Defects in mouse mammary gland development caused by conditional haploinsufficiency of Patched-1

Michael T. Lewis 0 2 Sarajane Ross 2 Phyllis A. Strickland 2 Charles W. Sugnet 2 Elsa Jimenez 2 Matthew P. Scott 1 Charles W. Daniel ) 2 0 Present address: Department of Physiology and Biophysics, University of Colorado School of Medicine , Box C240, Room 3802, Denver, CO 80262, USA 1 Departments of Developmental Biology and Genetics, Howard Hughes Medical Institute , 279 Campus Drive, Stanford University School of Medicine , Stanford, CA 94305, USA 2 Department of Biology, Sinsheimer Laboratories, University of California , Santa Cruz, CA 95064, USA - Defects in mouse mammary gland development caused by conditional haploinsufficiency of Patched-1 SUMMARY In vertebrates, the hedgehog family of cell signaling proteins and associated downstream network components play an essential role in mediating tissue interactions during development and organogenesis. Loss-of-function or misexpression mutation of hedgehog network components can cause birth defects, skin cancer and other tumors. The mammary gland is a specialized skin derivative requiring epithelial-epithelial and epithelialstromal tissue interactions similar to those required for development of other organs, where these interactions are often controled by hedgehog signaling. We have investigated the role of the Patched-1 (Ptc1) hedgehog receptor gene in mammary development and neoplasia. Haploinsufficiency at the Ptc1 locus results in severe histological defects in ductal structure, and minor morphological changes in terminal end buds in heterozygous postpubescent virgin animals. Defects are mainly ductal hyperplasias and dysplasias characterized by multilayered ductal walls and dissociated cells impacting ductal lumens. This phenotype is 100% penetrant. Mammary gland development (Fig. 1), like that of many organs, requires interactions between an epithelium and a surrounding mesenchyme (embryonic) or stroma (postnatal) (Cunha, 1994; Daniel and Silberstein, 1987; Howlett and Bissell, 1993; Imagawa et al., 1994; Russo and Russo, 1987; Sakakura, 1987; Schmeichel et al., 1998) and between epithelial cells themselves (Brisken et al., 1998). Such interactions control growth, govern overall patterning of the ductal tree, and influence the function of the gland. Most mammary development occurs in the subadult animal, where its embryonic-like growth characteristics can be readily examined and manipulated. This fact coupled with the similarities between tissue interactions critical to mammary gland development and those in other organs make the Remarkably, defects are reverted during late pregnancy and lactation but return upon involution and gland remodeling. Whole mammary gland transplants into athymic mice demonstrates that the observed dysplasias reflect an intrisic developmental defect within the gland. However, Ptc1-induced epithelial dysplasias are not stable upon transplantation into a wild-type epithelium-free fat pad, suggesting stromal (or epithelial and stromal) function of Ptc1. Mammary expression of Ptc1 mRNA is both epithelial and stromal and is developmentally regulated. Phenotypic reversion correlates with developmentally regulated and enhanced expression of Indian hedgehog (Ihh) during pregnancy and lactation. Data demonstrate a critical mammary role for at least one component of the hedgehog signaling network and suggest that Ihh is the primary hedgehog gene active in the gland. mammary gland an attractive model for the study of basic questions in developmental biology. Mouse mammary development begins at approximately embryonic day 10 (E10) (Fig. 1), with the definition of the nipple region and subsequent invasion of the underlying mammary mesenchyme by the presumptive mammary epithelium to establish a bulb of epithelial cells. After approximately E16, the bulb elongates and invades a second type of mesenchyme, the mammary fat pad precursor mesenchyme. The gland then initiates a small amount of ductal growth and branching morphogenesis, after which it becomes growth quiescent until puberty. Stimulated by ovarian hormones at puberty, the gland begins a proliferative phase of development, growing rapidly via the terminal end bud (TEB). The TEB is a bulb-like structure consisting of relatively undifferentiated epithelial cells at the tip Fig. 1. Phases of mammary gland development. Proliferative development in virgin animals is represented by the linear portion of the diagram from embryonic day 10 (E10) through maturity. Cyclical development initiated by pregnancy is represented by the circular portion of the diagram. of each growing duct, which invades and communicates with the fat pad stroma leaving differentiated ducts behind. In response to pregnancy, a cyclical phase of development is initiated in synchrony with the reproductive status of the animal. This cycle is characterized by growth and differentiation of secretory structures, lactation, and subsequent regression (involution) after weaning. At the end of involution, the morphology of the gland resembles that of the mature virgin animal. A promising candidate regulatory system for mediating the tissue interactions during mammary development is hedgehog signal transduction. In mammals, the genes encoding the hedgehog family of secreted signaling proteins (Sonic Hedgehog (Shh), Indian Hedgehog (Ihh), and Desert Hedgehog (Dhh)) and associated signaling network components are important regulators of cellular identity, patterning and tissue interactions during embryogenesis and organogenesis. These molecules are typically expressed in regions of inductive tissue interactions and are involved in diverse processes such as the development of skin, limbs, lung, eye, nervous system and tooth, the differentiation of cartilage and sperm, and the establishment of left-right asymmetry (Hammerschmidt et al., 1997; Ingham, 1998b; Levin, 1997). Whereas the range of vertebrate developmental processes dependent on hedgehog signaling testifies to its critical importance, the mechanics of hedgehog signaling are best understood from genetic studies in the fruitfly Drosophila melanogaster (Hammerschmidt et al., 1997; Ingham, 1998b). In flies, the signaling network consists of a single secreted hedgehog (HH) protein which binds to a receptor, patched (PTC), located in the membrane of nearby cells. In the absence of HH binding, PTC acts as a molecular brake to inhibit downstream signaling mediated by the smoothened (SMO) protein. Upon HH binding, PTC is inactivated allowing SMO to function. These events ultimately favor the conversion of a transcription factor, cubitus interruptus (CI) to a full-length activator form CI(act) over an alternative repressor form CI(rep). CI, in turn, controls expression of target genes that contribute to establishment of cell identity and to patterning of the fly body. In mammals the signaling network is more complex, with many of the fruitfly genes being duplicated to form multigene families (Ingham, 1998b). For example, instead of o (...truncated)