Gene-Environment Interactions Target Mitogen-activated Protein 3 Kinase 1 (MAP3K1) Signaling in Eyelid Morphogenesis

Gene-environment interactions determine the biological outcomes through mechanisms that are poorly understood. Mouse embryonic eyelid closure is a well defined model to study the genetic control of developmental programs. Using this model, we investigated how exposure to dioxin-like environmental pollutants modifies the genetic risk of developmental abnormalities. Our studies reveal that mitogen-activated protein 3 kinase 1 (MAP3K1) signaling is a focal point of gene-environment cross-talk. Dioxin exposure, acting through the aryl hydrocarbon receptor (AHR), blocked eyelid closure in genetic mutants in which MAP3K1 signaling was attenuated but did not disturb this developmental program in either wild type or mutant mice with attenuated epidermal growth factor receptor or WNT signaling. Exposure also markedly inhibited c-Jun phosphorylation in Map3k1^+/− embryonic eyelid epithelium, suggesting that dioxin-induced AHR pathways can synergize with gene mutations to inhibit MAP3K1 signaling. Our studies uncover a novel mechanism through which the dioxin-AHR axis interacts with the MAP3K1 signaling pathways during fetal development and provide strong empirical evidence that specific gene alterations can increase the risk of developmental abnormalities driven by environmental pollutant exposure.     

Kidney regeneration through nephron neogenesis in medaka

Although renal regeneration is limited to repair of the proximal tubule in mammals, some bony fish are capable of renal regeneration through nephron neogenesis in the event of renal injury. We previously reported that nephron development in the medaka mesonephros is characterized by four histologically distinct stages, generally referred to as condensed mesenchyme, nephrogenic body, relatively small nephron, and the mature nephron. Developing nephrons are positive for wt1 expression during the first three of these stages. In the present study, we examined the regenerative response to renal injury, artificially induced by the administration of sublethal amounts of gentamicin in adult medaka. Similar to previous reports in other animals, the renal tubular epithelium and the glomerulus of the medaka kidney exhibited severe damage after exposure to this agent. However, kidneys showed substantial recovery after gentamicin administration, and a significant number of developing nephrons appeared 14 days after gentamicin administration (P < 0.01). Similarly, the expression of wt1 in developing nephrons also indicated the early stages of nephrogenesis. These findings show that medaka has the ability to regenerate kidney through nephron neogenesis during adulthood and that wt1 is a suitable marker for detecting nephrogenesis.     

Development of an Automated Imaging Pipeline for the Analysis of the Zebrafish Larval Kidney

The analysis of kidney malformation caused by environmental influences during nephrogenesis or by hereditary nephropathies requires animal models allowing the in vivo observation of developmental processes. The zebrafish has emerged as a useful model system for the analysis of vertebrate organ development and function, and it is suitable for the identification of organotoxic or disease-modulating compounds on a larger scale. However, to fully exploit its potential in high content screening applications, dedicated protocols are required allowing the consistent visualization of inner organs such as the embryonic kidney. To this end, we developed a high content screening compatible pipeline for the automated imaging of standardized views of the developing pronephros in zebrafish larvae. Using a custom designed tool, cavities were generated in agarose coated microtiter plates allowing for accurate positioning and orientation of zebrafish larvae. This enabled the subsequent automated acquisition of stable and consistent dorsal views of pronephric kidneys. The established pipeline was applied in a pilot screen for the analysis of the impact of potentially nephrotoxic drugs on zebrafish pronephros development in the Tg(wt1b:EGFP) transgenic line in which the developing pronephros is highlighted by GFP expression. The consistent image data that was acquired allowed for quantification of gross morphological pronephric phenotypes, revealing concentration dependent effects of several compounds on nephrogenesis. In addition, applicability of the imaging pipeline was further confirmed in a morpholino based model for cilia-associated human genetic disorders associated with different intraflagellar transport genes. The developed tools and pipeline can be used to study various aspects in zebrafish kidney research, and can be readily adapted for the analysis of other organ systems.     

Disruption of Ah Receptor Signaling during Mouse Development Leads to Abnormal Cardiac Structure and Function in the Adult

The Developmental Origins of Health and Disease (DOHaD) Theory proposes that the environment encountered during fetal life and infancy permanently shapes tissue physiology and homeostasis such that damage resulting from maternal stress, poor nutrition or exposure to environmental agents may be at the heart of adult onset disease. Interference with endogenous developmental functions of the aryl hydrocarbon receptor (AHR), either by gene ablation or by exposure in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent AHR ligand, causes structural, molecular and functional cardiac abnormalities and altered heart physiology in mouse embryos. To test if embryonic effects progress into an adult phenotype, we investigated whether Ahr ablation or TCDD exposure in utero resulted in cardiac abnormalities in adult mice long after removal of the agent. Ten-months old adult Ahr ^-/- and in utero TCDD-exposed Ahr ^+/+ mice showed sexually dimorphic abnormal cardiovascular phenotypes characterized by echocardiographic findings of hypertrophy, ventricular dilation and increased heart weight, resting heart rate and systolic and mean blood pressure, and decreased exercise tolerance. Underlying these effects, genes in signaling networks related to cardiac hypertrophy and mitochondrial function were differentially expressed. Cardiac dysfunction in mouse embryos resulting from AHR signaling disruption seems to progress into abnormal cardiac structure and function that predispose adults to cardiac disease, but while embryonic dysfunction is equally robust in males and females, the adult abnormalities are more prevalent in females, with the highest severity in Ahr ^-/- females. The findings reported here underscore the conclusion that AHR signaling in the developing heart is one potential target of environmental factors associated with cardiovascular disease.     

Novel EGF pathway regulators modulate C. elegans healthspan and lifespan via EGF receptor,PLC‐γ, and IP3R activation

Improving health of the rapidly growing aging population is a critical medical, social, and economic goal. Identification of genes that modulate healthspan, the period of mid‐life vigor that precedes significant functional decline, will be an essential part of the effort to design anti‐aging therapies. Because locomotory decline in humans is a major contributor to frailty and loss of independence and because slowing of movement is a conserved feature of aging across phyla, we screened for genetic interventions that extend locomotory healthspan of Caenorhabditis elegans. From a group of 54 genes previously noted to encode secreted proteins similar in sequence to extracellular domains of insulin receptor, we identified two genes for which RNAi knockdown delayed age‐associated locomotory decline, conferring a high performance in advanced age phenotype (Hpa). Unexpectedly, we found that hpa‐1 and hpa‐2 act through the EGF pathway, rather than the insulin signaling pathway, to control systemic healthspan benefits without detectable developmental consequences. Further analysis revealed a potent role of EGF signaling, acting via downstream phospholipase C‐γplc‐3 and inositol‐3‐phosphate receptor itr‐1, to promote healthy aging associated with low lipofuscin levels, enhanced physical performance, and extended lifespan. This study identifies HPA‐1 and HPA‐2 as novel negative regulators of EGF signaling and constitutes the first report of EGF signaling as a major pathway for healthy aging. Our data raise the possibility that EGF family members should be investigated for similar activities in higher organisms.     

Novel cDNA sequences of aryl hydrocarbon receptors and gene expression in turtles (Chrysemys picta and Pseudemys scripta) exposed to different environments

Reproductive changes have been observed in painted turtles from a site with known contamination located on Cape Cod, MA, USA. We hypothesize that these changes are caused by exposure to endocrine-disrupting compounds and that genes involved in reproduction are affected. The aryl hydrocarbon receptor (AHR) is an orphan receptor that is activated by environmental contaminants. AHR mRNA was measured in turtles exposed to soil collected from a contaminated site. Adult turtles were trapped from the study site (Moody Pond, MP) or a reference site and exposed to laboratory environments containing soil from either site. The red-eared slider was used to assess neonatal exposure to soil and water from the sites. The environmental exposures occurred over a 13-month period. Juveniles showed an age-dependent increase in brain AHR1. Juvenile turtles exposed to the MP environment had elevated gonadal AHR1. Adult turtles exposed to the MP environment showed significantly decreased brain AHR2. The painted turtle AHR is the first complete reptile AHR cDNA sequence. Phylogenetic analysis of the painted turtle AHR showed that it clusters with other AHR2s. Partial AHR1 and partial AHR2 cDNA sequences were cloned from the red-eared slider. MEME analysis identified 18 motifs in the turtle AHRs, showing high conservation between motifs that overlapped functional regions in both AHR isoforms.     

Methylation of protein phosphatase 2A—Influence of regulators and environmental stress factors

Protein phosphatase 2A catalytic subunit (PP2A‐C) has a terminal leucine subjected to methylation, a regulatory mechanism conserved from yeast to mammals and plants. Two enzymes, LCMT1 and PME1, methylate and demethylate PP2A‐C, respectively. The physiological importance of these posttranslational modifications is still enigmatic. We investigated these processes in Arabidopsis thaliana by mutant phenotyping, by global expression analysis, and by monitoring methylation status of PP2A‐C under different environmental conditions. The lcmt1 mutant, possessing essentially only unmethylated PP2A‐C, had less dense rosettes, and earlier flowering than wild type (WT). The pme1 mutant, with 30% reduction in unmethylated PP2A‐C, was phenotypically comparable with WT. Approximately 200 overlapping genes were twofold upregulated, and 200 overlapping genes were twofold downregulated in both lcmt1 and pme1 relative to WT. Differences between the 2 mutants were also striking; 97 genes were twofold upregulated in pme1 compared with lcmt1, indicating that PME1 acts as a negative regulator for these genes. Analysis of enriched GO terms revealed categories of both abiotic and biotic stress genes. Furthermore, methylation status of PP2A‐C was influenced by environmental stress, especially by hypoxia and salt stress, which led to increased levels of unmethylated PP2A‐C, and highlights the importance of PP2A‐C methylation/demethylation in environmental responses.     

The effects of myocyte enhancer factor 2A gene on the proliferation, migration and phenotype of vascular smooth muscle cells

The genetic basis for the phenotypic switching of vascular smooth muscle cells (VSMCs) is unclear in atherosclerosis. Recent studies showed that the 21‐base pair deletion mutation (Δ21) in myocyte enhancer factor 2A (MEF2A) gene could be an inherited marker for coronary artery disease. MEF2A mutation may affect the phenotypic switching of VSMCs. Human aortic VSMCs were used. Four groups of VSMCs transfected with green fluorescent protein plasmid (control group), MEF2A wild‐type (WT) plasmid (WT group), MEF2A Δ21 plasmid (Δ21 group) or MEF2A siRNA (siRNA group) were studied. The proliferation of VSMCs was determined by methylthiazolyldiphenyl‐tetrazolium bromide, and the migration of VSMCs was measured by Millicell chamber. The protein expressions of MEF2A, smooth muscle α‐actin, SM22α, osteopontin and p38 mitogen‐activated protein kinase signaling pathway were detected by Western blotting. MEF2A protein expression was knockdown by siRNA transfection. MEF2A protein was overexpressed in WT and Δ21 groups. Δ21 and siRNA groups obviously showed more proliferation (methylthiazolyldiphenyl‐tetrazolium bromide, 0.63 vs 0.66 vs 0.31, P < 0.01) and migration (52.6 vs 58.0 vs 21.2, P < 0.01) of VSMCs as compared with the WT group. In addition, the transfection of Δ21 and siRNA could induce the down‐regulation of smooth muscle α‐actin and SM22α (P < 0.01) and the up‐regulation of osteopontin (P < 0.01) in VSMCs. The phosphorylated p38 signaling pathway expression was significantly enhanced in the Δ21 and siRNA groups as compared with that of the WT group (P < 0.01). These results suggest that MEF2A dominant negative mutation and RNA silence could induce the phenotypic switching of VSMCs, leading to its increased proliferation and migration, and p38 mitogen‐activated protein kinase signaling pathway may participate in it. Copyright © 2011 John Wiley & Sons, Ltd.     

Wnt signaling in kidney development and disease

The Wnt gene family, which encodes secreted growth and differentiation factors, has been implicated in kidney organogenesis. The Wnts control both ureteric bud development and signaling, but they also serve as inductive factors to regulate nephrogenesis in the mesenchcymal cells. Several of the Wnt genes are expressed in the developing kidney, and gene knock-out studies have revealed specific developmental functions for these. Consistent with this, changes in Wnt ligands and pathway components are associated with many kidney diseases, including kidney cancers, renal fibrosis, cystic kidney diseases, acute renal failure, diabetic nephropathy and ischaemic injury. It is these associations of the Wnt signaling system with kidney development and kidney diseases that form to topic of this review.Key words: Wnt signaling, tubule induction, ureter development, kidney diseases, kidney cancer     

Knockout of AtDjB1, a J‐domain protein from Arabidopsis thaliana,alters plant responses to osmotic stress and abscisic acid

AtDjB1 is a member of the Arabidopsis thaliana J‐protein family. AtDjB1 is targeted to the mitochondria and plays a crucial role in A. thaliana heat and oxidative stress resistance. Herein, the role of AtDjB1 in adapting to saline and drought stress was studied in A. thaliana. AtDjB1 expression was induced through salinity, dehydration and abscisic acid (ABA) in young seedlings. Reverse genetic analyses indicate that AtDjB1 is a negative regulator in plant osmotic stress tolerance. Further, AtDjB1 knockout mutant plants (atj1‐1) exhibited greater ABA sensitivity compared with the wild‐type (WT) plants and the mutant lines with a rescued AtDjB1 gene. AtDjB1 gene knockout also altered the expression of several ABA‐responsive genes, which suggests that AtDjB1 is involved in osmotic stress tolerance through its effects on ABA signaling pathways. Moreover, atj1‐1 plants exhibited higher glucose levels and greater glucose sensitivity in the post‐germination development stage. Applying glucose promoted an ABA response in seedlings, and the promotion was more evident in atj1‐1 than WT seedlings. Taken together, higher glucose levels in atj1‐1 plants are likely responsible for the greater ABA sensitivity and increased osmotic stress tolerance.     

AHR,a novel acute hypoxia‐response sequence,drives reporter gene expression under hypoxia in vitro and in vivo

ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs 1) is an early immediate gene. We have previously reported that ADAMTS1 was strongly induced by hypoxia. In this study, we investigated whether ADAMTS1 promoter‐driven reporter signal is detectable by acute hypoxia. We constructed the GFP (green fluorescent protein) expression vector [AHR (acute hypoxia‐response sequence)‐GFP] under the control of ADAMTS1 promoter and compared it with the constitutive GFP‐expressing vector under the control of CMV (cytomegalovirus promoter‐GFP). We transduced AHR‐GFP and examined whether GFP signals can be detected under the acute hypoxia. When the human umbilical vein [HUVEC (human umbilical vein endothelial cells)] was transduced under normoxia, there were few GFP signals, while CMV‐GFP showed considerable GFP signals. When HUVEC was stimulated with hypoxia, GFP signals from AHR‐GFP gene were induced under hypoxic conditions. Notably, the GFP signals peaked at 3 h under hypoxia. In ischaemic hind limb model, transduced AHR‐GFP showed hypoxic induction of GFP signals. In summary, we have demonstrated that the AHR system induced the reporter gene expression by acute hypoxia, and its induction is transient. This is the first report showing the unique acute hypoxia‐activated gene expression system.     

Mitogen‐activated protein kinase dependency in BRAF/RAS wild‐type melanoma: A rationale for combination inhibitors

Inhibitors targeting the mitogen‐activated protein kinase (MAPK) pathway and immune checkpoint molecules have dramatically improved the survival of patients with BRAF^V600‐mutant melanoma. For BRAF/RAS wild‐type (WT) melanoma patients, however, immune checkpoint inhibitors remain the only effective therapeutic option with 40% of patients responding to PD‐1 inhibition. In the present study, a large panel of 10 BRAF^V600‐mutant and 13 BRAF/RAS WT melanoma cell lines was analyzed to examine MAPK dependency and explore the potential utility of MAPK inhibitors in this melanoma subtype. We now show that the majority of BRAF/RAS WT melanoma cell lines (8/13) display some degree of sensitivity to trametinib treatment and resistance to trametinib in this melanoma subtype is associated with, but not mediated by NF1 suppression. Although knockdown of NF1 stimulates RAS and CRAF activity, the activation of CRAF by NF1 knockdown is limited by ERK‐dependent feedback in BRAF‐mutant cells, but not in BRAF/RAS WT melanoma cells. Thus, NF1 is not a dominant regulator of MAPK signaling in BRAF/RAS WT melanoma, and co‐targeting multiple MAP kinase nodes provides a therapeutic opportunity for this melanoma subtype.     

Polymorphism at the Clock gene predicts phenology of long‐distance migration in birds

Dissecting phenotypic variance in life history traits into its genetic and environmental components is at the focus of evolutionary studies and of pivotal importance to identify the mechanisms and predict the consequences of human‐driven environmental change. The timing of recurrent life history events (phenology) is under strong selection, but the study of the genes that control potential environmental canalization in phenological traits is at its infancy. Candidate genes for circadian behaviour entrained by photoperiod have been screened as potential controllers of phenological variation of breeding and moult in birds, with inconsistent results. Despite photoperiodic control of migration is well established, no study has reported on migration phenology in relation to polymorphism at candidate genes in birds. We analysed variation in spring migration dates within four trans‐Saharan migratory species (Luscinia megarhynchos; Ficedula hypoleuca; Anthus trivialis; Saxicola rubetra) at a Mediterranean island in relation to Clock and Adcyap1 polymorphism. Individuals with larger number of glutamine residues in the poly‐Q region of Clock gene migrated significantly later in one or, respectively, two species depending on sex and whether the within‐individual mean length or the length of the longer Clock allele was considered. The results hinted at dominance of the longer Clock allele. No significant evidence for migration date to covary with Adcyap1 polymorphism emerged. This is the first evidence that migration phenology is associated with Clock in birds. This finding is important for evolutionary studies of migration and sheds light on the mechanisms that drive bird phenological changes and population trends in response to climate change.