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Cropped, Drosophila transcription factor AP-4, controls tracheal terminal branching and cell growth.

Wong MM, Liu MF, Chiu SK - BMC Dev. Biol. (2015)

Bottom Line: Overexpressing the wild-type crp gene or a mutant that lacks the DNA-binding region in either the tracheal tissues or terminal cells led to a loss-of-function phenotype, implying that crp can affect terminal branching.Unexpectedly, the ectopic expression of cropped also led to enlarged organs, and cell-counting experiments on the salivary glands suggest that elevated levels of AP-4 increase cell size and organ size.We find that the branching morphogenesis of terminal cells of the tracheal tubes in Drosophila requires the dMyc-dependent activation of Cropped/AP-4 protein to increase the cell growth of terminal cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong, Hong Kong. kaychiu@cityu.edu.hk.

ABSTRACT

Background: Endothelial or epithelial cellular branching is vital in development and cancer progression; however, the molecular mechanisms of these processes are not clear. In Drosophila, terminal cell at the end of some tracheal tube ramifies numerous fine branches on the internal organs to supply oxygen. To discover more genes involved in terminal branching, we searched for mutants with very few terminal branches using the Kiss enhancer-trap line collection.

Results: In this analysis, we identified cropped (crp), encoding the Drosophila homolog of the transcription activator protein AP-4. Overexpressing the wild-type crp gene or a mutant that lacks the DNA-binding region in either the tracheal tissues or terminal cells led to a loss-of-function phenotype, implying that crp can affect terminal branching. Unexpectedly, the ectopic expression of cropped also led to enlarged organs, and cell-counting experiments on the salivary glands suggest that elevated levels of AP-4 increase cell size and organ size. Like its mammalian counterpart, cropped is controlled by dMyc, as ectopic expression of dMyc in terminal cells increased cellular branching and the Cropped protein levels in vivo.

Conclusions: We find that the branching morphogenesis of terminal cells of the tracheal tubes in Drosophila requires the dMyc-dependent activation of Cropped/AP-4 protein to increase the cell growth of terminal cells.

No MeSH data available.


Related in: MedlinePlus

Expression of dominant-negative Cropped leads to missing cells and disruption of organ development. (A) Overexpressing DN-crp by using btl-Gal4 in the tracheal system led to disruption of the formation of terminal and fusion cells (i and ii); the arrow heads point to the ends of DBs. (iii and iv) Larvae show a collapse in dorsal trunk. (i and iii) are fluorescence images of GFP-labeled tracheal cells and (ii and iv) are the same field under DIC optics. (B) Expression of DN-crp in the salivary glands driven by A9-Gal4 shrank the organs (ii) compared with control (i) under the same magnification. (C) Staining assay on the cells expressing crp and DN-crp in the salivary glands for apoptosis with changes in permeability by PI (i-iv) and breakage of DNA strands by the TUNEL assay (v-viii). DIC photomicrographs (i, iii, v, vii) and fluorescence microscopy (ii, iv, vi, viii) were taken under the same magnification. (D) Scanning electron micrographs of the ommatidia expressing DN-crp or WT crp driven by GMR-Gal4. (i-iv) The anterior of the fly is pointing to the left and the posterior to the right. DN-crp inhibited the formation of ommatidia and bristles in the posterior half of the eyes and WT crp caused overgrowth of groups of ommatidia. The white arrowheads indicate groups of protruding ommatidia in (ii and iii) and the yellow arrowheads points to the posterior end of the eye without any ommatidia. Scanning micrographs at higher magnification of the ommatidia from WT (v), WT crp- (vi), and DN-crp-expressing fly (vii) are shown. (vii) Electron micrograph taken from the dorsal side of the eye of DN-crp expressing fly and the anterior is to the left. Scale bars are 100 μm (A, B, and C), 200 μm (Di-iv), 20 μm (Dv,vi), and 50 μm (Dvii).
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Fig5: Expression of dominant-negative Cropped leads to missing cells and disruption of organ development. (A) Overexpressing DN-crp by using btl-Gal4 in the tracheal system led to disruption of the formation of terminal and fusion cells (i and ii); the arrow heads point to the ends of DBs. (iii and iv) Larvae show a collapse in dorsal trunk. (i and iii) are fluorescence images of GFP-labeled tracheal cells and (ii and iv) are the same field under DIC optics. (B) Expression of DN-crp in the salivary glands driven by A9-Gal4 shrank the organs (ii) compared with control (i) under the same magnification. (C) Staining assay on the cells expressing crp and DN-crp in the salivary glands for apoptosis with changes in permeability by PI (i-iv) and breakage of DNA strands by the TUNEL assay (v-viii). DIC photomicrographs (i, iii, v, vii) and fluorescence microscopy (ii, iv, vi, viii) were taken under the same magnification. (D) Scanning electron micrographs of the ommatidia expressing DN-crp or WT crp driven by GMR-Gal4. (i-iv) The anterior of the fly is pointing to the left and the posterior to the right. DN-crp inhibited the formation of ommatidia and bristles in the posterior half of the eyes and WT crp caused overgrowth of groups of ommatidia. The white arrowheads indicate groups of protruding ommatidia in (ii and iii) and the yellow arrowheads points to the posterior end of the eye without any ommatidia. Scanning micrographs at higher magnification of the ommatidia from WT (v), WT crp- (vi), and DN-crp-expressing fly (vii) are shown. (vii) Electron micrograph taken from the dorsal side of the eye of DN-crp expressing fly and the anterior is to the left. Scale bars are 100 μm (A, B, and C), 200 μm (Di-iv), 20 μm (Dv,vi), and 50 μm (Dvii).

Mentions: During the examination of tracheal branching phenotype of overexpressing DN-crp in the tracheal system with the btl-Gal4 driver, we found that 62% (n = 34) of the L2 and L3 larvae displayed a very severe phenotype in which the dorsal trunks of the larvae were collapsed (Figure 5Ai-iv). With the aid of the fluorescence signal from the nuclear GFP expressed in the whole tracheal system, there were just 3 to 4 cells along DB and the last cells could still attach to the somatic muscles (white arrow heads in Figure 5Ai, ii), but no terminal cells and fusion cells were observed. This indicates that DN-crp expressed in the tracheal system may have adversely affected the normal development of DB.Figure 5


Cropped, Drosophila transcription factor AP-4, controls tracheal terminal branching and cell growth.

Wong MM, Liu MF, Chiu SK - BMC Dev. Biol. (2015)

Expression of dominant-negative Cropped leads to missing cells and disruption of organ development. (A) Overexpressing DN-crp by using btl-Gal4 in the tracheal system led to disruption of the formation of terminal and fusion cells (i and ii); the arrow heads point to the ends of DBs. (iii and iv) Larvae show a collapse in dorsal trunk. (i and iii) are fluorescence images of GFP-labeled tracheal cells and (ii and iv) are the same field under DIC optics. (B) Expression of DN-crp in the salivary glands driven by A9-Gal4 shrank the organs (ii) compared with control (i) under the same magnification. (C) Staining assay on the cells expressing crp and DN-crp in the salivary glands for apoptosis with changes in permeability by PI (i-iv) and breakage of DNA strands by the TUNEL assay (v-viii). DIC photomicrographs (i, iii, v, vii) and fluorescence microscopy (ii, iv, vi, viii) were taken under the same magnification. (D) Scanning electron micrographs of the ommatidia expressing DN-crp or WT crp driven by GMR-Gal4. (i-iv) The anterior of the fly is pointing to the left and the posterior to the right. DN-crp inhibited the formation of ommatidia and bristles in the posterior half of the eyes and WT crp caused overgrowth of groups of ommatidia. The white arrowheads indicate groups of protruding ommatidia in (ii and iii) and the yellow arrowheads points to the posterior end of the eye without any ommatidia. Scanning micrographs at higher magnification of the ommatidia from WT (v), WT crp- (vi), and DN-crp-expressing fly (vii) are shown. (vii) Electron micrograph taken from the dorsal side of the eye of DN-crp expressing fly and the anterior is to the left. Scale bars are 100 μm (A, B, and C), 200 μm (Di-iv), 20 μm (Dv,vi), and 50 μm (Dvii).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4430030&req=5

Fig5: Expression of dominant-negative Cropped leads to missing cells and disruption of organ development. (A) Overexpressing DN-crp by using btl-Gal4 in the tracheal system led to disruption of the formation of terminal and fusion cells (i and ii); the arrow heads point to the ends of DBs. (iii and iv) Larvae show a collapse in dorsal trunk. (i and iii) are fluorescence images of GFP-labeled tracheal cells and (ii and iv) are the same field under DIC optics. (B) Expression of DN-crp in the salivary glands driven by A9-Gal4 shrank the organs (ii) compared with control (i) under the same magnification. (C) Staining assay on the cells expressing crp and DN-crp in the salivary glands for apoptosis with changes in permeability by PI (i-iv) and breakage of DNA strands by the TUNEL assay (v-viii). DIC photomicrographs (i, iii, v, vii) and fluorescence microscopy (ii, iv, vi, viii) were taken under the same magnification. (D) Scanning electron micrographs of the ommatidia expressing DN-crp or WT crp driven by GMR-Gal4. (i-iv) The anterior of the fly is pointing to the left and the posterior to the right. DN-crp inhibited the formation of ommatidia and bristles in the posterior half of the eyes and WT crp caused overgrowth of groups of ommatidia. The white arrowheads indicate groups of protruding ommatidia in (ii and iii) and the yellow arrowheads points to the posterior end of the eye without any ommatidia. Scanning micrographs at higher magnification of the ommatidia from WT (v), WT crp- (vi), and DN-crp-expressing fly (vii) are shown. (vii) Electron micrograph taken from the dorsal side of the eye of DN-crp expressing fly and the anterior is to the left. Scale bars are 100 μm (A, B, and C), 200 μm (Di-iv), 20 μm (Dv,vi), and 50 μm (Dvii).
Mentions: During the examination of tracheal branching phenotype of overexpressing DN-crp in the tracheal system with the btl-Gal4 driver, we found that 62% (n = 34) of the L2 and L3 larvae displayed a very severe phenotype in which the dorsal trunks of the larvae were collapsed (Figure 5Ai-iv). With the aid of the fluorescence signal from the nuclear GFP expressed in the whole tracheal system, there were just 3 to 4 cells along DB and the last cells could still attach to the somatic muscles (white arrow heads in Figure 5Ai, ii), but no terminal cells and fusion cells were observed. This indicates that DN-crp expressed in the tracheal system may have adversely affected the normal development of DB.Figure 5

Bottom Line: Overexpressing the wild-type crp gene or a mutant that lacks the DNA-binding region in either the tracheal tissues or terminal cells led to a loss-of-function phenotype, implying that crp can affect terminal branching.Unexpectedly, the ectopic expression of cropped also led to enlarged organs, and cell-counting experiments on the salivary glands suggest that elevated levels of AP-4 increase cell size and organ size.We find that the branching morphogenesis of terminal cells of the tracheal tubes in Drosophila requires the dMyc-dependent activation of Cropped/AP-4 protein to increase the cell growth of terminal cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong, Hong Kong. kaychiu@cityu.edu.hk.

ABSTRACT

Background: Endothelial or epithelial cellular branching is vital in development and cancer progression; however, the molecular mechanisms of these processes are not clear. In Drosophila, terminal cell at the end of some tracheal tube ramifies numerous fine branches on the internal organs to supply oxygen. To discover more genes involved in terminal branching, we searched for mutants with very few terminal branches using the Kiss enhancer-trap line collection.

Results: In this analysis, we identified cropped (crp), encoding the Drosophila homolog of the transcription activator protein AP-4. Overexpressing the wild-type crp gene or a mutant that lacks the DNA-binding region in either the tracheal tissues or terminal cells led to a loss-of-function phenotype, implying that crp can affect terminal branching. Unexpectedly, the ectopic expression of cropped also led to enlarged organs, and cell-counting experiments on the salivary glands suggest that elevated levels of AP-4 increase cell size and organ size. Like its mammalian counterpart, cropped is controlled by dMyc, as ectopic expression of dMyc in terminal cells increased cellular branching and the Cropped protein levels in vivo.

Conclusions: We find that the branching morphogenesis of terminal cells of the tracheal tubes in Drosophila requires the dMyc-dependent activation of Cropped/AP-4 protein to increase the cell growth of terminal cells.

No MeSH data available.


Related in: MedlinePlus