Figure 2.

Detrimental effect of PHOX2B-7Ala in the generation of RTN-like neurons in HBSOs

(A) Representative phase-contrast images of the day-60 control and PHOX2B-7Ala HBSOs. Bar chart showing the sizes of the day-60 control and PHOX2B-7Ala HBSOs (mean ± SEM). n ≥ 9 per group from three independent experiments. Unpaired t test; NS, non-significant.

(B) Immunostaining of PHOX2B, 5-HT in the day-60 control and PHOX2B-7Ala HBSOs.

(C) Bar charts showing the percentages of PHOX2B⁺ cells in the day-60 HBSOs derived from the control and PHOX2B-PARM mutants (mean ± SEM). n ≥ 8 per group from three independent experiments.

(D) qRT-PCR and (E) western blot analyses of PHOX2B expression in the day-60 control and PHOX2B-7Ala HBSOs. Data are represented as mean ± SEM. n ≥ 9 per group from three independent experiments. Unpaired t test.

(F) Immunostaining of PHOX2B, TH, and VGLUT2 in the day-60 control and PHOX2B-7Ala HBSOs. The inset on the bottom is an enlargement of a square region in the image. Filled and open arrow heads mark the RTN-like (PHOX2B⁺ VGLUT2⁺ TH^–) and PHOX2B⁺ TH⁺ neurons, respectively.

(G and H) Bar charts showing the percentages of (G) PHOX2B⁺VGLUT2⁺ and (H) PHOX2B⁺TH⁺ cells in the day-60 control and PHOX2B-7Ala HBSOs (mean ± SEM). n ≥ 8 per group from three independent experiments. Unpaired t test.

(I and J) Immunostaining of PHOX2B with (I) C-Casp3 and (J) TUJ1 (an enlargement of a square is shown in the right panel) in the day-60 control and PHOX2B-7Ala HBSOs. Mutant PHOX2B⁺ neurons that were viable (C-Casp3^–) and expressed pan-neuronal marker (TUJ1⁺) are marked by open and filled arrowheads, respectively. See also Figure S2.