Specification of the sea urchin embryo

The Definitive Territories of the Embryo

The zygotic mechanisms that lead to the formation of the sea urchin embryo can be defined as a set of territorial specification processes, followed by the downstream events of differentiation. By late cleavage each territory is a unique domain of gene expression, and the cells of each are the unique progenitors of a specific structure of the embryo. The territories constitute a spatially simple map, as shown in Fig. 3.1A-D. Except for some interterritorial boundary regions in which individual cell fates are sorted out only later, there are no overlapping domains of cell fate because the territories are all specified during cleavage, before there is any cell migration. Initially five territories are set up, viz, the oral and aboral ectoderm territories; the skeletogenic mesenchyme territory; the endomesodermal or "vegz" territory; and the "small micromere" territory, the cells of which in the late embryo become incorporated in the coelomic pouches. By the end of cleavage the endomesodermal territory has been further divided into a peripheral endodermal territory, which produces most of the gut or archenteron, and a central mesodermal territory, which produces several mesenchymal cell types and the remainder of the coelomic pouches. These territories and the structures to which each gives rise are indicated by color in the diagrams of Fig. 3-1.

The simplicity of the outcome is the key to the process: there are only about 12 differentiated cell types in the completed embryo or larva, and this consists mainly of single cell-thick structures. Though it is composed of a total of only a couple of thousand cells (1800 in S. purpuratus, the species after which Fig. 3-1 is drawn), this small organism is capable of feeding and of prolonged free-living existence in the wild. As shown in Fig. 3-1E-G, the completed embryo is equipped with a tripartite gut, consisting of a foregut which is connected to the mouth, a large midgut or stomach, and a hindgut terminated at the site of the original blastopore. The coelomic sacs protrude bilaterally from either side of the foregut (Fig. 3-IE, F). The blastocoel and its mesenchymal cell types are enclosed by the squamous aboral ectoderm, and at the top by the oral ectoderm, which surrounds the mouth. Within the blastocoel are skeletal rods that maintain the triangular shape of the larva and extend its cilia-bearing arms. At the intersection of the oral and aboral ectodermal territories is the ciliated band. This consists of some neurogenic precursors and of specialized cells that produce a dense belt of long cilia. A few neurons are also generated from elsewhere within the oral ectodermal territory.

For what follows a brief description of the main events leading up to the definition of the embryonic territories will be useful (some further details are in the legend to Fig. 3.1; see reviews of Davidson et al., 1998; Davidson, 1986, 1989; Wilt, 1987; and for the initial, "preterritorial" stages, Angerer and Angerer, 2000). At the outset of development the egg is polarized in the animal/vegetal (A/V) axis. This polarization is reflected in the diverse fates of the blastomeres inheriting given sectors of the maternal cytoarchitecture located along this axis. Thus oral and aboral ectoderm (green and yellow in Fig. 3-1) are formed from lineages arising from the upper (i.e., by convention toward the animal pole) two thirds or so of the egg, and endodermal and mesodermal cell types from the lower portion. An oral/aboral (O/A) polarization, which occurs after fertilization, sets up the second axis of the egg very early in development. This polarization has already taken place by third cleavage, since by then there have arisen founder blastomeres whose progeny will contribute exclusively to either oral or aboral lineages (Cameron et al., 1987; Henry et al., 1992; in S. purpuratus O/A specification is already set in train by 1st cleavage, as shown by Cameron et al, 1989). Much evidence indicates the presence of a complex assemblage of maternal transcription

Sea Urchin Embryo Animal Plate
Oral ectoderm □ Aboral ectoderm | Vegetal plate/endodefm B Skeletogenic mesoderm j" j Veg. plate mesoderm | Small micromeres Q Ciliated band ^ Larval spicules

FIGURE 3.1 Territorial origins of major embryonic structures in the sea urchin. (A) Fate map of the embryo, lateral view, projected on an external image of the blastula (~400-cell stage in S. purpuratus); see color key at lower right. The black lines indicate lineage compartments (Cameron et al., 1997, 1993; Horstadius, 1939; Logan and McClay, 1997; Ransick and Davidson, 1998; Ruffins and Ettensohn, 1993, 1996). No, Nl, and Na are 3rd cleavage blastomeres of the animal half (Cameron ei al., 1987); the veg\ and veg2 layers consist of the progeny of two 6th cleavage rings of eight cells each, which are sister cells; A, animal pole and V, vegetal pole; O, oral and Ab, aboral sides of the embryo. The skeletogenic (and barely visible small micromere) domains at the bottom consist of the progeny of the 4th cleavage micromeres. All cells are not finally allocated to the indicated territorial domains until the late blastula stage. The structures to which the territories give rise are shown in (E) and (F). (B-F) The diagrams show in color the already specified polyclonal domains at the respective stages. White areas in (B-D) denote border regions between territories the boundaries of which are not yet finally specified; i.e., progeny of different cells in the white regions may participate in both the adjacent programs of differentiation. (B) State of specification at 6th cleavage. The small micromere lineages (violet) have been set aside; the skeletogenic territory (red) is specified; and the veg2 cell ring is specified as endomesoderm (for lineage relationships see text and references therein). At least the central polar clones of oral (yellow) and aboral (green) ectoderm are by this stage specified, vegi is not, and its progeny will be allocated, by subsequent lineage-independent specification processes, both to endoderm and ectoderm territories. The bracket indicates the lineage constituents from which the vegetal plate (VP) will form. (C) Mesenchyme blastula stage, about 500 cells. The skeletogenic cells have ingressed into factors in the egg. But the subdivision of the egg into diverse territories cannot be attributed solely to regional sequestration of maternal factors, since blastomere recombination experiments show that with one exception, blastomeres from any region of the egg have the capacity to produce either endomesodermal or ectodermal cell types, depending on where they are placed and who their neighbors are (Horstadius, 1973; reviewed by Davidson, 1989; Davidson et al., 1998). The exception is the polyclonal skeletogenic domain which arises at the south pole of the egg (Fig. 3-1). These cells are autonomously specified and they are bound to execute their skeletogenic fate no matter where they are transplanted and even in culture, isolated from other cells. Specification of the remainder of the blastomeres involves both autonomous and conditional functions. An important mechanism of autonomous specification is regional nuclearization or regional activation of maternal transcription factors (recall, for example, the unequal nuclear distribution of the maternal SoxBl factor illustrated in Fig. 2.5). Conditional specification the blastocoel and can no longer be included in this external view. The vegetal plate derived from veg2 is now subdivided into a peripheral endodermal (blue) and a more central mesodermal (lavender) domain (Ruffins and Ettensohn, 1996). (D) Territorial domains of the vegetal plate viewed from the vegetal pole. The small micromere territory (violet) is at center. The secondary or vegetal plate mesodermal domain (lavender) will give rise to four mesodermal cell types: pigment cells, blastocoelar cells, muscle cells, and cells of the coelomic pouches (to which the small micromeres also contribute). Some vegi cells which have been recruited to the aboral ectoderm domain are also visible in (C). Most of the remaining veg] cells visible in (D) will subsequently be specified as endoderm. These will constitute the final population of future archenteron cells to invaginate at gastrulation, in the course of which they contribute mainly to the future hindgut and anus. (E, F) Final state of specification in early pluteus (about 1800 cells in S. purpuratus); (E) oral view; m, mouth; a, anus; (F) Lateral view. The ciliated band forms from cells at the oral/aboral interface, some of which are respecified from a prior state as aboral ectoderm (Cameron et al., 1993). Blastocoelar mesoderm cells, pigment cells lying along the ectodermal wall, and a coelomic pouch are indicated. The aboral ectoderm consists of a single squamous epithelial cell type. The gut is tripartite, consisting of esophagus, midgut, and hindgut. Skeletogenic cells are in process of generating the skeletal rods. The transverse skeletal rods can be seen in (E), as can the bilateral arrangement of the coelomic pouches, and the neurogenic oral epithelium and ciliated band. [(A-F) From Davidson etal. (1998) Development 125, 3269-3290 and The Company of Biologists, Ltd.] (G) Fluorescence photomicrograph of transgenic pluteus, lateral view. The embryo is expressing CAT protein under control of the ds-regulatory system of the sm50 skeletal matrix protein gene (cf. Fig. 2.4), here visualized with an anti-CAT antibody (Zeller et al., 1992). Only skeletogenic cells display fluorescence. Parts of embryo are indicated: abbreviations as above; and st, stomach; S, skeletal rod; M, skeletogenic mesenchyme cell; E, esophagus. [(G) From Zeller et al. (1992) Dev. Biol. 151, 382-390.]

in the early sea urchin embryo operates by short-range interblastomere signaling, and some particular interactions of this kind are touched upon in the following.

Very briefly, the origins and the fates of the definitive territories are as follows. The autonomous skeletogenic territory (red in Fig. 3-1) consists of the progeny of four 5th cleavage founder cells, which are the daughter cells of the four micro-meres separated from the macromeres by the unequally positioned 4th cleavage plane. At blastula stage the descendants of the skeletogenic micromeres ingress into the blastocoel: their progeny (32 in S. purpuratus, 64 in Lytechinus variega-tus) form a syncytium that is arranged in patterned lines against the blastocoel wall, thereby determining the positions of the skeletal rods that they secrete (Fig. 3.1G; McClay et al, 1992; Ettensohn, 1992; Davidson et al., 1998). The small micromere territory is founded by the 5th cleavage sister cells of the skeletogenic micromeres. After dividing once more they are carried into the interior by gastru-lar invagination. Ultimately, in postembryonic development, their progeny contribute to the mesoderm of the adult body plan, which derives from the embryonic coelomic sacs.

Both skeletogenic and coelomic sac constituents are mesodermal cell types, and the additional mesodermal cells of the embryo arise from progenitors that lie immediately peripheral to the small micromeres and the skeletogenic cells in the center of the vegetal plate (see Fig. 3.1). The mesoderm of the embryo (and of the postembryonic larva as well) thus all derives from blastomeres near the vegetal pole of the egg. But while the small micromere and skeletogenic territories are constituted of lineage elements which are autonomously specified and which segregate precociously during cleavage, the remaining mesodermal domain (lavender in Fig. 3.1) is defined only later in cleavage, by processes that are at least partly conditional. Notch signaling is required within the prospective mesodermal cells, and their specification depends on a late cleavage signal from the skeletogenic micromeres (Sherwood and McClay, 1997, 1999; Sweet et al., 1999; McClay et al, 2000).

Going back a step, we note that the endomesodermal territory as a whole consists of an invariant cleavage-stage lineage element, i.e., the vegz polyclone, though the later endodermal and mesodermal domains to which it gives rise are not defined according to lineage. That is, in every embryo, the vegi territory begins as a ring of eight 6th cleavage blastomeres which are the lower granddaughters (with respect to the A/V axis) of the 4th cleavage macromeres. Specification of the veg2 ring as endomesoderm is completed soon after these cells are born, and as we take up in more detail below, this event again depends on both autonomous and conditional functions.

The more peripheral cells of the veg2 endomesoderm territory become endo-derm (blue in Fig. 3.1). Additional endodermal components are derived from those vegi cells that happen to lie adjacent to the prospective veg2 endoderm (Logan and McClay, 1997; Ransick and Davidson, 1998). Late in gastrulation, following invagination of vegz endoderm, these cells roll inward and constitute the anus and hindgut, sometimes contributing to more anterior regions of the gut as well. The complex origins of the archenteron endoderm account for the endodermal fate map shown in Fig. 3-1 A.

There remain the oral and aboral ectoderm territories, which differ greatly in the diversity of the cell types their progeny produce. The squamous epithelial cell type (dark green in Fig. 3.IF) is the sole differentiated product of the aboral ectoderm (except for some contributions to the ciliated band at the margins; Cameron et al., 1993), but oral fates are more complex. From the oral ectoderm territory arise the mouth and esophagus; the columnar cells of the "face" of the late embryo (yellow in Fig. 3. IE, F); and neurogenic precursors located in the region surrounding the mouth and the ciliated band.

This will set the stage, and to begin we consider a little more extensively one of the most unusual aspects of Type 1 embryonic process, the precocious expression of territorial differentiation genes.

Early Transcriptional Activation of Cell Type-specific Genes

Many cell type-specific genes are activated during cleavage in sea urchin embryos, and some examples are listed in Table 3-1. As the territories of the cleavage and early blastula stage embryo each produce only one or a few cell types of the late embryo, the patterns of expression of these genes (where known) resemble the colored domains of Fig. 3-1A-F. That is, such genes are activated following specification in the early polyclonal territories, and they continue to be transcribed, often at stepped-up rates, within the differentiated morphological structure to which each territory gives rise. Table 3-1 includes genes encoding both differentiation proteins and cell type-specific transcription factors, expression of which ends up as a permanent feature of the respective differentiation states, as assayed in the late embryo.

Of course additional differentiation genes are called into play within each territory/differentiated structure as development proceeds, particularly those activated in response to novel intercellular interactions that come about as cells become motile and morphogenesis takes place. For example, several genes encoding proteins required for skeletogenesis begin to be expressed only as the skeletogenic precursors prepare to ingress into the blastocoel, and thereafter their rates of expression depend strongly on local skeletogenic activity (see review of Guss and Ettensohn, 1997). Nor is the expression of genes that in the end are utilized only in a given structure initially always territorial; e.g., the arylsulfatase gene is initially transcribed all over the embryo, then in all ectoderm territories, and finally only in the aboral ectoderm (Akasaka et al., 1990). So the examples in Table 3.1 are not necessarily illustrative of the way most cell type-specific genes expressed in the late embryo are mobilized. Rather, these examples tell us something about the specification mechanism itself: they imply that the network of regulatory interactions in the early embryo is evidently quite shallow. Genes such as SpKroxl and PlHbxl2 that are activated in early cleavage, as illustrated in Fig. 3.2A, B and Fig. 3.2C, D, respectively, are likely to be controlled directly by

TABLE 3.1 Territory- ancl Cell Type-specific Genes Activated During Cleavage or Early Blastula Stage in the Sea Urchin Embryo

Gene

Early territory

Final expression domain

Protein function

sped, spec2f,1 LpSlf1^

Aboral ectoderm

Aboral ectoderm

Ca+2 binding

cyllla'1

Aboral ectoderm

Aboral ectoderm

Cytoskeletal structure

metallotb ionein

nd12

Aboral ectoderm

Heavy metal binding

sm.50,6 sm3 7

Skeletogenic

Skeletogenic

Skeletal matrix

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