glial cells missing



Fly gliogenesis depends on the Glial-cell-deficient/Glial-cell-missing (Glide/Gcm) transcription factor. gcm expression is necessary and sufficient to induce the glial fate within and outside the nervous system, indicating that the activity of this gene must be tightly regulated. The current model is that Gcm activates the glial fate by inducing the expression of glial-specific genes that are required to maintain such a fate. Previous observations on the null gcmN7-4 allele evokes the possibility that another role of gcm might be to maintain and/or amplify its own expression. gcm does positively autoregulate in vitro and in vivo, although the GcmN7-4 protein is not able to do so. This is the first direct evidence of both a target and a regulator of Gcm. The gcm promoter was sequenced and a search was carried out for sites to which the protein would bind. The consensus Gcm binding site (GBS) is known to be 5'-ATGCGGGT-3'. Five GBS's are scattered throughout 6.5 kb upstream of the gcm transcription start site whereas none are found in >5 kb of sequence downstream of it. One of the GBSs corresponds to the octamer consensus sequence (site C), while the four other sites display a nucleotide change at the seventh (site A) or at the sixth position. Interestingly, the promoter of reverse polarity (repo), a putative target of gcm, also contains several GBSs. Two of the 11 binding sites correspond to the consensus, while all the others display a mismatch at one of the eight positions. Sites C and A induce the highest levels of reporter activity, 100- and 25-fold, respectively, compared with the activity observed with control reporter plasmids not containing the GBS or containing a 30mer of random DNA sequence. Sites B, D and E show poor activity, their levels of CAT activation being 2.4-, 5.2- and 2.2-fold, respectively, compared with the controls. From these data it is concluded that sites C and A are the main sites for transcriptional activation, and that this activation is mediated through the activity of the Gcm protein. Sites A and D reside in the opposite orientation with respect to that of B, C and E. Interestingly, sites present in the opposite orientation are also observed in three of the 11 GBSs found in the repo promoter. To determine whether activation of transcription is dependent on the orientation of the GBS, the transactivation potential of each site in its reverse orientation relative to the reporter gene was tested. The level of gcm-mediated transcriptional activation varies only slightly depending on the orientation of the binding site. gcm transcription is regulated at two distinct steps: initiation, which is gcm-independent, and maintenance, which requires gcm. Interestingly, autoregulation requires the activity of additional cell-specific cofactors (Miller, 1998).

A crucial step in gcm regulation occurs at the level of transcriptional initiation since ectopic gcm is sufficient to override the endogenous differentiation programs and to promote glial differentiation within and outside the nervous system. Subsequent to the first wave of gcm transcription, however, there is a second level of regulation that involves the maintenance of gcm activity. The requirement for this maintenance is strongly suggested by the phenotype of gcmN7-4 embryos, in which the gcm transcript rapidly decays. This mutation is due to a single amino acid substitution that renders the gcm protein unable to bind DNA and transactivate, eliminating any positive feedback loop. It is reasonable to assume that the maintained expression of certain essential regulators is necessary for the irreversible commitment of cells to a particular tissue phenotype. Indeed, direct and/or indirect autoregulation has been observed in other genes involved in the determination of a specific cell fate. Because the activity of early acting genes may be transient, autoregulation may be used to transduce short-lived signals into stable patterns of expression. For example, the autoregulatory binding sites in the promoter of pair-rule genes are necessary to maintain their striped pattern of expression. Strikingly, gcm activity seems to require several controls, as evidenced by its tight transcriptional regulation, its ability to autoregulate and its dependence on the cellular redox potential. Moreover, the presence of additional motifs, a PEST sequence and an instability element in the 3' untranslated region (UTR), as well as the presence of potential phosphorylation sites predict further levels of regulation. This strongly suggests that gcm activity imperatively must be shut off if not required, and that only the combination of transcriptional and post-transcriptional controls will ensure the strict regulation necessary for proper development (Miller, 1998 and references).

That gcm positively autoregulates has been determined in vivo by analyzing the gcmN7-4 mutation and by ectopically expressing gcm in different tissues. This confirms and extends the results obtained in vitro. Strikingly, autoregulation occurs at much higher levels in the neurogenic region than in the mesoderm or in the dorsal ectoderm. Since the UAS-gcm line used for ectopic expression in the different tissues is the same, it is likely that one or more cofactors necessary for gcm to autoregulate are differentially expressed in the embryo. Interestingly, a similar situation has been observed in the case of the fushi tarazu autoregulatory element. Indeed, the upstream element in the ftz promoter depends on ftz activity and acts to enhance the striped expression in the ectoderm. However, the same element does not exhibit ftz-dependent enhancer activity in the CNS, another tissue in which ftz is normally expressed and required. It is speculated that the ability to support gcm autoregulation reflects the competence of a given cell to adopt the glial fate. Cells of the nervous system may be loaded with cofactors that allow autoregulation, making cells more competent to take the glial fate than other cell types. Alternatively, cells outside the nervous system express inhibitory factors that do not allow autoregulation. The absence of the right combination of cell-specific factors can be compensated by high levels of gcm (such as those obtained using the GAL4 system) that render all cells competent to adopt the glial fate even in the absence of autoregulation. Indeed, although no autoregulation takes place in the mesoderm, gcm expression driven by the twi promoter does activate the glial fate in this derivative (Bernardoni, 1998). The need for cofactors may constitute an additional level of regulation of gene activity, in order to ensure that a given fate is adopted only in the proper cells (Miller, 1998).

Neuronal differentiation relies on proneural factors that also integrate positional information and contribute to the specification of the neuronal type. The molecular pathway triggering glial specification is not understood yet. In Drosophila, all lateral glial precursors and glial-promoting activity have been identified: this provides a unique opportunity to dissect the regulatory pathways controlling glial differentiation and specification. Although glial lineages are very heterogeneous with respect to position, time of differentiation, and lineage tree, they all express and require two homologous genes, gcm and gcm2, that act in concert, with gcm constituting the major glial-promoting factor. Glial specification resides in gcm transcriptional regulation. The gcm promoter contains lineage-specific elements as well as quantitative and turmoil elements scattered throughout several kilobases. Interestingly, there is no correlation between a specific regulatory element and the type of glial lineage. Thus, the glial-promoting factor acts as a naive switch-on button that triggers gliogenesis in response to multiple pathways converging onto its promoter. Both negative and positive regulation are required to control gcm expression, indicating that gliogenesis is actively repressed in some neural lineages (Ragone, 2003).

The gcm promoter contains lineage-specific elements as well as quantitative and temporal elements scattered throughout several kilobases. There is no correlation between a specific regulatory element and the mode of glia differentiation. For example, the shortest fragment rescues two neuroglioblasts (NGBs), the 1-1A, which arises ventrally, delaminates early, and produces CNS glia, and the 1-3, which arises very dorsally, delaminates late, and produces peripheral glia. Moreover, 1-1A glial-derived cells appear late, whereas those derived from the 1-3 are the first ones. Interestingly, distinct cis-regulatory elements responding to anteroposterior cues dictate the differentiation of glial precursors along the A/P axis. Indeed, all the lineages rescued by the 2-kb transgene are located anteriorly within each neuromere, while longer promoters result in the rescue of more posteriorly located precursors (Ragone, 2003).

Spatial regulation overlaps with temporal regulation, since 9 kb of upstream sequences allow for gcm expression in all lineages, but display delayed or precocious onset/decay in some lineages. The data show that temporal regulation does contribute to the normal profile of gcm expression and glial differentiation. The importance of gcm temporal regulation has also been demonstrated in the development of the adult nervous system. These data suggest that fly gliogenesis is a process in which several developmental pathways converge onto a single gene, gcm, which is at the crossroads between neurons and glia. Indeed, due to its uniform and delayed expression in all glial lineages, it is unlikely that gcm2 is critical for the induction of specific glial fates. Thus, the key for glia specification resides in the gcm promoter. Gliogenesis differs from neuronal development in the sense that the glial-promoting factor does not have a role in cell specification, whereas neurogenesis depends on factors that dictate both cell differentiation and specification. Whether these differences in developmental strategies reflect the fact that glial cells are more plastic in nature, as shown by the recent observation that mammalian glial cells may constitute a source of stem cells, remains to be elucidated (Ragone, 2003).

Spatial information is contained in the 9kb transgene, even though additional quantitative regulation may require the whole region between gcm and gcm2. This likely accounts for the lack of viability rescue even with the longest construct. Misexpression data have suggested that high Gcm levels are required for glial differentiation. Mesodermal Gcm leads to ectopic glia in that layer, however, the number and type of mesodermal cells that adopt the glial fate depend strictly on the levels of expression. These data might also indicate that cells outside the nervous system have different potentials to produce glia. The present study, however, clearly shows that high Gcm levels are necessary to produce glia. (1) Double dose of the same construct shows better rescue than single dose. (2) Transgenic lines carrying the same construct display different rescue potentials depending on the position of the insert. Dosage effect, however, cannot account for all rescue potential. Indeed, while large constructs show better rescue (higher penetrance and expressivity) when expressed in double dose as compared to single dose (two vs. one insert; homo- vs. hetero-zygous conditions for the same insert), this is not true for the shortest, 2kb transgene. Thus, qualitative information assigns lineage identity and mostly resides in the proximal region, whereas quantitative information strengthens the gliogenic potential and is scattered throughout many kilobases. An important need for qualitative regulation is also stressed by the fact that the glial lineages do not show major differences in the levels of gcm expression and yet they show different requirements with respect to the gcm promoter (Ragone, 2003).

High levels of gcm expression likely depend on the presence of several Gcm binding sites in the region between gcm and gcm2. They also depend on lineage-specific partners of gcm, as shown for the Prospero transcription factor in the 6-4 and 7-4 lineages. It is proposed that autoregulation and cofactor-mediated maintenance of Gcm play an important role in gliogenesis. Indeed, high and protracted expression may be necessary for late steps, such as glial proliferation, as previously suggested by the phenotype of gcm hypomorphic mutations (Ragone, 2003).

The use of gcm (rather than reporter gene)-containing constructs has allowed a comprehensive analysis to be carried out of the rescued glial phenotype and a functional assay (axonal phenotype) to be carried out. Moreover, the spatiotemporal profile of transgene expression has been assessed. This is specifically important for genes that, as gcm, are transiently expressed. Finally, possible involvement of coding and untranslated sequences can be taken into account, which is important in the case of gcm, a gene that autoregulates and displays asymmetric RNA distribution. All these conditions have allowed a definition of the spatial, temporal, and quantitative requirements of gcm (Ragone, 2003).

Neural stem cell specification requires both negative and positive regulation of gcm expression. gcm overexpression during embryogenesis produces glial cells in all the tissues in which it has been tested. This indicates that gcm is necessary and sufficient to induce the glial fate. It also suggests that gliogenesis requires gcm- positive regulation. Such mechanisms are indeed at work, since adding back wild- type copies of gcm into a mutant background restores both gcm expression and glial differentiation. Nevertheless, gcm also needs to be repressed in nongliogenic lineages, as seen in the 2-kb transgene (Ragone, 2003).

This first promoter analysis opens the perspective that glial differentiation occurs through negative regulation of the glial promoting activity. This finding also calls for a provocative view of a default glial state in some lineages of the nervous system. It is possible that a situation similar to the one observed in fly glia also takes place in vertebrate gliogenesis. Indeed, vertebrate proneural genes, which code for transcriptional activators, seem to inhibit the glial fate. This suggests that their role is to activate a repressor, which in turn shuts off the expression of yet unknown glial-promoting factors (Ragone, 2003).

Transcriptional Regulation

In many organisms, single neural stem cells can generate both neurons and glia. How are these different cell types produced from a common precursor? In Drosophila, glial cells missing is necessary and sufficient to induce glial development in the CNS. GCM mRNA has been reported to be asymmetrically localized to daughter cells during precursor cell division, allowing the daughter cell to produce glia while the precursor cell generates neurons. In this study, it has been shown that (1) GCM mRNA is uniformly distributed during precursor cell divisions; (2) the Prospero transcription factor is asymmetrically localized into the glial-producing daughter cell; (3) Prospero is required to upregulate gcm expression and induce glial development, and (4) mislocalization of Prospero to the precursor cell leads to ectopic gcm expression and the production of extra glia. A model for the separation of glia and neuron fates in mixed lineages is proposed in which the asymmetric localization of Prospero results in upregulation of gcm expression and initiation of glial development in only precursor daughter cells (Freeman, 2001).

In thoracic segments the neural precursor 6-4 generates both glia and neurons, and is referred to as NGB 6-4T; in abdominal segments the 6-4 precursor produces only glia, and so it is called GB 6-4A. The neural precursor 7-4 generates a lineage composed of both neurons and glia in all segments, and is referred to as NGB 7-4 (Freeman, 2001).

NGB 6-4T and GB 6-4A form at early embryonic stage 10 as part of the S3 wave of neuroblasts. The first division of NGB 6-4T is oriented along the apical-basal axis, producing a large apical post-divisional precursor (NGB 6-4T) and a smaller basal daughter cell (G). Gcm is expressed before this first division and both daughter cells inherit Gcm protein, which enters the nucleus in these cells immediately after NGB division. Interestingly, shortly after NGB 6-4T completes this division, the G daughter cell migrates from its basal position to a position just medial to the post-divisional NGB 6-4T, which may explain why this division was previously scored as mediolateral. By the end of the G cell medial migration, Gcm protein is downregulated in NGB 6-4T and maintained only in the G daughter cell. 6-4T continues to divide along the apical-basal axis and subsequently produces neuronal progeny. The G daughter cell maintains high levels of Gcm protein and produces three glial cells. These glia continue to express Gcm protein, activate the glial specific gene reversed polarity (repo), migrate medially, and differentiate into cell body glia (CBGs) (Freeman, 2001).

These results raise the question of how Gcm protein becomes asymmetrically restricted to the G daughter cell after the first division of NGB 6-4T. It has been proposed that GCM mRNA is asymmetrically partitioned into the G daughter cell during mitosis of NGB 6-4T. To test this model, GCM mRNA localization was scored by fluorescent in situ hybridization. Levels of GCM mRNA are observed in the predivisional NGB 6-4T, followed by uniform localization in the mitotic NGB 6-4T, and equal distribution to both NGB 6-4T and G sibling cells. After G cell migration GCM mRNA was observed in the G cell and down-regulation of GCM mRNA was observed in the post-divisional NGB 6-4T. gcm-expressing cells were scored throughout the CNS, and asymmetric localization of GCM mRNA has never been observed in any mitotic precursor cell. It is concluded that GCM mRNA is not asymmetrically localized in the mitotic NGB 6-4T, but rather that it becomes transcriptionally upregulated in the G daughter cell soon after it is born (Freeman, 2001).

Similar to NGB 6-4T, GB 6-4A divides along the apical-basal axis and its basal daughter cell rapidly migrates to a position medial to its apical sibling. In contrast to NGB 6-4T, GB 6-4A expresses high levels of Gcm protein and mRNA before its first division, and both daughter cells maintain gcm expression. These two cells subsequently express repo, migrate medially, and differentiate into cell body glia (CBG) (Freeman, 2001).

NGB 7-4 forms at late stage 8 as the most lateral En-positive S1 neuroblast. The first progeny from NGB 7-4 are Prospero positive and Gcm negative, and differentiate into neurons. At stage 10 (just before the formation of 6-4 neural precursors) NGB 7-4 begins producing several Prospero-positive, Gcm-positive daughter cells that make a total of six to seven glia. At stage 12, NGB 7-4 switches back to making Prospero-positive Gcm-negative daughter cells that develop into neurons. All divisions of NGB 7-4 are along the apico-basal axis; Gcm-positive progeny are budded off the basal surface of NGB 7-4 but then migrate extensively to their final positions; ultimately, two glia migrate along the ventral surface of the CNS and differentiate as a pair of En-positive midline channel glia, three remain on the ventral surface of the CNS near NGB 7-4 and develop into CBGs posterior to the En-positive stripe, and one or two migrate to a position slightly dorsal and lateral to NGB 7-4 and differentiate as lateral subperineurial glia. In contrast to the 6-4 neural precursors, GCM mRNA or protein is not detected in NGB 7-4, only in its glial progeny (Freeman, 2001).

In summary, glia-producing divisions of NGB 6-4T and GB 6-4A occur along the apicobasal axis, and these divisions are followed by medial migrations of glial progeny. Gcm mRNA and protein are present in the predivisional NGB 6-4T, and Gcm protein enters the NGB and daughter cell nuclei immediately after the first division of this NGB. In addition, no evidence is found for asymmetric localization of GCM mRNA in any glial lineage, including NGB 6-4T (Freeman, 2001).

If GCM mRNA and protein are equally distributed into NGB 6-4T and its first-born G daughter cell, how are GCM mRNA and protein levels upregulated in G but not NGB 6-4T? To address this issue, mitotic NGB 6-4T were assayed for proteins known to be asymmetrically localized along the apical-basal axis of neuroblasts. The goal was to identify candidate genes that could differentially regulate gcm expression in the NGB 6-4T lineage. Insc protein marks the apical side of most or all mitotic neuroblasts and is necessary and sufficient for apical-basal spindle orientation. In NGB 6-4T, Insc is localized as an apical crescent at all stages of mitosis and is partitioned into the apically-positioned NGB 6-4T following cytokinesis. The mitotic GB 6-4A also shows apical Insc localization. Because Insc is sufficient to orient the mitotic spindle in all neuroblasts and epithelial cells assayed, the apical localization of Insc in NGB 6-4T and GB 6-4A provides strong confirmation that both cells divide along their apical-basal axis (Freeman, 2001).

Miranda, Prospero, Staufen, and Numb proteins mark the basal side of many or all mitotic neuroblasts and regulate the fate of daughter cells or their neuronal progeny. In NGB 6-4T, Miranda, Prospero, Staufen and Numb all form basal crescents from metaphase through telophase, and are partitioned into the basally positioned G daughter cell of NGB 6-4T after cytokinesis. The mitotic GB 6-4A also shows basal localization of Miranda, Prospero, Staufen and Numb. These results further confirm the apical-basal division axis of NGB 6-4T and GB 6-4A during glial producing divisions, and show that all of the above proteins are candidates for regulating gcm expression in the basal G daughter cell of NGB 6-4T (Freeman, 2001).

To determine if miranda, prospero, staufen or numb are involved in the development of glia in the NGB 6-4T lineage, embryos mutant for each gene were scored for the number and position of mature glia derived from NGB 6-4T. The three glia from NGB 6-4T express repo and have distinctive positions within the CNS: two near the midline and one between NGB 6-4T and the midline. These are the only Repo-positive glia adjacent to the midline at the ventral surface of the CNS, and thus are easy to identify unambiguously. Mutations in staufen and numb have no effect on glial development in the NGB 6-4T lineage. By contrast, prospero mutant embryos show striking loss of NGB 6-4T-derived glia, while miranda mutant embryos have a similar but weaker phenotype. It is concluded that prospero and miranda, but not staufen or numb, are required for normal glial development in the NGB 6-4T lineage (Freeman, 2001).

To determine the earliest aspect of the prospero mutant phenotype in the NGB 6-4T lineage, whether gcm is expressed normally in the G daughter cell was assayed. In wild-type embryos, Gcm protein is detectable in the predivisional NGB 6-4T and in the sibling NGB 6-4T/G cells immediately after cytokinesis; subsequently, Gcm disappears from NGB 6-4T and is upregulated in the G cell and its progeny, which proceed to migrate medially and express repo. In prospero mutant embryos, gcm expression is activated normally in the predivisional NGB 6-4T and is detectable in the immediately post-mitotic NGB 6-4T and G cell. Therefore the early induction of gcm expression in these cells is clearly prospero-independent. However, Gcm protein levels subsequently decline in the G cell and its progeny and these cells fail to migrate to the midline or express repo. These data indicate that Prospero is required in the G daughter cell to maintain or upregulate gcm expression levels, induce medial migration, and activate repo expression. Surprisingly, these Gcm negative, Repo negative cells do not express the neuron-specific elav gene, and thus they appear unable to differentiate as glia or neurons (Freeman, 2001).

In prospero mutant embryos, gcm expression is also greatly reduced in the progeny of NGB 7-4. Low level expression of gcm is detectable in many NGB 7-4 progeny shortly after their birth, indicating that in this lineage (as in NGB 6-4T) the induction of gcm expression can occur in the absence of prospero function. However, gcm expression fades rapidly and these cells never express repo. Thus, prospero is essential for the maintenance of gcm expression and normal glial cell fate induction in both the NGB 6-4T and 7-4 lineages (Freeman, 2001).

prospero is clearly necessary for upregulation of gcm and glial cell fate induction in the 6-4T and 7-4 lineages, but is it sufficient to induce gcm expression in these lineages? In miranda mutant embryos, prospero mRNA and protein are delocalized during neural precursor cell division, resulting in similar concentrations of Prospero segregating to both NGBs and their daughter cells. Interestingly, in miranda mutants ectopic gcm expression is found in NGB6-4T at stage 13, a time when this NGB is normally making neuronal progeny. miranda mutants also show ectopic expression of gcm in NGB 7-4 lineage during its window of glial production. Thus, mislocalization of Prospero to the NGB by removal of miranda function is sufficient to induce ectopic gcm expression in these NGBs (Freeman, 2001).

Does the upregulation of gcm in NGBs drive the production of extra glial progeny? To address this question, Repo expression was assayed in the NGB6-4T lineage in miranda mutants because the entire NGB 6-4T lineage can be identified. In miranda mutants only four Repo positive cells are typically found in the entire NGB 6-4T lineage, but neuronal progeny are completely absent. This phenotype is interpreted to indicate that the G cell produces three glia as usual, but that its sibling NGB differentiates directly into a Repo-positive glia cell, resulting in a termination of the lineage. Thus, it appears that Prospero mislocalized to the NGB can potently activate gcm expression in the NGB and transform it into a glial cell (Freeman, 2001).

Two additional phenotypic classes in miranda mutants are found: (1) a variable number of Repo-positive glia are produced (between two and four) and subsequent neuronal progeny are generated normally (12% of hemisegments); or (2) the wild-type pattern of three Repo-positive glia and neuronal progeny are produced. These phenotypes indicate that low level Prospero in the NGB is not always sufficient to induce a glial fate, and that reduced Prospero in the G cell may lead to fewer glial progeny (Freeman, 2001).

Mislocalization of Prospero to NGB 7-4 by removal of miranda function can also induce Repo expression in this NGB (25% of hemisegments), showing that NGB 7-4 can also be partially transformed towards a glial fate. It is not known if this NGB differentiates as a glial cell (like the Pros-positive NGB 6-4T), generates extra glial progeny, or if it can eventually produce neurons. miranda, prospero double mutants do not show upregulation of gcm in NGBs 6-4T or 7-4, demonstrating that the upregulation of gcm in these NGBs in miranda mutant embryos is due to Prospero protein that is delocalized into the NGB. These results indicate that Miranda, by asymmetrically localizing Prospero to NGB daughter cells, restricts gcm upregulation and induction of the glial developmental program to the progeny of NGBs 6-4T and 7-4 during their phases of glial production (Freeman, 2001).

Previous reports have suggested that GCM mRNA is asymmetrically localized in a medial crescent in the mitotic NGB 6-4T, resulting in the selective partitioning of GCM mRNA into the medial glia-producing progeny of NGB 6-4T. These conclusions are likely to be in error for three main reasons: (1) the mitotic NGB 6-4T contains evenly distributed GCM mRNA, and this mRNA is partitioned equally between NGB 6-4T and its glial-producing G daughter cell after cytokinesis; (2) previous studies did not use a mitosis-specific marker together with probes for GCM mRNA localization to prove that the localization was being scored in mitotic NGBs; (3) NGB 6-4T always divides along the apical-basal axis, therefore a medial localization of GCM mRNA would not result in it being partitioned unequally into one daughter cell. Indeed, in a recent study use was made of more specific markers for mitotic stage and cell orientation. It was found that the first division of NGB 6-4T is not along the mediolateral axis, and that GCM mRNA is inherited by both the NGB and the G daughter cell. It is therefore concluded that the asymmetric localization of GCM mRNA is not the mechanism by which neuronal and glial lineages are separated in the NGB 6-4T lineage (Freeman, 2001).

Gcm protein has been reported to be absent from the predivisional NGB 6-4T, perhaps owing to translational repression of GCM mRNA prior to its first division. This is not the case, since Gcm protein is clearly present in the predivisional NGB 6-4T. In addition, it has been reported that Gcm protein is excluded from the nucleus in the postdivisional NGB 6-4T, and that an unidentified mechanism regulates nuclear entry of Gcm specifically in the G daughter cell. Robust Gcm protein expression, however, has been shown in the nucleus of NGB 6-4T, arguing strongly against the existence of such a mechanism (Freeman, 2001).

This study shows that gcm is induced properly in prospero mutant embryos, but that both the G daughter cell and the post-divisional NGB downregulate gcm expression with a similar timecourse. Thus, the induction of gcm expression in NGB 6-4T is prospero-independent. Interestingly, prospero mutant embryos also fail to upregulate gcm to high levels in NGB 7-4 daughter cells. This is consistent with a previous report that NGB 7-4-derived Repo-positive glia are absent in prospero mutants. Unlike NGB 6-4T, gcm expression was not detected in NGB 7-4, only in its new-born (pre-migration) daughter cells. Low level Gcm expression is still present in prospero mutant embryos; thus, the induction of gcm expression in this lineage also appears to be prospero-independent. It is proposed that prospero functions to upregulate low levels of gcm in these lineages, but is not sufficient to induce gcm expression on its own. Such a mechanism would explain why all neural stem cell progeny in the CNS express high levels of prospero but most never induce gcm and the glial developmental program. In agreement with this model, misexpression of low levels of gcm throughout the CNS requires Prospero to induce glia in a subset of lineages (Freeman, 2001).

How might the Prospero transcription factor upregulate low levels of gcm expression? Prospero is known to act as a co-factor to stimulate transcriptional activity of several DNA-binding proteins. Recent studies show that Gcm can positively autoregulate its own expression in neural tissues. It is possible that Prospero may act together with Gcm to stimulate expression levels of the gcm gene until they become sufficiently high for Gcm to positively autoregulate its own expression. However, embryos homozygous for the gcmN7-4 allele produce non-functional Gcm protein that is upregulated with normal kinetics in the NGB 6-4T and 7-4 lineages, indicating that Gcm function is dispensable for its own upregulation. It is proposed that a lineage-specific co-factor or extrinsic signal converges with Prospero function in these lineages to upregulate gcm expression (Freeman, 2001).

How does a NGB know when to make neurons or glia? For example, the first born daughter cell from NGB 6-4T gives rise to glia while all subsequent progeny are neuronal. By contrast, NGB 7-4 first produces several neuronal progeny, then switches to making glia, and finally switches back to making neurons. Interestingly, the window of developmental time during which these two neural precursors are making glia are strikingly similar: NBG 7-4 begins making glia at stage 10, shortly after this NGB 6-4T is born (late stage 10) and begins making glia; at stage 11 both precursors terminate glial production and switch to making neurons. The coordinate timing of glial production from these lineages may indicate that a temporally regulated extrinsic cue induces gcm expression in these NGBs or their newly born progeny (Freeman, 2001).

In prospero mutant embryos, NGB 6-4T and its progeny only transiently express low levels of gcm. What is the fate of these cells? They never express the glial-specific repo gene, and they also fail to express the neuron-specific elav gene, indicating that neither the glial or neuronal developmental program has been initiated. gcm is thought to transcriptionally activate genes that promote glial fate or repress neuronal fate. It is proposed that NGB 6-4T in prospero mutants produces enough Gcm protein to repress neuron-specific genes, yet insufficient amounts to robustly induce glial-specific genes. This in turn suggests that there may be different gcm thresholds for activating glial development (high threshold) and for repressing neuronal development (low threshold) (Freeman, 2001).

In miranda mutant embryos, Prospero protein is delocalized at mitosis, allowing NGB/daughter cell siblings to inherit equal concentrations of Prospero. In these embryos, ectopically upregulated gcm is frequently seen in NGBs 6-4T and 7-4, and extra glia are derived from NGB 6-4T. These data indicate that prospero is a potent activator of gcm expression in the NGB 6-4T and 7-4 lineages. The extra glia observed could come from an extension of the glial portion of the NGB 6-4T lineage, or from a transformation of this NGB into a purely glial progenitor. The latter model is favored, because neurons are never observed in the NGB 6-4T lineage when extra glia are observed. Moreover, high levels of Gcm are correlated with pure glial lineages such as GB 6-4A and the GP, and gcm is known to positively autoregulate which may commit precursors with high Gcm to a glial-producing fate (Freeman, 2001).

In miranda mutant embryos, the ectopic expression of gcm in NGB 6-4T and 7-4 is in fact due to delocalization of Prospero and not simply the absence of Miranda, because miranda; prospero double mutants fail to upregulate gcm in NGBs. In both the NGB 6-4T and 7-4 lineage, the delocalization of Prospero has relatively little effect on glial production by the daughter cells, presumably because there is sufficient Prospero protein in these daughter cells to upregulate gcm expression. Thus, with respect to glial cell fate induction, the asymmetric localization of Prospero may be more important for removing Prospero from the NGB than for enriching Prospero in the daughter cell (Freeman, 2001).

Specification of Drosophila hematopoietic lineage by conserved transcription factors

Two major classes of cells observed within the Drosophila hematopoietic repertoire are plasmatocytes/macrophages and crystal cells. The transcription factor Lz (Lozenge), which resembles human AML1 (acute myeloid leukemia- 1) protein, is necessary for the development of crystal cells during embryonic and larval hematopoiesis. Another transcription factor, Gcm (glial cells missing), is required for plasmatocyte development. Misexpression of Gcm causes crystal cells to be transformed into plasmatocytes. The Drosophila GATA protein Srp (Serpent) is required for both Lz and Gcm expression and is necessary for the development of both classes of hemocytes, whereas Lz and Gcm are required in a lineage-specific manner. Given the similarities of Srp and Lz to mammalian GATA and AML1 proteins, observations in Drosophila are likely to have broad implications for understanding mammalian hematopoiesis and leukemias (Lebestky, 2000).

Hemocytes of the Drosophila embryo are derived from the head mesoderm. The hemocyte precursors express the GATA factor Srp and give rise to two classes of cells: plasmatocytes and crystal cells. Plasmatocytes spread throughout the endolymph and act as macrophages, whereas crystal cells contain crystalline inclusions and are involved in the melanization of pathogenic material in the hemolymph. These cells can be first recognized in the late embryo, where they form a cluster around the proventriculus. Crystal cells are made clearly visible by the Black cell (Bc) mutation, which causes premature melanization of the crystalline inclusions (Lebestky, 2000 and refereces therein).

Lz is first detected in a small cluster of cells within the embryonic head mesoderm in a bilaterally symmetric pattern. Lz expression remains localized in bilateral clusters of 20 to 30 cells within the head mesoderm. At later stages, these crystal cell precursors (CCPs) form a loose cluster around the proventriculus. These cells have smooth, round morphology with large nuclei. The CCPs form a subset of the Srp-expressing hemocyte precursors (Lebestky, 2000).

The transcription factor Gcm promotes glial cell fate, and it also functions downstream of Srp in plasmatocyte differentiation. Lz expression is unaffected in gcm mutants. Gcm expression is initiated in a number of Srp-expressing hemocyte precursors, but Gcm is excluded from the CCPs. Consistent with their cell fate, the small subset of plasmatocytes derived from Lz-expressing progenitors do initiate Gcm expression. Gcm was misexpressed in the CCPs to assess whether exclusion of Gcm from these cells is essential for proper fate determination. This results in the transformation of CCPs into plasmatocytes. The converted cells exhibit morphological characteristics of plasmatocytes and express Croquemort. Moreover, in third-instar larvae, misexpression of Gcm in CCPs prevents the development of all crystal cells. These results suggest that the restricted expression of Gcm is required for the developmental program of embryonic plasmatocytes, and that its misexpression can override Lz-mediated crystal cell differentiation during both embryonic and larval hematopoiesis. The converse experiment of Lz misexpression in the entire hemocyte pool under the control of a heat shock promoter does not convert plasmatocytes into crystal cells. Vertebrate homologs of Gcm have been identified, but any role in hematopoiesis has not been investigated (Lebestky, 2000).

A model of Drosophila hematopoiesis is presented in which a pool of Srp-positive hemocyte precursors gives rise to a large population of Gcm-positive cells and a smaller subpopulation of Lz-positive cells. These results support a genetic hierarchy in which Srp, a Drosophila GATA factor, acts upstream of both Gcm and Lz, two mutually exclusive, lineage-specific transcription factors in hematopoiesis. Although the description of this hierarchy is incomplete in terms of the breadth of molecules involved, it does provide a theoretical framework for understanding how early hematopoietic progenitors in the embryo can differentiate and assume distinct cell fates (Lebestky, 2000).

A requirement for Notch in the genesis of a subset of glial cells in the Drosophila embryonic central nervous system which arise through asymmetric divisions

In the Drosophila CNS glial cells are known to be generated from glioblasts, which produce exclusively glia or neuroglioblasts that bifurcate to produce both neuronal and glial sublineages. The genesis of a subset of glial cells, the subperineurial glia (SPGs), involves a new mechanism and requires Notch. SPGs share direct sibling relationships with neurons and are the products of asymmetric divisions. This mechanism of specifying glial cell fates within the CNS is novel and provides further insight into regulatory interactions leading to glial cell fate determination. Furthermore, Notch signaling positively regulates glial cells missing expression in the context of SPG development (Udolph, 2001).

In order to better understand how a complete lineage of a specific NGB with all its progeny, including its glial cells, might be created, NB1-1 was chosen for a detailed analysis. NB1- 1 has been extensively used for cell fate specification studies and a sound basis of information about this NB lineage is available. NB1-1 is a NB that develops differential lineages in the thoracic versus the abdominal segments. Focus was placed on the abdominal NB1-1A because only these abdominal NB1-1 lineages contain glia. In addition to the aCC/pCC sibling neurons, which are the progeny of the first GMC produced from this lineage, NB1-1A generates 2 to 3 glial cells and 4 to 5 clustered interneurons (cN), yielding a total of 9 to 10 cells. The three glial cells belong to the group of subperineurial glia (SPG) that lie at the periphery of the nerve cord and enwrap the entire ventral nervous system. Two of the glia, the A- and B-SPGs, can be found in dorsal positions, with a third glia, the LV-SPG, located at ventral positions of the nerve cord. All SPGs, including the A- and B-SPG and LV-SPG of NB1-1A, are specifically labelled by two enhancer trap lines, M84 and P101 (Udolph, 2001 and references therein).

The expression patterns of the enhancer-trap lines M84 and P101 are indistinguishable in abdominal segments: both are expressed in the SPGs including the A- and B-SPG and LV-SPG cells produced from NB1-1A. To investigate the relationship between the three glial cells derived from NB1-1A, M84/P101 (a stock double homozygous for both the M84 and P101 insertions), embryos were stained with anti-ß-gal. In stage 12/13 embryos, a single M84/P101+ cell appears in dorsal positions of each hemi-neuromere. Embryos double labelled with anti-Even-skipped (anti-Eve) indicates that this cell is located posterior to the Eve+ cluster of NB1-1 (aCC/pCC) and NB7-1 progeny (CQ-neurons). Slightly later, a second M84/P101+ cell appears anterior to the first cell. According to the positioning within the developing nerve cord, the posterior cell represents the A-SPG, whereas the anterior cell is the B-SPG. A third glial cell, representing the LV-SPG, can be detected in ventral positions only after the A- and B-SPG are already present, suggesting that this cell is the last born glia within the lineage (Udolph, 2001).

As a first step toward elucidating the origin of the glial cells of the NB1-1A lineage, the effects of loss of function mutants in several genes, Notch, mastermind (mam) and numb, which are known to affect the resolution of distinct sibling cell fates, were tested for their effect on the development of A-, B- and LV-SPGs. Embryos hemizygous/homozygous for a conditional Notch allele, Nts1, and also carrying one copy each of M84 and P101 (Nts1/M84/P101) were subjected to the non-permissive temperature of 29°>C after 6 hours of development. This regime allows Notch to function during the singling out of NBs and removes Notch during the crucial period when it is required for sibling cell fate resolution. Double staining with anti-Eve and anti-ß-gal was performed. As expected, in most hemisegments, Nts1/M84/P101 embryos duplicate the RP2 neuron at the expense of its sibling cell. Moreover, in 96% of the hemisegments, M84/P101+ cells could not be found in typical dorsal or ventral positions. It is concluded that Notch function is required for the specification of the M84/P101 positive A-, B- and LV-SPGs. In wild-type embryos, M84/P101 is expressed in about eight SPGs per hemisegment, including the A- and B-SPGs and the LV-SPG (Udolph, 2001).

Removing Notch function results in the near complete abolishment of all M84/P101 expression, indicating a more general function for Notch in SPG specification. These findings prompted an investigation whether Notch is required in the specification of CNS glia in general. Experiments were performed in which glia-specific markers, either enhancer trap lines (gcm-lacZrA87 and pnt-lacZ) or an antibody (anti-Repo) were used. gcm-lacZrA87 is an insertion into the gcm gene and expresses ß-gal in the pattern of gcm. pointed (pnt) is specifically expressed in glial cells and has been reported to act downstream of gcm. Nts1embryos were shifted to the non-permissive temperature after 6 hours of development, and subsequently stained with anti-Eve and anti-ß-gal at late stage 16; taking into account the missing SPGs, both enhancer traps were expressed in a pattern reminiscent of wild-type embryos. However, an increase in the number of ß-gal-positive cells was observed, which could be in part due to a mild neurogenic phenotype caused by Nts1. Furthermore, it was found that the glial-specific protein Repo was widely expressed in the CNS of N55e11 (an amorphic N allele) embryos. Thus, general glial specific markers like gcm, pnt and repo are expressed in embryos that lack or have strongly reduced Notch function, indicating that although Notch is required for the formation of SPGs, there is not a global requirement for Notch in the specification of all CNS glia (Udolph, 2001).

Another neurogenic gene, mastermind, which has been linked to the Notch signaling pathway by its genetic interactions with Notch and its strikingly similar phenotype in early and late neurogenesis, was also tested. mam acts downstream of Notch during sibling cell fate specification in the embryonic nervous system. The hypomorphic mam345 allele used in this study shows only a mild hypertrophy of the nervous system but clearly has an effect on sibling cell fate specification. A severe reduction (94%) of P101+ cells was observed in mam345;P101 embryos similar to that seen with Nts1/M84/P101 embryos. These data suggest that both genes are strictly required for the specification of SPGs, most likely in a linear pathway. However, it is unclear how Notch acts in the specification of the SPGs. The possibility is considered that SPG glial cells could arise from a series of asymmetric cell divisions, with Notch being required to specify the glial daughters of these divisions (Udolph, 2001).

Based on its function as a negative regulator of Notch signaling, the expected numb phenotype is opposite that of Notch in terms of sibling cell fate transformation. The P101 expression pattern was tested in the background of a strong numb mutation. In contrast to Notch and mam, additional P101+ cells were found in the vicinity of the aCC/pCC position. In most of the examined hemi-neuromeres, up to four ß-gal-positive cells were detected in dorsal positions close to aCC/pCC. This is indicative of a duplication of the A- and B-SPGs. Additional P101+ cells with glial morphology were found in lateral and ventral positions of the nerve cord, presumably duplications of other SPGs. These findings are consistent with an asymmetric cell division model for the genesis of the SPGs (Udolph, 2001).

Clonal analysis demonstrates that the loss of Notch function results in the loss of SPGs and a concomitant gain of neuronal cells within the NB1-1A lineage, consistent with the notion that a sibling cell fate relationship exists between cluster neurons and glial cells in this lineage and that Notch is required for the asymmetric divisions that generate these postulated neuron/glia sibling pairs. The data support a hypothesis that glial cells and cluster neuron s share a sibling relationship within the NB1-1A lineage. Taken together, the data favour a model in which a series of three GMCs produced from NB1-1A can each divide asymmetrically to produce a neuron and a glia, with Notch signaling required to specify the glia fate, and Numb and the absence of Notch signaling required for the neuronal fate (Udolph, 2001).

It is known that gcm acts as a master regulator in gliogenesis because it has been described as a binary switch between glia and neurons. As expected, gcm appears to be required for the formation of SPGs. P101 marker gene expression is abolished in embryos homozygous for gcm. The similarity of gcm and Notch phenotypes suggests the possibility that both genes share a common pathway required for SPG specification. It was reasoned that if Notch acts downstream of gcm, then overexpression of activated N in a gcm minus genetic background should result in additional SPGs; however, if Notch acts upstream of gcm, then in a gcm minus background the overexpression of activated N should not result in the production of additional P101+ cells. In a genetic background lacking gcm function, activated N expression is unable to induce SPG development as indicated by the loss of marker gene expression. These findings are consistent with the notion that Notch functions upstream of gcm in the context of SPG development (Udolph, 2001).

Two types of neuroectodermally derived glial progenitors in the embryonic nervous system of Drosophila have been described. Glioblasts (GB) generate only glial progeny, and NGBs produce both neurons and glial cells within the same lineage. A mechanism by which both neurons and glia can arise within the lineage (NGB6-4T) of a thoracic NGB has recently been described. NGB6-4T represents a thoracic-specific NGB in which glial and neuronal sublineages bifurcate from each other during the first division of the parental NGB. Only one of the two daughters expresses Gcm, a master regulator of glial cell fate that is involved in regulating the expression of other glia-specific target genes. In the NGB6-4T lineage, the asymmetric distribution of Gcm results in a cell that is specified as a GB within a neuroglioblast lineage. The GB will exclusively generate the glial components, whereas its sibling will exclusively give rise to the neuronal components of the lineage (Udolph, 2001).

This study reports a novel mechanism by which glia can be generated during CNS development. The data suggest that NB1-1A gives rise to a set of three glial cells through a series of three GMC asymmetric divisions. Several lines of evidence support the notion that within a NGB lineage GMCs can produce both glial and neuronal cells. (1) Immunohistochemical analyses indicate that the A-, B- and LV-SPG arise at different times in development, and their non-simultaneous birth, in conjunction with the fact that their formation can be differentially affected by inactivation of Notch, suggests that they do not derive from a common precursor; (2) mutations in genes involved in specifying alternative sibling cell fates affect SPG development in a fashion that suggests these cells are siblings with non-glial components of the lineage; (3) transplantation experiments indicate that the 3 SPGs share sibling relationships with the neuronal components of the NB1-1A lineage, the cluster neurons (cN). Finally, analysis of two cell FLP-clones demonstrate that SPG glia and neurons share a direct sibling relationship (Udolph, 2001).

This proposed mechanism for the genesis of SPGs is fundamentally different from the ones described for GBs, e.g. anterior glioblast, and for NGBs, e.g. NGB6-4T and NGB5-6A. The first division of NB1-1A produces a neurogenic GMC that gives rise to a pair of sibling neuron s (aCC/pCC), and at the level of the first division of the parental NB no GB sublineage is bifurcated. In addition, the NB1-1A derived glial cells share a direct sibling relationship with neurons and does not involve the generation of a lineage internal GB at all. The asymmetric origin of glia described here provides a novel mechanistic framework of glial origin that might be used as a model system to gain further insights into the regulatory networks involved in glial cell fate specification in the CNS. It is concluded that the mechanisms leading to glial cell fate specification are complex and that multiple developmental mechanisms lead to glial cell fate specification. These mechanisms will be likely to require different molecular machinery that involve distinct sets of genes or genetic hierarchies; for example, Notch being required specifically for the SPGs but apparently not for other types of glia. However, most of the glial cells (except midline glia) strictly require the gcm gene. Thus, there seems to be a common molecular basis that is context and cell specifically regulated during development (Udolph, 2001).

This study provides the first evidence that Notch is a crucial component in the specification of a subclass of CNS glial cells, the subperineurial glia (SPG). As revealed by the transplantation experiments, Notch is not only required for the expression of the M84/P101 marker, but loss of Notch function leads to the loss of the SPG cell fate per se. In the case of NB1-1A, the data indicate that the loss of glia is accompanied by the conversion of these cells into neurons. In contrast, Notch gain of function results in the overproduction of SPG-like cells at the expense of the cluster neurons. Hence, Notch signaling is sufficient for the specification of these cells. A second gene, gcm, is also crucially required for the specification of the SPGs; in a gcm mutant background M84/P101 expression is completely absent. If an activated form of the Notch protein is ectopically expressed in the embryonic CNS of animals lacking gcm function, SPGs do not form; in addition, M84/P101 expression cannot be detected in the CNS of these embryos. This indicates that gcm function is strictly required for Notch-mediated SPG specification, suggesting that Notch functions upstream of gcm. It is interesting to note that Notch also appears to play an instructive role in gliogenesis for mammalian neural crest stem cells in culture (Udolph, 2001).

These results, in conjunction with what is already known about the origin of the aCC and pCC neurons, as well as the terminal lineage of NB1-1A, allow the following model to be proposed for the NB1-1A lineage: in total, NB1-1A gives rise to five pairs of sibling cells. The first pair comprises the aCC/pCC neurons resulting from GMC1-1a. In addition, three later born GMCs each give rise to one cluster neuron and one SPG-glia. A fifth GMC gives birth to two additional cluster neurons. The order of appearance and the position of the MP84/P101-labelled cells would suggest that, of the three glia-producing GMCs, the earliest born GMC gives rise to the A-SPG; the latest born GMC gives rise to the LV-SPG; and the GMC born in the middle gives rise to the B-SPG. The time of birth of the fifth GMC in this lineage, which is postulated to produce two cluster neurons, cannot be determined. Clearly, refinement and proof for this proposal will require experiments that provide direct temporal information (Udolph, 2001).

Context-dependent utilization of Notch activity in Drosophila glial determination

During Drosophila neurogenesis, glial differentiation depends on the expression of glial cells missing. Understanding how glial fate is achieved thus requires knowledge of the temporal and spatial control mechanisms directing gcm expression. In the adult bristle lineage, gcm expression is negatively regulated by Notch signaling. The effect of Notch activation on gliogenesis is context-dependent. In the dorsal bipolar dendritic (dbd) sensory lineage in the embryonic peripheral nervous system (PNS), asymmetric cell division of the dbd precursor produces a neuron and a glial cell, where gcm expression is activated in the glial daughter. Within the dbd lineage, Notch is specifically activated in one of the daughter cells and is required for gcm expression and a glial fate. Thus Notch activity has opposite consequences on gcm expression in two PNS lineages. Ectopic Notch activation can direct gliogenesis in a subset of embryonic PNS lineages, suggesting that Notch-dependent gliogenesis is supported in certain developmental contexts. Evidence is presented that POU-domain protein Nubbin/PDM-1 is one of the factors that provides such context (Umesono, 2002).

Notch signaling promotes glial fate during asymmetric division in the embryonic dbd lineage. Notch is specifically activated in the presumptive DBD support glia cell (DBDG) owing to the negative regulation by Numb in the sibling cell, and provides instructive information to induce gcm transcription and glial development. Expression of gcm occurs quickly after the artificial activation of Notch, even in cells that have initiated neuronal development. In gcm mutants, DBDG are transformed into neurons, although the activation of Notch, visualized by the Su(H)-reporter, is normal in the presumptive glia. Likewise, ectopic expression of gcm in presumptive dbd neurons causes neuron-to-glia transformation without affecting Notch activity. These findings suggest that gcm expression appears to be the sole target of Notch activation in establishing glial fate in the dbd lineage. Within the 3.5 kb region upstream of the gcm gene, two sequences have been identified that perfectly match the consensus core sites for Su(H). Thus, gcm could be a direct target of Su(H), downstream of the Notch signaling pathway (Umesono, 2002).

While the present data demonstrate a positive role for Notch in gliogenesis in the dbd lineage, other embryonic PNS glial cells do not require Notch activity for their formation. For example, in the adult bristle lineage Notch has an opposite function on gliogenesis; that of repressing gcm expression and glial development. Thus the role of Notch in the regulation of gcm expression is context-dependent. Notch has recently been shown to be a component of combinatorial signaling in cell fate determination in the Drosophila eye. It is possible that Notch signaling has different consequences depending on other factors that act on the same regulatory element (Umesono, 2002).

The context-dependent effect of Notch suggests that the gcm promoter may have a modular structure where each unit integrates different developmental signals. However, given the large diversity of glial subtypes in the nervous system, it is unlikely that each glial subtype has its own regulatory sequences and a unique mode of regulation. A model is favored in which gcm has a limited number of regulatory elements that respond to developmental signals that are present in multiple environments. Indeed a subset of glial subtypes respond in a similar way to Notch signals: in addition to the dbd lineage, the dda lineage can also induce gcm transcription upon Notch activation. Comparison of these two lineages offers hints on the nature of the developmental context in which Notch activation causes gcm transcription (Umesono, 2002).

One common feature that distinguishes dbd and dda lineages from other PNS lineages is the cell division pattern of their SOP. In dbd and dda lineages, SOPs divide to generate a neuron and a glial cell through an asymmetric division. In other gliogenic PNS lineages, the sibling cells of glial cells are not postmitotic neurons, but tertiary precursors that undergo further division to generate neurons and associated cells. These observations suggest that an interaction with the neuronal sibling may play a crucial part in promoting the Notch-dependent gcm activation during asymmetric cell division. Recently, Notch was shown to positively regulate gcm expression in the Neuroblast 1-1A lineage of the CNS, where the sibling pattern is identical to that of the dbd lineage. This also supports the idea that the cell division pattern provides a context that determines the effect of Notch activity (Umesono, 2002).

Coexpression of constitutively active Notch with Nubbin also generates ectopic glia outside dbd and dda lineages. This raises the possibility that Nubbin may be a part of the developmental context that allows Notch to promote gliogenesis. Within the embryonic PNS, dbd and dda neurons are the only two neurons that express Nubbin. In both lineages, Nubbin is present in both SOP daughter cells, at the time of glia versus neuron cell fate choice. Furthermore, temporal activation of Nubbin has been detected in presumptive glial cells derived from the NB1-1A lineage. Nubbin thus might create a permissive environment for the activation of gcm expression by the Notch signal. Since coexpression of Nubbin and constitutively active Notch does not cause glial transformation of all neurons, additional factors must exist that create a Notch-dependent gliogenic context (Umesono, 2002).

Nubbin is a POU-domain transcription factor with sequence-specific DNA-binding activity. The contextual role of Nubbin in Notch-dependent expression of gcm could employ a similar mechanism to the modulation of Notch activity in wing development, where Nubbin and Su(H) bind on the same enhancer element of Notch target genes. It will be interesting to further analyze the role of Nubbin in gliogenic lineages (Umesono, 2002).

DPP signaling controls gcm expression and the development of the lamina glia required for retinal axon targeting in the visual system of Drosophila

The Drosophila visual system consists of the compound eyes and the optic ganglia in the brain. Among the eight photoreceptor (R) neurons, axons from the R1-R6 neurons stop between two layers of glial cells in the lamina, the most superficial ganglion in the optic lobe. Although it has been suggested that the lamina glia serve as intermediate targets of R axons, little is known about the mechanisms by which these cells develop. DPP signaling has been shown to play a key role in this process. dpp is expressed at the margin of the lamina target region, where glial precursors reside. The generation of clones mutant for Medea, the DPP signal transducer, or inhibition of DPP signaling in this region results in defects in R neuron projection patterns and in the lamina morphology; these defects are caused by defects in the differentiation of the lamina glial cells. glial cells missing is expressed shortly after glia precursors start to differentiate and migrate. Its expression depends on DPP; gcm is reduced or absent in dpp mutants or Medea clones, and ectopic activation of DPP signaling induces ectopic expression of gcm and Repo. In addition, R axon projections and lamina glia development are impaired by the expression of a dominant-negative form of gcm, suggesting that gcm indeed controls the differentiation of lamina glial cells. These results suggest that DPP signaling mediates the maturation of the lamina glia required for the correct R axon projection pattern by controlling the expression of gcm (Yoshida, 2005).

dpp is expressed in the dorsal and ventral margins of the posterior region of the optic lobe, adjacent to the cells expressing wg, which induces dpp expression. Glial cells in the lamina target region arise from these regions and migrate into the lamina target region as they contact R axons. Axons from R1-R6 neurons stop between two rows of glial cell layers, the epithelial and marginal layers, and form the lamina plexus. The third row of glial cells, the medulla glia, is located just beneath the marginal glia. The homeodomain protein Repo is expressed in these glial cells (Yoshida, 2005).

The expression pattern of dpp-lacZ, an enhancer-trap allele of dpp, was compared with the expression pattern of Repo. At a stage prior to glia differentiation and migration, expression of the dpp reporter is detected in the dorsal and ventral margins of the lamina target region. dpp continues to be expressed at the margins of the lamina target region throughout the third larval instar (Yoshida, 2005).

wg at the posterior-most domain induces the expression of dpp and omb. Some wg-expressing cells extend projections towards the lamina target region. These cells extend scaffold axons along which the lamina glia migrate. Thus, it was possible that the wg signal is involved in the migration and/or differentiation of lamina glia. However, partial elimination of Wg activity with a wgts allele does not cause a specific defect in glia migration. Therefore, wg may play a role in organizing domains in the visual cortex by activating/repressing various genes, rather than contributing to the generation of specific cell types (Yoshida, 2005).

Medea is required for lamina glia development. Medea encodes a co-SMAD and mediates a range of DPP/BMP/TGFß signaling events. In addition to dpp, four related genes -- glass bottom boat (gbb), screw, activin and activin2 -- have been identified in Drosophila. GBB signals through TKV/Saxophone (SAX) and Wishful Thinking (WIT) type I and type II receptors, respectively. Activin uses Baboon as a type I receptor, and Punt and WIT as type II receptors. Brains mutant for gbb and wit were examined, but no defects in lamina glia development were observed. It is concluded that it is highly likely that dpp is the ligand responsible for lamina glia development. However, the possibility that one or more of the DPP-related ligands acts redundantly in this process cannot be excluded (Yoshida, 2005).

In the embryo, gcm initiates the specification of glial cells from neural cells of various lineages. gcm expression is strictly controlled to ensure the correct separation of glial versus neuronal cell fate. Analysis of the cis-regulatory elements of gcm suggests that gcm expression depends on multiple regulatory elements to allow the control of lineage-specific transcription and autoregulation. The analysis carried out in this study suggests that a different situation exists in the optic lobe; gcm is expressed in the glia and the lamina neuronal cells, and is required for the differentiation of these cell types. In addition, differentiation is controlled differently in the lamina and in the glia. In the lamina, gcm expression seems to be controlled by hh, and in the glia, by dpp. These results suggest that gcm is controlled and functioning in a different manner in the optic lobe. Uncovering the mechanisms of the control and function of gcm would probably prove an intriguing focus for future research (Yoshida, 2005).

DPP and its vertebrate homolog BMP play crucial roles in many aspects of development by controlling patterning, cell growth and differentiation. This analysis reveals a role for DPP signaling in lamina glia differentiation in the Drosophila visual system. DPP has also been reported to function in several aspects of visual center development; for instance, DPP signaling has been shown to be involved in the proliferation and migration of the subretinal glia in eye disc development, which plays an important role in the R axon navigation. In addition, defects have been reported in the medulla neuropile in dpp mutant animals, suggesting a role for dpp in neuronal fate specification. Furthermore, tkv is expressed in lamina precursor cells just ahead of the lamina furrow, where these cells meet R axons and start to differentiate. Although this possibility is one of the things that prompted an examination of the role of DPP signaling in lamina development, no defects were uncovered when Mad or Medea clones were generated in the OPC or the lamina. Moreover, dpp appears to be expressed in the inner proliferation center (IPC), which will form the lobula, in addition to its expression in the dorsal and ventral marginal domains. Thus, dpp may be required for some aspects of lobula development. Unfortunately, this cannot be easily addressed at this moment because of a lack of appropriate markers. Further study of the requirements for dpp in the lamina, the medulla, the lobula and other cell types could lead to a more comprehensive understanding of how DPP signaling controls differentiation and other events during development of the visual system (Yoshida, 2005).

Prospero, targeting gcm, acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells

Stem cells have the remarkable ability to give rise to both self-renewing and differentiating daughter cells. Drosophila neural stem cells segregate cell-fate determinants from the self-renewing cell to the differentiating daughter at each division. This study shows that one such determinant, the homeodomain transcription factor Prospero, regulates the choice between stem cell self-renewal and differentiation. The in vivo targets of Prospero have been identified throughout the entire genome. Prospero represses genes required for self-renewal, such as stem cell fate genes and cell-cycle genes. Surprisingly, Prospero is also required to activate genes for terminal differentiation. In the absence of Prospero, differentiating daughters revert to a stem cell-like fate: they express markers of self-renewal, exhibit increased proliferation, and fail to differentiate. These results define a blueprint for the transition from stem cell self-renewal to terminal differentiation (Choksi, 2006).

To identify sites within the Drosophila genome to which Prospero binds, use was made of an in vivo binding-site profiling technique, DamID. DamID is an established method of determining the binding sites of DNA- or chromatin-associated proteins. Target sites identified by DamID have been shown to match targets identified by chromatin immunoprecipitation (ChIP). DamID enables binding sites to be tagged in vivo and later identified on DNA microarrays. In brief, the DNA or chromatin-binding protein of interest is fused to an Escherichia coli adenine methyltransferase (Dam), and the fusion protein is expressed in vivo. The DNA-binding protein targets the fusion protein to its native binding sites, and the Dam methylates local adenine residues in the sequence GATC. The sequences near the protein-DNA interaction site are thereby marked with a unique methylation tag, over approximately 2-5 kilobase pairs (kb) from the binding site. The tagged sequences can be isolated after digestion with a methylation-sensitive restriction enzyme, such as DpnI (Choksi, 2006).

Dam was fused to the N terminus of Prospero, and transgenic flies were generated. The fusion protein is expressed from the uninduced minimal Hsp70 promoter of the UAS vector, pUAST, as high levels of expression of Dam can result in extensive nonspecific methylation and cell death. As a control for nonspecific Dam activity, animals expressing Dam alone were generated. To assess the sites to which Prospero binds during neurogenesis, genomic DNA was extracted from stage 10-11 embryos, approximately 4-7 hr after egg laying (AEL), expressing either the Dam-Prospero fusion protein or the Dam protein alone. The DNA was digested with DpnI and amplified by PCR. DNA from Dam-Prospero embryos was labeled with Cy3, and control DNA with Cy5. The samples were then cohybridized to genomic microarrays. Microarrays were designed that tile the entire euchromatic Drosophila genome. A 60 base oligonucleotide was printed for approximately every 300 bp of genomic DNA, resulting in roughly 375,000 probes on a single array (Choksi, 2006).

Log-transformed ratios from four biological replicates (two standard dye configurations plus two swapped dye configurations) were normalized and averaged. Regions of the genome with a greater than 1.4-fold log ratio (corresponding to approximately a 2.6-fold enrichment) of Dam-Prospero to the control over a minimum of four adjacent genomic probes were identified as in vivo Prospero binding sites. Using these parameters, a total of 1,602 in vivo Prospero binding sites were identified in the Drosophila genome. This work demonstrates that it is possible to map in vivo binding sites across the whole genome of a multicellular organism (Choksi, 2006).

Prospero is known to regulate the differentiation of photoreceptors in the adult eye, and recently sites have been characterized to which Prospero can bind upstream of two Rhodopsin genes, Rh5 and Rh6. A variant of the Prospero consensus sequence is found four times upstream of Rh5 and four times upstream of Rh6. Prospero was shown to bind this sequence in vitro, by band shift assay, and also by a 1-hybrid interaction assay in yeast. In addition, deletion analysis demonstrated that the consensus sequence is required for the Pros-DNA interaction both in vivo and in vitro. It was found that 67% of in vivo binding sites contain at least one Prospero binding motif. Combining in vivo binding-site data with searches for the Prospero consensus sequence reveals 1,066 distinct sites within the Drosophila genome to which Prospero binds during embryogenesis (Choksi, 2006).

A total of 730 genes have one or more of the 1,066 Prospero binding sites located within 1 kb of their transcription unit. Statistical analyses to determine GO annotation enrichment on the members of the gene list that had some associated annotation (519) was performed by using a web-based set of tools, GOToolbox. Using Biological Process (GO: 0008150) as the broadest classification, a list was generated of overrepresented classes of genes (Choksi, 2006).

The three most significant classes of genes enriched in the list of putative Prospero targets are Cell Fate Commitment, Nervous System Development, and Regulation of Transcription. Utilizing GO annotation, it was found that nearly 41% of all annotated neuroblast fate genes (11 of 27) are located near Prospero binding sites and that approximately 9% of known cell-cycle genes are near Prospero binding sites. These include the neuroblast genes achaete (ac), scute (sc), asense (ase), aPKC, and mira and the cell-cycle regulators stg and CycE. In addition, it was found that the Drosophila homolog of the mammalian B lymphoma Mo-MLV insertion region 1 (Bmi-1) gene, Posterior sex combs, is located near a Prospero binding site. Bmi-1 is a transcription factor that has been shown to regulate the self-renewal of vertebrate hematopoetic stem cells. It is concluded that Prospero is likely to regulate neuroblast identity and self-renewal genes as well as cell-cycle genes directly, repressing their expression in the GMC (Choksi, 2006).

Prospero enters the nucleus of GMCs, and its expression is maintained in glial cells but not in neurons . Therefore the list of targets was searched for genes annotated as glial development genes. Prospero binds near 45% of genes involved in gliogenesis. Among the glial genes, it was found that the master regulator of glial development, glial cells missing (gcm), and gilgamesh (gish), a gene involved in glial cell migration, are both near Prospero binding sites and are likely directly activated by Prospero in glia (Choksi, 2006).

In summary, Prospero binds near, and is likely to regulate directly, genes required for the self-renewing neural stem cell fate such as cell-cycle genes. It was also found that Prospero binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh) and to genes required for glial cell fate. The in vivo binding-site mapping experiments are supportive of a role for Prospero in regulating the fate of Drosophila neural precursors by directly controlling their mitotic potential and capacity to self-renew (Choksi, 2006).

The Drosophila ventral nerve cord develops in layers, in a manner analogous to the mammalian cortex. The deepest (most dorsal) layer of the VNC comprises the mature neurons, while the superficial layer (most ventral) is made up of the mitotically active, self-renewing neuroblasts. Neuroblast cell-fate genes and cell-cycle genes are normally expressed only in the most ventral cells, while Prospero is found in the nucleus of the more dorsally lying GMCs. If in GMCs, Prospero normally acts to repress neuroblast cell-fate genes and cell-cycle genes, then in a prospero mutant, expression of those genes should expand dorsally. Conversely, ectopically expressed Prospero should repress gene expression in the neuroblast layer.

The neuroblast genes mira, ase, and insc and the cell cycle genes CycE and stg show little or no expression in differentiated cells of wild-type stage 14 nerve cords. Expression of these neuroblast-specific genes was examined in the differentiated cells layer of prospero embryos and it was found that they are derepressed throughout the nerve cord of mutant embryos. mira, ase, insc, CycE, and stg are all ectopically expressed deep into the normally differentiated cell layer of the VNC. To check whether Prospero is sufficient to repress these genes, Prospero was expressed with the sca-GAL4 driver, forcing Prospero into the nucleus of neuroblasts. Prospero expression is sufficient to repress mira, ase, insc, CycE, and stg in the undifferentiated cell layer of the VNC. These data, combined with the Prospero binding-site data, demonstrate that Prospero is both necessary and sufficient to directly repress neuroblast genes and cell-cycle genes in differentiated cells. This direct repression of gene expression is one mechanism by which Prospero initiates the differentiation of neural stem cells (Choksi, 2006).

Having shown that Prospero directly represses genes required for neural stem cell fate, it was asked whether Prospero also directly activates GMC-specific genes. Alternatively, Prospero might regulate a second tier of transcription factors, which are themselves responsible for the GMC fate. Of the few previously characterized GMC genes, it was found that Prospero binds to eve and fushi-tarazu (ftz). In the list of targets several more GMC genes were expected to be found, but not genes involved in neuronal differentiation, since Prospero is not expressed in neurons. Surprisingly, however, it was foudn 18.8% of neuronal differentiation genes are located near Prospero binding sites (Choksi, 2006).

To determine Prospero's role in regulating these neuronal differentiation genes, in situ hybridization was carried out on prospero mutant embryos. Prospero was found to be necessary for the expression of a subset of differentiation genes, such as the adhesion molecules FasciclinI (FasI) and FasciclinII (FasII), which have roles in axon guidance and/or fasciculation. Netrin-B, a secreted protein that guides axon outgrowth, and Encore, a negative regulator of mitosis, also both require Prospero for proper expression. Therefore, in addition to directly repressing genes required for neural stem cell self-renewal, Prospero binds and activates genes that direct differentiation. These data suggest that Prospero is a binary switch between the neural stem cell fate and the terminally differentiated neuronal fate (Choksi, 2006).

To test to what extent Prospero regulates the genes to which it binds, genome-wide expression profiling was carried out on wild-type and prospero mutant embryos. While the DamID approach identifies Prospero targets in all tissues of the embryo, in this instance genes regulated by Prospero were assayed in the developing central nervous system. Small groups of neural stem cells and their progeny (on the order of 100 cells) were isolated from the ventral nerve cords of living late stage 12 embryos with a glass capillary. The cells were expelled into lysis buffer, and cDNA libraries generated by reverse transcription and PCR amplification. cDNA libraries prepared from neural cells from six wild-type and six prospero null mutant embryos were hybridized to full genome oligonucleotide microarrays, together with a common reference sample. Wild-type and prospero mutant cells were compared indirectly through the common reference (Choksi, 2006).

In the group of Prospero target genes that contain a Prospero consensus sequence within 1 kb of the transcription unit, 91 show reproducible differences in gene expression in prospero mutants. Seventy-nine percent of these genes (72) exhibit at least a 2-fold change in levels of expression. Many of the known genes involved in neuroblast fate determination and cell-cycle regulation (e.g., asense, deadpan, miranda, inscuteable, CyclinE, and string) show increased levels in a prospero mutant background, consistent with their being repressed by Prospero. Genes to which Prospero binds, but which do not contain an obvious consensus sequence, are also regulated by Prospero: CyclinA and Bazooka show elevated mRNA levels in the absence of Prospero, as does Staufen, which encodes a dsRNA binding protein that binds to both Miranda and to prospero mRNA (Choksi, 2006).

Expression of genes required for neuronal differentiation is decreased in the prospero mutant cells, consistent with Prospero being required for their transcription. These include zfh1 and Lim1, which specify neuronal subtypes, and FasI and FasII, which regulate axon fasciculation and path finding (Choksi, 2006).

The stem cell-like division of neuroblasts generates two daughters: a self-renewing neuroblast and a differentiating GMC. Prospero represses stem cell self-renewal genes and activates differentiation genes in the newly born GMC. In the absence of prospero, therefore, neuroblasts should give rise to two self-renewing neuroblast-like cells (Choksi, 2006).

The division pattern of individual neuroblasts was studied in prospero mutant embryos by labeling with the lipophilic dye, DiI. Individual cells were labeled at stage 6, and the embryos allowed to develop until stage 17. S1 or S2 neuroblasts were examined, as determined by their time of delamination. Wild-type neuroblasts generate between 2 and 32 cells, producing an average of 16.2 cells. Most of the clones exhibit extensive axonal outgrowth. In contrast, prospero mutant neuroblasts generate between 8 and 51 cells, producing an average of 31.8 cells. Moreover, prospero mutant neural clones exhibit few if any projections, and the cells are smaller in size. Thus, prospero mutant neuroblasts produce much larger clones of cells with no axonal projections, suggesting that neural cells in prospero mutants undergo extra divisions and fail to differentiate (Choksi, 2006).

Recently it was shown, in the larval brain, that clones of cells lacking Prospero or Brat undergo extensive cell division to generate undifferentiated tumors. Given that Prospero is nuclear in the GMC but not in neuroblasts, the expanded neuroblast clones in prospero mutant embryos might arise from the overproliferation of GMCs: the GMCs lacking Prospero may divide like neuroblasts in a self-renewing manner. It is also possible, however, that neuroblasts divide more frequently in prospero mutant embryos, giving rise to supernumerary GMCs that each divide only once. To distinguish between these two possibilities, the division pattern of individual GMCs was followed in prospero mutant embryos (Choksi, 2006).

S1 or S2 neuroblasts were labeled with DiI as before. After the first cell division of each neuroblast, the neuroblast was mechanically ablated, leaving its first-born GMC. All further labeled progeny derive, therefore, from the GMC. Embryos were allowed to develop until stage 17, at which time the number of cells generated by a single GMC was determined (Choksi, 2006).

To determine whether mutant GMCs are transformed to a stem cell-like state, stage 14 embryos were stained for the three neuroblast markers: Miranda (Mira), Worniu (Wor), and Deadpan (Dpn). In wild-type embryos at stage 14, the most dorsal layer of cells in the VNC consists mostly of differentiated neurons. As a result, few or none of the cells in this layer express markers of self-renewal. Mira-, Wor-, and Dpn- expressing cells are found on the midline only or in lateral neuroblasts of the differentiated cell layer of wild-type nerve cords. In contrast, a majority of cells in the differentiated cell layer of stage 14 prospero mutant embryos express all three markers: Mira is found cortically localized in most cells of the dorsal layer of prospero nerve cords; Wor is nuclear in most cells of mutant VNCs; Dpn is ectopically expressed throughout the nerve cord of prospero mutants (Choksi, 2006).

Expression of neuroblast markers in the ventral-most layer of the nerve cord (the neuroblast layer), to exclude the possibility that a general disorganization of cells within the VNC contributes to the increased number of Mira-, Wor-, and Dpn-positive cells in the dorsal layer. The number of neuroblasts in a prospero mutant embryo is normal in stage 14 embryos, as assayed by Wor, Dpn, and Mira expression. Thus, the increased expression of neuroblast markers in prospero mutants is the result of an increase in the total number of cells expressing these markers in the differentiated cell layer. It is concluded that prospero mutant neuroblasts divide to give two stem cell-like daughters. GMCs, which would normally terminate cell division and differentiate, are transformed into self-renewing neural stem cells that generate undifferentiated clones or tumors (Choksi, 2006).

Therefore, Prospero directly represses the transcription of many neuroblast genes and binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh), which regulate the timing of cell-fate specification in neuroblast progeny. Prospero maintains hb expression in the GMC, and it has been suggested that this is through regulation of another gene, seven-up (svp). Prospero not only regulates svp expression directly but also maintains hb expression directly. In addition, Prospero maintains Kr expression and is likely to act in a similar fashion on other genes of the temporal cascade. Intriguingly, Prospero regulates several of the genes that direct asymmetric neuroblast division (baz, mira, insc, aPKC). aPKC has recently been shown to be involved in maintaining the self-renewing state of neuroblasts (Choksi, 2006).

Prospero initiates the expression of genes necessary for differentiation. This is particularly surprising since prospero is transcribed only in neuroblasts, not in GMCs or neurons. Prospero mRNA and protein are then segregated to the GMC. Prospero binds near eve and ftz, which have been shown to be downstream of Prospero, as well as to genes required for terminal neuronal differentiation, including the neural-cell-adhesion molecules FasI and FasII. Prospero protein is present in GMCs and not neurons, suggesting that Prospero initiates activation of neuronal genes in the GMC. The GMC may be a transition state between the neural stem cell and the differentiated neuron, providing a window during which Prospero functions to repress stem cell-specific genes and activate genes required for differentiation. There may be few, or no, genes exclusively expressed in GMCs (Choksi, 2006).

Prospero acts in a context-dependent manner, functioning alternately to repress or activate transcription. This implies that there are cofactors and/or chromatin remodeling factors that modulate Prospero's activity. In support of this, although Prospero is necessary and sufficient to repress neuroblast genes, forcing Prospero into the nuclei of neuroblasts is not sufficient to activate all of the differentiation genes to which it binds (Choksi, 2006).

Neuroblasts decrease in size with each division throughout embryogenesis. By the end of embryogenesis, they are similar in size to neurons. A subset of these embryonic neuroblasts becomes quiescent and is reactivated during larval life: they enlarge and resume stem cell divisions to generate the adult nervous system. Neuroblasts in prospero mutant embryos divide to produce two self-renewing daughters but still divide asymmetrically with respect to size, producing a large apical neuroblast and a smaller basal neuroblast-like cell. The daughter may be too small to undergo more than three additional rounds of division during embryogenesis. prospero mutant cells eventually stop dividing, and a small number occasionally differentiate. This suggests that there is an inherent size limitation on cell division. The segregation of Brat, or an additional cell fate determinant, to the daughter cell may also limit the potential of the prospero mutant cells to keep dividing (Choksi, 2006).

The Prox family of atypical homeodomain transcription factors has been implicated in initiating the differentiation of progenitor cells in contexts as varied as the vertebrate retina, forebrain, and lymphatic system. Prospero/Prox generally regulates the transition from a multipotent, mitotically active precursor to a differentiated, postmitotic cell. In most contexts, Prox1 acts in a similar fashion to Drosophila Prospero: to stop division and initiate differentiation (Choksi, 2006).

It is proposed that Prospero/Prox is a master regulator of the differentiation of progenitor cells. Many of the vertebrate homologs of the Drosophila Prospero targets identified in this study may also be targets of Prox1 in other developmental contexts. Prospero directly regulates several genes required for cell-cycle progression, and it is possible that Prox1 will regulate a similar set of cell-cycle genes during, for example, vertebrate retinal development. In addition, numerous Prospero target genes have been identified whose orthologs may be involved in the Prox-dependent differentiation of retina, lens, and forebrain precursors (Choksi, 2006).

The cis-regulatory code of Hox function in Drosophila

Precise gene expression is a fundamental aspect of organismal function and depends on the combinatorial interplay of transcription factors (TFs) with cis-regulatory DNA elements. While much is known about TF function in general, understanding of their cell type-specific activities is still poor. To address how widely expressed transcriptional regulators modulate downstream gene activity with high cellular specificity, binding regions were identified for the Hox TF Deformed (Dfd) in the Drosophila genome. This analysis of architectural features within Hox cis-regulatory response elements (HREs) shows that HRE structure is essential for cell type-specific gene expression. It was also found that Dfd and Ultrabithorax (Ubx), another Hox TF specifying different morphological traits, interact with non-overlapping regions in vivo, despite their similar DNA binding preferences. While Dfd and Ubx HREs exhibit comparable design principles, their motif compositions and motif-pair associations are distinct, explaining the highly selective interaction of these Hox proteins with the regulatory environment. Thus, these results uncover the regulatory code imprinted in Hox enhancers and elucidate the mechanisms underlying functional specificity of TFs in vivo (Sorge, 2012).

In order to quantitatively identify genomic regions bound by the Hox TF Dfd in Drosophila, two complementing approaches were employed: ChIP-seq, which has been successfully applied previously to identify stage- and tissue-specific enhancer activities, and computational detection of clusters of TF binding sequences, which allows the identification of cis-regulatory modules irrespective of temporal and spatial context. To generate genome-wide maps of Dfd binding in vivo, ChIP was performed using stage 10-12 Drosophila embryos and a Dfd-specific antibod. Stage-independent in silico Dfd-specific Hox response elements (HREs) were identified by searching for clusters of conserved Dfd binding motifs, as defined by a position weight matrix (PWM), in the non-coding regions of the genomes of 12 distinct Drosophila species. By applying both approaches, 4526 genomic regions containing clusters of Dfd binding sites and 1079 Dfd ChIP-seq enrichment peaks were identified, including two out of the three well-characterized Dfd-HREs, namely rpr-4S3 and Dfd-EAE. To study the regulatory capacity of novel in silico and ChIP-seq detected HREs, cell culture-based enhancer assays were performed for 11 randomly selected HREs, and it was found that reporter expression driven by the identified genomic regions was in all cases dependent on Dfd binding. In vivo activity was tested of 21 arbitrarily selected enhancers in transgenic reporter lines, revealing that 7 out of 11 ChIP-identified and 5 out of 10 in silico-predicted Dfd-HREs recapitulate the spatio-temporal expression of adjacent genes). Most importantly, it was possible to demonstrate Dfd-dependent regulation of both transgenic reporter expression and endogenous gene expression, suggesting that they are bona fide direct Dfd target genes. Thus, the identified Dfd-HREs represent a data set of biologically relevant regulatory regions and an excellent resource to unravel sequence features within Hox responsive enhancers that might be essential for the highly selective Hox target gene regulation (Sorge, 2012).

Transcriptional regulation in many cases relies on the assembly of regulatory protein complexes mediated by closely spaced TF binding sites within a cis-regulatory module and previous studies have shown that Hox proteins employ this mechanism to control target gene activity in small subsets of cells. The novel HREs were systematically scanned for TF binding motifs appearing in close proximity to Dfd binding sites. Using a statistical test for pair-wise distance distributions, w11 overrepresented DNA motifs for known TFs were found adjoining to Dfd binding sites with 5 of the motifs occurring in both the ChIP-seq and in silico-identified Dfd-HREs. When the expression patterns of six of these transcriptional regulators known to bind to the 11 motifs that were identified were examined, colocalization with Dfd was found in different sub-populations of cells in all cases. Colocalization was already known for two TFs, whose binding sites were coupled to Dfd motifs, including Extradenticle (Exd) , which is known to cooperatively bind with Hox proteins to DNA and thereby increase Hox DNA-binding selectivity. It was next asked whether the short-distance arrangements in Dfd-HREs are of biological relevance and translated into the regulation of similar classes of target genes. To this end, the overrepresentation was statistically tested of expression and biological terms of genes associated with HREs harbouring specific combinations of Dfd and close-by motifs. This analysis revealed that only those Dfd-HREs with short distance intervals between the Dfd and adjacent motifs were coupled to similar gene classes, while random distance intervals did not show any correlation. Strikingly, genes associated with specific short-distance HREs had similar expression and functional annotations as the TFs interacting with the Hox adjoining motifs, suggesting that time and place of Hox action is dictated by spatio-temporally restricted co-regulators. Support for this hypothesis stems from the observation that one of the close-distance partners, Optix, regulates similar processes as Dfd, since Dfd and Optix mutants displayed comparable morphological defects in the head region, such as the absence of mouth hooks, a maxillary segment-derived structure known to be specified by Dfd. In addition, one of the genes associated with a Dfd-Optix HRE, the known Dfd target gene reaper (rpr), is expressed in the ventral epidermis primordium as predicted by its HRE architecture, and regulated by Dfd and Optix in ventral-maxillary cells, which also express these factors. A cell-culture assay using the well-established Dfd responsive module responsible for rpr expression in a few anterior-maxillary cells, the rpr-4S3 Dfd-HRE, with wild-type or mutated Dfd binding sites or reduction of Dfd levels by RNAi confirmed the requirement for simultaneous activity of Dfd and Optix on the rpr-4S3 Dfd-HRE for strong reporter gene induction. Optix binding to the rpr-4S3 Dfd-HRE was additionally confirmed by electrophoretic mobility shift assay (EMSA) experiments. Furthermore, transgenic reporter expression induced by the rpr-4S3 Dfd-HRE was lost in Optix mutant embryos or when the Optix binding sites were mutated. These results demonstrate that Optix, one of the newly identified factors, is a Dfd co-regulator required for proper regulation of the important Hox target gene rpr (Sorge, 2012).

Whether The precise spacing between Hox and adjacent binding sites plays a role for enhancer activity was explored. The rpr-4S3 Dfd HRE, which induces gene expression in a few anterior-maxillary cells, has previously been shown to be under the control of Dfd and Glial cells missing (Gcm), a Dfd co-regulator also identified in this study. Dfd and Gcm as well as Optix binding sites within the rpr-4S3 HRE are directly adjacent to each other, thus a 5- and 10-bp spacer was introduced to interfere with potential interactions of the proteins on the enhancer. In all cases, reporter gene expression was strongly reduced or completely abolished, showing that the close-distance arrangements between Dfd and Gcm as well as Dfd and Optix are required for the in vivo activity of the rpr-4S3 enhancer (Sorge, 2012).

While the results regarding the close-distance arrangement of Dfd and Gcm binding sites suggested the formation of a Dfd-Gcm protein complex, like in the case of Dfd and Exd, only independent binding of the two proteins to the rpr-4S3 enhancer was observed in EMSA experiments , supporting the idea of Hox proteins collaborating with other TFs on target HREs in the absence of physical contact. It has been shown before that Hox proteins together with other TFs that bind in the immediate vicinity recruit non-DNA binding cofactors to HREs. To test if such factors could interact with Dfd and the newly identified short distance binding TFs, the modENCODE data set was scanned and it was found that dCBP/Nej, a member of the CBP/p300 family of transcriptional co-activators bearing acetyltransferase activity, binds to the rpr-4S3 enhancer in vivo. As nej has been previously reported to genetically interact with Dfd, its function was examined in Dfd/Gcm-mediated transcriptional activation. Both factors, Dfd and Gcm, are required for transcriptional activation, since expression of Gcm in Drosophila D.Mel-2 cells, which have basal levels of Dfd activity, resulted in strong induction of reporter gene expression, while abolishing Dfd binding to the rpr-4S3 HRE by mutating all Dfd binding sites or by reducing Dfd protein levels in D.Mel-2 cells using RNAi, strongly reduced reporter gene expression in the presence of Gcm. Strikingly, Dfd- and Gcm-mediated reporter gene expression was strongly reduced in nej dsRNA-treated cells, whereas inhibition of protein deacetylation by Trichostatin A (TSA0) restored reporter gene expression. Consistently, rpr expression was abolished in nej mutant embryos. These results demonstrate that dCBP/Nej-mediated protein acetylation/histone modification is important for the combined activity of Dfd and Gcm on the rpr-4S3 HRE. While it was not possible to demonstrate that nej physically interacts with Dfd protein using various assays, EMSA experiments show that nej interacts with Gcm. Furthermore, acetylation of transiently transfected Gcm was detected in cultured Drosophila cells. Acetylation of Gcm is dependent on Nej, as it was reduced upon RNAi-mediated downregulation of nej. These results are consistent with published work demonstrating that in human cells CBP interacts with Gcma, resulting in its acetylation and stimulation of its transcriptional activity. Since about 10% of all Dfd and nej in vivo genomic binding events during embryonic stages 10-12 overlap, the functional interaction of Dfd and nej observed at the rpr locus does not seem an exception. This finding suggests that the interaction of co-activators (and co-repressors) with Hox proteins and close distance binding TFs on enhancer modules could be a commonly used mechanism to achieve highly specific spatio-temporal control of target gene activity. In this scenario, Hox proteins would control downstream genes by direct transcriptional and/or epigenetic regulation depending on HRE composition and thus cofactor identity and recruitment (Sorge, 2012)

Despite very similar DNA binding behaviour in vitro, Hox proteins regulate distinct morphological features along the anterior-posterior body axis in animal systems. To elucidate the mechanistic basis for the differences in their regulatory properties, Dfd-HREs identified in this study were compared to genomic regions bound by the Hox TF Ultrabithorax (Ubx) at identical developmental stages, as identified by the modENCODE consortium. Searching for overrepresented DNA motifs in both enriched ChIP regions, it was found that Dfd and Ubx bind to identical DNA sequences in vivo, reminiscent to in vitro systems. However, individual binding motifs seem to play only a minor role for Hox binding site selection in vivo, since this analysis revealed that Dfd and Ubx exclusively interact with non-overlapping genomic regions in embryonic stages 9-12. Consequently, Dfd- and Ubx-HREs were found to be associated with distinct classes of genes, revealing that genes with roles in the epidermis are primarily under the control of Dfd at the analysed embryonic stages while genes with mesoderm-related functions are predominantly regulated by Ubx. Consistently, it was found that the expression of tartan (trn), one of the genes associated with a Dfd-HRE, is regulated exclusively by Dfd, but not by Ubx, in epidermal cells, while parcas (pcs), one of the genes linked to a Ubx-HRE is under the selective control of Ubx in mesodermal cells. Furthermore, only Ubx-HREs were found to substantially overlap with cis-regulatory elements stage specifically bound by the mesoderm-specifying TFs Myocyte enhancer factor 2 (Mef2), Twist and Tinman. In contrast, the common ability of both Dfd and Ubx to regulate genes involved in nervous system development was underlined by comparable representations of binding motifs for the neuronal-specifying TFs Asense, Deadpan and Snail in Dfd- and Ubx-HREs (Sorge, 2012).

Strikingly, the basic design principles of Dfd- and Ubx-HREs were found to be similar: like in Dfd-HREs, six binding motifs for known TFs were located adjacent to Ubx binding sites and colocalization studies showed that they are expressed in subsets of Ubx-positive cells. Again, Ubx binding sites and motifs for potential co-regulators occurred most frequently in specific short intervals and only those Ubx-HREs with the preferred distance were associated with specific gene classes. This analysis also revealed that four of the six short-distance motifs were specific for Ubx-HREs, which is consistent with the data showing that Hox proteins interact with different and spatially restricted co-regulators to control target gene expression in selected cells. Importantly, in the cases of the close-distance motifs detected in both HREs, namely the binding sites for the TFs Ladybird early (Lbe) and Cut (Ct), the associated target genes were also expressed in non-overlapping tissues. This raised the question of how different Hox proteins can act on distinct target genes, even when their target HREs exhibit similar binding site compositions including short-distance arrangements. Since Lbe is active in both mesodermal and epidermal cells, one Dfd-Lbe and one Ubx-Lbe HRE was exemplarily analysed, and binding of Lbe protein was confirmed to both HREs by EMSAs. As predicted by the presence of Lbe binding sequences. Complex formation between the Hox protein and Lbe was observed in the case of Ubx and Lbe while Dfd and Lbe interact independently with the Dfd-Lbe HRE, indicating that the two Hox proteins employ different mechanisms for binding to the selected HREs. Lbe interaction with the Dfd-Lbe and Ubx-Lbe HREs is essential for in vivo activity, since in both cases ectopic reporter gene expression was observed when Lbe binding sites were mutated. Even more important, reporter gene expression was specifically changed only in segments in which either Dfd or Ubx is active, meaning in the case of the Dfd-Lbe HRE in maxillary cells and in the case of the Ubx-Lbe HRE in abdominal segments A1-A7. Taken together, these results demonstrate that the combined activity of Lbe and the Hox proteins Dfd or Ubx on selected HREs is critical for the precise spatiotemporal and segment-specific control of HRE activity. It was next asked whether additional (DNA- and non-DNA-binding) factors contribute to the predicted cell type-specific expression of the Dfd-Lbe and Ubx-Lbe HREs. Using the Drosophila Interactions Database (DroID; Murali, 2011) and published genome-wide DNA binding studies a search was carried out for unique Dfd-lbe and Ubx-lbe interactors. It was discovered that almost 20% of all Ubx-Lbe HREs but none of the Dfd-Lbe HREs were found to interact with the mesoderm-specifying factor Mef2 in vivo, while H3K9me3 histone marks, which are mediated by one of the unique Dfd-lbe interactors, Enhancer of zeste E(z), are enriched only within Dfd-Lbe HREs. Interestingly, E(z) modifies chromatin also by trimethylating H3K27 residues, a histone mark highly enriched at the genomic region spanning the ChIP-detected Dfd-Lbe HRE. Consistent with the repressive function of this histone modification, loss of Lbe binding to the Dfd-Lbe HRE results in ectopic reporter gene expression, suggesting that Lbe (and Dfd) recruits E(z) to the Dfd-Lbe HRE for cell type-specific target gene repression (Sorge, 2012).

Taken together, these results demonstrate that Hox proteins interact with different regulatory proteins on HREs, which allows them to differentially regulate their target genes despite their similar DNA binding properties. The fact that these interactions occur only in a few cells for a short period of time is very likely one of the major reasons why the identification of factors conferring regulatory precision and specificity to Hox function has met with little success so far (Sorge, 2012).

This study, has identified crucial features of HREs, which are essential for cell type-specific regulation of Hox target genes in vivo. In addition to motif composition the exact spatial arrangement of TF binding elements is critical to translate Dfd function into transcriptional regulation in vivo. These architectural features of Dfd-HREs alone accurately predict target gene function and expression patterns. Furthermore, it was found that epigenetic regulators bind to HREs on a genome-wide scale, suggesting that they generally collaborate with Hox proteins to achieve stable target gene regulation. This is in line with recent findings showing that chromatin modifications at enhancers strongly correlate with functional enhancer activity and tissue specificity. By comparing HREs regulated by Dfd and Ubx, two different Hox proteins with different embryonic regulatory specificities, this study shows that while similar design principles apply, specificity is encoded by distinct sets of co-occurring DNA motifs. Due to the highly dynamic regulatory output of Hox TFs in space and time, cell type-specific approaches are required in future to elucidate all relevant aspects of Hox-chromatin and Hox-cofactor interactions (Sorge, 2012).

Intrinsic dorsoventral patterning and extrinsic EGFR signaling genes control glial cell development in the Drosophila nervous system

Dorsoventral patterning and EGFR signaling genes are essential for determining neural identity and differentiation of the Drosophila nervous system. Their role in glial cell development in the Drosophila nervous system is not clearly established. This study demonstrates that the dorsoventral patterning genes, vnd, ind, and msh, are intrinsically essential for the proper expression of a master glial cell regulator, gcm, and a differentiation gene, repo, in the lateral glia. In addition, it was shown that esg is particularly required for their expression in the peripheral glia. These results indicate that the dorsoventral patterning and EGFR signaling genes are essential for identity determination and differentiation of the lateral glia by regulating proper expression of gcm and repo in the lateral glia from the early glial development. In contrast, overexpression of vnd, msh, spi, and Egfr genes repress the expression of Repo in the ventral neuroectoderm, indicating that maintenance of correct columnar identity along the dorsoventral axis by proper expression of these genes is essential for restrictive formation of glial precursor cells in the lateral neuroectoderm. Therefore, the dorsoventral patterning and EGFR signaling genes play essential roles in correct identity determination and differentiation of lateral glia in the Drosophila nervous system (Kim, 2015).

This study demonstrates that the DV patterning genes, ind, msh, and esg, are required for expression of the glial cell identity marker, gcm, and of the glial cell differentiation marker, Repo, in the proper region of the LTG in the Drosophila VNE. msh and esg acts locally in the formation and differentiation of the LG from the lateral column of the VNE, and esg strongly influences the formation and differentiation of the PG. ind is also locally involved in the initial formation and differentiation of the SG from the VNE. Considering that DV patterning genes, such as ind and msh, are required for the identity determination and formation of NBs in the intermediate and lateral columns along the DV axis, it is plausible that these two genes play essential roles in the proper development of the LTG in the corresponding columns. Interestingly, the zinc finger transcription factor, Esg, plays an important role in the formation and differentiation of the PG that originate from the lateral column, where esg is expressed. Although esg, together with snail and worniu, is required for the asymmetric division of NBs, the precise role of esg in embryonic CNS development has not been clearly determined. Thus, experimental results obtained in this study on esg's role in glial cell formation and differentiation is the first of its kind to analyze the role of esg in gliogenesis during embryonic CNS development (Kim, 2015).

Unexpectedly, vnd, which is essential for identity determination of the medial column NBs, showed the strongest influence on the proper formation and differentiation of all glia, including the LG, SG, and even PG in the VNE. Since the region of msh expression is ventrally expanded in the vnd mutant, disruption of the expression of gcm and Repo in the lateral column may have caused a decrease in the number of LG, LTG, and PG that originate from this region. In addition, the overexpression of vnd also repressed the expressions of Repo and MAPK in the Kr domain, presumably by promoting identity determination of the medial column in the intermediate and lateral columns. Original reports on the role of the vnd in formation and identity determination of the medial column NBs using the vnd target gene, NK6, showed that intermediate and lateral column identity markers are repressed by overexpression of vnd in the Kr-expression domain. One of the reasons for the wider influence of vnd in DV patterning than other DV patterning genes may be that vnd is expressed earliest among these genes, repressing expression of other DV patterning genes such as ind and msh in the medial column, in a process termed 'ventral dominance' (Kim, 2015).

The data revealed that the EGFR signaling receptor and ligand, Egfr and spi, play more global roles in glial cell development than do the DV patterning genes. Egfr and spi are required for initial glial cell formation as shown by reduced expression of gcm and Repo in the LGBs of the VNE. In addition, Repo expression in the differentiated glia was markedly reduced, especially in Egfr embryos, and in spi embryos, to a lesser degree. Interestingly, Repo expression is almost absent in the SG and remains only in the LGs of spi as well as of ind embryos. Since ind expression is activated by the EGFR signaling ligand, Spi, in the VNE to establish the identity of the intermediate column, it is plausible that glial phenotypes in spi and ind mutants are similar to each other. This result indicated that once the intermediate column identity is determined by ind-mediated repression of msh expression in the lateral column, EGFR signaling provides a consolidating extrinsic cue to make ind a repressor of some of the target genes in the intermediate column via MAPK-mediated phosphorylation. This interpretation is compatible with the results obtained by overexpression of Spi and Vn through Kr- and sca-Gal4 drivers, which show repressed Repo expression in the VNE due to the repressor activity of Ind, which in turn is activated by EGFR signaling. Thus, the results indicated that EGFR signaling globally activates many types of glial cell lineages in the VNE and delimits the area where glial cells originate by repressor activity that is chemically modified by EGFR signal transduction (Kim, 2015).

Establishment of proper identity along the DV axis by expression of the DV patterning and EGFR signaling genes is essential for correct formation and differentiation of glia from the VNE This study revealed that the DV patterning and EGFR signaling genes play important roles in the initial formation and differentiation of various types of glia in the Drosophila CNS. The DV patterning genes and EGFR signaling genes are locally and globally required, respectively, for glial cell formation and differentiation using loss-of function mutants of the genes. Unexpectedly, overexpression of the DV patterning and EGFR signaling genes also repressed the initial formation and differentiation of glia. Overexpression of vnd showed stronger repressor activity than msh on the Repo expression in most types of glial cells including the LG, whereas msh showed mild reduction in the Repo expression mainly in the SG, but not in the LG. The repressor activity of vnd started from the initial formation of the LGBs and continued until the glial cells differentiated into mature glia (Kim, 2015).

There are several possible explanations for the repressive effect in both loss-of-function and gain-of function mutants. First, vnd and EGFR signaling genes together play important roles in establishing identities of the medial and intermediate columns in DV patterning of the VNE. Therefore, overexpression of these genes also promote identities of the medial and intermediate in the lateral columns, where many glial cells, including the LG, PG, and some of the SG originate after neurons are formed. This identity change may block glial cell formation and differentiation from the lateral neuroectoderm. Second, overexpression of these genes may also promote neurogenesis over gliogenesis during developmental stages when overexpression was driven by Kr- and sca-Gal4. In addition, repressor activity appears to play a more dominant role than activator activity upon overexpression of vnd, considering that the DV patterning genes, vnd, int, and msh, act as successive repressors to establish and maintain their identity in the VNE. The results obtained using the loss-of-function and overexpression mutants demonstrate that the expression of a proper level of the DV patterning genes promote identity determination of neurons, while their overexpression represses formation of the glia in the VNE by default. In addition, repressor activity of the DV patterning genes appears to play a dominant role in the establishment of the three columnar divisions along the DV axis (Kim, 2015).

Similarly, overexpression of the EGFR signaling ligands, Spi and Vn, and the activated form of EGFR signaling receptor, EgfrAC, repressed Repo expression in all types of glial cells in the VNE. This may be due to the repressor activity of int, since activation of EGFR signaling induces phosphorylation of int and vnd to consolidate their repressor activity. In addition, since Egfr overexpression can cause expansion of vnd expression from the medial column to the lateral area, the intermediate and lateral columns may have acquired the medial identity, such that the LG and various types of other glia originating from the VNE are not generated after overexpression of Spi in the VNE (Kim, 2015).

These studies on the glial cell development in the Drosophila VNE revealed that the DV patterning and EGFR signaling genes play prominent roles in promoting neural identity, rather than glial identity during the early stages of CNS development, since their overexpression did not activate glial identity, but rather repressed it. Later, expression of the glial master gene, gcm, is required to promote glial cell identity in the VNE. It appears that the two-step mode of CNS development first ensures generation of a neural circuit and then provides supporting glial cells in the CNS. The results indicated that the DV patterning genes act locally to promote glial cell formation in their expression domains, but EGFR signaling genes act broadly throughout the VNE. Among the DV patterning genes, vnd, appears to influence glial cell formation and differentiation globally, since it represses int and msh to establish and maintain medial identity from the earliest developmental stage. It remains to be investigated how the DV patterning and EGFR signaling genes control the spatial and temporal regulation of glial cell formation and how they interact to promote glial identity in the CNS (Kim, 2015).

Targets of Activity

In gcm- mutant embryos, Prospero expression in neurons is almost normal, whereas its expression in glial cells is absent (Jones, 1995). gcm is epistatic (upstream) of repo (also known as RK2), a paired homeodomain protein involved in terminal glial differentiation (Hosoya, 1995 and Jones, 1995).

Binding site selection assays determine the motif 5'-AT(G/A)CGGGT-3' as the preferred binding site for GCM. Both the lack of homology to known proteins and the novel DNA binding specificity indicate that GCM contains a new type of DNA-binding domain. In transiently transfected cells, GCM also activates transcription from promoters consisting of the newly identified GCM-binding site and a TATA box. Thus, GCM is a novel type of transcription factor involved in early gliogenesis (Schreiber, 1997).

Members of the GCM family of transcription factors contain a DNA binding domain unrelated to any other known DNA binding domain and bind to a DNA sequence motif not recognized by any other known transcription factor. Positions 2, 3, 6 and 7 of the consensus GCM binding motif, 5'-ATGCGGGT-3', are particularly important for DNA binding; methylation of several G residues on the upper strand (but not on the lower strand) interfer with binding of GCM proteins. No differences were detected between the DNA binding of Drosophila GCM and mammalian mGCMa. Alanine scan mutagenesis of the DNA binding domain of mGCMa has identified the three conserved amino acids K74, C76 and C125 as being essential for DNA binding. Conserved cysteine residues are also found to be important for maintaining the overall integrity of the DNA binding domain and for mediating redox sensitivity of DNA binding. These cysteine residues are arranged in a symmetrical structure that bears no resemblance to other cysteine-containing structures, such as zinc fingers. In agreement with this, DNA binding of mGCMa is not dependent on zinc ions. These results give insights into the exact nature of the GCM binding sites expected in target genes and point to a role for redox regulation in the function of GCM proteins (Schreiber, 1998).

loco encodes an RGS protein required for Drosophila glial differentiation

Although glial cells are an important component in any complex nervous system, not much is known about the molecular mechanisms underlying glial development. In Drosophila, a number of gene functions and mechanisms required during glial development are emerging. Following lineage specification, terminal differentiation of glial cells is mediated by transcription factors encoded by repo and pointed. The identification of genes activated by pointed in glial cells should provide new insights in the molecular mechanisms underlying glial differentiation. loco , a regulator of G-protein signalling that functions as a GTPase-activating protein towards G-proteins) might represent such a pointed target gene. Analysis of the loco promotor region reveals the presence of GCM- and ETS-binding sites suggesting that loco might be a direct target of gcm as well. loco promotor-lacZ fusion constructs reveal a small promotor fragment that is capable of directing lacZ expression in almost all loco-expressing glial cells. This promotor fragment is indeed dependent on pointed function and ectopic pointed expression as well as ectopic gcm expression result in a corresponding ectopic lacZ expression. Sequence analysis and in vitro mutagenesis reveal both Gcm- and Pointed-binding sites within this element. These data, as well as the phenotypes observed in loco and pointed mutant embryos, suggest that loco is indeed a target of pointed. However, it is important to emphasize that loco expression in the tracheal system does not appear to depend on pointed function (Granderath, 1999).

gcm and pointed synergistically control glial transcription of the Drosophila gene loco

In Drosophila, lateral glial cell development is initiated by the transcription factor encoded by glial cells missing. gcm activates downstream transcription factors such as repo and pointed, which subsequently control terminal glial differentiation. The gene loco has been identified as a potential target gene of pointed and is involved in terminal glial differentiation. It encodes an RGS domain protein expressed specifically by the lateral glial cells in the developing embryonic CNS. The loco promoter and the control of the glial-specific transcription pattern has been analyzed. Using promoter-reporter gene fusions a 1.9 kb promoter element capable of directing the almost complete loco gene expression pattern has been identified. Sequence analysis suggests the presence of Gcm and Pointed DNA binding sites. Following in vitro mutagenesis of these sites their relevance in vivo has been demonstrated. The expression of loco is initially dependent on gcm. During subsequent stages of embryonic development Gcm and Pointed appear to activate loco transcription synergistically. In addition, at least two other factors appear to repress loco expression in the ectoderm and in the CNS midline cells (Granderath, 2000).

Two alternative modes are presented as to how loco transcription might be regulated. In the simple model, a linear array of transcriptional regulators results in the correct expression of loco. gcm acts on top of this cascade and activates pointed, which in turn leads to glial-specific loco expression. Alternatively, loco gene activation might be biphasic. Initially gcm concomitantly activates both loco and pointed. In a second phase, gcm and pointed act synergistically on the loco promoter to mediate high levels of glial-specific loco expression. The data favour the latter model (Granderath, 2000).

The 1.9 kb Rrk promoter element is capable of directing expression of a lacZ reporter in the complete loco expression domain. The Rrk fragment itself appears to contain more than one crucial regulatory element. The US1 construct, which overlaps the Rrk fragment, which harbors two gcm binding sites located in the 5' part of the Rrk fragment, directs glial expression resembling the expression of loco in a pointed mutant background. The 3' sequences of the Rrk fragment are found in the Nrk fragment. This promoter fragment, which harbours one gcm and one pointed binding site, is not able to confer any glial expression. Only the complete Rrk fragment is able to direct the entire loco transcriptional profile, pointing to synergistic effects of proteins binding to the 3' and 5' portions of the Rrk element. This notion is supported by the observation that pointed cannot activate the Rrk element when both Gcm binding sites GBS1 and GBS3 are deleted. Ectopic expression of either gcm or pointed alone within the neuroectoderm leads to sporadic activation of the Rrk enhancer, suggesting the presence of both gcm and pointed responsive elements. Coexpression of gcm and pointed in the rhomboid expression pattern shows two interesting results: (1) it is evident that cells within neuroectoderm activate the Rrk reporter fragment very strongly, showing that the two transcription factors act synergistically; (2) it is important to note that although comparably high levels of gcm and pointed are found in the CNS midline and the mesodermal cells, they never activate the Rrk reporter (Granderath, 2000).

Coexpression of gcm and pointed can also direct expression of the Nrk reporter. Within the Nrk fragment only one Gcm and one Pointed binding site are found, 370 bp apart. gcm is the master regulatory gene controlling lateral glial cell development. The gene pointed is not expressed in mutant gcm embryos suggesting that pointed expression depends on gcm. However, only coexpression of pointed and gcm leads to an efficient activation of the Rrk enhancer, indicating that gcm can not efficiently activate pointed transcription in the neuroectoderm. Despite the fact that pointedP1 is thought to act as a transcriptional activator it appears that cofactors such as gcm are required to allow full activation. This observation parallels results obtained in vertebrate systems, where it has been suggested that the binding of cofactors is a mechanism to relieve auto-inhibition of ETS proteins. pointed is expressed in many tissues during development and activates very different sets of genes (e.g., depending on the cells in which pointedP1 is expressed it activates tracheal, epidermal, neuronal or glial development). Thus, interaction with different tissue-specific coactivators might be an important step in selecting the appropriate downstream target genes (Granderath, 2000). Direct coactivation of glial target genes by both gcm and pointedP1 is possibly not confined to loco; the analysis of a second pointed-dependent enhancer element has revealed the presence of putative binding sites for both gcm and pointed (Granderath, S. and Klambt, C., unpublished data cited in Granderath, 2000).

The synergistic activation of loco by Gcm and Pointed could suggest that Pointed might be able to recruit or stabilize Gcm at the regulatory regions of terminal differentiation genes. This would lead to an increased expression of the respective genes but concomitantly could also disrupt the positive auto-regulatory feedback loop found for the gcm gene. This would provide a possible mechanism as to how the positive auto-regulation of gcm is terminated. How loco expression is maintained in vivo remains to be addressed (Granderath, 2000).

Terminal differentiation of glial cells is controlled by pointed. Two different isoforms are generated from the pointed locus, PointedP1 and PointedP2. They share the DNA binding domain and during embryonic CNS development they are expressed in the lateral glia (PointedP1) or the midline glia (PointedP2). Despite the common DNA binding activity, the two factors activate non-overlapping sets of target genes in the different glial cell types. The mechanism by which the selection of glial PointedP1 and PointedP2 target genes occurs appears to be complex. A simple model would be to postulate that specific, as yet unidentified cofactors are expressed either in the neuroectoderm or the CNS midline cells. However, in the midline, PointedP2 function can be substituted by PointedP1. This might be explained by postulating that PointedP1 is able to interact with a pointedP2 coactivator. Besides Gcm, additional factors appear to be required to specify PointedP1 target genes, because the coexpression of PointedP1 and Gcm in the CNS midline is not sufficient to evoke any Rrk reporter gene expression. Alternatively, the discrimination of PointedP1 and PointedP2 target genes might be mediated by transcriptional repressors. Two such proteins are known to be expressed in the CNS midline: Single minded and Abrupt. No potential Single minded binding sites were found in the Rrk construct. One potential Abrupt binding site (CTTAATTAA at position 1537-1547 of the Rrk fragment) was predicted by DNA sequence analysis. However, disruption of this site does not alter the reporter gene expression directed by the Rrk fragment in vivo. Thus, if Abrupt directly acts on the lococ1 promoter, it must bind to a different site in the Rrk fragment. Abrupt apparently represses Rrk-mediated expression (and possibly expression of other gcm-dependent genes) only in the apodemata, which might explain the muscle attachment defects observed in abrupt mutant embryos. In the CNS midline, however, the function of abrupt is not required for the repression of loco. Thus, additional experiments are required to determine which mechanisms are used in vivo to discriminate between lateral and midline glial gene expression (Granderath, 2000).

Gliogenesis in Drosophila: genome-wide analysis of downstream genes of glial cells missing in the embryonic nervous system

In Drosophila, the glial cells missing (gcm) gene encodes a transcription factor that controls the determination of glial versus neuronal fate. In gcm mutants, presumptive glial cells are transformed into neurons and, conversely, when gcm is ectopically misexpressed, presumptive neurons become glia. Although gcm is thought to initiate glial cell development through its action on downstream genes that execute the glial differentiation program, little is known about the identity of these genes. To identify gcm downstream genes in a comprehensive manner, genome-wide oligonucleotide arrays were used to analyze differential gene expression in wild-type embryos versus embryos in which gcm is misexpressed throughout the neuroectoderm. Transcripts were analyzed at two defined temporal windows during embryogenesis. During the first period of initial gcm action on determination of glial cell precursors, over 400 genes were differentially regulated. Among these are numerous genes that encode other transcription factors; this underscores the master regulatory role of gcm in gliogenesis. During a second later period, once glial cells have already differentiated, over 1200 genes were seen to be differentially regulated. Most of these genes, including many genes for chromatin remodeling factors and cell cycle regulators, were not differentially expressed at the early stage, indicating that the genetic control of glial fate determination is largely different from that involved in maintenance of differentiated cells. At both stages, glial-specific genes were upregulated and neuron-specific genes were downregulated, supporting a model whereby gcm promotes glial development by activating glial genes, while simultaneously repressing neuronal genes. In addition, at both stages, numerous genes that were not previously known to be involved in glial development were differentially regulated and, thus, identified as potential new downstream targets of gcm. For a subset of the differentially regulated genes, tissue-specific in vivo expression data were obtained that confirmed the transcript profiling results. This first genome-wide analysis of gene expression events downstream of a key developmental transcription factor presents a novel level of insight into the repertoire of genes that initiate and maintain cell fate choices in CNS development (Egger, 2002).

In stage 11 embryos, 26 genes encoding transcription factors are differentially regulated by targeted gcm misexpression (11 upregulated, 15 downregulated). The repo gene, a known direct target of gcm, has, next to gcm itself, the second highest increase in expression level (4.8-fold). Many of the other upregulated transcription factor genes such as zinc finger homeobox-2 (zhf-2), u-shaped (ush) and the Enhancer of split complex-member HLHm3 are known to act in different aspects of embryonic nervous system development. Genes of the Enhancer of split complex, for example, act during neural versus epidermal cell fate decision, and in the mouse, Enhancer of split members Hes1 and Hes5 have been shown to enhance glial cell fate. Among the transcription factors with decreased expression levels are engrailed (en) and ventral veins lacking/drifter (vvl/drf), which are expressed in a subset of neuronal precursor cells and are also involved in midline glial cell development, but not in lateral glial cell development. Other genes encoding transcription factors with decreased expression levels are sloppy paired 1 (slp1), goosecoid (gsc) and forkhead domain 96Cb (fd96Cb), which are expressed in subsets of neural precursor cells. Moreover, the scratch (scrt) transcription factor, a pan-neuronal gene encoding a zinc-finger protein that promotes neuronal development and can induce additional neurons when ectopically expressed, also shows decreased expression levels (Egger, 2002).

In stage 15/16 embryos, 38 genes encoding transcription factors are differentially regulated by targeted gcm misexpression (18 upregulated, 20 downregulated). As expected, gcm has the highest expression level increase (18.2-fold). In contrast to high REPO protein levels in stage 15/16 sca-gcm embryos, significant expression of repo transcripts is not detected at this stage. Several genes encoding transcription factors, which are expressed in specific neurons, such as eyeless (ey) and Ultrabithorax, are downregulated. Moreover, several members of the Enhancer of split complex such as HLHmbeta, HLHm7, and E(spl), are downregulated at stage 15/16, in contrast to stage 11; in addition to a role in early neurogenesis, these genes continue to be expressed in the normal developing nervous system of the wild type at later embryonic stages (Egger, 2002).

The marked increase in the number of affected transcripts at stage 15/16 is due in part to the fact that numerous genes encoding transcription factors belonging to the basal transcription machinery are differentially regulated at this stage. Among these are TfIIFbeta, Taf55, TfIIB, Taf60, Taf80 and Taf150. Moreover, among the upregulated genes encoding transcription factors, several are involved in chromatin remodeling, such as the brahma complex or associated genes [dalao (dalao), Brahma associated protein 60 kp (Bap60), Snf5-related 1 (Snr1) and absent, small or homeotic disc 2 (ash2)]. This suggests that the maintenance of glial cell differentiation at later embryonic stages involves chromatin remodeling as well as the regulation of global transcriptional processes (Egger, 2002).

In stage 11 embryos, 13 genes encoding kinases or phosphatases are differentially regulated by gcm misexpression (eight are upregulated, five are downregulated). Among the genes with increases in transcript abundance is heartless (htl), which encodes a fibroblast growth factor (FGF) receptor expressed in lateral glial cells. Conversely the Epidermal growth factor receptor (Egfr) shows a decrease in transcript abundance; the Egfr pathway is implicated in midline glial cell development. Decreased expression is also observed for shaggy (sgg), which encodes a protein kinase, and for skittles (sktl), which encodes a putative phosphatidylinositol-4-phosphate 5-kinase. Cells in sgg mutant embryos cannot adopt early epidermal fates and instead develop characteristics of CNS cells. Mutations in sktl cause abnormal development in the PNS (Egger, 2002).

In stage 15/16 embryos, 59 genes encoding kinases or phosphatases are differentially regulated by gcm misexpression (29 are upregulated, 30 are downregulated). Several genes involved in cell proliferation and mitotic division are upregulated. These included polo (polo), discs overgrown (dco), smallminded (smid) and Nek2 (Nek2). By contrast, genes involved in aspects of neuronal development, such as axogenesis and synaptogenesis are downregulated. Among these are derailed (drl), Neuron-specific kinase (Nrk) and Cdk5 activator-like protein (Cdk5alpha). The drl gene is involved in axonal guidance including routing across the midline. Nrk is specifically expressed in the embryonic CNS. Cdk5alpha controls multiple aspects of axon patterning. The only gene in this class that is known to be involved in glial differentiation is htl, which is upregulated at stage 11 and remains upregulated in stage 15/16 embryos, albeit at a lower level (Egger, 2002).

Ten genes encoding cell cycle regulators are differentially regulated by gcm misexpression (seven are upregulated, three are downregulated) in stage 15/16 embryos. For example, increases in transcript abundance are found for Cyclin B (CycB), Cyclin A (CycA) and Cyclin D (CycD). These genes encode regulators of cyclin-dependent kinases that act in different phases during mitotic cell cycles. By contrast, and rather unexpectedly, a marked decrease in transcript abundance is found for Cyclin E (CycE). CycE is essential for S-phase progression and its downregulation leads to the arrest of cell proliferation. Remarkably, in the earlier embryonic stage 11, none of the genes in the class of cell cycle regulators is influenced by gcm misexpression (Egger, 2002).

Several cases for gcm-dependent regulation of genes encoding cell adhesion molecules were observed. At stage 11, four genes encoding cell adhesion molecules are differentially regulated by gcm misexpression (two are upregulated, two are downregulated). At stage 15/16, 19 genes encoding cell adhesion molecules are differentially regulated by gcm misexpression (four are upregulated, 15 are downregulated). A striking example for a gene with a marked increased transcript level (13.6 fold) in stage 15/16 embryos is wrapper, which encodes a cell adhesion molecule that is expressed in midline glial cells and in late stages also in lateral glial cells. Genes with decreased transcript levels in stage 15/16 embryos that are mainly expressed in neurons are Tenascin major (Ten-m), Cadherin-N (CadN) and neuromusculin (nrm). All three act during axogenesis and synaptogenesis. The fact that most of the affected genes in the cell adhesion class show gcm-dependent decreased transcript levels could reflect the large diversity of cell adhesion molecules expressed by neurons (Egger, 2002).

In addition to its key role in gliogenesis, gcm also functions in a mesodermal lineage that gives rise to hematopoietic cells. When ectopically expressed in the early mesoderm, gcm can induce expression of Peroxidasin (Pxn), which is a marker for macrophage cells. Misexpression of gcm in cells of the neural lineage also gives rise to a few cells that express hemocyte markers, although most cells differentiate into glia. In accordance with these findings, transcript profiling of gcm misexpression embryos indicates that several genes encoding marker proteins for cells of the hemocyte lineage are differentially regulated. In stage 15/16 embryos, differential expression levels are detected for Pxn, serpent (srp) and the Scavenger receptor class C (type I) gene, all of which are expressed in hemocytes. Scavenger receptors play a crucial role in the phagocytosis of apoptotic cells and might also be able to mediate the direct recognition of microbial pathogens. It is noteworthy that the genes encoding Lysozyme B, Lysozyme C, Lysozyme D and Lysozyme E are all upregulated by gcm misexpression in stage 11 embryos. These four closely related lysozyme genes, clustered at locus 61F on the third chromosome, function as part of a system of inducible antibacterial immunity. These findings support the notion that the glial cell lineage and the hemocyte lineage, which give rise to cells involved in defense and immunity, may be molecularly related (Egger, 2002).

Terminal glial differentiation involves regulated expression of the excitatory amino acid transporters in the Drosophila embryonic CNS

The Drosophila excitatory amino acid transporters EAAT1 and EAAT2 are nervous-specific transmembrane proteins that mediate the high affinity uptake of L-glutamate or aspartate into cells. Both genes are expressed in discrete and partially overlapping subsets of differentiated glia and not in neurons in the embryonic central nervous system (CNS). In the PNS, EAAT2 is additionally expressed in several bilateral clusters of cells corresponding to sensory organs in the embryo head. Two of these clusters are most likely part of the dorsal and terminal organs of the antennomaxillary complex, but the other labeled sensory structures could not be identified with certainty. To assess the type of the EAAT2-expressing cells, anti-Elav, monoclonal 22C10, and anti-Repo antibodies were used. Elav is a nuclear marker for all Drosophila neurons and 22C10 labels all peripheral neurons. EAAT2 expression coincides exactly with 22C10 or Elav but not Repo expression. This indicates that the EAAT2 aspartate transporter is expressed in neurons in the PNS, in contrast to its glial localization in the CNS. Expression of these transporters is disrupted in mutant embryos deficient for the glial fate genes glial cells missing (gcm) and reversed polarity (repo). Conversely, ectopic expression of gcm in neuroblasts, which forces all nerve cells to adopt a glial fate, induces a ubiquitous expression of both EAAT genes in the nervous system. EAAT transcripts have been detected in the midline glia in late embryos and EAAT2 in a few peripheral neurons in head sensory organs. These results show that glia play a major role in excitatory amino acid transport in the Drosophila CNS and that regulated expression of the dEAAT genes contributes to generate the functional diversity of glial cells during embryonic development (Soustelle, 2002).

The dead ringer/retained transcriptional regulatory gene is required for positioning of the longitudinal glia in the Drosophila embryonic CNS

The Drosophila gene dead ringer (dri) [also known as retained (retn)] encodes a nuclear protein with a conserved DNA-binding domain termed the ARID domain (AT-rich interaction domain). dri is expressed in a subset of longitudinal glia in the Drosophila embryonic central nervous system and dri forms part of the transcriptional regulatory cascade required for normal development of these cells. Analysis of mutant embryos reveals a role for dri in formation of the normal embryonic CNS. Longitudinal glia arise normally in dri mutant embryos, but they fail to migrate to their final destinations. Disruption of the spatial organization of the dri-expressing longitudinal glia accounts for the mild defects in axon fasciculation observed in the mutant embryos. The axon phenotype includes incorrectly bundled and routed connectives, and axons that sometimes join the wrong bundle or cross from one tract to another. Consistent with the late phenotypes observed, expression of the glial cells missing (gcm) and reversed polarity (repo) genes was found to be normal in dri mutant embryos. However, from stage 15 of embryogenesis, expression of locomotion defects (loco) and prospero (pros) was found to be missing in a subset of LG. This suggests that loco and pros are targets of Dri transcriptional activation in some LG. It is concluded that dri is an important regulator of the late development of longitudinal glia (Shandala, 2003).

What is the molecular basis of the mutant phenotype found in dri mutants? Dri is a transcription factor, so the link between loss of dri function and the failure to differentiate properly is likely to be indirect, mediated through misregulation of dri targets required for normal longitudinal glial development. The most informative data came from an analysis of the position of dri in the glial transcriptional regulatory cascade. In general terms, dri activity was found to be downstream of gcm and repo, and independent of pnt and cut. It was also found to be upstream of two genes, loco and pros, which are essential for normal development of some glial cells. In this developmental context dri acts as an activator of downstream targets (Shandala, 2003).

Transcriptional regulation of the Drosophila glial gene repo

reversed polarity (repo) is a putative target gene of glial cells missing (gcm), the primary regulator of glial cell fate in Drosophila. Transient expression of Gcm is followed by maintained expression of repo. Multiple Gcm binding sites are found in repo upstream DNA. However, while repo is expressed in Gcm positive glia, it is not expressed in Gcm positive hemocytes. These observations suggest factors in addition to Gcm are required for repo expression. An analysis of the cis-regulatory DNA elements of repo was undertaken using lacZ reporter activity in transgenic embryos. A 4.2 kb DNA region upstream of the repo start site drives the wild-type repo expression pattern. Expression is dependent on multiple Gcm binding sites. Eleven sequences that match or have one mismatch from a consensus Gcm binding site (GBS) -- (A/G)CCCGCAT -- are located within the first four kb upstream of the repo transcription unit. By ectopically expressing Repo, it was shown that Repo can regulate its own enhancer. Finally, by systematically analyzing fragments of repo upstream DNA, expression is shown to be dependent on multiple elements that are responsible for activity in subsets of glia, as well as repressing inappropriate expression in the epidermis. These results suggest that Gcm acts synergistically with other factors to control repo transcription in glial cells (Lee, 2005).

Based on the presence of Gcm binding sites, repo is predicted to be a target of Gcm. Mutation of eleven binding sites results in significant loss of reporter expression in glia, demonstrating the direct regulation by Gcm. Mutation of these sites also demonstrates that 'imperfect' GBSs are responsible for a moderate level of repo expression. Because of two additional imperfect binding sites not included in these mutations, the result do not discount the possibility that Gcm activates a residual level of expression of repo −4.3ΔGBS11. However, a smaller −1.1 kb region driving expression in cell body glia (SPG) and subperineurial glia (SPG) still retains CBG expression even when all identifiable GBSs are mutated. This last result suggests other factors in addition to Gcm activate repo expression in glia (Lee, 2005).

One of these factors may be repo itself. Ubiquitously expressed Repo activates the reporter constructs. Interestingly, strong reporter activation was found in the epidermis but not in neurons, glia, or in any mesodermal tissue, and expression of repo was found to actually repress the glial expression driven by repo −4.3Δ11GBS-lacZ, suggesting Repo can act as a repressor in some contexts. repo's ability to act as a repressor was surprising given that previous studies have shown Repo to be a transcriptional activator, acting through ATTA DNA motifs. However, the current studies do not address whether or not Repo regulation of repo enhancer constructs is direct. While these studies show Repo can repress activation, they do not discount a scenario by which Repo auto-activates in the presence of unmutated Gcm binding sites (Lee, 2005).

The above observations and several lines of evidence suggest that negative factors are acting on the repo enhancer region to regulate its expression. (1) Despite expression of Gcm in hemocytes, neither endogenous repo nor the reporter constructs are ever expressed there. (2) Ectopic expression of repo-lacZ reporters by UAS-repo is permissive in the epidermis, but not in neurons. One possibility is that a pan-neural repressor prevents repo activation in neurons as well as glia in the absence of gcm. It is believed gcm would be able to displace the repressor to allow for activation by repo. If this model is correct, it may explain why repo reporters do not activate in glia in when GBSs are mutated, as repressors would still be present (Lee, 2005).

These studies show that regulation of repo by transcriptional repression is not limited to the CNS. Characterization of smaller regions spanning repo −4.3 reveals a repressor element (within repo −2.3/−1.9) that prevents inappropriate expression in the epidermis. repo −4.3/−2.3, which lacks the region containing this repressor element, shows expanded expression in the epidermal layer and also reveals the presence of an element (within repo −2.8/−2.3) that promotes epidermal expression. Activity is GBS independent, consistent with the observation that gcm is not expressed in the epidermis. These results show that the activity of factors expressed in the epidermis need to be repressed to maintain glial-specific expression of repo. This epidermal repression may also represent a mechanism to prevent activation of repo by factors shared between the epidermis and the CNS. Tight regulation and appropriate expression of repo is essential, since ectopic expression of repo in the epidermis results in lethality (Lee, 2005).

This analysis reveals that expression of repo in different glial subsets is promoted by other factors in addition to Gcm. Regions were found that promote expression in longitudingal glia (LG), peripheral glia (PG), SPG, and epidermis, and a proximal region was found that promotes expression in CBG. Activity is GBS dependent since mutation of GBSs reduce the strength of these specific glial activities. Despite having one or more GBSs, subfragments of the 4.2 kb region promote reporter expression in subsets of glia, rather than in all lateral glia where Gcm expression is found. This observation suggests Gcm acts with other factors to regulate spatial repo expression. Furthermore, the experiments show that mutating proximal GBSs affected the strength of glial-specific activities conferred by distal elements, suggesting that synergistic interactions between Gcm and other cis-acting factors can occur at some distance from one another on the DNA sequence (Lee, 2005).

These results extend observations that Gcm acts synergistically with glial-specific factors to control downstream genes. This study shows that repo regulation is dependent on several cis-regulatory elements that synergize with Gcm for activation and repression. Collectively, the results show that multiple factors promote repo expression in specific subsets of glia. Since Repo protein is expressed at equal levels in all Gcm positive glia, the question of why repo transcription depends on additional regulatory factors is subject to speculation. Glial cells have multiple functions that require transcriptional complexity for assignment and regulation. Moreover, failure to tightly regulate the expression of glial genes can result in neural dysfunction and lethality. While repo is involved in the terminal differentiation of all lateral glial cells, whether or not repo may contribute to the specification of glial cell diversity is not clear. This study represents a step towards understanding Gcm dependent glial-specific gene regulation and how expression is controlled in subsets of glial cells through multiple cis-regulatory elements and factors (Lee, 2005).

Expression profiling of glial genes during Drosophila embryogenesis

In the Drosophila CNS, the induction of the glial cell fate is dependent on gcm. Though a considerable number of other genes have been shown to be expressed in all or in subsets of glial cells, the course of glial cell differentiation and subtype specification is only poorly understood. This prompted the design of a whole genome microarray approach comparing gcm gain-of-function and, for the first time, gcm loss-of-function genetics to wildtype in time course experiments along embryogenesis. The microarray data were analyzed with special emphasis on the temporal profile of differential regulation. A comparison of both experiments enabled identification of more than 300 potential gcm target genes. Validation by in situ hybridization revealed expression in glial cells, macrophages, and tendon cells (all three cell types depend on gcm) for 70 genes, of which more than 50 had not been known to be under gcm control. Eighteen genes are exclusively expressed in glial cells, and their dependence on gcm was confirmed in situ. Initial considerations regarding the role of the newly discovered glial genes are discussed based on gene ontology and the temporal profile and subtype specificity of their expression. This collection of glial genes provides an important basis for the clarification of the genetic network controlling various aspects of glial development and function (Altenhein, 2006).

The majority of positive candidate genes from the GOF experiment showed expression in glial cells and neurons rather than exclusive expression in glial cells. Seven GOF candidate genes showed expression in glial cells exclusively, of which four are expressed in many if not all glial cells (CG15860, CG2893, CG3168, EG:22E5.11) and three are expressed in glial subsets only. These are cell body glia (CBG) (CG6218, CG6783) and peripheral glia (PG) (CG9336). The GOF resulted in only two genes specifically expressed in tendon cells (CG9796, CG1153), and no gene expressed in macrophages or cells of the hematopoietic lineage (Altenhein, 2006).

The LOF experiment resulted in a higher number of candidate genes with expression in certain glial subtypes and only two genes with expression in many if not all glial cells (CG2893, CG3408). Among the glial-subtype-specific expression, genes expressed in cell body glia (CBG) (CG6218, CG6783), subperineural glia (SPG) (CG5080), longitudinal glia (LG) (CG11910, CG7433) and peripheral glia (PG) (CG9336, CG9338) were found. Four of these glial-specific genes were also selected in the GOF experiment (CG2893, CG6218, CG6783, CG9336). Seven LOF candidate genes were expressed in tendon cells, and another eight genes showed expression in cells of the hematopoietic lineage including macrophages. One additional gene was selected which showed simultaneous expression in glial cells as well as in macrophages (CG16876) and one gene with expression in glial cells and tendon cells (CG15015) (Altenhein, 2006).

Taken together, five genes show expression in nearly all lateral glial cells, whereas 6 genes show staining in only a subset of glial cells, like CBG or LG. Additionally, seven genes were identified that are expressed in more than one glial subtype, e.g. in subperineural glia (SPG) and cell body glia (CBG). Furthermore, 9 genes are expressed in muscle tendon cells and 8 genes show expression in cells of the hematopoietic lineage. In addition, one gene was found simultaneously expressed in glial cells and in the blood cell lineage and one gene expressed in muscle tendon cells and in glia. The temporal expression profiles for most of the candidate genes in wildtype as well as in both mutant genetic backgrounds (as revealed by in situ hybridization) resemble the observed differential regulation in both the microarray experiments at least to some extent (Altenhein, 2006).

In order to analyze the expression of the candidate genes in glial cells in more detail, in situ hybridizations were performed in combination with anti-Repo antibody stainings. The dependence on gcm was tested by in situ hybridization in gain of function (Mz1060::gcm) and in gcm mutant backgrounds. As expected, all glial-cell-specific genes lack expression in gcm mutant embryos. Yet, not all of these genes show an increase in expression upon ectopic activation of gcm (Altenhein, 2006).

The temporal expression profile of the selected candidate genes was determined by in situ hybridization in wildtype embryos. For most of the selected genes, the expression in wildtype embryos is first detectable at stage 11 and continues until stage 16. For about 25% of the positively tested genes, expression was first detected at stage 12 to 13, and two genes are expressed in later stages only (Altenhein, 2006).

Filtering and validation steps enabled definition of clusters of genes with differential glial expression at specific time points or with specific regulation profiles. Most glial-specific genes in the GOF experiment showed differential regulation in stage 16, whereas in the LOF the glial-specific genes can be found throughout all stages. The profiles of differential regulation were compared with the temporal expression profiles as revealed by in situ hybridization in wildtype embryos. Both profiles do not necessarily match with each other. The regulation profile in the GOF depends on the Gal4 driver line rather than on endogenous expression. Hence, the profiles in the LOF experiment show a better overall accordance. Still, the discrepancies between differential regulation in both experiments and the endogenous in situ expression pattern cannot be explained. Astonishingly few genes showed antagonistic regulation in both experiments. Some of these genes (e.g., CG6218, CG6783) showed glial expression exclusively. The dependence on gcm was tested and nicely fits to the obtained microarray data. Most of the novel glial genes, however, were differentially regulated in only one of the two experiments. In situ hybridizations were performed in gcm mutant backgrounds as well as in Mz1060::gcm embryos, and expression patterns were compared to wildtype and to the obtained microarray data. With only few exceptions, most of the novel glial genes discovered from either experiment showed expression in the respective mutant background comparable to the observed microarray profiles. Especially those genes that are also expressed in neurons show an increase in expression upon ectopic activation of gcm, but no significant decrease in gcm mutants. Thus, the lack of glial-specific downregulation in the LOF experiment is masked by neuronal expression. Hence, these genes were only selected by upregulation in the GOF experiment. Conversely, some of the glial-specific downregulated genes in the LOF experiment are not differentially upregulated in the GOF. CG11910 for example does not show an increase in expression in situ upon ectopic gcm, even though it is expressed exclusively in longitudinal glial cells, suggesting that this gene requires factors in addition to Gcm to be activated. Hence, this gene was not previously discovered by either of the two gain-of-function microarray screens. The same observations were made when analyzing the expression of CG8965, which is also expressed in longitudinal glial cells. Apart from these two, all other genes with expression restricted to glial subtypes can be activated ectopically in Mz1060::gcm. This might correlate with the origin of the respective glial subtypes, indicating that spatial, temporal or even other specific cofactors are required for glial subtype specification. Still, some of the candidate genes of the LOF experiment were not differentially regulated on microarrays of the GOF experiment, though in situ hybridizations show an increase in expression for these genes upon ectopic activation of gcm in the CNS (e.g., CG11652, BG:DS05899.3). These discrepancies cannot be explained. Yet, all these observations demonstrate how carefully microarray experiments need to be designed and analyzed, especially with respect to the analysis of both gain- and loss-of-function genetics rather than only one situation (Altenhein, 2006).

GO annotation was used to categorize the putative or known function into eleven functional classes. Eighteen transcription factors (7%) were filtered, whose differential regulation starts in early stages (stages 9-12), which is compatible with a possible function in cell fate specification or early differentiation. Another 6% of the filtered genes encode ion transporters, antiporters, symporters or ion channels. Half of these show differential regulation at late stages only. This also appears reasonable as homeostatic control is believed to be one of the functions of glial cells. 13% of all filtered genes have putative enzymatic function and/or are involved in general metabolism. Whether this implies glial-specific or experimentally induced changes in metabolism remains unclear. Most of the filtered genes with negative in situ hybridization results were among those with putative metabolic function (Altenhein, 2006).

Astonishingly, neither repo nor pnt show differential regulation at any of the examined stages in both of the experiments and ttk appears downregulated only in the LOF at stage 11. With respect to lack of differential regulation of pnt and ttk, this can be explained by the fact that for both genes alternative splice variants are expressed in neurons or other cells. For every gene on the microarray, a single exon was amplified and spotted. For pnt and ttk, the spotted exons (exons 7 and 5, respectively) are present in all splice variants. Hence, expression outside of glial cells may mask the glial-specific expression and no strong differential regulation is observed. For repo, there is no such obvious explanation since repo is expressed in all lateral glial cells exclusively. The expression values for repo were extracted from all the microarray hybridizations and a strong expression of repo was found throughout the entire time course of both experiments without notable changes. Moreover, repo appears to be expressed on microarrays earlier and stronger than gcm (already at stage 9), which does not reflect the expression of repo in vivo (Altenhein, 2006).

A considerable number of the genes that were discovered in this study have already been identified: early determining factors such as gcm, repo and pnt, factors required for terminal glial cell differentiation and function like loco, EAAT1/2 or moody, and several genes expressed at various times in between. Most of these genes are expressed in subsets of glial cells only, and many are also expressed in other cells or tissues. Towards a first characterization of all these factors with respect to glial cell development, subtype specification and function, the candidate genes can be grouped for example according to (1) time point of expression in wildtype glial cells, (2) expression pattern in glial subtypes or (3) their annotated function. Most of the novel glial genes are expressed from stage 12/13 onwards, and the vast majority is only expressed in certain cells or subtypes. The primary determination of the glial cell fate in the embryo is achieved by Gcm, which is expressed early in development of all lateral glial cells. Further subtype specification obviously requires spatial and/or temporal cofactors. Together, they activate a variety of other genes, ranging from signaling or transporter molecules to proteins with putative metabolic function. The restriction of gene expression to particular glial cells includes all combinations of glial cell classes. Yet, some correlations can be observed with respect to the classification of glial cells according to morphological and positional criteria. Cell body glia and peripheral glia for example are best confined by subtype-specific gene expressions. Whether these subtypes require the respective genes for proper development or function remains to be shown. Apparently, peripheral glial cells, which migrate over long distances from their place of origin in the CNS into the periphery, require precise subtype-specific regulation. Many of the genes expressed in more than one subtype are also expressed in subperineurial glial cells. Recent publications deal with the function of these cells in blood–brain barrier formation and demonstrate the involvement of one particular gene, moody. moody is predicted to encode a G-protein-coupled receptor molecule. The requirement of proper signaling via heterotrimeric G-protein receptors for glial cell differentiation and formation of the blood–brain barrier has already proposed. One of the newly described glial genes, CG11910, is also predicted to encode a G-protein-coupled receptor molecule. It is expressed from stage 14 onwards in longitudinal glia, a glial cell type that is known to enwrap the longitudinal axonal connectives. Blood–brain barrier formation requires the tight connection of subperineurial glial cells by septate junctions. Some Drosophila genes are known to regulate the formation of septate junctions, for example, sinuous or neurexin. Both genes are differentially regulated on the microarray, too, and the latter is described to be required in blood–brain barrier formation. This suggests that the entire lists of differentially regulated genes of both the microarray experiments comprise more glial-specific candidates, which may be uncovered by further filtering with respect to certain functional annotations. Yet, the collection of glial genes identified so far provides an important basis for the clarification of the genetic network controlling various aspects of glial development and function (Altenhein, 2006).

A critical role for cyclin E in cell fate determination in the central nervous system of Drosophila; CycE functions upstream of pros and gcm

This study examined the process by which cell diversity is generated in neuroblast (NB) lineages in the central nervous system of Drosophila. Thoracic NB6-4 (NB6-4t) generates both neurons and glial cells, whereas NB6-4a generates only glial cells in abdominal segments. This is attributed to an asymmetric first division of NB6-4t, localizing prospero (pros) and glial cell missing (gcm) only to the glial precursor cell, and a symmetric division of NB6-4a, where both daughter cells express pros and gcm. This study shows that the NB6-4t lineage represents the ground state, which does not require the input of any homeotic gene, whereas the NB6-4a lineage is specified by the homeotic genes abd-A and Abd-B. They specify the NB6-4a lineage by down-regulating levels of the G1 cyclin, DmCycE (CycE). CycE, which is asymmetrically expressed after the first division of NB6-4t, functions upstream of pros and gcm to specify the neuronal sublineage. Loss of CycE function causes homeotic transformation of NB6-4t to NB6-4a, whereas ectopic CycE induces reverse transformations. However, other components of the cell cycle seem to have a minor role in this process, suggesting a critical role for CycE in regulating cell fate in segment-specific neural lineages (Berger, 2005).

In Drosophila, individual neuroblasts deriving from corresponding neuroectodermal positions among thoracic and abdominal segments generally acquire similar fates. However, some of these serially homologous neuroblasts produce lineages with segment-specific differences that contribute to structural and functional diversity within the CNS. The NB6-4 lineage was selected as a model to determine how this diversity evolves from a basic developmental ground state. As an experimental system, NB6-4 has an additional advantage, since Eagle (Eg) is expressed in all the cells of both thoracic and abdominal lineages and can thus be used as a lineage marker (Berger, 2005).

First the expression patterns of different homeotic genes were examined in thoracic and abdominal lineages of NB6-4. Antennapedia (Antp) is expressed in NB6-4t lineages of thoracic segments T1-T3. Abdominal A (Abd-A) is expressed in the NB6-4a lineage of abdominal segments A1-A6, whereas Abdominal B (Abd-B) is expressed in the NB6-4a lineage of segments A7-A8. Whereas loss of Antp function does not affect the NB6-4t lineage in any of the thoracic segments, loss-of-function mutations in abd-A and Abd-B cause NB6-4a-to-NB6-4t homeotic transformations in their corresponding segments. Interestingly, Ultrabithorax (Ubx), which is expressed in most of the cells of T3, is specifically absent in the NB6-4t lineage of that segment, and its loss-of-function alleles do not show any thoracic phenotypes. However, overexpression of Ubx as well as abd-A causes NB6-4t-to-NB6-4a transformations. Thus, it seems that the NB6-4t fate is the ground state and the NB6-4a state is imposed by the function of homeotic genes of the bithorax-complex (BX-C). This is consistent with previous reports that the T2 state is the ground state (for epidermis, including adult appendages) and other segmental identities are conferred by the function of homeotic genes (Berger, 2005).

The mechanism was examined by which abd-A or Abd-B specify the NB6-4a lineage compared with the NB6-4t lineage. As the mode and number of mitoses is the most obvious characteristic by which the NB6-4a lineage differs from NB6-4t, it was wondered whether factors regulating the cell cycle might be involved in controlling NB6-4 cell fate. One major factor that regulates the cell cycle is the G1 Cyclin CycE, which is needed for various aspects of the G1-to-S-phase transition (Berger, 2005).

To examine possible effects on cell fate decisions in the NB6-4t lineage, CycEAR95-mutant embryos were stained for gcm transcripts and Pros and Repo proteins. In wild-type embryos, gcm is initially distributed to both daughter cells during the first division of NB6-4t, but subsequently gets rapidly removed in the cell that functions as a neuronal precursor. Pros is transferred asymmetrically into only one cell, where it is needed to maintain and enhance the expression of gcm, thereby promoting glial cell fate. In CycEAR95 embryos, even at late stages (up to stage 14), gcm mRNA is strongly expressed in both daughter cells after the first division of NB6-4t. Even distribution of Pros was observed in both daughter cells, which could be the cause of continued expression of gcm. Furthermore, the glial marker Repo revealed that both cells differentiate as glial cells. The NB6-4a lineage is not affected in CycEAR95-mutant embryos, suggesting that the requirement for zygotic CycE is specific to NB6-4t (Berger, 2005).

Whether ectopic expression of CycE in abdominal lineages causes the opposite effect was tested. The sca-GAL4 line was used to drive UAS-CycE to achieve early expression in the neuroectoderm. An asymmetric distribution of Pros to one of the two progeny cells was observed just after the first division of NB6-4a. At later stages an increase was observed in the number of cells in the NB6-4a lineage (up to 5 cells). Some of these cells migrated medially, as NB6-4 glial cells normally do, maintaining Pros expression at a lower level. They also expressed Repo, which confirmed their glial identity. Other cells stayed in a dorso-lateral position and did not stain for Repo, suggesting neuronal identity (Berger, 2005).

To further investigate if ectopic CycE had indeed induced a neuronal sublineage in NB6-4a, and to test whether CycE can function cell-autonomously, a cell transplantation technique was employed. Single progenitor cells (stage 7) from the abdominal neuroectoderm of horseradish peroxidase (HRP)-labelled donor embryos overexpressing CycE were transplanted into the abdominal neuroectoderm of unlabelled wild-type hosts (at the same stage). The lineages produced by the transplanted cells were identified by morphological criteria. In all six cases, where cell clones were derived from NB6-4a, they were composed of both glial cells and neurons exhibiting their respective characteristic structures and positions. Because the clones are located in a wild-type abdominal environment, this experiment provides evidence that ectopic expression of CycE causes asymmetric division of NB6-4a and confers neuronal identity to one part of the lineage in a cell-autonomous manner. In these single-cell transplantation experiments, similar observations were made for NB1-1 and NB5-4, which also generate segment-specific lineages. Thus, CycE seems to have a general role in establishing segment-specific differences in neuroblast lineages (Berger, 2005).

Next whether the requirement for CycE to specify the neuronal lineage in NB6-4t is due to altered cell-cycle phases was examined. In string mutants, NB6-4t (whose proliferation is blocked before its first division) expresses gcm mRNA, as well as Pros and hunchback protein, although it does not differentiate as a glial cell. The composition of NB6-4t and NB6-4a lineages were further analysed in embryos mutant for other factors that interact with CycE in cell-cycle regulation. dacapo (dap) is the Drosophila homologue of members of the p21/p27Cip/Kip inhibitor family, which specifically block CycE-cdk complexes. Interestingly, in dap-null-mutant embryos an additional glial cell was observed in the NB6-4a lineage, but the appearance of any neuron-like cells was not observed. Consistent with these results, overexpression of Dap resulted in a reduction in the number of neurons in the NB6-4t lineage (from the normal number of 6 to 2-4), but homeotic transformation of the lineage did not occur. The number of glial cells was never affected, neither in the thorax nor in the abdomen. Ectopic expression of p21, the human homologue of the Drosophila dacapo gene, generated similar phenotypes (Berger, 2005).

The influence of the transcription factor dE2F, which mediates the activation of several genes needed for the initiation of S phase, was tested. In dE2F-mutant embryos, unlike in CycE mutants, no homeotic transformation of NB6-4t to NB6-4a was observed, although the number of neurons was reduced from 5-6 to 2-4. Ectopic expression of dE2F resulted in an increase in cell number in some abdominal hemisegments. In only a small percentage of those embryos, cells at lateral positions in abdominal segments did not show expression of gcm or Repo, suggesting their neuronal identity. Thus, although dE2F activation in the CNS depends on CycE, ectopic expression of dE2F cannot fully bypass the requirement for CycE in a NB6-4a-to-NB6-4t transformation. Similarly, ectopic expression of Rbf, a potent inhibitor of E2F target genes, did not cause any changes in the segregation of gcm mRNA, Pros or Repo in the NB6-4t lineage (Berger, 2005).

Whether interfering with another checkpoint of the cell cycle, the transition from G2 to M phase, affects NB6-4 cell fate was tested. Previous studies show that loss of CycA function prevents further mitosis after the first division of NB6-4t. However, the first division of NB6-4t follows the normal pattern; it gives rise to one glial and one neuronal cell. Similar effects were observed in CycA mutants. The cyclin-dependent kinase cdc2 heterodimerizes with CycA and CycB, and high levels of cdc2 expression have been shown to be required for maintaining the asymmetry of neuroblast divisions. In a cdc2 loss-of-function background, NB6-4t generated a normal lineage consisting of two glial cells and five to six dorso-lateral neurons. These observations show that NB6-4 cell fates do not change after manipulation of the transition from G2 to M phase (Berger, 2005).

These results suggest a critical role for CycE per se in regulating the NB6-4t lineage. Therefore whether CycE itself is differentially expressed between thoracic and abdominal NB6-4 lineages was tested. In situ hybridization with CycE RNA on wild-type embryos revealed that CycE is expressed just before the first division in NB6-4t. After the first division, CycE mRNA was detected in the neuronal precursor only and not in the glial precursor. In abdominal segments, no CycE expression was detected in NB6-4a before or after the division. Consistent with the role of CycE in specifying the NB6-4t lineage, notable levels of CycE transcripts were detected in the homeotically transformed NB6-4a lineages in abd-A-mutant embryos. Conversely, overexpression of abd-A caused down-regulation of CycE levels in thoracic segments and homeotic transformation of NB6-4t to NB6-4a. The importance of CycE in generating neuronal cells in the NB6-4t lineage was confirmed in an epistasis experiment involving abd-A and CycE mutants. As described above, loss of abd-A leads to transformation of NB6-4a to NB6-4t. Such homeotic transformation was suppressed by mutations in CycE, suggesting an absolute requirement for CycE in specifying the NB6-4t lineage. Finally, nine potential AbdA-binding sites (five of which are evolutionarily conserved in Drosophila pseudoobscura) were identified in a 5.0-kb enhancer fragment of CycE that is known to harbour cis-acting sequences for driving CycE expression in the CNS (Berger, 2005).

It is concluded that, in addition to its role in cell proliferation, CycE is necessary and sufficient for the specification of cell fate in the NB6-4 lineage. These results suggest that the function of CycE in regulating cell fate in NB6-4 lineages is independent, albeit partially, of its role in cell proliferation. The absence of any cell fate changes in the loss-of-function mutants of string, dap, cdc2 or CycA and in the Dap or Rbf gain-of-function genetic background may be attributed to the presence of CycE, which is strongly expressed in the NB6-4t, but not the NB6-4a, lineage. However, CycE may still function by controlling cell-cycle progression. NB6-4a, which does not express CycE, divides once followed by a cell-cycle arrest, presumably in G1. After the first division in the thorax, one daughter cell expresses high levels of CycE and divides roughly three times to generate neuronal cells. Therefore, this daughter cell presumably progresses through S phase. Chromatin reorganization during S phase might allow cell fate regulators to access their target genes, driving neuronal differentiation. Contrary to this interpretation, the other daughter cell of NB6-4t, which does not express CycE, divides twice but still generates glial cells. Thus, it remains to be investigated whether the role of CycE in neuronal cell fate determination is entirely independent of its role in cell proliferation. The results on the role of CycE in specifying neuronal compared with glial cell fate in the CNS are consistent with data from Xenopus on the role of cyclin-cdk complexes in specifying neuronal cell fate, inhibition of which promotes glial cell fate. In addition, this study shows that homeotic genes contribute to regional diversification of cell types in the CNS through the regulation of CycE levels (Berger, 2005).

Gcm/Glide-dependent conversion into glia depends on neural stem cell age, but not on division, triggering a chromatin signature that is conserved in vertebrate glia

Neurons and glia differentiate from multipotent precursors called neural stem cells (NSCs), upon the activation of specific transcription factors. In vitro, it has been shown that NSCs display very plastic features; however, one of the major challenges is to understand the bases of lineage restriction and NSC plasticity in vivo, at the cellular level. This study shows that overexpression of the Gcm transcription factor, which controls the glial versus neuronal fate choice, fully and efficiently converts Drosophila NSCs towards the glial fate via an intermediate state. Gcm acts in a dose-dependent and autonomous manner by concomitantly repressing the endogenous program and inducing the glial program in the NSC. Most NSCs divide several times to build the embryonic nervous system and eventually enter quiescence: strikingly, the gliogenic potential of Gcm decreases with time and quiescent NSCs are resistant to fate conversion. Together with the fact that Gcm is able to convert mutant NSCs that cannot divide, this indicates that plasticity depends on temporal cues rather than on the mitotic potential. Finally, NSC plasticity involves specific chromatin modifications. The endogenous glial cells, as well as those induced by Gcm overexpression display low levels of histone 3 lysine 9 acetylation (H3K9ac) and Drosophila CREB-binding protein (dCBP) Histone Acetyl-Transferase (HAT). Moreover, dCBP targets the H3K9 residue and high levels of dCBP HAT disrupt gliogenesis. Thus, glial differentiation needs low levels of histone acetylation, a feature shared by vertebrate glia, calling for an epigenetic pathway conserved in evolution (Flici, 2011).

Understanding the biology and the potential of stem cells of specific origins is a key issue in basic science and in regenerative medicine. This study shows that NSCs can be fully and stably redirected towards the glial fate in vivo, via a transient, intermediate, state, upon the expression of a single transcription factor. NSC plasticity is temporally controlled and quiescent NSCs cannot be converted; however, plasticity is independent of cell division. Finally, the acquisition of the glial fate involves low histone acetylation, a chromatin modification that is conserved throughout evolution, emphasizing the importance of this mark in glial cells (Flici, 2011).

NSCs produce the different types of neurons and glia that form the nervous system. These precursors can be converted into induced pluripotent cells and even into monocytes, a differentiated fate of an unrelated somatic lineage; however, the in vitro behavior may differ markedly from the in vivo situation. For example, the Achaete-Scute Complex homolog-like 1 transcription factor promotes the expression of oligodendrocyte features upon retroviral injection in the dentate gyrus, but promotes neuronal differentiation from the same progenitors in vitro. The use of NB-specific drivers, markers and conditional overexpression protocols, has led to the demonstration that a single transcription factor can fully convert NSCs into glia in a dose-dependent manner. High Gcm levels probably enable this transcription factor to counteract the endogenous transcriptional program and/or to compensate for the absence of cell-specific co-factors. Quantitative regulation is also required in physiological conditions; for example, the nuclear protein Huckebein enhances the gliogenic potential of Gcm upon triggering its positive autoregulation in a specific lineage. The present study therefore shows for the first time that NSCs can be completely and efficiently redirected in vivo towards a specific fate, also highlighting the importance of quantitative regulation in fate choices (Flici, 2011).

It is widely accepted that NSCs are multipotent precursors; however, their plastic features have not been investigated throughout their life at the cellular level. For example, the existence of a tri-potent NSC with the capacity to generate neurons, astrocytes and oligodendrocytes in the adult brain remains to be demonstrated in vivo. This study demonstrates that NSCs are more plastic at early embryonic stages than at the end of embryogenesis. Furthermore, the intrinsically defined program of quiescence is not compatible with fate conversion, even though quiescent cells are subsequently reactivated. As Drosophila glia are generated at different stages, it is unlikely that a general glial repressor arises late in development and specifically restricts the potential of Gcm. The data rather imply that temporal cues progressively limit NSC plasticity, a feature that may have important consequences in therapeutic applications (Flici, 2011).

It will be of great interest to determine whether such irreversible temporal restriction relies on external cues or whether it reflects an internal clock, as it has been shown for the acquisition of temporal identity, the process by which specific progenies are produced at different developmental stages (Flici, 2011).

Finally, the data show that Gcm does not reprogram neurons. Thus, although other somatic and even germ line cells can be reprogrammed into neurons, these post-mitotic cells seem endowed with an efficient brake to fate conversion. Interestingly, dorsal root ganglia neurons can transdifferentiate from one subtype into another in zebrafish, suggesting that, under some conditions, neurons can adopt a different, but closely related, phenotype. In addition, it cannot be formally excluded that a low percentage of immature neurons adopt a glial or a multipotent phenotype upon Gcm overexpression. Nevertheless, the data indicate that neurons are intrinsically different from other cell types, which may reflect a specific chromatin organization and/or expression of an efficient tumor suppressor molecular network. Transcriptome analyses will help characterizing the molecular signature responsible for the neuronal behavior (Flici, 2011).

Dedifferentiation and transdifferentiation of somatic cells can occur in the absence of mitosis, whereas NSCs plasticity has generally been associated to cell division, as a means to erase transcriptional programs and implement new ones. This study shows that, like terminally differentiated cells, NSCs can be efficiently redirected in the absence of cell division. The concomitant extinction of the endogenous program and activation of the glial program indicate that conversion occurs via an intermediate state, as has been described in B cell to macrophage experimental transdifferentiation. The acquisition of an intermediate state (partial reprogramming) has also been proposed for somatic cell reprogramming. The current findings raise a more general question as to whether intermediate states are common and unstable features of many plastic process including development. These states may reveal competing molecular pathways that in physiological conditions are alternatively consolidated or switched off in response to cell-specific signals. The development of tools enabling tracing these dynamic states will improve understanding of cell plasticity under physiological and experimental conditions (Flici, 2011).

Interestingly, altered tumor suppressor gene expression, which alters the proliferation pathway, leads to ambiguous cell identities, which may reflect the stabilization of intermediate fates. Similarly, Drosophila metastatic cells from brain tumors and several non-dividing NSC cells challenged with Gcm co-express the neuronal and the glial programs. It is proposed that the appropriate activation of the mitotic pathway is necessary for efficient consolidation/extinction of specific fates (Flici, 2011).

The interplay of extrinsic signals, transcription factors and chromatin modifications shape the identity of different cell types. The low and high levels of dCBP as well as H3K9ac truly represent a glial and neuronal signature, respectively. They both depend on gcm, which controls the fate choice, but not on genes downstream to Gcm, which are not sufficient to implement such choice. Thus, full fate conversion is accompanied by a cell-specific chromatin modification (Flici, 2011).

Interestingly, whereas dCBP accumulates at different levels in glia versus neurons and its overexpression or loss affects the levels of H3K9ac, the levels of dGCN5, another HAT that is able to acetylate the H3K9 residue in vivo, are similar in glia and neurons. Moreover, dGCN5 overexpression does not enhance H3K9ac levels nor does it affect the expression of glial genes. These data strongly suggest that the dCBP HAT specifically participates in setting up the H3K9ac signature. It should be noted that dGCN5 is a member of multiprotein complexes, which may explain why its overexpression cannot produce high HAT activity on its own. The balance between HATs and histone deacetylases (HDACs), enzymes with counteracting activities, is thought to be important in the regulation of histone acetylation levels. Although the investigation of histone deacetylation is not in the focus of this paper, the relevance of HDACs in the control of the glia-neuron histone acetylation signature cannot be excluded (Flici, 2011).

The tight regulation of histone acetylation in the nervous system seems to be evolutionarily conserved. Human neuronal disorders are frequently connected to downregulation of histone acetylation and HDAC inhibitors are good candidates as therapeutic tools. Histone acetylation is instrumental for mammalian memory formation and CBP plays an important role in long-term memory processes. Altogether, these data indicate that normal neuronal function requires high levels of histone acetylation (Flici, 2011).

This study shows that low HAT activity is necessary for glial differentiation. The increased levels of histone acetylation by overexpression of dCBP cause downregulation of the majority (but not all) of the tested glial genes, whereas the levels of general nuclear factors remain unchanged. The glial cells do not undergo apoptosis, indicating that high dCBP and histone acetylation levels influence specific pathways rather than generally affecting cell viability. The exact molecular mechanisms are not known, yet the behavior is similar in the mammalian CNS. Oligodendrocyte differentiation requires low levels of histone acetylation, resulting from high amounts of HDACs and low amounts of HATs (CBP and P300). The role of HDACs was further investigated, showing that such enzymes directly repress genes that prevent oligodendrocyte differentiation. Most probably an appropriate balance between HATs and HDACs is the key factor, which produces low levels of histone acetylation and regulates mammalian as well as Drosophila glial differentiation (Flici, 2011).

The broadly accepted model is that histone acetylation weakens the interaction between positively charged histone tails and negatively charged DNA, thereby contributing to transcriptional activation. The current data contradict this simple model. First, the levels of H3K4me3, a histone mark that is connected to actively transcribed genes, are similar in glia and neurons. Second, the total mRNA levels are not different in the two cell populations. Third, and most importantly, dCBP overexpression in glia specifically causes downregulation of a set of glial genes. It seems that the H3K9ac levels reflect specific functional differences between neurons and glia, rather than simply revealing general gene activation. Maybe neurons require more plastic and dynamic regulation of transcription than other cell types and this process requires higher capacity of histone acetylation. Supporting this theory is the finding that a large number of activity-regulated enhancers bind CBP in cortical neuronal cultures. The technological breakthrough will be to analyze the transcriptome and the chromatin landscape of a few cells, which will help understanding the mode of action of dCBP and HDACs in the control of Drosophila glial and neuronal differentiation (Flici, 2011).

The Gcm/Glide molecular and cellular pathway: New actors and new lineages

In Drosophila, the transcription factor Gcm/Glide plays a key role in cell fate determination and cellular differentiation. In light of its crucial biological impact, major efforts have been put for analyzing its properties as master regulator, from both structural and functional points of view. However, the lack of efficient antibodies specific to the Gcm protein precluded thorough analyses of its regulation and activity in vivo. In order to relieve such restraints, an epitope-tagging approach to 'FLAG'-recognize and analyze the functional protein was performed both in vitro (exogenous Gcm) and in vivo (endogenous protein). This study reveals a tight interconnection between the small RNA and the Gcm pathways. AGO1 and miR-1 are Gcm targets whereas miR-279 negatively controls Gcm expression. This study also identifies a novel cell population, peritracheal cells, expressing and requiring Gcm/Glide. Peritracheal cells are non-neuronal neurosecretory cells that are essential in ecdysis. In addition to emphasizing the importance of following the distribution and the activity of endogenous proteins in vivo, this study provides new insights and a novel frame to understand the Gcm biology (Laneve, 2013).

The pivotal role of Gcm in orchestrating cell fate specification involves a stringent regulation of its pathway, where multi-level control mechanisms converge. The FLAG-tagged Gcm tool constitutes a useful sensor to characterize post-transcriptional regulatory events (Laneve, 2013).

First, since several bands appear on Western Blots revealing the Gcm–FLAG product and the GCMa vertebrate protein is regulated by phosphorylation, which seems to affect its activity and stability, putative Gcm phosphorylation sites were sought in silico, and several were found. Then cell extracts overexpressing Gcm–FLAG (Act-Gal4) were inclubated with calf intestine phosphatase (CIP) prior to SDS-PAGE and observed a diminished band number and density, thus validating the Gcm phosphorylation prediction. Future studies will assess whether phosphorylation contributes to Gcm stability/activity (Laneve, 2013).

Second, miRNAs are endogenously expressed noncoding RNAs that represent key post-transcriptional regulators of gene expression. An online search carried for miRNAs predicted to target the gcm 3'UTR by miRanda, TargetScan and microCosm databases indicated miR-279/286/996 was the main family of putative effector miRNAs. miR-279, the most characterized member in Drosophila, was initially described for suppressing the formation of heterotopic olfactory neurons and as a component of a complex regulatory circuit orchestrated by the pleiotropic transcription factor Prospero (Pros). Finally, it was also shown to regulate the JAK/STAT pathway, driving rest: activity rhythms and modulating the response to morphogen gradients (Laneve, 2013).

To verify a possible role of miR-279, a sensor construct was developed in which the 3'UTR of gcm was inserted downstream to the FLAG-encoding cassette. Such construct (or its site-specific mutant derivative) was co-transfected in S2 cells along with a vector efficiently over-expressing the miRNA by mean of an Act-Gal4 driver (Laneve, 2013).

Western blot analysis demonstrates a specific downregulation exerted by miR-279 on gcm. Such modulation, abolished in a construct carrying a mutant target site, is specifically mediated by the gcm 3'UTR. Interestingly, ectopic expression of miR-286, a second member of the miR-279 family, diverging from mir-279 at the level of its 3′ sequence, failed to silence Gcm–FLAG. Since the 3′ region of microRNAs is known to play a role in the specificity of microRNA-target recognition, this accounts for the selectivity of gcm post-translational control by miR-279 in vitro (Laneve, 2013).

Finally, this study provides in vivo evidence for miRNA-mediated gcm regulation: (1) miR-279 was co-expressed in the embryonic neural territory (sca-Gal4 driver) in the presence of a Luciferase (Luc) reporter carrying its own 3′UTR or the gcm 3′UTR. By qRT-PCR analysis, a specific downregulation mediated by miR-279 on Luc RNA expression was detected only in the latter case, which parallels the in vitro data (2), an analogous gain-of-function strategy was used to analyze the expression of the endogenous gcm (gcm–FLAGBAC) in the presence or in the absence of overexpressed miR-279 at RNA and protein level by qRT-PCR and Western blot, respectively. Both approaches show a negative effect, thus validating miR-279 for targeting gcm in vivo in neurogenic territories. In sum, this study identified miR-279 as a negative modulator of gcm both in cell cultures and in embryos. The in vivo relevance of miR-279 on the gliogenic Gcm-dependent pathway was probed. Interestingly, when miR-279 was overexpressed (sca-Gal4) in hypomorphic gcm animals that contain reduced number of glia, a further decrease of glial cell number was expected, but the opposite result was found, and this was obtained upon using two allelic combinations. Thus, further regulatory steps compensate for the effects on gcm, thereby highlighting the complex network linked to small RNAs (Laneve, 2013).

Prompted by the above results, it was asked whether the interplay between Gcm and small RNA metabolism is more pervasive than emerged to date, upon establishing the role of Gcm in this pathway miR-1 was identified in an in vivo DAM ID screen and several GBSs were identified in the region upstream to the miR-1 transcription unit. miR-1 expression was evaluated in vivo upon hs-Gal4 driven Gcm activation and an up-regulation was found, where Gcm plays a crucial role for the development of blood cells. The gcm–Gal4 driver was used to overexpress Gcm and, to restrict the effects to the hemocyte precursor anlagen, and embryonic stages 5–9 were analyzed, when gliogenesis still has to start. An increase of miR-1 levels was confirmed and no effect was detected when Gcm expression was triggered by the sca-Gal4 driver. Interestingly, other miRNAs were identified as potential targets in the DAM ID screen; however their expression did not increase upon Gcm forced expression with any of the used drivers. This further validates the data obtained with miR-1 and suggests that the other miRNAs may work at different developmental stages. Finally, constitutive expression of Gcm in S2 cells, where miR-1 is not endogenously expressed, does not induce miR-1expression, likely due to the absence of appropriate co-activators. Overall, these data call for a cell-specific role of Gcm. Future studies will dissect the role of miR-1 in the Gcm pathways (Laneve, 2013).

Finally, the DAM ID screen also identified Argonaute 1 (AGO1) and an in silico inspection revealed the occurrence of several predicted GBSs mapping upstream to the AGO1 transcription unit. AGO1 a member of the Argonaute/PIWI protein family, involved in small RNA-mediated gene regulation. In Drosophila, AGO1 plays a specific role in miRNA biogenesis and function: it directs the unwinding of the intermediate duplex RNA generated during microRNA biosynthetic pathway and it selects one strand as mature microRNA loaded into the RISC (RNA-induced silencing complex) effector complex. AGO1 is broadly expressed in the embryo as well as in the imaginal discs and this, combined with the well known pleiotropic roles exerted by microRNA, accounts for its involvement in multiple developmental pathways. Interestingly, a genetic screen over a sensitized gcm background identified AGO1 as a putative interactor of gcm (Laneve, 2013).

In short, the Drosophila notum carries a fixed number of sensory organs called bristles. gcmPyx/+ flies ectopically express gcm in the larval notum, which triggers the differentiation of supernumerary bristles. gcmPyx/+ females show, in average, 18,5 bristles instead of the 11/heminotum typical of WT animals. This phenotype constituted the readout to identify putative gcm interacting genes in a dosage sensitive screen. The AGO1 mutation acts as a suppressor of the gcmPyx phenotype in double heterozygous conditions (genotype: gcmPyx/AGO108121), showing a positive genetic interaction with gcm (Laneve, 2013).

A microarray profiling also suggested AGO1 as a possible Gcm target in the neurogenic territories; however its upregulation upon Gcm forced expression as well as in gcm loss of function mutations made unclear the role of Gcm. Therefore (Act-Gal4 driver) Gcm or its tagged derivative was expressed in S2 cells, in which AGO1 is endogenously expressed, and this was found to induce AGO1 accumulation, reflecting and following the temporal accumulation of Gcm–FLAG itself. To further corroborate these data, increasing amounts of the Gcm–FLAG-expressing construct were transfected, and the amount of AGO1 was analyzed at the time-points of Gcm–FLAG highest expression (48–72 h after transfection). This revealed a clear correlation between the quantity of Gcm–FLAG and the expression of AGO1 . Furthermore, to exclude any unspecific influence of the FLAG epitope on target recognition, the same assay employing an untagged version of Gcm. Finally, it was verified AGO1 as a Gcm target in vivo: the UAS–gcm transgene was expressed under the control of the heat-shock (hs) inducible driver hs-Gal4 in Drosophila embryos: the Western blot demonstrates a clear up-regulation of AGO1 upon Gcm ectopic expression. In sum, this study provides significant genetic and molecular evidence for a positive interaction between AGO1 and gcm suggesting a mechanism of direct targeting. Interestingly, no modulation of AGO1 was observed upon Gcm forced expression in the nervous system using sca-Gal4. Since Gcm is required in different cell types, more efforts are required to clarify in which functional pathway Gcm controls AGO1. The data provide nevertheless first evidence for a cell-specific factor modulating the expression of AGO1. Indeed, the widespread distribution and function of miRNAs suggest a complex regulatory network controlling AGO1 expression/activity: Gcm can be proposed as a cell-specific component of the small RNA cascade (Laneve, 2013).

It is concluded that the transcriptional activator Gcm constitutes a paradigmatic example of master regulator, acting as a pivotal cell fate determinant and differentiation factor during Drosophila embryogenesis. It is therefore crucial to outline a trustworthy picture of Gcm biology, from expression to function. The present study provides the first characterization of Gcm at the protein level and reports a large set of data on gcm function, regulation and expression, collected both in vitro and in vivo. Specifically, small RNA metabolism was identified as an important element of the Gcm pathway and a novel gcm-dependent cell type essential in development was identified (Laneve, 2013).

Protein Interactions

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).

glial cells missing: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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