The evolution of a new cell type was associated with competition for a signaling ligand.

There is presently a very limited understanding of the mechanisms that underlie the evolution of new cell types. The skeleton-forming primary mesenchyme cells (PMCs) of euechinoid sea urchins, derived from the micromeres of the 16-cell embryo, are an example of a recently evolved cell type. All adult echinoderms have a calcite-based endoskeleton, a synapomorphy of the Ambulacraria. Only euechinoids have a micromere-PMC lineage, however, which evolved through the co-option of the adult skeletogenic program into the embryo. During normal development, PMCs alone secrete the embryonic skeleton. Other mesoderm cells, known as blastocoelar cells (BCs), have the potential to produce a skeleton, but a PMC-derived signal ordinarily prevents these cells from expressing a skeletogenic fate and directs them into an alternative developmental pathway. Recently, it was shown that vascular endothelial growth factor (VEGF) signaling plays an important role in PMC differentiation and is part of a conserved program of skeletogenesis among echinoderms. Here, we report that VEGF signaling, acting through ectoderm-derived VEGF3 and its cognate receptor, VEGF receptor (VEGFR)-10-Ig, is also essential for the deployment of the skeletogenic program in BCs. This VEGF-dependent program includes the activation of aristaless-like homeobox 1 (alx1), a conserved transcriptional regulator of skeletogenic specification across echinoderms and an example of a "terminal selector" gene that controls cell identity. We show that PMCs control BC fate by sequestering VEGF3, thereby preventing activation of alx1 and the downstream skeletogenic network in BCs. Our findings provide an example of the regulation of early embryonic cell fates by direct competition for a secreted signaling ligand, a developmental mechanism that has not been widely recognized. Moreover, they reveal that a novel cell type evolved by outcompeting other embryonic cell lineages for an essential signaling ligand that regulates the expression of a gene controlling cell identity.

Cadherin-6B proteolytic N-terminal fragments promote chick cranial neural crest cell delamination by regulating extracellular matrix degradation.

During epithelial-to-mesenchymal transitions (EMTs), chick cranial neural crest cells simultaneously delaminate from the basement membrane and segregate from the epithelia, in part, via multiple protease-mediated mechanisms. Proteolytic processing of Cadherin-6B (Cad6B) in premigratory cranial neural crest cells by metalloproteinases not only disassembles cadherin-based junctions but also generates shed Cad6B ectodomains or N-terminal fragments (NTFs) that may possess additional roles. Here we report that Cad6B NTFs promote delamination by enhancing local extracellular proteolytic activity around neural crest cells undergoing EMT en masse. During EMT, Cad6B NTFs of varying molecular weights are observed, indicating that Cad6B may be cleaved at different sites by A Disintegrin and Metalloproteinases (ADAMs) 10 and 19 as well as by other matrix metalloproteinases (MMPs). To investigate Cad6B NTF function, we first generated NTF constructs that express recombinant NTFs with similar relative mobilities to those NTFs shed in vivo. Overexpression of either long or short Cad6B NTFs in premigratory neural crest cells reduces laminin and fibronectin levels within the basement membrane, which then facilitates precocious neural crest cell delamination. Zymography assays performed with supernatants of neural crest cell explants overexpressing Cad6B long NTFs demonstrate increased MMP2 activity versus controls, suggesting that Cad6B NTFs promote delamination through a mechanism involving MMP2. Interestingly, this increase in MMP2 does not involve up-regulation of MMP2 or its regulators at the transcriptional level but instead may be attributed to a physical interaction between shed Cad6B NTFs and MMP2. Taken together, these results highlight a new function for Cad6B NTFs and provide insight into how cadherins regulate cellular delamination during normal developmental EMTs as well as aberrant EMTs that underlie human disease.

CRISPRz: a database of zebrafish validated sgRNAs.

CRISPRz (http://research.nhgri.nih.gov/CRISPRz/) is a database of CRISPR/Cas9 target sequences that have been experimentally validated in zebrafish. Programmable RNA-guided CRISPR/Cas9 has recently emerged as a simple and efficient genome editing method in various cell types and organisms, including zebrafish. Because the technique is so easy and efficient in zebrafish, the most valuable asset is no longer a mutated fish (which has distribution challenges), but rather a CRISPR/Cas9 target sequence to the gene confirmed to have high mutagenic efficiency. With a highly active CRISPR target, a mutant fish can be quickly replicated in any genetic background anywhere in the world. However, sgRNA's vary widely in their activity and models for predicting target activity are imperfect. Thus, it is very useful to collect in one place validated CRISPR target sequences with their relative mutagenic activities. A researcher could then select a target of interest in the database with an expected activity. Here, we report the development of CRISPRz, a database of validated zebrafish CRISPR target sites collected from published sources, as well as from our own in-house large-scale mutagenesis project. CRISPRz can be searched using multiple inputs such as ZFIN IDs, accession number, UniGene ID, or gene symbols from zebrafish, human and mouse.

Growth factor-mediated mesodermal cell guidance and skeletogenesis during sea urchin gastrulation.

Growth factor signaling pathways provide essential cues to mesoderm cells during gastrulation in many metazoans. Recent studies have implicated the VEGF and FGF pathways in providing guidance and differentiation cues to primary mesenchyme cells (PMCs) during sea urchin gastrulation, although the relative contributions of these pathways and the cell behaviors they regulate are not fully understood. Here, we show that FGF and VEGF ligands are expressed in distinct domains in the embryonic ectoderm of Lytechinus variegatus. We find that PMC guidance is specifically disrupted in Lv-vegf3 morphants and these embryos fail to form skeletal elements. By contrast, PMC migration is unaffected in Lv-fgfa morphants, and well-patterned but shortened skeletal elements form. We use a VEGFR inhibitor, axitinib, to show that VEGF signaling is essential not only for the initial phase of PMC migration (subequatorial ring formation), but also for the second phase (migration towards the animal pole). VEGF signaling is not required, however, for PMC fusion. Inhibition of VEGF signaling after the completion of PMC migration causes significant defects in skeletogenesis, selectively blocking the elongation of skeletal rods that support the larval arms, but not rods that form in the dorsal region of the embryo. Nanostring nCounter analysis of ∼100 genes in the PMC gene regulatory network shows a decrease in the expression of many genes with proven or predicted roles in biomineralization in vegf3 morphants. Our studies lead to a better understanding of the roles played by growth factors in sea urchin gastrulation and skeletogenesis.

Growth factors and early mesoderm morphogenesis: insights from the sea urchin embryo.

The early morphogenesis of the mesoderm is critically important in establishing the body plan of the embryo. Recent research has led to a better understanding of the mechanisms that underlie this process, and growth factor signaling pathways have emerged as key regulators of the directional movements of mesoderm cells during gastrulation. In this review, we undertake a comparative analysis of the various essential functions of growth factor signaling pathways in regulating early mesoderm morphogenesis, with an emphasis on recent advances in the sea urchin embryo. We focus on the roles of the vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) pathways in the migration of primary mesenchyme cells and the formation of the embryonic endoskeleton. We compare the functions of VEGF and FGF in sea urchins with the roles that these and other growth factors play in regulating mesoderm migration during gastrulation in Drosophila and vertebrates.

P58-A and P58-B: novel proteins that mediate skeletogenesis in the sea urchin embryo.

During sea urchin embryogenesis, the skeleton is produced by primary mesenchyme cells (PMCs). PMCs undergo a sequence of morphogenetic behaviors that includes ingression, directed migration, and cell-cell fusion. Ultimately, PMCs deposit the calcite-containing biomineral that forms the endoskeleton of the late embryo and early larva. The endoskeleton has a stereotypical structure and is the major determinant of the distinctive, angular shape of the larva. Although many candidate biomineralization proteins have been identified, functional data concerning these proteins are scant. Here, we identify and characterize two new biomineralization genes, p58-a and p58-b. We show that these two genes are highly conserved in Strongylocentrotus purpuratus and Lytechinus variegatus, two sea urchin species whose ancestors diverged approximately 100 mya. The p58-a and p58-b genes lie in tandem on the chromosome, suggesting that one of the two genes arose via a gene duplication event. The two genes encode closely related, type I transmembrane proteins. We have established by whole mount in situ hybridization that p58-a and p58-b are expressed specifically in the PMCs in both species. Knockdown of either gene by morpholino antisense oligonucleotides leads to profound defects in skeletogenesis, although skeletal elements are not completely eliminated. The P58-A and P58-B proteins do not appear to play a role in the specification, directed migration or differentiation of the PMCs, but most likely are directly involved in biomineralization during sea urchin embryonic development.