SummaryThe basement membrane (BM) is a thin layer of extracellular matrix (ECM) beneath nearly all epithelial cell types that is critical for cellular and tissue function. It is composed of numerous components conserved among all bilaterians [1]; however, it is unknown how all of these components are generated and subsequently constructed to form a fully mature BM in the living animal. Although BM formation is thought to simply involve a process of self-assembly [2], this concept suffers from a number of logistical issues when considering its construction in vivo. First, incorporation of BM components appears to be hierarchical [3, 4, 5], yet it is unclear whether their production during embryogenesis must also be regulated in a temporal fashion. Second, many BM proteins are produced not only by the cells residing on the BM but also by surrounding cell types [6, 7, 8, 9], and it is unclear how large, possibly insoluble protein complexes [10] are delivered into the matrix. Here we exploit our ability to live image and genetically dissect de novo BM formation during Drosophila development. This reveals that there is a temporal hierarchy of BM protein production that is essential for proper component incorporation. Furthermore, we show that BM components require secretion by migrating macrophages (hemocytes) during their developmental dispersal, which is critical for embryogenesis. Indeed, hemocyte migration is essential to deliver a subset of ECM components evenly throughout the embryo. This reveals that de novo BM construction requires a combination of both production and distribution logistics allowing for the timely delivery of core components.

Highlights d Labeled ECM in fly embryos can be examined from initiation to homeostasis d Quantifying ECM levels to homeostasis allows for modeling of basal turnover rate d Embryonic ECM has a half-life of 10 h, which was confirmed by pulse-chase analysis d Inhibiting MMPs or ECM interactions alters the basal turnover rate

Cell migration is hypothesised to involve a cycle of behaviours beginning with leading edge extension. However, recent evidence suggests that the leading edge may be dispensable for migration, raising the question of what actually controls cell directionality. Here we exploit the embryonic migration of Drosophila macrophages to bridge the different temporal scales of the behaviours controlling motility. This reveals that edge fluctuations during random motility are impersistent and weakly correlated with motion. In contrast, flow of the actin network behind the leading edge is highly persistent. Quantification of actin flow structure during migration reveals a stable organisation and asymmetry in the cell-wide flowfield that strongly correlates with cell Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Interactions between different cell types can induce distinct contact inhibition of locomotion (CIL) responses that are hypothesised to control population-wide behaviours during embryogenesis. However, our understanding of the signals that lead to cell-type specific repulsion and the precise capacity of heterotypic CIL responses to drive emergent behaviours is lacking. Using a new model of heterotypic CIL, we show that fibrosarcoma cells, but not fibroblasts, are actively repelled by epithelial cells in culture. We show that knocking down EphB2 or ERK in fibrosarcoma cells specifically leads to disruption of the repulsion phase of CIL in response to interactions with epithelial cells. We also examine the population-wide effects when these various cell combinations are allowed to interact in culture. Unlike fibroblasts, fibrosarcoma cells completely segregate from epithelial cells and inhibiting their distinct CIL response by knocking down EphB2 or ERK family proteins also disrupts this emergent sorting behaviour. These data suggest that heterotypic CIL responses, in conjunction with processes such as differential adhesion, may aid the sorting of cell populations.

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SummaryThe forces controlling tissue morphogenesis are attributed to cellular-driven activities and any role for extracellular matrix (ECM) is assumed to be passive. However, all polymer networks, including ECM, can theoretically develop autonomous stresses during their assembly. Here we examine the morphogenetic function of an ECM prior to reaching homeostatic equilibrium by analyzing de novo ECM assembly during Drosophila ventral nerve cord (VNC) condensation. Asymmetric VNC shortening and a rapid decrease in surface area correlate with exponential assembly of Collagen-IV (Col4) surrounding the tissue. Concomitantly, a transient developmentally-induced Col4 gradient leads to coherent long-range flow of ECM, which equilibrates the Col4 network. Finite element analysis and perturbation of Col4 network formation through the generation of dominant Col4-truncations that affect assembly, reveals that VNC morphodynamics is driven by a sudden increase in ECM-driven surface tension. These data highlight that ECM assembly stress and associated network instabilities can actively participate in tissue morphogenesis.

Summary Protein turnover rate is difficult to obtain experimentally. This protocol shows how to mathematically model turnover rates in an intervention-free manner given the ability to quantify mRNA and protein expression from initiation to homeostasis. This approach can be used to calculate production and degradation rates and to infer protein half-life. This model was successfully employed to quantify turnover during Drosophila embryogenesis, and we hypothesize that it will be applicable to diverse in vivo or in vitro systems. For complete details on the use and execution of this protocol, please refer to Matsubayashi et al. (2020) .

SummaryInteractions between different cell-types can induce distinct contact inhibition of 1 locomotion (CIL) responses that are hypothesized to control population-wide 2 behaviors during embryogenesis [1, 2]. However, our understanding of the signals 3 that lead to cell-type specific repulsion, and the precise capacity of heterotypic CIL 4 responses to drive emergent behaviors is lacking. Using a new in vitro model of 5 heterotypic CIL between epithelial and mesenchymal cells, we show that 6 fibrosarcoma cells (HT1080), but not fibroblasts (NIH3T3), are actively repelled by 7 epithelial cells in culture. We show that knocking down EphB2 in fibrosarcoma cells 8 specifically leads to disruption of the repulsion phase of CIL in response to 9 interactions with epithelial cells. Furthermore, this heterotypic interaction requires 10 ERK activation, downstream of EphB2 signaling. We also examine the population-11 wide effects when these various cell combinations, and their specific heterotypic CIL 12 responses, are allowed to interact in culture. Mixtures of fibrosarcoma and epithelial 13 cells -unlike fibroblasts and epithelial cells -lead to complete sorting and 14 segregation of the two populations, and inhibiting their distinct CIL response by 15 knocking down EphB2 or ERK in fibrosarcoma cells disrupts this emergent sorting 16 behavior. Our understanding of the mechanisms underlying developmental 17All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/373696 doi: bioRxiv preprint first posted online Jul. 20, 2018; 2 behaviors such as cell sorting is lacking as predominant sorting hypotheses, such as 18 differential adhesion, have recently been found inadequate in predicting the sorting 19 of mesenchymal cells [3, 4]. These data suggest that heterotypic CIL responses, in 20 conjunction with processes such as differential adhesion, may aid the sorting of cell 21 populations during embryogenesis. 22 23 Results and Discussion 24 25 Fibroblasts and fibrosarcoma cells exhibit distinct responses upon collision 26with an epithelial cell monolayer 27To study heterotypic cell-cell collisions, we developed a confrontation assay 28 whereby two different cell-types are separated by a barrier, which upon removal, 29 creates a uniform gap into which the different cell populations migrate and collide. 30Following a screen of a range of different epithelial versus mesenchymal cell-types, 31 an interesting and unexpected phenomenon was revealed. When a population of 32 migrating epithelial cells (HaCaT) encountered a population of migrating fibroblasts 33 (NIH3T3), both populations ceased their forward migration, forming a sharp border 34 ( Figure 1A, B and Video S1). This is in stark contrast to fibrosarcoma cells (HT1080) 35 which, upon collision with epithelial cells, seemed to undergo a complete repulsion 36 ( Figure 1A, B and Video ...