Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal to manufacture...
Reexamination Certificate
1999-07-23
2004-10-05
Reynolds, Deborah J. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal to manufacture...
C800S013000, C800S021000, C435S325000, C435S348000, C435S440000, C424S093100
Reexamination Certificate
active
06800790
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to members of the transforming growth factory-&bgr; family and their regulation of cell division, cell survival, and the specification of cell fates. Particularly, the invention relates to the bone morphogenetic protein (BMP)-2/4 homolog decapentaplegic (dpp) and its role in the maintenance of stem cells. For example, a dpp-based method for maintenance and controlling the division of germline stem cells, and a dpp-based method for defining a niche that controls germline stem cell proliferation are disclosed. Additionally, the invention provides a model of ovarian tumor development. The invention further relates to a dpp-based method for propagating stem cells in an undifferentiated state in vivo or by culturing in vitro.
2. Description of Related Art
In many adult tissues that undergo continuous cell turnover, a population of stem cells is responsible for replacing lost cells. Because of their pivotal role in controlling growth and neoplasia, the mechanisms regulating stem cell function are of great interest (reviewed by Potter and Loeffler, 1990; Doe and Spana, 1995; Lin, 1997; Morrison et al., 1997). Two mechanisms have been proposed to maintain stem cell divisions and regulate the differentiation of stem cell daughters: intrinsic factors and extracellular signals. Asymmetrically localized intrinsic factors help specify the fates of neuroblast daughters in Drosophila embryos (Doe and: Spana, 1995). Extracellular signals from surrounding cells mediated by cell surface-associated ligands and diffusible factors are frequently involved (Potter and Loeffler, 1990; Morrison et al., 1997). The identification of several of these factors has made it possible to culture some types of stem cell in vitro.
The Drosophila ovary presents an excellent system for studying two distinct groups of stem cells that remain active during much of adult life (reviewed by Spradling et al., 1997). The adult ovary contains 14-16 ovarioles each with a germarium at the tip, within which the germline and somatic stem cells are located. Two or three germline stem cells, located at the anterior tip of the germarium, divide asymmetrically to generate all germline cells in the ovariole (Wieschaus and Szabad, 1979; reviewed by Lin, 1997). Stem cell daughters known as cystoblasts undergo four rounds of synchronous division to produce groups of two, four, eight, and eventually 16 interconnected cystocytes, the precursors of ovarian follicles (reviewed by de Cuevas et al., 1997). Two somatic stem cells residing in the middle of the germarium give rise to all the somatic follicle cells (Margolis and Spradling, 1995); their equivalent in the testis are cyst progenitor cells. Three types of mitotically quiescent somatic cells are located in the vicinity of the stem cells: terminal filament and cap cells contact the germline stem cells, while inner sheath cells lie more posteriorly and contact both stem cell types.
Germline stem cell division is known to involve intrinsic mechanisms. This division and subsequent cystocyte divisions are physically unequal due to the segregation of fusomes rich in membrane skeleton proteins such as &agr;-spectrin and an adducin homolog encoded by hu-li tai shao (hts) (reviewed by de Cuevas et al., 1997). The round fusome (or “spectrosome”) characteristic of stem cells changes shape as cyst development proceeds, allowing cysts at different stages to be identified. The bag of marbles (bam) gene is highly expressed only in the stem cell daughter (McKearin and Spradling, 1990). The loss of Bam protein in cystoblasts prevents their differentiation, causing germline tumors to form (a “tumor” in Drosophila is a large clump of proliferating cells, the term does not imply these cells are cancerous). The genes pumilio (pum) and nanos (nos), encoding translational regulators, also play critical roles in the formation and maintenance of germline stem cells (Lin and Spradling, 1997; Forbes and Lehmann, 1998).
Less is known about the intercellular signals that control stem cell proliferation. Two important signaling molecules, Hedgehog (Hh) and Wingless (Wg) (reviewed by Perrimon, 1995; Cadigan and Nusse, 1997), are expressed in terminal filament and cap cells (Forbes et al., 1996a and 1996b). Hh signaling is critical for proliferation and differentiation of follicle cells, but it remained to be determined at the time the present invention was made whether somatic stem cells or their daughters are regulated (Forbes et al., 1996a and 1996b). The role of these signals in the germ line was even less clear because ectopic expression of hh did not appear to interfere with the function of germline stem cells (Forbes et al., 1996a).
Members of the transforming growth factor-&bgr;(TGF-&bgr;) family, including TGF-&bgr;s, activins, and the bone morphogenetic proteins (BMPs), elicit a broad range of cellular responses including the regulation of cell division, survival, and specification of cell fates (reviewed by Massague et al., 1996; Hogan, 1996a). TGF-&bgr;s were previously identified as repressing the proliferation of stem cells as assayed by either in vitro cultures or in vivo ectopic expression (Potter and Leoffler, 1990; Morrison et al., 1997). Inactivation of BMP-4 and its receptor BMPR in mice resulted in embryonic lethality for homozygous mutants (Winnier et al., 1995; Mishina et al., 1995), but no effect on stem cells was noted.
Similarly dpp, encoding a vertebrate BMP-2/4 homolog in Drosophila, functions as a local signal as well as a long-distance morphogen to pattern the early embryo and adult appendages by regulating cell proliferation and cell fate determination (Padgett et al., 1987; reviewed by Lawrence and Struhl, 1996). dpp is expressed in an anterior subset of follicle cells, and is required for establishing egg shape and polarity during late stages of oogenesis (Twombly et al., 1996). But an effect of dpp on maintaining and propagating stem cells, instead of causing their differentiation, has not been previously shown.
Major participants in the dpp signaling pathway have been identified: saxophone (sax) and thick veins (tkv) encode type I serine/threonine kinase transmembrane receptors, whereas punt encodes a type II serine/threonine kinase transmembrane receptor (Brummel et al., 1994; Nellen et al., 1994; Penton et al., 1994; Xie et al., 1994; Ruberte et al., 1995; Letsou et al., 1995). mothers against dpp (mad), Medea (Med), and Daughters against dpp (Dad) encode a family of conserved TGF-&bgr; transducers (Sekelsky et al., 1995; Tsuneizumi et al., 1997; Hudson et al., 1998; Wisotzkey et al., 1998; Das et al., 1998; Inoue et al., 1998), collectively known as Smads. Smads are proteins which transduce signals on behalf of TGF-&bgr; family members, or inhibit TGF-&bgr; signal transduction. A paradigm for TGF-&bgr; signal transduction has been developed from several experimental systems (Heldin et al., 1997). In Drosophila, Dpp binds both type I and II receptors to allow the constitutively active Punt kinase to phosphorylate and activate type I kinases, which phosphorylate Mad. The phosphorylated Mad brings Med into the nucleus as a transcriptional activator to stimulate dpp target gene expression.
Enhancing Dpp or other BMP-like signaling activities can be achieved by reducing the presence of Dad-like proteins, such as human Smad6 and Smad7. Vertebrate Smad6 and Smad 7 interact with type I receptors, and are known to inhibit both TGF-&bgr; and BMP signaling in cultured cells and frog embryos. Thus, disinhibition of TGF-&bgr; family members by inhibiting certain Smads promotes BMP-like signaling cascades. Additionally, Dpp or other BMP-like signaling activities may be increased by enhancing the function of Dpp or BMP receptors, such as Sax, Tkv, and Punt in Drosophila, and BMP receptors BMPR-II, ActR-II, Act-IIB, BMPR-IA, and ActR-I in humans. Other downstream positive regulators of Dpp or BMP signaling include Mad, Med, Dad, and Schnurri proteins in Drosophila, and Smad1, Smad4 and Smad5 in humans. See review by
Spradling Allan C.
Xie Ting
Carnegie Institution of Washington
Morgan & Lewis & Bockius, LLP
Reynolds Deborah J.
Woitach Joseph
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