UTILIZACIÓN DE ORGANOIDES CEREBRALES PARA EL ESTUDIO DE LA REGULACIÓN TRANSCRIPTIONAL DEL GEN TBR2 HUMANO DURANTE EL DESARROLLO DE LA CORTEZA CEREBRAL

Brain cortex development depends on the combined activity of a series of transcription factors and signaling pathways. The expression of these genes is finely regulated in both space and time, and even small alterations of these transcriptional profiles can result in severe brain/cognitive defects.

The TBR2 gene codes for a transcription factor specifically expressed in a population of precursors, called intermediate progenitors, which contribute essentially to the formation of the most superficial cortical layers of the cortex. The number and diversity of neurons in these layers, as well as the contribution of intermediate progenitors to cortical development, is particularly important in gyrencephalic animals (whose brains have convolutions) and especially in primates and humans. Besides, mutations that affect the non-coding genomic region of the TBR2 gene result in its silencing and in severe brain defects. However, although the functions of the TBR2 gene have been extensively investigated, the mechanisms that control its activation are poorly understood. This is largely due to the evolutionary divergence of the genomic region where the TBR2 gene is located, both in terms of non-coding sequences and synteny, as well as the requirement for complex transgenesis and genome editing experiments to study the transcriptional regulation of developmental genes, which also involve a large number of animals.

In this project we intend to address the study of the transcriptional regulation of the human TBR2 gene using brain organoids derived from human Induced Pluripotent Stem (iPS) cells as a model system. These organoids are 3D cultures that recapitulate the development and organization of the embryonic cerebral cortex. Epigenetic profiling and chromatin interaction analysis techniques will be combined to identify the putative regulatory sequences of the TBR2 gene. Net, The candidate sequences will be cloned into a reporter vector encoding the fluorescent protein GFP and transgenic human iPS cell lines will be established for each of them. These will be used to generate brain organoids and assess the functionaly of each putative TBR2 enhancer. Finally, compatible with the times of the TFM, a CRISPR/Cas9 approach will be used to eliminate specific enhancers (or groups of them) based on the results obtained in the initial screening and observe the effects on the transcription of the TBR2 gene.

The objectives of the current TFM proposal and the experimental approaches to be carried out by the selected TFM student are:

- Cloning of candidate enhancer sequences for the TBR2 gene: based on previously published epigenetic profiles, I have identified a total of 15 putative regulatory sequences for the TBR2 gene active in the human embryonic cerebral cortex. We will clone each of these sequences in a GFP reporter vector previously generated in the laboratory using basic molecular cloning techniques.

- Characterization of the epigenetic and chromatin interaction profiles of the genomic region of the TBR2 gene by ChIPseq and 4Cseq techniques.

- Generation of transgenic iPS cell lines: by electroporation of the generated plasmids and antibiotic resistance selection. Selected clones will be characterized by PCR and Sanger sequencing.

- Generation of brain organoids from transgenic iPS cell lines and analysis of GFP expression by confocal fluorescence microscopy. In addition, the activity of the enhancers that are positive in the different types of neuronal precursors will be analyzed based on specific markers by immunofluorescence techniques in cryosections.

- Generation of mutant iPS cell lines with deletions of enhancers or groups of enhancers by CRISPR/Cas9 electroporation in iPS cells. This last objective is subject to the progress of the project and its completion times.