A regulatory network of Sox and Six transcription factors initiate a cell fate transformation during hearing regeneration in adult zebrafish.

Using adult zebrafish inner ears as a model for sensorineural regeneration, we ablated the mechanosensory receptors and characterized the single-cell epigenome and transcriptome at consecutive time points during hair cell regeneration. We utilized deep learning on the regeneration-induced open chromatin sequences and identified cell-specific transcription factor (TF) motif patterns. Enhancer activity correlated with gene expression and identified potential gene regulatory networks. A pattern of overlapping Sox- and Six-family TF gene expression and binding motifs was detected, suggesting a combinatorial program of TFs driving regeneration and cell identity. Pseudotime analysis of single-cell transcriptomic data suggested that support cells within the sensory epithelium changed cell identity to a "progenitor" cell population that could differentiate into hair cells. We identified a 2.6 kb DNA enhancer upstream of the sox2 promoter that, when deleted, showed a dominant phenotype that resulted in a hair-cell-regeneration-specific deficit in both the lateral line and adult inner ear.

Major cardiorespiratory events do not increase after immunizations, eye exams, and other stressors in most very low birth weight infants.

BACKGROUND: Increased cardiorespiratory events with bradycardia and oxygen desaturation have been reported in very low birthweight (VLBW) infants following stressors such as immunizations. These events are difficult to quantify and may be mild. Our group developed an automated algorithm to analyze bedside monitor data from NICU patients for events with bradycardia and prolonged oxygen desaturation (BDs) and used this to compare BDs 24 hours before and after potentially stressful interventions. METHODS: We included VLBW infants from 2012-2017 with data available around at least one of four interventions: two-month immunizations, retinopathy of prematurity (ROP) examinations, ROP therapy, and inguinal hernia surgery. We used a validated algorithm to analyze electrocardiogram heart rate and pulse oximeter saturation data (HR, SpO2) to quantify BD events of HR < 100 beats/minute for≥4 seconds with oxygen desaturation < 80%SpO2 for≥10 seconds. BDs were analyzed 24 hours before and after interventions using Wilcoxon rank-sum tests. RESULTS: In 354 of 493 (72%) interventions, BD frequency stayed the same or decreased in the 24 hours after the event. An increase of at least five BD's occurred in 17/146 (12%) after immunizations, 85/290 (29%) after ROP examinations, 4/33 (12%) after ROP therapy, and 3/25 (12%) after hernia surgery. Infants with an increase in BDs after interventions had similar demographics compared to those without. More infants with an increase in BDs following immunizations were on CPAP or caffeine than those without. CONCLUSIONS: Most VLBW infants in our cohort had no increase in significant cardiorespiratory events in the 24 hours following potentially stressful interventions.

Vestibular and Auditory Hair Cell Regeneration Following Targeted Ablation of Hair Cells With Diphtheria Toxin in Zebrafish.

Millions of Americans experience hearing or balance disorders due to loss of hair cells in the inner ear. The hair cells are mechanosensory receptors used in the auditory and vestibular organs of all vertebrates as well as the lateral line systems of aquatic vertebrates. In zebrafish and other non-mammalian vertebrates, hair cells turnover during homeostasis and regenerate completely after being destroyed or damaged by acoustic or chemical exposure. However, in mammals, destroying or damaging hair cells results in permanent impairments to hearing or balance. We sought an improved method for studying hair cell damage and regeneration in adult aquatic vertebrates by generating a transgenic zebrafish with the capacity for targeted and inducible hair cell ablation in vivo. This model expresses the human diphtheria toxin receptor (hDTR) gene under the control of the myo6b promoter, resulting in hDTR expressed only in hair cells. Cell ablation is achieved by an intraperitoneal injection of diphtheria toxin (DT) in adult zebrafish or DT dissolved in the water for larvae. In the lateral line of 5 days post fertilization (dpf) zebrafish, ablation of hair cells by DT treatment occurred within 2 days in a dose-dependent manner. Similarly, in adult utricles and saccules, a single intraperitoneal injection of 0.05 ng DT caused complete loss of hair cells in the utricle and saccule by 5 days post-injection. Full hair cell regeneration was observed for the lateral line and the inner ear tissues. This study introduces a new method for efficient conditional hair cell ablation in adult zebrafish inner ear sensory epithelia (utricles and saccules) and demonstrates that zebrafish hair cells will regenerate in vivo after this treatment.

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

Spinal muscular atrophy (SMA) is the most common genetic disease in children. SMA is generally caused by mutations in the gene SMN1. The survival of motor neurons (SMN) complex consists of SMN1, Gemins (2-8), and Strap/Unrip. We previously demonstrated smn1 and gemin5 inhibited tissue regeneration in zebrafish. Here we investigated each individual SMN complex member and identified gemin3 as another regeneration-essential gene. These three genes are likely pan-regenerative, since they affect the regeneration of hair cells, liver, and caudal fin. RNA-Seq analysis reveals that smn1, gemin3, and gemin5 are linked to a common set of genetic pathways, including the tp53 and ErbB pathways. Additional studies indicated all three genes facilitate regeneration by inhibiting the ErbB pathway, thereby allowing cell proliferation in the injured neuromasts. This study provides a new understanding of the SMN complex and a potential etiology for SMA and potentially other rare unidentified genetic diseases with similar symptoms.

A model for reticular dysgenesis shows impaired sensory organ development and hair cell regeneration linked to cellular stress.

Mutations in the gene AK2 are responsible for reticular dysgenesis (RD), a rare and severe form of primary immunodeficiency in children. RD patients have a severely shortened life expectancy and without treatment die, generally from sepsis soon after birth. The only available therapeutic option for RD is hematopoietic stem cell transplantation (HSCT). To gain insight into the pathophysiology of RD, we previously created zebrafish models for Ak2 deficiencies. One of the clinical features of RD is hearing loss, but its pathophysiology and causes have not been determined. In adult mammals, sensory hair cells of the inner ear do not regenerate; however, their regeneration has been observed in several non-mammalian vertebrates, including zebrafish. Therefore, we used our RD zebrafish models to determine whether Ak2 deficiency affects sensory organ development and/or hair cell regeneration. Our studies indicated that Ak2 is required for the correct development, survival and regeneration of sensory hair cells. Interestingly, Ak2 deficiency induces the expression of several oxidative stress markers and it triggers an increased level of cell death in the hair cells. Finally, we show that glutathione treatment can partially rescue hair cell development in the sensory organs in our RD models, pointing to the potential use of antioxidants as a therapeutic treatment supplementing HSCT to prevent or ameliorate sensorineural hearing deficits in RD patients.

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

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.

CRISPRz: a database of zebrafish validated sgRNAs.

CRISPRz (http://research.nhgri.nih.gov/CRISPRz/) is a database of CRISPR/Cas9 target sequences that have been experimentally validated in zebrafish. Programmable RNA-guided CRISPR/Cas9 has recently emerged as a simple and efficient genome editing method in various cell types and organisms, including zebrafish. Because the technique is so easy and efficient in zebrafish, the most valuable asset is no longer a mutated fish (which has distribution challenges), but rather a CRISPR/Cas9 target sequence to the gene confirmed to have high mutagenic efficiency. With a highly active CRISPR target, a mutant fish can be quickly replicated in any genetic background anywhere in the world. However, sgRNA's vary widely in their activity and models for predicting target activity are imperfect. Thus, it is very useful to collect in one place validated CRISPR target sequences with their relative mutagenic activities. A researcher could then select a target of interest in the database with an expected activity. Here, we report the development of CRISPRz, a database of validated zebrafish CRISPR target sites collected from published sources, as well as from our own in-house large-scale mutagenesis project. CRISPRz can be searched using multiple inputs such as ZFIN IDs, accession number, UniGene ID, or gene symbols from zebrafish, human and mouse.