Wessel GM et al. (2020), Genetic manipulation of the pigment pathway in ...

ECB-ART-48597

Sci Rep 2020 Feb 06;101:1973. doi: 10.1038/s41598-020-58584-5.

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Genetic manipulation of the pigment pathway in a sea urchin reveals distinct lineage commitment prior to metamorphosis in the bilateral to radial body plan transition.

Wessel GM , Kiyomoto M , Shen TL , Yajima M .

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Echinoderms display a vast array of pigmentation and patterning in larval and adult life stages. This coloration is thought to be important for immune defense and camouflage. However, neither the cellular nor molecular mechanism that regulates this complex coloration in the adult is known. Here we knocked out three different genes thought to be involved in the pigmentation pathway(s) of larvae and grew the embryos to adulthood. The genes tested were polyketide synthase (PKS), Flavin-dependent monooxygenase family 3 (FMO3) and glial cells missing (GCM). We found that disabling of the PKS gene at fertilization resulted in albinism throughout all life stages and throughout all cells and tissues of this animal, including the immune cells of the coelomocytes. We also learned that FMO3 is an essential modifier of the polyketide. FMO3 activity is essential for larval pigmentation, but in juveniles and adults, loss of FMO3 activity resulted in the animal becoming pastel purple. Linking the LC-MS analysis of this modified pigment to a naturally purple animal suggested a conserved echinochrome profile yielding a pastel purple. We interpret this result as FMO3 modifies the parent polyketide to contribute to the normal brown/green color of the animal, and that in its absence, other biochemical modifications are revealed, perhaps by other members of the large FMO family in this animal. The FMO modularity revealed here may be important in the evolutionary changes between species and for different immune challenges. We also learned that glial cells missing (GCM), a key transcription factor of the endomesoderm gene regulatory network of embryos in the sea urchin, is required for pigmentation throughout the life stages of this sea urchin, but surprisingly, is not essential for larval development, metamorphosis, or maintenance of adulthood. Mosaic knockout of either PKS or GCM revealed spatial lineage commitment in the transition from bilaterality of the larva to a pentaradial body plan of the adult. The cellular lineages identified by pigment presence or absence (wild-type or knock-out lineages, respectively) followed a strict oral/aboral profile. No circumferential segments were seen and instead we observed 10-fold symmetry in the segments of pigment expression. This suggests that the adult lineage commitments in the five outgrowths of the hydropore in the larva are early, complete, fixed, and each bilaterally symmetric. Overall, these results suggest that pigmentation of this animal is genetically determined and dependent on a population of pigment stem cells that are set-aside in a sub-region of each outgrowth of the pentaradial adult rudiment prior to metamorphosis. This study reveals the complex chemistry of pigment applicable to many organisms, and further, provides an insight into the key transitions from bilateral to pentaradial body plans unique to echinoderms.

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Genes referenced: fmo1 gcml LOC100887844 LOC115925415 LOC583082 LOC586240
gRNAs referenced: LOC586240 gRNA2 LOC586240 gRNA3 LOC586240 gRNA4 LOC588806 gRNA23 LOC588806 gRNA24 LOC588806 gRNA25 LOC588806 gRNA26 LOC588806 gRNA27 gcml gRNA4 gcml gRNA5 gcml gRNA6 gcml gRNA7

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	Figure 1. Images of larvae (Hemicentrotus pulcherrimus) are shown at 10 days post fertilization. Larvae were fed algae and can be seen with full stomachs. Wild-type control animals also have deep red pigment cells throughout the body of the larva. Animals in which guide RNAs were injected for PKS, FMO3, or GCM develop, swim, and feed normally, but they have no pigmentation. Scale bar = 100 microns.
	Figure 2. Aboral and oral views of animals used in this study (Hemicentrotus pulcherrimus) are shown. Animals lacking functional PKS gene are lacking pigment throughout the test, spines, and tube feet. The same phenotype is seen for animals lacking GCM. Animals lacking FMO3 instead reveal pastel purple throughout their spines, but all other features of the adult are the same as in the wild-type control. Scale bar = 1 cm.
	Figure 3. Genome analysis of mutations.
	Figure 4. Close up of spines from wild-type, PKS- and FMO3-null animals. Wild-type spines have two sources of pigment, that in the individual cells throughout the surface (stroma) of the spine, and that throughout the complex latticework (stereom) of the spine. Note the variation in spine color in the wild-type spines (from one individual) and the cross-sectional patches of more or less pigment in the core of the spine. PKS-null spines have neither pigment in individual cells at the surface, nor in the core of the spine. FMO3 spines on the other hand have a pastel purple throughout the core of the spine and an occasional single cell of pigment at the surface. The overall morphology of the spines in each case is otherwise indistinguishable from each other, and GCM has the same phenotype as does PKS (data not shown). Scale bar = 50 microns.
	Figure 5. Tube feet are shown under polarized and DIC microscopy. Tube feet from control animals show the detailed patterning of the skeletal elements characteristic of this tissue as well as the intense red pigment of the pigment cells throughout the tip and base of each tube foot. The tube feet from animals lacking PKS also contain intricate skeletal elements in their tube feet, but have no detectable pigment in the tip, base, or elsewhere in the tube feet. This phenotype is indistinguishable from the GCM-null animals (data not shown). Tube feet of the FMO3-null animals appear indistinguishable from the wild-type tube feet. They have detailed skeletal elements, and pigment cells, seen in the tips of the tube feet by polarized light, and in the base of the tube feet seen by DIC. Scale bar = 50 microns.
	Figure 6. Pigment chemistry. LC-MS analysis of pigment extracted from spines of control and FMO3-null animals reveal that the ratios of each pigment expressed in spines changes dramatically when FMO3 is absent. The pigment changes and resulting purple color are consistent with the purple variant in a recent study of diversity of pigments in a different sea urchins, E. mateai.
	Figure 7. Coelomocytes were biopsied from adults and imaged by DIC. Wild-type animals have diverse cells types, include the intensely red-pigmented red spherule cells. This diverse population of coelomocytes and pigment cells is also present in the FMO3-null animals. PKS-null animals also have diverse coelomocytes but no pigment is present. Scale bar = 50 microns.
	Figure 8. Mosaic pigmentation was seen in PKS- and GCM-null animals. Shown are aboral, side, and oral views of representatives of the mosaics. Note that in segments lacking pigmentation, all external structures are lacking pigment, including the test, spines, and tube feet and that these regions extend longitudinally, from oral to aboral.
	Figure 9. Model of pigmentation mosaics in the transition from bilaterality to a radial body plan. A bilaterally symmetric larva adjacent to a test of H. pulcherrimus with spines and tube feet removed that reveals a major 5-fold symmetry characteristic of this phyla and a minor division of these of the five sections to yield 10 repeating segments. Each of the pentameric regions contain a bilateral region that may have different pigmentation. Pigmentation in each of the mosaics follows a 10-fold symmetry (5 times bilateral symmetry, enabling for example, half-pigmented animals, or a small slice (1/10) of albinism, or both, as present in this animal.

References [+] :

Brasseur, Identification and quantification of spinochromes in body compartments of Echinometra mathaei's coloured types. 2018, Pubmed, Echinobase