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doi: 10.1242/10.1242/dev.00440

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Altered myogenesis in Six1-deficient mice

¹ Département Génétique, Développement et Pathologie Moléculaire, Institut Cochin – INSERM 567, CNRS UMR 8104, Université Paris V, 24 Rue du Faubourg Saint Jacques, 75014 Paris, France
² Plateforme Recombinaison Homologue, Institut Cochin – INSERM 567, CNRS UMR 8104, Université Paris V, 24 Rue du Faubourg Saint Jacques, 75014 Paris, France
³ Plateforme Histologie, Institut Cochin – INSERM 567, CNRS UMR 8104, Université Paris V, 24 Rue du Faubourg Saint Jacques, 75014 Paris, France

View larger version (50K): [in a new window]   Fig. 1. Targeted disruption of the mouse Six1 gene. (A) Schematic representation of the wild-type allele, targeting vector (pPNT) and disrupted allele. The deleted sequence (starting in the 5' non coding region and extending to amino acid 178) codes for the N-terminal part of the Six1 protein, including the Six-domain and the Six-type homeodomain, both involved in specific DNA binding. The white and grey boxes represent the two exons of the Six1 gene with the coding region in grey; the blue box represents the ß-galactosidase reporter gene with the PGK-neomycin cassette downstream. The "NotI" site is a cloning site and thus is not present in the wild-type allele. (B) Phenotype of a newborn Six1–/– mouse (left) and wild-type littermate (right). (C) Southern blot analysis of genomic DNA digested with NcoI and hybridized with a 5' external probe (left) and a 3' external probe (right). (D) Gel-mobility shift assays performed with total protein extracts from E12.5 Six1+/+, Six1+/– and Six1–/– embryos, and with adult muscle nuclear extracts (amne) using a myogenin MEF3 probe. Different DNA/protein complexes can be identified. The amount of Six4 and Six5 DNA binding activity is not diminished in Six1–/– extracts when compared to wild type, while no Six1 DNA binding activity is detected in Six1–/– extracts. Six1 ab: added Six1 antibodies are able to displace specifically the Six1/MEF3 complex. ns: nonspecific protein/DNA interactions.

View larger version (66K): [in a new window]   Fig. 2. Skeletal defects of Six1–/– fetuses revealed by Alizarin Red (skeleton) and Alcian Blue (cartilage) staining. (A,C,E,F) Six1–/– fetuses, (B,D) normal littermates. (A-B) Ventral view and (C-D) lateral view of the trunk skeleton showing malformations including rib bifurcation, fusion of rib cartilage from adjacent ribs (arrows), truncated distal rib segments with defects in the attachment to the sternum (here, only two ribs reach the sternum (arrowhead), and disorganized ossification of the sternum. (E-F) Magnification of adjacent rib fusion and branching in two different Six1–/– fetuses (anterior is left). (E) The cartilage segment of the fifth right rib splits up (arrowhead) before fusing with the forth right rib (asterisk). (F) The sixth to the ninth rib are fused (asterisk). The seventh rib forks at the distal part of the bone segment (arrowhead).

View larger version (101K): [in a new window]   Fig. 3. Severe and selective muscular hypoplasia of Six1–/– fetuses. Histological sections of E18.5 Six1–/– (A,C,E,F,H,J,L,N) and Six1+/– (B,D,G,I,K,M,O) fetuses. (A,B) Haematoxylin-stained transverse sections at the distal forelimb level reveal a drastic reduction of the muscular masses in Six1–/–, compared to normal littermates: most dorsal and ventral masses are missing in Six1–/– embryos; R, radius; U, ulna. (C,D) Transverse sections of distal hindlimb stained with Haematoxylin and fast MHC (MY32) antibody reveal an important hypoplasia and absence of most ventral and intermediate muscle masses in Six1–/– fetus (2, medial and lateral gastrocnemius; 3, soleus), while most dorsal muscles are present but much smaller (1, tibialis anterior); T,- tibia; F, fibula. (C2-C4 and D2-D4) Higher magnifications of C1 and D1, respectively, at the tibialis anterior level. While the size of tibialis anterior is reduced by approximately 33% in Six1–/– fetuses, the density of the myogenic cells and their size are similar in Six1–/– and Six1+/– mice. As a result, the total number of myofibers is reduced by approximately 33%. (E) X-gal/Eosin-stained sagittal section at forelimb level of an E18.5 Six1–/– fetus: a strong hypoplasia characterizes both triceps brachii (4) and biceps brachii (5), but tendons (arrows) of these muscles seem correctly developed and attached. (F-I) Immunochemistry and Haematoxylin coloration of triceps muscle sections showing slow (F,G) and fast (H,I) MHC. Whereas this muscle is reduced in size in Six1–/– fetuses, slow and fast myofibers are present in equivalent relative proportions. (J-K) Haematoxylin/Eosin-stained sagittal sections at the thoracic level show that most superficial back muscles (11-13) are strikingly reduced and disorganized in Six1–/– fetuses, whereas intercostal muscles (9-10) are less affected. 9, intercostal interni, 10, intercostal externi, 11, spinotrapezius; 12, latissimus dorsi; 13, serratus dorsalis. (L,M) X-gal/Eosin-stained transverse sections at the head level. The tongue (6) is significantly reduced; the genioglossus muscle (7) is absent, but the masseter (8) seems correctly developed. Note also the Meckel's cartilage hypoplasia (arrows). (N,O) Fast myosin immunochemistry of diaphragm sections. In the Six1–/– embryo the diaphragm is reduced to a thin layer of connective tissue, without any detectable muscle fiber.

View larger version (81K): [in a new window]   Fig. 4. Altered primary myogenesis in Six1–/– embryos. X-gal staining of Six1–/– embryos (A,C,E,G,I,J) and Six1+/– littermates (B,D,F,H). (A,B) At E12.5, the staining observed in Six1–/– embryos is different to that in Six1+/– littermates in few restricted areas: at the limb level the blue staining is reduced in the distal anterior part (*), and at the interlimb level the ventral extension of epithelial somitic buds of the dermomyotome is reduced (arrows). (C,D) At E13.5 in Six1–/– embryos, all body muscles are either absent or severely disorganized, except some deep back and head muscles. (E,F) Transverse sections of E13.5 embryos at the trunk level (dashed line in C and D) show that the external myogenic layer, the cutaneus maximus (arrow), is absent and that the internal myogenic layer is reduced and disorganized. Also specific muscle areas such as the spinotrapezius (double arrow) are missing. (G,H) Detail of E13.5 Six1–/– embryos. Note the absence of most muscles at the shoulder level (single arrowhead), the disorganization of the latissimus dorsi (double arrowhead), and the strong reduction of abdominal and thoracic muscles (double arrow), whereas the deep back muscle, longissimus dorsi, appears correctly developed (arrow). (I,J) Detail of E14.5 Six1–/– embryos at deep back muscle (I) and head (J) levels showing blue myotubes correctly shaped at head level, but reduced in number at the body level.

View larger version (127K): [in a new window]   Fig. 5. Six1 is not required for myotomal differentiation or myogenic precursor cell migration. Immunochemistry experiments performed on transverse sections of E9.5 (A-C) and E11 (D-M) embryos did not show any differences between Six1–/–, Six1+/– and wild-type littermates. Presented here are only the results obtained with Six1–/– embryos (A-K), except for L and M which show the expression of Six4 and myogenin in Six1+/– embryos. D, dorsal; V, ventral. (A-C) Absence of Six1 does not impair early somitic differentiation: at E9.5 immunostaining with Myf5 (A), myogenin (B) and Pax3 (C) antibodies shows that Myf5 and myogenin accumulate correctly in the myotome (double arrowheads in A and B) and that Pax3 is normal in the dermomyotome (arrowhead in C). (D-F) Six1 does not control the expression of Pax3 in the dermomyotome and does not impair Pax3-dependent migration of hypaxial progenitor. (D) At E11, Pax3 expression is detected in the dermomyotome (arrowhead) and in migrating myogenic cells delaminating from the lateral edge of the dermomyotome at the forelimb level. (F) ß-galactosidase immunostaining revealed Six1-expressing cells in the myotome, in the lateral part of the dermomyotome and in migrating cells. (E) Double staining for Pax3 and ß-gal demonstrate that most of the Pax3-expressing cells in the lateral part of the dermomyotome, as well as most of the migrating precursors, also co-express Six1 (arrowhead), whereas differentiated myotomal cells express only Six1 (double arrowhead) and median dermomyotomal cells express only Pax3 (arrow). (G,H) Absence of Six1 does not impair Pax3-dependent migration into the limbs. Immunostaining with Pax3 antibodies shows that Pax3 accumulates correctly in migrating myogenic cells of both ventral and dorsal regions of the forelimb bud (G) at E11. (H) Six1 does not control the expression of Myf5 in limb buds: immunostaining with Myf5 antibodies shows that Myf5 can also be detected in dorsal and ventral myogenic regions of the forelimb bud at E11. (I-M) Six1 is not required for the activation of Myf5, Six4 and myogenin expression in the myotome. (I) Myf5 expression (double arrowheads) is not altered in Six1–/– myotome. (J,L) Six4 antibodies allow detection of Six4 accumulation in the dermomyotome (single arrowhead) and myotome (double arrowheads) in Six1–/– (J) and Six1+/– (L) embryos. Expression of Six4 in the myotome could compensate the absence of Six1 (K,M). Myogenin expression is detected with a specific antibody in both Six1–/– (K) and Six1+/– (M) embryos in the myotome (double arrowheads).

View larger version (80K): [in a new window]   Fig. 6. Absence of Six1 does not lead to an increase in apoptosis in Six1-expressing cells. TUNEL assays at the forelimb bud level of E11 Six1–/– and Six1+/– embryos (A,B,D,E). Six1-expressing cells are detected by an antibody against ß-galactosidase (B,C,E,F). (A-C) Apoptosis in Six1–/– embryos (A) is not increased in the dorsal and ventral aspects of the limb bud where Six1-positive migrating myogenic cells are detected (C), as revealed by double staining (B). (D-F) In Six1+/– embryos, similarly no massive apoptosis is detected in ventral and dorsal regions of the limbs (D) where Six1-positive cells accumulate (F), as revealed by double staining (E).

View larger version (51K): [in a new window]   Fig. 7. Six1 is needed for MyoD and myogenin expression in distal territories. Whole-mount in situ hybridization of Six1–/– embryos (A,C,E,G,I) and Six1+/– littermates (B,D,F,H,J) revealed that Six1-deficient mice fail to activate MyoD and myogenin genes in distal territories. (A-D) Hybridization with the MyoD mRNA probe shows the absence of MyoD expression in the limb buds (arrowheads) and the reduced ventrolateral extension of the dermomyotome (arrows), and the absence of MyoD expression in the epaxial most domain (double arrowheads). (E-H) Hybridization with the myogenin mRNA shows the absence of myogenin expression in the limb buds (arrowheads) and the altered organisation of the ventrolateral part of the dermomyotome (arrows). A broken line separates the epaxial and hypaxial myotome showing that myogenin expression is reduced in the epaxial most domain (double arrowheads). (I-J) At E12.5, hybridization with a MyoD mRNA probe reveals a decrease of MyoD-expressing cells at the shoulder level (arrowheads). (K-L) Detail of the Six1–/– (left) and Six1+/– (right) forelimbs (K) and hindlimbs (L) of E12.5 embryos showing that from this stage forelimb muscles are more affected than hindlimb muscles. (K) Dorsal view of forelimb buds shows a few MyoD-expressing cells in the Six1–/– forelimb (arrowhead). (L) Lateral view of hindlimb buds shows a few MyoD-expressing cells restricted to the dorsal region of Six1–/– hindlimb (arrowhead).

View larger version (41K): [in a new window]   Fig. 8. (A) Schematic representation of the myogenic phenotypes of Splotch mutant (similar phenotype is described for cMet–/– and Gab1–/– mice), and for Six1 and Lbx1 knockout mice and Mox2 (Bladt et al., 1995; Tremblay et al., 1998; Mankoo et al., 1999; Schafer and Braun, 1999; Brohmann et al., 2000; Gross et al., 2000; Sachs et al., 2000). Muscles not affected are in grey; green, orange, blue and purple muscles are missing (dark colours) or reduced (light colours) in Splotch, Six1–/–, Lbx1–/– and Mox2–/– mice respectively. A thin layer in the most dorsal region represents superficial back muscles. At the limb level, upper muscle mass represents dorsal muscles, lower muscle mass represents ventral muscles. (B) Schematic representation of the genetic mechanisms underlying myogenesis in the different myogenic compartments and at different times (adapted from Birchmeier and Brohmann, 2000). Six1 is expressed but plays no role in somite differentiation (1) and in the migration of myogenic precursor cells (2) (red in parenthesis). Migration is controlled by Pax3, and Lbx1 is required for migration at occipital and limb level only (*). (3) Instead Six1 is required for MyoD and myogenin expression in limb buds (bold red).

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