Correction: Spatial regulation of Drosophila ovarian Follicle Stem Cell division rates and cell cycle transitions.

[This corrects the article DOI: 10.1371/journal.pgen.1010965.].

Spatial regulation of Drosophila ovarian Follicle Stem Cell division rates and cell cycle transitions.

Drosophila ovarian Follicle Stem Cells (FSCs) present a favorable paradigm for understanding how stem cell division and differentiation are balanced in communities where those activities are independent. FSCs also allow exploration of how this balance is integrated with spatial stem cell heterogeneity. Posterior FSCs become proliferative Follicle Cells (FCs), while anterior FSCs become quiescent Escort Cells (ECs) at about one fourth the frequency. A single stem cell can nevertheless produce both FCs and ECs because it can move between anterior and posterior locations. Studies based on EdU incorporation to approximate division rates suggested that posterior FSCs divide faster than anterior FSCs. However, direct measures of cell cycle times are required to ascertain whether FC output requires a net flow of FSCs from anterior to posterior. Here, by using live imaging and FUCCI cell-cycle reporters, we measured absolute division rates. We found that posterior FSCs cycle more than three times faster than anterior FSCs and produced sufficient new cells to match FC production. H2B-RFP dilution studies supported different cycling rates according to A/P location and facilitated live imaging, showing A/P exchange of FSCs in both directions, consistent with the dynamic equilibrium inferred from division rate measurements. Inversely graded Wnt and JAK-STAT pathway signals regulate FSC differentiation to ECs and FCs. JAK-STAT promotes both differentiation to FCs and FSC cycling, affording some coordination of these activities. When JAK-STAT signaling was manipulated to be spatially uniform, the ratio of posterior to anterior division rates was reduced but remained substantial, showing that graded JAK-STAT signaling only partly explains the graded cycling of FSCs. By using FUCCI markers, we found a prominent G2/M cycling restriction of posterior FSCs together with an A/P graded G1/S restriction, that JAK-STAT signaling promotes both G1/S and G2/M transitions, and that PI3 kinase signaling principally stimulates the G2/M transition.

Single-cell expression profile of Drosophila ovarian follicle stem cells illuminates spatial differentiation in the germarium.

BACKGROUND: How stem cell populations are organized and regulated within adult tissues is important for understanding cancer origins and for developing cell replacement strategies. Paradigms such as mammalian gut stem cells and Drosophila ovarian follicle stem cells (FSC) are characterized by population asymmetry, in which stem cell division and differentiation are separately regulated processes. These stem cells behave stochastically regarding their contributions to derivative cells and also exhibit dynamic spatial heterogeneity. Drosophila FSCs provide an excellent model for understanding how a community of active stem cells maintained by population asymmetry is regulated. Here, we use single-cell RNA sequencing to profile the gene expression patterns of FSCs and their immediate derivatives to investigate heterogeneity within the stem cell population and changes associated with differentiation. RESULTS: We describe single-cell RNA sequencing studies of a pre-sorted population of cells that include FSCs and the neighboring cell types, escort cells (ECs) and follicle cells (FCs), which they support. Cell-type assignment relies on anterior-posterior (AP) location within the germarium. We clarify the previously determined location of FSCs and use spatially targeted lineage studies as further confirmation. The scRNA profiles among four clusters are consistent with an AP progression from anterior ECs through posterior ECs and then FSCs, to early FCs. The relative proportion of EC and FSC clusters are in good agreement with the prevalence of those cell types in a germarium. Several genes with graded profiles from ECs to FCs are highlighted as candidate effectors of the inverse gradients of the two principal signaling pathways, Wnt and JAK-STAT, that guide FSC differentiation and division. CONCLUSIONS: Our data establishes an important resource of scRNA-seq profiles for FSCs and their immediate derivatives that is based on precise spatial location and functionally established stem cell identity, and facilitates future genetic investigation of regulatory interactions guiding FSC behavior.

Division-independent differentiation mandates proliferative competition among stem cells.

Cancer-initiating gatekeeper mutations that arise in stem cells would be especially potent if they stabilize and expand an affected stem cell lineage. It is therefore important to understand how different stem cell organization strategies promote or prevent variant stem cell amplification in response to different types of mutation, including those that activate proliferation. Stem cell numbers can be maintained constant while producing differentiated products through individually asymmetrical division outcomes or by population asymmetry strategies in which individual stem cell lineages necessarily compete for niche space. We considered alternative mechanisms underlying population asymmetry and used quantitative modeling to predict starkly different consequences of altering proliferation rate: A variant, faster proliferating mutant stem cell should compete better only when stem cell division and differentiation are independent processes. For most types of stem cells, it has not been possible to ascertain experimentally whether division and differentiation are coupled. However, Drosophila follicle stem cells (FSCs) provided a favorable system with which to investigate population asymmetry mechanisms and also for measuring the impact of altered proliferation on competition. We found from detailed cell lineage studies that division and differentiation of an individual FSC are not coupled. We also found that FSC representation, reflecting maintenance and amplification, was highly responsive to genetic changes that altered only the rate of FSC proliferation. The FSC paradigm therefore provides definitive experimental evidence for the general principle that relative proliferation rate will always be a major determinant of competition among stem cells specifically when stem cell division and differentiation are independent.

Yorkie and Hedgehog independently restrict BMP production in escort cells to permit germline differentiation in the Drosophila ovary.

Multiple signaling pathways guide the behavior and differentiation of both germline stem cells (GSCs) and somatic follicle stem cells (FSCs) in the Drosophila germarium, necessitating careful control of signal generation, range and responses. Signal integration involves escort cells (ECs), which promote differentiation of the GSC derivatives they envelop, provide niche signals for FSCs and derive directly from FSCs in adults. Hedgehog (Hh) signaling induces the Hippo pathway effector Yorkie (Yki) to promote proliferation and maintenance of FSCs, but Hh also signals to ECs, which are quiescent. Here, we show that in ECs both Hh and Yki limit production of BMP ligands to allow germline differentiation. Loss of Yki produced a more severe germarial phenotype than loss of Hh signaling and principally induced a different BMP ligand. Moreover, Yki activity reporters and epistasis tests showed that Yki does not mediate the key actions of Hh signaling in ECs. Thus, both the coupling and output of the Hh and Yki signaling pathways differ between FSCs and ECs despite their proximity and the fact that FSCs give rise directly to ECs.

Contributions of Costal 2-Fused interactions to Hedgehog signaling in Drosophila.

The Drosophila kinesin-family protein Costal 2 (Cos2) and its mammalian ortholog Kif7 play dual roles in Hedgehog (Hh) signaling. In the absence of Hh, Cos2 and Kif7 contribute to proteolytic processing and silencing of the Hh-regulated transcription factors, Drosophila Cubitus interruptus (Ci) and mammalian Gli proteins. Cos2 and Kif7 are also necessary for full activation of full-length Ci-155 and Gli transcription factors in response to Hh proteins. Here, we use classical fused alleles and transgenic Cos2 products deficient for Fused (Fu) association to show that Cos2 must bind to Fu to support efficient Ci-155 processing. Residual Ci-155 processing in the absence of Cos2-Fu interaction did not require Suppressor of Fused, which has been implicated in processing mammalian Gli proteins. We also provide evidence that Cos2 binding to the CORD domain of Ci-155 contributes to both Ci-155 processing and Ci-155 silencing in the absence of Hh. In the presence of Hh, Ci-155 processing is blocked and Cos2 now promotes activation of Ci-155, which requires Fu kinase activity. Here, we show that normal Ci-155 activation by Hh requires Cos2 binding to Fu, supporting the hypothesis that Cos2 mediates the apposition of Fu molecules suitable for cross-phosphorylation and consequent full activation of Fu kinase. We also find that phosphorylation of Cos2 by Fu at two previously mapped sites, S572 and S931, which is thought to mediate Ci-155 activation, is not required for normal activation of Ci-155 by Hh or by activated Fu.

Regulation of mammalian Gli proteins by Costal 2 and PKA in Drosophila reveals Hedgehog pathway conservation.

Hedgehog (Hh) signaling activates full-length Ci/Gli family transcription factors and prevents Ci/Gli proteolytic processing to repressor forms. In the absence of Hh, Ci/Gli processing is initiated by direct Pka phosphorylation. Despite those fundamental similarities between Drosophila and mammalian Hh pathways, the differential reliance on cilia and some key signal transduction components had suggested a major divergence in the mechanisms that regulate Ci/Gli protein activities, including the role of the kinesin-family protein Costal 2 (Cos2), which directs Ci processing in Drosophila. Here, we show that Cos2 binds to three regions of Gli1, just as for Ci, and that Cos2 functions to silence mammalian Gli1 in Drosophila in a Hh-regulated manner. Cos2 and the mammalian kinesin Kif7 can also direct Gli3 and Ci processing in fly, underscoring a fundamental conserved role for Cos2 family proteins in Hh signaling. We also show that direct PKA phosphorylation regulates the activity, rather than the proteolysis of Gli in Drosophilia, and we provide evidence for an analogous action of PKA on Ci.

Regulation of Ci-SCFSlimb binding, Ci proteolysis, and hedgehog pathway activity by Ci phosphorylation.

Hedgehog (Hh) proteins signal by inhibiting the proteolytic processing of Ci/Gli family transcription factors and by increasing Ci/Gli-specific activity. When Hh is absent, phosphorylation of Ci/Gli triggers binding to SCF ubiquitin ligase complexes and consequent proteolysis. Here we show that multiple successively phosphorylated CK1 sites on Ci create an atypical extended binding site for the SCF substrate recognition component Slimb. GSK3 enhances binding primarily through a nearby region of Ci, which might contact an SCF component other than Slimb. Studies of Ci variants with altered CK1 and GSK3 sites suggest that the large number of phosphorylation sites that direct SCF(Slimb) binding confers a sensitive and graded proteolytic response to Hh, which collaborates with changes in Ci-specific activity to elicit a morphogenetic response. We also show that when Ci proteolysis is compromised, its specific activity is limited principally by Su(fu), and not by Cos2 cytoplasmic tethering or PKA phosphorylation.

Drosophila Smoothened phosphorylation sites essential for Hedgehog signal transduction.

The Hedgehog (Hh) signalling pathway is crucial for animal development and is aberrantly activated in several types of cancer. In Drosophila melanogaster, Hh signalling regulates target gene expression through the transcription factor Cubitus interruptus (Ci). Together, Protein Kinase A, Casein Kinase 1 and Glycogen Synthase Kinase 3 silence the pathway in the absence of ligand by phosphorylating Ci at a defined cluster of sites, thereby promoting its proteolytic conversion to a transcriptional repressor (Ci-75). In the presence of Hh, Ci-155 is no longer converted to Ci-75 and its ability to activate transcription is potentiated. All Hh responses require the seven transmembrane domain protein Smoothened, which itself becomes hyperphosphorylated during Hh signalling. Here we show that a cluster of protein kinase A and protein kinase A-primed casein kinase 1 phosphorylation sites in Smoothened, similarly distributed to those regulating Ci, are essential for Smoothened to transduce a Hh signal and for normal regulation of Smoothened protein levels.

Cyclin E-dependent protein kinase activity regulates niche retention of Drosophila ovarian follicle stem cells.

Whether stem cells have unique cell cycle machineries and how they integrate with niche interactions remains largely unknown. We identified a hypomorphic cyclin E allele WX that strongly impairs the maintenance of follicle stem cells (FSCs) in the Drosophila ovary but does not reduce follicle cell proliferation or germline stem cell maintenance. CycE(WX) protein can still bind to the cyclin-dependent kinase catalytic subunit Cdk2, but forms complexes with reduced protein kinase activity measured in vitro. By creating additional CycE variants with different degrees of kinase dysfunction and expressing these and CycE(WX) at different levels, we found that higher CycE-Cdk2 kinase activity is required for FSC maintenance than to support follicle cell proliferation. Surprisingly, cycE(WX) FSCs were lost from their niches rather than arresting proliferation. Furthermore, FSC function was substantially restored by expressing either excess DE-cadherin or excess E2F1/DP, the transcription factor normally activated by CycE-Cdk2 phosphorylation of retinoblastoma proteins. These results suggest that FSC maintenance through niche adhesion is regulated by inputs that normally control S phase entry, possibly as a quality control mechanism to ensure adequate stem cell proliferation. We speculate that a positive connection between central regulators of the cell cycle and niche retention may be a common feature of highly proliferative stem cells.

Investigating Adult Stem Cells Through Lineage analyses.

A simple, universal and fundamental definition of adult stem cell communities is proposed. Key principles of cell lineage methods for defining adult stem cell numbers, locations and behaviors are critically evaluated, emphasizing the imperatives of capturing the full spectrum of individual stem cell behaviors, examining a variety of experimental time periods and avoiding unwarranted assumptions. The focus is first on defining fundamentals and then addresses stem cell heterogeneity, potential hierarchies and how individual cells serve the function of a stem cell community.

Costal 2 interactions with Cubitus interruptus (Ci) underlying Hedgehog-regulated Ci processing.

Extracellular Hedgehog (Hh) proteins alter cellular behaviours from flies to man by regulating the activities of Gli/Ci family transcription factors. A major component of this response in Drosophila is the inhibition of proteolytic processing of the latent transcriptional activator Ci-155 to a shorter Ci-75 repressor form. Processing is thought to rely on binding of the kinesin-family protein Cos2 directly to Ci-155 domains known as CDN and CORD, allowing Cos2-associated protein kinases to phosphorylate Ci-155 efficiently and create a binding site for an E3 ubiquitin ligase complex. Here we show that the last three zinc fingers of Ci-155 also bind Cos2 in vitro and that the zinc finger region, rather than the CDN domain, functions redundantly with the CORD domain to promote Hh-regulated Ci-155 proteolysis in wing discs. We also find evidence for a unique function of Cos2 binding to CORD. Cos2 binding to CORD, but not to other regions of Ci, is potentiated by nucleotides and abrogated by the nucleotide binding variant Cos2 S182N. Removal of the CORD region alone enhances processing under a variety of conditions. Most strikingly, CORD region deletion allows Cos2 S182N to stimulate efficient Ci processing. We deduce that the CORD region has a second function distinct from Cos2 binding that inhibits Ci processing, and that Cos2 binding to CORD relieves this inhibition. We suggest that this regulatory activity of Cos2 depends on a specific nucleotide-bound conformation that may be regulated by Hh.

Hedgehog signaling: a smoothened conformational switch.

An intracellular conformational switch in the serpentine transmembrane protein Smoothened appears to underlie Hedgehog pathway activation. The switch is gated by electrostatic interactions that are regulated by multiple phosphorylations, potentially endowing a dose-dependent response.

Adult stem cells and niche cells segregate gradually from common precursors that build the adult Drosophila ovary during pupal development.

Production of proliferative follicle cells (FCs) and quiescent escort cells (ECs) by follicle stem cells (FSCs) in adult Drosophila ovaries is regulated by niche signals from anterior (cap cells, ECs) and posterior (polar FCs) sources. Here we show that ECs, FSCs, and FCs develop from common pupal precursors, with different fates acquired by progressive separation of cells along the AP axis and a graded decline in anterior cell proliferation. ECs, FSCs, and most FCs derive from intermingled cell (IC) precursors interspersed with germline cells. Precursors also accumulate posterior to ICs before engulfing a naked germline cyst projected out of the germarium to form the first egg chamber and posterior polar FC signaling center. Thus, stem and niche cells develop in appropriate numbers and spatial organization through regulated proliferative expansion together with progressive establishment of spatial signaling cues that guide adult cell behavior, rather than through rigid early specification events.

Opposing JAK-STAT and Wnt signaling gradients define a stem cell domain by regulating differentiation at two borders.

Many adult stem cell communities are maintained by population asymmetry, where stochastic behaviors of multiple individual cells collectively result in a balance between stem cell division and differentiation. We investigated how this is achieved for Drosophila Follicle Stem Cells (FSCs) by spatially-restricted niche signals. FSCs produce transit-amplifying Follicle Cells (FCs) from their posterior face and quiescent Escort Cells (ECs) to their anterior. We show that JAK-STAT pathway activity, which declines from posterior to anterior, dictates the pattern of divisions over the FSC domain, promotes more posterior FSC locations and conversion to FCs, while opposing EC production. Wnt pathway activity declines from the anterior, promotes anterior FSC locations and EC production, and opposes FC production. The pathways combine to define a stem cell domain through concerted effects on FSC differentiation to ECs and FCs at either end of opposing signaling gradients, and impose a pattern of proliferation that matches derivative production.

Adult organisms contain a variety of cells that are routinely replaced using adult stem cells which can generate the cells of a specific tissue. These stem cells are often clustered into small groups, where combinations of chemical signals from nearby cells can encourage each stem cell to divide or 'differentiate' into another type of cell. These different signals must somehow balance stem cell division and differentiation to maintain the size and shape of the community. The ovary of an adult fruit fly contains a group of adult stem cells called follicle stem cells, or FSCs for short. FSCs support the continual production of eggs by supplying two types of cell from opposite faces of the stem cell cluster: dividing follicle cells emerge from the back of the cluster and guide late egg development, while non-dividing escort cells come from the front and guide early egg development. Two of the signals that control FSCs are graded over the cluster. JAK-STAT signaling is strongest in the follicle cell territory and gradually declines towards the front, while Wnt signaling is strongest in escort cells and absent from early follicle cells. However, it was unclear how the gradients of these two signals maintain the FSC population and control the formation of follicle and escort cells. To answer this question, Melamed and Kalderon used genetic engineering to modify the strength of these two signals. The experiments measured how this affected the rate at which FSCs divide and are converted into follicle or escort cells. Melamed and Kalderon found that the strength of JAK-STAT signaling dictated division rates, which may explain why the rate cells divide varies across the FSC cluster and escort cells do not divide at all. JAK-STAT signaling also stimulated FSCs to become follicle cells and opposed their conversion to escort cells. Conversely, stronger Wnt signaling favored the production of escort cells and inhibited FSCs from transitioning to follicle cells. This suggests that the relative strength of these two opposing signals helps maintain thecorrect number of FSCs while also balancing the formation of follicle and escort cells. JAK-STAT, Wnt and other signals guide the development of many organisms, including humans, and have also been linked to cancer. Therefore, the principles and mechanisms uncovered may apply to other types of stem cells. Furthermore, this work highlights genetic changes that can allow a mutant stem cell to amplify and take over an entire stem cell community, which may play a role in cancer and other illnesses.

Drosophila hedgehog can act as a morphogen in the absence of regulated Ci processing.

Extracellular Hedgehog (Hh) proteins induce transcriptional changes in target cells by inhibiting the proteolytic processing of full-length Drosophila Ci or mammalian Gli proteins to nuclear transcriptional repressors and by activating the full-length Ci or Gli proteins. We used Ci variants expressed at physiological levels to investigate the contributions of these mechanisms to dose-dependent Hh signaling in Drosophila wing imaginal discs. Ci variants that cannot be processed supported a normal pattern of graded target gene activation and the development of adults with normal wing morphology, when supplemented by constitutive Ci repressor, showing that Hh can signal normally in the absence of regulated processing. The processing-resistant Ci variants were also significantly activated in the absence of Hh by elimination of Cos2, likely acting through binding the CORD domain of Ci, or PKA, revealing separate inhibitory roles of these two components in addition to their well-established roles in promoting Ci processing.

Morphogens play a crucial role in determining how cells are organized in developing organisms. These chemical signals act over a wide area, and the amount of signal each cell receives typically initiates a sequence of events that spatially pattern the multiple cells of an organ or tissue. One of the most well-studied groups of morphogens are the hedgehog proteins, which are involved in the development of many animals, ranging from flies to humans. In fruit flies, hedgehog proteins kickstart a cascade of molecular changes that switch on a set of 'target' genes. They do this by ultimately altering the activity of a protein called cubitus interruptus, which comes in two lengths: a long version called Ci-155 and a short version called Ci-75. When hedgehog is absent, Ci-155 is kept in an inactive state in the cytoplasm, where it is slowly converted into its shorter form, Ci-75: this repressor protein is then able to access the nucleus, where it switches 'off' the target genes. However, when a hedgehog signal is present, the processing of Ci into its shorter form is inhibited. Instead, Ci-155 becomes activated by a separate mechanism that allows the long form protein to enter the nucleus and switch 'on' the target genes. But it was unclear whether hedgehog requires both of these mechanisms in order to act as a morphogen and regulate the activity of developmental genes. To answer this question, Little et al. mutated the gene for Ci in the embryo of fruit flies, so that the Ci-155 protein could no longer be processed into Ci-75. Examining the developing wings of these flies revealed that the genes targeted by hedgehog are still activated in the correct pattern. In some parts of the wing, Ci-75 is required to switch off specific sets of genes. But when Little et al. blocked these genes, by adding a gene that constantly produces the Ci repressor in the presence or absence of hedgehog, the adult flies still developed normally structured wings. This suggests that hedgehog does not need to regulate the processing of Ci-155 into Ci-75 in order to perform its developmental role. Previous work showed that when one of the major mechanisms used by hedgehog to activate Ci-155 is blocked, fruit flies are still able to develop normal wings. Taken together with the findings of Little et al., this suggests that the two mechanisms induced by hedgehog can compensate for each other, and independently regulate the development of the fruit fly wing. These mechanisms, which are also found in humans, have been linked to birth defects and several common types of cancer, and understanding how they work could help the development of new treatments.

Processing of the Drosophila hedgehog signaling effector Ci-155 to the repressor Ci-75 is mediated by direct binding to the SCF component Slimb.

Signaling by extracellular Hedgehog (Hh) molecules is crucial for the correct allocation of cell fates and patterns of cell proliferation in humans and other organisms . Responses to Hh are universally mediated by regulating the activity and the proteolysis of the Gli family of transcriptional activators such that they induce target genes only in the presence of Hh . In the absence of Hh, the sole Drosophila Gli homolog, Cubitus interruptus (Ci), undergoes partial proteolysis to Ci-75, which represses key Hh target genes . This processing requires phosphorylation of full-length Ci (Ci-155) by protein kinase A (PKA), casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3), as well as the activity of Slimb . Slimb is homologous to vertebrate beta-TRCP1, which binds as part of an SCF (Skp1/Cullin1/F-box) complex to a defined phosphopeptide motif to target proteins for ubiquitination and subsequent proteolysis . Here, we show that phosphorylation of Ci at the specific PKA, GSK-3, and CK1 sites required in vivo for partial proteolysis stimulates binding to Slimb in vitro. Furthermore, a consensus Slimb/beta-TRCP1 binding site from another protein can substitute for phosphorylated residues of Ci-155 to direct conversion to Ci-75 in vivo. From this, we conclude that Slimb binds directly to phosphorylated Ci-155 to initiate processing to Ci-75. We also explore the phosphorylated motifs in Ci that are recognized by Slimb and provide some evidence that silencing of Ci-155 by phosphorylation may involve more than binding to Slimb.

Similarities between the Hedgehog and Wnt signaling pathways.

Hedgehog and Wnt proteins are signaling molecules that direct many aspects of metazoan development through signal transduction pathways that are just beginning to be understood. Recently, the common use of glycogen synthase kinase 3 and casein kinase 1 has been added to a growing list of straightforward similarities between Hedgehog and Wnt signaling pathways. These kinases silence both pathways by labeling a key transcription factor (Cubitus interruptus) or co-activator (beta-catenin) for proteolysis, and it is possible that reversal of these phosphorylation events is, in each case, central to pathway activation. This review compares the two pathways to explore whether our more extensive knowledge of Wnt pathways can be of predictive value for investigating Hedgehog signaling.

Publisher Correction: Live imaging of stem cells in the germarium of the Drosophila ovary using a reusable gas-permeable imaging chamber.

The version of this paper originally published contained an incorrect unit abbreviation in Step 21: "0.20 g/mL" should have been "0.20 mg/mL." In addition, the first sentence in Step 33 should have read "Use a second pipette with a cut-off pipette tip to add Matrigel to the center well," instead of "Use a second pipette to cut off the tip of the pipette and add Matrigel to the center well." These errors have been corrected in the PDF and HTML versions of the protocol.

Hedgehog signaling: an Arrestin connection?

Arrestins are best known for terminating signaling by G protein coupled receptors. New binding, localization and genetic studies suggest that Arrestins may also participate in the transduction of Hedgehog signals by the seven transmembrane domain protein, Smoothened.