Zebrafish reveal how two genes independently drive sensory organ regeneration

Scientists uncover how zebrafish switch between cell division and direct differentiation to rebuild sensory organs, challenging long-held views on tissue regeneration.

Study: Stem and progenitor cell proliferation are independently regulated by cell type-specific cyclinD genes. Image Credit: Anusorn Nakdee / Shutterstock.com

In a recent study published in the journal Nature Communications, researchers at the Stowers Institute for Medical Research in the USA investigated the mechanisms by which stem cell genes modulate regeneration in zebrafish lateral line neuromasts (sensory organs). Specifically, the study leveraged cutting-edge CRISPR gene editing, single-cell RNA sequencing, and live imaging to investigate how two distinct cyclin D genes, ccndx and ccnd2a, regulate distinct cell populations during the development and regeneration of these sensory organs.

Study findings reveal that ccndx and ccnd2a function via independent yet complementary pathways – the former modulates the division of progenitor cells, while the latter drives the amplification of stem cell proliferation. Even when one pathway is lost, the other can partially compensate, but regeneration proceeds via a limited and less robust mechanism. For example, in ccndx mutants, direct differentiation can generate hair cells without division; however, fewer cells and a pronounced polarity defect occur, with approximately 70% of regenerated hair cells exhibiting a posterior bias. This polarity defect is mechanistically linked to reduced expression of hes2.2, which normally inhibits Emx2, a master regulator of hair cell polarity. Together, these genes ensure robust tissue maintenance, offering new therapeutic directions for sensory regeneration research.

Background

Tissue turnover and regeneration are fundamental processes essential for maintaining life. These constantly occurring processes rely on delicate balances between populations of stem cells, which gradually replenish themselves over time, and progenitor cells, which mature into specialized forms in response to physiological requirements.

Decades of research have revealed that cyclin D proteins, known primarily for their crucial role in regulating cell cycle progression, have substantial modulatory effects on cell proliferation. Unfortunately, the mechanistic associations between specific stem cell populations and cyclin D protein use remain poorly understood. Unraveling these associations may open the door to novel regenerative therapies, especially relevant in today’s increasingly aging global population, which is experiencing a decline in sensory ability.

Recent research aims to leverage the zebrafish, a well-studied model organism renowned for its regenerative capabilities, to answer this question. In particular, the zebrafish’s sensory lateral line is known to regenerate cells rapidly following injury. This line consists of hair, support, and mantle cells, essential for detecting water movement. Given their functional analogy to the human inner ear, these models are used to gain a better understanding of sensory regeneration.

About the Study

The present study utilized several transgenic zebrafish larval strains to elucidate the mechanisms underlying regeneration in the lateral line sensory system. The study focuses on two cyclin D genes: ccndx and ccnd2a.

By leveraging CRISPR-Cas9 and CRISPR-Cas12a gene editing, transgenic zebrafish with ccndx and/or ccnd2a knocked out were then observed (via scRNA-seq) to elucidate their differences in gene expression and cell states. These assays were replicated over 10,000 cells and were complemented by RNA velocity modeling using the scVelo and partition-based graph abstraction (PAGA) algorithms.

Researchers began by treating zebrafish larvae with neomycin, an antibiotic (ototoxin) that triggers rapid hair death (and subsequent regeneration) along the lateral line organ. Observation via single-cell RNA-seq (scRNA-seq) assays in combination with 5-ethynyl-2′-deoxyuridine (EdU) labeling and time-lapse imaging (Nikon Ti2 Eclipse; 42 hours at 28.5 °C) resulted in the identification of two proliferating cell nuclear antigen (PCNA) expression-marked cell populations, one governed by ccndx and the other by ccnd2a.

Finally, protein modeling using the Robetta and Mol*3D Viewer platforms was conducted to compare the structural conservation of cyclin D proteins across transgenic zebrafish species. Statistical analyses comprised analysis of variance (ANOVA) and t-tests for inter-treatment difference significant reporting (p < 0.05).

Study Findings

Neomycin treatment assays revealed the presence of two independent cell populations involved in neuromast regeneration. scRNA-seq analysis revealed these populations: 1. Self-renewing amplifying stem cell populations that express ccnd2a, and 2. Progenitor cells that mature into sensory hair cells that express ccndx.

Notably, ccndx knockout strains demonstrated hair regeneration even in the absence of ccndx expression. Still, these hair cells were often misoriented, possibly because of altered hes2.2 and emx2 activity (genes involved in hair cell polarity). Importantly, expressing ccnd2a under the ccndx promoter, an artificial genetic rescue, was able to restore both the number and orientation of hair cells in ccndx-mutants (p < 0.0001), marking a mechanistic demonstration rather than a physiological compensation.

In contrast, ccnd2a-deficient mutants showed reduced amplifying stem cell proliferation during development, but regeneration was less affected, suggesting that other cyclins can compensate during tissue repair, without inducing polarity defects. Specifically, the study reports upregulation of ccnd1 during regeneration in ccnd2a mutants, potentially compensating for the loss of ccnd2a function (Supplementary Fig. 5N–P). This independent regulation suggests sensory regeneration can occur even if one population (pathway) is compromised. For example, five days post-fertilization (dpf), ccndx mutants showed significantly reduced EdU+ differentiating progenitor cells compared to their wild-type siblings (p = 0.0001), while amplifying cell counts remained unchanged (p = 0.35).

This finding that zebrafish hair cells can regenerate through direct differentiation without progenitor proliferation represents a paradigm shift in regenerative biology, challenging the long-standing view that proliferation is essential for tissue regeneration.

Finally, this study revealed the importance of ‘Notch signaling’ in sensory regeneration. This highly conserved cell-to-cell communication pathway, essential for development and tissue homeostasis, was found to suppress ccndx expression, with its inhibition leading to increased progenitor proliferation only in ccndx-intact fish, thereby confirming a tight regulatory loop between Notch activity, cyclin D expression, and regeneration capacity.

Conclusions

This study represents a significant advance in our understanding of regenerative biology, demonstrating for the first time that, contrary to prior assumptions, all proliferating cells in regenerative tissues are not regulated similarly. It highlights the future importance of tailored cell cycle regulation, leveraging both ccndx and ccnd2a for optimal sensory regeneration outcomes.

These findings could accelerate the development of therapies in regenerative medicine. However, it is important to note that ccndx is absent in mammals, so these findings are directly applicable only to non-mammalian species. As with all basic science studies in animal models, findings should be considered in the context of zebrafish biology, and their applicability to humans will require further research.