Role of Notch pathway in terminal follicle cell differentiation during Drosophila oogenesis

 During Drosophila oogenesis the body axes are determined by signaling between the oocyte and the somatic follicle cells that surround the egg chamber. A key event in the establishment of oocyte anterior-posterior polarity is the differential patterning of the follicle cell epithelium along the anterior-posterior axis. Both the Notch and epithelial growth factor (EGF) receptor pathways are required for this patterning. To understand how these pathways act in the process we have analyzed markers for anterior and posterior follicle cells accompanying constitutive activation of the EGF receptor, loss of Notch function, and ectopic expression of Delta. We find that a constitutively active EGF receptor can induce posterior fate in anterior but not in lateral follicle cells, showing that the EGF receptor pathway can act only on predetermined terminal cells. Furthermore, Notch function is required at both termini for appropriate expression of anterior and posterior markers, while loss of both the EGF receptor and Notch pathways mimic the Notch loss-of-function phenotype. Ectopic expression of the Notch ligand, Delta, disturbs EGF receptor dependent posterior follicle cell differentiation and anterior-posterior polarity of the oocyte. Our data are consistent with a model in which the Notch pathway is required for early follicle cell differentiation at both termini, but is then repressed at the posterior for proper determination of the posterior follicle cells by the EGF receptor pathway. Received: 5 November 1998 / Accepted: 14 December 1998     

The Drosophila ecdysone receptor (EcR) gene is required maternally for normal oogenesis

Oogenesis in Drosophila is regulated by the steroid hormone ecdysone and the sesquiterpenoid juvenile hormone. Response to ecdysone is mediated by a heteromeric receptor composed of the EcR and USP proteins. We have identified a temperature-sensitive EcR mutation, EcR(A483T), from a previously isolated collection of EcR mutations. EcR(A483T) is predicted to affect all EcR protein products (EcR-A, EcR-B1, and EcR-B2) since it maps to a common exon encoding the ligand-binding domain. In wild-type females, we find that both EcR-A and EcR-B1 are expressed in nurse cells and follicle cells throughout oogenesis. EcR mutant females raised at permissive temperature and then shifted to restrictive temperature exhibit severe reductions in fecundity. Oogenesis in EcR mutant females is defective, and the spectrum of oogenic defects includes the presence of abnormal egg chambers and loss of vitellogenic egg stages. Our results demonstrate a requirement for EcR during female reproduction and suggest that EcR is required for normal oogenesis.     

Drosophila tubulin-binding cofactor B is required for microtubule network formation and for cell polarity

Microtubules (MTs) are essential for cell division, shape, intracellular transport, and polarity. MT stability is regulated by many factors, including MT-associated proteins and proteins controlling the amount of free tubulin heterodimers available for polymerization. Tubulin-binding cofactors are potential key regulators of free tubulin concentration, since they are required for α-β-tubulin dimerization in vitro. In this paper, we show that mutation of the Drosophila tubulin-binding cofactor B (dTBCB) affects the levels of both α- and β-tubulins and dramatically destabilizes the MT network in different fly tissues. However, we find that dTBCB is dispensable for the early MT-dependent steps of oogenesis, including cell division, and that dTBCB is not required for mitosis in several tissues. In striking contrast, the absence of dTBCB during later stages of oogenesis causes major defects in cell polarity. We show that dTBCB is required for the polarized localization of the axis-determining mRNAs within the oocyte and for the apico-basal polarity of the surrounding follicle cells. These results establish a developmental function for the dTBCB gene that is essential for viability and MT-dependent cell polarity, but not cell division.     

The neurogenic locus brainiac cooperates with the Drosophila EGF receptor to establish the ovarian follicle and to determine its dorsal-ventral polarity.

We have characterized the function of a new neurogenic locus, brainiac (brn), during oogenesis. Homozygous brn females lay eggs with fused dorsal appendages, a phenotype associated with torpedo (top) alleles of the Drosophila EGF receptor (DER) locus. By constructing double mutant females for both brn and top, we have found that brn is required for determining the dorsal-ventral polarity of the ovarian follicle. However, embryos from mature brn eggs develop a neurogenic phenotype which can be zygotically rescued if a wild-type sperm fertilizes the egg. This is the first instance of a Drosophila gene required for determination of dorsal-ventral follicle cell fates that is not required for determination of embryonic dorsal-ventral cell fates. The temperature-sensitive period for brn dorsal-ventral patterning begins at the inception of vitellogenesis. The interaction between brn and DER is also required for at least two earlier follicle cell activities which are necessary to establish the ovarian follicle. Prefollicular cells fail to migrate between each oocyte/nurse cell complex, resulting in follicles with multiple sets of oocytes and nurse cells. brn and DER function is also required for establishing and/or maintaining a continuous follicular epithelium around each oocyte/nurse cell complex. These brn functions as well as the brn requirement for determination of dorsal-ventral polarity appear to be genetically separable functions of the brn locus. Genetic mosaic experiments show that brn is required in the germline during these processes whereas the DER is required in the follicle cells. We propose that brn may be part of a germline signaling pathway differentially regulating successive DER-dependent follicle cell activities of migration, division and/or adhesion and determination during oogenesis. These experiments indicate that brn is required in both tyrosine kinase and neurogenic intercellular signaling pathways. Moreover, the functions of brn in oogenesis are distinct from those of Notch and Delta, two other neurogenic loci that are known to be required for follicular development.     

Laminin A is required for follicle cell-oocyte signaling that leads to establishment of the anterior-posterior axis in Drosophila

The establishment of the anterior-posterior (AP) axis in Drosophila melanogaster requires signaling between the oocyte and surrounding somatic follicle cells during oogenesis [1] [2]. First, a signal from the oocyte (Gurken (Grk), a transforming growth factor-alpha (TGFalpha) homolog) is received by predetermined terminal follicle cells in which the epidermal growth factor receptor (EGFR) pathway is activated and a posterior fate is induced [2] [3] [4]. Later, the posterior follicle cells send an unidentified signal back to the oocyte, which leads to the reorganization of its cytoskeletal polarity. This reorganization is required for proper localization of maternal determinants, such as oskar (osk) and bicoid (bcd) mRNAs, that determine the AP polarity of the oocyte and the subsequent embryo [2]. We show here that when the gene lanA, which encodes the extracellular matrix component laminin A, is mutated in posterior follicle cells, localization of AP determinants is disrupted in the underlying oocyte. Posterior follicle-cell differentiation and follicle cell apical-basal polarity are unaffected in the lanA mutant cells, suggesting that laminin A is required for correct signaling from the posterior follicle cells that polarizes the oocyte. This is the first evidence that the extracellular matrix is involved in the establishment of a major body axis.     

Opposing effects of Notch-signaling in maintaining the proliferative state of follicle cells in the telotrophic ovary of the beetle Tribolium

ABSTRACT: INTRODUCTION: Establishment of distinct follicle cell fates at the early stages of Drosophila oogenesis is crucial for achieving proper morphology of individual egg chambers. In Drosophila oogenesis, Notch-signaling controls proliferation and differentiation of follicular cells, which eventually results in the polarization of the anterior-posterior axis of the oocyte. Here we analyzed the functions of Tribolium Notch-signaling factors during telotrophic oogenesis, which differs fundamentally from the polytrophic ovary of Drosophila. RESULTS: We found Notch-signaling to be required for maintaining the mitotic cycle of somatic follicle cells. Upon Delta RNAi, follicle cells enter endocycle prematurely, which affects egg-chamber formation and patterning. Interestingly, our results indicate that Delta RNAi phenotypes are not solely due to the premature termination of cell proliferation. Therefore, we monitored the terminal /stalk cell precursor lineage by molecular markers. We observed that upon Delta RNAi terminal and stalk cell populations were absent, suggesting that Notch-signaling is also required for the specification of follicle cell populations, including terminal and stalk precursor cells. CONCLUSIONS: We demonstrate that with respect to mitotic cycle/endocycle switch Notch-signaling in Tribolium and Drosophila has opposing effects. While in Drosophila a Delta-signal brings about the follicle cells to leave mitosis, Notch-signaling in Tribolium is necessary to retain telotrophic egg-chambers in an "immature" state. In most instances, Notch-signaling is involved in maintaining undifferentiated (or preventing specialized) cell fates. Hence, the role of Notch in Tribolium may reflect the ancestral function of Notch-signaling in insect oogenesis. The functions of Notch-signaling in patterning the follicle cell epithelium suggest that Tribolium oogenesis may - analogous to Drosophila - involve the stepwise determination of different follicle cell populations. Moreover, our results imply that Notch-signaling may contribute at least to some aspects of oocyte polarization and AP axis also in telotrophic oogenesis.     

Role of neurogenic genes in establishment of follicle cell fate and oocyte polarity during oogenesis in Drosophila

Oogenesis in Drosophila involves specification of both germ cells and the surrounding somatic follicle cells, as well as the determination of oocyte polarity. We found that two neurogenic genes, Notch and Delta, are required in oogenesis. These genes encode membrane proteins with epidermal growth factor repeats and are essential in the decision of an embryonic ectodermal cell to take on the fate of neuroblast or epidermoblast. In oogenesis, mutation in either gene leads to an excess of posterior follicle cells, a cell fate change reminiscent of the hyperplasia of neuroblasts seen in neurogenic mutant embryos. Furthermore, the Notch mutation in somatic cells causes mislocalization of bicoid in the oocyte. These results suggest that the neurogenic genes Notch and Delta are involved in both follicle cell development and the establishment of anterior-posterior polarity in the oocyte.     

Drosophila RalA is essential for the maintenance of Jak/Stat signalling in ovarian follicles

Small GTPases of the Ras-like (Ral) family are crucial for signalling functions in both normal and cancer cells; however, their role in a developing organism is poorly understood. Here, we identify the Drosophila Ral homologue RalA as a new key regulator of polar-cell differentiation during oogenesis. Polar cells have a crucial role in patterning the egg chamber and in recruiting border cells, which undergo collective and guided migration. We show that RalA function is essential for the maintenance of anterior and posterior polar-cell fate and survival. RalA is required cell autonomously to control the expression of polar-cell-specific markers, including the Jak/Stat ligand Unpaired. The loss of RalA also causes a cell non-autonomous phenotype owing to reduced Jak/Stat signalling in neighbouring follicle cells. As a result, border-cell assembly and migration as well as the polarization of the oocyte are defective. Thus, RalA is required in organizing centres to control proper patterning and migration in vivo.     

fringe and Notch specify polar cell fate during Drosophila oogenesis

fringe encodes a glycosyltransferase that modulates the ability of the Notch receptor to be activated by its ligands. We describe studies of fringe function during early stages of Drosophila oogenesis. Animals mutant for hypomorphic alleles of fringe contain follicles with an incorrect number of germline cells, which are separated by abnormally long and disorganized stalks. Analysis of clones of somatic cells mutant for a null allele of fringe localizes the requirement for fringe in follicle formation to the polar cells, and demonstrates that fringe is required for polar cell fate. Clones of cells mutant for Notch also lack polar cells and the requirement for Notch in follicle formation appears to map to the polar cells. Ectopic expression of fringe or of an activated form of Notch can generate an extra polar cell. Our results indicate that fringe plays a key role in positioning Notch activation during early oogenesis, and establish a function for the polar cells in separating germline cysts into individual follicles.     

Misexpression of argos, an inhibitor of EGFR signaling in oogenesis, leads to the production of bicephalic, ventralized, and lateralized Drosophila melanogaster eggs

Epidermal growth factor receptor (EGFR) signaling pathways are frequently involved in generating cell fate diversity in a number of organisms. During anterior-posterior and dorso-ventral polarity in the Drosophila egg chamber and eggshell, EGFR signaling leads to a number of determinative events in the follicle cell layer. A high level of Gurken signal leads to the expression of argos in dorsal midline cells. Lateral follicle cells, receiving a lower level of Gurken signal, can continue to express the Broad-Complex (BR-C) and differentiate into cells which produce chorionic appendages. Misexpression of argos in mid-oogenesis causes the midline cells to retain expression of BR-C, resulting in a single fused large appendage. Evidence that argos can directly repress Gurken-induced EGFR signaling is seen when premature expression of argos is induced earlier in oogenesis. It represses the Gurken signal at stage 5-6 of oogenesis which determines posterior follicle cells and occasionally leads to eggs with anteriors at both ends. We propose that the Gurken signal at stage 9 of oogenesis induces follicle cells to take on two fates, dorsal midline and lateral, each producing different parts of the eggshell and that argos is one of the key downstream genes required to select between these two fates.     

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.     

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.     

Dystroglycan is required for polarizing the epithelial cells and the oocyte in Drosophila

The transmembrane protein Dystroglycan is a central element of the dystrophin-associated glycoprotein complex, which is involved in the pathogenesis of many forms of muscular dystrophy. Dystroglycan is a receptor for multiple extracellular matrix (ECM) molecules such as Laminin, agrin and perlecan, and plays a role in linking the ECM to the actin cytoskeleton; however, how these interactions are regulated and their basic cellular functions are poorly understood. Using mosaic analysis and RNAi in the model organism Drosophila melanogaster, we show that Dystroglycan is required cell-autonomously for cellular polarity in two different cell types, the epithelial cells (apicobasal polarity) and the oocyte (anteroposterior polarity). Loss of Dystroglycan function in follicle and disc epithelia results in expansion of apical markers to the basal side of cells and overexpression results in a reduced apical localization of these same markers. In Dystroglycan germline clones early oocyte polarity markers fail to be localized to the posterior, and oocyte cortical F-actin organization is abnormal. Dystroglycan is also required non-cell-autonomously to organize the planar polarity of basal actin in follicle cells, possibly by organizing the Laminin ECM. These data suggest that the primary function of Dystroglycan in oogenesis is to organize cellular polarity; and this study sets the stage for analyzing the Dystroglycan complex by using the power of Drosophila molecular genetics.     

Investigating the function of follicular subpopulations during Drosophila oogenesis through hormone-dependent enhancer-targeted cell ablation

Although it is known that the establishment of polarity during Drosophila oogenesis is initiated by signalling from the oocyte to the overlying follicle cells, much less is understood about the role of specific follicular subpopulations. One powerful approach for addressing this question, toxigenic cell ablation of specific subpopulations, has not previously been applicable to studying follicular subpopulations because many of the genes and Gal4 enhancer trap insertions that are expressed in the ovary are also expressed at earlier times in development. To overcome this problem, we have utilized a fusion protein between Gal4 and the human estrogen receptor to achieve hormone-dependent, tissue-specific gene expression of UAS-linked transgenes in flies. We used this system to study the role of the polar subpopulations of follicle cells during oogenesis by expressing within them a modified form of diphtheria toxin that causes cell death. Our results confirmed previous functions ascribed to these cells, and also demonstrated a previously undescribed role for the border cells in facilitating the migration of the anterior Fasciclin III-expressing polar pair cells to the edge of the oocyte.     

Drosophila par-1 is required for oocyte differentiation and microtubule organization

BACKGROUND: Drosophila oocyte determination involves a complex process by which a single cell within an interconnected cyst of 16 germline cells differentiates into an oocyte. This process requires the asymmetric accumulation of both specific messenger RNAs and proteins within the future oocyte as well as the proper organization of the microtubule cytoskeleton, which together with the fusome provides polarity within the developing germline cyst. RESULTS: In addition to its previously described late oogenic role in the establishment of anterior-posterior polarity and subsequent embryonic axis formation, the Drosophila par-1 gene is required very early in the germline for establishing cyst polarity and for oocyte specification. Germline clonal analyses, for which we used a protein null mutation, reveal that Drosophila par-1 (par-1) is required for the asymmetric accumulation of oocyte-specific factors as well as the proper organization of the microtubule cytoskeleton. Similarly, somatic clonal analyses indicate that par-1 is required for microtubule stabilization in follicle cells. The PAR-1 protein is localized to the fusome and ring canals within the developing germline cyst in direct contact with microtubules. Likewise, in the follicular epithelium, PAR-1 colocalizes with microtubules along the basolateral membrane. However, in either case PAR-1 localization is independent of microtubules. CONCLUSIONS: The Drosophila par-1 gene plays at least two essential roles during oogenesis; it is required early in the germline for organization of the microtubule cytoskeleton and subsequent oocyte determination, and it has a second, previously described role late in oogenesis in axis formation. In both cases, par-1 appears to exert its effects through the regulation of microtubule dynamics and/or stability, and this finding is consistent with the defined role of the mammalian PAR-1 homologs.     

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.     

Functioning of the Drosophila orb gene in gurken mRNA localization and translation.

The orb gene encodes an RNA recognition motif (RRM)-type RNA-binding protein that is a member of the cytoplasmic polyadenylation element binding protein (CPEB) family of translational regulators. Early in oogenesis, orb is required for the formation and initial differentiation of the egg chamber, while later in oogenesis it functions in the determination of the dorsoventral (DV) and anteroposterior axes of egg and embryo. In the studies reported here, we have examined the role of the orb gene in the gurken (grk)-Drosophila epidermal growth factor receptor (DER) signaling pathway. During the previtellogenic stages of oogenesis, the grk-DER signaling pathway defines the posterior pole of the oocyte by specifying posterior follicle cell identity. This is accomplished through the localized expression of Grk at the very posterior of the oocyte. Later in oogenesis, the grk-DER pathway is used to establish the DV axis. Grk protein synthesized at the dorsal anterior corner of the oocyte signals dorsal fate to the overlying follicle cell epithelium. We show that orb functions in both the early and late grk-DER signaling pathways, and in each case is required for the localized expression of Grk protein. We have found that orb is also required to promote the synthesis of a key component of the DV polarity pathway, K(10). Finally, we present evidence that Orb protein expression during the mid- to late stages of oogenesis is, in turn, negatively regulated by K(10).