Analysis of compound heterozygotes reveals that the mouse floxed Pax6 tm1Ued allele produces abnormal eye phenotypes

Analysis of abnormal phenotypes produced by different types of mutations has been crucial for our understanding of gene function. Some floxed alleles that retain a neomycin-resistance selection cassette (neo cassette) are not equivalent to wild-type alleles and provide useful experimental resources. Pax6 is an important developmental gene and the aim of this study was to determine whether the floxed Pax6(tm1Ued) (Pax6(fl)) allele, which has a retained neo cassette, produced any abnormal eye phenotypes that would imply that it differs from the wild-type allele. Homozygous Pax6(fl/fl) and heterozygous Pax6(fl/+) mice had no overt qualitative eye abnormalities but morphometric analysis showed that Pax6(fl/fl) corneas tended be thicker and smaller in diameter. To aid identification of weak effects, we produced compound heterozygotes with the Pax6(Sey-Neu) (Pax6(−)) null allele. Pax6(fl/−) compound heterozygotes had more severe eye abnormalities than Pax6(+/−) heterozygotes, implying that Pax6(fl) differs from the wild-type Pax6(+) allele. Immunohistochemistry showed that the Pax6(fl/−) corneal epithelium was positive for keratin 19 and negative for keratin 12, indicating that it was abnormally differentiated. This Pax6(fl) allele provides a useful addition to the existing Pax6 allelic series and this study demonstrates the utility of using compound heterozygotes with null alleles to unmask cryptic effects of floxed alleles. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11248-016-9962-4) contains supplementary material, which is available to authorized users

Inherited liver shunts in dogs elucidate pathways regulating embryonic development and clinical disorders of the portal vein

Congenital disorders of the hepatic portal vasculature are rare in man but occur frequently in certain dog breeds. In dogs, there are two main subtypes: intrahepatic portosystemic shunts, which are considered to stem from defective closure of the embryonic ductus venosus, and extrahepatic shunts, which connect the splanchnic vascular system with the vena cava or vena azygos. Both subtypes result in nearly complete bypass of the liver by the portal blood flow. In both subtypes the development of the smaller branches of the portal vein tree in the liver is impaired and terminal branches delivering portal blood to the liver lobules are often lacking. The clinical signs are due to poor liver growth, development, and function. Patency of the ductus venosus seems to be a digenic trait in Irish wolfhounds, whereas Cairn terriers with extrahepatic portosystemic shunts display a more complex inheritance. The genes involved in these disorders cannot be identified with the sporadic human cases, but in dogs, the genome-wide study of the extrahepatic form is at an advanced stage. The canine disease may lead to the identification of novel genes and pathways cooperating in growth and development of the hepatic portal vein tree. The same pathways likely regulate the development of the vascular system of regenerating livers during liver diseases such as hepatitis and cirrhosis. Therefore, the identification of these molecular pathways may provide a basis for future proregenerative intervention

Inhibition of poly(ADP-ribose) polymerase-1 attenuates the toxicity of carbon tetrachloride

Carbon tetrachloride (CCl4) is routinely used as a model compound for eliciting centrilobular hepatotoxicity. It can be bioactivated to the trichloromethyl radical, which causes extensive lipid peroxidation and ultimately cell death by necrosis. Overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) can rapidly reduce the levels of (β-nicotinamide adenine dinucleotide and adenosine triphosphate and ultimately promote necrosis. The aim of this study was to determine whether inhibition of PARP-1 could decrease CCl4-induced hepatotoxicity, as measured by degree of poly(ADP-ribosyl)ation, serum levels of lactate dehydrogenase (LDH), lipid peroxidation,and oxidative DNA damage. For this purpose, male ICR mice were administered intraperitoneally a hepatotoxic dose of CCl4 with or without 6(5H)-phenanthridinone, a potent inhibitor of PARP-1. Animals treated with CCl4 exhibited extensive poly(ADP-ribosyl)ation in centrilobular hepatocytes, elevated serum levels of LDH, and increased lipid peroxidation. In contrast, animals treated concomitantly with CCl4 and 6(5H)-phenanthridinone showed significantly lower levels of poly(ADP-ribosyl) ation, serum LDH, and lipid peroxidation. No changes were observed in the levels of oxidative DNA damage regardless of treatment. These results demonstrated that the hepatotoxicity of CCl4is dependent on the overactivation of PARP-1 and that inhibition of this enzyme attenuates the hepatotoxicity of CCl4

Modeling the Effects of Cell Cycle M-phase Transcriptional Inhibition on Circadian Oscillation

Circadian clocks are endogenous time-keeping systems that temporally organize biological processes. Gating of cell cycle events by a circadian clock is a universal observation that is currently considered a mechanism serving to protect DNA from diurnal exposure to ultraviolet radiation or other mutagens. In this study, we put forward another possibility: that such gating helps to insulate the circadian clock from perturbations induced by transcriptional inhibition during the M phase of the cell cycle. We introduced a periodic pulse of transcriptional inhibition into a previously published mammalian circadian model and simulated the behavior of the modified model under both constant darkness and light–dark cycle conditions. The simulation results under constant darkness indicated that periodic transcriptional inhibition could entrain/lock the circadian clock just as a light–dark cycle does. At equilibrium states, a transcriptional inhibition pulse of certain periods was always locked close to certain circadian phases where inhibition on Per and Bmal1 mRNA synthesis was most balanced. In a light–dark cycle condition, inhibitions imposed at different parts of a circadian period induced different degrees of perturbation to the circadian clock. When imposed at the middle- or late-night phase, the transcriptional inhibition cycle induced the least perturbations to the circadian clock. The late-night time window of least perturbation overlapped with the experimentally observed time window, where mitosis is most frequent. This supports our hypothesis that the circadian clock gates the cell cycle M phase to certain circadian phases to minimize perturbations induced by the latter. This study reveals the hidden effects of the cell division cycle on the circadian clock and, together with the current picture of genome stability maintenance by circadian gating of cell cycle, provides a more comprehensive understanding of the phenomenon of circading gating of cell cycle

Over-expression of AhR (aryl hydrocarbon receptor) induces neural differentiation of Neuro2a cells: neurotoxicology study

BACKGROUND: Dioxins and related compounds are suspected of causing neurological disruption in human and experimental animal offspring following perinatal exposure during development and growth. The molecular mechanism(s) of the actions in the brain, however, have not been fully investigated. A major participant in the process of the dioxin-toxicity is the dioxin receptor, namely the aryl hydrocarbon receptor (AhR). AhR regulates the transcription of diverse genes through binding to the xenobiotic-responsive element (XRE). Since the AhR has also been detected in various regions of the brain, the AhR may play a key role in the developmental neurotoxicity of dioxins. This study focused on the effect of AhR activation in the developing neuron. METHODS: The influence of the AhR on the developing neuron was assessed using the Neuro2a-AhR transfectant. The undifferentiated murine neuroblastoma Neuro2a cell line (ATCC) was stably transfected with AhR cDNA and the established cell line was named N2a-Rα. The activation of exogenous AhR in N2a-Rα cells was confirmed using RNAi, with si-AhR suppressing the expression of exogenous AhR. The neurological properties of N2a-Rα based on AhR activation were evaluated by immunohistochemical analysis of cytoskeletal molecules and by RT-PCR analysis of mRNA expression of neurotransmitter-production related molecules, such as tyrosine hydroxylase (TH). RESULTS: N2a-Rα cells exhibited constant activation of the exogenous AhR. CYP1A1, a typical XRE-regulated gene, mRNA was induced without the application of ligand to the culture medium. N2a-Rα cells exhibited two significant functional features. Morphologically, N2a-Rα cells bore spontaneous neurites exhibiting axon-like properties with the localization of NF-H. In addition, cdc42 expression was increased in comparison to the control cell line. The other is the catecholaminergic neuron-like property. N2a-Rα cells expressed tyrosine hydroxylase (TH) mRNA as a functional marker of catecholaminergic neurotransmitter production. Thus, exogenous AhR induced catecholaminergic differentiation in N2a-Rα cells. CONCLUSION: The excessive activation of AhR resulted in neural differentiation of Neuro2a cells. This result revealed that dioxins may affect the nervous system through the AhR-signaling pathway. Activated AhR may disrupt the strictly regulated brain formation with irregular differentiation occurring rather than cell death

Experimental ‘Jet Lag’ Inhibits Adult Neurogenesis and Produces Long-Term Cognitive Deficits in Female Hamsters

Background: Circadian disruptions through frequent transmeridian travel, rotating shift work, and poor sleep hygiene are associated with an array of physical and mental health maladies, including marked deficits in human cognitive function. Despite anecdotal and correlational reports suggesting a negative impact of circadian disruptions on brain function, this possibility has not been experimentally examined. Methodology/Principal Findings: In the present study, we investigated whether experimental ‘jet lag ’ (i.e., phase advances of the light:dark cycle) negatively impacts learning and memory and whether any deficits observed are associated with reductions in hippocampal cell proliferation and neurogenesis. Because insults to circadian timing alter circulating glucocorticoid and sex steroid concentrations, both of which influence neurogenesis and learning/memory, we assessed the contribution of these endocrine factors to any observed alterations. Circadian disruption resulted in pronounced deficits in learning and memory paralleled by marked reductions in hippocampal cell proliferation and neurogenesis. Significantly, deficits in hippocampal-dependent learning and memory were not only seen during the period of the circadian disruption, but also persisted well after the cessation of jet lag, suggesting long-lasting negative consequences on brain function. Conclusions/Significance: Together, these findings support the view that circadian disruptions suppress hippocampal neurogenesis via a glucocorticoid-independent mechanism, imposing pronounced and persistent impairments on learnin