SUMMARYFibroblast growth factor (FGF)-dependent epithelial-mesenchymal transitions and cell migration contribute to the establishment of germ layers in vertebrates and other animals, but a comprehensive demonstration of the cellular activities that FGF controls to mediate these events has not been provided for any system. The establishment of the Drosophila mesoderm layer from an epithelial primordium involves a transition to a mesenchymal state and the dispersal of cells away from the site of internalisation in a FGF-dependent fashion. We show here that FGF plays multiple roles at successive stages of mesoderm morphogenesis in Drosophila. It is first required for the mesoderm primordium to lose its epithelial polarity. An intimate, FGF-dependent contact is established and maintained between the germ layers through mesoderm cell protrusions. These protrusions extend deep into the underlying ectoderm epithelium and are associated with high levels of E-cadherin at the germ layer interface. Finally, FGF directs distinct hitherto unrecognised and partially redundant protrusive behaviours during later mesoderm spreading. Cells first move radially towards the ectoderm, and then switch to a dorsally directed movement across its surface. We show that both movements are important for layer formation and present evidence suggesting that they are controlled by genetically distinct mechanisms.
Intercellular signalling via growth factors plays an important role in controlling cell differentiation and cell movements during the development of multicellular animals. Fibroblast Growth Factor (FGF) signalling induces changes in cellular behaviour allowing cells in the embryo to move, to survive, to divide or to differentiate. Several examples argue that FGF signalling is used in multi-step morphogenetic processes to achieve and maintain a transitional state of the cells required for the control of cell fate. In the genetic model Drosophila melanogaster, FGF signalling via the receptor tyrosine kinases Heartless (Htl) and Breathless (Btl) is particularly well studied. These FGF receptors affect gene expression, cell shape and cell–cell interactions during mesoderm layer formation, caudal visceral muscle (CVM) formation, tracheal morphogenesis and glia differentiation. Here, we will address the current knowledge of the biological functions of FGF signalling in the fly on the tissue, at a cellular and molecular level.
running title: Function of Drosophila Spindly key words: Drosophila, mitosis, cell migration, mitotic spindle, Dynein not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/199414 doi: bioRxiv preprint first posted online Oct. 6, 2017; 2 AbstractSpindly is a mitotic checkpoint protein originally identified as a specific regulator of Dynein activity at the kinetochore. In metaphase, Spindly recruits the Dynein/Dynactin complex, promoting the establishment of stable
24Microtubule polarity in axons and dendrites defines the direction of intracellular transport 25 in neurons. Axons contain arrays of uniformly polarized microtubules with plus-ends 26 facing the tips of the processes (plus-end-out), while dendrites contain microtubules with 27 minus-end-out orientation. It has been shown that cytoplasmic dynein, targeted to cortical 28 actin, removes minus-end-out microtubules from axons. Here we have identified Spindly, 29 a protein known for recruitment of dynein to kinetochores in mitosis, as a key factor 30 required for dynein-dependent microtubule sorting in axons of Drosophila neurons. 31Depletion of Spindly affects polarity of axonal microtubules in vivo and in primary neuronal 32cultures. In addition to these defects, depletion of Spindly in neurons causes major 33 collapse of axonal patterning in the third-instar larval brain as well as severe coordination 34 impairment in adult flies. These defects can be fully rescued by full-length Spindly, but 35 not by variants with mutations in its dynein-binding site. Biochemical analysis 36 demonstrated that Spindly binds F-actin, suggesting that Spindly serves as a link between 37 dynein and cortical actin in axons. Therefore, Spindly plays a critical role during 38 neurodevelopment by mediating dynein-driven sorting of axonal microtubules. 39 40 Significance Statement 41Neurons send and receive electrical signals through long microtubule-filled neurites called 42 axons and dendrites. One of the main structural differences between axons and dendrites 43 is how their microtubules are organized. Axons contains microtubules with their plus-ends 44 out while microtubules in dendrites are organized with mixed or plus-end-in orientation. 45Dynein, the main minus-end microtubule motor, anchored to cortical actin filaments in the 46 axons is responsible for the uniform microtubule polarity in axons. However, it is unknown 47 how dynein is recruited to the actin cortex in axons. The major finding of this work is that 48Spindly, a protein involved in anchoring dynein to kinetochores during cell division, has a 49 second important function in interphase cells recruiting dynein to the actin cortex in axons. 50 51 Introduction 52Neurons are post-mitotic cells that transmit electrical signals through long neurites called 53 axons and dendrites. Electric signals captured by dendrites are sent unidirectionally 54 through axons and transmitted to other cells. The polarity of microtubules is strikingly 55 different in these two types of neuronal processes. Axons contain microtubules oriented 56 predominantly with their plus-ends-out, while dendrites contain a large fraction of minus-57 end-out microtubules (1, 2). Failure in establishing correct polarity of microtubules results 58 in defects of cargo sorting (3). The differences in microtubule orientation between axons 59 and dendrites are gradually established during development. In cultured early stage 60 neurons, non-polarized neurites contain microtubules with mixed orientation. Later the 61...
Spindly was originally identified as a specific regulator of Dynein activity at the kinetochore. In early prometaphase, Spindly recruits the Dynein/Dynactin complex, promoting the establishment of stable kinetochore-microtubule interactions and progression into anaphase. While details of Spindly function in mitosis have been worked out in cultured human cells and in the C. elegans zygote, the function of Spindly within the context of an organism has not yet been addressed. Here, we present loss- and gain-of-function studies of Spindly using transgenic RNAi in Drosophila. Knock-down of Spindly in the female germ line results in mitotic arrest during embryonic cleavage divisions. We investigated the requirements of Spindly protein domains for its localisation and function, and found that the carboxy-terminal region controls Spindly localisation in a cell-type specific manner. Overexpression of Spindly in the female germ line is embryonic lethal and results in altered egg morphology. To determine whether Spindly plays a role in post-mitotic cells, we altered Spindly protein levels in migrating cells and found that ovarian border cell migration is sensitive to the levels of Spindly protein. Our study uncovers novel functions of Spindly and a differential, functional requirement for its carboxy-terminal region in Drosophila.
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