A lock on T Cell factor activity: from the protein complex conformation to its biological impacts on the Cerebellar Upper Rhombic Lip – LockOnURL

The cerebellum is vital in all jawed vertebrates for motor control and higher cognitive functions. Cerebellar neurons arise from two main germinative niches: the ventricular zone and the upper rhombic lip (URL), a specialized tissue with neural stem cell (NSC) niche properties. Recent studies have provided insights into the architecture of the human rhombic lip (RL) and how defects in its development contribute to various human syndromes. These findings challenge the rodent-centric view of URL development, highlighting the need for new experimental models and a deeper understanding of the pathways and gene sets that drive URL trajectories.
T Cell Factors (TCF) are essential transcription factors (TF) in the Wnt canonical pathway. TCF activity and the expression of proneural gene repressors Hairy and Enhancer of Split (Hes/Hey) are key markers of NSC self-renewal. TCF activity and Hes/Hey transcripts have been detected in the URL across species, but much remains unknown about their roles, as well as their regulatory interactions in the human cerebellar RL.
Our research indicates that amphibians are a relevant model for studying vertebrate URL development. We also discovered that BARHL TFs inhibit TCF activity and influence URL cell fate decisions, possibly via interactions with TCF and HES.
LockOnURL employs a transdisciplinary, multi-scale approach combining computational biology, artificial intelligence, multi-omics, bioinformatics, molecular biology, biochemistry, and developmental biology, spanning from Xenopus embryos to human organoids. The goal is to characterize the TCF/BARHL/HES interactive network, examining its 3D structure and transcriptional impact on cerebellar URL biology.
LockOnURL's objectives are:
1. To identify and validate the interacting surfaces and specificity determinants of TCF, GRO/TLE, BARHL, and HES proteins and develop a deep learning algorithm for predicting disordered protein interactions.
2. To define the transcriptomic profiles, conserved cell-state markers, and gene expression trajectories during early neurogenesis in amphibians using multi-omics techniques, including Single-Nucleus (SN) and Assay for Transposase-Accessible Chromatin (ATAC) sequencing, and compare them to mammals.
3. To examine how altered TCF activity impacts cellular differentiation and progression in the URL/EGL, including its role in cell fate specification, chromatin remodeling, and progenitor cell maintenance.
4. To study TCF activity in human-derived cerebellar organoids and assess the effects of TCF activation or inhibition, especially via BARHL interactions, on URL cell fate.
LockOnURL will document and validate the interactions, binding specificities, and conformational changes of TCF/LEF, Gro/TLE, BARHL, and HES transcriptional complexes both in vitro and in vivo. It will identify the structures, stoichiometries, molecular determinants, post-translational modifications, and potential interaction partners within these complexes. A computational tool will also be developed for large-scale proteome-wide screens to uncover potential TF interactions.
LockOnURL will track cell trajectories and gene regulatory networks in the developing amphibian cerebellum while conducting a cross-species comparison of URL trajectories. This will reveal conserved and unique pathways that govern URL cell behavior across species.
Ultimately, LockOnURL aims to establish a robust model system for studying harmful developmental changes in early cerebellar development. It will provide unprecedented insights into the dynamic role of TCF signaling in both amphibian and human cerebellar development at the cellular and molecular levels. The project will also offer essential information on the origins of cells and developmental hierarchies in the human RL, shedding light on dysregulated processes in cancer cells and informing potential therapeutic approaches.