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1.
Figure 8

Figure 8. From: The role of hypoxia and neurogenic genes (Mash-1 and Prox-1) in the developmental programming and maturation of pulmonary neuroendocrine cells in fetal mouse lung.

Analysis for expression of Mash-1 and Hes-1 in lung organ cultures. Western blot (a) and RT-PCR (b) were performed on E12 and E16 lungs in organ culture under normoxia and hypoxia. E12 lungs cultured under normoxia express Mash-1, whereas E12 lungs cultured under hypoxia show almost complete loss of Mash-1 expression. However, when these hypoxia-cultured lung explants were returned to normoxia for 24 h, there was a significant upregulation in Mash-1 expression (recovery). In contrast, E16 lungs did not exhibit any loss of Mash-1 expression under hypoxia. In comparison, Hes-1 expression was maintained under all conditions and gestational ages. Densitometric analysis of western blots shown indicates large differences for Mash-1 and some variance for Hes-1, all relative to positive controls for Mash-1 (brain) and Hes-1 (liver). Loading controls are tubulin for western blots and actin for RT-PCR.

Suzanne McGovern, et al. Lab Invest. ;90(2):180-195.
2.
Figure 4

Figure 4. From: The role of hypoxia and neurogenic genes (Mash-1 and Prox-1) in the developmental programming and maturation of pulmonary neuroendocrine cells in fetal mouse lung.

Long-term (6 days) explant organ culture of E12 lungs evaluated for branching morphogenesis and cell proliferation. Gross appearance of lung explants maintained in either normoxia (a, c, and e) or hypoxia (b, d, and f). Size of lungs increased more than twofold after 6 days in hypoxia (f) compared with normoxia (e). (g) Morphometric (n = 7) assessment of branching morphogenesis showed a significant and sustained increase in airway branching after 3 days in hypoxia compared with cultures maintained under normoxia. (h) Proliferative index (assessed by BrdU labeling; n = 4) of lung E12 organ cultures exposed to 6 days of hypoxia is significantly increased compared with normoxia (*P < 0.05).

Suzanne McGovern, et al. Lab Invest. ;90(2):180-195.
3.
Figure 5

Figure 5. From: The role of hypoxia and neurogenic genes (Mash-1 and Prox-1) in the developmental programming and maturation of pulmonary neuroendocrine cells in fetal mouse lung.

Immunohistochemical labeling of PNEC/NEB cells (a–d) and Clara cells (e, f) in E12 lung organ cultures maintained in normoxia or hypoxia for 6 days. In normoxia cultures (a, c), the size and number of PNEC/NEB cells (arrow) immunostained for PGP9.5 and neuroendocrine marker SV2 were markedly increased compared with that of cultures maintained in hypoxia (b, d) (original magnification × 400). Expression of Clara cell marker CC10 in airway epithelial cells (arrow) was comparable between cultures exposed to normoxia (e) or hypoxia (f) (original magnification × 200). (g) Quantification of immunoreactive PNEC/NEB cells per mm2 of lung tissue (n = 5) confirmed significant reduction (*P < 0.05) in these cells in lung explants maintained under hypoxia compared with those under normoxia.

Suzanne McGovern, et al. Lab Invest. ;90(2):180-195.
4.
Figure 6

Figure 6. From: The role of hypoxia and neurogenic genes (Mash-1 and Prox-1) in the developmental programming and maturation of pulmonary neuroendocrine cells in fetal mouse lung.

Effect of gestational age on responsiveness of PNEC/NEB to hypoxia. Comparison of immunohistochemical labeling for Mash-1 and Hes-1 in lung explants from E12 (a–d) and E16 (e–h) grown for 6 days under normoxia or hypoxia. In lung explants from E12 maintained for 6 days under normoxia, both Mash-1 (a) and Hes-1 (c) were strongly expressed. Arrow in panel c points to an NEB that is Hes-1 negative. In similar cultures exposed to hypoxia, NEB lacked expression of Mash-1, (b) but Hes-1 expression remained unaffected (d), as noted by epithelial cells showing positive nuclear staining for Hes-1 surrounding a Hes-1-negative NEB (arrow) (original magnification × 600). In contrast, organ cultures from E16 lungs exposed for 6 days to normoxia (e, g) or hypoxia (f, h) maintained strong expression of both Mash-1 and Hes-1 under either normoxia or hypoxia. Arrows point to NEB cells that are Mash-1 positive and Hes-1 negative. Note that in E16 + 6d cultures, the surrounding epithelium is positive for Hes-1 (original magnification × 400).

Suzanne McGovern, et al. Lab Invest. ;90(2):180-195.
5.
Figure 3

Figure 3. From: The role of hypoxia and neurogenic genes (Mash-1 and Prox-1) in the developmental programming and maturation of pulmonary neuroendocrine cells in fetal mouse lung.

Immunofluorescence labeling of Prox-1(−/−) lungs in situ at E14.5 (a, b) and in lungs from E13 after 2 days in explant culture (c–f). (a) Prox-1(−/−) E14.5 lung showing weak diffuse immunostaining for PGP9.5 in the cytoplasm of epithelial cells from a smaller distal airway including PNEC/NEB (green signal), with positive Mash-1 expression (red signal) in nuclei. Inset: higher magnification of a Mash-1-positive cell with background PGP9.5 staining. (b) The Prox-1(−/−) E14.5 lung dual immunostained for Mash-1 (red signal) showing positive NEB cell nuclei, whereas immunolabeling for Prox-1 (green signal) is negative (original magnification × 480). Wild-type (c, d) and Prox-1(−/−) (e, f) embryonic lungs grown in organ culture showing appropriate expression of PNEC/NEB cell cytoplasmic markers (green signal) PGP9.5 (c, e) and CGRP (d, f), whereas their nuclei are Mash-1 (red signal) positive. Arrows point to NEB (original magnification × 400).

Suzanne McGovern, et al. Lab Invest. ;90(2):180-195.
6.
Figure 1

Figure 1. From: The role of hypoxia and neurogenic genes (Mash-1 and Prox-1) in the developmental programming and maturation of pulmonary neuroendocrine cells in fetal mouse lung.

Experimental times, conditions, and summary of results comparing in vivo and explant organ cultures. (a) Experimental times and conditions (normoxia, NOX, large white arrows; hypoxia, HOX, large black arrows) for explant organ cultures are illustrated with respect to gestation period from E12 to P4. Symbols designate manipulations of explants under normoxia and hypoxia. The right-side column summarizes the expressions of neuroendocrine markers with respect to gestational times. Symbols: down arrow, start time of organ culture; circle, E12 + 6-day NOX; hatched circle, E16 + 6-day NOX; square, E12 + 6-day HOX; hatched square, E16 + 6-day HOX; triangle, E12 + 1-day NOX + 1-day NOX; diamond, E12 + 1-day HOX + 1-day NOX; oval, E12 + 6-day NOX + 1-day NOX; pentagon, E12 + 6-day HOX + 1-day NOX. (b) Results are summarized for expressions of these markers in PNEC/NEB relative to Mash-1-positive cells in mouse lungs during this gestational period. Each time point represents morphometric data obtained from five sections (five different pregnancies). A series of sections were immunostained and sections with the maximum number of Mash-1-positive cells were then enumerated for all other markers and expressed as a percentage relative to Mash-1-positive cells. Bars: horizontal black bars, Mash-1; white, Prox-1; gray stippled, PGP9.5; diagonal gray stripes, CGRP; black filled, SV2.

Suzanne McGovern, et al. Lab Invest. ;90(2):180-195.
7.
Figure 7

Figure 7. From: The role of hypoxia and neurogenic genes (Mash-1 and Prox-1) in the developmental programming and maturation of pulmonary neuroendocrine cells in fetal mouse lung.

Recovery of Mash-1 expression by switching of lung explants from hypoxia to normoxia (reoxygenation) in short-term (1 day, a–d) and long-term (6 day, e–h) cultures. (a) Positive immunoreactivity for PGP9.5 (green signal) and Mash-1 (red signal) in NEB cells (arrow) from E13 lung in normoxia culture for 1 day. (b) Developmental onset of a more mature PNEC/NEB marker CGRP (green signal, arrow), expressed along with Mash-1 (red signal) by day 2 in normoxia culture. (c) In contrast, in parallel organ cultures exposed to 1 day of hypoxia, there is loss of Mash-1 and PGP9.5 expression from NEB cells (arrow), whereas PGP9.5 immunoreactivity in adjacent ganglion cells is preserved (double arrows). (d) When hypoxia cultures are switched back for 1 day of normoxia, there is a robust recovery of Mash-1 expression (arrow) in NEB cell nuclei (red signal); however, note the absence of CGRP expression as compared with (b) (original magnification × 630). Long-term organ cultures initiated at E12 showed a strong expression of both PGP9.5 and Mash-1 in NEB cells (arrow) in cultures maintained in normoxia for 6 (e) or 7 days (f), whereas in organ cultures exposed to hypoxia (g) over the same time period, only very few Mash-1- and no PGP9.5-positive cells are evident. Double arrows indicate positive PGP9.5 staining in ganglia. (h) When these cultures are then switched back to normoxia for 1 day, there is a robust recovery of Mash-1 and PGP9.5 expression in NEB cells (arrow). Parallel control organ cultures in normoxia (f) maintain PGP9.5 and Mash-1 expression in NEB cells (arrow) (original magnification × 400).

Suzanne McGovern, et al. Lab Invest. ;90(2):180-195.
8.
Figure 2

Figure 2. From: The role of hypoxia and neurogenic genes (Mash-1 and Prox-1) in the developmental programming and maturation of pulmonary neuroendocrine cells in fetal mouse lung.

Expression of Mash-1 and Prox-1 in the developing mouse lung. Comparison of immunolocalization of (a–d) Mash-1 and (e–h) Prox-1 on adjacent paraffin sections from E12 to E15 mouse lung. Arrows point to positively stained colocalization of Mash-1 and Prox-1 in PNEC/NEB cells distributed along the airway epithelial basement membrane. The airway in panels a and e shows coexpression in PNEC/NEB, but as more evident in panels b and f, many Mash-1-positive cells did not express Prox-1 (original magnification × 200). (i–l) Confocal microscopy of dual immunofluorescence labeling for Mash-1 (red signal) and Prox-1 (green signal) on frozen sections of mouse lungs between E12 and E14. During early stages of lung development (i–k), only some Mash-1 + cells also coexpressed Prox-1 + (orange signal, arrow, as in panel k). However, by E15, all Mash-1 + cells were also Prox-1 + (l) (original magnification × 480). Higher magnification views of NEB in E14 (m, n) and E15 (o, p) mouse lungs with dual immunoflorescence for PGP9.5/Mash-1 and Mash-1/Prox-1. (m) Dual immunolabeling for Mash-1 in nuclei (red signal) and for neuroendocrine marker PGP9.5 (green signal) in the cytoplasm of NEB cells residing in a larger airway that is better differentiated. (n) Coexpression of Mash-1 and Prox-1 (orange signal) in nuclei of NEB cells located at airway bifurcation. (o) NEB cells showing localization of PGP9.5 (green signal) in the cytoplasm and developing submucosal nerve fibers entering the NEB base, whereas Mash-1 (red signal) is localized in nuclei. (p) A large NEB cell cluster with a uniform coexpression of Mash-1 and Prox-1 (orange signal) in all nuclei. (o) Note fine nerve fibers (arrow) underneath the NEB that label for PGP9.5 (original magnification × 640). (q) Western blot and (r) RT-PCR analyses of gestational series of lungs (E11–E18 and P4). Expression of Mash-1 protein and mRNA peaks at E15 correlating with the immunostaining shown above. No changes were noted in Hes-1 mRNA or protein expression during the same gestation period, except for an apparent increase at P4. Densitometry of western blots confirms a gradual increase in Mash-1 protein expression, peaking at E15 and then decreasing by P4 with minimal variation for Hes-1. All values are relative to positive controls for Mash-1 (brain) and Hes-1 (liver). Loading controls are tubulin for western blots and actin for RT-PCR.

Suzanne McGovern, et al. Lab Invest. ;90(2):180-195.

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