The origin of pigment cells in embryos of the sea urchin Strongylocentrotus purpuratus

DEVELOPMENTAL

BIOLOGY

107, 414-419

(19%)

The Origin of Pigment Cells in Embryos of the Sea Urchin Strongylocentrotus purpuratus ALLAN W. GIBSON AND ROBERT D. BURKE Department

of Biology, Received

University July

24,

of Victoria, 1984;

accepted

Victoria,

British

in revised

form

Columbia October

5% W ZYZ, Canada 2, 1984

A monoclonal antibody (SP1/20.3.1) that recognizes a cell surface epitope expressed by pigment cells in the pluteus larva of Strongylocentrotus purpuratus has been produced. Using indirect immunofluorescence, the epitope is first detected in nonpigmented cells of the vegetal plate after primary mesenchyme ingression. Between the beginning of gastrulation, and when the archenteron is one-third the distance across the blastocoel, SP1/20.3.1-positive cells are free within the blastocoel, at the tip of the archenteron, and dispersed within the blastoderm. Cells at the tip of the archenteron, and mesenchyme near the tip in later stages of gastrulation (secondary mesenchyme), do not express the SP1/20.3.1 antigen. By the completion of gastrulation all SP1/20.3.1-positive cells are dispersed throughout the epidermis. It has been concluded that in S. pwpuratus pigment cell precursors are released from the vegetal plate during the initial phase of gastrulation. The cells migrate first to the vegetal ectoderm, and subsequently disperse throughout the ectoderm and develop pigment granules. (cl 1985 Academic Press, Inc. INTRODUCTION

Mesenchymal cells in sea urchin embryos are categorized into two groups. Primary mesenchyme are derived from micromeres, and are the first cells to ingress into the blastocoel where they secrete the larval skeleton (Boveri, 1901; Okazaki, 1960,1965,1975a; Gustafson and Wolpert, 1967; Horstadius, 1973; Stearns, 1975). Secondary mesenchyme are derived from macromere descendants (Horstadius, 1973), and are released from the tip of the archenteron during the latter half of gastrulation (Gustafson and Kinnander, 1956, 1960; Dan and Okazaki, 1956; Gustafson, 1963, 1964). Before release the secondary mesenchyme extend filopodia to the ectoderm that pull or guide the archenteron across the blastocoel (Dan and Okazaki, 1956). After release the secondary mesenchyme forms a heterogeneous set of cells that are thought to differentiate into a variety of mesodermal tissues in the pluteus larva, including echinochrome-containing pigment cells, muscle, and coeloms. Here we describe a subset of mesenchyme that is released from the vegetal plate at the beginning of gastrulation and form the pigment cells of the pluteus. MATERIALS

Larval

AND

METHODS

Culture

Adult Strong~locentrotus purpuratus were collected from Sooke Harbor, British Columbia, Canada. Gametes were obtained by intracoelomic injection of 0.55 M KCl, and plutei were raised from fertilized eggs in 2-liter glass beakers at 9-11°C. Cultures were supplied 0012-1606/85 $3.00 Copyright All rights

0 1985 by Academic Press. Inc. of reproduction in any form reserved.

with a slow stream of air bubbles for agitation (Burke, 1983), and were fed daily with Lhnalliela salina (lo4 cells/ml). Production

of Mmmclmal

Antibodies

A crude antigen mixture was prepared from a 4% paraformaldehyde fixed, saline insoluble fraction of Dounce homogenized, 5-day-old plutei. After three washes in Dulbecco’s phosphate-buffered saline (PBS) the crude antigen was emulsified in a 1:l mixture of PBS and Freund’s complete adjuvant, and injected intraperitoneally into female BALB-c mice (50 mg wet wt./O.4 ml injection). Two booster innoculations were given at biweekly intervals (as above but with Freund’s incomplete adjuvant), and 4 days prior to fusion mice were given an intravenous injection of 25 mg unfixed, homogenized plutei in 0.2 ml PBS. Spleen cells were fused with SP-2/O myeloma cells following a procedure described by Galfre and Milstein (1981). The fusion culture was fractioned into 40 cultures, and grown in hypoxanthine/aminopterin/thymidine (HAT) medium to eliminate nonhybridoma cell lines. After 10 days tissue culture supernatants were assayed for anti-sea urchin antibodies (see below). Of 23 positive cultures 2 were cloned by limiting dilution, and selected hybridoma cell lines were recloned into 96-well plates. Monoclonal antibodies (McA) from one clone (SPV20.3.1) were produced from ascites tumors in BALB-c mice. To facilitate screening McA from large numbers of hybridoma cell lines small wells were made on poly-L414

GIBSON

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lysine coated glass slides by adhering 20 X 50 mm vinyl tape strips that had been punctured with a paperpunch (Jonak, 1980). A drop of filtered seawater (FSW) containing 200 to 400, 4% Formalin/FSW fixed, 5-day-old plutei was added to each well, and the larvae were allowed to settle. After rinsing off loose larvae and removing excess water by aspiration, immunoassays were carried out by adding 20 ~1 hybridoma supernatant to a well and incubating in a wet chamber for 1 hr. Slides were washed three times in PBS and incubated for 30 min in 20 ~1 fluorescein conjugated, anti-mouse IgG (Sigma, 1:25 dilution in PBS). Slides were rinsed three times for 5 min each in PBS, mounted in 50% glycerol/PBS, and viewed with a Zeiss Universal epifluorescence microscope with excitation at 450 to 490 nm, a 510 nm beam splitter, and a barrier filter at 520 nm. All immunoassay procedures were carried out at room temperature (RT). McA were named according to the nomenclature suggested by Pearson et al. (1977). Immun0cytochemistq.i Developmental stages from unfertilized eggs to 29day-old larvae were assayed for the SP1/20.3.1 epitope. Embryos or larvae were pelleted from FSW by centrifugation at 2009 for 3 min and fixed in 4% Formalin FSW for 4 hr at RT. To assay prehatched embryos eggs were fertilized in 10 n-& dithiothreitol/FSW (Epel et al, 1970) to inhibit formation of the fertilization envelope, which prevented antibody penetration. Assays were carried out in disposable glass tubes with washing by settlement and decantation. PBS was found to be a good immunoreaction buffer medium, although larval tissues tended to deteriorate, particularly in early embryos. Pigments were usually extracted. PBS made isotonic with seawater by adding glycine or glucose prevented extraction of pigments and allowed identification of any pigment cells that were not SP1/20.3.1positive, as well as unpigmented SP1/20.3.1-positive cells. Controls for nonspecific binding included primary incubation in nonrelevant hybridoma supernatant, myeloma supernatant, or buffer. To determine if the SP1/20.3.1 epitope was part of a cell surface molecule live tissues were assayed with FSW as the immunoreaction buffer. As well, all fixed developmental stages were immunostained with 0.1% Triton X-100 included in the McA solution and the staining pattern was compared to assays without detergent. Cell Counts To distinguish and count SP1/20.3.1-positive cells free within the blastocoel embryos at stages past oneeighth gastrula were dissociated into ectodermal and

Origin

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Cells

mesendodermal fractions by the procedure given by Harkey and Whiteley (1980). This treatment removes ectodermal cells and leaves a bag-like epidermal basal lamina enclosing the mesenchymal cells. Separated fractions were fixed and immunoassayed as above. RESULTS

Four hybridoma cell lines secreting tissue-specific McA were produced from the initial fusion of splenocytes and myeloma cells. McA from one cell line (SPl/ 20.3.1) were used in all experiments reported here. Tissue Specificity

Plutei all display a similar distribution of cells expressing the SP1/20.3.1 antigen. The cells are dispersed throughout the larval epidermis, as well as being concentrated at the arm tips and at the base of the body rods (apex) (Fig. 1A). This staining pattern corresponds to the distribution of pigment cells in live larvae. The dispersed cells are characteristically stellate, with three or four pseudopodia, and are similar in form to pigment cells in live larvae. In fixed larvae immunostained in PBS/glycine buffer, which does not extract pigment, the pigment cells are all SP1/20.3.1positive (Figs. 2B, C). A 1:l numerical correspondence between pigment cells and SP1/20.3.1-positive cells is apparent, except at the arm tips and apex where individual cells are difficult to distinguish. Control plutei incubated in nonrelevant hybridoma supernatant, myeloma supernatant, or PBS showed little nonspecific staining or autofluorescence. The pigment cells in live plutei, and in Formalin-fixed plutei in PBS/glycine buffer, were initially not fluorescent, but slowly (5 to 10 min) took on a dull fluorescence that is easily distinguished from fluorescein fluorescence. The SPli20.3.1 antigen appears to be a cell surface molecule. Although intact, live plutei will not stain with SP1120.3.1 McA, live, dissociated pigment cells will stain using FSW as the immunoreaction buffer. As well, the antigen is progressively removed from fixed larvae that are treated with increasing concentrations of Triton X-100 in the McA solution, suggesting that the molecule is associated with a lipid-soluble fraction of the cell. Various other species of sea urchins were assayed at the pluteus stage for cross-reactivity to SP1/20.3.1 McA. No cells in the plutei of the sand dollar Dendraster excentricus, or the red sea urchin S. franciscanus, cross-reacted with SPY20.3.1 McA. A population of pigment cells in plutei of the green sea urchin, S. drobachiensis, did cross-react with SP1/20.3.1, indicating similar antigenic determinants are present in both species. A second population of S. drobachiemk pigment

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DEVELOPMENTAL BIOLOGY

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FIG. 1. Distribution of SP1/20.3.1-positive cells in the pluteus larva of S. purpuratus. (A) The cells are stellate and dispersed among the larval epidermal cells. The stain intensity is greatest at the arm tips and apex (X175). (B, C) Fluorescence, and corresponding light micrograph of SPl/20.3.1-positive pigment cells (X200).

cells associated with developing cross-react with SPV20.3.1. Developmental

Appearance

pedicellariae

did not

of the SP1/20.3.1 Antigen

The SPV20.3.1 antigen is first detected by indirect immunofluorescence in vegetal plate cells of the 36-hr (11°C) mesenchyme blastula (Figs. 2A, B). At this stage the primary mesenchyme has ingressed, and the vegetal plate reformed as columnar cells. Unlike SPl/ 20.3.1-positive cells in older embryos, the membranes of stained cells at the mesenchyme blastula stage are indistinct, suggesting the antigen may be intracellular. Also indicating an intracellular antigen location is the increased stain intensity when 0.1% Triton X-100 is included in the McA solution. At the onset of gut invagination (38 hr) the antigen is still in some of the vegetal plate cells, as well as in some cells free within the blastocoel directly over the vegetal plate (Figs. 2C, D). When the gut is one-eighth to one-fourth the distance across the blastocoel (42-43 hr), of the 12 f 4 SP1/20.3.1-positive cells (Fig. 3), 8 + 2 (N = 20) are free in the blastocoel (Figs. 2E, F). The other SP1/20.3.1-positive cells are in the archenteron tip or within the vegetal blastodermal wall. At this stage the staining pattern is less granular and the cell boundaries of SP1/20.3.1-positive blastocoelar cells are sharply delineated, suggesting the antigen has

become associated with the cell surface. As well, 0.1% Triton X-100 in the McA solution reduces the stain intensity in embryos older than one-eighth gastrula. At the one-half gastrula stage (46 hr) there are 23 + ‘7 SP1/20.3.1-positive cells in the embryo, of which 2 + 1 (N = 20) are free blastocoelar cells (Figs. 2G, H; 3). Although SP1/20.3.1-positive cells in the archenteron tip are occasionally seen, the majority of stained cells are within the vegetal epidermis. As the archenteron approaches the animal plate (4950 hr), the number of SP1/20.3.1-positive cells has increased to 26 + 3 (Fig. 3), almost all of which are dispersed within the ectoderm (Figs. 21, J). In dissociated embryos at this stage, 1 f 1 (N = 20) SP1/20.3.1positive cells remain in the blastocoel. Blastocoelar cells near the tip of the archenteron that appear to have been recently released (secondary mesenchyme) are not SP1/20.3.1-positive (Figs. 21, J). At this stage in live embryos pigment granules can be seen in cells within the ectodermal layer. By the early pluteus stage (72 hr) 28 +- 5 SP1/20.3.1-positive cells are dispersed within the larval epidermis (Fig. 3) and each has one or two pseudopodia (Figs. 2K, L). In 72-hr plutei, assayed with PBS/glycine as the immunoreaction buffer, most stained cells contained pigment granules. Pigment cells continue to express the SP1/20.3.1 antigen for at least 29 days.

FIG. 2. Distribution of SPl/20.3.1-positive cells during S. pqnwatus embryogenesis. (A, B) SPl/20.3.1-positive cells are detected in the flattened vegetal plate of the 36-hr mesenchyme blastula. Some embryos have SP1/20.3.1-positive mesenchymal cells (arrowhead) at this stage; however, in most cases (C, D) invagination begins before SPU20.3.1 mesenchymal cells are seen. (E, F) The mean number of SPl/ 20.3.1-positive, mesenchymal cells reaches a maximum at the one-fourth gastrula stage. (G, H) By 44 hr, or the one-half gastrula stage, most of the SPl/20.3.1-positive cells are in the blastocoel or within the vegetal ectoderm. Note that most cells at the archenteron tip (persumptive secondary mesenchyme) are not SP1/20.3.1-positive. (I, J) At the three-fourth gastrula stage most stained cells are closely associated with the blastoderm. Cells in the archenteron tip, and mesenchymal cells near the tip (arrowheads) are not SP1/20.3.1-positive. (K, L) At the early pluteus stage SPl/20.3.1-positive cells are uniformly dispersed among the larval epidermal cells. Magnification: A to J, X300; K, L, X230. 417

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DEVELOPMENTAL BIOLOGY

30 SP1/2cl.3.1

T1

i

40

50 60 70 80 HR POST-FERTILIZATION

90

100

FIG. 3. Number of SP1/20.3.1-positive cells during S. ppuratus embryogenesis. Each point represents a mean from 20 embryos. Error bars indicate fl SD. Developmental stages are indicated at the top. MB, mesenchyme blastula; 1/4G, one-fourth gastrula; 1/2G, one-half gastrula; PR, prism stage; PL, pluteus stage.

DISCUSSION

The appearance of echinochrome-containing pigment cells in echinoid larvae varies between species. Pigment cells can be seen at the early gastrula stage of Lytechinus variegatus (Young, 1958), and Chaffee and Mazia (1963) extracted echinochrome from early gastrulae of S. purpratus, although pigment cells do not become visible until the late gastrula stage in this species. Paracentrotus lividus (Boveri, 1901; Monroy et al, 1951), S. fransiscanus (Chaffee and Mazia, 1963), and Psammechinus miliaris (Gustafson and Kinnander, 1956) all develop pigment cells at the late gastrula stage. In most cases pigment cells were first observed in the ectoderm, which led to the conclusion that they were of ectodermal origin (Boveri, 1901; Young, 1958). Studies of pigment cell lineage employing vital staining techniques have given conflicting results. Horstadius (1936, 1939, 1973) followed lineages in P. lividus and concluded that pigment cells were a subset of the secondary mesenchymal cells derived from veg, descendants. Using similar techniques with L. variegatus embryos Young (1958) concluded that pigment cells originated in the vegetal ectoderm of the gastrula from veg, descendants. Although species differences in pigment cell origins may exist, technical difficulties with staining single blastomeres in 32-cell embryos may provide an alternative explanation for these contradictory results. An apparently clear example of a mesenchymal origin is in a species of Echinocardium where pigment cells differentiate early, and are seen to ingress into the blastocoel from the vegetal plate prior to primary mesenchyme ingression (Gustafson and Wolpert, 1967). Although supported by anecdotal or circumstantial evidence only, in most species, a mesenchymal origin for pigment cells is now generally accepted (Gustafson and Wolpert, 1967; Stearns,

VOLUME lo?,1985

1975; Okazaki, 197513; Ryberg, 1979; Ryberg and Lundgren, 1979). SPV20.3.1 McA allows detection of pigment cell precursors in S. purpuratus 10 to 12 hr before pigment granules can be seen, and the antigen is expressed by pigment cells throughout larval life. During gastrulation the close proximity of many of the stained blastocoelar cells with the archenteron tip, and the progressive disappearance of stained cells from the tip during the initial phase of gastrulation, suggests that SP1/20.3.1-positive cells are released into the blastocoel between the onset of invagination and the one-half gastrula stage. The cells apparently migrate into the vegetal ectoderm, then disperse and develop pigment granules. The staining pattern of the SPV20.3.1 McA indicates that commonly employed procedures for separating ectoderm and mesendoderm fractions in sea urchin embryos may not yield homogeneous cell populations. Harkey and Whiteley (1980,1983) have used this technique to separate ectoderm from bags of skeletogenic mesenchyme at the one-fourth gastrula stage. The release of pigment cell precursors at the beginning of gastrulation suggests that mesenchyme at this stage is composed of cells with at least two fates. Ectodermal fractions from late gastrulae may also contain mesodermal derived pigment cells that have migrated into the epidermis. The fate of secondary mesenchyme has not been as well established as it has for primary mesenchyme. Okazaki (1960) considered only coelom-forming cells as secondary mesenchyme, yet in a later review (Okazaki, 197513) she suggested secondary mesenchyme formed pigment cells as well. Stearns (1975) considered mesenchyme forming the esophageal muscles as secondary mesenchyme, although Gustafson and Wolpert (1963) suggested muscle forming mesenchyme is a separate cell set. A common element of all descriptions of secondary mesenchyme is that they are the cells at the tip of the archenteron that extend contractile filopodia at the completion of the initial phase of gastrulation, and pull the archenteron across the blastocoel during the second phase of gastrulation (Dan and Okazaki, 1956; Gustafson and Kinnander, 1956, 1960; Gustafson and Wolpert, 1961, 1967). Although pigment cells are usually considered to be a subset of the secondary mesenchyme, in Echinocardium pigment cells ingress before archenteron invagination begins (Gustafson and Wolpert, 1967), and in S. purpuratus the majority of pigment cell precursors appear to be released prior to the second phase of gastrulation. Neither of these cell sets fit the common definition of secondary mesenchyme. Okazaki (1960) also described a set of nonskeletogenic mesenchyme in Clypeaster

GIBSON

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japonicus that are released prior to invagination, suggesting that other species also may have mesenchyme that are neither primary or secondary. Although the term secondary mesenchyme may be loosely used to describe all nonskeletogenic mesenchyme, more precise names based upon the fate of a set of cells in the pluteus, rather than the time of ingression, should be used when specific references are required. We thank Dr. T. Pearson for supplying the SP-2/O myeloma cell line, and G. Caine and D. O’Foighil for critically reading drafts of the manuscript. We also thank B. Bisgrove for valuable technical assistance. This work was supported by a Natural Sciences and Engineering Research Council (NSERC) fellowship and operating grant to R.D.B., and an NSERC postgraduate scholarship to A.W.G. REFERENCES BOVERI, T. (1901). Die polaritat

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OKAZAKI, K. (1975a). Spicule formation by isolated micromeres of the sea urchin embryo. Amer. Zool 15,567-581. OKAZAKI, K. (1975b). Normal development to metamorphosis. In “The Sea Urchin Embryo” (G. Czihak, ed.), pp. 175-232. SpringerVerlag, Berlin. PEARSON, T. W., GALFRE, G., ZEIGLER, A. and MILSTEIN, C. (1977). A myeloma hybrid producing antibody specific for an allotypic determinant on “IgD like” molecules of the mouse. Eur. J. Immunol 7, 684-690. RYBERG, E. (1979). Development and possible function of the red pigment in sea urchin larvae. In “Echinoderms, Past and Present” (M. Jangoux, ed.), A. A. Balkema, Rotterdam. RYBERG,E., and LUNDGREN, B. (1979). Some aspects on pigment cell distribution and function in the developing echinopluteus of Psammechinus miliaris. Dev. Growth D#er. 21,129-140. STEARNS, L. B. (1974). “Sea Urchin Development. Cellular and Molecular Aspects.” Dowden, Huchinson and Ross, Inc., Stroudsburg, Pennsylvania. YOUNG, R. S. (1958). Development of pigment in the larvae of the sea urchin, Lytechinus variegatus. Biol. Bull 114, 394-403.