The Dynamics and Regulation of Mesenchymal Cell Fusion in the Sea Urchin Embryo

AbstractCell–cell fusion occurs in a wide variety of developmental contexts, yet the mechanisms involved are just beginning to be elucidated. In the sea urchin embryo, primary mesenchyme cells (PMCs) fuse to form syncytial filopodial cables within which skeletal spicules are deposited. Taking advantage of the optical transparency and ease of micromanipulation of sea urchin embryos, we have developed methods for directly observing the dynamics of PMC fusionin vivo.A fraction of the PMCs was labeled with fluorescent dextran and transfer of the dye to unlabeled PMCs was followed by time-lapse, fluorescence microscopy. Fusion was first detected about 2 h after PMCs began to migrate within the blastocoel. Fusion proceeded in parallel with the assembly of the PMC ring pattern and was complete by the early gastrula stage. The formation of a single, extensive PMC syncytium was confirmed by DiI labeling of fixed embryos. When single micromeres were isolated and cultured in unsupplemented seawater, they divided and their progeny underwent fusion. This shows that the capacity to fuse is autonomously programmed in the micromere–PMC lineage by the 16-cell stage. PMC transplantations at late embryonic stages revealed that these cells remain fusion-competent long after their fusion is complete. At late stages, other mesenchyme cells (blastocoelar cells) are also present within the blastocoel and are migrating and fusing with one another. Fusion-competent blastocoelar cells and PMCs come into contact but do not fuse with one another, indicating that these two cell types fuse by distinct mechanisms. When secondary mesenchyme cells convert to a skeletogenic fate they alter their fusogenic properties and join the PMC syncytium, as shown by transfer of fluorescent dextran. Our analysis has provided a detailed picture of the cellular basis and regulation of mesodermal cell fusion and has important implications regarding molecular mechanisms that underlie fusion

A large-scale analysis of mRNAs expressed by primary mesenchyme cells of the sea urchin embryo

The primary mesenchyme cells (PMCs) of the sea urchin embryo have been an important model system for the analysis of cell behavior during gastrulation. To gain an improved understanding of the molecular basis of PMC behavior, a set of 8293 expressed sequenced tags (ESTs) was derived from an enriched population of mid-gastrula stage PMCs. These ESTs represented approximately 1200 distinct proteins, or about 15% of the mRNAs expressed by the gastrula stage embryo. 655 proteins were similar (P<10-7 by BLAST comparisons) to other proteins in GenBank, for which some information is available concerning expression and/or function. Another 116 were similar to ESTs identified in other organisms, but not further characterized. We conservatively estimate that sequences encoding at least 435 additional proteins were included in the pool of ESTs that did not yield matches by BLAST analysis. The collection of newly identified proteins includes many candidate regulators of primary mesenchyme morphogenesis, including PMC-specific extracellular matrix proteins, cell surface proteins, spicule matrix proteins and transcription factors. This work provides a basis for linking specific molecular changes to specific cell behaviors during gastrulation. Our analysis has also led to the cloning of several key components of signaling pathways that play crucial roles in early sea urchin development

The emergence of pattern in embryogenesis: regulation of beta-catenin localization during early sea urchin development.

The accumulation of beta-catenin in the nuclei of blastomeres at one pole of the early embryo is a highly conserved and essential feature of animal development. In the sea urchin, beta-catenin accumulates in the nuclei of vegetal blastomeres during early cleavage and activates gene regulatory networks that drive mesoderm and endoderm formation. Measurements of beta-catenin half-life in vivo have demonstrated a gradient in stability along the animal-vegetal axis. Dishevelled (Dsh), a protein that regulates beta-catenin turnover, is localized in the vegetal cortex, where it has an essential role in stabilizing beta-catenin and activating endomesodermal gene networks. Two motifs of Dsh are required for targeting to the vegetal cortex. Overexpression of Dsh in animal blastomeres does not alter their fate, which suggests that a localized activator of Dsh may be missing in these cells. Wnt signaling may be localized in the early sea urchin embryo, as it is in Xenopus, but findings point to possible differences in the initial polarizing signal in amphibians and echinoderms. Further studies will be required to determine the extent to which mechanisms that control beta-catenin nuclearization in early embryogenesis have been conserved during animal evolution.</p

Lessons from a gene regulatory network: echinoderm skeletogenesis provides insights into evolution, plasticity and morphogenesis.

Significant new insights have emerged from the analysis of a gene regulatory network (GRN) that underlies the development of the endoskeleton of the sea urchin embryo. Comparative studies have revealed ways in which this GRN has been modified (and conserved) during echinoderm evolution, and point to mechanisms associated with the evolution of a new cell lineage. The skeletogenic GRN has also recently been used to study the long-standing problem of developmental plasticity. Other recent findings have linked this transcriptional GRN to morphoregulatory proteins that control skeletal anatomy. These new studies highlight powerful new ways in which GRNs can be used to dissect development and the evolution of morphogenesis.</p

Horizontal transfer of the msp130 gene supported the evolution of metazoan biomineralization.

<p>It is widely accepted that biomineralized structures appeared independently in many metazoan clades during the Cambrian. How this occurred, and whether it involved the parallel co-option of a common set of biochemical and developmental pathways (i.e., a shared biomineralization "toolkit"), are questions that remain unanswered. Here, I provide evidence that horizontal gene transfer supported the evolution of biomineralization in some metazoans. I show that Msp130 proteins, first described as proteins expressed selectively by the biomineral-forming primary mesenchyme cells of the sea urchin embryo, have a much wider taxonomic distribution than was previously appreciated. Msp130 proteins are present in several invertebrate deuterostomes and in one protostome clade (molluscs). Surprisingly, closely related proteins are also present in many bacteria and several algae, and I propose that msp130 genes were introduced into metazoan lineages via multiple, independent horizontal gene transfer events. Phylogenetic analysis shows that the introduction of an ancestral msp130 gene occurred in the sea urchin lineage more than 250 million years ago and that msp130 genes underwent independent, parallel duplications in each of the metazoan phyla in which these genes are found.</p

Encoding anatomy: developmental gene regulatory networks and morphogenesis.

<p>A central challenge of developmental and evolutionary biology is to explain how anatomy is encoded in the genome. Anatomy emerges progressively during embryonic development, as a consequence of morphogenetic processes. The specialized properties of embryonic cells and tissues that drive morphogenesis, like other specialized properties of cells, arise as a consequence of differential gene expression. Recently, gene regulatory networks (GRNs) have proven to be powerful conceptual and experimental tools for analyzing the genetic control and evolution of developmental processes. A major current goal is to link these transcriptional networks directly to morphogenetic processes. This review highlights three experimental models (sea urchin skeletogenesis, ascidian notochord morphogenesis, and the formation of somatic muscles in Drosophila) that are currently being used to analyze the genetic control of anatomy by integrating information of several important kinds: (1) morphogenetic mechanisms at the molecular, cellular and tissue levels that are responsible for shaping a specific anatomical feature, (2) the underlying GRN circuitry deployed in the relevant cells, and (3) modifications to gene regulatory circuitry that have accompanied evolutionary changes in the anatomical feature.</p

The evolution of a new cell type was associated with competition for a signaling ligand.

There is presently a very limited understanding of the mechanisms that underlie the evolution of new cell types. The skeleton-forming primary mesenchyme cells (PMCs) of euechinoid sea urchins, derived from the micromeres of the 16-cell embryo, are an example of a recently evolved cell type. All adult echinoderms have a calcite-based endoskeleton, a synapomorphy of the Ambulacraria. Only euechinoids have a micromere-PMC lineage, however, which evolved through the co-option of the adult skeletogenic program into the embryo. During normal development, PMCs alone secrete the embryonic skeleton. Other mesoderm cells, known as blastocoelar cells (BCs), have the potential to produce a skeleton, but a PMC-derived signal ordinarily prevents these cells from expressing a skeletogenic fate and directs them into an alternative developmental pathway. Recently, it was shown that vascular endothelial growth factor (VEGF) signaling plays an important role in PMC differentiation and is part of a conserved program of skeletogenesis among echinoderms. Here, we report that VEGF signaling, acting through ectoderm-derived VEGF3 and its cognate receptor, VEGF receptor (VEGFR)-10-Ig, is also essential for the deployment of the skeletogenic program in BCs. This VEGF-dependent program includes the activation of aristaless-like homeobox 1 (alx1), a conserved transcriptional regulator of skeletogenic specification across echinoderms and an example of a "terminal selector" gene that controls cell identity. We show that PMCs control BC fate by sequestering VEGF3, thereby preventing activation of alx1 and the downstream skeletogenic network in BCs. Our findings provide an example of the regulation of early embryonic cell fates by direct competition for a secreted signaling ligand, a developmental mechanism that has not been widely recognized. Moreover, they reveal that a novel cell type evolved by outcompeting other embryonic cell lineages for an essential signaling ligand that regulates the expression of a gene controlling cell identity

P16 is an essential regulator of skeletogenesis in the sea urchin embryo

AbstractThe primary mesenchyme cells (PMCs) of the sea urchin embryo undergo a dramatic sequence of morphogenetic behaviors that culminates in the formation of the larval endoskeleton. Recent studies have identified components of a gene regulatory network that underlies PMC specification and differentiation. In previous work, we identified novel gene products expressed specifically by PMCs (Illies, M.R., Peeler, M.T., Dechtiaruk, A.M., Ettensohn, C.A., 2002. Identification and developmental expression of new biomineralization proteins in the sea urchin, Strongylocentrotus purpuratus. Dev. Genes Evol. 212, 419–431). Here, we show that one of these gene products, P16, plays an essential role in skeletogenesis. P16 is not required for PMC specification, ingression, migration, or fusion, but is essential for skeletal rod elongation. We have compared the predicted sequences of P16 from two species and show that this small, acidic protein is highly conserved in both structure and function. The predicted amino acid sequence of P16 and the subcellular localization of a GFP-tagged form of the protein suggest that P16 is enriched in the plasma membrane. It may function to receive signals required for skeletogenesis or may play a more direct role in the deposition of biomineral. Finally, we place P16 downstream of Alx1 in the PMC gene network, thereby linking the network to a specific “effector” protein involved in biomineralization

Mesenchymal cell fusion in the sea urchin embryo.

Mesenchymal cells of the sea urchin embryo provide a valuable experimental model for the analysis of cell-cell fusion in vivo. The unsurpassed optical transparency of the sea urchin embryo facilitates analysis of cell fusion in vivo using fluorescent markers and time-lapse three-dimensional imaging. Two populations of mesodermal cells engage in homotypic cell-cell fusion during gastrulation: primary mesenchyme cells and blastocoelar cells. In this chapter, we describe methods for studying the dynamics of cell fusion in living embryos. These methods have been used to analyze the fusion of primary mesenchyme cells and are also applicable to blastocoelar cell fusion. Although the molecular basis of cell fusion in the sea urchin has not been investigated, tools have recently become available that highlight the potential of this experimental model for integrating dynamic morphogenetic behaviors with underlying molecular mechanisms.</p