Notch signaling is required for the formation of mesangial cells from a stromal mesenchyme precursor during kidney development.

Mesangial cells are specialized pericyte/smooth muscle cells that surround and constrain the vascular network within the glomerulus of the kidney. They are derived from the stromal mesenchyme, a progenitor population distinct from nephron stem cells. Whether mesangial cells have a distinct origin from vascular smooth muscle cells (VSMCs) and the pathways that govern their specification are unknown. Here we show that Notch signaling in stromal progenitors is essential for mesangial cell formation but is dispensable for the smooth muscle and interstitial cell lineages. Deletion of RBPjk, the common DNA-binding partner of all active Notch receptors, with Foxd1(tgCre) results in glomerular aneurysm and perinatal death from kidney failure. This defect occurs early in glomerular development as stromal-derived, desmin-positive cells fail to coalesce near forming nephrons and thus do not invade the vascular cleft of the S-shaped body. This is in contrast to other mutants in which the loss of the mesangium was due to migration defects, and suggests that loss of Notch signaling results in a failure to specify this population from the stroma. Interestingly, Pdgfrb-positive VSMCs do not enter the vascular cleft and cannot rescue the mesangial deficiency. Notch1 and Notch2 act redundantly through γ-secretase and RBPjk in this process, as individual mutants have mesangial cells at birth. Together, these data demonstrate a unique origin of mesangial cells and demonstrate a novel, redundant function for Notch receptors in mesangial cell specification, proliferation or survival during kidney development.

The role of Notch signaling in kidney development and disease.

The kidney is the body's filter, responsible for the removal of metabolic waste and the excretion or reabsorption of electrolytes to control blood composition and pH balance. The functional unit of this filter is the nephron, whose segmented architecture has been largely conserved in form and function throughout eukaryotic evolution. Not surprisingly, the core developmental pathways that regulate the formation of the nephron have also been conserved. In particular, the Notch signaling pathway functions in both primitive and advanced nephrons to pattern domains required for the kidney's diverse functions. In this chapter, we will discuss the role that Notch plays in directing cell fate decisions during embryonic development of the pronephros and metanephros. We will go on to discuss the later role of Notch signaling as a cyst-suppressor and the consequences of aberrant or absent Notch activity in disease and cancer. The work discussed here highlights the fundamental importance of Notch during development and homeostasis of the kidney and underlies the need for mechanistic understanding of its role towards the treatment of human disease.

CITED1 expression in liver development and hepatoblastoma.

Hepatoblastoma, the most common pediatric liver cancer, consists of epithelial mixed embryonal/fetal (EMEF) and pure fetal histologic subtypes, with the latter exhibiting a more favorable prognosis. Few embryonal histology markers that yield insight into the biologic basis for this prognostic discrepancy exist. CBP/P-300 interacting transactivator 1 (CITED1), a transcriptional co-activator, is expressed in the self-renewing nephron progenitor population of the developing kidney and broadly in its malignant analog, Wilms tumor (WT). In this current study, CITED1 expression is detected in mouse embryonic liver initially on post-coitum day 10.5 (e10.5), begins to taper by e14.5, and is undetectable in e18.5 and adult livers. CITED1 expression is detected in regenerating murine hepatocytes following liver injury by partial hepatectomy and 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Importantly, while CITED1 is undetectable in normal human adult livers, 36 of 41 (87.8%) hepatoblastoma specimens express CITED1, where it is enriched in EMEF specimens compared to specimens of pure fetal histology. CITED1 overexpression in Hep293TT human hepatoblastoma cells induces cellular proliferation and upregulates the Wnt inhibitors Kringle containing transmembrane protein 1 (KREMEN1) and CXXC finger protein 4 (CXXC4). CITED1 mRNA expression correlates with expression of CXXC4 and KREMEN1 in clinical hepatoblastoma specimens. These data show that CITED1 is expressed during a defined time course of liver development and is no longer expressed in the adult liver but is upregulated in regenerating hepatocytes following liver injury. Moreover, as in WT, this embryonic marker is reexpressed in hepatoblastoma and correlates with embryonal histology. These findings identify CITED1 as a novel marker of hepatic progenitor cells that is re-expressed following liver injury and in embryonic liver tumors.

Notch pathway activation can replace the requirement for Wnt4 and Wnt9b in mesenchymal-to-epithelial transition of nephron stem cells.

The primary excretory organ in vertebrates is the kidney, which is responsible for blood filtration, solute homeostasis and pH balance. These functions are carried out by specialized epithelial cells organized into tubules called nephrons. Each of these cell types arise during embryonic development from a mesenchymal stem cell pool through a process of mesenchymal-to-epithelial transition (MET) that requires sequential action of specific Wnt signals. Induction by Wnt9b directs cells to exit the stem cell niche and express Wnt4, which is both necessary and sufficient for the formation of epithelia. Without either factor, MET fails, nephrons do not form and newborn mice die owing to kidney failure. Ectopic Notch activation in stem cells induces mass differentiation and exhaustion of the stem cell pool. To investigate whether this reflected an interaction between Notch and Wnt, we employed a novel gene manipulation strategy in cultured embryonic kidneys. We show that Notch activation is capable of inducing MET in the absence of both Wnt4 and Wnt9b. Following MET, the presence of Notch directs cells primarily to the proximal tubule fate. Only nephron stem cells have the ability to undergo MET in response to Wnt or Notch, as activation in the closely related stromal mesenchyme has no inductive effect. These data demonstrate that stem cells for renal epithelia are uniquely poised to undergo MET, and that Notch activation can replace key inductive Wnt signals in this process. After MET, Notch provides an instructive signal directing cells towards the proximal tubule lineage at the expense of other renal epithelial fates.

Organ culture and immunostaining of mouse embryonic kidneys.

The study of organogenesis in mammals allows investigation of a wide variety of basic cell biological processes in the context of the intact organ. This has become especially important in the age of genetics, as the consequences of gene deletion or mutation in the mouse can be directly linked to human congenital abnormalities. The ability to culture some organs ex vivo during development has emerged as an important tool to understand how tissues are constructed and the signaling pathways that regulate these processes. It has been especially useful in organs that grow via branching morphogenic mechanisms, such as the lung and kidney. Here we demonstrate isolation, ex vivo growth, and fluorescent immunostaining of mouse embryonic day 12.5 (E12.5) kidneys. To demonstrate nephron formation using live imaging, we have isolated and cultured kidneys from mice carrying a green fluorescent protein (GFP) transgene driven by the Hes 1 promoter, which is expressed early in the developing nephron. We also provide a protocol for robust imaging of multiple kidney structures in the whole-mount setting. These techniques serve as a basic platform for the analysis of branching morphogenesis and nephron formation in genetic mouse models or in response to exogenous factors, such as agonists or inhibitors, which can be directly added to the culture medium.

Endocardial cells are a distinct endothelial lineage derived from Flk1+ multipotent cardiovascular progenitors.

Identification of multipotent cardiac progenitors has provided important insights into the mechanisms of myocardial lineage specification, yet has done little to clarify the origin of the endocardium. Despite its essential role in heart development, characterization of the endocardial lineage has been limited by the lack of specific markers of this early vascular subpopulation. To distinguish endocardium from other vasculature, we generated an NFATc1-nuc-LacZ BAC transgenic mouse line capable of labeling this specific endothelial subpopulation at the earliest stages of cardiac development. To further characterize endocardiogenesis, embryonic stem cells (ESCs) derived from NFATc1-nuc-LacZ blastocysts were utilized to demonstrate that endocardial differentiation in vitro recapitulates the close temporal-spatial relationship observed between myocardium and endocardium seen in vivo. Endocardium is specified as a cardiac cell lineage, independent from other vascular populations, responding to BMP and Wnt signals that enhance cardiomyocyte differentiation. Furthermore, a population of Flk1+ cardiovascular progenitors, distinct from hemangioblast precursors, represents a mesodermal precursor of the endocardial endothelium, as well as other cardiovascular lineages. Taken together, these studies emphasize that the endocardium is a unique cardiac lineage and provides further evidence that endocardium and myocardium are derived from a common precursor.

Placental insufficiency associated with loss of Cited1 causes renal medullary dysplasia.

A number of studies have shown that placental insufficiency affects embryonic patterning of the kidney and leads to a decreased number of functioning nephrons in adulthood; however, there is circumstantial evidence that placental insufficiency may also affect renal medullary growth, which could account for cases of unexplained renal medullary dysplasia and for abnormalities in renal function among infants who had experienced intrauterine growth retardation. We observed that mice with late gestational placental insufficiency associated with genetic loss of Cited1 expression in the placenta had renal medullary dysplasia. This was not caused by lower urinary tract obstruction or by defects in branching of the ureteric bud during early nephrogenesis but was associated with decreased tissue oxygenation and increased apoptosis in the expanding renal medulla. Loss of placental Cited1 was required for Cited1 mutants to develop renal dysplasia, and this was not dependent on alterations in embryonic Cited1 expression. Taken together, these findings suggest that renal medullary dysplasia in Cited1 mutant mice is a direct consequence of decreased tissue oxygenation resulting from placental insufficiency.

The transcriptional activity of CITED1 is regulated by phosphorylation in a cell cycle-dependent manner.

CITED1 is the founding member of the CITED family of cofactors that are involved in regulating a wide variety of CBP/p300-dependent transcriptional responses. In the present study, we show that the phosphorylation status of CITED1 changes during the cell cycle and affects its transcriptional cofactor activity. Tryptic mapping and mutagenesis studies identified five phosphorylated serine residues in CITED1. Phosphorylation of these residues did not affect CRM1-dependent nuclear export, but did decrease CITED1 binding to p300 and inhibited CITED1-dependent transactivation of Smad4 and p300. These results suggest that CITED1 functions as a cell cycle-dependent transcriptional cofactor whose activity is regulated by phosphorylation.