Speakers
Description
Mutations in the SOX2 gene lead to defects in the development of multiple brain regions, causing blindness, intellectual disabilities, and seizures. SOX2 is a transcription factor that regulates many genes, some of which, when mutated, are involved in other neurodevelopmental disorders (NDDs).
Using RNA sequencing, we identified over a thousand genes that are downregulated following Sox2 deletion in neural stem cells derived from the developing mouse brain. Many of them have human homologs, and many belong to the ""T-Dark"" gene category, a group of genes of mostly unknown function.
We focused on 122 human T-Dark genes, whose expression is most reduced in Sox2-deleted mouse brain-derived neural stem cells. To explore their role in neurodevelopment, we applied a screening strategy based on growing in vitro human brain organoids derived from a previously established human embryonic stem cell line. This screening uses the CRISPR Lineage Tracing at Cellular Resolution in Heterogeneous Tissue (LICHT) technique, combining CRISPR-Cas9 mutagenesis and a dual barcode method. This approach allows us to identify mutated genes in underrepresented cell clones within brain organoids through genomic sequencing.
We designed and cloned a T-Dark guideRNAs library, and a control library that includes SOX2 and some of its known NDD-related targets. The libraries were then used to transduce the stem cells, from which brain organoids were differentiated. Afterwards, the organoids were dissociated to single cells, and DNA was extracted and prepared for sequencing, presently ongoing. Initial results will be discussed.
In parallel, we worked on optimizing this technique to make it more accessible and efficient for other laboratories.
We hypothesize that these T-Dark genes may act as functional effectors of SOX2. Identifying their neurodevelopmental roles will provide new insights into the SOX2-dependent regulatory network, not only in SOX2-related pathologies but also in other NDDs, with potential therapeutic perspective.
| Author(s) | G. Pozzolini, R. Baldi, A. Cascio, C. Marenco, D. Morciano, G. Antoniazzi, E. Zakirova, T. Lahoud, N. Alfano, G. Testa, C. Peano, S.E. Haendeler, P.C. Esk, V. Krenn and S.K. Nicolis |
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| Affiliation(s) | Dipartimento di Biotecnologie e Bioscienze , Università di Milano Bicocca, Milan, Italy , Human Technopole, Milano, Italy , University of Vienna and Medical University of Vienna, Vienna, Austria , Institute of Molecular Biology, University of Innsbruck, Innsbruck, Austria , Human Technopole Early Career Fellow, Dip. Biotecnologie e Bioscienze , Università degli Studi di Milano Bicocca, Milan, Italy |
