was shown in several ways. First, jing1.5-lacZ expression was absent in a homozygous sim mutant background. Second, the jing1.5-lacZ expression domain was expanded by activating the Spitz Egfr ligand thereby forcing midline glial survival. Lastly, MG characteristics, such as oblong shape and dorsal positions, are shown by some jing1.5-lacZ-expressing midline cells. Therefore, this enhancer is differentially activated in the CNS midline and trachea suggesting that there may be differences in the mechanism by which Sim/Tgo and Trh/Tgo heterodimers activate transcription. This is consistent with the differential abilities of Sim/Tgo and Trh/Tgo to associate with Dfr/Vvl in vitro and the inability of trh to induce ectopic CNS midline gene expression (Morozova, 2010). Strong CNS midline expression was also driven by the jing1.3 enhancer despite the absence of Sim/Tgo or Dfr/Vvl consensus DNA-binding sites. However, upon further characterization, the jing1.3-lacZ-expressing midline cells were found to express the segment polarity gene, engrailed (en). En-expressing CNS midline cells take up the posterior-most position within each VNC segment. Another En-positive midline cell lineage includes four to six MGP which are present at stage 13 but not at stage 17. The round shape of En-positive jing1.3-lacZ-expressing midline cells suggests that they belong to the MNB lineage and its progeny and do not belong to the MGP lineage. The mechanism of midline activation of jing1.3 is not known, but the ability of Jing to function as a repressor suggests that it may function combinatorially with En in segmental patterning. Further studies will be aimed at determining whether jing plays a role in segmental ectodermal patterning and its associated gene expression programs (Morozova, 2010). Enhancers are genomic cis-regulatory sequences that integrate spatiotemporal signals to control gene expression. Enhancer activity depends on the combination of bound transcription factors as well as - in some cases - the arrangement and spacing of binding sites for these factors. This study examined evolutionary changes to the sequence and structure of sparkling, a Notch/EGFR/Runx-regulated enhancer that activates the dPax2 gene in cone cells of the developing Drosophila eye. Despite functional and structural constraints on its sequence, sparkling has undergone major reorganization in its recent evolutionary history. The data suggest that the relative strengths of the various regulatory inputs into sparkling change rapidly over evolutionary time, such that reduced input from some factors is compensated by increased input from different regulators. These gains and losses are at least partly responsible for the changes in enhancer structure that were observe. Furthermore, stereotypical spatial relationships between certain binding sites ('grammar elements') can be identified in all sparkling orthologs - although the sites themselves are often recently derived. It was also found that low binding affinity for the Notch-regulated transcription factor Su(H), a conserved property of sparkling, is required to prevent ectopic responses to Notch in non-cone cells. It is concluded that rapid DNA sequence turnover does not imply either the absence of critical cis-regulatory information or the absence of structural rules. These findings demonstrate that even a severely constrained cis-regulatory sequence can be significantly rewired over a short evolutionary timescale (Swanson, 2011).
Because of spa's rapid structural evolution and binding-site
turnover, multispecies sequence alignments do not reveal many conserved features. Only the extreme 5' end of spa is unequivocally alignable across 12 Drosophila genomes. Given spa's complex regulatory circuitry and structure, its unusually rapid sequence divergence between D. mel and D. pse was surprising, especially because both orthologs of spa have identical cell-type specificities (Swanson, 2011).
This study demonstrated that even an enhancer that is
subject to structural constraints can be evolutionarily flexible;
therefore, an apparent lack of conserved cis-regulatory structure does not imply an absence of organizational rules within an enhancer (Swanson, 2011).
A model for the structural divergence of spa
between the melanogaster and obscura groups is proposed,
based on sequence analyses and experimental data. Although the remote control element (RCE) and its flanking Lz1-Ets1 pair are relatively stable, many other essential regulatory sites have been relocated. Within regions 4, 5, and 6a, putative novel regulatory motifs, essential for full-strength activation of both spa orthologs, have been identified whose movements are consistent with experimental data on spa's evolutionary restructuring (Swanson, 2011).
Important changes to the Lz/Ets/Su(H) inputs have also occurred: D. pse has fewer Su(H) and Lz sites, relative to the melanogaster group -- which can be compensated by newly acquired, functionally significant 5' Ets and epsilon (AGCCAG) sites. Meanwhile, the melanogaster group has gained a new Lz site and also has
a relative abundance of Su(H) sites, which may compensate for
relatively few epsilon and Ets sites (Swanson, 2011).
By tracking the reorganization of Su(H), Lz, Ets, and epsilon motifs
across multiple species, a speculative phylogeny of the spa enhancer within the genus Drosophila is proposed and the cis-regulatory content of the last common
ancestors (LCAs) of several species groups is predicted by reconstructing
the gain and loss of sites, and the changing strengths of transregulatory
inputs, in specific lineages. The main conclusions to be drawn from this evolutionary view of spa, informed by functional experiments, are: (1) significant enhancer rewiring has occurred since the divergence of the
mel and pse lineages; (2) this rewiring involves the loss and
gain of individual regulatory motifs, as well as compensatory changes in the overall strength of several trans-regulatory inputs through changes in binding-site number, position, and possibly affinity; (3) despite very rapid site turnover, characteristic configurations of sites ('grammar elements') can be identified;
(4) these grammar elements can be relocated within the
enhancer, suggesting that a specific arrangement of sites can
be more ancient than the individual sites that compose it.
These last two points, taken together, may explain how spa can continue to obey structural rules while being significantly reconfigured (Swanson, 2011).
A large proportion of the grammar elements that have been identified involve Lz/Runx and Ets motifs. Unlike the case of linked sites for Dorsal, Twist, and other factors in insect neurogenic enhancers, there is no single, clearly preferred
arrangement of Lz and Ets sites within spa: seven
distinct types of Lz/Ets grammar element were identified that are at least as
ancient as the LCA of the melanogaster group (Swanson, 2011).
Perhaps Runx and Ets factors, which are known to directly interact and to cooperatively activate transcription in flies and vertebrates, can synergize productively in several different spatial configurations. This is consistent with mapped Runx and Ets sites in vertebrate genomes, which are
frequently associated with one another in target enhancers,
but not with a single rigid arrangement or spacing (Swanson, 2011).
A nonstructural constraint on the sequence of spa was discovered: a requirement for nonconsensus, low-affinity Su(H) sites for proper cone-specific patterning. Because ectopic dPax2 expression in photoreceptor precursors causes
faulty cell fate specification and differentiation, resulting in
defective eye morphology, it is reasonable to suppose
that the expression pattern of spa[Su(H)-HiAff] would have
negative fitness consequences for the fly. Taken together
with previous work, the data presented in this study suggest that
spa requires input from Notch/Su(H) but also requires that
input to be attenuated at the cis-regulatory level, in order to
generate the proper levels and cell-type specificity of dPax2
expression in a tissue with widespread Notch signaling.
Like Notch/Su(H), EGFR/Ets signaling and Lz are also used
to specify multiple cell types in the retina, which presents
a challenge for combinatorial gene regulation: enhancers
must be able to make fine qualitative distinctions in regulatory
inputs and often must translate this information into relatively
sharp on/off decisions. These pressures could result in a cis-regulatory logic for genes like dPax2 in which many weak inputs are independently tuned (and spatially arranged) to maximize activation in the proper cell type, while minimizing ectopic activation. Previous studies of spa present a picture of an enhancer operating just above a functional threshold, such that the loss of a single regulatory site, or a loss of proper grammar, can result in transcriptional failure in cone cells. One of the main conclusions from this study is that, over a relatively short evolutionary timescale, a cis-regulatory module can find multiple solutions to this complex computational problem (Swanson, 2011).
The presence of weak, nonconsensus binding sites for
signal-regulated TFs is a common, but little remarked upon,
feature of developmental enhancers. Low-affinity TF
binding sites have well-documented functions in shaping a stripe of gene expression across a morphogen gradient and in determining temporal responses to developmental regulators. This study provides direct evidence supporting a role for weak signal response elements in preventing ectopic transcriptional responses to highly pleiotropic signaling pathways such as Notch (Swanson, 2011).
There is one striking question not addressed by this study:
why is this enhancer evolving at an unusually high rate, given
that its expression pattern is stable? Two plausible
explanations are given for which supporting data exist. First, dPax2
is on chromosome 4, the 'dot' chromosome of Drosophila,
which has a severely reduced recombination rate, resulting
in inefficient selection and relaxed sequence constraint.
No other cis-regulatory module on the fourth chromosome
has been subjected to an extensive evolutionary analysis,
nor are any as well-mapped as sparkling, but enhancers of
the fourth-chromosome genes eyeless and toy contain fairly
large blocks of sequence conservation, compared to spa. An alternative explanation for the rapid turnover observed
within spa involves the presence of nonconsensus, predicted
low-affinity sites for Su(H) and, in some cases, Lz and PntP2. For a typical TF, there are many more possible low-affinity binding sites than high-affinity sites: for example, the highest-affinity Su(H) consensus YGTGDGAAM
encompasses only 12 variants (TGTGGGAAA, etc.), whereas
the lower-affinity consensus of the same length nRTGDGWDn,
which accommodates all of the known Su(H) sites within spa,
contains 576 possible sequences. Accordingly, it is much
more likely that an enhancer will acquire a low-affinity binding
site via a single mutational event than a high-affinity site. Thus, an enhancer that does not require high-affinity binding sites for given trans-regulators may rapidly sample a variety of configurations of weak sites and may thereby undergo considerable sequence turnover without losing the input from that regulator. In other words, an enhancer such as spa, which must maintain a weak regulatory linkage with Notch/Su(H), may be less constrained than a high-affinity target with respect to the sequence, number, and position of its Su(H) binding sites. Whatever the reason for the rapid sequence divergence of spa, it provides an opportunity to examine in detail the evolutionary mechanisms by which a complex cis-regulatory module can be significantly reorganized, while still conforming to specific constraints of combinatorial logic and grammar (Swanson, 2011).
The insulin/IGF-activated AKT signaling pathway plays a crucial role in regulating tissue growth and metabolism in multicellular animals. Although core components of the pathway are well defined, less is known about mechanisms that adjust the sensitivity of the pathway to extracellular stimuli. In humans, disturbance in insulin sensitivity leads to impaired clearance of glucose from the blood stream, which is a hallmark of diabetes. This study presents the results of a genetic screen in Drosophila designed to identify regulators of insulin sensitivity in vivo. Components of the MAPK/ERK pathway were identified as modifiers of cellular insulin responsiveness. Insulin resistance was due to downregulation of insulin-like receptor gene expression following persistent MAPK/ERK inhibition. The MAPK/ERK pathway acts via the ETS-1 transcription factor Pointed. This mechanism permits physiological adjustment of insulin sensitivity and subsequent maintenance of circulating glucose at appropriate levels (Zhang, 2011).
The insulin signal transduction pathway is regulated by cross-talk from several other signaling pathways. This includes input from the amino-acid sensing TOR pathway into regulation of insulin pathway activity by way of S6 kinase regulating IRS. Signaling downstream of growth factor receptors has also been linked to regulation of insulin signaling. The active form of the small GTPase Ras can bind to the catalytic subunit of PI3K and promote its activity. Expression of a form of PI3K that cannot bind Ras allows insulin signaling, but at reduced levels. The work reported in this study provides evidence for a second mechanism through which growth factor receptor signaling through the MAPK/ERK pathway modulates insulin pathway activity. Transcriptional control of inr gene expression by EGFR signaling may provide a means to link developmental signaling to regulation of metabolism. In this context, a statistically significant correlation wass noted between EGFR target gene sprouty and inr gene expression at different stages during Drosophila development (Zhang, 2011).
Several steps of the insulin pathway can be regulated by phosphorylation. Given that the MAPK/ERK pathway is a kinase cascade, a priori, the possibility of phosphorylation-based interaction between these pathways would seem likely. However, this appears not to be the case. Acute pharmacological inhibition of the MAPK/ERK pathway proved to have no impact on insulin pathway activity. Thus short-term changes in MAPK/ERK pathway activity do not seem to be used for transient modulation of insulin pathway activity. Instead, the MAPK/ERK pathway acts through the ETS-1 type transcription factor Pointed to control expression of the inr gene. Transcriptional control of inr suggests a slower, less labile influence of the MAPK pathway. Taken together with the earlier studies, these findings suggest that growth factor signaling can regulate insulin sensitivity by both transient and long-lasting mechanisms (Zhang, 2011).
Why use both short-term and long-term mechanisms to modulate insulin responsiveness to growth factor signaling? The use of direct and indirect mechanisms that elicit a similar outcome is reminiscent of feed-forward network motifs. Although these motifs are often thought of in the context of transcriptional networks, the properties that they confer are also relevant in the context of more complex systems involving signal transduction pathways. In multicellular organisms, feed-forward motifs are often used to make cell fate decisions robust to environmental noise. The findings suggest a scenario in which a feed-forward motif is used in the context of metabolic control, linking growth factor signaling to insulin responsiveness. In this scenario, growth factor signaling acts directly via RAS to control PI3K activity and indirectly via transcription of the inr gene to elicit a common outcome: sensitization of the cell to insulin. This arrangement allows for a rapid onset of enhanced insulin sensitization, followed by a more stable long-lasting change in responsiveness. Thus a transient signal can both allow for an immediate as well as a sustained response. The transcriptional response also makes the system stable to transient decreases in steady-state growth factor activity. It is speculated that this combination of sensitivity and stability allows responsiveness while mitigating the effects of noise resulting from the intrinsically labile nature of RTK signaling. As illustrated by the data, failure of this regulation in the fat body leads to elevated circulating glucose levels, likely reflecting impaired clearance of dietary glucose from the circulation by the fat body. Maintaining circulating free glucose levels low is likely to be important due to the toxic effects of glucose. In contrast, circulating trehalose, glycogen or triglyceride levels showed no significant change in animals with reduced InR expression, suggesting that these aspects of energy metabolism can be maintained through compensatory mechanisms in conditions of moderately impaired insulin signaling (Zhang, 2011).
Earlier studies have shown that the transcription of the inr gene is under dynamic control. Activation of FOXO in the context of low insulin signaling leads to upregulation of inr transcription, thus constituting a feedback regulatory loop. Thus, InR expression appears to be under control of two receptor-activated cues, which have opposing activities: inr expression is positively regulated by the EGFR-MAPK/ERK module, but negatively regulated by its own activity on FOXO. In the setting of this study, the cross-regulatory input from the MAPK/ERK pathway was found to dominate over the autoregulatory FOXO-dependent mechanism. If conditions exist in which the FOXO-dependent mechanism was dominant, a limited potential for crossregulation by the MAPK/ERK pathway would be expected. Whether Pointed and FOXO display regulatory cooperativity at the inr promoter is an intriguing question for future study (Zhang, 2011).
The acquisition of flight contributed to the success of insects and winged forms are present in most orders. Key to understanding the origin of wings will be knowledge of the earliest postembryonic events promoting wing outgrowth. The Drosophila melanogaster wing is intensely studied as a model appendage, and yet little is known about the beginning of wing outgrowth. Vein (Vn) is a neuregulin-like ligand for the EGF receptor (Egfr), which is necessary for global development of the early Drosophila wing disc. vn is not expressed in the embryonic wing primordium and thus has to be induced de novo in the nascent larval wing disc. Decapentaplegic (Dpp), a Bone Morphogenetic Protein (BMP) family member, provides the instructive signal for initiating vn expression. The signaling involves paracrine communication between two epithelia in the early disc. Once initiated, vn expression is amplified and maintained by autocrine signaling mediated by the E-twenty six (ETS)-factor PointedP2 (PntP2). This interplay of paracrine and autocrine signaling underlies the spatial and temporal pattern of induction of Vn/Egfr target genes and explains both body wall development and wing outgrowth. It is possible this gene regulatory network governing expression of an EGF ligand is conserved and reflects a common origin of insect wings (Paul, 2012).
Deciphering gene regulatory networks (GRNs) is critical for understanding
the causation of development, and a large
network that explains endoderm development in the sea urchin
has been elaborated . The Drosophila early wing disc provides
a tractable system for GRN analysis because it is a relatively
small field of cells and many genes involved in the process
are known. This study has developed knowledge of the GRN involving Egfr signaling activated by its ligand Vn. It is a key circuit because Vn/Egfr
signaling induces the ap and iro-C genes, which are required for
development of the major territories of wing and body wall beginning
in the first instar. The spatial and temporal
control of vn expression involves two major inputs: initiation by
paracrine Dpp signaling and maintenance by a positive Vn/Egfr
autocrine feedback loop. These are direct, positive, early acting
inputs into the vn promoter. The Dpp signal emanates from the
peripodial epithelium and is unidirectional, activating vn only in
the disc proper. This directionality is important because ectopic
activation of Egfr in the peripodial epithelium antagonizes its
development and transforms the cells into columnar cells characteristic
of the disc proper, which develop into body wall structures. It is not known why Dpp signaling is apparently only active in cells across the lumen from the expression domain in the first instar. It is possible some factor required for Dpp signaling is
differentially expressed or Dpp signaling is mediated by cellular
processes as has been proposed for the early eye disc.
There are also negative inputs that operate slightly later in
development and function to limit the spatial extent of vn expression. Productive Vn/Egfr signaling is confined
to the future body wall because pntP2 expression focuses proximally
as development proceeds. There is also an early acting
negative input from Wg signaling that has been defined genetically. wg expression begins in distal cells in the first
instar wing disc, which is consistent with a role in repressing vn
expression in cells that will become the future wing (Paul, 2012).
The direct feedback loop involving vn
expression is an example of a regulatory circuit governing a
'community effect,' a term coined by Gurdon to describe the
change in transcription that occurs when cells are isolated from
their neighbors. This phenomenon suggested that
proximity to other cells serves as a mechanism to sustain expression
of particular genes important for a collective cell fate.
The term has been used to describe a developmental event
that is the result of a ligand inducing its own expression in a
neighboring cell. In this way, the signal is propagated
through a field of cells, which, as a 'community,' then express
a similar repertoire of target genes. Examples of this type of
subcircuit operating in pattern formation have been described
that involve TGF-β, FGF, and Wnt ligands.
This study reports a case in which an EGF ligand is implicated.
Adding an additional ligand class to the list supports the idea
that the strategy is widespread in development.
In a normal developmental context, a secreted EGF ligand, such
as Vn, can be produced as part of a positive feedback loop provided
there are tight controls that limit the operation of the loop both
spatially and temporally. Clearly, if there were no limits, a runaway
situation would result and too many cells would become part of the
'community,' or the state of activity would be perpetuated beyond
a certain developmental window. Indeed this seems to be the case in
some disease contexts involving EGF ligands, including neuregulin
(the vertebrate equivalent to Vn), where autocrine loops sustain the
continued growth of cancer cells (Paul, 2012).
It is widely thought that flight
contributed significantly to the success of insects and consequently
the evolution of the wing is of great interest. There are
two major theories; the first holds that wings derived from
a proximal branch of the leg and the second that wings derived
from a paranotal lobe extending from the dorsal body wall. In a combination of these ideas, it has been
proposed that the wing may have a leg origin but that the ability
to form a flat wing-like structure depends upon proximity to the
dorsal-lateral boundary in the side body (the paranotal lobe in
wingless forms). Dorsal appendages such as the tracheal gill
or stylus are like the Drosophila wing in that they express wg and vg
and may represent evolutionary precursors to the wing. Unlike
the wing, however, they do not form close to a region where
ap is also expressed, which may be essential for an outgrowth to
form a flat structure like the wing. Vn/Egfr signaling is upstream
of ap and therefore a prerequisite for wing formation.
This study trace the circuit regulating ap expression back earlier to
the induction of vn in a transient stripe triggered by Dpp signaling.
This initiates broad Vn/Egfr/PntP2 signaling that extends distally
and ap is induced throughout the dorsal compartment, where,
together with wg and vg, it acts to produce the wing. Continued
Egfr activation, however, blocks wing development, and hence the
domain of active signaling is shortly thereafter restricted to
proximal cells by the absence of PntP2 in distal cells. In proximal
cells where PntP2 persists, a feedback loop is established and body
wall development is promoted. The changing spatial domain of
vn (from a stripe to a proximal wedge) establishes a prepattern
that is permissive for both wing and body wall development. It will
be interesting to determine if Egfr signaling underlies body wall
formation in other species and if a similar transient spatial extension
of this activity correlates with winged morphs (Paul, 2012).
Although it is widely appreciated that a typical developmental control gene is regulated by multiple enhancers, coordination of enhancer activities remains poorly understood. This study proposes a mechanism for such coordination in Drosophila oogenesis>, when the expression of the transcription factor Broad (BR) evolves from a uniform to a two-domain pattern that prefigures the formation of two respiratory eggshell appendages. This change reflects sequential activities of two enhancers of the br gene, early and late, both of which are controlled by the epidermal growth factor receptor (EGFR) pathway. The late enhancer controls br in the appendage-producing cells, but the function of the early enhancer remained unclear. This study found that the early enhancer is essential for the activity of the late enhancer and induction of eggshell appendages. This requirement can be explained by a mechanism whereby the BR protein produced by the early enhancer protects the late enhancer from EGFR-dependent repression. This complex mechanism is illustrated using a computational model that correctly predicts the wild-type dynamics of BR expression and its response to genetic perturbations (Cheung, 2013).
Temporal control of transcription can be provided by changes in
the levels of inductive signals, by cross-regulatory interactions
between genes, and by dynamic use of different enhancers. For
example, the dynamic expression of rho in the early Drosophila
embryo results from sequential activities of two different rho
enhancers, responding to two different inductive cues. In
another control strategy, the early enhancer initiates expression,
and the late enhancer maintains it through positive autoregulation.
This mechanism controls Krox20 during the hindbrain
segmentation in vertebrates. Both of these scenarios are
different from the mechanism that coordinates br enhancers in
Drosophila oogenesis. First, both the early and late enhancers
respond to the same inductive signal. Second, the early enhancer
is needed not to the initiate the expression of the late enhancer,
but to protect it from ectopic and premature repression (Cheung, 2013).
In the wild-type egg chamber, brL, the late enhancer, is repressed only in cells
exposed to the maximal levels of GRK. In the absence of brE, the early enhancer,
signaling levels sufficient for repression are realized in the appendage
primordia, due to amplification of EGFR activation
resulting from ectopic expression of rho. This model is supported
by eggshell defects induced by the RNAi-based disruption of BR
expression by brE, and by ectopic expression of rho mRNA and
PNT-dependent loss of brL activity in the absence of BR. The requirement for the rho-dependent amplification
of EGFR signaling was tested computationally, by analyzing a simplified
model in which BR and PNT repress each other directly, without
feedback by rho. Extensive exploration of the
parameter space in this model could not identify a set of parameters that would be consistent with both the wild-type
expression of brL and its response to genetic perturbations.
Based on this, it is argued that amplification of EGFR signaling by
rho is essential for explaining the results (Cheung, 2013).
Going beyond br and rho, it is noted that dozens of genes regulated
by GRK are expressed in dynamic patterns. Some of
these patterns may be explained using the proposed computational
model based on the interplay of multiple enhancers and
dynamic signals. Although these models are more complex than
existing models of developmental patterning, their analysis is
essential for understanding temporal control of gene expression
in development (Cheung, 2013).
Intermediate neural progenitor cells (INPs) need to avoid differentiation and cell cycle exit while maintaining restricted developmental potential, but mechanisms preventing differentiation and cell cycle exit of INPs are not well understood. This study reports that the Drosophila homolog of mammalian Sp8 transcription factor Buttonhead (Btd) prevents premature differentiation and cell cycle exit of INPs in Drosophila larval type II neuroblast (NB) lineages. Loss of Btd leads to elimination of mature INPs due to premature differentiation of INPs into terminally dividing ganglion mother cells. Evidence is provided to demonstrate that Btd prevents the premature differentiation by suppressing the expression of the homeodomain protein Prospero in immature INPs. It was further shown that Btd functions cooperatively with the Ets transcription factor Pointed P1 to promote the generation of INPs. Thus, this work reveals a critical mechanism that prevents premature differentiation and cell cycle exit of Drosophila INPs (Xie, 2014).
This study shows that the Sp family transcription factor Btd is required to prevent the premature differentiation of INPs by suppressing the expression of Pros in immature INPs. Furthermore, evidence is provided to demonstrate that the combination of Btd and PntP1 is sufficient to specify type II NB lineages and promote the generation of INPs. Thus, this work reveals a critical mechanism that regulates INP generation (Xie, 2014).
The most striking phenotype resulting from the loss of Btd is the elimination of mature NPs. In addition, about 40% of btbmutant type II NB lineages ectopically express Ase in the NB and become type I-like NB lineages. However, although forced expression of Ase in type II NBs is sufficient to eliminate INPs in type II NB lineages, the loss of INPs is obviously not primarily due to the ectopic Ase expression or the transformation of type II NB lineages into type I-like NB lineage in that the loss of mature INPs occurs independently of the ectopic Ase expression in most btb mutant or Btd RNAi knockdown type II NB lineages. Instead, this study demonstrates that the loss of mature INPs in the absence of Btd is due to the premature differentiation of Ase+ immature INPs into GMCs. In Btd RNAi knockdown or btb mutant type II NB lineages without the ectopic Ase expression, Ase- immature INPs differentiate into Ase+ immature INPs normally as indicated by the expression of R9D11-mCD8-GFP, Mira, as well as PntP1 in Ase+ daughter cells next to the Ase- immature INPs. However, instead of differentiating into mature INPs, it is argued that Ase+ immature INPs prematurely differentiate into GMCs based on the following two pieces of evidence. First, Ase+ daughter cells eventually undergo terminal divisions as indicated by the positive pH3 staining and the position of the pH3-positive cells. Second, unlike mature INPs, the dividing Ase+ daughter cells do not form basal Mira crescent at metaphase. The terminal division and the lack of Mira crescent during the division are two unique features that distinguish GMCs from INPs in addition to the expression of nuclear Pros. Therefore, the elimination of mature INPs resulting from the loss of Btd is due to the premature differentiation of Ase+ immature INPs into GMCs (Xie, 2014).
Why does the loss of Btd lead to premature differentiation of INPs? The results show that the loss of Btd results in a reduction or loss of PntP1 in type II NBs and immature INPs as well as ectopic expression of Pros in early immature INPs. Previous studies show that PntP1 suppresses Ase in type II NBs and that inhibiting PntP1 activity leads to ectopic expression of Ase in type II NBs and elimination of INPs. Given that the ectopic Ase expression in btb mutant type II NBs is closely associated with the severe reduction or complete loss of PntP1 and that expression of UAS-pntP1 largely suppresses the ectopic Ase expression in btb mutant type II NBs, the severe reduction or loss of PntP1 most likely accounts for the ectopic Ase expression in btb mutant type II NBs. However, although the loss of PntP1 could lead to the loss of INPs, several lines of evidence are provided to demonstrate that the elimination of INPs in btb mutant or Btd RNAi knockdown type II NB lineages is primarily due to the ectopic activation of Pros in immature INPs rather than the reduction or loss of PntP1. First, ectopic nuclear Pros is consistently expressed in Ase- immature INPs when mature INPs are eliminated. Second, the loss of mature INPs can be fully rescued by Pros RNAi knockdown or even just by removing one wild type copy of pros. Third, Pros RNAi knockdown also rescues the reduction of PntP1 and suppresses the ectopic Ase expression in btb mutant type II NBs. In contrast, the expression of UAS-pntP1 fails to rescue mature INPs in most btb mutant type II NB lineages although it largely suppresses the ectopic Ase expression in the NBs. Furthermore, the complete elimination of mature INPs is also observed occasionally in btb mutant type II NB lineages without the reduction of PntP1. Therefore, the elimination of mature INPs resulting from the loss of Btd is primarily due to the ectopic Pros expression, which likely promotes premature differentiation of INPs into GMCs and cell cycle exit. The severe reduction or loss of PntP1 is responsible for the ectopic Ase expression in btb mutant type II NBs and is more likely a secondary effect due to the ectopic Pros expression and/or the loss of INPs. INPs and/or other progeny may provide feedback signals to the NBs as has been demonstrated in other systems (Xie, 2014).
The ectopic expression of Pros in Ase- immature INPs resulting from the loss of Btd suggests that Btd is critical for suppressing Pros expression in Ase- immature INPs. Btd was known as a head gap gene. It has been suggested that gap factors act largely as transcriptional repressors. Btd could directly suppress Pros by binding to the pros promoter as a transcriptional repressor. Alternatively, Btd could suppress Pros indirectly by regulating the expression or antagonizing the activity of factor(s) that activate(s) pros expression. The results show that ectopic/overly expression of Btd in type I NB lineages or mature INPs does not lead to overproliferation of type I NBs as observed in pros mutant type I NB lineages. Instead, ectopic expression of Btd promotes the generation of INP-like cells from type I NBs and transforms some type I NB lineages into type II-like NB lineages. Therefore, it is more likely that Btd suppresses Pros indirectly by regulating the expression or antagonizing the activity of pros activator(s). Previous studies have suggested that Ase, Daughterless, Numb, and Erm could activate pros expression. Since Ase and R9D11-Cd4-tdTomato, which is under the control of erm promoter, are not expressed in Ase- immature INPs in the absence of Btd, it is unlikely they are involved in the activation of pros in immature INPs. It would be interesting to investigate in the future if Numb or Daughterless could activate pros in immature INPs in the absence of Btd (Xie, 2014).
This study has provided several lines of evidence to demonstrate that Btd and PntP1function cooperatively to specify type II NB lineages and promote the generation of INPs. Results from this study as well as a previous study show that ectopic expression of UAS-pntP1 or UAS-btb alone can only promote the generation of INP-like cells in a subset of type I NB lineage, whereas ectopic expression of UAS- pntP1 in Btd-positive type I NB lineages or coexpression of UAS-btb and UAS-pntP1 can promote the generation of INP-like cells in nearly all type I NB lineages and transforms all these lineages into type II-like NB lineages. Consistently, the ability of PntP1 to promote the generation of INP-like cells in btb mutant type I NB lineages is largely impaired. These results suggest that the specification of type II NB lineages and the generation of INPs requires both PntP1 and Btd and that the combinatorial PntP1 and Btd is sufficient to promote the generation of INPs (Xie, 2014).
It is proposed that PntP1 and Btd function cooperatively but through different mechanisms to promote INP generation. PntP1 is responsible for the suppression of Ase in type II NBs. Meanwhile, PntP1 must be regulating the expression of other unknown target gene(s) that are/is essential for the generation of INPs, such as specification of immature INPs, because loss of Ase is not sufficient to promote the generation of INP-like cells in any type I NB lineages. Btd likely acts after PntP1 to mainly prevent premature differentiation of INPs into GMCs by indirectly suppressing pros in immature INPs. The role of Btd in suppressing Ase in type II NBs is minimal if there is any because unlike PntP1, which suppresses ase in nearly all type I NBs when it is ectopically expressed, overexpression of Btd only suppresses Ase in a small subset of type I NBs that produce INP-like cells in larval brains. Furthermore, Ase is expressed in Btd+ type I NBs, indicating Btd does not suppress Ase in type I NBs when it is expressed at normal levels. Studies in mammals as well as in Drosophila suggest that the Btd/Sp8 could functions downstream of Wnt signaling to regulate the expression of Fgf8 as well as Distal-less (Dll) and Headcase (Hdc) during the forebrain patterning as well as limb development. However, inhibiting Wnt signaling alone in type II NB lineages does not have any obvious phenotypes, indicating that Btd unlikely functions downstream of Wnt signaling in type II NB lineages (Xie, 2014).
Whether Fgf8, Dll, or Hdc could function downstream of Btd to regulate INP generation remains to be investigated in the future. In mammals, the Btd homolog Sp8 palys important roles in brain development. In the developing mouse forebrain, Sp8 is expressed in cortical progenitors in a mediolateral gradient across the ventricular zone as well as in the lateral ganglionic eminence (LGE) and medial ganglionic eminence (MGE). In developing human brains, Sp8 is abundantly expressed in the ventricular zone and the outer subventricular zone where RGs and oRGs reside. In addition to its roles in interneuron development and the patterning of developing mammalian brains and spinal cords, it was also shown that loss of Sp8 led to the reduction of the progenitor pool. The current results show that mammalian Sp8 can rescue the loss of mature INPs resulting from the loss of Btd in Drosophila, suggesting that Btd/Sp8 could have conserved functions across different species. It would be interesting to investigate if Sp8 has similar roles in promoting the generation of transient amplifying INPs, such as oRGs, in developing mammalian brains (Xie, 2014).
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