Title: A Human Yeast Artificial Chromosome Containing the Multiple Endocrine Neoplasia Type 2B Ret Mutation Does Not Induce Medullary Thyroid Carcinoma but Does Support the Growth of Kidneys and Partially Rescues Enteric Nervous System Development in Ret-Deficient Mice
Abstract: We generated a line of transgenic mice using a yeast artificial chromosome containing the Ret mutation responsible for the multiple endocrine neoplasia type 2B syndrome (MEN 2B). The resulting animals did not develop any of the expected neoplasms associated with MEN 2B. Transgenic animals were then bred with animals lacking murine Ret (RetM) to further evaluate the function of human mutated Ret (RetH2B) in the murine context. Whereas mice lacking RetM exhibit intestinal aganglionosis and the absence of kidneys with other genitourinary anomalies, expression of the RetH2B transgene in RetM-deficient mice allowed significant renal development with a partial rescue of the enteric nervous system. These RetH2B-positive/RetM-deficient mice exhibit normal Ret expression and survive longer than RetM-deficient mice, but still die at 3 to 5 days of age with evidence of enterocolitis. We conclude that the normal expression of a human Ret proto-oncogene with the MEN 2B mutation does not cause any features of MEN 2B in mice. Although the gene is normally expressed in the appropriate target tissues, there is incomplete phenotypic rescue in mice lacking murine Ret. These results suggest important interspecies differences between humans and mice in the function of the Ret oncogene. We generated a line of transgenic mice using a yeast artificial chromosome containing the Ret mutation responsible for the multiple endocrine neoplasia type 2B syndrome (MEN 2B). The resulting animals did not develop any of the expected neoplasms associated with MEN 2B. Transgenic animals were then bred with animals lacking murine Ret (RetM) to further evaluate the function of human mutated Ret (RetH2B) in the murine context. Whereas mice lacking RetM exhibit intestinal aganglionosis and the absence of kidneys with other genitourinary anomalies, expression of the RetH2B transgene in RetM-deficient mice allowed significant renal development with a partial rescue of the enteric nervous system. These RetH2B-positive/RetM-deficient mice exhibit normal Ret expression and survive longer than RetM-deficient mice, but still die at 3 to 5 days of age with evidence of enterocolitis. We conclude that the normal expression of a human Ret proto-oncogene with the MEN 2B mutation does not cause any features of MEN 2B in mice. Although the gene is normally expressed in the appropriate target tissues, there is incomplete phenotypic rescue in mice lacking murine Ret. These results suggest important interspecies differences between humans and mice in the function of the Ret oncogene. The Ret proto-oncogene encodes a receptor tyrosine kinase that regulates the growth and development within the neurological and excretory systems.1Avantaggiato V Dathan NA Grieco M Fabien N Lazzaro D Fusco A Simeone A Santoro M Developmental expression of the RET protooncogene.Cell Growth Dev. 1994; 5: 305-311PubMed Google Scholar, 2Durbec PL Larsson-Blomberg LB Schuchardt A Costantini F Pachnis V Common origin and developmental dependence on c-ret of subsets of enteric and sympathetic neuroblasts.Development. 1996; 122: 349-358PubMed Google Scholar, 3Pachnis V Mankoo B Costantini F Expression of the c-ret proto-oncogene during mouse embryogenesis.Development. 1993; 119: 1005-1017Crossref PubMed Google Scholar The gene was discovered using the NIH3T3 transfection assay and the name Ret derives from this discovery (rearranged during transfection).4Takahashi M Ritz J Cooper GM Activation of a novel human transforming gene, ret, by DNA rearrangement.Cell. 1985; 42: 581-588Abstract Full Text PDF PubMed Scopus (665) Google Scholar More recently, it was discovered that certain germ line mutations in the Ret proto-oncogene are associated with the multiple endocrine neoplasia (MEN) syndromes types 2A and 2B and familial medullary thyroid carcinoma (FMTC).5Carlson KM Dou S Chi D Scavarda N Toshima T Jackson CE Wells Jr, SA Goodfellow PJ Donis-Keller H Single missense mutation in the tyrosine kinase domain of the RET protooncogene is associated with multiple endocrine neoplasia type 2B.Proc Natl Acad Sci USA. 1994; 91: 1579-1583Crossref PubMed Scopus (579) Google Scholar, 6Donis-Keller H Dou S Chi D Carlson KM Toshima K Lairmore TC Howe JR Moley JF Goodfellow P Wells Jr, SA Mutations in the RET proto-oncogene are associated with MEN 2A and FMTC.Hum Mol Genet. 1993; 2: 851-856Crossref PubMed Scopus (1211) Google Scholar, 7Eng C Smith DP Mulligan LM Nagal MA Healey CS Ponder MA Gardner E Scheumann GFW Jackson CE Tunacliffe A Ponder BAJ Point mutation within the tyrosine kinase domain of the RET proto-oncogene in multiple endocrine neoplasia type 2B and related sporadic tumours.Hum Mol Genet. 1994; 3: 237-241Crossref PubMed Scopus (516) Google Scholar, 8Hofstra RMW Landsvater RM Ceccherini I Stulp RP Stelwagon T Luo Y Pasini B Hoppener JWM van Amstel HKP Romeo G Lips CJM Buys CHCM A mutation in the RET proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma.Nature. 1994; 367: 375-376Crossref PubMed Scopus (1068) Google Scholar, 9Mulligan LM Kwok JBJ Healey CS Elsdon MJ Eng C Gardner E Love DR Mole SE Moore JK Papi L Ponder MA Telenius H Tunnacliffe A Ponder BAJ Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A.Nature. 1993; 363: 458-460Crossref PubMed Scopus (1794) Google Scholar These syndromes are inherited in an autosomal dominant manner. Patients with MEN 2A develop medullary thyroid carcinomas (MTCs), pheochromocytomas, and hyperparathyroidism; similarly, those with MEN 2B also develop MTCs and pheochromocytomas, but additionally develop mucosal neuromas of the lips, oral mucosa, and alimentary tract. 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Indeed, inactivating Ret mutations are also the most common defined cause of Hirschsprung's disease in humans. The importance of Ret in the development of the nervous system was further demonstrated when the other Ret ligands persephin, artemin, and neuturin were characterized and demonstrated to interact with Ret to support the growth of neurons in the peripheral, central, and enteric nervous systems (ENSs).19Schuchardt A D'Agati V Larsson-Blomberg L Constantini F Pachnis V Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret.Nature. 1994; 367: 380-383Crossref PubMed Scopus (1447) Google Scholar, 20Baloh RH Tansey MG Lampe PA Fahrner TJ Enomoto H Simburger KS Leitner ML Araki T Johnson EM Milbrandt J Artemin, a novel member of the GDNF ligand family, supports peripheral and central neurons and signals through the GFRα3-RET receptor complex.Neuron. 1998; 21: 1291-1302Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 21Heuckeroth RO Enomoto H Grider JR Golden JP Hanke JA Jackman A Molliver DC Bardgett ME Snider WD Johnson EM Milbrandt J Gene targeting reveals a critical role for Neurturin in the development and maintenance of enteric, sensory, and parasympathetic neurons.Neuron. 1999; 2: 253-263Abstract Full Text Full Text PDF Scopus (280) Google Scholar, 22Kotzbauer PT Lampe PA Heukeroth RO Golden JP Creedon DJ Johnson Jr, EM Milbrandt J Neurturin, a relative of glial-cell-line-derived neurotrophic factor.Nature. 1996; 384: 467-470Crossref PubMed Scopus (660) Google Scholar, 23Milbrandt J de Sauvage FJ Fahrner TJ Baloh RH Leitner ML Tansey MG Lampe PA Heuckeroth RO Kotzbauer PT Simburger KS Golden JP Davies JA Vejsada R Kato AC Hynes M Sherman D Nishimura M Wang L-C Vandlen R Moffat B Klein RD Poulsen K Gray C Garces A Henderson CE Phillips HS Johnson EM Persephin, a novel neurotrophic factor related to GDNF and neuturin.Neuron. 1998; 20: 245-253Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar Recently, to study the dominant transforming activity of the MEN 2B mutation of Ret, investigators created a transgenic mouse line containing the equivalent codon 918 Met to Thr mutation in the mouse Ret gene.24Smith-Hicks CL Sizer KC Powers JF Tischler AS Costantini F C-cell hyperplasia, pheochromocytoma and sympathoadrenal malformation in a mouse model of multiple endocrine neoplasia type 2B.EMBO J. 2000; 19: 612-622Crossref PubMed Scopus (123) Google Scholar Interestingly, these mice did not precisely model the human phenotype. Whereas they developed thyroid C-cell hyperplasia and pheochromocytomas, the mice did not develop MTC. Moreover, the animals did not possess the neuromas of the lips, oral mucosa, or gastrointestinal tract found in humans with MEN 2B. These results suggest that there are species differences between humans and mice that are responsible for the different phenotypes associated with the MEN 2B mutation of Ret. However, Ret with the MEN 2B mutation does support normal development of the kidney and ENS, even in mice homozygous for MEN 2B.24Smith-Hicks CL Sizer KC Powers JF Tischler AS Costantini F C-cell hyperplasia, pheochromocytoma and sympathoadrenal malformation in a mouse model of multiple endocrine neoplasia type 2B.EMBO J. 2000; 19: 612-622Crossref PubMed Scopus (123) Google Scholar In contrast to the above mouse model, two other studies25Sweetser DA Froelick GJ Matsumoto AM Kafer KE Marck B Palmiter RD Kapur RP Ganglio neuromas and renal anomalies are induced by activated RETMEN2B in transgenic mice.Oncogene. 1999; 18: 877-886Crossref PubMed Scopus (40) Google Scholar, 26Gestblom C Sweetser DA Doggett B Kapur RP Sympathoadrenal hyperplasia causes renal malformations in RETMEN2B-transgenic mice.Am J Pathol. 1999; 155: 2167-2179Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar found that overexpression of a human MEN 2B transgene causes developmental renal malformations. In an attempt to create a mouse model of MEN 2B that recapitulates all of the sequelae of the human disease, we created a line of transgenic mice possessing the human Ret gene with the codon 918 Met to Thr mutation responsible for MEN 2B. To assure faithful expression of the gene through embryonic development and in the appropriate tissues, we used a yeast-associated chromosome (YAC) containing the human Ret gene with the MEN 2B mutation (designated RetH2B). This allows the inclusion of a considerable of amount of genetic material upstream and downstream from the coding elements of the gene, to ensure the inclusion of genetic elements responsible for the regulation of Ret expression. These transgenic animals were followed to see whether they developed the features of the human MEN 2B syndromes. To further evaluate the functionality of the human transgene, we bred these RetH2B transgenic mice to animals lacking one murine Ret allele (designated RetM), and then performed the genetic backcrosses to create mice that only express RetH2B. Our aims in this portion of the study were to determine whether the RetH2B gene corrects the phenotype associated with the RetM knockout and to investigate whether the human Ret gene with the MEN 2B mutation, expressed outside the context of the mouse Ret gene, impacts the development of tumors associated with MEN 2B.