Branching morphogenesis is a fundamental developmental process involved in the formation of a variety of organs, from fly trachea and air sacs to kidneys, vasculature, mammary and salivary glands, and the mammalian lungs (Affolter et al., 2003). Although there are general themes in how branches are generated and maintained, different cells and tissues form branches with distinct branch patterns defined by length, diameter, shape, and spacing.
During organogenesis of the vertebrate lung, a hollow bud emerges from the anterior foregut and subsequently undergoes a remarkably stereotyped branching pattern giving rise to thousands of terminal tubules (Metzger et al., 2008). Lung-branching mechanisms have been inferred through analysis of fixed samples, either at different gestational ages in vivo (Li et al., 2002, 2005; Liu et al., 2004; Lu et al., 2007; Tian et al., 2010) or after different times of organ culture in vitro (Nogawa et al., 1998). A comprehensive study of the branching pattern of embryonic lungs isolated over developmental time has defined three major branching modes (Metzger et al., 2008). These modes are (1) domain branching, where a new bud emerges from the epithelial tube; (2) planar bifurcation, the separation of a preexisting bud into two daughter buds within the plane of the culture; and (3) orthogonal bifurcation, the separation of a preexisting bud into four daughter buds, which form a rosette. Whereas these existing methods provide snapshots of the lung-branching process at a given time, they cannot describe the sequence of morphogenetic events, such as cell proliferation, cell migration, and cell shape changes in space and time, that may drive the formation of these different branch patterns. Over the past decade, there have been significant advances in our understanding of the genetic control of lung development, largely through studies of knockout and transgenic mice (Morrisey and Hogan, 2010). However, we still know very little about the cellular mechanisms involved in lung-branching morphogenesis nor whether specific molecules regulate these branching subroutines.
A full assessment of existing concepts and the genetic regulatory circuits necessitates a detailed description of the normal morphological events and the cellular behaviors that occur over time during lung development. Many of the paradigms of branching morphogenesis have come from analysis of other tubular systems such as the kidney (Costantini and Kopan, 2010), the mammary gland (Ewald et al., 2008), and the salivary gland (Harunaga et al., 2011). An understanding of the branching process in these models has been significantly advanced through the use of ex vivo approaches that allow a dynamic visualization of branching. Lung explants provide an accessible system to study the dynamics of lung morphogenesis and analysis of the cellular mechanisms involved in lung-branching morphogenesis. However, the potential of lung explants has been little exploited to explore the dynamics of lung branching.
In this study, we present a systematic time-lapse analysis of planar bifurcation and domain branching, by assessing the dynamics of lung epithelial cells primarily in lung explant systems and when appropriate, complemented with studies on organotypic lung epithelial cultures. To visualize the branching process, we imaged transgenic embryonic lungs that express (a) GFP in the distal lung epithelium under the human Surfactant-protein C promoter (Sftpc/GFP) or (b) GFP-labeled Histone 2B that can be induced in the lung epithelium by doxycycline (H2B-GFP). Our analysis reveals morphologically and molecularly distinct aspects of lung-branching morphogenesis that help to shape the branching buds and define a set of parameters that can be assessed in future studies of mutant lungs as well as to define the molecular and cellular processes underlying lung branching.
Bud Bifurcation in Lung Explants
Analysis of the growth of SftpC/GFP lungs from embryonic day 11.5 (E11.5) mouse embryos placed into explant culture and imaged over 24 hr demonstrates that planar bifurcation of a single bud into two buds is a continuous process but it can described by four morphological stages based on characteristic morphology (Fig.1A, B, Supp. Movie S1, which is available online). We term the first morphological phase as Stage 1 or the bud stage during which the circumference of the newly formed epithelial bud is relatively uniformly increased. Stage 2 is the flattened stage in which the round bud becomes flattened at its apex leading to a bud that is wider than it is high. Stage 3 is the cleft stage: epithelial cells on the two lateral sides of the bud begin to branch, whereas cells in the middle of the bud remain in position. Stage 4 is the outgrowth stage in which the two distinct branches continue to develop into two secondary buds, each of which repeats the cycle. These morphological stages were also observed in uncultured E12.5 embryonic lungs (Fig.1C, D). The dynamic imaging showed that the cleft forms in a position that divides the flattened bud symmetrically (Fig. 1E), suggestive of a highly regulative process.
Proliferation Is Uniform During Bud Bifurcation But Chromosome Orientation Helps to Drive Tissue Morphogenesis
Depending on the form and function of a given organ, a variety of cellular mechanisms are utilized to ensure its proper development and maintenance. During branching morphogenesis, proliferation supplies the growing tissue with increasing numbers of cells. Here we asked whether the coordination of proliferation rates in time and space may act instructively to result in directed tissue growth that would ultimately affect tissue morphology.
Previous in vitro studies using lung epithelial cells cultured in matrigel (organotypic cultures) demonstrated that cell proliferation rates were uniform between bud and non-bud areas until the buds were almost completely formed. These studies suggested that proliferation did not play an instructive role in the branching lung epithelium (Nogawa et al., 1998). Based on the recent proposal of three major branching subroutines in the lung (Metzger et al., 2008), we revisited this question to determine the rate, position, and contribution of proliferative cells during planar bifurcation and domain branch formation.
Lungs were isolated from E11.5 embryos and cultured as explants for 2 days. Phospho-Histone3 (p-H3) (Fig. 2A), which marks early to late prophase of the cell cycle, and mitotic events defined as cells with condensed chromosomes, were used to assess proliferation within the bud regions and the trunk regions. We first focused on buds undergoing planar bifurcation and compared these results with non-cultured E13.5 lungs. Our findings showed that proliferation in the bud regions exceeded that in the trunk regions by more than 2-fold (2.3 × in cultured lung explants, P = 0.004, Fig. 2B; 2.5 × in uncultured lungs, P = 0.02, Fig. 2C). Differential proliferation in the bud and trunk regions was evaluated further by following mitotic events over time. In order to broadly assess the proliferation events in the branching lung explant, we used the Sftpc driver to globally express H2B-GFP in all of the distal lung epithelium and performed 3D reconstructions of buds undergoing development through stages 1 and 2 to quantify the number of dividing cells (Fig. 2E, Supp. Movie S2). In this analysis, the division rate was 3.5 times higher in the budding region than the trunk region (P = 0.002). This differential proliferation is comparable to previous studies using BrdU uptake in organotypic cultures (Nogawa et al., 1998) as well as E12.5 lungs (Okubo et al., 2005), indicating that lung explants are a suitable model to study cellular behaviors during lung branching morphogenesis. Together, these observations demonstrate that proliferation is increased in the distal bud relative to the proximal trunk during planar bifurcation and therefore would be expected to lead to distal tissue outgrowth.
Regionalized cell division within the bud has been speculated to drive the emergence of the daughter buds during planar bifurcation. However, evaluation of proliferation events within the bud showed no difference between the middle and two lateral sides (Fig. 2E). Strikingly, live imaging did reveal a significant reorientation of the chromosomes during division. In many instances, at the onset of cell division the nucleus moved towards the lung epithelial lumen, divided and reintegrated back to the basal position within the epithelial monolayer (Supp. Movie S2), reminiscent of the observed interkinetic nuclear migration in the neuroepithelium (Tsai et al., 2010). Moreover, frequently the metaphase chromosomes rotated either by approximately 45° or 90° around their axes before re-integration of the nucleus into the plane of the epithelium (Fig. 2F, Supp. Movie S2). This process did not involve loss of epithelial polarity as assessed by the polarized expression of ZO-1 (Fig. 2D). This re-orientation of the chromosomes was observed throughout the tissue but was enriched in the lateral aspects of the bifurcating bud (Fig. 2F, Supp. Movie S2). Differences in the orientation of cell division have emerged as a fundamental mechanism specifying the structure and shape of organs (Castanon and Gonzalez-Gaitan, 2011). Whether re-orientation of chromosomes is crucial for the morphogenesis of the bifurcating bud remains to be determined. However, the notion that the chromosome orientation has an impact on lung branch morphology has recently been demonstrated in mice with increased Erk1/2 activity (Tang et al., 2011). This study revealed that division parallel to the airway longitudinal axis led to elongation of the tissue and that this bias is lost in mutants with increased ERK1/2 activity resulting in altered airway morphology. Based on this finding, we evaluated chromosomal division orientation during bud bifurcation in H2B-GFP lung explants. In both the trunk and what becomes the future cleft region, a cell division pattern that was parallel to the airway longitudinal axis was the most common pattern. This pattern was associated with elongation in the direction of the straight arrows in Figure 2G. In contrast, the incidence of parallel and perpendicular divisions was relatively similar on the lateral aspects of the dividing bud. This pattern was associated with bud growth in multiple directions (Fig. 2G, straight and curved arrows). In summary, proliferation is increased along the proximal to distal axis, whereas there is no difference within the bifurcating bud. However, the local regulation of chromosome orientation suggests a differential tissue expansion. Parallel oriented cell division along the longitudinal axis would lead to tissue elongation, whereas perpendicular orientation would favor tissue expansion.
Proliferation Is Localized During Domain Branching
Next we examined the proliferation profile in domain branches, defined by the formation of a new bud from the trunk region. This was studied in both E12.5 uncultured lungs and in H2B-GFP lung explants that were imaged over time. To determine the cell division events in uncultured lungs, we evaluated newly emerging domain branch profiles and subdivided the domain branch region into branching (bud region) and non-branching areas (regions immediately to the proximal and distal sides of the domain branch). We observed a 3-fold increase in proliferation in the domain bud relative to the non-branching region (Fig. 3A, B).
We then turned to live imaging of proliferation events leading to domain branch formation. Due to the lack of molecular markers, it has been difficult to study the earliest events leading to domain branch formation. Our live imaging approach, however, provides the ability to go back in time and examine the cellular behaviors that proceed the initiation of domain branch formation and its early progression. Similar to our results of planar bifurcation, nearly two-thirds of all proliferation events were localized to the new emerging bud whereas the remaining one-third occurred proximal and distal to the bud (Fig. 3C, Supp. Movie S3). Thus, proliferation seems to facilitate outgrowth of newly forming buds during domain branch formation. As noted during bud bifurcation, chromosome reorientation was frequent in the domain branch region (Fig. 3D). In addition, most of the cell divisions (61%) in the domain branch itself occurred along the proximal-distal axis of the newly forming bud parallel to the longitudinal axis of the epithelium, which would be expected to promote the elongation and outgrowth of the emerging bud (Fig. 3E). We also noticed more pronounced perpendicular cell division proximal to the domain branch (Fig. 3E, blue region) compared to a similar amount of perpendicular and parallel cell division distal to the domain branch (Fig. 3E, orange region). This would be expected to lead to an increase in diameter in the proximal airway versus a more equal distribution of growth both in diameter and length of the distal airway and this is reflective of the lung airway anatomy. Taken together, these data indicate that domain branching is associated with local increases in proliferation and oriented cell divisions that correlate with epithelial budding.
Epithelial Movements Help Shape the Airway Branches
The results above indicate that proliferation rate and orientation of the division plane significantly influence branching morphogenesis. However, it is likely that other cellular behaviors such as cell migration and movement help to shape the airway branches. Based on our live imaging observations in Figure 1A and Supp. Movie S1, areas of the epithelium such as the cleft region maintained their local position as if they were constrained from moving further distally, while the lateral aspects of the initial bud moved in different directions to form two laterally displaced buds. We examined whether the epithelial cells at the leading front of the branching epithelium had actin-rich protrusions, such as filopodia and lamellipodia (Mitchison and Cramer, 1996). Using primary lung epithelial cell cultures immunostained for actin filaments after 2 days of ongoing branching morphogenesis (Fig. 4A), we did not observe directed protrusions, cells that lie outside of the plane of the epithelium, or elongation of polarized epithelial cells into the surrounding environment. Similarly, in GFP/SftpC lung explants, we did not detect cell protrusions (Fig. 4B), suggesting that cell migration was not a prominent mechanism of branching in lungs.
To determine if a constant group of cells always remained at the bud tip to direct collective epithelial movement and outgrowth or whether cells change their position during the progression of branching morphogenesis, we conducted time-lapse video microscopy on E11.5 cultured lung explants with mosaic H2B-GFP labeling and followed individual H2B-GFP-labeled nuclei within the lung epithelium over time. During planar bifurcation (Fig. 5A, Supp. Movie S4), most of the H2B-GFP-labeled epithelial cells in the budding region moved in a distal direction and showed only minimal positional exchange with neighboring cells. Cells located towards the lateral sides of the initial bud demonstrated the greatest displacement over time (Fig. 5A, red, pink, yellow dots). In the middle of the bud (Fig. 4A, blue dot), the region that will become the cleft, there was little cell displacement. In fact, cells of the future cleft region retained their position, in contrast to the inward ingression movement of epithelial cells during cleft formation of the salivary gland (Larsen et al., 2006). Trunk cells moved very little and remained localized in the trunk region (orange dot). Moreover, the contribution of individual cells to the bud or cleft changed during the generation of new buds. Former tip cells become cleft cells (Fig. 5A, blue dot), whereas lateral cells (Fig. 5A, red and pink dots) end up at the tip of the daughter buds. Thus, there is not a constant set of cells that direct bud or cleft formation. This contrasts with the findings during kidney branching in which tip cells preferentially give rise to new tip cells (Costantini and Kopan, 2010). Instead, our observations suggest a tissue behavior–based mechanism, where a group of cells rather than individual specified cells, coordinate a given process. This behavior seems to be dependent on their current location and presumably on the biochemical environment.
Our studies above on domain branching indicated a localized increase in cell proliferation. However, an association with localized proliferation has not been seen in other budding systems such as in the Drosophila tracheal system (Affolter and Caussinus, 2008) or the budding of isolated lung endoderm in response to exogenous FGF (Nogawa et al., 1998). It was, therefore, suggested that bud initiation involved primarily changes in cell shape and cell-cell organization in vivo (Liu et al., 2004).
To answer whether initial bud formation (domain branching) is associated with extensive cell reorganization, we utilized lung explants with a mosaic expression pattern of H2B-GFP-labeled epithelial cells to assess the extent of cell movement from the trunk area into the new bud. As shown by video microscopy in Figure 5B and Supp Movie S5, we did not detect a significant contribution of surrounding GFP-positive epithelial cells into the emerging domain branch. This suggests that outgrowth is not primarily facilitated by collective movement of trunk epithelial cells into the new emerging branch. This was further supported by following the position of individual H2B-GFP labeled epithelial cells over time (Supp. Movie S6). Cells at the base of the bud (Fig. 5C, white dot) did not end up in the domain branch. In contrast, cells at the tip of the newly emerging bud (Fig. 5C, red dot) remained at the tip and became considerably displaced from their original position. Interestingly, cells at the perimeter of the domain branch (Fig. 5C, blue and yellow dots) remained at the base of the newly formed branch, but ended up closer together along the longitudinal axis of the trunk epithelium. At the same time, the distance between neighboring domain branches increased (Fig. 5C, yellow bar) without significant loss in diameter across the trunk (Fig. 5C, red bar). In conjunction with the proliferation profile around domain branches (Fig. 3), this observation raises the possibility that increase of epithelial cells along the trunk axis may help push the bulging domain branch epithelium together and thus indirectly contribute to domain branch outgrowth.
Myosin-Based Forces Transition the Distal Bud Through the Flattened Stage and Help to Regulate Domain Branch Formation
The observations above raise a number of questions including the mechanism controlling the outgrowth and location of domain branches and why certain cell populations move extensively while other cell populations maintain their position. One mechanism to control such behavior is by applying local mechanical forces, which would help to direct tissue growth and morphology (Aegerter-Wilmsen et al., 2010; Mammoto and Ingber, 2010). Rho-mediated changes in actin organization have been implicated in lung branching, leading to the suggestion that changes in cytoskeletal tension could generate spatial differences in tissue growth and patterning (Moore et al., 2005).
To assess the possible contribution of physical forces during different stages of planar bifurcation and domain branching, we examined the expression of phosphorylated non-muscle myosin light chain II (phospho-Myosin) in uncultured E12.5 lung tissue as an indirect indication of mechanical forces (Vicente-Manzanares et al., 2009). At the bud stage of planar bifurcation, phospho-Myosin is highly localized at the base and lateral sides of the growing bud but is present at much lower levels around the distal bud circumference (Figs. 6A, 7A). During the flattened stage, phospho-Myosin levels remain high along the trunk and the levels increase along the basal side of the distal epithelium (Figs. 6B, 7B). During the cleft stage, phospho-Myosin intensifies on the basal side of epithelial cells in the emerging cleft region and then is strongly expressed on the basal side in the cleft epithelium during the outgrowth stage (Figs. 6C, D, 7C, D). At the onset of domain branch formation (Fig. 6E, boxed, Fig. 7E), the emerging bud has low levels of phospho-Myosin expression, in contrast to the higher levels in the neighboring, lateral trunk area. As the domain branch transitions to the bud stage of planar bifurcation, it follows the same dynamic phospho-Myosin expression as indicated in Figure 6A–D. These findings raise the possibility that mechanical forces help to (1) support the tubular structure of the trunk epithelium, (2) influence the distal epithelium at the bud tip to transition into the flattened stage, and (3) retain cells in the cleft region restricting them from distal outgrowth. The local reduction in phospho-Myosin in domain branches and emerging buds suggests this may allow outgrowth of the epithelium.
Previous studies have demonstrated that myosin light chain kinase (MLCK) is involved in lung branching by genetic interference (Santos et al., 2007) or drug inhibition (Moore et al., 2005). These studies, however, did not address whether MLCK inhibition affects a particular stage of lung branching or whether it differentially affects domain branching or planar bifurcation. We, therefore, exploited lung explants to perturb MLCK function in a time-dependent manner and to determine whether specific transitions in the two different branching modes may be more sensitive to MLCK inhibition. ML-7, a cell-permeable and potent inhibitor of MLCK, was used as an efficient and fast way to interfere with MLCK function. Moreover, we coupled this with time-lapse video microscopy of the treated E11.5 H2B-GFP lung explants to visualize the branching dynamics over time. Buds expanded in the presence of ML-7; however, there was a striking failure of the epithelium to bifurcate when the inhibitor was applied at the bud stage (Fig. 8B, red asterisks, Supp. Movie S8). In fact, we never observed a bud transitioning to the flattened stage when MLCK-inhibitor was present. However, bifurcation still occurred when the MLCK-inhibitor was added after the bud had transitioned to the flattened stage (Fig. 8B, white asterisks, Supp. Movie S8) and bifurcation was similar to control DMSO-treated lung explants (Fig. 8A, Supp. Movie S7). Interestingly, domain branching occurred normally in the presence of MLCK-inhibitor (Fig. 8C, white arrows, Supp. Movie S9).
Inhibition of myosin ATPase activity by application of blebbistatin to explant cultures (Fig. 8D, E, Supp. Movie S10) caused a similar but even more dramatic result. Distal buds did not undergo flattening or bifurcation and instead the buds continued to expand in circumference. Intriguingly, we also saw ectopic buds from the trunk region (Fig. 8E, white arrows). As phospho-Myosin is normally highly expressed in the trunk and non-branching regions, our results suggest that inhibition of myosin-dependent forces is sufficient to elicit branching. Moreover, Hoechst staining showed numerous nuclei in the epithelial lumen (Fig. 8E) suggesting that the lumen of the epithelial tube was not maintained. Collectively, these results indicate that the mechanisms of planar bifurcation and domain branching are differentially regulated by the activity of MLCK.
Taken together, our study suggests that branching morphogenesis occurs primarily due to higher rates of cell proliferation in the bud relative to the trunk and mechanical forces that are locally applied and help to restrict or allow the tissue mass to expand in different directions. Thus, the observed cell and tissue movement during lung-branching morphogenesis is primarily an event based on cell and tissue displacement.
Morphogenesis is a four-dimensional process. Yet, much of our knowledge of morphogenetic processes relies on observations at single points in time, from which the preceding and subsequent developmental events can only be inferred. Ex vivo techniques, such as culturing denuded lung epithelia in matrigel (organotypic cultures) and following its branching behavior over time, have provided insights into the branching mechanisms. However, these cultures lack the complexity of an embryonic lung, most notably the influence of the surrounding mesenchyme. Whole lung explants maintain the tissue context and as shown here permit the continuous, real-time observation of the entire embryonic lung over several days of culture. The morphological resemblance of domain branching and bifurcation in lung explants to that of in vivo branching makes lung explants a powerful tool to explore the mechanisms underlying lung morphogenesis. To provide the first real-time analysis of the cellular behaviors during bud bifurcation and domain branching in embryonic lung explants, we exploited an imaging technique using two transgenic mouse lines that express GFP in the lung epithelium, Sftpc/GFP and H2B-GFP. Time-lapse visualization of the process of bifurcation has allowed us to describe a continuous process that is marked by four morphological phases. Moreover, our studies contribute to an understanding of the combinatorial roles of proliferation and mechanical forces in regulating lung-branching morphogenesis.
Planar Bifurcation in Four Stages
All terminal lung branching begins with the formation of a swollen bud at the distal end of the epithelial tube. During planar bifurcation, the bud expands (bud stage) before it ultimately separates into two daughter buds. After expansion, the bud flattens and extends in two opposite directions (flattened stage) similar to the characteristic “T” structure in the kidney (Watanabe and Costantini, 2004). This is followed by the cleft stage, in which the first morphological evidence of bifurcation becomes apparent. The position of the cleft can be predicted as the cleft predominantly forms midway along the longitudinal axis of the flattened bud and divides the bud symmetrically. During the outgrowth stage, each new daughter bud grows and expands before undergoing another round of branching.
Proliferation and Cell Division Orientation in Lung Branching Morphogenesis
Cell division is a predominant mechanism for tube growth and elongation in vertebrates. However, the role of cell proliferation during lung-branching morphogenesis has been little studied. Using time-lapse video microscopy, we assessed the role of proliferation with respect to planar bifurcation and domain branch formation in lung explants. Our studies showed a clear increase in proliferation in the bud relative to the stalk or trunk in both branch types. This is consistent with differential proliferation rates along the proximal to distal axis observed in earlier studies using sections from E12.5 lungs (Okubo et al., 2005) and organotypic lung cultures (Nogawa et al., 1998). In the bifurcating bud, there was a relatively uniform proliferation pattern (Fig. 2), in contrast to the concept that there may be preferential proliferation in the emerging buds. Domain branch formation was associated with a localized increase in cell division in the emerging bud relative to the trunk (Fig. 3). Additional mechanisms likely contribute to the spatial regulation of domain branch formation, and our studies suggest a local decrease in mechanical forces (Fig. 6). Furthermore, most cells maintain their position relative to their neighbors during lung outgrowth (Fig. 5). This contrasts with extensive cell rearrangements observed during branching morphogenesis of the kidney (Karner et al., 2009). Moreover, unlike the kidney we did not observe evidence for concomitant narrowing and lengthening of the lung epithelium that could assist either planar bifurcation or domain branching. This mechanism, also known as convergent extension, is regulated by members of the PCP pathway. In agreement with our live imaging results, recent studies of the PCP pathway proteins Vangl2 and Celsr1 found no evidence in support of convergent extension movements during lung branching (Yates et al., 2010; Tang et al., 2011). We also observed extensive movements of the non-cleft cells during bifurcation and outgrowth of the new buds. Moreover, the ability to track these cells showed that tip cells could become cleft cells in the next bifurcation, in contrast to the kidney in which tip cells preferentially remain as tip cells (Costantini and Kopan, 2010).
For both planar bifurcation and domain branching, the orientation of cell division appears to be important in the outgrowth and extension of the lung epithelium. Oriented mitosis in controlling tube shape has been demonstrated for the mouse kidney (Saburi et al., 2008; Karner et al., 2009), gut (Matsuyama et al., 2009), chick neural plate (Sausedo et al., 1997), and recently evidence was also obtained for the lung (Tang et al., 2011). Tang and colleagues demonstrated that the lung epithelial cell division plane is parallel to the airway longitudinal axis, a bias that was lost with increased ERK1/2 activity, leading to changes in airway morphology. Their studies reported a wide range of spindle angle distribution, whereas our studies showed three major angles, parallel, 45° and 90° with respect to the longitudinal axis of the lung epithelium (Figs. 2, 3). This inconsistency can likely be explained by the ability of time-lapse microscopy to follow the complete extent of spindle reorientation versus static images that may catch the process prior to completion.
Although the molecular mechanisms underlying spindle rotation during lung-branching morphogenesis are still poorly understood, the ability to assess the orientation by dynamic imaging allows us to speculate as to its role. Our findings related to planar bifurcation suggest that a bias of parallel division in the future cleft region would promote elongation of the distal bud and help facilitate transition into the flattened stage. This bias was not seen on the lateral sides of the flattening bud and thus divisions along both the parallel and perpendicular axes would be expected to promote three-dimensional expansion and bud growth. Unbiased orientations as seen in the airways of mutants with elevated ERK1/2 activity levels (Tang et al., 2011) would cause general, rather than localized, expansion of the bud. The extent and regional biases of spindle re-orientation observed in our studies could imply a significant level of regulation to ensure proper morphogenetic behaviors.
Mechanical cues may contribute to the predominately parallel oriented division along the longitudinal axis of the distal epithelium, where increased tension as reflected by phospho-Myosin might help to flatten the bud. This is in accordance with the recent proposal that cells divide according to cues provided by their mechanical micro-environment, aligning daughter cells with the external force field (Fink et al., 2011). An understanding of the genetic control of lung branching has increased substantially (Cardoso and Lu, 2006; Yates et al., 2010; Schnatwinkel and Niswander, 2012). It will be of interest to examine the impact of mutations in these genes on oriented cell division during lung branching to determine the molecular control of this morphogenetic process.
Mechanical Forces during Lung Branching Morphogenesis
Cell and tissue architecture is driven by internal forces that cells produce and that they exert on neighboring cells. For instance, contractile activity at the apical surface induces tissue folding and invagination (Chung and Andrew, 2008; Rohrschneider and Nance, 2009) and relies on myosin-II-dependent (Pollard, 1981) contraction of apically enriched actin-filaments (Sawyer et al., 2009). Although such mechanism may facilitate bud bifurcation, we additionally obtained data that would indicate a role for mechanical forces at the basal membrane in bud bifurcation. Such data includes (1) the localization of a dynamic basal myosin network that is associated with planar bifurcation progression (Fig. 6) and (2) the failure to bifurcate if the contractile regulation is inhibited by blebbistatin or an inhibitor of MLCK (Fig. 7). Whereas MLCK plays an important role in the mechanism of planar bifurcation, it does not affect the process of domain branch formation (Fig. 7C). This force-generating mechanism likely relies on the focal adhesion network, which connects the cell to the basal lamina and the surrounding mesenchyme.
Components of focal adhesions, such as integrins, also play a key role in lung development. The bronchi of mice lacking the integrin α3 subunit fail to branch into smaller bronchioles (Kreidberg et al., 1996). Mice lacking both integrin subunits α3 and α6 have marked lung hypoplasia (De Arcangelis et al., 1999). Finally, in cultured lung explants, neutralizing antibodies against integrin α5 β1 inhibited branching morphogenesis (Sakai et al., 2003). Given our findings, it would be interesting to revisit some of the mutant phenotypes with respect to a potential role in force-generating mechanisms.
What mechanisms might regulate mechanical forces in the lung epithelium and what signals lie upstream of MLCK? There is evidence for the involvement of molecules first identified for their role as attractant or repellant signals in axon guidance. For example, it has been proposed that lung branching is regulated by Slit, Robo, Semaphorins, and Plexins, molecules that are all dynamically expressed in the developing lung (Hinck, 2004). Moreover, most of these molecules have an impact on cytoskeletal dynamics including myosin activity (Gallo, 2006). Possible roles for these molecules in regulating cytoskeletal functions in the developing lung can be explored in the future using approaches defined here.
Many genes that govern tissue morphogenesis have been identified. However, we do not fully understand the interconnection between these genes that serve to drive the development of organs with specialized forms and functions. It is clear that mechanical forces exerted by cells are as important as genes and chemical signals in controlling development, morphogenesis, and tissue patterning (Mammoto and Ingber, 2010). In the mammalian lung, our time-lapse analysis has revealed that oriented cell division and myosin-dependent forces help the single-layered epithelium to form into the variety of branched structures.
Collective Cell Movement Versus a Mechanical-Proliferation Model
Recent time-lapse imaging studies have created models for the collective movement of cells, including neuronal precursors in the zebrafish lateral line (Lecaudey and Gilmour, 2006), epithelial cells during Drosophila dorsal closure (Jacinto et al., 2000), and border cell migration in Drosophila (Bianco et al., 2007; Prasad et al., 2009) All of these examples showed cells at the front extending cellular extensions or protrusions in the direction of movement. By contrast, examination of epithelial cells at the distal tip of lung branches did not reveal leading cellular extensions or actin-rich protrusions (Fig. 4), similar to what has been demonstrated at the front of elongating mammary ducts (Ewald et al., 2008). Here, we propose that a mechanically regulated outgrowth mechanism underlies branching morphogenesis in the lung. In this model, cell division has two functions: (1) to provide tissue mass to the growing epithelium and (2) to influence bud morphology through oriented cell division. Cell division parallel to the longitudinal axis would promote tissue extension, whereas perpendicular oriented cell division would direct general tissue expansion. In a scenario where no changes in mechanical forces were applied, the epithelium would expand, leading to an ever-growing sphere. However, application of local mechanical forces would cause the expanding tissue to bulge and grow out in areas of least resistance. Local mechanical forces in conjunction with the proximal to distal increase in proliferation and the cell division orientation would ensure unidirectional movement and growth of the developing lung buds. Together our studies provide new insight into the morphogenetic processes that help to shape the form and function of the embryonic lung.
GFP Reporter Lines
Animal experiments were approved by the Institutional Animal Care and Use Committee at University of Colorado School of Medicine. We used transgenic mice in which GFP was expressed in lung epithelial cells under the human Sftpc-promoter acquired from Dr. John Heath at University of Birmingham. Transgenic mice expressing H2B-GFP (Tumbar et al., 2004) were obtained from Jackson Laboratory (Bar Harbor, ME) and crossed with SPC-rtTA (Tichelaar et al., 2000) mice to obtain lung epithelial expression upon doxycycline addition to the lung explant cultures (see below).
Culture of Lung Explants
E11.5 embryonic lungs were dissected and cultured at the air liquid interface in BGJB medium + 3.5% serum + 1% v/v penicillin/streptomycin at 5% CO2, 5% O2, 37°C. GFP-H2B expression was induced at the start of ex vivo culture either by addition of 10 μg/ml Doxycycline to the culture media for 10 min followed by wash-out to induce mosaic expression or by addition of 10 μg/ml Doxycycline without wash-out to induce broader labeling. For time-lapse video microscopy, lung explants were cultured on Transwell filters (Corning, Lowell, MA) and mounted on a homemade tripod in a glass bottom Matek dish. Lung explants were cultured for one day prior to the start of each experiment to allow adaptation of the lung explants to the new environment. Inhibitors were used in a minimum of three separate experiments with similar results. Representative single experiments are shown. ML-7 (Calbiochem, San Diego, CA; 5 μM), and Blebbistatin (Calbiochem, 25 μM) were added prior to start of the live imaging experiment. All inhibitor experiments discussed in the text were done in full medium with DMSO as control when applicable.
Organotypic epithelial cultures were equilibrated in 25% sucrose in PBS for 1 hr, fixed in cold 1:1 methanol:acetone overnight at −20°C, then re-equilibrated in 25% sucrose in PBS for 1 hr. Lung explants were fixed for 30 min at RT in 4% PFA and permeabilized using 4:1 Methanol:DMSO at −20°C for at least 1 hr. Lung explants were fixed in 4% PFA overnight at 4°C, permeabilized in Methanol for 30 min, and equilibrated in TBST for 1 hr. All samples were blocked with 5% serum in TBST for 1 hr and incubated with primary antibody 2 hr to overnight at 4°C. Secondary antibodies (Molecular Probes Alexa series, Eugene, OR) were added for 1–4 hr. F-actin was stained with Alexa 488 or 568 Phalloidin, and nuclei were stained with Hoechst (Molecular Probes). Antibody stains were done at least 3 independent times. Primary antibodies were E-cadherin (Zymed, San Francisco, CA; 13–1900), ZO-1 (Chemicon, Temecula, CA; MAB1520), phospho-histone H3 and phosphor-myosinII (Cell Signalling, Danvers, MA).
Confocal time-lapse movies and images of antibody-stained samples were collected on a Zeiss (Thornwood, NY) LSM 510 Duo microscope by using a 10×/0.45 A-Plan-Apochromat and a 40×/0.6 LD-Plan-Neofluar objective lens. For time-lapse imaging, the temperature was held at 37°C and CO2 and O2 were held at 5% by using a CTI Controller 3700 and Temperature Control 37.2 combination. Confocal image sections (70 μm) were acquired between 4 and 10 min from three different locations to cover the entire bud using the MultiTime macro (Zeiss, Inc.). Time-lapse movies ranged in length from 16–48 hr. Images collected were analyzed by using Imaris (Bitplane, Zurich, Switzerland) and Zen software (Zeiss). Experiments on lung explants were repeated at least 4 times. Representative single experiments are shown.
Image Processing and Analysis
3D reconstitution images were assembled in ImageJ from a series of 5-μm confocal sections. Levels on images were equally adjusted for control and treated specimens in Adobe Photoshop. Fluorescent intensities were determined using ImageJ profile analysis.
Quantification of Proliferation Rates
Cell proliferation was determined by two methods: (1) the number of phospho-Histone3 stained nuclei and (2) the number of mitotic chromosomes. The numbers were normalized to the total number of nuclei in the given area (trunk, bud). For time-lapse image analysis, the number of mitotic chromosomes was determined in each of the indicated regions and presented as the percentage of the total mitotic cells in the total given area. For simplification, orientation of cell division was determined by its localization to the nearest longitudinal axis in a 2D superimposed image. Cell division along the epithelial longitudinal axis was counted as parallel, whereas orientation 90° to the nearest epithelial longitudinal axis was considered as perpendicular. A representative result from at least 4 independent experiments is shown.
We gratefully acknowledge Dr. John Heath from the University of Birmingham and Dr. Brigid Hogan from Duke University for providing the SftpC/GFP mice. We thank S. Reynolds and B. Appel for comments on the manuscript. L.N. is an investigator of the Howard Hughes Medical Institute. The authors state no conflict of interest.