-
Cell-state transitions and collective cell movement generate an endoderm-like region in gastruloids.ELife Apr 2022Shaping the animal body plan is a complex process that involves the spatial organization and patterning of the different germ layers. Recent advances in live imaging...
Shaping the animal body plan is a complex process that involves the spatial organization and patterning of the different germ layers. Recent advances in live imaging have started to unravel the cellular choreography underlying this process in mammals, however, the sequence of events transforming an unpatterned cell ensemble into structured territories is largely unknown. Here, using gastruloids -3D aggregates of mouse embryonic stem cells- we study the formation of one of the three germ layers, the endoderm. We show that the endoderm is generated from an epiblast-like homogeneous state by a three-step mechanism: (i) a loss of E-cadherin mediated contacts in parts of the aggregate leading to the appearance of islands of E-cadherin expressing cells surrounded by cells devoid of E-cadherin, (ii) a separation of these two populations with islands of E-cadherin expressing cells flowing toward the aggregate tip, and (iii) their differentiation into an endoderm population. During the flow, the islands of E-cadherin expressing cells are surrounded by cells expressing T-Brachyury, reminiscent of the process occurring at the primitive streak. Consistent with recent in vivo observations, the endoderm formation in the gastruloids does not require an epithelial-to-mesenchymal transition, but rather a maintenance of an epithelial state for a subset of cells coupled with fragmentation of E-cadherin contacts in the vicinity, and a sorting process. Our data emphasize the role of signaling and tissue flows in the establishment of the body plan.
Topics: Animals; Cadherins; Cell Differentiation; Cell Movement; Endoderm; Gastrulation; Germ Layers; Mammals; Mice
PubMed: 35404233
DOI: 10.7554/eLife.59371 -
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 -
Nature Communications Feb 2022During development, pseudostratified epithelia undergo large scale morphogenetic events associated with increased mechanical stress. Using a variety of genetic and...
During development, pseudostratified epithelia undergo large scale morphogenetic events associated with increased mechanical stress. Using a variety of genetic and imaging approaches, we uncover that in the mouse E6.5 epiblast, where apical tension is highest, ASPP2 safeguards tissue integrity. It achieves this by preventing the most apical daughter cells from delaminating apically following division events. In this context, ASPP2 maintains the integrity and organisation of the filamentous actin cytoskeleton at apical junctions. ASPP2 is also essential during gastrulation in the primitive streak, in somites and in the head fold region, suggesting that it is required across a wide range of pseudostratified epithelia during morphogenetic events that are accompanied by intense tissue remodelling. Finally, our study also suggests that the interaction between ASPP2 and PP1 is essential to the tumour suppressor function of ASPP2, which may be particularly relevant in the context of tissues that are subject to increased mechanical stress.
Topics: Actin Cytoskeleton; Animals; Apoptosis Regulatory Proteins; Caco-2 Cells; Cell Polarity; Dogs; Embryo Culture Techniques; Embryo, Mammalian; Epithelium; Female; Gastrulation; Germ Layers; Humans; Madin Darby Canine Kidney Cells; Mice; Mice, Transgenic; Morphogenesis; Mutation; Primitive Streak; Receptors, Neuropeptide Y; Stress, Mechanical; Tight Junctions; Tumor Suppressor Proteins
PubMed: 35177595
DOI: 10.1038/s41467-022-28590-4 -
Development (Cambridge, England) Mar 2022Despite previous intensive investigations on epiblast cell migration in avian embryos during primitive streak development before stage (st.) 4, this migration at later...
Despite previous intensive investigations on epiblast cell migration in avian embryos during primitive streak development before stage (st.) 4, this migration at later stages of brain development has remained uninvestigated. By live imaging of epiblast cells sparsely labeled with green fluorescence protein, we investigated anterior epiblast cell migration to form individual brain portions. Anterior epiblast cells from a broad area migrated collectively towards the head axis during st. 5-7 at a rate of 70-110 µm/h, changing directions from diagonal to parallel and forming the brain portions and abutting head ectoderm. This analysis revised the previously published head portion precursor map in anterior epiblasts at st. 4/5. Grafting outside the brain precursor region of mCherry-expressing nodes producing anterior mesendoderm (AME) or isolated AME tissues elicited new cell migration towards ectopic AME tissues. These locally convergent cells developed into secondary brains with portions that depended on the ectopic AME position in the anterior epiblast. Thus, anterior epiblast cells are bipotent for brain/head ectoderm development with given brain portion specificities. A brain portion potential map is proposed, also accounting for previous observations.
Topics: Animals; Birds; Brain; Cell Movement; Ectoderm; Gastrula; Germ Layers
PubMed: 35132990
DOI: 10.1242/dev.199999 -
Proceedings of the National Academy of... Feb 2022In warm-blooded vertebrate embryos (mammals and birds), the axial tissues of the body form from a growth zone at the tail end, Hensen's node, which generates neural,...
In warm-blooded vertebrate embryos (mammals and birds), the axial tissues of the body form from a growth zone at the tail end, Hensen's node, which generates neural, mesodermal, and endodermal structures along the midline. While most cells only pass through this region, the node has been suggested to contain a small population of resident stem cells. However, it is unknown whether the rest of the node constitutes an instructive niche that specifies this self-renewal behavior. Here, we use heterotopic transplantation of groups and single cells and show that cells not destined to enter the node can become resident and self-renew. Long-term resident cells are restricted to the posterior part of the node and single-cell RNA-sequencing reveals that the majority of these resident cells preferentially express G2/M phase cell-cycle-related genes. These results provide strong evidence that the node functions as a niche to maintain self-renewal of axial progenitors.
Topics: Animals; Body Patterning; Chick Embryo; Endoderm; Gastrula; Mesoderm; Nervous System; Notochord; Organizers, Embryonic; Stem Cell Niche; Stem Cells
PubMed: 35101917
DOI: 10.1073/pnas.2108935119 -
Frontiers in Cell and Developmental... 2021MicroRNAs have been explored in different organisms and are involved as molecular switches modulating cellular specification and differentiation during the embryonic...
MicroRNAs have been explored in different organisms and are involved as molecular switches modulating cellular specification and differentiation during the embryonic development, including the cardiovascular system. In this study, we analyze the expression profiles of different microRNAs during early cardiac development. By using whole mount hybridization in developing chick embryos, with microRNA-specific LNA probes, we carried out a detailed study of miR-23b, miR-130a, miR-106a, and miR-100 expression during early stages of embryogenesis (HH3 to HH17). We also correlated those findings with putative microRNA target genes by means of mirWalk and TargetScan analyses. Our results demonstrate a dynamic expression pattern in cardiac precursor cells from the primitive streak to the cardiac looping stages for miR-23b, miR-130a, and miR-106a. Additionally, miR-100 is later detectable during cardiac looping stages (HH15-17). Interestingly, the /inflow tract was shown to be the most representative cardiac area for the convergent expression of the four microRNAs. Through analysis we revealed that distinct Hox family members are predicted to be targeted by the above microRNAs. We also identified expression of several Hox genes in the at stages HH11 and HH15. In addition, by means of gain-of-function experiments both in cardiomyoblasts and explants, we demonstrated the modulation of the different Hox clusters, Hoxa, Hoxb, Hoxc, and Hoxd genes, by these microRNAs. Furthermore, we correlated the negative modulation of several Hox genes, such as Hoxa3, Hoxa4, Hoxa5, Hoxc6, or Hoxd4. Finally, we demonstrated through a dual luciferase assay that Hoxa1 is targeted by miR-130a and Hoxa4 is targeted by both miR-23b and miR-106a, supporting a possible role of these microRNAs in Hox gene modulation during differentiation and compartmentalization of the posterior structures of the developing venous pole of the heart.
PubMed: 35087828
DOI: 10.3389/fcell.2021.767954 -
Stem Cell Reports Feb 2022The formation of the primitive streak (PS) and the subsequent induction of neuroectoderm are hallmarks of gastrulation. Combining an in vitro reconstitution of this...
The formation of the primitive streak (PS) and the subsequent induction of neuroectoderm are hallmarks of gastrulation. Combining an in vitro reconstitution of this process based on mouse embryonic stem cells (mESCs) with a collection of knockouts in reporter mESC lines, we identified retinoic acid (RA) as a critical mediator of early neural induction triggered by TGFβ or Wnt signaling inhibition. Single-cell RNA sequencing analysis captured the temporal unfolding of cell type diversification, up to the emergence of somite and neural fates. In the absence of the RA-synthesizing enzyme Aldh1a2, a sensitive RA reporter revealed a hitherto unidentified residual RA signaling that specified neural fate. Genetic evidence showed that the RA-degrading enzyme Cyp26a1 protected PS-like cells from neural induction, even in the absence of TGFβ and Wnt antagonists. Overall, we characterized a multi-layered control of RA levels that regulates early neural differentiation in an in vitro PS-like system.
Topics: Aldehyde Dehydrogenase 1 Family; Animals; Benzamides; Cell Differentiation; Dioxoles; Ectoderm; Mice; Mouse Embryonic Stem Cells; Neurons; Primitive Streak; Retinal Dehydrogenase; Retinoic Acid 4-Hydroxylase; Signal Transduction; Tretinoin
PubMed: 35063128
DOI: 10.1016/j.stemcr.2021.12.014 -
PloS One 2022DNA methylation (DNAme; 5-methylcytosine, 5mC) plays an essential role in mammalian development, and the 5mC profile is regulated by a balance of opposing enzymatic...
DNA methylation (DNAme; 5-methylcytosine, 5mC) plays an essential role in mammalian development, and the 5mC profile is regulated by a balance of opposing enzymatic activities: DNA methyltransferases (DNMTs) and Ten-eleven translocation dioxygenases (TETs). In mouse embryonic stem cells (ESCs), de novo DNAme by DNMT3 family enzymes, demethylation by the TET-mediated conversion of 5mC to 5-hydroxymethylation (5hmC), and maintenance of the remaining DNAme by DNMT1 are actively repeated throughout cell cycles, dynamically forming a constant 5mC profile. Nevertheless, the detailed mechanism and physiological significance of this active cyclic DNA modification in mouse ESCs remain unclear. Here by visualizing the localization of DNA modifications on metaphase chromosomes and comparing whole-genome methylation profiles before and after the mid-S phase in ESCs lacking Dnmt1 (1KO ESCs), we demonstrated that in 1KO ESCs, DNMT3-mediated remethylation was interrupted during and after DNA replication. This results in a marked asymmetry in the distribution of 5hmC between sister chromatids at mitosis, with one chromatid being almost no 5hmC. When introduced in 1KO ESCs, the catalytically inactive form of DNMT1 (DNMT1CI) induced an increase in DNAme in pericentric heterochromatin and the DNAme-independent repression of IAPEz, a retrotransposon family, in 1KO ESCs. However, DNMT1CI could not restore the ability of DNMT3 to methylate unmodified dsDNA de novo in S phase in 1KO ESCs. Furthermore, during in vitro differentiation into epiblasts, 1KO ESCs expressing DNMT1CI showed an even stronger tendency to differentiate into the primitive endoderm than 1KO ESCs and were readily reprogrammed into the primitive streak via an epiblast-like cell state, reconfirming the importance of DNMT1 enzymatic activity at the onset of epiblast differentiation. These results indicate a novel function of DNMT1, in which DNMT1 actively regulates the timing and genomic targets of de novo methylation by DNMT3 in an enzymatic activity-dependent and independent manner, respectively.
Topics: 5-Methylcytosine; Animals; Cell Differentiation; DNA (Cytosine-5-)-Methyltransferase 1; DNA Methylation; DNA Methyltransferase 3A; DNA Modification Methylases; DNA-Binding Proteins; Genomic Imprinting; Heterochromatin; Mice; Mice, Knockout; Mouse Embryonic Stem Cells; Retroelements
PubMed: 34986190
DOI: 10.1371/journal.pone.0262277 -
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 -
Journal of Microbiology & Biology... Dec 2021In classroom studies of mammalian embryology, students must fully grasp the cellular and tissue remodeling needed to initiate gastrulation to ensure comprehension of...
Utilizing Playdoh in the Classroom to Construct a 3D Model Depicting Cellular Movements and Tissue Remodeling during Human Gastrulation, Early Organogenesis, and Embryonic Folding.
In classroom studies of mammalian embryology, students must fully grasp the cellular and tissue remodeling needed to initiate gastrulation to ensure comprehension of forthcoming developmental processes such as tissue specification and organogenesis. However, quickly and completely communicating three-dimensional concepts such as gastrulation, neurulation, and embryonic folding through common two-dimensional tools such as PowerPoint is challenging for students because this method lacks the spatial orientation needed to fully understand development. Therefore, professors can utilize active learning approaches with 3D-modeling clay to aid students in visualizing developmental changes. 3D-modeling of the developmental processes focused on cell and tissue movements for the initiation of gastrulation and organogenesis is limited in published literature/videos. Therefore, this activity fills in the modeling gap by focusing on the detachment and movement of the epiblast cell through the primitive streak to generate the three germ layers, neural tube folding, cardiogenesis, and the anatomical position of the early brain and heart to drive embryonic folding. The usage of this hands-on learning tool will assist lecturers in preventing early gaps in knowledge while students first construct the model and allows for correction in misunderstandings by utilizing the complete model in discussions after construction.
PubMed: 34804320
DOI: 10.1128/jmbe.00123-21