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Iron is an essential metal cofactor for enzymes involved in many cellular functions, such as energy generation and cell proliferation. However, excessive iron concentration leads to increased oxidative stress and toxicity. As such, iron homeostasis is strictly controlled by two RNA binding proteins known as Iron Regulatory Proteins (IRPs) that regulate the expression of iron management genes at post-transcriptional level. Despite this fine regulation, an impairment of iron homeostasis occurs during aging: iron progressively accumulates in several organs and, in turn, it exacerbates cellular vulnerability and tissue damage. Moreover, excessive iron accumulation within the CNS is observed in many neurodegenerative diseases. I investigated the age-dependent changes of iron homeostasis using the short lived fish Nothobranchius furzeri. Here, I show that: i) both iron content and expression of microRNA family miR-29 increase during adult life and aging in the N. furzeri brain; ii) iron up-regulates miR-29 expression in fish brain and murine neurons, while, in turn, miR-29 targets the 3’-UTR of IREB2 mRNA, reducing iron intake; iii) Transgenic fish with knock-down of miR-29 show significant adult-onset up- regulation of IRP2 and its target TFR1 in neurons and display enhanced age-dependent accumulation of brain iron; iv) miR-29 triggers a global gene expression response that partially overlaps with that induced by aging. My studies indicate that miR-29 modulates intracellular iron homeostasis and is up-regulated as an adaptive response to limit excessive iron accumulation and to contrast aging-induced processes.

Complete list of KEG pathway analysis. (PDF 839 kb)

Mir-29 target D. rerio and M. musculus Ireb2 3′-UTR. A Expression of GFP assessed by cytofluorometric analysis. Fusion with Ireb2 3′-UTR of D. rerio. The gray column indicates the baseline fluorescence intensity of the construct without miR-29 mimic. The middle blue column indicates the fluorescence with miR-29 mimic and the red column the fluorescence of a construct (Δ) where the putative binding site for miR-29 in the Ireb2 3′-UTR was mutated to destroy complementarity. Statistical significance of fluorescence difference between baseline and co-injection with miR-29 mimics was evaluated by Student’s t-test (** P

Neuronal-specific kif5a promoter expression. Representative double-labelling immunohistochemistry for eGFP (green) and neuronal marker HuC/D (pink) in optic tectum of 20-week-old kif5a:sponge-29 fish. Periventricular gray zone (PGZ), Tectum opticum (TeO). Scale bar: 100Â Îźm. (PDF 1014 kb)

Mir-29 predicted target DEGs identification in N. furzeri kif5a: sponge-29. A Intersection of D. rerio miR-29 predicted target genes (from TargetScanFish 6.2, score ≤ –0.30) with N. furzeri sponge-29 DEGs. A total of 38 predicted target genes were found deregulated in kif5a:sponge-29 fish, 28/38 upregulated (B) and 10/38 downregulated (C), respectively. Gray, red and green box represent a conserved binding site in the 3′-UTR of N. furzeri, M. musculus, and H. sapiens, respectively. Yellow colored genes lack a conserved binding site in N. furzeri. Light blue box indicate a physical interaction between target gene e and miR-29 observed by cross-linking immunoprecipitation (CLIP-seq). 1-3-4 numbers are relative to gene dataset type in Fig. 3e. (PDF 78 kb)

Age dependent regulation of IRP2 in wild-type and tg(kif5aa:eGFP-sponge-29). A, B IRP2 western blot analysis at different age point of wild-type (A) and tg(kif5aa:eGFP-sponge-29) brains. Three biological replicates were used for age point. Îą-tubulin was used as loading control. (PDF 163 kb)

Prediction of miR-29 binding site in D. rerio Elna1 and col11a1a mRNAs. Representative 3â ˛-UTR of Elna1 (ENSDARG00000069994.1) and Col11a1a (ENSDARG00000026165.1), respectively, with indication of the predicted binding sites (from TargetScanFish 6.2). Black circles shows miR-29 binding site and blue table shows the score of all predicted microRNA binding sites, miR-29 has the highest score in both the 3â ˛-UTR genes according to TargetScanFish 6.2. (PDF 531 kb)

Mir-29 upregulation coincides with Ireb2 and Tfrc downregulation in mouse brain. A Quantification of pri-miR-29 cluster (pri-miR-29b-a; pri-miR-29b-c) in postnatal day 0 and 60 mouse brain. Both primary transcripts dramatically increase during postnatal development (P

The short-lived turquoise killifish Nothobranchius furzeri (Nfu) is a valid model for aging studies. Here, we investigated its age-associated cardiac function. We observed oxidative stress accumulation and an engagement of microRNAs (miRNAs) in the aging heart. MiRNA-sequencing of 5 week (young), 12–21 week (adult) and 28–40 week (old) Nfu hearts revealed 23 up-regulated and 18 down-regulated miRNAs with age. MiR-29 family turned out as one of the most up-regulated miRNAs during aging. MiR-29 family increase induces a decrease of known targets like collagens and DNA methyl transferases (DNMTs) paralleled by 5´methyl-cytosine (5mC) level decrease. To further investigate miR-29 family role in the fish heart we generated a transgenic zebrafish model where miR-29 was knocked-down. In this model we found significant morphological and functional cardiac alterations and an impairment of oxygen dependent pathways by transcriptome analysis leading to hypoxic marker up-regulation. To get insights the possible hypoxic regulation of miR-29 family, we exposed human cardiac fibroblasts to 1% O2 levels. In hypoxic condition we found miR-29 down-modulation responsible for the accumulation of collagens and 5mC. Overall, our data suggest that miR-29 family up-regulation might represent an endogenous mechanism aimed at ameliorating the age-dependent cardiac damage leading to hypertrophy and fibrosis.

BACKGROUND: Annual killifishes inhabit temporary ponds and their embryos survive the dry season encased in the mud by entering diapause, a process that arrests embryonic development during hostile conditions. Annual killifishes are present within three clades distributed in Africa (one East and one West of the Dahomey gap) and South America. Within each of these phylogenetic clades, a non-annual clade is sister taxon to a annual clade and therefore represent an example of convergent evolution. Early cleavage of teleost embryos is characterized by a very fast cell cycle (15-30 minutes) and lack of G1 and G2 phases. Here, we decided to investigate rates of early cleavage in annual killifishes. In addition, we specifically tested whether also annual killifish embryos lack G1 and G2 phases. RESULTS: We used time lapse brightfield microscopy to investigate cell division kinetics during the first developmental stages of annual- and non-annual species belonging to the three different phylogenetic clades. Annual killifishes of all three clades showed cleavage times significantly longer when compared to their non-annual sister taxa (average 35 min vs. average 75 min). Using FUCCI fluorescent imaging of the cell cycle after microinjection in the annual species Nothobranchius furzeri, we demonstrate that the first 5 division are synchronous and do not show a G1 phase. Cell cycle synchronization is lost after the 5th cleavage division. CONCLUSIONS: Our results show, for the first time, that cell cycle rate during cleavage, a trait thought to be rather evolutionary conserved can undergo convergent evolutionary change in response to variations in life-history.

Background The annual fish Nothobranchius furzeri is characterized by a natural dichromatism with yellow-tailed and red-tailed male individuals. These differences are due to different distributions of xanthophores and erythrophores in the two morphs. Previous crossing studies have showed that dichromatism in N. furzeri is inherited as a simple Mendelian trait with the yellow morph dominant over the red morph. The causative genetic variation was mapped by linkage analysis in a chromosome region containing the Mc1r locus. However, subsequent mapping showed that Mc1r is most likely not responsible for the color difference in N. furzeri. To gain further insight into the molecular basis of this phenotype, we performed RNA-seq on F2 progeny of a cross between N. furzeri male and N. kadleci female. Results We identified 210 differentially-expressed genes between yellow and red fin samples. Functional annotation analysis revealed that genes with higher transcript levels in the yellow morph are enriched for the melanin synthesis pathway indicating that xanthophores are more similar to melanophores than are the erythrophores. Genes with higher expression levels in red-tails included xanthine dehydrogenase (Xdh), coding for a biosynthetic enzyme in the pteridine synthesis pathway, and genes related to muscle contraction. Comparison of DEGs obtained in this study with genes associated with pigmentation in the Midas cichlid (A. citrinellus) reveal similarities like involvement of the melanin biosynthesis pathway, the genes Ptgir, Rasef (RAS and EF-hand domain containing), as well as genes primarily expressed in muscle such as Ttn and Ttnb (titin, titin b). Conclusions Regulation of genes in the melanin synthetic pathway is an expected finding and shows that N. furzeri is a genetically-tractable species for studying the genetic basis of natural phenotypic variations. The current list of differentially-expressed genes can be compared with the results of fine-mapping, to reveal the genetic architecture of this natural phenotype. However, an evolutionarily-conserved role of muscle-related genes in tail fin pigmentation is novel finding and interesting perspective for the future. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-754) contains supplementary material, which is available to authorized users.