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Sea urchin larvae spend weeks to months feeding on plankton prior to metamorphosis. When handled in the laboratory they are easily injured, suggesting that in the plankton they are injured with some frequency. Fortunately, larval wounds are repaired through an efficient wound response with mesenchymal pigment cells and blastocoelar cells assisting as the epithelium closes. An injury to the epithelium leads to an immediate calcium transient that rapidly spreads around the entire larva and is necessary for activating pigment cell migration toward the wound. If calcium transport is blocked, the pigment cells fail to activate and remain in place. When activated, pigment cells initiate directed migration to the wound site from distances of at least 85 ​μm. Upon arrival at the wound site they participate in an innate immune response. Blastocoelar cells are recruited to the injury site as well, though the calcium transient is unnecessary for activating these cells. At the wound site, blastocoelar cells participate in several functions including remodeling the skeleton if it protrudes through the epithelium., (Copyright © 2022 Elsevier Inc. All rights reserved.)

This review reports recent findings on the specification and patterning of neurons that establish the larval nervous system of the sea urchin embryo. Neurons originate in three regions of the embryo. Perturbation analyses enabled construction of gene regulatory networks controlling the several neural cell types. Many of the mechanisms described reflect shared features of all metazoans and others are conserved among deuterostomes. This nervous system with a very small number of neurons supports the feeding and swimming behaviors of the larva until metamorphosis when an adult nervous system replaces that system., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Early in animal development many cells are conditionally specified based on observations that those cells can be directed toward alternate fates. The endomesoderm is so named because early specification produces cells that often have been observed to simultaneously express both early endoderm and mesoderm transcription factors. Experiments with these cells demonstrate that their progeny can directed entirely toward endoderm or mesoderm, whereas normally they establish both germ layers. This review examines the mechanisms that initiate the conditional endomesoderm state, its metastability, and the mechanisms that resolve that state into definitive endoderm and mesoderm., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In the sea urchin larva, most neurons lie within an ectodermal region called the ciliary band. Our understanding of the mechanisms of specification and patterning of these peripheral ciliary band neurons is incomplete. Here, we first examine the gene regulatory landscape from which this population of neural progenitors arise in the neuroectoderm. We show that ciliary band neural progenitors first appear in a bilaterally symmetric pattern on the lateral edges of chordin expression in the neuroectoderm. Later in development, these progenitors appear in a salt-and-pepper pattern in the ciliary band where they express soxC, and prox, which are markers of neural specification, and begin to express synaptotagminB, a marker of differentiated neurons. We show that the ciliary band expresses the acid sensing ion channel gene asicl, which suggests that ciliary band neurons control the larva's ability to discern touch sensitivity. Using a chemical inhibitor of MAPK signaling, we show that this signaling pathway is required for proper specification and patterning of ciliary band neurons. Using live imaging, we show that these neural progenitors undergo small distance migrations in the embryo. We then show that the normal swimming behavior of the larvae is compromised if the neurogenesis pathway is perturbed. The developmental sequence of ciliary band neurons is very similar to that of neural crest-derived sensory neurons in vertebrates and may provide insights into the evolution of sensory neurons in deuterostomes., Competing Interests: Declaration of competing interest The authors declare no competing interest or financial interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Gastrulation is arguably the most important evolutionary innovation in the animal kingdom. This process provides the basic embryonic architecture, an inner layer separated from an outer layer, from which all animal forms arise. An extraordinarily simple and elegant process of gastrulation is observed in the sea urchin embryo. The cells participating in sea urchin gastrulation are specified early during cleavage. One outcome of that specification is the expression of transcription factors that control each of the many subsequent morphogenetic changes. The first of these movements is an epithelial-mesenchymal transition (EMT) of skeletogenic mesenchyme cells, then EMT of pigment cell progenitors. Shortly thereafter, invagination of the archenteron occurs. At the end of archenteron extension, a second wave of EMT occurs to release immune cells into the blastocoel and primordial germ cells that will home to the coelomic pouches. The archenteron then remodels to establish the three parts of the gut, and at the anterior end, the gut fuses with the stomodaeum to form the through-gut. As part of the anterior remodeling, mesodermal coelomic pouches bud off the lateral sides of the archenteron tip. Multiple cell biological processes conduct each of these movements and in some cases the upstream transcription factors controlling this process have been identified. Remarkably, each event seamlessly occurs at the right time to orchestrate formation of the primitive body plan. This review covers progress toward understanding many of the molecular mechanisms underlying this sequence of morphogenetic events., (© 2020 Elsevier Inc. All rights reserved.)

The stereotypic cleavage pattern of sea urchin embryos provides a platform for dissection of early lineage decisions that lead to cell diversification. Cell transplantation provides a useful tool for understanding those decisions. The methods described in this paper provide a guide for how to produce embryonic mosaics in which either one cell is transplanted or an entire tier of cells are transplanted to a host embryo. Although the results of such a cut and paste experiment can be documented in many ways, one of the most useful approaches follows progeny of the transplanted cell as they go through morphogenesis using time-lapse imaging. Methods for mounting and imaging the embryos are provided., (© 2019 Elsevier Inc. All rights reserved.)

Background: Gastrulation is a complex orchestration of movements by cells that are specified early in development. Until now, classical convergent extension was considered to be the main contributor to sea urchin archenteron extension, and the relative contributions of cell divisions were unknown. Active migration of cells along the axis of extension was also not considered as a major factor in invagination., Results: Cell transplantations plus live imaging were used to examine endoderm cell morphogenesis during gastrulation at high-resolution in the optically clear sea urchin embryo. The invagination sequence was imaged throughout gastrulation. One of the eight macromeres was replaced by a fluorescently labeled macromere at the 32 cell stage. At gastrulation those patches of fluorescent endoderm cell progeny initially about 4 cells wide, released a column of cells about 2 cells wide early in gastrulation and then often this column narrowed to one cell wide by the end of archenteron lengthening. The primary movement of the column of cells was in the direction of elongation of the archenteron with the narrowing (convergence) occurring as one of the two cells moved ahead of its neighbor. As the column narrowed, the labeled endoderm cells generally remained as a contiguous population of cells, rarely separated by intrusion of a lateral unlabeled cell. This longitudinal cell migration mechanism was assessed quantitatively and accounted for almost 90% of the elongation process. Much of the extension was the contribution of Veg2 endoderm with a minor contribution late in gastrulation by Veg1 endoderm cells. We also analyzed the contribution of cell divisions to elongation. Endoderm cells in Lytechinus variagatus were determined to go through approximately one cell doubling during gastrulation. That doubling occurs without a net increase in cell mass, but the question remained as to whether oriented divisions might contribute to archenteron elongation. We learned that indeed there was a biased orientation of cell divisions along the plane of archenteron elongation, but when the impact of that bias was analyzed quantitatively, it contributed a maximum 15% to the total elongation of the gut., Conclusions: The major driver of archenteron elongation in the sea urchin, Lytechinus variagatus, is directed movement of Veg2 endoderm cells as a narrowing column along the plane of elongation. The narrowing occurs as cells in the column converge as they migrate, so that the combination of migration and the angular convergence provide the major component of the lengthening. A minor contributor to elongation is oriented cell divisions that contribute to the lengthening but no more than about 15%., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Most bilaterians exhibit a left-right asymmetric distribution of their internal organs. The sea urchin larva is notable in this regard since most adult structures are generated from left sided embryonic structures. The gene regulatory network governing this larval asymmetry is still a work in progress but involves several conserved signaling pathways including Nodal, and BMP. Here we provide a comprehensive analysis of Hedgehog signaling and it's contribution to left-right asymmetry. We report that Hh signaling plays a conserved role to regulate late asymmetric expression of Nodal and that this regulation occurs after Nodal breaks left-right symmetry in the mesoderm. Thus, while Hh functions to maintain late Nodal expression, the molecular asymmetry of the future coelomic pouches is locked in. Furthermore we report that cilia play a role only insofar as to transduce Hh signaling and do not have an independent effect on the asymmetry of the mesoderm. From this, we are able to construct a more complete regulatory network governing the establishment of left-right asymmetry in the sea urchin., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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.

In the sea urchin morphogenesis follows extensive molecular specification. The specification controls the many morphogenetic events and these, in turn, precede patterning steps that establish the larval body plan. To understand how the embryo is built it was necessary to understand those series of molecular steps. Here an example of the historical sequence of those discoveries is presented as it unfolded over the last 50 years, the years during which major progress in understanding development of many animals and plants was documented by CTDB. In sea urchin development a rich series of experimental studies first established many of the phenomenological components of skeletal morphogenesis and patterning without knowledge of the molecular components. The many discoveries of transcription factors, signals, and structural proteins that contribute to the shape of the endoskeleton of the sea urchin larva then followed as molecular tools became available. A number of transcription factors and signals were discovered that were necessary for specification, morphogenesis, and patterning. Perturbation of the transcription factors and signals provided the means for assembling models of the gene regulatory networks used for specification and controlled the subsequent morphogenetic events. The earlier experimental information informed perturbation experiments that asked how patterning worked. As a consequence it was learned that ectoderm provides a series of patterning signals to the skeletogenic cells and as a consequence the skeletogenic cells secrete a highly patterned skeleton based on their ability to genotypically decode the localized reception of several signals. We still do not understand the complexity of the signals received by the skeletogenic cells, nor do we understand in detail how the genotypic information shapes the secreted skeletal biomineral, but the current knowledge at least outlines the sequence of events and provides a useful template for future discoveries., (© 2016 Elsevier Inc. All rights reserved.)

Gene regulatory networks (GRNs) provide a systems-level orchestration of an organism's genome encoded anatomy. As biological networks are revealed, they continue to answer many questions including knowledge of how GRNs control morphogenetic movements and how GRNs evolve. The migration of the small micromeres to the coelomic pouches in the sea urchin embryo provides an exceptional model for understanding the genomic regulatory control of morphogenesis. An assay using the robust homing potential of these cells reveals a 'coherent feed-forward' transcriptional subcircuit composed of Pax6, Six3, Six1/2, Eya, and Dach1 that is responsible for the directed homing mechanism of these multipotent progenitors. The linkages of that circuit are strikingly similar to a circuit involved in retinal specification in Drosophila suggesting that systems-level tasks can be highly conserved even though the tasks drive unrelated processes in different animals.

The sea urchin larva is shaped by a calcite endoskeleton. That skeleton is built by 64 primary mesenchyme cells (PMCs) in Lytechinus variegatus. The PMCs originate as micromeres due to an unequal fourth cleavage in the embryo. Micromeres are specified in a well-described molecular sequence and enter the blastocoel at a precise time using a classic epithelial-mesenchymal transition. To make the skeleton, the PMCs receive signaling inputs from the overlying ectoderm, which provides positional information as well as control of the growth of initial skeletal tri-radiates. The patterning of the skeleton is the result both of autonomous inputs from PMCs, including production of proteins that are included in the skeletal matrix, and of non-autonomous dynamic information from the ectoderm. Here, we summarize the wealth of information known about how a PMC contributes to the skeletal structure. The larval skeleton is a model for understanding how information encoded in DNA is translated into a three-dimensional crystalline structure., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

It is a challenge to understand how the information encoded in DNA is used to build a three-dimensional structure. To explore how this works the assembly of a relatively simple skeleton has been examined at multiple control levels. The skeleton of the sea urchin embryo consists of a number of calcite rods produced by 64 skeletogenic cells. The ectoderm supplies spatial cues for patterning, essentially telling the skeletogenic cells where to position themselves and providing the factors for skeletal growth. Here, we describe the information known about how this works. First the ectoderm must be patterned so that the signaling cues are released from precise positions. The skeletogenic cells respond by initiating skeletogenesis immediately beneath two regions (one on the right and the other on the left side). Growth of the skeletal rods requires additional signaling from defined ectodermal locations, and the skeletogenic cells respond to produce a membrane-bound template in which the calcite crystal grows. Important in this process are three signals, fibroblast growth factor, vascular endothelial growth factor, and Wnt5. Each is necessary for explicit tasks in skeleton production., (Copyright © 2014 Wiley Periodicals, Inc.)

A relatively small number of signaling pathways govern the early patterning processes of metazoan development. The architectural changes over time to these signaling pathways offer unique insights into their evolution. In the case of Hedgehog (Hh) signaling, two very divergent mechanisms of pathway transduction have evolved. In vertebrates, signaling relies on trafficking of Hh pathway components to nonmotile specialized primary cilia. In contrast, protostomes do not use cilia of any kind for Hh signal transduction. How these divergent lineages adapted such dramatically different ways of activating the signaling pathway is an unanswered question. Here, we present evidence that in the sea urchin, a basal deuterostome, motile cilia are required for embryonic Hh signal transduction, and the Hh receptor Smoothened (Smo) localizes to cilia during active Hh signaling. This is the first evidence that Hh signaling requires motile cilia and the first case of an organism requiring cilia outside of the vertebrate lineage.

The Hedgehog pathway has been shown to be an important developmental signaling pathway in many organisms (Ingham and McMahon. Genes Dev 15:3059-3087, 2001). Recently that work has been extended to developing echinoderm embryos (Walton et al. Dev Biol 331(1):26-37, 2009). Here we describe several methods to perturb the Hedgehog signaling pathway in the sea urchin. These include microinjection of Morpholinos and mRNA constructs as well as treatments with small molecule inhibitors. Finally we provide simple methods for assaying Hedgehog phenotypes in the sea urchin embryo.

The border between the posterior ectoderm and the endoderm is a location where two germ layers meet and establish an enduring relationship that also later serves, in deuterostomes, as the anatomical site of the anus. In the sea urchin, a prototypic deuterostome, the ectoderm-endoderm boundary is established before gastrulation, and ectodermal cells at the boundary are thought to provide patterning inputs to the underlying mesenchyme. Here we show that a short-range Wnt5 signal from the endoderm actively patterns the adjacent boundary ectoderm. This signal activates a unique subcircuit of the ectoderm gene regulatory network, including the transcription factors IrxA, Nk1, Pax2/5/8 and Lim1, which are ultimately restricted to subregions of the border ectoderm (BE). Surprisingly, Nodal and BMP2/4, previously shown to be activators of ectodermal specification and the secondary embryonic axis, instead restrict the expression of these genes to subregions of the BE. A detailed examination showed that endodermal Wnt5 functions as a short-range signal that activates only a narrow band of ectodermal cells, even though all ectoderm is competent to receive the signal. Thus, cells in the BE integrate positive and negative signals from both the primary and secondary embryonic axes to correctly locate and specify the border ectoderm.

Gastrulation in the sea urchin begins with ingression of the primary mesenchyme cells (PMCs) at the vegetal pole of the embryo. After entering the blastocoel the PMCs migrate, form a syncitium, and synthesize the skeleton of the embryo. Several hours after the PMCs ingress the vegetal plate buckles to initiate invagination of the archenteron. That morphogenetic process occurs in several steps. The nonskeletogenic cells produce the initial inbending of the vegetal plate. Endoderm cells then rearrange and extend the length of the gut across the blastocoel to a target near the animal pole. Finally, cells that will form part of the midgut and hindgut are added to complete gastrulation. Later, the stomodeum invaginates from the oral ectoderm and fuses with the foregut to complete the archenteron. In advance of, and during these morphogenetic events, an increasingly complex input of transcription factors controls the specification and the cell biological events that conduct the gastrulation movements., (Copyright © 2011 Wiley Periodicals, Inc.)

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., Competing Interests: The authors have declared that no competing interests exist.

In the sea urchin, entry of β-catenin into the nuclei of the vegetal cells at 4th and 5th cleavages is necessary for activation of the endomesoderm gene regulatory network. Beyond that, little is known about how the embryo uses maternal information to initiate specification. Here, experiments establish that of the three maternal Wnts in the egg, Wnt6 is necessary for activation of endodermal genes in the endomesoderm GRN. A small region of the vegetal cortex is shown to be necessary for activation of the endomesoderm GRN. If that cortical region of the egg is removed, addition of Wnt6 rescues endoderm. At a molecular level, the vegetal cortex region contains a localized concentration of Dishevelled (Dsh) protein, a transducer of the canonical Wnt pathway; however, Wnt6 mRNA is not similarly localized. Ectopic activation of the Wnt pathway, through the expression of an activated form of β-catenin, of a dominant-negative variant of GSK-3β or of Dsh itself, rescues endomesoderm specification in eggs depleted of the vegetal cortex. Knockdown experiments in whole embryos show that absence of Wnt6 produces embryos that lack endoderm, but those embryos continue to express a number of mesoderm markers. Thus, maternal Wnt6 plus a localized vegetal cortical molecule, possibly Dsh, is necessary for endoderm specification; this has been verified in two species of sea urchin. The data also show that Wnt6 is only one of what are likely to be multiple components that are necessary for activation of the entire endomesoderm gene regulatory network.

Endomesoderm is the common progenitor of endoderm and mesoderm early in the development of many animals. In the sea urchin embryo, the Delta/Notch pathway is necessary for the diversification of this tissue, as are two early transcription factors, Gcm and FoxA, which are expressed in mesoderm and endoderm, respectively. Here, we provide a detailed lineage analysis of the cleavages leading to endomesoderm segregation, and examine the expression patterns and the regulatory relationships of three known regulators of this cell fate dichotomy in the context of the lineages. We observed that endomesoderm segregation first occurs at hatched blastula stage. Prior to this stage, Gcm and FoxA are co-expressed in the same cells, whereas at hatching these genes are detected in two distinct cell populations. Gcm remains expressed in the most vegetal endomesoderm descendant cells, while FoxA is downregulated in those cells and activated in the above neighboring cells. Initially, Delta is expressed exclusively in the micromeres, where it is necessary for the most vegetal endomesoderm cell descendants to express Gcm and become mesoderm. Our experiments show a requirement for a continuous Delta input for more than two cleavages (or about 2.5 hours) before Gcm expression continues in those cells independently of further Delta input. Thus, this study provides new insights into the timing mechanisms and the molecular dynamics of endomesoderm segregation during sea urchin embryogenesis and into the mode of action of the Delta/Notch pathway in mediating mesoderm fate.

The Hedgehog (Hh) signaling pathway is essential for patterning many structures in vertebrates including the nervous system, chordamesoderm, limb and endodermal organs. In the sea urchin, a basal deuterostome, Hh signaling is shown to participate in organizing the mesoderm. At gastrulation the Hh ligand is expressed by the endoderm downstream of the Brachyury and FoxA transcription factors in the endomesoderm gene regulatory network. The co-receptors Patched (Ptc) and Smoothened (Smo) are expressed by the neighboring skeletogenic and non-skeletogenic mesoderm. Perturbations of Hh, Ptc and Smo cause embryos to develop with skeletal defects and inappropriate non-skeletogenic mesoderm patterning, although initial specification of mesoderm occurs without detectable abnormalities. Perturbations of the pathway caused late defects in skeletogenesis and in the non-skeletogenic mesoderm, including altered numbers of pigment and blastocoelar cells, randomized left-right asymmetry of coelomic pouches, and disorganized circumesophageal muscle causing an inability to swallow. Together the data support the requirement of Hh signaling in patterning each of the mesoderm subtypes in the sea urchin embryo.

Dishevelled (Dsh) is a phosphoprotein key to beta-catenin dependent (canonical) and beta-catenin independent (noncanonical) Wnt signaling. Whereas canonical Wnt signaling has been intensively studied in sea urchin development, little is known about other Wnt pathways. To examine roles of these beta-catenin independent pathways in embryogenesis, we used Dsh-DEP, a deletion construct blocking planar cell polarity (PCP) and Wnt/Ca(2+) signaling. Embryos overexpressing Dsh-DEP failed to gastrulate or undergo skeletogenesis, but produced pigment cells. Although early mesodermal gene expression was largely unperturbed, embryos exhibited reduced expression of genes regulating endoderm specification and differentiation. Overexpressing activated beta-catenin failed to rescue Dsh-DEP embryos, indicating that Dsh-DEP blocks endoderm formation downstream of initial canonical Wnt signaling. Because Dsh-DEP-like constructs block PCP signaling in other metazoans, and disrupting RhoA or Fz 5/8 in echinoids blocks subsets of the Dsh-DEP phenotypes, our data suggest that noncanonical Wnt signaling is crucial for sea urchin endoderm formation and skeletogenesis., ((c) 2009 Wiley-Liss, Inc.)

The oral-aboral (OA) axis in the sea urchin is specified by the TGFbeta family members Nodal and BMP2/4. Nodal promotes oral specification, whereas BMP2/4, despite being expressed in the oral territory, is required for aboral specification. This study explores the role of Chordin (Chd) during sea urchin embryogenesis. Chd is a secreted BMP inhibitor that plays an important role in axial and neural specification and patterning in Drosophila and vertebrate embryos. In Lytechinus variegatus embryos, Chd and BMP2/4 are functionally antagonistic. Both are expressed in overlapping domains in the oral territory prior to and during gastrulation. Perturbation shows that, surprisingly, Chd is not involved in OA axis specification. Instead, Chd is required both for normal patterning of the ciliary band at the OA boundary and for development of synaptotagmin B-positive (synB) neurons in a manner that is reciprocal with BMP2/4. Chd expression and synB-positive neural development are both downstream from p38 MAPK and Nodal, but not Goosecoid. These data are summarized in a model for synB neural development.

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.

Recent work on the sea urchin endomesoderm gene regulatory network (GRN) offers many opportunities to study the specification and differentiation of each cell type during early development at a mechanistic level. The mesoderm lineages consist of two cell populations, primary and secondary mesenchyme cells (PMCs and SMCs). The micromere-PMC GRN governs the development of the larval skeleton, which is the exclusive fate of PMCs, and SMCs diverge into four lineages, each with its own GRN state. Here we identify a sea urchin ortholog of the Twist transcription factor, and show that it plays an essential role in the PMC GRN and later is involved in SMC formation. Perturbations of Twist either by morpholino knockdown or by overexpression result in defects in progressive phases of PMC development, including specification, ingression/EMT, differentiation and skeletogenesis. Evidence is presented that Twist expression is required for the maintenance of the PMC specification state, and a reciprocal regulation between Alx1 and Twist offers stability for the subsequent processes, such as PMC differentiation and skeletogenesis. These data illustrate the significance of regulatory state maintenance and continuous progression during cell specification, and the dynamics of the sequential events that depend on those earlier regulatory states.

Epithelial-mesenchyme transitions (EMTs) are familiar to all scholars of development. Each animal system utilizes an EMT to produce mesenchyme cells. In vertebrates, for example, there are a number of EMTs that shape the embryo. Early, entry of epiblast cells into the primitive streak is followed by the emergence of mesoderm via an EMT process. The departure of neural crest cells from the margin of the neural folds is an EMT process, and the delamination of cells from the endomesoderm to form the supporting mesenchyme of the lung, liver, and pancreas are EMTs. EMTs are observed in Drosophila following invagination of the ventral furrow, and even in Cnidarians, which have only two germ layers, yet mesoglial and stem cells delaminate from the epithelia and occupy the matrix between the ectoderm and endoderm. This review will focus on a classic example of an EMT, which occurs in the sea urchin embryo. The primary mesenchyme cells (PMCs) ingress from the vegetal plate of this embryo precociously and in advance of archenteron invagination. Because ingression is precisely timed, the PMC lineage precisely known, and the embryo easily observed and manipulated, much has been learned about how the ingression of PMCs works in the sea urchin. Though the focus of this review is the sea urchin PMCs, there is evidence that all EMTs share many common features at both cellular and molecular levels, and many of these mechanisms are also shown to be involved in tumor progression, especially metastasizing carcinomas., (Copyright 2008 Wiley-Liss, Inc.)

This paper reports a preliminary in silico analysis of the sea urchin kinome. The predicted protein kinases in the sea urchin genome were identified, annotated and classified, according to both function and kinase domain taxonomy. The results show that the sea urchin kinome, consisting of 353 protein kinases, is closer to the Drosophila kinome (239) than the human kinome (518) with respect to total kinase number. However, the diversity of sea urchin kinases is surprisingly similar to humans, since the urchin kinome is missing only 4 of 186 human subfamilies, while Drosophila lacks 24. Thus, the sea urchin kinome combines the simplicity of a non-duplicated genome with the diversity of function and signaling previously considered to be vertebrate-specific. More than half of the sea urchin kinases are involved with signal transduction, and approximately 88% of the signaling kinases are expressed in the developing embryo. These results support the strength of this nonchordate deuterostome as a pivotal developmental and evolutionary model organism.

The Hedgehog (Hh) and Notch signal transduction pathways control a variety of developmental processes including cell fate choice, differentiation, proliferation, patterning and boundary formation. Because many components of these pathways are conserved, it was predicted and confirmed that pathway components are largely intact in the sea urchin genome. Spatial and temporal location of these pathways in the embryo, and their function in development offer added insight into their mechanistic contributions. Accordingly, all major components of both pathways were identified and annotated in the sea urchin Strongylocentrotus purpuratus genome and the embryonic expression of key components was explored. Relationships of the pathway components, and modifiers predicted from the annotation of S. purpuratus, were compared against cnidarians, arthropods, urochordates, and vertebrates. These analyses support the prediction that the pathways are highly conserved through metazoan evolution. Further, the location of these two pathways appears to be conserved among deuterostomes, and in the case of Notch at least, display similar capacities in endomesoderm gene regulatory networks. RNA expression profiles by quantitative PCR and RNA in situ hybridization reveal that Hedgehog is produced by the endoderm beginning just prior to invagination, and signals to the secondary mesenchyme-derived tissues at least until the pluteus larva stage. RNA in situ hybridization of Notch pathway members confirms that Notch functions sequentially in the vegetal-most secondary mesenchyme cells and later in the endoderm. Functional analyses in future studies will embed these pathways into the growing knowledge of gene regulatory networks that govern early specification and morphogenesis.

Programmed cell death through apoptosis is a pan-metazoan character involving intermolecular signaling networks that have undergone substantial lineage-specific evolution. A survey of apoptosis-related proteins encoded in the sea urchin genome provides insight into this evolution while revealing some interesting novelties, which we highlight here. First, in addition to a typical CARD-carrying Apaf-1 homologue, sea urchins have at least two novel Apaf-1-like proteins that are each linked to a death domain, suggesting that echinoderms have evolved unique apoptotic signaling pathways. Second, sea urchins have an unusually large number of caspases. While the set of effector caspases (caspases-3/7 and caspase-6) in sea urchins is similar to that found in other basal deuterostomes, signal-responsive initiator caspase subfamilies (caspases-8/10 and 9, which are respectively linked to DED and CARD adaptor domains) have undergone echinoderm-specific expansions. In addition, there are two groups of divergent caspases, one distantly related to the vertebrate interleukin converting enzyme (ICE)-like subfamily, and a large clan that does not cluster with any of the vertebrate caspases. Third, the complexity of proteins containing an anti-apoptotic BIR domain and of Bcl-2 family members approaches that of vertebrates, and is greater than that found in protostome model systems such as Drosophila or Caenorhabditis elegans. Finally, the presence of Death receptor homologues, previously known only in vertebrates, in both Strongylocentrotus purpuratus and Nematostella vectensis suggests that this family of apoptotic signaling proteins evolved early in animals and was subsequently lost in the nematode and arthropod lineage(s). Our results suggest that cell survival is contingent upon a diverse array of signals in sea urchins, more comparable in complexity to vertebrates than to arthropods or nematodes, but also with unique features that may relate to specific requirements imposed by the biphasic life cycle and/or immunological idiosyncrasies of this organism.

The Wnt pathways are evolutionarily well-conserved signal transduction pathways that are known to play important roles in all Metazoans investigated to date. Here, we examine the Wnt pathway genes and target genes present in the genome of the echinoderm Strongylocentrotus purpuratus. Analysis of the Wnt genes revealed that eleven of the thirteen reported Wnt subfamilies are represented in sea urchin, with the intriguing identification of a Wnt-A ortholog thought to be absent in deuterostomes. A phylogenetic study of the Frizzled proteins, the Wnt receptors, performed throughout the animal kingdom showed that not all Frizzled subfamilies were present in the metazoan common ancestor, e.g. Fz3/6 emerged later during evolution. Using sequence analysis, orthologs of the vast majority of the cellular machinery involved in transducing the three types of Wnt pathways were found in the sea urchin genome. Furthermore, of about one hundred target genes identified in other organisms, more than half have clear echinoderm orthologs. Thus, these analyses produce new inputs in the evolutionary history of the Wnt genes in an animal occupying a position that offers great insights into the basal properties of deuterostomes.

In every organism, GTP-binding proteins control many aspects of cell signaling. Here, we examine in silico several GTPase families from the Strongylocentrotus purpuratus genome: the monomeric Ras superfamily, the heterotrimeric G proteins, the dynamin superfamily, the SRP/SR family, and the "protein biosynthesis" translational GTPases. Identified were 174 GTPases, of which over 90% are expressed in the embryo as shown by tiling array and expressed sequence tag data. Phylogenomic comparisons restricted to Drosophila, Ciona, and humans (protostomes, urochordates, and vertebrates, respectively) revealed both common and unique elements in the expected composition of these families. Galpha and dynamin families contain vertebrate expansions, consistent with whole genome duplications, whereas SRP/SR and translational GTPases are highly conserved. Unexpectedly, Ras superfamily analyses revealed several large (5+) lineage-specific expansions in the sea urchin. For Rho, Rab, Arf, and Ras subfamilies, comparing total human gene numbers to the number of sea urchin genes with vertebrate orthologs suggests reduced genomic complexity in the sea urchin. However, gene duplications in the sea urchin increase overall numbers such that total sea urchin gene numbers approximate vertebrate gene numbers for each monomeric GTPase family. These findings suggest that lineage-specific expansions may be an important component of genomic evolution in signal transduction.

The canonical Wnt pathway plays crucial roles in multiple developmental processes, including in axis specification. Throughout the animal kingdom, this pathway has been reported to drive patterning of axes as different as the animal-vegetal axis in echinoderms to the dorsal-ventral axis in vertebrates. Intriguingly enough, this pathway appears structurally and functionally well conserved during evolution. However, differences between these phyla are observed that explain how a same pathway can mediate establishment of two such apparently distinct axes. This review compares the axis specification processes used in two evolutionarily distant embryos, the sea urchin and Xenopus.

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.

Most eggs in the animal kingdom establish a primary, animal-vegetal axis maternally, and specify the remaining two axes during development. In sea urchin embryos, the expression of Nodal on the oral (ventral) side of the embryo is the first known molecular determinant of the oral-aboral axis (the embryonic dorsoventral axis), and is crucial for specification of the oral territory. We show that p38 MAPK acts upstream of Nodal and is required for Nodal expression in the oral territory. p38 is uniformly activated early in development, but, for a short interval at late blastula stage, is asymmetrically inactivated in future aboral nuclei. Experiments show that this transient asymmetry of p38 activation corresponds temporally to both oral specification and the onset of oral Nodal expression. Uniform inhibition of p38 prevents Nodal expression and axis specification, resulting in aboralized embryos. Nodal and its target Gsc each rescue oral-aboral specification and patterning when expressed asymmetrically in p38-inhibited embryos. Thus, our results indicate that p38 is required for oral specification through its promotion of Nodal expression in the oral territory.

Fringe proteins are O-fucose-specific beta-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 (LvN(neg)) injections or reported here for Notch MO (NMO) injections. Injecting a combination of LvN(neg) 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.

In the sea urchin embryo, specification of the endomesoderm is accomplished by the activity of a network of regulatory genes in the vegetal hemisphere, called the endomesoderm gene regulatory network (GRN). The activation of this network is mediated primarily through the activity of the Wnt pathway, though details of pathway activation remain unclear. To gain further insight into control of endomesoderm GRN activation, we have identified a sea urchin homologue of the co-repressor Groucho (LvGroucho) that has been shown to antagonize beta-catenin/Tcf activation complexes during Wnt signaling in other systems. Groucho functions by recruiting the histone deacetylase Rpd3 to the DNA template via interaction with site-specific transcription factors, resulting in localized chromatin condensation and transcriptional silencing. Our results show that the LvGroucho protein localizes to all nuclei throughout embryonic development. Interaction assays demonstrate that LvGroucho interacts with Tcf via both the Q and the WD domains of the protein. LvGroucho interacts with Tcf to antagonize the expression of key endomesoderm regulatory genes. Assays demonstrate that LvGroucho and n beta-catenin functionally compete for binding to Tcf as a major mechanism by which the Tcf-control switch is regulated. Functional analysis of the N-terminal AES197 domain of LvGroucho shows that it is sufficient to recapitulate the function of full-length LvGroucho. This finding strongly supports the conclusion that the effects of LvGro overexpression are due primarily to its interactions with Tcf and not other Groucho interacting partners, since Tcf is the only protein present in the sea urchin known to interact with AES197. Because the Q domain is unable to bind Rpd3, it was expected to behave as a dominant negative LvGroucho. Unexpectedly, overexpression of the Q domain gave functional results similar to LvGroucho and the AES197 domain. This is the first evidence for an inherent repressive function for the Q domain alone. Together, our results indicate that LvGroucho functionally competes with beta-catenin for Tcf binding, and this competitive mechanism regulates one of the earliest steps in the initiation of the sea urchin endomesoderm GRN.

The Strongylocentrotus purpuratus hnf6 (Sphnf6) gene encodes a new member of the ONECUT family of transcription factors. The expression of hnf6 in the developing embryo is triphasic, and loss-of-function analysis shows that the Hnf6 protein is a transcription factor that has multiple distinct roles in sea urchin development. hnf6 is expressed maternally, and before gastrulation its transcripts are distributed globally. Early in development, its expression is required for the activation of PMC differentiation genes such as sm50, pm27, and msp130, but not for the activation of any known PMC regulatory genes, for example, alx, ets1, pmar1, or tbrain. Micromere transplantation experiments show that the gene is not involved in early micromere signaling. Early hnf6 expression is also required for expression of the mesodermal regulator gatac. The second known role of hnf6 is its participation after gastrulation in the oral ectoderm gene regulatory network (GRN), in which its expression is essential for the maintenance of the state of oral ectoderm specification. The third role is in the neurogenic ciliated band, which is foreshadowed exactly by a trapezoidal band of hnf6 expression at the border of the oral ectoderm and where it continues to be expressed through the end of embryogenesis. Neither oral ectoderm regulatory functions nor ciliated band formation occur normally in the absence of hnf6 expression.

The entry of beta-catenin into vegetal cell nuclei beginning at the 16-cell stage is one of the earliest known molecular asymmetries seen along the animal-vegetal axis in the sea urchin embryo. Nuclear beta-catenin activates a vegetal signaling cascade that mediates micromere specification and specification of the endomesoderm in the remaining cells of the vegetal half of the embryo. Only a few potential target genes of nuclear beta-catenin have been functionally analyzed in the sea urchin embryo. Here, we show that SpWnt8, a Wnt8 homolog from Strongylocentrotus purpuratus, is zygotically activated specifically in 16-cell-stage micromeres in a nuclear beta-catenin-dependent manner, and its expression remains restricted to the micromeres until the 60-cell stage. At the late 60-cell stage nuclear beta-catenin-dependent SpWnt8 expression expands to the veg2 cell tier. SpWnt8 is the only signaling molecule thus far identified with expression localized to the 16-60-cell stage micromeres and the veg2 tier. Overexpression of SpWnt8 by mRNA microinjection produced embryos with multiple invagination sites and showed that, consistent with its localization, SpWnt8 is a strong inducer of endoderm. Blocking SpWnt8 function using SpWnt8 morpholino antisense oligonucleotides produced embryos that formed micromeres that could transmit the early endomesoderm-inducing signal, but these cells failed to differentiate as primary mesenchyme cells. SpWnt8-morpholino embryos also did not form endoderm, or secondary mesenchyme-derived pigment and muscle cells, indicating a role for SpWnt8 in gastrulation and in the differentiation of endomesodermal lineages. These results establish SpWnt8 as a critical component of the endomesoderm regulatory network in the sea urchin embryo., (Copyright 2004 Wiley-Liss, Inc.)

The Spdeadringer (Spdri) gene encodes an ARID-class transcription factor not previously known in sea urchin embryos. We show that Spdri is a key player in two separate developmental gene regulatory networks (GRNs). Spdri is expressed in a biphasic manner, first, after 12 h and until ingression in the skeletogenic descendants of the large micromeres; second, after about 20 h in the oral ectoderm, where its transcripts remain present at 30-50 mRNA molecules/cell far into development. In both territories, the periods of Spdri expression follow prior territorial specification events. The functional significance of each phase of expression was assessed by determining the effect of an alphaSpdri morpholino antisense oligonucleotide (MASO) on expression of 17 different mesodermal genes, 8 different oral ectoderm genes, and 18 other genes expressed specifically during endomesoderm specification. These effects were measured by quantitative PCR, supplemented by whole-mount in situ hybridization and morphological observations. Spdri is shown to act in the micromere descendants in the pathways that result in the expression of batteries of terminal skeletogenic genes. But, in the oral ectoderm, the same gene participates in the central GRN controlling oral ectoderm identity. Spdri is linked in the oral ectoderm GRN with several other genes encoding transcriptional regulators that are expressed specifically in various regions of the oral ectoderm. If its expression is blocked by treatment with alphaSpdri MASO, oral-specific features disappear and expression of the aboral ectoderm marker spec1 encompasses the whole of the ectoderm. In addition to disappearance of the oral ectoderm, morphological consequences of alphaSpdri MASO treatment include failure of spiculogenesis and of correct primary mesenchyme cell (pmc) patterning in the postgastrular embryo, and also failure of gastrulation. To further analyze these phenotypes, chimeric embryos were constructed consisting of two labeled micromeres combined with micromereless 4th cleavage host embryos; either the micromeres or the hosts contained alphaSpdri MASO. These experiments showed that, while Spdri expression is required autonomously for expression of skeletogenic genes prior to ingression, complete skeletogenesis also requires the expression of oral ectoderm patterning information. Presentation of this information on the oral side of the blastocoel in turn depends on Spdri expression in the oral ectoderm. Failure of gastrulation is not due to indirect interference with endomesodermal specification per se, since all endomesodermal genes tested function normally in alphaSpdri MASO embryos. Part of its cause is interference by alphaSpdri MASO with a late signaling function on the part of the micromere descendants that is needed to complete clearance of the Soxb1 repressor of gastrulation from the prospective endoderm, but in addition there is a nonautonomous oral ectoderm effect.

T-box family transcription factors have been identified in many organisms and are frequently associated with patterning events during embryonic development. With an interest in the molecular basis of patterning in the sea urchin embryo, we identified several members of the T-box family in Lytechinus variegatus. Here, we report the cloning and characterization of an ortholog of the Tbx2/3 subfamily, LvTbx2/3. To characterize the spatial distribution of LvTbx2/3 protein throughout sea urchin embryogenesis, a polyclonal antiserum was generated. Nuclear localization of LvTbx2/3 initiated at the mesenchyme blastula stage and protein was present into the pluteus stage. Localization was asymmetric throughout this period and costaining with marker genes indicated that asymmetry was about the oral/aboral (O/A) axis. Asymmetric distribution of LvTbx2/3 was observed in the aboral territories of all three germ layers. In the skeletogenic mesoderm lineage, LvTbx2/3 expression was dynamic because expression appeared initially in all skeletogenic mesenchyme cells (PMCs) but, subsequently, became refined solely to the aboral ones during skeletogenesis. To determine if the aboral expression of LvTbx2/3 is linked between germ layers, and to place LvTbx2/3 in the sequence of events that specifies the O/A axis, the effects of a series of perturbations to O/A polarity on LvTbx2/3 expression in each germ layer were examined. Preventing the nuclear localization of beta-catenin, pharmacological disruption of the O/A axis with NiCl(2), overexpression of BMP2/4 and disruption of the extracellular matrix all blocked LvTbx2/3 expression in all germ layers. This indicates that expression of LvTbx2/3 in the aboral territories of each germ layer is a common aspect of O/A specification, downstream of the molecular events that specify the axis. Furthermore, blocking the nuclear localization of beta-catenin, overexpression of BMP2/4 and disruption of the extracellular matrix also prevented the oral (stomodael) expression of LvBrachyury (LvBrac) protein, indicating that the O/A axis is established by a complex series of events. Last, the function of LvTbx2/3 in the formation of the O/A axis was characterized by examining the phenotypic consequences of ectopic expression of LvTbx2/3 mRNA on embryonic development and the expression of marker genes that identify specific germ layers and tissues. Ectopic expression of LvTbx2/3 produced profound morphogenetic defects in derivatives of each germ layer with no apparent loss in specification events in those tissues. This indicates that LvTbx2/3 functions as a regulator of morphogenetic movements in the aboral compartments of the ectoderm, endoderm and mesoderm.

We present the current form of a provisional DNA sequence-based regulatory gene network that explains in outline how endomesodermal specification in the sea urchin embryo is controlled. The model of the network is in a continuous process of revision and growth as new genes are added and new experimental results become available; see http://www.its.caltech.edu/~mirsky/endomeso.htm (End-mes Gene Network Update) for the latest version. The network contains over 40 genes at present, many newly uncovered in the course of this work, and most encoding DNA-binding transcriptional regulatory factors. The architecture of the network was approached initially by construction of a logic model that integrated the extensive experimental evidence now available on endomesoderm specification. The internal linkages between genes in the network have been determined functionally, by measurement of the effects of regulatory perturbations on the expression of all relevant genes in the network. Five kinds of perturbation have been applied: (1) use of morpholino antisense oligonucleotides targeted to many of the key regulatory genes in the network; (2) transformation of other regulatory factors into dominant repressors by construction of Engrailed repressor domain fusions; (3) ectopic expression of given regulatory factors, from genetic expression constructs and from injected mRNAs; (4) blockade of the beta-catenin/Tcf pathway by introduction of mRNA encoding the intracellular domain of cadherin; and (5) blockade of the Notch signaling pathway by introduction of mRNA encoding the extracellular domain of the Notch receptor. The network model predicts the cis-regulatory inputs that link each gene into the network. Therefore, its architecture is testable by cis-regulatory analysis. Strongylocentrotus purpuratus and Lytechinus variegatus genomic BAC recombinants that include a large number of the genes in the network have been sequenced and annotated. Tests of the cis-regulatory predictions of the model are greatly facilitated by interspecific computational sequence comparison, which affords a rapid identification of likely cis-regulatory elements in advance of experimental analysis. The network specifies genomically encoded regulatory processes between early cleavage and gastrula stages. These control the specification of the micromere lineage and of the initial veg(2) endomesodermal domain; the blastula-stage separation of the central veg(2) mesodermal domain (i.e., the secondary mesenchyme progenitor field) from the peripheral veg(2) endodermal domain; the stabilization of specification state within these domains; and activation of some downstream differentiation genes. Each of the temporal-spatial phases of specification is represented in a subelement of the network model, that treats regulatory events within the relevant embryonic nuclei at particular stages., ((c) 2002 Elsevier Science (USA).)

Development of the body plan is controlled by large networks of regulatory genes. A gene regulatory network that controls the specification of endoderm and mesoderm in the sea urchin embryo is summarized here. The network was derived from large-scale perturbation analyses, in combination with computational methodologies, genomic data, cis-regulatory analysis, and molecular embryology. The network contains over 40 genes at present, and each node can be directly verified at the DNA sequence level by cis-regulatory analysis. Its architecture reveals specific and general aspects of development, such as how given cells generate their ordained fates in the embryo and why the process moves inexorably forward in developmental time.

The developmental gene regulatory networks (dGRNs) of two sea urchin species, Lytechinus variegatus (Lv) and Strongylocentrotus purpuratus (Sp), have remained remarkably similar despite about 50 million years since a common ancestor. Hundreds of parallel experimental perturbations of transcription factors with similar outcomes support this conclusion. A recent scRNA-seq analysis suggested that the earliest expression of several genes within the dGRNs differs between Lv and Sp. Here, we present a careful reanalysis of the dGRNs in these two species, paying close attention to timing of first expression. We find that initial expression of genes critical for cell fate specification occurs during several compressed time periods in both species. Previously unrecognized feedback circuits are inferred from the temporally corrected dGRNs. Although many of these feedbacks differ in location within the respective GRNs, the overall number is similar between species. We identify several prominent differences in timing of first expression for key developmental regulatory genes; comparison with a third species indicates that these heterochronies likely originated in an unbiased manner with respect to embryonic cell lineage and evolutionary branch. Together, these results suggest that interactions can evolve even within highly conserved dGRNs and that feedback circuits may buffer the effects of heterochronies in the expression of key regulatory genes. [ABSTRACT FROM AUTHOR]

Using scRNA-seq coupled with computational approaches, we studied transcriptional changes in cell states of sea urchin embryos during development to the larval stage. Eighteen closely spaced time points were taken during the first 24 h of development of Lytechinus variegatus (Lv). Developmental trajectories were constructed using Waddington-OT, a computational approach to 'stitch' together developmental time points. Skeletogenic and primordial germ cell trajectories diverged early in cleavage. Ectodermal progenitors were distinct from other lineages by the 6th cleavage, although a small percentage of ectoderm cells briefly co-expressed endoderm markers that indicated an early ecto-endoderm cell state, likely in cells originating from the equatorial region of the egg. Endomesoderm cells also originated at the 6th cleavage and this state persisted for more than two cleavages, then diverged into distinct endoderm and mesoderm fates asynchronously, with some cells retaining an intermediate specification status until gastrulation. Seventy-nine out of 80 genes (99%) examined, and included in published developmental gene regulatory networks (dGRNs), are present in the Lv-scRNA-seq dataset and are expressed in the correct lineages in which the dGRN circuits operate. [ABSTRACT FROM AUTHOR]

Lytechinus variegatus is a camarodont sea urchin found widely throughout the western Atlantic Ocean in a variety of shallow-water marine habitats. Its distribution, abundance, and amenability to developmental perturbation make it a popular model for ecologists and developmental biologists. Here, we present a chromosomal-level genome assembly of L. variegatus generated from a combination of PacBio long reads, 10× Genomics sequencing, and HiC chromatin interaction sequencing. We show L. variegatus has 19 chromosomes with an assembly size of 870.4 Mb. The contiguity and completeness of this assembly are reflected by a scaffold length N50 of 45.5 Mb and BUSCO completeness score of 95.5%. Ab initio and transcript-informed gene modeling and annotation identified 27,232 genes with an average gene length of 12.6 kb, comprising an estimated 39.5% of the genome. Repetitive regions, on the other hand, make up 45.4% of the genome. Physical mapping of well-studied developmental genes onto each chromosome reveals nonrandom spatial distribution of distinct genes and gene families, which provides insight into how certain gene families may have evolved and are transcriptionally regulated in this species. Lastly, aligning RNA-seq and ATAC-seq data onto this assembly demonstrates the value of highly contiguous, complete genome assemblies for functional genomics analyses that is unattainable with fragmented, incomplete assemblies. This genome will be of great value to the scientific community as a resource for genome evolution, developmental, and ecological studies of this species and the Echinodermata. [ABSTRACT FROM AUTHOR]