Embryonic axis elongation is a complex multi-tissue morphogenetic process responsible for the formation of the posterior part of the amniote body. How movements and growth are coordinated between the different posterior tissues (e.g. neural tube, axial and paraxial mesoderm, lateral plate, ectoderm, endoderm) to drive axis morphogenesis remain largely unknown. Here, we use quail embryos to quantify cell behavior and tissue movements during elongation. We quantify the tissue-specific contribution to axis elongation using 3D volumetric techniques, then quantify tissue-specific parameters such as cell density and proliferation. To study cell behavior at a multi-tissue scale, we used high-resolution 4D imaging of transgenic quail embryos expressing fluorescent proteins. We developed specific tracking and image analysis techniques to analyze cell motion and compute tissue deformations in 4D. This analysis reveals extensive sliding between tissues during axis extension. Further quantification of tissue tectonics showed patterns of rotations, contractions and expansions, which are consistent with the multi-tissue behavior observed previously. Our approach defines a quantitative and multi-scale method to analyze the coordination between tissue behaviors during early vertebrate embryo morphogenetic events.

Embryogenesis is the coordinated assembly of tissues during morphogenesis through changes in individual cell behaviors and collective cell movements. Dynamic imaging, combined with quantitative analysis, is ideal for investigating fundamental questions in developmental biology involving cellular differentiation, growth control and morphogenesis. However, a reliable amniote model system that is amenable to the rigors of extended, high-resolution imaging and cell tracking has been lacking. To address this shortcoming, we produced a novel transgenic quail that ubiquitously expresses nuclear localized monomer cherry fluorescent protein (chFP). We characterize the expression pattern of chFP and provide concrete examples of how Tg(PGK1:H2B-chFP) quail can be used to dynamically image and analyze key morphogenetic events during embryonic stages X to 11.

Embryonic axis extension is a complex multi-tissue morphogenetic process responsible for the formation of the posterior part of the amniote body. Cells located in the caudal part of the embryo divide and rearrange to participate in the elongation of the different embryonic tissues (e.g. neural tube, axial and paraxial mesoderm, lateral plate, ectoderm, endoderm). We previously identified the paraxial mesoderm as a crucial player of axis elongation, but how movements and growth are coordinated between the different posterior tissues to drive morphogenesis remain largely unknown. Here we use the quail embryo as a model system to quantify cell behavior and movements in the various tissues of the elongating embryo. We first quantify the tissue-specific contribution to axis elongation by using 3D volumetric techniques, then quantify tissuespecific parameters such as cell density and proliferation at different embryonic stages. To be able to study cell behavior at a multi-tissue scale we used high-resolution 4D imaging of transgenic quail embryos expressing constitutively expressed fluorescent proteins. We developed specific tracking and image analysis techniques to analyze cell motion and compute tissue deformations in 4D. This analysis reveals extensive sliding between tissues during axis extension. Further quantification of "tissue tectonics" showed patterns of rotations, contractions and expansions, which are coherent with the multi-tissue behavior observed previously. Our results confirm the central role of the PSM in axis extension; we propose that the PSM specific cell proliferation and migration programs control the coordination of elongation between tissues during axis extension. Material and methodsQuail embryo and embryo culture Wild-type quail embryos (Japonica coturnix) were obtained from commercial sources and from the USC aviary. The PGK1:H2B-chFP quail line generation was described previously (Huss et al. 2015) and is maintained in the USC aviary. Embryos were staged according to (Ainsworth et al. 2010;Hamburger andHamilton 1992) Hamburger andHamilton, 1951). Embryos were cultured ex ovo with filter paper on albumen agar plates according to the EC (Early chick) technique (Chapman et al. 2001). Staining and immunodetectionsEmbryo were collected at the desired stages and fixed overnight in 4% formaldehyde [36% formaldehyde (47608, Sigma) diluted to 4% in PBS]. Blocking and tissues permeabilization were carried out for 2 hours in PBS/0.5% Triton/1% donkey serum. Primary antibodies against Sox2 (1/5000, millipore ab5603) and Bra (1/500, R&D AF2085) were incubated overnight at 4°C. After washing off primary antibody in PBT (PBS/O.1% Triton), embryos were incubated with secondary antibodies (donkey anti goat Alexa 594 and goat anti rabbit Alexa 488, 1/1000, Molecular probes) and DAPI (1/1000, D1306, Molecular probes) overnight at 4°C. The embryos were cleared in U2 scale (Hama et al. 2011) for at least 48h at 4°C and then mounted between slide and coverslip and imaged by confocal/2P microscopy. Proliferation ...

Louis-Jean et al. This is an open access article distributed under the terms of the Creative Commons Attribution License CC-BY 4.0., which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Stigmatizing language can negatively influence providers’ attitudes and care toward patients, but this has not been studied among physiatrists. An online survey was created to assess whether stigmatizing language can impact physical medicine and rehabilitation trainees’ attitudes toward patients. We hypothesized stigmatizing language would negatively impact trainees’ attitudes. Participants were randomized to a stigmatizing or neutral language vignette describing the same hypothetical spinal cord injury patient. Questions were asked about attitudes and assumptions toward the patient, pain management based on the vignette, and general views regarding individuals with disabilities. Between August 2021 and January 2022, 75 US physical medicine and rehabilitation residency trainees participated. Thirty-seven (49.3%) identified as women; 52 (69.3%) were White, and half (50.6%) received the stigmatized vignette. Participants exposed to stigmatizing language scored 4.8 points lower (P < 0.01) on the provider attitude toward patient scale compared with those exposed to neutral language. There were no significant differences in the disability attitude scores between the two groups (P = 0.81). These findings may indicate that stigmatizing language in the medical record may negatively affect physical medicine and rehabilitation trainees’ attitudes toward patients. Further exploration is needed to identify the best way to educate trainees and reduce the propagation of bias in the medical record.