Figure 6.

Specification of Hormonal Cells of the Pituitary In Vitro

(A) Bulk qRT-PCR analysis of day-60 cells patterned with FGF8, FGF8/BMP2, or BMP2 for 30 days. Patterning with BMP2 induced a more ventral cell identity (PIT1, GATA2, GH1, FSHB, and LHB) while FGF8 suppressed ventral cell types (FSHB). Data are plotted as mean ± SEM of two to four independent experiments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 (n.s., not significant) compared with the “default” pituitary differentiation on day 60.

(B) Unsupervised hierarchical clustering of FGF8, FGF8/BMP2, and BMP2 patterned cells using 34 primer pairs identified three larger clusters of cells, with cluster 2 mainly comprising cells patterned by FGF8 (or FGF8/BMP2) and cluster 3 mainly comprising cells patterned by BMP2 (or FGF8/BMP2).

(C) Quantification of hormonal transcripts per cell in different patterning conditions. Data are plotted as percentage of cells expressing the respective transcript (ct < 35 cycles in combination with a proper melting curve).

(D) Immunofluorescence analysis (representative images) of hormone expression in cells patterned with FGF8, FGF8/BMP2, or BMP2 on day 60 of differentiation. Scale bars, 50 μm.

(E) Quantification of hormone-expressing cells (per subtype) in different patterning conditions on day 60 of differentiation. High levels of FGF8 induced dorsal fate (ACTH) while intermediate levels of FGF8 and BMP2 induced dorsal/ventral fates (PRL and GH) compared with the default condition (E6 only). Data are plotted as mean ± SEM of two independent experiments. ^∗p < 0.05, ^∗∗p < 0.01 (n.s., not significant) compared with the “default” (E6 only) pituitary differentiation on day 60.

See also Figure S4.