Principles of human and mouse nephron development

Abstract

The mechanisms underlying kidney development in mice and humans is an area of intense study. Insights into kidney organogenesis have potential to guide our understanding of the origin of congenital anomalies, and enable the assembly of genetic diagnostic tools. A number of studies have delineated signaling nodes that regulate positional identities and cell-fates of nephron progenitor and precursor cells, whereas cross-species comparisons have markedly enhanced our understanding of conserved and divergent features of mammalian kidney organogenesis. New views into the complex cellular movements that occur as the proximal–distal axis is established, have challenged our understanding of nephron patterning, and provided important clues into the elaborate developmental context in which human kidney diseases can arise. Studies of kidney development in vivo have also facilitated efforts to recapitulate nephrogenesis in kidney organoids in vitro, by providing a detailed blueprint of signaling events, cell movements, and patterning mechanisms that are required for the formation of correctly patterned nephrons and maturation of physiologically functional apparatus that are responsible for maintaining human health.

INTRODUCTION

The kidney is an architecturally intricate organ system that is affected by a number of congenital anomalies. Approximately 3% of infants are born with kidney or genitourinary system abnormalities ¹. These developmental defects have been linked to a variety of genes (>200 genes thus far), many of which have unknown function within the kidney organogenic programme. The functional unit of the kidney is the nephron. These long structures maintain homeostatic processes by regulating blood composition, pressure, and the excretion of metabolic compounds and water. Human kidneys form around one million nephrons per kidney during development, whereas mice develop around 14,000 nephrons per kidney ², although these numbers vary considerably ^3,4. Importantly, low nephron endowment — which can result from premature birth, exposure to an adverse intrauterine environment or other causes of developmental anomalies — is a risk factor for kidney disease ^3,5–8. Approximately 9% of the world’s population are affected by chronic kidney disease (CKD); moreover, >15 million infants annually are born prematurely and are therefore at risk of developing CKD in later life ^9,10. The size of the challenge posed by the growing burden of CKD poses a considerable threat to health systems worldwide and requires the development of new therapeutic and diagnostic tools ⁸.

Nephrons form during embryonic and fetal development from three kidney structures: the pronephros, mesonephros, and metanephros. These developmental derivatives of the intermediate mesoderm develop sequentially from cell populations within the intermediate mesoderm — called the nephrogenic cord and the nephric duct — that are positioned along the anterior–posterior axis of the embryo¹¹. The metanephric kidney, which is the final and functional kidney to form in mammals, develops from embryonic and during the remainder of human fetal development ceasing shortly before birth; in the mouse, comparable developmental steps occur from embryonic day (E) 11.5 through to postnatal day 3–4 ¹². Differences also exist in the timing of anatomical changes to the nephrogenic niche between human and mouse, as would be expected given the differences in gestational length. For example, anatomical features of the second trimester human kidney display similarities to those of the postnatal mouse kidney, including reduced reduced progenitor cells per nephrogenic niche, fewer new branching events, and multiple nephrons within each niche ¹³. Once acquired, kidney function remains imperative to organismal life.

Our understanding of nephrogenesis has been aided by studies that have compared features of human and mouse nephrogenesis, and by advances in single-cell omics and high-resolution imaging technologies that have enabled higher resolution insights into the cellular diversity of the developing and mature nephron These studies complement findings from a wealth of genetic fate-mapping and gene perturbation experiments in animal models (Supplementary Table 1), to provide new views into the process of nephrogenesis and patterning. Detailed examination of nephrogenesis is critical for the development of novel therapeutic models and genetic diagnostic tools, and to pave the way for future regenerative therapies. Here, we describe our current understanding of nephron patterning and the processes by which nephron progenitors are recruited to the developing nephron.

Balancing renewal and differentiation

Nephrons are asynchronously and repeatedly generated from nephron progenitor cells (NPCs) — a self-renewing population of mesenchymal cells that exist during development — at the cortex of the growing kidney. In mammals, NPCs are depleted either shortly before or after birth, thereby ending nephrogenesis and finalizing nephron numbers ^2,12,14,15. To obtain a sufficient number of NPCs for nephron formation, NPCs balance their self-renewal and differentiation processes through the coordinated action of various signaling pathways, including those involving Gdnf, Wnt, Fgf, Bmp, Notch, Fat, Pi3k, and Mapk–Erk ^16–33 in concert with gene regulatory programmes driven by Six2, Pax2, Osr1, Eya1, Sall1, Wt1 and Ctnnb1–Tcf–Lef (Fig. 1a) ^34–42,43. Perturbations to these pathways can lead to premature NPC differentiation and loss of the NPC population, although removing specific components of these pathways can lead to different outcomes. For instance, the phenotypes caused by loss of Bmp7, or Fgf9 and Fgf20, are distinct from those caused by loss of Six2 ^18,34,44. Loss of Six2 results in a catastrophic loss of the ability of NPCs to maintain a progenitor-like state; accordingly, the NPCs differentiate en masse as they form at E11.5 in mice, giving rise to large quantities of nephrons fused together around the tips of the ureteric bud ³⁴. By contrast, Bmp7-null mice display a later onset phenotype after E14.4 ⁴⁵, resembling a moderate loss of NPCs and an inability to differentiate, whereas mice with mutations in Fgf9 and Fgf20 exhibit a dose-dependent loss of NPCs over time ¹⁸. Intriguingly the loss of single ligands can disrupt several potentially opposing aspects of NPC biology. Wnt9b and WNT9B are normally expressed in the collecting duct cells immediately outside the growing tip and collecting duct progenitors. Genetic deletion of Wnt9b results in a complex phenotype where the maintenance of the NPC state, cell cycling, and nephron induction are all perturbed, resulting in a severe and rapid loss in both NPC maintenance and differentiation ^27,33. When broken down into their basic components, the processes of NPC self-renewal and maintenance require control over several cell behaviors. Proliferation must be optimized to replenish the NPC population, avoid excess depletion and expansion and account for the proportion of cells that differentiate. In parallel, the state of multipotency must be sustained ⁴⁶. Disturbances to processes involved in the maintenance of progenitor numbers (achieved by deletion of Bmp7, Fgf9 or Fgf20, for example) are likely to result in milder phenotypes than disturbances that block the maintenance of the progenitor state (for example, caused by deletion of Six2) ^18,34,45. During normal kidney organogenesis, the number of NPCs surrounding each ureteric tip gradually reduce ², preceding cessation of nephron formation ^12,14,15. Elegant studies indicate that this reduction in NPC number results from aging of the nephron progenitor niche, whereby the NPCs modify their responsiveness to Wnt signals over time and eventually reach a tipping point where self-renewal processes are outweighed by signals that drive their differentiation ^15,47. Human nephrogenic niches (a single niche is here defined as a collecting duct tip and the surrounding NPCs and interstitial progenitor cells) also display a gradual reduction in nephron progenitor endowment, as evidenced by a vast reduction in the number of SIX1⁺ NPCs from ~1,500 per niche at week 11 of gestation to less than 400 by week 23 ¹³. Human nephrogenesis continues to around week 36 ¹⁴, which raises the question of how nephron progenitors are maintained until this time point and whether differences exist in the maintenance of late NPCs between humans and mice, in which nephrogenesis continues until around 3 days after birth. One distinct difference in human NPCs is the expression of SIX1, which is absent in mouse NPCs, which in part could generate a difference in the dynamics between human NPC maintenance and differentiation ⁴⁰. Another difference between human and mouse NPCs is the blurred lines between interstitial and nephrogenic progenitor mRNA signatures. A range of genes with functional significance in mouse interstitial progenitors, are expressed both in NPCs and interstial progenitors in humans ⁴⁸. Scrutiny of the molecular mechanisms that underpin the shift in responsiveness of NPCs to WNT late in the nephrogenic process and other potential differences between human and mouse may identify therapeutic approaches with which to boost nephrogenesis and nephron endowment.

Figure 1.

Nephron progenitor movements and a hierarchical ordering to the nephrogenic niche. a. Nephron progenitors positioned around collecting duct tips balance self-renewal and differentiation through complex gene regulatory networks and signaling pathways. Gene names are positioned to indicate the relative point within the niche where their function is best known. b. Mitosis-associated movements of nephron progenitor cells (NPCs). NPCs are normally positioned on the ureteric tip, to which they attach with a large foot-process. During mitosis the cells detach, mitose, disperse and reattach to the basal surface of the ureteric tip. adapted with permission from O’Brien et al., 2018 eLife ⁶¹. c. Cells within the nephrogenic niche are highly motile and undergo a variety of movements, including intra, inter, and ‘de-differentiation’.

Cellular movements in the nephrogenic niche

Fate-mapping experiments suggest that all cells in the mature nephron are derived from NPCs that express Six2 and Cited1 ^49,50. What is not yet known is whether the positions of NPCs within their niche biases their differentiation potential, thereby linking NPC position to cell fate decisions ^49,50. Findings from single-cell RNA sequencing (scRNA-seq) analyses of human and mouse NPCs suggest that the SIX2⁺ NPC pool comprises a diverse set of cell-states. However, computationally inferred differentiation trajectories of NPCs indicate that these progenitor states are not biased towards specific nephron cell fates ^51–55. Although scRNA-seq does not provide spatial information, these data are consistent with a model in which there are no spatial relationships between niche positions and final cell fates. This model would suggest that all NPCs are equally able to generate cell types along the proximal–distal axis of the nephron. However, analyses of mouse ^56,57 and human ^13,40,48,58 kidneys using in situ RNA hybridization and immunofluorescence are consistent with scRNA-seq and show that NPCs exist their progenitor state through a hierarchical order. Cited1 expression marks NPCs at the anatomical top of the niche whereas Six2 is expressed by NPCs from the anatomical top to the bottom of the niche ⁵⁹. This ordering of gene expression, with cells changing from a state characterized by Cited1⁺ Six2⁺ expression to one characterized by Cited1^low Six2⁺ expression coincides with changes in BMP signaling, with effects on self-renewal and differentiation ^20,59 as well as upregulation of genes indicating differentiation (Pax8, Lhx1, and Lef1)^59,60. Such a hierarchical order of NPC progression is attractive in its conceptual simplicity; however, this model is challenged by findings from studies performed using sophisticated genetic fate-mapping tools available in mice, which indicate that nephron progenitors are highly motile cells that can switch between states of differentiation and alter their positions within the niche. One instance of such cell movement occurs during cell division. NPCs are situated on the basal surface of collecting duct progenitors (CDPs) and, in a sequence of events that coordinates with cell divisions, they detach from the CDPs prior to and reattach after mitosis. The reattachment is dependent on Wnt11 expression in these CDPs ⁶¹ but the cycling itself is dependent on a range of other signaling such as the aforementioned Fgf9/20 and Wnt9b ligands (Fig. 1b). Surprisingly, this process of movement through cell division is mirrored by the epithelial CDPs. CDPs in the tips of the growing and branching collecting duct, divide and disperse from their original position by mitosis within the collecting duct lumen ⁶². A situation therefore occurs whereby progenitors from two lineages (ureteric/collecting duct and nephrogenic) extrude in opposite directions from their original positions and planes, undergo their respective cell divisions, before reinserting into their original plane. There is no evidence to suggest that these processes are synchronized, occur in anatomically matching/mirrored positions, or are co-regulated. However, it is nevertheless intriguing that two codependent progenitor populations (NPCs and CDPs require reciprocal signals) perform parallel ‘dispersions-by-mitosis’ events in opposite directions. Whether the physical movement of cells from their original position plays a role in the ability of cells to change from one expression-state to another is also unclear, but one would suspect it is at least a contributing factor.

Other examples of NPC movements also exist. Indeed, NPCs can migrate between ureteric tips by detaching from one tip, crossing the between-tip space — a region occupied by interstitial progenitors and endothelial precursors — to reattach at neighboring tips ⁶³. Again, the rationale for this movement is unclear. Lineage-tracing experiments have also shown that NPCs can move within the niche from positions where more differentiated cells (expressing differentiation markers) typically reside to areas of the niche in which self-renewing cells exist and resume expressing NPC markers ^63,64 (Fig. 1c). All these movements suggest that NPCs represent a highly dynamic and motile cell population. They also suggest that if the nephrogenic niche is hierarchically ordered as traditionally believed, that these states must be flexible — perhaps transient — and at least partially reversible given that the NPCs can so readily move between states and niche positions. Reconciling our understanding of the motility of NPCs with other factors, such as the effects of aging on the NPC niche, will be critical to understand how NPCs are maintained for the duration of development, and how they balance self-renewal with differentiation and motility. Of note, although NPCs in the human nephrogenic niche are arranged in a similar manner to those in the mouse, there is at present no direct data on cell motility associated with cell-cycling, tip-to-tip or intra-niche migrations in the human system. It is also unclear whether human NPCs age as they do in mice.

The gradual recruitment model

Comparative analyses of human and mouse nephron progenitor niches have provided a potential explanation for the motility of NPCs. A prominent feature of human nephrogenic niches is that NPCs situated on top of growing collecting duct tips are connected to nascent nephrons that are situated just below the collecting duct tips ^40,51,58. The NPC marker SIX2 persists briefly in cells located in the proximal renal vesicle. These cells have differentiated from their progenitor state and are structurally part of the forming nephron. ^12,40,57. In human nephrons, this expression of SIX2 is more pronounced than in mouse ^13,48,51,58, but even more conspicuous is the persistence and quantity of of SIX1 protein within the forming nephron ⁴⁰. The abundance of SIX1 protein decreases gradually in a distal-to-proximal direction through the nascent nephron ⁵¹ (Fig. 2), consistent with the downregulation of SIX1 as NPCs exit their niche. This observation initially led to the hypothesis that SIX1 might serve as a readout of when cells have existed their nephron progenitor state, and we now know that an order exists by which NPCs differentiate and that this order corresponds to the positioning of the cells along the distal-to-proximal axis of the nephron. This model, known as the gradual recruitment model, proposes that cells are first progressively aggregated into pretubular aggregates over a period of time and thereafter gradually incorporated into the forming and epithelializing nephron during the earliest stages of nephrogenesis. Cells that are added first generate the most distal segments. Cells added thereafter are incorporated in a distal-to-proximal direction ⁵¹.

Figure 2.

The gradual recruitment model of nephrogenesis. According to the gradual recruitment model, nephron progenitors are gradually added into the forming nephron. In the developing human kidney, expression of nephron progenitor markers, such as SIX1, are gradually lost whereas the abundance of the NOTCH ligand JAG1 on cell membranes, increases along a dynamic profile. a. In the pretubular aggregate, JAG1 is asymmetrically deposited in early recruits positioned adjacent to the ureteric epithelium. This deposition precedes the formation of a bona fide lumen evident in the renal vesicle b. Renal vesicles display strong JAG1 membrane labeling in the distal-most cells and weaker labeling in a band across the medial nephron. Nephron progenitor cells (NPCs) are gradually recruited to the proximal end of the renal vesicle. c. The late-stage renal vesicle and early Comma-shaped body nephron shows weaker JAG1 labeling in the distal-most cells and a strong JAG1 band across the middle of the nephron’s proximal-distal axis. JAG1 protein is absent in the proximal-most region. d. By the late Comma-shaped body stage to early S-shaped body stage, JAG1 labeling is strongest in the medial segment, where it coincides with the emergence of putative proximal precursors, and almost undetectable in distal domains. Adapted with permission from Lindström et al., 2021 Dev Cell ⁶⁷.

The existence of the cellular connection between the nephron progenitor niche and the forming nephron, into which the progenitors move, raises questions as to how this bridge of cells develops. Historic camera lucida drawings show that this connection exists in several species including the to mammals evolutionary distant chick ^65,66, suggesting conservation beyond the mammalian system. In mouse and human, this connection is observed as differentiating LEF1⁺, PAX8⁺ cells exit a CITED1⁺ NPC state and stream from the nephron progenitor niche to the nephron ⁵¹ (Fig. 2a–c). This cellular bridge ceases to exist by the mid-to-late comma-shaped body stage (Fig. 2d). Species-specific differences in developmental pace provide one explanation for the conspicuity of the bridge in human kidneys. Whereas mouse nephrogenesis is rapid and the progression from nephron progenitor to S-shaped body is approximately 24 hours, the equivalent human development is estimated to be 3–8 times slower ¹³. This slower pace lends temporal resolution to the gradual recruitment of NPCs. Consistent with the slower pace of each human nephron’s formation, early nephron anatomies are more readily annotated with precise anatomical stages and cells’ positions within nephrons are visually ordered in the human compared to the mouse ⁶⁷.

Time-lapse microscopy of differentiating NPCs ^21,63,64,68 has shown the movement of cells both towards and away from positions at which new nephrons are expected to form ^63,64, and movement of cells within the nephron progenitor-to-nephron bridge towards nascent nephrons ⁵¹. Fluorescent membrane-labelling studies show that cellular projections reminiscent of filopodia and lamellipodia extend towards the forming nephron ⁵¹. Live imaging studies confirmed that cells that are incorporated into early stages of pretubular aggregates and renal vesicles remain positioned distally, while those that arrive later bypass the already epithelializing nephron and integrate proximally ⁵¹. A combination of studies in human and mouse therefore provide a consistent model whereby the nephron proximal-distal axis is generated over time as a consequence of the gradual recruitment of NPCs (Fig. 2).

Developmental trajectories

Single-cell sequencing has provided insights into the transcriptional profiles across a range of differentiation time points in the developing nephrogenic, ureteric, interstitial, and vascular lineages, ^{51–54,67,69,70}. Such transcriptional profiles have been used to computationally model the developmental trajectories of NPCs as they differentiate into precursors within the early nephron. Of note, computational predictions of differentiation trajectories based on commonalities in transcriptional profiles ⁷¹, can be influenced by a number of factors, including the efficiency of mRNA transcript capture from the cells of interest and the extent to which the cells were dissociated and recovered in a tissue-representative manner, and a range of sample quality-metrics. However, the proposed developmental trajectories indicate that an initially common developmental trajectory leads to proximal and distal fates, whereas podocytes develop via a divergent path – possibly together with that of the parietal epithelium ^{51–54,67,69,70}. Single-cell chromatin accessibility analyses of developing nephrons are ⁶⁹ consistent with data from transcriptomic analyses. These data are further supported by live imaging studies of developing mouse nephrons and histology analyses of human and mouse nephrons, which show that podocytes and parietal cells are the last cells to be recruited into the nephron ⁵¹ (Fig. 2). These cells are incorporated into a nephron that already expresses genes coding for proteins with known roles in nephron patterning, as well as a host of ligands and receptors — that is, a different signal environment than that encountered by the first NPCs that clustering into the pretubular aggregates. Better understanding of the mechanisms by which diverging gene regulatory networks control whether differentiating NPCs generate components of the bona fide epithelial tubular domains of the nephron or cells in the renal corpuscle is important. Given that cell-cycling is an intrinsic component of self-renewal, it will also be important to determine whether and how proliferation contributes to nephron formation and patterning. The fact that nephrons can form and initiate early patterning in explant cultures treated with small-molecule inhibitors that block DNA synthesis (and therefore proliferation, indicates that proliferation is either not essential or that compensatory mechanisms exist ⁶⁸.

The distal nephron

The distal nephron comprises the loop of Henle, macula densa, distal convoluted tubule and nephron connecting tubule. These portions of the nephron function to concentrate urine through water and the reabsorption of ions ^72,73. The loop of Henle spans the cortical and medullary segments of the mammalian kidney. In the medulla, the loop of Henle has a vital role in generating the osmolality gradient required to drive water reabsorption in the descending limb of the loop of Henle. As the now-concentrated urine flows through the water-impermeable ascending loop of Henle, sodium chloride is regulated through apical ion channels ⁷⁴. Marking the transition between the loop of Henle and distal convoluted tubule is the macula densa. This sensory placode of cells detects sodium chloride and glucose metabolites in the filtrate coming from the loop of Henle. In response, it initiates a signaling cascade that is effected by the adjacent mesangium, which triggers tubuloglomerular feedback to regulate glomerular filtration rate and systemic blood pressure ^75–77.

Comprehensive analyses of the adult mouse kidney demonstrate remarkable cell heterogeneity even within segments of the distal nephron ^78,79. Cells in the descending and ascending limbs demonstrate unique gene signatures along the corticomedullary axis, raising the question of how positional identities in the nascent nephron contribute to patterning and maturation. As described below, available data suggest that the dose and duration of signaling factors are important for cell patterning in the early developing nephron.

Nephron aggregation and induction

The aggregation of nephron progenitors into pretubular aggregates coincides with a Wnt9b–β-catenin-mediated switch from a self-renewing to differentiating state (Fig. 3a). NPCs respond to ureteric epithelium-derived Wnt9b signals through β-catenin-mediated engagement and activation of enhancers adjacent to Wnt4, Pax8, and Lef1 – expression of these genes is activated in induced NPCs. β-catenin and Lef1 bind to a known Wnt4 enhancer to drive its expression ^{16,25,27,30,31,33,36,42}. Activation of Wnt4 expression is necessary for nephron epithelialization ⁸⁰ – which occurs via non-canonical signaling ^81,82. Lhx1 acting downstream of Wnt4, is required for further cell maturation, since Lhx1-mutant mice form renal vesicles that degenerate. Chimera analyses with Lhx1-mutant embryos injected into wildtype blastocysts demonstrated that Lhx1^−/− cells can contribute to early renal vesicle cells, but are absent in the epithelium of the developing distal nephron, further supporting the role of Lhx1 in distal maturation ⁸³. Pax8 expression is upregulated in NPCs treated with high doses of WNT agonist, CHIR99021, and alongside Pax2, is required for survival of nephron precursors and distal tubule formation ^35,42. The exact hierarchy of events downstream of β-catenin remains unclear. Fgf8-mutant mice form renal vesicles; however, they lack Wnt4 and Lhx1 expression and their renal vesicles do not progress beyond the S-shaped body stage, indicating a potential upstream role of this growth factor in nephron induction ⁸⁴. Further supporting an upstream role for Fgf8 in nephron induction, hypomorphs for Fgf8 display low levels of Wnt4 expression and partial nephrogenesis, but in both mild and severe hypomorphic conditions, tubule formation is significantly impaired, indicating that Fgf8 is needed for the survival of tubule precursors ⁸⁴.

Figure 3.

The gradual recruitment of cells into the nephron and formation of putative precursor domains. Colors indicate putative precursor-progeny relationships from Comma-shaped body nephrons through to adult nephrons as predicted by computational and anatomical — but not genetic fate-mapping — studies. a. The pretubular aggregate has a robust cellular connection with the nephron progenitor cell (NPC) population. WNT9B is thought to signal from the ureteric epithelium to NPCs and the nascent nephron, where cells are undergoing partial epithelialization. b. In the renal vesicle, recruitment of NPCs into the proximal renal vesicle is ongoing, and a separation of distal and medial domains is evident. β-catenin (β-cat) is active in a gradient. A slight proximal bend to the renal vesicle indicates where the glomerular cleft forms. MAFB — a transcription factor that regulates the development of podocytes — is first detected at this stage, as indicated. c. As the late-stage renal vesicle transitions to the Comma-shaped body nephron it elongates. Formation of the glomerular cleft is ongoing and a minor distal indentation emerges. Distal and medial cell-populations are separated, and the parietal epithelium exhibits slight flattening. Podocytes form, as evidenced by increased levels of MAFB. d. As the late Comma-shaped body transitions to the S-shaped body nephron, a distinct glomerular cleft and distal convolution is evident. Cell types evident at this stage include those of the emerging connecting tubule, the distal tubule and putative precursors of the loop of Henle, the medial segment, containing putative proximal tubule precursors, and parietal and podocyte precursors. e-f. The S-shaped body nephron continues to undergo elongation, morphogenesis, and separation of gene-expression profiles into distinct putative precursor populations. g. Proposed anatomy for the Capillary loop stage nephron and the association of the renal corpuscle with the distal segment of the tubule where the macula densa forms. h. mature juxtamedullary (left) and cortical (right) nephrons. Early developmental stages partially adapted with permission from Lindström et al., 2021 Dev Cell ⁶⁷.

Studies over the past decade have provided insights into the complex role of Notch signaling during early nephrogenesis; however, conflicting views remain. Expression of Notch gain-of-function mutants in NPCs — achieved by expression of its intracellular domain (NICD) — activates a mesenchymal-to-epithelial transition (MET) programme that can occur independent of Wnt4 signaling or activation ³². This finding suggests that Notch signaling is sufficient to replace Wnt activity as an inductive signal, at least in a model in which the NICD is strongly expressed. Consistent with the view that Notch is important in the initial epithelialization of NPCs, loss-of-function experiments in which Notch receptors were removed from Six2+ NPCs resulted in a failure of kidneys to form nephrons and led to reduced expression of the epithelial marker, E-cadherin ⁸⁵. These researchers also proposed that Notch activity is required to downregulate Six2 expression in nephron progenitors, allowing their exit from a progenitor state ⁸⁵. Indeed, removal of Six2 is sufficient to trigger epithelialization ³⁴; however, this process occurs within the normal framework of Wnt9b–β-catenin signaling. However, it remains to be determined whether downstream crosstalk exists between Notch and Wnt signaling pathways or whether the NICD can itself mediate nephrogenic MET at a gene expression level. Greater understanding of the roles of Notch, Wnt4 and Six2 signaling is therefore needed.

One point at which Notch and Wnt signaling pathways converge is through activation of the Notch ligand, Jagged1 (Jag1). In the hair follicle, for example, Jag1 acts as a target of the Wnt-effector, β-catenin, to induce active hair growth cycles and new hair follicle formation ^86,87. In both human and mouse kidneys, ⁵⁸ JAG1 first emerges in the distal segmentof the late pretubular aggregate (Fig 2); its requirement for full NPC epithelialization is evident from the severe reduction in nephron formation in Jag1 mutants ⁸⁸. The timing of JAG1 emergence — immediately preceding the detection of E-cadherin — fits with a role in epithelialization; however, how it might integrate with a Wnt4-driven MET is unclear. Wnt4 is thought to induce MET through calcium-driven NFAT signaling ^81,82, although what genes are targeted through this calcium–NFAT pathway during early nephrogenesis is also unclear. This uncertainty about the roles of Notch and Wnt signaling during early nephrogenesis shows that considerable effort will be required to disentangle the roles and potential interplay of these pathways.

Nephron distalization and patterning

As described earlier, the model of gradual recruitment suggests a mechanism whereby induced NPCs migrate from the nephrogenic niche into the pretubular aggregate and renal vesicle ⁵¹. The first recruits position themselves directly adjacent to the ureteric epithelium, whereas subsequent cells that arrive incorporate into the most proximal domain of the nascent nephron. Assuming this process establishes the proximal–distal axis, then one possibility is that the order of arrival positions the NPCs in distinct environments, which determines their fate. (Fig. 3a–c). The transcriptional activity of β-catenin in nascent nephrons displays a distal-to-proximal gradient; moreover, the positional identities of nephron precursor cells in the nephron are dependent on the appropriate level of β-catenin signaling ⁶⁸. Consistent with an important role for β-catenin in positional and cell-fate decisions, ectopic administration of Wnt3a to chick mesonephric tubules reorients their distal-to-proximal axes towards the Wnt source, with the most distal axis pointing towards the source of Wnt3a ⁸⁹. In the mammalian nephrogenic system, Wnt9b, which is expressed by the adjacent ureteric tip, might act to guide positional identities along the distal-proximal nephron axis ²⁷. In this scenario, the ureteric tip would serve as a signaling center from which Wnt9b acts as a morphogen, inducing strong β-catenin signaling in the earliest nephron progenitor recruits, with a decreasing gradient towards the medial region ^51,68. The earliest NPCs to arrive would be nearest to the ureteric bud and subjected to long and high Wnt9b exposure. Later arrivals would incorporate farther away from the Wnt9b source into an already forming and potentially auto-regulating nephron; self-organization in the nephron is evident from experiments in which nephrons spontaneously patterned following the induction of epithelialization in isolated metanephric mesenchyme ¹⁶. A role for Wnt9b in patterning ²⁷ is further supported by the finding that homozygous hypomorphic activity of Wnt9b in mouse embryos results in planar cell polarity defects; epithelial cells are elongated in a seemingly randomized direction, indicating a disrupted orientation of polarized cells ⁹⁰. β-catenin is also necessary for the maturation of both distal and — as described later —proximal tubules; perturbations in β-catenin signaling affecting the ability of NPCs to undergo differentiation and maturation ⁹¹. Genetic manipulation of β-catenin activity – either by its conditional deletion or stabilization of its encoding gene, Ctnnb1 – results in abnormal differentiation along the entire proximal–distal axis ⁹¹. Whether canonical Wnt ligands are able to affect long-range signals is, however, unclear. Work in the intestinal stem cell niche suggests that Wnt3 signals through a short-range gradient that requires direct cell contact ⁹². The premise that dose and duration of ligand exposure can induce diverse responses in recipient cells is also well illustrated by the example of spinal cord development, during which opposing morphogen gradients define gene expression domains along the dorsoventral axis of the spinal cord. The dose and duration of sonic hedgehog signaling determines the identities of the progenitor cells in the ventral neural tube, where each domain is defined by an emerging expression of mutually repressive transcription factors ⁹³. Signatures resembling these are also evident in the nephron, where transcription factors that are conserved between human and mouse emerge in a distal-to-proximal direction in the renal vesicle and form distinct gene signatures in proximal, medial and distal domains of the renal vesicle ^57,58,60,67. Computational models, based on in vivo anatomies and fluorescent stains, show that transcriptional boundaries emerging in the renal vesicle stage are less defined and become more so by the S-shaped body stages ⁶⁷. Although computational predictions of differentiation trajectories indicate that tubular proximal and distal fates share a common developmental trajectory, ^{51–54,67,69,70}, it is currently unclear whether both distal and proximal tubular precursors transition through the same transcriptional states, and if so, at which point in development they diverge.

Of the transcription factors that are expressed, Hnf1b is necessary for the development and survival of the medial and distal domain of the S-shaped body ⁹⁴. In humans, mutations in HNF1B cause renal cysts and in some cases, maturity-onset diabetes of the young, a rare form of genetic diabetes that occurs in adolescents and young adults ^95,96. Loss of Hnf1b results in reduced expression of distal transcription factor Pou3f3 (Supplementary Table 1). Of note, nephrons of Pou3f3-deficient mice mostly form normal renal corpuscles and proximal tubules, but lack mature distal segments, loops of Henle, and connecting tubules ⁹⁷. Whether the cells of these distal components are present and misprogrammed or whether these distal segments are severely truncated remains unclear, although cell proliferation is reduced in the early loop of Henle and analyses of histological sections indicate that cells of the distal components are indeed present ⁹⁷. These data suggest that proximal cell identities can form independently of distal POU3F3-mediated differentiation. HNF1B also regulates expression of Iroquois homeobox 1 and 2 (Irx1 and Irx2) in the medial segment of the S-shaped body ⁹⁸. The Iroquios family of transcription factors is broadly known to be transcriptionally repressive. Loss of Irx1 is thought to contribute to the ablation of the medial domain of the nascent nephron in Hnf1b-deficient animals ⁹⁴. Although Irx1, Irx2, and Irx3 have not been functionally analyzed in mammalian nephrons, morpholino knockdown of irx1 and irx2 in developing Xenopus does not induce a discernable pronephros phenotype ⁹⁹. Whether Irx1 and Irx2 are specifically linked to patterning of the mammalian loop of Henle and macula densa in the metanephric kidney, for which there are no known equivalent cell types in Xenopus nephrons, remains to be determined ^99,100. However, homozygous Irx2-mutant mice have no kidney phenotypes ¹⁰¹, suggesting redundancy in the roles of these transcriptional repressors, or that they are not required for mammalian kidney development. At the developmental boundary between medial and distal limb of the S-shaped body, Irx3 expression partially overlaps with that of Irx1 and Irx2 ⁹⁹. Unlike irx1 and 2, morpholino-directed knockdown of irx3 perturbed segmentation of the pronephros and caused loss of slc12a1 and clcnk — genes that are considered markers of intermediate segment development and are expressed in the adult ascending and distal straight components of the loop of Henle in mammals ^78,99. Whether Irx1, Irx2, and Irx3 have a role in generating a transcriptional boundary between presumptive distal and medial cell populations and their lineages remains to be determined.

Transcription factor boundaries are further restricted and refined as the nascent nephron transitions from the renal vesicle, Comma-shaped and S-shaped body stages, to the capillary loop-stage nephron ⁶⁷. The transcription factors, TFAP2A and TFAP2B, mark the distal limb of the S-shaped body, with TFAP2A spanning the distal and medial domain and forming a sharp boundary at the PAPPA2⁺ medial–distal boundary ⁶⁷. This border between the medial and distal limbs is replicated in the adult mouse, where Tfap2a expression spans distal cells from the mature Pappa2⁺ macula densa cells and beyond to the distal convoluted tubule and connecting tubule ⁷⁸. Immunostaining of TFAP2B in fetal human kidney tissue suggests a mosaic pattern of expression within this domain ⁶⁷. Knockout of Tfap2b in mouse nephrons resulted in the ablation of Parvalbumin (Pvalb) — a marker of differentiated distal convoluted tubule cells ¹⁰². However, POU3F3⁺ distal precursors formed normally, indicating that TFAP2B is required for maturation but not formation of distal convoluted tubules. Other distally restricted transcriptional regulators include the histone-lysine methyltransferase MDS1 and EVI1 Complex Locus (MECOM), which is present in distal segments of the S-shaped body through to the IRX1⁺–PAPPA2⁺ boundary ⁶⁷. Functional analyses of MECOM have not been performed in mammals; however, studies in zebrafish show that mecom, is required to maintain the early boundary between the proximal and distal tubule — a segment that is functionally similar to segment 3 of the proximal tubule and loop of Henle, respectively ¹⁰³. In zebrafish, emx1 is a target of mecom and is required for the formation of the distal-early and distal-late boundaries, which are functionally similar to the loop of Henle. EMX1 has not been analyzed in mammalian nephrons, but single-cell transcriptomic data of both mouse and human kidneys suggests that its expression is restricted to distal-most regions of the nephron ^67,78,79. GATA-binding protein 3 (GATA3) is present in both human and mouse nephrons, and forms a boundary between the connecting tubule and putative distal convoluted tubule precursors ⁵⁸. Loss of Gata3 has adverse effects on mesonephrogenesis, but its role in patterning during nephron patterning has not been established ^104,105. Although previously considered a marker of the ureteric lineage, studies in human and mouse have shown that GATA3 is localized to the connecting tubule of the nephron ^51,53,58,78. Findings from single-cell transcriptomics and cell fate mapping studies indicate that GATA3-expressing cells within the connecting tubule partially converge in their expression profiles and functions with cells in adjacently positioned segments of the nephron and collecting duct. Hoxb7 expression, initially restricted in the kidney from a cranial-caudal more anterior intermediate mesoderm population, expands into the distal nephron, where it had previously been thought to be absent ^51,53. Transcriptionally similar cells, even if they are from separate lineages, remain challenging to resolve in single-cell RNA-sequencing experiments where they tend to co-cluster, but use of Hox genes associated with the metanephric mesenchyme (HOX10/11) and their products has shown that these markers can be used to distinguish transcriptomes and cells from nephron and ureteric lineages ^{78,79,106,107}.

The relationships between distal precursors and their progeny beyond the S-shaped body — that is, to the Capillary loop stage and mature nephron — are not established by formal genetic fate-mapping. Efforts to trace precursor-progeny relationships are hampered by dynamic gene expression and changing morphogenesis. One such example is tracing from the Wnt-target Lgr5⁺, which is present in cells in the distal nascent nephron. Fate-mapping of Lgr5-expressing cells from the distal segment of the murine S-shaped body shows that these cells contribute to the thick ascending limb of the loop of Henle, macula densa, and the distal convoluted tubule ¹⁰⁸. However, human single-cell RNA-seq data shows that LGR5, is present in late renal vesicles to Comma-shaped body nephrons; potentially in much earlier segments than those found in the S-shaped body ⁶⁷. Consistent with this, another study using the same Lgr5-Cre mouse suggested that Lgr5 expression is present in the Comma-shaped body and thereafter gradually restricted to a domain adjacent to, and partially overlapping with the distal-most Jag1-expressing domain by the S-shaped body stage ⁶⁸. While such differences may seem trivial, they strongly influence efforts to understand when and where precursor–progeny relationships form and when fates are gradually refined. The Lgr5 example also highlights the difficulty in performing fate-mapping from nascent nephrons. Nephrons form across the whole kidney cortex in asynchronous fashion. This means Lgr5+ cells will be present in several nephrons at different stages and it is not possible to deconvolve which cells gave rise to what when the kidneys are eventually analyzed. While markers such as Lgr5 do not give definitive answers to precursor-progeny relationships they are nevertheless highly informative and can indicate early transcriptional domains linked to particular fates. For instance, within the human LGR5⁺ cell population, small plaques of PAPPA2⁺, IRX1⁺, POU3F3⁺ cells form in the S-shaped body ⁶⁷. This plaque is asymmetrically positioned with a bias towards the forming podocytes and renal corpuscle; it persists in this configuration through to nephron maturation, where its morphology adopts that of the macula densa ^67,76. Mature nephrons display this triple positive state in the macula densa ⁷⁸. Although not genetically traced, the positioning of the macula densa and renal corpuscle indicates a coordinated relationship that develops when these structures are anatomical neighbors as opposed to a model where they find each other at later stages of development ⁶⁷. Given that the macula densa is one of several derivatives of early Lgr5 exressing cells in mice therefore lends credibility to this relationship.

It is clear that canonical Wnt signaling also has a role in the maturation of distal cells, since Wnt7b is required for proliferation of the loop of Henle anlage ¹⁰⁹. Its expression is restricted to the bend of the descending limb of the loop of Henle in the adult nephrons of mice ⁷⁸, hinting at an integral role in the extension of the loop of Henle into the medulla. The Wnt7b-mutant phenotype – developmental arrest of the loop of Henle anlage-recapitulates the effects of loss of β-catenin expression in the murine developing nephron, whereby elongation of the loop of Henle is perturbed and the distal tubules fail to mature.

Computational predictions of precursor–progeny relationships

Efforts to computationally predict precursor–progeny relationships by assessing similarities between the total transcriptional signatures in the S-shaped body nephrons and adult nephrons, suggest that cell populations that remain in the cortex (for example, podocytes, proximal tubule and distal tubule) correlate more strongly with early developmental cell-profiles. By contrast, cell populations that are found in the medulla - for example within the loop of Henle are poorly correlated with what might be proposed to be their precursors ⁶⁷. Based on such computational predictions, nephron cell segments 1–3 in the proximal convoluted tubule – are derived from a single cluster in the S-shaped body, suggesting that domains that relate to the mature nephron do begin to form in the S-shaped body, but that the fine tuning that results in cell diversity occurs after the S-shaped body stage, as would be expected ⁶⁷. The positional relationships of putative precursors to adult nephrons can be modelled (Fig. 3e–h.

^1097891Although the field has begun to delineate the broad regulators of distal domain emergence, maintenance and border formation, the complexities of distal cell fate divergence from their common POU3F3⁺ precursors remain poorly characterized.

The proximal nephron

In the adult nephron, plasma filtrate passes through glomerular vasculature into the lumen of the proximal tubule. The proximal tubule is patterned into three transcriptionally, physiologically, and sexually dimorphic segments ⁷⁸. Proximal tubule cells use fatty acids and mitochondrial oxidative phosphorylation to generate energy that supports solute transport for the active reabsorption of nutrients. Perhaps as a consequence of their transport function supported by high metabolic activity, the cells of the proximal tubule are highly vulnerable to injury and dysregulation ^{110,111, 112}. In fact, the proximal tubule is the leading target of injury and key region affected in the progression of kidney disease ¹¹³. Despite its role as an essential and clinically relevant component of the kidney, how and where proximal tubule cell fates are established remains unclear.

Transcriptional regulation

Elegant loss-of-function and chromatin immunoprecipitation (ChIP) studies in mice have demonstrated an essential role for Hnf4a in proximal tubule cell differentiation ^114,115. Consistent with these data, patients with HNF4A-inactivating mutations present with maturity onset diabetes of the young ¹¹⁶, while heterozygous HNF4A R76W mutations are associated with Fanconi Renotubular Syndrome (FRTS) with nephrocalcinosis ¹¹⁷, indicating a conserved role for HNF4a in human proximal tubule function. HNF4a belongs to the hepatocyte nuclear factor family of transcriptional regulators, of which HNF4a, HNF4g, HNF1a, and HNF1b are expressed in the developing proximal tubules of mice and humans. Hnf1b is a direct transcriptional regulator of Hnf4a, and is expressed from the pretubular aggregate stage ^58,94,98 through to mature nephron tubules ^78,94. In mature mouse and human nephrons, HNF4a, HNF4g, and HNF1a are restricted to the proximal tubules, whereas HNF1b is expressed broadly but is absent in podocytes ^78,118,119. In the developing human nephron, HNF4A is first detected in a 2–3-cell-wide population within the JAG1⁺ WT1⁺ medial domain of the early S-shaped body ⁶⁷. The HNF4A⁺ domain then expands as the S-shaped body matures ⁶⁷, suggestive of a precursor-progeny relationship between these cells and all HNF4A⁺ cells in the mature proximal tubule (Figure 3). These data, combined with the direct regulation of Hnf4a by Hnf1b, suggest that combinations of signals are required for HNF4a activation

In mice, Hnf4a is required for the expression of various solute carriers and transporters for nutrients, toxins, and substrates, indicating it has a central role in imparting physiological functions to the proximal tubule ^114,115. In hepatocytes and the developing pancreas, HNF4a binds to and drives Hnf1a expression ^120,121. A similar relationship is plausible in the developing nephron, since HNF4A expression precedes that of HNF1A, which is then co-expressed with HNF4A ^67,78. By functioning to promote chromatin accessibility, HNF4a and its paralog HNF4g are redundantly required for the maturation of enterocytes in the intestine ¹²². In the mouse kidney, however, deletion of Hnf4a is sufficient to induce the loss of mature proximal tubules with fully formed brush borders, indicating HNF4a and HNF4g are not redundant in this context ^114,115. Whether this is also true in the human kidney remains to be validated. Hnf1a and Hnf4a are both expressed by E14.5 in the developing mouse kidney, and their expression levels decrease significantly in the absence of HNF1b ⁹⁸. Although details of the interplay between hepatocyte nuclear factors in the developing proximal tubule remain to be determined, ChIP-sequencing has shown that HNF4a binds to Hnf4a and Hnf1b in newborn mouse kidneys ¹¹⁵. In both humans and mice, HNF1B expression is upregulated in the medial segment of the S-shaped body where expression of HNF4A emerges ^58,67. Although co-staining studies are needed to definitively demonstrate co-localization, these data suggest the existence of a conserved, putative HNF1b–HNF4a feed-forward loop, in which functional HNF1B is required for the full activation and expression of HNF4A.

Hnf4a and its paralog Hnf4g function redundantly to promote chromatin accessibility of maturation genes in other organ systems, such as the intestine ¹²². Their ability to do so is dependent on a feed-forward loop with the Bmp/Smad signaling pathway ¹²². Hnf4 factors and Smad4 activate each other’s expression, and disruption of this module results in the loss of mature cells with fully formed brush borders in the intestine ¹²². While a pSMAD1/5/8 reporter BRE-Hspa1a-LacZ signaling reporter is active in the medial domain of the mouse S-shaped body nephron, the exact role of Bmp signaling in the developing nephron remains unclear. A role for Bmp signaling in nephrogenesis and patterning is largely limited to findings of genetic and pharmacological experiments ^68,123–126, ^127,128, highlighting another major developmental pathway that is to date inadequately understood within the context of nephron patterning and maturation.

All genetic experiments have thus far been performed in the mouse and specific divergent features between mice and human proximal tubule patterning have not been extensively studied and it remains to be determined whether HNF4A has a similar role in the developing human nephron. Moreover, given that the proximal tubule is consisting of multiple cell-types and is sexually dimorphic, it will be imperative to understand the mechanisms that drive these processes of cell-diversification and determine both HNF4A-dependent and indepndent developmental programs.

Notch in proximal nephron patterning

As outlined earlier, the model of gradual recruitment describes the progressive recruitment of mesenchymal progenitors into the developing nephron. In this model, proximal precursors are derivatives of NPCs that arrive after the distalizing nephron has begun to form (Fig. 2). These proximal precursor cells are marked by high expression of the Notch ligand JAG1 ⁵¹. Mutations in and/or haploinsufficiency of JAG1 causes Alagille syndrome — an autosomal dominant disorder, characterized by various symptoms, including kidney disease and delayed organ growth ^129–132. In the mouse, Hnf1b regulates Notch signaling components in the medial Comma-shaped and S-shaped body, and binds directly to the Notch ligands Lfng and Dll1 ⁹⁴. How Notch signaling controls nephron patterning is a topic of debate, and present studies offer inconsistent perspectives. Some studies ^{133, 134} have demonstrated that Notch signaling is primarily responsible for proximal cell-fate development. By contrast other work ¹³⁵ indicates that the formation of all nephron segments is Notch-dependent. Notch-receptors, ligands, and target genes are expressed along the proximal/distal axis of the developing kidney, with a bias toward the proximal lineage ^133,136; however, the exact mechanisms by which Notch influences cell fates is unclear ¹³². Studies involving manipulation of Notch signaling demonstrate that removal of Notch2 or expressing stabilized forms of Notch receptors (that is, NICDs) has the most consequential effects on kidney development ^133,134. The finding that normal expression levels of Notch1 cannot compensate for deletion of Notch2 ^88,133,134 has led to the suggestion that the strength of Notch2 signalling — as measured by the amount of NICD polypeptides produced from ligand-bound Notch receptors — may be more relevant than NICD composition in proximal–distal nephron patterning ^{133 88,137,138}. Pharmacologic inhibition of Notch with the γ-secretase inhibitor DAPT¹³⁹ supplemented to media of E12.5 ex vivo mouse kidney cultures modified TCF–Lef— β-catenin signalling, indicating cross-talk between Wnt–β-catenin and Notch signaling ⁶⁸. That nephrons partially recovered upon removal of DAPT suggests plasticity at these early developmental stages ⁶⁸.

As described earlier, JAG1 — which is the preferred ligand for Notch2 —is first detected in human nephrons in the distal-most end of the late pretubular aggregate ^38,51,57,60 (Fig. 2a and Fig. 4). How JAG1 is initially upregulated is unclear, but it is thought to be a direct target of β-catenin in systems such as the hair follicle ¹⁴⁰, and is likely also a Wnt–β-catenin target in the developing nephron, that is upregulated during NPC induction and early differentiation ⁴². Given the high levels of β-catenin activity in the nascent distal nephron ⁶⁸, Wnt–β-catenin may contribute to the activation of JAG1 in the distal pretubular aggregate. As the pretubular aggregate transitions into the renal vesicle – coinciding with the last-recruited mesenchymal progenitors that stream into the presumptive podocyte-precursor niche – the JAG1⁺ domain gradually expands proximally (Fig. 2b, Fig. 4). However, as the JAG1⁺ domain expands proximally, the distal-most cells of the now late-stage renal vesicle sharply decrease their abundance of JAG1 through an unknown mechanism ⁵¹ (Fig. 2c, Fig. 4). By this late-renal vesicle stage, separate populations of distal-most JAG1^low and medial JAG1^high have been established ⁵¹. This proximally directed spread of JAG1 fits with known mechanisms of Notch signaling to adjacent cell populations: Notch-mediated lateral inhibition is the process of promoting opposite or different cell fates to neighboring cells, whereas Notch-induced lateral induction induces neighboring cells to adopt a similar or common fate ¹⁴¹. These mechanisms, in conjunction with autoregulation – whereby a JAG1 ligand-induced Notch cascade results in upregulation of JAG1 – ¹⁴¹may explain JAG1 being downregulated distally as its high-abundance domain moves proximally. In a context-dependent manner, JAG1 can either inhibit or induce Notch signaling through a Dll1-Notch-Hes1 signaling pathway to alter the fates of pancreatic progenitor cells ¹⁴². Similar, but repetitive, oscillatory expression patterns of Notch signaling factors are important for numerous other developmental processes, including neurogenesis and somitogenesis, whereby the oscillatory expression of Notch target genes through Notch ligand-mediated cell-cell interactions controls gene expression during tissue morphogenesis^{143, 144}. It may be possible that a non-repetitive Notch-wave is present in the developing nephron. Indeed, the proximal expansion of JAG1 expression occurs as it is downregulated distally, resulting in a singular distal-to-proximal “wave” that originates during the pretubular aggregate nephron and persists through to the S-shaped body nephron ⁶⁷. Although JAG1 abundance is not a direct readout for Notch signaling activity, deletion of Jag1 perturbs nephron ⁸⁸ and collecting duct ¹⁴⁵ patterning. In the nephron, the loss of Jag1 compromises nephron numbers and proximal nephron development ⁸⁸. Transgenic overexpression of Jag1 in the collecting duct leads to hypoplastic kidneys with decreased nephron numbers¹⁴⁶. The Notch ligand Dll1 is also expressed alongside Jag1 in the medial S-shaped body, but Lotus tetragonolobus lectin binding cells in (a marker of proximal tubules) and Wt1⁺ podocytes are less sensitive to loss of Dll1 than they are to loss of Jag1 ⁸⁸ (Supplementary Table 1). The role of another Notch signaling component Lnfg, which alters the structure of Notch-receptors, is present in the nascent nephron but has not been fully characterized. In in vitro assays, overexpression of ^{88 147} Lfng enhanced the response of Notch1 to Dll1 while suppressing its response to Jag1. These and other studies¹³³ reporting high expression of Notch receptors, ligands, and targets in the transition zone of proximal/distal axis segmentation suggest a role in proximal fate selection

Figure 4.

Abundance of the Notch ligand, JAG1 abundance throughout nephron development as a lineage-map. Dashed lines around cells indicate cells that were JAG1⁺ but show dwindling levels of protein. The stepwise lineage tree predictions for nephrogenesis and JAG1 protein abundance are based on a cumulative view from mouse and human studies. The proposed relationships between cells, as indicated by arrows, are based on computational predictions from single-cell RNA-sequencing and immunofluorescent data from Lindström et al., 2018 Dev Cell ⁵¹ and Lindström et al., 2021 Dev Cell ⁶⁷. JAG1 abundance is based on human nephrons as shown in Lindström et al., 2018, and 2021 Dev Cell ^51,67. These approaches do not provide definitive precursor-progeny relationships and should be considered a model. Figure adapted with permission from ref. 71.

It is difficult to imagine how factors downstream of WNT9B–β-catenin signaling could mediate the proximal expansion of JAG1, if we assume that WNT9B–β-catenin signaling initiates JAG1 expression. The growing nephron deposits a basement membrane as it epithelializes, creating a scenario in which Wnt9b ligands would need to traverse several cellular compartments to reach recipient cells. TCF–LEF–β-catenin activities are detected throughout the distal-medial Comma-shaped body ⁶⁸, indicating that some form of signal propagation exists; however, the spread of JAG1 is more likely mediated through a lateral induction mechanism by which Notch signaling activity induces neighboring cells to adopt a common fate ¹⁴¹. By the Comma-shaped and early S-shaped body stages, JAG1 membrane deposition is highest in the medial domain where HNF4A was initially activated ⁶⁷. As the HNF4A⁺ domain emerges from within the JAG1⁺ population of cells, and then expands and elongates, the wave of JAG1 either retracts or is proximally downregulated with the consequence that JAG1 remains detectable in cells distally adjacent to the expanding proximal domain, which based on their anatomical position may be presumptive loop of Henle precursors ⁶⁷. This aligns with Jag1 expression being restricted to the descending loop of Henle in mature cortical nephrons ⁷⁸. In the developing human kidney, computational predictions from scRNA-seq and subsequent validations by in situ hybridization and immunolabelling enabled visualization of putative cellular relationships (Fig. 4)^51,67. Although such predictions do not mean that cell fates are fixed — indeed, early patterning is partially plastic so it is likely fates are not fully determined ⁶⁸ — the expression patterns and predicted relationships are in line with those observed in the mature nephron, where Jag1 is restricted to the descending loop of Henle and adjacent to HNF4a⁺ proximal tubules ⁷⁸. These data support a model in which JAG1 is activated in the distal-most pretubular aggregate and expands proximally as mesenchymal progenitors are recruited and the developing nephron elongates. The mechanisms that underpin the downregulation of JAG1 distally and the blocked expansion proximally beyond the HNF4A⁺ domain are not known. While it has been suggested that cell fates for the mature nephron begin to be specified in the S-shaped body nephron ^51,67, heterogeneity within this structure is likely more complex than previously thought. Moreover, although distinct boundaries of gene expression can stipulate lineage commitments, the apparent wave-like expression pattern of JAG1 suggests that the expression of genes can be gradually reduced or upregulated via processes such as lateral induction and lateral inhibition, thereby generating partially overlapping gene expression domains that may fine-tune cell fates.

The last recruits: podocytes and the parietal epithelium

Podocytes, the parietal epithelium, capillaries of the glomerular tuft, and mesangial cells collectively comprise the renal corpuscle — the filtration apparatus of the nephron. Podocytes and the parietal epithelium are derived from Six2⁺ NPCs ⁵⁰. According to the gradual recruitment model, these NPCs are the last to be recruited into the forming nephron ⁵¹ (Fig. 2 and Fig. 3). The transcriptional profile of podocyte-forming NPCs is distinct from that of early differentiating NPCs that incorporate into the forming tubular epithelium, with early and late transcriptional profiles being evident ^{51–55,67,70}. In human nephrogenesis, the first sign of podocyte differentiation is expression of OLFM3 followed by MAFB expression within a subset of OLFM3⁺ cells ^51,55. This expression occurs at the point at which NPCs stream in the proximal renal vesicle (Fig. 3b–c). Transcription factors that regulate podocyte development, such as TCF21 ^148,149, MAFB ¹⁵⁰ and WT1 ¹⁵¹, are expressed within the early profile and persist through maturation ⁵⁵. However, the nephron-to-NPC connection ceases as NPCs streaming from the top of the niche are physically contacting the developing and flattening parietal epithelial cells ^51,55. By the S-shaped body stage, the cell recruitment has ended and the strongest SIX2 signal comes from the forming parietal epithelium to which cell-streaming is last observed ⁶⁷. These later NPC recruits that likely form podocytes and the parietal epithelium, exhibit a rather unusual cell migration at a first glance. These NPCs stream past the already epithelializing and patterning renal vesicle to incorporate into the proximal nephron. At present we know nothing about how this process is regulated but NPCs that are attached to the collecting duct tip begin to extend long cell projections to the proximal end of the renal vesicle and eventually terminate their attachment to the tip ⁵¹. This would suggest active cell-migration, perhaps driven by a chemoattractive signal. Understanding this signal, the cell migration, and the consequences this has on the NPCs that drive them towards a podocyte or parietal fate, might provide insights to the exact environment required for the formation of these cells.

Conclusions and future challenges

The nephron progenitor niche represents a unique system in developmental biology. Progenitors are positioned on top of a rapidly growing and branching epithelial tubule that propels them through space. Outside these growing tubules, NPCs undergo considerable movement both between ureteric tips and niche positions and undergo cyclical movements, driven by their entry and exit through the cell-cycle. This range of complex movements are coupled to their differentiation and nephron patterning through mechanisms that are not yet fully elucidated. The developmental program that drives nephrogenesis in human, mouse, and other mammals generates shapes that closely resemble each other ^65,67,152. In other words, S-shaped body nephrons (Fig. 3e) that form in a human or a mouse can readily be identified as such. Moreover, the morphologies of stage-matched nephrons within the same species are sufficiently similar that they can be superimposed on each other without distorting their shapes ⁶⁷. These findings imply that each fold, bend, and twist taken during nephrogenesis might serve a function in the development of a functional nephron. However, insufficient molecular and anatomical data exist for us to fully understand the rationale of why precursors — for example, the HNF4A⁺ putative proximal precursors or the PAPPA2⁺ putative macula precursors, which are positioned at the crests of two separate bends of the S-shaped body — emerge in their specific positions. These uncertainties highlight the need for formal genetic fate-mapping to understand the relationships between precursors and their progeny and the environment in which these precursors arise.

Determining the impact of cell movements and the mechanisms underpinning these processes will require dissection of receptor-ligand interactions, potential chemotactic signals, and an understanding of how cells move from their niche into the forming nephron. Available studies have highlighted the potential importance of the dose and duration of signals in patterning of the nephron and the relevance of signaling boundaries, which are established by the growth of the nephron as it recruits progenitors. However, understanding of whether cells exit their niche by competition for space as in other systems - such as the intestinal crypt - or by active cell migration will provide insights to how the process of nephrogenesis can be more closely replicated in vitro in kidney organoids. Technological advances in single-cell profiling and imaging will pave the way for future research in this area.

Our understanding of in vivo developmental processes will improve the design of stem-cell based differentiation protocols that aim to replicate kidney cell-types in vitro ^153–156. Although it is entirely conceivable that future therapeutic devices, disease modeling platforms, and nephrotoxicity assays will look nothing like the convoluted tubular shapes found in a kidney, arguably, what is seen in vivo should be considered the benchmark for these efforts.