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First published online May 8, 2009
doi: 10.1242/10.1242/dev.026559

Development 136, 1929-1938 (2009)
Published by The Company of Biologists 2009
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Drosophila ptip is essential for anterior/posterior patterning in development and interacts with the PcG and trxG pathways

Ming Fang1,2,*, Hongyan Ren2, Jiabin Liu2, Ken M. Cadigan3, Sanjeevkumar R. Patel4 and Gregory R. Dressler1,*

1 Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA.
2 Department of Genetics and Developmental Biology, Southeast University Medical School, Nanjing, China 210009.
3 Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
4 Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA.


Figure 1
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  		Fig. 1. Conservation of ptip gene structure. (A) A phylogenetic tree approximating the evolutionary distance of presumptive ptip homologs across species. (B) Schematic alignment of mouse and Drosophila PTIP proteins. The BRCT domains are indicated by black boxes, the percentage amino acid identity and similarity (in parentheses) is shown below. The expanded glutamine-rich region is in gray. (C) Genomic structure of the Drosophila ptip gene; the position of the PiggyBacc04574 insertion is indicated. (D) Northern blot of total RNA isolated from the stages indicated and hybridized with a Drosophila ptip probe. Note the ptip germline null embryo clones have lost the full length 7 kb mRNA. Total 18S rRNA is shown by ethidium bromide staining below.

 

Figure 2
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  		Fig. 2. Analysis of Drosophila germline ptip mutants in development. Germline null and zygotic rescue embryos were generated by crossing PBacPTIP/FRT2A P[ovoD] females to Df(ptip)1/TM6 evelacZ males. Micrographs of cuticles from wild type (A), ptip maternal and ptip zygotic mutants (B) and zygotically rescued ptip maternal mutants (C) are shown. Note that the loss of maternal and zygotic ptip causes severe embryonic segmentation defects that could be partially rescued by providing a paternal ptip gene (arrow).

 

Figure 3
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  		Fig. 3. Patterning defects in ptip mutants. (A-E') The expression of maternal effect and segmentation genes is shown in wild type (A-E) and ptip germline null mutants (A'-E'). Whole-mount in situ hybridization for bicoid (A), oskar (B), eve (C) and ftz (D), and immunostaining for Engrailed (E). Note the normal posterior localization of bicoid mRNA in ptip mutants (A') but the loss of some pair rule gene stripes at the cellular blastoderm stage (C',D'). By the extended germband stage, Engrailed is barely detectable in ptip mutants (E').

 

Figure 4
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  		Fig. 4. Patterning defects in zygotic ptip null embryos. The imaginal discs from wild type (A,C,E,G) or zygotic ptip mutants with genotype PBacptip/Df(ptip)1 (B,D,F,H) were isolated from third instar larvae. (A,B) Wing imaginal discs stained with anti-Engrailed (red). (C,D) Leg imaginal discs stained with anti-Engrailed (red). (E,F) Micrographs of wing imaginal discs immunostained with antibodies against Wingless (red) and Distal-less (green). (G,H) Whole-mount in situ hybridization for cubitus interuptus (ci) expression in the wing imaginal discs. Note absence of ci expression in ptip mutants correlates with ectopic Engrailed protein in the anterior half of wing and leg discs.

 

Figure 5
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  		Fig. 5. Immunostaining for histone H3K4 trimethylation. (A-L) Salivary glands were dissected free from third instar larvae of wild type (A-C,G-I) or ptip zygotic nulls (D-F,J-L) and squashed onto slides for immunostaining with anti-histone H3K4me3 (A,D) or anti-histone H3K27me3 (G,J) antibodies (green). Nuclei were co-stained with propidium iodide (PI, red; B,E,H,K) and merged images are also shown (C,F,I,L). All micrographs were taken at manually set, equal exposure times. (M) Western blot of histones extracted from wild-type or zygotic ptip mutant larvae probed with the indicated antibodies. Relative protein levels normalized to total histone H3 are indicated; wild-type levels are arbitrarily designated as 1.0.

 

Figure 6
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  		Fig. 6. Mouse ptip rescues zygotic PBacptip lethality. (A) Western blot using an anti-mousePTIP antibody shows a dose response of mPTIP expression with the increased time of heat shock treatment (above) consistent with the RT-PCR results using a mouse ptip-specific primer pair (below). w1118, control flies; hs-, UAS-mPtip without heat shock; hs15, 15 minute heat shock; hs40, 40 minute heat shock; hs60, 60 minute heat shock. (B) Adult female and (C) adult male flies with the genotype P[hsGal4]/P[UAS-mPtip]; Df(ptip)1/PBacptip that were derived from heat shocked embryos as described in the Materials and methods section.

 

Figure 7
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  		Fig. 7. Derepression of Ubx in Drosophila Kc cells. (A) Kc cells were cultured for 8 days with control dsRNAs or with dsRNA against the indicated genes. Total RNA levels were analyzed by Northern blotting with probes against Ubx, Deformed, ptip and β-Tubulin. For ptip knockdowns, 3'- and 5'-specific dsRNAs were used. Relative mRNA levels are indicated below each band. Note, Ubx activation in response to PcG knockdown requires ptip, whereas Deformed activation does not. (B) Expression levels of the indicated genes were assayed by RT-PCR after the addition of dsRNAs (above) and 8 days in culture.

 

Figure 8
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  		Fig. 8. Microarray analysis of ptip knockdown in Kc cells. The charts summarize gene expression changes observed in Drosophila Kc cells after 7 days of treatment with ptip dsRNAs. A total of 52 genes were expressed at levels significantly higher (more than twofold) than controls, whereas 214 genes were expressed at twofold or lower levels. The complete dataset is presented in Table S1 in the supplementary material.

 

Figure 9
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  		Fig. 9. Microarray analysis of PcG target genes in Kc cells. (A) Expression heat diagram for the top 74 mRNA transcripts that demonstrate significant and greater than twofold upregulation of mRNA levels after PcG gene knockdown (red). Each row represents a gene. Each column represents relative expression levels after the combined RNAi treatments as indicated. The level of expression relative to that of control RNAi samples (in triplicates) of each gene is represented using a red-to-green color scale. For reference, expression values for housekeeping genes and RNA-related genes are also shown. Scatter graphs of mRNA expression levels, presented as fold change on a logarithmic scale, are shown to the right. The fold change for PcG knockdowns (green) is compared with the fold change for PcG plus PTIP knockdowns (red). The complete list of Polycomb target genes is shown in Table S2 in the supplementary material. The original array data is available at http://www.ncbi.nlm.nih.gov/geo (accession number GSE15092). (B) Averaged expression levels of datasets represented in A. P value is calculated by ANOVA analysis followed by Newman-Keul's multiple comparisons. (C) Northern analysis of Polycomb and ptip gene expression levels of samples used in A, rRNA levels are shown for loading controls. Relative mRNA levels of ptip, as determined by phosphoimage analysis, are indicated below each band.

 

Figure 10
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  		Fig. 10. Chromatin immunoprecipitation of mPTIP in Kc cells. (A) Chromatin was prepared from Kc cells transfected with control dsRNAs, dsRNAs against Pc and ph (ds-PcG), control dsRNAs and mPtip, or ds-PcG and mPtip. Western blot probed with antibodies against mPTIP (arrow) is shown. (B) ChIP experiment using antibodies against mPTIP and PCR primer pairs against the indicated genes. The chromatin preparations are as described in A. Data are normalized to control rabbit IgG and presented as the percentage input as calculated from standard curves. Note that mPTIP localizes to PcG target genes only when ds-PcG is present. Experiments were done in triplicate; error bars are one standard deviation from the mean. (C) ChIP experiment using antibodies against H3K4me3 and the chromatin preparations described in A. Note the increase in H3K4me3 levels when ds-PcG is present. This increase is enhanced with exogenous mPTIP. Experiments were done in triplicate; error bars are one standard deviation from the mean.

 

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© The Company of Biologists Ltd 2009