Use of Echinoderm Embryos to Study the Basic Mechanisms of Communication Among Cells

Developmental biology is a discipline studying the mechanisms regulating embryogenesis and it is one of the most attractive fields in rapid expansion among the biological sciences. Its study is becoming essential for the comprehension of any other fields in biology, since it combines molecular and cellular biology, physiology, anatomy, immunology, research on cancer and also evolution and ecology. Development is mainly devoted to the production and organisation of all the different cellular types constituting the adult organism. The generation of different types of cells is a process known as differentiation, while the organisation of differentiated cells in tissues and organs is performed during morphogenesis.

It is well known that cells do not behave as single entities, but rather their association in multicellular structures requires precise co-ordination between release and uptake of signals. Communication and interaction among living cells are, in fact, fundamental events required for the proper development of tissues and organs. Living cells continuously receive inputs from the environment and modify their behaviour throughout a complex network of signalling pathways. This communication occurs at various levels and,to obtain co-ordinated responses, the integration of exchanged information is essential. Malfunctioning of this network of signalling pathways is often connected with pathological conditions ranging from abnormal proliferation to cell death. Understanding the molecular basis of communication among living cells is a fundamental challenge for biologists, since, besides providing better understanding of the processes controlling growth, differentiation and death, it may increase the number of discoveries of new therapies against diseases caused by inappropriate signalling.

Historically, within the phylum of Echinodermata, the sea urchin embryo has been an excellent experimental system for investigating the cellular basis of development, principally because of its relatively simple organisation and because of its optical transparency that makes the observation of morphogenesis in vivo possible. Pioneering studies on development date back to 1892, when Driesch, following the experimental approach to embryology proposed by Roux about 10 years before, utilised the sea urchin embryo in his studies with important outcomes for embryology (Driesch 1892). On the basis of his results, Driesch proposed the very modern concept of nucleus-cytoplasm interaction as an essential event for development (Driesch 1894). Later, from 1928 to 1935, Horstadius performed some of the most remarkable experi ments in the history of embryology using the sea urchin embryo (Figs. 3,4). First, he separated each blastomere of the early embryo and followed their fates; then he was able to recombine different series of blastomeres and, from the results obtained, to propose the well-known theory on the existence of graded properties within the unfertilised egg and the early embryo (Horsta-dius 1939).

The sea urchin system was also one of the first in which time-lapse microscopy was exploited extensively. For example, the classic studies of Gustafson and Wolpert (1967) led to the identification of many of the basic behaviours exhibited by cells in the embryo during morphogenetic movements. At the time, these authors remarked, "we are, however, still ignorant about the final steps in the casual chain between the genes and the shapes they control" (Gustafson and Wolpert 1967). In the past 35 years, our knowledge of the molecular basis of developmental processes and the relationship between molecules and cell behaviour has advanced considerably. The classic studies

Fig. 3. Cover of the original review by Horstadius (1939) on development of the Para-centrotus lividus sea urchin embryo (Kindly provided by M. Delarue, Laboratoire Biolo gie et Multim├ędia, UPMC-P6, www.snv.jussieu.fr/bmedia/ sommaires/dvpt.htm)

THE MECHANICS OF SEA URCHIN DEVELOPMENT, STUDIED BY OPERATIVE METHODS

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Fig. 4. Diagram of the normal development of the P.lividus sea urchin embryo published in 1939 by Horstadius (Kindly provided by M. Delarue)

of Horstadius have been extended by the lineage studies performed by Davidson and colleagues, providing a more detailed picture of the establishment of tissue territories in the early embryo (see reviews by Cameron and Davidson 1991; Cameron et al. 1991). More recently, many other laboratories in the world, thanks to new technologies developed in the field of molecular biology, have succeeded in the identification and characterisation of control genes and their key target sites and the determination of their functional significance in the early embryo, allowing the proposal of complex gene regulatory networks (see review by Davidson et al. 2002). An equivalent remarkable progress has not been seen in the cell biology field, particularly in the characterisation of the proteins involved in cell communication and cell adhesion, although some studies date back to the early 1970s.

In the following text, molecules of the sea urchin embryo involved in cell communication, such as cell-matrix and cell-cell adhesion proteins (Pl-

nectin, fibronectin, laminin, collagen, integrin, toposome, cadherin), growth factors (BMP2/4, univin), signal transduction molecules (kinases), and their genes (when characterised) will be described. Our intention is not to review all the work done over the years in the field of cellular interaction in echino-derms, but rather we will focus on a few arguments on the assumption of re-examining some ideas and concepts.

Sea Urchin Embryo Transparency: A Living Laboratory for Studying Development and Morphogenesis

To facilitate the reader in the following discussion on the relationship between adhesion/signalling molecules and cell differentiation occurring during embryogenesis, the development of the Mediterranean sea urchin species Paracentrotus lividus will be briefly described. The time scale and embryo morphology apply, with very little modifications, to many other sea urchin species.

Fertilisation is the first event that gives rise to development and triggers instantaneously several changes within the egg. A transient decrease in the membrane potential, a slight increase in the intracellular pH and free calcium ions activate several metabolic processes, causing, among other things, the exocytosis of cortical granules and the consequent elevation of the fertilisation membrane (Fig. 5A). Cleavage stages begin within 1 h after fertilisation and are characterised by a number of cell divisions occurring about every 30 min (Fig. 5B,C). While the first three divisions give rise to eight equivalent blastomeres, the fourth one produces 16 cells of different sizes: eight mesomeres at the animal pole, four macromeres and four micromeres at the vegetal pole (Fig. 5D). Cameron and colleagues were able to describe extensively the cell lineage for each of the 16 blastomeres; in addition, they showed that most of the lineage founder cells for many tissues of the later embryo are established at the 64-cell stage (Cameron and Davidson 1991; Davidson et al. 1998). It is noteworthy to underline that the lineage boundaries are detected well before the appearance of any morphological difference in the embryo and the spatially restricted patterns of gene expression already described agree with these lineage boundaries. As cell divisions proceed, the embryo develops into a blastula, which consists of one layer of epithelial cells, with a typical apical-basal polarity, surrounding a filled cavity, the blastocoel (Fig. 5E). Later, the embryo hatches from the fertilisation envelope to become a free-swimming blastula. From this stage another phase of development starts, which is characterised by a number of morphogenetic movements that will completely rearrange the embryo. The first cells to move are the primary mesenchyme cells (PMCs) that detach from the vegetal plate and ingress into the blastocoel, where they migrate using short filopodia (mesenchyme blastula stage; Fig. 5F). Some PMCs migrate towards two ventro-lateral sites and

Embryo Blastocyst Grading
Fig. 5A-K. Development of the sea urchin embryo Paracentrotus lividus. A Fertilised egg; B two blastomeres; C four blastomeres; D 16 blastomeres; E early blastula; F mesenchyme blastula; G early gastrula; H middle gastrula; I late gastrula; J early pluteus; K pluteus

form clusters that are connected by other PMCs in a form of a subequatorial ring. The morphology of PMCs during migration was described for the first time by Gustafson and Wolpert using time-lapse microscopy (1961, 1967). Shortly after PMC ingression, the vegetal plate epithelium invaginates to form the archenteron, which is the future intestine. It is possible to distinguish at least three gastrula stages during the elongation of the archenteron towards the animal pole: early, middle and late gastrula (Fig. 5G-I). PMCs produce the skeleton, first in the form of triradiate spicules, which elongate and pattern in a complex species-specific manner. Skeleton is formed by calcium and magnesium carbonate, which deposit on an organic matrix constituted by a number of well-known spicule-specific proteins. In the laboratory, the larval stage of pluteus is obtained after 48 h post-fertilisation: it shows four arms, a complex patterned skeleton and a tripartite intestine, encircled by muscles undergoing peristaltic contractions (Fig. 5J,K). At this point, the larva, if correctly fed, is ready to continue development and form the juvenile sea urchin through metamorphosis.

From the developmental processes outlined above it is evident that cell-cell and cell-ECM contacts play a fundamental role in morphogenetic movements occurring during embryogenesis. Knowledge of the key actors involved as well as understanding of their complex interactions would eventually lead to the unravelling of the developmental machinery. Furthermore, it should be noted that cells forming the blastula stage monolayer exhibit a structural asymmetry of the cytoplasm and the plasma membrane is compartmen talised into distinct apical, basal and lateral domains, with characteristic lipid and protein compositions. Embryogenesis and morphogenesis are characterised by cell movements and complex cell rearrangements, which require appropriate interactions of cells with the underlying ECM by means of specific membrane receptors. For this reason, interest in the identification, purification and functional studies of ECM components, along with their ligands and other molecules involved in cell-matrix adhesion, has increased in recent years. In the following text, after a brief description of the appearance of the ECM upon embryo development, protein molecules found in different plasma membrane compartments of blastula cells will be described.

ECM Patterning in Echinoids During Embryo Development

In recent years, it has been shown that the ECM of the sea urchin embryo is a very complex structure, consisting of a number of layers organising during different developmental steps. Using specific antibodies, different storage compartments of the ECM components, like granules and vesicles, have been identified in the unfertilised egg cytoplasm. The protein contents of these compartments are exocytosed and assembled in a highly regulated fashion at different moments after fertilisation (for a review see McClay et al. 1990). The early event after sperm entry is the elevation of the fertilisation envelope above a water-filled perivitelline space (Fig. 5A; for more detailed description on ionic events occurring at this stage, see the chapter by Angelini et al., this Vol.). Then, a new ECM is secreted on the outer surface of the zygote, which gives rise to different layers, the most acknowledged of them being the apical lamina and the hyaline layer. During cleavage, other ECM molecules are also released into the newly forming basal lamina, which will underlie the blasto-coelic cavity from the stage of blastula (Fig. 5E). A great number of in vitro and in vivo studies suggest that ECM has an important role during morphogenesis, serving as a mechanical support to the embryo as well as a substrate for cell movements, and providing both spatial and temporal information to adherent cells. Thus the ECM is not a fixed structure but rather is able to respond to and effect changes in its local microenvironment.

Changes in ECM composition and regulated matrix remodelling are common events associated with embryogenesis and are carried on by matrix met-alloproteases (MMPs). These molecules are a growing family of metalloen-dopeptidases that act by cleaving the protein components of the ECM and thereby regulating its composition (for a review see Stamenkovic 2003). For many years MMPs were believed to function in facilitating cell migration simply by removing barriers such as collagen. However, recent discoveries have shed new light on the role of MMPs in embryology, physiology and disease. It is becoming increasingly clear, in fact, that MMPs are also involved in the functional regulation of non-ECM molecules, including growth factors and their receptors, cytokines and chemokines, adhesion receptors and cell-surface proteoglycans, and a variety of enzymes. MMPs therefore play an important role in the control of cellular interactions with their environment, promoting tissue turnover, both physiological, such as normal development, and pathological, such as inflammation and cancer. The sea urchin embryo was found to express a dynamic pattern of gelatinase activities associated with the external ECM, some expressed only in later stages of development, others expressed in the unfertilised egg and persisting throughout the course of embryonic development (Mayne and Robinson 1996; Robinson 1997; Flood et al. 2000). Their substrate specificity and metal ion requirements suggest that these molecules are members of the MMP class of ECM remodelling enzymes.

In recent years, a new family of molecules in the animal kingdom has been described, called ADAMs (A Disintegrin And Metalloprotease). These are multidomain transmembrane proteins containing a metalloproteinase and a disintegrin domain and all or some of the following structures: a signal peptide, a propeptide, a cysteine-rich and an epidermal growth factor (EGF)-like domain, a transmembrane region, and a cytoplasmic tail. As a consequence, ADAMs could have four potential functions: proteolysis, adhesion, fusion and intracellular signalling (Stone et al. 1999). Although the number of ADAM genes has grown rapidly, the biological functions of most members are still unclear. However, they seem to be key regulators of the cell-cell and cell-ECM interactions (see review by White 2003). Recently, a sea urchin SpADAM gene has been sequenced and the deduced protein sequence includes all the characteristic domains of vertebrate ADAMs. The structure and the types of cells in which SpADAM orthologues are expressed are then apparently conserved in deuterostomes (Rise and Burke 2002).

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