Introduction

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Mechanisms of vessel formation in the developing lung require the coordination of several processes common to all areas of embryonic cardiovascular development (1). Differentiation of endothelial cell precursors followed by migration and tube formation create a complex, highly organized network of vessels. This complicated scheme of vessel formation has been described as occurring by two mechanisms: angiogenesis, the budding and branching of new vessels from pre-existing vessels; and vasculogenesis, the de novo organization of blood vessels by differentiation of endothelial cells from mesoderm (2). Although angiogenesis is prominent in many processes in the adult, vasculogenesis appears to be restricted to the embryo.

Lung development has traditionally been divided into four or five stages based primarily on epithelial processes, as shown in Table 1 (comparison of mouse and human). The embryonic, or lung bud, stage (embryonic day [E]8 or E9 in the mouse) occurs as the foregut epithelium pushes out into, and becomes surrounded by, the splanchnic mesenchyme. The glandular stage (E9 to E16) has been characterized by rapid and extensive epithelial branching. The primary phase of vessel formation has been described as occurring during the canalicular stage (E16+) as vascular structures extend more peripherally, following the airway branches that have already formed. Two maturation stages the saccular, or newborn, stage and the alveolar stage (day 17 to maturation)have also been described, where thick-walled saccules that are capable of respiratory function eventually become thin and form the delicate septae of mature alveoli (9).

To date, descriptions of pulmonary arterial formation as occurring by angiogenesis or by a combination of angiogenesis and vasculogenesis have provided little conclusive data about the earliest stages of pulmonary vessel formation. Significant studies, performed after the proximal pulmonary arteries have canalized, provide anatomical information on the later stages of pulmonary vessel development (9, 12, 13). Recent work by deMello and colleagues correlated anatomic identification of vascular branching in the developing murine lung with morphologic changes at the cellular level (14). Using angiograms and casts of the vasculature, as well as light and electron microscopy, they supported the previously proposed concept of formation of central pulmonary vessels by angiogenic mechanisms, with formation of peripheral vessels by vasculogenesis. In their study, central-to-distal connections were first identified at 13 to 14 d.

The previous studies provide essential descriptions of the anatomic development of pulmonary vessels once canalization has occurred. However, further work is needed to identify endothelial cell precursors in both the distal lung mesenchyme and in the developing proximal pulmonary arteries to determine the actual mechanism of development, i.e., angiogenesis, vasculogenesis, or a combination of the two. In addition, a method to objectively quantify vessel development for in vivo analysis and organ culture studies would be advantageous.

The present study characterizes endothelial cell differentiation and vessel formation in the lung during mouse organogenesis and provides new information about the timing and mechanism of pulmonary artery formation. Fetal liver kinase-1 (flk, or vascular endothelial growth factor receptor-2) has been identified as the earliest member of a series of endothelial-specific receptor tyrosine kinases to be expressed on endothelial cells, and flk is required for endothelial cell differentiation and vessel formation (15- 18). In developing lung, flk is expressed in vessels whereas its ligand has been shown to be expressed in the adjacent epithelium (19, 20). In targeted disruption studies, a line of flk.LacZ mice was created by replacing the translated portion of the first coding exon, and the proximal part of the following intron, with a promoterless Escherichia coli gene for -galactosidase (-gal) (21). This construct resulted in endothelial-specific -gal expression under the control of the flk promoter. In the homozygous state, embryos died between E8.5 and E9.5 with defects in yolk-sac blood island formation and vasculogenesis. However, no defects in vascular development were detected in the heterozygous animals. In the present study, heterozygous flk.LacZ mice were used to identify endothelial cell precursors in the mesenchyme during early lung development, and to visualize vascular structures as differentiation and remodeling occurred. In contrast to earlier studies, we observed that continuity between endothelial cells of the branch pulmonary artery, and endothelial cells in the differentiating mesenchyme of the distal lung, was present from the earliest time point evaluated (E10.5). In addition, quantification of endothelial cell differentiation at each stage of lung development by a chemiluminescent reporter system allowed an objective assessment of ongoing vascular development. This assay provided evidence that pulmonary vessel formation is occurring at an earlier and more rapid pace than has previously been suspected, and provides a baseline to guide the study of normal and pathologic pulmonary vascular development.

	Materials and Methods

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Animals

Mice heterozygous for targeted insertion of LacZ into the flk locus were obtained from Jackson Labs (Bar Harbor, ME) (21). Male flk.LacZ mice were bred with wild-type CD1 female mice (Charles River, Portage, MI) and the morning of the vaginal plug was counted as E0.5. Developing lungs were evaluated at E10.5, E11.5, E12.5, E13.5, E14, and E15.5 to E16; at E18/birth; at 2 wk postnatal age; and in adulthood. All animal procedures conformed to institutional guidelines and to the NIH Guide for the Care and Use of Laboratory Animals.

Histochemical and Immunofluorescent Staining

For histochemical staining, whole embryos (E10.5), heart and lungs (E11.5), or whole lungs (all other stages) were rinsed in phosphate-buffered saline (PBS), fixed for 30 to 60 min in 4% paraformaldehyde (PFA) at room temperature, and rinsed twice in PBS with 2 mM magnesium chloride (MgCl₂). The embryos or organs were stained overnight in 1 mg/ml X-gal (4-chloro-5-bromo-3-indolyl--gal) in 5 mM K₄Fe(CN)₆, 5 mM K₃Fe(CN)₆, and 2 mM MgCl₂ at 4°C. Tissues were postfixed for several hours in 4% PFA, dehydrated through an ethanol gradient, placed in xylene, and embedded in paraffin. Seven-micron-thick sections were counterstained with eosin. For frozen sections, tissues were placed in 30% sucrose, then frozen in OCT (Sakura, Torrence, CA). Microscopic sections were photographed using a Nikon Eclipse E800 microscope (Nikon, Milville, NY) and an Image Pro Plus Digital Image System (Image Pro Plus, Glen Mills, PA).

To confirm endothelial expression of LacZ, double immunostaining was performed on 6-µm frozen sections of flk.LacZ lungs at E12.5 using monoclonal antibodies to platelet endothelial cell adhesion molecule-1 (PECAM-1) (rat antimouse, mAb390; PharMingen, San Diego, CA) at 1:200 dilution, and -gal (mouse anti--gal, GAL-13; Sigma, St. Louis, MO) diluted 1:500. Primary antibodies were detected using fluorescein isothiocyanate (FITC)- conjugated antirat and rhodamine-conjugated antimouse secondary antibodies, respectively.

Quantification of Endothelial -Gal Activity

Lungs from embryos and mice determined to be heterozygous for flk.LacZ were used to quantify the presence of endothelial cells by chemiluminescent detection of -gal activity. Lungs were dissected in cold PBS and immediately stored at 80°C and tails were stained in X-gal solution (as described earlier) to determine expression of transgene. Ten lungs expressing the LacZ gene were assayed for each embryonic stage and six LacZ lungs were used for 2-wk-stage analysis. Protein was extracted from individual lungs and, by means of a luminometer and a chemiluminescent reporter assay (Galactolight; Tropix, Bedford, MA), -gal activity was measured and compared with a known -gal standard (Boehringer Mannheim, Mannheim, Germany). Total protein per lung was determined (BCA; Pierce, Rockford, IL) and -gal activity was normalized to total protein. Averages of total lung protein, -gal activity/ lung, and -gal/protein for each stage were determined.

	Results

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Characterization of Pulmonary Vascular Development

Endothelial cell precursors of the distal lung mesenchyme and the branch pulmonary arteries were identified by expression of flk.LacZ in the developing lung at E10.5 (Figure 1). The outpouch of foregut epithelium forming the developing airway was seen surrounded by a layer of flk.LacZ-positive cells in the mesenchyme. There were no lacunae present in the mesenchyme at this stage. The endothelial cells forming the proximal or branch pulmonary artery were identified by flk.LacZ expression while they differentiated from mesenchyme. This primordial vessel, in which only the proximal portion contained a lumen, connected the aortic sac with the distal flk.LacZ-positive mesenchymal layer even by this early stage (Figure 1b). This is the earliest time point at which connections between proximal and distal vessels in the developing mouse lung have been described. Of note: development of the pulmonary vasculature by angiogenesis would be expected to result in a lack of continuity between the endothelial cells of the aortic sac and the endothelial cells of the distal lung mesenchyme; this lack was not observed at any stage.

View larger version (138K): [in this window] [in a new window]   Figure 1.   flk.LacZ expression in E10.5 mouse embryos. (a) Low-power view of sagittal section of an E10.5 flk.LacZ mouse after X-gal staining. (b) Magnified view of the lung bud and the area enclosed in the frame in a. Endothelial cells and their precursors are identified by blue staining of -gal protein activity. The developing pulmonary artery can be seen connecting the aortic sac with the differentiating endothelial cells in the distal mesenchyme surrounding the developing airway of the lung bud. The pulmonary artery can be seen to have a lumen forming in the proximal portion even by this early developmental stage. The epithelium of the airway is encircled by -gal-expressing mesenchymal cells that are endothelial cell precursors. (AoS = Aortic sac, DAo = Dorsal aorta, A = cardiac atrium, V = cardiac ventricle, Li = liver, PA = pulmonary artery, Mes = mesenchyme, Epi = airway epithelium.)

By E11.5, which corresponded to approximately 6 wk in human pulmonary vessel development, the endothelial cells of the developing right ventricular outflow tract, main pulmonary artery, aorta, and ductus arteriosus were identified by expression of flk.LacZ. The branch pulmonary arteries were seen connecting the ductus arteriosus and forming main pulmonary artery to the primary capillary plexus developing from the mesenchyme of the lung (Figures 2a and 2b). In the forming lung lobes, endothelial cells and precursors were identified by flk.LacZ expression as they encircled the budding airways. Microscopic sections (Figures 2c and 2d) showed proximal branch pulmonary arteries with patent lumens and vessels, which in other sections joined to form a confluence behind the heart, defining them as pulmonary veins. Both pulmonary arteries and veins contained cells that appeared to be of hematopoietic lineage. Clear microscopic intercellular connections were present between the differentiating endothelial cells of the distal capillary plexus and the endothelial cells of the lobar pulmonary arteries and veins (Figure 2d).

View larger version (92K): [in this window] [in a new window]   Figure 2.   flk.LacZ expression in E11.5 mouse embryos. (a) Anterior view of E11.5 flk.LacZ murine heart showing extensive LacZ expression with clear demarcation of the pulmonic, or right ventricular, outflow tract (RVOT) leading to the main pulmonary artery (MPA) (RV = right ventricle, LV = left ventricle). (b) Lateral view of heart and vasculature showing RVOT connection to the MPA, ductus arteriosus, and left pulmonary artery (LPA); the ductus arteriosus connects the main pulmonary artery to the aorta (DAo). The LPA extends to the distal mesenchyme (Mes). (c) Photomicrograph of coronal view of developing lung. There is increased tissue mass of both airway epithelium (Epi) and mesenchyme as compared with E10.5 mice. (d) Higher-power photomicrograph showing further branching of the airway epithelium into the mesenchyme, with vascular lumens seen in both the pulmonary artery (PA) and pulmonary vein (PV) as they connect proximal structures to the distal developing endothelium. Cells that appear to be hematopoietic in origin are seen within the lumen.

Analysis at E12.5 revealed second-generation vascular branches extending from the proximal pulmonary arteries, with a more complex vascular network present in the lung periphery (Figure 3a). The murine lung showed a single left lobe and four right lobes formed by this stage, with visible vascular branches to each lobe. The pulmonary arteries formed along the more lateral aspect of the lobe, whereas the pulmonary veins were present along the medial aspect and returned to a common region behind the heart to establish the pulmonary venous confluence. The most medial right lobe (referred to as the caudal lobe) was seen to have efferent and afferent vessels, extending toward the tip of the lobe as the pulmonary artery and returning as the pulmonary vein. Histologic evaluation (Figures 3b-3d) confirmed the widely patent lumen present at this stage, and showed the secondary branches connected to differentiating mesenchymal cells surrounding a primitive airway (Figure 3c). Direct connection between the proximal vessels and the distal vasculature was seen (Figure 3d) with lumens visible in the pulmonary artery and the secondary vascular branch, and starting to form in the periepithelial capillary plexus.

View larger version (150K): [in this window] [in a new window]   Figure 3.   Vascular development in E12.5 mouse lungs. (a) Whole-mount X-gal-stained flk.LacZ lung showing development of right and left pulmonary arteries (RPA and LPA, respectively) and pulmonary veins (PV). Secondary branches of pulmonary arteries (arrowheads) can be seen extending to each of the four right and the single left lobes. Early formation of the pulmonary venous confluence (asterisk) is seen where the medially located pulmonary veins converge. (b) Photomicrograph showing the presence of a lumen in the LPA as it connects to the distal mesenchyme. (c) High-power photomicrograph showing PA connection to endothelial cells differentiating in a periepithelial position (E = airway epithelium). A pulmonary vein is seen forming on the medial aspect of the lobe. (d) High-power view showing the origin of a secondary branch (arrowhead) from the LPA of the lung seen in b. The branch has a patent lumen and extends to the endothelial cells located in a periepithelial location showing direct connection of proximal and distal vascular structures.

Confirmation of flk.LacZ identification of endothelial cells was performed by double immunofluorescent staining for -gal protein and PECAM-1. Secondary labeling with a FITC-conjugated antibody to recognize bound anti-PECAM (Figure 4a) and rhodamine detection of anti--gal (Figure 4b) revealed identical patterns of labeling of cells in the mesenchyme in E12.5 lungs. The immunolabeling confirmed the sensitivity and specificity of flk.LacZ expression for endothelial cell identification during murine pulmonary vessel development.

View larger version (32K): [in this window] [in a new window]   Figure 4.   Confirmation of specificity of LacZ expression in endothelial cells. Immunofluorescent staining of E12.5 lungs with antibodies to the endothelial marker PECAM-1 (a) and to -gal (b) reveal nearly identical labeling of mesenchymal cells (Mes) with the absence of epithelial staining with both antibodies on the same lung section. The similar patterns of staining for the two antibodies confirm the expression of flk.LacZ in developing murine pulmonary vasculature in a sensitive and specific manner.

Analysis of whole lungs X-gal-stained at E13.5 showed further branching of the proximal pulmonary arteries with fine reticular networks of vessels in the periphery (Figure 5a). The pulmonary venous confluence was well formed by this stage with four pulmonary veins easily visible. On microscopic sections, large vascular structures were seen at the hilar area of the left lung, and secondary and tertiary branches were readily identified. The lacunae within the capillary plexus of the lung were expanded compared with those seen 1 d earlier, and were located at a constant distance from, and aligned parallel to, the developing airways (Figures 5b and 5c).

View larger version (72K): [in this window] [in a new window]   Figure 5.   Vascular development in E13.5 mouse lungs. (a) Whole-mount X-gal-stained flk.LacZ lung shows further branching of proximal arteries with ongoing development of a more extensive vascular plexus peripherally. Four well-formed pulmonary veins were seen returning from the lung lobes and formed a well-defined pulmonary venous confluence (asterisk). (b) Microscopic section showing lengthening of epithelial tubes with -gal-expressing endothelial cells, forming intercellular connections and lacunae (Lac) between and parallel to the airway tubes. (c) Higher-power view showing endothelial cell patterns in relation to the airway epithelium and to each other. Cells appear to make end-to-end connections to establish lumens parallel to the axes of the airways.

Whole-mount staining of 14-d lungs showed accentuation of -gal activity consistent with the increase in number of endothelial cells present by this stage (Figure 6a). It is notable that this large degree of vessel development occurred 48 h before the classically described vascular stage, the canalicular stage that begins at E16. On microscopic examination, a large number of airways were seen with accompanying vessels (Figures 6b and 6c) that were remodeled from the parallel lacunae seen 1 d earlier (Figure 5b). Many airways and arterioles were surrounded by smooth-muscle layers. The developing interlobular septae contained flk.LacZ expressing venous structures (Figure 6d).

View larger version (123K): [in this window] [in a new window]   Figure 6.   flk.LacZ expression in E14 mice. Further organization of the epithelial and vascular structures of the lung rapidly occurred in 24 h. (a) The depth of the blue color of LacZ expression on whole-mount X-gal staining was indicative of the intensity of vessel formation at this stage. This intense vessel formation occurred 48 h before the classically described canalicular stage of lung development. (b) Photomicrograph of developing lung at E14. A large amount of mesenchyme and developing endothelium (blue staining) was present surrounding the branching airways (Epi). Smooth muscle (Sm) was seen encircling airways and arterioles (Art) (shown in b, c, and e). Interlobular septae (Sep) have formed (c and d) and can be seen to contain developing pulmonary veins (PV).

Lungs evaluated at E15.5 to E16, which corresponded to approximately 17 wk in the human, showed continued lung growth with intense -gal activity, again consistent with the ongoing proliferation and differentiation of endothelial cells (Figure 7). At the microscopic level, extensive interconnecting vascular networks were seen throughout the lung, surrounding the increasingly branched airways (Figures 7a and 7b). Microscopic examination of newborn lung tissue showed flk.LacZ endothelial expression throughout the thickened septal walls during this saccular stage of development (Figures 7c and 7d). In the 2-wk-old mouse lungs, whole-mount staining continued to show extensive -gal activity (Figure 7e). The intensity of -gal activity was consistent with the accentuation of endothelial cell growth required for the capillary and alveolar-septal formation characteristic of perinatal and postnatal lung morphogenesis. On microscopic examination at 2 wk of age, the thin alveolar septae continued to show flk.LacZ expression (Figure 7f). Although the overall -gal activity remained high (as identified by histochemical staining) at 2 wk after birth, it should be noted that the flk-mediated expression of LacZ at this stage may have underestimated the endothelial cells present. Expression of flk is downregulated in the mature animal (18); consistent with this observation, we found that flk expression was downregulated in the adult mouse lung which showed no LacZ expression on gross or microscopic evaluations following X-gal staining (not shown).

View larger version (127K): [in this window] [in a new window]   Figure 7.   flk.LacZ expression in E15.5, E18 (newborn), and 2-wk-old mice. Low-power (a) and high-power (b) views of sections of E15.5 mouse lungs showing extensive vascular networks (vasc net) surrounding branching airways. The formation of vessel lumens can be seen within the networks. (c) By the newborn period, the thickness of the epithelial layer is decreased compared with 2 d earlier, although the septal wall remains relatively thick. Further branching of epithelial and mesenchymal structures brings airways and vessels into close approximation. Capillaries expressing flk.LacZ can be seen between the thick-walled septae. (d) High-power view shows that endothelial cells of capillaries contribute to formation of the septal walls. (e) Two weeks after birth, the lung remains very dark blue as a result of the extensive capillary expression of flk.LacZ. ( f ) Thinning of the septae and ongoing division have resulted in delicate alveolar walls (thin septae) that show expression of flk.LacZ in endothelial cells contributing to the blood-air interface.

Quantification of Pulmonary Vessel Growth

To quantify endothelial cell growth during pulmonary vascular development we performed a chemiluminescent assay for detection of enzymatic activity of -gal. The mice used for these experiments were heterozygotes with one copy of the gene for -gal (LacZ) inserted into a single flk allele, allowing endothelial specific expression of LacZ by the 5' regulatory regions of the flk gene (21). Therefore, -gal expression and activity were directly proportional to the number of endothelial cells present in the tissue at each stage of development. Overall lung growth was determined by total protein assessment of whole lungs. As total protein content of the lungs increased from 35 µg/lung at E12.5 to 6,456 µg/lung at 2 wk of age, there was a corresponding increase in -gal activity from 12 ng/lung to 3,215 ng/lung (Table 2). -Gal activity normalized to total protein showed the largest change between E12.5 (343 pg/µg) and E13.5 (450 pg/µg), after which the rate of increase in -gal was proportional to the increase in total lung protein. As noted with the morphologic evaluation of endothelial expression, these data confirm that accelerated endothelial proliferation occurred before the canalicular stage. Although there is ongoing development of the pulmonary vasculature during the canalicular stage, there appears to be no burst of vascular development at any developmental point but rather a steady continuum corresponding to overall lung growth.

	Discussion

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The LacZ gene under flk promoter control was used to mark early endothelial cell differentiation and vessel formation in the lung during murine embryonic development. Endothelial cell precursors were identified beginning at E10.5 as a layer of mesenchymal cells surrounding the invading epithelial tube. As ongoing differentiation occurred, there was an increase in the mass of flk.LacZ-expressing mesenchymal cells that subsequently transitioned into a primary capillary network. We speculate this network formed in a specified pattern relative to the branching epithelium as a result of signaling between epithelium and mesenchyme, as has been shown in studies of transplanted angioblasts into embryos (5). With continued remodeling, the primary capillary network developed into more definitive vascular structures forming the arterioles, veins, and capillaries of the lung.

Using flk promoter expression of LacZ we were able to identify continuity between endothelial cells of proximal and peripheral vessels much earlier than has been observed previously, and these results raise the possibility of vasculogenesis as the mechanism of proximal vessel formation. Prior developmental studies used intravascular injection techniques and concluded that patency corresponded to vessel formation. As development proceeded, the patency of the vessels extended more peripherally and therefore it was felt the vessels were growing by extension. With the marker used in this study, we identified connections present at the earliest time point evaluated, by E10.5, several days before patency found by contrast injection. The importance of this distinction is that it brings into question the accepted theory of formation by angiogenesis. The presence of differentiating endothelial cells before lumen formation raises the possibility of formation by vasculogenic mechanisms, as occurs in early development of the aorta (22, 23). A similar conclusion was reached after experiments performed in quail embryos using scanning electron microscopy and immunohistochemical techniques (7). Although the work presented in the current study does not prove vasculogenesis as the mechanism of proximal pulmonary vascular development, vasculogenesis is supported by the in situ differentiation of endothelial cells at the location of vessel formation and indicates that more work is required to clarify the actual mechanism.

In addition to enhancing morphologic evaluation of lung vessel development, this approach has allowed standardization of in vivo total protein content at stages of murine lung development and quantification of pulmonary vessel formation. Quantitative analysis of vascular development has previously relied on subjective techniques such as counting presumptive endothelial cells in a designated number of microscopic fields, counting numbers of vessel lumens, and measuring lengths of tubes in vitro. However, no quantification of vessel development has previously been performed in the developing mouse lung. In this study lungs from heterozygous mice having a single copy of the flk.LacZ gene present in each cell were used in a chemiluminescent assay to quantify endothelial cell differentiation. Previously, much of vascular development in the lung was thought to occur with a burst of vessel formation in the canalicular phase of lung growth. Our quantitative analysis showed a steady rate of increase in flk.LacZ expression, consistent with increased endothelial cell number, beginning during the pseudoglandular stage of development and continuing throughout all subsequent stages, including the alveolar stage. This increase in expression was parallel to the rate of overall increase in lung growth as assessed by protein content, indicating there was early and continuous development of the vasculature and not a specific period during which most of the peripheral vessels formed.

In summary, we used an early endothelial cell marker to identify primordial connections of proximal and distal vascular structures in the lung, and we provided evidence to support vasculogenesis as the mechanism of proximal as well as distal vessel formation. In addition, we quantified endothelial cell differentiation in the developing mouse lung and showed that vessel development is a continuous process that begins at the earliest stages of development, rather than occurring as a burst at later developmental stages. These data suggest that formation of the pulmonary vasculature is a process that is vulnerable to perturbation throughout in utero as well as perinatal development. Discerning the mechanism and timing of branch pulmonary artery formation will aid our understanding of the etiology of abnormal pulmonary vascular development in pulmonary atresia, diminutive and discontinuous pulmonary arteries, and disorders associated with multiple peripheral pulmonary vascular stenoses, such as Williams syndrome and Alagille syndrome. Correlation of changes in gene expression patterns with morphologic events will further our understanding of vascular ontogeny and will allow us to devise therapeutic strategies for these pathologic processes.