Limits...
The Drosophila melanogaster Mutants apblot and apXasta Affect an Essential apterous Wing Enhancer.

Bieli D, Kanca O, Gohl D, Denes A, Schedl P, Affolter M, Müller M - G3 (Bethesda) (2015)

Bottom Line: The role of Ap and its downstream effectors have been studied extensively.Using this system, we were able to demonstrate that the essential wing enhancer alone is not sufficient for normal wing development.The in situ rescue system will allow us to characterize the ap regulatory sequences in great detail at the endogenous locus.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, 4056 Basel, Switzerland.

No MeSH data available.


Related in: MedlinePlus

LacZ reporter assay and deletion analysis at the apterous locus. (A) Diagrammatic representation of the ap locus. As drawn at the top of the panel, it extends over roughly 50 kb. Its transcribed part is shown in green. ap is flanked by two genes indicated in blue: vulcan on the proximal and l(2)09851 on its distal side. Arrows above the genomic interval specify the direction of transcription of the three genes. Fragment apC, indicated in orange, has been reported to drive reporter expression in the dorsal compartment of the pouch, the hinge and the notum of the wing imaginal disc, where ap is normally expressed. Below, the relative positions and dimensions of nine fragments tested with our LacZ reporter assay are depicted. Fragments colored in orange (apO, apR, apOR, apOR3, and apRXa) elicit the same expression pattern as apC. Fragments depicted in gray (apP, apQ, apS, apOR2) do not drive reporter gene expression in the wing disc. (B) X-Gal staining in the wing disc of an apC-LacZ transgenic fly. Scale bar: 100 µm. (C) Deletions generated at the endogenous ap locus with FRT-containing inserts. At the top of the panel, triangles along the ap locus indicate the position of six different inserts. Pink arrowheads within them mark the orientation of the FRT sites according to the definition of Thibault et al. (2004). The location of the apRXa fragment is shown in orange. apDG3 deletes approximately 44 kb between inserts apf08090 to ape01573, thereby removing most of ap ORF and upstream sequences. apDG8 is a 20-kb deficiency that deletes the complete ap ORF from apf00878 to apD5f.1. apDG1 removes the complete intergenic spacer between apMM to ape01573. apDG11 deletes an 11-kb fragment from apMM to apDD35.34. Note that apD5f.1 and apMM have exactly the same insertion site. (D) Notum pictures of a wild-type fly and trans-heterozygous ap mutants. In the wild type, the wing and the haltere (arrowhead) are well formed and clearly visible. Df(2R)BSC696 is a large deletion at the base of 2R, deleting approximately 360 kb, including the whole ap locus. When Df(2R)BSC696 is crossed to apDG3 all wing and haltere structures are lost. Only small stumps of amorphic tissue remain at the actual attachment site of the wing (see arrow).Very similar phenotypes are observed in apDG8/apDG3, apDG1/apDG3 and apDG11/apDG3 flies. Scale bar: 25 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4478543&req=5

fig1: LacZ reporter assay and deletion analysis at the apterous locus. (A) Diagrammatic representation of the ap locus. As drawn at the top of the panel, it extends over roughly 50 kb. Its transcribed part is shown in green. ap is flanked by two genes indicated in blue: vulcan on the proximal and l(2)09851 on its distal side. Arrows above the genomic interval specify the direction of transcription of the three genes. Fragment apC, indicated in orange, has been reported to drive reporter expression in the dorsal compartment of the pouch, the hinge and the notum of the wing imaginal disc, where ap is normally expressed. Below, the relative positions and dimensions of nine fragments tested with our LacZ reporter assay are depicted. Fragments colored in orange (apO, apR, apOR, apOR3, and apRXa) elicit the same expression pattern as apC. Fragments depicted in gray (apP, apQ, apS, apOR2) do not drive reporter gene expression in the wing disc. (B) X-Gal staining in the wing disc of an apC-LacZ transgenic fly. Scale bar: 100 µm. (C) Deletions generated at the endogenous ap locus with FRT-containing inserts. At the top of the panel, triangles along the ap locus indicate the position of six different inserts. Pink arrowheads within them mark the orientation of the FRT sites according to the definition of Thibault et al. (2004). The location of the apRXa fragment is shown in orange. apDG3 deletes approximately 44 kb between inserts apf08090 to ape01573, thereby removing most of ap ORF and upstream sequences. apDG8 is a 20-kb deficiency that deletes the complete ap ORF from apf00878 to apD5f.1. apDG1 removes the complete intergenic spacer between apMM to ape01573. apDG11 deletes an 11-kb fragment from apMM to apDD35.34. Note that apD5f.1 and apMM have exactly the same insertion site. (D) Notum pictures of a wild-type fly and trans-heterozygous ap mutants. In the wild type, the wing and the haltere (arrowhead) are well formed and clearly visible. Df(2R)BSC696 is a large deletion at the base of 2R, deleting approximately 360 kb, including the whole ap locus. When Df(2R)BSC696 is crossed to apDG3 all wing and haltere structures are lost. Only small stumps of amorphic tissue remain at the actual attachment site of the wing (see arrow).Very similar phenotypes are observed in apDG8/apDG3, apDG1/apDG3 and apDG11/apDG3 flies. Scale bar: 25 µm.

Mentions: Constructs were introduced into the ap locus by RMCE into two docking sites, Mi{y[+mDint2]=MIC}MI02330 (Venken et al. 2011) and apc1.4b. DNA was injected at a concentration of 300 ng/µL in 1× phosphate-buffered saline (PBS) into early embryos of the genotype y w M{vas-int.Dm}zh-2A; MI02330/CyO or y w M{vas-int.Dm}zh-2A; apc1.4b/CyO. The relevant transgenic lines obtained in this way are apDD35.34 and apD5f.1, respectively. Their position and the orientation of the FRT are depicted in Figure 1C together with four other FRT containing transposon insertions. Five of the six stocks are homozygous and hemizygous viable. Their wings and halteres are of wild-type appearance. This is not the case for apf08090. The lethality of this chromosome cannot be reverted by excision of the PBac{WH}, indicating that it is associated with a second site lethal. Rare homozygous revertant escapers as well as frequent hemizygous revertants have normal wings. Therefore, the PBac{WH} insert is responsible for the strong phenotype in hemizygous apf08090 flies. However, this phenotype is not dependent on the gypsy insulator present in apf08090 because the wing phenotype is not suppressed in a su(Hw)− background (M. Müller, unpublished data).


The Drosophila melanogaster Mutants apblot and apXasta Affect an Essential apterous Wing Enhancer.

Bieli D, Kanca O, Gohl D, Denes A, Schedl P, Affolter M, Müller M - G3 (Bethesda) (2015)

LacZ reporter assay and deletion analysis at the apterous locus. (A) Diagrammatic representation of the ap locus. As drawn at the top of the panel, it extends over roughly 50 kb. Its transcribed part is shown in green. ap is flanked by two genes indicated in blue: vulcan on the proximal and l(2)09851 on its distal side. Arrows above the genomic interval specify the direction of transcription of the three genes. Fragment apC, indicated in orange, has been reported to drive reporter expression in the dorsal compartment of the pouch, the hinge and the notum of the wing imaginal disc, where ap is normally expressed. Below, the relative positions and dimensions of nine fragments tested with our LacZ reporter assay are depicted. Fragments colored in orange (apO, apR, apOR, apOR3, and apRXa) elicit the same expression pattern as apC. Fragments depicted in gray (apP, apQ, apS, apOR2) do not drive reporter gene expression in the wing disc. (B) X-Gal staining in the wing disc of an apC-LacZ transgenic fly. Scale bar: 100 µm. (C) Deletions generated at the endogenous ap locus with FRT-containing inserts. At the top of the panel, triangles along the ap locus indicate the position of six different inserts. Pink arrowheads within them mark the orientation of the FRT sites according to the definition of Thibault et al. (2004). The location of the apRXa fragment is shown in orange. apDG3 deletes approximately 44 kb between inserts apf08090 to ape01573, thereby removing most of ap ORF and upstream sequences. apDG8 is a 20-kb deficiency that deletes the complete ap ORF from apf00878 to apD5f.1. apDG1 removes the complete intergenic spacer between apMM to ape01573. apDG11 deletes an 11-kb fragment from apMM to apDD35.34. Note that apD5f.1 and apMM have exactly the same insertion site. (D) Notum pictures of a wild-type fly and trans-heterozygous ap mutants. In the wild type, the wing and the haltere (arrowhead) are well formed and clearly visible. Df(2R)BSC696 is a large deletion at the base of 2R, deleting approximately 360 kb, including the whole ap locus. When Df(2R)BSC696 is crossed to apDG3 all wing and haltere structures are lost. Only small stumps of amorphic tissue remain at the actual attachment site of the wing (see arrow).Very similar phenotypes are observed in apDG8/apDG3, apDG1/apDG3 and apDG11/apDG3 flies. Scale bar: 25 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4478543&req=5

fig1: LacZ reporter assay and deletion analysis at the apterous locus. (A) Diagrammatic representation of the ap locus. As drawn at the top of the panel, it extends over roughly 50 kb. Its transcribed part is shown in green. ap is flanked by two genes indicated in blue: vulcan on the proximal and l(2)09851 on its distal side. Arrows above the genomic interval specify the direction of transcription of the three genes. Fragment apC, indicated in orange, has been reported to drive reporter expression in the dorsal compartment of the pouch, the hinge and the notum of the wing imaginal disc, where ap is normally expressed. Below, the relative positions and dimensions of nine fragments tested with our LacZ reporter assay are depicted. Fragments colored in orange (apO, apR, apOR, apOR3, and apRXa) elicit the same expression pattern as apC. Fragments depicted in gray (apP, apQ, apS, apOR2) do not drive reporter gene expression in the wing disc. (B) X-Gal staining in the wing disc of an apC-LacZ transgenic fly. Scale bar: 100 µm. (C) Deletions generated at the endogenous ap locus with FRT-containing inserts. At the top of the panel, triangles along the ap locus indicate the position of six different inserts. Pink arrowheads within them mark the orientation of the FRT sites according to the definition of Thibault et al. (2004). The location of the apRXa fragment is shown in orange. apDG3 deletes approximately 44 kb between inserts apf08090 to ape01573, thereby removing most of ap ORF and upstream sequences. apDG8 is a 20-kb deficiency that deletes the complete ap ORF from apf00878 to apD5f.1. apDG1 removes the complete intergenic spacer between apMM to ape01573. apDG11 deletes an 11-kb fragment from apMM to apDD35.34. Note that apD5f.1 and apMM have exactly the same insertion site. (D) Notum pictures of a wild-type fly and trans-heterozygous ap mutants. In the wild type, the wing and the haltere (arrowhead) are well formed and clearly visible. Df(2R)BSC696 is a large deletion at the base of 2R, deleting approximately 360 kb, including the whole ap locus. When Df(2R)BSC696 is crossed to apDG3 all wing and haltere structures are lost. Only small stumps of amorphic tissue remain at the actual attachment site of the wing (see arrow).Very similar phenotypes are observed in apDG8/apDG3, apDG1/apDG3 and apDG11/apDG3 flies. Scale bar: 25 µm.
Mentions: Constructs were introduced into the ap locus by RMCE into two docking sites, Mi{y[+mDint2]=MIC}MI02330 (Venken et al. 2011) and apc1.4b. DNA was injected at a concentration of 300 ng/µL in 1× phosphate-buffered saline (PBS) into early embryos of the genotype y w M{vas-int.Dm}zh-2A; MI02330/CyO or y w M{vas-int.Dm}zh-2A; apc1.4b/CyO. The relevant transgenic lines obtained in this way are apDD35.34 and apD5f.1, respectively. Their position and the orientation of the FRT are depicted in Figure 1C together with four other FRT containing transposon insertions. Five of the six stocks are homozygous and hemizygous viable. Their wings and halteres are of wild-type appearance. This is not the case for apf08090. The lethality of this chromosome cannot be reverted by excision of the PBac{WH}, indicating that it is associated with a second site lethal. Rare homozygous revertant escapers as well as frequent hemizygous revertants have normal wings. Therefore, the PBac{WH} insert is responsible for the strong phenotype in hemizygous apf08090 flies. However, this phenotype is not dependent on the gypsy insulator present in apf08090 because the wing phenotype is not suppressed in a su(Hw)− background (M. Müller, unpublished data).

Bottom Line: The role of Ap and its downstream effectors have been studied extensively.Using this system, we were able to demonstrate that the essential wing enhancer alone is not sufficient for normal wing development.The in situ rescue system will allow us to characterize the ap regulatory sequences in great detail at the endogenous locus.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, 4056 Basel, Switzerland.

No MeSH data available.


Related in: MedlinePlus