Connecting the Segments
Authors: Roy Zent, Ambra Pozzi
Journal: Journal of the American Society of Nephrology (2012)

Tubular networks lined by epithelial cells, which are ancient structures found in all metazoan organisms, are vital components of many of our organs, including the gastrointestinal tract, lung, and kidney. Although there is commonality to the overall structure of tubes, their size, specific cell composition, and function are diverse, suggesting that they underwent distinct morphogenesis. Consistent with this hypothesis, different tube segments in branched organs have separate embryological origins and are connected together during development. A good example is in the kidney, where the nephrons derived from the metanephric mesenchyme join with the collecting system, which is derived from the ureteric bud. The process and molecular mechanisms where these two embryological distinct tube systems link together during development are poorly understood. Because of the complex process of tube morphogenesis and the inadequacy of tools to specifically label structures for visualization in vivo, the mechanisms of tubule formation have primarily been investigated in vitro and/or in lower animals. The two best-studied processes in model organisms are tracheal development in Drosophila and the excretory system in Caenorhabditis elegans, because they can be easily visualized. For example, advanced imaging was used to show that fibroblast growth factor is required to stimulate extension of bridge cells necessary for the formation of tracheal tubular structures in Drosophila,1 and Notch and Ras signaling regulates epithelial junctions, cell shape, cell identity, and interconnections between the large canal cell, smaller duct cell, and pore cell of the excretory system in C. elegans.2 A fundamental question in renal development in metazoa is how nephrons, which are derived from the metanephric mesenchyme, physically connect with the collecting system, which originates from the ureteric bud, to give rise to a functional kidney. Recent advances in Cre technology and development of mice expressing fluorescent label markers have facilitated the visualization of tubule formation in complex organisms, and therefore, we can study the mechanisms and timing of when cells organize into tubular networks. In JASN, the work by Kao et al.3 uses high-resolution image analysis in conjunction with genetic labeling of epithelia to define when and how nephrons connect to the kidney collecting system. The work by Kao et al.3 describes the development of a mouse with a myristoylated form of green fluorescent protein expressed in the developing metanephric mesenchyme, whereas all other cells constitutively express a myristoylated form of the Tomato fluorescent protein. In addition to this state of the art transgenic approach, the work by Kao et al.3 uses renal lumography with differentially charged fluorescent dextrans to label the tubule lumens. Three-dimensional reconstructions were performed to assemble images to answer some basic but critical questions as to how the two developing tubule systems within the developing kidney fuse. Using these methodologies, the work by Kao et al.3 shows that this interconnection takes place between renal vesicles and the ureteric epithelium at the late S-shaped body stage. At this developmental stage, precursor cells of the distal part of the renal vesicles in close proximity to the nascent collecting duct acquire an invasive-like phenotype, which is required for penetration and subsequent invasion into the ureteric epithelium. Interestingly, these distal nephron cells lack tight junction markers usually seen in the early S-shaped body stage; however, they do not express mesenchymal markers such as vimentin or Snail2. The work by Kao et al.3 shows that the process of tubule connection is highly dependent on silencing of the Notch pathways, because mice expressing the Notch intracellular domain selectively in renal vesicle cells, which leads to constitutive transcription of Notch targeted genes, are unable to form luminal interconnections. Altogether, these data support a model of specific molecular mechanisms differentiating distal nephron precursor cells so that they can invade the developing collecting duct to form a patent tubular connection between the nephron and collecting duct epithelia. Most importantly, this study suggests that interconnection between the nephron and the collecting duct is both temporally (late S-phase) and spatially (distal vesicle cells) controlled. The study by Kao et al.3 clearly shows the power of innovative imaging as a tool to visualize mammalian development in vivo. When these techniques are combined with more molecular assays, they have the potential to define the mechanics of normal tubule formation and patency as well as defects associated with tubular pathology. This finding could also greatly enhance our understanding of diseases, such as cystic kidney disease and tubular obstruction, where different gene mutations result in similar pathologies by radically different cellular mechanisms. These techniques do, however, have their limitations. They are very labor-intensive, and when used alone, they do not provide deep mechanistic insights into normal development or pathologic states. They require complex interbreeding of mice to introduce the transgene of interest into specific cell types and have limited access to immunostaining, because two fluorescent channels are used to simply visualize the mouse. Thus, the technique requires dedicated people with an exceptional microscopy facility that may not be available at all institutions. Nonetheless, where available, these techniques will greatly enhance our ability to understand various aspects of renal development and/or disease. It is unclear how renal tubules develop in vivo in mammals. Although the study by Kao et al.3 does not directly study mechanisms of tube formation, it shows that metanephric tubule cells are specifically differentiated to invade the ureteric bud and form a functional lumen by a process that does not require apoptosis. This finding contrasts with findings in Madin–Darby canine kidney (MDCK) cells, the most extensively used in vitro model of tubule formation, where lumen formation is mediated by partitioning-defective protein 6-atypical protein kinase C-Glycogen synthase kinase 3 β–regulated apoptosis.4 Although the observation in the work by Kao et al.3 may be specific for the site and/or type of segment connection, it suggests that renal tubule lumen formation occurs by mechanisms other than cell apoptosis. These mechanisms include wrapping, folding, invagination, or evagination of polarized epithelial cell sheets to generate a hollow lumen or conversion of loosely adherent mesenchymal cells into polarized epithelia by mesenchymal–epithelial transition.5 Mechanistically, many proteins and signaling cascades with divergent functions have been implicated in different stages of tube formation. These functions include pathways regulating establishment of epithelial cell polarity, formation of an apical cell surface, induction of lumen formation, hollowing and elongation, and finally, terminal differentiation of the tubule.6 Most mechanistic insights into renal tubule formation are based on in vitro studies with MDCK cells and to a lesser extent, collecting duct cells, because the phenotypes of mice, where proteins are often selectively deleted and/or overexpressed in specific nephron segments, are usually assessed by standard histology, which fails to provide information and/or details on tubule morphogenesis. This shortcoming may be changed by new imaging technologies similar to those technologies described in the work by Kao et al.,3 which can verify whether observations made in vitro are recapitulated in mammals. For example, the work by Kao et al.3 shows that the distal metanephric mesenchyme cells that invade the ureteric bud do not express occludin, zona occludens-1, and activated β-catenin, suggesting that they acquire mesenchymal features compared with the epithelial features found in other cells of the S-shaped body. These observations differ from the MDCK cell model of tubule and lumen formation, where there are tight connections between the cells and high expression of tight junction proteins.7 Another key finding in the paper by Kao et al.3 is that the invasive phenotype is highly dependent on Notch signaling. In addition to Notch, many molecular pathways have been shown to be critical for tubule lumen formation in MDCK and collecting duct cell models; however, whether the same mechanisms and/or pathways that occur in vitro also work in vivo is unclear. Functional disruption of integrin β1, the key molecule that regulates epithelial cell interactions with extracellular matrix, is a good example of this situation. Inhibition of the integrin β1 subunit in either MDCK8 or collecting duct cells9 in three-dimensional gels results in cyst formation with poorly formed lumens. In contrast, selective deletion of this subunit in the ureteric bud primarily results in decreased branching, whereas tubular lumens seem to form normally as assessed by histologic examination. These mice develop severe collecting duct dilatation after birth.9,10 It is possible that the tubular dilatation was caused by incomplete lumen formation, which could be assessed by the new imaging techniques and the state of the art tools described in the work by Kao et al.3 In conclusion, the rapid advances of sophisticated imaging techniques combined with the generation of transgenic mice expressing differentially fluorescently labeled cells allows us to visualize morphogenesis in vivo with improved resolution. These tools will help cross the wide gap that exists between in vivo and in vitro model systems by enabling us to verify whether the mechanisms controlling tubule formation in vivo are the same as those mechanisms identified in cell culture models. Understanding one of the most complex morphogenic processes in metazoans will help define basic mechanisms of abnormal tubule formation in pathologic conditions. Disclosure None.