SpKrl: An effector of the vegetal organizer that limits the range of SpAN protease expression in the Strongylocentrotus purpuratus embryo

Localization of nuclear beta-catenin initiates specification of vegetal fates in sea urchin embryos. I have identified SpKrl, a gene that is activated simultaneously with nuclear entry of beta-catenin. SpKrl is upregulated when nuclear beta-catenin activity is increased with LiCl. This effect is independent of protein synthesis, strongly suggesting that SpKrl is a direct target of beta-catenin and TCF. Embryos in which SpKrl translation is inhibited with morpholino antisense oligonucleotides lack endoderm. Conversely, injection of SpKrl mRNA rescues some vegetal structures in beta-catenin-deficient embryos. I show that SpKrl is a negative regulator that can repress the expression of the animalizing transcription factor, SpSoxB1. I propose that SpKrl functions in patterning the vegetal domain by suppressing animal regulatory activities.; SpSoxB1 is an important positive regulator of SpAN transcription. SpAN is expressed transiently in sea urchin animal blastomeres and encodes a member of a family of secreted, tethered proteases. Using two different polyclonal antibodies raised against non-overlapping regions of SpAN, I demonstrate that SpAN is present in the apical extracellular matrix, from which it can be isolated in active form. SpAN accumulation in the apical extracellular matrix begins at 10 h postfertilization and persists through mesenchyme blastula stage. A major component of the extracellular matrix is hyalin which, like SpAN, contains EGF-like and complement C1r/C1s protein interaction domains. SpAN can cleave hyalin in vivo within the repeat region of the protein. Since hyalin has been shown by antibody interference experiments to be required for the morphogenetic movements of gastrulation, I tested the idea that SpAN function may also be required for this process. A loss-of-function assay was developed by injecting into sea urchin eggs an mRNA encoding a single chain synthetic anti-SpAN antibody fragment (scFv-SpAN). In embryos expressing scFv-SpAN, the hyaline layer became swollen and detached from the embryos. I propose that SpAN activity is required for cells to establish appropriate interactions with the extracellular matrix that support morphogenesis.