Dissertation committee:
Γεώργιος Διαλλινάς, Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Σπυρίδων Ευθυμιόπουλος, Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Παναγιώτης Πολίτης, Ερευνητής Β, Ι.ΙΒ.Ε.Α.Α.
Παναγιώτα Παπαζαφείρη, Αναπλ. Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ
Δήμητρα Θωμαΐδου, Ερευνήτρια, Ελληνικό Ινστιτούτο Παστέρ
Ευαγγελία Εμμανουηλίδου, Επικ. Καθηγήτρια, Τμήμα Χημείας, ΕΚΠΑ
Ιωάννης Σεραφειμίδης, Ερευνητής Γ, Ι.ΙΒ.Ε.Α.Α.
Summary:
With the advent of new generation sequencing technologies, a growing list of formerly unknown regulatory RNA species have come into spotlight. Among them, long non-coding RNAs (lncRNAs) have been found to control stem cell pluripotency, carcinogenesis, development and function of several tissues and organs. Although thousands of lncRNAs are expressed in adult mammalian brain in a highly patterned and specific manner, they remain poorly characterized and their roles in brain development have not yet been studied.
To tackle this question, we initially performed RNA-Seq analysis in the developing nervous system of mouse embryo at embryonic day E12.5. Based on this analysis, we identified many lncRNAs highly expressed in neural cells. We focused on lncRNAs, which are transcribed from genomic loci in close proximity with protein coding genes, encoding for critical transcription factors (TFs) in brain development. We hypothesized that these lncRNAs may be implicated in the regulation of neighboring TF genes.
To this end, we characterized the changes in the expression profiles of a number of lncRNAs-TF pairs during development of mouse brain with real time-qPCR and in situ hybridizations. In this study, we focused firstly on the functional role of lncRNA TCONS_00034309, named by us as Lacuna, in the differentiation of neural stem cells and its relation to Tbr2 transcription factor, a critical regulator of neurogenesis, by ex vivo overexpression and knockdown studies.
More specifically, Lacuna gene is on chromosome 9, around 1kb away from Tbr2/Eomes gene. Its transcript consists of 3 exons and a total length of 1661 nt, as confirmed by our mapping strategy. Lacuna is differentially expressed in the developing mouse brain with higher expression during embryonic days E15 and E16. In situ hybridizations showed specific expression in the ventricular zone and cortical plate of the developing mouse cortex. Subcellular fractionation of neural stem cells and subsequent real time-qPCR revealed that Lacuna is found both in the cytosol and the nucleus, suggesting the possibility that it functions both in cis (affecting the neighboring gene) and in trans (affecting distal gene/genes or being involved in cytoplasmic processes).
To further clarify the functional role of Lacuna, we used a culture system for primary neural stem/progenitor cells, where progenitor/stem cells are isolated from mouse embryonic cortices of E14.5 and then, cultured appropriately to form neurospheres. In the presence of growth factors, neural stem cells (NSCs) are proliferating, whereas in the absence of growth factors, NSCs are differentiating into neurons and astrocytes. Using this culture system and upon overexpression of Lacuna, neurogenesis is significantly reduced (b-III tubulin and NeuN markers) and Olig2+ population is increased. On the other hand, astrogliogenesis doesn’t seem affected, as well as proliferation (BrdU+ index) and apoptosis (cleaved caspase 3+ index), but Nestin, a marker of neural cell stemness, is increased. Moreover, TBR2/EOMES+ population and Tbr2/Eomes expression are not affected by Lacuna overexpression, indicating that the effect on neurogenetic events is Tbr2 independent and suggesting a possible in trans action of Lacuna lncRNA.
In order to further elucidate this, we performed knock-down experiments in the same culture system, using a CRISPR-dCas9-KRAB Effector System to repress the transcription of Lacuna. After confirming the effectiveness of the technique (Lacuna is significantly repressed), we also confirmed that the system does not affect the locus in general (the neighboring genes Golga4 and Gm33460 are unaffected). Of note, knockdown of Lacuna in NSCs in the presence of growth factors results in dramatic downregulation of Tbr2/Eomes gene, suggesting that Lacuna is necessary for Tbr2 expression in NSCs.
Furthermore, in the absence of growth factors, Lacuna knockdown significantly promotes the differentiation of NSCs into both neurons (b-III tubulin+ index and NeuN+ index) and astrocytes (GFAP+ index), whereas the Olig2+ progenitor population and the Nestin+ cells are decreased. In this setup, Tbr2 is not expressed, as most of the cells have already been committed to a cell fate, suggesting that Lacuna has an in trans, differentiation-inhibiting action in NSCs.
Another lncRNA that drew our attention is Lockd, a lncRNA that was already studied in an erythroid cell line. Lockd is a 434 nt lncRNA located 4 kb away from Cdkn1b gene. Cdkn1b encodes for p27, a well studied cyclin-dependent kinase inhibitor. p27 is extensively studied in the nervous system, with established roles in promoting cell cycle exit, neuronal differentiation and migration. Intriguingly, both Lockd and p27 are differentially expressed during mouse brain development, but their expression profiles are opposite. Due to the proximity of Lockd and p27 loci and the involvement of p27 in cell cycle exit events, we first used N2A cells, a fast-growing mouse neuroblastoma cell line, to study Lockd and its relation to p27.
Indeed, upon overexpression of Lockd lncRNA in N2A cells, proliferation is significantly increased. Furthermore, under the same conditions, p27 expression is repressed, proposing that Lockd inhibits expression of p27, which in turn results in increased proliferation, as p27 physiologically promotes cell cycle exiting. As proliferation is a crucial process in brain development and neural stem cells, we also examined Lockd expression in NSCs cultures.
In fact, Lockd is expressed in NSC cultures, but more interestingly, it is significantly downregulated in minus growth factors conditions in comparison to plus growth factors conditions. Additionally, upon overexpression of Lockd in NSCs, proliferation is increased and p27 expression is repressed, as in N2A cells. These primary findings reveal an exciting relation of Lockd with p27, but also an important role of this lncRNA in the proliferation of N2A cells and neural stem cells.
To conclude, our data suggest that lncRNAs are new key players in differentiation and proliferation during brain development and we provided at least two such examples. Lacuna, a novel lncRNA, is necessary for Tbr2 expression and inhibits differentiation of NSCs and Lockd, an already studied lncRNA in another system, affects negatively p27 expression and promotes proliferation. Our study provides insights into the involvement of lncRNAs in organogenesis of the CNS and shows that lncRNAs and protein-coding genes form regulatory networks with important functions in neural stem cells and brain development.
