An ancestral Wnt-Brachyury feedback loop in axial patterning and recruitment of mesoderm-determining target genes.

Transcription factors are crucial drivers of cellular differentiation during animal development and often share ancient evolutionary origins. The T-box transcription factor Brachyury plays a pivotal role as an early mesoderm determinant and neural repressor in vertebrates; yet, the ancestral function and key evolutionary transitions of the role of this transcription factor remain obscure. Here, we present a genome-wide target-gene screen using chromatin immunoprecipitation sequencing in the sea anemone Nematostella vectensis, an early branching non-bilaterian, and the sea urchin Strongylocentrotus purpuratus, a representative of the sister lineage of chordates. Our analysis reveals an ancestral gene regulatory feedback loop connecting Brachyury, FoxA and canonical Wnt signalling involved in axial patterning that predates the cnidarian-bilaterian split about 700 million years ago. Surprisingly, we also found that part of the gene regulatory network controlling the fate of neuromesodermal progenitors in vertebrates was already present in the common ancestor of cnidarians and bilaterians. However, while several endodermal and neuronal Brachyury target genes are ancestrally shared, hardly any of the key mesodermal downstream targets in vertebrates are found in the sea anemone or the sea urchin. Our study suggests that a limited number of target genes involved in mesoderm formation were newly acquired in the vertebrate lineage, leading to a dramatic shift in the function of this ancestral developmental regulator.

Molecular evidence for a single origin of ultrafiltration-based excretory organs.

Excretion is an essential physiological process, carried out by all living organisms, regardless of their size or complexity.1-3 Both protostomes (e.g., flies and flatworms) and deuterostomes (e.g., humans and sea urchins) possess specialized excretory organs serving that purpose. Those organs exhibit an astonishing diversity, ranging from units composed of just few distinct cells (e.g., protonephridia) to complex structures, built by millions of cells of multiple types with divergent morphology and function (e.g., vertebrate kidneys).4,5 Although some molecular similarities between the development of kidneys of vertebrates and the regeneration of the protonephridia of flatworms have been reported,6,7 the molecular underpinnings of the development of excretory organs have never been systematically studied in a comparative context.4 Here, we show that a set of transcription factors (eya, six1/2, pou3, sall, lhx1/5, and osr) and structural proteins (nephrin, kirre, and zo1) is expressed in the excretory organs of a phoronid, brachiopod, annelid, onychophoran, priapulid, and hemichordate that represent major protostome lineages and non-vertebrate deuterostomes. We demonstrate that the molecular similarity observed in the vertebrate kidney and flatworm protonephridia6,7 is also seen in the developing excretory organs of those animals. Our results show that all types of ultrafiltration-based excretory organs are patterned by a conserved set of developmental genes, an observation that supports their homology. We propose that the last common ancestor of protostomes and deuterostomes already possessed an ultrafiltration-based organ that later gave rise to the vast diversity of extant excretory organs, including both proto- and metanephridia.

FGF signaling acts on different levels of mesoderm development within Spiralia.

FGF signaling is involved in mesoderm induction in members of deuterostomes (e.g. tunicates, hemichordates), but not in flies and nematodes, in which it has a role in mesoderm patterning and migration. However, we need comparable studies in other protostome taxa in order to decipher whether this mesoderm-inducing function of FGF extends beyond the lineage of deuterostomes. Here, we investigated the role of FGF signaling in mesoderm development in three species of lophophorates, a clade within the protostome group Spiralia. Our gene expression analyses show that the mesodermal molecular patterning is conserved between brachiopods and phoronids, but the spatial and temporal recruitment of transcription factors differs significantly. Moreover, the use of the inhibitor SU5402 demonstrates that FGF signaling is involved in different steps of mesoderm development, as well as in morphogenetic movements of gastrulation and axial elongation. Our findings suggest that the mesoderm-inducing role of FGF extends beyond the group of deuterostomes.

Molecular patterning during the development of Phoronopsis harmeri reveals similarities to rhynchonelliform brachiopods.

BACKGROUND: Phoronids, rhynchonelliform and linguliform brachiopods show striking similarities in their embryonic fate maps, in particular in their axis specification and regionalization. However, although brachiopod development has been studied in detail and demonstrated embryonic patterning as a causal factor of the gastrulation mode (protostomy vs deuterostomy), molecular descriptions are still missing in phoronids. To understand whether phoronids display underlying embryonic molecular mechanisms similar to those of brachiopods, here we report the expression patterns of anterior (otx, gsc, six3/6, nk2.1), posterior (cdx, bra) and endomesodermal (foxA, gata4/5/6, twist) markers during the development of the protostomic phoronid Phoronopsis harmeri. RESULTS: The transcription factors foxA, gata4/5/6 and cdx show conserved expression in patterning the development and regionalization of the phoronid embryonic gut, with foxA expressed in the presumptive foregut, gata4/5/6 demarcating the midgut and cdx confined to the hindgut. Furthermore, six3/6, usually a well-conserved anterior marker, shows a remarkably dynamic expression, demarcating not only the apical organ and the oral ectoderm, but also clusters of cells of the developing midgut and the anterior mesoderm, similar to what has been reported for brachiopods, bryozoans and some deuterostome Bilateria. Surprisingly, brachyury, a transcription factor often associated with gastrulation movements and mouth and hindgut development, seems not to be involved with these patterning events in phoronids. CONCLUSIONS: Our description and comparison of gene expression patterns with other studied Bilateria reveals that the timing of axis determination and cell fate distribution of the phoronid shows highest similarity to that of rhynchonelliform brachiopods, which is likely related to their shared protostomic mode of development. Despite these similarities, the phoronid Ph. harmeri also shows particularities in its development, which hint to divergences in the arrangement of gene regulatory networks responsible for germ layer formation and axis specification.

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.

Active mode of excretion across digestive tissues predates the origin of excretory organs.

Most bilaterian animals excrete toxic metabolites through specialized organs, such as nephridia and kidneys, which share morphological and functional correspondences. In contrast, excretion in non-nephrozoans is largely unknown, and therefore the reconstruction of ancestral excretory mechanisms is problematic. Here, we investigated the excretory mode of members of the Xenacoelomorpha, the sister group to Nephrozoa, and Cnidaria, the sister group to Bilateria. By combining gene expression, inhibitor experiments, and exposure to varying environmental ammonia conditions, we show that both Xenacoelomorpha and Cnidaria are able to excrete across digestive-associated tissues. However, although the cnidarian Nematostella vectensis seems to use diffusion as its main excretory mode, the two xenacoelomorphs use both active transport and diffusion mechanisms. Based on these results, we propose that digestive-associated tissues functioned as excretory sites before the evolution of specialized organs in nephrozoans. We conclude that the emergence of a compact, multiple-layered bilaterian body plan necessitated the evolution of active transport mechanisms, which were later recruited into the specialized excretory organs.

Echinoderm systems for gene regulatory studies in evolution and development.

One of the main challenges in Evolutionary Developmental Biology is to understand to which extent developmental changes are driven by regulatory alterations in the genomic sequence. In the recent years, the focus of comparative developmental studies has moved towards a systems biology approach providing a better understanding of the evolution of gene interactions that form the so called Gene Regulatory Networks (GRN). Echinoderms provide a powerful system to reveal regulatory mechanisms and within the past decade, due to the latest technological innovations, a great number of studies have provided valuable information for comparative GRN analyses. In this review we describe recent advances in evolution of GRNs arising from echinoderm systems, focusing on the properties of conserved regulatory kernels, circuit co-option events and GRN topological rearrangements.

Logics and properties of a genetic regulatory program that drives embryonic muscle development in an echinoderm.

Evolutionary origin of muscle is a central question when discussing mesoderm evolution. Developmental mechanisms underlying somatic muscle development have mostly been studied in vertebrates and fly where multiple signals and hierarchic genetic regulatory cascades selectively specify myoblasts from a pool of naive mesodermal progenitors. However, due to the increased organismic complexity and distant phylogenetic position of the two systems, a general mechanistic understanding of myogenesis is still lacking. In this study, we propose a gene regulatory network (GRN) model that promotes myogenesis in the sea urchin embryo, an early branching deuterostome. A fibroblast growth factor signaling and four Forkhead transcription factors consist the central part of our model and appear to orchestrate the myogenic process. The topological properties of the network reveal dense gene interwiring and a multilevel transcriptional regulation of conserved and novel myogenic genes. Finally, the comparison of the myogenic network architecture among different animal groups highlights the evolutionary plasticity of developmental GRNs.

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.

Myogenesis in the sea urchin embryo: the molecular fingerprint of the myoblast precursors.

BACKGROUND: In sea urchin larvae the circumesophageal fibers form a prominent muscle system of mesodermal origin. Although the morphology and later development of this muscle system has been well-described, little is known about the molecular signature of these cells or their precise origin in the early embryo. As an invertebrate deuterostome that is more closely related to the vertebrates than other commonly used model systems in myogenesis, the sea urchin fills an important phylogenetic gap and provides a unique perspective on the evolution of muscle cell development. RESULTS: Here, we present a comprehensive description of the development of the sea urchin larval circumesophageal muscle lineage beginning with its mesodermal origin using high-resolution localization of the expression of several myogenic transcriptional regulators and differentiation genes. A few myoblasts are bilaterally distributed at the oral vegetal side of the tip of the archenteron and first appear at the late gastrula stage. The expression of the differentiation genes Myosin Heavy Chain, Tropomyosin I and II, as well as the regulatory genes MyoD2, FoxF, FoxC, FoxL1, Myocardin, Twist, and Tbx6 uniquely identify these cells. Interestingly, evolutionarily conserved myogenic factors such as Mef2, MyoR and Six1/2 are not expressed in sea urchin myoblasts but are found in other mesodermal domains of the tip of the archenteron. The regulatory states of these domains were characterized in detail. Moreover, using a combinatorial analysis of gene expression we followed the development of the FoxF/FoxC positive cells from the onset of expression to the end of gastrulation. Our data allowed us to build a complete map of the Non-Skeletogenic Mesoderm at the very early gastrula stage, in which specific molecular signatures identify the precursors of different cell types. Among them, a small group of cells within the FoxY domain, which also express FoxC and SoxE, have been identified as plausible myoblast precursors. Together, these data support a very early gastrula stage segregation of the myogenic lineage. CONCLUSIONS: From this analysis, we are able to precisely define the regulatory and differentiation signatures of the circumesophageal muscle in the sea urchin embryo. Our findings have important implications in understanding the evolution of development of the muscle cell lineage at the molecular level. The data presented here suggest a high level of conservation of the myogenic specification mechanisms across wide phylogenetic distances, but also reveal clear cases of gene cooption.