Standardised imaging pipeline for phenotyping mouse laterality defects and associated heart malformations, at multiple scales and multiple stages

Abstract

20 asymmetry 21 Word count 7005 22 23 24 Desgrange et al. 2 Summary statement 25Laterality defects, which combine anomalies in several visceral organs, are challenging to phenotype. 26We have now developed a standardised approach for multimodality 3D imaging in mice, generating 27 quantifiable phenotypes. 28 29 Abstract 30 31 Laterality defects are developmental disorders resulting from aberrant left/right patterning. In 32 the most severe cases, such as in heterotaxy, they are associated with complex malformations of the 33 heart. Advances in understanding the underlying physiopathological mechanisms have been hindered 34 by the lack of a standardised and exhaustive procedure in mouse models, for phenotyping left/right 35 asymmetries of all visceral organs. Here, we have developed a multimodality imaging pipeline, which 36 combines non-invasive micro-ultrasound imaging, micro-CT and HREM, to acquire 3D images at 37 multiple stages of development and at multiple scales. Based on the position in the uterine horns, we 38 track, in a single individual, the progression of organ asymmetry, the situs of all visceral organs in 39 their thoracic or abdominal environment, together with fine anatomical left/right asymmetries of 40 cardiac segments. We provide reference anatomical images and organ reconstructions in the mouse, 41 and discuss differences with humans. This standardised pipeline, which we validated in a mouse 42 model of heterotaxy, offers a fast and easy-to-implement framework. The extensive 3D phenotyping 43 of organ asymmetry in the mouse uses the clinical nomenclature for direct comparison with patient 44 phenotypes. It is compatible with automated and quantitative image analyses, which is essential to 45 compare mutant phenotypes with incomplete penetrance and gain mechanistic insight into laterality 46 defects. 47 48 Desgrange et al. 3 Introduction 49 50 Laterality defects are developmental disorders, caused by impaired left-right patterning. They 51 affect collectively up to 1/2,000 live births, and comprise a spectrum of malformations ranging from 52 asymptomatic situs inversus or dextrocardia to severe heterotaxy (Desgrange et al., 2018; Lin et al., 53 2014). Heterotaxy corresponds to abnormal symmetry of the viscera (isomerism), and/or situs 54 discordance between visceral organs (Van Praagh, 2006). Many visceral organs (lung, spleen, 55 stomach, intestine, liver and pancreas) may be targeted and functionally impaired, with an abnormal 56 position in the abdominal or thoracic cavity (situs) or an impaired asymmetric shape. Diagnosis is 57 made on the combination of 3 out of 8 criteria, including abdominal situs abnormality, spleen 58 abnormality, isomerism of bronchi and the lungs, biliary atresia, intestinal malrotation, congenital 59 heart defects, isomerism of the atrial appendages, systemic venous anomalies (Lin et al., 2014). The 60 prognosis of heterotaxy mainly depends on the cardiac malformation, which can be complex, with 61 profound functional impacts, such as abnormal oxygen supply or obst...

Transient Nodal Signaling in Left Precursors Coordinates Opposed Asymmetries Shaping the Heart Loop

“…In the clinics, heterotaxy is a laterality defect associated with mutations affecting the formation or signaling of the left-right organizer (Guimier et al, 2015). The clinical picture is very variable, in terms of associations of left-right anomalies between different organs or between different heart segments (Desgrange et al, 2019;Lin et al, 2014). Even looking at a single structure such as the atria, the parameters of the anatomy of the appendages and of the connection of the inferior caval vein are not always concordant (Tremblay et al, 2017).…”

“…Embryos or hearts were collected and embedded in methacrylate resin (JB4) containing eosin and acridine orange as contrast agents (Le Garrec et al, 2017;Desgrange et al, 2019). One or two channel images of the surface of the resin block were acquired using the optical high-resolution episcopic microscope (Indigo Scientific) and a 1X Apo objective repeatedly after removal of 1.56-1.7 mm (embryos) and 2.34 mm (hearts) thick sections: the tissue architecture was imaged with a GFP filter and the staining of enzymatic precipitates with a RFP filter.…”

“…The authors declare no competing interests. The situs and anatomy of visceral organs were evaluated by micro-CT imaging (Quantum FX, perkin Elmer) after 72h of Lugol staining (Desgrange et al, 2019). The heart structure was phenotyped on 3D reconstruction of HREM images, based on the segmental approach (Van Praagh, 1972).…”

Mouse embryo phenotyping using X-ray microCT

“…Merchant et al (2016) measured the thickness of the myocardium of the left and right ventricle in hearts of E12.5 and E18.5 wild-type and p27 -/mutant mice. Desgrange et al (2019) used microCT together with in vivo micro-ultrasound imaging and HREM in a standardized imaging pipeline for phenotyping laterality defects and associated heart malformations. The pipeline involved microCT for imaging at E18.5 and included segmentation of thoracic and abdominal organs and the great vessels (Figure 6B).…”

Correlated Multimodal Imaging in Life Sciences: Expanding the Biomedical Horizon

“…Several workflows have so far been established that solved the above-mentioned challenges on sample preparation, relocalization of ROIs, and data correlation. Recent examples for multiscale combinations of in-vivo and ex-vivo imaging include the correlation of intravital microscopy, CT and EM to study single tumor cells in the cerebral vasculature [81]; correlation of X-ray holographic nano-tomography, EM and FM to disentangle dense neuronal circuitry in Drosophila melanogaster and mammalian central and peripheral nervous tissue [82]; correlation of local neuronal and capillary responses by two-photon microscopy with mesoscopic responses detected by ultrasound (US) and BOLD-fMRI [83]; or extended CMI pipelines that include the correlation of a variety of imaging technologies, such as non-invasive US, CT and highresolution episcopic microscopy (HREM) for phenotyping left/right asymmetries of all visceral organs in a mouse model of heterotaxy or combined OCT, PAI and HREM of chick embryos at multiple development stages [8,84,85]. Further examples of novel CMI pipelines that uncover biophysical or chemical information include the correlation of FM, molecular (MALDI MSI) and elemental imaging [X-ray fluorescence (XRF)] to analyze lipids and elements relevant to bone structures in the very same sample section of a chicken phalanx without tissue decalcification at the µm scales [86].…”