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First published online 13 December 2006
doi: 10.1242/dev.02746

Development 134, 381-392 (2007)
Published by The Company of Biologists 2007
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Comparative analysis of Hox downstream genes in Drosophila

Stefanie D. Hueber1,*, Daniela Bezdan1,*, Stefan R. Henz2, Martina Blank1, Haijia Wu1 and Ingrid Lohmann1,{dagger}

1 Max Planck Institute for Developmental Biology, Spemanstrasse 37-39, D-72076 Tübingen, Germany.
2 Department of Molecular Biology, Spemanstrasse 37-39, D-72076 Tübingen, Germany.


Figure 1
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  		Fig. 1. Identification of Hox downstream genes during early embryogenesis. (A) Outline of microarray analysis. Scanning electron micrographs of embryos at late stage 10-early stage 11, late stage 11-early stage 12, and late stage 12-early stage 13. arm::2xEGFP embryos demonstrate ubiquitous transgene expression beginning at stage 10. Time scale shows hours of embryogenesis. (B) Quantitative real-time PCR shows similar levels of overexpression of Dfd, Scr, Antp, Ubx, Abd-A and Abd-B transgenes. Numbers of genes regulated by the different Hox proteins are indicated. (C) Average fluorescent intensity (in arbitrary units) of 20 independent nuclei at different locations in wild-type, arm::Dfd and arm::Abd-B embryos stained either with {alpha}-Dfd or {alpha}-Abd-B antibody. Due to variable expression levels of endogenous Hox proteins, fluorescence from nuclei in different expression domains was measured (marked as `endo weak' and `endo strong'). (D) Representative embryo used for measuring fluorescent intensity of nuclei is shown. The upper two rows show wild-type and arm::Dfd embryos stained with {alpha}-Dfd antibody, the lower two rows wild-type and arm::Abd-B embryos stained with {alpha}-Abd-B antibody. Red boxes mark the areas used for fluorescence analysis. s, strong endogenous expression domain; w, weak endogenous expression domain. (E) Ubiquitous overexpression of Hox proteins in stage 11 and 12 was confirmed by antibody staining on arm::lacZ and arm::Hox embryos.

 

Figure 2
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  		Fig. 2. Verification of known Hox downstream genes identified by microarray analysis. (A) Relative expression levels of seven known Hox downstream genes identified in a microarray screen are shown. For three of the seven genes, in situ hybridizations were performed. Genes shown are: reaper (rpr), paired (prd), ß-Tubulin at 60D (ß-Tub60D), Distal-less (Dll), spalt major (salm), empty spiracles (ems) and nubbin (nub). (B) {alpha}-Dfd and {alpha}-Abd-B antibody stainings on embryos misexpressing different Hox genes confirmed posterior suppression as seen in the microarray experiment. Antibody stainings for all (Abd-B misexpression) or for some (Dfd misexpression) Hox proteins are shown.

 

Figure 3
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  		Fig. 3. Verification of newly identified Hox downstream genes by in situ hybridization. In situ hybridizations of the indicated genes in stage 11 and/or stage 12 arm::lacZ, arm::Hox, wild-type and Hox mutant embryos. Genes shown are: skl (A-D), CG5080 (E-H), CG7447 (I-L), sage (M-P), ImpL2 (Q-T) and spz (U-X). Hybridizations on embryos misexpressing Hox genes and on Hox mutant embryos were performed independently (with the respective arm::lacZ and wild-type controls). Differences in staining intensities are due to differences in the in situ hybridization procedures. Pictures of arm::lacZ and wild-type embryos were taken at different focal planes and thus expression patterns in these embryos appear slightly different. Red arrows mark expression domains changed in wild-type and Hox mutant embryos.

 

Figure 4
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  		Fig. 4. Confirmation of predicted direct Dfd downstream genes by EMSA. (A-D) EMSA for four predicted direct Dfd downstream genes tested using no protein, translation lysate only (L) and lysate with Dfd protein (D). To test the specificity of binding of Dfd protein to the DNA fragments, competitor oligonucleotides for the individual Dfd-binding sites (DBS) were used either in their wild-type (wt) or mutant (mt) sequence versions. The black arrowhead indicates the specific DNA-protein complex containing Dfd protein. Asterisks indicate the unbound labeled probe. Predicted Dfd response enhancers of the following genes were used: Eip63E (A), frizzled 2 (fz2) (B), wg (C) and CG5756 (D).

 

Figure 5
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  		Fig. 5. Specificity of Hox downstream gene regulation. (A) Classification of Hox downstream genes according to their regulation by one (unique), several (regional) and all (common) Hox proteins. Numbers of unique downstream genes for each Hox protein are shown on the left. The distribution of classes does not change when the Abd-A experiment, which was performed independently, is excluded from the analysis (shown in the table). (B) Distribution of the regulatory classes among all identified Dfd downstream genes and predicted direct Dfd target genes is very similar. No commonly regulated downstream genes are found among the predicted direct Dfd target genes. (C) Distribution of Hox downstream genes regulated at the two different stages analyzed. On the left side, the percentage of all Hox downstream genes regulated at the two stages are shown; on the right side, the distribution for each individual Hox protein is indicated. Numbers of genes are shown within bars. (D) In situ hybridizations of selected examples of genes regulated at specific stages (early-specifc, early and late, late-specific). Expression patterns of the following genes are displayed: Heat shock protein 23 (Hsp23), CG3097 and wrapper.

 

Figure 6
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  		Fig. 6. Functional classification of Hox downstream genes using GO categories. (A) Functional categories of downstream genes are shown for all newly identified Hox downstream genes (first column), for all identified Dfd downstream genes (second column), for all predicted direct Dfd target genes (third column) and for all known direct Hox target genes (fourth column). Numbers of genes for each category are indicated within bars. (B) Diagram showing subclasses of realizator genes, with numbers of genes for each class indicated. (C) Subclasses of realizators are often coordinately regulated, as shown here by three examples (apoptosis, cell adhesion, cell cycle or cell proliferation). Green arrows, increased expression; red arrows, reduced expression. (D) Morphological differences along the AP axis are reflected in the percentage of shared downstream genes regulated opposingly by two different Hox proteins. Light gray indicates similar morphologies; medium gray and dark gray indicate increasing differences in morphologies directed by the Hox genes compared. The scanning electron micrograph shows the morphology of a stage 13 embryo, with the expression domains of the different Hox proteins highlighted.

 

Figure 7
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  		Fig. 7. A framework for the morphogenesis of the maxillary segment in Drosophila. (A,C) Scanning electron micrographs of heads of stage 12 wild-type embryos. (B) Head of a stage 12 Dfd mutant embryo. The red arrow marks additional cells at the ventral side of the maxillary segment. (D) Head of a wg mutant embryo. The red arrow marks the size-reduced maxillary segment. (E,J) BrdU labeling of stage 12 wild-type and Dfd mutant embryos, respectively. Red arrows mark proliferating BrdU-positive cells at the ventral side of the maxillary segment. (F) Diagram of a stage 12 wild-type embryo. Mandibular (md), maxillary (mx) and labial (lb) segments are indicated; the box marks the area shown in G,H,I. (G,H,I) In stage 12 wild-type embryos, cells in the ventral part of the maxillary segment are round (G), whereas in Dfd mutant (H) and arm::hepact. (I) embryos, cells are elongated (marked in red). (K-T) skl (K,P), wg (L,Q), Eip63E (M,R), prd (N,S) and CG5080 (O,T) RNA expression in wild-type and Dfd mutant embryos, respectively. The red arrows indicate the expression of the respective genes that differs in wild-type and Dfd mutant embryos.

 

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