Post-metamorphic skeletal growth in the sea urchin Paracentrotus lividus and implications for body plan evolution

Background Understanding the molecular and cellular processes that underpin animal development are crucial for understanding the diversity of body plans found on the planet today. Because of their abundance in the fossil record, and tractability as a model system in the lab, skeletons provide an ideal experimental model to understand the origins of animal diversity. We herein use molecular and cellular markers to understand the growth and development of the juvenile sea urchin (echinoid) skeleton. Results We developed a detailed staging scheme based off of the first ~ 4 weeks of post-metamorphic life of the regular echinoid Paracentrotus lividus. We paired this scheme with immunohistochemical staining for neuronal, muscular, and skeletal tissues, and fluorescent assays of skeletal growth and cell proliferation to understand the molecular and cellular mechanisms underlying skeletal growth and development of the sea urchin body plan. Conclusions Our experiments highlight the role of skeletogenic proteins in accretionary skeletal growth and cell proliferation in the addition of new metameric tissues. Furthermore, this work provides a framework for understanding the developmental evolution of sea urchin body plans on macroevolutionary timescales. Supplementary Information The online version contains supplementary material available at 10.1186/s13227-021-00174-1.


Supplemental Materials and Methods (a) Culturing
The initial cultures were concentrated at approximately 10 larvae/ml. During the culturing period larvae were then diluted to an approximate final concentration of 1 larva/2ml in a large beaker. They were fed three times a week using a dropper with 70% Isocrysis, 30% Dunaliela tertiolecta and aquarium grade fatty acids (Snow Reef, SHG). The total number of algal cells used varied from 5.000-20,000/ml according to larval needs following microscopic observation of the stomach contents. The water was changed twice a week and larvae were kept at 18 0 C. In these conditions, the larvae became competent in approximately 3.5 weeks. Competent larvae metamorphosed onto the sides of the beaker in which they were cultured, or onto small, glass cover slips which were added to the beaker two to three days prior to metamorphosis.

(b) Immunohistochemistry
Juvenile and late stage P. lividus larvae were fixed in 4% paraformaldehyde (PFA) for 15 minutes. Individuals were then treated with ice cold methanol (MeOH) for one minute, and washed four to five times in 1X Phosphate Buffered Saline with 0.1% Tween (PBST). Most washes were for ~5 minutes in 200 µl. Samples were then stored in blocking buffer (BB; PBST with 4% goat or sheep serum and 1% BSA) at 4˚C, either overnight or for up to four months prior to incubation with primary antibodies. Samples were incubated with combinations of primary antibodies for 1.5 hours at 37˚C, or overnight at 4˚C, depending upon the primary antibody. Primary antibody type and dilutions are shown in Supplemental Table 1. Samples were then washed 4 to 5 times with PBST, and incubated with anti-rabbit, anti-rat and/or anti-mouse secondary antibodies (Thermo Fischer Scientific) conjugated with Alexa fluorophores (Supp. Table 1) for one hour using a dilution of 1/1000. Following removal of secondary antibodies, each sample was washed 4 to 5 times with PBST before addition of DAPI at a dilution of 1:10000 (of a stock solution of 5 mg/ml), for at least 15 minutes. To preserve skeleton intact, samples were intentionally not decalcified.

(c) Calcein staining
In order to visualize newly deposited CaC0 3 of the skeleton, live animals were incubated with Calcein (Sigma) at a dilution of 1:50 of a stock solution of 1.25 mg/ml in sea water. Calcein is light-sensitive, so incubation took place in the dark. Incubation took place in 4 ml wells with a coverslip placed at the bottom of each well. After 24 hours of incubation, calcein was washed out six times with fresh sea water. To understand the spatial incorporation of calcein relative to additional skeletal growth, pulse-chase experiments were carried out following incubation. 0hours chase animals were fixed immediately following calcein wash out and fixed following protocols for immunostaining up to the BB step and stored at 4˚C in the dark. 24-and 48-hours chase animals were reared in sea water in the dark prior to fixation and storage in BB in the dark at 4˚C. Following fixation, immunostaining against skeletogenic proteins was carried out as described above, except that all steps were carried out in the dark. Specimens were imaged using a Zeiss LSM 700 confocal microscope.

(d) Assays of cell proliferation
To understand the spatial distribution and quantity of proliferating cells during juvenile sea urchin growth assays of cell proliferation were carried out using the Click-iT® EdU Alexa Flour® 555HCS and (Life Technologies) and Click-iT TM EdU Cell Proliferation Kit for Imaging Alexa Flour TM 647 (Thermo Fisher Scientific). J2 animals were incubated with EdU in 7 ml wells at a dilution of 1:1000 (5 µM) in sea water for six hours. J5 and J6 animals were incubated with Edu for 63 hours. Edu was washed out six times following incubation, and 0-hours chase animals were immediately fixed in 4% PFA for 15 minutes, while 24-and 48 hours chase animals were further reared in wells until fixation. After fixation, samples were washed twice with PBST, and permeabilized in PBST+Triton for 30 minutes. They were then washed twice in PBST, permeabilized in MEOH for 1 minute, and washed a further two times before storage in BB overnight at 4˚C. Primary and secondary antibody incubations were carried out as described above, and following the PBST washes after secondary antibody incubation, samples were incubated in 50 µl of the Click-iT® Reaction cocktail, following the manufacturer's instructions for 30 minutes. Samples were then washed with 50µl of Click-iT® reaction Rinse Buffer for 30 minutes, before two washes in PBST, and addition of DAPI at a dilution of 1:10000 (of a stock solution of 5 mg/ml). Specimens were imaged using n Zeiss LSM 700 confocal microscope. For each image, five circles of identical size were used to define regions of interest (ROIs) on each image. The relative position of the circles on each image was not standard with respect to the image, thus resulting in a semi-random placement of each circle. This was done intentionally so as to not bias which portion of the animal surfaces were counted. EdU + and DAPI + nuclei were quantified in ImageJ by first converting images to 16-bit, then thresholding. Images were then converted to binary images using ProcessàBinary, and then Processà Binary àWatershed. Finally cells within the ROIs were counted using AnalyzeàAnalyze Particles. This is summarized in Supplemental Figure 11. The ratio of Edu + to DAPIstained nuclei was then counted for each image, and the ratios for the aboral and oral surfaces of each quantified individual were compared using a Mann-Whitney U test. R code to repeat analyses and plotting are in Supplemental File 1.

Supplemental Data (1) Staging Scheme
In order to make standardized comparisons across cultures and asynchronously developing individuals, we devised a staging scheme based off the presence and absence of specific morphological characters during the course of post-metamorphic development. Our staging scheme does not cover the most immediate stages of post-metamorphic growth, and for an account of those stages we refer the reader to Gosselin and Jangoux [1] (i) Stage Juvenile 1 (J1) -The earliest of our stages, J1 corresponds to approximately 1-3 days post-metamorphosis. At this stage, the post-metamorphic juvenile is readily characterized by the primary podia, and the interambulacral primary spines. There are four interambulacral spines in each interambulacral area, one on each interambulacral plate. Ambulacral regions are dominated by the primary podia, and two poorly developed secondary podia are present in each ambulacral area. The primary podia have well-formed disks and rosettes, while the secondary podia lack disks, and have not yet formed rosettes. There are two small ambulacral plates in each ambulacrum, and there are ten peristomial plates arranged inter-radially adoral to each interambulacrum. When viewed adorally, the five ocular plates, five genital plates, and an anal plate are visible, and two juveniles spines (or multified spines of Gosselin and Jangoux [1]) are present on each ocular plate. The ocular plates each bear a ledge, which extends slightly over top of the primary podia. Between one and two pedicellariae are present on two of the genital plates, and at least one juvenile spine is present on each genital plate. The plates of the periproct and genital plates do not appear well-sutured together. (ii) Stage Juvenile 2 (J2) -J2 animals correspond to approximately 4-7 days postmetamorphosis. This stage shows the additional growth on the oral surface relative to J1 individuals, and is primarily differentiated based upon the presence of five peristomial tube feet, and the development of disks and rosettes in the secondary podia of each ambulacrum. There are still four primary spines and interambulacral plates present in each interambulacrum. Ambulacral areas are still dominated by the primary podia, though secondary podia are much larger, about the same size as the primary podia. Secondary podia have well-developed disks and growing skeletal rosettes. Small, peristomial podia are present protruding through five of the peristomial plates. Ocular plates still bear two juvenile spines, and there are between one and two pedicellariae on two genital plates. Genital plates do not appear well-sutured. (iii) Stage Juvenile 3 (J3) -J3 stage animals are roughly 7-10 days postmetamorphosis. They are most readily characterized by the presence of newly added sphaeridia, spine-like sensory structures present along one of the most oral ambulacral plates in each ambulacrum. All plates of the test have grown larger at their margins, and interambulacral areas bear four plates and primary spines. Ambulacral plates have grown to abut one another and the interambulacral plates and a clear circular margin has formed around the peristome. The secondary podia bear well-developed disks and rosettes, and are about the same size as, and may appear larger than, the primary podia. Spine-like, elongate sphaeridia have started to grow towards the perradial margin of one ambulacral plate in each ambulacrum, but they lack the large bulb present in later stages. Peristomial podia have developed small disks. On the aboral surface, the genital and ocular plates have started to fuse via plate accretion, and the anal plate has grown larger and more distinct. The number of juvenile spines and pedicellariae remains unchanged from earlier stages. (iv) Stage Juvenile 4 (J4) -Stage J4 animals are approximately 8-12 days postmetamorphosis. They are differentiated from Stage J3 animals by having a welldeveloped bulb at the tip of sphaeridia, and by having begun to add a second row of secondary podia. Interambulacra consist of four plates, each with a primary spine. Ambulacra are dominated by secondary podia and sphaeridia. Sphaeridia are elongate and with a well-developed bulb at their tip. Aboral surface shows well-sutured genital and ocular plates.
Stage Juvenile 5 (J5) -Stage J5 animals are substantially older than earlier stages, being approximately 2.5-3.5 weeks post-metamorphosis. This gap in timing is reflected by the addition of numerous novel morphological structures relative to earlier stage animals. In J5 animals, the aboral surface is relatively smaller than the oral surface, and all news growth has taken place in axial tissues. There are still four interambulacral plates and primary spines in each interambulacral area, but interambulacral pedicellariae have been added to the most aboral interambulacral plate in each area. Ambulacral areas have welldeveloped, bulbous and glossy pedicellariae, and primary podia are no longer visible, presumably having been lost during the course of growth. There are two well-developed rows of secondary podia in each ambulacral area, with each podium bearing a disk and rosette. One or two secondary podia of the third row are growing adjacent to the ocular plate, but have yet to develop disks. Immediately aboral of the ocular plate, ambulacral areas are dominated by a large ambulacral primary spine. In the extraxial tissues on the aboral surface, very little growth has taken place. Juvenile spines are still present on the aboral surface, but they are not easily visible. By J5, juveniles have also opened their mouths. (vi) Stage Juvenile 6 (J6) -The final stage in our scheme, J6, corresponds to roughly 3-4 weeks post-metamorphosis. J6 animals are the most morphologically complex stage, being characterized primarily by the addition of interambulacral and ambulacral primary spines, as well as the addition of ambulacral pedicellariae adoral to the ambulacral primary spines. There are four or five interambulacral plates in J6 animals, however, the plate surfaces have grown, and new interambulacral primary spines have been added to the adapical most interambulacral plates in each area. There are thus five or six interambulacral primary spines in each interambulacrum. As in J5 animals, a single pedicellariae is also present in each interambulacral area. Pedicellariae have also formed on a single ambulacral plates adoral to the largest ambulacral primary spines (which was added in J5 animals) in some of the ambulacra. Well-developed sphaeridia are present in each ambulacrum. Like J5 animals, J6 animals have no primary podia, and their adoral ambualcra are dominated by ambulacral primary spines. There are two ambulacral primary spines in each ambulacrum. There are three well-developed rows of secondary podia in each ambulacrum, and the podia of a fourth row are growing. Like J5 animals, no growth of new structures is taking place in the extraxial tissues of the aboral surface. Fig. S1 Confocal images of pre-metamorphosis, late stage P. lividus larvae. Shows developing adult body plan within the rudiment stained for Msp130 using monoclonal antibody 6a9 (green), and Sm50 using an anti-Sm50 antibody (magenta). (a) Merged image of Sm50 and Msp130 and nuclear staining (DAPI, blue) showing developing skeletal tissues, within rudiment including spines, tubercles, and the rosette of a primary podium. DAPI (blue) Msp130 strongly labels the bodies and projections of cells tightly associated with the skeleton (skeletogenic cells), and the skeleton. Conversely, staining for Sm50 is stronger in the skeleton itself, and in structures such as the spines and rosette. (b) Interpretive drawing showing morphology

Supplemental Figures
Msp130 DAPI     . β-tubulin + cells are located throughout the animal, including in the tips of the podia. Abbreviations are as follows: ps, primary interambulacral spine; pp, primary podia; ped, pedicellariae; tub, tubercle; ipm, interpyrimidal muscle; pr, protractor muscle; ret, retractor muscle. All scale bars are 100 µm, except for (d'), which is 50 µm.      showing localization of Msp130 protein using 6a9. Localization is strongest in medial and distal portions of interambulacral spines, in the rosettes, in elongating sphaeridia and in the margins of peristomial plates. ps primary spine; js, juvenile spine; ped, pedicellariae; sph, sphaeridia, pp, primary podia; sp, secondary podia; hp, hemipyramid. These images are the same as those shown in Figure 3a-b of the main text. Scale bar in all images is 200 µm.         Supplemental Table Descriptions  Table S1. Antibodies and dilutions used in this study. [2] Formery, L., Orange, F., Formery, A., Yaguchi, S., Lowe, C.J., Schubert, M. & Croce, J.C. Neural anatomy of echinoid early juveniles and comparison of nervous system organization in echinoderms. Journal of Comparative Neurology.