Improving our resolution of kidney morphogenesis across time and space

Improving our resolution of kidney morphogenesis across time and space

Available online at ScienceDirect Improving our resolution of kidney morphogenesis across time and space Melissa H Little1,2 As...

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ScienceDirect Improving our resolution of kidney morphogenesis across time and space Melissa H Little1,2 As with many mammalian organs, size and cellular complexity represent considerable challenges to the comprehensive analysis of kidney organogenesis. Traditional analyses in the mouse have revealed early patterning events and spatial cellular relationships. However, an understanding of later events is lacking. The generation of a comprehensive temporospatial atlas of gene expression during kidney development has facilitated advances in lineage definition, as well as selective compartment ablation. Advances in quantitative and dynamic imaging have allowed comprehensive analyses at the level of organ, component tissue and cell across kidney organogenesis. Such approaches will enhance our understanding of the links between kidney development and final postnatal organ function. The final frontier will be translating this understanding to outcomes for renal disease in humans. Addresses 1 The Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia 2 Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, Victoria, Australia Corresponding author: Little, Melissa H ([email protected])

Current Opinion in Genetics & Development 2015, 32:135–143 This review comes from a themed issue on Developmental mechanisms, patterning and organogenesis Edited by Deborah J Andrew and Deborah Yelon 0959-437X/# 2015 Elsevier Ltd. All rights reserved.

Introduction The mammalian kidney is a complex organ comprising over 25 functionally and morphologically distinct cell types anatomically placed to deliver the filtration, reabsorption and secretion capacities essential for the maintenance of fluid homeostasis, nitrogenous waste regulation, blood pressure, red cell count and bone density. As congenital anomalies of the kidney and urinary tract are present in 40–50% of children with chronic kidney disease [1], the development of this organ has been the subject of extensive attention. The last 15 years have seen a revolution in our understanding of the molecular basis of kidney morphogenesis in the mouse, with

the capacity to tag specific cells facilitating temporospatial profiling of individual cellular subcompartments, the dissection of lineage relationships across development and the conditional alteration of gene expression. Coupled with advances in imaging across time and space the possibility of probing the nature of kidney organogenesis is improving. This article will focus on how these advances have changed our understanding of kidney development and the pertinence of these findings to the human kidney.

Fundamental events in kidney morphogenesis The final kidney in mammals, the metanephros, is mesodermal in origin and arises through reciprocal signalling between the nephric duct (ND) and the adjacent metanephric mesenchyme (MM) (Figure 1). Kidney morphogenesis can be divided into two key tubulogenetic events; the formation of a branching ureteric tree and nephron formation at the tips of this tree [2] (Figure 1). The first evidence of metanephros formation (gestational week 5 in humans, 10.5 dpc in mice) occurs as the ureteric bud (UB) branches from the ND, extending towards the MM in response to GDNF secretion. By 11.5 dpc, the UB begins to branch dichotomously to form the ureteric tree, which will become the collecting ducts through which urinary filtrate will exit. Exquisite timelapse imaging of UB branching in explant cultures has identified the requirement for ongoing expression of the GDNF receptor, RET, in the UB tips and the contribution of cells from the tips to subsequent tips as well as trailing and elongating branches [3]. RET signalling in the ureteric tips, in response to GDNF from the surrounding mesenchyme, upregulates RET and WNT11, the latter resulting in an upregulation of GDNF [7] (Figure 1c). Around this ureteric tree, the nephrons arise via a mesenchyme to epithelial transition triggered via Wnt9b from the adjacent ureteric tip [4] (Figure 1c). Grobstein identified the requirement for a ureteric signal to induce this event from the mesenchymal population [5]. It is now appreciated that the only the cap mesenchyme (CM) closely associated with each branching ureteric tip responds to form the nephrons. CM cells first condense to form a pretubular aggregate that, after a second Wnt4-mediated non-canonical signal [6], polarises to form the renal vesicle (RV) (Figure 1d). This structure is immediately patterned such that the side closest to the adjacent tip (distal RV) fuses with it [7]. After subsequent proliferation, elongation, segmentation and patterning, distinct functional domains form such that each nephron has a vascularised glomerulus, proximal tubule, loop of Henle Current Opinion in Genetics & Development 2015, 32:135–143

136 Developmental mechanisms, patterning and organogenesis

Figure 1

and distal tubule all connected back to the tips of the ureteric tree (Figure 1d). Regulation of this complex specialisation remains poorly investigated, although a growing number of compartment markers and key signalling pathways are being identified [2].

(a) Ureteric Budding nd

mm ub

A global molecular atlas of the developing mouse kidney

(b) Ureteric Branching

cap mesenchyme

ureteric tree

(c) Key Processes in the Nephrogenic Niche Branching

Self Renewal Fgf20


Wnt11 Gdnf









Bmp7 Smad


Differentiation (d) Nephrogenesis and Differentiation






Capillary Loop Nephron

nephron ureteric tree cap mesenchyme

distal tubule proximal tubule Mature Nephron loop of Henle

Current Opinion in Genetics & Development

Key inductive events in mammalian kidney morphogenesis. (a) Ureteric budding. Diagram showing the formation of the ureteric bud (UB) as a swelling of the nephric duct (ND) which grows towards the metanephric mesenchyme (mm) before undergoing initial bifurcation. (b) Ureteric branching. Diagrammatic view of the branching ureteric epithelium of the developing mouse kidney from 11.5 dpc (left) to 15.5 dpc (right) showing the ureteric tree and surrounding cap mesenchyme. (c) Key processes in the nephrogenic niche. Diagram of a nephrogenic niche illustrating the signalling pathways critical for branching (left) versus cap mesenchyme self renewal (top right) and Current Opinion in Genetics & Development 2015, 32:135–143

Elucidation of the molecular basis of these foundational morphogenetic events has largely been gene-by-gene, using knockout or overexpression studies in transgenic mice together with explant culture. The advent of microarrays for global transcriptional profiling represented a major change. Beginning with the separation of cellular compartments based on microdissection or laser capture, a global atlas of gene expression across time and space in the developing mouse kidney was generated [8]. In a process of iterative re-evaluation, gene expression was validated using RNA section in situ hybridisation with compartment specific markers then used to more specifically enrich via FACS [9–12] (Figure 2a). This led to the identification of smaller and smaller anatomical subdomains. The capacity to analyse all genes expressed at a given time and/or cell type facilitated network and pathway analyses previously not possible (Figure 2a,b). The advent of Next Generation sequencing allowed the identification of novel transcripts not necessarily present on microarrays and the capacity to sequence from single cells [13]. This has begun to reveal the transcriptional complexity present during kidney development, including the temporal and spatial variation in miRNA and non-coding RNA species. Within the CM/ureteric tip niche alone, almost all previously described genes show evidence for alternative exon splicing, 50 and 30 sequences and antisense transcripts [14] (Figure 2c). The comprehensive analysis of specific types of genes across kidney development has also been performed. The temporospatial expression of all transcription factors (TFs) across kidney development identified a subset of TFs enriched in key cellular compartments. Identifying other genes with comparable temporospatial expression patterns allowed the prediction of specific TF targets and gene-regulatory networks [15]. RNA sequencing is now being applied post chromatin immunoprecipitation (ChIP) to directly examine the action of specific TFs. For example, self-renewal of the CM requires Six2 expression, with b-catenin mediated canonical Wnt-signalling required for both CM survival and differentiation into nephron [4,16]. Six2 and b-catenin differentiation (bottom right). (d) Nephrogenesis and differentiation. Diagram showing the stages of nephron maturation from pretubular aggregate (PA) through renal vesicle (RV), comma-shaped body (CSB), S-shaped body (SSB), capillary loop nephron and mature nephron. The RV represents the point of transition from mesenchyme to a polarised epithelial state [7]. The formation of a connection between the forming nephron and the lumen of the adjacent ureteric epithelium occurs at late RV stage and is shown here at comma-shaped body.

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Figure 2





Sall1 Wt1 CM Bmp7




Wnt11 Emx2 Pax2 Wnt9b


Gdnf Six2 Eya1 Pax2 Hoxa11

RV Wnt4 Lhx1 Fgfrl1 Fgf8

Wnt4 Lhx1 Fgfrl1 Fgf8

Alternate long UTR Antisense non-coding transcripts Alternate splicing (c)

















200µm Current Opinion in Genetics & Development

Atlas of gene expression during kidney development. (a) Genitourinary Development Molecular Anatomy Project (GUDMAP) approach to temporospatial analysis of gene expression profiling in the developing mouse kidney. Cells were isolated using laser capture microdissection (LCM) and subjected to microarray based gene expression profiling. Genes showing regional or temporal specificity were validated using section in situ hybridisation and novel subdomains identified. The generation of transgenic animals driving reporters to these compartment was then subjected to LCM or FACS-based cell isolation for subsequent expression profiling [8–12]. (b) Identification of transcriptional complexity within the genes expressed in the cellular compartments of the nephrogenic niche based on Next Generation sequencing [14]. (c) Section in situ hybridisiation of genes identified as restricted to specific cellular compartments of the developing kidney. Eya1, cap mesenchyme; C230096N06, renal corpuscle; Spp2, proximal straight tubule; Umod, Loop of Henle; Upk3a, medullary collecting duct; Slco4c1, connecting segment. Adapted from [10].

ChIP of isolated CM identified distinct classes of genes associated with both the CM state and nephron induction, suggesting that in some instances Six2 and b-catenin cooperate while in other instances they compete [17]. This type of analysis will ultimately uncover how the balance between the progenitor and committed state is regulated.

Tracing lineage relationships The identification of cell specific gene expression patterns has facilitated temporal and spatial conditional gene knockout, lineage tracing and the ablation of specific cell populations. Lineage tracing has been critical to our understanding of both morphogenesis and the response of cells within the postnatal kidney to injury. Lineage Current Opinion in Genetics & Development 2015, 32:135–143

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tracing from the Six2 and Cited1 promoters has shown that the CM represents a self-renewing stem cell population able to give rise to all cell types within the nephron [18,19]. Lineage tracing from the same promoter also demonstrated that tubular repair in the postnatal organ in response to acute injury involved cells within the existing nephron (based on Six2-lineage) without any contribution from non-tubular locations [20]. This did not eliminate the possibility of cellular heterogeneity within mature nephrons such that repair was a function of a specific ‘stem/progenitor’ subpopulation. However, lineage tracing using a marker specific to the functionally mature proximal tubule, SLC34a1, shows that proliferation of such mature cells occurs in response to acute injury, suggesting this is a property of all tubular epithelium [21]. Similar lineage tracing has now been performed to examine the fate of the surrounding stroma, which is also considered to come from the initial MM. Based on Foxd1-lineage tracing, this mesenchyme has been shown to contribute to interstitial elements in both the cortex and medulla of the final kidney, notably the pericytes surrounding the vasculature [22]. Again, this lineage tracing model has contributed to our understanding of postnatal organ repair, illustrating that the fibrotic expansion seen in the chronically injured kidney arises from the stroma-derived pericytic population, not the tubular epithelium as had previously been proposed [23]. Lineage tracing from the Ren1 promoter revealed the relationship between the renin-expressing perivascular cells present during early development and the final renin-expressing population within the juxtaglomerular apparatus [24]. Using this same promoter, a transformation of Ren1+ cells into parietal epithelial cells and podocytes in response to damage has also been reported [25]. Lineage tracing has also begun elucidate the process of nephron segmentation. The medial Sshaped body of the early nephron begins to express Lgr5. Lineage tracing based on this promoter has revealed that this initial cell population proliferates clonally to form the entire loop of Henle [26]. This implies that while the Six2+ CM is the progenitor for all nephron segments, that specific progenitors subsequently arise to ensure the identity of individual segments. Strong evidence for this comes from a recent study in which lineage was randomly tagged using the confetti mouse strategy [27]. In this way, it was possible to evaluate the expansion of individual cells during kidney development. This confirmed the contribution of CM to all nephron segments, but also suggested that individual clones arising in a given nephron segment could proliferate in that segment but did not contribute to adjacent segments [27]. The identification of markers for such segment-specific progenitors will ultimately advance our understanding of nephron differentiation. Current Opinion in Genetics & Development 2015, 32:135–143

Quantitative temporospatial visualisation at a whole organ level Advances in live imaging are beginning to afford a more accurate view of the morphogenetic process at the level of individual cells. While time-lapse imaging has long been applied to the analysis of ureteric tree branching in explant cultures [28], the capacity to label individual cells within a population will provide new insights. The generation of transgenic lines driving fluorescent proteins under the control of inducible promoters has allowed labelling of cell shape (Hoxb7:myr-Venus) and cell cycle state (H2B-EYFP) [28]. Most recently, this has revealed a process of mitosis-associated cell dispersal (MACD) where mitosing cells within the ureteric tip delaminate from the epithelium and move into the lumen to divide before reinserting back into the epithelial layer [29]. Live imaging was also used to investigate how the distal RV invades the ureteric tip to form a connected lumen [30]. This reveals a loss of cell-cell junctions without the adoption of mesenchymal gene expression, and with these distal RV cells invading the ureteric tip lumen. Despite these informative studies, much remains to be visualised; the fate of individual cells within the CM across time, the process of cellular polarisation and constriction during nephron segmentation and convolution, the vascularisation of the Bowman’s capsule to form the initial capillary loop to name a few. Visualisation of many of these processes is not possible in explant cultures. Conversely, studies of the intact kidney are challenged by the complexity and size of the final structure. Gross anatomical and histological analyses of the mature kidney provide little detailed information about subtle morphogenetic defects, resulting in a large number of phenotypes described as hypoplastic or dysplastic with little resolution of the actual anomaly. The application of modalities such as MRI and intravital multiphoton fluorescence microscopy to the postnatal kidney have facilitated the dynamic visualisation of blood flow, protein uptake, cyst formation and progression and glomerular structure and function over time [31,32,33]. However, such approaches are not possible within the embryonic kidney. A major advance has come with the application of optical projection tomography (OPT) and high resolution confocal microscopy to catalogue kidney morphogenesis of the whole organ in the mouse [34]. Global visualisation of the entire ureteric compartment using OPT (Figure 3a) has provided an accurate 3D dataset amenable to quantification of volume, length, branch diameter, branchpoints, branch angle and branch generation across time [35]. Analysis of branching across the entire organ has revealed an underlying stereotypic lobe structure in the kidney that was previously unappreciated. It also confirms that branching is not a synchronous event and that final branch generation varies between different regions of the

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Figure 3







(f) 100


Niche no. CM pop. Nephron no.

80 60 40


% Maximum













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0 1 2 3 4 Current Opinion in Genetics & Development

Global quantification of tissue dynamics in the developing mouse kidney. (a) Optical projection tomograph (OPT) of the total 15.5 dpc mouse kidney stained for ureteric epithelium (Cytok, white) and cap mesenchyme (Six2, red). Scale bar 200 mm. (b) Confocal resolution image of individual niches within the 15.5 dpc mouse kidney stained for cap mesenchyme (Six1, red), ureteric epithelium (Cytok, white) and nuclei (DAPI, blue). One niche in the upper quadrant has been rendered, defining a single niche CM and tip domain. Scale bar 50 mm. (c) Isolated niche illustrating individual Six2+ nuclei within the CM and individual DAPI+ nuclei within the ureteric tip. (d) Spot count of individual cells within the CM and underlying ureteric tip. (e) Rendered OPT of ureteric T-shape (white) showing two (numbered in red) connecting nephrons (yellow). (f) Graph showing the temporal accumulation of niche number (Niche no.), total cap mesenchyme cell number (CM pop) and total nephrons per organ (Nephron no.), presented as a percentage of maximum, across the development of the mouse kidney. This is adapted from the data presented in Short et al. [34].

organ, explaining to some extent the final shape of the organ. Such comprehensive datasets allow quantitative global comparisons between strain, genotype and stage. Application of a graph discordance metric to such data can resolve in great detail subtle differences in ureteric tree patterning, as demonstrated in a comparison of normal and Tgfb+/ kidneys [36]. Of note, the ureteric branch patterning in this mutant is normal until late in development where branch elaboration fails, a defect unlikely to be evident in explant culture.

Understanding the relationship between branching, cap mesenchyme survival and nephron endowment Whole organ imaging has also been applied to the CM of the kidney [34]. OPT showed that broad domains of CM surround multiple initial branches but, as branching progresses, each tip becomes tightly surrounded by an

individual field of CM forming a tight CM/tip niche (Figure 3a). In the same study, high resolution confocal analysis was used to count individual cell numbers in tip and surrounding CM across time [34] (Figure 3b–d). The size of both tip and CM domains in each niche declines with time, but this reduction is more marked in the CM. An analysis of cell cycle length in both CM and UB tip populations, based upon relative EdU labelling across time, [37] showed a slowing of cell cycle in all compartments as development proceeded. By combining the global data on niche number, cells per niche and proliferation rate, a mathematical model of exit from the CM (differentiation into nephron) and exit from the tip (movement from tip to trunk) was constructed. The ureteric tree undergoes approximately 12 generations of branching in the mouse. While the rate of branching substantially slows after E15.5, there is a stable relationship of two nephrons per tip from 15.5 dpc until the end of Current Opinion in Genetics & Development 2015, 32:135–143

140 Developmental mechanisms, patterning and organogenesis

branching at birth (Figure 3e). Despite this, only approximately 50% of all nephrons are formed before branching ends (Figure 3f). Hence, while there is a clear relationship between branching and nephron number, there is considerable room for variation after branching ends. The regulation of nephrogenesis between birth and postnatal day 4, when the last RVs form, is not understood. Visualisation of this final stage of nephrogenesis has to date only involved reconstruction of serial sections [38]. However, expression profiling of the Six2+ population across the immediate postnatal period confirmed a dissociation between Six2 and self-renewal, with clear evidence of increased proliferation and differentiation of the CM despite persistent Six2 expression [39]. Hence, there is a last burst of nephron formation all around the remaining tip before the CM is lost. What triggers this shift in CM phenotype from selfrenewal to nephron formation? Does this mean it is possible to dissociate final nephron number from final tip number? Cell ablation studies in which approximately 40% of the CM was deleted from 12.5 dpc resulted in a 40% reduction in final nephron number and an overall reduction in cross-sectional area at birth [40]. However, the relative effect of this loss of CM on branching was not evaluated. The analysis of cell cycle length within the CM revealed the presence of two distinct populations cycling at different rates with a relative shift from fast to slow cycling cells across development [34]. CM heterogeneity is also implied by gene expression with a peripheral subdomain of the Six2+ CM also expressing Cited1 [41]. As Cited1-lineage tracing marks all cells of the nephron, it was assumed that the most self-renewing portion of the CM was the Cited1+ Six2+ domain with loss of Cited1 expression associating with commitment to nephrogenesis. Certainly Six2 protein intensity is highest in the more peripheral CM associated with a slow cell cycle [34]. However, the temporal depletion of the faster cycling population (lower Six2) would predict a situation where the CM remaining at birth was all selfrenewing uncommitted mesenchyme. This again suggests a requirement for an active trigger to initiate the final wave of nephron formation. Hence, the number of CM cells per tip at birth may be more important than final tip number in dictating final nephron complement. Evidence already exists for variation in nephron number between mouse strains, with a clear association between reduced nephron number and onset of chronic kidney disease in the inbred FVB/N strain [42] and oligosyndactyly (Os) [43] mice. The acquisition of quantitative whole organ datasets on these and other strains or genetic variants will ultimately facilitate mathematical modelling to predict how best to optimise final nephron number.

Relevance to the human kidney Research into kidney organogenesis in the mouse is hoped to assist in our understanding of human renal Current Opinion in Genetics & Development 2015, 32:135–143

disease. However, the mouse is an inaccurate model of the human and there has been little focus on understanding or reconciling these differences. Unlike the mouse, ureteric branching in the human ceases from gestational week 14–15 [44] (Figure 4). While the mouse is unipapillate, the human kidney is multipapillate, comprising 8–15 lobes [45], each containing a branching collecting duct tree that undergoes around 15 generations of branching [44] (Figure 4a,b). However, nephron induction next to individual ampullae, as seen in the mouse, accounts for only 10% of final nephron number, with the elaboration of additional nephrons (gestional week 16–36) occurring via arcade formation (multiple nephrons forming at the same tip and connecting to each other; approximately seven nephrons per arcade) and lateral nephron formation (attached to sides of an elongating duct; four to seven nephrons per stalk) [44,46] (Figure 4c). The equivalent period of nephron formation post completion of branching in the mouse occurs between birth and postnatal day 4, forms only 50% of all nephrons, accounts for approximately four to six nephrons per tip and continues only until the CM is lost [34,38] (Figure 4c). The continued existence of a CM in the human after trimester 1 is not established. In addition, this prolonged period of later nephron formation in the human results in a substantial shift in the overall prevalence of short versus long loops of Henle (1:3 in mouse versus 7:1 in human) [46] between these species. Evidence for long term effects on renal structure and hence function as a result of gestational insult (fetal programming) is well established in the mouse [47], with hypoxia [48], elevated glucocorticoids [47] and diabetes [49] all shown to reduce nephron number and alter renal function and blood pressure. Translation of these observations into the human setting is difficult to predict given the substantially different approach to kidney organogenesis seen between mouse and man. While lineage tracing, selective gene or cellular compartment ablation and advanced imaging, are not available in the human setting, the mouse has been predictive of most genes associated with congenital anomaly [50]. It is known, however, that nephron number in the human varies almost 10 fold between individuals (approximately 200 000 to 2 million/organ) [51] with evidence for variation based on age, race and gestational events such as birth weight. To date, this is inferred from stereological counting of nephrons number post death as no approaches have been developed for the in vivo quantification of nephron number in humans. Recently, infusion of cationic ferritin followed by MRI-based quantification of nephron number in live mice has been shown to agree with subsequent stereological quantification post collection of the same kidneys [52]. The applicability of such an approach to a patient will be dependant upon evidence that the imaging process itself does not damage subsequent renal function. However,

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Figure 4



C (b)








C=Cortex, OM=Outer Medulla (Inner & Oute stripes), IM=Inner Medulla (Papilla), P=Pelvis


HUMAN UB outgrowth (Week 5)


Nephron formation Branching

Gestation 38 - 42 weeks Trimester 1 Trimester 2 Trimester 3


Final nephrons initiated (postnatal day 4-6)

Birth UB outgrowth (10.5dpc) Nephron formation Branching

Gestation 19 days Current Opinion in Genetics & Development

Comparative timeline of kidney development between human and mouse. (a, b) Diagrams of the human (a) and mouse (b) kidney illustrating the anatomical differences. The human kidney consists of 8–15 lobes each with a branching ureteric tree and inner medullary (IM, papilla) while the mouse kidney is unipapillate. C, cortex; OM, outer medulla, IM, inner medulla; P, pelvis. (c) Comparative developmental timeline of human and mouse nephrogenesis identifying the duration of gestation, timing of initial UB outgrowth, period of ureteric branching and period of nephron formation. Note the prolonged period of nephron formation in the human after the end of branching in comparison to the mouse. Note also that final nephron formation in the mouse occurs in the immediate postnatal period.

were this feasible and safe, it would open up the prospect of evaluating total nephron number at birth in humans to detect individuals at risk in the absence of overt congential anomaly. This single advance would revolutionise the capacity to clinically intervene prior to a substantial loss of renal function, and herald an era of non-invasive evaluation of human kidney development of profound long term significance to the field.

NHMRC, Australian Research Council, Human Frontiers Science Program, Organovo Inc. and the National Institutes of Health (NIDDK), USA. I thank Dr. Alex Combes and Kylie Georgas for assistance with graphics and Associate Professor Jane Black for teaching me about human development.



MHL is an National Health and Medical Research Council (NHMRC) Senior Principal Research Fellow. Her research is supported by the

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest Vivante A, Kohl S, Hwang DY, Dworschak GC, Hildebrandt F: Single-gene causes of congenital anomalies of the kidney and urinary tract (CAKUT) in humans. Pediatr Nephrol 2014, 29:695-704. Current Opinion in Genetics & Development 2015, 32:135–143

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Little M, Georgas K, Pennisi D, Wilkinson L: Kidney development: two tales of tubulogenesis. Curr Top Dev Biol 2010, 90:193-229.


Chi X, Michos O, Shakya R, Riccio P, Enomoto H, Licht JD, Asai N, Takahashi M, Ohgami N, Kato M et al.: Ret-dependent cell rearrangements in the Wolffian duct epithelium initiate ureteric bud morphogenesis. Dev Cell 2009, 17:199-209.


Carroll TJ, Park JS, Hayashi S, Majumdar A, McMahon AP: Wnt9b plays a central role in the regulation of mesenchymal to epithelial transitions underlying organogenesis of the mammalian urogenital system. Dev Cell 2005, 9:283-292.


Grobstein C: Inductive epithelio-mesenchymal interaction in cultured organ rudiments of the mouse. Science 1953, 118:52-55.


Little MH, McMahon AP: Mammalian kidney development: principles, progress, and projections. Cold Spring Harb Perspect Biol 2012, 4.


Georgas K, Rumballe B, Valerius MT, Chiu HS, Thiagarajan RD, Lesieur E, Aronow BJ, Brunskill EW, Combes AN, Tang D, Taylor D et al.: Analysis of early nephron patterning reveals a role for distal RV proliferation in fusion to the ureteric tip via a cap mesenchyme-derived connecting segment. Dev Biol 2009, 332:273-286.



Brunskill EW, Aronow BJ, Georgas K, Rumballe B, Valerius MT, Aronow J, Kaimal V, Jegga AG, Yu J, Grimmond S et al.: Atlas of gene expression in the developing kidney at microanatomic resolution. Dev Cell 2008, 15:781-791. Harding SD, Armit C, Armstrong J, Brennan J, Cheng Y, Haggarty B, Houghton D, Lloyd-MacGilp S, Pi X, Roochun Y et al.: The GUDMAP database—an online resource for genitourinary research. Development 2011, 138:2845-2853.

10. Thiagarajan RD, Georgas KM, Rumballe BA, Lesieur E, Chiu HS, Taylor D, Tang DT, Grimmond SM, Little MH: Identification of anchor genes during kidney development defines ontological relationships, molecular subcompartments and regulatory pathways. PLoS ONE 2011, 6:e17286. 11. Brunskill EW, Sequeira-Lopez ML, Pentz ES, Lin E, Yu J, Aronow BJ, Potter SS, Gomez RA: Genes that confer the identity of the renin cell. J Am Soc Nephrol 2011, 22:2213-2225. 12. Brunskill EW, Georgas K, Rumballe B, Little MH, Potter SS: Defining the molecular character of the developing and adult kidney podocyte. PLoS ONE 2011, 6:e24640. 13. Brunskill EW, Park JS, Chung E, Chen F, Magella B, Potter SS:  Single cell dissection of early kidney development: multilineage priming. Development 2014, 141:3093-3101. This manuscript represents the first single cell expression profiling of cells from within the developing mouse kidney. 14. Thiagarajan RD, Cloonan N, Gardiner BB, Mercer TR, Kolle G, Nourbakhsh E, Wani S, Tang D, Krishnan K, Georgas KM et al.: Refining transcriptional programs in kidney development by integration of deep RNA-sequencing and array-based spatial profiling. BMC Genomics 2011, 12:441. 15. Yu J, Valerius MT, Duah M, Staser K, Hansard JK, Guo JJ,  McMahon J, Vaughan J, Faria D, Georgas K et al.: Identification of molecular compartments and genetic circuitry in the developing mammalian kidney. Development 2012, 139:1863-1873. This study catalogues the expression pattern of all transcription factors during kidney development then, based upon synexpression and a comparative analysis of transcription facto binding sites within evolutionarily conserved regions of minimal promoter, predicts targets of these transcription factors. 16. Karner CM, Das A, Ma Z, Self M, Chen C, Lum L, Oliver G, Carroll TJ: Canonical Wnt9b signaling balances progenitor cell expansion and differentiation during kidney development. Development 2011, 138:1247-1257. 17. Park JS, Ma W, O’Brien LL, Chung E, Guo JJ, Cheng JG,  Valerius MT, McMahon JA, Wong WH, McMahon AP: Six2 and Wnt regulate self-renewal and commitment of nephron progenitors through shared gene regulatory networks. Dev Cell 2012, 23:637-651. This study represents the first comprehensive analysis of transcription factor binding within a subcompartment of the developing mouse kidney. Current Opinion in Genetics & Development 2015, 32:135–143

Using ChIP for Six2 and beta-catenin, this identifies classes of regulated genes common to both transcriptional regulators but playing distinct roles in CM self-renewal versus differentiation. 18. Boyle S, Misfeldt A, Chandler KJ, Deal KK, Southard-Smith EM, Mortlock DP, Baldwin HS, de Caestecker M: Fate mapping using Cited1-CreERT2 mice demonstrates that the cap mesenchyme contains self-renewing progenitor cells and gives rise exclusively to nephronic epithelia. Dev Biol 2008, 313:234-245. 19. Kobayashi A, Valerius MT, Mugford JW, Carroll TJ, Self M, Oliver G, McMahon AP: Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development. Cell Stem Cell 2008, 3:169-181. 20. Humphreys BD, Valerius MT, Kobayashi A, Mugford JW, Soeung S, Duffield JS, McMahon AP, Bonventre JV: Intrinsic epithelial cells repair the kidney after injury. Cell Stem Cell 2008, 2:284-291. 21. Kusaba T, Lalli M, Kramann R, Kobayashi A, Humphreys BD:  Differentiated kidney epithelial cells repair injured proximal tubule. Proc Natl Acad Sci U S A 2014, 111:1527-1532. Addressing a major question within renal repair, this study shows that the cells contributing to tubular turnover post acute injury have a mature proximal tubule phenotype and hence are unlikely to represent a privileged stem cell population. 22. Kobayashi A, Mugford JW, Krautzberger AM, Naiman N, Liao J, McMahon AP: Identification of a multipotent self-renewing  stromal progenitor population during mammalian kidney organogenesis. Stem Cell Rep 2014, 3:650-662. In this study, lineage tracing based on the Foxd1 promoter established that the stroma, like the CM, is a self-renewing progenitor population but that it gives rise to the pericytic compartment of the kidney. It also validates the earlier relationship between tis Foxd1 mesenchyme and the Six2+ cap mesenchyme. 23. Humphreys BD, Lin SL, Kobayashi A, Hudson TE, Nowlin BT, Bonventre JV, Valerius MT, McMahon AP, Duffield JS: Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis. Am J Pathol 2010, 176:85-97. 24. Sequeira Lo´pez ML, Pentz ES, Nomasa T, Smithies O, Gomez RA: Renin cells are precursors for multiple cell types that switch to the renin phenotype when homeostasis is threatened. Dev Cell 2004, 6:719-728. 25. Pippin JW, Sparks MA, Glenn ST, Buitrago S, Coffman TM,  Duffield JS, Gross KW, Shankland SJ: Cells of renin lineage are progenitors of podocytes and parietal epithelial cells in experimental glomerular disease. Am J Pathol 2013, 183:542-557. A major shift in the field has been prior evidence that podocytes can turnover in response to injury with prior studies showing a transition between parietal epithelium and podocyte. This study shows that the stromal-derived, renin-expressing cells of the juxtaglomerular apparatus, a cell population not related to the podocyte, can also convert to podocyte in response to damage. 26. Barker N, Rookmaaker MB, Kujala P, Ng A, Leushacke M, Snippert H, van de Wetering M, Tan S, Van Es JH, Huch M et al.:  Lgr5(+ve) stem/progenitor cells contribute to nephron formation during kidney development. Cell Rep 2012, 2:540-552. While it is known that the cap mesenchyme gives rises to all segments of the final nephron, this study establishes, at least for the loop of Henle, that there are segment-specific progenitors. Here, clonal expansion of Lgr5+ cells within the early nephron was shown to form complete loops of Henle. 27. Rinkevich Y, Montoro DT, Contreras-Trujillo H, Harari-Steinberg O, Newman AM, Tsai JM, Lim X, Van-Amerongen R, Bowman A,  Januszyk M et al.: In vivo clonal analysis reveals lineagerestricted progenitor characteristics in mammalian kidney development, maintenance, and regeneration. Cell Rep 2014, 7:1270-1283. Supporting the observations seen in Barker et al. [26], this study suggests that each segment of the developing nephron contains progenitors capable of generating and repairing their own particular nephron segment. 28. Costantini F, Watanabe T, Lu B, Chi X, Srinivas S: Imaging kidney development. Cold Spring Harb Protoc 2011. pdb.top109.

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29. Packard A, Georgas K, Michos O, Riccio P, Cebrian C,  Combes AN, Ju A, Ferrer-Vaquer A, Hadjantonakis AK, Zong H, Little MH, Costantini F: Luminal mitosis drives epithelial cell dispersal within the branching ureteric bud. Dev Cell 2013, 27:319-330. This study, using high resolution imaging of developing kidneys (timelapse of explants in culture and confocal of fixed whole organs), reveals a unique mechanism of cell division in which the dividing cell extrudes from the epithelium into the lumen of the ureteric ampulla, undergoes cytokinesis, then shows reinsertion of mother and daughter cells at sites 1–3 cell diameters apart. This mitosis associated cell dispersal appears restricted to the branching tips and has never been previously described during normal development. 30. Kao RM, Vasilyev A, Miyawaki A, Drummond IA, McMahon AP: Invasion of distal nephron precursors associates with tubular interconnection during nephrogenesis. J Am Soc Nephrol 2012, 23:1682-1690. 31. Wilkinson L, Kurniawan ND, Phua YL, Nguyen MJ, Li J, Galloway GJ, Hashitani H, Lang RJ, Little MH: Association between congenital defects in papillary outgrowth and functional obstruction in Crim1 mutant mice. J Pathol 2012, 227:499-510. 32. Hackl MJ, Burford JL, Villanueva K, Lam L, Suszta´k K, Schermer B,  Benzing T, Peti-Peterdi J: Tracking the fate of glomerular epithelial cells in vivo using serial multiphoton imaging in new mouse models with fluorescent lineage tags. Nat Med 2013, 19:1661-1666. 33. Burford JL, Villanueva K, Lam L, Riquier-Brison A, Hackl MJ,  Pippin J, Shankland SJ, Peti-Peterdi J: Intravital imaging of podocyte calcium in glomerular injury and disease. J Clin Invest 2014, 124:2050-2058. These two studies push the resolution of intravital imaging in postnatal kidney to track cell fate across time and evaluate intracellular signalling in response to injury in the live animal. 34. Short KM, Combes AN, Lefevre J, Ju AL, Georgas KM,  Lamberton T, Cairncross O, Rumballe BA, McMahon AP, Hamilton NA, Smyth IM, Little MH: Global quantification of tissue dynamics in the developing mouse kidney. Dev Cell 2014, 29:188-202. This is the first global quantitative evaluation of the normal development of a mammalian organ. Applied to the developing kidney in the mouse, it catalogues imaging at the level of organ, tissue compartment and individual cell, generating a complete temporospatial dataset amenable to mathematical analysis that will underpin mathematical modelling of organogenesis. The capacity for this approach to identify subtle phenotypes provides an opportunity to reevaluate the effect of both genetics and environment on development. 35. Short K, Hodson M, Smyth I: Spatial mapping and quantification of developmental branching morphogenesis. Development 2013, 140:471-478. 36. Lamberton TO, Lefevre J, Short KM, Smyth IM, Hamilton NA:  Comparing and distinguishing the structure of biological branching. J Theor Biol 2014. pii: S0022-5193(14)00590-6. In this study, an approach to the comparison of complex branching structures is presented. This reveals an underlying stereotypy within the developing kidney previously hard to identify due to the fact that branching is dichotomous and asynchronous. 37. Lefevre J, Marshall DJ, Combes AN, Ju AL, Little MH, Hamilton NA: Modelling cell turnover in a complex tissue during development. J Theor Biol 2013, 338:66-79. 38. Rumballe BA, Georgas KM, Combes AN, Ju AL, Gilbert T, Little MH: Nephron formation adopts a novel spatial topology at cessation of nephrogenesis. Dev Biol 2011, 360:110-122. 39. Brunskill EW, Lai HL, Jamison DC, Potter SS, Patterson LT: Microarrays and RNA-Seq identify molecular mechanisms driving the end of nephron production. BMC Dev Biol 2011, 11:15. 40. Cebrian C, Asai N, D’Agati V, Costantini F: The number of fetal  nephron progenitor cells limits ureteric branching and adult nephron endowment. Cell Rep 2014, 7:127-137. In the first study of its type, Cebrian et al. investigates the effect of reducing CM size on overall kidney development. Using the Gdnf promoter to direct diphtheria-toxin mediated cell ablation, the ablation of 40% of the CM resulted in a 40% reduction in final nephron number.

41. Mugford JW, Yu J, Kobayashi A, McMahon AP: High-resolution gene expression analysis of the developing mouse kidney defines novel cellular compartments within the nephron progenitor population. Dev Biol 2009, 333:312-323. 42. Laouari D, Burtin M, Phelep A, Bienaime F, Noel LH, Lee DC,  Legendre C, Friedlander G, Pontoglio M, Terzi F: A transcriptional network underlies susceptibility to kidney disease progression. EMBO Mol Med 2012, 4:825-839. While focussed on identifying the genetic basis of the susceptibility of FVB./N mice to kidney disease, this study clearly illustrates the link between reduced nephron number and renal disease. This should prompt more careful evaluation of the basis of strain variation in renal function/ development. 43. El-Meanawy A, Schelling JR, Iyengar SK, Hayden P, Barathan S, Goddard K, Pozuelo F, Elashi E, Nair V, Kretzler M, Sedor JR: Identification of nephropathy candidate genes by comparing sclerosis-prone and sclerosis-resistant mouse strain kidney transcriptomes. BMC Nephrol 2012, 13:61. 44. Osathanondh V, Potter EL: Development of human kidney as shown by microdissection. Arch Pathol 1963, 76:290-302. 45. Treuting PM, Kowalewska J: Urinary system. In Comparative Anatomy and Histology: A Mouse and Human Atlas. Edited by Treuting PM, Dintzis S. San Diego: Academic Press; 2011:229-251. 46. Oliver J: Nephrons and Kidneys: A Quantitative Study of Developmental and Evolutionary Mammalian Renal Architectonics. New York: Hoeber Medical Division, Harper & Row; 1968. 47. Dorey ES, Pantaleon M, Weir KA, Moritz KM: Adverse prenatal  environment and kidney development: implications for programing of adult disease. Reproduction 2014, 147:R189-R198. This review represents a comprehensive overview of the evidence that gestational environment can affect postnatal renal function, presumably by altering the developmental program. 48. Wilkinson L, Neal C, Singh RR, Sparrow D, Kurniawan N, Ju A,  Grieve SM, Dunwoodie S, Moritz K, Little MH: Renal developmental defects resulting from in utero hypoxia are associated with suppression of ureteric beta-catenin signalling. Kidney Int 2015 [Epub ahead of print], PMID: 25587709. Describing the first analysis of the molecular basis of an adverse gestational environment on kidney development, this study also highlights the likelihood that the damage caused will vary with the timing, duration and severity of the insult. It also shows that normal development occurs at low oxygen tension (physiological hypoxia) and that further reductions in oxygen tension cannot be compensated for by induction of HIF proteins. 49. Hokke SN, Armitage JA, Puelles VG, Short KM, Jones L, Smyth IM,  Bertram JF, Cullen-McEwen LA: Altered ureteric branching morphogenesis and nephron endowment in offspring of diabetic and insulin-treated pregnancy. PLoS ONE 2013, 8:e58243. Applying more recent approaches to quantitative morphometrics, this study illustrates the effect of diabetes on kidney development. 50. Hwang DY, Dworschak GC, Kohl S, Saisawat P, Vivante A,  Hilger AC, Reutter HM, Soliman NA, Bogdanovic R, Kehinde EO et al.: Mutations in 12 known dominant disease-causing genes clarify many congenital anomalies of the kidney and urinary tract. Kidney Int 2014, 85:1429-1433. This large analysis of mutations in humans with congenital anomalies of the kidney and urinary tract (CAKUT) confirms the association between 12 genes known to affect kidney development and the mouse but suggests that mutations in other genes previously linked in humans to CAKUT are unlikely to be the causative events. 51. Puelles VG, Hoy WE, Hughson MD, Diouf B, Douglas-Denton RN, Bertram JF: Glomerular number and size variability and risk for kidney disease. Curr Opin Nephrol Hypertens 2011, 20:7-15. 52. Beeman SC, Cullen-McEwen LA, Puelles VG, Zhang M, Wu T,  Baldelomar EJ, Dowling J, Charlton JR, Forbes MS, Ng A et al.: MRIbased glomerular morphology and pathology in whole human kidneys. Am J Physiol Renal Physiol 2014, 306:F1381-F1390. The quantification of total nephron number using a non-invasive imaging modality would revolutionalise our understanding of disease in the mouse and the human. This study illustrates agreement between nephron counting performed using the current gold standard, stereological analyses of post-mortem tissue, and MRI imaging after infusion of ferritin. Current Opinion in Genetics & Development 2015, 32:135–143