Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press GHF- 1-promoter-targeted immortalization of a somatotropic progenitor cell results in dwarfism in transgenic mice Denise Lew, 1 Helen Brady, 2 Kay Klausing, 2 Katsuyuki Yaginuma, 2 Lars E. Theill, 2 Claudia Stauber, 1 Michael Karin, 2 and Pamela L. Mellon 1 The Departments of tReproductive Medicine, 1Neuroscience and 2Pharmacology and 1,2The Center for Molecular Genetics, University of California, San Diego, School of Medicine, La Jolla, CA 92093 USA; 1The Salk Institute ~or Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037 USA During pituitary development, the homeo domain protein GHF-1 is required for generation of somatotropes and lactotropes and for growth hormone (GH) and prolactin (PRL) gene expression. GHF-1 mRNA is detectable several days before the emergence of GH- or PRL-expressing cells, suggesting the existence of a somatotropic progenitor cell in which GHF-1 transcription is first activated. We have immortalized this cell type by using the GHF-1 regulatory region to target SV40 T-antigen (Tag) tumorigenesis in transgenic mice. The GHF-Tag transgene caused developmental entrapment of somatotropic progenitor cells that express GHF-I but not GH or PRL, resulting in dwarfism. Immortalized cell lines derived from a transgenic pituitary tumor maintain the characteristics of the somato/lactotropic progenitor in that they express GHF-1 mRNA and protein yet fail to activate GH or PRL transcription. Using these cells, we identified an enhancer that activates GHF-1 transcription at this early stage of development yet is inactive in cells representing later developmental stages of the somatotropic lineage or in other cell types. These experiments not only demonstrate the potential for immortalization of developmental progenitor cells using the regulatory regions from cell type-specific transcription factor genes but illustrate the power of such model systems in the study of developmental control. [Key Words: Transgenic mice; transcription factor GHF-1; POU homeo domain; progenitor cell; targeted oncogenesis.] November 9, 1992; revised version accepted January 27, 1993. Understanding the regulatory hierarchies that control Cells that express the three 13 subunits of the glycopro- development of cell lineages in mammalian organogen- tein hormones emerge in rum: thyrotropes expressing esis has been difficult owing to a paucity of tractable thyroid-stimulating hormone (TSH) [3 on el4, and gonad- experimental systems. Traditional approaches employed otropes expressing luteinizing hormone (LH) [3 on e15- in invertebrate systems, such as genetic analysis or sin- 16 and follicle-stimulating hormone (FSH) ~3 on e17. Two gle cell ablation, are often not practical in mammals, additional cell types, somatotropes, which express which therefore require novel strategies. growth hormone (GH) arising on e17 in the rat (e15 in The anterior pituitary is a useful model system for the mousel, and lactotropes, which express prolactin studying mammalian organogenesis {for review, see Voss (PRL) arising postnatally (Hoeffler et al. 1985; Leong et and Rosenfeld 1992). During the development of this al. 1985; Nogami et al. 1989; Doll6 et al. 1990; Simmons gland, five endocrine cell types arise in a specific tem- et al. 1990), are thought to be derived from a common poral pattern (Simmons et al. 1990). On embryonic day precursor, the somatomammotrope (for review, see 11 (el l J of the rat, prior to the emergence of Rathke's Karin et al. 1990). This cell, which exists during embry- pouch (the anlagen for the anterior pituitary) from an onic development and in small numbers in the adult, out-pocketing of the oral ectoderm, transcription of the transiently produces both GH and PRL before commit- gene encoding the a-subunit of the glycoprotein hor- ment to a final differentiated phenotype (somatotrope or mones becomes detectable. This represents the first dis- lactotrope). cemible step in commitment of the developing anterior The genes for GH and PRL evolved from a common pituitary. It is followed by expression of the pro-opiomel- ancestor iCooke et al. 1981; Cooke and Baxter 1982) and anocortin (POMC1 gene in developing corticotropes. share common developmental regulatory signals, in that both promoters have multiple binding sites for the pitu- Correspondingauthor. itary-restricted POU homeo domain transcription factor, GENES & DEVELOPMENT7:683-693 91993 by Cold Spring HarborLaboratory Press ISSN 0890-9369/93 $5.00 683 Downloaded from genesdev.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press Lew et al. GHF-1 (also termed Pit-l; Bodner and Karin 1987; Lefe- etic cell lineages. Oncogenesis can be targeted to specific vre et al. 1987; Bodner et al. 1988; Ingraham et al. 1988; cell types in transgenic mice using upstream transcrip- Nelson et al. 1988). Transfection analyses and in vitro tional regulatory sequences of specialized genes {Stewart transcription {Bodner and Karin 1987; Lefevre et al. 1987; et al. 1984; Hanahan 1989). Previously, we established Nelson et al. 1988; Mangalam et al. 1989) have shown immortal pituitary cell lines by directing expression of these sites to be important for tissue-specific expression the SV40 large T antigen (Tag) oncoprotein to anterior of both genes in somatotrope/lactotrope cell lines such pituitaries of transgenic mice using the regulatory region as GC, GH3, or 235 cells (Bancroft 1981; Reymond et al. of the earliest pituitary marker, the glycoprotein hor- 19841. The POU homeo domain family of transcriptional mone a-subunit gene (Windle et al. 1990), as well as a regulatory proteins includes several cell type-specific later marker of the gonadotrope lineage, the LH f~-sub- and ubiquitous transcriptional activators, many of unit gene (J.J. Windle, D.B. Whyte, and P.L. Mellon, in which are involved in cell fate determination {Herr et al. prep.). Remarkably, these cells are apparently frozen at 1988; Rosenfeld 1991). Several members of this class are the step in development at which the specific regulatory involved in developmental regulation of specific genes region is first activated and maintain the characteristics and act in concert with other factors to initiate commit- representing sequential developmental steps in the go- ment toward terminal differentiation (Ruvkun and nadotropic lineage (Sealfon et al. 1990; Windle et al. Finney 1991). 1990; Horn et al. 1991, 1992; Mellon et al. 1991; Schod- GHF-1 function is absolutely required for develop- erbek et al. 1992~ Tsutsumi et al. 1992). Given that im- ment of the somatotropic lineage. Mice with naturally mortalization targeted by the regulatory regions of genes occurring mutations or deletions of the GHF-1 gene fail for sequentially expressed known cellular products re- to develop somatotropes and lactotropes and exhibit an- sults in cells representing different steps in a develop- terior pituitary hypoplasia and dwarfism {Li et al. 1990; mental lineage, we reasoned that it might be possible to Castrillo et al. 1991). In addition to activation of the GH target successively earlier progenitor cells using the reg- and PRL genes, GHF-1 is also required for expansion of ulatory regions of genes encoding the transcriptional reg- the somatotropic lineage (Castrillo et al. 1991) and main- ulators that activate the expression of such cell type- tenance of thyrotropes (Li et al. 19901. During develop- specific markers. ment of the mouse anterior pituitary, GHF-I transcrip- To test this possibility, we targeted the putative soma- tion is detected at least 2 days before the emergence of totropic progenitor using the regulatory region of the somatomammotropic cells expressing GH and PRL tran- CHF-1 gene to express Tag. Transgenic animals express- scripts, indicating the potential existence of a GHF-1- ing a GHF-Tag transgene are severely dwarfed and de- expressing progenitor for this lineage. This progenitor velop pituitary tumors that express GHF-1 yet fail to cell expresses high levels of GHF-I transcripts and low express GH or PRL. Cell lines derived from such a pi- levels of GHF-1 protein (Doll6 et al. 1990), but the mech- tuitary tumor exhibited the characteristics of the soma- anism responsible for inefficient translation of GHF-1 totropic progenitor and provided a culture system that transcripts at this stage of development is not known. allowed the identification of a developmental stage-spe- Increased expression of GHF-1 protein correlates with cific enhancer controlling the expression of GHF-1, the onset of GH gene expression (Doll6 et al. 1990). which is inactive in later developmental stages. In addition to the GH and PRL genes, GHF-I is also involved in controlling its own transcription through positive autoregulation (Chen et al. 1990; McCormick et Results al. 1990). The minimal GHF-1 promoter fragment re- Targeting expression of Tag using the regulatory quired for maximal cell type-specific expression in com- region of GHF-1 mitted somatotrope cell lines (GC or GH 3 cells) consists of one GHF-l-binding site and another pituitary-specific To target oncogenesis to the putative somatotropie pro- element centered around the TATA box (McCormick et genitor, we utilized the 5'-flanking region of the GHF-1 al. 1991). Because CHF-1 transcription is detected before gene to direct expression of Tag in transgenic mice. Al- the appearance of GHF-1 protein (and, therefore, GHF-1 though transfections into a somatotropic