Limits...
Repulsion by Slit and Roundabout prevents Shotgun/E-cadherin-mediated cell adhesion during Drosophila heart tube lumen formation.

Santiago-Martínez E, Soplop NH, Patel R, Kramer SG - J. Cell Biol. (2008)

Bottom Line: Genetic evidence suggests that Robo and Shotgun (Shg)/E-Cad function together in modulating CB adhesion. robo and shg/E-Cad transheterozygotes have lumen defects.In robo loss-of-function or shg/E-Cad gain-of-function embryos, lumen formation is blocked because of inappropriate CB adhesion and an accumulation of E-Cad at the apical membrane.In contrast, shg/E-Cad loss-of-function or robo gain-of-function blocks lumen formation due to a loss of CB adhesion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA.

ABSTRACT
During Drosophila melanogaster heart development, a lumen forms between apical surfaces of contralateral cardioblasts (CBs). We show that Slit and its receptor Roundabout (Robo) are required at CB apical domains for lumen formation. Mislocalization of Slit outside the apical domain causes ectopic lumen formation and the mislocalization of cell junction proteins, E-cadherin (E-Cad) and Enabled, without disrupting overall CB cell polarity. Ectopic lumen formation is suppressed in robo mutants, which indicates robo's requirement for this process. Genetic evidence suggests that Robo and Shotgun (Shg)/E-Cad function together in modulating CB adhesion. robo and shg/E-Cad transheterozygotes have lumen defects. In robo loss-of-function or shg/E-Cad gain-of-function embryos, lumen formation is blocked because of inappropriate CB adhesion and an accumulation of E-Cad at the apical membrane. In contrast, shg/E-Cad loss-of-function or robo gain-of-function blocks lumen formation due to a loss of CB adhesion. Our data show that Slit and Robo pathways function in lumen formation as a repulsive signal to antagonize E-Cad-mediated cell adhesion.

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Examination of cell junction markers. All embryos are stage 17. (A) E-Cad localizes to CB apical surfaces (arrow). (B) In slit GOF embryos, E-Cad is also localized to CB basal surfaces (arrow). (C) In robo mutants, E-Cad accumulates at high levels at CB apical domains. (D) EM of an ena mutant. Arrow indicates a region where CBs fail to make contact. (E and F) E-Cad staining in cross-sectioned embryos as visualized by HRP staining. (E) in slit GOF embryos, E-Cad is enriched at sites of cell contact in both lumens (arrowheads). (F) In robo mutants, E-Cad accumulates at apical domains where CBs remain in contact (arrow). (G–I) Mef2 (magenta) and Ena (green), which concentrates at CB apical surfaces (arrow) in wild-type embryos (G). In slit GOF embryos (H), Ena is mislocalized to CB basal and lateral domains in discrete puncta (arrows). (I) In robo mutants, Ena is no longer concentrated at the apical domain of CBs. (J–L) Same as G–I showing Ena alone. Bar, 1 μm.
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fig4: Examination of cell junction markers. All embryos are stage 17. (A) E-Cad localizes to CB apical surfaces (arrow). (B) In slit GOF embryos, E-Cad is also localized to CB basal surfaces (arrow). (C) In robo mutants, E-Cad accumulates at high levels at CB apical domains. (D) EM of an ena mutant. Arrow indicates a region where CBs fail to make contact. (E and F) E-Cad staining in cross-sectioned embryos as visualized by HRP staining. (E) in slit GOF embryos, E-Cad is enriched at sites of cell contact in both lumens (arrowheads). (F) In robo mutants, E-Cad accumulates at apical domains where CBs remain in contact (arrow). (G–I) Mef2 (magenta) and Ena (green), which concentrates at CB apical surfaces (arrow) in wild-type embryos (G). In slit GOF embryos (H), Ena is mislocalized to CB basal and lateral domains in discrete puncta (arrows). (I) In robo mutants, Ena is no longer concentrated at the apical domain of CBs. (J–L) Same as G–I showing Ena alone. Bar, 1 μm.

Mentions: Based upon our findings, we hypothesize that Slit and Robo function to negatively regulate E-Cad–mediated cell adhesion between opposing CBs. Next, we examined the localization of E-Cad in our mutant backgrounds. In the wild type, E-Cad localizes to the specific sites of cell–cell contact between contralateral CBs (Fig. 3 F). The concentration of E-Cad at CB apical membranes can also be seen in dorsal view (Fig. 4 A). In slit GOF embryos where we observe ectopic lumen formation, we noticed changes in E-Cad expression. E-Cad fails to accumulate at high levels at the apical domain in CBs (Fig. 4 B). In XS, ectopic E-Cad accumulation is seen at sites of cell–cell contact surrounding the ectopic lumens (Fig. 4 E). However, in robo mutants, we observed higher–than–wild type levels of E-Cad at the apical membrane as seen in dorsal view and in XS (Fig. 4, C and F). These results are consistent with our EM studies revealing that in robo mutants, the CBs are tightly adhered (Fig. 3 H′). We obtained similar results when we looked at Baz localization (Fig. S1, K–O). Baz is a PDZ domain–containing protein that may provide a landmark for adherens junction assembly by recruiting E-Cad to sites of cell–cell contact (Harris and Peifer, 2005).


Repulsion by Slit and Roundabout prevents Shotgun/E-cadherin-mediated cell adhesion during Drosophila heart tube lumen formation.

Santiago-Martínez E, Soplop NH, Patel R, Kramer SG - J. Cell Biol. (2008)

Examination of cell junction markers. All embryos are stage 17. (A) E-Cad localizes to CB apical surfaces (arrow). (B) In slit GOF embryos, E-Cad is also localized to CB basal surfaces (arrow). (C) In robo mutants, E-Cad accumulates at high levels at CB apical domains. (D) EM of an ena mutant. Arrow indicates a region where CBs fail to make contact. (E and F) E-Cad staining in cross-sectioned embryos as visualized by HRP staining. (E) in slit GOF embryos, E-Cad is enriched at sites of cell contact in both lumens (arrowheads). (F) In robo mutants, E-Cad accumulates at apical domains where CBs remain in contact (arrow). (G–I) Mef2 (magenta) and Ena (green), which concentrates at CB apical surfaces (arrow) in wild-type embryos (G). In slit GOF embryos (H), Ena is mislocalized to CB basal and lateral domains in discrete puncta (arrows). (I) In robo mutants, Ena is no longer concentrated at the apical domain of CBs. (J–L) Same as G–I showing Ena alone. Bar, 1 μm.
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Related In: Results  -  Collection

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fig4: Examination of cell junction markers. All embryos are stage 17. (A) E-Cad localizes to CB apical surfaces (arrow). (B) In slit GOF embryos, E-Cad is also localized to CB basal surfaces (arrow). (C) In robo mutants, E-Cad accumulates at high levels at CB apical domains. (D) EM of an ena mutant. Arrow indicates a region where CBs fail to make contact. (E and F) E-Cad staining in cross-sectioned embryos as visualized by HRP staining. (E) in slit GOF embryos, E-Cad is enriched at sites of cell contact in both lumens (arrowheads). (F) In robo mutants, E-Cad accumulates at apical domains where CBs remain in contact (arrow). (G–I) Mef2 (magenta) and Ena (green), which concentrates at CB apical surfaces (arrow) in wild-type embryos (G). In slit GOF embryos (H), Ena is mislocalized to CB basal and lateral domains in discrete puncta (arrows). (I) In robo mutants, Ena is no longer concentrated at the apical domain of CBs. (J–L) Same as G–I showing Ena alone. Bar, 1 μm.
Mentions: Based upon our findings, we hypothesize that Slit and Robo function to negatively regulate E-Cad–mediated cell adhesion between opposing CBs. Next, we examined the localization of E-Cad in our mutant backgrounds. In the wild type, E-Cad localizes to the specific sites of cell–cell contact between contralateral CBs (Fig. 3 F). The concentration of E-Cad at CB apical membranes can also be seen in dorsal view (Fig. 4 A). In slit GOF embryos where we observe ectopic lumen formation, we noticed changes in E-Cad expression. E-Cad fails to accumulate at high levels at the apical domain in CBs (Fig. 4 B). In XS, ectopic E-Cad accumulation is seen at sites of cell–cell contact surrounding the ectopic lumens (Fig. 4 E). However, in robo mutants, we observed higher–than–wild type levels of E-Cad at the apical membrane as seen in dorsal view and in XS (Fig. 4, C and F). These results are consistent with our EM studies revealing that in robo mutants, the CBs are tightly adhered (Fig. 3 H′). We obtained similar results when we looked at Baz localization (Fig. S1, K–O). Baz is a PDZ domain–containing protein that may provide a landmark for adherens junction assembly by recruiting E-Cad to sites of cell–cell contact (Harris and Peifer, 2005).

Bottom Line: Genetic evidence suggests that Robo and Shotgun (Shg)/E-Cad function together in modulating CB adhesion. robo and shg/E-Cad transheterozygotes have lumen defects.In robo loss-of-function or shg/E-Cad gain-of-function embryos, lumen formation is blocked because of inappropriate CB adhesion and an accumulation of E-Cad at the apical membrane.In contrast, shg/E-Cad loss-of-function or robo gain-of-function blocks lumen formation due to a loss of CB adhesion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA.

ABSTRACT
During Drosophila melanogaster heart development, a lumen forms between apical surfaces of contralateral cardioblasts (CBs). We show that Slit and its receptor Roundabout (Robo) are required at CB apical domains for lumen formation. Mislocalization of Slit outside the apical domain causes ectopic lumen formation and the mislocalization of cell junction proteins, E-cadherin (E-Cad) and Enabled, without disrupting overall CB cell polarity. Ectopic lumen formation is suppressed in robo mutants, which indicates robo's requirement for this process. Genetic evidence suggests that Robo and Shotgun (Shg)/E-Cad function together in modulating CB adhesion. robo and shg/E-Cad transheterozygotes have lumen defects. In robo loss-of-function or shg/E-Cad gain-of-function embryos, lumen formation is blocked because of inappropriate CB adhesion and an accumulation of E-Cad at the apical membrane. In contrast, shg/E-Cad loss-of-function or robo gain-of-function blocks lumen formation due to a loss of CB adhesion. Our data show that Slit and Robo pathways function in lumen formation as a repulsive signal to antagonize E-Cad-mediated cell adhesion.

Show MeSH
Related in: MedlinePlus