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BioRxiv : the Preprint Server For... Nov 2023Emerging human pluripotent stem cell (hPSC)-based embryo models are useful for studying human embryogenesis. Particularly, there are hPSC-based somitogenesis models...
Emerging human pluripotent stem cell (hPSC)-based embryo models are useful for studying human embryogenesis. Particularly, there are hPSC-based somitogenesis models using free-floating culture that recapitulate somite formation. Somitogenesis involves intricately orchestrated bio-chemical and -mechanical events. However, none of the current somitogenesis models controls biochemical gradients or biomechanical signals in the culture, limiting their applicability to untangle complex biochemical-biomechanical interactions that drive somitogenesis. Here we report a new human somitogenesis model by confining hPSC-derived presomitic mesoderm (PSM) tissues in microfabricated trenches. Exogenous microfluidic morphogen gradients imposed on PSM cause axial patterning and trigger spontaneous rostral-to-caudal somite formation. A mechanical theory is developed to explain the size dependency between somites and PSM. The microfluidic somitogenesis model is further exploited to reveal regulatory roles of cellular and tissue biomechanics in somite formation. This study presents a useful microengineered, hPSC-based model for understanding the bio-chemical and -mechanical events that guide somite formation.
PubMed: 37961125
DOI: 10.1101/2023.10.29.564399 -
Frontiers in Cell and Developmental... 2023Maternal diabetes during pregnancy is well known to be associated with a higher risk for structural birth defects in the offspring. Recent searches for underlying...
Maternal diabetes during pregnancy is well known to be associated with a higher risk for structural birth defects in the offspring. Recent searches for underlying mechanisms have largely focused on aberrant processes in the embryo itself, although prior research in rodent models implicated dysfunction also of the visceral yolk sac. The objective of our research was to investigate both tissues within the conceptus simultaneously. We conducted unbiased transcriptome profiling by RNA sequencing on pairs of individual yolk sacs and their cognate embryos, using the non-obese diabetic (NOD) mouse model. The analysis was performed at gestational day 8.5 on morphologically normal specimen to circumvent confounding by defective development. Even with large sample numbers ( = 33 in each group), we observed considerable variability of gene expression, primarily driven by exposure to maternal diabetes, and secondarily by developmental stage of the embryo. Only a moderate number of genes changed expression in the yolk sac, while in the embryo, the exposure distinctly influenced the relationship of gene expression levels to developmental progression, revealing a possible role for altered cell cycle regulation in the response. Also affected in embryos under diabetic conditions were genes involved in cholesterol biosynthesis and NAD metabolism pathways. Exposure to maternal diabetes during gastrulation changes transcriptomic profiles in embryos to a substantially greater effect than in the corresponding yolk sacs, indicating that despite yolk sac being of embryonic origin, different mechanisms control transcriptional activity in these tissues. The effects of maternal diabetes on expression of many genes that are correlated with developmental progression (i.e. somite stage) highlight the importance of considering developmental maturity in the interpretation of transcriptomic data. Our analyses identified cholesterol biosynthesis and NAD metabolism as novel pathways not previously implicated in diabetic pregnancies. Both NAD and cholesterol availability affect a wide variety of cellular signaling processes, and can be modulated by diet, implying that prevention of adverse outcomes from diabetic pregnancies may require broad interventions, particularly in the early stages of pregnancy.
PubMed: 37928898
DOI: 10.3389/fcell.2023.1273641 -
Disease Models & Mechanisms Nov 2023Sonic hedgehog (Shh) signaling is the morphogen signaling that regulates embryonic craniofacial and neural tube development. G protein-coupled receptor 161 (Gpr161) is...
Sonic hedgehog (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 associated with craniofacial defects and neural tube defects. However, the structural defects of later embryonic stages and cell lineages underlying abnormalities have not been well characterized due to the limited lifespan of Gpr161 null mice. We found that embryos with Pax3 lineage-specific deletion of Gpr161 presented with tectal hypertrophy (anterior dorsal neuroepithelium), cranial vault and facial bone hypoplasia (cranial neural crest), vertebral abnormalities (somite) and the closed form of spina bifida (posterior dorsal neuroepithelium). In particular, the closed form of spina bifida was partly due to reduced Pax3 and Cdx4 gene expression in the posterior dorsal neural tubes of Gpr161 mutant embryos with decreased Wnt signaling, whereas Shh signaling was increased. We describe a previously unreported role for Gpr161 in the development of posterior neural tubes and confirm its role in cranial neural crest- and somite-derived skeletogenesis and midbrain morphogenesis in mice.
Topics: Mice; Animals; Neural Tube; Hedgehog Proteins; Transcription Factors; Spinal Dysraphism; Embryonic Development; Wnt Signaling Pathway; Neurogenesis; Spine
PubMed: 37885410
DOI: 10.1242/dmm.050277 -
Nature Communications Oct 2023Mutations of several genes cause incomplete penetrance and variable expressivity of phenotypes, which are usually attributed to modifier genes or gene-environment...
Mutations of several genes cause incomplete penetrance and variable expressivity of phenotypes, which are usually attributed to modifier genes or gene-environment interactions. Here, we show stochastic gene expression underlies the variability of somite segmentation defects in embryos mutant for segmentation clock genes her1 or her7. Phenotypic strength is further augmented by low temperature and hypoxia. By performing live imaging of the segmentation clock reporters, we further show that groups of cells with higher oscillation amplitudes successfully form somites while those with lower amplitudes fail to do so. In unfavorable environments, the number of cycles with high amplitude oscillations and the number of successful segmentations proportionally decrease. These results suggest that individual oscillation cycles stochastically fail to pass a threshold amplitude, resulting in segmentation defects in mutants. Our quantitative methodology is adaptable to investigate variable phenotypes of mutant genes in different tissues.
Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Zebrafish; Zebrafish Proteins; Somites; Phenotype; Gene Expression; Gene Expression Regulation, Developmental; Body Patterning
PubMed: 37838784
DOI: 10.1038/s41467-023-42220-7 -
Cells Sep 2023Purines are required for fundamental biological processes and alterations in their metabolism lead to severe genetic diseases associated with developmental defects whose...
Purines are required for fundamental biological processes and alterations in their metabolism lead to severe genetic diseases associated with developmental defects whose etiology remains unclear. Here, we studied the developmental requirements for purine metabolism using the amphibian as a vertebrate model. We provide the first functional characterization of purine pathway genes and show that these genes are mainly expressed in nervous and muscular embryonic tissues. Morphants were generated to decipher the functions of these genes, with a focus on the adenylosuccinate lyase (), which is an enzyme required for both salvage and de novo purine pathways. knockdown led to a severe reduction in the expression of the myogenic regulatory factors (MRFs: Myod1, Myf5 and Myogenin), thus resulting in defects in somite formation and, at later stages, the development and/or migration of both craniofacial and hypaxial muscle progenitors. The reduced expressions of and , which are two genes specific to the salvage and de novo pathways, respectively, resulted in similar alterations. In conclusion, our data show for the first time that de novo and recycling purine pathways are essential for myogenesis and highlight new mechanisms in the regulation of MRF gene expression.
Topics: Animals; Xenopus laevis; Muscle, Skeletal; Purines; Muscle Development
PubMed: 37830593
DOI: 10.3390/cells12192379 -
Development (Cambridge, England) Nov 2023Recent work shows that the developmental potential of progenitor cells in the HH10 chick brain changes rapidly, accompanied by subtle changes in morphology. This demands...
Recent work shows that the developmental potential of progenitor cells in the HH10 chick brain changes rapidly, accompanied by subtle changes in morphology. This demands increased temporal resolution for studies of the brain at this stage, necessitating precise and unbiased staging. Here, we investigated whether we could train a deep convolutional neural network to sub-stage HH10 chick brains using a small dataset of 151 expertly labelled images. By augmenting our images with biologically informed transformations and data-driven preprocessing steps, we successfully trained a classifier to sub-stage HH10 brains to 87.1% test accuracy. To determine whether our classifier could be generally applied, we re-trained it using images (269) of randomised control and experimental chick wings, and obtained similarly high test accuracy (86.1%). Saliency analyses revealed that biologically relevant features are used for classification. Our strategy enables training of image classifiers for various applications in developmental biology with limited microscopy data.
Topics: Animals; Deep Learning; Neural Networks, Computer; Brain; Microscopy; Wings, Animal
PubMed: 37830145
DOI: 10.1242/dev.202068 -
PeerJ 2023As a member of the immunoglobulin superfamily, hemolins play a vital role in insect development and defense against pathogens. However, the innate immune response of to...
BACKGROUND
As a member of the immunoglobulin superfamily, hemolins play a vital role in insect development and defense against pathogens. However, the innate immune response of to baculovirus infection varies among different insects.
METHODS AND RESULTS
In this study, the like gene from a Crambidae insect, , was cloned, and its role in insect development and baculovirus infection was analyzed. A 1,528 bp contig as potential -like gene of was reassembled from the transcriptome. Further, the complete sequence of () was cloned and sequenced. The cDNA of was 1,515 bp in length and encoded 408 amino acids. The deduced amino acid of CmHem has relatively low identities (41.9-62.3%) to various insect hemolins. However, it contains four Ig domains similarity to other insect hemolins. The expression level of was the highest in eggs, followed by pupae and adults, and maintained a low expression level at larval stage. The synthesized siRNAs were injected into mature larvae, and the transcription decreased by 51.7%. Moreover, the abdominal somites of larvae became straightened, could not pupate normally, and then died. Infection with a baculovirus, granulovirus (CnmeGV), the expression levels of in the midgut and fat body of significantly increased at 12 and 24 h, respectively, and then soon returned to normal levels.
CONCLUSIONS
Our results suggested that hemolin may be related to the metamorphosis of . Exposure to baculovirus induced the phased expression of gene in the midgut and fat body of , indicated that involved in the immune recognition of Crambidae insects to baculovirus.
Topics: Animals; Granulovirus; Amino Acid Sequence; Immunoglobulins; Moths; Larva; Baculoviridae
PubMed: 37810787
DOI: 10.7717/peerj.16225 -
Cureus Aug 2023Duplication of the alimentary tract is a rare malformation that can occur in any portion of the gastrointestinal tract. Rarely diagnosed in adulthood, it is usually an...
Duplication of the alimentary tract is a rare malformation that can occur in any portion of the gastrointestinal tract. Rarely diagnosed in adulthood, it is usually an incidental finding due to non-specific gastrointestinal symptoms. Approximately 80% of cases are diagnosed in infants less than two years old. The most common location is the ileum, and the least common location is the rectum. Embryological theories discussing the etiology of alimentary tract duplications include failure of internal vacuolization during the sixth week of fetal development and/or the yolk-sac dorsal protrusion may adhere to the ectoderm during somite development. Environmental factors such as trauma or hypoxia affecting various intestinal fragments receiving blood supply can evolve into duplication. Excision with consideration to the common blood supply to protect the native bowel is the preferred treatment approach. We present the case of an elderly 70-year-old male with a bowel obstruction complicated by sigmoid duplication. After preoperative assessments, the patient underwent a robotic sigmoidectomy. This case report highlights colonic duplication as a differential diagnosis in the bowel obstruction of an elderly patient.
PubMed: 37767241
DOI: 10.7759/cureus.44208 -
International Journal of Molecular... Sep 2023represents a type of single-transmembrane adaptor protein containing an N-terminal cysteine-rich domain and a proline-rich C-terminal region. Nine subfamily genes have...
represents a type of single-transmembrane adaptor protein containing an N-terminal cysteine-rich domain and a proline-rich C-terminal region. Nine subfamily genes have been proposed in most vertebrates; however, some might be species-specific. The number of genes present in zebrafish remains unclear. This study aimed to investigate the evolutionary relationships among family genes in zebrafish (TU strain) using phylogenetic and syntenic analyses. The function of was preliminarily examined via CRISPR/Cas13d-mediated knockdown. Following identification in zebrafish, 10 family genes, namely , , , , , , , , , and , were classified into three main clades and six subclades. Their encoding proteins contained a cysteine-rich N-terminal domain and a proline-rich C-terminal region containing different motifs. A specific syntenic block containing and was observed to be conserved across all species. Furthermore, all these genes were expressed during embryogenesis. was expressed in the presomitic mesoderm, somites, and so on. was identified as a regulator of the expression of the somite formation marker . Overall, our study provides new insights into the evolution of family genes and the control of over the convergent extension cells of somitic precursors in zebrafish.
Topics: Animals; Zebrafish; Zebrafish Proteins; Phylogeny; Cysteine; Membrane Proteins; Proline; Gene Expression Regulation, Developmental
PubMed: 37762365
DOI: 10.3390/ijms241814062 -
STAR Protocols Dec 2023The field of stem cell-based embryo-like models is rapidly evolving, providing in vitro models of in utero stages of mammalian development. Here, we detail steps to...
The field of stem cell-based embryo-like models is rapidly evolving, providing in vitro models of in utero stages of mammalian development. Here, we detail steps to first establish adherent spheroids composed of three cell types from mouse embryonic stem cells solely treated with a chemical inhibitor of SUMOylation. We then describe procedures for generating highly reproducible gastruloids from these dissociated spheroid cells, as well as embryo-like structures comprising anterior neural and trunk somite-like regions using an optimized microfluidics platform. For complete details on the use and execution of this protocol, please refer to Cossec et al. (2023)..
Topics: Animals; Mice; Mouse Embryonic Stem Cells; Sumoylation; Embryo, Mammalian; Microfluidics; Somites; Mammals
PubMed: 37721864
DOI: 10.1016/j.xpro.2023.102573