MEF2-dependent transcriptional control of Srpk3 in mouse embryos. (a) Schematic representation of Srpk3-HSP68-LacZ reporter constructs and results of F0 transgenic mouse analysis. The number of embryos that showed representative expression patterns and total transgenic embryos are shown on the right. Representative results are shown in panels b–e. The 2.9- and 0.4-kb fragments drove LacZ expression in the heart and somites in E11.5 embryos (b,c, respectively). The MEF2 site mutation (d), but not the E-box mutations (e), abolished the LacZ activity both in the heart and somites.

Muscle-specific expression of Srpk3. (a) Expression of the SRPK family in mouse embryos. Whole-mount and section in situ hybridization. Srpk3 is expressed exclusively in the developing heart, somites, and embryonic skeletal muscle. Srpk1 and Srpk2 are widely expressed in embryonic tissues, with enrichment in the neural tube and brain. Enlargements of the heart from each stage are shown to the right. (b) Expression of the SRPK family in adult mice. Srpk3 is specifically expressed in the heart and skeletal muscle, with a faint expression in the spleen. Testis-enriched expression of Srpk1 and Srpk2 is also shown below. With longer exposure, lower levels of Srpk1/-2 expression were observed ubiquitously.

Muscle defects in MCK–Srpk3 transgenic mice. (a,b) Muscle creatine kinase (MCK)–Srpk3 transgenic (Tg) mice showed significant growth retardation and muscle defects compared with nontransgenic (nTg) littermates at 1 mo of age. Bar in panel a represents 1 cm. (c) Histological analysis of MCK–Srpk3 Tg mice showed severe muscle degeneration with myofiber disarray, increase in centrally placed nuclei, and fibrosis. Hematoxylin & eosin (H&E), NADH-tetrazolium reductase (NADH-TR), and modified Gomori Trichrome (mGT) staining of gastrocnemius muscle. H&E staining of nTg control is also shown. Bar, 100 μm. (d) Muscle regeneration markers, Myh3 and Myh8, are up-regulated in the skeletal muscle of MCK–Srpk3 Tg mice. Similar results were obtained using F1 mice derived from two F0 founders of MCK–Srpk3 transgenic mice.

Identification of Stk23/Srpk3 as a novel SRPK. (a) Microarray analysis was performed using the hearts of E9.0 Mef2c-null and wild-type embryos. Down-regulation of the Stk23/Srpk3 expression in the Mef2c-null hearts was confirmed by RT–PCR. (Gapd) Glyceraldehyde-3-phosphate dehydrogenase expression as a control. (b) Amino acid structure of mouse Stk23/Srpk3. Srpk3 is a 566-amino-acid protein that contains a bipartite kinase domain (underlined), N terminus, and a hinge region. (c) Schematic representation of mouse SRPK family kinases. Srpk3 shows high sequence similarity to Srpk1 and Srpk2 in the kinase domain, but not in the N terminus and hinge region. The percentages in the boxes are identities of Srpk1 and Srpk2 to Srpk3 at an amino acid level. Srpk3 and Srpk2 have serine- and proline-rich sequences in the N terminus, respectively. Amino acid numbers are also shown. (d) Phosphorylation assays using SR domain proteins. Srpk3 phosphorylated SF2/ASF and the N-terminal region of Lamin B Receptor (LBR) in vitro.

MEF2-dependent muscle-specific transcription of Srpk3. (a) The structure of the mouse Srpk3 gene with schematics of the luciferase constructs and the results of luciferase assays are shown. Filled and open boxes indicate the exons for protein-coding and noncoding regions, respectively. MEF2C and MyoD strongly activate luciferase reporter expression controlled by the Srpk3 enhancer/promoter in C2C12 myoblasts. The 0.4-kb fragment is sufficient for the response. (b) Sequence of the 0.4-kb minimal muscle enhancer/promoter of Srpk3. The MEF2-binding site, E boxes, and GATA-binding sites are underlined. The putative transcriptional start site estimated by the most 5′-end of Srpk3 cDNA clones is indicated by an arrow. The translational start site (ATG) is italicized. (c) MEF2C and the myogenin/E12 complex bind to the fragments encompassing the MEF2 site and E boxes in the minimal muscle enhancer of Srpk3. Electrophoretic mobility shift assay. (Lane 1) Control. (Lane 2) MEF2C. (Lane 3) Myogenin and E12. (Lane 4) MEF2C, myogenin, and E12. (d) Mutation of the MEF2-binding site in the context of the 2.9- or 0.4-kb Srpk3 fragments significantly impairs the response to MEF2C and MyoD in luciferase assays. (e) Srpk3 expression is activated by 1 d after stimulation during myogenic conversion of C2C12 cells. Expression patterns of embryonic and perinatal myosins, Myh3 and Myh8, respectively, are also shown. (Hprt1) Hypoxanthine guanine phosphoribosyl transferase 1 as a control. (f) Activity of the Srpk3 luciferase reporters is markedly stimulated during myogenic conversion of C2C12 cells, while mutation of the MEF2-binding site almost abolishes it.

Expression profiles of muscle genes in Srpk3-null mice Expression of various muscle genes in the tibialis anterior muscle of Srpk3-null mice (KO) and wild-type control (WT). Ethidium bromide staining of RT–PCR products or phosphorimages of Northern blot hybridization are shown. (a) Slow- and fast-twitch myofiber markers. (b) Atrophy-related proteins. (c) Molecules related to calcium handling. At least three mice for each genotype were examined, and two representative results are shown. Gene names: (Atp2a1) ATPase, Ca⁺⁺ transporting, cardiac muscle, fast twitch 1 or Serca1; (Atp2a2) ATPase, Ca⁺⁺ transporting, cardiac muscle, slow twitch 2 or Serca2; (Capn1/-3) Calpain 1/-3; (Cilp), cartilage intermediate layer protein; (Ctsl) Cathepsin L; (Fbxo32) F-box-only protein 32, MAFbx or Atrogin-1; (Foxo3/-4) Forkhead box o3/o4; (Igfbp5) insulin-like growth factor-binding protein 5; (Mb) myoglobin; (Myl2) myosin, light polypeptide 2, regulatory, cardiac, slow; (Mylpf) myosin light chain, phosphorylatable, fast skeletal muscle; (Pln) Phospholamban; (Slc2a4) solute carrier family 2 (facilitated glucose transporter), member 4 or Glut4; (Sln) sarcolipin; (Tnni1) Troponin I, skeletal, slow 1; (Tnni2) Troponin I, skeletal, fast 2; (Tnnt1) Troponin T1, skeletal, slow; (Tnnt3) Troponin T3, skeletal, fast; (Trim63) tripartite motif-containing 63 or MuRF1.

Centronuclear myopathy of Srpk3-null mice (a) Gene targeting strategy. The mouse Srpk3 gene is shown with the targeting vector and the targeted allele. Parts of the exons 1 and 5, and the entire exons 2, 3, and 4 were replaced with the LacZ–neomycin resistance gene (Neo) cassette. Genotypes were determined by Southern blot analysis and PCR. The positions of the 5′ and 3′ Southern probes and PCR primers (5W, 3W, 3L) are shown. (TK) Thymidine kinase; (R) EcoRI; (H) HindIII; (S) SpeI; (V) EcoRV. (b) A representative result of PCR genotyping. (WT) Wild-type mouse; (Het) female heterozygous mouse; (KO) male hemizygously null mouse. (c) The Srpk3 mRNA expression is not detectable in the heart and skeletal muscle of Srpk3-null mice by Northern blot analysis. (d) The weight of various skeletal muscle groups significantly decreased in Srpk3-null mice. (BW) Body weight. Mean ± SD; n = 6; (*) p < 0.01. (E) H&E staining of various muscle groups of Srpk3-null mice. Bars, 100 μm. Percentages of centrally placed nuclei are also shown (n = 400–600). (f) The NADH-TR, ATPase and TUNEL staining, and the result of Evans Blue injection of Srpk3-null mice. ATPase activity stains type 1 fibers dark blue (open triangle), type 2B fibers blue (filled triangle), and type 2A fibers light blue (arrows). Cells in the bone marrow, but not skeletal myocytes, showed positive signals in TUNEL staining. Evans Blue injection showed no apparent abnormality of sarcolemmal integrity in Srpk3-null mice, in contrast to Mdx dystrophic mice shown as a positive control. Bar, 100 μm. Muscle groups: (EDL) extensor digitorum longus; (G/P) gastrocnemius and plantaris; (Quad) quadriceps; (Sol) soleus; (TA) tibialis anterior. (g) Expression of muscle regeneration markers, Myh3 and Myh8, is not activated in Srpk3-null mice, in contrast to Mdx mice. RT–PCR analysis.