During development and before birth, many organs develop from branched tubes. Whether forming the airways of the lungs, the collecting ducts of the kidneys or the milk ducts of the breast, there are many similarities between these structures. Given their shared tree-like structures, one possibility is that these tissues all form through the same general process.

A key challenge is understanding why branched networks develop and pattern in such a way as to assume their functional roles in the adult organ. A unifying theory, which proposes that certain tips stop growing in a random manner, has been proposed to solve this problem. In this theory, the branched mammary gland structures stop growing when the tips of the structure impinge on neighbouring branches. In the kidney, this cessation has been proposed to occur when nephrons – the structures that filter urine from blood – form near the end of the collecting ducts.

By growing kidneys in the laboratory and studying developing kidneys in mice, Short et al. investigated whether nephrons do affect collecting duct growth and branch development. The results of these experiments instead suggest that nephron formation has no effect on duct growth or branching. The nephrons also do not appear to affect how quickly the duct cells grow and divide. Moreover, there is no evidence that the cell proliferation in individual branch tips ceases randomly by any other mechanism.

Overall, the experiments Short et al. performed suggest that a unifying theory of branching in developing organs may not hold true, at least not in the way that has been envisioned previously.

Branching morphogenesis is an integral feature of the development of many organs including the kidneys, lungs and mammary gland. Branching typically begins with the formation of a bud like organ anlage which then proceeds to grow and divide, usually by bifurcation. The resulting network of interconnected epithelial tubules serves to break up the larger tissue masses in which they form, facilitating the exchange of oxygen (lungs) and nutrients (vasculature) and the transport of waste (kidneys) or secretions (glands). In some organs like the kidneys and lungs, the process of branching proceeds as a result of the orchestrated interaction of cells at the tips of the branching epithelium and specialized surrounding mesenchymal cells. In others, like the mammary gland, epithelial growth and arborisation occurs through collective cell migration in the absence of specific associated mesenchyma. In the kidneys, the ureteric bud (UB) elaborates (from embryonic day (E)11.5 in mice) within a field of specified cells known as the metanephric mesenchyme, which coalesces to form cap mesenchyme (CM) niches closely associated with each of the UB tips. These CM cells are nephron progenitors (Kobayashi et al., 2008), sub-populations of which sequentially commit to form nephrons as development progresses. They do so by first forming pre-tubular aggregates which undergo mesenchymal to epithelial transition to form renal vesicles (RV) that differentiate into comma- and S-shaped bodies that elongate and mature to form nephrons. Because the network established by the branching UB will ultimately form the urine collecting system, the nascent nephrons reconnect to the tip from which they were generated at the comma stage and establish a patent lumen through which urine can be transported (Kao et al., 2012; Georgas et al., 2009).

The pervasive role of branching morphogenesis in shaping the development of many organs poses the question as to whether it is governed by shared mechanisms or features. A recent paper by Hannezo et al. has proposed a ‘unifying theory of branching morphogenesis’ (Hannezo et al., 2017). This work, based principally on studies in the mammary gland, posits that the elongating tips of the branching mammary tree randomly explore their environment but cease branching and proliferation when in proximity to neighboring branches. It further proposes that developmental morphogenesis in many organs occurs by employing similar stochastic, self-organized approaches characterized by branching and annihilating random walks (BARW). In the kidney, the cessation of branching explicit in the BARW model was proposed to derive from the differentiation of nephrons at individual UB tips. We have investigated a central tenet of this theory - the premise that nephron formation triggers cessation of branching. In doing so, we have examined the broader impact of nephron formation and integration on the behavior of both UB tip and nephron progenitor cell niches. The relationship between these populations is critically important for establishing nephron endowment in the adult kidney; a metric increasingly thought to influence susceptibility to kidney disease and broader physiological measures such as blood pressure (Luyckx et al., 2013). We find no evidence that nephron formation can influence the extent of ureteric branching in the developing kidney nor does it alter tip or cap cell proliferation. This indicates that stochastic cessation of branching does not operate during kidney development and demonstrates that nephrogenesis has little to no impact on the progress of branching morphogenesis or the behavior of tip or CM progenitor cells in the developing organ.

We have previously profiled the size and cell behavior of the CM and UB tip niches in the developing kidney and correlated this with the rate of branching in the organ (Short et al., 2013; Short et al., 2014). This study showed that branching is most rapid in the period between E12.5 and E17.5 but did not examine, in detail, the relationship between nephron formation and branching itself. To do so, we used high resolution confocal microscopy to profile the number of connected nephrons per tip from E12.5 through to the cessation of nephron formation at postnatal day 3 (PN3). One prediction of the annihilation model in which nephron formation triggers cessation of branching (Hannezo et al., 2017), is that nephron formation should not precede the branching process (which the model predicts nephron formation will halt). Analysis of our confocal data (Figure 1A) indicates that almost all the tips of the branching ureteric tree carry an attached nephron 72 hr after branching initiates (87% at E14.5) and that further nephrons are added as development (and branching) progresses. We next utilized organ explant cultures to provide supporting evidence that ongoing branching of tips with associated nephrons was indeed possible. Developing kidneys grown in culture lose the 3D structure of an in vivo organ, but retain local CM-UB interactions including branching morphogenesis and nephron induction (Lindström et al., 2015; Combes et al., 2016; Watanabe and Costantini, 2004). Using a Hoxb7-GFP transgene to visualise the ureteric epithelium and bright field illumination to identify forming nephrons across time, we observed tips with attached nephrons undergoing bifurcation (Figure 1B, Figure 1—figure supplement 1).

Figure 1 with 1 supplement see all

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Figure 1—source data 1

Embryos were dissected at embryonic ages between E12.5 and E22.5 (the first column, embryonic day).

Confocal microscopy and Imaris software were used to view kidneys stained with anti-cytokeratin antibodies and count the number of nephron connections for multiple tips from multiple kidneys. The counts were binned into two groups; a single connection and more than one connection (the first column, nephron.connections). The proportion of tips counted with the number of nephron connections listed is in the third column.

https://doi.org/10.7554/eLife.38992.005

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Figure 1—source data 2

Embryos were dissected at embryonic ages between E11.5 and E21.5 (the Embryonic Day column), and staged using limb staging (the Stage Column).

Each row represents an analysis of a single kidney from an individual embryo. The kidneys were whole mount stained and analysed using Optical Projection Tomography and Tree Surveyor software, or confocal microscopy and Imaris software, as described in the methods. For stages 7 to 13, Tree Surveyor was used to reconstruct the ureteric trees, and provide a count of the tip number for each ureteric tree analysed. For stages 12 to 15, confocal microscopy was paired with Imaris analysis. Tips were identified using the property that a single cluster of cap mesenchyme cells associates with a single ureteric tree tip. In this counting method, clusters of cap mesenchyme were manually counted in Imaris to provide an estimate of tip number.

https://doi.org/10.7554/eLife.38992.006

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All of the UB tips in the developing kidney are localized to the surface of the organ, where they remain throughout development (Short et al., 2013; Short et al., 2014; Short et al., 2010). Notwithstanding the results derived from organ culture, this observation raises the formal possibility that nephron formation might still impede the branching of tips - but not their growth. In this scenario, for any given tip in which branching (but not growth) had ceased, it would be necessary for the branch to lengthen over time in order to maintain its position on the organ surface. If extension of non-branching, nephron-associated tips were continuing we would expect that the maximum tip length would grow with the size of the kidney. Since only a small proportion of tips at any given stage might cease branching, we used the maximum tip length per organ to assess this possibility (rather than the mean tip length, which might mask any such effects). Using this approach, no evidence of maximum tip length extension was observed relative to the increase in axial radius of the fetal organ with age (Figure 2A, p=0.95). Furthermore, if branching were to cease in a subpopulation of developing tips, their generation should remain static at subsequent stages. We therefore examined the distribution of tip branch generations (the number of branch segments between the root of the tree and each of the tips) but found no evidence of static branch generations. Instead, the distributions indicate continued branching of all tips (Figure 2B).

Figure 2

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Figure 2—source data 1

A single kidney from an E15.5 embryo was dissected, whole mount stained and analysed using light sheet microscopy and Imaris software, as described in the methods.

In Imaris, proliferative tips were identified by EdU staining, the number of nephrons counted by E-cadherin labelling and the branch generation determined by observing Trop2 stained ureteric tree branches back to the ureter. Fifty tips were assessed (tip number in column 1), and the number of associated nephrons (column 2) and branch generations were counted (column 3).

https://doi.org/10.7554/eLife.38992.008

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Figure 2—source data 2

Statistical analyses for Figures 2C and 3A, and 3B.

https://doi.org/10.7554/eLife.38992.009

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Simple measurements of nephron number per UB tip do not address how the presence of nephrons might have affected the historical branching behaviour of an individual tip. To better assess the relationship between the nephron ‘phenotype’ of a given tip and its branch history, we employed light sheet microscopy to image whole kidneys at E15.5 and then related the number of connected nephrons attached to a given tip with the number of bifurcations between that tip and the pelvis of the developing kidney (Video 1). Assessment of branch history of 50 tips in the kidney at this age found no evidence to support a relationship between the two measures (Figure 2D, Pearson’s r = 0.014, p=0.9210). Moreover co-labelling for EdU labelled proliferating cells found that high levels of cell division are apparent in tips with connected nephrons (Video 2.)

Video 1

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Video 2

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Taken together, these results provide no support for a ‘unifying theory’ in which nephron formation is responsible for the stochastic cessation of ureteric branching. We then examined the broader effects that nephron formation had on cell behaviors associated with nephron formation. In the mammary gland, proximity-associated annihilation of branching has been associated with a profound decrease in cell proliferation (Scheele et al., 2017). To examine whether stochastic or more subtle differences in tip cell division might be driven by nephron formation, proliferating cells in embryos at E13.5 and E17.5 and pups at P2 were labelled with a pulse of the nucleotide analogue EdU and kidneys collected and stained for subsequent confocal imaging of nephrogenic niches. This analysis allowed us to directly relate cell proliferation with the number of connected and associated nephron structures. We found that cell division in the tip cell niches decreases as development progresses (Figure 3A; p<1e⁻⁵, one-way ANOVA between any two stages), which is consistent with our previously reported studies (Short et al., 2014). However, we found little association between cell division and the number of total or connected nephron structures (at E13.5, p=0.0587/0.79 (connected/total nephron structures), n = 49; E17.5, p=0.155/0.0296, n = 50; P2, p=0.06687/0.4772, n = 50, one-way ANOVA). The only exception was at E17.5 where there was a weak but significant association between total associated structures, but this was not significant when examining connected nephrons. We then extended this analysis to profile the same measures in the nephron progenitors associated with each tip (Figure 3B) to determine whether the differentiation of nephrons might instead impact on the behavior of this cell niche. We found a similar (and previously described [Short et al., 2014]) time-dependent decrease in the rate of proliferation (p<1e⁻⁵, one way ANOVA), which is consistent with recent work detailing the progressive ageing of these cells and the accumulation of intrinsic characteristics, which affect their contribution to nephrogenic programs (Chen et al., 2015). However, we again found little relationship between the number of nephrons derived from and/or associated with a given cap cell niche and the rate of cell proliferation in that niche (E13.5, p=0.01827/0.3654 (connected/total nephron structures), n = 49; E17.5, p=0.3435/0.1503, n = 50; P2, p=0.06605/0.921, n = 50, one-way ANOVA). Together these analyses provide no compelling evidence for an association between nephrogenesis and tip/cap niche proliferation together at any one stage of development.

Figure 3

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Figure 3—source data 1

Kidneys samples (column 1) were dissected from embryos at embryonic ages (column 2), and stained for Edu, Six2, Trop2 and DAPI.

The kidneys were imaged using confocal microscopy and analysed using Imaris software. Multiple tips were assessed for each kidney (column 3). The number of attached nephron segments that are continuous with the ureteric tip (column 4) and the total number of nephric structures (both attached and non-attached, column 5) were counted. The same tip was assessed for tip cell number (column 6), Edu positive proliferating cells (column 7), the ratio of proliferative cells to total cell number (column 8). The clustered cap mesenchyme cells associated with the tip were also analysed for cap cell number (column 9), EdU positive proliferating cells (column 10), the ratio of proliferative cells to total cell number (column 11). The values for each tip.

https://doi.org/10.7554/eLife.38992.013

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In this study, we have examined whether nephron formation may contribute to a process of stochastic cessation proposed as a unifying theory of branching morphogenesis. Multiple lines of evidence suggest that this is not the case. Integrated nephrons are a feature of almost all tips in the actively branching kidney and live imaging of cultured organs highlights active branching in the presence of attached nephrons. Moreover, hallmarks of ceased branching are not evident in the analysis of terminal tip lengths, the progressive distribution of branch generations over time, rates of cell division or in the correlation between nephron number and branching history of a given tip. In particular, we have found little evidence for a relationship between nephron number and cell proliferation in either the tip cell niches (where the nephrons integrate) or in the nephron progenitor cells of the cap mesenchyme from which they derive. The latter observation suggests that nephron progenitors are fated to differentiate based on an internal ‘clock like’ mechanism which is consistent with recent studies (Chen et al., 2015). It remains to determine the manner in which individual nephrons remain associated with their tips of origin. Based on our findings, branching morphogenesis during kidney development is a process which differs considerably from the mammary gland. Although they develop in a superficially similar manner (i.e. through arborisation of an epithelium anlage) the cellular events which direct these processes and which shape branching are likely significantly different and not dictated by a ‘unifying’ developmental mechanism. Instead, we favour a model in which renal branching more closely mirrors that observed in the lung, another organ whose development is driven by reciprocal epithelial-mesenchymal interactions (Morrisey and Hogan, 2010).

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Kieran M Short

   Department of Anatomy and Developmental Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

2. Alexander N Combes

   1. Murdoch Children's Research Institute, Parkville, Australia
   2. Department of Anatomy and Neuroscience, School of Biomedical Sciences, University of Melbourne, Parkville, Australia

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

3. Valerie Lisnyak

   Department of Anatomy and Developmental Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia

   Contribution

   Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared

4. James G Lefevre

   Division of Genomics of Development and Disease, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

5. Lynelle K Jones

   Department of Anatomy and Developmental Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

6. Melissa H Little

   1. Murdoch Children's Research Institute, Parkville, Australia
   2. Department of Anatomy and Neuroscience, School of Biomedical Sciences, University of Melbourne, Parkville, Australia
   3. Department of Pediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Australia

   Contribution

   Conceptualization, Data curation, Supervision, Funding acquisition, Writing—review and editing

   Competing interests

   has consulted for and received research funding from Organovo Inc.

7. Nicholas A Hamilton

   Division of Genomics of Development and Disease, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

8. Ian M Smyth

   1. Department of Anatomy and Developmental Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia
   2. Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Australia

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   ian.smyth@monash.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1727-7829

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