The Olfactory Organ Is Populated by Neutrophils and Macrophages During Early Development

The immune system of vertebrates is characterized by innate and adaptive immunity that function together to form the natural defense system of the organism. During development innate immunity is the first to become functional and is mediated primarily by phagocytic cells, including macrophages, neutrophils, and dendritic cells. In the olfactory sensory system, the same sensory neurons in contact with the external environment have their first synapse within the central nervous system. This unique architecture presents a potential gateway for the entry of damaging or infectious agents to the nervous system. Here we used zebrafish as a model system to examine the development of the olfactory organ and to determine whether it shares immune characteristics of a host defense niche described in other tissues. During early development, both neutrophils and macrophages appear coincident with the generation of the primitive immune cells. The appearance of neutrophils and macrophages in the olfactory organs occurs as the blood and lymphatic vascular system is forming in the same region. Making use of the neurogenic properties of the olfactory organ we show that damage to the olfactory sensory neurons in larval zebrafish triggers a rapid immune response by local and non-local neutrophils. In contrast, macrophages, although present in greater numbers, mount a slower response to damage. We anticipate our findings will open new avenues of research into the role of the olfactory-immune response during normal neurogenesis and damage-induced regeneration and contribute to our understanding of the formation of a potential host defense immune niche in the peripheral nervous system.


INTRODUCTION
The olfactory sensory system is composed of the peripheral olfactory epithelium (OE), where the continually renewing olfactory sensory neurons (OSNs) are located. The axons of the OSNs reach the central nervous system (CNS) via the olfactory nerve (ON), where they make their first synapses in the olfactory bulb (OB) (Sakano, 2010;Whitlock, 2015). Thus, unlike other sensory systems, the first synapses of the OSNs lie within the CNS. This unique organization creates a potential pathway for chemical or biological agents to enter the CNS. Yet, although pathogens can enter the CNS via the OE (Pägelow et al., 2018), it is striking that our brains are not besieged by infections that enter through this direct olfactory portal.
Recently, it has been shown that in mammals "host defense niches" exist where myeloid cell types, such as neutrophils, remain associated with the tissue instead of patrolling the body (Yipp et al., 2017). These resident cells have been described in the lungs, a tissue that like the olfactory epithelia comes in contact with potential damaging airborne substances. In mammals, the airways of the nose and mouth have a network of lymphoid tissue in the pharynx and palate (tonsils), called nasopharynxassociated lymphoid tissue, which protect against invasion by neurotropic microorganisms, including viruses. Like mammals, zebrafish have the basic myeloid cell types including monocytes, neutrophils, eosinophils, mast cells, and dendritic cells, yet they do not have organized lymphoid structures such as tonsils/lymph nodes. Limited studies suggest that fish have a diffuse network of lymphoid and myeloid cells associated with the olfactory organ (OO) that may resemble mucosal immune tissues (Tacchi et al., 2014).
Similar to mammals, zebrafish generate blood/immune cells in successive waves during development. In zebrafish, during the first phase of hematopoiesis, precursors arise from the mesoderm generating the rostral blood island (RBI) and intermediate cell mass (ICM) before entering the circulation (Davidson and Zon, 2004). Myeloid cell precursors including monocytes/macrophages and granulocytes develop by 12 h postfertilization (hpf) (Lieschke et al., 2002), and functional macrophages and neutrophils are present by 30 hpf (Herbomel et al., 1999;Le Guyader et al., 2008). The RBI will also give rise to larval microglia via primitive macrophages (Herbomel et al., 1999(Herbomel et al., , 2001Xu et al., 2015). The larval zebrafish has been used to study immune system development and function because of the optical clarity, availability of reporter lines expressed in immune cell types, and sequenced genome (Mathias et al., 2006;Renshaw et al., 2006;Hall et al., 2007). Because of the regenerative properties of fish, in tissues such as the tail and fins, zebrafish are readily amenable to wounding studies (induced by cutting the tail for example) where the response of the innate immune system can be visualized and manipulated in intact living animals (Mescher et al., 2017). Here we make use of the peripheral olfactory sensory system to explore the early development of immune cells types and their potential association with the OO.
Previously, through microarray and RNAseq analyses of adult OE zebrafish (Harden et al., 2006;Calfun et al., 2016), we found that, in addition to OE specific genes, genes normally expressed in both the innate and the adaptive immune systems were also expressed. These findings prompted us to investigate the potential "immune architecture" of the OE. Because of the early development of innate immune system (Masud et al., 2017), we investigated the presence and dynamics of neutrophils and macrophages in the olfactory sensory system of developing zebrafish to better characterize the immune cells as well as understand their potential response to damage in the developing OO.

Copper Exposure
Initial dose-response analysis was performed based on previous work in zebrafish and salmon (Baldwin et al., 2003;Hernandez et al., 2011). A stock solution of 10 mM CuSO 4 was diluted in filtered embryo medium (Westerfield, 2007) for a final concentration of 10 µM CuSO 4 . Staged larvae were exposed to 10 µM CuSO 4 for 4 h and then washed out. The long-term effects of copper on neutrophil movement to the OO were quantified in individual larvae using adapted ChIn assay (d'Alençon et al., 2010).

Cryosectioning
Seven-dpf larvae were sacrificed and then fixed and embedded in 5% sucrose/1.5% agarose in mqH 2 O. Blocks were then submerged in 30% sucrose for 2 to 3 days and then stored covered by O.C.T. Compound (Tissue-Tek R ) in cryomolds at −20 • C. Twenty-five-micrometer cryosections were processed for immunofluorescense as described above; primary and secondary antibodies were incubated overnight.

TUNEL Labeling
Larvae were processed using in situ Cell Death Detection Kit, Fluorescein (Roche), according to manufacturer recommendations. Briefly, larvae were permeabilized for 1 h at 37 • C, washed twice, and labeled at the same temperature for 1 h. DAPI staining was used for nuclear labeling. Larvae were mounted in 2% low melting temperature agarose (Sigma) in an Attofluor Chamber for imaging (see below). Fluorescent signals in TUNEL-labeled preparations were quantified by mean pixel intensities from green (fluorescein from TUNEL staining), green (GFP from trpc2:GFP), and red (RFP from OMP:RFP) in OE and OB (selected as different ROIs in FIJI). Values were normalized by mean pixel intensity of the DAPI stained whole head (as another ROI).

Live Imaging
For live imaging of the olfactory sensory system, larvae were anesthetized (2% Tricaine Sigma) mounted in a cut tip of plastic Pasteur pipette in 2% low temperature agarose (Sigma) in embryo medium (Westerfield, 2007). The larvae were imaged in frontal view in an Attofluor Chamber (Thermo Fisher Scientific) filled with Embryo medium. The agarose covering the olfactory system was removed. Temperature was maintained at 26-28 • C, and images were captured using a Spinning disc confocal microscope (Olympus) with a 20 × 0.95 NA water immersion LUMPlanFL/IR objective.
Time-lapse videos of copper exposure: To generate the timelapse movies (Figures 5, 6, 8, 9), stacks of images were collected with 3 µm/optical section in a total depth of 150-µm depth.
All tracking data from time-lapse microscopy in control and copper-exposed larvae were processed using MTrackJ tracker in FIJI. Chemotactic index (CI) was calculated as described by Lämmermann et al. (2013), taking left or right OO as reference. Briefly, CI was defined as cos(α) with α as the angle between the distance vector to the damage site (OO) and the actual movement vector.

Image Analyses
For analysis of neutrophils and macrophages: Only cells within the boundaries of the sensory tissue were counted, and the values were given as the average of total number of mpx:GFP-positive or mpeg1:mCh-positive in both OOs with standard deviation. Values given for paired sensory structure are a sum of the individual sensory tissues. For time-lapse videos, all counts of neutrophils and macrophages in the two OOs were combined for each animal and the mean/SEM calculated for each time point.

Statistics
Data are presented as means ± standard deviations. Experiments number and statistical analysis were done using Prism 6 (GraphPad) and are indicated in each figure legend. Unpaired Student t-tests were performed unless otherwise indicated. Pvalues are indicated as follows: * P < 0.05, * * P < 0.01, * * * P < 0.001, * * * * P < 0.0001.

Phagocytic Cell Populations in the Developing Olfactory Organ
We first quantified phagocytic cells (neutrophils and macrophages) of the immune system to determine whether they were present in peripheral sensory systems during early development. We used the Tg(mpx:GFP) line to visualize neutrophils, a leukocyte subtype with strong myeloperoxidase (mpx) activity, and the Tg(mpeg1:mCh) line (macrophageexpressed gene, mpeg1.1, encodes perforin-2, a pore-forming protein associated with host defense against pathogens) to visualize macrophages in fixed whole-mount larvae (Figure 1). Olfactory sensory structures do not appear as a stratified epithelium until later in development; thus, we refer to the tissue as an OO (Figures 1A,E). At 7 dpf mpx:GFP+ neutrophils were found associated with the OOs (Figure 1B, green, arrows) and anti-Sox2-positive taste buds (Figure 1B, arrowheads). In contrast few neutrophils were directly associated with the ear (Figure 1C, green). When quantifying neutrophils in the developing sensory systems (Figure 1D), the olfactory sensory system has more neutrophils than other sensory systems (n = 30 animals per sensory system, one-way ANOVA, Tukey test, P < 0.05). No neutrophils were observed in the retina. In contrast to neutrophils, at 7 dpf there were many more macrophages in the OOs (Figure 1F), but not in the ear ( Figure 1G). Unlike the situation with neutrophils, the OO and eye had equal numbers of macrophages ( Figure 1H), yet the gustatory (mouth)  and auditory (ear) number remained lower (n = 30 animals per sensory system, one-way ANOVA, Tukey test, P < 0.05).

Blood Lymphatic System in the Developing Olfactory Organ
Recently, the lymphatic vasculature (LV) of the zebrafish brain has been described (Bower et al., 2017;van Lessen et al., 2017;Bower and Hogan, 2018), but little is known about the developing blood vasculature (BV) and LV associated with the olfactory sensory system. Using the Tg(lyve1b:DsRed); Tg(OMP:YFP) double-transgenic line at 5 dpf, we found LV on the dorsallateral surface of the telencephalon ( Figure 3A, red, arrows) extending around the region of the forming OB. By 7 dpf, the LV encircled the OB region ( Figure 3B, OB) where the axons of the OSNs terminate ( Figure 3B, asterisks, yellow). At 2 weeks postfertilization, the dorsal projections were maintained ( Figure 3C, red, arrows), and lyve1b:DsRed+ branches were apparent on the ventral side of the OOs (Figure 3C, red, arrowheads, G,G' , NL). We visualized the development of BV using the Tg(fli1a:EGFP) line ( Figures 3D-F, green). At 5 dpf, the BV was already apparent on the dorsal surface of the brain ( Figure 3D) and was found associated with the OOs before the LV. The nasal artery/nasal veins (NA/NVs) (Isogai et al., 2001) are the most rostrally projecting of vessels until at least 7 dpf ( Figure

Response of Neutrophils and Macrophages to Tissue Damage in the OO
Previously, it has been shown that copper exposure at concentrations ranging from 10 −9 to 10 −5 M (Tierney et al., 2010) damages the olfactory sensory epithelia of zebrafish and that the unique neurogenic characteristics of the OE allow for the replacement of the OSNs (Ma et al., 2018). In order to confirm that copper caused cell death in the developing OO, 5-dpf larvae were exposed to 10 µM CuSO 4 (Figure 4) and processed for TUNEL labeling (Figures 4A-C). At 5 dpf, whole-mount control fish showed no cell death ( Figure 4A). After exposure to 10 µM CuSO 4 , only the OOs were positive for TUNEL ( Figure 4B, green, arrows). Quantification of TUNEL fluorescence in control and treated animals showed a statistically significant increase in fluorescence in the OOs of copper-treated animals ( Figure 4C). The olfactory sensory system has several sensory cell types, and the Tg(OMP:RFP) reporter line is expressed only in ciliated OSNs, the most abundant sensory neuron type in the OO. Because differential sensitivity has been reported for ciliated and microvillous OSNs, we visualized the microvillous OSNs using the Tg(trpc2:GFP) line combined with Tg(lysc:DsRed) to visualize neutrophils in red (Figures 4D,E). Quantification of pixel intensity changes for Trpc2:GFP+ fluorescence confirmed that, unlike ciliated OSNs (Figure 5), microvillous OSNs were largely unaffected by copper exposure (Figures 4D,E, green; Figure 4F, gray bar). For all experiments (Figures 4D,E), 25 larvae were processed and examined (control and copperexposed). Of these, three different animals were analyzed from each treatment group. These results confirm that the damage caused by copper exposure is consistent and comparable with previous studies in zebrafish (Ma et al., 2018).

In vivo Neutrophil Response to Cell Damage
To confirm whether copper-induced damage affected the ciliated OSNs and triggered a neutrophil response, we exposed 5dpf Tg ( Analysis of the 2D path of individual neutrophils showed that copper exposure triggered the migration of non-local neutrophils from the dorsal and ventral sides of the head (Figures 5C ′′′ ,D ′ -D ′′′ , arrows), and these entered the OO and OB regions via pathways separate from the ON (Figure 5C ′′′ , arrows). During the time of copper exposure (4 h), the number of neutrophils increased from a pre-exposure average of 5.2 ± 1.7 to 15.0 ± 2.4 ( Figure 5E, data from analysis of 6 different time lapse videos). The mean speed of neutrophils after copper exposure increased from a pre-exposure velocity of 7.1 ± 0.2 µm/min, to a post-exposure velocity of 7.8 ± 0.1 µm/min (Figure 5F, n = 30 neutrophils; one representative video). Both velocities were in the range of the reported 11 µm/min for randomly migrating neutrophils in the ventral region of the head of 3dpf zebrafish larvae (Walters et al., 2010). Analysis of the CI ( Figure 5G) showed a significant increase in orientation toward the OO [CI of −0.05 (range, −0.21 to 0.15) to 0.24 (from −0.33 to 0.78)] but with a separation of groups, reflecting different patterns of movement of local neutrophils, which moved within the OO vs. those of non-local populations, which appeared to circulate in and out of the OO region (Figures 5D ′ -D ′′′ , arrows, G, red). The number of neutrophils in the OOs remained elevated in the continued presence of copper (9.8 ± 1.9) and decreased after washout (Figure 5H). A second increase is seen 24 h after copper treatment (8.1 ± 1.9; n = 48), which may be associated with the replacement of ciliated OSNs (Figures 5A-A ′′ , B), which have a lifelong program of cell renewal that is distinct from damage-induced regeneration. The number of neutrophils then returned to baseline at the end of the second-day postexposure ( Figure 5H), a time when the OSNs have recovered ( Figure 5B). Thus, consistent with previous reports, in juvenile zebrafish, damage triggers a rapid mobilization of neutrophils, and chemotaxis contributes to the migration of neutrophils to the site of damage (Mathias et al., 2006), which also correlates with the time course of neuronal regeneration.

Macrophage Response to Cell Damage
To better understand the dynamics of the macrophage response to OSN damage in the OO, we performed time lapse imaging in whole-mount preparations using a Tg(mpeg1:mCh); Tg(OMP:RFP) double transgenic line to visualize macrophages (Figures 6A-A ′′ ; red) and OSNs (Figures 6A-A ′′′ ; green; Supplementary Video 2). Before copper exposure, an extensive population of macrophages was found both associated with the OO (Figure 6A, green) and extending dorsal and ventral to the OOs (Figure 6A, red). In contrast to neutrophil migration induced during copper exposure, macrophages were closely associated with the OO (Figure 6A, arrows) and ON ( Figure 6A). During copper exposure, macrophages in the OOs (Figure 6A, red) were joined by macrophages located outside the OOs (Figures 6A ′ ,A ′′ , arrows; Supplementary Video 2). Analysis of the 2D path of only the macrophages associated with the OOs showed that copper exposure resulted in a statistically significant increase in the number of macrophages in the OOs (Figures 6B,B ′′′ ,C), with specific non-local macrophages moving . Neutrophils (red) in the OO, but unlike OSNs, microvillous OSNs were largely unaffected. (F) Quantification of microvillous OSNs (green, fluorescence) in control (gray) and copper-treated animals (red); no significant decrease in Trpc2:GFP fluorescence was observed. All fluorescence was normalized using DAPI (n = 3 larvae, two-way ANOVA, Tukey multiple-comparisons test, ****a = P < 0.0001, b = P < 0.01). All scale bars = 100 µm.
toward the OOs (Figures 6B ′ -B ′′′ , arrows). These macrophages entered the OO and OB via pathways separate from the ON (Figure 6A ′′′ , arrows). Similar to neutrophils, exposure to copper resulted in an increased number of macrophages ( Figure 6C; 11.5 ± 0.4) associated with the OO when compared to the controls (Figure 6C; 6.2 ± 0.3; data from analysis of six independent time lapse videos). In contrast to neutrophil dynamics, no significant difference in instantaneous velocity of the macrophages was observed (Figure 6D, pre-exposure velocity of 1.7 ± 0.7 µm/min to a post-exposure velocity of 2.2 ± 0.6 µm/min; n = 26 macrophages; 1 video), and both velocities were significantly slower than that observed for neutrophils after exposure to copper (pre 7.1 ± 0.2 µm/min/post 7.8 ± 0.1 "µm/min). When quantifying the total displacement of the tracked macrophages, there was a significant difference between two groups of macrophages ( Figure 6E): local macrophages that remained in close association with the OOs, moving <100 µm, and non-local (or wandering) macrophages that exceeded this total displacement (n = 51 macrophages, 16 local, 25 non-local, one representative time lapse). During copper exposure, there is a steady increase in macrophages associated with the OO (Figure 6F) that starts to decrease when copper is removed. Like neutrophils, 24 h post-exposure, there is a statistically significant increase of macrophages in the OOs that returns to baseline values at 48 h posttreatment ( Figure 5F).
To better understand the dynamics of macrophage movements in the OOs, we analyzed the movements of macrophages and neutrophils relative to copper-induced damage of the OSNs in Tg(mpeg1:mCh):Tg(mpx:EGFP):Tg(OMP:YFP) triple transgenic larvae (Figures 7A-C; Supplementary Video 3). As the OSNs degenerated, evidenced by the fading of green signal (Figures 7A-A ′′′ ), the macrophages associated with the OSNs (Figures 7A-A  In further analysis of the macrophage movements using cell tracking, two distinct populations were observed: fixed macrophages ( Figure 7B) (previously called local macrophages) that were always in the OOs, and "wandering" or non-local macrophages ( Figure 7C) that were able to enter the OOs when damage occurs, but were patrolling the head before olfactory damage. Therefore, the local or fixed macrophages, observed using the Tg(mpeg1:mCh) line, were found in the OO and their behavior contrasts sharply with the wandering phenotype, Control. (A ′ ) At 4 h post-copper exposure, there was an increase in neutrophils (green) in the OO and OSNs degenerated (red, asterisk). (A ′′ ) One day post treatment (dp) neutrophils decreased and the OSNs (red) were recovering. (B) Quantification of OSN (red fluorescence) in control (gray), copper-treated animals (red), 1 day (green bar) and 2 days posttreatment (blue bar). All fluorescence was normalized using DAPI (n = 3 larvae, two-way ANOVA, Tukey multiple-comparisons test, ****a = P < 0.0001, b = P < 0.01). All scale bars = 100 µm. (C-C ′′′ ) 5-dpf Tg(mpx: GFP);Tg(OMP:RFP) larva, frontal view. Imaging was initiated at time 0 ′ . At 37 ′ (C ′ ), larvae were exposed to 10 µM of CuSO 4 , and imaging continued at times indicated (see Supplementary Video 1 for sequence taken every minute). Boxed areas: Neutrophils (green, asterisks) associated with olfactory organ (OO, C-C ′′ ) and OMP:RFP+ OSNs in olfactory bulb (red, asterisk, C ′′′ ). (C ′′ ,C ′′′ ) Arrows in (C ′′′ ) non-local neutrophils that do not enter the OO near the ON (see Supplementary Video 4). (D-D ′′′ ) Individual 2D-cell tracking of neutrophils before, during, and after copper exposure. Each color represents a different neutrophil. (E) Number of neutrophils within the OO before and after copper exposure: analysis of six videos from different animals. Unpaired t-test, P < 0.0001). (F) Speed of neutrophils before and after copper exposure (n = 30 neutrophils; Unpaired t-test, P < 0.05). (G) Chemotactic index of neutrophils before and after copper exposure (n = 20 neutrophils; Unpaired t-test, P < 0.05). (H) Time course of neutrophil movement to the OO (n = 48 larvae. ANOVA, Kruskal-Wallis test, P < 0.0001). Scale bar C-C ′′′ = 150 µm. Tracking was done using the ImageJ plugin, MTrackJ.
perhaps reflecting different roles and subtypes of the phagocytic cells within the zebrafish head.
In response to copper-induced damage of the OSNs, both local and non-local macrophages changed their shape from a ramifiedstar-like shape (Figure 7D, red, arrowheads) to a rounded swollen morphology (Figure 7D ′ , red, arrowheads). In contrast to neutrophils, in response to copper, macrophages formed multiple vesicles and phagosome-like structures (Figures 7D,D ′ ) (Peri and Nusslein-Volhard, 2008), which were observed engulfing the OMP:YFP+ degenerating OSNs (Figure 7D ′ ,  arrowheads). Thus, the macrophages associated with the OOs during early development are greater in number, respond more slowly to copper-induced damage, and show distinct phagocytic behaviors.

Blood Lymphatic Vasculature and Neutrophil Migration
Because this was the first reported analysis of neutrophil responses in the OO, and our data on individual in vivo cell tracking suggested that neutrophils used preexisting pathways to reach the OOs in response to copper exposure, we further examined the neutrophil migration routes. To determine whether neutrophils migrated using BV and/or LV, we used Tg(mpx:GFP); Tg(lyve1b:DsRed) double-transgenic larvae to follow neutrophil movements associated with LV. Initially, there was no association of neutrophils with the developing rostral LV (data not shown), but in 7-dpf larvae, neutrophils were localized in the ventral-lateral OO (Figures 8A,A ′ , asterisks). After copper exposure the number of neutrophils increased (Figures 8A ′ ,B ′ , green, asterisks) and were found associated with the lyve1b:DsRed+ branch of the ventral-lateral OO (Figures 8A ′ , . Consistent with previous reports, we found that neutrophils showed a close association with the BV system in the developing embryo. In larvae exposed to copper, neutrophils moved along the NV on the medial side of the OO (Figures 8D,D ′ , asterisks, Supplementary Video 4). As the neutrophils migrate, they maintained intimate contact with the BV, often extending "feet" into the vasculature (Figures 8E-E ′′ ) as they moved (Supplementary Video 5). Thus, copper-induced damage to the developing OOs initiated neutrophil migration (Figure 8F, blue), which occurred along the medial NV (Figure 8F, green) at early stages and later included the ventral lateral OO associated with lyve1b:DsRed-positive LV branch ( Figure 3G).

Migration Route
Because the NA/NA are the primary routes for neutrophil migration to the OOs during early development and the classification as a vein or artery is unclear in the literature, we further analyzed the direction of blood flow in 5-dpf larvae. In Tg(fli1a:EGFP);Tg(gata1a:DsRed);Tg(mpx:GFP) triple transgenic larvae, we observed movement of erythrocytes ( Figure 9A, red, arrows) in the NV and with a net direction from ventral to dorsal. Analysis of videos taken with transmitted light of wholemount larvae in vivo confirmed the net direction as ventral to dorsal or "away" from the OO (Figure 9B, arrow). Furthermore, using Tg(fli1a:EGFP);Tg(lyve1b:DsRed) double-transgenic 15dpf larvae, we confirmed that the nasal lymphatic branch (Figure 9D, NL, arrow, lyve1b:DsRed+) appeared in association with the nasal BV ( Figure 9D, NV, arrow, fli1a:EGFP+ and lyve1b:DsRed+).

DISCUSSION
In this study, we examined the population of the developing OO by myeloid immune cells (neutrophils/macrophages) and their response to copper-induced damage. Key findings include the following: (1) both local and non-local neutrophils and macrophages are present in the developing OOs, where the local immune cells may play a role in the lifelong neurogenesis of the olfactory epithelia; (2) the appearance of the immune cells is correlated with the developing blood and LV of the OOs; (3) copper-induced damage triggers rapid but distinct responses from neutrophils and macrophages. Further studies are needed to determine the origin(s) of neutrophils and macrophages, as well as their different functions in developing and adult animals.

Phagocytic Cell Populations in the Developing Olfactory Organ
Neutrophils are essential players in the innate immune system as they are the first cells that respond to tissue damage and infection by rapidly migrating to the site of injury (swarming) (de Oliveira et al., 2016). We first detected neutrophils in the OO at 3 dpf, well after the 30 hpf when functional macrophages and neutrophils are present (Herbomel et al., 1999;Le Guyader et al., 2008). Perhaps consistent with the necessity of a strong immune defense, the OO had significantly greater number of neutrophils than the mouth or ear (the eye had no neutrophils). In contrast to the neutrophils, macrophage populations in the developing olfactory OO and the eye were much larger than the other sensory systems, and there was no significant difference in numbers of macrophages found in the OOs and eyes. While little is known about macrophages in the larval retina, damage to the adult retina in zebrafish triggers the rapid accumulation of immune cells including local microglia and extraretinally derived macrophages (Mitchell et al., 2018). Microglia appear to play a role in the regulation of neurogenesis (Salter and Beggs, 2014), and macrophages may play a critical role in regeneration of sensory organs (Denans et al., 2019). At this time we cannot determine whether the macrophage population we have described in the OOs also includes precursors of microglia that, in zebrafish, arise from the primitive macrophages (Ferrero et al., 2018). The finding that both the eyes and the OOs have large macrophage populations, coupled with their anatomically unique peripheral extension of the meninges, which contain a diverse array of immune cells (Rua and McGavern, 2018), supports a model we proposed where the olfactory epithelia are more like the retina of the eye than placodal-derived structures (Whitlock, 2008;Torres-Paz and Whitlock, 2014;Torres-Paz et al., 2020). The presence of microglia in the peripheral olfactory sensory system would argue that the OO shares more characteristics with the CNS than peripheral nervous system, and we are currently investigating macrophage and microglia populations in the adult olfactory system.

Neurogenic Response of Olfactory Organ
Unlike mammals, fish have the unique ability to maintain neurogenesis of sensory neurons throughout life. The exception to this difference is the olfactory epithelia where all vertebrates share the characteristic of ongoing sensory cell replacement (Bermingham-McDonogh and Reh, 2011). Copper, a heavy metal and pervasive environmental contaminant (Soller et al., 2005) is known to damage the olfactory sensory epithelia of fish, leading to loss of olfactory-driven behaviors (Sunderman, 2001;Baldwin et al., 2003;Matz and Krone, 2007), and to alter expression of genes involved in the olfactory signal transduction pathway in adult zebrafish (Tilton et al., 2008). Here we confirmed in larval zebrafish that exposure to copper resulted in OSN death (Lazzari et al., 2017;Ma et al., 2018) and the rapid recovery of the OSNs was accompanied by the influx of both neutrophils and macrophages. Neutrophil movements we observed in the OOs are consistent with earlier studies in juvenile zebrafish, where damage triggers a rapid mobilization of neutrophils, and directed chemotaxis contributes to the migration of more neutrophils to the site of damage (Mathias et al., 2006). Moreover, it has also been shown that in response to wounding induced inflammation neutrophils move rapidly (15 µm/min) toward the wound, whereas macrophage migration velocity was significantly slower (Ellett et al., 2011;Dudek et al., 2020). Here we found similar results where neutrophils increased their velocity in response to damage, whereas macrophages showed a slower response. In contrast to wound healing responses induced by tail cutting, here we found that the OOs contain populations of local neutrophils and macrophages who were joined by non-local neutrophils and wandering macrophages in response to damage. This difference is most likely due to the unusual characteristics of the OSNs. In contrast to the tail wounding where the response is an inflammatory response in a tissue capable of regeneration, the OO is a tissue that has ongoing sensory neurogenesis over which is imposed neural damage induced by copper. Recent studies suggest that macrophages are involved in the repair of different neural tissue. In larval zebrafish, copper-induced hair cell damage in both the lateral line (Carrillo et al., 2016) and spinal cord transsection (Tsarouchas et al., 2018) resulted in the recruitment of neutrophils and macrophages to the injury site where macrophages were correlated with repair and regeneration of neural tissue. Because both macrophages and microglia are suggested to play a role in neurogenesis, as well as regeneration, the fixed or local macrophages we describe here (and potentially the local neutrophils) may play a role in the ongoing turnover of FIGURE 10 | Summary of neutrophil and macrophage responses to copper-induced damage in the larval olfactory organ (OO). The blood vasculature (BV, green) wraps the olfactory organ (gray) and the lymphatic vasculature (LV, red) extends along the ventral posterior aspect. In untreated animals (before copper), there are local neutrophils (blue) and macrophages (pink) associated with the OO. In response to damage (after copper exposure) of the olfactory sensory neurons (OSNs, dark gray), non-local neutrophils and macrophages migrated to the OO. Neutrophils migrated in association with the BV, and both neutrophils and macrophages were seen associated with the LV. Macrophages changed to a more rounded morphology as they engulfed debris of dying OSNs.
OSNs. Thus, the presence of both local and non-local neutrophils and macrophages in the developing OOs suggests a dual response where the local immune cells protect against external challenges, and non-local immune cells arrive only once damage is detected.

Migration of Neutrophils and Macrophages
Studies in fish where a wounding response generated by tail and/or fin transsection (Ellett et al., 2011;Xu et al., 2012) have elucidated the role of macrophages and neutrophils in inflammation. In the wounding response, macrophages were found to patrol throughout the body, yet neutrophils were motile only in the head region of the larvae (Mathias et al., 2009;Deng et al., 2011;Ellett et al., 2011). While interstitial migration has been described for both neutrophils and macrophages (Barros-Becker et al., 2017), only neutrophils also use the blood-LV to migrate de Oliveira et al., 2016).
The LV has recently been "rediscovered" in the CNS of mammals (Aspelund et al., 2015;Louveau et al., 2015;Da Mesquita et al., 2018;Dolgin, 2020) and of zebrafish (Bower et al., 2017;van Lessen et al., 2017;Bower and Hogan, 2018), yet little is known about the development of the LV in the brain of vertebrates. Lymphatic endothelial cells are thought to arise from the BV system (Jung et al., 2017;Padberg et al., 2017); yet, to date, there are no detailed descriptions of the development of the BV and the LV in the olfactory sensory system. The development of the BV preceded the development of the LV in the OOs, and the primary route of neutrophil migration to the OOs was via the NVs whose development coincides with the first appearance of myeloid cells in the peripheral olfactory sensory system.
A fascinating question, brought to the fore by the current SARS-CoV2 pandemic, is how viruses gain access to the nervous system, and it is now apparent that the olfactory system is used by COVID-19 (coronavirus disease 2019) as an entry point to the nervous system (Desai and Oppenheimer, 2020;Divani et al., 2020). The study of the peripheral olfactory sensory system and the associated immune cells will allow us to better understand not only the rapid immune response to damage caused by toxic and infectious agents, but also how this neural immune interface may act as a host defense niche protecting the CNS.

CONCLUSIONS
During early development, at all times assayed, the OOs contain local populations of both neutrophils and macrophages, reminiscent of a potential host-defense niche described in other tissues where neutrophils are marginated (Yipp et al., 2017;Granton et al., 2018;Hidalgo et al., 2019) (Figure 10, Before Copper Exposure, blue). In response to damage non-local populations join local populations of neutrophils and macrophages as they mount a rapid immune response (Figure 10, After Copper Exposure, blue, pink). Neutrophils use the developing BV system (Figure 10, green) to access the OOs, and this may account for their greater velocity relative to macrophages.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

ACKNOWLEDGMENTS
We thank A. Moscoso, T. Ordenes and S. Alanis for care of the zebrafish, especially with the difficulties brought on by the pandemic.

604030/full#supplementary-material
Supplementary Video 1 | Neutrophils mobilize in response to copper induced damage of the developing olfactory organ. Time lapse of Tg(OMP:RFP0);Tg(mpx:GFP) 5dpf larva, maximum projection of 150 µm depth (3 µm optical sections), taken every minute for 4 h. A final concentration of 10 µM of CuSO 4 was added at minute 37 ( Figure 5). Scale bar= 150 µm. Time in minutes is indicated in upper left corner. Arrowheads indicate local neutrophils (green). After treatment neutrophils where observed to swarm, cluster and even divide, next to OSNs (red).
Supplementary Video 2 | Local and non-local macrophages respond to copper-induced damage in the olfactory organ. Time lapse of mpeg1:mCh;mpx:EGFP 5 dpf transgenic larva, maximum projection of 150 µm depth (3 µm optical sections), taken every 2 min for 1.7 h. A final concentration of 10 µM of CuSO 4 was added at minute 14 ( Figure 6). Scale bar= 150 µm. Time in minutes is indicated in upper left corner at the end of the time lapse macrophages are localized in the ventral and basal side of the olfactory organ, forming chain-like structures near branch of nasal lymphatics (Figures 3, 7).
Supplementary Video 3 | Macrophages respond differently to copper exposure. Time lapse of Tg(mpeg1:mCh);Tg(mpx:EGFP);Tg(OMP:YFP) in 5 dpf larva, maximum projection of 150 µm depth (3 µm optical sections), taken every minute for 2 h. A final concentration of 10 µM of CuSO 4 was added at minute 30 ( Figure 7). Scale bar= 50 µm. Time in minutes is indicated in upper left corner. Local (or fixed) macrophages do not move, but displayed a swollen morphology after copper-induced damage.
Supplementary Video 4 | Neutrophils move on the BV during early development. Time lapse of Tg(fli1a:EGFP;Tg(gata1:DsRed);Tg(mpx:GFP);Tg(OMP:RFP) quadruple transgenic larva at 5dpf, maximum projection of 300 µm depth with optical sections of 3 µm, taken every minute for 3.5 h. OSNs and erythrocytes in red; neutrophils and endothelial vasculature in green. A final concentration of 10 µM of CuSO 4 was added at minute 37 ( Figure 8). Scale= 150 µm. Time in minutes is indicated in upper left corner.
Supplementary Video 5 | The nasal vein as route for neutrophil migration to the olfactory organ. Magnified image of the olfactory organ in a time lapse (35 min extract) of a Tg(fli1a:EGFP;Tg(gata1:DsRed);Tg(mpx:GFP);Tg(OMP:RFP) quadruple transgenic larva at 5 dpf, during copper exposure. Maximum projection of 300 µm depth, taken every minute. Scale bar= 50 µm. Time in minutes is indicated in upper left corner.