jing: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
Gene name - jing
Cytological map position - 42B2-4
Function - transcription factor
Symbol - jing
FlyBase ID: FBgn0086655
Genetic map position -
Classification - C2H2 zinc finger
Cellular location - nuclear
The jing zinc-finger transcription factor, identified as a downstream target of slbo required for developmental control of border cell migration (Liu, 2001) also plays an essential role in controlling CNS midline and tracheal cell differentiation. The jing locus ('jing' means 'still' in Chinese) was initially identified in a screen for mutations that cause border cell migration defects in mosaic clones (Liu, 2001). Zygotically jing transcripts and protein accumulate from stage 9 in the CNS midline, trachea and in segmental ectodermal stripes. Jing protein localizes to the nuclei of CNS midline and tracheal cells implying a regulatory role during their development. Loss of jing-lacZ expression in homozygous single-minded (sim) mutants and induction of jing-lacZ by ectopic sim expression establish that jing is part of the CNS midline lineage. Embryonic recessive lethal jing mutations display genetic interactions in the embryonic CNS midline and trachea, with mutations in the bHLH-PAS genes sim and trachealess, and their downstream target genes (slit and breathless). Loss- and gain-of-function jing is associated with defects in CNS axon and tracheal tubule patterning. In jing homozygous mutant embryos, reductions in marker gene expression and inappropriate apoptosis in the CNS midline and trachea establish that jing is essential for the proper differentiation and survival of these lineages. These results establish that jing is a key component of CNS midline and tracheal cell development. Given the similarities between Jing and the vertebrate CCAAT-binding protein AEBP2 (He, 1999), it is proposed that jing regulates transcriptional mechanisms in Drosophila embryos and promotes cellular differentiation in ectodermal derivatives (Sedaghat, 2002a).
The jing expression pattern and gene dose effects in the CNS midline and trachea suggest that jing function may be important for the development of both systems. Therefore, CNS axon and tracheal tubule development was assessed in jing homozygous mutant embryos stained with monoclonal antibodies BP102 and 2A12, respectively. In jing3 homozygous mutant embryos, commissural growth cones are often absent in the midline at stage 12 when compared with wild type. By stage 14, homozygous jing3 mutants show losses of longitudinal connections and reduced commissures compared with wild type. Embryos double mutant for jing and sim display phenotypes similar to those of sim homozygotes. Therefore, the sim embryonic CNS axon phenotype is epistatic to that of jing, implying that jing functions downstream of sim (Sedaghat, 2002a).
The GAL4/UAS system was used to determine the effects of overexpressing jing in the CNS midline. Flies containing P[sim-GAL4] were crossed to flies containing P[jing-UAS] and their progeny stained with BP102 to assess CNS axon formation. Expression of one copy of P[jing-UAS] specifically in the CNS midline is sufficient to inhibit commissural and longitudinal axon formation. Therefore, the jing midline overexpression phenotype is similar to that resulting from jing loss of function, and phenotypes of jing and sim double heterozygotes. These results demonstrate that appropriate jing dose is a requirement for proper CNS axon development in the CNS midline. Interestingly, a similar CNS axon phenotype is observed after overexpression of sim in the CNS midline (Sedaghat, 2002a).
The homozygous jing CNS phenotype suggests an alteration in the mechanisms that guide CNS axons. Fasciclin 2 staining using 1D4 mAb, shows that longitudinal fascicles stall within segment boundaries, causing breaks in the longitudinal tracts in 95% of jing3 mutant segments. A subset of normally ipsilateral axons of the most medial fascicle project instead contralaterally in jing3 mutants. Since ipsilateral fascicles are prevented from crossing the midline in wild-type embryos, these results suggest that midline repulsive mechanisms are perturbed in jing mutant embryos (Sedaghat, 2002a).
jing's involvement in tracheal patterning was assessed. Embryos homozygous for a jing deficiency [Df(2R)ST1] and jing3 mutations are associated with losses of the dorsal trunk, severely disrupted transverse connectives and absences of the visceral branch. Embryos doubly mutant for jing and trh lack all tracheal tubules and display phenotypes identical to trh homozygous mutants. Therefore, trh loss-of-function is epistatic to jing loss-of-function, implying that jing functions downstream of trh (Sedaghat, 2002a).
To determine the effects of overexpressing jing in the trachea, flies containing the P[breathless (btl)-GAL4] driver were crossed to those containing P[jing-UAS]. Progeny from this cross were stained with 2A12 antibody and tracheal tubule development was analyzed by light microscopy. Overexpression of jing in the trachea is associated with defects in dorsal trunk fusion, as well as improper formation of the transverse connective, dorsal branch and visceral branch. Therefore, jing overexpression tracheal phenotypes are similar to jing loss-of-function tracheal phenotypes (Sedaghat, 2002a).
Cell type-specific markers were used to follow CNS midline development in homozygous jing mutant embryos. Midline cells were identified using anti-Sim and the glial-specific marker anti-Slit. Expression of sli was assessed in homozygous jing mutant embryos using the lacZ reporter P[1.0 HV, sli-lacZ]. There are reductions in the number of Sim-positive and sli-lacZ expressing midline cells in homozygous jing3 mutants compared with wild-type embryos during stage 9 and 11, respectively. This clearly demonstrates that the early differentiation of midline lineages requires jing function. By later stages of embryogenesis (stage 15), Sim and Sli immunoreactivity is drastically reduced in jing mutant nerve cords. The presence of Sli-positive cellular profiles in macrophages outside the VNC suggests that midline lineages are lost by cell death. Similar results were obtained using anti-Wrapper as a marker of glial identity (Sedaghat, 2002a).
To address whether midline glia enter apoptotic pathways, jing mutant embryos were double-labeled with anti-Sli and TUNEL, and the occurrence of apoptotic glia was monitored from stages 12 to 15. On average, there are one or two apoptotic midline glia within an entire nerve cord of a stage 12 wild-type embryo. By contrast, every nerve cord segment in jing3 mutant embryos contains apoptotic glia in addition to the presence of more TUNEL-positive profiles in the CNS. The increased occurrence of apoptotic glia correlates with reductions in Sli immunoreactivity in the midline of jing3 mutant embryos and establishes that jing function is required for midline glial survival (Sedaghat, 2002a).
Enhancer traps and antibodies were used to follow the development of individual motoneurons (VUMs, 22C10) and interneurons, such as the midline precursors (MP1, dMP2, vMP2; P223, anti-ODD and 22C10) and the median neuroblast (MNB; anti-Engrailed) in wild-type and homozygous jing3 mutant embryos. jing loss-of-function mutations are associated with reductions in the expression of all neuronal markers tested. There are absences of immunoreactivity in the VUMs, MNB and MP1 neuronal lineages in some VNC segments in jing3 mutant embryos. There is a loss of Odd immunoreactivity as early as stage 10 in MP neurons in homozygous jing3 mutants. Similar reductions in the number of immunoreactive vMP2 and dMP2 are observed by 22C10 staining of stage10 homozygous jing3 mutant embryos (Sedaghat, 2002a).
Within a particular VNC segment in jing3 mutants, there is a loss of Engrailed (EN)-positive neurons while the number of EN-expressing neuroectodermal cells remains equal to that in wild-type embryos. In addition, jing mutant embryos displaying reduced 22C10 staining of the VUMs in the CNS midline do not show any visible defects in peripheral nervous system development. These results strongly suggest that the primary site of jing CNS function is at the midline (Sedaghat, 2002a).
In summary, these results demonstrate that midline neuronal and glial populations do not differentiate without proper jing function and suggest a positive role for jing in promoting CNS midline cell development (Sedaghat, 2002a).
To determine the role of jing during tracheal development, a phenotypic analysis of homozygous jing mutant embryos was performed using antibodies to Trh as a marker of cell identity and to En for identifying the anterior border of the trachea. Initial defects in tracheal morphogenesis occur during tracheal placode stages in embryos homozygous mutant for all jing alleles. This correlates with the nuclear localization of Jing in tracheal placode cells. The number of Trh-positive precursors in stage 10 homozygous jing3 mutant embryos is approximately 22% of the expected number of wild-type cells. The relatively normal pattern of ectodermal segmentation in jing3 mutant embryos, as revealed by En staining, suggests that the improper differentiation of tracheal cells in these mutants is not likely to result from indirect effects of ectodermal patterning. These results also reveal that the positioning of tracheal placodes in jing3 mutants is not altered from that of wild-type embryos (Sedaghat, 2002a).
To determine the fate of tracheal lineages the pattern of cell death by double labeling wild-type and jing3 mutant stage 11 embryos with TUNEL and anti-Trh. Cell death is not common in the tracheal pits of wild-type stage 11 embryos. On average, there is a maximum of three TUNEL- and Trh-positive cells within an entire stage 11 wild-type embryo. By contrast, there is an average of 20 TUNEL- and Trh-positive precursors in stage 11 jing3 mutant embryos. There is also an increase in the number of apoptotic profiles surrounding the tracheal pits in jing3 compared with wild-type embryos. Cell death is observed by TUNEL labeling throughout embryogenesis in all tracheal branches in homozygous jing3 mutant embryos, suggesting that the requirement for jing function is not branch specific (Sedaghat, 2002a).
In jing3 homozygous mutant embryos, tracheal cells invaginate but the tracheal branches do not migrate properly to the anterior across En-positive stripes, as they do in wild-type embryos. In addition, fewer Trh-positive cells express En in homozygous jing3 mutant embryos compared with wild-type at stage 12. By stage 15 in jing3 mutant embryos, parts of the dorsal trunk, the dorsal branch and transverse connectives are missing and correlate with a loss of cells by apoptosis. In addition, the visceral branch does not form in jing3 mutant embryos. Therefore, the EGFR-dependent visceral and dorsal trunk branches appear more severely affected than the Dpp-dependent dorsal and ganglionic branches, as well as the transverse connectives in jing3mutant embryos. Despite the death of tracheal cells in jing mutant embryos, the overall embryonic pattern of cell death is not significantly altered by the end of embryogenesis from that of wild-type embryos. Therefore, the tracheal defects in jing mutants are not likely to result from widespread defects in embryonic differentiation (Sedaghat, 2002a).
The results presented here show that CNS midline neurons and glia do not differentiate properly in homozygous jing mutant embryos. Several lines of evidence support this. The expression of cell-type-specific markers of midline neuronal and glial identity is altered in jing mutants compared with that in wild-type embryos. For example, expression of the sli-lacZ 1.0 HV reporter initiates in six midline glia in each wild-type nerve cord segment during stage 11. By contrast, sli-lacZ 1.0 HV reporter expression in jing mutants initiates in only an average of three midline glia per nerve cord segment by stage 11. In addition, there are reductions in the number of Sim-positive midline cells and ODD-positive/22C10-positive MP neurons by stage 9 in jing3 homozygous mutant embryos, respectively. Therefore, early midline glial and neuronal differentiation is aberrant in homozygous jing mutant embryos. By the end of embryogenesis, many neuronal and glial cell type markers are barely detectable in homozygous jing mutant ventral nerve cords (Sedaghat, 2002a).
The loss of sim, sli, odd and 22C10/futsch expression in jing mutants may reflect improper activation/regulation of gene expression or may be secondary to cell loss. To address this issue, the pattern of cell death was analyzed in the CNS midline of jing mutant embryos. Apoptosis occurs in the midline glial lineage in wild-type embryos and begins during stage 12 to refine the number of cells from six to an average of three per nerve cord segment by the end of embryogenesis. In homozygous jing mutants, however, there are more apoptotic glia during stage 12 than in wild-type embryos and this correlates with the loss of Sli-positive glia. It is, therefore, likely that the loss in CNS midline gene expression in jing mutants results from a loss of cells. In summary, the loss in expression of cell identity markers and inappropriate cell death lead to the conclusion that midline neurons and glia do not differentiate properly in jing mutant embryos (Sedaghat, 2002a).
The arthropod ventral nerve cord is characterized by the ladder-like pattern of the major CNS axon tracts. The nerve cord is segmental and each neuromere is connected by longitudinal axons, which are separated by anterior and posterior commissures. Disruption of this pattern by jing gain-of-function specifically in the CNS midline reveals the requirement for proper jing function within these cells for axon patterning. In addition, homozygous mutant jing embryos display reductions in CNS midline cells while neuroectodermal and peripheral nervous system development is unperturbed. Together, these results show that jing mutations have strong effects on the CNS midline and that jing dosage is crucial for their development (Sedaghat, 2002a).
Genetic analysis of axon patterning in the Drosophila CNS has revealed the important role of neuron-glial function in this process. Mutations leading to reductions in midline neuron numbers correlate with a reduction in the number of commissural tracts, whereas mutations leading to reductions in midline glia numbers show fused commissure phenotypes. These observations are consistent with the hypothesis that midline neurons (such as the VUMs) are required to attract commissural growth cones initially to the CNS midline, whereas midline glia are required subsequently for the organization of commissural axons. Based on these observations, it is proposed that defects in the differentiation of midline neuronal precursors, such as the VUMs, in jing loss-of-function mutants, inhibit the attraction of commissural growth cones to the CNS midline during stage 12. As the attraction of commissural axons to the CNS midline precedes the separation of anterior from posterior commissures, the defects in midline neuronal differentiation and the associated lack of growth cones in the midline of jing mutants probably mask subsequent defects in glial-associated functions. During axon patterning, the MP1 interneurons participate in the formation of specific longitudinal pathways. Therefore, the defects in MP1 neuronal differentiation in jing mutants may account for the inhibition in the formation of the longitudinal connectives (Sedaghat, 2002a).
Signals generated by CNS midline cells control the commissural axon pattern by either guiding growth cones toward the midline or preventing them from crossing the midline. Defects in glial-associated functions occur in the CNS of homozygous jing mutant embryos. Reduced glial numbers and Sli production in jing mutants are consistent with the reduction in midline repulsion of longitudinal pathways as visualized by Fasciclin 2 staining. The remaining Sli protein product in stage 12 jing mutant nerve cords, however, is apparently sufficient to prevent a total collapse of the longitudinal connectives, as observed in homozygous sim and sli mutations (Sedaghat, 2002a).
This work has also identified multiple roles for jing in tracheal morphogenesis. The earliest function of jing is to allocate the correct number of cells to the tracheal placodes. Several lines of evidence support this. The number of tracheal placode cells is significantly reduced from wild-type in homozygous jing mutant embryos. In addition, tracheal precursors die in jing mutant embryos, suggesting that jing is essential for their differentiation. Since Jing localizes to the nuclei of tracheal placode cells and contains potential DNA-binding and transactivation domains, it is possible that it regulates genes essential for the differentiation and survival of tracheal precursors (Sedaghat, 2002a).
Although loss of jing function affects cellular differentiation in all tracheal lineages, it appears to have more severe effects on dorsal trunk and visceral branch development. The dorsal trunk and visceral branches derive from the same position in the tracheal placode and are induced by Epidermal growth factor receptior (Egfr). Egfr is activated in the central portion of the tracheal placodes by the restricted expression of rhomboid (rho). The defects in dorsal trunk and visceral branch formation in homozygous jing mutant embryos are similar to those in embryos homozygous mutant for Egfr signaling. Given that mutations in Egfr pathway genes do not affect tracheal placode cell numbers, it is proposed that jing may function prior to Egfr signaling (Sedaghat, 2002a).
Several lines of evidence suggest that jing functions specifically in tracheal cells: (1) Jing protein is detected within nuclei of tracheal precursors and differentiated lineages; (2) defective placodes in jing mutants are observed in hemisegments with normal en expression patterns indicating that defects in the metamerization process do not cause the jing tracheal phenotype. However, the possibility that Hedgehog signaling in segmental ectodermal stripes is affected by jing mutations cannot be ruled out. hh is required in determining proper tracheal placode numbers in some hemisegments. (3) The most severe defects in tracheal patterning in jing mutant embryos occur in the dorsal trunk and visceral branch, suggesting that there is some specificity to jing tracheal function, and (4) overexpression of jing specifically in the trachea results in defects in tracheal patterning that resemble jing loss-of-function phenotypes (Sedaghat, 2002a).
Based on genetic and phenotypic analyses, a role is proposed for jing downstream of sim and trh during CNS midline and tracheal development, respectively. (1) jing expression is not observed prior to that of either sim or trh in the CNS midline and trachea, respectively. jing expression is detected in the CNS midline during stage 9, which comes after the initiation of sim expression and establishment of midline fates. Jing protein is present in tracheal precursor nuclei, coincident with Trh during stage 10. (2) The CNS axon and tracheal phenotypes of homozygous jing mutations are less severe than those of homozygous sim and trh mutations, respectively. However, it cannot be rule out that maternal Jing may rescue the effects of zygotic jing mutations or that jing functions in a combinatorial fashion and therefore may not display severe phenotypes. (3) jing can be activated by ectopic expression of sim, suggesting that sim may regulate jing. The presence of three E-box ACGTG core sites in the 5' regulatory region of jing suggest that this regulation may be direct. (4) The sim and trh embryonic phenotypes are epistatic to that of jing, as shown by double mutant analysis. (5) jing mutations genetically interact with mutations in bHLH-PAS target genes such as sli and btl. The ventral displacement of midline cells in jing and sli double heterozygotes strongly suggests that jing is required for proper sli regulation (Sedaghat, 2002a).
In addition to its functions in CNS midline and tracheal cell development jing function is required for initiation of border cell migration during oogenesis. Epithelial to mesenchymal transitions and cell migration are important features of embryonic development and tumor metastasis. Expression of the basic-region/leucine zipper transcription factor, C/EBP, is required for the border cells to initiate their migration. jing locus was identified in a screen for mutations that cause border cell migration defects in mosaic clones. The jing mutant phenotype resembles that of slbo mutations, which disrupt the Drosophila C/EBP gene, but is distinct from other classes of border cell migration mutants. Expression of a jing-lacZ reporter in border cells requires C/EBP. Moreover, expression of jing from a heat-inducible promoter rescues the border cell migration defects of hypomorphic slbo mutants. The Jing protein is most closely related to a mouse protein, AEBP2 (He, 1999), which was identified on the basis of its ability to bind a small regulatory sequence within the adipocyte AP2 gene to which mammalian C/EBP also binds (Liu, 2001).
It appears that the border cell epithelial-to-mesenchymal transition requires changes in gene expression, some of which are mediated by C/EBP and Jing. DE-Cadherin is clearly one key downstream target of C/EBP, however it is not the only relevant downstream target. A thorough understanding of all of the changes required to convert a stationary epithelial cell to a migratory cell will require identification of more of the genes required for this process. It is striking that the three genes that have been identified to date in forward genetic screens for mutations that inhibit border cell migration, slbo, jing and taiman, have not been identified in any previous genetic screens. This raises the possibility that the genetic control of the epithelial-to-mesenchymal transition is significantly different from genetic control of pattern formation, eye development and other processes that have been subjected to extensive genetic analysis in Drosophila. However more extensive characterization of genes controlling border cell migration will be required before it is clear how different this process is from the others (Liu, 2001).
What are the similarities between control of border cell migration and adipocyte differentiation? It is proposed that the need to coordinate cell differentiation with nutritional status may be the link between mammalian adipocytes and Drosophila border cells that led to the conservation of C/EBP and AEBP2. Undoubtedly many genes are required for an epithelial cell to become motile; therefore it is striking that the jing locus encodes a protein with such a clear functional connection to C/EBP. Evolution, it seems, has conserved functional networks of transcriptional regulators, rather than individual genes. Mammalian proteins related to Jing and Slbo appear to be involved in the regulation of adipocyte differentiation, a process that bears little obvious resemblance to border cell migration. One similarity between adipocytes and border cells is that both cell types appear to coordinate their differentiation with nutritional status of the organism. In the case of adipocytes, at least two independent transcriptional regulatory pathways appear to be required. One pathway is the C/EBP pathway, which may also require AEBP2. A second pathway requires the activity of PPARgamma, a steroid hormone receptor-like molecule that is activated by circulating fatty acids whose levels depend upon diet. Drosophila ovarian development also responds to nutritional signals. Flies emerge from the pupal case with ovaries that contain only immature egg chambers. Further progression of oogenesis requires that the flies consume a rich diet. Application of lipophilic hormones, such as juvenile hormone or ecdysone, can bypass this requirement. The ecdysone receptor is required in the border cells for their migration. Thus border cell migration, like adipocyte differentiation, requires a hormonal signal, which reflects nutritional status, to be integrated with an intrinsic developmental program. It is this latter program that appears to be mediated by the C/EBP, AEBP2 and the Drosophila proteins Slbo and Jing (Liu, 2001 and references therein).
An interesting parallel between the ovarian and embryonic pathways involving jing is the activation of btl. btl is expressed in embryonic tracheal and midline glial cells, as well as border cells of the ovary, and is essential for their migration. btl is a direct target of C/EBP and Trh::Tgo heterodimers in vitro. Therefore, the strong dominant interactions between jing and btl in the embryonic trachea coupled with the role of jing in border cell migration implies an important link between the maternal and embryonic pathways involving jing. C/EBP is expressed in the embryonic trachea after btl expression begins. Therefore, C/EBP probably does not regulate jing or btl in the trachea, as it does in border cells. Furthermore, C/EBP expression is not sufficient to cause ectopic btl expression and therefore, it is proposed that this transcription factor carries out the gene expression program initiated by other factors. In this way, C/EBP can function in very different pathways, including fat metabolism in adipocytes, long-term memory in Aplysia neurons and cell migration in the ovarian border cells. The transcriptional capabilities of jing have not yet been tested and therefore it is not known whether jing co-operates with Tgo::Trh or Tgo::Sim heterodimers in activation of btl or other targets (Sedaghat, 2002a).
It is proposed that bHLH-PAS heterodimers may activate jing transcription by binding any or all of the three CNS midline elements (CMEs) present in the 5' regulatory region of jing. The initiation or maintenance of jing transcription may also require the function of additional transcription factors such as VVL in the CNS midline and trachea or Fish-hook in the CNS midline. The presence of a Fish-hook DNA-binding site (TACAAT) adjacent to the CMEs in the jing 5' regulatory region suggests this possibility (Sedaghat, 2002a).
Both genetic and phenotypic evidence suggest that jing functions in a different manner from either vvl or fish. vvl and fish have combinatorial regulatory roles in CNS midline or tracheal pathways, and therefore vvl and fish-null embryos do not show strong phenotypes. By contrast, the defects in cell numbers and increased cell death during stage 10 and 11 in jing mutants precede and are different from the defects in homozygous vvl and dfr-fish double mutants in the CNS midline or from dfr homozygotes in the trachea. Therefore, activation of jing transcription in the CNS midline or trachea cannot be controlled exclusively by Fish-hook or VVL (Sedaghat, 2002a).
jing encodes a putative DNA-binding protein with putative transcriptional regulatory domains, and its product can be seen in the nuclei of CNS midline and tracheal cells. Based on jing expression patterns and phenotypes, it is proposed that jing participates in the activation of genes downstream of both Sim::Tgo and Trh::Tgo in the CNS midline and trachea, respectively. Whether jing targets are also regulated by bHLH-PAS, POU or SOX combinatorial transcriptional activities remains to be determined. Nevertheless, Jing function is required to promote the differentiation of CNS midline and tracheal lineages, which, in the absence of jing function, do not differentiate and instead undergo apoptosis (Sedaghat, 2002a).
How does jing promote cellular differentiation? It is proposed that jing regulates the transcription of important survival factors including those of the Egfr pathway. For example, the rho gene product regulates the processing of the Egf receptor ligand Spitz and is expressed at stage 9/10 in the CNS midline and in the center region of the tracheal placodes. Proper function of Egfr pathway genes is required for the survival of cells most highly affected by jing mutations, including the CNS midline glia, the tracheal dorsal trunk and visceral branches. Furthermore, the rho regulatory region is controlled by Tgo:Trh:Dfr interactions and also contains multiple CAAT sites similar to those bound by AEBP2, the protein most related to Jing (He, 1999). This raises the possibility that jing may be involved in a combinatorial fashion in the regulation of bHLH-PAS target genes. However, this hypothesis does not account for the role of jing in EGFR-independent process such as survival of CNS midline neurons and dpp-dependent tracheal branches. One can then argue that jing function in the CNS midline and trachea is generic, and that Jing carries out the gene expression programs initiated by bHLH-PAS, POU domain and SOX transcription factors. If the transcriptional programs are not maintained by jing, cells enter default apoptotic pathways. Alternatively, Jing may be responsible for directly activating unknown survival factors in midline neurons and dpp-dependent tracheal branches (Sedaghat, 2002a).
Primary branching in the Drosophila trachea is regulated by the Trachealess (Trh) and Tango (Tgo) basic helix-loop-helix-PAS (bHLH-PAS) heterodimers, the POU protein Drifter (Dfr)/Ventral Veinless (Vvl), and the Pointed (Pnt) ETS transcription factor. The jing gene encodes a zinc finger protein also required for tracheal development. Three Trh/Tgo DNA-binding sites, known as CNS midline elements, in 1.5 kb of jing 5'cis-regulatory sequence (jing1.5) previously suggested a downstream role for jing in the pathway. This study shows that jing is a direct downstream target of Trh/Tgo and that Vvl and Pnt are also involved in jing tracheal activation. In vivo lacZ enhancer detection assays were used to identify cis-regulatory elements mediating embryonic expression patterns of jing. A 2.8-kb jing enhancer (jing2.8) drove lacZ expression in all tracheal cell lineages, the CNS midline and Engrailed-positive segmental stripes, mimicking endogenous jing expression. A 1.3-kb element within jing2.8 drove expression that was restricted to Engrailed-positive CNS midline cells and segmental ectodermal stripes. Surprisingly, jing1.5-lacZ expression was restricted to tracheal fusion cells despite the presence of consensus DNA-binding sites for bHLH-PAS, ETS, and POU domain transcription factors. Given the absence of Trh/Tgo DNA-binding sites in the jing1.3 enhancer, these results are consistent with previous observations suggesting a combinatorial basis to Trh-/Tgo-mediated transcriptional regulation in the trachea (Morozova, 2010).
In the developing Drosophila trachea, transcriptional regulation must be precisely coordinated with growth factor signaling to induce the appropriate cellular response. Studies of downstream transcriptional response elements in the transforming growth factor β (TGF-β) signaling pathway show the importance of discrete sequence changes differentiating an activation versus repressive response. Furthermore, such an activating enhancer element in the knirps gene in this pathway requires a cooperative effect with Trh and Tgo to possibly direct tissue specificity in the trachea. Tracheal gene expression is also controlled combinatorially by Trh/Tgo and Dfr/Vvl or either alone. Similarly, this study shows that Trh/Tgo response elements in the jing gene require additional elements to specify embryonic tracheal expression (Morozova, 2010).
Jing is implicated in transcriptional regulation in numerous biological processes, but its exact role is not known. This study extend previous observations of a role for jing in the trachea by establishing it as a direct downstream target of Trh/Tgo heterodimers. By analyzing jing 5' cis-regulatory regions, this study shows combinatorial basis to Trh/Tgo-mediated jing activation. A 2.8-kb jing enhancer recapitulates endogenous jing expression in the embryonic trachea, ectodermal stripes, and CNS midline. jing2.8 includes a distal 1.5-kb of genomic DNA that has three CMEs which are known for their involvement in combinatorial transcriptional regulation. The best evidence that Trh/Tgo complexes are able to directly activate the jing1.5 enhancer was gathered from Drosophila S2 cells by Luciferase reporter and ChIP assays. The CMEs in jing1.5-luc were required for activation by Trh/Tgo suggesting a protein-DNA interaction. Furthermore, Trh/Tgo heterodimers associated with and activated the jing1.5 enhancer. However, the combination of DNA-binding sites for bHLH-PAS, POU, and ETS transcription factors in jing1.5 is not capable of driving tracheal β-Gal expression in a pattern similar to that of endogenous jing. The jing1.3 enhancer cannot drive tracheal expression. Evidence is shown, in vitro and in vivo, that trh, pnt, and dfr/vvl regulate jing mRNA and even jing1.5-lacZ fusion cell expression. Given these results, along with the absence of additional CMEs and consensus POU domain-binding sites in jing1.3, it is proposed that trh and dfr/vvl regulate jing tracheal expression in combination with additional elements in jing1.3 (Morozova, 2010).
jing1.5 specifies a fusion cell component of jing expression that may instead be regulated by the bHLH-PAS transcription factors, Dys/Tgo. This is consistent with the presence of preferred and less preferred Dys/Tgo DNA-binding sites in the jing 1.5-lacZ enhancer. Prior to embryonic stage 12, trh is required for dys expression and then Dys and Archipelago downregulate trh specifically in fusion cells during stage 12. Therefore, Trh cannot activate jing1.5-lacZ in fusion cells from stage 12 which is consistent with the presence of fusion cell lacZ expression in embryos carrying CME deletions in jing1.5. The reductions in jing1.5-lacZ expression in the fusion cells of trh mutants may therefore result from subsequent reductions in dys expression (Morozova, 2010).
This study also characterized jing cis-regulatory elements controlling different aspects of jing expression in CNS glia and Engrailed-expressing midline neurons and segmental ectodermal cells. The midline expression of jing enhancers provided an opportunity to compare jing transcriptional regulation in two tissues. The data show that jing1.5 is sufficient to drive expression in MG and neurons where Jing is normally expressed. The CNS midline identity of jing1.5-lacZ-expressing cells was shown in several ways. First, jing1.5-lacZ expression was absent in a homozygous sim mutant background. Second, the jing1.5-lacZ expression domain was expanded by activating the Spitz Egfr ligand thereby forcing midline glial survival. Lastly, MG characteristics, such as oblong shape and dorsal positions, are shown by some jing1.5-lacZ-expressing midline cells. Therefore, this enhancer is differentially activated in the CNS midline and trachea suggesting that there may be differences in the mechanism by which Sim/Tgo and Trh/Tgo heterodimers activate transcription. This is consistent with the differential abilities of Sim/Tgo and Trh/Tgo to associate with Dfr/Vvl in vitro and the inability of trh to induce ectopic CNS midline gene expression (Morozova, 2010).
Strong CNS midline expression was also driven by the jing1.3 enhancer despite the absence of Sim/Tgo or Dfr/Vvl consensus DNA-binding sites. However, upon further characterization, the jing1.3-lacZ-expressing midline cells were found to express the segment polarity gene, engrailed (en). En-expressing CNS midline cells take up the posterior-most position within each VNC segment. Another En-positive midline cell lineage includes four to six MGP which are present at stage 13 but not at stage 17. The round shape of En-positive jing1.3-lacZ-expressing midline cells suggests that they belong to the MNB lineage and its progeny and do not belong to the MGP lineage. The mechanism of midline activation of jing1.3 is not known, but the ability of Jing to function as a repressor suggests that it may function combinatorially with En in segmental patterning. Further studies will be aimed at determining whether jing plays a role in segmental ectodermal patterning and its associated gene expression programs (Morozova, 2010).
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).
The expression pattern of jing was studied throughout embryogenesis with a jing-lacZ enhancer trap line (jing01094), digoxigenin-labeled jing DNA probes and a rat JING antibody. jing mRNA and protein product are first detected during precellular blastoderm stages, suggesting that Drosophila embryos contain a maternal supply of jing. A discernable jing expression pattern is apparent from stage 9, as jing transcripts and protein accumulate in the CNS midline, neuroectoderm and trachea (Sedaghat, 2002).
In the wild-type stage 9 CNS, jing mRNA is distributed in a dorsoventral pattern that is not continuous between segments. To determine the identity of the jing-expressing CNS cells, co-localization studies were performed using a jing-lacZ enhancer trap and confocal microscopy. Embryos carrying the jing-lacZ enhancer trap and stained with anti-ß-gal and anti-Sim show co-localization in subsets of CNS midline cells during stage 9. Since Sim localizes only to midline cells in the CNS, this result confirms the midline expression of jing. During stage 9, jing transcription also occurs in the neuroectoderm and in the supraoesophageal ganglion (Sedaghat, 2002).
During stage 10, Jing protein is present in the tracheal placodes. A pair of Jing-positive cells flank the tracheal placodes dorsally. The jing-lacZ enhancer trap is also expressed in Trh-positive tracheal cells in the anterior of each placode. The jing-lacZ enhancer trap is co-expressed with trh and tgo from stage 10 until stage 16 of embryogenesis. Jing protein is detected in all tracheal branches throughout embryonic tracheal development, consistent with a role for jing throughout tracheal tubulogenesis (Sedaghat, 2002).
During stage 12/3, jing transcripts and protein product are present in CNS midline cells and segmental ectodermal stripes. By stage 14, jing is strongly expressed in midline glia that occupy a characteristic dorsal position in the ventral nerve cord. Weaker jing expression is detected in ventrally positioned midline neurons. To determine the subcellular localization of Jing in the CNS, wild-type embryos were stained with anti-Jing and analyzed by confocal microscopy. By this method, Jing protein can be detected within the nuclei of the midline glia and to a lesser degree in midline neurons. Jing protein is not detectable by confocal microscopy in cells of the lateral neuroectoderm, as opposed to jing-lacZ expression (Sedaghat, 2002).
To further analyze jing-lacZ, its expression was assessed in homozygous jing and sim mutants using monoclonal anti-ß-gal. During stage 14, the jing-lacZ enhancer showed strong expression in CNS midline cells and weaker expression in lateral CNS cells. In homozygous jing01094 mutants carrying the jing-lacZ P element insertion, lacZ expression is reduced in the entire CNS suggesting that this insertion affects jing gene expression and that jing expression may be controlled by autoregulation. By contrast, in stage 15 simH9 homozygotes, jing-lacZ enhancer expression is absent in the CNS midline but still present in the lateral CNS and other areas of embryonic jing expression. This result confirms the midline identity of the jing-lacZ-expressing cells (Sedaghat, 2002).
To assess the midline identity of jing-lacZ enhancer expression further, whether sim activates the jing-lacZ enhancer was determined by in vivo ectopic expression experiments. The ability of sim to induce midline gene expression ectopically has been established. sim expression was targeted to the pair-rule ectodermal stripes of the paired (prd) gene using GAL4/UAS and by crossing flies containing the P[prd-GAL4] driver, and heterozygous for the jing-lacZ enhancer, with flies containing P[UAS-sim]. The progeny were stained with anti-Sim to confirm ectopic expression and with anti-ß-gal to identify ectopic jing-lacZ expression. Ectopic expression of sim is sufficient to activate jing-lacZ in ventrally positioned cells in pair-rule ectodermal stripes. The ventral activation of jing-lacZ by sim is consistent with results showing the activation of midline-specific genes by ectopic sim expression. In summary, the results shown here provide strong evidence that jing expression occurs in CNS midline cells (Sedaghat, 2002).
In a screen of 6,000 lines carrying new ethylmethane sulfonate (EMS) induced mutations, 20 mutations were identified, that fell into 16 lethal complementation groups, that caused border cell migration defects in mosaic clones. The phenotypes of the mutants in three complementation groups resembled slbo mutants in that the border cell migration defects were accompanied by reduction in border cell expression of ß-gal from the PZ enhancer trap line known as PZ6356. One of these mutations failed to complement the slbo null. Of the remaining four mutations, three (22F3, 31E6 and 47H6) were allelic and failed to complement the same overlapping deficiencies. Thus these three mutations define a new locus required for border cell migration, and have been named 'jing'. Despite the lack of staining for the PZ6356 marker, the border cells were still present in the mutant egg chambers, as detected by Nomarski optics imaging, rhodamine phalloidin staining and DAPI staining as well as by staining for the slbo-lacZ reporter from enhancer trap line PZ1310 which is also known as slbo1. The similarity between the jing and slbo mutant phenotypes suggested that jing might function in a common pathway with slbo. However, it did not appear that jing was an upstream factor required for slbo expression, since no reduction in ß-gal expression from the slbo enhancer trap was detected in jing mutant border cells (Liu, 2001).
Using GAL4-mediated expression of FLP, mosaic clones were generated exclusively within the follicle cell epithelium, therefore it was clear that jing function is required in follicle cells rather than germline cells. However, in order to rule out the possibility that the migration defects were an indirect consequence of defects in other follicle cells, the cell autonomy of jing within the follicle epithelium was determined. To do this, the wild-type chromosome arm was marked with an enhancer trap line PZ3050, which expressed ß-gal in border cells. In every mosaic egg chamber in which all of the border cells were ß-gal positive, they migrated normally. In every mosaic egg chamber in which the entire border cell cluster failed to stain for ß-gal activity, and therefore was homozygous mutant, migration failed. In these cases the location of the border cell cluster was determined by staining for filamentous actin and Fasciclin III, a cell adhesion molecule that is enriched in two cells within the border cell cluster. Thus, jing function is autonomous to the border cells (Liu, 2001).
Border cell clusters composed of a mixture of heterozygous and homozygous cells, exhibited several different types of behavior. Clusters containing a single ß-gal-positive (and therefore wild-type) cell failed to migrate, though the wild-type cell typically moved to the front of the cluster. Clusters containing a single mutant cell migrated normally. Clusters containing two or more wild-type cells frequently split into two groups, with the heterozygous cells detaching from the mutant cells and migrating partway towards the oocyte. In other instances mixed clusters stayed together as one group and migrated partway. Finally, in two cases the border cell cluster became very extended, with the wild-type cells migrating in a line and the mutant cells trailing behind (Liu, 2001).
This analysis of border cell clusters composed of mixtures of wild-type and homozygous mutant cells indicated that a single mutant cell can passively 'ride' along with wild-type clusters. However, a single wild-type cell does not appear to be capable of pulling multiple mutant cells. When a single wild-type cell occurs in an otherwise mutant cluster, the wild-type cell always appears to move to the front of the cluster. Border cell clusters composed of roughly half mutant and half wild-type cells frequently split into two clusters and the wild-type cells invade the nurse cell cluster. This behavior contrasts with the mixed-clone behavior reported for mutations in the shotgun (shg) locus, which encodes DE-cadherin. In this case the cells never seemed to split apart. It has been concluded that DE-cadherin can not be the only adhesion molecule responsible for holding the border cells together. Thus, there may be one or more additional adhesion molecules, whose expression is regulated by Jing, that are responsible for holding the border cells together as a cluster (Liu, 2001).
Dosage-sensitive genetic interactions between two loci are a good indicator that two gene products are functionally related. The l(2)01094 gene (BDGP) was identified in a search for P-element-induced mutations displaying severe CNS axon phenotypes in double heterozygous combination with null mutations in sim. The l(2)01094 gene has also been isolated in genetic screens and has been characterized for its role in border cell migration in Drosophila ovaries (Liu, 2001). The l(2)01094 P-element-induced allele is referred to as jing01094 (Sedaghat, 2002a).
To address whether jing dosage is important for CNS midline development, jing P-element insertion mutant alleles were placed in heterozygous combination with null mutations in genes whose primary effects arise from the CNS midline, including sim and sli mutations. Also tested were hypomorphic tgo mutations. CNS axon and midline cell development were assessed in double heterozygous embryos by BP102, anti-Sim or anti-Sli staining. jing01094 alleles perturb CNS axon formation and midline cell development in double heterozygous combination with simH9, tgo1, and sli1. For example, 54% of jing and sim double heterozygotes show improper commissural and longitudinal axon formation ('stalled axons'). A smaller percentage of jing01094 and simH9 double heterozygotes (7.7%) show 'collapsed axon' phenotypes similar to those of sim or sli homozygotes. The phenotypes of jing and sim double heterozygotes are insertion dependent since they revert to wild-type after precise excision of the P element in jing01094 flies (Sedaghat, 2002a).
Both CNS axon and midline cell development are perturbed in embryos triple heterozygous for jing, sim and tgo. However, unlike in homozygous sim mutants, the midline cells in jing, sim and tgo triple heterozygotes are specified but then fail to differentiate properly, as determined by their displacement from the ventral nerve cord (a feature characteristic of apoptotic cells) and loss of Sim immunoreactivity by stage 15. The ventral displacement of the CNS midline cells occurs after reduction of one copy of jing and sim, suggesting that these effects are specific for the midline. Triple heterozygotes also show alterations in repulsive signaling mechanisms. Fasciclin 2-positive longitudinal axons collapse into a single tract along the midline in jing01094; tgo1 simH9 triple heterozygotes stained with 1D4 monoclonal antibody. These phenotypes are, therefore, similar to those of homozygous mutations in sli1, which affect midline repulsion mechanisms and cause the ventral displacement of midline cells (Sedaghat, 2002a).
To characterize the relationship between jing and CNS midline further, one copy of both jing and sli were removed and the development of the CNS axons and midline cells were analyzed. Reducing one copy of both jing and sli is associated with collapsed axons (55%), the ventral displacement of Sim+ midline cells (38%) and reductions in Sli immunoreactivity (40%) in stage 14 embryonic nerve cords compared with wild type. By comparison, 57% of simH9 and sli1 double heterozygotes have collapsed axons and ventrally displaced midline cells (45%), which is consistent with the established regulatory role of sim. Comparison of Sim and Sli immunoreactivity in jing and sli double heterozygotes therefore reveals that although midline cells are present in these embryos, they do not adequately express sli. In summary, these results imply that jing dosage may be important for the regulation of sli (Sedaghat, 2002a).
Whether jing dosage is important for tracheal development was assessed by analyzing jing in trans-heterozygous combination with mutations in genes whose function is specific for the embryonic trachea. Tracheal tubule development was analyzed in double heterozygous embryos by staining with mAb 2A12, which in wild-type embryos stains the lumen of all tracheal tubules. Tracheal tubules do not form in homozygous trh mutants. Tracheal tubule formation is defective after both trh and jing are reduced by only one copy each. For example, 51% of embryos double heterozygous for jing01094 and trh1 show a significant loss of most tracheal branches by stage 15. In addition, jing01094 and trh1 double heterozygotes are sensitive to the dose of tgo, since 69% of embryos triple heterozygous for these mutations (jing01094; trh1 tgo1) show tracheal phenotypes. jing mutations also show dominant interactions with a direct target of Tgo and Trh heterodimers, the fibroblast growth factor receptor known as breathless (btl). Ninety eight percent of jing01094and btlH82Delta3 double heterozygotes show tracheal phenotypes that affect the formation of transverse connectives and visceral branches (Sedaghat, 2002a).
In conclusion, genetic analysis provides strong evidence that proper dose of jing in combination with that of trh or btl is important for tracheal tubule patterning. If jing functions in a parallel pathway to that of trh and btl these results would then indicate that the pathways must converge on a common component that is necessary for tracheal tubule formation (Sedaghat, 2002a).
Point mutations in jing were isolated by a chemical mutagenesis. From a screen of 6344 EMS-mutagenized second chromosomes, three novel jing mutations were isolated for failure to complement the embryonic lethality of jingK03404 genetically, therefore defining a single complementation group. jing EMS-induced mutations are homozygous embryonic lethal and are lethal in trans to jing P element-induced mutations and a deficiency Df(2R)ST1 covering the jing locus (Sedaghat, 2002a).
Based on phenotypic analysis of the CNS and trachea, the jing EMS-induced alleles were placed in the following allelic series of phenotypic severity: jing3>jing2>jing1. Molecular analysis of jing3 reveals a single nucleotide change in the coding region of this gene, confirming the identity of this complementation group. The jing3 mutation results in the conversion of tryptophan1200 (w1200) to a premature stop codon located in the middle of the second zinc-finger motif. Given the importance of the zinc-finger motifs and a nuclear localization signal to DNA binding, the molecular nature of the jing3 mutation is consistent with its strong loss-of-function and hemizygous phenotypes. The phenotype of jing3 mutant embryos is therefore shown in phenotypic analyses (Sedaghat, 2002a).
Loss- and gain-of-function studies have demonstrated a crucial role for the jing zinc finger transcription factor in neuronal and glial differentiation and survival in the embryonic central nervous system midline of Drosophila. The role of jing during embryonic brain development has been studied. Proper jing function is required for the formation of the primary brain axon scaffold. In homozygous jing3 mutant embryos the preoral commissure is not pioneered and never forms. Other axon pathways are pioneered but subsequently do not form properly, including the postoral tritocerebral commissure, the circumesophageal connectives and the pathways that connect the brain with the ventral nerve cord. To understand the cellular basis of the axon phenotype the jing expression pattern in the brain was characterized using a jing-lacZ enhancer trap. jing-lacZ enhancer trap expression occurs in glia and neurons in the brain midline and lateral clusters as determined by co-localization of the lacZ gene product with Repo and Castor, respectively. In addition, the jing-lacZ enhancer trap and the basic helix-loop-helix-PAS gene, single-minded (sim), are expressed in the only glial midline cluster present in stage-14 wild-type embryos. jing function is required for the differentiation of Repo-, Castor- and Sim-positive cells in the embryonic brain; each of these populations contain a reduced number of cells in homozygous jing3 mutant embryos. jing is required for neuronal and glial survival; repo- and castor-expressing cells undergo cell death in homozygous jing3 mutant embryos, as revealed by double labeling with Tunel. Expression of jing in sim-expressing cells in the brain disrupts the entire axon scaffold but most significantly results in loss of the preoral and postoral tritocerebral commissures. In addition, circumesophageal connectives are repelled after expression of two copies of UAS- jing in sim-expressing cells, suggesting the activation of axon repellent molecules. Over-expression of sim in the brain is also associated with loss of preoral and postoral tritocerebral commissures. Therefore, the proper dosage of jing and sim in the brain is critical for the formation of the primary axon scaffold. These results show that an important role for jing in the developing brain is the regulation of neuronal and glial differentiation and survival (Sedaghat, 2002b).
The results presented in this study allow for a comparative analysis of the role of the jing zinc finger transcription factor in the embryonic CNS. There are both similarities and differences in the fate of neurons and glia in the VNC and brain of homozygous jing3 mutants. However, in both cases, jing is essential for the terminal differentiation and survival of neurons and glia, suggesting that jing a conserved biological function. In the brain, proper formation of the commissures, longitudinal connectives and the connection between the brain and VNC depends on functional jing gene product. In loss-of-function jing mutants there is an absence of the preoral and postoral commissures and the axon connection between the brain and VNC as well as a thinning of the circumesophageal connectives. Therefore, jing function is required to establish the brain axon scaffold. The preoral brain commissure develops from neural extensions that originate from the medial edges of each hemisphere and project towards the midline. Along with these axonal projections are neuronal cell bodies and glial cells that reach the midline of the brain and establish an interhemispheric cell bridge. A row of glial cells extends across the midline and is associated with the preoral commissure. jing-lacZ is expressed in the row of glia that are associated with the preoral commissure. Loss of the preoral commissure in homozygous jing3 mutants may therefore result from improper differentiation of the glia and/or the neurons that make up the commissure (Sedaghat, 2002b).
The role of longitudinal glial cells in the formation of longitudinal axon tracts has been described in the VNC. In the wildtype brain, a row of longitudinal glial cells prefigures the circumesophageal connectives. In homozygous jing3 mutant brains the circumesophageal longitudinal connectives are pioneered but do not form properly. jing-lacZ is expressed in the longitudinal Repo positive glia that follow the circumesophageal longitudinal connectives and therefore it is possible that improper circumesophageal longitudinal formation in jing mutants results from defective longitudinal glial differentiation. Similarly, the absence of the axonal connection between the brain and VNC correlates with expression of jing-lacZ in the glia in this region. In addition, there is a severe reduction in the number of longitudinal glia at the connection between the brain and VNC in jing mutant embryos and many of the longitudinal glia in homozygous jing3 mutants undergo inappropriate apoptosis. These results support the notion that glia have a conserved role in prefiguring axon pathways in invertebrate and mammalian brains. To determine the role of jing in the brain, glial and neuronal fates in homozygous jing3 mutant embryos were analyzed (Sedaghat, 2002b).
Brain glia do not appear to require jing function for their specification; reductions in glial cell numbers or increased glial apoptosis are not observed in homozygous jing3 mutants until after stage 12. Therefore, terminal glial differentiation requires jing function; loss-of-function jing is associated with increased apoptosis in glial lineages. In contrast, brain neurons require jing function for their survival during early (stage 11) and late stages of differentiation. While apoptosis is barely detectable in Cas-positive neurons in wild-type stage-11 embryos it is significantly more prevalent in similarly staged homozygous jing3 mutant embryos. Many apoptotic Cas-positive neurons are present until the end of embryogenesis in homozygous jing3 mutant embryos compared to that in wild-type embryos. Therefore, jing plays important and different roles in glial and neuronal differentiation in the embryonic brain (Sedaghat, 2002b).
single-minded encodes a basic helix-loop-helix- PAS (bHLH-PAS) transcription factor that functions as a heterodimer with another bHLH-PAS transcription factor known as Tango (Tgo) to control downstream gene transcription in the embryonic VNC midline. Together, this transcriptional heterodimer acts as a lineage control switch. Because jing controls the differentiation and survival of Sim-positive neurons and glia in the VNC it was of interest to determine if it plays a similar role in the brain. First, the pattern of Sim protein localization in the embryonic brain was characterized. During stage 12, Sim is present in only one cluster in the posterior of each brain hemisphere: this is after the specification of neurons and glia. This suggests that sim is not likely involved in cellular specification in the brain as it is in the VNC. This analysis shows that Sim is found in five discrete clusters in each stage-15 brain hemisphere, suggesting that its expression is precisely regulated. Interestingly, Sim is also found in one midline cluster situated in the posterior region of the stage-15 brain in a location where Repo-positive glia are also found (Sedaghat, 2002b).
The function of sim in the embryonic brain appears to be different from its function in the VNC. In the VNC of homozygous sim mutants, commissures do not form and longitudinal axons run as a single tract down the midline. This is due to a loss of the midline cells and their associated repellent molecules such as Slit. In contrast, the preoral commissure of the brain does not require sim function for its formation while the circumesophageal connectives and postoral commissure are dependent on sim function. The axon phenotypes of jing and sim loss-of-function in the brain overlap and jing function is required for proper differentiation of Sim-positive cells. Since jing expression precedes that of sim in both neurons and glia in the brain jing is not likely a downstream component of bHLH-PAS pathways in the brain as it is in the VNC. jing and sim are both expressed in a midline glial cluster whose role in axon formation is not yet known. Targeted gene expression studies were performed to address whether the dosage of jing and sim is important for formation of the brain axon scaffold. The results show that jing and sim are expressed in the only group of Repo-positive glia present in the wild-type brain midline, while their co-localization in each hemisphere remains to be determined. Expression of one copy of UASjing in sim-expressing cells results in loss of the preoral and postoral commissures and aberrant circumesophageal formation. This phenotype resembles the jing loss-of-function phenotype and suggests that jing function is altered in the sim-expressing cells. Furthermore, this functional alteration suggests that jing dosage is critical in the cells that pattern the brain axon scaffold. Expression of two copies of UAS-jing in sim-expressing cells results in the repulsion of circumesophageal connectives from the midline suggesting the activation of axon repellent molecules. Over-expression of sim in the brain results in the same phenotype as targeted jing expression (Sedaghat, 2002b).
It remains to be determined whether jing is expressed in Sim-positive cells in the brain. However, these results show that an alteration of normal jing dosage in Sim-positive cells in the brain interferes with axonogenesis in a manner similar to that of sim over-expression. It is therefore possible that similar molecules are activated and/or repressed by expression of sim and jing in Sim-positive cells (Sedaghat, 2002b).
It is interesting that in the brain midline, the jing enhancer is activated in glia and Cas-expressing neurons as well as additional unidentified neurons. jing therefore has a more extensive midline expression than that of sim, revealing that regulation of jing expression in the midline of the brain and VNC is different. In the VNC, sim is required for jing expression and can ectopically activate the jing-lacZ enhancer in Drosophila embryos. Further differences exist in gene regulation between the brain and VNC midline. For example, Repo and Cas are present in the brain midline but are excluded from the VNC midline, while both proteins are found in the VNC neuroectoderm. Given the importance of jing function in cellular differentiation it will be important to determine its mechanism(s) of action (Sedaghat, 2002b).
The Drosophila jing gene encodes a zinc finger protein required for the differentiation and survival of embryonic CNS midline and tracheal cells. There is a functional relationship between jing and the Egfr pathway in the developing CNS midline and trachea. jing function is required for Egfr pathway gene expression and MAPK activity in both the CNS midline and trachea. jing over-expression effects phenocopy those of the Egfr pathway and require Egfr pathway function. Activation of the Egfr pathway in loss-of-function jing mutants partially rescues midline cell loss. Egfr pathway genes and jing show dominant genetic interactions in the trachea and CNS midline. Together, these results show that jing regulates signal transduction in developing midline and tracheal cells (Sonnenfeld, 2004).
The effect of a reduction in EGFR signaling on the jing gain-of-function phenotype was examined in the midline glia. sim-Gal4 and sli-Gal4 drivers were used to over-express jing specifically in the CNS midline in heterozygous and homozygous spi and S mutant backgrounds. The number of sli-lacZ-expressing midline glia in each nerve cord segment was quantified during stage 13 and compared to that in wild-type embryos over-expressing jing. Expression of two copies of the UAS-jing transgene in the midline glia of wild-type or heterozygous spi and S embryos resulted in an average of 12 midline glia instead of the normal 8 during stage 13. In contrast, UAS-jing transgene expression was unable to induce 12 midline glia in homozygous spi and S mutant backgrounds. In these embryos, there was an average of 1.5 midline glia in each nerve cord segment after jing over-expression; this is similar to the number of midline glia present in homozygous spi and S mutant embryos during stage 13 (Sonnenfeld, 2004).
To test the independent activity of jing, the effects of ectopic jing expression were examined in the Drosophila eye, which is a system that is functional for the Egfr pathway but not for jing or upstream regulators including single-minded (sim) or trachealess (trh). Analysis of jing01094 enhancer trap lacZ expression and of endogenous mRNA expression by in situ hybridization shows that jing is not expressed in third instar larval eye imaginal discs. Expression of wild-type jing in the eye, under regulation of the glass promoter (P[GMR-Gal4]), was associated with a rough appearance compared to P[GMR-Gal4] heterozygotes or wild-type. The rough eye consisted of highly disorganized ommatidia and mechanosensory bristles in 45% of flies and the number of ommatidia was reduced by 50% from that in wild-type and P[GMR-Gal4] heterozygous eyes. Therefore, the gain-of-function phenotypes of jing and Egfr both result in a significant reduction in ommatidia. Consistent with similar pathways, the rough eye phenotype of Egfr gain-of-function was not enhanced by that of jing. Out of 1000 flies scored, carrying P[GMR-Gal4] and both UAS-jing and UAS-ellipse, 100% showed the same eye phenotype as flies carrying only P[GMR-Gal4] and UAS-ellipse (Sonnenfeld, 2004).
The jing ectopic expression phenotype was dominantly suppressed by a 50% reduction in the levels of spi(spi1) and Df(2L)TW50 or Egfr deficiency [Df(2R)Egfr5]. After spi reduction, ommatidia were more organized and more abundant, although the position of the photoreceptors was not like that in controls. This interaction was not influenced by activation of the glass promoter in the heterozygous spi background (P[GMR-Gal4]/spi1). These results suggest that there is a dosage-sensitive interaction between the Egfr pathway and jing function in the eye, where increased jing activity can be suppressed by a reduction in downstream components such as spi and Egfr. Given that sim and trh are not expressed in third instar larval eye discs, these experiments suggest that jing can have an effect on the Egfr pathway in the absence of sim or trh and support the model that jing works as an independent regulator in bHLH-PAS pathways (Sonnenfeld, 2004).
Gene dosage experiments were used to determine the effects of simultaneously altering the levels of jing and genes of the Egfr pathway. Mutations in spi and its regulator Star, have been characterized for their midline and tracheal phenotypes. To determine whether jing and Egfr function is inter-dependent, the development of the CNS midline and trachea was analyzed in double heterozygotes of jing and S or spi. The basis for this experiment is that if the Egfr and jing pathways are inter-dependent then simultaneous reduction of only one copy of each gene should alter CNS midline and tracheal function. Multiple jing alleles balanced with wg-lacZ Cyo were crossed to SIIN23/wg-lacZ Cyo or spi1/wg-lacZ Cyo flies and their progeny were double stained with anti-Sim or anti-Trh and anti-β-Gal (Sonnenfeld, 2004).
The number of CNS midline cells was reduced from wild-type in embryos homozygous and double heterozygous for jing, spi or S and stained with anti-Sim. Since some of the Sim-positive nuclei appeared to be fragmenting, their fate was determined by TUNEL labeling to identify apoptotic cells. In wild-type embryos, cell death is uncommon in the CNS midline during stage 12 with an average of 6(±2) Sim-positive apoptotic nuclei per embryo. In contrast, in homozygous jing stage 12 mutant embryos, there was an average of 35(±3) Sim-positive apoptotic nuclei per embryo, therefore, displaying a significant increase over that in wild-type embryos. In embryos double heterozygous for mutations in jing and S or spi there was an average of 25(±2) and 30(±3) SIM-positive apoptotic nuclei per embryo during stage 12, respectively. This is consistent with the time period for the requirement of Egfr function in CNS midline glia. Embryos heterozygous for either jing, spi or S mutations did not alter the normal events of midline cell apoptosis. In summary, these results suggest that proper dosage of both jing and spi group gene function is required for midline cell survival (Sonnenfeld, 2004).
The jing gene was identified in two independent genetic screens for regulators of CNS midline development and border cell migration, two processes which are regulated by the EGFR. RTK signaling pathways have been implicated in multiple cell biological processes including proliferation, migration, differentiation and survival. How one MAPK pathway controls such different outcomes is a major area of research. Studies of Egfr function in the Drosophila adult eye suggest that signaling levels dictate the multiple cellular responses to the EGFR, such that differentiation requires the highest levels of signaling while mitosis and cell survival require less. Therefore, it is important to understand the mechanisms that control the expression of positive and negative regulators of this family of signaling molecules (Sonnenfeld, 2004).
Prior work has established the important role that the Egfr plays during the differentiation of midline glia (MG) and tracheal cells. Several lines of supportive evidence show that jing regulates Egfr signaling in the MG and trachea. (1) jing mutant embryos fail to maintain MAPK activity and Egfr expression in cells that clearly have midline and tracheal identities. (2) jing is required for and can induce Egfr pathway transcription in the CNS midline and trachea. (3) jing over-expression promotes midline glial survival in a similar fashion as over-expression of Egfr pathway genes. (4) jing-mediated over-expression phenotypes require Egfr pathway function in CNS midline glia and the adult eye. (5) A transgenic copy of either activated ras1, secreted spi or gain-of-function Egfr can partially rescue midline cell death in homozygous jing mutants. (6) Proper dosage of both pathways is essential for survival of midline glia and for proper tracheal morphogenesis. Together, these findings suggest that jing functions upstream in the Egfr/ras1 pathway. Future studies will be aimed at elucidating the nature of the relationship between jing and Egfr pathway genes and may help in the design of therapeutics to regulate over-active RTK pathways in oncogenic cells (Sonnenfeld, 2004).
jing is the only gene, other than those already characterized in the Egfr pathway, that can promote midline glial survival. jing over-expression, as driven by the sim and sli promoters, induces extra midline glia that express Egfr, slit and sim, and these glia appear to be rescued from apoptotic fates. The extra glia are observed during stage 13 which is consistent with the timing of Egfr pathway-induced extra glia. The absence of apoptotic glia and the wild-type midline neuronal numbers after jing over-expression suggest that the supernumerary glia are not likely recruited from neuronal populations and may represent glia rescued from death due to inappropriate Egfr expression. This effect phenocopies gain-of-function phenotypes in EGFR signaling in the CNS midline and suggests that jing-mediated cell survival may be carried out by the EGFR/RAS1 signaling pathway. In support, jing over-expression phenotypes in the CNS midline and eye are suppressed by reductions in Egfr function (Sonnenfeld, 2004).
The results suggest that jing is involved in both the differentiation and survival of cells in the embryonic CNS midline and trachea. In wild-type embryos, early MAPK activity controls midline glial (MG) differentiation through activation of the downstream Ets-type transcription factor pointed (pnt). The reductions in early MAPK activity and Egfr expression in the midline of jing mutants, therefore, reveals the requirement for jing function in MG differentiation. The similarities in gain- and loss-of-function midline glial phenotypes between pnt and jing are consistent with this model. In jing mutants, reduced MAPK activity occurs in midline and tracheal cells that express the sim and trh genes, respectively, indicating that the reductions in MAPK activity are not due to a general failure in cellular differentiation (Sonnenfeld, 2004).
It is possible that improper MG differentiation in jing mutants could be due to cells being committed to death. However, a loss of MAPK activity is detected prior to apoptosis in the CNS midline of homozygous jing mutants, suggesting that early MAPK inactivity in the CNS midline is independent of the apoptotic machinery. In support, the MG initially form in MAPK mutants and it is not until later stages, which are dependent on repression of hid, that the MG die. MG death in jing mutants may be due to a combined lack of the axon-glial contacts that are necessary for MAPK-mediated inactivation of hid as well as from reduced MAPK activity within the MG (Sonnenfeld, 2004).
During stage 10, EGFR signaling is activated in the central region of the tracheal placode by transcription of rhomboid resulting in the formation of anteroposterior branches including the dorsal trunk and visceral branch. Wingless (Wg) signaling originates in ectodermal cells adjacent to the tracheal placodes and causes Egfr-induced cells to form the dorsal trunk. jing is expressed in most tracheal cells and its protein product is localized to their nuclei suggesting that this C2H2-type zinc finger may have a regulatory role directly within these cells. Additional evidence that jing may have a role directly in tracheal cells comes from its perturbation of tracheal morphogenesis and alteration of Egfr/ras pathway gene expression profiles when over-expressed specifically in the trachea (Sonnenfeld, 2004).
jing affects branching morphogenesis and cellular survival in the tracheal system and its expression in the tracheal placodes coincides with that of Egfr pathway genes. jing and Egfr pathway mutants have similar tracheal phenotypes which include breaks in the dorsal trunk and reduced visceral branch formation. The reductions in Egfr-induced cells may explain the defects in dorsal trunk formation in jing homozygous mutant embryos and possibly in jing and Egfr pathway double heterozygotes. Alternatively, the dorsal trunk defects may arise from perturbations in Wg signaling in the ectoderm of jing mutants. spitz group tracheal mutant phenotypes do not reflect ectodermal patterning defects but this remains to be analyzed in more detail in jing mutants (Sonnenfeld, 2004).
The results indicate that proper Egfr pathway and jing function is required for midline and tracheal cell survival. This is the first evidence of such a survival role in the trachea and requires further investigation. However, this does not rule out the possibility that other processes involved in tracheal morphogenesis are not affected in double heterozygotes and jing homozygotes. Furthermore, in jing homozygotes and hemizygotes, truncated tubules are present in the transverse connectives, suggesting that the requirement for jing function is more global than that of Egfr/ras1. In support, jing is expressed in embryonic tissues that are not active in MAPK, suggesting that jing has additional functions (Sonnenfeld, 2004).
Compared to other midline and tracheal-expressed genes, those of the Egfr pathway are more highly expressed after jing over-expression (but not more than three-fold). Nevertheless, the effects of jing over-expression in the CNS midline can be seen by extra glia and Egfr expression establishing the importance of regulating jing expression during embryogenesis. Ectopic expression analyses suggest that jing is not sufficient to activate Egfr pathway gene expression. Therefore, these results suggest that in order to induce gene expression jing may require another protein, such as a cell-specific chromatin remodeling protein, that is not present in prd stripes but is present in the CNS midline, trachea and eye. The exact relationship between jing and Egfr pathway genes requires further analysis (Sonnenfeld, 2004).
The establishment of the proximo-distal (PD) axis in the legs of Drosophila melanogaster requires the expression of a nested set of transcription factors that are activated in discreet domains by secreted signaling molecules. The precise regulation of these transcription factor domains is critical for generating the stereotyped morphological characteristics that exist along the PD axis, such as the positioning of specific bristle types and leg joints. Evidence is provided that the Zn-finger protein encoded by the gene jing is critical for PD axis formation in the Drosophila leg. The data suggest that jing represses transcription and that it is necessary to keep the proximal gene homothorax (hth) repressed in the medial domain of the PD axis. jing is also required for alula and vein development in the adult wing. In the wing, Jing is required to repress another proximal gene, teashirt (tsh), in a small domain that will give rise to the alula. Interestingly, two other genes affecting alula development, Alula and elbow, also exhibit tsh derepression in the same region of the wing disc as jing- clones. Finally, jing is shown to genetically interact with several members of the Polycomb (Pc) group of genes during development. Together, these data suggest that jing encodes a transcriptional repressor that may participate in a subset of Pc-dependent activities during Drosophila appendage development (Culi, 2006).
A modifier screen was used to identify jing as a gene that genetically interacts with hth. Subsequent analysis of jing demonstrates that it plays a key role in forming the PD axis of the leg, a process that also depends on hth function. Moreover, jing behaves as a repressor of hth expression during leg development. Thus, although the genetic modifier assay (pigmentation of the adult male A4 tergite) is distinct from PD axis specification, the screen nevertheless successfully identified a new player in leg development. In addition to PD axis specification, Jing plays several other roles during adult development. It is required for the formation of the alula and for the correct specification of the tergites and for the differentiation of wing veins. This analysis strongly suggests that jing is used, in at least some contexts, as a transcriptional repressor. In the leg, the major target of repression is the gene hth, which is normally restricted to the proximal-most domain of the leg disc. tsh is also derepressed in jing minus clones in the leg, but only within a small region of the disc, just distal to the endogenous tsh domain. In the wing, the derepression of tsh was observed in jing- clones and more weakly, derepression of hth and wg. That Jing behaves as a transcriptional repressor is also supported by the finding that Jing-ZnfEnR (Jing's Zn finger domain fused to the repressor domain of Engrailed), but not Jing-ZnfE1A (the Jing Zn finger domain fused to the activation domain E1A) can rescue the tsh derepression observed in jing- clones in the wing (Culi, 2006).
The data also indicates that Jing requires the contribution of other factors to repress transcription, since jing is ubiquitously expressed in imaginal discs and its overexpression does not repress hth transcription in the proximal domain. One such factor could be Dac, a transcription factor expressed in an intermediate domain along the leg PD axis and that is also required to repress hth. The findings are also consistent with the characterization of a mammalian homolog of Jing, called AEBP2. The initial characterization of this gene demonstrated its potential to act as a transcriptional repressor and mapped the repression domain to one of the Zn fingers. More recently, AEBP2 has been found to be part of a Pc complex that contains a histone methyltransferase activity (Cao, 2002; Cao, 2004). These findings suggested that Jing may also be part of a Pc complex in Drosophila, a possibility that is supported by the current results. In particular, it was shown that jing genetically interacts with several members of the PcG. Accordingly, it is speculated that the derepression of hth and tsh observed in the absence of jing function may in part be due to the requirement of jing to maintain the repression of these genes in a Pc-dependent manner. Consistent with this view are findings demonstrating that, in the wing pouch, tsh repression is maintained by Pc-mediated silencing. In contrast to the situation in the wing, it appears that in the leg, hth (but not tsh) is repressed by Pc-mediated silencing, a finding that is also consistent with the data presented in this study. If Jing is a component of a Pc complex, one of the surprising findings described in this study is the degree of spatial specificity for the requirement for jing function. In the leg, phenotypes were observed only in jing- clones located in the medial domain along the PD axis. In these clones, hth was derepressed. However, in more distal clones, no affect on hth expression was observed. Similarly, in the wing, the predominant affect of jing- clones is on the development of the alula and the corresponding derepression of tsh in the presumptive alula region of the wing imaginal disc. No tsh derepression was observed in the remainder of the wing pouch. This observation is in contrast to the affect of Pc minus clones, which show derepression of tsh throughout the wing pouch. The underlying reason for the localized requirement is not clear, especially if jing is broadly expressed throughout leg and wing discs. One intriguing possibility is that distinct Pc complexes may be required to maintain gene silencing in different cell types (Culi, 2006).
Accordingly, jing may encode a more specialized component of some Pc complexes that help them achieve these cell-type specific repressor functions. The fact that Jing has a putative DNA binding domain, and thus could help target a subset of Pc-containing complexes, is also consistent with this proposal. A possible molecular mechanism is envisioned in which Jing binds the regulatory region of its target genes and, together with other transcription factors that provide regional specificity (e.g., Dac), represses their transcription. Subsequently, Jing may help recruit members of the Pc group complex and thus stabilize a repressed chromatin state. Ii is intriguing that jing has been found in several different modifier screens. Based on these observations, it appears as though jing-mediated repression may play a role in many cellular processes during development. In the future, it will be most interesting to biochemically confirm the interaction between Jing and Pc complexes in Drosophila and to better understand the basis of the functional specificity described in this study (Culi, 2006).
Many diverse animal species regenerate parts of an organ or tissue after injury. However, the molecules responsible for the regenerative growth remain largely unknown. The screen reported in this study aimed to identify genes that function in regeneration and the transdetermination events closely associated with imaginal disc regeneration using Drosophila melanogaster. A collection of 97 recessive lethal P-lacZ enhancer trap lines were screened for two primary criteria: first, the ability to dominantly modify wg-induced leg-to-wing transdetermination and second, for the activation or repression of the lacZ reporter gene in the blastema during disc regeneration. Of the 97 P-lacZ lines, six genes (Krüppelhomolog- 1, rpd3, jing, combgap, Aly and S6 kinase) were identified that met both criteria. Five of these genes suppress, while one enhances, leg-to-wing transdetermination and therefore affects disc regeneration. Two of the genes, jing and rpd3, function in concert with chromatin remodeling proteins of the Polycomb Group (PcG) and trithorax Group (trxG) genes during Drosophila development, thus linking chromatin remodeling with the process of regeneration (McClure, 2008).
There are three different mechanisms that organisms use to re-grow and replace lost or damaged body parts, and often, more than one mechanism can function within different tissues of the same organism. Muscle and bone, for example, repair themselves by activating a resident stem cell population, while the liver regenerates by compensatory proliferation of normally quiescent differentiated cells. Appendage/fin regeneration in lower vertebrates occurs by a process termed epimorphic regeneration, which proceeds in three distinct stages: (1) wound healing and migration of the surrounding epithelial cells to form the wound epidermis, (2) formation of the regeneration blastema -- a mass of undifferentiated and proliferating cells of mesenchymal origin and (3) regenerative outgrowth and pattern re-formation. Whether these diverse modes of regeneration share a common molecular and genetic basis is not known (McClure, 2008).
Regeneration in the Drosophila imaginal discs, the primordia of the adult fly appendages, closely parallels epimorphic limb/fin regeneration in lower vertebrates. Cells in the imaginal discs are rigidly determined to form specific adult structures (e.g., legs and wings) by the third larval instar. If the discs are fragmented at this time and cultured in vivo, they will regenerate. Disc regeneration begins 12 h after wounding, when transient heterotypic contacts are made between peripodial (squamous epithelium) and columnar cells (disc proper) near the cut edges of the wound. These initial contacts involve microvilli-like extensions and provide temporary wound closure. Then, approximately 24 h after wounding, homotypic cell contacts (between columnar or between squamous cells) are made involving the close apposition of cell membranes and cellular bridges, which eventually (48 h after wounding) restore the physical continuity of the disc. Before and during wound healing, cell division is randomly distributed throughout the disc. However, once completed (36-48 h after wounding), division is observed only in cells near the wound site. These cells are known as the regeneration blastema. Thus, like appendage regeneration in lower vertebrates, disc regeneration involves wound healing followed by blastema formation (McClure, 2008).
Blastema cells are responsible for the regeneration and repatterning of the entire missing disc fragment. Thus, these cells exhibit remarkable developmental plasticity. For example, in anterior- only leg disc fragments, some blastema cells will switch to posterior identity and establish a novel posterior compartment in the regenerate. This anterior/posterior conversion occurs during heterotypic wound healing, when hedgehog (hh)- expressing peripodial cells induce ectopic engrailed (en) expression in the apposing anterior columnar cells. In addition, the disc blastema, like its vertebrate counterpart, is able to form a normal regenerate (complete leg disc and adult leg) when isolated from the remaining disc fragment. Regenerative plasticity is also observed when a few blastema cells switch fate to that of another disc type (e.g., leg-to-wing), in a phenomenon known as transdetermination. Transdetermination events are closely associated with regenerative disc growth. Clonal analysis, for example, has shown that blastema cells first regenerate the missing disc structures, and only then, are they competent to transdetermine (McClure, 2008).
Little is known about how the regeneration blastema forms in the fragmented leg disc, although ectopic Wingless (Wg/Wnt1) expression is detected along the cut site, both prior to and during blastema formation. Wg is a developmental signal in many different tissues and animals; in flies Wg patterns all of the imaginal discs, functioning as both a morphogen and mitogen to regulate disc cell fate and growth. In lower vertebrates, Wnt ligands are key regulators of blastema formation during epimorphic regeneration. Thus, activation of Wg within the disc blastema is potentially important for regeneration. This idea is consistent with the observation that ubiquitous expression of wg during the second or third larval instars, in unfragmented leg discs, is sufficient to induce a regeneration blastema in the proximodorsal region of the disc, known as the weak point. Moreover, ubiquitous expression of wg mimics the pattern deviations associated with leg disc fragmentation and subsequent regeneration, including the duplication of ventral with concomitant loss of dorsal pattern elements and leg-to-wing transdetermination events. Thus, leg disc regeneration can be examined using two experimental protocols: fragmentation or ubiquitous wg expression. However, it is important to point out that only fragmentation-induced regeneration involves wound healing (McClure, 2008).
Precisely which molecules and signaling pathways are required for the process of regeneration remain poorly understood, partly because the organisms historically used to study regeneration (e.g., newts and salamanders) have been refractory to genetics and molecular manipulations. Recently, however, the use of new genetic techniques together with 'regeneration' model systems -- such as planarians, hydra and zebrafish have given researchers the opportunity to examine the mechanisms of regeneration and to identify the genes, proteins and signaling pathways that regulate different regenerative processes. For example, a large scale RNAi-based screen was performed to survey gene function in planarian tissue homeostasis and regeneration. Out of ~1000 genes examined, RNAi knock-down of 240 displayed regeneration-related phenotypes, including defects in wound healing, blastema formation and blastema cell differentiation. Despite these studies, however, it remains unclear whether regeneration requires only the modulation of genes expressed at the time of injury, the reactivation of earlier developmental genes and/or signaling pathways, or the activation of novel genes specific to the process of regeneration. Thus, a major interest in the field of regenerative biology is the identification of gene products that regulate blastema formation, blastema growth and regenerative cellular plasticity. A genetic screen, using wg-induced leg disc regeneration, aimed at identifying genes that regulate cellular plasticity and regeneration using Drosophila was carried out prothoracic leg discs. A collection of 97 recessive lethal P-element lacZ (PZ) insertion lines were screened for ectopic lacZ expression during wg-induced leg disc regeneration, and six genes were identified that function in wg-induced leg disc regeneration, including genes with functional ties to Wg signaling as well as chromatin remodeling proteins (McClure, 2008).
This study consisted of an enhancer trap screen designed to identify genes with changed gene expression during leg disc regeneration as well as required for regenerative proliferation and growth. The screen identified 19 genes that when heterozygous mutant (PZ/+), dominantly modify wg-induced leg-to-wing transdetermination, which serves as a functional assay for disc regeneration. Of the 19 genes, 37% are transcription factors or involved in transcriptional regulation (tai, Krh1, ken, jing, combgap (cg), rpd3 and Aly), 21% function in cell cycle regulation and growth (oho23B, S6k, polo and cycA), 10.5% play a role in protein secretion (Secβ61 and Syx13), and 31% are of other or unknown function [l(3)01629, CG30947, l(2)00248, l(3)05203, l(3)01344, Nup154]. The identification of transcription factors as the most frequent class of genes that modify wg-induced leg disc regeneration was similarly observed in a DNA microarray screen designed to identify genes enriched in leg disc cells that transdetermine to wing (Klebes, 2005). Together, these findings strongly suggest that transcription factors and their downstream targets play a prominent role in disc cell plasticity (McClure, 2008).
Using lacZ expression analyses, together with whole mount in situ hybridization experiments, the expression patterns of the 19 genes that modified wg-induced leg-to-wing transdetermination were verified. This analysis identified several different expression patterns upon wg-induced regeneration, including a loss of gene expression, ubiquitous expression and genes with expression limited to the regeneration blastema. Such observations indicate that a complex change of gene expression, both negative and positive, mediates the process of epimorphic regeneration. Six (jing, Alyi cg, rpd3, Kr-h1 and S6k) of the 19 modifiers displayed expression limited to the regeneration blastema, indicating that novel markers of regeneration and transdetermination have been identified. The blastema-specific expression patterns of jing, Aly, cg, Kr-h1, rpd3 and S6k raised the intriguing possibility that these genes may be functionally involved in the formation, cell proliferation or maintenance of the blastema during disc regeneration. Indeed, upon ubiquitous wg expression jing/+ animals rarely formed a regeneration blastema, indicating that two wild-type copies of jing are required for the initiation of the regenerative process. In contrast, Aly/+ and cg/+ animals formed a normal blastema, but only after a one-day delay. Therefore, two wild-type copies of the Aly and cg genes are required for the proper timing of regeneration. In addition, it was found that the frequency of blastema formation was reduced in rpd3/+ animals, implicating this gene in the process of regeneration. Interestingly, heterozygous mutations in all four of these genes (jing, Aly, cg and rpd3) strongly suppress wg-induced leg-to-wing transdetermination. It is speculated that the transdetermination frequency declines in these mutant animals because the initiation and/or timing of blastema formation is delayed. This idea is consistent with all previous work which has shown that blastema cells are only competent to transdetermine after they have regenerated the missing disc structures. Heterozygous mutations in Kr-h1 and S6k did not significantly alter the formation of the wg-induced regeneration blastema, however, these genes did affect regeneration-induced transdetermination. Such results suggest that Kr-h1 and S6k specifically function to modulate the cell fate changes that occur as a consequence of regeneration (McClure, 2008).
Investigations into the molecular basis of transdetermination have shown that inputs from the Wg, Decapentapelagic (Dpp) and Hedgehog (Hh) signaling pathways activate key selector genes out of their normal developmental context, such as ectopic Vg activation in the leg disc, which then drives cell-fate switches. Several of the genes identified in this screen have functional ties to Wg, Dpp and Hh signaling pathways. For example, Cg is a zinc-finger transcription factor that is required for proper transcriptional regulation of the Hh signaling effector gene Cubitus interruptus (Ci). In cg mutant wing and leg discs, Ci expression is lowered in the anterior compartment, resulting in the ectopic activation of wg and dpp and significant disc overgrowth. Another gene identified in this screen -- ken, functions in concert with Dpp to direct the development of the Drosophila terminalia. Further characterizations of whether these genes and other modifiers of transdetermination and regeneration affect Wg, Dpp and Hh expression and/or signaling may shed light on the regulation of regeneration and regeneration-induced proliferation and cell fate plasticity (McClure, 2008).
An important role in olfactory system development is played by transcription factors which act in sensory neurons or in their interneuron targets as cell autonomous regulators of downstream effectors such as cell surface molecules and signalling systems that control neuronal identity and process guidance. Some of these transcriptional regulators have been characterized in detail in the development of the neural elements that innervate the antennal lobe in the olfactory system of Drosophila. This study identified the zinc finger transcription factor Jing as a cell autonomously acting transcriptional regulator that is required both for dendrite targeting of projection neurons and local interneurons as well as for axonal targeting of olfactory sensory neurons in Drosophila olfactory system development. Immunocytochemical analysis shows that Jing is widely expressed in the neural cells during postembryonic development. MARCM-based clonal analysis of projection neuron and local interneuron lineages reveals a requirement for Jing in dendrite targeting; Jing loss-of-function results in loss of innervation in specific glomeruli, ectopic innervation of inappropriate glomeruli, aberrant profuse dendrite arborisation throughout the antennal lobe, as well as mistargeting to other parts of the CNS. ey-FLP-based MARCM analysis of olfactory sensory neurons reveals an additional requirement for Jing in axonal targeting; mutational inactivation of Jing causes specific mistargeting of some olfactory sensory neuron axons to the DA1 glomerulus, reduction of targeting to other glomeruli, as well as aberrant stalling of axons in the antennal lobe. Taken together, these findings indicate that Jing acts as a key transcriptional control element in wiring of the circuitry in the developing olfactory sensory system in Drosophila (Nair, 2013).
These findings place Jing among a small number of other transcription factors that have been shown to be required both for the development of the peripheral OSNs and for the development of their central targets, the PNs and LNs, during olfactory circuit formation. Thus, the POU domain transcription factor Acj6, the homeodomain transcription factor Ems and the zinc-finger transcription factor Jing are all required in OSNs as well as in PN/LN lineages. Moreover, all these transcription factors have dual roles in peripheral and central olfactory system development in that they are required for appropriate axonal/dendrite targeting of presynaptic/postsynaptic elements (Nair, 2013).
It is speculated that these, and other as yet unidentified transcription factors, could represent a transcription factor code that acts in a cell autonomous manner in specifying and interconnecting the presynaptic sensory neurons to their postsynaptic interneuronal targets in olfactory system development. A number of other transcription factors that have been shown to be required for proper wiring of the olfactory circuit viz., POU domain transcription factor Drifter, the LIM-homeodomain transcription factors Islet and Lim1, the LIM-cofactor Chip, the homeodomain transcription factors Cut and Ems, and the zinc finger transcription factors Squeeze and Lola could also be components of the different transcription factor codes or downstream transcriptional programs required for specification and wiring of different subsets of olfactory neurons. Such a transcriptional program could lead to the expression of comparable sets of cell surface recognition molecules (cell adhesion molecules and ligand/receptors) in the developing pre- and postsynaptic neurons and, hence, facilitate the formation of appropriate synaptic. Cell surface molecules such as Dscam, Robo, N-Cadherin, Semaphorin, Plexin A and leucine-rich repeat transmembrane protein Capricious have been shown to be required for dendrite/axonal targeting in the antennal lobe. An investigation of changes in the expression of these identified cell surface molecules following mutational alteration of the postulated Jing, Ems, Acj6 transcription factor code in OSNs and PNs/LNs should be both feasible and instructive (Nair, 2013).
Alternatively, given its ubiquitous expression in developing neural cells, the Jing transcription factor may not be an integral part of a putative transcription factor code for neuronal specificity and targeting, but rather be required for maintaining the stable expression of the more specific transcription factors that constitute the postulated code. Evidence for this type of a more general role for Jing in the determination of cell fate during development comes from experiments which indicate that Jing together with the Polycomb Group (PcG) and trithorax Group (trxG) genes can function in chromatin remodeling during development. Thus, like the Polycomb and Trithorax proteins, Jing could be involved in maintaining the lineage-specific transcriptional programs that specify cell identity during development through epigenetic modification of chromatin structure. A comparable functional role in the regulation of key developmental control genes through Polycomb Group proteins has been implicated for AEBP2, the mammalian homolog of Jing (Nair, 2013).
At the circuit level, the organization of olfactory system in insects and mammals has striking similarities. In both cases sensory cells that express the same olfactory receptor protein project their axons to the same glomeruli located either in the antennal lobe of insects or in the olfactory bulb of mammals. In these glomeruli, the olfactory sensory neurons make specific synaptic connections with the dendrites of second order projection neurons (PNs in insects; mitral and tufted cells in mammals) and local neurons (LNs in insects; periglomerular cells, granule cells, short axon cells in mammals). Given this structural similarity, it is intriguing that similar molecular control elements might operate in the formation of olfactory circuitry in insects and mammals during development. One example for this is represented by the Ems/Emx family of homeodomain transcription factors. Thus, the ems transcription factor is required for development of peripheral and central olfactory neurons in Drosophila, and the vertebrate homologs of ems, the Emx1/2 genes, are similarly required for the development of peripheral and central olfactory neurons in mammals. Given the role of Jing in peripheral and central olfactory system development in Drosophila, it will now be important to analyse the role of the homologous AEBP2 gene in mammalian olfactory system development (Nair, 2013).
Gene duplication, expansion and subsequent diversification are features of the evolutionary process. Duplicated genes can be lost, modified or altered to generate novel functions over evolutionary time scales. These features make gene duplication a powerful engine of evolutionary change. This study explores these features in the MADF-BESS family of transcriptional regulators. In Drosophila melanogaster, the family contains sixteen similar members, each containing an N-terminal, DNA binding MADF domain and a C-terminal, protein interacting, BESS domain. Phylogenetic analysis shows that members of the MADF-BESS family are expanded in the Drosophila lineage. Three members, that were named hinge1 (CG9437), hinge2 (CG8359) and hinge3 (CG13897) are required for wing development, with a critical role in the wing-hinge. hinge1 is a negative regulator of Wingless expression and interacts with core wing-hinge patterning genes such as teashirt, homothorax and jing. Double knockdowns along with heterologous rescue experiments are used to demonstrate that members of the MADF-BESS family retain function in the wing-hinge, in spite of expansion and diversification over 40 Million years. The wing-hinge connects the blade to the thorax and has critical roles in fluttering during flight. MADF-BESS family genes appear to retain redundant functions to shape and form elements of the wing-hinge in a robust and failsafe manner (Shukla, 2013).
Trait development results from the collaboration of genes interconnected in hierarchical networks that control which genes are activated during the progression of development. While networks are understood to change over developmental time, the alterations that occur over evolutionary times are much less clear. A multitude of transcription factors and a far greater number of linkages between transcription factors and cis-regulatory elements (CREs) have been found to structure well-characterized networks, but the best understood networks control traits that are deeply conserved. Fruit fly abdominal pigmentation may represent an optimal setting to study network evolution, as this trait diversified over short evolutionary time spans. However, the current understanding of the underlying network includes a small set of transcription factor genes. This study greatly expands this network through an RNAi-screen of 558 transcription factors. Twenty-eight genes were identified, including previously implicated abd-A, Abd-B, bab1, bab2, dsx, exd, hth, and jing, as well as 20 novel factors with uncharacterized roles in pigmentation development. These include genes which promote pigmentation, suppress pigmentation, and some that have either male- or female-limited effects. Many of these transcription factors control the reciprocal expression of two key pigmentation enzymes, whereas a subset controls the expression of key factors in a female-specific circuit. Pupal Abd-A expression pattern was conserved between species with divergent pigmentation, indicating diversity resulted from changes to other loci. Collectively, these results reveal a greater complexity of the pigmentation network, presenting numerous opportunities to map transcription factor-CRE interactions that structure trait development and numerous candidate loci to investigate as potential targets of evolution (Rogers, 2014).
A novel transcriptional repressor, AEBP2, has been identified that binds to a regulatory sequence (termed AE-1) located in the proximal promoter region of the aP2 gene that encodes the adipose fatty acid-binding protein. Sequence analysis of AEBP2 cDNA has revealed that it encodes a protein containing three Gli-Kruppel (Cys2-His2)-type zinc fingers. Northern blot analysis reveals two transcripts (4.5 and 3.5 kilobases) that are ubiquitously expressed in every mouse tissue examined. AEBP2 repressed transcription from the homologous aP2 promoter containing multiple copies of the AE-1 sequence. Moreover, a chimeric construct encoding a fusion AEBP2 protein with the Gal4 DNA-binding domain is able to repress the transcriptional activity of a heterologous promoter containing the Gal4-binding sequence. The transcriptional repression function of AEBP2 is completely abolished when one of the conserved histidine residues and a flanking serine residue in the middle zinc finger are replaced with an arginine residue. The defective transcriptional repression function of the mutant derivative is due neither to lack of expression nor to a failure to localize to the nucleus. Moreover, both the wild-type and mutant derivative of either the histidine-tagged recombinant AEBP2 proteins or the in vitro translated Gal4-AEBP2 fusion proteins are equally able to bind to the target DNA. These results suggest that a portion of the zinc finger structure may play a direct role in transcriptional repression function, but not in DNA binding (He, 1999).
Recent studies have revealed the intrinsic histone methyltransferase (HMTase) activity of the EED-EZH2 complex and its role in Hox gene silencing, X inactivation, and cancer metastasis. This study focuses on the function of individual components. It was found that the HMTase activity requires a minimum of three components -- EZH2, EED, and SUZ12 -- while AEBP2 (a mammalian Jing homolog) is required for optimal enzymatic activity. Using a stable SUZ12 knockdown cell line, it has been shown that SUZ12 knockdown results in cell growth defects, which correlate with genome-wide alteration on H3-K27 methylation as well as upregulation of a number of Hox genes. Chromatin immunoprecipitation (ChIP) assay identified a 500 bp region located 4 kb upstream of the HoxA9 transcription initiation site as a SUZ12 binding site, which responds to SUZ12 knockdown and might play an important role in regulating HoxA9 expression. Thus, this study establishes a critical role for SUZ12 in H3-lysine 27 methylation and Hox gene silencing (Cao, 2004).
AEBP2 is a zinc finger protein that has been shown to interact with the mammalian Polycomb Repression Complex 2 (PRC2). This study characterized this unknown protein and tested its potential targeting roles for the PRC2. AEBP2 is an evolutionarily well-conserved gene that is found in the animals ranging from flying insects to mammals. The transcription of mammalian AEBP2 is driven by two alternative promoters and produces at least two isoforms of the protein. These isoforms show developmental stage-specific expression patterns: the adult-specific larger form (51 kDa) and the embryo-specific smaller form (32 kDa). The AEBP2 protein binds to a DNA-binding motif with an unusual bipartite structure, CTT(N)15-23cagGCC with lower-case being less critical. A large fraction of AEBP2's target loci also map closely to the known target loci of the PRC2. In fact, many of these loci are co-occupied by the two proteins, AEBP2 and SUZ12. This suggests that AEBP2 is most likely a targeting protein for the mammalian PRC2 complex (Kim, 2009).
Aebp2 is a potential targeting protein for the mammalian Polycomb Repression Complex 2 (PRC2). A mutant mouse line disrupting the transcription of Aebp2 was generated to investigate Aebp2 in vivo roles. Aebp2-mutant homozygotes were found to be embryonic lethal while heterozygotes survived to adulthood with fertility. In developing mouse embryos, Aebp2 is expressed mainly within cells of neural crest origin. In addition, many heterozygotes display a set of phenotypes, enlarged colon and hypopigmentation, similar to those observed in human patients with Hirschsprung's disease and Waardenburg syndrome. These phenotypes are usually caused by the absence of the neural crest-derived ganglia in hindguts and melanocytes. ChIP analyses demonstrated that the majority of the genes involved in the migration and development process of neural crest cells are downstream target genes of AEBP2 and PRC2. Furthermore, expression analyses confirmed that some of these genes are indeed affected in the Aebp2 heterozygotes. Taken together, these results suggest that Aebp2 may regulate the migration and development of the neural crest cells through the PRC2-mediated epigenetic mechanism (Kim, 2011).
Polycomb Repressive Complex 2 (PRC2) is essential for gene silencing, establishing transcriptional repression of specific genes by tri-methylating Lysine 27 of histone H3, a process mediated by cofactors such as AEBP2. In spite of its biological importance, little is known about PRC2 architecture and subunit organization. This study presents the first three-dimensional electron microscopy structure of the human PRC2 complex bound to its cofactor AEBP2. Using a novel internal protein tagging-method, in combination with isotopic chemical cross-linking and mass spectrometry, all the PRC2 subunits and their functional domains have been localized and a detailed map of their interactions generated. The position and stabilization effect of AEBP2 suggests an allosteric role of this cofactor in regulating gene silencing. Regions in PRC2 that interact with modified histone tails are localized near the methyltransferase site, suggesting a molecular mechanism for the chromatin-based regulation of PRC2 activity (Ciferri, 2012).
Previous studies have shown that PRC2 favors di- and oligonucleosome substrates over mononucleosomes, octamers, or histone H3 peptides. Molecular explanations for this substrate preference have been largely hypothetical in the absence of any structural information. The positioning of the different subunits within the PRC2 structure suggests a model of how PRC2 could interact with a dinucleosome, by placing the regions interacting with histone tails in opposite sides of the complex, thus allowing interaction with two nucleosomes simultaneously, without any steric hindrance. A model of the structure suggests a possible arrangement illustrating this point that also agrees with the proposed binding of AEBP2 to nucleosomal DNA. In such arrangement, EED binding to one nucleosome would position the histone H3 tail from the second nucleosome in close proximity to the Ezh2 SET domain (see Mechanism and allosteric regulation of PRC2 during gene silencing). It is suggested that at loci of compact and repressed chromatin, H3K27-me3 marks are recognized by EED. This binding is signaled via the SANT domains to the SET domain increasing the methyl-transferase activity of Ezh2, strengthening the chromatin compaction. At loci of open and actively transcribed chromatin, H3K4me3 and H3K36me2,3 are recognized by the VEFS domain of Suz12 and transferred to Ezh2, with an allosteric regulation that blocks Ezh2's enzymatic activity (Ciferri, 2012).
In conclusion, the human PRC2 structure presented in this paper provides the first full picture of the molecular organization of this fundamental complex and offers an invaluable structural context to understand previous biochemical data. Furthermore, the functional mapping of different activities within the physical shape of the complex leads to novel, testable hypotheses on how PRC2 interacts with chromatin that should inspire future research of PRC2 function and regulation. Given the similarity in sequence between PRC2 components from different species, the molecular architecture that is seen for human PRC2 is expected to be conserved throughout higher eukaryotes (Ciferri, 2012).
Search PubMed for articles about Drosophila jing
Cao, R., et al. (2002). Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298(5595): 1039-43. 12351676
Cao, R. and Zhang, Y. (2004). SUZ12 is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex. Mol. Cell 15(1): 57-67. 15225548
Ciferri, C., Lander, G. C., Maiolica, A., Herzog, F., Aebersold, R. and Nogales, E. (2012). Molecular architecture of human polycomb repressive complex 2. Elife 1: e00005. PubMed ID: 23110252
Culi, J., Aroca, P., Modolell, J. and Mann, R. S. (2006). jing is required for wing development and to establish the proximo-distal axis of the leg in Drosophila melanogaster. Genetics 173(1): 255-66 . 16510782
He, G. P., Kim, S. and Ro, H. S. (1999). Cloning and characterization of a novel zinc finger transcriptional repressor. A direct role of the zinc finger motif in repression. J. Biol. Chem. 274: 14678-14684. 10329662
Kim, H., Kang, K. and Kim, J. (2009). AEBP2 as a potential targeting protein for Polycomb Repression Complex PRC2. Nucleic Acids Res 37: 2940-2950. PubMed ID: 19293275
Kim, H., Kang, K., Ekram, M. B., Roh, T. Y. and Kim, J. (2011). Aebp2 as an epigenetic regulator for neural crest cells. PLoS One 6: e25174. PubMed ID: 21949878
Klebes, A., et al. (2005). Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes. Development 132: 3753-3765. PubMed citation: 16077094
Liu, Y. and Montell, D. J. (2001). jing: a downstream target of slbo required for developmental control of border cell migration. Development 128: 321-330. 11152631
McClure, K. D. and Schubiger, G. (2008). A screen for genes that function in leg disc regeneration in Drosophila melanogaster. Mech. Dev. 125(1-2): 67-80. PubMed citation
Morozova, T., Hackett, J., Sedaghat, Y. and Sonnenfeld, M. (2010). The Drosophila jing gene is a downstream target in the Trachealess/Tango tracheal pathway. Dev. Genes Evol. 220(7-8): 191-206. PubMed Citation: 21061019
Nair, I. S., Rodrigues, V., Reichert, H., Vijayraghavan, K. (2013) The zinc finger transcription factor Jing is required for dendrite/axonal targeting in Drosophila antennal lobe development. Dev Biol 381: 17-27. PubMed ID: 23810656
Rogers, W. A., Grover, S., Stringer, S. J., Parks, J., Rebeiz, M. and Williams, T. M. (2014). A survey of the trans-regulatory landscape for Drosophila melanogaster abdominal pigmentation. Dev Biol 385: 417-432. PubMed ID: 24269556
Sedaghat, Y., Miranda, W. F. and Sonnenfeld, M. J. (2002a). The jing Zn-finger transcription factor is a mediator of cellular differentiation in the Drosophila CNS midline and trachea. Development 129: 2591-2606. 12015288
Sedaghat, Y. and Sonnenfeld, M. (2002b). The jing gene is required for embryonic brain development in Drosophila. Dev. Genes Evol. 212(6): 277-87. 12111212
Shukla, V., Habib, F., Kulkarni, A. and Ratnaparkhi, G. S. (2013). Gene duplication, lineage specific expansion and sub-functionalization in the MADF-BESS family patterns the Drosophila wing-hinge. Genetics 196(2): 481-96. PubMed ID: 24336749
Sonnenfeld, M. J., Barazesh, N., Sedaghat, Y. and Fan, C. (2004). The jing and ras1 pathways are functionally related during CNS midline and tracheal development. Mech. Dev. 121: 1531-1547. 15511644
Sun, X., Morozova, T. and Sonnenfeld, M. J. (2006). Glial and neuronal functions of the Drosophila homolog of the human SWI/SNF Gene, ATR-X (DATR-X), and the jing zinc finger gene specify the lateral positioning of longitudinal glia and axons. Genetics 173(3): 1397-415. 16648585
date revised: 10 February 2014
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