tramtrack
Notch targets tramtrack, downstream of Notch's interaction with Numb. Experimental evidence suggests that there is an alteration of ttk expression due to reduction or overexpression of Notch. ttk is normally expressed in the sheath cell, one of the products of the sensory organ precursor lineage, but not in the neural cell, the sister of the sheath cell. In Notch mutants, extra neurons have detected resulting from a transformation of sheath cells into neurons. ttk is expressed in most cells in the epidermis of the mutant embryo, but not in neurons, including the supernumerary neurons derived from transformation of sheath cells. Thus Notch function is required not only to specify the sheath cell but also to express ttk in this daughter cell of an asymmetric division. In a reciprocal experiment, overexpression of Notch turns on ttk expression in cells that normally do not express ttk. It is concluded that Notch targets ttk promoting non-neural fate (Guo, 1996).
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). 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).
Sev regulates pros in R7 cells by inactivating Ttk88, which otherwise represses pros through sequence elements distinct from the eye-specific enhancer. This is demonstrated by finding that overproduced Ttk88 blocks Sev from activating pros, and Sev can regulate the eye-specific enhancer only if it is linked with Ttk88 binding sites. It is not clear if the Sev signal is sufficient to inactivate Ttk88 without an Egfr input since the assay for Ttk88 activity is a reporter gene that includes the eye-specific enhancer. It is quite possible that Ttk88 inactivation in R7 cells requires both Sev and Egfr signals, since Ebi, acting downstream of Egfr to promote Ttk88 degradation, and Phyl/Sina, acting downstream of Sev, are both required to inactivate Ttk88 in 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).
Smooth muscle plays a prominent role in many fundamental processes and diseases, yet understanding of the transcriptional network regulating its development is very limited. The FoxF transcription factors are essential for visceral smooth muscle development in diverse species, although their direct regulatory role remains elusive. A transcriptional map of Biniou (a FoxF transcription factor) and Bagpipe (an Nkx factor) activity is presented as a first step to deciphering the developmental program regulating Drosophila visceral muscle development. A time course of chromatin immunoprecipitatation followed by microarray analysis (ChIP-on-chip) experiments and expression profiling of mutant embryos reveal a dynamic map of in vivo bound enhancers and direct target genes. While Biniou is broadly expressed, it regulates enhancers driving temporally and spatially restricted expression. In vivo reporter assays indicate that the timing of Biniou binding is a key trigger for the time span of enhancer activity. Although bagpipe and biniou mutants phenocopy each other, their regulatory potential is quite different. This network architecture was not apparent from genetic studies, and highlights Biniou as a universal regulator in all visceral muscle, regardless of its developmental origin or subsequent function. The regulatory connection of a number of Biniou target genes is conserved in mice, suggesting an ancient wiring of this developmental program (Jakobsen, 2007; full text of article).
The dynamic enhancer binding of Biniou suggested that the timing of Biniou occupancy is important for the timing of enhancer activity. To assess this in vivo, a number of regions from each of the three temporal clusters were linked to a GFP reporter. The timing of enhancer activity was assayed in vivo by in situ hybridization in transgenic embryos, to avoid time delays due to GFP protein folding and protein perdurance. All regions examined drive expression in a subset of Biniou-expressing cells and recapitulate all or part of the target genes' expression. This study focused on their temporal activity (Jakobsen, 2007).
The initiation of enhancer activity closely matches the first time point of Biniou binding for >90% of enhancers examined (10 of 11 CRMs). The early-bound enhancers (ttk, fd64a-e, lame duck (lmd), bap3) drive expression at stages 10-11, reflecting the binding of Biniou at these stages of development. Similarly, all four continuous-bound enhancers (HLH54F, otk, mib2, bap-FH) initiate expression at the first time period when Biniou binds. The two late-bound enhancers, in contrast, do not initiate expression at stages 10 or 11 of development, matching the lack of Biniou binding during these stages. Instead, the expression of the fd64a late enhancer initiates at stage 13, while the ken enhancer initiates VM expression at stage 14. This shift in the initiation of activity mirrors Biniou binding to these enhancers at stages 12-13 and 13-14, respectively. The only exception is the CG2330 enhancer, which initiates expression at stage 11, while Biniou enhancer binding was first detected at stage 13-14). As the expression of endogenous CG2330 does not initiate until stage 13, the apparent discrepancy in enhancer activity may simply reflect the exclusion of some regulatory motifs within the limits of the cloned region (Jakobsen, 2007).
Remarkably, the duration of enhancer activity is also tightly correlated with the time span of Biniou binding in 10 out of 11 CRMs examined. This is particularly striking in the early-bound enhancers: When Biniou ceases to bind to these CRMs (lmd, ttk, fd64a early, and bap3), their ability to regulate expression is lost. The converse is also true. Continuous Biniou binding correlates with continuous enhancer activity, specifically for bap-FH, HLH54F, and otk. The exception is the mib2 enhancer. In the context of this module Biniou binding it is not sufficient to maintain enhancer activity in the VM at late developmental time points (Jakobsen, 2007).
Taken together, these data indicate that the timing of Biniou enhancer binding is predictive for temporal enhancer activity in the large majority of cases (Jakobsen, 2007).
tramtrack was first identified as a protein that binds to the promoter of the pair-rule gene fushi tarazu (Harrison, 1990). ttk expression completely represses ftz. Mutations in the ftz promoter that disrupt binding of TTK result in ectopic ftz expression unusually early in development, during the third nuclear division, suggesting ftz requires active repression early in development (Brown, 1991).
Ectopic expression of the 69 kDa ttk protein significantly represses even-skipped, odd-skipped, hairy and runt. The 88 kDa form does not similarly repress these genes (Brown, 1993).
The 88 kDa form of ttk is expressed in the eyes, where it represses genes that are not appropriate for eye differentiation, possibly engrailed (Xiong, 1993).
futsch expression was analyzed in mutant backgrounds that specifically alter the 22C10 expression pattern. The Zn finger transcription factor Tramtrack acts as a repressor of 22C10 antigen expression, and in tramtrack mutant embryos, high levels of 22C10 antigen are found in the mesoderm (Giesen, 1997). Consistent with Futsch being the 22C10 antigen, the expression of futsch mRNA is also drastically increased in tramtrack mutant embryos (Himmel, 2000).
The Drosophila homeobox gene fushi tarazu (ftz) is expressed in a highly dynamic striped pattern in early embryos. A key
regulatory element that controls the ftz pattern is the ftz proximal enhancer, which mediates positive autoregulation via multiple
binding sites for the Ftz protein. In addition, the enhancer is necessary for stripe establishment prior to the onset of
autoregulation. Nine binding sites for multiple Drosophila nuclear proteins have been identifed in a core 323-bp region of
the enhancer. Three of these nine sites interact with the same cohort of nuclear proteins in vitro. The
nuclear receptor Ftz-F1 interacts with this repeated module. Additional proteins interacting with this module were purified
from Drosophila nuclear extracts. Peptide sequences of the zinc finger protein Tramtrack and the transcription factor Adh transcription factor 1 (Adf-1) were
obtained. While Ttk is thought to be a repressor of ftz stripes, both Adf-1 and Ftz-F1 have been shown to activate transcription
in a binding site-dependent fashion. These two proteins are expressed ubiquitously at the time ftz is expressed in stripes,
suggesting that either may activate striped expression alone or in combination with the Ftz protein. The roles of the nine nuclear
factor binding sites were tested in vivo, by site-directed mutagenesis of individual and multiple sites. The three Ftz-F1/Adf-1/Ttk binding sites are functionally redundant and essential for stripe expression in transgenic
embryos. Thus, a biochemical analysis has identified cis-acting regulatory modules that are required for gene expression in vivo.
The finding of repeated binding sites for multiple nuclear proteins underscores the high degree of redundancy built into
embryonic gene regulatory networks (Han, 1998).
It was proposed several years ago that Ttk acts as a repressor of ftz stripes, since the protein is present before
and after ftz is expressed in stripes but is not detected during the time that ftz is expressed in stripes (Harrison, 1990).
The proximal enhancer used in the current studies contains multiple binding sites for Ttk. Therefore, an initial
intention was to test the role of Ttk as a repressor of ftz stripes by simultaneously mutating multiple Ttk binding
sites. It was expected that fusion gene expression would initiate earlier and/or persist later in the absence of
repression by Ttk. Fusion genes 12 and 13 carry mutations in four Ttk sites, while all five sites are
mutated in fusion gene 14. However, three of the five Ttk binding sites overlap with binding sites for activator
proteins that are necessary to activate expression of fusion genes (fusion gene 11). Therefore, it was
not possible to test whether Ttk represses through its proximal enhancer binding sites, since mutations result in
loss of activation due to this overlap. Currently, the role of Ttk in regulating ftz is unclear. Mutation of Ttk
binding sites in the zebra element results in premature activation of ftz gene expression, and ectopic
expression of Ttk at later stages causes a decrease in ftz expression levels. However, given the
observation that most Ttk binding sites also interact with other nuclear proteins, it is difficult to know whether
these observations are a result of direct negative regulation of ftz by Ttk. Preliminary results suggest that Ttk can act as a transcriptional activator, raising the possibility
either that Ttk interacts with a corepressor to decrease ftz expression levels or that observed effects of Ttk
overexpression in embryos are indirect (Han, 1998).
Regulatory genes directing embryonic development are expressed in complex patterns. The Drosophila homeobox gene fushi tarazu (ftz) is expressed in a striped pattern that is controlled by several discrete and large cis- regulatory elements. One key cis-element is the ftz proximal enhancer, which is required for stripe establishment and which mediates autoregulation by direct binding of Ftz protein. To identify the trans-acting factors that regulate ftz expression and autoregulation, a modified yeast two hybrid screen, the Double Interaction Screen (DIS), was developed. The DIS was designed to isolate both DNA binding transcriptional regulators that interact with the proximal enhancer and proteins that interact with Ftz itself when it is bound to the enhancer. The screen identified two candidate Ftz protein cofactors as well as activators and repressors of ftz transcription that bind directly to the enhancer. One of these [Tramtrack (Ttk)] is known to bind to at least five sites in the proximal enhancer; genetic studies suggest that Ttk acts as a repressor of ftz in the embryo. In yeast cells, Ttk protein strongly activates transcription, suggesting that yeast may be missing a necessary co-repressor that is present in Drosophila embryos. Also characterized was the activity of a second candidate ftz repressor isolated in the screen: the product of the pair-rule gene sloppy paired, a member of the forkhead family. Slp1 is shown in this study to be a DNA binding protein. A high affinity binding site for Slp1 in the ftz proximal enhancer was identified. Slp1 represses transcription via this binding site in yeast cells, consistent with its role as a direct repressor of ftz stripes in interstripe regions during late stages of embryogenesis. The DIS should be a generally useful method used to identify DNA binding transcriptional regulators and protein partners of previously characterized DNA binding proteins (Yu, 1999).
Neurons and glia are often derived from common multipotent stem cells. In Drosophila, neural identity appears to be the default fate of these precursors. Stem cells that generate either neurons or glia transiently express neural stem
cell-specific markers. Further development as glia requires the activation of glial-specific regulators. However, this
must be accompanied by simultaneous repression of the alternate neural fate. The Drosophila transcriptional repressor Tramtrack is a key repressor of neuronal fates. It is expressed at high levels in all mature
glia of the embryonic central nervous system. Analysis of the temporal profile of Tramtrack expression in glia shows that it follows that of existing
glial markers. When expressed ectopically before neural stem cell formation, Tramtrack represses the neural stem cell-specific genes asense and
deadpan. Surprisingly, Tramtrack protein levels oscillate in a cell cycle-dependent manner in proliferating glia, with expression dropping before
replication, but re-initiating after S phase. Overexpression of Tramtrack blocks glial development by inhibiting S-phase and repressing expression of
the S-phase cyclin, cyclin E. Conversely, in tramtrack mutant embryos, glia are disrupted and undergo additional rounds of replication. It is proposed that
Tramtrack ensures stable mature glial identity by both repressing neuroblast-specific genes and controlling glial cell proliferation (Badenhorst, 2001).
The 69 kDa Tramtrack isoform (Ttk69) is the only transcriptional regulator known to be expressed in all mature glia of the
Drosophila embryonic CNS. At late stages of embryonic development Ttk69 is expressed in both midline and lateral glia. As a first step to determine the function of Ttk69 in glia the temporal profile of Ttk69 expression was examined.
Confocal microscopy has shown that Ttk69 is expressed late in glial development. In lateral glia, double labeling shows that Ttk69 is expressed after either Gcm, the ETS protein PointedP1 or Repo. For example, when Repo is first detected in the longitudinal glioblast, Ttk69 can not be detected. Later, however, low level expression of Ttk69 commences in this progenitor (Badenhorst, 2001).
After the longitudinal glioblast divides, Ttk69 continues to be expressed at a low level while the daughters divide and migrate towards the midline. However, once these glia adopt their correct positions overlying the longitudinal tracts, high levels of Ttk69 are detected. This expression profile is recapitulated by the glioblast derived from neuroblast 3-1. Initially, Ttk69 is not expressed. Ttk69 expression initiates later and finally is expressed at high-levels in the progeny glia (Badenhorst, 2001).
In contrast, glia which express Repo and do not undergo further division express high levels of Ttk69 from the moment Repo can first be identified. An example is the cell body glia. In no case, was the expression of Ttk69 detected before that of Repo (Badenhorst, 2001).
The timing of Ttk69 shows that it does not initiate glial determination. It has been proposed that Ttk69 is expressed in glia to repress neural identity genes. Lateral glioblasts transiently express neural stem cell markers during their development and can adopt the neuronal fate when the glial-determining pathway is not initiated in gcm mutants. Stable glial identity could require neuronal repression. To determine neuronal-specific genes repressed by Ttk69, an analysis was carried out of how ectopic expression of Ttk69 at various stages of nervous system development affects expression of the hierarchy of neuronal markers. This included the proneural genes of the achaete-scute complex, the pan-neural genes (for example asense) and the mature neuronal markers Elav and the antigen 22C10 (Badenhorst, 2001).
Ectopic expression of Ttk69 at any stage does not prevent neuroblast formation. Thus, expression of Ttk69 before neuroblast formation using Kr-Gal4 does not repress the proneural genes achaete or lethal of scute. Strikingly, however, it does inhibit the pan-neural genes asense, dpn and scratch. Consequently, further neuronal development is inhibited and expression of both mature neuronal markers Elav and 22C10 is ablated. Equivalent results were obtained by ectopically expressing Ttk69 in neuroblasts and their progeny using the sca-Gal4 driver. Such expression almost completely inhibits the normal expression of dpn in the embryonic CNS (Badenhorst, 2001).
If, however, Ttk69 is ectopically expressed after the normal neuroblast expression of asense and deadpan, neurons are not ablated. Thus, directed expression of Ttk69 using elav-Gal4 (which is expressed in all post-mitotic neurons after the phase of pan-neural gene expression does not repress the neural markers Elav, 22C210 or Fasciclin II. This indicates that the neural stem cell-specific genes asense and deadpan are the principal targets of Ttk69 repression in the hierarchy of neural determination. Moreover, neural identity, once conferred, cannot be reversed by Ttk69 overexpression, since Ttk69 expression cannot switch neurons to the alternative glial fate (Badenhorst, 2001).
Ectopic Ttk69 expression in mature neurons does, however, disrupt nervous system organization. Specifically, neurons show pathfinding and fasciculation defects. The normal pattern of the three longitudinal fascicles revealed when using antibodies against Fasciclin II fails to form when Ttk69 is expressed in mature neurons. The Fascicles are fused and fail to separate. Pathfinding defects are also observed in the segmental nerve. These abnormalities are confirmed by mAb 22C10 staining (Badenhorst, 2001).
In the Drosophila embryonic and larval PNS, ectopic expression of Ttk69 can convert neurons into non-neuronal cells, while, conversely, loss of ttk leads to the opposite transformation. In the CNS, however, neuron loss induced by ectopic Ttk69 expression does not lead to increased glial number. In fact, ectopic expression of Ttk69 in the embryonic CNS also blocks glial development. Normal glial development is inhibited by early overexpression of Ttk69. Thus, ectopic expression of Ttk69 before precursor formation, using Kr-Gal4, extinguishes the lateral glial markers Gcm and Repo in the domain of expression (Badenhorst, 2001).
The block to glial development is not simply caused by the failure of stem cells to develop. Later ectopic Ttk69 expression also can inhibit glial development. Thus, longitudinal glia are ablated when Ttk69 is only expressed after the formation of the LGB using the sca-Gal4 driver. sca-Gal4 does not direct high levels of expression in the longitudinal glia when they are first formed. Consequently, Repo expression in the LGB is unaffected. High level expression starts later and, by stage 12, glial number as revealed by Repo staining is reduced relative to the wild type. These results were confirmed using another driver, MZ1580-Gal4 that induces Ttk69 expression in the LGB and its progeny. When Ttk69 is ectopically expressed under the control of this driver, longitudinal glial number is reduced although there is segmental variability in the penetrance of longitudinal glial suppression due to variability in expression levels (Badenhorst, 2001).
To understand why early ectopic Ttk69 expression inhibits glial development, the temporal profile of Ttk69 expression in the CNS was re-examined. Specifically, Ttk69 expression was analyzed in the longitudinal glia relative to replication marked by BrdU incorporation. The longitudinal glioblast delaminates from the lateral edges of the neuroepithelium and divides symmetrically at least three times, the progeny migrating medially towards the ventral midline. Ttk69 protein is undetectable in longitudinal glial cells when they undergo DNA replication. Although the LGBs express Ttk69, Ttk69 is absent from their daughters when undergoing replication. However, later, when these glia exit S phase, Ttk69 is again expressed at low levels. The longitudinal glia divide synchronously once more and appear to enter S phase shortly thereafter. Again, during DNA replication Ttk69 is undetectable, but after S phase is complete, Ttk69 expression reinitiates. After this division, glia do not incorporate BrdU, and consistently express high levels of Ttk69 (Badenhorst, 2001).
The absence of Ttk69 from replicating glia implies that ectopic expression of Ttk69 may block normal glial development by inhibiting cell cycle progression. Whether ectopic expression of Ttk69 blocks entry into S-phase was examined. BrdU incorporation is inhibited by ectopic expression of Ttk69 using the Kr-Gal4 driver. In Ttk69-expressing segments of a Stage 10 embryo, the normal BrdU incorporation in the ventral neuroectoderm is inhibited. In the embryonic nervous system, entry into S phase is driven by bursts of transcription of S-phase cyclins -- specifically cyclin E. Heat-shock induced overexpression of Ttk69 blocks zygotic transcription of cyclin E. At earlier stages, maternally deposited cyclin E transcripts are unaffected by Ttk69 overexpression, indicating that Ttk69 affects transcript synthesis rather than stability, consistent with its role as a transcription repressor. Ectopic expression of Ttk69 was induced by a 1 hour heat-shock, after which embryos were processed immediately for in situ hybridization. The rapidity of repression of cyclin E and the presence of multiple Ttk69 consensus recognition sites in the cyclin E promoters both suggest that repression is direct (Badenhorst, 2001).
Since ectopic Ttk69 expression blocks replication, an examination was performed to see if, conversely, loss of Ttk69 induces glial proliferation. Mutant embryos in which only Ttk88 function is affected (ttk1) are homozygous viable and have no obvious defects in their CNS development. Mutants affecting only Ttk69 or both Ttk69 and Ttk88 are embryonic lethal and the CNS is disorganized. In mutant embryos, glial development initiates correctly but, by later stages of development, glia are disorganized. In the case of the longitudinal glia, the LGBs are formed correctly and expression of Gcm and Repo is normal. However, BrdU incorporation shows that glia undergo ectopic rounds of replication. For example, at stage 12 in wild-type embryos, the LGB has divided twice to generate four longitudinal glial precursors that can be trapped undergoing replication. In contrast, in equivalent stage ttk mutant embryos, between 6-7 glia can be observed to undergo DNA replication. In wild-type animals, these glia divide once more to produce the final eight glia which intermingle with at least three glia derived from other sources and migrate along the longitudinal connectives. Confocal analysis of anti-Repo stained wild-type embryos indicates that in each hemisegment there are 9.9±0.76 glia associated with the longitudinal connectives. In the equivalent stage ttk mutant embryos, longitudinal glial number in each hemi-segment, as revealed by anti-Repo staining, is increased to 17.8±2.23. Thus, loss of Ttk69 results in additional glial cells being generated in the longitudinal glial lineage (Badenhorst, 2001).
The widespread expression of Ttk69 and its timing are consistent with Ttk69 maintaining glial identity rather than initiating glial determination. Previous investigations have shown that the two main populations of Drosophila CNS glia, midline glia and lateral glia, are specified by distinct mechanisms. For example, lateral glia require Gcm to initiate development, while midline glia do not. Yet Ttk69 is expressed in both populations -- the only transcription factor expressed in common. It has been suggested that glial determination requires two independent steps: first, a proglial function, for example, Gcm, that initiates glial development, and second, an anti-neural activity that downregulates the previous potential to form neurons. The ability of Ttk69 to repress to neuron development shows that it performs the later role and in this report the targets of neuronal repression have been identifed (Badenhorst, 2001).
In the Drosophila CNS, neural development requires the expression of a cascade of transcription regulators, initiating with high level expression of the proneural genes of the Achaete-Scute complex (AS-C), leading to expression of neural stem cell-specific genes asense and deadpan, and ultimately a battery of specific neural identity genes. Repressing any of these factors could inhibit neural development. Staged overexpression of Ttk69 shows that, in the CNS, Ttk69 inhibits expression of asense and deadpan. In this manner, the ability to adopt neuronal fate is blocked (Badenhorst, 2001).
To date, in mammals, a master regulator of glial development analogous to Gcm has not been identified. However, recently, Notch signaling has been shown to allow some multipotent stem cells in the nervous system to differentiate preferentially into glia. Two downstream components of the Notch pathway have been implicated: the Hairy-E(Spl)-related bHLH repressors -- Hes1 and Hes5. Both are thought to act as repressors of the neuronal specific bHLH genes Mash1, NeuroD and Math3. These studies, however, have not clarified whether Notch signaling acts instructively to promote glial development or whether repression of the neuronal specific bHLH genes permits multipotent stem cells to respond to other instructive signals that induce gliogenesis. If Hes1 and Hes5 act permissively, the parallels between vertebrate and Drosophila gliogenesis would be striking. In Drosophila, Ttk69 can be induced by Notch signaling. Ttk69 represses the neuronal-specific bHLH gene asense. Ttk69 itself does not induce glial fate but blocks the alternative neuronal fate (Badenhorst, 2001).
It is clear from the current study that, at least in some glial populations, Ttk69 has the ability to regulate proliferation. In the Drosophila glioblasts Ttk69 is expressed at low levels soon after glial specification, blocking neural genes. This expression is not constant, though, but oscillates during the cell cycle. Significantly, Ttk69 can not be detected when glia enter S phase and commence DNA replication. Like neuroblasts, glioblasts appear to delaminate in G2 of the cell cycle. The timing of BrdU incorporation indicates that immediately after mitosis they enter S phase and undergo DNA replication. Although Ttk69 is expressed in glia in G2, Ttk69 is not detected in glia that incorporate BrdU. Since Ttk69 can repress cyclin E expression, the absence of Ttk69 allows replication to occur. Once replication is complete, Ttk69 is expressed again. The BrdU incorporation experiments indicate that the LGB undergoes three synchronous cell divisions to produce eight longitudinal glia. This agrees well with a recent estimate of between 7-9 longitudinal glia obtained by DiI labeling of the longitudinal glioblast. After the third mitosis, longitudinal glia do not undergo replication but instead express higher levels of Ttk. By inhibiting cyclin E, high levels of Ttk69 would keep glia in G1 of the cell cycle. Similarly, differentiation of oligodendrocytes and Müller glia is accompanied by high levels of the cyclin-dependent kinase inhibitor p27, blocking re-entry into the cell cycle (Badenhorst, 2001).
Longitudinal glial number was determined in ttk mutant embryos at embryonic stage 14, as the longitudinal glia first extend along the longitudinal axonal tracts. At later stages of development, glia in ttk mutant embryos migrate inappropriately. The regular array of glia along the longitudinal connectives is lost as glia collapse towards the midline. Antibody staining against the nuclear glial antigen Repo indicates nuclear fragmentation, which is evidence of possible cell death. It is feasible that inappropriate proliferation triggers apoptosis (Badenhorst, 2001).
In its ability to repress pan-neural genes and control proliferation, Ttk69 resembles the homeodomain protein Prospero. Prospero is not detected in the nuclei of neuroblasts but is detected in the nuclei of their daughter ganglion mother cells (GMCs). GMCs divide once and terminally differentiate. In GMCs, Prospero appears to be required to repress the pan-neural genes asense and deadpan, thus enforcing the transition from neuroblast to GMC. Recently, it has been shown that loss of Prospero leads to ectopic proliferation in the CNS, suggesting that it is required for exit of GMCs from the cell cycle. Ttk69 appears to play an analagous function in glia (Badenhorst, 2001).
The oscillation in Ttk69 protein levels during the cell cycle demonstrates that there is dynamic control of Ttk expression. Several mechanisms may regulate Ttk69 protein levels. Ttk69 has PEST sequences characteristic of short-lived proteins. Moreover, both isoforms of Ttk have been shown to be targeted for ubiquitin-dependent proteolysis by a complex containing the ring-finger proteins Sina and Phyllopod. It is possible that Ttk69 is destroyed by regulated proteolysis prior to initiation of S-phase. It is interesting in this regard that Ebi, a modifier of over-proliferation phenotypes associated with E2F/DP overexpression, has been shown to interact with Sina and Phyllopod and targets destruction of at least one Ttk isoform in vitro. Another dimension is added by the recent report that Ttk69 translation can be repressed by the RNA-binding protein Musashi. Translational repression by Musashi is not constitutive but appears to be regulated, in part, by signaling through the Notch pathway. The intersection between regulated translational repression and targeted proteolysis of residual protein provides exquisite control of protein levels (Badenhorst, 2001).
In conclusion, these results demonstrate that Ttk69 maintains glial differentiation. This is achieved in two ways. (1) Ttk69 represses the neural stem cell-specific genes: this prevents the reprogramming of glia into neurons. (2) Ttk69 inhibits inappropriate proliferation of glia. In this way CNS organization is preserved (Badenhorst, 2001).
During embryogenesis, the activated Torso receptor tyrosine kinase (TOR RTK) pathway activates tailless (tll) expression by a relief-of-repression mechanism. Various lines of evidence have suggested that multiple factors are required for this repression. Tramtrack69 (TTK69) binds to two sites within tll
cis-regulatory DNA, TC2 and TC5, and that TTK69 is phosphorylated by mitogen activated protein kinase. These two sites are similar to the TTK69 consensus
binding sites identified in the eve gene (GGTCCTGC) and in the fushi tarazu (ftz) zebra element (AnGTCCTnGCA).
TTK69 binding sequences from both eve and ftz regulatory regions share a core sequence, TCCT. Three additional sites containing the core
sequence were found in the tll-MRRe fragment, designated TC1, TC3 and TC4.
In embryos lacking maternal ttk69 activity, the
expression of both endogenous tll and lacZ driven by the tll minimal regulatory region (tll-MRR) are expanded. Further, in wild-type embryos, the tll-MRR mutated in TC5 drives uniform lacZ expression before late stage 4. It is concluded that TTK69 is required for early (before the end of stage 4) repression of tll transcription (Chen, 2002).
In the absence of activation of the TOR RTK pathway, tll is repressed throughout the embryo. Several lines of evidence presented in this study indicate that TTK69 plays a role in this repression, and that its repressive effect is down-regulated by TOR RTK signaling. First, TTK69 activity is required to repress both endogenous tll expression and lacZ expression mediated by the tll-MRR. Second, TTK69 binds to two sites in the tll MRRe, TC2 and TC5. Third, mutation in TC5 causes the tll-MRR to drive uniform early expression of lacZ. Finally, TTK69 is phosphorylated in vitro by MAPK, a target of the TOR pathway. These data indicate that TTK69 functions early during the period when tll is repressed in the central domain of the embryo, i.e. before and during embryonic stage 4 (Chen, 2002).
TTK69 has been shown to act as a transcriptional repressor in a number of contexts during Drosophila development. TTK69 was first identified by its binding to a repression element in the upstream enhancer of the ftz gene, and subsequently shown to act as a transcriptional repressor to regulate expression of additional pair-rule genes, namely eve, odd-skipped, runt and hairy. Later in development, TTK69 acts as an inhibitory regulator to down-regulate the cellular response to neuralizing signals. In the developing eye, TTK69 inhibits photoreceptor cell fate by interacting with and enhancing the activity of the general repressor Yan. The function of TTK69 as a repressor of tll activity in the early embryo is thus consistent with other described roles of the protein during Drosophila development. There are presently at least five known proteins ¯ Gaga, Grainyhead, Groucho, Capicua, and TTK69 ¯ that appear to play a role in the TOR RTK-modulated repression of tll (Chen, 2002).
Protein interaction studies suggest that at least some of these proteins are part of a larger complex that represses tll. Cic and Gro have been shown to interact with each other in vitro. In addition to interacting with each other directly, some of the described tll repressors are likely to be associated with each other through their interactions with other proteins. Both Gro and TTK69 have been shown to interact with histone deacetylase RPD3; in addition, TTK69 interacts with the co-repressors CtBP and Sin3A. Therefore, in addition to the transcriptional regulators with a described role in tll repression, RPD3, CtBP and Sin3A may also be involved in the tor-modulated repression of tll, and are possible members of a multiprotein tll repression complex (Chen, 2002).
Several features of the organization of binding sites in the tll-MRRe lend further support to the idea of a tll repression complex. First, at either end of the tll-MRRe, there is a tor-RE flanked on one side by a demonstrated TTK69 binding site and on the other by a TTK69 core binding site (although these core sites did not bind TTK69 in the in vitro studies, they might do so in vivo, in the presence of other transcriptional regulators), suggesting that adjacent binding sites may promote functional interactions between TTK69 and transcription factors binding to tor-RE (tor-REB). Secondly, tll-MRR with base substitution in either tor-REa or TC5 drives uniform lacZ expression during early stage 4, indicating that both tor-REa and TC5 are required for repression. Thirdly, the tor-REb containing region, including TC2, has partial repression activity; whether this repression activity is a function of tor-REb, TC2, or both sites remains to be determined (Chen, 2002).
Assembly of the repression complex must involve binding of proteins (possibly including GAGA and/or NTF-1) to tor-RE, since repression cannot be established in the absence of this site. Since tll expression is observed only at the poles of the embryo 90 min after fertilization, the repression complex must be assembled by this time. TTK69 is likely to play an accessory role in establishing the complex, but is not absolutely required (since repression can be established later in the absence of TTK69). TTK69 presumably binds to TC5 and may also interact with other proteins, such as GAGA or other BTB domain proteins (since the BTB domain proteins tend to form multimeric aggregates). Consistent with the detection of TTK69 uniformly throughout stage 3, but not in stage 5 embryos, TTK69 is assumed to be displaced from the repression complex by late stage 4 and degraded (Chen, 2002).
Multiple lines of genetic and biochemical evidence converge to support the idea that TTK69 is regulated by RTK pathway activity. When ttk69 is ectopically expressed in the early embryo, the level of TTK69 is reduced at the poles of the embryo, where the TOR pathway is active. Genetic analysis in the developing eye has shown that repression activity of TTK69 is regulated by an RTK pathway. Further, uniform expression in the developing eye of a constitutively active ras, rasV12, causes a dramatic reduction in TTK69 levels, suggesting that TTK69 is degraded where ras is active. The demonstration that TTK69 is phosphorylated by MAP kinase provides further in vitro evidence to support the notion that the activity of TTK69 is modulated by RTK signaling. Phosphorylation of TTK69 might lead to its degradation by a pathway similar to that described for its isoform TTK88. Degradation of TTK88 is initiated when it complexes with Phyllopod (Phyl) and Seven in absentia (Sina); Sina interacts with a ubiquitin conjugating enzyme UBCD1, resulting in ubiquitination of TTK88 and its targeting for proteolytic destruction. The conjugation of TTK69 with dSMT3, a ubiquitin-like protein, has been shown to be correlated, both in vitro and in vivo, with the extent of TTK69 phosphorylation. It is concluded that the activation of tll expression at the poles of the embryo is mediated in part by MAP kinase inactivation of TTK69 repression (Chen, 2002).
The neural selector gene cut, a homeobox transcription factor, is required for the specification of the correct identity of external (bristle-type) sensory organs. Targets of cut function, however, have not been described. bereft (bft) mutants exhibit loss or malformation of a majority of the interommatidial bristles of the eye and cause defects in other external sensory organs. These mutants were generated by excising a P element located at chromosomal location 33AB, the enhancer trap line E8-2-46, indicating that a gene near the insertion site is responsible for this phenotype. Similar to the transcripts of the gene nearest to the insertion, reporter gene expression of E8-2-46 coincides with Cut in the support cells of external sensory organs, which secrete the bristle shaft and socket. Although bft transcripts do not obviously code for a protein product, bereft's expression is abolished in bft deletion mutants, and the integrity of the bft locus is required for (interommatidial) bristle morphogenesis. This suggests that disruption of the bft gene is the cause of the observed bristle phenotype. Attempts were made to determine what factors regulate the expression of bft and the enhancer trap line. The correct specification of individual external sensory organ cells involves not only cut, but also the lineage genes numb and tramtrack. Mutations of these three genes affect the expression levels at the bft locus. Furthermore, cut overexpression is sufficient to induce ectopic bft expression in the PNS and in nonneuronal epidermis. On the basis of these results, it is proposed that bft acts downstream of cut and tramtrack to implement correct bristle morphogenesis (Hardiman, 2002).
In an effort to identify and characterize genes that might integrate information from the selector gene cut and lineage gene ttk, bereft was cloned. bft is expressed in es (mechano- and chemo-receptors), but not in ch (chordotonal) support cells. Analysis of flies with deletions of the bft locus, together with the es support cell-specific expression pattern, suggest that bft function is required for correct morphogenesis of the cuticular structure forming support cells, in particular those of the interommatidial bristles of the eye. Moreover, bft expression in es organs is reduced in cut and ttk mutants, and cut and ttk interact genetically with bft. These data are consistent with the idea that bft is a target for cut and ttk in the implementation of es organ-specific structures (Hardiman, 2002).
Targets of both cut and ttk were sought on the basis of the expression pattern of candidate genes within the PNS. cut is expressed in all the cells of es organs (at higher levels in support cells), whereas ttk is found in three es and two ch support cells, but not the neurons. Thus, the support cells of es organs express both cut and ttk, suggesting that genes responsive to these two pathways (i.e., the pathways leading to organ identity specification and lineage decisions, respectively) should also be expressed in these cells. An enhancer trap line, E8-2-46, was identified in which the lacZ reporter gene is expressed primarily in the support cells of es organs within the PNS, on the basis of position, morphology, and cut expression. Although E8-2-46 is expressed in both es support cells (as identified by high levels of cut expression), the level of expression is lower in one of them. To determine which of the two cell support cells express the reporter gene more strongly, the dorsal-most abdominal es organ (desD) were examined. DesD are aligned in a stereotyped linear fashion: tormogen, trichogen, thecogen, and neuron (from dorsal to ventral). Strong reporter activity is observed in the bristle shaft-forming trichogen cell, whereas cut expression predominates in the shaft-forming tormogen cell (Hardiman, 2002).
E8-2-46 reporter gene expression was examined in mutations of the lineage genes numb and ttk. In numb1 mutants, the number of cells expressing the reporter gene increases as would be expected if the neural pIIb secondary precursors are transformed into pIIa, the support cell precursors. Similarly, Keilin organ cells are also increased. In ttk mutants the opposite phenotype is expected, since ttk is required for support cell development. Indeed, in ttk702/7 mutants E8-2-46 expression per hemisegment is reduced, similar in extent to the reduction observed in cut mutants (Hardiman, 2002).
Low-level ectopic expression of the Runt transcription factor blocks activation of the Drosophila melanogaster segmentation gene engrailed (en) in odd-numbered parasegments and is associated with a lethal phenotype. By using a genetic screen for maternal factors that contribute in a dose-dependent fashion to Runt-mediated repression, it is shown that there are two distinct steps in the repression of en by Runt. The initial establishment of repression is sensitive to the dosage of the zinc-finger transcription factor Tramtrack. By contrast, the co-repressor proteins Groucho and dCtBP, and the histone deacetylase Rpd3, do not affect establishment but instead maintain repression after the blastoderm stage. The distinction between establishment and maintenance is confirmed by experiments with Runt derivatives that are impaired specifically for either co-repressor interaction or DNA binding. Other transcription factors can also establish repression in Rpd3-deficient embryos: this indicates that the distinction between establishment and maintenance may be a general feature of eukaryotic transcriptional repression (Wheeler, 2002).
Each sensory organ of the Drosophila peripheral nervous system is derived from a single sensory organ precursor cell (SOP). These originate in territories defined by expression of the proneural genes of the Achaete-Scute complex (AS-C). Formation of ectopic sensilla outside these regions is prevented by transcriptional repression of proneural genes. The BTB/POZ-domain transcriptional repressor Tramtrack (Ttk) co-operates in this repression. Ttk is expressed ubiquitously, except in proneural clusters and SOPs. Ttk over-expression represses proneural genes and sensilla formation. Loss of Ttk enhances bristle-promoting mutants. Using neural repression as an assay, functional domains of Ttk have been dissected, confirming the importance of the Bric-a-brac-Tramtrack-Broad complex (BTB) motif. The Ttk BTB domain is a protein-protein interaction motif mediating tetramer formation (Badenhorst, 2002).
Ttk is expressed in the non-neuronal cells of sensory organs. Ttk expression prevents these cells from adopting the alternate neuronal fate. However, it is speculated that Ttk also functions earlier in sensory organ formation, during SOP formation. To determine if Ttk can control SOP recruitment and, thus, sensilla number in the adult PNS, the expression of both isoforms of Ttk was analyzed relative to the proneural genes and SOP markers in third instar wing imaginal discs. Immunostaining using isoform-specific antibodies directed against either Ttk69 or Ttk88 shows that both are expressed ubiquitously in wing imaginal discs, except in proneural clusters, SOPs and SOP daughter cells that have retained the ability to form neurons. Ttk is not detected in SOPs labelled using the enhancer-trap line A101. Later, however, Ttk is observed in the non-neuronal progeny of SOPs. Cells that express the proneural genes do not react with antibodies against either Ttk isoform. Thus, in territories which give rise to the dorsal (dCh) and ventral (vCh) chemosensory bristles of the wing margin, Ttk is absent from the high-expressing Achaete-positive cells destined to form SOPs. Moreover, Ttk is not detected in cells that express the neural precursor gene asense (Badenhorst, 2002).
Since Ttk is excluded from proneural territories tests were perfomed to see if ectopic expression of Ttk could repress proneural genes and, hence, sensilla formation. Ttk isoforms were over-expressed prior to the formation of SOPs using the Gal4-UAS system under the control of MS1096-Gal4. Ectopic expression of Ttk69 removes the external structures (bristles and sockets) of all wing sensilla with the exception of the ventral mechanosensory bristles (these sensilla arise during pupal stages, at a time and in an area in which MS1096-Gal4 drives negligible expression). Antibody staining of pupal wings using mAb 22C10 shows that loss of external structures is accompanied by neuron ablation. Furthermore, in third instar larval wing discs, SOPs are ablated (revealed using A101). Over-expression of Ttk88 using MS1096-Gal4 has equivalent, albeit milder, effects on sensory organ formation. Ttk88 over-expression removes all dCh bristles but only reduces vCh and medial mechanosensory bristle numbers (Badenhorst, 2002).
The ablation of SOPs is caused by the repression of proneural genes. Ectopic expression of Ttk69 under the control of a heat-shock promoter inhibits achaete and scute transcription. Accumulation of Asense protein is also blocked. Over-expression of Ttk88 also perturbs achaete, scute and asense expression showing that both isoforms of Ttk can repress the AS-C. Significantly, though, the extent of repression is lower. This could reflect differences in protein stability of the two isoforms. Both are targeted for ubiquitin-dependent proteolysis. However, Ttk69, unlike Ttk88, is post-translationally modified by the small ubiquitin-like molecule dSmt3. This modification has been shown to protect IkappaBalpha from ubiquitin-dependent degradation (Badenhorst, 2002).
Over-expression of Ttk69 also reduces the wing blade area. BrdU incorporation reveals that Ttk69 over-expression blocks cell proliferation. In embryos, over-expression of Ttk69 blocks BrdU incorporation and cyclinE expression. Ttk88 has no effect on cell proliferation or wing blade area (Badenhorst, 2002).
To dissect functional domains of Ttk required for transcriptional repression, the ability of Ttk69 to ablate sensilla and reduce wing blade area when over-expressed using MS1096-Gal4 was exploited. Analysis of adult wing over-expression phenotypes highlights the critical importance of the BTB domain for Ttk69-mediated repression. Mutation of conserved residues within the BTB motif (Ttk-D32A, Ttk-D32N, Ttk-K46E, Ttk-S53A), or removal of the BTB domain (Ttk-DeltaBTB, Ttk-R113), almost completely cripples Ttk69 function. None of the effects on the area of the wing blade area are seen and only the dorsal chemosensory (dCh) bristles are reduced in number. The sensitivity of these sensilla reflects their emergence at the peak of MS1096-Gal4-directed expression. Using the SOP marker A101, it was confirmed that the effects on these sensilla are due to the suppression of SOPs and not cell-fate transformations in the sensilla cell lineages. Over-expression of the D32A BTB point mutant slightly reduces dCh SOPs. Ectopic expression of the Ttk BTB motif alone in wild-type backgrounds (Ttk-BTB) has no effect on wing development (Badenhorst, 2002).
A series of internal and C-terminal Ttk69 deletions was constructed to define other domains required for repression. Deletion of the C-terminal domain predicted to interact with the co-repressor dCtBP (Ttk-DeltaSA) has no effect on Ttk69-mediated repression in this assay. Deletion of a region including the internal dMi-2-interacting motif (Ttk-286-ZFS) yields a construct that still retains some repression activity but is less effective at preventing sensilla development and has milder effects on cell proliferation. When both domains are deleted in a minimal Ttk69 variant that contains only the BTB motif and the zinc finger domains (Ttk-BTB-ZF), partial Ttk69 activity is observed. Directed expression of this construct ablates most dCh bristles and SOPs within the domain of expression, although it only partially suppresses medial mechanosensory bristles and has no effect on cell proliferation. It is inferred that the BTB motif and the zinc fingers alone are sufficient to confer at least some repressive activity, independent of other domains (Badenhorst, 2002).
One function that has been ascribed to the BTB domain is protein multimerization and self-association. The BTB domain has been described as a protein-protein interaction motif, while crystal structures show that the domain can form homodimers and possibly higher order oligomers. To determine the stoichiometry of the interactions, the Ttk-BTB motif was over-expressed in Escherichia coli and purified to homogeneity. Although the domain is mostly insoluble when expressed alone, when fused to glutathione-S-transferase (GST), a small fraction (approximately 2%) is soluble and can be purified. Electron microscopy of the purified fusion protein shows that most GST-BTB moieties form tetramers. Size-exclusion data confirm the fact that the BTB motif exists in solution as a tetramer. This strong preference for tetramer formation is indicative of a unique higher order structure and is not obviously consistent with the linear arrays of homodimers. Ttk BTB-mediated interactions were confirmed using the yeast two-hybrid system (Badenhorst, 2002).
Although wild-type BTB domains interact profitably, mutation of conserved residues within the Ttk BTB domain completely abolishes protein-protein interactions. None of the mutant BTB-LexA fusions interact with the corresponding mutant BTB-B42 fusion protein, the wild type Ttk BTB-B42 fusion or any of the other mutant BTB-B42 fusions. This applies equally to non-conservative substitutions such as K46E and conservative changes such as A44G, A44V and Y88F. However, like the wild-type Ttk BTB-LexA fusion, the mutant Ttk BTB-LexA fusions are still targeted to the nucleus and are able to bind DNA as assayed by the GAL1 repression assay (Badenhorst, 2002).
Both the expression profile of Ttk and its ability to repress proneural genes when expressed ectopically suggest that Ttk functions like the transcriptional repressors hairy and emc as a global regulator to limit AS-C expression to proneural clusters. To confirm this, tests were made to determined if loss, or reduction, of Ttk induces excess SOP production. Dominant interactions between ttk and known bristle-promoting mutants were sought. A series of ttk alleles were used: mutations that reduce both Ttk69 and Ttk88. Mutations that affect both isoforms of Ttk exacerbate ectopic bristle production seen in excess function achaete mutations. acHw1 and acHw49c induce ectopic Ac expression and cause the development of extra bristles, particularly along the wing veins L2, L3 and L5. Reduction of both Ttk69 and Ttk88 levels enhances these phenotypes. In contrast, slightly elevating Ttk69 levels through basal expression from a hs-ttk69 transgene decreases the strength of the acHw49c phenotype (Badenhorst, 2002).
ttk mutants that affect both isoforms of Ttk also interact dominantly with hairy and emc alleles to cause a phenotype that mimics the gain-of-function acHw mutations. Adult emc/ttk transheterozygous flies develop ectopic bristles on the wing blade. Similarly, hrM730, h/ttk transheterozygotes exhibit many ectopic bristles on the L2, L3 and L5 wing veins and a variable number of additional dorsocentral and scutellar bristles. However, reduction of both Ttk69 and Ttk88 is required for ectopic bristle production (Badenhorst, 2002).
Ectopic wing bristle production was used as an assay to show that dCtBP interacts with both ttk and hairy. ttk/dCtBP and hairy/dCtBP transheterozygotes develop ectopic bristle on the wing blade, arguing that dCtBP is required for transcription repression of the proneural genes (Badenhorst, 2002).
The ability of the BTB domain to compromise wild-type Ttk function was explored. Over-expression of the Ttk BTB domain in a wild-type background has no effect on bristle development. However, when levels of either Ttk or Hairy are reduced by half, over-expression of the BTB leads to the development of ectopic bristles on the wing. This suggests that the BTB domain can form non-profitable multimers with wild-type Ttk protein to titrate its function (Badenhorst, 2002).
Previous studies have demonstrated that Ttk is required for the development of the adult and embryonic PNS. It was shown that Ttk acts in the daughter cells of SOPs to distinguish non-neuronal cells from their neuronal siblings. In ttk mutants, non-neuronal cells are transformed into neurons leading to neuron duplication in each sensillum. These studies reveal that Ttk plays an additional function during sensilla development, namely to control the number of cells that are initially recruited as SOPs. ttk interacts genetically with hairy and emc. Analogous to hairy and emc, Ttk acts as a blanket transcriptional repressor to inhibit the formation of ectopic SOPs and sensilla outside of proneural regions. Like Hairy, Ttk acts as a factor that patterns AS-C expression, limiting proneural gene expression to the proneural clusters, thus committing cells that lie outside these domains to the epidermal fate (Badenhorst, 2002).
However, Ttk might also participate in the process of SOP singularization, and repress proneural expression in those cells of the proneural cluster that are not destined to form SOPs. When over-expressed, Ttk has the ability to repress achaete expression in all cells of the wing imaginal disc, including proneural clusters and the presumptive SOPs. This distinguishes Ttk from Hairy. Ectopically expressed Hairy only blocks achaete expression outside proneural clusters, while expression in the presumptive SOPs is unaffected (Badenhorst, 2002).
Ttk blocks SOP recruitment by repressing transcription of the proneural genes. In the developing PNS, Ttk completely inhibits achaete and asense expression and blocks part of the scute expression profile. Surprisingly, in the embryonic central nervous system (CNS), Ttk over-expression only represses asense but has no effect on achaete. Inspection of the promoters of the proneural genes reveals that the immediate 5' promoter region of asense contains many clustered consensus Ttk69-binding sites, suggesting that Ttk inhibits asense by directly repressing the proximal promoter. In contrast, the upstream promoter region of achaete does not contain large numbers of consensus sites. A cluster of Ttk69-recogntion sites is found downstream of achaete. It is conceivable that specific repression of achaete in the PNS is achieved by blocking PNS-specific enhancers while not affecting regulatory elements required for expression in the CNS. The existence of separate enhancers directing expression of achaete and scute in the CNS and PNS has been inferred from deletions and inversions that affect subsets of the achaete expression profile (Badenhorst, 2002).
Alternatively, Ttk may repress achaete in cooperation with other factors that are only present in the wing imaginal disc and not expressed in the CNS. Ttk69 binds to the dMi-2 subunit of NURD - the nucleosome remodeling deacetylase . Recruitment of histone deacetylases to the Ttk69-binding sites downstream of achaete could establish an acetylation-free domain covering achaete if deacetylase activity spreads from the initial site of recruitment to modify flanking nucleosomes. Such deacetylation may be required to allow other factors to repress achaete (Badenhorst, 2002).
Over-expression of either isoform of Ttk can repress achaete and asense expressions. However, Ttk69 consistently has stronger effects. One explanation for this difference is that the isoforms may have different protein stabilities. Both isoforms are subject to ubiquitin-dependent proteolysis, but Ttk69 is also modified by the ubiquitin-like protein Smt3. Smt3-modification has been proposed to protect target proteins from ubiquitin-dependent proteolysis. Further evidence that Ttk69 and Ttk88 co-operate to repress proneural genes is provided by the genetic interactions between ttk and bristle-promoting mutants. Only ttk alleles that reduce expression of both Ttk69 and Ttk88 levels show a strong interaction. ttk1e11, which only affects Ttk69 but does not enhance phenotypes significantly. Flies mutant for ttk1, which reduces Ttk88 expression, are homozygous viable and do not show ectopic wing bristles. Although this mutation also has a slight Ttk69 gain-of-function phenotype caused inappropriate translation of Ttk69 in some microchaete daughter cells, this effect is largely confined to abdominal and thoracic microchaete and wing sensilla are unaffected (Badenhorst, 2002).
Temporal and spatial regulation of proliferation and differentiation by signaling pathways is essential for animal development. Drosophila follicular epithelial cells provide an excellent model system for the study of temporal regulation of cell proliferation. In follicle cells, the Notch pathway stops proliferation and promotes a switch from the mitotic cycle to the endocycle (M/E switch). This study shows that zinc-finger transcription factor Hindsight mediates the role of Notch in regulating cell differentiation and the switch of cell-cycle programs. Hindsight is required and sufficient to stop proliferation and induce the transition to the endocycle. To do so, it represses string, Cut, and Hedgehog signaling, which promote proliferation during early oogenesis. Hindsight, along with another zinc-finger protein, Tramtrack, downregulates Hedgehog signaling through transcriptional repression of cubitus interruptus. These studies suggest that Hindsight bridges the two antagonistic pathways, Notch and Hedgehog, in the temporal regulation of follicle-cell proliferation and differentiation (Sun, 2007).
How developmental signals coordinate to control cell proliferation and differentiation remains largely unknown. These data reveal a molecular mechanism that links signal-transduction pathways and the cell-cycle machinery. Hnt is induced by Notch signaling and mediates most, if not all, Notch functions in the downregulation of Hh signaling and the M/E switch in follicle cells during midoogenesis. Loss of hnt function in follicle cells results in an extra round of the mitotic cycle after stage 6 and a delayed entry into the endocycle. In contrast, misexpression of Hnt at an earlier stage causes the follicle cells to differentiate prematurely and enter the endocycle. Hnt suppresses both stg and Cut, whose expression must be downregulated to ensure the M/E switch. In addition, Notch signaling appears to act through Hnt to downregulate Hh signaling by suppressing ci transcription, so Hnt links the two antagonistic signaling pathways in follicle-cell development. The transcriptional repression of ci is probably not mediated by Hnt alone, because ttk exhibited a similar defect in transcriptional regulation of ci and stg (Sun, 2007).
Studies have shown that downregulation of Cut mediates part of Notch function during the M/E switch. Specifically, Cut promotes cell proliferation and maintains an immature-cell fate, but Stg, the Cdc25 homolog, is not regulated by Cut. To induce the mitotic division ectopically during midoogenesis in follicle cells, both Cut and Stg must be misexpressed. The current study suggests that both Cut and Stg are suppressed by Hnt. Without Stg activity, a major regulator of G2/M transition, follicle cells are arrested before they enter the M phase, and downregulation of Cut allows accumulation of Fzr, causing degradation of CycA and CycB by the UPS, thus lowering CDK activity. This process allows endocycling follicle cells to by-pass the M phase and enter the next S phase. Repeated gap phases and S phases constitute the endocycle (Sun, 2007).
The finding that hnt follicle cells enter the endocycle after one additional round of the mitotic cycle suggests that hnt mutation causes a delay in the M/E switch. Mutations of the Notch pathway may also result in only a delay in entering the endocycle. In Notch mosaics, the cell number in mutant clones is approximately twice that of the twin spots, suggesting that an additional cell cycle also takes place. Further testing of this hypothesis requires a detailed analysis of the DNA content and clone size in Notch pathway mutants. Alternatively, Hnt may not be the sole mediator of the Notch effect; for example, Su(H)-independent Notch signaling may also be required in the M/E switch. Although hnt mutant cells can enter the endocycle late, they could not enter the chorion-gene-amplification program even much later, suggesting that Hnt function is also required for chorion-gene amplification (Sun, 2007).
The removal of negative components of the Hh pathway such as ptc causes overproliferation in follicle cells. Loss-of-function analyses of fu, a positive regulator of the pathway, revealed fewer cells in the mutant clones than in twin spots. The nuclear sizes of fu mutant cells were similar to those of the wild-type at the same developmental stage, and no fragmentation of the chromosomes was observed. Hh signaling therefore promotes cell proliferation in follicle cells during early oogenesis. Thus, Hnt-mediated downregulation of Hh signaling through suppression of ci transcription plays an important role in the M/E switch. Hh signaling is probably not involved in regulating Cut or Stg expression, because ectopic expression of Ci-155 in follicle cells during midoogenesis did not extend Stg-lacZ or Cut expression beyond stage 6, and fu mutant follicle cells showed normal Cut expression during early oogenesis. Other factors may therefore mediate the role of Hh signaling to modulate proliferation of follicle cells (Sun, 2007).
Hnt is not only required to mediate the role of Notch in regulating the M/E switch in follicle cells, but it is also sufficient to drive premature entry into the endocycle. Only a few cells misexpressing Hnt at the early stages of oogenesis were recovered, consistent with the role of Hnt in terminating the mitotic phase. In an extreme case, a stage-4 egg chamber contained only ~20 follicle cells, most of which misexpressed Hnt. Hnt misexpression suppresses Cut and stg-lacZ expression, suggesting that Hnt acts as a transcriptional repressor. Consistent with this interpretation, the mammalian homolog of Hnt, RREB1, also acts as a transcriptional repressor in several cellular contexts (Sun, 2007).
An interesting observation from these studies is that ttk clones have a phenotype similar to that of hnt clones. As in Notch regulation of Hnt, ttk is possibly downstream of Notch, but the current analysis of Notch mutants in stage-1 to stage-10 egg chambers showed no obvious change in Ttk expression. It was also found that Hnt has no role in regulating ttk expression. The findings that ttk expression is not regulated by Hnt or Notch during midoogenesis is perhaps not surprising given that Ttk69 is evenly expressed throughout early and midoogenesis. The phenotypic similarity between hnt and ttk mutants suggests that ttk and hnt act cooperatively to suppress gene expression at the M/E transition. Ttk may act as a permissive signal for Hnt to regulate Ci expression and the M/E switch. In the absence of either one, the M/E switch cannot take place properly. Consistent with this hypothesis, Ttk is known to act as a transcriptional repressor in the Drosophila eye. Whether Hnt and Ttk bind directly to the regulatory sequence of the cell-cycle genes and/or ci remains unclear (Sun, 2007).
Several lines of evidence suggest that the role of Hnt in promoting the M/E switch is not universal. First, during embryogenesis, a hnt-deficiency line enters the G1 arrest normally after cycle 16 in epidermal cells and undergoes normal M/E switch in the salivary gland, although Fzr is required for this process. Second, nurse-cell endoreplication does not require Hnt; no obvious defect was detected in hnt germline clones. The specific role of Hnt in follicle-cell-cycle regulation may stem from its role in regulating cell differentiation. For example, Hnt expression may cause upregulation of Fzr through the downregulation of Cut. This indirect role of Hnt suggests that the cell-cycle regulation may be a by-product of cell differentiation (Sun, 2007).
Both Notch and Hh signaling pathways are implicated in the regulation of differentiation and proliferation, but precisely how the two interact in regulating cellular processes is poorly understood. Depending on the cellular environment, their effects on proliferation and differentiation differ. In Drosophila eye imaginal discs, Notch triggers the onset of proliferation during the second mitotic wave (SMW), the opposite of its role in follicle-cell development. In the SMW, Notch positively affects dE2F1 and CycA expression and promotes S phase entry. In these cells, Hh signaling, along with Dpp, activates Dl expression, thereby activating the Notch pathway. Hh and Notch therefore act sequentially and positively during the SMW, whereas, in follicle cells, they act antagonistically. Hh signaling is active in the mitotic follicle cells in early oogenesis, but it is downregulated during the M/E switch when Notch signaling is activated. Notch appears to be superimposable on Hh signaling; mutation of the negative regulator of the Hh pathway, ptc, in follicle cells cannot interfere with the activation of Notch signaling as long as these cells are in direct contact with the germline cells. These ptc mutant cells show no accumulation of Ci-155, consistent with the finding that Notch signaling suppresses ci transcription through Hnt. The ptcS2 cells that were out of contact with germline cells remained in the mitotic cycle because they could not receive Dl signaling from them, suggesting that Hh signaling is sufficient to keep these cells in the undifferentiated and mitotically active state (Sun, 2007).
Notch-dependent activation of Hnt and downregulation of Ci may be involved in another follicle-cell process, the migration of a specialized group of anterior follicle cells toward the border between the nurse cells and the oocyte at stage 9. These so-called border cells showed downregulation of ci during migration. When slbo-Gal4 was used to drive Ci overexpression in border cells, ~66% of egg chambers showed defects in border-cell migration. Notch signaling, as well as ttk, has been reported to be required for border-cell migration. Hnt was found to be expressed in the border cells and depended on Notch signaling. The occasional hnt border-cell clones observed also showed defects in border-cell migration, so the crosstalk between Hh and Notch through Hnt may go beyond the regulation of the M/E switch in follicle cells (Sun, 2007).
In the developing Drosophila eye, the precursors of the neuronal photoreceptor cells R1/R6/R7 and non-neuronal cone cells share the same developmental potential and constitute the R7 equivalence group. It is not clear how cells of this group elaborate their distinct fates. This study shows that both TTK88 and D-Pax2 play decisive roles in cone cell development and act in concert to transform developing R1/R6/R7 into cone cells: while TTK88 blocks neuronal development, D-Pax2 promotes cone cell specification. In addition, ectopic TTK88 in R cells induces apoptosis, which is suppressed by ectopic D-Pax2. It was further demonstrated that Phyllopod (Phyl), previously shown to promote the neuronal fate in R1/R6/R7 by targeting TTK for degradation, also inhibits D-Pax2 transcription to prevent cone cell specification. Thus, the fates of R1/R6/R7 and cone cells are determined by a dual mechanism that coordinately activates one fate while inhibiting the other (Shi, 2009).
Members of the R7 equivalence group have the developmental potential to become a neuronal R1/R6/R7 or a non-neuronal cone cell. TTK88 and D-Pax2 are both specifically expressed in developing cone cells. The absence of cone cells in ttk1; spapol double mutants and their presence in ttk1 and spapol single mutants strongly suggest that both D-Pax2 and TTK88 function in cone cell development. This study has shown that (1) blocking the neuronal fate by TTK88 and (2) promoting the non-neuronal fate by D-Pax2 are simultaneous and coordinated steps in the transformation of R cells into cone cells. The TTK88 function of blocking the neuronal fate is largely redundant with that of TTK69 because ectopic Phyl, which down-regulates D-Pax2 and TTK88 as well as TTK69, is sufficient to transform cone cells into R7 cells, whereas removal of only TTK88 and D-Pax2 in ttk1; spapol double mutants results in the loss of cone cells but not their conversion into R cells. In contrast, during R1/R6/R7 development, Phyl promotes the neuronal fate not only (1) through targeting TTK (both TTK88 and TTK69) for degradation, thereby releasing the inhibition of R cell specification, but also (2) by down-regulating D-Pax2 to block the cone cell fate. Therefore, it is proposed that the cell fates of the R7 equivalence group are determined by a dual mechanism that coordinately promotes the fate of one cell type and blocks that of the other. For cone cell development, it is not sufficient to provide D-Pax2 that activates the cone cell fate, the alternative neuronal fate has to be blocked as well by TTK protein. Conversely, for R cell development, it is not sufficient to specify this fate by removing its TTK block, but inhibition of the alternative cone cell fate by preventing D-Pax2 activation is equally important (Shi, 2009).
In third instar larval and early pupal eye discs, TTK88 protein is initially detected in all undifferentiated basal cells, but later restricted to cone cells. TTK88 blocks neuronal R cell differentiation, but is unable to promote non-neuronal cone cell specification. Thus, TTK88 serves as a safeguard in basal cells that maintains them in an undifferentiated state. The binary switch between R1/R6/R7 and cone cell fates is regulated by Phyl, which is present in the former but absent from the latter. Activation of phyl depends on Svp in R1/R6, or on high levels of Ras signaling in R7 where svp is repressed by Lz. This follows from the observation that Phyl is absent from cone cells, while ectopic Svp or high levels of Ras signaling in cone cells transforms these into R7 cells in a Phyl-dependent manner. Moreover, it has been shown that phyl is activated by Sev-induced Ras signaling in R7 precursor cells. Since Svp is absent from cone cells and Ras signaling too low because Sev is not activated, phyl is inactive in and its product absent from cone cells (Shi, 2009).
In R1/R6/R7 precursors, Phyl forms a complex with Ebi and Sina, activating a ubiquitin-proteasome machinery that targets TTK for degradation and hence releases the block of the neuronal fate in these cells. In addition to TTK, D-Pax2 must be absent from R cells because ectopic D-Pax2 in R cells of GMR-D-Pax2 flies causes frequent loss of R cells or their strong deformation, even though early R cell development seems normal as judged by staining for Elav. It is proposed that the same ubiquitin-proteasome machinery also targets an activator X of D-Pax2 transcription for degradation and thus indirectly down-regulates D-Pax2 in R1/R6/R7 precursors. Therefore, the cone cell fate is blocked in these cells while R1/R6/R7 specification begins (Shi, 2009).
In developing cone cells, TTK is not degraded because Phyl is absent. Strong N signaling in cone cell precursors is activated by high concentrations of the N ligand, Dl, on neighboring R cells. As a consequence, D-Pax2 transcription is activated in cone cells by the combinatorial effect of Lz, N-activated Su(H), and the concomitant activation of PntP2 and inhibition of Yan by EGFR-activated Ras signaling. Thus, as TTK and D-Pax2 are both present, the R cell fate is blocked and cone cell specification is initiated (Shi, 2009).
Absence of TTK88 in ttk1 or of D-Pax2 in spapol mutants does not result in the transformation of cone cells into R7 cells. According to the model see A dual mechanism determines cell fates in the R7 equivalence group, efficient transformation depends on the absence of both TTK (TTK88/TTK69) and D-Pax2. This is achieved by ectopic expression of Phyl in cone cells under control of the sev enhancer or by combining the homozygous ttk1 mutation with a heterozygous null allele of yan, which results in the derepression of phyl in cone cells. Similarly, homozygous hypomorphic yanP mutations combined with heterozygous strong ttk alleles results in a large fraction of ommatidia with supernumerary R7 cells. In this case, Phyl levels induced in cone cells suffice to unblock the neuronal fate by further reducing TTK levels and to down-regulate D-Pax2 levels to an extent that cone cells are transformed efficiently into R7 cells. Ectopic Phyl transforms cone cells into R7 rather than R1/R6 photoreceptor cells because Svp, which specifies the R1/R6 versus the R7 cell fate, is not expressed in cone cells. Thus, the model is in good agreement with earlier observations that ttk and yan mutations act synergistically to alter the fate of cone cell precursors to that of R7 cells. It further confirms an earlier suggestion that TTK88 functions in a pathway distinct from and parallel to Ras signaling (Shi, 2009).
In addition, the reverse situation was shown to be true as well. Ectopic expression of both D-Pax2 and TTK88 in R cell precursors transforms R1/R6/R7 with a much higher efficiency into cone cells than R2-R5. For when D-Pax2 and TTK88 are expressed under the control of the sev enhancer, which results in their ectopic expression in R3/R4/R7 precursors, only one additional cone cell appears with high efficiency. By contrast, when ectopic expression occurs in all R cell precursors under the control of the GMR enhancer, only three supernumerary cone cells appear with high efficiency although up to 7 additional cone cells were observed. Moreover, ectopic co-expression in R1/R6/R7 of TTK88 and D-Pax2 in phyl mutants efficiently transforms these R cell precursors into supernumerary cone cells. The evidence, therefore, suggests the existence of a dual mechanism regulated by a binary switch between the non-neuronal cone cell and neuronal R cell fate in the R7 equivalence group. The state of this switch depends on the presence or absence of Phyl that coordinately regulates TTK and D-Pax2 levels through ubiquitin-directed proteolysis (Shi, 2009).
The model might suggest Sina as an alternative switch to Phyl. However, this possibility is excluded because Sina is expressed in photoreceptors as well as cone cells. Moreover, in sina mutants only R7 is transformed into a cone cell even though Sina is expressed in all photoreceptors. This observation has been explained by a redundancy of sina function with that of musashi in the down-regulation of TTK in R1 and R6. It is not known how TTK is down-regulated in photoreceptor precursors different from R1/R6 and R7. However, it appears that this mechanism is not only independent of Sina but also independent of Phyl, as Phyl is not expressed in photoreceptors different from R1/R6/R7 (Shi, 2009).
Degradation of TTK is mediated through an E3 ubiquitin ligase complex, including Phyl/Sina/Ebi, which targets TTK to the proteasome. The results suggest that the same complex also functions to down-regulate D-Pax2 transcription. It is therefore conceivable that this complex targets one or several of the activators of D-Pax2 for degradation (X in the model). It is improbable that TTK is this activator because TTK is only known to act as repressor (Shi, 2009).
Transcription of D-Pax2 in cone cells is regulated by the combinatorial action of Lz, N-activated Su(H), and the EGFR-regulated effectors PntP2 and Yan. EGFR signaling and Lz are both active in R1/R6 and R7 precursors. Ectopic expression in R7 of the constitutively active intracellular domain of N, NIC, activates D-Pax2. It follows that the Phyl/Sina/Ebi-dependent proteasome machinery down-regulates D-Pax2 transcription in R7 by antagonizing N signaling. Consistent with this conclusion, it has been suggested that the E3 ubiquitin ligase complex Phyl/Sina/Ebi, targeting TTK for degradation, may inhibit the transcription-activating activity of Su(H). It is thus attractive to speculate that Su(H) is the target of this complex that may not include all of its components and thus may not degrade Su(H) but only inhibit its activity required in a complex with NIC to activate D-Pax2. Such an interpretation is consistent with the observation that Su(H) levels in R1/R6/R7 are indistinguishable from those in cone cells. However, it cannot be excluded that other targets like NIC might be modified rather than degraded by the Phyl/Sina/Ebi complex, since the concentration of NIC, when expressed under the control of the sev enhancer, is independent of co-expression with Phyl (Shi, 2009).
The mechanism by which TTK blocks neuronal development is unclear. However, since TTK encodes a transcriptional repressor, it may repress one or several genes (Y in the model) that are required for neuronal development. Down-regulation of TTK would then derepress the Y genes, which act to specify neuronal development. One of the factors encoded by Y is the transcription factor Prospero (Pros), which is required for proper development of R7 since ectopic expression of TTK88 in R7 precursors abolishes the elevation of Pros. Since loss-of-function alleles of pros do not affect other R cells, other Y factors must exist (Shi, 2009).
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