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Department of Human Genetics (L.T.R., B.S.W., S.A.C.), University of Michigan, Ann Arbor, Michigan 48109-0638; Murdoch Children’s Research Institute (S.A.R., P.Q.T.), Royal Children’s Hospital, Melbourne, Victoria 3052, Australia; and Department of Paediatrics (P.Q.T.), University of Melbourne, Victoria 3010, Australia
Address all correspondence and requests for reprints to: Sally A. Camper, 4909 Buhl Building. Ann Arbor, Michigan 48109-0618. E-mail: scamper{at}umich.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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GSU subunit promoter to express activated NOTCH2 persistently in pre-gonadotropes and pre-thyrotropes of transgenic mice. At birth, there is a small reduction in the population of fully differentiated thyrotropes and almost no fully differentiated gonadotropes. The temporal and spatial expression of Hey1 suggests that it could be a mediator of this effect. Gonadotropes complete their differentiation program eventually, although expression of LH and FSH is mutually exclusive with NOTCH2 transgene expression. This demonstrates that activated Notch2 is sufficient to delay gonadotrope differentiation, and it supports the hypothesis that Notch2 regulates progenitor cell differentiation in the pituitary gland. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Multiple pituitary hormone deficiency is a genetically heterogeneous disease that can result from mutations in several different transcription factors. Mutations in the prophet of PIT1 gene, called PROP1, are a common genetic cause of multiple pituitary hormone deficiency in humans (3, 4, 5, 6). In Ames dwarf mice, a missense mutation in Prop1 causes a phenotype that includes hypoplasia of the anterior pituitary with nearly complete loss of somatotropes, lactotropes, and thyrotropes (7, 8, 9, 10), and reduced gonadotropin production (11). The Ames dwarf phenotype is identical with that of mice with a deletion in Prop1 generated by gene targeting, when compared on the same genetic background (12). Portions of the Prop1-deficient phenotype can be attributed to the failure of the mutant Prop1 to activate Pit1 because Pit1 is required for thyrotrope, somatotrope, and lactotrope cell fates (7, 13). However, Prop1- and Pit1-deficient mice differ substantially in pituitary morphology, cell death, and vascularization (10, 14). At birth, the pituitaries of Pit1-deficient mice appear normal in morphology and size, whereas pituitaries of Prop1 mutants are extremely dysmorphic, giving the appearance that proliferating cells are unable to migrate into the ventral area of the gland where differentiation markers are normally detected. This difference indicates that PROP1 has important transcriptional target genes in the pituitary besides Pit1 (9). The characterization of Prop1 target genes and determining how all the pathways intersect is critical for elucidating the function of Prop1.
Recently, a number of additional molecular changes in Prop1-deficient mice have been discovered. These include prolonged Hesx1 expression (9) and spatially expanded expression of the corepressor transducin-like enhancer of split (Tle3) and the gonadotrope-specific transcription factor Nr5a1, or Sf1 (15, 16). The temporal and spatial expression patterns of these genes make them less likely candidates for explaining the profound dysmorphology of Prop1 mutant pituitaries than the NOTCH2 receptor. Notch2 is expressed in the progenitor cells of the anterior pituitary at embryonic d 12.5 (e12.5) through e14.5 (17). At these times, expression is found in the luminal cells of Rathke’s pouch that are undergoing rapid proliferation but not in the differentiated cells expressing GSU (-subunit of glycoprotein hormones, encoded by Cga). Notch2 expression decreases as pituitary development proceeds, indicating that it may need to be extinguished for cell differentiation to occur. The timing and location of Prop1 and Notch2 expression appear coincident, and no Notch2 expression is detectable in Prop1 mutant pituitaries (17). There are several Notch family members expressed in the pituitary in similar spatio-temporal patterns to Notch2, although their expression is maintained in Prop1 mutants. These include Notch3, the Notch ligand Dll1, and Hes1, a downstream target gene of Notch. Hes1 expression overlaps with Notch expression in undifferentiated cells in Rathke’s pouch (17). The normal location of Notch2 expression at the juncture between highly proliferating and differentiating cells, and the lack of expression in Prop1 mutants, make it a provocative candidate for explaining the profound organ dysmorphology characteristic of Prop1 mutant mice.
Notch is an evolutionarily conserved signaling system that influences precursor maintenance and cell fate selection in many organ systems, including some endocrine cell types. In the enteroendocrine system, Notch signaling represses differentiation of the endoderm into endocrine cells. Development of the endocrine cells in the pancreas is accelerated by disruption of the Notch signaling pathway (18), and persistent Notch1 expression in pancreatic precursor cells prevents their differentiation into endocrine and exocrine cells (19, 20). Similarly, Notch receptors regulate the transition from proliferation to differentiation in the cerebellum. Notch1 deficiency causes premature appearance of differentiated neurons at the expense of undifferentiated cells (21) and persistent activation of Notch2 maintains granule neuron precursor cells in a proliferative state (22). Notch signaling can also define the differentiated fate of cells. For example, neural crest stem cells and retinal progenitors rely on Notch signaling to switch production from neurons to glia (23, 24). Notch also functions downstream of VEGF in vasculogenesis to specify differentiation into arteries rather than veins (25, 26, 27). These examples are the precedents for our hypothesis that Notch signaling in the pituitary controls the transition from proliferation to differentiation and potentially, through lateral inhibition, influences the differentiation of cells into the five hormone producing cell types.
Notch2 knockout mice exhibit early embryonic lethality, and there is potential for redundancy of Notch2 and Notch3 function because of their overlapping expression patterns. Thus, it is difficult to discern the function of Notch2 in pituitary development with simple loss of function approaches. As an alternative, we tested the functional role of Notch2 in pituitary development by generating mice that express the activated form of Notch2 in differentiated pituitary cells, well beyond the normal developmental window of Notch2 expression. This strategy was successful in demonstrating the importance of Notch signaling in the development of pancreatic endocrine cells and cerebellar neurons. We found that persistent expression of activated Notch2 interferes with development of gonadotropes and, to a lesser extent, thyrotropes. Moreover, fully differentiated gonadotropes extinguish Notch2 transgene expression, suggesting that Notch2 can inhibit cell differentiation in the pituitary gland just as it does in the nervous system and pancreatic endocrine cells (19, 22). In contrast, the proliferation of pituitary cells expressing activated Notch2 is not affected. This study is the first evidence that the Notch pathway can affect pituitary cell fate specification.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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GSU promoter (28, 29, 30) to express the constitutively active Notch2 intracellular domain in differentiating gonadotrope and thyrotrope cells of the anterior pituitary (Fig. 1A
). Six lines containing the Notch2 transgene were generated, and two of the three lines with high levels of expression at postnatal d 1 (p1) were chosen for characterization (Fig. 1B). NOTCH2 protein is detectable in the anterior lobe of transgenic mice, but not in wild-type pituitaries. Both lines have identical phenotypes, and growth and fertility of the mice bearing the Notch2 transgene are not obviously affected.
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). Pituitaries of wild-type mice were stained with antibodies specific for Cyclin D2 and NOTCH2 at e12.5. Cyclin D2 marks dividing cells at the G1 to S phase transition and is normally detected in the dorsal cells of Rathke’s pouch. NOTCH2 is also present in the cells of Rathke’s pouch at e12.5, but NOTCH2 immunoreactive cells are located slightly more ventrally. No cellular colocalization of NOTCH2 and Cyclin D2 is evident at e12.5. This pattern suggests that NOTCH2 marks the transition from proliferation to onset of differentiation in normal pituitaries. At e15.5, endogenous Notch2 expression is not detectable (Ref. 17 and data not shown), but transgenic mice express activated NOTCH2 in the ventral portion of the anterior lobe (Fig. 1G). Proliferating, Cyclin D2-positive cells predominate around the lumen of Rathke’s pouch (Fig. 1F), and there is no colocalization of Cyclin D2 and transgenic NOTCH2 expression (Fig. 1H). Similar results were obtained using bromo-deoxyuridine to detect cells in S-phase of the cell cycle (see figure published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). These results demonstrate that the constitutively active Notch2 transgene is not sufficient to induce proliferation of anterior lobe cells that have already initiated differentiation.
Persistent Notch2 Expression Delays Gonadotrope Differentiation
To test the effect of NOTCH2 expression on differentiation, we examined hormone expression in the anterior pituitary of activated Notch2 transgenics using immunohistochemistry on the day of birth (p1), p5, and adult (Fig. 2). The size and morphology of the anterior lobe of transgenic mice is similar to wild-type mice at all ages examined. We assessed the presence of gonadotropes using the ß-subunits of FSH and LH as markers. In wild-type mice at p1, FSH (Fig. 2B) and LH (Fig. 2C) positive cells are readily detectable in the ventral aspect of the anter- ior lobe. In contrast, the anterior lobe of the Notch2 transgenics is devoid of FSH-positive cells (Fig. 2E) and only possesses a few LH-positive cells at p1 (Fig. 2F). The paucity of LHß and lack of FSHß protein in Notch2 transgenics at p1 is consistent with the reduced mRNA level measured by quantitative RT-PCR analysis (Fig. 3). The PCR cycle at which the amplification product is detected is normalized against the cycle at which hypoxanthine phospho-ribosyl transferase is detected and compared between wild-type and transgenic pituitaries to determine whether there is a statistically significant change. RT-PCR also revealed deficits in two other important gene products: a 4-fold decrease in transcripts for GnRH receptor (Gnrhr), a pre-gonadotrope marker, and a 2-fold decrease in transcripts for the transcription factor GATA2, which is necessary for gonadotrope and thyrotrope function (31, 32). Other transcription factors known to influence gonadotrope differentiation exhibited a trend toward reduced expression in transgenic mice relative to normal mice, but the differences were not statistically significant (Fig. 3). This includes early growth response gene 1 (Egr1), which is necessary for LHß-subunit production and pituitary transcription factor 2 (Pitx2), which causes negligible expression of Egr1, Nr5a1, Gnrhr, Lhb, and Fshb when it is reduced to less than 50% the normal level (33).
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) and show more LH expressing cells than detected at p1 (Fig. 2L), although the transgenic mice still have fewer FSH and LH immunoreactive cells than wild type (Fig. 2, H and I, respectively). By adulthood, the size of the fully differentiated gonadotrope population appears similar in activated NOTCH2 transgenic mice (Fig. 2, Q and R) and wild-type mice (Fig. 2, N and O). The ectopic, persistent expression of activated NOTCH2 delays gonadotrope differentiation, but it is not sufficient to block it completely. Despite the approximately 1-wk postponement in gonadotrope differentiation, Notch2 transgenic mice exhibit normal gonadal development. At 4 and 6 wk of age, there are no differences in size or histology of wild-type and transgenic testes, seminal vesicles, or ovaries (data not shown). Both male and female Notch2 transgenic mice are fertile.
Notch2 Reduces Thyrotropes But Does Not Affect GSU-Negative Lineages
We tested whether persistent, activated Notch2 expression influences thyrotrope differentiation using immunohistochemistry for TSHß-subunit (TSH). Compared with wild-type pituitaries (Fig. 4A), fewer TSH immunoreactive cells are present in NOTCH2 transgenics at p1 (Fig. 4B). We confirmed that this modest reduction is evident and statistically significant at the mRNA level by RT-PCR (Fig. 3). In contrast, corticotropes have normal levels of proopiomelanocortin (Pomc) transcripts (Fig. 3) and exhibit normal POMC immunohistochemical staining (Fig. 4D). Immunostaining for the transcription factor PIT1 is identical between wild-type (Fig. 4E) and transgenic mice (Fig. 4F) and prolactin and GH immunostaining are unchanged (data not shown). These data demonstrate that the presence of activated NOTCH2 can inhibit differentiation of pre-gonadotropes and pre-thyrotropes, albeit to a different degree, and there is no effect on other pituitary cell types.
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GSU LineageGSU (green cells, Fig. 5A) also express the activated NOTCH2 transgene (red nuclei, Fig. 5A), but by adulthood very few cells expressing GSU express transgenic NOTCH2 (Fig. 5D). Similarly, at p1 most TSH-positive cells express transgenic NOTCH2 (Fig. 5C), but in adults the majority of TSH-positive cells do not express the NOTCH2 transgene (Fig. 5E). We confirmed the lack of FSHß immunoreactivity at p1 in transgenic mice (Fig. 5C), and the dramatic increase in FSH immunoreactive cells in the pituitaries of adult transgenic mice (Fig. 5F), and noted that most FSH-positive cells in adult pituitaries do not express activated NOTCH2. This suggests that gonadotropin ß-subunit expression is incompatible with activated NOTCH2 expression.
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). Values were normalized to glyceraldehyde 3 phosphate dehydrogenase (GAPDH) transcript levels and depicted as fold change in relation to the normalized transcript level in wild-type pituitaries. The Notch2 transgene is expressed at significantly higher levels than endogenous Notch2 mRNA level at p1 and in adults. The potential Notch targets Hes1, Hes6, and Hes7 (Fig. 6 and data not shown) are expressed, but transgenic and normal mice have indistinguishable levels at p1 and in adults. Hey1 expression tends to be higher in transgenic mice than normal mice at p1, although the difference does not reach significance with the most rigorous statistical tests. In adult mice, levels of Hey1 expression are identical in transgenic and normal mice, despite the persistently elevated Notch2 transgene expression. Perhaps activated NOTCH2 can increase Hey1 expression in conjunction with essential cofactors like RbpJ
at p1 but not in adults (36).
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and 1D). By e13.5, Hey1 is detected in the developing anterior lobe cells (Fig. 7B) where it persists at e14.5 (Fig. 7C). Expression in the prospective intermediate lobe is low at both e13.5 and e14.5. Hey1 transcripts are not detectable at e16.5 (Fig. 7D), which is just before the normal appearance of Lhb and Fshb transcripts. This demonstrates that Hey1 expression is diminished in normal pituitary development before gonadotropes complete their differentiation process. The pattern of Hey1 expression suggests that it could influence the differentiation of other cell types not tested in this transgenic expression experiment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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GSU promoter did not drive cell proliferation, but it prevented the timely completion of gonadotrope, and to a lesser extent, thyrotrope differentiation. The Notch target gene Hey1 may be a mediator of this effect based on the pattern and timing of Hey1 expression in normal mice relative to that of Notch2. These data support the idea that the functional role of NOTCH2 signaling in the pituitary is to repress differentiation of hormone-producing cells. Notch signaling is an evolutionarily conserved pathway controlling proliferation and differentiation in many different organ systems. The developing pituitary expresses a variety of ligands including DLL1 and DLL3, the receptors NOTCH2 and NOTCH3, and the target genes HES1, HES6, and HEY1. Because some of these genes have mutually exclusive spatial expression patterns, it is likely that Notch signaling is affecting multiple stages of pituitary development. We find that NOTCH2 is localized in undifferentiated cells of Rathke’s pouch that lie between the cells with the highest mitotic index and those expressing differentiation markers (17, 39). During normal pituitary development cells appear to cease proliferation and activate NOTCH2 as they move into a transitional zone and then extinguish NOTCH2 as they migrate further to colonize the anterior lobe. Cells fail to leave Rathke’s pouch and migrate to the anterior lobe in Prop1 mutants, which are also NOTCH2 deficient (10), indicating that Notch signaling may play a role in migration of pituitary precursors.
Ectopic expression of activated Notch2 is sufficient to delay cell specification. This suggests a role for Notch signaling in pituitary development that is similar to its role in enteroendocrine cell development. Ectopic activation of Notch1 in pancreatic progenitor cells, using the Pdx1 promoter, prevented endocrine and exocrine cell differentiation, without having an effect on cell proliferation (19, 20). In contrast, mice deficient in Notch signaling such as those lacking the Notch ligand Dll1, the signaling mediator Rbpj, or the downstream effecter Hes1 show accelerated pancreatic endocrine cell differentiation (18, 40), demonstrating that Notch signaling normally prevents early cell specification in pancreatic development. Consistent with this paradigm, Hes1 null mice exhibit precocious and excessive endocrine cell development in the stomach and small intestine (40). Thus, a major function of Notch is to prevent premature cell specification in the developing endocrine cells of the gut and pituitary gland.
The gonadotrope differentiation program appears to be a step-wise process that involves activation of GSU expression at e11.5 (41), followed by expression of the transcription factor NR5A1 (also known as steroidogenic factor 1) at e13.5 (42) and activation of Gnrhr expression at e14.5 (43). Finally, expression of Lhb and Fshb are detected at e16.5 and e17.5, respectively (41). The delay between Nr5a1 expression and the detection of Lhb and Fshb transcripts probably reflects the requirement for additional transcription factors, such as early growth response protein 1 (EGR1, also known as Krox-24 and NGFI-A), which is essential for Lhb transcription, and is first activated at e14.5, or possibly Gata2 (31) (44, 45). In addition, NUPR1 (also known as P8) and OTX1 may have roles in gonadotrope differentiation in late gestation and after birth, respectively (31, 46, 47). Misexpression of activated NOTCH2 leads to delayed appearance of LH and FSH in pituitary gonadotropes, but it does not block GSU expression or Gnrhr, suggesting that Notch2 signaling disrupts function of factors expressed from e12.5 to e14.5, although we detected no statistically significant change in expression of Gata2 or Egr1. It is also possible that HEY1 interferes with transcription of the ß-subunit genes directly.
The delayed gonadotropin expression in Notch2 transgenics bears some similarities to transient gonadotropin deficiency in other genetically modified mice, although the time courses of recovery are varied. Mice homozygous for an Otx1 null allele exhibit a transient loss of GH, FSH and LH that requires 3 months for recovery (46), and persistent expression of Prop1 delays gonadotrope differentiation by approximately 1 wk (48, 49). In contrast, the recovery of gonadotrope differentiation is more efficient in Notch2 transgenics, and there is no obvious effect on gonadotropin target organs. The mechanisms of compensation in Otx1-deficient mice and Prop1 overexpressing transgenic mice are not known. The hypogonadotropic hypogonadism in Prop1 overexpressing transgenic mice does not appear to involve any of the gonadotrope differentiation factors, signaling molecules or receptors that are already identified (48, 49). Notch2 transgenics exhibit discordant expression of the transgene and gonadotropins, suggesting that silencing of transgene expression is necessary for activation of the differentiation program. Prop1 transgenic mice maintainProp1 expression throughout the period of compensatory changes and beyond, and clearly time does not reverse the genetic Otx1 deficiency. Each of these models provides an opportunity to discover the molecular players that serve as the underpinnings of gonadotrope differentiation.
The observed recovery of gonadotrope cells in the Notch2 transgenic pituitaries indicates that there is a mechanism by which gonadotropes differentiate despite the presence of activated NOTCH2. The recovery could be attributable to the reduction in the number of cells expressing the Notch2 transgene with time. There may be a small group of cells that initially escape the expression of the transgene, and it is this group of cells that differentiate and are responsible for the gonadotrope recovery. Another idea is that expression of activated NOTCH2 is insufficient to drive expression of a downstream repressor such as HEY1 after birth. Regardless of the mechanism, the similarities between these three mouse models of delayed gonadotrope differentiation indicate that the temporal and spatial regulation of several different genes is essential for completion of the gonadotrope differentiation process. They also highlight that there must be redundant mechanisms promoting LH and FSH expression because gonadotropin recovery is a common theme.
HEY1 is a bHLH domain-containing transcriptional repressor regulated by Notch1, 2 and 3 in various systems (34). One important function of Hey genes during development is to promote endothelial cell proliferation, migration, and organization in vessel formation (50, 51). Hey1 and Hey2 also influence neuronal development. Misexpression studies show that early ectopic expression of Hey1 and Hey2 maintains neural precursors, whereas later in development misexpression can bias cell fate by inhibiting the transcription of the proneural genes Mash1 and Math3 (52). We speculate that Hey1 and Hes1 are downstream targets of Notch2, based on their expression patterns in the pituitary gland and their activity in other contexts (53, 54, 55).
These studies are the first to demonstrate that Notch signaling can influence pituitary development. Activated NOTCH2 expression in pre-gonadotropes can cause a profound delay in gonadotrope differentiation. These studies also support the hypothesis that the failure of multiple cell types to differentiate in Prop1 mutants might be attributable to failure of the Notch2 signaling pathway. Hypogonadotrophic hypogonadism (HH) is marked by lowered levels of serum gonadotropins, delayed puberty, and infertility. The genetic causes of HH are heterogeneous, with some pituitary based cases attributed to mutations in Gnrhr (56), Nr0b1 (DAX1) (57), and Nr5a1 (58). The results reported here raise the possibility that some cases of gonadotrope dysfunction in HH might be due to aberrant activation of Notch2 signaling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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GSU-Notch2 construct the Notch2 intracellular domain fragment (22) was inserted into pPoly III plasmid containing a ß-globin polyadenylation sequence. A 4.6-kb fragment containing the mouse -subunit promoter and enhancer (28) used to direct expression in the gonadotropes and thyrotropes of the anterior pituitary. Restriction enzyme mapping and partial sequencing were used to verify the identity of the construct. Before microinjection, the construct was released from the vector by a restriction enzyme digest with Kpn1 and Cla1.
Generation, Genotyping, and Breeding of Transgenic Mice
The purified insert was microinjected into F2 zygotes from F1 (C57BL/6J x SJL/J) parents (The Jackson Laboratory, Bar Harbor, ME) by the University of Michigan Transgenic Animal Model Core. Embryos at the two-cell stage were implanted in pseudopregnant CD-1 females at 0.5 d post coitum. Genotyping to identify the presence of the transgene was conducted using genomic DNA isolated from tail biopsy samples. Oligonucleotides were designed to amplify a region spanning the GSU promoter and the Notch2 coding sequence (5'-TCA ACT TTC AGG ATG TTT TGT GTA A-3' and 5'-ATG GAG TGT GGC TGA TGT CTG C-3'). A standard reaction mixture was used containing the following concentrations of reagents per reaction: primers 12.5 pmol, BSA 5 µg, and Taq 5 U. PCR amplification was conducted for 29 cycles of denaturing at 92 C for 30 sec, annealing at 55 C for 30 sec, and elongating at 72 C for 30 sec with a final elongation step conducted at 72 C for 10 min.
Transgenic founders and their male progeny were backcrossed for two generations to the C57BL/6J background (The Jackson Laboratory) to establish lines, and data were gathered from two of the six lines. These lines are officially named TgN (Cga-Notch2)1Sac and TgN (Cga-Notch2)3Sac. All procedures involving the use of mice were approved by the University of Michigan Committee on the Use and Care of Animals. All experiments were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guidelines for the Care and Use of Experimental Animals.
Histology, Immunohistochemistry, and in Situ Hybridization
Wild-type and Notch2 transgenic embryos and adult pituitaries were fixed for 2–24 h in 10% formalin in PBS (pH 7.2), dehydrated, and embedded in paraffin. Sagittal or coronal sections of 6 µm were then prepared for immunostaining. For NOTCH2 immunostaining, slides were boiled in 10 mM citric acid (pH 6), for 10 min and then incubated with the mouse monoclonal NOTCH2 antibody (1:1500, C651.6DbHN Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) which was diluted in PBS containing BSA (3%), Tween 20 (0.5%), and normal goat serum (5% wt/vol) as previously described (17). The PerkinElmer TSA kit was used for antibody detection. Antibodies from the National Hormone and Pituitary Program were used to detect hormones: ACTH (1:1000), LH (1:1500), FSH (1:1800), and TSH (1:1000). Vectastain kits (Vector Laboratories, Burlingame, CA) were used for signal amplification and the Sigma Fast 3,3-Diaminobenzidine Tablet Sets (Sigma, St. Louis, MO) were used for antibody detection. Before mounting, slides were counterstained with methyl green (Vector Laboratories). For double immunostaining with NOTCH2 and Cyclin D2 (1:200; Santa Cruz Laboratories, Santa Cruz, CA) or hormones, the slides were boiled in citrate (see above) for 5 min. Antibodies (same as above) were diluted using the mouse on mouse antibody kit (Vector Laboratories) and the slides were incubated in a biotinylated antimouse secondary antibody. Cyclin D2 and hormone antibodies were detected with antirabbit-cy2 (1:200; Jackson ImmunoResearch, West Grove, PA), whereas NOTCH2 antibodies were detected with an avidin-cy3 (1:200; Jackson ImmunoResearch). Before mounting, slides were washed in 70% ethanol containing Sudan Black (0.1% wt/vol; Sigma) for 45 min followed by washing with PBS for 10 min to control autofluorescence.
For Hey1 in situ hybridization, embryos were collected and embedded in paraffin as they were for immunohistochemistry. Gene expression was detected using digoxigenin-labeled riboprobes as previously described (17). The Hey1 in situ probe, provided by Manfred Gessler (University of Wuerzburg, Germany), corresponds to the last 550 bp of the coding region of Hey1 (59).
Quantitative PCR
RNA was prepared from single adult pituitaries or by pooling three p1 pituitaries of each genotype and processing the tissue with an IKA Ultraturax homogenizer. RNA was isolated using the Rnaqueos Micro Kit, following the manufacturer’s protocol (Ambion, Austin, TX) from eight pools (p1) or eight individual (adult) pituitaries of each genotype. RNA was eluted in 26 µl and treated with deoxyribonuclease I (Ambion). RNA was converted to cDNA using the Invitrogen (Carlsbad, CA) cDNA synthesis system and random hexamers. PCR was performed using 10 ng of cDNA, TaqMan Universal PCR Master Mix and TaqMan probes specific for the genes of interest. Amplification and detection was carried out on an ABI Prism 7000 Sequence Detection system (Applied Biosystems, Foster City, CA). To analyze the data, the 
CT method (60) is employed. Eight separate RNA samples from each genotype were analyzed by quantitative PCR. The cycle at which the PCR product level reaches a threshold value (CT) for each transcript of interest is averaged, and the SD calculated. These CT values were normalized against the average CT for a reference gene such as hypoxanthine phospho-ribosyl transferase or GAPDH, yielding a CT value. CT represents relative normalized expression of the gene of interest in transgenic compared with wild type, i.e. CT wt = CTwt 
CTwt and CT tg = CTtg CTwt. The SD of the CT is calculated by taking the square root of the sum of the SD of the gene of interest squared and the SD of the reference gene squared. The fold change in normalized transgene expression relative to normalized wild-type expression is calculated by assuming that each PCR cycle should generate a 2-fold increase in the amount of PCR product. Consequently the fold change is equal to 2 to the power of CT. To determine the minimal and maximal ranges of a CT value, 2 is taken to the power of the quantity (CT ± SD of CT). Nonoverlapping minimal and maximal ranges are considered statistically significant.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Current address for P.Q.T.: School of Molecular and Biomedical Science, University of Adelaide, Adelaide 5005, Australia.
Current address for L.T.R.: Molecular and Integrative Physiology, University of Illinois-Urbana, Champlain, Illinois 61801.
Current address for S.A.R.: Monash Institute of Medical Research, Monash Medical Centre, VIC 3168, Australia.
None of the authors have anything to disclose.
First Published Online July 13, 2006
Abbreviations: CT, Threshold value; e1, embryonic d 1; Egr1, early growth response gene 1; GAPDH, glyceraldehyde 3 phosphate dehydrogenase; GSU, -subunit of glycoprotein hormones, encoded by Cga; HH, hypogonadotrophic hypogonadism; p1, postnatal d 1; Pomc, proopiomelanocortin.
Received for publication September 27, 2005. Accepted for publication June 20, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-subunit transgene in thyrotropes, gonadotropes, and developing pituitary gland. Mol Endocrinol 8:1420–1433-subunit gene requires multiple interacting DNA elements in transgenic mice and cultured cells. Mol Endocrinol 12:622–633-dependent Notch function regulates Gata2 and is essential for the formation of intra-embryonic hematopoietic cells. Development 132:1117–1126
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  J. Chen, L. Gremeaux, Q. Fu, D. Liekens, S. Van Laere, and H. Vankelecom Pituitary Progenitor Cells Tracked Down by Side Population Dissection Stem Cells, May 1, 2009; 27(5): 1182 - 1195. [Abstract] [Full Text] [PDF]
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 K. S Alatzoglou, D. Kelberman, and M. T Dattani The role of SOX proteins in normal pituitary development J. Endocrinol., March 1, 2009; 200(3): 245 - 258. [Abstract] [Full Text] [PDF]
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 X. Zhu, A. S. Gleiberman, and M. G. Rosenfeld Molecular Physiology of Pituitary Development: Signaling and Transcriptional Networks Physiol Rev, July 1, 2007; 87(3): 933 - 963. [Abstract] [Full Text] [PDF]
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