A novel method for obtaining semi-thin cross sections of the Drosophila heart and their labeling with multiple antibodies

Harpaz, Nofar; Volk, Talila

doi:10.1016/j.ymeth.2011.09.017

Cited by 11 publications

(6 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…GFP antibody stains were performed as above using PBS instead of PBT. Antibodies used: guinea pig α-Laminin (1:400) (Harpaz and Volk, 2012), rabbit α-Trol (1:1000) (Friedrich et al, 2000), rabbit α-GFP (1:200, Molecular Probes A21311 and A31852). Fluorescent images were obtained using Zeiss LSM 510 or LSM 880 confocal microscopes.…”

Section: Methodsmentioning

confidence: 99%

Rab10-Mediated Secretion Synergizes with Tissue Movement to Build a Polarized Basement Membrane Architecture for Organ Morphogenesis

2016

View full text Add to dashboard Cite

Summary Basement membranes (BMs) are planar protein networks that support epithelial function. Regulated changes to BM architecture can also contribute to tissue morphogenesis, but how epithelia dynamically remodel their BMs is unknown. In Drosophila, elongation of the initially spherical egg chamber correlates with the generation of a polarized network of fibrils in its surrounding BM. Here, we use live imaging and genetic manipulations to determine how these fibrils form. BM fibrils are assembled from newly synthesized proteins in the pericellular spaces between the egg chamber’s epithelial cells, and undergo oriented insertion into the BM by directed epithelial migration. We find that a Rab10-based secretion pathway promotes pericellular BM protein accumulation and fibril formation. Finally, by manipulating this pathway, we show that BM fibrillar structure influences egg chamber morphogenesis. This work highlights how regulated protein secretion can synergize with tissue movement to build a polarized BM architecture that controls tissue shape.

show abstract

Section: Methodsmentioning

confidence: 99%

Rab10-Mediated Secretion Synergizes with Tissue Movement to Build a Polarized Basement Membrane Architecture for Organ Morphogenesis

2016

View full text Add to dashboard Cite

show abstract

“…Quantification of relative fluorescent intensity for ECM proteins was performed in ImageJ via measurement of mean pixel intensity within a region of interest (ROI) selected over the heart tube (Figure 2C) and normalized to F-actin average intensity. The following antibodies were used: mouse anti-Pericardin (1:40, EC11-s, DSHB), guinea pig anti-Laminin A (1:500) [52], rabbit anti-GFP (1:500, ab6556, Abcam), rabbit anti-Collagen IV (1:500, ab6586, Abcam), and Rhodamin Phalloidin (1:1000, Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Extracellular matrix downregulation in the Drosophila heart preserves contractile function and improves lifespan

et al. 2017

View full text Add to dashboard Cite

Aging is associated with extensive remodeling of the heart, including basement membrane (BM) components that surround cardiomyocytes. Remodeling is thought to impair cardiac mechanotransduction, but the contribution of specific BM components to age-related lateral communication between cardiomyocytes is unclear. Using a genetically tractable, rapidly aging model with sufficient cardiac genetic homology and morphology, e.g. Drosophila melanogaster, we observed differential regulation of BM collagens between laboratory strains, correlating with changes in muscle physiology leading to cardiac dysfunction. Therefore, we sought to understand the extent to which BM proteins modulate contractile function during aging. Cardiac-restricted knockdown of ECM genes Pericardin, Laminin A, and Viking in Drosophila prevented age-associated heart tube restriction and increased contractility, even under viscous load. Most notably, reduction of Laminin A expression correlated with an overall preservation of contractile velocity with age and extension of organismal lifespan. Global heterozygous knockdown confirmed these data, which provides new evidence of a direct link between BM homeostasis, contractility, and maintenance of lifespan.

show abstract

“…Egg chambers were mounted in SlowFade Antifade (Invitrogen). Antibodies used: rabbit α-HA (1:200, Rockland), rabbit α-SPARC (1:400) (Martinek et al, 2002), guinea pig α-laminin (1:400) (Harpaz and Volk, 2012). All images were obtained using a Zeiss LSM 510 or LSM 880 confocal microscope, except 1F and S1F, which were obtained with a Leica FluoIII microscope with Canon rebel camera and 4E, N–P, S1A, and S5A, C–D, which were obtained using a Leica DM550B microscope with a Leica DFC425C camera.…”

Section: Methodsmentioning

confidence: 99%

Dynamic regulation of basement membrane protein levels promotes egg chamber elongation in Drosophila

Isabella

Horne‐Badovinac

2015

Developmental Biology

View full text Add to dashboard Cite

Basement membranes (BMs) are sheet-like extracellular matrices that provide essential support to epithelial tissues. Recent evidence suggests that regulated changes in BM architecture can direct tissue morphogenesis, but the mechanisms by which cells remodel BMs are largely unknown. The Drosophila egg chamber is an organ-like structure that transforms from a spherical to an ellipsoidal shape as it matures. This elongation coincides with a stage-specific increase in Type IV Collagen (Col IV) levels in the BM surrounding the egg chamber; however, the mechanisms and morphogenetic relevance of this remodeling event have not been established. Here, we identify the Collagen-binding protein SPARC as a negative regulator of egg chamber elongation, and show that SPARC down-regulation is necessary for the increase in Col IV levels to occur. We find that SPARC interacts with Col IV prior to secretion and propose that, through this interaction, SPARC blocks the incorporation of newly synthesized Col IV into the BM. We additionally observe a decrease in Perlecan levels during elongation, and show that Perlecan is a negative regulator of this process. These data provide mechanistic insight into SPARC’s conserved role in matrix dynamics and demonstrate that regulated changes in BM composition influence organ morphogenesis.

show abstract

A novel method for obtaining semi-thin cross sections of the Drosophila heart and their labeling with multiple antibodies

Cited by 11 publications

References 19 publications

Rab10-Mediated Secretion Synergizes with Tissue Movement to Build a Polarized Basement Membrane Architecture for Organ Morphogenesis

Rab10-Mediated Secretion Synergizes with Tissue Movement to Build a Polarized Basement Membrane Architecture for Organ Morphogenesis

Extracellular matrix downregulation in the Drosophila heart preserves contractile function and improves lifespan

Dynamic regulation of basement membrane protein levels promotes egg chamber elongation in Drosophila

Contact Info

Product

Resources

About