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 serine/threonine kinase dPak is required for polarized assembly of F-actin bundles and apical-basal polarity in the Drosophila follicular epithelium

During epithelial development cells become polarized along their apical-basal axis and some epithelia also exhibit polarity in the plane of the tissue. Mutations in the gene encoding a Drosophila Pak family serine/threonine kinase, dPak, disrupt the follicular epithelium that covers developing egg chambers during oogenesis. The follicular epithelium normally exhibits planar polarized organization of basal F-actin bundles such that they lie perpendicular to the anterior-posterior axis of the egg chamber, and requires contact with the basement membrane for apical-basal polarization. During oogenesis, dPak becomes localized to the basal end of follicle cells and is required for polarized organization of the basal actin cytoskeleton and for epithelial integrity and apical-basal polarity. The receptor protein tyrosine phosphatase Dlar and integrins, all receptors for extracellular matrix proteins, are required for polarization of the basal F-actin bundles, and for correct dPak localization in follicle cells. dpak mutant follicle cells show increased beta(Heavy)-spectrin levels, and we speculate that dPak regulation of beta(Heavy)-spectrin, a known participant in the maintenance of membrane domains, is required for correct apical-basal polarization of the membrane. We propose that dPak mediates communication between the basement membrane and intracellular proteins required for polarization of the basal F-actin and for apical-basal polarity.     

Requirements of Lgl in cell differentiation and motility during Drosophila ovarian follicular epithelium morphogenesis

The epithelial follicle cell layer over the egg chamber in Drosophila ovary undergoes patterning and morphogenesis at oogenesis. These developmental processes are essential for constructing the eggshell and establishing the body axes of the egg and resultant embryo, thereby being crucial for the egg development. We have previously shown that lethal(2)giant larvae (lgl), a Drosophila neoplastic tumor suppressor gene (nTSG) is required for the posterior follicle cell (PFC) fate induction during antero-posterior pattern formation of the follicular epithelium. In this report, we further characterize lgl in this epithelium patterning and the morphogenetic changes of specified border cells. Genetic interactions of lgl with discs large (dlg) and scribble (scrib), another two nTSGs in specifying the PFC fate reveal a cooperative role of this group of genes. Meanwhile, we find that loss of lgl function causes failure of follicle cells at the anterior to differentiate properly. The clonal analysis further indicates that lgl is necessary not only for the border cell differentiation, but also for control of the collective border cell migration via presumably modulating the apico-basal polarity and cell adhesion. Overall, we identify Lgl as an essential factor in regulating differentiation and morphogenetic movement of the ovarian epithelial follicle cells.     

Requirements of Lgl in cell differentiation and motility during Drosophila ovarian follicular epithelium morphogenesis

The epithelial follicle cell layer over the egg chamber in Drosophila ovary undergoes patterning and morphogenesis at oogenesis. These developmental processes are essential for constructing the eggshell and establishing the body axes of the egg and resultant embryo, thereby being crucial for the egg development. We have previously shown that lethal(2)giant larvae (lgl), a Drosophila neoplastic tumor suppressor gene (nTSG) is required for the posterior follicle cell (PFC) fate induction during antero-posterior pattern formation of the follicular epithelium. In this report, we further characterize lgl in this epithelium patterning and the morphogenetic changes of specified border cells. Genetic interactions of lgl with discs large (dlg) and scribble (scrib), another two nTSGs in specifying the PFC fate reveal a cooperative role of this group of genes. Meanwhile, we find that loss of lgl function causes failure of follicle cells at the anterior to differentiate properly. The clonal analysis further indicates that lgl is necessary not only for the border cell differentiation, but also for control of the collective border cell migration via presumably modulating the apico-basal polarity and cell adhesion. Overall, we identify Lgl as an essential factor in regulating differentiation and morphogenetic movement of the ovarian epithelial follicle cells.     

Misshapen decreases integrin levels to promote epithelial motility and planar polarity in Drosophila

Complex organ shapes arise from the coordinate actions of individual cells. The Drosophila egg chamber is an organ-like structure that lengthens along its anterior–posterior axis as it grows. This morphogenesis depends on an unusual form of planar polarity in the organ’s outer epithelial layer, the follicle cells. Interestingly, this epithelium also undergoes a directed migration that causes the egg chamber to rotate around its anterior–posterior axis. However, the functional relationship between planar polarity and migration in this tissue is unknown. We have previously reported that mutations in the Misshapen kinase disrupt follicle cell planar polarity. Here we show that Misshapen’s primary role in this system is to promote individual cell motility. Misshapen decreases integrin levels at the basal surface, which may facilitate detachment of each cell’s trailing edge. These data provide mechanistic insight into Misshapen’s conserved role in cell migration and suggest that follicle cell planar polarity may be an emergent property of individual cell migratory behaviors within the epithelium.     

Actin bundles in human hair follicles as revealed by confocal laser microscopy

A confocal laser microscope was used to examine the distribution pattern of actin bundles in whole-mounts of human hair follicles stained with fluorescently labeled phalloidin. Actin bundles were found exclusively in the epithelial outer root sheath of the lower and middle portions of the follicle. In the growth stage, the lower follicle was characterized by well-developed actin bundles arranged circumferentially in the innermost and outermost cell layers of the outer root sheath. Actin bundles in the innermost cells were aligned end-to-end so that they formed complete circular bands surrounding the inner root sheath. In the outermost cells, actin bundles ran underneath the basal plasma membrane to which they attached at both ends. In contrast, in the quiescent stage, actin bundles in the lower follicle were disposed radially toward the follicle surface where they terminated perpendicular to the basal plasma membrane. In the middle follicle, circumferential actin bundles were found only in the intermediate layer of the outer root sheath throughout the hair cycle. Immunofluorescent anti-myosin and anti-α-actinin staining showed a striated pattern along actin bundles. Vinculin was localized at both ends of actin bundles, corresponding to the cell-to-cell or cell-to-substrate adherens junctions. Glycerinated follicles changed in shape on the addition of MgATP, suggesting a contraction of actin bundles. From these observations, we conclude that actin bundles in the hair follicle are comparable to stress fibers and that they serve as a tensile scaffold for the growth and integrity of the follicle. Received: 6 May 1995 / Accepted: 25 October 1995     

Dynamics of apoptosis in the ovarian follicle cells during the late stages of Drosophila oogenesis

In the present study, we demonstrate the apoptotic events of the ovarian follicle cells during the late stages of oogenesis in Drosophila melanogaster. Follicle cell morphology appears normal from stage 10 up to stage 14, exhibiting a euchromatic nucleus and a well-organized cytoplasm. First signs of apoptosis appear at the anterior pole of the egg chamber at stage 14A. They are characterized by loss of microvilli at the apical cell membrane, alterations in nuclear morphology, such as chromatin condensation and convolution of the nuclear membrane, and also by condensation and vacuolization of the cytoplasm. During the following stage 14B, the follicle cell nuclei contain fragmented DNA as is demonstrated by acridine orange staining and TUNEL (TdT-mediated dUTP nick end-labeling) assay. Finally, the apoptotic follicle cells seem to detach from the eggshell when the mature egg chamber exits the ovariole. The detached follicle cells exhibit condensed nuclear chromatin, a disorganized cytoplasm with crowded organelles and are surrounded by epithelial cells. The above results seem to be associated with the abundant phagocytosis that we observed at the entry of the lateral oviducts, where the epithelial cells contain apoptotic cell bodies. Additionally, we tested the effect of etoposide treatment in the follicular epithelium and found that it induces apoptosis in a stage- and site-specific manner. These observations suggest a possible method of absorption of the apoptotic follicle cells that prevents the blockage of the ovarioles and helps the regular production of mature eggs.     

Methods for identifying and averaging variable molecular conformations in tomograms of actively contracting insect flight muscle

During active muscle contraction, tension is generated through many simultaneous, independent interactions between the molecular motor myosin and the actin filaments. The ensemble of myosin motors displays heterogeneous conformations reflecting different mechanochemical steps of the ATPase pathway. We used electron tomography of actively contracting insect flight muscle fast-frozen, freeze substituted, Araldite embedded, thin-sectioned and stained, to obtain 3D snapshots of the multiplicity of actin-attached myosin structures. We describe procedures for alignment of the repeating lattice of sub-volumes (38.7 nm cross-bridge repeats bounded by troponin) and multivariate data analysis to identify self-similar repeats for computing class averages. Improvements in alignment and classification of repeat sub-volumes reveals (for the first time in active muscle images) the helix of actin subunits in the thin filament and the troponin density with sufficient clarity that a quasiatomic model of the thin filament can be built into the class averages independent of the myosin cross-bridges. We show how quasiatomic model building can identify both strong and weak myosin attachments to actin. We evaluate the accuracy of image classification to enumerate the different types of actin–myosin attachments.     

Programmed cell death of follicular epithelium during the late developmental stages of oogenesis in the fruit flies Bactrocera oleae and Ceratitis capitata (Diptera, Tephritidae) is mediated by autophagy

In the present study, we describe the features of programmed cell death of ovarian follicle cells, occurring during the late developmental stages of oogenesis in the olive fruit fly, Bactrocera oleae and the medfly, Ceratitis capitata. During stage 14, the follicle cells contain autophagic vacuoles, and they do not exhibit caspase activity in all parts of the egg chamber. Their nuclei are characterized by condensed chromatin, accompanied with high- but not low-molecular weight DNA fragmentation events exclusively detected in distinct cells of the anterior pole. These data argue for the presence of an autophagy-mediated cell death program in the ovarian follicle cell layer in both species. The above results are likely associated with the abundant phagocytosis observed at the entry of the lateral oviducts, where numerous cell bodies are massively engulfed by epithelial cells. We strongly believe that during the termination of the above Dipteran oogenesis, an efficient mechanism of absorption of the degenerated follicle cells is selectively activated, in order to prevent the blockage of the ovarioles and thus robustly support the physiological completion of the ovulation process.     

An EM autoradiographic study on ovarian follicle cells of Drosophila melanogaster with special reference to the formation of egg coverings.

Ovarian follicle cells of Drosophila melanogaster have been studied by ultrastructural and autoradiographic analyses. During their migration through the germarium, follicle cells undergo several structural changes and, of these, the most conspicuous one occurs at the level of the nucleolus. By the time the first ovarian chamber is formed, follicle cells have formed a layer of uniform thickness all around a cluster or nurse cells and the oocyte. Following the initiation of vitellogenesis, the follicle cells overlaying the oocyte become columnar while those over the nurse cells become very thin. During stages 9-10, the columnar follicle cells are involved in the formation of the vitelline membrane, while from stages 11 to 13 these cells produce the endochorion. An EM autoradiographic analysis has shown that the rate of 3H-uridine incorporation in follicle cells nuclei is low in previtellogenic chambers, while it becomes very high in nuclei of stage 9-10 chambers. After short exposure to uridine, silver grains are located predominantly over nucleoli. Evidence from incorporation studies with 3H-lysine indicates that the columnar follicle cells and the region of the various egg coverings are highly labelled within an hour of incubation in the tracer. The observations confirm that columnar follicle cells are the only cells in the chamber involved in the formation of materials which make up the egg coverings.     

Regulation of cell proliferation and patterning in Drosophila oogenesis by Hedgehog signaling

The localized expression of Hedgehog (Hh) at the extreme anterior of Drosophila ovarioles suggests that it might provide an asymmetric cue that patterns developing egg chambers along the anteroposterior axis. Ectopic or excessive Hh signaling disrupts egg chamber patterning dramatically through primary effects at two developmental stages. First, excess Hh signaling in somatic stem cells stimulates somatic cell over-proliferation. This likely disrupts the earliest interactions between somatic and germline cells and may account for the frequent mis-positioning of oocytes within egg chambers. Second, the initiation of the developmental programs of follicle cell lineages appears to be delayed by ectopic Hh signaling. This may account for the formation of ectopic polar cells, the extended proliferation of follicle cells and the defective differentiation of posterior follicle cells, which, in turn, disrupts polarity within the oocyte. Somatic cells in the ovary cannot proliferate normally in the absence of Hh or Smoothened activity. Loss of protein kinase A activity restores the proliferation of somatic cells in the absence of Hh activity and allows the formation of normally patterned ovarioles. Hence, localized Hh is not essential to direct egg chamber patterning.     

Visualization of stimulus‐specific heterogeneous activation of individual vascular smooth muscle cells in aortic tissues

Intercellular communication among autonomic nerves, endothelial cells (ECs), and vascular smooth muscle cells (VSMCs) plays a central role in an uninterrupted regulation of blood flow through vascular contractile machinery. Impairment of this communication is linked to development of vascular diseases such as hypertension, cerebral/coronary vasospasms, aortic aneurism, and erectile dysfunction. Although the basic concept of the communication as a whole has been studied, the spatiotemporal correlation of ECs/VSMCs in tissues at the cellular level is unknown. Here, we show a unique VSMC response to ECs during contraction and relaxation of isolated aorta tissues through visualization of spatiotemporal activation patterns of smooth muscle myosin II. ECs in the intimal layer dictate the stimulus‐specific heterogeneous activation pattern of myosin II in VSMCs within distinct medial layers. Myosin light chain (MLC) phosphorylation (active form of myosin II) gradually increases towards outer layers (approximately threefold higher MLC phosphorylation at the outermost layer than that of the innermost layer), presumably by release of an intercellular messenger, nitric oxide (NO). Our study also demonstrates that the MLC phosphorylation at the outermost layer in spontaneously hypertensive rats (SHR) during NO‐induced relaxation is quite high and approximately 10‐fold higher than that of its counterpart, the Wister–Kyoto rats (WKY), suggesting that the distinct pattern of myosin II activation within tissues is important for vascular protection against elevated blood pressure.     

The Hippo pathway controls polar cell fate through Notch signaling during Drosophila oogenesis

During Drosophila oogenesis, the somatic follicle cells form an epithelial layer surrounding the germline cells to form egg chambers. In this process, follicle cell precursors are specified into polar cells, stalk cells, and main-body follicle cells. Proper specification of these three cell types ensures correct egg chamber formation and polarization of the anterior–posterior axis of the germline cells. Multiple signaling cascades coordinate to control the follicle cell fate determination, including Notch, JAK/STAT, and Hedgehog signaling pathways. Here, we show that the Hippo pathway also participates in polar cell specification. Over-activation of yorkie (yki) leads to egg chamber fusion, possibly through attenuation of polar cell specification. Loss-of-function experiments using RNAi knockdown or generation of mutant clones by mitotic recombination demonstrates that reduction of yki expression promotes polar cell formation in a cell-autonomous manner. Consistently, polar cells mutant for hippo (hpo) or warts (wts) are not properly specified, leading to egg chamber fusion. Furthermore, Notch activity is increased in yki mutant cells and reduction of Notch activity suppresses polar cell formation in yki mutant clones. These results demonstrate that yki represses polar cell fate through Notch signaling. Collectively, our data reveal that the Hippo pathway controls polar cell specification. Through repressing Notch activity, Yki serves as a key repressor in specifying polar cells during Drosophila oogenesis.     

An EM autoradiographic study on ovarian follicle cells of Drosophila melanogaster with special reference to the formation of egg coverings

Summary Ovarian follicle cells of Drosophila melanogaster have been studied by ultrastructural and autoradiographic analyses.During their migration through the germarium, follicle cells undergo several structural changes and, of these, the most conspicuous one occurs at the level of the nucleolus. By the time the first ovarian chamber is formed, follicle cells have formed a layer of uniform thickness all around a cluster or nurse cells and the oocyte. Following the initiation of vitellogenesis, the follicle cells overlying the oocyte become columnar while those over the nurse cells become very thin. During stages 9–10, the columnar follicle cells are involved in the formation of the vitelline membrane, while from stages 11 to 13 these cells produce the endochorion.An EM autoradiographic analysis has shown that the rate of ³H-uridine incroporation in follicle cell nuclei is low in previtellogenic chambers, while it becomes very high in nuclei of stage 9–10 chambers. After short exposure to uridine, silver grains are located predominantly over nucleoli.Evidence from incorporation studies with ³H-lysine indicates that the columnar follicle cells and the region of the various egg coverings are highly labelled within an hour of incubation in the tracer.The observations confirm that columnar follicle cells are the only cells in the chamber involved in the formation of materials which make up the egg coverings.This work was partly supported by C.N.R. (Italy)I am indebted to Dr. J. Jacob from the Institute of Animal Genetics (Edinburgh) for introducing me to the use of EM autoradiography     

Upright Imaging of Drosophila Egg Chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.     

The development of the hexagonally structured egg envelope of the C-O sole (Pleuronichthys coenosus)

The surface of a mature, pelagic C-O sole egg is composed of polygonal chambers having four to eight sides, most of which are hexagonally shaped. This honeycomb pattern initially appears on primary oocytes as a thin layer of compact, electron-dense material. Discrete thickenings begin to develop on the envelope of perinuclear stage oocytes. The thickenings lengthen and thin to form the hexagonal walls of the envelope in oocytes undergoing yolk vesicle formation. The walls of each hexagonal chamber occur in an area corresponding to the lateral margins of the adjacent follicle cell, suggesting that the hexagonal walls are produced by the follicle cells. The hexagonal layer is nearly complete at the beginning of vitellogenesis, and as vitellogenesis continues, a striated envelope layer composed of fibrillar lamellae develops between the oocyte and the hexagonal layer. The striated layer appears to be secreted by the oocyte. After vitellogenesis is completed, oocytes are ovulated and double in size during a period of maturation. Concurrently, the striated primary envelope stretches and thins into eight to nine horizontal lamellae. On the mature egg surface, the polygonal chambers are about 24–31 μm in diameter. Within each chamber there is a subpattern of polygonal areas; each polygon is 1.5–2.0 μm in diameter, and circumscribes a pore canal opening. This exceptional envelope may furnish the egg with some degree of protection, resiliency, and buoyancy, but its specific functions are not known.     

Eyes absent,a key repressor of polar cell fate during Drosophila oogenesis

Throughout Drosophila oogenesis, specialized somatic follicle cells perform crucial functions in egg chamber formation and in signaling between somatic and germline cells. In the ovary, at least three types of somatic follicle cells, polar cells, stalk cells and main body epithelial follicle cells, can be distinguished when egg chambers bud from the germarium. Although specification of these three somatic cell types is important for normal oogenesis and subsequent embryogenesis, the molecular basis for establishment of their cell fates is not completely understood. Our studies reveal the gene eyes absent (eya) to be a key repressor of polar cell fate. EYA is a nuclear protein that is normally excluded from polar and stalk cells, and the absence of EYA is sufficient to cause epithelial follicle cells to develop as polar cells. Furthermore, ectopic expression of EYA is capable of suppressing normal polar cell fate and compromising the normal functions of polar cells, such as promotion of border cell migration. Finally, we show that ectopic Hedgehog signaling, which is known to cause ectopic polar cell formation, does so by repressing eya expression in epithelial follicle cells.     

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.     

Using optogenetics to link myosin patterns to contractile cell behaviors during convergent extension

Distinct patterns of actomyosin contractility are often associated with particular epithelial tissue shape changes during development. For example, a planar-polarized pattern of myosin II localization regulated by Rho1 signaling during Drosophila body axis elongation is thought to drive cell behaviors that contribute to convergent extension. However, it is not well understood how specific aspects of a myosin pattern influence the multiple cell behaviors, including cell intercalation, cell shape changes, and apical cell area fluctuations, that simultaneously occur during morphogenesis. Here, we developed two optogenetic tools, optoGEF and optoGAP, to activate or deactivate Rho1 signaling, respectively. We used these tools to manipulate myosin patterns at the apical side of the germband epithelium during Drosophila axis elongation and analyzed the effects on contractile cell behaviors. We show that uniform activation or inactivation of Rho1 signaling across the apical surface of the germband is sufficient to disrupt the planar-polarized pattern of myosin at cell junctions on the timescale of 3–5 min, leading to distinct changes in junctional and medial myosin patterns in optoGEF and optoGAP embryos. These two perturbations to Rho1 activity both disrupt axis elongation and cell intercalation but have distinct effects on cell area fluctuations and cell packings that are linked with changes in the medial and junctional myosin pools. These studies demonstrate that acute optogenetic perturbations to Rho1 activity are sufficient to rapidly override the endogenous planar-polarized myosin pattern in the germband during axis elongation. Moreover, our results reveal that the levels of Rho1 activity and the balance between medial and junctional myosin play key roles not only in organizing the cell rearrangements that are known to directly contribute to axis elongation but also in regulating cell area fluctuations and cell packings, which have been proposed to be important factors influencing the mechanics of tissue deformation and flow.