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The ecologically significant shift in developmental strategy from planktotrophic (feeding) to lecithotrophic (nonfeeding) development in the sea urchin genus Heliocidaris is one of the most comprehensively studied life history transitions in any animal. Although the evolution of lecithotrophy involved substantial changes to larval development and morphology, it is not known to what extent changes in gene expression underlie the developmental differences between species, nor do we understand how these changes evolved within the context of the well-defined gene regulatory network (GRN) underlying sea urchin development. To address these questions, we used RNA-seq to measure expression dynamics across development in three species: the lecithotroph Heliocidaris erythrogramma, the closely related planktotroph H. tuberculata, and an outgroup planktotroph Lytechinus variegatus. Using well-established statistical methods, we developed a novel framework for identifying, quantifying, and polarizing evolutionary changes in gene expression profiles across the transcriptome and within the GRN. We found that major changes in gene expression profiles were more numerous during the evolution of lecithotrophy than during the persistence of planktotrophy, and that genes with derived expression profiles in the lecithotroph displayed specific characteristics as a group that are consistent with the dramatically altered developmental program in this species. Compared to the transcriptome, changes in gene expression profiles within the GRN were even more pronounced in the lecithotroph. We found evidence for conservation and likely divergence of particular GRN regulatory interactions in the lecithotroph, as well as significant changes in the expression of genes with known roles in larval skeletogenesis. We further use coexpression analysis to identify genes of unknown function that may contribute to both conserved and derived developmental traits between species. Collectively, our results indicate that distinct evolutionary processes operate on gene expression during periods of life history conservation and periods of life history divergence, and that this contrast is even more pronounced within the GRN than across the transcriptome as a whole. [ABSTRACT FROM AUTHOR]

Epithelial-mesenchymal transition (EMT) is a fundamental cell state change that transforms epithelial to mesenchymal cells during embryonic development, adult tissue repair and cancer metastasis. EMT includes a complex series of intermediate cell state changes including remodeling of the basement membrane, apical constriction, epithelial de-adhesion, directed motility, loss of apical-basal polarity, and acquisition of mesenchymal adhesion and polarity. Transcriptional regulatory state changes must ultimately coordinate the timing and execution of these cell biological processes. A wellcharacterized gene regulatory network (GRN) in the sea urchin embryo was used to identify the transcription factors that control five distinct cell changes during EMT. Single transcription factors were perturbed and the consequences followed with in vivo time-lapse imaging or immunostaining assays. The data show that five different sub-circuits of the GRN control five distinct cell biological activities, each part of the complex EMT process. Thirteen transcription factors (TFs) expressed specifically in pre-EMT cells were required for EMT. Three TFs highest in the GRN specified and activated EMT (alx1, ets1, tbr) and the 10 TFs downstream of those (tel, erg, hex, tgif, snail, twist, foxn2/3, dri, foxb, foxo) were also required for EMT. No single TF functioned in all five sub-circuits, indicating that there is no EMT master regulator. Instead, the resulting sub-circuit topologies suggest EMT requires multiple simultaneous regulatory mechanisms: forward cascades, parallel inputs and positive-feedback lock downs. The interconnected and overlapping nature of the sub-circuits provides one explanation for the seamless orchestration by the embryo of cell state changes leading to successful EMT. [ABSTRACT FROM AUTHOR]

Abstract: Fringe proteins are O-fucose-specific β-1,3 N-acetylglucosaminyltransferases that glycosylate the extracellular EGF repeats of Notch and enable Notch to be activated by the ligand Delta. In the sea urchin, signaling between Delta and Notch is known to be necessary for specification of secondary mesenchyme cells (SMCs). The Lytechinus variegatus Fringe homologue is expressed in both the signaling and receiving cells during this first Delta–Notch signal. Perturbation of Fringe expression through morpholino antisense oligonucleotide (MO) injection results in fewer SMCs but also causes decreased and delayed archenteron invagination. Partial endoderm specification occurs but expression of some endoderm genes is compromised. The data are consistent with a Fringe-requiring Notch signal as one upstream component of archenteron morphogenesis. Finally, Fringe perturbations result in more severe phenotypes than those previously reported for Notch dominant-negative (LvNneg) injections or reported here for Notch MO (NMO) injections. Injecting a combination of LvNneg and NMO results in a more severe phenotype than either treatment alone, and this combination phenocopies the fringe MO embryos. Taken together, the results show that Fringe is necessary both for maternal and zygotic Notch signals, and these Notch signals affect specification of mesoderm and endoderm. [Copyright &y& Elsevier]

Bilateral animals, including humans and most metazoans, are not perfectly symmetrical. Some internal structures are distributed asymmetrically to the right or left side. A conserved Nodal and BMP signaling system directs molecular pathways that impart the sidedness to those asymmetric structures. In the sea urchin embryo, one such asymmetrical structure, oddly enough, is the entire adult, which grows out of left sided structures produced in the larva. In a paper just published in PLOS Biology, BMP signaling is shown to be necessary early in larval development to initiate the asymmetric specification of one of those left-sided structures, called the left coelomic pouch. This study reports that BMP signaling activates a group of transcription factors asymmetrically in the left coelomic pouch only, which launch the pathway that eventually leads to the formation of the adult that emerges from the larva at metamorphosis. [ABSTRACT FROM AUTHOR]

An abstract of the article "Frizzled1/2/7 signaling β-catenin nuclearisation and initiates endoderm specification in macromeres during sea urchin embryogenesis" is presented.

Activation of the Notch signaling pathway segregates the non-skeletogenic mesoderm (NSM) from the endomesoderm during sea urchin embryo development. Subsequently, Notch signaling helps specify the four subpopulations of NSM, and influences endoderm specification. To gain further insight into how the Notch signaling pathway is regulated during these cell specification events, we identified a sea urchin homologue of Numb (LvNumb). Previous work in other model systems showed that Numb functions as a Notch signaling pathway antagonist, possibly by mediating the endocytosis of other key Notch interacting proteins. In this study, we show that the vegetal endomesoderm expresses lvnumb during the blastula and gastrula stages, and that the protein is localized to the presumptive NSM. Injections of lvnumb mRNA and antisense morpholinos demonstrate that LvNumb is necessary for the specification of mesodermal cell types, including pigment cells, blastocoelar cells and muscle cells. Functional analysis of the N-terminal PTB domain and the C-terminal PRR domain of LvNumb shows that the PTB domain, but not the PRR domain, is sufficient to recapitulate the demonstrable function of full-length LvNumb. Experiments show that LvNumb requires an active Notch signal to function during NSM specification and that LvNumb functions in the cells responding to Delta and not in the cells presenting the Delta ligand. Furthermore, injection of mRNA encoding the intracellular domain of Notch rescues the LvNumb morpholino phenotype, suggesting that the constitutive intracellular Notch signal overcomes, or bypasses, the absence of Numb during NSM specification. [ABSTRACT FROM AUTHOR]

The foxa gene is an integral component of the endoderm specification subcircuit of the endomesoderm gene regulatory network in the Strongylocentrotus purpuratus embryo. Its transcripts become confined to veg2, then veg1 endodermal territories, and, following gastrulation, throughout the gut. It is also expressed in the stomodeal ectoderm. gatae and otx genes provide input into the pregastrular regulatory system of foxa, and Foxa represses its own transcription, resulting in an oscillatory temporal expression profile. Here, we report three separate essential functions of the foxa gene: it represses mesodermal fate in the veg2 endomesoderm; it is required in postgastrular development for the expression of gut-specific genes; and it is necessary for stomodaeum formation. If its expression is reduced by a morpholino, more endomesoderm cells become pigment and other mesenchymal cell types, less gut is specified, and the larva has no mouth. Experiments in which blastomere transplantation is combined with foxa MASO treatment demonstrate that, in the normal endoderm, a crucial role of Foxa is to repress gcm expression in response to a Notch signal, and hence to repress mesodermal fate. Chimeric recombination experiments in which veg2, veg1 or ectoderm cells contained foxa MASO show which region of foxa expression controls each of the three functions. These experiments show that the foxa gene is a component of three distinct embryonic gene regulatory networks. [ABSTRACT FROM AUTHOR]

Wnt signaling pathways play key roles in numerous developmental processes both in vertebrates and invertebrates. Their signals are transduced by Frizzled proteins, the cognate receptors of the Wnt ligands. This study focuses on the role of a member of the Frizzled family, Fz5/8, during sea urchin embryogenesis. During development, Fz5/8 displays restricted expression, beginning at the 60-cell stage in the animal domain and then from mesenchyme blastula stage, in both the animal domain and a subset of secondary mesenchyme cells (SMCs). Loss-of-function analyses in whole embryos and chimeras reveal that Fz5/8 is not involved in the specification of the main embryonic territories. Rather, it appears to be required in SMCs for primary invagination of the archenteron, maintenance of endodermal marker expression and apical localization of Notch receptors in endodermal cells. Furthermore, among the three known Wnt pathways, Fz5/8 appears to signal via the planar cell polarity pathway. Taken together, the results suggest that Fz5/8 plays a crucial role specifically in SMCs to control primary invagination during sea urchin gastrulation. [ABSTRACT FROM AUTHOR]