: 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 corresponding to (a). (c) Magenta channel showing staining for Sm50. Stronger staining of Sm50 is identified at the base of the spines and rosette, where deposition of skeletal tissue is active. In contrast, the larval skeleton is not strongly labeled. (d) Green channel showing staining for Msp130 using monoclonal antibody 6a9. Note the strong staining in the larval skeleton, and the staining of the skeletogenic cells. sc, skeletogenic cells; ls, larval skeleton; pp, primary podium; ros, rosette. Scale bar is 50 μm. Fig. S2. Post-metamorphic P. lividus juveniles. Light microscope images showing post-metamorphic juvenile P. lividus. (a) Oral view of stage J4 juvenile. Sphaeridium is well developed and oral surface is dominated by tube feet. Pigmented cells are abundant in the distal disk of the tube feet. (b) Lateral view of J5 animal. Animal is oblong in lateral view, and the aboral surface bears far fewer structures than the oral surface. (c) Oral view of same animal as (b). Note well-developed, circular peristomial margin. Pedicellariae are present on interambulacral plates, and well-developed, glossy sphaeridia are present on ambulacral plates in addition to ambulacral spines. (d) Aboral surface as same animal in (b)-(c). The aboral surface bears few structures, except for the juvenile spines present on genital and ocular plates. Darkened crescent is the gut, visible inside of the animal. sp, secondary podia; sph, sphaeridia; ps, primary interambulacral spine; js, juvenile spine; ped, pedicellariae; as, ambulacral spine. Fig. S3. Skeleton and Musculature of P. lividus juveniles. Light sheet microscope images showing staining in J1 individual for Myosin Heavy Chain (MHC) using an anti-MHC antibody and Msp130 using monoclonal antibody 6a9. (a) Lateral view showing staining for MHC (Magenta), Msp130 (Green), and nuclei using DAPI (Blue). Msp130 strongly stains the interambulacral primary spines and the juvenile spines, while MHC is localized in the muscular tissue of the primary podia, in longitudinal bands surrounding the tubercles, in bundles within the spines, and in the bases of the pedicellariae valves. (a’) Magenta channel from (a) showing only MHC. (a’’) Magenta and blue channels showing MHC and DAPI. Staining for MHC is strongest in the bases of the pedicellariae valves. (b) Close-up of primary interambulacral spine showing bundles of MHC + cells within the lumen of skeletal spines. (b’) Magenta channel from (b), showing bundles of MHC + cells. This image is the same as Fig. 2b in the main text. (b’’) Bundles of MHC + cells relative to DAPI staining, indicating that each MHC + circle stains multiple cells as opposed to a single cell. (c) Enlargement of pedicellarial staining. The most intense staining for MHC in the animal is the base of the pedicellariae pincers (valves). (d) Close-up from (a) with only magenta and green channels showing longitudinal bands of MHC + cells surrounding the tubercles of primary interambulacral spines and muscles along the primary podia. ps, primary interambulacral spine; js, juvenile spine; pp, primary podia; ped, pedicellariae. Scale bars in (a)-(a’’) are 100 μm, rest are 25 μm. Fig. S4. Musculature and morphology of P. lividus juveniles. Confocal microscope images showing the distribution of MHC + tissues relative to tissues immunoreactive to an anti-β-tubulin antibody and cell nuclei, marked with DAPI. (a) Image showing a lateral view of MHC immunoreactive cells relative to tissues marked with anti-β-tubulin and DAPI in a J1 individual. MHC + tissues are present throughout the animal, with strong immunoreactivity in the pedicellariae, in longitudinally elongate-cells in the tubercles, and in longitudinal bands alongside the interior of the podia. This image is the same as Fig. 2a in the main text. (b) Oral view showing location of MHC + and β-tubulin + tissues in a J2 P. lividus. As in the lateral view, MHC immunoreactive cells are clearly seen around the tubercles and alongside the interior of the podia. In the oral view, MHC + cells are also seen in latitudinal bands around the opening of the mouth in the peristomial membrane. Additionally, muscles involved in the protraction and retraction of the Aristotle’s lantern are visible. (c) Same image as (a), showing only the channel with MHC. Note, a few MHC + cells in the spines. (d) Single-channel image showing MHC channel from (b). Protractor and retractor muscles are visible within the animal, as well as interpyrimidal muscles of the Aristotle’s lantern. (d’) Close-up showing single MHC + cells in the primary spines. Compare with Figure (S3b). (e) Lateral view showing the R-tubulin channel from (a). β-tubulin marks cilia on the outside of the animal, as well as tissues of the nervous system throughout the animal, including around the tubercles. (f) Oral view showing R-tubulin + cells. R- tubulin channel from (b). 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. Fig. S5. Localization of neuronal markers in early juveniles. Confocal microscope images showing staining in J1 individual for synaptotagmin using 1E11 and serotonin using an antiserotonin antibody. (a) Oral view of J1 individual showing immunoreactivity for synaptotagmin (green), and serotonin (magenta). Nuclei are stained with DAPI (blue). Synaptotagmin is localized within the interior of the primary podia, and in bands surrounding the tubercles and bases of the spines. Synaptotagmin and serotonin co-localize in single cells arranged proximodistally along the lumen of the primary interambulacral spines. This is the same as Fig. 2d in the main text. (b) Close-up of (a). (c) Aboral view of same individual as (a). (d) Green channel from (a) showing synaptotagmin. (e) Green channel from (b) showing synaptotagmin. Note the synaptotagmin + cells lining the lumen of the primary interambulacral spines. (f) Green channel from c showing synaptotagmin. (g) Magenta channel from a showing serotonin. (h) Magenta channel from (b) showing serotonin. Note the synaptotagmin + cells lining the lumen of the primary interambulacral spines, which we interpret to be serotonergic neurons. (i) Magenta channel from (c) showing Serotonin. (j) Green and blue channels from (a) showing synaptotagmin and DAPI. (k) Green and blue channels from (b) showing synaptotagmin and DAPI. (l) Green and blue channels from (c) showing synaptotagmin and DAPI. All scale bars in (c), (f), (i), (l) are 50 μm, rest are 100 m. Fig. S6. Neuronal and skeletal markers in early juveniles. Confocal images showing localization of ELAV and Msp130 in J2 individual. (a) Staining for ELAV using an anti-ELAV antibody (Magenta), Msp130 using monoclonal antibody 6a9 (Green), and DAPI (blue). Msp130 is strongly localized in the primary and juvenile spines, and the rosettes while ELAV is localized in cells in the primary and secondary podia. (b) Close-up of same individual as (a) showing ELAV + cells, interpreted to be neurons, in the secondary podia. In the interambulacral primary spines, Msp130 is localized in the cell bodies of cells that we interpret to be skeletogenic cells. This image is the same as Fig. 2c in the main text. (c) Magenta and green channels of (a), showing staining of ELAV and Msp130. (d) Magenta and green channels of (b). Importantly Msp130 and ELAV are differentially localized in the secondary podia, with ELAV marking neurons, and Msp130 staining the growing rosette. (e) Magenta and blue channels of (a), showing ELAV and DAPI. (f) Magenta and blue channels of (b), showing ELAV and DAPI. Cell nuclei are marked by DAPI, but ELAV stains the surrounding cell body. (g) Green and blue channels of a, showing MSP130 and DAPI. (h) Green and blue channels of (b), showing Msp130 and DAPI. As with ELAV, Msp130 stains the cell body, while nuclei of the same cells are marked with DAPI. n, neuron; ros, rosette; sc, skeletogenic cell. Scale bars in (a), (c), (e), (g) are 200 μm, scale bars in (b), (d), (f), (h) are 100 μm. Fig. S7. Specific and pan-neuronal markers. Confocal images showing localization of ELAV and Synaptotagmin in J1 individual. (a) Staining for synaptotagmin (green). Notice rings of nerves surrounding tubercles and in bundles of neurons in the primary spines. (b) Stainingfor ELAV. Notice bundles of neurons in primary spines. (c) DAPI staining showing distribution of cell nuclei. (d) Merge showing co-localization of Synaptotagmin and ELAV. Of importance is the fact that co-localization is most strong in the bundles of neurons at the base of the primary spines. Scale bars in (a)-(c) are 200 μm, (d) is 50 μm. Fig. S8 Localization of acetylated tubulin. Confocal images showing staining for acetylated tubulin and DAPI in J1 animals. (a) Merge showing staining for acetylated tubulin (green) using an anti-acetylated tubulin antibody and for cell nuclei using DAPI (blue) on the oral surface. Cilia cover the entire surface of the animal and the lateral edges of the spines, as shown in Gosselin and Jangoux [1]. Acetylated tubulin is also found in the interior of the primary and secondary podia, which we interpret to be tissue of the nervous system in agreement with a recent study [2]. (b) Staining for acetylated tubulin and cell nuclei on the aboral surface of a different J1 individual. Cilia are found across the surface of the animal, and show a high density on pedicellariae. The individual from (a) is shown in the bottom of the image. (c) Green channel from a showing only staining for acetylated tubulin. Note the cilia lining the margin of the discs of the primary podia. (d) Green channel from (b) showing staining of acetylated tubulin. pp, primary podia; sp, secondary podia; js, juvenile spine; ps, primary interambulacral spine; ped, pedicellariae. Scale bar in all images is 200 μm. Fig. S9 Localization and co-localization of skeletal proteins in young juveniles. Localization of the skeletogenic proteins Msp130 (green) and Sm50 (purple) in a J1 P. lividus juvenile. (a)-(c)- show localization of Msp130 and Sm50 relative to DAPI (blue), which stains for nuclei. (d)-(f) show both distinct localization, and co-localization of Msp130 and Sm50 in skeletal tissues. (g)-(i) show localization of Sm50, staining is strongest around the margins of plates, in median portions of spines, and in milled-rings of spines. (j)-(l) Staining of Msp130 in skeletal tissues. Staining is strongest in the tips of spines. pp, primary podia; sp, secondary podia, ps, primary interambulacral spine; ped, pedicellariae. These images are the same as those shown in Fig. 2il in the main text. Scale bars 200 μm. Fig. S10. Calcein localization relative to skeletal proteins. Confocal microscopy showing staining of Sm50 using an anti-Sm50 antibody (magenta), and Msp130 using monoclonal antibody 6a9 (green), as well as the incorporation of fluorescent calcein (yellow) into the growing J3 sea urchin test. (a) Aboral surface showing incorporation of calcein into the margins of accreting genital, anal, ocular and interambulacral plates. Msp130 and Sm50 are also shown, and reveal distinct patterns of localization and co-localization. (b) Oral surface of same individual shown in (a). Calcein is shown incorporated into margins of accreting ambulacral and interambulacral plates, elongating sphaeridia, growing hemipyramids, and rosettes. There is co-localization of Sm50 and Msp130 in sites of calcein incorporation, implicating Sm50 and Msp130 in active skeletogenesis. (c) Same as (a), with calcein removed. Co-localization of Msp130 and Sm50 is indicated by greyish-white color. This is evident at the margins of genital and interambulacral plates, as well as in the tubercles. Compared with (a) and (e), it is evident that strong co-localization of these skeletogenic genes occurs in sites of accretion as identified using calcein. (d) Same as (b), with calcein removed. Co-localization of Msp130 and Sm50 is evident in the margins of accreting peristomial, ambulacral, and interambulacral plates, as well as in the elongating sphaeridia and rosettes of secondary podia. Compared with (a), and (f), this co-localization is evident in sites of calcein incorporation. (e) Yellow channel from (a), showing sites of calcein incorporation in genital, interambulacral and anal plate margins. Compare with co-localization of skeletogenic proteins in (c). (f) Yellow channel from (b), showing sites of calcein incorporation in margins of accreting peristomial, ambulacral, and interambulacral plates, and growing hemipyramids, rosettes and sphaeridia. Compare with sites of skeletogenic protein localization in (d), (h), and (j). (g) Purple channel from (a), showing localization of Sm50 protein. Immunoreactivity is stronger in the bases of spines and tubercles, and in the margins of genital, anal and interambulacral plates. (h) Purple channel from (b), showing localization of SM50. Immunoreactivity is stronger in margins of peristomial plates, elongating sphaeridia, and rosettes of secondary podia. (i) Green channel from a showing localization of Msp130 proteins using 6a9. Immunoreactivity is stronger in the medial and distal portions of interambulacral spines, and in the distal portions of juvenile spines and pedicellariae. (j) Green channel from (b) 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 Fig. 3a-b of the main text. Scale bar in all images is 200 μm. Fig. S11. Skeletal growth over time. Localization of Msp130 (green), Sm50 (purple), and incorporation of calcein (yellow) into growing skeleton in J3 individuals at 0 (a-a’’’), 24 (b-b’’’), and 48 (c–c’’’) hours chase. Description of staining given in the main text. a’, b’, c’ and a’’’, b’’’, and c’’’ are zoomed in images of the above showing incorporation of calcein. Orange bars indicate gap between calcein-marked plated due to subsequent accretion. ps primary spine; js, juvenile spine; op, ocular plate, gp, genital plate; ap, anal plate; sph, sphaeridia. Images are the same as individuals in Figs. 3a,b,d,e in the main text. Scale bars in a-c and a’’-c’’ 200 μm, a’, b’’’, c’, c’’ 25 μm, b’ and a’’’ 50 μm. Fig. S12. Calcein and Sm50. Confocal microscopy showing staining for Sm50 using an anti-Sm50 antibody (magenta), and the incorporation of fluorescent calcein (yellow) into the J3 sea urchin test at 0, 24, and 48 h chase. (a) Aboral surface from 0 h chase animal showing incorporation of calcein) relative to localization of Sm50. Image is the same as Fig. 3a in the main text, with green channel (MSP130) removed. Sm50 is localized throughout all visible skeletal structures, including localization in areas of calcein incorporation such as the margins of plates, shown in whitish-grey. (a’) Yellow channel from (a), showing sites of calcein incorporation in aboral surface of 0 h chase animal. (a’’) Oral surface from 0 h chase animal, showing incorporation of calcein into skeleton relative to localization of Sm50. Sm50 stains the entire skeleton except for the teeth, and is localized in sites of calcein incorporation in the sphaeridia, rosettes, and margins of peristomial, ambulacral, and interambulacral plates. Image is the same as Fig. 3b in the main text, with green channel (Msp130) removed. (a’’’) Yellow channel from (a’’) showing incorporation into oral surface of 0 h chase animal. (b) Aboral surface of 24 h chase animal showing incorporation of calcein relative to staining for Sm50. Additional skeletogenesis at the margin of plates has taken place in the 24 h since calcein incubation, as evident in the gaps between calcein-incorporated skeleton in adjacent plates. Sm50 is still strongly localized in some areas of calcein incorporation, such as the tubercles and margins of plates. Image is the same as Fig. 3d in the main text, with green channel (Msp130) removed. (b’) Yellow channel from (b), showing incorporation of calcein into aboral surface of 24 h chase animal. (b’’) Incorporation of calcein and localization of Sm50 protein in oral surface of 24 h chase animal. Sm50 is localized where calcein was incorporated into the skeleton in the ambulacral, peristomial and interambulacral plates, but strong Sm50 localization is also evident in skeleton that has been deposited subsequent to incubation with calcein. This is most evident in the margins of the peristomial plates, in the elongating sphaeridia, and in the rosettes of secondary podia. (b’’’) Yellow channel showing incorporation of calcein in the skeleton of the oral surface of a 24 h chase animal. (c) Incorporation of calcein and localization of Sm50 in the skeleton of the aboral surface of a 48 h chase animal. Gaps between sites of calcein incorporation in adjacent plates (of maximum size of 7.5 μm) are clearly visible on the aboral surface. Additionally, strong Sm50 localization in these gaps, and in other structures such as the tubercles, bases of primary spines, and margins of plates are indicative of further biomineralization involving Sm50. (c’) Yellow channel from c showing incorporation of calcein into aboral skeleton of 48 h chase animal. Image is the same as zoom of Fig. 3e in the main text. (c’’) Oral surface of 48 h chase animal showing incorporation of calcein and localization of Sm50. Substantial growth has taken place in the 48 h since the incorporation of calcein, which is evident in the strong localization of Sm50 in the sphaeridia, in themargins of the peristomial plates, and in the rosettes of secondary podia. Additionally, gaps between areas of calcein incorporation are present in ambulacral and interambulacral plates. (c’’’) Yellow channel of (c’’) showing incorporation of calcein into oral surface of 48 h chase animal. iamb, interambulacral plate; op, ocular plate; sph, sphaeridia; pp, primary podia; sp, secondary podia. Scale bar in all images is 200 μm. Fig. S13. Skeletal growth in older (J6) juvenile. Localization of SM50 (purple), MSP130 (green) and incorporation of calcein (yellow) into J6 animal at 0 h chase. (a) shows staining of MSP130, SM50, and incorporation of calcein into oral surface. This is the same image as Fig. 3c in the main text. (b) is yellow channel from (a) showing calcein in the Aristotle’s lantern, rosettes, and spines. (c) is the aboral surface of the same J6 animal from (a). (d) shows the yellow channel with calcein from (c). (e–f) are close-ups of the aboral surface from (c) showing incorporation of calcein into growing spines and tubercles relative to MSP130 and SM50 (e) and only SM50 (f). (e) is the same as Fig. 3f from the main text. ps primary spine; sp, secondary podia; sph, sphaeridia; ped, pedicellariae; aps, ambulacral primary spine. Scale bars in a-d 200 μm and e–f 50 μm. Fig S14. Images showing different stages of the strategy used to quantify EdU + nuclei relative to all nuclei. (a) and (b) Show raw images for EdU + (a) and DAPI + cells (b). (c-d) Binary images for quantification. (e–f) Five regions of interest of binary cells for quantification. See expanded details in Supplemental Methods. Image is the same as Fig. 4b from the main text. Fig. S15. Cell proliferation pulse-chase in early juvenile stages. Confocal microscope images showing the incorporation of EdU into the oral surface of growing J2 sea urchins, as well as staining for Sm30 using an anti-SM30 antibody. (a) Staining for EdU (magenta), Sm30 (gray), and cell nuclei using DAPI (blue) in the oral surface of a 0-h chase individual. Most proliferation takes place in the secondary podia and growing peristomial podia. Additionally, immediately adoral to the primary podia, there is a zone of cell proliferation corresponding to the zone where new plates are added. Of interest, there is no proliferation in the primary podia themselves, which form in the rudiment and atrophy within a few weeks following metamorphosis [1]. (a-a- ‘’) are the same image as Fig. 4b from the main text. (a’) ray channel from a showing localization of Sm30. Protein is localized in all skeletal tissue except for the teeth and is most strongly localized in the primary interambulacral spines. (a’’) Localization of Sm30 relative to proliferating cells marked by EdU. There is very little proliferation in the primary spines and in the peristomial test plates. (b) Staining for EdU, Sm30, and cell nuclei using DAPI in a 24 h chase individual. As in the 0 h chase individuals, most proliferating cells are located in the secondary and peristomial podia, and the proliferative zone adoral to the primary podia. (b’) Gray channel from (b), showing localization of Sm30. (b’’) Localization of Sm30 relative to proliferative cells marked by EdU. (c) Staining for EdU, Sm30, and cell nuclei using DAPI in a 48 h chase individual. Localization of EdU + cells does not differ substantially from 0 and 24 h chase individuals. Presence of cell doublets indicates that rates of cell division are relatively slow, and that not much cell division has taken place in the 48 h since incubation with EdU. (c’) Gray channel showing localization of Sm30. (c’’) Location of EdU + cells relative to skeletal tissues identified using Sm30. ps, primary interambulacral spine; pp, primary podia; sp, secondary podia. Scale bar in all images is 200 μm. Fig. S16. Cell proliferation in J5 Juvenile. Incorporation of EdU into a 0 h chase J5 individual after 63 h of incubation with EdU, and staining for S530 using an anti-SM50 antibody. (a) Aboral surface of 0 h chase animal showing localization of Sm50 relative to proliferating cells marked by EdU (magenta-white). There is relatively little cell proliferation present in extraxial tissues on the aboral surface such as the genital and periproctal (anal) plates. Most cell proliferation seen from this view is associated with novel structures, such as newly added secondary podia and ambulacral spines. (b) Proliferating cells marked by EdU and localization of Sm50. Proliferation is extensive on the oral surface in axial tissues, especially when compared to the extraxial tissues of the aboral surface. High degrees of cell proliferation are found in the disks of the secondary podia, as well as in the peristomial podia and sphaeridia. Most proliferating cells on spines are located more proximally towards the base of the spine. (c) Magenta channel from (a), showing location of proliferating cells on aboral surface. (d) Magenta channel from (b), showing location of proliferating cells on oral surface. (e) Close-up view of cell proliferation associated with growth of a newly added ambulacral primary spine. Most proliferating cells are localized towards the base of the spine and the tubercle. Strong staining of Sm50 is also located towards the base of the spine, though we interpret most distal growth takes place via skeletogenesis. (e) is the same as Fig. 4i in the main text. (f) Cell proliferation associated with a growing primary spine and secondary podia. As in (e), most proliferating cells are located nearer to the base of the spine. High degrees of cell proliferation are associated with the growth of the secondary podia, as is also seen in J2 individuals in Fig. 4b. (f) is the same as Fig. 4j in the main text. (g) On the oral surface, high degrees of cell proliferation are associated with the tube feet. In particular, the margins of the tube feet disk show high degrees of cell proliferation, potentially associated with sensory motor neurons. ps, primary interambulacral spine; aps, ambulacral primary spine; sp, secondary podia; ped, pedicellariae; m, mouth. Scale bars in (a)-(d) are 200 μm, scale bars in (e)-(g) are 100 μm. Fig. S17. Cell proliferation in a J6 Juvenile. Incorporation of EdU into a 48 h chase growing J6 sea urchin after 63 h of incubation with EdU. (a) EdU stained nuclei (magenta) relative to all cell nuclei marked with DAPI (gray) on the aboral surface. Note the low abundance of proliferating cells on extraxial tissue (genital and anal plates). Most cell proliferation in this view is associated with the addition of new structures, such as ambulacral and interambulacral primary spines. (b) Cell proliferation marked using EdU on the oral surface of a J6 animal. High degrees of cell proliferation are associated with newly added pedicellariae. Proliferating cells are also found in the circular margins of the disks of the secondary podia. (c) Magenta channel of (a), showing location of cells marked with EdU. (d) Magenta channel of (b), showing EdU marked cells on oral surface. (e) Close-up of newly added ambulacral and interambulacral spines, secondary podia, and associated cell proliferation. (f) Close-up of proliferating cells in newly added secondary podia and spines. (g) Close-up of ambulacral pedicellariae and associated cell proliferation. ps, primary interambulacral spine; aps, ambulacral primary spine; sp, secondary podia; ped, pedicellariae; m, mouth. Scale bars in (a)-(d) are 200 μm, scale bars in (e)-(g) are 100 μm.