Characterization of morphogenes and new transcription factors involved in the development of striatal medium spiny neurons

Abstract

The striatum is one of the brain structures that controls body movement. In humans the striatum consists of two nuclei, the caudate and putamen which are separated by the internal capsule, while in mice it is a single structure. Its neuronal population is defined by two types of cells: Medium spiny neurons (MSNs) and Interneurons. MSNs comprise 90-95% of the striatal cell population, and can be classified as striatonigral or striatopallidal neurons.

During telencephalic development, the generation of striatal MSNs occurs in the Lateral Ganglionic Eminence (LGE) where there are two defined zones: namely the Germinal Zone (GZ) that consists of progenitor cells, and the Mantle Zone (MZ) where the progenitors have differentiated into postmitotic cells. Several transcription factors have been described to be involved in the generation of striatal MSNs, such as Gsx1/2, Ascl1, Dlx1/2, Dlx5/6, Ebf1 and Ikaros. However, there are still a lot of factors that may participate in the striatal development which function or expression has not been yet elucidated.

Thus, the main objective of this thesis is to study the striatal development characterizing the function and expression of some striatal genes as well as finding out new candidate genes and mechanisms that might be controlling this process.

First, we characterized the role of Nolz1, showing a high expression in the progenitors of the SVZ and with less intensity in the MZ of the LGE. Its expression together with several experiments indicates this gene could be playing a dual role promoting the progenitors to exit the cell cycle and inducing their differentiation into neurons. Furthermore, Nolz1 might be promoting the differentiation of the neural precursors through the retinoic acid signalling pathway.

We continue characterizing the striatal genes studying Helios, which expression was detected between the SVZ and MZ border, although the higher levels were observed in the postmitotic region. First, Helios might arrest neural precursors in the G1 phase of the cell cycle, and second, it would also be promoting the differentiation into striatopallidal MSNs. In Helios knockout mice, it is observed an alteration of the striatal neurogenesis, accompanied by a reduction in the number of born neurons and consequently increasing the number of proliferative precursors. These alterations during development promote an aberrant neuronal differentiation, causing cell death and a reduction in the adult striatopallidal MSNs.

In order to explain the complex network of genes that control the striatal development, we performed a transcriptomic analysis of striatal samples during different developmental stages (E12.5, E14.5, E16.5 and E18.5) and separating both striatal regions (GZ and MZ).

From the analysis we determined that the striatum can be explained by six gene expression profiles or categories, each of them represented by a group of genes that develop specific striatal functions. These categories are: Early LGE progenitors, Late GZ progenitors, Fate specification, Neuronal differentiation, Maturation and Reorganization.

To establish the relation between human and mouse striatum, we compared our mice samples with human striatal samples from a data base, showing similitude between species. From the comparison we could determine new candidate genes to participate in the differentiation of mouse and human striatal MSNs, such as Zfp521/ZNF521 and FOXO1. The characterization of Zfp521/ZNF521 in mouse and human determined it is expressed by striatal postmitotic neurons, and specifically in mouse we could observe that it is expressed by striatonigral MSNs. On the other hand, FOXO1 is expressed in both progenitors and human striatal MSNs.

In conclusion, with the proposal of a striatal model and the characterization of the expression and function of some striatal genes such as Nolz1, Helios, Zfp521/ZNF521 and FOXO1, we bring one more piece to comprehend the complex puzzle that forms the striatal development.

L’estriat és un nucli subcortical del cervell que juga un paper vital en el control del moviment corporal. En l’humà, l’estriat està format de dos nuclis, el caudat i el putamen, separats per la càpsula interna; mentre que en ratolí forma una única estructura. La seva població neuronal es defineix per dos tipus de cèl·lules: les neurones estriatals de projecció (MSNs, de l’anglès Medium Spiny Neurons) i les interneurones. Les més abundants són les MSNs, representant el 90-95% de les neurones estriatals, i que poden classificar-se en estriatopalidals i estriatonigrals. Aquestes neurones es generen en la Eminència ganglionar lateral (LGE, del ingés Lateral ganglionic eminece), on s’hi distingeixen dues zones: la zona germinal (GZ) o proliferativa i la zona del mantell (MZ, del inglés Mantle zone) o postmitòtica. Hi ha diferents factors de transcripció que s’han relacionat amb el desenvolupament de MSNs, no obstant, l’expressió i funció de tots ells encara no està definida i podría haver-hi altres gens involucrats en aquest complex procés. Per tant, l’objectiu d’aquesta tesi és profunditzar en l’estudi del desenvolupament estriatal determinant la funció d’alguns d’aquests gens estriatals així com descrivint l’entramat de gens i mecanismes que regulen aquest procés.

Primer hem caracteritzat el paper de Nolz1, que s’expressa principalment en la SVZ i els estudis ens mostren que estaria jugant un doble paper induint la sortida de cicle cel·lular dels progenitors i promovent la seva diferenciació neuronal a través de la via de l’àcid retinoic. Un altre gen que hem estudiat és Helios, i els resultats mostren que també tindria un doble paper, primer modulant la proliferació dels progenitors i segon promovent la diferenciació de neurones striatopalidals.

Per tal d’explicar l’entramat de gens que controlen el desenvolupament estriatal, hem realitzat un anàlisi transcriptòmic de mostres estriatals durant diferents estadis del desenvolupament (E12.5, E14.5, E16.5 i E18.5) i separant les dues regions estriatals (GZ i MZ). Els resultats de l’anàlisi han mostrat que l’estriat pot explicar-se a través de sis perfils d’expressió genètica o categories, cadascun dels quals està representat per un grup de gens que desenvolupen funcions estriatals específiques. Aquestes categories són: Progenitors de la LGE que s’expressen d’hora, Progenitors tardans de la GZ, Especificació neural, Diferenciació neuronal, Maduració i Reorganització.

Finalment, a través de comparar mostres estriatals de ratolí i humanes vam poder determinar gens candidats a participar en la generació de MSNs durant el desenvolupament striatal humà i de ratolí, el Zfp521/ZNF521 i FOXO1.

Per tant, amb la proposta del model estriatal juntament amb la caracterització de l’expressió i funció d’alguns gens estriatals com Nolz1, Helios, Zfp521/ZNF521 i FOXO1, aportem una peça més del complex trencaclosques que conforma el desenvolupament estriatal.