Design of sgRNA and ssODN to edit in the TBX19 K146R mutation

A, Illustration of the TBX19 gene (HGNC: 11596; ENSEMBL: ENSG00000143178; Human GRCh38) and TBX19 protein.

B, Wild-type (WT) sequence containing target site, cut site, target and PAM sequences.

C, ssODN design to edit in a missense K146R mutant of TBX19 using CRISPR/Cas9.

D, Sequence analysis of a TBX19 KI hiPSC clone 63 obtained by Sanger sequencing after screening by CAPS. This clone was subsequently used in this work to differentiate into pituitary organoids (see below).

E, Summary of our strategy procedure:

Step 1: Production of the knock-in hiPSC lines by CRISPR/Cas9 genome editing.

Step 2: Differentiation into pituitary organoids from mutant hiPSC lines in parallel with the isogenic WT line using 3D culture, followed by comparison of the development of organoids between the two groups.

Time course of organoid growth and gene expression in WT and TBX19 KI organoids

A, Culture protocol and outline to generate pituitary organoids in three-dimensional (3D) culture from hiPSC. Organoids were collected at days (d) 0, 6, 18, 27, 48, 75, 105 during differentiation to analyze.

B, Bright-field microscopy views of WT organoid examples at different time points throughout differentiation. Scale bars indicated in each image.

C, Number of replicates per time point and per genotype in experiments depicted in following graphs. Asterisks indicate that 7-8 organoids were grouped for each sample. For other points, each sample consists of a single organoid.

D-H, Relative quantification (RQ) mRNA expression analysis for key markers of pituitary organoids during differentiation: WT (in black line) and TBX19 KI organoids (in red line). Relative quantification of each target gene was obtained by the 2−ΔΔCt method from qRT-PCR results (see Methods). Data show means ± standard error of the mean (SEM, Mann-Whitney t-test [unpaired, two-tailed, nonparametric]). p < 0.05 (*), p< 0.01 (**).

D, Relative quantification of HESX1 expression, the earliest pituitary placode marker assessed. The expression of HESX1 is significantly downregulated in TBX19 KI organoids vs. WT at d18 and d27.

E, Relative quantification expression of PITX1, a pituitary progenitor marker. PITX1 was significantly downregulated in TBX19 KI organoids by d48 and d75.

F, Relative quantification expression of LHX3, a pituitary progenitor marker. LHX3 was significantly lower in TBX19 KI organoids as compared to WT from d48 onwards.

G, Relative quantification expression of TBX19, a critical transcriptional determinant for corticotroph differentiation. TBX19 expression is higher in TBX19 KI organoids at d105. H, Relative quantification expression of POMC, a corticotroph marker. POMC was significantly downregulated in TBX19 KI organoids from d48 onwards.

Impairment of corticotroph development in TBX19 KI organoids as compared with controls at the protein level

A, B, Immunostaining of LHX3 and CDH1 (E-cadherin) expression in epithelial cells, typical of Rathke’s pouch ectoderm in early pituitary primordia, was reduced in TBX19 KI organoid vs WT on day 48 (n=10 organoids for each group). Scale bars: 10 μm.

C, D, Immunostaining showed that ACTH and TBX19 expressions were reduced in TBX19 KI organoid vs WT on day 105 (n=10 organoids for each group). Scale bars: 10 μm.

3D reconstruction of whole wild-type (WT) and TBX19 KI organoids on day 105

A, A representative image of whole-mount immunostaining against ACTH in a cleared WT organoid on d105 using light-sheet microscopy. Scale bar: 200 μm.

B, A representative image of whole-mount immunostaining against ACTH in a cleared TBX19 KI organoid on d105 as above, showing impaired corticotroph differentiation. Scale bar: 200 μm.

C, The number of corticotroph cells per mm3 was significantly decreased in TBX19 KI organoids (* p= 0.0159). Means ± SEM for n=4 WT, n=5 TBX19 KI organoids. Mann-Whitney test (unpaired, two-tailed, nonparametric).

Design of sgRNA and ssODN to introduce a NFKB2 D865G mutation

A, Illustration of the NFKB2 gene (HGNC: 7795; ENSEMBL: ENSG00000077150; Human GRCh38).

B, WT sequence containing target site, cut site, target and PAM sequence.

C, ssODN design to introduce the missense mutation D865G into NFKB2 using CRISPR/Cas9.

D, Sequence analysis of the NFKB2 KI hiPSC clone 7, obtained by Sanger sequencing, after screening by CAPS. This clone was subsequently used in this work to differentiate into pituitary organoids (see below).

Time course of organoid growth and gene expression in WT and NFKB2 KI organoids

A, Number of replicates per time point and per genotype in experiments depicted in following graphs. Asterisks indicate that 7-8 organoids were grouped for each sample. For other point, each sample consists of a single organoid.

B-F, Relative quantification (RQ) mRNA expression analysis for key markers of pituitary organoids during differentiation: WT (black line and dots) and NFKB2 KI organoids (red line and dots). Data show means ± standard error of the mean (SEM);. Mann-Whitney t-test (unpaired, two-tailed, nonparametric). p < 0.05 (*), p< 0.01 (**).

B, Relative quantification expression of HESX1, the earliest pituitary placode marker assessed. The expression of HESX1 was upregulated in NFKB2 KI organoids vs. WT between d27 and d75.

C, Pituitary progenitor marker P I T X 1was significantly downregulated in NFKB2 KI organoids at d48 and d75.

D, Pituitary progenitor marker LHX3 was significantly lower in NFKB2 KI organoids as compared to WT from d48 onwards.

E, Relative quantification expression of TBX19, a corticotroph marker. TBX19 was significantly increased in NFKB2 KI organoids by d105.

F, Relative quantification expression of POMC, a corticotroph marker. POMC was significantly downregulated in NFKB2 KI organoids from d75.

G, Volume of organoids (mm3) on d105, calculated using Imaris software (see in methods). There was no significant difference in volume between WT and NFKB2 KI organoids (p= 0.6126). Data show means ± SEM; n=7 in the WT group, n=8 in the mutant group. Mann-Whitney test (unpaired, two-tailed).

Impairment of pituitary development in NFKB2 KI organoids

A, B, Immunostaining for LHX3 (green) and p100/p52 (red) in the early pituitary-type epithelium was reduced in NFKB2 KI organoids vs WT on day 48 (n=10 organoids for each group). Stronger expression of p100/p52 was observed in pituitary progenitors, but expression in the hypothalamic part of the organoid cannot be excluded. Scale bar: 10 μm

C, D, by day 105, although nuclear TBX19 was detectable in both mutants, these cells failed to co-express ACTH in NFKB2 KI organoids (n=10 organoids for each group). Scale bars: 10 μm

3D reconstruction of whole Wild type (WT) and NFKB2 KI organoids on day 105

A, A representative image of whole-mount immunostaining against ACTH in a cleared WT organoid on d105 using light-sheet microscopy. Scale bar: 200 μm.

B, A representative image of whole-mount immunostaining against ACTH in a cleared NFKB2 KI organoid on d105 as above, showing impaired corticotroph differentiation. Scale bar: 100 μm.

C, The number of corticotroph cells per mm3 was significantly decreased in NFKB2 KI organoids (* p= 0.0007). Means ± SEM for n=6 organoids WT, n=8 NFKB2 KI organoids. Mann-Whitney test (unpaired, two-tailed, nonparametric).

Whole-transcriptome profiles of WT vs NFKB2 KI organoids on day 48

A, Heat map showing global differential gene expression between WT vs NFKB 2KI organoids (n=5 for each group).

B, Volcano plot of genes showing differential gene expression between WT vs NFKB2 KI organoids (expressed in Log2 fold-change). Blue dots in the left panel indicate 144 genes involved in hypothalamic-pituitary development. Grey dots indicate all other genes detected in RNA-seq.

C, Volcano plot of genes showing differential gene expression between WT vs NFKB2 KI organoids. Green dots indicate genes coding for 39 key transcription factors whose expression is significantly changed (padj < 0.05). Red dots indicate key transcription factors with non-significant changes (padj> 0.05). Grey dots indicate all other genes detected in RNA-seq.

RNA-seq expression data for a list of 144 genes known from the literature to have a functional influence on pituitary-hypothalamic development. Differentially expressed genes (padj < 0.05) in NFKB2 KI organoids are highlighted in green when upregulated and in pink when downregulated.

Differential gene expression analysis for genes associated with different stages of epithelial-to-mesenchymal transition in NFKB2 KI vs WT organoids. Significantly decreased and increased expressions are highlighted in pink and green, respectively.

Impaired pituitary progenitor development is correlated with changes in growth factor expression in NFKB2 KI organoids

A, RT-qPCR experiments at d48 showing that HESX1 expression is upregulated NFKB2 KI organoids, compared to WT and TBX19 KI, whereas PITX1 and LHX3 are downregulated in both mutants.

B, BMP4 expression is increased whereas and FGF8 and FGF10 are decreased in NFKB2 mutants, but unchanged in TBX19 KI organoids.

Data show means ± SEM; n=17, 12 and 11 for WT, TBX19 KI and NFKB2 KI respectively. Mann-Whitney t-test (unpaired, two-tailed, nonparametric). p < 0.05 (*), p< 0.01 (**), p<0.005 (***),

Impaired differentiation of corticotrophs in TBX19 KI an dNFKB2 KI organoids

A, RT-qPCR measurements of TBX19 expression show a late (d105) increase in both mutants, with stronger effect in TBX19 KI organoids.

B, Corticotroph terminal differentiation marker NR4A2 was decreased in both mutants at d48 and d75, but only in NFKB2 mutants at d105.

C, PCSK1 downregulation is only observed in NFKB2 KI at d48, and in both models at d75 and d102.

D, A decrease in POMC expression is observed in both mutants from d48 onwards.

Data show means ± SEM; d48: n=17, 12 and 11; d75, n=9, 7 and 6; d105: n=9, 5 and 5 for WT, TBX19 KI and NFKB2 KI organoids respectively. Mann-Whitney t-test (unpaired, two-tailed, nonparametric). p < 0.05 (*), p< 0.01 (**), p<0.005 (***),

POU1F1-dependent lineages are affected in TBX19 KI an dNFKB2 KI organoids

A, Strong downregulation of PROP1 expression is observed at d48 in both mutants and persists at d75.

B, Expression of POU1F1-dependant lineage progenitors and mature somatotrophs marker NEUROD4 is decreased at d48 inNFKB2 organoids, and in both models at d105.

C, ZBTB20, a marker for POU1F1-dependant lineage progenitors and mature lactotrophs, is less expressed in NFKB2 KI organoids at d48, but at normal levels by d75. In TBX19 KI mutants, its expression is normal at d48 but decreased by d75.

D, On d75, POU1F1 expression is barely detectable in either model.

Data show means ± SEM; d48: n=17, 12 and 11; d75: n=9, 7 and 6 for WT, TBX19 KI and NFKB2 KI organoids respectively. Mann-Whitney t-test (unpaired, two-tailed, nonparametric). p < 0.05 (*), p< 0.01 (**), p<0.005 (***).

Identification of genes potentially under direct p52 regulation

Venn diagram showing the total number and intersections of genes identified as p52 targets, genes differentially expressed at d48 between WT and NFKB2 KI organoids, and a literature-curated list of genes particularly implicated in hypothalamus-pituitary development. The table on the right shows the identity of the 23 genes (circled in red), with color code indicating whether they are upregulated (green) or downregulated (pink) in NFKB2 KI mutants at d48.

Changes in gene expression induced by NFKB2 D865G/D865G mutation during hypothalamic-pituitary organoid development

Diagram illustrating changes in gene expression (black arrows) associated with different stages of normal hypothalamic-pituitary organoids development and cell differentiation. The NFKB2D865G/D865G mutation perturbs these changes (red arrow), possibly through direct transcriptional control for genes indicated in blue.