della Gaspera B et al. (2009), The Xenopus MEF2 gene family: evidence of a rol...

XB-ART-39599

Dev Biol 2009 Apr 15;3282:392-402. doi: 10.1016/j.ydbio.2009.01.039.

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The Xenopus MEF2 gene family: evidence of a role for XMEF2C in larval tendon development.

della Gaspera B , Armand AS , Sequeira I , Lecolle S , Gallien CL , Charbonnier F , Chanoine C .

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MEF2 transcription factors are well-established regulators of muscle development. In this report, we describe the cloning of multiple splicing isoforms of the XMEF2A and XMEF2C encoding genes, differentially expressed during Xenopus development. Using whole-mount in situ hybridization, we found that the accumulation of XMEF2C mRNA in the tadpole stages was restricted to intersomitic regions and to the peripheral edges of hypaxial and cranial muscle masses in contrast to XMEF2A and XMEF2D, characterized by a continuous muscle cell expression. The XMEF2C positive cells express the bHLH transcription factor, Xscleraxis, known as a specific marker for tendons. Gain of function experiments revealed that the use of a hormone-inducible XMEF2C construct is able to induce Xscleraxis expression. Furthermore, XMEF2C specifically cooperates with Xscleraxis to induce tenascin C and betaig-h3, two genes preferentially expressed in Xenopus larval tendons. These findings 1) highlight a previously unappreciated and specific role for XMEF2C in tendon development and 2) identify a novel gene transactivation pathway where MEF2C cooperates with the bHLH protein, Xscleraxis, to activate specific gene expression.

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Species referenced: Xenopus laevis
Genes referenced: col12a1 col1a2 col5a1 mef2a mef2c mef2d myf6 myh4 myod1 pax3 scx tgfbi tnc tnmd

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	Fig. 2. (A) Whole-mount in situ hybridization with labeled antisense probes against XMEF2D, XMEF2A and XMEF2C during embryonic development. The numbers refer to the developmental stages. The anterior side of the embryos is on the left. Lateral view for all embryos except dorsal view for XMEF2D stages 12, 16 and 20; XMEF2A stages 16 and 20; and anterior view for XMEF2C stage 19. (B) XMEF2A, XMEF2D and XMEF2C expression at stages 37–38 in head muscle anlagens as indicated by XMRF4 mRNAs localization. Lateral view on the left. ba, brachial arches; cm, cranio-facial muscle; fhr, frontal head region; nc, neural crest; h, heart; hm, hypaxial muscles. Arrowhead: hypaxial migratory cells.
	Fig. 3. Comparison of the localization of XMEF2C mRNA with the muscle markers by whole-mount in situ hybridization. (A) Expression of MyHC E3 and XMEF2C (left and middle respectively) at stages 39–40 (left). Double in situ hybridization of XMEF2C (purple) and E3 (blue) shows that XMEF2C mRNA is expressed in cells localized between streams of hypaxial muscle (right, stage 41). (B) XMEF2C is expressed in intersomitic space. Comparison of hybridization with XMRF4 (left top), XMEF2C (left middle), and cohybridization with XMRF4 and XMEF2C (left bottom). The thin XMEF2C expression is distinct from the thick XMRF4 expression. Double in situ hybridization shows that XMRF4 or XMEF2A mRNA (purple) accumulates around the large and centrally localized nuclei of mononucleated muscle cells (Kiełbówna, and Daczewska, 2005) while XMEF2C mRNA (blue) is restricted to intersomitic space. Frontal section indicates that XMEF2C mRNA is found throughout the intersomitic space (C) XMEF2C is strongly expressed in the head at the muscle-border zone corresponding to connective tissue associated with muscle at stage 41. XMEF2A and XMEF2D are expressed in muscle cells as indicated by XMRF4 expression. (D) Comparison of the localization of XMEF2C mRNA and pax3 mRNA at stages 37–38. Lateral view shows that pax3 mRNA (left) is more concentrated in the anterior and posterior side of somites while MEF2C mRNA (middle) is restricted to the intersomitic region. Frontal section (right) indicates that pax3 expression is limited to the external border of somites. The probe is indicated in each image. Arrows indicate XMEF2C expression except in D where they show intersomitic space.
	Fig. 4. Comparison of the localization of XMEF2C and Xscleraxis mRNA by whole-mount in situ hybridization. (A) Stage 38 (a–d). At stage 40 (e and f) XMEF2C begins to be expressed in the border of muscle masses of the head while scleraxis expression is faint and more diffuse around muscle masses. Lateral view (a and b). Ventral view (c–f). In somite region, dorsal views (g and h) indicate that XMEF2C expression is restricted to the intersomitic space while Xscleraxis expression appears faint and more diffuse. In lateral view of hypaxial region, XMEF2C mRNA begins to be expressed at stage 40 from the bottom (i) to the top (k) of connective tissue between muscle cells. No Xscleraxis expression was detected at these stages (j and l). (B) XMEF2C and Xscleraxis are largely colocalized in stage 43 embryo in the borders of muscle masses as indicated by MRF4 staining (c). (a), (b) and (c): ventral view of the head region, (d) and (e): lateral view of the head, (f) and (g): lateral view of the hypaxial region. The probe is indicated in each image. Arrows indicate Scleraxis expression in connective tissue associated with hypaxial muscles. (C) Scleraxis mRNA begins to be expressed in intersomitic space at stage 43. Embryos have been hybridized with XMEF2C, XMRF4 and Xscleraxis, and cohybridized with XMRF4 and Xscleraxis. XMRF4 mRNA accumulates around the large and centrally localized nuclei of muscle cells. At this stage, muscle cells are mononucleated and span the entire somite. The myotome presents a convex side on dorsal view. Arrow head indicates the intersomitic space between two convex myotome segments. The thin XMEF2C and Xscleraxis expression are distinct from the thick MRF4 expression and is localized between the convex myotome. Dorsal view on the top, higher magnification of dorsal view in the middle and lateral view at the bottom. The probe is indicated under each image. Arrows indicate Xscleraxis expression in intersomitic space. (D) single (a–d) or double (e–g) in situ hybridization of XMEF2C mRNA (blue) with XMRF4, XMEF2A or Xscleraxis mRNA (purple) in the ventral view of the head embryo at stage 41. XMEF2C mRNA is not colocalized with XMRF4 and XMEF2A mRNA while Xscleraxis begins to be strongly expressed in most of the XMEF2C expressing cells.
	Fig. 5. Comparison of the localization of XMEF2C, Xscleraxis, betaig-h3, tenascin C and tenomodulin mRNA by whole-mount in situ hybridization. (A) Betaig-h3 mRNA begins to be expressed in connective tissue associated with hypaxial muscle at stage 41. (B) XMEF2C, Xscleraxis, betaig-h3, tenascin C and tenomodulin mRNA are largely colocalized at stage 45 in head region (ventral view, top) intersomitic space as indicated by staining between the convex myotome (dorsal view, middle). Connective tissue associated with hypaxial muscles expresses XMEF2C, Xscleraxis, betaig-h3 and tenascin C, but we cannot detect tenomodulin mRNA (lateral view, bottom). (C) Col1a2, col5a1 and col12a1 mRNA is also expressed in intersomitic space at stage 45 (D) Comparison of MEF2C and scleraxis mRNA accumulation 3 days after feeding. MEF2C mRNA is always expressed at this stage while scleraxis mRNA appears stronger. Arrows indicate connective tissue associated with hypaxial muscles expression. Numbers indicate developmental stages.
	Fig. 6. (A) Western blot of late gastrula embryos injected into the two blastomeres at the two-cell stage with 300 pg of MyoDF, 100 pg of MEF2AF, MEF2DF and MEF2CF mRNA. Proteins were detected with anti-flag antibody. (B) Embryos were injected unilaterally at the two-cell stage at the level of marginal zone with the quantities of mRNA described for western blot. Embryos were fixed at stages 18–19 and submitted to whole-mount in situ hybridization with desmin probe. The injected mRNAs are indicated in each image. mRNA encoding the lineage tracer β-galactosidase (blue) was used to identify the injected side of embryos (e–g) except for embryos injected with synthetic mRNA of MyoD where the ectopic expression of desmin is easily detected (a–d).
	Fig. 7. (A) Western blot of late gastrula embryos injected into the two blastomeres at the two-cell stage with 300 pg of MEF2DGRF, MEF2CGRF or ScleraxisF synthetic mRNA. Proteins were detected with anti-flag antibody. Molecular weight markers are indicated in kilodalton. (B) Xscleraxis expression at stage 31. (C) Left: detection of Xscleraxis mRNA by whole mount in situ hybridization after unilateral injection of MEF2CGRF synthetic mRNA (+ MEF2CGRF) at the two-cell stage and induction by dexamethasone at stage 20. Right: Detection of Xscleraxis mRNA by real-time PCR after bilateral injection of mock (mock), MEF2CGRF or MEF2DGRF synthetic mRNA at the two-cell stage and induction by dexamethasone at stage 20. (D) Betaig-h3 expression at stage 31 (left) and comparison of betaig-h3 with XMRF4 expression (betaig-h3 + MRF4) indicates that betaig-h3 is expressed in intersomitic space (right, arrows). (E) Embryos were injected unilaterally at the two-cell stage, at the marginal zone level with the quantities of mRNA used for western blot. Embryos were injected by MEF2CGRF synthetic mRNA (+ MEF2CGRF), scleraxisF synthetic mRNA (+ scleraxisF) or by a mix of MEF2CGRF and scleraxisF synthetic mRNA (+ MEF2CGRF + scleraxisF). Embryos were induced by 10 μM dexamethasone at stage 20, fixed at stages 30–31 and submitted to whole-mount in situ hybridization to detect betaig-h3. (F) Tenascin C expression at stage 32 (left) and comparison of tenascin C with XMEF2C expression (tenascin C + MEF2C) indicates that tenascin C is expressed in somitic cells (right, arrows). (G) Embryos were injected unilaterally at the two-cell stage as described in (E) and analyzed for tenascin C expression. mRNA encoding the lineage tracer β-galactosidase (blue) was used to identify the injected side in panels C, E and G. Inj., injected side; uninj., uninjected side. In panels C, E and G, injected side at the top, non-injected side in the middle, and dorsal view at the bottom. No variations were observed in uninduced embryos injected by MEF2CGRF synthetic mRNA.
	mef2c (MADS box transcription enhancer factor 2, polypeptide C (myocyte enhancer factor 2C)) expression in Xenopus laevis, NF stage 40, lateral view, anterior left.
	mef2a (myocyte enhancer factor 2A) gene expression in Xenopus laevis, assayed via in situ hybridization, NF stage 40, lateral view, anterior left.
	mef2d (myocyte enhancer factor 2D) gene expression in Xenopus laevis, assayed via in situ hybridization, NF stage 40, lateral view, anterior left.
	mef2a (myocyte enhancer factor 2A) gene expression in Xenopus laevis, assayed via in situ hybridization, NF stage 30, lateral view, anterior left.
	mef2c (MADS box transcription enhancer factor 2, polypeptide C (myocyte enhancer factor 2C)) expression in Xenopus Laevis, stage 28, lateral view, anterior left.
	mef2d (myocyte enhancer factor 2D) expression in Xenopus laevis,assayed via in situ hybridization, NF stage 30, lateral view, anterior left.
	mef2a (myocyte enhancer factor 2A) expression in Xenopus laevis, in situ hybridization, stage 20, dorsal view, anterior left.
	mef2c (MADS box transcription enhancer factor 2, polypeptide C (myocyte enhancer factor 2C)) expression in Xenopus laevis, NF stage 19, anterior view, dorsal up.
	mef2d (myocyte enhancer factor 2D) gene expression in Xenopus laevis, assayed via in situ hybridization, NF stage 20, dorsal view,anterior right.




	scxa (scleraxis homolog A) gene expression in Xenopus laevis, assayed via in situ hybridization, NF stage 45 embryo, dorsal view, head and trunk region only, anterior up.
	tnc (tenascin C ) gene expression in Xenopus laevis embryos, NF stage 32, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
	tnc (tenascin C) gene expression in Xenopus laevis embryos, NF stage 45, assayed by in situ hybridization, dorsal view,anterior up.
	col5a1 (collagen type 5, alpha 1) gene expression in Xenopus laevis embryos, NF stage 45, assayed by in situ hybridization, dorsal view, anterior up.
	col12a1 (collagen type 12, alpha 1) gene expression in Xenopus laevis embryos, NF stage 45, assayed by in situ hybridization, dorsal view, anterior up.
	col1a2 (collagen type 1, alpha 2) gene expression in Xenopus laevis embryos, NF stage 45, assayed by in situ hybridization, dorsal view, anterior up.
	myh4 (myosin, heavy chain 4, skeletal muscle) gene expression in Xenopus laevis embryos, NF stage 40, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.