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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 required for fold formation and for the expression of RhoGap71E and RhoGef2.(A and B) Sagittal view of a wild type (A) and dys2/dys3 (B) prepupal leg disc epithelium, stained with Phallodin (Phal) (red) and E(spl)mβ-CD2 (green). The joints between tarsal segments 2/3 and 3/4 are shown in A (arrows), and the corresponding region in B (arrows). Note the apical constriction in cells immediately distal to the E(spl)mβ-CD2 expression domain in the wild type leg and its absence in dys2/dys3 mutant legs. Single channels for Phal (A′ and B′) and E(spl)mβ-CD2 (B′ and B″) are displayed below. (C and D) RhoGap71E-Z (C, red) and RhoGef2-Z (D, red) expression compared to Dys (green). (C′ and D′) Sagittal view of a single joint where the last dys expressing cell is marked by an arrow. Note that both genes, RhoGap71E-Z and RhoGef2-Z, are expressed in a single row of Dys-positive cells and in a row of adjacent Dys-negative cells. (C″ and D″) Single channels for RhoGap71E-Z and RhoGef2-Z. (E) en-Gal4, UAS-GFP; UAS-dys-RNAi (green) and (F) hh-Gal4, UAS-GFP; UAS-dys-RNAi (green) downregulates RhoGap71E-Z and RhoGef2-Z expression (red, arrows), respectively. Dlg is in blue. Single channels for RhoGap71E-Z (E′) and RhoGef2-Z (F′) are displayed below.
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pgen-1004621-g004: dys is required for fold formation and for the expression of RhoGap71E and RhoGef2.(A and B) Sagittal view of a wild type (A) and dys2/dys3 (B) prepupal leg disc epithelium, stained with Phallodin (Phal) (red) and E(spl)mβ-CD2 (green). The joints between tarsal segments 2/3 and 3/4 are shown in A (arrows), and the corresponding region in B (arrows). Note the apical constriction in cells immediately distal to the E(spl)mβ-CD2 expression domain in the wild type leg and its absence in dys2/dys3 mutant legs. Single channels for Phal (A′ and B′) and E(spl)mβ-CD2 (B′ and B″) are displayed below. (C and D) RhoGap71E-Z (C, red) and RhoGef2-Z (D, red) expression compared to Dys (green). (C′ and D′) Sagittal view of a single joint where the last dys expressing cell is marked by an arrow. Note that both genes, RhoGap71E-Z and RhoGef2-Z, are expressed in a single row of Dys-positive cells and in a row of adjacent Dys-negative cells. (C″ and D″) Single channels for RhoGap71E-Z and RhoGef2-Z. (E) en-Gal4, UAS-GFP; UAS-dys-RNAi (green) and (F) hh-Gal4, UAS-GFP; UAS-dys-RNAi (green) downregulates RhoGap71E-Z and RhoGef2-Z expression (red, arrows), respectively. Dlg is in blue. Single channels for RhoGap71E-Z (E′) and RhoGef2-Z (F′) are displayed below.

Mentions: Epithelial cells at the presumptive joints undergo apical constriction and form characteristic folds that prefigure the future joint [22], [23]. The formation of epithelial folds involve cells immediately distal to the E(spl)mβ-CD2 expression domain (Figure 4A). In contrast, in dys mutant prepupal legs these cells distal to the E(spl)mβ-CD2 domain fail to reproduce these shape changes compared to control legs (compare Figure 4B and 4A). Two processes help sculpt the joint structure, namely JNK-mediated apoptosis driven by the pro-apoptotic gene rpr[20] and cell shape changes mediated by the Rho-family of GTPases [27]. The expression of RhoGef2 and RhoGap71E is restricted to the tarsal segments where they are coexpressed with a single row of Dys-positive cells and are extended distally to a row of Dys-negative cells (Figure 4C and 4D). Downregulation of Dys levels with a dys-RNAi in the engrailed (en) or the hedgehog (hh) domains lead to compartment cell autonomous loss of RhoGap71E and RhoGef2 expression, respectively (Figure 4E and 4F). Next, we compared the expression of dys with the pro-apoptotic genes rpr and hid. We found that the expression of these two genes in the prepupal leg discs is restricted to the distal end of each tarsal segments, where they are coexpressed with dys (Figure 5A, S3A) [20], [34]. Interestingly, as previously described for RhoGap71E and RhoGef2, we observed that rpr and hid are also expressed in a row of cells distal to dys expression (Figure 5A and S3A). Next we investigated if the expression of rpr and hid depends on dys. The downregulation of Dys levels in anterior cells along the PD axis with a dpp-Gal4 line or in the posterior compartment with en-Gal4, strongly reduced or eliminated the expression of rpr and hid, respectively (Figure 5B and S3B). Furthermore, forced expression of dys in the posterior compartment for 24 hrs using the tubGal80ts technique is sufficient to promote cell autonomously rpr and hid expression in the inter-joint domain (Figure 5C and S3C). This ectopic activation of the pro-apoptotic genes rpr and hid is accompanied by an increase of cell death in larval and prepupal leg discs, as visualized by the presence of activated Caspase-3 (DCas-3) (Figure 5D and S3D). To test if the downregulation in the expression of the pro-apoptotic genes observed after reducing Dys levels is associated to a decrease in cell death, we compared the number of DCas-3 positive cells in wild type and dys mutant legs (see Material and Methods). To determine the precise location of the apoptotic cells during joint formation, we separately counted DCas-3 positive cells within the E(spl)mβ domain and at the gap between two E(spl)mβ domains (termed here as “fold” domain). In wild type prepupal legs we found a significant increase in the number of DCas-3 positive cells in the “fold” domain compared to the E(spl)mβ one, while in dys mutant legs such significant difference was not observed (Figure 5E–G). Interestingly, while the total number of apoptotic cells per segment was comparable between wild type (average = 13,8) and dys mutant legs (average = 14,8), the distribution of DCas-3-positive cells in the joint was altered. dys mutant legs have approximately the same number of apoptotic cells in the E(spl)mβ than in the “fold” domains (Figure 5E–G).


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 required for fold formation and for the expression of RhoGap71E and RhoGef2.(A and B) Sagittal view of a wild type (A) and dys2/dys3 (B) prepupal leg disc epithelium, stained with Phallodin (Phal) (red) and E(spl)mβ-CD2 (green). The joints between tarsal segments 2/3 and 3/4 are shown in A (arrows), and the corresponding region in B (arrows). Note the apical constriction in cells immediately distal to the E(spl)mβ-CD2 expression domain in the wild type leg and its absence in dys2/dys3 mutant legs. Single channels for Phal (A′ and B′) and E(spl)mβ-CD2 (B′ and B″) are displayed below. (C and D) RhoGap71E-Z (C, red) and RhoGef2-Z (D, red) expression compared to Dys (green). (C′ and D′) Sagittal view of a single joint where the last dys expressing cell is marked by an arrow. Note that both genes, RhoGap71E-Z and RhoGef2-Z, are expressed in a single row of Dys-positive cells and in a row of adjacent Dys-negative cells. (C″ and D″) Single channels for RhoGap71E-Z and RhoGef2-Z. (E) en-Gal4, UAS-GFP; UAS-dys-RNAi (green) and (F) hh-Gal4, UAS-GFP; UAS-dys-RNAi (green) downregulates RhoGap71E-Z and RhoGef2-Z expression (red, arrows), respectively. Dlg is in blue. Single channels for RhoGap71E-Z (E′) and RhoGef2-Z (F′) are displayed below.
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Related In: Results  -  Collection

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pgen-1004621-g004: dys is required for fold formation and for the expression of RhoGap71E and RhoGef2.(A and B) Sagittal view of a wild type (A) and dys2/dys3 (B) prepupal leg disc epithelium, stained with Phallodin (Phal) (red) and E(spl)mβ-CD2 (green). The joints between tarsal segments 2/3 and 3/4 are shown in A (arrows), and the corresponding region in B (arrows). Note the apical constriction in cells immediately distal to the E(spl)mβ-CD2 expression domain in the wild type leg and its absence in dys2/dys3 mutant legs. Single channels for Phal (A′ and B′) and E(spl)mβ-CD2 (B′ and B″) are displayed below. (C and D) RhoGap71E-Z (C, red) and RhoGef2-Z (D, red) expression compared to Dys (green). (C′ and D′) Sagittal view of a single joint where the last dys expressing cell is marked by an arrow. Note that both genes, RhoGap71E-Z and RhoGef2-Z, are expressed in a single row of Dys-positive cells and in a row of adjacent Dys-negative cells. (C″ and D″) Single channels for RhoGap71E-Z and RhoGef2-Z. (E) en-Gal4, UAS-GFP; UAS-dys-RNAi (green) and (F) hh-Gal4, UAS-GFP; UAS-dys-RNAi (green) downregulates RhoGap71E-Z and RhoGef2-Z expression (red, arrows), respectively. Dlg is in blue. Single channels for RhoGap71E-Z (E′) and RhoGef2-Z (F′) are displayed below.
Mentions: Epithelial cells at the presumptive joints undergo apical constriction and form characteristic folds that prefigure the future joint [22], [23]. The formation of epithelial folds involve cells immediately distal to the E(spl)mβ-CD2 expression domain (Figure 4A). In contrast, in dys mutant prepupal legs these cells distal to the E(spl)mβ-CD2 domain fail to reproduce these shape changes compared to control legs (compare Figure 4B and 4A). Two processes help sculpt the joint structure, namely JNK-mediated apoptosis driven by the pro-apoptotic gene rpr[20] and cell shape changes mediated by the Rho-family of GTPases [27]. The expression of RhoGef2 and RhoGap71E is restricted to the tarsal segments where they are coexpressed with a single row of Dys-positive cells and are extended distally to a row of Dys-negative cells (Figure 4C and 4D). Downregulation of Dys levels with a dys-RNAi in the engrailed (en) or the hedgehog (hh) domains lead to compartment cell autonomous loss of RhoGap71E and RhoGef2 expression, respectively (Figure 4E and 4F). Next, we compared the expression of dys with the pro-apoptotic genes rpr and hid. We found that the expression of these two genes in the prepupal leg discs is restricted to the distal end of each tarsal segments, where they are coexpressed with dys (Figure 5A, S3A) [20], [34]. Interestingly, as previously described for RhoGap71E and RhoGef2, we observed that rpr and hid are also expressed in a row of cells distal to dys expression (Figure 5A and S3A). Next we investigated if the expression of rpr and hid depends on dys. The downregulation of Dys levels in anterior cells along the PD axis with a dpp-Gal4 line or in the posterior compartment with en-Gal4, strongly reduced or eliminated the expression of rpr and hid, respectively (Figure 5B and S3B). Furthermore, forced expression of dys in the posterior compartment for 24 hrs using the tubGal80ts technique is sufficient to promote cell autonomously rpr and hid expression in the inter-joint domain (Figure 5C and S3C). This ectopic activation of the pro-apoptotic genes rpr and hid is accompanied by an increase of cell death in larval and prepupal leg discs, as visualized by the presence of activated Caspase-3 (DCas-3) (Figure 5D and S3D). To test if the downregulation in the expression of the pro-apoptotic genes observed after reducing Dys levels is associated to a decrease in cell death, we compared the number of DCas-3 positive cells in wild type and dys mutant legs (see Material and Methods). To determine the precise location of the apoptotic cells during joint formation, we separately counted DCas-3 positive cells within the E(spl)mβ domain and at the gap between two E(spl)mβ domains (termed here as “fold” domain). In wild type prepupal legs we found a significant increase in the number of DCas-3 positive cells in the “fold” domain compared to the E(spl)mβ one, while in dys mutant legs such significant difference was not observed (Figure 5E–G). Interestingly, while the total number of apoptotic cells per segment was comparable between wild type (average = 13,8) and dys mutant legs (average = 14,8), the distribution of DCas-3-positive cells in the joint was altered. dys mutant legs have approximately the same number of apoptotic cells in the E(spl)mβ than in the “fold” domains (Figure 5E–G).

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