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Molecular Endocrinology, doi:10.1210/me.2005-0409
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Molecular Endocrinology 20 (6): 1378-1390
Copyright © 2006 by The Endocrine Society

Cell Proliferation and Vascularization in Mouse Models of Pituitary Hormone Deficiency

Robert D. Ward, Brandon M. Stone, Lori T. Raetzman and Sally A. Camper

Graduate Program in Cellular and Molecular Biology (R.D.W., S.A.C.), and Department of Human Genetics (B.M.S., L.T.R., S.A.C.), University of Michigan, Ann Arbor, Michigan 48109-0618

Address all correspondence and requests for reprints to: Sally A. Camper, Graduate Program in Cellular and Molecular Biology, Department of Human Genetics, 4909 Buhl Building, East Catherine Street, Ann Arbor, Michigan 48109-0618. E-mail: scamper{at}umich.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutations in the transcription factors PIT1 (pituitary transcription factor 1) and PROP1 (prophet of Pit1) lead to pituitary hormone deficiency and hypopituitarism in mice and humans. To determine the basis for this, we performed histological analysis of Pit1- and Prop1-deficient dwarf mouse pituitaries throughout fetal and postnatal development. Pit1-deficient mice first exhibit pituitary hypoplasia after birth, primarily caused by reduced cell proliferation, although there is some apoptosis. To determine whether altered development of the vascular system contributes to hypopituitarism, we examined vascularization from embryonic d 14.5 and throughout development. No obvious differences in vascularization are evident in developing Pit1-deficient pituitaries. In contrast, the Prop1-deficient mouse pituitaries are poorly vascularized and dysmorphic, with a striking elevation in apoptosis. At postnatal d 11, apoptosis-independent caspase-3 activation occurs in thyrotropes and somatotropes of normal but not mutant pituitaries. This suggests that Prop1 and/or Pit1 may be necessary for caspase-3 expression. These studies provide further insight as to the mechanisms of Prop1 and Pit1 action in mice.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MULTIPLE PITUITARY hormone deficiency (MPHD) is caused by a variety of transcription factor mutations. The majority of genetic MPHD cases result from mutations in the transcription factors, PROP1 and POU1F1 (PIT1), whereas mutations in HESX1, LHX3, and LHX4 are more rare (1, 2, 3, 4, 5, 6, 7, 8). Mutations in PIT1 produce deficiencies in GH, prolactin (PRL), and TSH and pituitary hypoplasia (9). PROP1 mutations cause progressive deficiencies in the same hormones as PIT1, with additional reductions in the gonadotropins, as well as ACTH loss (10, 11, 12, 13). These affected individuals exhibit a high level of variation in the age of onset and the severity of the disease (14). Transient hyperplasia progressing to hypoplasia is observed commonly in PROP1-deficient patients but not PIT1-deficient patients. A single adult LHX3-deficient MPHD patient exhibited an enlarged anterior pituitary, but, to date, no follow up study on pituitary growth has been reported (7). The relationship between pituitary growth and MPHD in PROP1 patients is not understood.

Several mouse models have been used to dissect the mechanism of PROP1 and PIT1 action in pituitary development. The Ames dwarf (Prop1df) and the Prop1null mouse models recapitulate the human MPHD phenotype in that they have deficiencies in TSH, GH, PRL, and low gonadotropins, as well as profound adult pituitary hypoplasia (15, 16, 17, 18, 19). There is no evidence for progressive hormone deficiency, however. Mouse models with mutations in Pit1 include Snell’s dwarf (Pit1dw) and Pit1dwJ, and they lack GH, PRL, and TSH and exhibit adult pituitary hypoplasia (20, 21, 22). These mouse models have been used extensively to learn more about the causes of MPHD as well as to study the mechanism of action of PROP1 and PIT1 in basic pituitary development. Few studies have undertaken a direct comparison of pituitary development in Prop1- and Pit1-deficient mice.

In mice, pituitary development begins at approximately embryonic d 9 (e9) with a thickening of the oral ectoderm to form the pituitary placode, which grows and invaginates to produce Rathke’s pouch and eventually forms the intermediate and anterior lobes of the pituitary. This process requires the direct contact of Rathke’s pouch with the neural ectoderm of the ventral diencephalon, which, in turn, evaginates to form the infundibulum that is the future posterior lobe of the pituitary gland and pituitary stalk (23, 24). By e12.5, Rathke’s pouch has separated from the oral ectoderm and has begun to expand to form the anterior lobe as proliferating cells migrate ventrally. Prop1 is expressed from about e10 until e16, with peak expression at e12.5 in a dorsal to ventral gradient (16). Prop1 deficiency results in the failure to transactivate Pit1, which is normally expressed from e14.5 throughout adulthood and is essential for specification and differentiation of TSH-, GH-, and PRL-producing cells (19, 20, 21, 25). In humans, however, little is known about the timing and expression pattern of PROP1 and PIT1. Pituitary specification takes place in the first trimester of pregnancy, in contrast to mice, in which substantial pituitary differentiation is occurring during late gestation and early postnatal life. Prop1-deficient pituitaries exhibit a failure of precursor cell migration from Rathke’s pouch to the developing anterior lobe. This results in a dysmorphic pouch and hypoplastic anterior lobe by e14.5, a defect that is not observed in the Pit1-deficient pituitaries (17, 19). The Prop1-deficient mouse pituitary also exhibits decreased proliferation with enhanced apoptosis during postnatal development that ultimately results in a pituitary that fails to grow beyond 7–10 d of postnatal development (17). It is not known whether these defects are solely the consequence of the Prop1 deficiency or result from the failure to activate Pit1.

Angiogenesis is essential for postnatal growth, morphogenesis, and other biological processes, and inadequate vascularization can lead to apoptosis (26, 27, 28, 29, 30). Angiogenesis in the rodent pituitary begins as the vessel network from the surrounding tissues invades the contact zone between the developing Rathke’s pouch and the ventral diencephalon. This process causes a separation in the contact zone as the primary portal vessel system forms. The separation of the developing pituitary from the oral ectoderm is accompanied by the invasion of a capillary network into the developing anterior lobe (31). The adult anterior lobe receives its blood supply from the portal vessels into the dense capillary network of the anterior lobe, although the intermediate lobe as well as the residual lumen of Rathke’s pouch is poorly vascularized (31, 32, 33). The vascular network is necessary for the secreted hormones to be carried from the pituitary to the endocrine target organs, as well as for transportation of negative feedback signals that act on the pituitary directly (34). Little is known about the genetic regulation of pituitary vascularization or its effect on cell specification.

The vascular network is important for the differentiation of endocrine cell types of the pancreas (26, 35, 36, 37). It is not known whether vascular development stimulates differentiation of other types of endocrine cells. The timing of pituitary vascularization is coincident with evidence of cell differentiation, suggesting that it could exert an influence on this process. Early studies on the Snell dwarf (Pit1dw) suggested that the vessel network may be developmentally delayed, which could contribute to the failed differentiation and function of several hormone secreting cell types (38). The conclusiveness of this study is weakened by the inability to genotype mice for the Pit1 mutation at that time. Thus, investigation of vascular development in dwarf mice merits reexamination.

In this study, we compare pituitary development and vascularization in Prop1df/df,Pit1dw/dw, and wild-type mice. We find vascular abnormalities in Prop1 but not Pit1 mutants. Both mouse mutants exhibit a decrease in proliferating pituitary cells after birth, resulting in a profoundly hypoplastic organ by 1 wk of postnatal development. This proliferation decrease is due to the failure of the PIT1 lineage, which normally begins to divide during postnatal development. The Pit1 mutant pituitary exhibits elevated apoptosis compared with wild-type mice after birth, but Prop1 mutants have substantially more apoptosis than Pit1-deficient mice. A limitation in the study of pituitary organogenesis is the lack of appropriate molecular markers for specific developmental stages. During the course of this study, we discovered that normal thyrotropes and somatotropes exhibit apoptosis-independent caspase-3 activation after birth. This suggests a role for proteolysis in function and/or expansion of these cell types. This study provides basic information about how the pituitary gland forms and enhances our understanding of the mechanism of PROP1 as well as PIT1 action.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pit1dw/dw Pituitaries Exhibit Hypoplasia after Birth
Pit1 is downstream of Prop1 in the transcriptional hierarchy; therefore, we sought to determine which pituitary phenotypes are similar or different between the Prop1df/df and the Pit1dw/dw mice. We selected the Prop1df/df mice instead of the Prop1null mutants for this study because the Prop1null mutants are on a genetic background that causes a high incidence of lethality that is secondary to the pituitary hormone deficiency (15). We previously reported that the Prop1-deficient anterior lobe is hypoplastic at e14.5, although the overall size of the organ is similar to wild type until birth (17). In addition, Prop1-deficient pituitaries are extremely dysmorphic at postnatal d 1 (P1) (Fig. 1Go, A and C). We expected that the Pit1dw/dw mice would exhibit hypoplasia soon after e14.5 because that is when Pit1 normally becomes transcriptionally active. However, the overall size of the Pit1dw/dw pituitary is similar to wild type at P1, and it does not exhibit any dysmorphology (Fig. 1Go, G and E). By P11, however, both Prop1df/df and the Pit1dw/dw pituitary glands are obviously smaller than normal littermates, and the difference is due to reduced growth of the anterior lobes (Fig. 1Go, compare D with B and H with F). Volumetric analysis confirmed these observations (data not shown) (17). The Pit1 and Prop1 mutant mice are the same size as their normal littermates at birth (15, 19). For both mutants, the size difference just begins to become apparent by around 2 wk of age, and it is reliably visible by weaning at 3 wk. By adulthood, the mutant mice are approximately one third to one fourth the size of their littermates, yet the difference in volume and weight of the normal and mutant adult pituitaries is 10-fold (17, 39). Furthermore, the pituitary glands of the mutants essentially fail to grow much after birth even though the mice continue to grow through weaning. Thus, the growth of the anterior lobe of the pituitary is disproportionately retarded relative to the body growth of the animal.


Figure 1
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  		Fig. 1. Prop1df and Pit1dw Pituitaries Are the Same Size as Wild-Type Pituitaries at Birth but Develop Hypoplasia during Postnatal Development

Prop1+/+ (A and B), Prop1df/df (C and D), Pit1+/+ (E and F), and Pit1dw/dw (G and H) neonates were sectioned in the coronal plane and comparable pituitary sections were stained with hematoxylin and eosin to examine the pituitary morphology and size at P1 (A, C, E, and G) and P11 (B, D, F, and H). At P1, pituitaries from Prop1-deficient mice (C) have a dysmorphic appearance compared with wild-type littermates (A), although the overall size is similar. The pituitaries of Pit1-deficient mice (G) are similar in morphology and size to wild-type littermates at this age (E), and the proportion of cells in the posterior, intermediate, and anterior lobes are normal. At P11, the Prop1-deficient pituitaries (D) retain the dysmorphic appearance and exhibit overall organ hypoplasia when compared with wild-type littermates (B). Although the Pit1-deficient pituitaries (H) do not exhibit dysmorphology at P11, they are hypoplastic when compared with wild-type pituitaries (F) at this age. The organ hypoplasia is attributable to the reduced size of the anterior lobe. P, Posterior lobe; I, intermediate lobe; A, anterior lobe. Scale bar, 100 µm.

 
Most Proliferating Anterior Pituitary Cells Are Not Fully Differentiated
Reduced expression of Pit1 is sufficient to reduce cell proliferation in somatotrophic cell lines (40), but the correspondence between Pit1 expression and cell proliferation has not been investigated in intact animals. To determine whether PIT1-positive cells are actively dividing late in embryonic and/or early postnatal development, we used immunohistochemistry to detect colocalization of PIT1 protein and a proliferation marker: either Ki67 or bromodeoxyuridine (BrdU). Ki67 is expressed in actively dividing cells, but not quiescent cells, and it is likely important for ribosome biosynthesis (41, 42). The thymidine analog, BrdU, is incorporated into newly synthesized DNA strands during the S-phase of the cell cycle (43, 44). At e17.5, there are few, if any, PIT1-positive cells that express Ki67, which suggests that cells of the PIT1 lineage are not actively dividing at this time (Fig. 2AGo). The PIT1-positive cells are also mutually exclusive from Ki67 cells at P1 (Fig. 2BGo). Furthermore, we did not detect any colocalization of Ki67 or BrdU with antibodies against GH, FSHß, LHß, ACTH, or TSHß in P1 pituitaries (data not shown). Taken together, these data suggest that the majority of PIT1-positive cells and hormone-expressing cells are not actively dividing during embryonic development through the time of birth. Analysis at P8 revealed that many PIT1-positive cells were colabeled with Ki67, indicating that the PIT1 lineage begins to divide in postnatal development, coinciding with the age at which the pituitary growth deficiency becomes apparent in the Prop1 and Pit1 mutants.


Figure 2
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  		Fig. 2. PIT1 Lineage Undergoes Active Proliferation at P8, whereas Pit1-Deficient Pituitaries Exhibit Decreased Proliferation by 1 wk of Postnatal Development

Wild-type pituitaries were coimmunostained with Ki67 (green) and PIT1 (red) at e17.5 (A), P1 (B), and P8 (C) to determine the age at which the PIT1 lineage begins to actively divide. At e17.5 (A) as well as P1 (B), PIT1-positive cells are mutually exclusive of Ki67-positive cells indicating that the PIT1 lineage is not actively dividing at this time. At P8 (C), there are many PIT-positive/Ki67-positive cells (yellow), indicating that the PIT1 lineage is actively dividing. Immunostaining for Ki67 or BrdU was assessed in Pit1+/+ (D, G, and J) and Pit1dw/dw (E, H, and K) neonates at P1 (D and E) and P8 (G, H, J, and K). Immunostaining for Ki67 (green) shows that there are no obvious differences in the number of proliferating cells in Pit1dw/dw pituitaries (E) compared with their wild-type littermates (D) at birth. Immunostaining for Ki67 (green, G and H) or BrdU (green, J and K) shows that the Pit1dw/dw anterior lobes (H and K) have fewer proliferating cells than wild-type littermates (G and J) by P8. Red cells in panel H are red blood cells that autofluoresce. Original magnification, x200. F, The average number of apoptotic cells at P1 is significantly increased in Pit1dw/dw pituitaries (gray bar, P < 0.005) compared with the wild type (white bar), but not to the extent of apoptosis exhibited by the Prop1df/df pituitaries (black bar, P < 0.001). I, The average number of apoptotic cells is increased at P8 in the Prop1df/df pituitaries (black bar, P < 0.001) compared with the wild-type (white bar) but not the Pit1dw/dw pituitaries (gray bar). The number of animals analyzed at each time point is as follows (n = +/+, dw/dw, and df/df): P1, n = 6, 2, and 3; P8, n = 9, 3, and 8. I, intermediate lobe; A, anterior lobe.

 
Pit1 Deficiency Results in Decreased Proliferation by P8 and Some Apoptosis
We compared cell proliferation in wild-type and Pit1dw/dw pituitaries at P1 and P8 (Fig. 2Go). There is no obvious difference in the fraction of cells that are expressing Ki67 at P1 (Fig. 2Go, compare E and D). At P8, however, the Pit1dw/dw pituitary has fewer Ki67- and BrdU-positive cells than wild type, indicating a decrease in proliferation (Fig. 2Go, compare H to G and K to J) as a result of the Pit1 deficiency. Previous studies revealed that Prop1df/df pituitaries exhibit both decreased proliferation and increased apoptosis at P8 (17). The Pit1dw/dw pituitaries showed a slight increase in apoptosis at P1, relative to wild type, 31 ± 18 vs. 18 ± 7, but the difference was not as profound as observed for Prop1 mutants, 47 ± 15 vs. 18 ± 7 (Fig. 2FGo). There was no increase in apoptosis observed in the P8 Pit1dw/dw pituitaries compared with wild type, although the Prop1df/df pituitary exhibits enhanced apoptosis at this time (Fig. 2IGo).

Aberrant Vegfa Expression and Inadequate Vascularization in Prop1df/df But Not Pit1dw/dw Pituitaries
Previous studies suggested that vascularization of the pituitary takes place during the time that cell specification becomes evident, and that this process might be delayed in Pit1dw/dw mice (32, 33, 38). We compared the vascularization of Prop1df/df and Pit1dw/dw pituitaries with their normal littermates throughout embryonic development by immunohistochemical staining for platelet endothelial cell adhesion molecule (PECAM). At e14.5, the developing anterior lobe has begun to show evidence of blood vessels, but Rathke’s pouch is poorly vascularized (Fig. 3Go, A, C, D, and F). The tightly stratified structure of the luminal cells may prevent invasion of the vessels. At this stage, there is no difference in PECAM staining between Prop1df/df mice and normal littermates. By e17.5, the normal pituitary anterior lobe has a dense vascular network, whereas cells in the intermediate lobe and those that line the lumen in the anterior lobe remain poorly vascularized (Fig. 3Go, G and K). In contrast, the Prop1df/df pituitary has decreased vascular density. Although the small anterior lobe has vessel structures, the majority of the dysmorphic pituitary is poorly vascularized (Fig. 3Go, H and L).


Figure 3
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  		Fig. 3. Aberrant VEGF Expression and Poor Vascularization in Fetal Prop1-Deficient Pituitaries

Immunohistochemical staining for PECAM (red) and VEGF (green) was carried out on sagittal pituitary sections from e14.5 wild-type (A–C) and Prop1df/df (D–F) as well as coronal pituitary sections from e17.5 wild type (G, I, and K) and Prop1df/df (H, J, and L). At e14.5, PECAM staining surrounds Rathke’s pouch and is present within the developing anterior lobe in both the wild-type and Prop1df/df pituitaries (compare A and C with D and F). VEGFA staining is intense within the luminal cells of Rathke’s pouch and dispersed throughout the developing anterior lobe in both the wild-type and Prop1df/df pituitaries (compare B and C with E and F). At e17.5, PECAM staining is apparent throughout the posterior and anterior lobes in the wild-type pituitary, although the intermediate lobe and luminal cells of the residual Rathke’s pouch are devoid of it (G, bracket, and K), and VEGFA staining is prominent in the intermediate and anterior lobes of wild-type mice (I and K). At e17.5, Prop1df/df pituitaries have more stratified cells that are poorly stained with PECAM compared with wild type (compare H, bracket and L with G, bracket and K). In contrast, VEGFA staining is intense throughout the luminal cells of the mutant dysmorphic pituitary at e17.5, in a pattern that resembles e14.5 (compare J and L with E and F). INF, Infundibulum; RP, Rathke’s pouch; P, posterior lobe; I, intermediate lobe; A, anterior lobe. Original magnification, x200.

 
The pituitaries of normal and Prop1df/df mice were also examined for the presence of VEGFA (vascular endothelial growth factor A), an important signaling factor for vascular development and a cell survival factor in some systems (45, 46). At e14.5, dense VEGFA immunohistochemical staining is normally apparent on the luminal side of the Rathke’s pouch as well as speckled throughout the developing anterior lobe (Fig. 3Go, B and C). As embryonic development proceeds, VEGFA expression becomes more dispersed, and expression is dense throughout the anterior lobe by e17.5 (Fig. 3Go, I and K). The expression pattern of PECAM and VEGFA remains the same from e17.5 through P8 (Fig. 4Go, A, D, and G). The VEGFA immunostaining appears normal in Prop1df/df pituitaries at e14.5 (Fig. 3Go, E and F), but the dense luminal expression of VEGFA is abnormally retained at e17.5 (Fig. 3Go, J and L), suggesting that these cells have not differentiated beyond e14.5. This aberrant VEGFA expression pattern persists throughout postnatal development of Prop1df/df pituitaries (Fig. 4Go, E and H). In addition, the anterior lobes of Prop1df/df mice remain poorly vascularized (Fig. 4Go, compare B and H with A and D). The PECAM and VEGFA expression in Pit1dw/dw pituitaries is normal at P1 (data not shown) and P8 (Fig. 4Go, C, F, and I), suggesting that these pituitaries are normally vascularized.


Figure 4
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  		Fig. 4. Aberrant VEGF Expression and Poor Vascularization in Postnatal Prop1df/df But Not Pit1dw/dw Pituitaries

PECAM and VEGFA immunostaining were carried out on coronal sections of P8 wild-type (A, D, and G), Prop1df/df (B, E, and H), and Pit1dw/dw (C, F, and I) pituitaries. PECAM immunoreactivity is detected in the posterior and anterior lobe but not the intermediate lobe or luminal cells of the anterior lobe in normal mice (A, bracket, and G). Mislocalized luminal cells in Prop1df/df pituitaries have decreased PECAM immunoreactivity (B, brackets, and H). The Pit1dw/dw pituitary has normal PECAM immunoreactivity compared with wild type (C, bracket, and I) at this age. VEGFA immunostaining is aberrant in the P8 Prop1df/df pituitary (E and H) compared with wild type (D and G) or the Pit1dw/dw (F and I). P, Posterior lobe; I, intermediate lobe; A, anterior lobe; WT, wild type. Original magnification, x200.

 
Apoptosis-Independent Caspase-3 Activation in Normal P11 Pituitaries
There are very few apoptotic cells in normal pituitaries during postnatal development. However, activated caspase-3 immunoreactivity is prominent in healthy cells in the anterior lobes of wild-type mice (Fig. 5AGo). In contrast, no caspase-3 immunostaining is detectable in either the Prop1df/df or Pit1dw/dw anterior lobes at P8 and P11 (Fig. 5Go, B and C, and data not shown). To determine the nature of these apoptosis-independent activated caspase-3-positive cells, we performed coimmunolocalization studies with caspase-3 antibodies and antibodies directed against pituitary hormones, PIT1, Ki67, and the folliculo-stellate marker, S100. The majority of the caspase-3-positive cells are colabeled with PIT1 (Fig. 5Go, D and E) and Ki67 (data not shown), but not S100 (data not shown). This suggested that apoptosis-independent caspase-3 activation is a normal characteristic of a portion of the PIT1 lineage. Caspase-3 immunoreactivity colocalizes with TSH- and GH-positive cells, but not PRL, FSH, or ACTH (Fig. 5Go, F–I, and data not shown). This result suggests an apoptosis-independent caspase-3 activation pathway is turning on between P8 and P11.


Figure 5
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  		Fig. 5. Apoptosis-Independent Caspase-3 Activation in Normal But Not Mutant Pituitaries

Immunohistochemical staining for activated caspase-3 (green) revealed its expression in the cytoplasm of nonapoptotic cells of the anterior lobe in the P11 normal pituitary (A, inset). No immunoreactivity was detected in either the Prop1df/df (B) or the Pit1dw/dw (C) pituitaries. Normal P11 pituitaries were coimmunostained with activated caspase-3 (green, cytoplasmic) and PIT1 (red, nuclear). PIT1 and activated caspase-3 colocalized in many of the nonapoptotic cells (D and E). Coimmunostaining was carried out on P11 normal pituitaries for activated caspase-3 (green, cytoplasmic) and a hormone. No colocalization was detected for caspase-3 and FSHß (F) or ACTH (G), (red, cytoplasmic). Caspase-3 colocalizes with GH (H, yellow) and TSHß (I, yellow). Original magnification, x200 (A–D). WT, Wild type.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Mutations in both PROP1 and PIT1 cause MPHD in humans and mice. The manifestations are similar, except that PROP1 deficiency causes progressive, and more extensive hormone loss and hyperplasia with regression to hypoplasia. Prop1 and Pit1 mutations in mice are useful models for understanding human MPHD. Adult Prop1- and Pit1-deficient mice are approximately one third the size of littermates, with hypoplastic anterior lobes deficient in GH, PRL, and TSH hormone-producing cells (15, 16, 17, 19, 20, 21, 22). Prop1 allows for the differentiation and ventral migration of progenitors from the proliferative zone of Rathke’s pouch into the developing anterior lobe. The failure of precursor cells to stream away from Rathke’s pouch in Prop1 mutants causes dysmorphology that may give the impression of a larger organ (Fig. 6Go). These trapped precursor cells undergo apoptosis, which may mimic the regression of pituitary hyperplasia in PROP1 patients. Prop1 mutants have a hypoplastic anterior lobe early in development. In contrast, fetal Pit1dw pituitaries are indistinguishable from normal pituitaries, indicating that PIT1 is dispensable for initial expansion of the anterior lobe. One week after birth Pit1dw mutant pituitaries are obviously smaller than normal primarily because of reduced cell proliferation, although there is some apoptosis. PIT1-positive cells normally begin to proliferate after birth, at the same time that pituitary hypoplasia is first apparent in Pit1dw pituitaries. Thus, failure of the PIT1 lineage contributes to the smaller adult organs characteristic of both mutants.


Figure 6
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  		Fig. 6. Model of Pituitary Growth in Normal, Prop1-, and Pit1-Deficient Mice

Pituitary development begins in normal mice as an invagination of the oral ectoderm to form Rathke’s pouch. At e12.5, the peak of Prop1 expression, progenitors migrate ventrally into the developing anterior lobe and differentiate. At e14.5, Pit1 activation signals the specification of the PIT1 lineage. After birth, from P1 to P11, the PIT1 lineage undergoes active proliferation and the anterior lobe expands laterally. In the Prop1df/df animal, ventral migration is blocked, resulting in Rathke’s pouch dysmorphology and anterior lobe hypoplasia by e14.5. At birth, this dysmorphology remains evident, and the overall organ size is normal. Pit1 is not activated, and the PIT1 lineage fails to differentiate. This causes failure of the organ to expand during postnatal development. The hypoplasia is also partially attributable to enhanced apoptosis of the mislocalized, stratified luminal cells, possibly because of poor vascularization. The Pit1dw/dw pituitary does not exhibit any dysmorphology or altered vascularization. Cells evidently migrate from the pouch into the anterior lobe normally, but the PIT1 lineage fails to differentiate. This causes decreased proliferation during postnatal development and pituitary hypoplasia that is accompanied by an increase in apoptosis and subsequent pituitary hypoplasia. Prop1 and Pit1 expression profile is shown as black bars. Yellow, developing posterior lobe; red, stratified luminal cells; blue, anterior lobe cells.

 
One-week-old mice normally have a substantial pool of PIT1-positive proliferating cells. Although a small fraction of GH cells are also proliferating, most pituitary hormone-secreting cell types are not dividing. This indicates that most of the dividing PIT1-positive cells are precursors that have undergone lineage commitment but not yet completely differentiated into hormone producing cells. Activation of hormone gene expression is not associated with permanent quiescence, however, because somatotropes actively proliferate after the first week of life in the mouse (47). Actively dividing GH, PRL, and TSH cells are evident in postnatal rat pituitaries (48, 49). This differs from other endocrine cell types, such as the enteroendocrine cells of the gut, which seem to be derived predominantly from hormone-negative stem cell pools (50). Identification of the signals that regulate cell proliferation at different stages of pituitary differentiation will shed light on the normal organogenesis and may contribute to understanding the etiology of pituitary adenomas.

There are several candidates for signals that trigger the expansion of the PIT1 lineage and/or its derivatives after birth. Hypothalamic releasing hormones constitute one class of candidates for stimulating proliferation. In addition to regulating the production and secretion of anterior lobe hormones, these hormones act as specific mitogens for individual cell types. For example, mice deficient in the hypothalamic signals or their receptors exhibit decreased numbers of hormone secreting cells types in the anterior lobe after birth (51, 52, 53, 54, 55). GHRH cannot account for all of the stimulation of somatotrope proliferation, however, because Ghrh knockout mice and Ghrhrlit/lit mutants have a sizeable somatotrope population (52, 56). Another class of candidates includes the fibroblast growth factors, which act as cell survival factors, mitogens, and differentiation-inducing factors. Fibroblast growth factor (FGF) 4, FGF10, and specific FGF receptors have profound effects on pituitary cell differentiation and proliferation (57, 58, 59, 60, 61). PIT1-positive, hormone-negative cells can be induced to differentiate into lactotropes after exposure to FGF2 (62). A variety of signals may be important for proliferation of the PIT1 lineage and its derivatives during postnatal development.

The progressive nature of hormone deficiency in PROP1-deficient human patients is an interesting feature not found in patients with PIT1 mutations. We propose that the role of PROP1 in progenitor cell generation extends for a longer period in humans than in mice (17). There are several lines of evidence that support of this idea. First, PROP1 transcripts have been detected in adult pituitaries of the human, pig, and rat, suggesting that there is a role for Prop1 in pituitary cell maintenance in some species (63, 64, 65, 66, 67, 68). Second, human GHRH-expressing transgenic giant rats undergo somatotrope hyperplasia and progression to adenomas with corresponding increases in Prop1 mRNA levels, suggesting that Prop1 has an ongoing role in the Pit1 lineage in rodents (69). Third, overexpression of Prop1 in transgenic mice results in increased susceptibility to pituitary adenomas (70). Taken together, these results suggest that PROP1 may normally play a role in stimulating pituitary stem cells to renew the cells of the adult organ; and aberrant expression of Prop1 may be sufficient to overstimulate pituitary growth. Another possibility is that PROP1 is not absolutely essential for PIT1 activation in humans. In support of this idea, there is a 2-d lag between peak Prop1 expression and detection of Pit1 transcripts in mice, which suggests that other factors are involved in activation of Pit1 expression. Furthermore, Prop1-deficient mice show limited differentiation of the Pit1 lineage on a mixed genetic background but not on an inbred background like C57BL/6J (15, 71). Perhaps the genetic background in humans is even more supportive of limited PIT1 (POU1F1) expression in the absence of PROP1 than the mixed genetic background in mice.

Previous studies showed that as pituitary development proceeds, a pervasive capillary network is formed throughout the anterior and posterior lobes, but the intermediate lobe and luminal cells of the anterior lobe that represent the residual Rathke’s pouch remain poorly vascularized, presumably due to their tight adhesion (31, 32, 33). These studies used India Ink injections and fluorescently labeled gelatin to visualize the blood vessel network. We examined vascular development in dwarf and normal mouse pituitaries via immunohistochemical staining for platelet endothelial cell adhesion molecule (PECAM/CD-31) and the signaling molecule VEGFA. PECAM mediates cell-cell adhesion of the blood vessel endothelial cells. VEGFA is essential for recruitment and development of blood vessels, and in some systems it acts as a cell survival factor independent of vascularization. We found that the stratified cells of the Prop1-deficient pituitary are poorly vascularized, similar to the intermediate lobe cells of normal mice. The majority of the stratified cells in Prop1df mice do not express intermediate lobe markers, suggesting that these cells undergo apoptosis because of failed differentiation. Inadequate vascularization may also contribute to the apoptosis, as it has been implicated in hypoxia-induced apoptosis (27). Furthermore, VEGFA is essential for recruitment and development of blood vessels, and in some systems it acts as a cell survival factor independent of vascularization. For example, the growth plates of the long bones express Vegfa but are not vascularized. Vegfa is required for survival of the chondrocytes in the growth plate because without it, these cells exhibit massive apoptosis (45, 46). Although the Prop1df mice exhibit apoptosis with no decrease in Vegfa expression, the pattern of Vegfa expression is abnormal, giving the appearance of being arrested at e14.5. Both the inadequate vascularization and/or the failure of the mislocalized cells to differentiate may contribute to the enhanced apoptosis in the Prop1df pituitary.

The Pit1dw pituitary does not exhibit a block in cell migration or subsequent dysmorphology, although the overall organ becomes hypoplastic after birth. The pituitaries of the Pit1-deficient mice appear to have normal vascular architecture and limited apoptosis. In addition, these mice do not exhibit aberrant Vegfa expression characteristic of Prop1 mutants. A subtle delay in vascular development was reported in Pit1dw mice (38), but we did not observe any abnormalities. There are two points relevant to this. First, we cannot rule out a subtle change in the timing of blood vessel fenestration because we did not examine the vascular network at the electron microscopic level. Second, we used normal heterozygous mothers for the matings and normal littermates as controls, and the previous study used mutant mothers and fathers with hormone replacement and nonlittermate controls. Imperfect hormone replacement could contribute to developmental delay in the fetuses carried by the mutant mothers. In any case, our data clearly show that vascular development in Pit1dw mutants is more similar to normal mice than Prop1df. This is supported by organ volume, organ shape, VEGFA expression, and PECAM expression.

Pit1dw pituitaries have a larger anterior lobe than Prop1df at birth, indicating that Prop1 stimulates the production of ample precursor cells, even if there is no functional Pit1. The genes regulated by Prop1 that control this process are not known. Other than Pit1, the only genes whose expression is reported to depend on Prop1 are Hesx1, Nnat, Pou3f4, and Notch2 (16, 19, 72). Another difference between the two mutants is that Prop1df mouse pituitaries exhibit extensive apoptosis after birth, whereas Pit1dw have more limited apoptosis (17). We considered three plausible explanations for this difference. First, the Prop1df pituitary is severely dysmorphic, apparently due to a cell migration defect, and these mislocalized cells may be targeted for apoptosis in the same way that primordial germ cells that fail to migrate to the genital ridge undergo programmed cell death (73). The second possibility is that the failure to differentiate may result in targeted cell death, as described for undifferentiated PC12 cells and human epidermal keratinocytes (74, 75). Prop1df precursors are trapped in an earlier stage of differentiation than Pit1dw precursors, which could make them more sensitive to apoptosis. Third, the lack of vascularization in Prop1df mice may contribute to the sensitivity to apoptosis.

The activated caspase-3 expression in the cytoplasm of nonapoptotic cells in normal P11 pituitaries is surprising. This apoptosis-independent caspase-3 activation colocalizes with the TSH- and GH-secreting cells in normal mice and is absent in both Prop1df and Pit1dw pituitaries. Apoptosis-independent caspase-3 activation has been reported in PC12 cells, where it is hypothesized to be important for cleavage of the cytoskeletal stabilization protein, TAU, allowing for cell dispersal (76). Caspase-3 activation is involved in T-cell proliferation, the differentiation of erythroid and skeletal muscle cells, and the dispersal and migration of neuronal cells to their final destination (77, 78, 79, 80). Calpains, which are similar to the caspases in that they are calcium-dependent proteases, function in the cleavage of various cytoskeletal proteins and focal adhesion components (81). These examples suggest that caspase-3 activation in GH and TSH cells may allow them to assume their characteristic cellular shape and/or to disperse and migrate throughout the anterior lobe. Consistent with this idea, hormone-secreting cell types are specified in distinct locations of the developing anterior lobe, but they are interspersed throughout the anterior lobe of the adult organ (82). Recent imaging work suggests that dispersed cells may actually be connected through cellular processes that facilitate coordinated hormone secretion (83, 84, 85). Therefore, we suggest that caspase-3 is responsible for important thyrotrope and/or somatotrope functions beginning at P11. Other proteases may also be important in postnatal development. We recently identified induction of Prss28 after birth in association with gonadotrope differentiation (86).

Interestingly, Prop1 deficiency results in the absence of Notch2 expression, which normally defines the boundary between the proliferative zone of Rathke’s pouch and the developing anterior lobe. Notch2 has been implicated in the commitment and lineage-specific differentiation of progenitor cells in the embryonic pituitary (72). Thus, PROP1 action in cell migration may involve the Notch signaling pathway as well as various cell adhesion components. Prop1 is essential for the activation of Pit1, which specifies the PIT1 lineage (somatotropes, lactotropes, and thyrotropes). The PIT1 lineage proliferates starting during the first week of postnatal life, and it is the failure of the PIT1 lineage specification that results in the subsequent pituitary hypoplasia characteristic of both Prop1df and Pit1dw mouse mutants. The Prop1df developing pituitary is poorly vascularized and contains excess stratified cells that appear to be trapped at an early stage of differentiation with persistent, dense, luminal Vegfa expression. Thus, failure to differentiate and inadequate vascularization may contribute to the enhanced apoptosis observed in these mutants. Finally, we have discovered an apoptosis-independent caspase-3 activation in thyrotropes and somatotropes. This study provides a mechanism for the pituitary growth defect in both the Pit1dw and the Prop1df mouse models. It also suggests why PROP1-deficient MPHD patients often exhibit regression of pituitary volume by magnetic resonance imaging, whereas PIT1 MPHD patients generally do not.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mice
Ames dwarf mice (DF/B-df/df) that were originally obtained from Dr. A. Bartke (Southern Illinois University, Carbondale, IL) and Snell dwarf mice (DW/J Mlphln Pit1dw; The Jackson Laboratory, Bar Harbor, ME) have been maintained at the University of Michigan through heterozygous matings. The morning after conception is designated e0.5 and the day of birth is designated as P1. All mice were housed in a 12-h light, 12-h dark cycle in ventilated cages with unlimited access to tap water and Purina 5020 chow. All procedures using mice were approved by the University of Michigan Committee on Use and Care of Animals, and all experiments were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guidelines of the Care and Use of Experimental Animals.

PCR Genotyping
The genotype of Prop1df mice was determined by PCR amplification with the forward primer 5'-GAGCTGGGGAGACCTAAGCTTTGCC-3' and reverse primer 5'-GCCCAGATGTCAGGATACTG-3' to produce a 135-bp band. Restriction digestion with HinfI generates 97- and 40-bp bands specific to the wild-type allele, whereas the Prop1df mutant allele is uncut. The genotype of Pit1dw mice was determined by PCR amplification as previously described (19).

Histology and Immunohistochemistry
Embryos and mouse heads were fixed for 2–24 h in 4% paraformaldehyde in PBS (pH 7.2). All samples were washed in PBS, dehydrated in a graded series of ethanol, and embedded in paraffin. Six-micrometer sections were prepared and either stained with hematoxylin and eosin or processed as described below.

To detect cell proliferation at P1, Ki67 was examined with a rabbit polyclonal anti-Ki67 antibody (1:500; NovoCastra, Newcastle, UK) after antigen retrieval with 10 mM citrate boiling and detected with a biotin-conjugated goat antirabbit IgG (1:400; Jackson ImmunoResearch Laboratories, West Grove, PA) using either the tyramide signal amplification (TSA) fluorescein isothiocyanate (FITC) kit (according to the manufacturer’s protocol; PerkinElmer, Boston, MA) or a streptavidin-conjugated Cy2 fluorophore (1:200; Jackson ImmunoResearch). To detect cell proliferation at P8, individual mouse pups were injected ip with BrdU at 100 µg/g body weight, 2 h before the mice were killed. After epitope retrieval in 2 N HCl, BrdU incorporation was examined with a rat monoclonal anti-BrdU antibody (1:200; Caltag, Burlingame, CA) and detected with a FITC-labeled antirat IgG secondary antibody (1:400; Jackson ImmunoResearch). PIT1 was examined with a rabbit polyclonal antirat PIT1 antibody (1:400; S. Rhodes, Indiana University/Purdue University, Indianapolis, IN) after antigen retrieval with 10 mM citrate boiling and detected with a biotin-conjugated goat antirabbit IgG (1:500; Jackson ImmunoResearch) followed by either the TSA tetramethylrhodamine (TRITC) kit (PerkinElmer) or a streptavidin-conjugated TRITC fluorophore (1:400; Jackson ImmunoResearch). PECAM was examined with a rat monoclonal antimouse PECAM/CD31 antibody (1:25; BD Biosciences Pharmingen, Franklin Lakes, NJ) and detected with a biotin-conjugated donkey antirat IgG secondary antibody (1:400; Jackson ImmunoResearch) and using the TSA TRITC kit (PerkinElmer). VEGF-A was examined with a rabbit polyclonal antihuman VEGF-A antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and detected with the biotin-conjugated antirabbit IgG secondary antibody using the TSA FITC kit (PerkinElmer). PECAM was examined with a rat monoclonal antimouse PECAM/CD31 antibody (1:25; BD Biosciences Pharmingen, Franklin Lakes, NJ) and detected with a biotin-conjugated donkey antirat IgG secondary antibody (1:400; Jackson ImmunoResearch) and using the TSA TRITC kit (PerkinElmer). VEGF-A was examined with a rabbit polyclonal antihuman VEGF-A antibody (1:100; Santa Cruz Biotechnology) and detected with the biotin-conjugated antirabbit IgG secondary antibody using the TSA FITC kit (PerkinElmer). Activated caspase-3 was examined with a rabbit polyclonal antihuman cleaved caspase-3 antibody (1:50; Cell Signaling Technology, Inc., Beverly, MA) after antigen retrieval in 10 mM citrate boiling and detected with the biotin-conjugated antirabbit IgG secondary antibody using the TSA FITC kit (PerkinElmer). Hormones were examined using the rabbit antirat TSHß antibody [1:1600; National Institute of Diabetes and Digestive Kidney Diseases (NIDDK), Torrance, CA], monkey antirat GH antibody (1:1000; NIDDK), rabbit antirat FSHß antibody (1:1800; NIDDK), and rabbit antihuman ACTH antibody (1:1800; NIDDK) and detected with either the biotin-conjugated antirabbit IgG (1:400; Jackson ImmunoResearch) or biotin-conjugated antimonkey IgG (1:200; from the Human Vectastain ABC kit; Vector Laboratories, Burlingame, CA) secondary antibodies using the TSA TRITC kit (PerkinElmer).

Programmed cell death in the pituitaries was detected by the terminal deoxyuridine triphosphate nick end labeling method using the in situ cell detection kit peroxidase detection POD (Roche, Indianapolis, IN). Briefly, this kit detects cells with DNA strand breaks by end labeling the free 3'OH groups in nicked genomic DNA with terminal transferase and fluorescein-labeled deoxyuridine triphosphate. The incorporated fluorescein is detected with a ß-peroxidase POD-conjugated antifluorescein antibody. Apoptotic cells were counted on three to eight pituitary sections per animal. Student’s t test was used to determine statistical significance.

All sections in which a TRITC fluorophore was used for detection were incubated with 0.1% Sudan Black B (Fisher Scientific, Pittsburgh, PA) in 70% ethanol for 30 min before coverslip mounting to prevent auto fluorescence of red blood cells.

Microscopy
All images were captured with a Leitz DMRB microscope (W. Nuhsbaum, Inc., McHenry, IL) and an Optronics (Goleta, CA) camera.


   ACKNOWLEDGMENTS
 
We thank Simon Rhodes for the generous gift of the PIT1 antibody and Phil Gage for his suggestions.


   FOOTNOTES
 
This work was funded by the National Institutes of Health [T32 GM07863 and T32 GM07315 (to R.D.W.), National Research Service Award F32 DK60306 (to L.T.R.), and R37 HD30428 (to S.A.C.)] and The Endocrine Society (Undergraduate Research Fellowship to B.M.S.).

Current address for L.T.R.: Department of Molecular and Integrative Physiology, University of Illinois, Urbana, Illinois 61801.

Disclosure statement: None of the authors have anything to disclose.

First Published Online March 23, 2006

Abbreviations: BrdU, Bromodeoxyuridine; e, embryonic day; FGF, fibroblast growth factor; FITC, fluorescein isothiocyanate; MPHD, multiple pituitary hormone deficiency; P, postnatal day; PECAM, platelet endothelial cell adhesion molecule; PRL, prolactin; TRITC, tetramethylrhodamine; TSA, tyramide signal amplification.

Received for publication October 12, 2005. Accepted for publication March 13, 2006.


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