BIOLOGICAL OVERVIEW

Repo protein is a glial specific homeodomain protein. Though not required for early glial determination, it is required for aspects of glial differentiation, in particular the expression of late glial markers. repo function is required for neural cell viability, mediated by glial cells. Mutants exhibit a reversed polarity for the electrophysiological response of photoreceptor cells to light. Axonogenesis is initially normal in repo mutants, but later embryos have a disordered longitudinal axon bundle. Since glia are often used as a substrate for axon migration, death of glial cells would disrupt this process.

In Drosophila, cell-fate determination of all neuroectoderm-derived glial cells depends on the transcription factor Glial cells missing (Gcm), which serves as a binary switch between the neuronal and glial cell fates. Because the expression of Gcm is restricted to the early phase of glial development, other factors must be responsible for the terminal differentiation of glial cells. Expression of three transcription factors, Reversed polarity, Tramtrack p69 (Ttk69) and PointedP1 (PntP1), is induced by Gcm in glial cells. Repo is a paired-like homeodomain protein, expressed exclusively in glial cells, and is required for the migration and differentiation of embryonic glial cells. To understand how Repo functions in glial terminal differentiation, the mechanism of gene regulation by Repo was analyzed. Repo is shown to act as a transcriptional activator through the CAATTA motif in glial cells, and three genes are defined whose expression in vivo depends on Repo function. In different types of glial cells, Repo can act alone, or cooperate with either Ttk69 or PntP1 to regulate different target genes. Coordination of target gene expression by these three transcription factors may contribute to the diversity of glial cell types. In addition to promoting glial differentiation, it was found that Repo is also necessary to suppress neuronal development, cooperating with Ttk69. It is proposed that Repo plays a key role in both glial development and diversification (Yuasa, 2003).

To test whether Repo indeed functions as a transcriptional activator, the transcriptional regulatory activity of Repo in culture cells was tested. A luciferase reporter gene was constructed with two Repo-binding sites (CAATTA-luc) and its transcriptional activity was tested in the Drosophila S2 cell line. Co-transfection with a Repo-expressing plasmid (pACT-repo) causes a sevenfold increase in luciferase activity compared with co-transfection with the vector alone (pACT). An altered Repo protein, lacking most of its homeodomain, but retaining a putative nuclear localization signal located from amino acid 1 to 4 of the homeobox (pACT-repoDeltabox), is unable to activate transcription of the reporter gene, despite being localized to the nucleus. Furthermore, transcriptional activation by Repo is dependent on the presence of the CAATTA motif in the reporter gene; a single base substitution in this motif results in a complete loss of Repo-dependent transcription. It is concluded that Repo is a transcriptional activator that can act through the CAATTA motif (Yuasa, 2003).

To identify the transcriptional activation domain of Repo, fusion proteins of various segments of Repo with the DNA-binding domain of GAL4 were expressed in S2 cells, and their transcriptional activation activity was assayed by measuring the enzymatic activities of the UAS-luciferase reporter gene. Fusion of the full-length Repo to the GAL4 DNA-binding domain caused a 2.4-fold activation of the reporter gene compared with the GAL4 DNA-binding domain alone. Two non-overlapping segments of Repo (amino acids 1-219, 452-612) were identified that have significant levels of transactivation activity. Deletion of either segment retains the transcriptional activation activity present in the full-length fusion, suggesting that Repo may contain regions that inhibit the function of its activation domains. Indeed, deleting the N-terminal 124 amino acids causes an increase in activation that is more than 20-fold, indicating that a strong inhibitory domain is present in the N terminus. The presence of multiple functional domains suggests that Repo may employ different mechanisms of transcriptional regulation depending on the cellular context (Yuasa, 2003).

To test whether Repo activates transcription through CAATTA sites in vivo, the expression pattern of the ftz HDS lacZ reporter gene was examined in the repo mutant background. This reporter gene (2x21F) carries two copies of a 21 mer containing the CAATTA motif, and is expressed in all glial cells in the PNS and a subset of CNS glia. The glial expression of the ftz HDS reporter gene in the PNS overlaps precisely with Repo-expressing cells. The loss of repo function abolishes lacZ expression in both the PNS and CNS glia, even though glial cells are still present in stage 16 repo mutant embryos. Expression of the ftz HDS reporter gene in the antenno-maxillary complex and posterior spiracles, where Repo is not expressed, is unaffected in repo mutants. These results indicate that Repo acts through the CAATTA site to drive transcription in glial cells (Yuasa, 2003).

To address whether Repo is sufficient to activate transcription of the ftz HDS reporter gene, Repo was expressed ectopically in the presumptive ventral neurogenic region and the dorsal epidermis. Such embryos expressed lacZ in non-glial cells within the dorsal epidermis, adjacent to the PNS. Thus, Repo is sufficient for the expression of the ftz HDS reporter gene in specific cellular contexts (Yuasa, 2003).

Although ectopic Repo induces the appearance of many non-glial lacZ-expressing cells in the dorsal epidermis, cells within the CNS do not respond to ectopic Repo. In fact, even in the wild-type background, not all Repo-positive glia in the CNS expressed the ftz HDS reporter. This suggests that the mechanism by which Repo regulates transcription may be different in the CNS from the one for peripheral glia. One possible scenario is that the functions of Repo in the CNS require cooperation with one or more other factors, and that these interactions preclude Repo from acting through the CAATTA motif. Ttk69 and PntP1 are good candidates for such co-factors, because ttk and pointed are both required for the development of CNS glial cells. Although repo, ttk and pointed are expressed in overlapping subsets of CNS glial cells, their expression is mutually independent; Repo continues to be expressed in the ttk or pointed mutant background, and lacZ expression levels in enhancer-trap lines of ttk or pointed are unaffected in repo mutant embryos. Moreover, ectopic expression of Repo in the entire neuroectoderm does not increase the expression of pointed P1 mRNA or Ttk69, nor does ectopic expression of either Ttk69 or PntP1 affect Repo expression. All three genes are most probably regulated independently, downstream of the glial determinant Gcm (Yuasa, 2003).

Although glial specification by Gcm is well established, how the characteristics of individual glial cells are determined is poorly understood. Gcm expression is confined to the early stage of glial development, suggesting that Gcm itself does not participate in the terminal differentiation of glia. Moreover, Gcm also directs blood cell development; Gcm is expressed in macrophage precursors and ectopic expression of Gcm in crystal cell precursors causes the transformation of crystal cells to macrophages. These results clearly show that the expression of Gcm does not always lead to the determination and terminal differentiation of glia. In glial cells, Gcm induces the expression of three transcription factors, Repo, Ttk69, and PntP1, and the loss of these proteins causes abnormal glial development, although Gcm expression remains normal. Although gcm can direct repo expression in various contexts, repo is not expressed endogenously in blood cells, but is confined to Gcm-positive glial cells, lasting even after gcm expression has ceased. In repo mutant embryos, the migration, survival and terminal differentiation of glial cells are abnormal. This study shows that Repo activates gene expression in glia, and also demonstrates that Repo mediates the suppression of neuronal differentiation. These results suggest that Repo is the major factor that is necessary for glial development (Yuasa, 2003).

Despite the glia-specific expression of the ftz HDS reporter gene, the CAATTA motif is not Repo specific. Ftz and En bind the CAATTA motif, which also resembles the consensus binding sequence for Antp and Ubx. Why do other homeodomain proteins fail to drive the ftz HDS reporter gene, a target of Repo, in vivo? Recent results show that homeodomain proteins require co-factors to activate the transcription of their target genes. Co-factors, such as Exd and FtzF1, are also DNA-binding proteins that require specific binding sites in the target gene. Homeodomain proteins other than Repo may be incapable of activating the ftz HDS reporter gene because this reporter does not have binding sites for required co-factors (Yuasa, 2003).

The behavior of the ftz HDS reporter gene suggests that the requirement for co-factors may also apply to Repo. Although the ectopic expression of Repo induced the ectopic expression of the ftz HDS reporter gene in the periphery, it did not affect the expression pattern in the CNS. Thus, Repo cannot be the single factor responsible for the activation of the ftz HDS reporter gene. Indeed, the expression pattern of the ftz HDS reporter gene is altered by changing nucleotides outside the CAATTA motif, indicating that ftz HDS contains binding sites for factors other than Repo. The simplest interpretation is that such factors are present in the periphery, but not in the CNS (Yuasa, 2003).

Using additional target genes of Repo, further evidence was provided that the transcriptional regulation by Repo involves co-factors. Although the enhancer-trap line M84 and the loco gene are both dependent on Repo function, and can be expressed precociously and ectopically upon mis-expression of Repo, much stronger responses are obtained when Repo is co-expressed with Ttk69 or PntP. Endogenous expression of M84 and the loco gene occurs in cells that co-express Repo and Ttk69 or PntP1, respectively. Ttk69 and PntP1 are thus good candidates for Repo co-factors. These results show that Repo and PntP1 cooperate on loco expression through their binding sites in the loco promoter. Likewise the synergism between Repo and Ttk69 may also occur on the promoter of their target genes (Yuasa, 2003).

The conclusion that the expression of M84 and loco are achieved by a cooperation of Repo and Ttk69/PntP1 does not rule out the possibility that these genes are also direct targets of Gcm. In fact, reporter genes driven by loco enhancer elements are expressed normally in stage 14 repo mutant embryos, indicating that other factor(s) activate their transcription at the onset of gliogenesis. Because the loco enhancer element contains Gcm-binding sites, Gcm can directly regulate loco. However, since the expression of Gcm in glia is transient, transcription initiated by Gcm must be sustained by other factors. Repo and PntP1 are the best candidates for factors that maintain loco expression throughout glial development and functioning (Yuasa, 2003).

The synergistic effect of Repo and Ttk69 on M84 marker expression suggests a positive role of Ttk69 on glial differentiation. Since the major function of Ttk69 has been thought to be the inhibition of neuronal differentiation through transcriptional repression, the positive action of Ttk69 on glial gene expression could be an indirect effect through repressing transcription of a repressor for M84 expression. However, Ttk69 can activate transcription in yeast cells, suggesting that Ttk69 may also promote transcription, depending on the cellular context. Recent studies also implicate a role for Ttk69 in cell proliferation, through controlling the expression of cell cycle regulators. Overexpression of Ttk69 results in the inhibition of glial development, accompanied by the repression of the S-phase cyclin and glial proliferation. Since an increase in the number of cells that express M84 glial marker is observed upon co-expression of Ttk69 and Repo, the result cannot be accounted for by the ability of Ttk69 to inhibit glial cell cycle. Whereas ectopic expression of Ttk69 reduces the expression of the endogenous repo gene, the misexpression paradigm provides exogenous Repo through the GAL4/UAS control. Thus the existence of Repo might modify the activity of Ttk69, so that it plays a positive role on glial development (Yuasa, 2003 and references therein).

Glial fate determination involves not only the promotion of glial differentiation but also the suppression of neuronal properties. Because ectopic Gcm can induce neurogenesis in certain contexts, it is unlikely that Gcm directly represses neuronal differentiation. Ttk69 has been proposed to inhibit neuronal differentiation, mainly because of its loss-of-function phenotype in the sensory organ. Here, it has been shown that the co-expression of Repo and Ttk69 has a potent neuron-suppressing activity, and further demonstrated that the repo mutant permits neuronal differentiation even when Gcm is overexpressed. This strongly suggests that Repo functions not only to activate the transcription of glial genes, but also to prevent the neuronal differentiation of presumptive glial cells (Yuasa, 2003).

If glia and neuron represent two mutually exclusive cell states that must be chosen between early in development, it is somewhat strange that suppression of neuronal development should be carried out by proteins that are expressed throughout glial differentiation. The existence of continuous suppression of neuronal properties in glia suggests that cells within the nervous system may retain the potential to become neurons or glia throughout their cellular history. This idea is supported by the observation that Gcm is able to transform post-mitotic neurons into glia. Conversely, in the vertebrate nervous system, glial cells (astrocytes and oligodendrocyte-precursor) can respond to environmental signals and function as neural stem cells, generating neurons. The role of Repo and Ttk69 may be to suppress the ability of glia to respond to cues that would cause them to change into neurons or neural precursors (Yuasa, 2003).

cDNA clone length - 3.2 kb

Bases in 5' UTR - 515

Exons - two

Bases in 3' UTR - 851

PROTEIN STRUCTURE

Amino Acids - 612

Structural Domains

Repo is a PRD-like protein since it has a PRD-type homeodomain, but lacks a PRD box (Xiong, 1994).

Evolutionary Homologies

Other paired homeodomains, including those of Aristaless, Bicoid and UNC-4 of C. elegans, share common features of a PRD homeodomain. Repo's DNA binding specificity deviates from that of the standard paired class homeodomains (Campbell, 1994 and Xiong, 1994).

Promoter Structure

The Glial cells missing protein is a novel DNA-binding protein. Its DNA-binding activity is localized in the N-terminal 181 amino acids. It binds with high specificity to the nucleotide sequence, (A/G)CCCGCAT, which is a novel sequence among known targets of DNA-binding proteins. Eleven such GCM-binding sequences are found in the 5' upstream region of the repo gene, whose expression in early glial cells is dependent on gcm. This suggests that the GCM protein is a transcriptional regulator directly controlling repo (Akiyama, 1996).

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).

Targets of Activity

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 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).

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of repo at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

Repo first appears in glioblasts at stage 9. repo is expressed in all developing glia in both the central and peripheral nervous systems, excluding midline glia and two of three segmental nerve root glial cells (Campbell, 1994 and Halter, 1995).

repo is required for proper differentiation of glia in the visual system (Xiong, 1995). Survival of laminar neurons in the optic lobe depends on repo expression in the laminar glia, indicating that the laminar glia supply factors required for neural survival. repo mutant glia also undergo cell death, suggesting that either the laminar neurons are required for survival of the glia or that repo expression is required to suppress an intrinsic cell suicide program. Subsequent to the laminar cell death, the retinal cells in repo mutants also degenerate (Xiong, 1995).

Glial cells differentiate from the neuroepithelium. In flies, gliogenesis depends on the expression of glial cell deficient/glial cell missing (glide/gcm). The phenotypes for glide/gcm loss- and gain-of-function mutations suggest that gliogenesis occurs in cells that, by default (that is without gcm intervention), would differentiate into neurons. gcm is demonstrated in this work to be able to induce cells to activate the glial developmental program, even from the mesoderm, a distinct germ layer. This demonstrates that gliogenesis does not require a ground neural state. Ectopic gcm expression leads to Repo expression at the positon of the heart, a tissue of mesodermal origin. Anti-Repo labeling is also observed at the position of midline cells, which have a mesectodermal origin. The lateral glial identity of Repo-positive cells at the position of the midline is conserved within two enhancer trap lines that specifically label midline cells. Ectopic expression of gcm in another line results in the activation of glial markers in many more cells. For example, ectopic Repo-positive cells are detected at the postion of the pharynx and in metameric stripes along the dorsoventral axis, at the position of somatic muscles. It is estimated that roughly half the muscle cells express the Repo glial marker upon mesodermal gcm activation. The competence to express glial-specific genes becomes restricted during development, since many fewer cells adopt a glial fate when gcm is expressed late. Mesodermal gcm expression inhibits the muscle fate as determined by examination of mesodermal markers Mef-2 and the Myosin heavy chain. A close inspection of mutant embryos reveals that the muscle layer is severely disrupted. Embryos expressing gcm ectopically lack most muscle fibers and display a significant number of round, unfused muscle cells. Ectopic expression of gcm in the dorsal ectoderm results in Repo expression in dorsal epidermal cells. Dorsal closure does not occur in these embryos and Repo-positive cells display a typical elongated glial cell morphology. These findings challenge the common view of the establishment of cell diversity in the nervous system. Strikingly, ectopic gcm overrides positional information by repressing the endogenous developmental program. These findings also indicate that glial differentiation tightly depends on gcm transcriptional regulation. It is likely that gcm homologs have similar actions during vertebrate gliogenesis (Bernardoni, 1998).

Neurons and glia are produced in stereotyped patterns after neuroblast cell division during development of the Drosophila central nervous system. The first cell division of thoracic neuroblast 6-4 (NB6-4T) is asymmetric, giving rise to a glial precursor cell and a neuronal precursor cell. In contrast, abdominal NB6-4 (NB6-4A) divides symmetrically to produce two glial cells. To understand the relationship between cell division and glia-neuron cell fate determination, the effects of known cell division mutations on the NB6-4T and NB6-4A lineages were examined and compared. Based on observation of expression of glial fate determination and early glial differentiation genes, the onset of glial differentiation occurs in NB6-4A but not in NB6-4T when both cell cycle progression and cytokinesis are genetically arrested. In contrast, glial differentiation starts in both lineages when cytokinesis is blocked with intact cell cycle progression. These results show that NB6-4T, but not NB6-4A, requires cell cycle progression for acquisition of glial fate, suggesting that distinct mechanisms trigger glial differentiation in the different lineages (Akiyama-Oda, 2000).

Cell division mutants stg, cycA, and pbl were used to investigate the relationship between cell division and glia- neuron cell fate determination in NB6-4. To determine the effects of cell cycle arrest on cell fate of NB6-4, expression of a glial fate determination protein, Gcm, and an early glial marker protein, Repo, were examined in stg mutant embryos. In normal development of NB6-4T, these proteins are detectable in the medial daughter cell after the first cell division and in its progeny cells, while they are expressed in both daughter cells of NB6-4A. In stg mutant embryos, neither Gcm nor Repo is detectable in NB6-4T, whereas both proteins are expressed in NB6-4A at levels comparable to those of wild-type embryos. This indicates that stg activity is required for the onset of glial differentiation in neuroglioblast NB6- 4T, but not in glioblast NB6-4A. GCM mRNA was examined to determine whether the lack of Gcm expression in stg mutant NB6-4T resulted from loss of transcription or failure of posttranscriptional regulation. In contrast to wild-type embryos, in which GCM mRNA is detected in both the NB6-4T and the NB6-4A lineages, GCM mRNA is detected in NB6-4A, but not in NB6-4T of stg mutant embryos. This indicates that transcription of gcm in NB6-4T does not occur or occurs at only a very low level in the mutant embryos (Akiyama-Oda, 2000).

There are at least two possibilities that explain how stg mutation affects the onset of glial differentiation in NB6-4T: (1) that phosphatase activity of Stg protein is directly required for Gcm expression and (2) that stg-mediated cell cycle progression is needed for function and/or distribution of some factors that are necessary for Gcm expression. The latter possibility is favored, since not all the stg-induced cells expressed the glial proteins in the rescue experiments. The normal expression of glial markers in cycA mutant embryos, in which the first cell division is normal, is consistent with the notion that the first cell division is a critical point for the onset of glial differentiation in the NB6-4T lineage (Akiyama-Oda, 2000).

In pbl mutant embryos, which lack cytokinesis, all the nuclei of NB6-4T express the glial proteins, suggesting that cytokinesis is not required for the onset of glial differentiation in the NB6-4T lineage. It has been suggested that cytokinesis may be required for negative regulation of glial differentiation, since more than three nuclei in the pbl mutant NB6-4T, in contrast to three glial cells in the wild-type, express Repo. In wild-type embryos, expression of Gcm protein becomes prominent in one of the daughter cells shortly after the first cell division of NB6-4T. Gcm protein is not detected in NB6-4T of the stg mutant, while beta-gal is detected, although rather weakly, in the cell of stg mutants bearing gcm^p-lacZ (gcm promoter regulating lacZ expression). This indicates that the gcm promoter may be initially activated even when cell cycle progression is arrested by stg mutation (Akiyama-Oda, 2000).

Cell cycle progression of NB6-4T appears to be more closely related to up-regulation of Gcm expression. This regulatory mechanism may lead to a sufficient level of Gcm expression, which enables transcription of downstream glia-specific genes including repo. GCM mRNA is present from before the first cell division of NB6-4T in wild-type embryos. The level of Gcm protein in the NB6-4T lineage is possibly controlled by several steps of regulation, including transcription, stability of mRNA, and translation. The findings of this study suggest that such regulatory mechanisms involve stg-mediated cell cycle progression (Akiyama-Oda, 2000).

NB6-4T and NB6-4A are the corresponding cells in thoracic and abdominal segments that share expression of some marker genes, including en and eg. However, these cells show distinct patterns of proliferation and cell fate. NB6-4T produces three glial cells and four to six neuronal cells, while NB6-4A produces only two glial cells. Despite this difference, glial fate arises from both lineages. The analyses using stg mutant embryos reveals that the effects of the mutation on glial differentiation in the NB6-4T and NB6-4A lineages are distinct. In the mutant, expression of Gcm and Repo is detected in NB6-4A but not in NB6-4T. This indicates that the start of glial differentiation in NB6-4T is dependent on stg-mediated cell cycle progression but that in NB6-4A this is not the case (Akiyama-Oda, 2000).

This raises the question of whether the different regulatory mechanisms for glial differentiation in these cells are reflected by their distinct cell types: neuroglioblast and glioblast. In the other glioblast GP, Gcm and Repo expression are detected in the stg mutant, indicating that glial differentiation in the glioblasts is independent of cell cycle progression. In addition to these glioblasts, a few cells expressing the glial proteins are observed in the mutant. These cells might have been neuroglioblasts, suggesting that there may be another mechanism to control the onset of glial differentiation in neuroglioblast lineages (Akiyama-Oda, 2000).

In the NB6-4T lineage, the first cell division is a critical step for triggering glial differentiation. Coincident with the onset of glial differentiation is the occurence of cell fate bifurcation. In the cell division rescue experiments using eg-GAL4, Gcm-positive and Gcm-negative cells appear after the first cell division, although surrounding cells are still stg mutant. The cell fate bifurcation is probably regulated cell intrinsically and coupled to cell division. In contrast, all the nuclei of NB6-4T in the pbl mutant express Gcm and Repo. This may be because these proteins contain the nuclear localization signal that enables them to enter the nuclei within the single cell after translation, even if asymmetry might initially appear within the cell (Akiyama-Oda, 2000).

During cell division of NBs, the transcription factor Prospero is asymmetrically segregated to ganglion mother cells, in which this protein has a role in specification of cell identity. The first cell division of NB6-4T shows some similarity to such NB division, since the transcription factor Gcm is expressed preferentially in one daughter cell after cell division to start sublineage-specific differentiation. There may be a repressor and/or an activator of Gcm expression, which should be segregated to or expressed only in the neuronal daughter cell and the glial daughter cell (Akiyama-Oda, 2000).

Larval

repo is expressed in glial cells of eye, wing and leg discs (Campbell 1994 and Xiong, 1994).

The arrival of retinal axons in the Drosophila brain triggers the assembly of glial and neuronal precursors into a neurocrystalline array of lamina synaptic cartridges. Retinal axons arriving from the eye imaginal disc trigger the assembly of neuronal and glial precursors into precartridge ensembles in the crescent-shaped lamina target field. In the eye disc, photoreceptor cells assemble into ommatidial clusters behind the morphogenetic furrow (mf) as it moves to the anterior. The ommatidial clusters project their axon fascicles into the crescent-shaped lamina. Neuronal precursor cells of the lamina (LPCs) are incorporated into the axon target field at its anterior margin, which is demarcated by a morphological depression known as the lamina furrow. Glia precursor cells (GPCs) are generated in two domains that lie at the dorsal and ventral anterior margins of the prospective lamina. These glial precursors migrate into the lamina along an axis perpendicular to that of LPC entry. Postmitotic LPCs within the lamina axon target field express the nuclear protein Dac, as revealed by anti-Dac antibody staining. Like the eye, lamina differentiation occurs in a temporal progression on the anterioposterior axis. Axon fascicles from new ommatidial R-cell clusters arrive at the anterior margin of the lamina (adjacent to the lamina furrow) and associate with neuronal and glia precursors in a vertical lamina column assembly. At the anterior of the lamina, at the trough of the lamina furrow, LPCs await a retinal axon-mediated signal in G1-phase and enter their terminal S-phase at the posterior margin of the furrow. Postmitotic (Dac-positive) LPCs assemble into columns at the posterior margin of the furrow. In older columns at the posterior of the lamina, a subset of postmitotic LPCs express definitive neuronal markers as they become specified as the lamina neurons L1-L5. Lamina neurons L1-L4 form a stack in a superficial layer, while L5 neurons reside in a medial layer near the R1-R6 axon termini. These neurons arise at cell-type specific positions along the column's vertical axis. Lamina glial cells take up cell-type positions in the precartridge assemblies. Epithelial (E-glia) and marginal (Ma-glia) glia are located above and below the R1-R6 termini, respectively. Satellite glia are interspersed among the neurons of the L1-L4 layer. The Ma-glia and E-glia layers, both located ventral to the neuronal precursor column, sandwich the R1-R6 axon termini. The medulla neuropil serves as the target for R7/8 axons and is separated from the lamina by the medulla glia, situated just below the Ma-glia (Huang, 1998 and references).

Hedgehog, a secreted protein, is an inductive signal delivered by retinal axons for the initial steps of lamina differentiation. In the development of many tissues, Hedgehog acts in a signal relay cascade via the induction of secondary secreted factors. Lamina neuronal precursors respond directly to Hedgehog signal reception by entering S-phase, a step that is controlled by the Hedgehog-dependent transcriptional regulator Cubitus interruptus. The terminal differentiation of neuronal precursors and the migration and differentiation of glia appear to be controlled by other retinal axon-mediated signals. Thus retinal axons impose a program of developmental events on their postsynaptic field utilizing distinct signals for different precursor populations (Huang, 1998).

A number of markers distinguish glial and neuronal precursor cells from the corresponding mature cell types. The expression of optomotor-blind (omb) labels both glial precursors in the dorsal and ventral anlagen and mature glia that have migrated into the lamina target field. The glia cell marker Repo and the enhancer-trap lacZ insertion 3-109 are expressed by glia once they have entered the lamina target field. Cubitus interruptus (Ci), a transcriptional mediator of Hh signaling is expressed by LPCs anterior of the lamina furrow and by the postmitotic neuronal precursors within the lamina. The nuclear protein Dachshund is expressed only by neuronal precursors that have begun terminal differentiation and lie posterior to the lamina furrow. Thus, Omb and Ci label the glial and neuronal precursors, respectively, while the mature cells, following their interaction with retinal axons, additionally express Repo and Dac. In the lamina target field of eyeless mutants (mutants that project no neurons toward the optic disc), such as eyes absent (eya) or sine oculis (so), Dac expression is not detected and Repo expression is greatly diminshed. The migration and early differentiation of lamina glia are independent of Hh. Enhanced transcription of the putative Hh receptor, patched (ptc) is a universal characteristic of Hh signal reception. All classes of glia in the lamina region upregulate ptc expression in an hh-dependent fashion. These cells are thus Hh-responsive. All three classes of lamina glia, as well as medulla glia, that express a ptc-lacZ reporter construct are in close proximity to Hh-bearing retinal axons. Glia cell ptc reporter gene expression is not observed in hh- animals. This raises the question of whether Hh signal reception is responsible for the migration and/or subsequent maturation of glia cells. To determine whether the migration of glial precursors into the lamina target field is Hh-dependent, the distribution of Omb-positive cells was examined in hh- animals. In the wild type, a trail of Omb-positive cells delineates a path of glia migration from the dorsal and vental anlagen. Is glia precursor migration Hh-dependent? This was investigated by examining the distribution of Omb-positive cells in hh¹ mutant animals. hh¹ is a regulatory mutation that specifically affects hh expression in the visual system. In hh¹ animals, approximately 12 columns of ommatidia initiate differentiation in the eye imaginal disc before the anterior progression of the morphogenetic furrow ceases. hh¹ retinal axons lack Hh immunoreactivity by the time they reach the lamina target field and thus the Hh-dependent steps of LPC maturation fail to occur in hh¹ animals. Omb staining reveals a relatively normal number of glia precursors in the lamina target field of hh¹ animals, despite the absence of Dac induction. The Omb-positive cells are distributed uniformly along the dorsoventral axis among the retinal axon fascicles, but appear more closely spaced than in the wild type. A likely explanation for this spacing defect is the absence of the neuronal precursors that would constitute the majority of lamina cells at this point in development. To determine whether the glial precursors that enter the lamina target field in hh- animals express a retinal innervation-dependent marker, their expression of Repo was examined. In hh¹ animals, the Omb-positive cells within the lamina also express Repo. Moreover, the Repo-positive cells occupy proper layers above and below the R1-R6 axon termini expected for satellite, marginal and epithelial glia, though the lack of markers specific for these three glia types precludes an unambiguous determination of glial cell type. The presence of marginal and epithelial glia is consistent with the observation that R1-R6 growth cones terminate in their proper positions between these layers in hh- animals. The ectopic expression of Hh in the brains of `eyeless' animals is sufficient to induce the initial steps of LPC maturation in the absence of retinal axons. However, neither Hh nor the Hh-mediated events of LPC maturation are sufficient for glia cell migration and maturation (Huang, 1998).

Effects of mutation or deletion

Weak repo alleles are viable but affect glia in the optic lobe. This results in a reversal of polarity for the electrophysiological response to light in the adult. Strong repo alleles cause defects in embryonic glia and are lethal to embryos (Xiong, 1994). repo deficient glial cells do not migrate properly and by stage 16 many have died. Expression of late glial markers is severely reduced in repo mutants (Haller 1995 and Xiong, 1994).

REFERENCES

Akiyama, Y., et al. (1996). The gcm-motif: A novel DNA-binding motif conserved in Drosophila and mammals. Proc. Natl. Acad. Sci. 93: 14912-14916. 8962155

Akiyama-Oda, Y., et al. (2000). Distinct mechanisms triggering glial differentiation in Drosophila thoracic and abdominal neuroblasts 6-4. Dev. Biol. 222: 429-439. 10837130

Bernardoni, R., Miller, A. A. and Giangrande, A. (1998). Glial differentiation does not require a neural ground state. Development 125(16): 3189-3200.

Campbell , G., et al. (1994). RK2, a glial-specific homeodomain required for embryonic nerve cord condensation and viability in Drosophila. Development 120: 2957-2966. 7607085

Halter, D.A., Uban, J., Rickert, C., Ner, S.S., Ito, K., Travers, A.A. and Technau, G.M. (1995). The homeobox gene repo is required for the differentiation and maintenance of glia function in the embryonic nervous system of Drosophila melanogaster. Development 121: 317-332

Huang, Z. and Kunes, S. (1998). Signals transmitted along retinal axons in Drosophila: Hedgehog signal reception and the cell circuitry of lamina cartridge assembly. Development 125: 3753-3764. 9729484

Lee, B. P. and Jones, B. W. (2005). Transcriptional regulation of the Drosophila glial gene repo. Mech. Dev. 122(6): 849-62. 1593923

Shandala, T., Takizawa, K. and Saint, R. (2003). The dead ringer/retained transcriptional regulatory gene is required for positioning of the longitudinal glia in the Drosophila embryonic CNS. Development 130: 1505-1513. 12620977

Soustelle, L., et al. (2002). Terminal glial differentiation involves regulated expression of the excitatory amino acid transporters in the Drosophila embryonic CNS. Dev. Bio. 248: 294-306. 12167405

Xiong, W.C., Okano, H., Patel, N.H., Blendy, J.A. and Montell, C. (1994). repo encodes a glial-specific homeodomain protein required in the Drosophila nervous system. Genes & Dev. 8: 981-994

Xiong, W.C. and Montell, C. (1995). Defective glia induce neuroal apoptosis in the repo visual system of Drosophila. Neuron 14: 581-90

Yuasa, Y., et al. (2003). Drosophila homeodomain protein REPO controls glial differentiation by cooperating with ETS and BTB transcription factors. Development 130: 2419-2428. 12702656

date revised: 10 August 2005 

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