Primary differentiation and ectoderm-specific gene expression in the animalized sea urchin embryo

Primary differentiation in sea urchin embryos, animalized by zinc, has been gauged by the formation of characteristic endodermal and mesodermal tissue derivatives and by the accumulation of the ectoderm-specific Spec 1 mRNA. Increasing the dosage of zinc diminishes the differentiation of secondary mesenchyme, primary mesenchyme, endoderm, and ectoderm, in decreasing order. Treatment is effective only during the blastula stages, involving successive periods of sensitivity for these tissues. Removal of zinc with chelator results in the resumption of differentiation to increasing degree for this series of tissues. The developmental initiation of Spec 1 gene expression, normally at the earliest blastula stage, can be delayed by zinc for at least 30 hr before being implemented by treatment of the animalized embryos with a chelator. We conclude (1) that those processes in the blastula which are required for differentiation and are suppressed by zinc are distinguishable from the determinative processes, which are not affected by the animalizing agent and occur earlier during midcleavage; (2) that animalization by zinc involves a graded failure of primary tissues to form; and (3) that animalization involves a pause in the schedule of differentiation, which can be reinstated by removal of the animalizing agent, thereby providing a survival value inherent in a flexible schedule of development.     

Mosaic incorporation and regulated expression of an exogenous gene in the sea urchin embryo

A fusion gene construct in which the bacterial chloramphenicol acetyltransferase (CAT) gene is controlled by CyIIIa actin gene cis-regulatory sequences was injected into unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. The distribution of CAT DNA sequences was measured directly by in situ hybridization in squashed 24-hr blastula preparations derived from these eggs. Earlier studies had shown that stable mosaic incorporation of the exogenous DNA occurs during cleavage, after which the exogenous sequences replicate at approximately the pace of the host cell genomes. The fractions of embryonic cells observed in this study to include CAT DNA sequences imply that their stable incorporation into a replicating nuclear form occurs most often in a single cell at the 3rd or 4th cleavage stages, though it may occur as early as 2nd cleavage, or as late as 7th cleavage. Corroborative measurements were carried out by the same method on squashed preparations of embryos at earlier stages, and by in situ hybridizations of CAT mRNA, both in dissociated embryos and in cytological sections of 72-hr pluteus-stage embryos. Hybridizations to CAT mRNA and to CAT DNA were carried out on alternate sections of several embryos. The results confirm unequivocally that although CAT mRNA appears only in the aboral ectoderm in embryos derived from eggs injected with the CyIIIa.CAT fusion gene, the exogenous sequences are indeed present, though silent, in the various other cell types of the late embryo.     

Spatial patterns of metallothionein mRNA expression in the sea urchin embryo

Metallothioneins (MTs) are small, cysteine-rich proteins that bind heavy metals which induce their synthesis. Tissue fractionation of embryos at pluteus stage previously demonstrated that in the absence of added zinc, basal expression of MT mRNA is confined to ectoderm, whereas induction by zinc results in increased expression in the endoderm + mesoderm tissue fraction. Using in situ hybridization we now show that expression in the pluteus larva is restricted almost exclusively to the single cell type comprising the aboral ectoderm. Induction by Zn results in a marked accumulation of MT mRNA in gut and oral ectoderm to levels at least as high as that in aboral ectoderm. MT mRNA is also expressed in presumptive aboral ectoderm at earlier stages of normal development. In addition it is transiently expressed at variable levels in oral ectoderm and, to a lesser extent, in presumptive gut.     

Modularity and design principles in the sea urchin embryo gene regulatory network

The gene regulatory network (GRN) established experimentally for the pre-gastrular sea urchin embryo provides causal explanations of the biological functions required for spatial specification of embryonic regulatory states. Here we focus on the structure of the GRN which controls the progressive increase in complexity of territorial regulatory states during embryogenesis; and on the types of modular subcircuits of which the GRN is composed. Each of these subcircuit topologies executes a particular operation of spatial information processing. The GRN architecture reflects the particular mode of embryogenesis represented by sea urchin development. Network structure not only specifies the linkages constituting the genomic regulatory code for development, but also indicates the various regulatory requirements of regional developmental processes.     

R11: a cis-regulatory node of the sea urchin embryo gene network that controls early expression of SpDelta in micromeres

A gene regulatory network (GRN) controls the process by which the endomesoderm of the sea urchin embryo is specified. In this GRN, the program of gene expression unique to the skeletogenic micromere lineage is set in train by activation of the pmar1 gene. Through a double repression system, this gene is responsible for localization of expression of downstream regulatory and signaling genes to cells of this lineage. One of these genes, delta, encodes a Notch ligand, and its expression in the right place and time is crucial to the specification of the endomesoderm. Here we report a cis-regulatory element R11 that is responsible for localizing the expression of delta by means of its response to the pmar1 repression system. R11 was identified as an evolutionarily conserved genomic sequence located about 13 kb downstream of the last exon of the delta gene. We demonstrate here that this cis-regulatory element is able to drive the expression of a reporter gene in the same cells and at the same time that the endogenous delta gene is expressed, and that temporally, spatially, and quantitatively it responds to the pmar1 repression system just as predicted for the delta gene in the endomesoderm GRN. This work illustrates the application of cis-regulatory analysis to the validation of predictions of the GRN model. In addition, we introduce new methodological tools for quantitative measurement of the output of expression constructs that promise to be of general value for cis-regulatory analysis in sea urchin embryos.     

Determination and morphogenesis in the sea urchin embryo

The study of the sea urchin embryo has contributed importantly to our ideas about embryogenesis. This essay re-examines some issues where the concerns of classical experimental embryology and cell and molecular biology converge. The sea urchin egg has an inherent animal-vegetal polarity. An egg fragment that contains both animal and vegetal material will produce a fairly normal larva. However, it is not clear to what extent the oral-aboral axis is specified in embryos developing from meridional fragments. Newly available markers of the oral-aboral axis allow this issue to be settled. When equatorial halves, in which animal and vegetal hemispheres are separated, are allowed to develop, the animal half forms a ciliated hollow ball. The vegetal half, however, often forms a complete embryo. This result is not in accord with the double gradient model of animal and vegetal characteristics that has been used to interpret almost all defect, isolation and transplantation experiments using sea urchin embryos. The effects of agents used to animalize and vegetalize embryos are also due for re-examination. The classical animalizing agent, Zn2+, causes developmental arrest, not expression of animal characters. On the other hand, Li+, a vegetalizing agent, probably changes the determination of animal cells. The stability of these early determinative steps may be examined in dissociation-reaggregation experiments, but this technique has not been exploited extensively. The morphogenetic movements of primary mesenchyme are complex and involve a number of interactions. It is curious that primary mesenchyme is dispensable in skeleton formation since in embryos devoid of primary mesenchyme, the secondary mesenchyme cells will form skeletal elements. It is likely that during its differentiation the primary mesenchyme provides some of its own extracellular microenvironment in the form of collagen and proteoglycans. The detailed form of spicules made by primary mesenchyme is determined by cooperation between the epithelial body wall, the extracellular material and the inherent properties of primary mesenchyme cells. Gastrulation in sea urchins is a two-step process. The first invagination is a buckling, the mechanism of which is not understood. The secondary phase in which the archenteron elongates across the blastocoel is probably driven primarily by active cell repacking. The extracellular matrix is important for this repacking to occur, but the basis of the cellular-environmental interaction is not understood.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fibronectin in the developing sea urchin embryo

The presence of fibronectin in developing sea urchin embryos was studied uing immunofluorescence staining. The fluorescence pattern indicates that fibronectin is found on the cell surfaces and between cells in the blastula and gastrula stages, indicating that it plays a role in cell adhesion. Its presence on invaginating cells also suggests its involvement in morphogenesis during early development.     

Matrix metalloproteases of the developing sea urchin embryo

Abstract. A distinct group of metalloproteases has been identified in the developing sea urchin embryo by gelatin substrate gel zymography, a highly sensitive protease detection assay. The developing Arbacia embryo exhibited four prominent bands of gelatinase activity with apparent molecular masses of 55, 50, 42 and 38 kDa. The activity of the 55, 42 and 38 kDa tissue gelatinases increased and that of the 50 kDa tissue gelatinase decreased during embryonic development. All four enzymes were EDTA- and 1,10-phenanthroline sensitive and phenyl methyl sulphonyl fluoride (PMSF) insensitive. None of the enzymes had detectable caseinolytic activity in casein substrate gels. Although the Arbacia enzymes possessed a number of properties that are characteristic of the mammalian matrix metalloprotease family, they did not appear to be converted to lower molecular weight forms by organomercurial treatment and are distinct in this aspect. The Arbacia metalloproteases are candidate enzymes for the tissue and matrix remodeling that occurs during sea urchin embryo development.     

Matrix metalloproteases of the developing sea urchin embryo

Abstract. A distinct group of metalloproteases has been identified in the developing sea urchin embryo by gelatin substrate gel zymography, a highly sensitive protease detection assay. The developing Arbacia embryo exhibited four prominent bands of gelatinase activity with apparent molecular masses of 55, 50, 42 and 38 kDa. The activity of the 55, 42 and 38 kDa tissue gelatinases increased and that of the 50 kDa tissue gelatinase decreased during embryonic development. All four enzymes were EDTA- and 1,10-phenanthroline sensitive and phenyl methyl sulphonyl fluoride (PMSF) insensitive. None of the enzymes had detectable caseinolytic activity in casein substrate gels. Although the Arbacia enzymes possessed a number of properties that are characteristic of the mammalian matrix metalloprotease family, they did not appear to be converted to lower molecular weight forms by organomercurial treatment and are distinct in this aspect. The Arbacia metalloproteases are candidate enzymes for the tissue and matrix remodeling that occurs during sea urchin embryo development.