High-throughput hyperdimensional vertebrate phenotyping.

Most gene mutations and biologically active molecules cause complex responses in animals that cannot be predicted by cell culture models. Yet animal studies remain too slow and their analyses are often limited to only a few readouts. Here we demonstrate high-throughput optical projection tomography with micrometre resolution and hyperdimensional screening of entire vertebrates in tens of seconds using a simple fluidic system. Hundreds of independent morphological features and complex phenotypes are automatically captured in three dimensions with unprecedented speed and detail in semitransparent zebrafish larvae. By clustering quantitative phenotypic signatures, we can detect and classify even subtle alterations in many biological processes simultaneously. We term our approach hyperdimensional in vivo phenotyping. To illustrate the power of hyperdimensional in vivo phenotyping, we have analysed the effects of several classes of teratogens on cartilage formation using 200 independent morphological measurements, and identified similarities and differences that correlate well with their known mechanisms of actions in mammals.

Large-scale analysis of neurite growth dynamics on micropatterned substrates.

During both development and regeneration of the nervous system, neurons display complex growth dynamics, and several neurites compete to become the neuron's single axon. Numerous mathematical and biophysical models have been proposed to explain this competition, which remain experimentally unverified. Large-scale, precise, and repeatable measurements of neurite dynamics have been difficult to perform, since neurons have varying numbers of neurites, which themselves have complex morphologies. To overcome these challenges using a minimal number of primary neurons, we generated repeatable neuronal morphologies on a large scale using laser-patterned micron-wide stripes of adhesive proteins on an otherwise highly non-adherent substrate. By analyzing thousands of quantitative time-lapse measurements of highly reproducible neurite growth dynamics, we show that total neurite growth accelerates until neurons polarize, that immature neurites compete even at very short lengths, and that neuronal polarity exhibits a distinct transition as neurites grow. Proposed neurite growth models agree only partially with our experimental observations. We further show that simple yet specific modifications can significantly improve these models, but still do not fully predict the complex neurite growth behavior. Our high-content analysis puts significant and nontrivial constraints on possible mechanistic models of neurite growth and specification. The methodology presented here could also be employed in large-scale chemical and target-based screens on a variety of complex and subtle phenotypes for therapeutic discoveries using minimal numbers of primary neurons.