Lunatic Fringe-mediated Notch signaling is required for lung alveogenesis.

Distal lung development occurs through coordinated induction of myofibroblasts, epithelial cells, and capillaries. Lunatic Fringe (Lfng) is a beta(1-3) N-acetylglucosamine transferase that modifies Notch receptors to facilitate their activation by Delta-like (Dll1/4) ligands. Lfng is expressed in the distal lung during saccular development, and deletion of this gene impairs myofibroblast differentiation and alveogenesis in this context. A similar defect was observed in Notch2(beta-geo/+)Notch3(beta-geo/beta-geo) compound mutant mice but not in Notch2(beta-geo/+) or Notch3(beta-geo/beta-geo) single mutants. Finally, to directly test for the role of Notch signaling in myofibroblast differentiation in vivo, we used ROSA26-rtTA(/+);tetO-CRE(/+);RBPJkappa(flox/flox) inducible mutant mice to show that disruption of canonical Notch signaling during late embryonic development prevents induction of smooth muscle actin in mesenchymal cells of the distal lung. In sum, these results demonstrate that Lfng functions to enhance Notch signaling in myofibroblast precursor cells and thereby to coordinate differentiation and mobilization of myofibroblasts required for alveolar septation.

Ectopic notch activation in developing podocytes causes glomerulosclerosis.

Genetic evidence supports an early role for Notch signaling in the fate of podocytes during glomerular development. Decreased expression of Notch transcriptional targets in developing podocytes after the determination of cell fate suggests that constitutive Notch signaling may oppose podocyte differentiation. This study determined the effects of constitutive Notch signaling on podocyte differentiation by ectopically expressing Notch's intracellular domain (NOTCH-IC), the biologically active, intracellular product of proteolytic cleavage of the Notch receptor, in developing podocytes of transgenic mice. Histologic and molecular analyses revealed normal glomerular morphology and expression of podocyte markers in newborn NOTCH-IC-expressing mice; however, mice developed severe proteinuria and showed evidence of progressive glomerulosclerosis at 2 wk after birth. Features of mature podocytes were lost: Foot processes were effaced; expression of Wt1, Nphs1, and Nphs2 was downregulated; cell-cycle re-entry was induced; and the expression of Pax2 was increased. In contrast, mice with podocyte-specific inactivation of Rbpsuh, which encodes a protein essential for canonical Notch signaling, seemed normal. In addition, the damaging effects of NOTCH-IC expression were prevented in transgenic mice after simultaneous conditional inactivation of Rbpsuh in murine podocytes. These results suggest that Notch signaling is dispensable during terminal differentiation of podocytes but that constitutive (or inappropriate) Notch signaling is deleterious, leading to glomerulosclerosis.

Expression of Hairy/Enhancer of Split genes, Hes1 and Hes5, during murine nephron morphogenesis.

Hairy/Enhancer of Split (Hes) genes encode transcriptional repressors that function as downstream targets of activated Notch receptors in cell fate decisions during tissue development. During nephrogenesis, multiple Notch pathway genes are co-expressed in multi-potent epithelial progenitors (i.e. pre-tubular aggregates), but demonstrate distinct expression patterns in early nephrons (i.e. S-shaped bodies), suggesting that Notch signaling functions in patterning epithelial cell fate during nephron morphogenesis. To define the spatial activation of the Notch pathway in developing nephrons, we analyzed the expression of Hes1 and Hes5 by mRNA in situ hybridization in cryosections of developing kidneys, and compared their spatiotemporal expression with the expression of other Notch pathway genes in nephron progenitors. Hes1, and to a lesser extent Hes5, were expressed in pre-tubular aggregates and comma-shaped bodies of embryonic day (E) 13.5 and newborn kidneys. In S-shaped bodies, Hes1 expression was detected in the middle part which gives rise to the proximal tubule, but also extended into the lower and upper parts which give rise to the glomerulus and distal tubule, respectively, and was similar to the proximal-distal expression patterns for Notch1 and Jagged1 in these nephrogenic structures. In contrast, strong Hes5 expression was restricted to the middle segment of S-shaped bodies, and resembled Delta-like 1 expression. These data show that Hes1 and Hes5 expression are independently regulated along the proximal-distal axis of the developing nephron. Consequently, the differential, spatial regulation of Hes1 and Hes5 gene expression by the Notch signaling pathway in developing nephrons may be a mechanism for patterning cell fate decisions during nephron morphogenesis.