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.

Timeline of Developmental Defects Generated upon Genetic Inhibition of the Retinoic Acid Receptor Signaling Pathway

“…As anticipated, Rarabg ΔE9. 5 mutants reproduced all the defects present in Rarabg ΔE10.5 mutants at the same developmental stages [4]. They also displayed additional abnormalities which are detailed below.…”

“…# These abnormalities are completely penetrant. § These abnormalities are equal in frequency and severity to those present in Rarabg ΔE10.5 mutants [4]. * From the phenotypes of Rara −/− ;Rarb −/− , Rara −/− ;Rarg −/− and Rarb −/− ;Rarg −/− KO mutants at late foetal stages ∆E9.5 Embryos and Comparison with Rarabg ∆E10.…”

“…5) ubiquitously expressed recombinase (cre/ERT 2 ) that can be activated by tamoxifen (TAM) to conditionally invalidate the Rara and Rarg genes in the Rarb −/− background [3]. This approach previously allowed us to demonstrate that the RAR signalling pathway is required for many developmental processes that are determined between embryonic day 10.5 (E10.5) and E11.5 [4]. Here, by analysing the phenotypic consequences of ablation of all 3 Rar genes at E8.5 and E9.5 on 3-dimensional reconstructions from high-resolution episcopic microscopy (HREM) images, we define critical windows of time during which the RAR signalling is required during early development for proper organogenesis.…”

High Resolution Episcopic Microscopy for Qualitative and Quantitative Data in Phenotyping Altered Embryos and Adult Mice Using the New “Histo3D” System

“…The field of view and slice thickness could be adapted to approach at best the isotropic voxels for every size of the samples and especially for smaller-sized samples. It is noteworthy that with Histo3D, the spatial resolution (measure of detail found in the image) obtained with the bigger samples and by collecting 1000 sections over 3.5 h, permitted the segmentation and reconstruction of structures as thin as cranial nerves (vagus or hypoglossal nerves, for example) or vessels [59] (Mark et al, 2021, this issue), which is comparable to previous reports of cardiovascular or nervous system studies [22,23,27,30,69] using smaller numerical resolution (2 × 2 × 2 µm 3 ) and generating more than 2000 to 4000 single sections in 4 to 8 h of sectioning.…”

“…Mesonephric tubules were still identified (Figure 4f) even though it is generally accepted that the mesonephros has degenerated at E14.5 [57,58]. Interestingly, a similar analytical approach allowed the identification of congenital defects in the urogenital system of retinoic acid receptor-knockout mice [59] (Mark et al, 2021, this issue).…”