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M Mochii

Publications and source records attributed to M Mochii.

At least 19 recordsLinked to original sources
PubMed2001

Hypodermal expression of Caenorhabditis elegans TGF-beta type I receptor SMA-6 is essential for the growth and maintenance of body length.

There are several transforming growth factor-beta (TGF-beta) pathways in the nematode Caenorhabditis elegans. One of these pathways regulates body length and is composed of the ligand DBL-1, serine/threonine protein kinase receptors SMA-6 and DAF-4, and cytoplasmic signaling components SMA-2, SMA-3, and SMA-4. To further examine the molecular mechanisms of body-length regulation in the nematode by the TGF-beta pathway, we examined the regional requirement for the type-I receptor SMA-6. Using a SMA-6::GFP (green fluorescent protein) reporter gene, sma-6 was highly expressed in the hypodermis, unlike the type-II receptor DAF-4, which is reported to be ubiquitously expressed. We then examined the ability of SMA-6 expression in different regions of the C. elegans body to rescue the sma-6 phenotype (small) and found that hypodermal expression of SMA-6 is necessary and sufficient for the growth and maintenance of body length. We also demonstrate that GATA sequences in the sma-6 promoter contribute to the hypodermal expression of sma-6.

Animals↗
PubMed2001

Use of cDNA subtraction and RNA interference screens in combination reveals genes required for germ-line development in Caenorhabditis elegans.

Caenorhabditis elegans is an ideal organism for the study of the molecular basis of fundamental biological processes such as germ-line development, especially because of availability of the whole genome sequence and applicability of the RNA interference (RNAi) technique. To identify genes involved in germ-line development, we produced subtracted cDNA pools either enriched for or deprived of the cDNAs from germ-line tissues. We then performed differential hybridization on the high-density cDNA grid, on which about 7,600 nonoverlapping expressed sequence tag (EST) clones were spotted, to identify a set of genes specifically expressed in the germ line. One hundred and sixty-eight clones were then tested with the RNAi technique. Of these, 15 clones showed sterility with a variety of defects in germ-line development. Seven of them led to the production of unfertilized eggs, because of defects in spermatogenesis (4 clones), or defects in the oocytes (3 clones). The other 8 clones led to failure of oogenesis. These failures were caused by germ-line proliferation defect (Glp phenotype), meiotic arrest, and defects in sperm--oocyte switch (Mog phenotype) among others. These results demonstrate the efficacy of the screening strategy using the EST library combined with the RNAi technique in C. elegans.

Animals↗
PubMed2001

Spontaneous mutation in Mitf gene causes osteopetrosis in silver homozygote quail.

Silver homozygous quail was recently reported to have mutations in Mitf gene. Although numerous mutations in Mitf gene have been reported in mice, no mutations corresponding to the mutation in the homozygous silver (B/B) quail in Mitf gene have been reported to cause defects in pigmentation and bone. Therefore, we investigated the bones of the B/B homozygotes. Comparison of the bones of the B/B homozygotes with those of wild-type by X-ray examination revealed osteopetrosis in the long bones of B/B homozygotes. However, osteopetrosis in B/B homozygotes was less severe than that observed in mi/mi mice. Histological examination showed that there were less TRAP-positive multinucleated cells in the trabecular bones in B/B homozygote tibia than in the wild type. In vitro osteoclastogenesis study also suggested that formation of TRAP-positive multinucleated cell was suppressed in the marrow cells of the long bones of the B/B homozygotes. Furthermore, overexpression of chicken Mitf via retroviral transfection into B/B homozygote bone marrow cells in cultures increased the number of TRAP-positive cells 2-3 fold more than that in control. These results indicated that in addition to the previously reported defect in melanogenesis, osteoclastogenesis was inhibited in B/B homozygotes. These results indicate that the novel mutations in Mitf gene observed in the B/B homozygote quail impair osteoclastic bone resorption.

Aging↗
PubMed2001

Visualization of endogenous BMP signaling during Xenopus development.

The TGF-beta superfamily of growth factors is known to transmit signals to the nucleus mainly through the Smads, intracellular signaling components that are highly conserved from nematodes to humans. The signaling activity of the Smads is regulated by their ligand-stimulated phosphorylation through Ser/Thr kinase receptors. Here, to examine the in vivo role of BMP, we investigated the spatio-temporal activation of BMP-regulated signals during Xenopus development, using a polyclonal antibody that specifically recognizes the phosphorylated form of BMP-regulated Smads. BMP signaling was observed uniformly in embryos as early as stage 7, but was restricted to the ventral side of the embryo at the late blastula stage, supporting the proposed role of BMP4 as a ventralizing factor in Xenopus embryos. In addition, localized staining was detected in several developing organs, consistent with the predicted function of BMP family members in organogenesis.

Animals↗
PubMed1999

Identification of transforming growth factor-beta- regulated genes in caenorhabditis elegans by differential hybridization of arrayed cDNAs.

Members of the transforming growth factor-beta family play critical roles in body patterning, in both vertebrates and invertebrates. One transforming growth factor-beta-related gene, dbl-1, has been shown to regulate body length and male ray patterning in Caenorhabditis elegans. We screened arrayed cDNAs to identify downstream target genes for the DBL-1 signaling by using differential hybridization. C. elegans cDNAs representing 7,584 independent genes were arrayed on a nylon membrane at high density and hybridized with (33)P-labeled DNA probes synthesized from the mRNAs of wild-type, dbl-1, sma-2, and lon-2 worms. Signals for all the spots representing hybridized DNA were quantified and compared among strains. The screening identified 22 and 2 clones, which were positively and negatively regulated, respectively, by the DBL-1 signal. Northern hybridization confirmed the expression profiles of most of the clones, indicating good reliability of the differential hybridization using arrayed cDNAs. In situ hybridization analysis revealed the spatial and temporal expression patterns of each clone and showed that at least four genes, including the gene for the type I receptor for DBL-1, sma-6, were transcriptionally regulated by the DBL-1 signal.

Animals↗
PubMed1999

Lens regeneration in Xenopus is not a mere repeat of lens development, with respect to crystallin gene expression.

The spatio-temporal expression of three crystallin genes (alpha A, beta B1 and gamma) in lenses of Xenopus laevis was studied by in situ hybridization to compare the process of lens formation in embryonic development with that of lens regeneration from cornea that occurs in the tadpole. During embryonic lens development, all three crystallin transcripts were initially detected at the same stage of lens placode formation, and subsequently their signals became restricted to the presumptive lens fiber region. At later stages, the three crystallin genes were expressed in primary and secondary lens fibers, but not in lens epithelium. During lens regeneration, alpha A- and beta B1-crystallin signals were first detected in the presumptive lens fiber region of the lens vesicle. The expression of gamma-crystallin, however, appeared later than the other two crystallin genes and was detected only in morphologically discernible lens fibers. In the later stages of lens regeneration, expression of these crystallins was observed only in the lens fiber region, similar to embryonic lens development. These results reveal that lens regeneration from the inner layer of the outer cornea is not simply a repetition of embryonic lens development, when examined at the level of crystallin gene transcription.

Amino Acid Sequence↗
PubMed1999

Pax-6 and Prox 1 expression during lens regeneration from Cynops iris and Xenopus cornea: evidence for a genetic program common to embryonic lens development.

Lens regeneration from non-lens ocular tissues has been well documented in amphibians, from the dorsal iris in the newt and from the outer cornea in Xenopus. To understand the early molecular events which govern lens regeneration, we examined the expression of two early marker genes of normal lens development, Pax-6 and Prox 1. In both Cynops (newt) iris and Xenopus cornea, Pax-6 is expressed soon after lentectomy in a region broader than that giving rise to the regenerating lens, indicative of an important role for Pax-6 in determination of the regeneration potential. Then Prox 1 expression begins within the Pax-6-expressing tissue, and these Prox 1-expressing cells give rise to the regenerating lens. This sequence of events also takes place in the lens placode of the embryo, indicating that the presence of the same genetic program operates in both embryonic lens development and lens regeneration, at least partly. In the Cynops iris, Pax-6 expression occurs initially in the entire marginal region of the iris after lentectomy but then becomes restricted to the dorsal region. Further studies are expected to elucidate the mechanism of this long-standing problem of the dorsal-restriction of lens regeneration from the newt iris.

Amino Acid Sequence↗
PubMed1998

Spontaneous transdifferentiation of quail pigmented epithelial cell is accompanied by a mutation in the Mitf gene.

An ectopic neural retina is formed at the outer layer of the retina in the silver homozygote (B/B) of the Japanese quail. In situ hybridization and immunohistochemical analysis revealed that cells in the outer layer of retina first expressed a pigment-cell-specific gene, mmp115, and then began to express a neural marker in B/B embryos, indicating that the ectopic neural retina is formed via transdifferentiation of differentiated pigmented epithelial cells (PECs). An in vitro study revealed that cultured retinal PECs (rPECs) from B/B embryos exhibit less pigment granule and a higher growth rate than cells from heterozygotes (B/+). B/+ PECs stopped proliferating when confluency was reached, while B/B PECs continued to proliferate. Some B/B cells overlaid other B/B cells and formed lentoid bodies. Immunological analysis revealed that B/B rPECs transdifferentiated to lens cells and neural cells in vitro with no addition of basic FGF (bFGF), while B/+ rPECs required bFGF to transdifferentiate. Expression of PEC-specific genes, mmp115, tyrosinase, and TRP-1, was downregulated, but that of Mitf and pax6 was upregulated in B/B PECs. Antibody against Mitf stained the nucleus of B/+ PECs but not that of B/B cells, suggesting that the normal Mitf is not present in the silver homozygote due to mutation. Sequence analysis revealed that Mitf from the silver homozygote has an amino acid substitution in the basic region and is truncated in the C-terminal region. Transient transfection analysis revealed that Mitf from the silver homozygote exhibits a lower level of activity than wild-type Mitf with respect to transactivation of the mmp115 promoter. Furthermore, overexpression of chicken Mitf induced normal pigmentation in B/B rPECs. These results strongly suggest that the silver phenotype is caused by the mutation of Mitf and that Mitf plays a critical role in rPEC differentiation and transdifferentiation.

Amino Acid Sequence↗
PubMed1998

Role of Mitf in differentiation and transdifferentiation of chicken pigmented epithelial cell.

Mitf encodes a basic helix-loop-helix-leucine-zipper (bHLHzip) protein that is known to function in the development of melanocytes, pigmented epithelial cells (PECs), osteoclasts, and mast cells. In this paper, we report on the isolation, expression, and overexpression of the chicken Mitf and discuss the role of its protein product in the differentiation and transdifferentiation of PECs. Northern blotting showed that chicken Mitf is predominantly expressed in embryonic retinal pigmented epithelium (PE), but is expressed at low levels in other tissues. A 5' RACE analysis revealed differences in the 5' region Mitf nRNA in PE and other tissues. Immunological analysis revealed that Mitf, the protein encoded by Mitf, is first detected in the nuclei of the optic vesicle cells at embryonic stage 13 in a restricted region covered with mesenchymal cells. From stage 14 to 24, the specific staining is observable in the PE and precursor of the PE, the outer layer of the optic cup. In embryos at stages later than stage 29 the signals for Mitf in the future iris, ciliary body, and posterior retinal regions become faint. These results show that expression of Mitf starts at the optic vesicle stage at which no other marker genes for PECs such as mmp115 and tyrosinase are expressed. Dedifferentiation of cultured retinal PECs (rPECs) was induced by phenylthiourea and testicular hyaluronidase, bFGF, or TGF-beta. Mitf expression was inhibited by these factors and reactivated during redifferentiation of the dedifferentiated cells into rPECs, showing the correlation between Mitf expression and rPEC differentiation. Retrovirus-mediated overexpression of Mtif inhibited bFGF-induced dedifferentiation and transdifferentiation of rPECs to both lens and neural cells. These findings showed that downregulation of Mitf expression is essential for the transdifferentiation of rPEC. Mitf overexpression caused hyperpigmentation in cultured rPECs and suppressed the changes in gene expression induced by bFGF. Mitf overexpression promoted expression of mmp115 and tyrosinase in bFGF-treated rPECs suggesting a critical role for Mitf in rPEC differentiation. Mitf overexpression, however, did not promote expression of another rPEC-specific gene, pP344, in bFGF-treated rPECs. This result suggests the presence of other regulatory genes promoting rPEC differentiation. The expression patterns of pax6 and Mitf are complementary both in vivo in vitro. Overexpression of Mitf inhibited expression of pax6 in cultured rPECs. These observations suggest that Mitf regulates pax6 expression negatively.

Amino Acid Sequence↗
PubMed1998

A critical role for the optic vesicle in lens development; a reinvestigation of free lens formation in Cynops pyrrhogaster.

The role of the optic vesicle in lens development was reinvestigated in Cynops pyrrhogaster. To study the necessity for the optic vesicle in early lens development, the optic anlages of stage 17-27 embryos were ablated and the frequency of free lens formation was examined with lens specific markers. Free lens formation was not observed when operations were performed prior to contact between the head surface epidermis and the optic vesicle (stages 17-18). On the contrary, free lens formation occurred in all cases where the optic vesicles were removed after the initiation of lens placode formation in the head surface epidermis (stage 27). However, no lens fiber formation was observed in these free lenses as judged by the absence of lens fiber specific gene expression, namely gamma-crystallin, at stages when secondary lens fiber formation could be found in the control lenses of the unoperated sides. The pattern of expression of alpha A-crystallin in the developing free lens also differed from that of the normally developing lens. This paper is the first report to indicate that the coordinated and sequential expression of crystallin genes are influenced by the optic vesicle; the optic vesicle is required for proper regulation of the alpha A- and gamma-crystallin but not beta B1-crystallin genes.

Animals↗
PubMed1998

Avian neural crest-derived neurogenic precursors undergo apoptosis on the lateral migration pathway.

Neural crest cells of vertebrate embryos disperse on distinct pathways and produce different derivatives in specific embryonic locations. In the trunk of avian embryos, crest-derived cells that initially migrate on the lateral pathway, between epidermal ectoderm and somite, produce melanocytes but no neuronal derivatives. Although we found that melanocyte precursors are specified before they disperse on the lateral pathway, we also observed that a few crest-derived neuronal cells are briefly present on the same pathway. Here, we show that neuronal cells are removed by an episode of apoptosis. These observations suggest that localized environmental factor(s) affect the distribution of fate-restricted crest derivatives and function as a 'proof-reading mechanism' to remove 'ectopic' crest-derived cells.

Animals↗
PubMed1996

Isolation of a novel chick homolog of Serrate and its coexpression with C-Notch-1 in chick development.

Intercellular signaling mediated by the transmembrane proteins, Notch as receptor and its ligands, Delta and Serrate, plays essential roles in the developmental fate decision of many cell types in Drosophila. The Notch genes are highly conserved both in invertebrates and in vertebrates, suggesting that Notch pathway regulates cell fate decisions during vertebrates development. Notch, Delta and Serrate homologs in chicken have been cloned (Henrique et al., Nature 375: 787-790, 1995; Myat et al., Dev. Biol. 174: 233-247, 1996). We isolated a novel chick homolog of Drosophila Serrate, named C-Serrate-2, and examined its expression patterns during the early chick development using whole-mount in situ hybridization. C-Serrate-2 transcripts were detected in several tissues including the forebrain, the myotome and the apical ectodermal ridge (AER) of the limb bud of a 4-day-old chick embryo. In most of the regions where C-Serrate-2 was expressed, C-Notch-1 was also expressed. Our observations suggest that Serrate-2-Notch-1 signaling plays a role in a variety of morphogeneses during the chick development.

Amino Acid Sequence↗
PubMed1995

Gene transfer into circulating primordial germ cells of quail embryos.

During early stages in avian embryogenesis primordial germ cells (PGCs) show a unique migration pathway toward the gonadal anlage through the circulation. In the present study, liposomes consisting of plasmid DNA (pMiwZ; containing lacZ as a reporter) and Lipofectin were injected into the marginal veins of quail embryos during the stages PGCs were circulating in the blood vessels. The lacZ expression was then histochemically detected in the gonads at later embryonic stages, indicating the expression of the injected DNA in PGCs.

Animals↗
PubMed1994

A complete culture system for avian transgenesis, supporting quail embryos from the single-cell stage to hatching.

We report here a method to produce quail hatchlings by culture in vitro from the single-cell stage. The culture is composed of three steps. In the first step, the fertilized ovum surrounded by thick albumen obtained from the magnum is cultured for 24 hr at 41.5 degrees C in a tightly sealed 20-ml plastic cup with chicken thin albumen added to the equator level of the ovum (System Q1). In the second step, a quail egg shell, cut horizontally and emptied, is used as a bed shell. After the thick albumen is removed, the embryo with egg yolk is transferred to the bed shell and thin albumen from chicken eggs is added to fill the shell. Then, the embryo is cultured for an additional 52 hr at 37.5 degrees C while being rocked at an angle of 90 degrees at 30-min intervals (System Q2). The embryo is transferred again to a chicken bed shell and cultured at 37.5 degrees C with rocking at a 30-degree angle (System Q3). Just before hatching, the rocking of embryos is stopped. The procedure yielded a hatchability of 25%. For transgenesis, a plasmid construct containing a beta-actin-lacZ hybrid gene (pMiwZ) is microinjected into the ovum at the single-cell stage, which is cultured in vitro for 85-90 hr using Systems Q1 and Q2 consecutively. Seven out of 17 surviving embryos exhibited lacZ gene expression in embryonic tissues as detected by histochemistry. The procedure described here should be highly applicable for the production of transgenic birds.

Actins↗
PubMed1994

Predominant melanogenesis and lentoidogenesis in vitro from multipotent pineal cells by dimethyl sulfoxide and hexamethylene bisacetamide.

Pineal cells of the 8-day embryonic quail are multipotent cells which differentiate in vitro into skeletal muscle fibers, pigmented epithelial cells (PECs), lens cells and neurons. However, it was not yet clear whether precursor cells which gave such a wide repertoire of differentiation were single type or not. The present culture studies revealed that pineal cells were exclusively directed to ocular differentiation pathways by dimethyl sulfoxide (DMSO) and hexamethylene bisacetamide (HMBA), suggesting a single type of precursor cell in the pineal body. DMSO directed pineal cells to differentiate into PECs. Co-administration of basic fibroblast growth factor (bFGF) with DMSO partially inhibited PEC differentiation and promoted lens cell differentiation. Northern blot analysis using cDNAs specific to PEC and lens cell confirmed this morphological observation. HMBA completely inhibited pigmentation of cultured pineal cells and markedly promoted lens cell differentiation. Ocular differentiation of pineal cells was accompanied with the loss of myogenicity. We discuss three possible pathways of lens cell differentiation from pineal cells. The agents which affect pineal cell differentiation seemed to modulate the cell-substrate interaction. And the interaction was suggested to be one of the environmental cues in the differentiation.

Acetamides↗
PubMed1993

Genetic characterization of the multipotent dedifferentiated state of pigmented epithelial cells in vitro.

Retinal pigmented epithelial cells (PECs) of chicken embryos extensively and almost synchronously transdifferentiate into lens cells in medium containing phenylthiourea and testicular hyaluronidase, passing through the bipotent dedifferentiated state. We have isolated genes that are expressed specifically by either pigment or lens cells and analyzed their expression in the transdifferentiation process. The expression of some proto-oncogenes was also studied. In the dedifferentiation process, expression of the c-myc gene was enhanced and the transcription of PEC-specific genes (MMP115, pP344) was completely repressed. However, transcription of lens-specific genes (alpha-, beta- and delta-crystallins genes) remained silent in dedifferentiated pigment cells. Activation of len- or PEC-specific genes occurred only in conditions permissive for lens or PEC differentiation, respectively. These results indicated that lens transdifferentiation from PECs proceeds through a multipotent (or at least bipotent) intermediate cell state in which the c-myc gene is activated, but neither PEC- nor lens-specific genes are expressed.

Animals↗
PubMed1992

Tissue-specific response of estrogen receptor gene expression to estrogen in chick.

We have developed a sensitive assay system by RT (reverse transcription)-PCR(polymerase chain reaction) to detect the low level of chicken estrogen receptor (cER) transcript. Using this system, the differential expression of cER gene in chick tissues was observed. Moreover, we found that the oviduct cER transcript levels in chick was not affected, albeit the remarkable growth of this tissue, by exogenous estrogen. In contrast, estrogen enhanced the hepatic cER transcript level several folds. Thus, the present study clearly showed a novel tissue-specific response of cER gene expression to estrogen in chick.

Animals↗