Molecular and Cellular Characterization of the TH Pathway in the Sea Urchin Strongylocentrotus purpuratus.

Thyroid Hormones (THs) are a class of signaling molecules produced by coupling iodine with tyrosine residues. In vertebrates, extensive data support their important role in a variety of processes such as metabolism, development and metamorphosis. On the other hand, in invertebrates, the synthesis and role of the THs have been, so far, poorly investigated, thus limiting our understanding of the function and evolution of this important animal signaling pathway. In sea urchins, for example, while several studies focused on the availability and function of external sources of iodotyrosines, preliminary evidence suggests that an endogenous TH pathway might be in place. Here, integrating available literature with an in silico analysis, various homologous genes of the vertebrate TH molecular toolkit have been identified in the sea urchin Strongylocentrotus purpuratus. They include genes involved in the synthesis (Sp-Pxdn), metabolism (Sp-Dios), transport (Sp-Ttrl, Sp-Mct7/8/10) and response (Sp-Thr, Sp-Rxr and Sp-Integrin αP) to thyroid hormones. To understand the cell type(s) involved in TH synthesis and/or response, we studied the spatial expression of the TH toolkit during urchin development. Exploiting single-cell transcriptomics data in conjunction with in situ hybridization and immunohistochemistry, we identified cell types that are potentially producing or responding to THs in the sea urchin. Finally, growing sea urchin embryos until the larva stage with and without a source of inorganic iodine, we provided evidence that iodine organification is important for larval skeleton growth.

Methodology for Whole Mount and Fluorescent RNA In Situ Hybridization in Echinoderms: Single, Double, and Beyond.

Identifying the location of a specific RNA in a cell, tissue, or embryo is essential to understand its function. Here we use echinoderm embryos to demonstrate the power of fluorescence in situ RNA hybridizations to localize sites of specific RNA accumulation in whole mount embryo applications. We add to this technology the use of various probe-labeling technologies to colabel multiple RNAs in one application and we describe protocols for incorporating immunofluorescence approaches to maximize the information obtained in situ. We offer alternatives for these protocols and troubleshooting advice to identify steps in which the procedure may have failed. Overall, echinoderms are wonderfully suited for these technologies, and these protocols are applicable to a wide range of cells, tissues, and embryos.

ATAC-Seq for Assaying Chromatin Accessibility Protocol Using Echinoderm Embryos.

Cis-regulatory elements (CREs) and transcription factors (TFs) associated with them determine temporal and spatial domains of gene expression. Therefore, identification of these CREs and TFs is crucial to elucidating transcriptional programs across taxa. With chromatin accessibility facilitating transcription factor access to DNA, the identification of regions of open chromatin sheds light both on the function of the regulatory elements and their evolution, thus allowing the recognition of potential CREs. Buenrostro and colleagues have developed a novel method for exploring chromatin accessibility: assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), which can be used for the purpose of identifying putative CREs. This method was shown to have considerable advantages when compared to traditional methods such as sequence conservation analyses or functional assays. Here we present the adaptation of the ATAC-seq method to echinoderm species and discuss how it can be used for CRE discovery.

Unravelling the evolutionary history of kisspeptin.

Experiments in sea cucumbers reveal how the physiological responses regulated by a neuropeptide called kisspeptin have evolved.

Development and evolution of gut structures: from molecules to function.

The emergence of a specialized system for food digestion and nutrient absorption was a crucial innovation for multicellular organisms. Digestive systems with different levels of complexity evolved in different animals, with the endoderm-derived one-way gut of most bilaterians to be the prevailing and more specialized form. While the molecular events regulating the early phases of embryonic tissue specification have been deeply investigated in animals occupying different phylogenetic positions, the mechanisms underlying gut patterning and gut-associated structures differentiation are still mostly obscure. In this review, we describe the main discoveries in gut and gut-associated structures development in echinoderm larvae (mainly for sea urchin and, when available, for sea star) and compare them with existing information in vertebrates. An impressive degree of conservation emerges when comparing the transcription factor toolkits recruited for gut cells and tissue differentiation in animals as diverse as echinoderms and vertebrates, thus suggesting that their function emerged in the deuterostome ancestor.

Using ATAC-seq and RNA-seq to increase resolution in GRN connectivity.

Echinoderms have some of the most complete reconstructed developmental gene regulatory networks (GRN) of any embryo, accounting for the formation of most embryo tissues and organs. Yet, many nodes (genes and regulators) and their regulatory interactions are still to be uncovered. Traditionally, knockdown/knockout experiments are performed to determine regulator-gene interactions, which are individually validated by cis-regulatory analysis. Differential RNA-seq, combined with perturbation analysis, allows for genome-wide reconstruction of a GRN around given regulators; however, this level of resolution cannot determine direct interactions. ChiP-chip or ChIP-seq is better equipped for determining, genome-wide, whether binding of a given transcription factor (TF) to cis-regulatory elements occurs. Antibodies for the TFs of interest must be available, and if not, this presents a limiting factor. ATAC-seq identifies regions of open chromatin, that are typically trimethylated at H3K4, H3K36 and H3K79 (Kouzarides, 2007), for a given time point, condition, or tissue. This technology combined with RNA-seq and perturbation analysis provides high resolution of the possible functional interactions occurring during development. Additionally, ATAC-seq is less expensive than ChIP-seq, requires less starting material, and provides a global view of regulatory regions. This chapter provides detailed steps to identify potential regulatory relationships between the nodes of a GRN, given a well assembled genome, annotated with gene models, and ATAC-seq data combined with RNA-seq and knockdown experiments.

A conceptual history of the "regulatory genome": From Theodor Boveri to Eric Davidson.

The formalization of the idea of "Regulatory Genome" is a recent one. However, it stems from a long tradition in the study of how the genetic information is transferred between generations. Theodore Boveri suggested for the first time that the whole genome participates in the shaping of individuals. Through a long lineage of researchers, we have learned how this whole-genome activity is regulated, in space and time. It is, however, due to the insights and experimental approaches taken by different researchers, among them Eric Davidson and associates, that we understand the mechanistic basis of this regulation. Whole batteries of regulatory genes interact through their cis-regulatory modules, generating a precise pattern of cross-controlled gene activity (Gene Regulatory Networks). How these genes are deployed in development and evolution has become an area of vibrant research. Here we revisit the history of this intellectual endeavour, taking as key defining points along this historical trajectory the contributions of Theodor Boveri and Eric Davidson.

New Neuronal Subtypes With a "Pre-Pancreatic" Signature in the Sea Urchin Stongylocentrotus purpuratus.

Neurons and pancreatic endocrine cells have a common physiology and express a similar toolkit of transcription factors during development. To explain these common features, it has been hypothesized that pancreatic cells most likely co-opted a pre-existing gene regulatory program from ancestral neurons. To test this idea, we looked for neurons with a "pre-pancreatic" program in an early-branched deuterostome, the sea urchin. Only vertebrates have a proper pancreas, however, our lab previously found that cells with a pancreatic-like signature are localized within the sea urchin embryonic gut. We also found that the pancreatic transcription factors Xlox/Pdx1 and Brn1/2/4 co-localize in a sub-population of ectodermal cells. Here, we find that the ectodermal SpLox+ SpBrn1/2/4 cells are specified as SpSoxC and SpPtf1a neuronal precursors that become the lateral ganglion and the apical organ neurons. Two of the SpLox+ SpBrn1/2/4 cells also express another pancreatic transcription factor, the LIM-homeodomain gene islet-1. Moreover, we find that SpLox neurons produce the neuropeptide SpANP2, and that SpLox regulates SpANP2 expression. Taken together, our data reveal that there is a subset of sea urchin larval neurons with a gene program that predated pancreatic cells. These findings suggest that pancreatic endocrine cells co-opted a regulatory signature from an ancestral neuron that was already present in an early-branched deuterostome.

The crowns have eyes: multiple opsins found in the eyes of the crown-of-thorns starfish Acanthaster planci.

BACKGROUND: Opsins are G protein-coupled receptors used for both visual and non-visual photoreception, and these proteins evolutionarily date back to the base of the bilaterians. In the current sequencing age, phylogenomic analysis has proven to be a powerful tool, facilitating the increase in knowledge about diversity within the opsin subclasses and, so far, at least nine types of opsins have been identified. Within echinoderms, opsins have been studied in Echinoidea and Ophiuroidea, which do not possess proper image forming eyes, but rather widely dispersed dermal photoreceptors. However, most species of Asteroidea, the starfish, possess true eyes and studying them will shed light on the diversity of opsin usage within echinoderms and help resolve the evolutionary history of opsins. RESULTS: Using high-throughput RNA sequencing, we have sequenced and analyzed the transcriptomes of different Acanthaster planci tissue samples: eyes, radial nerve, tube feet and a mixture of tissues from other organs. At least ten opsins were identified, and eight of them were found significantly differentially expressed in both eyes and radial nerve, with R-opsin being the most highly expressed in the eye. CONCLUSION: This study provides new important insight into the involvement of opsins in visual and nonvisual photoreception. Of relevance, we found the first indication of an r-opsin photopigment expressed in a well-developed visual eye in a deuterostome animal. Additionally, we provided tissue specific A. planci transcriptomes that will aid in future Evo Devo studies.

A challenging diagnosis of dyspnea: A case report of contralateral reexpansion pulmonary edema.

Reexpansion pulmonary edema (RPE) is an uncommon complication of thoracentesis or chest drainage. It occurs in the ipsilateral or contralateral lung. Causes, pathogenesis and therapy are not well understood especially for contralateral RPE. We describe a case of fatal contralateral RPE in a 59-years-old woman with right lung cancer underwent ultrasound-guided thoracentesis for massive pleural effusion and severe dyspnea. Pathogenesis of contralateral RPE is probably multifactorial and in this case is mostly due to the overperfusion of the healthy lung and consequent capillary damage. The right therapy for this condition is not known.

Omics approaches to study gene regulatory networks for development in echinoderms.

Gene regulatory networks (GRNs) describe the interactions for a developmental process at a given time and space. Historically, perturbation experiments represent one of the key methods for analyzing and reconstructing a GRN, and the GRN governing early development in the sea urchin embryo stands as one of the more deeply dissected so far. As technology progresses, so do the methods used to address different biological questions. Next-generation sequencing (NGS) has become a standard experimental technique for genome and transcriptome sequencing and studies of protein-DNA interactions and DNA accessibility. While several efforts have been made toward the integration of different omics approaches for the study of the regulatory genome in many animals, in a few cases, these are applied with the purpose of reconstructing and experimentally testing developmental GRNs. Here, we review emerging approaches integrating multiple NGS technologies for the prediction and validation of gene interactions within echinoderm GRNs. These approaches can be applied to both 'model' and 'non-model' organisms. Although a number of issues still need to be addressed, advances in NGS applications, such as assay for transposase-accessible chromatin sequencing, combined with the availability of embryos belonging to different species, all separated by various evolutionary distances and accessible to experimental regulatory biology, place echinoderms in an unprecedented position for the reconstruction and evolutionary comparison of developmental GRNs. We conclude that sequencing technologies and integrated omics approaches allow the examination of GRNs on a genome-wide scale only if biological perturbation and cis-regulatory analyses are experimentally accessible, as in the case of echinoderm embryos.

Reconstructing SALMFamide Neuropeptide Precursor Evolution in the Phylum Echinodermata: Ophiuroid and Crinoid Sequence Data Provide New Insights.

The SALMFamides are a family of neuropeptides that act as muscle relaxants in echinoderms. Analysis of genome/transcriptome sequence data from the sea urchin Strongylocentrotus purpuratus (Echinoidea), the sea cucumber Apostichopus japonicus (Holothuroidea), and the starfish Patiria miniata (Asteroidea) reveals that in each species there are two types of SALMFamide precursor: an L-type precursor comprising peptides with a C-terminal LxFamide-type motif and an F-type precursor solely or largely comprising peptides with a C-terminal FxFamide-type motif. Here, we have identified transcripts encoding SALMFamide precursors in the brittle star Ophionotus victoriae (Ophiuroidea) and the feather star Antedon mediterranea (Crinoidea). We have also identified SALMFamide precursors in other species belonging to each of the five echinoderm classes. As in S. purpuratus, A. japonicus, and P. miniata, in O. victoriae there is one L-type precursor and one F-type precursor. However, in A. mediterranea only a single SALMFamide precursor was found, comprising two peptides with a LxFamide-type motif, one with a FxFamide-type motif, five with a FxLamide-type motif, and four with a LxLamide-type motif. As crinoids are basal to the Echinozoa (Holothuroidea + Echinoidea) and Asterozoa (Asteroidea + Ophiuroidea) in echinoderm phylogeny, one model of SALMFamide precursor evolution would be that ancestrally there was a single SALMFamide gene encoding a variety of SALMFamides (as in crinoids), which duplicated in a common ancestor of the Echinozoa and Asterozoa and then specialized to encode L-type SALMFamides or F-type SALMFamides. Alternatively, a second SALMFamide precursor may remain to be discovered or may have been lost in crinoids. Further insights will be obtained if SALMFamide receptors are identified, which would provide a molecular basis for experimental analysis of the functional significance of the "cocktails" of SALMFamides that exist in echinoderms.

Pattern and process during sea urchin gut morphogenesis: the regulatory landscape.

The development of the endoderm is a multistage process. From the initial specification of the endodermal domain in the embryo to the final regionalization of the gut, there are multiple stages that require the involvement of complex gene regulatory networks. In one concrete case, the sea urchin embryo, some of these stages and their genetic control are (relatively) well understood. Several studies have underscored the relevance of individual transcription factor activities in the process, but very few have focused the attention on gene interactions within specific gene regulatory networks (GRNs). Sea urchins offer an ideal system to study the different factors involved in the morphogenesis of the gut. Here we review the knowledge gained over the last 10 years on the process and its regulation, from the early specification of endodermal lineages to the late events linked to the patterning of functional domains in the gut. A lesson of remarkable importance has been learnt from comparison of the mechanisms involved in gut formation in different bilaterian animals; some of these genetic mechanisms are particularly well conserved. Patterning the gut seems to involve common molecular players and shared interactions, whether we look at mammals or echinoderms. This astounding degree of conservation reveals some key aspects of deep homology that are most probably shared by all bilaterian guts.

C-opsin expressing photoreceptors in echinoderms.

Today's progress in molecular analysis and, in particular, the increased availability of genome sequences have enabled us to investigate photoreceptor cells (PRCs) in organisms that were formerly inaccessible to experimental manipulation. Our studies of marine non-chordate deuterostomes thus aim to bridge a gap of knowledge regarding the evolution of deuterostome PRCs prior to the emergence of vertebrates' eyes. In this contribution, we will show evidence for expression of a c-opsin photopigment, which, according to our phylogenetic analysis, is closely related to an assemblage of chordate visual c-opsins. An antibody raised against sea urchins' c-opsin protein (Sp-Opsin1) recognizes epitopes in a variety of tissues of different echinoderms. While in sea urchins this c-opsin is expressed in locomotory and buccal tube feet, spines, pedicellaria, and epidermis, in brittlestars and starfish we found the immuno-reaction to be located exclusively in cells within the animals' spines. Structural characteristics of these c-opsin+ PRC types include the close vicinity/connection to nerve strands and a, so far unexplored, conspicuous association with the animals' calcite skeleton, which previously has been hypothesized to play a role in echinoderm photobiology. These features are discussed within the context of the evolution of photoreceptors in echinoderms and in deuterostomes generally.

Two ParaHox genes, SpLox and SpCdx, interact to partition the posterior endoderm in the formation of a functional gut.

We report the characterization of the ortholog of the Xenopus XlHbox8 ParaHox gene from the sea urchin Strongylocentrotus purpuratus, SpLox. It is expressed during embryogenesis, first appearing at late gastrulation in the posterior-most region of the endodermal tube, becoming progressively restricted to the constriction between the mid- and hindgut. The physiological effects of the absence of the activity of this gene have been analyzed through knockdown experiments using gene-specific morpholino antisense oligonucleotides. We show that blocking the translation of the SpLox mRNA reduces the capacity of the digestive tract to process food, as well as eliminating the morphological constriction normally present between the mid- and hindgut. Genetic interactions of the SpLox gene are revealed by the analysis of the expression of a set of genes involved in endoderm specification. Two such interactions have been analyzed in more detail: one involving the midgut marker gene Endo16, and another involving the other endodermally expressed ParaHox gene, SpCdx. We find that SpLox is able to bind Endo16 cis-regulatory DNA, suggesting direct repression of Endo16 expression in presumptive hindgut territories. More significantly, we provide the first evidence of interaction between ParaHox genes in establishing hindgut identity, and present a model of gene regulation involving a negative-feedback loop.

Opsins and clusters of sensory G-protein-coupled receptors in the sea urchin genome.

Rhodopsin-type G-protein-coupled receptors (GPCRs) contribute the majority of sensory receptors in vertebrates. With 979 members, they form the largest GPCR family in the sequenced sea urchin genome, constituting more than 3% of all predicted genes. The sea urchin genome encodes at least six Opsin proteins. Of these, one rhabdomeric, one ciliary and two G(o)-type Opsins can be assigned to ancient bilaterian Opsin subfamilies. Moreover, we identified four greatly expanded subfamilies of rhodopsin-type GPCRs that we call sea urchin specific rapidly expanded lineages of GPCRs (surreal-GPCRs). Our analysis of two of these groups revealed genomic clustering and single-exon gene structures similar to the most expanded group of vertebrate rhodopsin-type GPCRs, the olfactory receptors. We hypothesize that these genes arose by rapid duplication in the echinoid lineage and act as chemosensory receptors of the animal. In support of this, group B surreal-GPCRs are most prominently expressed in distinct classes of pedicellariae and tube feet of the adult sea urchin, structures that have previously been shown to react to chemical stimuli and to harbor sensory neurons in echinoderms. Notably, these structures also express different opsins, indicating that sea urchins possess an intricate molecular set-up to sense their environment.

The sea urchin kinome: a first look.

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.

Identification and developmental expression of the ets gene family in the sea urchin (Strongylocentrotus purpuratus).

A systematic search in the available scaffolds of the Strongylocentrotus purpuratus genome has revealed that this sea urchin has 11 members of the ets gene family. A phylogenetic analysis of these genes showed that almost all vertebrate ets subfamilies, with the exception of one, so far found only in mammals, are each represented by one orthologous sea urchin gene. The temporal and spatial expression of the identified ETS factors was also analyzed during embryogenesis. Five ets genes (Sp-Ets1/2, Sp-Tel, Sp-Pea, Sp-Ets4, Sp-Erf) are also maternally expressed. Three genes (Sp-Elk, Sp-Elf, Sp-Erf) are ubiquitously expressed during embryogenesis, while two others (Sp-Gabp, Sp-Pu.1) are not transcribed until late larval stages. Remarkably, five of the nine sea urchin ets genes expressed during embryogenesis are exclusively (Sp-Ets1/2, Sp-Erg, Sp-Ese) or additionally (Sp-Tel, Sp-Pea) expressed in mesenchyme cells and/or their progenitors. Functional analysis of Sp-Ets1/2 has previously demonstrated an essential role of this gene in the specification of the skeletogenic mesenchyme lineage. The dynamic, and in some cases overlapping and/or unique, developmental expression pattern of the latter five genes suggests a complex, non-redundant function for ETS factors in sea urchin mesenchyme formation and differentiation.

Genetic organization and embryonic expression of the ParaHox genes in the sea urchin S. purpuratus: insights into the relationship between clustering and colinearity.

The ANTP family of homeodomain transcription factors consists of three major groups, the NKL, the extended Hox, and the Hox/ParaHox family. Hox genes and ParaHox genes are often linked in the genome forming two clusters of genes, the Hox cluster and the ParaHox cluster, and are expressed along the major body axis in a nested fashion, following the relative positions of the genes within these clusters, a property called colinearity. While the presences of a Hox cluster and a ParaHox cluster appear to be primitive for bilaterians, few taxa have actually been examined for spatial and temporal colinearity, and, aside from chordates, even fewer still manifest it. Here we show that the ParaHox genes of the sea urchin Strongylocentrotus purpuratus show both spatial and temporal colinearity, but with peculiarities. Specifically, two of the three ParaHox genes-discovered through the S. purpuratus genome project-Sp-lox and Sp-Cdx, are expressed in the developing gut with nested domains in a spatially colinear manner. However, transcripts of Sp-Gsx, although anterior of Sp-lox, are detected in the ectoderm and not in the gut. Strikingly, the expression of the three ParaHox genes would follow temporal colinearity if they were clustered in the same order as in chordates, but each ParaHox gene is actually found on a different genomic scaffold (>300 kb each), which suggests that they are not linked into a single coherent cluster. Therefore, ParaHox genes are dispersed in the genome and are used during embryogenesis in a temporally and spatially coherent manner, whereas the Hox genes, now fully sequenced and annotated, are still linked and are employed as a complex only during the emergence of the adult body plan in the larva.

A MAPK pathway is involved in the control of mitosis after fertilization of the sea urchin egg.

Activation and role of mitogen-activated protein (MAP) kinase (MAPK) during mitosis are still matters of controversy in early embryos. We report here that an ERK-like protein is present and highly phosphorylated in unfertilized sea urchin eggs. This MAPK becomes dephosphorylated after fertilization and a small pool of it is transiently reactivated during mitosis. The phosphorylated ERK-like protein is localized to the nuclear region and then to the mitotic poles and the mitotic spindle. Treatment of eggs after fertilization with two different MEK inhibitors, PD 98059 and U0126, at low concentrations capable to selectively induce dephosphorylation of this ERK-like protein, or expression of a dominant-negative MEK1/2, perturbed mitotic progression. Our results suggest that an ERK-like cascade is part of a control mechanism that regulates mitotic spindle formation and the attachment of chromosomes to the spindle during the first mitosis of the sea urchin embryo.