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Sea urchins (echinoids) are common model organisms for research in developmental biology and for their unusual transition from a bilaterally organized larva into a post-metamorphic adult with pentaradial body symmetry. The adult also has a calcareous endoskeleton with a multimetameric pattern of continuously added elements, among them the namesake of this phylum, spines. Nearly all echinoids have both large primary spines, and an associated set of smaller secondary spines. We hypothesize that the secondary spines of the tropical variegated urchin species, Lytechinus variegatus, are morphologically and molecularly distinct structures from primary spines and not just small versions of the large spines. To test this premise, we examined both spine types using light microscopy, micro-CT imaging, lectin labeling, transcriptomics, and fluorescence in situ hybridization (FISH). Our findings reveal basic similarities between the two spine types in mineral and cellular anatomy, but with clear differences in growth patterns, genes expressed, and in the profile of various expressed genes. In particular, secondary spines have non-overlapping, longitudinally concentrated growth bands that lead to a blunt and straight profile, and a distinct transcriptome involving the upregulation in many genes in comparison to the primary spines. Neural, ciliary, and extracellular matrix interacting factors are implicated in the differentially expressed gene (DEG) dataset, including two genes-ONECUT2 and an uncharacterized discoidin- and thrombospondin-containing protein. We show spine type-specific localizations by FISH, which will be of interest to ongoing work in urchin spine patterning. These results demonstrate that primary and secondary spines of L. variegatus have overlapping but distinct molecular and biomineralization characteristics, suggesting unique developmental, regenerative, and representation in this spiny dermal phylum.
Fig. 1. Secondary and primary spines of interambulacral areas in Lytechinus variegatus (Lv; A,C), and Strongylocentrotus purpuratus for comparison (Sp; B,D). Primary (P), secondary (S), and miliary (M) tubercles are indicated on live animals (A,B), and on tests (C,D). In both species, primary spines grow in rows from the skeletal plates, with groups of secondary spines surrounding them. The arrangement of secondary and miliary tubercles in S. purpuratus is tighter, forming a clear scrobicular ring compared to L. variegatus. Transverse sections of primary (E) and secondary (F) spines of L. variegatus are shown in brightfield. The spine types are visually distinct by size and septa number, with the larger primaries having more septa. (G) Diagrammatic representation of spine anatomy (after Heatfield and Travis, 1975b). Scale bars represent 1 mm on A—D; 250 μm on E; 125 μm on F.
Fig. 2. Micro-CT reconstructions of primary (P) and secondary (S) spines of L. variegatus, digitally sliced in the longitudinal (A–C) and transverse (D) planes. (A) and (D) show primaries and secondaries from the same individual to scale, while (B) and (C) show closeups of isolated spines and are not to scale. Because micro-CT reconstruction compiles a series of planar X-ray images, translucent areas are dark and dense areas bright. Notice the secondary in (D; arrow), which has one subtle concentric ring (arrowhead). Scale bars represent 1 mm.
Fig. 3. Wheat germ agglutinin and concanavalin A staining of primary (proximal, A; distal, B) and secondary spines (C,D). Nuclei, stained with DAPI, cluster in rows between the septa (see Fig. 1). Arrowheads mark ConA-positive cellular bodies. (E) shows a primary spine with a deep Z field, which allows visualization of the nuclei of these ConA-positive globular cells. Scale bars represent 100 μm.
Fig. 4. Summarized results of differential gene expression analysis from RNA-seq of 4 replicate urchins, from the white color morph group (A). (B) PCA plot comparing expression levels of all genes. Primary spines (PS) and secondary spines (SS) assort along the PC1 axis. (C): Volcano plot shows the distribution of differentially expressed genes, secondaries relative to primaries. (D) Mainstream hierarchical clustering analysis heatmap of DEGs. Each row represents the expression level (FPKM, Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced) of a DEG and is homogenized; rows can be compared horizontally but not vertically.
Fig. 5. Sm50 (A) proximal and distal secondaries, (B) proximal—distal transition zone in a primary spine. Scale bars represent 100 μm.
Fig. 6. FISH images labeling NOTCH2-like (A proximal secondary, B,C distal secondary, D proximal primary, E distal primary). Scale bars represent 100 μm.
Fig. 7. FISH images of DiscTSR (A) proximal secondary, (B) distal secondary, (C) medial secondary in partial longitudinal section, (D) proximal primary, (E) distal primary. Scale bars represent 100 μm.
Fig. 8. FISH images labeling of ONECUT2 (M proximal secondary, N distal secondary, O P medial secondary in surface view and longitudinal section, Q proximal primary, R distal primary). Scale bars represent 100 μm.
