Chapter Fourteen - Cell–Cell Interactions Driving Kidney Morphogenesis

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Abstract

The mammalian kidney forms via cell–cell interactions between an epithelial outgrowth of the nephric duct and the surrounding nephrogenic mesenchyme. Initial morphogenetic events include ureteric bud branching to form the collecting duct (CD) tree and mesenchymal-to-epithelial transitions to form the nephrons, requiring reciprocal induction between adjacent mesenchyme and epithelial cells. Within the tips of the branching ureteric epithelium, cells respond to mesenchyme-derived trophic factors by proliferation, migration, and mitosis-associated cell dispersal. Self-inhibition signals from one tip to another play a role in branch patterning. The position, survival, and fate of the nephrogenic mesenchyme are regulated by ECM and secreted signals from adjacent tip and stroma. Signals from the ureteric tip promote mesenchyme self-renewal and trigger nephron formation. Subsequent fusion to the CDs, nephron segmentation and maturation, and formation of a patent glomerular basement membrane also require specialized cell–cell interactions. Differential cadherin, laminin, nectin, and integrin expression, as well as intracellular kinesin and actin-mediated regulation of cell shape and adhesion, underlies these cell–cell interactions. Indeed, the capacity for the kidney to form via self-organization has now been established both via the recapitulation of expected morphogenetic interactions after complete dissociation and reassociation of cellular components during development as well as the in vitro formation of 3D kidney organoids from human pluripotent stem cells. As we understand more about how the many cell–cell interactions required for kidney formation operate, this enables the prospect of bioengineering replacement structures based on these self-organizing properties.

Introduction

Tissue development relies upon local cellular interactions mediated by secreted morphogens, cell–extracellular matrix, or direct cell–cell interactions. Whether applied to invertebrate or vertebrate development, the final arrangement of differentiated cells relies upon locally acting signals driving competence, induction, cellular identity, and ultimately functional specialization. This combination of inherent locally acting interactions between cells enables the formation of complex structures without a template or scaffold. Such an internally regulated process is referred to as self-organization. As in other tissues, organogenesis of the mammalian permanent kidney, the metanephros, involves such self-organizing cell–cell interactions.

The mammalian kidney is mesodermal in origin, positioned at a specific rostrocaudal and mediolateral position of the embryo and arises from the intermediate mesoderm (reviewed by Kopan et al., 2014, Little and McMahon, 2012). Indeed, three paired excretory organs arise in a rostrocaudal sequence during mammalian embryogenesis: the pronephros, mesonephros, and finally the metanephros. The formation of all these structures is preceded by the initiation of nephric duct formation from the intermediate mesoderm at the level of the forelimb at around E8.5 in the mouse (reviewed by Dressler, 2009, Saxen, 1987). The nephric duct (also called the mesonephric duct or the Wolffian duct) extends in a caudal direction as the embryo develops, with the tubular components of the pronephros, mesonephros, and finally the metanephros forming from an adjacent mesodermal population referred to as the nephric cord. The permanent mammalian excretory organ, the metanephros, is this third set of paired structures and we focus on the development of this organ in this chapter. Until recently, the origin of the metanephric mesenchyme (MM) was regarded to be the same as that of the temporally earlier mesonephros. However, distinctions in gene expression in this more caudal mesenchymal population (Challen et al., 2004, Wellik et al., 2002), as well as lineage analyses (Taguchi et al., 2014; Xu et al., 2014), suggest that the mesenchyme giving rise to the caudal mesonephric tubules and the metanephros represents a more caudal or temporally distinct region of the intermediate mesoderm.

In the E10–10.5 mouse, the nephric duct at the level of the hindlimb begins to swell and a single side-branch, the ureteric bud (UB), arises from the duct and migrates toward the MM (reviewed by Costantini & Kopan, 2010). This outbudding is a response to the production of glial-derived neurotrophic factor (Gdnf) by the mesenchyme. Gdnf acts as a long-distance diffusible inducer, being detected within the nephric duct population via the Ret tyrosine kinase receptor (Ret) and a coreceptor, GFRα1. The UB elongates as an unbranched epithelial duct until it reaches the MM, where branching is initiated. The positioning of UB outgrowth and suppression of premature branching is regulated via the balance between bone morphogenic protein (BMP4) and Gremlin signaling along the nephric duct and extending UB (Michos et al., 2007). UB branching has been regarded as a simple dichotomous branching event lacking the apparent stereotypic patterning of organs such as the lung (Metzger, Klein, Martin, & Krasnow, 2008). However, the kidney achieves a specific shape and orientation, highlighting the fact that branching is not spatially uniform or synchronized. Recent comprehensive imaging of the branching ureteric tree has revealed an underlying initial lobe patterning and the frequent presence of trifurcations early in organogenesis when the proliferation rate is highest, with each of these representing an asynchronous branching event that resolves to bifurcations with time (Short et al., 2014; Fig. 1A). Time-lapse imaging and population modeling show that cells within the ureteric tip contribute both to subsequent tips as well as the intervening branches (Chi et al., 2009, Short et al., 2014).

Once the UB reaches the MM, a subpopulation of MM cells closest to the ureteric epithelium condenses to form the Gdnf+Six2+ cap mesenchyme (CM). The CM is a self-renewing progenitor population (Boyle et al., 2008, Kobayashi et al., 2008) also competent to form the second epithelial compartment of the kidney, the nephrons (Little, Georgas, Pennisi, & Wilkinson, 2010). The interaction between the ureteric tip and the adjacent CM is classically regarded as a reciprocal inductive event (reviewed by Kopan et al., 2014). While the CM requires support from the UB to survive and to form nephrons, conversely the identity, proliferation rate, and branching of the UB requires continued Gdnf production by the CM. Ret signaling in response to ligand binding induces the expression of Wnt11 by the ureteric tip which in turn is thought to increase Gdnf expression within the MM (Majumdar, Vainio, Kispert, McMahon, & McMahon, 2003).

As noted above, the CM can be induced by signals from the ureteric tip to form the nephrons. This induction event represents a mesenchyme-to-epithelial transition (MET), a relatively rare event in organogenesis. Both the survival of the CM as a progenitor population and its induction to form nephrons via MET are directed by signals from the adjacent ureteric tip (Barak et al., 2012, Carroll et al., 2005, Karner et al., 2011). The first visible evidence of nephron induction is the formation of a pretubular aggregate, an event triggered by canonical Wnt signaling, which begins to express markers such as Wnt4, Fgfrl1, and Fgf8. This subsequently undergoes MET as a response to Wnt4-mediated noncanonical Wnt signaling (Burn et al., 2011, Tanigawa et al., 2011) to form an epithelial renal vesicle (RV). The RV is defined as the first stage of nephron formation (defined as such in Georgas et al., 2009, Little et al., 2007). As soon as the RV forms, the cells within the RV show evidence of positional identity with distinct molecular profiles defining the proximal pole, furthest from the adjacent UB tip, and the distal pole, adjacent to the tip. By late RV, the distal pole invades and fuses with the adjacent ureteric tip to form a contiguous tubular lumen (Georgas et al., 2009). After substantial elongation and segmentation, the proximal pole will ultimately vascularize to form the filtering glomerulus.

RV polarization represents the beginning of the process of nephron segmentation that results in a precisely patterned tube comprising more than a dozen distinct functional cell types, including components of the glomerulus, proximal tubule (PT), loop of Henle (LoH), and distal tubule (Costantini and Kopan, 2010, Little et al., 2010). This early patterning is accompanied by differential expression of a variety of cell–cell adhesion molecules (Cho et al., 1998, Goto et al., 1998, Mah et al., 2000). The precise shape (convolution) and alignment of the forming nephrons with respect to the advancing and elongating collecting duct (CD) and the surrounding interstitial elements, including vasculature, are essential for ultimate renal function. While the signals directing this patterning remain imprecisely understood, this again involves long- and short-range signals between adjacent cell populations, as well as the formation of specialized cell–cell and cell–matrix interactions. Specification of the PT involves notch signaling (Cheng et al., 2007), while nephron and CD elongation requires canonical and noncanonical Wnt signaling between interstititum and epithelium as well as between one epithelial element and another (Karner et al., 2009, Lienkamp et al., 2012, Yu et al., 2009). At the cell–cell interface, differential cadherin expression across space as time is involved in nephron segmentation and specific integrin and laminin subunit interactions regulate appropriate CM survival and glomerular formation and maturation (Chen et al., 2004, Goto et al., 1998, Kanwar et al., 2004, Mathew et al., 2012, Miner, 2012, Yang, Zimmerman, et al., 2013).

While the identity and lineage relationships of most of the cell types involved in kidney development have now been defined, along with a number of the critical growth factor/receptor interactions and their downstream subcellular consequences, much remain a mystery. At the subcellular level, what produces the spatially constrained branching CD or the precise formation and fusion of RVs around the ureteric tips? How does a patent glomerular basement membrane (GBM) result from the cell–cell interactions between the podocytes and glomerular endothelial cells? How is the balance between self-renewal and nephron induction regulated to produce a final organ? In this chapter, we will focus on what we know about the nature of critical cell–cell interactions during the formation of key components of the developing kidney and how we may ultimately be able to use this understanding to recapitulate organogenesis in vitro for the purposes of regenerative medicine.

Section snippets

Cell–Cell Interactions Within the Developing Ureteric Epithelium

In vitro models of epithelial biology have been used to great effect to study the molecular mechanisms underlying cell–cell interactions, polarization, lumen formation, and disease (Debnath and Brugge, 2005, Rodriguez-Fraticelli and Martin-Belmonte, 2014, Roignot et al., 2013, Shewan et al., 2011). In parallel, developmental biologists have been investigating the formation of branched epithelial structures in vivo with a focus on the mechanisms regulating the broader morphogenic program (

The Nephrogenic Niche—Balancing Self-Renewal and Differentiation

The CM, which represents the nephron progenitor population of the kidney, exists in a histologically distinct domain around each ureteric tip. The CM, together with the underlying tip epithelium and surrounding stroma, comprises the nephrogenic niche (Fig. 2A and B). For quantification purposes, a niche can be defined as the region surrounding or underlying a spatially distinct cluster of CM cells. This definition differs from tip number as, before 14.5 dpc, cap domains are broad and can overlay

Mediators of CM Integrity, Identity, and Morphology

The CM is a derivative of the MM. While it is described as a mesenchyme and does not show evidence of classical epithelial morphology, these cells do show some anatomical signs of alignment around the ureteric tip, suggesting an integrity requiring cell–cell interactions both within the CM and between the CM and the ureteric tip.

A number of cell adhesion components or proteins that associate with cell–cell junctions contribute to CM morphology or maintenance. The CM produces neural cell

Differential Cell–Cell Adhesion in Nephron Formation, Fusion, Patterning, and Segmentation

The transition from pretubular aggregate to RV includes a profound alteration in the nature of the tissue, from a mesenchyme to an epithelium. This involves a substantial change in both the identity and the placement of the adhesion molecules involved. Before MET has begun, cell–cell adhesion molecule expression is dominated by NCAM and by three cadherins, OB-cadherin (Cdh11), N-cadherin (Cdh2), and R-cadherin (Chd4; Goto et al., 1998, Klein et al., 1998). OB-cadherin is typically mesenchymal

The Adhesion–Cytoskeleton–Signaling Axis in Kidney Tubulogenesis

Whether within the CD epithelium or the many mesenchyme-derived segments of the nephron, final morphology results from the intracellular transduction of external signals. External cell–cell and cell–substrate junctions link via the inner face of the plasma membrane with the microfilament or intermediate filament cytoskeletal systems of the cell. Structurally, this creates a network of cytoskeletal and junctional elements that enables tissues to withstand mechanical loads. The junctional and

Formation of the Glomerular Filter

The filter of the glomerulus has three main components: (i) the fenestrated endothelium of the glomerular capillaries, the 60–80 nm pores through which molecules can pass but not cells; (ii) the three-layered (or perhaps five-layered: Salmon, Neal, & Harper, 2009) GBM, which provides the modest filtration function; and (iii) the slit diaphragm between podocytes of the nephron, which provides the finest filtering.

Endothelial cells, most of which differentiate from renal stroma (Hyink et al., 1996

In Vitro Self-Organization Generates Kidney Organoids

As described above, kidney organogenesis involves temporospatially defined reciprocal induction events, cell migration, and proliferation in response to secreted and cell adhesion-based signals. As such, the component cells involved in kidney organogenesis appear capable of elaborating a highly complex architecture without a scaffold or template, suggestive of a self-organizing structure (Camazine et al., 2001, Sasai et al., 2012). The formation of functional tissues and organisms by

Self-Organization in Directed Differentiation to Kidney

In the twenty-first century, self-assembly or self-organization is being seen by some tissue engineers as a powerful method for construction of complex tissues without a scaffold. The power of morphogenesis via self-organization has recently been highlighted by observations in studies of the directed differentiation of human pluripotent stem cells. The approach commonly taken to direct a pluripotent cell source to a specific mature cell type is to recapitulate in a stepwise fashion the process

Application of Cell–Cell and Cell–Matrix Interactions Kidney Tissue Engineering

The power of self-organization raises the prospect of the generation of replacement organs based around the spontaneous recapitulation of organogenesis without the need for positional direction or scaffolding. The capacity to direct the differentiation of pluripotent human cells toward kidney by capitalizing on the critical cell–cell communication that normally occurs during development also raises the prospect of the generation of large amounts of starting material for template-based

Conclusion

In conclusion, we have presented here the existing understanding of how kidney morphogenesis occurs at the level of cell–cell interactions. This represents the intersection between what observational anatomical developmental biology and mouse genetics has revealed at the organ level for many decades and what systems biology and gene expression studies tell us are the key components at the molecular level. With the advent of reporters of intracellular signaling, cytoskeletal structure and

Acknowledgments

M. H. L. is a National Health and Medical Research Council of Australia Senior Principal Research Fellow. M. H. Little's research is supported by the National Health and Medical Research Council of Australia, the Australian Research Council, the Human Frontiers Science Program and Organovo Inc. J. A. Davies’ research is supported by BBSRC, British Heart Foundation, European Union, Leverhulme Trust, MRC, NIH/NIDDK, and The Wellcome Trust. A. N. C. is a DECRA Fellow of the Australian Research

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