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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2010.09.011 Byline: Shunsuke Yaguchi, Junko Yaguchi, Zheng Wei, Kogiku Shiba, Lynne M. Angerer, Kazuo Inaba Keywords: Ankyrin; Cilia; Ciliogenesis; FoxQ2; NK2.1; Nodal; Animal plate Abstract: In sea urchin embryos, the apical tuft forms within the neurogenic animal plate. When FoxQ2, one of the earliest factors expressed specifically in the animal plate by early blastula stage, is knocked down, the structure of the apical tuft is altered. To determine the basis of this phenotype, we identified FoxQ2-dependent genes using microarray analysis. The most strongly down-regulated gene in FoxQ2 morphants encodes a protein with ankyrin repeats region in its N-terminal domain. We named this gene ankAT-1, Ankyrin-containing gene specific for Apical Tuft. Initially its expression in the animal pole region of very early blastula stage embryos is FoxQ2-independent but becomes FoxQ2-dependent beginning at mesenchyme blastula stage and continuing in the animal plate of 3-day larvae. Furthermore, like FoxQ2, this gene is expressed throughout the expanded apical tuft region that forms in embryos lacking nuclear [beta]-catenin. When AnkAT-1 is knocked-down by injecting a morpholino, the cilia at the animal plate in the resulting embryos are much shorter and their motility is less than that of motile cilia in other ectoderm cells, and remains similar to that of long apical tuft cilia. We conclude that AnkAT-1 is involved in regulating the length of apical tuft cilia. Article History: Received 20 July 2010; Revised 16 September 2010; Accepted 16 September 2010

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2010.08.009 Byline: Shunsuke Yaguchi, Junko Yaguchi, Robert C. Angerer, Lynne M. Angerer, Robert D. Burke Keywords: TGFbeta signaling; Neurogenesis; Ciliary band; Oral signaling; Nodal; BMP; Lefty; Smad; Alk; Urchin Abstract: The ciliary band is a distinct region of embryonic ectoderm that is specified between oral and aboral ectoderm. Flask-shaped ciliary cells and neurons differentiate in this region and they are patterned to form an integrated tissue that functions as the principal swimming and feeding organ of the larva. TGF[beta] signaling, which is known to mediate oral and aboral patterning of the ectoderm, has been implicated in ciliary band formation. We have used morpholino knockdown and ectopic expression of RNA to alter TGF[beta] signaling at the level of ligands, receptors, and signal transduction components and assessed the differentiation and patterning of the ciliary band cells and associated neurons. We propose that the primary effects of these signals are to position the ciliary cells, which in turn support neural differentiation. We show that Nodal signaling, which is known to be localized by Lefty, positions the oral margin of the ciliary band. Signaling from BMP through Alk3/6, affects the position of the oral and aboral margins of the ciliary band. Since both Nodal and BMP signaling produce ectoderm that does not support neurogenesis, we propose that formation of a ciliary band requires protection from these signals. Expression of BMP2/4 and Nodal suppress neural differentiation. However, the response to receptor knockdown or dominant-negative forms of signal transduction components indicate signaling is not acting directly on unspecified ectoderm cells to prevent their differentiation as neurons. Instead, it produces a restricted field of ciliary band cells that supports neurogenesis. We propose a model that incorporates spatially regulated control of Nodal and BMP signaling to determine the position and differentiation of the ciliary band, and subsequent neural patterning. Article History: Received 1 February 2010; Revised 17 June 2010; Accepted 6 August 2010

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.034 Byline: Zheng Wei, Robert C. Angerer, Lynne M. Angerer Keywords: Gene prediction; Microarray; Genscan Abstract: We present an initial characterization of a database that contains temporal expression profiles of sequences found in 35,282 gene predictions within the sea urchin genome. The relative RNA abundance for each sequence was determined at 5 key stages of development using high-density oligonucleotide microarrays that were hybridized with populations of polyA+ RNA sequence. These stages were two-cell, which represents maternal RNA, early blastula, the time at which major tissue territories are specified, early and late gastrula, during which important morphogenetic events occur, and the pluteus larva, which marks the culmination of pre-feeding embryogenesis. We provide evidence that the microarray reliably reports the temporal profiles for the large majority of predicted genes, as shown by comparison to data for many genes with known expression patterns. The sensitivity of this assay allows detection of mRNAs whose concentration is only several hundred copies/embryo. The temporal expression profiles indicate that 5% of the gene predictions encode mRNAs that are found only in the maternal population while 24% are embryo-specific. Further, we find that the concentration of >80% of different mRNAs is modulated by more than a factor of 3 during development. Along with the annotated sea urchin genome sequence and the whole-genome tiling array (the transcriptome, Samanta, M., Tongprasit, W., Istrrail, S., Cameron, R., Tu, Q., Davidson, E., Stolc, V., in press. A high-resolution transcriptome map of the sea urchin embryo. Science), this database proves a valuable resource for designing experiments to test the function of specific genes during development. Author Affiliation: National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA Article History: Received 5 June 2006; Revised 10 August 2006; Accepted 15 August 2006

Pl-nectin is an ECM protein located on the apical surface of ectoderm cells of Paracentrotus lividus sea urchin embryo. Inhibition of ECM-ectoderm cell interaction by the addition of McAb to Pl-nectin to the culture causes a dramatic impairment of skeletogenesis, offering a good model for the study of factor(s) involved in skeleton elongation and patterning. We showed that skeleton deficiency was not due to a reduction in the number of PMCs ingressing the blastocoel, but it was correlated with a reduction in the number of Pl-SM30-expressing PMCs. Here, we provide evidence on the involvement of growth factor(s) in skeleton morphogenesis. Skeleton-defective embryos showed a strong reduction in the levels of expression of Pl-univin, a growth factor of the TGF-[beta] superfamily, which was correlated with an equivalent strong reduction in the levels of Pl-SM30. In contrast, expression levels of Pl-BMP5-7 remained low and constant in both skeleton-defective and normal embryos. Microinjection of horse serum in the blastocoelic cavity of embryos cultured in the presence of the antibody rescued skeleton development. Finally, we found that misexpression of univin is also sufficient to rescue defects in skeleton elongation and SM30 expression caused by McAb to Pl-nectin, suggesting a key role for univin or closely related factor in sea urchin skeleton morphogenesis. Keywords: Sea urchin embryo; Growth factor; Extracellular matrix; Skeletogenesis; Ecto-mesoderm induction

Previous studies in sea urchin embryos have demonstrated that nuclearization of [beta]-catenin is essential for initial steps in the specification of endoderm and mesenchyme, which are derived from vegetal blastomeres. This process begins at the 4th and extends through the 9th cleavage stage, an interval in which the SpSoxB1 transcription regulator is downregulated by [beta]-catenin-dependent gene products that include the transcription repressor SpKr1. These observations raise the possibility that SpSoxB1 removal is required to allow vegetal development to proceed. Here we show that elevated and ectopic expression of this factor suppresses differentiation of all vegetal cell types, a phenotype that is very similar to that caused by the suppression of [beta]-catenin nuclear function by cadherin overexpression. Suppression of vegetal fates involves interference at the protein-protein level because a mutation of SpSoxB1 that prevents its binding to DNA does not significantly reduce this activity. Reduction in SpSoxB1 level results in elevated TCF/Lef-[beta]-catenin-dependent expression of a luciferase reporter gene in vivo, indicating that in the normal embryo this protein suppresses the primary vegetal signaling mechanism that is required for specification of mesenchyme and endoderm. Surprisingly, normal expression of SpSoxB1 is required for gastrulation and endoderm differentiation, as shown by both morpholino-mediated translational interference and expression of a dominant negative protein. Similar gain-of-function and loss-of-function assays of a closely related factor, SpSoxB2, demonstrate that it, too, is required for gastrulation and that its overexpression can suppress vegetal development. However, significant phenotypic differences are apparent in the two perturbations, indicating that SpSoxB1 and SpSoxB2 have at least some distinct developmental functions. The results of all these studies support a model in which the concentration of SpSoxB factors must be tightly regulated along the animal-vegetal axis of the early sea urchin embryo to allow [beta]-catenin-dependent specification of endoderm and mesenchyme cell fates as well as to activate target genes required for gastrulation. Keywords: Morpholino; Misexpression; Animal-vegetal axis

We discuss recent progress in understanding how cell fates are specified along the animal-vegetal axis of the sea urchin embryo. This process is initiated by cell-autonomous, maternally directed, mechanisms that establish three unique gene-regulatory domains. These domains are defined by distinct sets of vegetalizing (beta-catenin) and animalizing transcription factor (ATF) activities and their region of overlap in the macromeres, which specifies these cells as early mesendoderm. Subsequent signaling among cleavage-stage blastomeres further subdivides fates of macromere progeny to yield major embryonic tissues. Zygotically produced Wnt8 reinforces maternally regulated levels of nuclear (beta)-catenin in vegetal derivatives to down regulate ATF activity and further promote mesendoderm fates. Signaling through the Notch receptor from the vegetal micromere lineages diverts adjacent mesendoderm to secondary mesenchyme fates. Continued Wnt signaling expands the vegetal domain of (beta)-catenin's transcriptional regulatory activity and competes with animal signaling factors, including BMP2/4, to specify the endoderm-ectoderm border within veg(sub 1) progeny. This model places new emphasis on the importance of the ratio of maternally regulated vegetal and animal transcription factor activities in initial specification events along the animal-vegetal axis.

We have shown previously by in situ hybridization that a gene encoding a fibroblast growth factor receptor (SpFGFR) is transcribed in many cell types during the initial phases of sea urchin embryogenesis (Strongylocentrotus purpuratus) (McCoon et al., J. Biol. Chem. 271, 20119-20195, 1996). Here we demonstrate by immunostaining with affinity-purified antibody that SpFGFR protein is detectable only in muscle cells of the embryo and appears at a time suggesting that its function is not in commitment to a muscle fate, but instead may be required to support the proliferation, migration, and/or differentiation of myoblasts. Surprisingly, we find that SpFGFR transcripts are enriched in embryo nuclei, suggesting that lack of processing and/or cytoplasmic transport in nonmuscle cells is at least part of the posttranscriptional regulatory mechanism. Western blots show that SpFGFR is also specifically expressed in adult lantern muscle, but is not detectable in other smooth muscle-containing tissues, including tube foot and intestine, or in coelomocytes, despite the presence of SpFGFR transcripts at similar concentrations in all these tissues. We conclude that in both embryos and adults, muscle-specific SpFGF receptor synthesis is controlled primarily at a posttranscriptional level. We show by RNase protection assays that transcripts encoding the Ig3S variant of the ligand binding domain of the receptor, previously shown to be enriched in embryo endomesoderm fractions, are the predominant, if not exclusive, SpFGFR transcripts in lantern muscle. Together, these results suggest that only a minority of SpFGFR transcripts are processed, exported, and translated in both adult and embryonic muscle cells and these contain predominantly, if not exclusively, Ig3S ligand binding domain sequences. Key Words: posttranscriptional regulation; secondary mesenchyme; nuclear RNA; muscle.

Orthodenticle-related (Otx) proteins are a highly conserved class of homeobox-containing transcription factors found in a wide range of organisms. They function in numerous developmental events, most prominently, anterior head patterning in insects and vertebrates. In the sea urchin, Strongylocentrotus purpuratus, an orthodenticle-related protein called SpOtx is believed to direct the activation of the aboral ectoderm-specific Spec2a gene and more generally the differentiation of aboral ectoderm cells. To learn more about the structure, expression, and function of SpOtx and compare its properties with those of orthologs from other species, we isolated cDNA and genomic clones containing SpOtx sequences. Here, we report that SpOtx exists in two forms ([Alpha] and [Beta]) that are generated by alternative RNA splicing from a single SpOtx gene. SpOtx([Alpha]) and SpOtx([Beta]) had identical C-termini and homeoboxes but were entirely different in their N-terminal domains. SpOtx([Alpha]) mRNAs were transcribed from a single start site and accumulated in all cells during cleavage, but were gradually concentrated in oral ectoderm and vegetal plate territories during gastrulation. In contrast, three distinct SpOtx([Beta]) mRNAs resulted from two separate transcriptional initiation events, and these transcripts began to accumulate at mesenchyme blastula stage primarily in ectoderm and then later were largely restricted to oral ectoderm and vegetal plate territories. DNA-binding activity for SpOtx([Beta]) appeared later in development than SpOtx([Alpha]). Overexpression of SpOtx([Alpha]) and SpOtx([Beta]) induced in sea urchin embryos by mRNA injection demonstrated that SpOtx([Alpha]) was able to repress the accumulation of SpOtx([Beta]) transcripts, whereas SpOtx([Beta]) had no effect on the accumulation of SpOtx([Alpha]) transcripts. These results demonstrate that novel forms of Otx are produced in sea urchins by differential promoter utilization and alternative splicing. It may be that similar regulatory mechanisms lead to diverse forms of Otx in vertebrates.

The mechanism that establishes the maternally determined animal - vegetal axis of sea urchin embryos is unknown. We have analyzed the cis-regulatory elements of the SpHE gene of Strongylocentrotus purpuratus, which is asymmetrically expressed along this axis, in an effort to identify components of maternal positional information. Previously, we defined a regulatory region that is sufficient to provide correct nonvegetal expression of a [Beta]-galactosidase reporter gene (Wei, Z., Angerer, L.M., Gagnon, M. L., and Angerer, R. C., Dev. Biol. 171, 195-211, 1995). We have now analyzed this region intensively in order to determine if the spatial pattern is controlled by nonvegetal-positive activities or by vegetal-negative activities. The regulatory sequences, except the basal promoter, were mutated by either deletion or sequence replacement. None of these mutations resulted in ectopic [Beta]-gal expression in vegetal cells, showing that no single negative cis element is responsible for the lack of vegetal SpHE transcription. Surprisingly, even short segments of the regulatory region containing only several identified cis elements also direct nonvegetal expression. Furthermore, the SpHE basal promoter functions effectively in vegetal cells in combination with cis-acting elements derived from the PMC-specific gene, SM50. We conclude that the spatial pattern of SpHE transcription is achieved by multiple positive activities concentrated in nonvegetal cells. The vegetal expression of SM50 also is regulated only by positive activities (Makabe, K. W., Kirchhamer, C. V., Britten, R. J., and Davidson, E. H., Development 121, 1957-1970, 1995). A chimeric promoter containing both SpHE and SM50 regulatory sequences is active ubiquitously, suggesting that these regulators are not reciprocally repressive. These observations suggest a model in which the SpHE and SM50 genes are activated by separate sets of positive maternal activities concentrated, respectively, in nonvegetal and vegetal domains of the early embryo.

In order to investigate how the maternally specified animal-vegetal axis of the sea urchin embryo is established, we have examined the molecular basis of regulation of several genes transcribed differentially in nonvegetal and vegetal domains of the very early blastula. Here we present an initial characterization of the regulatory region of one of these, SpAN, which encodes a protease in the astacin family related to Drosophila tolloid and vertebrate BMP-1 (Reynolds et el., Development 114, 769-786). Tests of SpAN promoter function in vivo show that high-level activity and correct not-vegetal expression are mediated by sequences within 300 bp upstream of the basal promoter. In vitro studies have identified six protein binding sites serviced by at least five different proteins. Comparison of the structure of the SpAN promoter to that of SpHE, whose expression pattern is identical, shows that both promoters contain multiple positively acting upstream elements close to the basal promoter. We show that two elements are critical for high-level transcription of SPAN, since exact replacement of either results in 10- to 20-fold reduction in promoter strength. These shared elements are, however, not essential for spatially correct SpHE gene transcription. We conclude that the coordinate strong activities of the SpAN and SpHE promoters in the nonvegetal domain of the embryo rely primarily on different transcription factor activities.

A Strongylocentrotus purpuratus hatching enzyme cis regulatory region, characterized by a proximal promoter/enhancer, is found to have the ability to regulate low levels of transcriptional activity in nonvegetal cells of early sea urchin blastulae. These cis elements are Otx (an orthodenticle homologue), CCAAT, ets-related and three unidentified motifs. The subsets of these elements associate in different patterns to obtain levels of transcriptions that are compared to the unaltered regulatory region.

An orthodenticle-related protein-encoding cDNA clone SpOtx from the sea urchin Strongylocentrotus purpuratus exhibits an orthodenticle homeobox but differs from its mouse and fly equivalents in sequence. Band-shift analysis reveals that SpOtx, with a binding specificity for the TAATCC/T sequence of DNA, binds to the Spec2a gene that contains multiple sites with this motif. The dividing embryo exhibits SpOtx transcripts initially in all cells, which is limited to oral endoderm and ectoderm cells in the later stages.

PCR cloning, library screening and RNA blotting procedures reveal a new gene univin, encoding the transformation growth factor beta (TGF-beta) and belonging to the bone morphogenetic protein group in the sea urchin Stronglyocentrotus purpuratus. The cDNA clone of the gene contains a 383-amino acid residue protein with seven cysteine residues and a cluster of basic residues. The spatial and temporal patterns of the gene vary at different developmental stages and are similar to those of SpAN gene, suggesting a correlation between SpAN and univin in regulating developmental stages in sea urchin embryo.

Sequencing and cloning of embryonic messenger RNA and genomic cDNA clones in Tripneustes gratilla reveal an actin-encoding gene, Tg616, that is orthologous to CyI actin gene in Strongylocentrotus purpuratus. Nucleotide sequences of the regions encoding proteins, the highly conserved 150 nucleotide sequence at the transcription start site and the nucleotide sequences of the untranslated mRNA regions are similar in both genes. The occurrence of Tg616 mRNA expression in the blastula stage, during gut differentiation and pluteus stage, indicates that its localization is distinct from that of SpCyI gene.

The mRNAs of the sea Urchin gastrula polyadenylated RNA population were selected at random and examined to study the temporal and spatial patterns of accumulation. Three different assays revealed that the most common pattern of expression was a large increase in mRNA between the egg and gastrula phases. The mRNAs were normally present in the maternal group and are found in blots as single mature-sized messages. Several numbers of high-molecular-weight, and heterodisperse transcripts with the same sequences, are also present in the egg cytoplasm.

During embryogenesis the sea urchin early pluteus larva differentiates 40-50 neurons marked by expression of the pan-neural marker synaptotagmin B (SynB) that are distributed along the ciliary band, in the apical plate and pharyngeal endoderm, and 4-6 serotonergic neurons that are confined to the apical plate. Development of all neurons has been shown to depend on the function of Six3. Using a combination of molecular screens and tests of gene function by morpholino-mediated knockdown, we identified SoxC and Brn1/2/4, which function sequentially in the neurogenic regulatory pathway and are also required for the differentiation of all neurons. Misexpression of Brn1/2/4 at low dose caused an increase in the number of serotonin-expressing cells and at higher dose converted most of the embryo to a neurogenic epithelial sphere expressing the Hnf6 ciliary band marker. A third factor, Z167, was shown to work downstream of the Six3 and SoxC core factors and to define a branch specific for the differentiation of serotonergic neurons. These results provide a framework for building a gene regulatory network for neurogenesis in the sea urchin embryo. [ABSTRACT FROM AUTHOR]