Background: Members of the ZFP36 family of RNA-binding proteins regulate gene expression post-transcriptionally by binding to AU-rich elements in the 3’UTR of mRNA and stimulating mRNA degradation. The proteins within this family target different transcripts in different tissues. In particular, ZFP36 targets myogenic transcripts and may have a role in adult muscle stem cell quiescence. Our study examined the requirement of ZFP36L1 and ZFP36L2 in adult muscle cell fate regulation. Methods: We generated single and double conditional knockout mice in which Zfp36l1 and/or Zfp36l2 were deleted in Pax7-expressing cells. Immunostained muscle sections were used to analyse resting skeletal muscle, and a cardiotoxin-induced injury model was used to determine the regenerative capacity of muscle. Results: We show that ZFP36L1 and ZFP36L2 proteins are expressed in satellite cells. Mice lacking the two proteins in Pax7-expressing cells have reduced body weight and have reduced skeletal muscle mass. Furthermore, the number of satellite cells is reduced in adult skeletal muscle and the capacity of this muscle to regenerate following muscle injury is diminished. Conclusion: ZFP36L1 and ZFP36L2 act redundantly in myogenesis. These findings add further intricacy to the regulation of the cell fate of Pax7-expressing cells in skeletal muscle by RNA-binding proteins. Keywords: RNA, RNA-binding proteins, Satellite cells, Skeletal muscle stem cells, Regeneration, Injury, Zfp36L1, Zfp36L2 Background skeletal muscle, satellite cells express Pax7 and they are Paired box transcription factors 3 and 7 (Pax3 and Pax7, the adult stem cell population . They largely reside in respectively) are co-expressed in the dermomyotome a mitotically quiescent state between the basal lamina during mouse embryonic development [1, 2]. Their ex- and plasma membrane of myofibres [6, 7]. Typically, in pression gives rise to myogenic progenitor cells which response to growth cues, tissue injury or disease, signals are necessary for myogenic development. As critical reg- from the surrounding environment, and from within the ulators, Pax3 is required for embryonic myogenesis, specialised stem cell niche, promote exit from the quies- whilst Pax7 is predominantly required for adult myogen- cent state and induce satellite cell activation [8–11]. esis and specifies the adult stem cell population in Following activation, satellite cells proliferate and either muscle [3–5]. The identity of the cells that arise from enter the myogenic differentiation programme or the myogenic progenitor cells during embryonic and self-renew to re-establish the quiescent satellite cell pool adult myogenesis may be determined by the expression [8–10]. Knowledge of the mechanisms that maintain of myogenic regulatory factors (MRFs) that are adult stem cells within tissues in an undifferentiated expressed downstream of Pax3 and Pax7. In adult state, and control the differentiation or self-renewal fate of these cells, is important for understanding tissue homeostasis and may have application in the emerging * Correspondence: email@example.com; firstname.lastname@example.org Deceased field of regenerative medicine. Laboratory of Cell Signalling, The Babraham Institute, Cambridge, UK Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, UK Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bye-A-Jee et al. Skeletal Muscle (2018) 8:37 Page 2 of 12 Genetic programmes act transcriptionally and post- Pax7-expressing cells during the normal myogenic transcriptionally to achieve stringent regulation of the process, and we provide additional insight into the com- development, specificity and replenishment of skeletal plexity of the regulation of skeletal muscle myogenesis muscle. At the level of transcription, MRFs bind to by RBPs. the promoters of myogenic determination genes to initiate gene expression and thus govern the transi- Methods tion from the muscle precursor cell or satellite cell to Mice multinucleated myofibre throughout development [12, Mice were bred and maintained in the Babraham Insti- 13]. Post-transcriptional regulation by alternative spli- tute Biological Support Unit under Specific Opportunis- cing and polyadenylation, or by control of mRNA tic Pathogen Free (SOPF) conditions. After weaning, translation and degradation, also regulates gene ex- mice were transferred to individually ventilated cages pression in satellite cells. Specifically, the interaction with 1–5 mice per cage. Mice were fed CRM (P) VP diet between cis-acting elements within the mRNA and (Special Diet Services) ad libitum and received environ- trans-acting elements, such as microRNAs and mental enrichment. Mario Capecchi (University of Utah) RNA-binding proteins (RBPs), regulates transcript sta- provided transgenic mice expressing Cre-recombinase bility and translation. There is accumulating evidence under the control of the Pax7 promoter (Pax7Cre) . tm1.1Tnr tm1.1Tnr to demonstrate that RBPs are necessary for normal The Zfp36l1 and Zfp36l2 targeted condi- muscle physiology [14, 15]. In particular, RBPs includ- tional alleles were generated at the Babraham Institute ing HuR, AUF1, ZFP36 and KSRP bind to AU-rich el- . Interbreeding generated mice on the C57BL/6 ements to modulate the expression of numerous genetic background in which either or both floxed alleles myogenic regulatory factors that function at different were present with Pax7Cre. For simplicity, these will be phases of myogenesis to ensure normal skeletal referred to as Zfp36L1-P, Zfp36L2-P and Zfp36L1/L2-P fl/fl fl/fl muscle development. The overlapping roles of these for Pax7CreZfp36l1 , Pax7CreZfp36l2 and Pax7- fl/fl fl/fl RBPs indicate that the coordination between RBPs is Cre-Zfp36l1 Zfp36l2 , respectively. All experiments necessary for the normal progression of myogenesis were carried out without blinding or randomisation on [16, 17]. Collectively, this highlights the importance 3–12-week-old mice. of RBPs in establishing cell identity during the normal myogenic process. Satellite cell isolation Zfp36 encodes tristetraprolin (TTP) the prototype of a Satellite cells were isolated from hind limb skeletal mus- small family of RBPs, called the ZFP36 family, that are cles from mice using a protocol adapted from characterised by highly conserved tandem CCCH Woodhouse et al. . Briefly, 6–12-week-old mice were zinc-finger RNA-binding domains . ZFP36 is a RBP sacrificed by rising CO and/or cervical dislocation that promotes RNA decay and negatively regulates the according to Schedule One of the Home Office Protocol expression of the myogenic regulatory factor MyoD by for the sacrifice of mice. Hind limb skeletal muscles binding to the 3’UTR of MyoD mRNA . Mouse satel- (including gastrocnemius, plantaris, soleus, tibialis anter- lite cells from Zfp36-deficient mice express increased ior (TA), extensor digitorum longus and quadriceps) amounts of MyoD and display impaired satellite activa- were dissected from the mice. To isolate individual cells, tion, demonstrating a role for ZFP36 in the maintenance the muscles were mechanically homogenised and further of quiescence . The functions of the ZFP36L1 and digested in a DMEM solution containing 0.12% (w/v) ZFP36L2 family members have not been evaluated in type II collagenase (Lorne Laboratories) and 2 mg/ml skeletal muscle stem cell fate, but have been shown to dispase II (Roche) at 37 °C with intermittent trituration act redundantly to promote quiescence during lympho- using a 19G needle during the incubation. To remove cyte development [19–21]. ZFP36L1 has been implicated excess debris, PBS was added and the slurry was passed in the persistence of the marginal zone B lymphocyte through a 40-μM filter. The filtrate was centrifuged at population  and ZFP36L2 in the maintenance of the 1500×g, and the supernatant was discarded. Red blood haematopoietic stem cell pool . To date, the roles of cells were lysed in a solution consisting of 140 mM ZFP36L1 and ZFP36L2 in specific myogenic cell popula- NH Cl, 2 mM Tris-HCl at pH 7.2. Lysis was stopped by tions have not been studied. Here, we demonstrate that addition of PBS. The remaining cells were extensively loss of both ZFP36L1 and ZFP36L2 in Pax7-expressing washed, and non-specific antibody binding sites were cells results in a reduced number of satellite cells in blocked with a PBS solution containing 2% foetal bovine adult mice, and furthermore, provide evidence that these serum (PAA; PBS/FBS). Cells in PBS/FBS were incubated genes promote regeneration following muscle injury. with the following primary antibodies in the dark: Mechanistically, we suggest that ZFP36L1 and ZFP36L2 anti-CD31-PE (Abcam), anti-CD45-PE (BD Biosciences), may act redundantly in determining the cell fate of anti-Sca1-PE (BD Biosciences), anti-VCAM1-biotin (BD Bye-A-Jee et al. Skeletal Muscle (2018) 8:37 Page 3 of 12 Biosciences) and anti-CD34-APC (eBioscience). The haematoxylin (TCS Biosciences) and Van Gieson solu- VCAM1-biotin antibody was visualised by staining with tion (Sigma-Aldrich) to analyse fibrosis, according to streptavidin-AlexaFluor488 conjugate (Life Technolo- standard protocols. Sections were dehydrated in etha- gies). Cells were finally stained with DAPI (2.5 μg/ml; nol, immersed in xylene (VWR), mounted with a glass Life Technologies) and sorted using a BD FACSAria III coverslip in Entellen (Merck) and air-dried under a cell sorter (BD Bioscience). Antibodies against CD31, laminar flow cupboard. CD45 and Sca-1 were used to gate the lineage-negative cells. VCAM1- and CD34-positive satellite cells were Immunofluorescence of cryosections gated and collected into Eppendorf tubes containing a TA cryosections on glass slides were fixed and per- high serum medium as described above. Satellite cells meabilised. Non-specific binding sites were blocked were subsequently cultured or lysed for protein with an IgG blocking reagent (M.O.M; Vector Labora- extraction. tories) and subsequently with a PBS solution contain- ing 3%BSA, 5%goatserum and 0.2% (w/v)Triton Primary satellite cell culture X-100 (blocking buffer). The following primary anti- Primary satellite cells were plated at a density of 1.5 × 10 bodies were prepared in the blocking buffer and incu- per ml and cultured in a high serum medium containing bated overnight at 4 °C: anti-Pax7 (DSHB), anti-MyoD 20% foetal bovine serum (PAA), 2 mM L-glutamine (Santa Cruz), anti-Myogenin (Santa Cruz) and (Invitrogen), 2 ml antibiotic anti-mycotic solution anti-laminin α-2 chain (Enzo Life Sciences). Sections (Promega) and 2.5 ng/ml basic fibroblast growth factor were incubated in the dark at room temperature with (Promega) in DMEM (Invitrogen). All cells were main- the fluorescence-conjugated secondary antibodies tained using standard tissue culture techniques at 37 °C in AlexaFluor 488 (donkey anti-rabbit; Life Technolo- 5% CO at arelativehumidity of 100%. gies), AlexaFluor 568 (goat anti-mouse; Life Technolo- gies) and/or AlexaFluor 633 (goat anti-rat; Life Muscle regeneration model Technologies) in blocking buffer. Sections were Muscle injury and regeneration was characterised in 6– washed in PBS/TX. To stain the nuclei, DAPI was di- 12-week-old mice using cardiotoxin (CTX; Sigma-Aldrich). luted in PBS/TX and incubated with the sections in Mice were anaesthesised with isofluorane according to the dark at room temperature. The sections were standard protocols. Ten micromolar CTX was pre- washed in PBS/TX and then mounted with glass cov- pared in PBS and 100 μl was injected into the TA erslips using Vectashield, a hard set mounting muscle of isofluorane-anaesthetised mice. To act as medium (Vector Laboratories). Slides were dried in an internal vehicle control, 100 μlofPBS wasinjected the dark at room temperature. into the contralateral TA. Following the procedure, mice were housed and cared for as described above. Western blotting Mice were sacrificed 1, 5, 10 and 25 days following Proteins were extracted and Western blotting was per- injury by rising CO and/or cervical dislocation ac- formed as described in . The following primary anti- cording to Schedule One of the Home Office Protocol body dilutions were prepared in a PBS solution (0.1 M for the sacrifice of mice and the TA muscles were phosphate buffer was used throughout) containing 0.2% harvested. I-Block (Applied Biosystems) and 0.05% Tween-20: anti-BRF1/2 (Cell Signalling Technologies), anti-pan Histology ZFP36-family (SB1/30.13) and anti-β-actin (Abcam). The Mice were sacrificed as described above and the TA was following secondary antibodies were diluted in a PBS so- dissected from the ventral crest of the tibia and mounted lution containing 0.05% Tween-20: goat anti-rabbit-HRP vertically with the largest part of the TA on the base of a conjugated (Biorad) or donkey anti-rat-HRP conjugated cork disc (Fischer). The proximal tendons of the TA (Abcam). were used to balance the TA upright against a needle to enable transverse cross-sectioning. The TA was coated in Tissue Tek (Leica) and snap frozen in liquid Statistical analysis and imaging nitrogen-cooled iso-pentane (VWR). Transverse The statistical tests used are indicated in the figure cross-sections (10 μm) were generated on a Leica Cryo- legends. A minimum of three replicates were per- stat (Leica) and collected on pre-treated positively- formed for each experiment, and data are presented charged glass microscope slides (VWR). Transverse as means ± SEM. All images were acquired using a × cross-sections of the TA were stained with filtered Gill’s 40 objective lens on an Olympus confocal microscope, haematoxylin solution (Sigma-Aldrich) and eosin and quantification was performed using ImageJ soft- (VWR) to analyse tissue morphology, or Weigert’s ware (NIH). Bye-A-Jee et al. Skeletal Muscle (2018) 8:37 Page 4 of 12 Results and discussion Zfp36l2, and support whole body and skeletal muscle The role of RNA-binding proteins in controlling cell fate growth. is emerging as an important mechanism. In mice, the Transverse cross sections of the TA from Zfp36L1/ transcripts encoding the three ZFP36 family members L2-P mice stained with haemotoxylin and eosin (H and are expressed in the skeletal muscle system and, more E), and immunostained with anti-laminin, indicated that specifically, are expressed to varying degrees in quiescent the myofibre organisation and architecture were similar and activated satellite cells . By the Western blotting to controls, and although this was not quantified, we ob- of proteins from isolated satellite cells from mouse adult served a mixture of small and large myofibres (Fig. 1d). skeletal muscle, we found that both ZFP36L1 and Furthermore, all nuclei were peripheral and located ZFP36L2 are expressed in this myogenic cell population around the myofibres, and similarly to controls, there (Fig. 1a; the results of a second independent experiments was no apparent on-going muscle degeneration or re- are shown in Additional file 1). Germline knockout mice generation. Quantification of myofibre size and the dis- for Zfp36l1 and Zfp36l2 exhibit severe developmental tribution of myofibre types may provide further and growth defects and as a result Zfp36l1 knockout understanding of the roles of ZFP36L1 and ZFP36L2 on mice die in utero between E8 and E12 and Zfp36l2 adult skeletal muscle architecture. Taken together, these knockout mice die within 2 weeks of birth [23, 27–29]. data are consistent with a requirement for Zfp36l1 and Therefore, we adopted a conditional tissue-specific Zfp36l2 to promote whole body growth and sustain skel- knockout approach and used Pax7Cre to delete Zfp36l1 etal muscle development in adults. Studies have demon- and/or Zfp36l2 in Pax7-expressing cells. In mice, skeletal strated that Pax7-expressing cells contribute little to muscle progenitor cells arise in the dermomyotome dur- embryonic development, but show that Pax7 expression ing E9 and E12 of embryonic development, and specific- is required for adult myogenesis and specification of sat- ally, Pax7 is first expressed in muscle progenitor cells in ellite cells [3, 5, 32]. Further work is required to deter- the central regions of the dermomyotome at around E10 mine whether the observed phenotypes are due to [2, 4, 30, 31]. In our model, Zfp36l1 and/or Zfp36l2 defects in the Pax7 cell population at earlier develop- would therefore be deleted in Pax7-expressing progeni- mental stages. tor cells during the development of the dermomyotome, In adults, studies suggest that a decrease in myofi- as well as in Pax7-expressing cells in adults. bre volume may be related to a decline in nuclei Mice with targeted mutation of either RBP in their number and more specifically a decrease in satellite Pax7-expressing cells were viable and did not display cells, which could delay myonuclei turnover . As any whole body growth or muscle mass defects (Add- satellite cells are required for post-natal growth, we itional file 2). Thus, the expression and activity of explored this possibility by assessing whether Zfp36l1 Pax7Cre, or the loss of Zfp36l1 or Zfp36l2, do not pre- and Zfp36l2 could regulate the satellite cell popula- vent mouse development or affect whole body growth. tion. Transverse cross sections of the TA muscle Mice lacking both Zfp36l1 and Zfp36l2 in were immunostained with anti-Pax7 and were used Pax7-expressing cells (hereafter called Zfp36L1/L2-P) to quantify the number of satellite cells per field of were viable, but were severely growth-retarded com- view (Fig. 2 and Additional file 2). Quantifying and pared to Cre-negative littermates (hereafter called con- investigating satellite cell fate decisions on single trol; Fig. 1b). Whole body growth retardation was myofibres is a well-established and useful method of apparent from 3 weeks of age and continued to adult- analysing a range of satellite cell dynamics [34–36]. hood in both male and female mice (Fig. 1b). Further- However, it was not possible to determine the num- more, both the TA and gastrocnemius muscles from ber of satellite cells or to examine satellite cell fate Zfp36L1/L2-P mice were significantly reduced in weight in single myofibres isolated from Zfp36L1/L2-P mice. compared to the same muscles from the control mice. In our experiments, we found that Zfp36L1/L2-P Satellite cells isolated from Zfp36L1/L2-P adult mice myofibres could not be successfully cultured and sat- contained no detectable ZFP36L1 or ZFP36L2 protein ellite cells seemed to ‘wash away’. This could be in- indicating effective ablation of both proteins (Fig. 1a and dicative of an intrinsic inability of satellite cells from Additional file 1). However, further examination of the Zfp36L1/L2-P mice to associate with the myofibre embryonic developmental stages from when Pax7 is first under these conditions. Further work is needed to expressed is required to determine when precisely the investigate this more thoroughly. Zfp36l1 and Zfp36l2 genes are deleted and any effects of The deletion of Zfp36l1 alone in Pax7-expressing cells this on the developing embryo. We did not establish did not result in a change in satellite cell number in TA whether ZFP36 was expressed in the isolated satellite cross sections, and deletion of Zfp36l2 had little, if any, cells from Zfp36L1/L2-P mice, but at the genetic level it effect on the number of satellite cells (Additional file 2). was unable to compensate for the loss of Zfp36l1 and Due to the whole body growth difference in the Bye-A-Jee et al. Skeletal Muscle (2018) 8:37 Page 5 of 12 Fig. 1 ZFP36L1 and ZFP36L2 are both required for whole body growth. Characterisation of Zfp36L1/L2-P mice. Controls represent Cre-negative littermates. a Western blot showing the ablation of ZFP36L1 and ZFP36L2 in isolated satellite cells from Zfp36L1/L2 mice (see also Additional file 1). b Weights of male and female Zfp36L1/L2-P and control mice measured from 10 days to 45 days of age. Error bars represent SEM, p < 0.0001**** measured by two-way ANOVA with Tukey’s multiple comparison test, n =10. c Weights of tibialis anterior (TA) and gastrocnemius hind limb skeletal muscle from 3- to 4-week-old Zfp36L1/L2-P and control mice in milligrammes. Significance was measured by unpaired, two-tailed Mann Whitney test, n = 8 for control; n = 7 for Zfp36L1/L2-P. d Transverse cross sections of TA muscles were stained with haematoxylin (H; myofibre; pink) and eosin (E; nuclei; purple), and immunostained with antibodies against laminin (green), an extracellular matrix protein to delineate the periphery of the individual muscle fibres and with DAPI (blue), a nuclear marker. Scale bars: 100 μm. H and E images were acquired using a × 20 objective lens, and immunofluorescence images were acquired using a × 40 objective lens. Images representative of n =3 Zfp36L1/L2-P mice, the number of satellite cells was compared to controls (Fig. 2). Whilst we only focused quantified at two post-natal developmental time points on the adult population of Pax7-expressing cells, it is to determine if there was a change in the number of sat- feasible that reduced muscle mass and reduced satellite ellite cells with age. At both 3 and 7 weeks of age, the cell number in adult skeletal muscle may be due to de- number of satellite cells per field of view was signifi- fects and depletion of the Pax7-expressing cell pool early cantly decreased in TAs from the Zfp36L1/L2-P mice in embryonic development. Nevertheless, we conclude Bye-A-Jee et al. Skeletal Muscle (2018) 8:37 Page 6 of 12 Fig. 2 Deleting ZFP36L1 and ZFP36L2 in Pax7-expressing cells reduces the number of satellite cells in TA muscles. Transverse cross sections from 3- to 4-week-old Zfp36L1/L2-P and control mice. Controls represent Cre-negative littermates. a Sections were immunostained with antibodies against Pax7 (red), a marker of adult satellite cells, laminin (green), an extracellular matrix protein to delineate the periphery of the individual muscle fibres, and with DAPI (blue), a nuclear marker. Open arrows indicate Pax7+ cells. Scale bars: 100 μm. b Average number of satellite cells in TAs from control and Zfp36L1/L2-P mice. Error bars represent SEM, significance measured by unpaired two-tailed Student’s t test, n = 3. Calculations from 10 fields of view per experiment that ZFP36L1 and ZFP36L2 are not absolutely required for the progression through the myogenic programme. to establish skeletal muscle and suggest that these RBPs Redundancy between Zfp36l1 and Zfp36l2 has been may have a role in Pax7-expressing cells in adult skeletal demonstrated in the development of lymphocytes muscle. Further work is required to establish the myo- [19–21]. ZFP36L1 and ZFP36L2 both contain two highly genic potential of the mutant Pax7-expressing cells at all conserved zinc finger domains with similar RNA-binding stages of development. The requirement for Zfp36l1 and properties . Their homology and presumably similar Zfp36l2 in satellite cell function is unresolved. The data mRNA-binding specificities provide the simplest explan- suggest there is a redundant requirement for Zfp36l1 ation for the observed redundancy, but this requires and Zfp36l2 for the expansion of progenitor cells and/or formal demonstration. Bye-A-Jee et al. Skeletal Muscle (2018) 8:37 Page 7 of 12 In addition to a requirement for post-natal growth, and there was no visible change in myofibre arrange- satellite cellsalsohavearolein muscleregeneration ment or obvious fibrosis throughout the injury time following injury [25, 37–40]. To examine the regen- course (Fig. 3 and Additional file 3). TAs injected erative capacity of skeletal muscle from Zfp36L1/L2-P with the vehicle control was thus valid uninjured con- mice, 6–12-week-old mice were challenged with cardi- trols. H and E staining of CTX-injured TAs from otoxin (CTX), an agent that induces muscle degener- mice lacking either Zfp36l1 or Zfp36l2 in Pax7-ex- ation, but leaves satellite cells intact (Fig. 3 and pressing cells was indistinguishable from controls Additional file 3). CTX was administered locally into (Additional file 3). the TA muscle, and a PBS “vehicle control” was ad- TAs from control and Zfp36L1/L2-P mice injected ministered into the contralateral muscle. Muscles with CTX demonstrated striking morphological were recovered at 1, 5, 10, or 25 days following treat- changes characterised by myofibre degeneration and ment, and transverse cross sections were stained with an increased number of nuclei, indicative of infiltrat- H and E to assess the extent of damage to the skel- ing leukocytes and fibrosis (Fig. 3). Typically, in the etal muscle architecture (Fig. 3a and Additional file 3), injured control TAs at 1 day post-injury, myofibres and Van Gieson’s stain, which stains collagen and is dissociated from their regular arrangement; at 5 and an indicator of fibrosis (Fig. 3b). TA muscles from 10 days post-injury, there was further myofibre both mutant and control mice that had been injected disruption and increased leukocyte infiltration (in- with the vehicle control did not exhibit any injury ferred from the staining of nuclei) as the muscle Fig. 3 Deleting ZFP36L1 and ZFP36L2 in Pax7-expressing cells impairs skeletal muscle regeneration. The TA muscles of Zfp36L1/L2-P mice were injected with either CTX or PBS. Muscles were harvested at 1, 5, 10 or 25 days following treatment and transverse cross sections were generated. Controls represent Cre-negative littermates. a Sections were stained with haematoxylin (H; myofibre; pink) and eosin (E; nuclei; purple) to assess the skeletal muscle architecture. Open arrows identify centrally located nuclei, an indication of muscle regeneration. b Sections were Van Gieson’s stain to determine the extent of fibrosis. Closed arrows indicate fibrotic tissue (red). Insets of respective PBS-injected, uninjured contralateral TA muscles are in the left-hand corner of each image. Scale bars: 100 μm. Images representative of n = 3 at each time point Bye-A-Jee et al. Skeletal Muscle (2018) 8:37 Page 8 of 12 degenerated; and at 25 days post-injury, myofibre re- impaired regeneration may be due to defects in the generation was apparent by the presence of myofi- function of myogenic cells derived from Pax7-expressing bres that harboured nuclei, which were centrally progenitor cells. located, and fewer nuclei of infiltrating cells (Fig. 3). To investigate the requirement for Zfp36l1 and TAs from Zfp36L1/L2-P mice at day 1 following Zfp36l2 further, transverse cross sections from injured muscle injury, appeared similar to injured TAs from control and Zfp36L1/L2-P were immunostained with control mice. At 5 and 10 days post-injury, muscle antibodies against myogenin (Fig. 4a), which is required regeneration appeared delayed and there was in- for myoblasts to differentiate further into myofibres. In creased collagen staining in between myofibres com- injured TAs from control and Zfp36L1/L2-P mice, there pared to controls (Fig. 3b). At 25 days post-injury, was a peak in the number of myogenin+ cells at the 5 there were centrally located nuclei within myofibres, day time point (Fig. 4b). At the 10 and 25 day time indicating regeneration. However, this regeneration points, the numbers of myogenin+ cells in the injured appeared to be reduced in comparison to control TAs from Zfp36L1/L2-P mice were similar to those in TAs (Fig. 3a). As we deleted ZFP36L1 and ZFP36L2 the injured control TAs (Fig. 4b). Whilst we are unable in Pax7-expressing cells, it is possible that the to exclude the possibility that a difference might have Fig. 4 Terminal differentiation can occur independently of ZFP36L1 and ZFP36L2. Transverse cross sections of injured TAs from Zfp36L1/L2-P mice were immunostained with antibodies for Pax7 (red), myogenin (myog; turquoise), laminin (lam; green), and counterstained with the nuclear marker DAPI (blue). Controls represent Cre-negative littermates. a Representative images of injured control and Zfp36L1/L2-P TAs. Insets represent PBS-injected, uninjured contralateral TA muscles. Open arrows indicate myogenin+ myogenic cells (myog; turquoise). Scale bars: 100 μm. b Average number of myogenin+ cells present in injured control and Zfp36L1/L2-P TAs during the injury time course. Images were acquired using a × 40 objective lens. Calculations were based on 10 fields of view per experiment. Error bars represent the SEM, two-way ANOVA adjusted for multiple comparisons with Sidak’s post hoc test, n =3 Bye-A-Jee et al. Skeletal Muscle (2018) 8:37 Page 9 of 12 Fig. 5 (See legend on next page.) Bye-A-Jee et al. Skeletal Muscle (2018) 8:37 Page 10 of 12 (See figure on previous page.) Fig. 5 Deleting ZFP36L1 and ZFP36L2 in Pax7-expressing cells impairs expansion of the satellite cell pool during skeletal muscle regeneration. Transverse cross sections of injured TAs from Zfp36L1/L2-P mice were immunostained with antibodies for Pax7 (red), MyoD (turquoise), Laminin (lam; green), and counterstained with the nuclear marker DAPI (blue). Activated cells were co-stained with Pax7 and MyoD (white). Controls represent Cre-negative littermates. a Representative images of injured control and Zfp36L1/L2-P TAs. Insets represent PBS-injected, uninjured contralateral TA muscles. Open arrows indicate Pax7+ satellite cells (red); closed arrows indicate MyoD+ myogenic cells (turquoise), and dotted arrows indicate Pax7 +MyoD+ activated satellite cells (white). Scale bars: 100 μm. b Average number of satellite cells present in injured control and Zfp36L1/L2-P TAs during the injury time course. c Average number of activated cells present in injured control and Zfp36L1/L2-P TAs during the injury time course. d Average number of MyoD+ cells present in injured control and Zfp36L1/L2-P TAs during the injury time course. Images were acquired using a × 40 objective lens. Calculations from 10 fields of view per experiment. Error bars represent the SEM, two-way ANOVA adjusted for multiple comparisons with Sidak’s post hoc test, n =3 been found with a larger sample size, there was little utilisation of genetic tools which permit elective dele- effect, if any, of the deletion of Zfp36l1 and Zfp36l2 on tion of Zfp36l1 and Zfp36l2 in Pax7-expressing cells terminal differentiation at day 5. We show that muscle [41, 42] would offer an opportunity to clearly define from Zfp36L1/L2-P mice has a diminished regenerative the functional properties of the adult satellite cell capacity and suggest that this may reflect the reduced population independently of developmental effects. availability or function of satellite cells. Furthermore, the measurement of mRNA abundance To assess the satellite cell population, CTX-injured could reveal the effect these proteins have on gene TAswerestained during theinjurytime coursewith expression and allow for mechanisms of action to be anti-Pax7 to identify quiescent satellite cells and ascertained. We did not establish mRNA targets for self-renewed satellite cells, and anti-MyoD to identify ZFP36L1 and ZFP36L2 in our system. Interestingly, the muscle lineage committed cells (myoblasts). Cells closely related ZFP36 RBP has been shown to regulate the co-staining with anti-Pax7 and anti-MyoD were expression of MyoD mRNA , and it is feasible that considered to be activated (Pax7+MyoD+) satellite ZFP36L1 and ZFP36L2 also regulate MyoD expression in cells (Fig. 5a). At each time point, we quantified the myogenic cells. The application of methods that measure number of Pax7+, MyoD+ and Pax7+MyoD+ cells in RNA-protein interactions in intact cells could be sections from injured and uninjured TAs in control optimised and applied to satellite cells and their myogenic and Zfp36L1/L2-P mice. In the TAs from control derivatives from adults to identify the direct targets and mice at days 5 and 10 post-injury, the Pax7+ satellite thereby develop the understanding of gene regulation by cellpoolexpanded(Fig. 5b), the number of activated RBPs in skeletal muscle. (Pax7+MyoD+) satellite cells increased (Fig. 5c), and there was an increased number of MyoD+ cells (Fig. 5d). Conclusions At 25 days post-injury, the number of Pax7+ satellite We show that Zfp36l1 and Zfp36l2 act redundantly to cells returned to uninjured levels and the number of determine the number of functional Pax7-expressing activated (Pax7+MyoD+) satellite cells and MyoD+ cells in adult mice. The lack of a phenotypic effect of the cells reduced to nearly that of uninjured TAs. This targeting by Pax7cre of Zfp36l1 or Zfp36l2 alone on suggests the return of satellite cells to quiescence and skeletal muscle argues against the phenotypes being due almost complete regeneration (Fig. 5b). By contrast, to the non-specific effects of cre. The requirement for in injured TAs from Zfp36L1/L2-P mice, the pool of Zfp36l1 or Zfp36l2 in Pax7-expressing cells is important Pax7+ satellite cells did not expand, and there were for the growth and regenerative capacity of adult skeletal negligible numbers of activated (Pax7+MyoD+) satellite muscle. The mechanisms by which these RBPs regulate cells (Fig. 5b, c). There was an increase in the number of the function and fate of Pax7-expressing cells remains to MyoD+ cells at the 5 day post-injury time point (Fig. 5d). be established, but in other systems, these RBP regulate This increase in MyoD+ cells may contribute to the quiescence  and cellular identity [22, 44]. Our find- capacity of injured TAs from Zfp36L1/L2-P mice to ings should prompt further experiments designed to undergo regeneration. understand how ZFP36L1 and ZFP36L2 control cell fate In our model, Zfp36l1 and Zfp36l2 are deleted in decisions and the kinetics of cell turnover. Pax7-expressing cells throughout development; thus, we can not rule out the possibility that the regenera- Additional files tive defect could be as a result of a requirement for the RBPs in Pax7-expressing cells in the dermomyo- Additional file 1: Confirmation of ablation of ZFP36L1 and ZFP36L2 in tome and/or Pax7-derived myogenic cells. Here, we satellite cells of Zfp36L1/L2-P mice. Western blot determining the expression of ZFP36L1 and ZFP36L2 in isolated satellite cells from Zfp36L1/L2-P and used thePax7Cremouselinetoaddress rolesfor control mice. (TIF 76 kb) ZFP36L1 and ZFP36L2 in myogenesis. In future, the Bye-A-Jee et al. Skeletal Muscle (2018) 8:37 Page 11 of 12 Publisher’sNote Additional file 2: Characterisation of mice lacking either Zfp36l1 or Zfp36l2 Springer Nature remains neutral with regard to jurisdictional claims in (referred to as Zfp36L1-P or Zfp36L2-P mice). A. Weights of male and female published maps and institutional affiliations. mice measured from 10 to 45 days. Error bars represent SEM, two- way ANOVA with Tukey’s multiple comparison test, n = 10. B. TA Author details and gastrocnemius muscle weights from 7-week-old male and female mice. Laboratory of Cell Signalling, The Babraham Institute, Cambridge, UK. Significance was measured by unpaired two-tailed Mann Whitney test; n =6 Laboratory of Lymphocyte Signalling and Development, The Babraham for Zfp36L1-P and its respective control, and n = 8 for Zfp36L2-P and Institute, Cambridge, UK. Department of Pharmacology, University of its control. Red data points indicate female mice and blue data points indicate Cambridge, Tennis Court Road, Cambridge, UK. Present address: European male mice for the Zfp36L2-P graph. C. Average number of satellite cells in Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), cross-sections of TAs from 7-week-old control, Zfp36L1-P and Zfp36L2-P mice. Wellcome Genome Campus, Hinxton, Cambridge, UK. Present address: Error bars represent SEM, significance was measured by unpaired two-tailed Illumina Cambridge Ltd, Chesterford Research Park, Little Chesterford, Saffron Student’s t test; n = 5 for Zfp36L1-P and its respective control, and n =4 for Walden, Essex, UK. Present address: University of Oxford Medical School, Zfp36L2-P and its control. Controls represent Cre-negative littermates. Calculations Medical Sciences Division, University of Oxford, John Radcliffe Hospital, based on 10 fields of view per experiment. (TIF 201 kb) Oxford, UK. Present address: Human Genetics, Wellcome Trust Sanger Additional file 3: Transverse cross sections of the TA muscles from mice Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK. lacking either Zfp36l1 or Zfp36l2 (referred to as Zfp36L1-P or Zfp36L2-P mice), and their respective controls, recovered at 1, 5, 10 and 25 days Received: 19 July 2018 Accepted: 22 November 2018 following injection with either CTX or PBS, stained with haematoxylin (H; myofibre; pink) and eosin (E; nuclei; purple) to assess the skeletal muscle architecture. Open arrows identify centrally located nuclei, an indication of muscle regeneration. Controls represent Cre-negative littermates. References Scale bars: 100 μm. Representative of n = 3. (TIF 843 kb) 1. Hausburg MA, Doles JD, Clement SL, Cadwallader AB, Hall MN, Blackshear PJ, Lykke-Andersen J, Olwin BB. Post-transcriptional regulation of satellite cell quiescence by TTP-mediated mRNA decay. eLife. 2015;4:e03390. Abbreviations 2. Relaix F, Rocancourt D, Mansouri A, Buckingham M. A Pax3/Pax7- CTX: Cardiotoxin; Lam: Laminin; Myog: Myogenin; RBP: RNA-binding proteins; dependent population of skeletal muscle progenitor cells. Nature. 2005; TA: Tibialis anterior; TTP: Tristetraproline 435(7044):948–53. 3. Hutcheson DA, Zhao J, Merrell A, Haldar M, Kardon G. Embryonic and fetal Acknowledgements limb myogenic cells are derived from developmentally distinct progenitors Jennifer Pell passed away before the submission of the final version of this and have different requirements for beta-catenin. Genes Dev. 2009;23(8): manuscript. We thank Dr. Geoffrey Butcher and Dr. Anne Corcoran for 997–1013. comments on the manuscript, Mario Capecci for the Pax7-Cre mouse strain, 4. Kassar-Duchossoy L, Giacone E, Gayraud-Morel B, Jory A, Gomes D, the Babraham Institute’s Biological Support Unit, Geoffrey Morgan and Arthur Tajbakhsh S. Pax3/Pax7 mark a novel population of primitive myogenic cells Davies in the Flow Cytometry Core Facility, Dr. Anne Segonds-Pichon for the during development. Genes Dev. 2005;19(12):1426–31. assistance with the statistics and Dr. Simon Walker in the Imaging Facility for 5. Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, Rudnicki MA. the expert technical advice and assistance. Pax7 is required for the specification of myogenic satellite cells. Cell. 2000; 102(6):777–86. Funding 6. Mauro A. Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol. This work was funded by the Biotechnology and Biological Sciences 1961;9:493–5. Research Council grants BBS/E/B/000C0427 and BBS/E/B/000C0433. 7. Schultz E, Gibson MC, Champion T. Satellite cells are mitotically quiescent in mature mouse muscle: an EM and radioautographic study. J Exp Zool. 1978; Availability of data and materials 206(3):451–6. The datasets used and/or analysed during the current study are available 8. Collins CA, Olsen I, Zammit PS, Heslop L, Petrie A, Partridge TA, Morgan JE. from the corresponding authors on reasonable request. Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell. 2005;122(2):289–301. 9. Yin H, Price F, Rudnicki MA. Satellite cells and the muscle stem cell niche. Authors’ contributions Physiol Rev. 2013;93(1):23–67. HB designed and implemented all aspects of the study and drafted and edited 10. Tedesco FS, Dellavalle A, Diaz-Manera J, Messina G, Cossu G. Repairing the manuscript. DP assisted in the muscle tissue collection and satellite cell skeletal muscle: regenerative potential of skeletal muscle stem cells. J Clin isolation and provided the protocols and experimental design advice. Invest. 2010;120(1):11–9. SW optimised and provided the protocols and experimental design 11. Wozniak AC, Anderson JE. Nitric oxide-dependence of satellite stem cell advice. PB provided experimental design advice. RW provided the activation and quiescence on normal skeletal muscle fibers. Dev Dynamics. protocols and experimental design advice. MT provided the conditional knockout 2007;236(1):240–50. mice, SB1/30.13 antibodies, protocols and study advice and drafted and edited 12. Braun T, Gautel M. Transcriptional mechanisms regulating skeletal muscle the manuscript. JP designed the study and provided the resources and laboratory differentiation, growth and homeostasis. Nat Rev Mol Cell Biol. 2011;12(6): for the implementation of the study. JP passed away before the production of the 349–61. final manuscript, but all other authors, including the family of JP, approved the 13. Cooper RN, Tajbakhsh S, Mouly V, Cossu G, Buckingham M, Butler-Browne final manuscript. GS. In vivo satellite cell activation via Myf5 and MyoD in regenerating mouse skeletal muscle. J Cell Sci. 1999;112(Pt 17):2895–901. Ethics approval and consent to participate 14. Farina NH, Hausburg M, Betta ND, Pulliam C, Srivastava D, Cornelison D, Procedures were approved by the Babraham Institute Animal Welfare, Olwin BB. A role for RNA post-transcriptional regulation in satellite cell Experimentation and Ethics Committee and licenced under the Animals activation. Skelet Muscle. 2012;2(1):21. (Scientific procedures) Act 1986 and EU Directive 2010/63/EU. 15. Apponi LH, Corbett AH, Pavlath GK. RNA-binding proteins and gene regulation in myogenesis. Trends Pharmacol Sci. 2011;32(11):652–8. Consent for publication 16. Cammas A, Sanchez BJ, Lian XJ, Dormoy-Raclet V, van der Giessen K, Lopez Consent for the publication of the manuscript was given by Caitlin Beddows, de Silanes I, Ma J, Wilusz C, Richardson J, Gorospe M, et al. Destabilization of the daughter of the deceased Principal Investigator, Dr. Jennifer Pell. nucleophosmin mRNA by the HuR/KSRP complex is required for muscle fibre formation. Nat Commun. 2014;5:4190. Competing interests 17. Linker K, Pautz A, Fechir M, Hubrich T, Greeve J, Kleinert H. Involvement The authors declare that they have no competing interests. of KSRP in the post-transcriptional regulation of human iNOS Bye-A-Jee et al. Skeletal Muscle (2018) 8:37 Page 12 of 12 expression-complex interplay of KSRP with TTP and HuR. Nucleic Acids 38. Sambasivan R, Yao R, Kissenpfennig A, Van Wittenberghe L, Paldi A, Gayraud- Res. 2005;33(15):4813–27. Morel B, Guenou H, Malissen B, Tajbakhsh S, Galy A. Pax7-expressing satellite 18. Lai WS, Carballo E, Thorn JM, Kennington EA, Blackshear PJ. Interactions of cells are indispensable for adult skeletal muscle regeneration. Development. CCCH zinc finger proteins with mRNA. Binding of tristetraprolin-related zinc 2011;138(17):3647–56. finger proteins to Au-rich elements and destabilization of mRNA. J Biol 39. Finnerty CC, McKenna CF, Cambias LA, Brightwell CR, Prasai A, Wang Y, El Chem. 2000;275(23):17827–37. Ayadi A, Herndon DN, Suman OE, Fry CS. Inducible satellite cell depletion attenuates skeletal muscle regrowth following a scald-burn injury. J Physiol. 19. Hodson DJ, Janas ML, Galloway A, Bell SE, Andrews S, Li CM, Pannell R, Siebel 2017;595(21):6687–701. CW, MacDonald HR, De Keersmaecker K, et al. Deletion of the RNA-binding 40. Zhu H, Xiao F, Wang G, Wei X, Jiang L, Chen Y, Zhu L, Wang H, Diao Y, proteins ZFP36L1 and ZFP36L2 leads to perturbed thymic development and T Wang H, et al. STAT3 regulates self-renewal of adult muscle satellite cells lymphoblastic leukemia. Nat Immunol. 2010;11(8):717–24. during injury-induced muscle regeneration. Cell Rep. 2016;16(8):2102–15. 20. Galloway A, Saveliev A, Lukasiak S, Hodson DJ, Bolland D, Balmanno K, 41. Murphy MM, Lawson JA, Mathew SJ, Hutcheson DA, Kardon G. Satellite Ahlfors H, Monzon-Casanova E, Mannurita SC, Bell LS, et al. RNA-binding cells, connective tissue fibroblasts and their interactions are crucial for proteins ZFP36L1 and ZFP36L2 promote cell quiescence. Science. 2016; muscle regeneration. Development. 2011;138(17):3625–37. 352(6284):453–9. 42. Lepper C, Fan CM. Generating tamoxifen-inducible Cre alleles to investigate 21. Vogel KU, Bell LS, Galloway A, Ahlfors H, Turner M. The RNA-binding myogenesis in mice. Methods Mol Biol. 2012;798:297–308. proteins Zfp36l1 and Zfp36l2 enforce the Thymic beta-selection checkpoint 43. Ule J, Hwang HW, Darnell RB. The future of cross-linking and by limiting DNA damage response signaling and cell cycle progression. J immunoprecipitation (CLIP). Cold Spring Harb Perspect Biol. 2018;10(8): Immunol. 2016;197(7):2673–85. a032243. 22. Newman R, Ahlfors H, Saveliev A, Galloway A, Hodson DJ, Williams R, Besra 44. Dumdie JN, Cho K, Ramaiah M, Skarbrevik D, Mora-Castilla S, Stumpo DJ, GS, Cook CN, Cunningham AF, Bell SE, et al. Maintenance of the marginal- Lykke-Andersen J, Laurent LC, Blackshear PJ, Wilkinson MF, et al. Chromatin zone B cell compartment specifically requires the RNA-binding protein modification and global transcriptional silencing in the oocyte mediated by ZFP36L1. Nat Immunol. 2017;18(6):683–93. the mRNA decay activator ZFP36L2. Dev Cell. 2018;44(3):392–402 e397. 23. Stumpo DJ, Broxmeyer HE, Ward T, Cooper S, Hangoc G, Chung YJ, Shelley WC, Richfield EK, Ray MK, Yoder MC, et al. Targeted disruption of Zfp36l2, encoding a CCCH tandem zinc finger RNA-binding protein, results in defective hematopoiesis. Blood. 2009;114(12):2401–10. 24. Keller C, Hansen MS, Coffin CM, Capecchi MR. Pax3:Fkhr interferes with embryonic Pax3 and Pax7 function: implications for alveolar rhabdomyosarcoma cell of origin. Genes Dev. 2004;18(21):2608–13. 25. Woodhouse S, Pugazhendhi D, Brien P, Pell JM. Ezh2 maintains a key phase of muscle satellite cell expansion but does not regulate terminal differentiation. J Cell Sci. 2013;126(Pt 2):565–79. 26. Paxton CW, Cosgrove RA, Drozd AC, Wiggins EL, Woodhouse S, Watson RA, Spence HJ, Ozanne BW, Pell JM. BTB-Kelch protein Krp1 regulates proliferation and differentiation of myoblasts. Am J Physiol Cell physiol. 2011;300(6):C1345–55. 27. Stumpo DJ, Byrd NA, Phillips RS, Ghosh S, Maronpot RR, Castranio T, Meyers EN, Mishina Y, Blackshear PJ. Chorioallantoic fusion defects and embryonic lethality resulting from disruption of Zfp36L1, a gene encoding a CCCH tandem zinc finger protein of the Tristetraprolin family. Mol Cell Biol. 2004; 24(14):6445–55. 28. Bell SE, Sanchez MJ, Spasic-Boskovic O, Santalucia T, Gambardella L, Burton GJ, Murphy JJ, Norton JD, Clark AR, Turner M. The R ng protein Zfp36l1 is required for normal vascularisation and post-transcriptionally regulates VEGF expression. Dev Dynamics. 2006;235(11):3144–55. 29. Ramos SB, Stumpo DJ, Kennington EA, Phillips RS, Bock CB, Ribeiro-Neto F, Blackshear PJ. The CCCH tandem zinc-finger protein Zfp36l2 is crucial for female fertility and early embryonic development. Development. 2004; 131(19):4883–93. 30. Fan CM, Tessier-Lavigne M. Patterning of mammalian somites by surface ectoderm and notochord: evidence for sclerotome induction by a hedgehog homolog. Cell. 1994;79(7):1175–86. 31. Tajbakhsh S, Cossu G. Establishing myogenic identity during somitogenesis. Curr Opin Genet Dev. 1997;7(5):634–41. 32. von Maltzahn J, Jones AE, Parks RJ, Rudnicki MA. Pax7 is critical for the normal function of satellite cells in adult skeletal muscle. Proc Natl Acad Sci U S A. 2013;110(41):16474–9. 33. Brack AS, Bildsoe H, Hughes SM. Evidence that satellite cell decrement contributes to preferential decline in nuclear number from large fibres during murine age-related muscle atrophy. J Cell Sci. 2005;118(Pt 20):4813–21. 34. Brun CE, Wang YX, Rudnicki MA. Single EDL myofiber isolation for analyses of quiescent and activated muscle stem cells. Methods Mol Biol. 2018;1686:149–59. 35. Pasut A, Jones AE, Rudnicki MA. Isolation and culture of individual myofibers and their satellite cells from adult skeletal muscle. J Visualized Exp. 2013;73:e50074. 36. Gallot YS, Hindi SM, Mann AK, Kumar A. Isolation, culture, and staining of single myofibers. Bio-protocol. 2016;6(19):e1942. 37. Lepper C, Partridge TA, Fan CM. An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration. Development. 2011;138(17):3639–46.
Skeletal Muscle – Springer Journals
Published: Dec 7, 2018