Browsing by Author "Edgar, Allison"

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    Embryo microinjection of the lecithotrophic sea urchin Heliocidaris erythrogramma.
    (Journal of biological methods, 2019-01) Edgar, Allison; Byrne, Maria; Wray, Gregory A
    Microinjection is a common embryological technique used for many types of experiments, including lineage tracing, manipulating gene expression, or genome editing. Injectable reagents include mRNA overexpression, mis-expression, or dominant-negative experiments to examine a gene of interest, a morpholino antisense oligo to prevent translation of an mRNA or spliceoform of interest and CRISPR-Cas9 reagents. Thus, the technique is broadly useful for basic embryological studies, constructing gene regulatory networks, and directly testing hypotheses about cis-regulatory and coding sequence changes underlying the evolution of development. However, the methods for microinjection in typical planktotrophic marine invertebrates may not work well in the highly modified eggs and embryos of lecithotrophic species. This protocol is optimized for the lecithotrophic sea urchin Heliocidaris erythrogramma.
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    Specification of Mesodermal Lineages Was Altered in the Evolution of the Lecithotrophic Sea Urchin Heliocidaris erythrogramma
    (2019) Edgar, Allison

    A striking feature of sea urchin development is specification of molecularly, behaviorally, and morphologically distinct types of mesoderm prior to gastrulation. The echinoderm gene regulatory network (GRN) for mesoderm specification and differentiation has become a key system for understanding GRN evolution. Its details – including transient early signaling, several temporal layers of sub-circuits for cell fate commitment, and terminal differentiation gene batteries – are known in detail for euechinoid sea urchins and from comparative studies for several other echinoderm groups. From these studies, it appears that GRNs were surprisingly stable over several hundred million years of evolution, particularly early in development.

    The euechinoid urchin Heliocidaris erythrogramma exhibits a highly derived mode of development in which embryogenesis and juvenile metamorphosis co-occur, entailing extensive changes to its axial patterning, gastrulation, and fate map. These changes arose over only ~4 million years since its divergence with planktotrophic urchins. I have performed the first detailed GRN analysis on H. erythrogramma mesoderm. I analyzed gene expression by in situ hybridization and analysis of published whole-transcriptome data. I manipulated embryos by inhibiting cell signaling and mRNA translation using reagents commonly employed in other sea urchins to construct a partial developmental GRN that can be compared to the ancestral planktotrophic sea urchin and to other echinoderm outgroups. I found that H. erythrogramma has lost precocious specification of larval skeletogenic mesoderm and shows extensive changes from planktotrophic euechinoids in transcription factor expression, cell signaling, and cell behavior.

    I found that a key early GRN sub-circuit that specifies larval skeletogenic mesoderm has been lost in H. erythrogramma. Skeletogenic cells arise differently in H. erythrogramma, with skeletogenic markers restricted to the archenteron and coelomic pouches and excluded from ingressed mesenchyme, in sharp contrast to other euechinoids with larval skeletons. Furthermore, the role of early signaling that is coordinated by this cell type has changed extensively. H. erythrogramma has lost a requirement for Delta signaling to induce nonskeletogenic mesenchymal mesoderm. These and other dramatic changes to a deeply conserved network in a short evolutionary time frame suggest strong selective pressure, rather than developmental system drift, altered the GRN. The echinoderm GRN stability over long evolutionary time scales thus suggests stabilizing selection rather than resistance to change arising from network complexity. Knowing which elements of the GRN have changed will permit formal tests for selection at relevant genomic loci and comparisons with the GRNs in independently lecithotrophic urchins to better understand how development evolves.