Mesogenin 1 is a master regulator of paraxial presomitic mesoderm differentiation

Abstract

Neuromesodermal (NM) stem cells generate neural and paraxial presomitic mesoderm (PSM) cells, which are the respective progenitors of the spinal cord and musculoskeleton of the trunk and tail. The Wnt-regulated basic helix-loop-helix (bHLH) transcription factor mesogenin 1 (Msgn1) has been implicated as a cooperative regulator working in concert with T-box genes to control PSM formation in zebrafish, although the mechanism is unknown. We show here that, in mice, Msgn1 alone controls PSM differentiation by directly activating the transcriptional programs that define PSM identity, epithelial-mesenchymal transition, motility and segmentation. Forced expression of Msgn1 in NM stem cells in vivo reduced the contribution of their progeny to the neural tube, and dramatically expanded the unsegmented mesenchymal PSM while blocking somitogenesis and notochord differentiation. Expression of Msgn1 was sufficient to partially rescue PSM differentiation in Wnt3a(-/-) embryos, demonstrating that Msgn1 functions downstream of Wnt3a as the master regulator of PSM differentiation. Our data provide new insights into how cell fate decisions are imposed by the expression of a single transcriptional regulator.

Keywords: Differentiation; EMT; Embryonic stem cell; Motility; Mouse; PSM; Paraxial mesoderm; Somite; Wnt; bHLH transcription factor.

Fig. 1.

Msgn1 promotes paraxial mesoderm formation. (A-H) WISH analysis of Tbx6 (A-D) and T (E-H) mRNA expression in E8.5 control (A,E, lateral view; B,F ventral view) and Msgn1^−/− (C,G, lateral view; D,H, ventral view) embryos. Scale bars: 200 μm. (I) iF-Msgn1 EBs treated with (+) or without (−) Dox from 0-72 h are analyzed for the expression of Tbx6, Pax3 and Osr1 by semi-quantitative RT-PCR. Cyclophilin A (PpiA) is used as a loading control. (J) Density plots of flow cytometry analysis for the expression of Pdgfrα and Flk1 in iF-Msgn1 and iF-Tbx6 EBs treated with (+) or without (−) Dox for 24 h. Numbers indicate the percentage of cells within each quadrant. Arrowheads indicate the section containing Pdgfrα-expressing paraxial mesoderm cells.

Fig. 2.

Identification of the Msgn1 transcriptome in differentiating iF-Msgn1 EBs. (A) Hierarchical clustering of differentially expressed mesoderm-associated genes induced by F-Msgn1 (+Dox) for 24 h (P≤0.05; fold change ≥1.5). The color bar represents log₂ intensity values. (B) Pathway and tissue expression analysis of all differentially expressed genes (P≤0.05, fold change≥1.5) induced by F-Msgn1 for 24 h. The x-axis shows the P-value (−log₁₀). (C-H) qPCR analysis of select differentially expressed mesoderm genes in iF-Msgn1 EBs treated with (+) or without (−) Dox for 12 h and 24 h. The y-axis shows the normalized fold change. Results are mean±s.e.m.

Fig. 3.

ChIP-seq analysis of iF-Msgn1 EBs. (A) Genomic distribution of 4087 Msgn1 peaks (pink) compared with those from genome background (blue). For genome background, percentages of peaks from random sampling of similar length reads across the genome background are used. A binomial exact test was used to calculate the P-values. TSS, transcription start site; TES, transcription end site. (B) Msgn1 peaks within 25 kb upstream or downstream of the TSS are plotted. (C) Venn diagram depicting the intersection of 1860 genes bound by Msgn1 (blue circle) with 332 differentially expressed genes at the 24-h time point (green circle). The overlap is statistically significant (P≤4.6×10⁻⁸⁰, hypergeometric probability test). (D) GO biological processes associated with genes bound by F-Msgn1 and analyzed by GREAT. The x-axis shows the P-value (−log₁₀). (E) Discriminative DNA motif discovery (DREME) analysis of Msgn1 peaks identifies an E-box (underlined), CCATHTGB, as the top motif (E-value=7.6×10^–279). The motif logo displays nucleotide conservation at that position (measured in bits) and height of the symbol reflect the relative frequency at that position.

Fig. 4.

The paraxial mesoderm regulators Tbx6 and Pdgfra are direct target genes of Msgn1. (A) Msgn1-binding peaks (red) and input control (blue) at the Tbx6 locus for the indicated genomic intervals (mm9 coordinates). The schematic (below) illustrates the four E-boxes (E1-4, red rectangles) identified in the Tbx6+3913 enhancer peak. Arrowheads reflect gene orientation and the y-axis indicates the peak height corresponding to the normalized fold enrichment. (B) Luciferase reporter assay of Tbx6+3913 enhancer co-transfected with pCS2 Control (green) or pCS2-Msgn1 (orange) expression constructs. The y-axis shows the normalized fold change; error bars show the s.d.; n=3. (C) ChIP-qPCR analysis of F-Msgn1 binding to Tbx6+3913 enhancer using control ‘C’ and anti-Msgn1 ‘αM’ ascites in iF-Msgn1 EBs. (D) Msgn1 binds the Pdgfra locus at −2140 bp, relative to TSS. (E) Luciferase reporter assay of the Pdgfra −2140 wild-type (WT) and E-box mutant enhancers co-transfected with control or Msgn1 expression constructs. The y-axis shows the normalized fold change; error bars show the s.d.; n=3. (F-I) WISH analysis of Pdgfra mRNA expression in control and Msgn1^−/− embryos. G and I are cross sections through the PS [dashed white lines in F and H (ventral view)]. Scale bars: 200 μm (F,H), 50 μm (G,I).

Fig. 5.

The EMT master regulator Snai1 is a direct target of Msgn1. (A) Semi-quantitative PCR analysis of Snai1 expression in iF-Msgn1 EBs treated with (+) or without (−) Dox from 0-72 h. Cyclophilin A (PpiA) is used as a loading control. (B) Msgn1 binds to the +5148 bp enhancer of Snai1. The schematic (below) shows the two E-boxes (red rectangles) identified in the Snai1 +5148 enhancer peak. Arrowheads indicate orientation and y-axis denotes peak height, corresponding to the normalized fold enrichment. (C) Luciferase reporter assay of Snai1 +5148 enhancer co-transfected with control or Msgn1 expression constructs. The y-axis shows the normalized fold change; error bars show the s.d.; n=3. (D) ChIP-qPCR analysis of Msgn1 enrichment on Snai1 +5148 locus using control ‘C’ and anti-Msgn1 ‘αM’ ascites in E9.5 PSM and head extracts and in iF-Msgn1 EBs. (E-H) WISH analysis of Snai1 mRNA in E8.5 control and Msgn1^−/− embryos (E,G, lateral view; F,H, ventral view). Scale bars: 200 μm.

Fig. 6.

Msgn1 is required for EMT and motility. (A) qPCR analysis of epithelial and mesenchymal genes expressed in Dox-treated (+Dox) iF-Msgn1 EBs after 48 h. The y-axis shows the normalized fold change; error bars are the s.d. (B,C) Histological analysis (H&E) of PS cross-sections of control (n=7) and Msgn1^−/− (n=9) embryos at E8.5. (D,E) Rhodamine-phalloidin staining of control (D, n=21) and Msgn1^−/− (E, n=14) tail bud explants dissected at the 18-22 somite stage and cultured for 48 h. The distance migrated from explant (E) to periphery (P) is indicated by white line. (F) The average distance migrated in control (n=13) and Msgn1^−/− (n=7) explants is shown on y-axis. *P≤9.88×10⁻⁵, two-sample unequal variance Student's t-test. (G-N) Immunostaining of control and Msgn1^−/− explants cultured for 48 h and stained with Rhodamine-phalloidin, and anti-E-cadherin and anti-vimentin antibodies. The dashed white line demarcates the explant (E) from periphery (P) region. Scale bars: 100 μm.

Fig. 7.

Ectopic expression of F-Msgn1 in NM stem cells excludes cells from the neural tube and specifies PSM fates. (A-F) Immunodetection of EGFP (A,D) and F-Msgn1 (B,E) in cross-sections through the trunk neural tube (dashed lines) of E10.5 control (T-Cre^tg/+; R26-flox-rtTA-Ires-EGFP/⁺) (A-C) and T-cre-F-Msgn1^GOF mutant embryos (T-Cre^tg/+; R26-flox-rtTA-Ires-EGFP/⁺; lacZ-TRE-F-Msgn1^tg/+) (D-F). (G) Quantitative lineage tracing analysis of T-Cre labeled cells in neural tubes of control (A, n=5) and T-cre-F-Msgn1^GOF mutant (D, n=5) are shown. *P≤−2.18×10⁻⁴, two-sample unequal variance Student's t-test. (H-M) Immunodetection of F-Msgn1 and Tbx6 protein in cross-sections through the PSM of E9.5 control (H-J) and Tcre-F-Msgn1^GOF mutant (K-M) embryos. NT, neural tube; PM, paraxial mesoderm; N, notochord; H, hindgut. Arrowheads denote the notochord. (N) Quantitative analysis of Tbx6⁺ cells in control (I, n=5) and Tcre-F-Msgn1^GOF mutants (L, n=5). *P≤−7.38×10⁻⁶. (O-T) WISH analysis of Msgn1 (O,R), Tbx6 (P,S) and Dll3 (Q,T), with the posterior somite marker Uncx4.1 (orange in O,P,R and S; blue in Q and T) in E9.5 controls and Tcre-F-Msgn1^GOF mutants. (U-Z) WISH of T mRNA expression in control (U-W) and Tcre-F-Msgn1^GOF mutants (X-Z). V,W, cross-sections in U and Y; Z, cross-sections in X at the indicated embryonic locations. Scale bars: 100 μm (D,Y), 50 μm (K), 200 μm (R,X).

Fig. 8.

Msgn1 rescues paraxial mesoderm in Wnt3a^−/− mutants. (A) Flow cytometry analysis of iF-Msgn1 or control iCre EBs treated with (+) or without (−) Dkk1 and/or Dox for 48 h for the expression of Pdgfrα and Flk1. The arrowheads indicate Pdgfrα⁺ cells. (B-G) β-Gal staining of E9.5 control (B), Wnt3a^−/− (C), and Wnt3a^−/−; Tcre-F-Msgn1^GOF (D) mutants. (E-G) These panels represent corresponding cross sections of embryos depicted in B-D, taken at the indicated location (black line, B). Arrowheads indicate paraxial mesoderm. (H-K) WISH analysis of Meox1 in control (H), Tcre-F-Msgn1^GOF (I), Wnt3a^−/− (J), and Wnt3a^−/−; Tcre-F-Msgn1^GOF (K) mutants. Curved white line in J,K highlights the rescued posterior paraxial mesoderm domain. Scale bars: 200 μm (B,H), 100 μm (E).

Fig. 9.

Msgn1 is a master regulator of PSM differentiation. Bipotential NM stem cells, defined by Sox2 and T expression, generate the progenitors of the trunk spinal cord (NPC, neural progenitor cells) and musculoskeletal system (MPC, mesodermal progenitor cells). Wnt3a maintains both the NM stem cell and the MPC. In our model, the Wnt3a/β-catenin pathway activates the target genes T and Msgn1 in NM stem cells in the PS to specify paraxial mesoderm fate (Yamaguchi et al., 1999; Dunty et al., 2008; Chalamalasetty et al., 2011). Msgn1 initiates the genetic programs that define PSM identity and cell behavior by activating downstream master regulators of EMT and motility (Snai1) and neural suppression (Tbx6) among others. Msgn1 and Tbx6 expression must be extinguished for PSM cells to undergo MET and segmentation to form somites and, ultimately, to differentiate into skeletal muscle, bone, cartilage, tendons, endothelial cells and dermis.