Much progress has been made recently in delineating the cellular and molecular mechanisms driving tube morphogenesis. In this review, we discuss the major outstanding questions regarding the formation, elongation and elaboration of epithelial tubular networks. We pay particular attention to emerging common themes and differences between tube formation processes in Drosophila and mammals. We focus on those experiments that have revealed the underlying cellular and molecular mechanisms utilized during tube formation and branching morphogenesis. Finally, we consider whether present data argue for common cellular or molecular programs for tube morphogenesis or whether each organ utilizes a specific spatial and temporal configuration of more fundamental cellular and molecular mechanisms. It is our hope that this review will stimulate future work focused on resolving tube morphogenesis into a series of molecularly regulated changes in the discrete properties and behaviors of individual cells.
Tubular organization is a common feature of many developing tissues. Tubes can represent a transient phase of organ development. The vertebrate neural tube initially forms as a simple columnar epithelium, but ultimately gives rise to the complex architecture of the brain and spinal cord. Tubular organization is quite useful and can serve many important physiologic roles, including: control and delivery of gases, nutrients, waste and hormones, compartmentalization of organ function, and barrier function between the organism and its environment. The respiratory, circulatory, and secretory organs are all built of networks of interconnected tubes. Tubular epithelial organs arise from each of the germ layers, ectoderm (e.g. mammary gland), mesoderm (e.g. kidney), and endoderm (e.g. liver). Many of the same cellular and molecular mechanisms are utilized during the formation, elongation and elaboration of endothelial tubes, a topic that is reviewed in detail in this issue and elsewhere (Risau and Flamme, 1995; Beck and D’Amore, 1997; Carmeliet, 2000; Ellertsdottir et al., 2009). In this review, we focus specifically on epithelial tubular organs.
Epithelial tubes are only one of the constituent tissues of an organ, and are themselves surrounded by a complex mixture of supporting cells and extracellular matrix (ECM), collectively referred to as a stroma or mesenchyme. Mature epithelia are distinguished by a few key features, including close cell-cell contact and adhesion, strong apico-basal polarity, specialized intercellular junctions connecting neighboring cells, and a basally located basement membrane or basal lamina (). During the formation of an epithelial tube, each of these features can be independently modulated. Also, during morphogenesis and in the very early embryo, the organization of the epithelium of a given organ can vary significantly from its mature quiescent organization. Furthermore, there are some characteristic interspecies differences in epithelial organization (Knust and Bossinger, 2002
). Chief among these differences is the organization and location of the barrier junctional complex. In mammals, tight or occluding junctions are composed of claudins and sit apical to the adherens junction (). In Drosophila melanogaster
, the septate junction is also composed of claudins, but sits basal to the adherens junction. Both tight and septate junctions are responsible for controlling paracellular permeability (Knust and Bossinger, 2002
Figure 1 All epithelial tubes comprise polarized cells surrounding a central lumen, with the apical surfaces of the cells facing the lumen and the basal surface contacting either a basal lamina or other cells (A). Apical surfaces often contain microvilli, actin-rich, (more ...)
As we consider the different model systems for studying tubular epithelia, it is worth noting that epithelia can have very different, but organ-specific, differentiated architecture and organization. Classically, the epithelia are subdivided into types, based first on the number of cell layers (). Single layered epithelia are termed simple and multilayered epithelia are termed stratified. Epithelia in which the nuclei occupy more than one layer, but all cells have direct contact with the basement membrane, are considered pseudo-stratified. In cases where the epithelium has several organizations in close juxtaposition, it is collectively considered to be a transitional epithelium. This basic classification scheme can be further refined to indicate cell shapes within the epithelium, whereby cells with similar height to width ratios are termed cuboidal, those with greater height than width are columnar, and those with greater width than height are squamous. Many of the secretory epithelia are further enveloped in a layer of cells expressing muscle markers such as alpha-smooth muscle actin. If these cells are between the epithelial cells and the basement membrane, they are termed myoepithelial cells, and if they are outside the basement membrane, they are termed myofibroblasts (). For any given tissue, there is a characteristic adult differentiated epithelial type, but the organization can be drastically different in early development or during developmental remodeling. For example, the mammary gland luminal epithelium lines and defines the luminal space and has a simple epithelial organization, but transiently reorganizes into a stratified epithelium during embryonic and postnatal branching morphogenesis (Hinck and Silberstein, 2005
; Ewald et al., 2008
;). We might expect that different strategies are employed to build different types and architectures of tubular networks.
In contrast to epithelia, mesenchymal tissues are much looser and are characterized by a predominance of single cells dispersed through extensive ECM. Integrin based cell-matrix adhesions are relatively more common than cadherin based intercellular adhesions. These distinctions are not absolute however, as epithelial tissues can undergo an epithelial to mesenchymal transition (EMT) and give rise to dispersed single cells. Prominent physiologic examples of EMT include the vertebrate neural crest, the ingressing cells of the chick epiblast during gastrulation, and the sclerotome and associated derivatives of the ventral portion of the somite. The converse process of mesenchymal to epithelial transition (MET) can also occur, most notably in the condensation of mesodermally-derived mesenchymal cells in the formation of the vertebrate kidney. At present, it is unclear whether epithelium and mesenchyme should be considered to be two alternative organizations or two opposing poles that define a continuum of organization (O’Brien et al., 2002
). As we begin to understand the cellular processes underlying epithelial morphogenesis, we need to pay close attention to the extent to which epithelial organization is maintained throughout the process. Perhaps the most critical aspect of this organization is the degree of apico-basal polarity. It may be easier and more reliable to monitor and dissect the regulation of apico-basal polarity specifically rather than epithelial organization more broadly.
Major questions in tubular morphogenesis
As we try to understand tube morphogenesis, we should start with: what needs to be accomplished by the tissue? Then, how do the cells do it? Then, what are the molecules that mediate/direct/specify the cell behaviors? Finally, which cellular and molecular mechanisms are general across tissues or across phyla? There are several distinct subprocesses that must occur for an undifferentiated early embryonic tissue to become a mature differentiated tubular network. The precise order of these events can vary dramatically between tissues, but each of these subprocesses must occur at least once.
First, the cells of an epithelial tube must be specified and made molecularly distinct from the surrounding cells that will give rise to other tissue types. This epithelial identity then needs to be maintained, though systems vary greatly in the extent to which it is continuously maintained or iteratively de- and re-differentiated. It is also possible for tubular morphogenesis to include a full or partial EMT followed by reestablishment of full epithelial identity. Once the future epithelial tissue is specified, a lumen needs to be built, as a tube is defined by the existence of a specialized luminal space (). As with epithelial identity, different systems may vary in the extent to which the lumen is permanently maintained once established. Tubes then need to elongate and elaborate (). Three conceptually distinct mechanisms exist for tube elaboration. The end of the tube can split, with further elongation in both directions, a process known as bifurcation. New tubes can initiate from sites distant from either tube end, a process referred to as side branching. The tube can separate and exchange cell-cell contacts for cell matrix contacts without significant elongation, a process referred to as clefting. Additionally, both the diameter and length of the tube can be regulated and the final tissue and cell type specific differentiation program needs to be completed (). It is important to distinguish to what extent these subprocesses of tube specification, lumen formation, elongation, and elaboration occur as a temporally distinct series of events or whether one or more of these subprocesses occurs simultaneously.
Figure 2 Epithelial tube formation involves dynamic cellular changes, beginning with epithelial polarization to form the cell layer that surrounds the central lumen (A). The geometry of cells in the tube primordium, coupled with the order and arrangement of cells (more ...)
A critical starting point in understanding the elongation and elaboration of tubular networks is to define the simplest functional unit of morphogenesis. To what extent is the process of epithelial branching morphogenesis meaningfully autonomous to the epithelium? What is the minimum set of cell types and extracellular matrix that is sufficient to build the form and differentiated function of a given epithelial tube? In the mammary gland, there is clear coordination between epithelial cells and stromal cells (Van Nguyen and Pollard, 2002
; Ingman et al., 2006
), as well as coordinate motility in different epithelial cell types such as the luminal and myoepithelial cells (Ewald et al., 2008
). In the mouse lung, it is also clear that important patterning information is exchanged between the epithelial and mesenchymal compartments, with the lung vasculature playing an important signaling role (Warburton et al., 2000
). It is important to determine whether epithelial tube morphogenesis can be fully captured based on an understanding of the actions of the epithelial cells alone, or whether it is necessary to understand the epithelium in its normal context of extracellular matrix and stromal cells (Nelson and Bissell, 2006
). It is clear in the mammalian kidney (Constantini, 2006
; Dressler, 2006
; Nigam and Shah, 2009
), lung (Metzger et al., 2008
), salivary gland (Patel et al., 2006
) and mammary gland (Lu and Werb, 2008
) that there is a close juxtaposition of many stromal cell types basal to the epithelium during branching. There are also changes in the stromal composition and differentiation during branching. In the mammary gland, loss of specific stromal cell populations has a significant effect on ductal elongation (Ingman et al., 2006
). Further work is required in each of these systems to determine the extent to which epithelial-stromal communication is a refining mechanism or a central source of patterning information.
Model systems for studying tubular morphogenesis
In an ideal world, we would have a single model system that developed and elaborated tubular networks through conserved mechanisms and that was ideally suited for genetics, cell biology and imaging. Instead, we have a number of different model systems with different strengths and limitations. Some reagents and techniques work best in specific systems. Simplified cell culture models with immortalized cells have provided critical insights into the molecular mechanisms of lumen formation and apico-basal polarity (Debnath and Brugge, 2005
; O’Brien et al., 2002
). These models have been more limited for modeling the 3D complexity of organ architecture, but have provided mechanistic insights relevant to in vivo development.
Recently, techniques have been developed in multiple labs to model the development of epithelial organs in a more organotypic fashion, with successful protocols established for mammary development (Simian et al., 2001
; Wiseman et al., 2003
; Meyer et al., 2004
; Fata et al., 2007
, Ewald et al., 2008
), submandibular gland (Steinberg et al., 2005
), and lung (Liu et al., 2004
). These cultures are alternately referred to as organotypic, organoid, or mesenchyme-free epithelial cultures. All share conceptually similar isolation protocols in which the epithelial compartment of the gland is separated from the stromal compartment and then cultured in 3D gels of ECM proteins. It is worth noting that these preparations are rarely completely mesenchyme free, as there are tightly attached stromal cells that can readily be carried along with the epithelial fragments. These cultures are modular in that they allow arbitrary recombinations of epithelium, stroma, and ECM. This experimental flexibility allows the role of complex factors to be tested in an organotypic context. For example, it is difficult in vivo to eliminate whole classes of stromal cells or stromal proteins without systemic effects elsewhere in the animal, whereas it is comparatively easy in 3D culture. It is also valuable that similar techniques can be used with primary human epithelium (Yu et al., 2007
) and so insights from model systems can be readily and directly tested in human disease states.
It is also possible to culture the entire epithelial organ (Sakai and Onodera, 2008
), most notably in salivary gland (Larsen et al., 2003
), kidney (Srinivas et al., 1999
), lung (Bellusci et al., 1997
), and mammary gland ( Topper et al., 1975
; Gallo-Hendrikx et al., 2001
). Since the epithelium is cultured in its normal stromal environment, development is often even more similar to normal in vivo development. These cultures are a major advantage when the molecules being tested are broadly required in the early embryo (e.g. E-cadherin or fibronectin). Importantly, organ cultures make mammalian organ development observable. Branching morphogenesis in mammalian tissues occurs inside an embryo inside the mother (salivary, lung, etc) or inside an adolescent mouse over weeks (mammary gland). A major advantage to externally developing embryos is that organ development can often be directly imaged in the intact organism. Drosophila in particular has been a rich experimental system for understanding tube morphogenesis as the genetics are well developed and live imaging in vivo is practical due to the clarity of the embryo and the relatively close proximity of relevant tissues to the embryo surfaces (Ribeiro et al., 2002
; Kato et al., 2004
; Caussinus et al., 2008
; Cheshire et al., 2008
). Similarly, in vivo imaging of organ development in zebrafish is beginning to provide additional insights into the mechanisms of tube morphogenesis, most notably during kidney development (Vasilyev et al., 2009
). The availability of these various model systems increases the importance of the validation of in vitro results in vivo and also of the cross-validation of results from one model system to others and to human development and disease.
Experimental approaches to understanding tubular morphogenesis
The morphogenesis of epithelial tubes is intrinsically a question of the morphogenesis of a tissue, not a single cell. This requires both coordination among the cells of the epithelium and between the epithelial compartment and surrounding stromal cells. We seek to resolve complex tissue transformations (e.g. mammary or tracheal branching morphogenesis) into a series of discrete changes in the properties and behaviors of the individual constituent cells of that tissue. We expect that the apparently complex tissue behavior of branching morphogenesis in different systems can be explained as unique configurations of simpler, conserved subprocesses. Essentially the problem is: what happens when and how is it regulated?
Imaging provides the foundational description of the cellular and molecular events that occur during branching morphogenesis. Live cell imaging is particularly important as it enables accurate reconstruction of the sequence of these events. In highly stereotyped or geometrically simple tissues, a series of fixed samples can provide a useful starting point, but there is no substitute for watching the same cells over time. When you are additionally interpreting the phenotypes of molecular perturbations with less than 100% penetrance or some degree of mosaicism, the problem is compounded further. Even a single high-resolution movie can unambiguously resolve the sequence of cellular processes involved. Recent improvements in computer hardware, software and microscope automation enable simultaneous collection of dozens to hundreds of time-lapse movies. This multiplicity enables researchers to reconstruct most or all of the different normal developmental trajectories. Coupled with recent advances in molecular imaging, it is now a reasonable goal to image branching morphogenesis in each of the major models systems, with distinct channels of information for the dynamics of actin, microtubules, junctional complexes and various signaling molecules.
With an ever increasing understanding of the spatial and temporal dynamics of cells driving tube morphogenesis, attention naturally turns to identifying the critical molecular regulators for these cell behaviors. Forward and reverse genetic approaches have been and will continue to be used to isolate and identify molecular regulators governing these changes in cell behaviors and properties. Mammalian and Drosophila studies have diverged significantly in their technical approaches to these questions, with forward genetic screens dominating in Drosophila and reverse genetic approaches, chiefly gene deletion by homologous recombination, providing the mainstay of murine genetic approaches to branching morphogenesis. The challenge today is to integrate the combined insights of imaging and genetics to achieve a cellular resolution understanding of tube morphogenesis and its regulation. There is a critical role for mammalian systems with close physiologic similarity to humans and for simpler model systems with unique experimental advantages.