ABSTRACT
Fracture healing complications increase with age, with higher rates of delayed unions and nonunions and an associated increase in morbidity and mortality in older adults. Macrophages have a dynamic role in fracture healing, and we have previously demonstrated that age-related changes in macrophages are associated with attenuated fracture repair in old mice. Here, we provide a single cell characterization of the immune cells involved in the early phase of fracture healing. We show that there were multiple transcriptionally distinct macrophage subpopulations present simultaneously within the healing tissue. Fracture healing was attenuated in old mice compared to young, and macrophages from the fracture callus of old mice demonstrated a pro-inflammatory phenotype compared to young. Interestingly, Trem2 expression was decreased in old macrophages compared to young. Young mice lacking Trem2 demonstrated attenuated fracture healing and inflammatory dysregulation similar to old mice. Trem2 dysregulation has previously been implicated in other age-related diseases, but its role in fracture healing is unknown. This work provides a robust characterization of the macrophage subpopulations involved in fracture healing, and further reveals the important role of Trem2 in fracture healing and may be a potential driver of age-related inflammatory dysregulation. Future work may further examine macrophages and Trem2 as potential therapeutic targets for management of fracture repair in older adults.
ABSTRACT
Background: Bone fracture is one of the most globally prevalent injuries, with an estimated 189 million bone fractures occurring annually. Delayed union or nonunion occurs in up to 15% of fractures and involves the interruption or complete failure of bone continuity following fracture. Preclinical testing is essential to support the translation of novel strategies to promote improved fracture repair treatment, but there is a paucity of small animal models that recapitulate clinical attributes associated with delayed fracture healing. This study explores whether the Zmpste24 -/- (Z24 -/- ) knockout mouse model of Hutchinson-Gilford progeria syndrome presents with delayed fracture healing. Leveraging the previously characterized Z24 -/- phenotype of genomic instability, epigenetic changes, and fragility, we hypothesize that these underlying alterations will lead to significantly delayed fracture healing relative to age-matched wild type (WT) controls. Methods: WT and Z24 -/- mice received intramedullary fixed tibia fractures at â¼12 weeks of age. Mice were sacrificed throughout the time course of repair for the collection of organs that would provide information regarding the local (fracture callus, bone marrow, inguinal lymph nodes) versus peripheral (peripheral blood, contralateral tibia, abdominal organs) tissue microenvironments. Analyses of these specimens include histomorphometry, µCT, mechanical strength testing, protein quantification, gene expression analysis, flow cytometry for cellular senescence, and immunophenotyping. Results: Z24 -/- mice demonstrated a significantly delayed rate of healing compared to WT mice with consistently smaller fracture calli containing higher proportion of cartilage and less bone after injury. Cellular senescence and pro-inflammatory cytokines were elevated in the Z24 -/- mice before and after fracture. These mice further presented with a dysregulated immune system, exhibiting generally decreased lymphopoiesis and increased myelopoiesis locally in the bone marrow, with more naïve and less memory T cell but greater myeloid activation systemically in the peripheral blood. Surprisingly, the ipsilateral lymph nodes had increased T cell activation and other pro-inflammatory NK and myeloid cells, suggesting that elevated myeloid abundance and activation contributes to an injury-specific hyperactivation of T cells. Conclusion: Taken together, these data establish the Z24 -/- progeria mouse as a model of delayed fracture healing that exhibits decreased bone in the fracture callus, with weaker overall bone quality, immune dysregulation, and increased cellular senescence. Based on this mechanism for delayed healing, we propose this Z24 -/- progeria mouse model could be useful in testing novel therapeutics that could address delayed healing. The Translational Potential of this Article: This study employs a novel animal model for delayed fracture healing that researchers can use to screen fracture healing therapeutics to address the globally prevalent issue of aberrant fracture healing.
ABSTRACT
Sonic hedgehog (SHH) signaling from the frontonasal ectodermal zone (FEZ) is a key regulator of craniofacial morphogenesis. Along with SHH, pre-B-cell leukemia homeobox (PBX) transcription factors regulate midfacial development. PBXs act in the epithelium during fusion of facial primordia, but their specific interactions with SHH have not been fully investigated. We hypothesized that PBX1/3 regulate SHH expression in the FEZ by activating or repressing transcription. The hypothesis was tested by manipulating PBX1/3 expression in chick embryos and profiling epigenomic landscapes at early developmental stages. PBX1/3 expression was perturbed in the chick face beginning at stage 10 (HH10) using RCAS viruses, and the resulting SHH expression was assessed at HH22. Overexpressing PBX1 expanded SHH expression, while overexpressing PBX3 decreased SHH expression. Conversely, reducing PBX1 expression decreased SHH expression, but reducing PBX3 induced ectopic SHH expression. We performed ATAC-seq and mapped binding of PBX1 and PBX3 with ChIP-seq on the FEZ at HH22 to assess direct interactions of PBX1/3 with the SHH locus. These multi-omics approaches uncovered a 400 bp PBX1-enriched element within intron 1 of SHH (chr2:8,173,222-8,173,621). Enhancer activity of this element was demonstrated by electroporation of reporter constructs in ovo and luciferase reporter assays in vitro . When bound by PBX1, this element upregulates transcription, while it downregulates transcription when bound by PBX3. The present study identifies a cis- regulatory element, named SFE1, that interacts with PBX1/3 to modulate SHH expression in the FEZ and establishes that PBX1 and PBX3 play complementary roles in SHH regulation during embryonic development.
ABSTRACT
Fractures continue to be a global economic burden as there are currently no osteoanabolic drugs approved to accelerate fracture healing. In this study, we aimed to develop an osteoanabolic therapy which activates the Wnt/ß-catenin pathway, a molecular driver of endochondral ossification. We hypothesize that using an mRNA-based therapeutic encoding ß-catenin could promote cartilage to bone transformation formation by activating the canonical Wnt signaling pathway in chondrocytes. To optimize a delivery platform built on recent advancements in liposomal technologies, two FDA-approved ionizable phospholipids, DLin-MC3-DMA (MC3) and SM-102, were used to fabricate unique ionizable lipid nanoparticle (LNP) formulations and then tested for transfection efficacy both in vitro and in a murine tibia fracture model. Using firefly luciferase mRNA as a reporter gene to track and quantify transfection, SM-102 LNPs showed enhanced transfection efficacy in vitro and prolonged transfection, minimal fracture interference and no localized inflammatory response in vivo over MC3 LNPs. The generated ß-cateninGOF mRNA encapsulated in SM-102 LNPs (SM-102-ß-cateninGOF mRNA) showed bioactivity in vitro through upregulation of downstream canonical Wnt genes, axin2 and runx2. When testing SM-102-ß-cateninGOF mRNA therapeutic in a murine tibia fracture model, histomorphometric analysis showed increased bone and decreased cartilage composition with the 45 µg concentration at 2 weeks post-fracture. µCT testing confirmed that SM-102-ß-cateninGOF mRNA promoted bone formation in vivo, revealing significantly more bone volume over total volume in the 45 µg group. Thus, we generated a novel mRNA-based therapeutic encoding a ß-catenin mRNA and optimized an SM-102-based LNP to maximize transfection efficacy with a localized delivery.
ABSTRACT
CD47 is a ubiquitous and pleiotropic cell-surface receptor. Disrupting CD47 enhances injury repair in various tissues but the role of CD47 has not been studied in bone injuries. In a murine closed-fracture model, CD47-null mice showed decreased callus bone volume, bone mineral content, and tissue mineral content as assessed by microcomputed tomography 10 days post-fracture, and increased fibrous volume as determined by histology. To understand the cellular basis for this phenotype, mesenchymal progenitors (MSC) were harvested from bone marrow. CD47-null MSC showed decreased large fibroblast colony formation (CFU-F), significantly less proliferation, and fewer cells in S-phase, although osteoblast differentiation was unaffected. However, consistent with prior research, CD47-null endothelial cells showed increased proliferation relative to WT cells. Similarly, in a murine ischemic fracture model, CD47-null mice showed reduced fracture callus bone volume and bone mineral content relative to WT. Consistent with our In vitro results, in vivo EdU labeling showed decreased cell proliferation in the callus of CD47-null mice, while staining for CD31 and endomucin demonstrated increased endothelial cell mass. Finally, WT mice administered a CD47 morpholino, which blocks CD47 protein production, showed a callus phenotype similar to that of non-ischemic and ischemic fractures in CD47-null mice, suggesting the phenotype was not due to developmental changes in the knockout mice. Thus, inhibition of CD47 during bone healing reduces both non-ischemic and ischemic fracture healing, in part, by decreasing MSC proliferation. Furthermore, the increase in endothelial cell proliferation and early blood vessel density caused by CD47 disruption is not sufficient to overcome MSC dysfunction.
ABSTRACT
CD47 is a ubiquitous and pleiotropic cell-surface receptor. Disrupting CD47 enhances injury repair in various tissues but the role of CD47 has not been studied in bone injuries. In a murine closed-fracture model, CD47-null mice showed decreased callus bone volume, bone mineral content, and tissue mineral content as assessed by microcomputed tomography 10 days post-fracture, and increased fibrous volume as determined by histology. To understand the cellular basis for this phenotype, mesenchymal progenitors (MSC) were harvested from bone marrow. CD47-null MSC showed decreased large fibroblast colony formation (CFU-F), significantly less proliferation, and fewer cells in S-phase, although osteoblast differentiation was unaffected. However, consistent with prior research, CD47-null endothelial cells showed increased proliferation relative to WT cells. Similarly, in a murine ischemic fracture model, CD47-null mice showed reduced fracture callus bone volume and bone mineral content relative to WT. Consistent with our in vitro results, in vivo EdU labeling showed decreased cell proliferation in the callus of CD47-null mice, while staining for CD31 and endomucin demonstrated increased endothelial cell mass. Finally, WT mice administered a CD47 morpholino, which blocks CD47 protein production, showed a callus phenotype similar to that of non-ischemic and ischemic fractures in CD47-null mice, suggesting the phenotype was not due to developmental changes in the knockout mice. Thus, inhibition of CD47 during bone healing reduces both non-ischemic and ischemic fracture healing, in part, by decreasing MSC proliferation. Furthermore, the increase in endothelial cell proliferation and early blood vessel density caused by CD47 disruption is not sufficient to overcome MSC dysfunction.
ABSTRACT
Craniosynostosis is a common yet complex birth defect, characterized by premature fusion of the cranial sutures that can be syndromic or nonsyndromic. With over 180 syndromic associations, reaching genetic diagnoses and understanding variations in underlying cellular mechanisms remains a challenge. Variants of FGFR2 are highly associated with craniosynostosis and warrant further investigation. Using the missense mutation FGFR2W290R , an effective mouse model of Crouzon syndrome, craniofacial features were analyzed using geometric morphometrics across developmental time (E10.5-adulthood, n = 665 total). Given the interrelationship between the cranial vault and basicranium in craniosynostosis patients, the basicranium and synchondroses were analyzed in perinates. Embryonic time points showed minimal significant shape differences. However, hetero- and homozygous mutant perinates and adults showed significant differences in shape and size of the cranial vault, face, and basicranium, which were associated with cranial doming and shortening of the basicranium and skull. Although there were also significant shape and size differences associated with the basicranial bones and clear reductions in basicranial ossification in cleared whole-mount samples, there were no significant alterations in chondrocyte cell shape, size, or orientation along the spheno-occipital synchondrosis. Finally, shape differences in the cranial vault and basicranium were interrelated at perinatal stages. These results point toward the possibility that facial shape phenotypes in craniosynostosis may result in part from pleiotropic effects of the causative mutations rather than only from the secondary consequences of the sutural defects, indicating a novel direction of research that may shed light on the etiology of the broad changes in craniofacial morphology observed in craniosynostosis syndromes.
ABSTRACT
The genetic architecture of trait variance has long been of interest in genetics and evolution. One of the earliest attempts to understand this architecture was presented in Lerner's Genetic Homeostasis (1954). Lerner proposed that heterozygotes should be better able to tolerate environmental perturbations because of functional differences between the alleles at a given locus, with each allele optimal for slightly different environments. This greater robustness to environmental variance, he argued, would result in smaller trait variance for heterozygotes. The evidence for Lerner's hypothesis has been inconclusive. To address this question using modern genomic methods, we mapped loci associated with differences in trait variance (vQTL) on 1,101 individuals from the F34 of an advanced intercross between LG/J and SM/J mice. We also mapped epistatic interactions for these vQTL in order to understand the influence of epistasis for the architecture of trait variance. We did not find evidence supporting Lerner's hypothesis, that heterozygotes tend to have smaller trait variances than homozygotes. We further show that the effects of most mapped loci on trait variance are produced by epistasis affecting trait means and that those epistatic effects account for about a half of the differences in genotypic-specific trait variances. Finally, we propose a model where the different interactions between the additive and dominance effects of the vQTL and their epistatic partners can explain Lerner's original observations but can also be extended to include other conditions where heterozygotes are not the least variable genotype.
Subject(s)
Epistasis, Genetic , Models, Genetic , Mice , Male , Animals , Phenotype , Genotype , Mice, Inbred Strains , Heterozygote , HomozygoteABSTRACT
Introduction: Currently, there are no non-surgical FDA-approved biological approaches to accelerate fracture repair. Injectable therapies designed to stimulate bone healing represent an exciting alternative to surgically implanted biologics, however, the translation of effective osteoinductive therapies remains challenging due to the need for safe and effective drug delivery. Hydrogel-based microparticle platforms may be a clinically relevant solution to create controlled and localized drug delivery to treat bone fractures. Here, we describe poly (ethylene glycol) dimethacrylate (PEGDMA)-based microparticles, in the shape of microrods, loaded with beta nerve growth factor (ß-NGF) for the purpose of promoting fracture repair. Methods: Herein, PEGDMA microrods were fabricated through photolithography. PEGDMA microrods were loaded with ß-NGF and in vitro release was examined. Subsequently, bioactivity assays were evaluated in vitro using the TF-1 tyrosine receptor kinase A (Trk-A) expressing cell line. Finally, in vivo studies using our well-established murine tibia fracture model were performed and a single injection of the ß-NGF loaded PEGDMA microrods, non-loaded PEGDMA microrods, or soluble ß-NGF was administered to assess the extent of fracture healing using Micro-computed tomography (µCT) and histomorphometry. Results: In vitro release studies showed there is significant retention of protein within the polymer matrix over 168 hours through physiochemical interactions. Bioactivity of protein post-loading was confirmed with the TF-1 cell line. In vivo studies using our murine tibia fracture model show that PEGDMA microrods injected at the site of fracture remained adjacent to the callus for over 7 days. Importantly, a single injection of ß-NGF loaded PEGDMA microrods resulted in improved fracture healing as indicated by a significant increase in the percent bone in the fracture callus, trabecular connective density, and bone mineral density relative to soluble ß-NGF control indicating improved drug retention within the tissue. The concomitant decrease in cartilage fraction supports our prior work showing that ß-NGF promotes endochondral conversion of cartilage to bone to accelerate healing. Discussion: We demonstrate a novel and translational method wherein ß-NGF can be encapsulated within PEGDMA microrods for local delivery and that ß-NGF bioactivity is maintained resulting in improved bone fracture repair.
ABSTRACT
Morphogenesis requires highly coordinated, complex interactions between cellular processes: proliferation, migration, and apoptosis, along with physical tissue interactions. How these cellular and tissue dynamics drive morphogenesis remains elusive. Three dimensional (3D) microscopic imaging poses great promise, and generates elegant images. However, generating even moderate through-put quantified images is challenging for many reasons. As a result, the association between morphogenesis and cellular processes in 3D developing tissues has not been fully explored. To address this critical gap, we have developed an imaging and image analysis pipeline to enable 3D quantification of cellular dynamics along with 3D morphology for the same individual embryo. Specifically, we focus on how 3D distribution of proliferation relates to morphogenesis during mouse facial development. Our method involves imaging with light-sheet microscopy, automated segmentation of cells and tissues using machine learning-based tools, and quantification of external morphology via geometric morphometrics. Applying this framework, we show that changes in proliferation are tightly correlated to changes in morphology over the course of facial morphogenesis. These analyses illustrate the potential of this pipeline to investigate mechanistic relationships between cellular dynamics and morphogenesis during embryonic development.
ABSTRACT
Introduction: Clinical and preclinical data suggest accelerated bone fracture healing in subjects with an additional traumatic brain injury (TBI). Mechanistically, altered metabolism and neuro-endocrine regulations have been shown to influence bone formation after combined fracture and TBI, thereby increasing the bone content in the fracture callus. However, the early inflammatory response towards fracture and TBI has not been investigated in detail so far. This is of great importance, since the early inflammatory phase of fracture healing is known to be essential for the initiation of downstream regenerative processes for adequate fracture repair. Methods: Therefore, we analyzed systemic and local inflammatory mediators and immune cells in mice which were exposed to fracture only or fracture + TBI 6h and 24h after injury. Results: We found a dysregulated systemic immune response and significantly fewer neutrophils and mast cells locally in the fracture hematoma. Further, local CXCL10 expression was significantly decreased in the animals with combined trauma, which correlated significantly with the reduced mast cell numbers. Discussion: Since mast cells and mast cell-derived CXCL10 have been shown to increase osteoclastogenesis, the reduced mast cell numbers might contribute to higher bone content in the fracture callus of fracture + TBI mice due to decreased callus remodeling.
Subject(s)
Brain Injuries, Traumatic , Fractures, Bone , Animals , Mice , Bony Callus/metabolism , Brain Injuries, Traumatic/immunology , Fracture Healing , Fractures, Bone/immunology , Osteogenesis/physiologyABSTRACT
Older adults suffer more bone fractures with higher rates of healing complications and increased risk of morbidity and mortality. An improved understanding of the cellular and molecular mechanism of fracture healing and how such processes are perturbed with increasing age may allow for better treatment options to manage fractures in older adults. Macrophages are attractive therapeutics due to their role in several phases of fracture healing. After injury, bone marrow-derived macrophages are recruited to the injury and propagate the inflammatory response, contribute to resolution of inflammation, and promote bone regeneration. A tissue resident population of macrophages named osteal macrophages are present in the periosteum and are directly associated with osteoblasts and these cells contribute to bone formation. Here, we utilized bulk RNA sequencing to analyze the transcriptional activity of osteal macrophages from old and young mice present in primary calvarial cultures. Macrophages demonstrated a diverse transcriptional profile, expressing genes involved in immune function as well as wound healing and regeneration. Periostin was significantly downregulated in macrophages from old mice compared to young. Periostin is an extracellular matrix protein with important functions that promote osteoblast activity during bone regeneration. An age-related decrease of periostin expression was verified in the fracture callus of old mice compared to young. Young periostin knockout mice demonstrated attenuated fracture healing outcomes that reflected what is observed in old mice. This study supports an important role of periostin in fracture healing, and therapeutically targeting the age-related decrease in periostin may improve healing outcomes in older populations.
Subject(s)
Fracture Healing , Fractures, Bone , Mice , Animals , Fracture Healing/physiology , Bony Callus , Osteogenesis/physiology , Bone Regeneration , Osteoblasts , Mice, KnockoutABSTRACT
Background: Cardiac injuries following trauma are associated with a worse clinical outcome. So-called trauma-induced secondary cardiac injuries have been recently described after experimental long bone fracture even in absence of direct heart damage. With the progressive aging of our society, the number of elderly trauma victims rises and therefore the incidence of hip fractures increases. Hip fractures were previously shown to be associated with adverse cardiac events in elderly individuals, which have mainly been attributed to pre-conditioned cardiac diseases. The aim of the present study was to investigate the effect of hip fractures on the heart in healthy young and middle-aged mice. Materials and Methods: Young (12-week-old) and middle-aged (52-week-old) female C57BL/6 mice either received an intramedullary stabilized proximal femur fracture or sham treatment. The observation time points included 6 and 24 h. Systemic levels of pro-inflammatory mediators as well as local inflammation and alterations in myocardial structure, metabolism and calcium homeostasis in left ventricular tissue was analyzed following hip fracture by multiplex analysis, RT-qPCR and immunohistochemistry. Results: After hip fracture young and middle-aged mice showed increased systemic IL-6 and KC levels, which were significantly elevated in the middle-aged animals. Furthermore, the middle-aged mice showed enhanced myocardial expression of HMGB1, TLR2/4, TNF, IL1ß and NLRP3 as well as considerable alterations in the myocardial expression of glucose- and fatty acid transporters (HFABP, GLUT4), calcium homeostasis proteins (SERCA) and cardiac structure proteins (desmin, troponin I) compared to the young animals following hip fracture. Conclusion: Young and middle-aged mice showed local myocardial alterations, which might predispose for the development of secondary cardiac injury following hip fracture. Age and the age-associated phenomenon of 'inflammaging' seemed to be an independent risk factor aggravating and accelerating cardiac alterations following hip fracture.
Subject(s)
HMGB1 Protein , Hip Fractures , Animals , Calcium , Desmin , Fatty Acids , Female , Glucose , Hip Fractures/etiology , Inflammation Mediators , Interleukin-6 , Mice , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein , Risk Factors , Toll-Like Receptor 2 , Troponin IABSTRACT
BACKGROUND: Asymmetries in craniofacial anomalies are commonly observed. In the facial skeleton, the left side is more commonly and/or severely affected than the right. Such asymmetries complicate treatment options. Mechanisms underlying variation in disease severity between individuals as well as within individuals (asymmetries) are still relatively unknown. RESULTS: Developmental reductions in fibroblast growth factor 8 (Fgf8) have a dosage dependent effect on jaw size, shape, and symmetry. Further, Fgf8 mutants have directionally asymmetric jaws with the left side being more affected than the right. Defects in lower jaw development begin with disruption to Meckel's cartilage, which is discontinuous. All skeletal elements associated with the proximal condensation are dysmorphic, exemplified by a malformed and misoriented malleus. At later stages, Fgf8 mutants exhibit syngnathia, which falls into two broad categories: bony fusion of the maxillary and mandibular alveolar ridges and zygomatico-mandibular fusion. All of these morphological defects exhibit both inter- and intra-specimen variation. CONCLUSIONS: We hypothesize that these asymmetries are linked to heart development resulting in higher levels of Fgf8 on the right side of the face, which may buffer the right side to developmental perturbations. This mouse model may facilitate future investigations of mechanisms underlying human syngnathia and facial asymmetry.
Subject(s)
Branchial Region , Heart , Animals , Fibroblast Growth Factor 8/genetics , Humans , Jaw Abnormalities , Maxilla , Mice , Mouth AbnormalitiesABSTRACT
Complex morphological traits are the product of many genes with transient or lasting developmental effects that interact in anatomical context. Mouse models are a key resource for disentangling such effects, because they offer myriad tools for manipulating the genome in a controlled environment. Unfortunately, phenotypic data are often obtained using laboratory-specific protocols, resulting in self-contained datasets that are difficult to relate to one another for larger scale analyses. To enable meta-analyses of morphological variation, particularly in the craniofacial complex and brain, we created MusMorph, a database of standardized mouse morphology data spanning numerous genotypes and developmental stages, including E10.5, E11.5, E14.5, E15.5, E18.5, and adulthood. To standardize data collection, we implemented an atlas-based phenotyping pipeline that combines techniques from image registration, deep learning, and morphometrics. Alongside stage-specific atlases, we provide aligned micro-computed tomography images, dense anatomical landmarks, and segmentations (if available) for each specimen (N = 10,056). Our workflow is open-source to encourage transparency and reproducible data collection. The MusMorph data and scripts are available on FaceBase ( www.facebase.org , https://doi.org/10.25550/3-HXMC ) and GitHub ( https://github.com/jaydevine/MusMorph ).
Subject(s)
Databases, Factual , Mice , Animals , Brain , Mice/anatomy & histology , X-Ray MicrotomographyABSTRACT
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
Realistic mappings of genes to morphology are inherently multivariate on both sides of the equation. The importance of coordinated gene effects on morphological phenotypes is clear from the intertwining of gene actions in signaling pathways, gene regulatory networks, and developmental processes underlying the development of shape and size. Yet, current approaches tend to focus on identifying and localizing the effects of individual genes and rarely leverage the information content of high-dimensional phenotypes. Here, we explicitly model the joint effects of biologically coherent collections of genes on a multivariate trait - craniofacial shape - in a sample of n = 1145 mice from the Diversity Outbred (DO) experimental line. We use biological process Gene Ontology (GO) annotations to select skeletal and facial development gene sets and solve for the axis of shape variation that maximally covaries with gene set marker variation. We use our process-centered, multivariate genotype-phenotype (process MGP) approach to determine the overall contributions to craniofacial variation of genes involved in relevant processes and how variation in different processes corresponds to multivariate axes of shape variation. Further, we compare the directions of effect in phenotype space of mutations to the primary axis of shape variation associated with broader pathways within which they are thought to function. Finally, we leverage the relationship between mutational and pathway-level effects to predict phenotypic effects beyond craniofacial shape in specific mutants. We also introduce an online application that provides users the means to customize their own process-centered craniofacial shape analyses in the DO. The process-centered approach is generally applicable to any continuously varying phenotype and thus has wide-reaching implications for complex trait genetics.
Subject(s)
Face/anatomy & histology , Skull/anatomy & histology , Multivariate Analysis , PhenotypeABSTRACT
Canonical Wnt signaling plays multiple roles critical to normal craniofacial development while its dysregulation is known to be involved in structural birth defects of the face. However, when and how Wnt signaling influences phenotypic variation, including those associated with disease, remains unclear. One potential mechanism is via Wnt signaling's role in the patterning of an early facial signaling center, the frontonasal ectodermal zone (FEZ), and its subsequent regulation of early facial morphogenesis. For example, Wnt signaling may directly alter the shape and/or magnitude of expression of the sonic hedgehog (SHH) domain in the FEZ. To test this idea, we used a replication-competent avian sarcoma retrovirus (RCAS) encoding Wnt3a to modulate its expression in the facial mesenchyme. We then quantified and compared ontogenetic changes in treated to untreated embryos in the three-dimensional (3D) shape of both the SHH expression domain of the FEZ, and the morphology of the facial primordia and brain using iodine-contrast microcomputed tomography imaging and 3D geometric morphometrics (3DGM). We found that increased Wnt3a expression in early stages of head development produces correlated variation in shape between both structural and signaling levels of analysis. In addition, altered Wnt3a activation disrupted the integration between the forebrain and other neural tube derivatives. These results show that activation of Wnt signaling influences facial shape through its impact on the forebrain and SHH expression in the FEZ, and highlights the close relationship between morphogenesis of the forebrain and midface.
ABSTRACT
BACKGROUND: Trauma is the leading cause of death and disability worldwide, especially in the young population. Cardiac injuries are an independent predictor for a poor overall outcome after trauma. The aim of the present study was to analyze systemic inflammation as well as local cardiac inflammation after experimental limb-, neuro- and combined trauma in mice. METHODS: Male C57BL/6 mice received either a closed tibia fracture (Fx), isolated traumatic brain injury (TBI) or a combination of both (Fx â+ âTBI). Control animals underwent sham procedure. After 6 and 24 âh, systemic levels of inflammatory mediators were analyzed, respectively. Locally, cardiac inflammation and cardiac structural alterations were investigated in left ventricular tissue of mice 6 and 24 âh after trauma. RESULTS: Mice showed enhanced systemic inflammation after combined trauma, which was manifested by increased levels of KC, MCP-1 and G-CSF. Locally, mice exhibited increased expression of inflammatory cytokines (IL-1ß, TNF) in heart tissue, which was probably mediated via toll-like receptor (TLR) signaling. Furthermore, mice demonstrated a redistribution of connexin 43 in cardiac tissue, which appeared predominantly after combined trauma. Besides inflammation and structural cardiac alterations, expression of glucose transporter 4 (GLUT4) mRNA was increased in the heart early after TBI and after combination of TBI and limb fracture, indicating a modification of energy metabolism. Early after combination of TBI and tibia fracture, nitrosative stress was increased, manifested by elevation of nitrotyrosine in cardiac tissue. Finally, mice showed a trend of increased systemic levels of cardiac troponin I and heart-fatty acid binding protein (HFABP) after combined trauma, which was associated with a significant decrease of troponin I and HFABP mRNA expression in cardiac tissue after TBI and combination of TBI and limb fracture. CONCLUSION: Mice exhibited early cardiac alterations as well as alterations in cardiac glucose transporter expression, indicating a modification of energy metabolism, which might be linked to increased systemic- and local cardiac inflammation after limb-, neuro- and combined trauma. These cardiac alterations might predispose individuals for secondary cardiac damage after trauma that might compromise cardiac function after TBI and long bone fracture. TRANSLATIONAL POTENTIAL STATEMENT: Injuries to the head and extremities frequently occur after severe trauma. In our study, we analyzed the effects of closed tibia fracture, isolated TBI, and the combination of both injuries with regard to the development of post-traumatic secondary cardiac injuries.