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The jing Zn-finger transcription factor is a mediator of cellular differentiation in the Drosophila CNS midline and trachea

Yalda Sedaghat^*, Wilson F. Miranda^* and Margaret J. Sonnenfeld

Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada
^* These authors contributed equally to this work

View larger version (61K): [in a new window]   Fig. 1. Genetic interactions in the CNS. (A-F) Preparations of stage 14 ventral nerve cords stained with mAb BP102. Anterior is upwards. (G,H) Stage 15 embryos stained with mAb 1D4 (anti-Fasciclin 2). (A) Wild-type CNS axon scaffold with anterior and posterior commissures (AC and PC) separating longitudinal connectives (LC). (B) Collapsed axon phenotype of a simH9 homozygote. (C) ‘Stalled axon’ phenotype of jing01094; simH9 double heterozygote. Arrow and arrowhead indicate improper commissural and longitudinal formation, respectively. (D) The axon scaffold develops properly in embryos heterozygous for simH9 and a jing01094 P element revertant (jingrev). (E) The phenotype of embryos heterozygous for tgo1 and jing01094 resembles weak sim phenotypes (arrow) and spitz group phenotypes (arrowhead). (F) Severe ‘collapsed axon’ phenotype in jing01094, tgo1 simH9 triple heterozygote. (G) Three wild-type 1D4-positive longitudinal fascicles run parallel to the midline. (H) Fusion of longitudinal fascicles in jing01094, tgo1 simH9 triple heterozygotes. (I) Quantification of jing genetic interactions. Double and triple heterozygotes (genotype) were examined for CNS axon scaffold formation using mAb BP102. Data are presented as the percentage of embryos with stalled, collapsed and fused axon phenotypes (n>50 embryos scored in all cases). Note that reduction of one copy of tgo in a jing01094; sim double heterozygote causes a shift in distribution from embryos with stalled axon phenotypes toward those with collapsed axon phenotypes (jing01094, tgo1 simH9). jing01094 and simH9 mutations show similar dose sensitivities to sli1.

View larger version (96K): [in a new window]   Fig. 2. Genetic interactions alter CNS midline cell development. Whole-mount embryos stained with anti-SIM (A-C,F-I) and anti-SLI (D,E,J,K). (A-E,H,I) Sagittal section with anterior towards the left and (F,G,J,K) frontal views with anterior upwards. (A) In stage 13 wild-type embryos, SIM-positive cells are distributed ventrally to dorsally (arrow) and are only occasionally outside the nerve cord (arrowhead). (B) In stage 13 jing01094, tgo1 simH9 triple heterozygotes, the midline cells are displaced dorsally (arrowhead) and ventrally (arrow). (C) In stage 14 jing01094, simH9 double heterozygotes, midline cells are displaced ventrally. Arrow denotes absence of dorsal midline cells. (D) In stage 13 wild-type embryos, SLI-positive glia are present dorsally. (E) In stage 13 jing01094, tgo1 simH9 triple heterozygotes, SLI immunoreactivity is displaced to segment boundaries (arrow) and ventrally. (F) Stage 15 wild-type embryo showing SIM+ cells. (G) Absence of SIM immunoreactivity in stage 15 jing01094, tgo1 simH9 triple heterozygotes. (H) SIM+ midline cells are displaced ventrally in stage 14 simH9/sli1 double heterozygotes. (I) Midline cells are displaced ventrally (arrow) in jing01094/sli1 double heterozygotes. Arrowhead denotes absence of cells. (J) Wild-type midline SLI immunoreactivity. Stage 14. (K) Reduced SLI immunoreactivity in stage 14 jing01094/sli1 double heterozygotes. Arrows in J,K indicate SLI immunoreactivity.

View larger version (68K): [in a new window]   Fig. 3. Genetic interactions in the trachea. Stage 15 embryos stained with mAb 2A12 to visualize tracheal tubules and shown with anterior left in sagittal view. (A) Wild-type embryo. (B) Absence of tracheal tubules in homozygous trh1 mutants. (C) Severe loss of tracheal tubules in jing01094; trh1 double heterozygotes. (D) Reductions in tracheal branches in jing01094; btlH823 double heterozygotes. (E) Quantification of jing genetic interactions in the trachea. Double and triple heterozygous combinations (genotype). Data show the percentage of embryos with loss of tracheal branches. Tracheal development in jing01094; trh1 double heterozygotes is sensitive to the dose of tgo (jing01094; tgo1 trh1). jing01094; btlH823 double heterozygotes show the highest percentage of embryos with tracheal tubule defects. In all cases more than 65 embryos were scored. DB, Dorsal branch; VB, visceral branch; DT, dorsal trunk; TC, transverse connective; LTa, lateral trunk anterior; LTp, lateral trunk posterior.

View larger version (61K): [in a new window]   Fig. 4. jing genomic structure and embryonic expression. (A) Genomic interval containing jing ESTs, lethal P element insertions and adjacent gene (LD10015). DNA-binding sites of bHLH-PAS (CMEs) and SOX HMG protein Dichaete (TACAAT) are present in the jing 5' regulatory region. (B) jing exon/intron structure and point mutation. Exons (filled boxes), introns (lines) and protein motifs (colored boxes). Exon 2 contains repeats of poly-glutamine (Q), poly-serine (S) and alanine-glutamine (AQ). C2H2-type zinc fingers (Zn). The jing3 mutation is a G-to-A change at nucleotide 3806 of cDNA LD36562 converting W1200 to a stop codon. (C-O) Embryonic expression of jing. (C,I,L) In situ hybridization of jing digoxigenin-labeled DNA probes to wild-type embryos. (D,G,J,M) jing-lacZ enhancer trap expression detected via anti-ß-gal staining and confocal microscopy. (E,F,H,K,N,O) Anti-JING immunostaining of wild-type embryos. Confocal microscopy (F,H,N), light microscopy (E,K,O). (C-E) By stage 9, jing transcripts and protein product are present in the CNS midline (arrows) and neuroectoderm (small arrowheads) and supraesophageal ganglion (arrowheads). (D) Colocalization of jing-lacZ product (anti-ß-gal, green) and SIM (red) in a subset of CNS midline precursors at stage 9, as detected via double-label immunostaining (arrow). (F) Stage 10 embryo showing JING localization in tracheal placodes (arrow) and two adjacent cells (small arrowhead). In this focal plane, JING is observed in three MP neurons in the CNS (larger arrowhead). (G) Merged confocal image showing co-localization of jing-lacZ product (anti-ß-gal, green) and TRH (red) in tracheal placodes (arrow) detected via double-label immunostaining of wild-type stage 10 embryos. (H) Localization of JING to all tracheal branches in a stage 15 wild-type embryo. Note nuclear localization of JING. (I-K) At stage 12/3, jing transcription occurs in the CNS midline (arrows) and segmental ectodermal stripes (arrowheads). Small arrowhead in J indicates Sim+ muscle cells. jing transcription (L,M) and protein product (N) are highest in midline glia (arrows), as shown in a stage 15 wild-type embryo. Large arrowheads in L-N show midline neurons; small arrowheads in L,M show jing-negative midline glia. (O) anti-JING immunostaining of homozygous embryos carrying a deletion in the jing gene (Df(2R)ST1) to reveal the specificity of the antisera.

View larger version (107K): [in a new window]   Fig. 5. The jing-lacZ enhancer is expressed in the midline. lacZ expression in whole-mount embryos heterozygous for the jing-lacZ enhancer trap and stained with anti-ß-gal. (A) Strong midline jing-lacZ expression (arrow) and weaker neuroectodermal expression in stage 14 jing01094 heterozygotes (arrowhead). (B) Reduced jing-lacZ expression in the midline and neuroectoderm of jing01094 homozygotes suggests autoregulation. (C) In simH9/simH9 null mutants, jing-lacZ midline expression is not detectable (arrow) but lacZ expression still occurs in the neuroectoderm (arrowhead). (D-F) Embryos heterozygous for the jing-lacZ enhancer trap, P[prd-Gal4] and P[UAS-sim] and stained with anti-SIM (D, red), anti-ß-gal (E, green). (E) Ectopic jing-lacZ activation (arrows). (F) Merged images of D,E. (F) Activation of jing-lacZ in the ventral region of prd pair-rule stripes (arrows).

View larger version (137K): [in a new window]   Fig. 6. jing loss- and gain-of-function phenotypes in the CNS and trachea. Frontal views showing CNS axon scaffolds (BP102 stain) (A-F) and longitudinal fascicles (mAb 1D4 stain; anti-Fasciclin 2) (G,H). Sagittal views of stage 15 whole-mount embryos stained with mAb 2A12 to visualize tracheal tubules (I-L). (A,B) Pioneering growth cones do not approach the midline during stage 12 in homozygous jing3 mutants (B) as they do in wild-type (A) (arrowheads). (C,D) In stage 14 jing3 mutants, there are variable absences of either anterior or posterior commissures (arrow) and longitudinal connectives (arrowhead) (D) compared with wild type (C). (E) jing3;simH9 double mutants show collapsed axon phenotypes. (F) Overexpression of jing in the CNS midline (sim-GAL4/jing-UAS) disrupts commissural (arrow) and longitudinal axon formation (arrowhead). (G) Ipsilateral projection of wild-type Fasciclin 2-positive longitudinal bundles. (H) In jing3 homozygotes, longitudinal fascicles inappropriately cross the midline (arrows). Longitudinal fascicles stall within segments (arrowhead). (I,J) Embryos homozygous for a deficiency covering jing (Df(2R)ST1) and jing3 mutations show defects in the formation of all tracheal branches, including the dorsal trunk (arrowhead) and transverse connectives (TC) (arrow). Note absence of the visceral branch (compare with Fig. 3A). (K) jing3; trh1 double mutants show loss of all tracheal tubules. (L) Overexpression of jing in the trachea (btl-GAL4/jing-UAS) disrupts all aspects of tracheal tubule development. Branch fusion in the dorsal trunk does not occur (arrowhead) and the dorsal branch and transverse connectives are reduced (arrows). The visceral branch is absent. DB, Dorsal branch; VB, visceral branch; DT, dorsal trunk; TC, transverse connective; LTa, lateral trunk anterior; LTp, lateral trunk posterior.

View larger version (99K): [in a new window]   Fig. 7. jing mutations disrupt CNS midline glial differentiation. (A-D) Embryos stained with anti-SIM; (E,F) ß-gal expression in embryos carrying sli reporter P[sli 1.0 HV-lacZ]; (G,H) anti-SLI stain to visualize midline glia; (I-J') Stage 12 embryos double labeled with anti-SLI (green) and TUNEL (red), and confocal images of 1 µm serial sections. Sagittal views with anterior towards the left. (A,B) sim expression is reduced (arrow) in stage 9 jing3 homozygotes (B) compared with wild-type (A). (C,D) Reduced SIM immunoreactivity and small size of midline neurons (arrows) and glia (arrowheads) in stage 15 jing3 homozygotes (D) compared with wild-type (C). (E) Wild-type stage 11 expression of P[sli 1.0 HV-lacZ] in six midline glia. (F) P[sli 1.0 HV-lacZ] expression in average of 3.2 midline glia/segment in stage 11 jing3 homozygotes. (G) Wild-type SLI-positive glia during stage 15. (H) Reduced SLI immunoreactivity (arrowhead) and detection in macrophages outside the nerve cord (arrow) in stage 15 jing3 homozygotes. (I) Stage 12 wild-type sli-lacZ expression (green). TUNEL-positive profiles (red) are present only outside the CNS (arrow). (I') Close-up view of I. SLI- and TUNEL-positive glia (arrowhead). (J) Loss in SLI immunoreactivity (arrowheads) in stage 12 jing3 homozygotes. Note increase in TUNEL labeling compared with wild type. (J') Close-up view of J (arrows, dead glia). TUNEL-positive profiles in the nerve cord (arrowhead).

View larger version (132K): [in a new window]   Fig. 8. jing mutations disrupt midline neuronal differentiation. Wild-type (A,C,E,G,I) and jing3 mutant embryos (B,D,F,H,J) stained with neuronal specific antibodies. Sagittal views of whole-mount stage 14 (A,B) and stage 10 embryos (I,J). (C-H) Frontal views of dissected stage 15 nerve cords with anterior up. (A) mAb 22C10 stains the VUM neuron cell bodies and axons projecting dorsally (arrow) in each nerve cord segment. (B) Absence of VUM cell bodies and axons in some jing3 segments (arrow). (C) Six wild-type Engrailed (EN)-positive neurons (detecting the VUMs and MNB). (D) Absence of EN-positive neurons in the CNS midline of some jing3 segments (arrow) and reductions in others. Neuroectodermal EN-positive neurons are not reduced from wild-type. (E,F) Wild-type P223 enhancer trap expression in the MP1, vMP2 and dMP2 neurons. (F) Downregulation of P223 expression in MP lineages of jing3 homozygotes. (G) Wild-type Odd-skipped (ODD) expression in MP1 and dMP2 neurons. (H) Reduced ODD expression in jing3 MP1 and dMP2 neurons. (I) Stage 10 embryo double-labeled with anti-ODD and TUNEL. Apoptotic MP lineages are not detectable. (J) Stage 10 jing3 homozygous mutant embryo double-labeled with anti-ODD and TUNEL. Note that ODD immunoreactivity is significantly reduced compared with wild type. TUNEL-positive MP neurons are not present in this embryo.

View larger version (82K): [in a new window]   Fig. 9. jing tracheal phenotype. (A) Wild-type stage 10 embryo. Tracheal nuclei are visualized with anti-Trachealess (TRH, red) antibodies and anti-Engrailed (EN, green) references the anterior tracheal border. A subset of tracheal cells express EN (arrows, yellow cells). (B) In jing3 stage 10 homozygous mutant embryos, the number of TRH-positive precursors is reduced. However, the segmental EN-positive stripes and positioning of the placodes appear normal. (C) Wild-type stage 11 embryo stained with anti-TRH (green) and TUNEL (red) showing a rare apoptotic tracheal cell (arrow). (D) Stage 11 jing3 mutant embryo stained with anti-TRH (green) and TUNEL (red). Many apoptotic tracheal cells are present (arrows) and more apoptotic cells surround the pits than in wild-type embryos (arrowhead). (E) Wild-type embryo at stage 12 shows migration of primary branches across EN-positive stripes. A domain of EN-positive tracheal cells encompasses a region of the segmental stripe (bracket). (F) The domain of EN-positive migrating tracheal cells is smaller (bracket) in some stage 12 jing3 mutant segments and absent in others (arrowhead). (G,H) Wild-type stage 15 embryo stained with both anti-TRH (green, G) and TUNEL (red, H). (I,J) jing3 stage 15 homozygous mutant embryo stained with anti-TRH (green, I) and TUNEL (red, J). (I) In jing3 mutant embryos the EGFR-dependent branches are most severely affected. The visceral branch does not form and regions of the dorsal branch are absent (arrows). The number of cells in the DPP-dependent branches (DB and TC) is reduced compared with wild type. (J) By contrast, the overall apoptotic pattern in jing3 mutant embryos is not significantly elevated from wild type. DB, dorsal branch; DT, dorsal trunk; VB, visceral branch; TC, transverse connective; LTa, lateral trunk anterior; LTp, lateral trunk posterior.

View larger version (25K): [in a new window]   Fig. 10. Model for the role of the JING Zn-finger transcription factor in CNS midline and tracheal cell differentiation. Heterodimers of TGO:SIM and TGO:TRH may bind to the CMEs in the 5' regulatory region of jing. In the CNS midline, the SOX HMG protein Dichaete (FISH in the figure) may also bind its target site, TACAAT, to promote jing transcription. JING enters the nucleus of CNS midline and tracheal cells where it may perform a regulatory role. This regulatory role is required for the differentiation and survival of CNS midline and tracheal precursors, possibly through the control of EGFR signaling and/or other uncharacterized survival factors. Alternatively, jing may function to maintain the transcriptional output initiated by bHLH-PAS, POU and SOX regulatory molecules, and failure to do so results in cell death. JING function is required throughout CNS midline and tracheal development for the formation of a functional central nervous system and tracheal tubule network. Question marks denote that direct interactions have not been demonstrated.