2017 Jun 30
IGFN1_v1 Is Required for Myoblast Fusion and Differentiation
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IGFN1_v1 Is Required for Myoblast Fusion and Differentiation
Igfn1 is a complex locus that codes for multiple splicing variants of Immunoglobulin- and Fibronectin-like domain containing proteins predominantly expressed in skeletal muscle. To reveal possible roles for Igfn1, we applied non-selective knock-down by shRNAs as well as specific targeting of Igfn1 exon 13 by CRISPR/Cas9 mutagenesis in C2C12 cells. Decreased expression of Igfn1 variants via shRNAs against the common 3'-UTR region caused a total blunting of myoblast fusion, but did not prevent expression of differentiation markers. Targeting of N-terminal domains by elimination of exon 13 via CRISPR/Cas9 mediated homologous recombination, also resulted in fusion defects as well as large multinucleated cells. Expression of IGFN1_v1 partially rescued fusion and myotube morphology in the Igfn1 exon 13 knock-out cell line, indicating a role for this variant in myoblast fusion and differentiation. However, in vivo overexpression of IGFN1_v1 or the Igfn1 Exon 13 CRISPR/Cas9 targeting vector did not result in significant size changes in transfected fibres.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Fig 1. Knock-down of IGFN1 provokes cell detachment and fusion impairment.
A) DNA sequence of
Igfn1 3’UTR genomic region showing the coding sequence in bold. The sequence targeted by sh-2-IGFN1 within the 3’UTR is indicated in bold underlined. The sequence targeted by sh-1-IGFN1 is boxed in gray. B) As labelled, cultures of C2C12 cells transiently transduced with scrambled shRNA (7 days in DF medium), sh1-IGFN1 (4 days in DF medium) and selected clone Igfn1KD1 (4 days in DF medium). Note detachment of Sh1-IGFN1 transiently transduced and stably selected (Clone Igfn1KD1) cells. C) Igfn1KD2, a clone selected following transduction with sh-2-IGFN1, shows detachment upon switching to DF medium, whilst the control C2C12 cell line forms myotubes.
Fig 2. Knock-down of IGFN1 isoforms by shRNAs does not prevent expression of differentiation markers.
A) Analysis of IGFN1 variants, as identified with antibodies against IGFN1 Kip2b and Kip1, and markers of differentiation throughout in vitro differentiation of C2C12 myoblasts as indicated. The indicated Igfn1KD1 lane corresponds to a stable cell line selected with sh-1-IGFN1 targeting the 3’UTR of Igfn1 collected at 4 days in DF medium. Note depletion of the largest IGFN1 protein variants. B) Two knock down clones, obtained with sh-2-IGFN1 targeting a different sequence of IGFN1 3’ UTR, showing decreased complexity of IGFN1 products compared to the C2C12 cell line, as labelled.
Fig 3. Characterization of Igfn1KD1 cell line.
A) Confocal images of Igfn1KD1 and C2C12 cells obtained with Ab-US43 antibodies against IGFN1, as labelled. Note a diminished signal in the Igfn1KD1 cell line. Images were obtained with identical confocal settings. B) Igfn1KD1 fails to form myotubes on collagen coated dishes. Brightfield images of Igfn1KD1 and C2C12 at day 0 and day 3 in DF medium as labelled. C) Confocal images of Igfn1KD1 and C2C12 cells stained with phalloidin (red) at Day 2 of differentiation. White arrows point at nuclei within a single myotube identified by the uninterrupted actin filaments. Images are representative of three independent experiments and obtained with identical confocal settings. Nuclei stained with DAPI (blue). Size bar = 30μm. D) Immunofluorescence images of Igfn1KD1 and control C2C12 cells maintained for 14 days in DF medium and labelled with antibodies against alpha-actinin, as indicated. White arrow points at striations within a single Igfn1KD1 mononucleated cell that survived detachment. E) Western blots (top panels) and quantification (histogram) of relative active Rac1 levels in control and Igfn1KD1 cell lines. The histogram shows the average amount of active Rac1-GTP normalised to the total amount of Rac1 by densitometry analysis for two independent experiments. Similar levels of active Rac1 are observed in the Igfn1KD1 cell line and the control. F) Quantification analysis of apoptosis in control and Igfn1KD1 cell lines at day 4 of differentiation. The histogram shows the average quantification of number of cells undergoing apoptosis from six independent experiments, expressed as the ratio of apoptotic/non apoptotic cells (see Methods for details). (*) Differences are significant (p<0.001): t = 6.11.
Fig 4. Expression of recombinant IGFN1:GFP or IGFN1_v1:GFP in the Igfn1KD cell lines does not result in rescue of myoblast fusion.
Confocal images show representative examples of transfected cells in cultures maintained in DF medium for a week. Transfected cells contained one or two nuclei. Phalloidin staining (red) was used on cells transfected with IGFN1_v1:GFP only.
Fig 5. Design and validation of an
Igfn1 exon 13 CRISPR/Cas9 targeting vector.
A) Genomic structure of
Igfn1 and Igfn1_v1 and predicted domain composition of IGFN1. The first exon, exon 13 and the last exon are indicated. The SMART ( http://smart.embl-heidelberg.de) predicted domain composition of full length IGFN1 is shown, with vertical lines indicating exon boundaries. Note that exon 13 codes for part of the second globular domain of IGFN1. B) Genomic sequence surrounding Igfn1 exon 13. Exon 13 is indicated in capital letters. The CRISPR/CAS targeting sequence is indicated in bold. Left and right homologous recombination arms are boxed in grey. C) Validation of Igfn1 exon 13 CRISPR/Cas9 targeting vector. A T7 endonuclease I digestion of the 852bp amplicon encompassing the targeted region. Note that cleavage products of the expected sizes (505bp and 347bp) are only obtained in digested (+ T7E) sample from transfected 3T3 cells (+ Igfn1 exon 13 CRISPR/Cas9).
Fig 6. Targeting of
Igfn1 exon 13 by CRISPR/Cas9 mutagenesis.
A) Schematics of the expected genomic arrangements that follows repair of double strand breaks either by homologous recombination or non-homologous end joining (NHEJ), as indicated. Homologous recombination using left (“Left arm”) and right (“Right arm”) homologous region results in replacement of exon 13 by a cassette containing GFP and puromycin reporter and selection markers, respectively. NHEJ results in random mutations inserted in exon 13, represented by asterisks. The position and labels of the screening primers are indicated. B) Screening of C2C12 derived puromycin resistant clones. PCR products of the correct size obtained with BF_L + HR_L and HR_R + AF_R indicate the presence of at least one homologous recombined allele. Non-recombined alleles produce a product of 852bp with primers Arm_L + Arm_R. Note that only clone 19 failed to produce a product for the non-recombined allele, indicating that both alleles have been recombined in this clone. C) Example of NHEJ allele (clone 28) showing a single nucleotide deletion (asterisk) aligned to the control non-targeted allele. D) Validation of qRT-PCR primers. The position of the primers on exon 11 and 12 is indicated by black arrows on the relevant segment of the
Igfn1 genomic structure (top). cDNA from C2C12 cells produced a single product of the expected size (141 bp) in a standard PCR assay, as labelled. E) qRT-PCR results from clones carrying disruptive mutations in exon 13 (C14, C19, C28, C33) and C212 control, expressed as fold change relative to the levels of C2C12, as indicated. Error bars represent the standard deviation of three technical replicates. (*) indicates significant difference (P≤0.05) to the C2C12 level.
Fig 7. Disruption of
Igfn1 exon 13 in C2C12 cells causes aberrant differentiation patterns.
A) Fluorescence images of proliferating clones carrying disruptive mutations on both alleles and stained for phalloidin (green), as labelled. B) Plot of average cell area of proliferating cells for each selected clone, as labelled. Clones 19 and 33 were significantly smaller in size than C2C12 (t-test, *P≤0.05). Error bars represent the standard deviation of each mean. C) Bright field microscopy images of
Igfn1 exon 13 knockout clones and C2C12s after 7 days in differentiation medium. Note prominent round cells (closed arrows) and wide myotubes (open arrows) in all selected clones. D) Fluorescence images of alpha-actinin stained (red) Igfn1 exon 13 knockout clones after 7 days in differentiation medium. Note the presence of an alpha-actinin-rich ring in single cells (closed white arrows) and wide multinucleated myotubes (open white arrows). E) Plot of average myotube diameter for each selected clone, as labelled. All clones were significantly wider than the C2C12 control (t-test, *P≤0.05).
Fig 8. The KO19 cell line displays fusion and differentiation defects partially rescued by expression of IGFN1_v1.
A) Representative fluorescence images of myoblasts differentiated for 10 days for C2C12, KO19 and KO19 transfected with IGFN1_v1 coding plasmid (KO19+V1). Cells were stained for alpha-actinin (red) and DAPI (blue). Two examples per cell line shown, as labelled. Scale bar represents 50μm. B) Graph showing the differentiation index for the indicated cell lines, calculated as the proportion of nuclei within an alpha-actinin expressing cell to the total number of nuclei within the same field. Note that KO19 cells have a significantly lower differentiation index than wildtype and rescued cells. C) Fusion index calculated as the percentage of alpha-actinin positive cells with three or more nuclei. KO19 cells have significantly lower fusion index than wildtype and rescued cells. D) Mean diameter of alpha-actinin positive cells containing three or more nuclei, average diameter is significantly higher in knockout cells compared to both the wildtype and the rescue. (**) p<0.01, (*) p<0.05.
Fig 9. Overexpression of IGFN1_V1:tdTomato in mouse skeletal muscle.
(A) Confocal image of a whole mount longitudinal view of mouse EDL/TA muscle fibres electroporated with IGFN1-tdTomato. IGFN1_v1-tdtomato (red) shows nuclear expression and colocalization with alpha-actinin (green) at the Z-disc. The insets (white outlined squares) are shown magnified. The overlay includes DAPI (blue). B) Cross sections of electroporated muscles. The panels show fluorescence images of EDL/TA muscle sections from muscles electroporated with either IGFN1_v1:tdTomato or tdTomato from three male siblings, as indicated. Note that pMAXGFP (GFP) was co-electroporated to facilitate identification of positively transfected fibres. Fibres have been manually labelled with yellow (transfected) or blue (untransfected) outlines for area measurement purposes. (C) Box and whisker plots showing the upper and lower quartiles of cross sectional area measurements for IGFN1_v1:tdTomato+pMAXGFP transfected (IGFN1_v1-tdTom/GFP) and non-transfected fibres and for pMAXGFP transfected (GFP) and non-transfected fibres. The data was pulled from the three electroporated mice illustrated in B. Horizontal and vertical bars represent the median and range of values, respectively. Mann-Whitney U test showed no significant difference between transfected and non-transfected fibres.
Fig 10. Expression of
Igfn1 exon 13 CRISPR/Cas9 in vivo does not cause significant fibre size change.
A) Fluorescence images of cross sections from whole EDL/TA muscles electroporated with
Igfn1 exon 13 CRISPR/Cas9 +tdTomato or tdTomato alone, as indicated. B) Box and whisker plots showing the upper and lower quartiles of cross sectional area measurements for Igfn1 exon 13 CRISPR/Cas9+tdTomato transfected (IGFN1 CRISPR/td) and non-transfected fibres and for tdTomato (td) transfected and non-transfected fibres. Quantifications were carried out for three mice independently, as indicated. Horizontal and vertical bars represent the median and range of values, respectively. Mann-Whitney U test showed no significant difference between transfected and non-transfected fibres.
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Cell Differentiation / physiology
Clustered Regularly Interspaced Short Palindromic Repeats
Muscle, Skeletal / cytology
Support was provided by the Medical Research Council (MC_U142684168 to Andy Haynes) and Biotechnology and Biological Sciences Research Council (BB/M011151/1 to Tobias Cracknell).
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