New Neurons for the Inner Ear: Neurogenesis in the Zebrafish Statoacoustic Ganglion during Growth, Homeostasis and Regeneration

The vertebrate inner ear is a remarkable sensory organ, harboring two different senses: the auditory system, responsible for hearing, and the vestibular system, responsible for balance. Even though the anatomical structure of the vertebrate inner ear is very complex, only three different cell types are mainly involved on a cellular level in the perception of sound as well as balance and movement: sensory hair cells that are surrounded by supporting cells receive the stimulus and transfer it via sensory neurons to the brain. Worldwide, millions of people suffer from sensorineural hearing loss, caused by the loss of sensory hair cells and/or their innervating neurons within the inner ear. In mammals, including humans, both cell types are only produced during fetal stages making loss of these cells and the resulting consequences irreversible. In contrast, it is known that zebrafish produce sensory hair cells throughout life and additionally possess the remarkable capacity to regenerate them upon lesion. However, it is unknown whether new sensory neurons are also formed throughout life in the zebrafish statoacoustic ganglion (SAG), which transduces signals from the inner ear to the brain. Moreover, it is unknown whether sensory neurons are replaced upon loss. Hence, the first aim of this study was to investigate whether new sensory neurons are produced beyond larval stages. To this end, analysis of different transgenic lines combined with immunohistochemistry against known markers for neuronal stem and progenitor cells, neurons, glia and myelinating cells as well as markers for proliferation were used to identify distinct cell populations and anatomical landmarks in the juvenile and adult SAG. In the juvenile SAG, a pool of highly proliferating Neurod/Nestin-positive neuronal progenitors produces large amounts of new sensory neurons. In contrast, at adult stages this neurogenic niche transitions to a quiescent state, in which Neurod/Nestin-positive neuronal progenitor cells are no longer proliferating and the neurogenesis rate is very low. Moreover, BrdU pulse chase experiments revealed the existence of a proliferative but otherwise marker-negative cell population that replenishes the Neurod/Nestinpositive progenitor pool throughout life, indicating a neural stem cell-like cell population upstream of the neuronal progenitor cell pool. Additionally, expression of glia markers and a switch in the myelination pattern was found to mark the peripheral and central nervous system transitional zone (PCTZ) as a prominent landmark of the SAG. To further study the nature of the proliferating but otherwise unknown stem cell-like cell population replenishing the Neurod/Nestin-positive neuronal progenitor pool, the transcriptome of proliferating cells and their progeny of the juvenile and adult SAG was analyzed via single cell RNA-sequencing using the Smart-Seq2 technology. Therefore, a pipeline including preparation of the SAG as well as cell dissociation followed by fluorescence-activated cell sorting was established to obtain single cells from the SAG. The fluorescent reporters Tg(pcna:GFP) and Tg(nestin:mCherry-CreERT2) were used to label proliferating cells (GFP-only positive), proliferating progenitors (GFP/mCherry-double positive as well as nonproliferating progenitor cells (mCherry-positive). Additionally, based on the perdurance of the fluorophores in the progeny of the cells expressing the reporter constructs, this sorting strategy also enables to sort the progeny of proliferating cells differentiating into neuronal progenitor cells (GFP/mCherry-double positive but not expressing pcna) to trace back the putative stem cell-like cell population replenishing the Neurod/Nestin-positive progenitor population. Similar, the sorting strategy also included newborn neurons as the progeny of neuronal progenitors (mCherry-positive but not expressing nestin). In the transcriptome data obtained from the juvenile SAG, the majority of the analyzed cells could be assigned to the neuronal lineage, reflecting the neuronal differentiation trajectory from neuronal progenitor cells transitioning to newborn neurons and even further differentiating into mature neurons. Additionally, two different putative neuronal stem cell-like cell clusters were identified which are currently under validation. In contrast, in the adult transcriptome data the majority of cells were identified as cells from the sensory lineage, including cells expressing markers specific for hair cells and the sensory epithelium. Only a minority of cells came from the neuronal lineage, with the group of newborn and differentiating neurons clustering together in one cluster. Very few cells were identified as neuronal progenitor cells and did not cluster together, whereas both putative stem cell-like cell populations could be identified as distinct cluster. However, validation of the putative stem cell population remains subject to further studies. The second aim of this thesis was to investigate the regenerative capacity of the adult SAG and to study whether the neurogenic progenitor cell niche can be reactivated and to give rise to new sensory neurons upon damage. Therefore, a lesion paradigm using unilateral injections into the otic capsule was established. Upon lesion, mature SAG neurons undergo apoptosis and a massive infiltration with immune cells was found. Importantly, the Neurod-positive progenitor cells reentered the cell cycle displaying a peak in proliferation at 8 days post lesion before they returned to homeostatic levels at 57 days post lesion. In parallel to reactive proliferation, an increase in neurogenesis from the Neurod-positive progenitor pool was observed. Reactive neurogenesis started at around 4 days post lesion, peaked at 8 days post lesion decreased again to low homeostatic levels at 57 days post lesion. The administration of the thymidine analog BrdU to label proliferating cells and their progeny revealed the generation of new sensory neurons from proliferating neuronal progenitor cells within 19 days post lesion. Interestingly, reactive proliferation as well as an increased neurogenesis rate were also detected in the unlesioned SAG, revealing a systemic effect of the unilateral lesions. Taken together, this study is the first to show that neurogenesis in the zebrafish SAG persists way beyond larval stages. New neurons descend from a population of Neurod/Nestin-positive neuronal progenitor cells that is highly proliferative during juvenile stages but turn quiescent at adulthood. Nevertheless, this neuronal progenitor cell pool is replenished throughout life by a currently unknown neuronal stem cell-like cell population. Additional this study reveals the regenerative capacity of the adult SAG: upon lesion Neurod/Nestin-positive progenitor cells are reactivated to re-enter the cell cycle, proliferate and give rise to new neurons leading to an increased neurogenesis rate to replace lost mature neurons. Studying the underlying genes and pathways in zebrafish compared to mammalian species will hopefully provide valuable insights that will help developing cures for auditory and vestibular neuropathies in the future