Fig. 3. Arrest of loop of Henle (HL) elongation at the primitive loop stage in Brn1-deficient kidney. (A-C) Classification of HL developmental stages; anlage (A), primitive loop (B) and immature loop (C). Each developing HL stage is outlined by arrowheads. (D) Schematic drawing of the anlage, primitive loop and immature loop of Henle. (E,F) Quantitation of the numbers of primitive loops and immature loops of Henle in Brn1-deficient kidneys. All observable independent HLs were counted within one median longitudinal section from each animal at E16.5. The number of primitive loops of Henle in Brn1^-/- kidneys increased significantly from the levels seen in Brn1^+/+ kidneys (E). However, no immature loops of Henle were identified in Brn1^-/- kidneys (F). Data are shown as the mean±s.e.m. (n=3 or 4). ^*P<0.05 compared with Brn1^+/+ kidney (ANOVA). (G-L) BrdU labeling in the anlage and primitive loops of Henle at E16.5. No significant difference in the numbers of BrdU-incorporated cells between Brn1^+/+ and Brn1^-/- kidneys was detectable at the anlage stage (G-I). At the primitive loop stage, the number of cells incorporating BrdU was significantly decreased in Brn1^-/- kidney in comparison with the Brn1^+/+ kidney (J-L). Data are shown as the mean±s.e.m. (n=3 or 4). ^*P<0.05 compared with Brn1^+/+ kidney (ANOVA). (M-P) TUNEL analysis of primitive and immature loops of Henle at P0. In the Brn1^+/+ kidney, TUNEL-positive cells were detected in the bend of the immature loop of Henle, near the papillary tip of medulla (O, arrow). TUNEL-positive cells were never observed in the primitive loop (M). In the Brn1^-/- kidney, TUNEL-positive cells were present near the bend of primitive loop (N, arrows). The HL could not be identified in the papilla of Brn1^-/- kidneys (P). (Q-T) Active caspase 3 immunostaining of primitive and immature loops of Henle at P0. In the Brn1^+/+ kidney, active caspase 3-positive cells were detected in the bend of the immature loop of Henle, near the papillary tip of medulla (S, arrow). Active caspase 3-positive cells were never observed in the primitive loop (Q). In the Brn1^-/- kidney, active caspase 3-positive cells were present near the bend of primitive loop (R, arrow). The HL could not be identified in the papilla of Brn1^-/- kidneys (T). Scale bars: 20 µm.