Defects in heart and lung development in compound heterozygotes for two different targeted mutations at the N-myc locus

Cecilia B. Moens 0 2 Brian R. Stanton 1 Luis F. Parada 1 Janet Rossant 0 2 0 Department of Molecular and Medical Genetics, University of Toronto 1 Molecular Embryology Section, ABL-Basic Research Program, National Cancer Institute - Frederick Cancer Research and Development Center , Frederick, Maryland 21702-1201 , USA 2 Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, 600 University Ave , Toronto, Ontario, M5G 1X5 , Canada *Author for correspondence - Defects in heart and lung development in compound heterozygotes for two different targeted mutations at the N-myc locus Two types of mutant allele, one leaky and one null, have been generated by gene targeting at the N-myc locus in embryonic stem cells and the phenotypes of mice homozygous for these mutations have been described. These mutations have shown that N-myc has a number of functions during development, including a role in branching morphogenesis in the lung, which manifests itself at birth in mice homozygous for the leaky allele, and roles in the development of the mesonephric tubules, the neuroepithelium, the sensory ganglia, the gut and the heart, which become evident at midgestation in embryos homozygous for the null allele. In an attempt to define roles for N-myc at other stages of development, we have combined the two types of N-myc mutant allele in a compound heterozygote that as a result contains approximately 15% of normal levels of N-Myc protein. Compound heterozygotes died during gestation at a time N-myc is a member of the myc family of proto-oncogenes, which includes N-myc, c-myc, and L-myc. Myc proteins are site-specific DNA-binding proteins (Blackwell et al., 1990; Prendergast and Ziff, 1991; Alex et al., 1992), belonging to the basic-helix-loop-helix class of transcription factors, which includes genes that control cell fate determination in such diverse processes as myogenesis, neurogenesis and sex determination (reviewed in Garrell and Campuzano, 1991). Deregulated expression of myc genes has been implicated in the genesis or progression of a number of naturally occurring tumours, in the transformation of cells in culture and in the formation of tumours in transgenic mice (reviewed in DePinho et al., 1991). In general, the sites of expression of a given myc gene in vivo reflect the types of tumours associated with its elevated expression. Thus Nmyc is expressed predominantly in the embryo where it is restricted to undifferentiated subsets of cells in the central and peripheral nervous system, lung, kidney and eye intermediate to the times of death of embryos homozygous for either mutation individually, and their death appeared to result from cardiac failure stemming from hypoplasia of the compact subepicardial layer of the myocardium. Investigation of the expression pattern of N-myc and various markers of differentiation in wildtype and compound heterozygote mutant hearts has suggested that N-myc may function in maintaining the proliferation and/or preventing the differentiation of compact layer myocytes. This study illustrates the importance of generating different mutations at a given locus to elucidate fully the function of a particular gene during development. (Mugrauer et al., 1988; Hirning et al., 1991; Zimmerman et al., 1986) and overexpression of N-myc has been associated with tumours of embryonic origin such as neuroblastoma (Kohl et al., 1983; Schwab et al., 1983), small-cell lung cancer (Nau et al., 1986; Wong et al., 1986), Wilms tumour (Nisen et al., 1986) and retinoblastoma (Lee et al., 1984). N-myc is also expressed in the skin (Mugrauer et al., 1988), in the epithelial layer of the intestine (Hirning et al., 1991) and, earlier in development, in the heart, sclerotome and visceral arches (Katoh et al., 1991). The functioning of a Myc protein in vivo should depend not only on its own level of expression, but also on the levels of Max, a protein which, like the Myc proteins, possesses a basic-helix loop helix-leucine zipper (bHLH-LZ) domain (Blackwood and Eisenman, 1991), and which associates with N-Myc, L-Myc and c-Myc proteins in vivo (Blackwood et al., 1992; Wenzel et al., 1991; Mukherjee et al., 1992). Max is required for specific DNA binding by Myc proteins (Blackwood and Eisenman, 1991; Prendergast and Ziff, 1991; Kato et al., 1992; Barrett et al., 1992), and has been shown to be required for transcriptional activation (Amati et al., 1992) and transformation (Amati et al., 1993) by c-myc. Unlike Myc proteins, Max is able to form homodimers in vitro and thereby to bind the myc-binding site (Prendergast and Ziff, 1991; Kato et al., 1992). However Max does not transactivate downstream genes on its own (Amati et al., 1992; Kretzner et al., 1992) because it lacks a transactivation domain (Kato et al., 1992) which Myc proteins possess (Kato et al., 1990). Both transformation of fibroblasts by c-myc and N-myc (Mukherjee et al., 1992; Makela et al., 1992; Prendergast et al., 1992) and transcriptional transactivation by c-myc (Amati et al., 1992; Kretzner et al., 1992) have been shown to be enhanced by low levels of Max and inhibited by excess Max, suggesting that Myc function is indeed influenced by levels of Max. Recently, bHLH-LZ proteins have been isolated which also bind the Myc recognition site and which suppress transcription as heterodimers with Max (Ayer et al., 1993; Zervos et al., 1993). One of these, Mad (Ayer et al., 1993), has been shown to compete with Myc for Max in vitro and in transfected cells. Thus high levels of expression of Max dimerization partners may indirectly affect Myc function in vivo by competing for available Max protein and for DNA-recognition sites. Finally, Myc function in vivo may be affected by the levels of other Myc proteins (Mukherjee et al., 1992; Resar et al., 1993). In an effort to understand the function of myc genes in embryogenesis, leaky and null mutations were made in Nmyc in embryonic stem cells by homologous recombination (Charron et al., 1990; Stanton et al., 1990; Sawai et al., 1991; Moens et al., 1992). These mutations have allowed a description of the function of N-myc at different stages of development. Mice homozygous for the null mutations die at midgestation (Stanton et al., 1992; Charron et al., 1992) while mice homozygous for the leaky mutation survive until birth, when they die due to a defect in lung branching morphogenesis (Moens et al., 1992). The latter phenotype is consistent with the normal expression of N-myc in the developing lung epithelium, and has led us to postulate that Nmyc plays a role in the response of the lung epithelium to the signals from the lung mesenchyme that induce epithelial branching. There have been two detailed reports of the phenotype of mice carrying null mutations in N-myc (Stanton et al., 1992; Charron et al., 1992). In the homozygous condition, these mutations cause embryonic death at around 11.5 days p.c. and cause hypoplasia in a number of co (...truncated)