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Figure 2 Conditional expression of bmp2b causes activation of BMP signaling, elevated expression of id1 and an increased proliferation of NSCs. (A–C’’) Immunostaining with GS (green) and pSmad1/5/9 (red) antibodies on sections of WT telencephala, treated with heat-shock (A–A’’, +HS) and Tg(hs:bmp2b) telencephala without heat-shock (B–B’’, -HS) or with heat-shock (C–C’’, +HS). pSmad1/5/9 immunoreactivity was detected in GS+ NSCs exclusively in heat-shocked telencephala of the Tg(hs:bmp2b) line. (C’’) inset: a magnified view of a pSmad+/GS+ NSC. Note the nuclear localization of pSmad in contrast to cytoplasmic localization of GS. White arrows show individual NSCs, yellow arrows indicate the processes of the same NSCs (A–C’’). (D–F) ISH on telencephalic cross- sections indicating increased id1 expression after heat-shock-induced bmp2b expression. Black arrows show the expression of id1 in the ventricular zone of WT after heat-shock (D), Tg(hs:bmp2b) after heat-shock (F) and Tg(hs:bmp2b) without heat-shock (E). (G) RT-qPCR analysis of id1 mRNA expression in WT telencephala with heat-shock (WT +HS) and Tg(hs:bmp2b) telencephala without heat-shock (-HS) and with heat-shock (+HS). (H–J’) Immunohistochemistry with antibodies against the NSC marker S100β (blue) and the proliferation marker PCNA (green) on telencephalic transverse sections from WT with heat-shock (H,H’) and Tg(hs:bmp2b) without heat-shock (I,I’) and with heat-shock (J,J’) focusing on the ventricular zone of the dorsal telencephalon. Note that the number of proliferating NSCs (PCNA+/S100β+ cells, dashed, white boxed areas) is reduced in heat-shocked telencephala of the Tg(hs:bmp2b) line (J,J’). (K) Quantification of the PCNA+/S100β+ + cells in telencephala of the Tg(hs:bmp2b) line showing a significant reduction in the heat-shocked (+HS) telencephala compared to the control group without heat-shock. Significance is indicated by asterisks: ns, not significant; *** p < 0.001. n = 3 brains (G), n = 15 sections (K). Scale bar = 20 μm (A–C’’,H–J’), 100 μm (D–F). HS: heat-shock.