Early expression onset of tissue-specific effector genes during the specification process in sea urchin embryos.

BACKGROUND: In the course of animal developmental processes, various tissues are differentiated through complex interactions within the gene regulatory network. As a general concept, differentiation has been considered to be the endpoint of specification processes. Previous works followed this view and provided a genetic control scheme of differentiation in sea urchin embryos: early specification genes generate distinct regulatory territories in an embryo to express a small set of differentiation driver genes; these genes eventually stimulate the expression of tissue-specific effector genes, which provide biological identity to differentiated cells, in each region. However, some tissue-specific effector genes begin to be expressed in parallel with the expression onset of early specification genes, raising questions about the simplistic regulatory scheme of tissue-specific effector gene expression and the current concept of differentiation itself. RESULTS: Here, we examined the dynamics of effector gene expression patterns during sea urchin embryogenesis. Our transcriptome-based analysis indicated that many tissue-specific effector genes begin to be expressed and accumulated along with the advancing specification GRN in the distinct cell lineages of embryos. Moreover, we found that the expression of some of the tissue-specific effector genes commences before cell lineage segregation occurs. CONCLUSIONS: Based on this finding, we propose that the expression onset of tissue-specific effector genes is controlled more dynamically than suggested in the previously proposed simplistic regulation scheme. Thus, we suggest that differentiation should be conceptualized as a seamless process of accumulation of effector expression along with the advancing specification GRN. This pattern of effector gene expression may have interesting implications for the evolution of novel cell types.

Nonstationary footprints of ENSO in the Mekong River Delta hydrology.

The Mekong River Delta (MRD) is an essential agricultural area for the worldwide rice supply. Floods and droughts triggered by El Niño southern oscillation (ENSO) have been threatening sustenance in the MRD. Sustainable food supplies require understanding the response of the MRD hydrology to the changing ENSO behaviour in recent decades. Here, we reconstructed the annual rainfall maxima in the MRD using the oceanic paleoclimate proxy from coral skeletons and compared them with ENSO indexes. Annual minima of coral-based seawater oxygen isotope (δ18Osw) correlated with annual rainfall maxima, which allowed to extend rainfall data from 1924 to the recent. The annual rainfall maxima based on δ18Osw negatively correlated with the central Pacific El Niño index. This suggested that La Niña and central Pacific El Niño events lead to heavy and light rainy seasons. The heavy rainy season had more serious impacts in recent decades, which likely increases the flood risk. In contrast, the frequency and rainfall amount of the light rainy season has not changed significantly, although a catastrophic drought has hit the MRD. Our finding concludes that the impact of the ENSO event on MRD hydrology is inconsistent in the past century.

Gene regulation of adult skeletogenesis in starfish and modifications during gene network co-option.

The larval skeleton of the echinoderm is believed to have been acquired through co-option of a pre-existing gene regulatory network (GRN); that is, the mechanism for adult skeleton formation in the echinoderm was deployed in early embryogenesis during echinoderm diversification. To explore the evolutionary changes that occurred during co-option, we examined the mechanism for adult skeletogenesis using the starfish Patiria pectinifera. Expression patterns of skeletogenesis-related genes (vegf, vegfr, ets1/2, erg, alx1, ca1, and clect) suggest that adult skeletogenic cells develop from the posterior coelom after the start of feeding. Treatment with inhibitors and gene knockout using transcription activator-like effector nucleases (TALENs) suggest that the feeding-nutrient sensing pathway activates Vegf signaling via target of rapamycin (TOR) activity, leading to the activation of skeletogenic regulatory genes in starfish. In the larval skeletogenesis of sea urchins, the homeobox gene pmar1 activates skeletogenic regulatory genes, but in starfish, localized expression of the pmar1-related genes phbA and phbB was not detected during the adult skeleton formation stage. Based on these data, we provide a model for the adult skeletogenic GRN in the echinoderm and propose that the upstream regulatory system changed from the feeding-TOR-Vegf pathway to a homeobox gene-system during co-option of the skeletogenic GRN.

Human disease-associated extracellular matrix orthologs ECM3 and QBRICK regulate primary mesenchymal cell migration in sea urchin embryos.

Sea urchin embryos have been one of model organisms to investigate cellular behaviors because of their simple cell composition and transparent body. They also give us an opportunity to investigate molecular functions of human proteins of interest that are conserved in sea urchin. Here we report that human disease-associated extracellular matrix orthologues ECM3 and QBRICK are necessary for mesenchymal cell migration during sea urchin embryogenesis. Immunofluorescence has visualized the colocalization of QBRICK and ECM3 on both apical and basal surface of ectoderm. On the basal surface, QBRICK and ECM3 constitute together a mesh-like fibrillar structure along the blastocoel wall. When the expression of ECM3 was knocked down by antisense-morpholino oligonucleotides, the ECM3-QBRICK fibrillar structure completely disappeared. When QBRICK was knocked down, the ECM3 was still present, but the basally localized fibers became fragmented. The ingression and migration of primary mesenchymal cells were not critically affected, but their migration at later stages was severely affected in both knock-down embryos. As a consequence of impaired primary mesenchymal cell migration, improper spicule formation was observed. These results indicate that ECM3 and QBRICK are components of extracellular matrix, which play important role in primary mesenchymal cell migration, and that sea urchin is a useful experimental animal model to investigate human disease-associated extracellular matrix proteins.

pmar1/phb homeobox genes and the evolution of the double-negative gate for endomesoderm specification in echinoderms.

In several model animals, the earliest phases of embryogenesis are regulated by lineage-specific genes, such as Drosophila bicoid Sea urchin (echinoid) embryogenesis is initiated by zygotic expression of pmar1, a paired-class homeobox gene that has been considered to be present only in the lineage of modern urchins (euechinoids). In euechinoids, Pmar1 promotes endomesoderm specification by repressing the hairy and enhancer of split C (hesC) gene. Here, we have identified the basal echinoid (cidaroid) pmar1 gene, which also promotes endomesoderm specification but not by repressing hesC A further search for related genes demonstrated that other echinoderms have pmar1-related genes named phb Functional analyses of starfish Phb proteins indicated that, similar to cidaroid Pmar1, they promote activation of endomesoderm regulatory gene orthologs via an unknown repressor that is not HesC. Based on these results, we propose that Pmar1 may have recapitulated the regulatory function of Phb during the early diversification of echinoids and that the additional repressor HesC was placed under the control of Pmar1 in the euechinoid lineage. This case provides an exceptional model for understanding how early developmental processes diverge.

cis-Regulatory analysis for later phase of anterior neuroectoderm-specific foxQ2 expression in sea urchin embryos.

The specification of anterior neuroectoderm is controlled by a highly conserved molecular mechanism in bilaterians. A forkhead family gene, foxQ2, is known to be one of the pivotal regulators, which is zygotically expressed in this region during embryogenesis of a broad range of bilaterians. However, what controls the expression of this essential factor has remained unclear to date. To reveal the regulatory mechanism of foxQ2, we performed cis-regulatory analysis of two foxQ2 genes, foxQ2a and foxQ2b, in a sea urchin Hemicentrotus pulcherrimus. In sea urchin embryos, foxQ2 is initially expressed in the entire animal hemisphere and subsequently shows narrower expression restricted to the anterior pole region. In this study, as a first step to understand the foxQ2 regulation, we focused on the later restricted expression and analyzed the upstream cis-regulatory sequences of foxQ2a and foxQ2b by using the constructs fused to short half-life green fluorescent protein. Based on deletion and mutation analyses of both foxQ2, we identified each of the five regulatory sequences, which were 4-9 bp long. Neither of the regulatory sequences contains any motifs for ectopic activation or spatial repression, suggesting that later mRNA localization is regulated in situ. We also suggest that the three amino acid loop extension-class homeobox gene Meis is involved in the maintenance of foxQ2b, the expression of which is dominant during embryogenesis.

Cidaroids, clypeasteroids, and spatangoids: Procurement, culture, and basic methods.

This chapter describes practical methods and key points for using non-camarodont echinoids including cidaroids (Order Cidaroida), clypeasteroids (also known as sand dollars; Order Clypeasteroida), and spatangoids (also known as heart urchins; Order Spatangoida) as experimental subjects for biological studies. The content described here is based on six Japanese species of echinoids (Astriclypeus manni, Clypeaster japonicus, Echinocardium cordatum, Peronella japonica, Prionocidaris baculosa, and Scaphechinus mirabilis). Specific topics addressed in this chapter include the collection and maintenance of adults, embryonic culture, and experimental procedures for micromanipulations, whole mount in situ hybridization, and immunological experiments.

Oman coral δ18O seawater record suggests that Western Indian Ocean upwelling uncouples from the Indian Ocean Dipole during the global-warming hiatus.

The Indian Ocean Dipole (IOD) is an interannual mode of climate variability in the Indian Ocean that has intensified with 20th century global-warming. However, instrumental data shows a global-warming hiatus between the late-1990s and 2015. It is presently not clear how the global-warming hiatus affects modes of climate variability such as the IOD, and their basin-wide ocean-atmosphere teleconnections. Here, we present a 26-year long, biweekly record of Sr/Ca and δ18O from a Porites coral drilled in the Gulf of Oman. Sea surface temperature (SSTanom) is calculated from Sr/Ca ratios, and seawater δ18O (δ18Osw-anom) is estimated by subtracting the temperature component from coral δ18O. Our δ18Osw-anom record reveals a significant regime shift in 1999, towards lower mean δ18Osw values, reflecting intensified upwelling in the western Indian Ocean. Prior to the 1999 regime shift, our SSTanom and δ18Osw-anom show a clear IOD signature, with higher values in the summer of positive-IOD years due to weakened upwelling. The IOD signature in SSTanom and δ18Osw-anom disappears with the overall intensification of upwelling after the 1999 regime shift. The inferred increase in upwelling is likely driven by an intensified Walker circulation during the global-warming hiatus. Upwelling in the Western Indian Ocean uncouples from the IOD.

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.

Past summer upwelling events in the Gulf of Oman derived from a coral geochemical record.

We used a high-resolution oxygen isotope (δ18Ocoral), carbon isotope (δ13Ccoral) and Sr/Ca ratios measured in the skeleton of a reef-building coral, Porites sp., to reveal seasonal-scale upwelling events and their interannual variability in the Gulf of Oman. Our δ13Ccoral record shows sharp negative excursions in the summer, which correlate with known upwelling events. Using δ13Ccoral anomalies as a proxy for upwelling, we found 17 summer upwelling events occurred in the last 26 years. These anomalous negative excursions of δ13Ccoral result from upwelled water depleted in 13C (dissolved inorganic carbon) and decreased water-column transparency. We reconstructed biweekly SSTs from coral Sr/Ca ratios and the oxygen isotopic composition of seawater (δ18OSW) by subtracting the reconstructed Sr/Ca-SST from δ18Ocoral. Significant δ18OSW anomalies occur during major upwelling events. Our results suggest δ13Ccoral anomalies can be used as a proxy for seasonal upwelling intensity in the Gulf of Oman, which, driven by the Indian/Arabian Summer Monsoon, is subject to interannual variability.

Chronic active Epstein-Barr virus infection with marked pericardial effusion successfully treated with allogeneic peripheral blood stem cell transplantation.

A 23-year-old woman presented with a persistent fever and shortness of breath. Computed tomography showed marked pericardial effusion, hepatosplenomegaly, and cervical and mediastinal lymph node swelling. Epstein-Barr virus (EBV) antibody titers were abnormally elevated, and the copy number of EBV-DNA was increased in peripheral blood. Based on these observations, she was diagnosed with chronic active EBV infection (CAEBV). The EBV-infected cells in her peripheral blood were CD4(+)T lymphocytes. Fever and pericardial effusion improved following treatment with a combination of prednisolone, etoposide, and cyclosporine; however, peripheral blood EBV-DNA levels remained high. The patient underwent allogeneic peripheral blood stem cell transplantation from an EBV-seronegative, HLA-matched sibling donor, with fludarabine and melphalan conditioning. The post-transplantation course was uneventful, except for mild skin acute graft-versus-host disease (grade 2). EBV-DNA became undetectable in peripheral blood 98 days post transplantation. She has since been in good health without disease recurrence. CAEBV is a potentially fatal disease caused by persistent EBV infection of T lymphocytes or natural killer cells, thus requiring prompt treatment and allogeneic transplantation. Pericardial effusion is rarely observed in CAEBV and can impede its diagnosis. Therefore, we should be aware that patients may present with marked pericardial effusion as an initial manifestation of CAEBV.

Roles of hesC and gcm in echinoid larval mesenchyme cell development.

To understand the roles of hesC and gcm during larval mesenchyme specification and differentiation in echinoids, we performed perturbation experiments for these genes in two distantly related euechinoids, Hemicentrotus pulcherrimus and Scaphechinus mirabilis. The number of larval mesenchyme cells increased when the translation of hesC was inhibited, thereby suggesting that hesC has a general role in larval mesenchyme development. We confirmed previous results by demonstrating that gcm is involved in pigment cell differentiation. Simultaneous inhibition of the translation of hesC and gcm induced a significant increase in the number of skeletogenic cells, which suggests that gcm functions in skeletogenic fate repression. Based on these observations, we suggest that: (i) hesC participates in some general aspects of mesenchymal cell development; and (ii) gcm is involved in the mechanism responsible for the binary specification of skeletogenic and pigment cell fates.

Successful Allogeneic Stem Cell Transplantation for Severe Aplastic Anemia after Treatment of Lymphoproliferative Disorder Caused by Rabbit Antithymocyte Globulin.

Immunosuppressive therapy (IST) with a combination of antithymocyte globulin (ATG) and cyclosporine (CsA) is an effective therapeutic modality for patients with aplastic anemia (AA) who are not eligible for allogeneic stem cell transplantation (Allo-SCT) from a human leukocyte antigen-identical sibling donor. However, there have been reports of some patients developing lymphoproliferative disorder (LPD) after IST for AA. We herein report a case of a 26-year-old man with severe AA (SAA) complicated by LPD after a single course of IST, who was successfully treated with Allo-SCT from an unrelated donor. Two months after starting IST for SAA, he developed LPD in the stomach. CsA was reduced, however, his neutrophil counts decreased, and CsA could not be discontinued. The patient was treated with rituximab monotherapy, and LPD resulted in complete remission. However, he failed IST for SAA and underwent Allo-SCT with reduced-intensity conditioning to recover his hematopoiesis. The patient has achieved complete hematopoietic recovery without the recurrence of LPD for five years after transplantation. This is the first report of successful Allo-SCT for SAA after the treatment of LPD caused by the use of rabbit ATG. This case provides useful information for the management of SAA with the development of LPD after IST.

Expession patterns of mesenchyme specification genes in two distantly related echinoids, Glyptocidaris crenularis and Echinocardium cordatum.

The molecular mechanism of the larval mesenchyme cell specification in echinoids has been well analyzed. However, most of the data have been provided by studies of a single group of echinoids, the order Camarodonta. Little is known about this mechanism in other echinoid orders. We examined the expression patterns of mesenchyme specification genes, micro1, hesC, alx1, tbr, ets1, cyp1, and gcm, in the two non-Camarodonta echinoids, Glyptocidaris crenularis and Echinocardium cordatum. We found that the expression patterns of some genes contained characteristics that were unique to one of the species; others were shared by the two species. Some of the shared characteristics of G. crenularis and E. cordatum are not found in the species belonging to Camarodonta, suggesting the derived status of this order. The expression of ets1 in E. cordatum aboral ectoderm is one of the molecular level modifications possibly related to an evolutionarily novel larval structure, the posterior process. Our results suggest that a considerable number of modifications in the mesenchyme specification mechanisms have been introduced during the echinoid evolution.

Efficacy of myeloablative allogeneic hematopoietic stem cell transplantation in adult patients with MLL-ELL-positive acute myeloid leukemia.

Acute myeloid leukemia (AML) with mixed lineage leukemia-eleven-nineteen lysine-rich leukemia (MLL-ELL) is a rare subtype of MLL-rearranged AML. The outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for patients with this disease remains unknown. In the present study, we retrospectively investigated the efficacy of allo-HSCT in eight adult MLL-ELL-positive AML patients. Although all eight patients achieved first complete remission (CR1), three (37.5 %) patients experienced relapse after induction therapy. Five (62.5 %) patients underwent allo-HSCT during CR1, whereas two (25.0 %) underwent allo-HSCT during disease relapse, and one (12.5 %) during CR2. All three patients who received allo-HSCT beyond CR1 died due to AML progression after allo-HSCT. Of the five patients who received allo-HSCT during CR1, three (60.0 %) remained alive at study conclusion. The overall survival rate at five years was 50.0 %. Intriguingly, clonally expanded non-leukemic cells expressing MLL-ELL during consolidation therapy were found to be eradicated after allo-HSCT during the monitoring of minimal residual disease in one patient; this indicates that allo-HSCT is efficacious for eliminating pre-leukemic cells resistant to chemotherapy. In conclusion, allo-HSCT soon after CR1 represents a promising therapeutic option for adult AML patients with MLL-ELL, although the outcome of allo-HSCT for patients beyond CR1 was dismal.

Early development and neurogenesis of Temnopleurus reevesii.

Sea urchins are model non-chordate deuterostomes, and studying the nervous system of their embryos can aid in the understanding of the universal mechanisms of neurogenesis. However, despite the long history of sea urchin embryology research, the molecular mechanisms of their neurogenesis have not been well investigated, in part because neurons appear relatively late during embryogenesis. In this study, we used the species Temnopleurus reevesii as a new sea urchin model and investigated the detail of its development and neurogenesis during early embryogenesis. We found that the embryos of T. reevesii were tolerant of high temperatures and could be cultured successfully at 15-30°C during early embryogenesis. At 30°C, the embryos developed rapidly enough that the neurons appeared at just after 24 h. This is faster than the development of other model urchins, such as Hemicentrotus pulcherrimus or Strongylocentrotus purpuratus. In addition, the body of the embryo was highly transparent, allowing the details of the neural network to be easily captured by ordinary epifluorescent and confocal microscopy without any additional treatments. Because of its rapid development and high transparency during embryogenesis, T. reevesii may be a suitable sea urchin model for studying neurogenesis. Moreover, the males and females are easily distinguishable, and the style of early cleavages is intriguingly unusual, suggesting that this sea urchin might be a good candidate for addressing not only neurology but also cell and developmental biology.

Early-Onset Severe Diffuse Alveolar Hemorrhage after Bortezomib Administration Suggestive of Pulmonary Involvement of Myeloma Cells.

Severe acute lung injury is a rare but life-threatening complication associated with bortezomib. We report a patient with multiple myeloma who developed a severe diffuse alveolar hemorrhage (DAH) immediately after the first bortezomib administration. The patient was suspected to have pulmonary involvement of myeloma, which caused DAH after rapidly eradicating myeloma cells in the lungs with bortezomib. Rechallenge with bortezomib was performed without recurrent DAH. In patients with multiple myeloma who manifest abnormal pulmonary shadow, we should be aware of early-onset severe DAH after bortezomib administration, which might be due to pulmonary involvement of myeloma cells.

Acute myeloid leukemia concurrent with spinal epidural extramedullary myeloid sarcoma accompanied by a high CD25 expression and the FLT3-ITD mutation.

Myeloid sarcoma (MS) is an extramedullary myeloid tumor that sometimes presents with antedating systemic leukemia, leading physicians to the misdiagnosis of lymphoma. CD25 is expressed in 13% of patients with acute myeloid leukemia (AML), and its expression is associated with FLT3-ITD mutations, an elevated serum soluble interleukin 2 receptor (sIL-2R) level and a lower survival rate. However, there are no reports concerning the relationship between MS and the CD25 expression. We herein report a case of AML accompanied by thoracic epidural MS with a high CD25 expression, the FLT3-ITD mutation and an extremely elevated serum sIL-2R level in a 59-year-old man who presented with paraplegia.