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Lhx4 and Prop1 are required for cell survival and expansion of the pituitary primordia

¹ Department of Human Genetics and
² Graduate Program in Cell and Molecular Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0638, USA

View larger version (171K): [in a new window]   Fig. 1. Prop1 and Lhx4 are necessary for normal temporal and spatial activation of Lhx3 and Isl1. (A-D) Embryos collected at E12.5 were genotyped as described in Materials and Methods, sectioned in the sagittal plane, and stained with Hematoxylin and Eosin (H&E). (E-P) Immunohistochemistry was used to detect LHX3 (E-H), ISL1 (I-L), and PITX1 (M-P) in sections from each genotype. LHX3 is present throughout Rathke’s pouch in wild-type and Prop1df/df mice (E,F), but only a few LHX3-positive cells were present in Lhx4 mutants (arrow and inset, G), and no positive cells were detected in double mutants (H). (I-L) ISL1 is restricted to the ventral part of the anterior lobe in wild-type and Prop1df/df mice (I,J) but it is expressed in the most dorsal area of Lhx4 mutant pituitaries (K) and in double mutants (L). (M-P) PITX1 is present in the pituitaries of all the genotypes examined.

View larger version (139K): [in a new window]   Fig. 2. Prop1 normally represses dorsal growth and activates ventral expansion. Midsagittal sections of embryos collected at E14.5 were stained with H&E to reveal the dorsal overgrowth in Prop1df/df (B) relative to wild type (A) and Lhx4–/– (C). The anterior lobe is marked with a bracket. Limited anterior lobe expansion is evident in Prop1 and Lhx4 mutants, but no anterior lobe development is detected in Prop1df/df, Lhx4–/– double mutants (D-G) in sections spanning the lateral through midsagittal regions. The number of lumens is increased in double mutants relative to any of the other genotypes.

View larger version (150K): [in a new window]   Fig. 3. Corticotropes and GSU expressing cells fail to develop in double mutants. Immunohistochemical detection of LHX3 (A-D), ACTH (E-H), and GSU (I-L) in sagittal sections of embryos of each genotype at E14.5 reveal the lack of cell specification in double mutants. LHX3 immunoreactivity is present throughout the entire Rathke’s pouch of wild type (A) Prop1df/df (B), Lhx4–/– (C) and double mutants (D). However, ACTH is not detectable in the double mutants (H) compared to the other genotypes (E-G). Similarly GSU is absent in double mutants (L) but is present in the other genotypes (I-K). In these sections, anterior lobe is marked with a bracket and intermediate lobe with a curved bracket.

View larger version (144K): [in a new window]   Fig. 4. Double mutants express an intermediate lobe marker and a pregonadotrope marker. H&E-stained mid-sagittal sections of pituitaries at P0 reveals the stunted anterior lobe in Prop1df/df, Lhx4–/– double mutants relative to normal mice and single mutants (A-D). p, posterior lobe; I, intermediate lobe; a, anterior lobe. (E-H) ACTH immunoreactivity is normally detected in intermediate lobe melanotropes and anterior lobe corticotropes (E). By P0, ACTH is present in double mutant pouches (H), but it is confined to the dorsal aspect of the primordia in the prospective melanotropes. (I-L) GSU protein is easily detected in thyrotropes and gonadotropes of wild-type (I) and Prop1df/df pituitaries (J). Only isolated cells in Lhx4–/– pituitaries express GSU (inset, K), and GSU is not evident in any sections of double mutants (L). (M-P) SF1-positive pre-gonadotropes lie along the ventral surface of wild-type anterior lobes (bracket, M). The expression domain of SF1 is greatly expanded dorsally in Prop1df/df mice, including areas within the dysmorphic pouch (bracket, N). There are a few isolated SF1-positive cells in Lhx4–/– pituitaries (inset and arrowhead, O) and readily detectable SF1 expressing cells in double mutants (inset, P).

View larger version (90K): [in a new window]   Fig. 5. Proliferation in Rathke’s pouch at E12.5 and E14.5 is unaffected in mice lacking Prop1, Lhx4, or in double mutants. Immunostaining of BrdU-injected embryos was performed to assess proliferation levels in the pituitary mutants at E12.5 (A-D) and at E14.5 (E-G). Wild-type (A,E), Prop1df/df (B,F), Lhx4–/– (C,G) and Prop1df/dfLhx4–/– (D) mice all had dividing cells in the pituitary primordium. The anterior lobe is indicated with a bracket.

View larger version (109K): [in a new window]   Fig. 6. Rathke’s pouch is smaller in Lhx4–/– mice owing to cell death at E12.5. Tunnel assay at E12.5 (A-D) and at E14.5 (E-H) demonstrated that only a few cells are dying (arrows) in the pituitary of wild-type (A) and Prop1df/df (B) mice. However, numerous dying cells were detected in Rathke’s pouch at E12.5 in Lhx4–/– (C) and Prop1df/dfLhx4–/– (D) mice. At E14.5 no cell death was detected in any of the genotypes: wild type (E), Prop1df/df (F), Lhx4–/– (G) and Prop1df/dfLhx4–/– (H).

View larger version (20K): [in a new window]   Fig. 7. Lhx4 and Prop1 function together in promoting anterior pituitary formation. Analysis of Lhx4 mutants revealed that this gene is necessary for initial activation of LHX3 expression at the appropriate time and for cell survival. Ames dwarf mutant analysis showed that Prop1 is essential for repressing dorsal expansion of the primordium and SF1 expression, as well as for the expansion of the anterior lobe. In the absence of both of these genes, no specification of corticotropes, gonadotropes or thyrotropes occurs in the anterior lobe. The schematic of the defects in the Lhx4 mutant pituitary is depicted at E12.5 and the Prop1 mutant pituitary at P0.