bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and embryonic expression of Hrsna, a snail family gene of the ascidian Halocynthia roretzi: Implication in the origins of mechanisms for mesoderm specification and body axis formation in chordates
Authors: Wada Shuichi , Saiga Hidetoshi
Published: March 2003

snail family genes encode a transcription factor with specific zinc finger motifs. In vertebrates, they are expressed in the entire mesoderm in early embryogenesis and later in the paraxial mesoderm and the tail‐bud, suggesting roles in specification and morphogenesis of the paraxial mesoderm. In the present study, a snail family gene Hrsna from a member of the chordates, an ascidian (Halocynthia roretzi), was cloned to obtain an insight into the origin of the mechanisms of mesoderm specification and body axis formation as observed in vertebrates. Expression of Hrsna during ascidian embryogenesis was found to be quite similar to that of vertebrate snail genes. First, before gastrulation, Hrsna was initially expressed in most precursors of mesodermal tissues including the notochord where As‐T, the ascidian homolog of brachyury, was expressed. Hrsna expression persisted in the paraxial mesoderm, the mesenchyme and muscle, but not in the notochord precursors. Also, just as vertebrate snail family genes are expressed in the border of the neural plate that develops into dorsal neural tube and neural crest cells, so Hrsna expression was detected in the precursors of lateral and dorsal regions of the neural tube. However, Hrsna expression was not detected in the tip of the tail, unlike in vertebrate counterparts. In the light of the present findings, similarity and dissimilarity of mechanisms governing mesoderm specification and body axis formation between ascidians and vertebrates are discussed.
Self‐renewal and differentiation of a basic fibroblast growth factor‐dependent multipotent hematopoietic cell line derived from embryonic stem cells
Authors: Anzai Hiroko , Nagayoshi Mariko , Obata Masanori , Ikawa Yoji , Atsumi Tadao
Published: March 2003

Despite the accumulation of information on the origin of hematopoietic stem cells, it is still unclear how these cells are generated in ontogeny. Isolation of cell lines equivalent to early embryonic hematopoietic progenitor cells can be helpful. A multipotent hematopoietic progenitor cell line, A‐6, was isolated from H‐1 embryonic stem (ES) cells. The self‐renewal of A‐6 cells was supported by basic‐fibroblast growth factor (b‐FGF) and their differentiation into definitive erythroid cells, granulocytes and macrophages was induced after co‐culture with ST‐2 stromal cells. A‐6 cells were positive for the surface markers of hematopoietic stem cell, c‐kit, CD31, CD34, Flt3/Flk2, PgP‐1, and HSA, but were negative for that of the differentiated cells. Reverse transcription‐polymerase chain reaction analysis showed that A‐6 cells produced mRNA from SCL/tal‐1 and GATA‐2 genes. Among various cytokines examined, only stem cell factor (SCF) and Flt3/Flk2 ligand (FL) supported the proliferation of A‐6 cells instead of b‐FGF. The FL, as well as b‐FGF, supported the self‐renewal of A‐6 cells, whereas SCF induced differentiation into myeloid cells. A‐6 cells will be useful for the characterization of hematopoietic progenitor cells derived from ES cells and provide a model system to realize the control mechanisms between self‐renewal and differentiation of hematopoietic stem cells.
Neural inducing factors from Xenopus laevis eggs and embryos
Authors: Tiedemann Heinz , Tiedemann Hildegard
Published: March 2003

Large foreheads can be induced by ribonucleoprotein particles from Xenopus laevis eggs and embryos. The host embryos develop only a rudimentary primary axis. A neural inducing factor from the cytosol of gastrula–neurula stages has been partially purified. The factors are associated with other proteins in larger complexes.
Mosaic analysis with oep mutant reveals a repressive interaction between floor‐plate and non‐floor‐plate mutant cells in the zebrafish neural tube
Authors: Shinya Minori , Furutani‐Seiki Makoto , Kuroiwa Atsushi , Takeda Hiroyuki
Published: March 2003

The floor plate is located at the ventral midline of the neural tube in vertebrates. Floor‐plate development is severely impaired in zebrafish one‐eyed pinhead (oep) mutants. oep encodes a membrane‐bound protein with an epiblast growth factor (EGF) motif and functions autonomously in floor‐plate precursors. To understand the cell behavior and cell–cell interaction during floor‐plate development, the distribution and gene expression of wild‐type and oep mutant cells in genetic mosaics were examined. When mutant shield cells were transplanted into a wild‐type host, an ectopic neural tube with a floor plate was induced. However, the floor plate of the secondary axis was consistently devoid of mutant cells while its notochord was composed entirely of mutant cells. This indicates that oep shield cells adopt only a notochord fate in a wild‐type environment. In reciprocal transplants (wild to oep), however, grafted shield cells frequently contributed to part of the floor‐plate region of the secondary neural tube and expressed floor‐plate markers. Careful examination of serial sections revealed that a mutant neural cell, when located next to the wild‐type cells at the ventral midline, inhibited floor‐plate differentiation of the adjacent wild‐type cells. This inhibition was effective over an area only one‐ or two‐cells wide along the anteroposterior axis. As the cells located at the ventral midline of the oep neural tube are thought to possess a neural character, similar to those located on either side of the floor plate in a wild‐type embryo, this inhibition may play an important role during normal development in restricting the floor‐plate region into the ventral‐most midline by antagonizing homeogenetic signals from the floor‐plate cells.
Significant role of laminin‐1 in branching morphogenesis of mouse salivary epithelium cultured in basement membrane matrix
Authors: Hosokawa Yukio , Takahashi Yu , Kadoya Yuichi , Yamashina Shohei , Nomizu Motoyoshi , Yamada Yoshihiko , Nogawa Hiroyuki
Published: March 2003

Mouse submandibular epithelium shows branching morphogenesis in mesenchyme‐free conditions when covered with a basement membrane matrix (Matrigel) in medium supplemented with epidermal growth factor. In the present study, the role of laminin‐1 (LN1), a major glycoprotein of Matrigel, in this culture system was defined. When the epithelium was cultured in a LN1‐nidogen gel, the epithelium showed much branching, comparable to that observed with Matrigel. By electron microscopy, only a felt‐like matrix was formed on the epithelial surface in the LN1‐nidogen gel cultures, while an organized basal lamina structure was formed on the epithelial surface in direct or transfilter recombination cultures with mesenchyme. Next, the epithelium covered with Matrigel was cultured in medium containing either biologically active peptides from LN1, IKVAV‐including peptide (2097–2108), AG10 (2183–2194), AG32 (2370–2381) or AG73 (2719–2730) from the α1 chain, or YIGSR‐including peptide (926–933) from the β1 chain. Only AG73 (RKRLQVQLSIRT from the α1 chain carboxyl‐terminal globular domain) inhibited the epithelial branching in Matrigel. These results suggest that LN1‐nidogen can support the branching morphogenesis of submandibular epithelium even if LN1‐nidogen is not assembled into an intact basal lamina, and that the AG73 sequence is an important site on LN1, which interacts with submandibular epithelial cells.
Noggin upregulates Fos expression by a calcium‐mediated pathway in amphibian embryos
Authors: Leclerc Catherine , Duprat Anne‐Marie , Moreau Marc
Published: March 2003

In amphibia, noggin, one of the neural inducers expressed in the Spemann organizer, acts by neutralizing the effects of bone morphogenetic protein‐4 (BMP‐4). It is shown that noggin is able to activate L‐type calcium channels. The fos proto‐oncogene is known to be induced within minutes by calcium signaling. Here it is reported that in animal cap explants of the amphibian Pleurodeles waltl, noggin can induce upregulation of a FOS‐related protein in a calcium‐dependent manner. Specific inhibition of the dihydropyridine sensitive L‐type calcium channels blocked both calcium influx and the induction of FOS‐related protein. When animal cap explants were treated with caffeine in order to release calcium from an internal store or with a specific agonist of the L‐type calcium channels, FOS‐related protein could be detected in cell nuclei by 5 or 15 min, respectively. Additionally, the calcium calmodulin kinase inhibitor, KN62, could block the upregulation of FOS‐related protein induced by agents that increased intracellular calcium ([Ca2+]i). The present results suggest that transcription factors from the FOS family are downstream targets of neural inducer noggin.
Generation and characterization of a monoclonal antibody, mH1, raised against mitotic HeLa cells
Authors: Okubo Masa‐Aki , Chiba Shota , Nishikata Takahito , Matsuno Akira , Hosoya Hiroshi
Published: March 2003

Hybridoma cell lines were prepared from spleen cells of mouse immunized with mitotic HeLa cells. A monoclonal antibody (mH1), which intensively reacted with cleavage furrows of dividing HeLa cells in immunofluorescence, was obtained. In interphase, this antibody diffusely stained whole HeLa cells. Immunoelectron microscopy showed that mH1 antigens were localized at microvillus projections at the surface of dividing HeLa cells, but definite localization of that antigen was not observed in interphasic cells. Immunoblot analysis showed that mH1 is reactive to 42‐kDa and 130‐kDa components. Further, the 42‐kDa component was identified as a gamma‐actin homolog by N‐terminal amino acid sequence analysis.
Vitelline‐coat lysin: Comparison of lysins among three species in the genus Tegula
Authors: Haino‐Fukushima Kazu , Shimoirisa Masaki , Yamakawa Yoshio
Published: March 2003

Vitelline‐coat lysins from two species of marine mollusc of the genus Tegula, Tegula lischkei and Tegula sp., were purified and their properties compared with those of Tegula pfeifferi. Cross‐reaction tests among these three species proved that the lysin action on the vitelline coat was species specific. Each of the lysins from these three species is a single, basic polypeptide with a molecular weight of 16 000–17 000 and an isoelectric point of pH 10.5. All of them share a common antibody recognition site(s) for the anti‐T. pfeifferi lysin antibody. Their amino acid sequences were analyzed using intact lysins and peptides separated by reverse phase high‐performance liquid chromatography after V8 proteinase digestion. The amino acid sequences of the N‐terminus (Nos 1–66) from the three species showed 98% homology, and those of the C‐terminus (Nos 91–118) showed 89% homology. It was concluded that the species specificity of the vitelline coat lysin appears to be determined by a sequence of 24 residues (Nos 67–90) in the middle region of the molecule.
Cause of the decreased number of PGC in albino Xenopus: Analysis of the number and position of pPGC in albino embryos during and after cleavage
Authors: Ogiso‐Ono Yurie , Ikenishi Kohji
Published: March 2003

In order to understand the cause for the decreased number of primordial germ cells (PGC) in Xenopus albino (ap/ap) tadpoles, the number of presumptive PGC (pPGC) in the albino and wild‐type embryos at Nieuwkoop and Faber’s stages 6–37/38 were examined using the antibody specific to germ plasm. The positions of pPGC in the endodermal cell mass in embryos of both types at stages 28 and 33/34 were also observed to learn the migratory behavior of pPGC. The number of pPGC in the albino increased up to stage 28 with development, but decreased thereafter. In contrast, the number in the wild‐type increased to stage 33/34 as development proceeded, and the number of pPGC in stage 33/34 embryos reached nearly that of PGC of the feeding tadpoles in the same batches. Judging from the positions of pPGC, the migration of pPGC from the median part through the lateral to the dorsal part of the endodermal cell mass in the albino was suspected to be somewhat later than that in the wild‐type. These results, together with the results in previous studies, suggest that the decreased number of PGC in the albino would be closely related to the sudden decrease in number of pPGC at stage 33/34, as well as to the ectopic position of pPGC in endodermal cell mass, the latter of which had already been demonstrated to be responsible for the differentiation into PGC.
Brachyury (T) expression in embryonal carcinoma P19 cells resembles its expression in primitive streak and tail‐bud but not that in notochord
Authors: Yamaguchi Hirotake , Niimi Tomoaki , Kitagawa Yasuo , Miki Kiyoshi
Published: March 2003

Brachyury (T) is involved in mesoderm induction during early mouse development. T expression in embryonal carcinoma P19 cells, which differentiate into mesoderm derivatives in vitro, was studied. Endogenous T expression in P19 cells was transiently induced when the cells were allowed to form aggregates. This expression was enhanced by dimethyl sulfoxide. In situ hybridization showed that T expressing cells formed clusters on the aggregates. Transfection of plasmids encoding reporter genes under the control of the upstream region of T showed that the sequence up to – 351 bp can resemble the differentiation‐dependent expression of T in P19 cells. To define the promoter region regulating T expression, transgenic mice carrying LacZ under the control of the upstream region were prepared. The region up to – 351 bp is sufficient to direct the expression in the primitive streak and tail‐bud. The upstream region up to – 2400 bp does not support expression in the notochord. The sequence between – 987 and – 585 bp enhances expression in the primitive streak and tail‐bud. In the tail‐bud where new cells for elongation of the anteroposterior axis were continuously supplied, the sequence up to – 987 bp drove lacZ expression in gut endoderm and prospective neuroectoderm as well as in mesoderm derivatives.
Lim1 related homeobox gene (HpLim1) expressed in sea urchin embryos
Authors: Kawasaki Takahiko , Mitsunaga‐Nakatsubo Keiko , Takeda Koichi , Akasaka Koji , Shimada Hiraku
Published: March 2003

A cDNA clone for the LIM class homeobox gene (HpLim1) of the sea urchin, Hemicentrotus pulcherrimus, was isolated. HpLim1 contains two LIM domains and a LIM‐class homeodomain, and amino acid sequences of these three domains are highly homologous to corresponding domains of Lim1 of other animals. Accumulation of HpLim1 transcripts begins at hatching, and declines after the mesenchyme blastula stage. HpLim1 mRNA was localized in the vegetal plates of hatched blastulae, but it was not detectable in primary mesenchyme cells (PMC) ingressed into the blastocele. HpLim1 mRNA‐injected embryos became spherical with markedly reduced gut formation, failed to express marker proteins for aboral ectoderm and mesoderm, and mainly expressed an oral ectoderm marker. These results imply that while short‐term expression of HpLim1 in the vegetal plate is needed for differentiation of aboral ectoderm, endoderm and PMC, ectopic expression of HpLim1 suppresses normal differentiation directing all embryonic cells to differentiate into oral ectoderm.
Sox regulates transcription of the sea urchin arylsulfatase gene
Authors: Ogawa Mari , Akasaka Koji , Mitsunaga‐Nakatsubo Keiko , Shimada Hiraku
Published: March 2003

A 50 bp region from – 194 bp to – 144 bp of the arylsulfatase gene (HpArs) of the sea urchin, Hemicentrotus pulcherrimus, is related to the temporally regulated expression of this gene. This region contains a Sox (Sry‐related HMG box)‐binding site, and the introduction of sequence mutations to this site significantly reduced the activity of the HpArs promoter, even in the presence of the C15 enhancer, which consists of HpOtx and CAAT motifs. A protein that binds to the Sox‐binding site in the 50 bp region of the HpArs gene was detected in nuclear extracts of mesenchyme blastulae and a protein synthesized in vitro using SoxB1 cDNA of another sea urchin, Strongylocentrotus purpuratus, also bound to this Sox site. These results suggest that HpSox, which is maternally expressed and remains abundant by the pluteus stage, is clearly implicated in regulation of the HpArs gene. The presence of a negatively acting cis element in this 50 bp region has also been detected.
Human truncated Smad 6 (Smad 6s) inhibits the BMP pathway in Xenopus laevis
Authors: Krishnan Priya , King Michael W. , Neff Anton W. , Sandusky George E. , Bierman Kristin L. , Grinnell Brian , Smith Rosamund C.
Published: March 2003

A previously identified truncated form of the human Smad 6 gene containing a unique 12 amino acid motif at its N‐terminus was studied. We have named this truncated form of the gene Smad 6s, for ‘short‐form’, to distinguish it from the full‐length form (Smad 6fl). Reverse transcription–polymerase chain reaction and immunohistochemistry revealed that Smad 6s has a unique pattern of expression in human coronary tissue and is upregulated in diseased heart tissue. We used the expression of human Smad 6s in Xenopus laevis as a model system to assess Smad 6s function. Injection of Smad 6fl RNA (4‐cell embryos, 2 × ventral) produced tadpoles with partial secondary axes. In contrast, Smad 6s RNA injected in a similar manner produced tadpoles with a severe ‘head‐only’ phenotype with no morphological appearance of a secondary axis. Mutant Smad 6s RNA lacking the unique 12 amino acids at the N‐terminus of the Smad 6s isoform produced no embryonic phenotype, suggesting that this region is important in conferring biological activity. Ectodermal explant assays show that Smad 6s has activity consistent with being a BMP antagonist and can synergize with and enhance the activities of the activin and fibroblast growth factor pathways, all of which are novel findings in this study.
Comparative study of sequential expression of the organizer‐related genes in normal Cynops pyrrhogaster embryos and mesodermalized ectoderm
Authors: Tabata Toshiaki , Sakaguchi Kouji , Tajima Tatsuya , Suzuki Akio S.
Published: March 2003

An artificially mesodermalized ectoderm (mE) of early Cynops pyrrhogaster gastrula acquires the organizer property; the mE is able to induce the secondary axis. The expression of organizer‐related genes was investigated during the mesodermalizing process of the mE. The expression of C. pyrrhogaster organizer‐related genes, such as bra, gsc, lim‐1, chd and noggin, were analyzed. Cynops pyrrhogaster shh expression was also investigated. The organizer‐related genes were activated by 12 h after the mesoderm‐inducing stimulus. It was noted that there was a temporal gap in the expression of each gene. The expression of bra and gsc seemed to be more quickly activated during the mesodermalizing process. While expression of lim‐1 and noggin was activated later than that of bra and gsc, lim‐1 expression was earlier than chd and noggin expression. Shh expression was activated later than lim‐1/noggin. The present study suggests the possibility that the bra/gsc, lim‐1, chd, noggin and shh genes are expressed one by one in that order during the mesodermalizing of the presumptive ectoderm. It also indicates that the sequence is not always consistent with that of the whole embryo during normal embryogenesis. The meaning of the discrepancy will be discussed in connection with the cascade of certain genes expressed during the mesodermalizing process.
An RGDS peptide‐binding receptor, FR‐1R, localizes to the basal side of the ectoderm and to primary mesenchyme cells in sand dollar embryos
Authors: Katow Hideki , Sofuku Shosuke
Published: March 2003

Immunoblotting using polyclonal antibodies (pAb) raised against an FR‐1 receptor (FR‐1R), a 57 kDa Arg‐Gly‐Asp‐Ser (RGDS)‐binding protein, of the sand dollar Clypeaster japonicus showed that the pAb monospecifically bound to the protein. FR‐1R was present in purified plasma membrane, suggesting that the protein is a membrane‐bound protein. The molecular structure of FR‐1R did not change throughout the early embryogenesis, whereas its expression changed significantly during this period. FR‐1R was present in the cortex of unfertilized eggs and was then transferred to the hyaline layer soon after the fertilization. The hyaline layer retained FR‐1R immunoreactivity during early embryogenesis. FR‐1R appeared on the basal side of the ectoderm at the morula stage and was retained basolaterally, at least, to the early gastrula stage. In mesenchyme blastulae, FR‐1R was also present on the surface of primary mesenchyme cells (PMC). FR‐1R was localized on the basal side of the ectoderm in early gastrulae, exclusively at the place where PMC formed ventrolateral aggregates, and at the apical tuft ectoderm. In vitro, PMC bound to FR‐1R and its binding was inhibited in the presence of a synthetic RGDS peptide or the pAb. The pAb introduced into the blastocoele perturbed PMC migration and gastrulation. FR‐1R was weakly recognized by antihuman integrin β5 subunit pAb.
Pluripotent cells (stem cells) and their determination and differentiation in early vertebrate embryogenesis†
Authors: Tiedemann H. , Asashima M. , Grunz H. , Knöchel W.
Published: March 2003

Mammalian embryonic stem cells can be obtained from the inner cell mass of blastocysts or from primordial germ cells. These stem cells are pluripotent and can develop into all three germ cell layers of the embryo. Somatic mammalian stem cells, derived from adult or fetal tissues, are more restricted in their developmental potency. Amphibian ectodermal and endodermal cells lose their pluripotency at the early gastrula stage. The dorsal mesoderm of the marginal zone is determined before the mid‐blastula transition by factors located after cortical rotation in the marginal zone, without induction by the endoderm. Secreted maternal factors (BMP, FGF and activins), maternal receptors and maternal nuclear factors (β‐catenin, Smad and Fast proteins), which form multiprotein transcriptional complexes, act together to initiate pattern formation. Following mid‐blastula transition in Xenopus laevis (Daudin) embryos, secreted nodal‐related (Xnr) factors become important for endoderm and mesoderm differentiation to maintain and enhance mesoderm induction. Endoderm can be induced by high concentrations of activin (vegetalizing factor) or nodal‐related factors, especially Xnr5 and Xnr6, which depend on Wnt/β‐catenin signaling and on VegT, a vegetal maternal transcription factor. Together, these and other factors regulate the equilibrium between endoderm and mesoderm development. Many genes are activated and/or repressed by more than one signaling pathway and by regulatory loops to refine the tuning of gene expression. The nodal related factors, BMP, activins and Vg1 belong to the TGF‐β superfamily. The homeogenetic neural induction by the neural plate probably reinforces neural induction and differentiation. Medical and ethical problems of future stem cell therapy are briefly discussed.
N‐Methyl‐D‐aspartate receptor‐dependent and ‐independent cytotoxic effects of Dictyostelium discoideum differentiation‐inducing factor‐1 on rat cortical neurons
Authors: Kanno Toshio , Kubohara Yuzuru , Goto Fumio , Takata Kuniaki , Hayashi Kensuke
Published: March 2003

Differentiation‐inducing factor‐1 (DIF‐1) is a chlorinated alkylphenone (small lipophilic hormone) that induces stalk cell formation in the cellular slime mold Dictyostelium discoideum. Recent studies have revealed that DIF‐1 inhibits growth and induces the differentiation of mammalian tumor cells. The present study examines the effects of DIF‐1 on rat cortical neurons in primary culture. We found that DIF‐1 induced rapid neuronal cell death. The release of lactate dehydrogenase (LDH), as an indicator of cell death, increased dose‐dependently with DIF‐1. The release of LDH was inhibited by the N‐methyl‐D‐aspartate (NMDA) receptor antagonists MK801 and AP5, suggesting that the NMDA receptor is involved in the induction of cell death by DIF‐1. However, glutamate cytotoxicity could not explain the entire action of DIF‐1 on neurons because the estimated concentration of glutamate around DIF‐1‐treated neurons was below 50 μM and DIF‐1 caused more severe cell death than 500 μM glutamate. We discovered that another portion of DIF‐1 cytotoxicity is independent of the NMDA receptor; that is, coaddition of DIF‐1 and MK801 induced dendritic beading and increased expression of the immediate early genes c‐fos and zif/268. These results indicate that DIF‐1 induces rapid cell death via both NMDA receptor‐dependent and ‐independent pathways in rat cortical neurons..
Ectopically localized HNK‐1 epitope perturbs migration of the midbrain neural crest cells in Pax6 mutant rat
Authors: Nagase Takashi , Nakamura Shun , Harii Kiyonori , Osumi Noriko
Published: March 2003

Small eye rats, which have a mutation in a gene encoding transcription factor Pax6, exhibit impaired migration of the midbrain neural crest cells, thereby showing severe craniofacial defects. Orthotopic grafting of the midbrain neural crest cells taken from the wild‐type into Pax6 mutant embryos has suggested environmental defects along the migratory pathway of the midbrain crest cells. In the present study we found that the HNK‐1 carbohydrate epitope was ectopically localized in the frontonasal epithelium of Pax6 mutant embryos. The GlcAT‐P gene, encoding an enzyme for the synthesis of the HNK‐1 epitope, was also expressed ectopically in the frontonasal epithelium of the mutant. In explant cultures, the migration rate of neural crest cells from the midbrain, but not from the forebrain, was significantly less in HNK‐1‐coated dishes than in non‐coated dishes. These results suggest that the arrested migration of the midbrain crest cells in Pax6 mutant embryos may, at least in part, be due to the inhibitory effect of the HNK‐1 epitope ectopically localized in the frontonasal epithelium.
Region‐specific gastrointestinal Hox code during murine embryonal gut development
Authors: Kawazoe Yasuhiro , Sekimoto Tomohisa , Araki Masatake , Takagi Katsumasa , Araki Kimi , Yamamura Ken‐ichi
Published: March 2003

Hox genes encode transcription factors, and they are involved in the specification of each body part along the anteroposterior (AP) body axis during embryogenesis. To clarify AP pattern formation of the digestive tract, the expression patterns of Hox genes belonging to paralogous groups 4 and 5, and parts of groups 6 and 7, were systematically examined by whole‐mount and section in situ hybridization. The Hox gene expression pattern of paralogous groups 4–9 in the developing gut at 12.5 days post‐coitum was fully examined. All HoxA and HoxB genes in paralogous groups 4–8 were expressed in the stomach, in contrast to the HoxC and HoxD genes. In the midgut region, all Hox cluster genes showed colinear expression within each cluster, yielding the Hox code; the more 3′ located genes were expressed more rostrally and the 5′ group genes more caudally. The colinear expression of HoxA and HoxB cluster genes started from the duodenum, that of HoxC cluster genes started from the jejunum, and HoxD cluster genes were expressed in the caudal part of the midgut, ileum and cecum. In the hindgut region, HoxD cluster genes and Abd‐B family genes were expressed. Thus, a different Hox code seems to exist in each subdomain of developing gut (foregut, midgut and hindgut). The visceral mesoderm restricted expression also suggested that the Hox code primarily functions in mesenchymal specification, and then leads to the regional differentiation of gut subdomains as the result of epithelial– mesenchymal interactions.
Post‐nodal mesoblast caudalizes the host axis and inhibits cell population growth, and induces new primitive streaks in chick embryos
Authors: Joshi‐Banka Deepti , Paranjape Sharayu , Ghaskadbi Surendra , Modak Sohan P.
Published: March 2003

One to eight post‐nodal fragments (PN) or Hensen’s nodes (HN) from full primitive streak stage chick embryos were transplanted onto the area pellucida or area opaca of stage 4 embryos and cultured for 20 h. Thirteen morphological and numerical parameters were affected in the host embryos and analyzed by multiple logistic regression for parametric hierarchy. In the area pellucida, both PN and HN transplants inhibited cell population growth while only PN caudalized the host axis and induced supernumerary primitive streaks expressing the mesoderm‐specific gene Brachyury. In the area opaca, neither grafts influenced host axis morphogenesis, but PN inhibited the cell population growth significantly. Tracking [3H]TdR labeled grafts showed that PN cells migrated towards the host axis and participated in the formation of supernumerary somites and hearts. When placed near the host axis, PN caudalized it and inhibited cell population growth.
Basic transcription element binding protein is a thyroid hormone‐regulated transcription factor expressed during metamorphosis in Xenopus laevis
Authors: Hoopfer Eric D. , Huang Liyue , Denver Robert J.
Published: March 2003

Basic transcription element binding protein (BTEB) is a member of the Krüppel family of zinc finger transcription factors. It has been shown that BTEB plays a role in promoting neuronal process formation during postembryonic development. In the present study, the biochemical properties, transactivation function, and the developmental and hormone‐regulated expression of BTEB in Xenopus laevis (xBTEB) are described. xBTEB binds the GC‐rich basic transcription element (BTE) with high affinity and functions as a transcriptional activator on promoters containing multiple or single GC boxes. xBTEB mRNA levels increase in the tadpole brain, intestine and tail during metamorphosis, and are correlated with tissue‐specific morphological and biochemical transformations. xBTEB mRNA expression can be induced precociously in premetamorphic tadpole tissues by treatment with thyroid hormone. In situ hybridization histochemistry showed that thyroid hormone upregulates xBTEB mRNA throughout the brain of premetamorphic tadpoles, with the highest expression found in the subventricular zones of the telencephalon, diencephalon, optic tectum, cerebellum and spinal cord. xBTEB protein parallels changes in its mRNA, and it was found that xBTEB is not expressed in mitotic cells in the developing brain, but is expressed just distal to the proliferative zone, supporting the hypothesis that this protein plays a role in neural cell differentiation.
Sucrose stimulates branching morphogenesis of embryonic mouse lung in vitro: A problem of osmotic balance between lumen fluid and culture medium
Authors: Nogawa Hiroyuki , Hasegawa Yoko
Published: March 2003

In organ cultures of lung rudiments from 11‐day mouse embryos, it was found that addition of sucrose to the culture medium stimulated branching morphogenesis and reduced lumen distension. Two possible roles of sucrose were postulated: one as a nutrient and another as a generator of osmotic pressure inducing osmosis of water from the lumen fluid to the culture medium across a simple columnar epithelial cell layer. To assess which was the case, branching morphogenesis was investigated in lung rudiments cultured in medium in which osmotic pressure was increased by the addition of lactose or NaCl rather than sucrose: similar acceleration of branching was observed in both. In another experiment, lumen fluid of cultured lung rudiments was mechanically drained each day, and significantly stimulated branching morphogenesis was observed even when sucrose was not added to the culture medium. Heparin is known to induce abnormal lumen distension and inhibits branching morphogenesis. Heparin‐induced abnormal morphogenesis was prevented either by the addition of sucrose to the culture medium or by the mechanical drainage of lumen fluid. These results suggest that lumen distension caused by the accumulation of lumen fluid disrupts lung branching morphogenesis in vitro, even when the mechanism of branching morphogenesis is intact.
Expression and role of retinoic acid receptor alpha in lens regeneration
Authors: Tsonis Panagiotis A. , Tsavaris Melissa , Call Mindy K. , Chandraratna Roshantha A. S. , Del Rio‐Tsonis Katia
Published: March 2003

The role of retinoids in eye development has been well studied. Retinoids and their receptors regulate gene expression and morphogenesis of the eye. In this study, a highly specific antagonist of retinoic acid receptor (RAR)‐α was used in an attempt to study its function in lens regeneration. It was found that this antagonist inhibited lens regeneration and lens fiber differentiation. It was also shown that RAR‐α is expressed in the lens during the process of regeneration. These results indicate that different RAR might have unique as well as redundant effects and patterns of expression in the regenerating lens.
Chromosome mapping of Xenopus tropicalis using the G‐ and Ag‐bands: Tandem duplication and polyploidization of larvae heads
Authors: Uehara Mariko , Haramoto Yoshikazu , Sekizaki Hiroyuki , Takahashi Shuji , Asashima Makoto
Published: March 2003

Developmental cytogenetic analyses of Xenopus tropicalis larvae from two origins were performed on stage 27–34 heads treated with colchicine. Standard G‐band karyotyping using trypsin and chromosome mapping of 184 bands were examined. Although the main karyotype was 2n = 20, polyploidy (3n = 30 or 4n = 40) and aneuploidy were detected in each individual treated with colchicine, even those treated for only 1 h. The percentage of polyploid karyotypes was 10–20% across the total of measured metaphases. The mean mitotic index was 0.10. Chromosomal breaks and exchanges were detected at the secondary constriction of chromosomes 5 or 6. Ag‐band detection showed clearly positive staining at the secondary constriction of chromosome 5, which corresponds to the nucleolar organizer region. Tandem duplication of negative G‐bands at the secondary constriction of chromosome 6 and the short arm of chromosome 10 was suggested by this study. X. tropicalis thus provides a good model to study the mechanism and effects of chromosomal abnormalities, gene mapping and tissue specific gene expression in the developmental process.
Expression of FTZ‐F1α in transgenic Xenopus embryos and oocytes
Authors: Asahi Hirotaka , Takase Minoru , Yuge Masahiro , Matsui Kumi , Mori Mitsuaki , Fujita Takashi , Nakamura Masahisa
Published: March 2003

Fushi tarazu transcription factor‐1 (FTZ‐F1) was originally found as a regulator of fushi tarazu gene expression in Drosophila. The frog homologue (FTZ‐F1α) and the 3.5 kb 5′‐flanking region of the FTZ‐F1α gene have been cloned, and it has been shown by reverse transcription–polymerase chain reaction that FTZ‐F1α expression begins in embryos at stage 11 and becomes stronger after that. By in situ hybridization analysis, the FTZ‐F1α mRNA was also found in immature frog oocytes. In this study, immunohistology revealed that the product of FTZ‐F1α was localized in the cytoplasm of the immature oocyte. To analyze the promoter activity of the Rana rugosa FTZ‐F1α gene, transgenic Xenopus were produced carrying the fusion construct, consisting of truncated 5′‐flanking regions (3.0, 1.8 and 0.3 kb) of the FTZ‐F1α gene and the green fluorescent protein (GFP) open reading frame. The 0.3 kb 5′‐flanking region could drive GFP expression in Xenopus embryos at stage 20 and in immature oocytes in the ovary 2 months after metamorphosis. Gel mobility shift assay was used to test whether proteins in extracts from Xenopus embryos and ovaries bound to the 0.3 kb DNA. The extract from embryos at stage 11 formed one retarded band. The extract from ovaries formed a different retarded band. The results, taken together, indicate that production of transgenic Xenopus is very useful for the analysis of the promoter activity of genes in amphibians. The results also suggest that at least two proteins (one in the embryo and the other in the ovary of 2‐month‐old postmetamorphosing Xenopus) bind the 0.3 kb 5′‐flanking region of the FTZ‐F1α gene. These proteins may be involved in the regulation of FTZ‐F1α gene expression in amphibians.
Physiological tolerance of Camellia rusticana leaves to heavy snowfall environments: The effects of prolonged snow cover on evergreen leaves
Authors: KUME ATSUSHI , TANAKA CHIKAKO , MATSUMOTO SHUNICHI , INO YOSHIO
Published: March 2003

Camellia
rusticana is an evergreen broad‐leaved shrub found only in areas of heavy snowfall in Japan. The ecophysiological tolerance of this species to prolonged snow cover was studied in comparison with those of Camellia japonica found in areas of light snowfall. The shoots of C. rusticana and C. japonica were stored under a simulated snow cover for 1 year. During the experimental period, about 20% of the leaves of C. japonica defoliated after 90–140 days of storage and more than 80% of the leaves browned within 360 days. The proliferation of fungi was observed on all shoots of C. japonica after 200 days. In contrast, the leaves of C. rusticana showed no visible changes throughout the experimental period. The decline in the rate of soluble carbohydrate content in C. rusticana was about one‐sixth of that in C.␣japonica. The photosynthetic capacity (O2 exchange rates at saturated light and CO2 at 20°C) of C.␣japonica dropped to about half its initial value after 140 days, while that of the C. rusticana did not change even after 360 days. Under dark conditions, the stomata of C. rusticana were always closed through the experiment, but those of C. japonica were not completely closed after 90 days. The differences in stomata characteristics and the consumption rate of soluble carbohydrates under snow‐covered conditions between the two Camellia species are important factors for determining their habitat segregation by snow depth.
Identification of conservation measures to protect the Japanese endangered plant species Aster kantoensis
Authors: INOUE KEN , KURAMOTO NOBORU , MAKI MASAYUKI , MASUDA MICHIKO , WASHITANI IZUMI
Published: March 2003

To identify the factors responsible for degrading the habitat of the endangered plant species Aster kantoensis, as well as the vulnerable life stage where this occurs, we carried out sowing experiments. Two natural habitats were simulated, being situated along the floodplains of the Tama River in central Japan. Seeds collected from a natural habitat were sown in two apparently suitable locations (Tomoda and Ishida sites). Germination, survival, growth, and seed production were subsequently monitored from 1993 through to 1997. The Tomoda site was a gravel bar in floodplains formed by flooding in 1991, while the Ishida site (two plots) was one gravel bar where several plants were growing sparsely and another where a population had become extinct in 1992. Seed cohorts completed their life cycle within 3 years at the Ishida site and within 5 years at the Tomoda site. Monitored parameters at Ishida were substantially lower than those at Tomoda. In addition, estimates of population growth indicated an increase at Tomoda and a rapid decrease at Ishida. However, degradation of habitats seemed to occur at Tomoda over the monitored periods. In view of our results, we conclude that natural germination of about 0.13% is needed for increasing population size. The major factors for decreasing population size are believed to be the lack of safe sites for germination and seedling establishment in old habitats (> 10 years). Conservation measures are suggested based on these findings.
Regulation of food provisioning and parental body condition in Leach’s storm‐petrels,Oceanodroma leucorhoa: Experimental manipulation of offspring food demand
Authors: Takahashi Akinori , Niizuma Yasuaki , Watanuki Yutaka
Published: March 2003

We examined the effects of offspring food demand on parental regulation of food provisioning and body condition in a small long‐lived seabird, Leach’s storm‐petrels (Oceanodroma leucorhoa). In one experimental group, food demand of chicks on their parents was increased by removing one parent (‘single’), and in another group these food demands were decreased by supplementary feeding of chicks (‘supplement’). A further unmanipulated group provided a ‘control’. Feeding frequencies by one parent were higher in the single but lower in the supplement than in the control group, in accordance with the food demand of chicks. The size of meals appeared to be not different among the experimental groups. However, as single parents did not compensate perfectly for the increase of chick food demand by food provisioning, single chicks grew at slower rates and fledged at smaller masses than control chicks. Supplement chicks grew at similar rates and fledged at similar masses as control chicks, because parents decreased food provisioning and food processing capacity of the chicks might be limited. The body condition of parents, which was determined by body mass loss and feather regrowth rate, did not differ among the groups. These results indicate that feeding frequency was regulated by parental decision in this storm‐petrel species. Parents may adjust their food provisioning to match the food demand of chicks but within a certain range so as not to deteriorate their own body condition.
Within‐ and between‐patch variability of predation intensity on the mussel Mytilus trossulus Gould on a rocky intertidal shore in Oregon, USA
Authors: Noda Takashi
Published: March 2003

Variability of predation intensity is an important cause of spatial differences of community structure and organization in the intertidal rocky shore. Field experiments were conducted to evaluate the within‐ and between‐patch variability of the effects of different types of predators (small invertebrates and birds) on Mytilus trossulus Gould, which occupies an intermediate position in the competitive hierarchy among sessile organisms in disturbance patches within a California mussel (Mytilus californianus Conrad) bed community on the central Oregon Coast. Predation by birds did not significantly affect the mortality of M. trossulus. On the contrary, predation by small invertebrate whelks (Nucella spp.) had a significant effect on M. trossulus mortality. Predation by whelks also caused between‐ and within‐patch variability of mortality of M. trossulus. Within patches, M. trossulus mortality at patch margins was significantly higher than at patch centers only when invertebrate predators were present. Wave exposure did not cause between‐patch variability of predation intensity. Between‐and within‐patch variability of predation intensity may be caused by the variability of supply of whelks from the surrounding mussel mat. The movement of predators between patches and surrounding matrices may play an important role in the patch dynamics of these communities.
A record of acorn eating by sika deer in western Japan
Authors: Weerasinghe Udayani Rose , Takatsuki Seiki
Published: March 2003

The rumen contents of 14 sika deer (Cervus nippon) collected in November 1996 in Yamaguchi Prefecture, the western end of Honshu, Japan, were analyzed by the point‐frame method. The contents were composed of high‐quality foods like deciduous and evergreen browse and acorns. The composition, which consisted of a significant amount of acorns (27.8 ± 22.5%, mean ± SD), was in contrast to that of northern sika deer populations which exclusively eat graminoid leaves including dwarf bamboos. This is the first record of acorns being a major contributor to diet for wild sika deer. By the point‐frame method, acorns were underestimated, and the weight contribution was much greater.
Modeling associative learning with generalization for a case of warning signals
Authors: Higashi Shigeo Yachi and Masahiko
Published: March 2003

Animals’ associative learning plays a crucial role in many intraspecific or interspecific interactions, involving an animal’s use of information on its interacting counterparts. Here, we present a theoretical model that captures the basic features of an animal’s associative learning, which may involve generalization, for a simplest case of warning signals. Specifically, we derive formulae for the average level of associative memory as functions of a few parameters that reflect the population density of prey, predator’s efficiency of prey detection, and the properties of predator’s associative learning, including generalization and memory decay. This average level of associative memory is of central importance in determining prey’s fitness and, thus, the evolution of warning signals (i.e. aposematism). The derived formula also shows that another species with similar signal enhances the fitness of an aposematic species of concern as long as their signal is similar enough for generalization to occur. The model developed here can be extended to more complicated cases and the basic idea can be applied to modeling other interactions involving associative learning with generalization.
Impacts of dry and hazy weather in 1997 on a tropical rainforest ecosystem in West Sumatra, Indonesia
Authors: Yoneda Tsuyoshi , Nishimura Sen , Chairul
Published: March 2003

An equatorial rainforest in the coastal region of West Sumatra suffered a long period of dry and hazy weather in 1997. Canopy trees with higher growth rates before the event had reduced growth rates during the 8 months from June 1997 to an average of 23% of their 1996 growth rate. The size‐dependency of growth depression in forest trees greater than 5 cm in stem diameter could be observed during this period. The weather induced defoliation, and high litterfall rates of leaves continued throughout the period. The impacts of the weather on growth rates were analyzed by using Saeki’s mathematical model for community production. This model suggested that the great reduction in growth‐rates of active canopy trees would be caused mainly by the interacting effects of potential photosynthetic rate and leaf biomass because of their sensitive responses to drought. In the severe weather conditions, these variables were estimated to have decreased to more than 50% of their 1996 values.
Differential salt tolerance of two Artemisia species growing in contrasting coastal habitats
Authors: Ishikawa Shin‐Ichi , Kachi Naoki
Published: March 2003

To investigate factors determining the differences in their salt tolerance, growth and germination, experiments were conducted on two plant species belonging to genus Artemisia: Artemisia fukudo Makino, a biennial salt marsh plant and Artemisia stelleriana Bess, a perennial coastal hind dune plant. Growth experiments revealed that salinity (100 and 300 m
M NaCl) inhibited the relative growth rate (RGR) in A. stelleriana significantly but not in A. fukudo. These specific differences in salt tolerance were mainly attributed to differential responses of net assimilation rate (NAR). That is, the reduction in RGR in A. stelleriana was mainly due to the reduction in NAR, whereas no significant reduction in NAR was observed in A. fukudo. The reduction in RGR in A. stelleriana in the salt treatment was also attributable to a reduced leaf area ratio (LAR). Specific leaf area (SLA) in the two species decreased in the 300 m
M treatment. The decrease in SLA in A. fukudo was, however, compensated for partly by an increase in leaf weight ratio (LWR). Germination experiments also showed that A. fukudo has a higher salt tolerance than does A. stelleriana. These results are consistent with the differences in the salinity conditions between the native habitats of the two species.