Pathogenesis of Anorectal Malformations in Retinoic Acid Receptor Knockout Mice Studied by HREM

Abstract

Anorectal malformations (ARMs) are relatively common congenital abnormalities, but their pathogenesis is poorly understood. Previous gene knockout studies indicated that the signalling pathway mediated by the retinoic acid receptors (RAR) is instrumental to the formation of the anorectal canal and of various urogenital structures. Here, we show that simultaneous ablation of the three RARs in the mouse embryo results in a spectrum of malformations of the pelvic organs in which anorectal and urinary bladder ageneses are consistently associated. We found that these ageneses could be accounted for by defects in the processes of growth and migration of the cloaca, the embryonic structure from which the anorectal canal and urinary bladder originate. We further show that these defects are preceded by a failure of the lateral shift of the umbilical arteries and propose vascular abnormalities as a possible cause of ARM. Through the comparisons of these phenotypes with those of other mutant mice and of human patients, we would like to suggest that morphological data may provide a solid base to test molecular as well as clinical hypotheses.

Keywords: Fraser syndrome; congenital malformations; mesenteric artery; mouse embryonic development; umbilical artery; vitamin A deficiency.

Figure 1

External appearance of control (a–d) and Rarabg^ΔE10.5 mutant (e–h) littermates at E14.5 and E15.5, as indicated. The cryptophthalmos (e,g), the generalised oedema (arrowheads in f), the shortening of the snout (g) and the complete syndactyly (h) are characteristic features of the Rarabg^ΔE10.5 mutant phenotype. Note that: (i) the two E14.5 littermates (a,e) are fixed in Bouin’s fluid, which accounts for their yellow and opaque skin, allowing a better visualisation of the cryptophthalmos; (ii) these two E14.5 foetuses were subsequently analysed by HREM (see Figure 2); (iii) the two littermates at E15.5 (b–d and f–h) are fixed in paraformaldehyde, which accounts for their translucent skin, allowing visualisation of the subcutaneous oedema (white arrowhead). F, forelimbs; H, hindlimbs.

Figure 2

Three-dimensional reconstructions of the rectum and posterior portion of the urogenital tract in a control (a,c) and a Rarabg^ΔE10.5 mutant (b,d) foetus at E14.5. (a,b) Ventrolateral views of the left side and (c,d) projections of left lateral views on sagittal HREM sections. (a,c) In the control foetus, the urogenital sinus comprises 2 distinct portions: the urinary bladder (UB), which is located anterior to the openings of the ureters (U), and the pelvic urethra (PU) into which the Wolffian ducts (WD) terminate. The pelvic urethra communicates with the amniotic cavity (AC) at the site of disintegration of the cloacal membrane (CM). The rectum (R) is continuous with the tip of the ectodermal groove (E) located between the genital tubercle and the tail. (b,d) In the mutant foetus, the rectum ends blindly (R*) at a distance from the ectoderm; the urogenital sinus is markedly hypoplastic and lacks the portion corresponding to the urinary bladder. Neither the ureters nor the Wollfian ducts make contact with the urogenital sinus, and the Wolffian ducts give rise to irregular outgrowths (arrowheads in d) corresponding to supernumerary ureteric buds; the Müllerian duct (MD) is missing. GT, genital tubercle; R and Rm, lumen and mesenchyme of the rectum, respectively; T, tail; UB and UBm, lumen and mesenchyme of the urinary bladder, respectively. Same magnifications in (a,b) and in (c,d). Scale bar in (d): 150 µm (c,d).

Figure 3

Left lateral views of 3D reconstructions of the urogenital system and gut in control (a,c,e) and Rarabg^ΔE10.5 mutant (b,d,f) littermates at E11.5, E12.5 and E13.5, as indicated. CD, cloacal duct; CL, lumen of the cloaca; CM, site of disintegration of the cloacal membrane; E, ectodermal groove between the tail and genital tubercle; hG and mG, lumen of the hindgut and midgut, respectively; Go, gonad; K, metanephric mesenchyme (kidney mesenchyme); R and R*, lumen of the rectum and blind ending of the mutant rectum; U, ureteric buds (a,b) or ureters (c–f); UGS, lumen of the urogenital sinus; WD, Wolffian duct. Arrowheads point to abnormal outgrowths of the Wolffian ducts. Same magnifications in (a,b), in (c,d) and in (e,f).

Figure 4

Left lateral views of the urogenital sinus (UGS) and gut in control (a,c) and Rarabg^ΔE10.5 mutant (b,d) littermates at E12.5 and E13.5 as indicated, with projections on sagittal HREM sections. Note that the layer of mesoderm separating the cloacal duct (CD) and/or the rectum from the base of the genital tubercle is much thicker in mutant embryos than in the controls (dotted lines), whereas the intra-cloacal mesenchyme (ICM), sandwiched between the UGS and rectum, is much less developed in the mutants than in the controls. CM, site of disintegration of the cloacal membrane; E, ectodermal groove between the genital tubercle and the tail; GT, genital tubercle; R and R*, lumen of the rectum and blind ending of the mutant rectum; T, tail; UGS, lumen of the urogenital sinus; V, vertebrae. Scale bar (in d): 150 µm (a–d).

Figure 5

Left lateral (a,c) and ventral (b,d) views of the cloaca, kidneys and arteries in control and Rarabg^ΔE10.5 mutant embryos at E11.5. (a,b) In the control, the left and the right umbilical arteries are equal in calibre; their roots have taken a lateral course and bypass the kidneys on each side of the midline; the principal arteries to the hindlimbs arise from their lateral sides. (c,d) In the mutant, the umbilical roots still originate from the ventral wall of the aorta and pass medial to the kidneys; the left umbilical artery is hypoplastic and the principal artery to the left hindlimb connects directly to the dorsal aorta. AMA, anterior mesenteric artery; CA, coeliac artery; DA, midline dorsal aorta; CL, lumen of the cloaca; K, metanephric mesenchyme; fPA, left artery to the hindlimb bud; r.root and f.root, roots of the right and left umbilical arteries; rUA, fUA and mUA, right, left and middle umbilical arteries, respectively. The asterisk designates the remnant of the medial umbilical roots. Same magnification in (a–d).

Figure 6

Left lateral (a,c) and ventral (b,d) views of the urogenital sinus (UGS), kidneys, gonads and arteries in control and Rarabg^ΔE10.5 mutant embryos at E12.5. (a,b) In the control, the left and the right umbilical arteries have similar calibres; their roots have yielded the common iliac arteries. (c,d) In the mutant, the right umbilical root has regressed; the left root is in a median position and is hyperplastic as it carries the whole umbilical circulation; the principal artery to the right hindlimb bud connects directly to the dorsal aorta. AMA, anterior mesenteric artery; CA, coeliac artery; DA, midline dorsal aorta; Go, gonad; K, metanephric mesenchyme; rIA and fIA, right and left common iliac arteries; rPA and fPA, right and left arteries to the hindlimb buds; root, root of the single umbilical artery in the mutant; rUA and fUA, right and left umbilical arteries. UGS, lumen of the urogenital sinus. Same magnification in (a–d).