Patterning and early cell lineage decisions in the developing kidney: the role of Pax genes

Abstract

Specification of the intermediate mesoderm and the epithelial derivatives that will make the mammalian kidney depends on the concerted action of many transcription factors and signaling proteins. Among the earliest genes expressed in the nephric duct and surrounding mesenchyme is Pax2, whose function is essential for making and maintaining the epithelium. The Pax2 protein is subject to phosphorylation in response to signals that activate the c-Jun N-terminal kinase pathway, including Wnts and BMPs. In cell culture systems, Pax2 is know to recruit components of a histone H3 lysine 4 methyltransferase complex to specific DNA sites to alter the pattern of histone modifications and determine gene expression. This epigenetic function may underlie the ability of Pax2 and similar proteins to maintain cell lineages during development.

Introduction

One the most important papers published in the last 30 years, in my admittedly biased view, was one that I first came across as a graduate student in the early 1980s but did not really appreciate until nearly a decade later. The paper reported research carried out by Nuesslein and Wieschaus [1] and was the first to describe a genetic screen in the fruit fly Drosophila for mutations that affect body patterning, with an emphasis on segmentation and segment polarity along the anterior–posterior axis. This work has led to the identification of some of the most interesting developmental regulatory gene families and the first conceptualization of the fundamental principles of sequential compartmentalization of a complex developing organism. For their pioneering work, Nuesslein and Wieschaus, together with Ed Lewis whose work on homeotic mutations also warranted recognition, were awarded the Nobel Prize in Medicine in 1995.

The work of Nusslein and Wieschaus proved immensely important as many mammalian homologues of Drosophila developmental regulatory genes were quickly identified. Genes such as hedgehog and patch, paired, hunchback, runt, and wingless proved to be the prototypes for families of proteins that controlled not only a whole variety of developmental processes, but were also critical for cancer initiation and progression and other abnormalities. This work was important to me personally, as I began my postdoctoral fellowship in the lab of Peter Gruss, trying to identify the first mouse homologues of the paired gene family. Three of the segmentation genes found in the fly, namely, paired, gooseberry-proximal, and gooseberry-distal, contained a new type of DNA binding domain that was highly conserved across divergent species and allowed us to identify mouse and human genes merely by low-stringency hybridization. Genes encoding this domain were called Pax genes (for paired-box) and were subsequently associated with mammalian development.

In mice and humans, there are nine Pax genes that encode proteins with amino terminal DNA binding, i.e., paired domains. The proteins are classified into four subfamilies according to the presence of additional downstream elements, including an octapeptide repressor domain and a homeobox DNA binding domain (Table 1). All of the Pax genes are expressed in developing structures and control the early specification of specific cell types or the compartmentalization of the embryo into specific regions. Pax genes are among the many types of prototypical DNA binding proteins that evolved exclusively in metazoans and are likely to be important for cell lineage specification in multicellular organisms [2]. For example, Pax6, the human aniridia gene, was proposed to encode a master regulatory switch for eye development because of its ability to induce ectopic eyes in Drosophila [3] and in vertebrates [4]. While this may be an oversimplification, the idea that Pax genes are genetic switches for cell lineage decisions is supported by many lines of evidence. For example, in the lymphoid lineage, Pax5 determines the B cell phenotype [5], whereas in the kidney Pax2 appears to determine the epithelial cell phenotype.

Table 1. The Pax (paired-box) gene family.

Gene	Chromosome		Structural Features*			Mutalions		Expression Patterns
	Mouse	Human	PD	OD	HD	Mouse	Man
Pax1	2	20p				Undulated		vertebral column, thymus
Pax2	19	10q				Renal Coloboma		kidney, CNS, eye, ear
Pax3	1	2q				Splotch	Wardenburg	CNS, neural crest, nose, muscle
Pax4	6	7
Pax5	4	9p						CNS, B-lymphocytes
Pax6	2	11p				Small eye	Aniridia	CNS, eye, pituitary
Pax7	4	1p						CNS muscle, nose
Pax8	2	2q						CNS, kidney, thyroid
Pax9	12	14q						vertebral column, tooth buds

PD, paired domain; OD, octapeptide domain; HD, homeodomain; CNS, cental nervous system

Pax genes in kidney development

In the kidney, Pax2 is essential for initiating the development of the mesonephros and metanephros from the intermediate mesoderm. After gastrulation, the mesoderm is compartmentalized along the medio-lateral axis into axial, intermediate, and lateral plate mesoderm (for more comprehensive reviews of kidney development, see [6, 7]). Among the earliest markers specific for the intermediate mesoderm are the expression of the Pax2 and Pax8 genes, which are considered to be redundant at embryonic day 9.5 (E9.5) in the mouse [8]. However, at later stages, Pax2—but not Pax8—mutant mice exhibit complete agenesis of both kidneys and ureters [9]. In humans, the loss of one Pax2 allele can result in renal hypoplasia, vesicoureteral reflux, and optic nerve colobomas [10-12]. These phenotypes are consistent with the Pax2 expression pattern in the epithelial components of the mesonephros, the Wolffian duct, and the ureteric bud and in the developing renal tubules [13, 14]. Both in vivo and in vitro, Pax2 is required for the conversion of renal mesenchymal cells to a primitive, proliferating epithelium [15]. Prior to induction, Pax2 protein expression demarcates the metanephric mesenchyme, activates glial-derived neurotrophic factor (GDNF) expression such that ureteric bud epithelial outgrowth can occur, and controls the response to inductive signals [16]. In the developing nephrons, Pax2 expression persists in the condensing cap mesenchyme around the ureteric bud tips and in the comma and s-shaped bodies, being down-regulated first in the most proximal loop of the s-shaped body and then in the epithelial cells of the proximal and distal tubules (Fig. 1a).

Fig. 1.

Expression of paired-box 2 (Pax2) and groucho-related 4 (Grg4) protein in the embryonic kidney. Immunostaining for proteins in serial sagittal, sections from an embryonic day 16.5 (E16.5) mouse kidney. a The Pax2 protein is shown in red and E-cadherin is given in green. Note high levels of Pax2 protein in the peripheral nephrogenic zone, especially the cap mesenchyme (arrows). Note also that Pax2 levels decrease in the s-shaped bodies as the podocyte progenitors are specified (arrowheads). b The Grg4 protein is shown in red and E-cadherin is given in green. Note the low levels in nuclei of mesenchymal cells (arrowheads) and high levels in podocyte progenitor cells (arrowheads)

The cumulative genetic and expression data point to an essential role for Pax2 in patterning the early intermediate mesoderm such that renal epithelial cells can be generated. In the chick embryo, ectopic Pax2 could expand the region of intermediate mesoderm [8], whereas in the Xenopus embryo Pax2 and lim1 could achieve similar effects [17]. Thus, within the context of the developing mesoderm, Pax2 may be sufficient to define the boundaries of the intermediate mesoderm and the kidney progenitor field along the medio-ventral axis.

Pax genes in disease

In addition to their fundamental roles during embryonic development, Pax genes also contribute to the initiation and progression of disease. In cancer cells, Pax2 is aberrantly expressed in human Wilms’ tumors [14], in adult renal cell carcinoma [18, 19], and in prostate carcinomas [20], and it may be a necessary for tumor growth [18]. Pax2 expression is also linked to endometrial cancer in response to tamoxifen and estrogen [21], consistent with the early expression and function of Pax2 in the Mullerian ducts [9].

Persistent Pax2 expression is observed in other diseases characterized by the aberrant proliferation of renal epithelial cells, such as polycystic kidney disease (PKD) [22] and infantile cystic–dysplastic kidneys [23]. In two different mouse models of PKD, reduced Pax2 gene dosage slows renal cyst growth by increasing the rate of apoptosis in cystic epithelia [22, 24], suggesting that Pax2 regulates essential survival factors for proliferating epithelia. Unlike the proximal and distal tubules, in adult inner medullary collecting ducts, Pax2 expression persists and is up-regulated under hyperosmotic conditions to promote survival [25]. In contrast, persistent expression in proximal tubules and glomerular podocytes in transgenic mice results in microcystic kidneys and nephrotic syndrome [26]. While Pax2 expression in mature non-dividing proximal tubular epithelial cells is undetectable, Pax2 expression persists in other urogenital epithelia, such as the endometrial glands of the uterus where its loss, together with the tumor suppressor PTEN, is associated with endometrial precancerous lesions [27]. Thus, both the gain or loss of Pax expression can be deleterious in developing and in adult organisms.

The biochemistry of Pax protein function

While the genetics of Pax genes has clearly established their biological roles in morphogenesis, how the proteins achieve their biochemical functions is still unclear. The Pax proteins interact with DNA through the characteristic 128 amino acid paired domain, located at the amino-terminus of the protein that binds a bipartite DNA recognition sequence [28-31]. The crystal structure of the Drosophila paired protein bound to DNA [32] shows three amino-terminal α-helices that resemble a homeo-domain, followed by a carboxyl terminal region with three smaller α-helices. The amino terminal α-helices contact the 3′ part of the bipartite target sequence, whereas the carboxyl terminal tail recognizes the 5′ end of the DNA sequence. Furthermore, interactions between the paired domain and the DNA recognition sequences can change the conformation of the Pax protein and the target DNAs [33, 34]. The size and complexity of the DNA binding domain found in Pax proteins has made it difficult to discern consensus binding sites, although several have been proposed.

Genes thought to be up regulated by Pax2 include Wt1 [35], gdnf [16], Wnt4 [36], and sfrp2 [37] in the developing kidney and engrailed-2 [38] in the developing hindbrain. Yet, these may represent only a small fraction of the potential Pax2 target genes. In cell culture systems, Pax2-dependent transcription activation requires the serine–threonine-rich carboxyl-terminal domain [39], which can be phosphorylated by the c-Jun N-terminal kinase (JNK) to enhance transactivation of Pax2 reporter genes [40]. JNK translocates to the nucleus in response to a variety of external signals, including Wnt proteins, and phosphorylates specific nuclear transcription factors [41]. In the kidney, the most relevant upstream activators of JNK may be Wnt4 and Wnt9b, which are known regulators of kidney development [42, 43]. The activation of JNK has been observed in cultured nephron progenitor cells [44] and also in response to bone morphogenetic proteins (BMPs) in whole embryonic kidney organ cultures [45]. The Pax2 protein also associates with the JNK scaffolding protein Jip1, and this interaction is enhanced upon activation of the JNK signaling module [40]. Compound mutants of JNK1, 2, and 3 show renal hypoplasia and optic nerve coloboma very similar to Pax2 hypomorphic phenotype [46]. Furthermore, inhibition of JNK activity by pharmacological inhibitors can inhibit renal development in vitro (Fig. 2). JNK inhibition results in Pax2-positive metanephric mesenchymal cells that fail to aggregate around the tips of the ureteric buds. Clearly, JNK activation can result in the phosphorylation of many other nuclear effector proteins, such as c-Jun and ATF. Yet these data suggest an attractive model whereby Wnt signaling drives increased phosphorylation of the Pax2 transactivation domain to stimulate Pax2-dependent gene expression in response to inductive signals. However, the identification of specific sites of phosphorylation on the Pax2 protein and confirmation that such sites are indeed phosphorylated in vivo will be necessary to define the mechanism of JNK function in the developing kidney.

Fig. 2.

Effect of c-Jun N-terminal kinase (JNK) inhibitors on kidney development in vitro. Kidney rudiments dissected from E11.5 mouse embryos were cultured for 2 days either with control media (a) or with 10 μM of SP600125, a specific JNK inhibitor (b). Whole rudiments were stained with antibodies against Pax2 (red) or cytokeratin (green). Cytokeratin marks the ureteric bud and its branches. Note the condensation of Pax2-positive cells around the ureteric bud tips in a (arrows). Upon JNK inhibition, Pax2-positive cells are diffuse and fail to aggregate (b, arrows)

Pax proteins also suppress gene expression by interactions with Groucho (Grg/Tle) proteins [47]. Grg4 suppresses Pax2-dependent gene activation in cell culture, can form a complex at the DNA binding site, and can completely suppress phosphorylation of the Pax2 transactivation domain, even in the presence of activated JNKs [48]. Long-term repression by Groucho is thought to involve interactions with histone deacetylases and the chromatin silencing machinery [49, 50]. However, deacetylation of histones is not the only possible mechanism of Groucho-mediated repression, as experiments with deletion mutants [51], deacetylase inhibitors [49], and anti-HDAC antibodies [52] would suggest. Grg4 expression is found at low levels in the kidney mesenchyme, including Pax2-positive cap mesenchyme (Fig. 1b). However, as nephron development progresses, Grg4 is up-regulated in the s-shaped bodies in podocyte progenitor cells and persists in the more mature podocytes.

The Pax proteins and histone modification

Despite the well-characterized genetics and structural biology, few Pax proteins have been directly linked to the transcription machinery. Until recently, most assays for Pax activity in cells utilized transient reporters outside the context of chromatin. However, it is now possible to to model Pax2 activity along different lines (Fig. 3). An emerging hypothesis is that Pax proteins mark specific regions of chromatin for histone modification. Such epigenetic marks can delineate both active and inactive regions of the genomes in a stable and heritable manner Thus, cell lineage restriction becomes fixed at the level of chromatin structure, thereby establishing chromatin in an active or repressed state such that transcription or silencing can be maintained in subsequent generations of daughter cells.

Fig. 3.

Model of Pax2-mediated chromatin remodeling. Pax2 binds to a target DNA sequence and interacts with the adaptor protein PTIP when Pax2 is phosphorylated. PTIP recruits a KMT2 and promotes H3K4 methylation. This enables nucleosome remodeling factors (NuRFs) to keep the chromatin open and allow for nucleosome movement. Under conditions of increasing Groucho (Grg/Tle) concentration, Pax2 binds to a Grg/Tle complex, which dephosphorylates the Pax2 carboxy-terminal domain and inhibits PTIP interactions. The Grg/Tle complex may then promote silencing by recruiting chromatin condensation proteins such as HP1

Chromatin biology and the study of epigenetic pathways have progressed due to the remarkable progress made in defining the patterns of histone modifications and identifying the enzymes responsible for the methylation and acetylation of histone tails. While the Polycomb and Trithorax group of genes were defined genetically as repressors and activators, respectively, in flies, the true biochemical function of these proteins was not evident until the purification of histone methyltransferases and their associated proteins in yeast and tetrahymena (for review see [53]). Thus, many of the Polycomb group proteins are part of complexes that methylate histone H3 at lysine 9 or lysine 27, or histone H4 at lysine 20; these modifications correlate with gene silencing and heterochromatin formation. The Trithorax group of proteins generally belong to complexes associated with histone H3 lysine 4 (H3K4) or lysine 36 (H3K36) methylation, modifications usually associated with active gene expression and euchromatin. These specific lysine residues can be mono-, di-, or trimethylated, further increasing the combinatorial complexity of nucleosome modifications.

The modification of histones was first linked to cell lineage decisions in embryonic stem cells by large-scale genomic chromatin immunoprecipitation analyses with antibodies specific for trimethylation at H3K4 and K3K27. In pluripotent embryonic stem (ES) cells, many key regulatory genes had low levels of both types of methylation marks, even though the genes were not expressed [54, 55]. These so-called bivalent epigenetic marks were then resolved into high levels of trimethyl H3K4 or H3K27 depending on whether the genes were expressed or not in subsequent differentiated derivatives. These data are consistent with a model of cell lineage decision-making that requires the compartmentalization of the genome into active and inactive domains. In mammalian development, the loss of pluripotency occurs as the epiblast undergoes gastrulation to form the primary germ layers. Strikingly, many of the epigenetic regulatory genes, including Polycomb and Trithorax homologues and de novo DNA methylases, show embryonic defects at this time.

If an accumulation of positive and negative epigenetic marks is essential for differentiation along cell lineage pathways, then specific proteins must control the locus and tissue specificity for Polycomb and Trithorax complexes. In flies, the Polycomb response elements (PREs) are known cis-acting DNA sequences that bind directly to the complex [56, 57]. However, in mammals, PREs have not been described, nor is it clear how histone methyltransferase complexes recognize individual genes at the right time.

The identification of the Pax2 interacting protein PTIP led us to re-examine the biochemical function of Pax proteins in development. PTIP is a ubiquitously expressed nuclear protein that is part of an H3K4 methylation complex and contains a carboxy-terminal phospho-serine binding domain [58-61]. In cell culture, Pax2 binding to DNA recruits PTIP and a KMT2C/D complex that methylates H3K4 at that site [61]. The KMT2s are SET domain proteins that are the mammalian homologues of the Drosophila epigenetic regulator Trithorax. The recruitment of KMT2D requires PTIP, suggesting that the Pax–PTIP interaction is a rate-limiting step in nucleating the complex (Fig. 3). Thus, PTIP acts as an adaptor protein that links the H3K4 methylation machinery to a sequence-specific binding protein. Although there are multiple Trithorax homologues in mammals, PTIP was not found in the KMT2A (MLL1) complex that was first purified, rather it seems to co-purify with the KMT2C/D complexes, suggesting that there may be other adaptor molecules yet to be discovered.

Mice homozygous for a PTIP null allele are post-gastrulation lethal, disorganized, and developmentally arrested [62]. This phenotype is more severe than any of the KMT2 mutations described to date. By E8.5 there is a global reduction in detectable levels of H3K4 di- and trimethylation. These data suggest that PTIP must interact with other DNA binding proteins beyond the Pax family. This is confirmed in the Xenopus gastrula where a PTIP homologue was shown to mediate activin signaling by binding to P-Smad2 [63]. In the fly embryo, a PTIP homologue is also essential for early patterning, including the correct expression of segmentation genes prior to gastrulation and the activation of many Polycomb group target genes once repression is relieved [64]. We have also studied PTIP in embryonic stem cells, where it is necessary for retaining pluripotency during in vitro culture [65].

The Pax2 gene has a close relative, Pax5, whose paired domain is identical and whose biochemical function is likely to be very similar because the two proteins can complement each other [66]. Given that Pax5 specifies B cell fate in the lymphoid lineage, we recently examined the function of PTIP in B cells. Consistent with the previous data linking PTIP to H3K4 methylation, PTIP-deficient B cells are unable to methylate the promoter regions necessary for immunoglobulin class switch recombination [67], a process that requires Pax5 protein and long-range chromatin interactions.

Our hypothesis is that Pax proteins mark specific regions of chromatin through the modification of histones (Fig. 3). These epigenetic marks can delineate both active and inactive regions of the genomes in a stable and heritable manner through potential interactions with nucleosome remodeling complexes and other chromatin effectors [68]. In the case of Pax2, we propose that interactions with PTIP specify active regions of chromatin, whereas interactions with the Grg/Tle family or repressors could delineate silent regions of chromatin. One advantage of such a model is that a given gene could be activated early, when levels of Grg/Tle are low, and silenced as Grg/Tle levels rise. In the developing intermediate mesoderm and the kidney, this could establish chromatin domains in an active state such that embryonic gene transcription can occur in epithelial progenitors and subsequent generations of proliferating cells. Thus, the Pax2-positive cap-mesenchyme, surrounding the ureteric bud tips, could be imprinted with an epithelial fate in response to inductive signals. Since PTIP and the KMT2 proteins are expressed in all cells, the control is achieved by the activation of a DNA binding proteins that targets the histone methylation complex to specific genes. In order to examine this hypothesis, new tools and methods must be developed such that Pax2 target genes can be identified and analyzed at the level of chromatin structure.

Summary

The specification of cell lineages and patterning in the embryo occurs sequentially as specific regions are increasingly restricted in their developmental fates. When and how this occurs is still not entirely clear. Nevertheless, the role of epigenetic regulatory genes in partitioning the genome into active and inactive domains is evident in a variety of organisms and is highly conserved through evolution. The function of Pax2 in the kidney has been inferred by the phenotypic analysis of loss-of-function mutants in mice, fish, and humans. Our recent data suggest that Pax proteins help establish the renal epithelial fate by delineating chromatin domains through histone modification. The novel protein PTIP is a key adaptor that links Pax proteins and possibly many other types of DNA binding proteins to a histone H3K4 methyltransferase complex. If such modifications are established in development, we now need to address the effects of epigenetic changes on renal disease states, on the stability of the terminal epithelial phenotype, and in the aging cell.