Abelson tyrosine-protein kinase 2 regulates myoblast proliferation and controls muscle fiber length
- NYU Medical School, United States
Figures
Figure 1 with 1 supplement
Diaphragm muscle fibers in E18.5 Abl2 mutant mice are extraordinarily long, and the central tendon is diminished in size.
Whole mounts of muscle were stained with antibodies to myosin heavy chain (MHC). (A) Costal muscle fibers in E18.5 embryonic diaphragm muscle normally extend from the ribcage and attach medially to …
Figure 1—figure supplement 1
Embryonic development of Abl2−/− diaphragm.
Embryos were collected between E12.5-E14.5 to detect differences in diaphragm muscle development between Abl2 mutant and control mice. β-galactosidase activity was detected in myoblasts and muscle …
Figure 2 with 1 supplement
Muscle fibers are excessively long in intercostal, levator auris, and diaphragm muscles in adult Abl2 mutant mice.
Whole mounts of adult muscle were stained with antibodies to myosin heavy chain (MHC) and GFP. Cross-sections of muscle were stained with antibodies to Laminin. (A) Intercostal muscle fibers …
Figure 2—figure supplement 1
Limb muscles appear normal in muscle length and cross-sectional area.
Hind limbs were fixed in 4% PFA, and tibialis anterior (TA) muscles were dissected at full length from three wild type and 3 Abl2−/− mice. Sections were stained with antibodies to Laminin to outline …
Figure 3
The increase in muscle fiber length is accompanied by an increase in myonuclei number.
(A) Single fibers dissociated from the ventral quadrant of the costal diaphragm muscle were stained with Phalloidin to label F-actin and DAPI to label nuclei. (B) The scatter plot shows the number …
Figure 4 with 1 supplement
Abl2 acts in myoblasts to regulate muscle development.
(A,B) Abl2 mRNA is highly expressed in the muscle but not within the central tendon (CT) of the diaphragm. (C) Muscle and tendon development appear normal in Abl2flox/+ control mice (Abl2f/+). …
Figure 4—figure supplement 1
Abl2 expression is enriched in proliferating myoblasts.
(A) Western blot analysis of wild type (wt) and Abl2−/− primary cultures. Cadherin levels (pan-Cad) were measured in cell lysates, and Abl2 was immunoprecipitated (IP) from lysates containing equal …
Figure 5 with 1 supplement
The exercise endurance of Abl2 null and Abl2 muscle-conditional mutant mice is impaired.
(A) During a twenty-hour period, Abl2−/− and Abl2f/−; MyodiCre conditionally mutant mice run less than wild type (wt) mice. (B) The limb strength of Abl2−/−, Abl2f/−; MyodiCre and wild type mice are …
Figure 5—figure supplement 1
Ultrastructural appearance of muscle-tendon junctions and muscle fibers is normal in Abl2 mutant mice.
(A) Deep muscle membrane specializations form at the myotendinous junction in wild type and Abl2−/− mice. (B) The scatter plot shows the sarcomere lengths for 12 fibers from two wild type and two Abl…
Figure 6 with 1 supplement
Myoblast proliferation is enhanced by a loss of Abl2.
(A,B) In vivo labeling of E13.5-E14.5 diaphragm muscles shows that EdU incorporation is greater in Abl2−/− myoblasts than wild type (wt) myoblasts. (C) Representative images of cultured diaphragm …
Figure 6—figure supplement 1
The number of Pax7+cells is increased in proportion to the increased length of myofibers.
Serial cross-sections of the diaphragm were stained with antibodies to Laminin to outline muscle fibers and with antibodies to Pax7 to label satellite cells. The number of Pax7+ cells was divided by …
Figure 7
Enhanced myoblast proliferation in Abl2−/− mice leads to enhanced myoblast fusion.
Primary cultures from the diaphragm muscle of E18.5 mice were stained with antibodies to myosin heavy chain (MHC) to label differentiated muscle fibers and MyoD to label myoblasts. (A) …
Figure 8 with 1 supplement
Ectopic muscle islands, which are found in the central tendon of mice that are heterozygous for Abl2, induce tendon cell differentiation.
(A,B) Whole mount diaphragms, stained with antibodies to myosin heavy chain (MHC), revealed 1 to 4 ectopic islands in the central tendon of Abl2+/− mice. (C) Scx RNA expression is enhanced at the …
Figure 8—figure supplement 1
The orientation of ectopic muscle fibers correlates with the orientation of myofibers in the nearby main costal diaphragm muscle.
Representative images of the central tendon area in whole mounts of the diaphragm from Abl2+/− mice. The cartoon shows the method for measuring and quantifying the orientation of myofibes in ectopic …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| gene (Mus musculus) | Abl2 | The Jackson Laboratory | RRID:MGI:2653897 | |
| gene (Mus musculus) | Myod iCre | The Jackson Laboratory | RRID:IMSR_JAX:014140 | |
| gene (Mus musculus) | Mck-Cre | The Jackson Laboratory | RRID:IMSR_JAX:006475 | |
| gene (Mus musculus) | Pax3 Cre | The Jackson Laboratory | RRID:IMSR_JAX:005549 | |
| gene (Mus musculus) | Rosa 26 LacZ | The Jackson Laboratory | RRID:IMSR_JAX:003474 | |
| gene (Mus musculus) | Scx::Cre | MGI:5317938 | n/a | Ronen Schweitzer lab |
| gene (Mus musculus) | Scx::GFP | PMID: 17497702 | n/a | Ronen Schweitzer lab |
| strain, strain background (Mus musculus) | C57BL/6J | The Jackson Laboratory | RRID:IMSR_JAX000664 | |
| genetic reagent (Mus musculus) | Targeting Vector: Abl2tm1a(EUCOMM)Hmgu | EUCOMM | RRID:SCR_003104 | European Conditional Mouse Mutagenesis Program |
| cell line (Mus musculus) | C2C12 skeletal muscle cells | Burden lab/ATCC | Cat# CRL-1772, RRID:CVCL_0188 | |
| antibody | Rabbit anti-Abl2 | Proteintech Group | Cat# 17693–1-AP RRID:AB_2289025 | 1:1000 |
| antibody | Goat anti-Arg (C-20) | Santa Cruz Biotechnology | Cat# sc-6356 RRID:AB_2221106 | 1:200 |
| antibody | Rabbit anti-β-Actin (13E5) | Cell Signaling Technology | Cat # 4970 | 1:1000 |
| antibody | Mouse anti-Myosin (MY-32) | Sigma-Aldrich | Cat# M7523 RRID:AB_260649 | 1:1000 |
| antibody | Chicken anti-GFP | Abcam | Cat# ab92456 RRID:AB_10561923 | 1:3000 |
| antibody | Mouse anti-Myosin (NOQ7.5.4D) | Sigma-Aldrich | Cat# M8421 RRID:AB_477248 | 1:1000 |
| antibody | Rabbit anti-Laminin | Sigma-Aldrich | Cat# L9393 RRID:AB_477163 | 1:1000 |
| antibody | Mouse anti-Pax7 | Santa Cruz Biotechnology | Cat# sc-81648 RRID:AB_2159836 | 1:500 (bioreactor) |
| antibody | Rabbit anti-MyoD C-20 | Santa Cruz Biotechnology | Cat# sc-304 RRID:AB_631992 | 1:1000 |
| antibody | Mouse anti-MyoD (5.8A) | ThermoFisher Scientific | Cat# MA1-21816 RRID:AB_560242 | 1:1000 |
| antibody | Donkey anti-mouse (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor 594 | ThermoFisher Scientific | Cat# A-21203 RRID:AB_2535789 | 1:2000 |
| antibody | Donkey anti-rabbit (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor 594 | ThermoFisher Scientific | Cat# A-21207 also A21207 RRID:AB_141637 | 1:2000 |
| antibody | Donkey anti-mouse (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor 488 | ThermoFisher Scientific | Cat# A-21202 RRID:AB_141607 | 1:2000 |
| antibody | Donkey anti-rabbit (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor 488 | ThermoFisher Scientific | Cat# A-21206 also A21206 RRID:AB_2535792 | 1:2000 |
| antibody | Goat anti-Rat IgG (H + L) Cross-adsorbed Secondary Antibody Alexa Fluor 488 | ThermoFisher Scientific | Cat# A-11006 also A11006 RRID:AB_2534074 | 1:2000 |
| commercial assay or kit | Click-iT Plus EdU Alexa Fluor 488 Imaging Kit | ThermoFisher Scientific | Cat# C10637 | |
| software, algorithm | Prism 7.0 | http://www.graphpad.com/scientific-software/prism/ | RRID:SCR_002798 |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.29905.016
