Progenitor translatome changes coordinated by Tsc1 increase perception of Wnt signals to end nephrogenesis.

Mammalian nephron endowment is determined by the coordinated cessation of nephrogenesis in independent niches. Here we report that translatome analysis in Tsc1+/- nephron progenitor cells from mice with elevated nephron numbers reveals how differential translation of Wnt antagonists over agonists tips the balance between self-renewal and differentiation. Wnt agonists are poorly translated in young niches, resulting in an environment with low R-spondin and high Fgf20 promoting self-renewal. In older niches we find increased translation of Wnt agonists, including R-spondin and the signalosome-promoting Tmem59, and low Fgf20, promoting differentiation. This suggests that the tipping point for nephron progenitor exit from the niche is controlled by the gradual increase in stability and possibly clustering of Wnt/Fzd complexes in individual cells, enhancing the response to ureteric bud-derived Wnt9b inputs and driving synchronized differentiation. As predicted by these findings, removing one Rspo3 allele in nephron progenitors delays cessation and increases nephron numbers in vivo.

Notch dimerization and gene dosage are important for normal heart development, intestinal stem cell maintenance, and splenic marginal zone B-cell homeostasis during mite infestation.

Cooperative DNA binding is a key feature of transcriptional regulation. Here we examined the role of cooperativity in Notch signaling by CRISPR-mediated engineering of mice in which neither Notch1 nor Notch2 can homo- or heterodimerize, essential for cooperative binding to sequence-paired sites (SPS) located near many Notch-regulated genes. Although most known Notch-dependent phenotypes were unaffected in Notch1/2 dimer-deficient mice, a subset of tissues proved highly sensitive to loss of cooperativity. These phenotypes include heart development, compromised viability in combination with low gene dose, and the gut, developing ulcerative colitis in response to 1% dextran sulfate sodium (DSS). The most striking phenotypes-gender imbalance and splenic marginal zone B-cell lymphoma-emerged in combination with gene dose reduction or when challenged by chronic fur mite infestation. This study highlights the role of the environment in malignancy and colitis and is consistent with Notch-dependent anti-parasite immune responses being compromised in Notch dimer-deficient animals.

A bigenic mouse model of FSGS reveals perturbed pathways in podocytes, mesangial cells and endothelial cells.

Focal segmental glomerulosclerosis is a major cause of end stage renal disease. Many patients prove unresponsive to available therapies. An improved understanding of the molecular basis of the disease process could provide insights leading to novel therapeutic approaches. In this study we carried out an RNA-seq analysis of the altered gene expression patterns of podocytes, mesangial cells and glomerular endothelial cells of the bigenic Cd2ap+/-, Fyn-/- mutant mouse model of FSGS. In the podocytes we observed upregulation of many genes related to the Tgfß family/pathway, including Gdnf, Tgfß1, Tgfß2, Snai2, Vegfb, Bmp4, and Tnc. The mutant podocytes also showed upregulation of Acta2, a marker of smooth muscle and associated with myofibroblasts, which are implicated in driving fibrosis. GO analysis of the podocyte upregulated genes identified elevated protein kinase activity, increased expression of growth factors, and negative regulation of cell adhesion, perhaps related to the observed podocyte loss. Both podocytes and mesangial cells showed strong upregulation of aldehyde dehydrogenase genes involved in the synthesis of retinoic acid. Similarly, the Cd2ap+/-, Fyn-/- mesangial cells, as well as podocytes in other genetic models, and the glomeruli of human FSGS patients, all show upregulation of the serine protease Prss23, with the common thread suggesting important functionality. Another gene with strong upregulation in the Cd2ap+/-, Fyn-/- mutant mesangial cells as well as multiple other mutant mouse models of FSGS was thrombospondin, which activates the secreted inactive form of Tgfß. The Cd2ap+/-, Fyn-/- mutant endothelial cells showed elevated expression of genes involved in cell proliferation, angioblast migration, angiogenesis, and neovasculature, all consistent with the formation of new blood vessels in the diseased glomerulus. The resulting global definition of the perturbed molecular pathways in the three major cell types of the mutant glomerulus provide deeper understanding of the molecular pathogenic pathways.

Hamartin regulates cessation of mouse nephrogenesis independently of Mtor.

Nephrogenesis concludes by the 36th week of gestation in humans and by the third day of postnatal life in mice. Extending the nephrogenic period may reduce the onset of adult renal and cardiovascular disease associated with low nephron numbers. We conditionally deleted either Mtor or Tsc1 (coding for hamartin, an inhibitor of Mtor) in renal progenitor cells. Loss of one Mtor allele caused a reduction in nephron numbers; complete deletion led to severe paucity of glomeruli in the kidney resulting in early death after birth. By contrast, loss of one Tsc1 allele from renal progenitors resulted in a 25% increase in nephron endowment with no adverse effects. Increased progenitor engraftment rates ex vivo relative to controls correlated with prolonged nephrogenesis through the fourth postnatal day. Complete loss of both Tsc1 alleles in renal progenitors led to a lethal tubular lesion. The hamartin phenotypes are not dependent on the inhibitory effect of TSC on the Mtor complex but are dependent on Raptor.

Intrinsic Age-Dependent Changes and Cell-Cell Contacts Regulate Nephron Progenitor Lifespan.

During fetal development, nephrons of the metanephric kidney form from a mesenchymal progenitor population that differentiates en masse before or shortly after birth. We explored intrinsic and extrinsic mechanisms controlling progenitor lifespan in a transplantation assay that allowed us to compare engraftment of old and young progenitors into the same young niche. The progenitors displayed an age-dependent decrease in proliferation and concomitant increase in niche exit rates. Single-cell transcriptome profiling revealed progressive age-dependent changes, with heterogeneity increasing in older populations. Age-dependent elevation in mTor and reduction in Fgf20 could contribute to increased exit rates. Importantly, 30% of old progenitors remained in the niche for up to 1 week post engraftment, a net gain of 50% to their lifespan, but only if surrounded by young neighbors. We provide evidence in support of a model in which intrinsic age-dependent changes affect inter-progenitor interactions that drive cessation of nephrogenesis.

The intracellular domains of Notch1 and Notch2 are functionally equivalent during development and carcinogenesis.

Although Notch1 and Notch2 are closely related paralogs and function through the same canonical signaling pathway, they contribute to different outcomes in some cell and disease contexts. To understand the basis for these differences, we examined in detail mice in which the Notch intracellular domains (N1ICD and N2ICD) were swapped. Our data indicate that strength (defined here as the ultimate number of intracellular domain molecules reaching the nucleus, integrating ligand-mediated release and nuclear translocation) and duration (half-life of NICD-RBPjk-MAML-DNA complexes, integrating cooperativity and stability dependent on shared sequence elements) are the factors that underlie many of the differences between Notch1 and Notch2 in all the contexts we examined, including T-cell development, skin differentiation and carcinogenesis, the inner ear, the lung and the retina. We were able to show that phenotypes in the heart, endothelium, and marginal zone B cells are attributed to haploinsufficiency but not to intracellular domain composition. Tissue-specific differences in NICD stability were most likely caused by alternative scissile bond choices by tissue-specific γ-secretase complexes following the intracellular domain swap. Reinterpretation of clinical findings based on our analyses suggests that differences in outcome segregating with Notch1 or Notch2 are likely to reflect outcomes dependent on the overall strength of Notch signals.

Pathogenic pathways are activated in each major cell type of the glomerulus in the Cd2ap mutant mouse model of focal segmental glomerulosclerosis.

BACKGROUND: Mutations in several genes expressed in podocytes, including Cd2ap, have been associated with focal segmental glomerulosclerosis in humans. Mutant mouse models provide an opportunity to better understand the molecular pathology that drives these diseases. METHODS: In this report we use a battery of transgenic-GFP mice to facilitate the purification of all three major cell types of the glomerulus from Cd2ap mutant mice. Both microarrays and RNA-seq were used to characterize the gene expression profiles of the podocytes, mesangial cells and endothelial cells, providing a global dual platform cross-validating dataset. RESULTS: The mesangial cells showed increased expression of profibrotic factors, including thrombospondin, Tgfb2 and Tgfb3, as well as the angiogenesis factor Vegf. They also showed upregulation of protective genes, including Aldh1a2, involved in retinoic acid synthesis and Decorin, a Tgfb antagonist. Of interest, the mesangial cells also showed significant expression of Wt1, which has generally been considered podocyte specific. The Cd2ap mutant podocytes showed upregulation of proteases as well as genes involved in muscle and vasculature development and showed a very strong gene expression signature indicating programmed cell death. Endothelial cells showed increased expression of the leukocyte adhesion associated factors Vcam1 and Sele, as well as Midkine (promoting angiogenesis), endothelin and many genes responsive to cytokines and interferons. CONCLUSIONS: This study provides a comprehensive analysis of the changing properties of the three cell types of the glomerulus in Cd2ap mutants, identifying activated and repressed pathways and responsible genes, thereby delivering a deeper molecular understanding of this genetic disease.

Second-generation Notch1 activity-trap mouse line (N1IP::CreHI) provides a more comprehensive map of cells experiencing Notch1 activity.

We have previously described the creation and analysis of a Notch1 activity-trap mouse line, Notch1 intramembrane proteolysis-Cre6MT or N1IP::Cre(LO), that marked cells experiencing relatively high levels of Notch1 activation. Here, we report and characterize a second line with improved sensitivity (N1IP::Cre(HI)) to mark cells experiencing lower levels of Notch1 activation. This improvement was achieved by increasing transcript stability and by restoring the native carboxy terminus of Cre, resulting in a five- to tenfold increase in Cre activity. The magnitude of this effect probably impacts Cre activity in strains with carboxy-terminal Ert2 fusion. These two trap lines and the related line N1IP::Cre(ERT2) form a complementary mapping tool kit to identify changes in Notch1 activation patterns in vivo as the consequence of genetic or pharmaceutical intervention, and illustrate the variation in Notch1 signal strength from one tissue to the next and across developmental time.

Single cell dissection of early kidney development: multilineage priming.

We used a single cell RNA-seq strategy to create an atlas of gene expression patterns in the developing kidney. At several stages of kidney development, histologically uniform populations of cells give rise to multiple distinct lineages. We performed single cell RNA-seq analysis of total mouse kidneys at E11.5 and E12.5, as well as the renal vesicles at P4. We define an early stage of progenitor cell induction driven primarily by gene repression. Surprising stochastic expression of marker genes associated with differentiated cell types was observed in E11.5 progenitors. We provide a global view of the polarized gene expression already present in the renal vesicle, the first epithelial precursor of the nephron. We show that Hox gene read-through transcripts can be spliced to produce intergenic homeobox swaps. We also identify a surprising number of genes with partially degraded noncoding RNA. Perhaps most interesting, at early developmental times single cells often expressed genes related to several developmental pathways. This provides powerful evidence that initial organogenesis involves a process of multilineage priming. This is followed by a combination of gene repression, which turns off the genes associated with most possible lineages, and the activation of increasing numbers of genes driving the chosen developmental direction.

A gene expression atlas of early craniofacial development.

We present a gene expression atlas of early mouse craniofacial development. Laser capture microdissection (LCM) was used to isolate cells from the principal critical microregions, whose development, differentiation and signaling interactions are responsible for the construction of the mammalian face. At E8.5, as migrating neural crest cells begin to exit the neural fold/epidermal ectoderm boundary, we examined the cranial mesenchyme, composed of mixed neural crest and paraxial mesoderm cells, as well as cells from adjacent neuroepithelium. At E9.5 cells from the cranial mesenchyme, overlying olfactory placode/epidermal ectoderm, and underlying neuroepithelium, as well as the emerging mandibular and maxillary arches were sampled. At E10.5, as the facial prominences form, cells from the medial and lateral prominences, the olfactory pit, multiple discrete regions of underlying neuroepithelium, the mandibular and maxillary arches, including both their mesenchymal and ectodermal components, as well as Rathke's pouch, were similarly sampled and profiled using both microarray and RNA-seq technologies. Further, we performed single cell studies to better define the gene expression states of the early E8.5 pioneer neural crest cells and paraxial mesoderm. Taken together, and analyzable by a variety of biological network approaches, these data provide a complementing and cross validating resource capable of fueling discovery of novel compartment specific markers and signatures whose combinatorial interactions of transcription factors and growth factors/receptors are responsible for providing the master genetic blueprint for craniofacial development.

Building an atlas of gene expression driving kidney development: pushing the limits of resolution.

Changing gene expression patterns is the essential driver of developmental processes. Growth factors, micro-RNAs, long intergenic noncoding RNAs, and epigenetic marks, such as DNA methylation and histone modifications, all work by impacting gene expression. The key features of developing cells, including their ability to communicate with others, are defined primarily by their gene-expression profiles. It is therefore clear that a gene-expression atlas of the developing kidney can provide a useful tool for the developmental nephrology research community. Toward this end, the GenitoUrinary Development Molecular Anatomy Project (GUDMAP) consortium has worked to create an atlas of the changing gene-expression patterns that drive kidney development. In this article, the global gene-expression profiling strategies of GUDMAP are reviewed. The initial work used laser-capture microdissection to purify multiple compartments of the developing kidney, including cap mesenchyme, renal vesicle, S-shaped bodies, proximal tubules, and more, which were then gene-expression profiled using microarrays. Resolution of the atlas was then improved by using transgenic mice with specific cell types labeled with green fluorescent protein (GFP), allowing their purification and profiling. In addition, RNA-Seq replaced microarrays. Currently, the atlas is being pushed to the single-cell resolution using microfluidic approaches that allow high-throughput RNA-Seq analysis of hundreds of individual cells. Results can identify novel types of cells and define interesting heterogeneities present within cell populations.

SP8 regulates signaling centers during craniofacial development.

Much of the bone, cartilage and smooth muscle of the vertebrate face is derived from neural crest (NC) cells. During craniofacial development, the anterior neural ridge (ANR) and olfactory pit (OP) signaling centers are responsible for driving the outgrowth, survival, and differentiation of NC populated facial prominences, primarily via FGF. While much is known about the functional importance of signaling centers, relatively little is understood of how these signaling centers are made and maintained. In this report we describe a dramatic craniofacial malformation in mice mutant for the zinc finger transcription factor gene Sp8. At E14.5 they show facial prominences that are reduced in size and underdeveloped, giving an almost faceless phenotype. At later times they show severe midline defects, excencephaly, hyperterlorism, cleft palate, and a striking loss of many NC and paraxial mesoderm derived cranial bones. Sp8 expression was primarily restricted to the ANR and OP regions during craniofacial development. Analysis of an extensive series of conditional Sp8 mutants confirmed the critical role of Sp8 in signaling centers, and not directly in the NC and paraxial mesoderm cells. The NC cells of the Sp8 mutants showed increased levels of apoptosis and decreased cell proliferation, thereby explaining the reduced sizes of the facial prominences. Perturbed gene expression in the Sp8 mutants was examined by laser capture microdissection coupled with microarrays, as well as in situ hybridization and immunostaining. The most dramatic differences included striking reductions in Fgf8 and Fgf17 expression in the ANR and OP signaling centers. We were also able to achieve genetic and pharmaceutical partial rescue of the Sp8 mutant phenotype by reducing Sonic Hedgehog (SHH) signaling. These results show that Sp8 primarily functions to promote Fgf expression in the ANR and OP signaling centers that drive the survival, proliferation, and differentiation of the NC and paraxial mesoderm that make the face.

Changes in the gene expression programs of renal mesangial cells during diabetic nephropathy.

BACKGROUND: Diabetic nephropathy is the leading cause of end stage renal disease. All three cell types of the glomerulus, podocytes, endothelial cells and mesangial cells, play important roles in diabetic nephropathy. In this report we used Meis1-GFP transgenic mice to purify mesangial cells from normal mice and from db/db mice, which suffer diabetic nephropathy. The purpose of the study is to better define the unique character of normal mesangial cells, and to characterize their pathogenic and protective responses during diabetic nephropathy. METHODS: Comprehensive gene expression states of the normal and diseased mesangial cells were defined with microarrays. By comparing the gene expression profiles of mesangial cells with those of multiple other renal cell types, including podocytes, endothelial cells and renal vesicles, it was possible to better define their exceptional nature, which includes smooth muscle, phagocytic and neuronal traits. RESULTS: The complete set of mesangial cell expressed transcription factors, growth factors and receptors were identified. In addition, the analysis of the mesangial cells from diabetic nephropathy mice characterized their changes in gene expression. Molecular functions and biological processes specific to diseased mesangial cells were characterized, identifying genes involved in extracellular matrix, cell division, vasculogenesis, and growth factor modulation. Selected gene changes considered of particular importance to the disease process were validated and localized within the glomuerulus by immunostaining. For example, thrombospondin, a key mediator of TGFß signaling, was upregulated in the diabetic nephropathy mesangial cells, likely contributing to fibrosis. On the other hand the decorin gene was also upregulated, and expression of this gene has been strongly implicated in the reduction of TGFß induced fibrosis. CONCLUSIONS: The results provide an important complement to previous studies examining mesangial cells grown in culture. The remarkable qualities of the mesangial cell are more fully defined in both the normal and diabetic nephropathy diseased state. New gene expression changes and biological pathways are discovered, yielding a deeper understanding of the diabetic nephropathy pathogenic process, and identifying candidate targets for the development of novel therapies.

RNA-Seq defines novel genes, RNA processing patterns and enhancer maps for the early stages of nephrogenesis: Hox supergenes.

During kidney development the cap mesenchyme progenitor cells both self renew and differentiate into nephrons. The balance between renewal and differentiation determines the final nephron count, which is of considerable medical importance. An important goal is to create a precise genetic definition of the early differentiation of cap mesenchyme progenitors. We used RNA-Seq to transcriptional profile the cap mesenchyme progenitors and their first epithelial derivative, the renal vesicles. The results provide a global view of the changing gene expression program during this key period, defining expression levels for all transcription factors, growth factors, and receptors. The RNA-Seq was performed using two different biochemistries, with one examining only polyadenylated RNA and the other total RNA. This allowed the analysis of noncanonical transcripts, which for many genes were more abundant than standard exonic RNAs. Since a large fraction of enhancers are now known to be transcribed the results also provide global maps of potential enhancers. Further, the RNA-Seq data defined hundreds of novel splice patterns and large numbers of new genes. Particularly striking was the extensive sense/antisense transcription and changing RNA processing complexities of the Hox clusters.

Laser capture.

This chapter describes detailed methods used for laser capture microdissection (LCM) of discrete subpopulations of cells. Topics covered include preparing tissue blocks, cryostat sectioning, processing slides, performing the LCM, and purification of RNA from LCM samples. Notes describe the fine points of each operation, which can often mean the difference between success and failure.

Genes that confer the identity of the renin cell.

Renin-expressing cells modulate BP, fluid-electrolyte homeostasis, and kidney development, but remarkably little is known regarding the genetic regulatory network that governs the identity of these cells. Here we compared the gene expression profiles of renin cells with most cells in the kidney at various stages of development as well as after a physiologic challenge known to induce the transformation of arteriolar smooth muscle cells into renin-expressing cells. At all stages, renin cells expressed a distinct set of genes characteristic of the renin phenotype, which was vastly different from other cell types in the kidney. For example, cells programmed to exhibit the renin phenotype expressed Akr1b7, and maturing cells expressed angiogenic factors necessary for the development of the kidney vasculature and RGS (regulator of G-protein signaling) genes, suggesting a potential relationship between renin cells and pericytes. Contrary to the plasticity of arteriolar smooth muscle cells upstream from the glomerulus, which can transiently acquire the embryonic phenotype in the adult under physiologic stress, the adult juxtaglomerular cell always possessed characteristics of both smooth muscle and renin cells. Taken together, these results identify the gene expression profile of renin-expressing cells at various stages of maturity, and suggest that juxtaglomerular cells maintain properties of both smooth muscle and renin-expressing cells, likely to allow the rapid control of body fluids and BP through both contractile and endocrine functions.

Defining the molecular character of the developing and adult kidney podocyte.

BACKGROUND: The podocyte is a remarkable cell type, which encases the capillaries of the kidney glomerulus. Although mesodermal in origin it sends out axonal like projections that wrap around the capillaries. These extend yet finer projections, the foot processes, which interdigitate, leaving between them the slit diaphragms, through which the glomerular filtrate must pass. The podocytes are a subject of keen interest because of their key roles in kidney development and disease. METHODOLOGY/PRINCIPAL FINDINGS: In this report we identified and characterized a novel transgenic mouse line, MafB-GFP, which specifically marked the kidney podocytes from a very early stage of development. These mice were then used to facilitate the fluorescent activated cell sorting based purification of podocytes from embryos at E13.5 and E15.5, as well as adults. Microarrays were then used to globally define the gene expression states of podocytes at these different developmental stages. A remarkable picture emerged, identifying the multiple sets of genes that establish the neuronal, muscle, and phagocytic properties of podocytes. The complete combinatorial code of transcription factors that create the podocyte was characterized, and the global lists of growth factors and receptors they express were defined. CONCLUSIONS/SIGNIFICANCE: The complete molecular character of the in vivo podocyte is established for the first time. The active molecular functions and biological processes further define their unique combination of features. The results provide a resource atlas of gene expression patterns of developing and adult podocytes that will help to guide further research of these incredible cells.

The GUDMAP database--an online resource for genitourinary research.

The GenitoUrinary Development Molecular Anatomy Project (GUDMAP) is an international consortium working to generate gene expression data and transgenic mice. GUDMAP includes data from large-scale in situ hybridisation screens (wholemount and section) and microarray gene expression data of microdissected, laser-captured and FACS-sorted components of the developing mouse genitourinary (GU) system. These expression data are annotated using a high-resolution anatomy ontology specific to the developing murine GU system. GUDMAP data are freely accessible at www.gudmap.org via easy-to-use interfaces. This curated, high-resolution dataset serves as a powerful resource for biologists, clinicians and bioinformaticians interested in the developing urogenital system. This paper gives examples of how the data have been used to address problems in developmental biology and provides a primer for those wishing to use the database in their own research.

Microarrays and RNA-Seq identify molecular mechanisms driving the end of nephron production.

BACKGROUND: The production of nephrons suddenly ends in mice shortly after birth when the remaining cells of the multi-potent progenitor mesenchyme begin to differentiate into nephrons. We exploited this terminal wave of nephron production using both microarrays and RNA-Seq to serially evaluate gene transcript levels in the progenitors. This strategy allowed us to define the changing gene expression states following induction and the onset of differentiation after birth. RESULTS: Microarray and RNA-Seq studies of the progenitors detected a change in the expression profiles of several classes of genes early after birth. One functional class, a class of genes associated with cellular proliferation, was activated. Analysis of proliferation with a nucleotide analog demonstrated in vivo that entry into the S-phase of the cell cycle preceded increases in transcript levels of genetic markers of differentiation. Microarrays and RNA-Seq also detected the onset of expression of markers of differentiation within the population of progenitors prior to detectable Six2 repression. Validation by in situ hybridization demonstrated that the markers were expressed in a subset of Six2 expressing progenitors. Finally, the studies identified a third set of genes that provide indirect evidence of an altered cellular microenvironment of the multi-potential progenitors after birth. CONCLUSIONS: These results demonstrate that Six2 expression is not sufficient to suppress activation of genes associated with growth and differentiation of nephrons. They also better define the sequence of events after induction and suggest mechanisms contributing to the rapid end of nephron production after birth in mice.

Defining the genetic blueprint of kidney development.

Thousands of genes show differential expression patterns during kidney development, suggesting that the genetic program driving this process is complex. While great progress has been made in defining the outline of the genetic basis of nephrogenesis, it is clear that much remains to be learned. A global atlas of the gene expression profiles of the multiple elements of the developing kidney would allow the identification of novel growth factor-receptor interactions, identify additional molecular markers of distinct components, facilitate the generation of compartment specific GFP-CRE transgenic mouse tools, lend insights into the genetic regulatory circuits governing nephron formation, and fully characterize the waves of gene expression that impel nephrogenesis. Both microarrays and next generation deep sequencing of cDNA libraries can be used to define comprehensive, sensitive, and quantitative gene expression profiles. In addition, laser capture microdissection and transgenic GFP mice can be used to isolate specific compartments and pure cell types from the developing kidney. Advancing technologies are even allowing robust gene expression profiling of single cells. The final goal is the production of an exquisitely detailed atlas of the gene expression program that drives kidney development.