Thursday, November 21, 2024
11/21/2024
Project Summary Hearing loss and vestibular dysfunction are caused by death of sensory hair cells that in mammals fail to regenerate. Therefore, there is an urgent need to develop techniques for regenerating hair cells in humans. Promising progress has been made but the regenerated hair cells do not fully mature. It is therefore essential that we gain a detailed understanding of the gene regulatory networks (GRNs) regulating hair cell differentiation. In mice several transcription factors (TFs) regulate hair cell subtypes, such as TBX2 that specifies inner ear hair cells (IHCs) and INSM1 and IKZF2 that are important for outer hair cell (OHC) fate determination. How these TFs are regulated, how they interact with each other and what their direct targets are is unknown. Also, as these factors are not sufficient to completely convert hair cell fates, additional co-factors must exist. Zebrafish possess hair cells not only in their ears but also in the skin as part of the sensory lateral line (LL) system. We and others have developed the zebrafish LL into a powerful model system to interrogate the molecular mechanisms underlying hair cell regeneration. Our preliminary scRNA-Seq and TF motif analyses show that the GRNs that regulate zebrafish lateral line versus ear hair cell identities share genes with the mouse GRN that regulates IHCs versa OHCs, such as Insm1, Ikzf2 and Tbx2. In addition, we identified prdm1a as a new regulatory factor controlling hair cell differentiation. Strikingly, loss of prdm1a leads to a conversion of LL hair cells into ear hair cells via the activation of tbx2. Prdm1 is also expressed in mouse progenitor cells but is turned off in hair cells. We hypothesize that the genes involved in the lineage decisions that induce cells to differentiate from a common hair cell progenitor into organ-specific hair cell types are regulated by similar core GRNs in zebrafish and mammals. Here we are proposing to characterize the GRNs consisting of insm1a/b, ikzf2, prdm1a and tbx2a/b underlying hair cell subtype decisions in the lateral line and ear, identify additional genes in the GRNs and functionally test key genes and enhancers. Importantly, building cell type-specific GRNs during the regeneration time course will identify the injury-responsive upstream regulators of the hair cell type-specifying genes. We will integrate single cell gene expression and chromatin accessibility from the same cells at several regeneration time points to predict TF binding motifs in cis- regulatory elements, linking TFs to enhancers and target genes. We will then computationally infer gene regulatory networks and key TFs that control hair cell lineage commitment events in the LL and ear. TFs predicted to play key roles will be functionally tested. In summary, our proposed experiments will construct GRNs of hair cell subtype differentiation in the zebrafish in a regenerative context, which will yield candidate factors essential for directing the differentiation of the correct hair cell types in various mouse sensory epithelia during regeneration and organoid production. In addition, the comparison of the GRNs between the LL and the ear will provide insights into how changes in regulatory elements drive the evolution of different hair cell types.
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