Protocol for analysis of integrin-mediated cell adhesion of lateral plate mesoderm cells isolated from zebrafish embryos

Summary Lateral plate mesoderm (LPM) cells differentiate into various cell types including endothelial and hematopoietic cells. In zebrafish embryos, LPM cells migrate toward the midline along the ventral surfaces of somites during which their cell fate specification depends upon efficient integrin-mediated cell adhesion and migration. Herein, we present a protocol for analysis of integrin-mediated cell adhesion of LPM cells isolated from zebrafish embryos. This allows the study of the molecular mechanisms underlying integrin activation required for LPM cell fate specification. For complete details on the use and execution of this protocol, please refer to Rho et al. (2019).


SUMMARY
Lateral plate mesoderm (LPM) cells differentiate into various cell types including endothelial and hematopoietic cells. In zebrafish embryos, LPM cells migrate toward the midline along the ventral surfaces of somites during which their cell fate specification depends upon efficient integrin-mediated cell adhesion and migration. Herein, we present a protocol for analysis of integrin-mediated cell adhesion of LPM cells isolated from zebrafish embryos. This allows the study of the molecular mechanisms underlying integrin activation required for LPM cell fate specification. For complete details on the use and execution of this protocol, please refer to Rho et al. (2019).

BEFORE YOU BEGIN Zebrafish
To visualize the LPM cells, we have used the Tg(fli1a:EGFP) y1 zebrafish line in which EGFP is expressed in LPM cells during somitogenesis and in endothelial cells at a later stage (Lawson and Weinstein, 2002) (Figure 1). The Tg(fli1a:EGFP) y1 zebrafish are intercrossed to obtain embryos exhibiting strong EGFP fluorescence in the LPM cells. In our study, we also used rap1b ncv124 mutant fish with the background of the Tg(fli1a:EGFP) y1 zebrafish line to investigate the role of Rap1b in integrin-mediated cell adhesion of LPM cells (Rho et al., 2019).
CRITICAL: Using embryos exhibiting strong EGFP fluorescence in LPM cells is important for obtaining clear images of the LPM cells adherent to the fibronectin-coated culture dish. The paper describing the generation of Tg(fli1a:EGFP) zebrafish line indicated that the level of EGFP expression correlates with transgene copy numbers and that the Tg(fli1a:EGFP) y1 line used in our study carries more than 25 copies of transgene (Lawson and Weinstein, 2002). Thus, using the zebrafish lines carrying high copy numbers of the transgene provides clear images.
Note: In the Tg(fli1a:EGFP) y1 line, expression of EGFP in LPM cells and endothelial cells does not alter cellular physiology and behavior, because hematopoiesis and vascular development normally occur in this transgenic line (Rho et al., 2019;Lawson and Weinstein, 2002).
Alternatives: Instead of the Tg(fli1a:EGFP) y1 zebrafish line, you may be able to use transgenic zebrafish lines expressing various types of fluorescence proteins under control of the fli1a promoter to visualize cell morphology and cellular structures such as the actin cytoskeleton. For instance, using Tg(fli1a:Myr-EGFP) and Tg(fli1a:Lifeact-mCherry) lines allows us to image LPM cell morphology and the actin cytoskeleton, respectively (Wakayama et al., 2015). However, embryos exhibiting strong fluorescence must be used to obtain clear images. It may also be possible to use zebrafish mutants and morphants to investigate the mechanisms of integrin-mediated cell adhesion and migration of LPM cells.

Fibronectin-coated glass base dish
Timing: 1 day Coat the 35-mm-diameter glass-base dishes with 200 mL of 10 mg/mL fibronectin in PBS at 4 C and leave them for 12-16 h. Wash the dishes with PBS or sterile water before use.
Note: Fibronectin-coated dishes washed with sterile water can be stored at 2-8 C under sterile conditions for several days.

KEY RESOURCES TABLE
Alternatives: Cell culture grade fibronectin can be obtained from the alternative companies such as Sigma-Aldrich. The 35-mm-diameter dishes with 12-mm diameter glass bottom can also be purchased from the alternative companies such as Thermo Scientific Nunc.
Alternatives: Although we used FluoView FV1000 confocal upright microscope to image the LPM cells, equivalent confocal microscope can be used. REAGENT  Make 1 mL/tube aliquots in 1.5 mL-Eppendorf tubes and store at À20 C To prepare working solution, add 1 mL of 10 mg/mL Pronase to 9 mL of 0.03% sea salt solution.
CRITICAL: Directly add 1 mL of sterile water into the vial containing 5 mg Liberase and gently agitate the vial at 4 C until 30 min. Make the aliquots and store at À20 C. Do not repeat freeze/thaw cycles.
To prepare working solution of 50 mg/mL Liberase in DPBS, add 50 mL of 1003 stock solution to 450 mL DPBS. 1. Transfer the Tg(fli1a:EGFP) y1 zebrafish into the breeding tank in which the male and female fish are separated by a divider during the afternoon or evening ( Figure 2A).

Note:
To stimulate good quality egg production, the day-night light cycle (14 h light/10 h dark cycle) is controlled with an automatic timer, because zebrafish reproduction depends on the light cycle. 2. When the light comes on the following morning, remove the divider separating the male and female fish to initiate breeding ( Figure 2B).
Note: Breeding tanks have a removable insert with holes to prevent adult fish from eating their eggs.
Note: Reducing the amount of water inside the tank facilitates zebrafish breeding.
3. Remove the adult fish 15-30 min after the beginning of spawning. 4. Collect the embryos by pouring the water and the embryos through a mesh tea strainer. 5. Rinse the embryos on the strainer well with 0.03% sea salt solution ( Figures 2C and 2D). 6. Wash the embryos off into a 10-cm petri dish using E3 medium ( Figure 2E).

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7. Keep the embryos in petri dishes containing E3 medium at a density of less than 30 embryos per dish until 17 hpf at 28 C.
Note: Do not incubate the embryos at a density of more than 30 embryos per dish to prevent developmental delay.

Preparation of the cells dissociated from the embryos
Timing: 1.5-2 h The Tg(fli1a:EGFP) y1 embryos are dissociated into a single cell suspension. Screen the embryos with a fluorescence stereo microscope in advance, selecting the brightest embryos.  Figure 2H). e. Pipet the embryos back and forth in E3 medium using plastic Pasteur pipette to remove the chorions. f. Collect the dechorionated embryos in the 10-cm petri dish containing E3 medium ( Figure 2I). 9. Enzymatic dissociation of the embryos a. Transfer fifteen embryos into a 2 mL-Eppendorf tube.
Note: If possible, select embryos at the same stage by counting the number of somites. b. Wash the embryos with 1 mL of PBS once. c. Incubate the embryos with 500 mL of 13 Liberase solution at 37 C for 1 h.
Note: During the incubation, gently mix the embryos every 20 min using a P1000 single channel Pipette. d. After the incubation, add 1 mL of Liberase termination solution to the tube and mix to terminate the enzymatic reaction. e. Dissociate the embryonic cells by gentle pipetting using a P1000 Pipette.  Note: The cells derived from the embryos contain not only GFP-positive LPM cells but also GFP-negative cells.
Optional: While the GFP-positive LPM cells can be isolated using a fluorescence-activated cell sorter, this step might result in cell damage.
14. Incubate the cells, thereby allowing them to adhere to the fibronectin-coated dish, at 28 C for 13 h.

Staining of the LPM cells adhering to the fibronectin-coated dish
Timing: 2 days The cells adhering to the fibronectin-coated dish are stained with rabbit anti-GFP and mouse antivinculin antibodies followed by Alexa Fluor 488-conjugated anti-rabbit and Alexa Fluor 564-conjugated anti-mouse secondary antibodies and with Alexa Fluor 633-conjugated phalloidin to analyze cell spreading and the formation of focal adhesions in the LPM cells. Pause point: The stained cells can be stored in the dark at 4 C for several days.

Fluorescence imaging of the LPM cells adhering to the fibronectin-coated dish
Timing: 1-2 h Fluorescence images of the LPM cells adhering to the fibronectin-coated dish are obtained with a FluoView FV1000 confocal upright microscope system equipped with a water-immersion 603 lens (LUMFL N 603/1.10 W), 473-, 559-, and 635-nm laser lines, and a GaAsP photomultiplier tube controlled with FluoView ASW software ( Figure 3A).

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27. We set the confocal imaging parameters as follows, but you will need to optimize these to achieve reasonable signal-to-noise ratio in your image without saturating the detector, as well as taking care to use Nyquist sampling: a. Scanning speed; 10 ms/pixel b. Sampling; 640 3 640 pixels, 0.094 mm/pixel c. Zoom; 3.53 for a FOV of 60 3 60 mm d. Laser power; 473 nm 1.0%-1.5%, 559 nm 4.0%-5.0%, 635 nm 1.0%-2.5%  Figure 3B).

Quantitative analysis of cell spreading and focal adhesion formations in the LPM cells adhering to the fibronectin-coated dish
Timing: 3 h Cell spreading area and the number and size of focal adhesions were analyzed using ImageJ software according, basically, to the protocol described previously (Horzum et al., 2014) (Figure 4).

EXPECTED OUTCOMES
If this protocol is followed, most of the GFP-positive LPM cells derived from the Tg(fli1a:EGFP) y1 zebrafish embryos form integrin-mediated focal adhesions and efficiently adhere to the fibronectin-coated dish after 13 h of culture, as shown in Figure 5A (Figures 5B and 5C). Thus, this protocol enables us to quantitatively analyze formation of integrin-mediated cell adhesion of LPM cells derived from zebrafish embryos.
By utilizing this protocol, we previously investigated the role of Rap1b, a small GTPase that belongs to the Ras superfamily, in integrin-mediated cell adhesion of LPM cells involved in hematopoietic stem cell development ( Figure 5) (Rho et al., 2019). In this study, we found that LPM cells derived from rap1b ncv124 mutant embryos exhibited less spreading on the fibronectin-coated dish than the wild type cells. We also showed the number and size of focal adhesions to be significantly smaller in rap1b ncv124 mutant-derived LPM cells than in wild type cells ( Figures 5B and 5C). Notably, rap1b ncv124 mutant-derived LPM cells exhibited a significant decrease in the number of mature focal adhesions ( Figure 5D). By performing the experiments using this protocol, we have succeeded in The mutant zebrafish can be used to investigate the role of a gene of interest in integrin-mediated cell adhesion of LPM cells. However, wild type and mutant fish need to be intercrossed to obtain wild type LPM cells and those derived from the mutants, respectively, because we are unable to perform genotyping of each embryo at 17 hpf before cell dissociation. Alternatively, the embryos injected with control morpholino oligonucleotide and with that targeting a gene of interest can be used, although it would be necessary to confirm that observed effects are specific to the mutants used.

TROUBLESHOOTING
Problem 1 Insufficient number of embryos collected (step 2).

Potential solution
Maintaining adult zebrafish in a healthy condition is important to get good quality and large number of embryos. Therefore, it is advised to feed the fish more food than usual for several days before breeding. In addition, the breeding fish should be between 3 and 12 months of age for production of large number of embryos, although zebrafish reach sexual maturity in 2 to 3 months.

Problem 2
Abnormal development and low fertilization rates (step 7).

Potential solution
As mentioned above, use of adult zebrafish in a healthy condition is important to get good quality embryos. Thus, refer to the potential solution for ''problem 1''. In addition, keeping zebrafish embryos in a clean environment is necessary for the normal development. Therefore, when collecting the embryos, rinse them on the strainer thoroughly to remove feces and residual food (step 5) (Figure 2C). In addition, remove the abnormal embryos from the petri dish during the incubation.

Potential solution
Incubating the embryos with the Pronase solution at 28 C instead of 20-25 C facilitates the dechorionation. But, be careful to prevent the embryos to be damaged. For that, carefully check the condition of the chorions during the incubation and control the incubation time. Alternatively, the chorions can be manually removed with fine forceps. But, take great care not to damage the embryos, because they are fragile.

Problem 4
Drying of cells on the glass-base dish by evaporation of Leibovitz's L-15 medium (step 13).

Potential solution
Incubation of the cells plated onto a glass-base dish in a fish incubator at 28 C may result in evaporation of the medium. To prevent medium evaporation and drying of the cells, place the dishes inside a 150 mm petri dish in which papers soaked with distilled water have been placed. Careful addition of the medium to the dish 9 h after the plating of cells is also feasible.

Problem 5
Too few GFP-positive cells in field of view (step 28).

Potential solution
The cells isolated from the embryos showing weak GFP fluorescence may be difficult to detect. Therefore, when selecting the Tg(fli1a:EGFP) y1 zebrafish embryos at 17 hpf under the fluorescence stereomicroscope in step 8, it is advised that the embryos exhibiting strong GFP fluorescence be collected. This allows LPM cells expressing a high level of GFP to be obtained.

Problem 6
Weak fluorescence signal obtained from the stained cells (step 29).

Potential solution
Incubation with Alexa Fluor-labeled secondary antibodies at 4 C for 12-16 h instead of that for 2 h at 15-25 C may increase fluorescence signal (step 22). In addition, staining of the cells with higher concentration of primary antibodies may also improve the signal (step 20).

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Shigetomo Fukuhara (s-fukuhara@nms.ac.jp).

Materials availability
The Tg(fli1a:EGFP) y1 zebrafish line, which was originally developed by Nathan Lawson and Brant Weinstein (Lawson and Weinstein, 2002), used in this study can be obtained from the Zebrafish International Resource Center (https://zebrafish.org/fish/lineAll.php). The rap1b ncv124 mutant zebrafish will be deposited at the National BioResource Project Zebrafish in Japan (https://shigen.nig. ac.jp/zebra/index_en.html).

Data and code availability
This study generated neither any unique datasets nor code.