See the embryonic expression pattern of jing at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
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).
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date revised: 30 July 2008
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