餅井 真

Amputation of the larval tail of Xenopus injures the notochord, spinal cord, muscle masses, mesenchyme, and epidermis, induces the growth and differentiation of cells in those tissues, and results in tail regeneration. A dorsal incision in the larval tail injures the same tissues and induces cell growth and differentiation, but never results in the formation of any extra appendages. The first sign of tail regeneration is the multilayered wound epidermis and Xwnt-5a expression in the distal region, neither of which is observed in the recovering region after a dorsal incision. To evaluate the role of Xwnt-5a in tail regeneration, Xwnt-5a was overexpressed in the recovering region. When an animal cap injected with Xwnt-5a mRNA was grafted into the dorsal incision, an ectopic protrusion was formed. Morphological and molecular analyses revealed that the protrusion was an ectopic larval tail, which was equivalent to the regenerating tail but different from the tail that develops from the embryonic tail bud. Lineage labeling revealed that the major differentiated structures of the ectopic tail were formed from host cells, suggesting that Xwnt-5a induced host cells to make a complete tail. The ectopic tail was not induced by Xwnt-8 or Xwnt-11, demonstrating the specificity of Xwnt-5a in this process. A pharmacological study showed that JNK signaling is required in tail regeneration. These results support the proposition that Xwnt-5a plays an instructive role in larval tail regeneration via Wnt/JNK signaling. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

We examined several candidate posterior/mesodermal inducing molecules using permanent blastula-type embryos (PBEs) as an assay system. Candidate molecules were injected individually or in combination with the organizer factor chordin mRNA. Injection of chordin alone resulted in a white hemispherical neural tissue surrounded by a large circular cement gland, together with anterior neural gene expression and thus the development of the anterior-most parts of the embryo, without mesodermal tissues. When VegT, eFGF or Xbra mRNAs were injected into a different blastomere of the chordin-injected PBEs, the embryos elongated and formed eye, muscle and pigment cells, and expressed mesodermal and posterior neural genes. These embryos formed the full spectrum of the anteroposterior embryonic axis. In contrast, injection of CSKA-Xwnt8 DNA into PBEs injected with chordin resulted in eye formation and expression of En2, a midbrain/hindbrain marker, and Xnot, a notochord marker, but neither elongation, muscle formation nor more posterior gene expression. Injection of chordin and posteriorizing molecules into the same cell did not result in elongation of the embryo. Thus, by using PBEs as the host test system we show that (i) overall anteroposterior neural development, mesoderm (muscle) formation, together with embryo elongation can occur through the synergistic effect(s) of the organizer molecule chordin, and each of the 'overall posteriorizing molecules' eFGF, VegT and Xbra; (ii) Xwnt8-mediated posteriorization is restricted to the eye level and is independent of mesoderm formation; and (iii) proper anteroposterior patterning requires a separation of the dorsalizing and posteriorizing gene expression domains.

Tail regeneration in urodeles is dependent on the spinal cord (SC), but it is believed that anuran larvae regenerate normal tails without the SC. To evaluate the precise role of the SC in anuran tail regeneration, we developed a simple operation method to ablate the SC completely and minimize the damage to the tadpole using Xenopus laevis. The SC-ablated tadpole regenerated a twisted and smaller tail. These morphological abnormalities were attributed to defects in the notochord (NC), as the regenerated NC in the SC-ablated tail was short, slim and twisted. The SC ablation never affected the early steps of the regeneration, including closure of the amputated surface with epidermis and accumulation of the NC precursor cells. The proliferation rate of the NC precursor cells, however, was reduced, and NC cell maturation was retarded in the SC-ablated tail. These results show that the SC has an essential role in the normal tail regeneration of Xenopus larvae, especially in the proliferation and differentiation of the NC cells. Gene expression analysis and implantation of a bead soaked with growth factor showed that fibroblast growth factor-2 and -10 were involved in the signaling molecules, which were expressed in the SC and stimulated growth of the NC cells.

The tail of the Xenopus tadpole contains major axial structures, including a spinal cord, notochord and myotomes, and regenerates within 2 weeks following amputation. The tail regeneration in Xenopus can provide insights into the molecular basis of the regeneration mechanism. The regenerated tail has some differences from the normal tail, including an immature spinal cord and incomplete segmentation of the muscle masses. Lineage analyses have suggested that the tail tissues are reconstructed with lineage-restricted stem cells derived from their own tissues in clear contrast to urodele regeneration, in which multipotent blastema cells derived from differentiated cells play a major role. Comprehensive gene expression analyses resulted in the identification of a panel of genes involved in sequential steps of the regeneration. Manipulation of genes' activities suggested that the tail regeneration is regulated through several major signaling pathways.

We succeeded in visualization of the primordial germ cells (PGCs) in a living Xenopus embryo. The mRNA of the reporter Venus protein, fused to the 3' untranslated region (UTR) of DEADSouth, which is a component of the germ plasm in Xenopus eggs, was micro-injected into the vegetal pole of fertilized eggs and then the cells with Venus fluorescence were monitored during development. The behavior of the cells was identical to that previously described for PGCs. Almost all Venus-expressing cells were Xdazl-positive in the stage 48 tadpoles, indicating that they were PGCs. In addition, we found three sub-regions (A, B and C) in the 3' UTR, which were involved in the PGC-specific expression of the reporter protein. Sub-region A, which was identified previously as a localization signal for the germ plasm during oogenesis, participated in anchoring of the mRNA at the germ plasm and the degradation of the mRNA in the somatic cells. Sub-regions B and C were also involved in anchoring of the mRNA at the germ plasm. Sub-region B participated in the enhancement of translation. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

In many organisms, proper embryo development depends on the asymmetrical distribution of mRNA in the cytoplasm of the egg. Here we report comprehensive screening of RNA localized in the animal or vegetal hemisphere of the Xenopus egg. Macroarrays including over 40000 independent embryonic cDNA clones, representing at least 17000 unigenes, were differentially hybridized with labeled probes synthesized from the mRNA of animal or vegetal blastomeres. After two rounds of screening, we identified 33 clones of transcripts that may be preferentially distributed in the vegetal region of the early stage embryo, but transcripts localized in the animal region were not found. To assess the array results, we performed northern blot and quantitative real-time reverse transcription-polymerase chain reaction analysis. As a result, 21 transcripts of the 33 were confirmed to be localized in the vegetal region of the early stage embryo. Whole-mount in situ hybridization analysis revealed that 11 transcripts, including 7 previously reported genes, were localized in the vegetal hemisphere of the egg. These 11 transcripts were categorized into three groups according to their expression patterns in the egg. The first group, which contained four transcripts, showed uniform expression in the vegetal hemisphere, similar to VegT. The second group, which contained three transcripts, showed gradual expression from the vegetal pole to the equator, similar to Vg1. The last group, which contained three transcripts, was expressed at the germ plasm, similar to Xdazl. One transcript, Xwnt11, showed both the second and the third expression patterns.

Xenopus larvae possess a remarkable ability to regenerate their tails after they have been severed. To gain an understanding of the molecular mechanisms underlying tail regeneration, we performed a cDNA macroarray-based analysis of gene expression. A Xenopus cDNA macroarray representing 42,240 independent clones was differentially hybridized with probes synthesized from the total RNA of normal and regenerating tails. Temporal expression analysis revealed that the up-regulated genes could be grouped into early or late responding genes. A comparative expression analysis revealed that most genes showed similar expression patterns between tail development and regeneration. However, some genes showed regeneration-specific expression. Finally, we identified 48 up-regulated genes that fell into several categories based on their putative functions. These categories reflect the various processes that take place during regeneration, such as inflammation response, wound healing, cell proliferation, cell differentiation, and control of cell structure. Thus, we have identified a panel of genes that appear to be involved in the process of regeneration. (c) 2005 Wiley-Liss, Inc.

The regeneration of the amputated tail of Xenopus laevis larvae is an excellent model system for regeneration research. The wound left by the amputated tail is covered with epidermis within 24 h. Then, the cell number increases near the amputation plane at the notochord, spinal cord and muscle regions. An apparently complete tail with notochord, muscle and spinal cord is regenerated within two weeks. To reveal whether the molecular mechanism underlying the tail regeneration is the same as that in embryonic tail development, the gene expression patterns of the embryonic tail bud and the regenerating tail were compared by in situ hybridization and reverse transcription-polymerase chain reaction. Most genes analyzed were expressed at similar levels in both tissues, whereas two bone morphogenetic protein (BMP)-antagonists, chordin and noggin, were detected only in the embryonic tail bud. The regenerating tail also lacked expression of Xshh in the floor plate and expression of Xdelta-1 in the spinal cord and presomitic mesoderm. These results show that there are some differences in gene expression between the two processes. Furthermore, when the tail of Xenopus larvae is amputated, the regenerating tail has a gene expression pattern similar to the distal portion of the larval tail rather than the embryonic tail bud, suggesting that the cut larval tail does not make a new embryonic tail bud, but rather a new larval tail tip for regeneration.

It has been known that rotenone and 1-methyl-4-phenylpyridinium ion (MPP+, a metabolite of MPTP), which inhibit mitochondrial complex I, are useful tools for parkinsonian models in vertebrates such as primates and rodents. Planarian, an invertebrate flatworm, has a high potential for regeneration, and dopamine plays a key role in its behavior. In the present study, we examined a cloned planarian, the GI strain from Dugesia japonica. Planarians that were treated with rotenone or MPTP underwent autolysis and individual death in a concentration and time-dependent manner. In addition, these effects induced by rotenone or MPTP were inhibited by several antiparkinsonian drugs and caspase inhibitors. These results suggest that the degeneration of planarian dopaminergic system induced by rotenone or MPTP may be mediated through caspase-like activation.

The plumage on the dorsal trunk of normal quail embryos exhibits longitudinal black and brown stripes of pigments produced by melanocytes. However, this pigmentation pattern disappeared in Bh (black at hatch) heterozygous and homozygous embryos because of overall black and brown pigmentation of plumages, respectively. To investigate the mechanisms of the pigment pattern formation of plumage and clarify the roles of the Bh locus in the pattern formation, we examined the expression pattern of genes relating to melanocyte development (Mitf, MelEM antigen, Kitl, Kit and EdnrB2) and melanin pigment production (Dct, Tyrp1, Tyr and Mmp115) in Bh mutant and wild-type embryos throughout development. As a result, we found that MelEM antigen was expressed in melanoblasts committed to produce black pigment before apparent melanogenic gene expression, and that Bh heterozygotes and homozygotes showed abnormal expression patterns of the MelEM antigen. These results indicate that MelEM antigen is a good marker for melanoblasts committed to produce black pigment, and suggests that the Bh locus directs melanocytes to produce eumelanin in proper positions. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

The spatio-temporal expression of three crystallin genes (alphaA, betaB1 and gamma) in the developing and regenerating lenses of newt was compared by in situ hybridization in lens differentiation in normal development with during regeneration. In normal development, all crystallin transcripts were first detected at the same stage in the posterior region of the lens vesicle (McDevitt's lens development stage V) and continued during lens fiber differentiation of the posterior cells into the primary lens fiber cell differentiation (McDevitt's lens development stage VII-VIII). At later stages, the expression of the three genes was restricted to the secondary lens fibers and gradually became undetectable in primary lens fibers (McDevitt's lens development stage X). The signal for gamma-crystallin was never detected in lens epithelium at any stage, whereas signals for alphaA- and betaB1-crystallin were detected in the lens epithelium at the stage when the primary lens fiber mass was formed. During lens regeneration, signals for the three crystallins were first detected at the same stage at the ventral margin of a regenerating lens vesicle (Sato's lens regeneration stage IV). The expression patterns of three crystallin genes were similar to those in normal development (Sato's lens regeneration stage V-X). The expression pattern of the crystallin genes in normal lens development fundamentally resembles that during lens regeneration, suggesting the absence of unique expression programs of crystallin genes for lens regeneration not found in ontogeny.

The spatio-temporal expression of three crystallin genes (alphaA, betaB1 and gamma) in the developing and regenerating lenses of newt was compared by in situ hybridization in lens differentiation in normal development with during regeneration. In normal development, all crystallin transcripts were first detected at the same stage in the posterior region of the lens vesicle (McDevitt's lens development stage V) and continued during lens fiber differentiation of the posterior cells into the primary lens fiber cell differentiation (McDevitt's lens development stage VII-VIII). At later stages, the expression of the three genes was restricted to the secondary lens fibers and gradually became undetectable in primary lens fibers (McDevitt's lens development stage X). The signal for gamma-crystallin was never detected in lens epithelium at any stage, whereas signals for alphaA- and betaB1-crystallin were detected in the lens epithelium at the stage when the primary lens fiber mass was formed. During lens regeneration, signals for the three crystallins were first detected at the same stage at the ventral margin of a regenerating lens vesicle (Sato's lens regeneration stage IV). The expression patterns of three crystallin genes were similar to those in normal development (Sato's lens regeneration stage V-X). The expression pattern of the crystallin genes in normal lens development fundamentally resembles that during lens regeneration, suggesting the absence of unique expression programs of crystallin genes for lens regeneration not found in ontogeny.

Using cDNA-based array analysis combined with double-stranded RNA interference (dsRNAi), we have identified yk298h6 as a target gene of Caenorhabditis elegans TGF-beta signaling. Worms overexpressing dbl-1, a TGF-beta ligand, are 16% longer than wild type. Array analysis shows yk298h6 to be one of several genes suppressed in such worms. Disruption of yk298h6 function by dsRNAi also resulted in long worms, suggesting that it is a negative regulator of body length. yk298h6 was then mapped to, and shown to be identical to, lon-1, a known gene that affects body length. lon-1 encodes a 312 amino acid protein with a motif sequence that is conserved from plants to humans. Expression studies confirm that LON-1 is repressed by DBL-1, suggesting that LON-1 is a novel downstream component of the C. elegans TGF-beta growth regulation pathway. Consistent with this, LON-1 is expressed mainly in the larval and adult hypodermis and has dose-dependent effects on body length associated with changes in hypodermal ploidy, but not hypodermal cell proliferation.

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.

Caenorhabditis elegans is an ideal organism for the study of the molecular basis of fundamental biological processes such as germline 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.

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 (BIB) 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 BIB homozygotes, However, osteopetrosis in BIB 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 BIB 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 BIB homozygotes, Furthermore, overexpression of chicken Mitf via retroviral transfection into BIB 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 BIB homozygotes, These results indicate that the novel mutations in Mitf gene observed in the B/B homozygote quail impair osteoclastic bone resorption. (C) 2001 Wiley-Liss, Inc.

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.

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 P-33-labeled DNA probes synthesized from the mRNAs of wild-type, dbl-1, sma-2 and Ion-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 DBL1, sma-6 were transcriptionally regulated by the DBL-1 signal.

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 P-33-labeled DNA probes synthesized from the mRNAs of wild-type, dbl-1, sma-2 and Ion-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 DBL1, sma-6 were transcriptionally regulated by the DBL-1 signal.

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.

The spatio-temporal expression of three crystallin genes (αA, βB1 and γ) 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, αA- and βB1-crystallin signals were first detected in the presumptive lens fiber region of the lens vesicle. The expression of γ-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.

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.

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 PB l-crystallin genes.

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 BIB PECs continued to proliferate. Some BIB cells overlaid other BIB cells and formed lentoid bodies. Immunological analysis revealed that BIB rPEGs transdifferentiated to lens cells and neural cells in vitro with no addition of basic FGF (bFGF), while B/+ rPEGs required bFGF to transdifferentiate. Expression of PEG-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 Bl 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. (C) 1998 Academic Press.

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 Miff 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 of Mitf mRNA in PE and other tissues. Immunological analysis revealed that Mitf, the protein encoded by Miff, 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 25, 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. Miff expression was inhibited by these factors and reactivated during redifferentiation of the dedifferentiated cells into rPECs, showing the correlation between Miff expression and rPEC differentiation. Retrovirus-mediated overexpression of Miff inhibited bFGF-induced dedifferentiation and transdifferentiation of rPECs to both lens and neural cells. These findings showed that downregulation of Miff expression is essential for the transdifferentiation of rPEC. Miff overexpression caused hyperpigmentation in cultured rPECs and suppressed the changes in gene expression induced by bFGF. Miff overexpression promoted expression of mmp115 and tyrosinase in bFGF-treated rPECs suggesting a critical role for Mitf in rPEC differentiation. Miff 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 and in vitro. Overexpression of Miff inhibited expression of pax6, in cultured rPECs. These observations suggest that Mitf regulates pax6 expression negatively. (C) 1998 Academic Press.

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 ef 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.

When retinal pigmented epithelial cells (PEG) of chick embryos are cultured under appropriate conditions, the phenotype changes to that of lens cells through a process known as transdiiferentiation. The first half of the process, characterized by dedifferentiation of PEG, is accompanied by a marked decrease in adhesiveness of PEC to collagen type I- or type IV-coated dishes. To understand the underlying mechanisms of this change, we analyzed the expression of integrins, which are major receptors for extracellular matrix components. Northern blot analysis with cDNA probes for chicken alpha 3, alpha 6, alpha 8, alpha v, beta 1 and beta 5 integrin mRNA showed that the genes for all these integrins are transcribed at similar levels in PEC and dedifferentiated PEC (dePEC). Further analysis of beta 1 integrin, which is a major component of integrin heterodimers, showed that although the protein amount of beta 1 integrin was not changed, its localization at focal contacts seen in PEC was lost in dePEC. When anti-beta 1 integrin antibody was added to the PEC culture medium, a decrease of cell-substrate adhesiveness occurred, followed by a gradual change in both morphology and gene expression patterns to ones similar to those of dePEC. These findings suggest that an appropriate distribution of beta 1 integrin plays an essential role in maintaining the differentiated state of PEC through cell-substrate adhesion.

We first examined the pH change of the albumen of quail (Cotumix cotumix japonica) eggs before and after they were laid, as well as that of laid eggs. The pH rose rapidly after laying and continued to increase gradually in storage. Incubation at 37.5 degrees C accelerated the increase in the pH of infertile eggs, while that of fertile eggs remained low during incubation. Referring to these results, we obtained a protocol for producing quail hatchlings by culture in vitro from naked ova, The naked ovum was filled with chicken (Gallus domesticus) thick albumen, the pH of which had been adjusted to 7.2-7.4. The ovum was cultured at 41.5 degrees C in 20% CO2 in air for the first 24 h. Then the embryo was moved to a surrogate quail egg shell that had been filled with non-pH-adjusted chicken thin albumen and cultured for a further 48 h in 100% air. The embryo was transferred again to a surrogate chicken egg shell and cultured under the same conditions until hatching. The culture yielded quail chicks with a hatchability of 19.4%. The method proposed here should be applicable to the production of transgenic birds.