The forkhead transcription factor, Foxd1, is necessary for pituitary luteinizing hormone expression in mice

Abstract

The pituitary gland regulates numerous physiological functions including growth, reproduction, temperature and metabolic homeostasis, lactation, and response to stress. Pituitary organogenesis is dependent on signaling factors that are produced in and around the developing pituitary. The studies described in this report reveal that the forkhead transcription factor, Foxd1, is not expressed in the developing mouse pituitary gland, but rather in the mesenchyme surrounding the pituitary gland, which is an essential source of signaling factors that regulate pituitary organogenesis. Loss of Foxd1 causes a morphological defect in which the anterior lobe of the pituitary gland protrudes through the cartilage plate that is developing ventral to the pituitary at embryonic days (e)14.5, e16.5, and e18.5. The number of proliferating pituitary cells is increased at e14.5 and e16.5. Loss of Foxd1 also results in significantly decreased levels of Lhb expression at e18.5. This decrease in Lhb expression does not appear to be due to a change in the number of gonadotrope cells in the pituitary gland. Previous studies have shown that loss of the LIM homeodomain factor, Lhx3, which is activated by the FGF signaling pathway, results in loss of LH production. Although there is a difference in Lhb expression in Foxd1 null mice, the expression pattern of LHX3 is not altered in Foxd1 null mice. These studies suggest that Foxd1 is indirectly required for normal Lhb expression and cartilage formation.

Figure 1. Foxd1 is expressed in the mesenchyme surrounding the developing pituitary gland.

X-gal staining of mid-sagittal sections from Foxd1^+/LacZ embryos shows Foxd1 expression (blue) in the mesenchyme (arrows) surrounding the developing pituitary gland at e10.5 (A), e12.5 (B), and e14.5 (C). Foxd1 expression is not apparent in the pituitary gland itself (dotted lines) during development. Whole mount β-galactosidase staining was performed at e10.5 (A). At later ages, embryos were frozen and sections were stained for β-galactosidase (B, C). Pictures were taken at 200X and scale bars represent 100 µm.

Figure 2. Loss of Foxd1 expression results in pituitary dysmorphology.

Hematoxylin and eosin staining of mid-sagittal sections (A–J) and coronal sections (K–L) revealed that the developing pituitary protrudes through the cartilage plate by e14.5 in Foxd1^LacZ/LacZ embryos (arrows). Pictures were taken at 200X (A–F) or 100X (G–L). Scale bars represent 100 µm.

Figure 3. Loss of Foxd1 expression results in disruption of the developing cartilage plate.

Mid-sagittal (A–F) and coronal (G–H) sections from Foxd1^LacZ/LacZ embryos and wild type littermates were stained with Gomori’s aldehyde fuchsin to visualize cartilage. (A–B) The prospective pituitary gland and surrounding tissue appears normal at e12.5. (C–H) A break in the cartilage plate ventral to the pituitary is apparent in Foxd1^LacZ/LacZ embryos by e14.5 (arrows). Pictures were taken at 100X and scale bars represent 100 µm.

Figure 4. Expression of luteinizing hormone is reduced in Foxd1^LacZ/LacZ embryos.

(A) Real time RT-PCR revealed that expression of Lhb is significantly reduced in embryos lacking Foxd1 expression compared to wild type littermates at e18.5 (P<0.05). Expression of other pituitary hormones was not significantly reduced. (B–K) No apparent differences in GH, TSHB, POMC, FSHB, or CGA were observed. (L–M) The intensity of LHB staining is reduced in mutant pituitary glands. (N) The number of LHB-positive cells was counted manually and set relative to wild type controls. No significant difference was detected in Foxd1^LacZ/LacZ pituitaries as compared to wild type. Cell nuclei are stained with DAPI (blue). Pictures were taken at 200X (B–K) or 630X (L–M). Scale bars represent 100 µm.

Figure 5. Apoptosis is not different in Foxd1^LacZ/LacZ embryos.

TUNEL was performed to label apoptotic cells in mid-sagittal sections from Foxd1^LacZ/LacZ embryos and wild type littermates. (A–F) No loss of apoptosis was observed. (A–B) Arrows indicate regions of pituitary cell apoptosis. Pictures were taken at 200X and scale bars represent 100 µm.

Figure 6. Pituitary cell proliferation is increased in Foxd1^LacZ/LacZ embryos.

(A–J) Actively dividing cells were labeled with bromodeoxyuridine (BrdU). Immunohistochemistry was performed to detect BrdU in mid-sagittal sections from Foxd1^LacZ/LacZ embryos and wild type littermates. (K) The number of BrdU-positive cells was manually counted in pituitary sections from Foxd1^LacZ/LacZ embryos and wild type littermates. The number of BrdU-positive cells is significantly higher in Foxd1^LacZ/LacZ embryonic pituitaries at e14.5 and e16.5 as compared to wild type littermates (P<0.01).

Figure 7. LHX3 patterns are unchanged in Foxd1^LacZ/LacZ embryos.

Immunohistochemistry was performed to observe LHX3 patterns in mid-sagittal (A–J) and coronal (K–L) sections of embryonic pituitary glands from Foxd1^LacZ/LacZ embryos and wild type littermates. No difference in LHX3 protein distribution patterns was observed. Pictures were taken at 200X (A–J) or 100X (K–L). Scale bars represent 100 µm.