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Pitx2 is required at multiple stages of pituitary organogenesis: pituitary primordium formation and cell specification

¹ Neuroscience Program and
² Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109-0638, USA
³ Institut de Recherches Cliniques de Montreal, Montréal Québec, Canada

View larger version (13K): [in a new window]   Fig. 1. Three Pitx2 alleles. (A) Genomic organization of the Pitx2 locus. Among three expressed Pitx2 cDNA isoforms, Pitx2a and Pitx2b arise by alternative splicing and Pitx2c utilizes an alternative promoter. Arrows above exons indicate transcriptional initiation sites. (B) Pitx2neo is a hypomorphic allele containing a neo resistance gene within the intronic site of Pitx2. (C) Pitx2 null allele results from the excision of exon 4, which encodes most homeodomain sequences. Black boxes, coding sequences; gray boxes, non-coding sequences; a, Pitx2a-specific exon; b, Pitx2b-specific exon; c, transcriptional start site of Pitx2c isoform; HD, homeodomain.

View larger version (92K): [in a new window]   Fig. 2. Pituitary organ size diminishes with reduced PITX2 dosage. (A) Diagram of pituitary morphology. INF, infundibulum, the prospective posterior lobe; INT, intermediate lobe; ANT, anterior lobe. Immunohistochemical examination shows that the expression of PITX1 (B) and PITX2 (C) overlaps in the anterior lobe and intermediate lobe, but not in the posterior lobe (D). Histological examination of E12.5 embryo sagittal sections of Pitx2 wild type (E), Pitx2neo/neo (F), Pitx2neo/– (G) and Pitx2–/– (H) shows that the size of Rathke’s pouch decreases as the Pitx2 level is reduced. Note that the Pitx2neo/– embryo has an intermediate pouch size.

View larger version (108K): [in a new window]   Fig. 3. Expression of pituitary hormone genes is affected in Pitx2 hypomorphs. In situ hybridization using pituitary cell-specific markers was performed in Pitx2neo homozygotes and wild-type littermates at P1. The expression of gonadotrope-specific markers Lhb (A,B), Fshb (C,D) and Gnrhr (E,F) is almost undetectable in Pitx2neo/neo pituitaries. Gh (G,H), Ghrhr (I,J) and Tshb (K,L) in situ hybridization reveals the reduction in somatotropes and thyrotropes, respectively. POMC-producing corticotropes appear to develop normally in Pitx2neo/neo mice (M,N).

View larger version (100K): [in a new window]   Fig. 4. Reduced Pitx2 causes decreased expression of lineage-specific transcription factors. In situ hybridization or immunohistochemistry was performed to examine the change of transcription factor expression in Pitx2neo/neo mice and wild-type littermates at E12.5 and P1. Gata2 expression initiates normally at early stages (A,B), but fails to be maintained (C,D). The expression of Egr1 (E,F) and SF1 (G,H) is nearly undetectable in Pitx2neo/neo animals. PITX1 (I,J and K,L) and PROP1 (M,N) expression was unchanged in homozygotes for Pitx2neo. Pit1 in situ hybridization shows a reduction in the number of Pit1-producing cells (O,P). Note Gata2 (A,B), PITX1 (I,J) and PROP1 (M,N) expression at E12.5. Arrowhead, rostral tip of pituitary gland; the brackets in C-F indicate the ventral and caudomedial aspect of the pituitary gland.

View larger version (13K): [in a new window]   Fig. 5. PITX2 enhances Gata2 transcription. The fold increase (y-axis) of luciferase gene activity compared to control (transfection of empty vectors only) is shown. Three PITX2 isoform expression vectors were individually cotransfected with Gata2 luciferase construct into T3-1 (A) or CV-1 cells (B). PITX2 showed a significant induction of Gata2 luciferase expression in both cell lines (A,B; P<0.05). Different PITX2 isoforms may have different transactivation properties on Gata2 promoter (A). Asterisk indicates statistically significant induction compared to basal level (transfection of reporter construct only without PITX2 expression vector, P<0.05). Results from more than 4 independent experiments were averaged.

View larger version (66K): [in a new window]   Fig. 6. Pitx1 and Pix2 have functional overlaps in pituitary ontogeny. Histological examinations of double mutants carrying Pitx1– and Pitx2neo alleles show the normal formation of Rathke’s pouch in all genotypes (A-C), but not in Pitx1–/–;Pitx2neo/neo mice at E12.5 (D). Note that Rathke’s pouch in Pitx1–/–;Pitx2neo/neo mice does not expand (D). Immunoreactive LHX3 is present all genotypes (E-H).

View larger version (27K): [in a new window]   Fig. 7. Pitx2 functions at multiple developmental stages. Analysis of Pitx2–/– mice revealed that Pitx2 is required for Rathke’s pouch expansion at early stages of embryogenesis, and studies from Pitx2neo/neo homozygotes showed that Pitx2 is also required for cell lineage specification at later stages of organogenesis, influencing expression of downstream transcription factors. In addition to Pitx1 function in pituitary hormone transcription, Pitx1 acts synergistically with Pitx2 at an early stage of pituitary development.

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