Michelle Starz-Gaiano

Group choreography: mechanisms orchestrating the collective movement of border cells

Cell movements are essential for animal development and homeostasis but also contribute to disease. Moving cells typically extend protrusions towards a chemoattractant, adhere to the substrate, contract and detach at the rear. It is less clear how cells that migrate in interconnected groups in vivo coordinate their behaviour and navigate through natural environments. The border cells of the Drosophila melanogaster ovary have emerged as an excellent model for the study of collective cell movement, aided by innovative genetic, live imaging, and photomanipulation techniques. Here we provide an overview of the molecular choreography of border cells and its more general implications.

Phagocytic ability declines with age in adult Drosophila hemocytes

Most multicellular organisms show a physiological decline in immune function with age. However, little is known about the mechanisms underlying these changes. We examined Drosophila melanogaster, an important model for identifying genes affecting innate immunity and senescence, to explore the role of phagocytosis in age‐related immune dysfunction. We characterized the localized response of immune cells at the dorsal vessel to bacterial infection in 1‐week‐ and 5‐week‐old flies. We developed a quantitative phagocytosis assay for adult Drosophila and utilized this to characterize the effect of age on phagocytosis in transgenic and natural variant lines. We showed that genes necessary for bacterial engulfment in other contexts are also required in adult flies. We found that blood cells from young and old flies initially engulf bacteria equally well, while cells from older flies accumulate phagocytic vesicles and thus are less capable of destroying pathogens. Our results have broad implications for understanding how the breakdown in cellular processes influences immune function with age.

Interpretation of the UPD/JAK/STAT morphogen gradient in Drosophila follicle cells.

We are using Drosophila follicle cells to study the mechanisms that promote cell motility. Using genetics we identified a gene regulatory network that controls the dynamic pattern of activation of JAK/STAT in anterior follicle cells. Under the influence of a graded signal, Unpaired (UPD), JAK/STAT becomes activated first in a graded fashion. STAT, in turn, locally activates its own repressor, Apontic (APT), a new feedback regulator of JAK/STAT signaling. High levels of JAK/STAT also activate Slow Border Cells (SLBO), which undermines APT-mediated repression. In this way, cells that achieve a high JAK/STAT level maintain SLBO expression and form border cells, which then migrate out of the cell layer. Cells with lower JAK/STAT activity express more APT than SLBO, ultimately lose STAT activity, and remain in the follicular epithelium. To better understand how the graded signal is converted to an all-or-none decision to move or stay, we developed a mathematical model. Simulations using the model reproduce the observed dynamics of JAK/STAT expression in the wild type and in several mutant situations. By combining biological experiments and mathematical modeling, we can achieve a more sophisticated understanding of how cells interpret molecular gradients.

Socs36E attenuates STAT signaling to optimize motile cell specification in the Drosophila ovary.

The Janus kinase/Signal transducers and activators of transcription (JAK/STAT) pathway determines cell fates by regulating gene expression. One example is the specification of the motile cells called border cells during Drosophila oogenesis. It has been established that too much or too little STAT activity disrupts follicle cell identity and cell motility, which suggests the signaling must be precisely regulated. Here, we find that Suppressor of cytokine signaling at 36E (Socs36E) is a necessary negative regulator of JAK/STAT signaling during border cell specification. We find when STAT signaling is too low to induce migration in the presumptive border cell population, nearby follicle cells uncharacteristically become invasive to enable efficient migration of the cluster. We generated a genetic null allele that reveals Socs36E is required in the anterior follicle cells to limit invasive behavior to an optimal number of cells. We further show Socs36E genetically interacts with the required STAT feedback inhibitor apontic (apt) and APTs downstream target, mir-279, and provide evidence that suggests APT directly regulates Socs36E transcriptionally. Our work shows Socs36E plays a critical role in a genetic circuit that establishes a boundary between the motile border cell cluster and its non-invasive epithelial neighbors through STAT attenuation.

Age- and diet-specific effects of variation at S6 kinase on life history, metabolic, and immune response traits in Drosophila melanogaster.

Life history theory hypothesizes that genetically based variation in life history traits results from alleles that alter age-specific patterns of energy allocation among the competing demands of reproduction, storage, and maintenance. Despite the important role that alleles with age-specific effects must play in life history evolution, few naturally occurring alleles with age-specific effects on life history traits have been identified. A recent mapping study identified S6 kinase (S6k) as a candidate gene affecting lipid storage in Drosophila. S6k is in the target of rapamycin pathway, which regulates cell growth in response to nutrient availability and has also been implicated to influence many life history traits from fecundity to life span. In this article, we used quantitative complementation tests to examine the effect of allelic variation at S6k on a range of phenotypes associated with metabolism and fitness in an age-, diet-, and sex-specific manner. We found that alleles of S6k have pleiotropic effects on total protein levels, glycogen storage, life span, and the immune response and demonstrate that these allelic effects are age, diet, and sex specific. As many of the genes in the target of rapamycin pathway are evolutionarily conserved, our data suggest that genes in this pathway could play a pivotal role in life history evolution in a wide range of taxa.

A mathematical model of collective cell migration in a three-dimensional, heterogeneous environment.

Cell migration is essential in animal development, homeostasis, and disease progression, but many questions remain unanswered about how this process is controlled. While many kinds of individual cell movements have been characterized, less effort has been directed towards understanding how clusters of cells migrate collectively through heterogeneous, cellular environments. To explore this, we have focused on the migration of the border cells during Drosophila egg development. In this case, a cluster of different cell types coalesce and traverse as a group between large cells, called nurse cells, in the center of the egg chamber. We have developed a new model for this collective cell migration based on the forces of adhesion, repulsion, migration and stochastic fluctuation to generate the movement of discrete cells. We implement the model using Identical Math Cells, or IMCs. IMCs can each represent one biological cell of the system, or can be aggregated using increased adhesion forces to model the dynamics of larger biological cells. The domain of interest is filled with IMCs, each assigned specific biophysical properties to mimic a diversity of cell types. Using this system, we have successfully simulated the migration of the border cell cluster through an environment filled with larger cells, which represent nurse cells. Interestingly, our simulations suggest that the forces utilized in this model are sufficient to produce behaviors of the cluster that are observed in vivo, such as rotation. Our framework was developed to capture a heterogeneous cell population, and our implementation strategy allows for diverse, but precise, initial position specification over a three- dimensional domain. Therefore, we believe that this model will be useful for not only examining aspects of Drosophila oogenesis, but also for modeling other two or three-dimensional systems that have multiple cell types and where investigating the forces between cells is of interest.

Circuitous Genetic Regulation Governs a Straightforward Cell Migration

Drosophila border cells undergo a straightforward and stereotypical collective migration during egg development. However, a complex genetic program underlies this process. A variety of approaches, including biochemical, genetic, and imaging strategies have identified many regulatory components, revealing layers of control. This complexity suggests that the active processes of evaluating the environment, remodeling the cytoskeleton, and coordinating movements among cells, demand rapid systems for modulating cell behaviors. Multiple signaling inputs, nodes of integration, and feedback loops act as molecular rheostats to fine-tune gene expression levels and physical responses. Since key genetic regulators of border cell migration have been shown to be required in other types of cell migration, this model system continues to provide an important avenue for genetic discovery.

Socs36E limits STAT signaling via Cullin2 and a SOCS-box independent mechanism in the Drosophila egg chamber.

The Suppressor of Cytokine Signaling (SOCS) proteins are critical, highly conserved feedback inhibitors of signal transduction cascades. The family of SOCS proteins is divided into two groups: ancestral and vertebrate-specific SOCS proteins. Vertebrate-specific SOCS proteins have been heavily studied as a result of their strong mutant phenotypes. However, the ancestral clade remains less studied, a potential result of genetic redundancies in mammals. Use of the genetically tractable organism Drosophila melanogaster enables in vivo assessment of signaling components and mechanisms with less concern about the functional redundancy observed in mammals. In this study, we investigated how the SOCS family member Suppressor of Cytokine Signaling at 36E (Socs36E) attenuates Janus Kinase/Signal Transducer and Activator of Transcription (Jak/STAT) activation during specification of motile border cells in Drosophila oogenesis. We found that Socs36E genetically interacts with the Cullin2 (Cul2) scaffolding protein. Like Socs36E, Cul2 is required to limit the number of motile cells in egg chambers. We demonstrated that loss of Cul2 in the follicle cells significantly increased nuclear STAT protein levels, which resulted in additional cells acquiring invasive properties. Further, reduction of Cul2 suppressed border cell migration defects that occur in a Stat92E-sensitized genetic background. Our data incorporated Cul2 into a previously described Jak/STAT-directed genetic regulatory network that is required to generate a discrete boundary between cell fates. We also found that Socs36E is able to attenuate STAT activity in the egg chamber when it does not have a functional SOCS box. Collectively, this work contributes mechanistic insight to a Jak/STAT regulatory genetic circuit, and suggests that Socs36E regulates Jak/STAT signaling via a Cul2-dependent mechanism, as well as by a Cullin-independent manner, in vivo.

Culturing Drosophila Egg Chambers and Investigating Developmental Processes Through Live Imaging.

Drosophila oogenesis provides many examples of essential processes in development. A myriad of genetic tools combined with recent advances in culturing egg chambers ex vivo has revealed several surprising mechanisms that govern how this tissue develops, and which could not have been determined in fixed tissues. Here we describe a straightforward protocol for dissecting ovaries, culturing egg chambers, and observing egg development in real time by fluorescent microscopy. This technique is suitable for observation of early- or late-stage egg development, and can be adapted to study a variety of cellular, molecular, or developmental processes. Ongoing analysis of oogenesis in living egg chambers has tremendous potential for discovery of new developmental mechanisms.

Identification of Novel Regulators of the JAK/STAT Signaling Pathway that Control Border Cell Migration in the Drosophila Ovary

The Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway is an essential regulator of cell migration both in mammals and fruit flies. Cell migration is required for normal embryonic development and immune response but can also lead to detrimental outcomes, such as tumor metastasis. A cluster of cells termed “border cells” in the Drosophila ovary provides an excellent example of a collective cell migration, in which two different cell types coordinate their movements. Border cells arise within the follicular epithelium and are required to invade the neighboring cells and migrate to the oocyte to contribute to a fertilizable egg. Multiple components of the STAT signaling pathway are required during border cell specification and migration; however, the functions and identities of other potential regulators of the pathway during these processes are not yet known. To find new components of the pathway that govern cell invasiveness, we knocked down 48 predicted STAT modulators using RNAi expression in follicle cells, and assayed defective cell movement. We have shown that seven of these regulators are involved in either border cell specification or migration. Examination of the epistatic relationship between candidate genes and Stat92E reveals that the products of two genes, Protein tyrosine phosphatase 61F (Ptp61F) and brahma (brm), interact with Stat92E during both border cell specification and migration.