Constitutive metanephric mesenchyme-specific expression of interferon-gamma causes renal dysplasia by regulating Sall1 expression

<div><p>Transplacental viral and parasitic infections have been shown to initiate an innate response in the mammalian embryo by increasing the expression of pro-inflammatory cytokines such as interferon-gamma (Ifng). However, the developmental consequences of an activated innate immunity and, in particular, the effects of induction of Ifng expression independent of infection have been largely overlooked. Here, we demonstrate <i>in vivo</i> that the conditional overexpression of Ifng in metanephric mesenchymal (MM) progenitors results in renal agenesis or hypoplasia. Cell death was observed in and around the MM region of E10.5–11.5 mutants where Ifng was constitutively expressed during early kidney development and resulted in a retardation of branching morphogenesis. Furthermore, isolated mutant or normal Ifng-treated metanephroi replicated this phenotype in culture, demonstrating the inherent nature of the aberrant morphogenesis. The expression of renal progenitor marker Sall1 was significantly decreased in the MM of mutant kidneys, suggesting that a reduction in <i>Sall1</i> may be the cause of cell death in the MM during early kidney development and that, in turn, retards UB branching in the mutants. Therefore, the aberrant induction of Ifng expression, as part of an innate immune response, may contribute to renal agenesis or hypoplasia during early metanephric development by regulating the MM progenitor population.</p></div

Selective In Vitro Propagation of Nephron Progenitors Derived from Embryos and Pluripotent Stem Cells

Nephron progenitors in the embryonic kidney propagate while generating differentiated nephrons. However, in mice, the progenitors terminally differentiate shortly after birth. Here, we report a method for selectively expanding nephron progenitors in vitro in an undifferentiated state. Combinatorial and concentration-dependent stimulation with LIF, FGF2/9, BMP7, and a WNT agonist is critical for expansion. The purified progenitors proliferated beyond the physiological limits observed in vivo, both for cell numbers and lifespan. Neonatal progenitors were maintained for a week, while progenitors from embryonic day 11.5 expanded 1,800-fold for nearly 20 days and still reconstituted 3D nephrons containing glomeruli and renal tubules. Furthermore, progenitors generated from mouse embryonic stem cells and human induced pluripotent cells could be expanded with retained nephron-forming potential. Thus, we have established in vitro conditions for promoting the propagation of nephron progenitors, which will be essential for dissecting the mechanisms of kidney organogenesis and for regenerative medicine

Preferential Propagation of Competent SIX2+ Nephronic Progenitors by LIF/ROCKi Treatment of the Metanephric Mesenchyme

Understanding the mechanisms responsible for nephrogenic stem cell preservation and commitment is fundamental to harnessing the potential of the metanephric mesenchyme (MM) for nephron regeneration. Accordingly, we established a culture model that preferentially expands the MM SIX2+ progenitor pool using leukemia inhibitory factor (LIF), a Rho kinase inhibitor (ROCKi), and extracellular matrix. Passaged MM cells express the key stem cell regulators Six2 and Pax2 and remain competent to respond to WNT4 induction and form mature tubular epithelia and glomeruli. Mechanistically, LIF activates STAT, which binds to a Stat consensus sequence in the Six2 proximal promoter and sustains SIX2 levels. ROCKi, on the other hand, attenuates the LIF-induced differentiation activity of JNK. Concomitantly, the combination of LIF/ROCKi upregulates Slug expression and activates YAP, which maintains SIX2, PAX2, and SALL1. Using this novel model, our study underscores the pivotal roles of SIX2 and YAP in MM stem cell stability

Proliferation and cell death during embryonic kidney development.

<p>A. Proliferating cells were detected with phosphohistone 3 antibody in kidneys at E14.5. Proliferation rates over comparable areas are similar between normal and mutant kidneys. B. TUNEL analysis of kidney at E14.5. The incidence of TUNEL-+ cells at this stage over comparable areas is similar between normal and mutant kidneys. C. immunostaining for anti-phosphohistone 3 in proliferating cells in kidneys at E11.5. The dotted line encircles comparable portions of metanephroi. D. TUNEL analysis of kidney at E10.5 (upper) and E11.5 (lower). The kidney is stained with a rabbit anti-Pax2 antibody to demarcate the MM/UB area. The epithelial structure is the nephric duct (ND), and the adjacent Pax2+ tissue delineates the MM region. The UB is also marked with Hoxb7/myr-venus (A, B, D). TUNEL+ cells are numerous in mutant MMs. nor—normal, mu—mutant.</p

Overexpression of <i>Ifng</i> in MM progenitor cells results in renal agenesis or hypoplasia.

<p>49% of mutant embryos have two small kidneys (mu-left), 30% of mutant embryos have one small kidney (mu-middle), and 21% of mutant embryos have no kidneys at E14.5 (mu-right). nor—normal, mu—mutant.</p

Expression of Ifng increases in the kidneys of the <i>Ifng</i> GOF mouse.

<p>A. Schematic depicting the Ifng GOF mouse line. Murine Ifng is ectopically expressed in the Pax3-expressing domain after Cre-mediated excision of a LoxP-CAT-Stop cassette. B. The amount of Ifng in kidneys increases about 13 fold in mutant vs. normal kidneys, when <i>Ifng</i> expression is targeted to the MM. The amount of Ifng was measured by ELISA. C. Ifng expression (red) is elevated in the MM-derived areas. The UB is marked with Hoxb7/myr-venus (green). Scale bar = 20 μm. D. <i>Ifng</i> mRNA is elevated in kidneys from <i>Ifng</i> GOF mice. mRNA is measured by semi-quantitative RT-PCR. <i>Ifngr1</i> and <i>Ifngr2</i> are also detected in mouse embryonic kidneys. E. Stat1 protein is activated in mutant kidneys, as determined by phosphorylation of Y701 and S727 in immunoblots. As a loading control, β-Actin was used. All these experiments used E14.5 kidneys. nor—normal, mu—mutant.</p

UB branching morphogenesis is retarded in Ifng GOF mutants.

<p>A. WISH for <i>c-Ret</i> at E10.5 (upper) or at E11.5 (lower). UB tips are marked with black arrows. ND—nephric duct, CND—common nephric duct. B. Calbindin staining of kidney explants cultured for 24h. C. UB tip numbers are significantly reduced in mutants relative to normal embryos during development. Hoxb7/myr-venus containing kidneys were isolated at E11.5, E12.5 and E13.5 and UB tips were counted. D. Hoxb7/myr-venus-expressing cultured kidney explants after treatment with mouse recombinant Ifng (20ng/ml). The fluorescent (upper) or bright field (lower) images were taken from cultures at 5h, 24h and 48h. For explant cultures, E11.5 kidneys were used (B & D). nor—normal, mu—mutant.</p

The expression of Sall1 and its potential targets are modulated in mutant kidneys.

<p>E14.5 kidneys were used for all analyses. A. semi-quantitative RT-PCR for <i>Sall1</i>. The expression level of <i>Sall1</i> mRNA is decreased in mutants. B. Immunostaining of Sall1. Sall1 protein expression is also decreased in mutants. Lower: higher magnification of areas shown in the white rectangles. C. Active β-Catenin is determined by immunoblotting with a β-Catenin antibody that detects unphosphorylated residues at Ser33/Ser37/Thr41, which target the molecule for ubiquitination. β-Catenin is activated in mutants. D. RT-PCR of <i>Wnt9b</i> and <i>Axin2</i>. The mRNA levels of <i>Wnt9b</i> and <i>Axin2</i>, a target gene of Wnt/β-Catenin signaling, are increased in mutants. E. semi-quantitative RT-PCR for <i>Kif26b</i>. The mRNA level of <i>Kif26b</i>, a target gene for Sall1, is reduced in mutants. nor—normal, mu—mutant.</p

SIX2 and CITED1, markers of nephronic progenitor self-renewal, remain active in primitive elements of Wilms\u27 tumor

Purpose: SIX2 and CITED1 are transcriptional regulators that specify self-renewing nephronic progenitor cells of the embryonic kidney. We hypothesized that SIX2, which promotes and maintains this stem cell population, and CITED1 remain active in Wilms\u27 tumor (WT). Methods: To evaluate expression domains and the pathogenic significance of SIX2 and CITED1 across WT, the Children\u27s Oncology Group provided 40 WT specimens of stages I to IV (n = 10 per stage), which were enriched for unfavorable histology (n = 20) and treatment failure (relapse or death, n = 20). SIX2 and CITED1 protein expression was evaluated qualitatively (immunohistochemistry) and quantitatively (Western blot, or WB). Gene transcription was estimated using quantitative real-time polymerase chain reaction (qRT-PCR). Results: SIX2 was visualized by immunohistochemistry in 36 (94.7%) of 38 specimens. Protein and messenger RNA expression of SIX2 were quantitatively similar across all stages of disease (P = .48 WB; P = 0.38 qPCR), in favorable or unfavorable histology (P = 0.51 WB; P = 0.58 qPCR), and in treatment failure or success (P = 0.86 WB; P = 0.49 qPCR). Although CITED1 expression paralleled SIX2 qualitatively, no quantitative correlation between SIX2 and CITED1 expression was observed (Spearman correlation coefficient, 0.28; P = 0.08). As in the fetal kidney, overlapping, but also distinct, WT cellular expression domains were observed between SIX2 and CITED1. Conclusion: SIX2 and CITED1 remain active across all disease characteristics of WT. Activity of these genes in WT potentially identifies a population of self-renewing cancer cells that exhibit an embryonic, stemlike phenotype. Taken together, these transcriptional regulators may be fundamental to WT cellular self-renewal and may represent targets for novel therapies that promote terminal differentiation

Mesoderm-specific <i>Stat3</i> deletion affects expression of <i>Sox9</i> yielding Sox9-dependent phenotypes

<div><p>To date, mutations within the coding region and translocations around the <i>SOX9</i> gene both constitute the majority of genetic lesions underpinning human campomelic dysplasia (CD). While pathological coding-region mutations typically result in a non-functional SOX9 protein, little is known about what mechanism(s) controls normal <i>SOX9</i> expression, and subsequently, which signaling pathways may be interrupted by alterations occurring around the <i>SOX9</i> gene. Here, we report the identification of Stat3 as a key modulator of <i>Sox9</i> expression in nascent cartilage and developing chondrocytes. <i>Stat3</i> expression is predominant in tissues of mesodermal origin, and its conditional ablation using mesoderm-specific <i>TCre</i>, <i>in vivo</i>, causes dwarfism and skeletal defects characteristic of CD. Specifically, <i>Stat3</i> loss results in the expansion of growth plate hypertrophic chondrocytes and deregulation of normal endochondral ossification in all bones examined. Conditional deletion of <i>Stat3</i> with a <i>Sox9Cre</i> driver produces palate and tracheal irregularities similar to those described in <i>Sox9</i>^<i>+/-</i> mice. Furthermore, mesodermal deletion of <i>Stat3</i> causes global embryonic down regulation of <i>Sox9</i> expression and function <i>in vivo</i>. Mechanistic experiments <i>ex vivo</i> suggest Stat3 can directly activate the expression of <i>Sox9</i> by binding to its proximal promoter following activation. These findings illuminate a novel role for Stat3 in chondrocytes during skeletal development through modulation of a critical factor, <i>Sox9</i>. Importantly, they further provide the first evidence for the modulation of a gene product other than <i>Sox9</i> itself which is capable of modeling pathological aspects of CD and underscore a potentially valuable therapeutic target for patients with the disorder.</p></div