To determine whether jing might be a downstream target of C/EBP, the expression of jing enhancer trap elements rH623 was examined in slbo mutant egg chambers. Expression of ß-gal in nurse cells was unchanged, however expression of ß-gal in the border cells was dramatically reduced. This was not due to absence of the border cells in the slbo mutant because the cells are still present, as revealed by staining for the slbo enhancer trap line PZ1310, which is also known as slbo1 (Liu, 2001).
To demonstrate that the AEBP2-related protein is indeed responsible for the border cell migration defects that are observed in jing mosaic clones, transgenic flies were generated expressing the putative Jing protein under the control of the heat inducible hsp70 promoter (hs-jing). When Jing was expressed from the transgene, border cell migration was restored in jing mosaic egg chambers. Partial migration was observed even in the absence of heat shock, possibly due to leaky expression from the hsp70 promoter at 25°C. Migration was complete in all stage 10 egg chambers examined, when flies were subjected to a 1-hour heat pulse and then incubated overnight at 18°C to allow migration to occur. However, heat inducible expression of Slbo was not able to rescue the jing migration defect. While hs-jing rescues the migration defect, it does not appear to provide the proper level or timing of expression to restore PZ6356 expression. The reduction in border cell expression of jing-lacZ in slbo mutant egg chambers suggested that jing might be a downstream target of slbo. Therefore whether heat inducible expression of jing could restore migration in slbo1 mutant egg chambers was tested. Although no rescue was observed in the absence of heat shock, border cell migration was complete in all stage 10 egg chambers observed, following a one-hour heat shock and an overnight incubation at 18°C . P[hs-slbo] also rescues the slbo migration defect fully, as expected. The P[hs-jing] transgene rescues border cell migration in two different combinations of slbo alleles, slbo1/slbo1 and slbo1/slbory7. However, expression of jing does not rescue border cell migration in slbo1/slboe7b, the null allele, whereas P[hs-slbo] does rescue border cell migration in slbo1/slboe7b. Therefore over-expression of jing is able to compensate for reduced levels of Slbo protein that are observed in slbo1 and slbory7, but not for the more severe reduction in Slbo protein that is found in slbo1/slboe7b (Liu, 2001).
It is concluded that the jing locus functions in the slbo pathway, based on several lines of evidence. (1) The phenotypes of slbo and jing are similar in that of border cell migration defects and are accompanied by loss of expression of the PZ6356 marker. (2) Expression of jing in the border cells depends upon wild-type slbo function. This regulation appears to be at a transcriptional level, since reduction in lacZ reporter gene expression from the jing enhancer trap line is evident on slbo mutant egg chambers. Further evidence that jing and slbo function in a common pathway is that expression of Jing from a heat-inducible transgene can rescue the border cell migration defects associated with hypomorphic slbo alleles. This result also indicates that Jing is a critical downstream target of slbo. Jing is likely to cooperate with Slbo in activating transcription from downstream target genes. The evidence for this is that, in vivo, both jing and slbo are normally required for PZ6356 expression and over-expression of Jing can compensate for reduced levels of Slbo. Moreover, the mammalian protein most related to Jing, AEBP2, was identified in a screen for proteins that bind to the same enhancer element as C/EBP, in the adipose P2 gene (He, 1999). AEBP2 was originally reported to encode a 300 amino acid protein with transcriptional repressor activity. However, the mRNA for AEBP2 is 4 kb in length whereas the published cDNA was only 2 kb in length. Also, the AEBP2 cDNA sequence does not contain an in-frame stop codon upstream of the reported open reading frame. Therefore it is quite likely that the reported protein sequence is incomplete and represents only the C-terminal DNA binding domain of AEBP2. The protein expressed from such a truncated clone exhibits repressor activity, but the full-length protein may in fact be an activator. The Jing protein is considerably longer than the reported AEBP2, and the loss of PZ6356 expression in the jing mutant background would be consistent with the proposal that Jing functions as an activator in vivo (Liu, 2001).
Analysis of genomic DNA sequence (GenBank accession number, AF285778) surrounding two lethal P-element insertions in jing reveals that there are three putative DNA binding sites for Tgo::Sim and Tgo::Trh (CMEs), and one for the HMG SOX protein called Fish-hook (also known as Dichaete, D) (TACAAT) in the 5' regulatory region of jing. This raises the possibility that jing may be a direct transcriptional target of bHLH-PAS heterodimers and SOX proteins including Tgo:Sim, Tgo:Trh or Fish-hook (Sedaghat, 2002a).
At the time of normal border cell migration, expression of Drosophila E-cadherin (DE-cadherin: Shotgun) increases within the border cells, and Drosophila C/EBP is required for this elevation of DE-cadherin expression. Drosophila ß-catenin, known as Armadillo (Arm), colocalizes with DE-cadherin in both wild-type and mutant egg chambers. To determine whether jing function is also required for proper accumulation of DE-cadherin and Arm, egg chambers containing jing mutant border cells were stained with antibodies against DE-cadherin or Arm, and the staining was compared to wild-type and slbo mutant border cells. In wild-type border cell clusters, staining for DE-cadherin and Arm is strongest in the central cells known as polar cells, which express FASIII and in the junctions between border cells. The staining is somewhat less intense and punctate in appearance at the interfaces between border cells and nurse cells. In slbo mutant clusters, DE-cadherin and Arm staining is only detected in the central polar cells. Border cells mutant for jing exhibit normal expression of both DE-cadherin and Arm. Thus jing function, unlike slbo, is not required for either DE-cadherin or Arm expression. In all cases FASIII staining is normal, indicating normal polar cell fate (Liu, 2001).
DE-cadherin expression is required for border cell migration and is reduced in slbo mutant border cells, but not in jing mutant border cells. Yet expression of Jing is able to rescue the migration defect associated with the slbo hypomorph. DE-cadherin expression in the P[hs-jing];slbo/slbo egg chambers was examined to determine whether the cells were able to migrate despite the absence of DE-cadherin expression or, alternatively, whether high levels of Jing were able to restore DE-cadherin expression. DE-cadherin expression in the border cells is restored in early stage 9 slbo;hs-jing egg chambers, following expression of Jing, but not at later stages (Liu, 2001).
Thus DE-Cadherin expression is not affected in jing mutant clones, though it is reduced in slbo mutants. DE-cadherin expression may require the presence of either jing or slbo. In slbo mutants, expression of a jing-lacZ reporter is also reduced, and DE-cadherin expression is affected. However in jing mutants, slbo expression does not appear to be reduced and DE-cadherin expression is unaffected. However DE-cadherin expression may require that some Slbo protein is present since over-expression of Jing does not rescue the strong female sterile combination of slbo alleles (LY6/e7b) even though it does rescue the weaker allele (slbo1). This selective rescue has also been observed with hs-breathless, which rescues the mild but not the strong female sterile slbo alleles. To date only hs-slbo has been observed to rescue the border cell migration defects associated with the strongest female sterile alleles of the slbo locus. Thus jing cannot completely substitute for slbo, consistent with the observation that there are multiple downstream targets of slbo with essential roles in border cell migration (Liu, 2001).
Neuronal-glial communication is essential for constructing the orthogonal axon scaffold in the developing Drosophila central nervous system (CNS). Longitudinal glia (LG) guide extending commissural and longitudinal axons while pioneer and commissural neurons maintain glial survival and positioning. However, the transcriptional regulatory mechanisms controlling these processes are not known. The midline function of the jing C2H2-type zinc finger transcription factor has been shown to be only partially required for axon scaffold formation in the Drosophila CNS. A screen was performed for gain-of-function enhancers of jing gain-of-function in the eye; the Drosophila homolog DATR-X (also termed XNP) of the disease gene of human alpha-thalassemia/mental retardation X-linked (ATR-X) was identified, as well as other genes with potential roles in gene expression, translation, synaptic transmission and cell cycle. jing and DATR-X reporter genes are expressed in both CNS neurons and glia including the longitudinal glia. Co-expression of jing and DATR-X in embryonic neurons synergistically affects longitudinal connective formation. During embryogenesis, jing and DATR-X have autonomous and non-autonomous roles in the lateral positioning of LG, neurons and longitudinal axons as shown by cell-specific knock-down of gene expression. jing and DATR-X are also required autonomously for glial survival. jing and DATR-X mutations show synergistic effects during longitudinal axon formation, suggesting they are functionally related. These observations support a model in which downstream gene expression, controlled by a potential DATR-X-Jing complex, facilitates cellular positioning and axon guidance, ultimately allowing for proper connectivity in the developing Drosophila CNS (Sun, 2006).
Of the candidates from the screen, DATR-X was chosen for study due to a possible involvement in Jing CNS function and disease relevance. Mutations in the human ATR-X gene are associated with several X-linked mental retardation phenotypes that lead to cognitive delay, facial dysmorphism, microcephaly, skeletal and genital abnormalities and neonatal hypotonia. 87% of mental retardation (MR) genes have a fruit fly homolog and 76% have a candidate functional ortholog revealing a remarkable conservation between humans and Drosophila melanogaster. Some orthologs of human MR genes have cellular phenotypes involving neurons, glia and neural precursor cells and arise from defects in proliferation, migration and process extension or arborization. For example, targeted mutation of ATR-X to the early forebrain in mice leads to cortical progenitor cell death and reduced forebrain size. In addition, mutations in genes controlling the identity of forebrain neuronal precursors can result in holoprosencephaly where the brain hemispheres do not separate. An increased understanding of the molecular and cellular bases for hereditary MR is critical for the generation of drug treatments (Sun, 2006).
ATR-X belongs to the SWI/SNF group of chromatin remodeling proteins that use the energy provided by ATP hydrolysis to disrupt histone-DNA associations and move nucleosomes to different positions. This chromatin modulation allows for the access of activators or repressors to their DNA binding sites in their target genes. The helicase C and SNF2N domains of ATR-X have been shown to have DNA translocase and nucleosome-remodeling activities. Accordingly, mutations in ATR-X have been mapped to the helicase C and SNF2N domains which show approximately 60% homology with those in DATR-X and have been conserved from C. elegans to humans. This conservation supports a conserved role for Drosophila ATR-X in chromatin remodeling (Sun, 2006).
Vertebrate ATR-X has a C2C2 zinc finger motif in the amino terminus that is similar to a plant homeodomain (PHD) finger previously identified in proteins involved in chromatin-mediated transcriptional regulation. Interestingly, D. melanogaster and C. elegans ATR-X proteins do not contain the zinc finger domain, suggesting that these structures may have been acquired through evolution due to a necessity in vertebrate chromatin remodeling mechanisms (Sun, 2006).
Given the absence of the zinc finger domains in DATR-X, it is postulated that invertebrate DATR-X proteins may be complexed with proteins containing a nuclear targeting and DNA-binding motif in order to regulate gene expression at the proper regulatory sites. This may be a role for Jing since it has a very similar embryonic expression pattern as well as mutant and over-expression phenotypes as those of DATRX. Therefore, it seems that the ATPase domain of DATR-X has been conserved through evolution and that the other regions of the protein may have evolved to suit the specific needs of the cell. In summary, different mechanisms of ATR-X function and different binding partners across species may account for the divergence of sequence with respect to the amino terminal and Q-rich repeats while the main chromatin remodeling aspects of ATR-X remain similar (Sun, 2006).
Jing encodes a nuclear protein with putative DNA-binding and transcriptional regulatory domains. The C2H2 zinc fingers of Jing are most similar (50% identical) to those of the mouse adipocyte enhancer binding protein 2 (AEBP2) and also show 25% homology to those of the Kruppel family of transcription factors including those encoding gli and ZIC2. AEBP2 function is implicated in chromatin remodeling events and has strong expression in the brain. Genetic screening identifies a related group of jing-interacting genes. A background sensitive to jing function was used to conduct a genetic screen in the eye. For the GOF screen, it was hypothesized that mis-expression of jing in the eye in combination with other genes involved in jing transcriptional regulation would lead to alterations in gene expression and consequently disrupt ommatidial formation. The genetic relationship between DATR-X and jing in embryonic neurons and glia shows that the screen was successful in identifying genes whose function in adult neuronal cells is relevant to jing function in the embryonic CNS (Sun, 2006).
EP(3)3084 contains a transposon in proximity to a novel gene known by its Flybase transcript identifier as CG15507. Despite strong effects of EP(3)3084 expression in the eye these were specifically strongly enhanced after co-expression with jing, DAtx2 and JIGR1. Furthermore, each gene specifically interacted with the other three, but not with randomly chosen EP lines, suggesting a functional relationship among the four genes. The EP elements in these lines are located in the 5' untranslated region of the downstream genes suggesting these elements may result in over-expression. Given the regulatory role of MADF domains, it is possible that JIGR1 regulates gene expression with Jing and DATR-X. Alternatively, JIGR1 may regulate the expression of a Jing/DATR-X target gene. Likewise, DAtx2 may by involved in regulating the translation of a protein that is an essential component of a Jing/DATR-X/JIGR1 complex or a downstream target of these genes. A role for the orthologs of translational regulators in mental retardation has been shown for the Drosophila ortholog of fragile X mental retardation 1 (Dfmr1). Dfmr1 regulates the MAP1B homolog of Futsch to control synaptic structure and function in the embryonic Drosophila CNS. Therefore, genetic screening and phenotypic analysis in Drosophila has the power to decipher pathways and the cellular bases of MR genes (Sun, 2006).
In wild-type Drosophila embryos, longitudinal glia assume characteristic positions and do not cross the midline or into adjacent VNC segments. This is due to multiple mechanisms at different stages of development including response to repulsive and attractive molecules, cell-cell contact, trophic support and axon contact. A disruption in any of these processes will perturb formation of the glial and axonal scaffolds. The expression of jing and DATR-X reporter genes in longitudinal glia correlates with the longitudinal glial phenotypes associated with mutations in these genes. During stage 12, Robo present on the LG responds to repulsive midline Slit molecules to maintain lateral positioning. The medial misplacement of Robo- and Repo-positive LG during stage 12 after jing and DATR-X glial-specific knockdown suggests that there may have been a breakdown in Robo-dependent repulsive mechanisms. However, the fact that Robo protein was present after jing and DATR-X glial and neuronal knockdown suggests that robo expression may not be regulated by Jing and DATR-X. Alternatively, Jing and DATR-X may regulate the expression of a factor that controls how Robo 'reads' the Slit signal. In support, misrouting of axons across the midline in the presence of Robo occurs in calmodulin and Son of sevenless mutants where these proteins are required to process the Sli signal. It is also possible that jing and DATR-X regulate the expression of factors controlling glial and neuronal positioning in a Robo-independent fashion (Sun, 2006).
jing and DATR-X mutations clearly affect more than Robo-mediated LG positioning. (1) Glial survival is not affected in robo mutant embryos whereas glia die despite continuous axonal contact in jing and DATR-X glial-specific mutants. Therefore, the loss of CNS glia may reflect a breakdown in an intrinsic survival pathway mediated by jing and DATR-X. The expression of jing and DATR-X reporter genes in glia is consistent with such a role. Furthermore, both jing and DATR-X/ATR-X have been implicated in cell survival processes in the CNS midline and tracheal cells and in cortical progenitors, respectively. (2) In robo mutants only the central pCC/MP2 fascicle but not the outer two longitudinal fascicles are affected. However, in jing and DATR-X glial and neuronal mutants the outer fascicles are fused, often broken and can be seen crossing the midline (Sun, 2006).
These defects are similar to those after ablation of neurons or glia and after genetic loss of glia as in gcm mutants. These observations suggest that multiple biological processes require the proper function of these genes and are consistent with an important upstream role for jing and DATR-X in glial and neuronal differentiation. Evidence is accumulating that chromatin accessibility plays a key role in the transcriptional regulation of cell-type-specific gene expression in the CNS. The conservation in ATPase domains along with the similar phenotype of DATR-X and jing mutations and in their expression patterns raises the possibility that Jing is involved in the targeting of a chromatin remodeling complex containing DATR-X to transcriptional target genes whose products are required for the response of longitudinal growth cones and glia to guidance cues. In summary, a group of genes have been identified that pertain to jing function and specifically genetically interact in adult neuronal cells. The results show that specific neural and glial developmental defects underlie the problems in axon guidance associated with mutations in DATR-X and jing. More studies using targeted mutations of MR genes will alleviate the view that brain phenotypes result from generic effects due to a heightened sensitivity of the brain (Sun, 2006).
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