Peptidoglycan recognition proteins（PGRPs） are a family of pattern recognition receptors（PRRs） of the immune system,which bind and hydrolyze bacterial peptidoglycan.Here,a long type PGRP（PGRP-L） was first cloned ...Peptidoglycan recognition proteins（PGRPs） are a family of pattern recognition receptors（PRRs） of the immune system,which bind and hydrolyze bacterial peptidoglycan.Here,a long type PGRP（PGRP-L） was first cloned in the lower vertebrate species Xenopus tropicalis（Xt）.The XtPGRP-L possessed a conserved genomic structure with five exons and four introns.The alignment and phylogenetic analysis indicated that XtPGRP-L might be a type of amidase-like PGRP.The 3-D model showed that XtPGRP-L possessed a conserved structure compared with the Drosophila PGRP-Lb.During embryonic development,XtPGRP-L was not expressed until the 72 h tadpole stage.In adult tissues,it was strongly expressed in the liver,lung,intestine,and stomach.Furthermore,after LPS stimulation,the expression of XtPGRP-L was up-regulated significantly in the liver,intestine and spleen,indicating that XtPGRP-L may play an important role in the innate immunity of Xenopus tropicalis.展开更多
A vector-based RNAi expression system was developed using the Xenopus tropicalis U6 promoter, which transcribes small RNA genes by RNA polymerase Ⅲ. The system was first validated in a Xenopus laevis cell line, desig...A vector-based RNAi expression system was developed using the Xenopus tropicalis U6 promoter, which transcribes small RNA genes by RNA polymerase Ⅲ. The system was first validated in a Xenopus laevis cell line, designing a short hairpin DNA specific for the GFP gene. Co-transfection of the vector-based RNAi and the GFP gene into Xenopus XR1 cells significantly decreased the number of GFP-expressing cells and overall GFP fluorescence. Vector-based RNAi was subsequently validated in GFP transgenic Xenopus embryos. Sperm nuclei from GFP transgenic males and RNAi construct-incubated-sperm nuclei were used for fertilization, respectively. GFP mRNA and protein were reduced by -60% by RNAi in these transgenic embryos compared with the control. This transgene-driven RNAi is specific and stable in inhibiting GFP expression in the Xenopus laevis transgenic line. Gene silencing by vector-based RNAi and Xenopus transgenesis may provide an alternative for ＇repression of gene function＇ studies in vertebrate model systems.展开更多
We describe the temporal and spatial expression pattern of Sox 1 gene during Xenopus laevis early development and compare the expression patterns of Sox 1-3 in the developing eye and brain. Alignment of Sox 1-3 amino ...We describe the temporal and spatial expression pattern of Sox 1 gene during Xenopus laevis early development and compare the expression patterns of Sox 1-3 in the developing eye and brain. Alignment of Sox 1-3 amino acid sequences shows a high conservation within the HMG-box DNA binding domains. RT-PCR analysis indicates that Sox 1 is expressed throughout development from the unfertilized egg to at least the tadpole stage, although at different expression levels. The transcripts of XSox 1 are detected in the animal pole at cleavage and blastrula stages and mainly in the central nervous system （CNS） and the developing eye at neurula stages. The study of the developmental expression of XSox 1 will aid in the elucidation of the function of SoxB 1 subgroup genes in vertebrate neurogenesis.展开更多
Accumulated evidence indicates that the activating transcription factor 4（atf4） is a developmentally relevant gene.Here,we report on the characterization of atf4 in Xenopus embryos,which is differentially expressed ...Accumulated evidence indicates that the activating transcription factor 4（atf4） is a developmentally relevant gene.Here,we report on the characterization of atf4 in Xenopus embryos,which is differentially expressed in the central nervous system,eyes,blood,and the pronephros,as well as in developing endodermal organs such as the stomach,duodenum,liver,and pancreas.Ectopic expression of atf4 in the animal hemisphere of Xenopus embryos had no obvious effects on the induction of neural progenitors,but suppressed neurogenesis and eye formation without promoting apoptosis.Our data suggest that tightly controlled atf4 activities may be crucial for normal neurogenesis and early eye patterning.展开更多
Neural tube defects （NTDs） are severe congenital malformation diseases, which occur in 1 out of 1000 births in human. In Xenopus, several tissue movements are involved in the neural tube closure process. Immediately...Neural tube defects （NTDs） are severe congenital malformation diseases, which occur in 1 out of 1000 births in human. In Xenopus, several tissue movements are involved in the neural tube closure process. Immediately after the neural tube fusion, the neural crest cells get monopolar protrusion toward dorsal midline and migrate to form the roof of the neural tube. At the same time, radial intercalation takes place from the ventral neural tube and forces it to be single-layered. Here, we physically block the neural tube closure to test the cell movements and the following patterning in Xenopus laevis explants. The results show that the single-layered neural tube fails to form and the neural crest cells remain at the lateral regions in the explants with NTDs. However, the patterning of the neural tube is not affected as indicated by the normal expression of the preneural genes. These results indicate a requirement of the neural tube fusion for the radial intercalation and the dorsal midline directed neural crest migration, but not for the dorsal-ventral patterning of the neural tube.展开更多
Many chemicals are released into the environment, and chemical contamination has been suggested as a contributing factor to amphibian declines. To add to a growing body of knowledge about the impact of individual chem...Many chemicals are released into the environment, and chemical contamination has been suggested as a contributing factor to amphibian declines. To add to a growing body of knowledge about the impact of individual chemicals on non-target organisms, we examined the specificity of deformities induced by exposure to four pesticides （atrazine, 2,4-dichloropheoxyacetic acid （2,4-D）, triadimefon, and glyphosate） in the model amphibian species, Xenopus laevis. We focused on the period of organ morphogenesis, as it is frequently found to be particularly sensitive to chemical exposure yet also commonly overlooked. We found similar levels of intestine malformations and edemas, as well as disruption of skeletal muscle, in atrazine and triadimefon exposed tadpoles. The effects of 2,4-D were only apparent at the highest concentrations we examined; glyphosate did not induce dramatic malformations at the concentrations tested. While researchers have shown that it is important to understand how chemical mixtures affect non-target organisms, our results suggest that it is first crucial to determine how these chemicals act independently in order to be able to identify consequences of individual pesticide exposure.展开更多
T3-induced Xenopus metamorphosis is an ideal model for detecting thyroid hormone（TH）signaling disruption of chemicals. To optimize the T3-induced Xenopus assay and improve its sensitivity and reproducibility, we int...T3-induced Xenopus metamorphosis is an ideal model for detecting thyroid hormone（TH）signaling disruption of chemicals. To optimize the T3-induced Xenopus assay and improve its sensitivity and reproducibility, we intend to develop quantitatively morphological endpoints and choose appropriate concentrations and exposure durations for T3 induction.Xenopus laevis at stage 52 were exposed to series of concentrations of T3（0.31–2.5 nmol/L）for 6 days. By comparing morphological changes induced by T3, we propose head area,mouth width, unilateral brain width/brain length, and hindlimb length/snout-vent length as quantitative parameters for characterizing T3-induced morphological changes, with body weight as a parameter for indicating integrated changes. By analyzing time-response curves, we found that following 4-day exposure, T3-induced grossly morphological changes displayed linear concentration–response curves, with moderate morphological changes resulting from 1.25 nmol/L T3 exposure. When using grossly morphological endpoints to detect TH signaling disruption, we propose 4 days as exposure duration of T3, with concentrations close to 1.25 nmol/L as induction concentrations. However, it is appropriate to examine morphological and molecular changes of the intestine on day 2 due to their early response to T3. The quantitative endpoints and T3 induction concentrations and durations we determined would improve the sensitivity and the reproducibility of the T3-induced Xenopus metamorphosis assay.展开更多
We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspensioncultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA f...We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspensioncultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA fragments. The effects of different divalent cations and cycloheximide on DNA cleavage in this system were assessed.The fact that nuclei of plant cells can be induced to undergo apoptosis in a cell-free animal system suggests that animals and plants share a common signal transduction pathway triggering in the initiation stage of apoptosis.展开更多
Decabromodiphenyl ether （decaBDE）,as a flame retardant,is widely produced and used.To study the thyroid disruption by technical decaBDE at low concentrations,Xenopus laevis tadpoles were exposed to technical decaBDE...Decabromodiphenyl ether （decaBDE）,as a flame retardant,is widely produced and used.To study the thyroid disruption by technical decaBDE at low concentrations,Xenopus laevis tadpoles were exposed to technical decaBDE mixture DE-83R （1-1000 ng/L） in water from stage 46/47 （free swimming larvae,system of Nieuwkoop and Faber） to stage 62.DE-83R at concentration of 1000 ng/L significantly delayed the time to metamorphosis （presented by forelimb emergence,FLE）.Histological examination showed that DE83R at all tested concentrations caused histological alterations-multilayer follicular epithelial cell and markedly increased follicle size accompanied by partial colloid depletion and increase in the peripheral colloid vacuolation,in thyroid glands.All tested concentrations of DE-83R also induced a down-regulation of thyroid receptor mRNA expression.These results demonstrated that technical decaBDE disrupted the thyroid system in X.laevis tadpoles.Analysis of polybrominated diphenyl ethers （PBDEs） （sum of 39 congeners） in X.laevis indicated that mean concentrations of total PBDEs in X.laevis exposed to 1,10,100,1000 ng/L were 11.0,128.1,412.1,1400.2 ng/g wet weight,respectively.Considering that PBDEs burden of X.laevis tadpoles was close to PBDEs levels in amphibians as reported in previous studies,our study has raised new concerns for thyroid disruption in amphibians of technical decaBDE at environmentally relevant concentrations.展开更多
We developed the T3-induced Xenopus metamorphosis assay, which is supposed to be able to sensitively detect thyroid hormone（TH） signaling disruption of chemicals. The present study aimed to validate the T3-induced X...We developed the T3-induced Xenopus metamorphosis assay, which is supposed to be able to sensitively detect thyroid hormone（TH） signaling disruption of chemicals. The present study aimed to validate the T3-induced Xenopus metamorphosis assay by re-evaluating the TH signaling antagonism of tetrabromobisphenol A（TBBPA）, a known TH signaling disruptor. According to the assay we developed, Xenopus tadpoles at stage 52 were exposed to 10–500 nmol/L TBBPA in the presence of 1 nmol/L T3. After 96 hr of exposure, TBBPA in the range of 10–500 nmol/L was found to significantly inhibit T3-induced morphological changes of Xenopus tadpoles in a concentration-dependent manner in term of body weight and four morphological endpoints including head area（HA）, mouth width（MW）, unilateral brain width/brain length（ULBW/BL）, and hind-limb length/snout-vent length（HLL/SVL）.The results show that these endpoints we developed are sensitive for characterizing the antagonistic effects of TBBPA on T3-induced metamorphosis. Following a 24-hr exposure,we found that TBBPA antagonized expression of T3-induced TH-response genes in the tail,which is consistent with previous findings in the intestine. We propose that the tail can be used as an alternative tissue to the intestine for examining molecular endpoints for evaluating TH signaling disruption. In conclusion, our results demonstrate that the T3-induced Xenopus metamorphosis assay we developed is an ideal in vivo assay for detecting TH signaling disruption.展开更多
Inositol requiring enzyme-1 （IRE1） is highly conserved from yeasts to humans. Upon the endoplasmic reticu- lum （ER） stress, IRE1 activates X-box-binding protein 1 （XBP1） by unconventionally splicing XBP1 mRNA, w...Inositol requiring enzyme-1 （IRE1） is highly conserved from yeasts to humans. Upon the endoplasmic reticu- lum （ER） stress, IRE1 activates X-box-binding protein 1 （XBP1） by unconventionally splicing XBP1 mRNA, which activates the unfolded protein response （UPR） to restore ER homeostasis. In mice, IREla inactivity leads to embryonic death and IREla plays an essential role in extraembryonic tissues and the placenta. However, its precise action in the embryo proper is still unknown. In this study, the loss of function ana/ysis was performed to investigate the function of Xenopus IREla （xlREla） during pancreas development. Firstly, the complete open reading frame of xIRE1α was amplified and the expression pattern was detected. The effects of Xenopus IRE1α and XBP1 during embryo development were detected with whole-mount in situ hybridization. The results demonstrated that xIRE1α was much closer to human IREla when compared with their sequence alignment, xlREla was expressed strongly in developing pancreas and the knockdown of xIREla inhibited the differentiation and specification of the pancreas, xlREltt, which was required for cytoplasmic splicing of XBP1 pre-mRNA and XB- P1MO, also showed inhibitory effects on pancreas development. These results suggest that xlREla is essential for pancreas development during embryogenesis and functions via the XBP1 dependent pathway.展开更多
Tributyltin（TBT）,a biocide used in antifouling paints,has shown strong teratogenic effects on Xenopus tropicalis embryos at environmentally relevant concentrations.X.tropicalis embryos were exposed to 50,100 and 200...Tributyltin（TBT）,a biocide used in antifouling paints,has shown strong teratogenic effects on Xenopus tropicalis embryos at environmentally relevant concentrations.X.tropicalis embryos were exposed to 50,100 and 200 ng/L tributyltin chloride for 72 hr.The histological changes were further observed on abnormal eyes,enlarged trunks,enlarged proctodaeums and absence of fins induced by TBT.The lens and the retinal layers of abnormal eyes were slightly or barely differentiated,and that the pigment epithelium was neither continuous nor smooth.The abdomens were full of undifferentiated gut tissue with yolk-rich inclusions in the tadpoles with enlarged trunks.The proctodaeums formed a bump-like or columnar structure.The mass of yolk-rich cells occupied the lumen,blocked the opening and even turned inside out of the proctodaeum.Both the ventral and dorsal fins in trunks and tails became narrow or even disappeared totally.Our results suggest that great changes of histology took place corresponding to the unique phenotypes.The gut tissue was poorly differentiated,which led to the failed elongation of the guts and subsequently the enlarged trunks.The enlarged proctodaeums were due to the undifferentiation of inner layer,the expansion of outer epidermal part and the absence of fins around them.In brief,the histological observations provided insights into the reason of the unique external malformations in some degree.展开更多
AIM:To investigate the role of inositol-requiring enzyme 1α(IRE1α) in gut development of Xenopus lavies embryos.METHODS:Xenopus embryos were obtained with in vitro fertilization and cultured in 0.1 × MBSH.One a...AIM:To investigate the role of inositol-requiring enzyme 1α(IRE1α) in gut development of Xenopus lavies embryos.METHODS:Xenopus embryos were obtained with in vitro fertilization and cultured in 0.1 × MBSH.One and half nanogram of IRE1α,1 ng of IRE1α-GR mRNA,1 ng of IRE1αΔC-GR mRNA,and 50 ng of IRE1α morpholino oligonucleotide(MO) or XBP1(C)MO were injected into four blastomeres at 4-cell stage for scoring the phenotype and marker gene analysis.To rescue the effect of IRE1α MO,1 ng of IRE1α-GR mRNA was coinjected with 50 ng of MO.For the activation of the GR-fusion proteins,dexamethasone was prepared as 5 mmol/L stock solutions in 100% ethanol and applied to the mRNA injected embryos at desired stages in a concentration of 10 μmol/L in 0.1 × MBSH.Embryos were kept in dexamethasone up to stage 41.Whole-mount in situ hybridization was used to determine specific gene expression,such as IRE1α,IRE1β,Xbra and Xsox17α.IRE1α protein expression during Xenopus embryogenesis was detected by Western blotting.RESULTS:In the whole-mount in situ hybridization analysis,xenopus IRE1α and IRE1β showed quite different expression pattern during tadpole stage.The relatively higher expression of IRE1α was observed in the pancreas,and significant transcription of IRE1β was found in the liver.IRE1α protein could be detected at all developmental stages analyzed,from stage 1 to stage 42.Gain-of-function assay showed that IRE1α mRNA injected embryos at tailbud stage were nearly normal and the expression of the pan-mesodermal marker gene Xbra and the endodermal gene Xsox17α at stage 10.5 was not significantly changed in embryos injected with IRE1α mRNA as compared to uninjected control embryos.And at tadpole stage,the embryos injected with IRE1α-GR mRNA did not display overt phenotype,such as gut-coiling defect.Loss-of-function assay demonstrated that the IRE1α MO injected embryos were morphologically normal before the tailbud stages.We did not observe a significant change of mesodermal and endodermal marker gene expression,while after stage 40,about 80% of the MO injected embryos exhibited dramatic gut defects in which the guts did not coil,but other structures outside the gastrointestinal tract were relatively normal.To test if the phenotypes were specifically caused by the knockdown of IRE1α,a rescue experiment was performed by co-injection of IRE1α-GR mRMA with IRE1α MO.The data obtained demonstrated that the gut coiling defect was rescued.The deletion mutant of IRE1α was constructed,consisting of the N-terminal part without the C-terminal kinase and RNase domains named IRE1αΔC,to investigate the functional domain of IRE1α.Injection of IRE1αΔCGR mRNA caused similar morphological alterations with gut malformation by interfering with the function of endogenous xIRE1α.In order to investigate if IRE1α/XBP1 pathway was involved in gut development,50 ng of XBP1 MO was injected and the results showed that knockdown of XBP1 resulted in similar morphological alterations with gut-coiling defect at tadpole stage.CONCLUSION:IRE1α is not required for germ layer formation but for gut development in Xenopus lavies and it may function via XBP1-dependent pathway.展开更多
DEAR EDITOR Protein arginine methyltransferases (PRMTs)are involved in many cellular processes via the arginine methylation of histone or non-histone proteins.We examined the expression patterns of prmt4,prmt7,and prm...DEAR EDITOR Protein arginine methyltransferases (PRMTs)are involved in many cellular processes via the arginine methylation of histone or non-histone proteins.We examined the expression patterns of prmt4,prmt7,and prmt9 during embryogenesis in Xenopus using whole-mount in situ hybridization and quantitative reverse transcription polymerase chain reaction (RT-PCR).Xenopus prmt4 and prmt7 were expressed in the neural crest,brain,and spinal cord,and also detected in the eye,branchial arches,and heart at the tailbud stage.Specific print9 signals were not detected in Xenopus embryos until the late tailbud stage when weak expression was observed in the branchial arches.Quantitative RT-PCR indicated that the expression of prmt4 and prmt7 was up-regulated during the neurula stage,whereas prmt9 maintained its low expression until the late tailbud stage,consistent with the whole-mount in situ hybridization results.Thus,the developmental expression patterns of these three print genes in Xenopus embryos provide a basis for further functional study of such genes.展开更多
Tissue inhibitors of metalloproteinases (TIMPs) modulate extracellular matrix remodeling during embryonic develop- ment and disease. TIMP-3 expression was examined during Xenopus laevis embryogenesis: TIMP-3 transcrip...Tissue inhibitors of metalloproteinases (TIMPs) modulate extracellular matrix remodeling during embryonic develop- ment and disease. TIMP-3 expression was examined during Xenopus laevis embryogenesis: TIMP-3 transcripts detected in the maternal pool of RNA increased at the mid-blastula transition, decreased dramatically during gastrulation and increased again during neurulation and axis elongation. Interestingly, the decrease during gastrulation was not seen in LiCl treated (dorsalized) embryos. Whole mount in situ hybridization of TIMP-3 using DIG-labeled RNA probes demonstrated that the transcripts were present in all dorsal tissues during embryogenesis, but were prominent only in head structures starting at stage 35. Overexpression of TIMP-3 through transgenesis and RNA injections led to devel- opmental abnormalities and death. Both overexpression strategies resulted in post-gastrulation perturbation including those to neural and head structures, as well as truncated axes. However, RNA injections resulted in more severe early defects such as failure of neural tube closure, and transgenesis caused truncated axes and head abnormalities. No transgenic embryo expressing TIMP-3 survived past stage 40.展开更多
Kruppel-like factor 4（Klf4） is a zinc finger transcription factor and plays crucial roles in Xenopus embryogenesis.However, its regulation during embryogenesis is still unclear. Here, we report that Tcf711, a key do...Kruppel-like factor 4（Klf4） is a zinc finger transcription factor and plays crucial roles in Xenopus embryogenesis.However, its regulation during embryogenesis is still unclear. Here, we report that Tcf711, a key downstream transducer of the Wnt signaling pathway, could promote Klf4 transcription and stimulate Klf4 promoter activity in early Xenopus embryos. Furthermore, cycloheximide treatment showed a direct effect on Klf4 transcription facilitated by Tcf711. Moreover, the dominant negative form of Tcf711（dnTcf711）, which lacks N-terminus of the β-catenin binding motif, could still activate Klf4 transcription, suggesting that this regulation is Wnt/β-catenin independent.Taken together, our results demonstrate that Tcf711 lies upstream of Klf4 to maintain its expression level during Xenopus embryogenesis.展开更多
There is a pressing need for developing in vivo or ex vivo assays to screen the glucocorticoid（GC） signaling disruption of chemicals. Thus, we aimed to establish an ex vivo assay for screening GC signaling disruptio...There is a pressing need for developing in vivo or ex vivo assays to screen the glucocorticoid（GC） signaling disruption of chemicals. Thus, we aimed to establish an ex vivo assay for screening GC signaling disruption based on the GC-response gene transcription in Xenopus laevis tails cultured ex vivo. Firstly, we investigated effects of corticosterone（CORT, a main GC in frogs） on GC-response gene expression, and determined the six genes as molecular endpoints for assaying the GC signaling disruption. CORT in the range of 1.56–400 nmol/L was found to up-regulate transcription of the six GC-response genes, exhibiting comparable or higher sensitivity than previously reported assays. To validate this ex vivo assay, then, we examined effects of dexamethasone（a known GC signaling agonist） on GC-response gene expression. Dexamethasone displayed an agonistic action in a concentration-dependent manner, further demonstrating the efficiency of the established assay. Finally, we applied the ex vivo assay to evaluate the GC signaling disruption of bisphenol A（BPA）. In accordance with previous reports, we found a concentration-dependent agonistic activity of BPA,showing that the established assay is effective for detecting the GC signaling disrupting activity of environmental chemicals. Correspondingly, the GC signaling agonistic actions of CORT and BPA in ex vivo tails accorded with the observations in vivo, indicating that the ex vivo assay is able to detect the actions of chemicals in vivo. Overall, we established an ex vivo assay that can effectively screen GC signaling disruption of environmental chemicals.展开更多
The homozygous inv （inversion of embryonic turning） mouse mutant shows situs inversus and polycystic kidney disease, both of which result from the lack of the inv gene. Previously, we suggested that inv may be impor...The homozygous inv （inversion of embryonic turning） mouse mutant shows situs inversus and polycystic kidney disease, both of which result from the lack of the inv gene. Previously, we suggested that inv may be important for the left-right axis formation, not only in mice but also in Xenopus, and that calmodulin regulates this inv protein function. Here, we isolated and characterized two Xenopus laevis homologs （Xinv-1 and Xinv-2） of the mouse inv gene, and performed functional analysis of the conserved IQ motifs that interact with calmodulin. Xinv-1 expresses early in development in the same manner as mouse inv does. Unexpectedly, a full-length Xenopus inv mRNA did not randomize cardiac orientation when injected into Xenopus embryos, which is different from mouse inv mRNA. Contrary to mouse inv mRNA, Xenopus inv mRNA with mutated IQ randomized cardiac orientation. The present study indicates that calmodulin binding sites （IQ motifs） are crucial in controlling the biological activity of both mouse and Xenopus inv proteins. Although mouse and Xenopus inv genes have a quite similar structure, the interaction with calmodulin and IQ motifs ofXenopus inv and mouse inv proteins may regulate their function in different ways.展开更多
Anti-keratin monoclonal antibody AF5 was introduced into fertilized eggs of Xenopus laevis., and its effects on embryonic development were studied. Survival rate of the antikeratin-injected embryos was much lower (onl...Anti-keratin monoclonal antibody AF5 was introduced into fertilized eggs of Xenopus laevis., and its effects on embryonic development were studied. Survival rate of the antikeratin-injected embryos was much lower (only 35.76% at gastrula) than that of the control (74.85% at gastrula), in which embryos were injected with mouse IgG. Most of survivors in the experimental series showed aberrant external appearance. On the other hand, in cleavage stage, ie 2-7 h after fertilization, immunohistochemi-cal staining of embryos showed that the experimental embryos were mostly keratin negative, while embryos of the control ones were keratin positive. When introducing this antikeratin into one cell of a 2-cell embryo, only the unin-jected half of the embryo continued its development while the other half could not develop at all. These results suggested that intact keratin cytoskeleton in early embryos is indispensable to the embryonic development of Xenopus laevis.展开更多
The complex transformation of a tadpole to a frogduring amphibian development is under the control of thyroid hormone (T3). T3 is known to regulate gene transcription through its nuclear receptors. We have previouslyi...The complex transformation of a tadpole to a frogduring amphibian development is under the control of thyroid hormone (T3). T3 is known to regulate gene transcription through its nuclear receptors. We have previouslyisolated many genes which are up-regulated by T3 in theintestine of Xenopus laevis tadpoles. We have now cloneda full- length cDNA for one such gene (IU12). Sequenceanalysis shows that the IU12 cDNA encodes a plasmamembrane protein with 12 transmembrane domains andhomologous to a mammalian gene associated with cell activation and organ development. Similarly, we have foundthat IU12 is activated during intestinal remodeling whenboth cell death and proliferation take place. Furthermore,IU12 is an early T3-response gene and its expression in theintestine during T3-induced metamorphosis mimics thatduring normal development. These results argue for a roleof IU12 in the signal transduction pathways leading to intestinal metamorphosis.展开更多
基金supported by the Project from the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (10KJB240001)the Foundation for Talent Recruitment of Yancheng Institute of Technology (XKR2011007)the National Natural Science Foundation of China (30830083)
文摘Peptidoglycan recognition proteins（PGRPs） are a family of pattern recognition receptors（PRRs） of the immune system,which bind and hydrolyze bacterial peptidoglycan.Here,a long type PGRP（PGRP-L） was first cloned in the lower vertebrate species Xenopus tropicalis（Xt）.The XtPGRP-L possessed a conserved genomic structure with five exons and four introns.The alignment and phylogenetic analysis indicated that XtPGRP-L might be a type of amidase-like PGRP.The 3-D model showed that XtPGRP-L possessed a conserved structure compared with the Drosophila PGRP-Lb.During embryonic development,XtPGRP-L was not expressed until the 72 h tadpole stage.In adult tissues,it was strongly expressed in the liver,lung,intestine,and stomach.Furthermore,after LPS stimulation,the expression of XtPGRP-L was up-regulated significantly in the liver,intestine and spleen,indicating that XtPGRP-L may play an important role in the innate immunity of Xenopus tropicalis.
文摘A vector-based RNAi expression system was developed using the Xenopus tropicalis U6 promoter, which transcribes small RNA genes by RNA polymerase Ⅲ. The system was first validated in a Xenopus laevis cell line, designing a short hairpin DNA specific for the GFP gene. Co-transfection of the vector-based RNAi and the GFP gene into Xenopus XR1 cells significantly decreased the number of GFP-expressing cells and overall GFP fluorescence. Vector-based RNAi was subsequently validated in GFP transgenic Xenopus embryos. Sperm nuclei from GFP transgenic males and RNAi construct-incubated-sperm nuclei were used for fertilization, respectively. GFP mRNA and protein were reduced by -60% by RNAi in these transgenic embryos compared with the control. This transgene-driven RNAi is specific and stable in inhibiting GFP expression in the Xenopus laevis transgenic line. Gene silencing by vector-based RNAi and Xenopus transgenesis may provide an alternative for ＇repression of gene function＇ studies in vertebrate model systems.
文摘We describe the temporal and spatial expression pattern of Sox 1 gene during Xenopus laevis early development and compare the expression patterns of Sox 1-3 in the developing eye and brain. Alignment of Sox 1-3 amino acid sequences shows a high conservation within the HMG-box DNA binding domains. RT-PCR analysis indicates that Sox 1 is expressed throughout development from the unfertilized egg to at least the tadpole stage, although at different expression levels. The transcripts of XSox 1 are detected in the animal pole at cleavage and blastrula stages and mainly in the central nervous system （CNS） and the developing eye at neurula stages. The study of the developmental expression of XSox 1 will aid in the elucidation of the function of SoxB 1 subgroup genes in vertebrate neurogenesis.
基金supported in part by funds from the Key Project of Knowledge Innovation Program of the Chinese Academy of Sciences(KSCX2-YW-R-083)the National Basic Research Program of China(2009CB941202)
文摘Accumulated evidence indicates that the activating transcription factor 4（atf4） is a developmentally relevant gene.Here,we report on the characterization of atf4 in Xenopus embryos,which is differentially expressed in the central nervous system,eyes,blood,and the pronephros,as well as in developing endodermal organs such as the stomach,duodenum,liver,and pancreas.Ectopic expression of atf4 in the animal hemisphere of Xenopus embryos had no obvious effects on the induction of neural progenitors,but suppressed neurogenesis and eye formation without promoting apoptosis.Our data suggest that tightly controlled atf4 activities may be crucial for normal neurogenesis and early eye patterning.
基金supported by grants from the National Natural Science Foundation of China (30425011 30530380)the Innovation Project of the Chinese Academy of Sciences (KSCX2-YW-R-090)~~
文摘Neural tube defects （NTDs） are severe congenital malformation diseases, which occur in 1 out of 1000 births in human. In Xenopus, several tissue movements are involved in the neural tube closure process. Immediately after the neural tube fusion, the neural crest cells get monopolar protrusion toward dorsal midline and migrate to form the roof of the neural tube. At the same time, radial intercalation takes place from the ventral neural tube and forces it to be single-layered. Here, we physically block the neural tube closure to test the cell movements and the following patterning in Xenopus laevis explants. The results show that the single-layered neural tube fails to form and the neural crest cells remain at the lateral regions in the explants with NTDs. However, the patterning of the neural tube is not affected as indicated by the normal expression of the preneural genes. These results indicate a requirement of the neural tube fusion for the radial intercalation and the dorsal midline directed neural crest migration, but not for the dorsal-ventral patterning of the neural tube.
基金NSF REU (DBI 0649190)Tufts Summer Scholars and Marshall Awards for funding
文摘Many chemicals are released into the environment, and chemical contamination has been suggested as a contributing factor to amphibian declines. To add to a growing body of knowledge about the impact of individual chemicals on non-target organisms, we examined the specificity of deformities induced by exposure to four pesticides （atrazine, 2,4-dichloropheoxyacetic acid （2,4-D）, triadimefon, and glyphosate） in the model amphibian species, Xenopus laevis. We focused on the period of organ morphogenesis, as it is frequently found to be particularly sensitive to chemical exposure yet also commonly overlooked. We found similar levels of intestine malformations and edemas, as well as disruption of skeletal muscle, in atrazine and triadimefon exposed tadpoles. The effects of 2,4-D were only apparent at the highest concentrations we examined; glyphosate did not induce dramatic malformations at the concentrations tested. While researchers have shown that it is important to understand how chemical mixtures affect non-target organisms, our results suggest that it is first crucial to determine how these chemicals act independently in order to be able to identify consequences of individual pesticide exposure.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(No.XDB14040102)the National Natural Science Foundation of China(No.21377153)
文摘T3-induced Xenopus metamorphosis is an ideal model for detecting thyroid hormone（TH）signaling disruption of chemicals. To optimize the T3-induced Xenopus assay and improve its sensitivity and reproducibility, we intend to develop quantitatively morphological endpoints and choose appropriate concentrations and exposure durations for T3 induction.Xenopus laevis at stage 52 were exposed to series of concentrations of T3（0.31–2.5 nmol/L）for 6 days. By comparing morphological changes induced by T3, we propose head area,mouth width, unilateral brain width/brain length, and hindlimb length/snout-vent length as quantitative parameters for characterizing T3-induced morphological changes, with body weight as a parameter for indicating integrated changes. By analyzing time-response curves, we found that following 4-day exposure, T3-induced grossly morphological changes displayed linear concentration–response curves, with moderate morphological changes resulting from 1.25 nmol/L T3 exposure. When using grossly morphological endpoints to detect TH signaling disruption, we propose 4 days as exposure duration of T3, with concentrations close to 1.25 nmol/L as induction concentrations. However, it is appropriate to examine morphological and molecular changes of the intestine on day 2 due to their early response to T3. The quantitative endpoints and T3 induction concentrations and durations we determined would improve the sensitivity and the reproducibility of the T3-induced Xenopus metamorphosis assay.
文摘We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspensioncultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA fragments. The effects of different divalent cations and cycloheximide on DNA cleavage in this system were assessed.The fact that nuclei of plant cells can be induced to undergo apoptosis in a cell-free animal system suggests that animals and plants share a common signal transduction pathway triggering in the initiation stage of apoptosis.
基金supported by the Knowledge Innovation Program of Chinese Academy of Sciences(No. KZCX2-YW-420-3,KZCX2-YW-Q-02-05)the National Natural Science Foundation of China (No.20437020,20677074)
文摘Decabromodiphenyl ether （decaBDE）,as a flame retardant,is widely produced and used.To study the thyroid disruption by technical decaBDE at low concentrations,Xenopus laevis tadpoles were exposed to technical decaBDE mixture DE-83R （1-1000 ng/L） in water from stage 46/47 （free swimming larvae,system of Nieuwkoop and Faber） to stage 62.DE-83R at concentration of 1000 ng/L significantly delayed the time to metamorphosis （presented by forelimb emergence,FLE）.Histological examination showed that DE83R at all tested concentrations caused histological alterations-multilayer follicular epithelial cell and markedly increased follicle size accompanied by partial colloid depletion and increase in the peripheral colloid vacuolation,in thyroid glands.All tested concentrations of DE-83R also induced a down-regulation of thyroid receptor mRNA expression.These results demonstrated that technical decaBDE disrupted the thyroid system in X.laevis tadpoles.Analysis of polybrominated diphenyl ethers （PBDEs） （sum of 39 congeners） in X.laevis indicated that mean concentrations of total PBDEs in X.laevis exposed to 1,10,100,1000 ng/L were 11.0,128.1,412.1,1400.2 ng/g wet weight,respectively.Considering that PBDEs burden of X.laevis tadpoles was close to PBDEs levels in amphibians as reported in previous studies,our study has raised new concerns for thyroid disruption in amphibians of technical decaBDE at environmentally relevant concentrations.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(No.XDB14040102)the National Natural Science Foundation of China(No.21377153)
文摘We developed the T3-induced Xenopus metamorphosis assay, which is supposed to be able to sensitively detect thyroid hormone（TH） signaling disruption of chemicals. The present study aimed to validate the T3-induced Xenopus metamorphosis assay by re-evaluating the TH signaling antagonism of tetrabromobisphenol A（TBBPA）, a known TH signaling disruptor. According to the assay we developed, Xenopus tadpoles at stage 52 were exposed to 10–500 nmol/L TBBPA in the presence of 1 nmol/L T3. After 96 hr of exposure, TBBPA in the range of 10–500 nmol/L was found to significantly inhibit T3-induced morphological changes of Xenopus tadpoles in a concentration-dependent manner in term of body weight and four morphological endpoints including head area（HA）, mouth width（MW）, unilateral brain width/brain length（ULBW/BL）, and hind-limb length/snout-vent length（HLL/SVL）.The results show that these endpoints we developed are sensitive for characterizing the antagonistic effects of TBBPA on T3-induced metamorphosis. Following a 24-hr exposure,we found that TBBPA antagonized expression of T3-induced TH-response genes in the tail,which is consistent with previous findings in the intestine. We propose that the tail can be used as an alternative tissue to the intestine for examining molecular endpoints for evaluating TH signaling disruption. In conclusion, our results demonstrate that the T3-induced Xenopus metamorphosis assay we developed is an ideal in vivo assay for detecting TH signaling disruption.
文摘Inositol requiring enzyme-1 （IRE1） is highly conserved from yeasts to humans. Upon the endoplasmic reticu- lum （ER） stress, IRE1 activates X-box-binding protein 1 （XBP1） by unconventionally splicing XBP1 mRNA, which activates the unfolded protein response （UPR） to restore ER homeostasis. In mice, IREla inactivity leads to embryonic death and IREla plays an essential role in extraembryonic tissues and the placenta. However, its precise action in the embryo proper is still unknown. In this study, the loss of function ana/ysis was performed to investigate the function of Xenopus IREla （xlREla） during pancreas development. Firstly, the complete open reading frame of xIRE1α was amplified and the expression pattern was detected. The effects of Xenopus IRE1α and XBP1 during embryo development were detected with whole-mount in situ hybridization. The results demonstrated that xIRE1α was much closer to human IREla when compared with their sequence alignment, xlREla was expressed strongly in developing pancreas and the knockdown of xIREla inhibited the differentiation and specification of the pancreas, xlREltt, which was required for cytoplasmic splicing of XBP1 pre-mRNA and XB- P1MO, also showed inhibitory effects on pancreas development. These results suggest that xlREla is essential for pancreas development during embryogenesis and functions via the XBP1 dependent pathway.
基金supported by the National Natural Science Foundation of China (No. 20877023,20507007)
文摘Tributyltin（TBT）,a biocide used in antifouling paints,has shown strong teratogenic effects on Xenopus tropicalis embryos at environmentally relevant concentrations.X.tropicalis embryos were exposed to 50,100 and 200 ng/L tributyltin chloride for 72 hr.The histological changes were further observed on abnormal eyes,enlarged trunks,enlarged proctodaeums and absence of fins induced by TBT.The lens and the retinal layers of abnormal eyes were slightly or barely differentiated,and that the pigment epithelium was neither continuous nor smooth.The abdomens were full of undifferentiated gut tissue with yolk-rich inclusions in the tadpoles with enlarged trunks.The proctodaeums formed a bump-like or columnar structure.The mass of yolk-rich cells occupied the lumen,blocked the opening and even turned inside out of the proctodaeum.Both the ventral and dorsal fins in trunks and tails became narrow or even disappeared totally.Our results suggest that great changes of histology took place corresponding to the unique phenotypes.The gut tissue was poorly differentiated,which led to the failed elongation of the guts and subsequently the enlarged trunks.The enlarged proctodaeums were due to the undifferentiation of inner layer,the expansion of outer epidermal part and the absence of fins around them.In brief,the histological observations provided insights into the reason of the unique external malformations in some degree.
基金Supported by The National Natural Science Foundation of China,No.30971680A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, No.JX10131801101
文摘AIM:To investigate the role of inositol-requiring enzyme 1α(IRE1α) in gut development of Xenopus lavies embryos.METHODS:Xenopus embryos were obtained with in vitro fertilization and cultured in 0.1 × MBSH.One and half nanogram of IRE1α,1 ng of IRE1α-GR mRNA,1 ng of IRE1αΔC-GR mRNA,and 50 ng of IRE1α morpholino oligonucleotide(MO) or XBP1(C)MO were injected into four blastomeres at 4-cell stage for scoring the phenotype and marker gene analysis.To rescue the effect of IRE1α MO,1 ng of IRE1α-GR mRNA was coinjected with 50 ng of MO.For the activation of the GR-fusion proteins,dexamethasone was prepared as 5 mmol/L stock solutions in 100% ethanol and applied to the mRNA injected embryos at desired stages in a concentration of 10 μmol/L in 0.1 × MBSH.Embryos were kept in dexamethasone up to stage 41.Whole-mount in situ hybridization was used to determine specific gene expression,such as IRE1α,IRE1β,Xbra and Xsox17α.IRE1α protein expression during Xenopus embryogenesis was detected by Western blotting.RESULTS:In the whole-mount in situ hybridization analysis,xenopus IRE1α and IRE1β showed quite different expression pattern during tadpole stage.The relatively higher expression of IRE1α was observed in the pancreas,and significant transcription of IRE1β was found in the liver.IRE1α protein could be detected at all developmental stages analyzed,from stage 1 to stage 42.Gain-of-function assay showed that IRE1α mRNA injected embryos at tailbud stage were nearly normal and the expression of the pan-mesodermal marker gene Xbra and the endodermal gene Xsox17α at stage 10.5 was not significantly changed in embryos injected with IRE1α mRNA as compared to uninjected control embryos.And at tadpole stage,the embryos injected with IRE1α-GR mRNA did not display overt phenotype,such as gut-coiling defect.Loss-of-function assay demonstrated that the IRE1α MO injected embryos were morphologically normal before the tailbud stages.We did not observe a significant change of mesodermal and endodermal marker gene expression,while after stage 40,about 80% of the MO injected embryos exhibited dramatic gut defects in which the guts did not coil,but other structures outside the gastrointestinal tract were relatively normal.To test if the phenotypes were specifically caused by the knockdown of IRE1α,a rescue experiment was performed by co-injection of IRE1α-GR mRMA with IRE1α MO.The data obtained demonstrated that the gut coiling defect was rescued.The deletion mutant of IRE1α was constructed,consisting of the N-terminal part without the C-terminal kinase and RNase domains named IRE1αΔC,to investigate the functional domain of IRE1α.Injection of IRE1αΔCGR mRNA caused similar morphological alterations with gut malformation by interfering with the function of endogenous xIRE1α.In order to investigate if IRE1α/XBP1 pathway was involved in gut development,50 ng of XBP1 MO was injected and the results showed that knockdown of XBP1 resulted in similar morphological alterations with gut-coiling defect at tadpole stage.CONCLUSION:IRE1α is not required for germ layer formation but for gut development in Xenopus lavies and it may function via XBP1-dependent pathway.
基金supported by grants from the Research Grants Council of Hong Kong CUHK14167017,CUHK24100414 to H.Z.the Shenzhen Innovation Committee of Science and Technology grants(JCYJ20150331101823691)Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research(2017B030301018)to Y.D
文摘DEAR EDITOR Protein arginine methyltransferases (PRMTs)are involved in many cellular processes via the arginine methylation of histone or non-histone proteins.We examined the expression patterns of prmt4,prmt7,and prmt9 during embryogenesis in Xenopus using whole-mount in situ hybridization and quantitative reverse transcription polymerase chain reaction (RT-PCR).Xenopus prmt4 and prmt7 were expressed in the neural crest,brain,and spinal cord,and also detected in the eye,branchial arches,and heart at the tailbud stage.Specific print9 signals were not detected in Xenopus embryos until the late tailbud stage when weak expression was observed in the branchial arches.Quantitative RT-PCR indicated that the expression of prmt4 and prmt7 was up-regulated during the neurula stage,whereas prmt9 maintained its low expression until the late tailbud stage,consistent with the whole-mount in situ hybridization results.Thus,the developmental expression patterns of these three print genes in Xenopus embryos provide a basis for further functional study of such genes.
文摘Tissue inhibitors of metalloproteinases (TIMPs) modulate extracellular matrix remodeling during embryonic develop- ment and disease. TIMP-3 expression was examined during Xenopus laevis embryogenesis: TIMP-3 transcripts detected in the maternal pool of RNA increased at the mid-blastula transition, decreased dramatically during gastrulation and increased again during neurulation and axis elongation. Interestingly, the decrease during gastrulation was not seen in LiCl treated (dorsalized) embryos. Whole mount in situ hybridization of TIMP-3 using DIG-labeled RNA probes demonstrated that the transcripts were present in all dorsal tissues during embryogenesis, but were prominent only in head structures starting at stage 35. Overexpression of TIMP-3 through transgenesis and RNA injections led to devel- opmental abnormalities and death. Both overexpression strategies resulted in post-gastrulation perturbation including those to neural and head structures, as well as truncated axes. However, RNA injections resulted in more severe early defects such as failure of neural tube closure, and transgenesis caused truncated axes and head abnormalities. No transgenic embryo expressing TIMP-3 survived past stage 40.
基金supported by the Start-up Funding of Henan University of Science and Technology(13480027) to Q. C.the Key Science Foundation of Nanjing Medical University(2015NJMUZD002)+2 种基金the Natural Science Foundation of Higher Education Institutions of Jiangsu Province(16KJB-180020)Natural Science Foundation of Jiangsu Province (BK20171053)National Natural Science Funds of China (81702747) to C.L
文摘Kruppel-like factor 4（Klf4） is a zinc finger transcription factor and plays crucial roles in Xenopus embryogenesis.However, its regulation during embryogenesis is still unclear. Here, we report that Tcf711, a key downstream transducer of the Wnt signaling pathway, could promote Klf4 transcription and stimulate Klf4 promoter activity in early Xenopus embryos. Furthermore, cycloheximide treatment showed a direct effect on Klf4 transcription facilitated by Tcf711. Moreover, the dominant negative form of Tcf711（dnTcf711）, which lacks N-terminus of the β-catenin binding motif, could still activate Klf4 transcription, suggesting that this regulation is Wnt/β-catenin independent.Taken together, our results demonstrate that Tcf711 lies upstream of Klf4 to maintain its expression level during Xenopus embryogenesis.
基金supported by the National Natural Science Foundation of China (No. 21377153)the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDB14040102)
文摘There is a pressing need for developing in vivo or ex vivo assays to screen the glucocorticoid（GC） signaling disruption of chemicals. Thus, we aimed to establish an ex vivo assay for screening GC signaling disruption based on the GC-response gene transcription in Xenopus laevis tails cultured ex vivo. Firstly, we investigated effects of corticosterone（CORT, a main GC in frogs） on GC-response gene expression, and determined the six genes as molecular endpoints for assaying the GC signaling disruption. CORT in the range of 1.56–400 nmol/L was found to up-regulate transcription of the six GC-response genes, exhibiting comparable or higher sensitivity than previously reported assays. To validate this ex vivo assay, then, we examined effects of dexamethasone（a known GC signaling agonist） on GC-response gene expression. Dexamethasone displayed an agonistic action in a concentration-dependent manner, further demonstrating the efficiency of the established assay. Finally, we applied the ex vivo assay to evaluate the GC signaling disruption of bisphenol A（BPA）. In accordance with previous reports, we found a concentration-dependent agonistic activity of BPA,showing that the established assay is effective for detecting the GC signaling disrupting activity of environmental chemicals. Correspondingly, the GC signaling agonistic actions of CORT and BPA in ex vivo tails accorded with the observations in vivo, indicating that the ex vivo assay is able to detect the actions of chemicals in vivo. Overall, we established an ex vivo assay that can effectively screen GC signaling disruption of environmental chemicals.
文摘The homozygous inv （inversion of embryonic turning） mouse mutant shows situs inversus and polycystic kidney disease, both of which result from the lack of the inv gene. Previously, we suggested that inv may be important for the left-right axis formation, not only in mice but also in Xenopus, and that calmodulin regulates this inv protein function. Here, we isolated and characterized two Xenopus laevis homologs （Xinv-1 and Xinv-2） of the mouse inv gene, and performed functional analysis of the conserved IQ motifs that interact with calmodulin. Xinv-1 expresses early in development in the same manner as mouse inv does. Unexpectedly, a full-length Xenopus inv mRNA did not randomize cardiac orientation when injected into Xenopus embryos, which is different from mouse inv mRNA. Contrary to mouse inv mRNA, Xenopus inv mRNA with mutated IQ randomized cardiac orientation. The present study indicates that calmodulin binding sites （IQ motifs） are crucial in controlling the biological activity of both mouse and Xenopus inv proteins. Although mouse and Xenopus inv genes have a quite similar structure, the interaction with calmodulin and IQ motifs ofXenopus inv and mouse inv proteins may regulate their function in different ways.
文摘Anti-keratin monoclonal antibody AF5 was introduced into fertilized eggs of Xenopus laevis., and its effects on embryonic development were studied. Survival rate of the antikeratin-injected embryos was much lower (only 35.76% at gastrula) than that of the control (74.85% at gastrula), in which embryos were injected with mouse IgG. Most of survivors in the experimental series showed aberrant external appearance. On the other hand, in cleavage stage, ie 2-7 h after fertilization, immunohistochemi-cal staining of embryos showed that the experimental embryos were mostly keratin negative, while embryos of the control ones were keratin positive. When introducing this antikeratin into one cell of a 2-cell embryo, only the unin-jected half of the embryo continued its development while the other half could not develop at all. These results suggested that intact keratin cytoskeleton in early embryos is indispensable to the embryonic development of Xenopus laevis.
文摘The complex transformation of a tadpole to a frogduring amphibian development is under the control of thyroid hormone (T3). T3 is known to regulate gene transcription through its nuclear receptors. We have previouslyisolated many genes which are up-regulated by T3 in theintestine of Xenopus laevis tadpoles. We have now cloneda full- length cDNA for one such gene (IU12). Sequenceanalysis shows that the IU12 cDNA encodes a plasmamembrane protein with 12 transmembrane domains andhomologous to a mammalian gene associated with cell activation and organ development. Similarly, we have foundthat IU12 is activated during intestinal remodeling whenboth cell death and proliferation take place. Furthermore,IU12 is an early T3-response gene and its expression in theintestine during T3-induced metamorphosis mimics thatduring normal development. These results argue for a roleof IU12 in the signal transduction pathways leading to intestinal metamorphosis.
