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Fig. 1. Results of genetic crosses: Cross (A; Cross #35 in Table 1) A homozygous white spined urchin crossed with a purple spined urchin produced all white spined F1 offspring. In the F2 generation, purple urchins returned in a 3:1 ratio. Cross (B; Cross #37 in Table 2) A homozygous white urchin crossed with a patterned green urchin produced all-white patterned offspring (similar in appearance to urchin a in F2 generation). In the F2 generation both patterned and nonpatterned white and colored offspring: (a) white, patterned; (b) white, nonpatterned; (c) purple-green, patterned; (d) purple-green, nonpatterned. Cross (C; Cross #15 in Table 3) Entirely purple cross produced purple F1 and F2 offspring. Cross (D; Cross #36 in Table 2) Purple urchin crossed with a patterned green urchin produce bicolored patterned F1 offspring. In the F2 generation both patterned and nonpatterned offspring: (a) purple, patterned; (b) purple-green, patterned; green, patterned (not seen); (c) purple, nonpatterned; (d) purple-green, nonpatterned.
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Fig. 2. (A) Post-metamorphic juvenile approx. 2 mm in HD. The red granules are red pigment cells that accumulate in areas of rapid growth, usually around the sutures, the apical plate and genital pores. There is a higher concentration of these cells around the sutures of the ambulacral plates; all primary spines have a purple band roughly midway down the spine. All post-metamorphic juveniles look very similar irrespective of adult color phenotype. (B) Later stage juvenile that will become a purple urchin. The purple along the spines accumulates and will eventually cover the entire spine and the test will become progressively darker. (C) Later stage juvenile that will become a white urchin. The purple band along the spine becomes fainter until it disappears altogether. The test may become light as it in this individual or may become darker. (D) Later stage bicolored juvenile that displays both spine and test patterning. The spatial differentiation of green and purple on the spines is clearly visible as is the difference in color between the ambulacral and interambulacral sectors.
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Fig. 3. Juvenile Lytechinus variegatus of wild-type (A,B) and gene knock out of polyketide synthase (PKS1; C,D). Regardless of the final colormorph phenotype in the adult, juvenile spines all initially have 1–2 purple pigment bands along the nascent skeleton (white arrows). Wildtype animals also have pigmented immune cells (red spherule cells) densely packed throughout the body (asterisk). Animals whose PKS1 genes have been inactivated by Cas9/sgRNA do not have detectable pigment in either of these cells (C,D). The slight green regions in the bodies of A and C is algae in the digestive system. The juveniles at this stage are less than 2 millimeters in diameter.
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Fig. 4. High Resolution micro Computed Tomography (microCT). A and B are low magnification images of LvWhite spines. All other images are transverse tomography sections of spines from S. purpuratus, LvWhite, LvGreen, and LvRed, taken approximately 25% from the base of the spine (top row) and 25% from the tip of the spine (bottom row). Spines from Lv are approximately 1 millimeter in diameter whereas in Sp they are ~ 1.5 millimeters, all depending on where in the spine the cross section is taken. .
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Fig. 5. Number of septa as a function of color and position along the oral/aboral axis of adults.
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Fig. 6. Cells of the spine revealed by scanning electron microscopy (SEM). (A) Low magnification view of two intact spines along their length showing the septa (mineralized ridges) and interseptal spaces (between the septa). (B) Higher magnification of a spine where left–right orientation is proximal-distal for the spine, respectively. Each of the septa have proximal-distal oriented ridges and are covered by the external epithelium. (C) The epithelium (*) covers the mineralized skeleton (#) with tightly adherent junctions (arrows) as well as projections through pores internally. (D) With most of the epithelium removed from the skeleton, except for the row identified by the asterisk (*), the cellular projections and extracellular matrix are apparent. Bar: A = 500 microns. B = 100 microns. C = 20 microns. D = 50 microns.
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Fig. 7. Top row, live adult spines labeled with the lectins Concanavalin A (Green) and Wheat germ agglutinin (Red). Note distinct boundaries in the epithelium expressing a rich source of terminal α-D-mannosyl and α-D-glucosyl groups, or to N-acetyl-D-glucosamine and sialic acid, respectively. Bottom row are spines from a 2-week old juvenile, similarly labeled and showing that the distinct domains seen in adults are already formed in a young juvenile. The WGA labeling appears predominantly pericellular at this stage.
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Fig. 8. Genes whose expression is enriched in the immune pigmented cells of the larvae (red spherule cells) are expressed in the epithelium of the spine as seen by fluorescence in situ RNA hybridization. PKS = polyketide synthase; Sm50 = spicule matrix protein of 50 kDa (is not in pigmented cells of the larva but is an important control for hybridization and profiling); note that Sm50 mRNA is enriched at the tip of the spine; Mif5 = macrophage migration inhibitory factor #5, which is involved in a variety of immune functions in mammals; FMO3 = Flavin-containing monooxygenase #3; gLNC = Long non-coding RNA enriched in green spines; ABCg11 = ATP-binding cassette protein - #11 in the g family of ABC proteins. Each of the genes are expressed broadly in the spine epithelial cells with enrichment transitions seen in FMO3 and SM50. The most distinct labeling profile is in gLNC, which appears perinuclearly in select epithelial cells. Bars in each image = 50 micrometers
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Fig. 9. (A,B) PKS immunolabeling at base of spine and (C,D) at spine tip (distal). Note how the tip is highly enriched for the PKS protein, relative to the base. Bar = 100 microns
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Fig. 10. (A,B) FMO3 immunolabeling at base of spine and (C,D) at spine tip (distal). Notice the dramatic enrichment in the tip. Scale bar = 10 microns.
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Fig. 11. Quantitation of larval pigment gene expression in adult spines of red/white/green colormorphs of Lytechinus variegatus using qPCR (A). Values were normalized to ubiquitin and then to the values of the red colormorph. Significance measurements are shown in the graph (B).
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Fig. 12. Spine cells are replicative. (EdU incorporated nuclei). Pulsing spines for 0.5–3 h cultured in vitro resulted in significant EdU labeling. The epithelium looks remarkably tightly bound so how these divisions occur must include dynamic restructuring of the mitotic cells. Scale bar = 8 microns
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Fig. 13. (A) Immune cells were counted in fifty-micron swaths of demineralized spines at the base, in the middle, and at the tip, and the percent of these counted cells in each region was calculated. (B) Velocity of individual presumptive red-spherule cells in intact spines from 5 spines of different Lytechinus variegatus individuals. Each dot on the graph represents an individual cell’s average velocity over the course of the time-lapse video. Significant p values between spines by One-way ANOVA are indicated with stars (*= 0.0282, **= 0.0023, ****= < 0.0001).
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