Hoxb-5 expression in the developing mouse lung suggests a role in branching morphogenesis and epithelial cell fate

 Hoxb-5 is one of the few homeobox genes strongly expressed in the developing mouse lung. To explore the hypothesis that Hoxb-5 acts to regulate epithelial cell fate and branching morphogenesis in the developing lung, we studied the temporal, spatial, and cell-specific expression of Hoxb-5 from gestational day (d) 13.5 to postnatal day (P) 2. Immunocytochemistry demonstrated regional localization of Hoxb-5 protein to developing conducting airways and surrounding mesenchyme. The cellular expression pattern changed from diffusely positive nuclei of mesenchymal cells on d13.5 to become more localized to nuclei of subepithelial fibroblasts and some adjacent columnar and cuboidal epithelial cells on d14.5. After d14.5, Hoxb-5 protein expression continued to decrease in mesenchymal cells distal from developing airways, but persisted in fibroblasts underlying conducting airways. Hoxb-5 protein expression persisted in nuclei of columnar and cuboidal epithelial cells on d16.5 and d17.5, with expression in low cuboidal epithelial cells as well from d17.5 to P2. Western blot analysis showed temporal and quantitative changes in Hoxb-5 protein expression with peak expression on d14.5–15.5. We conclude that Hoxb-5 protein is developmentally regulated in a temporal, spatial, and cell-specific manner throughout the pseudoglandular, canalicular, and terminal saccular periods of lung development in the mouse. This localization and expression pattern suggests that Hoxb-5 may influence branching morphogenesis, cell–cell communication, cell fate, and differentiation of conducting airway epithelia. Accepted: 5 May 1997     

Suppression of embryonic lung branching morphogenesis by antisense oligonucleotides against HOM/C homeobox factors

The role of HOM/C homeobox genes on rat embryonic lung branching morphogenesis was investigated using the lung bud explant culture system in an air/liquid interface. Knock down of homeobox b3 and b4 expression by antisense oligonucleotide treatment repressed airway branch formation, while antisense oligonucleotide against homeobox a3 showed no effect. Addition of antisense Hoxb3 oligonucleotide resulted in upregulation of collagen type III mRNA and fibroblast growth factor 10 mRNA, while that of the T-box regulatory factor-4 was decreased. Consequently, expression of Clara cell-specific secretory protein was decreased. These results suggest a critical role for homeobox b3 and b4 genes in lung airway branching morphogenesis.     

Stage-dependent responses of the developing lung to retinoic acid signaling

Morphological analysis of vitamin A-deficient rat fetuses and of retinoic acid receptor (RAR and RXR) mutant mice have demonstrated that retinoic acid (RA) is essential for lung development. To gainfurther insight into RA signaling pathways during primary lung budformation and lung branching, we have investigated the effects of RA and of a pan-RAR antagonist in cultures of whole embryos and lung explants. Treatment of E8.0 embryos with the pan-RAR antagonist inhibits the formation of the primitive respiratory system. On the other hand, treatment of E11.75 and E12.5 lung explants with RA inhibits branching morphogenesis, whereas treatment with the pan-RAR antagonist at the same developmental stages stimulates formation of distal buds. The inhibitory effect of RA on branching is strongly decreased in RARbeta null lungs, while enhancement of budding by the pan-RAR antagonist is not affected by an RARgamma null mutation. Additionally, cellular retinol binding protein one (CRBPI) null lungs are more sensitive than wild type lungs to the pan-RAR antagonist-induced stimulation of branching. These data indicate that retinoid signaling is indispensable for the formation of primary lung buds and the oesophagotracheal septum from the primitive foregut. They also suggest that at the pseudoglandular stage, RA signaling through RARbeta, but not RARgamma, inhibits distal bud formation thereby promoting the formation of conducting airways. Moreover, the level of CRBPI in the pseudoglandular lung appears to participate in the control of branching morphogenesis.     

Smad1 expression and function during mouse embryonic lung branching morphogenesis

Bone morphogenetic protein (BMP) 4 plays very important roles in regulating developmental processes of many organs, including lung. Smad1 is one of the BMP receptor downstream signaling proteins that transduce BMP4 ligand signaling from cell surface to nucleus. The dynamic expression patterns of Smad1 in embryonic mouse lungs were examined using immunohistochemistry. Smad1 protein was predominantly detected in peripheral airway epithelial cells of early embryonic lung tissue [embryonic day 12.5 (E12.5)], whereas Smad1 protein expression in mesenchymal cells increased during mid-late gestation. Many Smad1-positive mesenchymal cells were localized adjacent to large airway epithelial cells and endothelial cells of blood vessels, which colocalized with a molecular marker of smooth muscle cells (alpha-smooth muscle actin). The biological function of Smad1 in early lung branching morphogenesis was then studied in our established E11.5 lung explant culture model. Reduction of endogenous Smad1 expression was achieved by adding a Smad1-specific antisense DNA oligonucleotide, causing approximately 20% reduction of lung epithelial branching. Furthermore, airway epithelial cell proliferation and differentiation were also inhibited when endogenous Smad1 expression was knocked down. Therefore, these data indicate that Smad1, acting as an intracellular BMP signaling pathway component, positively regulates early mouse embryonic lung branching morphogenesis.     

Regulation of retinoic acid signaling during lung morphogenesis

Little is known about how retinoic acid (RA) synthesis, utilization and metabolism are regulated in the embryonic lung and how these activities relate to lung pattern formation. Here we report that early lung bud formation and subsequent branching morphogenesis are characterized by distinct stages of RA signaling. At the onset of lung development RA signaling is ubiquitously activated in primary buds, as shown by expression of the major RA-synthesizing enzyme, RALDH-2 and activation of a RARE-lacZ transgene. Nevertheless, further airway branching appears to require downregulation of RA pathways by decreased synthesis, increased RA degradation in the epithelium via P450RAI-mediated metabolism, and inhibition of RA signaling in the mesenchyme by COUPTF-II expression. These mechanisms controlling local RA signaling may be critical for normal branching, since we show that manipulating RA levels in vitro to maintain RA signaling activated as in the initial stage, leads to an immature lung phenotype characterized by failure to form typical distal buds. We show that this phenotype likely results from RA interfering with the establishment of a distal signaling center, altering levels and distribution of Fgf10 and Bmp4, genes that are essential for distal lung formation. Furthermore, RA upregulates P450RAI expression, suggesting the presence of feedback mechanisms controlling RA availability. Our study illustrates the importance of regional mechanisms that control RA availability and utilization for correct expression of pattern regulators and normal morphogenesis during lung development.     

Coordinated expression of Hoxb genes and signaling molecules during development of the chick respiratory tract

To elucidate the molecular mechanism for regulating the region-specific morphogenesis of the chicken respiratory tract, we analyzed the spatiotemporal expression patterns of the Hoxb genes, Bmp-2, Bmp-4, Wnt-5a, and Wnt-11 in the developing respiratory tract. We found region-specific expression of these genes in the mesenchymal layer of the respiratory tract. Before bronchial branching proceeds, Hoxb genes show nested expression patterns around the ventral-distal tip of the lung bud. As morphogenesis proceeds, these expression domains correspond to the morphological subdivisions of the chick respiratory tract. Hoxb-5 and Hoxb-6 expression domains demarcate the trachea, bronchial tree, and air sacs. Particularly the expression domains of Hoxb-6 to -9 correspond to the morphological subdivisions of the air sacs along the proximodistal axis. Bmp-4 and Bmp-2 are expressed throughout the entire pulmonary mesenchyme and its dorsal half, respectively. Wnt-5a and Wnt-11 are expressed in the tracheal mesenchyme. Interestingly, the expression domain of Bmp-2 is complementary to the Hoxb-6 domain. The respiratory mesenchyme influences the process of epithelial branching during morphogenesis. By tissue recombination experiments, we found that the dorsal and the ventral pulmonary mesenchyme, demarcated by Hoxb-6 expression, have different inductive capacities toward the tracheal epithelium. These observations suggest the possibility that Hoxb genes are involved in the system specifying regional differences in morphogenesis and cytodifferentiation of respiratory tract. In addition, it is possible that BMPs and WNTs mediate region-specific epithelial-mesenchymal interaction in this system.     

Iroquois genes influence proximo-distal morphogenesis during rat lung development

Lung development is a highly regulated process directed by mesenchymal-epithelial interactions, which coordinate the temporal and spatial expression of multiple regulatory factors required for proper lung formation. The Iroquois homeobox (Irx) genes have been implicated in the patterning and specification of several Drosophila and vertebrate organs, including the heart. Herein, we investigated whether the Irx genes play a role in lung morphogenesis. We found that Irx1-3 and Irx5 expression was confined to the branching lung epithelium, whereas Irx4 was not expressed in the developing lung. Antisense knockdown of all pulmonary Irx genes together dramatically decreased distal branching morphogenesis and increased distention of the proximal tubules in vitro, which was accompanied by a reduction in surfactant protein C-positive epithelial cells and an increase in beta-tubulin IV and Clara cell secretory protein positive epithelial structures. Transmission electron microscopy confirmed the proximal phenotype of the epithelial structures. Furthermore, antisense Irx knockdown resulted in loss of lung mesenchyme and abnormal smooth muscle cell formation. Expression of fibroblast growth factors (FGF) 1, 7, and 10, FGF receptor 2, bone morphogenetic protein 4, and Sonic hedgehog (Shh) were not altered in lung explants treated with antisense Irx oligonucleotides. All four Irx genes were expressed in Shh- and Gli(2)-deficient murine lungs. Collectively, these results suggest that Irx genes are involved in the regulation of proximo-distal morphogenesis of the developing lung but are likely not linked to the FGF, BMP, or Shh signaling pathways.     

Thyroid hormone affects embryonic mouse lung branching morphogenesis and cellular differentiation

Although thyroid hormone (T(3)) influences epithelial cell differentiation during late fetal lung development, its effects on early lung morphogenesis are unknown. We hypothesized that T(3) would alter embryonic lung airway branching and temporal-spatial differentiation of the lung epithelium and mesenchyme. Gestational day 11.5 embryonic mouse lungs were cultured for 72 h in BGJb serum-free medium without or with added T(3) (0.2, 2.0, 10.0, or 100 nM). Evaluation of terminal bud counts showed a dose- and time-dependent decrease in branching morphogenesis. Cell proliferation was also significantly decreased with higher doses of T(3). Morphometric analysis of lung histology showed that T(3) caused a dose-dependent decrease in mesenchyme and increase in cuboidal epithelia and airway space. Immunocytochemistry showed that with T(3) treatment, Nkx2.1 and surfactant protein SP-C proteins became progressively localized to cuboidal epithelial cells and mesenchymal expression of Hoxb5 was reduced, a pattern resembling late fetal lung development. We conclude that exogenous T(3) treatment during early lung development accelerated epithelial and mesenchymal cell differentiation at the expense of premature reduction in new branch formation and lung growth.     

Dexamethasone-induced changes in lung function are not prevented by concomitant treatment with retinoic acid

Alveolarization is impaired in rats treated with dexamethasone (Dex) on postnatal days 4-13, but concomitant treatment with all-trans retinoic acid (RA) increases alveolar number. To determine whether morphological changes induced by Dex and/or RA predict changes in lung function at 1 mo, we assessed resting breathing parameters, dynamic compliance, ventilation required to maintain O(2) saturation at > or = 90%, and pressure-volume curves of air-filled lungs. During resting breathing, mean tidal volume per gram was greater in Dex + RA-treated rats than in controls (P < 0.05). Dynamic compliance was also greater in Dex- and Dex + RA-treated rats than in controls or RA-treated rats (P < 0.02). In Dex- and Dex + RA-treated rats, we observed increased hysteresis ratios (P < or = 0.006), air trapping (P < 0.05), and lung volumes at 5 and 13.5 cmH(2)O pressure (P < 0.001) and decreased elastic recoil (P < 0.007). The effect of Dex on elastic recoil was greater in female than in male rats (P = 0.006). Despite impaired septation, O(2) saturation was not compromised in Dex- or Dex + RA-treated rats. Thus lung function changes induced by Dex treatment during alveolarization were not prevented by concomitant treatment with RA.     

Targeted expression of a dominant negative FGF receptor blocks branching morphogenesis and epithelial differentiation of the mouse lung.

Mouse lung development begins when two lung buds sprout from the epithelium of the embryonic gut. Patterning of the airways is then accomplished by the outgrowth and repetitive branching of the two lung buds, a process called branching morphogenesis. One of the four fibroblast growth factor (FGF) receptor genes, FGFR2, is expressed in the epithelium of a number of embryonic organs including the lung buds. To block the function of FGFR2 during branching morphogenesis of the lung without affecting its function in other embryonic tissues, the human surfactant protein C promoter was used to target expression of a dominant negative FGFR2 exclusively to lung bud epithelium in transgenic mice. Newborn mice expressing the transgene were completely normal except that instead of normally developed lungs they had two undifferentiated epithelial tubes that extended from the bifurcation of the trachea down to the diaphragm, a defect that resulted in perinatal death. Thus, the dominant negative FGF receptor completely blocked airway branching and epithelial differentiation, without prohibiting outgrowth, establishing a specific role for FGFs in branching morphogenesis of the mammalian lung.     

Gremlin negatively modulates BMP-4 induction of embryonic mouse lung branching morphogenesis

Bone morphogenetic protein-4 (BMP-4) is a key morphogen for embryonic lung development that is expressed at high levels in the peripheral epithelium, but the mechanisms that modulate BMP-4 function in early mouse lung branching morphogenesis are unclear. Here, we studied the BMP-4 antagonist Gremlin, which is a member of the DAN family of BMP antagonists that can bind and block BMP-2/4 activity. The expression level of gremlin in embryonic mouse lungs is highest in the early embryonic pseudoglandular stage [embryonic days (E) 11.5-14.5] and is reduced during fetal lung maturation (E18.5 to postnatal day 1). In situ hybridization indicates that gremlin is diffusely expressed in peripheral lung mesenchyme and epithelium, but relatively high epithelial expression occurs in branching buds at E11.5 and in large airways after E16.5. In E11.5 lung organ culture, we found that exogenous BMP-4 dramatically enhanced peripheral lung epithelial branching morphogenesis, whereas reduction of endogenous gremlin expression with antisense oligonucleotides achieved the same gain-of-function phenotype as exogenous BMP-4, including increased epithelial cell proliferation and surfactant protein C expression. On the other hand, adenoviral overexpression of gremlin blocked the stimulatory effects of exogenous BMP-4. Therefore, our data support the hypothesis that Gremlin is a physiologically negative regulator of BMP-4 in lung branching morphogenesis.     

Evidence that SPROUTY2 functions as an inhibitor of mouse embryonic lung growth and morphogenesis

Experimental evidence is rapidly emerging that the coupling of positive regulatory signals with the induction of negative feedback modulators is a mechanism of fine regulation in development. Studies in Drosophila and chick have shown that members of the SPROUTY family are inducible negative regulators of growth factors that act through tyrosine kinase receptors. We and others have shown that Fibroblast Growth Factor 10 (FGF10) is a key positive regulator of lung branching morphogenesis. Herein, we provide direct evidence that mSprouty2 is dynamically expressed in the peripheral endoderm in embryonic lung and is downregulated in the clefts between new branches at E12.5. We found that mSprouty2 was expressed in a domain restricted in time and space, adjacent to that of Fgf10 in the peripheral mesenchyme. By E14.5, Fgf10 expression was restricted to a narrow domain of mesenchyme along the extreme edges of the individual lung lobes, whereas mSprouty2 was most highly expressed in the subjacent epithelial terminal buds. FGF10 beads upregulated the expression of mSprouty2 in adjacent epithelium in embryonic lung explant culture. Lung cultures treated with exogenous FGF10 showed greater branching and higher levels of mSpry2 mRNA. Conversely, Fgf10 antisense oligonucleotides reduced branching and decreased mSpry2 mRNA levels. However, treatment with exogenous FGF10 or antisense Fgf10 did not change Shh and FgfR2 mRNA levels in the lungs. We investigated Sprouty2 function during lung development by two different but complementary approaches. The targeted overexpression of mSprouty2 in the peripheral lung epithelium in vivo, using the Surfactant Protein C promoter, resulted in a low level of branching, lung lobe edges abnormal in appearance and the inhibition of epithelial proliferation. Transient high-level overexpression of mSpry2 throughout the pulmonary epithelium by intra-tracheal adenovirus microinjection also resulted in a low level of branching. These results indicate for the first time that mSPROUTY2 functions as a negative regulator of embryonic lung morphogenesis and growth.     

Functional diversity of notch family genes in fetal lung development

In Drosophila, developmental signaling via the transmembrane Notch receptor modulates branching morphogenesis and neuronal differentiation. To determine whether the notch gene family can regulate mammalian organogenesis, including neuroendocrine cell differentiation, we evaluated developing murine lung. After demonstrating gene expression for notch-1, notch-2, notch-3, and the Notch ligands jagged-1 and jagged-2 in embryonic mouse lung, we tested whether altering expression of these genes can modulate branching morphogenesis. Branching of embryonic day (E) 11.5 lung buds increased when they were treated with notch-1 antisense oligodeoxynucleotides in culture compared with the corresponding sense controls, whereas notch-2, notch-3, jagged-1, or jagged-2 antisense oligos had no significant effect. To assess cell differentiation, we immunostained lung bud cultures for the neural/neuroendocrine marker PGP9.5. Antisense to notch-1 or jagged-1 markedly increased numbers of PGP9.5-positive neuroendocrine cells alone without affecting neural tissue, whereas only neural tissue was promoted by notch-3 antisense in culture. There was no significant effect on cell proliferation or apoptosis in these antisense experiments. Cumulatively, these observations suggest that interactions between distinct Notch family members can have diverse tissue-specific regulatory functions during development, arguing against simple functional redundancy.     

M2 macrophage polarisation is associated with alveolar formation during postnatal lung development

Background

Macrophages are traditionally associated with inflammation and host defence, however a greater understanding of macrophage heterogeneity is revealing their essential roles in non-immune functions such as development, homeostasis and regeneration. In organs including the brain, kidney, mammary gland and pancreas, macrophages reside in large numbers and provide essential regulatory functions that shape organ development and maturation. However, the role of macrophages in lung development and the potential implications of macrophage modulation in the promotion of lung maturation have not yet been ascertained.

Methods

Embryonic day (E)12.5 mouse lungs were cultured as explants and macrophages associated with branching morphogenesis were visualised by wholemount immunofluorescence microscopy. Postnatal lung development and the correlation with macrophage number and phenotype were examined using Colony-stimulating factor-1 receptor-enhanced green fluorescent protein (Csf1r-EGFP) reporter mice. Structural histological examination was complemented with whole-body plethysmography assessment of postnatal lung functional maturation over time.Flow cytometry, real-time (q)PCR and immunofluorescence microscopy were performed to characterise macrophage number, phenotype and localisation in the lung during postnatal development. To assess the impact of developmental macrophage modulation, CSF-1 was administered to neonatal mice at postnatal day (P)1, 2 and 3, and lung macrophage number and phenotype were assessed at P5. EGFP transgene expression and in situ hybridisation was performed to assess CSF-1R location in the developing lung.

Results

Macrophages in embryonic lungs were abundant and densely located within branch points during branching morphogenesis. During postnatal development, structural and functional maturation of the lung was associated with an increase in lung macrophage number. In particular, the period of alveolarisation from P14-21 was associated with increased number of Csf1r-EGFP+ macrophages and upregulated expression of Arginase 1 (Arg1), Mannose receptor 1 (Mrc1) and Chemokine C-C motif ligand 17 (Ccl17), indicative of an M2 or tissue remodelling macrophage phenotype. Administration of CSF-1 to neonatal mice increased trophic macrophages during development and was associated with increased expression of the M2-associated gene Found in inflammatory zone (Fizz)1 and the growth regulator Insulin-like growth factor (Igf)1. The effects of CSF-1 were identified as macrophage-mediated, as the CSF-1R was found to be exclusively expressed on interstitial myeloid cells.

Conclusions

This study identifies the presence of CSF-1R+ M2-polarised macrophages localising to sites of branching morphogenesis and increasing in number during the alveolarisation stage of normal lung development. Improved understanding of the role of macrophages in lung developmental regulation has clinical relevance for addressing neonatal inflammatory perturbation of development and highlights macrophage modulation as a potential intervention to promote lung development.     

Endothelial monocyte activating polypeptide II inhibits lung neovascularization and airway epithelial morphogenesis

Neovascularization is crucial to lung development and is mediated through a variety of angiogenic and anti-angiogenic factors. Herein, we show that excess Endothelial Monocyte Activating Polypeptide (EMAP) II, an anti-angiogenic protein, not only inhibits fetal lung neovascularization, but also significantly alters airway epithelial morphogenesis. In a murine allograft model of lung neovascularization and morphogenesis, embryonic lungs transplanted under the skin of immunocompromised mice receiving intraperitoneal EMAP II, had a 56% reduction in vessel density (P<0.0001) compared to control. EMAP II treated lung transplants also exhibited a marked alteration in lung morphogenesis, including lack of type II alveolar cell formation, determined by markedly decreased expression of surfactant protein C, and increased apoptosis. In contrast, lung implants in animals receiving an EMAP II blocking antibody had an increase in vessel density of 50% (P<0.0001) and increased expression of surfactant protein C mRNA in distal epithelium. These studies demonstrate that EMAP II negatively modulates lung neovascularization as well as leading to the arrest of lung airway epithelial morphogenesis and apoptosis.     

Fetal Calcium Regulates Branching Morphogenesis in the Developing Human and Mouse Lung: Involvement of Voltage-Gated Calcium Channels

Airway branching morphogenesis in utero is essential for optimal postnatal lung function. In the fetus, branching morphogenesis occurs during the pseudoglandular stage (weeks 9–17 of human gestation, embryonic days (E)11.5–16.5 in mouse) in a hypercalcaemic environment (∼1.7 in the fetus vs. ∼1.1–1.3 mM for an adult). Previously we have shown that fetal hypercalcemia exerts an inhibitory brake on branching morphogenesis via the calcium-sensing receptor. In addition, earlier studies have shown that nifedipine, a selective blocker of L-type voltage-gated Ca²⁺ channels (VGCC), inhibits fetal lung growth, suggesting a role for VGCC in lung development. The aim of this work was to investigate the expression of VGCC in the pseudoglandular human and mouse lung, and their role in branching morphogenesis. Expression of L-type (Ca_V1.2 and Ca_V1.3), P/Q type (Ca_V2.1), N-type (Ca_V2.2), R-type (Ca_V2.3), and T-type (Ca_V3.2 and Ca_V3.3) VGCC was investigated in paraffin sections from week 9 human fetal lungs and E12.5 mouse embryos. Here we show, for the first time, that Ca_v1.2 and Ca_v1.3 are expressed in both the smooth muscle and epithelium of the developing human and mouse lung. Additionally, Ca_v2.3 was expressed in the lung epithelium of both species. Incubating E12.5 mouse lung rudiments in the presence of nifedipine doubled the amount of branching, an effect which was partly mimicked by the Ca_v2.3 inhibitor, SNX-482. Direct measurements of changes in epithelial cell membrane potential, using the voltage-sensitive fluorescent dye DiSBAC₂(3), demonstrated that cyclic depolarisations occur within the developing epithelium and coincide with rhythmic occlusions of the lumen, driven by the naturally occurring airway peristalsis. We conclude that VGCC are expressed and functional in the fetal human and mouse lung, where they play a role in branching morphogenesis. Furthermore, rhythmic epithelial depolarisations evoked by airway peristalsis would allow for branching to match growth and distension within the developing lung.