Embryonic development is coordinated by networks of evolutionary conserved regulatory genes encoding transcription factors and components of cell signalling pathways. In the sea urchin embryo, a number of genes encoding transcription factors display territorial restricted expression. Among these, the zygotic Hbox12 homeobox gene is transiently transcribed in a limited number of cells of the animal-lateral half of the early Paracentrotus lividus embryo, whose descendants will constitute part of the ectoderm territory. To obtain insights on the regulation of Hbox12 expression, we have explored the cis-regulatory apparatus of the gene. In this paper, we show that the intergenic region of the tandem Hbox12 repeats drives GFP expression in the presumptive aboral ectoderm and that a 234 bp fragment, defined aboral ectoderm (AE) module, accounts for the restricted expression of the transgene. Within this module, a consensus sequence for a Sox factor and the binding of the Otx activator are both required for correct Hbox12 gene expression. Spatial restriction to the aboral ectoderm is achieved by a combination of different repressive sequence elements. Negative sequence elements necessary for repression in the endomesoderm map within the most upstream 60 bp region and nearby the Sox binding site. Strikingly, a Myb-like consensus is necessary for repression in the oral ectoderm, while down-regulation at the gastrula stage depends on a GA-rich region. These results suggest a role for Hbox12 in aboral ectoderm specification and represent our first attempt in the identification of the gene regulatory circuits involved in this process.

The sea urchin early histone repeating unit contains one copy of each of the five histone genes whose coordinate expression during development is regulated by gene-specific elements. To learn how within the histone repeating unit a gene-specific activator can be prevented to communicate with the heterologous promoters, we searched for domain boundaries by using the enhancer blocking assay. We focused on the region near the 3′ end of the H2A gene where stage-specific nuclease cleavage sites appear upon silencing of the early histone genes. We demonstrated that a DNA fragment of 265 bp in length, defined as sns (for silencing nucleoprotein structure), blocked the enhancer activity of the H2A modulator in microinjected sea urchin embryos only when placed between the enhancer elements and the promoter. We also found that sns silenced the modulator elements even when placed at 2.7 kb from the promoter. By contrast, the enhancer activity of the modulator sequences, located downstream to the coding region, was not affected when sns was positioned in close proximity to the promoter. Finally, the H2A sns fragment placed between the simian virus 40 regulative region and the tk promoter repressed chloramphenicol acetyltransferase expression in transfected human cell lines. We conclude that 3′ end of the H2A gene contains sequence elements that behave as functional barriers of enhancer function in the enhancer blocking assay. Furthermore, our results also indicate that the enhancer blocking function of sns lacks enhancer and species specificity and that it can act in transient assays.

In the sea urchin embryo skeletogenesis is the result of a complex series of molecular and cellular events that coordinate the morphogenetic process. Past and recent evidence strongly indicate that skeletal initiation and growth are strictly dependent on signals emanating from the oral ectodermal wall. As previously suggested, Orthopedia (Otp), a homeodomain-containing transcription factor specifically expressed in a small subset of oral ectoderm cells, might be implicated in this signalling pathway. In this study, we utilize three different strategies to address the issue of whether Otp is an upstream regulator of sketelogenesis. We describe the effects of microinjection of Otp morpholino-substituted antisense oligonucleotides and dominant-negative Otp-engrailed mRNA in Paracentrotus lividus embryos. We demonstrate that inhibition of Otp expression completely abolishes skeletal synthesis. By contrast, coinjection of Otp mRNA and the morpholino antisense oligonucleotide specifically rescues the skeletogenic program. In addition, localized ectodermal expression of the Otp-GFP fusion gene construct driven by the hatching enzyme promoter, induces ectopic and abnormal spiculogenesis. We further show that an indirect target of this homeoprotein is the skeletogenic specific gene SM30, whose expression is known to be under the strict control of the oral ectoderm territory. Based on these results, we conclude that Otp triggers the ectoderm-specific signal that promotes skeletogenesis.

In the sea urchin embryo, the lineage founder cells whose polyclonal progenies will give rise to five different territories are segregated at the sixth division. To investigate the mechanisms by which the fates of embryonic cells are first established, we looked for temporal and spatial expression of homeobox genes in the very early cleavage embryos. We report evidence that PlHboxl2, a paired homeobox-containing gene, is expressed in the embryo from the 4-cell stage. The abundance of the transcripts reaches its maximum when the embryo has been divided into the five polyclonal territoriesnamely at the 64-cell stage-and it abruptly declines at later stages of development. Blastomere dissociation experiments indicate that maximal expression ofPlHboxl2 is dependent on intercellular interactions, thus suggesting that signal transduction mechanisms are responsible for its transcriptional activation in the early cleavage embryo. Spatial expression of PlHboxl2 was determined by whole-mount in situ hybridization. PlHboxl2 transcripts in embryos at the fourth, fifth, and sixth divisions seem to be restricted to the conditionally specified ectodermal lineages. These results suggest a possible role of the PlHboxl2 gene in the early events of cell specification of the presumptive ectodermal territories.In metazoan organisms, commitment of cells to a particular fate or set of fates takes place by three known modes. Syncitial specification is the mechanism used by Drosophila and most insect embryos. Blastomere specification is largely conditional in most invertebrate embryos, and conditional specification is also the major mechanism operating after cellularization in Drosophila. Finally, in most invertebrate embryos the fates of some blastomeres are mostly determined by autonomous specification processes (1, 2). In the sea urchin embryo, specification of cell fates is both cell autonomous and conditional. Only the four micromeres that arise at the vegetal pole at the fourth division appear to be autonomously specified (3). If removed from the embryo and cultured, the micromeres will in fact differentiate in skeletogenic mesenchyme cells, form spicules, and express the cell-lineage marker genes (4-7).Founder cells that are conditionally specified constitute a large fraction of the sea urchin embryo (3). Lithium and phorbol 12-myristate 13-acetate, which are known to affect the inositol phosphate and the protein kinase C second messenger pathways (8-10), respectively, alter cell fate during development. Therefore, signal transduction mechanisms, activated by ligand-receptor interactions, are most probably involved in the specification of adjacent blastomeres. Initial specification of founder cells ends at the sixth cleavage. After segregation of the lineages, the sea urchin embryo at the 64-cell stage can be divided into five polyclonal territories that will differentiate into various structures of the pluteus (11,12).The molecular details of blastomere specification in the sea urchin remain to be elucidated. To clarify ...

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Field burning of post‐harvest residue has been an established management practice for economical production of Kentucky bluegrass (Poa pratensis L.) seed. Fields not burned soon after harvest usually have poor seed production in the following year. Residue remaining on fields shades, and thereby restricts tiller growth and subsequent seed yield. To explore the hypothesis, polyethylene shade screens which excluded 30 and 67% of sunlight were placed over September field‐burned ‘Baron’ Kentucky bluegrass plants for 75 and 130 days. Other treatments included mechanical vacuum clipping at 2.5, 7.6, and 15.2 cm levels, field burning of residue and no residue removal. The grass was seeded on Thatuna and Naff silt loam series (fine‐silty, mixed, mesic Xeric Argialbolls and fine‐silty, mixed, mesic Ultic Argixerolls, respectively). Tiller numbers from plants where residue was removed to the 2.5, 7.6, and 15.2 cm levels in 1978‐1979 were comparable to tiller numbers from plants shaded at the 30% level. Tiller numbers for plants shaded at 67% were comparable to tiller numbers where residue was removed at 15.2 cm, or where no residue was removed. Leaf and sheath length were generally inversely related to the level of residue removal. Panicle numbers for shading at 67% in 1979‐1980 were comparable to residue removal at 15.2 cm and no removal of residue. Seed yields from artificially shaded plants in 1978‐1979 were 51 to 55% less than yields from plants where residue was not removed. In 1979‐1980, seed yields from shaded plants at the 67% light exclusion for 130 days were 76% of yields from plants of open‐field burn but did not differ from yields from plants where residue was not removed or mechanically clipped to heights of 7.6 or 15.2 cm. It was concluded that reduced Light penetration into the canopy could change plant growth and reduce seed production potential.

Two homeobox-containing genes that belong to different homeodomain classes have been isolated from a sea urchin genomic library. One, PlHbox11, is the sea urchin homologue of the human and mouse Hox B3 gene, the other, PlHbox12, shows about 55% identity with paired class genes. Expression profile analysis of the two sea urchin Hbox genes suggests that they play different roles during embryogenesis. In fact, PlHbox11 transcripts are rare and are detected only in the pluteus larva and in the Aristotle's lantern and intestine of the adult. The PlHbox12 gene is, on the contrary, transiently expressed in the very early embryo already at the four cell stage; it accumulates at the 64 cell stage and disappears at later stages of development. In situ hybridization experiments to 16 and 32 cell stage whole mount embryos showed localization of the PlHbox12 mRNA to part of the mesomere-macromere region of the early cleavage embryo. These observations suggest a possible role of this gene in early events of cell specification.

Most of the current knowledge concerning fundamental genetic mechanisms, evolutionary processes and development, cellular physiology, and pathogenesis comes from studies of different animal model systems. Whereas mice, rats, and other small mammals are generally thought of as the typical model systems used by researchers in biomedical studies, aquatic models including both freshwater and marine organisms have long proved to be essential for the study of basic biological processes. For over a century, biologists have used the sea urchin embryo as a prototype for the investigation of developmental mechanisms that contribute to building the embryo body plan. Here we highlight the contribution of the sea urchin embryo as a simple model for studying aging and age-associated neurodegenerative diseases, as well as the pathways and mechanisms involved in cell survival and death. Moreover, we point out the role of this embryonic system as a potent and affordable tool for learning about developmental effects and toxicity responses to environmental contaminants and chemical compounds.