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).

Inhibiting glutamate activity during consolidation suppresses age-related long-term memory impairment in Drosophila

In Drosophila, long-term memory (LTM) formation requires increases in glial gene expression. Klingon (Klg), a cell adhesion molecule expressed in both neurons and glia, induces expression of the glial transcription factor, Repo. However, glial signaling downstream of Repo has been unclear. This study demonstrates that Repo increases expression of the glutamate transporter, EAAT1, and EAAT1 is required during consolidation of LTM. The expressions of Klg, Repo, and EAAT1 decrease upon aging, suggesting that age-related impairments in LTM are caused by dysfunction of the Klg-Repo-EAAT1 pathway. Supporting this idea, overexpression of Repo or EAAT1 rescues age-associated impairments in LTM. Pharmacological inhibition of glutamate activity during consolidation improves LTM in klg mutants and aged flies. Altogether, the results indicate that LTM formation requires glial-dependent inhibition of glutamate signaling during memory consolidation, and aging disrupts this process by inhibiting the Klg-Repo-EAAT1 pathway (Matsuno, 2019).

Changes in glial transcription due to neuronal activity have been studied previously, but a specific role of glial transcription in LTM has been less characterized. Expression of the glial transcription factor, Repo, increases shortly after spaced training, and this increase is required for LTM formation. This report has identified Eaat1 as a Repo-regulated glial gene required for LTM consolidation. Eaat1 encodes a glial glutamate transporter that removes glutamate from synaptic sites and transports it into astrocytes. Thus, the data indicate that glutamate signaling needs to be inhibited during LTM consolidation (Matsuno, 2019).

To identify Eaat1, a screen was performed for various genes regulating glial physiology for altered expression during LTM formation. Expression of Eaat1 and crammer was found to increase after spaced training. As Eaat1, but not crammer, is expressed exclusively in glia, focus was placed on Eaat1 as a likely Repo-regulated gene. Indeed, spaced-training-induced increases in EAAT1 depend on Repo and Klg activity. Interestingly, expression of the glial gene, genderblind, which encodes another glial glutamate transporter, required Repo activity for expression, but was not affected by spaced training, suggesting that other transcriptional regulatory factors besides Repo are likely necessary to differentially regulate genes required for memory consolidation from those required for other glial functions (Matsuno, 2019).

Because only screened selected genes were screened, it is possible that Repo induces the expression of other unidentified genes after spaced training. However, somewhat unexpectedly, it was found that overexpression of Eaat1 alone in glial cells is sufficient to rescue the LTM defects of klg and repo mutants. This indicates that the major function of the Klg/Repo signaling pathway is to induce glial expression of Eaat1. It further suggests that one function of astrocytes is to decrease glutamate signaling during LTM consolidation (Matsuno, 2019).

Combined with results from previous studies, this work identifies a putative pathway linking neuronal activity to glial inhibition of glutamate signaling. In flies, the homophilic cell adhesion molecule, Klingon, is expressed in both neurons and glia, and needs to be expressed in both cell types for normal LTM (Matsuno, 2015). Repo expression normally increases after spaced training, whereas it fails to do so in klg mutants, indicating that Klg-mediated neuron-glia communication is necessary for this increase (Matsuno, 2015). Thus, it is proposed that spaced training increases neuronal activity, which induces signaling to glia via the cell adhesion molecule Klg. This results in increased Repo activity in glia, which increases Eaat1 expression, and subsequently decreases glutamate signaling (Matsuno, 2019).

Previous work from various groups including has shown that glutamate signaling through NMDA-type receptors (NRs) is necessary for learning and memory. Overexpression of NRs in mice enhances learning and memory formation, and it has been shown that glial production of D-serine, a neuromodulator that functions as a coactivator of NRs, is necessary for short-lasting memory. In the current study, focus was placed on glutamate activity specifically during memory consolidation, instead of during initial learning and memory formation. Considering the current findings with those of previous studies, it is proposed that NR-dependent glutamate signaling needs to be initially high, during formation of short-lasting memories, but low during a later phase where short-lasting memories are consolidated into LTM. This suggests that glia play at least two roles in memory. They produce D-serine that contributes to high NR activity during memory formation and also produce EAAT1 after learning, which functions to reduce glutamate signaling during memory consolidation (Matsuno, 2019).

Age-related impairments in Drosophila memory do not consist of a general decrease in all forms of learning and memory, but instead consist of decreases in two specific phases of memory, MTM and LTM. The current results suggest that both these memory effects are caused by age-related glial dysfunction. Glia in young flies are able to produce sufficient amounts of D-serine for normal MTM, whereas D-serine amounts decrease 2-fold in aged flies. This decrease is responsible for age-related impairments in 1-h memory, because increasing glial production of D-serine, or directly feeding of D-serine to aged flies, rescues this impairment. Likewise, glial dysfunction is also responsible for age-related impairments in LTM because aged glia are unable to inhibit glutamate signaling during consolidation. Thus, in contrast to young flies, aged flies are unable to modulate glutamate activity during learning and consolidation, leading to defects in the two memory phases (Matsuno, 2019).

The model that EAAT1 inhibits glutamate activity during consolidation stems from EAAT1's role in clearing glutamate from synaptic sites and transporting it into astrocytes. This model is consistent with several mammalian studies that demonstrated decreased expression of astrocytic glutamate transporters upon aging, with a consequent reduction of glutamate uptake. Further supporting this model, it was found that feeding flies memantine or MK801, NMDA receptor antagonists, after spaced training, restores normal LTM in klg mutants and restores LTM in aged flies to youthful levels. This effect requires feeding after training during the consolidation phase. Similar results were obtained by feeding riluzole, a glutamate modulator, which decreases glutamate release and increases astrocytic glutamate uptake. Riluzole has also been reported to ameliorate age-related cognitive decline in mammals, suggesting that the mechanisms of AMI may be conserved between species. In contrast, this study found that D-serine feeding, which rescues age-related declines in short-lasting (1-h) memory, does not improve declines in LTM, but rather attenuates it. This is consistent with the model wherein declines in short-lasting memory and LTM are caused by distinct or opposing mechanisms and glutamate signaling needs to be suppressed during consolidation. Somewhat unexpectedly, it was also found that (s)-4C3HPG, the mGluR1 antagonist/mGluR2 agonist, also ameliorated age-related impairments in LTM. This result indicates that glutamate activity through both ionotropic and metabotropic glutamate receptors antagonizes memory consolidation (Matsuno, 2019).

Currently, it is unclear why glutamate signaling needs to be inhibited during consolidation, but a previous study has shown that Mg2+ block mutations in NMDA-type glutamate receptors (NRs) cause specific defects in LTM in Drosophila. Although Mg2+ block mutations have various effects, one effect is to increase NR activity. Increased NR activity results in increased activity of dCREB2b, an inhibitory isoform of CREB. CREB-dependent gene expression is required during consolidation of LTM, suggesting that consolidation may be preferentially sensitive to NR activity (Matsuno, 2019).

Alternatively, it is possible that neuronal activity needs to be inhibited globally during memory consolidation. Sleep is known to be important for LTM. Sleep deprivation during consolidation prevents LTM formation, whereas artificially inducing sleep after training has been reported to improve LTM. Thus a second possibility is suggested that inhibition of glutamate signaling after spaced training may be a brain-wide phenomenon that promotes consolidation by inducing the organism to sleep. Thus far, gross alterations in sleep duration in klg and repo mutants have not been detected, although this does not preclude minor disruptions in sleep quality that may not be detectable by motion-based sleep assays. Finally, a third possibility is envisioned wherein neuronal inhibition may be required as a neuroprotective mechanism that may be necessary to prevent cell death in neurons that were extensively stimulated during spaced training (Matsuno, 2019).

Mapping the glutamatergic neurons whose activity is inhibited during consolidation will be of great interest in the future. As aversive olfactory memories are formed and stored in the Drosophila MBs, it is possible that specific glutamatergic MB output neurons (MBONs) are inhibited during consolidation. Several glutamatergic MBONs are involved in feedback networks with the lobes of the MBs, suggesting that altering the activity of these neurons may modulate memory consolidation and memory-associated behavioral responses (Matsuno, 2019).

This study has demonstrate that increased expression of Eaat1 is required for LTM consolidation. Based on numerous results from other groups, it is hypothesized that Eaat1 functions to reduce glutamate signaling, and support for this model is provided by demonstrating that pharmacological inhibition of glutamate signaling during consolidation improves LTM under various conditions. However, due to technical limitations, it was not possible to actually measure glutamate concentrations at synapses during memory consolidation and it is not known where and how much glutamate signaling has to be inhibited for optimal LTM consolidation (Matsuno, 2019).

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).

Transcriptional Regulation

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

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).

Glial wingless/Wnt regulates glutamate receptor clustering and synaptic physiology at the Drosophila neuromuscular junction

Glial cells are emerging as important regulators of synapse formation, maturation, and plasticity through the release of secreted signaling molecules. This study used chromatin immunoprecipitation along with Drosophila genomic tiling arrays to define potential targets of the glial transcription factor Reversed polarity (Repo). Unexpectedly, wingless (wg), encoding a secreted morphogen that regulates synaptic growth at the Drosophila larval neuromuscular junction (NMJ), was identified as a potential Repo target gene. Repo regulates wg expression in vivo, and local glial cells secrete Wg at the NMJ to regulate glutamate receptor clustering and synaptic function. This work identifies Wg as a novel in vivo glial-secreted factor that specifically modulates assembly of the postsynaptic signaling machinery at the Drosophila NMJ (Kerr, 2014).

The diversity of genes directly regulated by Repo-a critical transcriptional regulator of glial cell development in Drosophila-has not been thoroughly explored. ChIP studies from Drosophila S2 cells identified several potential Repo targets that have been shown to govern fundamental aspects of glial development or function. For example, known targets were identified that actively promote glial cell fate specification (e.g., pointed, distalless;, blood-brain barrier formation (e.g., gliotactin, loco, coracle, Nrv1, engulfment activity (e.g., dCed-6), neurotransmitter metabolism (e.g., EAAT1, Gs2), ionic homeostasis (e.g., fray), and neuron-glia signaling during nervous system morphogenesis (e.g., Pvr). For at least two potential targets, gs2 and Cp1, this study demonstrated a key requirement for Repo in their transcriptional activation during development (Kerr, 2014).

Given the broad roles of this collection of genes in glial cell biology, this work supports the hypothesis that Repo transcriptionally regulates a diverse class of genes that modulate many aspects of glial cell development. For instance, Pointed, which is now a predicted Repo target, is a key glial factor that activates glial fate at very early developmental stages. Likewise, Repo appears to regulate Gliotactin, Coracle, and Nrv1, which are molecules essential for formation of the pleated septate junction-based blood-brain barrier at mid to late embryogenesis in Drosophila. At the same time, EAAT1 and GS2 are activated late in the embryonic glial program, with expression being retained even in fully mature glia, and these transporters are critical for synaptic neurotransmitter recycling. Since EAAT1 and GS2 are both activated by Repo, and primarily expressed in CNS glia, these data argue that Repo is directly upstream of multiple key glial factors required for glutamate clearance from CNS synapses (Kerr, 2014).

Mammalian excitatory glutamatergic synapse formation is modulated by multiple soluble glia-derived factors including TSPs, Hevin/Sparc, and glypicans 4 and 6. These factors, along with other secreted glial factors that remain to be identified, are essential for initial synapse formation and (with the exception of TSPs) can promote postsynaptic differentiation through membrane insertion and clustering of AMPA receptors. This study identified Wg as a novel glia-derived factor essential for postsynaptic structure and function in vivo at the Drosophila glutamatergic NMJ. Combined with previous findings that NMJ glia can also release a TGF-β family member to regulate presynaptic growth in a retrograde manner (Fuentes-Medel, 2012), these studies provide compelling evidence that Drosophila glia function as a major integrator of synaptic signals during developmen (Kerr, 2014).

Previous work has demonstrated that Wg/Wnt signaling potently modulates the coordinated assembly of both presynaptic and postsynaptic structures at the Drosophila NMJ (Speese, 2007). Loss of Wg, or its receptor DFz2, leads to a dramatic decrease in synaptic boutons and disrupted clustering of postsynaptic glutamate receptors (Packard, 2002). Although previous studies supported evidence implicating motor neurons in Wg release, the presence of alternative cellular sources remained an open and important question. The surprising discovery of Wg as a candidate Repo target gene by ChIP led to an exploration of the possibility that NMJ glia could act as an additional in vivo source of NMJ Wg. Consistent with this idea, this study found that peripheral glia expressed Wg, SPGs were able to deliver Wg::GFP to the NMJ, and knockdown of SPG Wg significantly reduced NMJ Wg levels and led to a partial phenocopy of wg mutant phenotypes (Kerr, 2014).

Interestingly, it was found that loss of glia-derived Wg could account for some, but not all, wg loss-of-function phenotypes. For example, whereas depletion of glia-derived Wg disrupted clustering of postsynaptic glutamate receptors, it had no effect on the formation of synaptic boutons. In contrast, depletion of neuronal Wg led to defects in both glutamate receptor clustering as well as bouton formation. Although only neuronal Wg regulated bouton growth, these data argue that both glial and neuronal Wg are capable of modulating the assembly of glutamate receptor complexes. Thus, this study has identified two in vivo sources of Wg at the NMJ: the presynaptic neuron and local glial cells (Kerr, 2014).

Regarding the modulation of neurotransmission, both glial and neuronal Wg was found to have important roles, which, as in the case of the development of synaptic structure, were only partially overlapping. Loss of glial or neuronal Wg resulted in postsynaptic defects in neurotransmission, including increased mEJP amplitude (a postsynaptic property), decreased nerve-evoked EJPs, and decreased quantal content. Consistent with Repo regulating glial Wg expression, these phenotypes were mimicked by loss of repo function. The most notable difference in functional requirements for glial versus neuronal Wg is in mEJP frequency (a presynaptic function): depletion of glial Wg resulted in a dramatic increase in mEJP frequency, whereas manipulating neuronal Wg had no effect. Thus both glial and neuronal Wg are critical regulators of synaptic physiology in vivo, likely modulating NMJ neurotransmission in a combinatorial fashion, although glial Wg has the unique ability to modulate presynaptic function (Kerr, 2014).

The increase in mEJP amplitude is consistent with findings that GluR cluster size was increased upon loss of glia- or neuron-derived Wg, and that in general this was accompanied by minor changes in GluRIIA signal intensity. A potential explanation is that neuron- and glia-derived Wg regulate the levels of GluRIIA subunits. Previously, it was demonstrated that downregulation of the postsynaptic Frizzled Nuclear Import (FNI) pathway also increased GluRs at the NMJ (Speese, 2012). This suggests that glia- and neuron-derived Wg may act in concert via the FNI pathway to stabilize the synapse by regulating GluR expression (Kerr, 2014).

An important property of the larval NMJ is the ability to maintain constant synaptic function throughout development via structural and functional modifications. The combined functions of glial and neuronal Wg likely contribute to this mechanism, as together they positively regulate synaptic growth and function as well as organize postsynaptic machinery. However, a previous study suggested that the transcription factor Gooseberry (Gsb), in its role as positive regulator of synaptic homeostasis in neurons, may be antagonized by Wg function (Marie, 2010). Mutations in gsb block the increase in neurotransmitter release observed when postsynaptic GluRs are downregulated. Furthermore, Marie (2010) showed that the gsb mutant defect can be rescued by a heterozygous wg mutant allele. However, the specific role of Gsb in this process is unclear, as rapid synaptic homeostasis was normal in the mutant, and defects appeared restricted to a long-term decrease in GluR function. It will be important to define the specific role of Gsb in synaptic homeostasis and to manipulate Wg function in alternative ways before a clear relationship between Wg and Gsb can be established (Kerr, 2014).

How could neuronal versus glial Wg differ in regulating NMJ development and physiology? One possibility is that the level or site of Wg delivery by each cell type is different. For example, since SPGs invade the NMJ only intermittently (Fuentes-Medel, 2009), it is possible that they release most of their Wg outside of the NMJ, whereas the presynaptic neuron, which is embedded in the muscle cell, delivers it more efficiently and directly to the postsynaptic muscle cell. Alternatively, the Wg morphogen released by glia versus that released by neurons could be qualitatively different through alternative post-translational modifications such as glycosylation. Either mechanism would allow for glia to modulate specific aspects of NMJ physiology independently from neuronal Wg, perhaps in an activity-dependent manner (Kerr, 2014).

Although glia-derived Wg does not modulate NMJ growth, Drosophila glia can indeed regulate synaptic growth at the NMJ in vivo. It has been demonstrated previously that Drosophila glia release the TGF-β ligand Maverick to modulate TGF-β/BMP retrograde signaling at the NMJ and thereby the addition of new synaptic boutons (Fuentes-Medel, 2012). The discovery that glia-derived Wg can exert significant control over the physiological properties of NMJ synapses expands the mechanisms by which Drosophila glia can control NMJ synapse development and function. In the future it will be important to understand how glial Wg and TGF-β signaling integrate to promote normal NMJ growth, physiology, and plasticity (Kerr, 2014).

DEVELOPMENTAL BIOLOGY

Embryonic

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).

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date revised: 15 August 2023 

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