Figure 1.
Line3-2 reveals lung hypoplasia and limb syndactyly.
(A–H) Embryos dissected at E18.5 (A) reveal that line3-2 mutants are slightly reduced in size and have significantly smaller lung lobes (B–F). (G–H) E18.5 embryos fixed and stained for bone (red) and cartilage (blue) in the forelimb demonstrate gross malformation and reduction in the number of digits in the line3-2. (I–N) Histological analysis of E18.5 lung lobes show decreased airway number, increased airway diameter and defects in distal lung epithelial morphology in line3-2. (I–L) E18.5 lungs were dissected, paraffin embedded and sectioned at 10 um. (M) Airway number and (N) the mean linear intercept (MLI) of wild-type and mutant lungs were determined from 4 different embryos at E18.5. Error bars are indicated as standard error of the mean (SEM).
Figure 2.
Line3-2 mutants show a reduction in distal epithelial cell marker Sftpc but normal proximal differentiation.
(A–J) Lung sections from E18.5 embryos were analyzed by immunohistochemistry for the indicated proximal differentiation markers (A–H) as well as for the distal epithelial cell marker Sftpc (I–J) revealing no significant changes in proximal epithelial differentiation but a marked decrease in Sftpc expression (insets in each panel show higher magnification views). (K) Expression of Sftpc was quantified by qPCR at the indicated stages, confirming the reduced expression of Sftpc in mutant lungs. Error bars represent SEM from 4 different embryos.
Figure 3.
Line3-2 mutation perturbs cell proliferation and causes increased apoptosis in the lung.
(A–I) In mutant lungs, cell proliferation is decreased during the pseudoglandular stage in mutant lungs, but is higher at the terminal sac stage as detected by EdU incorporation in lung sections. (I) Data presented as percentage of total nuclei. The results are representatives of 6 different sections. (J–R) Apoptosis is markedly increased in mutant lungs. (J–R) Lungs from E14.5 embryos were sectioned and processed for TUNEL staining. (R) The number of TUNEL positive cells were determined on sections and presented as percentage of total nuclei. The results are representatives of 7 different sections. Error bars are presented as SEM. (High) indicates higher magnification view.
Figure 4.
Line3-2 contains two missense mutations in the Nubp1 gene.
(A) Schematic representation of the genomic region around the Nubp1 gene, which contains two missense mutations at nucleotide position 743 and 803 of the coding sequence. The mutations leads to a Threonine to Lysine and Proline to Glutamine change in the amino acid sequence of Nubp1. (B) Protein sequence alignment of the indicated genes show the mutations in line3-2 to be in a highly conserved region. Red boxes and asterisks highlight mutated regions. (C–H) E14.5 embryos from a complementation cross between line3-2 and Nubp1 knock-out reveals similar defects in lung (H) and limb development (G) as lin3-2 homozygous mutants (E, F).
Figure 5.
Localization, expression and interaction of Nubp1.
(A–E) Nubp1 and its homolog Nubp2 are expressed in the distal lung epithelium. (A, C) In situ hybridization of Nubp1 and Nubp2 on E11.5 lung whole mounts. (B, D) Q-PCR for Nubp1 and Nubp2 at different developmental stages reveals no expression differences in mutant versus wild-type lungs. (E) PCR amplification of the indicated genes on FACS sorted E18.5 distal lung epithelial cells (GFP +) versus remaining GFP (-) cells. (F) Nubp2 interacts with Nubp1 but not with the mutant protein (Nubp1-m1Nisw). A549 cells overexpressing the indicated genes were processed for immunoprecipitation and analyzed by western blotting. (G–H) Mutation in the Nubp1 gene (Nubp1-m1Nisw) does not alter its cellular localization. A549 cells were stably transfected with GFP-Nubp1 or GFP-Nubp1(m1Nisw) and processed for immunofluorescence.
Figure 6.
Knock-down of Nubp1 causes centrosome multiplication and alteration in centrsome dynamics.
(A–B) A549 cells stably transfected with GFP-Centrin were either transfected with control or Nubp1 specific siRNA. Represented is a time sequence showing the movement of the centriole pair in control cells and the aberrant numbers, increased and irregular centrosome movements in Nubp1 knock-down cells. (C–F) Knock-down of Nubp1 leads to multiple microtubule organization points and decreased acetylated-tubulin. A549 cells were transfected with either control or Nubp1 siRNA and stained for the indicated antigens.
Figure 7.
Nubp1(m1Nisw) lungs show altered distribution of Par3 and Numb1.
(A–R) E16.5 lungs were sectioned and stained by immunohistochemistry for the indicated proteins. (A–H) The cellular localization of the polarity protein Par3 is significantly disrupted in Nubp1(m1Nisw) mutant lung. (I–R) The mutation in Nubp1(m1Nisw) disrupts the cellular localization of Numb1. Note the defect is restricted to distal epithelial cells. Images K-R are higher magnifications of the regions indicated in images I and J.