During the development of the face, tissues move, change shape, and fuse in tightly orchestrated patterns to create all the parts of a normal face. These shape changes are driven by factors such as ...cell signaling, migration, proliferation, and apoptosis. However, the contributions of each of these drivers to morphogenesis are poorly studied. Here, we explore differential cell proliferation as a driver of mouse facial morphogenesis. We quantify patterns in both the spatial distribution and orientation of proliferation in the developing face in 3D over a critical period of murine facial development (E9.5‐E11.5). We use immunostaining with light sheet microscopy (LSM) to capture total and proliferating nuclei. To compare proliferative density in facial tissues, we segment these images using a novel convolutional neural network. We then generate atlases of average proliferation at each half‐day age point within our range and use these to identify relationships between morphology and cell proliferation. We show that regions with more dense proliferation tend to undergo more intensive shape changes. We then simulate outgrowth of the maxillary process using a cell simulation engine, PhysiCell, to demonstrate that differential proliferation is necessary to maintain expected morphology in growing tissues. In addition to differential proliferation, localized orientation of cell division could also affect morphology. In plants and some animal tissues, including murine limb buds, preferentially oriented cell proliferation drives shape change by causing tissue elongation in specific directions. We explore the orientation and distribution of cell proliferation using LSM: we inject pregnant dams with a synthetic nucleotide, EdU, 5 minutes before harvest to mark the daughter cells of proliferative events occurring in the interim. We then compare the angles of the proliferative axes for each pair of daughters relative to the primary direction of tissue growth. Preliminary results suggest that cell proliferation in the maxillary and nasal processes is oriented preferentially towards the axis of tissue growth. These results suggest that both the distribution and orientation of cell proliferation play a role in murine facial morphogenesis. Understanding the mechanisms underlying morphogenesis is important to guide future research that could lead to earlier and more robust diagnosis and treatment of syndromes and facial abnormalities.
A variety of genetic mutations affect cell proliferation during organism development, leading to structural birth defects. However, the mechanisms by which these alterations influence the development ...of the face remain unclear. Cell proliferation and its relation to shape variation can be studied using Light-Sheet Microscopy (LSM) imaging across a range of developmental time points using mouse models. The aim of this work was to develop and evaluate accurate automatic methods based on convolutional neural networks (CNNs) for: (i) tissue segmentation (neural ectoderm and mesenchyme), (ii) cell segmentation in nuclear-stained images, and (iii) segmentation of proliferating cells in phospho-Histone H3 (pHH3)-stained LSM images of mouse embryos. For training and evaluation of the CNN models, 155 to 176 slices from 10 mouse embryo LSM images with corresponding manual segmentations were available depending on the segmentation task. Three U-net CNN models were trained optimizing their loss functions, among other hyper-parameters, depending on the segmentation task. The tissue segmentation achieved a macro-average F-score of 0.84, whereas the inter-observer value was 0.89. The cell segmentation achieved a Dice score of 0.57 and 0.56 for nuclear-stained and pHH3-stained images, respectively, whereas the corresponding inter-observer Dice scores were 0.39 and 0.45, respectively. The proposed pipeline using the U-net CNN architecture can accelerate LSM image analysis and together with the annotated datasets can serve as a reference for comparison of more advanced LSM image segmentation methods in future.
Abstract only
There is a long‐standing prediction that small changes in proliferation and apoptosis during the time frame of facial morphogenesis act to shape the face. Further, many studies show ...genetic alterations that cause structural birth defects affect local proliferation or apoptosis. Yet, it is unclear how much of local change in regional proliferation would be necessary to cause a defect. Here, we set out to understand the relationship between growth, morphology and proliferation and test that prediction that targeted proliferation shapes the developing face by quantifying proliferation and apoptosis in 3D and relating it to the growth of the face. We use whole mount staining for proliferation and apoptosis markers, whole tissue clearing methods, lightsheet microscopy and atlas and machine learning based quantification methods to identify individual proliferating or apoptotic nuclei within a 3D tissue structure at a set time point. We also employee geometric morphometric analysis of the same tissue structure to quantify overall morphology. By collecting data at various time points across facial development (E9.5–E11.5) and quantifying the age of each embryo, we are able to relate cell biological level growth to tissue level growth and morphological change and relate these two parameters in a way not performed previously.
Support or Funding Information
NIH NIDCR R01‐DE019638 to RM and BH, NSERC Discovery to BH, and CIHR Foundation grant to BH and RM, CIHR postdoctoral fellowship to RMG.
Atlas based quantification of proliferation: A) Maximum Projection of the embryo highlighting the external morphology ‐ lateral view. B) Proliferation staining (phospho‐Histone H3) ‐ lateral view. C–D) Surface morphology of the atlas (n=5) C ‐ anterior view, D ‐ Lateral view. E–F) Heat map of proliferating cells (no density correction) E ‐ anterior view, F ‐ Lateral view.
Figure 1
One common phenotype observed in response to many developmental perturbations is a change in proliferation or apoptosis. Further, it is often predicted that small changes in proliferation or ...apoptosis can explain the development of a structural birth defect. One flaw with this logic is that little is known about the relationships between proliferation and morphology in the face. Does proliferation actually play a role in the normal directional outgrowth and morphological changes which pattern the developing face? Here, we set out to understand the spatial distribution of proliferation in the developing mouse face and relate regional proliferation to the growth of the face over a small span of developmental time (E10‐E11). We use light sheet microscopy to capture total and proliferating nuclei in 30 E10.5 to 11.5 mouse embryo heads. Cells are quantified using a convolutional neural network methodology that has similar accuracy in cell identification to the between observer error. From these images, we then generate an atlas using linear and non‐linear transformation and perform analysis of embryo morphology and distribution of proliferation relative to total cells. Models of proliferation and its ability to alter morphology are generated in PhysiCell (www.physicell.org). We identify regions where there is both change in proliferation and morphology that relates to changes in the number of tail somites. We also use the spatial data gathered from these to inform a model of growth of the maxillary prominence to determine how much proliferation is likely to contribute to the directional growth of the maxilla.
The crystal structure of gpD, the capsid-stabilizing protein of bacteriophage lambda, was solved at 1.1 A resolution. Data were obtained from twinned crystals in space group P21 and refined with ...anisotropic temperature factors to an R-factor of 0.098 (Rfree = 0. 132). GpD (109 residues) has a novel fold with an unusually low content of regular secondary structure. Noncrystallographic trimers with substantial intersubunit interfaces were observed. The C-termini are well ordered and located on one side of the trimer, relatively far from its three-fold axis. The N-termini are disordered up to Ser 15, which is close to the three-fold axis and on the same side as the C-termini. A density map of the icosahedral viral capsid at 15 A resolution, obtained by cryo-electron microscopy and image reconstruction, reveals gpD trimers, seemingly indistinguishable from the ones seen in the crystals, at all three-fold sites. The map further reveals that the side of the trimer that binds to the capsid is the side on which both termini reside. Despite this orientation of the gpD trimer, fusion proteins connected by linker peptides to either terminus bind to the capsid, allowing protein and peptide display.
For the crystallization of proteins under microgravity conditions, a Chinese re-entry system was used, in which 101 experiments of 25 different biological macromolecules were accommodated. From the ...results obtained we conclude that under microgravity conditions crystal growth can only be expected under those crystallization conditions which also permit crystal growthon earth. A number of space-grown crystals were larger in size and of a better quality in their ability to diffract X-rays than the corresponding ground control crystals grown at the Chinese launch site. However, the space-grown crystals have not reached the X-ray diffraction quality of the crystals obtained under optimal conditions in the home laboratories.
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