Int J Stem Cells.  2020 Nov;13(3):342-352. 10.15283/ijsc20037.

Cdo Is Required for Efficient Motor Neuron Generation of Embryonic Stem Cells

Affiliations
  • 1Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, Korea
  • 2Department of Physiology, Sungkyunkwan University School of Medicine, Suwon, Korea
  • 3Single Cell Network Research Center, Sungkyunkwan University School of Medicine, Suwon, Korea

Abstract

Background and Objectives
The directed differentiation of pluripotent stem cells into motor neurons is critical for the development of disease modelling and therapeutics to intervene degenerative motor neuron diseases. Cell surface receptor Cdo functions as a coreceptor for Sonic hedgehog (Shh) with Boc and Gas1 in the patterning of ventral spinal cord neurons including motor neurons. However, the discrete function of Cdo is not fully understood.
Methods and Results
In this study, we examined the role of Cdo in motor neuron generation by utilizing in vitro differentiation of Cdo+/+ and Cdo−/− embryonic stem cells (ESCs). In response to Shh, Cdo−/− ESCs exhibited impaired expression of motor neuron specification markers while dorsal interneuron specification markers were significantly increased, compared to Cdo+/+ ESCs. Reactivation of Shh signalling pathway with Smoothened (Smo) agonist (SAG) restored motor neuron specification in Cdo−/− ESCs. In addition, electrophysiological analysis revealed the immature electrical features of Cdo−/− ESCs-derived neurons which was restored by SAG.
Conclusions
Taken together, these data suggest that Cdo as a Shh coreceptor is required for the induction of motor neuron generation by fully activating Shh signalling pathway and provide additional insights into the biology of motor neuron development.

Keyword

Cdo; Shh coreceptors; Shh signalling pathway

Figure

  • Fig. 1 The expression of genes implicated in specification and differentiation of ESCs into motor neurons. (a) The schematic representation of motor neuron differentiation used in this study. The detailed description of the procedure is presented in Materials and Methods. (b∼d) qRT-PCR analysis for the expression of stemness genes (Nanog and Oct4) (b), motor neuron differentiation markers (Olig2, Nkx6.1, Hb9 and Isl1) (c) and Shh signaling components (Shh, Ptch1, Gli1, Gli2, Gli3, Cdo, Boc and Gas1) (d) during specification and differentiation of mESCs into motor neuron. L32 was selected as a reference gene. Data represent means±SEM. *p<0.05, **p<0.01, ***p<0.001 (n=3, each).

  • Fig. 2 Cdo deficiency altered gene expression in motor neuron specification of ES cells. (a) Scatter plot images of differentially expressed genes. Red and green colors indicate up- and down-regulated genes in Cdo−/− cells relative to Cdo+/+ cells, respectively (1.5-fold change, p<0.05). Black dots indicate genes with no significant change of RNA expression level (less than 1.5-fold change). (b) The bar charts indicate NES of the gene sets contributing to neuronal development in motor neuron specification-induced Cdo+/+ and Cdo−/− cells. (c) Heat map for the relative levels of the gene sets related to dorsal-, ventral-interneuron and spinal cord motor neuron in Cdo−/− cells compared to Cdo+/+ cells at MNS-3. Red=Up-regulated genes, Blue=Down-regulated genes. (d) qRT-PCR analysis for dorsal interneuron regulators (Pax3, Daam2 and Uncx) and motor neuron specific gene (Nkx6.1) in Cdo+/+ and Cdo−/− cells at MNS-3. Each value was normalized to the level of L-32, an endogenous control. Data represent means±SEM. *p<0.05, **p<0.01, ***p<0.001.

  • Fig. 3 Cdo−/− ESCs displayed impaired motor neuronal specification. (a, b) qRT-PCR analysis for the levels of ventral and motor neuron specification markers (Pax6, Olig2, Nkx6.1, Hb9, Isl1 and Chx10) (a) and dorsal interneuron specification markers (Brn3a, Pax2, Lbx1 and Pax3) (b) in Cdo+/+ and Cdo−/− mESCs at MNS-5 and Elong stages. L32 was selected as a reference gene. Data represent means±SEM. *p<0.05, **p<0.01, ***p<0.001. (n=3, each) (c) Immunofluorescent staining for motor neuronal markers (Olig2 and Nkx6.1) in Cdo+/+ and Cdo−/− EBs at MNS-5. Nuclei were visualized by DAPI. Size bar=25μm. (d) Quantification of Olig2- and Nkx6.1-positive cells shown in panel c. Data represent means±SD. **p<0.01, ***p<0.001. (n=6) (e) Immunofluorescent staining for motor neuronal markers (Hb9 and Isl1) in Cdo+/+ and Cdo−/− cells at Elong stage. NF was also immunostained as a neuronal differentiation marker. Nuclei were visualized by DAPI. Size bar=10μm. (f) Quantification of Hb9- and Isl1-positive cells shown in panel e. Data represent mean±SD. ***p<0.001 (n=6).

  • Fig. 4 Shh-independent activation by SAG restored motor neuron differentiation in Cdo-deficient ESCs. To activate Shh signaling pathway in Shh-independent way, Cdo+/+ and Cdo−/− ESCs were treated with 1 μM SAG instead of Shh during MNS stage. (a, b) qRT-PCR analysis for genes related to ventral neural subtypes and motor neuron specification (Pax6, Olig2, Nkx6.1, Hb9, Isl1 and Chx10) (a) and dorsal interneuron specification (Brn3a, Pax2, Lbx1 and Pax3) (b) in Cdo+/+ and Cdo−/− ESCs at MNS-5 and Elong. L32 was selected as a reference gene. Data represent means±SEM. *p<0.05, **p<0.01, ***p<0.001. (n=3, each) (c) Immunostaining for Olig2/Nkx6.1or Hb9/Isl1 in Cdo+/+ and Cdo−/− EBs at MNS-5. DAPI was used to visualize nuclei. Size bar=25μm. (d) Quantification of Olig2-, Nkx6.1-, Hb9- and Isl1-positive cells shown in panel c. Data represent means±SD. *p<0.05. (n=6, each) (e) Immunostaining for Hb9 or Isl1 in Cdo+/+ and Cdo−/− cells at Elong. NF was immunostained indicating neuronal differentiation. Nuclei were visualized by DAPI. Size bar=10μm. (f) Quantification of Hb9- or Isl1-positive cells shown in panel e. Data represent means±SD. *p<0.05 (n=6).

  • Fig. 5 SAG restored functional maturation of Cdo−/− ESC-derived motor neurons. (a∼c) Differential interference contrast (DIC) images and representative trace of a whole-cell current clamp recording from Cdo+/+ ESC-derived motor neurons, which were differentiatated with Shh (a) or Cdo−/− ESC-derived motor neurons, which were exposed to Shh (b) or SAG (c) for differentiation. (n=13 for Cdo+/+ (+Shh), n=9 for Cdo−/− (+Shh), n=8 for Cdo−/− (+SAG)) Scale bars=10 μm. (d∼i) Evoked action potentials generated in response to a depolarizing current injection. Graphs comparing the passive membrane properties. The sample size (n-number) is indicated in each graph. Values are means±SEM. *p<0.05, **p<0.01, ***p<0.001.


Reference

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