Unique spatial and cellular expression patterns of Hoxa5, Hoxb4, and Hoxb6 proteins in normal developing murine lung are modified in pulmonary hypoplasia

Abstract

Background: Hox transcription factors modulate signaling pathways controlling organ morphogenesis and maintain cell fate and differentiation in adults. Retinoid signaling, key in regulating Hox expression, is altered in pulmonary hypoplasia. Information on pattern-specific expression of Hox proteins in normal lung development and in pulmonary hypoplasia is minimal. Our objective was to determine how pulmonary hypoplasia alters temporal, spatial, and cellular expression of Hoxa5, Hoxb4, and Hoxb6 proteins compared to normal lung development.

Methods: Temporal, spatial, and cellular Hoxa5, Hoxb4, and Hoxb6 expression was studied in normal (untreated) and nitrofen-induced hypoplastic (NT-PH) lungs from gestational day 13.5, 16, and 19 fetuses and neonates using Western blot and immunohistochemistry.

Results: Modification of protein levels and spatial and cellular Hox expression patterns in NT-PH lungs was consistent with delayed lung development. Distinct protein isoforms were detected for each Hox protein. Expression levels of the Hoxa5 and Hoxb6 protein isoforms changed with development and were altered further in NT-PH lungs. Compared to normal lungs, GD19 and neonatal NT-PH lungs had decreased Hoxb6 and increased Hoxa5 and Hoxb4. Hoxa5 cellular localization changed from mesenchyme to epithelia earlier in normal lungs. Hoxb4 was expressed in mesenchyme and epithelial cells throughout development. Hoxb6 remained mainly in mesenchymal cells around distal airways.

Conclusions: Unique spatial and cellular expression of Hoxa5, Hoxb4, and Hoxb6 participates in branching morphogenesis and terminal sac formation. Altered Hox protein temporal and cellular balance of expression either contributes to pulmonary hypoplasia or functions as a compensatory mechanism attempting to correct abnormal lung development and maturation in this condition.

Figure 1. Hoxa5, Hoxb4 and Hoxb6 western blots and densitometric protein levels in normal and NT-PH lungs

As shown by representative western blots, Hoxa5 (A), Hoxb4 (B), and Hoxb6 (C) were each detected as a cluster of three protein bands at approximate molecular weights of 32–37 kDa. Summary densitometry analysis on the cluster of protein isoforms detected for each Hox protein (from five separate blots for each protein from five experiments) is shown in (D). Densitometry of the lower molecular weight protein isoforms are shown for Hoxa5 (E) and Hoxb6 (F). Prior to Gd19, normal lung (D, left side of graph) had higher Hoxa5 and Hoxb4 than Hoxb6 followed by higher Hoxb6 compared to Hoxa5 and Hoxb4 after Gd19. This change in relative protein levels of Hoxa5, Hoxb4 and Hoxb6 did not occur in NT-PH lungs (D, Right side of graph) with Gd19 and Neo protein levels of these three Hox proteins resembling an exaggerated pattern to that seen in earlier stages of lung development. Compared to normal lungs, Gd19 and neonatal NT-PH lungs showed a trend towards increased Hoxa5 protein by at least 40% (p=0.06, N=5, Mean ± SEM) and in neonatal lungs, a similar increase of Hoxb4 by 40%. Neonatal NT-PH lungs had a significant decrease in Hoxb6 by 45% (*p = 0.006, N = 5, Mean ± SEM, Neo normal control verses Neo NT-PH). When evaluating changes in the smaller protein isoforms identified for Hoxa5 and Hoxb6, the 32 kDa isoform for Hoxa5 (E) contributed to a significant increase in the total protein levels of Hoxa5 in neonatal NT-PH lungs compared to normal controls (*p = 0.004, N = 5, Mean ± SEM, Neo NT-PH lungs verses Neo normal controls) whereas the relative proportion of densitometric signal for the smaller bands for Hoxb6 (F) was only slightly increased with advancing gestation in NT-PH lungs.

Figure 1. Hoxa5, Hoxb4 and Hoxb6 western blots and densitometric protein levels in normal and NT-PH lungs

As shown by representative western blots, Hoxa5 (A), Hoxb4 (B), and Hoxb6 (C) were each detected as a cluster of three protein bands at approximate molecular weights of 32–37 kDa. Summary densitometry analysis on the cluster of protein isoforms detected for each Hox protein (from five separate blots for each protein from five experiments) is shown in (D). Densitometry of the lower molecular weight protein isoforms are shown for Hoxa5 (E) and Hoxb6 (F). Prior to Gd19, normal lung (D, left side of graph) had higher Hoxa5 and Hoxb4 than Hoxb6 followed by higher Hoxb6 compared to Hoxa5 and Hoxb4 after Gd19. This change in relative protein levels of Hoxa5, Hoxb4 and Hoxb6 did not occur in NT-PH lungs (D, Right side of graph) with Gd19 and Neo protein levels of these three Hox proteins resembling an exaggerated pattern to that seen in earlier stages of lung development. Compared to normal lungs, Gd19 and neonatal NT-PH lungs showed a trend towards increased Hoxa5 protein by at least 40% (p=0.06, N=5, Mean ± SEM) and in neonatal lungs, a similar increase of Hoxb4 by 40%. Neonatal NT-PH lungs had a significant decrease in Hoxb6 by 45% (*p = 0.006, N = 5, Mean ± SEM, Neo normal control verses Neo NT-PH). When evaluating changes in the smaller protein isoforms identified for Hoxa5 and Hoxb6, the 32 kDa isoform for Hoxa5 (E) contributed to a significant increase in the total protein levels of Hoxa5 in neonatal NT-PH lungs compared to normal controls (*p = 0.004, N = 5, Mean ± SEM, Neo NT-PH lungs verses Neo normal controls) whereas the relative proportion of densitometric signal for the smaller bands for Hoxb6 (F) was only slightly increased with advancing gestation in NT-PH lungs.

Figure 1. Hoxa5, Hoxb4 and Hoxb6 western blots and densitometric protein levels in normal and NT-PH lungs

As shown by representative western blots, Hoxa5 (A), Hoxb4 (B), and Hoxb6 (C) were each detected as a cluster of three protein bands at approximate molecular weights of 32–37 kDa. Summary densitometry analysis on the cluster of protein isoforms detected for each Hox protein (from five separate blots for each protein from five experiments) is shown in (D). Densitometry of the lower molecular weight protein isoforms are shown for Hoxa5 (E) and Hoxb6 (F). Prior to Gd19, normal lung (D, left side of graph) had higher Hoxa5 and Hoxb4 than Hoxb6 followed by higher Hoxb6 compared to Hoxa5 and Hoxb4 after Gd19. This change in relative protein levels of Hoxa5, Hoxb4 and Hoxb6 did not occur in NT-PH lungs (D, Right side of graph) with Gd19 and Neo protein levels of these three Hox proteins resembling an exaggerated pattern to that seen in earlier stages of lung development. Compared to normal lungs, Gd19 and neonatal NT-PH lungs showed a trend towards increased Hoxa5 protein by at least 40% (p=0.06, N=5, Mean ± SEM) and in neonatal lungs, a similar increase of Hoxb4 by 40%. Neonatal NT-PH lungs had a significant decrease in Hoxb6 by 45% (*p = 0.006, N = 5, Mean ± SEM, Neo normal control verses Neo NT-PH). When evaluating changes in the smaller protein isoforms identified for Hoxa5 and Hoxb6, the 32 kDa isoform for Hoxa5 (E) contributed to a significant increase in the total protein levels of Hoxa5 in neonatal NT-PH lungs compared to normal controls (*p = 0.004, N = 5, Mean ± SEM, Neo NT-PH lungs verses Neo normal controls) whereas the relative proportion of densitometric signal for the smaller bands for Hoxb6 (F) was only slightly increased with advancing gestation in NT-PH lungs.

Figure 1. Hoxa5, Hoxb4 and Hoxb6 western blots and densitometric protein levels in normal and NT-PH lungs

As shown by representative western blots, Hoxa5 (A), Hoxb4 (B), and Hoxb6 (C) were each detected as a cluster of three protein bands at approximate molecular weights of 32–37 kDa. Summary densitometry analysis on the cluster of protein isoforms detected for each Hox protein (from five separate blots for each protein from five experiments) is shown in (D). Densitometry of the lower molecular weight protein isoforms are shown for Hoxa5 (E) and Hoxb6 (F). Prior to Gd19, normal lung (D, left side of graph) had higher Hoxa5 and Hoxb4 than Hoxb6 followed by higher Hoxb6 compared to Hoxa5 and Hoxb4 after Gd19. This change in relative protein levels of Hoxa5, Hoxb4 and Hoxb6 did not occur in NT-PH lungs (D, Right side of graph) with Gd19 and Neo protein levels of these three Hox proteins resembling an exaggerated pattern to that seen in earlier stages of lung development. Compared to normal lungs, Gd19 and neonatal NT-PH lungs showed a trend towards increased Hoxa5 protein by at least 40% (p=0.06, N=5, Mean ± SEM) and in neonatal lungs, a similar increase of Hoxb4 by 40%. Neonatal NT-PH lungs had a significant decrease in Hoxb6 by 45% (*p = 0.006, N = 5, Mean ± SEM, Neo normal control verses Neo NT-PH). When evaluating changes in the smaller protein isoforms identified for Hoxa5 and Hoxb6, the 32 kDa isoform for Hoxa5 (E) contributed to a significant increase in the total protein levels of Hoxa5 in neonatal NT-PH lungs compared to normal controls (*p = 0.004, N = 5, Mean ± SEM, Neo NT-PH lungs verses Neo normal controls) whereas the relative proportion of densitometric signal for the smaller bands for Hoxb6 (F) was only slightly increased with advancing gestation in NT-PH lungs.

Figure 1. Hoxa5, Hoxb4 and Hoxb6 western blots and densitometric protein levels in normal and NT-PH lungs

As shown by representative western blots, Hoxa5 (A), Hoxb4 (B), and Hoxb6 (C) were each detected as a cluster of three protein bands at approximate molecular weights of 32–37 kDa. Summary densitometry analysis on the cluster of protein isoforms detected for each Hox protein (from five separate blots for each protein from five experiments) is shown in (D). Densitometry of the lower molecular weight protein isoforms are shown for Hoxa5 (E) and Hoxb6 (F). Prior to Gd19, normal lung (D, left side of graph) had higher Hoxa5 and Hoxb4 than Hoxb6 followed by higher Hoxb6 compared to Hoxa5 and Hoxb4 after Gd19. This change in relative protein levels of Hoxa5, Hoxb4 and Hoxb6 did not occur in NT-PH lungs (D, Right side of graph) with Gd19 and Neo protein levels of these three Hox proteins resembling an exaggerated pattern to that seen in earlier stages of lung development. Compared to normal lungs, Gd19 and neonatal NT-PH lungs showed a trend towards increased Hoxa5 protein by at least 40% (p=0.06, N=5, Mean ± SEM) and in neonatal lungs, a similar increase of Hoxb4 by 40%. Neonatal NT-PH lungs had a significant decrease in Hoxb6 by 45% (*p = 0.006, N = 5, Mean ± SEM, Neo normal control verses Neo NT-PH). When evaluating changes in the smaller protein isoforms identified for Hoxa5 and Hoxb6, the 32 kDa isoform for Hoxa5 (E) contributed to a significant increase in the total protein levels of Hoxa5 in neonatal NT-PH lungs compared to normal controls (*p = 0.004, N = 5, Mean ± SEM, Neo NT-PH lungs verses Neo normal controls) whereas the relative proportion of densitometric signal for the smaller bands for Hoxb6 (F) was only slightly increased with advancing gestation in NT-PH lungs.

Figure 1. Hoxa5, Hoxb4 and Hoxb6 western blots and densitometric protein levels in normal and NT-PH lungs

As shown by representative western blots, Hoxa5 (A), Hoxb4 (B), and Hoxb6 (C) were each detected as a cluster of three protein bands at approximate molecular weights of 32–37 kDa. Summary densitometry analysis on the cluster of protein isoforms detected for each Hox protein (from five separate blots for each protein from five experiments) is shown in (D). Densitometry of the lower molecular weight protein isoforms are shown for Hoxa5 (E) and Hoxb6 (F). Prior to Gd19, normal lung (D, left side of graph) had higher Hoxa5 and Hoxb4 than Hoxb6 followed by higher Hoxb6 compared to Hoxa5 and Hoxb4 after Gd19. This change in relative protein levels of Hoxa5, Hoxb4 and Hoxb6 did not occur in NT-PH lungs (D, Right side of graph) with Gd19 and Neo protein levels of these three Hox proteins resembling an exaggerated pattern to that seen in earlier stages of lung development. Compared to normal lungs, Gd19 and neonatal NT-PH lungs showed a trend towards increased Hoxa5 protein by at least 40% (p=0.06, N=5, Mean ± SEM) and in neonatal lungs, a similar increase of Hoxb4 by 40%. Neonatal NT-PH lungs had a significant decrease in Hoxb6 by 45% (*p = 0.006, N = 5, Mean ± SEM, Neo normal control verses Neo NT-PH). When evaluating changes in the smaller protein isoforms identified for Hoxa5 and Hoxb6, the 32 kDa isoform for Hoxa5 (E) contributed to a significant increase in the total protein levels of Hoxa5 in neonatal NT-PH lungs compared to normal controls (*p = 0.004, N = 5, Mean ± SEM, Neo NT-PH lungs verses Neo normal controls) whereas the relative proportion of densitometric signal for the smaller bands for Hoxb6 (F) was only slightly increased with advancing gestation in NT-PH lungs.

Figure 2. Ratio of Hoxa5 to Hoxb6, Hoxb4 to Hoxb6, and Hoxa5 to Hoxb4 protein levels

Densitometry analysis showed that Hoxa5 to Hoxb6 ratio (A) gradually decreased throughout gestation in normal controls. However in NT-PH lungs, Hoxa5 to Hoxb6 ratio increased throughout gestation and was significantly increased in neonatal NT-PH lungs (*P<0.05, N=5, Mean ± SEM, Neo normal controls verses Neo NT-PH). On or before Gd19, Hoxb4/Hoxb6 ratio (B) was not different in normal verses NT-PH lungs but became significantly greater in neonatal NT-PH lungs (*P<0.05, N=5, Mean ± SEM, Neo normal controls verses Neo NT-PH). Normal lung Hoxa5 to Hoxb4 ratio (C) was unchanged across gestation but significantly elevated in Gd19 NT-PH lungs compared to normal controls (*P<0.05, N=5, Mean ± SEM, Gd19 Normal Controls verses Gd19 NT-PH).

Figure 2. Ratio of Hoxa5 to Hoxb6, Hoxb4 to Hoxb6, and Hoxa5 to Hoxb4 protein levels

Densitometry analysis showed that Hoxa5 to Hoxb6 ratio (A) gradually decreased throughout gestation in normal controls. However in NT-PH lungs, Hoxa5 to Hoxb6 ratio increased throughout gestation and was significantly increased in neonatal NT-PH lungs (*P<0.05, N=5, Mean ± SEM, Neo normal controls verses Neo NT-PH). On or before Gd19, Hoxb4/Hoxb6 ratio (B) was not different in normal verses NT-PH lungs but became significantly greater in neonatal NT-PH lungs (*P<0.05, N=5, Mean ± SEM, Neo normal controls verses Neo NT-PH). Normal lung Hoxa5 to Hoxb4 ratio (C) was unchanged across gestation but significantly elevated in Gd19 NT-PH lungs compared to normal controls (*P<0.05, N=5, Mean ± SEM, Gd19 Normal Controls verses Gd19 NT-PH).

Figure 2. Ratio of Hoxa5 to Hoxb6, Hoxb4 to Hoxb6, and Hoxa5 to Hoxb4 protein levels

Densitometry analysis showed that Hoxa5 to Hoxb6 ratio (A) gradually decreased throughout gestation in normal controls. However in NT-PH lungs, Hoxa5 to Hoxb6 ratio increased throughout gestation and was significantly increased in neonatal NT-PH lungs (*P<0.05, N=5, Mean ± SEM, Neo normal controls verses Neo NT-PH). On or before Gd19, Hoxb4/Hoxb6 ratio (B) was not different in normal verses NT-PH lungs but became significantly greater in neonatal NT-PH lungs (*P<0.05, N=5, Mean ± SEM, Neo normal controls verses Neo NT-PH). Normal lung Hoxa5 to Hoxb4 ratio (C) was unchanged across gestation but significantly elevated in Gd19 NT-PH lungs compared to normal controls (*P<0.05, N=5, Mean ± SEM, Gd19 Normal Controls verses Gd19 NT-PH).

Figure 3. Hoxa5 protein immunolocalization in normal developing mouse lung and in NT-PH lungs. Bar = 50μ in Panels A–H and 20μ in corresponding inserts

In Gd13.5 normal controls (A) and NT-PH (B) lungs, Hoxa5 protein was mostly localized to mesenchyme (arrowheads) with minimal expression in epithelial cells of columnar-lined airways (arrows). In Gd16 normal lungs (C), Hoxa5 protein localization changed being mostly restricted to epithelial cells of branching airway tips (arrows) (see higher magnification insert). This change was not seen in Gd16 NT-PH lungs where Hoxa5 remained mostly diffusely expressed in mesenchyme (arrowheads). Hoxa5 protein remained mostly localized to epithelial cells (arrows) in Gd 19 normal controls (E). Gd19 NT-PH lungs (F) also had persistent expression in thicker mesenchyme (arrowheads) whereas epithelial cells (arrows) especially at septal tips remained negative. In neonatal normal controls (G), Hoxa5 epithelial cell expression was less intense as compared to strong Hoxa5 mesenchymal (arrowheads) and epithelial expression (arrows) especially at septal tips in hypoplastic lungs (H).

Figure 4. Hoxb4 protein immunolocalization in normal developing mouse lung and in NT PH lungs, Bar = 50μ in Panels A–H and 20μ in corresponding inserts

Hoxb4 protein was localized to both mesenchymal (arrowheads) and epithelial cells (arrows) in Gd13.5 normal controls (A) and NT-PH lungs (B) but epithelial cell expression was more intense. Gd16 normal controls (C) and NT-PH lungs (D) had strong epithelial cell expression, but Hoxb4 mesenchymal cell (arrowheads) expression was more intense in NT-PH lungs than in the normal controls. Gd19 normal controls (E) and NT-PH lungs (F) had similar Hoxb4 mesenchymal (arrowheads) and epithelial (arrows) cell localization, but NT-PH lungs had increased intensity of Hoxb4 in epithelial cells (arrows), especially in bronchiolar airways as well as somewhat increased mesenchymal expression (arrowheads). Compared to normal neonatal lungs (G), epithelial cell (arrow) Hoxb4 staining remained stronger in neonatal NT-PH lungs (H). Thicker mesenchyme (arrowhead) in NT-PH lungs continued to have greater intensity of Hoxb4 positive cells than seen in the normal lungs.

Figure 5. Hoxb6 protein immunolocalization in normal developing mouse lung and in NT-PH lungs, Bar = 100μ in A and C and 50μ in B, D, E and F through K

Hoxb6 protein was mostly localized to mesenchyme (arrowheads) at Gd13.5 in normal control lungs (A, B) but decreased in NT-PH lungs (C, D). Epithelial cells of proximal more central airways (arrows in B and D) were mostly negative. Gd16 normal lungs (F) had Hoxb6 positive mesenchymal cells (arrowheads) around distal airways with cuboidal epithelial cells (long arrow in F). Although some cuboidal epithelial cells (long arrow in F) were positive for Hoxb6 at Gd16, columnar epithelial cells of proximal airways (short arrow in F) remained mostly negative. Mesenchymal expression (arrowheads in I) remained decreased around less well-developed airways in Gd16 NT-PH lungs (I). In normal lungs at Gd19 (G), Hoxb6 epithelial (arrow) and mesenchymal cell (arrowhead) expression was present and more intense compared to Gd19 NT-PH lungs (J). In normal neonatal lungs (H), mesenchymal Hoxb6 protein expression (arrowheads) was more diffuse around developing saccules. In neonatal NT-PH lungs (K) saccules were less developed with thicker surrounding mesenchyme (arrowhead) that had clusters of Hoxb6 positive mesenchymal cells and epithelial (arrow) expression remained minimal.

Figure 5. Hoxb6 protein immunolocalization in normal developing mouse lung and in NT-PH lungs, Bar = 100μ in A and C and 50μ in B, D, E and F through K

Hoxb6 protein was mostly localized to mesenchyme (arrowheads) at Gd13.5 in normal control lungs (A, B) but decreased in NT-PH lungs (C, D). Epithelial cells of proximal more central airways (arrows in B and D) were mostly negative. Gd16 normal lungs (F) had Hoxb6 positive mesenchymal cells (arrowheads) around distal airways with cuboidal epithelial cells (long arrow in F). Although some cuboidal epithelial cells (long arrow in F) were positive for Hoxb6 at Gd16, columnar epithelial cells of proximal airways (short arrow in F) remained mostly negative. Mesenchymal expression (arrowheads in I) remained decreased around less well-developed airways in Gd16 NT-PH lungs (I). In normal lungs at Gd19 (G), Hoxb6 epithelial (arrow) and mesenchymal cell (arrowhead) expression was present and more intense compared to Gd19 NT-PH lungs (J). In normal neonatal lungs (H), mesenchymal Hoxb6 protein expression (arrowheads) was more diffuse around developing saccules. In neonatal NT-PH lungs (K) saccules were less developed with thicker surrounding mesenchyme (arrowhead) that had clusters of Hoxb6 positive mesenchymal cells and epithelial (arrow) expression remained minimal.