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Oct 2020
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Isolation of Myofibres and Culture of Muscle Stem Cells from Adult Zebrafish
成年斑马鱼肌纤维的分离和肌肉干细胞的培养   

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Abstract

Skeletal muscles generate force throughout life and require maintenance and repair to ensure efficiency. The population of resident muscle stem cells (MuSCs), termed satellite cells, dwells beneath the basal lamina of adult myofibres and contributes to both muscle growth and regeneration. Upon exposure to activating signals, MuSCs proliferate to generate myoblasts that differentiate and fuse to grow or regenerate myofibres. This myogenic progression resembles aspects of muscle formation and development during embryogenesis. Therefore, the study of MuSCs and their associated myofibres permits the exploration of muscle stem cell biology, including the cellular and molecular mechanisms underlying muscle formation, maintenance and repair. As most aspects of MuSC biology have been described in rodents, their relevance to other species, including humans, is unclear and would benefit from comparison to an alternative vertebrate system. Here, we describe a procedure for the isolation and immunolabelling or culture of adult zebrafish myofibres that allows examination of both myofibre characteristics and MuSC biology ex vivo. Isolated myofibres can be analysed for morphometric characteristics such as the myofibre volume and myonuclear domain to assess the dynamics of muscle growth. Immunolabelling for canonical stemness markers or reporter transgenes identifies MuSCs on isolated myofibres for cellular/molecular studies. Furthermore, viable myofibres can be plated, allowing MuSC myogenesis and analysis of proliferative and differentiative dynamics in primary progenitor cells. In conclusion, we provide a comparative system to amniote models for the study of vertebrate myogenesis, which will reveal fundamental genetic and cellular mechanisms of MuSC biology and inform aquaculture.


Graphic abstract:


Schematic of Myofibre Isolation and Culture of Muscle Stem Cells from Adult Zebrafish.


Keywords: Zebrafish (斑马鱼), Muscle fibre (肌肉纤维), Myofibre (肌纤维), Stem cell (干细胞), Skeletal muscle (骨骼肌), Myonucleus (肌核), Pax7 (Pax7), MuSC (MuSC), Adult (成人), Satellite cell (卫星细胞)

Background

Skeletal musculature provides lifelong body support and movement through coordinated contraction of myofibres, highly specialised syncytial cells containing hundreds of post-mitotic myonuclei. Myofibres constantly adapt to both exogenous and endogenous stimuli in part thanks to resident muscle stem cells (MuSCs), also known as satellite cells, located underneath the basal lamina of most myofibres (Katz, 1961; Mauro, 1961; Relaix and Zammit, 2012; Purohit and Dhawan, 2019). In response to exercise or damage, quiescent MuSCs quickly activate and become muscle progenitor cells (MPCs) called myoblasts, which proliferate, differentiate and fuse either to pre-existing multinucleated myofibres or to one another to form new myofibres (Fukada et al., 2020). Isolation of rodent myofibres and culture of associated MuSCs is a well-established tool to explore muscle stem cell biology, providing not only an understanding of MuSC behaviour and regulation of quiescence, activation, proliferation, self-renewal and differentiation (Zammit et al., 2004), but also yielding insights into both embryonic muscle development and adult myofibre growth and maintenance (Buckingham and Relaix, 2015). However, mechanisms underlying murine MuSC biology may not fully resemble those found in human myogenesis. Therefore, alternative vertebrate models to study adult/MuSC myogenesis are desirable to consolidate findings of ancestral mechanisms in vertebrate muscle. Notably, despite substantial overlap of molecular pathways shared in myogenesis between zebrafish and amniotes (Hammond et al., 2007; Hinits et al., 2009 and 2011; Ganassi et al., 2018; Osborn et al., 2020), the complementary study of adult muscle homeostasis in zebrafish is limited to mechanical trituration of bulk muscle, hindering the purity of myogenic yield (Alexander et al., 2011; Froehlich et al., 2014). Adult myofibre isolation in fish has permitted the study of their physical or contractile properties (Johnston and Altringham 1988; Davies 1995; Johnston et al., 2004), and some studies have more recently exploited it to investigate zebrafish MuSC biology (Anderson et al., 2012; Zhang and Anderson, 2014; Ganassi et al., 2018 and 2020).


Here, we describe how to isolate single viable myofibres and associated MuSCs through enzymatic digestion and fine trituration of the trunk musculature of adult zebrafish. This method is adapted from the standard mouse protocol (Bischoff, 1975; Cardasis and Cooper, 1975a and 1975b; Bekoff and Betz, 1977a and 1977b; Rosenblatt et al., 1995; Moyle and Zammit, 2014). We explain how to plate myofibres to study MuSC activation and progression through myogenesis in vitro, ensuring a virtually pure myogenic population (Ganassi et al., 2018 and 2020). Our protocol provides an appropriate toolbox for comparative analysis of adult myogenesis across vertebrates and has been recently developed and used to explore the function of the transcription factor Myogenin in adult MuSC activation, proliferation and differentiation (Ganassi et al., 2018 and 2020). Myofibre isolation, culture, and analysis from adult fish exploits the advantages of the zebrafish model, such as the spatial segregation of slow and fast myofibres that facilitates fibre-type specific studies (Blagden et al., 1997; Pipalia et al., 2016; Hromowyk et al., 2020), and provides insight useful to aquaculture. As an alternative to the classical rodent procedure, the analysis of fish MuSC also offers an independent benchmark to verify genetic and cellular mechanisms identified using rodent models. Therefore, application of our techniques to adult zebrafish muscle has the potential to contribute to understanding genetic, molecular, and cellular mechanisms maintaining and adapting human musculature.


The method is simple, efficient, and cost-effective and permits the study of 1) myofibre characteristics ex vivo, 2) MuSC-derived myoblasts/myotubes ex vivo and 3) mechanisms of adult muscle formation, development, and maintenance.

Materials and Reagents

Materials required for dissection and dissociation of adult muscle

  1. Deep Petri dishes (150 mm and 100 mm) sterile, cell culture grade (Corning, catalog numbers: 430599 and 430167)

  2. Glass Pasteur pipettes (22 cm), sterile (Volac, catalog number: D812)

  3. 0.45 μm and 0.2 μm sterile syringe filters (ThermoFisher, catalog numbers: 15216869 and 15206869)

  4. Sterile syringe, 50 ml (Terumo, catalog number: SS+50ES1)

  5. Aluminium foil

  6. Bijou tubes, 7 ml

  7. Tricaine methanesulfonate (MS-222) solution (Sigma-Aldrich, catalog number: E10521)

  8. 70% Ethanol solution (in deionised water) (70% EtOH) (Ethanol absolute; Sigma-Aldrich, catalog number: 1024282500)

  9. 5% Bovine serum albumin (BSA) (powder, Sigma-Aldrich, catalog number: A7906)

  10. Collagenase from Clostridium histolyticum (Sigma-Aldrich, catalog number: C0130)

  11. Dulbecco’s modified Eagle’s medium (DMEM), high glucose, GlutaMAX, Pyruvate (ThermoFisher, catalog number: 31966)

  12. Phosphate-buffered saline Ca2+ and Mg2+ free (PBS), sterile (Oxoid, catalog number: BR0014G)

  13. Penicillin and Streptomycin solution (Sigma-Aldrich, catalog number: P0781)

  14. 1% Virkon solution (in deionised water) (powder, 3S Healthcare) (see Recipes)

  15. P/S-PBS (see Recipes)

  16. BSA-PBS (see Recipes)

  17. cDMEM (complete DMEM) (see Recipes)

  18. Collagenase-cDMEM (see Recipes)


Materials required for myofibre and MuSC-derived cell immunolabelling

  1. Cover glasses 50 mm × 22 mm (Academy, catalog number: 400-04-17)

  2. Glass Slides (Fisher, catalog number: 1157-2203)

  3. Crystal-clear plastic microcentrifuge tubes, 2 ml (Starlab, catalog number: S-1620-2700)

  4. Transparent nail varnish

  5. Paraformaldehyde (PFA) solution, 4% in PBS (PFA-PBS) (Alfa Aesar, catalog number: J61899)

  6. Liquid blocker super pap pen (Pyramid Innovation, catalog number: R62002-E)

  7. Triton X-100 detergent solution (Sigma-Aldrich, catalog number: X100)

  8. Chicken anti-GFP (RRID:AB_300798; Abcam, catalog number: 13970; use 1:400)

  9. Goat anti-chicken IgY (H+L), Alexa Fluor® 488 (RRID:AB_2534091; Thermo Fisher Scientific, catalog number: A11032, use 1:1000)

  10. Hoechst 33342 solution (ThermoFisher, catalog number: H3570, use 1:1000)

  11. Normal goat serum (NGS) (Agilent, catalog number: x0907)

  12. Glycerol-based mounting medium (Agilent, catalog number: 50001)

  13. PBSTx (see Recipes)


Materials required for myofibre and MuSC-derived cell culture

  1. 24-well plates cell culture grade (ThermoFisher, catalog number: 142475)

  2. Fetal bovine serum (FBS), heat inactivated (ThermoFisher, catalog number: 10500-064)

  3. Horse serum (HS) (ThermoFisher, catalog number: 26050088)

  4. Matrigel (Corning, catalog number: 354263)

  5. 5-ethynyl-2’-deoxyuridine (EdU) solution (From Click-iT EdU kit; ThermoFisher, catalog number: C10646)

  6. Gentamicin (Gibco, catalog number: 15750-060)

  7. Matrigel solution (see Recipes)

  8. Proliferation Medium (PM) (see Recipes)

  9. Differentiation Medium (DM) (see Recipes)

Equipment

Dissection and dissociation of adult muscle

  1. Tissue culture hood or lamina flow cabinet

  2. Tissue culture incubator (humidified, 28.5°C, 5% CO2)

  3. Cork dissection board (IKEA, catalog number: 870.777.00)

  4. Dissection metal pins

  5. Fine forceps, one pair (Idealtek, No. 5A.s)

  6. Sterile disposable scalpels No. 10 (Swann-Morton, catalog number: 0501)

  7. Bunsen burner

  8. Diamond-tipped pen (VWR, catalog number: 201-0392)

  9. Dissection microscope with transmission illumination (Zeiss Stemi SV6 and Leica M50)

Software

  1. Image Analysis: Fiji; NIH (www.Fiji.sc)

  2. Data presentation: GraphPad Prism 8 (https://www.graphpad.com/scientific-software/prism/)

Procedure

  1. Muscle Dissection

    Where possible, perform steps under sterile conditions in a tissue culture hood or laminar flow cabinet.

    1. Euthanise the fish by immersion in ice-cold 0.3 mg/ml tricaine solution. Immerse fish in chilled tricaine solution aliquoted into a 50 ml tube for the required amount of time. To minimise animal distress, keep the tube on ice during incubation. Please note that the duration of tricaine incubation must be determined empirically, depending on fish size and age as described before (Westerfield, 2000; Harper and Lawrence, 2011) (see Note 1).

    2. Remove fish carcass from tricaine solution and immerse it in 25 ml of 1% Virkon (see Recipe 1) solution in a 100 mm dish. Incubate for 5 min to kill bacteria and fungi.

    3. Use clean forceps to transfer the fish carcass to a new 100 mm dish containing 25 ml P/S-PBS (see Recipe 2) and incubate for 5 min.

    4. Transfer fish carcass into a new empty 100 mm Petri dish. Use a disposable scalpel to remove scales. To increase scaling efficiency, position the blade perpendicular to the antero-posterior axis of the fish body and gently scrub the skin surface from tail to head (see Note 2).

    5. Wash the descaled carcass into a new 100 mm dish containing 25 ml of fresh P/S-PBS for 5 min. Meanwhile, carefully wipe dissection metal pins, corkboard and fine forceps with 70% EtOH to reduce chances of contamination.

    6. Move fish carcass to a new 100 mm dish, gently dry residual P/S-PBS with cloth and spray with 70% EtOH on both sides.

    7. Move fish to the dissecting corkboard and place one pin passing through tissue just behind the gill operculum and a second pin penetrating the tissue just anterior to the base of the caudal fin (Figure 1A).

    8. Use the scalpel to cut fins as close as possible to the fish body. Removed fins can be processed to extract genomic DNA for fish genotyping (Figures 1A and 1B) (see Note 3).

    9. Make a curved incision along the ventral side of the carcass to facilitate evisceration using blade and fine forceps (Figure 1A).

    10. At this point, different portions of the carcass can be collected for required analyses. As indicated in Figure 1B: i) fins are useful for retrospective genotyping, ii) the muscle region near the tail tip is usually damaged by the dissecting pin but can be used for whole muscle RNA/Protein analysis, iii) the adjacent 5 mm section of muscle can be cryopreserved for histological analysis and iv) most of the trunk musculature is processed for myofibre isolation.

    11. Use the scalpel to make a light incision on the skin just behind the gill operculum and perpendicular to the antero-posterior axis, carefully avoiding incision of the muscle beneath. Use the fine forceps to gently pinch and lift the skin along the incision edge. Carefully grab and pull the skin toward the fish tail to expose the underlying muscle (Figure 1C).

    12. Continue to pull gently until reaching the pin positioned close to the tail (Figure 1C). Slow muscle is strongly attached to the overlying skin, so pull very gently to avoid damaging the slow myofibres. Most of the trunk musculature should now be exposed.

    13. Use the same procedure to remove skin from the contralateral side.

    14. When skinning is completed, unpin the fish and rotate it 90° onto its back so that the ventral side (belly) points upward towards the operator (Figures 1D-1F).

    15. Re-pin the fish to the corkboard in the new position, using one pin passing through the lower jaw and head and the second at the base of the tail. The vertebral column should be visible and accessible through the opening in the belly (Figures 1E and 1F).

    16. Use the blade to cut on the right of the vertebral column along the entire antero-posterior axis to create two muscle fillets, one bearing the associated vertebral column and spinal cord and the other without. It is important to angle the scalpel so that its tip points toward the dorsal midline, penetrating the anterior-most part of the muscle tissue close to the vertebral column (Figure 1E). Draw the blade posteriorly until the tail pin is reached, leaving the ribs in the fillet (Figure 1E’). Stopping or hesitating whilst cutting along the column can lead to varying fillet thickness and damage the medial-most muscle.

    17. Use scalpel to remove the fish head and fully release the two muscle fillets. The fish fillets display slow and fast muscle compartments (Figure 1G). The spinal cord should be visible in the right fillet and can be removed with the scalpel, but this is not essential. We usually do not remove it to reduce possible damage to the surrounding muscle tissue.

    18. Muscle dissection should require 30 min and can be performed on multiple fish in parallel.


  2. Myofibre Dissociation and Isolation

    1. Rinse one 150 mm and two 100 mm new sterile Petri dishes per fish with BSA-PBS solution (see Recipe 3) to prevent myofibre adhesion to the dish. Remove excess BSA-PBS solution and add 25 ml and 10 ml of complete DMEM (cDMEM, see Recipe 4) to the 150 mm and 100 mm dishes, respectively. Place dishes in a 28.5°C 5% CO2 incubator for at least 30 min to allow the cDMEM to warm.

    2. Place the freshly dissected fillets in the bijou tube with Collagenase-cDMEM solution (see Recipe 5 and Note 4), apply cap loosely and incubate at 28.5°C in 5% CO2 incubator for 120 min with occasional (every 30 min) very gentle swirling of the tube (Figure 1H).

    3. Meanwhile, use a diamond pen to score two glass Pasteur pipettes per fish and create openings with diameters of approximately 1 and 3-4 mm, respectively (Figure 1I). Use a Bunsen burner to melt the glass around the opening to smoothly polish any sharp edges (Figure 1I and Video 1). Test the polishing by circling the pipette edge on aluminium foil. No cut/tear should be produced. Quickly flame the prepared glass pipettes to sterilise, wrap in aluminium foil and store in the tissue culture hood until use.


      Video 1. Glass Pasteur pipette cut and heat-polish process

    4. When incubation is complete, place the bijou tube in the tissue culture hood. A well-digested muscle looks slightly swollen and, under the microscope, hair-like myofibres appear dislodged around the edge of the muscle mass (Figures 1J and J’ and see Note 5). Also collect the 150 mm dish with warm cDMEM from the incubator and place it in the culture hood.

    5. Gently decant and discard most of Collagenase-cDMEM solution from the bijou tube. Rapidly invert the bijou tube to pour the muscle fillets into the 150 mm Petri containing cDMEM. Return the Petri dish with fillets to the incubator for 20-30 min. This allows the muscle to rest and dilute the Collagenase, promoting inactivation of the enzyme.

    6. Place the dissecting microscope in the culture hood, if possible; otherwise, use a clean area away from doors, windows and draughts or other contamination sources. Collect the 150 mm dish with fillets and place under the lens of the microscope.

    7. Rinse the heat-polished glass pipettes with BSA-PBS solution to prevent myofibre adhesion.

    8. Using the pipette with the larger diameter (~3-4 mm), direct cDMEM onto the fillets repeatedly for at least 10 min, pipetting up and down to expel a continuous stream of liquid (Figure 1K). Tissue dissociation can be enhanced by carefully passing the fillets once or twice in and out of the glass pipette, but not continuously as this will damage the myofibres and reduce the final yield. Myofibres are visible hair-like structures that will be released from the muscle bulk (Figures 1K’ and 1L).

    9. Continue the trituration process until most myofibres have been released. The procedure will also result in the release of debris, including fat droplets and hypercontracted myofibres (Figure 1L), which will increase the turbidity of the medium. If trituration is prolonged, allow a further 5-10 min incubation at 28.5°C, 5% CO2 to re-equilibrate the temperature and pH of the medium. If medium reaches below the range of physiological temperatures (22-29°C) for an extended time, myofibers will hypercontract and die.

    10. Place the 150 mm plate back in the incubator for 10-15 min to allow released myofibres to rest and sink to the bottom.

    11. Using the glass pipette with the smaller diameter (~1 mm), carefully collect intact myofibres and transfer them onto a 100 mm dish with fresh cDMEM (Figure 1K’). If needed, the remaining muscle bulk can be further processed to enhance the release of residual myofibres, but not longer than 30-60 min as this will reduce the viability of residual myofibres.

    12. Place the 100 mm dish containing cleaned myofibres back in the incubator for another 10-15 min to allow them to rest and sink to the bottom.

    13. Viable myofibres appear translucent and with a smooth surface (Figure 1M). If needed, myofibres can be transferred into a new cDMEM-containing 100 mm dish to clean further. If significant debris is still present in the dish, repeat Steps B11 and B12.

    14. The entire muscle dissection procedure should require 180 min.



      Figure 1. Dissection and Isolation of Myofibres from Adult Zebrafish. A. Representative picture of an 8-month-old adult zebrafish depicts pin positioning (orange dots) to anchor fish carcass to the dissecting board. Red dashed lines indicate cuts to remove fins and to perform ventral incision for evisceration. B. Summary of analysis performable from different portions of the fish (ext; extraction). C. Blue dashed line and arrowheads indicate position of the skin incision, pinch and pulling direction for skinning with forceps. D. After skinning, 90° rotation of the carcass on its dorsal side exposes the ventral incision upward. Red dashed line and orange dots indicate cut direction and pin positioning, respectively. E, E’. Diagram of scalpel angle and cut direction during fish filleting. Red dashed arrows indicate inclination toward the dorsal midline (E) and cut direction towards tail pin (E’). Dashed black line shows position of spinal cord (E’). F. View of the ventral incision upward. Dashed red line and arrow indicate cut direction for filleting, with the spinal cord used as a guide (dashed white line). Antero-posterior orientation is indicated (h; head, t; tail). G. Slow (dark arrowhead) and fast (light arrowhead) muscle portions are visible in the dissected fillets (h; head, t; tail). H. Fillet is incubated with Collagenase-cDMEM solution for 120 min in the bijou tube at 28.5°C. I. Diagram of Pasteur glass pipettes cut and heat-polished to obtain two pipettes with wide (3-4 mm, pink) and small diameter (1 mm, purple) apertures, for muscle trituration and single muscle fibre handling, respectively. Purple (cut for 1 mm opening) or pink (cut for 3-4 mm opening) dashed lines indicate cut position on each pipette with diamond pen and heat polishing with a flame. Desired result for cut and edge heat-polish is shown in bottom pictures (see Video 1). J, J’. Representative images of muscle fillet after 120 min incubation in Collagenase-cDMEM solution (J). Note the hair-like myofibres dislodged around the edge of the muscle mass (J’, zoomed area in green). Position of ribs and spinal cord is also indicated. K, K’. Schematic of fillet trituration (K) and single myofibre isolation and wash (K’) with estimated duration in minutes. L. Representative images of single myofibres during washes. Yellow arrowheads denote intact viable myofibres, while the magenta arrowhead and asterisks indicate damaged or hypercontracted myofibres, respectively. Estimated time for fish preparation/muscle dissection (C-F) and myofibre dissociation/isolation (G-J) are reported beside panels. M. Representative single viable myofibre following muscle dissection and isolation.


  3. Analysis of Isolated Myofibres

    1. Isolated myofibres are now ready for analysis, such as morphometrical measurements. Myofibres can be photographed prior to fixation using a brightfield microscope, which allows exclusion of those that are hypercontracted or damaged. Depending on the microscope used, damaged myofibres that are not yet fully hypercontracted appear shorter and more opaque, with a rough and irregular surface (Figure 1L).

    2. Nuclear counting and subsequent analyses may require myofibre fixation. Under the microscope, use the BSA-PBS pre-rinsed glass pipette with smaller diameter (Figures 1I and 1K’) to collect isolated myofibres and place them in a 2 ml clear round-bottomed microcentrifuge tube that has been rinsed with BSA-PBS to prevent myofibre adhesion. Gently swirl the dish to gather all myofibres at the centre of the plate to reduce the volume of cDMEM medium collected with myofibres. We suggest limiting the number of myofibres to 40 per tube to avoid clumping and possible damage.

    3. Leave the collection microcentrifuge tube standing upright for 5 min at room temperature to allow the myofibres to sink to the bottom of the tube.

    4. Carefully remove the medium above the myofibres with a pipette and replenish the tube with 1 ml of 4% PFA in PBS solution (PFA-PBS) by gentle trickling down the side of the tilted tube. Incubate for 10-15 min at room temperature.

    5. Remove PFA-PBS solution and gently replenish with 1.5 ml of PBS to wash the myofibres. Incubate for 5 min and repeat the wash with fresh PBS. Lysine or BSA may be added to more efficiently inhibit the PFA. Fixed myofibres can be stored at 4°C for at least 2 weeks.

    6. Remove PBS, wash and replace with freshly prepared Hoechst 33342 dye solution diluted in PBS to stain myofibre nuclei. Incubate for 15 min and replace with fresh PBS (as in Step C5). Myofibres are now ready to be mounted on glass slides for detailed analysis.

    7. Use a water-repellent pap pen to outline a rectangular area (size depending on the size of the coverslip, e.g., 50 mm × 22 mm) on several glass slides. Under the microscope, use a clean smaller diameter pipette, pre-rinsed with BSA-PBS to collect myofibres from the 2 ml tube and transfer them onto the prepared glass slide. We suggest limiting to 10/15 myofibres per slide to facilitate handling.

    8. Remove as much PBS as possible from the glass slide with a pipette to ease myofibre adhesion and reduce the risk of damage and/or loss. A 200 µl micropipette tip wrapped in aluminium foil and pre-immersed in BSA-PBS can be used to carefully reposition myofibres after PBS removal. We suggest being quick as residual liquid, along with immersed myofibres, will dry out rapidly.

    9. Place two/three drops of glycerol-based mounting medium on the glass slide, position a 50 mm × 22 mm coverslip with an edge on the slide touching glycerol and gently lower the coverslip to avoid trapping air bubbles that can mis-position or sweep away myofibres. Wait 5 min to allow the mounting medium to spread beneath the coverslip.

    10. Seal the coverslip and secure to the glass slide by brushing on a small amount of nail varnish, first at the corners and then seal the edges.

    11. Mounted myofibres can be photographed using an epifluorescence or confocal microscope.

    12. Using imaging software, measure myofibre width (diameter) at a minimum of two different positions along each myofibre. Concomitantly, measure myofibre length and count the number of nuclei using Hoechst (Figure 2A).

    13. Myofibre volume can be calculated with the formula reported in the Data analysis section and Figure 2A. An example of myofibre volume in Myog+/- adults from Ganassi et al. (2020) is shown (Figure 2B). Alternatively, confocal scanning may give full myofibre morphology profile (see Ganassi et al., 2020).


  4. Myofibre Immunolabelling

    1. Fixed myofibres (Step C5) can be processed for immunolabelling. Here, we deploy the transgenic fish TgBAC(pax7a:GFP)t32239Tg (Nüsslein-Volhard C.; MPI Tübingen) (Mahalwar et al., 2014) and provide a template immunolabelling protocol using anti-GFP antibody to detect MuSCs. Although GFP fluorescence encoded by the pax7a:GFP transgene does resist PFA fixation, we suggest enhancement using the anti-GFP antibody, especially if co-labelling with multiple antibodies and fluorophores (Ganassi et al., 2020).

    2. Remove the PBS from the fixed myofibres with the BSA-PBS pre-rinsed smaller-diameter glass pipette and replace with 0.5% Triton-X100 detergent in PBS (PBSTx; see Recipe 6). Incubate for 15 min to permeabilise the cell membranes of both myofibres and associated MuSCs.

    3. Remove PBSTx and gently add a blocking solution of 10% normal goat serum (NGS) in PBS to block non-specific antibody binding. Incubate for at least 30 min, occasionally tilting the tube (see Note 6). Alternatively, 5% NGS in PBS solution can be used to incubate for 1 h.

    4. Prepare antibody solution by diluting the anti-GFP primary antibody in 0.1% Triton-X100 detergent PBS solution (PBSTx0.1) containing 2% NGS. Remove blocking solution from tube and gently add the primary antibody solution. Incubate overnight (16 h) at 4°C.

    5. Remove the primary antibody solution and replace with fresh PBSTx0.1 to wash myofibres for 5 min (see Note 7). Primary antibody solution can be stored at 4°C and re-used reliably within one week (perhaps longer if 0.002% sodium azide in PBS is added).

    6. Wash myofibres three times for 5 min each using PBSTx0.1 with occasional gentle tilting of the tube.

    7. Dilute fluorochrome-conjugated (e.g., Alexa Fluor 488) secondary antibodies and Hoechst 33342 dye solution (10 µg/ml final) in PBSTx0.1 and incubate for at least 60 min at room temperature, protected from light, with occasional tube swirling. Secondary antibody solution can be stored at 4°C in the dark and re-used reliably within one week or longer if 0.002% sodium azide in PBS is added.

    8. Transfer myofibres onto a prepared glass slide and mount under a coverslip as described in Steps C7-C11. An example of a pax7a:GFP MuSC on a myofibre immunolabelled for GFP is shown in Figure 2C and can be found in Ganassi et al. (2020). Store slides at 4°C in the dark; GFP fluorescence lasts for up to 14 days.


  5. Myofibre-derived MuSC Culture and Immunolabelling

    1. Coat the desired number of wells of a 24-well plate by rinsing with Matrigel solution (see Recipe 7). Be sure to completely cover the surface of each well. Immediately remove excess solution using a sterile pipette and return the Matrigel solution to 4°C to avoid precocious gelling. Place the prepared plate in a 28.5°C 5% CO2 incubator for 30-45 min to allow Matrigel gelling.

    2. Prepare the proliferation medium (PM; see Recipe 8) and pre-warm at 28.5°C in the incubator prior to aliquoting 200 µl per Matrigel-coated well.

    3. Transfer myofibres with a pipette into a new 100 mm dish containing 5 ml of 40% FBS-cDMEM solution. Each transfer of myofibres leads to concomitant carry-over of nearly 150-200 µl of cDMEM from the original dish that dilutes the serum concentration. At the end of the transfer, the final 100 mm dish now contains about 10 ml of DMEM with approximately 20% FSB, thus mimicking PM.

    4. Gently swirl the 100 mm dish to gather myofibres at its centre. Use the small diameter glass pipette BSA-PBS pre-rinsed to transfer approximately 90-100 freshly isolated myofibres into each Matrigel-coated well. Ensure that the myofibres are evenly spaced across the well by placing the plate on a flat surface and moving laterally in cross-like orthogonal directions several times.

    5. Place the 24-well plate(s) in the incubator and culture the myofibres undisturbed for at least 48 h. During the initial 24 h, myofibres can easily be dislodged, impacting MuSC activation, proliferation, migration and adhesion to the culture plate. Even opening/closing the incubator door can cause vibrations that dislodge myofibres.

    6. While a fraction of MuSCs activates within 24 h after plating, migrate off the myofibre and adhere to the Matrigel coating (see Note 8), we suggest waiting 48 h before analysis on MuSC. MPCs/myoblasts can be followed in culture and assayed for proliferation or differentiation capacity over time (Figure 2D).

    7. At 48 h, cells can be immunolabelled with anti-Desmin antibody to confirm the purity of the myogenic population. Myofibre plating usually yields almost pure myogenic progenitors in culture (Figure 2E). The original data is presented in Figure 4-figure supplement 1 (Ganassi et al., 2020), and a similar calculation can be found in Supplementary Figure 6g (Ganassi et al., 2018). Cell fixation is described below (Step E12).

    8. Cells can be collected at desired time point(s) for RNA extraction and gene expression analysis by RT-qPCR (see Note 9 and Figure 4 in Ganassi et al., 2020).

    9. Analysis of MuSC proliferation using 5-ethynyl-2′-deoxyuridine (EdU) is best performed no earlier than 2 days (48 h) from myofibre plating. Dilute EdU to a final concentration of 10 µM in fresh pre-warmed PM (EdU-PM).

    10. Remove PM from culture well and rinse vigorously twice with freshly prepared PM to remove plated myofibres. At this time point, most myofibres should be either floating or loosely adhering to Matrigel and thus easily removed.

    11. Remove PM and quickly rinse twice with PBS. Replace PBS with EdU-PM solution and place for 2-8 h in 28.5°C 5% CO2 incubator. Duration of incubation can be changed according to the experimental design; the example shown here refers to 8 h treatment.

    12. At the end of incubation, remove EdU-PM solution, wash vigorously twice with PBS and fix with PFA-PBS (4% PFA in PBS) for 15 min. If next step is immunolabelling, continue as described above for myofibres (Steps D2-D6).

    13. After the final wash in PBSTx0.1, perform click chemistry to reveal EdU incorporation following the manufacturer’s instructions.

    14. Remove PBS, wash and replace with freshly prepared Hoechst 33342 dye solution diluted in PBS to stain nuclei. Incubate for 15 min.

    15. Wash twice with fresh PBS, then replenish each well with 300 µl of PBS. The cells are now ready to be visualised using an inverted epifluorescence microscope (Figure 2F). Cells can be stored at 4°C (2-4 weeks) by replacing PBS with PBS containing 0.002% sodium azide to prevent microbial growth.

    16. Dynamics of MuSC/MPC proliferation can be followed over time, as reported in Figure 2F (adapted from Figures 4E and 4F [Ganassi et al., 2020]).

    17. Zebrafish myoblasts can also be induced to differentiate to evaluate the myogenic program. After 96 h from initial plating, remove PM and wash twice with sterile PBS to eliminate serum residues. Remove PBS and add 500 µl of differentiation medium (DM; see Recipe 9) to each well. Replace DM every 48 h. We previously assessed myogenic differentiation by culturing zebrafish primary myoblasts in low serum medium for 5 days prior to immunolabelling for structural components such as Myosins (Figure 2D). Figure 2G shows myotubes differentiated for 5 days and co-immunolabelled for myosin heavy chain (MyHC) using MF20 and A4.1025 concomitantly (Blagden et al., 1997) and counterstained with Hoechst 33342 to assess differentiation and cell fusion (original data is in Figure 8 in Ganassi et al., 2018) and Figure 4 - Supplement 1E (Ganassi et al., 2020).



      Figure 2. Analysis of Isolated Myofibres and Cultured MuSCs. A. Diagram of myofibre average diameter (myofibre width, red arrowed dashed line) and unit length (blue arrowed dashed line) measurements to calculate myofibre volume. Avg, indicates arithmetic mean of diameter measure across myofibre length (D1 and D2). B. Example results of 8-month-old adult myofibre volume (adapted from Ganassi et al., 2020). Symbols represent average values from 20-30 myofibres from each of three different fish. C.TgBAC(pax7a:GFP)t32239Tg (pax7a:GFP) myofibre immunolabelled for GFP (green) reveals the position of a MuSC (green rectangle, magnified at right) near the myofibre-end and myofibre nuclei counterstained with Hoechst 33342 (white). D. Schematic of myofibre plating, MuSCs/myoblasts proliferation (PM; proliferation medium), expansion and myotube differentiation protocol (DM; differentiation medium) with indicated timing, medium change and analysis. E. Example calculation of fraction of cells that were Desmin+ (myoblasts) two days after myofibre plating. F. MuSC-derived myoblasts can be EdU pulsed (red) two days after myofibre plating in proliferation medium. Nuclei were counterstained with Hoechst 33342 (blue). G. Representative image of differentiated multinucleated myotubes or mononucleated cells containing MyHC (red) after 5 days of culture in differentiation medium, coloured arrowheads indicate nuclei within the same cell (from Ganassi et al., 2020). Cyan dashed rectangle indicates the magnified area in the right panel. Nuclei were counterstained with Hoechst 33342 (white). Example results of fusion index (adapted from Ganassi et al., 2018). All graphs report mean ± SEM, and symbols represent biological replicates.

Data analysis

  1. Use the measured average myofibre width (avg diameter) and length to calculate the volume of the myofibre following the formula:

    [(Length × 𝜋) × [(average Diameter/2)2] (Figure 2A),

    where ‘average’ is the arithmetic mean of diameter measurements at a minimum of two different positions along the myofibre length. Values can be compared with chosen statistical analysis (e.g., unpaired two-tailed t-test). Graphs were produced in GraphPad Prism 8 (see Software).

  2. Myofibre volume and number of nuclei can be combined to calculate the myonuclear domain using the formula: [(Length × 𝜋) × [(average Diameter/2)2]/Number of myofibre nuclei]. Alternatively, average myofibre width and length can be used to calculate the surface area of the myofibre following the formula: Length × 𝜋 × avg Diameter. The Surface Area Domain Size (SADS), the notional SA occupied by each myofibre nucleus, is calculated using the formula: SA/number of myofibre nuclei. Examples of the calculation are available in Ganassi et al. (2020) or in Brack et al. (2005) for mouse myofibres.

Notes

  1. For zebrafish weighing 0.3-0.6 g, euthanasia is usually reached within 5-10 min after incubation in ice-cold 0.3 mg/ml tricaine solution. However, this is only indicative timing and must be determined empirically and according to local guidelines.

  2. It is essential to wash and thoroughly sterilise equipment and dissecting tools to avoid microbial contamination.

  3. Removing fish fins is not essential but facilitates handling and reduces the risk of microbial contamination.

  4. It is important to batch-test replacement reagents, such as Collagenase, against existing, optimised components. There are variable amounts of proteases in batches of Collagenase, but Collagenase with neutral protease around 53 U and clostripain at approximately 0.6 U is ideal, as described before (Rosenblatt et al., 1995).

  5. Adult (8-15 months old) zebrafish trunk muscle is usually digested after 2 h. Although longer incubations (≥ 3 h) have a marginal effect on myofibre viability, shorter incubation may reduce digestion efficiency, hindering the isolation of viable myofibers. The precise time depends upon both the age and size of the fish and the activity of the batch of Collagenase used and should be determined empirically.

  6. The normal serum used for blocking should derive from the species in which the secondary antibody was raised.

  7. Myofibres can be stained with fluorochrome-conjugated toxins to detect the subcellular structure, such as filamentous actin (F-actin) using Phalloidin (ThermoFisher, A12379) or neuromuscular junction (acetylcholine receptor) using α-Bungarotoxin (ThermoFisher, B35451).

  8. If myofibres/MuSCs are to be cultured for longer periods, replace half the volume of the medium with fresh medium every 48 h.

  9. For gene expression analysis, collect cells by removing medium and washing twice with PBS. Incubate with the appropriate volume (e.g., 200 µl for a 24-well plate well) of Accutase® reagent to detach the cells from Matrigel for 10 min (or until complete detachment of all cells; check under a microscope, but this should not take longer than 15 min) at 28.5°C, 5% CO2 (see Ganassi et al., 2020). Collect cells in a 1.5 ml clear tube, pellet by centrifugation at 200 × g at 4°C and wash once in PBS. Pelleted cells are now ready for RNA extraction and analysis or can be stored at -80°C.

Recipes

  1. 1% Virkon

    Prepare 1% Virkon by dissolving 5 g of powder in 500 ml of deionised sterile water.

    1% Virkon solution can be aliquoted and stored at -20°C for at least 2 months. Virkon powder can be stored at room temperature.

  2. P/S-PBS

    Prepare 10% vol/vol Penicillin/Streptomycin solution in PBS (P/S-PBS) and aliquot 25 ml into a 100 mm plastic Petri plate.

  3. BSA-PBS

    Prepare 5% BSA solution in sterile PBS (BSA-PBS) and heat-inactivate at 60°C for 60 min before filtering through a 0.45 μm syringe filter.

  4. cDMEM (complete DMEM)

    Prepare cDMEM by adding Penicillin/Streptomycin solution at 1% vol/vol and gentamicin to 50 µg/ml to DMEM. Prepared cDMEM can be stored at 4°C for 2-3 weeks.

  5. Collagenase-cDMEM

    Immediately before dissection, prepare a 0.2% collagenase solution in cDMEM. In the tissue culture hood, filter-sterilise the Collagenase-cDMEM solution using a sterile syringe with a 0.2 μm filter. Consider approximately 2 ml Collagenase-cDMEM for each fish (two fillets) and aliquot into separate 7 ml bijou tubes.

  6. PBSTx

    Prepare PBSTx (0.5%) by diluting 100% Triton X100 in sterile PBS. Solutions containing Triton X-100 can be prepared in advance, but for long term storage, use PBS containing 0.002% sodium azide instead of PBS and wrap the tube/bottle in aluminium foil to protect from light.

  7. Matrigel solution

    Defrost Matrigel stock overnight at 4°C. Dilute it to 1 mg/ml in DMEM and aliquot into 2 ml micro-centrifuge tubes. It is essential to complete this step on ice or the Matrigel will gel and form lumps. Aliquots of diluted Matrigel can be stored at 4°C for up to 2 weeks or frozen at -20°C for longer term storage.

  8. Proliferation Medium (PM)

    Prepare PM by supplementing DMEM with 1% Penicillin/Streptomycin, 10 µg/ml gentamicin and 20% FBS. Pre-warm PM to 28.5°C in the incubator. We recommend preparing PM fresh prior to starting muscle dissection/dissociation and to use within 2 to 3 days.

  9. Differentiation Medium (DM)

    Prepare DM by supplementing DMEM with 1% Penicillin/Streptomycin, 10 µg/ml gentamicin and 2% horse serum. Pre-warm DM to 28.5°C in the incubator. We recommend preparing fresh DM as needed and using it within 2 to 3 days.

Acknowledgments

We thank all members of the Hughes lab and Bruno Correia da Silva and his staff for fish care. This work is supported by grants from the Medical Research Council to S.M.H. (MRC Programme Grants G1001029 and MR/N021231/1) and P.S.Z. (MR/P023215/1 and MR/S002472/1). The present protocol was developed and used in Ganassi et al. (2018) and Ganassi et al. (2020).

Competing interests

The authors declare that they have no competing interests.

Ethics

All procedures were performed on adult zebrafish in accordance with the PPL license held under the UK Animals (Scientific Procedures) Act 1986, and later modifications conformed to all relevant guidelines and regulations. All lines used were reared at King's College London on a 14/10 h light/ dark cycle at 28.5°C, with staging and husbandry as described before (Westerfield, 2000).

References

  1. Alexander, M. S., Kawahara, G., Kho, A. T., Howell, M. H., Pusack, T. J., Myers, J. A., Montanaro, F., Zon, L. I., Guyon, J. R. and Kunkel, L. M. (2011). Isolation and transcriptome analysis of adult zebrafish cells enriched for skeletal muscle progenitors. Muscle Nerve 43(5): 741-750.
  2. Anderson, J. E., Wozniak, A. C. and Mizunoya, W. (2012). Single muscle-fiber isolation and culture for cellular, molecular, pharmacological, and evolutionary studies. Methods Mol Biol 798: 85-102.
  3. Bekoff, A. and Betz, W. (1977a). Properties of isolated adult rat muscle fibres maintained in tissue culture. J Physiol 271(2): 537-547.
  4. Bekoff, A. and Betz, W. J. (1977b). Physiological properties of dissociated muscle fibres obtained from innervated and denervated adult rat muscle. J Physiol 271(1): 25-40.
  5. Bischoff, R. (1975). Regeneration of single skeletal muscle fibers in vitro. Anat Rec 182(2): 215-235.
  6. Blagden, C. S., Currie, P. D., Ingham, P. W. and Hughes, S. M. (1997). Notochord induction of zebrafish slow muscle mediated by Sonic hedgehog. Genes Dev 11(17): 2163-2175.
  7. Brack, A. S., Bildsoe, H. and Hughes, S. M. (2005). Evidence that satellite cell decrement contributes to preferential decline in nuclear number from large fibres during murine age-related muscle atrophy. J Cell Sci 118(Pt 20): 4813-4821.
  8. Buckingham, M. and Relaix, F. (2015). PAX3 and PAX7 as upstream regulators of myogenesis. Semin Cell Dev Biol 44: 115-125.
  9. Cardasis, C. A. and Cooper, G. W. (1975a). An analysis of nuclear numbers in individual muscle fibers during differentiation and growth: a satellite cell-muscle fiber growth unit. J Exp Zool 191(3): 347-358.
  10. Cardasis, C. A. and Cooper, G. W. (1975b). A method for the chemical isolation of individual muscle fibers and its application to a study of the effect of denervation on the number of nuclei per muscle fiber. J Exp Zool 191(3): 333-346.
  11. Davies, M. L., Johnston, I.A. and van de Wal, J. (1995). Muscle Fibers in Rostral and Caudal Myotomes of the Atlantic Cod (Gadus morhua L.) Have Different Mechanical Properties. Physiol Zool 68(4): 673-697.
  12. Froehlich, J. M., Seiliez, I., Gabillard, J. C. and Biga, P. R. (2014). Preparation of primary myogenic precursor cell/myoblast cultures from basal vertebrate lineages. J Vis Exp(86): 51354.
  13. Fukada, S. I., Akimoto, T. and Sotiropoulos, A. (2020). Role of damage and management in muscle hypertrophy: Different behaviors of muscle stem cells in regeneration and hypertrophy. Biochim Biophys Acta Mol Cell Res 1867(9): 118742.
  14. Ganassi, M., Badodi, S., Ortuste Quiroga, H. P., Zammit, P. S., Hinits, Y. and Hughes, S. M. (2018). Myogenin promotes myocyte fusion to balance fibre number and size. Nat Commun 9(1): 4232.
  15. Ganassi, M., Badodi, S., Wanders, K., Zammit, P. S. and Hughes, S. M. (2020). Myogenin is an essential regulator of adult myofibre growth and muscle stem cell homeostasis. Elife 9: e60445.
  16. Hammond, C. L., Hinits, Y., Osborn, D. P., Minchin, J. E., Tettamanti, G. and Hughes, S. M. (2007). Signals and myogenic regulatory factors restrict pax3 and pax7 expression to dermomyotome-like tissue in zebrafish. Dev Biol 302(2): 504-521.
  17. Harper, C. and Lawrence, C. (2011). The Laboratory Zebrafish. Boca Raton: CRC Press.
  18. Hinits, Y., Osborn, D. P. and Hughes, S. M. (2009). Differential requirements for myogenic regulatory factors distinguish medial and lateral somitic, cranial and fin muscle fibre populations. Development 136(3): 403-414.
  19. Hinits, Y., Williams, V. C., Sweetman, D., Donn, T. M., Ma, T. P., Moens, C. B. and Hughes, S. M. (2011). Defective cranial skeletal development, larval lethality and haploinsufficiency in Myod mutant zebrafish. Dev Biol 358(1): 102-112.
  20. Hromowyk, K.J., Talbot, J.C., Martin, B.L., Janssen, P.M. and Amacher, S.L. (2020). Cell fusion is differentially regulated in zebrafish post-embryonic slow and fast muscle. Dev Bol 462(1): 85-100.
  21. Johnston, I. and Altringham, J. (1988). Muscle contraction in polar fishes: experiments with demembranated muscle fibres. Comp Biochem Physiol 90B(3): 547-555.
  22. Johnston, I. A., Abercromby, M., Vieira, V. L., Sigursteindottir, R. J., Kristjansson, B. K., Sibthorpe, D. and Skulason, S. (2004). Rapid evolution of muscle fibre number in post-glacial populations of Arctic charr Salvelinus alpinus. J Exp Biol 207(Pt 25): 4343-4360.
  23. Katz, B. (1961). The termination of the afferent nerve fibre in the muscle spindle of the frog. Philos Trans R Soc Lond B Biol Sci 243(703): 221-240.
  24. Mahalwar, P., Walderich, B., Singh, A. P. and Nüsslein-Volhard, C. (2014). Local reorganization of xanthophores fine-tunes and colors the striped pattern of zebrafish. Science 345(6202): 1362-1364.
  25. Mauro, A. (1961). Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 9: 493-495.
  26. Moyle, L. A. and Zammit, P. S. (2014). Isolation, culture and immunostaining of skeletal muscle fibres to study myogenic progression in satellite cells. Methods Mol Biol 1210: 63-78.
  27. Osborn, D. P. S., Li, K., Cutty, S. J., Nelson, A. C., Wardle, F. C., Hinits, Y. and Hughes, S. M. (2020). Fgf-driven Tbx protein activities directly induce myf5 and myod to initiate zebrafish myogenesis. Development 147(8): dev184689.
  28. Pipalia, T. G., Koth, J., Roy, S. D., Hammond, C. L., Kawakami, K. and Hughes, S. M. (2016). Cellular dynamics of regeneration reveals role of two distinct Pax7 stem cell populations in larval zebrafish muscle repair. Dis Model Mech 9(6): 671-684.
  29. Purohit, G. and Dhawan, J. (2019). Adult Muscle Stem Cells: Exploring the Links Between Systemic and Cellular Metabolism. Front Cell Dev Biol 7: 312.
  30. Relaix, F. and Zammit, P. S. (2012). Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage. Development 139(16): 2845-2856.
  31. Rosenblatt, J. D., Lunt, A. I., Parry, D. J. and Partridge, T. A. (1995). Culturing satellite cells from living single muscle fiber explants. In Vitro Cell Dev Biol Anim 31(10): 773-779.
  32. Westerfield, M. (2000). The Zebrafish Book - A guide for the laboratory use of zebrafish (Danio rerio). University of Oregon Press.
  33. Zammit, P. S., Golding, J. P., Nagata, Y., Hudon, V., Partridge, T. A. and Beauchamp, J. R. (2004). Muscle satellite cells adopt divergent fates: a mechanism for self-renewal? J Cell Biol 166(3): 347-357.
  34. Zhang, H. and Anderson, J. E. (2014). Satellite cell activation and populations on single muscle-fiber cultures from adult zebrafish (Danio rerio). J Exp Biol 217(Pt 11): 1910-1917.

简介

[摘要]骨骼肌终生产生力量,需要维护和修理以确保效率。居民肌肉干细胞(MuSCs),被称为卫星细胞的人口,停留小号成人myofibres的基底层下方,有助于小号到两个肌肉生长和再生。在暴露于激活信号后,MuSCs 增殖产生成肌细胞,该成肌细胞分化和融合以生长或再生肌纤维。这种肌源性进展类似于胚胎发生过程中肌肉形成和发育的各个方面。因此,在MuSCs及其相关myofibres的研究允许的探索 肌肉干细胞生物学,包括肌肉形成、维持和修复的细胞和分子机制。由于 MuSC 生物学的大多数方面已在啮齿动物中进行了描述,因此它们与其他物种(包括人类)的相关性尚不清楚,并且将受益于与替代脊椎动物系统的比较。在这里,我们描述了一个过程的成年斑马鱼myofibres的隔离和免疫标记或文化,使两者肌纤维特性和麝香生物学检查体外。分离myofibres可以分析形态特征,如在肌纤维体积和myonuclear域,以评估所述肌肉生长的动力学。经典干细胞标记或报告基因的免疫标记可识别分离的肌纤维上的 MuSC,用于细胞/分子研究。此外,可以接种可行的肌纤维,允许 MuSC 肌生成和分析原代祖细胞的增殖和分化动力学。总之,我们提供了一个用于研究脊椎动物肌生成的羊膜模型的比较系统,它将揭示 MuSC 生物学的基本遗传和细胞机制,并为水产养殖提供信息。


图文摘要:
成年斑马鱼肌肉干细胞的肌纤维分离和培养示意图。


[背景]骨骼肌肉组织通过肌纤维(包含数百个有丝分裂后肌核的高度特化合胞细胞)的协调收缩提供终生的身体支持和运动。Myofibres不断适应于外源性和内源性刺激在由于常驻肌肉干细胞(MuSCs),也被称为卫星细胞,位于部分最myofibres的基底层下方(卡茨,1961;毛罗,1961; Relaix和扎米特,2012; Purohit 和 Dhawan ,2019 年)。响应运动或损伤,静止的 MuSC 迅速激活并成为称为成肌细胞的肌肉祖细胞 (MPC) ,其增殖、分化并融合为预先存在的多核肌纤维或彼此融合以形成新的肌纤维(Fukada等人,2020 年) ) 。啮齿动物肌纤维的分离和相关 MuSC 的培养是探索肌肉干细胞生物学的成熟工具,不仅提供了对 MuSC行为的理解以及对静止、激活、增殖、自我更新和分化的调节(Zammit等人,2004),但也产生了对胚胎肌肉发育和成人肌纤维生长和维持的见解(Buckingham 和 Relaix ,2015)。然而,小鼠 MuSC 生物学的潜在机制可能并不完全类似于人类肌生成中发现的机制。因此,研究成体/MuSC 肌生成的替代脊椎动物模型对于巩固脊椎动物肌肉祖先机制的发现是可取的。值得注意的是,尽管斑马鱼和羊膜动物在肌生成中共享的分子途径存在大量重叠(Hammond等人,2007 年;Hinits等人,2009 年和2011 年;Ganassi等人,2018 年;Osborn等人,2020 年),补充研究斑马鱼成年肌肉稳态的影响仅限于大块肌肉的机械研磨,阻碍了肌原产量的纯度(Alexander等人,2011 年;Froehlich等人,2014 年)。在鱼成人肌纤维隔离已经允许了他们的物理或收缩特性的研究(约翰斯顿和奥尔特灵厄姆1988年,1995年戴维斯;约翰斯顿等人,2004年),以及一些研究最近利用它来研究斑马鱼麝香生物学(安德森等人。, 2012; Zhang 和 Anderson , 2014; Ganassi等人, 2018和2020) 。

在这里,我们描述了如何通过酶消化和对成年斑马鱼的躯干肌肉进行精细研磨来分离单个可行的肌纤维和相关的 MuSC。该方法是从标准鼠标协议适配(Bischoff的,; Cardasis和Cooper 1975 ,1975和1975b ;贝科夫和贝兹,1977 a和1977b ;布拉特等人,1995;和莫伊尔扎米特,2014) 。我们解释了如何接种肌纤维以通过体外肌生成研究 MuSC 的激活和进展,确保几乎纯的肌源性群体(Ganassi等人,2018 年和2020 年)。我们的协议提供了一个合适的工具箱,用于比较分析脊椎动物的成体肌细胞生成,最近开发并用于探索转录因子肌细胞生成素在成体 MuSC 激活、增殖和分化中的功能(Ganassi等人,2018和2020)。肌纤维分离,培养,并分析从成年鱼利用了斑马鱼的模型的优点,如慢速和快速myofibres的空间隔离有利于纤维类型特异性的研究(Blagden等人,1997年; Pipalia等人,2016; Hromowyk等人,2020 年),并提供对水产养殖有用的见解。作为经典啮齿动物程序的替代方法,鱼 MuSC 的分析还提供了一个独立的基准来验证使用啮齿动物模型识别的遗传和细胞机制。因此,应用我们的技术,以成年斑马鱼的肌肉有利于理解遗传,分子的潜力,维持并适应人体肌肉组织和细胞机制。

该方法是简单,有效,和成本效益的,并且允许所述的研究1)肌纤维的特性离体,2)MUSC衍生的成肌细胞/肌管体外和3)的成体肌肉形成,发展机制,和维护。

关键字:斑马鱼, 肌肉纤维, 肌纤维, 干细胞, 骨骼肌, 肌核, Pax7, MuSC, 成人, 卫星细胞


材料和试剂

 
材料- [R equired为d issection和d的issociation一个dult米uscle
深培养皿(150 毫米和 100毫米)无菌,细胞培养级(康宁,目录号:430599 和 430167)
玻璃巴斯德移液器(22 cm),无菌(Volac,目录号:D812)
0.45微米,0.2微米的无菌注射器过滤器(赛默飞,目录号小号:15216869和15206869)
无菌注射器,50 ml(Terumo,目录号:SS+50ES1)
铝箔
Bijou 管,7 毫升
三卡因甲磺酸盐(MS-222)溶液(Sigma - Aldrich,目录号:E10521)
70% 乙醇溶液(在去离子水中)(70% EtOH)(绝对乙醇;Sigma - Aldrich,目录号:1024282500)
5% 牛血清白蛋白(BSA)(粉末,Sigma - Aldrich,目录号:A7906)
来自溶组织梭菌的胶原酶(Sigma - Aldrich,目录号:C0130)
Dulbecco 改良 Eagle 培养基(DMEM),高葡萄糖,GlutaMAX,丙酮酸盐(ThermoFisher,目录号:31966)
磷酸盐缓冲盐水 Ca 2+和 Mg 2+游离(PBS),无菌(Oxoid,目录号:BR0014G)
青霉素和链霉素溶液(Sigma - Aldrich,目录号:P0781)
1% Virkon 溶液(在去离子水中)(粉末,3S Healthcare)(见配方)
P/S-PBS (见食谱)
BSA-PBS (见食谱)
cDMEM(完整的 DMEM)(见食谱)
胶原酶-cDMEM (见食谱)
 
材料- [R equired为米yofibre和麝香衍生细胞我mmunolabelling
盖玻片 50 毫米× 22 毫米(学院,目录号:400-04-17)
载玻片(Fisher,目录号:1157-2203)
水晶般透明的塑料微量离心管,2 ml(Starlab,目录号:S-1620-2700)
透明指甲油
多聚甲醛(PFA)溶液,PBS(PFA-PBS)中的4%(Alfa Aesar,目录号:J61899)
液体阻滞剂超级纸笔(Pyramid Innovation,目录号:R62002-E)
Triton X-100 洗涤剂溶液(Sigma - Aldrich,目录号:X100)
鸡抗GFP(RRID:AB_300798 ;Abcam,目录号:13970;使用1:400)
山羊抗-鸡IgY(H + L),Alexa氟® 488(RRID:AB_2534091 ;赛默飞世尔科技,产品目录号:A11032,使用1:1000)
Hoechst 33342 溶液(ThermoFisher,目录号:H3570,使用 1:1000)
正常山羊血清(NGS)(安捷伦,目录号:x0907)
基于甘油的封固剂(安捷伦,目录号:50001)
PBST x (见食谱)
 
材料- [R equired为米yofibre和MUSC来源的细胞培养物
24 孔板细胞培养级(ThermoFisher,目录号:142475)
胎牛血清(FBS),热灭活(ThermoFisher,目录号:10500-064)
马血清(HS)(ThermoFisher,目录号:26050088)
Matrigel(康宁,目录号:354263)
5-乙炔基-2'-脱氧尿苷(EdU)溶液(来自 Click-iT EdU 试剂盒;ThermoFisher,目录号:C10646)
庆大霉素(Gibco,目录号:15750-060)
基质胶溶液(见配方)
增殖培养基(PM)(见配方)
分化培养基(DM)(见食谱)
 
设备
 
解剖和d的issociation一个dult米uscle
组织培养罩或层流柜
组织培养箱(加湿,28.5°C,5% CO 2 )
软木解剖板(宜家,目录号:870.777.00)
解剖金属针
细镊子,一对(Idealtek,No. 5A.s)
无菌一次性手术刀 No. 10(Swann-Morton,目录号:0501)
本生灯
菱形笔(VWR,目录号:201-0392)
带透射照明的解剖显微镜(Zeiss Stemi SV6 和 Leica M50)
 
软件
 
图像分析:斐济;NIH ( www.Fiji.sc )
数据展示:Graph P ad Prism 8 ( https://www.graphpad.com/scientific-software/prism/ )
 
程序
 
肌肉解剖
在可能的情况下,在组织培养罩或层流柜中的无菌条件下执行步骤。
Euthani小号E中的鱼通过在冰冷的0.3毫克/毫升三卡因溶液浸泡。将鱼浸入冷冻的三卡因溶液中,等分到 50 毫升管中所需的时间。为了尽量减少动物的痛苦,在孵化过程中将管子放在冰上。请注意,在持续时间牛逼ricaine孵化必须凭经验确定,根据鱼的大小和所描述的时代之前(韦斯特,2000;哈珀和劳伦斯,2011) (见注1 )。
从三卡因溶液中取出鱼尸体,然后将其浸入100毫米培养皿中的 25毫升1% Virkon(参见配方 1 )溶液中。我ncubate 5分钟杀死细菌和真菌。
使用干净的镊子将鱼尸体转移到一个新的 100毫米盘中,其中包含 25 毫升P/S-PBS(参见配方 2 )并孵育 5分钟。
将鱼尸体转移到新的 100 毫米培养皿中。使用一次性手术刀去除鳞片。为了提高缩放效率,将刀片垂直于鱼体的前后轴放置,并从尾部到头部轻轻擦洗皮肤表面(见注 2 )。
将除垢后的尸体洗入一个新的 100毫米盘中,其中含有 25 毫升新鲜的 P/S-PBS 5 分钟。同时,用70% EtOH仔细擦拭解剖金属针、软木板和细镊子,以减少污染的机会。 
将鱼尸体移到新的 100毫米盘子中,用布轻轻擦干残留的 P/S-PBS,并在两侧喷洒 70% 乙醇。
将鱼移到解剖软木板,并放置一个穿过鳃盖后面组织的针,第二个针穿过尾鳍基部前面的组织(图 1A )。
使用手术刀尽可能靠近鱼体切割鳍。除去翅片可以被处理,以提取用于基因分型鱼(基因组DNA图小号1A和1个乙)(见注3 )。
沿着尸体的腹侧做一个弯曲的切口,以方便使用刀片和细钳进行内脏(图 1A )。
此时,可以收集屠体的不同部分以进行所需的分析。正如所指示图1B :1)散热片是有用的用于回顾性基因分型,ii)所述尾尖端附近的肌肉区通常通过在解剖针损坏,但可用于整个肌肉RNA /蛋白质分析,iii)所述相邻5个15mm截面肌肉可以冷冻保存用于组织学分析和iv) 大部分躯干肌肉组织被处理用于肌纤维分离。
使用手术刀在鳃盖后面的皮肤上做一个轻切口,并垂直于前后轴,小心地避免切开下方的肌肉。使用细镊子轻轻捏住并沿切口边缘提起皮肤。小心地抓住并将皮肤拉向鱼尾,以暴露下面的肌肉(图 1C )。
继续轻轻拉动,直到到达靠近尾部的销(图 1C )。慢肌牢固地附着在上面的皮肤上,因此要非常轻柔地拉动,以免损坏慢肌纤维。现在应该暴露大部分躯干肌肉组织。
使用相同的步骤,从去除皮肤的对侧。
当剥皮完成后,取消固定鱼和旋转90°到它的背面,以使腹侧(腹部)点向上朝向操作者(图小号1D- 1 ˚F )。
重新销的鱼在新位置上的公告板,使用一个销穿过的下颚和头和第二在尾的基部。在V ertebral列应通过在腹部开口(可见和可访问图小号1E和1 ˚F )。
使用刀片沿整个前后轴在脊柱右侧切割以创建两个肌肉圆角,一个带有相关的脊柱和脊髓,另一个没有。重要的是将手术刀倾斜,使其尖端指向背中线,穿透靠近脊柱的肌肉组织的最前部(图 1E)。向后拉刀片,直到到达尾销,将肋骨留在圆角中(图 1E')。沿着柱子切割时停止或犹豫会导致不同的圆角厚度并损伤最内侧的肌肉。
用手术刀取下鱼头,完全松开两块肌肉鱼片。鱼片显示慢速和快速肌肉隔室(图 1G )。脊髓应该在右侧圆角中可见,并且可以用手术刀移除,但这不是必需的。我们通常不会去除它以减少对周围肌肉组织的可能损害。
肌肉解剖应需要 30 分钟,并且可以在多条鱼上并行进行。
 
肌纤维分离和分离
1.每条鱼用BSA-PBS 溶液冲洗一个 150毫米和两个 100毫米的新无菌培养皿(参见配方 3 ),以防止肌纤维粘附在培养皿上。去除多余的 BSA-PBS 溶液,分别在 150毫米和 100毫米培养皿中加入 25毫升和 10毫升完整的 DMEM(cDMEM,参见配方 4 )。将菜肴放入 28.5°C 5% CO 2培养箱中至少 30分钟,让 cDMEM 升温。          
2.将新鲜切下的鱼片放入装有胶原酶-cDMEM 溶液的 bijou 管中(参见配方 5和注 4 ),松松地盖上盖子,在 28.5°C 的 5% CO 2培养箱中孵育 120 分钟,偶尔(每 30 分钟)非常轻柔地旋转管子(图 1H )。      
3.同时,使用金刚石笔得分两个玻璃巴斯德每鱼移液管和创建具有直径开口小号的约1和3-4毫米,分别(图1I )。使用本生灯熔化开口周围的玻璃,以平滑抛光任何锋利的边缘(图 1I和视频 1 )。通过在铝箔上盘旋移液管边缘来测试抛光。Ñ ø剪切/撕裂应来制造。将准备好的玻璃移液管快速火焰消毒,用铝箔包裹并储存在组织培养罩中直至使用。      
 
 
视频 1. 玻璃巴斯德吸管切割和热抛光过程
 
4.孵育完成后,将 bijou 管放入组织培养罩中。良好的消化的肌肉看起来为轻微的溶胀,并且在显微镜下观察,发状myofibres周围出现的肌肉质量的边缘脱落(图小号1J和J”和见注5 )。还收集150毫米的菜用温水cDMEM从所述的培养罩培养箱,并将其放置。      
5.从 bijou 管中轻轻倒出并丢弃大部分胶原酶-cDMEM 溶液。快速倒置 bijou 管,将肌肉鱼片倒入含有 cDMEM的 150毫米培养皿中。将带鱼片的培养皿放回培养箱中 20-30 分钟。这允许肌肉休息并稀释胶原酶,促进酶的失活。      
6.如有可能,将解剖显微镜置于培养罩中;否则,请使用远离门窗和气流或其他污染源的清洁区域。收集带有圆角的 150毫米盘子并放置在显微镜的镜头下。      
7.用 BSA-PBS 溶液冲洗热抛光玻璃移液管以防止肌纤维粘附。      
8.使用具有移液管的直径较大(〜3-4毫米),直接cDMEM到圆角反复至少10分钟,上下移液排出液体(的连续流图1K )。组织离解可以通过仔细使鱼片一次或进出玻璃吸管的两次来增强,但不是连续,因为这会损坏myofibres和减少的最终产率。Myofibres是将从肌肉本体被释放可见毛发状结构(图1K”和1大号)。      
9.继续研磨过程直到大部分肌纤维被释放。该过程还将导致碎片的释放,包括脂肪滴和过度收缩的肌纤维(图 1L ),这将增加培养基的浊度。如果研磨时间延长,请在 28.5°C、5% CO 2下再孵育5-10分钟,以重新平衡培养基的温度和 pH 值。如果培养基长时间低于生理温度范围(22-29°C),肌纤维将过度收缩并死亡。        
10.将 150 毫米板放回培养箱中 10-15 分钟,让释放的肌纤维静止并沉入底部。   
11.使用与玻璃吸管的直径较小(〜1mm)的,小心收集完好myofibres,并将它们转移到100毫米的菜新鲜cDMEM(图1K” )。如果需要,可以进一步处理剩余的肌肉块以增强残留肌纤维的释放,但不能超过 30-60 分钟,因为这会降低残留肌纤维的活力。   
12.将含有清洁过的肌纤维的 100 毫米培养皿放回培养箱中再放置 10-15 分钟,让它们静置并沉入底部。   
13.活肌纤维呈现半透明且表面光滑(图 1M )。如果需要,可以将肌纤维转移到一个新的含有 cDMEM 的 100 毫米盘子中以进一步清洁。如果显著碎片仍然存在于盘中,重复小号TEPS乙1 1和乙12。   
14.整个肌肉解剖过程需要 180 分钟。   
 
 
图 1.成年斑马鱼肌纤维的解剖和分离。A. 8个月的代表性图片-老成年斑马鱼描绘销定位(橙色点),以锚鱼尸体在解剖板。红色虚线表示切割以去除鳍并进行腹侧切口以进行内脏。乙。可从鱼的不同部位进行的分析总结(提取;提取)。Ç 。蓝色虚线和箭头表示用钳子剥皮的皮肤切口、捏和拉方向的位置。D.剥皮后,将尸体背侧旋转 90°,向上暴露腹侧切口。红色虚线和橙色点分别表示切割方向和引脚定位。E,E' 。鱼片切割过程中的手术刀角度和切割方向图。红色虚线箭头表示朝向背中线 ( E ) 的倾斜度和朝向尾销( E' ) 的切割方向。黑色虚线显示脊髓(E' )的位置。˚F 。腹侧切口向上的视图。红色虚线和箭头表示切肉的方向,以脊髓为指导(白色虚线)。指示了前后方向(h;头,t;尾)。格。在解剖的鱼片 (h; head, t; tail) 中可以看到慢 (暗箭头) 和快 (亮箭头) 肌肉部分。H . 鱼片与胶原酶-cDMEM 溶液在 bijou 管中在 28.5°C 下孵育 120 分钟。我。巴斯德玻璃移液管经过切割和热抛光以获得两个具有宽(3-4 毫米,粉红色)和小直径(1 毫米,紫色)孔径的移液器,分别用于肌肉研磨和单肌纤维处理。紫色(切割 1 毫米开口)或粉红色(切割 3-4 毫米开口)虚线表示每个移液器上的切割位置,用钻石笔和火焰加热抛光。切割和边缘热抛光所需的结果显示在底部图片中(参见视频 1 )。J,J' 。在胶原酶-cDMEM 溶液中孵育 120 分钟后肌肉鱼片的代表性图像( J )。注意在肌肉块边缘移动的毛发状肌纤维(J' ,绿色放大区域)。还标明了肋骨和脊髓的位置。K,K' 。鱼片研磨 ( K ) 和单个肌纤维分离和洗涤 ( K' ) 的示意图,估计持续时间以分钟为单位。升。洗涤过程中单个肌纤维的代表性图像。黄色箭头表示完整的存活myofibres ,而品红慈姑星号表示损坏或hypercontracted myofibres ,分别。鱼准备/肌肉解剖 ( CF ) 和肌纤维分离/隔离 ( GJ ) 的估计时间在面板旁边报告。米。肌肉解剖和分离后具有代表性的单个可行肌纤维。
 
分离肌纤维的分析
分离的肌纤维现在可以进行分析,例如形态测量。肌纤维可以在固定前使用明场显微镜拍摄,这样可以排除过度收缩或受损的肌纤维。根据所使用的显微镜,损坏被尚未完全hypercontracted出现短myofibres和更不透明的,具有粗糙和不规则的表面(图1L )。
核计数和随后的分析可能需要肌纤维固定。在显微镜下,使用较小直径的BSA-PBS 预冲洗玻璃移液管(图s 1I和 1 K' )收集分离的肌纤维并将它们放入已用 BSA-冲洗过的 2 ml 透明圆底微量离心管中PBS 以防止肌纤维粘连。轻轻旋转培养皿,将所有肌纤维聚集在盘子中央,以减少用肌纤维收集的 cDMEM 培养基的体积。我们建议将每管肌纤维的数量限制为 40,以避免结块和可能的损坏。
让收集微量离心管在室温下直立 5 分钟,让肌纤维沉入管底。
小心地取出上面的介质的用移液管myofibres并通过温和地流下倾斜管的侧面用1ml的PBS溶液4%PFA(PFA-PBS)的补充管。在室温下孵育 10-15 分钟。
去除 PFA-PBS 溶液并轻轻补充 1.5 ml PBS 以清洗肌纤维。孵育 5 分钟,然后用新鲜的 PBS 重复洗涤。可以添加赖氨酸或 BSA 以更有效地抑制 PFA。固定的肌纤维可以在 4°C 下储存至少 2 周。
取出 PBS,清洗并更换为新鲜制备的 Hoechst 33342 染料溶液,用 PBS 稀释以染色肌纤维核。孵育15分钟,并用新鲜的PBS替换(如在小号TEP Ç 5 )。肌纤维现在可以安装在载玻片上进行详细分析。
使用防水PAP笔勾勒出一个矩形区域(大小取决于盖玻片的大小,例如,50毫米× 22毫米)在几个载玻片上。在显微镜下,使用干净的小直径移液器,用 BSA-PBS 预冲洗,从 2 ml 管中收集肌纤维,并将它们转移到准备好的载玻片上。我们建议将每张载玻片的肌纤维限制为 10/15,以方便处理。
用移液器从载玻片中尽可能多地去除 PBS,以减轻肌纤维粘附并降低损坏和/或丢失的风险。A 200微升微量移液器尖端包裹在铝箔和预浸渍在BSA-PBS可被用于去除PBS后小心复位myofibres。我们建议尽快,因为残留的液体以及浸入的肌纤维会迅速变干。
将两/三滴基于甘油的安装介质放在载玻片上,放置 50 mm × 22 mm 的盖玻片,在载玻片上的边缘接触甘油,然后轻轻降低盖玻片以避免捕获可能错误定位或扫走的气泡肌纤维。等待 5 分钟,让安装介质在盖玻片下方扩散。
密封盖玻片并通过刷上少量指甲油将其固定到载玻片上,首先在角落,然后密封边缘。
安装的肌纤维可以使用落射荧光或共聚焦显微镜拍摄。
使用成像软件,在每个肌纤维的至少两个不同位置测量肌纤维宽度(直径)。同时,使用 Hoechst 测量肌纤维长度并计算细胞核数(图 2A )。
肌纤维体积可与报道的公式来计算所述数据分析部分和图2A 。电子在肌纤维体积的xample生成素+/-从成人加纳西等。( 2020)显示 (图 2B )。或者,共聚焦扫描可以提供完整的肌纤维形态轮廓(参见Ganassi等人,2020 年)。
 
肌纤维免疫标记
可以处理固定的肌纤维(步骤C5 )以进行免疫标记。在这里,我们部署了转基因鱼TgBAC(pax7a:GFP) t32239Tg (Nüsslein-Volhard C.;MPI Tübingen)(Mahalwar等人,2014 年),并提供了使用抗 GFP 抗体检测 MuSC 的模板免疫标记协议。尽管由pax7a:GFP转基因编码的 GFP 荧光确实抵抗 PFA 固定,但我们建议使用抗 GFP 抗体进行增强,特别是如果与多种抗体和荧光团共标记(Ganassi等,2020)。
用BSA-PBS预冲洗过的小直径玻璃移液管从固定的肌纤维中取出 PBS,并用PBS 中的0.5% Triton-X100 清洁剂(PBST x ;参见配方 6 )替换。孵育15分钟,以permeabilise的两个肌纤维细胞膜小号和相关MuSCs。
去除 PBSTx 并在 PBS 中轻轻添加10% 正常山羊血清 (NGS)的封闭溶液以阻止非特异性抗体结合。孵育至少 30分钟,偶尔倾斜试管(见注 6 )。或者,可使用 PBS 溶液中的 5% NGS 孵育 1 小时。
通过在含有 2% NGS 的0.1% Triton-X100 洗涤剂 PBS 溶液(PBST x 0.1)中稀释抗 GFP 一抗来制备抗体溶液。从管中取出封闭液,轻轻加入一抗溶液。在 4°C 下孵育过夜(16 小时)。
去除一抗溶液并用新鲜的 PBST x 0.1代替以清洗肌纤维 5 分钟(见注 7 )。一抗溶液可以在 4°C 下储存并在一周内可靠地重复使用(如果在 PBS 中加入 0.002% 叠氮化钠,可能会更长)。
使用 PBST x 0.1清洗肌纤维 3 次,每次 5 分钟,偶尔轻轻倾斜试管。
稀荧光染料缀合的(例如,Alexa氟488)的第二抗体和Hoechst 33342染料溶液(10μg/ ml的终浓度)在PBST X 0.1孵育在室温下至少60分钟,避光,偶有管涡旋。如果在 PBS 中加入 0.002% 叠氮化钠,二抗溶液可以在 4°C 下避光保存,并在一周或更长时间内可靠地重复使用。
如在所述的转印myofibres到准备好的载玻片上并安装在盖玻片下小号TEPS C7- Ç 11.一种Ë一个的xample pax7a:GFP MUSC上免疫标记的GFP一个肌纤维被示出在图2C和中可以找到(加纳西等al. , 2020) 。将载玻片储存在 4°C 下避光;GFP 荧光可持续长达 14 天。
 
肌纤维衍生的 MuSC 培养和免疫标记
用Matrigel溶液冲洗,涂上所需数量的 24 孔板孔(参见配方 7 )。确保完全覆盖每个孔的表面。立即使用无菌移液管去除多余的溶液,并将 Matrigel溶液放回4°C 以避免早熟凝胶。放置所制备的板在一个28.5℃,5%CO 2培养箱中30-45分钟,以允许基质胶凝胶化。
准备增殖培养基(PM;参见配方 8 )并在培养箱中在 28.5°C 下预热,然后在每个 Matrigel 包被的孔中分装 200 µl。
用移液器将肌纤维转移到含有 5 ml 40% FBS-cDMEM 溶液的新 100 mm 培养皿中。肌纤维的每次转移都会导致来自稀释血清浓度的原始培养皿中近 150-200 µl cDMEM 的伴随结转。在的端部的转印中,最终100mm皿现在包含约10的10ml DMEM与大约20%的FSB,从而模拟PM。
轻轻旋转 100 毫米培养皿以在其中心收集肌纤维。使用预先冲洗过的小直径玻璃吸管 BSA-PBS将大约 90-100 个新鲜分离的肌纤维转移到每个 Matrigel 涂层井中。通过将板放置在平坦的表面上并在类似交叉的正交方向横向移动几次,确保肌纤维均匀分布在井中。
将 24 孔板放入培养箱中,不受干扰地培养肌纤维至少 48 小时。在最初的 24 小时内,肌纤维很容易脱落,影响 MuSC 的激活、增殖、迁移和粘附到培养板上。即使打开/关闭培养箱门也会引起振动,从而使肌纤维脱落。
虽然一小部分 MuSCs 在电镀后 24 小时内激活,从肌纤维上迁移并粘附在基质胶涂层上(见注 8 ),但我们建议在对 MuSC 进行分析之前等待 48 小时。可以在培养中跟踪 MPC/成肌细胞,并随着时间的推移检测增殖或分化能力(图 2D )。
在 48 小时后,可以用抗结蛋白抗体对细胞进行免疫标记,以确认生肌群的纯度。中号yofibre电镀通常会产生在培养物(几乎是纯生肌祖图2E) 。Ť他原始数据中介绍图4图1的补充(加纳西等人,2020) ,和一个类似的计算可以在补充图6G中找到(加纳西等人,2018) 。细胞固定描述如下(步骤E12 )。
可以在所需时间点收集细胞,用于通过 RT-qPCR 进行 RNA 提取和基因表达分析(参见Ganassi等人,2020 年的注释 9和图 4 )。
使用 5-乙炔基-2'-脱氧尿苷 (EdU) 分析 MuSC 增殖最好在肌纤维电镀后 2 天(48 小时)内进行。在新鲜预热的 PM (EdU-PM) 中将EdU 稀释到 10 μM的最终浓度。 
从培养物中取出 PM,并用新鲜制备的 PM 用力冲洗两次,以去除电镀的肌纤维。此时,大多数肌纤维应漂浮或松散地粘附在基质胶上,因此很容易去除。
去除 PM 并用 PBS 快速冲洗两次。用 EdU-PM 溶液替换 PBS,并在 28.5°C 5% CO 2培养箱中放置 2-8 小时。温育持续时间可根据被改变的实验设计; 此处显示的示例是指 8 小时处理。
孵化结束时,去除 EdU-PM 溶液,用 PBS 剧烈洗涤两次,并用 PFA-PBS(PBS 中的 4% PFA)固定 15 分钟。如果下一个步骤是免疫标记,如上所述继续用于myofibres(小号TEPS D2- d 6 )。
在PBST最后一次洗涤后X 0.1,执行点击化学揭示的EdU掺入以下的制造商的说明。
取出 PBS,清洗并更换为新鲜制备的 Hoechst 33342 染料溶液,在 PBS 中稀释以染色细胞核。孵育 15 分钟。
洗涤两次,用新鲜的PBS,然后补充各孔用300μl的PBS中。现在可以使用倒置落射荧光显微镜对细胞进行可视化(图 2F )。用含有 0.002% 叠氮化钠的 PBS 代替 PBS 以防止微生物生长,可以将细胞储存在 4°C(2-4 周)。
MUSC / MPC增殖的动态可以随时间跟踪,如报道图2F (改编自图小号4E和4 ˚F [加纳西等人,2020 ] )。
也可以诱导斑马鱼成肌细胞分化以评估肌原性程序。从初始电镀 96 小时后,去除 PM 并用无菌 PBS 清洗两次以消除血清残留物。去除 PBS 并在每个孔中加入 500 µl 分化培养基(DM;参见配方 9 )。每 48 小时更换 DM。我们之前通过在低血清培养基中培养斑马鱼原代成肌细胞 5 天来评估肌原性分化,然后再对肌球蛋白等结构成分进行免疫标记(图 2D )。图2G分化的肌管示出了5天,共免疫标记用于使用MF20和A4.1025伴随肌球蛋白重链(MyHC的)(Blagden等人,1997)并用Hoechst 33342复以评估分化和细胞融合(原始数据是在图8中加纳西。等人,2018)以及图4 -增补1E (加纳西等人,2020) 。
 
 
图 2.分离的肌纤维和培养的 MuSC 的分析。A.计算肌纤维体积的肌纤维平均直径(肌纤维宽度,红色箭头虚线)和单位长度(蓝色箭头虚线)测量图。Avg,表示跨肌纤维长度(D1 和 D2)的直径测量的算术平均值。乙。8个月的实施例的结果-旧成人肌纤维体积(改编自加纳西等人,2020 )。符号代表从20平均值-从每个的三个不同的鱼30个myofibres。Ç 。TgBAC(pax7a:GFP) t32239Tg ( pax7a:GFP ) 肌纤维免疫标记为 GFP(绿色)显示了靠近肌纤维末端的 MuSC(绿色矩形,在右侧放大)的位置,肌纤维核用 Hoechst 33342(白色)复染。d 。肌纤维电镀、MuSCs/成肌细胞增殖(PM;增殖培养基)、扩张和肌管分化方案(DM;分化培养基)的示意图,具有指定的时间、培养基变化和分析。乙。肌纤维电镀两天后结蛋白+ (成肌细胞)的细胞分数的示例计算。˚F 。MuSC 衍生的成肌细胞可以在增殖培养基中的肌纤维电镀两天后进行 EdU 脉冲(红色)。细胞核用 Hoechst 33342(蓝色)复染。格。在分化培养基中培养 5 天后,含有 MyHC(红色)的分化多核肌管或单核细胞的代表性图像,彩色箭头表示同一细胞内的细胞核(来自Ganassi等人,2020 年)。青色虚线矩形表示右侧面板中的放大区域。细胞核用 Hoechst 33342(白色)复染。融合指数的示例结果(改编自Ganassi等人,2018)。所有的图表报告平均值±SEM ,和S ymbols代表生物复制。
 
数据分析
 
使用测量的平均肌纤维宽度(平均直径)和长度来计算肌纤维的体积,如下公式:
[(长度× 𝜋 ) × [(平均直径/2) 2 ](图2A ) ,
其中“平均”是直径测量的算术平均值在至少沿两个不同位置的肌纤维长度。可以将值与选择的统计分析(例如,未配对的双尾t检验)进行比较。图形是在 Graph P ad Prism 8 中生成的(参见软件)。
肌纤维体积和细胞核数可以结合使用以下公式计算肌核域:[(长度× 𝜋 ) × [(平均直径/2) 2 ]/肌纤维细胞核数]。或者,可以使用平均肌纤维宽度和长度来计算肌纤维的表面积,公式如下:长度× 𝜋 ×平均直径。表面积域大小 (SADS),即每个肌纤维核占据的名义 SA,使用以下公式计算:SA/肌纤维核数。Ganassi等人提供了计算示例。( 2020)或在Brack等人中。( 2005)用于小鼠肌纤维。
 
笔记
 
对于重 0.3-0.6克的斑马鱼,在冰冷的 0.3 毫克/毫升三卡因溶液中孵育后,通常会在 5-10分钟内达到安乐死。然而,这只是指示性时间,必须根据经验和当地指南确定。
重要的是要进行彻底sterili小号机电设备及解剖工具,以避免微生物污染。
去除鱼翅不是必需的,但有助于处理并降低微生物污染的风险。
这是批量测试更换试剂,如胶原酶重要,对现有的,优化解小号编组件。胶原酶的批次中存在不同数量的蛋白酶,但如前文所述,具有约 53 U 中性蛋白酶和约 0.6 U梭菌蛋白酶的胶原酶是理想的(Rosenblatt等,1995)。
成年(8-15 个月大)斑马鱼躯干肌肉通常在2 小时后被消化。尽管更长的温育(≥ 3 ħ )有一个上肌纤维生存能力边际效应,更短的孵育可减少消化效率,阻碍了可行的肌纤维隔离。精确时间取决于鱼的年龄和大小以及所用胶原酶批次的活性,应根据经验确定。
用于封闭的正常血清应来自产生二抗的物种。
Myofibres可与染色荧光染料缀合的毒素来检测所述使用亚细胞结构,诸如丝状肌动蛋白(F-肌动蛋白)鬼笔环肽(赛默飞,A12379)或神经肌肉接头(乙酰胆碱受体),使用α银环蛇毒素(赛默飞,B35451)。
如果myofibres / MuSCs是更长的时间来进行培养,更换一半的每48小时的新鲜培养基的介质的体积。
对于基因表达分析,通过去除培养基并用 PBS 洗涤两次来收集细胞。孵育与所述适当体积(例如,200微升对于24孔板)的Accutase的®试剂以分离从基质胶的细胞10分钟(或直到所有细胞完全脱离;在显微镜下检查,但是这不应该采取长于 15 分钟)在28.5°C、5% CO 2 (参见Ganassi等人,2020 年)。收集细胞在1.5 ml的透明管,丸粒,通过离心,在200 ×克一吨4℃和在PBS中洗涤一次。沉淀的细胞现在可以用于 RNA 提取和分析,也可以储存在 -80°C。
 
食谱
 
1% 维康
将5 g粉末溶解在 500 ml去离子无菌水中,制备 1% Virkon 。
1% Virkon 溶液可分装并在 -20°C 下至少保存 2 个月。Virkon 粉末可以在室温下储存。
P/S-PBS
在 PBS ( P/S-PBS) 中制备 10% vol/vol青霉素/链霉素溶液,并将 25 ml 等分到 100 mm 塑料培养皿中。
BSA-PBS
制备在无菌PBS(BSA-PBS)5%BSA溶液和热-通过0.45μm注射器式过滤器过滤之前失活,在60℃进行60分钟。
cDMEM (完整的 DMEM)
通过在 DMEM 中加入 1% vol/vol 的青霉素/链霉素溶液和庆大霉素至 50 µg/ml 来制备 cDMEM。制备好的 cDMEM 可在4°C 下保存 2-3 周。
胶原酶-cDMEM
在解剖之前,立即在 cDMEM 中制备 0.2% 胶原酶溶液。在组织培养罩中,使用带有 0.2 μm 过滤器的无菌注射器对胶原酶-cDMEM 溶液进行过滤消毒。考虑为每条鱼(两条鱼片)加入大约 2 ml 胶原酶-cDMEM,然后等分到单独的 7 ml bijou 管中。
PBST ×
通过在无菌 PBS 中稀释 100% Triton X100 来制备 PBST x (0.5%)。含的Triton X-100的溶液可以预先准备,但对于长期储存,含0.002%叠氮化钠的PBS,而不是使用PBS和包裹在所述管/瓶铝箔以避光。
基质胶溶液
在 4°C 下解冻 Matrigel 库存过夜。在 DMEM 中将其稀释至 1 毫克/毫升,然后分装到 2 毫升微量离心管中。必须在冰上完成此步骤,否则 Matrigel 会凝胶并形成团块。稀释的 Matrigel 等分试样可在 4°C 下储存长达 2 周,或在 -20°C 下冷冻以进行更长时间的储存。
扩散的Medi嗯(PM)
通过用1% 青霉素/链霉素、10 µg/ml 庆大霉素和20% FBS补充 DMEM 来制备 PM 。在培养箱中将PM 预热至 28.5° C。我们建议在开始肌肉解剖/分离之前准备新鲜的 PM,并在 2 到 3 天内使用。
分化的Medi嗯(DM)
通过用1% 青霉素/链霉素、10 µg/ml 庆大霉素和2% 马血清补充 DMEM 来制备 DM 。在培养箱中将DM 预热至 28.5° C 。我们建议准备新鲜DM为需要编辑,并使用其内2至3天。
 
致谢
 
我们感谢休斯实验室的所有成员和布鲁诺科雷亚达席尔瓦及其工作人员对鱼类的照顾。这项工作得到了医学研究委员会对 SMH(MRC 计划补助金 G1001029 和 MR/N021231/1)和 PSZ(MR/P023215/1 和 MR/S002472/1)的资助。本协议是在Ganassi等人中开发和使用的。( 2018)和Ganassi等人。( 2020) 。
 
利益争夺
 
作者声明他们没有竞争利益。
 
伦理
 
与根据对成年斑马鱼进行所有程序的PPL许可证英国动物(科学规程)1986年法令举行,后来修改符合编到所有相关的准则和条例。使用的所有线路在伦敦国王学院饲养在14/10 ħ在28.5℃下光照/黑暗周期,用所描述的分段和饲养之前(韦斯特,2000) 。
 
参考
 
1. Alexander, MS, Kawahara, G., Kho, AT, Howell, MH, Pusack, TJ, Myers, JA, Montanaro, F., Zon, LI, Guyon, JR 和 Kunkel, LM (2011)。富含骨骼肌祖细胞的成年斑马鱼细胞的分离和转录组分析。肌肉神经43(5):741-750。      
2. Anderson, JE、Wozniak, AC 和 Mizunoya, W. (2012)。用于细胞、分子、药理学和进化研究的单肌纤维分离和培养。方法 Mol Biol 798:85-102。                    
3. Bekoff, A. 和 Betz, W. (1977 a )。在组织培养中维持的分离的成年大鼠肌肉纤维的特性。生理学杂志271(2): 537-547。      
4. Bekoff, A. 和 Betz, WJ (1977 b )。从受神经支配和去神经支配的成年大鼠肌肉中获得的分离肌纤维的生理特性。生理学杂志271(1): 25-40。      
5. Bischoff, R. (1975)。体外单个骨骼肌纤维的再生。Anat Rec 182(2): 215-235。                    
6. Blagden, CS, Currie, PD, Ingham, PW 和 Hughes, SM (1997)。声波刺猬介导的斑马鱼慢肌的脊索诱导。基因开发11(17):2163-2175。      
7. Brack, AS, Bildsoe, H. 和 Hughes, SM (2005)。证据表明,在小鼠年龄相关的肌肉萎缩期间,卫星细胞减少导致大纤维的核数优先下降。J Cell Sci 118(第 20 篇):4813-4821。      
8. Buckingham, M. 和 Relaix, F. (2015)。PAX3 和 PAX7 作为肌生成的上游调节剂。精细胞开发生物学44:115-125。      
9. Cardasis, CA 和 Cooper, GW (1975 a )。分化和生长过程中单个肌肉纤维中核数的分析:卫星细胞 - 肌肉纤维生长单位。J Exp Zool 191(3): 347-358。                    
10. Cardasis, CA 和 Cooper, GW (1975 b )。一种化学分离单个肌肉纤维的方法及其在研究去神经支配对每个肌肉纤维的细胞核数量的影响中的应用。J Exp Zool 191(3): 333-346。                 
11.戴维斯,ML ,约翰斯顿,IA和范·德·沃,J 。(1995)。    大西洋鳕鱼 ( Gadus morhua L.) 的头端和尾端肌节中的肌肉纤维具有不同的机械特性。生理学动物园l 68(4): 673-697。              
12. Froehlich, JM, Seiliez, I., Gabillard, JC 和 Biga, PR (2014)。从基础脊椎动物谱系制备原代肌源性前体细胞/成肌细胞培养物。J Vis Exp (86) : 51354。   
13. Fukada, SI, Akimoto, T. 和 Sotiropoulos, A. (2020)。肌肉肥大中损伤和管理的作用:肌肉干细胞在再生和肥大中的不同行为。Biochim Biophys Acta Mol Cell Res 1867(9): 118742。   
14. Ganassi, M., Badodi, S., Ortuste Quiroga, HP, Zammit, PS, Hinits, Y. 和 Hughes, SM (2018)。肌细胞生成素促进肌细胞融合以平衡纤维数量和大小。国家通讯社9(1): 4232。   
15. Ganassi, M., Badodi, S., Wanders, K., Zammit, PS 和 Hughes, SM (2020)。肌细胞生成素是成人肌纤维生长和肌肉干细胞稳态的重要调节剂。Elife 9 :e60445。   
16. Hammond, CL, Hinits, Y., Osborn, DP, Minchin, JE, Tettamanti, G. 和 Hughes, SM (2007)。信号和生肌调节因子限制 pax3 和 pax7 表达在斑马鱼的皮肤肌瘤样组织中。开发生物学302(2):504-521。   
17. Harper, C.和Lawrence, C. (2011)。实验室斑马鱼。博卡拉顿:CRC 出版社。                 
18. Hinits, Y.、Osborn, DP 和 Hughes, SM (2009)。对生肌调节因子的不同要求区分内侧和外侧体节、颅骨和鳍肌纤维群。发展136(3):403-414。   
19. Hinits, Y., Williams, VC, Sweetman, D., Donn, TM, Ma, TP, Moens, CB 和 Hughes, SM (2011)。Myod 突变斑马鱼的颅骨发育缺陷、幼虫致死率和单倍体不足。开发生物学358(1):102-112。   
20. Hromowyk, KJ, Talbot, JC, Martin, BL, Janssen, PM 和 Amacher, SL (2020)。细胞融合在斑马鱼胚胎后慢肌和快肌中受到差异调节。Dev Bol 462 (1): 85-100。   
21. Johnston, I. 和 Altringham, J. (1988)。极地鱼类的肌肉收缩:脱膜肌肉纤维实验。Comp Biochem Physiol 90B(3):547-555。                 
22. Johnston, IA, Abercromby, M., Vieira, VL, Sigursteindottir, RJ, Kristjansson, BK, Sibthorpe, D. 和 Skulason, S. (2004)。北极红点鲑(Salvelinus alpinus)冰后种群肌肉纤维数量的快速演化。J Exp Biol 207(第 25 篇):4343-4360。   
23. Katz, B. (1961)。青蛙肌梭中传入神经纤维的终止。Philos Trans R Soc Lond B Biol Sci 243(703) :221-240。                 
24. Mahalwar, P.、Walderich, B.、Singh, AP 和 Nüsslein-Volhard, C.(2014 年)。叶黄素的局部重组对斑马鱼的条纹图案进行微调和着色。科学345(6202):1362-1364。                 
25. Mauro, A. (1961)。骨骼肌纤维的卫星细胞。J Biophys Biochem Cytol 9:493-495。   
26. Moyle, LA 和 Zammit, PS (2014)。骨骼肌纤维的分离、培养和免疫染色以研究卫星细胞的肌源性进展。方法 Mol Biol 1210:63-78。   
27. Osborn, DPS, Li, K., Cutty, SJ, Nelson, AC, Wardle, FC, Hinits, Y. 和 Hughes, SM (2020)。Fgf 驱动的 Tbx 蛋白活动直接诱导 myf5 和 myod 启动斑马鱼的肌生成。开发147(8) :dev184689。   
28. Pipalia, TG, Koth, J., Roy, SD, Hammond, CL, Kawakami, K. 和 Hughes, SM (2016)。再生的细胞动力学揭示了两种不同的 Pax7 干细胞群在斑马鱼幼虫肌肉修复中的作用。Dis Model Mech 9(6): 671-684。                 
29. Purohit, G. 和 Dhawan, J. (2019)。成人肌肉干细胞:探索全身代谢和细胞代谢之间的联系。Front Cell Dev Biol 7:312。   
30. Relaix, F. 和 Zammit, PS (2012)。卫星细胞对于骨骼肌再生至关重要:边缘的细胞返回中心舞台。发展139(16):2845-2856。   
31. Rosenblatt, JD, Lunt, AI, Parry, DJ 和 Partridge, TA (1995)。从活的单肌纤维外植体中培养卫星细胞。体外细胞开发生物学动画31(10): 773-779。   
32. Westerfield, M. (2000)。斑马鱼手册 - 斑马鱼 ( Danio rerio )实验室使用指南。俄勒冈大学出版社。   
33. Zammit, PS, Golding, JP, Nagata, Y., Hudon, V., Partridge, TA 和 Beauchamp, JR (2004)。肌肉卫星细胞采用不同的命运:一种自我更新的机制?J Cell Biol 166(3): 347-357。                 
34. Zhang, H. 和 Anderson, JE (2014)。来自成年斑马鱼 ( Danio rerio ) 的单肌纤维培养物的卫星细胞活化和种群。J Exp Biol 217(第 11 篇):1910-1917。   
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Copyright Ganassi et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Ganassi, M., Zammit, P. S. and Hughes, S. M. (2021). Isolation of Myofibres and Culture of Muscle Stem Cells from Adult Zebrafish. Bio-protocol 11(17): e4149. DOI: 10.21769/BioProtoc.4149.
  2. Ganassi, M., Badodi, S., Wanders, K., Zammit, P. S. and Hughes, S. M. (2020). Myogenin is an essential regulator of adult myofibre growth and muscle stem cell homeostasis. Elife 9: e60445.
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