Modeling Mammary Branching Morphogenesis in Organotypic Culture
Mammary ductal elongation in vivo occurs over the course of weeks, making it difficult to monitor directly. Accordingly, we imaged individual cells over time in an organotypic culture model of mammary branching morphogenesis (Fata et al., 2007
; Simian et al., 2001
; Sternlicht et al., 2005
; Wiseman et al., 2003
). We cultured fragments of freshly isolated mammary epithelium (organoids) in 3D Matrigel gels and collected over 200 bright-field movies and over 400 3D confocal movies to determine the basic sequence of events involved in branching morphogenesis. In minimal medium, the epithelial pieces all formed “simple cysts” with a bilayered organization and a clear lumen (). Addition of FGF2 to the medium induced initiation and elongation of new ducts (Movie S1
). We considered an organoid branched if it had three ducts, as previously established (Fata et al., 2007
). There was no correlation between the initial structure of the organoid and the sites of ductal initiation in culture.
Keratin-14-GFP-Actin Mice Enable Dynamic Visualization of Myoepithelial Cell Behavior
In vivo, the mammary epithelium consists of keratin-8- and -18-positive LE cells and smooth muscle α-actin (SMA)- and keratin-14-positive ME cells (Gudjonsson et al., 2005
). In adult mammary glands, SMA-positive (SMA+) cells are basally located and generally align with the long axis of quiescent ducts. However, during pubertal branching morphogenesis, SMA+ cells adopt a characteristic stellate morphology (Figures S2A–S2D
). These two different morphologies can coexist in adjacent regions of the same duct and imply a change in the behavior of SMA+ cells.
To visualize ME behavior during branching morphogenesis, we utilized a reporter mouse in which the keratin-14 promoter drives expression of an actin-EGFP fusion protein (K14-GFP-actin; Vaezi et al., 2002
). K14-GFP-actin cells in the intact mammary gland were also SMA+ (Figure S2E
) and basally located. Based on these markers, we refer to K14-GFP-actin+ or SMA+ cells as ME cells and K14-GFP-actin− or SMA− epithelial cells as LE cells.
Luminal Clearance and Luminal Filling Precede Ductal Initiation
To determine the relative contributions of ME cells and LE cells to mammary branching morphogenesis, we stained all cells with CellTracker Red and then collected a series of 134 long-term confocal movies of organoids from K14-GFP-actin mice. The organoids began as multilayered epithelia and progressively resolved into bilayered structures with large, clear lumens and a single, complete basal ME layer (; Movie S2
). Once the lumens were cleared, the organoids filled their lumens again, predominately with LE cells (; Movie S3
). We termed this solid structure a “complex cyst” ().
Complex cysts had a single basal ME layer and multiple LE layers, but they also had rare internal SMA+ cells (), similar to the basal-type body cells in TEBs (Mailleux et al., 2007). Epithelial polarity was also reduced, with atypical protein kinase C-ζ (APKC-ζ and β-catenin () present at essentially all sites of cell-cell contact, instead of being restricted into distinct apical and basolateral domains. Complex cysts still retained polarity at a tissue level, however, with apically localized zona occludens 1 (ZO-1) marking luminal surfaces and basally localized β1-integrin marking the cell-matrix border and some sites of cell-cell contact (). The complex cyst stage in culture more closely resembled the TEB in vivo than the quiescent duct (Figure S1
). This transition from a multilayered epithelium to a bilayered epithelium and back to a multilayered epithelium () was characteristic of all organoids examined.
New Ducts Initiate from a Multilayered Epithelium
Ductal initiation from complex cysts was a transition from an essentially filled lumen to a progressively clearing lumen, with new ducts initiating and elongating at gaps in ME coverage (; Movies S4 and S5
). The fronts of the elongating ducts were always multilayered, but there was often thinning and reversion to a bilayer at adjacent sites. LE cells appeared adherent to each other, yet they moved chaotically in the direction of elongation, whereas ME cells moved both toward and counter to the direction of ductal elongation.
Myoepithelial Cells Move Actively during Ductal Elongation and Bifurcation
Branching morphogenesis resulted from the interplay between ME and LE motility. We observed LE ducts elongate past ME coverage, followed by ME migration to restore coverage. We observed ME cells fully covering () or partially covering an elongating LE duct (). Ducts typically stopped elongating after full coverage by ME cells, whereas ducts typically bifurcated after partial coverage by ME cells. We verified that the location of SMA+ cells was similar to that of K14-GFP-actin+ cells, and we frequently observed SMA+ cells at sites of bifurcation (). We could not distinguish whether ME cells induced bifurcations or responded to a separate decision to bifurcate, but ME motility closely correlated with changes in the shape of elongating ducts (Movie S5
Cessation of elongation was accompanied by a transition in the tip of the duct from a multilayered epithelium to a single or bilayered epithelium, shown in an organoid derived from a transgenic reporter mouse with GFP expressed in both LE and ME (β-actin-EGFP; ; Movie S6
). The tips of ducts in growth-arrested organoids were often free of SMA+ cells (). The timing and extent of ductal elongation varied among organoids, but each eventually stopped elongating and reverted to a bilayered organization.
Mammary Epithelium Is Multilayered and Incompletely Polarized during Morphogenesis
Cells within Elongating Ducts Migrate Collectively, but Remain Epithelial
Since the multilayered tips of elongating ducts were organized quite differently from quiescent ducts, we sought to determine if they remained epithelial during morphogenesis. On the basis of close cellular association and the consistent localization of E-cadherin and β-catenin to surfaces of intercellular contact throughout branching (), we conclude that ducts remained epithelial. Epithelia also characteristically have basement membranes at their basal surface. We chose to stain for laminin 332, as it is a component of mammary epithelial basement membrane, but not of Matrigel. Unbranched structures, whether simple or complex cysts, displayed complete coverage with laminin 332 (), whereas the tips of actively elongating ducts were generally free of laminin 332 (). We could not distinguish whether laminin 332 was proteolytically degraded, displaced, or simply not synthesized at these sites.
During Collective Epithelial Migration, Cells Are Incompletely Polarized
To determine whether apicobasal polarity was retained during elongation, we used four molecular markers: β-catenin to mark basolateral cell surfaces, APKC-ζ to mark apical cell surfaces, β1-integrin to mark cell-matrix interactions, and ZO-1 to mark luminal surfaces.
At the cellular level, APKC-ζ and β-catenin were both detected at all lateral cell surfaces in the multilayered region during the complex cyst phase and during active elongation. APKC-ζ colocalized with ZO-1, whereas β-catenin was distinct from ZO-1. We conclude that cells in the multilayered region are incompletely polarized and do not have distinct apical and basolateral domains ().
At the tissue level, we observed β1-integrin localized to the cell-matrix interface () and sometimes also to lateral sites of cell-cell contact (, yellow). ZO-1 localized to luminal surfaces and to small pockets and channels within the multilayered region. We conclude that the multilayered epithelium is polarized at the tissue level, but its constituent cells are incompletely polarized relative to cells within quiescent regions of mammary epithelium.
Proliferation Is Required to Form Complex Cysts
Previous work showed that growth factor-induced proliferation is highest just before ductal initiation (Fata et al., 2007
). We confirmed those results by staining for mitotic cells with phosphohistone H3 (H3-P) and observed frequent mitotic cells in the complex cyst and early branching stages (); there were fewer mitotic cells in simple cysts or late branching organoids. H3-P+ cells were present in all regions of organoids and showed no obvious correlation between the location of proliferation and the direction of extension.
Proliferation could be required to initiate branching, or to provide new cells to enlarge a branched structure. To distinguish whether inhibition of proliferation would produce arrest at the simple cyst stage or produce small, branched organoids, we inhibited DNA polymerase A by using aphidicolin. When added 24 or 48 hr into culture, aphidicolin completely blocked branching. At doses of 5 or 10 μM, all organoids arrested at the simple cyst stage (Figure S2
); therefore, proliferation is required to form the multilayered, complex cyst.
Ductal Elongation Is Accomplished by a Multilayered Epithelium
We sought to determine the cellular mechanism of ductal initiation from complex cysts. To observe the cellular basis of ductal initiation, we collected 127 confocal time-lapse movies of initiating and elongating ducts. We observed that new ducts always initiated as multilayered epithelial structures (N = 394/394 ducts in 127 movies; ; Movies S4–S8
). Significantly, we never observed basally directed protrusions, cell migration out of the luminal epithelium, or initiation and elongation of fully polarized epithelial ducts (N = 0/394 ducts), ruling out chain migration as the mechanism for tubulogenesis in this system. Instead, ductal elongation was accomplished by a collectively migrating, dynamically rearranging, multilayered epithelium at the ductal tip. This structure reverted to a bilayered epithelium surrounding a simple lumen in the trailing duct (). The thinning of the trailing duct from multiple to a single luminal layer also contributed to ductal elongation.
Ductal Elongation Occurs without Forward-Oriented Actin Protrusions in a Rearranging, Multilayered Cell Population
Ductal Elongation Occurs without Leading Cellular Extensions
Leading cell extensions or specialized actin-rich protrusions, such as filopodia and lamellipodia, are typically observed at the leading edge of individual migratory cells (Mitchison and Cramer, 1996
) and of collectively migrating groups of cells, ranging from Drosophila
border cells (Fulga and Rorth, 2002
; Prasad and Montell, 2007
) to zebrafish neural precursors (Lecaudey and Gilmour, 2006
). In contrast to these previous studies, we never observed cellular extensions or actin-rich protrusions at the front of advancing mammary ducts (, N > 250). Instead, F-actin was enriched along lateral cell surfaces and colocalized with ZO-1 at luminal surfaces.
Cells Dynamically Rearrange during Collective Epithelial Migration
We next asked if a constant group of cells remained at the invasion front or if cells exchanged positions during invasion (). To distinguish these possibilities, we followed individual cells during ductal extension by exploiting the mosaic expression of EGFP within the mammary epithelium of mice in which GFP was knocked into the Sca-1 locus (Sca-1-EGFP). To provide context, all cells were stained with CellTracker Red.
Cells in the multilayered region of the extending duct continuously exchanged positions (N = 394 ducts in 127 movies; see Movies S7 and S8
). This cell rearrangement was characteristic of both newly initiated ducts () and later ducts with well-defined lumens (, ′, and 4C′). We verified this conclusion by using three different GFP reporter lines: K14-GFP-actin (), Sca-1-EGFP (), and β-actin-EGFP transgenic mice (). In all cases, the cells at the tip of the extending ducts dynamically rearranged.
Duct Initiation Requires Rac-1 and Myosin Light Chain Kinase
What are the molecular regulators of ductal initiation? Previous studies in mammary epithelium (Vargo-Gogola et al., 2006
), ureteric bud (Meyer et al., 2006
), 3D MDCK morphogenesis (Yu et al., 2003
), and epidermis (Vaezi et al., 2002
) identified Rac- and Rho-type GTPases as critical regulators of epithelial morphogenesis. Accordingly, we tested the role of these pathways in mammary epithelial branching morphogenesis, by using soluble inhibitors: Rac-1 Inhibitor (NSC23766) for Rac, Y27632 for Rho kinase (ROCK), and ML7 for myosin light chain kinase (MLCK).
Rac inhibition resulted in a strong dose-dependent inhibition of ductal initiation (). Rac-inhibited cysts were covered externally by ME cells, but they also had convoluted internal tubules, with associated internal ME cells () and differed markedly from the normal complex cysts (). These internal tubules were not observed in control organoids (). Movies of organoids from the K14-GFP-actin (; Movie S9
; 24 movies total) or β-actin-EGFP (not shown) transgenic mice revealed that the cysts were relatively static and had extensive ME coverage, internal ME cells, and persistently filled lumens.
Rac-1 and MLCK Are Required for Ductal Initiation
MLCK inhibition also prevented ductal initiation in a dose-dependent fashion (). Maximal inhibition was achieved at doses ranging from 4 to 10 μM, depending on the experiment. We did not observe internal tubules in MLCK-inhibited cysts. Long-term confocal movies of K14-GFP-actin organoids (; Movie S10
) and β-actin-EGFP organoids (not shown) revealed that MLCK-inhibited organoids transiently formed complex cysts, but then gradually cleared their lumens and restored a largely bilayered epithelial structure (19 movies total). MLCK-inhibited organoids remained persistently enclosed in ME cells.
ROCK Is Required to Restore Bilayered Epithelial Architecture
As ducts ceased elongating, they transitioned from a multilayered epithelium to a single luminal layer (). We sought to understand the molecular regulation of this transition. Recent work identified p190-B Rho-GTPase as a regulator of mammary epithelial architecture, with its inactivation resulting in hyperplasia and disorganized myoepithelial coverage (Vargo-Gogola et al., 2006
). Accordingly, we used Y-27632 (Uehata et al., 1997
) to inhibit the related molecules ROCK1 and ROCK2. ROCK inhibitor-treated organoids branched at equal or higher rates than controls, but they were heterogeneous and poorly patterned (; Figure S3; Movies S12 and S14–S17
). The effects were acutely reversible when ROCK inhibitor was washed out (Figure S3E; Movie S17
ROCK Signaling Is Required to Restore Simple Ductal Architecture
Our data indicate that ME location, behavior, and migration reflect, and appear to influence, the pattern of branching morphogenesis ( and ). ROCK inhibition resulted in disorganized, incomplete coverage by ME cells. After ROCK inhibition, ME cells were often spindle shaped, with no obvious correlation between ME position and final organoid structure ().
In control organoids, there was a sharp organizational difference between the multilayered, elongating end and the trailing duct behind it (). The trailing ducts of control organoids had clear, ZO-1-lined lumens, apically enriched F-actin, and a simple, bilayered structure. ROCK-inhibited organoids had no central lumen and instead localized ZO-1 and F-actin to small foci and had a persistent, disorganized, multilayered structure. No ROCK inhibitor-treated organoids were observed to revert to a bilayered architecture. LE cell shapes were also rounder, but E-cadherin remained localized to lateral LE surfaces in ROCK inhibitor-treated organoids, suggesting that the cells remained adherent and epithelial (). ROCK-inhibited organoids had abnormally positioned ME cells, abnormal LE and ME cells shapes, no central lumens, and a persistent failure to revert from a multilayered to a bilayered architecture.
To characterize the kinetics and cell behavioral basis of the ROCK inhibitor phenotype, we collected 67 long-term confocal movies from K14-GFP-actin (; Movie S12
), β-actin-EGFP (Figure S3C; Movie S15
), and Sca-1-EGFP (Figure S3D; Movie S16
). The effect of ROCK inhibition was acute, with changes in ME coverage and LE organization observable within 12 hr. A major question was the degree to which the abnormal architecture of the LE related to the loss of ME coverage ().
ME cells appear to play a restraining role in normal organoids and seem to limit and pattern LE mobility (″; Movies S4 and S5
). We examined the acute effects of ROCK inhibition on ME behavior by filming in parallel control and ROCK inhibitor-treated organoids from the same K14-GFP-actin+ mouse 1 hr after ROCK inhibition (; Movies S11 and S12
). In control organoids, ducts initiated through gaps in ME coverage, elongated through the combined motility of LE cells and ME cells, and reverted to a bilayered architecture. Conversely, in ROCK-inhibited organoids, ME cells quickly changed shape, and their motility had little relationship to the vigorous growth of the largely LE organoid.
We next sought to distinguish whether LE behavior was altered in ROCK-inhibited organoids, by using GFP reporters that highlighted LE behavior. In control organoids, LE cells rearrange vigorously in the multilayered ends of ducts, but they are more restrained in the trailing duct region (). In ROCK-inhibited organoids, LE cells rearranged vigorously throughout the multilayered epithelium (Sca-1-EGFP, Figure S3D
; Movie S16
; β-actin-EGFP, Figure S3; Movie S15
). Taken together, these findings demonstrate that ROCK inhibition induces acute changes in LE and ME cell shape and cell behavior, abnormal epithelial architecture, and collapse of the lumen. ROCK inhibition results in a hyperbranched epithelium with disorganized ME coverage and prevents restoration of bilayered epithelial architecture.
Normal and Neoplastic Mammary Epithelial Morphogenesis In Vivo Occurs via a Multilayered Epithelial State
Does the multilayered epithelium we observe in culture resemble the in vivo epithelial organization of mammary ducts during morphogenesis? We selected two examples of in vivo epithelial morphogenesis: (1) TEBs during normal pubertal morphogenesis and (2) hyperplasias from a pathologically validated mouse model of luminal-type breast cancer (Herschkowitz et al., 2007
; Lin et al., 2003
), in which polyoma virus middle T oncogene is expressed under the control of the mouse mammary tumor virus promoter (MMTV-PymT) (Guy et al., 1992
We first compared TEBs and quiescent ducts during normal morphogenesis in vivo to the multilayered epithelial organization of elongating ducts in culture (Figure S1
). TEBs were multilayered and were enclosed in a layer of SMA+ cap cells. In the multilayered body cell region of TEBs, β-catenin, APKC-ζ, and β1-integrin all localized to lateral cell surfaces. ZO-1 localized apically, but largely to finger-like projections and isolated pockets within the multilayered body cell region. The lumens of quiescent ducts in vivo were simple and were lined by a single luminal epithelial layer, with apically localized ZO-1 and APKC-ζ, basolaterally localized β-catenin, and basally localized β1-integrin. TEBs were also hyperproliferative relative to quiescent ducts. The most notable differences between TEBs in vivo and elongating ducts of organoids in culture were that TEBs were composed of significantly more cells and encountered a much more diverse cellular stroma. Therefore, the epithelial organization of TEBs in vivo and elongating ducts in culture was similar.
In early MMTV-PymT hyperplasias, we observed a progressive loss of SMA+ ME cells, with fewer ME cells correlating with less normal ductal morphology (), consistent with previous reports (Lin et al., 2003
). Regions with little or no ME coverage were always multilayered. ZO-1 in multilayered hyperplasias was localized in small pockets (). In later lesions, SMA+ cells were rare or absent. In multilayered hyperplasias, β-catenin localized to all sites of cell-cell contact, indicating incomplete apicobasal polarity (). Therefore, normal and neoplastic epithelia during morphogenesis share a similar multilayered epithelial organization ().
Mammary Epithelia Are in a Multilayered State during Morphogenesis