Kinky Tail Research Articles

Deletion of Pax1 scoliosis-associated regulatory elements leads to a female-biased tail abnormality

Adolescent idiopathic scoliosis (AIS), a sideways curvature of the spine, is sexually dimorphic, with increased incidence in females. A genome-wide association study identified a female-specific AIS susceptibility locus near the PAX1 gene. Here, we use mouse enhancer assays, three mouse enhancer knockouts, and subsequent phenotypic analyses to characterize this region. Using mouse enhancer assays, we characterize a sequence, PEC7, which overlaps the AIS-associated variant, and find it to be active in the tail tip and intervertebral disc. Removal of PEC7 or Xe1, a known sclerotome enhancer nearby, or deletion of both sequences lead to a kinky tail phenotype only in the Xe1 and combined (Xe1+PEC7) knockouts, with only the latter showing a female sex dimorphic phenotype. Extensive phenotypic characterization of these mouse lines implicates several differentially expressed genes and estrogen signaling in the sex dimorphic bias. In summary, our work functionally characterizes an AIS-associated locus and dissects the mechanism for its sexual dimorphism.

Impact of glyphosate residues in sow diets on neonatal piglets: tail kinks, stillborn and diarrhoea

Among the applications, glyphosate-based herbicides are used for pre-harvest crop desiccation and weed management in glyphosate resistant crops. Due to these applications glyphosate residues have been found in feedstuff for conventional livestock. Besides its herbicidal property, glyphosate has documented mineral chelating and antimicrobial properties. Previous studies have found glyphosate residuals in animal organs, and glyphosate may cause adverse effects in animals during the embryonic or neonatal period. The present study has two endpoints; (1) whether glyphosate concentration in the diets fed to sows during early gestation, was associated with the frequency of congenital tail kinks and stillborn piglets, and (2) whether the glyphosate concentration in diets fed to lactating sows, is associated with the frequency of diarrhoea in neonatal piglets. The concentration of glyphosate in 78 gestation diets obtained from seven Danish conventional sow herds ranged from 0.08 to 1.52 mg/kg dry matter (DM) and in 34 lactation diets from 0.06 to 0.87 mg/kg DM. When correcting for an average DM content of 88.3% in gestation diets and 89.0% in lactation diets the concentration of glyphosate ranged from 0.07 to 1.34 and 0.05 to 0.77 mg/kg feed, respectively. In the first study, 28,336 docked tails from 2 to 4-day-old piglets were assessed for congenital tail kinks and the number of kinky tails per herd per week was linked to the glyphosate concentration in sow diets during week 1 to 6 of gestation. Percentage of kinky tails and stillborn piglets ranged from 0 to 2.60% and 0.7 to 3.2% per week, respectively. No associations between the glyphosate concentration in sow gestation diets and the frequency of kinky tails or stillborn piglets were found. In the second study, faeces consistency of 5,526 piglets from 386 litters was linked to the glyphosate concentration in sow lactation diets. A proportion of 0.15 of the 0- to 5-day-old piglets had diarrhoea. No association was found between the concentration of glyphosate in sow lactation diets and the frequency of diarrhoea in neonatal piglets.

Correction of Vertebral Bone Development in Ectodysplasin A1-Deficient Mice by Prenatal Treatment With a Replacement Protein.

X-linked hypohidrotic ectodermal dysplasia with the cardinal symptoms hypodontia, hypotrichosis and hypohidrosis is caused by a genetic deficiency of ectodysplasin A1 (EDA1). Prenatal EDA1 replacement can rescue the development of skin appendages and teeth. Tabby mice, a natural animal model of EDA1 deficiency, additionally feature a striking kink of the tail, the cause of which has remained unclear. We studied the origin of this phenomenon and its response to prenatal therapy. Alterations in the distal spine could be noticed soon after birth, and kinks were present in all Tabby mice by the age of 4 months. Although their vertebral bones frequently had a disorganized epiphyseal zone possibly predisposing to fractures, cortical bone density was only reduced in vertebrae of older Tabby mice and even increased in their tibiae. Different availability of osteoclasts in the spine, which may affect bone density, was ruled out by osteoclast staining. The absence of hair follicles, a well-known niche of epidermal stem cells, and much lower bromodeoxyuridine uptake in the tail skin of 9-day-old Tabby mice rather suggest the kink being due to a skin proliferation defect that prevents the skin from growing as fast as the skeleton, so that caudal vertebrae may be squeezed and bent by a lack of skin. Early postnatal treatment with EDA1 leading to delayed hair follicle formation attenuated the kink, but did not prevent it. Tabby mice born after prenatal administration of EDA1, however, showed normal tail skin proliferation, no signs of kinking and, interestingly, a normalized vertebral bone density. Thus, our data prove the causal relationship between EDA1 deficiency and kinky tails and indicate that hair follicles are required for murine tail skin to grow fast enough. Disturbed bone development appears to be partially pre-determined in utero and can be counteracted by timely EDA1 replacement, pointing to a role of EDA1 also in osteogenesis.

Trichostatin A mitigates radiation-induced teratogenesis in C57Bl/6 mice.

Radiation exposure in utero is known to lead to serious concerns to both the mother and children, including developmental anomalies in the children. In the recent past, trichostatin A, an HDAC (histone deacetylase) inhibitor and epigenetic modifier, has been shown to mitigate radiation-induced anomalies in the male reproductive system of C57BL/6 mice. Therefore, the current study was undertaken to evaluate the mitigating effects of trichostatin A (TSA) against radiation-induced developmental anomalies in mice. Foetuses of in utero whole-body gamma-irradiated mice during the active organogenesis period were examined for developmental anomalies at 8.5 and 18.5 days of gestation. In utero radiation exposure caused developmental anomalies like microcephaly, microphthalmia, gastroschisis and kinky tail besides prenatal mortality. TSA administration post-irradiation was observed to reduce 50% of prenatal mortality at E18.5 by reducing congenital and developmental anomalies. Observation of such results could be corroborated with the HDAC inhibitory potential of TSA knowing that developmental anomalies may have epigenetic origin. TSA, therefore, can be considered as a potential radiomitigator.

Loss of Chondroitin Sulfate Modification Causes Inflammation and Neurodegeneration in skt Mice.

One major aspect of the aging process is the onset of chronic, low-grade inflammation that is highly associated with age-related diseases. The molecular mechanisms that regulate these processes have not been fully elucidated. We have identified a spontaneous mutant mouse line, small with kinky tail (skt), that exhibits accelerated aging and age-related disease phenotypes including increased inflammation in the brain and retina, enhanced age-dependent retinal abnormalities including photoreceptor cell degeneration, neurodegeneration in the hippocampus, and reduced lifespan. By positional cloning, we identified a deletion in chondroitin sulfate synthase 1 (Chsy1) that is responsible for these phenotypes in skt mice. CHSY1 is a member of the chondroitin N-acetylgalactosaminyltransferase family that plays critical roles in the biosynthesis of chondroitin sulfate, a glycosaminoglycan (GAG) that is attached to the core protein to form the chondroitin sulfate proteoglycan (CSPG). Consistent with this function, the Chsy1 mutation dramatically decreases chondroitin sulfate GAGs in the retina and hippocampus. In addition, macrophage and neutrophil populations appear significantly altered in the bone marrow and spleen of skt mice, suggesting an important role for CHSY1 in the functioning of these immune cell types. Thus, our study reveals a previously unidentified impact of CHSY1 in the retina and hippocampus. Specifically, chondroitin sulfate (CS) modification of proteins by CHSY1 appears critical for proper regulation of immune cells of the myeloid lineage and for maintaining the integrity of neuronal tissues, since a defect in this gene results in increased inflammation and abnormal phenotypes associated with age-related diseases.

Mutation of Murine Rpl5 reveals a New Model for Diamond Blackfan Anemia Characterized By Defective Erythropoiesis

Background Ribosome dysfunction is implicated in several abnormal developmental conditions and diseases in humans. Heterozygous germline ribosomal protein (RP) variants are found in the majority of individuals with Diamond Blackfan anemia (DBA) with RPL5 (uL18) being the second most frequently mutated gene. Patients with RPL5 mutations are for unknown reasons at increased risk of congenital defects when compared to other genotypes. There is currently no available Rpl5 mouse model with anemia and therefore hematopoiesis in this genotype has not been well studied. Methods We characterized a novel mutant mouse with a growth and kinky tail defect, and increased mortality. The mouse was generated in a chromosome 11 balancer ENU mutagenesis screen. Following genetic mapping and whole exome sequencing, we identified a heterozygous intronic Rpl5 missense variant, which segregated with all affected mice. We first performed molecular analyses to validate the impact of this variant on RPL5 and ribosome levels, and then examined the development and physiology of multiple tissues by histology, micro-CT, echocardiogram and measurement of erythroid differentiation to determine the etiology of the observed increased mortality in newborn Rpl5 mutant mice. Results This variant led to decreased Rpl5 mRNA and protein, and consequential polysome defects (decreased 60S and 80S ribosome subunits) consistent with RPL5 haploinsufficiency. All Rpl5+/- neonates exhibited deficient terminal erythroid differentiation. A fraction of the mutants progressed to severe anemia leading to increased mortality while others appeared to undergo spontaneous remission. Surviving adult/aged mice exhibited no anemia although preliminary studies indicate reduced numbers of hematopoietic progenitors. Most newborn mutant mice had large ventricular septal defects (VSD) while surviving adult mice had small VSDs on echocardiogram. Rpl5+/- mice were significantly smaller without craniofacial abnormalities as assessed by micro-CT or histology. Bone and cartilage staining of newborn mice revealed that the kinky tail defect resulted from delayed endochondral ossification. Conclusion This model recapitulates the major developmental and erythroid differentiation defects of DBA in an animal model. We propose that this novel Rpl5 mutant provides an opportunity to study the mechanism of the variable penetrance of anemia and the congenital defects normally encountered in DBA. Disclosures Singh: Emmaus Medical, Inc: Consultancy.

Context-specific regulation of cell survival by a miRNA-controlled BIM rheostat.

Knockout of the ubiquitously expressed miRNA-17∼92 cluster in mice produces a lethal developmental lung defect, skeletal abnormalities, and blocked B lymphopoiesis. A shared target of miR-17∼92 miRNAs is the pro-apoptotic protein BIM, central to life-death decisions in mammalian cells. To clarify the contribution of miR-17∼92:Bim interactions to the complex miR-17∼92 knockout phenotype, we used a system of conditional mutagenesis of the nine Bim 3' UTR miR-17∼92 seed matches. Blocking miR-17∼92:Bim interactions early in development phenocopied the lethal lung phenotype of miR-17∼92 ablation and generated a skeletal kinky tail. In the hematopoietic system, instead of causing the predicted B cell developmental block, it produced a selective inability of B cells to resist cellular stress; and prevented B and T cell hyperplasia caused by Bim haploinsufficiency. Thus, the interaction of miR-17∼92 with a single target is essential for life, and BIM regulation by miRNAs serves as a rheostat controlling cell survival in specific physiological contexts.

Insertion of a chimeric retrotransposon sequence in mouse Axin1 locus causes metastable kinky tail phenotype

BackgroundTransposable elements (TEs) make up > 50% of the human genome, and the majority of retrotransposon insertions are truncated and many are located in introns. However, the effects of retrotransposition on the host genes remain incompletely known.ResultsWe report here that insertion of a chimeric L1 (cL1), but not IAP solo LTR, into intron 6 of Axin1 using CRIPSR/Cas9 induced the kinky tail phenotype with ~ 80% penetrance in heterozygous AxincL1 mice. Both penetrant (with kinky tails) and silent (without kinky tails) AxincL1 mice, regardless of sex, could transmit the phenotype to subsequent generations with similar penetrance (~ 80%). Further analyses revealed that a longer Axin1 transcript isoform containing partial cL1-targeted intron was present in penetrant, but absent in silent and wild type mice, and the production of this unique Axin1 transcript appeared to correlate with altered levels of an activating histone modification, H3K9ac.ConclusionsThe mechanism for AxincL1 mice is different from those previously identified in mice with spontaneous retrotransposition of IAP, e.g., AxinFu and Avy, both of which have been associated with DNA methylation changes. Our data suggest that Axin1 locus is sensitive to genetic and epigenetic alteration by retrotransposons and thus, ideally suited for studying the effects of new retrotransposition events on target gene function in mice.

Homozygous Dkk1 Knockout Mice Exhibit High Bone Mass Phenotype Due to Increased Bone Formation.

Wnt antagonist Dkk1 is a negative regulator of bone formation and Dkk1 +/- heterozygous mice display a high bone mass phenotype. Complete loss of Dkk1 function disrupts embryonic head development. Homozygous Dkk1 -/- mice that were heterozygous for Wnt3 loss of function mutation (termed Dkk1 KO) are viable and allowed studying the effects of homozygous inactivation of Dkk1 on bone formation. Dkk1 KO mice showed a high bone mass phenotype exceeding that of heterozygous mice as well as a high incidence of polydactyly and kinky tails. Whole body bone density was increased in the Dkk1 KO mice as shown by longitudinal dual-energy X-ray absorptiometry. MicroCT analysis of the distal femur revealed up to 3-fold increases in trabecular bone volume and up to 2-fold increases in the vertebrae, compared to wild type controls. Cortical bone was increased in both the tibiae and vertebrae, which correlated with increased strength in tibial 4-point bending and vertebral compression tests. Dynamic histomorphometry identified increased bone formation as the mechanism underlying the high bone mass phenotype in Dkk1 KO mice, with no changes in bone resorption. Mice featuring only Wnt3 heterozygosity showed no evident bone phenotype. Our findings highlight a critical role for Dkk1 in the regulation of bone formation and a gene dose-dependent response to loss of DKK1 function. Targeting Dkk1 to enhance bone formation offers therapeutic potential for osteoporosis.

Evaluation of developmental toxicity of guaifenesin using pregnant female rats.

Objectives:Guaifenesin possesses expectorant, muscle relaxant, and anticonvulsive properties. To the best of our knowledge, the promising data regarding the developmental toxicity of guaifenesin are scarce. The current study investigates the developmental toxic effects of guaifenesin in detail using female rats.Materials and Methods:Twenty-five dams were divided into five groups. Group 1 served as a control, while Group-2, -3, -4, and -5 were administered with 250, 350, 500, and 600 (mg/kg b.w.) doses of guaifenesin, respectively, starting from gestation day 6 to day 17. Half of the total recovered fetuses was subjected to morphologic and morphometric analysis, while other half was subjected to skeletal examination.Results:A significant reduction in maternal weight, and food/water intake, was observed, however, no mortality and morbidity were observed. About 14 dead fetuses were found in Group-3 and -4 each, while 26 in Group 5. Morphological analysis revealed 21.2%, 45.4%, 67.2%, and 86.9% of total fetuses having hemorrhagic spots in Group-2, -3, -4, and -5, respectively. Dropping wrist/ankle and kinky tail were found in Group-4 and -5 only. Morphometric analysis showed a significant decline in fetal weight, full body length, skull length, forelimb length, hindlimb length, and tail length in all guaifenesin treated groups. Skeletal examination displayed that only Group 5 fetuses had increased intercostal space between 7th and 8th rib. We also observed improper development of carpals, metacarpals, tarsals, and metatarsals of the Group 5 fetuses.Conclusion:Guaifenesin showed a significant developmental toxicity at selected test doses; therefore, a careful use is suggested during pregnancy.

Retrotransposon Activates Ectopic Ptf1 Expression: A Short Tail

Retrotransposon Activates Ectopic Ptf1 Expression: A Short Tail

Effects of Schistosoma mansoni infection and Mirazid treatment on pregnant mice and their fetuses

A total of 48 female albino mice, with average weight 23 g, have been used in this study, divided into 4 groups (12 mice for each group). Groups I & II served as uninfected groups. Group II was subjected to pregnancy and received an oral dose of Mirazid (300 mg/Kg) (Pharco Pharmaceuticals, Alexandria, Egypt) for three consecutive days. Groups III and IV were infected subcutaneously with 60 ±10 Schistosoma mansoni cercariae (Egyptian strain) and were subjected to pregnancy. Group IV received an oral dose of Mirazid (300 mg/Kg). The results showed that the distribution of implantation sites of fetuses in the uterine horns was found unequal in infected groups which completed the pregnancy. The mean number of fetuses was smaller in infected, treated or both groups when compared with control groups. Also, there were many abortion cases, especially when infection progressed. In the present study, infection was found to induce some growth retardation among fetuses as compared with the control ones. Growth retardation was indicated by highly significant decrease in their body weight and length, abnormal skin and limbs, kinky tail, kyphotic body and hematoma formation. Treatment with Mirazid did not improve these malformation as well as control ones. In conclusion, more studies are needed regarding the relationship between the pregnancy and schistosomiasis as the present work reflects the deleterious effect of schistosomiasis on the pregnant mice and on the fetal outcomes. In addition, the results reflected, for the first time, the unsafely using of Mirazid during pregnancy where announcement should be clear to avoid Mirazid in treatment of schistosome-infected pregnant women.

Mutation in Pxdn encoding peroxidasin causes small lenses and kinky tails in the mouse

AbstractPurpose The mouse mutant KTA48 was identified within the recessive Munich ENU Project among offspring of treated C3HeB/FeJ mice. It is characterized by a kinky tail, white belly spot and small dull eyes.Methods Linkage analysis was performed using SNP‐based technology and microsatellite markers. Sequence analysis, histology and in‐situ hybridization were performed according to standard techniques.Results The genome‐wide linkage analysis mapped the KTA48 mutation on chromosome 12; positional candidate gene analysis detected a mutation in the Pxdn gene (encoding peroxidasin) co‐segregating with the mutation. The Pxdn‐mRNA of the KTA48 mutants contains a T‐>A mutation at pos. 3816 (T3816A) creating a new Alw26I restriction site; the mutation converts the Cys at codon 1272 into a stop codon (Cys1272X). The mutation co‐segregates with the phenotype in the breeding colony; it is not present in several wild‐type strains. The lens phenotype is slightly variable. During an early stage of eye development (E12.5), Pxdn is expressed in the posterior part of the lens and in the central part of the retina without obvious changes. At the end of embryonic development (E17.5), we observed small lenses outside the optical axis and rupture of the lens capsule. Later, microphagia is accompanied by deformed cornea and multilayered retina. At P21, the small lens is attached to the cornea, and in some cases, the eye is completely disorganized..Conclusion The recessive mouse mutation KTA48 is caused by a premature stop codon of the Pxdn gene. The function of Peroxidasin is still unknown; there is no mutation known neither in humans nor in mice.

The efficiency of Trifolium pratense seed extract against the deleterious effects caused by nicotine in the liver of rat

The aim of this study was to determine the adverse effects of maternal nicotine treatment during pregnancy and on rat liver of newborns. Also to determine a protective effect of Trifolium Pratenseseed extract against the deleterious effects of nicotine. All groups were treated from the 6th day of gestation till parturition. The dams and their newborns were sacrificed and piece of liver from each subjected to the histological studies. The morphological findings of the newborns revealed marked signs of retardation in growth with malformations including superficial haematoma, shortened neck region, kinky tail, uni-and bilateral deformity of both fore and hind limbs and abnormal skin. The histopathological studies on the liver of the maternal nicotine treated and their newborns revealed disruption of hepatic cords arrangement. Blood sinusoids were dilated. The hepatic tissue exhibited inflammatory cells with signs of early cellular granulomatous lesions. Some hepatocytes obliterated and others with karyolysis nuclei. Kupffer cells become hypertrophied and dispersed throughout the hepatic cords. Fatty vacuoles and infiltrations were detected in many of the hepatocytes. The magnitude of these changes was dose dependent. TPSE supplementation was found to ameliorate an prevent the adverse effects of maternal nicotine treatment morphologically and histologically.

Kinked tail mutation results in notochord defects in heterozygotes and distal visceral endoderm defects in homozygotes

Proper formation of the anterior-posterior (AP) axis in the developing embryo is critical for the correct patterning and often survival of the organism. In the mouse, an initial step in axis establishment is the specification and migration of the distal visceral endoderm (DVE). We have identified a semi-dominant spontaneous mutation in mouse, named kinked tail (knk), which when heterozygous results in a kinky tail phenotype due to fusions and dysmorphology of the tail vertebrae. Vertebral fusions appear to be a secondary effect of notochord thickening and branching in the tail region. Homozygosity for knk results in early embryonic lethality by embryonic day 8.5 due to improper timing of DVE specification and migration, and subsequent failure to establish the AP axis.

Truncation of the Catalytic Domain of the Cylindromatosis Tumor Suppressor Impairs Lung Maturation

Cyld encodes a 956-amino acid deubiquitinating enzyme (CYLD), which is a negative regulator of nuclear factor κB and mitogen-activated protein kinase pathways. Mutations that truncate and inactivate the carboxyl-terminal deubiquitinating domain of CYLD underlie the development of skin appendage tumors in humans, whereas down-regulation of Cyld expression has been associated with the development of various types of human malignancies including lung cancer. To establish an animal model of human CYLD inactivation and characterize the biological role of CYLD in vivo, we generated mice carrying a homozygous deletion of Cyld exon 9 (CyldΔ9/Δ9 mice) using a conditional approach. Deletion of exon 9 would cause a carboxyl-terminal truncation of CYLD and inactivation of its deubiquitinating activity. In accordance with previous studies, fibroblasts from CyldΔ9/Δ9 embryos had hyperactive nuclear factor κB and c-Jun kinase pathways compared with control fibroblasts. CyldΔ9/Δ9 newborn mice were smaller than wild-type littermates with a short and kinky tail and nomajor developmental defects. However, CyldΔ9/Δ9 mice died shortly after birth from apparent respiratory dysfunction. Histological examination of E18.5 CyldΔ9/Δ9 lungs demonstrated an immature phenotype characterized by hyperplasic mesenchyme but apparently normal epithelial, smooth muscle. and endothelial structures. Our study identifies an important role of CYLD in lung maturation, which may underlie the development of many cases of lung cancer.

Chondroitin 4-O-sulfotransferase-1 is required for somitic muscle development and motor axon guidance in zebrafish

CS (chondroitin sulfate) has been implicated in a variety of biological processes during development. Its biological functions are closely associated with characteristic sulfated structures. Here, we report the characterization of a zebrafish counterpart of C4ST-1 (chondroitin 4-O-sulfotransferase-1) and its functional importance in embryogenesis. Recombinant C4ST-1 showed a substrate preference for chondroitin and catalysed the 4-O-sulfation of GalNAc residues, a highly frequent modification of CS in the embryos of zebrafish as well as other vertebrates. Whole-mount in situ hybridization revealed that C4ST-1 showed a distinct spatiotemporal expression pattern in the developing zebrafish embryo. During the segmentation stages, strong expression was observed along the body axis including the notochord and somites. Functional knockdown of C4ST-1 with specific antisense morpholino-oligonucleotides led to a marked decrease in the 4-O-sulfation and amount of CS in the embryos. Consistent with the preferential expression in the rostrocaudal axis, C4ST-1 morphants displayed morphological defects exemplified by a ventrally bent trunk and a curled and/or kinky tail, largely due to misregulated myotomal myod expression, implying perturbation of axial muscle differentiation in somites. Furthermore, the aberrant projection of spinal motor axons, which extended ventrally at the interface between the notochord and individual somites, was also observed in C4ST-1 morphants. These results suggest that 4-O-sulfated CS formed by C4ST-1 is essential for somitic muscle differentiation and motor axon guidance in zebrafish development.

Generation and characterization of EphA1 receptor tyrosine kinase reporter knockout mice

Eph receptor tyrosine kinases (RTKs) are a highly conserved family of signaling proteins with functions in cellular migration, adhesion, apoptosis, and proliferation during both adult and embryonic life. Here, we describe a knock-in mouse in which EphA1 expression is disrupted via the insertion of an internal ribosome entry site (IRES)-human placental alkaline phosphatase (ALPP) reporter cassette into exon II of the EphA1 gene. This was shown to successfully knockout expression of endogenous EphA1 and enforce expression of the ALPP reporter by the EphA1 promoter. Staining for the ALPP reporter protein demonstrated an epithelially restricted expression pattern in mouse tissues. In EphA1 null mice, two separate phenotypes were identified: abnormal tail development manifesting as a kinky tail was found in approximately 80% of homozygous adults. A second, distinct abnormality present in approximately 18% of females was characterized by imperforate uterovaginal development with hydrometrocolpos and caused by a resistance of cells to apoptosis during reproductive tract canalization. These results indicate a possible role for EphA1 in tissue patterning and hormone-induced apoptotic processes.

Protein Encoded by the AxinFu Allele Effectively Down-regulates Wnt Signaling but Exerts a Dominant Negative Effect on c-Jun N-terminal Kinase Signaling

Axin plays an architectural role in many important signaling pathways that control various aspects of development and tumorigenesis, including the Wnt, transforming growth factor-beta, MAP kinase pathways, as well as p53 activation cascades. It is encoded by the mouse Fused (Fu) locus; the Axin(Fu) allele is caused by insertion of an IAP transposon. Axin(Fu/Fu) mice display varying phenotypes ranging from embryonic lethality to relatively normal adulthood with kinky tails. However, the protein product(s) has not been identified or characterized. In the present study, we conducted immunoprecipitation using brain extracts from the Axin(Fu) mice with specific antibodies against different regions of Axin and found that a truncated Axin containing amino acids 1-596 (designated as Axin(Fu-NT)) and the full-length complement of Axin (Axin(WT)) can both be generated from the Axin(Fu) allele. When tested for functionality changes, Axin(Fu-NT) was found to abolish Axin-mediated activation of JNK, which plays a critical role in dorsoventral patterning. Together with a proteomics approach, we found that Axin(Fu-NT) contains a previously uncharacterized dimerization domain and can form a heterodimeric interaction with Axin(WT). The Axin(Fu-NT)/Axin(WT) is not conducive to JNK activation, providing a molecular explanation for the dominant negative effect of Axin(Fu-NT) on JNK activation by wild-type Axin. Importantly, Axin(Fu-NT) exhibits no difference in the inhibition of Wnt signaling compared with Axin(WT) as determined by reporter gene assays, interaction with key Wnt regulators, and expression of Wnt marker genes in zebrafish embryos, suggesting that altered JNK signaling contributes, at least in part, to the developmental defects seen in Axin(Fu) mice.

TRANSGENERATIONAL EPIGENETIC ALTERATIONS IN MICE PRODUCED BY IN VITRO CULTURE

In mammals, one of the two main periods of epigenetic reprogramming by demethylation occur during preimplantation development. Moreover, the regulation of many genes expressed during preimplantation development is controlled by retrotransposable elements. We have reported that in vitro culture (IVC) of mice embryos in presence of serum produces alterations in the transcription pattern of imprinted and non-imprinted genes, and it also induces a long-term effect on postnatal development, growth, and behaviour. Transgenerational effects in response to particular foetal environments are well recognised and have been associated with the abnormal establishment of epigenetic regulation of the foetus, however, no relationship has been established between alterations in the epigenetic reprogramming during preimplantation and transgenerational responses. The objective of our study was to evaluate the ability of in vitro culture during 4 days, in presence of serum (FCS) to induce epigenetic alteration in the Axin-fused (AxinFu) metastable epialleles. We used mice carrying the AxinFu allele, where the presence or absence of its characteristic phenotype, a kinked tail, correlates with differential DNA methylation at an IAP retrotransposon within AxinFu. In these mice the epigenetic status of AxinFu can be inherited transgenerationally. To investigate if the inheritance of AxinFu phenotype is affected by superovulation, embryo transfer, IVC in KSOM, and IVC in KSOM+FCS, we analyzed the proportions of offspring phenotypes arising from penetrant (kinky tail) and silent (normal tail) sires crossed with wild type dams. The phenotype of the offspring was quantified according to the level of curvature and size of the tail from 0 to 5. The IVC, and, more significantly, the presence of FCS increased the severity of the phenotype in the offspring generated (p<0.01, ANOVA one way analysis), and this severity of the phenotype was transmitted to the next generation. These results demonstrate that suboptimal IVC procedures are capable of inducing epigenetic alteration in metastable epialleles that can be generationally inherited. (platform)