Michael J. Fairchild

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

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

Analysis of integrin turnover in fly myotendinous junctions

Transient (short-term) cell adhesion underlies dynamic processes such as cell migration, whereas stable (long-term) cell adhesion maintains tissue architecture. Ongoing adhesion complex turnover is essential for transient cell adhesion, but it is not known whether turnover is also required for maintenance of long-term adhesion. We used fluorescence recovery after photobleaching to analyze the dynamics of an integrin adhesion complex (IAC) in a model of long-term cell-ECM adhesion, myotendinous junctions (MTJs), in fly embryos and larvae. We found that the IAC undergoes turnover in MTJs and that this process is mediated by clathrin-dependent endocytosis. Moreover, the small GTPase Rab5 can regulate the proportion of IAC components that undergo turnover. Also, altering Rab5 activity weakened MTJs, resulting in muscle defects. In addition, growth of MTJs was concomitant with a decrease in the proportion of IAC components undergoing turnover. We propose that IAC turnover is tightly regulated in long-term cell-ECM adhesions to allow normal tissue growth and maintenance.

Talin Autoinhibition Is Required for Morphogenesis

The establishment of a multicellular body plan requires coordinating changes in cell adhesion and the cytoskeleton to ensure proper cell shape and position within a tissue. Cell adhesion to the extracellular matrix (ECM) via integrins plays diverse, essential roles during animal embryogenesis and therefore must be precisely regulated. Talin, a FERM-domain containing protein, forms a direct link between integrin adhesion receptors and the actin cytoskeleton and is an important regulator of integrin function. Similar to other FERM proteins, talin makes an intramolecular interaction that could autoinhibit its activity. However, the functional consequence of such an interaction has not been previously explored in vivo. Here, we demonstrate that targeted disruption of talin autoinhibition gives rise to morphogenetic defects during fly development and specifically that dorsal closure (DC), a process that resembles wound healing, is delayed. Impairment of autoinhibition leads to reduced talin turnover at and increased talin and integrin recruitment to sites of integrin-ECM attachment. Finally, we present evidence that talin autoinhibition is regulated by Rap1-dependent signaling. Based on our data, we propose that talin autoinhibition provides a switch for modulating adhesion turnover and adhesion stability that is essential for morphogenesis.

In vivo functional analysis reveals specific roles for the integrin-binding sites of talin

Adhesion receptors play diverse roles during animal development and require precise spatiotemporal regulation, which is achieved through the activity of their binding partners. Integrins, adhesion receptors that mediate cell attachment to the extracellular matrix (ECM), connect to the intracellular environment through the cytoplasmic adapter protein talin. Talin has two essential functions: orchestrating the assembly of the intracellular adhesion complex (IAC), which associates with integrin, and regulating the affinity of integrins for the ECM. Talin can bind to integrins through two different integrin-binding sites (IBS-1 and IBS-2, respectively). Here, we have investigated the roles of each in the context of Drosophila development. We find that although IBS-1 and IBS-2 are partially redundant, they each have specialized roles during development: IBS-1 reinforces integrin attachment to the ECM, whereas IBS-2 reinforces the link between integrins and the IAC. Disruption of each IBS has different developmental consequences, illustrating how the functional diversity of integrin-mediated adhesion is achieved.

Distinct developmental roles for direct and indirect talin-mediated linkage to actin

Transmembrane adhesion receptors, such as integrins, mediate cell adhesion by interacting with intracellular proteins that connect to the cytoskeleton. Talin, one such linker protein, is essential to connect extracellular matrix-bound integrins to the cytoskeleton. Talin can connect to the cytoskeleton either directly, through its actin-binding motifs, or indirectly, by recruiting other actin-binding proteins. Talins carboxy-terminal end contains a well-characterized actin-binding domain (ABD). We tested the role of the C-terminal ABD of talin in integrin function in Drosophila. We found that introduction of mutations that reduced actin binding in vitro into the isolated C-terminal Talin-ABD impaired actin binding in vivo. Moreover, when engineered into full-length talin, these mutations disrupted a subset of integrin-mediated adhesion-dependent developmental events. Specifically, morphogenetic processes that involve dynamic, short-term integrin-mediated adhesion were particularly sensitive to impaired function of the C-terminal Talin-ABD. We propose that during development talin connects integrins to the cytoskeleton in distinct ways in different types of integrin-mediated adhesion: directly in transient adhesions and indirectly in stable long-lasting adhesions. Our results provide insight into how a similar array of molecular components can contribute to diverse adhesive processes throughout development.

A somatic permeability barrier around the germline is essential for Drosophila spermatogenesis

Interactions between the soma and germline are essential for gametogenesis. In the Drosophila testis, differentiating germ cells are encapsulated by two somatic cells that surround the germline throughout spermatogenesis. chickadee (chic), the fly ortholog of Profilin, mediates soma-germline interactions. Knockdown of Chic in the soma results in sterility and severely disrupted spermatogenesis due to defective encapsulation. To study this defect further, we developed a permeability assay to analyze whether the germline is isolated from the surrounding environment by the soma. We find that germline encapsulation by the soma is, by itself, insufficient for the formation of a permeability barrier, but that such a barrier gradually develops during early spermatogenesis. Thus, germline stem cells, gonialblasts and early spermatogonia are not isolated from the outside environment. By late spermatocyte stages, however, a permeability barrier is formed by the soma. Furthermore, we find that, concomitant with formation of the permeability barrier, septate junction markers are expressed in the soma and localize to junctional sites connecting the two somatic cells that surround the germline. Importantly, knockdown of septate junction components also disrupts the permeability barrier. Finally, we show that germline differentiation is delayed when the permeability barrier is compromised. We propose that the permeability barrier around the germline serves an important regulatory function during spermatogenesis by shaping the signaling events that take place between the soma and the germline. Higlighted article: In the Drosophila testis, profilin and septate junction components are required for encapsulation and isolation of the germline by a somatic permeability barrier.

The Talin Head Domain Reinforces Integrin-Mediated Adhesion by Promoting Adhesion Complex Stability and Clustering

Talin serves an essential function during integrin-mediated adhesion in linking integrins to actin via the intracellular adhesion complex. In addition, the N-terminal head domain of talin regulates the affinity of integrins for their ECM-ligands, a process known as inside-out activation. We previously showed that in Drosophila, mutating the integrin binding site in the talin head domain resulted in weakened adhesion to the ECM. Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies. Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development. Here, we describe results suggesting that the talin head domain reinforces and stabilizes the integrin adhesion complex by promoting integrin clustering distinct from its ability to support inside-out activation. Specifically, we show that an allele of talin containing a mutation that disrupts intramolecular interactions within the talin head attenuates the assembly and reinforcement of the integrin adhesion complex. Importantly, we provide evidence that this mutation blocks integrin clustering in vivo. We propose that the talin head domain is essential for regulating integrin avidity in Drosophila and that this is crucial for integrin-mediated adhesion during animal development.

Distinct regulatory mechanisms control integrin adhesive processes during tissue morphogenesis

Cell adhesion must be precisely regulated to enable both dynamic morphogenetic processes and the subsequent transition to stable tissue maintenance. Integrins link the intracellular cytoskeleton and extracellular matrix, relaying bidirectional signals across the plasma membrane. In vitro studies have demonstrated that multiple mechanisms control integrin‐mediated adhesion; however, their roles during development are poorly understood. We used mutations that activate or deactivate specific functions of vertebrate β‐integrins in vitro to investigate how perturbing Drosophila βPS‐integrin regulation in developing embryos regulation affects tissue morphogenesis and maintenance. We found that morphogenetic processes use various β‐integrin regulatory mechanisms to differing degrees and that conformational changes associated with outside‐in activation are essential for developmental integrin functions. Long‐term adhesion is also sensitive to integrin dysregulation, suggesting integrins must be continuously regulated to support stable tissue maintenance. Altogether, in vivo phenotypic analyses allowed us to identify the importance of various β‐integrin regulatory mechanisms during different morphogenetic processes. Developmental Dynamics 240:36–51, 2011.

Identification of genetic networks that act in the somatic cells of the testis to mediate the developmental program of spermatogenesis

Spermatogenesis is a dynamic developmental process requiring precisely timed transitions between discrete stages. Specifically, the germline undergoes three transitions: from mitotic spermatogonia to spermatocytes, from meiotic spermatocytes to spermatids, and from morphogenetic spermatids to spermatozoa. The somatic cells of the testis provide essential support to the germline throughout spermatogenesis, but their precise role during these developmental transitions has not been comprehensively explored. Here, we describe the identification and characterization of genes that are required in the somatic cells of the Drosophila melanogaster testis for progress through spermatogenesis. Phenotypic analysis of candidate genes pinpointed the stage of germline development disrupted. Bioinformatic analysis revealed that particular gene classes were associated with specific developmental transitions. Requirement for genes associated with endocytosis, cell polarity, and microtubule-based transport corresponded with the development of spermatogonia, spermatocytes, and spermatids, respectively. Overall, we identify mechanisms that act specifically in the somatic cells of the testis to regulate spermatogenesis.

Occluding Junctions Maintain Stem Cell Niche Homeostasis in the Fly Testes

Stem cells can be controlled by their local microenvironment, known as the stem cell niche. The Drosophila testes contain a morphologically distinct niche called the hub, composed of a cluster of between 8 and 20 cells known as hub cells, which contact and regulate germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Both hub cells and CySCs originate from somatic gonadal precursor cells during embryogenesis, but whereas hub cells, once specified, cease all mitotic activity, CySCs remain mitotic into adulthood [1, 2]. Cyst cells, derived from the CySCs, first encapsulate the germline and then, using occluding junctions, form an isolating permeability barrier [3]. This barrier promotes germline differentiation by excluding niche-derived stem cell maintenance factors. Here, we show that the somatic permeability barrier is also required to regulate stem cell niche homeostasis. Loss of occluding junction components in the somatic cells results in hub overgrowth. Enlarged hubs are active and recruit more GSCs and CySCs to the niche. Surprisingly, hub growth results from depletion of occluding junction components in cyst cells, not from depletion in the hub cells themselves. Moreover, hub growth is caused by incorporation of cells that previously expressed markers for cyst cells and not by hub cell proliferation. Importantly, depletion of occluding junctions disrupts Notch and mitogen-activated protein kinase (MAPK) signaling, and hub overgrowth defects are partially rescued by modulation of either signaling pathway. Overall, these data show that occluding junctions shape the signaling environment between the soma and the germline in order to maintain niche homeostasis.