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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Localization of Slit, Robo, and Robo2 in the heart. All embryos are wild type. (A–C) EMs of embryos in XS. (A) Early stage 16 embryo showing two CBs initiating contact at their dorsal leading edges (arrows). (B) At late stage16, the dorsal edge has made contact (asterisk) and the CBs are initiating contact ventrally (arrow). (C) At stage 17, the lumen (arrow) is formed between two contralateral CBs joined at dorsal and ventral attachment points (asterisks). (D) Schematic of the heart in XS showing CBs joined at dorsal and ventral attachments (arrows). (E–G) Confocal images of stage 16 embryos in a dorsal view. (E) Mef2 labels CB nuclei (magenta), and Slit (green) localizes to the apical side of CBs. (F) Mef2 (magenta) and Robo (green). Robo localizes to the apical side of CBs, alary muscles (asterisks), and PCs (arrow). (G) Mef2 (magenta) and Robo2 (green), which localizes to the PCs (arrow). (H) Schematic of the heart in dorsal view showing the position of CBs (green) and PCs (yellow). A single CB is shown making contact with one contralateral and two ipsilateral CBs (arrows). (I–K) XSs of stage 17 embryos stained with HRP (brown). (I) Slit accumulates at the apical surface of the lumen (arrow). (J) Robo localizes to apical lumenal surface and the alary muscles (asterisk). (K) Robo2 is restricted to the PCs (arrow). Bar, 2 μm.
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fig1: Localization of Slit, Robo, and Robo2 in the heart. All embryos are wild type. (A–C) EMs of embryos in XS. (A) Early stage 16 embryo showing two CBs initiating contact at their dorsal leading edges (arrows). (B) At late stage16, the dorsal edge has made contact (asterisk) and the CBs are initiating contact ventrally (arrow). (C) At stage 17, the lumen (arrow) is formed between two contralateral CBs joined at dorsal and ventral attachment points (asterisks). (D) Schematic of the heart in XS showing CBs joined at dorsal and ventral attachments (arrows). (E–G) Confocal images of stage 16 embryos in a dorsal view. (E) Mef2 labels CB nuclei (magenta), and Slit (green) localizes to the apical side of CBs. (F) Mef2 (magenta) and Robo (green). Robo localizes to the apical side of CBs, alary muscles (asterisks), and PCs (arrow). (G) Mef2 (magenta) and Robo2 (green), which localizes to the PCs (arrow). (H) Schematic of the heart in dorsal view showing the position of CBs (green) and PCs (yellow). A single CB is shown making contact with one contralateral and two ipsilateral CBs (arrows). (I–K) XSs of stage 17 embryos stained with HRP (brown). (I) Slit accumulates at the apical surface of the lumen (arrow). (J) Robo localizes to apical lumenal surface and the alary muscles (asterisk). (K) Robo2 is restricted to the PCs (arrow). Bar, 2 μm.

Mentions: The alignment of cardioblasts (CBs) into rows on either side of the midline is the first of several steps during heart tube assembly. After alignment, the two rows of CBs, which are each flanked by a row of pericardial cells (PCs), migrate dorsally. Prior to merging at the midline, CBs undergo a mesenchyme-to-epithelium transition and acquire apical-basal polarity (Fremion et al., 1999). At the dorsal midline, contralateral pairs of CBs make specific dorsal and ventral contacts between their opposing apical membranes to form the lumen (Fig. 1, D and H). To better understand the steps leading to lumen formation, we performed EM on cross sections (XSs) of wild-type embryos at three steps during the late stages of heart development. At early embryonic stage 16, each CB initiates contact with its contralateral counterpart at the dorsalmost leading edge of the apical membrane (Fig. 1 A). After dorsal contact, CBs undergo a shape change and make contact at their ventral apical surfaces (Fig. 1 B). In this way, a lumen is formed between two opposing CBs (Fig. 1 C). The fact that CBs specifically make contact at dorsal and ventral attachment points (Fig. 1 D) suggests that an inhibitory mechanism may prevent the centralmost apical surfaces between these points from coming into contact.


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)

Localization of Slit, Robo, and Robo2 in the heart. All embryos are wild type. (A–C) EMs of embryos in XS. (A) Early stage 16 embryo showing two CBs initiating contact at their dorsal leading edges (arrows). (B) At late stage16, the dorsal edge has made contact (asterisk) and the CBs are initiating contact ventrally (arrow). (C) At stage 17, the lumen (arrow) is formed between two contralateral CBs joined at dorsal and ventral attachment points (asterisks). (D) Schematic of the heart in XS showing CBs joined at dorsal and ventral attachments (arrows). (E–G) Confocal images of stage 16 embryos in a dorsal view. (E) Mef2 labels CB nuclei (magenta), and Slit (green) localizes to the apical side of CBs. (F) Mef2 (magenta) and Robo (green). Robo localizes to the apical side of CBs, alary muscles (asterisks), and PCs (arrow). (G) Mef2 (magenta) and Robo2 (green), which localizes to the PCs (arrow). (H) Schematic of the heart in dorsal view showing the position of CBs (green) and PCs (yellow). A single CB is shown making contact with one contralateral and two ipsilateral CBs (arrows). (I–K) XSs of stage 17 embryos stained with HRP (brown). (I) Slit accumulates at the apical surface of the lumen (arrow). (J) Robo localizes to apical lumenal surface and the alary muscles (asterisk). (K) Robo2 is restricted to the PCs (arrow). Bar, 2 μm.
© Copyright Policy
Related In: Results  -  Collection

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fig1: Localization of Slit, Robo, and Robo2 in the heart. All embryos are wild type. (A–C) EMs of embryos in XS. (A) Early stage 16 embryo showing two CBs initiating contact at their dorsal leading edges (arrows). (B) At late stage16, the dorsal edge has made contact (asterisk) and the CBs are initiating contact ventrally (arrow). (C) At stage 17, the lumen (arrow) is formed between two contralateral CBs joined at dorsal and ventral attachment points (asterisks). (D) Schematic of the heart in XS showing CBs joined at dorsal and ventral attachments (arrows). (E–G) Confocal images of stage 16 embryos in a dorsal view. (E) Mef2 labels CB nuclei (magenta), and Slit (green) localizes to the apical side of CBs. (F) Mef2 (magenta) and Robo (green). Robo localizes to the apical side of CBs, alary muscles (asterisks), and PCs (arrow). (G) Mef2 (magenta) and Robo2 (green), which localizes to the PCs (arrow). (H) Schematic of the heart in dorsal view showing the position of CBs (green) and PCs (yellow). A single CB is shown making contact with one contralateral and two ipsilateral CBs (arrows). (I–K) XSs of stage 17 embryos stained with HRP (brown). (I) Slit accumulates at the apical surface of the lumen (arrow). (J) Robo localizes to apical lumenal surface and the alary muscles (asterisk). (K) Robo2 is restricted to the PCs (arrow). Bar, 2 μm.
Mentions: The alignment of cardioblasts (CBs) into rows on either side of the midline is the first of several steps during heart tube assembly. After alignment, the two rows of CBs, which are each flanked by a row of pericardial cells (PCs), migrate dorsally. Prior to merging at the midline, CBs undergo a mesenchyme-to-epithelium transition and acquire apical-basal polarity (Fremion et al., 1999). At the dorsal midline, contralateral pairs of CBs make specific dorsal and ventral contacts between their opposing apical membranes to form the lumen (Fig. 1, D and H). To better understand the steps leading to lumen formation, we performed EM on cross sections (XSs) of wild-type embryos at three steps during the late stages of heart development. At early embryonic stage 16, each CB initiates contact with its contralateral counterpart at the dorsalmost leading edge of the apical membrane (Fig. 1 A). After dorsal contact, CBs undergo a shape change and make contact at their ventral apical surfaces (Fig. 1 B). In this way, a lumen is formed between two opposing CBs (Fig. 1 C). The fact that CBs specifically make contact at dorsal and ventral attachment points (Fig. 1 D) suggests that an inhibitory mechanism may prevent the centralmost apical surfaces between these points from coming into contact.

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