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1.
J Morphol ; 285(6): e21711, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38840450

ABSTRACT

The histological origin of podocysts in scyphozoans has long been undetermined, with uncertainty whether they arise from mesenchymal amoebocytes or stalk and pedal disc ectoderm in polyps. Histological investigation on the pedal disc was difficult due to the settlement of polyps on hard substrates. In this study, we investigated the histological characteristics of polyps during podocyst production in Asian moon jelly (Aurelia coerulea) with utilizing those attached on thin polystyrene substrates. Fine histological features of the pedal disc became possible after the substrates were decomposed during histological processing. Our findings unequivocally demonstrate that the cell mass of podocysts originates from the ectoderm of the pedal disc and the stalk without the involvement of amoebocytes in the mesoglea. Preceding the podocyst formation, the pedal disc undergoes enlargement facilitated by the elongated stalk ectodermal cells, which attach to a substrate. Subsequently, the pedal disc ectoderm give rise to the primary podocyst cells with accumulating nutrient granules in the cytoplasm and forming the cyst capsule cooperatively with the invaginated pedal disc ectoderm. Direct transformation from the ectodermal cells to podocyst cells suggests that podocyst formation involves tissue dedifferentiation. Throughout the period of podocyst production, the gastrodermis of polyps is physically separated from the ectoderm by the mesoglea and shows no histological changes, and no amoebocytes appear in the mesoglea. These histological properties are totally different from those in other modes of asexual reproduction, which incorporate the endoderm of polyps, suggesting the developmental and evolutionary differences between these asexual reproductions and podocyst production in Scyphozoa.


Subject(s)
Ectoderm , Scyphozoa , Animals , Cell Dedifferentiation
2.
Theriogenology ; 225: 89-97, 2024 Sep 01.
Article in English | MEDLINE | ID: mdl-38796961

ABSTRACT

The first cell differentiation event that occurs in the embryo determines the inner cell mass (ICM) and the trophectoderm (TE). In the mouse, glucose (GLC) is essential for this process, while oxygen tension (O2) also interferes with TE formation. The roles of GLC and O2 in this event in bovine embryos are not completely elucidated. We hypothesized that the absence of glucose and a higher O2 tension negatively impact ICM and TE cell allocation in the bovine embryo. The objective of this study was to evaluate the effect of GLC within different O2 levels on the formation of the TE. In vitro-produced embryos were cultured in serum-free KSOM medium and randomly submitted to treatments on the day of IVC, according to a 2x2 factorial model, in which GLC (present [+GLC] or absent [-GLC]) and O2 (low [5%O2] or high [20%O2]) were the independent variables. Cleavage and blastocyst rates were obtained at D4 and D8, respectively. Embryos at D8 were subjected to autofluorescence analysis to quantitate NADH and FAD + or fixed for GATA3 and YAP1 immunostaining using a laser scanning confocal microscope. Total, TE, and ICM cell counts were obtained. Embryos were also harvested for gene expression quantification of GATA3, YAP1, SOX2, CDX2, TFAP2C and OCT4. Results indicate that there was an effect of O2 (p = 0.018) on cleavage rates, although no differences were observed in blastocyst rates. NADH was higher in -GLC compared to + GLC (p = 0.014) and no differences in FAD+ were observed. Total cell count data were not different between variables. There was an increase in the ICM cell count in the +GLC 5%O2 condition compared to the other three conditions. No effects of GLC, O2, or their interactions were observed on TE cell count or the TE/total cell ratio. CDX2 (p = 0.007) and TFAP2C (p = 0.038) were increased in -GLC 20%O2 compared to + GLC 20%O2. SOX2 was decreased in +GLC 20%O2 compared to + GLC 5%O2 (p = 0.027) or compared to -GLC 20%O2 (p = 0.005). GATA3, YAP1, and OCT4 genes did not present differences among conditions. In conclusion, both GLC and high oxygen tension did not impair TE formation and TE cell number, although a +GLC-low oxygen environment led to a higher number of ICM cells. Interestingly, the expression of TE-related gene CDX2 was increased in the absence of glucose within higher O2 tension. Our results implicate that according to the oxygen tension used in IVC, glucose can exert different effects on blastocyst cell allocation or gene expression.


Subject(s)
Embryo Culture Techniques , Glucose , Oxygen , Animals , Cattle/embryology , Oxygen/metabolism , Oxygen/pharmacology , Glucose/pharmacology , Embryo Culture Techniques/veterinary , Embryo, Mammalian , Fertilization in Vitro/veterinary , Embryonic Development/drug effects , Ectoderm/metabolism , Gene Expression Regulation, Developmental , Blastocyst Inner Cell Mass/metabolism
3.
Zebrafish ; 21(2): 171-176, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38621215

ABSTRACT

The transgenic (TG) zebrafish allows researchers to bio-image specific biological phenomena in cells and tissues in vivo. We established TG lines to monitor changes in the ovaries of live fish. The original TG line with ovarian fluorescence was occasionally established. Although the cDNA integrated into the line was constructed for the expression of enhanced green fluorescent protein (EGFP) driven by the medaka ß-actin promoter, the expression of EGFP is restricted to the oocytes and gills in adult fish. Furthermore, we found that germinal vesicles (GVs) in oocytes of the established line can be observed by relatively strong fluorescence around the GV. In this study, we tried to capture the dynamic processes of germinal vesicle breakdown (GVBD) during meiotic cell division using the GV fluorescent oocytes. As a result, GV migration and GVBD could be monitored in real time. We also succeeded in observing actin filaments involved in the migration of GV to the animal pole. This strain can be used for education in the process of oocyte meiotic cell division.


Subject(s)
Ectoderm/embryology , Embryonic Structures , Ovary , Zebrafish , Female , Animals , Oocytes , Animals, Genetically Modified , Cell Division
4.
Dev Cell ; 59(8): 941-960, 2024 Apr 22.
Article in English | MEDLINE | ID: mdl-38653193

ABSTRACT

In recent years, the pursuit of inducing the trophoblast stem cell (TSC) state has gained prominence as a compelling research objective, illuminating the establishment of the trophoblast lineage and unlocking insights into early embryogenesis. In this review, we examine how advancements in diverse technologies, including in vivo time course transcriptomics, cellular reprogramming to TSC state, chemical induction of totipotent stem-cell-like state, and stem-cell-based embryo-like structures, have enriched our insights into the intricate molecular mechanisms and signaling pathways that define the mouse and human trophectoderm/TSC states. We delve into disparities between mouse and human trophectoderm/TSC fate establishment, with a special emphasis on the intriguing role of pluripotency in this context. Additionally, we re-evaluate recent findings concerning the potential of totipotent-stem-like cells and embryo-like structures to fully manifest the trophectoderm/trophoblast lineage's capabilities. Lastly, we briefly discuss the potential applications of induced TSCs in pregnancy-related disease modeling.


Subject(s)
Cell Differentiation , Cell Lineage , Trophoblasts , Trophoblasts/cytology , Trophoblasts/metabolism , Animals , Humans , Mice , Female , Pregnancy , Ectoderm/metabolism , Ectoderm/cytology , Embryonic Development , Cellular Reprogramming
5.
PLoS Biol ; 22(4): e3002611, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38683880

ABSTRACT

As tissues grow and change shape during animal development, they physically pull and push on each other, and these mechanical interactions can be important for morphogenesis. During Drosophila gastrulation, mesoderm invagination temporally overlaps with the convergence and extension of the ectodermal germband; the latter is caused primarily by Myosin II-driven polarised cell intercalation. Here, we investigate the impact of mesoderm invagination on ectoderm extension, examining possible mechanical and mechanotransductive effects on Myosin II recruitment and polarised cell intercalation. We find that the germband ectoderm is deformed by the mesoderm pulling in the orthogonal direction to germband extension (GBE), showing mechanical coupling between these tissues. However, we do not find a significant change in Myosin II planar polarisation in response to mesoderm invagination, nor in the rate of junction shrinkage leading to neighbour exchange events. We conclude that the main cellular mechanism of axis extension, polarised cell intercalation, is robust to the mesoderm invagination pull. We find, however, that mesoderm invagination slows down the rate of anterior-posterior cell elongation that contributes to axis extension, counteracting the tension from the endoderm invagination, which pulls along the direction of GBE.


Subject(s)
Drosophila melanogaster , Ectoderm , Gastrulation , Mesoderm , Myosin Type II , Animals , Mesoderm/embryology , Mesoderm/cytology , Gastrulation/physiology , Ectoderm/cytology , Ectoderm/embryology , Ectoderm/metabolism , Myosin Type II/metabolism , Drosophila melanogaster/embryology , Cell Polarity , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Embryo, Nonmammalian , Morphogenesis , Body Patterning/physiology , Drosophila/embryology
6.
Cell Rep ; 43(5): 114136, 2024 May 28.
Article in English | MEDLINE | ID: mdl-38643480

ABSTRACT

Embryos, originating from fertilized eggs, undergo continuous cell division and differentiation, accompanied by dramatic changes in transcription, translation, and metabolism. Chromatin regulators, including transcription factors (TFs), play indispensable roles in regulating these processes. Recently, the trophoblast regulator TFAP2C was identified as crucial in initiating early cell fate decisions. However, Tfap2c transcripts persist in both the inner cell mass and trophectoderm of blastocysts, prompting inquiry into Tfap2c's function in post-lineage establishment. In this study, we delineate the dynamics of TFAP2C during the mouse peri-implantation stage and elucidate its collaboration with the key lineage regulators CDX2 and NANOG. Importantly, we propose that de novo formation of H3K9me3 in the extraembryonic ectoderm during implantation antagonizes TFAP2C binding to crucial developmental genes, thereby maintaining its lineage identity. Together, these results highlight the plasticity of the chromatin environment in designating the genomic binding of highly adaptable lineage-specific TFs and regulating embryonic cell fates.


Subject(s)
CDX2 Transcription Factor , Cell Lineage , Chromatin , Gene Expression Regulation, Developmental , Transcription Factor AP-2 , Animals , Chromatin/metabolism , Mice , Cell Lineage/genetics , Transcription Factor AP-2/metabolism , Transcription Factor AP-2/genetics , CDX2 Transcription Factor/metabolism , CDX2 Transcription Factor/genetics , Nanog Homeobox Protein/metabolism , Nanog Homeobox Protein/genetics , Blastocyst/metabolism , Blastocyst/cytology , Transcription Factors/metabolism , Transcription Factors/genetics , Female , Histones/metabolism , Cell Differentiation/genetics , Ectoderm/metabolism , Ectoderm/cytology , Embryonic Development/genetics
7.
Int J Mol Sci ; 25(8)2024 Apr 09.
Article in English | MEDLINE | ID: mdl-38673725

ABSTRACT

Human-induced pluripotent stem cells (hiPSCs) offer a promising source for generating dental epithelial (DE) cells. Whereas the existing differentiation protocols were time-consuming and relied heavily on growth factors, herein, we developed a three-step protocol to convert hiPSCs into DE cells in 8 days. In the first phase, hiPSCs were differentiated into non-neural ectoderm using SU5402 (an FGF signaling inhibitor). The second phase involved differentiating non-neural ectoderm into pan-placodal ectoderm and simultaneously inducing the formation of oral ectoderm (OE) using LDN193189 (a BMP signaling inhibitor) and purmorphamine (a SHH signaling activator). In the final phase, OE cells were differentiated into DE through the application of Purmorphamine, XAV939 (a WNT signaling inhibitor), and BMP4. qRT-PCR and immunostaining were performed to examine the expression of lineage-specific markers. ARS staining was performed to evaluate the formation of the mineralization nodule. The expression of PITX2, SP6, and AMBN, the emergence of mineralization nodules, and the enhanced expression of AMBN and AMELX in spheroid culture implied the generation of DE cells. This study delineates the developmental signaling pathways and uses small molecules to streamline the induction of hiPSCs into DE cells. Our findings present a simplified and quicker method for generating DE cells, contributing valuable insights for dental regeneration and dental disease research.


Subject(s)
Cell Differentiation , Epithelial Cells , Induced Pluripotent Stem Cells , Morpholines , Purines , Pyrimidines , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/drug effects , Cell Differentiation/drug effects , Epithelial Cells/metabolism , Epithelial Cells/cytology , Epithelial Cells/drug effects , Tooth/cytology , Ectoderm/cytology , Ectoderm/metabolism , Cells, Cultured , Bone Morphogenetic Protein 4/metabolism , Bone Morphogenetic Protein 4/pharmacology , Pyrazoles/pharmacology , Signal Transduction/drug effects , Small Molecule Libraries/pharmacology
8.
Int J Dev Biol ; 68(1): 25-37, 2024.
Article in English | MEDLINE | ID: mdl-38591691

ABSTRACT

In vertebrate development, ectoderm is specified into neural plate (NP), neural plate border (NPB), and epidermis. Although such patterning is thought to be achieved by molecular concentration gradients, it has been revealed, mainly by in vitro analysis, that mechanical force can regulate cell specification. During in vivo patterning, cells deform and migrate, and this applies force to surrounding tissues, shaping the embryo. However, the role of mechanical force for cell specification in vivo is largely unknown. In this study, with an aspiration assay and atomic force microscopy, we have demonstrated that tension on ectodermal cells decreases laterally from the midline in Xenopus early neurula. Ectopically applied force laterally expanded the neural crest (NC) region, a derivative of the NPB, whereas force relaxation suppressed it. Furthermore, force application activated both the FGF and Wnt pathways, which are required for NC formation during neuroectodermal patterning. Taken together, mechanical force is necessary for NC formation in order to regulate signaling pathways. Furthermore, molecular signals specify the NP and generate force on neighboring tissue, the NPB, with its closure. This force activates signals, possibly determining the appropriate width of a narrow tissue, the NC.


Subject(s)
Neural Crest , Xenopus Proteins , Animals , Neural Crest/physiology , Xenopus laevis/metabolism , Xenopus Proteins/metabolism , Ectoderm/metabolism , Wnt Signaling Pathway , Gene Expression Regulation, Developmental
9.
Curr Top Dev Biol ; 157: 67-82, 2024.
Article in English | MEDLINE | ID: mdl-38556459

ABSTRACT

Transplantation experiments have shown that a true organizer provides instructive signals that induce and pattern ectopic structures in the responding tissue. Here, we review craniofacial experiments to identify tissues with organizer properties and signals with organizer properties. In particular, we evaluate whether transformation of identity took place in the mesenchyme. Using these stringent criteria, we find the strongest evidence for the avian foregut ectoderm. Transplanting a piece of quail foregut endoderm to a host chicken embryo caused ectopic beaks to form derived from chicken mesenchyme. The beak identity, whether upper or lower as well as orientation, was controlled by the original anterior-posterior position of the donor endoderm. There is also good evidence that the nasal pit is necessary and sufficient for lateral nasal patterning. Finally, we review signals that have organizer properties on their own without the need for tissue transplants. Mouse germline knockouts of the endothelin pathway result in transformation of identity of the mandible into a maxilla. Application of noggin-soaked beads to post-migratory neural crest cells transforms maxillary identity. This suggests that endothelin or noggin rich ectoderm could be organizers (not tested). In conclusion, craniofacial, neural crest-derived mesenchyme is competent to respond to tissues with organizer properties, also originating in the head. In future, we can exploit such well defined systems to dissect the molecular changes that ultimately lead to patterning of the upper and lower jaw.


Subject(s)
Chickens , Ectoderm , Chick Embryo , Animals , Mice , Jaw , Neural Crest , Endothelins , Body Patterning
10.
Results Probl Cell Differ ; 72: 61-80, 2024.
Article in English | MEDLINE | ID: mdl-38509252

ABSTRACT

Studies using early-stage avian embryos have substantially impacted developmental biology, through the availability of simple culture methods and easiness in tissue manipulation. However, the regulations underlying brain and head development, a central issue of developmental biology, have not been investigated systematically. Yoshihi et al. (2022a) devised a technique to randomly label the epiblast cells with a green fluorescent protein before their development into the brain tissue. This technique was combined with grafting a node or node-derived anterior mesendoderm labeled with a cherry-colored fluorescent protein. Then cellular events were live-recorded over 18 hours during the brain and head development. The live imaging-based analyses identified previously undescribed mechanisms central to brain development: all anterior epiblast cells have a potential to develop into the brain tissues and their gathering onto a proximal anterior mesendoderm forms a brain primordium whereas the remaining cells develop into the covering head ectoderm. The analyses also ruled out the direct participation of the node's activity in the brain development. Yoshihi et al. (2022a) also demonstrate how the enigmatic data from classical models can be reinterpreted in the new model.This chapter was adapted from Yoshihi K, Iida H, Teramoto M, Ishii Y, Kato K, Kondoh H. (2022b). Epiblast cells gather onto the anterior mesendoderm and initiate brain development without the direct involvement of the node in avian embryos: Insights from broad-field live imaging. Front Cell Dev Biol. 10:1019845. doi: 10.3389/fcell.2022.1019845.


Subject(s)
Gastrula , Germ Layers , Germ Layers/metabolism , Ectoderm/metabolism , Embryonic Development , Brain
11.
Sci Adv ; 10(9): eadh7748, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38427729

ABSTRACT

Mechanisms specifying amniotic ectoderm and surface ectoderm are unresolved in humans due to their close similarities in expression patterns and signal requirements. This lack of knowledge hinders the development of protocols to accurately model human embryogenesis. Here, we developed a human pluripotent stem cell model to investigate the divergence between amniotic and surface ectoderms. In the established culture system, cells differentiated into functional amnioblast-like cells. Single-cell RNA sequencing analyses of amnioblast differentiation revealed an intermediate cell state with enhanced surface ectoderm gene expression. Furthermore, when the differentiation started at the confluent condition, cells retained the expression profile of surface ectoderm. Collectively, we propose that human amniotic ectoderm and surface ectoderm are specified along a common nonneural ectoderm trajectory based on cell density. Our culture system also generated extraembryonic mesoderm-like cells from the primed pluripotent state. Together, this study provides an integrative understanding of the human nonneural ectoderm development and a model for embryonic and extraembryonic human development around gastrulation.


Subject(s)
Ectoderm , Pluripotent Stem Cells , Humans , Ectoderm/metabolism , Cell Differentiation/genetics , Mesoderm
12.
Dev Biol ; 508: 64-76, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38190932

ABSTRACT

Feathers originate as protofeathers before birds, in pterosaurs and basal dinosaurs. What characterizes a feather is not only its outgrowth, but its barb cells differentiation and a set of beta-corneous proteins. Reticula appear concomitantly with feathers, as small bumps on plantar skin, made only of keratins. Avian scales, with their own set of beta-corneous proteins, appear more recently than feathers on the shank, and only in some species. In the chick embryo, when feather placodes form, all the non-feather areas of the integument are already specified. Among them, midventral apterium, cornea, reticula, and scale morphogenesis appear to be driven by negative regulatory mechanisms, which modulate the inherited capacity of the avian ectoderm to form feathers. Successive dermal/epidermal interactions, initiated by the Wnt/ß-catenin pathway, and involving principally Eda/Edar, BMP, FGF20 and Shh signaling, are responsible for the formation not only of feather, but also of scale placodes and reticula, with notable differences in the level of Shh, and probably FGF20 expressions. This sequence is a dynamic and labile process, the turning point being the FGF20 expression by the placode. This epidermal signal endows its associated dermis with the memory to aggregate and to stimulate the morphogenesis that follows, involving even a re-initiation of the placode.


Subject(s)
Ectoderm , Feathers , Animals , Chick Embryo , Feathers/metabolism , Ectoderm/metabolism , Biological Evolution , Birds , Keratins/metabolism , Morphogenesis
13.
Nan Fang Yi Ke Da Xue Xue Bao ; 44(1): 119-128, 2024 Jan 20.
Article in Chinese | MEDLINE | ID: mdl-38293983

ABSTRACT

OBJECTIVE: To investigate the potential value of exosomes derived from rat ectoderm mesenchymal stem cells (EMSCs-exo) for repairing secondary spinal cord injury. METHODS: EMSCs-exo were obtained using ultracentrifugation from EMSCs isolated from rat nasal mucosa, identified by transmission electron microscope, nanoparticle tracking analysis (NTA), and Western blotting, and quantified using the BCA method. Neonatal rat microglia purified by differential attachment were induced with 100 µg/L lipopolysaccharide (LPS) and treated with 37.5 or 75 mg/L EMSCs-exo. PC12 cells were exposed to 400 µmol/L H2O2 and treated with EMSCs-exo at 37.5 or 75 mg/L. The protein and mRNA expressions of Arg1 and iNOS in the treated cells were determined with Western blotting and qRT- PCR, and the concentrations of IL- 6, IL-10, and IGF-1 in the supernatants were measured with ELISA. The viability and apoptosis of PC12 cells were detected using CCK-8 assay and flow cytometry. RESULTS: The isolated rat EMSCs showed high expressions of nestin, CD44, CD105, and vimentin. The obtained EMSCs-exo had a typical cup-shaped structure under transmission electron microscope with an average particle size of 142 nm and positivity for CD63, CD81, and TSG101 but not vimentin. In LPS-treated microglia, EMSCs-exo treatment at 75 mg/L significantly increased Arg1 protein level and lowered iNOS protein expression (P < 0.05). EMSCs-exo treatment at 75 mg/L, as compared with the lower concentration at 37.5 mg/L, more strongly increased Arg1 mRNA expression and IGF-1 and IL-10 production and decreased iNOS mRNA expression and IL-6 production in LPS-induced microglia, and more effectively promoted cell survival and decreased apoptosis rate of H2O2-induced PC12 cells (P < 0.05). CONCLUSION: EMSCs-exo at 75 mg/L can effectively reduce the proportion of M1 microglia and alleviate neuronal apoptosis under oxidative stress to promote neuronal survival, suggesting its potential in controlling secondary spinal cord injury.


Subject(s)
Exosomes , Mesenchymal Stem Cells , Spinal Cord Injuries , Rats , Animals , Microglia/metabolism , Lipopolysaccharides/adverse effects , PC12 Cells , Interleukin-10 , Hydrogen Peroxide/metabolism , Insulin-Like Growth Factor I/metabolism , Ectoderm/metabolism , Oxidative Stress , Spinal Cord Injuries/metabolism , RNA, Messenger/metabolism
14.
Genesis ; 62(1): e23532, 2024 Feb.
Article in English | MEDLINE | ID: mdl-37435631

ABSTRACT

Ectodermal appendages in mammals, such as teeth, mammary glands, sweat glands and hair follicles, are generated during embryogenesis through a series of mesenchymal-epithelial interactions. Canonical Wnt signaling and its inhibitors are implicated in the early steps of ectodermal appendage development and patterning. To study the activation dynamics of the Wnt target and inhibitor Dickkopf4 (Dkk4) in ectodermal appendages, we used CRSIPR/Cas9 to generate a Dkk4-Cre knock-in mouse (Mus musculus) line, where the Cre recombinase cDNA replaces the expression of endogenous Dkk4. Using Cre reporters, the Dkk4-Cre activity was evident at the prospective sites of ectodermal appendages, overlapping with the Dkk4 mRNA expression. Unexpectedly, a predominantly mesenchymal cell population in the embryo posterior also showed Dkk4-Cre activity. Lineage-tracing suggested that these cells are likely derived from a few Dkk4-Cre-expressing cells in the epiblast at early gastrulation. Finally, our analyses of Dkk4-Cre-expressing cells in developing hair follicle epithelial placodes revealed intra- and inter-placodal cellular heterogeneity, supporting emerging data on the positional and transcriptional cellular variability in placodes. Collectively, we propose the new Dkk4-Cre knock-in mouse line as a suitable model to study Wnt and DKK4 inhibitor dynamics in early mouse development and ectodermal appendage morphogenesis.


Subject(s)
Hair Follicle , Wnt Signaling Pathway , Mice , Animals , Prospective Studies , Hair Follicle/metabolism , Ectoderm/metabolism , Morphogenesis , Mammals
15.
Nature ; 626(7998): 357-366, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38052228

ABSTRACT

Recently, several studies using cultures of human embryos together with single-cell RNA-seq analyses have revealed differences between humans and mice, necessitating the study of human embryos1-8. Despite the importance of human embryology, ethical and legal restrictions have limited post-implantation-stage studies. Thus, recent efforts have focused on developing in vitro self-organizing models using human stem cells9-17. Here, we report genetic and non-genetic approaches to generate authentic hypoblast cells (naive hPSC-derived hypoblast-like cells (nHyCs))-known to give rise to one of the two extraembryonic tissues essential for embryonic development-from naive human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble with naive hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of additional naive hPSC-derived analogues of the second extraembryonic tissue, the trophectoderm, the efficiency of bilaminoid formation increases from 20% to 40%, and the epiblast within the bilaminoids continues to develop in response to trophectoderm-secreted IL-6. Furthermore, we show that bilaminoids robustly recapitulate the patterning of the anterior-posterior axis and the formation of cells reflecting the pregastrula stage, the emergence of which can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We have therefore successfully modelled and identified the mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis.


Subject(s)
Embryonic Development , Germ Layers , Pluripotent Stem Cells , Humans , Cell Differentiation , Embryo Implantation , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Embryonic Development/genetics , Embryonic Development/physiology , Germ Layers/cytology , Germ Layers/embryology , Germ Layers/metabolism , Pluripotent Stem Cells/cytology , Interleukin-6/metabolism , Gastrula/cytology , Gastrula/embryology , Amnion/cytology , Amnion/embryology , Amnion/metabolism , Ectoderm/cytology , Ectoderm/embryology , Ectoderm/metabolism , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Otx Transcription Factors/genetics , Otx Transcription Factors/metabolism
16.
Dev Biol ; 506: 20-30, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38052294

ABSTRACT

Cranial placodes are transient ectodermal thickenings that contribute to a diverse array of organs in the vertebrate head. They develop from a common territory, the pre-placodal region that over time segregates along the antero-posterior axis into individual placodal domains: the adenohypophyseal, olfactory, lens, trigeminal, otic, and epibranchial placodes. These placodes terminally differentiate into the anterior pituitary, the lens, and contribute to sensory organs including the olfactory epithelium, and inner ear, as well as several cranial ganglia. To study cranial placodes and their derivatives and generate cells for therapeutic purposes, several groups have turned to in vitro derivation of placodal cells from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs). In this review, we summarize the signaling cues and mechanisms involved in cranial placode induction, specification, and differentiation in vivo, and discuss how this knowledge has informed protocols to derive cranial placodes in vitro. We also discuss the benefits and limitations of these protocols, and the potential of in vitro cranial placode modeling in regenerative medicine to treat cranial placode-related pathologies.


Subject(s)
Ectoderm , Skull , Animals , Humans , Vertebrates , Cell Differentiation , Signal Transduction , Gene Expression Regulation, Developmental
17.
Dev Biol ; 506: 85-94, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38040078

ABSTRACT

The gill slits of fishes develop from an iterative series of pharyngeal endodermal pouches that contact and fuse with surface ectoderm on either side of the embryonic head. We find in the skate (Leucoraja erinacea) that all gill slits form via a stereotypical sequence of epithelial interactions: 1) endodermal pouches approach overlying surface ectoderm, with 2) focal degradation of ectodermal basement membranes preceding endoderm-ectoderm contact; 3) endodermal pouches contact and intercalate with overlying surface ectoderm, and finally 4) perforation of a gill slit occurs by epithelial remodelling, without programmed cell death, at the site of endoderm-ectoderm intercalation. Skate embryos express Fgf8 and Fgf3 within developing pharyngeal epithelia during gill slit formation. When we inhibit Fgf signalling by treating skate embryos with the Fgf receptor inhibitor SU5402 we find that endodermal pouch formation, basement membrane degradation and endodermal-ectodermal intercalation are unaffected, but that epithelial remodelling and gill slit perforation fail to occur. These findings point to a role for Fgf signalling in epithelial remodelling during gill slit formation in the skate and, more broadly, to an ancestral role for Fgf signalling during pharyngeal pouch epithelial morphogenesis in vertebrate embryos.


Subject(s)
Ectoderm , Gills , Animals , Endoderm , Vertebrates , Morphogenesis
18.
Genesis ; 62(1): e23553, 2024 Feb.
Article in English | MEDLINE | ID: mdl-37735882

ABSTRACT

The neural crest is a stem cell population that originates from the ectoderm during the initial steps of nervous system development. Neural crest cells delaminate from the neuroepithelium by undergoing a spatiotemporally regulated epithelial-mesenchymal transition (EMT) that proceeds in a coordinated wave head-to-tail to exit from the neural tube. While much is known about the transcriptional programs and membrane changes that promote EMT, there are additional levels of gene expression control that neural crest cells exert at the level of RNA to control EMT and migration. Yet, the role of post-transcriptional regulation, and how it drives and contributes to neural crest EMT, is not well understood. In this mini-review, we explore recent advances in our understanding of the role of post-transcriptional regulation during neural crest EMT.


Subject(s)
Epithelial-Mesenchymal Transition , Neural Crest , Neural Crest/metabolism , Epithelial-Mesenchymal Transition/genetics , Ectoderm , Neural Tube , Cell Movement/physiology , Gene Expression Regulation, Developmental
19.
Cell Prolif ; 57(4): e13577, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38041497

ABSTRACT

Cell fate determination in mammalian development is complex and precisely controlled and accumulating evidence indicates that epigenetic mechanisms are crucially involved. N4-acetylcytidine (ac4C) is a recently identified modification of messenger RNA (mRNA); however, its functions are still elusive in mammalian. Here, we show that N-acetyltransferase 10 (NAT10)-mediated ac4C modification promotes ectoderm differentiation of human embryonic stem cells (hESCs) by acetylating nuclear receptor subfamily 2 group F member 1 (NR2F1) mRNA to enhance translation efficiency (TE). Acetylated RNA immunoprecipitation sequencing (acRIP-seq) revealed that levels of ac4C modification were higher in ectodermal neuroepithelial progenitor (NEP) cells than in hESCs or mesoendoderm cells. In addition, integrated analysis of acRIP-seq and ribosome profiling sequencing revealed that NAT10 catalysed ac4C modification to improve TE in NEP cells. RIP-qRT-PCR analysis identified an interaction between NAT10 and NR2F1 mRNA in NEP cells and NR2F1 accelerated the nucleus-to-cytoplasm translocation of yes-associated protein 1, which contributed to ectodermal differentiation of hESCs. Collectively, these findings point out the novel regulatory role of ac4C modification in the early ectodermal differentiation of hESCs and will provide a new strategy for the treatment of neuroectodermal defects diseases.


Subject(s)
Human Embryonic Stem Cells , Animals , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ectoderm/metabolism , Cell Differentiation , Base Sequence , Mammals/metabolism , COUP Transcription Factor I/genetics , COUP Transcription Factor I/metabolism , N-Terminal Acetyltransferases/genetics , N-Terminal Acetyltransferases/metabolism
20.
Dev Biol ; 507: 20-33, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38154769

ABSTRACT

The neural tube, the embryonic precursor to the brain and spinal cord, begins as a flat sheet of epithelial cells, divided into non-neural and neural ectoderm. Proper neural tube closure requires that the edges of the neural ectoderm, the neural folds, to elevate upwards and fuse along the dorsal midline of the embryo. We have previously shown that members of the claudin protein family are required for the early phases of chick neural tube closure. Claudins are transmembrane proteins, localized in apical tight junctions within epithelial cells where they are essential for regulation of paracellular permeability, strongly involved in apical-basal polarity, cell-cell adhesion, and bridging the tight junction to cytoplasmic proteins. Here we explored the role of Claudin-3 (Cldn3), which is specifically expressed in the non-neural ectoderm. We discovered that depletion of Cldn3 causes folic acid-insensitive primarily spinal neural tube defects due to a failure in neural fold fusion. Apical cell surface morphology of Cldn3-depleted non-neural ectodermal cells exhibited increased membrane blebbing and smaller apical surfaces. Although apical-basal polarity was retained, we observed altered Par3 and Pals1 protein localization patterns within the apical domain of the non-neural ectodermal cells in Cldn3-depleted embryos. Furthermore, F-actin signal was reduced at apical junctions. Our data presents a model of spina bifida, and the role that Cldn3 is playing in regulating essential apical cell processes in the non-neural ectoderm required for neural fold fusion.


Subject(s)
Ectoderm , Neural Crest , Chick Embryo , Animals , Ectoderm/metabolism , Neural Crest/metabolism , Chickens/metabolism , Claudin-3/metabolism , Neural Tube , Claudins/genetics , Claudins/metabolism , Tight Junctions/metabolism
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