Interactive Fly, Drosophila




Transcriptional control of the Drosophila terminal gap gene huckebein (hkb) depends on Torso (Tor) receptor tyrosine kinase (RTK) signaling and the Rel/NFB homolog Dorsal (Dl). Dl acts as an intrinsic transcriptional activator in the ventral region of the embryo, but under certain conditions, such as when it is associated with the non-DNA-binding co-repressor Groucho (Gro), Dl is converted into a repressor. Gro is recruited to the enhancer element in the vicinity of Dl by sequence-specific transcription factors such as Dead Ringer (Dri). The interplay between Dl, Gro and Dri on the hkb enhancer was examined and it was shown that when acting over a distance, Gro abolishes rather than converts Dl activator function. However, reducing the distance between Dl- and Dri-binding sites switches Dl into a Gro-dependent repressor that overrides activation of transcription. Both of the distance-dependent regulatory options of Gro -- quenching and silencing of transcription -- are inhibited by RTK signaling. These data describe a newly identified mode of function for Gro when acting in concert with Dl. RTK signaling provides a way of modulating Dl function by interfering either with Gro activity or with Dri-dependent recruitment of Gro to the enhancer (Hader, 1999).

The cis-acting element has been identified that mediates expression of the Drosophila gene hkb, which is necessary for terminal pattern formation and to size the mesoderm anlage in the blastoderm embryo. Deletion analysis of this element reveals a 162 base pair (bp) sub-element that integrates the activities of the Tor-dependent RTK signaling cascade and the morphogen Dl. This element, termed hkb ventral element (VE), comprises a 112 bp ventral activator element (VAE) and a 50 bp ventral repressor element (VRE) (Hader, 1999).

The VAE contains a Dl-binding site, identified in vitro, and mediates gene activation along the ventral side of the embryo. VAE-mediated gene expression is absent in embryos lacking Dl activity and extends throughout Toll10b mutants, in which Dl is present in all nuclei of the embryo. The expression pattern is not altered in embryos lacking snail and twist, the zygotic mediators of Dl. It is also not affected in embryos that lack Tor or express constitutively active TorY9, which causes RTK signaling throughout the embryo. In contrast, the VE fails to activate in the absence of Tor and mediates broad ventral expression in torY9 embryos not seen in the absence of Dl activity. This indicates that VAE mediates transcriptional activation by Dl, that the VRE, which by itself fails to activate transcription, is necessary to prevent Dl-dependent activation in the central region of the embryo, and that the activity of the unknown repressor, mediated by the VRE, is relieved by RTK signaling (Hader, 1999).

The evolutionarily conserved co-repressor Gro acts as a repressor of Dl activity, since both hkb expression and VE-driven gene expression expand along the ventral side of embryos lacking groucho (gro) activity. However, VAE-driven gene expression and the terminal expression domains of hkb are not significantly affected by lack of Gro. Thus, Gro functions as a repressor of VAE-directed, Dl-dependent transcriptional activation in the ventral region of the embryo and must act through the VRE (Hader, 1999).

Previous results have shown that Gro switches the transcriptional activator Dl into a potent silencer of transcription. This requires the formation of a multiprotein repressor complex of which Dl and Gro are obligatory components. Complex formation requires that Gro is recruited next to Dl by sequence-specific transcription factors such as Cut or Dri. Lack of either Gro or Dri activity results in VE-driven gene expression along the ventral axis of the embryo, indicating that both factors are necessary for repression of Dl-dependent activation. A single binding site has been identified for Dri in the VRE. Replacement of 5 bp in this site (VE-DRI) results in loss of repression in the central region of the embryo, indicating that Dri is necessary for recruitment of Gro to the VE (Hader, 1999).

The VE differs from the cis-acting elements of the genes zerknullt (zen) and decapentaplegic (dpp), both of which mediate long-range Dl-dependent transcriptional silencing by Gro. In these elements, binding sites for Dri and Dl are directly adjacent, whereas in the VE they are some 90 bp apart. This distance suggested the possibility that Gro cannot associate with Dl on the VE, implying that Gro must prevent Dl-dependent activation by a means other than formation of a long-range silencing complex, for example, by short-range quenching. This proposal was tested by monitoring gene expression patterns directed by a cis-acting activator element of the gene knirps (kni-element) to which the VRE, the VAE, the VE or molecularly defined variants of the VE were fused (Hader, 1999).

The kni-element drives gene expression throughout the embryo except in the posterior pole region. It mediates activation in response to the transcriptional activators Bicoid (Bcd) and Caudal (Cad) and acts in a Dl-independent fashion. Addition of the VRE to the kni-element does not cause ventral repression, nor does addition of the VE or the VAE. This indicates that within the VE, Gro abolishes the activator function of Dl instead of converting Dl into a long-range repressor that interferes with transcriptional activation by Bcd and Cad (Hader, 1999).

To investigate whether this action of Gro on Dl is determined by the arrangement of Dri- and Dl-binding sites in the VE, the transcription patterns driven by a modified VE-kni-element were examined in which the normal distance of 91 bp between the binding sites was reduced to 45 bp. This reduction results in Dl-dependent repression along the ventral side of wild-type embryos. Repression is not observed in the absence of Gro or Dl or in embryos expressing the constitutively active TorY9 protein. In contrast, the repression domain expands anteriorly in tor mutant embryos, which lack RTK signaling, and is found to be Dl-dependent. This suggests that the spatial arrangement of the Dl- and Dri-binding sites dictates the mechanism by which Gro and Dl act within the enhancer element. In one case, Dl is suppressed by Gro, in the other, Dl is converted into a potent silencer of transcription that can override activation by Bcd and Cad. Both modes of repression are controlled by Tor-dependent RTK signaling (Hader, 1999).

In the zen and dpp cis-acting elements, Gro causes Dl-mediated long-range silencing. Gro functions either by inhibiting the assembly and function of the core RNA polymerase II complex, by positioning nucleosomes over the core promoter and/or by recruiting the histone deacetylase Rpd3 to the template, where the enzyme can modulate local chromatin structure. However, in the VE, Gro only inhibits Dl-dependent activation without converting Dl into a repressor. The different modes of Gro function, that is, long-range silencing and short-range quenching, as shown here, are dependent on the distance between the Dl- and Dri-binding sites and/or their orientation on the enhancer, since shortening of the spacer distance converts the VE into a dpp- or zen-like element. This suggests that the way in which Gro regulates Dl activity depends on whether or not the two proteins can directly interact in vivo. Furthermore, both regulatory options of Gro on Dl are abolished by RTK signaling, a phenomenon that corresponds to the observation that Dl-dependent repression of dpp and zen is relieved by local Tor activity in the pole regions of the embryo. RTK-dependent phosphorylation may therefore interfere with the binding of Dri to the DNA template, the recruitment of Gro, or with both. Phosphorylation of the vertebrate Gro homolog TLE1 has been demonstrated, and many potential phosphorylation sites have been noted in Dri. Thus, local RTK-dependent phosphorylation may render one or both factors inactive, preventing Gro-dependent repression of Dl in the termini of the wild-type embryo (Hader, 1999).

These results establish that the cooperation between two maternal signaling systems, which determines the spatial limits of the Drosophila mesoderm anlage through hkb expression, is based on the management of the ubiquitously distributed factors Gro and Dri by local RTK signaling and that Gro can act through different modes on Dl. Lack of dead ringer (dri) activity does not result in an overt expansion of hkb expression on the ventral side of the embryo. However, as has been observed for VE-dependent gene expression, it causes only weak defects in mesoderm formation as compared with Gro-deficient embryos or embryos that express hkb under the control of the VAE. Thus, the interactions shown here represent only the Dri-dependent aspect of Gro's effect on hkb expression. The full picture of hkb control is likely to involve additional and redundantly acting factor(s) that recruit Gro to sites flanking the VE within the hkb control region (Hader, 1999).

Transcriptional Regulation

Activation of the receptor tyrosine kinase (RTK) torso defines the spatial domains of expression of the transcription factors tailless and huckebein in the posterior. Torso regulates tailless and huckebein through the elements of the ras pathway. Transcription of tll during early embryogenesis is apparently activated via a phosphorylation cascade initiated by Torso and the serine-threonine kinase Raf homolog pole hole. Corkscrew, a tyrosine phosphatase, in the Torso pathway also helps regulate tailless transcription. The MEK homolog (downstream of raf1) is involved in both positive and negative regulation of tailless and by extrapolation, of huckebein (Tsuda, 1993). Raf, required for expression of tailless and huckebein can be activated by the RTK in a Ras-independent pathway (Hou, 1995). huckebein anterior expression is controlled by Torso and by Bicoid through anterior and dorsal/ventral patterning systems (Bronner, 1994).

Mutations in torso and trunk that express low levels of the respective protein have differential affects on the expression of tailless and huckebein. For example a reduced amount of TRK can trigger signaling of TOR to levels required to activate tll but not hkb. For a given number of TOR receptors, an increase in the amount of TRK results in the appearance of more structures of the most posterior segment (A8) (Furriols, 1996).

The terminal portions of the Drosophila body pattern are specified by the localized activity of the receptor tyrosine kinase Torso (Tor) at each pole of the early embryo. Tor activity elicits the transcription of two 'gap' genes, tailless (tll) and huckebein (hkb), in overlapping but distinct domains by stimulating the Ras signal transduction pathway. Quantitative variations in the level of Ras activity can specify qualitatively distinct transcriptional and morphological responses. Low levels of Ras activity at the posterior pole direct tll but not hkb transcription; higher levels drive transcription of both genes. Correspondingly, low levels of Ras activity specify a limited subset of posterior terminal structures, whereas higher levels specify a larger subset. When a constitutively active 1X RasV12 gene is expressed in torso mutant embryos, brachyenteron (byn) is expressed in a small terminal cap, whereas the domain of expression in 2X RasV12 is much broader. Because both the activation and repression of terminal byn expression is known to depend, respectively, on tll and hkb, it is surmised that higher levels of Ras activity are required at the posterior of wild-type (ras+) embryos to drive sufficiently high levels of Hkb expression to repress byn expression (a phenomenon not observed with even 2X RasV12 ectopic expression). 1X RasV12 forms the least terminal of the posterior terminal structures: the eighth abdominal dentical band and the posterior spiracles. The extent of restoration is considerably greater in 2X RasV12 embryos: these form additional terminal structures such as the anal tuft and anal pads.The response to Ras activity is not uniform along the body. Instead, levels of Ras activity that suffice to drive tll and hkb transcription at the posterior pole fail to drive their expression in more central portions of the body, apparently due to repression by other gap gene products. The levels of Huckebein and/or Kruppel through the embryo might be responsible for a failure to express hkb in response to moderate RasV12 activity. It is concluded that tll and hkb transcription, as well as the terminal structures, are specified by two inputs: a gradient of Ras activity, which emanates from the pole, and the opposing influence of more centrally deployed gap genes, which repress the response to Ras (Greenwood, 1997).

capicua is involved in gene repression in Drosophila terminal and dorsoventral patterning. Given the similarities between torso gain of function mutations (torgof) and cic1 phenotypes, the expression patterns of tailless and huckebein were examined in capicua1 (cic1) embryos. Expression of both genes expands toward the center of such embryos, predominantly in the posterior domain. The expanded expression of tll and hkb is very similar to that observed in torgof and groucho mutant embryos. The expression of a lacZ transgene under the control of a tor-RE from the tll promoter that drives terminal-specific transcription has also been examined. In cic1 mutant embryos, expression of this construct is derepressed toward the middle of the embryo. Together, these results suggest that the cic gene is normally required to restrict tll and hkb expression to the embryonic poles (Jimenez, 2000).

cic could affect tll and hkb expression by restricting Tor signaling to the embryonic poles (e.g., by limiting the domain of Tor receptor activation, or the domain of Tor signal transduction inside of the embryo). Alternatively, cic could function, like gro, as a repressor of tll and hkb downstream of the Tor pathway. To help distinguish between these possibilities, epistasis analyses were performed using loss-of-function mutations in tor, Draf, and Dsor (encoding a Drosophila MAPK kinase homolog) that normally cause a phenotype complementary to that of cic1, that is, absence of terminal structures. Embryos from females homozygous for cic1 and tor are identical to those from cic1 females alone. Likewise, cic1 females carrying loss-of-function clones of Draf or Dsor in the germ line produce embryos that display the cic phenotype. Thus, cic acts genetically downstream of Draf and Dsor. In addition, the domain of Tor signal activity was examined directly using a monoclonal antibody against the active, diphosphorylated form of Drosophila MAPK (known as Erk) and a normal pattern of Erk activation was found in cic1 embryos. This shows that derepression of tll and hkb in cic1 mutant embryos is not due to an expanded domain of Tor signaling, suggesting that cic is part of the activity that represses tll and hkb in the central region of the embryo and is inhibited by Tor signaling at the embryonic poles (Jimenez, 2000).

The similar effects of cic and gro on terminal patterning raise the possibility that cic is necessary for Gro corepressor activity in general. However, two lines of evidence argue against this idea: (1) Gro participates in many developmental processes, whereas the role of cic appears restricted to terminal and dorsoventral patterning; (2) Gro-dependent repression by Hairy in a sex determination assay does not require cic function. These results indicate that cic does not generally affect Gro activity (Jimenez, 2000).

In Drosophila, the gradient of the Bicoid (Bcd) morphogen organizes the anteroposterior axis while the ends of the embryo are patterned by the maternal terminal system. At the posterior pole, expression of terminal gap genes is mediated by the local activation of the Torso receptor tyrosine kinase (Tor). At the anterior, terminal gap genes are also activated by the Tor pathway but Bcd contributes to their activation. Evidence is presented that Tor and Bcd act independently on common target genes in an additive manner. Furthermore, the terminal maternal system is shown not to be required for proper head development, since high levels of Bcd activity can functionally rescue the lack of terminal system activity at the anterior pole. This observation is consistent with a recent evolution of an anterior morphogenetic center consisting of Bcd and anterior Tor function (Schaeffer, 2000).

In the posterior region of the embryo, the tor pathway activates the zygotic effectors tll and hkb, which are sufficient to specify the most posterior anlagen and the gut of the larva. At the anterior, the function of the terminal system is more difficult to interpret and, in tor mutants, hkb expression is only reduced. It actually requires bcd;tsl double mutants to lose all anterior hkb expression, which indicates additive functions of the anterior and terminal systems on this common target gene. hkb seems particularly interesting in this context, as its function is required for the formation of the labrum: reduction of hkb expression, as observed in terminal mutant background leads to the deletion of this particular structure (Schaeffer, 2000).

Therefore, it was asked whether the rescue of anterior structures (e.g. the labrum) mediated by high levels of Bcd in terminal system mutants is correlated with the restoration of the hkb expression pattern. Expression of hkb is first detected in the terminal regions (anterior and posterior) of the syncytial blastoderm. In terminal mutant embryos, the posterior domain is absent, whereas the anterior domain is reduced. In a tsl background with four or six copies of bcd, however, hkb expression extends further towards the posterior. Hence, the level of hkb expression can be regained by increasing the levels of Bcd in a terminal system mutant, even though its exact expression domain cannot be restored. It is likely that fate-map shifts are able to absorb the slightly changed expression domain of hkb. This suggests that the lack of terminal system activity at the anterior can simply be overcome by another system through enhancement of transcriptional activation of common target genes (Schaeffer, 2000).

Tor has been shown to antagonize Groucho-mediated repression of genes such as hkb and tll, probably by acting on the HMG-box transcription factor Capicua. Therefore, it is likely that Tor enhances Bcd activity by derepression, i.e. the inactivation of potential repressors of Bcd target genes, and thereby rendering any transcriptional activator more potent. As the cis-regulatory control regions of most developmental genes comprise both repressor and activator sites, the inactivation of potential repressors should lead to enhanced expression, or enlarged expression domains, as observed for several bcd target genes in a tor gain-of-function background (Schaeffer, 2000).

The hedgehog gene product, secreted from engrailed-expressing neuroectoderm, is required for the formation of post-S1 neuroblasts in rows 2, 5 and 6. The Hedgehog protein functions not only as a paracrine but also as an autocrine factor and its transient action on the neuroectoderm 1-2 hours (at 18¡C) prior to neuroblast delamination is necessary and sufficient to form normal neuroblasts. In contrast to epidermal development, hedgehog expression required for neuroblast formation is regulated by neither engrailed nor wingless. hedgehog and wingless, at virtually the same time, bestow composite positional cues on the neuroectodermal regions for S2-S4 neuroblasts and, consequently, post-S1 neuroblasts in different rows can acquire different positional values along the anterior-posterior axis. As with wingless, huckebein expression in putative proneural regions for certain post-S1 neuroblasts is under the control of hedgehog. hedgehog and wingless are involved in separate, parallel pathways and loss of either is compensated for by the other in NB 7-3 formation (Matsuzaki, 1996).

How is neuroblast-specific gene expression established? This paper's focus was on the huckebein gene, because it is expressed in a subset of neuroblasts and is required for aspects of neuronal and glial determination. hkb is required within the neuroblast 1-1, 2-2 and 4-2 lineages for proper axon pathfinding of interneurons and motoneurons and for proper muscle target recognition by motoneurons. In addition, hkb is required for expression of repo in A and B glia derived from the neuroblast 1-1 lineage, for expression of even-skipped in the RP2 motorneuron derived from the NB 4-2 lineage, and is required for the production of serotonin in neurons derived from the NB 7-3 lineage.

Huckebein is a nuclear protein first detected in small clusters of neuroectodermal cells and then in a subset of neuroblasts. The temporal pattern of Hkb is highly stereotyped. Each half segment is divided into three longitudinal columns and seven transverse rows. At stage 8, hkb is detected in small clusters of neuroectodermal cells in medial rows 1/2; later it is found in row 7 and intermediate rows 4/5, and still later in a few cells in row 5. The small early clusters expand to give rise to cells that express hkb later and each region gives rise to the different classes of neuroblasts in which Hkb is expressed. The secreted Wingless and Hedgehog proteins activate huckebein expression in distinct but overlapping clusters of neuroectodermal cells and neuroblasts, whereas the nuclear Engrailed and Gooseberry proteins repress huckebein expression in specific regions of neuroectoderm or neuroblasts. Hedgehog activates hkb in rows 1/2 and 7 (giving rise to the 5HT expressing NB 7-3 lineage), while Wingless activates hkb in row 4 (giving rise to the eve expressing NB 4-2 motorneuron lineage). Wingless and Hedgehog activate hkb in the neuroectoderm of row 5. Early-forming neuroblasts of rows 5 and 6 never express hkb even though they develop from Hkb+ neuroectoderm (row 5). Gooseberry functions to repress hkb expression in row 5 neuroblasts while Engrailed represses hkb expression in rows 6/7 neuroectoderm. Integration of these activation and repression inputs is required to establish the precise neuroectodermal pattern of huckebein, which is subsequently required for the development of specific neuroblast cell lineages (McDonald, 1997).

During early Drosophila and C. elegans development, the germ cell precursors undergo a period of transcriptional quiescence. Germ cell-less (Gcl), a germ plasm component necessary for the proper formation of 'pole cells', the germ cell precursors in Drosophila, is required for the establishment of this transcriptional quiescence. While control embryos silence transcription prior to pole cell formation in the pole cell-destined nuclei, this silencing does not occur in embryos that lack Gcl activity. The failure to establish quiescence is tightly correlated with failure to form the pole cells. Furthermore, Gcl can repress transcription of at least a subset of genes in an ectopic context, independent of other germ plasm components. These results place Gcl as the earliest gene known to act in the transcriptional repression of the germline. Gcl's subcellular distribution on the nucleoplasmic surface of the nuclear envelope (Jongens, 1994) and its effect on transcription suggest that it may act to repress transcription in a manner similar to that proposed for transcriptional silencing of telomeric regions (Leatherman, 2002).

gcl is required to repress transcription during the establishment of the germ cell lineage. To determine if this activity is dependent or independent of other germ plasm components, the effect of ectopically localizing Gcl on transcription was examined. Replacement of the 3'UTR of the gcl transcript with the 3'UTR of bicoid results in the anterior localization of gcl mRNA and protein to the anterior pole of the embryo. In these 'hgb' embryos, a slightly variable but consistent decrease was found in the intensity of H5 staining (H5 is a monoclonal antibody that recognizes a phosphorylated form of RNA polymerase II that is associated with active transcription) in the anterior nuclei compared to control embryos throughout the syncytial blastoderm stage, and this decrease indicates that Gcl is sufficient to repress transcription ectopically. However, the anterior expression of Gcl clearly does not lead to complete silencing of the anterior nuclei, since some H5 staining persists (Leatherman, 2002).

The reduced H5 staining observed in the anterior of the hgb embryos could be due to global partial repression of all genes, or it could result from a specific subset of genes being severely repressed while others are unaffected. To distinguish between these possibilities, the expression was examined of specific genes whose expression pattern includes the anterior of the embryo, including sisA, sisB (scute), tailless, huckebein, hunchback, and knirps. These genes are all independently activated by maternally contributed factors, so any effects on their transcription are likely to be direct rather than a consequence of an earlier defect. By using in situ hybridization, it was found that the early anterior expression domains of sisA, sisB, tailless, and huckebein are severely repressed in all of the hgb embryos examined, but no effect was seen on hunchback and knirps expression. These data suggest that the transcriptionally repressive effect of Gcl is not global, but rather specific to a subset of genes. Gcl is also present in a variety of tissues later in development, at times when transcription is active, which further suggests a non-global mode of silencing (Leatherman, 2002).

Formation of tubes of the correct size and shape is essential for viability of most organisms, yet little is understood of the mechanisms controlling tube morphology. A new allele of hairy has been identified in a mutagenesis screen. hairy mutations cause branching and bulging of the normally unbranched salivary tube, in part through prolonged expression of huckebein (hkb). Hkb controls polarized cell shape change and apical membrane growth during salivary cell invagination via two downstream target genes, crumbs (crb), a determinant of the apical membrane, and klarsicht (klar), which mediates microtubule-dependent organelle transport. In invaginating salivary cells, crb and klar mediate growth and delivery of apical membrane, respectively, thus regulating the size and shape of the salivary tube (Myat, 2002).

Capicua integrates input from two maternal systems in Drosophila terminal patterning

In Drosophila, the maternal terminal system specifies cell fates at the embryonic poles via the localised stimulation of the Torso receptor tyrosine kinase (RTK). Signalling by the Torso pathway relieves repression mediated by the Capicua and Groucho repressors, allowing the restricted expression of the zygotic terminal gap genes tailless and huckebein. This study reports a novel positive input into tailless and huckebein transcription by maternal posterior group genes, previously implicated in abdomen and pole cell formation. Absence of a subset of posterior group genes, or their overactivation, leads to the spatial reduction or expansion of the tailless and huckebein posterior expression domains, respectively. The terminal and posterior systems converge, and exclusion of Capicua from the termini of posterior group mutants is ineffective, accounting for reduced terminal gap gene expression in these embryos. It is proposed that the terminal and posterior systems function coordinately to alleviate transcriptional silencing by Capicua, and that the posterior system fine-tunes Torso RTK signalling output, ensuring precise spatial domains of tailless and huckebein expression (Cinnamon, 2004).

Terminal gap gene expression must be tightly regulated for the correct specification of terminal cell fates at the nonsegmented poles. Clearly, the Tor pathway plays a key role in driving tll and hkb transcription, given that terminal gap genes are not expressed at the posterior end of terminal group mutants, and as a result terminal structures such as the terminal filzkorper (FK) do not form. In this paper, a novel biological role is unraveled for the maternal posterior system, showing that members of this group, in particular Nos, positively regulate transcription of the zygotic subordinate genes of the terminal system. Torso response elements (TREs) in the tll upstream regulatory region, which are derepressed in cic mutants, also respond to alterations in maternal osk dosage, and the Cic repressor is not excluded from the termini of posterior group mutants. These results are consistent with the posterior system feeding into the Tor signalling pathway, upstream of or at the level of the Cic repressor. It is suggested that the concerted activities of both the terminal and posterior systems, in their spatially overlapping zones of action, generate accurate domains of terminal gap gene expression at the posterior (Cinnamon, 2004).

It was originally proposed that the four maternal systems that pattern the early Drosophila embryo act largely independently of each other. Recent work, however, demonstrated interactions between the Tor pathway and the anterior and D/V systems. For example, tll has been shown to respond to the anterior determinant Bicoid (Bcd) even when Tor signalling is genetically blocked. Indeed, cis-acting DNA elements responsive to these three maternal systems have been found in the tll upstream regulatory region. The current results now link the terminal and posterior systems, previously thought to be independent of each other, in terminal gap gene regulation, reinforcing the idea that maternal systems that pattern the early embryo act in a coordinated manner (Cinnamon, 2004 and references therein).

Why has the positive input, by posterior group genes into terminal patterning, been largely overlooked to date? Classical segmentation studies mostly involved phenotypic analyses at the cuticular level. For this reason, and when taking into account the primary contribution of the terminal system, the delicate input by the posterior group has gone unnoticed. Thus, the unextended FK that develops in posterior group mutant background, which may arise from decreased terminal gap gene expression, had largely been attributed to pleiotropic effects arising from abdominal defects. It has been possible to detect the relatively subtle changes in tll and hkb gene expression patterns only by investigating terminal gap gene regulation at the molecular level. In fact, at least one other molecular study had previously reported reduced terminal gap gene expression in osk mutant embryos (Cinnamon, 2004 and references therein).

One emerging concept is that, for the refinement of the expression levels and spatial extents of RTK signalling targets, it is also imperative to integrate accurately information originating from other, non-RTK sources. In many cases this integration occurs at the level of target gene enhancers, with various effectors of distinct signalling pathways binding to specific DNA elements to regulate transcription. For example, D-Pax2 expression in the cone and pigment cells of the developing eye is regulated by effectors of the EGFR RTK pathway, such as Pointed P2 and Yan, and also by the Notch signalling component Suppressor of Hairless, as well as by the transcription factor Lozenge. The current study shows that terminal gap gene expression requires not only Tor RTK pathway activity but also a contribution from the posterior system. In this instance, inputs from these two maternal coordinate systems are interpreted and linked not at the level of terminal gap gene promoters but at the level of the Cic repressor. Thus, Cic functions as an integrator of multiple regulatory inputs, with both the posterior and terminal systems acting to relieve transcriptional silencing mediated by this repressor (Cinnamon, 2004).

Surprisingly, anterior tll and hkb expression is also reduced in posterior group mutants. Similarly, others have reported prolonged bcd expression and head defects in pum mutants. It is speculated that low levels of Osk and Nos, which escape translational repression, similarly regulate terminal gap gene expression via Cic removal at the anterior. In accordance with this, the dismissal of Cic from the anterior pole of posterior group mutants is also ineffective (Cinnamon, 2004).

How does Nos, which has been assigned the role of a translational repressor, positively regulate tll and hkb transcription? The results suggest that Nos does so indirectly, by downregulating the accumulation of the Cic repressor at the termini. The exact mechanism by which the Tor pathway mediates the exclusion of Cic from terminal regions has not been established, but one model argues that phosphorylation of Cic by MAPK causes degradation of the protein, as in the case of Yan. Thus, Nos could be affecting this process in one of several possible ways, at the level or downstream of MAPK. For example, Nos could be facilitating the translocation of phosphorylated MAPK into the nucleus. In posterior group mutants, then, activated MAPK would remain in the cytoplasm rather than enter the nucleus, impeding Cic phosphorylation and degradation. Alternatively, Nos may be modulating MAPK activity, or regulating adaptor proteins that promote Cic phosphorylation by nuclear MAPK. Nos may also be controlling the translation of factors that are involved in the nuclear trafficking (import/export) or degradation of Cic, or perhaps may even be acting on the cic message itself. Future studies will distinguish between these possibilities, and may shed new light on the molecular mechanisms underlying role of Nos in other developmental processes, for example, the establishment/maintenance of transcriptional quiescence in pole cells. The positive input by the posterior group genes is viewed as evolving to modulate terminal pathway activity, merging with other varied modes of Tor regulation to ultimately ensure accurate tll and hkb expression and, consequently, precise cell fate determination (Cinnamon, 2004).

The Tor signal transduction pathway is under multiple tiers of regulation, outside and inside the nucleus. For instance, internalisation and trafficking of the activated Tor receptor to the lysosome for degradation attenuates the signal, as evident by the spatial broadening and temporal prolonging of Tor activation in mutants for hrs, a component of the endosomal recycling machinery (Lloyd. 2002). Yet another level of control is provided by the tyrosine phosphatase corkscrew, which sharpens the gradient of Tor activity. Additionally, multiple cytoplasmic adaptor proteins take part in transducing the Tor signal, conceivably buffering against surplus or deficiency in signalling (Cinnamon, 2004).

In the nucleus, tll and hkb are subjected to silencing by several repressors. Derepression of tll is observed in grainy-head and tramtrack69 (ttk69) mutants, and the proteins encoded by these genes bind tll promoter sequences. Cic and Gro appear to play a leading role in terminal gap gene silencing, given that mutations in cic and gro bring about a significant expansion of the tll and hkb expression domains. Intriguingly, however, tll expression never reaches the middle of the embryo in these mutants. tll is uniformly expressed, albeit weakly, throughout the embryo only when both the developmental corepressors Gro and CtBP are removed concomitantly. This broadened tll expression likely stems from the fact that there is a redundancy in the activities that normally restrict terminal gap gene transcription from inappropriately spreading into the central portion of the embryo; by jointly removing the Gro and CtBP coregulators, activity of the above repressors is compromised. Alternatively, CtBP might be acting in conjunction with a novel, unidentified repressor that prevents tll transcription in the middlemost region of the embryo (Cinnamon, 2004).

So what is the purpose of the input by the posterior group genes into tll and hkb transcription? Quantitative differences in Tor receptor activity have to be eventually interpreted and translated into distinct cell fates at the termini. Strong Tor activation induces both hkb and tll expression, whereas weaker Tor activation only brings about tll expression. It is surmised that the precision endowed by the Tor RTK cascade may not suffice for the complex patterning of the termini, given that mere two-fold fluctuations in Tor signalling result in defective embryonic development. For example, mutants with reduced Tor RTK activity show partial tll expression and the complete loss of hkb. These mutants consequently develop incomplete terminal structures and die at the larval stage. Conversely, overactivation of the Tor pathway leads to anterior expansion of the posterior tll expression domain, perturbing segmentation in central body parts, likely as a result of downregulation of abdominal gap genes by the Tll protein. Thus, the precise spatial confinement of terminal gap gene expression domains requires the coordinated integration of regulatory inputs, coming from two maternal systems and converging on the same effector protein, Cic (Cinnamon, 2004).

Targets of Activity

HKB sets the anterior and the posterior borders of the ventral furrow, but employs different modes of regulation. In the posterior part of the blastoderm, HKB represses the expression of snail in the endodermal primordium. In the anterior part, HKB antagonizes the activation of target genes by Twist and SNA. Here, Bicoid permits the co-expression of hkb, sna and twi, which are all required for the development of the anterior digestive tract. Mesodermal fate is determined in locations where no hkb is expressed but wheresna and twi are expressed. In the anterior, HKB and SNA together determine endodermal fate. HKB together with SNA and TWI are required for foregut development (Reuter, 1994b).

Both ectopic huckebein and tailless are able to repress the activity of the central gap gene giant, thus interfering with segmentation in the ectodermal trunk region (Bronner, 1994).

Anterior repression of orthodenticle is carried out by Huckebein which in turn receives input for the torso system, from Dorsal and from Bicoid. Dorsal functions in the anterior repression of otd expression. The repression function of Dorsal is mediated, at least in part, through Huckebein, since anterior hkb expression is lost in dorsal mutants. Contrary to early models of embryonic pattern formation, high levels of Bicoid are not required for otd activation or for the establishment of anterior head structures (Gao, 1996).

The gene serpent plays a key role in the development of the gut endoderm. serpent mutant embryos lack the entire midgut and do not show endodermal differentiation. serpent appears to act as a homeotic gene downstream of huckebein to promote morphogenesis and differentiation of anterior and posterior midgut (Reuter, 1994a).

Terminal gap genes tailless and huckebein direct the formation of the posterior hunchback stripe. The TLL protein binds in vitro to specific sites within the 1.4 kb posterior enhancer region, providing the first direct evidence for activation of gene expression by TLLl. The anterior border of the posterior hb stripe is determined by TLL concentration in a manner analogous to the activation of anterior hb expression by Bicoid (Margolis, 1995).

The effects of mutations in five anterior gap genes (hkb, tll, otd, ems and btd) on the spatial expression of segment polarity genes wg and hh were analyzed at the late blastoderm stage and during subsequent development. Both wg and hh are normally expressed at blastoderm stage in two broad domains anterior to the segmental stripes of the trunk region. At the blastoderm stage, each gap gene acts specifically to regulate the expression of either wg or hh in the anterior cephalic region: hkb, otd and btd regulate the anterior blastoderm expression of wg, while tll and ems regulate hh blastoderm expression (Mohler, 1995).

huckebein regulates aspects of ganglion mother cell and neuronal identity required for proper motoneuron axon pathfinding in the NB 4-2 lineage. It is expressed in a subset of Drosophila CNS precursors, including the NB 4-2/GMC 4-2a/RP2 cell lineage. In huckebein mutant embryos, GMC 4-2a does not express the cell fate marker even-skipped. Conversely, huckebein overexpression produces a duplicate even-skipped-positive GMC 4-2a. Loss of huckebein does not affect the number, position, or type of neurons in the NB 4-2 lineage; however, all motoneurons show axon pathfinding defects and never terminate at the correct muscle (Chu-LaGraff, 1995).

By examining expression of arc in different mutant embryos, it was determined that transcription factors known to be required for patterning and maintenance of various developing epithelia control arc expression in those domains. tll and hkb, which are required to pattern the posterior 15% of the embryo, control arc expression in the posterior midgut primordium. fkh, which appears to act as a maintenance, or permissive, transcription factor, is required for expression of arc throughout the gut. byn, which is required for hindgut development and specifies its central domain (the large intestine), controls expression of arc in the elongating hindgut. Kr and cut, required for evagination and extension of the Malpighian tubule buds control expression of arc in the tubule primordia (Liu, 2000).

Identification and expression of Ima, a novel Ral-interacting Drosophila protein; Ima is a target of Hkb

This study reports the identification of Ima (FlyBase name: Magi), a novel Drosophila MAGUK-like protein, which contains two WW and four PDZ protein interaction domains and interacts with the small GTPase dRal in the yeast two-hybrid system and pull-down assays. The gene is expressed in distinct spatiotemporal patterns throughout embryonic development. Overexpression of Ima interferes with normal Drosophila development, indicating that the gene functions in a tissue specific manner (Beller, 2002).

The finding that ima is expressed in the posterior terminal region of the embryo, which is established in response to torso-dependent Ras/Raf signaling, prompted an inquiry as to whether and how ima expression is controlled by the activity of this pathway. ima expression was examined in embryos that lack the activity of key components of the maternal terminal organizer system or its mediators such as the terminal gap genes tailless and huckebein. Embryos from females which were homozygous for the torso loss-of-function allele torpm failed to express the posterior terminal ima expression domain, whereas the dorsal expression domain of ima was not affected. This result shows that activated components of the torso signaling pathway are necessary for the activation of posterior terminal ima gene expression (Beller, 2002).

In order to place ima within the torso-dependent signaling cascade, it was asked whether ima is a direct control target of the maternal components of the pathway or whether its expression is regulated in response to their zygotic mediators, namely the zygotic gap genes tailless and huckebein. Posterior terminal expression was unchanged in embryos homozygous for the tailless loss-of-function allele taillessl10–22 but absent from embryos homozygous for a huckebein loss-of-function allele (hkb2). Posterior terminal ima expression is not affected in homozygous hindsight mutant embryos, which lack a transcription factor that acts downstream and in response to huckebein activity. torso-dependent control of ima expression is therefore mediated by huckebein activity and represents a direct or indirect target of the huckebein encoded transcription factor and does not require hindsight activity (Beller, 2002).

In order to test whether lack of ima activity interferes, for example, with huckebein-dependent aspects of embryonic development, homozygous embryos were examined for the deletion Df(2R)CC2 which uncovers the genomic region 2R 57C2-57C5 as assessed by in situ hybridization to polytene salivary gland chromosomes. It deletes ima (2R 57C2,3) as well as several other genes. Homozygous Df(2R)CC2 embryos develop no obvious body patterning defects but die in the egg shell. These observations indicate that the lack of ima does not cause a scorable morphological defect (Beller, 2002).

ima gain-of-function experiments were performed by employing the Gal4/UAS system to examine the effect of a UAS-dependent Ima expression. Using the T80-Gal4 and daG32-Gal4 drivers, ima was ubiquitously expressed. In addition, ima was expressed in en-Gal4-driven stripes along the longitudinal axis of gastrulating embryos and by responding to the C155 elav-Gal4 driver in neurons. en stripe expression of ima had no scorable effect on development, whereas ubiquitous and neural overexpression of Ima consistently caused a rough eye phenotype and supernumerary, misoriented bristles on the notum. None of these effects were observed when Ima deletion mutants lacking either the WW domains or the PDZ domains were expressed under otherwise identical conditions as full-size Ima. Collectively, these observations indicate that misexpression of Ima interferes in a tissue-specific manner with normal Drosophila development and that this activity requires both the WW and the PDZ domains (Beller, 2002).

Specification of motoneuron fate in Drosophila: Integration of positive and negative transcription factor inputs by a minimal eve enhancer

The mechanisms that generate neuronal diversity within the Drosophila central nervous system (CNS), and in particular in the development of a single identified motoneuron called RP2, are of great interest. Expression of the homeodomain transcription factor Even-skipped (Eve) is required for RP2 to establish proper connectivity with its muscle target. The mechanisms by which eve is specifically expressed within the RP2 motoneuron lineage have been examined. Within the NB4-2 lineage, expression of eve first occurs in the precursor of RP2, called GMC4-2a. A small 500 base pair eve enhancer has been identified that mediates eve expression in GMC4-2a. Four different transcription factors (Prospero, Huckebein, Fushi tarazu, and Pdm1) are all expressed in GMC4-2a, and are required to activate eve via this minimal enhancer; one transcription factor (Klumpfuss) represses eve expression via this element. All four positively acting transcription factors act independently, regulating eve but not each other. Thus, the eve enhancer integrates multiple positive and negative transcription factor inputs to restrict eve expression to a single precursor cell (GMC4- 2a) and its RP2 motoneuron progeny (McDonald, 2003).

GMC4-2a forms at stage 9, becomes Eve+ at stage 11, and generates the Eve+ RP2/sib neurons at late stage 11. The second-born Eve-negative GMC4-2b forms at stage 10, and generates an unknown pair of neurons. The first transcription factors detected in GMC4-2a are Pros and Hkb, due to inheritance of the proteins from the neuroblast. The next transcription factors detected in GMC4-2a are Ftz and Pdm1. Ftz is first detected at stage 10, and Pdm1 is first detected at stage 11. The de novo expression of Pdm1 is distinct from its inheritance in GMCs produced by Pdm+ neuroblasts during the assignment of temporal identity. The last protein to be detected is Eve, which appears only at late stage 11. Pros, Hkb, Ftz, and Pdm1 are each expressed transiently in the RP2/sib neurons at stage 12, but by stage 16 none of these proteins is detectable in the mature RP2 neuron. It is concluded that there is a temporal sequence of transcription factor expression in GMC4-2a: first Pros and Hkb, then Ftz, then Pdm1, and that Eve is detected only after all of these proteins are present (McDonald, 2003).

GMC4-2b forms at late stage 10, never expresses Eve, and generates two unknown Eve-negative neurons. Three transcription factors that positively regulate eve expression are detected in GMC4-2b: Pros, Ftz, and Hkb. The pattern of Pdm1 expression is too complex to score at the time GMC4-2b is born. The negative regulator Klu is detected in GMC4-2b but not GMC4-2a. It is concluded that GMC4-2b expresses at least three of the four positively acting transcription factors that are required to activate eve (Pros, Ftz, Hkb), and at least one negative regulator of eve expression (Klu). The absence of eve expression is likely due to the presence of Klu, rather than the absence of a positive regulator, because klu mutants can activate eve transcription in GMC4-2b (McDonald, 2003).

The sequential expression of Pros, Hkb, Ftz, Pdm1, and Eve in GMC4-2a raises the possibility that these four transcription factors act in a linear pathway to regulate eve expression. If so, then a mutant in an early-acting gene should lead to loss of expression of all later-acting genes in the pathway. Alternatively, the four transcription factors could all act directly to activate eve transcription, with expression of eve occurring only after all transcription factors are present. In this case, mutants in one gene should have no effect on any other gene except eve. To distinguish between these two models, pros, hkb, ftz, and pdm1 mutants were examined for expression of all four transcription factors and eve. Pdm1 is detected in GMC4-2a in all mutant genotypes: Ftz is detected in GMC4-2a in all mutant genotypes: pros, hkb, and pdm1, and Hkb is detected in GMC4-2a in all mutant genotypes. Finally, Pros is observed in GMC4-2a in all mutant genotypes, as expected because Pros is transcribed and translated in neuroblasts and is asymmetrically partitioned into each GMC. Taken together, these data support the model that all four transcription factors act directly to activate eve transcription, with expression of eve occurring only after all transcription factors are present (McDonald, 2003).

To test the model that Pros, Hkb, Ftz, and Pdm1 transcription factors directly regulate eve expression, the eve cis-regulatory DNA that confers regulated expression in the NB4-2 lineage was identified. Eve is expressed in a subset of neurons in the embryonic CNS, including the aCC/pCC neurons derived from NB1-1, the U1-5 neurons derived from NB7-1, the EL neurons derived from NB 3-3, and the RP2/sib neurons derived from NB4-2. An eve cis-regulatory element [R79R92; from ~7.9 and ~9.2 kilobase pair (kb) on the eve genomic map] has been defined that accurately directs lacZ expression to the Eve+ cells within two NB lineages: GMC4-2a and its RP2 progeny and GMC1-1a and its aCC/pCC progeny. The properties of this element are examined in this study in detail. When the R79R92 eve element was truncated to ~7.9 to ~8.6 kb (R79N86), lacZ expression in RP2 and aCC was normal, whereas expression in the pCC neuron was reduced. Truncation of the eve element to ~7.9 to ~8.4 kb (R79S84) almost completely abolished expression of lacZ in pCC, although occasionally expression in pCC was observed at low levels, whereas expression in RP2 and aCC remained high. Further truncation of the left end point to ~8.0 kb (S80S84) resulted in a reduction of expression in both aCC and RP2. Addition of the region ~8.4 to ~8.6 kb to this fragment (S80N86) increased the level of expression. However, because the region ~8.4 to ~9.2 kb (S84R92) did not show any ability to activate lacZ, the region ~8.4 to ~8.6 kb is apparently insufficient on its own to direct expression, and thus serves an auxiliary function. The removal of ~8.2 to ~8.4 kb from P80N86 abolished expression (SNdeltaSC). Together with the fact that each of the fragments ~7.9 to ~8.2 kb (S79C82) and ~8.2 to ~9.2 kb (C82R92) failed to activate lacZ, this indicates that both of the regions ~7.9 to ~8.2 kb and ~8.2 to ~8.4 kb are necessary to direct expression, and that neither alone is sufficient. Consistent with this, two tandem copies of ~8.2 to ~8.4 kb failed to activate lacZ (C82S84x2), suggesting that the two regions may provide qualitatively different activities. In summary, the critical eve cis-regulatory element for the GMC4-2a and RP2 lies in a 0.5 kb fragment of genomic DNA between ~7.9 and ~8.4 kb (McDonald, 2003).

Do the genes that activate or repress eve expression in the NB4-2 lineage work through the minimal 500 bp RP2/aCC eve enhancer? Expression of R79S84-lacZ was assayed in pros, ftz, hkb, pdm1, and klu mutant embryos, and whether it was regulated identically to the endogenous eve gene was tested. ftz, pdm1, and hkb mutant embryos show loss of R79S84-lacZ in the RP2 neuron but not the aCC neuron, identical to the pattern of endogenous eve expression in these mutants. pros mutants show loss of eve-lacZ in both RP2 and aCC, identical to the pattern of endogenous eve expression in pros mutants. In embryos lacking klu, R79S84-lacZ is expressed in two cells at the RP2 position, whereas expression in aCC is normal; this matches the pattern of endogenous eve expression in klu mutant embryos. It is concluded that the R79S84 minimal eve cis-regulatory element precisely reproduces the pattern of endogenous eve expression within the NB4-2 lineage, and that transcription factors regulating eve in GMC4-2a can act through this enhancer to activate or repress eve expression (McDonald, 2003).

Expression of eve is not detected in GMC4-2b in wild-type embryos, but mutations in the klu gene result in ectopic expression of eve in GMC4-2b. Klu contains four predicted zinc fingers, one of which is highly homologous to the WT1 zinc finger domain. The consensus binding site for the WT1 zinc finger transcription factor is a ten nucleotide sequence, 5'-(C/G/T)CGTGGG( A/T)(G/T)(T/G)-3', with variable nucleotides shown in parentheses. It was reasoned that if Klu directly binds to the eve enhancer to repress expression in GMC4-2b, one or more WT1 consensus binding sites should be found in the minimal eve enhancer R79S84. Three conserved putative Klu-binding sites were found in the R79S84 sequence: site 1, GGGTGGGGAG at nucleotides ~8066 to ~8075; site 2, GCGTGGGTGA at nucleotides ~8090 to ~8099; and site 3, TCGCCCACCA at ~8262 to ~8271. Based on the fact that altering the C2, G3, G5, G6, and G7 to T or T4 to A in the WT1-consensus binding site abolished WT1 binding, nucleotide substitutions were made in the three putative Klu-binding sites. In sites 1 and 2, As were substituted for T4, G6, and G7. In site 3, which is a reversed binding site, Ts were substituted for C4, C6, and A7. These substitutions were made at all three sites; transgenic lines were constructed expressing the mutant enhancer driving lacZ (eveK123-lacZ), and the pattern of lacZ expression was examined in the CNS of wild-type embryos and embryos misexpressing Klu protein in the NB4-2 lineage (McDonald, 2003).

In wild-type embryos, the eveK123-lacZ transgene is expressed in the aCC and RP2 neurons, similar to the wild-type (R79S84) eve-lacZ transgene. However, in one or two hemisegments per embryo, an extra cell expressing eveK123-lacZ adjacent to the RP2 neuron was observed. This phenotype is very similar to wild-type (R79S84) eve-lacZ expression in klu mutant embryos, although slightly less penetrant. It is concluded that the eveK123-lacZ transgene mimics the klu mutant phenotype, and it is proposed that Klu represses eve expression via direct binding to one or more of these sites (McDonald, 2003).

To further test this hypothesis, gain of function experiments were used to test whether ectopic Klu in GMC4-2a can repress eve-lacZ expression via these sites. Expression of a wild-type (R79S84) eve-lacZ transgene was compared with a transgene containing three mutated Klu consensus binding sites (eveK123-lacZ) in embryos where Scabrous-Gal4 (Sca-Gal4) drives ectopic expression of UAS-klu in all neuroblast lineages. The wild-type (R79S84) eve-lacZ expression is partially repressed by ectopic Klu expression, but the eveK123-lacZ transgene with mutated Klu sites is repressed to a lesser extent. This difference in repression is only observed when the levels of transgene expression are lowered by raising the embryos at 18°C; when the transgenes are more strongly expressed (by raising the embryos at 23°C) no detectable repression was observed. Taken together, Klu loss of function and misexpression studies indicate that Klu acts partly, but not completely, through three predicted Klu-binding sites to repress eve expression in the NB4-2 lineage (McDonald, 2003).

In summary, hkb, ftz, pdm1, and pros are independently required to activate eve expression in GMC4-2a. This suggests that the eve enhancer is capable of integrating the input of all four of these transcription factors to activate transcription. Hb and Ind are also necessary for eve expression in GMC4-2a, but it is not known if they act directly on the eve element or via one of the four transcription factors described in this study. Putative binding sites were found for each of the positively acting transcription factors within the minimal eve element, but mutation of these sites had no effect on expression of the eve-lacZ transgene in embryos (M. Fujioka, J.A. McDonald, and C.Q. Doe, unpublished results reported in McDonald, 2003). It remains to be determined whether Pros, Hkb, Ftz, or Pdm1 activate eve transcription via direct binding to the minimal eve element, or indirectly by activating or facilitating the binding of other transcriptional activators (McDonald, 2003).

Based on functional dissection of the RP2/aCC/pCC eve element, it seems to be composed of three parts. The regions ~7.9 to ~8.2 kb and ~8.2 to ~8.4 kb are each necessary to direct the expression pattern (together they comprise the minimal element for expression in RP2 and aCC), while the region ~8.4 to ~8.6 kb enhances the level of expression. Expression in the pCC neuron is further enhanced by the region extending to ~9.2 kb. The two regions within the minimal element seem to be regulated by different factors, because two copies of ~8.2 to ~8.4 kb (increasing the number of activator binding sites within this region by twofold) could not substitute for the function of the region ~7.9 to ~8.2 kb. This is consistent with the fact that at least four factors are independently required to activate eve in RP2 neurons. How does Klu repress eve expression in GMC4-2b? Negative regulation of eve expression by Klu is due to direct binding to the eve minimal element. (1) It is shown that klu mutants exhibit similar derepression of the eve minimal element transgene and the endogenous eve gene in the NB4-2 lineage; (2) three consensus binding sites are detected for Klu in the eve minimal element (comparison of Drosophila virilis and Drosophila melanogaster shows that the three identified sites are highly conserved); (3) mutation of these sites results in ectopic expression of eve-lacZ in the NB4-2 lineage in wild-type, and (4) mutation of these sites impairs repression of eve-lacZ by ectopic Klu in the NB4-2 lineage. The predicted Klu binding sites (K123) are probably only a subset of relevant Klu binding sites, however, because mutation of the sites gives only partially penetrant phenotypes (McDonald, 2003).

Surprisingly, it was not possible to separate the GMC4-2a/ RP2 element from the GMC1-1a/aCC/pCC element. In both NB 1-1 and NB 4-2 lineages, eve is expressed in the first-born GMC and its neuronal progeny. Both first-born GMCs share expression of several transcription factors, including Pros and Ftz. However, many other transcription factors are differentially expressed, such as the GMC1-1a specific expression of Vnd and Odd-skipped, and the GMC4-2a specific expression of Hkb, Pdm1, and Ind. It is possible that one or more commonly expressed transcription factors are required for expression of eve in both GMC1-1a and GMC4-2a, such as Pros, and this is why the elements cannot be subdivided (McDonald, 2003).

Spatial regulation of microRNA gene expression in the Drosophila embryo: The 8-miR enhancer is regulated by the localized Huckebein repressor, whereas miR-1 is activated by Dorsal and Twist

MicroRNAs (miRNAs) regulate posttranscriptional gene activity by binding to specific sequences in the 3' UTRs of target mRNAs. A number of metazoan miRNAs have been shown to exhibit tissue-specific patterns of expression. This study investigated the possibility that localized expression is mediated by tissue-specific enhancers, comparable to those seen for protein-coding genes. Two miRNA loci in Drosophila melanogaster are investigated, the mir-309–6 polycistron (8-miR) and the mir-1 gene. The 8-miR locus contains a cluster of eight distinct miRNAs that are transcribed in a common precursor RNA. The 8-miR primary transcript displays a dynamic pattern of expression in early embryos, including repression at the anterior and posterior poles. An 800-bp 5' enhancer was identified that recapitulates this complex pattern when attached to a RNA polymerase II core promoter fused to a lacZ-reporter gene. The miR-1 locus is specifically expressed in the mesoderm of gastrulating embryos. Bioinformatics methods were used to identify a mesoderm-specific enhancer located ~5 kb 5' of the miR-1 transcription unit. Evidence is presented that the 8-miR enhancer is regulated by the localized Huckebein repressor, whereas miR-1 is activated by Dorsal and Twist. These results provide evidence that restricted activities of the 8-miR and miR-1 miRNAs are mediated by classical tissue-specific enhancers (Biemar, 2005).

The 8-miR complex is located between two predicted protein-coding genes, CG15125 and CG11018, in the 56E region on the right arm of chromosome 2. To determine the approximate transcription start site of the 8-miR transcription unit, 5' RACE was used. Several independent experiments were carried out, and RACE products corresponding to two different start sites were isolated several times. Consensus sequences for both an initiator and a TATA box are appropriately spaced upstream of the identified start sites. The alignment of this genomic interval with the corresponding regions of the most divergent Drosophilids indicates strong conservation of each of the individual miRNAs within the 8-miR complex (Biemar, 2005).

A digoxigenin-labeled 8-miR antisense RNA probe was hybridized to staged embryos to determine the expression profile of the precursor transcript during development. Expression is initially detected in all of the nuclei of precellular embryos. As expected, staining is restricted to nuclei and not seen in the cytoplasm. The first indication of differential spatial regulation occurs at the midpoint of cellularization, when 8-miR transcripts are lost at the posterior pole. By the completion of cellularization, this loss in staining expands and there is also reduced expression in anterior regions. Staining persists at the anterior tip but is lost from subterminal regions of the anterior pole (Biemar, 2005).

During gastrulation there is both dorsal-ventral and anterior-posterior modulation of the 8-miR-staining pattern. Staining is first lost from the presumptive mesoderm and neurogenic ectoderm in ventral and lateral regions. There are transient stripes of 8-miR expression in the dorsal ectoderm, but they rapidly give way to a single band of staining in central regions. By the onset of the rapid phase of germband elongation, staining is essentially lost except for residual expression at the anterior tip and dorsal ectoderm (Biemar, 2005).

The early loss of staining at the posterior pole suggests that Huckebein (Hkb) might repress 8-miR transcription in the early embryo. To investigate this possibility, colocalization assays were done with snail, which is selectively expressed in the presumptive mesoderm of cellularizing and gastrulating embryos. The posterior border of the snail pattern is established by the localized Hkb repressor. The 8-miR pattern displays a similar posterior border, and there is an expansion of both the snail and 8-miR patterns in hkb-/hkb- mutant embryos (Biemar, 2005).

Further evidence for repression by Hkb was obtained by analyzing torso dominant (torD) mutants. tor encodes a receptor tyrosine kinase that is normally activated only at the poles, where it is required for the localized expression of tailless (tll) and hkb. torD encodes a constitutively activated form of the receptor tyrosine kinase that results in expanded expression of hkb and tll at the poles. This expansion in Hkb causes a severe shift in the posterior border of both the snail and 8-miR expression patterns. The identification of a sequence-specific transcriptional repressor, Hkb, as a likely regulator of 8-miR expression suggests that the dynamic staining pattern is probably controlled at the level of de novo transcription (Biemar, 2005).

Direct support for this possibility was obtained by the identification of an 8-miR enhancer. An ~800-bp genomic DNA fragment extending from the miR-3 region of the 8-miR complex to the predicted start site of CG11018 was attached to a lacZ-reporter gene containing the minimal eve promoter sequence. The resulting fusion gene recapitulates most aspects of the endogenous 8-miR expression pattern. In particular, lacZ transcripts are initially detected throughout precellular embryos but sequentially lost from the posterior pole and anterior regions during cellularization. At the onset of gastrulation, expression is diminished in ventral regions, and the staining detected in the dorsal ectoderm exhibits segmental modulation. Thus, the 5' 8-miR enhancer contains repression elements that mediate silencing by Hkb (and possibly Tll) at the termini in response to Tor signaling (Biemar, 2005).

The preceding analysis provides evidence that cell-specific enhancers regulate miRNA gene expression, as seen for protein coding genes. Further support was obtained by analyzing a second miRNA that displays localized expression in the early Drosophila embryo, miR-1. The mir-1 gene is highly conserved in different animal groups and displays localized expression in a variety of mesodermal lineages, including cardiac mesoderm in vertebrates. The Drosophila mir-1 gene is first expressed in the presumptive mesoderm during the final phases of cellularization. Expression persists in differentiating mesodermal tissues during gastrulation, germband elongation, and segmentation. Mutant embryos that contain the constitutively activated Toll10B receptor display ubiquitous expression of miR-1, concomitant with the transformation of all of the tissues into mesoderm (Biemar, 2005).

Whole-genome tiling arrays were used to obtain an estimate of the miR-1 transcription unit. These high-density oligonucleotide arrays contain 25-nt oligomers spaced on average every 36 bp and cover the entire nonrepetitive Drosophila genome, from one end of each chromosome to the other. Total RNA was extracted from three different mutant strains. Embryos derived from pipe-/pipe- females lack Toll-signaling activity and thereby lack a Dorsal nuclear gradient. As a result, genes normally activated by high, intermediate, and low levels of the gradient are silent, and there is a loss of mesoderm and neurogenic ectoderm. Instead, genes that are repressed by the Dorsal gradient, and normally restricted to the dorsal ectoderm, are now expressed throughout the embryo, causing the transformation of mesoderm and neurogenic ectoderm into dorsal ectoderm. Previous microarray assays have shown that genes expressed in the dorsal ectoderm are overexpressed in mutant embryos derived from pipe-/pipe- embryos. As expected, such mutants display little or no expression of the miR-1 transcription unit. Similarly, embryos derived from Tollrm9/Tollrm10 mutants contain weak Toll signaling and low levels of nuclear Dorsal everywhere. These low levels are insufficient for the activation of mesoderm genes, but are sufficient for the activation of neurogenic genes and the repression of dorsal ectoderm genes. Again, these mutants fail to express miR-1. Toll10B embryos contain strong, ubiquitous Toll signaling and high levels of Dorsal, which activate mesoderm genes throughout the embryo. These embryos display strong expression of the miR-1 transcription unit. The tiling array suggests that the gene is ~2.9 kb in length. The mature, processed miRNA is located roughly in the center of the inferred transcription unit (Biemar, 2005).

The early expression of the miR-1 primary transcript in the mesoderm raises the possibility that the gene might be regulated by the Dorsal gradient. Approximately one-half of all Dorsal-target enhancers also contain binding sites for the basic helix-loop-helix Twist activator. A 50-kb interval encompassing the miR-1 locus was surveyed for clusters of Dorsal and Twist binding sites. The best cluster was identified ~5 kb upstream of the miR-1 start site. There are a total of three Dorsal- and four Twist-binding sites contained over an interval of ~1.1 kb in this distal 5' region (Biemar, 2005).

A genomic DNA fragment encompassing these sites was attached to a lacZ-reporter gene and expressed in transgenic embryos. The reporter gene exhibits localized expression in the ventral mesoderm, beginning at the onset of gastrulation. Expression persists during germband elongation. These observations suggest that miR-1 is directly activated by Dorsal and Twist. However, lacZ transcripts expressed from the miR-1::lacZ transgene are detected somewhat later than the endogenous miR-1 primary transcript, which first appears before the completion of cellularization. It is conceivable that the miR-1 locus contains a second enhancer that directs earlier expression (Biemar, 2005).

The preceding analysis provides evidence that dynamic patterns of miRNA gene expression are controlled by tissue-specific enhancers, and not by the differential processing of miRNA precursor RNAs. Both the 8-miR and miR-1 enhancers produce authentic patterns of lacZ-reporter gene expression when attached to the core promoter region of the eve gene. The 8-miR enhancer appears to be regulated by the Hkb repressor, whereas miR-1 is activated by Dorsal and Twist (Biemar, 2005).

The miR-1 enhancer is somewhat unusual among 'type 1' Dorsal target enhancers, in that it contains a large number of Snail repressor sites. Type 1 enhancers are activated by high levels of the Dorsal gradient in the ventral mesoderm. Previous studies have identified six such enhancers. They all contain multiple low-affinity Dorsal binding sites, but essentially lack Snail repressor sites. The general absence of Snail sites permits activation of type 1 genes in the ventral mesoderm where there are high levels of the repressor. An exception is the type 1 intronic enhancer that regulates Heartless (Htl), one of the two FGF receptor genes in the Drosophila genome (Biemar, 2005).

The htl intronic enhancer is ~800 bp in length and contains two low-affinity Dorsal binding sites and two optimal Twist sites. Each Twist site overlaps a Snail repressor site, but the enhancer nonetheless activates lacZ-reporter gene expression in the presumptive mesoderm before the completion of cellularization. The htl enhancer fails to mediate expression in the neurogenic ectoderm because it lacks the arrangement of optimal Dorsal and Twist sites required for activation by intermediate levels of the Dorsal gradient (type 2 enhancers) (Biemar, 2005).

The miR-1 enhancer contains three weak Dorsal sites, four optimal Twist sites (CACATGT; Kate Senger, unpublished results cited in Biemar, 2005), and five Snail repressor sites (three of the sites overlap the optimal Twist sites and two occur at separate sites). Perhaps the relative increase in the number of Snail repressor sites in the miR-1 enhancer (vs. the htl enhancer) causes late onset of miR-1::lacZ transgene expression. The Snail repressor is transiently expressed in the ventral mesoderm during cellularization but disappears after invagination. It is during the time when Snail levels subside that the miR-1 enhancer first becomes active (Biemar, 2005).

Previous studies have emphasized the importance of the Snail repressor in defining spatially localized patterns of gene expression. Dorsal target genes activated by intermediate (type 2) and low (type 3) levels of the gradient contain Snail repressor sites that keep the genes off in the ventral mesoderm and restricted to the neurogenic ectoderm. The present identification of the distal miR-1 enhancer raises the possibility that Snail also influences the timing of gene expression (Biemar, 2005).

The similarities in miR-1 and Htl regulation raise the possibility that the miR-1 miRNA attenuates the activity of one or more components of the FGF-signaling pathway. FGF is essential for the migration of the invaginated mesoderm along the inner surface of the neurogenic ectoderm. It is also important for the activation of cardiac genes in the dorsal-most mesoderm that forms the heart. miR-1 might attenuate one or more target mRNAs engaged in mesoderm migration and/or heart induction. The mammalian miR-1 miRNA has been shown to attenuate Hnd2 expression, which is essential for the differentiation of ventricular cardiomyocytes (Zhao, 2005). Despite the conservation of the miR-1 miRNA sequence, and a potential role in suppressing heart formation in both flies and mice, it would appear that distinct mechanisms of regulation are used in the two systems: Dorsal and Twist activate miR-1 in flies, whereas distinct regulatory factors, SRF and MyoD, activate miR-1 in the mouse embryo. It is possible however, that later phases of miR-1 expression depend on nautilus (nau), the Drosophila homolog of MyoD (Biemar, 2005).

Huckebein is part of a combinatorial repression code in the anterior blastoderm

The hierarchy of the segmentation cascade responsible for establishing the Drosophila body plan is composed by gap, pair-rule and segment polarity genes. However, no pair-rule stripes are formed in the anterior regions of the embryo. This lack of stripe formation, as well as other evidence from the literature that is further investigated in this study, led to a hypothesis that anterior gap genes might be involved in a combinatorial mechanism responsible for repressing the cis-regulatory modules (CRMs) of hairy (h), even-skipped (eve), runt (run), and fushi-tarazu (ftz) anterior-most stripes. This study investigated huckebein (hkb), which has a gap expression domain at the anterior tip of the embryo. Using genetic methods deviations from the wild-type patterns of the anterior-most pair-rule stripes were detected in different genetic backgrounds, consistent with Hkb-mediated repression. Moreover, an image processing tool was developed that, for the most part, confirmed the assumptions. Using an hkb misexpression system, specific repression on anterior stripes was detected. Furthermore, bioinformatics analysis predicted an increased significance of binding site clusters in the CRMs of h 1, eve 1, run 1 and ftz 1 when Hkb was incorporated in the analysis, indicating that Hkb plays a direct role in these CRMs. Hkb and Slp1, which is the other previously identified common repressor of anterior stripes, might participate in a combinatorial repression mechanism controlling stripe CRMs in the anterior parts of the embryo and define the borders of these anterior stripes (Andrioli, 2012).

The aim of this study was to understand the mechanisms underlying the regulation of the anterior pair-rule stripes. The model tested was first proposed for eve 2 regulation. Transcriptional activators do not give enough patterning information, and the presence of repressors is instructive for determining the precise positioning of a particular stripe. The hypothesis was that transcription repressors could be working in a combinatorial manner to determine the correct positioning of the anterior stripes and prevent, in a spatial and temporal manner, the expression of stripe CRMs in the more anterior regions of the embryo by counteracting the activity of activators. There is plenty of evidence supporting this hypothesis, which was further confirmed in this study (Andrioli, 2012).

Regarding activators, computational analysis predicted Bcd, Hb and Btd binding sites are part of significant clusters in the anterior-most stripe CRM. These predictions agree well with previous genetic data and in vivo DNA binding data from ChIP/chip experiments. Thus, Btd, and above all the widely spread maternal factors Bcd and Hb, might activate anterior stripe CRMs early in the anterior blastoderm. Alternatively, the early broad expression patterns of pair-rule genes could be under the control of dedicated CRMs, although no such elements have yet been reported. It is possible that other regulatory elements could contribute to the expression detected early in the anterior blastoderm, for instance, the CRM responsible for the expression of h head patch or the CRMs responsible for eve 3, eve 5 and h 5, which were proposed to be activated by the maternal factor DSTAT (Drosophila Signal Transducer and Activator of Transcription), which is ubiquitously expressed in the embryo (Andrioli, 2012).

The expression of several gap domains covering all of the anterior regions of the embryo ahead of the seven-striped patterns is consistent with the expected subsequent local repression of pair-rule CRMs activated in the head region. Of these gap domains, Slp1 is a common repressor for anterior pair-rule stripes, but other repressors besides Slp1 were predicted to be necessary for correctly determining the borders of the anterior-most stripes. This study investigated hkb, which, in addition to tll, is the other major gap gene target of the Torso signaling regulation in the terminal system. In the anterior region, hkb is required for the proper formation of the foregut and midgut. Its domain at the anterior tip coincides with the region where the diffused early expression patterns of pair-rule genes first fade. These observations are consistent with local repression roles of Hkb. However, it was not possible to detect derepression of pair-rule genes in the anterior pole of hkb- embryos. One possibility is that the progressive non-detection of the expression of pair-rule genes might correspond to a failure in activation. In fact, Bcd activation was shown to be down-regulated by the Torso-signaling cascade at the anterior tip. Nevertheless, other data suggest that the Torso pathway might induce a repression mechanism at the anterior tip that would be parallel and redundant with Torso-induced inhibition of Bcd. Thus, one might predict that another repressor might still able to act on Hkb targets in the absence of Hkb protein (Andrioli, 2012).

Although no pair-rule derepression was detected in the anterior pole, it was possible to detect subtle deviations in the positioning of eve 1 in hkb- embryos, which was confirmed by morphological measurements using the image processing tool. Enhanced derepression effects were also detected for all anterior-most stripes investigated in slp-;hkb- double-mutant embryos compared to the effects observed in slp- embryos; these results were statistically significant. With the hkb misexpression system, repression effects were detected for h 1, eve 1, run 1 and ftz 1. With the exception of gt repression, no other gap domain disruption was detected in these assays. These results strongly suggest direct repression by Hkb on the CRMs of these stripes. In vivo binding data confirms this possibility. Moreover, with the bioinformatics analysis it was verified that Hkb, along with putative activators, increased the already high significance values of predicted clusters for activators that match these stripe CRMs. Therefore, the combined data suggest that Hkb acts as a repressor for a specific group of anterior pair-rule stripes (Andrioli, 2012).

These data also suggest that there is another possible mechanism underlying the repression that involves the activity of repressors further away from their original sources. One example of this mechanism is expression detected for the ectopic hkb domain, demonstrating that target CRMs are sensitive to Hkb-mediated repression even in the presence of low expression levels of Hkb. The prediction is that low concentrations of Hkb that have diffused away from its endogenous domain could still repress these CRMs. For this mechanism, repressors could fulfill additive repression roles at different anterior subdomains or even contribute to the definition of the anterior borders of stripes that are distantly positioned from where gap domains are detected. Thus, the increased derepression observed in slp-;hkb- embryos would be expected if a combinatorial additive mechanism existed in which each repressor had a small contribution to the overall repression. Following the same rationale, one can predict that at least one other repressor is still responsible for setting anterior border stripes in slp-;hkb- embryos (Andrioli, 2012).

The complexity of the regulation of genes involved in early patterning was postulated to be a condition that is necessary for sensing relatively small differences in the concentrations and combinations of many regulatory factors, which is likely the environment found in the syncytial blastoderm. In agreement with that hypothesis, recent studies revealed that the protein gradients of factors such as Bcd and Dorsal alone are not sufficient to determine all of the spatial limits of target gene expression and that these gradients might combine with other factors to pattern the early embryo. In the head region, it has been suggested that Bcd and the terminal system-mediated activities interact at the level of the target CRMs to generate the proper patterning for the head region of the embryo. In contrast to these studies that focused on gap genes, the current data shed light on a mechanism that is involved in the regulation of the anterior stripe CRMs, with the putative participation of hkb (Andrioli, 2012).

The correct positioning of the anterior pair-rule stripes must be a critical issue in the early developmental patterning of the fly. Even a slightly incorrect positioning of the anterior stripes, for instance, results in the non-formation of the mandibular segment in the slp null mutant. Thus, a complex repression mechanism is necessary to shape the stripes and to avoid inappropriate expression of their CRMs. Therefore, Hkb, Slp1 and other repressors are likely involved in a combinatorial repressive activity in the CRMs of the anterior stripes. Other experiments are necessary to test this hypothesis further and to reveal the underlying molecular mechanisms involved in this regulation (Andrioli, 2012).

Protein Interactions

Since the genetic data indicates that Grunge/Atrophin functions closely with even-skipped and huckebein, tests were performed for possible physical interactions using a GST pull-down assay. It was found that the full-length radiolabeled Atro can bind to a full-length Eve (GST-Eve) or Hkb (GST-Hkb), but not to GST alone (Zhang, 2001).

The binding data, along with the genetic data, suggest a possible mechanism in which Atro functions as a corepressor for site-specific repressors like Eve and Hkb. One function of Eve and Hkb might be to recruit Atro to the promoter site where Atro can exert its repressive activities. This hypothesis would predict that Atro can directly repress transcription when it is tethered to DNA via a heterologous DNA binding domain. To test this, an Atro-GAL4 fusion was generated and its function was examined in the Kreggy/NEE-LacZ system. Briefly, full-length Atro was fused to the C terminus of the Gal4 DNA binding domain (Gal4DB::Atro), and the chimeric gene was placed under the control of the Kruppel promoter (Kr-Gal4DB::Atro), which drives gene expression in a broad band in the central region of the blastoderm-stage embryo. The LacZ reporter gene (UAS-NEE)-LacZ, which is driven by a modified rhomboid NEE enhancer that contains three UAS sites for Gal4 binding, is normally expressed in the ventral side of the same stage embryos. However, when the (UAS-NEE)-LacZ flies are crossed with the Kr-Gal4DB::Atro transgenic animals, their progeny show a repressed LacZ transcription in the central region where the Gal4DB::Atro fusion protein is expressed. This result suggests that the full-length Atro protein can behave as a transcriptional corepressor in vivo (Zhang, 2001).

Given the sequence similarity between Atro and human Atrophin-1, it is possible that human Atrophin-1 also functions as a transcriptional corepressor in vivo. Thus, this possibility was tested using the Kreggy/NEE-LacZ system in fly embryos. Interestingly, when tethered to Gal4 DNA binding domain, human Atrophin-1 can repress LacZ transcription in (UAS-NEE)-LacZ reporter embryos, since more than 75% of the examined embryos exhibited a repressed LacZ transcription, suggesting that the function of Atrophin family proteins are evolutionarily conserved. Human Atrophin-1 was further tested with a poly-Q expansion in the same system and it was found that only about 18% of the examined embryos displayed a repressed LacZ transcription, suggesting that poly-Q expansion alters Atrophin's transcriptional repressive activity (Zhang, 2001).

Huckebein-mediated autoregulation of Glide/Gcm triggers glia specification

Cell specification in the nervous system requires patterning genes dictating spatio-temporal coordinates as well as fate determinants. In the case of neurons, which are controlled by the family of proneural transcription factors, binding specificity and patterned expression trigger both differentiation and specification. In contrast, a single gene, glial cell missing (gcm), is sufficient for all fly lateral glial differentiation. How can different types of cells develop in the presence of a single fate determinant? That is, how do differentiation and specification pathways integrate and produce distinct glial populations? By following an identified lineage, this study shows that glia specification is triggered by gcm expression levels, mediated by cell-specific protein-protein interactions. Huckebein (Hkb), a lineage-specific factor, provides a molecular link between gcm and positional cues. Importantly, Hkb does not activate transcription; rather, it physically interacts with Gcm thereby triggering its autoregulation. These data emphasize the importance of fate determinant cell-specific quantitative regulation in the establishment of cell diversity (De Iaco, 2006).

Each segment of the fly embryonic ventral cord contains about 60 stereotypically organized lateral glial cells, most of which arise from neuroglioblasts (NGBs), mixed precursors producing both neurons and glia. In the absence of Gcm, glia are absent and transform into neurons. This phenotype is also observed in hkb embryos, but restricted to lateral glia derived from NGB1-1A. These data suggest that Hkb and Gcm work in the same pathway in NGB1-1A, prompting this lineage to be used as a model to understand the bases of glia specification (De Iaco, 2006).

NGB1-1A lineage contains six to eight neurons and three glial cells of the subperineural glia (SPG) class. NGB1-1A first produces aCC and pCC motoneurons and subsequently gives rise to several ganglion mother cells (GMCs), each producing a neuron and a glial cell that do not divide further. hkb transcripts are first detected at the time gliogenesis starts and colocalize with gcm RNA within the NGB1-1A lineage. Expression of both mRNAs starts at stage 11 in a single, dividing, cell. By late stage 11, two cells express gcm; one of them is somewhat larger, more apically located than the other, and corresponds to NGB1-1A (II. This cell expresses both gcm and hkb, whereas the basal, small, cell corresponds to ganglion mother cell II (GMC II) and only expresses gcm. Repo glial-specific marker starts being detected in one cell of the lineage by mid stage 12, three cells per hemisegment being labeled at late stage 12 (De Iaco, 2006).

This study shows that Hkb controls glia specification by binding Gcm glia promoting factor and inducing high levels of gcm expression. Thus, cell-specific autoregulation of a fate determinant coordinates patterning and differentiation and is crucial for the establishment of cell diversity (De Iaco, 2006).

Fate determinant levels are important for glial differentiation: (1) the number of supernumerary glia depends on the amount of ectopic Gcm; (2) gcm RNA is unequally distributed in the dividing NGB, the presumptive neuroblast inheriting less RNA than the presumptive glioblast; (3) gcm contains a PEST motif, characteristic of proteins at high turn over, and an mRNA instability element (IE) in the 3' untranslated region, both of which are conserved throughout evolution. The present data show that gcm levels control not only the number but also the type of induced glia, demonstrating for the first time that autonomous cue quantitative regulation controls cell specification within a neural lineage (De Iaco, 2006).

gcm autoregulation in NGB1-1A does not depend on cooperative activation of two transcriptional pathways. Rather, the presence of the Gcm binding site (GBS) is sufficient in vitro and in vivo for cell-specific gcm autoregulation, thus pointing to a novel molecular strategy controlling fate determinant levels (De Iaco, 2006).

The role of Hkb is to sustain gcm-dependent transcription by acting on Gcm binding and transactivation potential. This is also supported by three observations: (1) Hkb works on different GBSs; (2) mutagenized GBSI is active, although at low levels, in the presence of Hkb and Gcm, but not in the presence of Gcm alone; (3) gcm overexpression does produce ectopic SPG in an hkb context, since this overcomes the need for autoregulation. In the establishment of terminal patterning in the fly embryo, hkb works as a repressor via interaction of its Nt region with Groucho protein. This region, however, is neither relevant for Hkb binding to Gcm nor for activating gcm autoregulation. Thus, hkb works either as a repressor or as a coactivator by interacting with different proteins (De Iaco, 2006).

Glia specificity (e.g., longitudinal versus SPG) could rely on genes that are activated by a pathway independent of Gcm and impose specification on postmitotic cells otherwise displaying a default 'pan-glia' fate. Specification of postmitotic cells does also take place during the development of neurons, where it refines/maintains early decisions taken in precursor cells and allows for further diversification. While establishing the possible need for postmitotic specification still awaits the identification of novel glial markers, present data demonstrate the importance of cues working in glial precursors (De Iaco, 2006).

Integrating cell differentiation and specification via Gcm-Hkb interaction allows a single fate determinant to generate different types of glia. It will be interesting to determine whether Hkb also acts on gcm targets, as it is known that some of these targets are pan-glial (Repo), whereas others are lineage specific (Loco). It will also be interesting to determine whether gcm autoregulation mediated by cell-specific factors is necessary in other glial lineages (De Iaco, 2006).

These data show that NGB1-1A glia specification requires two equally important regulatory steps. The first one is gcm independent and results in low levels of gcm expression, whereas the second one is gcm and hkb dependent and induces high levels of gcm. Previous data show that hkb expression is induced by columnar genes, which control D/V regionalization in the nervous system. Thus, cell-specific factors such as Hkb provide an intermediate step between patterning genes and fate determinants, thereby triggering the identity of neural precursors (De Iaco, 2006).

Supernumerary P101 positive cells induced by Hkb overexpression are all located close to endogenous SPG. The central and abdominal position of supernumerary SPG suggests that cells within the NGB1-1A lineage are 'competent' to express the SPG fate. Furthermore, hkb gliogenic activity is also temporally restricted, since the first division is never affected and always produces aCC/pCC sibling neurons. These data call for additional factors regulating gcm expression and SPG specification and, indeed, homeotic as well as temporal genes are known to control the NGB1A-A lineage. Thus, a grid of positional cues along spatial and temporal axes works through gcm, thereby triggering differentiation of the appropriate type of glia. Understanding the interplay of these cues will be the matter of further studies; however, the data already demonstrate that cofactor-mediated quantitative regulation plays a pivotal role in cell specification. Such fine-tuning and accurate orchestration of events highlights the complexity underlying nervous system differentiation. Integrating qualitative and quantitative regulation via cell-specific autoregulation likely applies to other developmental processes in which a single fate determinant triggers different phenotypes (De Iaco, 2006).

huckebein: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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