Alx1, a member of the Cart1/Alx3/Alx4 subfamily of Paired-class homeodomain proteins, is an essential component of the gene network controlling skeletogenic fate specification in the sea urchin embryo

In the sea urchin embryo, the large micromeres and their progeny function as a critical signaling center and execute a complex morphogenetic program. We have identified a new and essential component of the gene network that controls large micromere specification, the homeodomain protein Alx1. Alx1 is expressed exclusively by cells of the large micromere lineage beginning in the first interphase after the large micromeres are born. Morpholino studies demonstrate that Alx1 is essential at an early stage of specification and controls downstream genes required for epithelial-mesenchymal transition and biomineralization. Expression of Alx1 is cell autonomous and regulated maternally through ß-catenin and its downstream effector, Pmar1. Alx1 expression can be activated in other cell lineages at much later stages of development, however, through a regulative pathway of skeletogenesis that is responsive to cell signaling. The Alx1 protein is highly conserved among euechinoid sea urchins and is closely related to the Cart1/Alx3/Alx4 family of vertebrate homeodomain proteins. In vertebrates, these proteins regulate the formation of skeletal elements of the limbs, face and neck. Our findings suggest that the ancestral deuterostome had a population of biomineral-forming mesenchyme cells that expressed an Alx1-like protein

Identification and developmental expression of new biomineralization proteins in the sea urchin Strongylocentrotus purpuratus.

The endoskeleton of the sea urchin larva is a network of calcareous rods secreted by primary mesenchyme cells (PMCs). In this study, we identified seven new biomineralization-related proteins through an analysis of a large database of gene products expressed by PMCs. The proteins include three new spicule matrix proteins (SpSM29, SpSM32, and SpC-lectin), two proteins related to the PMC-specific cell surface glycoprotein MSP130 (MSP130-related-1 and -2), and two novel proteins (SpP16 and SpP19). The genes encoding these proteins are expressed specifically by cells of the large micromere-PMC lineage and are activated zygotically beginning at the blastula stage, prior to PMC ingression. Several of the mRNAs show regulated patterns of expression within the PMC syncytium that correlate with the pattern of skeletal rod growth. This work identifies new proteins that may regulate the process of biomineralization in this tractable model system.</p

Differential stability of β-catenin along the animal-vegetal axis of the sea urchin embryo mediated by dishevelled

beta-Catenin has a central role in the early axial patterning of metazoan embryos. In the sea urchin, beta-catenin accumulates in the nuclei of vegetal blastomeres and controls endomesoderm specification. Here, we use in-vivo measurements of the half-life of fluorescently tagged beta-catenin in specific blastomeres to demonstrate a gradient in beta-catenin stability along the animal-vegetal axis during early cleavage. This gradient is dependent on GSK3beta-mediated phosphorylation of beta-catenin. Calculations show that the difference in beta-catenin half-life at the animal and vegetal poles of the early embryo is sufficient to produce a difference of more than 100-fold in levels of the protein in less than 2 hours. We show that dishevelled (Dsh), a key signaling protein, is required for the stabilization of beta-catenin in vegetal cells and provide evidence that Dsh undergoes a local activation in the vegetal region of the embryo. Finally, we report that GFP-tagged Dsh is targeted specifically to the vegetal cortex of the fertilized egg. During cleavage, Dsh-GFP is partitioned predominantly into vegetal blastomeres. An extensive mutational analysis of Dsh identifies several regions of the protein that are required for vegetal cortical targeting, including a phospholipid-binding motif near the N-terminus

Differential stability of beta-catenin along the animal-vegetal axis of the sea urchin embryo mediated by dishevelled.

beta-Catenin has a central role in the early axial patterning of metazoan embryos. In the sea urchin, beta-catenin accumulates in the nuclei of vegetal blastomeres and controls endomesoderm specification. Here, we use in-vivo measurements of the half-life of fluorescently tagged beta-catenin in specific blastomeres to demonstrate a gradient in beta-catenin stability along the animal-vegetal axis during early cleavage. This gradient is dependent on GSK3beta-mediated phosphorylation of beta-catenin. Calculations show that the difference in beta-catenin half-life at the animal and vegetal poles of the early embryo is sufficient to produce a difference of more than 100-fold in levels of the protein in less than 2 hours. We show that dishevelled (Dsh), a key signaling protein, is required for the stabilization of beta-catenin in vegetal cells and provide evidence that Dsh undergoes a local activation in the vegetal region of the embryo. Finally, we report that GFP-tagged Dsh is targeted specifically to the vegetal cortex of the fertilized egg. During cleavage, Dsh-GFP is partitioned predominantly into vegetal blastomeres. An extensive mutational analysis of Dsh identifies several regions of the protein that are required for vegetal cortical targeting, including a phospholipid-binding motif near the N-terminus.</p