Pituitary Stem Cell Regulation by Zeb2 and BMP Signaling

Abstract

Epithelial to mesenchymal transition (EMT) is important for many developing organs, and for wound healing, fibrosis, and cancer. Pituitary stem cells undergo an EMT-like process as they migrate and initiate differentiation, but little is known about the input of signaling pathways or the genetic hierarchy of the transcriptional cascade. Prop1 mutant stem cells fail to undergo changes in cellular morphology, migration, and transition to the Pou1f1 lineage. We used Prop1 mutant mice to identify the changes in gene expression that are affiliated with EMT-like processes. BMP and TGF-β family gene expression was reduced in Prop1 mutants and Elf5, a transcription factor that characteristically suppresses EMT, had elevated expression. Genes involved in cell-cell contact such as cadherins and claudins were elevated in Prop1 mutants. To establish the genetic hierarchy of control, we manipulated gene expression in pituitary stem cell colonies. We determined that the EMT inducer, Zeb2, is necessary for robust BMP signaling and repression of Elf5. We demonstrated that inhibition of BMP signaling affects expression of target genes in the Id family, but it does not affect expression of other EMT genes. Zeb2 is necessary for expression of the SHH effector gene Gli2. However, knock down of Gli2 has little effect on the EMT-related genes, suggesting that it acts through a separate pathway. Thus, we have established the genetic hierarchy involved in the transition of pituitary stem cells to differentiation.

Keywords: ELF5; GLI2; ID gene family; Rathke's pouch; epithelial to mesenchymal-like transition; transcription factor PROP1.

Figure 1.

Claudin 10 and keratin 8 are expressed in pituitary progenitors. CLDN10 immunostaining was carried out on sagittal (A, A’, B, B’, C, C’) and coronal (D, D’, E, E’) sections from normal (A, B, C, D, E) and Prop1 mutant pituitaries (A’, B’, C’, D’, E’) at the indicated embryonic and postnatal days. KRT8 immunostaining was carried out on coronal sections from normal (F) and Prop1 mutant pituitaries (F’) at P1. SOX2 is a pituitary progenitor marker. Coimmunostaining for SOX2 (green) and CLDN10 (red) shows that the ectopic CLDN10 in the Prop1 mutant postnatal mouse pituitary is expressed in many SOX2-positive cells (G, G’, H, H’). Cell nuclei are stained with DAPI (blue). White arrow, CLDN10 and SOX2 positive cells; yellow arrow, CLDN10 only cell. (A, A’) 20× (scale bar = 200 μM), (B, B’) 40× (scale bar = 100 μM), (C, C’) 10× (scale bar = 200 μM), (D, D’) 20× (scale bar = 200 μM), (E, E’) 20× (scale bar = 200 μM), (F, F’) 10× (scale bar = 200 μM), and (G, H) 20× (scale bar = 200 μM).

Figure 2.

Reduced phospho-SMAD expression in Prop1 mutant pituitary primordia. Sagittal sections from normal (A-C) and Prop1 mutant (D-F) pituitaries collected at e13.5 were immunostained for pSMAD (green). Cell nuclei are stained with DAPI (blue). Each panel (A-G) represents a different individual animal. White arrows are pointing to pSMAD expression boundary in dorsal and ventral lumen. Double immunohistochemistry for pSMAD (green) and PROP1 (red) in sagittal e13.5 pituitary gland. Cell nuclei are stained with DAPI (blue). (A-G) 20× (200 μM scale bar); (H) 63× (100 μM). (I) Average percentage of pSMAD positive cells in Prop1^+/+ and Prop1^df/df e13.5 pituitary gland. N = 3 for each genotype. *P < 0.05, Student unpaired t test.

Figure 3.

Prop1 mutant embryos and CFCs exhibit reduced Id gene expression. In situ hybridization for Id2 (A, C) and Id3 (B, D) in normal (A, B) and Prop1 mutant (C, D) on sagittal sections at e14.5. (J-M) 10× (200 μM scale bar). RT-qPCR was used to determine relative mRNA expression of Id genes in P11 pituitary CFCs in normal and Prop1 mutant mice (N = 3), (E). Id1, Id2, and Id3 were significantly reduced in Prop1 mutant CFCs. *P < 0.05, multiple unpaired t test.

Figure 4.

Reduced BMP signaling does not affect the EMT-like process in stem cell colonies. CFCs were prepared from P11 pituitary glands and treated with vehicle (DMSO; A-C) or dorsomorphin (D-F), stained for pSMAD, and developed with fluorescence, and cell nuclei were stained with DAPI (blue) (A, B, D, E) and photographed in bright field (C, F). Colonies were scored as pSMAD positive (A, D) or pSMAD negative (B, E). Images are 20× (scale bar = 200 μM). Dorsomorphin treatment caused a significant reduction in the percentage of pSMAD-positive CFCs (G). N = 4 for each treatment. **P < 0.01. Student unpaired t test was used, and values represent SEM (G). Gene expression was analyzed by RT-qPCR (H, I). Transcripts for Id1, Id2, and Id3 were significantly reduced (H). There were no changes in expression of Cdh1, Gli2, Zeb2, or Cldn23 (I). N = 4. ** P < 0.01. Multiple unpaired t test was used, and values represent SEM.

Figure 5.

Zeb2 activates BMP signaling and suppresses Elf5 expression. CFCs were prepared from P11 pituitary glands and treated with a control siRNA (A) or Zeb2 siRNA (B). Colonies were stained with antibodies to pSMAD (and developed with fluorescence), and cell nuclei are stained with DAPI (blue). Images are 20× (scale bar = 200 μM). (A, B). RT-qPCR was carried out to measure Zeb2, Id1, Id2, and Id3 transcripts relative to Hprt (C). Zeb2 transcripts were efficiently knocked down, and Id2 and Id3 were also reduced (N = 3). Inhibition of Zeb2 expression permitted elevated expression of Elf5, but not Axin2, Cldn10, or Ehf. N = 4. * P < 0.05) and ** P < 0.01. Multiple unpaired t test was used, and values represent SEM.

Figure 6.

Depleting Gli2 does not affect the EMT-like process in stem cell colonies. CFCs were prepared from P11 pituitary glands and treated with Gli2 siRNA for a scrambled sequence. RT-qPCR was carried out to measure Id1-3 (A) and for EMT genes Gli2, Ecad, Cldn23, and Zeb2, relative to Hprt (B). N = 4. Gli2 transcripts were significantly reduced (P < 0.05) (B), but there was no effect on expression of the EMT genes (B) or on expression of Id1, Id2, or Id3 (A). N = 3 for each. ** P <0.01. Multiple unpaired t test was used, and values represent SEM.

Figure 7.

Genetic hierarchy of pituitary stem cells transitioning to differentiation. SOX2 is expressed in pituitary stem cells, and pituitary-specific fate is stimulated by expression of Lhx3, Lhx4, Pitx1, and Pitx2. Prop1 suppresses expression of Sox2 and Hesx1. It activates expression of Notch2 in a positive feedback loop. Notch signaling activates expression of Snai2, an activator of EMT, and Hey1. Prop1 also activates expression of Zeb2, which is necessary to suppress Cdh1 and Elf5, and to activate Gli2 and BMP signaling. Gli2 and BMP signaling are not necessary for the EMT-like process. After progenitors undergo EMT, they initiate expression of POU1F1 and progress to differentiate into hormone producing cells that produce GH, PRL, and TSH.