Alx1, a member of the Cart1/Alx3/Alx4 subfamily of Paired-class homeodomain proteins, is an essential component of the gene network controlling skeletogenic fate specification in the sea urchin embryo

In the sea urchin embryo, the large micromeres and their progeny function as a critical signaling center and execute a complex morphogenetic program. We have identified a new and essential component of the gene network that controls large micromere specification, the homeodomain protein Alx1. Alx1 is expressed exclusively by cells of the large micromere lineage beginning in the first interphase after the large micromeres are born. Morpholino studies demonstrate that Alx1 is essential at an early stage of specification and controls downstream genes required for epithelial-mesenchymal transition and biomineralization. Expression of Alx1 is cell autonomous and regulated maternally through ß-catenin and its downstream effector, Pmar1. Alx1 expression can be activated in other cell lineages at much later stages of development, however, through a regulative pathway of skeletogenesis that is responsive to cell signaling. The Alx1 protein is highly conserved among euechinoid sea urchins and is closely related to the Cart1/Alx3/Alx4 family of vertebrate homeodomain proteins. In vertebrates, these proteins regulate the formation of skeletal elements of the limbs, face and neck. Our findings suggest that the ancestral deuterostome had a population of biomineral-forming mesenchyme cells that expressed an Alx1-like protein

Neurogenesis during Brittle Star Arm Regeneration Is Characterised by a Conserved Set of Key Developmental Genes

Neural regeneration is very limited in humans but extremely efficient in echinoderms. The brittle star Amphiura filiformis can regenerate both components of its central nervous system as well as the peripheral system, and understanding the molecular mechanisms underlying this ability is key for evolutionary comparisons not only within the echinoderm group, but also wider within deuterostomes. Here we characterise the neural regeneration of this brittle star using a combination of immunohistochemistry, in situ hybridization and Nanostring nCounter to determine the spatial and temporal expression of evolutionary conserved neural genes. We find that key genes crucial for the embryonic development of the nervous system in sea urchins and other animals are also expressed in the regenerating nervous system of the adult brittle star in a hierarchic and spatio-temporally restricted manner

The cis-regulatory system of the tbrain gene: Alternative use of multiple modules to promote skeletogenic expression in the sea urchin embryo

The genomic cis-regulatory systems controlling regulatory gene expression usually include multiple modules. The regulatory output of such systems at any given time depends on which module is directing the function of the basal transcription apparatus, and ultimately on the transcription factor inputs into that module. Here we examine regulation of the Strongylocentrotus purpuratus tbrain gene, a required activator of the skeletogenic specification state in the lineage descendant from the embryo micromeres. Alternate cis-regulatory modules were found to convey skeletogenic expression in reporter constructs. To determine their relative developmental functions in context, we made use of recombineered BAC constructs containing a GFP reporter and of derivatives from which specific modules had been deleted. The outputs of the various constructs were observed spatially by GFP fluorescence and quantitatively over time by QPCR. In the context of the complete genomic locus, early skeletogenic expression is controlled by an intron enhancer plus a proximal region containing a HesC site as predicted from network analysis. From ingression onward, however, a dedicated distal module utilizing positive Ets1/2 inputs contributes to definitive expression in the skeletogenic mesenchyme. This module also mediates a newly discovered negative Erg input which excludes non-skeletogenic mesodermal expression

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

Precambrian Animal Life: Probable Developmental and Adult Cnidarian Forms from Southwest China

The evolutionary divergence of cnidarian and bilaterian lineages from their remote metazoan ancestor occurred at an unknown depth in time before the Cambrian, since crown group representatives of each are found in Lower Cambrian fossil assemblages. We report here a variety of putative embryonic, larval, and adult microfossils deriving from Precambrian phosphorite deposits of Southwest China, which may predate the Cambrian radiation by 25–45 million years. These are most probably of cnidarian affinity. Large numbers of fossilized early planula-like larvae were observed under the microscope in sections. Though several forms are represented, the majority display remarkable conformity, which is inconsistent with the alternative that they are artifactual mineral inclusions. Some of these fossils are preserved in such high resolution that individual cells can be discerned. We confirm in detail an earlier report of the presence in the same deposits of tabulates, an extinct crown group anthozoan form. Other sections reveal structures that most closely resemble sections of basal modern corals. A large number of fossils similar to modern hydrozoan gastrulae were also observed. These again displayed great morphological consistency. Though only a single example is available, a microscopic animal remarkably similar to a modern adult hydrozoan is also presented. Taken together, the new observations reported in this paper indicate the existence of a diverse and already differentiated cnidarian fauna, long before the Cambrian evolutionary event. It follows that at least stem group bilaterians must also have been present at this time

An ancient role for Gata-1/2/3 and Scl transcription factor homologs in the development of immunocytes

AbstractAlthough vertebrate hematopoiesis is the focus of intense study, immunocyte development is well-characterized in only a few invertebrate groups. The sea urchin embryo provides a morphologically simple model for immune cell development in an organism that is phylogenetically allied to vertebrates. Larval immunocytes, including pigment cells and several blastocoelar cell subtypes, emerge from a population of non-skeletal mesodermal (NSM) precursors that is specified at the blastula stage. This ring of cells is first partitioned into oral and aboral fields with distinct blastocoelar and pigment cell gene regulatory programs. The oral field is subsequently specified into several distinct immune and non-immune cell types during gastrulation. Here we characterize the oral NSM expression and downstream function of two homologs of key vertebrate hematopoietic transcription factors: SpGatac, an ortholog of vertebrate Gata-1/2/3 and SpScl, an ortholog of Scl/Tal-2/Lyl-1. Perturbation of SpGatac affects blastocoelar cell migration at gastrulation and later expression of immune effector genes, whereas interference with SpScl function disrupts segregation of pigment and blastocoelar cell precursors. Homologs of several transcription regulators that interact with Gata-1/2/3 and Scl factors in vertebrate hematopoiesis are also co-expressed in the oral NSM, including SpE-protein, the sea urchin homolog of vertebrate E2A/HEB/E2-2 and SpLmo2, an ortholog of a dedicated cofactor of the Scl–GATA transcription complex. Regulatory analysis of SpGatac indicates that oral NSM identity is directly suppressed in presumptive pigment cells by the transcription factor SpGcm. These findings provide part of a comparative basis to understand the evolutionary origins and regulatory biology of deuterostome immune cell differentiation in the context of a tractable gene regulatory network model

High regulatory gene use in sea urchin embryogenesis: Implications for bilaterian development and evolution

A global scan of transcription factor usage in the sea urchin embryo was carried out in the context of the Strongylocentrotus purpuratus genome sequencing project, and results from six individual studies are here considered. Transcript prevalence data were obtained for over 280 regulatory genes encoding sequence-specific transcription factors of every known family, but excluding genes encoding zinc finger proteins. This is a statistically inclusive proxy for the total “regulome” of the sea urchin genome. Close to 80% of the regulome is expressed at significant levels by the late gastrula stage. Most regulatory genes must be used repeatedly for different functions as development progresses. An evolutionary implication is that animal complexity at the stage when the regulome first evolved was far simpler than even the last common bilaterian ancestor, and is thus of deep antiquity

Repression of mesodermal fate by foxa, a key endoderm regulator of the sea urchin embryo

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

Vasa Protein Expression is Restricted to the Small Micromeres of the Sea Urchin, but is Inducible in Other Lineages Early in Development

Vasa is a DEAD-box RNA helicase that functions in translational regulation of specific mRNAs. In many animals it is essential for germ line development and may have a more general stem cell role. Here we identify vasa in two sea urchin species and analyze the regulation of its expression. We find that vasa protein accumulates in only a subset of cells containing vasa mRNA. In contrast to vasa mRNA, which is present uniformly throughout all cells of the early embryo, vasa protein accumulates selectively in the 16-cell stage micromeres, and then is restricted to the small micromeres through gastrulation to larval development. Manipulating early embryonic fate specification by blastomere separations, exposure to lithium, and dominant-negative cadherin each suggest that, although vasa protein accumulation in the small micromeres is fixed, accumulation in other cells of the embryo is inducible. Indeed, we find that embryos in which micromeres are removed respond by significant up-regulation of vasa protein translation, followed by spatial restriction of the protein late in gastrulation. Overall, these results support the contention that sea urchins do not have obligate primordial germ cells determined in early development, that vasa may function in an early stem cell population of the embryo, and that vasa expression in this embryo is restricted early by translational regulation to the small micromere lineage.Organismic and Evolutionary Biolog

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