Quantifying spatial position in a branched structure in immunostained mouse tissue sections.

We have developed a protocol to quantify the position of a cell in a branched structure based on two-dimensional microscopy images of tissue sections. Biological branched structures include organs such as the lungs, kidneys, and pancreas. In these organs, cell fate has been correlated with position, based on a qualitative estimate. However, a quantitative means of evaluating the cell position has been lacking. With this protocol, the correlation between cell fate and cell position was measured in mouse embryonic pancreas. For complete details on the use and execution of this protocol, please refer to Nyeng et al. (2019).

Cooperation between melanoma cell states promotes metastasis through heterotypic cluster formation.

Melanomas can have multiple coexisting cell states, including proliferative (PRO) versus invasive (INV) subpopulations that represent a "go or grow" trade-off; however, how these populations interact is poorly understood. Using a combination of zebrafish modeling and analysis of patient samples, we show that INV and PRO cells form spatially structured heterotypic clusters and cooperate in the seeding of metastasis, maintaining cell state heterogeneity. INV cells adhere tightly to each other and form clusters with a rim of PRO cells. Intravital imaging demonstrated cooperation in which INV cells facilitate dissemination of less metastatic PRO cells. We identified the TFAP2 neural crest transcription factor as a master regulator of clustering and PRO/INV states. Isolation of clusters from patients with metastatic melanoma revealed a subset with heterotypic PRO-INV clusters. Our data suggest a framework for the co-existence of these two divergent cell populations, in which heterotypic clusters promote metastasis via cell-cell cooperation.

p120ctn-Mediated Organ Patterning Precedes and Determines Pancreatic Progenitor Fate.

The mechanism of how organ shape emerges and specifies cell fate is not understood. Pancreatic duct and endocrine lineages arise in a spatially distinct domain from the acinar lineage. Whether these lineages are pre-determined or settle once these niches have been established remains unknown. Here, we reconcile these two apparently opposing models, demonstrating that pancreatic progenitors re-localize to establish the niche that will determine their ultimate fate. We identify a p120ctn-regulated mechanism for coordination of organ architecture and cellular fate mediated by differential E-cadherin based cell sorting. Reduced p120ctn expression is necessary and sufficient to re-localize a subset of progenitors to the peripheral tip domain, where they acquire an acinar fate. The same mechanism is used re-iteratively during endocrine specification, where it balances the choice between the alpha and beta cell fates. In conclusion, organ patterning is regulated by p120ctn-mediated cellular positioning, which precedes and determines pancreatic progenitor fate.

Microenvironment-derived factors driving metastatic plasticity in melanoma.

Cellular plasticity is a state in which cancer cells exist along a reversible phenotypic spectrum, and underlies key traits such as drug resistance and metastasis. Melanoma plasticity is linked to phenotype switching, where the microenvironment induces switches between invasive/MITFLO versus proliferative/MITFHI states. Since MITF also induces pigmentation, we hypothesize that macrometastatic success should be favoured by microenvironments that induce a MITFHI/differentiated/proliferative state. Zebrafish imaging demonstrates that after extravasation, melanoma cells become pigmented and enact a gene expression program of melanocyte differentiation. We screened for microenvironmental factors leading to phenotype switching, and find that EDN3 induces a state that is both proliferative and differentiated. CRISPR-mediated inactivation of EDN3, or its synthetic enzyme ECE2, from the microenvironment abrogates phenotype switching and increases animal survival. These results demonstrate that after metastatic dissemination, the microenvironment provides signals to promote phenotype switching and provide proof that targeting tumour cell plasticity is a viable therapeutic opportunity.

Why do bacteria regulate public goods by quorum sensing?-How the shapes of cost and benefit functions determine the form of optimal regulation.

Many bacteria secrete compounds which act as public goods. Such compounds are often under quorum sensing (QS) regulation, yet it is not understood exactly when bacteria may gain from having a public good under QS regulation. Here, we show that the optimal public good production rate per cell as a function of population size (the optimal production curve, OPC) depends crucially on the cost and benefit functions of the public good and that the OPC will fall into one of two categories: Either it is continuous or it jumps from zero discontinuously at a critical population size. If, e.g., the public good has accelerating returns and linear cost, then the OPC is discontinuous and the best strategy thus to ramp up production sharply at a precise population size. By using the example of public goods with accelerating and diminishing returns (and linear cost) we are able to determine how the two different categories of OPSs can best be matched by production regulated through a QS signal feeding back on its own production. We find that the optimal QS parameters are different for the two categories and specifically that public goods which provide accelerating returns, call for stronger positive signal feedback.

A Quantitative System for Studying Metastasis Using Transparent Zebrafish.

Metastasis is the defining feature of advanced malignancy, yet remains challenging to study in laboratory environments. Here, we describe a high-throughput zebrafish system for comprehensive, in vivo assessment of metastatic biology. First, we generated several stable cell lines from melanomas of transgenic mitfa-BRAF(V600E);p53(-/-) fish. We then transplanted the melanoma cells into the transparent casper strain to enable highly quantitative measurement of the metastatic process at single-cell resolution. Using computational image analysis of the resulting metastases, we generated a metastasis score, µ, that can be applied to quantitative comparison of metastatic capacity between experimental conditions. Furthermore, image analysis also provided estimates of the frequency of metastasis-initiating cells (∼1/120,000 cells). Finally, we determined that the degree of pigmentation is a key feature defining cells with metastatic capability. The small size and rapid generation of progeny combined with superior imaging tools make zebrafish ideal for unbiased high-throughput investigations of cell-intrinsic or microenvironmental modifiers of metastasis. The approaches described here are readily applicable to other tumor types and thus serve to complement studies also employing murine and human cell culture systems.

Exploiting social evolution in biofilms.

Bacteria are highly social organisms that communicate via signaling molecules, move collectively over surfaces and make biofilm communities. Nonetheless, our main line of defense against pathogenic bacteria consists of antibiotics-drugs that target individual-level traits of bacterial cells and thus, regrettably, select for resistance against their own action. A possible solution lies in targeting the mechanisms by which bacteria interact with each other within biofilms. The emerging field of microbial social evolution combines molecular microbiology with evolutionary theory to dissect the molecular mechanisms and the evolutionary pressures underpinning bacterial sociality. This exciting new research can ultimately lead to new therapies against biofilm infections that exploit evolutionary cheating or the trade-off between biofilm formation and dispersal.

Coexistence of phage and bacteria on the boundary of self-organized refuges.

Bacteriophage are voracious predators of bacteria and a major determinant in shaping bacterial life strategies. Many phage species are virulent, meaning that infection leads to certain death of the host and immediate release of a large batch of phage progeny. Despite this apparent voraciousness, bacteria have stably coexisted with virulent phages for eons. Here, using individual-based stochastic spatial models, we study the conditions for achieving coexistence on the edge between two habitats, one of which is a bacterial refuge with conditions hostile to phage whereas the other is phage friendly. We show how bacterial density-dependent, or quorum-sensing, mechanisms such as the formation of biofilm can produce such refuges and edges in a self-organized manner. Coexistence on these edges exhibits the following properties, all of which are observed in real phage-bacteria ecosystems but difficult to achieve together in nonspatial ecosystem models: (i) highly efficient virulent phage with relatively long lifetimes, high infection rates and large burst sizes; (ii) large, stable, and high-density populations of phage and bacteria; (iii) a fast turnover of both phage and bacteria; and (iv) stability over evolutionary timescales despite imbalances in the rates of phage vs. bacterial evolution.

Acyl-homoserine lactone-dependent eavesdropping promotes competition in a laboratory co-culture model.

Many Proteobacteria use acyl-homoserine lactone (AHL)-mediated quorum sensing to activate the production of antibiotics at high cell density. Extracellular factors like antibiotics can be considered public goods shared by individuals within a group. Quorum-sensing control of antibiotic production may be important for protecting a niche or competing for limited resources in mixed bacterial communities. To begin to investigate the role of quorum sensing in interspecies competition, we developed a dual-species co-culture model using the soil saprophytes Burkholderia thailandensis (Bt) and Chromobacterium violaceum (Cv). These bacteria require quorum sensing to activate the production of antimicrobial factors that inhibit growth of the other species. We demonstrate that quorum-sensing-dependent antimicrobials can provide a competitive advantage to either Bt or Cv by inhibiting growth of the other species in co-culture. Although the quorum-sensing signals differ for each species, we show that the promiscuous signal receptor encoded by Cv can sense signals produced by Bt, and that this ability to eavesdrop on Bt can provide Cv an advantage in certain situations. We use an in silico approach to investigate the effect of eavesdropping in competition, and show conditions where early activation of antibiotic production resulting from eavesdropping can promote competitiveness. Our work supports the idea that quorum sensing is important for interspecies competition and that promiscuous signal receptors allow eavesdropping on competitors in mixed microbial habitats.

Sustainability of virulence in a phage-bacterial ecosystem.

Virulent phages and their bacterial hosts represent an unusual sort of predator-prey system where each time a prey is eaten, hundreds of new predators are born. It is puzzling how, despite the apparent effectiveness of the phage predators, they manage to avoid driving their bacterial prey to extinction. Here we consider a phage-bacterial ecosystem on a two-dimensional (2-d) surface and show that homogeneous space in itself enhances coexistence. We analyze different behavioral mechanisms that can facilitate coexistence in a spatial environment. For example, we find that when the latent times of the phage are allowed to evolve, selection favors "mediocre killers," since voracious phage rapidly deplete local resources and go extinct. Our model system thus emphasizes the differences between short-term proliferation and long-term ecosystem sustainability.