sparkling
In spa(pol) mutants, the deletion of an enhancer abolishes Spa expression in cone and primary pigment cells and results in the severely disturbed development of non-neuronal ommatidial cells. Because Spa is not expressed in R7 cells, its expression in newly recruited cone cells distinguishes their fate from that of R7 cells. Lozenge may be the transcription factor whose synthesis would have to precede that of Spa, which is required for the specification of the R7 equivalence group, including R1/R6, R7 and the cone cells. Lozenge helps define the R7 equivalence group by repressing seven-up (Fu, 1997).
Dominant mutations provide invaluable tools for Drosophila geneticists. The
dominant eye mutation Glazed (Gla), described by T. H. Morgan more than 50 years ago, has now been analyzed. Gla causes the loss of photoreceptor cells during pupal stages, in a process reminiscent of
apoptosis, with a concomitant overproduction of eye pigment. Ommatidial bristles are missing in the anterior-ventral part where the Gla mutant phenotype is generally more pronounced. Most of the eye appears to consist of pigment cells since pigment granules are highly abundant over the entire surface. Pigment cell shape is predominantly rectangular, suggesting that most of the pigment cells have adopted a tertiary rather than a secondary pigment cell fate. It is only between 30 and 40 h of pupal development that mutant and wild-type discs differ. In pupal discs older than 40 h, no more Elav-positive photoreceptor cells are found in mutant clones. This phenotype is very similar to that
caused by the loss of D-APC, a negative regulator of Wingless (Wg) signal transduction. However, genetic
analyses reveal that the Gla gain-of-function phenotype can be reverted to wild-type. By
generating a P-element-induced revertant of Gla, it has been demonstrated that Gla is allelic to wg. The
molecular lesion in Gla indicates that the insertion of a roo retrotransposon leads to ectopic expression
of wg during pupal stages. The Gla phenotype is similar to that caused by ectopic
expression of Wg driven by the sevenless (sev) enhancer. In both cases Wg exerts its effect, at least
in part, by negatively regulating the expression of the Pax2 homolog sparkling (spa). Ectopic expression of wg in sev-wg discs occurs early enough to block the formation of interommatidial bristles by reducing spa expression. In Gla mutants, however, ectopic wg may be expressed too late to interfere with spa expression in the bristle precursor cells, and the sensory organ precursors of interommatidial bristles are formed normally. Ectopic Wg might inhibit a process that normally protects the developing photoreceptor cells from undergoing programmed cell death. Gla represents not
only the first dominant allele of wg, but it may also be the first allele ever described for wg (Brunner, 1999).
Runx proteins have been implicated in acute myeloid leukemia, cleidocranial dysplasia, and stomach cancer. These proteins control key developmental processes in which they function as both transcriptional activators and repressors. How these opposing regulatory modes can be accomplished in the in vivo context of a cell has not been clear. The developing cone cell in the Drosophila visual system was used to elucidate the mechanism of positive and negative regulation by the Runx protein Lozenge (Lz). A regulatory circuit is described in which Lz causes transcriptional activation of the homeodomain protein Cut, which can then stabilize a Lz repressor complex in the same cell. Whether a gene is activated or repressed is determined by whether the Lz activator or the repressor complex binds to its upstream sequence. This study provides a mechanistic basis for the dual function of Runx proteins that is likely to be conserved in mammalian systems (Canon, 2003).
Interestingly, D-Pax2, which is directly activated by Lz, is
needed to activate cut in cone cells.
Therefore, although indirectly, Lz positively regulates cut.
This presents an interesting developmental circuit in which Lz, acting
as a transcriptional activator, causes expression of a cofactor that then binds with Lz to convert it into a direct repressor of
transcription. Both the presence of the cofactor and binding
sites for this cofactor in the controlling regions of an Lz target gene are required for Lz-mediated repression (Canon, 2003).
This model was then tested in R7 cells where both Dpn and Lz are
coexpressed. Here, Lz does not repress dpn, presumably because Cut is absent from R7. Consistent with this notion, mis-expression of
Cut in R7 cells using lz-Gal4 causes repression of
dpn in these cells. This is not a secondary result
of a change in cell fate because the expression of the R7 cell-specific
marker Prospero remains unchanged in this genetic background (Canon, 2003).
These results add another level of complexity to recent studies
demonstrating a combinatorial code whereby a relatively small number of
signaling pathways and activated transcription factors work together to
generate unique cell fates. In cone cells, the
Notch and EGFR pathways are required along with Lz to activate
D-Pax2, and therefore cut. In contrast, the combination of these few inputs is not right for activation of cut in the R7 neurons, and therefore dpn is not
repressed. The circuit described here demonstrates a higher order of
sophistication necessary for a cell to choose between a neuronal and
nonneuronal fate using a very limited number of inputs. Using a
self-regulated circuit and just two signaling pathways, a single Runx
protein is capable of causing opposing effects on different enhancers in the same cell, resulting in a unique fate (Canon, 2003).
The induction of cone cells in the Drosophila larval eye disc by the determined R1/R6 photoreceptor precursor cells requires integration of the Delta-Notch and EGF receptor signaling pathways with the activity of the Lozenge transcription factor. This study demonstrates that the zinc-finger transcription factor Hindsight (Hnt) is required for normal cone-cell induction. R-cells in which hindsight levels are knocked down using RNAi show normal subtype specification, but these cells have lower levels of the Notch ligand Delta. HNT functions in the determined R1/R6 precursor cells to allow Delta transcription to reach high enough levels at the right time to induce the cone-cell determinants Prospero and D-Pax2 in neighboring cells. The Delta signal emanating from the R1/R6 precursor cells is also required to specify the R7 precursor cell by repressing seven-up. As hindsight mutants have normal R7 cell-fate determination, it is inferred that there is a lower threshold of Delta required for R7 specification than for cone-cell induction (Pickup, 2009).
This study shows that Hnt function is necessary to elevate the Dl ligand
in the R1/R6 precursor cells to a level high enough to achieve cone-cell
induction. Notably, Hnt is not an on/off switch for Dl expression;
rather it potentiates the level of Dl transcription in the R1/R6
precursor cells. The data suggest that this modulation is likely to be
independent of Chn, which is itself a transcriptional repressor of Dl. Although this paper does not show that this Hnt effect is due to direct action, the exact sequence for two Hnt binding sites was found in the upstream and
intronic sequences of the Delta transcription unit (Pickup, 2009).
Earlier reports describing Hnt function in the ovary show that Hnt
expression is regulated by the Notch signaling pathway and controls follicle
cell proliferation and differentiation. This
paper reports that Hnt acts upstream of Notch activation by regulating Dl
ligand expression levels. These two modes of regulation are not necessarily
mutually exclusive, but it is not thought that Notch activates the hnt
gene in the eye. (1) Hnt is expressed in all the R-cell precursors in the
eye, whereas the Notch pathway is activated at high levels only in a subset of
these precursors, as well as in the accessory cone and pigment cell
precursors, where Hnt is not expressed at all. (2) When Notch activity is attenuated by using the Nts mutant, Hnt expression in the furrow expands to all cells that now acquire a neuronal fate. This result cannot be interpreted as a simple repression of
Hnt expression by Notch activation in non-neuronal cells, as Hnt expression is
not complementary to Notch activation in the eye disc. (3) Notch activation cannot be sufficient to induce Hnt expression in the eye disc, since no expansion of Hnt expression into adjacent, non-determined cells is seen when Dl is ectopically expressed early in the cone-cell precursors (with the lz-Gal4 driver). (4) It was shown that the expression of Dl in the R-cell precursors is partly dependent on Hnt function. Others have clearly demonstrated that this late Dl expression does not require Notch activity, since it is unaffected in a Nts1 mutant (Pickup, 2009).
The two-signal model of R7 fate hypothesizes that R7 determination requires
a strong RTK signal (achieved by the additive effects of Sevenless and EGFR
activation) together with Notch activation. These signals are necessary to activate pros and repress svp expression, respectively. Since the cone-cell precursor cells do not contact the determined R8 cell at the appropriate time, they will not 'see' the SEV ligand BOSS. Cone cell precursors, then, will not ordinarily activate their Sev receptors. In this model, different fates have been reinforced in the R7/cone equivalence group by adding a second, activating ligand for EGFR (Pickup, 2009).
This paper suggests a further level of complexity. It was shown, by
manipulating the level of Dl in the R1/R6 signaling cells, that
activation of the key players in cone-cell determination requires high levels
of the Notch activation in the cone-cell precursor cell. Several lines of
evidence support the idea that the level of the Dl ligand is translated into
cell-fate differences in a responding R precursor cell. Since there is low Dl
expression in the R7 precursor cell and only late expression of Dl in the
cone-cell precursor cell, the adjacent R1/R6 precursor cells never activate their
Notch receptors. Both the R7 precursor and the cone-cell precursor cells
receive their ligand signal from the R1/R6 precursor cells. In this hypothesis, the R7 precursor cell requires only a low level of ligand signal to activate the R7-like program: turning on pros and off svp (Pickup, 2009).
It is suggested that the cone-cell precursor requires a high level of ligand
signal to activate the cone-cell program. Expressing a dominant-negative form
of Dl in the R1/R6 signaling cells prevents cone-cell, but not R7-cell,
determination. Since both the cone and R7 precursor cells receive their Dl input from the same R1/R6 cells, it is possible that an intrinsic feature of the R7 precursor cell - possibly the high RTK activation - antagonizes N signaling, so that D-Pax2 transcription does not occur in that cell. The transcriptional repressor, Lola, may also be involved in this distinction, since it is known to bias precursor cells towards R7-over cone-cell fate (Pickup, 2009).
Although a role for Notch signaling in cone-cell induction has been shown
to be necessary for D-Pax2 expression, it has
not been directly demonstrated as necessary for pros regulation in
cone cells. The experiments presented in this study suggest that high levels of Notch signaling may indirectly or directly be required for Pros expression in the cone-precursor cells. This requirement is independent of the role of SU(H) in inducing D-Pax2, since there are normal levels of Pros in the cone-cell
precursors of a D-Pax2 null mutant.
Ectopically activating the Notch pathway in the R1/R6 precursor cells
occasionally induces ectopic Pros (but eliminates ELAV) in these cells.
Although this effect on Pros expression may be a secondary result of a
cell-fate transformation, it could also be interpreted as a more direct effect
of Notch signaling on pros transcription. In a different context,
Pros expression has been shown to be affected by Dl-activated Notch signaling
in a subset of glial cells in the embryonic CNS (Pickup, 2009).
Why would there be two Dl thresholds for different cell fates? There is
some preliminary work that suggests different mechanisms for Notch-activated
transcriptional readout in the responding cell, depending on the level of
signal received. In the cone-cell equivalence group, the cone-cell
determination pathway requires that D-PAX2 and Pros be expressed. It is
hypothesized that D-Pax2 may require a higher level of Notch
activation than Pros, which is also required for R7 determination.
These experiments indicate that there may be coordinated regulation of both
D-Pax2 and Pros expression in the cone cells. It is
postulated that the mechanism of Pros-gene induction in the cone cells
is different from pros regulation in R7. By potentiating the level of
Dl gene expression in the R1/R6 signaling cells, it is possible to
overlay the cone-cell fate over the transcriptional module necessary for
R7-cell fate. This simple change has, thus, allowed for the elaboration of
very different cell fates from the same equivalence group (Pickup, 2009).
Sparkling expression is required for activation of cut in cone cells and of the Bar locus in primary pigment cells. Cut expression is strongly reduced in cone cell of spa(pol) mutants, as compared to wild type. Interestingly, Cut expression recovers; by 45 hours after pupariation it has risen to levels even above those of wild-type. The lack of Spa protein in cone cells appears to delay the development of the cells, since the shape of their nuclei and the nuclear accumulation of Cut resemble those of earlier stages in wild-type pupal discs. This delay may be caused by a late larval and early pupal requirement of Spa for cut activation, which later becomes independent of Spa. Expression of cut in bristle cells, many of which are mispositioned, appears unaffected during these stages. Expression of both Bar proteins in primary pigment cells is abolished completely in spa(pol) mutants. However, it remains unaffected in the irregularly positioned bristle cells, which continue to express Spa protein. Thus Spa exerts at least part of its control of primary pigment cell development through its regulation of Bar expression. Bar is also expressed in R1 and R6 precuror cells, where Lozenge rather than Spa is one of its activators. It is suggested that close functional analogies exist between Spa and Pax2 in the development of the insect and vertebrate eye. In the absence in Pax2, the optic stalk epithelium develops into pigmented retina and fails to proliferate and differentiate into glial cells, which populate the optic nerve and are essential for the guidance of the retinal axons. Thus the cone cell in Drosophila might be considered as a kind of neuronal support, or glial -- a cell that may have evolved from a more primitive ancestral glial cell. In favor of such a hypothesis, it is observed that spa is expressed in glial cells in the developing PNS (Fu, 1997).
Pax5 (BSAP) functions as both a transcriptional activator and repressor during midbrain patterning, B-cell development and
lymphomagenesis. Pax5 exerts its repression function by recruiting members of the Groucho corepressor
family. In a yeast two-hybrid screen, the groucho-related gene product Grg4 was identified as a Pax5 partner protein. Both proteins
interact cooperatively via two separate domains: the N-terminal Q and central SP regions of Grg4, and the octapeptide motif and
C-terminal transactivation domain of Pax5. The phosphorylation state of Grg4 is altered in vivo upon Pax5 binding. Moreover, Grg4
efficiently represses the transcriptional activity of Pax5 in an octapeptide-dependent manner. Similar protein interactions resulting in
transcriptional repression were also observed between distantly related members of both the Pax2/5/8 and Groucho protein families. In
agreement with this evolutionary conservation, the octapeptide motif of Pax proteins functions as a Groucho-dependent repression domain in Drosophila embryos.
These data indicate that Pax proteins can be converted from transcriptional activators to repressors through interaction with corepressors of the Groucho protein
family (Eberhard, 2000).
Three groucho-related genes coding for full-length Grg proteins (Grg1, 3a and 4) have been identified to date in the mouse genome. Using transient transfection assays, all three murine Grg proteins have been shown to be phosphorylated in a Pax5-dependent manner and can repress
the transcriptional activity of Pax5 efficiently. Even the distantly related Groucho protein of Drosophila is able to interact with Pax5 and to down-modulate the activity of this transcription factor in heterologous mammalian cells.
Furthermore, GST pull-down assays have demonstrated that the mouse Pax8 and Drosophila Pax2/5/8 proteins can bind full-length Grg4 with an affinity similar to that of
human Pax5. Moreover, the transcriptional activity of the mouse Pax8, zebrafish Pax2.1 and Drosophila Pax2/5/8 proteins could be repressed efficiently
by Grg4 in transfected plasmacytoma cells. These different Pax proteins are also able to promote additional phosphorylation of Grg4 in
transfected COP-8 fibroblasts. Collectively, these data demonstrate, therefore, that the interaction between distantly related members of the Pax2/5/8
and Groucho protein families has been conserved in evolution (Eberhard, 2000).
Inspired by the high evolutionary conservation of the Groucho-Pax2/5/8 protein interaction, an investigation was carried out to see whether the octapeptide motif can function in vivo as a repression domain during Drosophila development. Based on the transcriptional regulation of the Sex lethal (Sxl) gene in Drosophila embryos, a repression assay was employed. Sxl is a key regulator of sex determination and dosage compensation: Sxl transcription is initiated only in female blastoderm embryos. In male embryos, Sxl expression is prevented by the transcriptional repressor Deadpan (Dpn), which is a member of the Hairy-related basic helix-loop-helix (bHLH) protein family. The negative effect of Dpn can be mimicked in female embryos by ectopic expression of the related Hairy protein at the time of sex determination. Premature Hairy expression under the control of the hunchback (hb) promoter represses Sxl transcription in the anterior part of female embryos, which leads to female-specific lethality. Repression of Sxl by Hairy depends on the interaction of its C-terminal WRPW motif with Groucho and, consequently, does not occur in embryos deprived of maternal Groucho function. Moreover, substitution of the C-terminal Hairy sequences by a heterologous repression domain still leads to down-regulation of Sxl expression, thus providing a convenient assay for the study of Groucho-dependent repression domains in vivo (Eberhard, 2000 and references therein).
This assay was used to examine the in vivo function of the octapeptide motif by replacing the C-terminal region of Hairy with a sequence encompassing the 90 amino acids located between the paired domain and partial homeodomain of zfPax2.1. The octapeptide motif is the only conserved element that is shared between this zebrafish Pax2.1 sequence and the corresponding region of the Drosophila Pax2/5/8 protein. Expression of the chimeric HairyPax2.1 protein under the control of the hb promoter results in significant reduction of Sxl expression in the anterior half of transgenic female embryos, as compared with the uniform Sxl staining of wild-type embryos. Moreover, the repression of Sxl by HairyPax2.1 is dependent on Groucho, as it is not observed in embryos lacking maternal gro function. However, the HairyPax2.1 protein is clearly less active in repressing the Sxl gene than a HairyGsc protein containing the GEH motif of Goosecoid (Gsc) as a potent repression domain. This difference in repression activity is also reflected by the fact that ectopic expression of HairyGsc caused female lethality, whereas HairyPax2.1 doesnot significantly affect female viability. These data indicate that the octapeptide motif of the zebrafish Pax2.1 protein can function as a weak Groucho-dependent repression domain in Drosophila embryos (Eberhard, 2000).
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