BIOLOGICAL OVERVIEW

Finding a transcription factor that acts close to a branch point in a developmental pathway can be rather exciting. Glial cells missing acts near the point at which neural cells and glia diverge. What is the basis for the generation of two cell fates from a single precursor?

In many cases glia and neurons have a common precursor: the neuroblast. glial cells missing is expressed in almost all glia as soon as they are born. If gcm is lacking, presumptive glial cells are transformed into neurons. If gcm is expressed in presumptive neurons, these cells transform into glia. gcm does not provide an immediate explanation for the establishment of alternate cell fates, as does the action of Prospero and Numb, but its effects occur close enough to the point at which neural and glial cells diverge so that an excess or deficiency of GCM can cause a reversal of cell fate. Is gcm conserved throughout evolution. How many genes distinguish the developmental pathways of neurons and glia, and what is the pathway of their activation. Where does Glial cells missing fit into the developmental hierarchy? (Hosoya, 1995 and Jones, 1995).

Two targets of GCM have been identified in activation of glial cell fate in the longitudinal glia. The embryonic Drosophila CNS has two classes of glia: midline glial cells and lateral glial cells. Midline glial development is triggered by EGF-receptor signaling, whereas lateral glial development is controlled by the glial cells missing (gcm) gene. Subsequent glial cell differentiation depends partly on pointed . tramtrack (ttk) is required for all CNS glia development. Mutant ttk embryos are characterized by an embryonic CNS axon pattern phenotype of fused segmental commissures, indicating a requirement of ttk during midline glial development. In ttk embryos, longitudinal axon tract formation is impaired and the connectives appear thinner. This phenotype is indicative of a defect in the longitudinal glia (Giesen, 1997).

tramtrack encodes two zinc-finger proteins, one of which, Ttkp69, is expressed in all non-neuronal CNS cells. ttk expression in the ventral cord is restricted to lateral and midline glial cells. All cells that express the glial marker Repo also express Ttkp69. The transverse nerve exit glial cells (or DM cells) express Ttkp69. In the CNS of stage 16 ttk mutants, there are about 20% less lateral glial cells than a wild-type CNS. In mutants, although the midline glial cells are initially present in normal number and position, they fail to perform their normal migration. Therefore ttk is required for normal glial development. The exit glial cells in mutant ttk embryos are slightly enlarged, but they are still able to ensheath both the segmental and intersegmental axon bundles. Like ttk, pointed is expressed in glial cells. However, unlike ttk, pointed is required for glial cell development. Ectopic ttkp69 expression in the neuroectoderm leads to a partial block of neuronal development as indicated by substantially reduced expression of the neuronal Elav antigen as well as other neuronal markers examined (Giesen, 1997).

Both Ttkp69 and pointed are downstream of gcm. gcm, however, is not expressed in midline glia, and ttkp69 as well as pointed expression in midline cells is normal in gcm mutants. pointed and ttkp69 are both expressed under the control of gcm in lateral glial cells; the expression of these genes appears to be independent of one another. Thus the two targets of gcm appear to act in parallel. Glial cell differentiation may depend on a dual process, requiring the activation of glial differentiation by pointed and the concomitant repression of neuronal development by tramtrack (Giesen, 1997).

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. glide/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 glide/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 glide/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 glide/gcm activation. The competence to express glial-specific genes becomes restricted during development, since many fewer cells adopt a glial fate when glide/gcm is expressed late. Mesodermal glide/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 glide/gcm ectopically lack most muscle fibers and display a significant number of round, unfused muscle cells. Ectopic expression of glide/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 glide/gcm overrides positional information by repressing the endogenous developmental program. These findings also indicate that glial differentiation tightly depends on glide/gcm transcriptional regulation. It is likely that glide/gcm homologs have similar actions during vertebrate gliogenesis (Bernardoni, 1998).

The finding that ectopic glide/gcm triggers transcription of downstream glial-specific targets raises an important question about the mechanisms governing cell-specific transcription. How can genes that are normally silent in mesoderm be switched on by the activity of a single transcription factor? An appealing explanation is that, like other activators, glide/gcm may recruit coactivators that remodel nucleosomes in the regions containing glide/gcm targets and activate the transcription of such genes. Interestingly, glide/gcm is less efficient when induced in mesoderm at late stages, which may be due to the reduced accessibility of the chromatin of glide/gcm targets with development over time. It is proposed that, as the mesodermal program is activated, a more stable repressive state in those chromatin regions is established and propagated throughout the cell cycle. The establishment of a stable structural difference on chromosomal domains has already been observed during vertebrate development. A role for the proliferative state in the establishment of a repressed chromatin state is also in agreement with the different degree of glial fate induction observed upon glide/gcm expression in pre- versus post-mitotic cells of the nervous system. Thus, understanding the glide/gcm mode of action represents an extremely important step toward unravelling the general mechanisms of cell differentiation at a molecular level (Bernardoni, 1998).

In the development of the Drosophila central nervous system, some of the neuroblasts designated as neuroglioblasts generate both glia and neurons. Little is known about how neuroglioblasts produce these different cell types. NB6-4 in the thoracic (T) segment (NB6-4T) is a neuroglioblast, although the corresponding cell in the abdominal (A) segment (NB6-4A) produces only glia. The cell divisions in the NB6-4T lineage are described, following changes in cell number and cell arrangement. These cell divisions occur in the proliferative zone, underlying the epidermis, after neuroblast delamination. In the thoracic segment, mitosis gn1 (generating glial and neural precursors) occurs in NB6-4T, oriented parallel to the embryonic surface with the production of M1 (medial) and L1 (lateral) cells. Initially, GCM mRNA is evenly distributed, but becomes localized to the medial half of the NB6-4T cell and is inherited primarily by M1, which then expresses Gcm protein. M1 is a glioblast that divides twice (mitosis g2 and g3) to give rise to three glial cells, MMM3, MML3 and ML2. They correspond to MM-CBGs and M-CBG. Mitoses g2 and g3 are also oriented parallel to the epithelial plane. L1 generate smaller Prospero-positive ganglion mother cells through mitoses n2 and n3 from the basal side. In the abdominal segment, NB6-4A divides once in the mediolateral orientation, producing two glial cells. During this cell division, GMC mRNA is distributed evenly and segregated into both daughter cells. Gcm protein is also detected during division and in both daughter cells. These observations suggest that mechanisms regulating GCM mRNA expression and its translation play an important role in glial and neuronal lineage bifurcation that results from asymmetric cell division (Akiyama-Oda, 1999).

Thus a correlation exists between orientation of mitoses and cell identities in the NB6-4 lineage. The first division of NB6-4T and the subsequent cell divisions of its glial (medial) progeny are oriented parallel to the embryonic surface. These cell divisions are in contrast to those of many other neuroblasts, which are oriented perpendicular to the epithelial plane. It has been shown that two glioblasts, GP and NB6-4A, which give rise to only glial cells, divide parallel to the embryonic surface. Similar observations have been obtained in studies of the median neuroblast division in the glasshopper embryo. Thus, mitoses that produce glia or glial precursors are oriented parallel to the embryonic surface, at least as so far observed in the CNS. Although the mitotic orientation in the NB6-4 lineage could not be determined in gcm overexpressing embryos, orientation in gcm mutants appears normal. This suggests that orientation of the glia-producing mitoses is not a result of gcm expression. L1 divides in an orientation parallel to the apicobasal axis of the surface epithelium. This mitotic orientation is perpendicular to those of NB6-4T and M1, indicating that the spindle orientation must be rotated through 90 degrees in the L1 cell and that the mitotic axes of M1 and L1 are differentially determined, as well as their cell fate. How mitotic orientation is regulated remains to be studied (Akiyama-Oda, 1999).

The first cell division of NB6-4T (mitosis gn1) bifurcates glial and neuronal lineages. Expression of GCM mRNA is closely associated with this bifurcation. The mRNA starts to be synthesized before mitosis and is distributed evenly throughout the entire cytoplasm. However, it is inherited by the M1 cell after mitosis gn1. This asymmetric inheritance of GCM mRNA is the primary event separating the glial from neuronal progeny in the NB6-4T lineage. The asymmetric distribution of the mRNA may be due to spatial control of stability, directed transport or anchoring to localized RNA-binding factors. As for Prospero mRNA localization in neuroblasts, RNA-binding protein Staufen has been shown to be required in this process. Continuous overexpression of Gcm in the NB6-4T lineage causes all the progeny cells to follow the glial fate. It is possible that regulation at the mRNA level as well as at the transcriptional level plays an important role in preventing gcm expression in the neuronal lineage and facilitating sufficient gcm expression in the glial lineage. This strict regulation seems important, since the gcm activity is highly effective in inducing glial fate (Akiyama-Oda, 1999 and references).

Characterization of a second Gcm gene in Drosophila

The fly glial cell missing gene codes for a transcription factor that induces gliogenesis. Lack of its product eliminates lateral glial cells in the embryonic nervous system. A second gene, glide2, termed gcm2 in FlyBase, has been identified that is homologous to gcm in the binding domain and is also necessary and sufficient to promote glial differentiation. glide2 codes for a transcription factor that displays a weaker and delayed expression compared with gcm. The two genes, which are located 27 kb apart and share cis-regulatory elements, are able to auto- and cross-regulate, indicating that they form a gene complex. Finally, it has been shown that lack of both products eliminates all lateral glial cells, which means that the two genes contain all the fly lateral glial promoting activity (Kammerer, 2001).

The existence in the fly genome of a 141 bp fragment homolog to the gcm motif prompted an analysis of the putative role and profile of expression of the gene containing this sequence. The glide2 gene maps at 30B and is located 27 kb 5' to the gcm gene, the two genes being disposed head to head. Some 480 bp downstream of glide2 is located a gene homologous to thioredoxin, whereas two predicted genes are located between gcm and glide2. No ESTs have been recovered for such genes in embryonic libraries. Their predicted open reading frames (ORFs) have some similarity with esterase-like proteins and are not related to the gcm family. One member of this protein family, Gliotactin, is required for the formation of the blood-brain barrier, but not to promote gliogenesis (Kammerer, 2001).

Computational analysis on the glide2 genomic sequence predicts the existence of a gene containing three exons, however, the 5' end of the cDNA clone maps upstream of the predicted gene. To determine the structure and the 5' end of glide2, reverse transcription PCR (RT-PCR) was performed on RNA from wild-type embryos using different oligonucleotide sets. This revealed the existence of a glide2 cDNA containing four exons; the length of the transcript is ~2.4 kb. A putative Initiator element (Inr) is present 58 bp 5' to oligo nucleotide P1. Such cDNA contains a predicted ORF of 606 amino acids, with a deduced molecular weight of 65 kDa. This protein sequence differs from the 613 amino acid protein proposed in GadFly in the first four amino acid residues. The first exon contains an untranslated region of 433 bp (relative to the putative start site in the Inr), as well as the first 11 coding nucleotides. The second exon contains the gcm motif as well as the PEST motif, typical of rapidly degraded proteins. Interestingly, the latter motif is conserved throughout evolution in the Gcm family even though the position and the number of PEST motifs varies. Stretches of homopolymeric repeats of glutamine, serine, alanine, glycine, tyrosine, histidine and proline are located in the first 60 and in the last 250 amino acids. Surprisingly, while it has been shown that gcm codes for a transcription factor, no clear nuclear localization signal (NLS) could be identified in Glide2. The only other member of the Gcm family devoid of NLS is hGcmA (Kammerer, 2001).

The degree of conservation between Gcm or Glide2 and the different family members is very similar, which makes it difficult to assign a specific ancestor for vertebrate gcmA and gcmB. The homology between the two fly genes within the gcm motif, however, is stronger (69%) than that found between any of them and the other members of the family (56%-64%). These data suggest that indepedent duplications have taken place in arthropod and vertebrate lineages. Within the motif, Glide2 retains the four cysteines and the seven histidines displaying the same relative spacing H-X₁₁-H-X₈-C-X₅-C-X₃-C-X₁₄-C-X₁₁-C-X_2-4-C-X_8-9-C-X₂-H-X₂₃-H as that found in Gcm. Cysteines within the gcm motif have been shown to be either essential for DNA binding and transactivation or to play a role in the regulation of redox sensitivity to DNA binding (Kammerer, 2001).

To understand the mode of action of glide2, its expression during development was analyzed. RT-PCRs on total RNA from different stages produced amplification products of the expected size. glide2 expression is first detected in embryos: it decreases during larval stages and peaks again in 1-day pupae. Almost no RNA is present in adult flies. In situ hybridization was performed on wild-type embryos using a glide2-specific riboprobe. Transcripts are first detected at around stage nine in the procephalic mesoderm, the region from which hemocytes take origin, and in one cell per hemisegment, at the position of the neuroglioblast 1-3 lineage. The levels of expression, as well as the number of expressing cells in the trunk, increase as development proceeds. By stage 12, most transcripts are restricted to the nervous system, at the position of glial lineages. Some expression is also detectable in ectodermal stripes located laterally at the position of apodemal cells. No expression was found in midline cells (Kammerer, 2001).

The profiles of glide2 and gcm are very similar, even though glide2 is expressed at much lower level than gcm. Using probes of similar length and the same experimental conditions for the two reactions, glide2 expression was detected after 4 h, whereas gcm expression was detected after a few minutes. This is in agreement with the observation that the glide2-specific signal cannot be detected on Northern blots containing 10 µg of poly(A)⁺ RNA. Overall, glide2 expression is delayed compared with that of gcm, which is already detected at the end of the blastoderm stage and fades by stage 15 (Kammerer, 2001).

glide2 expression profile suggests a role in glial differentiation; therefore whether glide2 is able to induce gliogenesis was tested. Using scabrous (sca-Gal4) and twist (twi-Gal4) drivers, glide2 was expressed throughout the neurogenic region and in the mesoderm, respectively. In both cases, ectopic gliogenesis, as revealed by labeling with anti-Repo, an antibody that recognizes all lateral embryonic glial cells, was observed. Thus, as for Gcm, Glide2 is sufficient to induce the glial fate, irrespective of the cell type in which it is expressed (Kammerer, 2001).

By using a gcm-specific probe it was found that glide2 expression in the neurogenic region does induce gcm whereas mesodermal glide2 expression does not, indicating that the Glide2 protein is sufficient to promote gliogenesis. To confirm that glide2 does not require gcm, ectopic expression was induced in a null mutant, gcm^N7-4, which misses most lateral glial cells. Indeed, neuroblast glide2 expression leads to massive ectopic Repo in the CNS of gcm^N7-4 embryos. Interestingly, Gcm induces more glial cells than Glide2. These results altogether demonstrate that (1) glide2 is sufficient to induce gliogenesis, (2) glide2 can induce gcm expression, and (3) glide2 can induce gliogenesis directly, without gcm (Kammerer, 2001).

gcm codes for a transcription factor that binds to the Gcm binding site (GBS) and thereby activates target genes. Given the structure of Glide2 and its ability to induce gcm, it was asked whether it behaves as a transcription factor. To do this, a glide2 expression vector was co-transfected with a chloramphenicol acetyl transferase (CAT) reporter containing a GBS (5'-ATGCGGGT-3') in front of the thymidine kinase (tk) promoter fused to the CAT coding sequence. Transactivation was quantified by measuring the amounts of CAT protein. The results were compared with those obtained with an expression vector containing gcm coding sequences or with an expression vector that does not contain any insert. Transactivation assays were performed by using different amounts of expression vector. As expected, gcm expression results in the activation of transcription, which was arbitrarily given a 100% value. Glide2 also induces transcription, compared with the results obtained with the 'empty' transcription vector. Glide2, however, is weaker compared with Gcm. Moreover, its transactivation potential decreases as the amount of protein increases: 0.5 µg of glide2 expression vector lead to a congruous activation (25%) whereas 5 µg of the same vector leads to very weak activation (<10%) (Kammerer, 2001).

In a second co-transfection assay, a fragment containing three GBSs was used. In this context, Glide2 and Gcm show similar transactivation potentials when >2.5 µg of expression vector are transfected. The 1.96 kb fragment used in this test is located in the glide2 promoter, suggesting that glide2 expression depends on a feedback loop and/or on cross-regulation from Gcm. Indeed, the 27 kb fragment separating the two genes contains many GBS consensus [5'-AT(G/A)CGGG(T/C)-3'], most of which are located closer to glide2 than the gcmtranscription start site. Although the presence of binding sites cannot be taken as an evidence for direct regulation, it is interesting to note that the two predicted genes are located between sites 3 and 5 and are separated by site 4. Further analyses will be required to define the profile of expression of such genes and to determine whether they are targets of Glide-like molecules (Kammerer, 2001).

In order to see if gcm regulates glide2 in vivo, the effects of loss- and gain-of-function gcm mutations on the expression of glide2 were examined. By performing RT-PCR, an increase of glide2 expression was found in embryos that express gcm throughout the neurogenic region. The increase of glide2 expression is comparable to that observed with repo. The activation of glide2 by gcm is further confirmed by in situ experiments with glide2-specific probes on wild-type and sca-glide/gcm embryos. Massive ectopic glide2 expression is present in sca-glide/gcm embryos, the labeling being limited to the neurogenic region, in which gcm has been induced. These results, together with the transfection assays, strongly suggest that gcm directly induces glide2 expression. They also explain why glide2 expression is slightly delayed compared with that of gcm (Kammerer, 2001)

glide2 expression was analyzed in two gcm null mutations, gcm^N7-4 and gcm²⁶. glide2 expression is not severely affected in gcm^N7-4 embryos, while it is increased (4-fold induction) in gcm²⁶. These intriguing results prompted an analysis of the spatial distribution of gcm transcripts by in situ hybridization. Surprisingly, most of the glide2-specific signal is absent in the ventral cord of gcm^N7-4 embryos, whereas it is less affected in gcm²⁶. In gcm^N7-4, very few cells still express glide2, the level being much lower than in the wild type. In gcm²⁶ embryos, more cells express it; in addition, the level of glide2 expression is rather high, as confirmed by the fact that some of them enter the glial pathway and also express repo. These in situ results also indicate that glide2 regulation is tissue-specific, since the expression in the lateral stripes is not induced by gcm. Indeed, lateral stripes show stronger labeling in gcm²⁶ than in wild-type embryos, which explains why glide2 RNA levels are increased in RT-PCR experiments. Thus, gcm at least partially controls glide2 expression (Kammerer, 2001).

By performing PCR on genomic DNA, it was established that glide2 resides within the Df(2L)132 . This deficiency spans ~115 kb and covers two lethal complementation groups, gcm^N7-4 and, more proximally, l(2)DA2. No known genes affecting glial differentiation are contained within the deficiency. Df(2L)132 completely lacks Repo labeling in embryonic PNS and CNS whereas gcm null mutants, which partially affect glide2 expression, still retain some Repo labeling. In particular, 36 and 40 repo-positive cells were found, respectively, in the cord and in the head of gcm²⁶ embryos whereas three and seven were found in gcm^N7-4 embryos. This further confirms that glide2 is required for gliogenesis. Interestingly, the amount of Repo-positive cells directly correlates with the levels of glide2 expression previously observed in the mutants, the lowest value being in the deficiency, the highest in gcm²⁶. Moreover, gcm²⁶ and glide/gcm^N7-4 embryos also display weaker axonal defects compared with those observed in the deficiency, as shown by labeling with several axon-specific antibodies. Indeed, breaks in the longitudinal commissures, as well as abnormal fasciculation in the intersegmental and segemental nerves, were more often seen in the deficiency than in gcm mutants. This substantiates the role of glide2 in the differentiation of glial cells, which are necessary to maintain the axon scaffold. Although the possibility that genes contained in the deficiency might somehow act in glial differentiation and axonal navigation cannot be formally excluded, the results obtained with this mutant are in agreement with all the other in vivo and in vitro data and support the hypothesis that glide2 and gcm act in concert to promote glial differentiation (Kammerer, 2001).

The characterization of two null mutations, gcm²⁶ and gcm^N7-4, has allowed the nature of promoter interactions at 30B to be clarified and also to explain the different behavior of the two alleles. The gcm^N7-4 mutation produces an inactive protein, as a consequence, glide2 transcription is severely impaired. However, the deletion of gcm promoter and transcribed sequences (as seen in gcm²⁶) has a milder effect on glide2. The most likely interpretation of these results is that glide2 expression is activated by gcm via the gcm binding sites and through a regulatory element that is common to gcm and glide2. In a wild-type embryo, this element preferentially activates gcm by interacting with its proximal promoter. In gcm²⁶ embryos, however, the proximal promoter of gcm is not available. In its absence, the common regulatory element can only work on the glide2 promoter and activates it more efficiently. This is why glide2 transcription is less affected in gcm²⁶ than in gcm^N7-4. Such promoter competition is even more evident outside the nervous system. In the lateral stripes of cells, glide2 expression is higher in gcm²⁶ than in wild-type embryos. Promoter competition also explains why glide2 expression increases as gcm expression declines (Kammerer, 2001).

GENE STRUCTURE

Exons - two

PROTEIN STRUCTURE

Amino Acids - 504

Structural Domains

GCM has a distinct nuclear targeting sequence, a basic cysteine rich region, PEST-like sequences that assist in rapid protein degradation, and three repeats of the sequence MPVP in the N-terminal region (Hosoya, 1995 and Jones, 1995).

A transactivation function has been detected within the C-terminal part of GCM. In addition to this transactivation domain a sequence-specific DNA-binding domain maps within the N-terminal part of the GCM protein in close proximity to a bipartite nuclear localization signal. Both the lack of homology to known proteins and the novel DNA binding specificity indicate that GCM contains a new type of DNA-binding domain. Thus, GCM is a novel type of transcription factor involved in early gliogenesis (Schreiber, 1997).

The gcm2 cDNA clone has an ORF that encodes a protein of 613 amino acids, a 928-bp 5' UTR, and a 171-bp 3' UTR with poly(A) tail. Comparison with the published genomic sequence has revealed the gcm2 transcription unit has 3 exons and a location 27.9 kb 5' to the gcm gene, in opposing orientation. Located between gcm2 and gcm are two predicted genes encoding for carboxylesterases (CG3841 and CG4382). Immediately 3' of gcm2 is the thioredoxin gene, one of several Thioredoxin-like genes in Drosophila. The predicted amino acid sequence and exon structure of gcm2 are in agreement with the prediction of the Genome Project, differing in amino acid sequence at only six residues. A second cDNA has a fourth, alternative 5' exon, located upstream, that replaces the first 11 residues of the first predicted protein with four different residues. These results suggest that gcm2 has two separate promoters with alternate splicing at the 5' end. Gcm2 protein is similar in structure to other Gcm family members, sharing a highly conserved N-terminal gcm-motif of 156 amino acids. While all Gcm family members are very similar in the gcm-motif, Gcm2 and Gcm are more similar to each other (69% identity; 83% similarity) than to their two vertebrate counterparts Gcm1/GCMa and Gcm2/GCMb (55%-64% identity; 70%-78% similarity to human Gcm1 and Gcm2) (Alphonso, 2002).

EVOLUTIONARY HOMOLOGS

The GCM 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. Homologous genes from human and mouse have been identified whose products share a highly conserved N-terminal region with Drosophila GCM. At least one of these has been shown to have DNA-binding activity similar to that of GCM. By comparing the deduced amino acid sequences of these gene products, a "gcm motif," has been defined. There are 3 absolutely conserved stretches of 9 or 10 amino acid residues, and seven conserved cysteine and four conserved histidine residues. The evolutionarily conserved motif possesses the DNA-binding activity. There is evidence for the existence of additional gcm-motif genes in mouse as well as in Drosophila. The gcm-motif, therefore, forms a family of novel DNA-binding proteins, and may function in various aspects of cell fate determination (Akiyama, 1996).

Glia cell missing (GCM) transcription factors form a small family of transcriptional regulators in metazoans. The prototypical Drosophila GCM protein directs the differentiation of neuron precursor cells into glia cells, whereas mammalian GCM proteins are involved in placenta and parathyroid development. GCM proteins share a highly conserved 150 amino acid residue region responsible for DNA binding, known as the GCM domain. The crystal structure of the GCM domain from murine GCMa bound to its octameric DNA target site is presented at 2.85 Å resolution. The GCM domain exhibits a novel fold consisting of two domains tethered together by one of two structural Zn ions. The novel use of a ß-sheet in DNA recognition is observed, whereby a five- stranded ß-sheet protrudes into the major groove perpendicular to the DNA axis. The structure combined with mutational analysis of the target site and of DNA-contacting residues provides insight into DNA recognition by this new type of Zn-containing DNA-binding domain (Cohen, 2003).

The pharyngeal arches give rise to multiple organs critical for diverse processes, including the thymus, thyroid and parathyroids. Several molecular regulators of thymus and thyroid organogenesis are strikingly conserved between mammals and zebrafish. However, land animals have parathyroids whereas fish have gills. The murine transcription factor Glial cells missing 2 (Gcm2) is expressed specifically in the parathyroid primordium in the endodermal epithelium of the third pharyngeal pouch, and in both mice and humans is required for normal development of parathyroid glands. The molecular regulation of fish gill organogenesis remains to be described. gcm2 is expressed in the zebrafish pharyngeal epithelium, and Hox group 3 paralogs are required for gcm2 expression. Strikingly, zebrafish gcm2 is expressed in the ectodermal portion of the pharyngeal epithelium and is required for the development of the gill filament buds, precursors of fish-specific gill filaments. This study identifies yet another role for a GCM gene in embryonic development and indicates a role for gcm2 during the evolution of divergent pharyngeal morphologies (Hogan, 2004).

The glial cells missing (gcm) gene in Drosophila encodes a transcription factor that determines the choice between glial and neuronal fates. Two mammalian gcm homologs, Gcm1 and Gcm2, have been isolated and their expression patterns characterized during embryonic development. Although Gcm2 is expressed in neural tissues at a low level, the major sites of expression for both of the mammalian genes are nonneural, suggesting that the functions of the mammalian homologs have diverged and diversified. Gcm1 is detected in a subset of cells in the placenta. The location of the positive cells within the placenta suggests that they are labyrinthine trophoblasts. No other tissue examined was positive for Gcm1 transcripts by in situ hybridization. The expression of Gcm2 also appears to be highly restricted. Only parathyroid tissue was positive for Gcm2. A comparison to PTH gene expression on adjacent sections indicates that the cells expressing Gcm2 are PTH-secreting cells. The expression of Gcm2 in parathyroid tissue is consistent with the isolation of a human Gcm2 cDNA from an adult parathyroid adenoma. A developmental time course analysis of Gcm2 expression shows that it is expressed as early as E10, preceding the expression of PTH. When expressed ectopically, Gcm1 can substitute functionally for Drosophila gcm by transforming presumptive neurons into glia. Thus, certain biochemical properties, although not the specificity of the tissue in which the gene is expressed, have been conserved through the evolution of the Gcm gene family (Kim, 1998).

A novel human homolog (GCMB) of the Drosophila glial cells missing gene (gcm) was isolated using RACE. GCMB contains a gcm motif sequence and a nuclear targeting sequence similar to that of gcm and mouse GCMb. GCMB is located within chromosome 6p24.2. Transcripts of GCMB are detected in fetal brain, normal adult kidney, and several medulloblastomas, gliomas and non-neuroepithelial tumor cell lines. These data suggest that humans have two homologs of gcm, as do mice, and that human gcm genes form a novel family which may function not only during fetal development but also in the postnatal or pathological stage (Kanemura, 1999).

During neural development of Drosophila melanogaster, Glial cells missing (Gcm), functions as a binary switch that promotes glial cell fate while simultaneously inhibiting the neuronal fate. Sequence similarities between Gcm and the recently identified mouse protein mGCMa are strictly limited to the aminoterminal DNA-binding domain. mGCMa efficiently activates transcription in Drosophila cells just as Drosophila Gcm activates transcription in mammalian cells. Transactivation potential is present in two separate regions of mGCMa outside the DNA-binding domain. One of them maps to the carboxyterminal 88 amino acids, a location corresponding exactly to the transactivation domain of Gcm. Similarities between Gcm and mGCMa were also observed in vivo. Overexpression of mGCMa in the developing nervous system of Drosophila embryos leads to an increase in glial-like cells at the expense of neurons. Outside the neurogenic region, mGCMa interfers with epidermal development, as evident from changes in cell morphology and marker expression. Thus, mGCMa function is at least partially independent of a cell's predisposition to a neural fate. The potent activity of mGCMa in Drosophila and its extensive functional similarities to Gcm make mGCMa a candidate for a regulator of mouse glial development (Reifegerste, 1999).

A new cis-element, trophoblast-specific element 2 (TSE2) is located in the placenta-specific enhancer of the human aromatase gene that dictates aromatase tissue-specific expression. In the minimum enhancer region, an element similar to the trophoblast-specific element (TSE) also exists; this was originally described for the human chorionic gonadotropin alpha-subunit gene. The dual presence of TSE and TSE2 is required to direct trophoblast-specific expression driven by a heterologous thymidine kinase promoter. A 2562-base pair cDNA clone encoding a 436-amino acid protein that binds to TSE2 was isolated from a human placental cDNA library using a yeast one-hybrid system with the TSE2 as a reporter sequence. The protein is identical to hGCMa, a mammalian homolog of the Drosophila Gcm (Glial cells missing) protein. Expression of hGCMa is restricted to the placenta. The protein also binds to PLE1 in the leptin promoter among other cis-elements reported to confer placenta-specific expression, suggesting that hGCMa is a placenta-specific transcription regulator, possibly involved in the expression of multiple placenta-specific genes (Yamada, 1999).

The molecular organization of the murine Gcm1 is reported: its spatio-temporal pattern of expression in developing placenta, and its map position at E1-E3 on murine chromosome 9. The murine gene is composed of at least 6 exons. The promoter region contains an 'initiation sequence' and is GC rich, characteristics of the promoters of several transcription factors. The mRNA has a modest 5'UTR (ca. 200 bases) but an extensive 3' UTR (ca. 2 kb). Northern blot and mRNA in situ hybridization studies have shown that Gcm1 expression is readily detectable only in the placenta. It begins at embryonic day 7.5 within trophoblast cells of the chorion and continues to about embryonic day 17.5 within a subset of labyrinthine trophoblast cells. Comparison with other transcription factors reveals that Gcm1 expression defines a unique subset of trophoblast cells (Basyuk, 1999).

The glial cells missing (GCM) family of transcription factors consists of Drosophila GCM and the mammalian proteins GCMa and GCMb. A detailed structure-function analysis of the mouse GCMb (mGCMb) protein has been undertaken. DNA-binding specificity is very similar to that of other GCM proteins. Nevertheless, mGCMb is only a weak transcriptional activator in a number of different tissue culture systems. Interestingly, this is not due to an intrinsic absence of transactivation potential. Two separate transactivation domains have been identified within mGCMb: one is carboxyl-terminally adjacent to the DNA-binding domain and the other is located within the extreme carboxyl terminus. Activity of both transactivation domains is, however, modulated by an inhibitory region unique to mGCMb and located between the two transactivation domains. Furthermore, pulse-chase experiments prove that the mGCMb protein has a half-life approximately four times shorter than mGCMa. Introduction of the inhibitory domain of mGCMb into mGCMa shortens the half-life of mGCMa to a value typical of mGCMb with a concomitant reduction in transactivation potential. Given the strong correlation between protein stability and transactivation potential, functional differences between the two mammalian GCM homologs are likely due to differences in stability with a single inhibitory region in mGCMb being involved in the reduction of both (Tuerk, 2000).

The formation of the labyrinth layer is a critical step of placental development. The transcription factor Gcm1 plays a pivotal role in labyrinth development, but the sequence of events controlling its expression has not been identified yet. Gcm1 expression occurs in three distinct phases during placental development, each specific to a particular stage of chorio-allantois interaction. In the first, the pre-fusion phase, Gcm1 mRNA is expressed in isolated clusters of chorionic cells, but not efficiently translated. Contact with the allantois is required for the translation of Gcm1 mRNA and subsequent development of the initial clusters. Upon allantois-chorion fusion, the second phase (Gcm1 expression) is greatly induced in clusters of chorionic cells separated by non-expressing cells and the Gcm1 mRNA is translated to protein. In the third phase, the labyrinth formation, cells expressing Gcm1 proliferate, involute in the chorionic plate and branched villi formation begins (Stecca, 2002).

Brief ectopic expression of Gcm1 in mouse embryonic tail bud profoundly affects the development of the nervous system. All mice from 5 independently derived transgenic lines exhibit either one or both of two types of congenital spinal cord pathologies: failure of the neural tube to close (spina bifida) and multiple neural tubes (diastematomyelia). Because the transgene is expressed only in a restricted caudal region and only for a brief interval (E8.5 to E13.5), there was no evidence of embryonic lethality. The dysraphisms develop during the period and within the zone of transgene expression. Evidence is presented that these dysraphisms result from an inhibition of neuropore closure and a stimulation of secondary neurulation. After transgene expression ceases, the spinal dysraphisms are progressively resolved and the neonatal animals, while showing signs of scarring and tissue resorption, have a closed vertebral column. The multiple spinal cords remain but are enclosed in a single spinal column as in the human diastematomyelia. The animals live a normal life time, are fertile and do not exhibit any obvious weakness or motor disabilities. This study shows that ectopic expression of Gcm1 in multipotential mesenchymal cells induces the down regulation of two factors normally required for mesodermal differentiation, Notch1 and Tbx6, in cells assuming neurepithelial cell type. In Drosophila, Gcm-has been shown to induce interference with mesodermal differentiation (Nait-Oumesmar, 2002).

The potential to induce glial differentiation is conserved between Drosophila and mammalian glial cells missing genes

Drosophila glial cells missing (gcm) is a key gene that determines the fate of stem cells within the nervous system. Two mouse gcm homologs have been identified, but their function in the nervous system remains to be elucidated. To investigate their function, retroviral vectors harboring Drosophila gcm and two mouse Gcm genes were constructed. Expression of these genes appears to influence fibroblast features. In particular, mouse Gcm1 induces the expression of astrocyte-specific Ca²⁺-binding protein, S100ß, in those cells. Introduction of the mouse Gcm1 gene in cultured cells from embryonic brains resulted in the induction of an astrocyte lineage. This effect was also observed by in utero injection of retrovirus harboring mouse Gcm1 into the embryonic brain. However, cultures from mouse Gcm1-deficient mouse brains did not exhibit significant reductions in the number of astrocytes. Furthermore, in situ hybridization analysis of mouse Gcm1 mRNA revealed distinct patterns of expression in comparison with other well-known glial markers. The mammalian homolog of Drosophila gcm, mouse Gcm1, exhibits the potential to induce gliogenesis, but may function in the generation of a minor subpopulation of glial cells (Iwasaki, 2003).

The present study provides several important insights into the function of mammalian Gcm in the nervous system. First, it has been shown that mouse Gcm1 and mouse Gcm2 are expressed in the embryonic brain throughout development by real time PCR. Next, forced expression studies using a retroviral vector indicated that mouse Gcm1 indeed promotes astrocyte lineage and suppresses neuronal lineage in cultured cells from E12 mouse brains. This induction was so prompt that GFAP⁺ cells appeared only 2 days after infection. The induction of astrocytes by mouse Gcm1 was also detected by in utero injection of the retroviral vector into embryonic brains. Previous reports have demonstrated that ectopic expression of mouse Gcm1 and mouse Gcm2 in the mouse retina failed to cause neuron-to-glia transformation. This discrepancy with the current results may be explained by the differences in Müller cells in the retina and in astrocytes referred to in these studies. Drosophila has two types of glia -- longitudinal and midline glia -- but gcm is involved only in longitudinal glial differentiation. Mammalian astrocytes exhibit a large heterogeneity differing in morphology, distribution, molecule types expressed, function and cell lineage, including gray matter astrocytes, white matter astrocytes, Müller glia in the retina, Bergman glia in the cerebellum and radial glia. Similar to Drosophila gcm, differences in Gcm involvement may occur among these cells (Iwasaki, 2003).

Although glial cells missing (gcm) genes are known as glial determinants in the fly embryo, the role of vertebrate orthologs in the central nervous system is still under debate. The chicken ortholog of fly gcm (herein referred to as c-Gcm1), is expressed in early neuronal lineages of the developing spinal cord and is required for neural progenitors to differentiate as neurons. Moreover, c-Gcm1 overexpression is sufficient to trigger cell cycle exit and neuronal differentiation in neural progenitors. Thus, c-Gcm1 expression constitutes a crucial step in the developmental cascade that prompts progenitors to generate neurons: c-Gcm1 acts downstream of proneural (neurogenin) and progenitor (Sox1-3) factors and upstream of NeuroM neuronal differentiation factor. Strikingly, this neurogenic role is not specific to the vertebrate gene, since fly gcm and gcm2 are also sufficient to induce the expression of neuronal markers. Interestingly, the neurogenic role is restricted to post-embryonic stages and two novel brain neuronal lineages expressing and requiring gcm genes were identified. Finally, fly gcm and the chick and mouse orthologs induce expression of neural markers in HeLa cells. These data, which demonstrate a conserved neurogenic role for Gcm transcription factors, call for a re-evaluation of the mode of action of these genes during evolution (Soustelle, 2007).

date revised: 2 February 2023

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