SUMMARYNotch signaling is known to regulate the proliferation and differentiation of intestinal stem and progenitor cells; however, direct cellular targets and specific functions of Notch signals had not been identified. We show here in mice that Notch directly targets the crypt base columnar (CBC) cell to maintain stem cell activity. Notch inhibition induced rapid CBC cell loss, with reduced proliferation, apoptotic cell death and reduced efficiency of organoid initiation. Furthermore, expression of the CBC stem cellspecific marker Olfm4 was directly dependent on Notch signaling, with transcription activated through RBP-J binding sites in the promoter. Notch inhibition also led to precocious differentiation of epithelial progenitors into secretory cell types, including large numbers of cells that expressed both Paneth and goblet cell markers. Analysis of Notch function in Atoh1-deficient intestine demonstrated that the cellular changes were dependent on Atoh1, whereas Notch regulation of Olfm4 gene expression was Atoh1 independent. Our findings suggest that Notch targets distinct progenitor cell populations to maintain adult intestinal stem cells and to regulate cell fate choice to control epithelial cell homeostasis.

The MLL family of histone methyltransferases maintains active chromatin domains by methylating histone H3 on lysine 4 (H3K4). How MLL complexes recognize specific chromatin domains in a temporal and tissue-specific manner remains unclear. We show that the DNA-binding protein PAX2 promotes assembly of an H3K4 methyltransferase complex through the ubiquitously expressed nuclear factor PTIP (pax transcription activation domain interacting protein). PTIP copurifies with ALR, MLL3, and other components of a histone methyltransferase complex. PTIP promotes assembly of the ALR complex and H3K4 methylation at a PAX2-binding DNA element. Without PTIP, Pax2 binds to this element but does not assemble the ALR complex. Embryonic lethal ptip-null mutants and conditional mutants both show reduced levels of methylated H3K4. Thus, PTIP bridges DNA-binding developmental regulators to histone methyltransferase-dependent epigenetic regulation.

The bone morphogenetic proteins (BMPs) profoundly affect embryonic development, differentiation and disease. BMP signaling is suppressed by cysteine-rich domain proteins, such as chordin, that sequester ligands from the BMP receptor. We describe a novel protein, KCP, with 18 cysteine-rich domains. Unlike chordin, KCP enhances BMP signaling in a paracrine manner. Smad1-dependent transcription and phosphorylated Smad1 (P-Smad1) levels are increased, as KCP binds to BMP7 and enhances binding to the type I receptor. In vivo, Kcp(-/-) mice are viable and fertile. Because BMPs have a pivotal role in renal disease, we examined the phenotype of Kcp(-/-) mice in two different models of renal injury. Kcp(-/-) animals show reduced levels of P-Smad1, are more susceptible to developing renal interstitial fibrosis, are more sensitive to tubular injury and show substantial pathology after recovery. The data indicate an important role for KCP in attenuating the pathology of renal fibrotic disease.

The reduction in podocyte density to levels below a threshold value drives glomerulosclerosis and progression to ESRD. However, technical demands prohibit high-throughput application of conventional morphometry for estimating podocyte density. We evaluated a method for estimating podocyte density using single paraffin-embedded formalin-fixed sections. Podocyte nuclei were imaged using indirect immunofluorescence detection of antibodies against Wilms' tumor-1 or transducin-like enhancer of split 4. To account for the large size of podocyte nuclei in relation to section thickness, we derived a correction factor given by the equation CF=1/(D/T+1), where T is the tissue section thickness and D is the mean caliper diameter of podocyte nuclei. Normal values for D were directly measured in thick tissue sections and in 3-to 5-mm sections using calibrated imaging software. D values were larger for human podocyte nuclei than for rat or mouse nuclei (P,0.01). In addition, D did not vary significantly between human kidney biopsies at the time of transplantation, 3-6 months after transplantation, or with podocyte depletion associated with transplant glomerulopathy. In rat models, D values also did not vary with podocyte depletion, but increased approximately 10% with old age and in postnephrectomy kidney hypertrophy. A spreadsheet with embedded formulas was created to facilitate individualized podocyte density estimation upon input of measured values. The correction factor method was validated by comparison with other methods, and provided data comparable with prior data for normal human kidney transplant donors. This method for estimating podocyte density is applicable to high-throughput laboratory and clinical use. Pagtalunan et al. used the term podocyte density to describe the key relationship between podocyte number and glomerular tuft volume. 1 Model systems have proven the causative relationship between podocyte depletion (resulting from reduced podocyte number or dysfunction and/or glomerular enlargement) and glomerulosclerosis and progression to ESRD. 2-9 Groundbreaking kidney morphometric biopsy reports from type 1 and 2 diabetes, IgA nephropathy, and hypertensive kidney biopsies in humans support the concept that reduced podocyte number and density is associated with development of glomerulosclerosis and progression, 1,10-15 and strongly imply that podocyte density estimation could help guide clinical decision making.The importance of avoiding simplistic podocyte counting strategies and using appropriate stereologic considerations for estimating podocyte number and density have recently been re-emphasized. [16][17][18][19][20] Optimal research methods for estimating podocyte density, such as the disector/fractionator approach, are too technically demanding for high-throughput