Gene targeting by ‘ends-out’ homologous recombination enables the deletion of genomic sequences and concurrent introduction of exogenous DNA with base-pair precision without sequence constraint. In Drosophila, this powerful technique has remained laborious and hence seldom implemented. We describe a targeting vector and protocols that achieve this at high frequency and with very few false positives in Drosophila, either with a two-generation crossing scheme or by direct injection in embryos. The frequency of injection-mediated gene targeting can be further increased with CRISPR-induced double-strand breaks within the region to be deleted, thus making homologous recombination almost as easy as conventional transgenesis. Our targeting vector replaces genomic sequences with a multifunctional fragment comprising an easy-to-select genetic marker, a fluorescent reporter, as well as an attP site, which acts as a landing platform for reintegration vectors. These vectors allow the insertion of a variety of transcription reporters or cDNAs to express tagged or mutant isoforms at endogenous levels. In addition, they pave the way for difficult experiments such as tissue-specific allele switching and functional analysis in post-mitotic or polyploid cells. Therefore, our method retains the advantages of homologous recombination while capitalising on the mutagenic power of CRISPR.

Tissue morphogenesis requires coordination of multiple force-producing components. During dorsal closure in fly embryogenesis, an epidermis opening closes. A tensioned epidermal actin/MyosinII cable, which surrounds the opening, produces a force that is thought to combine with another MyosinII force mediating apical constriction of the amnioserosa cells that fill the opening. A model proposing that each force could autonomously drive dorsal closure was recently challenged by a model in which the two forces combine in a ratchet mechanism. Acute force elimination via selective MyosinII depletion in one or the other tissue shows that the amnioserosa tissue autonomously drives dorsal closure while the actin/MyosinII cable cannot. These findings exclude both previous models, although a contribution of the ratchet mechanism at dorsal closure onset remains likely. This shifts the current view of dorsal closure being a combinatorial force-component system to a single tissue-driven closure event.

The closure of epidermal openings is an essential biological process that causes major developmental problems such as spina bifida in humans if it goes awry. At present, the mechanism of closure remains elusive. Therefore, we reconstructed a model closure event, dorsal closure in fly embryos, by large-volume correlative electron tomography. We present a comprehensive, quantitative analysis of the cytoskeletal reorganization, enabling separated epidermal cells to seal the epithelium. After establishing contact through actin-driven exploratory filopodia, cells use a single lamella to generate 'roof tile'-like overlaps. These shorten to produce the force, 'zipping' the tissue closed. The shortening overlaps lack detectable actin filament ensembles but are crowded with microtubules. Cortical accumulation of shrinking microtubule ends suggests a force generation mechanism in which cortical motors pull on microtubule ends as for mitotic spindle positioning. In addition, microtubules orient filopodia and lamellae before zipping. Our 4D electron microscopy picture describes an entire developmental process and provides fundamental insight into epidermal closure.

Magnetic tweezers are mainly divided into two classes depending on the ability of applying torque or forces to the magnetic probe. We focused on the second category and designed a device composed by a single electromagnet equipped with a core having a special asymmetric profile to exert forces as large as 230 pN-2.8 μm Dynabeads at distances in excess of 100 μm from the magnetic tip. Compared to existing solutions our magnetic tweezers overcome important limitations, opening new experimental paths for the study of a wide range of materials in a variety of biophysical research settings. We discuss the benefits and drawbacks of different magnet core characteristics, which led us to design the current core profile. To demonstrate the usefulness of our magnetic tweezers, we determined the microrheological properties inside embryos of Drosophila melanogaster during the syncytial stage. Measurements in different locations along the dorsal-ventral axis of the embryos showed little variation, with a slight increase in cytoplasm viscosity at the periphery of the embryos. The mean cytoplasm viscosity we obtain by active force exertion inside the embryos is comparable to that determined passively using high-speed video microrheology.

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Cell-type-specific F-actin structures and myosin motors are key generators of the forces that drive tissue morphogenesis in developing organisms. These cytoskeletal elements mediate defined cell deformation and control the arrangement of cell-cell contacts. This SnapShot presents a selection of morphogenetic processes, the analysis of which has pioneered specific types of F-actin/myosin-mediated force generation in development.

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Tissue morphogenesis integrates cell type-specific biochemistry and architecture, cellular force generation and mechanisms coordinating forces amongst neighbouring cells and tissues.We use finite element-based modelling to explore the interconnections at these multiple biological scales in embryonic dorsal closure, where pulsed actomyosin contractility in adjacent Amnioserosa (AS) cells powers the closure of an epidermis opening. Based on our in vivo observations, the model implements F-actin nucleation periodicity that is independent of MyoII activity. Our model reveals conditions, where depleting MyoII activity nevertheless indirectly affects oscillatory F-actin behaviour, without the need for biochemical feedback. In addition, it questions the previously proposed role of Dpp-mediated regulation of the patterned actomyosin dynamics in the AS tissue, suggesting them to be emergent. Tissue-specific Dpp interference supports the model's prediction. The model further predicts that the mechanical properties of the surrounding epidermis tissue feed back on the shaping and patterning of the AS tissue. Finally, our model's parameter space reproduces mutant phenotypes and provides predictions for their underlying cause. Our modelling approach thus reveals several unappreciated mechanistic properties of tissue morphogenesis.