Notochord

Fig 1 illustrates experimental perturbations (above the timeline), and associated analyses (below the timeline). Adult Tropical 5D zebrafish were housed at Sinnhuber Aquatic Research Laboratory at Oregon State University. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Oregon State University. Each tank was kept at 28°C on a 14h light/ 10h dark photoperiod. Group spawns of adult zebrafish were set up the night prior, and embryos were collected and staged [13 (link)]. Embryo chorions were enzymatically removed using pronase (90 μL of 25.3 U/μl; Roche, Indianapolis, In, USA) at 4 hours post fertilization (hpf) using a custom automated dechorionator and protocol described in Mandrell et al. [14 (link)]. Six hpf dechorinated embryos were placed individually into the wells of two 96-well plates per chemical using the automated embryo placement systems (AEPS) [14 (link)]. Chemicals were added to final well concentrations of 0, 0.0064, 0.064, 0.64, 6.4, and 64uM, with 0.64% DMSO included as the vehicle. Thus, there are 32 embryos per chemical per concentration. The layout of each concentration within a plate is shown in Fig 1. At 24hpf, an embryonic photomotor response (EPR) test was implemented [8 ]. After EPR, all exposed plates were wrapped with alumnium foil to prevent photodegradation, kept in a 28°C incubator, and statically exposed until 120hpf.
At 120hpf, zebrafish larvae movement was recorded in Viewpoint Zebrabox (Viewpoint Life Sciences, Lyon, France) during a 7-minute period of light followed by an 8-minute period of dark, then evaluated for 18 distinct morphological endpoints. The 18 morphological endpoints recorded for developmental assessment were Mortality (MORT), Yolk sac edema (YSE), Body axis (AXIS), Eye defect (EYE), Snout (SNOU), Jaw (JAW), Otic vesicle (OTIC), Pericardial edema (PE), Brain (BRAI), Somite (SOMI), Pectoral fin (PFIN), Caudal fin (CFIN), Pigmentation (PIG), Circulation (CIRC), Truncated body (TRUN), Swim bladder (SWIM), Notochord & Bent tail (NC), and Touch response (TR). Each morphological endpoint was recorded as a binary presence/absence according to the protocol detailed in [12 (link)]. All data were recorded by the Zebrafish Acquisition and Analysis Program (ZAAP) [12 (link)]. The current manuscript primarily focuses on the 120hpf behavioral assessment (see right-most ‘Light Condition Exposure’ portion of Fig 1).

Six weeks after implantation, rats were sacrificed, and discs were harvested for histological analysis to demonstrate proteoglycan distribution in the IVD. The disc with the adjacent vertebral body was fixed in 10% neutral buffered formalin for 1 week and decalcified in Rapid Cal Immuno (BBC Biochemical, Mount Vernon, WA, USA) for 2 weeks. Tissues were then processed for paraffin embedding and sectioning into coronal sections (10 µm) using a microtome (Leica, Wetzlar, Germany). The obtained sections were dewaxed, rehydrated, and stained with safranin-O (Sigma, St. Louis, MO, USA) to analyze the quantity and distribution of proteoglycan content. Finally, the sections were mounted using mounting media and scanned with an Olympus C-mount camera adapter (U-TVO.63XC, Tokyo, Japan).
Likewise, the histological scoring was done by using a comprehensive 16-point scale for the assessment of IVD based on safranin-O staining. The scoring was based on the NP morphology, NP cellularity, AF morphology, endplate morphology, and the boundary between the NP and AF, resulting in five subcategories. Briefly, the NP and AF morphology each include two degenerative features since they were ranked to be highly crucial in the study. As alterations in notochordal cell morphology are an important and easily notable feature in degenerative rat IVDs, so the NP cellularity category was also weighted twice with two features. Briefly, in terms of NP morphology, analyses on the NP shape and total NP area were performed. Similarly, cell number and cellular morphology were analyzed to measure NP cellularity. The border appearance was observed in order to differentiate no interruption, minimal interruption, and no distinction between NP and AF. Furthermore, to examine AF morphology in IVD, lamellar organization and any tears/fissures/disruptions in the AF region were identified. Finally, in terms of endplate, any appearance of disruptions, microfractures, osteophyte, or ossifications were examined to produce the histological scores. Thus, non-degenerative characteristics were represented as 0, mild degenerative characteristics as 1, and severe degenerative changes as 2. The sum of the separate scores ranged from 0 (normal) to 16 (most severe) The NP, AF, endplate, and boundary between the NP and AF of the IVD were scored and added together for a total IVD score [74 (link)]. Two independent observers who were completely blinded to the sample information conducted the histological analysis of all samples.
Furthermore, the obtained sections were dewaxed, rehydrated, and stained with H&E for analysis of the tissue morphology and proteoglycan distribution in IVDs. The disc NP-cell number and H&E positive area were measured using ImageJ software (https://imagej.nih.gov/ij/ (accessed on 10 November 2022)). Briefly, we created binary images at a fixed intensity level and measured the area between vertebral endplates [13 (link)].

For EdU labeling, larvae were incubated in 2.5 mg/ml EdU (VWR) in PBS for 30 min. Larvae were fixed in 4% PFA in PBS overnight at 4°C and rinsed 5× for 20 min in PBTx. Larvae were equilibrated in 100 mM Tris-HCl pH 8 for 2 min and EdU was detected by using a copper-catalyzed azide-alkyne click reaction (25 µM Alexa Fluor 647 Azide (Invitrogen, A10277), 100 mM Tris, 1 mM CuSO4, 100 mM ascorbic acid, pH 8) with 30 min incubation time. Subsequently, larvae were rinsed 5× for 20 min in PBTx. The GFP signal was recovered by using an anti-GFP antibody. Immunostaining was performed as described for anti-pS6, with the following antibodies and dilutions: rabbit anti-GFP antibody (GeneTex, GTX113617; 1:200), goat anti-rabbit antibody conjugated to Alexa Fluor 488 (Invitrogen, A-11070; 1:500). For quantification of EdU⁺ caudal fin mesenchyme, cells within 300 µm anterior of the notochord tip were counted.