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.

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.

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.

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 or (coding for hamartin, an inhibitor of Mtor) in renal progenitor cells.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

Microdissection of the gene expression codes driving nephrogenesis.

The kidney represents an excellent model system for learning the principles of organogenesis. It is intermediate in complexity, and employs many commonly used developmental processes. As such, kidney development has been the subject of intensive study, using a variety of techniques, including in situ hybridization, organ culture and gene targeting, revealing many critical genes and pathways.

Gene expression programs of mouse endothelial cells in kidney development and disease.

Endothelial cells are remarkably heterogeneous in both morphology and function, and they play critical roles in the formation of multiple organ systems. In addition endothelial cell dysfunction can contribute to disease processes, including diabetic nephropathy, which is a leading cause of end stage renal disease. In this report we define the comprehensive gene expression programs of multiple types of kidney endothelial cells, and analyze the differences that distinguish them.

Analysis of early nephron patterning reveals a role for distal RV proliferation in fusion to the ureteric tip via a cap mesenchyme-derived connecting segment.

While nephron formation is known to be initiated by a mesenchyme-to-epithelial transition of the cap mesenchyme to form a renal vesicle (RV), the subsequent patterning of the nephron and fusion with the ureteric component of the kidney to form a patent contiguous uriniferous tubule has not been fully characterized. Using dual section in situ hybridization (SISH)/immunohistochemistry (IHC) we have revealed distinct distal/proximal patterning of Notch, BMP and Wnt pathway components within the RV stage nephron. Quantitation of mitoses and Cyclin D1 expression indicated that cell proliferation was higher in the distal RV, reflecting the differential developmental programs of the proximal and distal populations.

Atlas of gene expression in the developing kidney at microanatomic resolution.

Kidney development is based on differential cell-type-specific expression of a vast number of genes. While multiple critical genes and pathways have been elucidated, a genome-wide analysis of gene expression within individual cellular and anatomic structures is lacking. Accomplishing this could provide significant new insights into fundamental developmental mechanisms such as mesenchymal-epithelial transition, inductive signaling, branching morphogenesis, and segmentation.

Genetic manipulation of periostin expression reveals a role in cardiac hypertrophy and ventricular remodeling.

The cardiac extracellular matrix is a dynamic structural support network that is both influenced by, and a regulator of, pathological remodeling and hypertrophic growth. In response to pathologic insults, the adult heart reexpresses the secreted extracellular matrix protein periostin (Pn). Here we show that Pn is critically involved in regulating the cardiac hypertrophic response, interstitial fibrosis, and ventricular remodeling following long-term pressure overload stimulation and myocardial infarction.

Cardiac-specific ablation of G-protein receptor kinase 2 redefines its roles in heart development and beta-adrenergic signaling.

G-protein receptor kinase 2 (GRK2) is 1 of 7 mammalian GRKs that phosphorylate ligand-bound 7-transmembrane receptors, causing receptor uncoupling from G proteins and potentially activating non-G-protein signaling pathways. GRK2 is unique among members of the GRK family in that its genetic ablation causes embryonic lethality. Cardiac abnormalities in GRK2 null embryos implicated GRK2 in cardiac development but prevented studies of the knockout phenotype in adult hearts.

Distinct pathways regulate proapoptotic Nix and BNip3 in cardiac stress.

Up-regulation of myocardial Nix and BNip3 is associated with apoptosis in cardiac hypertrophy and ischemia, respectively. To identify mechanisms of gene regulation for these critical cardiac apoptosis effectors, the determinants of Nix and BNip3 promoter activation were elucidated by luciferase reporter gene expression in neonatal rat cardiac myocytes. BNip3 transcription was increased by hypoxia but not by phenylephrine (10 microM), angiotensin II (100 nM), or isoproterenol (10 microM).