Morphogenetic forces planar polarize LGN/Pins in the embryonic head during Drosophila gastrulation.

Spindle orientation is often achieved by a complex of Partner of Inscuteable (Pins)/LGN, Mushroom Body Defect (Mud)/Nuclear Mitotic Apparatus (NuMa), Gαi, and Dynein, which interacts with astral microtubules to rotate the spindle. Cortical Pins/LGN recruitment serves as a critical step in this process. Here, we identify Pins-mediated planar cell polarized divisions in several of the mitotic domains of the early Drosophila embryo. We found that neither planar cell polarity pathways nor planar polarized myosin localization determined division orientation; instead, our findings strongly suggest that Pins planar polarity and force generated from mesoderm invagination are important. Disrupting Pins polarity via overexpression of a myristoylated version of Pins caused randomized division angles. We found that disrupting forces through chemical inhibitors, depletion of an adherens junction protein, or blocking mesoderm invagination disrupted Pins planar polarity and spindle orientation. Furthermore, directional ablations that separated mesoderm from mitotic domains disrupted spindle orientation, suggesting that forces transmitted from mesoderm to mitotic domains can polarize Pins and orient division during gastrulation. To our knowledge, this is the first in vivo example where mechanical force has been shown to polarize Pins to mediate division orientation.

The nature of cell division forces in epithelial monolayers.

Epithelial cells undergo striking morphological changes during division to ensure proper segregation of genetic and cytoplasmic materials. These morphological changes occur despite dividing cells being mechanically restricted by neighboring cells, indicating the need for extracellular force generation. Beyond driving cell division itself, forces associated with division have been implicated in tissue-scale processes, including development, tissue growth, migration, and epidermal stratification. While forces generated by mitotic rounding are well understood, forces generated after rounding remain unknown. Here, we identify two distinct stages of division force generation that follow rounding: (1) Protrusive forces along the division axis that drive division elongation, and (2) outward forces that facilitate postdivision spreading. Cytokinetic ring contraction of the dividing cell, but not activity of neighboring cells, generates extracellular forces that propel division elongation and contribute to chromosome segregation. Forces from division elongation are observed in epithelia across many model organisms. Thus, division elongation forces represent a universal mechanism that powers cell division in confining epithelia.

An RNAi based screen in Drosophila larvae identifies fascin as a regulator of myoblast fusion and myotendinous junction structure.

BACKGROUND: A strength of Drosophila as a model system is its utility as a tool to screen for novel regulators of various functional and developmental processes. However, the utility of Drosophila as a screening tool is dependent on the speed and simplicity of the assay used. METHODS: Here, we use larval locomotion as an assay to identify novel regulators of skeletal muscle function. We combined this assay with muscle-specific depletion of 82 genes to identify genes that impact muscle function by their expression in muscle cells. The data from the screen were supported with characterization of the muscle pattern in embryos and larvae that had disrupted expression of the strongest hit from the screen. RESULTS: With this assay, we showed that 12/82 tested genes regulate muscle function. Intriguingly, the disruption of five genes caused an increase in muscle function, illustrating that mechanisms that reduce muscle function exist and that the larval locomotion assay is sufficiently quantitative to identify conditions that both increase and decrease muscle function. We extended the data from this screen and tested the mechanism by which the strongest hit, fascin, impacted muscle function. Compared to controls, animals in which fascin expression was disrupted with either a mutant allele or muscle-specific expression of RNAi had fewer muscles, smaller muscles, muscles with fewer nuclei, and muscles with disrupted myotendinous junctions. However, expression of RNAi against fascin only after the muscle had finished embryonic development did not recapitulate any of these phenotypes. CONCLUSIONS: These data suggest that muscle function is reduced due to impaired myoblast fusion, muscle growth, and muscle attachment. Together, these data demonstrate the utility of Drosophila larval locomotion as an assay for the identification of novel regulators of muscle development and implicate fascin as necessary for embryonic muscle development.

Aplip1, the Drosophila homolog of JIP1, regulates myonuclear positioning and muscle stability.

During muscle development, myonuclei undergo a complex set of movements that result in evenly spaced nuclei throughout the muscle cell. In Drosophila, two separate pools of Kinesin and Dynein work in synchrony to drive this process. However, how these two pools are specified is not known. Here, we investigate the role of Aplip1 (the Drosophila homolog of JIP1, JIP1 is also known as MAPK8IP1), a known regulator of both Kinesin and Dynein, in myonuclear positioning. Aplip1 localizes to the myotendinous junction and has genetically separable roles in myonuclear positioning and muscle stability. In Aplip1 mutant embryos, there was an increase in the percentage of embryos that had both missing and collapsed muscles. Via a separate mechanism, we demonstrate that Aplip1 regulates both the final position of and the dynamic movements of myonuclei. Aplip1 genetically interacts with both Raps (also known as Pins) and Kinesin to position myonuclei. Furthermore, Dynein and Kinesin localization are disrupted in Aplip1 mutants suggesting that Aplip1-dependent nuclear positioning requires Dynein and Kinesin. Taken together, these data are consistent with Aplip1 having a function in the regulation of Dynein- and Kinesin-mediated pulling of nuclei from the muscle end.This article has an associated First Person interview with the first author of the paper.

Emery-Dreifuss muscular dystrophy-linked genes and centronuclear myopathy-linked genes regulate myonuclear movement by distinct mechanisms.

Muscle cells are a syncytium in which the many nuclei are positioned to maximize the distance between adjacent nuclei. Although mispositioned nuclei are correlated with many muscle disorders, it is not known whether this common phenotype is the result of a common mechanism. To answer this question, we disrupted the expression of genes linked to Emery-Dreifuss muscular dystrophy (EDMD) and centronuclear myopathy (CNM) in Drosophila and evaluated the position of the nuclei. We found that the genes linked to EDMD and CNM were each necessary to properly position nuclei. However, the specific phenotypes were different. EDMD-linked genes were necessary for the initial separation of nuclei into distinct clusters, suggesting that these factors relieve interactions between nuclei. CNM-linked genes were necessary to maintain the nuclei within clusters as they moved toward the muscle ends, suggesting that these factors were necessary to maintain interactions between nuclei. Together these data suggest that nuclear position is disrupted by distinct mechanisms in EDMD and CNM.