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Nature Communications Sep 2023Understanding of the molecular drivers of lineage diversification and tissue patterning during primary germ layer development requires in-depth knowledge of the dynamic...
Understanding of the molecular drivers of lineage diversification and tissue patterning during primary germ layer development requires in-depth knowledge of the dynamic molecular trajectories of cell lineages across a series of developmental stages of gastrulation. Through computational modeling, we constructed at single-cell resolution, a spatio-temporal transcriptome of cell populations in the germ-layers of gastrula-stage mouse embryos. This molecular atlas enables the inference of molecular network activity underpinning the specification and differentiation of the germ-layer tissue lineages. Heterogeneity analysis of cellular composition at defined positions in the epiblast revealed progressive diversification of cell types. The single-cell transcriptome revealed an enhanced BMP signaling activity in the right-side mesoderm of late-gastrulation embryo. Perturbation of asymmetric BMP signaling activity at late gastrulation led to randomization of left-right molecular asymmetry in the lateral mesoderm of early-somite-stage embryo. These findings indicate the asymmetric BMP activity during gastrulation may be critical for the symmetry breaking process.
Topics: Animals; Mice; Gastrulation; Functional Laterality; Gastrula; Germ Layers; Mesoderm
PubMed: 37709743
DOI: 10.1038/s41467-023-41482-5 -
ELife Sep 2023Development of the dorsal aorta is a key step in the establishment of the adult blood-forming system, since hematopoietic stem and progenitor cells (HSPCs) arise from...
Development of the dorsal aorta is a key step in the establishment of the adult blood-forming system, since hematopoietic stem and progenitor cells (HSPCs) arise from ventral aortic endothelium in all vertebrate animals studied. Work in zebrafish has demonstrated that arterial and venous endothelial precursors arise from distinct subsets of lateral plate mesoderm. Here, we profile the transcriptome of the earliest detectable endothelial cells (ECs) during zebrafish embryogenesis to demonstrate that tissue-specific EC programs initiate much earlier than previously appreciated, by the end of gastrulation. Classic studies in the chick embryo showed that paraxial mesoderm generates a subset of somite-derived endothelial cells (SDECs) that incorporate into the dorsal aorta to replace HSPCs as they exit the aorta and enter circulation. We describe a conserved program in the zebrafish, where a rare population of endothelial precursors delaminates from the dermomyotome to incorporate exclusively into the developing dorsal aorta. Although SDECs lack hematopoietic potential, they act as a local niche to support the emergence of HSPCs from neighboring hemogenic endothelium. Thus, at least three subsets of ECs contribute to the developing dorsal aorta: vascular ECs, hemogenic ECs, and SDECs. Taken together, our findings indicate that the distinct spatial origins of endothelial precursors dictate different cellular potentials within the developing dorsal aorta.
Topics: Chick Embryo; Animals; Zebrafish; Arteries; Hematopoietic Stem Cells; Hemangioblasts; Aorta
PubMed: 37695317
DOI: 10.7554/eLife.58300 -
Ecotoxicology and Environmental Safety Oct 2023Tris(2-chloroisopropyl) phosphate (TCIPP) is an organophosphate flame retardant detected in the environment and eggs, feathers, and livers. Early-developmental-stage...
Tris(2-chloroisopropyl) phosphate (TCIPP) is an organophosphate flame retardant detected in the environment and eggs, feathers, and livers. Early-developmental-stage avian embryos are vulnerable to the toxic effects of chemicals. However, studies on the specific effects of TCIPP on avian embryonic development are limited. We aimed to investigate the toxicity of TCIPP in early chicken embryos using a previously developed shell-less incubation system. Fertilized chicken (Gallus gallus domesticus) eggs (n = 220) were exposed to 50 or 500 nmol TCIPP/(g egg) or dimethyl sulfoxide (DMSO) as a vehicle control on Day 0 of incubation. Development of 198 embryos was monitored from Days 3-9 of incubation, and 22 embryos on Day 4 and 74 embryos on Day 9 were dissected. Messenger RNA expression levels for several genes were measured in embryos on Day 4. Both TCIPP-exposed groups showed a significant reduction in survival rate. Imaging analyses revealed significant decreases in body length, head and bill length, eye diameter, and forelimb and hindlimb length in both TCIPP-treated groups. TCIPP exposure significantly impaired the development of extraembryonic blood vessels and the production of red blood cells. A TCIPP-dose-dependent decreasing trend in heart rate was observed on Days 4-7. The somitic angle increased significantly on Days 4-6, and embryos with curved somites showed cleavage in the back and gaps between somites, resulting in asymmetrical somite formation. A significant correlation was found between the somitic angle and FGF8 expression levels, suggesting that TCIPP exposure affects somite formation through an altered FGF-signaling pathway. Embryos with somitic deformities in TCIPP-exposed groups had significantly reduced survival rates, indicating that abnormal segment formation directly increased mortality. Finally, eye weight and ocular luminosity values were significantly reduced, suggesting that TCIPP may also affect eye development. Overall, these findings highlight severe toxic effects of TCIPP on avian embryonic development, including in vascularization, cardiac function, and somite and ocular development.
Topics: Animals; Chick Embryo; Chickens; Phosphates; Flame Retardants; Organophosphorus Compounds; Organophosphates
PubMed: 37690177
DOI: 10.1016/j.ecoenv.2023.115445 -
International Journal of Molecular... Sep 2023Sialidases remove terminal sialic acids residues from the non-reducing ends of glycoconjugates. They have been recognized as catabolic enzymes that work within different...
Sialidases remove terminal sialic acids residues from the non-reducing ends of glycoconjugates. They have been recognized as catabolic enzymes that work within different subcellular compartments and can ensure the proper turn-over of glycoconjugates. Four mammalian sialidases (NEU1-4) exist, with different subcellular localization, pH optimum and substrate specificity. In zebrafish, seven different sialidases, with high homology to mammalian counterparts, have been identified. Zebrafish Neu3.2 is similar to the human cytosolic sialidase NEU2, which is involved in skeletal muscle differentiation and exhibits a broad substrate specificity toward gangliosides and glycoproteins. In zebrafish , mRNA is expressed during somite development, and its enzymatic activity has been detected in the skeletal muscle and heart of adult animals. In this paper, 1-4-cell-stage embryos injected with splice-blocking morpholino showed severe embryonic defects, mainly in somites, heart and anterior-posterior axis formation. and expressions were altered in morphants, and impaired musculature formation was associated with a defective locomotor behavior. Finally, the co-injection of mouse mRNA in morphants rescued the phenotype. These data are consistent with the involvement of cytosolic sialidase in pathologies related to muscle formation and support the validity of the model to investigate the pathogenesis of the diseases.
Topics: Animals; Down-Regulation; Muscle Development; Muscle, Skeletal; Neuraminidase; Zebrafish; Zebrafish Proteins
PubMed: 37686385
DOI: 10.3390/ijms241713578 -
IScience Sep 2023Adducin 1 (Add1) is known as a membrane cytoskeletal protein, but its nuclear function remains unclear. In this study, we generated -deficient zebrafish to investigate...
Adducin 1 (Add1) is known as a membrane cytoskeletal protein, but its nuclear function remains unclear. In this study, we generated -deficient zebrafish to investigate its role in hematopoiesis. Lack of impaired both primitive and definitive hematopoiesis, preventing healthy erythrocyte development. RNA sequencing revealed activation of the p53 pathway in -depleted erythroblast cells, leading to apoptosis at the 14-somites stage and 24 hpf. Interestingly, partial rescue of the anemic phenotype and apoptosis was observed with insufficiency. Mechanistically, ADD1 was found to regulate promoter activity. These findings demonstrate that Add1 plays a crucial role in zebrafish erythropoiesis, involving the p53-mediated apoptotic pathway, expanding its regulatory role beyond cytoskeletal functions.
PubMed: 37636049
DOI: 10.1016/j.isci.2023.107516 -
Frontiers in Cell and Developmental... 2023Morphological phenotyping of the mouse embryo is described at neurulation stages, primarily as a guide to evaluating the outcome of whole embryo cultures between... (Review)
Review
Morphological phenotyping of the mouse embryo is described at neurulation stages, primarily as a guide to evaluating the outcome of whole embryo cultures between embryonic days 8.5 and 9.5. During this period, neural tube closure is initiated and progresses to completion in the cranial region. Spinal closure is still underway at the end of the culture period. The focus of this article is particularly on phenotyping that can be performed at the bench, using a stereomicroscope. This involves assessment of embryonic health, through observation and scoring of yolk sac blood circulation, measurement of developmental stage by somite counting, and determination of crown-rump length as a measure of growth. Axial rotation ("turning") can also be assessed using a simple scoring system. Neural tube closure assessment includes: 1) determining whether closure has been initiated at the Closure 1 site; 2) evaluating the complex steps of cranial neurulation including initiation at Closure sites 2 and 3, and completion of closure at the anterior and hindbrain neuropores; 3) assessment of spinal closure by measurement of posterior neuropore length. Interpretation of defects in neural tube closure requires an appreciation of, first, the stages that particular events are expected to be completed and, second, the correspondence between embryonic landmarks, for example, somite position, and the resulting adult axial levels. Detailed embryonic phenotyping, as described in this article, when combined with the versatile method of whole embryo culture, can form the basis for a wide range of experimental studies in early mouse neural development.
PubMed: 37601098
DOI: 10.3389/fcell.2023.1223849 -
BioRxiv : the Preprint Server For... Jul 2023The sclerotome in vertebrates comprises an embryonic population of cellular progenitors that give rise to diverse adult tissues including the axial skeleton, ribs,...
The sclerotome in vertebrates comprises an embryonic population of cellular progenitors that give rise to diverse adult tissues including the axial skeleton, ribs, intervertebral discs, connective tissue, and vascular smooth muscle. In the thorax, this cell population arises in the ventromedial region of each of the segmented tissue blocks known as somites. How and when sclerotome adult tissue fates are specified and how the gene signatures that predate those fates are regulated has not been well studied. We have identified a previously unknown role for Ca /calmodulin-dependent protein kinase II (CaMKII) in regulating sclerotome patterning in zebrafish. Mechanistically, CaMKII regulates the activity of parallel signaling inputs that pattern sclerotome gene expression. In one downstream arm, CaMKII regulates distribution of the established sclerotome-inductive morphogen sonic hedgehog (Shh), and thus Shh-dependent sclerotome genes. In the second downstream arm, we show a previously unappreciated inductive requirement for Bmp signaling, where CaMKII activates expression of and consequently Bmp activity. Bmp activates expression of a second subset of stereotypical sclerotome genes, while simultaneously repressing Shh-dependent markers. Our work demonstrates that CaMKII promotes parallel Bmp and Shh signaling as a mechanism to first promote global sclerotome specification, and that these pathways subsequently regionally activate and refine discrete compartmental genetic programs. Our work establishes how the earliest unique gene signatures that likely drive distinct cell behaviors and adult fates arise within the sclerotome.
PubMed: 37503202
DOI: 10.1101/2023.07.21.550086 -
Developmental Cell Oct 2023The mammalian body plan is shaped by rhythmic segmentation of mesoderm into somites, which are transient embryonic structures that form down each side of the neural...
The mammalian body plan is shaped by rhythmic segmentation of mesoderm into somites, which are transient embryonic structures that form down each side of the neural tube. We have analyzed the genome-wide transcriptional and chromatin dynamics occurring within nascent somites, from early inception of somitogenesis to the latest stages of body plan establishment. We created matched gene expression and open chromatin maps for the three leading pairs of somites at six time points during mouse embryonic development. We show that the rate of somite differentiation accelerates as development progresses. We identified a conserved maturation program followed by all somites, but somites from more developed embryos concomitantly switch on differentiation programs from derivative cell lineages soon after segmentation. Integrated analysis of the somitic transcriptional and chromatin activities identified opposing regulatory modules controlling the onset of differentiation. Our results provide a powerful, high-resolution view of the molecular genetics underlying somitic development in mammals.
Topics: Pregnancy; Female; Mice; Animals; Somites; Embryonic Development; Mesoderm; Cell Differentiation; Chromatin; Mammals
PubMed: 37499658
DOI: 10.1016/j.devcel.2023.07.003 -
BioRxiv : the Preprint Server For... Jul 2023Shh signaling is the morphogen signaling that regulates embryonic craniofacial and neural tube development. G protein-coupled receptor 161 (Gpr161) is a negative...
Shh signaling is the morphogen signaling that regulates embryonic craniofacial and neural tube development. G protein-coupled receptor 161 (Gpr161) is a negative regulator of Shh signaling, and its inactivation in mice results in embryo lethality with craniofacial and neural tube defects (NTDs). However, the structural defects of later embryonic stages in null mice and cell lineages underlying abnormalities were not well characterized due to their limited lifespan. We found the lineage-specific deletion of in mice presented with tectal hypertrophy (anterior dorsal neuroepithelium), cranial vault and facial bone hypoplasia (cranial neural crest (CNC)), vertebral abnormalities (somite), and the closed form of spina bifida (posterior dorsal neuroepithelium). In particular, the closed form of spina bifida is partly due to the reduced and gene expression of the posterior dorsal neural tubes of mutant embryos involving decreased Wnt signaling whereas Shh signaling was increased. This study provides the novel role of Gpr161 in the posterior neural tube development and confirms its role on CNC- and somite-derived skeletogenesis and midbrain morphogenesis in mice.
PubMed: 37461574
DOI: 10.1101/2023.07.07.548129 -
Scientific Reports Jul 2023The establishment of left-right patterning in mice occurs at a transient structure called the embryonic node or left-right organizer (LRO). Previous analysis of the LRO...
The establishment of left-right patterning in mice occurs at a transient structure called the embryonic node or left-right organizer (LRO). Previous analysis of the LRO has proven challenging due to the small cell number and transient nature of this structure. Here, we seek to overcome these difficulties to define the transcriptome of the LRO. Specifically, we used single cell RNA sequencing of 0-1 somite embryos to identify LRO enriched genes which were compared to bulk RNA sequencing of LRO cells isolated by fluorescent activated cell sorting. Gene ontology analysis indicated an enrichment of genes associated with cilia and laterality terms. Furthermore, comparison to previously identified LRO genes identified 127 novel LRO genes, including Ttll3, Syne1 and Sparcl1, for which the expression patterns were validated using whole mount in situ hybridization. This list of novel LRO genes will be a useful resource for further studies on LRO morphogenesis, the establishment of laterality and the genetic causes of heterotaxy.
Topics: Animals; Mice; Transcriptome; Cell Count; Cell Separation; Cilia; RNA; Calcium-Binding Proteins; Extracellular Matrix Proteins
PubMed: 37393374
DOI: 10.1038/s41598-023-36862-2