Pituitary, Pancreatic and Gut Neuroendocrine Defects in Protein Tyrosine Phosphatase- Sigma-Deficient Mice

Jane Batt, Sylvia Asa, Chris Fladd and Daniela Rotin

The Hospital for Sick Children (J.B., C.F., D.R.), Program in Cell Biology, and Institute of Medical Science and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada, M5G 1X8; and Departments of Pathology (S.A.), University of Toronto and Laboratory Medicine and Pathobiology, University Health Network, Toronto, Ontario, Canada, M5G 2C4

Address all correspondence and requests for reprints to: Dr. Daniela Rotin, Program in Cell Biology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada, M5G 1X8. E-mail: drotin{at}sickkids.on.ca


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The expression of receptor protein tyrosine phosphatase sigma (PTP{sigma}) is developmentally regulated in neuronal and neuroendocrine tissues. We have previously shown that mice deficient in PTP{sigma} demonstrate nervous system abnormalities, pituitary hypoplasia, increased neonatal mortality (60%), and death from a wasting syndrome at 2–3 wk of age (38%). We have now examined the role of PTP{sigma} on pituitary, pancreas and enteroendocrine cytodifferentiation, hormone production, and development. The adenohypophyses of PTP{sigma}(-/-) mice were small and exhibited reduced GH and PRL immunoreactivity. Cells containing TSH, LH, FSH, ACTH, pituitary-specific POU homeodomain factor (Pit-1), ER, and steroidogenic factor 1 were found in normal proportions and distributions. The diminished expression of GH and PRL was not associated with apoptosis of somatotrophs or lactotrophs. Pit-1-positive TSH-negative cells were detected, suggesting that impaired GH and PRL synthesis was not attributable to Pit-1 deficiency. In the knockout mice, pancreatic islets were hypoplastic with reduced insulin immunoreactivity, and there was also variable expression of gut hormones. Functionally, the GH deficiency was associated with hypoglycemia and death in the PTP{sigma}(-/-) neonate and accordingly, ip administration of GH rescued the PTP{sigma}(-/-) neonate and normalized the blood glucose. These data indicate that PTP{sigma} plays a major role in differentiation and development of the neuroendocrine system.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PROTEIN TYROSINE PHOSPHATASE sigma (PTP{sigma}) is a receptor tyrosine phosphatase and a member of the mammalian leukocyte common antigen-related (LAR) family. It consists of a cell adhesion-like extracellular domain composed of Ig and fibronectin type III repeats, a transmembrane domain, and two tandemly repeated catalytic domains (1, 2). The Drosophila homolog DLAR is expressed in pioneer neurons in the central nervous system, and in collaboration with the tyrosine kinase abl and its substrate enabled, controls motor axon guidance during Drosophila embryogenesis (3, 4). Although its ligand, substrate(s) and signaling pathway(s) are unknown, mammalian expression of PTP{sigma} is highly developmentally regulated in the central and peripheral nervous systems, pituitary, and epithelial tissues, suggesting a critical role in development (5, 6, 7, 8, 9). In view of this highly regulated expression, and the key role DLAR was found to play in the genesis of the Drosophila nervous system, we (10) and others (11) have generated PTP{sigma} knockout mice. These mice demonstrate increased mortality, developmental delay with growth retardation, abnormalities of both the central and peripheral nervous systems, and pituitary dysplasia. In this study we define the neuroendocrine requirement for PTP{sigma} in pituitary, pancreas and enteroendocrine cytodifferentiation, and hormone production. Our results show that PTP{sigma} is specifically required for the development of the somatotroph/lactotroph lineage of the anterior pituitary. GH and PRL production is impaired in PTP{sigma}(-/-) mice. The GH deficiency is associated with hypoglycemia and death in PTP{sigma}(-/-) neonates. Intraperitoneal administration of GH normalizes blood glucose (BG) and rescues the PTP{sigma}(-/-) neonates. In addition, the islets of the endocrine pancreas are hypoplastic, with diminished insulin immunoreactivity. These abnormalities result from the loss of trophic support normally conferred by GH since exogenous GH administration improves islet morphology and insulin immunoreactivity. PTP{sigma}(-/-) mice also exhibit marked abnormalities in the expression of the gut enteroendocrine hormones. These data indicate that PTP{sigma} plays a major role in differentiation and development of the neuroendocrine system.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The PTP{sigma}(-/-) animals consist of three cohorts: 1) 60% die as neonates within hours of birth; 2) 37.5% demonstrate growth retardation and succumb to a wasting syndrome by 2 to 3 wk of age, characterized by dehydration, an empty gut at autopsy, and extreme cachexia; 3) 2.5% survive to adulthood, appear healthy, and are fertile but remain 30–50% smaller by weight than their PTP{sigma}(+/+) or PTP{sigma}(+/-) littermates (10).

We have analyzed neuroendocrine tissues in PTP{sigma} knockout mice. The adrenal medulla and pulmonary neuroendocrine bodies (NEBs) appeared normal in PTP{sigma}(-/-), PTP{sigma}(+/-), and PTP{sigma}(+/+) mice, based on gross appearance at autopsy, histological evaluation, and in the case of pulmonary NEBS, on immunohistochemistry with calcitonin gene-related peptide (data not shown). Subsequent evaluation focused on the abnormalities noted in the pituitary, endocrine pancreas, and enteroendocrine system of the gut.

Pituitary Histology and Immunohistochemistry
The anterior pituitary was hypoplastic relative to the intermediate lobe in all three cohorts of PTP{sigma}(-/-) animals. As we previously reported, in many of the PTP{sigma}(-/-) neonates the posterior pituitary was also hypoplastic (10). A prominent intermediate cleft was still evident in all PTP{sigma}(-/-) newborns, as compared with wild type (WT) controls (Fig. 1Go, A and B). The cleft forms during embryonic development of the adenohypophysis but by birth has become negligible. Its marked persistence in the PTP{sigma}(-/-) neonate suggests developmental delay of the adenohypophysis.



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Figure 1. Immunostaining of the Adenohypophysis of a PTP{sigma}(-/-) Neonate (A) and a PTP{sigma}(+/+) Neonate (B) with Anti-GH Antibody

The PTP{sigma}(-/-) mouse demonstrates a significant decrease in the number of GH-immunoreactive cells (brown). In addition, the intermediate cleft (arrowhead) is prominent in the PTP{sigma}(-/-) neonate, compared with the PTP{sigma}(+/+) neonate, suggesting developmental delay of the adenohypophysis of the knockout animal (magnification, 200x). C, Western blot of serum GH of a representative PTP{sigma}(-/-) and(+/+) neonatal sibling pair. Fifty micrograms of serum protein were loaded per lane. Media from GH-secreting GH4 cells was used as a positive control. In PTP{sigma}(-/-) neonates (n = 6), serum GH levels were 34.3 ± 21% less than wild-type littermate controls. D, Immunostaining of the adenohypophysis of a wasting 3-wk-old PTP{sigma}(-/-) mouse and (E) a PTP{sigma}(+/+) sibling with anti-PRL antibodies (brown staining) demonstrates an absence of PRL immunoreactivity in the PTP{sigma}(-/-) mouse (magnification, 400x).

 
As the knockout cassette replaces PTP{sigma} with the ß-galactosidase gene, we analyzed the pattern of PTP{sigma} expression by Lac Z staining. Lac Z staining of newborn PTP{sigma}(-/-) mice demonstrates the expression pattern of PTP{sigma} in the anterior and intermediate pituitary (Fig. 2Go, A and B). Lac Z expression is still evident in the wasting 2- to 3-wk-old PTP{sigma}(-/-) animals, albeit significantly decreased when compared with expression in the newborn mouse (data not shown). Lac Z is also expressed in adult PTP{sigma}(-/-) pituitaries (data not shown).



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Figure 2. Lac Z Staining of Newborn (A) PTP{sigma}(+/+) and (B) PTP{sigma}(-/-) Pituitaries and (D) PTP{sigma}(+/+) and (E) PTP{sigma}(-/-) Pancreases

The pattern of PTP{sigma} expression in both the anterior and intermediate pituitary, and the islet of the pancreas, is denoted by the blue Lac Z staining in the PTP{sigma}(-/-) mice. Costaining for (C) GH (red-brown) and Lac Z reveals cells staining both blue and red-brown, which demonstrates that PTP{sigma} is expressed in somatotrophs. F, Costaining for insulin (red-brown) and Lac Z reveals PTP{sigma} expression in B cells. Magnification: panels A and B, 100x; inset, 400x; panel C, 600x; panels D and E, 100x; inset, 400x; panel F, 600x.

 
The anterior lobe of the adenohypophysis consists of corticotrophs, gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs, which synthesize and secrete their respective signature hormones, ACTH, FSH/LH, TSH, GH, and PRL. The newborn PTP{sigma}(-/-) animals revealed a decrease in the percentage of anterior pituitary cellular area immunopositive for GH (Fig. 1Go, A and B, and Fig. 3AGo). Accordingly, there was a variable but significant decrease in serum GH levels in the knockout neonates (Fig. 1CGo). Lac Z and GH costaining demonstrated the expression of PTP{sigma} in the somatotrophs (Fig. 2CGo). The remaining hormones, TSH, FSH, LH, and ACTH, were normal in both the PTP{sigma}(-/-) and (+/+) neonates (Fig. 3AGo). The {alpha}- subunit of glycoprotein hormones and the transcription factors ER and steroidogenic factor 1 (SF-1) were also normal in both the PTP{sigma}(-/-) and (+/+) neonates (data not shown). As expected, PRL was not expressed in the newborn mice because lactotroph terminal differentiation and maturation does not normally occur until hours to days postnatally.



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Figure 3. Morphometric Analyses of Immunostaining for Adenohypophyseal Hormones in (A) Newborn, (B) Wasting 2- to 3-wk-Old, and (C) Adult PTP{sigma}(-/-) and PTP{sigma}(+/+ and +/-) Pituitaries

The mean hormone-immunopositive cellular area is expressed as a percentage of the total pituitary cellular area ± SD (n = 2 to 5 mice/genotype/hormone). GH is significantly decreased in newborn PTP{sigma}(-/-) mice compared with sibling PTP{sigma}(+/+ or +/-) controls (P < 0.05). PRL is significantly decreased in 2- to 3- wk-old PTP{sigma}(-/-) mice compared with sibling controls (P < 0.05).

 
Lactotrophs and somatotrophs derive from a common somatotroph stem cell during adenohypophyseal cytodifferentiation (12, 13). In view of the neonatal reduction of GH immunoreactivity, it was therefore not surprising to find a significant decrease in PRL immunostaining in the wasting 2- to 3-wk-old PTP{sigma}(-/-) mice (Fig. 1Go, D and E, and Fig. 3BGo). Immunostaining for all the other anterior pituitary hormones and transcription factors was normal in this cohort (Fig. 3BGo). Immunostaining of the PTP{sigma}(-/-) adult anterior pituitaries for all the above hormones and transcription factors was also normal (Fig. 3CGo).

To determine whether the significantly decreased immunopositivity for GH or PRL in the young knockout animals was attributable to apoptosis of somatotrophs and lactotrophs, we performed TUNEL assays (DNA nick end labeling of tissue sections). In neither the GH-deficient PTP{sigma}(-/-) neonates, nor the PRL- depleted 2- to 3-wk-old PTP{sigma}(-/-) mice, was apoptosis detected in the pituitaries (data not shown).

Pit-1 is a transcription factor that is necessary for the development of the somatotroph stem cell and its subsequent differentiation into thyrotrophs, lactotrophs, and somatotrophs (12, 14). To determine whether the decreased GH and PRL immunoreactivity was secondary to a reduction in Pit-1 expression and the subsequent failure of somatotroph/lactotroph lineage development, we examined Pit-1 immunoreactivity. The relative number of Pit-1-positive cells was not reduced in those PTP{sigma}(-/-) mice, which showed markedly decreased GH, and absent PRL immunoreactivity (Fig. 4Go, A and B). Double immunostaining for Pit-1 and TSH in these PTP{sigma}(-/-) cohorts revealed a population of cells immunoreactive for Pit-1 only, showing that not all Pit-1-positive cells are thyrotrophs (Fig. 4CGo). This suggests that in the PTP{sigma}(-/-) neonate and the 2- to 3-wk-old wasting PTP{sigma}(-/-) mice there are committed somatotrophs and/or lactotrophs incapable of hormone expression. Alternatively, arrest in pituitary differentiation may occur just before the emergence of somatotrophs and lactotrophs in these PTP{sigma}(-/-) mice.



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Figure 4. Pit-1 Immunostaining

A, Neonatal and (B) a wasting 3-wk-old PTP{sigma}(-/-) adenohypophysis. Pit-1 immunoreactivity is intact (dark brown) in both cohorts of mice. C, Double immunostaining of a wasting 3-wk-old PTP{sigma}(-/-) adenohypophysis with anti-Pit-1 and anti-TSH antibodies. The Pit-1 immunoreactivity is represented by the dark purple nuclear staining. The TSH immunoreactivity is respresented by the red cytosolic staining. Thyrotrophs are immunoreactive for both Pit-1 and TSH. A population of Pit-1-positive, TSH-negative cells represents cells destined for the somatotroph/lactotroph lineage. Magnification: panels A and B, 400x; panel C, 350x.

 
Neonatal Blood Glucose
The decreased GH levels in the PTP{sigma}(-/-) newborn led us to hypothesize that hypoglycemia may contribute to the high neonatal mortality. BG levels were therefore determined for a series of neonates, and the pituitaries were immunostained for GH. Severe hypoglycemia (BG = 0.7 ± 0.0 mmol/liter) was evident in PTP{sigma}(-/-) neonates with markedly decreased immunoreactivity for GH compared with PTP{sigma}(+/+ or +/-) sibling controls (BG = 3.8 ± 0.1 mmol/liter).

To prove that the GH deficiency was a major contributing factor to the neonatal mortality, we administered human GH (Humatrope, Eli Lilly & Co., Indianapolis, IN) to a series of newborn litters (Fig. 5AGo). Survival analysis demonstrated that the PTP{sigma}(-/-) neonatal mortality rate was significantly reduced when animals were treated with GH compared with saline control, or when left untreated (Fig. 5BGo). Histological analysis of PTP{sigma}(-/-) pituitaries at the completion of therapy (5 d of age) revealed normal GH immunoreactivity (data not shown). BG levels of Humatrope-treated PTP{sigma}(-/-) neonates that completed the treatment course were also normal (5.6 ± 1.0 mmol/liter). Therefore, it appears that PTP{sigma}(-/-) neonatal death is secondary to hypoglycemia, which is contributed to by GH deficiency.



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Figure 5. Kaplan-Meier Survival Curves of GH Treated PTP{sigma}(-/-) Newborn Mice

A, Newborn litters were treated with GH (Humatrope) 0.06 mg/kg/d or saline ip daily for 4 d, or received no treatment. B, Kaplan Meier survival analysis revealed a significant decrease in neonatal mortality and improvement in long-term survival in GH-treated, as compared with saline-treated PTP{sigma}(-/-) mice or PTP{sigma}(-/-) mice receiving no therapy. P < 0.05.

 
Pancreas Histology And Immunohistochemistry
Histological assessment of the pancreas revealed hypoplasia of islets in the PTP{sigma}(-/-) neonates and in the wasting 2- to 3-wk-old PTP{sigma}(-/-) mice (Fig. 6Go, A and B). Lac Z staining of the pancreas revealed the expression of PTP{sigma} in the islets (Fig. 2Go, D and E).



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Figure 6. H & E Staining of the Pancreas

A wasting 3-wk-old PTP{sigma}(-/-) mouse (panel A) demonstrates islet hypoplasia in comparison to the islet of a 3-wk-old PTP{sigma}(+/+) control (panel B). Insulin immunostaining (dark brown) of the islets of a wasting 3-wk-old PTP{sigma}(-/-) mouse (panel C) and a 3-wk-old PTP{sigma}(+/+) control (panel D) demonstrates a decrease in the number of insulin-immunoreactive cells in the PTP{sigma}(-/-) animal (magnification, 250x).

 
The islets of the endocrine pancreas are composed of four cell types, A, B, PP, and D, that synthesize and secrete glucagon, insulin, pancreatic polypeptide (PP), and somatostatin, respectively. The insulin-containing cell mass was reduced in the hypoplastic islets of the wasting 2- to 3-wk-old PTP{sigma}(-/-) animal (Fig. 6Go, C and D, Fig. 7BGo), while glucagon immunoreactivity was intact in this cohort. Serum insulin levels were also significantly reduced in this cohort of PTP{sigma}(-/-) mice (0.08 ± 0.12 ng/ml) compared with PTP{sigma}(+/+) controls (0.492 ± 0.23 ng/ml). Despite their small size, immunoreactive cells containing insulin and glucagon were present in normal proportions in the islets of neonatal PTP{sigma}(-/-) mice (Fig. 7AGo). Both insulin and glucagon immunoreactivities were normal in the adult PTP{sigma}(-/-) animals (Fig. 7CGo). Lac Z and insulin costaining revealed the expression of PTP{sigma} in the B cell of the islet of newborn mice (Fig. 2FGo).



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Figure 7. Morphometric Analyses of Immunostaining For Pancreatic Hormones in (A) Newborn, (B) Wasting 2- to 3-wk-Old, and (C) Adult PTP{sigma}(-/-) and Control PTP{sigma}(+/+ and +/-) Mice

The mean hormone-immunopositive cellular area is expressed as a percentage of the total islet cellular area ± SD (n = 2 to 5 mice per genotype per hormone). Insulin immunoreactivity is significantly decreased (P < 0.05) in the 2- to 3-wk-old cohort of PTP{sigma}(-/-) mice.

 
Although there was marked variability in PP and somatostatin immunoreactivity in the islets of all three cohorts of PTP{sigma}(-/-) mice, there was no significant difference in the immunopositive cell area compared with PTP{sigma}(+/+) controls (Fig. 7Go, A, B, and C).

To determine whether the reduction in islet mass was due to apoptosis, we performed a TUNEL assay on representative animals from each of the PTP{sigma}(-/-) cohorts. There was no increased rate of apoptosis in the PTP{sigma}(-/-) mice (data not shown).

To determine whether the islet hypoplasia and decreased insulin in the PTP{sigma}(-/-) mice were the result of GH deficiency, we assessed islet histology and insulin immunoreactivity in the GH-treated animals. Both insulin immunoreactivity and islet morphology normalized in the PTP{sigma}(-/-) newborns and the 2- to 3-wk-old wasting PTP{sigma}(-/-) mice receiving GH (data not shown).

BG And Serum Bicarbonate Levels in 2- to 3-wk-Old Animals
In view of the hypoinsulinemia noted in the wasting 2- to 3-wk-old PTP{sigma}(-/-) mice, we speculated that they might be succumbing to diabetic ketoacidosis or a hyperglycemic hyperosmolar state. We therefore measured BG levels and serum bicarbonate levels via arterial blood gas analysis. Serum ketone levels, anion gap, and serum osmolality could not be determined because of an insufficient volume of serum available from the wasting PTP{sigma}(-/-) mice. BG levels of the PTP{sigma}(-/-) mice were 4.6 ± 0.40 mmol/liter; BG levels of the PTP{sigma}(+/+) controls were 9.0 ± 1.48 mmol/liter. Serum bicarbonate levels of the PTP{sigma}(-/-) mice were 19.2 ± 2.38 meq/liter and were not significantly different from PTP{sigma}(+/+) controls (20.7 ± 2.02 meq/liter). Therefore, despite the hypoinsulinemia, enough insulin was present to prevent hyperglycemia and the initiation of ketogenesis.

The BG level of the PTP{sigma}(+/+) mice represents a random fed level. The wasting 2- to 3-wk-old PTP{sigma}(-/-) mice had no milk in the gut at autopsy. Thus, the BG level of the PTP{sigma}(-/-) mice in this experiment represents a fasting level and is therefore lower than that of the fed controls.

Gut Histology And Immunohistochemistry
On hematoxylin-eosin (H & E) section, mild villous trophy was occasionally evident in the small and large intestine of the PTP{sigma}(-/-) mice (data not shown). Immunohistochemistry revealed significant variability in the expression of gut hormones by the enteroendocrine system (Table 1Go). Most profoundly affected were the wasting 2- to 3-wk-old PTP{sigma}(-/-) mice. This entire cohort (100%) exhibited a decrease in cells containing serotonin, some with total lack of immunoreactivity for this gut hormone (Fig. 8Go). Secretin, gastrin, somatostatin, glucagon, and cholecystokinin (CCK) were decreased in 50% to 75% of mice. Peptide YY (PYY) was the only hormone with intact staining in the 2- to 3-wk-old wasting PTP{sigma}(-/-) cohort.


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Table 1. PTP{sigma}(-/-) Enteroendocrine Immunohistochemistry

 


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Figure 8. Serotonin Immunostaining of the Gut

A wasting 3-wk-old PTP{sigma}(-/-) (panel A) and PTP{sigma}(+/+) control animal (panel B) demonstrates almost total absence of serotonin-immunoreactive cells (dark brown) in the knockout animal. Magnification, 200x.

 
Newborn PTP{sigma}(-/-) mice revealed decreased secretin, gastrin, serotonin, glucagon, and somatostatin immunoreactivity in 20–60% of mice. CCK and PYY immunostaining were intact (Table 1Go). Adult PTP{sigma}(-/-) immunostaining was normal with the exception of somatostatin and glucagon (Table 1Go). Glucagon-like peptide-1 (GLP-1) and glucagon-like peptide (GLP-2) immunoreactivity paralleled glucagon immunoreactivity.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The process of adenohypophyseal development and cell lineage differentiation follows a highly specific pattern and temporal sequence, which is dictated by a number of transcription-regulatory proteins (15, 16, 17, 18). These same factors then act alone, or in concert with one another and putative repressors, to initiate expression of the hormone product of the mature anterior pituitary cell. All adenohypophyseal endocrine cells are thought to derive from a common stem cell in Rathke’s pouch (12, 15, 16). Three separate pathways of cytodifferentiation have been tentatively delineated, one resulting in corticotrophs, another gonadotrophs, and the third, a somatotroph stem cell that ultimately gives rise to somatotrophs, mammosomatotrophs, lactotrophs, and thyrotrophs (12, 19, 20, 21, 22) (Fig. 9Go). These lineages do not develop concurrently, but rather sequentially. In the mouse the corticotrophs are the first adenohypophyseal cell type to emerge at embryonic day 15, and the mature PRL-secreting lactotrophs are the last, appearing shortly after birth (12, 15). It is the somatotroph/lactotroph lineage that has been affected by the loss of the PTP{sigma} protein.



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Figure 9. Cytodifferentiation of the Adenohypophyseal Lineages

All anterior pituitary cell types derive from a Rathke’s pouch stem cell. Three separate pathways sequentially arise, resulting in corticotrophs, gonadotrophs, and a somatotroph stem cell that ultimately differentiates into somatotrophs, lactotrophs, and thyrotrophs. Pit-1 expression is essential for the differentiation of the somatotrophs, lactotrophs, and thyrotrophs. Loss of PTP{sigma} results in abnormal development of the somatotroph/lactotroph lineage.

 
The differentiation of the somatotroph stem cell from Rathke’s pouch stem cell is determined by the expression of the homeodomain protein Pit-1. Some evidence suggests that in the absence of any other transcription factors, the somatotroph stem cell will retain somatotroph morphology and function (12). However, other investigations have found that Pit-1 expression alone is insufficient to activate the GH gene, indicating that cooperative factor(s) are required for GH expression (23, 24, 25). Potential candidates include Zn-15 and Zn-16, zinc finger transcription factors that bind the GH promoter (25, 26). GHRF is a key regulator of the pulsatile release of GH from the pituitary but is not essential for somatotroph cytodifferentiation. In the absence of GHRF, e.g. in anencephalics, GH-secreting somatotrophs still develop (27).

It is well established that Pit-1, while necessary, is not sufficient for the terminal differentiation of thyrotrophs, mammosomatotrophs, or lactotrophs. For example, coexpression of the transcription factor GATA-2 along with Pit-1 is required for differentiation of the thyrotroph (28, 29). In cooperation with Pit-1, estrogen binding to its receptor leads to weak activation of the PRL gene and induces differentiation of the mammosomatotroph (30). Members of the ETS transcription factor family are also involved in the synergistic activation of the PRL promoter when coexpressed with Pit-1 (31, 32). The current model of cytodifferentiation hypothesizes that additional repressor(s) acting on the mammosomatotroph down-regulate GH gene transcription, thus permitting terminal differentiation of the mature lactotroph (12).

GH and PRL immunostaining was significantly diminished in PTP{sigma}(-/-) newborns and 2- to 3-wk-old mice, respectively. All animals revealed normal Pit-1 and TSH immunoreactivity. These results suggest appropriate adenohypophyseal development to the emergence of the somatotroph stem cell in PTP{sigma}(-/-) mice. Possible explanations for the subsequent downstream abnormalities of the somatotroph and lactotroph include the divergence of the majority of Pit-1-positive cells to a thyrotroph phenotype, leaving a diminished number to retain the somatotroph phenotype, and none to proceed to a mature lactotroph. However, coimmunostaining for Pit-1 and TSH proved this hypothesis wrong by demonstrating a population of cells immunoreactive for Pit-1 but not for TSH. Selective depletion of the Pit-1-positive TSH-negative cells via apoptosis was not evident, as the TUNEL assay was negative. Appropriate specification of the somatotrophs and lactotrophs, but delayed expansion, could account for the observed phenotype. Normally, somatotroph terminal differentiation is complete by the end of gestation, and proliferation begins before birth. Terminal differentiation, maturation, and expansion of the lactotroph population all occur postnatally over hours to days. In the PTP{sigma}(-/-) mouse, developmental delay of the adenohypophysis could result in terminal differentiation and expansion of both somatotrophs and lactotrophs postnatally. However, eventually all adenohypophyseal cell lines would emerge. This hypothesis is supported by the persistence of a prominent embryonic cleft in the newborn PTP{sigma}(-/-) mice, normal immunoreactivity for all anterior pituitary hormones in the PTP{sigma}(-/-) mice that survive to adulthood, and the presence of developmental delay in the peripheral nervous system of PTP{sigma}(-/-) mice (10). In addition, all surviving GH-treated neonates revealed normal pituitary GH immunoreactivity at 5 d of age, implying that maturation and proliferation of the somatotrophs will occur postnatally if the animal survives.

An alternative explanation that must be considered, however, is that the terminal cytodifferentiation of the somatotroph and lactotroph is indeed intrinsically impaired in the newborn and wasting 2- to 3-wk-old PTP{sigma}(-/-) mice. We do not know the reason for the presence of three distinct phenotypic cohorts of the PTP{sigma}(-/-) mice. Possible explanations for this phenotypic diversity include variability of PTP{sigma} penetrance, effects of modifier genes, and varying levels of compensatory contribution from other related genes (e.g. the PTP{sigma} close relative LAR, which is also expressed in the pituitary) (8). Any of these events could potentially alter development of the somatotroph/lactotroph lineage in the affected cohorts.

A second PTP{sigma} knockout mouse, generated by the targeted disruption of the intracellular catalytic domains by Tremblay and colleagues (11), demonstrated similar adenohypophyseal hypoplasia. While GH immunoreactivity or serum levels were not measured in these mice, IGF-I levels were found to be significantly decreased by 25–40% in the PTP{sigma}(-/-) animals compared with PTP{sigma}(+/+ and +/-) controls. This result is in agreement with the decreases in GH immunoreactivity noted in our PTP{sigma}(-/-) animals. Similarly, the Tremblay knockout mice demonstrated a decrease in PRL immunoreactivity. However, in contrast to our findings, decreases in the percentage of TSH, LH, and FSH and increases in the percentage of ACTH-immunopositive cells were noted in their PTP{sigma}(-/-) mice. These differences in phenotype may be due to residual expression of the extracellular domain of PTP{sigma} in those mice. Analysis of the pancreas, adrenals, NEBs, and the enteroendocrine gut was not reported in the Tremblay mice.

The substantial decrease in GH immunoreactivity in newborn PTP{sigma}(-/-) animals led us to speculate that the high neonatal mortality rate may be secondary to hypoglycemia. GH is one of the counterregulatory hormones involved in glucose homeostasis. In times of fasting, GH, in concert with glucagon, epinephrine, and ACTH, maintains BG levels via stimulation of glycogenolysis, ketogenesis, and gluconeogenesis. The gluconeogenic and ketogenic pathways do not mature in the human newborn liver until 12–24 h of age (33). Thus, the neonate is susceptible to transient hypoglycemia. Further challenge resulting from deficiencies in one of the counterregulatory hormones can result in profound, sustained hypoglycemia, as seen in panhypopituitarism and isolated GH deficiency in the human newborn. BG levels in PTP{sigma}(-/-) newborns with severely diminished GH immunostaining were less than 1.0 mmol/liter which, if sustained, is incompatible with life. ACTH immunoreactivity was normal in all mice. Intraperitoneal administration of GH to newborn PTP{sigma}(-/-) mice significantly decreased the neonatal mortality rate. Treated PTP{sigma}(-/-) mice killed at 5 d of age demonstrated normal GH immunoreactivity and BG levels. This suggests that the neonatal mortality was due to profound hypoglycemia induced, at least in part, by GH insufficiency. The rescue achieved with GH administration, however, was not 100%. Those neonates that were administered GH but still died may have received inadequate GH supplementation, or a second phenomenon may have contributed to their death. Unfortunately, the pituitaries and pancreases of the GH-treated PTP{sigma}(-/-) newborns that failed therapy were unavailable for postmortem analysis, due to the fact that the mother expediently disposes of the body.

Histological assessment of the endocrine pancreas revealed islet hypoplasia in the newborn and wasting 2- to 3-wk-old PTP{sigma}(-/-) animals. While insulin immunostaining was intact in the small islets of the neonates, there was a marked decrease in insulin immunoreactivity in the islets of the 2- to 3-wk-old PTP{sigma}(-/-) animals. Measurement of serum insulin levels supported the immunohistopathology. The 2- to 3-wk-old knockout animals had extremely low insulin levels that were significantly decreased compared with controls. In the healthy adult PTP{sigma}(-/-) animals, islet morphology and insulin immunostaining were normal. Throughout all three cohorts of PTP(-/-) animals, marked variability of PP and somatostatin immunostaining was evident. Glucagon immunoreactivity was intact in all animals.

While the islet is composed of four cell types, which include insulin-producing B cells, glucagon-producing A cells, somatostatin-producing D cells, and pancreatic polypeptide-producing PP cells, its principal component is the B cell (34). Rapid growth of the endocrine pancreas occurs in the late fetal and early weeks of life, after which growth of the exocrine pancreas ensues (35). This makes the finding of hypoplasia of the islets at 2–3 wk of age even more striking, as the endocrine/exocrine tissue ratio should be at its peak at that time. TUNEL assay revealed that loss of islet mass was not secondary to apoptosis, but was indeed due to true hypoplasia. Numerous proteins, including differentiation factors such as activin, classic growth factors such as hepatocyte growth factor, and hormones such as GH and PRL, have been variably implicated in islet cell differentiation and proliferation, as well as activation of expression of insulin by the B cell (35, 36, 37, 38, 39, 40). In rats bearing GH- and PRL-producing tumors, and human GH-expressing transgenic mice, B cell mass increases and insulin synthesis and secretion are enhanced (40, 41). Thus, while there are many possible candidates to explain the islet hypoplasia and decreased insulin immunoreactivity observed in the PTP{sigma}(-/-) mice, the most likely contributory cause is GH deficiency. Indeed, islet morphology and insulin immunoreactivity significantly improved in the PTP{sigma}(-/-) mice treated with GH. Therefore, islet hypoplasia and decreased insulin immunoreactivity are likely secondary to the pituitary abnormalities noted in the PTP{sigma}(-/-) animals.

The low insulin levels and clinical picture of the wasting 2- to 3-wk-old PTP{sigma}(-/-) animal suggested that diabetic ketoacidosis or a hyperglycemic hyperosmolar state may be the cause of death in this cohort. However, hyperglycemia was not evident in these mice, and serum bicarbonate levels were not significantly different from PTP{sigma}(+/+) controls. Despite the hypoinsulinemia, adequate amounts were present for the prevention of ketogenesis. Therefore, ketoacidosis was not responsible for the death of our PTP{sigma} knockout mice.

The PTP{sigma}(-/-) cohort lost at 2–3 wk of age from a wasting syndrome revealed severe loss of serotonin-immunopositive cells in the proximal gut, compared with control animals. With the exception of this consistent finding, the PTP{sigma}(-/-) mice displayed a significant heterogeneity between and within cohorts with respect to the level of expression of the various gut peptide hormones. Differentiation of the intestinal epithelium from a pluripotent stem cell to a terminally committed enteroendocrine cell is a highly regulated and complex process (42, 43, 44). In view of this complexity, it is difficult to hypothesize the mechanism(s) by which lack of PTP{sigma} would translate into a specific loss of serotonin-producing cells in the gut of a cohort of animals destined to die, and a variable decrease in the expression of other hormones in the other two cohorts of PTP{sigma}(-/-) animals. We can speculate that the loss of serotonin expression leading to abnormal gut motility, combined with impaired absorptive capacity due to mild villous atrophy, may be responsible for the wasting syndrome noted in the 2- to 3-wk-old knockout animals.

Overall, the mechanism by which the lack of PTP{sigma} results in the complex phenotype observed is not fully understood. While abl and disabled were genetically identified as collaborative proteins in the developing Drosophila nervous system, ligand(s) and substrate(s) in the mammal have yet to be identified. The cell adhesion molecule-like ectodomain and tyrosine phosphatase enzymatic activity likely engage PTP{sigma} in a number of signaling networks that could likely affect development and function of the endocrine system.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PTP{sigma} Knockout Mice and Experimental Cohorts
Inactivation of the full-length PTP{sigma} gene was performed and mice were characterized as previously described (10). The PTP{sigma}(+/-) mice are phenotypically indistinguishable from PTP{sigma}(+/+) mice. The PTP{sigma}(-/-) animals consist of three cohorts: 1) most (60%) die as neonates within hours of birth; 2) 37.5% demonstrate growth retardation and succumb to a wasting syndrome by 2 to 3 wk of age; and 3) 2.5% survive to adulthood.

All animal experimentation was conducted in accordance with accepted standards of humane animal care.

Histological Analysis
Representative animals from the three cohorts of PTP{sigma}(-/-) and control PTP{sigma}(+/-),(+/+) mice were killed, and examined by complete autopsy. Neuroendocrine tissues, including the pituitary, pancreas, gut, adrenals, as well as lungs, were fixed and embedded in paraffin, sectioned (5 µm), and stained with H & E for histological evaluation. The pituitaries were also stained with the Gordon-Sweet silver method to demonstrate the reticulin fiber network. Thyroid and parathyroid were not systematically examined due to difficulty of reliably harvesting these tissues from the mice.

Immunocytochemical stains to localize hormones and other cellular antigens were performed using the avidin- biotin-peroxidase complex technique and visualized using 3,3'-diaminobenzidine (DAB). For pituitary, the duration of exposure to primary antiserum was 30 min at room temperature. The primary polyclonal antisera were directed against the following antigens and were used at the specified dilutions: ACTH (DAKO Corp., Carpinteria, CA); rat GH, PRL, ßTSH, ßFSH, and LH [all donated by the National Hormone and Pituitary Program (NHPP), National Institute of Diabetes, Digestive and Kidney Disease (NIDDK), Bethesda, MD], at 1:2,000, 1:15,000, 1:3,000, 1:1,000, 1:500, and 1:400, respectively. Nuclear transcription factors Pit-1, SF-1, and ER were localized after microwave antigen retrieval and detected with the avidin-biotin-peroxidase Elite technique (Vector Laboratories, Inc., Burlingame, CA). For Pit-1 a polyclonal antiserum raised against a synthetic peptide corresponding to the epitope depicted by residues 214–230 of rat pit-1 (BabCo, Berkeley Antibody Company, Richmond, CA) was used at 1:600 for 1 h at room temperature. For ER a monoclonal antisera (Novocastra, Newcastle upon Tyne, UK), was used at a 1:70 dilution for 1 h. A polyclonal antisera against SF-1 (Upstate Biotechnology, Inc., Lake Placid, NY) was used at a 1:600 dilution for 1 h. For colocalization of nuclear transcription factors and cytoplasmic hormones, the nuclear stain was performed as described and detected using cobalt DAB. The cytoplasmic antigen was identified using a peroxidase-conjugated secondary antibody method to avoid cross-reaction and visualized with DAB. For gut and pancreas, primary antisera and antibodies were directed against the following antigens and were used at the specified dilutions and incubation durations: insulin (monoclonal antibody from BioGenex Laboratories, Inc., San Ramon, CA) 1:40 for 30 min; glucagon (polyclonal antiserum from Immunon, Pittsburg, PA) 1:200 for 30 min: GLP-1 (polyclonal antiserum donated by Dr. D. J. Drucker, Toronto, Ontario, Canada) 1:1,500 for 30 min; GLP-2 (polyclonal antiserum donated by Dr. D. J. Drucker) 1:2,500 for 30 min; peptide YY (PYY) (polyclonal antiserum from Peninsula Laboratories, Inc., Belmont, CA) 1:2,000 for 30 min; somatostatin (polyclonal antiserum from DAKO Corp.) prediluted preparation further diluted 1:40 overnight; pancreatic polypeptide (PP) (polyclonal antiserum from DAKO Corp.) 1:6,000 for 30 min, CCK (polyclonal antiserum from Serotec, Oxford, UK) 1:1,000 for 30 min; serotonin (monoclonal antibody from DAKO Corp.) 1:50 for 60 min; secretin (polyclonal antiserum from Biogenex Laboratories, Inc.) prediluted preparation further diluted 1:5 overnight; gastrin (polyclonal antiserum from Zymed, San Francisco, CA) 1:150 for 60 min. For the lungs, calcitonin gene-related peptide, a polyclonal antisera (Chemicon, Temecula, CA), was used at a dilution of 1:400 overnight at 4 C. Appropriate positive and negative (omission of the primary antibody) controls were performed in each case.

Morphometric Analysis
The hormone content of representative pituitary glands or pancreatic islets was quantified using MCID software (Imaging Research, Inc., St. Catherine’s, Canada) (n = 2 to 5 mice/hormone/cohort). The area of immunopositive anterior pituitary cells was determined and expressed as a percentage of the total adenohypophyseal area. Representative islets were assessed for determination of pancreatic hormone content. The area of immunopositive cells was determined and expressed as a percentage of islet area.

An experienced endocrine pathologist, blinded to the PTP{sigma} mouse genotype and cohort, graded islet size as being normal, increased, or decreased. Due to the heterogeneity of islet distribution throughout the pancreas, MCID quantitation of total islet area relative to total pancreatic area was not performed as the entire pancreas could not realistically be scanned for analysis. The heterogeneous distribution of islets precluded selection of a representative area and therefore any attempts at quantitation would have been biased and inaccurate. Similarly, due to the large lengths and surface area of small and large bowel, immunopositivity for the enteroendocrine hormones was assessed by an experienced pathologist, blinded to mouse genotype or cohort, and graded as being normal, increased, or decreased.

TUNEL Assay
Nuclei of tissue sections were stripped from proteins by incubation with 20 µg/ml proteinase K (Sigma, St. Louis, MO) for 15 min at room temperature, and the slides were then washed in double distilled water (DDW) for 2 min (x4). Endogenous peroxidase was inactivated by covering the sections with 2% H2O2 for 5 min at room temperature. The sections were rinsed with DDW, and immersed in TDT buffer (Oncor) (30 mM Trizma base, pH 7.2, 140 mM sodium cacodylate, 1 mM cobalt chloride). TDT (0.3 equivalent units/µl) and biotinylated dUTP in TDT buffer (Oncor, Gaithersburg, MD) were added to cover the sections and then incubated in humid atmosphere at 37 C for 60 min. The reaction was terminated by transferring the slides to TB buffer (300 mM sodium chloride, 30 mM sodium citrate) for 15 min at room temperature. The sections were rinsed with DDW, covered with 2% aqueous solution of BSA for 10 min at room temperature, rinsed in DDW, and immersed in PBS for 5 min. The sections were covered with streptavidin peroxidase, diluted 1:10–1:20 in water, incubated for 30 min at 37 C, washed in DDW, immersed for 5 min in PBS, and stained with DAB for about 30 min at 37 C.

Lac Z Staining
Animals were killed and the pituitaries and pancreas were harvested and fixed in a 0.1 M sodium phosphate buffer (pH 7.9) containing 1% formaldehyde, 0.1% glutaraldehyde, 2 mM MgCl2, and 5 mM EGTA for 6 h. Organs were washed for 2 h with four exchanges of a wash buffer (2 mM MgCl2, 0.01% deoxycholate, 0.02% Nonidet P-40 in 0.1 M sodium phosphate buffer, pH 7.9) at room temperature. The tissue was then incubated in PBS containing 5 mM ferricyanide, 5 mM ferrocyanide, 2 mM MgCl2 and X-gal (Roche, Indianapolis, IN) 0.1 mg/ml overnight at 37 C. Tissues were subsequently rinsed in 70% EtOH, paraffin embedded, and sectioned.

Blood and Serum Chemistry
BG levels were determined using the One Touch Glucometer (Johnson & Johnson, New Brunswick, NJ) on venous blood obtained via tail clip (older mice) and decapitation (newborn). Arterial blood for blood gas analysis was obtained via cardiac puncture on mice anesthetized with isoflurane 2.5%. Samples were collected in a heparinized syringe, transported on ice, and immediately analyzed on a Nova Stat Profile Plus 9 (Nova Medical, Wakefield, MA). Serum insulin levels were measured with the Rat Insulin ELISA Kit (Crystal Chem, Inc., Chicago, IL) according to manufacturer instructions, but with two modifications. Purified mouse insulin (Crystal Chem, Inc.) instead of rat, was used to generate the standard curve. Serum samples with insulin levels below the sensitivity of the assay were spiked with a known quantity of purified mouse insulin, to ensure absorbance of the sample fell within the exponential portion of the standard curve. Serum GH levels were assessed by Western blot analysis. Fifty micrograms of serum protein were separated by 12% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-GH antibodies at a dilution of 1:5,000 (polyclonal antisera donated by Dr. S. Ezzat, Toronto, Ontario, Canada). Relative band intensities were quantified by charge coupled device camera detection of enhanced chemiluminescence and the serum GH levels of PTP{sigma}(-/-) mice expressed as a percentage of control PTP{sigma}(+/+) littermates. Quantitation of serum GH levels by RIA or ELISA was not possible due to the small and limiting volumes of PTP{sigma}(-/-) sera attainable.

All statistic analyses are represented as mean ± SD.

GH Replacement Therapy
This experiment was designed as a two-phase study using human GH, as murine GH is not available for commercial use. In the first phase recombinant human GH (Humatrope) was administered daily to all animals within newborn litters for 4 d, at a dose of 0.06 mg/kg/d ip. Control litters were administered saline daily ip for 4 d or received no treatment. Innate survival of animals was monitored and mortality was documented. Data were subjected to Kaplan Meier Survival Analysis for determination of statistical significance.

After completion of survival analyses (phase 1), a phase 2 study was initiated to determine the effects of GH rescue on the pituitary and pancreatic pathology in the PTP{sigma}(-/-) mice. Litters were again treated with Humatrope, saline, or received no treatment as described above. A portion of PTP{sigma}(-/-) mice that completed treatment were euthanized at 5 d of age, blood glucose was measured as previously outlined, and pituitaries were immunostained for GH and morphometric analysis as previously outlined. PTP{sigma}(-/-) wasting 2- to 3-wk-old mice were euthanized, and the pancreases were dissected for insulin immunostaining and morphometry as previously described.


    ACKNOWLEDGMENTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We wish to thank Kelvin So and Veronica Wong for assistance with the immunocytochemistry of the pituitary and the pulmonary neuroendocrine bodies; Trudey Nicklee and Jennifer Roo for assistance with the morphometric analyses; and Susie Tsai for performing the TUNEL assays. We would also like to thank A. Griffin, D. Stephens, and Dr. L. Doering for his guidance regarding the Lac Z staining.


    FOOTNOTES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
This work was supported by grants from the Canadian Institute of Health Research (CIHR) (MGP-15274) to D.R. and S.A. D.R. is a recipient of a CIHR Scientist Award, and J.B. is supported by a CIHR Fellowship.

Abbreviations: BG, Blood glucose; CCK, cholecystokinin; DAB, 3,3'-diaminobenzidine; DDW, double distilled water; GLP, glucagon-like peptide; H & E, hematoxylin-eosin; LAR, leukocyte common antigen-related; NEBs, neuroendocrine bodies; Pit1, pituitary-specific POU homeodomain factor; PP, pancreatic polypeptide; PTP, protein tyrosine phosphatase; PYY, peptide YY; SF-1, steroidogenic factor 1; TUNEL, DNA nick end labeling of tissue sections.

Received for publication August 1, 2000. Accepted for publication September 21, 2001.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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