3-Dimensional morphometric analysis of murine bladder development and dysmorphogenesis

Abstract

Background: Disorders of the urinary tract represent a major cause of morbidity and impaired quality of life. To better understand the morphological events responsible for normal urinary tract development, we performed 3-D reconstructive analysis of developing mouse bladders in control, mgb-/-, and Fgfr2(Mes-/-) mice.

Results: Detrusor smooth muscle differentiation initiated in the bladder dome and progressed caudally with the leading edge extending down the right posterior surface of the bladder. Gender-specific differences in detrusor smooth muscle development were observed during early embryonic development. Bladder trigone morphology transitioned from an isosceles to equilateral triangle during development due to the preferential lengthening of the urethra to ureter distance. The primary defect observed in mgb-/- bladders was a significant reduction in detrusor smooth muscle differentiation throughout development. Deviations from normal trigone morphology correlated best with VUR development in Fgfr2(Mes-/-) mice, while alterations in intravesicular tunnel length did not.

Conclusions: Multivariate morphometric analysis provides a powerful tool to quantify and assess urinary tract development.

Fig. 1

3-dimensional reconstruction (A, C, E, G & I) and representative H&E stained sections (B, D, F, H, & J) of embryonic (E) day 13, 14, 15, 16 and 17 control (C) and mutant (M) female (F) and male (M) bladders. Outlined structures include bladder lumen (green), urothelium (purple), smooth muscle (red), ureter (blue), and umbilical artery (gold).

Fig. 2

Comparison of total bladder volume for control and mgb−/− mice from embryonic (E) day 13 through E17. *P=0.0007, **P=0.0005. The transformation of data to meet statistical assumptions resulted in interval units being reported in (microns³)^1/6.

Fig. 3

Anterior, right side and caudal views of 3-D reconstruction of the smooth muscle layer in control bladders at embryonic (E) days 13, 14, 15, 16 and 17. E13 male (M) and female (F) bladders are shown for comparison.

Fig. 4

Anterior, right side and caudal views of 3-D reconstruction of the smooth muscle layer in mgb−/− bladders at embryonic (E) days 13, 14 and 15. E13 male (M) and female (F) bladders are shown for comparison. E16 and E17 mgb−/− bladders were not reconstructed because they develop in utero megabladder.

Fig. 5

Bladder trigone schematic showing internal bladder trigone (ABC), external bladder trigone (CDE) and intravesicular trapezoid (ABED). The individual lines and angles generated by this schematic are labeled 1–8 and a–j respectively.

Fig. 6

A. Bladder trigone geometry during development. Line numbers represent lengths in microns × 100 (Table 2). B. Comparison of total bladder perimeter and area in control mice. Note biphasic response with break point occurring at E16. *P=0.0009; **P=0.0013; ***P=0.0036; #P=0.0032; #P=0.0002; #P=0.0001 comparing each developmental time point to E13. Both perimeter and area have significant MANOVA p-values of <0.0125. The transformation of data to meet statistical assumptions resulted in interval units being reported in (area)^1/6.

Fig. 7

3-D reconstruction of normal (A) and Fgfr^Mes−/− (B) bladders showing orientation of the left (L) and right (R) ureters and urethra. Note low positioning of the right refluxing ureter in the Fgfr^Mes−/− bladder compared to control. Outlined structures include detrusor smooth muscle (red), bladder lamina propria (white) urethral lumen (blue) and ureteral smooth muscle (red).

Fig. 8

Modified box plot comparing line lengths (A) and angles (B) of the internal bladder trigone, external bladder trigone, and intravesicular trapezoid in embryonic day 15 (E15) control (−) and Fgfr^Mes−/− (x) bladders. The black bar for each parameter represents the normal control range ± standard deviation.

Fig. 9

Modified box plot comparing line lengths (A) and angles (B) of the internal bladder trigone, external bladder trigone, and intravesicular trapezoid in postnatal day 1 (P1) control (−) and Fgfr^Mes−/− (x) bladders. The black bar for each parameter represents the normal control range ± standard deviation.

Fig. 10

Schematic diagram of the developing bladder showing division of the intravesicular tunnel (1–3) into submucosal tunnel (1–2) and intramural tunnel (2–3). Histological sections stained with α-smooth muscle isoactin show corresponding ureteral landmarks (1, 2, & 3) used to identify the various segments of the intravesicular tunnel.

Fig. 11

Modified box plot comparing the length of the intravesicular tunnel, submucosal tunnel and intramuscular tunnel in embryonic day 15 (E15; A) and postnatal day 1 (P1; B) in control (−) and Fgfr^Mes−/− (x) bladders. The black bar for each parameter represents the normal control range ± standard deviation. Yellow circle in panel B represents a P1 Fgfr^Mes−/− non-refluxing mutant.