Tube formation by complex cellular processes in Ciona intestinalis notochord

In the course of embryogenesis multicellular structures and organs are assembled from constituent cells. One structural component common to many organs is the tube, which consists most simply of a luminal space surrounded by a single layer of epithelial cells. The notochord of ascidian Ciona forms a tube consisting of only 40 cells, and serves as a hydrostatic “skeleton” essential for swimming. While the early processes of convergent extension in ascidian notochord development have been extensively studied, the later phases of development, which include lumen formation, have not been well characterized. Here we used molecular markers and confocal imaging to describe tubulogenesis in the developing Ciona notochord. We found that during tubulogenesis each notochord cell established de novo apical domains, and underwent a mesenchymal–epithelial transition to become an unusual epithelial cell with two opposing apical domains. Concomitantly, extracellular luminal matrix was produced and deposited between notochord cells. Subsequently, each notochord cell simultaneously executed two types of crawling movements bi-directionally along the anterior/posterior axis on the inner surface of notochordal sheath. Lamellipodia-like protrusions resulted in cell lengthening along the anterior/posterior axis, while the retraction of trailing edges of the same cell led to the merging of the two apical domains. As a result, the notochord cells acquired endothelial-like shape and formed the wall of the central lumen. Inhibition of actin polymerization prevented the cell movement and tube formation. Ciona notochord tube formation utilized an assortment of common and fundamental cellular processes including cell shape change, apical membrane biogenesis, cell/cell adhesion remodeling, dynamic cell crawling, and lumen matrix secretion.     

Epithelial tube morphogenesis during Drosophila tracheal development requires Piopio,a luminal ZP protein

The formation of branched epithelial networks is fundamental to the development of many organs, such as the lung, the kidney or the vasculature. Little is known about the mechanisms that control cell rearrangements during tubulogenesis and regulate the size of individual tubes. Recent studies indicate that whereas the basal surface of tube cells interacts with the surrounding tissues and helps to shape the ramification pattern of tubular organs, the apical surface has an important role in the regulation of tube diameter and tube growth. Here we report that two proteins, Piopio (Pio) and Dumpy (Dp), containing a zona pellucida (ZP) domain are essential for the generation of the interconnected tracheal network in Drosophila melanogaster. Pio is secreted apically, accumulates in the tracheal lumen and possibly interacts with Dp through the ZP domains. In the absence of Pio and Dp, multicellular tubes do not rearrange through cell elongation and cell intercalation to form narrow tubes with autocellular junctions; instead they are transformed into multicellular cysts, which leads to a severe disruption of the branched pattern. We propose that an extracellular matrix containing Pio and Dp provides a structural network in the luminal space, around which cell rearrangements can take place in an ordered fashion without losing interconnections. Our results suggest that a similar structural role might be attributed to other ZP-domain proteins in the formation of different branched organs.     

Drosophila Knickkopf and Retroactive are needed for epithelial tube growth and cuticle differentiation through their specific requirement for chitin filament organization

Precise epithelial tube diameters rely on coordinated cell shape changes and apical membrane enlargement during tube growth. Uniform tube expansion in the developing Drosophila trachea requires the assembly of a transient intraluminal chitin matrix, where chitin forms a broad cable that expands in accordance with lumen diameter growth. Like the chitinous procuticle, the tracheal luminal chitin cable displays a filamentous structure that presumably is important for matrix function. Here, we show that knickkopf (knk) and retroactive (rtv) are two new tube expansion mutants that fail to form filamentous chitin structures, both in the tracheal and cuticular chitin matrices. Mutations in knk and rtv are known to disrupt the embryonic cuticle, and our combined genetic analysis and chemical chitin inhibition experiments support the argument that Knk and Rtv specifically assist in chitin function. We show that Knk is an apical GPI-linked protein that acts at the plasma membrane. Subcellular mislocalization of Knk in previously identified tube expansion mutants that disrupt septate junction (SJ) proteins, further suggest that SJs promote chitinous matrix organization and uniform tube expansion by supporting polarized epithelial protein localization. We propose a model in which Knk and the predicted chitin-binding protein Rtv form membrane complexes essential for epithelial tubulogenesis and cuticle formation through their specific role in directing chitin filament assembly.     

Drosophila tracheal morphogenesis: intricate cellular solutions to basic plumbing problems

The cellular architecture of tubular organs suggests striking similarities in the mechanisms of tubulogenesis between species. The formation of the Drosophila respiratory organ (trachea) highlights the basic principles of branch patterning and tube growth that generate a highly elaborate but stereotyped epithelial tubular network. Oriented cell migration, changes in cell shape, selective growth of the apical cell membrane and intracellular lumen formation are essential events in this process. These morphogenetic processes build four structurally distinct classes of tubes that facilitate optimal airflow and gas exchange with target tissues. The molecular players in these plots include attractant and repellent signals, differentiation factors that cause a high diversity of cell fates within the epithelium, and determinants of tube formation and dimensions.     

The transmembrane receptor Uncoordinated5 (Unc5) is essential for heart lumen formation in Drosophila melanogaster

Transport of liquids or gases in biological tubes is fundamental for many physiological processes. Our knowledge on how tubular organs are formed during organogenesis and tissue remodeling has increased dramatically during the last decade. Studies on different animal systems have helped to unravel some of the molecular mechanisms underlying tubulogenesis. Tube architecture varies dramatically in different organs and different species, ranging from tubes formed by several cells constituting the cross section, tubes formed by single cells wrapping an internal luminal space or tubes that are formed within a cell. Some tubes display branching whereas others remain linear without intersections. The modes of shaping, growing and pre-patterning a tube are also different and it is still not known whether these diverse architectures and modes of differentiation are realized by sharing common signaling pathways or regulatory networks. However, several recent investigations provide evidence for the attractive hypothesis that the Drosophila cardiogenesis and heart tube formation shares many similarities with primary angiogenesis in vertebrates. Additionally, another important step to unravel the complex system of lumen formation has been the outcome of recent studies that junctional proteins, matrix components as well as proteins acting as attractant and repellent cues play a role in the formation of the Drosophila heart lumen. In this study we show the requirement for the repulsively active Unc5 transmembrane receptor to facilitate tubulogenesis in the dorsal vessel of Drosophila. Unc5 is localized in the luminal membrane compartment of cardiomyocytes and animals lacking Unc5 fail to form a heart lumen. Our findings support the idea that Unc5 is crucial for lumen formation and thereby represents a repulsive cue acting during Drosophila heart tube formation.     

The first "Slit" is the deepest: the secret to a hollow heart

Tubular organs are essential for life, but lumen formation in nonepithelial tissues such as the vascular system or heart is poorly understood. Two studies in this issue (Medioni, C., M. Astier, M. Zmojdzian, K. Jagla, and M. Sémériva. 2008. J. Cell Biol. 182:249-261; Santiago-Martínez, E., N.H. Soplop, R. Patel, and S.G. Kramer. 2008. J. Cell Biol. 182:241-248) reveal unexpected roles for the Slit-Robo signaling system during Drosophila melanogaster heart morphogenesis. In cardioblasts, Slit and Robo modulate the cell shape changes and domains of E-cadherin-based adhesion that drive lumen formation. Furthermore, in contrast to the well-known paracrine role of Slit and Robo in guiding cell migrations, here Slit and Robo may act by autocrine signaling. In addition, the two groups demonstrate that heart lumen formation is even more distinct from typical epithelial tubulogenesis mechanisms because the heart lumen is bounded by membrane surfaces that have basal rather than apical attributes. As the D. melanogaster cardioblasts are thought to have significant evolutionary similarity to vertebrate endothelial and cardiac lineages, these findings are likely to provide insights into mechanisms of vertebrate heart and vascular morphogenesis.     

A transient luminal chitinous matrix is required to model epithelial tube diameter in the Drosophila trachea

Epithelial tubes are found in many vital organs and require uniform and correct tube diameters for optimal function. Tube size depends on apical membrane growth and subapical cytoskeletal reorganization, but the cues that coordinate these events to ensure functional tube shape remain elusive. We find that epithelial tubes in the Drosophila trachea require luminal chitin polysaccharides to attain the correct diameter. Tracheal chitin forms a broad transient filament within the tubes during the restricted period of expansion. Loss of chitin causes tubular constrictions and cysts associated with irregular subapical cytoskeletal organization, without affecting epithelial integrity and polarity. Analysis of previously identified tube expansion mutants in genes encoding septate junction proteins further suggests that septate junction components may function in tubulogenesis through their role in luminal matrix assembly. We propose that the transient luminal protein/polysaccharide matrix is sensed by the epithelial cells and coordinates cytoskeletal organization to ensure uniform lumen diameter.     

A Luminal Glycoprotein Drives Dose-Dependent Diameter Expansion of the Drosophila melanogaster Hindgut Tube

An important step in epithelial organ development is size maturation of the organ lumen to attain correct dimensions. Here we show that the regulated expression of Tenectin (Tnc) is critical to shape the Drosophila melanogaster hindgut tube. Tnc is a secreted protein that fills the embryonic hindgut lumen during tube diameter expansion. Inside the lumen, Tnc contributes to detectable O-Glycans and forms a dense striated matrix. Loss of tnc causes a narrow hindgut tube, while Tnc over-expression drives tube dilation in a dose-dependent manner. Cellular analyses show that luminal accumulation of Tnc causes an increase in inner and outer tube diameter, and cell flattening within the tube wall, similar to the effects of a hydrostatic pressure in other systems. When Tnc expression is induced only in cells at one side of the tube wall, Tnc fills the lumen and equally affects all cells at the lumen perimeter, arguing that Tnc acts non-cell-autonomously. Moreover, when Tnc expression is directed to a segment of a tube, its luminal accumulation is restricted to this segment and affects the surrounding cells to promote a corresponding local diameter expansion. These findings suggest that deposition of Tnc into the lumen might contribute to expansion of the lumen volume, and thereby to stretching of the tube wall. Consistent with such an idea, ectopic expression of Tnc in different developing epithelial tubes is sufficient to cause dilation, while epidermal Tnc expression has no effect on morphology. Together, the results show that epithelial tube diameter can be modelled by regulating the levels and pattern of expression of a single luminal glycoprotein.     

Ciona intestinalis notochord as a new model to investigate the cellular and molecular mechanisms of tubulogenesis

Biological tubes are a prevalent structural design across living organisms. They provide essential functions during the development and adult life of an organism. Increasing progress has been made recently in delineating the cellular and molecular mechanisms underlying tubulogenesis. This review aims to introduce ascidian notochord morphogenesis as an interesting model system to study the cell biology of tube formation, to a wider cell and developmental biology community. We present fundamental morphological and cellular events involved in notochord morphogenesis, compare and contrast them with other more established tubulogenesis model systems, and point out some unique features, including bipolarity of the notochord cells, and using cell shape changes and cell rearrangement to connect lumens. We highlight some initial findings in the molecular mechanisms of notochord morphogenesis. Based on these findings, we present intriguing problems and put forth hypotheses that can be addressed in future studies.     

The anchor cell initiates dorsal lumen formation during C. elegans vulval tubulogenesis

Tubulogenesis and lumen formation are critical to the development of most organs. We study Caenorhabditis elegans vulval and uterine development to probe the complex mechanisms that mediate these events. Development of the vulva and the ventral uterus is coordinated by the inductive cell-signaling activity of a gonadal cell called the anchor cell (AC). We demonstrate that in addition to its function in specifying fate, the AC directly promotes dorsal vulval tubulogenesis. Two types of mutants with defective anchor cell behavior reveal that anchor cell invasion of the vulva is important for forming the toroidal shape of the dorsal vulval cell, vulF. In fos-1 mutants, where the AC cannot breakdown the basement membranes between the gonad and the vulva, and in mutants in unc-6 netrin or its receptor unc-40, which cause AC migration defects, the AC fails to invade the vulva and no lumen is formed in vulF. By examining GFP markers of dorsal vulval cell fate, we demonstrate that fate specification defects do not account for the aberrant vulF shape. We propose that the presence of the AC in the center of the developing vulF toroid is required for dorsal vulval lumen formation to complete vulval tubulogenesis.     

Epithelial dynamics of pancreatic branching morphogenesis

The mammalian pancreas is a highly branched gland, essential for both digestion and glucose homeostasis. Pancreatic branching, however, is poorly understood, both at the ultrastructural and cellular levels. In this article, we characterize the morphogenesis of pancreatic branches, from gross anatomy to the dynamics of their epithelial organization. We identify trends in pancreatic branch morphology and introduce a novel mechanism for branch formation, which involves transient epithelial stratification and partial loss of cell polarity, changes in cell shape and cell rearrangements, de novo tubulogenesis and epithelial tubule remodeling. In contrast to the classical epithelial budding and tube extension observed in other organs, a pancreatic branch takes shape as a multi-lumen tubular plexus coordinately extends and remodels into a ramifying, single-lumen ductal system. Moreover, our studies identify a role for EphB signaling in epithelial remodeling during pancreatic branching. Overall, these results illustrate distinct, step-wise cellular mechanisms by which pancreatic epithelium shapes itself to create a functional branching organ.     

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.     

bullwinkle is required for epithelial morphogenesis during Drosophila oogenesis

Many organs, such as the liver, neural tube, and lung, form by the precise remodeling of flat epithelial sheets into tubes. Here we investigate epithelial tubulogenesis in Drosophila melanogaster by examining the development of the dorsal respiratory appendages of the eggshell. We employ a culture system that permits confocal analysis of stage 10-14 egg chambers. Time-lapse imaging of GFP-Moesin-expressing egg chambers reveals three phases of morphogenesis: tube formation, anterior extension, and paddle maturation. The dorsal-appendage-forming cells, previously thought to represent a single cell fate, consist of two subpopulations, those forming the tube roof and those forming the tube floor. These two cell types exhibit distinct morphological and molecular features. Roof-forming cells constrict apically and express high levels of Broad protein. Floor cells lack Broad, express the rhomboid-lacZ marker, and form the floor by directed cell elongation. We examine the morphogenetic phenotype of the bullwinkle (bwk) mutant and identify defects in both roof and floor formation. Dorsal appendage formation is an excellent system in which cell biological, molecular, and genetic tools facilitate the study of epithelial morphogenesis.     

Divide and polarize: recent advances in the molecular mechanism regulating epithelial tubulogenesis

Epithelial organs are generated from groups of non-polarized cells by a combination of processes that induce the acquisition of cell polarity, lumen formation, and the subsequent steps required for tubulogenesis. The subcellular mechanisms associated to these processes are still poorly understood. The extracellular environment provides a cue for the initial polarization, while cytoskeletal rearrangements build up the three-dimensional architecture that supports the central lumen. The proper orientation of cell division in the epithelium has been found to be required for the normal formation of the central lumen in epithelial morphogenesis. Moreover, recent data in cellular models and in vivo have shed light into the underlying mechanisms that connect the spindle orientation machinery with cell polarity. In addition, recent work has clarified the core molecular components of the vesicle trafficking machinery in epithelial morphogenesis, including Rab-GTPases and the Exocyst, as well as an increasing list of microtubule-binding and actin-binding proteins and motors, most of which are conserved from yeast to humans. In this review we will focus on the discussion of novel findings that have unveiled important clues for the mechanisms that regulate epithelial tubulogenesis.     

Luminal matrices: An inside view on organ morphogenesis

Tubular epithelia come in various shapes and sizes to accommodate the specific needs for transport, excretion and absorption in multicellular organisms. The intestinal tract, glandular organs and conduits for liquids and gases are all lined by a continuous layer of epithelial cells, which form the boundary of the luminal space. Defects in epithelial architecture and lumen dimensions will impair transport and can lead to serious organ malfunctions. Not surprisingly, multiple cellular and molecular mechanisms contribute to the shape of tubular epithelial structures. One intriguing aspect of epithelial organ formation is the highly coordinate behavior of individual cells as they mold the mature lumen. Here, we focus on recent findings, primarily from Drosophila, demonstrating that informative cues can emanate from the developing organ lumen in the form of solid luminal material. The luminal material is produced by the surrounding epithelium and helps to coordinate changes in shape and arrangement of the very same cells, resulting in correct lumen dimensions.     

From fate to function: the Drosophila trachea and salivary gland as models for tubulogenesis

Tube formation is a ubiquitous process required to sustain life in multicellular organisms. The tubular organs of adult mammals include the lungs, vasculature, digestive and excretory systems, as well as secretory organs such as the pancreas, salivary, prostate, and mammary glands. Other tissues, including the embryonic heart and neural tube, have requisite stages of tubular organization early in development. To learn the molecular and cellular basis of how epithelial cells are organized into tubular organs of various shapes and sizes, investigators have focused on the Drosophila trachea and salivary gland as model genetic systems for branched and unbranched tubes, respectively. Both organs begin as polarized epithelial placodes, which through coordinated cell shape changes, cell rearrangement, and cell migration form elongated tubes. Here, we discuss what has been discovered regarding the details of cell fate specification and tube formation in the two organs; these discoveries reveal significant conservation in the cellular and molecular events of tubulogenesis.     

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.     

C. elegans daf-6 encodes a patched-related protein required for lumen formation

Sensory organs are often composed of neuronal sensory endings accommodated in a lumen formed by ensheathing epithelia or glia. Here we show that lumen formation in the C. elegans amphid sensory organ requires the gene daf-6. daf-6 encodes a Patched-related protein that localizes to the luminal surfaces of the amphid channel and other C. elegans tubes. While daf-6 mutants display only amphid lumen defects, animals defective for both daf-6 and the Dispatched gene che-14 exhibit defects in all tubular structures that express daf-6. Furthermore, DAF-6 protein is mislocalized, and lumen morphogenesis is abnormal, in mutants with defective sensory neuron endings. We propose that amphid lumen morphogenesis is coordinated by neuron-derived cues and a DAF-6/CHE-14 system that regulates vesicle dynamics during tubulogenesis.