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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 -
Nature Feb 2023The vertebrate body displays a segmental organization that is most conspicuous in the periodic organization of the vertebral column and peripheral nerves. This metameric...
The vertebrate body displays a segmental organization that is most conspicuous in the periodic organization of the vertebral column and peripheral nerves. This metameric organization is first implemented when somites, which contain the precursors of skeletal muscles and vertebrae, are rhythmically generated from the presomitic mesoderm. Somites then become subdivided into anterior and posterior compartments that are essential for vertebral formation and segmental patterning of the peripheral nervous system. How this key somitic subdivision is established remains poorly understood. Here we introduce three-dimensional culture systems of human pluripotent stem cells called somitoids and segmentoids, which recapitulate the formation of somite-like structures with anteroposterior identity. We identify a key function of the segmentation clock in converting temporal rhythmicity into the spatial regularity of anterior and posterior somitic compartments. We show that an initial 'salt and pepper' expression of the segmentation gene MESP2 in the newly formed segment is transformed into compartments of anterior and posterior identity through an active cell-sorting mechanism. Our research demonstrates that the major patterning modules that are involved in somitogenesis, including the clock and wavefront, anteroposterior polarity patterning and somite epithelialization, can be dissociated and operate independently in our in vitro systems. Together, we define a framework for the symmetry-breaking process that initiates somite polarity patterning. Our work provides a platform for decoding general principles of somitogenesis and advancing knowledge of human development.
Topics: Humans; Body Patterning; Cell Culture Techniques, Three Dimensional; In Vitro Techniques; Somites; Spine; Biological Clocks; Epithelium
PubMed: 36543321
DOI: 10.1038/s41586-022-05655-4 -
Journal of Korean Neurosurgical Society Jul 2023Chiari II malformation (CM II) is still the main cause of severe morbidity and mortality in children with open neural tube defects (ONTDs). The goal of this study was to...
OBJECTIVE
Chiari II malformation (CM II) is still the main cause of severe morbidity and mortality in children with open neural tube defects (ONTDs). The goal of this study was to validate a CM II model in late-stage chick embryos with surgically induced ONTDs.
METHODS
To make the chick embryo model of ONTD, their neural tubes were opened for a length of 5-6 somites at the thoracic level in Hamburger and Hamilton stage 18 chick embryos (n=150). They were reincubated in ovo. up to a total age of 17-21 days. A total of 19 embryos survived and were assigned to either the postoperative day (POD) 14-15 group (n=6) or the POD 17-18 group (n=13). Magnetic resonance imaging (MRI) and histopathologic findings of embryo heads with spinal ONTDs were compared with age-matched normal chick embryos.
RESULTS
The chick embryos with ONTDs demonstrated definite and constant structural changes, such as downward displacement of the cerebellum to just above the foramen magnum and narrow and small cerebrospinal fluid spaces in the crowded small posterior fossa. These morphologic features were more prominent in the POD 17-18 group than in the POD 14-15 group.
CONCLUSION
This is the first description of CM II with spinal ONTD in a late-stage chick embryo model with MRI and histopathological analysis. The morphological changes of the posterior fossa in this study mimic those of CM II associated with spinal ONTD in humans. This model will facilitate investigation of the pathogenesis of CM II.
PubMed: 36537033
DOI: 10.3340/jkns.2022.0144 -
Nature Jan 2023Sequential segmentation creates modular body plans of diverse metazoan embryos. Somitogenesis establishes the segmental pattern of the vertebrate body axis. A molecular...
Sequential segmentation creates modular body plans of diverse metazoan embryos. Somitogenesis establishes the segmental pattern of the vertebrate body axis. A molecular segmentation clock in the presomitic mesoderm sets the pace of somite formation. However, how cells are primed to form a segment boundary at a specific location remains unclear. Here we developed precise reporters for the clock and double-phosphorylated Erk (ppErk) gradient in zebrafish. We show that the Her1-Her7 oscillator drives segmental commitment by periodically lowering ppErk, therefore projecting its oscillation onto the ppErk gradient. Pulsatile inhibition of the ppErk gradient can fully substitute for the role of the clock, and kinematic clock waves are dispensable for sequential segmentation. The clock functions upstream of ppErk, which in turn enables neighbouring cells to discretely establish somite boundaries in zebrafish. Molecularly divergent clocks and morphogen gradients were identified in sequentially segmenting species. Our findings imply that versatile clocks may establish sequential segmentation in diverse species provided that they inhibit gradients.
Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Body Patterning; Gene Expression Regulation, Developmental; Somites; Zebrafish; Zebrafish Proteins; Biological Clocks; Periodicity; Extracellular Signal-Regulated MAP Kinases
PubMed: 36517597
DOI: 10.1038/s41586-022-05527-x -
Cell Death Discovery Dec 2022Chromosome stability relies on bipolar spindle assembly and faithful chromosome segregation during cell division. Kinesin-5 Eg5 is a plus-end-directed kinesin motor...
Chromosome stability relies on bipolar spindle assembly and faithful chromosome segregation during cell division. Kinesin-5 Eg5 is a plus-end-directed kinesin motor protein, which is essential for spindle pole separation and chromosome alignment in mitosis. Heterozygous Eg5 mutations cause autosomal-dominant microcephaly, primary lymphedema, and chorioretinal dysplasia syndrome in humans. However, the developmental roles and cellular mechanisms of Eg5 in organogenesis remain largely unknown. In this study, we have shown that Eg5 inhibition leads to the formation of the monopolar spindle, chromosome misalignment, polyploidy, and subsequent apoptosis. Strikingly, long-term inhibition of Eg5 stimulates the immune responses and the accumulation of lymphocytes in the mouse spleen through the innate and specific immunity pathways. Eg5 inhibition results in metaphase arrest and cell growth inhibition, and suppresses the formation of somite and retinal development in zebrafish embryos. Our data have revealed the essential roles of kinesin-5 Eg5 involved in cell proliferation, chromosome stability, and organogenesis during development. Our findings shed a light on the cellular basis and pathogenesis in microcephaly, primary lymphedema, and chorioretinal dysplasia syndrome of Eg5-mutation-positive patients.
PubMed: 36513626
DOI: 10.1038/s41420-022-01281-1 -
Biomedicine & Pharmacotherapy =... Feb 2023Targeted gene therapy has shown durable efficacy in non-neoplastic and neoplastic patients. Therefore, finding a suitable target has become a key area of research.... (Review)
Review
Targeted gene therapy has shown durable efficacy in non-neoplastic and neoplastic patients. Therefore, finding a suitable target has become a key area of research. Mesenchyme homeobox 1 (MEOX1) is a transcriptional factor that plays a significant role in regulation of somite development. Evidence indicates that abnormalities in MEOX1 expression and function are associated with a variety of pathologies, including non-neoplastic and neoplastic diseases. MEOX1 expression is upregulated during progression of most diseases and plays a critical role in maintenance of the cellular phenotypes such as cell differentiation, cell cycle arrest and senescence, migration, and proliferation. Therefore, MEOX1 may become an important molecular target and therapeutic target. This review will discuss the current state of knowledge on the role of MEOX1 in different diseases.
Topics: Humans; Homeodomain Proteins; Transcription Factors; Gene Expression Regulation; Cell Differentiation; Neoplasms; Cellular Senescence; Gene Expression Regulation, Neoplastic
PubMed: 36495659
DOI: 10.1016/j.biopha.2022.114068 -
Systematic Parasitology Apr 2023Two new species of copepods assigned to the genus Acusicola Cressey, 1970 (Cyclopoida: Ergasilidae) are proposed based on post-metamorphic adult females, parasitizing...
Two new species of copepods assigned to the genus Acusicola Cressey, 1970 (Cyclopoida: Ergasilidae) are proposed based on post-metamorphic adult females, parasitizing the gills of two actinopterygian fish off Brazil namely, the Tripletail Lobotes surinamensis (Bloch) (Lobotidae), collected in the coastal zone of the State of Pará, near Curuçá Municipallity, and the Swordspine snook Centropomus ensiferus Poey (Centropomidae) collected in Sepetiba Bay, State of Rio de Janeiro, Brazil. Acusicola iamarinoi n. sp. parasite of L. surinamensis, differs from its closet congeners based on the first segment of the antennule armed with 10 setae, the presence of a maxillule armed with four elements and a pair of blunt processes dorsally on the fourth pedigerous somite. Acusicola pasternakae n. sp., collected from C. ensiferus, can be distinguished from its closest congeners based on the membranous sheath of the first endopodal segment of antenna with horizontal marks, the first segment of the antennule armed with 11 setae and a spine on the last exopodal segment of leg 2. This is the first report of representatives of Acusicola parasitizing fish of the families Lobotidae and Centropomidae as well as new geographical records of the genus in the coast of State of Pará and in Sepetiba Bay, Brazil.
Topics: Female; Animals; Copepoda; Parasites; Brazil; Gills; Species Specificity; Fishes; Perciformes; Fish Diseases
PubMed: 36471195
DOI: 10.1007/s11230-022-10076-y -
Nature Communications Dec 2022Although integrins are known to be mechanosensitive and to possess many subtypes that have distinct physiological roles, single molecule studies of force exertion have...
Although integrins are known to be mechanosensitive and to possess many subtypes that have distinct physiological roles, single molecule studies of force exertion have thus far been limited to RGD-binding integrins. Here, we show that integrin α4β1 and RGD-binding integrins (αVβ1 and α5β1) require markedly different tension thresholds to support cell spreading. Furthermore, actin assembled downstream of α4β1 forms cross-linked networks in circularly spread cells, is in rapid retrograde flow, and exerts low forces from actin polymerization. In contrast, actin assembled downstream of αVβ1 forms stress fibers linking focal adhesions in elongated cells, is in slow retrograde flow, and matures to exert high forces (>54-pN) via myosin II. Conformational activation of both integrins occurs below 12-pN, suggesting that post-activation subtype-specific cytoskeletal remodeling imposes the higher threshold for spreading on RGD substrates. Multiple layers of single integrin mechanics for activation, mechanotransduction and cytoskeleton remodeling revealed here may underlie subtype-dependence of diverse processes such as somite formation and durotaxis.
Topics: Integrin beta1; Actins; Mechanotransduction, Cellular; Integrin alpha4beta1; Oligopeptides
PubMed: 36463259
DOI: 10.1038/s41467-022-35173-w -
Frontiers in Endocrinology 2022COMP (Cartilage Oligomeric Matrix Protein), also named thrombospondin-5, is a member of the thrombospondin family of extracellular matrix proteins. It is of clinical...
COMP (Cartilage Oligomeric Matrix Protein), also named thrombospondin-5, is a member of the thrombospondin family of extracellular matrix proteins. It is of clinical relevance, as in humans mutations in COMP lead to chondrodysplasias. The gene encoding zebrafish Comp is located on chromosome 11 in synteny with its mammalian orthologs. Zebrafish Comp has a domain structure identical to that of tetrapod COMP and shares 74% sequence similarity with murine COMP. Zebrafish is expressed from 5 hours post fertilization (hpf) on, while the protein is first detectable in somites of 11 hpf embryos. During development and in adults is strongly expressed in myosepta, craniofacial tendon and ligaments, around ribs and vertebra, but not in its name-giving tissue cartilage. As in mammals, zebrafish Comp forms pentamers. It is easily extracted from 5 days post fertilization (dpf) whole zebrafish. The lack of Comp expression in zebrafish cartilage implies that its cartilage function evolved recently in tetrapods. The expression in tendon and myosepta may indicate a more fundamental function, as in evolutionary distant Drosophila muscle-specific adhesion to tendon cells requires thrombospondin. A sequence encoding a calcium binding motif within the first TSP type-3 repeat of zebrafish Comp was targeted by CRISPR-Cas. The heterozygous and homozygous mutant Comp zebrafish displayed a patchy irregular Comp staining in 3 dpf myosepta, indicating a dominant phenotype. Electron microscopy revealed that the endoplasmic reticulum of myosepta fibroblasts is not affected in homozygous fish. The disorganized extracellular matrix may indicate that this mutation rather interferes with extracellular matrix assembly, similar to what is seen in a subgroup of chondrodysplasia patients. The early expression and easy detection of mutant Comp in zebrafish points to the potential of using the zebrafish model for large scale screening of small molecules that can improve secretion or function of disease-associated COMP mutants.
Topics: Adult; Humans; Mice; Animals; Cartilage Oligomeric Matrix Protein; Zebrafish; CRISPR-Cas Systems; Phenotype; Thrombospondins; Mammals
PubMed: 36452329
DOI: 10.3389/fendo.2022.1000662 -
Cells Nov 2022Bioelectricity is defined as endogenous electrical signaling mediated by the dynamic distribution of charged molecules. Recently, increasing evidence has revealed that...
Bioelectricity is defined as endogenous electrical signaling mediated by the dynamic distribution of charged molecules. Recently, increasing evidence has revealed that cellular bioelectric signaling is critical for regulating embryonic development, regeneration, and congenital diseases. However, systematic real-time dynamic electrical activity monitoring of whole organisms has been limited, mainly due to the lack of a suitable model system and voltage measurement tools for biology. Here, we addressed this gap by utilizing a genetically stable zebrafish line, Tg (: ASAP1), and ASAP1 (Accelerated sensor of action potentials 1), a genetically encoded voltage indicator (GEVI). With light-sheet microscopy, we systematically investigated cell membrane potential (Vm) signals during different embryonic stages. We found cells of zebrafish embryos showed local membrane hyperpolarization at the cleavage furrows during the cleavage period of embryogenesis. This signal appeared before cytokinesis and fluctuated as it progressed. In contrast, whole-cell transient hyperpolarization was observed during the blastula and gastrula stages. These signals were generally limited to the superficial blastomere, but they could be detected within the deeper cells during the gastrulation period. Moreover, the zebrafish embryos exhibit tissue-level cell Vm signals during the segmentation period. Middle-aged somites had strong and dynamic Vm fluctuations starting at about the 12-somite stage. These embryonic stage-specific characteristic cellular bioelectric signals suggest that they might play a diverse role in zebrafish embryogenesis that could underlie human congenital diseases.
Topics: Animals; Humans; Middle Aged; Zebrafish; Electrophysiological Phenomena; Gastrula; Embryonic Development; Blastomeres
PubMed: 36429015
DOI: 10.3390/cells11223586