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Nature Communications Sep 2023Generating primordial germ cell-like cells (PGCLCs) from human pluripotent stem cells (hPSCs) advances studies of human reproduction and development of infertility...
Generating primordial germ cell-like cells (PGCLCs) from human pluripotent stem cells (hPSCs) advances studies of human reproduction and development of infertility treatments, but often entails complex 3D aggregates. Here we develop a simplified, monolayer method to differentiate hPSCs into PGCs within 3.5 days. We use our simplified differentiation platform and single-cell RNA-sequencing to achieve further insights into PGCLC specification. Transient WNT activation for 12 h followed by WNT inhibition specified PGCLCs; by contrast, sustained WNT induced primitive streak. Thus, somatic cells (primitive streak) and PGCLCs are related-yet distinct-lineages segregated by temporally-dynamic signaling. Pluripotency factors including NANOG are continuously expressed during the transition from pluripotency to posterior epiblast to PGCs, thus bridging pluripotent and germline states. Finally, hPSC-derived PGCLCs can be easily purified by virtue of their CXCR4PDGFRAGARP surface-marker profile and single-cell RNA-sequencing reveals that they harbor transcriptional similarities with fetal PGCs.
Topics: Humans; Germ Cells; Cell Differentiation; Embryonic Development; Fetus; RNA
PubMed: 37709760
DOI: 10.1038/s41467-023-41302-w -
Stem Cell Reports Jan 2024While studied extensively in model systems, human gastrulation remains obscure. The scarcity of fetal biological material as well as ethical considerations limit our...
While studied extensively in model systems, human gastrulation remains obscure. The scarcity of fetal biological material as well as ethical considerations limit our understanding of this process. In vitro attachment of natural blastocysts shed light on aspects of the second week of human development in the absence of the morphological manifestation of gastrulation. Stem cell-derived blastocyst models, blastoids, provide the opportunity to reconstitute pre- to post-implantation development in vitro. Here we show that upon in vitro attachment, human blastoids self-organize a BRA population and undergo gastrulation. Single-cell RNA sequencing of these models replicates the transcriptomic signature of the human gastrula. Analysis of developmental timing reveals that in both blastoid models and natural human embryos, the onset of gastrulation as defined by molecular markers, can be traced to timescales equivalent to 12 days post fertilization. In all, natural human embryos and blastoid models self-organize primitive streak and mesoderm derivatives upon in vitro attachment.
Topics: Humans; Gastrulation; Gastrula; Embryonic Development; Blastocyst; Mesoderm
PubMed: 38101401
DOI: 10.1016/j.stemcr.2023.11.005 -
Developmental Dynamics : An Official... May 2022Defects in secondary neurulation play an important role in neural tube defects. Researchers have investigated the processes of secondary neurulation and caudal body...
BACKGROUND
Defects in secondary neurulation play an important role in neural tube defects. Researchers have investigated the processes of secondary neurulation and caudal body formation mainly by microscopic observations and molecular experiments. Although conventional histology is a powerful tool for observing the details of morphology, it has limitations in the presentation of gross three-dimensional (3D) configurations of small embryos. The goal of this study was to visualize secondary neurulation and related structures in chick embryos in Hamburger and Hamilton (HH) stages 10-22 using microCT.
RESULTS
The gross morphology of the chick embryo of various developmental stages was well visualized using microCT. Also, the detailed structures of the caudal cell mass (CCM) were presented starting from HH stage 12 to stage 16. The spatiotemporal relationship of CCM with the floor plate of the neural tube and notochord was shown. The dynamic changes of the chordoneural hinge, the cavitation of the secondary neural tube, and the primitive streak were described throughout the early stages of secondary neurulation.
CONCLUSIONS
By utilizing the advantages of the microCT technique, our study shed light on the secondary neurulation in early-stage chick embryos and this can be the 3D reference for related structures.
Topics: Animals; Chick Embryo; Imaging, Three-Dimensional; Neural Tube; Neurulation; Notochord; X-Ray Microtomography
PubMed: 34811830
DOI: 10.1002/dvdy.441 -
Mechanisms of Development Sep 1997We review the early stages of chick embryogenesis, in particular the formation of the hypoblast, and the ingression of endoderm and mesoderm through the primitive... (Review)
Review
We review the early stages of chick embryogenesis, in particular the formation of the hypoblast, and the ingression of endoderm and mesoderm through the primitive streak. The formation of a trilaminar embryo during gastrulation is accompanied by the specification of body axes. The first axis is already present in the unfertilized egg and runs from the cytoplasmatic animal to the yolk rich vegetal pole. Already within the uterus a second axis conveys bilateral symmetry to the embryo. It extends from a dorsal/anterior to a ventral/posterior position. These axial poles segregate during gastrulation to form the classical coordinates, a dorsal-ventral and an anterior-posterior axis. The establishment of axes is accompanied by the expression of specific combinations of homeobox genes during gastrulation in the chick, as in other metazoa. We review the avian specific information and compare it with findings in other species. A combinatorial homeobox code for the specification of identities during development is discussed.
Topics: Animals; Avian Proteins; Body Patterning; Chick Embryo; Endoderm; Gastrula; Gene Expression Regulation, Developmental; Genes, Homeobox; Homeodomain Proteins; Mesoderm; Nerve Tissue Proteins; Otx Transcription Factors; Trans-Activators; Transcription Factors
PubMed: 9347911
DOI: 10.1016/s0925-4773(97)00102-0 -
ELife Jul 2021In classical descriptions of vertebrate development, the segregation of the three embryonic germ layers completes by the end of gastrulation. Body formation then...
In classical descriptions of vertebrate development, the segregation of the three embryonic germ layers completes by the end of gastrulation. Body formation then proceeds in a head to tail fashion by progressive deposition of lineage-committed progenitors during regression of the primitive streak (PS) and tail bud (TB). The identification by retrospective clonal analysis of a population of neuromesodermal progenitors (NMPs) contributing to both musculoskeletal precursors (paraxial mesoderm) and spinal cord during axis formation challenged these notions. However, classical fate mapping studies of the PS region in amniotes have so far failed to provide direct evidence for such bipotential cells at the single-cell level. Here, using lineage tracing and single-cell RNA sequencing in the chicken embryo, we identify a resident cell population of the anterior PS epiblast, which contributes to neural and mesodermal lineages in trunk and tail. These cells initially behave as monopotent progenitors as classically described and only acquire a bipotential fate later, in more posterior regions. We show that NMPs exhibit a conserved transcriptomic signature during axis elongation but lose their epithelial characteristicsin the TB. Posterior to anterior gradients of convergence speed and ingression along the PS lead to asymmetric exhaustion of PS mesodermal precursor territories. Through limited ingression and increased proliferation, NMPs are maintained and amplified as a cell population which constitute the main progenitors in the TB. Together, our studies provide a novel understanding of the PS and TB contribution through the NMPs to the formation of the body of amniote embryos.
Topics: Animals; Body Patterning; Cell Differentiation; Chick Embryo; Gene Expression Regulation, Developmental; Mesoderm; Neural Stem Cells; Primitive Streak
PubMed: 34227938
DOI: 10.7554/eLife.64819 -
Developmental Biology May 2022T is the founding member of the T-box family of transcription factors; family members are critical for cell fate decisions and tissue morphogenesis throughout the animal...
T is the founding member of the T-box family of transcription factors; family members are critical for cell fate decisions and tissue morphogenesis throughout the animal kingdom. T is expressed in the primitive streak and notochord with mouse mutant studies revealing its critical role in mesoderm formation in the primitive streak and notochord integrity. We previously demonstrated that misexpression of Tbx6 in the paraxial and lateral plate mesoderm results in embryos resembling Tbx15 and Tbx18 nulls. This, together with results from in vitro transcriptional assays, suggested that ectopically expressed Tbx6 can compete with endogenously expressed Tbx15 and Tbx18 at the binding sites of target genes. Since T-box proteins share a similar DNA binding domain, we hypothesized that misexpressing T in the paraxial and lateral plate mesoderm would also interfere with the endogenous Tbx15 and Tbx18, causing embryonic phenotypes resembling those seen upon Tbx6 expression in the somites and limbs. Interestingly, ectopic T expression led to distinct embryonic phenotypes, specifically, reduced-sized somites in embryos expressing the highest levels of T, which ultimately affects axis length and neural tube morphogenesis. We further demonstrate that ectopic T leads to ectopic expression of Tbx6 and Mesogenin 1, known targets of T. These results suggests that ectopic T expression contributes to the phenotype by activating its own targets rather than via a straight competition with endogenous T-box factors.
Topics: Animals; Ectopic Gene Expression; Embryonic Development; Gene Expression Regulation, Developmental; Mesoderm; Mice; Somites; T-Box Domain Proteins
PubMed: 35276131
DOI: 10.1016/j.ydbio.2022.02.010 -
Cell Aug 2011
Topics: Animals; Embryo, Mammalian; Epithelial-Mesenchymal Transition; Gastrula; Mice; Primitive Streak
PubMed: 21816280
DOI: 10.1016/j.cell.2011.07.028 -
Mechanisms of Development Sep 2020During mouse embryonic development a mass of pluripotent epiblast tissue is transformed during gastrulation to generate the three definitive germ layers: endoderm,... (Review)
Review
During mouse embryonic development a mass of pluripotent epiblast tissue is transformed during gastrulation to generate the three definitive germ layers: endoderm, mesoderm, and ectoderm. During gastrulation, a spatiotemporally controlled sequence of events results in the generation of organ progenitors and positions them in a stereotypical fashion throughout the embryo. Key to the correct specification and differentiation of these cell fates is the establishment of an axial coordinate system along with the integration of multiple signals by individual epiblast cells to produce distinct outcomes. These signaling domains evolve as the anterior-posterior axis is established and the embryo grows in size. Gastrulation is initiated at the posteriorly positioned primitive streak, from which nascent mesoderm and endoderm progenitors ingress and begin to diversify. Advances in technology have facilitated the elaboration of landmark findings that originally described the epiblast fate map and signaling pathways required to execute those fates. Here we will discuss the current state of the field and reflect on how our understanding has shifted in recent years.
Topics: Animals; Body Patterning; Cell Differentiation; Cell Lineage; Ectoderm; Embryonic Development; Endoderm; Female; Gastrula; Gastrulation; Germ Layers; Mesoderm; Mice; Organ Specificity; Pregnancy
PubMed: 32473204
DOI: 10.1016/j.mod.2020.103617 -
Developmental Biology Dec 2005The prevalent model for the generation of axial polarity in mouse embryos proposes that a radial to a linear transition in the expression of primitive streak markers...
The prevalent model for the generation of axial polarity in mouse embryos proposes that a radial to a linear transition in the expression of primitive streak markers precedes the formation of the primitive streak on one side of the epiblast. This model contrasts with the models of mesoderm formation in other vertebrates as it suggests that the primitive streak is initially established in a radial pattern rather than a localized region of the epiblast. Here, we examine the proposed correlation between the expression of Brachyury and Wnt3, two genes reported as expressed radially in the proximal epiblast, with the movements of proximal anterior epiblast cells at stages leading to the formation of the primitive streak. Our results reveal that neither Brachyury nor Wnt3 forms a ring of expression in the proximal epiblast as previously thought. In embryos dissected between 5.5 and 6.5 dpc, Brachyury is first expressed in the distal extra-embryonic ectoderm and subsequently on one side of the epiblast. Wnt3 expression is evident first in the posterior visceral endoderm of 5.5 dpc embryos and later in the posterior epiblast. Lineage analysis shows that the movements of the proximal epiblast do not restrict Brachyury expression to the posterior epiblast. Our data suggest a model whereby the localized expression of these genes in the posterior epiblast, and hence the formation of the primitive streak, is the result of local cell-cell interactions in the future posterior portion of the egg cylinder rather than regionalization of a radial pattern of expression in proximal epiblast cells.
Topics: Animals; Body Patterning; Cell Communication; Cell Movement; Ectoderm; Endoderm; Female; Fetal Proteins; Gastrula; Gene Expression Regulation, Developmental; Male; Mice; Models, Biological; T-Box Domain Proteins; Wnt Proteins; Wnt3 Protein
PubMed: 16289026
DOI: 10.1016/j.ydbio.2005.09.012 -
PloS One 2019Cul4b-null (Cul4bΔ/Y) mice undergo growth arrest and degeneration during the early embryonic stages and die at E9.5. The pathogenic causes of this lethality remain...
Cul4b-null (Cul4bΔ/Y) mice undergo growth arrest and degeneration during the early embryonic stages and die at E9.5. The pathogenic causes of this lethality remain incompletely characterized. However, it has been hypothesized that the loss of Cul4b function in extraembryonic tissues plays a key role. In this study, we investigated possible causes of death for Cul4b-null embryos, particularly in regard to the role of embryonic Cul4b. First, we show that the loss of embryonic Cul4b affects the growth of the inner cell mass in vitro and delays epiblast development during the gastrulation period at E6.5~E7.5 in vivo, as highlighted by the absence of the epiblastic transcription factor Brachyury from E6.5~E7.5. Additionally, at E7.5, strong and laterally expanded expression of Eomes and Fgf8 signaling was detected. Sectioning of these embryos showed disorganized primitive streak layer cells. Second, we observed that Mash2-expressing cells were present in the extraembryonic tissues of Cul4b-deficient embryos at E6.5 but were absent at E7.5. In addition, the loss of Cul4b resulted in decreased expression of cyclin proteins, which are required for the cell cycle transition from G1 to S. Taken together, these observations suggest that the embryonic expression of Cul4b is important for epiblast growth during E6.5~E7.5, and the loss of Cul4b results in either delayed growth of the epiblast or defective localization of primitive streak layer cells. As a result, the signaling activity mediated by the epiblast for subsequent ectoplacental cone development is affected, with the potential to induce growth retardation and lethality in Cul4bΔ/Y embryos.
Topics: Animals; Blastocyst Inner Cell Mass; Cullin Proteins; Embryo, Mammalian; Female; Fetal Proteins; Gastrulation; Gene Expression Regulation, Developmental; Germ Layers; Heterozygote; Male; Mice; Mice, Knockout; Models, Animal; Primitive Streak; T-Box Domain Proteins
PubMed: 31260508
DOI: 10.1371/journal.pone.0219221