Animals

Large white domestic crossbred pigs (Sus scrofa) were used in this study. An atlas focused on pig cardiovascular development was previously published using these same animals6. All animal work was humanely conducted under an approved University of Missouri IACUC protocol and according to ARRIVE guidelines. All wildtype animals studied, including both fetuses and newborns, were generated by Landrace Large White cross parent gilt with semen from Choice USA Genetics (Choice USA, West Des Moines, IA). All pigs used for this study were raised on an approved farm facility and then moved into a University of Missouri animal facility for sample collection. All facilities are approved for biomedical pigs by the University of Missouri Animal Care and Use Committee and followed the Guide for the Care and Use of Laboratory Animals and the program is AAALAC accredited. The Sap130 mutant pig used in this study was generated previously with RRID, NSRRC:00816.

Breeding and harvesting of fetal and newborn pigs was carried out as previously described6. Briefly, wild type gilts were bred by artificial insemination with wild type semen. Day 0 of gestation was classified as the first day of detectable estrus, and pregnant female pigs were humanely euthanized on day 20, 26, 30, 35, 42, 64, 90 or 115 of gestation (referred to as D20, D26, D30, D35, D42, D64, D90, D115). Specimens up to 35 GA are referred to as embryos as they are largely indistinguishable between mouse, pig, human, but those at 42days GA and beyond are referred to as fetuses, as at these stages they have craniofacial and limb features distinct for the pig. The stages selected for the present study are based on our earlier analysis of the temporal profile of cardiovascular development in the pig, with D20 corresponding to early heart development comprising the looped heart tube developmental stage6. At this stage, neither ventricular, atrial, nor outflow septation has occurred. As the heart is the first organ system to form, this provided a reasonable starting point for profiling development of the abdominal organs, which are initiated after formation of the heart. From our previous study, we had determined spacing at~56day intervals can maximize what can be learned regarding the developmental progression of organogenesis at these early stages. However, the exact day of collection varied by a day or two, determined by availability of staff for collection of the specimen. Beyond day 35, we collected and analyzed fetuses at increasingly larger intervals that spanned Day 42, Day 64, Day 90 and Day 115 (newborn). By Day 42, most abdominal organs are fully formed, except for the gonads whose development continues at Day 64, but is completed by day 90.

For the embryo/fetus collection, the uterus was opened on the antimesometrial side and fetuses were removed. The whole fetus from each stage was then drop-fixed in 4% paraformaldehyde at room temperature. For fetuses at D42, D64 and D90, a small opening on the side of each fetus was introduced to allow fixative to permeate the chest cavity. Newborn piglets were kept on ice until dissection of all organs. Collected organs were photographed and placed in fixative. Embryos/fetuses were fixed in 4% PFA or 10% buffered formalin for 25days. At minimum, three embryos/fetuses per stage were analyzed. D20 to D42 embryos/fetuses were necropsied by using a stereomicroscope with digital images captured using the Kontron Progres digital camera. MRI scans were conducted followed by histological reconstructions using episcopic confocal microscopy. Newborn pigs (D115) and 2-day old pigs were analyzed by gross dissections and individual organs were separated and further analyzed by MRI. All animal work was humanely conducted under an approved University of Missouri IACUC Protocol.

The SAP130 mutant piglet was generated by a het x het mating of male 243 and female 244. The male (243) contains a 7bp deletion and the female (244) contains a 4bp deletion in SAP130. Six piglets were born. The piglets were genotyped in the same manner as Gabriel et al.6. After the piglets were genotyped, it was identified that one of the founder pigs was mosaic and contained a second modified allele, a 6bp deletion with 2bp mutation. It is not clear, which founder pig contained the third allele. Only one SAP130 mutant piglet was born from this litter containing an allele with a 4bp deletion and an allele with a 7bp deletion which resulted in a SAP130 null genotype. The mutant piglet was identified by external phenotype and euthanized for analysis at term.

For embryos at D20, D26, D30, and D35, following necropsy and MRI, the whole fetus or only the abdominal section of the fetus was embedded in paraffin for episcopic confocal microscopy (ECM). Paraffin embedded samples were sectioned using a Leica SM2500 sledge microtome and serial confocal images of the block face were captured using a Leica LSI scanning confocal macroscope mounted above the sample block as previously described6. The 2D serial image stacks collected were visualized using the OsiriX Dicom viewer11 (https://www.osirix-viewer.com). These image stacks could be digitally re-sectioned in multiple imaging planes and 3D reconstructed for optimal viewing of the abdominal organs.

Prior to MRI scanning, embryos/fetuses were fixed and stained with a gadolinium (Gd)-based contrast agent to shorten the tissue T1. Briefly, after fixation embryos/fetuses were immersed in 1:200 MultiHance23 (gadobenate dimeglumine, 529mg/ml, Bracco Diagnostic, Inc. Monroe Twp, NJ) diluted with phosphate-buffered saline (PBS) at 40C for at least 48h. After staining, small embryos were secured on a tongue depressor (McKesson Medical-Surgical, Irving, TX) with Webglue surgical adhesive (n-butyl cyanoacrylate, Patterson Veterinary, Devens, MA). The embryos/fetuses were then immersed in Fomblin Y (perfluoropolyether, Sigma-Aldrich Millipore) to eliminate the susceptibility artifact at the tissue-air interface and to avoid dehydration during imaging.

MRI was carried out as previously described, with special emphases on abdominal structures, using a Bruker Biospec 7T/30 system (Bruker Biospin MRI, Billerica,MA) with a 35-mm or 72-mm quadrature coil for both transmission and reception6. 3D MRI was acquired with a fast spin echo sequence, the Rapid Acquisition with Refocusing Echoes (RARE), with the following parameters: effective echo time (TE) 24.69ms,RARE factor 8, repetition time (TR) 900ms. We used RARE also known as Fast Spin-Echo (FSE) or Turbo Spin-Echo (TSE) pulse sequence for high-resolution 3D imaging with T2-weighted contrast. It generates similar T2-weighted contrast as the Half-Fourier-Acquired Single-shot Turbo spin Echo (HASTE), a Turbo spin-echo technique that is used for sequential acquisition of high-resolution T2-weighted images. However, the strategy for fast spin echo is different. Our RARE condition with RARE factor 8 uses 8 echoes as 8 phase-encoded k-space lines to accelerate acquisition; whereas HASTE uses a single-shot technique or segmented multiple shorts to cover sufficient k-space from a single TR. HASTE although commonly used in human scanners, it is not available in the Bruker preclinical scanner used in this study. RARE provides flexible T2-weighting conditions by changing RARE factors depending on the tissue types of interest. We have tested various RARE factors, TE, and TR combinations to optimize the contrast, signal to noise ratio (SNR), and scan time used in this study.

The field of view (FOV), acquisition matrix and voxel sizes varied based on the sample size. The typical spatial resolution for D26, D30, and D35 embryos ranged from 39m to 46m, that of D42, D64 and D90 fetuses ranged from 45m to 62m. The FOV, matrix, resolution, echo time, RARE factor, and other MR parameters used for imaging at the different GAs are provided in Supplemental Spread Sheet 1. The 3D MRI imaging stacks were exported with DICOM format and could be re-oriented to any viewing angle with Horos Dicom Viewer (Horosproject.org).

Necropsies were performed as previously described on D42, D64, D90, D105, D115 and 2-day old wildtype normal pigs which showed no external malformations6. Briefly the thoracic, abdominal, and pelvic viscera were examined in situ for malformations, the heart, great vessels, and lungs were removed as a block and examined using the sequential segmental analytical method24,25. Following examination of thoracic organs, the abdominal-pelvic visceral blocks were removed as a block and dissected and examined from behind (dorsal in the pig). Because pigs are quadrupeds, structures, which in bipedal mammals are described as inferior in pigs are described as posterior or caudal, for example the inferior caval vein can be referred to as the posterior or caudal caval vein in the pig. However, to better align the pig to the bipedal mammal, we have chosen, like others, to describe the abdominal organs of pigs as in bipedal mammals26. The abdominal organs in mammals obtain their basic gross appearance before term but continue to develop after birth by increasing in size or length as well as at the cellular and biochemical levels. In addition, in the very early embryo the organs begin by cell differentiation, and they lack the expected configuration that is seen in the fetus. In this study we focus on the assessment of the basic gross appearance of the organs before term.

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Profiling development of abdominal organs in the pig | Scientific Reports - Nature.com

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