The contribution of Notch1 to nephron segmentation in the developing kidney is revealed in a sensitized Notch2 background and can be augmented by reducing Mint dosage

Abstract

We previously determined that Notch2, and not Notch1 was required for forming proximal nephron segments. The dominance of Notch2 may be conserved in humans, since Notch2 mutations occur in Alagille syndrome (ALGS) 2 patients, which includes renal complications. To test whether mutations in Notch1 could increase the severity of renal complications in ALGS, we inactivated conditional Notch1 and Notch2 alleles in mice using a Six2-GFP∷Cre. This BAC transgene is expressed mosaically in renal epithelial progenitors but uniformly in cells exiting the progenitor pool to undergo mesenchymal to epithelial transition. Although delaying Notch2 inactivation had a marginal effect on nephron numbers, it created a sensitized background in which the inactivation of Notch1 severely compromised nephron formation, function and survival. These and additional observations indicate that Notch1 in concert with Notch2 contributes to the morphogenesis of renal vesicles into S-shaped bodies in a RBP-J dependent manner. A significant implication is that elevating Notch1 activity could improve renal functions in ALGS2 patients. As proof of principle, we determined that conditional inactivation of Mint, an inhibitor of Notch-RBP-J interaction, resulted in a moderate rescue of Notch2 null kidneys, implying that temporal blockage of Notch signaling inhibitors downstream of receptor activation may have therapeutic benefits for ALGS patients.

Reduced Notch Signaling Leads to Renal Cysts and Papillary Microadenomas

“…17 Finally, when Notch2 deletion is delayed until after nephrogenesis has begun, a sensitized background forms in which the contribution of Notch1 to nephron segmentation is revealed. 13 These findings demonstrated that canonical Notch signals acting early in the RV specify the proximal nephron fates, and Notch2 contributes the lion share of this signal. However, these experiments do not address whether Notch signaling is required again in nephron differentiation, maintenance, or physiology.…”

“…Notch1, Notch2, and genes coding for their ligands Dll1 and Jag1 are all expressed by renal vesicle (RV) cells. 13,14 After ligand binding, the juxtamembrane negative regulatory region (NRR) unfolds, permitting metalloproteases to cleave Notch. Ectodomain shedding is followed by intramembrane cleavage mediated by ␥-secretase, leading to the release of the Notch intracellular domain (NICD).…”

“…This transgene is expressed mosaically in the renal epithelial progenitor population, 13 allowing many progenitor cells to retain the floxed allele. In the induced mesenchyme 26 and in pretubular aggregates, uniform high levels of Cre expression are detected, leading to complete (Ͼ99%) gene inactivation before RV formation.…”

“…In the induced mesenchyme 26 and in pretubular aggregates, uniform high levels of Cre expression are detected, leading to complete (Ͼ99%) gene inactivation before RV formation. 13 Because Notch2 expression begins as Cre expression becomes uniform (Supplementary Figure S1) f/f kidneys. Although some nephron loss-and visibly smaller kidneys-is detected in 30% of newborn mice, viability, fertility, and fecundity are not affected.…”

Chromatin-based Mechanisms of Renal Epithelial Differentiation

“…36,37 During the conversion of RV into an S-shaped structure, a middle compartment is established in a Notch-dependent manner. Whereas Notch2 is expressed throughout the RV, 38 Dll1, a ligand capable of activating Notch receptors, is restricted to the distal compartment. 32 Notch2 activation is likely to initially occur at the boundary between distal Dll1 ϩ and proximal Dll1 Ϫ segments of the RV because ligand/receptor interaction within the same cell can be inhibitory.…”