Interaction of Wnt3a, Msgn1 and Tbx6 in neural versus paraxial mesoderm lineage commitment and paraxial mesoderm differentiation in the mouse embryo

Abstract

Paraxial mesoderm is the tissue which gives rise to the skeletal muscles and vertebral column of the body. A gene regulatory network operating in the formation of paraxial mesoderm has been described. This network hinges on three key factors, Wnt3a, Msgn1 and Tbx6, each of which is critical for paraxial mesoderm formation, since absence of any one of these factors results in complete absence of posterior somites. In this study we determined and compared the spatial and temporal patterns of expression of Wnt3a, Msgn1 and Tbx6 at a time when paraxial mesoderm is being formed. Then, we performed a comparative characterization of mutants in Wnt3a, Msgn1 and Tbx6. To determine the epistatic relationship between these three genes, and begin to decipher the complex interplay between them, we analyzed double mutant embryos and compared their phenotypes to the single mutants. Through the analysis of molecular markers in mutants, our data support the bipotential nature of the progenitor cells for paraxial mesoderm and establish regulatory relationships between genes involved in the choice between neural and mesoderm fates.

Figure 1. Wnt3a^−/−, Msgn1^−/− and Tbx6^−/− exhibit a profound loss of paraxial mesoderm and the posterior somites

Expression analysis of the paraxial mesoderm marker Mox1 in wild-type (A, E) Wnt3a^−/− (B, F), Msgn1^−/− (C, G) and Tbx6^−/− embryos (D, H) at 16–18ss (A–D) and 10ss (E–H) reveals lack of posterior somites in mutants. Higher magnification of the boxed areas in A–H show posterior somite region (A′–H′). Asterisks indicate position of somite 7, considered the last anterior somite. Brackets indicate one somite. som, somite; ss, somite stage.

Figure 2. Expression analysis of Wnt3a, Msgn1 and Tbx6 in wild-type embryos

Dynamic expression patterns of Wnt3a (A–C, J–L), Msgn1 (D–F, M–O) and Tbx6 (G–I, P– R) at early bud (EB) (A, D, G), late bud (LB) (B, E, H), headfold stage (HF) (C, F, I), 3–4ss (J, M, P), 8–9ss (K, N, Q) and 19ss (L, O, R). Lateral views of embryo (A, B, C1, D, E, F1, G, H, I1, J1, K1, L1, M1, N1, O1, P1, Q1 and R1). Ventral views (J2, M2, P2). Posterior views (C2, F2, I2) of embryos in C1, F1 and I1, showing expression of Wnt3a, Msgn1 and Tbx6 in the primitive streak (ps) and in the presomitic mesoderm region. Transverse sections (C3, F3, I3) through HF stage embryos showing Wnt3a expression in the epiblast (C3) and Msgn1 (F3) and Tbx6 (I3) expression in the presomitic mesoderm. Higher magnifications of dashed boxes in C3, F3 and I3 (C4, F4 and I4). Schematic representations of Wnt3a, Msgn1 and Tbx6 expression at the HF stage (C4′, F4′ and I4′). Dorsal views (K2, N2, Q2) of tail region of early somite stage embryos in K1, N1 and Q1 show expression of Wnt3a in the primitive streak region. Expression of Msgn1 and Tbx6 extends anterior into the presomitic mesoderm. Transverse sections through tail region of 8–9ss embryos (K3, N3 and Q3). Schematic representations of Wnt3a, Msgn1 and Tbx6 expression at the early somite stage (K3′, N3′ and Q3′). Higher magnifications of tail region of 19ss embryos show Wnt3a expression restricted to the most posterior part of the tail region and more anteriorly expanded expression of Msgn1 and Tbx6 (L2, O2 and R2). Transverse sections through tail regions shown in L2, O2 and R2 (L3, O3 and R3). Dashed lines indicate the plane of section. Red arrows mark the boundary between presomitic mesoderm and the segmented somites (som). ab, allantoic bud; hf, head fold; hg, hind gut; np, neural plate.

Figure 3. Gross morphology of single and double Wnt3a^−/−, Msgn1^−/− and Tbx6^−/− mutants

Morphology of a wild-type embryo showing a normal formed neural tube and properly segmented somites (som) (A). Wnt3a^−/− showing loss of posterior somites (B1) and a reduced tail region (B2). Msgn1^−/− (C) showing a kinked neural tube (nt), (black arrowhead), lack of paraxial mesoderm (red arrowhead) (C1) and an expanded tail region (C2). Asterisk marks a bleb resulting from defects in the vasculature of the mutant (C1). Tbx6^−/− lacking posterior somites, showing aberrant anterior somites, a slightly kinked nt (black arrowhead) (D1) and an expanded tail region (D2). Wnt3a^−/−; Msgn1^−/− double mutants (E) with loss of posterior somites (E1) and a reduced tail region (E2). Wnt3a^−/−; Tbx6^−/− (F) displaying severely kinked nt (black arrowhead) as well as a greater reduction in mesoderm tissue (F1). Msgn1^−/−; Tbx6^−/− (G) exhibit a severely kinked nt (black arrowhead), loss of posterior somites, severely aberrant anterior somites, loss of paraxial mesoderm (red arrowhead) (G1) and expanded tail region (G2). Lateral views of embryos (A–G). Dorsal view of embryos (A1-G1). Higher magnification of tail region (A2-G2).

Figure 4. Histological analysis of single and double Wnt3a^−/−, Msgn1^−/− and Tbx6^−/− mutants

Whole mount and indicated transverse sections (A–G) through somite and tail regions in an anterior to posterior sequence of 12–14ss wild-type embryo (A), Wnt3a^−/− (B), Msgn1^−/− (C), Tbx6^−/− (D), Wnt3a^−/−; Msgn1^−/− (E), Wnt3a^−/−; Tbx6 ^−/− (F) and Msgn1^−/−; Tbx6^−/− (G). Sections show major hallmarks of the phenotype of single and double mutants: Wnt3a^−/− (B) normal anterior somites (B1), loss of posterior somites (B2) and reduced tail region (B3); Msgn1^−/− (C) normal anterior somites (C1), lack of posterior somites, reduced paraxial mesoderm (C2) and massively expanded tail region (C3); Tbx6^−/− (D) normal anterior somites (D1), lack of posterior somites, formation of lateral ectopic neural tubes (green arrowheads) (D2) and expanded tail region (D3); Wnt3a^−/−; Msgn1^−/− (E) normal anterior somites, cell cluster on top of the neural tube (E1) as well as ventral to neural tube (black arrowheads) (E2) and reduced tail region (E3); Wnt3a^−/−; Tbx6^−/− (F) aberrant anterior (F1) and loss of posterior somites (F2) and reduced tail region(F3); Msgn1^−/−; Tbx6^−/− (G) severely kinked neural tube (black arrow) (G1), cell cluster lateral to neural tube (black arrowheads) (G1 and G2), loss of posterior somites and paraxial mesoderm (G2), and expanded tail region (G3). da, dorsal aorta; hg, hindgut; m, mesoderm; no, notochord; nt, neural tube; ps, primitive streak; som, somites. Scale bar in A1 and A2: 50 μm. Scale bar in A3: 100 μm

Figure 5. Expression of Wnt3a, Msgn1 and Tbx6 in single and double mutants during posterior somite formation

Expression patterns of Wnt3a (A, F, H, and J), Msgn1 (B, D, I, and K) and Tbx6 (C, E, G and L), respectively, in wild-type (A–C), Wnt3a^−/− (D–E), Msgn1^−/− (F–G), Tbx6^−/− (H–I) Wnt3a^−/−; Msgn1^−/− (L), Wnt3a^−/−; Tbx6^−/− (K) and Msgn1^−/−; Tbx6^−/− (J) mutants showing downregulation of Msgn1 and Tbx6 in Wnt3a^−/− (D and E), upregulation of Wnt3a and loss of Tbx6 in Msgn1^−/− (F and G), upregulation of Wnt3a and downregulation of Msgn1 in Tbx6^−/− (H and I), loss of Tbx6 expression in Wnt3a^−/−; Msgn1^−/− (L), loss of Msgn1 in Wnt3a^−/−; Tbx6^−/− (K) and upregulation of Wnt3a in Msgn1^−/−; Tbx6^−/− (J). Lateral views of whole mount embryos (A1-L1). Higher magnifications of tail region of embryos (A2-L2). Frontal section of a Msgn1^−/−; Tbx6^−/− double mutant embryo showing overexpression of Wnt3a in the neural tube (nt) (J3). Transverse sections through Tbx6^−/− (H3-H4) and Msgn1^−/−; Tbx6^−/− (J4-J6) showing upregulation of Wnt3a expression in areas of the expanded tail region as well as in cell accumulations close to the neural tube (nt) of Msgn1^−/−; Tbx6^−/− (J6) (red arrowhead). By comparison, transverse sections of Wnt3a expression in the wild-type (A3-A4). Cartoon of a 10–12ss embryo showing approximate planes of sections depicted in A3-A4, H3-H4, J3-J6 (M).

Figure 6. Upregulation of Sox2 in the tail region of 12ss Tbx6^−/− and Msgn1^−/−; Tbx6^−/− mutant embryos

Whole mount views of 10–12 somite stage (ss) wild-type (A), Wnt3a^−/− (B), Msgn1 ^−/− (C), Tbx6^−/− (D) Wnt3a^−/−; Msgn1^−/− (E), Wnt3a^−/−; Tbx6^−/− (F) and Msgn1^−/−; Tbx6^−/− (G) embryos. Dashed lines indicate plane of sections taken. Transverse sections through primitive streak/tail region, stained for Sox2 and Hoechst (A1-G1) and Sox2 (A2-G2). Transverse sections through region of posterior somites stained with Sox2 and Hoechst to label nuclei (A3-G3) and Sox2 (A4-G4). Green arrowheads point to ectopic neural tubes. Yellow arrow highlights Sox2 expression in the epiblast in the wild-type (A2). Scale bars in A1 and A3: 100 μm.

Figure 7. Upregulation of Sox2 in later stage Tbx6^−/− and Msgn1^−/−; Tbx6^−/− mutant embryos

Whole mount views of 20–25 ss wild-type (A), Wnt3a^−/− (B), Msgn1^−/− (C), Tbx6^−/− (D), Wnt3a^−/−; Msgn1^−/− (E), Wnt3a^−/−; Tbx6^−/− (F) and Msgn1^−/−; Tbx6^−/− (G) embryos. Dashed lines indicate plane of sections taken. Transverse sections through tail bud region of the embryo with immunofluorescent staining for Sox2 protein and Hoechst to label nuclei (A1-G1) and Sox2 (A2-G2). Transverse sections through region of posterior somites stained with Sox2 and Hoechst to label nuclei (A3-G3) and Sox2 (A4-G4). Green arrowheads point to ectopic (lateral and ventral) neural tubes. Yellow arrow in A1 highlights Sox2 expression in the epiblast of the wild-type. Scale bars in A1 and A3: 100 μm.

Figure 8. Ectopic neuralization and apoptosis in tail buds of Msgn1^−/−; Tbx6^−/− mutants

Transverse sections of a 20ss Msgn1^−/−; Tbx6 ^−/− embryo in anterior to posterior sequence stained with Phalloidin to label F-Actin and Hoechst to label nuclei (A1-D1), Sox2 (A2-D2), and Sox2 and Hoechst (A3-D3). Sox2 is expressed in the neural tube as well as ectopically expressed in cells enclosed within, or on top of, the neural tube (yellow arrowheads). Transverse sections through the tail bud of a 20ss Msgn1^−/−; Tbx6^−/− embryo reveal death of cells that have downregulated Sox2. H&E stained sections (E1) and higher magnification of boxed area in E1 (E2) showing cell debris. Tail bud stained for Sox2 and Hoechst labeling nuclei (F) and immunodetection of activated Caspase (G) counterstained with Hoechst, reveal cells that undergo apoptosis have lower levels of Sox2 expression. F2 and G2 are higher magnifications of boxed areas in F1 and G1.

Figure 9. Summary of sequential steps in paraxial mesoderm specification and morphogenesis affected in mutants

Schematic representation depicting the sequential steps necessary for proper paraxial mesoderm specification and morphogenesis in wild-type embryos, and details of which are affected in Wnt3a^−/−, Msgn1^−/− and Tbx6^−/− mutants, respectively.

Figure 10. Working model for paraxial mesoderm specification and differentiation in the mouse embryo

Schematic representation with details of the genetic regulatory network of three critical factors, Wnt3a, Msgn1 and Tbx6, controlling paraxial mesoderm specification. This working model suggests a complex network in which Wnt3a functions at the top of the hierarchy inducing several processes that involve the activation of Tbx6 and Msgn1, several feedback loops and the involvement of each factor in possible parallel pathways for the proper specification of neural ectoderm and paraxial mesoderm, during posterior somite formation. Anterior somite formation is independent of Wnt3a function. This model focuses on paraxial mesoderm and therefore, for simplicity, does not include factors such as T/Brachyury. TBE, T-Box binding element.