Ameliorative effects of Magnolia sieboldii buds hexane extract on experimental reflux esophagitis.

Gastroesophageal reflux disease (GERD) is a disease that stomach contents continually refluxing into esophagus causes symptoms and/or complications. The study was working to find natural plant extracts with good effects and small side effects to treat reflux esophagitis (RE). The anti-inflammatory effects of hexane extract of Magnolia sieboldii (MsHE) were conducted on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. The ameliorative effects of MsHE on esophageal damage in rats induced by gastric acid reflux was explored in vivo. The results showed that MsHE decreased the production of nitric oxide (NO) and expression levels of iNOS, COX-2 and TNF-α on LPS-stimulated RAW 264.7 cells and MsHE treatment ameliorated the rats' esophageal tissue damage induced by gastric acid and inhibited the increase of inflammatory mediators and pro-inflammatory cytokines by regulating NF-κB signaling pathway. In addition, MsHE protected the function of barrier of epithelial cells against inflammatory conditions through increasing the expression of tight junctions. Furthermore, liquid chromatography-mass spectrometry analysis was used for determine the active ingredients contained in MsHE. The results show that MsHE can alleviate experimental rat RE by regulating NF-κB signaling pathway. In summary, MsHE may be used as a source material of drug candidate for the treatment of RE.

Twist2 contributes to termination of limb bud outgrowth and patterning through direct regulation of Grem1.

Twist1 has been demonstrated to play critical roles in the early development of neural crest and mesodermally derived tissues including the limb. Twist2 has been less well characterised but its relatively late onset of expression suggests specific roles in the development of a number of organs. Expression of Twist2 within the developing limbs begins after formation of the limb bud and persists within the peripheral mesenchyme until digital rays condense. We have used RCAS-mediated overexpression in chick to investigate the function of Twist2 in limb development. Viral misexpression following injection into the lateral plate mesoderm results in a spectrum of hypoplastic limb phenotypes. These include generalized shortening of the entire limb, fusion of the autopod skeletal elements, loss of individual digits or distal truncation resulting in complete loss of the autopod. These phenotypes appear to result from a premature termination of limb outgrowth and manifest as defective growth in both the proximal-distal and anterior-posterior axes. In situ hybridisation analysis demonstrates that many components of the Shh/Grem1/Fgf regulatory loop that controls early limb growth and patterning are downregulated by Twist2 overexpression. Grem1 has a complementary expression pattern to Twist2 within the limb primordia and co-expression of both Grem1 and Twist2 results in a rescue of the Twist2 overexpression phenotype. We demonstrate that Twist proteins directly repress Grem1 expression via a regulatory element downstream of the open reading frame. These data indicate that Twist2 regulates early limb morphogenesis through a role in terminating the Shh/Grem1/Fgf autoregulatory loop.

Intermittent PTH (1-34) injection rescues the retarded skeletal development and postnatal lethality of mice mimicking human achondroplasia and thanatophoric dysplasia.

Achondroplasia (ACH) and thanatophoric dysplasia (TD) are caused by gain-of-function mutations of fibroblast growth factor receptor 3 (FGFR3) and they are the most common forms of dwarfism and lethal dwarfism, respectively. Currently, there are few effective treatments for ACH. For the neonatal lethality of TD patients, no practical effective therapies are available. We here showed that systemic intermittent PTH (1-34) injection can rescue the lethal phenotype of TD type II (TDII) mice and significantly alleviate the retarded skeleton development of ACH mice. PTH-treated ACH mice had longer naso-anal length than ACH control mice, and the bone lengths of humeri and tibiae were rescued to be comparable with those of wild-type control mice. Our study also found that the premature fusion of cranial synchondroses in ACH mice was partially corrected after the PTH (1-34) treatment, suggesting that the PTH treatment may rescue the progressive narrowing of neurocentral synchondroses that cannot be readily corrected by surgery. In addition, we found that the PTH treatment can improve the osteopenia and bone structure of ACH mice. The increased expression of PTHrP and down-regulated FGFR3 level may be responsible for the positive effects of PTH on bone phenotype of ACH and TDII mice.

Effects of pomegranate extracts on cartilage, bone and mesenchymal cells of mouse fetuses.

Pomegranate is a rich source of polyphenols, which are believed to be responsible for the oestrogenic activities of extracts of this fruit in mice. One of these potential activities is the prevention of bone loss. The objectives of the present study were to determine the effects of pomegranate extract on chondrogenesis and osteogenesis in mouse embryos in vivo and limb bud cultures in vitro. A total of fifty pregnant Balb/c mice were given vehicle, pomegranate juice extract (PJE), pomegranate husk extract (PHE) or a mixture of husk and juice extract (PME). Their embryos were stained with alizarin red S and alcian blue, and the length of the femur, tibia and their ossification zones were measured on day 19 of gestation. Bone Ca content in pregnant mice was also measured. Mice treated with PJE showed an increase in bone Ca content. Dietary supplementation with all extracts significantly increased embryo femur length and osteogenesis index. Mesenchymal cells from fetal limb buds were cultured and exposed to 10, 100, 1000 and 10 000 µg/ml of PJE, PHE or PME. The number of viable cells was greater in cultures exposed to the extracts than in control cultures. The number of cartilage nodules and their diameters were greater in extract-treated cell cultures, a finding which reflected increased cell proliferation and differentiation rates. In conclusion, the findings of the present study suggest that pomegranate is able to enhance bone formation.

Toxic effects of Elaeagnus angustifolia fruit extract on chondrogenesis and osteogenesis in mouse limb buds.

OBJECTIVES: We determined the effect of Elaeagnus angustifolia extract on chondrogenesis and osteogenesis in mouse embryo limb buds in vitro and in vivo. Limb bud mesenchyme from day 12.5 embryos were used for high-density micromass cultures. Water/alcohol extract was added to culture media at 10, 100, 1000 and 10000 µg/L. Cytotoxicity was tested with neutral red. Chondogenesis was detected by alcian blue and osteogenesis was detected by alizarin red S and alkaline phosphatase activity. For in vivo experiments, 40 pregnant mice were given 0.5, 5.0 or 50.0 mg/kg of the extract between days 8 and 18 of gestation. Embryos were stained with alizarin red S and alcian blue to measure femur and ossified region lengths. Total bone mass volume was measured stereometrically. Data were compared with ANOVA and LSD. RESULTS: In limb bud cultures 10 µg/mL of extract reduced chondrogenesis but not osteogenesis. Higher concentrations had no effect on chondrogenesis or osteogenesis. In pregnant mice 50 mg/kg of the extract significantly increased fetal femur and ossified zone length, but significantly decreased bone and cartilage volumes. SIGNIFICANCE: The extract had no favorable effects on chodrification or ossification and appeared to reduce chondrogenesis. This is in apparent contradiction to its empirical effects in human adults.

SHH propagates distal limb bud development by enhancing CYP26B1-mediated retinoic acid clearance via AER-FGF signalling.

The essential roles of SHH in anteroposterior (AP) and AER-FGF signalling in proximodistal (PD) limb bud development are well understood. In addition, these morphoregulatory signals are key components of the self-regulatory SHH/GREM1/AER-FGF feedback signalling system that regulates distal progression of limb bud development. This study uncovers an additional signalling module required for coordinated progression of limb bud axis development. Transcriptome analysis using Shh-deficient mouse limb buds revealed that the expression of proximal genes was distally extended from early stages onwards, which pointed to a more prominent involvement of SHH in PD limb axis development. In particular, retinoic acid (RA) target genes were upregulated proximally, while the expression of the RA-inactivating Cyp26b1 enzyme was downregulated distally, pointing to increased RA activity in Shh-deficient mouse limb buds. Further genetic and molecular analysis established that Cyp26b1 expression is regulated by AER-FGF signalling. During initiation of limb bud outgrowth, the activation of Cyp26b1 expression creates a distal 'RA-free' domain, as indicated by complementary downregulation of a transcriptional sensor of RA activity. Subsequently, Cyp26b1 expression increases as a consequence of SHH-dependent upregulation of AER-FGF signalling. To better understand the underlying signalling interactions, computational simulations of the spatiotemporal expression patterns and interactions were generated. These simulations predicted the existence of an antagonistic AER-FGF/CYP26B1/RA signalling module, which was verified experimentally. In summary, SHH promotes distal progression of limb development by enhancing CYP26B1-mediated RA clearance as part of a signalling network linking the SHH/GREM1/AER-FGF feedback loop to the newly identified AER-FGF/CYP26B1/RA module.

Combined developmental toxicity of bisphenol A and genistein in micromass cultures of rat embryonic limb bud and midbrain cells.

Bisphenol A (BPA), widely used in industry and dentistry, and genistein (GEN), the predominant component of soy product, are both known environmental estrogen. In the present study, we investigated the developmental toxicity of BPA and GEN and their combined effect using micromass test, which is one of three standard alternative developmental toxicity tests recommended by European Center for the Validation of Alternative Methods (ECVAM). The results showed that IC50-P (cell proliferation) and IC50-D (cell differentiation) of BPA and GEN were approximately 20 and 5 µg/ml, respectively. No observed adverse effect level (NOAEL) of BPA and GEN were 10 and 0.94 µg/ml, respectively. The manifestation of BPA as a teratogen was insufficient, although the "low dose" effect should be paid attention to. While the evidence of GEN as a teratogen was solid, especially with the consideration of "high dose" application in clinical treatment. The combined effect of BPA and GEN was generally additive action except that in MB proliferation.

Coordinated and sequential activation of neutral and acidic DNases during interdigital cell death in the embryonic limb.

Interdigital tissue regression during embryonic development is one of the most representative model systems of morphogenetic cell death, but the degenerative cascade accounting for this process awaits clarification. Although the canonical apoptotic caspase pathway appears to be activated in the interdigital mesenchyme committed to die, neither genetic nor chemical blockage of caspases or their downstream effectors, is sufficient to prevent cell death. Hence, alternative and/or complementary dying pathways must also be responsible for this degenerative process. In this work we have chosen to study the endonucleases during the regression of the interdigital tissue of avian embryos to gain insights into the molecular mechanisms accounting for programmed cell death in this system. We show that caspase activated DNase, which is a neutral DNase associated with the caspase apoptotic pathway, appears to be the main endonuclease only at an initial phase of interdigit regression. However at peak stages of the degenerative process, the acidic DNases L-DNase II and lysosomal DNase IIB become predominant in the system and markers for cell autophagy become moderately up-regulated. Consistent with the activation of acidic endonucleases we observed that microenvironmental pH value in the interdigits decreased to levels only appropriate for acidic enzymes. Furthermore, we found that overexpression of lysosomal DNase IIB in embryonic limb mesoderm promoted cell death, which was also accompanied by up-regulation and activation of L-DNase II. Up-regulation of acidic DNases was maintained in interdigits explanted to culture dishes, where the participation of exogenous professional phagocytes of hematopoietic origin is avoided. Finally, and consistent with all our findings, up-regulation of acidic DNases was much reduced in the webbed interdigits of duck embryos, characterized by a rudimentary interdigital degenerative process. We conclude that the regression of the interdigital tissue involves a coordinated and sequential activation of the caspase and lysosomal degenerative molecular cascades.

[Effects of extracts from Patrinia heterophylla on gene expression patterns during morphogenesis of chicken limb buds in vivo].

OBJECTIVE: To study the effects of the extracts from Patrinia heterophylla on gene expression patterns during morphogenesis of chicken limb buds in vivo. METHODS: Implanted a bead into an chicken embryo, which was soaked in the extracts from Patrinia heterophylla. Detected the extracts-induced morphogenesis changes (Myf5, Myod and PCNA). RESULTS: The extracts from Patrinia heterophylla (200 mg/mL) could affect limb bud development, reduce gene expression of MyfS, MyoD and PCNA. CONCLUSION: The extracts from Patrinia heterophylla can inhibit cell differentiation and proliferation.

Comprehensive survey of carapacial ridge-specific genes in turtle implies co-option of some regulatory genes in carapace evolution.

The turtle shell is an evolutionary novelty in which the developmental pattern of the ribs is radically modified. In contrast to those of other amniotes, turtle ribs grow laterally into the dorsal dermis to form a carapace. The lateral margin of carapacial primordium is called the carapacial ridge (CR), and is thought to play an essential role in carapace patterning. To reveal the developmental mechanisms underlying this structure, we systematically screened for genes expressed specifically in the CR of the Chinese soft-shelled turtle, Pelodiscus sinensis, using microbead-based differential cDNA analysis and real-time reverse transcription-polymerase chain reaction. We identified orthologs of Sp5, cellular retinoic acid-binding protein-I (CRABP-I), adenomatous polyposis coli down-regulated 1 (APCDD1), and lymphoid enhancer-binding factor-1 (LEF-1). Although these genes are conserved throughout the major vertebrate lineages, comparison of their expression patterns with those in chicken and mouse indicated that these genes have acquired de novo expression in the CR in the turtle lineage. In association with the expression of LEF-1, the nuclear localization of beta-catenin protein was detected in the CR ectoderm, suggesting that the canonical Wnt signaling triggers carapace development. These findings indicate that the acquisition of the turtle shell did not involve the creation of novel genes, but was based on the co-option of pre-existing genes.

PCNA in situ hybridization: a novel and reliable tool for detection of dynamic changes in proliferative activity.

In order to investigate developmental processes, several methods have been established that allow the visualization of local proliferation zones and to follow their dynamics during morphogenesis. In this study we present a detailed description of transitory and continuous proliferation zones in the developing chick embryo. By tracing the S-phase marker proliferating cell nuclear antigen (PCNA) at the mRNA level we were able to identify the initiation and termination of proliferation programs. This approach provides additional information in comparison to the well-known BrdU incorporation or the PCNA immunostaining, which exclusively labels cells that contain PCNA protein. By means of PCNA in situ hybridization we analyzed the normal expression pattern in the 2- to 5-day-old chick embryo. We furthermore monitored the effects on PCNA expression after various manipulations such as removal of the apical ectodermal ridge (AER), the zone of polarizing activity (ZPA), and the surface ectoderm. In addition, we applied morphogens, such as fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), and retinoic acid (RA), and subsequently analyzed changes in the pattern of PCNA expression. While ablation of ZPA, AER, or ectoderm are known to reduce cell proliferation and were paralleled by loss of PCNA expression, neither BMP-2 nor BMP-4 affected PCNA expression. Upregulation of PCNA expression could be achieved by application of RA or FGFs, factors known to induce cell proliferation during limb bud outgrowth. The PCNA in situ hybridization data presented here clearly show that this method offers a novel, very sensitive tool for tracing cell proliferation and for visualizing the dynamic patterns arising due to the initiation and termination of the proliferation program.

Evaluation of the teratogenicity of fennel essential oil (FEO) on the rat embryo limb buds culture.

The use of FEO as a remedy for control of primary dysmenorrhea increases concern about its potential teratogenicity due to its estrogen-like activity. Limb bud mesenchymal cells, when grown in high-density cultures, can be differentiated into a number of cell types including cartilage and muscle. These cells have been used extensively for in vitro studies of chondrogenesis. Therefore, we used limb bud cells and Alcian blue staining method that is specific for staining cartilage proteoglycan, to determine the teratogenic effect of FEO. Limb bud cells obtained from day 13 rat embryo were cultivated and exposed to various concentrations of FEO for 5 days at 37 degrees C and the number of differentiated foci were counted. Retinoic acid (90 microg/ml) was chosen as positive standard control. The differentiation was also evaluated using limb bud micromass culture using immunocytochemical techniques and BMP-4 antibody. The results showed that FEO at concentration as low as 0.93 mg/ml produced a significant reduction in the number of stained differentiated foci. However, this reduction was due to cell loss, determined by neutral red cell viability assay, rather than to be related to decrease in cell differentiation. These findings suggest that the FEO at the studied concentrations may have toxic effect on fetal cells, but there was no evidence of teratogenicity.

The Krüppel-like factor epiprofin is expressed by epithelium of developing teeth, hair follicles, and limb buds and promotes cell proliferation.

We identified a cDNA clone for epiprofin, which is preferentially expressed in teeth, by differential hybridization using DNA microarrays from an embryonic day 19.5 mouse molar cDNA library. Sequence analysis revealed that this cDNA encodes a member of the Krüppel-like factor family containing three characteristic C2H2-type zinc finger motifs. The full-length cDNA was obtained by the 5' Cap capture method. Except for its 5'-terminal sequence, the epiprofin mRNA sequence is almost identical to the predicted sequence of Krüppel-like factor 14/Sp6 (specificity protein 6), which was previously identified in expressed sequence tag data bases and GenBank by an Sp1 zinc finger DNA-binding domain search (Scohy, S., Gabant, P., Van Reeth, T., Hertveldt, V., Dreze, P. L., Van Vooren, P., Riviere, M., Szpirer, J., and Szpirer, C. (2000) Genomics 70, 93-101). This sequence difference is due to differences in the assignment of the location of exon 1. In situ hybridization revealed that epiprofin mRNA is expressed by proliferating dental epithelium, differentiated odontoblast, and also hair follicle matrix epithelium. In addition, whole mount in situ hybridization showed transient expression of epiprofin mRNA in cells of the apical ectodermal ridge in developing limbs and the posterior neuropore. Transfection of an epiprofin expression vector revealed that this molecule is localized in the nucleus and promotes cell proliferation. Thus, epiprofin is a highly cell- and tissue-specific nuclear protein expressed primarily by proliferating epithelial cells of teeth, hair follicles, and limbs that may function in the development of these tissues by regulating cell growth.

Comparison of embryotoxicity of ESBO and phthalate esters using an in vitro battery system.

Epoxidized soy bean oil (ESBO) and phthalate esters have been used as a plasticizer in polyvinyl chloride products. In this study, the embryotoxicity of ESBO and phthalate esters, namely, diethyl hexyl phthalate (DEHP), butylbenzyl phthalate (BBP) and dibutyl phthalate (DBP) was evaluated using short-term in vitro battery system, such as the whole embryo, midbrain and limb bud culture systems. Whole embryos at gestation day 9.5 were cultured for 48 h and the morphological scoring was measured. The cytotoxic effect and differentiation for mid-brain (MB) and limb bud (LB) cell were assessed by 50% inhibition concentration (IC(50)) with neutral red uptake and hematoxylin-stained foci (MB) or Alcian Blue staining (LB), respectively. In the whole embryo culture assay, ESBO (83, 250 and 750 microg/ml) exerted no toxic effect on growth and development of the embryo, whereas phthalate esters (1, 10, 100 microg/ml for DEHP, 10, 100, 1,000 microg/ml for BBP and DBP) inhibited growth and development dose dependently. In mid-brain and limb bud culture, the IC(50) of differentiation and cytotoxicity in BBP was 412.24 and 231. 76 microg/ml for mid-brain, and 40.13 and 182.38 microg/ml for limb bud, respectively. The IC(50) of differentiation and cytotoxicity in DBP was 27.47 and 44.53 microg/ml for mid-brain, and 21.21 and 25.54 microg/ml for limb bud cells, respectively. The lower IC(50) in both cells was obtained from DBP when compared to BBP. From these results, limb bud cells responded more sensitively to BBP and DBP than mid-brain cells. The IC(50) of limb bud cell differentiation and cytotoxicity in DBP is 1.9 and 7.1 less than that of BBP. However, any alteration in cytotoxicity and differentiation was observed with ESBO treatment. These studies suggested that ESBO is not embryotoxic; however, DEHP, BBP and DBP exhibit embryotoxic potential at high concentration.

Embryonic retinoic acid synthesis is required for forelimb growth and anteroposterior patterning in the mouse.

Numerous studies, often performed on avian embryos, have implicated retinoic acid (RA) in the control of limb bud growth and patterning. Here we have investigated whether the lack of endogenous RA synthesis affects limb morphogenesis in mutant mouse embryos deficient for the retinaldehyde dehydrogenase 2 (Raldh2/Aldh1a2). These mutants, which have no detectable embryonic RA except in the developing retina, die at E9.5-E10 without any evidence of limb bud formation, but maternal RA supplementation through oral gavage from E7.5 can extend their survival. Such survivors exhibit highly reduced forelimb rudiments, but apparently normal hindlimbs. By providing RA within maternal food, we found both a stage- and dose-dependency for rescue of forelimb growth and patterning. Following RA supplementation from E7.5 to 8.5, mutant forelimbs are markedly hypoplastic and lack anteroposterior (AP) patterning, with a single medial cartilage and 1-2 digit rudiments. RA provided until E9.5 significantly rescues forelimb growth, but cannot restore normal AP patterning. Increasing the RA dose rescues the hypodactyly, but leads to lack of asymmetry of the digit pattern, with abnormally long first digit or symmetrical polydactyly. Mutant forelimb buds are characterized by lack of expression or abnormal distal distribution of Sonic hedgehog (Shh) transcripts, sometimes with highest expression anteriorly. Downregulation or ectopic anterior expression of Fgf4 is also seen. As a result, genes such as Bmp2 or Hoxd genes are expressed symmetrically along the AP axis of the forelimb buds, and/or later, of the autopod. We suggest that RA signaling cooperates with a posteriorly restricted factor such as dHand, to generate a functional zone of polarizing activity (ZPA).

Differential expression of chicken CYP26 in anterior versus posterior limb bud in response to retinoic acid.

Multiple studies indicate that quantitative control of the levels of all-trans-retinoic acid (RA) in the vertebrate embryo is necessary for correct development. The function of RA in cells is regulated by a number of coordinated mechanisms. One of those mechanisms involves controls on the rate of RA catabolism. Recently, enzymes capable of catabolizing RA were found to constitute a new family, called CYP26, within the cytochrome P450 superfamily. CYP26 homologues have been isolated from human, mouse, zebra fish, and recently from the chick. In this study, we examined the regulation of chicken CYP26 (cCYP26) expression by RA during the early phase of chick limb outgrowth. In the anterior limb mesenchyme and apical ectodermal ridge (AER), cCYP26 expression was induced in a concentration dependent manner by implanting beads soaked in 0.1, 1, and 5 mg/ml RA. The RA-induced expression of cCYP26 in anterior limb mesenchyme and the AER was detected as early as 1 hr after treatment and was not affected by the presence of cycloheximide. In contrast to the anterior limb, the induction of cCYP26 was dramatically reduced (or absent) when RA beads were implanted in the posterior limb mesenchyme. Furthermore, induction of cCYP26 expression in the anterior mesenchyme was inhibited by transplantations of the zone of polarizing activity (ZPA) and by Shh-soaked beads. Our data suggest that different mechanisms regulate retinoid homeostasis in the AER and mesenchyme during limb bud outgrowth. J. Exp. Zool. 290:136-147, 2001.

Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb.

Ci/Gli zinc finger proteins mediate the transcriptional effects of Hedgehog protein signals. In Drosophila, Ci action as transcriptional repressor or activator is contingent upon Hedgehog-regulated, PKA-dependent proteolytic processing. We demonstrate that PKA-dependent processing of vertebrate Gli3 in developing limb similarly generates a potent repressor in a manner antagonized by apparent long-range signaling from posteriorly localized Sonic hedgehog protein. The resulting anterior/posterior Gli3 repressor gradient can be perturbed by mutations of Gli3 in human genetic syndromes or by misregulation of Gli3 processing in the chicken mutant talpid2, producing a range of limb patterning malformations. The high relative abundance and potency of Gli3 repressor suggest specialization of Gli3 and its products for negative Hedgehog pathway regulation.

The use of a retinoid receptor antagonist in a new model to study vitamin A-dependent developmental events.

Multiple fetal anomalies occur in vitamin A deficient animals as well as in retinoic acid receptor gene 'knockout' mice, indicating that retinoic acid (an active metabolite of vitamin A) performs some essential functions in normal development. Additional approaches are needed to probe directly the stages and sites in the embryo where a presence of endogenous retinoic acid is indispensable. We have employed a new strategy for this purpose which involved an intervention in retinoic acid receptor (RAR)-dependent functions at specific developmental stages by means of a highly effective RAR antagonist, AGN 193109. We report that in an in vitro cell differentiation bioassay, AGN 193109 completely reversed the inhibitory action of a potent RAR agonist, AGN 190121. In pregnant mice, a single oral 1 mg/kg dose of the antagonist given on 8 day post coitum (dpc) produced a severe craniofacial anomaly (median cleft face or frontonasal dysplasia) and eye malformations in virtually all exposed fetuses. On the other hand, treatment on 11 dpc, a time in development when RARs are strategically expressed in the limb bud primordium, no limb anomalies could be induced by the antagonist. Even after a high dose of 100 mg/kg, limb development progressed normally in spite of the fact that measurable concentrations of the antagonist were present. Because retinoids are long known to influence skin morphology, we next monitored the effects of the antagonist on skin development. When given late in gestation, on 14 dpc, we found that the antagonist delayed differentiation and maturation of the fetal skin and hair follicles. We conclude that this model provides a convenient and pertinent system which enables us to seek and clarify true functions of retinoic acid and its cognate receptors in embryogenesis and in adult animals.

[Application of mouse limb bud culture to study the influence of zinc on teratogenesis induced by cadmium].

In this study, an in vitro method of mouse limb bud culture in self-made rotator with continuous supplementation of gas mixture was employed in studying the teratogenic potential of cadmium and the influence of zinc on the teratogenesis induced by cadmium. Image analysis on the area and the form of the bone analgen of the cultured limb was used to evaluate quantitatively their teratogenic potentials. Different amounts of cadmium were directly added to culture medium. As cadmium concentrations were increased from 0.1 to 1.0 microgram/ml, the degree of morphogenetic differentiation and the area of the bone anlagen of limbs culture were significantly decreased. The paws and long bones were affected seriously. Cadmium had a greater effect on chondrogenic tissue than on soft tissue. Then various levels of Zn, together with cadmium (1.0 microgram/ml medium), were added into the culture media. As Zn concentrations increased from 1.0 to 10.0 micrograms/ml, the degree of morphogenetic differentiation and the area of cartilaginous bone anlagen of limbs culture were improved or increased. The long bones were better ameliorated as compared with the paw.

Multigenic control of the localization of the zone of polarizing activity in limb morphogenesis in the mouse.

We report here that in three preaxial polydactylous mutants in the mouse, namely, 1st, 1x, and Xp1, ectopic expression of the Shh and Fgf-4 genes can be detected at the anterior margin of limb buds. These and three other mutants, namely, Rim4, Hx, and Xt1, which we described in our previous study, all appeared to form a duplicated zone of polarizing activity (ZPA) at the anterior margin of the limb bud. We studied the spatial and temporal pattern of expression of the Gli3 gene, which is affected in a loss-of-function type of mutation, Xt1. The expression domain of Gli3 appeared to be complementary to the ZPA region and the gene was expressed prior to Shh. The results support the hypothesis that GLI3 functions in the anterior portion of limb mesoderm to suppress the expression of Shh. In Drosophila, the gene ci, the fly homologue of Gli, functions to repress hh, suggesting that the negative regulation of the expression of hedgehog by genes belonging to the GLI-kruppel family has been conserved from flies to mice. Finally, we found that the polydactylous phenotype of the mutants Rim4, Xt, 1st, and 1x could be abrogated by the crossing with an inbred strain derived from wild mouse, MSM, whereas the phenotype of Xp1 could not. These results indicate the presence of a modifier gene(s) that can influence the mutant phenotype and also that the mutations could be classified into two categories with regard to the mode of interaction with the modifier gene(s). Thus, this study revealed a multigenic control in the establishment of the anteroposterior axis in mouse limb development.