All Hormone-Producing Cell Types of the Pituitary Intermediate and Anterior Lobes Derive From Prop1-Expressing Progenitors

Abstract

Mutations in PROP1, the most common known cause of combined pituitary hormone deficiency in humans, can result in the progressive loss of all hormones of the pituitary anterior lobe. In mice, Prop1 mutations result in the failure to initiate transcription of Pou1f1 (also known as Pit1) and lack somatotropins, lactotropins, and thyrotropins. The basis for this species difference is unknown. We hypothesized that Prop1 is expressed in a progenitor cell that can develop into all anterior lobe cell types, and not just the somatotropes, thyrotropes, and lactotropes, which are collectively known as the PIT1 lineage. To test this idea, we produced a transgenic Prop1-cre mouse line and conducted lineage-tracing experiments of Prop1-expressing cells. The results reveal that all hormone-secreting cell types of both the anterior and intermediate lobes are descended from Prop1-expressing progenitors. The Prop1-cre mice also provide a valuable genetic reagent with a unique spatial and temporal expression for generating tissue-specific gene rearrangements early in pituitary gland development. We also determined that the minimal essential sequences for reliable Prop1 expression lie within 10 kilobases of the mouse gene and demonstrated that human PROP1 can substitute functionally for mouse Prop1. These studies enhance our understanding of the pathophysiology of disease in patients with PROP1 mutations.

Figure 1.

Prop1 genomic regions and conservation, Prop1-cre generation, and HsPROP1 transgenic rescue. A, Schematic diagram of mouse Prop1 genomic region on chromosome 11 (black) and human PROP1 on chromosome 5 (blue) as visualized using the UCSC Genome Browser (http://genome.ucsc.edu). Black brackets above the genomic schematic indicate the portion of mouse chromosome 11 contained in the original rescue BAC, RP23-250I22, the BAC used for generating the Prop1-cre, RP23-479M11, and 2 rescue plasmids of 25 and 10 kb. The blue bracket below the genomic schematic indicates the portion of human chromosome 5 contained in HsPROP1 BAC RP11-452O4. B, VISTA plot (http://genome.lbl.gov/vista/mvista/submit.shtml) comparing conserved genomic regions between mouse, human, cow, dog, and rat genomes in the region of the 10-kb rescue plasmid. Pink peaks indicate CNSs and purple peaks represent conserved coding sequences. Also indicated is the location where the cre cassette was inserted into BAC RP23-479M11 to generate Prop1-cre. C, Three-week-old HsPROP1; Prop1^df/df transgenic and Prop1^df/df mice. D, Graph of average weight at 3 weeks for wild-type, Prop^df/df, and HsProp1; PROP1^df/df mice. Brackets indicate P values for 2-tailed, t tests between wild-type (n = 37) and Prop1^df/df (n = 11) and between Prop1^df/df and HsPROP1; Prop1^df/df (n = 18) mice. E, Pituitary from a 3-week-old Prop1-cre; Rosa^mT/mG mouse. Green fluorescence indicates recombination from Prop1-cre, and red fluorescence indicates no recombination. Scale bar, 500 μm.

Figure 2.

Pituitary-specific Prop1-cre activity and PROP1 immunostaining. A and B, Whole mount X-gal-stained e11.5 Prop1-cre; Rosa^stop-Lacz embryo. Blue indicates LacZ activity and cre expression. A, Lateral view. B, Dorsal view. Arrow indicates the developing pituitary gland as seen through the hindbrain. C and D, PROP1 expression (green) assayed by immunostaining, and counterstained with DAPI (blue), on e12.5 sagittal sections. C, Wild type. Arrow indicates PROP1 expression in Rathke's pouch. D, Prop1^−/−. Scale bars, 500 μm (A and B) and 100 μm (C and D).

Figure 3.

Prop1-cre activity compared with PROP1 expression. A–D, e10.5 sagittal sections. E–H, e11.5 sagittal sections. I–L, e12.5 sagittal sections. M–P, e14.5 sagittal sections. Q–S, e16.5 coronal sections. T–V, Postnatal day 21 (P21) pituitary coronal sections. A, E, I, M, Q, R, and S, Prop1-cre; Rosa^stop-LacZ embryos X-gal stained (blue) to indicate LacZ activity caused by cre recombination, and counterstained with neutral red. Arrow in E indicates small region of LacZ activity. Arrow in I indicates LacZ activity in the rostral tip. B, F, J, and N, Prop1-cre; Rosa^mT/mG embryos where red fluorescence indicates no cre activity and green fluorescence indicates cre recombination. Arrow in F indicates a small region of cre activity. Arrow in J indicates cre activity in the rostral tip. Arrow in N indicates a region with no cre activity (red). C, G, K, and O, PROP1 immunostaining (purple) on the same sections as B, F, J, and N. Arrow in K indicates no expression of PROP1 in the rostral tip. D, Overlay of Prop1-cre; Rosa^mT/mG and PROP1 immunostaining images. H, L, and P, Overlay of eGFP fluorescence and PROP1 immunostaining. Arrow in P indicates a small region with no cre activity and no PROP1 expression in the pituitary anterior lobe. R and S, Boxed regions in Q, presented at higher magnification. Double arrow in R indicates the intermediate lobe. T, Fluorescence from Prop1-cre; Rosa^mT/mG tdTOMATO (red) and eGFP (green). U, Same image as T, with the red channel removed and immunostained for PECAM1 (blue). V, Same image as T with all 3 channels, demonstrating colocalization of PECAM1 with tdTOMATO. Sale bars, 100 μm.

Figure 4.

All anterior and intermediate lobe hormone-expressing cell types are derived from a Prop1-cre progenitor. A, D, G, J, M, and P, Gray scale images of immunostaining for hormones from Prop1-cre; Rosa^mT/mG pituitaries. GH (A), PRL (D), TSHβ (G), POMC in the anterior lobe (J), POMC in the intermediate lobe (M), and LHβ (P). (B, E, H, K, N, and Q) Gray scale images of eGFP detection of same images as A, D, G, J, M, and P. C, F, I, L, O, and R, Overlay of hormone (red) and eGFP (green) images. Arrows indicate the same cell in each image. Immunostainings for A–L and P–R were performed on P21 pituitaries, while immunostainings for M–O were performed on e18.5 pituitaries. Scale bars, 20 μm.