Disruption of tph1 genes demonstrates the importance of serotonin in regulating ventilation in larval zebrafish (Danio rerio)

Highlights

• Tph1a is expressed in skin and & pharyngeal arch neuroepithelial cells (NECs), and in pharyngeal arch Merkel-like cells (MLCs).

• Tph1b is expressed predominately in pharyngeal arch MLCs and sporadically in skin NECs.

• Knockout of tph1 resulted in similar changes of serotonergic cell density in tph1a^−/−, tph1b^−/− and tph1a^−/−b^-/- mutants.

• tph1a^−/− and tph1b^−/− mutants showed markedly different ventilatory response when exposed to hypoxia.

Abstract

Serotonergic neuroepithelial cells (NECs) in larval zebrafish are believed to be O₂ chemoreceptors. Serotonin (5-HT) within these NECs has been implicated as a neurotransmitter mediating the hypoxic ventilatory response (HVR). Here, we use knockout approaches to discern the role of 5-HT in regulating the HVR by targeting the rate limiting enzyme for 5-HT synthesis, tryptophan hydroxylase (Tph). Using transgenic lines, we determined that Tph1a is expressed in skin and pharyngeal arch NECs, as well as in pharyngeal arch Merkel-like cells (MLCs), whereas Tph1b is expressed predominately in MLCs. Knocking out the two tph1 paralogs resulted in similar changes in detectable serotonergic cell density between the two mutants, yet their responses to hypoxia (35 mmHg) were different. Larvae lacking Tph1a (tph1a^−/− mutants) displayed a higher ventilation rate when exposed to hypoxia compared to wild-types, whereas tph1b^−/− mutants exhibited a lower ventilation rate suggesting that 5-HT located in locations other than NECs, may play a dominant role in regulating the HVR.

Introduction

The ability to sense O₂ levels in the environment to initiate physiological responses (e.g. ventilatory adjustments) aimed at maintaining O₂ homeostasis is a fundamental requirement for the survival of multicellular organisms. In adult mammals, O₂ chemoreception is mediated by peripheral chemoreceptor cells (type I or glomus cells) residing in the carotid body (Nurse, 2010). Upon exposure to hypoxia, K⁺ channels are inhibited in type I cells leading to, or facilitating, membrane depolarization, Ca²⁺ entry through voltage-gated channels and neurosecretion to initiate downstream responses including hyperventilation (Nurse, 2010).

In teleost fish, the functional analogs of the peripheral respiratory chemoreceptors of mammals are termed neuroepithelial cells (NECs) (Jonz and Nurse, 2009; Milsom and Burleson, 2007). The NECs of the adult gill are characterized by an abundance of dense core vesicles containing serotonin (5-HT) (Dunel-Erb et al., 1982; Jonz et al., 2004). Serotonergic NECs also are found on the skin of larval fish, where they are thought to play an important role in O₂ chemoreception before the maturation of gill NECs (Coccimiglio and Jonz, 2012). Similar to carotid body type I cells, isolated gill NECs respond to acute hypoxia with K⁺ channel inhibition, membrane depolarization, a rise in intracellular Ca²⁺ levels and increased synaptic vesicle activity (Burleson et al., 2006; Jonz et al., 2004; Qin et al., 2010; Zachar et al., 2017). However, unlike type I cells that contain multiple putative neurotransmitters within the same cell, including 5-HT, catecholamines and acetylcholine, most NECs contain predominantly 5-HT (Porteus et al., 2013). Thus in fish, 5-HT is speculated to be an important neurochemical in O₂ chemoreception and for mediating the hypoxic ventilatory response (HVR) initiated by O₂ chemoreceptors.

The earliest studies examining the role of 5-HT in O₂ chemoreception and the regulation of ventilation demonstrated that in isolated gill arch preparations of both the spiny dogfish (Squalus acanthias) (Poole and Satchell, 1979) and rainbow trout (Oncorhynchus mykiss) (Burleson and Milsom, 1995a), addition of 5-HT to the perfusion media activated afferent nerve fibers of the gill filaments. Subsequent studies reported that application of 5-HT via intra-arterial injections or bathing in 5-HT solutions caused increases in ventilation or aquatic surface respiration in several species including rainbow trout (Burleson and Milsom, 1995b; Fritsche et al., 1992), European eel (Anguilla Anguilla) (Janvier et al., 1996), toadfish (Opsanus beta) (McDonald et al., 2010), and zebrafish (Danio rerio) (Abdallah et al., 2015; Shakarchi et al., 2013). Although these studies demonstrated a potential overall role for 5-HT in O₂ chemoreception and the control of breathing, they could not distinguish the effects mediated by 5-HT released from peripheral NECs versus 5-HT acting at one or more upstream or downstream locations. To address this issue directly, Kermorgant et al. (2014a) injected 5-HT into the brain of rainbow trout and observed a potent stimulatory effect on ventilation. Similar increases in ventilation were observed when fluoxetine, a selective 5-HT reuptake inhibitor, was injected into the brain (Kermorgant et al., 2014b). Despite these previous studies, the exact sites at which 5-HT acts to stimulate ventilation remain unknown. To date, all evidence is indirect, either showing 5-HT containing cells in gills and skin of fish, or demonstrating ventilatory responses to exogenous 5-HT. The absence of definitive data, in part, reflects the technical challenges associated with blocking/activating site-specific 5-HT receptors. However, with the recent advances in genetic manipulation techniques such as CRISPR knockout (Chang et al., 2013; Hwang et al., 2013; Zimmer et al., 2019), new approaches are available to address these questions in zebrafish, an amenable model species.

The first and rate-limiting step in the biosynthesis of 5-HT is catalyzed by tryptophan hydroxylase (Tph), and thus Tph is often used as a marker for 5-HT synthesis and serotoninergic activity. In zebrafish, three paralogs of tph genes are present; tph1a, tph1b and tph2. All three paralogs are expressed in the central nervous system (CNS) (Lillesaar, 2011), while data on the peripheral expression of the three paralogs is scarce, with some data suggesting that tph1b is expressed in Merkel-like cells (MLCs) at the base of taste bud cells (Kapsimali et al., 2011; Soulika et al., 2016). In mammals, tph2 is the neuronal-specific enzyme that controls brain 5-HT synthesis whereas tph1 is responsible for peripheral, non-neuronal 5-HT synthesis (Côté et al., 2003; Zhang et al., 2006). In this study we chose to examine the peripheral expression of tph1a and tph1b to determine whether either gene is responsible for 5-HT presence in NECs. In addition, we generated/obtained tph1a and tph1b knockout zebrafish to examine the effects of 5-HT originating from the different paralogs, on ventilation.