Journal: PLoS Biology
Article Title: In Vitro Generation of Neuromesodermal Progenitors Reveals Distinct Roles for Wnt Signalling in the Specification of Spinal Cord and Paraxial Mesoderm IdentityDevelopment of Spinal Cord Neurons in Delicate Balance
Figure Lengend Snippet: Generation of neural cells with specific AP identities from ESCs. (A) Schematic representation of differentiation conditions used for the generation of NPCs with specific Anterior (N A ), Hindbrain (N H ) and Spinal cord (N P ) identities. (B) Relative expression levels of the indicated genes from N A , N H and N P cells at day 5 (D5) of differentiation indicate that N A , N H and N P cells express distinct sets of genes. The standard scores (z-scores) of the indicated genes from mRNA-seq analysis reveals that N A cells express high levels of forebrain markers including Otx1 and Otx2 ; N H cells express genes characteristic of hindbrain including Mafb and Hoxa2 genes; N P cells express high levels of posterior 5′ Hox genes including Hoxc8 and Hoxc9 . The individual Z-score for each replicate is indicated on the graph with circles, triangles and squares. (C) Time course of Hoxb and Hoxc cluster activation in cells cultured in N H and N P conditions showing fold change compared to D1. Posterior Hox genes are selectively activated only in the N P conditions and show temporal colinearity with the induction of anterior Hox genes prior to posterior Hox genes  . In N H cells Hoxb1 and Hoxb2 are induced prior to Hoxc4 . However, the more posterior Hox genes are not induced. By contrast, in N P conditions the 5′ Hox genes Hoxc6 , Hoxc8 and Hoxc9 are induced at D4 and their expression is maintained at day 5. (Note log 2 scale). (D) Immunohistochemistry indicates that N H cells analysed at D8 differentiate into MNs of hindbrain identity coexpressing Hoxb4 and Phox2b. (E) N P cells exposed to SAG generate spinal neurons coexpressing Hoxc6 and Hoxc9 with b-tubulin (Tuj1). These were not detected in N H conditions. Coexpression of Hoxc6 and Hoxc9 with Islet1 indicates the generation of spinal MNs of forelimb and thoracic identity, respectively. These MNs also expressed Lim3/Raldh2 and HB9. (F) Graph showing the standard scores (z-scores) of Zfp42 (Rex1), Pou5F1 (Oct3/4) and Fgf5 from the mRNA-seq from D1 to D3. The kinetics of gene expression indicate that ESCs progressively lose their stem cell identity and acquire a transient epiblast identity at D2. (G) Hoxc6/Tuj1 and Hoxc9/Tuj1 positive cells were quantified in independent fields of D8 cells differentiated in N P conditions. All data used to generate the plots of Figure 1 can be found in Data S1 .
Article Snippet: The following primary antibodies were used: mouse anti-Hoxc6 (1∶10) (DSHB), mouse anti-Hoxc9 (1∶10) (gift of T. Jessell), mouse anti-Hoxc10 (1∶50) (DSHB), rat anti-Hoxb4 (1∶100) (gift of A. Gould), rabbit anti-Phox2b (1∶200), mouse anti-Tuj1 (1∶1000) (Covance), rabbit anti-Tuj1 (1∶500) (Covance), rabbit anti-Olig2 (1∶500) (Chemicon), mouse anti-Sox2 (1∶200) (ab92494, Abcam), rabbit anti-Sox2 (1∶200) (Millipore), goat anti-Sox2 (1∶100) (R & D), goat anti-Tbx6 (1∶200) (R & D) or rabbit anti-Tbx6 (0.6 µg/ml) (ab38883, Abcam), goat anti-Brachyury (1∶500) (R & D), rabbit anti-RALDH2 (1∶500) (Sigma), rabbit anti-Desmin (1∶500) (Abcam), mouse anti-MyoD1 (1∶200) (DAKO), mouse anti-Islet1 (1∶2000) (gift of T. Jessell), mouse anti-Lim3 (1∶10) (DSHB), mouse anti-HB9 (1∶100) (DSHB), anti-Foxa2 (1 mg/ml) (Santa Cruz; sc-6554), anti-GFP (10 µg/ml) (Abcam; ab13970), anti-Pax3 (1∶20) (DSHB);. anti-Nanog (2.5 µg/ml) (14-5761-80, eBioscience); anti-Oct4 (1 µg/ml) (N-19, Santa Cruz), HoxC8 (5 µg/ml) (Abcam).
Techniques: Expressing, Activation Assay, Cell Culture, Immunohistochemistry