The vertebrate kidney derives from the intermediate mesoderm (IM), which represents an anatomically defined region along the dorsoventral axis—positioned between the paraxial mesoderm, forming the somites in the dorsal part of the embryo, and the lateral plate mesoderm, which differentiates into ventrally located skeletomuscular and vascular tissues. Mammalian kidneys develop in three successive stages, generating three distinct excretory structures known as the pronephros, mesonephros, and metanephros ( Fig. 1.2 ). The pronephros and mesonephros are vestigial structures in mammals and degenerate before birth; the metanephros is the definitive mammalian kidney. Many of the signaling pathways and genes that play important roles in the metanephric kidney appear to play parallel roles during the development of the pronephros and mesonephros. The pronephros consists of pronephric tubules and the pronephric duct (also known as the precursor to the Wolffian duct) and develops from the rostral-most region of the urogenital ridge at 22 days’ gestation in humans and 8.5 days post coitum (embryonic stage E8.5) in mice ( Table 1.1 ). Throughout the rest of this chapter, most timelines of kidney development are with reference to the mouse. The pronephros serves as the principal excretory organ of the larval stages of fishes and amphibians. The mesonephros develops caudal to the pronephric tubules in the midsection of the urogenital ridge. The mesonephros becomes the functional excretory apparatus in lower vertebrates (adult fish and amphibians) and may perform a filtering function during embryonic life in mammals. Before its degeneration, endothelial, peritubular myoid, and steroidogenic cells from the mesonephros migrate into the adjacent adrenogonadal primordia, which ultimately form the adrenal gland and gonads. Abnormal mesonephric migration leads to gonadal dysgenesis, a fact that underscores the intricate association between these organ systems during development and explains the common association of gonadal and renal defects in congenital syndromes. ^,

Three types of kidney primordia during mammalian development.

Schematic of mouse E11.5 embryo. The pronephros and mesonephros develop in a rostral-to-caudal direction, and the tubules are aligned adjacent to the Wolffian or nephric duct (WD) . The metanephros develops by interaction of the ureteric bud epithelium (UB), outgrowth from the caudal end of the WD, and metanephric mesenchyme (MM) . The pronephros and mesonephros are vestigial structures in mice and humans and are regressed during development.

The metanephros, which is formed at the caudal end of the urogenital ridge, is the third and final kidney stage, representing the definitive adult kidney of higher vertebrates. The metanephros development relies on triad interactions among the three types of progenitors: the nephron progenitor (NP) and stromal progenitor that reside in the metanephric mesenchyme (MM) and the ureteric bud (UB) ^, (see Fig. 1.2 ). The NP in the MM forms the nephron (i.e., glomerular podocytes, parietal epithelial cells, proximal tubules, loops of Henle, distal tubules, and connecting tubules), while the UB gives rise to collecting ducts and the ureter. The stromal progenitor in the MM differentiates into cortical and medullary interstitial cells, as well as mesangial cells and renin-producing cells. The UB is first visible as an outgrowth at the caudal end of the Wolffian duct approximately between the fourth and fifth weeks of gestation in humans or E10.5 in mice. The MM becomes histologically distinct from the surrounding mesenchyme and is found adjacent to the UB. Upon invasion of the UB into the MM, signals from the MM cause the UB to branch into a T-shaped form (at around E11.5 in mice) and then undergo iterative dichotomous branching, giving rise to the urinary collecting duct system ( Fig. 1.3 ). Simultaneously, the UB sends reciprocal signals to NPs in the MM, which are induced to condense along the surface of the UB tips.

Ureteric branching morphogenesis.

Rapid reiterative branching of the ureteric bud epithelium within a 5-day period in mice as imaged with a pan-cytokeratin antibody using optical projection tomography. By E16.5, the renal pelvis, formed by the widening and coalescence of the earliest branches of the ureteric tree, is already apparent.

Reproduced with permission from Short KM, Smuth I. Imaging, analyzing and interpreting branching morphogenesis in the developing kidney. Results Probl Cell Differ. 2017;60:233–256.

After condensation, a subset of NP aggregates adjacent to and below the tips of the branching UB. These cell clusters, known as pretubular aggregates, undergo mesenchymal-to-epithelial conversion to become the renal vesicle ( Fig. 1.4 ).

Overview of nephrogenesis.

Reciprocal interaction among the nephron progenitor, stromal progenitor, and ureteric bud within the nephrogenic zone enables continuous branching of the ureteric epithelium, self-renewal of nephron progenitor, and differentiation into epithelialized nephron structures. Successive proximo-distal patterning within the nascent nephron structure results in the formation of an S-shaped body with distinct gene expression patterning coding for the future nephron domains. CSB, comma-shaped body; PA, pretubular aggregate; RV, renal vesicle; SSB, S-shaped body.

The renal vesicle undergoes patterned segmentation and proceeds through a series of morphologic changes that include gradual recruitment of NPs to form the glomerulus and components of the nephrogenic tubules from the proximal convoluted tubule, loop of Henle, and distal tubule. The renal vesicles undergo differentiation, passing through morphologically distinct stages starting from the comma-shaped stage and proceeding to the S-shaped body, capillary loop, and mature stages. Each step involves precise proximal-to-distal patterning and structural transformations, ultimately forming the glomerulus and components of the nephrogenic tubules from the proximal tubule, loop of Henle, and distal tubule (see Fig. 1.4 ). Remarkably, this process is repeated 600,000 to 1 million times in each developing human kidney as new nephrons are sequentially born at the tips of the UB, namely the nephrogenic zone, throughout fetal life.

The nephrogenic zone is an active site of nephrogenesis. It is morphologically identified as a narrow band beneath the renal capsule where the branching UB tips are found together with nascent nephrons (pretubular aggregates, renal vesicles, comma-shaped bodies, and S-shaped bodies), self-renewing NPs, and stromal progenitors (see Fig. 1.4 ). Kidney growth and nephrogenesis occur in a radial fashion, with new branches of the ureteric tree and newer nephrons added at the outermost periphery of the developing cortex. Accordingly, within the developing kidney, the most mature nephrons are found in the innermost layers of the cortex and the most immature nephrons are found in the most peripheral regions (see Fig 1.1 ). The nephrogenic zone becomes progressively thinner with the gradual depletion of nephrogenic progenitors and disappears when the remaining NPs have completely epithelialized by 36 weeks’ gestation in humans and postnatal day 3 (P3) in mice. The kidney eventually divides into two major compartments: an outer region called the cortex and an inner region called the medulla. The glomeruli and proximal and distal tubules are in the cortex, along with the distal part of the nephron, which connects directly to the collecting ducts. The loop of Henle and rest of the collecting duct network comprise the epithelial structures found in the medulla.

The glomerulus develops from the most proximal end of the renal vesicle, farthest from the tip of the UB. ^, Distinct cell types of the glomerulus are first identified in the S-shaped stage, where presumptive podocytes appear as a columnar epithelial cell layer (see Fig. 1.4 ). Parietal epithelial cells differentiate and flatten to form the Bowman capsule, a structure that surrounds the urinary space and is continuous with the proximal tubular epithelium. A vascular cleft develops, separating the presumptive podocyte layer from more distal cells that will form the proximal tubule. Simultaneously, endothelial cells migrate into the cleft to form capillary loops. The tubular portion of the nephron is segmented in a proximal-distal order into the proximal convoluted tubule, descending and ascending loops of Henle, and distal convoluted tubule. The distal tubule is adjacent to the collecting duct, a derivative of the UB.

Although all segments of the nephron are present at birth and filtration occurs before birth, maturation of the tubule continues in the postnatal period. Increased expression levels of transporters, switches in transporter isoforms, alterations in paracellular transport mechanisms, and permeability and biophysical properties of tubular membranes have all been observed to occur postnatally.

The collecting duct system is composed of hundreds of tubules through which the filtrate produced by the nephrons is conducted out of the kidney, to the ureter, and then to the bladder. Water and salt resorption and excretion, ammonia transport, and H ⁺ ion secretion required for acid-base homeostasis also occur in the collecting ducts, under different regulatory mechanisms and using different transporters and channels that are active along tubular portions of the nephron. The collecting ducts are the product of branching morphogenesis from the single UB, which enables the formation of arborized structure with a single exit (see Fig. 1.3 ). Considerable remodeling is involved in forming collecting ducts from branches of the UB. The branching is highly patterned, with the first several rounds of branching being somewhat symmetric, followed by additional rounds of asymmetric branching, in which a main trunk of the collecting duct continues to extend toward the nephrogenic zone, while smaller buds branch as they induce new nephrons within the nephrogenic zone. The mouse kidney has a single papilla and calyx, whereas a human kidney has 8 to 10 papillae, each of which drains into a minor calyx, with several minor calyces draining into a smaller number of major calyces.

For decades in classic embryologic studies of kidney development, emphasis has been placed on the reciprocal inductive signals between MM and UB. However, in recent years, interest in the stromal cell as a key regulator of nephrogenesis has arisen. ^, The stromal progenitor in the MM differentiates into cortical and medullary interstitial cells, as well as mesangial cells and renin-producing cells ( Fig. 1.5 ). The stromal progenitor cells also provide signals required for UB branching and patterning of the developing kidney. Disruption or loss of these stromal cells leads to impairment of UB branching, reduction in nephron number, disrupted nephron patterning with failure of cortical-medullary boundary formation, and maldevelopment of the renal vasculature. A reciprocal signaling loop from the UB exists to properly pattern stromal cell populations. Loss of these UB-derived signals leads to a buildup of stromal cells beneath the capsule that are several layers thick. As nephrogenesis proceeds, stromal cells differentiate into peritubular interstitial cells and pericytes that are required for vascular remodeling and the production of extracellular matrix responsible for proper nephron formation.

The nephron and stromal progenitors.

Lineage tracing analysis in E14.5 mouse kidneys showing derivatives of Six2 -expressing nephron progenitors (A, C, and E) and the Foxd1 -expressing stromal progenitors (B, D, and F), stained for β-galactosidase activity (blue). a, Adrenal gland; cd, collecting duct; ci, renal cortical interstitium; cm, cap mesenchyme; cs, renal cortical stroma; gal, galactosidase; gc, glomerular capillary; k, kidney; mi, renal medullary interstitium; ms, mesangium; nt, nephrogenic tubule; pe, parietal epithelium; rv, renal vesicle; sb, S-shaped body; ue, ureter epithelium; um, ureter mesenchyme; ut, ureteric tip; ve, visceral epithelium (podocyte).

Reproduced with permission from Kobayashi A, Mugford JW, Krautzberger Am, et al. Identification of a multipotent self-renewing stromal progenitor population during mammalian kidney organogenesis. Stem Cell Reports . 2014;3:650–662.

The microcirculations of the kidney include the specialized glomerular capillary system, responsible for production of the ultrafiltrate and the vasa recta bundles and peritubular capillaries involved in the countercurrent mechanism for urine osmoregulation. At E11.5 in mice, the UB is tracked by a primitive vessel that elaborates in synchrony with both UB branching and nephrogenesis. Capillaries form networks around the developing nephric tubules by E13.5, and the hilar artery and first-order interlobar renal artery branches can be identified by E14.5. These branches will form the corticomedullary arcades and interlobular arteries that branch from these arcades. Further branching produces the glomerular afferent arterioles. From E13.5 onward, endothelial cells migrate into the vascular cleft of developing glomeruli, where they undergo differentiation to form the glomerular capillary loops. The presence of endothelial cell–containing glomeruli becomes apparent at E14.5. The efferent arterioles carry blood away from the glomerulus to a system of fenestrated peritubular capillaries that are in close contact with the adjacent tubules and receive filtered water and solutes reabsorbed from the filtrate. These capillaries have few pericytes. In comparison, the vasa rectae, which surround the medullary tubules and are involved in urinary concentration, are also fenestrated but have more pericytes. They arise from the efferent arterioles of deep glomeruli. The peritubular capillary system surrounding the proximal tubules is well developed in the late fetal period, whereas the vasa rectae mature 1 to 3 weeks postnatally.

Many of the genetic pathways regulating kidney development are conserved between mice and humans. Due to the relative ease of manipulating the genome of the mouse, its small size, and shorter gestation period, the mouse has become the primary model organism for the study of mechanism of human development and diseases. Anatomic and functional features of the human kidney are highly similar in the mouse, albeit at a smaller scale, while a number of genes identified as essential for normal mouse kidney development are also known to be associated with congenital anomalies of the kidney and urinary tract (CAKUT) and other kidney diseases.

The wealth of our understanding of metanephric kidney development over the past few decades is owed to the harnessing of homologous gene recombination to introduce targeted mutations and novel alleles into specific genes in cultured mouse embryonic stem cells (ESCs). This paved the way for the creation of numerous genetically engineered mice that have become highly valuable tools for the study of renal developmental biology and the etiology of certain genetic diseases of the kidneys and urinary tract ( Table e1.1 ). The technology is applied in many ways. Its simplest way is creation of null mutations within the germline that generate gene knockout mouse models. The limitation of this method is that certain genes are essential for early development and their inactivation in the germline can cause premature lethality, thus precluding the analysis of the function of those genes in organogenesis. An improvement to this is the use of a conditional gene-targeting strategy, allowing for the creation of conditional alleles. This involves introducing small recognition sites for recombinase enzymes of which the Cre recombinase is the most routine now ( Fig. 1.7 ). A conditional “floxed” allele of the target gene locus is created by incorporating two loxP sites within two separate introns, flanking the exons that can be excised or recombined. In principle, normal transcription from the locus is expected before recombination of the floxed allele. The Cre recombinase is engineered under the control of a tissue-specific promoter. Breeding between tissue-specific transgenic Cre animals and those harboring a conditional allele for a particular gene ultimately results in a cell- or tissue-type specific inactivation of the gene of interest. This strategy has also been refined in some cases, so the Cre expression is temporally regulated using a drug such as doxycycline or tamoxifen. A number of Cre lines are now available to target genes specifically within different subpopulations of renal cells and progenitors. The transgenic Cre lines driven by the Hoxb7, Six2, Foxd1, and Nphs2 promoters are among the most highly cited gene excision drivers for targeted genetic inactivation in the ureteric, nephrogenic, stromal, and podocyte cell lineages. ^, Lately, the introduction of the more precise CRISPR-Cas9 gene editing tool, an ingenious application of the adaptive immune response of prokaryotes against bacteriophages, is facilitating the way researchers create customized mutant cells and animal models to study various aspects of renal development ( Fig. 1.8 ). The complex of guide RNAs and Cas9 proteins can be introduced directly into fertilized oocytes to rapidly generate mutant mice, so there is no need to manipulate mouse ESCs. This method is becoming increasingly popular, although great care must be taken to avoid off-target mutations.

Cre-lox homologous gene recombination system.

Simplified overview of Cre recombinase–mediated homologous recombination for generation of tissue-specific conditional null mutations of a target gene. Cre recombinase expression is engineered to be driven under the control of tissue-specific promoter (promoter ^TS ). The target gene (or typically certain exons within the target chromosomal locus) is flanked with loxP sites (recognition sites for Cre recombinase). Cre activity in specific cell types mediates the excision of the loxP-flanked (floxed) target gene, creating a null allele.

Gene editing using the CRISPR-Cas9 system.

Simplified overview for targeted introduction of mutations using the CRISPR-Cas9 system. Site-specific cleavage of a target gene locus with the Cas9 endonuclease creates a double-strand DNA break. The nicked target gene can be repaired by an error-prone, nonhomologous repair mechanism resulting in nucleotide insertion, deletion, or frameshift mutations. Introduction of a homologous engineered donor DNA results in a homologous substitution into the target locus, which then results in a precisely edited gene.

Significant innovations in high-throughput gene sequencing are also facilitating the identification of monogenic mutations associated with heritable kidney diseases. ^, Putative disease-causing genes identified from genome-wide association studies can then be validated in corresponding gene-targeted mouse models. Similarly, a gene whose mutation has been identified to cause kidney maldevelopment and dysfunction in mice can be investigated for incidence of mutations across the human genome (e.g., Arhgdia in nephrotic syndrome). Other genome-wide approaches that have led to the discovery of novel genes in kidney development and disease include gene trap consortia ^, and genome-wide transcriptome and proteome projects.

Reporter mouse lines are animal models engineered to express transgenes encoding a fluorescent protein tag or an enzyme (e.g., bacterial β-galactosidase) in a cell lineage–specific manner. In the simplest model, a single transgene comprising the reporter coding region is placed downstream of a cell-specific promoter. Such a model is useful to identify and follow distinct cell lineages where the chosen promoter is active. Hoxb7-EGFP was the first fluorescent transgene developed to visualize renal development. Enhanced green fluorescent protein (EGFP), placed under the control of the Hoxb7 promoter, specifically labels the Wolffian duct and the ureteric epithelial lineage. Hoxb7-EGFP has therefore proven to be invaluable in studying the rates and pattern of ureteric branching morphogenesis and ureteral development, including disruption of these events in the context of particular mutant backgrounds. ^, An alternative model uses two separate transgenes, namely a cell-specific Cre driver and an independent reporter transgene (e.g., R26R). With the R26R reporter model, expression of fluorescent protein gene (e.g., tdTomato) or β-galactosidase transgene is switched on by a Cre-mediated removal of an upstream stop codon. Inducible reporter systems incorporate genetic regulatory elements that allow for both cell-specific promoter- and drug-dependent activation of the reporter gene. The most commonly used strategies for controlled transgene expression in mice are the Tet-operon/repressor bi-transgenic and the estrogen receptor ligand-binding domain (Cre-ERT2) systems. These inducible reporter systems allow for timed pulse labeling of cell lineages, resulting in permanent tagging of progenitor cells and their direct derivatives. Inducible reporter systems have become valuable tools for the mapping of cell fates. Inducible reporter systems have been used to establish that the Six2 ⁺ cells are the progenitors of all epithelial structures of the mature nephron (see Fig. 1.5 ). In some cases, the Cre transgene is engineered with an associated fluorophore expression cassette (e.g., GFP-Cre fusion gene) under the control of a shared promoter, thus allowing easy identification of cells with targeted gene mutations. A wide variety of reporter and Cre transgenes (see Table e1.1 ) are now available to characterize the development and organization of multiple compartments of the kidney.

Reporter mice expressing fluorescent tags also come in handy for segregating and isolating particular cell types on the basis of fluorescence, facilitating global gene expression analysis, elucidation of transcriptional regulatory networks, and epigenetic interactions that orchestrate particular morphogenetic events ( Table 1.2 ). Purified populations of fluorescence-tagged cells can be used for high-throughput discovery of transcriptomes, genome-wide DNA-binding protein interactions, histone modifications, and nucleosome complexes through next-generation sequencing methods such as RNA-seq (RNA sequencing) and ChIP-seq (chromatin immunoprecipitation sequencing).

Recent advances in synthetic biology using an “understanding by creating” approach have enabled the ex vivo generation of kidney tissues from pluripotent stem cells (PSCs), namely kidney organoids ( Fig. 1.9 ). These PSC-derived kidney organoids can form many renal structures including the glomerulus, proximal tubule, loop of Henle, distal tubule, collecting ducts, interstitium, and a primitive endothelial network. This breakthrough, in turn, has served as a tool to validate the efficacy of lineage-specifying signaling molecules identified through in vivo studies. The feasibility of screening using in vitro experimental setups has significantly enhanced our understanding of kidney lineage specification with unprecedented resolution, particularly in analyzing signal concentration, combinations, timing, and duration aspects that are not readily apparent in in vivo analyses. In addition, organoids with gene-edited PSCs using CRISPR-Cas9 technology (see Fig. 1.8 ) provide a unique opportunity for a time- and cost-effective strategy to interrogate novel gene functions relevant to embryonic kidney development in not only mice but also humans. In addition, the selective induction protocol of either NP or UB lineage allowed the cell-autonomous role of commonly expressed genes, such as PAX2, in nephron and the collecting duct development to be addressed. ^, Such gene-edited organoids or patient PSC-derived organoids are being used to model various genetic diseases including podocyte diseases, ciliopathies, and polycystic diseases. These efforts will eventually lead to discovery of curative drugs. Another long-term goal of kidney organoid research is kidney replacement therapy. However, the currently available kidney organoids are immature and lack ureters and a proper vascular network. ^, A deeper understanding of the developmental processes in vivo will be required to generate transplantable kidneys ex vivo.

Generation of kidney organoid based on development.

Representative-directed differentiation protocols of pluripotent stem cells (PSCs) toward nephron progenitor (NP), ureteric bud (UB), and stromal progenitor (SP) lineages are summarized. Induced progenitors are further differentiated to form nephron organoids (right upper panel) or ureteric bud organoids (right middle panel), or they are reassembled to reconstitute higher-order kidney structures (right lower panel). Commonly used signaling molecules among multiple reports are shown in bold. A factor specifically used in human PSC differentiation is shown in red. AIM, Anterior intermediate mesoderm; ECM, extracellular matrix; PIM, posterior intermediate mesoderm; PS, primitive streak; PSC, pluripotent stem cell; RA, retinoic acid; TBM, tail bud mesenchyme; WD, Wolffian duct.

The kidney, including the embryonic kidney, contains numerous cell types, with each cell type consisting of a limited number of cells. Conventional transcriptomic techniques, which usually require thousands of cells, result in the averaged property of the whole kidney or sorted limited fractions of cell types. The newly emerging scRNA-seq technology has revolutionized the field of kidney development research. It can measure transcriptomic information of a few tens of thousands of cells, with a depth of a few thousand genes per single cell, facilitating the collection of transcriptomic data from entire tissues or embryos. Moreover, advances in bioinformatics tools have enabled computational retrospective recovery of cell types, eliminating the need to presort the target cell of interest and even allowing identification of previously unknown subpopulations. ^, Indeed, this technique has allowed the identification of multiple cell types in the embryonic and adult kidney and many representative genes expressed in each cell type including not only nephrons and collecting ducts but also interstitial cells, mesangial cells, and the vasculature. ^, The scope of the analysis has been extended from mouse to human embryonic kidney so that in addition to genes conserved across species, many human-specific gene features have been identified. Taking advantage of the relatively slow pace of human development, scRNA-seq analysis more precisely identifies the transitional cell states from NPs to nephron formation via S-shaped bodies. scRNA-seq analysis has also been applied to human kidney organoids, allowing precise identification of cell types in the organoids. Comparison of these data with human embryonic kidney data revealed the common or differentially expressed genes in the organoids and showed that the currently available organoids are immature and correspond to the embryonic stage. Thus scRNA-seq has become one of the standard gene expression analyses for renal research.

The weakness of scRNA-seq is the loss of positional information, as dissociation of the tissue into individual cells is required for analysis. Some spatial scRNA-seq techniques, which detect gene expression in histological sections, have been developed and applied to some renal research including kidney injury models. However, current spatial scRNA-seq has not achieved analysis at the single-cell resolution. Instead, new hybridization techniques using a few hundred or thousands of known gene probes have been developed. Further advances in single-cell analysis are expected in the next decade.

Organisms separated by millions of years of evolution from humans still provide useful models to study the genetic basis and function of mammalian kidney development. This stems from the fact that all these organisms possess excretory organs designed to remove metabolic wastes from the body and that genetic pathways involved in other aspects of invertebrate development may serve as templates to dissect pathways in mammalian kidney development. The excretory organs of invertebrates differ greatly in their structure and complexity and range in size from a few cells in Caenorhabditis elegans to several hundred cells in Drosophila, to the more recognizable kidneys in amphibians, birds, and mammals. In C. elegans, the excretory system consists of a single large H-shaped excretory cell, a pore cell, a duct cell, and a gland cell. ^, C. elegans provides many benefits as a model system: the availability of powerful genetic tools including “mutants by mail,” a short life and reproductive cycle, a publicly available genome sequence and resource database ( http://www.wormbase.org ), the ease of performing genetic enhancer-suppressor screens in worms, and the fact that they share many genetic pathways with mammals. Major contributions to our understanding of the function of polycystic and cilia-related genes have been made from studying C. elegans. The Pkd1 and Pkd2 homologs, LOV1 and LOV2 of C. elegans, are involved in cilia development and mating behavior. ^, Elucidation of the genetic interactions of the Kirrel (Neph1) ortholog and nephrin-like molecules SYG1 and SYG2 in synapse formation in C. elegans also provided clues to the roles of these genes in glomerular and slit diaphragm formation and function in mammals.

Similar to C. elegans, the relative ease of large-scale genetic screens and phenotypic characterization in Drosophila makes it another valuable complementary model for understanding the genetic basis of developmental processes. The excretory system of Drosophila consists of two parts, the nephrocytes and the malpighian tubules, which are functionally analogous to podocytes and renal tubules, respectively. A fundamental difference from vertebrate kidneys is that the nephrocytes and malpighian tubules are not physically connected. Nephrocytes either surround the heart (epicardial nephrocytes) or the esophagus (garland cells) and have elaborate membrane invaginations that closely resemble the glomerular filtration barrier. Remarkably, mutations of Drosophila homologs of genes known to be essential to form slit diaphragms and maintain podocyte functions also impair nephrocyte morphology and filtration functions. Similarly, conserved genes that regulate normal patterning and function of Malpighian tubules and vertebrate renal tubules have been identified. Functional readouts such as impaired nephrocyte filtration or uptake of tracers, ultrastructural analysis, and mortality screens can be executed efficiently in Drosophila, which can facilitate the characterization of novel gene functions that are vital for renal filtration.

The pronephros is the functional kidney of the larva of some fishes (except for jawless fishes, which only develop the pronephros) and amphibians, while the mesonephros serve as the kidney in adults of these aquatic animals. The pronephros of the zebrafish (Danio rerio) larva consists of two tubules connected to a fused, single, midline glomerulus. The zebrafish pronephric glomerulus expresses many of the same genes found in mammalian glomeruli (e.g., Vegfa, Nphs1, Nphs2, and Wt1 ) and contains podocytes and fenestrated endothelial cells. Advantages to the zebrafish as a model system include its short reproductive cycle, transparency of the larvae with easy visualization of defects in pronephric development without sacrificing the organism, the availability of the genome sequence, the ability to rapidly knockout/knockdown gene function, and the ability to perform functional studies of filtration using fluorescently tagged labels of varying sizes. These features make zebrafish amenable to both forward and reverse genetic screens. The pronephros of the clawed frog Xenopus laevis has also been used as a simple model to study early events in nephrogenesis. ^, Similar to the fish, the pronephros of the clawed frog consists of a single glomus, paired tubules, and a duct. The fact that X. laevis embryos develop rapidly outside the body (all major organ systems are formed by 6 days of age); the ease of injecting DNA, mRNA, and protein; ability to perform grafting; and in vitro culture experiments establish the frog as a valuable model system to dissect early inductive and patterning cues. ^, The availability of transgenic zebrafish and Xenopus lines that express the fluorescent protein EGFP in pronephric and mesonephric kidneys provides an opportunity to visualize real-time kidney development and function.

Because many of the transcription factors in the MM are required to maintain GDNF expression, their deletion often leads to insufficient UB attraction toward the MM. Two exceptions are the Osr1 (Odd1 )- and Eya1 -mutant embryos, where the MM itself is absent, suggesting that Osr1 and Eya1 represent early determinants of the MM (see Fig. 1.11 ). ^, Osr1 marks the IM from which the mesenchymal cells within the mesonephros and metanephros are derived and is subsequently downregulated upon epithelial differentiation. Mice lacking Osr1 do not form the MM and do not express several other factors required for metanephric kidney formation including Eya1, Six2, Pax2 , Sall1, or Gdnf .

Eya1 -mediated specification of the MM cell fate is thought to occur via interaction with another transcription factor, Six1 . EYA1 and SIX1 mutations are found in humans with branchiootorenal (BOR) syndrome. Eya1 and Six1 form a regulatory complex that is involved in transcriptional regulation. ^, Interestingly, Eya1 was shown to have an intrinsic phosphatase activity that regulates the activation of the Eya1/Six1 complex. ^, Moreover, Eya and Six family genes are coexpressed in several tissues in mammals, Xenopus and Drosophila, further supporting a functional interaction between these genes. ^{, , , ,} Direct transcriptional targets of this complex appear to include the pro-proliferative factor Myc . In the Eya1 -deficient urogenital ridge the putative MM is completely absent. Consistent with this finding, Six1 is either absent or poorly expressed in the presumptive location of the MM of Eya1 -null embryos. ^, Eya1 is expressed in the Six1 -null mesenchyme, suggesting that Eya1 is upstream of Six1 . ^,

The transcription factor Wt1 is another essential regulator of early MM development. Wt1 expression is weak in the uninduced MM but increases in the condensed cap mesenchyme surrounding the branching UB tips. Wt1 expression remains throughout nephrogenesis but eventually becomes restricted to the presumptive podocytes at the proximal end of the S-shaped nephron. Mature podocytes continue to express Wt1 at high levels. Genetic loss of Wt1 in mice prevents UB outgrowth and causes apoptosis of the MM, whereas human mutations of WT1 have been linked to renal tumors. ^, Among the numerous identified transcriptional targets of Wt1 known to be required for kidney development are Bmp7, Pax2, and Sall1 . It has also been shown that Wt1 regulates antagonistic fibroblast growth factor (FGF) and bone morphogenetic protein (BMP)/SMAD (contraction of homologous Sma and MAD genes of worms and fruitflies) signaling pathways, effectively promoting the proliferation and survival of the MM. ^, The absence of Wt1 significantly downregulates the expression of the genes for several FGF ligands, including Fgf8, Fgf10, Fgf16, and Fgf20, which support mesenchymal proliferation. ^, The impairment of FGF signaling upon loss of Wt1 is exacerbated by the upregulation of BMP/SMAD signaling, which promotes apoptosis. This is thought to occur through the loss of Bmper expression, a direct target of Wt1, which inhibits BMP4 signaling.

At the signaling factor level, compound loss of FGFR1 and FGFR2 in the MM leads to renal agenesis, presumably originating from the misactivation of critical transcription factors such as Six2, Sall1, and Pax2 as early as E10.5.

The discovery of the distinct origins of UB and nephron/stromal progenitors has significantly advanced research on the directed differentiation of PSCs ( Fig 1.9 ). The T+ tailbud mesenchyme at E8.5, redefined as the source of NPs, was known to differentiate and be maintained under active Wnt signaling in vivo ( Fig. 1.10 ). Accordingly, T+ tailbud mesenchyme-like cells were induced from mouse ESCs by prolonged exposure to high concentrations of a Wnt signaling agonist (60 hours). The subsequent combined application of activin, retinoic acid, Bmp, and a moderate level of Wnt agonist was found to synergistically induce the expression of Osr1, Wt1, and Hox11, the representative markers of the posterior IM. Subsequent treatment with low levels of Wnt agonists and Fgf9 activated the expression of NP markers. When these NPs were co-cultured with the embryonic spinal cord, acting as exogenous Wnt signaling sources in place of the UB, they differentiated into self-organized epithelial nephron structures that included glomerular podocytes, parietal epithelia (Bowman capsule), and renal tubules. The signaling factors used in mouse ESC differentiation were also effective in inducing human nephron structures from human-induced PSCs (iPSCs), with adjustments made for the interspecies developmental rate. For example, Wnt agonist treatment was extended from 60 hours in mice to 144 hours in humans, corresponding to the approximately 2.5 times longer period of early gastrulation processes. While there are recognizable variations in individual induction protocols, most subsequent reports have adopted sequential induction steps involving prolonged high-Wnt exposure followed by withdrawal of Wnt and addition of FGF signaling to induce the NP lineage ^, (see Fig. 1.9 ).

Lineage Specification Toward the Nephric Duct/Ureteric Bud

Several components of the genetic network supporting the development of the nephric duct and the UB have been identified (see Fig. 1.11 ). Pax2 and Pax8 are required to maintain the expression of Lhx1 . Nephric duct specification fails in Pax2/Pax8 mutants, while conditional deletion of Lhx1 impairs maintenance of the nephric duct epithelia. Pax2, Pax8, and Lhx1 altogether likely coordinate the expression of Gata3, which is necessary for elongation of the nephric duct. Gata3 and Emx2, which are required for the expression of Ret in the nephric duct, are both regulated by β-catenin (Ctnnb1), an effector of the canonical Wnt signaling pathway. ^{, ,} Unlike these genes that are expressed in the nephric duct epithelia, Aldh1a2 (Raldh2), a gene in the retinoic acid synthesis pathway, is expressed in the surrounding mesenchyme and likely acts in parallel with Gata3 to maintain Ret expression in the nephric duct epithelia. Therefore the absence of Gata3 or Aldh1a2 causes misguided elongation of the nephric duct, terminating into either blind-ended ureters or abnormal connections between the bladder and urethra. The absence of Aldh1a2 also leads to formation of ectopic ureters and hydronephrotic kidneys. Ret does not affect the nephric duct fate but has importance in later UB development and insertion of the nephric duct to the cloaca. Furthermore, scRNA-seq analysis of the developing nephric duct shows that it is divided into several subdomains from rostral to caudal and Tfap2b is expressed in the midregion. Deletion of Tfap2a and Tfap2b leads to defects in nephric duct morphogenesis and reduction of Gata3 + caudal domains, while Gata3 deletion results in the absence of the Aldh1a3 + Ret + caudalmost domain including the nephric duct tips ( Fig. 1.12 ).

Spatial organization of nephric duct progenitors.

Spatial development of the nephric duct. Representative genes expressed in each region are shown.

Modified with permission from Sanchez-Ferras O, Pacis A, Sotiropoulou M, et al. A coordinated progression of progenitor cell states initiate urinary tract development. Nat Commun. 2021;12:2627.

The Wolffian duct is derived from the E8.5 anterior IM, which differentiates from the T+ mesoderm earlier than the NP lineage (see Fig. 1.10 ). Consistent with the in vivo lineage separation process, a shorter exposure (36 hours vs. 60 hours in NP induction) of mouse ESCs to high concentrations of Wnt signaling followed by administration of retinoic acid, Fgf, and a transforming growth factor-beta (Tgfb) inhibitor induced the anterior IM to express Osr1/Pax2/Lhx1/Gata3 (see Fig. 1.9 ). The effect of Tgfb inhibitor in inducing anterior IM was in sharp contrast to the requirement of Tgfb superfamily agonist: activin signaling in posterior IM differentiation. Subsequent treatment with a moderate concentration of Wnt agonist, retinoic acid, and Fgf induced committed Wolffian duct precursors expressing Lhx1/Pax2/Ret and exhibiting cell surface molecules Cxcr4+/Kit+. Further continuous treatment with retinoic acid, Wnt signaling, and increased administration of Gdnf signaling induced Wolffian duct maturation and eventual formation of bud structures. Induction of the UB lineage in human iPSCs revealed that a 36-hour Wnt signaling treatment (vs. 144 hours for NP induction) for T+ mesoderm induction and the addition of a Bmp signaling inhibitor to the mouse anterior IM induction factors were critical. These observations suggest a commonly critical feature of the Wnt signal exposure period for the selective induction of either anterior or posterior IM-derived kidney lineages, as well as slight differences in signal requirements between species. Accordingly, subsequent reports on human UB lineage induction commonly used an initial short exposure (24 to 48 hours) of the Wnt signaling for the primitive streak/mesoderm differentiation, and then the anterior IM was induced with the combination of Fgf, retinoic acid, and dual inhibition of Tgfb and Bmp signaling. The induced ureteric buds in both mice and humans show branching capacity in vitro when embedded in the extracellular matrix in the presence of Wnt agonist and Gdnf (see Fig. 1.9 ).

In many cases of renal agenesis, a failure of the GDNF-Ret signaling axis has been identified. GDNF, a member of the TGF-β superfamily and secreted by the MM, activates the Ret-GFRα1 receptor complex that is expressed by cells of the nephric duct and the UB. Activation of the Ret tyrosine kinase is of central importance to ureteric budding and branching. Most mutant embryos lacking Gdnf, Ret, or Gfra1 exhibit partial or complete renal agenesis due to severe impairment of UB sprouting while exogenous GDNF is sufficient to induce sprouting of ectopic buds from the nephric duct. ^{, , ,} Consistently, other genes linked to renal agenesis are known to regulate the normal expression of GDNF. These include transcription factors (e.g., Eya1, Pax2, Six1, Hox11 paralogues, and Sall1 ) and proteins required to stimulate or maintain GDNF expression (e.g., GDF11, Kif26b, nephronectin, α8β1-integrin, and Fras1) (see Fig. 1.11 ). ^{, , , ,}

As described earlier, Eya1 mutants fail to form the MM. Pax2 is a transcriptional regulator of the paired box family and is expressed widely during the development of both UB and mesenchymal components of the urogenital system. ^, Through a combination of molecular and in vivo studies, it has been demonstrated that Pax2 appears to act as a transcriptional activator of Gdnf and regulates the expression of Ret . ^, Pax2 also appears to regulate kidney formation through epigenetic control because it is involved in the assembly of a histone H3–lysine 4 methyltransferase complex through the ubiquitously expressed nuclear factor PTIP, which regulates histone methylation. The Hox genes are conserved in all metazoans and specify positional information along the body axis. Hox11 paralogues include Hoxa11, Hoxc11, and Hoxd11 . Mice carrying mutations in any one of these genes do not have kidney abnormalities; however, triple-mutant mice for these genes demonstrate a complete absence of metanephric kidney induction. Eya1, Pax2, and Hox11 appear to form a complex to coordinately regulate the expression of G dnf . However, the GDNF reduction may not always be mediated by direct transcription. For example, total amount of secreted GDNF can be reduced secondarily when the maintenance of NPs is affected due to the impaired gene regulatory network consisting of multiple transcription factors. Indeed, there are many “super-enhancers,” which are co-occupied by Six2, Hoxd11, Osr1, Wt1, and Sall1, for the genes involved in the progenitor program, and these transcription factors appear to activate one another to maintain the progenitors.

Other possibilities include impaired adhesive interactions within the NPs and between the NP and ureteric bud. For example, Sall1 is necessary for the expression of the kinesin Kif26b by the MM cells, and in the absence of either Sall1 or Kif26b, the α8β1-integrin expressed by the MM is downregulated. The loss of Itga8 (α8 integrin) or Itgb1 (β1-integrin), as well as deletion of their ligand Npnt (nephronectin) expressed in the UB tips, compromises the adhesion of the MM cells to the UB tips, ultimately causing loss of G dnf expression and failure of UB outgrowth. ^{, ,} A MM-derived secreted protein Isthmin-1 (Ism1) also acts on integrin α8β1 and promotes cell–cell adhesion of the MM, thereby regulating GDNF expression UB attraction. Genetic inactivation of basement membrane proteins associated with Fraser syndrome ( Fras1, Frem1, and Frem2 ) also leads to renal agenesis characterized by severe downregulation of Gdnf expression. ^{, ,} It has been proposed that the Fras1/Frem1/Frem2 ternary complex anchors nephronectin to the UB basement membrane, thus stabilizing engagement with α8β1 integrin expressed by the MM ( Fig. 1.13 ). Grip1, a PDZ-domain protein known to interact with Fras1, is required to localize the Fras1/Frem1/Frem2 complex on the basal aspect of the UB epithelium. Grip1 mutations phenocopy Fraser syndrome, including renal agenesis, thus further highlighting the importance of the strategic localization of nephronectin on the UB surface toward the opposing MM.

Molecular model of renal defect in Fraser syndrome.

(A) Adhesion to the ureteric bud (UB) epithelium positively regulates the expression of glial cell–derived neurotrophic factor (GDNF) by the metanephric mesenchyme (MM) . Adhesion and GDNF expression are impaired in the absence of (B) nephronectin (expressed by the UB), (C) α8β1 integrin (expressed by the MM), and (D) or the Fras1/Frem1/Frem2 complex. Fras1, Frem1, and Frem2 are implicated in Fraser syndrome and are believed to coordinately anchor nephronectin to the UB basement membrane and stabilize the conjugation with α8β1 integrin.

Modified from Kiyosumi, Takeichi M, Nakano I, et al. Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome-associated proteins. J Cell Biol. 2012;197:677–689.

UB sprouting and subsequent branching requires a unique spatial organization of Ret signaling. The bulbous UB tip is a region enriched with proliferative ureteric epithelial cells, in contrast to the emerging stalk regions of the developing ureteric tree. ^, It is well appreciated that receptor tyrosine kinase signaling primarily through Ret is key to the proliferation of UB tip epithelia. Exogenous GDNF supplemented in explanted embryonic kidneys can cause expansion of the UB tip region toward the source of the ligand. ERK kinase activation is prominent within the ampullary UB terminals where Ret expression is elevated. Consistently, chimera analysis in mice reveals that Ret-deficient cells do not contribute to the formation of the UB tips. Altogether, these studies underscore the importance of strategic levels of Ret expression and activation of proliferative signaling pathways in the stereotypical sculpting of the nascent collecting duct network.

A ligand-receptor complex formed by GDNF, GFRα1, and Ret is necessary for autophosphorylation of Ret on its intracellular tyrosines ( Fig. 1.14 ). A number of downstream adaptor molecules and effectors have been identified to interact with active phosphorylated Ret, including Grb2, Grb7, Grb10, ShcA, Frs2, PLCγ1, Shp2, Src, and Dok adaptor family members (Dok4/5/6). These downstream Ret effectors altogether are likely contributors to the activation of the Ras/SOS/ERK and PI3K/Akt pathways supporting the proliferation, survival, and migratory behavior of the UB epithelium. ^{, ,} Knock-in mutations of the interaction site for Shc/Frs2/Dok adaptors on the short isoform of Ret lead to the formation of rudimentary kidneys. ^, Specific mutation of the PLCγ1 docking site on Ret leads to renal dysplasia and ureter duplications. The loss of Shp2 and upstream ERK regulators Map2k1 (Mek2) and Map2k2 (Mek1) in the UB lineage also cause severe renal hypoplasia phenocopying that is observed in occasional Ret-deficient kidneys. ^, UB-specific inactivation of Pten , a target of the PI3K/Akt pathway, disrupts UB branching. These findings underscore the significance of Ret signaling in normal UB branching.

Ret signaling pathway.

Ret is activated and becomes autophosphorylated on intracellular tyrosine residues upon association with glial cell–derived neurotrophic factor (GDNF) and GFRα1. Signaling molecules such as Grb2, Shc, FRS2, PLCγ1, and Shp2 bind directly to the phosphorylated tyrosine residues within the intracellular domain of Ret. Recruitment of Shc, FRS2, and Grb2 leads to activation of extracellular signal-regulated kinase (ERK) and PI3K/Akt pathways. GDNF-Ret signaling leads to specific activation of a host of genes, some of which are strongly dependent on the upregulation of the transcription factors Etv4 and Etv5 (solid arrows). Etv4/Etv5 activation requires activation of the PI3K/Akt but not the ERK pathway. Sox8 and Sox9 are believed to act in parallel to reinforce transcriptional responses to GDNF-Ret engagement. Some of these pathways are shared with the FGF7/10-FGFR2 receptor signaling system. Spry1 and Spred2 negatively regulate ERK signaling, whereas Dusp6 likely mitigates the dephosphorylation of the Ret receptor, thus acting as part of a negative feedback regulatory loop.

A number of transcriptional targets of Ret activation in UBs stimulated with GDNF have been elucidated (see Fig. 1.14 ). Among these are Ret itself and Wnt11, which stimulates Gdnf expression in the MM, suggesting that a positive feedback loop exists for the GDNF-Ret signaling pathway. Ret activation also positively regulates the ETS transcription factors Etv4 and Etv5, which are also necessary for normal UB branching morphogenesis. Etv4 -null homozygous mutants and compound heterozygous mutants for Etv4 and Etv5 manifest severe renal hypoplasia or renal agenesis, suggesting that these transcription factors are indispensable targets of Ret for proper UB development. In chimeric animals, Etv4/Etv5 -deficient cells, like Ret -deficient cells, fail to integrate within the UB tip domain. ^,

The gene Sprouty was identified as a general antagonist of receptor tyrosine kinases and was discovered for inhibiting the FGF and EGF signaling pathways. Of the four mammalian Sprouty homologs, Spry1 is expressed at the UB tips. Sprouty molecules are thought to uncouple receptor tyrosine kinases with the activation of ERK pathways either through competitive binding with the Grb2/SOS complex or the kinase Raf, effectively repressing ERK activation. Interestingly, its expression is upregulated upon GDNF-mediated Ret activation. This suggests that Ret activates a negative feedback mechanism via Spry1 to control activated ERK levels and modulate cell proliferation in the UB. ^, Thus Spry1 deficiency leads to ectopic UB induction and it can partially rescue renal development in the absence of either GDNF or Ret. ^, The transcriptional targets of Ret, such as Etv4, Etv5, and Wnt11, are retained in Gdnf/Spry1 or Ret/Spry1 compound null mutants. ^, Other signaling including FGF7/FGF10 and FGFR2 receptors is likely to partially compensates the GDNF-Ret pathway.

The generation of a sufficient number of nephrons requires a highly regulated balance between the expansion of NP compartments and the differentiation toward epithelialized renal vesicles ( Fig. 1.15 ). This has important clinical implications as the impaired renewal of nephron precursors or their perturbed differentiation can ultimately cause a wide range of renal pathologies due to significant paucity of functional nephrons. Here, the extracellular signaling molecules such as Wnt, Bmp, and Fgf, as well as the downstream transcription factor Six2, regulate the delicate balance between progenitor self-renewal and differentiation.

Tripartite inductive interactions regulating ureteric branching, nephron progenitor self-renewal, and nephrogenesis.

Six2 and Cited1 are expressed in the self-renewing nephron progenitors within the cap mesenchyme (CM) surrounding the ureteric bud. The UB tip domains express high levels of Ret, which is activated by glial cell–derived neurotrophic factor (GDNF) from the surrounding CM. Wnt11 is upregulated in response to Ret activation and stimulates GDNF synthesis in the CM. Wnt9b expressed by the UB and Fat4 by the Foxd1-positive stroma are required to initiate nephrogenesis from a subset of the CM. This results in the formation of a transient renal vesicle expressing FGF8 and Wnt4, factors that sustain epithelialization. The stroma expresses Aldh1a2, a gene required for retinoic acid synthesis, and genes for the retinoic acid receptors ( Rara and Rarb ). Retinoic acid signaling stimulates elevated expression of Ret in the UB tip domain while at the same time suppressing Ret expression in the UB cleft domain via Rara/Rarb2 and Ecm1 in the stroma to initiate bifurcation of the UB tip to generate new branches. Foxd1 in the cortical stroma also represses Dcn, thus relieving the Dcn-mediated suppression of BMP7-dependent signaling, which results in phosphorylation of SMAD1/5/8 (pSMAD1/5/8) and epithelialization of the cap mesenchyme.

NPs differentiate into epithelial nephron structures through mesenchymal-to-epithelial transition. The UB-secreted factor Wnt9b activates the canonical β-catenin–dependent pathway and induces the expression of Wnt4 and Fgf8 in part of the cap mesenchyme, called pretubular aggregates. The Wnt4 expressed in pretubular aggregates, in turn, cell-autonomously acts to form epithelialized renal vesicles (see Fig. 1.15 ). Canonical Wnt signaling pathway is necessary and sufficient for the early inductive actions of Wnt9b and Wnt4, although it is also known that Wnt4 can activate a noncanonical alternative pathway during the final phase of epithelialization. ^,

Although the Wnt signal was originally recognized as the differentiation-inductive signal in the NPs more recently, its role in the maintenance and proliferation of NPs especially through the interaction with transcription factor Six2, has become apparent in both vivo and in vitro studies.

The transcription factor Six2 is exclusively expressed in the undifferentiated NPs, called cap mesenchyme, which condenses adjacent to the UB and is downregulated once this cap mesenchyme differentiates into pretubular aggregates. Complete loss of Six2 causes premature ectopic formation of renal vesicles and the untimely depletion of nephron precursors. ^, In contrast, overexpression of Six2 prevented epithelialization of the cap mesenchyme. Therefore Six2 is required to keep these NP cells in a naïve, proliferative precursor state.

At the molecular level, Six2 has been demonstrated to function as both a transcriptional activator that maintains NPs and as part of a repressor complex silencing differentiation-related genes. ^, For the maintenance of NPs, Six2 synergizes with multiple transcription factors including Osr1, Hoxd11, Wt1, and Sall1 to promote the transcription of relevant downstream genes (e.g., Six2, Eya1, and Sall1 ), forming a positive feedback loop. To prevent nephron differentiation, Six2 interacts with Tcf (Lef) and Aes (Groucho/TLE) to form a repressor complex that suppresses genes related to epithelialization (e.g., Fgf8 and Wnt4 ). ^,

The interaction between Wnt signaling pathways and Six2 appears to be intricately dosage, duration, and context dependent. ^{, , ,} When Six2 expression remains high, Wnt signaling promotes progenitor renewal. In this situation, the stabilization of β-catenin promotes the association of Six2 downstream target Myc with β-catenin, favoring precursor proliferation. ^, Indeed, deletion of the agonistic Wnt signaling molecules R-spondins ( Rspo1 and Rspo3 ) leads to a rapid decline of NPs in mice. In vitro studies using primary cultures of mouse NPs further indicate the dose-dependent role of Wnt signaling, where weak activation favors proliferation, while strong action drives differentiation.

On the other hand, with a sustained canonical Wnt-signaling pathway, β-catenin accumulates and displaces Groucho family repressors converting the Tcf complex into a differentiation driver. The stabilization of β-catenin eventually interferes with Six2 expression, thus attenuating Six2 expression. Although the continued activation of β-catenin can initiate early tubulogenesis, as evidenced by the expression of Wnt4 and Fgf8, it fails to complete the mesenchymal-to-epithelial transition. Therefore the proper nephron differentiation requires the shutdown of canonical Wnt signaling after a transient activation. The more precise resolution of this time window has been addressed by in vitro nephron differentiation studies using chemical Wnt signaling activators, such as Gsk3β inhibitors. ^{, , ,}

Notch signaling is required to prime NPs for differentiation and contributes to the silencing of Six2 expression. All nephron segments fail to form when Notch signaling is lost within the Six2 ⁺ precursor lineage.

The growth factor BMP7 is also required for the formation of the nephrogenic compartment. Activation of the Jnk pathway mediates the proliferative effect of BMP7 in uncommitted NPs. ^, BMP7, through activation of the p38-MAPK, causes upregulation of the transcriptional repressor Trps1 . Loss of Trps1 severely impairs the formation of renal vesicles. It has been speculated that Trps1 may indirectly relieve repression of Cdh1 expression. Additionally, BMP7-dependent phosphorylation and nuclear translocation of SMAD1/5/8 are required for Wnt9b-induced epithelialization. Thus BMP7 has dual essential roles in promoting both progenitor replenishment and priming for epithelialization. How these pathways integrate with Six2 -dependent signaling complexes remains poorly understood.

FGF8, FGF9, and FGF20, and their cognate receptors FGFR1 and FGFR2 are essential to form nephrons. FGF9 and FGF20 are required to maintain the multipotency and proliferative state of NPs. FGF8 is not needed for the NP maintenance or formation of renal vesicles but is required for the survival of the newly formed nephrogenic epithelia. Renal vesicles lacking Fgf8 fail to express Wnt4 and Lhx1 and do not progress into S-shaped intermediate nephrons. ^, Potential downstream targets of FGFR1/FGFR2 relevant to nephrogenesis are the closely related MAGUK family proteins encoded by the genes Cask and Dlg1 . The absence of Cask and Dlg1 causes impaired proliferation and cell death in nephron precursors, with a distinctive dampening of the Ras/ERK signaling pathway.

On the basis of these observations, the protocols to expand the mouse NPs in vitro have been established and applied to human organoid-derived progenitors. Many of the resumes include BMP, FGF, a Notch inhibitor, and a Wnt activator at low concentrations because too much Wnt stimulation leads to progenitor differentiation. LIF is also shown to be effective for in vitro expansion, but the physiological role of LIF in progenitor maintenance in vivo remains unclear.