A morphogen gradient of Bone Morphogenetic Protein (BMP) signaling patterns the dorsoventral embryonic axis of vertebrates and invertebrates. The prevailing view in vertebrates for BMP gradient formation is through a counter-gradient of BMP antagonists, often along with ligand shuttling to generate peak signaling levels. To delineate the mechanism in zebrafish, we precisely quantified the BMP activity gradient in wild-type and mutant embryos and combined these data with a mathematical model-based computational screen to test hypotheses for gradient formation. Our analysis ruled out a BMP shuttling mechanism and a bmp transcriptionally-informed gradient mechanism. Surprisingly, rather than supporting a counter-gradient mechanism, our analyses support a fourth model, a source-sink mechanism, which relies on a restricted BMP antagonist distribution acting as a sink that drives BMP flux dorsally and gradient formation. We measured Bmp2 diffusion and found that it supports the source-sink model, suggesting a new mechanism to shape BMP gradients during development.
- Joseph Zinski
- Mary Mullins
- Joseph Zinski
- Ye Bu
- Xu Wang
- Wei Dou
- David Umulis
- Mary Mullins
- Joseph Zinski
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#803105, #804931) of the University of Pennsylvania Perelman School of Medicine.
- Robb Krumlauf, Stowers Institute for Medical Research, United States
© 2017, Zinski et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Cerebellar climbing fibers convey diverse signals, but how they are organized in the compartmental structure of the cerebellar cortex during learning remains largely unclear. We analyzed a large amount of coordinate-localized two-photon imaging data from cerebellar Crus II in mice undergoing ‘Go/No-go’ reinforcement learning. Tensor component analysis revealed that a majority of climbing fiber inputs to Purkinje cells were reduced to only four functional components, corresponding to accurate timing control of motor initiation related to a Go cue, cognitive error-based learning, reward processing, and inhibition of erroneous behaviors after a No-go cue. Changes in neural activities during learning of the first two components were correlated with corresponding changes in timing control and error learning across animals, indirectly suggesting causal relationships. Spatial distribution of these components coincided well with boundaries of Aldolase-C/zebrin II expression in Purkinje cells, whereas several components are mixed in single neurons. Synchronization within individual components was bidirectionally regulated according to specific task contexts and learning stages. These findings suggest that, in close collaborations with other brain regions including the inferior olive nucleus, the cerebellum, based on anatomical compartments, reduces dimensions of the learning space by dynamically organizing multiple functional components, a feature that may inspire new-generation AI designs.
Drug resistance is a challenge in anticancer therapy. In many cases, cancers can be resistant to the drug prior to exposure, i.e., possess intrinsic drug resistance. However, we lack target-independent methods to anticipate resistance in cancer cell lines or characterize intrinsic drug resistance without a priori knowledge of its cause. We hypothesized that cell morphology could provide an unbiased readout of drug resistance. To test this hypothesis, we used HCT116 cells, a mismatch repair-deficient cancer cell line, to isolate clones that were resistant or sensitive to bortezomib, a well-characterized proteasome inhibitor and anticancer drug to which many cancer cells possess intrinsic resistance. We then expanded these clones and measured high-dimensional single-cell morphology profiles using Cell Painting, a high-content microscopy assay. Our imaging- and computation-based profiling pipeline identified morphological features that differed between resistant and sensitive cells. We used these features to generate a morphological signature of bortezomib resistance. We then employed this morphological signature to analyze a set of HCT116 clones (five resistant and five sensitive) that had not been included in the signature training dataset, and correctly predicted sensitivity to bortezomib in seven cases, in the absence of drug treatment. This signature predicted bortezomib resistance better than resistance to other drugs targeting the ubiquitin-proteasome system. Our results establish a proof-of-concept framework for the unbiased analysis of drug resistance using high-content microscopy of cancer cells, in the absence of drug treatment.