Studio del ruolo del gene xpdzrn3 nello sviluppo del pronefro di Xenopus laevis

Il gene pdzrn3 (PDZ domain containing ring finger 3) codifica per una proteina contenente domini specializzati per mediare le interazioni con altre proteine: un dominio RING finger coinvolto in attività ubiquitinasica di tipo E3 e due domini PDZ. La sua sequenza è conservata in tutti i Vertebrati. Nel topo è espresso nel sistema nervoso centrale (SNC), nella regione del cuore, nei muscoli scheletrici, nel fegato e nel rene. Finora, i dati presenti in letteratura mettono in evidenza un ruolo di pdzrn3 nello sviluppo muscolare, in particolare nella rigenerazione dopo lesione, e nella plasticità sinaptica del SNC. L’ obiettivo della mia tesi è quello di studiare la funzione di pdzrn3 nello sviluppo del rene; per comprendere la funzione di questo gene abbiamo utilizzato come organismo modello Xenopus laevis poichè possiede un rene embrionale molto semplice chiamato pronefro. Nei Vertebrati il pronefro è il rene primitivo embrionale che, durante le fasi precoci di sviluppo, svolge sia la funzione escretoria sia la funzione di regolazione omeostatica degli ioni. Nell’adulto il pronefro verrà sostituito da un organo simile ma più complesso, il mesonefro, che, negli amnioti, subirà un ulteriore sviluppo che porterà al metanefro funzionale. Il pronefro si origina a partire dal mesoderma ed è costituito da tre componenti principali: il glomus, i tubuli e il dotto. Il glomus, che costituisce la porzione vascolare, ha la funzione di filtrare dalla circolazione gli scarti prodotti dall’organismo che poi verranno riversati nel nefrocele. I tubuli, che si originano a partire dal mesoderma laterale intermedio, hanno il compito di riassorbire dal nefrocele, tramite i nefrostomi, soluti organici e ioni, rilasciando invece nel dotto pronefrico i prodotti di rifiuto che verranno escreti tramite la cloaca. Grazie alla sua organizzazione anatomica il pronefro può essere considerato come l’unità funzionale minima del rene. In Xenopus laevis, il pronefro si forma a partire dallo stadio embrionale 12,5 (gastrula tardiva) ed è completamente funzionale a stadi embrionali più tardivi (stadio 37-38). L’analisi di espressione del gene xpdzrn3 (l’omologo di pdzrn3 di Xenopus) durante lo sviluppo, mostra un segnale specifico a livello di differenti regioni embrionali; già a partire dagli stadi più precoci analizzati (stadio 22-23) risulta espresso a livello dei somiti e del territorio presuntivo dell’occhio. Embrioni a stadio 23 mostrano inoltre espressione di xpdzrn3 a livello delle isole ematiche e nel territorio presuntivo di formazione del pronefro. Stadi più tardivi (fino a stadio 37-38), mostrano, oltre alle regioni embrionali sopra riportate, un segnale specifico anche in alcune regioni del SNC, nella regione dell’occhio, negli archi branchiali, nella vescicola otica, nel pronefro e nella cloaca. Sono stati effettuati esperimenti di perdita di funzione utilizzando un oligonucleotide “morpholino” diretto contro una regione di mRNA di xpdzrn3, lingo circa 20 nucleotidi contenente il codone di inizio traduzione “ATG”. Il morpholino Mo-xpdzrn3, avente funzione di reprimere la traduzione di Xpdzrn3, è stato iniettato in embrioni a stadio di 2 cellule a livello della zona marginale, che è la zona presuntiva di formazione mesodermica. Gli embrioni iniettati sono stati ibridati tramite l’uso di alcuni marcatori pronefrici. A stadio 37-38, abbiamo osservato una alterazione del “looping” dei tubuli prossimali e del posizionamento nefrostomico, nel lato iniettato rispetto al lato di controllo, messa in evidenza dai geni pax8, hnf1 e dal marcatore pan-pronefrico Na+/K+ ATPase. A stadio 22-23 i marcatori precoci del pronefro, pax8 e hnf1mostrano un'alterazione del loro dominio di espressione nell’area presuntiva di formazione del pronefro in seguito a iniezione di morfolino diretto contro xpdzrn3. Esperimenti di controllo effettuati con un morpholino “standard” disegnato su una sequenza umana non omologa a Xenopus, non hanno evidenziato alcuna significativa alterazione fenotipica a livello del pronefro, indicando la specificità dei fenotipi ottenuti con Mo-pdzrn3. Inoltre i saggi di “rescue”, effettuati utilizzando il trascritto pdzrn3 di Danio rerio, hanno evidenziato il recupero delle alterazioni osservate con i marcatori sopra citati. In conclusione possiamo sostenere che xpdzrn3 ha un’espressione regolata e dinamica durante lo sviluppo del pronefro di Xenopus . Con i dati ottenuti dagli esperimenti di ” loss of function” ipotizziamo un ruolo di xpdrn3 nella regolazione dello sviluppo precoce del pronefro ( st. 22-23) e, a stadi più tardivi ( st.37-38), un possibile coinvolgimento di questo gene nello sviluppo delle anse prossimali dei tubuli pronefrici

pdzrn3 is required for pronephros morphogenesis in Xenopus laevis

Pdzrn3, a multidomain protein with E3-ubiquitin ligase activity, has been reported to play a role in myoblast and osteoblast differentiation and, more recently, in neuronal and endothelial cell development. The expression of the pdzrn3 gene is developmentally regulated in various vertebrate tissues, including muscular, neural and vascular system. Little is known about its expression during kidney development, although genetic polymorphisms and alterations around the human pdzrn3 chromosomal region have been found to be associated with renal cell carcinomas and other kidney diseases. We investigated the pdzrn3 spatio-temporal expression pattern in Xenopus laevis embryos by in situ hybridization. We focused our study on the development of the pronephros, which is the embryonic amphibian kidney, functionally similar to the most primitive nephric structures of human kidney. To explore the role of pdzrn3 during renal morphogenesis, we performed loss-of-function experiments, through antisense morpholino injections and analysed the morphants using specific pronephric markers. Dynamic pdzrn3 expression was observed in embryonic tissues, such as somites, brain, eye, blood islands, heart, liver and pronephros. Loss of function experiments resulted in specific alterations of pronephros development. In particular, at early stages, pdzrn3 depletion was associated with a reduction of the pronephros anlagen and later, with perturbations of the tubulogenesis, including deformation of the proximal tubules. Rescue experiments, in which mRNA of the zebrafish pdzrn3 orthologue was injected together with the morpholino, allowed recovery of the kidney phenotypes. These results underline the importance of pdzrn3 expression for correct nephrogenesis

Transcriptome Adaptation of the Ovine Mammary Gland to Dietary Supplementation of Extruded Linseed

Several dietary strategies were adopted to reduce saturated fatty acids and increase beneficial fatty acids (FA) for human health. Few studies are available about the pathways/genes involved in these processes. Illumina RNA-sequencing was used to investigate changes in the ovine mammary gland transcriptome following supplemental feeding with 20% extruded linseed. Comisana ewes in mid-lactation were fed a control diet for 28 days (control period) followed by supplementation with 20% DM of linseed panel for 28 days (treatment period). Milk production was decreased by 30.46% with linseed supplementation. Moreover, a significant reduction in fat, protein and lactose secretion was also observed. Several unsaturated FAs were increased while short and medium chain saturated FAs were decreased by linseed treatment. Around four thousand (1795 up- and 2133 down-regulated) genes were significantly differentially regulated by linseed supplementation. The main pathways affected by linseed supplementation were those involved in the energy balance of the mammary gland. Principally, the mammary gland of fed linseed sheep showed a reduced abundance of transcripts related to the synthesis of lipids and carbohydrates and oxidative phosphorylation. Our study suggests that the observed decrease in milk saturated FA was correlated to down-regulation of genes in the lipid synthesis and lipid metabolism pathways

Discovering the Repeatome of Five Species Belonging to the Asteraceae Family: A Computational Study

Genome divergence by repeat proliferation and/or loss is a process that plays a crucial role in species evolution. Nevertheless, knowledge of the variability related to repeat proliferation among species of the same family is still limited. Considering the importance of the Asteraceae family, here we present a first contribution towards the metarepeatome of five Asteraceae species. A comprehensive picture of the repetitive components of all genomes was obtained by genome skimming with Illumina sequence reads and by analyzing a pool of full-length long terminal repeat retrotransposons (LTR-REs). Genome skimming allowed us to estimate the abundance and variability of repetitive components. The structure of the metagenome of the selected species was composed of 67% repetitive sequences, of which LTR-REs represented the bulk of annotated clusters. The species essentially shared ribosomal DNA sequences, whereas the other classes of repetitive DNA were highly variable among species. The pool of full-length LTR-REs was retrieved from all the species and their age of insertion was established, showing several lineage-specific proliferation peaks over the last 15-million years. Overall, a large variability of repeat abundance at superfamily, lineage, and sublineage levels was observed, indicating that repeats within individual genomes followed different evolutionary and temporal dynamics, and that different events of amplification or loss of these sequences may have occurred after species differentiation

Genome-wide identification and characterisation of exapted transposable elements in the large genome of sunflower (Helianthus annuus L.)

Transposable elements (TEs) are an important source of genome variability, playing many roles in the evolution of eukaryotic species. Besides well-known phenomena, TEs may undergo the exaptation process and generate the so-called exapted transposable element genes (ETEs). Here we present a genome-wide survey of ETEs in the large genome of sunflower (Helianthus annuus L.), in which the massive amount of TEs, provides a significant source for exaptation. A library of sunflower TEs was used to build TE-specific Hidden Markov Model profiles, to search for all available sunflower gene products. In doing so, 20,016 putative ETEs were identified and further investigated for the characteristics that distinguish TEs from genes, leading to the validation of 3,530 ETEs. The analysis of ETEs transcription patterns under different stress conditions showed a differential regulation triggered by treatments mimicking biotic and abiotic stress; furthermore, the distribution of functional domains of differentially regulated ETEs revealed a relevant presence of domains involved in many aspects of cellular functions. A comparative genomic investigation was performed including species representative of Asterids and appropriate outgroups: the bulk of ETEs resulted specific to the sunflower, while few ETEs presented orthologues in the genome of all analysed species, making the hypothesis of a conserved function. This study highlights the crucial role played by exaptation, actively contributing to species evolution

How Quercus ilex L. saplings face combined salt and ozone stress: a transcriptome analysis

Background: Similar to other urban trees, holm oaks (Quercus ilex L.) provide a physiological, ecological and social service in the urban environment, since they remove atmospheric pollution. However, the urban environment has several abiotic factors that negatively influence plant life, which are further exacerbated due to climate change, especially in the Mediterranean area. Among these abiotic factors, increased uptake of Na + and Cl − usually occurs in trees in the urban ecosystem; moreover, an excess of the tropospheric ozone concentration in Mediterranean cities further affects plant growth and survival. Here, we produced and annotated a de novo leaf transcriptome of Q. ilex as well as transcripts over- or under-expressed after a single episode of O3 (80 nl l-1, 5 h), a salt treatment (150mM for 15 days) or a combination of these treatments, mimicking a situation that plants commonly face, especially in urban environments. Results: Salinity dramatically changed the profile of expressed transcripts, while the short O3 pulse had less effect on the transcript profile. However, the short O3 pulse had a very strong effect in inducing over- or under-expression of some genes in plants coping with soil salinity. Many differentially regulated genes were related to stress sensing and signalling, cell wall remodelling, ROS sensing and scavenging, photosynthesis and to sugar and lipid metabolism. Most differentially expressed transcripts revealed here are in accordance with a previous report on Q. ilex at the physiological and biochemical levels, even though the expression profiles were overall more striking than those found at the biochemical and physiological levels. Conclusions: We produced for the first time a reference transcriptome for Q. ilex, and performed gene expression analysis for this species when subjected to salt, ozone and a combination of the two. The comparison of gene expression between the combined salt + ozone treatment and salt or ozone alone showed that even though many differentially expressed genes overlap all treatments, combined stress triggered a unique response in terms of gene expression modification. The obtained results represent a useful tool for studies aiming to investigate the effects of environmental stresses in urban-adapted tree species

Notulae to the Italian alien vascular flora: 14

In this contribution, new data concerning the distribution of vascular flora alien to Italy are presented. It includes new records, confirmations, and status changes for Italy or for Italian administrative regions. Nomenclatural and distribution updates, published elsewhere, and corrections are provided as Suppl. materia

Notulae to the Italian alien vascular flora: 14

In this contribution, new data concerning the distribution of vascular flora alien to Italy are presented. It includes new records, confirmations, and status changes for Italy or for Italian administrative regions. Nomenclatural and distribution updates, published elsewhere, and corrections are provided as Suppl. material

Transcriptome analysis of plant-fungus interaction: RNA-seq approach on sunflower (Helianthus annuus L.) mycorrhizal roots.

Arbuscular mycorrhizal (AM) symbiosis is a beneficial interaction between plant and fungus occurring in almost 80% of land plants, AM fungi can penetrate the root creating a tree-shaped structure called arbuscule within the host cell. Fungus eases host nutrients uptake from the soil, especially phosphorus (P) and nitrogen (N), in exchange it receives sugars deriving from plant photosynthesis. AM symbiosis in agriculture reduce the impact of fertilizer for crops. Sunflower (Helianthus annuus L.) is a worldwide cultivated oilseed crop belonging to the asteraceae family, the most numerous amongst angiosperms. Recently has been assessed its mycorrhizal status in wild and cultivated genotypes but no molecular data are available for symbiosis in this plant. H.annuus genome (inbred line XRQ) has been recently sequenced and represents a complex challenge due to its size (about 3.3 Giga bp) and composition, made of 80% of repetitive elements represented especially by retrotransposons. Next generation sequencing RNA-seq allows exploration of wide transcriptome changes in cells or tissues under altered status as for instance during plant-fungus interaction. This method has been exploited to asses gene expression during AM symbiosis only in leguminosae and solanaceae but barely is known for other species. Aim of this work is to exploit RNA-seq approach on mycorrhizal roots of sunflower during early and late phase of symbiosis in order to investigate both gene and retrotransposons expression. For the experimental procedures several seeds of H.annuus (inbred line HA412-HO) were germinated then one-week old plantlets were split in two groups, one of control and the other inoculated with the AM fungus Rhizoglomus irregulare. In order to establish symbiosis progression staining analyses have been performed on inoculated roots. Roots of control and mycorrhizal plantlets were harvested at 4 and 16 days post inoculation, representing early and late stage of symbiosis,respectively. Three biological replicates for treatment and harvest point were retained and mRNA extracted from harvested roots. Overall 12 cDNA libraries were sequenced by Illumina sequencer. In the first part of the thesis sequence reads deriving from the libraries were aligned on sunflower transcriptome reference (inbred line XRQ). Raw counts were normalized and statistical analyses on pairwise comparison between control and inoculated roots at early and late stage of colonization were carried out to identify differentially expressed genes (DEGs). Overall 19 up-regulated DEGs were found during the early phase of symbiosis while 694 and 13 respectively up and down-regulated transcripts have been retrieved in the late stage of colonization. Whole DEGs detected during early phase were differentially expressed also in the late phase of symbiosis suggesting a pivotal role of these genes for onset and maintenance of plant-fungus interaction. Transcripts differentially expressed during late phase of symbiosis were principally involved in biological functions such as signaling, cell-wall and membrane remodeling, transcription, hormone biosynthesis and transport. Besides known genes related to symbiosis even some brand new transcripts were detected in plant-fungus interaction as for instance encoding several BAHD-acyltransferases, an oxoglutarate dependent dioxygenase and a stellacyanin-like protein. In the second part of the thesis, in order to analyse retrotransposon expression during AM symbiosis, reads from the libraries were mapped on reverse transcriptase (RT) retrotranspons domain sequences extracted from whole genome assembly of sunflower inbred line HA412-HO. Corresponding expression values of raw counted reads were analyzed in order to investigate RT expression in mycorrhizal and control libraries at early and late phase of symbiosis. Globally 46 RTs, mostly belonging to Copia superfamily, resulted expressed in control and inoculated samples. Amongst these 4 Copia were differentially expressed between control and inoculated roots after 16 days of symbiosis. In addition, at genomic sequence level, expressed RTs showed a significant higher number of genes placed in the proximity (100,000 bp up and down-stream) of the sequence compared to not expressed ones, suggesting that RT expression could be related to epigenetic control of gene expression. Finally, considering the retrotranpsosons activation after environmental stimuli, the expression of the 46 RTs was compared to other biotic (GA3, SA, IAA, Strigolactones, Brassinosteroids, Ethylene, Kinetin) and abiotic (Salt, PEG) stimuli on sunflower roots. After comparison 25 RTs were found to be expressed in at least one of the treatments, 17 were expressed specifically during fungal symbiosis and 4 were differentially expressed uniquely after plant-fungus interaction. In conclusion AM symbiosis between H. annuus and R. Irregulare leads to a wide remodeling of plant transcriptome concerning both genes and non-genes sequences such as retrotransposons, that may possibly cause rearrangement of host genome

A computational comparative study of the repetitive DNA in the genus Quercus L

An overall picture comparing the repetitive components of the genomes of three Quercus species was obtained by genome skimming with Illumina sequence reads. Read sets of Q. lobata, Q. robur, and Q. suber species were subjected to hybrid clustering in order to assemble a repeatome of the Quercus genus and to annotate it. The repeatome was composed of 8573 clusters. The abundance of repeated sequences in the three species was assessed by mapping Illumina reads of each species onto the repeatome. The repetitive portion of the genome was similar among the three species. The most abundant repetitive sequences were long terminal repeat-retrotransposons. Copia elements were overrepresented when compared with Gypsy ones. The most abundant retrotransposon lineages were SIRE for the Copia superfamily and Ogre/TAT for the Gypsy superfamily. Some of the clusters belonging to these lineages showed different transpositional time profiles among the three species. Ribosomal DNAs accounted for 3.64–6.75% of the repetitive component. Satellite DNAs were much more abundant in Q. lobata (8.68% of the genome) than in the two other species. However, different satellite DNAs showed large variations in their abundances. Overall, the composition of the repetitive portion of the genome showed some differences among oak species, suggesting a possible role of repeats for Quercus species differentiation. In the cases of Q. lobata and Q. robur, both of which belong to the Quercus section of the Quercus genus, such differences may be related to the different geographical origins of the species