An ENU-Mutagenesis Screen in the Mouse: Identification of Novel Developmental Gene Functions

BACKGROUND: Mutagenesis screens in the mouse have been proven useful for the identification of novel gene functions and generation of interesting mutant alleles. Here we describe a phenotype-based screen for recessive mutations affecting embryonic development. METHODOLOGY/PRINCIPAL FINDINGS: Mice were mutagenized with N-ethyl-N-nitrosourea (ENU) and following incrossing the offspring, embryos were analyzed at embryonic day 10.5. Mutant phenotypes that arose in our screen include cardiac and nuchal edema, neural tube defects, situs inversus of the heart, posterior truncation and the absence of limbs and lungs. We isolated amongst others novel mutant alleles for Dll1, Ptprb, Plexin-B2, Fgf10, Wnt3a, Ncx1, Scrib(Scrib, Scribbled homolog [Drosophila]) and Sec24b. We found both nonsense alleles leading to severe protein truncations and mutants with single-amino acid substitutions that are informative at a molecular level. Novel findings include an ectopic neural tube in our Dll1 mutant and lung defects in the planar cell polarity mutants for Sec24b and Scrib. CONCLUSIONS/SIGNIFICANCE: Using a forward genetics approach, we have generated a number of novel mutant alleles that are linked to disturbed morphogenesis during development.

NOTCH signaling promotes the survival of irradiated basal airway stem cells

Radiation-induced lung injury to normal airway epithelium is a frequent side-effect and dose-limiting factor in radiotherapy of tumors in the thoracic cavity. NOTCH signaling plays key roles in self-renewal and differentiation of upper airway basal lung stem cells during development, and the NOTCH pathway is frequently deregulated in lung cancer. In preclinical lung cancer models, NOTCH inhibition was shown to improve the radiotherapy response by targeting tumor stem cells, but the effects in combination with irradiation on normal lung stem cells are unknown. NOTCH/gamma-secretase inhibitors are potent clinical candidates to block NOTCH function in tumors, but their clinical implementation has been hampered by normal tissue side-effects. Here we show that NOTCH signaling is active in primary human-and murine-derived airway epithelial stem cell models and when combined with radiation NOTCH inhibition provokes a decrease in S-phase and increase in G(1)-phase arrest. We show that NOTCH inhibition in irradiated lung basal stem cells leads to a more potent activation of the DNA damage checkpoint kinases pATM and pCHK2 and results in an increased level of residual 53BP1 foci in irradiated lung basal stem cells reducing their capacity for self-renewal. The effects are recapitulated in ex vivo cultured lung basal stem cells after in vivo whole thorax irradiation and NOTCH inhibition. These results highlight the importance of studying normal tissue effects that may counteract the therapeutic benefit in the use of NOTCH/gamma-secretase inhibitors in combination with radiation for antitumor treatment

Age-Related Changes in the Cellular Composition and Epithelial Organization of the Mouse Trachea

<div><p>We report here senescent changes in the structure and organization of the mucociliary pseudostratified epithelium of the mouse trachea and main stem bronchi. We confirm previous reports of the gradual appearance of age-related, gland-like structures (ARGLS) in the submucosa, especially in the intercartilage regions and carina. Immunohistochemistry shows these structures contain ciliated and secretory cells and Krt5+ basal cells, but not the myoepithelial cells or ciliated ducts typical of normal submucosal glands. Data suggest they arise de novo by budding from the surface epithelium rather than by delayed growth of rudimentary or cryptic submucosal glands. In old mice the surface epithelium contains fewer cells per unit length than in young mice and the proportion of Krt5+, p63+ basal cells is reduced in both males and females. However, there appears to be no significant difference in the ability of basal stem cells isolated from individual young and old mice to form clonal tracheospheres in culture or in the ability of the epithelium to repair after damage by inhaled sulfur dioxide. Gene expression analysis by Affymetrix microarray and quantitative PCR, as well as immunohistochemistry and flow sorting studies, are consistent with low-grade chronic inflammation in the tracheas of old versus young mice and an increase in the number of immune cells. The significance of these changes for ARGL formation are not clear since several treatments that induce acute inflammation in young mice did not result in budding of the surface epithelium.</p></div

Basal cells in young and old tracheal epithelium.

<p>Tracheas from 3 young (3 month) and 3 old (22 month) male mice were fixed and midline longitudinal sections stained for nuclei (DAPI, blue) and Krt5 (green). Areas between cartilages 4 and 10 were photographed and a montage prepared. (A,B) Typical distribution of Krt5 basal cells in young versus old tracheas. (C) Total cells (blue) and total Krt5 + cells (red) present between cartilages 4 and 10. (D) Krt5+ cells as a percentage of total DAPI+ cells. The values are shown as average and SEM and p = <0.05 Scale bar: 100 um.</p

Age-related changes in the organization of tracheal epithelium.

<p>Longitudinal midline sections of tracheas from young (4–7month) and old (24–26 month) male C57Bl/6 mice were stained with haematoxylin and eosin. (A, B) Low power view showing ARGLS in the intercartilage regions (arrows). (A′, B′) High power view of boxed regions. (C, D) Note high density of ARGLS in the carina of an old mouse and the direct connection between an ARGL and the airway lumen. (E, F) Section through distal trachea showing an ARGLS (dotted line) within a submucosal gland. The cystic structure is engorged with extracellular material and there is apparent discharge of this material into the airway lumen (arrow). By contrast normal glands (asterix) are not enlarged and are connected to the airway lumen by ducts (arrowhead) Scale bars: A–D  = 500 μm; E–G  = 100 μm.</p

Age dependence of ARGLs.

<p>Presence of ARGLs in longitudinal midline sections of entire trachea and main stem bronchi of male and female C57Bl/6 mice aged 4 (n = 10), 7–10 (n = 10), 12–16 (n = 5) and 24–28 (n = 10) months. - no ARGLs, + 1–2 ARGLs/section, ++ 3–6 ARGLs/section, +++ 6–8 ARGLs/section and ++++ more than 8 ARGLS/section. No difference was noted between males and females or between mice from difference sources.</p

Immune cells in young and old tracheas.

<p>Tracheal cells harvested from 3 young (4 month) and three old (23 month) female C57Bl/6 mice that had only recently been imported from vendors (NIA Charles River labs in MD) were analyzed with 11 color flow cytometry. (Left panel) There was a significant increase in the percentage of CD45 cells in the aged compared with young trachea (25.87±2.30% versus 14.12±2.34%, n = 3, p<0.05). (Middle panel) There was also an increase in the percentage of cells of lymphoid lineage, such as T and B cells (p = <0.05) in old versus young tracheas. (Right panel) There was no significant change in the distribution of myeloid cell populations (polymorphonuclear leukocytes, monocytes, macrophages and dendritic cells) between the two groups.</p