Homeobox-containing gene transiently expressed in a spatially restricted pattern in the early sea urchin embryo.

Abstract

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 ...

Structure, regulation, and function of micro1 in the sea urchin Hemicentrotus pulcherrimus

“…1). PlHbox12 was isolated from a cDNA library of 32/64-cell stage embryos of P. lividus by using an oligonucleotide probe encoding the most conserved region of helix III of the homeodomain (Di Bernardo et al 1995), while pmar1 was cloned from a S. purpuratus library, using PlHbox12 cDNA as a probe (Oliveri et al 2002). Compared with micro1, PlHbox12 and Pmar1 proteins have 11-15 substitutions (75-82% identity) and 5-9 substitutions (85-92% identity) in their homeodomains, respectively.…”

Enhancer blocking activity located near the 3′ end of the sea urchin early H2A histone gene

“…Nucleic acid samples from a pool of 50-70 microinjected embryos, at morula (5 h of development) or early gastrula (20 h of development) stages, were digested with RNase-free DNase I and hybridized with 32 P-labeled antisense CAT RNA transcribed in vitro from the H2A-INSERT-CAT plasmid (28). Hybridization conditions, RNase digestion, and gel fractionation of the RNase-resistant hybrids were as described (30). Transfection was carried out by incubating the human cell lines, HeLa and U-2 OS, with calcium phosphate precipitates containing 20 g of recombinant SV40 CAT plasmids and 2 g of the internal control ␤-galactosidase expression plasmid pON1 (31).…”

Symmetry Breaking and Establishment of Dorsal/Ventral Polarity in the Early Sea Urchin Embryo

“…In particular, in S. purpuratus the DV axis passes through a plane about 45° clockwise from the first cleavage furrow [7], indicating that secondary axis specification is initiated between fertilization and first cleavage, which is consistent with the asymmetric distribution of mitochondria within eggs and early embryos of this species [25]. Classical studies in P. lividus embryos instead demonstrate that DV axis is randomly oriented with respect to the first cleavage plane, and that it is established between the fifth and eighth cleavage [8], which broadly corresponds to the peak of hbox12 transcription [51,53].…”

“…Available evidence indeed indicates that there is no hbox12 orthologue in S. purpuratus, and that the closest homolog of hbox12 in the latter species is pmar1 [51,53], which is instead involved in micromere lineage specification [86]. This strongly suggests that DV axis specification in S. purpuratus cannot depend on hbox12, since the gene probably does not exist in that species.…”

“…In this regard, we have shown in P. lividus that the Hbox12 homeodomain repressor is expressed by prospective dorsal ectoderm cells, spatially facing and preceding the onset of nodal transcription, where it acts preventing the ectopic activation of nodal expression [51][52][53][54]. Functional analysis revealed that expression of hbox12 and nodal genes are mutually exclusive and both required for DV polarization.…”