Assembly of continuous high-resolution draft genome sequence of Hemicentrotus pulcherrimus using long-read sequencing.

The update of the draft genome assembly of sea urchin, Hemicentrotus pulcherrimus, which is widely studied in East Asia as a model organism of early development, was performed using Oxford nanopore long-read sequencing. The updated assembly provided ~600-Mb genome sequences divided into 2,163 contigs with N50 = 516 kb. BUSCO completeness score and transcriptome model mapping ratio (TMMR) of the present assembly were obtained as 96.5% and 77.8%, respectively. These results were more continuous with higher resolution than those by the previous version of H. pulcherrimus draft genome, HpulGenome_v1, where the number of scaffolds = 16,251 with a total of ~100 Mb, N50 = 143 kb, BUSCO completeness score = 86.1%, and TMMR = 55.4%. The obtained genome contained 36,055 gene models that were consistent with those in other echinoderms. Additionally, two tandem repeat sequences of early histone gene locus containing 47 copies and 34 copies of all histone genes, and 185 of the homologous sequences of the interspecifically conserved region of the Ars insulator, ArsInsC, were obtained. These results provide further advance for genome-wide research of development, gene regulation, and intranuclear structural dynamics of multicellular organisms using H. pulcherrimus.

The crucial role of CTCF in mitotic progression during early development of sea urchin.

CCCTC-binding factor (CTCF), an insulator protein with 11 zinc fingers, is enriched at the boundaries of topologically associated domains (TADs) in eukaryotic genomes. In this study, we isolated and analyzed the cDNAs encoding HpCTCF, the CTCF homolog in the sea urchin Hemicentrotus pulcherrimus, to investigate its expression patterns and functions during the early development of sea urchin. HpCTCF contains nine zinc fingers corresponding to fingers 2-10 of the vertebrate CTCF. Expression pattern analysis revealed that HpCTCF mRNA was detected at all developmental stages and in the entire embryo. Upon expressing the HpCTCF-GFP fusion protein in early embryos, we observed its uniform distribution within interphase nuclei. However, during mitosis, it disappeared from the chromosomes and subsequently reassembled on the chromosome during telophase. Moreover, the morpholino-mediated knockdown of HpCTCF resulted in mitotic arrest during the morula to blastula stage. Most of the arrested chromosomes were not phospholylated at serine 10 of histone H3, indicating that mitosis was arrested at the telophase by HpCTCF depletion. Furthermore, impaired sister chromatid segregation was observed using time-lapse imaging of HpCTCF-knockdown embryos. Thus, HpCTCF is essential for mitotic progression during the early development of sea urchins, especially during the telophase-to-interphase transition. However, the normal development of pluteus larvae in CRISPR-mediated HpCTCF-knockout embryos suggests that disruption of zygotic HpCTCF expression has little effect on embryonic and larval development.

TrBase: A genome and transcriptome database of Temnopleurus reevesii.

Sea urchins have a long history as model organisms in biology, but their use in genetics is limited because of their long breeding cycle. In sea urchin genetics, genome editing technology was first established in Hemicentrotus pulcherrimus, whose genome has already been published. However, because this species also has a long breeding cycle, new model sea urchins that are more suitable for genetics have been sought. Here, we report a draft genome of another Western Pacific species, Temnopleurus reevesii, which we established as a new model sea urchin recently since this species has a comparable developmental process to other model sea urchins but a short breeding cycle of approximately half a year. The genome of T. reevesii was assembled into 28,742 scaffold sequences with an N50 length of 67.6 kb and an estimated genome size of 905.9 Mb. In the assembled genome, 27,064 genes were identified, 23,624 of which were expressed in at least one of the seven developmental stages. To provide genetic information, we constructed the genome database TrBase (https://cell-innovation.nig.ac.jp/Tree/). We also constructed the Western Pacific Sea Urchin Genome Database (WestPac-SUGDB) (https://cell-innovation.nig.ac.jp/WPAC/) with the aim of establishing a portal site for genetic information on sea urchins in the West Pacific. This site contains genomic information on two species, T. reevesii and H. pulcherrimus, and is equipped with homology search programs for comparing the two datasets. Therefore, TrBase and WestPac-SUGDB are expected to contribute not only to genetic research using sea urchins but also to comparative genomics and evolutionary research.

Hp-s1 Ganglioside Suppresses Proinflammatory Responses by Inhibiting MyD88-Dependent NF-κB and JNK/p38 MAPK Pathways in Lipopolysaccharide-Stimulated Microglial Cells.

Hp-s1 ganglioside is isolated from the sperm of sea urchin (Hemicentrotus pulcherrimus). In addition to neuritogenic activity, the biological function of Hp-s1 in neuroinflammation is unknown. In this study, we investigated the anti-neuroinflammatory effect of Hp-s1 on lipopolysaccharide (LPS)-stimulated microglial cells. MG6 microglial cells were stimulated with LPS in the presence or absence of different Hp-s1 concentrations. The anti-inflammatory effect and underlying mechanism of Hp-s1 in LPS-activated microglia cells were assessed through a Cell Counting kit-8 assay, Western blot analysis, and immunofluorescence. We found that Hp-s1 suppressed not only the expression of inducible nitric oxide synthase and cyclooxygenase-2 but also the expression of proinflammatory cytokines, such as TNF-α, IL-1ß, and IL-6. Hp-s1 inhibited the LPS-induced NF-κB signaling pathway by attenuating the phosphorylation and translocation of NF-κB p65 and by disrupting the degradation and phosphorylation of inhibitor κB-α (IκBα). Moreover, Hp-s1 inhibited the LPS-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Hp-s1 also reduced the expression of myeloid differentiation factor 88 (MyD88) and TNF receptor-associated factors 6 (TRAF6), which are prerequisites for NF-κB and MAPKs activation. These findings indicated that Hp-s1 alleviated LPS-induced proinflammatory responses in microglial cells by downregulating MyD88-mediated NF-κB and JNK/p38 MAPK signaling pathways, suggesting further evaluation as a new anti-neuroinflammatory drug.

Involvement of Netrin/Unc-5 Interaction in Ciliary Beating and in Pattern Formation of the Ciliary Band-Associated Strand (CBAS) in the Sea Urchin, Hemicentrotus pulcherrimus.

The GABAergic neural circuit is involved in the motile activities of both larval and juvenile sea urchins. Therefore, its function is inherited beyond metamorphosis, despite large scale remodeling of larval organs during that period. However, the initial neural circuit formation mechanism is not well understood, including how glutamate decarboxylase-expressing blastocoelar cells (GADCs) construct the neural circuit along the circumoral ciliary band (a ciliary band-associated strand, CBAS) on the larval body surface. In this study, using whole-mount immunohistochemistry and 3D reconstructed imaging, the ontogenic process of CBAS patterning was studied by focusing on Netrin and the interaction with its receptor, Unc-5. During the early 2-arm pluteus stage, a small number of GADCs egress onto the apical surface of the larval ectoderm. Then, they line up on the circumoral side of the ciliary band, and by being inserted by a further number of GADCs, form longer multicellular strands along the Netrin stripe. Application of a synthetic peptide, CRFNMELYKLSGRKSGGVC of Hp-Netrin, that binds to the immunoglobulin domain of Unc-5 during the prism stage, causes stunted CBAS formation due to inhibition of GADC egression. This also results in reduced ciliary beating. Thus, the Netrin/Unc-5 interaction is involved in the construction and function of the CBAS.

Meis transcription factor maintains the neurogenic ectoderm and regulates the anterior-posterior patterning in embryos of a sea urchin, Hemicentrotus pulcherrimus.

Precise body axis formation is an essential step in the development of multicellular organisms, for most of which the molecular gradient and/or specifically biased localization of cell-fate determinants in eggs play important roles. In sea urchins, however, any biased proteins and mRNAs have not yet been identified in the egg except for vegetal cortex molecules, suggesting that sea urchin development is mostly regulated by uniformly distributed maternal molecules with contributions to axis formation that are not well characterized. Here, we describe that the maternal Meis transcription factor regulates anterior-posterior axis formation through maintenance of the most anterior territory in embryos of a sea urchin, Hemicentrotus pulcherrimus. Loss-of-function experiments revealed that Meis is intrinsically required for maintenance of the anterior neuroectoderm specifier foxQ2 after hatching and, consequently, the morphant lost anterior neuroectoderm characteristics. In addition, the expression patterns of univin and VEGF, the lateral ectoderm markers, and the mesenchyme-cell pattern shifted toward the anterior side in Meis morphants more than they did in control embryos, indicating that Meis contributes to the precise anteroposterior patterning by regulating the anterior neuroectodermal fate.

Cooperative Wnt-Nodal Signals Regulate the Patterning of Anterior Neuroectoderm.

When early canonical Wnt is experimentally inhibited, sea urchin embryos embody the concept of a Default Model in vivo because most of the ectodermal cell fates are specified as anterior neuroectoderm. Using this model, we describe here how the combination of orthogonally functioning anteroposterior Wnt and dorsoventral Nodal signals and their targeting transcription factors, FoxQ2 and Homeobrain, regulates the precise patterning of normal neuroectoderm, of which serotonergic neurons are differentiated only at the dorsal/lateral edge. Loss-of-function experiments revealed that ventral Nodal is required for suppressing the serotonergic neural fate in the ventral side of the neuroectoderm through the maintenance of foxQ2 and the repression of homeobrain expression. In addition, non-canonical Wnt suppressed homeobrain in the anterior end of the neuroectoderm, where serotonergic neurons are not differentiated. Canonical Wnt, however, suppresses foxQ2 to promote neural differentiation. Therefore, the three-dimensionally complex patterning of the neuroectoderm is created by cooperative signals, which are essential for the formation of primary and secondary body axes during embryogenesis.

HpBase: A genome database of a sea urchin, Hemicentrotus pulcherrimus.

To understand the mystery of life, it is important to accumulate genomic information for various organisms because the whole genome encodes the commands for all the genes. Since the genome of Strongylocentrotus purpratus was sequenced in 2006 as the first sequenced genome in echinoderms, the genomic resources of other North American sea urchins have gradually been accumulated, but no sea urchin genomes are available in other areas, where many scientists have used the local species and reported important results. In this manuscript, we report a draft genome of the sea urchin Hemincentrotus pulcherrimus because this species has a long history as the target of developmental and cell biology in East Asia. The genome of H. pulcherrimus was assembled into 16,251 scaffold sequences with an N50 length of 143 kbp, and approximately 25,000 genes were identified in the genome. The size of the genome and the sequencing coverage were estimated to be approximately 800 Mbp and 100×, respectively. To provide these data and information of annotation, we constructed a database, HpBase (http://cell-innovation.nig.ac.jp/Hpul/). In HpBase, gene searches, genome browsing, and blast searches are available. In addition, HpBase includes the "recipes" for experiments from each lab using H. pulcherrimus. These recipes will continue to be updated according to the circumstances of individual scientists and can be powerful tools for experimental biologists and for the community. HpBase is a suitable dataset for evolutionary, developmental, and cell biologists to compare H. pulcherrimus genomic information with that of other species and to isolate gene information.

Effects of monocrotophos pesticide on cholinergic and dopaminergic neurotransmitter systems during early development in the sea urchin Hemicentrotus pulcherrimus.

During early development in sea urchins, classical neurotransmitters, including acetylcholine (ACh), dopamine (DA), and serotonin (5-HT), play important roles in the regulation of morphogenesis and swimming behavior. However, the underlying mechanisms of how organophosphate pesticides cause developmental neurotoxicity by interfering with different neurotransmitter systems are unclear. In this study, we investigated the effects of 0.01, 0.10, and 1.00mg/L monocrotophos (MCP) pesticide on the activity of acetyltransferase (ChAT), acetylcholinesterase (AChE), monoamine oxidase, the concentration of DA, dopamine transporter, and the transcription activity of DA receptor D1 and tyrosine hydroxylase, during critical periods in cholinergic and dopaminergic nervous system development in sea urchin (Hemicentrotus pulcherrimus) embryos and larvae. At the blastula stages, MCP disrupted DA metabolism but not 5-HT metabolism, resulting in abnormal development. High ChAT and AChE activity were observed at the gastrulation-completed stage and the two-armed pluteus stage, respectively, MCP inhibited ChAT activity and AChE activity/distribution and resulted in developmental defects of the plutei. From the gastrula stage to the two-armed pluteus stage, we found ubiquitous disrupting effects of MCP on ACh, DA, and 5-HT metabolism, particularly at critical periods during the development of these neurotransmitter systems. Therefore, we propose that this disruption is one of the main mechanisms of MCP-related developmental neurotoxicity, which would contribute better understanding insight into the mechanism of MCP pesticide's toxic effects.

The 3'UTR of nanos2 directs enrichment in the germ cell lineage of the sea urchin.

Nanos is a translational regulator required for the survival and maintenance of primordial germ cells during embryogenesis. Three nanos homologs are present in the genome of the sea urchin Strongylocentrotus purpuratus (Sp), and each nanos mRNA accumulates specifically in the small micromere (sMic) lineage. We found that a highly conserved element in the 3' UTR of nanos2 is sufficient for reporter expression selectively in the sMic lineage: microinjection into a Sp fertilized egg of an RNA that contains the GFP open reading frame followed by Sp nanos2 3'UTR leads to selective reporter enrichment in the small micromeres in blastulae. The same result was seen with nanos2 from the sea urchin Hemicentrotus pulcherrimus (Hp). In both species, the 5'UTR alone is not sufficient for the sMic localization but it always increased the sMic reporter enrichment when present with the 3'UTR. We defined an element conserved between Hp and Sp in the nanos2 3'UTR which is necessary and sufficient for protein enrichment in the sMic, and refer to it as GNARLE (Global Nanos Associated RNA Lability Element). We also found that the nanos2 3'UTR is essential for the selective RNA retention in the small micromeres; GNARLE is required but not sufficient for this process. These results show that a combination of selective RNA retention and translational control mechanisms instills nanos accumulation uniquely in the sMic lineage.

A homologous series of α-glucans from Hemicentrotus pulcherrimus and their immunomodulatory activity.

Seven macromolecular polysaccharides (HPP-2S-HPP-8S) were purified from the gonads of sea urchin Hemicentrotus pulcherrimus. They were characterized as α-glucan homologues, sharing the same α-1,4-glucan backbone substituted at C-6 positions by glucose with HPP-1S that occurs as the major polysaccharide in H. pulcherrimus, while with higher degrees of branching, and additionally possessing minor amounts of mannose and ribose. The branching degree and amounts of non-glucose branches showed a generally increasing tendency across HPP-2S - HPP-8S. These polysaccharides exhibited significant macrophage-activating effects by augmenting the secretion of NO, TNF-α and IL-6, which probably involves the activation of NF-κB and MAPKs signaling pathways. Notably, the polysaccharides with a higher degree of branching exhibited markedly enhanced immunomodulatory capacity with a lowest effective concentration of 1.95 µg/mL. This work provides new cases of bioactive α-glucans and reveals their potential application as immunomodulating agents.

Zinc finger homeobox is required for the differentiation of serotonergic neurons in the sea urchin embryo.

Serotonergic neurons differentiate in the neurogenic animal plate ectoderm of the sea urchin embryo. The regulatory mechanisms that control the specification or differentiation of these neurons in the sea urchin embryo are not yet understood, although, after the genome was sequenced, many genes encoding transcription factors expressed in this region were identified. Here, we report that zinc finger homeobox (zfhx1/z81) is expressed in serotonergic neural precursor cells, using double in situ hybridization screening with a serotonergic neural marker, tryptophan 5-hydroxylase (tph) encoding a serotonin synthase that is required for the differentiation of serotonergic neurons. zfhx1/z81 begins to be expressed at gastrula stage in individual cells in the anterior neuroectoderm, some of which also express delta. zfhx1/z81 expression gradually disappears as neural differentiation begins with tph expression. When the translation of Zfhx1/Z81 is blocked by morpholino injection, embryos express neither tph nor the neural marker synaptotagminB in cells of the animal plate, and serotonergic neurons do not differentiate. In contrast, Zfhx1/Z81 morphants do express fez, another neural precursor marker, which appears to function in the initial phase of specification/differentiation of serotonergic neurons. In addition, zfhx1/z81 is one of the targets suppressed in the animal plate by anti-neural signals such as Nodal as well as Delta-Notch. We conclude that Zfhx1/Z81 functions during the specification of individual anterior neural precursors and promotes the expression of tph and synaptotagminB, required for the differentiation of serotonergic neurons.

Regulations of microtubule sliding by Ca2+ and cAMP and their roles in forming flagellar waveforms.

The function of Ca(2+) and cAMP in extruding doublet microtubules from sea urchin sperm axoneme and generating flagellar waves was investigated in order to clarify the regulatory mechanism of microtubule sliding and the formation mechanism of beating patterns of cilia and flagella. Almost all potentially asymmetric spermatozoa that were demembranated with Triton in the absence of Ca(2+) and reactivated with MgATP(2-) (Gibbons, B.H. and Gibbons, I.R. (1980). J. Cell Biol., 84: 13-27), beat with planar waves closely resembling those of the intact spermatozoa, whereas potentially symmetric spermatozoa, in which axonemal calmodulin was removed by detergent extraction in the presence of millimolar Ca(2+) (Brokaw, C.J. and Nagayama, S.M. (1985). J. Cell Biol., 100: 1875-1883), beat with three-dimensional waves if they were reactivated with low MgATP(2-). At a high MgATP(2-), almost all demembranated spermatozoa beat with planar waves. cAMP enhanced the three-dimensionality of the flagellar waves at a low Ca(2+). These changes in the flagellar waves were caused by different regulations of the microtubule sliding by calcium, cAMP, and MgATP(2-).

Development of the GABA-ergic signaling system and its role in larval swimming in sea urchin.

The present study aimed to elucidate the development and γ-amino butyric acid (GABA)-ergic regulation of larval swimming in the sea urchin Hemicentrotus pulcherrimus by cloning glutamate decarboxylase (Hp-gad), GABAA receptor (Hp-gabrA) and GABAA receptor-associated protein (Hp-gabarap), and by performing immunohistochemistry. The regulation of larval swimming was increasingly dependent on the GABAergic system, which was active from the 2 days post-fertilization (d.p.f.) pluteus stage onwards. GABA-immunoreactive cells were detected as a subpopulation of secondary mesenchyme cells during gastrulation and eventually constituted the ciliary band and a subpopulation of blastocoelar cells during the pluteus stage. Hp-gad transcription was detected by RT-PCR during the period when Hp-Gad-positive cells were seen as a subpopulation of blastocoelar cells and on the apical side of the ciliary band from the 2 d.p.f. pluteus stage. Consistent with these observations, inhibition of GAD with 3-mercaptopropioninc acid inhibited GABA immunoreactivity and larval swimming dose dependently. Hp-gabrA amplimers were detected weakly in unfertilized eggs and 4 d.p.f. plutei but strongly from fertilized eggs to 2 d.p.f. plutei, and Hp-GabrA, together with GABA, was localized at the ciliary band in association with dopamine receptor D1 from the two-arm pluteus stage. Hp-gabarap transcription and protein expression were detected from the swimming blastula stage. Inhibition of the GABAA receptor by bicuculline inhibited larval swimming dose dependently. Inhibition of larval swimming by either 3-mercaptopropionic acid or bicuculline was more severe in older larvae (17 and 34 d.p.f. plutei) than in younger ones (1 d.p.f. prism larvae).

Vibrio hemicentroti sp. nov., an alginate lyase-producing bacterium, isolated from the gut microflora of sea urchin (Hemicentrotus pulcherrimus).

An alginate lyase-producing bacterium, designated AlyHP32(T), was isolated from the gut of sea urchin (Hemicentrotus pulcherrimus) obtained from the South Sea, Republic of Korea. Cells of strain AlyHP32(T) were Gram-reaction-negative and motile with a single polar flagellum. The strain grew with 1-6 % (w/v) NaCl (optimum 2-4 %) and at 4-30 °C (optimum 15-25 °C). Phylogenetic analysis based on sequences of the 16S rRNA gene and five housekeeping genes (atpA, pyrH, recA, rpoA and rpoD) revealed that strain AlyHP32(T) belonged to the genus Vibrio and formed a compact clade with the Vibrio splendidus group. However, DNA-DNA hybridization and fingerprints using the repetitive primers BOX and REP indicated that strain AlyHP32(T) was distinct from closely related species of the genus Vibrio. The major fatty acids were summed feature 3 (C16:1ω7c and/or C16:1ω6c) and C16:0. The DNA G+C content was 44.1 mol%. The predominant quinone was ubiquinone Q-8. Based on genotypic, phenotypic and DNA-DNA hybridization analysis, strain AlyHP32(T) represents a novel species of the genus Vibrio; the name Vibrio hemicentroti sp. nov. (type strain AlyHP32(T) = KCTC 32085(T) = DSM 26178(T)) is proposed for this novel taxon.

Salegentibacter echinorum sp. nov., isolated from the sea urchin Hemicentrotus pulcherrimus.

A novel Gram-negative, strictly aerobic, heterotrophic, non-motile and yellow-pigmented bacterial strain, designated HD4(T), was isolated from the sea urchin Hemicentrotus pulcherrimus collected from the Yellow Sea in China. Optimal growth of the strain was observed at 28-30 °C, pH 6.8-7.3, and in the presence of 3-5 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain HD4(T) exhibited high similarity with the members of Salegentibacter (92.3-95.4 %). The DNA G+C content was 37.0 mol%, MK-6 was the main respiratory quinone and summed feature 3 (comprising iso-C15:0 2-OH/C16:1ω7c), iso-C15:0, iso-C17:0 3-OH and anteiso-C15:0 were the major cellular fatty acids. The predominant polar lipids in strain HD4(T) were phosphatidylethanolamine and two unknown lipids (L2, L4). Based on the phylogenetic, physiological and biochemical characteristics, strain HD4(T) should be classified as a novel species within the genus Salegentibacter, for which the name Salegentibacter echinorum sp. nov. is proposed. The type strain is HD4(T) (=CICC 10466(T) = NRRL B-59666(T)).

Binding properties of thyroxine to nuclear extract from sea urchin larvae.

We previously reported that thyroid hormones are involved in the formation of the adult rudiment and adult-type skeleton in sea urchin larvae, as well as in the resorption of larval tissues. In the present study, to search for the presence of thyroid hormone receptor in sea urchin larvae, we performed a ligand-binding assay between radiolabeled thyroid hormones and nuclear extracts from the larvae of the sea urchin Hemicentrotus pulcherrimus. The presence of binding sites with a high affinity to thyroxine (T4) was detected in the nuclear extract, but not in the cytoplasmic fraction. The dissociation constants for the T4 binding to the nuclear extracts were estimated to be about 18 pM from the mesenchyme-blastula stage to the four-armed pluteus stage. The quantity of T4 binding sites in the nuclear extracts increased during larval development. These results suggest that the binding affinity to T4 in the nuclear extracts was caused by a putative nuclear thyroid hormone receptor in sea urchin larvae.

Structure elucidation and antitumor activity of a water soluble polysaccharide from Hemicentrotus pulcherrimus.

Sea urchin nowadays serves as a delicacy around the world, and its gonads accumulate abundant polysaccharides before gametogenesis. However, the structure and bioactivity of these polysaccharides remain less well understood. Herein, a water soluble polysaccharide (HPP-1S) with a molecular weight of 2.996 × 107 Da was purified from the gonads of Hemicentrotus pulcherrimus. Chemical, spectroscopic and oligosaccharide sequencing analyses revealed that HPP-1S was a highly homogeneous polysaccharide featuring a linear backbone of 1,4-linked α-d-glucose with 1,6-α-d-glucose and 1,6-α-D-glucuronic acid side chains grafted on the backbone in an alternating pattern. In vitro, HPP-1S can arrest the cell cycle at G2/M and sub-G1 phases, and induce apoptosis in Hela cells potentially by increasing expression ratio of Bax/Bcl-2. In vivo, HPP-1S exhibited obvious antitumor efficacy in Hela xenograft-bearing nude mice with low toxicity. These findings indicated that HPP-1S might serve as a potential low toxic antitumor agent.

ankAT-1 is a novel gene mediating the apical tuft formation in the sea urchin embryo.

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 ß-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.

HpSumf1 is involved in the activation of sulfatases responsible for regulation of skeletogenesis during sea urchin development.

Sulfatases such as arylsulfatase and heparan sulfate 6-O-endosulfatase play important roles in morphogenesis during sea urchin development. For the activation of these sulfatases, Cα-formylglycine formation by sulfatase modifying factor (Sumf) is required. In this study, to clarify the regulatory mechanisms for the activation of sulfatases during sea urchin development, we examined the expression and function of the Hemicentrotus pulcherrimus homologs of Sumf1 and Sumf2 (HpSumf1 and HpSumf2, respectively). Expression of HpSumf1 but not HpSumf2 mRNA was dynamically changed during early development. Functional analyses of recombinant HpSumf1 and HpSumf2 using HEK293T cells expressing mouse arylsulfatase A (ArsA) indicated that HpSumf1 and HpSumf2 were both able to activate mammalian ArsA. Knockdown of HpSumf1 using morpholino antisense oligonucleotides caused abnormal spicule formation in the sea urchin embryo. Injection of HpSumf2 mRNA had no effect on skeletogenesis, while injection of HpSumf1 mRNA induced severe supernumerary spicule formation. Taken together, these findings suggest that HpSumf1 is involved in the activation of sulfatases required for control of skeletogenesis.