Skeletogenesis in the sea urchin embryo

Skeletogenesis, a key morphogenetic event in the embryonic development of vertebrates, is also a very important, albeit a transient, milestone in the development of a marine invertebrate, the sea urchin. In recent years this biological system, in which the biomineralization process can be investigated in a well-defined, single cell type (the primary mesenchyme cell), has received considerable study. The objective of this review is to summarize our current knowledge about skeletogenesis in the sea urchin embryo and highlight the outstanding questions that remain to be answered. It will become apparent that there are two broad sets of questions: one set pertains to cell differentiation and morphogenesis; the other is concerned with basic cellular processes that are common to most cell types in a developing organism, such as cell-cell interactions, membrane fusion, ion transport and exocytosis. The former set of questions has been reviewed on numerous occasions, most recently by Morrill & Santos, 1985; Davidson, 1986; Solursh, 1986; Wilt, 1987 and McClay & Ettensohn, 1987. Therefore, coverage of these issues will be restricted to points that pertain to the basic cellular processes involved in spiculogenesis.One reason the skeleton of the sea urchin embryo has attracted attention is its remarkable structure, which is readily apparent upon microscopic examination of the embryo. This is illustrated in Fig. 1 which shows the embryo of Strongylocentrotus purpuratus at the pluteus stage, viewed by polarized light (Nomarski optics). The skeleton grows from two triradiate spicules that increase in complexity as the embryo progresses from the late gastrula to the pluteus stage. The inorganic component of the spicules is the mineral calcite, CaCO3, plus a small amount (5 %) of MgCO3.The most distinctive feature of the skeleton is its apparently crystalline nature. Thus, although the skeleton is a complex, branched structure, the individual spicules behave ‘optically as though chiseled out of a single crystal of calcite’ (Okazaki & Inoue, 1976; Inoue & Okazaki, 1977). In fact, each spicule can be considered to have a polycrystalline structure, made of stacks of contiguous microcrystals. However, as pointed out most clearly by Okazaki & Inoue (1976), such microcrystals do not define the overall shape of the spicule. Rather, the morphology of the spicule is dictated by the syncytial structure produced by the primary mesenchyme cells.Both optical birefringence and X-ray diffraction (Donnay & Pawson, 1969) indicate that the spicules are crystalline; however, no crystalline structure is apparent upon scanning electron microscopic examination of the intact or fractured spicules (Millonig, 1970). Even in the absence of obvious crystalline faces, the isolated spicule can serve as a template for growth of calcite crystals upon addition of CaCl2 and NaHCO3, and the crystals that grow on the spicule under these conditions are aligned parallel to each other and follow the form of the spicule (Okazaki & Inoue, 1976; Inoue & Okazaki, 1977). Thus, despite the absence of some of the expected features of a polycrystalline structure, there is little doubt that this extraordinary structure is composed of calcite crystals.A second reason for interest in the spicule relates to its ontogeny. About a century ago three investigators, Selenka, Semar and Theel, independently carried out investigations on the origin of the spicule. In 1882, Theel wrote“The manner in which the calcareous deposits originate in the Echinoderms, and the subsequent mode of increase of the skeleton are by no means clear and offer a great deal of interest. When studying these processes, the investigator soon perceives that the calciferous cells, though free in the blastocoel, act by no means without method, and that they cooperate for a common goal as if they were conscious.”One hundred years later this ‘cooperative’ behaviour of the ‘conscious’, ‘calciferous cells’ is no less remarkable, and only slightly better understood. What is clear now is that the cells responsible for assembly of the skeleton are primary mesenchyme cells and that they originate from a subset of the micromeres formed in the 16-cell embryo (Fig. 2). At early blastula stage, after numerous rounds of cell division, the cells whose progenitors were the micromeres are indistinguishable from the other cells comprising the blastodermal layer of the embryo. Only upon formation of the vegetal plate of the blastula-stage embryo do these 30—50 cells emerge as a distinguishable cell type. At this point in the developmental program, the primary mesenchyme cells leave the blastoderm, enter the blastocoel and form a cell cluster at the vegetal pole. Subsequently, migratory behaviour is initiated and the primary mesenchyme cells extend filopodia and move along the blastocoelic wall.The appearance of the primary mesenchyme cells in the vegetal half of the early-gastrula-stage embryo is shown by scanning electron microscopy in Fig. 3. At this stage of development the primary mesenchyme cells cease their migration in the blastocoel and become positioned as two cell clusters that are formed bilaterally with respect to the site where the endoderm invaginates. Subsequently, as shown in Fig. 4, an annular pattern of cells is formed and two loci become readily evident by light microscopy. The cells at each locus have fused with each other and, within the syncytial structures that are formed, the birefringent, polycrystalline spicules grow. Upon reaching pluteus stage, an abundance of extracellular matrix is also found associated with the syncytia and blastocoelic wall (Fig. 5). Throughout development from gastrula to pluteus stages the skeleton grows in both size and complexity. Its existence, however, is only transient in the overall life cycle of the sea urchin because it is “lost” when the pluteus larva undergoes metamorphosis to form the juvenile sea urchin. For more comprehensive coverage of issues pertaining to the ontogeny of the spicule-forming cells, the reader is referred to the important studies of Horstadius (1973), the recent review by Wilt (1987) and the monograph by Davidson (1986).One important finding was that isolated micromeres (but not meso- or macromeres) cultivated in sea water containing serum could differentiate in vitro into spicule-forming primary mesenchyme cells (Okazaki, 1975a). This observation suggested that isolated micromeres are already programmed for the differentiation process and that external cues provided by other blastomeres are not necessary. This idea was supported by the findings that primary mesenchyme cells isolated from mesenchyme-blastula-stage embryos by virtue of their selective adhesiveness to the surface of tissue culture dishes (Venkatasubramanian & Solursh, 1984; Carson et al. 1985) or to the immobilized & spicules in the apparent absence of other cell This is also in more complex, in vitro of primary mesenchyme cells by the of the mesenchyme-blastula-stage embryo that has been of cells & In three primary mesenchyme cells or primary mesenchyme cells within the of the spicules formed are they the form found in the intact embryo. in three the sea water not spicule formation it is with although in the of blastula a of spicule formation can be in the absence of for serum is of the for that have been in the process of the embryos (Okazaki, However, such have to be on this apparent that a in serum the of that form to & the of serum is in the blastocoel, as are and & in the of these in the stages to formation of the spicule is of the spicules is the skeleton for each of sea urchin has a structure with cells in vitro indicate that conditions cell formation of spicules & Okazaki, However, it is clear that the structures formed in are by more At the early stage of spicule the overall structure of the spicule is by the of the primary mesenchyme cells in the the of these What cues the primary mesenchyme cells to cease migration at a site in the is that the is not and that of serve as for the migratory primary mesenchyme This of the blastodermal cells, these cells are to the of the this idea is not no for of the However, et al. studying the ontogeny of the that individual extracellular matrix are into this structure at stages of of that blastodermal cells, an in the of primary mesenchyme cells in that define the shape of the spicule. & studying spicule formation by primary mesenchyme cells within found that in the of a of blastodermal cells the spicules formed by primary mesenchyme cells were However, when the of blastodermal cells was of the and the morphology of the spicules formed that of spicules made in the embryo. the of these and in with the of & and Okazaki & that filopodia of the primary mesenchyme cells can with cells that the In if such to of the primary mesenchyme cells, one that blastodermal cells are not and that some the the that biomineralization is a cellular process that in the development of both the and invertebrate, there is a great deal of basic of this respect to the skeleton of the sea it is not clear define the mineral type that the mineral in the sea urchin spicule could in be or the calcite that is In as there are in the mineral in the that it is if the calcite in a single spicule is a single crystal or a of crystal a polycrystalline of (Okazaki & Inoue, Thus, as reviewed by Okazaki & Inoue (1976), the of light microscopy and X-ray diffraction are with a single crystal structure, by electron microscopy a polycrystalline the of an by studying has become an important component of of biomineralization for the of these in the biomineralization growth of the have In the the serve as a template to define both the overall shape and the crystal form of the However, there are basic the crystal be by In the second the the matrix not serve as a template a within which the In this the overall shape of the is by the the of the is is not respect to the it is important to that its with respect to the spicule is not will be the spicule is in a shown in Fig. when this membrane is by with and and the mineral is by addition of or an in the shape of the spicule et al. this of the or was of it associated with the of the membrane and at the of studies it clear that are in the mineral they are to that have been on the surface of the mineral to be the of the matrix in of the the is that because the spicule is a of calcite and it less and more it if made of calcite the not only in of the also in that a more on the overall of the matrix and an of the of the in it will be in these issues pertaining to the of the some has been made in this on the matrix of tissue of a of suggested that in some are in in and are the et al. studies by & have the of in in both the and spicule matrix of the Subsequently, the of of these by with was & this was by a of for individual the that matrix in the of the and and two from appearance in the to in the by Wilt and and their have recently provided the on individual of the matrix of the sea urchin spicule. et al. that after of isolated an matrix that an overall structure to the The in this matrix not have the of apparently was in within the of the of this matrix et al. the of This of was shown to albeit with et al. findings on the of matrix were by & et al. (1987) a to these spicule matrix and it to a isolated by this was to a that a to the in the of isolated from the matrix et al. studies that the this at late from the blastula stage its in an In the in primary mesenchyme cells into the blastocoel of the which clearly that a matrix is in primary mesenchyme cells, were in to define the structure of the and the et al. The from the structure of the the of a and an as expected for a The most feature of the is that half of the is composed of a of the this and was second feature of interest is the abundance out of three of in an of this and a of was however, the of these features with respect to the biomineralization process to be questions about the process of were not in the the a of have in recent years on individual in the overall process of intact embryos or a of in vitro of of the are on the of Okazaki that micromeres cultivated in vitro in the of serum apparent differentiation to primary mesenchyme cells and produced studies et al. that although was by the the of primary mesenchyme cells into the blastocoel was not a in the addition of to by of embryos in the of et al. a in in the & Solursh, in a in Thus, to little on of or studies by & that the in the overall process of the of a in the vegetal plate and the of from these shape in which the blastodermal cells into the blastocoel, their and the McClay and & & 1985; McClay & Ettensohn, very with these the primary mesenchyme cells their to to and to other cells and the to the into the after the blastocoel, the primary mesenchyme cells studies have on the of the on the surface of the primary mesenchyme cells or in the blastocoel that be involved in the migratory of the of cell-cell and is which is to have for a cell surface and & In fact, has been found to be associated with the primary mesenchyme cells the stage in which they migratory behaviour et al. It is not that the of to the cells is by a cell surface of the type shown in other to the in For such a has been shown to be in and et al. For the to in the migration process it be expected in addition to with the primary mesenchyme cells, it with or of the of in the The although is not involved in cell migration & 1987 and In to migration of primary mesenchyme cells is by the of et al. 1986; & et al. or the absence of in the sea water & Solursh, & that primary mesenchyme cells in embryos in the behaviour of the and cellular that in the migration of these in vitro studies with isolated primary mesenchyme cells these (Venkatasubramanian & Solursh, of the apparent of in the blastocoel in the migration these were in both purpuratus and embryos at the mesenchyme blastula stage of development & studies the of the in the of and studies on the of that not to in the of this et al. This that the in cell migration are intact In this it is of interest that & have shown that when the filopodia of isolated primary mesenchyme cells with an of extracellular matrix the cell this is clear although considerable has been made in the migration of these cells, the intact have not been a that is on of on the surface of these cells to on the surface of the blastocoel not the that although these cells a of they at is clear from the studies that the of the blastocoel a in the overall of primary mesenchyme cell from to spicule of this and for the idea that the be the of development is provided by the of et al. and the important recent studies of McClay and & 1986; McClay & Ettensohn, carried out microscopic of primary mesenchyme cells that been into the blastocoel of an embryo. found that the behaviour of the primary mesenchyme cells is on the stage of the that primary mesenchyme cells into the blastocoel of a blastula embryo to to the vegetal remain the for At that they the primary mesenchyme In primary mesenchyme cells into embryos no can and to the vegetal pole. Thus, the blastodermal surface to the of for migration of primary mesenchyme the migration of the primary mesenchyme cells within the blastocoel with their by to form a syncytial that the of the spicule. of the event have been In early & that both micromeres and their the primary mesenchyme cells, have a to & studying isolated primary mesenchyme cells in by not only that these cells with each that the syncytial formed can into or cells are remarkable in their they with each other differentiation and cells scanning electron primary mesenchyme cells that apparently have a common is shown in Fig. this it be that recently in membrane in formation from & in cells and in cells & the in sea urchin et al. and sea urchin et al. this can now be primary mesenchyme cell because very recent studies indicate that spicule formation by primary mesenchyme cells in culture and of the cells to the culture is not by these the most site of is the event that is a to spicule and, more are biological processes that are of great respect to the sea urchin embryo because the overall process of formation of a spicule a of studies on the that such as and be the of the primary mesenchyme cells that out the process is the process can be with a in vitro of primary mesenchyme cells in sea water containing to define individual in the process cells in the of Okazaki of micromeres that differentiate in vitro to primary mesenchyme cells, not in our cells for a cell of primary mesenchyme cells and blastodermal cells by embryos at the mesenchyme blastula stage et al. a of the cells formed and after spicules were formed in of these of no on spicule formation or on the of cells in the of these are with the idea that differentiation and that cell increase in of and into the cells was at the as spicule of into and into spicules was by a of of This observation suggested a for in spicule In one or more to be by the primary mesenchyme cells formation of This not to have the of it not and it was not by However, its and to be for spicule formation of and three involved in of in spicule studies in an in vitro of cell types with intact embryos et al. & that the of is a for skeleton studies this and of also suggested that a in be a for differentiation to spicule-forming cells & studies & that these cells not the stages of differentiation and form spicules in the of of the process if was This finding a for in spicule as a component of an extracellular The this observation and a at the blastula stage in an a type and its later to be et al. for a of purpuratus primary mesenchyme cells was by of the that primary mesenchyme cells to culture dishes blastodermal cells do not & Solursh, 1984; Carson et al. The appearance of the primary mesenchyme cells and associated spicules as by light or scanning microscopy is shown in Fig. This has been in a of studies at an of the of the with purpuratus embryos a that on primary mesenchyme cells at the mesenchyme blastula stage et al. interest was that the of a to the of primary mesenchyme cells in culture spicule by light microscopy or of of the the findings are illustrated in Fig. et 1985) of the of the it was suggested that the of the to the surface Subsequently, it was shown et al. 1985) in cells from the culture of to was under these conditions the a finding with the idea that the is involved in the in its subsequent to developmental of the was also in both purpuratus and embryos and in primary mesenchyme cells isolated from purpuratus embryos et al. In the in vitro it was found that the of the with in and as as it was found that the on the was in the purpuratus and in in cell types of the embryo to the gastrula stage. this stage, and of the was found to be to the primary mesenchyme cells (Fig. In in the was in or in embryos the primary mesenchyme cells were embryos from purpuratus with a pattern of indistinguishable from that of the the that early of the in purpuratus embryos was to later of this in primary an et al. (1987) the of a primary mesenchyme that a was a containing the of the This a on the surface of primary mesenchyme to the this in a increase in the of at and subsequent to primary mesenchyme cell within the Subsequently, the by this et al. was both a it and the to the The that the was and that the only the form of the and not the by This observation is with the idea that the by is the which is to be In a recent of these studies et al. it was that this is in and is to a subset of the In with these et al. the found that the in the is to the however, in this the was found in of the et al. found that upon the is with the other of the only of it are found in the However, upon appearance of the primary mesenchyme cells in the blastocoel, the is in and is found in the and at the surface of the cells and spicule for the et al. that the of the is with in the and the primary mesenchyme cells of the embryo. & have that the is a and have shown that the which the is is the This which is has an apparent of It which in its such a in to be these from two that the primary mesenchyme cell a cell surface this its is involved in the process to be other that to be to primary mesenchyme cells have been of a that is at the of primary mesenchyme cell in is found associated with the spicule & micromeres from in it was shown that a in the of a with conditions that of the spicules shown to be in embryos of of sea and purpuratus under conditions of spicule was found to & important overall feature of the spicule formation process that be individual can be is the site of of and the of growth of the spicule. a century ago it was that a evident within one primary mesenchyme cell in each of the two clusters of primary mesenchyme cells in the this is to the spicules that grow in with the syncytial cells found in the embryo and in primary mesenchyme cell however, it that of spicule growth at these within the some of these electron microscopic were to the primary mesenchyme cells and spicules formed in culture et al. This that the cell and the filopodia and formed to in the blastocoel of the intact embryo (Fig. It was that the spicule was by a by of However, apparently this not the spicules from the external because the mineral could be in or cells by a of or by the of the In deposits in the cell to such that they were to that the and to the electron microscopy and X-ray that the deposits in the cell and common to the spicule. electron microscopic examination of the spicule (Fig. after of the spicule mineral in the within the membrane and a matrix that with this spicule and to matrix on these it was that of the spicule are and that spicule in an extracellular et al. It that and associated matrix are to this a However, at there is no for an for the and of or with to the in the the site of spicule growth was in the primary mesenchyme cell culture & The where are the of growth of the this with by found that in this cell culture system, in which most of the spicules are there were two points of growth and the growth by addition of of to both of each spicules more two a pattern of were from this study. because the in the spicule the it not be in a and not readily with in the culture if such of the the have been the that there are two growth loci in spicules means that one of the spicule is not in a with respect to the other In the that the of the two of these spicules are in that growth at an in the observation that the of the with respect to the spicule was the in and spicules that a common for is in both respect to the of the it is important to point out that it is clear from studies with a of that is a cellular process that is by in the the and and the to the it is important to that the in sea water is and it that a in the membrane of primary mesenchyme cells to the this is the the of at the of be by of this In this it is that have been to into the spicule & more the in be to the of that in the primary mesenchyme cells the assembly of the skeleton in the sea urchin embryo has been for a only in the have considerable in the cellular and of this This is the of at two the of cell culture to with a single cell type and the of and biological that can be as a of these are as differentiation into a spicule-forming cell in the of cell and of cells filopodia to form a syncytial template for spicule The spicules grow within this and the the of a formation the of one or more of which are cell surface are also found in the spicule of a for the is also the our of the that are spicule formation light on a of basic cellular processes, cell cell fusion, and and Thus, for it is that as more on cell such as and their a will emerge that basic of the migratory behaviour by primary mesenchyme cells the the of and biological will one to the of that in the basic involved in The will be to to and to the of these in each of these component biological this is a very considerable one that has because of the cellular processes are to have in a of biological in the was supported by of and and a to is a of The are to the of their for are to of for and and for the scanning electron shown in and and provided Fig. is for with The of is also has been for