nmyc misexpression in lung (gain of function)

Understanding how lung progenitor cells balance proliferation against differentiation is relevant to clinical disorders such as bronchopulmonary dysplasia of premature babies and lung cancer. Previous studies have established that lung development is severely disrupted in mouse mutants with reduced levels of the proto-oncogene Nmyc, but the precise mechanisms involved have not been explored. We show here that Nmyc expression in the embryonic lung is normally restricted to a distal population of undifferentiated epithelial cells, a high proportion of which are in the S phase of the cell cycle. Overexpression of NmycEGFP in the epithelium under the control of surfactant protein C (Sftpc) regulatory elements expands the domain of S phase cells and upregulates numerous genes associated with growth and metabolism, as shown by transcriptional microarray. In addition, there is marked inhibition of differentiation, coupled with an expanded domain of expression of Sox9 protein, which is also normally restricted to the distal epithelial compartment. By contrast, conditional deletion of Nmyc leads to reduced proliferation, epithelial differentiation and high levels of apoptosis in both epithelium and mesenchyme. Unexpectedly, about 50% of embryos in which only one copy of Nmyc is deleted die perinatally, with similarly abnormal lungs. We propose a model in which Nmyc is essential in the developing lung for maintaining a distal population of undifferentiated, proliferating progenitor cells. Keywords: genetic modification: mutant vs control To generate Nmyc1EGF fusion protein, mouse Nmyc1 cDNA was inserted into the SmaI site of pEGFP (BectonDickinson), excised by SalI and EcoRI, and inserted into a vector containing a 3.7 kb promoter/enhancer of the human SFTPC gene (Wert et al., 1993). Transfection of 293 cells shows that the fusion protein localizes to the nucleus (see Fig. S1 in supplementary material). Four transgenic embryos were generated by pronuclear injection into (C57BL/6J and DBA/2J) F2 fertilized eggs. They were collected at E18.5, so it is not known if they would have survived postnatally. Duplicate samples are biological replicates.