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Innexin 3, a new gene required for dorsal closure in Drosophila embryo.

Giuliani F, Giuliani G, Bauer R, Rabouille C - PLoS ONE (2013)

Bottom Line: Accordingly, in addition to the known interaction of Inx2 with DE-cadherin, we show that Inx3 can bind to DE-cadherin.Furthermore, Inx3-GFP overexpression recruits DE-cadherin from its wildtype plasma membrane domain to typical Innexin plaques, strengthening the notion that they form a complex.Finally, we show that Inx3 stability is directly dependent on tissue tension.

View Article: PubMed Central - PubMed

Affiliation: Hubrecht Institute for Developmental Biology and Stem Cell Research, Utrecht, The Netherlands ; UMC Utrecht, Utrecht, The Netherlands.

ABSTRACT

Background: Dorsal closure is a morphogenetic event that occurs during mid-embryogenesis in many insects including Drosophila, during which the ectoderm migrates on the extraembryonic amnioserosa to seal the embryo dorsally. The contribution of the ectoderm in this event has been known for a long time. However, amnioserosa tension and contractibility have recently been shown also to be instrumental to the closure. A critical pre-requisite for dorsal closure is integrity of these tissues that in part is mediated by cell-cell junctions and cell adhesion. In this regard, mutations impairing junction formation and/or adhesion lead to dorsal closure. However, no role for the gap junction proteins Innexins has so far been described.

Results and discussion: Here, we show that Innexin 1, 2 and 3, are present in the ectoderm but also in the amnioserosa in plaques consistent with gap junctions. However, only the loss of Inx3 leads to dorsal closure defects that are completely rescued by overexpression of inx3::GFP in the whole embryo. Loss of Inx3 leads to the destabilisation of Inx1, Inx2 and DE-cadherin at the plasma membrane, suggesting that these four proteins form a complex. Accordingly, in addition to the known interaction of Inx2 with DE-cadherin, we show that Inx3 can bind to DE-cadherin. Furthermore, Inx3-GFP overexpression recruits DE-cadherin from its wildtype plasma membrane domain to typical Innexin plaques, strengthening the notion that they form a complex. Finally, we show that Inx3 stability is directly dependent on tissue tension. Taken together, we propose that Inx3 is a critical factor for dorsal closure and that it mediates the stability of Inx1, 2 and DE-cadherin by forming a complex.

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inx3::GFP overexpression rescues dorsal closure defects of Df(3R)BSC789.A: Western blot detection of Inx3 upon Inx3 depletion by RNAi using UASwizinx3 and actGAL4. Note that the overexpression of UASwizinx3 is not enough to deplete Inx3 when compared to siblings. B, B': Brightfield micrographs of dorso-lateral cuticle preparation of actGAL4; UASwizinx3 (B) and UASwizinx3; AS>GAL4 larvae (B'). The first cross yields 50% larvae carrying both the transgene and the driver but none of them exhibit dorsal closure defects (0/115). The second cross yields 100% larvae carrying both the transgene and the driver and none of them show developmental defects. C–E': Brightfield micrographs of dorso-lateral cuticle preparation of heterozygous Df(3R)BSC789 larvae expressing UAS-inx3::GFP under the control of actGAL4 (GFP positive, A), homozogous Df(3R)BSC789 larvae not expressing inx3::GFP (GFP negative, B), homozogous Df(3R)BSC789 larvae expressing inx3::GFP under the control of actGAL4 (GFP positive, C and C'). Larvae in C and C' are smaller and sometimes have defects in the head or in the rear but do not exhibit any dorsal closure defects. F: Quantification of the rescue of the dorsal closure defects [1] by inx3::GFP driven actGAL4[2] and AS>GAL4[3]. The rescue cross (w; +; Df(3R)BSC789, UAS-inx3::GFP/TM3, Sb x w; actGal4/CyO; Df(3R)BSC789/TM3, Sb) was set. GFP positive and negative embryos were sorted before letting the larvae develop to first instar. The red color number indicates the number of larvae in [2] in which dorsal closure defects due to the deficiency are rescued by inx3::GFP expression under the control of actGAL4. Note that they represent about ½ of the GFP positive population of embryos as expected (see Materials and Methods for predicted outcome). Note that it is not the case in cross [3]. The blue color number indicated the numbers of larvae in which dorsal closure defects due to the deficiency are not rescued by inx3::GFP expression under the control of an amnioserosa specific driver (AS>GAL4). G,G': Homozogous Df(3R)BSC789 larvae expressing inx3::GFP under the control of AS>GAL4 (GFP positive). They exhibit dorsal closure defects (kink (G) and holes (G')) similar to the homozogous Df(3R)BSC789 not expressing inx3::GFP.
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pone-0069212-g004: inx3::GFP overexpression rescues dorsal closure defects of Df(3R)BSC789.A: Western blot detection of Inx3 upon Inx3 depletion by RNAi using UASwizinx3 and actGAL4. Note that the overexpression of UASwizinx3 is not enough to deplete Inx3 when compared to siblings. B, B': Brightfield micrographs of dorso-lateral cuticle preparation of actGAL4; UASwizinx3 (B) and UASwizinx3; AS>GAL4 larvae (B'). The first cross yields 50% larvae carrying both the transgene and the driver but none of them exhibit dorsal closure defects (0/115). The second cross yields 100% larvae carrying both the transgene and the driver and none of them show developmental defects. C–E': Brightfield micrographs of dorso-lateral cuticle preparation of heterozygous Df(3R)BSC789 larvae expressing UAS-inx3::GFP under the control of actGAL4 (GFP positive, A), homozogous Df(3R)BSC789 larvae not expressing inx3::GFP (GFP negative, B), homozogous Df(3R)BSC789 larvae expressing inx3::GFP under the control of actGAL4 (GFP positive, C and C'). Larvae in C and C' are smaller and sometimes have defects in the head or in the rear but do not exhibit any dorsal closure defects. F: Quantification of the rescue of the dorsal closure defects [1] by inx3::GFP driven actGAL4[2] and AS>GAL4[3]. The rescue cross (w; +; Df(3R)BSC789, UAS-inx3::GFP/TM3, Sb x w; actGal4/CyO; Df(3R)BSC789/TM3, Sb) was set. GFP positive and negative embryos were sorted before letting the larvae develop to first instar. The red color number indicates the number of larvae in [2] in which dorsal closure defects due to the deficiency are rescued by inx3::GFP expression under the control of actGAL4. Note that they represent about ½ of the GFP positive population of embryos as expected (see Materials and Methods for predicted outcome). Note that it is not the case in cross [3]. The blue color number indicated the numbers of larvae in which dorsal closure defects due to the deficiency are not rescued by inx3::GFP expression under the control of an amnioserosa specific driver (AS>GAL4). G,G': Homozogous Df(3R)BSC789 larvae expressing inx3::GFP under the control of AS>GAL4 (GFP positive). They exhibit dorsal closure defects (kink (G) and holes (G')) similar to the homozogous Df(3R)BSC789 not expressing inx3::GFP.

Mentions: A–C: Immunolocalisation of endogenous Inx1 (A), Inx2 (B) and Inx3 (C) in wildtype (WT) Drosophila embryos (stage 11–13). A'–C': Details of Inx1-3 localisation in plaques at the plasma membrane of the amnioserosa cells (blowup from dashed areas in A–C) D–F: Immunolocalisation of Inx1 in ogreko (D), Inx2 in kropfg43 (E) and Inx3 in Df(3R)BSC789 (F) homozygous mutants, showing the antibodies specificity. The lack of labeling is not due to loss of tissue (see Figure 2 for ogreko and kropfg43 and Figure 4 for Df(3R)BSC789). G–G”: Immunolocalisation of endogenous Inx1 (G, red) and DE-cadherin (G', green) in amnioserosa cells of WT stage 13 embryos. Scale bars: 25 µm.


Innexin 3, a new gene required for dorsal closure in Drosophila embryo.

Giuliani F, Giuliani G, Bauer R, Rabouille C - PLoS ONE (2013)

inx3::GFP overexpression rescues dorsal closure defects of Df(3R)BSC789.A: Western blot detection of Inx3 upon Inx3 depletion by RNAi using UASwizinx3 and actGAL4. Note that the overexpression of UASwizinx3 is not enough to deplete Inx3 when compared to siblings. B, B': Brightfield micrographs of dorso-lateral cuticle preparation of actGAL4; UASwizinx3 (B) and UASwizinx3; AS>GAL4 larvae (B'). The first cross yields 50% larvae carrying both the transgene and the driver but none of them exhibit dorsal closure defects (0/115). The second cross yields 100% larvae carrying both the transgene and the driver and none of them show developmental defects. C–E': Brightfield micrographs of dorso-lateral cuticle preparation of heterozygous Df(3R)BSC789 larvae expressing UAS-inx3::GFP under the control of actGAL4 (GFP positive, A), homozogous Df(3R)BSC789 larvae not expressing inx3::GFP (GFP negative, B), homozogous Df(3R)BSC789 larvae expressing inx3::GFP under the control of actGAL4 (GFP positive, C and C'). Larvae in C and C' are smaller and sometimes have defects in the head or in the rear but do not exhibit any dorsal closure defects. F: Quantification of the rescue of the dorsal closure defects [1] by inx3::GFP driven actGAL4[2] and AS>GAL4[3]. The rescue cross (w; +; Df(3R)BSC789, UAS-inx3::GFP/TM3, Sb x w; actGal4/CyO; Df(3R)BSC789/TM3, Sb) was set. GFP positive and negative embryos were sorted before letting the larvae develop to first instar. The red color number indicates the number of larvae in [2] in which dorsal closure defects due to the deficiency are rescued by inx3::GFP expression under the control of actGAL4. Note that they represent about ½ of the GFP positive population of embryos as expected (see Materials and Methods for predicted outcome). Note that it is not the case in cross [3]. The blue color number indicated the numbers of larvae in which dorsal closure defects due to the deficiency are not rescued by inx3::GFP expression under the control of an amnioserosa specific driver (AS>GAL4). G,G': Homozogous Df(3R)BSC789 larvae expressing inx3::GFP under the control of AS>GAL4 (GFP positive). They exhibit dorsal closure defects (kink (G) and holes (G')) similar to the homozogous Df(3R)BSC789 not expressing inx3::GFP.
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Show All Figures
getmorefigures.php?uid=PMC3722180&req=5

pone-0069212-g004: inx3::GFP overexpression rescues dorsal closure defects of Df(3R)BSC789.A: Western blot detection of Inx3 upon Inx3 depletion by RNAi using UASwizinx3 and actGAL4. Note that the overexpression of UASwizinx3 is not enough to deplete Inx3 when compared to siblings. B, B': Brightfield micrographs of dorso-lateral cuticle preparation of actGAL4; UASwizinx3 (B) and UASwizinx3; AS>GAL4 larvae (B'). The first cross yields 50% larvae carrying both the transgene and the driver but none of them exhibit dorsal closure defects (0/115). The second cross yields 100% larvae carrying both the transgene and the driver and none of them show developmental defects. C–E': Brightfield micrographs of dorso-lateral cuticle preparation of heterozygous Df(3R)BSC789 larvae expressing UAS-inx3::GFP under the control of actGAL4 (GFP positive, A), homozogous Df(3R)BSC789 larvae not expressing inx3::GFP (GFP negative, B), homozogous Df(3R)BSC789 larvae expressing inx3::GFP under the control of actGAL4 (GFP positive, C and C'). Larvae in C and C' are smaller and sometimes have defects in the head or in the rear but do not exhibit any dorsal closure defects. F: Quantification of the rescue of the dorsal closure defects [1] by inx3::GFP driven actGAL4[2] and AS>GAL4[3]. The rescue cross (w; +; Df(3R)BSC789, UAS-inx3::GFP/TM3, Sb x w; actGal4/CyO; Df(3R)BSC789/TM3, Sb) was set. GFP positive and negative embryos were sorted before letting the larvae develop to first instar. The red color number indicates the number of larvae in [2] in which dorsal closure defects due to the deficiency are rescued by inx3::GFP expression under the control of actGAL4. Note that they represent about ½ of the GFP positive population of embryos as expected (see Materials and Methods for predicted outcome). Note that it is not the case in cross [3]. The blue color number indicated the numbers of larvae in which dorsal closure defects due to the deficiency are not rescued by inx3::GFP expression under the control of an amnioserosa specific driver (AS>GAL4). G,G': Homozogous Df(3R)BSC789 larvae expressing inx3::GFP under the control of AS>GAL4 (GFP positive). They exhibit dorsal closure defects (kink (G) and holes (G')) similar to the homozogous Df(3R)BSC789 not expressing inx3::GFP.
Mentions: A–C: Immunolocalisation of endogenous Inx1 (A), Inx2 (B) and Inx3 (C) in wildtype (WT) Drosophila embryos (stage 11–13). A'–C': Details of Inx1-3 localisation in plaques at the plasma membrane of the amnioserosa cells (blowup from dashed areas in A–C) D–F: Immunolocalisation of Inx1 in ogreko (D), Inx2 in kropfg43 (E) and Inx3 in Df(3R)BSC789 (F) homozygous mutants, showing the antibodies specificity. The lack of labeling is not due to loss of tissue (see Figure 2 for ogreko and kropfg43 and Figure 4 for Df(3R)BSC789). G–G”: Immunolocalisation of endogenous Inx1 (G, red) and DE-cadherin (G', green) in amnioserosa cells of WT stage 13 embryos. Scale bars: 25 µm.

Bottom Line: Accordingly, in addition to the known interaction of Inx2 with DE-cadherin, we show that Inx3 can bind to DE-cadherin.Furthermore, Inx3-GFP overexpression recruits DE-cadherin from its wildtype plasma membrane domain to typical Innexin plaques, strengthening the notion that they form a complex.Finally, we show that Inx3 stability is directly dependent on tissue tension.

View Article: PubMed Central - PubMed

Affiliation: Hubrecht Institute for Developmental Biology and Stem Cell Research, Utrecht, The Netherlands ; UMC Utrecht, Utrecht, The Netherlands.

ABSTRACT

Background: Dorsal closure is a morphogenetic event that occurs during mid-embryogenesis in many insects including Drosophila, during which the ectoderm migrates on the extraembryonic amnioserosa to seal the embryo dorsally. The contribution of the ectoderm in this event has been known for a long time. However, amnioserosa tension and contractibility have recently been shown also to be instrumental to the closure. A critical pre-requisite for dorsal closure is integrity of these tissues that in part is mediated by cell-cell junctions and cell adhesion. In this regard, mutations impairing junction formation and/or adhesion lead to dorsal closure. However, no role for the gap junction proteins Innexins has so far been described.

Results and discussion: Here, we show that Innexin 1, 2 and 3, are present in the ectoderm but also in the amnioserosa in plaques consistent with gap junctions. However, only the loss of Inx3 leads to dorsal closure defects that are completely rescued by overexpression of inx3::GFP in the whole embryo. Loss of Inx3 leads to the destabilisation of Inx1, Inx2 and DE-cadherin at the plasma membrane, suggesting that these four proteins form a complex. Accordingly, in addition to the known interaction of Inx2 with DE-cadherin, we show that Inx3 can bind to DE-cadherin. Furthermore, Inx3-GFP overexpression recruits DE-cadherin from its wildtype plasma membrane domain to typical Innexin plaques, strengthening the notion that they form a complex. Finally, we show that Inx3 stability is directly dependent on tissue tension. Taken together, we propose that Inx3 is a critical factor for dorsal closure and that it mediates the stability of Inx1, 2 and DE-cadherin by forming a complex.

Show MeSH
Related in: MedlinePlus