Limits...
The bHLH-PAS transcription factor dysfusion regulates tarsal joint formation in response to Notch activity during drosophila leg development.

Córdoba S, Estella C - PLoS Genet. (2014)

Bottom Line: This novel Dys function depends on its obligated partner Tango to activate the transcription of target genes.We also identified a dedicated dys cis-regulatory module that regulates dys expression in the tarsal presumptive leg joints through direct Su(H) binding.All these data place dys as a key player downstream of Notch, directing distal versus proximal joint morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid (UAM), Madrid, Spain.

ABSTRACT
A characteristic of all arthropods is the presence of flexible structures called joints that connect all leg segments. Drosophila legs include two types of joints: the proximal or "true" joints that are motile due to the presence of muscle attachment and the distal joints that lack musculature. These joints are not only morphologically, functionally and evolutionarily different, but also the morphogenetic program that forms them is distinct. Development of both proximal and distal joints requires Notch activity; however, it is still unknown how this pathway can control the development of such homologous although distinct structures. Here we show that the bHLH-PAS transcription factor encoded by the gene dysfusion (dys), is expressed and absolutely required for tarsal joint development while it is dispensable for proximal joints. In the presumptive tarsal joints, Dys regulates the expression of the pro-apoptotic genes reaper and head involution defective and the expression of the RhoGTPases modulators, RhoGEf2 and RhoGap71E, thus directing key morphogenetic events required for tarsal joint development. When ectopically expressed, dys is able to induce some aspects of the morphogenetic program necessary for distal joint development such as fold formation and programmed cell death. This novel Dys function depends on its obligated partner Tango to activate the transcription of target genes. We also identified a dedicated dys cis-regulatory module that regulates dys expression in the tarsal presumptive leg joints through direct Su(H) binding. All these data place dys as a key player downstream of Notch, directing distal versus proximal joint morphogenesis.

No MeSH data available.


Related in: MedlinePlus

dys is a direct target of the Notch pathway.(A) Schematic representation of dys cis-regulatory region. Horizontal bars represent the DNA elements available in the Janelia database that maps around and within the dys gene. Blue bars correspond to the dys-CRMs identified by Jiang et al., 2010 [40] that drove reporter gene expression in fusion tracheal cells. Two Janelia lines (GMR_13D07 and GMR_13B03, green bars) drove reporter gene expression in the tarsal segments of the leg. The 640 bp overlapping sequence (dys640, red bar), DNA conservation between other Drosophilds and the two overlapping fragments (dys640A and dys640B) are also represented. (B–D) Prepupal leg discs stained for Dys (green) and for (B) dys640-Z, (C) dys640A-Z and (D) dys640B-Z. Note the perfect co-localization between dys640-Z and Dys (B). Single lacZ channels are displayed below. (E) DNA sequence of various Drosophilid species surrounding the two identified Su(H) binding sites (red shade) is shown. Asterisks mark perfect DNA conservation between species. Observe that both Su(H) sites are conserved (F) EMSA to assess binding of Su(H) to probes containing wild-type (WT) or mutated (mut) binding sites (see Material and Methods for sequences). Arrows indicate protein-DNA complexes, while asterisk indicate a non-specific band present in both wild type and mutant probe. (G–J) Prepupal leg discs stained for Dys (green) and for (G) dys640-Z, (H) dys640Su(H)-1-Z, (I) dys640Su(H)-2-Z and (J) dys640Su(H)-1+2-Z. All constructs have been inserted in the same genomic location and images were obtained keeping the confocal settings constant in the merge image. Single channels are displayed below, and for dys640Su(H)-1+2–Z the gain has been increase for visualization purposes.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4199481&req=5

pgen-1004621-g007: dys is a direct target of the Notch pathway.(A) Schematic representation of dys cis-regulatory region. Horizontal bars represent the DNA elements available in the Janelia database that maps around and within the dys gene. Blue bars correspond to the dys-CRMs identified by Jiang et al., 2010 [40] that drove reporter gene expression in fusion tracheal cells. Two Janelia lines (GMR_13D07 and GMR_13B03, green bars) drove reporter gene expression in the tarsal segments of the leg. The 640 bp overlapping sequence (dys640, red bar), DNA conservation between other Drosophilds and the two overlapping fragments (dys640A and dys640B) are also represented. (B–D) Prepupal leg discs stained for Dys (green) and for (B) dys640-Z, (C) dys640A-Z and (D) dys640B-Z. Note the perfect co-localization between dys640-Z and Dys (B). Single lacZ channels are displayed below. (E) DNA sequence of various Drosophilid species surrounding the two identified Su(H) binding sites (red shade) is shown. Asterisks mark perfect DNA conservation between species. Observe that both Su(H) sites are conserved (F) EMSA to assess binding of Su(H) to probes containing wild-type (WT) or mutated (mut) binding sites (see Material and Methods for sequences). Arrows indicate protein-DNA complexes, while asterisk indicate a non-specific band present in both wild type and mutant probe. (G–J) Prepupal leg discs stained for Dys (green) and for (G) dys640-Z, (H) dys640Su(H)-1-Z, (I) dys640Su(H)-2-Z and (J) dys640Su(H)-1+2-Z. All constructs have been inserted in the same genomic location and images were obtained keeping the confocal settings constant in the merge image. Single channels are displayed below, and for dys640Su(H)-1+2–Z the gain has been increase for visualization purposes.

Mentions: We have screened 11 DNA fragments derived from the Janelia Gal4 data base that cover the 5′ region and the introns of the dys genomic locus for expression in the leg imaginal disc [33]. Only two overlapping sequences, located between exons 2 and 3, GMR_13D07 and GMR_13B03, drive expression of a GFP reporter in a ring-like pattern that resembles dys expression in the leg (Figure 1A and S1). The overlapping sequence (640 bp long), cloned in a nuclear lacZ reporter vector (see Material and Methods), contains the information necessary to reproduce dys expression pattern in the tarsus (Figure 7B). We have previously shown that Notch acts upstream of dys and, in agreement with our genetic results, dys640-lacZ expression is disrupted in Notch knockdown prepupal leg discs (Figure S4A). Moreover, ectopic expression of NotchICD activates dys640-lacZ expression, although this activation is restricted to the tarsus, just as described for dys endogenous expression (Figure S5B).


The bHLH-PAS transcription factor dysfusion regulates tarsal joint formation in response to Notch activity during drosophila leg development.

Córdoba S, Estella C - PLoS Genet. (2014)

dys is a direct target of the Notch pathway.(A) Schematic representation of dys cis-regulatory region. Horizontal bars represent the DNA elements available in the Janelia database that maps around and within the dys gene. Blue bars correspond to the dys-CRMs identified by Jiang et al., 2010 [40] that drove reporter gene expression in fusion tracheal cells. Two Janelia lines (GMR_13D07 and GMR_13B03, green bars) drove reporter gene expression in the tarsal segments of the leg. The 640 bp overlapping sequence (dys640, red bar), DNA conservation between other Drosophilds and the two overlapping fragments (dys640A and dys640B) are also represented. (B–D) Prepupal leg discs stained for Dys (green) and for (B) dys640-Z, (C) dys640A-Z and (D) dys640B-Z. Note the perfect co-localization between dys640-Z and Dys (B). Single lacZ channels are displayed below. (E) DNA sequence of various Drosophilid species surrounding the two identified Su(H) binding sites (red shade) is shown. Asterisks mark perfect DNA conservation between species. Observe that both Su(H) sites are conserved (F) EMSA to assess binding of Su(H) to probes containing wild-type (WT) or mutated (mut) binding sites (see Material and Methods for sequences). Arrows indicate protein-DNA complexes, while asterisk indicate a non-specific band present in both wild type and mutant probe. (G–J) Prepupal leg discs stained for Dys (green) and for (G) dys640-Z, (H) dys640Su(H)-1-Z, (I) dys640Su(H)-2-Z and (J) dys640Su(H)-1+2-Z. All constructs have been inserted in the same genomic location and images were obtained keeping the confocal settings constant in the merge image. Single channels are displayed below, and for dys640Su(H)-1+2–Z the gain has been increase for visualization purposes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199481&req=5

pgen-1004621-g007: dys is a direct target of the Notch pathway.(A) Schematic representation of dys cis-regulatory region. Horizontal bars represent the DNA elements available in the Janelia database that maps around and within the dys gene. Blue bars correspond to the dys-CRMs identified by Jiang et al., 2010 [40] that drove reporter gene expression in fusion tracheal cells. Two Janelia lines (GMR_13D07 and GMR_13B03, green bars) drove reporter gene expression in the tarsal segments of the leg. The 640 bp overlapping sequence (dys640, red bar), DNA conservation between other Drosophilds and the two overlapping fragments (dys640A and dys640B) are also represented. (B–D) Prepupal leg discs stained for Dys (green) and for (B) dys640-Z, (C) dys640A-Z and (D) dys640B-Z. Note the perfect co-localization between dys640-Z and Dys (B). Single lacZ channels are displayed below. (E) DNA sequence of various Drosophilid species surrounding the two identified Su(H) binding sites (red shade) is shown. Asterisks mark perfect DNA conservation between species. Observe that both Su(H) sites are conserved (F) EMSA to assess binding of Su(H) to probes containing wild-type (WT) or mutated (mut) binding sites (see Material and Methods for sequences). Arrows indicate protein-DNA complexes, while asterisk indicate a non-specific band present in both wild type and mutant probe. (G–J) Prepupal leg discs stained for Dys (green) and for (G) dys640-Z, (H) dys640Su(H)-1-Z, (I) dys640Su(H)-2-Z and (J) dys640Su(H)-1+2-Z. All constructs have been inserted in the same genomic location and images were obtained keeping the confocal settings constant in the merge image. Single channels are displayed below, and for dys640Su(H)-1+2–Z the gain has been increase for visualization purposes.
Mentions: We have screened 11 DNA fragments derived from the Janelia Gal4 data base that cover the 5′ region and the introns of the dys genomic locus for expression in the leg imaginal disc [33]. Only two overlapping sequences, located between exons 2 and 3, GMR_13D07 and GMR_13B03, drive expression of a GFP reporter in a ring-like pattern that resembles dys expression in the leg (Figure 1A and S1). The overlapping sequence (640 bp long), cloned in a nuclear lacZ reporter vector (see Material and Methods), contains the information necessary to reproduce dys expression pattern in the tarsus (Figure 7B). We have previously shown that Notch acts upstream of dys and, in agreement with our genetic results, dys640-lacZ expression is disrupted in Notch knockdown prepupal leg discs (Figure S4A). Moreover, ectopic expression of NotchICD activates dys640-lacZ expression, although this activation is restricted to the tarsus, just as described for dys endogenous expression (Figure S5B).

Bottom Line: This novel Dys function depends on its obligated partner Tango to activate the transcription of target genes.We also identified a dedicated dys cis-regulatory module that regulates dys expression in the tarsal presumptive leg joints through direct Su(H) binding.All these data place dys as a key player downstream of Notch, directing distal versus proximal joint morphogenesis.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid (UAM), Madrid, Spain.

ABSTRACT
A characteristic of all arthropods is the presence of flexible structures called joints that connect all leg segments. Drosophila legs include two types of joints: the proximal or "true" joints that are motile due to the presence of muscle attachment and the distal joints that lack musculature. These joints are not only morphologically, functionally and evolutionarily different, but also the morphogenetic program that forms them is distinct. Development of both proximal and distal joints requires Notch activity; however, it is still unknown how this pathway can control the development of such homologous although distinct structures. Here we show that the bHLH-PAS transcription factor encoded by the gene dysfusion (dys), is expressed and absolutely required for tarsal joint development while it is dispensable for proximal joints. In the presumptive tarsal joints, Dys regulates the expression of the pro-apoptotic genes reaper and head involution defective and the expression of the RhoGTPases modulators, RhoGEf2 and RhoGap71E, thus directing key morphogenetic events required for tarsal joint development. When ectopically expressed, dys is able to induce some aspects of the morphogenetic program necessary for distal joint development such as fold formation and programmed cell death. This novel Dys function depends on its obligated partner Tango to activate the transcription of target genes. We also identified a dedicated dys cis-regulatory module that regulates dys expression in the tarsal presumptive leg joints through direct Su(H) binding. All these data place dys as a key player downstream of Notch, directing distal versus proximal joint morphogenesis.

No MeSH data available.


Related in: MedlinePlus