A question of identity

The mammalian Pax genes are homologues of the Drosophila gene paired, and are a family of transcription factors harbouring a highly conserved DNA-binding paired-box domain. The Pax genes have been found to be involved in organizing numerous aspects of development, including development of the central nervous system and diverse organs such as the pancreas and the kidneys. Pax2 belongs to this family and has for a long time been known to be important in mammalian renal development (as well as other tissues).  It is expressed in the pro- and mesonephros as well as the earliest condensing metanephric mesenchyme, and thereafter in the cap mesenchyme, renal vesicles, developing nephrons and ureteric bud. The essential requirement for Pax2 in kidney development is underlined by renal agenesis in Pax2 null mice, and by human mutations that cause pathology including renal malformations. Although known to be involved in normal renal development, the function of Pax2 in different renal cell types has not been established.

Two recent papers, one in mouse and one in human iPSC-derived organoids, now begin to shed light on the function of Pax2 in the metanephric mesenchyme.

A loss of function mutation for Pax2 leads to renal agenesis, making it impossible to dissect the contribution of Pax2 to the specification of different renal cell types. To solve this, Naiman et al created a floxed conditional compound heterozygote, with a non-functional truncated second allele. By crossing to a Six2-GFP-Cre line, Pax2 was conditionally removed only in the Six2-positive population that marks a subset of the metanephric mesenchyme – the cap mesenchyme. In this system, the kidneys were severely hypoplastic and only developed a small ureteric bud tree, underlining the importance of Pax2. Perhaps surprisingly, however, the cap mesenchyme did initially form, and CM cells persisted for 2 days. It could be that Pax2 is not directly required for specifying this subset of metanephric mesenchymal cells. However, deleting Pax2 in cells that are expressing Six2 means that the nephron progenitor cells have already begun to be specified as a subset of the MM when Pax2 is removed (because Six2 is a marker of the cap mesenchyme). This leads to a kind of chicken and egg question and therefore this study does not yet answer whether or not Pax2 is required for specification of the cap mesenchymal cells.

Nevertheless, the persistence of the cap cells for some time before apparently disappearing begged the question what was happening to the cap mesenchyme cells when Pax2 was absent. The authors surmised that there were two possibilities: either they were dying, or they were changing their identity. Transdifferentiation is a process whereby cells that are already committed to a cell fate can be persuaded to take on the identity of a different cell type, without first being taken back to an earlier state. To test whether the Six2-positive cap mesenchyme cells were transdifferentiating or simply dying, Naiman et al used a LacZ-Cre reporter to lineage trace specifically the Pax2-deficient, Six2-expressing cells. What they found was that the cap mesenchymal cells in the Pax2 mutants were not simply dying: they were changing their identity towards renal interstitial cell fate. This is striking because previous work has shown that the lineage of Six2+ nephron progenitor population and the Foxd1+ stromal progenitor population are committed very soon after the onset of metanephric development. Furthermore, the authors show that in wild type metanephric mesenchyme, Pax2 and Foxd1 are usually not co-expressed. Yet, analysis of the expression pattern of the transdifferentiating cells showed that cells starting out as Six2+Foxd1-, are briefly Six2+Foxd1+ before settling into their new identities as stromal progenitors and gaining a Six2-Foxd1+ signature.

So what does this tell us about the role of Pax2 in renal development, at least in mice? Clearly, Pax2 is required for maintenance of the nephron progenitor population. Six2 is also required for maintenance of this population by repressing premature MET. Unlike Six2, however, Pax2 appears to maintain this population by repressing stromal identity. With Waddington’s epigenetic landscape for cell fate determination in mind, this means that removal of Pax2, even after the marble has rolled down the cap mesenchyme “trough” leads to cells reverting to a default identity of renal interstitium. It seems as though cap mesenchyme cells are desperate to leave their identity behind, but this is thwarted by Pax2 and Six2 as they block escape to a new identity, one stromal and one epithelial.

Whatever light has been shed by the Naiman paper, however, some questions remain about the role of Pax2 in the MM. Hot on the heels of the work by Naiman et al is another paper, demonstrating that PAX2 appears to be dispensable for nephron formation, and therefore nephron progenitor cell maintenance, in human iPS cells. Kaku et al have taken human iPS cells, knocked out PAX2 (replacing it with GFP), and used their protocol to differentiate to metanephric mesenchyme. This mesenchyme exhibited robust tubulogenesis when co-cultured with murine spinal cord, despite the absence of PAX2. Using cell type specific markers, the authors then show that this differentiated cell population is indeed metanephric mesenchyme and identify two distinct sub-populations within it: ITGa8+/PDGFRA-, and ITGa8-/PDGFRA-. PDGFRα is a cell surface receptor that is associated with renal interstitial cells and which has been identified previously by the authors as a negative selection marker for nephron progenitors. The ITGa8+/PDGFRA- population sorted from both wild type and PAX2-deleted hiPSCs-derived MM was shown to induce tubulogensis whilst the ITGa8- population did not. Proximo-distal polarity was not impaired, and glomerular structures were observed. The only phenotype they observed in their PAX2 knockout organoids was a morphological change in the parietal epithelial cells of the glomerulus.

On the one hand, Naiman et al show quite convincingly that Pax2 is essential for maintaining the cap mesenchyme population and repressing a shift towards a different identity. On the other hand, Kaku et al suggest that it might be dispensable for nephron progenitor maintenance in human cells in vitro. One possible explanation to reconcile these opposing findings is that there is a species difference. It would be interesting to see if Pax2 remains dispensable for nephron formation from mouse iPS or ES cell-derived MM. Another possibility raised by Kaku et al is that PAX8, which is thought to have some redundancy with PAX2 based on the more severe phenotype of the double Pax2/Pax8 knockout mice, might be compensating for the lack of PAX2. They show that PAX8 expression is expanded in the epithelia of their hiPSC-derived PAX2 null organoids. If this is the case then there must indeed be some species differences since PAX8 remained intact in the Naiman conditional knockout. And in humans with mutations in PAX2, the serious kidney hypoplasia seen does not support a strong compensatory role for PAX8.

Clearly, in vitro generation from iPS cells and in vivo development are very different developmental processes. Another possible explanation for the different outcomes upon removal of PAX2 is that an organoid derived from a single renal cell type is not equal to an organ developed in vivo. In the human iPSC PAX2 knockout, the protocol differentiates only MM without UB and spinal cord is used to induce tubulogenesis. Could there be a signal from the UB telling the MM to become stromal progenitors, with PAX2 repressing this signal? If so, then in the absence of UB, PAX2 would indeed be dispensable for nephron formation since the message to follow the stromal lineage would be non-existent. Or perhaps the protocol for nephron formation in vitro does not faithfully reproduce the situation in vivo; there may be compensatory mechanisms at play due to this difference and therefore extrapolation to human in vivo development should be approached cautiously.

What is clear is that the role of Pax2 in mammalian renal development in vivo is critical. Its role in maintaining the identity of the nephron progenitor population within the developing renal environment in mice is a fascinating developmental mechanism, without which the cells undergo a sort of identity crisis leading to failure of the kidney to develop. Many questions persist including possible species difference, the mechanism of repression of stromal identity by Pax2 in mice, and the role of Pax2 in the collecting duct. It will be interesting to see how the story of Pax2 unfolds and the differences between species and in vitro/in vivo development.

Melanie Lawrence

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More information on the 20th UK/EU nephrogenesis workshop

Registration for the nephrogenesis workshop on 22 June in The Roslin Institute, University of Edinburgh is still open. A preliminary program is now available. Registration and coffee will start at 9 pm, with the first talk at 9:30. The day is expected to end at 16:30 pm.

Submission of abstracts, from which the majority of talks will be selected, will be closed on 9 June 2017.  Two keynote talks will be given by Andreas Schedl and Barry Denholm.

For all other information about the workshop, please see this previous blog entry.

The easiest kidney research fundraising you’ve ever done…

Not directly linked to kidney development, but still pretty close… On 22 June the International Kidney Cancer Coalition is organising the World Kidney Cancer Q&A Day. As part of this, the Q&A day quiz is now online. Not only very informative but for every completed quiz $5 is donated to kidney cancer research. So go there now, spend 2 minutes on the quiz, and help kidney cancer fundraising.

 

Peter Hohenstein

Registration UK/EU nephrogenesis workshop now open

On 22 June 2017 the 20th UK/EU nephrogenesis workshop will be held at The Roslin Institute, University of Edinburgh. Registration is now open.

As always, we have tried to keep registration fees as low as possible. PhD students and technicians will get free registration, post-docs and PIs will have a registration fee of £30. Registrations include all tea, coffee breaks and lunch breaks. These low registrations costs have been made possible by generous support from Kidney Research UK,  Transnetyx and IDT Integrated DNA Technologies. Note that if you want to use the free registration option you will need to supply a letter from your supervisor or line manager to confirm your status. This letter needs to be uploaded at the time of registration.

Keynote speakers will be Andreas Schedl from the University of Nice in France and Barry Denholm, Centre for Integrative Physiology, University of Edinburgh. Other talks will be selected from submitted abstracts. Abstracts can also be uploaded at the moment of registration.

The Roslin Institute is on Easter Bush Campus, south of Edinburgh. It is served by bus routes 15, 37, X47 and 67 from Lothian Buses. Taxi costs from the city centre to the institute are approximately £20 and take 20-30 minutes. Finding accommodation in Edinburgh is not a problem, and hotels in all price categories are available.

 

Peter Hohenstein

Kidney reaggregation and improved kidney imaging in the special issue on organoids in Development

The journal Development published a special issue this week on organoids. Besides a wide variety of papers on organoid systems for different tissues, two papers are focusing on the developing kidney.

Since the demonstration by Unbekandt and Davies in 2010 that the classic Auerbach and Grobstein aggregation system could be adapted to only require cell types from the embryonic kidney itself, this aggregation method or derivatives from it have formed the basis of many seminal kidney organoid papers. However, the mechanism underlying this remarkable self-organization have remained unclear. Now Lefevre et al from the Little lab, using time-lapse and confocal imaging as well as mathematical modelling, study the dynamics of this reaggregation process.They use time-lapse imaging of reaggregated kidneys to study the kinetics of the spontaneous nephron initiation. Clusters of ureteric epithelium cells formed after 8 hours, which after 48 hours was followed by the formation of cap mesenchyme clusters around them. Mathematical modelling based on these time-lapse data suggested that at least in the first 24 hours differential adhesion between cells could account for the formation of the UE clusters. Finally, the authors hypothesise that homophilic cadherin interactions could explain this clustering, and using blocking antibodies they show that P-cadherin, but not E-cadherin, is likely involved in this clustering of UE cells in the first 24 hours. In all, this is a very interesting study on the mechanism behind these aggregation experiments.

A second kidney paper in this special issue is from the lab of Seppo Vainio. Saarela et al present a method to improve the confocal time-lapse imaging of kidney rudiments which they refer to as ‘fixed z-direction’ or ‘FiZD’ imaging.  They limit the growth of the embryonic kidney in the z-direction by growing the kidney under a porous membrane but on a glass slide, with the two separated by glass beads that determine the space the growing kidney can get:

f1-large

The system allows for very good development, including the formation of Loops of Henle, as had previously been observed in the Sebinger low-volume culture method. It would be interesting to see how these two different culture conditions allow this better development that the conventional method does not allow. The (confocal) imaging using the FiZD cultures is indeed superb and allows for computer-assisted cell segmentation and morphometric analysis. The FiZD system will no doubt find useful uses in the time-lapse and confocal imaging of developing kidneys.

Peter Hohenstein

Serum-free UB cell maintenance, proliferation and branching in vitro

From patterning of the vasculature and neuronal networks to the development of diverse organs such as the lungs, mammary glands, and kidneys, branching morphogenesis is a common phenomenon observed during animal development. Perturbations in normal branching caused by genetic or exogenous factors can lead to disease, or abnormalities in surrounding tissues. In the kidney, correct patterning of the collecting duct is central to renal function, allowing appropriate filtration and drainage into the bladder. In addition, the ureteric bud and tip cells that will become the mature collecting duct are essential for the development of the kidney’s main processing unit, the nephron. Thus, perturbations in the process of branching morphogenesis in the developing kidney can lead to numerous pathologies and so studying the mechanisms behind this process are of great value. A platform to study branching of ureteric bud cells in vitro is a useful way of dissecting some of these mechanisms, and may also be a key part of advancing renal regenerative medicine.

Previous work has shown that it is possible to coax ureteric bud tips in vitro to propagate and branch in the absence of metanephric mesenchyme (MM) or MM-conditioned media. This was in itself an important step forward, since proliferation and branching of the ureteric bud normally requires signaling from the MM. However, serum was still required in addition to growth factors and signaling factors that would normally be secreted by the mesenchymal population. This is a problem for studying the process of branching in vitro, since serum contains numerous known and unknown factors that can act as confounding variables.

In their recent Stem Cell Reports publication, Yuri et al. have used a systematic approach to reveal a defined set of factors for the maintenance, proliferation and branching of dissected UB tips as well as dispersed ureteric bud (UB) cells. Crucially, their culture method does not require serum, making it ideal for studying pathways involved in branching. The authors began by culturing UBs from e11.5 mouse kidneys in factors known to be important for UB cell maintenance – specifically, GDNF and FGF1. These two factors together, or FGF1 alone, allowed the UB cells to survive and proliferate but did not allow the survival of tip cells or allow branching – this was a problem since the tip cells represent the stem cells of the collecting duct. It has previously been shown buy Bridewater et al and  Marose et al that Wnt / β-catenin signaling is necessary for maintaining UB tip cell identity, so the authors hypothesized that addition of the GSK-B inhibitor CHIR99021 might encourage tip cell proliferation and maintenance. In fact, addition of all three factors (GDNF, FGF1 and CHIR) induced extensive branching and led to the enrichment of tip cells. Interestingly, direct inclusion of Wnts as a substitute for the WNT activator CHIR99021 did not recapitulate the effects, suggesting a more global pathway. To investigate this, the authors turned to a mouse knockout of an R-spondin protein receptor with abnormal UB branching. Rspondin (RSPO) proteins are known agonists of WNT-B-catenin signaling so they hypothesized that RSPO proteins may be able to activate WNT in their culture system similarly to CHIR99021.  In fact they found that substituting either RSPO1 or RSPO3 for CHIR99021 in the culture system preserved the maintenance of tip cell identity and ability to branch extensively.

Continuing their systematic approach to identifying the defined factors required for in vitro propagation of branching UBs, the authors then investigated the role or retinoic acid (RA). RA is known to be crucial to kidney development as shown by the Mendelsohn lab (Batourina et al and Rosselot et al) and indeed the authors found that culturing UBs in RA alone allowed the cells to survive – but not branch. Adding GDNF to the RA encouraged proliferation and maintenance of tip markers; this highlights the essential role of RA in maintaining tip cell identity and the interplay of RA and GDNF/Ret signaling in proliferation and branching. This is of potentially crucial importance for renal regenerative medicine; RA is mainly secreted by the stromal cells and so inclusion of this population of cells in any approaches to renal tissue engineering may be required.

In addition to defining serum-free factors that allow the proliferation and branching of UBs in vitro, the authors proceeded to identify a defined set of factors that would allow single (dispersed) UB cells to propagate. This would be a useful addition to the toolkit of renal biologists, allowing dispersed UB cells to be manipulated (e.g. transfected with plasmids) and then their proliferation and branching studied either alone or in the context of other cell types. Stunningly, the authors were able to combine the factors above – FGF1, GDNF, RA – to not only allow proliferation but also branching of single UB cells, and inclusion of CHIR99021 into the cocktail induced further branching in an additive manner. Just as impressively, the authors mixed UB branched structures formed from single, dispersed UB cells with metanephric mesenchyme, and showed that they were still able to induce the mesenchyme to form nephrons, demonstrating the potential power of this approach.

This work is an impressive step forward in the propagation of UB cells, opening up avenues for studies of branching morphogenesis and kidney development in a serum-free manner. It will be interesting to see if this approach will hold true in human cells differentiated from pluripotent stem cells – if so it could be a valuable addition to the field of renal regenerative medicine.

Melanie Lawrence

Direct reprogramming into tubule cells

Many regenerative medicine approaches are based on the reprogramming of somatic cells to iPS cells, followed by directed differentiation to the desired cell type. This is a powerful possibility, in the decade since the first publication on iPS cells the reprogramming has become pretty much routine, and the directed differentiation protocols are rapidly becoming more efficient, more specific and more diverse. Differentiation towards renal fates is no exception to this. However, this method of generating cells of a specific type is, of course, indirect, so will take longer and every step needed that does not have 100% efficiency (and what in stem cell biology is 100% efficient?) will lead to reduced overall efficiency. Moreover, going via a fully pluripotent state followed by directed differentiation will always run the risk of incomplete differentiation and teratoma or other types of cancer development. In this respect, it is also important to remember that incomplete in vivo reprogramming with the Yamanaka factors leads to tumours resembling Wilms’ tumours.

The alternative is to transdifferentiate cells directly from a somatic cell type to the desired type without a pluripotent intermediate.Several examples of this are now known. Previously, the lab of Melissa Little presented data on the transdifferentiation of HK2 cells, an adult human proximal tubule cell line, into CITED1-positive nephron progenitors. However, so far there have been no follow-up publications, and especially the demonstration of this on primary cells will be essential for therapeutic use. And obviously, for this additional routes to use nephron progenitors in a therapeutic setting are required.

Now, in the December issue of Nature Cell Biology, Kaminski et al identify a method to transdifferentiate mouse and human fibroblasts into kidney tubule cells, which they refer to as induced tubular renal epithelial cells, or iRECs. In a reprogramming factor discovery approach reminiscent of the original iPS publication they first use a bioinformatics approach to identify 55 candidate reprogramming factors, which using expression analysis in Xenopus and involvement in human congenital renal disease and mouse phenotypes was further reduced to 13 candidates. These 13 were tested in their capacity to active GFP expression from a Ksp/Cdh16-Cre driven reporter MEFs. Use of all 13 factors gave a low (0.1%) efficiency of reporter activation. Subsequent removal of factors identified four factors, Emx2Hnf1b, Hnf4a and Pax8 which combined were sufficient to increase efficiency to 0.6% after transduction and 11.2% after 31 days of culture. Further optimisation led to efficiencies of 23.8% after 5 weeks or  5.4% after one week, although the latter required co-expression of SV-largeT which might not be desirable for therapeutic purposes.  The iRECs showed different epithelial characteristics and their expression profile resembled that of different segments of primary tubules. Interestingly,  further removal of some of the factors resulted in more cells resembling more specific markers. Functionally, iRECs closely resemble tubule cells, they are polarised with correct expression of different polarity markers, they take up fluorescent albumin and they are sensitive to nephrotoxins. the cells can integrate into tubules of disaggregated embryonic kidneys, and they could form tubules in decellularized kidneys. Finally, as icing on the cake, the authors showed it is possible to make iRECs from postnatal (tail) mouse fibroblasts and human fibroblasts.

Ther data presented in this paper convincingly show that it is possible to directly reprogram fibroblasts into renal tubular cells. This gives important new opportunities for regenerative medicine and further illustrates the plasticity of fully differentiated cells when transfected with the right reprogramming factors.

Peter Hohenstein