Neuron, Vol. 1, 605-613, September, 1988, Copyright 0 1988 by Cell Press Glucagon Gene Regulatory Region Directs Oncoprotein Expression to Neurons and Pancreatic a Cells Shimon Efrat: Gladys Teitelman,t Muhammad Anwar,+ David Ruggiero,+ and Douglas Hanahan* *Cold Spring Harbor Laboratory Cold Spring Harbor, New York 11724 + Division of Neurobiology Cornell University Medical College 411 East 69th Street New York, New York 10021 Summary The regulatory region of the rat preproglucagon gene targets expression of the SV40 large T oncoprotein to two cell types in transgenic mice, the pancreatic a cells and a set of neurons localized in the hindbrain, both of which normally produce preproglucagon. Addi- tional neurons in the forebrain and midbrain stain for T antigen but do not express the endogenous glucagon gene. Synthesis of T antigen in endocrine a cells results in the heritable development of pancreatic glucago- nomas. In brains of transgenic mice from three inde- pendent lineages, expression of the hybrid gene begins at embryonic day 12 in neuroblasts of the hindbrain, where it continues throughout adult life, most notably in the medulla. Remarkably, oncoprotein expression in both proliferating neuroblasts and mature neurons has no apparent consequences, either phenotypic or tumor- igenic. Expression of the hybrid glucagon gene in both neurons and islet cells supports a possible interrelation- ship between these cell types. Introduction Mature postmitotic neurons of the CNS originate from precursor neuroblasts in the neuroepithelium of the neural tube. After a certain number of cell divisions, which varies between regions of the tube, neuroblasts lose their pro- liferative ability and migrate into the mantle layer, where they differentiate into neurons (Jacobson, 1978). Thus, during embryonic development, the proliferative ability of each neuroblast in the CNS is determined in a manner that establishes the subsequent number, distribution, and synaptic connectivity of mature neurons. This pro- grammed transitory cell proliferation raises questions about the nature of the regulatory signals that control neuroblast proliferation, as well as the mechanism of their terminal differentiation into a postmitotic condi- tion. In contrast to the CNS, the control of cell division in neurons of the peripheral nervous system appears to be more relaxed. Peripheral neurons, which derive from the neural crest, remain capable of cell division after their differentiation (Rothman et al., 1980) and, moreover, can undergo neoplastic transformation. All known neu- roblastomas originate from neuroblasts of neural crest origin (Bolande, 1967). Tumors of neuroblasts originat- ing from the neural tube are virtually unknown, as are tumors of mature neurons in the CNS. This suggests that CNS neuroblasts and neurons may be unusually resis- tant to factors which promote continuous cell prolifera- tion and neoplastic transformation. In recent years, transgenic mice have been employed to study tumorigenesis in vivo by targeting expression of oncogenes to particular cell types using cell-specific promoters (for review see Hanahan, 1986, 1988; Palmiter and Brinster, 1986; Cory and Adams, 1988). Although a number of genes expressed in neurons in the CNS have been isolated, the lack of characterized regulatory ele- ments that reliably direct neuron-specific transgene ex- pression (Rosenfeld et al., 1988) has thus far precluded the extention of oncogene studies to these ceils. This re- port evaluates the consequences of oncogene expres- sion in neurons of the CNS, given that we have observed that the regulatory region of the rat preproglucagon gene will target expression of a linked gene to a specific distri- bution of neurons. The T antigen (Tag) oncogene of SV40 has been employed in this study since it encodes a novel antigen that is also a potent oncoprotein shown to trans- form a wide variety of cell types in transgenic mice (re- viewed by Hanahan, 1986, 1988). The glucagon gene encodes a preprohormone that contains, in addition to glucagon, two glucagon-like pep- tides, GLP-I and GLP-II (Bell et al., 1983a, 1983b). Pro- teolytic processing in the a cells of the endocrine pan- creas gives rise primarily to glucagon and GLP-I. Both peptides are involved with insulin in maintaining serum glucose levels and in regulating carbohydrate metabo- lism. In several mammalian species, expression of pre- proglucagon is observed in rare cells of the intestinal mucosa (Hoist, 19831, where the major peptide prod- ucts are glycentin (a larger form of glucagon), GLP-I, and GLP-II (Mojsov et al., 1986). Glucagon peptides, similar to those found in the intestine, have also been detected in various regions of the brain using immunohistochemi- cal analysis (Tager et al., 1980; Hatton et al., 1982; Tager, 1984), which cannot unambiguously distinguish synthe- sis from uptake or cross-reactivity. In situ RNA hybridiza- tion has confirmed preproglucagon expression in only one of these regions, namely in neurons of the nucleus tractus solitarii (NTS), which are located in the brainstem (Han et al., 1986). Here we report that a fragment of the glucagon gene extending 850 bp upstream of the cap site directs cell-specific expression of Tag to neurons in the NTS and several other regions of the brain, as well as to pancreatic a cells. While expression in the pancreas re- sults in development of tumors, no abormalities have been observed in the CNS. Results Generation of CluZ-Tag Transgenic Mice A hybrid oncogene (GluZ-Tag) composed of the 5’- flanking region ofthe rat preproglucagon gene (Heinrich