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. 2023 Jun 22;18(7):1500–1515. doi: 10.1016/j.stemcr.2023.05.020

Figure 4.

Figure 4

PHOX2B-PARMs disrupt the formation of respiratory center-like region

(A) Immunostaining of PHOX2B and markers of forebrain (TBR1), midbrain (FOXA2), and hindbrain (ISL1) in the day-60 control HCOs.

(B) Immunostaining of different markers of the respiratory center-like region in the day-60 control HCOs. Yellow arrows mark the RTN-like (PHOX2B+ VGLUT2+ TH−) neurons.

(C) Schematic showing the additional Ala repeats added to the PHOX2B-PARM mutant hPSC lines.

(D) Representative phase-contrast images of day-60 HCOs derived from the control and the PHOX2B-PARM mutants. Bar chart showing the sizes of day-60 HCOs derived from the control and the PHOX2B-PARM mutants (mean ± SEM). n ≥ 36 per group from four independent experiments. One-way ANOVA; NS, non-significant.

(E) Immunostaining of markers of A5 domain (PHOX2B+; TH+; VGLUT2−), C1 domain (PHOX2B+; TH+; VGLUT2+), and RTN neurons (PHOX2B+; TH−; VGLUT2+) in the day-60 HCOs derived from the control and the PHOX2B-PARM mutants. Yellow arrows mark the RTN-like (PHOX2B+ VGLUT2+ TH−) neurons.

(F and G) Bar charts showing the percentages of (F) PHOX2B+ and (G) PHOX2B+; VGLUT2+ double-positive cells in respiratory center-like region of the day-60 HCOs derived from the control and PHOX2B-PARM mutants. n ≥ 3 per group from three independent experiments. One-way ANOVA; NS, non-significant. All the insets on the bottom of the immunostaining image are the enlargement of the respective square regions in the images. See also Figure S4.