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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

The tracheal phenotypes of the cropped mutant compared with the blistered/pruned mutant. (A) A pair of dorsal branches (DB, arrowheads) in a wild-type third-instar larva. The air-filled terminal branches ramify extensively on the dorsal muscles. (B) The same view of a blisteredex84 homozygote. Note the absence of terminal branches beyond the arrowheads where the nuclei of terminal cells are. (C) A crpk10415 homozygote. Note the absence of terminal branches beyond the arrowheads, as in the blistered mutant. (D) A pair of lateral trunk LG branches (arrowheads) in a wild-type third-instar larva. (E) The same view of a blisteredex84 homozygote. Note the absence of terminal branches beyond the arrowheads. (F) A crpk10415; btl-Gal4/UAS-GFP larva. (G) A fluorescence image of (F) showing the GFP-labeled tracheal cell cytoplasmic extensions. (H) A dorsal branch of a stage 16/17 wild-type embryo stained with mAb 2A12 to show the tracheal lumen. The arrowhead indicates the position of the terminal nucleus. The dashed line shows the continuation of the base of the dorsal branch out of the plane of focus. (I) The same view of a blisteredex84 mutant. (J) A crpk10415 mutant. The scale bars for A-D = 30 μm, for E-J = 10 μm.
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Fig1: The tracheal phenotypes of the cropped mutant compared with the blistered/pruned mutant. (A) A pair of dorsal branches (DB, arrowheads) in a wild-type third-instar larva. The air-filled terminal branches ramify extensively on the dorsal muscles. (B) The same view of a blisteredex84 homozygote. Note the absence of terminal branches beyond the arrowheads where the nuclei of terminal cells are. (C) A crpk10415 homozygote. Note the absence of terminal branches beyond the arrowheads, as in the blistered mutant. (D) A pair of lateral trunk LG branches (arrowheads) in a wild-type third-instar larva. (E) The same view of a blisteredex84 homozygote. Note the absence of terminal branches beyond the arrowheads. (F) A crpk10415; btl-Gal4/UAS-GFP larva. (G) A fluorescence image of (F) showing the GFP-labeled tracheal cell cytoplasmic extensions. (H) A dorsal branch of a stage 16/17 wild-type embryo stained with mAb 2A12 to show the tracheal lumen. The arrowhead indicates the position of the terminal nucleus. The dashed line shows the continuation of the base of the dorsal branch out of the plane of focus. (I) The same view of a blisteredex84 mutant. (J) A crpk10415 mutant. The scale bars for A-D = 30 μm, for E-J = 10 μm.

Mentions: In a genetic screen for mutations that affect the outgrowth of tracheal terminal branches, we identified three independent P[lacZ] enhancer trap mutations that caused a substantial reduction in the number of terminal branches in larvae. One of these mutants, (l(2)k10415), which we call cropped (crp), is described in this paper. In wild-type animals, the first terminal branches sprout at the end of embryogenesis, and, these branches ramify into extensive networks of fine branches throughout larval life (Figure 1A). In homozygous crpk10415 mutants, the early stages of tracheal development were normal, but most of the terminal branches seldom extend beyond the cell body (Figure 1C), similar to what is observed in blistered/pruned mutants (Figure 1B). The counting of dorsal branches included only from segments 3 to 9 because they were clearly visible under the microscope. In 54% of the dorsal branches (total DBs counted; n = 229), only one terminal branch remained, and the ones that did develop (14%, DBs; n = 229) were much shorter than normal; the remainder of the DBs exhibited a less severe and variable reduction in branching. The visceral, ganglionic, and lateral trunk terminal branches were affected similarly; 67% of the lateral G (LG) branch split into two branches but with few finer terminal branches (Figure 1F and G). A tracheal cytoplasmic GFP marker showed that terminal cells in the crpk10415 mutant extended fewer (N = 2.3 ± 0.68) cytoplasmic processes than normal (N = 15.7 ± 2.7) (Figure 1G) and that many of the processes that formed did not develop an air-filled lumen (Figure 1F vs. Figure 1D). The larval terminal branching defect of crpk10415 resembles that of blistered mutants phenotypically (Figure 1B and E), and like blistered mutants, the crpk10415 mutant also affected the outgrowth of the first terminal branches of DB that form at the end of embryogenesis (Figure 1I and J), suggesting that cropped is necessary for the embryonic and larval tracheal terminal branching. Tracheal terminal branches appear to be the only tissue affected, as the other tissues, including the muscles, gut, central nervous system, salivary glands, and imaginal discs, were grossly intact in the mutants.Figure 1


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

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

The tracheal phenotypes of the cropped mutant compared with the blistered/pruned mutant. (A) A pair of dorsal branches (DB, arrowheads) in a wild-type third-instar larva. The air-filled terminal branches ramify extensively on the dorsal muscles. (B) The same view of a blisteredex84 homozygote. Note the absence of terminal branches beyond the arrowheads where the nuclei of terminal cells are. (C) A crpk10415 homozygote. Note the absence of terminal branches beyond the arrowheads, as in the blistered mutant. (D) A pair of lateral trunk LG branches (arrowheads) in a wild-type third-instar larva. (E) The same view of a blisteredex84 homozygote. Note the absence of terminal branches beyond the arrowheads. (F) A crpk10415; btl-Gal4/UAS-GFP larva. (G) A fluorescence image of (F) showing the GFP-labeled tracheal cell cytoplasmic extensions. (H) A dorsal branch of a stage 16/17 wild-type embryo stained with mAb 2A12 to show the tracheal lumen. The arrowhead indicates the position of the terminal nucleus. The dashed line shows the continuation of the base of the dorsal branch out of the plane of focus. (I) The same view of a blisteredex84 mutant. (J) A crpk10415 mutant. The scale bars for A-D = 30 μm, for E-J = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: The tracheal phenotypes of the cropped mutant compared with the blistered/pruned mutant. (A) A pair of dorsal branches (DB, arrowheads) in a wild-type third-instar larva. The air-filled terminal branches ramify extensively on the dorsal muscles. (B) The same view of a blisteredex84 homozygote. Note the absence of terminal branches beyond the arrowheads where the nuclei of terminal cells are. (C) A crpk10415 homozygote. Note the absence of terminal branches beyond the arrowheads, as in the blistered mutant. (D) A pair of lateral trunk LG branches (arrowheads) in a wild-type third-instar larva. (E) The same view of a blisteredex84 homozygote. Note the absence of terminal branches beyond the arrowheads. (F) A crpk10415; btl-Gal4/UAS-GFP larva. (G) A fluorescence image of (F) showing the GFP-labeled tracheal cell cytoplasmic extensions. (H) A dorsal branch of a stage 16/17 wild-type embryo stained with mAb 2A12 to show the tracheal lumen. The arrowhead indicates the position of the terminal nucleus. The dashed line shows the continuation of the base of the dorsal branch out of the plane of focus. (I) The same view of a blisteredex84 mutant. (J) A crpk10415 mutant. The scale bars for A-D = 30 μm, for E-J = 10 μm.
Mentions: In a genetic screen for mutations that affect the outgrowth of tracheal terminal branches, we identified three independent P[lacZ] enhancer trap mutations that caused a substantial reduction in the number of terminal branches in larvae. One of these mutants, (l(2)k10415), which we call cropped (crp), is described in this paper. In wild-type animals, the first terminal branches sprout at the end of embryogenesis, and, these branches ramify into extensive networks of fine branches throughout larval life (Figure 1A). In homozygous crpk10415 mutants, the early stages of tracheal development were normal, but most of the terminal branches seldom extend beyond the cell body (Figure 1C), similar to what is observed in blistered/pruned mutants (Figure 1B). The counting of dorsal branches included only from segments 3 to 9 because they were clearly visible under the microscope. In 54% of the dorsal branches (total DBs counted; n = 229), only one terminal branch remained, and the ones that did develop (14%, DBs; n = 229) were much shorter than normal; the remainder of the DBs exhibited a less severe and variable reduction in branching. The visceral, ganglionic, and lateral trunk terminal branches were affected similarly; 67% of the lateral G (LG) branch split into two branches but with few finer terminal branches (Figure 1F and G). A tracheal cytoplasmic GFP marker showed that terminal cells in the crpk10415 mutant extended fewer (N = 2.3 ± 0.68) cytoplasmic processes than normal (N = 15.7 ± 2.7) (Figure 1G) and that many of the processes that formed did not develop an air-filled lumen (Figure 1F vs. Figure 1D). The larval terminal branching defect of crpk10415 resembles that of blistered mutants phenotypically (Figure 1B and E), and like blistered mutants, the crpk10415 mutant also affected the outgrowth of the first terminal branches of DB that form at the end of embryogenesis (Figure 1I and J), suggesting that cropped is necessary for the embryonic and larval tracheal terminal branching. Tracheal terminal branches appear to be the only tissue affected, as the other tissues, including the muscles, gut, central nervous system, salivary glands, and imaginal discs, were grossly intact in the mutants.Figure 1

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