Disparate progenitor cell populations contribute to maintenance and repair neurogenesis in the zebrafish olfactory epithelium

Abstract

Olfactory sensory neurons (OSNs) undergo constant turnover under physiological conditions but also regenerate efficiently following tissue injury. Maintenance and repair neurogenesis in the olfactory epithelium (OE) have been attributed to the selective activity of globose (GBCs) and horizontal basal cells (HBCs), respectively. In zebrafish, cells with GBC-like properties are localized to the peripheral margins of the sensory OE and contribute to OSN neurogenesis in the intact OE, while cells that resemble HBCs at the morphological and molecular level are more uniformly distributed. However, the contribution of these cells to the restoration of the injured OE has not been demonstrated. Here, we provide a detailed cellular and molecular analysis of the tissue response to injury and show that a dual progenitor cell system also exists in zebrafish. Zebrafish HBCs respond to the structural damage of the OE and generate a transient population of proliferative neurogenic progenitors that restores OSNs. In contrast, selective ablation of OSNs by axotomy triggers neurogenic GBC proliferation, suggesting that distinct signaling events activate GBC and HBC responses. Molecular analysis of differentially expressed genes in lesioned and regenerating OEs points toward an involvement of the canonical Wnt/β-catenin pathway. Activation of Wnt signaling appears to be sufficient to stimulate mitotic activity, while inhibition significantly reduces, but does not fully eliminate, HBC responses. Zebrafish HBCs are surprisingly active even under physiological conditions with a strong bias toward the zones of constitutive OSN neurogenesis, suggestive of a direct lineage relationship between progenitor cell subtypes.

HB-EGF Promotes Horizontal Basal Cell Proliferation and Olfactory Sensory Neuron Regeneration in the Zebrafish Olfactory Epithelium

“…OSNs undergo rapid degeneration upon nasal irrigation with the detergent Triton X-100 (TrX; Iqbal and Byrd-Jacobs, 2010), which reaches a peak at 24 h post lesion (hpl) and is followed by gradual repopulation of the OE with HuC/D-positive sensory neurons over a consecutive 4 to 6 d period. Analysis of RNAseq data (Kocagöz et al, 2022; additional data included in this study) revealed that genes typically expressed by OSNs are downregulated over the first 24 hpl but show partial or full recovery by 120 hpl, consistent with the degeneration and reestablishment of OSNs over the same period (Figure 1A). Expression of the general neuronal markers embryonic lethal abnormal visual system-like 3 (elavl3; HuC), microtubule-associated protein 2 (map2), and neuronal cell adhesion molecule 1b (ncam1b) is lowest at 24 hpl but rebounds to pre-injury levels by 120 hpl.…”

“…Unlike mammals, the zebrafish OE contains multiple morphologically and molecularly distinct OSN subtypes, of which the olfactory receptor/trace amine-associated-(OR/TAAR-) expressing ciliated and the V2R-(= OlfC-) expressing microvillous cells are the most abundant (Sato et al, 2005;Korsching, 2020). Ciliated OSNs appear to recover more efficiently than apically located microvillous and the fish-specific crypt (ora4a), pear (adora2c), and ompa-or ora1-expressing chemosensory cells as indicated by the temporal expression profiles of cell type-specific marker genes (Sato et al, 2005 The tissue response to injury is characterized by an early activation of HBCs, which generate a transient population of ascl1-positive GBC-like progenitors as regenerative OSN neurogenesis unfolds (Kocagöz et al, 2022). At the transcriptional level, the HBC markers keratin4 (krt4) and krt5 are highly upregulated (3.9 ± 0.1-and 6.7 ± 2.0-fold, respectively) at 24 hpl consistent with HBC activation and increased cell proliferation (Figure 1B).…”

“…Conversely, the generation of HBCs from activated GBCs has also been suggested to occur in the mouse OE (Lin et al, 2017). Either way, it seems to be important that resident HBCs and GBCs GBC-like cells also overlap across the sensory region (Kocagöz et al, 2022), which exposes them to a similar molecular environment. Yet, differences in matrix attachment and cell-cell contacts, which further define their respective microenvironment (Carter et al, 2004) are likely to exist.…”

“…In contrast, restoration of the injured OE depends on horizontal basal cells (HBCs), which are largely quiescent in the intact tissue and serve as ‘reserve stem cells’ that are selectively activated by structural damage ( Schnittke et al, 2015 ; Herrick et al, 2017 ). In zebrafish, GBCs are restricted to the interlamellar curves (ILCs) and the sensory/non-sensory border (SNS) at the central and peripheral margins of the region that is occupied by olfactory sensory neurons (OSNs), respectively, while HBCs are uniformly distributed across basal layers of nearly the entire tissue ( Bayramli et al, 2017 ; Demirler, 2020 ; Kocagöz et al, 2022 ). Accordingly, sustained cell proliferation and OSN generation can be observed at the edges of the sensory OE under physiological conditions ( Bayramli et al, 2017 ), whereas the response to injury is characterized by a transient increase in OSN neurogenesis from within the sensory region between the sites of maintenance neurogenesis ( Iqbal and Byrd-Jacobs, 2010 ; Kocagöz et al 2022 ).…”

“…positions 3.615 -4.064), and the ascl1a (422 nt fragment ofENSDART00000056005.5; positions 638 -1060;Kocagöz et al, 2022) genes that were amplified from zebrafish cDNA using specific primer pairs. The hybridized slides were washed in descending concentrations of sodium-sodium citrate (SSC) buffer (5x SSC, 2x SSC/50% formamide, 2x SSC, 0.2x SSC) at the hybridization temperature.…”