PerspectiveDevelopment

Deciphering the Underlying Mechanism of Specification and Differentiation: The Sea Urchin Gene Regulatory Network

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Science's STKE  14 Nov 2006:
Vol. 2006, Issue 361, pp. pe47
DOI: 10.1126/stke.3612006pe47

Abstract

The regulatory genome functions as a vast information processor through development. It processes the initial conditions that are set by asymmetric distributions of cellular components in the egg and translates them into the onset of spatially localized specification states. It regulates the timely differential activation of signaling molecules and transcription factors that divide the emerging domains into subdomains. It also governs the activation of groups of differentiation genes, the genes that encode, at the protein level, the functional and the structural properties of a cell type. The sea urchin endomesoderm gene regulatory network provides a window into the different levels of the regulatory apparatus. It demonstrates how the static physical genomic components define functional connections between the various regulatory genes that act together to conduct the dynamical developmental program.

A single cell, the fertilized egg, gives rise to a tremendous diversity of cell types to create the various tissues and organs that eventually form the adult body plan. The DNA can be thought of as both the hardware and the software of this developmental program, and it also serves a memory function to convey the program from one generation to the next. How this information is encoded and how this program is executed are two fundamental questions in biology. A first step toward answering these questions is to understand the mechanisms that control differential gene expression and lead to the formation of the body plan.

The instructions for cell specification and differentiation are primarily encoded in the regulatory regions of the genomic DNA (1, 2). Thus, every gene contains regulatory sequences necessary to control when and where it is expressed (Fig. 1). These sequences, cis-regulatory modules, can be located upstream, downstream, and in the introns of the gene they control. Each cis-regulatory module contains a cluster of binding sites for transcription factors. The modules function as information processors: The input that they "read" is the concentration of different transcription factors in the cell nucleus. The output is the expression level of the gene that they control. Various cis-regulatory modules become active at distinct times and in different locations in the embryo, depending on many elements, such as cell lineage. Similar mechanisms control the expression of transcription factors and cell signaling molecules, the major informational molecules of the cell. Many of these regulatory genes are functionally interconnected through their cis-regulatory modules to form complex structures such as network motifs, network subcircuits, and, eventually, gene regulatory networks.

Fig. 1.

The regulatory hierarchy: The cis-regulatory module of gene A contains binding sites for both gene A and gene B. Gene A contains multiple such modules (pink boxes) that control its expression at different embryonic domains and at different times in development. Gene A is a part of a positive-feedback motif with gene B—gene A activates itself and gene B, and gene B activates gene A. This corresponds to the cis-regulatory module that is depicted above. This network motif is a part of a network subcircuit that is responsible for domain A specification. The subcircuit is a part of a gene regulatory network that describes the interaction of many such subcircuits.

A network motif is a small set of regulatory genes that are connected to each other in typical and well-defined ways (3, 4)—for example, positive-feedback loops (Fig. 1) (5), feedforward loops (6), and autorepressors (7). The functional unit that brings about a complete developmental task is the network subcircuit, a higher level of the regulatory hierarchy (Fig. 1). A network subcircuit is a specific combination of multiple network motifs that gives rise to a dynamic spatial expression pattern. A gene regulatory network comprises multiple subcircuits and controls how different emerging domains interact with each other, how domain boundaries are set, and how differentiation takes place. A thorough understanding of the regulatory apparatus can only be obtained by stepping back and forth in the different levels of the regulatory hierarchy and understanding how one defines the next.

The sea urchin is one of the classic model systems of development, with many characteristics that make it an excellent system for the molecular age. Classic experiments such as blastomere isolation (8) and balstomere transplantation (9) were first conducted in sea urchins about a hundred years ago. However, understanding these experiments at the molecular level became feasible only in the last 15 years owing to the advent of molecular biology and the ability to perform discrete perturbation assays and quantitative expression experiments. These new techniques enable the construction of a map of the gene regulatory network that governs the early endomesoderm specification in sea urchin embryo (Fig. 2) (10). By analyzing the sea urchin gene regulatory network, we can identify some general characteristics of developmental systems.

Fig. 2.

Diagram of the gene regulatory network for pregastrular endomesoderm specification in sea urchins. Time proceeds from top to bottom. The network was initially presented by Davidson et al. (10) and is periodically updated (this snapshot captured 6 October 2006) at http://sugp.caltech.edu/endomes/ where the time course in the different domains and much of the underlying experimental data and a current list of supporting references are maintained. The network is separated into blocks that correspond to the three lineages: the primary mesenchyme cells (PMC), the secondary mesenchyme cells (SMC), and the endoderm. The genes that are active in the progenitor endomesoderm field and their descendants, the SMC and the endoderm, are shown in the light green box. Genes that are active only in the SMC are indicated in light blue boxes, genes that are active only in endoderm cells are indicated in yellow boxes, and genes that are active in both cell lineages are indicated in yellow boxes framed by light blue. The upper part of the diagram is the regulatory network where the nodes are transcription factors and signaling molecules. The lower separate boxes are the batteries of differentiation genes, the proteins which actually execute the differentiation program. The thick lines indicate links that were verified by cis-regulatory analysis. R of mic, repressor of micromeres; GSK-3, glycogen synthase kinase 3; VEGFR, vascular endothelial growth factor receptor; abo, aboral; Ubiq, ubiquitous; Mat, maternal; activ, activator; rep, repressor; unkn, unknown; Nucl., nuclearization; X, β-catenin source; nβ, TCF-nuclearized β-catenin–TCF1 complex; ES, early signal; ECNS, early cytoplasmic nuclearization system; Zyg. N., zygotic Notch; SU(H), suppressor of hairless; NIC, Notch intracellular part; PMC, primary mesenchyme cells; Skel, skeleton; Mes, mesoderm; end, endoderm; Veg1 Endo, Veg1 ring of cells that becomes endoderm.

The earliest cues that initiate localized specification in the sea urchin embryo are asymmetric distributions of proteins, mRNA, and cellular components in the egg, termed "maternal anisotropies" (1). They form the "initial conditions" that define how the specification program progresses differentially in the various emerging domains. These maternal anisotropies are translated by cis-regulatory modules of regulatory genes into the onset of specification states. An example of such maternal anisotropy is the asymmetric distribution of the Dishevelled protein in the vegetal pole of the egg (11).

The Dishevelled protein is a key component of the β-catenin–Wnt8 pathway, and in the sea urchin egg it is localized in the vegetal pole (11). As a result, β-catenin is stabilized initially only in the vegetal-most cells of the embryo, the micromeres, and only in these cells does it enter the nucleus (12, 13). Once in the nucleus, β-catenin binds to the transcription factor TCF (T cell factor) instead of Groucho, a dominant repressor that is globally expressed (Fig. 3A) (14). β-catenin–TCF forms a permissive complex that allows the activation of downstream genes. One of the downstream genes that is activated by the β-catenin–TCF complex is the transcription factor blimp1 (1518). The β-catenin–TCF permissive complex, together with Blimp1, activates the expression of the Wnt8 signaling molecule (Fig. 3A) (16, 19, 20). The Wnt8 signal is received by the neighboring cells and induces further stabilization of β-catenin and therefore causes further activation of blimp1 and wnt8. This community effect is a mechanism to amplify the initial asymmetry and converts it into a new localized domain. Reception of the Wnt8 signal by the next tier of cells activates the same subcircuit there and leads to its expansion toward the animal pole. On the other hand, Blimp1 shuts this subcircuit off in the domains through which it has already passed (15, 18). Blimp1 represses transcription of its own gene and, because its input is required for the expression of wnt8, the wnt8 expression is also stopped (Fig. 3A). The resulting dynamic pattern of nucleus-localized β-catenin and of wnt8 and blimp1 expression forms a ring that moves from the vegetal pole toward the animal pole (18). This subcircuit turns on key regulatory genes in the domains that it passes and eventually controls the specification of the endoderm (5).

Fig. 3.

Prominent subcircuits in the network. (A) Wnt8–β-catenin–blimp1 subcircuit (16, 19, 20). The inactive state (right): In the absence of nuclearized β-catenin, Groucho binds to TCF, and together they form a repressing complex. Active state (left): When β-catenin is stabilized, it enters the nucleus where it binds to TCF and forms an activation complex. This activation complex turns on blimp1, and then both β-catenin–TCF and Blimp1 activate wnt8 expression. Wnt8 binds to the neighboring cells and enhances further stabilization of β-catenin. The subcircuit is turned off by Blimp1, which shuts itself down, leading to a down-regulation of wnt8. (B) Pmar1 subcircuit (21). Maternal Otx and nuclearized β-catenin turn on expression of pmar1 in the micromeres. Pmar1 down-regulates a ubiquitous repressor so that key regulatory genes are turned on. (C) Delta-Notch signaling turns suppressor of hairless [Su(H)] from a repressor to an activator so that gcm, an important pigment cell transcription factor, is turned on. Gcm locks itself on and down-regulates foxa expression in the pigment cells (probably indirectly), to prevent endodermal fate (25, 26). (D) Endoderm specification. A positive-feedback loop is formed between otxβ and gatae, and both turn on key regulatory genes including foxa (5). FoxA represses gcm in the endoderm to exclude pigment cell fate (7).

The β-catenin–wnt8–blimp1 subcircuit is composed of the following motifs: a coherent feed-forward loop, a positive-feedback loop, and an autorepressor. A feed-forward loop is a three-component motif in which one component regulates the other and together the two regulate the third. "Coherent," in this context, means that the direct and the indirect regulation have the same sign (3, 4). In this case, β-catenin–TCF activates blimp1, and together they activate wnt8. A positive-feedback loop is formed between β-catenin–TCF and wnt8: β-catenin–TCF activates expression of the wnt8 gene, and Wnt8 reception enhances nuclear localization of β-catenin. An autorepressor is a transcription factor that represses its own gene, such as Blimp1 (Fig. 3A). Every motif has a specific role in this subcircuit—the feed-forward loop increases the activation fidelity, the positive feedback amplifies the maternal asymmetry and moves the expression pattern forward, and the autorepressor shuts the subcircuit down. The specific combination of these building blocks in this subcircuit accounts for the dynamic expression patterns of these genes and their downstream genes.

The presence of β-catenin in the nucleus of the vegetal plate cells is transformed by another network subcircuit to give rise to the skeletogenic lineage. In the micromere, the vegetal-most cells of the embryo, β-catenin–TCF, together with a ubiquitous activator, Otx, activate the expression of a repressor, pmar1 (Fig. 3B) (21, 22). Pmar1 represses a ubiquitous repressor and permits the expression of many micromere-specific regulatory genes. One of the regulatory genes that Pmar1 activity turns on is the signaling molecule Delta (23, 24). Delta is received by its receptor, Notch, in the neighboring tier of cells (Fig. 3C). As a result, the intracellular part of the Notch receptor enters the cell’s nucleus and turns Suppressor of Hairless [Su(H)] from a repressor into an activator (25). This activates the expression of transcription factors that are necessary for specification of the pigment cell (e.g., gcm; see below) (25, 26).

Global repressors that are repressed or inhibited by local activators are typical for the early stages of development. Once specification states have been established, they are maintained by transcriptional positive-feedback loops and the activation of local repressors that prevent ambiguity of specification (2, 7). There are two examples of positive-feedback loops in the network. The first example is mediated by the transcription factor gcm (glial cells missing), which positively autoregulates itself and induces pigment cell specification (Fig. 3C) (2527). A second example is a positive-feedback loop between the genes otxβ and gatae that control specification and differentiation of the endoderm (5, 28). One of the downstream genes that receives input from Otxβ and GataE is the repressor foxa. FoxA represses gcm in the endoderm, whereas Gcm down-regulates foxa in the pigment cells (7). This mutual exclusion is critical to the correct specification of these two neighboring cell lineages that are born from the same tier of cells.

The sea urchin gene regulatory network reveals the multiple levels of the regulatory apparatus and the way they are interconnected. The physical components that carry the code are the static cis-regulatory modules. They define the functional connections between the regulatory genes and signaling molecules and the complex structure of the different subcircuits. On the basis of these connections and the initial conditions in the egg, a dynamic program takes place: Asymmetries are amplified to set spatial domains, spatial domains are subdivided by responding to different signaling cues, and eventually, the body plan emerges. Thus, we have entered an era in which it is possible to specify the molecular basis of the mechanism that forms a well-organized diversity from a single cell.

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