Dynamics of branched tissue assembly
© BioMed Central Ltd 2012
Published: 31 October 2012
The assembly of cells into tissues is a complex process controlled by numerous signaling pathways to ensure the fidelity of the final structure. Tissue assembly is also very dynamic, as exemplified by the formation of branched organs. Here we present two examples of tissue assembly in branched systems that highlight this dynamic nature: formation of the tracheal network in Drosophila melanogaster and the ducts of the mammary gland in mice. Extension of the branches during tracheal development is a stereotyped process that produces identical organ geometries across individuals, whereas elongation of the ducts of the pubertal mammary gland is a non-stereotyped process that produces unique patterns. By studying these two organs, we can begin to understand the dynamic nature of development of other stereotyped and non-stereotyped branching systems, including the lung, kidney, and salivary gland.
The self assembly of cells into tissues and organs is an elegant and intricate process that is vital for development and homeostasis. During organogenesis, the assembly of cells is controlled genetically as well as through cues from cell-cell and cell-matrix interactions [1, 2]. Cells assemble into higher-order patterns that are either stereotyped such that there is little variation in the timing and final architecture between individuals, or non-stereotyped, which leads to differences in the details . The branching of the ducts of the pubertal mammary gland is a non-stereotyped process in which the interactions between the ducts and their surrounding microenvironment produce unique tree-like architectures. In contrast, branching of the trachea in Drosophila melanogaster is a stereotyped process that is under strict genetic control. These two systems serve as excellent models with which to investigate the dynamic interplay between cells during tissue formation, under two different modes of control.
Formation of the trachea in D. melanogaster
The DB consists of approximately six cells and these cells migrate dorsally away from the sac toward the morphogen Branchless (Bnl), which acts as a chemoattractant secreted by the surrounding cells . However, the branch is able to extend normally even when all but one of the cells is devoid of Btl, the receptor for Bnl . The cell with functional Btl receptors acts as a leader and is able to guide the rest of the cells. In wild-type embryos, a leader cell emerges even when every cell expresses Btl. At this stage of development, the cells of the DB are nearly identical and there is no predetermined leader; however, there are small differences in the expression level of Btl. By exploiting these small differences, the cells with the highest expression levels of Btl jockey for the lead position in the newly forming branch, while simultaneously amplifying the difference in the activation of Btl among the population through Notch-mediated lateral inhibition [14–17]. The activation of Btl by Bnl induces the production of Delta, a ligand that binds to and activates Notch in the neighboring cells. Activated Notch inhibits signaling downstream of the Btl receptor and production of Delta within that cell. Over time, this results in one cell with substantially higher Btl activity than its neighbors [16, 17]. The successful cell assumes the role of the leader at the tip of the branch while the other cells form the stalk. This establishes a hierarchy and the leader cell is able to efficiently move the branch up the Bnl gradient. As the dorsal branch extends, one of the cells of the branch comes in contact with a cell from the neighboring branch and binds to it, thus fusing the branches to form a contiguous airway . Notch signaling again plays a similar role by aiding Bnl and Dpp and ordaining the fusion cell fate [16, 18, 19].
During metamorphosis, the trachea once again undergoes significant remodeling. Posterior tracheal branches are lost while anterior branches expand to cover that space and new cells replace most of the cells in the existing branches. Progenitor cells called tracheoblasts that have remained quiescent during embryonic and larval development begin to proliferate and travel along the TC and VB under the guidance of Btl and replace the old cells [20, 21]. However, the DBs are replaced not by progenitor cells but by differentiated DB cells that reenter the cell cycle and proliferate to replace the existing branch .
Formation of the mammary ducts
Estrogen and growth hormone initiate ductal elongation by activating the estrogen receptor (ERα) and the growth hormone receptor, respectively, in the stromal cells surrounding the epithelium [27–29]. The activation of growth hormone receptor enables TEB formation through the insulin-like growth factor pathway . Estrogen signaling leads to the activation of progesterone receptors in the epithelium, which induces epithelial proliferation and ductal movement. Estrogen signaling also stimulates the stromal cells to activate matrix metalloproteinases (MMPs) through amphiregulin and epidermal growth factor receptor . Stromal cells activate several MMPs, including MMP3 and MMP14. MMP14 activates MMP2, which aids in ductal elongation by preventing apoptosis of the cells within the TEB. MMP2 and MMP3 also play a role in determining sites of lateral branching along the duct. High MMP2 prevents the initiation of lateral branches by stimulating the production of transforming growth factor (TGF)β, whereas MMP3 enables lateral branching by degrading the basement membrane and allowing the cells to extend through the fat pad .
Unlike tracheal branching, there is no evidence for a global chemoattractant that guides the TEBs through the fatpad . The TEBs weave through the fatpad, extending lateral branches and bifurcating along the way, by integrating the global stimulatory cues with more local cues from neighboring cells, their environment and their own geometry . Patterns of endogenous mechanical stresses arise along the ducts due to asymmetries in their geometry coupled with isometric contraction of the cells and their ability to transmit these forces to their neighbors through adherens junctions . These mechanical stress patterns appear to regulate the sites at which branches initiate. Indeed, three-dimensional organotypic culture models revealed that branches initiate from sites of highest mechanical stress , and that cells in these regions of high mechanical stress adopt a more motile mesenchymal phenotype [34, 35], increase signaling through focal adhesion kinase (FAK) , and increase production of MMPs to aid in degradation of surrounding matrix, all of which enable branch extension . However, while high mechanical stress is necessary for branch initiation, it is not sufficient . Branch initiation and extension are also controlled by the local concentration of morphogens such as TGFβ . High concentrations of TGFβ prevent branching of the ducts, and autocrine secretion of this inhibitory morphogen by the epithelial cells is thought to prevent aberrant branching and maintain proper spacing between ducts [37, 38]. By responding to these various stimulatory and inhibitory cues, the TEBs navigate through the stroma until they reach the limits of the fat pad, where they regress and form terminal ducts. However, the ducts still maintain a certain population of undifferentiated stem cells that are capable of self-renewal and repopulation of the entire mammary gland [39, 40]. These undifferentiated cells are thought to aid in the remodeling of the mammary gland during and after pregnancy, where further ductal expansion and regression occur, respectively .
Branching of the trachea and the mammary gland high-light the dynamic process of tissue formation. These two systems show how stem cells, progenitor cells and differentiated cells come together, and through a combination of global and local guidance cues specify the final architecture of the tissue. During puberty in mammals, the pluripotent cap cells combined with other cell types of the TEB and through the regulation of the micro-environment produce the unique pattern of the ductal tree. In the formation of D. melanogaster trachea, even though the final architecture is fixed, the cells of the invaginated tracheal placodes have a high degree of freedom to determine the leaders, the fusion cells and those that will remain quiescent during the various branch formations. In addition to normal development, the dynamics of stem cells also come into play during tumor formation. Cancer stem cells (CSCs) are specialized cells within tumors that are capable of self-renewal and initiation of tumors . CSCs have all the characteristics of normal stem cells and are even capable of generating non-malignant structures [42, 43]. The origin of CSCs is still hotly debated. While the similarities between CSCs and normal stem cells might strongly suggest that CSCs arise through aberrant differentiation of normal stem cells, there is also evidence to suggest that CSCs might arise from progenitors or even through dedifferentiation of fully differentiated cells. Understanding the dynamics of cells during normal development will help in understanding CSCs and malignant development in general, as many of the processes involved in normal development are conserved in malignant development.
Thanks in large part to advances in technology, we are finally able to glimpse at the complexities of developmental processes. However, better in vivo imaging approaches are needed to fully answer most of the open questions, as it is near impossible to investigate dynamic processes using still images. Advances in live imaging could help, for example, to study the formation and movement of the TEBs. Studies in culture have suggested that organization of the TEBs might involve cellular sorting to optimize the structure for invasion through the fatpad ; however, this has yet to be shown in vivo. Furthermore, the mechanism by which the TEB moves through the fatpad is still unclear . In addition to imaging, advances in techniques to manipulate in vivo microenvironments might prove to be the most fruitful venture. The mechanics of the microenvironment integrate with molecular cues in the regulation of cell division, motility, branching, and stem cell differentiation . Laser ablation studies in D. melagonaster have been used to show the involvement of tensile forces in tracheal branching [47–49]. At present, however, culture models still provide one of the best means of studying the effects of the mechanics of the microenvironment on these processes in a controlled and quantifiable manner. While they do suffer from drawbacks of artificiality, sophisticated three-dimensional culture models can aid to bridge the gap between the plastic world and the in vivo world, and could play an important role in studying the interplay between multiple cell types during development [50, 51]. A better appreciation of the dynamic nature of tissue assembly will have a great impact on the study of normal and malignant development and may also will help advance the field of tissue engineering.
This article is part of a thematic series on Physical influences on stem cells edited by Gordana Vunjak-Novakovic. Other articles in the series can be found online at http://stemcellres.com/series/physical
cancer stem cell
terminal end bud
transforming growth factor
Work from the authors' lab was supported in part by the NIH (CA128660, GM083997, and HL110335), Susan G Komen for the Cure, the David and Lucile Packard Foundation, and the Alfred P Sloan Foundation. CMN holds a Career Award at the Scientific Interface from the Burroughs Wellcome Fund.
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