-
BMC Biology Feb 2023Skeletal muscle development is a multistep process whose understanding is central in a broad range of fields and applications, from the potential medical value to human...
BACKGROUND
Skeletal muscle development is a multistep process whose understanding is central in a broad range of fields and applications, from the potential medical value to human society, to its economic value associated with improvement of agricultural animals. Skeletal muscle initiates in the somites, with muscle precursor cells generated in the dermomyotome and dermomyotome-derived myotome before muscle differentiation ensues, a developmentally regulated process that is well characterized in model organisms. However, the regulation of skeletal muscle ontogeny during embryonic development remains poorly defined in farm animals, for instance in pig. Here, we profiled gene expression and chromatin accessibility in developing pig somites and myotomes at single-cell resolution.
RESULTS
We identified myogenic cells and other cell types and constructed a differentiation trajectory of pig skeletal muscle ontogeny. Along this trajectory, the dynamic changes in gene expression and chromatin accessibility coincided with the activities of distinct cell type-specific transcription factors. Some novel genes upregulated along the differentiation trajectory showed higher expression levels in muscular dystrophy mice than that in healthy mice, suggesting their involvement in myogenesis. Integrative analysis of chromatin accessibility, gene expression data, and in vitro experiments identified EGR1 and RHOB as critical regulators of pig embryonic myogenesis.
CONCLUSIONS
Collectively, our results enhance our understanding of the molecular and cellular dynamics in pig embryonic myogenesis and offer a high-quality resource for the further study of pig skeletal muscle development and human muscle disease.
Topics: Animals; Mice; Cell Differentiation; Chromatin; Chromatin Immunoprecipitation Sequencing; Gene Expression Regulation, Developmental; Muscle Development; Muscle, Skeletal; Single-Cell Analysis; Single-Cell Gene Expression Analysis; Swine
PubMed: 36726129
DOI: 10.1186/s12915-023-01519-z -
Frontiers in Cell and Developmental... 2022During vertebrate development, symmetry breaking occurs in the left-right organizer (LRO). The transfer of asymmetric molecular information to the lateral plate mesoderm...
During vertebrate development, symmetry breaking occurs in the left-right organizer (LRO). The transfer of asymmetric molecular information to the lateral plate mesoderm is essential for the precise patterning of asymmetric internal organs, such as the heart. However, at the same developmental time, it is crucial to maintain symmetry at the somite level for correct musculature and vertebrae specification. We demonstrate how left-right signals affect the behavior of zebrafish somite cell precursors by using live imaging and fate mapping studies in dand5 homozygous mutants compared to wildtype embryos. We describe a population of cells in the vicinity of the LRO, named Non-KV Sox17:GFP+ Tailbud Cells (NKSTCs), which migrate anteriorly and contribute to future somites. We show that NKSTCs originate in a cluster of cells aligned with the midline, posterior to the LRO, and leave that cluster in a left-right alternating manner, primarily from the left side. Fate mapping revealed that more NKSTCs integrated somites on the left side of the embryo. We then abolished the asymmetric cues from the LRO using dand5-/- mutant embryos and verified that NKSTCs no longer displayed asymmetric patterns. Cell exit from the posterior cluster became bilaterally synchronous in dand5-/- mutants. Our study revealed a new link between somite specification and Dand5 function. The gene dand5 is well known as the first asymmetric gene involved in vertebrate LR development. This study revealed a new link for Dand5 as a player in cell exit from the maturation zone into the presomitic mesoderm, affecting the expression patterns of myogenic factors and tail size.
PubMed: 36699016
DOI: 10.3389/fcell.2022.989615 -
Frontiers in Cell and Developmental... 2022Neural tube closure is a fundamental process during vertebrate embryogenesis, which leads to the formation of the central nervous system. Defective neural tube closure...
Neural tube closure is a fundamental process during vertebrate embryogenesis, which leads to the formation of the central nervous system. Defective neural tube closure leads to neural tube defects which are some of the most common human birth defects. While the intrinsic morphogenetic events shaping the neuroepithelium have been studied extensively, how tissues mechanically coupled with the neural plate influence neural tube closure remains poorly understood. Here, using embryos, live imaging in combination with loss of function experiments and morphometric analysis of fixed samples we explore the reciprocal mechanical communication between the neural plate and the somitic mesoderm and its impact on tissue morphogenesis. We show that although somitic mesoderm convergent extension occurs independently from neural plate morphogenesis neural tube closure depends on somitic mesoderm morphogenesis. Specifically, impaired somitic mesoderm remodelling results in defective apical constriction within the neuroepithelium and failure of neural tube closure. Last, our data reveal that mild abnormalities in somitic mesoderm and neural plate morphogenesis have a synergistic effect during neurulation, leading to severe neural tube closure defects. Overall, our data reveal that defective morphogenesis of tissues mechanically coupled with the neural plate can not only drastically exacerbate mild neural tube defects that may arise from abnormalities within the neural tissue but can also elicit neural tube defects even when the neural plate is itself free of inherent defects.
PubMed: 36699010
DOI: 10.3389/fcell.2022.1091629 -
Frontiers in Genetics 2022In 2002 we published an article describing a population of vessel-associated progenitors that we termed mesoangioblasts (MABs). During the past decade evidence had... (Review)
Review
In 2002 we published an article describing a population of vessel-associated progenitors that we termed mesoangioblasts (MABs). During the past decade evidence had accumulated that during muscle development and regeneration things may be more complex than a simple sequence of binary choices (e.g., dorsal vs. ventral somite). LacZ expressing fibroblasts could fuse with unlabelled myoblasts but not among themselves or with other cell types. Bone marrow derived, circulating progenitors were able to participate in muscle regeneration, though in very small percentage. Searching for the embryonic origin of these progenitors, we identified them as originating at least in part from the embryonic aorta and, at later stages, from the microvasculature of skeletal muscle. While continuing to investigate origin and fate of MABs, the fact that they could be expanded (also from human muscle) and cross the vessel wall, suggested a protocol for the cell therapy of muscular dystrophies. We tested this protocol in mice and dogs before proceeding to the first clinical trial on Duchenne Muscular Dystrophy patients that showed safety but minimal efficacy. In the last years, we have worked to overcome the problem of low engraftment and tried to understand their role as auxiliary myogenic progenitors during development and regeneration.
PubMed: 36685855
DOI: 10.3389/fgene.2022.1056114 -
Toxicology and Applied Pharmacology Feb 20232,4-di-tert-butylphenol (2,4-DTBP) is a synthetic antioxidant used in polyethylene crosspolymer (PEX) water distribution pipes and food-related plastics. 2,4-DTBP can...
2,4-di-tert-butylphenol (2,4-DTBP) is a synthetic antioxidant used in polyethylene crosspolymer (PEX) water distribution pipes and food-related plastics. 2,4-DTBP can leach from plastic materials and has been found in breast milk, cord blood, and placental tissue, giving rise to the concern that this compound may interfere with fetal development. The objective of this study is to assess the impacts of 2,4-DTBP on cellular differentiation. Human induced pluripotent stem (HiPS) cells were differentiated into osteoblasts or myoblasts over 40 days, and analyzed for markers of somite, dermomyotome, sclerotome, myoblast, and osteoblast development. When cultured as stem cells, 2,4-DTBP did not alter cell viability and expression of markers (NANOG, OCT4). However, upon differentiation into somite-like cells, 2,4-DTBP had reduced levels of MEOX1 and TBX6 transcripts, while NANOG and OCT4 were in turn upregulated in a dose-dependent manner. At the sclerotome-like stage, PAX9 mRNA decreased by 2-fold in the 0.5 μM and 1.0 μM 2,4-DTBP exposure groups. After 40 days of differentiation into an osteoblast-like lineage, exposure to 2,4-DTBP significantly reduced expression of the osteogenesis transcripts RUNX2 and OSX in a dose-dependent manner. Further, Alizarin Red staining of calcium deposits was decreased in the 0.5 μM and 1.0 μM treatment groups. In contrast, myogenesis was not affected by 2,4-DTBP exposure. Interestingly, KEAP1 expression was significantly increased in the sclerotomal-like cells, but decreased in the dermomytomal-like cells, which may suggest a mechanism of action. Overall, this study shows that 2,4-DTBP can delay key processes during sclerotome and osteoblast development, leading to a potential for bone developmental issues in exposed individuals.
Topics: Female; Pregnancy; Humans; Osteogenesis; Kelch-Like ECH-Associated Protein 1; NF-E2-Related Factor 2; Placenta; Cell Differentiation; Plastics; Osteoblasts; Cells, Cultured
PubMed: 36682590
DOI: 10.1016/j.taap.2023.116386 -
Cell Feb 2023Axial development of mammals involves coordinated morphogenetic events, including axial elongation, somitogenesis, and neural tube formation. To gain insight into the...
Axial development of mammals involves coordinated morphogenetic events, including axial elongation, somitogenesis, and neural tube formation. To gain insight into the signals controlling the dynamics of human axial morphogenesis, we generated axially elongating organoids by inducing anteroposterior symmetry breaking of spatially coupled epithelial cysts derived from human pluripotent stem cells. Each organoid was composed of a neural tube flanked by presomitic mesoderm sequentially segmented into somites. Periodic activation of the somite differentiation gene MESP2 coincided in space and time with anteriorly traveling segmentation clock waves in the presomitic mesoderm of the organoids, recapitulating critical aspects of somitogenesis. Timed perturbations demonstrated that FGF and WNT signaling play distinct roles in axial elongation and somitogenesis, and that FGF signaling gradients drive segmentation clock waves. By generating and perturbing organoids that robustly recapitulate the architecture of multiple axial tissues in human embryos, this work offers a means to dissect mechanisms underlying human embryogenesis.
Topics: Animals; Humans; Body Patterning; Embryonic Development; Gene Expression Regulation, Developmental; Mammals; Mesoderm; Morphogenesis; Somites; Wnt Signaling Pathway; Organoids
PubMed: 36657441
DOI: 10.1016/j.cell.2022.12.042 -
Open Biology Jan 2023Studies over the past decade have shown how stem cells representing embryonic and extra-embryonic tissues of the mouse can self-assemble in the culture dish to...
Studies over the past decade have shown how stem cells representing embryonic and extra-embryonic tissues of the mouse can self-assemble in the culture dish to recapitulate an astonishing part of early embryonic development. A systematic analysis has demonstrated how pluripotent embryonic stem cells can be induced to behave like the implanting epiblast; how they can interact with trophectoderm stem cells to form a patterned structure resembling the implanting embryo prior to gastrulation; and how the third stem cell type-extra-embryonic endoderm cells-can be incorporated to generate structures that undergo the cell movements and gene expression patterns of gastrulation. Moreover, such stem cell-derived embryo models can proceed to neurulation and establish progenitors for all parts of the brain and neural tube, somites, beating heart structures and gut tube. They develop within extra-embryonic yolk sacs that initiate haematopoiesis. Here we trace this journey of discovery.
Topics: Pregnancy; Female; Mice; Animals; Embryo, Mammalian; Embryonic Development; Endoderm; Embryonic Stem Cells; Brain; Cell Differentiation
PubMed: 36630196
DOI: 10.1098/rsob.220325 -
STAR Protocols Mar 2023Mouse embryonic stem cells (mESCs) can be directed to acquire cell-lineage-specific genetic programs and phenotypes by stepwise exposure to defined factors, allowing the...
Mouse embryonic stem cells (mESCs) can be directed to acquire cell-lineage-specific genetic programs and phenotypes by stepwise exposure to defined factors, allowing the development of in vitro models for studying disease and tissue generation. In this protocol, we describe the use of cultured mESCs to generate presomitic-like mesoderm cells undergoing further specification towards myogenic progenitors (MPs). Further, we describe here a procedure to obtain, dissect, and fluorescence-activated cell sorting (FACS)-isolate somitic cells from genetically labeled Pax7; Rosa26 embryos. For complete details on the use and execution of this protocol, please refer to Khateb et al..
Topics: Animals; Mice; Flow Cytometry; Cell Lineage
PubMed: 36607809
DOI: 10.1016/j.xpro.2022.102001 -
Developmental Dynamics : An Official... Apr 2023Hox genes encode transcription factors that are important for establishing the body plan. Hoxa5 is a member of the mammalian Hox5 paralogous group that regulates the...
BACKGROUND
Hox genes encode transcription factors that are important for establishing the body plan. Hoxa5 is a member of the mammalian Hox5 paralogous group that regulates the patterning and morphology of the cervical-thoracic region of the axial skeleton. Hoxa5 also plays crucial functions in lung morphogenesis.
RESULTS
We generated a Hoxa5eGFP reporter mouse line using CRISPR technology, allowing real-time visualization of Hoxa5 expression. Hoxa5eGFP recapitulates reported embryonic Hoxa5 mRNA expression patterns. Specifically, Hoxa5eGFP can be visualized in the developing mouse neural tube, somites, lung, diaphragm, foregut, and midgut, among other organs. In the stomach, posteriorly biased Hoxa5eGFP expression correlates with a drastic morphological reduction of the corpus in Hox5 paralogous mutants. Expression of Hoxa5eGFP in the lung continues in all lung fibroblast populations through postnatal and adult stages.
CONCLUSIONS
We identified cell types that express Hoxa5 in postnatal and adult mouse lungs, including various fibroblasts and vascular endothelial cells. This reporter line will be a powerful tool for studies of the function of Hoxa5 during mouse development, homeostasis, and disease processes.
Topics: Mice; Animals; Endothelial Cells; Homeodomain Proteins; Transcription Factors; Lung; Genes, Homeobox; Organogenesis; Phosphoproteins; Mammals
PubMed: 36577717
DOI: 10.1002/dvdy.563 -
Nature Communications Dec 2022Classic microsurgical techniques, such as those used in the early 1900s by Mangold and Spemann, have been instrumental in advancing our understanding of embryonic...
Classic microsurgical techniques, such as those used in the early 1900s by Mangold and Spemann, have been instrumental in advancing our understanding of embryonic development. However, these techniques are highly specialized, leading to issues of inter-operator variability. Here we introduce a user-friendly robotic microsurgery platform that allows precise mechanical manipulation of soft tissues in zebrafish embryos. Using our platform, we reproducibly targeted precise regions of tail explants, and quantified the response in real-time by following notochord and presomitic mesoderm (PSM) morphogenesis and segmentation clock dynamics during vertebrate anteroposterior axis elongation. We find an extension force generated through the posterior notochord that is strong enough to buckle the structure. Our data suggest that this force generates a unidirectional notochord extension towards the tailbud because PSM tissue around the posterior notochord does not let it slide anteriorly. These results complement existing biomechanical models of axis elongation, revealing a critical coupling between the posterior notochord, the tailbud, and the PSM, and show that somite patterning is robust against structural perturbations.
Topics: Animals; Zebrafish; Robotics; Morphogenesis; Somites; Mesoderm; Notochord; Micromanipulation; Body Patterning
PubMed: 36566327
DOI: 10.1038/s41467-022-35632-4