Project Outcomes Report

Intellectual Merit:

1. Overview This project focused on uncovering the mechanism for skeletal patterning in sea urchin embryos. Understanding how patterns form in developing embryos is important for learning how birth defects arise and how we might prevent or treat such defects; this is especially important for skeletal birth defects that are the second-most common in humans. Studying simple, rapidly developing animals like the sea urchin accelerates discovery; because developmental processes are highly conserved, what we learn in a simple animal like a sea urchin can teach us a lot about how more complex animals like humans develop. During this award period, we have published two peer-reviewed manuscripts (Hogan et al., 2020, Rodriguez-Sastre et al., 2022), collaborated on two others (Li et al., 2020, Leong et al., 2021), published two book chapters describing methods (Rodriguez-Sastre et al., 2019, Zuch and Bradham, 2019), and published four additional preprints (Schatzberg et al., 2021; Hawkins et al., 2022; Thomas et al., 2022, Descoteaux et al., 2022) that are currently progressing to peer-reviewed publications. This award also supported three additional manuscripts that we expect to submit within the next 6 months. 

2. Transcriptomics and Genomics. Our developmental transcriptome for the sea urchin species, Lytechinus variegatuswas published in Developmental Biology in 2020. The results unexpectedly and intriguingly show that gene expression changes are abrupt and occur at specific developmental timepoints (Fig. 1). We also published two collaborative papers with Naoki Irie (U. Tokyo) that focused on comparative genomic analyses of echinoderms. 

3. Pattern Formation. We use sea urchin embryos as a simple model for pattern formation. In these embryos mesenchymal cells called PMCs secrete a mineralized endoskeleton, while the adjacent ectodermal tissue provides information to instruct the positioning of the mesenchymal cells and thereby control the pattern of the skeleton. We found that ethanol (EtOH) exposure elicits teratogenic effects of on sea urchins that are hallmarked by skeletal patterning defects (Fig. 2A) (Developmental Biology, 2023). A separate study identified voltage-gated sodium channels (VGSCs) as required for normal skeletal patterning (Fig. 2B); this preprint is currently under peer review.  A separate manuscript now in preparation focuses on the effects of polyfluorinated alkyl substances (PFAS) on sea urchins, and found that that both PFOA and GenX, an ostensibly safer PFAS alternative, are each potent teratogens that disrupt skeletal patterning in sea urchins. We are interested in defining and studying the cues that regulate PMC positioning. We've also found that the 5' lipoxygenase gene (LOX) is required for normal skeletal patterning; the corresponding manuscript is currently under major revision in response to peer review. One of our major projects is understanding how the PMCs respond to skeletal patterning cues, and we have approached this work using single cell transcriptomics and machine-learning based automated 3-D PMC detection. This project's experimental work is nearly complete, and we expect its submission in the spring of 2023. To overcome analytical obstacles in this project, we devised a novel approach for scRNA-seq analysis that we named ICAT for "identifying cells after treatment" (Fig. 3). A preprint describing ICAT has been published and the manuscript is currently resubmitted. We have also recently developed a new technique for serial pulse-chase live fluorescent labeling of the skeleton ("polychrome labeling") using differently colored calcium-binding fluorochromes that provides previously unavailable temporal insight into skeletal formation dynamics (Fig. 4); this preprint is currently under peer review. We also published an invited book chapter that describes our experimental approach for spatial analysis of PMC positions in 3-D space.

4. Bioelectricity. Finally, we are interested in the role that bioelectrical changes play in development and pattern formation. Along with the VGSC study described above, we have identified the v-ATPase proton pump (VHA) as required for dorsal specification (Fig. 5). The VHA preprint is currently under major revision following peer review. We also published an invited book chapter that details our approach for the detection of voltage and ion levels in live embryos with fluorescent reporters.

Broader Impacts:

The Principal Investigator (PI) mentored 9 graduate students (7 PhD students and 2 Masters students), and 13 undergraduate research students during the course of this award. The PI's lab hosted high school and students and teachers for short-term research projects, along with undergraduate students for both wet-lab and bioinformatics projects, each summer. The PI and the students attended conferences regularly, including the International Conference for the Developmental Biology of the Sea Urchin, which the PI organized in 2018, the Society for Developmental Biology, and the Gordon Conference on Biomineralization, except during the pandemic. The PI taught courses on Developmental Biology and Advanced Cell Biology each year. The PI also co-taught the Echinoderm Module at the Embryology Course at the Marine Biology Laboratory at Woods Hole MA (2017-2021). 

Last Modified: 12/23/2022
Modified by: Cynthia Bradham