Identification of ephrin-A3 and novel genes specific to the midbrain-MHB in embryonic zebrafish by ordered differential display.

Development of the tectum and the cerebellum is induced by a reciprocal inductive signaling between their respective primordia, the midbrain and the midbrain/hindbrain boundary (MHB). We set out to identify molecules that function in and downstream of this reciprocal signaling. Overexpression of LIM domain of the transcription factor Islet-3 (LIM(Isl-3)) leads to inhibition of this reciprocal signaling and to resultant defects in tectal and cerebellar development. We therefore searched for genes that may be either up- or down-regulated by overexpression of LIM(Isl-3) by comparing the gene expression profiles in the midbrain and the MHB of normal embryos and embryos in which Islet-3 function was repressed, using a combination of ordered differential display and whole-mount in situ hybridization. Among genes identified in this search, two cDNA fragments encoded Wnt1 and FGF8, which are already known to be essential for the reciprocal signaling between the midbrain and the MHB, confirming the effectiveness of our strategy. We identified four other partial cDNA clones that were specifically expressed around the MHB, ten cDNAs specifically expressed in the tectum, and three cDNAs expressed in neural crest cells including those derived from the midbrain level. The ephrin-A3 gene was specifically expressed in posterior tectum in a gradient that decreased anteriorly. Although ephrin-A2 and ephrin-A5 have been reported to be expressed in the corresponding region in mouse embryos, the superior/inferior colliculi, mouse ephrin-A3 is not expressed prominently in this region, suggesting that the role of ephrin-A3 in brain development may have been altered in the process of brain evolution.

Transcriptional regulation of the gene for epidermal growth factor-like peptides in sea urchin embryos.

Exogastrula-inducing peptides (EGIPs), which are epidermal growth factor-related peptides of the sea urchin Anthocidaris crassispina, are substances that elicit abnormal gastrulation (exogastrulation) during embryogenesis of the sea urchin. In the present study we have examined the regulation of the expression of the EGIP precursor gene (EGIP) in sea urchin embryos. Whole mount in situ hybridization showed that EGIP is zygotically expressed afterthe onset of gastrulation in subdomains of the embryonic and larval ectoderm. The expression is confined in early gastrulae to small ectodermal regions adjoining the vegetal plate, which progressively expand to almost the entire ectoderm except the oral hood and postoral tips of the arms in later stages. In adults the expression is restricted to the ovary. Zygotic EGIP expression is sensitive to dissociation of embryonic cells, as well as to disruption of the extracellular matrix (ECM) with 5-cis-hydroxyproline, suggesting requirements for interaction with neighboring cells and/or with the ECM. The expression of reporter genes (chloramphenicol acetyl transferase and green fluorescent protein) under the regulation of the 4.6 kb upstream region of EGIP is temporally and spatially similar to that of the endogenous gene, showing that EGIP expression is regulated at the transcription level during embryogenesis by the cis-elements within the 4.6 kb upstream region.

Cloning and characterization of cDNA for syndecan core protein in sea urchin embryos.

The cDNA for the core protein of the heparan sulfate proteoglycan, syndecan, of embryos of the sea urchin Anthocidaris crassispina was cloned and characterized. Reverse transcription-polymerase chain reaction (RT-PCR) was used with total ribonucleic acid (RNA) from late gastrula stage embryos and degenerate primers for conserved regions of the core protein, to obtain a 0.1 kb PCR product. A late gastrula stage cDNA library was then screened using the PCR product as a probe. The clones obtained contained an open reading frame of 219 amino acid residues. The predicted product was 41.6% identical to mouse syndecan-1 in the region spanning the cytoplasmic and transmembrane domains. Northern analysis showed that the transcripts were present in unfertilized eggs and maximum expression was detected at the early gastrula stage. Syndecan mRNA was localized around the nuclei at the early cleavage stage, but was then found in the ectodermal cells of the gastrula embryos. Western blotting analysis using the antibody against the recombinant syndecan showed that the proteoglycan was present at a constant level from the unfertilized egg stage through to the pluteus larval stage. Immunostaining revealed that the protein was expressed on apical and basal surfaces of the epithelial wall in blastulae and gastrulae.

Zebrafish Mesp family genes, mesp-a and mesp-b are segmentally expressed in the presomitic mesoderm, and Mesp-b confers the anterior identity to the developing somites.

Segmentation of a vertebrate embryo begins with the subdivision of the paraxial mesoderm into somites through a not-well-understood process. Recent studies provided evidence that the Notch-Delta and the FGFR (fibroblast growth factor receptor) signalling pathways are required for segmentation. In addition, the Mesp family of bHLH transcription factors have been implicated in establishing a segmental prepattern in the presomitic mesoderm. In this study, we have characterized zebrafish mesp-a and mesp-b genes that are closely related to Mesp family genes in other vertebrates. During gastrulation, only mesp-a is expressed in the paraxial mesoderm at the blastoderm margin. During the segmentation period, both genes are segmentally expressed in one to three stripes in the anterior parts of somite primordia. In fused somites (fss) embryos, in which all early somite boundary formation is blocked, initial mesp-a expression at the gastrula stage remains intact, but the expression of mesp-a and mesp-b is not detected during the segmentation period. This suggests that these genes are downstream targets of fss at the segmentation stage. Comparison with her1 expression (Müller, M., von Weizsäcker, E. and Campos-Ortega, J. A. (1996) Development 122, 2071-2078) suggests that, like her1, mesp genes are not expressed in primordia of the first several somites. Furthermore, we found that zebrafish her1 expression oscillates in the presomitic mesoderm. The her1 stripe, which first appears in the tailbud region, moves in a caudal to rostral direction, and it finally overlaps the most rostral mesp stripe. Thus, in the trunk region, both her1 and mesp transcripts are detected in every somite primordium posterior to the forming somites. Ectopic expression of Mesp-b in embryos causes a loss of the posterior identity within the somite primordium, leading to a segmentation defect. These embryos show a reduction in expression of the posterior genes, myoD and notch5, with uniform expression of the anterior genes, FGFR1, papc and notch6. These observations suggest that zebrafish mesp genes are involved in anteroposterior specification within the presumptive somites, by regulating the essential signalling pathways mediated by Notch-Delta and FGFR.

Association of the sea urchin EGF-related peptide, EGIP-D, with fasciclin I-related ECM proteins from the sea urchin Anthocidaris crassispina.

Exogastrula-inducing peptides (EGIP) of the sea urchin Anthocidaris crassispina are endogenous peptides related to epidermal growth factor (EGF), which induce exogastrulation in the embryo. Recently, a protein(s) from sea urchin embryos that binds to one of the EGIP, EGIP-D (EGIP-D-binding protein, EBP) was purified. The isolation and characterization of the cDNA clones for two EBP proteins (EBP-alpha and EBP-beta) is reported. The two EBP proteins were highly similar in structure to each other; both possessed putative cell-binding sites and two repeated sequences characteristically seen in the insect neuronal cell adhesion protein, fasciclin I. The EBP showed similarity with other sea urchin proteins HLC-32, Bep1, and Bep4. It has been confirmed that bacterially expressed EBP proteins associate with EGIP-D as does native EBP, suggesting the interaction between EGF-related proteins and fasciclin I-related proteins. An EBP transcript of 1.4 kb was strongly expressed in immature ovaries but not in immature testes. A somewhat lower level of the transcript existed in unfertilized eggs and the amount gradually declined to an almost undetectable level by the pluteus stage. The EBP proteins were present throughout embryonic development at nearly constant levels. Although most of the proteins were distributed rather evenly in the cytoplasm, a small portion was detected on the apical surface of blastomeres and ectodermal cells, showing that EBP are components of the hyaline layer.

Expression of a src-type protein tyrosine kinase gene, AcSrc1, in the sea urchin embryo.

By screening a cDNA library and 3'-rapid amplification of cDNA ends, the cDNA for a non-receptor type protein tyrosine kinase from the sea urchin Anthocidaris crassispina was analyzed. The deduced protein (AcSrc1) with the highest identity of about 60% to mammalian Src family kinases shows the characteristic features of the Src family. AcSrc1 mRNA is maternally expressed in unfertilized eggs, while zygotic expression is first detected in blastulae and continues through the pluteus stage. Zygotic mRNA expression, visualized by in situ hybridization, is detected specifically in archenteron at the gastrula stage, while it is restricted in plutei to the midgut and hindgut, suggesting specific roles for AcSrcl in the formation and/or functions of the digestive tract. Meanwhile, western blot analysis has shown that the AcSrc1 protein is constantly expressed throughout embryogenesis. By immunostaining, it was found that the protein (distributed evenly in the cytoplasm of unfertilized eggs) is translocated to the membrane after fertilization. All through the following development, AcSrcl was localized to the peripheries of different embryonic cells, although at a relatively low level of localization at the boundaries between adjacent cells.

Functional organization of DNA elements regulating SM30alpha, a spicule matrix gene of sea urchin embryos.

The SM30a gene encodes a protein in the embryonic endoskeleton of the sea urchin Strongylocentrotus purpuratus, and is specifically expressed in the skeletogenic primary mesenchyme cell lineage. To clarify the mechanism for the differentiation of this cell lineage, which proceeds rather autonomously in the embryo, regulation of the SM30alpha gene was investigated previously and it was shown that the distal DNA region upstream of this gene from - 1.6 to - 1.0 kb contained numerous negative regulatory elements that suppressed the ectopic expression of the gene in the gut. Here we study the influence of the proximal region from - 303 to + 104 bp. Analysis of the expression of reporter constructs indicated that a strong positive enhancer element existed in the region from -142 to -105bp. This element worked both in forward and reverse orientations and additively when placed tandemly upstream to the reporter gene. In addition, other weaker positive and negative regulatory sites were also detected throughout the proximal region. Electrophoretic gel mobility shift analyses showed that multiple nuclear proteins were bound to the putative strong enhancer region. One of the proteins binding to this region was present in ear y blastulae, a time when the SM30 gene was still silent, but it was not in prism embryos actively expressing the gene. The binding region for this blastula-specific protein was narrowed down to the region from - 132 to -122 bp, which included the consensus binding site for the mammalian proto-oncogene product, Ets. Two possible SpGCF1 binding sites were identified in the vicinity of the enhancer region. This information was used to make a comparison of the general regulatory architecture of genes that contribute to the formation of the skeletal spicule.

Induction of metamorphosis in the sand dollar Peronella japonica by thyroid hormones.

The larva of the sand dollar Peronella japonica lacks a mouth and gut, and undergoes metamorphosis into a juvenile sand dollar without feeding. In the present study, it was found that thyroid hormones accelerate the metamorphosis of P. japonica larvae. The contents of thyroid hormones in larvae increased gradually during development. Thiourea and potassium perchlorate, inhibitors of thyroid hormone synthesis, delayed larval metamorphosis and simultaneously repressed an increase in the content of thyroxine in the larval body. These results suggest that the P. japonica larva has a system for synthesis of thyroid hormones that act as factors for inducing metamorphosis.

Purification of EGIP-D-binding protein from the embryos of the sea urchin Anthocidaris crassispina.

Exogastrula-inducing peptides (EGIPs) are intrinsic factors that are present in eggs and embryos of the sea urchin Anthocidaris crassispina. They induce exogastrulation when added exogenously to the embryos. In the present study, we isolated an EGIP-D-binding protein (EBP) from a homogenate of mesenchyme blastulae. EBP had an apparent molecular weight of 33,000. The N-terminal amino acid sequence of EBP had a sequence homology to HLC-32 and bep4 identified in other sea urchin embryos. In addition to its ability of binding to EGIP-D, EBP also inhibited exogastrulation induced by EGIP-D. These results suggest that EBP plays an essential role in EGIP-D-induced exogastrulation.

The protein tyrosine kinases of the sea urchin Anthocidaris crassispina.

In order to know the function of protein tyrosine kinases (PTKs) in the development of sea urchin embryos, we performed reverse transcription-polymerase chain reaction (RT-PCR) to obtain partial cDNA fragments for PTK genes using primers to highly conserved regions of the PTK family. A total of seven PTK sequences were identified, two of which represented receptor PTK (RTK1 and RTK2), and five of which were non-receptor PTKs (NRTK1-5). RTK1 was highly similar to FGF receptor and Ret kinase, while RTK2 showed features of the insulin receptor family. NRTK1 and 2 belonged to the Src family and could be involved in egg activation at fertilization. NRTK3 showed the features of the Btk family kinases, while NRTK4 seemed to be a member of the Syk/ZAP70 family. NRTK5 is the Csk-type kinase of the sea urchin, which is known to negatively regulate the Src family kinases. RTK1 was not detected in unfertilized eggs and was activated after blastula stage. All the other PTK genes were expressed both maternally in unfertilized eggs and zygotically after fertilization, though each gene showed distinct temporal patterns.

Molecular cloning of a cDNA that encodes the precursor to several exogastrula-inducing peptides, epidermal-growth-factor-related polypeptides of the sea urchin Anthocidaris crassispina.

Complementary DNA clones for exogastrula-inducing peptides (EGIPs) of the sea urchin Anthocidaris crassispina, which are related to epidermal growth factor (EGF), were obtained from a cDNA library of late gastrula embryos using, as probe, the partial cDNA for one of the EGIP (EGIP-D) obtained by the reverse-transcription PCR method. The longest cDNA was composed of 1662 bp, and encoded a protein of approximately 36 kDa with a region that resembled a signal sequence. The deduced protein contains the sequences of EGIP-C, EGIP-D, and EGIP-A in that order, followed by the sequence for an unidentified EGIP-like polypeptide. When expressed in Escherichia coli as a fusion protein with beta-galactosidase, the product for the cDNA was specifically recognized by a rabbit antibody raised against EGIP-D that had been purified from embryos. Characteristic amino acid residues were found around the N-terminus and the C-terminus of each EGIP sequence, suggesting a specific processing mechanism for the generation of the individual EGIPs from the precursor. RNA-blot analysis revealed the presence of EGIP mRNA in unfertilized eggs. The level of this mRNA decreased gradually after fertilization, began to increase dramatically after the onset of gastrulation, and continued to increase through the pluteus stage. Genomic Southern-blot analysis suggested that this gene is present as a single copy. A homology search showed that the EGIP cDNA has a similarity to the cDNA for SpEGF2 which was cloned as a gastrula-specific gene in another sea urchin, Strongylocentrotus purpuratus.

Genomic organization of a gene encoding the spicule matrix protein SM30 in the sea urchin Strongylocentrotus purpuratus.

We report the characterization of a genomic clone containing portions of two tandemly arranged genes that encode a spicule matrix protein, SM30, of the sea urchin Strongylocentrotus purpuratus. The isolated 18.4-kilo-base genomic clone contains the complete genomic sequence of one SM30 gene, designated SM30-alpha, and a portion of another SM30 gene, designated SM30-beta. Southern blot analysis shows that SM30 protein is encoded by a small gene family of two to four members. RNase protection assays indicate that the SM30-alpha gene is expressed at the time of spicule formation in the sea urchin embryo. In addition, mapping of SM30-alpha shows that a large single intron interrupts the coding sequence. Comparison of the nucleic acid and amino acid sequences of the SM30-alpha genomic sequence and the previously isolated SM30 cDNA reveals them to be very similar, but not identical. We also demonstrate that 2.6 kilobases of upstream sequence of SM30-alpha are sufficient to direct primary mesenchyme cell-specific expression of a reporter gene construct.

Formation of the adult rudiment of sea urchins is influenced by thyroid hormones.

After the sea urchin embryo has developed to a pluteus larva, the adult rudiment (sea urchin rudiment) is formed, with other adult structures, on the left side of the larva and finally the juvenile sea urchin is formed after metamorphosis. We report here that thyroid hormones (THs) are involved in the formation of the adult rudiment and the adult-type skeleton and the resorption of larval tissues of the sea urchin. The contents of THs in the larval body were determined by radioimmunoassay after the separation of individual THs by HPLC. We confirmed the presence of THs in the larval body and in algae on which the larvae feed. The THs accumulate gradually following the development of the larva and reach maximum levels at the eight-armed stage when the adult rudiment is completed. These results suggest that the development of the larva is influenced by THs accumulated in the larval body. However, inhibitors for the synthesis of THs do not affect the development of the larval body, suggesting a supply of THs from algae.

Conservation of the dimeric unit of H2A and H2B histones during the replication cycle.

The behavior of H2A and H2B histones during the replication cycle has been investigated. H2A-H2B dimer fractions obtained from MH-134SC cells labeled with suitable precursors were fractionated by rate zonal centrifugation in sucrose gradients containing 2 M NaCl. Labeling for one round of replication cycle with an amino acid mixture enriched with dense isotopes and [3H]lysine as a radioactive marker yielded a distinct peak of dense dimer that sedimented faster than the normal dimer. Similar density labeling of cells did not cause measurable alteration in the sedimentation profile of the preexisting normal dimer marked with [14C]lysine. The data suggest conservation of the bulk of the dimeric unit during the replication cycle. Biological significance of the dimeric behavior of H2A and H2B is discussed in favor of the partially conservative model of histone octamer assembly.

Activation of the systemic production of tumor necrosis factor after exposure to acute stress.

Tumor necrosis factor (TNF), which was originally identified as a tumoricidal factor, is now regarded as one of the main regulators of inflammation and various immune systems. Thus it has been considered to be mobilized in case of emergency. However, we assume that TNF and the cytokine network driven by the monokine also function under normal condition for homeostasis of the animal body which is exposed to various kinds of physiological stress. To test this possibility, we exposed C3H/He mice for up to 3 days to five types of acute stress: food deprivation, drinking water deprivation, sleep deprivation, swimming, and physical restraint. After release from the stress, the level of priming for systemic production of TNF was examined using OK-432 (a streptococcal preparation) as a trigger. Priming of TNF production was not observed immediately after 2-day exposure to most of the stressors. Sleep deprivation alone tended to induce a primed state especially when the stress period was lengthened to 3 days. On the other hand, by keeping mice in a normal condition for a 2-day restorative interval after 2-day exposure to the stressors, systemic production of TNF was consistently primed for all the stress examined. The time course of the priming effect was examined in detail for water-deprivation stress. The effect was detected as early as 3 hours after release from stress, was sustained for 2 days, and returned to the basal level by 4 days after the release. Based on these results, we discussed the role of the TNF-driven cytokine circuit in adaptation to stress.

Conservative segregation of tetrameric units of H3 and H4 histones during nucleosome replication.

We have specifically investigated the behavior of H3 and H4 histones during the replication cycle of MH-134SC cells. Mononucleosomes obtained from cells density-labeled with IdU or dense amino acids in the presence of appropriate radiolabeled precursors were applied to sucrose gradients containing 0.3 M NaCl and 4 M urea for rate zonal centrifugation. This allowed the resolution of dense and normal subnucleosome particles composed of DNA and two molecules each of H3 and H4 without any measurable interparticle histone exchange. On labeling with dense amino acids and radiolabeled lysine, a distinct peak of radiolabeled dense particles was obtained. In contrast, pre-radiolabeled H3 and H4 remained in the normal subnucleosome peak region even after one generation time of culturing with dense amino acids. These data indicate the formation of (H3-H4)2 tetramers composed entirely of new H3 and H4 molecules as well as the conservation of pre-existing tetramers. Density labeling for 1 h with IdU in the presence of radiolabeled lysine yielded a distinct peak of radiolabeled dense particles, indicating the deposition of new tetramers on newly replicated DNA. Similar rate zonal analysis of subnucleosome particles obtained from cells prelabeled for 1 h with radiolabeled lysine followed by various IdU-labeling schedules in nonisotopic media yielded data suggesting that tetramers once deposited do not move about randomly during the replication cycle. A possible mode of nucleosome replication is discussed in the light of the present data.

Secretion of tumor necrosis factor during fetal and neonatal development of the mouse: ontogenic inflammation.

Inflammation plays an important role in homeostasis of the body. We therefore can assume that an inflammatory state occurs during ontogenesis of animals. To address this problem, we examined the ability of tumor necrosis factor (TNF), one of the inflammatory mediators, to be secreted by mouse cells during development. We cultured cells prepared from various parts of fetuses (10-19 days of gestation) and postnatal brains by collagenase digestion and assayed the secreted TNF activity by the L-929 cytotoxicity test. We found TNF activity by fetal cells without any stimulation. The spontaneous secretion of TNF was relatively high at around 13-15 days of gestation. The secretion was enhanced by lipopolysaccharide (LPS), showing that fetal cells are in an activated state for TNF secretion. These TNF activities were neutralized completely by rabbit anti-murine TNF antibody. Spontaneous and LPS-enhanced secretion by postnatal brain cells reached a peak around 7 days after birth, and thereafter declined rapidly. This time course was well correlated to the increase in the weight of brain. The producing cells were negative in macrophage marker surface antigen, and heterogeneous in relation to adherence and phagocytic activity, showing that TNF is secreted by various types of cells in the fetal body. These results suggest the presence of an inflammation-like state during ontogenesis. We consider that this "ontogenic inflammation" may be the prototype of inflammation, which can regulate homeostasis of the adult body.

Reassembly of nucleosomal histone octamers during replication of chromatin.

We have examined critically whether or not new and old histones mix in the octameric units of nucleosomes during chromatin replication. MH-134SC cells were density-labeled by culturing with amino acid mixtures enriched with dense isotopes 2H, 13C, and 15N. Mononucleosomes obtained from labeled cells were fractionated by rate zonal sedimentation through a sucrose gradient in heavy water (Senshu et al. (1985) Eur J. Biochem. 150, 575-580). The fractionation can be performed under conditions that do not destabilize nucleosomes. Density-labeling yielded heterogeneous mononucleosome species which showed higher sedimentation rates than normal mononucleosomes. However, they were indistinguishable with respect to their protein compositions, electrophoretic mobilities, electrophoretic patterns of single-stranded DNA fragments liberated by DNase I digestion, electrophoretic mobilities and sedimentation velocities of the DNA moieties, and metabolic stabilities of the histone moieties. These data suggest that the heterogeneity of density-labeled mononucleosomes resulted from the formation of histone octamers density-substituted to different degrees. This would be an inevitable consequence of mixing of new and old histones in the octameric unit of nucleosomes.

Fractionation of newly replicated nucleosomes by density labeling and rate zonal centrifugation for the analysis of the deposition sites of newly synthesized nucleosomal core histones.

We have found that partial resolution of newly replicated nucleosomes can be achieved by rate zonal centrifugation through sucrose density gradients preformed in heavy water. Nucleosome samples were obtained from MH-134SC cells density labeled with 5-iododeoxyuridine in the presence of suitable isotopic precursors. The method is simple and can be performed under conditions that do not destabilize the nucleosome structure. This gave us an exciting opportunity to study the deposition sites of newly synthesized histones. Nucleosomes were obtained from cells pulse-labeled simultaneously with 5-iododeoxyuridine and [3H]lysine for the rate zonal analysis. Proteins in the resulting fractions were resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and visualized by silver staining and fluorography. The distribution of newly synthesized H2A and H2B coincided closely with that of bulk nucleosomes. The distribution of newly synthesized H3 and H4 was shifted to the bottom sides of the bulk nucleosome peaks, but not so far as to the putative peaks of newly replicated (dense) nucleosomes. This means that newly synthesized histones are deposited on DNA in disproportionate amounts and that their sites of deposition are not restricted to newly replicated DNA.