Molecular Patterning along the Sea Urchin Animal-Vegetal Axis

The molecular regulatory mechanisms underlying primary axis formation during sea urchin development have recently been identified. Two opposing maternally inherited systems, one animalizing and one vegetalizing, set up the animal-vegetal (A–V) axis. The vegetal system relies in part on the Wnt–β-catenin-Tcf/Lef signaling pathway and the animal system is based on a cohort of animalizing transcription factors that includes members of the Ets and Sox classes. The two systems autonomously define three zones of cell-type specification along the A–V axis. The vegetalmost zone gives rise to the skeletogenic mesenchyme lineage; the animalmost zone gives rise to ectoderm; and the zone in which the two systems overlap generates endoderm, secondary mesenchyme, and ectoderm. Patterning along the A–V also depends on cellular interactions involving Wnt, Notch, and BMP signaling. We discuss how these systems impact the formation of the second axis, the oral-aboral axis; how they connect to later developmental events; and how they lead to cell-type-specific gene expression via cis-regulatory networks associated with transcriptional control regions. We also discuss how these systems may confer on the embryo its spectacular regulatory capacity to replace missing parts.

Introduction

A century ago, Boveri, 1901a, Boveri, 1901b recognized and began experimental investigations on the animal–vegetal (A–V) polarity within the sea urchin egg. Like eggs of many animals, sea urchin eggs have a firmly established, maternally imposed A–V polarity. Axes of polarity provide a three-dimensional coordinate system that patterns the embryo. There is much variation in the timing of establishment of different axes within species and between species and also in the mechanisms that underlie polarization (Goldstein and Freeman, 1997). In sea urchins, the ectoderm forms from the animal part of the egg (the animal pole is defined as the point of polar body extrusion), whereas the mesoderm and endoderm are derived from the vegetal part (the vegetal pole is defined as the pole opposite the animal pole). An oral–aboral (O–Ab) axis orthogonal to the A–V axis is specified in the early embryo, but it is not fixed until gastrulation (Cameron et al., 1990, Hardin et al., 1992, Davidson et al., 1998). Many experiments indicate that patterning along the A–V axis depends on localized maternal determinants and cellular interactions, whereas differentiation of oral and aboral ectoderm requires interactions with vegetal cells. Although several speculative models have been advanced, the molecular mechanisms involved in axial patterning of the echinoid embryo have remained obscure until recently. New methods for assessing and modulating gene expression, combined with experimental manipulations of embryos, have provided much exciting new information. Here, we summarize the experimental results that provide our current understanding of the mechanisms of axial polarization and pattern formation in the sea urchin embryo, with an emphasis on important recent advances (Angerer and Angerer, 1999, Angerer and Angerer, 2000, Ettensohn and Sweet, 2000).

We review evidence that indicates that the establishment of the A–V axis involves cell autonomous activation of maternally specified vegetalizing and animalizing transcriptional regulatory factors. During early cleavages, maternally encoded animalizing factors, including orthologs of mammalian SoxB1, SoxB2, and Ets4, become localized exclusively to the nuclei of cells in the animal zone of the embryo destined to become ectodermal cell types. During the same time period, β-catenin becomes localized in nuclei of the cells close to the vegetal pole, cells whose descendents will produce skeletogenic and coelomic mesoderm. These animalizing and vegetalizing transcription systems overlap in nuclei of cells having an intermediate position along the A–V axis that are destined to become mesoderm, endoderm, and ectoderm. In turn, the maternally encoded transcription factors regulate the zygotic transcription of genes having spatially restricted domains of expression along the A–V axis. Some of the responding genes encode transcription factors, including those of the Krox–Krüpple class, that are involved in refining the pattern. Other responding genes encode proteins involved in intercellular signaling that have important roles in patterning the embryo. These include a Wnt8 ortholog that appears to reinforce the role of β-catenin in vegetal patterning, a BMP2/4 extracellular signal that promotes ectoderm differentiation, and a ligand for the Notch receptor that is produced by the vegetalmost blastomeres and is involved in specification of secondary mesenchyme cells, distinguishing them from endoderm.

In the intact sea urchin embryo, there is a strong correspondence between cell lineage and cell fate. Expression of “marker” genes in clones of cells sharing fates is generally activated early in development, implying that specification of fate has occurred. We use specification as defined by Logan and McClay (1999) to indicate processes that result in distinctive cellular identities that predict their fates. A stricter definition of specification requires that clonal expression of marker genes be maintained when the specified embryo fragments are cultured in isolation (Davidson, 1989). This has sometimes been confirmed for fragments of sea urchin embryos, but regulation (in this context, the expression of genes normally restricted to other lineages) is often observed as well. Most urchin embryonic cells remain uncommitted to a fate until late in development, as assayed by cell transplantation experiments. That is, the developmental potentials of most cells remain broader than their fates in the intact embryo until late stages. Micromeres are the prominent exception to this generalization.

The cellular interactions involved in specification of cell fates in the sea urchin can be either evocative or repressive. Genes whose expression in embryos is associated with cellular specification are often temporally activated by general activating transcription factors. Negative spatial regulatory transcription factors then restrict expression to appropriate embryonic territories. The late commitment to a fate by most cells and the robust and counterbalancing regulatory networks involved in patterning the sea urchin embryo account for its remarkable capacity to regulate in response to experimental perturbations that disrupt normal cellular interactions.