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Effetti del knockdown del recettore degli estrogeni Î22 (esr2a) sullo sviluppo di Danio rerio
Get PDFABSTRACT
This thesis work is focused on the study of the role of the maternal mRNA for the estrogen receptor erβ2 (esr2a) on the long-term epigenetic modulation of development in zebrafish (Danio rerio).
Experimental evidence indicates that steroid hormones may participate in the maternal programming of the development in fish (Auperin and Geslin, 2008) and mammals (Darnaudéry and Maccari, 2007). In particular, estrogens secreted by the granulosa cells during vitellogenesis may be incorporated in fish oocytes (Hines et al, 1999) with possible significant effects on the subsequent ontogeny of the offspring.
In the present work on zebrafish, the expression levels of the mRNA coding for the Esr2a receptor were found to be high in ovulated oocytes and early embryos. The concentration of these transcripts decreased along the 8 hours post-fertilization (hpf) and then returned to rise with the onset of embryonic transcription.
The gene knockdown by morpholino antisense technology was applied to investigate the functional role of the esr2a in zebrafish development. Two different morpholinos were used: the first, designed on the ATG start translation codon, determines the knockdown by blocking the translation of both maternal and zygotic transcripts (esr2aATGMO); the second interferes with the correct splicing process to block only the post-transcriptional maturation of the zygotic messenger (esr2aSPLICMO).
The embryos microinjected with esr2aATGMO died between 12 and 14 days after fertilization (dpf) and displayed several morphological alterations compared to non-injected embryos (WT) or to controls microinjected with a standard unrelated morpholino (StdMO). The morphant phenotype of fish treated with esr2aATGMO was characterized by delayed body growth, curved shape, abnormal development of brain and splanchnocranium, enlarged and hemorrhagic pericardial cavity, uninflated swim bladder, rudimental caudal fin and abnormal circular swimming.
The specificity of the effects was verified by co-injecting the esr2aATGMO with a morpholino inactivating the p53 protein. The phenotype obtained with the co-injection corresponded to that of fish injected with esr2aATGMO alone. This shows that the effects of the morpholino for esr2a are specific and not due to the hyperactivation of p53 caused by the microinjection. Resumption of the WT phenotype was achieved by co-injecting esr2aATGMO with the transcript for zebrafish Ers2a bearing 8 silent mutations in the region complementary to the morpholino. This demonstrates that the binding of the oligonucleotide to its target was very specific.
To further investigate the role of the gene esr2a, an experiment of gain of function was performed by increasing the concentration of the receptor transcript. Developmental abnormalities were also monitored during first days after fertilization following the microinjection of the mRNA for esr2a into eggs permeated with a solution of 17β-estradiol (E2).
The embryos treated with esr2aSPLICMO were comparable to controls. This morpholino interferes in the process of maturation of the zygotic transcript, leading to the loss of the third exon and to a translated protein that doesn't work. Significantly, this result suggests that the observed phenotypic changes after treatment with esr2aATGMO are mainly due to the inactivation of the maternal transcript.
The in situ hybridization analysis using developmental markers highlighted a possible up-regulation of empty spiracles homeobox 1 (emx1) and of sine oculis homeobox homolog 3a (six3a) at 24 and 48 hpf and of sonic hedgehog (shha) at 48 hpf. Staining with alcian blue revealed an altered pattern of cranial cartilage organization at 6 dpf in fish treated with esr2aATGMO. Subsequent hybridization with cranial markers showed a slightly different expression profiles between WT and morphants of the gene distal-less homeobox gene 2a (dlx2a) at 48 hpf and neurogenic differentiation (NeuroD) at 24 hpf.
Two-color microarray analysis of the total genome of zebrafish was used to study gene expression in embryos at 8 and 48 hpf after treatment with esr2aATGMO compared to controls injected with StdMO. Analysis at 8 hpf highlights changes in gene expression due mainly to the maternal receptor knockdown. The analysis at 48 hpf allows to detect morpholino effects due to the inactivation of embryonic transcripts as well. At 8 hpf, 237 transcripts were significantly up-regulated, while 219 were down-regulated as a result of the absence of the Esr2a receptor in embryos injected with esr2aATGMO. At 48 hpf, 165 and 124 transcripts, presumably of zygotic origin, were up- and down-regulated, respectively. Among these, only 8 were in common with those up-regulated at 8 hpf and 17 with the down-regulated ones.
Interestingly, transcripts involved in the negative regulation of cell proliferation were up-regulated. It can be assumed that the absence of Esr2a leads to a decrease in cell proliferation, perhaps due to an increased apoptosis. This would be in keeping with the TUNEL analysis on the rate of cell death, which showed a greater number of apoptotic cells in the brain of morphant fish at 24 and 48 hpf as compared to controls.
The analysis of embryonic vascularization in the transgenic line TG (fli1:EGFP) at 3 and 6 dpf, based on the vascular activity of endogenous alkaline phosphatase, evidenced a marked decrease in subintestinal vessel formation and derangement in the distribution patterns of trunk and tail vessels in fish treated with esr2aATGMO.
These results suggest that the development of zebrafish is influenced by epigenetic control dependent on maternal esr2a mRNA and interaction with the inheritance of maternal estrogens.RIASSUNTO
Il mio lavoro di tesi si è focalizzato sullo studio del ruolo svolto dagli mRNA ovocitari di origine materna codificanti la forma erβ2 (esr2a) del recettore degli estrogeni nella modulazione epigenetica a lungo termine dello sviluppo in zebrafish (Danio rerio).
Crescenti evidenze indicano che gli ormoni steroidei possono partecipare alla programmazione materna nei processi di sviluppo dei pesci (Auperin e Geslin, 2008) e dei mammiferi (Darnaudéry e Maccari, 2007). In particolare, gli estrogeni secreti dalle cellule della granulosa durante la vitellogenesi possono essere accumulati negli ovociti dei pesci (Hines et al., 1999) con un possibile ruolo di programming iniziale avente effetti profondi sulla ontogenesi successiva.
Nel presente lavoro sullo zebrafish, è stato osservato mediante ibridazione in situ che i livelli di espressione degli mRNA che codificano per il recettore Esr2a sono alti negli oociti ovulati e nell'embrione subito dopo la fecondazione. La concentrazione di questi trascritti diminuisce nelle 8 ore che seguono la fecondazione (hpf), per poi riprendere a crescere dopo l'inizio della trascrizione embrionale, confermando precedenti dati di qRT-PCR (Pikulkaew et al., 2010).
Il ruolo funzionale del gene esr2a è stato studiato mediante silenziamento genico ottenuto con la tecnologia del morfolino antisenso utilizzando due diversi morfolini microiniettati nelle uova appena deposte: il primo, progettato sull'ATG d'inizio traduzione, determina il knockdown mediante blocco della traduzione dei trascritti di origine materna e zigotica (esr2aATGMO), mentre il secondo interferisce nel corretto processo di splicing per bloccare la maturazione post-trascrizionale del solo messaggero zigotico (esr2aSPLICMO).
Gli embrioni ottenuti dopo microiniezione di esr2aATGMO presentano numerose alterazioni morfologiche rispetto agli embrioni non iniettati (WT) o microiniettati con morfolino standard di controllo (StdMO) e muoiono tra i 12 e i 14 giorni dopo la fecondazione (dpf). Il fenotipo morfante è caratterizzato da: ritardo nella crescita corporea, forma ricurva, anormale sviluppo di cervello e splancnocranio, cavità pericardica ampia ed emorragica, vescica natatoria non insufflata, pinna caudale rudimentale e anomala natazione circolare.
La specificità degli effetti ottenuti con la microiniezione è stata verificata anche mediante coiniezione, con l'esr2aATGMO, di un morfolino inattivante la proteina p53. Il fenotipo ottenuto silenziando anche p53 corrisponde a quello morfante; questo dimostra che gli effetti di esr2aATGMO sono specifici e non collegati ad un'iperattivazione di p53 causata dall'iniezione. E' stato possibile inoltre riottenere il fenotipo WT mediante la coiniezione con l'esr2aATGMO del trascritto di zebrafish per Esr2a mutato nella regione complementare al morfolino. Questo dimostra che il legame dell'oligonucleotide al target è estremamente specifico.
Per approfondire in maniera più dettagliata la funzione del gene esr2a ho eseguito un esperimento di gain of function con il quale ho aumentato la concentrazione del trascritto del recettore. In un esperimento successivo ho iniettato l'mRNA per esr2a e trattato le uova con 17β-estradiolo (E2) per osservare eventuali anomalie nei primi giorni dopo la fecondazione.
Dalla microiniezione di esr2aSPLICMO si ottengono embrioni con fenotipo paragonabile ai controlli. Questo morfolino interferisce nel processo di maturazione del messaggero zigotico, portando alla perdita del terzo esone e a una proteina tradotta non funzionante. Il risultato che ho ottenuto E' un dato importante, perchè suggerisce come le variazioni osservabili nel fenotipo esr2aATGMO siano imputabili principalmente all'inattivazione del trascritto materno.
Un'analisi mediante ibridazione in situ utilizzando marcatori di sviluppo ha evidenziato nei morfanti esr2aATGMO una possibile up-regolazione di empty spiracles homeobox 1 (emx1) e sine oculis homeobox homolog 3a (six3a) a 24 e 48 hpf e di sonic hedgehog a (shha) a 48 hpf. La colorazione con alcian blu delle cartilagini craniche ha rivelato un pattern alterato a 6 dpf. Le successive analisi mediante in situ di alcuni marcatori della regione cranica hanno evidenziato un profilo di espressione lievemente differente tra WT e morfanti per il gene distal-less homeobox gene 2a (dlx2a) a 48 hpf e di neurogenic differentiation (neurod) a 24 hpf.
Un'analisi di microarray two-color sul genoma totale di zebrafish è stata utilizzata per studiare l'espressione genica su embrioni di 8 e 48 hpf trattati con esr2aATGMO rispetto ai controlli iniettati con StdMO. Con l'analisi microarray a 8 hpf, si possono evidenziare le variazioni nell'espressione dovute principalmente al knockdown del recettore materno. L'analisi a 48 hpf permette invece di rilevare effetti del morfolino dovuti all'inattivazione anche dei trascritti embrionali.
L'analisi pangenomica di microarray a 8 hpf, ha individuato 237 trascritti significativamente up-regolati e 219 down-regolati a causa dell'assenza del recettore Esr2a materno negli embrioni iniettati con esr2aATGMO. A 48 hpf, 165 e 124 trascritti, presumibilmente zigotici, sono rispettivamente up-e down-regolati. Tra questi solo 8 sono in comune con quelli up-regolati a 8 hpf e 17 con quelli down-regolati.
E' interessante osservare la presenza di un'up-regolazione dei trascritti implicati nella regolazione negativa della proliferazione cellulare. Si può ipotizzare che l'assenza di Esr2a porti ad una diminuzione della proliferazione cellulare, forse dovuta ad un aumento dell'apoptosi. Il dato è in accordo con i risultati del saggio di morte cellulare, mediante TUNEL, che ha evidenziato un numero di cellule apoptotiche nei morfanti notevolmente aumentato e concentrato nell'area cerebrale a 24 e 48 hpf.
L'analisi della vascolarizzazione embrionale sfruttando l'attività vasale della fosfatasi alcalina endogena e mediante l'utilizzo della linea transgenica TG(fli1:EGFP) a 3 e 6 dpf ha mostrato nei morfanti esr2aATGMO una netta diminuzione dei vasi subintestinali ed un pattern di distribuzione dei vasi lungo il tronco e la coda meno definito.
I risultati ottenuti suggeriscono che nello sviluppo di zebrafish sia presente un controllo epigenetico dipendente dall'mRNA materno di Esr2a e da un'interazione con gli estrogeni di eredità materna
Short-term inhibition of TERT induces telomere length-independent cell cycle arrest and apoptotic response in EBV-immortalized and transformed B cells
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