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First published online 16 February 2005
doi: 10.1242/dev.01678

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Nmyc plays an essential role during lung development as a dosage-sensitive regulator of progenitor cell proliferation and differentiation

¹ Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA
² Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA

View larger version (71K): [in a new window]   Fig. 7. Conditional deletion of Nmyc results in severely abnormal lung development. (A) Gross morphology of lungs at E18.5. The lung that is cre(+);Nmycflox/+ (middle panel) has a mosaic phenotype with some normal tissue (white box). By contrast, all cre(+);Nmycflox/flox lungs (right panel) had a much more uniform abnormal phenotype. (B) Sections of E18.5 lungs after staining with Hematoxylin and Eosin. In this case, two examples of cre(+);Nmycflox/+ lungs are shown (middle panels): the one on the left has a normal morphology and resembles a cre(-) lung (far left), whereas the one on the right is severely abnormal and resembles a cre(+);Nmycflox/flox lung (far right), except that regions of normal tissue are present (black box enlarged in inset). (C,D) Sections of wild-type and abnormal heterozygous cre(+);Nmycflox/flox or Nmycflox/+ lungs at E14.5 (C) and E15.5 (D). (E) Level of Nmyc RNA in conditional mutant lungs as measured by RT-PCR of total RNA. Note the significant decrease in RNA levels at E14.5-E16.5. Asterisk indicates abnormal cre(+);Nmycflox/+ lung at E15.5. (F) Nmyc antibody staining of wild-type and cre(+); Nmycflox/flox lungs at E18.5. Note there is only weak staining of cells in which Nmyc has been deleted. Scale bars: in B, 200 µm; in C,D, 100 µm; in F, 50 µm.

View larger version (88K): [in a new window]   Fig. 1. Nmyc expression and BrdU incorporation in the wild-type lung. (A) RT-PCR shows that Nmyc transcripts decrease during lung development, whereas those for differentiated epithelial cell markers (Sftpc, Sftpa and Aqp5) increase. (B) Immunohistochemistry at E12.5, E14.5 and E16.5 shows higher Nmyc expression in the distal compared with the proximal epithelium. Blue arrows mark the boundary between the two regions. At E18.5, low levels are seen in presumed type II cells (insets). (C) Immunostaining for Nmyc, BrdU, cyclin D1 and phosphohistone H3 (PHH3) in adjacent sections of E12.5 lung. (D) Percentage of BrdU-positive nuclei in Nmyc-high distal endoderm and Nmyc-low proximal endoderm of E12.5 lungs, as determined by counting nuclei in cross sections of tubules, where the identification of individual cells is unambiguous. *, difference is significant (P<0.001). Scale bars: 50 µm.

View larger version (122K): [in a new window]   Fig. 8. Evidence for increased apoptosis and reduced proliferation in conditional mutant lungs. (A-F) Sections of cre(-) and cre(+) lungs at E14.5, E15.5 and E18.5. Note the presence of cellular debris (arrowheads), and the irregular shape and smaller size of the epithelial cells in the conditional mutant lungs (D-F). (G-L) Immunofluorescence for cleaved caspase 3 and DAPI (nuclei). Note abundant apoptotic cells in the lumen, epithelium and mesenchyme of cre(+);Nmycflox/+ and cre(+);Nmycflox/flox lungs at E15.5, E16.5 and E18.5 (arrowheads). The faint fluorescence seen in J is from red blood cells. (M-O) BrdU labeling. Most distal epithelial cells in the E15.5 wild-type lung are positive for BrdU after a 1-hour pulse. By contrast, note the reduced epithelial labeling of mutant lungs at E15.5 and E16.5 (arrowheads). Scale bars: 50 µm.

View larger version (101K): [in a new window]   Fig. 2. Morphology of Sftpc-NmycEGFP transgenic and normal lungs at E18.5. (A) External appearance of wild-type and transgenic lungs. (B) The NmycEGFP fusion protein is expressed throughout the distal transgenic epithelium (lower panel). Fluorescence in wild type is background (upper panel). (C) RT-PCR analysis confirming Nmyc overexpression in E18.5 transgenic lungs. (D-I) Low and high power images of sections stained with Haematoxylin and Eosin from normal (D,G) and transgenic lungs (E,F,H,I) with least severe (E,H) and most severe (F,I) phenotypes. Note the cuboidal undifferentiated epithelium, extensive mesenchyme, and lack of apposition of blood vessels to the epithelium in the transgenic lungs. (J-L) Electron micrographs of wild-type (J) and transgenic (K,L) lungs. In J, note the blood vessels (asterisk) close to the lumen of the alveolar sac, and the thin walled type I alveolar cells and two type II cells that have lost their glycogen (arrows). In K, the epithelial cells resemble immature type II cells, as they contain both glycogen and lamellar bodies (inset, arrowheads). Lamellar bodies are also in the lumen (asterisk). (L) Densely packed undifferentiated epithelial cells with glycogen but no lamellar bodies. Scale bars: 50 µm.

View larger version (123K): [in a new window]   Fig. 3. Cell proliferation and apoptosis in Sftpc-NmycEGFP transgenic lungs. Sections of wild-type (A,C,E) and transgenic (B,D,F) E18.5 lung after a 1-hour pulse in vivo with BrdU followed by immunohistochemistry (A,B), or after staining with antibody to PHH3 (C,D) or cleaved caspase 3 (E,F). Note much higher rate of cell proliferation in both mesenchyme and epithelium of the transgenic lung, and the epithelial cells undergoing apoptosis (arrowheads). Scale bars: 50 µm.

View larger version (100K): [in a new window]   Fig. 4. High levels of Nmyc inhibit epithelial cell differentiation. (A,B) Sections of normal and severely affected E18.5 transgenic lungs hybridized with riboprobes for Scgb1a1 (a marker for differentiated Clara cells) and Sftpc (a marker, when expressed at high levels, for type II cells). (A,C) In wild type, a sharp boundary is present (arrowheads) at the BADJ between bronchioles lined with Clara cells (red) and the future alveoli, lined by type II cells (green). In the transgenic lung the boundary is not sharp (white bracket). (C,D) Sections of the same lungs after staining with antibody to Sox2. In normal lungs (C), Sox2 is restricted to nuclei of proximal epithelial cells, with a sharp boundary at the BADJ (arrowheads). In the transgenic lung (D), the boundary is not sharp (white bracket). Nuclei are stained with DAPI. (E) Gene expression assayed by RT-PCR of total RNA extracted from two normal and two transgenic lungs. Representative genes are lung differentiation markers (Aqp5, SftpA, SftpB, SftpC, and Scgb1a1), upregulated genes from microarray data (Ppan, Nol5a, Rog, cyclin D2 and claudin 6), and Sox9. Scale bars: 50 µm.

View larger version (100K): [in a new window]   Fig. 5. Expression of Sox9 in normal, transgenic and conditional mutant lungs. (A,C,E) Immunohistochemistry for Sox9 in wild-type lungs. Note high expression of Sox9 in undifferentiated distal epithelial cells at E14.5 and E16.5, and downregulation in alveolar epithelium at E18.5 (E,G). In cre(+);Nmycflox/flox lungs at E14.5 and E16.5, the number of Sox9-positive cells is greatly reduced (B,D). By contrast, there are numerous Sox9-positive epithelial cells, including small clusters (arrowheads), in the E18.5 Sftpc-NmycEGFP lung (F). (G,H) Double immunohistochemistry for Sox9 and Scgb1a1. Note the sharp junction between proximal and distal domains (arrowhead in G), and low Sox9 expression, in wild-type lung. In transgenic lung (H), note the absence of a clear boundary and the high Sox9 expression, even in bronchiolar regions (br) where Scgb1a1 is expressed. DAPI staining marks nuclei. Scale bars: 50 µm.

View larger version (12K): [in a new window]   Fig. 6. Strategy for conditional deletion of Nmyc in the developing lung. (A) Breeding strategy to generate Sftpc-cre transgenic mice either homozygous or heterozygous for Nmycflox. (B) Typical result of genotyping lung and liver DNA. Note that the recombined Nmycflox allele was detected in the lung and not the liver. The unrecombined Nmycflox allele in the cre(+) lungs presumably derives from the mesoderm in which cre is not expressed.

View larger version (77K): [in a new window]   Fig. 9. Differentiation of epithelial cells in conditional mutant lungs. Double in situ hybridization for Sftpc and Scgb1a1 in cre(-);Nmycflox/+ (A) and cre(+);Nmycflox/flox (B) lungs at E18.5. Note the paucity of type II cells in the mutant lung. (C,D) Immunofluorescence for Gp38 expressed on the apical surface of type I cells at this stage. Note the presence of attenuated presumptive type I cells lining the large sacs. (E) RT-PCR for Aqp5, SftpA and actin. Expression of Aqp5 is prematurely increased in cre(+);Nmycflox/flox lung at E16.5. Scale bars: 50 µm.

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