Live-imaging of the <em>Drosophila</em> Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

dissection-drosophila-pupal-retina-for-immunohistochemistry-western

Dissection of the <em>Drosophila</em> Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Wesleyan University

Developing tissues require cells to undergo intricate processes to shift into appropriate niches. This requires a functional connection between adhesion-mediating events at the cell surface and a cytoskeletal reorganization to permit directed movement. A small number of proteins are proposed to link these processes. Here, we identify one candidate, Cindr, the sole Drosophila melanogaster member of the CD2AP/CIN85 family (this family has been previously implicated in a variety of processes). Using D. melanogaster retina, we demonstrate that Cindr links cell surface junctions (E-cadherin) and adhesion (Roughest) with multiple components of the actin cytoskeleton. Reducing cindr activity leads to defects in local cell movement and, consequently, tissue patterning and cell death. Cindr activity is required for normal localization of Drosophila E-cadherin and Roughest, and we show additional physical and functional links to multiple components of the actin cytoskeleton, including the actin-capping proteins capping protein alpha and capping protein beta. Together, these data demonstrate that Cindr is involved in dynamic cell rearrangement in an emerging epithelium.

The Drosophila CD2AP/CIN85 orthologue Cindr regulates junctions and cytoskeleton dynamics during tissue patterning.

Correct cellular patterning is central to tissue morphogenesis, but the role of epithelial junctions in this process is not well-understood. The Drosophila pupal eye provides a sensitive and accessible model for testing the role of junction-associated proteins in cells that undergo dynamic and coordinated movements during development. Mutations in polychaetoid (pyd), the Drosophila homologue of Zonula Occludens-1, are characterized by two phenotypes visible in the adult fly: increased sensory bristle number and the formation of a rough eye produced by poorly arranged ommatidia. We found that Pyd was localized to the adherens junction in cells of the developing pupal retina. Reducing Pyd function in the pupal eye resulted in mis-patterning of the interommatidial cells and a failure to consistently switch cone cell contacts from an anterior-posterior to an equatorial-polar orientation. Levels of Roughest, DE-Cadherin and several other adherens junction-associated proteins were increased at the membrane when Pyd protein was reduced. Further, both over-expression and mutations in several junction-associated proteins greatly enhanced the patterning defects caused by reduction of Pyd. Our results suggest that Pyd modulates adherens junction strength and Roughest-mediated preferential cell adhesion.

Polychaetoid controls patterning by modulating adhesion in the Drosophila pupal retina.

The Drosophila pupal eye has become a popular paradigm for understanding morphogenesis and tissue patterning. Correct rearrangement of cells between ommatidia is required to organize the ommatidial array across the eye field. This requires cell movement, cell death, changes to cell-cell adhesion, signaling and fate specification.

A quantitative method to analyze Drosophila pupal eye patterning.

Here we investigate the structural and functional basis of the interactions between Notch and Wingless signalling in Drosophila. Using yeast-two-hybrid and pull-down assays we show that Notch can bind directly a form of Dishevelled that is stabilized upon Wingless signalling. Moreover, we show that the mechanism by which Wingless signalling is able to downregulate Notch is by promoting its ligand-independent traffic to a compartment where it is degraded and that this activity depends on Dishevelled.

Wingless modulates the ligand independent traffic of Notch through Dishevelled.

We present a computer simulation and associated experimental validation of assembly of glial-like support cells into the interweaving hexagonal lattice that spans the Drosophila pupal eye. This process of cell movements organizes the ommatidial array into a functional pattern. Unlike earlier simulations that focused on the arrangements of cells within individual ommatidia, here we examine the local movements that lead to large-scale organization of the emerging eye field. Simulations based on our experimental observations of cell adhesion, cell death, and cell movement successfully patterned a tracing of an emerging wild-type pupal eye. Surprisingly, altering cell adhesion had only a mild effect on patterning, contradicting our previous hypothesis that the patterning was primarily the result of preferential adhesion between IRM-class surface proteins. Instead, our simulations highlighted the importance of programmed cell death (PCD) as well as a previously unappreciated variable: the expansion of cells' apical surface areas, which promoted rearrangement of neighboring cells. We tested this prediction experimentally by preventing expansion in the apical area of individual cells: patterning was disrupted in a manner predicted by our simulations. Our work demonstrates the value of combining computer simulation with in vivo experiments to uncover novel mechanisms that are perpetuated throughout the eye field. It also demonstrates the utility of the Glazier-Graner-Hogeweg model (GGH) for modeling the links between local cellular interactions and emergent properties of developing epithelia as well as predicting unanticipated results in vivo.

Computer simulation of cellular patterning within the Drosophila pupal eye.

Patterning of the Drosophila pupal eye is characterized by precise cell movements. In this paper, we demonstrate that these movements require an Arf regulatory cycle that connects surface receptors to actin-based movement. dArf6 activity-regulated by the Arf GTPase-activating proteins (ArfGAPs) dAsap and dArfGAP3 and the Arf GTP exchange factors Schizo and dPsd-promoted large cellular extensions; time-lapse microscopy indicated that these extensions presage cell rearrangements into correct epithelial niches. During this process, the Drosophila eye also requires interactions between surface Neph1/nephrin adhesion receptors Roughest and Hibris, which bind the adaptor protein Cindr (CD2AP). We provide evidence that Cindr forms a physical complex with dArfGAP3 and dAsap. Our data suggest this interaction sequesters ArfGAP function to liberate active dArf6 elsewhere in the cell. We propose that a Neph1/nephrin-Cindr/ArfGAP complex accumulates to limit local Arf6 activity and stabilize adherens junctions. Our model therefore links surface adhesion via an Arf6 regulatory cascade to dynamic modeling of the cytoskeleton, accounting for precise cell movements that organize the functional retinal field. Further, we demonstrate a similar relationship between the mammalian Cindr orthologue CD2AP and Arf6 activity in cell motility assays. We propose that this Cindr/CD2AP-mediated regulation of Arf6 is a widely used mechanism in emerging epithelia.

Role for a Cindr-Arf6 axis in patterning emerging epithelia.

Morphogenetic modeling of tissues requires coordinated regulation of adhesion. For its correct patterning, the Drosophila pupal eye requires several Immunoglobulin superfamily cell adhesion molecules (IgCAMs) and the adaptor protein Cindr. Orthologs of these proteins are essential components of specialized junctions of the vertebrate kidney; the Cindr ortholog Cd2ap is essential for the integrity of this structure.

ArticleTotal : 2	Year
Publication title Cited by 7	2015
Publication title Cited by 11	2019

Article	Year
The Drosophila CD2AP/CIN85 orthologue Cindr regulates junctions and cytoskeleton dynamics during tissue patterning. The Journal of cell biology\| PubMed ID: 18362180	2008
Polychaetoid controls patterning by modulating adhesion in the Drosophila pupal retina. Developmental biology\| PubMed ID: 18423436	2008
A quantitative method to analyze Drosophila pupal eye patterning. PloS one\| PubMed ID: 19753121	2009
Wingless modulates the ligand independent traffic of Notch through Dishevelled. Fly\| PubMed ID: 20495361	2010
Computer simulation of cellular patterning within the Drosophila pupal eye. PLoS computational biology\| PubMed ID: 20617161	2010
Role for a Cindr-Arf6 axis in patterning emerging epithelia. Molecular biology of the cell\| PubMed ID: 21976699	2011
Interactions between Drosophila IgCAM adhesion receptors and cindr, the Cd2ap/Cin85 ortholog. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 23027549	2012

Ruth I. Johnson

Biography

JoVE Journal Publications

Other Publications