Simmons et al., 1990). In mouse, corticotropes, producing ...... thyrotropes, somatotropes and lactotropes (Lew et al., 1993). Each of the three differentiated cell ...
Development 122, 151-160 (1996) Printed in Great Britain © The Company of Biologists Limited 1996 DEV4599
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Anterior pituitary cells defective in the cell-autonomous factor, df, undergo cell lineage specification but not expansion Philip J. Gage, Michelle L. Roller, Thomas L. Saunders, Lori M. Scarlett and Sally A. Camper* Department of Human Genetics, University of Michigan Medical School, Medical Science II M3816, Ann Arbor, MI 48109-0618, USA *Author for correspondence
SUMMARY The Ames dwarf mouse transmits a recessive mutation (df) resulting in a profound anterior pituitary hypocellularity due to a general lack of thyrotropes, somatotropes and lactotropes. These cell types are also dependent on the pituitary-specific transcription factor, Pit-1. We present evidence that expression of Pit-1 and limited commitment to these cell lineages occurs in df/df pituitaries. Thus, the crucial role of df may be in lineage-specific proliferation, rather than cytodifferentiation. The presence of all three Pit-1-dependent cell types in clonally derived clusters provides compelling evidence that these three lineages share a common, pluripotent precursor cell. Clusters con-
taining different combinations of Pit-1-dependent cell types suggests that the Pit-1+ precursor cells choose from multiple developmental options during ontogeny. Characterization of df/df↔+/+ chimeric mice demonstrated that df functions by a cell-autonomous mechanism. Therefore, df and Pit-1 are both cell-autonomous factors required for thyrotrope, somatotrope and lactotrope ontogeny, but their relative roles are different.
INTRODUCTION
adrenocorticotrophpic hormone (ACTH), appear on embryonic day 12.5, followed by thyrotropes, producing thyroid stimulating hormone (TSH), at e14.5; somatotropes, producing growth hormone (GH), at e15.5; gonadotropes, producing luteinizing and follicle stimulating hormones (LH and FSH, respectively), at e16.5-17.5; and lactotropes, producing prolactin (PRL), at e18.5 (Japon et al., 1994). The pituitaryspecific transcription factor, Pit-1 (GHF-1), is first expressed on e14.5 (Lin et al., 1994) subsequent to the early period of intense cell proliferation within Rathke’s pouch. Analysis of classical mouse mutants, ablation experiments and human pituitary tumors have each provided important insights into the developmental relationships that exist between the five anterior pituitary cell types. Anterior pituitary glands from Ames (df) and Pit-1-defective Snell (dw) dwarf mice are profoundly hypocellular due to a general absence of thyrotropes, somatotropes and lactotropes (Bartke, 1965; Cheng et al., 1983; Gage et al., 1995; Phelps et al., 1993). The existence of these two nonallelic mutations that both impact the same three pituitary cell types is consistent with the hypothesis that these three lineages are developmentally related. In addition, transgene ablation experiments have shown that most lactotropes derive initially from somatotropes (Behringer et al., 1988; Borrelli et al., 1989), via a GH+, PRL+ intermediate cell (Hoeffler et al., 1985; Lloyd et al., 1988). Detection of TSH+, GH+ cells following chemical ablation of the thyroid is consistent with a close relationship between TSH and GH cells (Horvath et al., 1990). Finally, many human thyrotrope
The specification and expansion of diverse cell lineages from one or a few stem cell populations are fundamental processes required for the generation of complex, multicellular tissues and organisms. The mammalian anterior pituitary gland provides an excellent model for the study of cell lineage specification and organogenesis. At embryonic day 8.5 (e8.5) in the mouse, the anterior and intermediate lobe anlagen begin to arise from an invagination of oral ectoderm, termed Rathke’s Pouch, posterior to the anterior neuropore (Schwind, 1928). The posterior lobe arises simultaneously from an evagination of neuroectoderm. Thickening of Rathke’s pouch occurs concomitantly with separation of the pouch from the oral ectoderm at e12.5 (Schwind, 1928). Intense cell proliferation within the ventral wall of Rathke’s pouch from e12.5-14 initiates formation of the nascent anterior pituitary gland (Ikeda and Yoshimoto, 1991). A second round of increased cell proliferation occurs within the developing anterior pituitary gland from e16.5-17.5 (Ikeda and Yoshimoto, 1991). Cell proliferation and subsequent cytodifferentiation within the nascent anterior lobe depend on contact with the overlying neuroectoderm, implying the existence of local inductive influences (Daikoku et al., 1982, 1983). The ontogeny of the five anterior pituitary cell types, defined by activation of their respective polypeptide hormone gene(s), is temporally and spatially regulated (Japon et al., 1994; Simmons et al., 1990). In mouse, corticotropes, producing
Key words: cell differentiation, cell autonomous, pituitary gland, anterior, dwarfism, somatostatin, thyrotropin, prolactin
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adenomas also produce growth hormone or prolactin, further supporting a relationship between the three cell types (Kannan, 1987). The similarity between the pituitary phenotypes of Ames and Snell dwarf mice demonstrates that the normal products at the df and dw loci participate in a common regulatory hierarchy that is essential for thyrotrope, somatotrope and lactotrope ontogeny. The df mutation has been localized on mouse chromosome 11 but the nature of the defective gene is unknown (Buckwalter et al., 1991). The Snell phenotype results from a mutation within the Pit-1 gene that functionally inactivates the Pit-1 protein (Camper et al., 1990; Li et al., 1990). A member of the POU-homeodomain family of transcription factors (Ruvkun and Finney, 1991), Pit-1 binds to, and transcriptionally activates, the promoter-regulatory elements from several pituitary genes, including those for TSHβ, GH (Rhodes et al., 1994), PRL, GRFR and Pit-1 (Chen et al., 1990; Rhodes et al., 1993). The absence of detectable TSH, GH, PRL, GRFR and Pit-1 transcripts in pituitaries from adult dw/dw mice establishes that functional Pit-1 is essential for expression of these genes. The hypocellularity of dw/dw pituitaries is likely to result from the role of Pit-1 in cell proliferation (Castrillo et al., 1991). Pituitary development is regulated by both cell-autonomous and non-cell-autonomous factors. The product of the dw locus was shown to be cell autonomous by pituitary transplantation experiments, prior to its identification as Pit-1 (Carsner and Rennels, 1960). Non-cell-autonomous factors are likely to be responsible for the inductive interactions that influence early pituitary development (Daikoku et al., 1982, 1983; Kawamura and Kikuyama, 1995; Watanabe, 1982a,b). In addition, numerous extrinsic factors have been shown to affect proliferation of individual pituitary cell types in adult animals, including thyroid hormone (Horvath et al., 1990a), growth hormone releasing factor (Guillemin et al., 1982; Mayo et al., 1988) and estrogen (Lieberman et al., 1983). However, no extrinsic factors affecting all three cell types deficient in Ames dwarf mice have been described. Determining the mechanism of df action is crucial for understanding the function of this factor in pituitary ontogeny. In the present report, we critically assess the effect of the df mutation on pituitary development, particularly with respect to the three Pit-1-dependent cell types. We also use chimeric mice to test whether df functions as a cell-autonomous or non-cellautonomous factor. MATERIALS AND METHODS Mice DF/B-df/df mice were kindly provided by Dr A. Bartke (Southern Illinois University; Carbondale, IL) in 1988 and have been bred and maintained at the University of Michigan. Stain 83 mice carry 1000 copies of a β-globin transgene (Tg) (Lo, 1986). The Tg marker was bred onto the DF/B-df/+ background to generate Tg/Tg,df/df mice. Homozygous dwarf males (df/df or Tg/Tg,df/df) used for mating were injected with 2 µg thyroid hormone (T4, Sigma T-0397) and 50 µg ovine growth hormone (National Hormone Pituitary Program) three times a week for three weeks to enhance fertility (O’Hara et al., 1988). Dwarf mice were also fed a diet containing 25 mg thyroid powder per kg chow (AIN-76A, US Biochemicals), (Eicher and Beamer, 1980). CD-1 and FVB females were purchased from Charles River Labs.
These experiments were approved by the University of Michigan Committee on Use and Care of Animals (AAALAC accredited, Animal Welfare Assistance no. A3114-01) and all mice were housed and cared for according to NIH guidelines. Genotyping The genotype at the df locus was deduced from the dwarf phenotype (df/df) or by test mating (+/+ or df/+). To assay for the transgene marker, genomic DNA (250 ng) isolated from tail biopsies (Miller et al., 1988) was amplified by polymerase chain reaction (PCR) using the primer sequences (5′-CCAATCTGCTCACACAGGATAGAGAGGGC AGG-3′ and 5′-CCTTGAGGCTGTCCAAGTGATTCAGGCCATCG-3′). In samples containing 1000/2000 copies of the transgene, 20 amplification cycles were sufficient to visualize the specific product; in non-transgenic animals 25 cycles were required to generate a signal from the endogenous β-globin gene. Generation of aggregation chimeras Morulae were obtained from Tg/Tg,df/df × Tg/Tg,df/df matings and matings of albino, non-transgenic CD1 or FVB/N mice. The fertility of Tg/Tg,df/df mice was enhanced with hormone replacement therapy (O’Hara et al., 1988). Females were superovulated by injection with 5 I.U. pregnant mare serum gonadotropin, followed 46 hours later with 5 I.U. human chorionic gonadotropin (Hogan et al., 1986). Morulae were isolated 2.5 days post coitum (dpc) and Tg/Tg,df/df and normal CD-1 or FVB/N embryos were aggregated (Hogan et al., 1986). Based on the proportion of chimeric mice produced, CD-1 and FVB/N morulae fused with df/df morulae at equal efficiency. Generation of df/df morulae proved to be the significant ratelimiting step in these experiments due to the infertility of both males and females. Although the hormone-treated Tg/Tg,df/df males were prescreened for fertility by breeding, only 55% of superovulated Tg/Tg,df/df females mated with Tg/Tg,df/df males. In contrast, CD-1 and FVB/N females plugged at a rate of 75% and 98%, respectively. In situ hybridization histochemistry Pituitaries from adult chimeric and control mice were fixed with 4% paraformaldehyde and embedded into TissuePrep 2 (Fisher Scientific). Embedded pituitaries were sectioned at a thickness of 3 µm (immunohistochemistry only) or 1.5 µm (combined in situ and immunohistochemistry). In situ hybridization histochemistry to detect the Tg marker was performed using elements from several protocols (Lo, 1986; Thomson and Solter, 1988). A biotinylated β-globin probe was generated by nick-translation of pMBd2 (Lo, 1986) using a BioNick Translation kit (BRL). Sections were deparaffinized in Histoclear (National Diagnostics, Inc.) and xylene, rehydrated through graded alcohols, deproteinated in 0.2 N HCl and acetylated in 0.1 M triethanolamine/0.25% (v/v) acetic anhydride. Target DNA sequences were denatured by incubating sections at 70°C. Sections were incubated with an empirically determined amount of biotinylated probe. Specifically bound probe was detected by incubation of slides with strepavidin-horse radish peroxidase (Detek-HRP, Enzo Diagnostics), followed by a chromagen solution containing diaminobenzidine (0.0125%), NiCl2 (0.015%) and H2O2 (0.06%), which yielded a blue-black reaction product. The percent of labeled nuclei in each of the three pituitary lobes was determined after scoring the numbers of labeled and total nuclei in prints from randomly photographed fields of the pituitary sections. Labeled nuclei were never observed in pituitaries from non-transgenic negative control animals. Labeling was observed in approximately 80% of the nuclei from non-chimeric Tg/Tg-positive control pituitaries, consistent with previously reported results (Thomson and Solter, 1988). Therefore, the percent contribution of df/df cells was calculated by dividing the fraction of labeled nuclei in experimental sample by the fraction of labeled nuclei in Tg/Tg control samples processed in parallel.
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Immunohistochemistry Tissues for immunohistochemistry were fixed and processed as described above. Immunohistochemistry with primary antisera directed against human ACTH (Dako Corp.), rat TSHβ (AFP1274789), rat GH (AFP 4112), rat PRL (AFP425-10-91), rat LHβ (AFP-222387) or rat Pit-1 (Howard and Maurer, 1994) was performed as described previously (Kendall et al., 1991). In experiments designed to detect Pit-1, sections were pretreated with 5 µg pepsin; 0.01 N HCl (pH 8.0) followed by 5 µg saponin in H2O. Bound primary antibody was visualized using the ABC method (Vector) with diaminobenzidine as the chromogen. Sections were counterstained with hematoxylin. Staining for each cell type could be blocked by preincubation of the primary antiserum with the relevant hormone (data not shown).
(Jackson and Bennett, 1990; Kwon et al., 1988)). Reaction products were labeled by substituting α-[32P]-dATP for dATP in the PCR amplification and were digested overnight with DdeI to distinguish the albino (130 and 30 bp) and normal (165 bp) Tyr alleles (Fig. 6). The fragments were fractionated through 7% polyacrylamide and quantitated using a phosphorimager (Molecular Dynamics) and ImageQuant analysis software. To account for the difference in the number of adenosine sites between the two allele-specific fragments, the raw quantitation for the 130 bp mutant-specific band was multiplied by a factor of 1.18. The contribution of df/df cells to a tissue was calculated as: cts/minute in 165 bp product divided by cts/minute in 130 bp product × 1.18. Each tissue sample was measured in triplicate PCR experiments.
Quantitative polymerase chain reaction Brain, kidney, liver, lung and spleen isolated from chimeric and control animals were homogenized briefly at low speed with a Polytron (Brinkman) to disrupt the tissues and the homogenates were digested overnight at 37°C in 400 µg/ml proteinase K; 50 mM Tris•HCl, (pH 8.0); 2 mM EDTA and 0.5% Tween 20 (Ramakrishnan et al., 1994). Samples were heated at 95°C for 10 minutes to denature residual proteinase K activity and immediately added to polymerase chain reaction (PCR) amplifications. PCR (primers: 5′TCAAAGGGGTGGATGACCG-OH and 5′-GACACATAGTAATGCATCC) was used to amplify a 340 bp region from within the coding region for the tyrosinase gene (Tyr) (nucleotides +212 to +552,
RESULTS
TSH
Immunodetection of Pit-1-dependent cell lineages in df/df pituitaries While df/df pituitaries are hypocellular and generally devoid of the three Pit-1-dependent cell types, the demonstration of rare somatotropes in df/df mice suggested that thyrotropes and lactotropes might also be present (Gage et al., 1995). Normal anterior pituitary glands immunostained for the presence of TSH, GH and PRL contain 2-5% thyrotropes, 30-40% somatotropes and 20-40% lactotropes (Fig. 1a-c) ((Kendall et al.,
GH
PRL
wt
df
df
Fig. 1. Identification of rare thyrotropes, somatotropes and lactotropes in df/df pituitaries. (a-c) Normal and (d-i) df/df pituitaries were immunostained to detect the presence of the pituitary hormones TSH (a,d,g), GH (b,e,h), PRL (c,f,i), or an individual hormone and Pit-1 (g-i). In experiments designed to detect a single hormone (a-f), DAB was used as chromogen (brown). For double immunostaining experiments (g-i), DAB and VectorVIP (violet) were used as chromagens to detect Pit-1 and hormone, respectively. Although the normal and mutant pituitary sections were photographed at the same magnification, only a portion of the anterior lobe is visible in the normal pituitary, but all three lobes of the hypocellular df/df pituitary are depicted. Cells staining for TSH, GH or PRL were consistently observed in df/df pituitaries (arrowheads, df). However, the frequency of these cells was rare compared to wild-type pituitaries (compare a-c versus d-f). All TSH+, GH+, or PRL+ cells having an identifiable nucleus present in the section also expressed Pit-1. Bar, 100 µm (a-f) or 10 µm (g-i).
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P. J. Gage and others Table 1. Content of hormone-positive clusters suggests multiple terminal differentiation pathways
Positive Cells Per 1/3 Pituitary
140 120 100 80 60
1
2
3
Total clusters
TSH GH PRL TSH, GH GH, PRL TSH, PRL TSH, GH, PRL
7 1 4 1 2 2 1
3 3 3 0 1 1 2
5 3 0 0 1 1 1
15 7 7 1 4 4 4
*Hormones present within cells of an individual cluster. No cells staining for multiple hormones were observed. †Number of clusters for each category in three different individuals.
40 20 0
df/df pituitary†
Hormones present*
1
2
3
4
Fig. 2. Quantitation of thyrotropes, somatotropes and lactotropes in dwarf pituitaries. Pituitaries from Tg/Tg,df/df (1-4) mice were sectioned in their entirety and immunostained with antisera specific for TSH, GH or PRL. For each animal, 1/3 of the sections, taken from throughout the organ, were immunostained for each hormone. The total number of hormone-expressing cells for each lineage was then scored. Key: black, TSH; gray, GH; white, PRL.
1991) and S. Kendall, unpublished results). Pituitary sections from four Ames dwarf animals revealed the presence of occasional cells immunostained for TSH, GH or PRL in each of the animals (Fig. 1d-f). These rare cells occurred either individually or in small clusters of 2 to 20 cells per section. Quantitation of the rare hormone-positive cells revealed that each df/df pituitary contains several hundred thyrotropes and somatotropes, as well as a few lactotropes (Fig. 2). These numbers represent less than 1% of the normal component of these cells ((Kendall et al., 1991; Sasaki and Sano, 1982), P. Gage, data not shown). Thus, commitment to all three Pit-1-dependent cell lineages can occur in df/df pituitaries. Consistent with previous reports (Lin et al., 1994; Slabaugh et al., 1981), these cells were never observed dw/dw pituitaries (data not shown). The previous demonstration that Pit-1 is required for hormone-positive thyrotropes, somatotropes and lactotropes in adults prompted us to determine whether Pit-1 was present in these rare cells in df/df animals. Sections were immunostained for both Pit-1 and the hormones TSH, GH and PRL. Cells expressing both Pit-1 and each of these hormones were observed in pituitary sections from multiple df/df animals (n=3) (Fig. 1g-i). Although not a quantitative assay, the intensity of staining for Pit-1 and the various hormones appeared to be equivalent, on a per cell basis, between df/df and normal cells. There were no examples of hormone-positive cells that lacked Pit-1. Thus, the df/df thyrotropes, somatotropes and lactotropes appear to be Pit-1-dependent and arise via normal developmental pathways. Clusters of Pit-1+ cells staining positively for an individual hormone frequently contained Pit-1+ cells that were negative for that hormone (Fig. 1g-i), suggesting that clusters of Pit-1+ cells might consist of more than one differentiated cell lineage. This hypothesis was confirmed by examining df/df pituitary sections immunostained for the presence of all three hormones,
using a different chromogen to detect each. Clusters contained one, two or three of the Pit-1-dependent cell lineages (Table 1). In clusters containing two of the cell lineages, each possible pairwise combination was observed. Generation of chimeric mice We tested whether df is cell-autonomous or non-cell autonomous by assaying for the behavior of df/df cells in aggregation chimeric mice consisting of df/df and normal cells (+/+). If df is non-cell autonomous, the number of df/df cells that populate the thyrotrope, somatotrope and lactotrope lineages should be enhanced by the presence of normal cells in the chimera. If df is cell autonomous, the intrinsic defect would prohibit any increase in df/df cell differentiation. Chimeric mice were constructed such that the two cell types could be distinguished and quantitated both histologically and genetically (Table 2). A 1000-copy β-globin transgene (Tg) detectable by in situ hybridization (Lo, 1986) was introduced onto the DF/B-df/df background from which the df/df-cell donor embryos were isolated. The utility of this transgene as a histological marker for cell lineage analyses in chimeric animals has been established previously (Thomson and Solter, 1988). All known albino mouse strains carry a G to C transversion in codon 103 of the tyrosinase (Tyr) gene (Jackson and Bennett, 1990; Yokoyama et al., 1990) that also leads to a Dde I restriction fragment length polymorphism (RFLP). Since the normal cells were derived from albino mice this RFLP was exploited to discriminate between the wild-type and mutant tyr alleles (Jackson and Bennett, 1990) present in df/df and normal cells, respectively (Table 2). Chimeric mice were constructed by morulae aggregation. A total of 16 chimeric mice were obtained. Coat color chimerism is a good indication of the relative contribution of cells from
Table 2. Cell types used to construct chimeric mice Cell type Locus df tyrosinase* Marker transgene†
Dwarf
Normal
df/df Pigmented (C/C) Tg/Tg
+/+ Albino (c/c) nontransgenic
*Used for phenotypic and genotypic quantitation of df/df cell contribution to chimeric tissues. †1000-copy β-globin transgene present in df/df cells.
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Fig. 3. Underrepresentation of df/df cells in the anterior lobe (ant) relative to the posterior (post) and intermediate (int) lobes. Paraffin-embedded chimeric pituitaries were sectioned at approximately the thickness of one cell (1.5-3 µm) and processed by in situ hybridization histochemistry to detect the transgene tag present in df/df cells. Specifically bound probe was detected by incubation of sections with a strepavidin-horse radish peroxidase conjugate (Enzo Diagnostics) followed by DAB and NiCl2. Nuclei of df/df cells contained two dark, black foci of reaction product corresponding to the two Tgmarked chromosomes (Tg/Tg) (arrowheads). Results shown are from chimera 1.
each parent to the tissues of the chimeric animal (Vogt et al., 1987). Based on this criterion, several mice contained high df/df cell contributions, demonstrating that df/df cells were able to contribute to chimeric mice with high efficiency. All chimeras displayed normal growth and adult body size, regardless of the df/df cell contribution. Six chimeric mice with medium to high df/df cell contributions were examined in detail. The anterior pituitary glands from these mice were normal in size and appearance. Therefore, the overall contribution of even an apparently small number of normal cells to the chimeric mice could rescue both the somatic growth defect and the hypopituitarism characteristic of non-chimeric df/df mice. Histology of chimeric pituitaries Pituitary glands were examined by in situ hybridization histochemistry designed to detect the transgene marker present in df/df cells. Consistent with previously reported results (Kusakabe et al., 1988), the two parental cell types were dispersed essentially randomly throughout the pituitary glands of the chimeras. The number of Tg-marked df/df cells in the anterior pituitary gland was dramatically reduced relative to the posterior and intermediate lobes (Fig. 3). The percent contribution of df/df cells to each lobe of these pituitaries was quantified by in situ analysis (Fig. 4). In chimera 1, df/df cells represented only 30% of the anterior lobe versus 90% for the posterior and intermediate lobes. Similar results were obtained in each of the other chimeras except number 5. The df/df cell contribution was reduced in both the anterior and intermediate lobes of chimera 5, possibly reflecting the common origin of these lobes from Rathke’s pouch. The anterior pituitary gland of all six chimeras was composed primarily of wild-type cells. These results demonstrate that df/df pituitary cells have an intrinsic defect in cell proliferation or survival, consistent with the profound hypocellularity of non-chimeric df/df anterior pituitary glands. In order to classify the Tg-marked df/df cells into one of the five anterior pituitary cell lineages, in situ-labeled sections were immunostained with antisera specific for the individual
anterior pituitary hormones. Most df/df cells were corticotropes or gonadotropes (data not shown). Thyrotropes, somatotropes, and lactotropes originating from df/df cells were only rarely observed, even in chimeras with exceptionally high overall df/df cell contributions (Fig. 5). There was no increase in the number of these cells per section relative to that observed in non-chimeric df/df mice. Therefore, the normal cells within the chimeric animals did not enhance the contribution of df/df cells to the thyrotrope, somatotrope and lactotrope lineages. Quantitation of df/df cell contribution to peripheral tissues Quantitative PCR was used to confirm that the under representation of df/df cells was specific to the anterior pituitary gland (Fig. 6). This approach exploited the DdeI RFLP between the wild-type and mutant tyrosinase alleles present in the df/df and normal cells, respectively. The df/df cell contributions to the brain, kidney, liver, lung and spleen from the six mice were quantitated. The df/df cell contribution to the five peripheral organs, as well as to the posterior and intermediate lobes of the pituitary gland, were all approximately equivalent (Fig. 4). These quantitative results also matched estimates of df/df cell contribution to each mouse based on coat color chimerism (data not shown). However, the df/df cell contribution to an animal’s anterior pituitary gland was significantly reduced relative to the other organs. For example, the df/df cell contribution to the anterior pituitary gland of chimera 1 was only 32% but the df/df cell contribution to all of the other organs ranged from 75-98% and averaged 90% (Fig. 4). This established that the lack of expansion by df/df cells was specific to the anterior pituitary gland. DISCUSSION Specification and expansion of differentiated cell lineages within the mammalian anterior pituitary gland is controlled by a hierarchy of genes encoding both cell-autonomous and noncell-autonomous factors. The study of classical mouse mutants
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% Contribution by df/df-Derived Cells
Chimera 1
Chimera 2
100 90
80 70
60 50 40
30
20 10 0
Br Ki Li Lu Sp
P
I
A
Chimera 4
% Contribution by df/df-Derived Cells
Chimera 3
Br Ki Li Lu Sp
P
I
A
Chimera 5
Br Ki Li Lu Sp
P
I
A
P
I
A
Chimera 6
100 90 80
70
60 50
40
30 20 10 0
Br Ki Li Lu Sp
GH
P
I
A
Br Ki Li Lu Sp
P
I
A
TSH
Br Ki Li Lu Sp
Fig. 4. The df/df cell contribution to the anterior pituitary is low relative to the posterior and intermediate pituitaries, and five peripheral tissues Results for PCR (open columns) and in situ (shaded columns). Quantitation of df/df cell contributions to the 6 chimeric mice studied are shown. Results from each data set were derived from triplicate PCR experiments. Bars represent the standard deviation of the mean. Br, brain; Ki, Kidney; Li, liver; Lu, lung; Sp, spleen; P, posterior pituitary; I, intermediate pituitary; A, anterior pituitary.
PRL
Fig. 5. Demonstration that df/df thyrotropes, somatotropes and lactotropes are rare in chimeric pituitaries. Sections of chimeric pituitaries previously in situ-stained to detect the Tg marker in df/df cells were subsequently immunostained for the presence of GH, TSH or PRL using DAB as the chromogen. Doubly-stained cells (arrowheads) containing both the blue-black nuclear in situ signal and the brown immunostaining signal were rare, but detectable, in all 6 chimeric pituitaries. Results shown are from chimera 5. Most df/df cells observed were corticotropes and gonadotropes (data not shown).
has not only resulted in the molecular identification of several factors within this hierarchy but has also established their functional relevance in vivo. For example, the functional role of the
cell-autonomous factor, Pit-1, in pituitary cell lineage specification and proliferation was demonstrated through analysis of the Snell dwarf mouse (Camper et al., 1990; Li et al., 1990).
Intrinsic block in pituitary cell expansion
Fig. 6. PCR assay used to quantitate df/df cell contribution to peripheral tissues. The df/df cell contribution to five peripheral tissues from the chimeric mice was quantitated. A DdeI polymorphism between the wild-type (C/C) and mutant (c/c) tyrosinase alleles (Yokoyama et al., 1990) present in df/df and normal cells, respectively, was exploited to quantitate the contribution of each cell type to peripheral tissues. PCR amplification of a 340 bp target sequence spanning the polymorphic site and digestion of the resulting products with DdeI (arrows) generates fragments diagnostic for both alleles. An autoradiograph demonstrating results from experiments programmed with genomic DNA from control animals (lanes 1-9) and tissues from chimera 5 (lanes 10-15) is shown. Quantification of the wild-type (165 bp) and mutant (130 bp) tyrosinase allele-specific bands in experiments programmed with control DNAs at varying ratios (df/df:+/+) (lanes 4-9) yielded the expected ratios for the two cell types and established that the assay was quantitative over the range used. The predominance of the wild-type-allele-specific 165 bp band demonstrates that the brain (Br), liver (Li), lung (Lu), spleen (Sp) and kidney (Ki) were derived largely from df/df cells.
Analysis of the less severe murine little mutation established the importance of an extrinsic factor, the hypothalamic neuropeptide growth hormone-releasing factor, and its receptor for proliferation of committed somatotropes (Godfrey et al., 1993; Lin et al., 1992). The Ames dwarf mouse offers another opportunity for insight into anterior pituitary ontogeny since the df mutation defines a factor that is crucial in the development of the three Pit-1-dependent cell lineages. The near identity of the Ames and Snell dwarf phenotypes has provided compelling evidence that df and Pit-1 participate in a common developmental program during pituitary ontogeny. We have demonstrated that df is a cell-autonomous factor that is required for proliferation, but not commitment, of cells within the thyrotrope, somatotrope and lactotrope lineages. The detection of rare Pit-1+ cells expressing TSH, GH or PRL in df/df pituitaries represents a fundamental distinction between the effects of the df and dw mutations on thyrotrope, somatotrope and lactotrope ontogeny. If the df mutation is a complete loss-of-function allele, then these data demonstrate that df is not absolutely required for lineage specification, including activation of Pit1 or the relevant hormone genes. Rather, its major role must be in the expansion or survival of
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these three lineages. Ectopic programmed cell death does not appear to account for the hypocellularity of df/df anterior pituitaries since we have observed no evidence of apoptotic nuclei. Thus, df likely plays a central role in efficient cell proliferation within the thyrotrope, somatotrope and lactotrope lineages. Alternatively, if the df mutation is a partial loss-offunction mutation, then df may have a role in lineage specification and proliferation. An example of such a partial loss-offunction allele is provided by the point mutation within the Pit1 gene that destroys the ability of Pit-1 to transactivate the GH and PRL genes, but leaves the TSHβ activation and cell proliferation functions intact (Pfaffle et al., 1992). We considered the possibility that the rare committed cell types in df/df mice result from genetic mechanisms such as somatic reversion or suppression, phenomena observed for several types of mutations, including retroviral insertion (Copeland et al., 1983), DNA duplication (Brilliant et al., 1992), small insertions and deletions, and point mutations (Greenspan et al., 1988). However, the frequencies of these events are at least an order of magnitude less than the frequencies of commitment that we have documented in Ames dwarf mice. Moreover, we have never observed df/df animals with near normal growth as would be expected if reversion occurred early in gestation (Melvold, 1971). Thus these explanations seem less likely than the idea that the clusters represent committed cells arrested in development due to the lack of a lineage-specific cell proliferation factor, df. The Pit-1+ cells in df/df pituitaries occurred within small, defined clusters, suggestive of a clonal origin. Some clusters were homogeneous, consisting of a single Pit-1-dependent cell type. Heterogeneous clusters containing each possible pairwise combination or all three cell types were also detected. Over 20% of the clusters contained thyrotropes together with somatotropes, lactotropes, or both. This is consistent with the hypothesis that the three Pit-1-dependent cell lineages share a common progenitor (Fig. 7). The heterogeneity of the clusters also suggests that each Pit-1+ progenitor did not follow the same, rigidly defined developmental program. Rather, distinct clusters, and the individual cells within them, must have the potential to complete different developmental program(s). The numerous examples of Pit-1+ clusters composed of only thyrotropes, somatotropes or lactotropes indicates that each cell type has the capacity to differentiate directly from the Pit-1+ progenitor independently of the others (Fig. 7). The direct differentiation to lactotropes without a GH+ intermediate may not be the primary mechanism for generation of lactotropes. The potential for more than one route could explain the inability to completely eliminate lactotropes via somatotrope ablation (Behringer et al., 1988; Borrelli et al., 1989). The hypothesis that somatotropes, lactotropes and thyrotropes derive from a common progenitor is consistent with examples in other systems (Anderson, 1989). Mammalian neural crest cell differentiation and hematopoesis, and Drosophila neurogenesis each involve the derivation of multiple cell lineages from a common precursor. However other relationships between the Pit-1+ precursor and the three differentiated cell types have been proposed. These range from the strictly linear (see e.g. (Rosenfeld, 1991)) to the complex (see e.g., Karin et al., 1990). The most attractive alternative hypothesis to the common progenitor model is one in which thyrotropes arise from a set of Pit-1+ precursors that are distinct
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Fig. 7. Model for ontogeny of thyrotropes, somatotropes and lactotropes. Two periods of intensified cell proliferation occur during anterior pituitary ontogeny (bars). df is crucial for the later proliferative phase and may impact the earlier phase as well. Because df is cell autonomous, it must be expressed within the proliferating cells. A progenitor cell expresses Pit-1 and at least one additional, unknown factor in order to generate the fully differentiated thyrotropes, somatotropes and lactotropes (Lew et al., 1993). Each of the three differentiated cell types can be generated directly from a common progenitor by stochastic and/or extrinsically determined processes. However, most lactotropes are likely to derive from somatotropes via a GH+, PRL+ intermediate cell. Proliferation and function of thyrotropes, somatotropes and lactotropes are stimulated by thyrotropin releasing hormone (TRH) (Horvath et al., 1990), growth hormone-releasing factor (GRF) (Guillemin et al., 1982; Hammer et al., 1985) and estrogen (E2) (Lieberman et al., 1983; Lloyd, 1983), respectively. The df mutation renders the cells unable to respond to these extrinsic cues.
from those that produce somatotropes and lactotropes (Borrelli, 1994; Karin et al., 1990). If this is the case, the frequent appearance of thyrotropes with somatotropes and lactotropes in df/df pituitaries may suggest local interactions which are facilitated by aggregation of the two lineages. Nevertheless, the high frequency with which somatotrope and/or lactotropes appear independently of thyrotropes implies that these interactions are either not essential or transient. The interdependence of thyrotrope, somatotrope and lactotrope differentiation is supported by the analysis of mice generated by gene targeting that are deficient in the pituitary hormones TSH, LH and FSH. These dwarf mice exhibit extreme hyperplasia of thyrotropes, apparently at the expense of somatotrope and lactotrope proliferation (Kendall et al., 1995). One interpretation of this data is that when Pit-1+ precursor cells are recruited to produce thyrotropes, fewer somatotropes and lactotropes can be formed (Fig. 7). Alternatively, thyrotropin or thyroid hormone might be required in order to attain normal numbers of somatotropes and lactotropes. The importance of thyrotropes for somatotrope and
lactotrope differentiation could be resolved by examining the effects of thyrotrope ablation. Regardless of whether the differentiation of thyrotropes, somatotropes and lactotropes involves a single common, or multiple progenitor(s), it is clear that the pathway involves more steps than are defined by the current mouse mutants (Fig. 7). The atypical cells detected in df/df and dw/dw pituitaries by ultrastructural analysis may represent undifferentiatied precursors that accumulate prior to the expression of df and Pit1 (Cheng et al., 1983). Activation of Pit-1 expression is an intermediate step in the cytodifferentiation pathway. A Pit-1+ cell line that fails to express any of the hormone genes reveals that other factors in addition to Pit-1 are required for progression through the pathway and provides a model for the study of one intervening step (Lew et al., 1993). The next step, diversification into individual differentiated phenotypes, is probably regulated by lineage-specific transcriptional activation and repression events downstream of the Pit-1+ precursor cell. Distinct transcriptional activators are involved in Pit-1dependent activation of the TSHβ, GH and PRL promoters (Rhodes et al., 1994). The size of the anterior pituitary gland and proportion of each of the three differentiated cell types is probably regulated by extrinsic factors such as TRH and thyroid hormone, growth hormone releasing factor and estrogen (Fig. 7). Some examples of factors that regulate the expansion of distinct sublineages by stimulating lineage-specific proliferation of precursor cells include the lymphokines in hematopoiesis and both extracellular matrix and the pituitary hormone α-MSH in neural crest differentiation (Anderson, 1989). A particularly striking feature of the aggregation chimeric mice was the ability of the wild-type cells to generate a normal-sized anterior pituitary gland composed of the appropriate ratios of the five endocrine cell types. This was achieved even in chimeric mice composed almost entirely of df/df cells, suggesting that relatively few progenitors are sufficient to populate the entire lobe and that the extrinsic cues effectively regulate the proportions of each cell type. It is important to note that this compensation by normal cells is restricted to the Pit-1-dependent cell types since corticotrope and gonadotrope pools in these animals are composed primarily of df/df cells. The colonization of the liver by normal cells in c-jun−/−↔wild-type chimeric mice is similar to the phenomena that we observed in the anterior pituitaries of df/df↔+/+ chimeras (Hilberg et al., 1993). However, not all organogenesis defects can be compensated so fully by normal cells. For example, mice chimeric for the cellautonomous staggerer mutation had underdeveloped cerebellums due to the fact that wild-type cells could only partially compensate for the Purkinje cell defect (Herrup and Mullen, 1979). Thus, the precise control of anterior pituitary cell number and cell type composition by extrinsic cues is remarkable. It may reflect the fact that these pituitary cell types retain their proliferative capacity into adulthood (Bach et al., 1995; Horvath et al., 1990). In addition, adult mice retain the ability to respond to changing demands, such as pregnancy and lactation, by altering the proportion of individual pituitary cell types. The inability of df/df cells to recognize or respond to these proliferative signals demonstrates that df plays a central role in mediating these events, potentially in both fetal and adult mice. The limited number of Pit-1+ cells in df/df mice suggests that
Intrinsic block in pituitary cell expansion df is essential for the second period of intensified pituitary cell proliferation at e15.5-16.5 that occurs after the activation of Pit-1 at e14.5 (Ikeda and Yoshimoto, 1991). Given that Pit-1 is important for proliferation in cell culture, the small clusters observed in df/df mice may result directly from Pit-1 expression (Castrillo et al., 1991), however df may be important for efficient Pit-1-mediated cell proliferation. Many important developmental control genes are required at more than one time during ontogeny. For example genes encoding the helix-loop-helix proteins that are crucial for initating sex determination in early Drosophila embryogenesis are also required later in development for the initiation of neurogenesis (Jan and Jan, 1990). Thus, it is possible that df is also required for the earlier wave of cell proliferation that occurs prior to the activation of Pit-1 (
