Modeling Bosch-Boonstra-Schaaf Optic Atrophy Syndrome in 3D human neural organoids to elucidate cell type-specific expression and function of NR2F1.

• Applicant: Magdalena Laugsch, Ph.D.
• Subject Area: Human Genetics
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 450057775

The transcription factor NR2F1 regulates the expression of various genes that control the development of neural progenitor cells (NPC) and cortical neurons (CN) in the brain as well as neural crest cells (CNCC) in the craniofacial formation (head and face). NR2F1 mutations or deletions cause Bosch-Boonstra-Schaaf Optic Atrophy Syndrome (BBSOAS) characterized by delayed development, intellectual disability, optic atrophy, and craniofacial abnormalities. All reported BBSOAS cases with NR2F1 deletions encompass also the locus of the adjacent long non-coding RNA lncNR2F1. While current data indicate that the lncNR2F1 transcript does not regulate NR2F1 expression, the impact of regulatory elements within the lncNR2F1 locus on NR2F1 expression remains unexplored. In our ongoing DFG-funded project, we have elucidated the effect of lncNR2F1 deletion on NR2F1 expression and BBSOAS. We generated a novel panel of CRISPR/Cas9 engineered induced pluripotent stem cell (hiPSC) lines, with NR2F1 and lncNR2F1 deletion, and differentiated them into CNCC. Utilizing state-of-the-art techniques from epigenomics, transcriptomics, and bioinformatics, we showed that the lncNR2F1 deletion reduces tissue-specific NR2F1 expression suggesting CNCC-specific enhancers at the lncNR2F1 locus. Importantly, dysregulated NR2F1 level was shown to affect bone development. Our pending studies will reveal whether analysis of these regulatory elements should be included in the molecular diagnosis of BBSOAS. Although our study focuses on CNCC development, we have provided the first experimental evidence that in NPC and CN the lncNR2F1 locus does not enhances but silences NR2F1 expression and aim to investigate this relationship in more detail. NR2F1 binds to thousands of targets (genes and regulatory elements) to achieve its function. Hence, we are determining NR2F1-dependent regulatory networks essential for CNCC differentiation. In the follow-up project we plan to map NR2F1-dependent regulatory networks in NPC/CN. So far, we have employed two-dimensional (2D) cell culture, which is a robust and commonly used model to study NPC/CN but does not reflect the architecture and cell heterogeneity of otherwise unavailable human live tissue. In contrast, 3D cell aggregated (organoids) closely mimic the complex tissue organization. Since NR2F1 gradient is crucial for proper patterning and defined cellular composition of the developing forebrain, we will study the impact of the silencer on NR2F1 expression in forebrain organoids with NR2F1 and lncNR2F1 deletion. Simultaneous interrogation of single-cell transcriptomics and epigenomics data from the same cell will allow novel and contextual integration of NR2F1 expression and function across multiple cell types within the human forebrain tissue in a dish. Our genome-wide data will significantly deepen the understanding of the multifaceted role NR2F1 biology and may contribute to improvement and development of novel BBSOAS treatment.

• DFG Programme: Research Grants