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Molecular Endocrinology 16(1):155–169
Copyright © 2002 by The Endocrine Society
Pituitary, Pancreatic and Gut Neuroendocrine
Defects in Protein Tyrosine PhosphataseSigma-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
The expression of receptor protein tyrosine phosphatase sigma (PTP␴) is developmentally regulated
in neuronal and neuroendocrine tissues. We have
previously shown that mice deficient in PTP␴ 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␴ on
pituitary, pancreas and enteroendocrine cytodifferentiation, hormone production, and development.
The adenohypophyses of PTP␴(ⴚ/ⴚ) 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 ex-
pression of GH and PRL was not associated with
apoptosis of somatotrophs or lactotrophs. Pit-1positive 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␴(ⴚ/ⴚ) neonate and accordingly, ip administration of GH
rescued the PTP␴(ⴚ/ⴚ) neonate and normalized
the blood glucose. These data indicate that PTP␴
plays a major role in differentiation and development of the neuroendocrine system. (Molecular
Endocrinology 16: 155–169, 2002)
ROTEIN TYROSINE PHOSPHATASE sigma (PTP␴)
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␴ is
highly developmentally regulated in the central and peripheral nervous systems, pituitary, and epithelial tissues,
suggesting a critical role in development (5–9). 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␴ knockout mice. These mice demonstrate in-
creased 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␴ in pituitary, pancreas and enteroendocrine cytodifferentiation, and hormone production. Our results show
that PTP␴ is specifically required for the development of
the somatotroph/lactotroph lineage of the anterior pituitary. GH and PRL production is impaired in PTP␴(⫺/⫺)
mice. The GH deficiency is associated with hypoglycemia and death in PTP␴(⫺/⫺) neonates. Intraperitoneal
administration of GH normalizes blood glucose (BG) and
rescues the PTP␴(⫺/⫺) 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␴(⫺/⫺) mice also exhibit marked abnormalities in the
expression of the gut enteroendocrine hormones. These
data indicate that PTP␴ plays a major role in differentiation and development of the neuroendocrine system.
P
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.
RESULTS
The PTP␴(⫺/⫺) animals consist of three cohorts: 1)
60% die as neonates within hours of birth; 2) 37.5%
155
156
Mol Endocrinol, January 2002, 16(1):155–169
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␴(⫹/⫹) or PTP␴(⫹/⫺) littermates
(10).
We have analyzed neuroendocrine tissues in PTP␴
knockout mice. The adrenal medulla and pulmonary
neuroendocrine bodies (NEBs) appeared normal in
PTP␴(⫺/⫺), PTP␴(⫹/⫺), and PTP␴(⫹/⫹) 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␴(⫺/⫺)
animals. As we previously reported, in many of the
PTP␴(⫺/⫺) neonates the posterior pituitary was also
hypoplastic (10). A prominent intermediate cleft was
still evident in all PTP␴(⫺/⫺) newborns, as compared
with wild type (WT) controls (Fig. 1, A and B). The cleft
forms during embryonic development of the adenohypophysis but by birth has become negligible. Its
marked persistence in the PTP␴(⫺/⫺) neonate suggests developmental delay of the adenohypophysis.
As the knockout cassette replaces PTP␴ with the
␤-galactosidase gene, we analyzed the pattern of PTP␴
expression by Lac Z staining. Lac Z staining of newborn
PTP␴(⫺/⫺) mice demonstrates the expression pattern of
PTP␴ in the anterior and intermediate pituitary (Fig. 2, A
and B). Lac Z expression is still evident in the wasting
2- to 3-wk-old PTP␴(⫺/⫺) animals, albeit significantly
decreased when compared with expression in the newborn mouse (data not shown). Lac Z is also expressed in
adult PTP␴(⫺/⫺) pituitaries (data not shown).
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␴(⫺/⫺)
animals revealed a decrease in the percentage of anterior pituitary cellular area immunopositive for GH
(Fig. 1, A and B, and Fig. 3A). Accordingly, there was
a variable but significant decrease in serum GH levels
in the knockout neonates (Fig. 1C). Lac Z and GH
costaining demonstrated the expression of PTP␴ in
the somatotrophs (Fig. 2C). The remaining hormones,
TSH, FSH, LH, and ACTH, were normal in both the
PTP␴(⫺/⫺) and (⫹/⫹) neonates (Fig. 3A). The ␣subunit of glycoprotein hormones and the transcription factors ER and steroidogenic factor 1 (SF-1) were
also normal in both the PTP␴(⫺/⫺) and (⫹/⫹) neonates (data not shown). As expected, PRL was not
expressed in the newborn mice because lactotroph
Batt et al. • Defects in PTP␴-Deficient Mice
terminal differentiation and maturation does not normally occur until hours to days postnatally.
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␴(⫺/⫺)
mice (Fig. 1, D and E, and Fig. 3B). Immunostaining for
all the other anterior pituitary hormones and transcription factors was normal in this cohort (Fig. 3B). Immunostaining of the PTP␴(⫺/⫺) adult anterior pituitaries
for all the above hormones and transcription factors
was also normal (Fig. 3C).
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␴(⫺/⫺) neonates, nor the PRLdepleted 2- to 3-wk-old PTP␴(⫺/⫺) 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␴(⫺/⫺) mice, which showed
markedly decreased GH, and absent PRL immunoreactivity (Fig. 4, A and B). Double immunostaining for
Pit-1 and TSH in these PTP␴(⫺/⫺) cohorts revealed a
population of cells immunoreactive for Pit-1 only,
showing that not all Pit-1-positive cells are thyrotrophs
(Fig. 4C). This suggests that in the PTP␴(⫺/⫺) neonate
and the 2- to 3-wk-old wasting PTP␴(⫺/⫺) 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␴(⫺/⫺) mice.
Neonatal Blood Glucose
The decreased GH levels in the PTP␴(⫺/⫺) 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␴(⫺/⫺) neonates with markedly decreased immunoreactivity for GH compared with PTP␴(⫹/⫹ 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., Indianap-
Batt et al. • Defects in PTP␴-Deficient Mice
Mol Endocrinol, January 2002, 16(1):155–169
157
Fig. 1. Immunostaining of the Adenohypophysis of a PTP␴(⫺/⫺) Neonate (A) and a PTP␴(⫹/⫹) Neonate (B) with Anti-GH
Antibody
The PTP␴(⫺/⫺) mouse demonstrates a significant decrease in the number of GH-immunoreactive cells (brown). In addition, the
intermediate cleft (arrowhead) is prominent in the PTP␴(⫺/⫺) neonate, compared with the PTP␴(⫹/⫹) neonate, suggesting
developmental delay of the adenohypophysis of the knockout animal (magnification, 200⫻). C, Western blot of serum GH of a
representative PTP␴(⫺/⫺) 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␴(⫺/⫺) 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␴(⫺/⫺) mouse and (E)
a PTP␴(⫹/⫹) sibling with anti-PRL antibodies (brown staining) demonstrates an absence of PRL immunoreactivity in the
PTP␴(⫺/⫺) mouse (magnification, 400⫻).
olis, IN) to a series of newborn litters (Fig. 5A). Survival
analysis demonstrated that the PTP␴(⫺/⫺) neonatal
mortality rate was significantly reduced when animals
were treated with GH compared with saline control, or
when left untreated (Fig. 5B). Histological analysis of
PTP␴(⫺/⫺) pituitaries at the completion of therapy (5 d
of age) revealed normal GH immunoreactivity (data not
shown). BG levels of Humatrope-treated PTP␴(⫺/⫺)
neonates that completed the treatment course were
also normal (5.6 ⫾ 1.0 mmol/liter). Therefore, it ap-
158 Mol Endocrinol, January 2002, 16(1):155–169
Batt et al. • Defects in PTP␴-Deficient Mice
Fig. 2. Lac Z Staining of Newborn (A) PTP␴(⫹/⫹) and (B) PTP␴(⫺/⫺) Pituitaries and (D) PTP␴(⫹/⫹) and (E) PTP␴(⫺/⫺)
Pancreases
The pattern of PTP␴ 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␴(⫺/⫺) mice. Costaining for (C) GH (red-brown) and Lac Z reveals cells staining both blue and
red-brown, which demonstrates that PTP␴ is expressed in somatotrophs. F, Costaining for insulin (red-brown) and Lac Z reveals
PTP␴ expression in B cells. Magnification: panels A and B, 100⫻; inset, 400⫻; panel C, 600⫻; panels D and E, 100⫻; inset, 400⫻;
panel F, 600⫻.
Batt et al. • Defects in PTP␴-Deficient Mice
Mol Endocrinol, January 2002, 16(1):155–169
159
Fig. 3. Morphometric Analyses of Immunostaining for Adenohypophyseal Hormones in (A) Newborn, (B) Wasting 2- to 3-wk-Old,
and (C) Adult PTP␴(⫺/⫺) and PTP␴(⫹/⫹ 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␴(⫺/⫺) mice compared with sibling PTP␴(⫹/⫹ or
⫹/⫺) controls (P ⬍ 0.05). PRL is significantly decreased in 2- to 3- wk-old PTP␴(⫺/⫺) mice compared with sibling controls
(P ⬍ 0.05).
pears that PTP␴(⫺/⫺) neonatal death is secondary to
hypoglycemia, which is contributed to by GH
deficiency.
Pancreas Histology And Immunohistochemistry
Histological assessment of the pancreas revealed hypoplasia of islets in the PTP␴(⫺/⫺) neonates and in the
wasting 2- to 3-wk-old PTP␴(⫺/⫺) mice (Fig. 6, A and
B). Lac Z staining of the pancreas revealed the expression of PTP␴ in the islets (Fig. 2, D and E).
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␴(⫺/⫺) animal (Fig. 6, C
and D, Fig. 7B), while glucagon immunoreactivity was
intact in this cohort. Serum insulin levels were also
significantly reduced in this cohort of PTP␴(⫺/⫺) mice
(0.08 ⫾ 0.12 ng/ml) compared with PTP␴(⫹/⫹) 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␴(⫺/⫺) mice (Fig. 7A). Both insulin and
160 Mol Endocrinol, January 2002, 16(1):155–169
Batt et al. • Defects in PTP␴-Deficient Mice
Fig. 4. Pit-1 Immunostaining
A, Neonatal and (B) a wasting 3-wk-old PTP␴(⫺/⫺) adenohypophysis. Pit-1 immunoreactivity is intact (dark brown) in both
cohorts of mice. C, Double immunostaining of a wasting 3-wk-old PTP␴(⫺/⫺) 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, 400⫻; panel
C, 350⫻.
glucagon immunoreactivities were normal in the adult
PTP␴(⫺/⫺) animals (Fig. 7C). Lac Z and insulin
costaining revealed the expression of PTP␴ in the B
cell of the islet of newborn mice (Fig. 2F).
Although there was marked variability in PP and
somatostatin immunoreactivity in the islets of all three
cohorts of PTP␴(⫺/⫺) mice, there was no significant
difference in the immunopositive cell area compared
with PTP␴(⫹/⫹) controls (Fig. 7, 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␴(⫺/⫺)
cohorts. There was no increased rate of apoptosis in
the PTP␴(⫺/⫺) mice (data not shown).
Batt et al. • Defects in PTP␴-Deficient Mice
Mol Endocrinol, January 2002, 16(1):155–169
161
Fig. 5. Kaplan-Meier Survival Curves of GH Treated PTP␴(⫺/⫺) 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␴(⫺/⫺) mice or PTP␴(⫺/⫺) mice receiving no therapy. P ⬍ 0.05.
To determine whether the islet hypoplasia and decreased insulin in the PTP␴(⫺/⫺) 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␴(⫺/⫺) newborns and the 2- to
3-wk-old wasting PTP␴(⫺/⫺) mice receiving GH (data
not shown).
BG And Serum Bicarbonate Levels in 2- to 3-wkOld Animals
In view of the hypoinsulinemia noted in the wasting 2to 3-wk-old PTP␴(⫺/⫺) 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␴(⫺/⫺) mice. BG levels of the
PTP␴(⫺/⫺) mice were 4.6 ⫾ 0.40 mmol/liter; BG levels
of the PTP␴(⫹/⫹) controls were 9.0 ⫾ 1.48 mmol/liter.
Serum bicarbonate levels of the PTP␴(⫺/⫺) mice were
19.2 ⫾ 2.38 meq/liter and were not significantly different from PTP␴(⫹/⫹) 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␴(⫹/⫹) mice represents
a random fed level. The wasting 2- to 3-wk-old
PTP␴(⫺/⫺) mice had no milk in the gut at autopsy.
Thus, the BG level of the PTP␴(⫺/⫺) 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␴(⫺/⫺) mice (data not shown). Immunohistochemistry revealed significant variability in the expres-
162
Mol Endocrinol, January 2002, 16(1):155–169
Batt et al. • Defects in PTP␴-Deficient Mice
Fig. 6. H & E Staining of the Pancreas
A wasting 3-wk-old PTP␴(⫺/⫺) mouse (panel A) demonstrates islet hypoplasia in comparison to the islet of a 3-wk-old
PTP␴(⫹/⫹) control (panel B). Insulin immunostaining (dark brown) of the islets of a wasting 3-wk-old PTP␴(⫺/⫺) mouse (panel
C) and a 3-wk-old PTP␴(⫹/⫹) control (panel D) demonstrates a decrease in the number of insulin-immunoreactive cells in the
PTP␴(⫺/⫺) animal (magnification, 250⫻).
sion of gut hormones by the enteroendocrine system
(Table 1). Most profoundly affected were the wasting 2to 3-wk-old PTP␴(⫺/⫺) mice. This entire cohort (100%)
exhibited a decrease in cells containing serotonin, some
with total lack of immunoreactivity for this gut hormone
(Fig. 8). 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␴(⫺/⫺) cohort.
Newborn PTP␴(⫺/⫺) mice revealed decreased
secretin, gastrin, serotonin, glucagon, and somatostatin immunoreactivity in 20–60% of mice. CCK
and PYY immunostaining were intact (Table 1). Adult
PTP␴(⫺/⫺) immunostaining was normal with the
exception of somatostatin and glucagon (Table 1).
Glucagon-like peptide-1 (GLP-1) and glucagonlike peptide (GLP-2) immunoreactivity paralleled
glucagon immunoreactivity.
Batt et al. • Defects in PTP␴-Deficient Mice
Mol Endocrinol, January 2002, 16(1):155–169
163
Fig. 7. Morphometric Analyses of Immunostaining For Pancreatic Hormones in (A) Newborn, (B) Wasting 2- to 3-wk-Old, and (C)
Adult PTP␴(⫺/⫺) and Control PTP␴(⫹/⫹ 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␴(⫺/⫺) mice.
DISCUSSION
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 (15–18).
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, 19–22) (Fig. 9). 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␴ protein.
Batt et al. • Defects in PTP␴-Deficient Mice
164 Mol Endocrinol, January 2002, 16(1):155–169
Table 1. PTP␴(⫺/⫺) Enteroendocrine Immunohistochemistry
Neonate (n ⫽ 6)
2- to 3-wk (n ⫽ 5)
Adults (n ⫽ 5)
Serotonin
Secretin
Somatostatin
Gastrin
Glucagon
CCK
PYY
2 or N
U
N
2 or N
2 or N
N
2 or N
2 or N
2 or N
2 or N
2 or N
N
2 or N
2 or N
2 or N
N
2 or N
N
N
N
N
N, Normal; U, undetectable; arrows indicate increase or decrease.
Fig. 8. Serotonin Immunostaining of the Gut
A wasting 3-wk-old PTP␴(⫺/⫺) (panel A) and PTP␴(⫹/⫹) control animal (panel B) demonstrates almost total absence of
serotonin-immunoreactive cells (dark brown) in the knockout animal. Magnification, 200⫻.
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 (23–25). 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 downregulate GH gene transcription, thus permitting terminal differentiation of the mature lactotroph (12).
GH and PRL immunostaining was significantly diminished in PTP␴(⫺/⫺) 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␴(⫺/⫺)
mice. Possible explanations for the subsequent downstream abnormalities of the somatotroph and lactotroph include the divergence of the majority of Pit1-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
Batt et al. • Defects in PTP␴-Deficient Mice
Mol Endocrinol, January 2002, 16(1):155–169
165
Fig. 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␴
results in abnormal development of the somatotroph/lactotroph lineage.
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␴(⫺/⫺) 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␴(⫺/⫺)
mice, normal immunoreactivity for all anterior pituitary
hormones in the PTP␴(⫺/⫺) mice that survive to adulthood, and the presence of developmental delay in the
peripheral nervous system of PTP␴(⫺/⫺) 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␴(⫺/⫺) mice. We do not know the reason for the
presence of three distinct phenotypic cohorts of the
PTP␴(⫺/⫺) mice. Possible explanations for this phenotypic diversity include variability of PTP␴ penetrance, effects of modifier genes, and varying levels
of compensatory contribution from other related
genes (e.g. the PTP␴ 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␴ 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␴(⫺/⫺) animals compared with PTP␴(⫹/⫹ and ⫹/⫺) controls. This result is
in agreement with the decreases in GH immunoreactivity noted in our PTP␴(⫺/⫺) 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␴(⫺/⫺) mice.
These differences in phenotype may be due to residual
expression of the extracellular domain of PTP␴ in
those mice. Analysis of the pancreas, adrenals, NEBs,
and the enteroendocrine gut was not reported in the
Tremblay mice.
166
Mol Endocrinol, January 2002, 16(1):155–169
The substantial decrease in GH immunoreactivity in
newborn PTP␴(⫺/⫺) 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␴(⫺/⫺) 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␴(⫺/⫺) mice significantly decreased the neonatal
mortality rate. Treated PTP␴(⫺/⫺) 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␴(⫺/⫺) 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 2to 3-wk-old PTP␴(⫺/⫺) 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␴(⫺/⫺) 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␴(⫺/⫺) 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
Batt et al. • Defects in PTP␴-Deficient Mice
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 (35–40).
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␴(⫺/⫺) mice, the most likely contributory cause is
GH deficiency. Indeed, islet morphology and insulin
immunoreactivity significantly improved in the
PTP␴(⫺/⫺) mice treated with GH. Therefore, islet hypoplasia and decreased insulin immunoreactivity are
likely secondary to the pituitary abnormalities noted in
the PTP␴(⫺/⫺) animals.
The low insulin levels and clinical picture of the
wasting 2- to 3-wk-old PTP␴(⫺/⫺) 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␴(⫹/⫹) controls. Despite the
hypoinsulinemia, adequate amounts were present for
the prevention of ketogenesis. Therefore, ketoacidosis
was not responsible for the death of our PTP␴ knockout mice.
The PTP␴(⫺/⫺) cohort lost at 2–3 wk of age from a
wasting syndrome revealed severe loss of serotoninimmunopositive cells in the proximal gut, compared
with control animals. With the exception of this consistent finding, the PTP␴(⫺/⫺) 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 (42–44). In view of this complexity, it is difficult to hypothesize the mechanism(s) by
which lack of PTP␴ 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␴(⫺/⫺) 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␴
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)
Batt et al. • Defects in PTP␴-Deficient Mice
in the mammal have yet to be identified. The cell
adhesion molecule-like ectodomain and tyrosine
phosphatase enzymatic activity likely engage PTP␴ in
a number of signaling networks that could likely affect
development and function of the endocrine system.
MATERIALS AND METHODS
PTP␴ Knockout Mice and Experimental Cohorts
Inactivation of the full-length PTP␴ gene was performed and
mice were characterized as previously described (10). The
PTP␴(⫹/⫺) mice are phenotypically indistinguishable from
PTP␴(⫹/⫹) mice. The PTP␴(⫺/⫺) 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␴(⫺/⫺)
and control PTP␴(⫹/⫺),(⫹/⫹) 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 ␮m), 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 avidinbiotin-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,
␤TSH, ␤FSH, 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
Mol Endocrinol, January 2002, 16(1):155–169
167
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␴ 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 ␮g/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 (⫻4). 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/␮l)
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.
168 Mol Endocrinol, January 2002, 16(1):155–169
Batt et al. • Defects in PTP␴-Deficient Mice
Lac Z Staining
Acknowledgments
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.
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.
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␴(⫺/⫺) mice expressed as a percentage of control PTP␴(⫹/⫹) littermates. Quantitation of serum
GH levels by RIA or ELISA was not possible due to the small
and limiting volumes of PTP␴(⫺/⫺) 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␴(⫺/⫺)
mice. Litters were again treated with Humatrope, saline, or
received no treatment as described above. A portion of
PTP␴(⫺/⫺) 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␴(⫺/⫺)
wasting 2- to 3-wk-old mice were euthanized, and the pancreases were dissected for insulin immunostaining and morphometry as previously described.
Received August 1, 2000. Accepted September 21, 2001.
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: [email protected].
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.
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