Globins and nitric oxide homeostasis in fish embryonic development

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Globins and nitric oxide homeostasis in fish embryonic development Elizabeth R. Rochona, Paola Cortia,b, a b

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Heart, Lung, Blood, and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA 15213, USA Division of Cardiology, Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA

A R T I C LE I N FO

A B S T R A C T

Keywords: NO Zebrafish Embryo Nitrite Cytoglobin Globin X

Since the discovery of new members of the globin superfamily such as Cytoglobin, Neuroglobin and Globin X, in addition to the most well-known members, Hemoglobin and Myoglobin, different hypotheses have been suggested about their function in vertebrates. Globins are ubiquitously found in living organisms and can carry out different functions based on their ability to bind ligands such as O2, and nitric oxide (NO) and to catalyze reactions scavenging NO or generating NO by reducing nitrite. NO is a highly diffusible molecule with a central role in signaling important for egg maturation, fertilization and early embryonic development. The globins ability to scavenge or generate NO makes these proteins ideal candidates in regulating NO homeostasis depending on the micro environment and tissue NO demands. Different amounts of various globins have been found in zebrafish eggs and developing embryos where it's unlikely that they function as respiratory proteins and instead could play a role in maintaining embryonic NO homeostasis. Here we summarize the current knowledge concerning the role of NO in adult fish in comparison to mammals and we discuss NO function during embryonic development with possible implications for globins in maintaining embryonic NO homeostasis.

1. Introduction Nitric oxide (NO) is an important signaling molecule in many cell types and is involved in many physiological processes including cell proliferation, differentiation, apoptosis, macrophage activity and neurotransmission (Tejero et al., 2019). The action of NO in the cardiovascular system is well documented, especially in mammals where NO is a very potent regulator of vascular tone and blood pressure (Tejero et al., 2019; Stuart-Smith, 2002). NO diffuses from endothelial cells into the vascular smooth muscle cells and activates soluble guanylyl cyclase (sGC), which in turn generates cGMP and mediates vasodilation (Denninger and Marletta, 1999). NO can be produced through the action of NO synthase (NOS) enzymes. In the presence of molecular oxygen and several cofactors (including NADPH and tetrahydrobiopterin (BH4)), the various NOS isoforms present in different mammalian tissues will convert the amino acid L-arginine to NO and L-citrulline (Forstermann and Sessa, 2012). In low oxygen environments, NO production by NOS enzymes is inefficient and other systems are involved (Thomas et al., 2015). In conditions of low oxygen and/or low pH, globins can act as nitrite reductases and generate NO. The reaction requires an electron and results in oxidation of the heme from its reduced ferrous (Fe2+) state to the oxidized ferric (Fe3+) state (Fig. 1). Nitrite is generated in mammals by

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reduction of nitrate by bacteria. In these conditions, serial reduction of inorganic nitrate (NO3−) and nitrite (NO2−) anions are an important source of NO (Lundberg et al., 2008; Gladwin et al., 2005). NO has an extremely low half-life and its diffusion gradient is limited by scavenging reactions. As such, nitrate and nitrite serve as inert bioavailable reservoirs in the blood and tissues, where they can be converted into NO during hypoxia by nitrite reductases. Regulating NO production and consumption is key for NO signaling processes in the cell. At levels within the picomolar to nanomolar range, NO is able to activate its target sGC. While higher levels of NO can be important for activating wound healing, it can lead to oxidative stress with negative consequences for living cells (Thomas et al., 2015). Excess NO can react with superoxide to form peroxynitrite (ONOO−). In addition to depleting NO bioavailability, peroxynitrite can react with lipids, DNA, protein thiols and oxidize cysteine residues. These amino acid modifications affect protein structure and function, altering catalytic activity of enzymes, affecting signal transduction and cytoskeletal arrangement. For these reasons, it is imperative for the cell to have multiple mechanisms in place that can fine tune NO levels depending on the needs of the cell. Globins also play a key role in scavenging excess NO (Tejero and Gladwin, 2014). In conditions where the oxyferrous species of the globin is prevalent, NO dioxygenation is the most common reaction with the heme group of globins, where the oxy heme

Corresponding author at: Pittsburgh Heart, Lung, Blood, and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA 15213, USA. E-mail address: [email protected] (P. Corti).

https://doi.org/10.1016/j.margen.2019.100721 Received 8 August 2019; Received in revised form 7 October 2019; Accepted 18 October 2019 1874-7787/ © 2019 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

Please cite this article as: Elizabeth R. Rochon and Paola Corti, Marine Genomics, https://doi.org/10.1016/j.margen.2019.100721

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Fig. 1. Nitric oxide (NO) is produced by globin proteins in the conditions of low oxygen and/or pH. The ferrous (Fe2+) deoxyGlobin reacts with nitrite (NO2−) and hydrogen ions (H+) to produce ferric (Fe3+) metGlobin, hydroxide (OH−) and NO. In conditions of high oxygen and/or pH, globin proteins scavenge NO. Ferrous oxyGlobin reacts with NO to form ferric metGlobin and inert nitrate (NO3−). The ferric metGlobin is reverted back to its active ferrous globin form through a reduction reaction carried about by cytochrome b5 (Cyb5)/cytochrome b5 reductase 3 (Cyb5R3) in the presence of NADH.

In this review, we compare how mammals and fish species maintain NO homeostasis, both through NOS enzymes and globins. We summarize the roles for NO signaling focusing on fish reproduction and development and we discuss the current state of knowledge regarding the variety of globin proteins expressed in zebrafish tissues and their hypothesized developmental roles.

oxides to its ferric form (Fe3+) reacting with NO and converting NO to nitrate effectively consuming NO (Fig. 1). Recently new globin proteins of ancient origin and related to Hemoglobin and Myoglobin by a common ancestor, have been discovered: Cytoglobin (Kawada et al., 2001; Burmester et al., 2002; Trent 3rd and Hargrove, 2002), Neuroglobin (Burmester et al., 2000), Androglobin (Hoogewijs et al., 2012), Globin X (Roesner et al., 2005), Globin E (Fuchs et al., 2006) and Globin Y (Kugelstadt et al., 2004) with yet unidentified physiological functions. These globins are expressed in all vertebrate organs in different tissues and in relatively low amounts suggesting that they may have evolutionarily conserved functions beyond oxygen binding. Many teleost species are tetraploid due to genome duplication events and thus have an even larger diversity of globin genes (Hartley and Horne, 1984; Johnson et al., 1987; Glasauer and Neuhauss, 2014; Fraser et al., 2006) and their detection very early during fish development (Tiedke et al., 2011) renders an uncertain respiratory function for these globins. The most recent findings in mammalian models support a function in maintaining NO balance (Liu et al., 2017; Alvarez et al., 2017; Straub et al., 2012). The shift between nitrite reduction and NO dioxygenation varies according to globin oxygen affinity, oxygen levels and the availability of a reducing system in the environment. The reducing system in place in mammalian tissues restores the oxidized ferric heme to the reduced ferrous form to allow continuous NO scavenging (Smagghe et al., 2008; Amdahl et al., 2017). Similar to mammals, fish NO homeostasis is preserved and while changing in different tissues, NO metabolites generally stay constant with significant but non-extreme changes in oxygen tension, suggesting that NO regulators are at play (Hansen and Jensen, 2010a). NO signaling has been studied in fish to further understand a variety of processes in adults and embryos including (but not limited to) angiogenesis (Vimalraj et al., 2019), neural development (Bradley et al., 2010) and reproduction (Li et al., 2018). Zebrafish have emerged as a leading model for studying developmental processes due to their small size, optical transparency during development, ease of genetic manipulation and potential for drug discovery. Additionally, NO can be directly imaged in live zebrafish embryos during development, providing a powerful tool for analysis of NO spatial and temporal localization in living cells, responses to external stimuli and drug treatments, response to injury and changes throughout development (Lepiller et al., 2007). Taking advantage of their small size, the embryos rely highly on passive diffusion for oxygen intake, ruling out the need for globins in oxygen absorption (Barrionuevo and Burggren, 1999). Interestingly, distinct Hemoglobin chains are expressed during teleost embryonic development prior to the beginning of circulation (16 somite stage) (Ganis et al., 2012). At this time the embryo takes advantage of passive diffusion of oxygen for its demand (Grillitsch et al., 2005) and it's not until days later (4–7 days post fertilization) that Hemoglobin becomes fundamental for oxygen supply, suggesting important non-respiratory roles for hemoglobin during development.

2. NO production and homeostasis in mammals In mammalian systems NO is mainly generated by NOS proteins from L-arginine in the presence of oxygen (Daff, 2010). There are three isoforms of NOS: neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3). Both nNOS and eNOS are constitutively expressed and their activity is regulated by Ca2+/Calmodulin binding (Bredt and Snyder, 1990; Busse and Mulsch, 1990). nNOS is negatively regulated by NO, generating a feedback loop and pulsatile NO production, which may be important for synaptic transmission (Salerno, 2008; Salerno and Ghosh, 2009; Abu-Soud et al., 1995; Stuehr et al., 1995). Besides its strong expression in neurons, nNOS is also present in vascular smooth muscle cells and adventitial fibroblasts (Boulanger et al., 1998; Buchwalow et al., 2002; Schwarz et al., 1999). Murine models suggest that nNOS is also important for regulating cerebral blood flow and is protective against the development of atherosclerotic lesions (Kuhlencordt et al., 2006; Pelligrino et al., 1993). eNOS is predominantly expressed in endothelial cells but has also been found in red blood cells (Wood et al., 2013). NO produced by eNOS is important for regulating blood flow, platelet activation and the adhesion of inflammatory cells onto the endothelial cell surface (Huang et al., 1995; Monica et al., 2016). A fine balance of NO produced by eNOS is key to maintaining vascular homeostasis. Both knockout and overexpression mouse models have detrimental cardiovascular effects (Ozaki et al., 2002; Kuhlencordt et al., 2001). Unlike nNOS and eNOS, iNOS is regulated at the transcriptional level, and with a high calmodulin binding affinity, it is active even at very low concentrations of calcium (Cho et al., 1992). iNOS is expressed in the airway epithelium, neural cells, macrophages and in hepatocytes in response to inflammation (Lundberg et al., 1995; Voraphani et al., 2014; Zhao et al., 2013) where it produces high levels of NO. The need to balance production of NO by iNOS is highlighted by the negative effect excess iNOS generated NO has on atherosclerosis. However, activation of iNOS can also be protective following ischemia/reperfusion injury (Bolli, 2001; Kanno et al., 2000; West et al., 2008). The route to generate NO through the NOS activities becomes highly ineffective in conditions of low oxygen due to the oxygen requirement by the NOS enzymes. In these conditions NO production is largely dependent on other pathways relying on nitrite reduction where the globin heme can catalyze electron transfer reactions in presence of an electron source such as NADH [reviewed in (Lundberg et al., 2008)]. The main source of nitrite for mammals is through dietary consumption 2

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suggesting other mechanisms of NO production are utilized when Nos function is inhibited. The function of nitrite and NO in fish metabolism has only been recently explored (Hansen and Jensen, 2010a; Fago, 2017; Hansen et al., 2016; Sandvik et al., 2012). In aquatic habitats, nitrate anions, along with ammonia cations and nitrite anions, form an inorganic nitrogen reservoir pool available to fish making them potentially more susceptible to toxicity from high levels of nitrogen compounds compared to mammals. The NO cycle from nitrogen compound disposal is enhanced in low oxygen conditions while at the same time NO concentrations must be kept under tight control to avoid toxicity. These observations suggest that fish also exploit additional mechanisms to preserve NO homeostasis. Decreases in oxygen concentration increases the intracellular concentrations of nitrite, which serve as a reservoir of NO for signaling (Hansen and Jensen, 2010b). In mammals, nitrite is endogenously formed with the reaction of NO with molecular oxygen but can also enter the body through the diet (MacArthur et al., 2007). Freshwater fish have an additional route for nitrite uptake, the direct uptake of ambient nitrite through the gills via the branchial Cl−/HCO3– exchanger that can concentrate nitrite in the blood against the gradient (Sandvik et al., 2012; Jensen, 2003). Endogenous concentrations of nitrite similar to mammals could be detected in fish blood and cardiac tissue (Hansen et al., 2016; Jensen, 2007a). Upon exposure to high levels of nitrite in the water, fresh water fish experience a large increase in nitrite concentrations (from picomolar-micromolar concentrations to millimolar concentrations) (Hansen and Jensen, 2010a; Jensen, 2003; Jensen et al., 2017) with negative consequences on hemoglobin and oxygen binding capacity. The nitrite reduction by deoxyHemoglobin (Fe2+) is favored in the presence of low oxygen and low pH with consequent formation of NO and ferric metHemoglobin (Fe3+) which is unable to bind oxygen (Jensen, 2007b). However, similar to mammals, at low nitrite concentrations this reaction generates NO and may be important for hypoxic vasodilation and other NO driven mechanisms in the fish (Gladwin et al., 2005; Gladwin et al., 2006). Nitrite is known to be toxic at high concentrations in fish but the nitrite effect at physiological concentrations appears to be protective in the heart (Lahnsteiner, 2008; Voslarova and Svobodova, 2006). Zebrafish is a hypoxia semi-tolerant species and it is related to goldfish and carp. The latter is capable of surviving and being fully functional at very low levels of environmental oxygen (van der Meer et al., 2005). The crucian carp have developed unique strategies to survive hypoxia and it maintains normal cardiac output during anoxia-exposed periods. In these conditions, high levels of endogenous nitrite accumulate in the anoxic heart and during anoxia/reoxygenation it is shifted and acts as a NO donor to protect the cardiac muscle from reoxygenation (Sandvik et al., 2012). In addition to the cadre of globin proteins expressed in mammals (Hemoglobin, Myoglobin, Cytoglobin, Neuroglobin and Androglobin) that act as possible nitrite reductases, fish have additional globin proteins not present in mammals that can reduce nitrite to form NO, including Globin X and Cytoglobin 1 (Roesner et al., 2005; Burmester and Hankeln, 2014). Globin X is a six-coordinate globin present in fish, and other lower vertebrates including amphibians and reptiles. Six-coordinate globins have two histidines of the polypeptidic chain binding to the heme cofactor, as opposed to penta-coordinate globins with only one histidine bound to the heme. Erythrocyte expression of globin X has been demonstrated in zebrafish and striped bass. Interestingly, Globin X has an extremely fast nitrite reductase rate (26.7 M−1 s−1), approximately 200-fold faster than human Hemoglobin (Corti et al., 2016b). This may account for the high NO production in fish in the presence of nitrite. Globin X is a globin of ancient origin and among globins it is the one that best mimics the common ancestor of all globins, uncovering a role in concert with nitrite that is possibly conserved among other globins. Zebrafish have two cytoglobin genes, most likely due to the genome wide duplication event in the teleost lineage (Fuchs et al., 2005; Hoffmann et al., 2011). While Cytoglobin 2 is closely related to

of nitrate. Mammals rely on commensal bacteria within the gastrointestinal tract to reduce nitrate to nitrite through the action of nitrate reductase enzymes (Lundberg et al., 2008; Duncan et al., 1995). Once in the blood, nitrite acts as an endocrine reservoir, capable to being reduced to NO during hypoxia. This reaction has been shown to be physiologically relevant in human red blood cells where in hypoxia, Hemoglobin acts as a nitrite reductase to generate NO with consequent vasodilation (Grubina et al., 2007; Huang et al., 2005a; Helms and KimShapiro, 2013). Myoglobin is the main nitrite reductase enzyme in the heart and production of NO by Myoglobin leads to inhibition of mitochondrial respiration and cardioprotection (Shiva et al., 2007a; Hendgen-Cotta et al., 2008; Rassaf et al., 2007; Shiva et al., 2007b; Gonzalez et al., 2008) in conditions of low oxygen (Hendgen-Cotta et al., 2008; Rassaf et al., 2007; Gonzalez et al., 2008; Shiva and Gladwin, 2009; Dezfulian et al., 2007). In addition to Myoglobin and Hemoglobin, Neuroglobin and Cytoglobin have been reported to efficiently reduce nitrite to NO in various mammalian tissues during hypoxia with significant metabolic consequences such as vasodilation and cytoprotection (Tejero et al., 2019; Shiva et al., 2007b; Huang et al., 2005b; Tiso et al., 2011; Li et al., 2012; Corti et al., 2016a; Reeder and Ukeri, 2018). As NO levels are critical to exert normal physiological function as opposed to harming cells, NO homeostasis has to be maintained in a fine balance. An example of tightly regulated NO levels is observed in blood vessels. Endothelial cells produce NO through activation of eNOS and endothelial Hemoglobin α can increase or scavenge NO depending on the redox status of the heme (Fe2+ versus Fe3+), highlighting the eloquent mechanisms cells use oxygen availability and reducing systems to control NO bioavailability. In the presence of oxygen, ferrous (Fe2+) oxyHemoglobin α scavenges NO, producing nitrate and ferric (Fe3+) Hemoglobin α, limiting eNOS produced NO diffusion from the endothelial cell to the vascular smooth muscle cell (Straub et al., 2012). The oxidized, ferric form of Hemoglobin α is not able to scavenge NO, allowing it to freely diffuse, bind to sGC and results in vasodilation. Recently, Cytoglobin has also been found in vascular smooth muscle cells where it catabolizes NO, with Cytoglobin knockout mice having excess NO and lower blood pressures (Liu et al., 2017). It is interesting to note the repeated use of globin proteins, Hemoglobin α in endothelial cells (Straub et al., 2012), Myoglobin in cardiomyocytes (Flogel et al., 2001) and Cytoglobin in vascular smooth muscle cells (Liu et al., 2017) in regulating NO homeostasis in a variety of cardiovascular cell types through similar mechanisms. 3. NO production and homeostasis in adult fish In fish, NO production by the NO synthases enzymes is reported. Zebrafish have three nos genes: nos1 (nNos), nos2a (iNosa) and nos2b (iNosb), the latter two resulting from a genome duplication event in the teleost lineage. In the adult, nNOS has been identified in various regions of the brain, with strong expression present in proliferation zones (Holmqvist et al., 2000). iNosa has variable expression in adult organs with strong expression in the spleen, kidney, muscle, gut, ovary and skin and is notably absent from the heart, liver and testis (Lepiller et al., 2009). iNos2b is found throughout all adult organs tested. It should be noted that while a nos3 (eNos) gene has not been identified in the zebrafish genome, iNos2b seems to have subfunctionalized and may act in ways more similar to mammalian eNOS than iNOS. It is constitutively expressed, does not localize to immune cells and its expression is not well induced during an immune response (Poon et al., 2008). Additionally, similar to mammalian eNOS, it contains a myristoylation site on its n-terminus (Lepiller et al., 2009). Inducible forms of NOS have also been identified in the adult goldfish (Laing et al., 1996), rainbow trout (Laing et al., 1999; Campos-Perez et al., 2000) and carp (Saeij et al., 2000). Similar to mammals, fish Nos enzymes are dependent on oxygen but the NO metabolites are maintained at comparable levels during normoxia and hypoxia (Hansen and Jensen, 2010a), 3

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biological systems, small amounts of NO are necessary for sperm motility (via signaling through sGC), while high amounts of NO decrease sperm motility and also result in sperm with abnormal morphology (Wu et al., 2004). Mammalian sperm express all three NOS enzymes and NOS activity correlates with sperm maturation (Herrero et al., 2003; de Lamirande and Lamothe, 2009; de Lamirande et al., 2009). nNos, iNos as well as NO has been reported in the testis of catfish and zebrafish (Lepiller et al., 2009; nee Pathak and Lal, 2010) and the effect of NO on sperm motility in catfish has been tested with analogous results to mammalian studies (Barman et al., 2013) suggesting evolutionarily conserved mechanisms for NO and sperm function. Androglobin is a newly identified globin protein that is found in most metazoans including mammals and fish. It is not a typical globin, containing a globin fold with an N-terminal calpain-like domain and an IQ calmodulin binding motif (Hoogewijs et al., 2012). In mice, Androglobin transcript is found highly expressed in the testes, beginning at postnatal day 25, a time corresponding to an increase in postmeiotic spermatid abundance (Hoogewijs et al., 2012). The function of Androglobin is not known and the precise localization in fish is not yet identified. However, given its strong localization to mammalian testes and the need to maintain NO homeostasis for proper sperm maturation and function, a potential role for Androglobin may be in maintaining NO balance in male gonads. Due to the high conservation of globins among vertebrates, this function may be conserved from fish to mammals.

mammalian Cytoglobin in structure and biochemical function, Cytoglobin 1 has evolved unique functions (Corti et al., 2016a). Unlike mammalian Cytoglobin and Cytoglobin 2, Cytoglobin 1 has a pentacoordinate heme group, with expression reported in red blood cells, and other tissues including the kidney, liver, gut and eye (Corti et al., 2016a). It can efficiently reduce nitrite to form NO at a rate of 14.2 M−1 s−1 (compared to 0.40 M−1 s−1 and 0.31 M−1 s−1 for mammalian Cytoglobin and Cytoglobin 2, respectively) (Corti et al., 2016a). How fish regulate NO levels is not well understood. Similar to mammals, the Cytochrome b5/Cytochrome b5 reductase system is in place and reduces the ferric (Fe3+) iron back to its active ferrous (Fe2+) state, capable of reducing nitrite (Amdahl et al., 2017; Amdahl et al., 2019) (Fig. 1). Alternatively, in the presence of oxygen, globin proteins present in fish are able to convert NO to inert nitrate (Amdahl et al., 2017). These reactions occur quickly and effectively reduce NO diffusion and bioavailability (Helms and Kim-Shapiro, 2013). Further work is required to understand how fish species regulate and cope with the large amounts of NO produced in the presence of nitrite, especially during hypoxic periods. At the same time, NO is a critical signaling molecule necessary for physiological processes and in development. 4. NO role in fish gametes maturation and reproduction Notably NO plays a crucial role in reproduction at every level in the organism. Similar to mammals, during oocyte maturation, NO levels increase in follicular cells but remain constant in oocytes, while cGMP levels increase in the follicular cell layer but decrease in oocytes. Based on this, a dual role for the NO/sGC/cGMP pathway in either activation or meiotic maturation of oocytes has been proposed, where different NO concentrations appear to determine different functions (Li et al., 2018; Nath et al., 2018). The biphasic nature of NO function during reproduction has been noted in many studies (Thaler and Epel, 2003). The “right” amount of NO stimulates and enhances the reproductive events, but either lack of NO or too much NO has negative consequences arguing for the need for fine regulation of NO levels during these events. Collectively, the functional relevance of the Nos isoforms in zebrafish gamete maturation is demonstrated (Li et al., 2018) but how NO levels are regulated to maintain NO homeostasis is still not understood. NO involvement in reproduction is also key in egg activation upon fertilization through increased calcium levels. This calcium rise is a prerequisite for the activation of the egg upon fertilization and it is seen in all eggs studied, from jellyfish to mammals (Stricker, 1999). Analysis of zebrafish maternal transcript reported no detection of the NO synthase enzymes in eggs and embryos until 6 h post fertilization (Trapnell et al., 2010). Since fertilization in fish occurs externally, an internal source of NO in gametes seems necessary to fertilize eggs and for the initial zygotic divisions. Despite the requirement of NO during egg fertilization, it is unclear what the source of this NO is, leading to the hypothesis that some other NO producer must be at play in the egg (Harvey et al., 2013). As globins can produce or consume NO based on their intrinsic biophysics and the amount of environmental oxygen, it is reasonable to propose that globins are possible candidates for maintaining NO homeostasis during egg fertilization. Analysis of the zebrafish maternal transcript revealed high levels of globin X expression with a dramatic drop in the expression levels a few hours post fertilization suggesting a key role during fertilization (Tiedke et al., 2011; Trapnell et al., 2010). Among all the globins, globin X is detectable at significant levels, with maternal hemoglobin βa1, hemoglobin ζ and androglobin transcripts present at very low levels (Lee et al., 2013). As previously described, Globin X has been shown to be a very efficient nitrite reductase (Corti et al., 2016b) and may be generating NO to regulate egg activation. In mammals, NO is known to be important also for several aspects of sperm function, including motility, viability, maturation, hyperreaction and oocyte fusion (reviewed in (Buzadzic et al., 2015)). Similar to other

5. NO role in fish embryonic development Taking advantage of the small size, NO has been directly detected and imaged live in zebrafish embryos providing evidence that NO production changes throughout development and in response to stressful stimuli (Lepiller et al., 2007). The strongest NO positive sites in 5 day post fertilization larvae are the notochord, the bulbous arteriosus (analogous to the mammalian outflow tract) of the heart and cranial bones (Lepiller et al., 2007). While NOS inhibitors did decrease the NO signal in developing embryos, it was not completely abrogated, suggesting that there are additional sources of NO in the developing embryo that are NOS independent (Lepiller et al., 2007). In mammals NO has been shown to be a potent regulator of vascular tone, and this holds true in zebrafish during vascular development. While a specific vascular source of NO is missing, application of NO donors results in a significant increase in both the venous and arterial vessel diameters and NO scavengers constrict axial vessels suggesting regulation of vascular tone by a complex interaction of vasoactive substances generated locally by vascular endothelial cells (Fritsche et al., 2000; Pelster et al., 2005; North et al., 2009). NO signaling influences angiogenesis and hematopoiesis through similar mechanisms. Klf2a, a flow responsive transcription factor that is key in modulating angiogenesis, has been shown to regulate the transcription of both nos1 and nos2b in the zebrafish (Wang et al., 2011). In support of a role in angiogenesis, NO donors accelerated angiogenesis early in development, while NO scavengers decreased angiogenesis (Pelster et al., 2005). Additionally, NO production is decreased in embryos that do not express klf2a or have no flow (a transcriptional requirement for klf2a expression) (Wang et al., 2011; Parmar et al., 2006). Klf2a also regulates hematopoietic stem cell development. Pharmacological inhibition of NO or knockdown of nos1 in the zebrafish (or NOS3 in mice) blocked hematopoietic stem cell development (North et al., 2009). These data suggest that NO signaling downstream of Klf2a is a key mediator of both angiogenic and hematopoietic developmental programs. A specific source of NO in the embryonic vasculature and blood stream has not been identified and an involvement of globins in NO homeostasis during angiogenesis is a possibility. In zebrafish, expression of myoglobin has been reported in venous endothelial cells and embryos with myoglobin knockdown experience pericardial edema and angiogenic defects (Vlecken et al., 2009). The mechanism behind this defect has not yet been explored and could 4

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environmental oxygen status and pH, redox status of the iron atom, and reducing systems within the cell, globin proteins can store and deliver oxygen, act as oxygen sensing molecules, participate in peroxidase reactions or participate in electron transfer reactions, reducing nitrite to NO in hypoxic conditions or scavenge NO to form nitrate in the presence of oxygen, as described above. In recent years, significant work has gone into characterizing the biochemical properties of and determining the physiological functions of newly identified globin proteins, generating a rich pool of literature. From this work globins have been implicated in a myriad of processes including (but not limited to) cardiovascular health and homeostasis (Liu et al., 2017; Alvarez et al., 2017; Straub et al., 2012; Hendgen-Cotta et al., 2008), skeletal muscle regeneration (Singh et al., 2014), and regulation of cell proliferation (Braganza et al., 2019; Chen et al., 2014; Halligan et al., 2009). However, there remains a large gap in knowledge regarding the role of globins during embryonic development. In this review we have briefly highlighted the current knowledge regarding NO signaling during development using zebrafish as a model system. Based on the ability of globins to regulate NO levels, globin proteins may also be involved in some or all of these processes. Many globins are expressed developmentally. Besides erythrocyte specific hemoglobin, several hemoglobin subunits are expressed before the onset of hematopoiesis in zebrafish including hemoglobin βa1 and hemoglobin ζ, which are weakly maternally expressed, and hemoglobin αadult 1 is zygotically expressed (Lee et al., 2013). Zebrafish myoglobin is zygotically expressed (Tiedke et al., 2011; Lee et al., 2013). In situ hybridization also demonstrates larval myoglobin expression within a variety of structures including: the eye, otic vesicle, cephalic musculature, mandible, branchial arches, heart, fin rays, intestine, floor plate, roof plate, select venous endothelial cells and regions of the brain including the telencephalon, midbrain and hindbrain (Vlecken et al., 2009). Based on qRT-PCR results, neuroglobin has similar larval expression levels to myoglobin, although in situ hybridization data is missing and precise cellular localization is unknown (Tiedke et al., 2011). During development, cytoglobin 1 has consistently higher expression levels than cytoglobin 2 (Tiedke et al., 2011). In situ hybridization of cytoglobin 1 reveals expression in specific brain regions and somites beginning at approximately the 19 somite stage (Rauch et al., 2003), while in situ hybridization data for cytoglobin 2 is not yet available. Precise data on the embryonic and larval expression of globin x and androglobin is also lacking, however both have been identified in mRNA sequencing experiments, with globin x expression having strong maternally contributed expression to the egg and androglobin being weakly maternally expressed (Lee et al., 2013).

potentially be related to myoglobin's ability to modulate NO levels through nitrite reduction or deoxygenation reactions. Alternatively, the effect on angiogenesis may be an indirect effect and further research is required to elucidate this aspect. NO signaling is also important for cardiac progenitor migration, determining proper heart looping. Canonical NO signaling (NO activates sGC, catalyzing the synthesis of cGMP) targets a number of proteins including Bone Morphogenetic Protein-4 (Bmp4) which is involved in determining the positioning of the heart during embryonic development. In zebrafish embryos, cardiac progenitor cell migration is regulated by Bmp4 which represses migration on the left side of the body axis. This results in an increase of cardiac cell migration on the right side and consequent formation of heart tube on that side (Veerkamp et al., 2013). This process is affected by NO overproduction in chick embryos with occurrence of situs inversus in 20–30% of the embryos treated with the NO donor, DEAN (Siamwala et al., 2019). NO addition reverses the heart looping only during a critical window of time corresponding to the differentiation of cardiac progenitor cells involved in blood island formation. NO treatment increases BMP4 expression on the left side of the chick embryo impairing the cardiac progenitor cell migration through the SMAD signaling pathway (Siamwala et al., 2019). Further work is needed in the zebrafish to determine if the relationship between Bmp4 expression, cardiac progenitor cell migration and NO overproduction is relevant in this model. Additionally, the source of NO is unclear in this scenario and may be generated from Nos enzymes or a variety of developmentally expressed globin proteins. NO is not only a potent neurotransmitter, it is also involved in axon development, both in vertebrate and invertebrate systems [reviewed by (Cossenza et al., 2014)]. In the immature nervous system, nNos regulates motor axon development and exogenous elevation of NO/cGMP levels suppresses motor axon branching (Bradley et al., 2010). Additionally, cranial neural crest cells are NO responsive, pluripotent cells that give rise to nerve cells, facial bone and cartilage, the eye, otic placode and additional structures. The enteric nervous system regulates gut function and is derived from cranial neural crest cells (Ganz, 2018; Olsson et al., 2008). Migrating cranial neural crest cells express nos, and Nos inhibition results in aberrant cranial neural crest migration, defects in cell differentiation through a mechanism that involves NO altering histone acetylation and transcriptional regulation of hox genes (Kong et al., 2014). It should be noted that Neuroglobin is expressed in neural stem cells, and while there is still debate surrounding its role, evidence suggests that it is important for neural differentiation, proliferation and protection against stroke (Yu et al., 2018; Haines et al., 2013). Nitrergic neurons make up the most prominent neural subtype within the enteric nervous system (Ganz, 2018). Endogenous NO concentrations within the developing zebrafish gut range from 1 to 3 μM depending on the location within the gut (Dumitrescu et al., 2018). While nos1 and nos2a are both expressed in the gut and contribute to NO production, treatment with the Nos inhibitor L-NAME reduced but did not fully abrogate NO gut levels, suggesting additional Nos-independent sources of NO (Holmberg et al., 2006). Excess NO inhibits gut motility (Holmberg et al., 2006), also suggesting a need to carefully regulate the balance of NO within the gut to maintain proper function. Interestingly, myoglobin is expressed in the gut of the developing zebrafish where its function is not yet determined. Myoglobin knockdown results in a decrease in gut size in approximately 30% of embryos, however, how and why this occurs is not yet understood (Vlecken et al., 2009). In light of the role of NO in gut development and motility, a potential role for Myoglobin may be in regulating this process through NO homeostasis.

7. Conclusions Uncovering the roles of these globin proteins during development will surely reveal new, unknown functions of this class of proteins, NO signaling and genetic interactions. Mouse Myoglobin knockout models revealed interesting genetic compensatory mechanisms that were able to overcome the loss of this protein while maintaining normal myocardial oxygen function (Meeson et al., 2001). Similarly, Neuroglobin knockout mice revealed no striking phenotype, but interesting changes in gene expression related to HIF1α and the glycolytic pathway were noted that may be related to compensatory mechanisms (Hundahl et al., 2011). While much work has focused on identifying phenotypes in Cytoglobin mouse knockout models, it is unclear if there is a developmental or compensatory phenotype. While these interactions are complicated and make these studies difficult to interpret, they add a wealth of important knowledge that is applicable to understanding both general biology and human health and disease. Using zebrafish as a model to understand the developmental roles of globin proteins may yield important insights in the role of this highly conserved class of proteins in mammalian development.

6. Globins in development Globins are conserved ancient proteins that are found in archaea, bacteria and eukaryotes. Depending on heme coordination, 5

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Declaration of Competing Interest

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