Egg Chamber Elongation Research Articles

Distinct contributions of ECM proteins to basement membrane mechanical properties in Drosophila.

The basement membrane is a specialized extracellular matrix (ECM) that is crucial for the development of epithelial tissues and organs. In Drosophila, the mechanical properties of the basement membrane play an important role in the proper elongation of the developing egg chamber; however, the molecular mechanisms contributing to basement membrane mechanical properties are not fully understood. Here, we systematically analyze the contributions of individual ECM components towards the molecular composition and mechanical properties of the basement membrane underlying the follicle epithelium of Drosophila egg chambers. We find that the Laminin and Collagen IV networks largely persist in the absence of the other components. Moreover, we show that Perlecan and Collagen IV, but not Laminin or Nidogen, contribute greatly towards egg chamber elongation. Similarly, Perlecan and Collagen, but not Laminin or Nidogen, contribute towards the resistance of egg chambers against osmotic stress. Finally, using atomic force microscopy we show that basement membrane stiffness mainly depends on Collagen IV. Our analysis reveals how single ECM components contribute to the mechanical properties of the basement membrane controlling tissue and organ shape.

Spinning the Matrix – Mechanisms of Basement Membrane Secretion and Assembly

Basement membranes (BMs) are sheet‐like extracellular matrices that line the basal surfaces of epithelial tissues and surround muscles, adipose tissue, and nerves. My lab studies how regulated changes to BM architecture contribute to tissue morphogenesis. In Drosophila, elongation of the initially spherical egg chamber coincides with the generation of a polarized network of fibrils in its surrounding BM. We found that 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 planar BM network by directed epithelial migration. We further found that a Rab10‐dependent secretion pathway ensures that a subset of new BM proteins exit the cell through a basal region of the lateral membrane for fibril formation. Rab10 recruits kinesin motors to BM secretory vesicles and transports them to this location on a polarized microtubule array. Finally, by manipulating Rab10 function, we showed for the first time that BM fibrils play an instructive role in egg chamber elongation. This work identifies a novel intracellular trafficking pathway for BM proteins and highlights how regulated protein secretion can synergize with tissue movement to build a polarized BM architecture that controls tissue shape.

Cell-matrix adhesion and cell-cell adhesion differentially control basal myosin oscillation and Drosophila egg chamber elongation

Pulsatile actomyosin contractility, important in tissue morphogenesis, has been studied mainly in apical but less in basal domains. Basal myosin oscillation underlying egg chamber elongation is regulated by both cell–matrix and cell–cell adhesions. However, the mechanism by which these two adhesions govern basal myosin oscillation and tissue elongation is unknown. Here we demonstrate that cell–matrix adhesion positively regulates basal junctional Rho1 activity and medio-basal ROCK and myosin activities, thus strongly controlling tissue elongation. Differently, cell–cell adhesion governs basal myosin oscillation through controlling medio-basal distributions of both ROCK and myosin signals, which are related to the spatial limitations of cell–matrix adhesion and stress fibres. Contrary to cell–matrix adhesion, cell–cell adhesion weakly affects tissue elongation. In vivo optogenetic protein inhibition spatiotemporally confirms the different effects of these two adhesions on basal myosin oscillation. This study highlights the activity and distribution controls of basal myosin contractility mediated by cell–matrix and cell–cell adhesions, respectively, during tissue morphogenesis.

A Mutation in fat2 Uncouples Tissue Elongation from Global Tissue Rotation.

Global tissue rotation was proposed as a morphogenetic mechanism controlling tissue elongation. In Drosophila ovaries, global tissue rotation of egg chambers coincides with egg chamber elongation. Egg chamber rotation was put forward to result in circumferential alignment of extracellular fibers. These fibers serve as molecular corsets to restrain growth of egg chambers perpendicular to the anteroposterior axis, thereby leading to the preferential egg chamber elongation along this axis. The atypical cadherin Fat2 is required for egg chamber elongation, rotation, and the circumferential alignment of extracellular fibers. Here, we have generated a truncated form of Fat2 that lacks the entire intracellular region. fat2 mutant egg chambers expressing this truncated protein fail to rotate yet display normal extracellular fiber alignment and properly elongate. Our data suggest that global tissue rotation, even though coinciding with tissue elongation, is not a necessary prerequisite for elongation.

Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis.

Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies.

Influence of ovarian muscle contraction and oocyte growth on egg chamber elongation in Drosophila.

Organs are formed from multiple cell types that make distinct contributions to their shape. The Drosophila egg chamber provides a tractable model to dissect such contributions during morphogenesis. Egg chambers consist of 16 germ cells (GCs) surrounded by a somatic epithelium. Initially spherical, these structures elongate as they mature. This morphogenesis is thought to occur through a 'molecular corset' mechanism, whereby structural elements within the epithelium become circumferentially organized perpendicular to the elongation axis and resist the expansive growth of the GCs to promote elongation. Whether this epithelial organization provides the hypothesized constraining force has been difficult to discern, however, and a role for GC growth has not been demonstrated. Here, we provide evidence for this mechanism by altering the contractile activity of the tubular muscle sheath that surrounds developing egg chambers. Muscle hypo-contraction indirectly reduces GC growth and shortens the egg, which demonstrates the necessity of GC growth for elongation. Conversely, muscle hyper-contraction enhances the elongation program. Although this is an abnormal function for this muscle, this observation suggests that a corset-like force from the egg chamber's exterior could promote its lengthening. These findings highlight how physical contributions from several cell types are integrated to shape an organ.

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

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.

Epithelial rotation promotes the global alignment of contractile actin bundles during Drosophila egg chamber elongation

Tissues use numerous mechanisms to change shape during development. The Drosophila egg chamber is an organ-like structure that elongates to form an elliptical egg. During elongation the follicular epithelial cells undergo a collective migration that causes the egg chamber to rotate within its surrounding basement membrane. Rotation coincides with the formation of a “molecular corset”, in which actin bundles in the epithelium and fibrils in the basement membrane are all aligned perpendicular to the elongation axis. Here we show that rotation plays a critical role in building the actin-based component of the corset. Rotation begins shortly after egg chamber formation and requires lamellipodial protrusions at each follicle cell’s leading edge. During early stages, rotation is necessary for tissue-level actin bundle alignment, but it becomes dispensable after the basement membrane is polarized. This work highlights how collective cell migration can be used to build a polarized tissue organization for organ morphogenesis.

The Drosophila Egg Chamber--A New Spin on How Tissues Elongate

During development, tissues undergo complex cellular rearrangements and changes in shape that produce a diversity of body plans and the functional organs therein. The Drosophila egg chamber has emerged as an exciting and highly tractable model in which to investigate novel mechanisms driving the elongation of tissues. Egg chambers are multicellular assemblies within flies' ovaries that will each give rise to a single egg. Although initially spherical, these simple organ-like structures lengthen as they grow. This transformation depends on an unusual form of planar polarity in the egg chamber's outer epithelial layer, in which arrays of linear actin bundles and fibril-like structures in the basement membrane both align perpendicular to the axis of elongation. The resulting circumferential arrangement of structural molecules is then thought to act as a "molecular corset" that directionally biases growth of the egg chamber. I will explore four fundamental questions about this system: (1) How is the circumferential pattern generated in the follicular epithelium? (2) What is the physical nature of the corset? (3) How does a corset-type mechanism lead to the cellular rearrangements necessary for the elongation of tissues? and (4) To what extent are the cellular mechanisms controlling egg chamber elongation conserved in other systems? For each topic, I will present insights gleaned from the recent literature and highlight fertile areas for future investigation.

Microtubule Polarity Predicts Direction of Egg Chamber Rotation in Drosophila

Whole-tissue rotations have recently been recognized as a widespread morphogenetic process important for tissue elongation [1-4]. In Drosophila ovaries, elongation of the egg chamber involves a global rotation of the follicle epithelium along the anterior-posterior axis [5]. Individual egg chambers rotate either in a clockwise or counterclockwise direction; however, how the symmetry of egg chambers is broken to allow rotation remains unknown. Here we show that at the basal side of follicle cells, microtubules are preferentially aligned perpendicular to the anterior-posterior axis of the egg chamber. Microtubule depolymerization stalls egg chamber rotation and egg chamber elongation. The preferential alignment of microtubules and egg chamber rotation depend on the atypical cadherin Fat2 and the planar polarized Fat2 localization depends on intact microtubules. Moreover, by tracking microtubule plus-end growth invivo using EB1::GFP, we find that microtubules are highly polarized in the plane of the follicle epithelium. Polarization of microtubules precedes the onset of egg chamber rotation and predicts the direction of rotation. Our data suggest a feedback amplification mechanism between Fat2 localization and microtubule polarity involved in breaking symmetry and directing egg chamber rotation.

A Rab10-Dependent Mechanism for Polarized Basement Membrane Secretion during Organ Morphogenesis

Basement membranes (BMs) are specialized extracellular matrices that are essential for epithelial structure and morphogenesis. However, little is known about how BM proteins are delivered to the basal cell surface or how this process is regulated during development. Here, we identify a mechanism for polarized BM secretion in the Drosophila follicle cells. BM proteins are synthesized in a basal endoplasmic reticulum (ER) compartment from localized mRNAs and are then exported through Tango1-positive ER exit sites to basal Golgi clusters. Next, Crag targets Rab10 to structures in the basal cytoplasm, where it restricts protein delivery to the basal surface. These events occur during egg chamber elongation, a morphogenetic process that depends on follicle cell planar polarity and BM remodeling. Significantly, Tango1 and Rab10 are also planar polarized at the basal epithelial surface. We propose that the spatial control of BM production along two tissue axes promotes exocytic efficiency, BM remodeling, and organ morphogenesis.

A Screen for Round Egg Mutants inDrosophilaIdentifies Tricornered, Furry, and Misshapen as Regulators of Egg Chamber Elongation

The elongation of tissues and organs during embryonic development results from the coordinate polarization of cell behaviors with respect to the elongation axis. Within the Drosophila melanogaster ovary, initially spherical egg chambers lengthen dramatically as they develop to create the elliptical shape of the mature egg. This morphogenesis depends on an unusual form of planar polarity within the egg chamber’s outer epithelial cell layer known as the follicle cells. Disruption of follicle cell planar polarity leads to the production of round rather than elongated eggs; however, the molecular mechanisms that control this tissue organization are poorly understood. Starting from a broadly based forward genetic screen, we have isolated 12 new round egg complementation groups, and have identified four of the mutated genes. In mapping the largest complementation group to the fat2 locus, we unexpectedly discovered a high incidence of cryptic fat2 mutations in the backgrounds of publicly available stocks. Three other complementation groups correspond to the genes encoding the cytoplasmic signaling proteins Tricornered (Trc), Furry (Fry), and Misshapen (Msn). Trc and Fry are known members of an NDR kinase signaling pathway, and as a Ste20-like kinase, Msn may function upstream of Trc. We show that all three proteins are required for follicle cell planar polarity at early stages of egg chamber elongation and that Trc shows a planar polarized distribution at the basal follicle cell surface. These results indicate that this new mutant collection is likely to provide novel insight into the molecular mechanisms controlling follicle cell planar polarity and egg chamber elongation.

Genetic evidence for antagonism between Pak protein kinase and Rho1 small GTPase signaling in regulation of the actin cytoskeleton during Drosophila oogenesis.

During Drosophila oogenesis, basally localized F-actin bundles in the follicle cells covering the egg chamber drive its elongation along the anterior-posterior axis. The basal F-actin of the follicle cell is an attractive system for the genetic analysis of the regulation of the actin cytoskeleton, and results obtained in this system are likely to be broadly applicable in understanding tissue remodeling. Mutations in a number of genes, including that encoding the p21-activated kinase Pak, have been shown to disrupt organization of the basal F-actin and in turn affect egg chamber elongation. pak mutant egg chambers have disorganized F-actin distribution and remain spherical due to a failure to elongate. In a genetic screen to identify modifiers of the pak rounded egg chamber phenotype several second chromosome deficiencies were identified as suppressors. One suppressing deficiency removes the rho1 locus, and we determined using several rho1 alleles that removal of a single copy of rho1 can suppress the pak phenotype. Reduction of any component of the Rho1-activated actomyosin contractility pathway suppresses pak oogenesis defects, suggesting that Pak counteracts Rho1 signaling. There is ectopic myosin light chain phosphorylation in pak mutant follicle cell clones in elongating egg chambers, probably due at least in part to mislocalization of RhoGEF2, an activator of the Rho1 pathway. In early egg chambers, pak mutant follicle cells have reduced levels of myosin phosphorylation and we conclude that Pak both promotes and restricts myosin light chain phosphorylation in a temporally distinct manner during oogenesis.

The receptor-like tyrosine phosphatase lar is required for epithelial planar polarity and for axis determination within drosophila ovarian follicles.

The follicle cell monolayer that encircles each developing Drosophila oocyte contributes actively to egg development and patterning, and also represents a model stem cell-derived epithelium. We have identified mutations in the receptor-like transmembrane tyrosine phosphatase Lar that disorganize follicle formation, block egg chamber elongation and disrupt Oskar localization, which is an indicator of oocyte anterior-posterior polarity. Alterations in actin filament organization correlate with these defects. Actin filaments in the basal follicle cell domain normally become polarized during stage 6 around the anterior-posterior axis defined by the polar cells, but mutations in Lar frequently disrupt polar cell differentiation and actin polarization. Lar function is only needed in somatic cells, and (for Oskar localization) its action is autonomous to posterior follicle cells. Polarity signals may be laid down by these cells within the extracellular matrix (ECM), possibly in the distribution of the candidate Lar ligand Laminin A, and read out at the time Oskar is localized in a Lar-dependent manner. Lar is not required autonomously to polarize somatic cell actin during stages 6. We show that Lar acts somatically early in oogenesis, during follicle formation, and postulate that it functions in germarium intercyst cells that are required for polar cell specification and differentiation. Our studies suggest that positional information can be stored transiently in the ECM. A major function of Lar may be to transduce such signals.