Determining the roles of ROS and CMTM4 in sea urchin skeletal patterning with a novel polychrome labeling method for post hoc analysis
Embargo Date
2026-03-14
OA Version
Citation
Abstract
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, labeling protocols are optimized for additional calcium-binding fluorochromes and it is demonstrated that these fluorochromes can be used individually or in nested pulse-chase experiments to understand the temporal dynamics of skeletogenesis and patterning in sea urchin larvae. Using a pulse-chase approach, it is shown that the initiation of skeletogenesis begins synchronously in control embryos around 15 hours post fertilization but that triradiate formation is delayed and asynchronous in embryos ventralized via treatment with either nickel or chlorate. The extent of fluorochrome incorporation at several time points is also measured in three-pulse embryos to show that skeletal elements elongate much more slowly in axitinib-treated embryos, and that axitinib treatment is sufficient to induce abnormal orientation of the triradiates. Of the mineralization markers tested, alizarin red (AZ) was the only fluorochrome that significantly perturbed skeletal patterning in sea urchin embryos. To characterize the effects of AZ on skeletal patterning, it is first shown that the effects of AZ are dose-dependent and that intermediate doses of AZ produce abnormal rotation, branching, and loss of many skeletal elements. The development of AZ-treated embryos is also delayed, as evident by delayed initiation of biomineralization and migration of the primary mesenchyme cells (PMCs) which secrete the skeleton. While AZ treatment does not perturb gross ectodermal dorsal-ventral (DV) or neural specification, it does perturb the connectivity and patterning of serotonergic neurons, which are abnormally absent from the midline. The effects of AZ on skeletal patterning are shown to be at least partially due to elevation of reactive oxygen species due to catalase inhibition.
Lastly, our polychrome labeling approaches are applied to a novel patterning cue: chemokine-like factor MARVEL transmembrane domain-containing protein 4 (CMTM4). First, the spatiotemporal dynamics of CMTM4 expression are characterized and CMTM4 is confirmed to be a membrane-bound protein. Normal CMTM4 levels are shown to be required for normal skeletal patterning, and polychrome labeling is used to reveal delayed initiation of biomineralization in CMTM4-perturbed embryos as well as left-right asymmetries in the progression of skeletogenesis. Finally, CMTM4 is shown to have a role in PMC migration but not gross ectodermal DV specification. Together, this work defines a new use for calcium-binding fluorochromes in the context of sea urchin skeletogenesis and applies this method to known and novel skeletal patterning perturbations to reveal new insights into the progression and regulation of skeletal patterning and biomineralization.
Description
2024