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During embryogenesis, morphogens form a concentration gradient in responsive tissue, which is then translated into a spatial cellular pattern. The mechanisms by which morphogens spread through a tissue to establish such a morphogenetic field remain elusive. Here, we investigate by mutually complementary simulations and in vivo experiments how Wnt morphogen transport by cytonemes differs from typically assumed diffusion-based transport for patterning of highly dynamic tissue such as the neural plate in zebrafish. Stochasticity strongly influences fate acquisition at the single cell level and results in fluctuating boundaries between pattern regions. Stable patterning can be achieved by sorting through concentration dependent cell migration and apoptosis, independent of the morphogen transport mechanism. We show that Wnt transport by cytonemes achieves distinct Wnt thresholds for the brain primordia earlier compared with diffusion-based transport. We conclude that a cytoneme-mediated morphogen transport together with directed cell sorting is a potentially favored mechanism to establish morphogen gradients in rapidly expanding developmental systems.

Signaling filopodia, termed cytonemes, are dynamic actin-based membrane structures that regulate the exchange of signaling molecules and their receptors within tissues. However, how cytoneme formation is regulated remains unclear. Here, we show that Wnt/planar cell polarity (PCP) autocrine signaling controls the emergence of cytonemes, and that cytonemes subsequently control paracrine Wnt/β-catenin signal activation. Upon binding of the Wnt family member Wnt8a, the receptor tyrosine kinase Ror2 becomes activated. Ror2/PCP signaling leads to the induction of cytonemes, which mediate the transport of Wnt8a to neighboring cells. In the Wnt-receiving cells, Wnt8a on cytonemes triggers Wnt/β-catenin-dependent gene transcription and proliferation. We show that cytoneme-based Wnt transport operates in diverse processes, including zebrafish development, murine intestinal crypt and human cancer organoids, demonstrating that Wnt transport by cytonemes and its control via the Ror2 pathway is highly conserved in vertebrates.

The evolution of drug resistance in microbial pathogens provides a paradigm for investigating evolutionary dynamics with important consequences for human health. Candida albicans, the leading fungal pathogen of humans, rapidly evolves resistance to two major antifungal classes, the triazoles and echinocandins. In contrast, resistance to the third major antifungal used in the clinic, amphotericin B (AmB), remains extremely rare despite 50 years of use as monotherapy. We sought to understand this long-standing evolutionary puzzle. We used whole genome sequencing of rare AmB-resistant clinical isolates as well as laboratory-evolved strains to identify and investigate mutations that confer AmB resistance in vitro. Resistance to AmB came at a great cost. Mutations that conferred resistance simultaneously created diverse stresses that required high levels of the molecular chaperone Hsp90 for survival, even in the absence of AmB. This requirement stemmed from severe internal stresses caused by the mutations, which drastically diminished tolerance to external stresses from the host. AmB-resistant mutants were hypersensitive to oxidative stress, febrile temperatures, and killing by neutrophils and also had defects in filamentation and tissue invasion. These strains were avirulent in a mouse infection model. Thus, the costs of evolving resistance to AmB limit the emergence of this phenotype in the clinic. Our work provides a vivid example of the ways in which conflicting selective pressures shape evolutionary trajectories and illustrates another mechanism by which the Hsp90 buffer potentiates the emergence of new phenotypes. Developing antibiotics that deliberately create such evolutionary constraints might offer a strategy for limiting the rapid emergence of drug resistance.