Tlx3 Promotes Glutamatergic Neuronal Subtype Specification through Direct Interactions with the Chromatin Modifier CBP
Fig 5
Effects of Tlx3 homeodomain deletion on glutamatergic neuronal marker proteins in ES-derived neurons.
(A) Immunoblot analysis to evaluate the expression levels of glutamatergic neuronal marker proteins (NeuroD, Ngn1, GluR2, GluR4, and VGLUT2) and the pan-neuronal marker protein (Tau) in response to forced expression of Tlx3 or Tlx3ΔHD in ES cells. Whole lysates from ES cells stably transfected with the empty vector served as the negative control. Lysates from undifferentiated ES cells (ES), EBs, and ES-derived cells on day 4 (NI-4) or 7 (NI-7) after neural induction were analyzed by immunoblotting. As a control for protein loading, the blots were analyzed with antiactin antibodies (lower panel). (B) Immunocytochemical analysis of the expression of glutamate receptor subunits (GluR2 and GluR4; red) and pan-neuronal markers (Tuj1 and neurofilament; blue) in ES-derived cells at NI-7. Transgenic ES cells exhibited eGFP fluorescence (green). (C) Representative flow cytometric scatter plots of Tlx3- or Tlx3ΔHD-expressing ES-derived cells at NI-7. Cells stably transfected with an empty vector served as the negative control. Cells were stained with primary antibodies for glutamate receptor subunits (GluR2/3) or a pan-neuronal marker (Tuj1), followed by staining with AlexaFluor 568-conjugated secondary antibodies. The specificity of the primary antibodies was confirmed based on a comparison with the isotype control IgG. The fluorescence intensity of eGFP (marking transgenic ES cell-derived cells) is indicated on the x-axis and the fluorescence intensities of GluR2/3 and Tuj1 immunostaining on the y-axis. (D) Mean ratio of GluR2/3-positive relative to Tuj1-positive cells in GFP-positive cell populations. The values were normalized against Tuj1 (arbitrarily set to 1), and they are presented as the mean ± SD (n cells = 10,000, *p< 0.05, one-way ANOVA). ES, undifferentiated embryonic stem cells; NI-4, ES-derived cells at neural induction day 4; NI-7, ES-derived cells at neural induction day 7. Scale bar: 20μm.