O2 is essential to many animals. Vertebrate species rely on specialized chemoreceptive cells to “sense” O2 changes in order to make appropriate physiological adjustments to maintain homeostasis. Aquatic vertebrates are especially prone to fluctuations in environmental O2 availability and have adapted respiratory and cardiovascular responses to cope with hypoxia, a condition characterized by a low level of O2. In teleost fish, such as zebrafish (Danio rerio), neuroepithelial cells (NECs) present in the gill epithelium are the putative O2 chemoreceptors that mediate hypoxic signals to facilitate such responses. NECs contain the neurotransmitter serotonin (5-HT) and exhibit extensive neural innervation. They are sensitive to hypoxia, as isolated NECs undergo membrane depolarization and vesicular recycling when exposed to acute hypoxia. Other neurotransmitters, such as dopamine (DA), acetylcholine (ACh) and adenosine triphosphate (ATP), have also been suggested to regulate the ventilatory responses to hypoxia. However, the presence of these neurotransmitters or targeted receptors in gills are not well explored. In my PhD studies, I identified cellular and molecular components involved in chemotransduction and transmission for hypoxic signals in NECs of zebrafish through various experimental approaches. First, using the existing transgenic zebrafish line, ETvmat2:GFP, I established a method to reliably identify gill NECs. I showed that these cells could be distinguished based on their high expressions of the reporter gene GFP in vitro, in situ and in cytometric analyses. GFP-labeled NECs also displayed increases in cell size and population in response to chronic hypoxia. Second, using immunohistochemistry and confocal microscopy, I localized cholinergic cells and dopaminergic cells, sources of DA and ACh secretion respectively, in the gills. These cells present distinct populations from serotonergic NECs. In addition, I found purinergic P2X3 receptors, targets of ATP, to be present in gill NECs and other iv neurons. These findings offered different avenues in which hypoxic signals could be regulated. Lastly, using the single cell RNA sequencing approach, I determined the transcriptomic profile of NECs. NECs showed high expressions of G protein regulators, similar to those found in the mammalian O2 chemoreceptors, and they expressed high levels of genes likely to be involved in O2 signal transduction and transmission. Within the gill cell atlas generated using the single cell sequencing data, I localized a number of 5-HT, ACh and DA receptors in various gill cell populations, providing evidence for the 5-HT fast synaptic excitatory neurotransmission, paracrine and endocrine regulation of the signal. The studies overall provide compelling evidence to support a role for NECs as the primary O2 chemoreceptor in zebrafish, and further our understanding of signal modulation in the hypoxic response.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/42656 |
| Date | 13 September 2021 |
| Creators | Pan, Wen |
| Contributors | Jonz, Michael |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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