Guided genetic screen to identify genes essential in the regeneration of hair cells and other tissues

Abstract

Regenerative medicine holds great promise for both degenerative diseases and traumatic tissue injury which represent significant challenges to the health care system. Hearing loss, which affects hundreds of millions of people worldwide, is caused primarily by a permanent loss of the mechanosensory receptors of the inner ear known as hair cells. This failure to regenerate hair cells after loss is limited to mammals, while all other non-mammalian vertebrates tested were able to completely regenerate these mechanosensory receptors after injury. To understand the mechanism of hair cell regeneration and its association with regeneration of other tissues, we performed a guided mutagenesis screen using zebrafish lateral line hair cells as a screening platform to identify genes that are essential for hair cell regeneration, and further investigated how genes essential for hair cell regeneration were involved in the regeneration of other tissues. We created genetic mutations either by retroviral insertion or CRISPR/Cas9 approaches, and developed a high-throughput screening pipeline for analyzing hair cell development and regeneration. We screened 254 gene mutations and identified 7 genes specifically affecting hair cell regeneration. These hair cell regeneration genes fell into distinct and somewhat surprising functional categories. By examining the regeneration of caudal fin and liver, we found these hair cell regeneration genes often also affected other types of tissue regeneration. Therefore, our results demonstrate guided screening is an effective approach to discover regeneration candidates, and hair cell regeneration is associated with other tissue regeneration.

A subset of SMN complex members have a specific role in tissue regeneration via ERBB pathway-mediated proliferation

“…In a previous study (10), we showed that mutations in smn1 and gemin5 negatively impacted the ability of zebrafish to regenerate different tissues after injury. Regenerative medicine is a rapidly expanding field of science that focuses on replacing or regenerating organs damaged by injury, aging, or degenerative conditions.…”

“…Our previous large-scale mutagenesis screen showed smn1 and gemin5, two SMN complex members, were essential for tissue regeneration (10). In this study, we expanded our mutagenesis screen to systemically examine the potential role of each of the nine members of the SMN complex in tissue regeneration.…”

“…Hearing loss is one of the common sensory disorders negatively affecting the quality of life for hundreds of millions of people worldwide (11). In a search for novel genes involved in hearing regeneration, we previously performed a large-scale mutagenesis screen in zebrafish and identified smn1 and gemin5 as essential genes for hair cell regeneration (10). Both Smn1 and Gemin5 belong to the SMN complex, a multiprotein complex functioning in the biosynthesis of small nuclear ribonucleoproteins (snRNP).…”

“…Regeneration of different tissues can be achieved by utilizing a common set of molecular pathways and many genes are pan-regenerative in that they are induced and essential regardless of the specific injured tissue (17). Both smn1 and gemin5 genes were involved in regulating the regeneration of multiple tissues including neuromasts, caudal fins, and livers (10). To determine whether gemin3 had similar phenotypes, we examined the regeneration of caudal fin and liver in gemin3 mutants.…”

“…Support cells in the zebrafish neuromast are further surrounded by mantle cells, which resemble quiescent stem or progenitor cells (13). Hair cell regeneration studies in zebrafish have uncovered numerous genetic factors and molecular pathways that are associated with the regeneration of hair cells (14); conversely, random mutagenesis studies revealed that mutations in only a small number of genes actually significantly alter hair cell regeneration specifically (10,13,15,16). There is consistently a gap between the number of genes transcriptionally associated with regeneration and the number of genes essential for regeneration in other tissues as well (17,18).…”