Hes1 is required for pituitary growth and melanotrope specification

Abstract

Rathke's pouch contains progenitor cells that differentiate into the endocrine cells of the pituitary gland. It gives rise to gonadotrope, thyrotrope, somatotrope, corticotrope and lactotrope cells in the anterior lobe and the intermediate lobe melanotropes. Pituitary precursor cells express many members of the Notch signaling pathway including the downstream effector gene Hes1. We hypothesized that Hes1 regulates the timing of precursor differentiation and cell fate determination. To test this idea, we expressed Hes1 in differentiating pituitary cells and found that it can inhibit gonadotrope and thyrotrope differentiation. Pituitaries of Hes1 deficient mice have anterior lobe hypoplasia. All cells in the anterior lobe are specified and differentiate, but an early period of increased cell death and reduced proliferation causes reduced growth, evident as early as e14.5. In addition, cells within the intermediate lobe differentiate into somatotropes instead of melanotropes. Thus, the Hes1 repressor is essential for melanotrope specification. These results demonstrate that Notch signaling plays multiple roles in pituitary development, influencing precursor number, organ size, cell differentiation and ultimately cell fate.

Fig. 1

Hes1 is expressed in the developing pituitary in a temporally and spatially restricted pattern. Hes1 mRNA is detected by in situ hybridization in Rathke's Pouch, present in mid-sagittal sections of pituitaries, at e10.5 (A) and e11.5 (B). Hes1 expression wanes at e14.5 (C) and is undetectable by e16.5 (D). To increase the length of time Hes1 is expressed and to force its expression in differentiating cells, transgenic mice were generated with a construct (E) containing 4.6 kb of the mouse αGSU promoter and enhancer sequences, the Hes1 open reading frame cDNA, and mouse protamine 1 intron and polyadenylation sequences.

Fig. 2

Mis-expression of Hes1 inhibits gonadotrope and thyrotrope differentiation. Coronal sections of pituitaries from e18.5 mice were probed for Hes1 expression by in situ hybridization. Hes1 in not detected in the anterior lobe of wild-type mice at this time (A), but transcripts are present in two independent, transient transgenic anterior pituitary lobes (B,C). The α subunit common to the dimeric hormones LH, FSH, and TSH is reduced in transgenic (E, F) pituitaries relative to wild-type (D). Both LHβ and TSHβ are also absent or substantially reduced in Hes1 transgenics (LH H, I; TSH K, L) compared to wild-type (LH G; TSH J). POMC, a hormone marker unrelated to α subunit, is expressed similarly in wild-type (M) and transgenic (N,O) pituitaries.

Fig. 3

Pituitary size is reduced in Hes1 mutants. Hematoxylin and eosin (H&E) staining reveals the morphology of wild-type (A-D) and Hes1 null (E-H) pituitaries during development. In e11.5 sagittal sections, the pouch of the Hes1 mutant is formed, but has not completely separated from the underlying oral ectoderm (arrow, E) compared with the wild-type sagittal section (A). At e14.5, wild-type pituitaries have a nearly fused cartilage plate underneath Rathke's pouch (B), but Hes1 mutants exhibit a large gap between the rostral and caudal aspects of the cartilage (F). Coronal sections at e14.5 demonstrate that the size of the wild-type anterior pituitary (C, bracket) is substantially greater than the Hes1 mutant (G, bracket). This size difference between the wild-type (D, bracket) and Hes1 mutant (H, bracket) is even more pronounced in coronal sections of e18.5 pituitaries.

Fig. 4

Decreased cell proliferation and increased cell death in Rathke's pouch of Hes1 mutants. Proliferation was assessed by BrdU immunohistochemistry at e11.5 (A, C) and Cyclin D2 immunohistochemistry at e12.5 (E,G). There is an area of decreased density of proliferating cells in the Hes1 mutants at e11.5 (C, white bracket), compared to the wild-type Rathke's pouch, where proliferating cells are evenly spacing throughout (A). At e12.5 proliferating cells are located throughout Rathke's pouch of both wild-type (E) and Hes1 mutants (G). Cell death was detected by the TUNEL method in sagittal sections of wild-type (B, F) and Hes1 mutant (D, H) pituitaries. Dying cells are labeled green and all nuclei are marked blue with DAPI. At e11.5, wild-type pituitaries have no dying cells within Rathke's pouch, but Hes1 mutants have many dying cells (D, white bracket). At e12.5, no cell death is detected in Rathke's pouch of wild-type (F) or Hes1 mutants (H).

Fig. 5

Hes1 is necessary for cell survival and LHX3 expression. Cell death (A, B), LHX3 (C, D) and ISL1 (E, F) expression were examined in wild-type and Hes1 mutant embryos collected at e10.5. Six6 (G, H) and Fgf10 (I, J) mRNA expression was examined by in situ hybridization on sagittal sections of e11.5 embryos. At e10.5, wild-type embryos exhibit cell death at the junction between Rathke's pouch and the attached oral ectoderm (A, green stained cells). Hes1 mutants have ectopic dying cells in the caudal aspect of Rathke's pouch (B, white bracket) that corresponds with lack of LHX3 expression (D, white bracket). Six6 expression in the diencephalon (arrow) and Rathke's pouch is equivalent in wild-type (G) and Hes1 mutants (H). Fgf10 expression in the infundibulum (arrow) is similar in wild-type (I) and Hes1 mutants (J).

Fig. 6

Intermediate lobe melanotropes are lost in the absence of Hes1. Coronal sections of wild-type (A, C, E, G) and Hes1 null (B, D, F, H) pituitaries were examined at e18.5. An antibody that recognizes POMC derivatives in the intermediate lobe (IL) and anterior lobe (AL) shows that in the wild-type pituitary, the posterior lobe (PL) is devoid of staining whereas the IL and AL contain many stained cells. In Hes1 mutants, the AL contains POMC stained cells, but the IL has a dramatic reduction in staining. MSH and PC2 immunoreactivity is apparent in the intermediate lobe of wild-type pituitaries (C, E, respectively) but not in Hes1 mutants (D, F, respectively). T-PIT immunoreactivity is similar in wild-type (G) and Hes1 mutant anterior and intermediate lobes (H).

Fig. 7

Hes1 deficiency causes intermediate lobe cell fate switch to somatotrope. PIT1 immunoreactive cells are normally detected only in the anterior lobe of wild-type mice at e16.5 (A) and e18.5 (C). The PIT1 negative intermediate lobe is denoted with a bracket. Hes1mutant mice express PIT1 becomes expressed in the caudal part of the intermediate lobe at e16.5 (B, bracket), and PIT1 is expressed throughout by e18.5 (D, bracket). Additionally, GH is expressed in the intermediate and anterior lobes of Hes1 mutants (F, bracket), but only in the anterior lobe of wild-type mice (E, bracket). There is no change in TSHβ, LHβ or αGSU immunostaining between wild-type (G, I, K, respectively) and Hes1 mutant (H, J, L, respectively) pituitaries at e18.5.

Figure 8

Genetic model of pituitary development and dependence on Hes1. During initial organogenesis, Hes1 is necessary for survival and proliferation of Rathke's pouch precursors (A). Hes1 is also necessary for intermediate lobe cells to become melanotropes and not PIT1-containing somatotropes (B).