SpSoxB1, an early transcriptional regulator of differentiation along the animal-vegetal axis in the sea urchin embryo.

摘要

In the developing sea urchin embryo's animal-vegetal (AV) axis, maternal regulatory activities establish three tissue territories: pre-ectoderm animally, primary mesenchyme vegetally, and mesendoderm in between. SpAN is a metalloprotease whose transcription is activated cell autonomously along the animal-vegetal axis in the more animal two of these territories. SpSoxB1 polyclonal antiserum supershifts a specific complex formed between nuclear extract and an essential sox regulatory motif in the SpAN promoter.; Developmental immunoblots and EMSAs show that SpSoxB1 is present at low levels in eggs and accumulates during cleavage. SpSoxB1 transcripts are uniformly distributed in the egg; likewise, its protein accumulates to high concentrations in all nuclei of 4- and 8-cell embryos. However, at fourth cleavage, micromeres have reduced nuclear SpSoxB1, while high levels persist in macromeres and mesomeres. The vegetal region lacking nuclear SpSoxB1 expands so that, after blastula stage, SpSoxB1 expression retracts to delineate the ectoderm-endoderm border.; Several experiments suggest that SpSoxB1 is an essential architectural regulator of SpAN transcription. Multimerized sox-binding motifs from the SpAN promoter fail to activate transcription from a basal promoter in vivo. Additional in vivo transcription assays with the promoter's CCAAT-binding site brought closer to the SpAN basal promoter, however, produce wild type levels of transcription. Furthermore, in vitro bending assays demonstrate that SpSoxB1 folds DNA at the SpAN promoter sox motif upon binding.; To examine SpSoxB1 function in cell fate specification along the AV axis, full-length SpSoxB1, a putative dominant negative form (SoxB1-HMG), or a repressive form (SoxB1-En) were overexpressed by transcript microinjection. Furthermore, loss of function for SpSoxB1 was generated using an SpSoxB1-specific, morpholine-substituted antisense oligonucleotide. Overexpressed SpSoxB1 results in embryos devoid of vegetally derived structures. Overexpression of SoxB1-HMG produces embryos that contain normal levels of vegetal pole primary mesenchyme cells (PMCs) but that lack differentiated ectoderm and endoderm derivatives. Repressive SpSoxB1 converts almost all cells to PMCs. Interestingly, loss of SpSoxB1 function produced embryos lacking endoderm and having altered orientation of oral and aboral territories. These data support a model in which SpSoxB1 is required for the specification of the endoderm region of the embryo early in development and for ectoderm patterning.