Polychrome labeling reveals skeletal triradiate and elongation dynamics and abnormalities in patterning cue-perturbed embryos

The larval skeleton of the sea urchin Lytechinus variegatus is an ideal model system for studying skeletal patterning; however, our understanding of the etiology of skeletal patterning in sea urchin larvae is limited due to the lack of approaches to live-image skeleton formation. Calcium-binding fluorochromes have been used to study the temporal dynamics of bone growth and healing. To date, only calcein green has been used in sea urchin larvae to fluorescently label the larval skeleton. Here, we optimize labeling protocols for four other calcium-binding fluorochromes-alizarin red, xylenol orange, tetracycline, and calcein blue- and demonstrate that these fluorochromes can be used individually or in nested pulse-chase experiments to understand the temporal dynamics of skeletogenesis and patterning. Using this pulse-chase approach we reveal that the initiation of skeletogenesis begins around 15 hours post fertilization, which is earlier than previously thought. We also explore the timing of triradiate formation in embryos treated with a range of patterning perturbagens, and demonstrate that triradiates form late and asynchronously in embryos ventralized via treatment with either nickel at early gastrula stage or with chlorate from fertilization. Finally, we measure the extent of fluorochrome incorporation in triple-labeled embryos to determine the elongation rate of numerous skeletal elements throughout early skeletal patterning and compare this to the rate of skeletal growth in axitinib-treated embryos. We find that skeletal elements elongate much more slowly in axitinib-treated embryos, and that axitinib treatment is sufficient to induce abnormal orientation of the triradiates. Highlights Calcium-binding fluorochromes selectively label the sea urchin larval skeleton Pulsed, nested polychrome labeling offers temporal insight into skeletal patterning Triradiate formation is delayed and asynchronous in ventralized embryos VEGFR inhibition slows skeletal elongation and perturbs triradiate orientation