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BioRxiv : the Preprint Server For... May 2024Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples....
UNLABELLED
Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, we designed a robust pipeline to perform single-cell and nuclei analysis on mouse embryos from E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to WT perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, we present a methodology designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with FAST genotyping protocol (3 hours) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. We also include guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.
SUMMARY
We establish a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.
PubMed: 38746120
DOI: 10.1101/2024.04.29.591777 -
Stem Cell Research May 2024Y chromosome deletion and karyotype abnormalities are commonly associated with congenital non-obstructive azoospermia, impairing spermatogenesis. Specifically, the...
Y chromosome deletion and karyotype abnormalities are commonly associated with congenital non-obstructive azoospermia, impairing spermatogenesis. Specifically, the deletion of the Y chromosome Azoospermia factor a (AZFa) has been identified in infertile males with severely impaired spermatogenesis. AZFa, encompassing megabase-scale of the Y chromosome region, poses challenges in modeling AZFa deletion-related male infertility using gene editing tools. Here, we successfully created an AZFa-deleted human embryonic stem cell line utilizing the CRISPR/Cas9 gene editing tool. Our analysis indicates the AZFa-deleted stem cell line holds promise for differentiation into ectoderm, mesoderm, and endoderm, highlighting its potential for further comprehensive study.
PubMed: 38733811
DOI: 10.1016/j.scr.2024.103436 -
Diagnostics (Basel, Switzerland) Apr 2024Oligodontia can be isolated or syndromic, associated with other ectodermal abnormalities. The aim of the study was to perform hair examination in orthodontic patients...
Oligodontia can be isolated or syndromic, associated with other ectodermal abnormalities. The aim of the study was to perform hair examination in orthodontic patients diagnosed with oligodontia with a low clinical expression of symptoms of ectodermal origin. All available orthodontic patients diagnosed with oligodontia in the permanent dentition were enrolled. Hair examination included clinical evaluation of the patients' hair, trichoscopy, trichogram and evaluation of the hair shafts under a polarized light microscope. In total, 25 patients, 18 males and 7 females, aged 6 to 24 years were evaluated for the presence of dental and hair abnormalities. The number of congenitally absent teeth ranged from 6 to 24 teeth and diastemas, microdontia, taurodontism and altered tooth shape were found in 23 patients. Hair disorders were found in 68% of the subjects. Hypotrichosis, the heterogeneity of shaft color and loss of pigment, androgenetic alopecia, telogen effluvium, trichoschisis, pili canaliculi, trichorrhexis nodosa and pseudomoniletrix were observed. Trichoscopy and trichogram are valid non-invasive diagnostic tests which could be used to differentiate between isolated and syndromic oligodontia in patients with a low clinical expression of ectodermal symptoms.
PubMed: 38732359
DOI: 10.3390/diagnostics14090945 -
EvoDevo May 2024Spiders are a diverse order of chelicerates that diverged from other arthropods over 500 million years ago. Research on spider embryogenesis, particularly studies using...
Spiders are a diverse order of chelicerates that diverged from other arthropods over 500 million years ago. Research on spider embryogenesis, particularly studies using the common house spider Parasteatoda tepidariorum, has made important contributions to understanding the evolution of animal development, including axis formation, segmentation, and patterning. However, we lack knowledge about the cells that build spider embryos, their gene expression profiles and fate. Single-cell transcriptomic analyses have been revolutionary in describing these complex landscapes of cellular genetics in a range of animals. Therefore, we carried out single-cell RNA sequencing of P. tepidariorum embryos at stages 7, 8 and 9, which encompass the establishment and patterning of the body plan, and initial differentiation of many tissues and organs. We identified 20 cell clusters, from 18.5 k cells, which were marked by many developmental toolkit genes, as well as a plethora of genes not previously investigated. We found differences in the cell cycle transcriptional signatures, suggestive of different proliferation dynamics, which related to distinctions between endodermal and some mesodermal clusters, compared with ectodermal clusters. We identified many Hox genes as markers of cell clusters, and Hox gene ohnologs were often present in different clusters. This provided additional evidence of sub- and/or neo-functionalisation of these important developmental genes after the whole genome duplication in an arachnopulmonate ancestor (spiders, scorpions, and related orders). We also examined the spatial expression of marker genes for each cluster to generate a comprehensive cell atlas of these embryonic stages. This revealed new insights into the cellular basis and genetic regulation of head patterning, hematopoiesis, limb development, gut development, and posterior segmentation. This atlas will serve as a platform for future analysis of spider cell specification and fate, and studying the evolution of these processes among animals at cellular resolution.
PubMed: 38730509
DOI: 10.1186/s13227-024-00224-4 -
Science Advances May 2024Polycomb repressive complexes 1 and 2 (PRC1 and 2) are required for heritable repression of developmental genes. The cis- and trans-acting factors that contribute to...
Polycomb repressive complexes 1 and 2 (PRC1 and 2) are required for heritable repression of developmental genes. The cis- and trans-acting factors that contribute to epigenetic inheritance of mammalian Polycomb repression are not fully understood. Here, we show that, in human cells, ectopically induced Polycomb silencing at initially active developmental genes, but not near ubiquitously expressed housekeeping genes, is inherited for many cell divisions. Unexpectedly, silencing is heritable in cells with mutations in the H3K27me3 binding pocket of the Embryonic Ectoderm Development (EED) subunit of PRC2, which are known to disrupt H3K27me3 recognition and lead to loss of H3K27me3. This mode of inheritance is less stable and requires intact PRC2 and recognition of H2AK119ub1 by PRC1. Our findings suggest that maintenance of Polycomb silencing is sensitive to local genomic context and can be mediated by PRC1-dependent H2AK119ub1 and PRC2 independently of H3K27me3 recognition.
Topics: Humans; Histones; Ubiquitination; Gene Silencing; Polycomb-Group Proteins; Polycomb Repressive Complex 2; Polycomb Repressive Complex 1; Genome, Human; Epigenesis, Genetic; Mutation
PubMed: 38718120
DOI: 10.1126/sciadv.adl4529 -
BMC Pediatrics May 2024Aplasia cutis congenita (ACC) is a congenital disorder that can be classified into nine types, with Type I ACC being the most common. Type V ACC associated with fetus...
Aplasia cutis congenita (ACC) is a congenital disorder that can be classified into nine types, with Type I ACC being the most common. Type V ACC associated with fetus papyraceus is a rare subtype of ACC. We report the case of a Type V ACC in a male newborn with extensive abdominal skin defects. The patient received conservative treatment using hydrogel foam and silicone foam dressings. Approximately five weeks later, the patient was discharged when more than 60% of the skin had completed epithelialization. After discharge from West China Second University Hospital, Chengdu , the patient continued to be followed up regularly at the Burns and Plastic Surgery Clinic at local hospital in Gansu. We followed up the child by telephone. After 4 months of follow-up, scar tissue formation was observed in the trunk area. The infant is 2 years and 5 months old now, physical examination did not reveal any organ problems.
Topics: Humans; Male; Ectodermal Dysplasia; Infant, Newborn; Conservative Treatment; Bandages
PubMed: 38714985
DOI: 10.1186/s12887-024-04777-0 -
Scientific Reports May 2024Current approaches to diagnosing male infertility inadequately assess the complexity of the male gamete. Beyond the paternal haploid genome, spermatozoa also deliver...
Current approaches to diagnosing male infertility inadequately assess the complexity of the male gamete. Beyond the paternal haploid genome, spermatozoa also deliver coding and non-coding RNAs to the oocyte. While sperm-borne RNAs have demonstrated potential involvement in embryo development, the underlying mechanisms remain unclear. In this study, 47 sperm samples from normozoospermic males undergoing fertility treatment using donor oocytes were sequenced and analyzed to evaluate associations between sperm RNA elements (exon-sized sequences) and blastocyst progression. A total of 366 RNA elements (REs) were significantly associated with blastocyst rate (padj < 0.05), some of which were linked to genes related to critical developmental processes, including mitotic spindle formation and both ectoderm and mesoderm specification. Of note, 27 RE-associated RNAs are predicted targets of our previously reported list of developmentally significant miRNAs. Inverse RE-miRNA expression patterns were consistent with miRNA-mediated down-regulation. This study provides a comprehensive set of REs which differ by the patient's ability to produce blastocysts. This knowledge can be leveraged to improve clinical screening of male infertility and ultimately reduce time to pregnancy.
Topics: Humans; Male; Infertility, Male; Spermatozoa; MicroRNAs; Adult; Female; Blastocyst; RNA; Embryonic Development
PubMed: 38705876
DOI: 10.1038/s41598-024-60586-6 -
Neural Development May 2024The evolution of central nervous systems (CNSs) is a fascinating and complex topic; further work is needed to understand the genetic and developmental homology between...
BACKGROUND
The evolution of central nervous systems (CNSs) is a fascinating and complex topic; further work is needed to understand the genetic and developmental homology between organisms with a CNS. Research into a limited number of species suggests that CNSs may be homologous across Bilateria. This hypothesis is based in part on similar functions of BMP signaling in establishing fates along the dorsal-ventral (D-V) axis, including limiting neural specification to one ectodermal region. From an evolutionary-developmental perspective, the best way to understand a system is to explore it in a wide range of organisms to create a full picture.
METHODS
Here, we expand our understanding of BMP signaling in Spiralia, the third major clade of bilaterians, by examining phenotypes after expression of a dominant-negative BMP Receptor 1 and after knock-down of the putative BMP antagonist Chordin-like using CRISPR/Cas9 gene editing in the annelid Capitella teleta (Pleistoannelida).
RESULTS
Ectopic expression of the dominant-negative Ct-BMPR1 did not increase CNS tissue or alter overall D-V axis formation in the trunk. Instead, we observed a unique asymmetrical phenotype: a distinct loss of left tissues, including the left eye, brain, foregut, and trunk mesoderm. Adding ectopic BMP4 early during cleavage stages reversed the dominant-negative Ct-BMPR1 phenotype, leading to a similar loss or reduction of right tissues instead. Surprisingly, a similar asymmetrical loss of left tissues was evident from CRISPR knock-down of Ct-Chordin-like but concentrated in the trunk rather than the episphere.
CONCLUSIONS
Our data highlight a novel asymmetrical phenotype, giving us further insight into the complicated story of BMP's developmental role. We further solidify the hypothesis that the function of BMP signaling during the establishment of the D-V axis and CNS is fundamentally different in at least Pleistoannelida, possibly in Spiralia, and is not required for nervous system delimitation in this group.
Topics: Animals; Biological Evolution; Bone Morphogenetic Protein Receptors, Type I; Body Patterning; Signal Transduction
PubMed: 38698415
DOI: 10.1186/s13064-024-00181-7 -
European Journal of Medical Genetics Apr 2024To date 11 patients with Coffin-Siris syndrome type 7 (OMIM 618027) have been described since the first literature report. All reported patients carried de novo variants...
To date 11 patients with Coffin-Siris syndrome type 7 (OMIM 618027) have been described since the first literature report. All reported patients carried de novo variants with presumed dominant negative effect, which localized in the PHD1/PHD2 domains of DPF2. Here we report on the first familial case of Coffin-Siris syndrome type 7. The index patient presented during the 1st year of life with failure to thrive and ectodermal anomalies. The genetic analysis using whole exome sequencing showed a likely pathogenic missense variant in the PHD1 region. The family analysis showed that the mother as well as the older brother of the index patient also carried the detected DPF2 variant in heterozygous state. The mother had a history of school difficulties but no history of failure to thrive and was overall mildly affected. The brother showed developmental delay with autistic features, ectodermal anomalies and overlapping morphologic features but did not have a history of growth failure problems. To our knowledge this is the first report of an inherited likely pathogenic variant in DPF2, underlining the variability of the associated phenotype as well as the importance of considering inherited DPF2 variants during the variant filtering strategy of whole exome data.
PubMed: 38697389
DOI: 10.1016/j.ejmg.2024.104945 -
Cell Transplantation 2024Multipotent mesenchymal stem cells (MSCs) have high self-renewal and multi-lineage differentiation potentials and low immunogenicity, so they have attracted much... (Review)
Review
Multipotent mesenchymal stem cells (MSCs) have high self-renewal and multi-lineage differentiation potentials and low immunogenicity, so they have attracted much attention in the field of regenerative medicine and have a promising clinical application. MSCs originate from the mesoderm and can differentiate not only into osteoblasts, cartilage, adipocytes, and muscle cells but also into ectodermal and endodermal cell lineages across embryonic layers. To design cell therapy for replacement of damaged tissues, it is essential to understand the signaling pathways, which have a major impact on MSC differentiation, as this will help to integrate the signaling inputs to initiate a specific lineage. Hedgehog (Hh) signaling plays a vital role in the development of various tissues and organs in the embryo. As a morphogen, Hh not only regulates the survival and proliferation of tissue progenitor and stem populations but also is a critical moderator of MSC differentiation, involving tri-lineage and across embryonic layer differentiation of MSCs. This review summarizes the role of Hh signaling pathway in the differentiation of MSCs to mesodermal, endodermal, and ectodermal cells.
Topics: Mesenchymal Stem Cells; Hedgehog Proteins; Humans; Cell Differentiation; Signal Transduction; Animals; Multipotent Stem Cells
PubMed: 38695366
DOI: 10.1177/09636897241244943