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

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 two additional calcium-binding fluorochromes: xylenol orange 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 a pulse-chase approach, we show that the initiation of skeletogenesis begins around 15 ​h post fertilization. We also assess the timing of triradiate formation in embryos treated with a range of patterning perturbagens and demonstrate that triradiate formation is delayed and asynchronous in embryos ventralized via treatment with either nickel or chlorate. 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 embryos treated with axitinib to inhibit VEGFR. 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.