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, 138 (2), 371-84

Connective Tissue Fibroblasts and Tcf4 Regulate Myogenesis

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Connective Tissue Fibroblasts and Tcf4 Regulate Myogenesis

Sam J Mathew et al. Development.

Abstract

Muscle and its connective tissue are intimately linked in the embryo and in the adult, suggesting that interactions between these tissues are crucial for their development. However, the study of muscle connective tissue has been hindered by the lack of molecular markers and genetic reagents to label connective tissue fibroblasts. Here, we show that the transcription factor Tcf4 (transcription factor 7-like 2; Tcf7l2) is strongly expressed in connective tissue fibroblasts and that Tcf4(GFPCre) mice allow genetic manipulation of these fibroblasts. Using this new reagent, we find that connective tissue fibroblasts critically regulate two aspects of myogenesis: muscle fiber type development and maturation. Fibroblasts promote (via Tcf4-dependent signals) slow myogenesis by stimulating the expression of slow myosin heavy chain. Also, fibroblasts promote the switch from fetal to adult muscle by repressing (via Tcf4-dependent signals) the expression of developmental embryonic myosin and promoting (via a Tcf4-independent mechanism) the formation of large multinucleate myofibers. In addition, our analysis of Tcf4 function unexpectedly reveals a novel mechanism of intrinsic regulation of muscle fiber type development. Unlike other intrinsic regulators of fiber type, low levels of Tcf4 in myogenic cells promote both slow and fast myogenesis, thereby promoting overall maturation of muscle fiber type. Thus, we have identified novel extrinsic and intrinsic mechanisms regulating myogenesis. Most significantly, our data demonstrate for the first time that connective tissue is important not only for adult muscle structure and function, but is a vital component of the niche within which muscle progenitors reside and is a critical regulator of myogenesis.

Figures

Fig. 1.
Fig. 1.
Tcf4 is highly expressed in muscle connective tissue fibroblasts. In the embryonic (A-C), neonatal (D-G) and adult (H-K) limb, Tcf4+ fibroblasts are associated with Pax7/MyoD+ myoblasts (A-C), MyHC+ myofibers (D-F) and laminin-ensheathed myofibers (H-J). By P0 and in the adult mouse, Tcf4+ fibroblasts (arrows) lie within the Sirius Red+ connective tissue (G,K) outside the MyHC+ myofibers (D-F) and laminin+ muscle basal lamina (H-J). In the neonate, some myonuclei (arrowheads) express low levels of Tcf4. Asterisks in A and D indicate enlarged inset regions in A-G. Scale bars: in A, 200 μm in A-G; in A inset and H, 50 μm in A-G insets and H-K.
Fig. 2.
Fig. 2.
Tcf4 is highly expressed and Tcf4GFPCre+neo genetically labels muscle connective tissue fibroblasts in culture. (A-D) Adherent fibroblasts derived from neonatal limb muscles express Tcf4 and αSMA (arrows, A-C) and vimentin (D). (E-G) In a preparation of adherent cells from limb muscles from Tcf4GFPCre+neo/+;R26RYFP/+ mice, 58% of Tcf4+ fibroblasts are genetically labeled with YFP (arrows, E-G). (H-J) In a preparation of total cells from limb muscles from Tcf4GFPCre+neo/+;R26RYFP/+ mice, MyHC+ myogenic cells are not labeled by YFP. Scale bar: in J, 100 μm for A-J.
Fig. 3.
Fig. 3.
Tcf4GFPCre+neo genetically labels muscle connective tissue fibroblasts in vivo. (A-J) In the embryonic limb of Tcf4GFPCre+neo/+;R26RlacZ/+ mice (A-F) and neonatal limb of Tcf4GFPCre+neo/+;R26RmTmG/+ mice (G-J) β-gal+ (A-F) or membrane bound GFP+ (G-I) Tcf4-derived cells lie associated with, but interstitial to, Pax7/MyoD+ myoblasts (A-F) or laminin-ensheathed myogenic cells (G-I) and in Sirius Red+ regions (J). (K-M) In whole-mount preparations, β-gal+ cells (blue) are present throughout the tibialis anterior and gastrocnemius muscles and concentrated in the gastrocnemius aponeurosis of Tcf4GFPCre+neo/+;R26RlacZ/+ mice. (N) Drawing of the gastrocnemius muscle. Asterisks in A, D and G indicate enlarged regions in D-F, insets in D-F and insets in G-J, respectively. Scale bars: in A, 50 μm for A-C; in D, 12.5 μm for D-F; in D inset, 3.125 μm for D-F insets; in G, 100 μm for G-J; in G inset, 25 μm for G-J insets.
Fig. 4.
Fig. 4.
Germline loss of Tcf4 leads to a reduction in slow MyHCI and fast MyHCIIb and an increase in developmental MyHCemb in many muscles. (A-Y) Loss of Tcf4 in Tcf4GFPCre/GFPCre leads to a reduction in the percent MyHCI+/total laminin+ myofibers in the extensor digitorum longus (EDL; F-J), tibialis anterior (TA; K-O), soleus (P-T) and diaphragm (U-Y), but not in the rectus femoris (RF; A-E). (Z-BB) All muscles in Tcf4+/+ and Tcf4GFPCre/GFPCre mice express MyHCemb (Z,AA), but overall MyHCemb protein levels (by western blot of P0 hind limbs) are increased with loss of Tcf4 (BB). The medial head of gastrocnemius is truncated with loss of Tcf4 (arrows, Z,AA). (CC-EE) By qPCR, MyHCI transcript levels are significantly reduced, whereas MyHCemb levels are increased in Tcf4GFPCre/GFPCre quadriceps (CC), TA (DD) and diaphragm (EE). MyHCIIb levels are significantly reduced in the Tcf4GFPCre/GFPCre TA and diaphragm (DD,EE). Data are expressed as mean ± s.e.m. Scale bars: in A, 200 μm for A-D, F-I, K-N, P-S, U-X; in Z, 400 μm for Z,AA.
Fig. 5.
Fig. 5.
Myogenic loss of Tcf4 leads to a reduction in slow MyHCI and MyHCIIb in limb and diaphragm muscles. (A,B,D,E,G-J) Loss of Tcf4 in embryonic and fetal muscles (Pax3Cre/+;Tcf4del/fl) or fetal muscles only (Pax7Cre/+;Tcf4del/fl) leads to a reduction in the percent MyHCI+/total laminin+ myofibers in the TA (D,E), soleus (G,H) and diaphragm (I,J), but not in the EDL (A,B). (C,F,K) By qPCR, MyHCI and MyHCIIb levels are reduced in the quadriceps, TA and diaphragm of Pax3Cre/+;Tcf4del/fl mice (C,F,K). MyHCemb levels are increased in the diaphragm of Pax3Cre/+;Tcf4del/fl mice (K). Data are expressed as mean ± s.e.m.
Fig. 6.
Fig. 6.
Reduction of Tcf4 in connective tissue fibroblasts leads to a reduction in slow MyHCI in fetal limb muscles and an increase in MyHCemb in neonatal limb and diaphragm muscles. (A,C,F,I) Deletion of Tcf4 in ∼60% of connective tissue fibroblasts in Tcf4GFPCre+neo/fl mice leads to a reduction in the percent MyHCI+/total laminin+ myofibers in the TA (C) and soleus (F), but not in the EDL (A) or diaphragm (I). (B,D,G,J) By qPCR, MyHCI levels are reduced in neonatal quadriceps and TA and adult soleus (B,D,G), whereas MyHCemb levels are increased in adult quadriceps, TA, soleus and diaphragm muscles of Tcf4GFPCre+neo/fl mice (B,D,G,J). (E,H) Ablation of ∼50% of the Tcf4+ fibroblasts in Tcf4GFPCre+neo/+;R26RDTA/+ mice leads to a reduction in the percent MyHCI+ myofibers in the soleus (H) but not the TA (E). Data are expressed as mean ± s.e.m.
Fig. 7.
Fig. 7.
Myoblasts cultured in the presence of Tcf4+ fibroblasts differentiate into myofibers with increased slow MyHCI expression and more nuclei. (A-G) After 7 days of differentiation, myoblasts cultured in the presence of Tcf4+ fibroblasts had a greater percent MyHCI+/total YFP+ myofibers (A-D,G) and an increased frequency of more multinucleate myofibers (E) and a higher fusion index (F). Data in F and G are expressed as mean ± s.e.m. (H) Model of the role of Tcf4 and Tcf4+ fibroblasts in regulating muscle fiber type during development. Tcf4 intrinsically regulates MyHCI and MyHCIIb, probably by direct binding of Tcf4 to their enhancers and activation by β-catenin in fetal myofibers. Tcf4-dependent signals in fibroblasts extrinsically promote MyHCI and repress MyHCemb in myofibers.

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