Abstract: The prechordal cranium, or the anterior half of the neurocranial base, is a key structure for understanding the development and evolution of the vertebrate cranium, but its embryonic configuration is not well understood. It arises initially as a pair of cartilaginous rods, the trabeculae, which have been thought to fuse later into a single central stem called the trabecula communis (TC). Involvement of another element, the intertrabecula, has also been suggested to occur rostral to the trabecular rods and form the medial region of the prechordal cranium. Here, we examined the origin of the avian prechordal cranium, especially the TC, by observing the craniogenic and precraniogenic stages of chicken embryos using molecular markers, and by focal labeling of the ectomesenchyme forming the prechordal cranium. Subsequent to formation of the paired trabeculae, a cartilaginous mass appeared at the midline to connect their anterior ends. During this midline cartilage formation, we did not observe any progressive medial expansion of the trabeculae. The cartilages consisted of premandibular ectomesenchyme derived from the cranial neural crest. This was further divided anteroposteriorly into two portions, derived from two neural crest cell streams rostral and caudal to the optic vesicle, called preoptic and postoptic neural crest cells, respectively. Fate-mapping analysis elucidated that the postoptic neural crest cells were distributed exclusively in the lateroposterior part of the prechordal cranium corresponding to the trabeculae, whereas the preoptic stream of cells occupied the middle anterior part, differentiating into a cartilage mass corresponding to the intertrabecula. These results suggest that the central stem of the prechordal cranium of gnathostomes is composed of two kinds of distinct cartilaginous modules: a pair of trabeculae and a median intertrabecula, each derived from neural crest cells populating distinct places of the craniofacial primordia through specific migratory pathways.
Abstract: 17beta-Estradiol (E2) plays important roles in the development and differentiation of the gonad and central nervous systems, but little is known regarding the effects of exogenous E2 on chondrogenesis in skeletal development. In the present study, we found that treatment with E2 1-5 days post-fertilization (dpf) at concentrations above 1.5x10(-5)M increased the mortality rate in zebrafish embryos. Morphological analysis showed that treatment with E2 1-5dpf caused abnormal cartilage formation in a dose-dependent manner at concentrations above 5x10(-6)M. E2 1-5dpf at 1.5x10(-5)M caused defects of the ethmoid plate, parallel cleft of the trabecular cartilage, and hypoplasia of Meckel's cartilage and the ceratohyal cartilage. The sensitivity of embryos to E2 depended on the developmental stage. In early chondrogenesis (1-2dpf), the embryos were highly sensitive to E2, leading to hypoplasia of the cartilage. In situ hybridization studies showed that expression levels of patched1 (ptc1) and patched2 (ptc2) receptor mRNAs were markedly decreased by exposure to 2x10(-5)M E2 1-2dpf. However, the expression levels of sonic hedgehog (shh) and tiggywinkle hedgehog (twhh) mRNAs were constant in the E2-treated embryos. In addition, the estrogen receptor antagonist ICI 182,780 did not completely abolish the effects of E2, suggesting that E2 may not inhibit chondrogenesis through its nuclear estrogen receptor. These results suggest that exposure to exogenous E2 possibly inhibits chondrogenesis via inhibition of the hedgehog (Hh) signal transduction system.
Abstract: Developing vertebrate limbs are often utilized as a model for studying pattern formation and morphogenetic cell death. Herein, we report that conditional deletion of Rac1, a member of the Rho family of proteins, in mouse limb bud mesenchyme led to skeletal deformities in the autopod and soft tissue syndactyly, with the latter caused by a complete absence of interdigital programmed cell death. Furthermore, the lack of interdigital programmed cell death and associated syndactyly was related to down-regulated gene expression of Bmp2, Bmp7, Msx1, and Msx2, which are known to promote apoptosis in the interdigital mesenchyme. Our findings from Rac1 conditional mutants indicate crucial roles for Rac1 in limb bud morphogenesis, especially interdigital programmed cell death.
Abstract: 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 'verall 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.
Abstract: Freshwater sponges include six extant families which belong to the suborder Spongillina (Porifera). The taxonomy of freshwater sponges is problematic and their phylogeny and evolution are not well understood. Sequences of the ribosomal internal transcribed spacers (ITS1 and ITS2) of 11 species from the family Lubomirskiidae, 13 species from the family Spongillidae, and 1 species from the family Potamolepidae were obtained to study the phylogenetic relationships between endemic and cosmopolitan freshwater sponges and the evolution of sponges in Lake Baikal. The present study is the first one where ITS1 sequences were successfully aligned using verified secondary structure models and, in combination with ITS2, used to infer relationships between the freshwater sponges. Phylogenetic trees inferred using maximum likelihood, neighbor-joining, and parsimony methods and Bayesian inference revealed that the endemic family Lubomirskiidae was monophyletic. Our results do not support the monophyly of Spongillidae because Lubomirskiidae formed a robust clade with E. muelleri, and Trochospongilla latouchiana formed a robust clade with the outgroup Echinospongilla brichardi (Potamolepidae). Within the cosmopolitan family Spongillidae the genera Radiospongilla and Eunapius were found to be monophyletic, while Ephydatia muelleri was basal to the family Lubomirskiidae. The genetic distances between Lubomirskiidae species being much lower than those between Spongillidae species are indicative of their relatively recent radiation from a common ancestor. These results indicated that rDNA spacers sequences can be useful in the study of phylogenetic relationships of and the identification of species of freshwater sponges.
Abstract: BACKGROUND: Oral leukoplakia is a precancerous change developed in the oral mucosa, and the mechanism that oral leukoplakia becomes malignant through atypical epithelium is not known. Here we compared the beta-catenin expression detected by immunohistochemical staining in the normal oral epithelium and in the oral leukoplakia with or without dysplasia. RESULTS: The normal oral epithelium showed beta-catenin expression only in the cell membrane, but not in the nuclei. In the oral leukoplakia without dysplasia, 7 out of 17 samples (41%) showed beta-catenin expression in the cell membrane, and 5 samples (29%) showed expression in the nuclei. In the oral leukoplakia with dysplasia, nuclear expression of beta-catenin was shown in 11 out of 12 samples (92%). Incidence of nuclear beta-catenin expression was significantly different between dysplasia and normal oral epithelium (P < 0.01), and also between oral leukoplakia with dysplasia and those without dysplasia (P < 0.01). Wnt3 expression was detected in the epithelial cell membrane or cytoplasm in oral leukoplakia where nuclear expression of beta-catenin was evident, but not in epithelial cells without nuclear expression of beta-catenin. CONCLUSION: The components of canonical Wnt pathway, such as Wnt3, beta-catenin, and cyclin D1, were detected, implying that this pathway is potentially involved in the progression of dysplasia in oral leukoplakia.
Abstract: The direct effects of Wnt4 on myogenic proliferation and differentiation of skeletal muscle precursors are examined. Wnt4 cDNA was misexpressed in the presumptive limb fields on the right side of stage 16 chick embryos. Muscle development was evaluated at stage 37 with hematoxylin-eosin staining and immunohistochemical staining for fast and slow types of the myosin heavy chain (MyHC). Overexpression of Wnt4 resulted in up-regulation of Pax7 and MyoD1 expression. The muscle mass showed a significant increase compared with that of the control limb. The area for fast MyHC-expressing cells showed a significant increase, whereas a slight decrease was observed for slow MyHC-expressing cells. Wnt4 acted as a stimulator during myogenic proliferation and differentiation, especially, for fast-type muscle in C2C12 cells. The present results are identical to those of myostatin knockout, suggesting that Wnt4 is acting against myostatin as an antagonizing signal for myostatin.
Abstract: Wnt/beta-catenin signaling is involved in the formation of the apical ectodermal ridge (AER) during vertebrate limb development. Although Wnt3a is a potent ligand for chick AER formation, whether chick Wnt3a can induce Fgf8 expression in chick embryos is unclear and the Wnt ligand involved in chick AER formation remains unknown. Here, we examined whether another Wnt3a isoform is expressed in the AER, and whether Wnt3 contributes to AER formation in chick as well as mouse embryos. We found that chick Wnt3 was not expressed in the presumptive limb ectoderm at the early stages of AER formation. Using 5'-rapid amplification of cDNA ends, we isolated another chick Wnt3a transcript. This novel variant, Wnt3a variant 2, induced Fgf8 in the limb ectoderm and activated the beta-catenin pathway in vivo and in vitro. These data showed that Wnt3a variant 2 is an active form of chick Wnt3a that regulates chick AER formation.
Abstract: alpha-Enolase and c-myc promoter binding protein 1 are encoded by a single gene, ENO1, and are synthesized from the same transcript through alternative use of translational start sites. We have investigated the localization of ENO1 gene transcripts detected as proteins with an immunohistochemical method and also as mRNA with an in situ hybridization method on tissue sections of oral epithelium and oral squamous cell carcinoma, and demonstrated the differential distribution of the gene transcripts in normal oral epithelium and oral squamous cell carcinoma in humans. Expression of the ENO1 transcript was detectable in the region from the basal cell layers to the lower granular cell layers. Three patterns of ENO1 localization were observed with immunostaining in the epithelia: cytoplasm, nuclei, and both nuclei and cytoplasm. These patterns were observed randomly within the same specimen. In contrast to normal oral epithelium, ENO1 protein was not detectable in the nuclei of carcinoma cells. Our results indicate that differential subcellular localization of ENO1 products may be closely related to carcinogenesis of the oral epithelium.
Abstract: Although it is known that sustained activation of classical mitogen-induced protein kinase (MAPK, also known as ERK) induced by nerve growth factor (NGF) plays an important role in the induction of neurite outgrowth, the role of p38 MAPK in neural cell function is still not clear. We developed two neuronal cell lines from PC12 cells, PC12m3 and PC12m32, in which NGF-induced neurite outgrowth is impaired and that show neurite outgrowth in response to hyperosmotic shock. The frequencies of neurite outgrowth of PC12m3 and PC12m32 cells induced by osmotic shock were approximately 10- and 12-fold greater, respectively, than that in PC12 parental cells. The p38 MAPK pathway inhibitor SB203580 but not the ERK pathway blocker U0126 inhibited the ability of PC12m3 and PC12m32 cells to induce neurite outgrowth in response to osmotic shock. Furthermore, expression of a nonactivable form of p38 but not that of wild-type p38 significantly blocked neurite outgrowth induced by osmotic shock. The extent of phosphorylation of p38 MAPK induced by osmotic shock in PC12m32 cells was much greater than that in PC12 parental cells. The upstream kinases MKK3 and MKK6, which phosphorylate and activate p38 MAPK, also showed higher levels in PC12m32 cells than in PC12 parental cells when treated with osmotic shock. Inhibition of p38 MAPK by SB203580 resulted in inhibition of the activity of the transcription factor CREB, which is activated by osmotic shock. These findings indicate that activation of CREB mediated by a p38 pathway distinct from the NGF signaling pathway may be required for neurite outgrowth.
Abstract: In the process of limb development, various signaling molecules are produced in the organizing center of the limb bud. These molecules regulate pattern formation and the morphogenesis of the limb. Members of the bone morphogenetic protein (BMP) family and their receptors are expressed in the limb bud in spatiotemporal specific patterns. BMP signaling regulates chondrogenesis of limb mesenchyme, and promotes apoptosis of the interdigital soft tissue. Recently, this signaling was found to be involved in establishment of dorsoventral polarity of the limb bud and in regulation of limb growth via maintenance of the apical ectodermal ridge. BMPs also regulate digit morphology. In these events, BMPs work together with other signaling molecules, but they sometimes act as negative regulators of these other molecules so that limb morphogenesis can proceed normally.
Abstract: The apical ectodermal ridge (AER) is indispensable for vertebrate limb development and requires Wnt/beta-catenin signaling for induction and maintenance. We report identification and involvement of Wnt10a in AER formation during chick limb development. Chicken Wnt10a has 82% identity with mouse Wnt10a in the amino acid sequence. The Wnt10a gene was expressed broadly in the surface ectoderm from as early as stage 10. By stage 15, the expression was restricted to the surface ectoderm overlying the lateral plate mesoderm. Wnt10a expression became intensified in the presumptive limb ectoderm during AER formation, and subsequently intense expression signals persisted in the AER. Wnt10a misexpression led to ectopic Fgf8 expression in the developing limb ectoderm and induced translocation of beta-catenin in chick embryo fibroblasts. These results suggest that Wnt10a is involved in AER formation in the chick limb bud through the Wnt/beta-catenin signaling pathway.
Abstract: Fibroblast growth factor (FGF) signaling is crucial for the induction and growth of the ear, a sensory organ that involves intimate tissue interactions. Here, we report the abnormality of Fgf10 null ear and the identification of a cis-regulatory element directing otic expression of Fgf10. In Fgf10 null inner ears, we found that the initial development of semicircular, vestibular, and cochlear divisions is roughly normal, after which there are abnormalities of semicircular canal/cristae and vestibular development. The mutant semicircular disks remain without canal formation by the perinatal stage. To elucidate regulation of the Fgf10 expression during inner ear development, we isolated a 6.6-kb fragment of its 5'-upstream region and examined its transcriptional activity with transgenic mice, using a lacZ-reporter system. From comparison of the mouse sequences of the 6.6-kb fragment with corresponding sequences of the human and chicken Fgf10, we identified a 0.4-kb enhancer sequence that drives Fgf10 expression in the developing inner ear. The enhancer sequences have motifs for many homeodomain-containing proteins (e.g., Prx, Hox, Nkx), in addition to POU-domain factors (e.g., Brn3), zinc-finger transcription factors (e.g., GATA-binding factors), TCF/LEF-1, and a SMAD-interacting protein. Thus, FGF10 signaling is dispensable for specification of otic compartment identity but is required for hollowing the semicircular disk. Furthermore, the analysis of a putative inner ear enhancer of Fgf10 has disclosed a complicated regulation of Fgf10 during inner ear development by numerous transcription factors and signaling pathways.
Abstract: The proper patterning of somites to give rise to sclerotome, dermomyotome, and myotome involves the coordination of many different cellular processes, including lineage specification, cell proliferation, cell death, and differentiation, by intercellular signals. One such family of secreted signaling proteins known to influence somite patterning is the Wnt family. Although the participation of Wnt-3a in the patterning of dorsal structures in the somite is well established, no clear consensus has emerged about the cellular processes that are governed by Wnt-3a in the somite. The recent demonstration that Wnt-3a has a proliferative role in the neural tube [Development 129 (2002) 2087] suggested that Wnt-3a might also act to regulate proliferation in somites. To test this hypothesis, we first analyzed the effects of Wnt-3a on segmental plate and somite explants (from Hamburger and Hamilton stage 10 chick embryos) grown in culture. These studies indicate that Wnt-3a is capable of maintaining and/or inducing expression of both Pax-3 and Pax-7, transcription factors that have been implicated in proliferation. To directly test for a role in proliferation, explants were immunostained with antibodies against phospho-histone H3. Explants treated with Wnt-3a show an increase in the percentage of cells expressing phospho-histone H3 as compared to controls. To test the proliferative effect of Wnt-3a in vivo, we ectopically expressed Wnt-3a in chick neural tubes via electroporation. Consistent with previous studies, ectopic expression of Wnt-3a in vivo results in a mediolateral expansion of the dermomyotome and myotome. We now show that proliferation of dorsal/dermomyotomal cells is significantly enhanced by ectopic Wnt-3a. Collectively, our explant and in vivo studies indicate that an increase in proliferation plays an important role in the expansion of the dermomyotome and myotome in Wnt-3a-treated embryos. Furthermore, our results demonstrate that small changes in proliferation can dramatically influence patterning and morphogenesis.
Abstract: We investigated the role of the p38 mitogen-activated protein kinase (MAPK) pathway in heat-shock-induced neurite outgrowth of PC12 mutant cells in which nerve growth factor (NGF)-induced neurite outgrowth is impaired. When cultures of the PC12 mutant (PC12m3) cells were exposed to heat stress at 44 degrees C for 10 min, activity of p38 MAPK increased and neurite outgrowth was greatly enhanced. The neurite extension was inhibited by the p38 MAPK inhibitor BS203580. Longer heat treatment of PC12m3 cells provoked cell death, which was enhanced by SB203580. These findings suggest that heat-induced activation of p38 MAPK is responsible for the neurite outgrowth and survival of PC12m3 cells.
Abstract: Paraquat (PQ), a quaternary nitrogen herbicide, is highly toxic to humans and animals. Acute poisoning and death due to PQ exposure have been reported over the past few decades. Excessive production of oxygen free radicals has been proposed to play an important role in the pulmonary pathology. The aim of the present work was to evaluate the implications for genes that are regulated by oxidative stress at the early stage of PQ exposure in rat lungs. We performed differential display RT-PCR (DD-PCR) on total RNA extracted from rat lungs after injection of 20mg per kg body weight. The experimental DD-PCR conditions, primer length and annealing temperature, were adjusted to improve reproducibility, and 19 differentiated clones were isolated. Sequence analysis followed by conventional RT-PCR and real-time RT-PCR analyses were used to confirm the results. Four clones were finally determined to be significantly affected. These genes were mRNAs for plasma phospholipid transfer protein (PLTP), CL1BA protein, (latrophilin: LPH), and alphaII-spectrin as well as one unknown gene. We demonstrated the distribution of mRNA expression of one gene, LPH, in lung tissues. The present study suggests that 20mg per kg intraperitoneal PQ affects the expression of numerous genes in the lung at 3 h, the onset of pulmonary injury, and that the four genes specified may be major contributors to serious lung injury due to PQ exposure.
Abstract: The T-box gene family encodes a set of transcription factors that are involved in various developmental processes. We isolated tbx20 gene from chick embryos and examined in detail its expression patterns during heart development. In situ hybridization showed that tbx20 was expressed in the lateral plate mesoderm and subsequently in the primitive heart tube. At stages of looped heart, tbx20 was localized in the outflow tract (OT) and atrioventricular (AV) canal, in which valvuloseptal endocardial cushion develops. At later stages, although tbx20 was expressed predominantly in the nascent right ventricle, transcripts of tbx20 were down-regulated in the left ventricle. These results suggest that tbx20 may play important roles in a variety of developmental processes in cardiogenesis, such as chamber-specification and septation.
Abstract: Bone morphogenetic protein-3 and 3b (BMP-3 and BMP-3b) together represent a unique subgroup of the BMP family. BMP-3b shares 82% amino acid identity with BMP-3 in the mature region (ligand domain), but only 37% in the pro-region (pro-domain). In osteoblasts, BMP-3 and 3b have similar antagonistic activity against BMP-2, but they are differentially regulated. In developing embryos, BMP-3 and 3b have different dorsalizing activities. BMP-3b triggers secondary head formation in an autonomous manner, whereas BMP-3 induces aberrant tail formation. Loss-of-function analysis demonstrates that coordinated activity of xBMP-3b and cerberus, a head inducer, are required for head formation in Xenopus embryos. At the molecular level, BMP-3b antagonizes both nodal-like proteins (Xnr1 and derriere) and ventralizing BMPs (BMP-2 and ADMP), whereas BMP-3 only antagonizes ventralizing BMPs. Moreover, BMP-3b, but not BMP-3, associates with the monomeric form of Xnr1, a nodal-like protein. These molecular features of BMP-3 and 3b are due to their distinct pro-regions. These findings suggest that the processing of precursor regions and assembly of BMP-3 and 3b are important in various developmental processes and organogenesis.
Abstract: The limb musculature arises by delamination of premyogenic cells from the lateral dermomyotome. Initially the cells express Pax3 but, upon entering the limb bud, they switch on the expression of MyoD and Myf5 and undergo terminal differentiation into slow or fast fibres, which have distinct contractile properties that determine how a muscle will function. In the chick, the premyogenic cells express the Wnt antagonist Sfrp2, which is downregulated as the cells differentiate, suggesting that Wnts might regulate myogenic differentiation. Here, we have investigated the role of Wnt signalling during myogenic differentiation in the developing chick wing bud by gain- and loss-of-function studies in vitro and in vivo. We show that Wnt signalling changes the number of fast and/or slow fibres. For example, in vivo, Wnt11 decreases and increases the number of slow and fast fibres, respectively, whereas overexpression of Wnt5a or a dominant-negative Wnt11 protein have the opposite effect. The latter shows that endogenous Wnt11 signalling determines the number of fast and slow myocytes. The distinct effects of Wnt5a and Wnt11 are consistent with their different expression patterns, which correlate with the ultimate distribution of slow and fast fibres in the wing. Overexpression of activated calmodulin kinase II mimics the effect of Wnt5a, suggesting that it uses this pathway. Finally, we show that overexpression of the Wnt antagonist Sfrp2 and DeltaLef1 reduces the number of myocytes. In Sfrp2-infected limbs, the number of Pax3 expressing cells was increased, suggesting that Sfrp2 blocks myogenic differentiation. Therefore, Wnt signalling modulates both the number of terminally differentiated myogenic cells and the intricate slow/fast patterning of the limb musculature.
Abstract: Bone morphogenetic proteins (BMPs) and their antagonists are involved in the axial patterning of vertebrate embryos. We report that both BMP-3b and BMP-3 dorsalize Xenopus embryos, but act as dissimilar antagonists within the BMP family. BMP-3b injected into Xenopus embryos triggered secondary head formation in an autonomous manner, whereas BMP-3 induced aberrant tail formation. At the molecular level, BMP-3b antagonized nodal-like proteins and ventralizing BMPs, whereas BMP-3 antagonized only the latter. These differences are due to divergence of their pro-domains. Less BMP-3b than BMP-3 precursor is proteolytically processed in embryos. BMP-3b protein associated with a monomeric form of Xnrl, a nodal-like protein, whereas BMP-3 did not. These molecular features are consistent with their expression profiles during Xenopus development. XBMP-3b is expressed in the prechordal plate, while xBMP-3 is expressed in the notochord. Using antisense morpholino oligonucleotides, we found that the depletion of both xBMP-3b and cerberus, a head inducer, caused headless Xenopus embryos, whereas the depletion of both xBMP-3 and cerberus affected the size of the somite. These results revealed that xBMP-3b and cerberus are essential for head formation regulated by the Spemann organizer, and that xBMP-3b and perhaps xBMP-3 are involved in the axial patterning of Xenopus embryos.
Abstract: In normal New Zealand white rabbits, immunization with rabbit lung ACE (angiotensin converting enzyme) induced atherosclerotic retinal changes, and glomerular changes similar to those seen in diabetic nephropathy. Also, in genetically diabetogenic rats, immunization with the rabbit lung ACE induced diabetic nephropathy and retinopathy.
Abstract: In a random sample of 200 patients with type 2 diabetes mellitus, immunoreactivities to ACE (angiotensin converting enzyme) were measured by ELISA. Immunoreactivities were positive for 129 (64.5%) patients, and were positive in 30 (83.3%) out of 36 patients in the early stage of clinical diabetic nephropathy. Serum ACE activity in rabbits immunized with ACE decreased to 50% of the control level after 7 months (78.0 +/- 3.8 IU/L/37 degrees C, basal, 42.0 +/- 5.0 at 7 months and 33.3 +/- 3.5 IU/L/37 degrees C at 8 months, respectively). When rabbit serum containing antiACE antibodies was mixed, after heat-treatment at 56 degrees C for 30 min, with normal human serum, the ACE activity was reduced in a concentration-dependent manner. These results suggested that anti-ACE autoantibody may be present in patients with type 2 diabetes mellitus. However, the absence of data on the epitope for the antibody does not allow any conclusion except that the immunoreactivities to ACE are higher in type 2 diabetic patients than in non-diabetic individuals.
Abstract: In the developing limb bud, mesenchymal cells show position-specific affinity, suggesting that the positional identity of the cells is represented as their surface properties. Since the affinity is regulated by glycosylphosphatidylinositol (GPI)-anchored cell surface proteins, and by EphA4 receptor tyrosine kinase, we hypothesized that the GPI-anchored ligand, the ephrin-A family, also contributes to the affinity. Here, we describe the role of ephrin-A2 in the chick limb bud. Ephrin-A2 protein is uniformly distributed in the limb bud during early limb development. As the limb bud grows, expression of ephrin-A2 is strong in its proximal-to-intermediate regions, but weak distally. The position-dependent expression is maintained in vitro, and is regulated by FGF protein, which is produced in the apical ectodermal ridge. To investigate the role of ephrin-A2 in affinity and in cartilage morphogenesis of limb mesenchyme, we ectopically expressed ephrin-A2 in the limb bud using the retrovirus vector, RCAS. Overexpressed ephrin-A2 modulated the affinity of the mesenchymal cells that differentiate into autopod elements. It also caused malformation of the autopod skeleton and interfered with cartilage nodule formation in vitro without inhibiting chondrogenesis. These results suggest that ephrin-A2 regulates the position-specific affinity of limb mesenchyme and is involved in cartilage pattern formation in the limb.
Abstract: The Oka vaccine strain induces immunity against the varicellazoster virus (VZV) without clinical manifestations. However, the molecular basis of virulence in VZV is not known. VZV glycoproteins induce cellular and humoral immunity against VZV after infection. To investigate the molecular basis of viral attenuation and distinguish the Oka vaccine strain from the wild strains, we compared the DNA sequences of seven glycoproteins between the Oka vaccine and the parental Oka strains. The DNA sequences in gE, gB, gH, gI, gC, gL and gK of the Oka vaccine strain were completely consistent with those of the parental Oka strain. The Oka vaccine strain induced immunity level similar to the parental Oka strain. One-point mutation was detected at the stop-codon of gC in the Oka vaccine strain. This could be useful for distinguishing the Oka vaccine strain from the parental Oka strain by DNA sequence analysis.
Abstract: Myostatin, which is a member of the TGF-beta superfamily, is a negative regulator of skeletal muscle formation. Double-muscled Piedmontese cattle have a C313Y mutation in myostatin and show increased skeletal muscle mass which resulted from an increase of myofiber number (hyperplasia) without that of myofiber size (hypertrophy). To examine whether this mutation in myostatin gene affects muscle development in a dominant negative manner, we generated transgenic mice overexpressing the mutated gene. The transgenic mice exhibited dramatic increases in the skeletal muscle mass resulting from hyperplasia without hypertrophy. In contrast, it has been reported that a myostatin mutated at its cleavage site produces hypertrophy without hyperplasia in the muscle. Thus, these results suggest that (1) the myostatin containing the missense mutation exhibits a dominant negative activity and that (2) there are two types in the dominant negative form of myostatin, causing either hypertrophy or hyperplasia.
Abstract: We obtained a drug-hypersensitive PC12 mutant cell (PC12m3), in which neurite outgrowth was strongly stimulated by various drugs such as FK506, calcimycin and cAMP, under the condition of NGF treatment. The frequency of neurite outgrowth stimulated by FK506 was approximately 40 times greater than by NGF alone. The effects of FK506 on neurite outgrowth in PC12m3 cells were inhibited by rapamycin, an FK506 antagonist, and by calcimycin, a calcium ionophore. PC12m3 cells had a strong NGF-induced MAP kinase activity, the same as PC12 parental cells. However, FK506-induced MAP kinase activity was detected only in PC12m3 cells. The activation of MAP kinase by FK506 in PC12m3 cells was markedly inhibited by rapamicin and calcimycin. FK506-induced MAP kinase activity was also inhibited by MAP kinase inhibitor U0126. These results demonstrate that drug-hypersensitive PC12m3 cells have a novel FK506-induced MAP kinase pathway for neuritogenesis.
Abstract: The development of avian cutaneous appendages, feathers and scales, is known to arise from the epithelial-mesenchymal interaction. Here we show that FGF10 is associated with this developmental process as an early signal from mesenchymal cells underlying nascent cutaneous placodes. Expression of Fgf10 was detected in the mesenchymal cells underneath the developing placodes. Forced expression of Fgf10 in the femoral skin suppressed expression of Shh and a zinc finger gene snail-related (cSnR), while induced expression of Bmp2 in the interbud region, resulting in thickening of the epidermal layer. Furthermore, forced expression of Fgf10 in the foot skin caused marked ingrowings of the epidermis. The cells in the epidermal ingrowings expressed beta-catenin, proliferating cell nuclear antigen, and an epidermal stem cell marker p63. These results support the idea that FGF10 is a mesenchymally derived stimulator of epidermal development through crosstalk with bone morphogenetic protein (BMP), beta-catenin, and other signaling pathways.
Abstract: We examined the complex short tandem repeat (STR) locus at the 3'-flanking region of the neurotensin receptor (NTR) gene. The polymorphism of this locus was first reported as a simple tetranucleotide repeat variation by Le et al., but it also offers a surprisingly informative variation, that permits reliable individual identification by two complementary strategies: fluorescent-labelled polymerase chain reaction (PCR)/electrophoresis and direct sequencing of the PCR products. We determined the alleles in 203 Japanese by fluorescent-labelled PCR/electrophoresis. Determination was based on their length with a reliability of +/-1 bp, and the frequency of each allele was very low. Sequencing analysis further grouped these alleles in detail. Sequencing demonstrated that the locus varied by six repetitive units and three insertion/deletion positions of nucleotide fragments. We detected multiple alleles having different structures even in the same allele length. We found structural differences in homozygous alleles having the same base pair size. We also determined that apparently homozygous alleles were heterozygous from sequencing electropherograms showing an overlap of nucleotides or +/-1 bp difference. These results indicate that this locus is structurally hypervariable in addition to having allelic length variations, promising a great advance in individual identification in forensic practice.
Abstract: During continuous culture of neural PC12 cells, we obtained a drug-hypersensitive PC12 mutant cell that showed high stimulation of neurite outgrowth by various drugs. When several Chinese medicines such as shu-jing-huo-xie-tang and Wu-Ling-San were provided to these PC12 mutant cells, the frequency of nerve growth factor (NGF)-induced neurite outgrowth increased approximately 30-fold compared to NGF alone. Neurite outgrowth induced by NGF in PC12 cells is accompanied by sustained activation of mitogen-activated protein kinase (MAPK); however, these Chinese medicines did not induce MAPK activity. The findings thus indicate that certain Chinese medicines may induce neurite outgrowth by a novel mechanism which is distinct from the NGF-activated pathway in PC12 mutant cells.
Abstract: The Wnt antagonist Frzb-1 is expressed during limb skeletogenesis, but its roles in this complex multistep process are not fully understood. To address this issue, we determined Frzb-1 gene expression patterns during chick long bone development and carried out gain- and loss-of-function studies by misexpression of Frzb-1, Wnt-8 (a known Frzb-1 target), or different forms of the intracellular Wnt mediator LEF-1 in developing limbs and cultured chondrocytes. Frzb-1 expression was quite strong in mesenchymal prechondrogenic condensations and then characterized epiphyseal articular chondrocytes and prehypertrophic chondrocytes in growth plates. Virally driven Frzb-1 misexpression caused shortening of skeletal elements, joint fusion, and delayed chondrocyte maturation, with consequent inhibition of matrix mineralization, metalloprotease expression, and marrow/bone formation. In good agreement, misexpression of Frzb-1 or a dominant-negative form of LEF-1 in cultured chondrocytes maintained the cells at an immature stage. Instead, misexpression of Wnt-8 or a constitutively active LEF-1 strongly promoted chondrocyte maturation, hypertrophy, and calcification. Immunostaining revealed that the distribution of endogenous Wnt mediator beta-catenin changes dramatically in vivo and in vitro, from largely cytoplasmic in immature proliferating and prehypertrophic chondrocytes to nuclear in hypertrophic mineralizing chondrocytes. Misexpression of Frzb-1 prevented beta-catenin nuclear relocalization in chondrocytes in vivo or in vitro. The data demonstrate that Frzb-1 exerts a strong influence on limb skeletogenesis and is a powerful and direct modulator of chondrocyte maturation, phenotype, and function. Phases of skeletogenesis, such as terminal chondrocyte maturation and joint formation, appear to be particularly dependent on Wnt signaling and thus very sensitive to Frzb-1 antagonistic action.
Abstract: The Wnt family of growth factors are important regulators of several developmental processes including skeletogenesis. To further investigate the role of Wnts we analysed their expression in the developing chick limb and performed functional analyses in vivo and in vitro. We found that Wnt5b and Wnt11 are restricted within the prehypertrophic chondrocytes of the cartilage elements, Wnt5a is found in the joints and perichondrium, while Wnt4 is expressed in the developing joints and, in some bones, a subset of the hypertrophic chondrocytes. These Wnts mediate distinct effects on the initiation of chondrogenesis and differentiation of chondrocytes in vitro and in vivo. Wnt4 blocks the initiation of chondrogenesis and accelerates terminal chondrocyte differentiation in vitro. In contrast, Wnt5a and Wnt5b promote early chondrogenesis in vitro while inhibiting terminal differentiation in vivo. As Wnt5b and Wnt11 expression overlaps with and appears after Indian hedgehog (Ihh), we also compared their effects with Ihh to see if they mediate aspects of Ihh signalling. This showed that Ihh and Wnt5b and Wnt11 control chondrogenesis in parallel pathways.
Abstract: Fibroblast growth factor 10 (FGF10) is known to be expressed in limb mesenchymal cells and to function as a mesenchymal signaling factor involved in epithelial-mesenchymal interactions during limb development. To elucidate regulation of Fgf10 expression, we isolated the promoter region of Fgf10 containing its 2.0 kb upstream 5'-fragment from the initiation codon and its 0.9 kb downstream fragment. Transcriptional activity of the fragment was examined with transgenic mice, using a lacZ-reporter system. Although no significant expression of the reporter gene was observed for the 0.2 kb 5'-fragment, expression was detected in the apical ectodermal ridge of the limb bud and developing cartilage of the limb for the 2.0 kb and 0.7 kb 5'-fragments, respectively. From comparison of the mouse sequences of the 2.0 kb fragment with corresponding sequences of human and chicken Fgf10, we identified 17 conserved putative enhancer motifs for AER expression and other unidentified expressions. For limb cartilage expression, we found putative enhancer sequences conserved among the three species in the 0.7 kb 5'-fragment. In the fragment, three DNA binding motifs were identified in the mouse and human sequence, although they are not conserved in the corresponding chicken sequence.
Abstract: Increased formation of reactive oxygen species is a cause of paraquat (PQ)-induced injury and also provides a link between the signaling pathways and transcriptional events that regulate the expression of a large number of genes. However, the molecular mechanisms involved in PQ-induced injury remain unclear. To investigate the changes in gene expression at the onset of PQ injury, we used the differential display-polymerase chain reaction (PCR) method. Rats were treated intraperitoneally with 20 mg/kg PQ, and after 3 h the lungs were immediately excised. Samples of mRNA from normal and treated rats were used to prepare radiolabeled cDNAs, which were electrophoresed. Then the transcription levels were compared. We isolated 26 fragments of cDNA that were potentially affected by PQ, and determined their nucleotide sequences. Six clones of interest were selected and analyzed further. The reverse transcript-PCR based on their sequence information confirmed the differential expression for five clones: four clones were up-regulated and one was down-regulated. We were particularly interested in two genes that had homology with the known gene: TATA box-binding protein-associated factor, RNA polymerase II, B, 150 kDa (TAFIIB), and a candidate gene for lipodystrophy, Lpin2. Both genes were significantly up-regulated within 3 h of PQ intake and the stimulation continued during our 24-h observation period. In addition, up-regulation of Lpin2 was observed in the lungs, but not in the liver and kidneys. In situ hybridization using lung sections showed that the expression of both genes was strongly visualized in Clara cells and in alveolar macrophages. These findings suggest a stimulation of transcription levels and changes in lipid metabolism in Clara cells and in macrophages in the lungs, which result in their playing a crucial role at the onset of PQ-driven pulmonary injury.
Abstract: We have isolated a chick cDNA for p63, a member of the p53 transcription factor family. This cDNA encodes a protein of 582 amino acids for an alpha isoform in the C-terminal region, while lacking the N-terminal transactivation domain. The chick p63 gene is first expressed in the prospective cutaneous ectoderm at stage 6 and later in the developing epithelia. The p63 expression is intense in specialized epithelial structures, such as apical ectodermal ridge of the limb bud, epithelia of branchial arches and feather buds. Furthermore, we have found that the transcripts are detected in the interdigital epithelium, intersomite epithelium, and epaxial dermamyotome.
Abstract: The bone morphogenetic protein (BMP) family, comprising multifunctional peptide growth factors, regulates many developmental processes in a variety of tissues. We examined the spatiotemporal expression of BMP5 by in situ hybridization in chick embryonic hearts from stages 5 to 33. The BMP5 gene was first expressed in the endoderm underlying the precardiac mesoderm at stages 5 to 8. Thereafter, BMP5 expression was restricted to the myocardium of the atrioventricular (AV) canal and outflow tract (OT) regions, where the valvuloseptal endocardial cushion tissue is induced. These results suggest that BMP5 may play important roles not only in myocardial differentiation, but also in the formation and maintenance of endocardial cushion tissue.
Abstract: The interactions of Sonic hedgehog (Shh) and fibroblast growth factor (FGF) play important roles in vertebrate limb pattern formation. In the posterior region of the chick limb bud, Shh and FGF-4 each maintain expression in a positive feedback loop. In the anterior region, Shh can also induce Fgf-4 expression in the anterior apical ectodermal ridge. However, the possibility of Shh induction by FGF protein is unclear. Because many experiments to analyze gene expression have been carried out by using the forelimb bud of the chick embryo, we investigated gene expression of the cells in the anterior region of the chick hindlimb bud after FGF-4 application and compared the results with those for the forelimb bud. When an FGF-4-containing bead was implanted into the anterior region of a stage 20 hindlimb bud, ectopic expression of Shh was induced in the mesenchyme beneath the anterior end of the apical ectodermal ridge at 36 hr after implantation. Subsequent to Shh activation, Hoxd13 was also observed in the anterior-distal region of the limb bud. Furthermore, FGF-4 implantation to the hindlimb bud caused additional digit formation accompanying respecification of positional value in the anterior tissue. Ectopic Shh was induced in cells located distal to the FGF-4 bead, and the cells of the flank region did not contribute to ectopic Shh induction. On the other hand, no ectopic Shh and Hoxd13 expression was detected by grafting an FGF-4 bead into the forelimb bud. Although FGF-4 implantation to the forelimb bud occasionally induced extra digit 2 formation, no embryos had an extra digit 3 or digit 4, and many specimens exhibited normal skeletal pattern. These results demonstrate the difference between the fore- and hindlimb buds in the cell competence of Shh induction in response to FGF-4, suggesting the possibility that the responsiveness of mesenchymal cells in signaling molecules is not the same in the fore- and hindlimb buds.
Abstract: During continuous culturing, PC12 cells are subject to spontaneous mutations. We obtained PC12m3 cells, clone cells in which outgrowth of neuronal processes (dendrites and axons) under the condition of nerve growth factor (NGF) treatment was highly stimulated by various inducers, such as cyclic adenosine monophosphate (cAMP), calcium ionophore, steroid and high osmolarity. The number of cells with neuronal processes in the presence of cAMP was approximately twenty-fold greater than PC12 parental cells and other PC12 mutant cells. In PC12m3 cells, NGF-induced outgrowth of neuronal processes was reduced by cytotoxic solanine, whereas the effect of NGF was unaffected by hyaluronic acid. In PC12m3 cells, various inducers of neurite outgrowth, such as cAMP, calcium ionophore and high osmolarity, activated mitogen activated protein (MAP) kinase, whereas solanine and hyaluronic acid did not cause any significant activation of MAP kinase. However, PC12m3 cells, in which NGF-induced outgrowth of neuronal processes were impaired, had strong NGF-induced MAP kinase activity as PC12 parental cells had. These findings suggest that cAMP, calcium influx and high osmolarity induce outgrowth of neuronal processes in PC12m3 cells through activation of the downstream target of MAP kinase or through a novel pathway independent of NGF activation.
Abstract: Paraquat (PQ) is a well-known pneumotoxicant that exerts its toxic effect by elevating intracellular levels of superoxide. In addition, production of pro-inflammatory cytokines has possibly been linked to PQ-induced inflammatory processes through reactive oxygen species (ROSs) and nitric oxide (NO). However, the role of NO in PQ-induced cell injury has been controversial. To explore this problem, we examined the effect of NO on A549 cells by exposing them to the exogenous NO donor NOC18 or to cytokines; tumor necrosis factor-alpha, interleukin-1 beta and interferon-gamma, as well as PQ. Although the exogenous NO donor on its own had no effect on the release of lactate dehydrogenase (LDH), remarkable release was observed when the cells were exposed to high concentrations of NOC18 and PQ. This cellular damage caused by 1 mM NOC18 plus 0.2 mM PQ was ascertained by phase contrast microscopy. On the other hand, NO derived from 25-50 microM NOC18 added into the medium improved the MTT reduction activity of mitochondria, suggesting a beneficial effect of NO on the cells. Incubation of A549 cells with cytokines increased in inducible NO synthase (iNOS) expression and nitrite accumulation, resulting in LDH release. PQ further potentiated this release. The increase in nitrite levels could be completely prevented by NOS inhibitors, while the leakage of LDH was not attenuated by the inhibition of NO production with them. On the other hand, ROS scavenging enzymes, superoxide dismutase and catalase, inhibited the leakage of LDH, whereas they had no effect on the increase in the nitrite level. These results indicate that superoxide, not NO, played a key role in the cellular damage caused by PQ/cytokines. Our in vitro models demonstrate that NO has both beneficial and deleterious actions, depending on the concentrations produced and model system used.
Abstract: Sonic hedgehog (Shh) and Indian hedgehog (Ihh) are important regulators of skeletogenesis, but their roles in this complex multistep process are not fully understood. Recent studies have suggested that the proteins participate in the differentiation of chondrogenic precursor cells into chondrocytes. In the present study, we have tested this possibility more directly. We found that implantation of dermal fibroblasts expressing hedgehog proteins into nude mice induces ectopic cartilage and bone formation. Immunohistological and reverse-transcription polymerase chain reaction (RT-PCR) analyses revealed that the ectopic tissues derived largely if not exclusively from host cells. We found also that treatment of clonal prechondrogenic RMD-1 and ATDC5 cells in culture with Ihh or recombinant amino half of Shh (recombinant N-terminal portion of Shh [rShh-N]) induced their differentiation into chondrocytes, as revealed by cytoarchitectural changes, Alcian blue staining and proteoglycan synthesis. Induction of RMD-1 cell differentiation by Ihh or rShh-N was synergistically enhanced by cotreatment with bone morphogenetic protein 2 (BMP-2) but was blocked by cotreatment with fibroblast growth factor 2 (FGF-2). Our findings indicate that hedgehog proteins have the ability to promote differentiation of chondrogenic precursor cells and that their action in this process can be influenced and modified by synergistic or antagonist cofactors.
Abstract: The embryonic gut of vertebrates consists of endodermal epithelium, surrounding mesenchyme derived from splanchnic mesoderm and enteric neuronal components derived from neural crest cells. During gut organogenesis, the mesenchyme differentiates into distinct concentric layers around the endodermal epithelium forming the lamina propria, muscularis mucosae, submucosa and lamina muscularis (the smooth muscle layer). The smooth muscle layer and enteric plexus are formed at the outermost part of the gut, always some distance away from the epithelium. How this topographical organization of gut mesenchyme is established is largely unknown. Here we show the following: (1) Endodermal epithelium inhibits differentiation of smooth muscle and enteric neurons in adjacent mesenchyme. (2) Endodermal epithelium activates expression of patched and BMP4 in adjacent non-smooth muscle mesenchyme, which later differentiates into the lamina propria and submucosa. (3) Sonic hedgehog (Shh) is expressed in endodermal epithelium and disruption of Shh-signaling by cyclopamine induces differentiation of smooth muscle and a large number of neurons even in the area adjacent to epithelium. (4) Shh can mimic the effect of endodermal epithelium on the concentric stratification of the gut. Taken together, these data suggest that endoderm-derived Shh is responsible for the patterning across the radial axis of the gut through induction of inner components and inhibition of outer components, such as smooth muscle and enteric neurons.
Abstract: During attempts to transform a normal human fibroblast strain (GM730) by X-irradiation, we obtained a partially transformed cell strain (GM730pt) which demonstrates several aspects of the transformed phenotype including morphological changes, increased saturation density, growth in soft agar, and focus formation in long-term cultures. When GM730pt cells were transfected with the feline c-myc gene, morphology of the cells changed dramatically following seven days of expression. Transfection of other plasmid DNAs or oncogenes such as pUC8, pSV2neo, src, sis, and H-ras had little or no effects on the phenotype of GM730pt cells. On the other hand, a gel purified, small fragment of c-myc DNA had a complete cell alteration activity. Furthermore, Bal 31 deletion and M13 sequencing experiments showed that the alteration seen in GM730pt cells is delimited to a 24 nucleotide stretch (active myc element) from the second intron of the feline c-myc gene that contains a T-rich sequence.
Abstract: The dorsal ectoderm of the limb bud is known to regulate anterior-posterior patterning as well as dorsal-ventral patterning during vertebrate limb morphogenesis. Wnt-7a, expressed in the dorsal ectoderm, encodes a key molecule implicated in these events. In the present study, chicken frizzled-10 (Fz-10) encoding a Wnt receptor was used to study mechanisms of Wnt-7a signaling during chick limb patterning, because its expression is restricted to the posterior-distal region of the dorsal limb bud. Fz-10 transcripts colocalize with Sonic hedgehog (Shh) in the dorsal side of stages 18-23 chick limb buds. It was demonstrated that Fz-10 interacts with Wnt-7a to induce synergistically the expression of Wnt-responsive genes, such as Siamois and Xnr3, in Xenopus animal cap assays. In the chick limb bud, Fz-10 expression is regulated by Shh and a signal from the dorsal ectoderm, presumably Wnt-7a, but not by signals from the apical ectodermal ridge. These results suggest that Fz-10 acts as a receptor for Wnt-7a and has a positive effect on Shh expression in the chick limb bud.
Abstract: We have identified chick frizzled (Fz)-10, encoding a Wnt receptor, and examined the expression pattern during embryogenesis. Fz-10 is expressed in the region posterior to the Hensen's node at stage 6. Fz-10 expression is detected in the dorsal domain of the neural tube and the central nervous system of the developing embryo. In the developing limb, Fz-10 expression starts at stage 18 in the posterior-dorsal region of the distal mesenchyme, and gradually expands to the anterior-distal region. Fz-10 is also expressed in the feather bud and branchial arch. Implantation of Sonic hedgehog (Shh)-expressing cells into the anterior margin of the limb bud resulted in the induction of Fz-10 expression in anterior-dorsal mesenchyme.
Abstract: Members of the frizzled (Fz) family are involved in Wnt signaling during embryogenesis in the vertebrate. We identified chicken cognates of Fz-2, Fz-3, Fz-4, Fz-6 and Fz-8, and examined spatial and temporal expression patterns in the chick embryos by whole-mount in situ hybridization. Fz-4 is intensely expressed in the apical ectodermal ridge and distal mesenchyme of the limb bud at stages 20 to 27. The transcripts are confined to the posterior-distal end of the digit-forming region at stages 25 to 27. Fz-2 is weakly expressed in the proximal limb mesenchyme at stages 25 to 27, while Fz-3 and Fz-6 expressions are uniform in the limb bud at these stages. Fz-2 is also expressed in the dermatomyotome. No expression signal for Fz-8 is detectable in the embryo at stages 20 to 27. The differential expression patterns of the Fz family, together with spatially restricted expression of the Wnt family members in the developing limb, suggest distinct but overlapping functions to transduce Wnt signal implicated in cellular interaction during embryogenesis.
Abstract: Members of the Wnt family are known to play diverse roles in the organogenesis of vertebrates. The full-coding sequences of chicken Wnt-5a were identified and the role it plays in limb development was examined by comparing its expression pattern with that of two other Wnt members, Wnt-4 and Wnt-11, and by misexpressing it with a retrovirus vector in the limb bud. Wnt-5a expression is detected in the limb-forming region at stage 14, and in the apical ectodermal ridge and distal mesenchyme of the limb bud. The signal was graded along the proximal-distal axis at stages 20-28 and also along the anterior-posterior axis during early stages. It disappeared in the cartilage-forming region after stage 26, and was restricted to the region surrounding the phalanges at stage 34. Wnt-4 and Wnt-11, other members of the Wnt-5a-subclass, were expressed with a distinct spatiotemporal pattern during the later phase. Wnt-4 was expressed in the articular structure and Wnt-11 was expressed in the dorsal and ventral mesenchyme adjacent to the ectoderm. Wnt-5a expression was partially reduced after apical ectodermal ridge removal, whereas Wnt-11 expression was down-regulated by dorsal ectoderm removal. Therefore, expression of these Wnt was differentially regulated by the ectodermal signal. Misexpression of Wnt-5a in the limb bud with the retrovirus resulted in truncation of long bones predominantly in the zeugopod because of retarded chondrogenic differentiation. Distal elements, such as the phalanges and metacarpals, were not significantly reduced in size. These results suggest that Wnt-5a is involved in pattern formation along the proximal-distal axis by regulation of chondrogenic differentiation.
Abstract: Basic fibroblast growth factor (FGF2) is generally known to induce proliferation of cultured mesangial cells and is expressed in proliferative mesangial cells in anti-Thy1.1 mesangial proliferative glomerulonephritis (anti-Thy1.1 GN). The distribution of the FGF receptor (FGFR) has not been studied in anti-Thy1.1 GN, so we used in situ hybridization to determine whether cells expressing FGFR1-4 mRNAs could be detected. In normal rats, all glomeruli were negative for FGFR1-4 mRNA, but those of the mesangial proliferative phase expressed FGFR1-4 mRNA in proliferative mesangial cells. Proliferation of mesangial cells has not been observed in normal rats injected with FGF2( )but it has been noted in anti-Thy1.1 rats injected with FGF2. These data and our results demonstrate that mesangial cells produce and release FGF2( )after injury and that during the proliferative phase these cells upregulate FGFR in vivo. This study is the first to demonstrate expression of FGFR1-4 mRNAs in pathological glomeruli of anti-Thy1.1 GN. The FGF2 and FGFR1-4 genes were expressed in the proliferative mesangial cells. Upregulation of FGFR is necessary for mesangial proliferation by FGF2.
Abstract: In developing limb bud, mesenchymal cells form cellular aggregates called "mesenchymal condensations". These condensations show the prepattern of skeletal elements of the limb prior to cartilage differentiation. Roles of various signaling molecules in chondrogenesis in the limb bud have been reported. One group of signaling factors includes the Wnt proteins, which have been shown to have an inhibitory effect on chondrogenesis in the limb bud. Therefore, regulation of Wnt activity may be important in regulating cartilage differentiation. Here we show that Frzb-1, which encodes a secreted frizzled-related protein that can bind to Wnt proteins and can antagonize the activity of some Wnts, is expressed in the developing limb bud. At early stages of limb development, Frzb-1 is expressed in the ventral core mesenchyme of the limb bud, and later Frzb-1 expression becomes restricted to the central core region where mesenchymal condensations occur. At these stages, a chondrogenic marker gene, aggrecan, is not yet expressed. As limb development proceeds, expression of Frzb-1 is detected in cartilage primordial cells, although ultimately Frzb-1 expression is down-regulated. Similar results were obtained in the recombinant limb bud, which was constructed from dissociated and re-aggregated mesenchymal cells and an ectodermal jacket with the apical ectodermal ridge. In addition, Frzb-1 expression preceded aggrecan expression in micromass cultures. These results suggest that Frzb-1 has a role in condensation formation and cartilage differentiation by regulating Wnt activity in the limb bud.
Abstract: HoxD expression and cartilage pattern formation were compared after application of a recombinant amino-terminal peptide of Sonic hedgehog protein (Shh-N) and implantation of cells expressing the Sonic hedgehog (Shh) gene. During digit duplication after implantation of a Shh-N-soaked bead, BMP-2 and Patched expression was transiently induced in the anterior limb mesenchyme 20 h after grafting, but was reduced to the basal level 48 h after grafting. On the contrary, when Shh-expressing cells were grafted to the anterior limb bud, expression domains of the BMP-2 and Patched genes were initially induced in the restricted region in close proximity to the grafted cells. Induced expression of BMP-2 and Patched was maintained in the anterior-peripheral region of the limb bud for 42 h after grafting. In either case, HoxD12 and HoxD13 were consistently induced in the anterior-distal limb mesenchyme, accompanying mirror-image duplication of the digit pattern. Induction and maintenance of HoxD expression were consistent with the resultant digit pattern. A steep gradient of Shh activity provided by Shh-expressing cells is most adequate to induce complete digit pattern, as compared to the shallow gradient provided by Shh-N protein released from a bead. These results suggest that positional identity is respecified by Shh-N activity within the first 24 h during digit duplication, and that Shh-N on its own is not acting as a long-range signaling molecule to determine positional identity at a distance in the limb bud.
Abstract: Sonic hedgehog (Shh) gene encodes a secreted protein that acts as an important mediator of cell-cell interactions. A detailed analysis of Shh expression in the digestive organs of the chicken embryo was carried out. Shh expression in the endoderm begins at stage 7, when the formation of the foregut commences, and is found as narrow bands in the midgut. Shh expression around the anterior intestinal portal at stage 15 is restricted to the columnar endoderm lined by the thick splanchnic mesoderm, suggesting that the existence of thick splanchnic mesoderm might be necessary for Shh expression in the columnar endoderm. After the gut is closed, Shh expression is found universally in digestive epithelia, including the cecal epithelium. However, its expression ceases in the epithelium of the proventricular glands, the ductus choledochus and ductus pancreaticus that protrude from the main digestive duct. When the gizzard epithelium differentiated into glands under the influence of the proventricular mesenchyme, the glandular epithelium lost the ability to express Shh. These findings suggest that Shh expression in the epithelium may be regulated by surrounding mesenchyme throughout organogenesis of the digestive organs and is closely involved in epithelial-mesenchymal interactions in developing digestive organs.
Abstract: Vertebrate limb is used as a model system to understand the mechanism of pattern formation in development. Mesenchymal cells of the limb bud are differentiated into chondrogenic cells or fibroblastic cells. The chondrogenic cells form bifurcated and segmented cartilage structure. This cartilage pattern is regulated by many signaling molecules and transcriptional factors. In the early stage of cartilage differentiation, mesenchymal cells aggregate into suitable region in the limb bud, and the aggregates form prepattern of skeletal elements. Cell adhesion molecules have been shown of their involvement in this cell aggregate formation and the cartilage differentiation process. Expression of these cell adhesion molecules and other cell surface molecules may be regulated by signaling molecules or transcriptional factors, although no regional specificities of these molecules have been reported. In this review, we describe about regional differences of cell affinity of limb bud mesenchyme. We show the differential cell affinity represents the positional identity of the mesenchyme in limb bud, and glycosylphosphatidylinositol (GPI) anchored cell surface proteins are involved in this different cell affinity. From these results, we discuss the importance of the cell affinity in pattern formation of limb bud.
Abstract: Soluble signaling factors are involved in morphogenetic events during vertebrate limb development. They belong to the Hedgehog family, the bone morphogenetic protein (BMP) family, the fibroblast growth factor (FGF) family and the Wnt family. FGF-8 and FGF-10 play central roles to specify the limb field and promote initial outgrowth. In the established limb bud, FGF-4, FGF-8 and BMP-2 are secreted in the apical ectodermal ridge and control proximal-distal pattern formation. In the zone of polarizing activity Sonic hedgehog is produced and pattern along the anterior-posterior axis. Members of the BMP family may be the secondary signals in this patterning. Wnt-7a from the dorsal ectoderm dorsalizes limb mesenchyme and controls dorsal-ventral patterning. These factors expressed in the signaling centers in limb buds influence gene expression each other and coordinate limb morphogenesis.
Abstract: Although regional differences in mesenchymal cell affinity in the limb bud represent positional identity, the molecular basis for cell affinity is poorly understood. We found that treatment of the cell surface with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) could change cell affinity in culture. When PI-PLC was added to the culture medium, segregation of the progress zone (PZ) cells from different stage limb buds was inhibited. Similarly, sorting out of the cells from different positions along the proximodistal (PD) axis of the same stage limb buds was disturbed. Since PI-PLC can remove glycosylphosphatidylinositol (GPI)-anchored membrane bound proteins from the cell surface, the GPI-anchored cell surface proteins may be involved in sorting out. To define the GPI-anchored molecules that determine the segregation of limb mesenchymal cells, we examined the effect of neutralizing antibody on the EphA4 receptor that binds to GPI-anchored cell surface ligands, called ephrin-A. Sorting out of the PZ cells at different stages could be inhibited by the neutralizing antibody to EphA4. These results suggest that EphA4 and its GPI-anchored ligands are, at least in part, involved in sorting out of limb mesenchymal cells with different proximal-distal positional values, and that GPI-anchored cell surface proteins play important roles in determining cell affinity in the limb bud.
Abstract: Homeobox genes are expressed both temporally and spatially during vertebrate development, and regulate the tissue-specific expression of other genes. A Xenopus paired-related homeobox- 1 (Xprx-1) cDNA was cloned. Xprx-1 had a paired-related homeodomain, but did not contain a paired-box. The sequence of Xprx-1 had a high level of homology with K-2(mouse) and Prx-1 (chicken), thus Xprx-1 is assumed to be the Xenopus homolog of these genes. Xprx-1 transcripts were maternally restricted, in Xenopus embryos, and a decrease in the late blastula stage was followed by an increase in zygotic transcripts after gastrulation. The transcripts were localized to the animal hemisphere of the late blastula and were concentrated in the branchial arches of the tail-bud stage embryo. In animal cap experiments, Activin A dose-dependently induced Xprx-1 gene expression. These results suggest that Xprx-1 plays a role in early Xenopus development similar to other species.
Abstract: To examine the role of bone morphogenetic protein (BMP) signaling in chondrocytes during endochondral ossification, the dominant negative (DN) forms of BMP receptors were introduced into immature and mature chondrocytes isolated from lower and upper portions of chick embryo sternum, respectively. We found that control sternal chondrocyte populations expressed type IA, IB, and II BMP receptors as well as BMP-4 and -7. Expression of a DN-type II BMP receptor (termed DN-BMPR-II) in immature lower sternal (LS) chondrocytes led to a loss of differentiated functions; compared with control cells, the DN-BMPR- II-expressing LS chondrocytes proliferated more rapidly, acquired a fibroblastic morphology, showed little expression of type II collagen and aggrecan genes, and upregulated type I collagen gene expression. Expression of DN-BMPR-II in mature hypertrophic upper sternal (US) chondrocytes caused similar effects. In addition, the DN-BMPR-II-expressing US cells exhibited little alkaline phosphatase activity and type X collagen gene expression, while the control US cells produced both alkaline phosphatase and type X collagen. Both DN-BMPR-II-expressing US and LS chondrocytes failed to respond to treatment with BMP-2 . When we examined the effects of DN forms of types IA and IB BMP receptors, we found that DN-BMPR-IA had little effect, while DN-BMPR-IB had similar but weaker effects compared with those of DN-BMPR-II. We conclude that BMP signaling, particularly that mediated by the type II BMP receptor, is required for maintenance of the differentiated phenotype, control of cell proliferation, and expression of hypertrophic phenotype.
Abstract: In the early chick embryo, an apical ectodermal ridge (AER) is formed from the overlying ectoderm of the presumptive limb bud region at the dorsal-ventral (DV) boundary. We report here that the ectopic DV boundary formed in the presumptive wing, flank, and leg fields induces an ectopic AER structure. Dorsal tissue (ectoderm and mesoderm) from the presumptive wing field of stage 10 to 17 embryos was inserted into a slit in the somatopleure of the future ventral side of host embryos. The same method was used to implant ventral tissue into the future dorsal side of host embryos. After the implantation, ectopic AER was induced and an additional limb or limb-like structure developed. In related experiments, ectoderm-free presumptive wing tissue was implanted, which resulted in a considerably decreased frequency of ectopic AER formation. Further analysis of chick and quail chimeras suggests that the ectopic AER was formed from the ectodermal cells overlying the boundary of host and graft mesodermal cells. These results indicate that the DV boundary organizes the AER structure in the limb bud field of early-stage chick embryos and that the ectoderm of the grafted tissues plays an important role in this process.
Abstract: We have isolated chicken JNK2-alpha1 encoding a c-Jun N-terminal kinase, and examined the expression during embryogenesis. The kinase domain sequence is well conserved between chicken and mammals, but carboxy-terminal sequence of JNK is divergent from subtype 1 and 2, possibly derived from alternative splicing. The JNK2-alpha1 gene is preferentially expressed in the neuroepithelium of developing brain at stages 16-26, and transcripts are not detectable in the other region including spinal cord. These results suggest that JNK2-alpha1 is involved in development of the central nervous system as a mediator of stress-activated protein kinase pathway conferring competence to the external stimuli such as growth factors.
Abstract: Vertebrate limb formation has been known to be initiated by a factor(s) secreted from the lateral plate mesoderm. In this report, we provide evidence that a member of the fibroblast growth factor (FGF) family, FGF10, emanates from the prospective limb mesoderm to serve as an endogenous initiator for limb bud formation. Fgf10 expression in the prospective limb mesenchyme precedes Fgf8 expression in the nascent apical ectoderm. Ectopic application of FGF10 to the chick embryonic flank can induce Fgf8 expression in the adjacent ectoderm, resulting in the formation of an additional complete limb. Expression of Fgf10 persists in the mesenchyme of the established limb bud and appears to interact with Fgf8 in the apical ectoderm and Sonic hedgehog in the zone of polarizing activity. These results suggest that FGF10 is a key mesenchymal factor involved in the initial budding as well as the continuous outgrowth of vertebrate limbs.
Abstract: We have isolated the chicken homeobox genes LH-2A and LH-2B encoding two related LIM domain-containing homeodomain proteins and examined the expression pattern during chick limb development. LH-2A is most closely related to human and rat LH-2, while LH-2B is less conserved. Although both LH-2A and LH-2B are expressed in the limb mesenchyme throughout stage 16 to stage 32, LH-2A transcripts are detectable in the distal limb bud and LH-2B transcripts are detectable in the anterior limb bud. Signals from the apical ectodermal ridge positively regulate LH-2A expression, since removal of the apical ectoderm resulted in the rapid reduction of LH-2A expression in the distal limb mesenchyme. Ectopic expression of the sonic hedgehog gene in the anterior margin of the limb bud resulted in the rapid reduction of LH-2B expression accompanying respecification of the positional value to the posterior phenotype. These results suggest that LH-2A and LH-2B play important roles in the determination and specification of the proximal-distal and anterior-posterior positional values, respectively.
Abstract: In the present study, two series of experiments were done with PAN nephropathy rats given fibroblast growth factor 2 (FGF2) or FGF2 neutralizing antibodies. In the first series of experiments, a dose of 10 micrograms of FGF2 (FGF2 group), 40 micrograms of an FGF2 neutralizing antibody (Anti-FGF2 group) or an equal volume of physiological saline (Control group) was administered for four days after PAN injection. Urinary protein increased more in the FGF2 group than in the other two groups. PCNA (+) glomerular cells were found in decreasing order in groups FGF2, Control and Anti-FGF2. Most of the PCNA (+) cells were podocytes and epithelial cells of Bowman's capsule. Staining for desmin, a marker of podocyte injury, was significantly reduced in the Anti-FGF2 group. Glomerular adhesive lesions were found in decreasing order in groups FGF2, Control and Anti-FGF2. The second series of experiments was designed to study the effects of FGF2 neutralizing antibody (40 micrograms for 5 days after PAN injection, in MoAb group) on severely damaged podocytes caused by repeated (two courses) injections in the PAN nephropathy rats. The results were the same as those in series 1. An increase in urinary protein excretion was observed in both groups, but on the 40th day, the level of proteinuria in the MoAb group decreased abruptly. It was observed that the MoAb group had few adhesive glomeruli compared to the IgG group (administration of mouse IgG) and the PCNA (+) epithelial cells of Bowman's capsule were also few. It was supposed that FGF2 would promote the formation of adhesive lesions by stimulating the proliferation of podocytes and epithelial cells of Bowman's capsule. Additionally, FGF2 itself was thought to impair podocytes because of the increasing desmin score and proteinuria.
Abstract: We analyzed a Japanese chick wingless mutant (Jwg) to know a molecular mechanism underlying wing development. We observed expression patterns of eleven marker genes to characterize the mutant. Expressions of dorsoventral (DV) and mesenchymal marker genes were intact in nascent Jwg limb buds. However, expression of Fgf8, a marker gene for the apical ectodermal ridge (AER), was delayed and shortly disappeared in the wing regressing AER. Later on, ventral expression of dorsal marker genes of Wnt7a and Lmx1 indicated that the wing bud without the AER became bi-dorsal. In addition, the posterior mesoderm became defective, as deduced from the impaired expression patterns of Sonic hedgehog (Shh), Msx1, and Prx1. We attempted to rescue a wing by implanting Fgf8-expressing cells into the Jwg wing bud. We found that FGF8 can rescue outgrowth of the wing bud by maintaining Shh expression. Thus, the Jwg gene seems to be involved in maintenance of the Fgf8 expression in the wing bud. Further, it is suggested that the AER is required for maintenance of the DV boundary and the polarizing activity of the established wing bud.
Abstract: Members of the fibroblast growth factor (FGF) family play important roles in various developmental processes in vertebrates. Since two genes closely related to the vertebrate FGF receptor (FGFR) genes DFR1 and DFR2/breathless have already been reported in Drosophila, the existence of a Drosophila FGF has been predicted. In the present study, we examined whether DFR1 is functionally interchangeable with a vertebrate FGFR in the Xenopus system. First, we found that the expression of DFR1 promoted Ca2+ efflux in response to human basic (b)FGF in Xenopus oocytes, whereas the coexpression of a dominant negative form of DFR1 (ΔDFR1) with a chick FGFR1/cek1 inhibited promotion of Ca2+ efflux induced by the expression of cek1 in the oocyte. Second, the expression of ΔDFR1 was observed to induce a defect in the posterior structure of the Xenopus embryo at stage 30, as observed with a dominant negative form of cek1 (Δcek1). Third, we found that the expression of ΔDFR1 inhibited the expression of FGF-regulated genes such as Xbra, Xnot, and Xshh in Xenopus embryos at stage 11, while the coexpression of DFR1 with ΔDFR1 could rescue the inhibited expression of FGF-regulated genes. These results indicate that DFR1 acts as an FGFR in Xenopus embryos and that an FGF is likely to exist in Drosophila.
Abstract: Sonic hedgehog (Shh) is a vertebrate gene homologous to a Drosophila segment polarity gene, hedgehog, and functions as a secreted signaling molecule in limb pattern formation, differentiation of motor neurons, and sclerotome induction. We found that Shh is also expressed in epithelia of the developing whisker, hair, tooth, rugae, and thyroid primordium of mouse embryos. In whisker and hair development, Shh is expressed in epithelial cells before condensation of the underlying mesenchymal cells and then in the placode. The expression of Shh continues in the hair bulb surrounding the dermal papilla. Shh is also expressed in epithelial cells of the tooth bud, then localized to the enamel knot. The Shh expression continues in developing ameloblasts. These results suggest that SHH is an essential epithelial signaling molecule in epithelio-mesenchymal interactions for the terminal differentiation.
Abstract: Tooth development involves reciprocal epithelial-mesenchymal interactions, polarized growth, mesenchyme condensation, and complex morphogenetic events. Because these processes bear similarities to those occurring in the developing limb, we asked whether morphogenetic signals found in the limb also occur in the developing tooth. We grafted mouse embryo tooth germs to the anterior margin of host chick embryo wing buds and determined whether the dental tissues had polarizing activity. Indeed, the grafts induced supernumerary digits. Activity of both molar and incisor tooth germs increased from bud to cap stages and was maximal at late bell stage in newborn. With further development the polarizing activity began to decrease, became undetectable in adult molar mesenchyme but persisted in incisor mesenchyme, correlating with the fact that incisors grow throughout postnatal life while molars do not. When different portions of neonatal incisors were assayed, a clear proximo-distal gradient of activity was apparent, with maximal activity restricted to the most proximal portion where undifferentiated mesenchyme and enamel organ reside. In situ hybridizations demonstrated that prior to induction of supernumerary digits, the tooth germ grafts induced expression in host tissue of Hoxd-12 and Hoxd-13. In addition, whole-mount in situ hybridizations and immunohistochemistry showed that developing tooth germs express Sonic hedgehog (Shh). Shh expression was first detected in bud stage tooth germs; at later stages Shh transcripts were prominent in enamel knot and differentiating ameloblasts at the cuspal region. We concluded that tooth germs possess polarizing activity and produce polarizing factors such as Shh. As in the limb, these factor(s) and activity probably play key roles in establishing polarity and regulating morphogenesis during early tooth development. Given its subsequent association with differentiating ameloblasts, Shh probably participates also in cytogenetic events during odontogenesis.
Abstract: To examine the role of BMP signaling during limb pattern formation, we isolated chicken cDNAs encoding type I (BRK-1 and BRK-2) and type II (BRK-3) receptors for bone morphogenetic proteins. BRK-2 and BRK-3, which constitute dual-affinity signaling receptor complexes for BMPs, are co-expressed in condensing precartilaginous cells, while BRK-1 is weakly expressed in the limb mesenchyme. BRK-3 is also expressed in the apical ectodermal ridge and interdigital limb mesenchyme. BRK-2 is intensely expressed in the posterior-distal region of the limb bud. During digit duplication by implanting Sonic hedgehog-producing cells, BRK-2 expression is induced anteriorly in the new digit forming region as observed for BMP-2 and BMP-7 expression in the limb bud. Dominant-negative effects on BMP signaling were obtained by over-expressing kinase domain-deficient forms of the receptors. Chondrogenesis of limb mesenchymal cells is markedly inhibited by dominant-negative BRK-2 and BRK-3, but not by BRK-1. Although the bone pattern was not disturbed by expressing individual dominant-negative BRK independently, preferential distal and posterior limb truncations resulted from co-expressing the dominant-negative forms of BRK-2 and BRK-3 in the whole limb bud, thus providing evidence that BMPs are essential morphogenetic signals for limb bone patterning.
Abstract: Normal human fibroblasts have a finite proliferative capacity in vitro. Thus, immortalization of human cells is associated with cellular aging. We have established an immortalization-sensitive cell line from fibroblasts of Wilms' tumor patients which have a partial deletion of chromosome 1 1p. This cell line was easily immortalized by introducing SV4OT. By differential hybridization using both SV4OT-introduced crisis cells and young cells, we cloned a gene that was highly expressed in 1 1p-cells at the time of the crisis and named this gene C-1. Nucleotide sequence analysis of C-1 revealed that it contains a helix-loop-helix domain, indicating that it may be a transcription factor. Expression of the C-1 gene was transiently induced early in the G0-to-S phase transition in two normal human (OUMS-24 and HSF-412) and a non-tumorigenic immortal human (OUMS-24F) fibroblast cell lines, while the other immortal SUSM-1 cells highly expressed the C-1 gene in the middle G1 phase. These results suggest that the C-1 gene product may function as a transcription factor related to the cell cycle.
Abstract: It has been shown that mirror-image duplications of the zeugopodia and digits are formed when MRC-5 fibroblasts producing hepatocyte growth factor (HGF) are applied to the anterior region of the chick limb bud (Yonei et al. [1993] Dev. Biol. 160:246-253). To evaluate the role of HGF in limb development, we observed the expression pattern of the HGF gene using in situ hybridization. The HGF gene was expressed in the mesoderm of the limb bud and in the central core region of mandibular arch and maxillary processes at stages 17 to 24. When both wing and leg buds begin to extend distally, the HGF gene is expressed in the mesenchymal cells, but not in the ectodermal cells and somites. Concomitant with establishment of the apical ectodermal ridge, distal mesenchymal cells of the limb bud express the HGF gene intensely with a gradient higher in the distal region. The HGF expression is later confined to the ventral and subapical mesenchyme of the limb bud, although no signal is detectable in the apical and non-ridge ectoderm. However, signal for the c-met proto-oncogene encoding the HGF receptor is not detectable in the limb bud at stages 17 to 24. These results suggest that HGF produced in the limb mesoderm may be involved in initial induction and maintenance of the apical ectoderm during limb development.
Abstract: To elucidate what initiates formation of the limb, we have attempted to induce an additional limb from the flank of the chick embryo by infecting retrovirus or implanting cells. We report here that an additional limb can be formed from the flank when we implant fibroblast growth factor 4 (Fgf4)-expressing cells into the lateral plate mesoderm at the pre-limb bud stage. In a newly formed limb bud, expressions of both Sonic hedgehog and chick Fgf4, which are authentic morphogenetic signals from the zone of polarizing activity and the apical ectodermal ridge, respectively, are induced by the implanted cells. Thus, it is concluded that the competence for limb development is present along the flank of the chick embryo and that FGF4 applied ectopically at the pre-limb bud stage can alter the developmental fate of flank cells to become limb cells. The present experimental system will contribute to a further elucidation on how the limb is formed.
Abstract: We have isolated a new member of the Wnt gene family, Wnt-11, from chick embryo cDNA library and examined the expression pattern during embryogenesis by in situ hybridization. The Wnt-11 gene encodes Cys-rich secretory protein distantly related to Wnt-1 through Wnt-8 from the mouse and Xenopus. Expression of the Wnt-11 gene became evident at stage 14 in the dorsolateral region of somites and gradually restricted to the dermatome at stage 19 and later. In contrast to the other Wnt genes, Wnt-11 was not expressed in the neuroepithelium throughout stages 14-26. At stage 24 and later, Wnt-11 was expressed in the subectodermal mesenchyme of the limb and feather buds. The unique expression pattern of Wnt-11 in the paraxial mesoderm and dermatome suggests that Wnt-11 may play an important role in dermal development.
Abstract: BRK-3 is a vertebrate type II receptor for BMP-4 distantly related to invertebrate type II receptors for BMP-2/BMP-4/dpp, such as daf-4 and punt. BRK-3 has a long carboxy-terminal sequence following intracellular kinase domain and is capable of forming a high-affinity complex with a type I receptor, BRK-2. To examine the role of BRK-2 + BRK-3 receptor complex in BMP signaling during early embryogenesis, the dominant-negative form of BRK-3 was ectopically expressed in the Xenopus embryos. A secondary body axis expressing the Sonic hedgehog and N-CAM genes is induced by injecting mRNA encoding truncated form of BRK-3 into ventral marginal region, implicating the BMP signaling in axial mesoderm induction. Formation of the secondary axis depends on whether the deletion extends into the kinase domain, not into the carboxy-terminal tail, suggesting that the kinase domain, but not the tail region, is essential for BMP signaling.
Abstract: Bone morphogenetic proteins (BMPs) comprise the largest subfamily of TGF-beta-related ligands and are known to bind to type I and type II receptor serine/threonine kinases. Although several mammalian BMP type I receptors have been identified, the mammalian BMP type II receptors have remained elusive. We have isolated a cDNA encoding a novel transmembrane serine/threonine kinase from human skin fibroblasts which we demonstrate here to be a type II receptor that binds BMP-4. This receptor (BRK-3) is distantly related to other known type II receptors and is distinguished from them by an extremely long carboxyl-terminal sequence following the intracellular kinase domain. The BRK-3 gene is widely expressed in a variety of adult tissues. When expressed alone in COS cells, BRK-3 specifically binds BMP-4, but cross-linking of BMP-4 to BRK-3 is undetectable in the absence of either the BRK-1 or BRK-2 BMP type I receptors. Cotransfection of BRK-2 with BRK-3 greatly enhanced affinity labeling of BMP-4 to the type I receptor, in contrast to the affinity labeling pattern observed with the BRK-1 + BRK-3 heteromeric complex. Furthermore, a subpopulation of super-high affinity binding sites is formed in COS cells upon cotransfection only of BRK-2 + BRK-3, suggesting that the different heteromeric BMP receptor complexes have different signaling potential.
Abstract: To elucidate the molecular mechanisms of chick feather formation, we observed expression patterns of the Sonic hedgehog (Shh) gene, which is one of the vertebrate homologs of the Drosophila segment polarity gene, hedgehog, and encodes a signaling molecule functioning in limb pattern formation and motor neuron induction. We found that the Shh gene is also expressed in the apical region of the feather placodes and then in nine to eleven longitudinal stripes along feather filaments. The stripe was found to correspond to one of the outer marginal zones of each barb ridge, termed the zone of Shh expression. No significant expression signal was detected in the scale bud of developing legs. Thus, Shh is likely to function as an epithelial signaling molecule in epithelio-mesenchymal interaction during feather formation. Furthermore, since genes of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-4 (FGF-4) are coexpressed with Shh during feather formation as observed in limb morphogenesis, interactions among FGF-4, Shh and BMP-2 may be involved in formation of feather filaments and barbs in a similar fashion as elucidated in limb pattern formation.
Abstract: We have isolated two members of the Wnt gene family, Wnt-4 and Wnt-11, from chick embryo cDNA library, and determined the entire coding sequences. The Wnt-4 and Wnt-11 genes encode secretory proteins composed of 351 and 354 amino acids, respectively, both having 24 Cys residues conserved among other Wnt family members. Alignment of the deduced amino acid sequences reveals that chicken Wnt-4 and Wnt-11 are most similar to Xenopus Wnt-4 and mouse Wnt-11; respectively. Northern blot analysis indicates the Wnt-4 expression at 1.5 kilobase and the Wnt-11 expression at 2.0 kilobase in the chick embryo.
Abstract: An expression vector, pKK233-2 [Amann and Brosius, Gene 40 (1985) 183-190], was modified by replacing the trc promoter with the T7 phi 10 promoter and introducing a strong transcriptional terminator upstream from this promoter. This modification successfully suppressed background transcription and resulted in strict dependence upon induction.
Abstract: Members of the Wnt gene family code for cysteine-rich, secreted proteins, which are differentially expressed in the developing brain and possibly act as an intercellular signaling molecule. A Wnt gene, e.g., Wnt-1 is known to be essential for specification of the midbrain cell fate. Since other members of the Wnt genes are likely to be also involved in development of the brain, we searched chick Wnt members expressing in specific domains of the brain. We found several novel Wnt members expressing specifically in the central nervous system of chick embryos. One of the genes was intensely expressed in a segment of the diencephalon at stages 14 and 20 and in a dorsal region of the spinal cord at stage 20. Although the expression patterns differ from those of mouse or Xenopus Wnt-4, the gene is highly homologous to the mouse or frog Wnt-4. Thus, we designated the gene as Cwnt-4 (chick Wnt-4). Our results suggested that Cwnt-4 is involved in segmentation of the forebrain into the neuromere D2 and in differentiation of the dorsal region of the spinal cord.
Abstract: In facultative anaerobes, the anaerobic expression of respiratory genes is regulated by a transcriptional activator, FNR. Transcription in vitro of the E. coli fnr gene was repressed by its product, FNR. The transcription of the E. coli narX gene encoding the nitrate sensor protein was likewise repressed. DNA truncation experiments for fnr and narX genes indicated that multiple anaero-boxes in each promoter region are essential for repression by the FNR protein, but they also suggest that factor-independent upstream activation signals are operating with these promoters.
Abstract: We have recently reported fluctuations in the expression of the period repeat sequence, pp2.5, during light-dark cycles in the suprachiasmatic nucleus (SCN) of rat. Presently, we performed in situ hybridization which shows that the fluctuation of pp2.5 expression continues during constant darkness conditions in the SCN of rat. The light exposure during subjective night but not subjective day triggered its elevated expression in a time-dependent manner which is parallel to that of c-fos expression. In this review, the cloning and characterization of multiple per repeat sequences from mouse genom and rat brain mRNA were summarized. The abundance of a novel per repeat mRNA (designated as RB15) fluctuates during a light-dark cycle in the SCN. These findings suggest that per repeat sequence may have a role for the mammalian circadian rhythms. The evolutionary relationship between the mammarian per repeat sequence and the Drosophila period gene is also discussed.
Abstract: Androgen-induced growth factor (AIGF/FGF-8) has purified from a mouse mammary carcinoma cell line and its cDNA cloning revealed that AIGF is the eighth member of the fibroblast growth factor family. To clarify a biological role of FGF-8, Northern blot analysis of various adult tissues was carried out, but no expression was found. However, whole mount in situ hybridization of 7.5- to 14.5-day gestation (E7.5-14.5) embryos revealed six predominant expression domains: [1]primitive streak region (E7.5-9.75); [2]midbrain-hindbrain border (E8.0-10.5); [3]rostral forebrain (E8.0-10.5); [4]limb ectoderm and apical ectodermal ridge (E9.25-13.5); [5]nasal placode and epithelium (E9.5-12.5); and [6]branchial arch ectoderm (E8.5-11.5). The embryonic expression pattern suggests a unique role of FGF-8 in mouse development, especially in gastrulation, brain development, and limb and facial morphogenesis.
Abstract: The expression patterns of two distinct types of fibroblast growth factor receptor (FGFR) genes, FGFR1 and FGFR2, were compared during early chick eye development. In situ hybridization was performed with riboprobes synthesized from cDNA fragments of FGFRs cloned by the polymerase chain reaction method. FGFR1 was expressed in the prospective lens, neural retina, pigment epithelium and mandibular mesenchyme. In contrast, FGFR2 was expressed predominantly in the periocular mesenchyme of a 2.5 day-old embryo. In the 5.5-day-old embryo, transcripts of FGFR2 were detected in the prospective corneal epithelium. The results suggest that expression patterns of FGFR1 and FGFR2 are complementary and ligands of each FGFR might be involved differentially in early chick eye development. It is concluded that the action of FGFs on pigment epithelium and lens cells reported so far, probably occurs through FGFR1, and both types of FGFR are involved in head mesenchymal development.
Abstract: When a mouse zone of polarizing activity (ZPA) at the posterior margin of the limb bud was grafted into the anterior margin of the chick limb bud, the expressions of the chick homeobox genes HoxD12 and D13 were induced prior to the formation of chick extra digits. This induction was observed in a restricted domain close to both the grafted mouse ZPA and the chick apical ectodermal ridge (AER). When the posterior half of the AER was removed, the normal expression was diminished in the distaloposterior region. Thus, it is likely that at least two distinct factors, one from the ZPA and the other from the AER, act cooperatively to provide positional information to induce the sequential expression of the HoxD genes.
Abstract: Expression patterns of three fibroblast growth factor receptor genes, FGFR1 (cek1), FGFR2 (cek3) and FGFR3 (cek2), were observed in limb and feather morphogenesis. Expression of FGFR1 was observed in the mesoderm of limb bud, in the mesenchyme just underneath the feather placode, and then in the anterior mesenchyme of the feather bud. While expression of FGFR2 was observed in both surface ectoderm and mesenchymal aggregates corresponding to the future bones of the limb, the mesenchyme between the feather placode, and surface ectoderm of feather buds. Expression of FGFR3 was observed rather ubiquitously over mesoderm of limb and feather buds. Differential expression of these FGF receptor genes suggested that differential roles of these receptors in epithelia-mesenchymal interactions of limb and feather morphogenesis.
Abstract: The anteroposterior (A-P) axis pattern of the chick limb is likely to be controlled by a small region of mesenchyme cells at the posterior margin of the limb bud (ZPA, zone of polarizing activity). In this study, we found that MRC-5 fibroblast cells had the capacity for duplicated-pattern formation of the chick limb along the A-P axis when grafted to the anterior region of the limb bud. MRC-5 cells were effective only during pre-limb bud stages, and the leg bud was more responsive than the wing bud. Grafted cells remained at the base of the limb bud when limb development proceeded. These results suggest that the products of MRC-5 cells are involved in three possible processes of duplicated-pattern formation: induction of the polarizing activity; maintenance of this activity, which is present weakly at pre-limb bud stages; and determination of A-P axis as the ZPA factor(s). By allowing the use of a small number of cells of embryonic tissues with polarizing activity, the analysis of duplicate formation with the MRC-5 cell line provides a powerful tool for elucidating the molecular nature of the ZPA.
Abstract: The sequential transphosphorylation from autophosphorylated nitrate-sensing protein (NarX) to the transcriptional regulator protein (NarL), both operating in signal transduction to control the expression of the respiratory nitrate reductase (nar) operon in E. coli, was demonstrated with an in vitro reconstructed system to function similarly to other bacterial two-component regulatory systems. Over-expression system established by means of the pT7 promoter/polymerase provided both NarX and NarL proteins to reconstruct the in vitro transphosphorylation system. The phosphorylated NarL was detected, and the unstable phosphorylated group was directly assigned to acyl phosphate in the in vitro system by 31P-NMR spectroscopy.
Abstract: We identified a homeobox-containing gene, Prx-1, isolated from the chick limb bud cDNA library. The homeodomain sequence is related to Drosophila paired and goose-berry and mouse Pax-3, Pax-6, and Pax-7. The deduced amino acid sequence of the Prx-1 gene product reveals the absence of a paired-box sequence and extensive similarity to mouse S8 and MHox homeodomain proteins, thus constituting a new class of homeobox gene. Using an in situ hybridization method, the Prx-1 gene is shown to be expressed predominantly in the limb bud and visceral arches. At early stages of limb development, distal mesodermal cells express the homeobox gene with an apparent gradient along the proximal-distal axis. The signal is absent in the apical and nonridge ectoderm. Removal of the apical ectodermal ridge had no apparent effect on the subsequent expression of Prx-1 in the limb mesenchyme. The Prx-1-expressing cells are later confined to the interdigital and perichondrial regions. The Prx-1 transcripts are also detectable in the mesenchyme of the visceral arches and facial primordia subjacent to the ectoderm. The Prx-1 gene is weakly expressed in somites and condensing vertebrae. No signal is detectable in neural tube and ectodermal epithelium. These results suggest that the Prx-1 homeodomain protein is involved in the differentiation of bone, muscle, and other tissues of mesodermal origin during limb development.
Abstract: Activin is known to induce axial mesoderm during early development in Xenopus embryo. Activin receptor was recently identified to be a member of transmembrane serine/threonine kinase family. We have studied the role of activin-mediated signaling in the limb morphogenesis by identifying the target cells. We isolated cDNAs encoding chicken activin receptors, cARIIA and cARIIB, and examined their expression patterns during chick embryogenesis. The cARIIA gene is expressed in the apical ectoderm of the limb bud at stage 20-21, whereas the cARIIB gene is expressed uniformly in the limb mesenchyme. Expression of the cARIIA gene is confined to dorsal and ventral mesenchyme at stage 23, and later confined to precartilaginous cells. Transcripts of the cARIIA gene are found in developing neuroepithelium of spinal cord, brain and eye, surface ectoderm differentiating to epidermis, and myoblasts differentiating to muscle. The IIB receptor gene is highly expressed in the developing brain. These results suggest that the activins and their receptors are implicated in the limb development, especially, in differentiation of muscle, skin and bone.
Abstract: We have isolated RPK-2 cDNA from chick embryo cDNA library that encodes a receptor protein kinase of the TGF-beta receptor subfamily. The deduced amino acid sequence reveals the presence of a hydrophobic transmembrane helix and a kinase domain distantly related to type II receptor for TGF-beta. The kinase domain sequence is most similar to RPK-1 identified recently in the chick embryo. Several cysteine residues are contained in the amino-terminal ectodomain, suggesting the protein as a receptor for a peptide growth factor of the TGF-beta family.
Abstract: We have isolated cDNA encoding a new member of protein kinase family from chicken embryo cDNA library. The deduced amino acid sequence comprises a cysteine-rich extracellular domain, a single hydrophobic transmembrane domain, and a cytoplasmic serine/threonine kinase domain. Two short inserts are contained in the kinase domain. The primary structure shows that it belongs to the receptor-type serine/threonine kinase subfamily and is most similar to Daf-1. Conserved cysteine residues in the ectodomain suggest the protein as a receptor for a peptide growth factor of TGF-beta family.
Abstract: When a mouse zone of polarizing activity (ZPA) at the posterior margin of the limb bud was grafted at the anterior margin of the chick limb bud, we found that expression of the chick homeobox genes, Chox-4.7 and -4.8, was induced prior to the formation of chick extra digits. The induction was observed in the restricted domain close to both grafted mouse ZPA and the chick apical ectodermal ridge (AER). When the posterior half of the AER was removed, the normal expression was diminished in the posterodistal region. Thus, it is likely that at least two distinct factors, one from the ZPA and the other from the AER, provide positional information to induce cooperatively the sequential expression of the Chox-4 genes.
Abstract: To elucidate target cells of activins during embryogenesis we isolated cDNAs of chick activin receptor type II (cActR-II) and studied expression patterns of the cActR-II gene by in situ hybridization. Transcripts of cActR-II were observed in neuroectoderm developing to spinal cord, brain and eyes, in surface ectoderm differentiating to epidermis, and in myotomes differentiating to muscles. The expression patterns of cActR-II suggest that activin and its receptor are involved in differentiation of chick neural tissues, muscle and skin after inducing the dorsal mesoderm.
Abstract: We have isolated two closely related cDNAs, Chox-7 and Chox-8, encoding homeodomain-containing proteins homologous to Drosophila msh. The Chox-7 and Chox-8 genes are chicken cognates of mouse Hox-7.1 and Hox-8.1, respectively. In situ hybridization using 3' regions of the cDNAs as probes revealed that the Chox-7 gene is highly expressed in the mesenchyme subjacent to the apical ectodermal ridge whereas Chox-8 expression is localized in the anterodistal mesenchymal region at early stages of limb formation, suggesting different roles during limb development. At later stages, both genes are expressed in the anterior and posterior mesenchymes and in the interdigital mesenchyme where programmed cell death occurs.
Abstract: We integrated our information on morphogenetic roles of retinoic acid, focusing on development of chick limb. Retinoic acid has been considered to be a putative morphogen released from the ZPA. However, since exogenous retinoic acid induces expression of RAR beta, but not grafted ZPA, the ZPA is unlikely to produce retinoic acid. From the result that retinoic acid-treated cells induce digit duplication, retinoic acid converts anterior cells into ZPA cells. We found that retinoic acid also induces expression of homeobox genes indirectly and that bFGF enhances expression of the homeobox genes. Thus, we considered that retinoic acid and growth factors cooperate with each other to activate homeobox genes. A morphogenetic role of retinoic acid is to coordinate developmental stage of each cells by controlling expression of homeobox genes.
Abstract: Maternal treatment with 100 mg/kg of retinoic acid (RA) on day 9 of gestation in mice caused craniofacial abnormalities of the mandibulofacial dysostosis type. The abnormal morphology was attributed to the excessive cell death in the dorsal aspects of the maxillary and mandibular prominences of the first pharyngeal arch and in the proximal region of the mandibular prominence. To investigate the expression of the RA receptor (RAR) genes in abnormal face morphogenesis, in situ hybridization was performed. The distribution patterns of RAR α and γ transcripts were not altered in the treated embryos. By contrast, the teratogenic dose of RA increased the level of RAR β transcripts, as early as 3 hr after RA-treatment, in the regions where the RAR β expression is at a low level in normal development. The increase of RAR β transcripts was detected by 12 hr, and declined to the low level within 24 hr after the treatment. The regions where ectopic expression of RAR β gene was observed included the areas where the excessive cell death occurred 9–12 hr after RA-treatment. These results suggest that ectopic induction of RAR β by RA may lead to the excessive cell death, threfore may cause abnormal morphogenesis.
Abstract: Retinoic acid is known to play an important role during development of central nervous system. In order to clarify function of retinoic acid during the development, we investigated expression pattern of the chick retinoic acid receptor subtype beta gene by an in situ hybridization method. We found that expression of the beta gene is localized in neural tube at stages 16-20, then is turned to be restricted to developing motoneurons at stages 23-29. These results suggested that retinoic acid and its receptor beta are involved in differentiation of the motoneurons in spinal cord.
Abstract: We have isolated and identified four chicken homeobox genes in the upstream region of the Chox-4 complex. The Chox-4g and -4f genes, at the 5' extremity of the complex, were expressed locally in the vicinity of the zone of polarizing activity (ZPA) at early stages of limb development, substantiating the involvement of the genes in anteroposterior axis determination. To confirm their function, we implanted a bead containing retinoic acid, or the ZPA itself, in the anterior margin of the limb bud, leading to formation of mirror-image duplicated digits, and observed the resultant change in gene expression. Expression of the Chox-4g and -4f genes was induced in the new digit-forming region. Those results suggest that positional information assigned by a ZPA morphogen is imprinted on cellular memory by expression of the Chox-4 genes to maintain positional signaling along the anteroposterior axis in the limb field.
Abstract: The cDNA sequences of isoforms of retinoic acid receptor beta from the chick have been determined. The sequence is different from that reported previously only in the 5' region, suggesting a product of alternative splicing and differential usage of promoters. One of them, the novel RAR-beta 4 isoform, is presumed to encode an amino-terminal truncated region A of retinoic acid receptor beta.
Abstract: Retinoic acid is a putative morphogen in limb formation in the chick and other vertebrates. In chick limb formation, it is thought that retinoic acid is released from the zone of polarizing activity (ZPA) and the concentration gradient of retinoic acid formed from the posterior to the anterior provides positional cues for digit formation. Implantation of a bead containing retinoic acid at the anterior margin of the limb bud induces a mirror-image symmetrical duplication of the digit pattern similar to that observed when the ZPA is grafted into the anterior margin of the host limb bud. Also, the level of endogenous retinoic acid (25 nM on average) is higher in the posterior one third of the limb bud. We found that when the bead containing either retinoic acid or an analogue but not the ZPA, was implanted in the anterior margin of the chick limb bud, expression of the retinoic acid receptor type-beta gene was induced around the bead within 4 h. These results indicate that exogenous retinoic acid is not identical with the ZPA morphogen. As the anterior tissue exposed to retinoic acid has polarizing activity, we conclude that the primary function of exogenous retinoic acid is to induce polarizing activity in the limb bud.
Abstract: 'Per repeat is a rodent genomic DNA fragment which is homologous to the Gly-Thr repetitive amino acid sequence of the Drosophila clock gene period'. This study examined the temporal and spatial expression of the per repeat mRNA in rat brain. Northern blot analysis showed that the level of per repeat mRNA species fluctuates under light-dark cycle conditions in rat brain. Furthermore, the fluctuation of per repeat mRNA was clearly observed in the suprachiasmatic nucleus, but not so in other regions including the hippocampus as shown by in situ hybridization. The above results suggest that the Gly-Thr repetitive sequence also plays an important role in the circadian rhythm of mammals.
Abstract: Retinoic acid (RA) is known as a teratogen that induces abnormalities in facial structures which are made up mainly of neural crest-derived mesenchyme. We investigated expression patterns of RA receptor (RAR) genes (subtypes alpha, beta, gamma) during mouse facial development. The expression of the RAR beta gene is specific for the mesenchyme around developing eyes and nose, whereas the RAR gamma gene is expressed in the mesenchyme differentiating to facial cartilages and bones. In contrast, the RAR alpha gene is expressed weakly and uniformly over the facial region. These results suggest that crucial roles of endogenous RA in facial development depend on differential functions of the RAR subtypes.
Abstract: Hepatocyte growth factor (HGF) has been demonstrated to be synthesized and secreted by non-parenchymal liver cells for liver regeneration after hepatic injury. We performed in situ hybridization to identify HGF-producing cell types in rat liver hepatitis induced by administrating carbon tetrachloride as a hepatotoxin. We found that transcripts of the HGF gene are localized in the Kupffer and endothelial cells in normal livers and increased remarkably in the Kupffer cells of the damaged livers. Thus, HGF is concluded to be synthesized in the Kupffer and endothelial cells to repair the liver tissue in paracrine fashion. No significant increase in the transcripts of the HGF gene was observed in livers after partial hepatectomy, indicating that a mechanism on liver regeneration after the hepatectomy differs from that on liver repairs. Since the HGF gene expression was also found in lung and kidney, HGF may be a ubiquitous factor for tissue repairs.
Abstract: Although the retinal angiogenic and mitogenic factors have been identified to be acidic and basic fibroblast growth factors (aFGF and bFGF), little information has so far been available about the cells producing them and their function in retinal tissues. We found, by in situ hybridization, that the expression pattern of the aFGF gene differed remarkably from that of the bFGF gene in adult rat eyes. Our results demonstrated that the aFGF gene was produced by photoreceptor visual cells, neuronal cells in the inner nuclear layer and ganglion cells of the retina, in addition to pigment epithelial cells of the choroid, iris and ciliary body, and epithelial cells of the cornea, conjunctiva and lens, while bFGF was synthesized solely by the photoreceptor visual cells.
Abstract: The nucleotide sequence of a 4.4-kilobase SacII-SspI fragment encoding the narXL operon and a part of the narK gene of Escherichia coli has been determined. The narX and narL genes encode proteins of molecular weight 67,275 and 23,927, respectively, and are transcribed from a common promoter, narXp, locating within 429 bases upstream of narX. Transcription from narXp is not significantly induced by nitrate under anaerobiosis, whereas transcription from narK promoter, which overlaps narXp region and is transcribed divergently, is fully induced by nitrate. The N-terminal two-thirds of the NarL protein has extensive homology with those of a diverse set of prokaryotic regulatory proteins, including OmpR, PhoB, SfrA, UhpA, CheY, CheB, NtrC, DctD, FixJ, VirG, SpoOF, and SpoOA. A segment locating in the C-terminal half of the NarL protein seems to have potential most likely to form the helix-turn-helix structure characteristic of a class of DNA-binding protein. The protein is considered to play a role as a transcriptional activator of the nitrate reductase operon, narCHJI, and the narK gene. The C-terminal region of the NarX protein also has homology with other regulatory proteins known as counterparts of two-component regulatory systems, such as EnvZ, PhoR, PhoM, CpxA, NtrB, DctB, FixL, and VirA. Presence of two copies of hydrophobic segments in the N-terminal half of the NarX protein suggests the role as a transmembrane receptor sensing nitrate.
Abstract: To determine the involvement of 3'-end structure of mRNA in the regulation of gene expression in eukaryotic cell, a series of pSCAT plasmids was constructed with chloramphenicol acetyltransferase (CAT) gene and the 3'-regulatory elements from various eukaryotic genes. As a result of determination of the CAT activities and the mRNA level generated from these plasmids, both results were well correlated. Furthermore, the treatment of transfected cells with phorbol ester (TPA) revealed that the polyadenylated CAT mRNAs form some genes were stabilized, in contrast, the mRNAs bearing 3'-end structure of histone or C-myc genes were not.
Abstract: The nucleotide sequence of the Escherichia coli narK gene, which is located in the upstream region of the narCHJI operon, was determined. The narK gene encodes a very hydrophobic protein with 463 amino acid residues (Mr 49,693). A narK deletion mutant, under conditions for the induction of nitrate respiration, was unable to perform nitrate transport. Loss of transport activity was recovered by transforming the mutant with a narK+ plasmid. Thus, we conclude that the narK gene encodes a transmembrane protein participating in nitrate transport. In the narK promoter region, we defined a unique sequence that we designate as a 'nitrate box', functioning as a putative NarL-binding site, in addition to the consensus sequence of the 'anaero-box'.
Abstract: Spatial and temporal expression pattern of retinoic acid receptor (RAR) genes was investigated in mouse finger bones during development by an in situ hybridization method with riboprobes synthesized from a human cDNA of the RAR-alpha. We found that the RAR genes are expressed intensively and specifically in calcifying fronts of the mouse finger bones, whereas the expression pattern is rather uniform in the limb buds and cartilage matrices of the embryonic fingers. Our findings are consistent with the fact that vitamin A is essential for normal mammalian bone development.
Abstract: We found, by an in situ hybridization method with riboprobes synthesized from human cDNA of the retinoic acid receptor (RAR), that the RAR genes (predominantly gamma-subtype) are intensively expressed in the epidermis of normal and psoriasic human skins, and also in keratinizing fronts of 4-day-old mouse skins, nail matrices and hair follicles. Thus, target cells of retinoic acid in the skins are concluded to be keratinocytes, which is quite consistent with the fact that retinoic acid regulates keratinization of epidermis in vivo and also modulates expression of the keratin gene in vitro.
Abstract: The nucleotide sequence of a HinPI-HpaII restriction nuclease fragment which complemented a delta chlE strain of Escherichia coli was determined. Two open reading frames were deduced to be the structural genes for ChlE and ChlN proteins, which have molecular weights of 44,067 and 26,719, respectively. Both proteins were required for complementing a chromosomal deletion of the chlE locus. The chlE and chlN genes were transcribed from a common promoter, chlEp, located upstream of chlE. Transcriptional and translational signal sequences were recognized in this region.
Abstract: Experiments were carried out to examine the role of the host defence mechanism in the proliferation of androgen-dependent Shionogi carcinoma 115 in the DS mouse. Stimulation of tumor growth by dexamethasone was synergistic with androgen action. Enhanced host immunity caused by bacterial infection resulted in remarkable inhibition of tumor growth. Not only androgen but also the host defence mechanism is one of the major factors that determine overall growth rate of the androgen-dependent tumor in this strain of mice.
Abstract: The glutamine permease operon encoding the high-affinity transport system of glutamine in Escherichia coli could be cloned in one of the mini F plasmids, but not in pBR322 or pACYC184, by selection for restoration of the Gln+ phenotype, the ability to utilize glutamine as a sole carbon source. We determined the nucleotide sequence of the glutamine permease operon, which contains the structural gene of the periplasmic glutamine-binding protein (glnH), and indispensable component of the permease activity. The N-terminal amino acid sequence and the overall amino acid composition of the purified glutamine-binding protein were in good agreement with those predicted from the nucleotide sequence, if the N-terminal 22 amino acid residues were discounted. The latter comprised two Lys residues (nos. 2 and 6) followed by 16 hydrophobic amino acid residues and was assumed to be a signal peptide for transport into the periplasmic space. There were two additional reading frames (glnP and glnQ) downstream of glnH sharing a common promoter. It was concluded that the glnP and glnQ proteins as well as the glnH protein are essential for glutamine permease activity.
Abstract: The effects of KSCN and other chaotropic salts on the androgen receptor in cytosol of Shionogi carcinoma 115 were studied by means of charcoal adsorption assay and sucrose gradient centrifugation. When KSCN or NaSCN was added to the [3H]-dihydrotestosterone-cytosol mixture at the final concentration of 0.5 M, the androgen binding to the cytosol receptor was considerably inhibited. The inhibition reached maximum within 5 h at 0 degrees C and was dependent on the kind of chaotropic anion added: the potency of inhibitory effect in descending order was KSCN greater than KI greater than KBr greater than KCl. The inhibition was not observed in the estradiol-receptor interaction with KSCN or NaSCN up to 0.5 M. When 0.5 M KSCN-treated androgen-cytosol mixture was subjected to gel filtration or (NH4)2SO4 fractionation to remove the salt, a partial recovery (30-40%) in specific binding activity was observed. The binding activity of androgen receptor was unaffected by a treatment with KSCN up to 0.1 M and the androgen-receptor complex sedimented in a 5S form in 0.1-0.3 M KSCN, 0.5 M KCl or 0.5 M KBr. These results suggest that the binding activity of androgen receptor is more susceptible than that of estrogen receptor to chaotropic salts which cause impairment in intramolecular hydrophobic interactions.
Abstract: Although adriamycin (1 mg/kg) and 5-fluorouracil (25 mg/kg) were equally effective in inhibiting the growth of Ehrlich carcinoma, both ascites and solid forms, in the mouse, the growth of Shionogi carcinoma 115 in the male DS mouse was relatively resistant to the treatment with adriamycin as compared to 5-fluorouracil. However, direct cytotoxic activity of adriamycin in cultured Shionogi carcinoma 115 (SC 115) cells was about 1000-fold molar potent when compared with 5-fluorouracil. The apparent ineffectiveness of adriamycin against SC 115 in the DS mouse seems to be, at least in part, due to the depression of host defence mechanisms.
Abstract: The effects of dexamethasone and indomethacin on the growth of androgen-dependent Shionogi carcinoma 115 were studied. When castrated male DS mice were treated with dexamethasone for 2 weeks at daily doses of 1-5 mg/kg starting from 3 days before tumor inoculation, the tumor weight as well as the transplantability was significantly increased. The tumor grown in dexamethasone-treated mice had morphological, biochemical and biological characteristics similar to those of the original Shionogi carcinoma 115. Indomethacin at daily doses of 2-10 mg/kg also stimulated the tumor growth in castrated DS mice, but the effect was less pronounced than that of dexamethasone. In allogeneic male ICR mice, dexamethasone promoted tumor growth, but indomethacin had no effect. These results suggest that tumor growth in the mouse is stimulated not only by androgens but also by glucocorticoids through the suppression of immune responses of the host and direct action on the tumor cells.
Abstract: Experiments were performed to observe the role of estrogen receptor in the proliferation of androgen-dependent mouse tumor, Shionogi carcinoma 115. Estradiol and diethylstilbestrol inhibited tumor growth as well as the weight gain of seminal vesicles and prostate glands in intact male mice. Tamoxifen decreased the tumor weight in intact males. Both nitromifene given to intact mice and tamoxifen given to castrated androgenized mice decreased the weight of seminal vesicles, but increased tumor weight. Estradiol was bound to the androgen receptor of the tumor cytosol with relatively high affinity, whereas diethylstilbestrol, tamoxifen and nitromifene were not. These were effective competitors in the estrogen receptor present in the tumor cytosol. These results suggest that the estrogen receptor system in Shionogi carcinoma 115 inhibits the proliferation of tumor cells.
Abstract: Estrogen receptors in the nuclei of rat uterus were determined 1 or 6 h after the injection of [3H]estradiol. Ratio of 0.4 M KCl-extractable to -resistant receptor-estradiol complex was constant during this time interval. Three consecutive extractions took out about 95% of receptor-estradiol complex either 1 or 6 h after injection. Extraction with KCl before the exchange assay reduced the amount of salt-resistant nuclear receptor-estradiol complex. However, when exchange incubation with [3H] estradiol was done before KCl extraction, salt-resistant receptor-estradiol complex was significantly increased, even after three consecutive extractions.
Abstract: The characteristics of the androgen receptor in the cytoplasmic fraction of Shionogi carcinoma 115 were studied in vitro by means of charcoal adsorption assay, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis. The equilibrium dissociation constant for [3H]dihydrotestosterone (Kd = (2-5) X 10(-11) M) was estimated from independently determined rates of association and dissociation, and was lower by one order of magnitude than the value obtained by saturation analysis (Kd = (2-8) X 10(-10) M). Evaluation of the effect of temperature on receptor binding of androgen allowed the estimation of several thermodynamic parameters, including activation energies of association (4 kcal/mol) and dissociation (14 kcal/mol), the apparent free energy (-13 kcal/mol), enthalpy (-9 kcal/mol), and entropy (+14 cal/mol per K). The receptor was greatly stabilized when bound with androgen. The results indicate how the lability of the unbound receptor and slow rate of dihydrotestosterone-receptor interaction can influence the estimation of dissociation constants by usual saturation analysis. The sedimentation coefficient of androgen receptor in freshly prepared cytosol was 6S, and became 7S after storage for 2 months at -80 degrees C. The 7S conversion of the receptor was reversed by treatment with heparin. In all cases, a single 5S peak was obtained in the presence of 0.5 M KCl. On electrophoresis in heparin-containing polyacrylamide gel, protein-bound radioactive androgen migrated as a single peak (Rf = 0.5 in 5% gel). Differences in reported values for the sedimentation coefficient of androgen receptor in cytosol of Shionogi carcinoma 115 appear to be derived from aggregation of the receptor protein during the assay procedure.
Abstract: Trans-4-hydroxy-3-methoxycinnamic acid (ferulic acid, FA) antagonized the effect of exogenous androgens on the ventral prostate (VP) in castrated rats as well as the effect of endogenous androgens in intact rats. FA, however, had no effect on the seminal vesicles (SV) and levator ani muscle (LAM), nor oestrogenic effect in female rats and mice. FA did not antagonize the receptor binding of testosterone nor inhibit the conversion of testosterone into 5 alpha-dihydrotestosterone (DHT).
Abstract: Experiments were carried out in an attempt to elucidate the mechanism of fasting-induced natriuresis in conscious rats. Female animals were given low sodium diet and saline in a fixed concentration for at least three days, and deprived of food thereafter. The sodium balance significantly shifted to negative independently of potassium supply in intact rats. Such was also observed in the dexamethasone-replaced, adrenalectomized rats and was not affected by further administration of aldosterone. In addition, the diuretic effect of insulin in fasted intact rats was not evident in the fed diabetic rats in which diabetes had been induced by streptozotocin. Such findings suggested participation of factors other than insulin. The natriuresis of fasting in intact rats appears to involve two factors, one of which is independent of the sodium intake level. Dependence on the sodium intake level may be derived from alteration of the solute diuresis-like effect of drinking saline w hen animals are fasted. These results suggest that neither aldosterone nor insulin is a major causal factor involved in fasting-induced natriuresis.
