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Molecular Endocrinology, doi:10.1210/me.2007-0039
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Molecular Endocrinology 21 (6): 1458-1466
Copyright © 2007 by The Endocrine Society

Hes1 and Hes5 Control the Progenitor Pool, Intermediate Lobe Specification, and Posterior Lobe Formation in the Pituitary Development

Aya Kita1, Itaru Imayoshi1, Masato Hojo, Masashi Kitagawa, Hiroshi Kokubu, Ryosuke Ohsawa, Toshiyuki Ohtsuka, Ryoichiro Kageyama and Nobuo Hashimoto

Department of Neurosurgery (A.K., M.H., M.K., N.H.), Kyoto University Graduate School of Medicine, and Institute for Virus Research (A.K., I.I., M.K., H.K., R.O., T.O., R.K.), Kyoto University, Kyoto 606-8507, Japan

Address all correspondence and requests for reprints to: Masato Hojo, Department of Neurosurgery, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: mhojo{at}kuhp.kyoto-u.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The pituitary gland is composed of two distinct entities: the adenohypophysis, including the anterior and intermediate lobes, and the neurohypophysis, known as the posterior lobe. This critical endocrine organ is essential for homeostasis, metabolism, reproduction, and growth. The pituitary development requires the control of proliferation and differentiation of progenitor cells. Although multiple signaling molecules and transcription factors are required for the proper pituitary development, the mechanisms that regulate the fate of progenitor cells remain to be elucidated. Hes genes, known as Notch effectors, play a crucial role in specifying cellular fates during the development of various tissues and organs. Here, we report that mice deficient for Hes1 and Hes5 display severe pituitary hypoplasia caused by accelerated differentiation of progenitor cells. In addition, this hypoplastic pituitary gland (adenohypophysis) lacks the intermediate lobe and exhibits the features of the anterior lobe only. Hes1 and Hes5 double-mutant mice also lack the neurohypophysis (the posterior lobe), probably due to incomplete evagination of the diencephalon. Thus, Hes genes control not only maintenance of progenitor cells but also intermediate vs. anterior lobe specification during the adenohypophysis development. Hes genes are also essential for the formation of the neurohypophysis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE PITUITARY GLAND is composed of two distinct entities: the adenohypophysis and the neurohypophysis. The adenohypophysis includes the anterior and intermediate lobes, whereas the neurohypophysis constitutes the posterior lobe (1, 2, 3, 4). The mature adenohypophysis contains six distinct cell types that arise from common progenitors. The anterior lobe contains five hormone-producing cell types: corticotropes secreting ACTH, thyrotropes secreting TSH, gonadotropes secreting LH and FSH, somatotropes secreting GH, and lactotropes secreting prolactin, and the intermediate lobe contains one hormone-producing cell type: melanotropes secreting {alpha}MSH (1, 2, 3). ACTH and {alpha}MSH are both processed from the same precursor, proopiomelanocortin (POMC). POMC is proteolytically cleaved by prohormone convertases PC1 and PC2. Interestingly, PC2 is specifically expressed in melanotropes, suggesting that PC2 is the key convertases in the processing of POMC into {alpha}MSH (5). In contrast to the adenohypophysis, the neurohypophysis does not contain endocrine cells, but secretes oxytocin and vasopressin (3).

The adenohypophysis and the neurohypophysis develop from two different sources: the adenohypophysis derives from Rathke’s pouch, a specialized region of the oral roof ectoderm, whereas the neurohypophysis derives from the infundibulum, an evagination of the ventral diencephalon (4). The pituitary development starts at embryonic d 8.5 (E8.5) in mice. The oral ectoderm thickens and invaginates to form Rathke’s pouch. The dorsal portion of Rathke’s pouch directly contacts the ventral diencephalon, which evaginates at E10 to form the infundibulum. The apposition of Rathke’s pouch and the infundibulum is maintained throughout the early stages of pituitary organogenesis, suggesting that the inductive interactions are involved in the pituitary development. Rathke’s pouch separates from the oral ectoderm and further develops and differentiates, and the six endocrine cell types emerge in a temporally and spatially specific fashion from E12.5–E17.5 (1, 2, 3, 4). Meanwhile, the infundibulum, evaginated from the ventral diencephalon, develops and forms the neurohypophysis (the posterior lobe) (3, 4). The pituitary organogenesis involves the initial proliferation process of progenitor cells and the subsequent differentiation process into distinct cell types. Although multiple signaling molecules and transcription factors are required for the proper pituitary development (1, 2, 3, 6, 7), the mechanisms that regulate the fate of progenitor cells remain to be elucidated.

The basic helix-loop-helix (bHLH) genes Hes1 and Hes5 are essential effectors for Notch signaling, which regulate the maintenance of progenitor cells and the timing of their differentiation in various tissues and organs (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). Notch is a transmembrane protein and activated by its ligands such as Delta. Upon activation, the intracellular domain of Notch is cleaved off and transferred into the nucleus, where the Notch intracellular domain forms a complex with the DNA-binding protein RBP-J. This complex induces expression of Hes1 and Hes5, which antagonize bHLH genes such as Mash1, Math, and Neurogenin, and thereby inhibit the differentiation of progenitor cells (8). In the nervous system, both Hes1 and Hes5 are essential for the regulation of neural stem cells. In the endocrine system, Hes1 controls pancreatic cell differentiation by repressing Neurogenin3 (15, 16, 17). The analysis of mice deficient for Prop1 has indirectly implicated involvement of Notch pathway in the pituitary development, but its precise role remains to be determined (20). Here, to test the hypothesis that Hes genes control the fate determination of pituitary progenitors, we analyzed Hes-mutant mice.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Compensatory Expression of Hes1 and Hes5 in the Developing Pituitary Gland
We first examined Hes1 and Hes5 expression in the developing pituitary gland by in situ hybridization. It is known that the expression patterns of these two genes are complementary to each other in the developing nervous system (11). At E10.5–E12.5, Hes1 was strongly expressed throughout the entire Rathke’s pouch and the ventral diencephalon (Fig. 1Go, A and B). After E12.5, Hes1 expression was gradually down-regulated in the ventral region (presumptive anterior lobe) and restricted to the dorsal region of Rathke’s pouch. At E14.5, Hes1 expression occurred at the periluminal side of the dorsal region of Rathke’s pouch, which contains progenitor cells (Fig. 1CGo, arrowheads). By E17.5, Hes1 expression was further down-regulated and observed mainly at the periluminal side of Rathke’s pouch (Fig. 1DGo). We also examined the expression of Hes1 protein in the developing pituitary gland. The expression pattern of Hes1 protein was similar to that of Hes1 mRNA (Fig. 1Go, E–H). To characterize Hes1– cells, we examined the expression of POMC by double staining. Interestingly, differentiated hormonal cells (POMC+ cells) did not express Hes1 protein (Fig. 1IGo). These results suggest that Hes1 is expressed by immature progenitor cells, but not by differentiated hormonal cells in the developing pituitary. In contrast, Hes5 was not detectable in the developing pituitary of wild-type embryo (Fig. 1JGo). However, Hes5 expression was observed in Rathke’s pouch of Hes1-null embryo (Fig. 1KGo), suggesting that Hes5 could compensate Hes1 for the pituitary development. Thus, to understand the roles of Hes genes in the pituitary development, we decided to analyze Hes1; Hes5 double-mutant mice. However, because almost all Hes1; Hes5 double-mutant embryos die by E11, before overt pituitary development (9), we examined conditional Hes1; Hes5 double-mutant mice.


Figure 1
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  		Fig. 1. Hes1 and Hes5 Expression in the Developing Pituitary Gland

A–D, In situ hybridization of Hes1. At E10.5–E12.5, Hes1 is strongly expressed throughout the entire Rathke’s pouch and the ventral diencephalon (A and B). After E12.5, Hes1 expression is gradually down-regulated in the ventral region (presumptive anterior lobe) and restricted to the dorsal region of Rathke’s pouch (B). At E14.5, Hes1 expression occurs at the periluminal side of the dorsal region of Rathke’s pouch, which contains progenitor cells (C, arrowheads). By E17.5, Hes1 expression is further down-regulated and observed mainly at the periluminal side of Rathke’s pouch (D). Boxed region is enlarged in the inset (D, inset). In contrast, the sense probes did not detect any specific signals (data not shown). E–H, Immunostaining with anti-Hes1 antibody. The expression pattern of Hes1 protein is similar to that of Hes1 mRNA. Boxed region is enlarged in the inset (H, inset). I, Immunostaining with anti-Hes1 (green) and anti-POMC (red) antibodies. Hes1 is not expressed by POMC+ cells. J and K, In situ hybridization of Hes5 at E13.5. Hes5 is not expressed in wild-type Rathke’s pouch (J). However, in the absence of Hes1, Hes5 expression is observed (K). VD, Ventral diencephalon; RP, Rathke’s pouch. Bars, 100 µm (A–D, H); 50 µm (E–G, I–K).

 
Generation of Conditional Hes1; Hes5 Double-Mutant Mice
Emx1 is a mouse homolog of the Drosophila homeobox gene empty spiracles (ems), and is expressed mainly in the dorsal telencephalon (21). In this study, we used Emx1-Cre mice to generate conditional Hes1; Hes5 double-mutant mice, with the Cre/loxP system (22, 23). Hes genes were deleted in the developing pituitary gland by crossing Emx1+/Cre; Hes1+/–; Hes5–/– mice with Hes1fl/fl; Hes5–/– mice. To examine the Cre recombinase activity in the developing pituitary gland, Emx1-Cre mice were crossed with the ROSA26 reporter mice (24, 25). Cre recombinase activity was detected throughout the entire Rathke’s pouch and the ventral diencephalon of E11.5 Emx1-Cre mice (Fig. 2AGo). To show the direct evidence of Hes1 protein ablation in the conditional Hes1; Hes5 double-mutant pituitary gland, immunohistochemistry with anti-Hes1 antibody was performed (Fig. 2Go, B–E). No Hes1 expression was detected in the pituitary gland and the ventral diencephalon of conditional Hes1; Hes5 double-mutant mice at E12.5 (Fig. 2EGo). This result shows that Hes1 was successfully disrupted in the conditional Hes1; Hes5 double-mutant Rathke’s pouch and ventral diencephalon. To investigate the function of Hes genes in the developing pituitary gland, we examined both Hes1 single-mutant and conditional Hes1; Hes5 double-mutant mice.


Figure 2
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  		Fig. 2. Premature Pituitary Differentiation in Hes-Mutant Mice

A–E, Direct evidence of ablation of Hes1 protein in Rathke’s pouch by Emx1-Cre transgene. A, X-gal staining of a midsagittal section from an E11.5 Emx1-Cre/ROSA-26 embryo. The entire Rathke’s pouch and the ventral diencephalon are strongly stained. B–E, Immunostaining of E12.5 embryos with anti-Hes1 antibody (B and C, 4',6-diamidino-2-phenylindole; D and E, anti-Hes1 antibody). Hes1 is expressed in Rathke’s pouch and the ventral diencephalon of control mice (D). In contrast, Hes1 is not expressed in conditional Hes1; Hes5 double-mutant Rathke’s pouch and the ventral diencephalon (E). F–Q, Morphological and immunohistological analysis of Hes-mutant pituitary glands at E12.5. F–H, Midsagittal sections were stained with hematoxylin and eosin (HE). In Hes1-null and conditional Hes1; Hes5 double-mutant mice (G and H), Rathke’s pouch is smaller than the control (F). The evagination of the infundibulum is disturbed in conditional Hes1; Hes5 double-mutant mice (H, arrowhead) compared with the control (F, arrow) and Hes1-null mice (G, arrow). n = 8 (control), 4 (Hes1-null mice), and 3 (conditional Hes1; Hes5 double-mutant mice). I–K, Immunostaining with anti-Ki67 antibody. Ki67+ cells are located in Rathke’s pouch, but not detectable in the ventral presumptive anterior lobe in both control (I) and Hes-mutant mice (J and K). However, Ki67+ cells are decreased in the ventrocaudal region of Hes-mutant Rathke’s pouch (J and K, bracket) compared with the control (I). n = 11 (control), 4 (Hes1-null mice), and 3 (conditional Hes1; Hes5 double-mutant mice). L–N, Immunostaining with anti-POMC antibody. POMC+ cells are detected in the ventrocaudal region of Hes-mutant Rathke’s pouch, where Ki67+ cells are reduced [M and N, bracket; compare with J and K (serial sections)]. This phenotype is more prominent in conditional Hes1; Hes5 double mutant mice than Hes1-null mice. n = 9 (control), 4 (Hes1-null mice), and 4 (conditional Hes1; Hes5 double-mutant mice). O–Q, TUNEL assay. Both control (O) and Hes-mutant pituitary glands (P and Q) are negative for TUNEL assay. n = 6 (control), 3 (Hes1-null mice), and 3 (conditional Hes1; Hes5 double-mutant mice). VD, Ventral diencephalon; RP, Rathke’s pouch. Bars, 50 µm (A–E, I–Q); 100 µm (F–H).

 
Premature Pituitary Differentiation in Hes-Mutant Mice
We first examined the pituitary gland at E12.5. We were able to obtain about 40, 25, and 20 sections (16-µm thickness) of the pituitary gland from each embryo of the control, Hes1-null, and conditional Hes1; Hes5 double-mutant mice, respectively (at least three embryos were examined in each genotype). Thus, the size of Rathke’s pouch was reduced in Hes1-null and conditional Hes1; Hes5 double-mutant mice, compared with the control, although the difference was not obvious in the sagittal sections (Fig. 2Go, F–H). To investigate the mechanism of hypoplasia of Hes-mutant Rathke’s pouches, we analyzed cell proliferation and death. Although Ki67+ cells were detected throughout Rathke’s pouch in control mice (Fig. 2IGo), Ki67+ cells were reduced in number in the ventrocaudal region of both Hes1-null and conditional double-mutant Rathke’s pouches (Fig. 2Go, J and K, bracket) compared with the control (Fig. 2IGo). To determine the extent of cell death, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed. Both control and Hes-mutant Rathke’s pouches were negative for TUNEL assay (Fig. 2Go, O–Q). These results indicate that progenitors do not die but prematurely exit from the cell cycle in the absence of Hes genes. To characterize the cells that become postmitotic, the expression of POMC, the early hormonal marker, was examined (26). In Hes-mutant Rathke’s pouches, POMC+ cells prematurely differentiated in the region where Ki67+ cells were decreased (Fig. 2Go, M and N, bracket), compared with the control (Fig. 2LGo). This phenotype was more prominent in conditional Hes1; Hes5 double-mutant mice than in Hes1 single-mutant mice (Fig. 2Go, M and N). These results show that, in the absence of Hes genes, cell differentiation is accelerated in the developing pituitary gland. Thus, Hes genes are essential for the maintenance of progenitor cells in Rathke’s pouch during the pituitary development.

Pituitary Hypoplasia and Loss of Intermediate Lobe in Conditional Hes1; Hes5 Double-Mutant Mice
We next examined the pituitary gland at E18. We were able to obtain about 100, 60, and 30 sections (16-µm thickness) of the pituitary gland from each embryo of the control, Hes1-null, and conditional Hes1; Hes5 double-mutant mice, respectively (at least five embryos were examined in each genotype). At this stage, Hes1-null pituitary was hypoplastic but morphologically normal compared with the control (Fig. 3Go, A–D, F, and G). Both the wild-type and Hes1-null pituitaries consist of round and columnar cells, which are characteristic of the anterior and intermediate lobe cells, respectively (Fig. 3Go, C, D, F, and G). However, in conditional Hes1; Hes5 double-mutant mice, the pituitary was not only severely hypoplastic but also morphologically abnormal compared with both control and Hes1-null mice: the conditional double-mutant pituitary was sphere-shaped and lacked the structure of the lumen of Rathke’s pouch (Fig. 3Go, E and H). In addition, the conditional double-mutant pituitary was composed of round cells only and lacked columnar cells (Fig. 3Go, C–H). These data suggest that the conditional Hes1; Hes5 double-mutant pituitary lacks the structure of the intermediate lobe. To confirm these findings, we performed immunostaining with anti-POMC and PC2 antibodies. PC2 is a good immunohistochemical marker of the intermediate lobe due to its specific expression pattern (5), whereas POMC is expressed by both corticotropes of the anterior lobe and melanotropes of the intermediate lobe (26). In the wild-type and Hes1-null pituitaries, there were two types of POMC+ cells, round cells in the anterior lobe and columnar cells in the intermediate lobe (Fig. 3Go, I and J). Furthermore, the columnar cells expressed PC2 (Fig. 3Go, L and M). In contrast, although POMC+ cells were detected in conditional double-mutant pituitaries, they were round in shape, the morphological feature characteristic of the anterior lobe cells (Fig. 3KGo). Furthermore, no PC2+ cell was detected in conditional double-mutant pituitaries (Fig. 3NGo). These data demonstrate that, in conditional Hes1; Hes5 double-mutant pituitary gland, none of progenitors differentiated into the intermediate lobe cells. To investigate the mechanism of hypoplasia in Hes-mutant pituitaries, we also analyzed cell proliferation and death at this stage (Fig. 3Go, O–T). To analyze cell proliferation, we counted the number of Ki67+ cells in the sections of pituitary glands. At least three independent embryos of each genotype were examined. For each embryo, at least three sections were examined. Ki67+ cells were 333 ± 10.3, 207 ± 5.60, 176 ± 15.4 in control, Hes1-null, and conditional Hes1; Hes5 double-mutant mice, respectively (Figs. 3Go, O–Q, and 4). Although there are still many progenitors (Ki67+ cells) in both control and Hes-mutant mice at E18, the number of progenitors was significantly reduced in Hes-mutant pituitaries (P < 0.0001) (Fig. 4Go). Furthermore, both control and Hes-mutant pituitaries were negative for TUNEL assay (Fig. 3Go, R–T), suggesting that apoptosis is not the cause of hypoplasia and lack of intermediate lobe cells in Hes-mutant pituitaries. These results raise the possibility that the cells that should normally become the intermediate lobe adopted the anterior lobe fate in the absence of Hes genes.


Figure 3
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  		Fig. 3. Pituitary Hypoplasia and Loss of Intermediate and Posterior Lobes in Conditional Hes1; Hes5 Double-Mutant Mice

A–H, Coronal (A and B), midsagittal (C–E), and parasagittal (F–H) sections derived from E18 embryos and stained with hematoxylin and eosin (HE). The Hes1-null pituitary gland (B, D, and G) is reduced in size but morphologically normal compared with the control (A, C, and F). In conditional Hes1; Hes5 double-mutant mice, the pituitary gland is severely hypoplastic and dysmorphic (E and H). This double-mutant pituitary gland contains no columnar cell, which is characteristic of the intermediate lobe cells, and lacks the lumen of Rathke’s pouch (E and H, inset). This double-mutant pituitary gland also lacks the posterior lobe (E and H). n = 11 (control), 9 (Hes1-null mice), and 5 (conditional Hes1; Hes5 double-mutant mice). I–N, Immunostaining with anti-POMC and anti-PC2 antibodies. There is no significant difference in the expression patterns of POMC and PC2 between Hes1-null pituitary glands and the control (I, J, L, and M). In contrast, in conditional Hes1; Hes5 double-mutant pituitary gland, POMC+ cells exhibit morphologically the feature of the anterior lobe cells (K). In addition, no PC2+ cell is detected in the conditional double mutant pituitary gland (N). n = 17 (control), 5 (Hes1-null mice), and 6 (conditional Hes1; Hes5 double-mutant mice). O–Q, Immunostaining with anti-Ki67 antibody. Although there are still many Ki67+ cells in both control and Hes-mutant mice, the number of Ki67+ cells is significantly reduced in Hes-mutant pituitaries. n = 16 (control), 7 (Hes1-null mice), and 6 (conditional Hes1; Hes5 double-mutant mice). R–T, TUNEL assay. Both Hes-mutant and control pituitary glands are negative for TUNEL assay. n = 5 (control), 3 (Hes1-null mice), and 3 (conditional Hes1; Hes5 double-mutant mice). AL, Anterior lobe; IL, intermediate lobe; PL, posterior lobe. Bars, 50 µm (C–T); 200 µm (A and B).

 

Figure 4
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  		Fig. 4. Decrease of Progenitors in Hes-Mutant Pituitaries

The number of Ki67+ cells in the sections of pituitary glands. At least three independent embryos of each genotype were examined. For each embryo, at least three sections were examined. The number of progenitors (Ki67+ cells) was significantly reduced in Hes-mutant pituitaries. *, Changes compared with the control are statistically significant (t test, P < 0.0001).

 
To characterize Hes-mutant anterior pituitaries, other cell lineages were examined. GH-producing cells were slightly increased (Fig. 5Go, A–C), whereas TSH- and LH-producing cells were not affected in conditional double-mutant mice (Fig. 5Go, D–I). Because the pituitary gland is severely hypoplastic, it is not possible to determine which cell types are adopted by the intermediate lobe cells in the absence of Hes genes.


Figure 5
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  		Fig. 5. Expression of GH, TSH, and LH in Hes-Mutant Pituitary Glands at E18

A–I, Immunostaining with anti-GH (A–C), anti-TSH (D–F), and anti-LH (G–I) antibodies. GH-producing cells are slightly increased, whereas LH- or TSH-producing cells are not affected in conditional Hes1; Hes5 double-mutant mice (C, F, and I), compared with both control (A, D, and G) and Hes1-null mice (B, E, and H). n = 10 (A), 3 (B), 4 (C), 16 (D), 5 (E), 4 (F), 13 (G), 4 (H), and 3 (I). AL, Anterior lobe; IL, intermediate lobe. Bars, 50 µm.

 
Conditional Hes1; Hes5 Double-Mutant Mice Also Lack the Neurohypophysis
In addition to the adenohypophysis, we also found the defects of the neurohypophysis in conditional Hes1; Hes5 double-mutant mice. At E12.5, the evagination of the infundibulum was affected in conditional Hes1; Hes5 double-mutant mice compared with the control (Fig. 2Go, F and H, arrow and arrowhead). At E18, the neurohypophysis formed normally in both the wild-type and Hes1-null mice (Fig. 3Go, A–D). In contrast, the neurohypophysis was lost in conditional Hes1; Hes5 double-mutant mice (Fig. 3Go, E and H). These data show that Hes genes are also essential for the formation of the neurohypophysis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Hes1 and Hes5 Control the Progenitor Pool and Intermediate Lobe Specification in the Adenohypophysis Development
We found that, in the absence of Hes genes, the pituitary gland is severely hypoplastic and dysmorphic due to accelerated differentiation of progenitors and lacks the intermediate lobe. Our results demonstrate that Hes genes regulate maintenance of adenohypophysis progenitor cells and control intermediate vs. anterior lobe specification during the pituitary development (Fig. 6Go). In the nervous system, Hes genes are essential for maintenance of neural stem cells. In the absence of Hes genes, neural stem cells prematurely differentiate into neurons only and become depleted without generating other cell types. In addition to maintenance of the progenitor pool, it has been shown that Hes genes regulate binary cell fate decision in many systems: Hes1 promotes glial vs. neuronal (8), T cell vs. B cell (14), and exocrine vs. endocrine cell fate decision (15, 16, 17). Similarly, it is likely that Hes1 and Hes5 promote intermediate vs. anterior lobe specification in the adenohypophysis development. Further investigations, such as rescue studies, will be necessary to understand the mechanism for the regulation of pituitary progenitor cells by Hes genes.


Figure 6
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  		Fig. 6. Proposed Roles of Hes Genes in the Pituitary (Adenohypophysis) Development

A, Schematic Hes1 expression pattern in the developing adenohypophysis. Blue shows Hes1 expression. In the early stage, Hes1 is expressed in all progenitors. In the middle stage, Hes1 expression is restricted mainly to the progenitors located in the presumptive intermediate lobe. In the late stage, Hes1 expression is restricted to the periluminal proliferative zone. RP (orange), Rathke’s pouch; AL (red), presumptive anterior lobe; IL (yellow), presumptive intermediate lobe; RT (green), rostral tip. B, Proposed roles of Hes genes in the adenohypophysis development. Hes genes control the maintenance of adenohypophysis progenitors, and the specification of the intermediate lobe during the pituitary development.

 
In the neural development, Hes genes regulate the timing of differentiation of neural stem cells by repressing premature onset of activator-type bHLH genes, such as Mash1, Math, and Neurogenin (8, 11, 27). The Notch-Hes pathway also controls the pancreatic cell differentiation by repressing Neurogenin3 (15, 16, 17). In the pituitary development, the involvement of bHLH genes such as Mash1, NeuroD, and Neurogenin has been previously reported (28, 29). In addition, the involvement of GATA2, a hematopoietic transcription factor, has been also reported (1, 6, 30). Furthermore, a recent report shows that Notch pathway regulates GATA2 in hematopoietic stem cells (31). Although we have no definitive evidence, it is possible that these transcription factors will act as the downstream of Hes genes in the pituitary development.

Hes1 and Hes5 Are Essential for the Formation of the Neurohypophysis
We also found the defects of the neurohypophysis (the posterior lobe) in the absence of Hes1 and Hes5. In contrast to the adenohypophysis, the neurohypophysis arises from the ventral diencephalon. The evagination of the diencephalon is the first step in the formation of the neurohypophysis. During neural development, in the absence of Hes genes, progenitor cells cannot be maintained and prematurely differentiate into neurons (8). At E10.5, the neural tube is severely disorganized in Hes1 and Hes5 double-mutant mice (11). In the wall of the diencephalon where the infundibulum arises, Hes1 is strongly expressed. Hence, in the absence of Hes genes, the evagination of the diencephalon may be affected due to the accelerated differentiation of the progenitor cells of the diencephalon.

Implications of Simultaneous Loss of the Intermediate and Posterior Lobes during the Pituitary Development
The ventral diencephalon has direct contact with the intermediate lobe through the posterior lobe in the pituitary system. It is well described that signaling molecules expressed in the ventral diencephalon provide instructive cues for cell proliferation to form the pituitary gland (1, 2, 3). In Hes1 single-mutant mice, the pituitary gland was hypoplastic but both the intermediate and posterior lobes formed normally. In conditional Hes1; Hes5 double-mutant mice, although rare, we occasionally observed the intermediate lobe. In these cases, the posterior lobe also formed (data not shown). In contrast, in most of Hes1; Hes5 double-mutant mice, both the intermediate and posterior lobes were lost, suggesting that the mutual interaction between Rathke’s pouch and the ventral diencephalon has an essential role in the development of both the intermediate and posterior lobes.

Notch-Independent Regulation of Hes Genes
During preparation of this manuscript, two independent studies on Notch-Hes pathway in the pituitary development have been reported. Raetzman et al. (32) reported the analysis of Hes1 single-mutant mice, and their results are different from those of our Hes1-null mice but are similar to those of our Hes1; Hes5 conditional knockout mice. The reason why we do not observe the loss of melanotropes in Hes1-null mice could be due to the difference of the mouse strains. Raetzman et al. used C56BL6, whereas we used ICR, suggesting that compensation by Hes5 is more effective in ICR. Zhu et al. (33) analyzed conditional RBP-J-mutant mice and reported that melanotropes are increased in the intermediate lobe of conditional RBP-J-mutant mice. This result is different from our data of Hes1; Hes5 conditional knockout mice. It has been shown that Hes1 expression does not solely depend on the Notch-RBP-J pathway. Hes1 expression occurs before Notch and Delta are expressed (8, 11). Furthermore, the analysis of RBP-J and Notch1 mutants revealed no change in Hes1 expression at E9.0, suggesting that the contribution of these genes to the regulation of Hes1 in the early embryo is minimal (34). It has been also reported that c-Jun N-terminal protein kinase signaling can modulate Hes1 expression in a Notch-independent manner (35). The difference in phenotype between RBP-J and Hes mutants may be explained by the Notch-independent regulation of Hes genes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mice
All animals used in this study were maintained and handled in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Floxed Hes1 mice contain floxed Hes1 (exon 2–4) allele, and the detail will be described elsewhere (I. Imayoshi and R. Kageyama, unpublished data). Emx1-Cre mice were obtained from RIKEN BioResource Center (Ibaragi, Japan) (22). Genotypes of Emx1-Cre, Hes1 and Hes5 mutant mice were determined as previously described (13, 19, 22, 27). Hes1fl/fl; Hes5–/– and Emx1+/Cre; Hes1+/–; Hes5–/– mice were obtained by crossbreeding. Conditional Hes1; Hes5 double-mutant mice were obtained by crossing Hes1fl/fl; Hes5–/– and Emx1+/Cre; Hes1+/–; Hes5–/– mice. All analyses were performed between littermates. Hes1+/fl; Hes5–/– mice were found to have no significant phenotype in the developing pituitary gland and were therefore used as controls. For analysis of Cre recombinase activity, Emx1-Cre mice were crossed with ROSA26 reporter mice (generous gift from P. Sariano, Fred Hutchinson Cancer Research Center, Seattle, WA) and analyzed by X-gal staining as previously described (24, 25).

In Situ Hybridization
Digoxigenin-labeled antisense RNA probes corresponding to fragments of Hes1 and Hes5 cDNAs were synthesized in vitro. In situ hybridization was performed as previously described (13, 27). Briefly, 16-µm-thick cryosections were treated with proteinase K, refixed with 0.2% glutaraldehyde and 4% paraformaldehyde, washed with 0.1% Tween 20 in PBS, and hybridized with RNA probe in 50% formamide, 5x standard saline citrate (SSC), 1% sodium dodecyl sulfate, 50 µg/ml heparin, and 50 µg/ml tRNA solution at 65 C overnight. After hybridization, sections were washed at 65 C in 50% formamide, 5x SSC, 1% sodium dodecyl sulfate, treated with ribonuclease, washed in 50% formamide and 2x SSC, washed in Tris-buffered saline, and incubated with alkaline phosphatase-conjugated antibody against digoxigenin at 4 C overnight. After incubation, the sections were washed with 0.1% Tween 20 in Tris-buffered saline three times, and in 100 mM NaCl, 100 mM Tris-HCl (pH 9.5), 50 mM MgCl2, and 0.1% Tween 20 solution once. For a color development reaction, 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate were used as substrates. Labeled preparations were imaged using a Carl Zeiss (Oberkochen, Germany) Axiophoto microscope equipped with a charge-coupled device camera.

Immunostaining
Immunostaining was performed with the following antibodies: rabbit anti-POMC (ACTH) (1:4000; Chemicon, Temecula, CA), rabbit anti-GH (1:4000; Chemicon), mouse anti-TSH (1:2000; DakoCytomation, Carpinteria, CA), mouse anti-LH (1:2000; DakoCytomation), rabbit anti-PC2 (1:200; Chemicon), and mouse anti-Ki67 (1:100; BD Pharmingen, San Diego, CA) antibodies. Affinity-purified guinea pig anti-Hes1 antibody was raised against the bacterially expressed recombinant Hes1 protein (86–278) and used at a 1:200 dilution. The detail of this anti-Hes1 antibody will be described elsewhere (I. Imayoshi and R. Kageyama, unpublished data). Cryosections were incubated in 5% normal goat serum and 0.1% Triton X-100 at room temperature for 1 h, and then incubated with primary antibodies at 4 C overnight or for 2 d (for Hes1). A biotinylated antibody against mouse IgG, rabbit IgG and guinea pig IgG (1:200; Vector Laboratories, Burlingame, CA) were used for a secondary antibody. Fluorescein isothiocyanate-avidin D (1:1000; Vector Laboratories) was added to detect the signal. Samples were then treated with 4',6-diamidino-2-phenylindole and were mounted with Fluoromount G (Southern Biotechnology, Birmingham, AL). For immunostaining with anti-TSH and -LH antibodies, MOM blocking reagent (Vector Laboratories) was added in the blocking solution. For immunostaining with anti-Ki67 and -Hes1 antibodies, the antigen retrieval procedure was performed: sections were boiled in 0.01 M Na-citrate buffer (pH 6.0) at 90 C for 5 min. For all antibodies, no significant signal was detected in negative controls. Labeled preparations were also imaged using a Carl Zeiss Axiophoto microscope equipped with a charge-coupled device camera. TUNEL assay was performed with a detection kit as indicated in the protocol provided by the manufacturer (Roche, Basel, Switzerland) (13, 27).


   ACKNOWLEDGMENTS
 
We thank P. Soriano for providing ROSA26 reporter mice, and H. Shimojo, A. Ishii, and S. Sakamoto for technical help.


   FOOTNOTES
 
This work was supported by the Japan Society for the Promotion of Science (17659443 to M.H.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 10, 2007

1 A.K. and I.I. contributed equally to this work. Back

Abbreviations: bHLH, Basic helix-loop-helix; E8.5, embryonic d 8.5; PC1, prohormone convertase 1; POMC, proopiomelanocortin; SSC, standard saline citrate; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

Received for publication January 22, 2007. Accepted for publication March 30, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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U.-M. Fiuza and A. M. Arias
Cell and molecular biology of Notch
J. Endocrinol., September 1, 2007; 194(3): 459 - 474.
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