Abstract
To determine the role of L-Myc in normal mammalian development and its functional relationship to other members of the Myc family, we determined the normal patterns of L-myc gene expression in the developing mouse by RNA in situ hybridization and assessed the phenotypic impact of L-Myc deficiency produced through standard gene targeting methodology. L-myc transcripts were detected in the developing kidney and lung as well as in both the proliferative and the differentiative zones of the brain and neural tube. Despite significant expression of L-myc in developing mouse tissue, homozygous null L-myc mice were found to be viable, reproductively competent, and represented in expected frequencies from heterozygous matings. A detailed histological survey of embryonic and adult tissues, characterization of an embryonic neuronal marker, and measurement of cellular proliferation in situ did not reveal any congenital abnormalities. The lack of an apparent phenotype associated with L-Myc deficiency indicates that L-Myc is dispensable for gross morphological development and argues against a unique role for L-Myc in early central nervous system development as had been previously suggested. Although overlapping expression patterns among myc family members raise the possibility of complementation of L-Myc deficiency by other Myc oncoproteins, compensatory changes in the levels of c- and/or N-myc transcripts were not detected in homozygous null L-myc mice.
Full Text
The Full Text of this article is available as a PDF (2.1 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Amati B., Brooks M. W., Levy N., Littlewood T. D., Evan G. I., Land H. Oncogenic activity of the c-Myc protein requires dimerization with Max. Cell. 1993 Jan 29;72(2):233–245. doi: 10.1016/0092-8674(93)90663-b. [DOI] [PubMed] [Google Scholar]
- Amati B., Dalton S., Brooks M. W., Littlewood T. D., Evan G. I., Land H. Transcriptional activation by the human c-Myc oncoprotein in yeast requires interaction with Max. Nature. 1992 Oct 1;359(6394):423–426. doi: 10.1038/359423a0. [DOI] [PubMed] [Google Scholar]
- Auffray C., Rougeon F. Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur J Biochem. 1980 Jun;107(2):303–314. doi: 10.1111/j.1432-1033.1980.tb06030.x. [DOI] [PubMed] [Google Scholar]
- Barrett J., Birrer M. J., Kato G. J., Dosaka-Akita H., Dang C. V. Activation domains of L-Myc and c-Myc determine their transforming potencies in rat embryo cells. Mol Cell Biol. 1992 Jul;12(7):3130–3137. doi: 10.1128/mcb.12.7.3130. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Benvenisty N., Leder A., Kuo A., Leder P. An embryonically expressed gene is a target for c-Myc regulation via the c-Myc-binding sequence. Genes Dev. 1992 Dec;6(12B):2513–2523. doi: 10.1101/gad.6.12b.2513. [DOI] [PubMed] [Google Scholar]
- Berberich S. J., Cole M. D. Casein kinase II inhibits the DNA-binding activity of Max homodimers but not Myc/Max heterodimers. Genes Dev. 1992 Feb;6(2):166–176. doi: 10.1101/gad.6.2.166. [DOI] [PubMed] [Google Scholar]
- Bernard O., Drago J., Sheng H. L-myc and N-myc influence lineage determination in the central nervous system. Neuron. 1992 Dec;9(6):1217–1224. doi: 10.1016/0896-6273(92)90079-s. [DOI] [PubMed] [Google Scholar]
- Birrer M. J., Raveh L., Dosaka H., Segal S. A transfected L-myc gene can substitute for c-myc in blocking murine erythroleukemia differentiation. Mol Cell Biol. 1989 Jun;9(6):2734–2737. doi: 10.1128/mcb.9.6.2734. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Birrer M. J., Segal S., DeGreve J. S., Kaye F., Sausville E. A., Minna J. D. L-myc cooperates with ras to transform primary rat embryo fibroblasts. Mol Cell Biol. 1988 Jun;8(6):2668–2673. doi: 10.1128/mcb.8.6.2668. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blackwell T. K., Kretzner L., Blackwood E. M., Eisenman R. N., Weintraub H. Sequence-specific DNA binding by the c-Myc protein. Science. 1990 Nov 23;250(4984):1149–1151. doi: 10.1126/science.2251503. [DOI] [PubMed] [Google Scholar]
- Blackwood E. M., Eisenman R. N. Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science. 1991 Mar 8;251(4998):1211–1217. doi: 10.1126/science.2006410. [DOI] [PubMed] [Google Scholar]
- Blackwood E. M., Lüscher B., Eisenman R. N. Myc and Max associate in vivo. Genes Dev. 1992 Jan;6(1):71–80. doi: 10.1101/gad.6.1.71. [DOI] [PubMed] [Google Scholar]
- Büeler H., Fischer M., Lang Y., Bluethmann H., Lipp H. P., DeArmond S. J., Prusiner S. B., Aguet M., Weissmann C. Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature. 1992 Apr 16;356(6370):577–582. doi: 10.1038/356577a0. [DOI] [PubMed] [Google Scholar]
- Charron J., Malynn B. A., Fisher P., Stewart V., Jeannotte L., Goff S. P., Robertson E. J., Alt F. W. Embryonic lethality in mice homozygous for a targeted disruption of the N-myc gene. Genes Dev. 1992 Dec;6(12A):2248–2257. doi: 10.1101/gad.6.12a.2248. [DOI] [PubMed] [Google Scholar]
- Chiu F. C., Barnes E. A., Das K., Haley J., Socolow P., Macaluso F. P., Fant J. Characterization of a novel 66 kd subunit of mammalian neurofilaments. Neuron. 1989 May;2(5):1435–1445. doi: 10.1016/0896-6273(89)90189-x. [DOI] [PubMed] [Google Scholar]
- Davis A. C., Wims M., Spotts G. D., Hann S. R., Bradley A. A null c-myc mutation causes lethality before 10.5 days of gestation in homozygotes and reduced fertility in heterozygous female mice. Genes Dev. 1993 Apr;7(4):671–682. doi: 10.1101/gad.7.4.671. [DOI] [PubMed] [Google Scholar]
- DePinho R. A., Hatton K. S., Tesfaye A., Yancopoulos G. D., Alt F. W. The human myc gene family: structure and activity of L-myc and an L-myc pseudogene. Genes Dev. 1987 Dec;1(10):1311–1326. doi: 10.1101/gad.1.10.1311. [DOI] [PubMed] [Google Scholar]
- DePinho R. A., Legouy E., Feldman L. B., Kohl N. E., Yancopoulos G. D., Alt F. W. Structure and expression of the murine N-myc gene. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1827–1831. doi: 10.1073/pnas.83.6.1827. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DePinho R. A., Schreiber-Agus N., Alt F. W. myc family oncogenes in the development of normal and neoplastic cells. Adv Cancer Res. 1991;57:1–46. doi: 10.1016/s0065-230x(08)60994-x. [DOI] [PubMed] [Google Scholar]
- Downs K. M., Martin G. R., Bishop J. M. Contrasting patterns of myc and N-myc expression during gastrulation of the mouse embryo. Genes Dev. 1989 Jun;3(6):860–869. doi: 10.1101/gad.3.6.860. [DOI] [PubMed] [Google Scholar]
- Evan G. I., Littlewood T. D. The role of c-myc in cell growth. Curr Opin Genet Dev. 1993 Feb;3(1):44–49. doi: 10.1016/s0959-437x(05)80339-9. [DOI] [PubMed] [Google Scholar]
- Galinović-Schwartz V., Peng D., Chiu F. C., Van de Water T. R. Temporal pattern of innervation in the developing mouse inner ear: an immunocytochemical study of a 66-kD subunit of mammalian neurofilaments. J Neurosci Res. 1991 Sep;30(1):124–133. doi: 10.1002/jnr.490300114. [DOI] [PubMed] [Google Scholar]
- Halazonetis T. D., Kandil A. N. Determination of the c-MYC DNA-binding site. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6162–6166. doi: 10.1073/pnas.88.14.6162. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hellsten E., Vesa J., Speer M. C., Mäkelä T. P., Järvelä I., Alitalo K., Ott J., Peltonen L. Refined assignment of the infantile neuronal ceroid lipofuscinosis (INCL, CLN1) locus at 1p32: incorporation of linkage disequilibrium in multipoint analysis. Genomics. 1993 Jun;16(3):720–725. doi: 10.1006/geno.1993.1253. [DOI] [PubMed] [Google Scholar]
- Jolly R. D., Martinus R. D., Palmer D. N. Sheep and other animals with ceroid-lipofuscinoses: their relevance to Batten disease. Am J Med Genet. 1992 Feb 15;42(4):609–614. doi: 10.1002/ajmg.1320420436. [DOI] [PubMed] [Google Scholar]
- Kato G. J., Barrett J., Villa-Garcia M., Dang C. V. An amino-terminal c-myc domain required for neoplastic transformation activates transcription. Mol Cell Biol. 1990 Nov;10(11):5914–5920. doi: 10.1128/mcb.10.11.5914. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kato G. J., Lee W. M., Chen L. L., Dang C. V. Max: functional domains and interaction with c-Myc. Genes Dev. 1992 Jan;6(1):81–92. doi: 10.1101/gad.6.1.81. [DOI] [PubMed] [Google Scholar]
- Kaye F., Battey J., Nau M., Brooks B., Seifter E., De Greve J., Birrer M., Sausville E., Minna J. Structure and expression of the human L-myc gene reveal a complex pattern of alternative mRNA processing. Mol Cell Biol. 1988 Jan;8(1):186–195. doi: 10.1128/mcb.8.1.186. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kerkhoff E., Bister K., Klempnauer K. H. Sequence-specific DNA binding by Myc proteins. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4323–4327. doi: 10.1073/pnas.88.10.4323. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kohl N. E., Kanda N., Schreck R. R., Bruns G., Latt S. A., Gilbert F., Alt F. W. Transposition and amplification of oncogene-related sequences in human neuroblastomas. Cell. 1983 Dec;35(2 Pt 1):359–367. doi: 10.1016/0092-8674(83)90169-1. [DOI] [PubMed] [Google Scholar]
- Kohl N. E., Legouy E., DePinho R. A., Nisen P. D., Smith R. K., Gee C. E., Alt F. W. Human N-myc is closely related in organization and nucleotide sequence to c-myc. Nature. 1986 Jan 2;319(6048):73–77. doi: 10.1038/319073a0. [DOI] [PubMed] [Google Scholar]
- Kretzner L., Blackwood E. M., Eisenman R. N. Myc and Max proteins possess distinct transcriptional activities. Nature. 1992 Oct 1;359(6394):426–429. doi: 10.1038/359426a0. [DOI] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- Land H., Parada L. F., Weinberg R. A. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature. 1983 Aug 18;304(5927):596–602. doi: 10.1038/304596a0. [DOI] [PubMed] [Google Scholar]
- Legouy E., DePinho R., Zimmerman K., Collum R., Yancopoulos G., Mitsock L., Kriz R., Alt F. W. Structure and expression of the murine L-myc gene. EMBO J. 1987 Nov;6(11):3359–3366. doi: 10.1002/j.1460-2075.1987.tb02657.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lüscher B., Eisenman R. N. New light on Myc and Myb. Part I. Myc. Genes Dev. 1990 Dec;4(12A):2025–2035. doi: 10.1101/gad.4.12a.2025. [DOI] [PubMed] [Google Scholar]
- Ma A., Moroy T., Collum R., Weintraub H., Alt F. W., Blackwell T. K. DNA binding by N- and L-Myc proteins. Oncogene. 1993 Apr;8(4):1093–1098. [PubMed] [Google Scholar]
- Mackem S., Mahon K. A. Ghox 4.7: a chick homeobox gene expressed primarily in limb buds with limb-type differences in expression. Development. 1991 Jul;112(3):791–806. doi: 10.1242/dev.112.3.791. [DOI] [PubMed] [Google Scholar]
- Mansour S. L., Thomas K. R., Capecchi M. R. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature. 1988 Nov 24;336(6197):348–352. doi: 10.1038/336348a0. [DOI] [PubMed] [Google Scholar]
- Moens C. B., Auerbach A. B., Conlon R. A., Joyner A. L., Rossant J. A targeted mutation reveals a role for N-myc in branching morphogenesis in the embryonic mouse lung. Genes Dev. 1992 May;6(5):691–704. doi: 10.1101/gad.6.5.691. [DOI] [PubMed] [Google Scholar]
- Morgenbesser S. D., DePinho R. A. Use of transgenic mice to study myc family gene function in normal mammalian development and in cancer. Semin Cancer Biol. 1994 Feb;5(1):21–36. [PubMed] [Google Scholar]
- Morgenbesser S. D., Schreiber-Agus N., Bidder M., Mahon K. A., Overbeek P. A., Horner J., DePinho R. A. Contrasting roles for c-Myc and L-Myc in the regulation of cellular growth and differentiation in vivo. EMBO J. 1995 Feb 15;14(4):743–756. doi: 10.1002/j.1460-2075.1995.tb07053.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mugrauer G., Alt F. W., Ekblom P. N-myc proto-oncogene expression during organogenesis in the developing mouse as revealed by in situ hybridization. J Cell Biol. 1988 Oct;107(4):1325–1335. doi: 10.1083/jcb.107.4.1325. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mugrauer G., Ekblom P. Contrasting expression patterns of three members of the myc family of protooncogenes in the developing and adult mouse kidney. J Cell Biol. 1991 Jan;112(1):13–25. doi: 10.1083/jcb.112.1.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mukherjee B., Morgenbesser S. D., DePinho R. A. Myc family oncoproteins function through a common pathway to transform normal cells in culture: cross-interference by Max and trans-acting dominant mutants. Genes Dev. 1992 Aug;6(8):1480–1492. doi: 10.1101/gad.6.8.1480. [DOI] [PubMed] [Google Scholar]
- Möröy T., Fisher P. E., Lee G., Achacoso P., Wiener F., Alt F. W. High frequency of myelomonocytic tumors in aging E mu L-myc transgenic mice. J Exp Med. 1992 Feb 1;175(2):313–322. doi: 10.1084/jem.175.2.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Möröy T., Fisher P., Guidos C., Ma A., Zimmerman K., Tesfaye A., DePinho R., Weissman I., Alt F. W. IgH enhancer deregulated expression of L-myc: abnormal T lymphocyte development and T cell lymphomagenesis. EMBO J. 1990 Nov;9(11):3659–3666. doi: 10.1002/j.1460-2075.1990.tb07577.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nau M. M., Brooks B. J., Battey J., Sausville E., Gazdar A. F., Kirsch I. R., McBride O. W., Bertness V., Hollis G. F., Minna J. D. L-myc, a new myc-related gene amplified and expressed in human small cell lung cancer. Nature. 1985 Nov 7;318(6041):69–73. doi: 10.1038/318069a0. [DOI] [PubMed] [Google Scholar]
- Peterson G. L. Determination of total protein. Methods Enzymol. 1983;91:95–119. doi: 10.1016/s0076-6879(83)91014-5. [DOI] [PubMed] [Google Scholar]
- Prendergast G. C., Lawe D., Ziff E. B. Association of Myn, the murine homolog of max, with c-Myc stimulates methylation-sensitive DNA binding and ras cotransformation. Cell. 1991 May 3;65(3):395–407. doi: 10.1016/0092-8674(91)90457-a. [DOI] [PubMed] [Google Scholar]
- Prendergast G. C., Ziff E. B. Methylation-sensitive sequence-specific DNA binding by the c-Myc basic region. Science. 1991 Jan 11;251(4990):186–189. doi: 10.1126/science.1987636. [DOI] [PubMed] [Google Scholar]
- Rapola J., Järvelä I., Peltonen L. The neuronal ceroid lipofuscinoses: unfolding the genetic defect. Pediatr Pathol. 1991 Nov-Dec;11(6):799–806. doi: 10.3109/15513819109065477. [DOI] [PubMed] [Google Scholar]
- Rudnicki M. A., Braun T., Hinuma S., Jaenisch R. Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development. Cell. 1992 Oct 30;71(3):383–390. doi: 10.1016/0092-8674(92)90508-a. [DOI] [PubMed] [Google Scholar]
- Saga Y., Yagi T., Ikawa Y., Sakakura T., Aizawa S. Mice develop normally without tenascin. Genes Dev. 1992 Oct;6(10):1821–1831. doi: 10.1101/gad.6.10.1821. [DOI] [PubMed] [Google Scholar]
- Sawai S., Shimono A., Wakamatsu Y., Palmes C., Hanaoka K., Kondoh H. Defects of embryonic organogenesis resulting from targeted disruption of the N-myc gene in the mouse. Development. 1993 Apr;117(4):1445–1455. doi: 10.1242/dev.117.4.1445. [DOI] [PubMed] [Google Scholar]
- Schmid P., Schulz W. A., Hameister H. Dynamic expression pattern of the myc protooncogene in midgestation mouse embryos. Science. 1989 Jan 13;243(4888):226–229. doi: 10.1126/science.2911736. [DOI] [PubMed] [Google Scholar]
- Schreiber-Agus N., Horner J., Torres R., Chiu F. C., DePinho R. A. Zebra fish myc family and max genes: differential expression and oncogenic activity throughout vertebrate evolution. Mol Cell Biol. 1993 May;13(5):2765–2775. doi: 10.1128/mcb.13.5.2765. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schreiber-Agus N., Torres R., Horner J., Lau A., Jamrich M., DePinho R. A. Comparative analysis of the expression and oncogenic activities of Xenopus c-, N-, and L-myc homologs. Mol Cell Biol. 1993 Apr;13(4):2456–2468. doi: 10.1128/mcb.13.4.2456. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schwab M., Alitalo K., Klempnauer K. H., Varmus H. E., Bishop J. M., Gilbert F., Brodeur G., Goldstein M., Trent J. Amplified DNA with limited homology to myc cellular oncogene is shared by human neuroblastoma cell lines and a neuroblastoma tumour. Nature. 1983 Sep 15;305(5931):245–248. doi: 10.1038/305245a0. [DOI] [PubMed] [Google Scholar]
- Schwab M., Varmus H. E., Bishop J. M. Human N-myc gene contributes to neoplastic transformation of mammalian cells in culture. Nature. 1985 Jul 11;316(6024):160–162. doi: 10.1038/316160a0. [DOI] [PubMed] [Google Scholar]
- Sheiness D. K., Hughes S. H., Varmus H. E., Stubblefield E., Bishop J. M. The vertebrate homolog of the putative transforming gene of avian myelocytomatosis virus: characteristics of the DNA locus and its RNA transcript. Virology. 1980 Sep;105(2):415–424. doi: 10.1016/0042-6822(80)90042-2. [DOI] [PubMed] [Google Scholar]
- Sheiness D., Bishop J. M. DNA and RNA from uninfected vertebrate cells contain nucleotide sequences related to the putative transforming gene of avian myelocytomatosis virus. J Virol. 1979 Aug;31(2):514–521. doi: 10.1128/jvi.31.2.514-521.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stanton B. R., Perkins A. S., Tessarollo L., Sassoon D. A., Parada L. F. Loss of N-myc function results in embryonic lethality and failure of the epithelial component of the embryo to develop. Genes Dev. 1992 Dec;6(12A):2235–2247. doi: 10.1101/gad.6.12a.2235. [DOI] [PubMed] [Google Scholar]
- Stanton L. W., Fahrlander P. D., Tesser P. M., Marcu K. B. Nucleotide sequence comparison of normal and translocated murine c-myc genes. Nature. 1984 Aug 2;310(5976):423–425. doi: 10.1038/310423a0. [DOI] [PubMed] [Google Scholar]
- Torres R., Schreiber-Agus N., Morgenbesser S. D., DePinho R. A. Myc and Max: a putative transcriptional complex in search of a cellular target. Curr Opin Cell Biol. 1992 Jun;4(3):468–474. doi: 10.1016/0955-0674(92)90013-3. [DOI] [PubMed] [Google Scholar]
- Tso J. Y., Sun X. H., Kao T. H., Reece K. S., Wu R. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res. 1985 Apr 11;13(7):2485–2502. doi: 10.1093/nar/13.7.2485. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vesa J., Hellsten E., Verkruyse L. A., Camp L. A., Rapola J., Santavuori P., Hofmann S. L., Peltonen L. Mutations in the palmitoyl protein thioesterase gene causing infantile neuronal ceroid lipofuscinosis. Nature. 1995 Aug 17;376(6541):584–587. doi: 10.1038/376584a0. [DOI] [PubMed] [Google Scholar]
- Wenzel A., Cziepluch C., Hamann U., Schürmann J., Schwab M. The N-Myc oncoprotein is associated in vivo with the phosphoprotein Max(p20/22) in human neuroblastoma cells. EMBO J. 1991 Dec;10(12):3703–3712. doi: 10.1002/j.1460-2075.1991.tb04938.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Xu L., Wallen R., Patel V., DePinho R. A. Role of first exon/intron sequences in the regulation of myc family oncogenic potency. Oncogene. 1993 Sep;8(9):2547–2553. [PubMed] [Google Scholar]
- Yancopoulos G. D., Nisen P. D., Tesfaye A., Kohl N. E., Goldfarb M. P., Alt F. W. N-myc can cooperate with ras to transform normal cells in culture. Proc Natl Acad Sci U S A. 1985 Aug;82(16):5455–5459. doi: 10.1073/pnas.82.16.5455. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yin Y., Tainsky M. A., Bischoff F. Z., Strong L. C., Wahl G. M. Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles. Cell. 1992 Sep 18;70(6):937–948. doi: 10.1016/0092-8674(92)90244-7. [DOI] [PubMed] [Google Scholar]
- Zimmerman K. A., Yancopoulos G. D., Collum R. G., Smith R. K., Kohl N. E., Denis K. A., Nau M. M., Witte O. N., Toran-Allerand D., Gee C. E. Differential expression of myc family genes during murine development. 1986 Feb 27-Mar 5Nature. 319(6056):780–783. doi: 10.1038/319780a0. [DOI] [PubMed] [Google Scholar]