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Vaccari, Enrico (2009) Ruolo dei geni prep nella formazione dello scheletro facciale di Danio rerio. [Tesi di dottorato]

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Abstract (inglese)

The aim of the present work was the determination of the role of prep1.1 and prep 1.2 genes in the craniofacial skeleton development of Danio rerio. Prep genes encode homeoproteins belonging to the family of Meinox, a subclass of TALE proteins (Three Amino acid Loop Extension). Meinox proteins associate with members of the Pbx protein family to constitute the major partner of Hox proteins: in turn, the trimeric complex Meinox-Pbx –Hox binds to specific target genes. Due to the teleost genome duplication, zebrafish has two prep1 proteins. Indeed, considering the partial redundant function of paralogue genes, zebrafish constitute a good model to study genes with embryological pleiotropic effect. Thanks to a combined genetic, molecular and hystochemical approach we elucidated a different role of prep1.1 and prep1.2 in ruling the constitution of the craniofacial skeleton. As prep1.1 is fundamental to the differentiation of the neural crest cells (NCC) in chondrocytes, prep1.2 regulates the differentiation of the NCC in a non cell-autonomous way through the control of the pharyngeal endoderm segmentation. Employing the luciferase reporter gene and qRT-PCR we were able to highlight in vivo that prep1.2 transcription is positively regulated by RA (Retinoic Acid) by means of a 3’ -RARE (RA- Responsive Element) located in the first intron. Furthermore we stressed the ability of Prep1.2 to control the RA synthesis , directly regulating the expression of the enzyme responsible of its own production and establishing an autoregulative loop. Our results demonstrate the in vivo interaction among Meinox genes and RA. Moreover we have been able to provide clear evidence of the functional specialization of prep1 genes in zebrafish craniofacial development.

Abstract (italiano)

In questo lavoro è stata analizzata la funzione nella formazione dello scheletro facciale dei geni prep1.1 e prep1.2 di Danio rerio. I geni prep codificano per omeoproteine facenti parte della famiglia Meinox una classe delle proteine TALE (Three Amino acid Loop Extension). Le proteine Meinox in associazione con le proteine della famiglia Pbx, oltre ad essere i principali partner delle proteine Hox, possiedono numerose altre funzioni. La presenza di due proteine prep1 in zebrafish, dovuta alla duplicazione del suo genoma, ha reso possibile lo studio delle loro funzioni embrionali in un modello meno affetto da pleiotropia vista la parziale ridondanza funzionale dei geni paraloghi. Attraverso metodiche genetiche, molecolari ed istochimiche si è riusciti a stabilire che i due geni controllano la formazione dello scheletro facciale in modi differenti. Mentre prep1.1 è necessario per il differenziamento delle cellule delle creste neurali craniali in condrociti, prep1.2 regola il differenziamento delle CCN in maniera cellulo indipendente attraverso il controllo della segmentazione dell’endoderma faringeo. Usando il gene reporter della luciferasi e qRT-PCR abbiamo dimostrato che i livelli trascrizionali di prep1.2 sono regolati positivamente dall’AR attraverso una regione 3’-RARE (Retinoic Acid Responsive Element) presente all’interno del suo primo introne. Abbiamo inoltre evidenziato la capacità di Prep1.2 di controllare la sintesi dell’AR attraverso il controllo dell’espressione dell’enzima responsabile della sua sintesi entrando così a far parte di un loop auto regolativo. I risultati ottenuti oltre ad essere la prima dimostrazione in vivo dell’interazione tra geni Meinox ed AR dimostrano una forte specializzazione funzionale dei geni prep di zebrafish durante lo sviluppo dello scheletro cranio facciale.

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Tipo di EPrint:Tesi di dottorato
Relatore:Argenton, Francesco
Dottorato (corsi e scuole):Ciclo 21 > Scuole per il 21simo ciclo > BIOSCIENZE > GENETICA E BIOLOGIA MOLECOLARE DELLO SVILUPPO
Data di deposito della tesi:02 Febbraio 2009
Anno di Pubblicazione:31 Gennaio 2009
Parole chiave (italiano / inglese):zebrafish prep1.1 prep1.2 morpholino
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/11 Biologia molecolare
Struttura di riferimento:Dipartimenti > Dipartimento di Biologia
Codice ID:1912
Depositato il:02 Feb 2009
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