scholarly journals Embryonic regeneration by relocalization of the Spemann organizer during twinning in Xenopus

2018 ◽  
Vol 115 (21) ◽  
pp. E4815-E4822 ◽  
Author(s):  
Yuki Moriyama ◽  
Edward M. De Robertis

The formation of identical twins from a single egg has fascinated developmental biologists for a very long time. Previous work had shown that Xenopus blastulae bisected along the dorsal–ventral (D-V) midline (i.e., the sagittal plane) could generate twins but at very low frequencies. Here, we have improved this method by using an eyelash knife and changing saline solutions, reaching frequencies of twinning of 50% or more. This allowed mechanistic analysis of the twinning process. We unexpectedly observed that the epidermis of the resulting twins was asymmetrically pigmented at the tailbud stage of regenerating tadpoles. This pigment was entirely of maternal (oocyte) origin. Bisecting the embryo generated a large wound, which closed from all directions within 60 minutes, bringing cells normally fated to become Spemann organizer in direct contact with predicted ventral-most cells. Lineage-tracing analyses at the four-cell stage showed that in regenerating embryos midline tissues originated from the dorsal half, while the epidermis was entirely of ventral origin. Labeling of D-V segments at the 16-cell stage showed that the more pigmented epidermis originated from the ventral-most cells, while the less-pigmented epidermis arose from the adjoining ventral segment. This suggested a displacement of the organizer by 90°. Studies with the marker Chordin and phospho-Smad1/5/8 showed that in half embryos a new D-V gradient is intercalated at the site of the missing half. The displacement of self-organizing morphogen gradients uncovered here may help us understand not only twin formation in amphibians, but also rare cases of polyembryony.

2015 ◽  
Vol 27 (1) ◽  
pp. 132
Author(s):  
L. P. Sepulveda-Rincon ◽  
D. Dube ◽  
P. Adenot ◽  
L. Laffont ◽  
S. Ruffini ◽  
...  

The first lineage specification occurs during pre-implantation mammalian development. At the blastocyst stage, 2 cell lineages can be distinguished: the inner cell mass (ICM) and the trophectoderm (TE). The exact timing when embryo cells are skewed to these lineages is not clearly determined in mammalian species. In murine embryos, it has been suggested that the first cleavage plane might be related to the embryonic-abembryonic (Em-Ab) axis at blastocyst stage. Thus, the daughter cells of the 2-cell embryo might already be predisposed to a specific cell lineage further on development. The objective of the present study was to observe how the first cleavage in bovine embryos may be related to cell lineage allocation at the blastocyst stage, using a noninvasive tracing approach. Bovine oocytes were harvested, in vitro matured, and fertilised. At the 2-cell stage, embryos were injected in one blastomere with the membrane tracer DiI. At the blastocyst stage, embryos (n = 346) were classified as orthogonal when the Em-Ab axis was orthogonally divided by the borderline between labelled and non-labelled cells; as deviant if the borderline was overlapping the Em-Ab axis; and as random when the labelled and non-labelled cells were randomly distributed. Total cell count (TCC) and the ICM/TE ratio was allowed by DNA staining with 4′,6-diamidino-2-phenylindole (DAPI) and by immunostaining of the ICM with Sox2 antibody. Analysis of variance was performed by one-way ANOVA employing IBM SPSS v21 (SPSS Inc., Chicago, IL, USA) to determine any difference between the cell lineage allocation patterns, TCC, and the ICM/TE ratio. P-values = 0.05 were considered significant. All values are reported as mean ± standard error of mean. Within 40 repetitions, the blastocyst classification was as follows: orthogonal 14.9% (±2.32, n = 56), deviant 22.2% (±2.58, n = 80), and random 62.9% (±2.64, n = 210). A significant difference was found in the incidence between the random group against the orthogonal and deviant, but not between the latter two. Regarding TCC, a significant difference was observed only between the orthogonal (99.6 ± 11.7 cells, n = 15) and deviant (135 ± 7.3 cells, n = 25) groups, but not with random embryos (116 ± 5.5 cells, n = 42). Finally, no significant difference was found among the groups concerning the ICM/TE ratio (0.43 ± 0.07 for orthogonal, n = 7; 0.54 ± 0.06 for deviant, n = 14; and 0.40 ± 0.03 for random embryos, n = 26). In conclusion, bovine embryos present a marked tendency for a random distribution of the daughter cells derived from the 2-cell blastomeres. However, around 37% of the blastocysts present a patterned cell division, where the daughter cells remain together through pre-implantation development. The effect of these cell lineage allocation patterns on implantation and further embryo development needs to be addressed.The authors acknowledge Laboratoire d'Excellence Revive (Investissement d'Avenir, ANR-10-LABX-73) and CONACyT Mexico for funding.


2008 ◽  
Vol 20 (1) ◽  
pp. 133
Author(s):  
M. Katayama ◽  
R. M. Roberts

Fertile adults and occasionally twins have been derived from murine blastomeres at the 2-cell stage, indicating that such blastomeres may be equivalently totipotent, but there are conflicting reports that individual blastomeres from 2-cell stage murine conceptuses make different contributions to the embryonic and abembryonic regions of the blastocyst, implying that they differ in developmental potential. Here, we have re-examined this subject using 2 mouse strains, CF1 and NIH Swiss (SW), and 2 experimental approaches, random blastomere destruction at the 2-cell stage by repeated insertion of a needle into its nucleus and lineage tracing with the dye, DiI-CM. The manipulated conceptuses and untreated controls were cultured in KSOM-AA to morula and blastocyst stages (84 or 108 h pc, respectively), fixed, and immunostained for Oct4 and Cdx2. Antigen distribution, number of nuclei (stained by 42,6-diamidino-2-phenylindole), and cell progeny labeled with DiI-CM were examined by confocal laser scanning microscopy. Cell numbers are means � SD and were analyzed by a Student t-test. Cells positive for Cdx2 were assumed to represent trophectoderm or trophectoderm precursors, ones positive for Oct4 but negative for Cdx2 (Oct+Cdx–) inner cell mass. Ablation of a blastomere failed to prevent developmental progression in either strain, but the total number of cells at both morula (SW 11.4 � 3.3 v. 19.2 � 7.1; CF1 10.1 � 2.5 v. 22.1 � 6.4) and blastocyst (SW 48.6 � 7.4 v. 69.4 � 9.9; CF1 24.8 � 6.2 v. 53.8 � 13.5) was significantly reduced. In SW, the average fraction of Oct+Cdx– cells after blastomere ablation was significantly lower (P < 0.05) than in controls in morulae (0.47 � 0.2 v. 0.65 � 0.1) but not in blastocysts (0.33 � 0.1 and 0.34 � 0.1). In CF1, the fraction of Oct+Cdx– cells was lower (P < 0.05) than controls in both morulae and blastocysts (0.31 � 0.2 v. 0.58 � 0.2 and 0.18 � 0.1 v. 0.27 � 0.04, respectively). The CF1 morulae fell mainly into 2 groups, one low fraction (≤0.3, 54%) of Oct+Cdx– cells and the other with a more normal fraction (0.3 to 0.8, 43%) relative to controls. A majority of NIH Swiss morulae had an Oct+Cdx– cell fraction >0.4 and in this respect resembled controls. We then examined these strain differences by lineage tracing. The majority of SW blastocysts (65%, n = 34) demonstrated a random localization of DiI-labeled cell progeny (i.e., there was no preferential distribution of labeled cells to either the embryonic or abembryonic poles). By contrast, in CF1 (n = 38), 32% of blastocysts had labeled cells confined to their embryonic end and 42% with DiI-labeled, Cdx2-positive cells clustered at the abembryonic locale. A random localization was observed in 26% of blastocysts. In conclusion, these data confirm that there is plasticity in early mouse development but also suggest that in CF1, but not in SW conceptuses, blastomeres at the 2-cell stage differ in their abilities to contribute to the embryonic pole. Similar strain differences may explain the disagreements among studies on lineage tracing in early cleavage stage conceptuses.


2005 ◽  
Vol 93 (5) ◽  
pp. 2940-2949 ◽  
Author(s):  
Colin G. Evans ◽  
Adarli Romero ◽  
Elizabeth C. Cropper

We are studying afferent transmission from a mechanoafferent, B21, to a follower, B8. During motor programs, afferent transmission is regulated so that it does not always occur. Afferent transmission is eliminated when spike propagation in B21 fails, i.e., when spike initiation is inhibited in one output region-B21's lateral process. Spike initiation in the lateral process is inhibited by the B52 and B4/5 cells. Individual B52 and B4/5-induced inhibitory postsynaptic potentials (IPSPs) in B21 differ. For example, the peak amplitude of a B4/5-induced IPSP is four times the amplitude of a B52 IPSP. Nevertheless, when interneurons fire in bursts at physiological (i.e., low) frequencies, afferent transmission is most effectively reduced by B52. Although individual B52-induced IPSPs are small, they have a long time constant and summate at low firing frequencies. Once IPSPs summate, they effectively block afferent transmission. In contrast, individual B4/5-induced IPSPs have a relatively short time constant and do not summate at low frequencies. B52 and B4/5 therefore differ in that once synaptic input from B52 becomes effective, afferent transmission is continuously inhibited. In contrast, periods of B4/5-induced inhibition are interspersed with relatively long intervals in which inhibition does not occur. Consequently, the probability that afferent transmission will be inhibited is low. In conclusion, it is widely recognized that afferent transmission can be regulated by synaptic input. Our experiments are, however, unusual in that they relate specific characteristics of postsynaptic potentials to functional inhibition. In particular we demonstrate the potential importance of the IPSP time constant.


2019 ◽  
Vol 23 ◽  
pp. 233121651987139 ◽  
Author(s):  
Melissa J. Polonenko ◽  
Ross K. Maddox

The frequency-specific tone-evoked auditory brainstem response (ABR) is an indispensable tool in both the audiology clinic and research laboratory. Most frequently, the toneburst ABR is used to estimate hearing thresholds in infants, toddlers, and other patients for whom behavioral testing is not feasible. Therefore, results of the ABR exam form the basis for decisions regarding interventions and hearing habilitation with implications extending far into the child’s future. Currently, responses are elicited by periodic sequences of toneburst stimuli presented serially to one ear at a time, which take a long time to measure multiple frequencies and intensities, and provide incomplete information if the infant wakes up early. Here, we describe a new method, the parallel ABR (pABR), which uses randomly timed toneburst stimuli to simultaneously acquire ABR waveforms to five frequencies in both ears. Here, we describe the pABR and quantify its effectiveness in addressing the greatest drawback of current methods: test duration. We show that in adults with normal hearing the pABR yields high-quality waveforms over a range of intensities, with similar morphology to the standard ABR in a fraction of the recording time. Furthermore, longer latencies and smaller amplitudes for low frequencies at a high intensity evoked by the pABR versus serial ABR suggest that responses may have better place specificity due to the masking provided by the other simultaneous toneburst sequences. Thus, the pABR has substantial potential for facilitating faster accumulation of more diagnostic information that is important for timely identification and treatment of hearing loss.


2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Xin Yu ◽  
Ying Wang ◽  
Yilin Luo ◽  
Long Yin

Limited studies in recent years have shown that asphalt pavement subject to seawater in coastal regions or deicing salt in cold regions may be seriously damaged after being soaked in saline water for a long time. However, there is limited research into the influence of salt on rheological properties of asphalt after long-term aging. In this study, rheological properties of unmodified and polymer-modified asphalt after long-term aging were tested after being soaked in different concentrations of salt (0.3%~5%) for different durations (1 day~30 days). Orthogonal array based on the Taguchi method was used for experimental design. The frequency sweep tests were performed on the specimens of aged asphalt after being soaked for complex modulus and phase angle master curves and ultimate fatigue temperature. BBR tests were performed for stiffness. The test results indicate that saline water appears to reduce low temperature properties and fatigue resistance properties and improved high temperature properties of aged asphalt, and it also affects the sensitivity of complex modulus and phase angles at low frequencies.


Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-32
Author(s):  
Sara A. Rubin ◽  
Chloé S. Baron ◽  
Song P. Yang ◽  
Aaron McKenna ◽  
Leonard I. Zon

Developmental hematopoietic stem and progenitor cells (HSPCs) have been thought to be largely replaced by adult hematopoietic stem cells (HSCs) after birth. Here, we performed simultaneous genetic lineage tracing by cellular barcoding and transcriptional profiling of zebrafish hematopoiesis to investigate HSPC clonal dynamics and gain insight into the developmental origin of adult blood cells. We took advantage of a recently developed dynamic lineage tracing system, scGESTALT, a CRISPR-based approach that allows for inducible cellular barcoding at two different time points during zebrafish embryonic development. We induced the first stage of lineage recording in the early embryo via microinjection at the one-cell stage and selected 28 hours post-fertilization, just before the birth of definitive blood stem cells, for additional recording activity. In this way, we were able to barcode HSCs, which will pass their unique barcodes on to their progeny, thereby enabling lineage tracing. To study the clonal dynamics and developmental origins of adult blood cells, we dissected paired adult kidney marrow and thymi tissues from barcoded fish between 3 and 7 months of age. We then performed single-cell RNA sequencing (scRNA-seq) in combination with lineage tracing by specifically amplifying the GESTALT barcode and sequencing both transcriptional and GESTALT barcode libraries. In total, we recovered transcriptional profiles from 71,109 cells across 9 fish. By optimizing promoter choice, we improved barcode recovery by almost 3-fold (17.7% vs. 6.5%) for detection in cells from the zebrafish kidney marrow. In two of these more efficient lineage recording fish, we detected 61 and 72 unique HSPC clones that contributed to adult hematopoiesis. The majority of these HSPC clones did not exhibit significant lineage biases; however, we demonstrated that 6/61 and 4/72 clones from the two fish, respectively, were significantly enriched in the myeloid lineage and 8/61 and 8/72 clones were significantly enriched in the lymphoid lineage (p&lt; 0.05). These findings demonstrate that myeloid and lymphoid biased clones arise during normal development. From our paired thymus and kidney marrow sample, we identified 51 unique kidney marrow clones, 10 shared, and 2 unique thymus clones. This latter finding of unique thymus clones was surprising and suggests that long-lived embryonic T cell progenitors persist and contribute to adult T cell production in the zebrafish. Taken together, we have demonstrated how scGESTALT can uncover complex lineage relationships in blood, mapping the origins and contributions of HSCs and embryonic progenitors to the adult hematopoietic system. Disclosures Zon: Amagma Therapeutics: Current equity holder in private company, Other: Founder; Celularity: Consultancy; Cellarity: Consultancy; CAMP4 Therapeutics: Current equity holder in private company, Other: Founder; Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder.


Development ◽  
2000 ◽  
Vol 127 (16) ◽  
pp. 3467-3474 ◽  
Author(s):  
M.A. Ciemerych ◽  
D. Mesnard ◽  
M. Zernicka-Goetz

Recent studies suggest early (preimplantation) events might be important in the development of polarity in mammalian embryos. We report here lineage tracing experiments with green fluorescent protein showing that cells located either near to or opposite the polar body at the 8-cell stage of the mouse embryo retain their same relative positions in the blastocyst. Thus they come to lie on either end of an axis of symmetry of the blastocyst that has recently been shown to correlate with the anterior-posterior axis of the postimplantation embryo (see R. J. Weber, R. A. Pedersen, F. Wianny, M. J. Evans and M. Zernicka-Goetz (1999). Development 126, 5591–5598). The embryonic axes of the mouse can therefore be related to the position of the polar body at the 8-cell stage, and by implication, to the animal-vegetal axis of the zygote. However, we also show that chimeric embryos constructed from 2-cell stage blastomeres from which the animal or the vegetal poles have been removed can develop into normal blastocysts and become fertile adult mice. This is also true of chimeras composed of animal or vegetal pole cells derived through normal cleavage to the 8-cell stage. We discuss that although polarity of the postimplantation embryo can be traced back to the 8-cell stage and in turn to the organisation of the egg, it is not absolutely fixed by this time.


Development ◽  
1995 ◽  
Vol 121 (12) ◽  
pp. 3979-3988 ◽  
Author(s):  
K. Itoh ◽  
T.L. Tang ◽  
B.G. Neel ◽  
S.Y. Sokol

Shaggy is a downstream component of the wingless and Notch signaling pathways which operate during Drosophila development. To address the role of glycogen synthase kinase 3 beta (GSK3 beta), a mammalian homologue of Shaggy, in vertebrate embryogenesis, it was overexpressed in Xenopus embryos. Microinjection of rat GSK3 beta mRNA into animal ventral blastomeres of 8-cell-stage embryos triggered development of ectopic cement glands with an adjacent anterior neural tissue as evidenced by in situ hybridization with Xotx2, a fore/midbrain marker, and NCAM, a pan-neural marker. In contrast, animal dorsal injection of the same dose of GSK3 beta mRNA caused eye deficiencies, whereas vegetal injections had no pronounced effects on normal development. Using several mutated forms of rat GSK3 beta, we demonstrate that the observed phenotypes are dose-dependent and tightly correlate with GSK3 beta enzymatic activity. Lineage tracing experiments showed that the effects of GSK3 beta are cell autonomous and that ectopic cement glands and eye deficiencies arose directly from cells containing GSK3 beta mRNA. Molecular marker analysis of ectodermal explants overexpressing GSK3 beta has revealed activation of Xotx2 and of cement gland marker XAG-1, but expression of NCAM and XIF-3 was not detected. Phenotypic effects of mRNA encoding a Xenopus homologue of GSK3 beta were identical to those of rat GSK3 beta mRNA. We hypothesize that GSK3 beta mediates the initial steps of neural tissue specification and modulates anteroposterior ectodermal patterning via activation of Otx2 transcription. Our observations implicate GSK3 beta in signaling pathways operating during neural tissue development and during specification of anterior ectodermal cell fates.


Development ◽  
1987 ◽  
Vol 101 (3) ◽  
pp. 565-582 ◽  
Author(s):  
J.C. Chisholm ◽  
E. Houliston

The timing, spatial distribution and control of cytokeratin assembly during mouse early development has been studied using a monoclonal antibody, TROMA-1, which recognizes a 55,000 Mr trophectodermal cytokeratin (ENDO A). This protein was first detected in immunoblots at the 4-cell stage, and became more abundant at the 16-cell stage and later. Immunofluorescence analysis revealed assembled cytokeratin filaments in some 8-cell blastomeres, but not at earlier stages. At the 16-cell stage, filaments were found in both polarized (presumptive trophectoderm; TE) and apolar (presumptive inner cell mass; ICM) cells in similar proportions, although polarized cells possessed more filaments than apolar cells. By the late 32-cell, early blastocyst, stage, all polarized (TE) cells contained extensive filament networks whereas cells positioned inside the embryo tended to have lost their filaments. The presence of filaments in inside cells at the 16-cell stage and in ICM cells was confirmed by immunoelectron microscopy. Lineage tracing techniques demonstrated that those cells in the ICM of early blastocysts which did possess filaments were almost exclusively the progeny of polar 16-cell blastomeres, suggesting that these filaments were directly inherited from outside cells at the 16- to 32-cell transition. Inhibitor studies revealed that proximate protein synthesis but not mRNA synthesis is required for filament assembly at the 8-cell stage. These results demonstrate that there are quantitative rather than qualitative differences in the expression of cytokeratin filaments in the inner cell mass and trophectoderm cells of the mouse embryo.


1877 ◽  
Vol 25 (171-178) ◽  
pp. 124-131

In making experiments on the sensitiveness of supersaturated solutions to air and greasy surfaces, I was much annoyed by the solutions so frequently crystallizing on the removal of the cotton-wool, as this necessitated boiling the flask again and waiting till it was cool. I noticed that frequently part of the cotton-wool adhered to the mouth of the flask; and it struck me that in removing this some fibres must get detached and fall in, carrying with them in all probability crystals of the salt. I soon convinced myself that this was the case, and that cottonwool is perhaps the worst material that could be chosen for covering these solutions. I now always use paper or tinfoil; and I find that these can be removed many times from the same solution without inducing crystallization. I then found that even the most sensitive solutions could be taken up in a clean glass tube and dropped on a clean glass plate without crystallizing; and that they will remain liquid exposed to the air for a very long time, often, in fact, till they dry up by evaporation in modified forms. Twenty drops on a plate give twenty experiments on the effect of air, clean and unclean surfaces, and evaporation; then the plate is cleaned, and more drops are taken from the original solution till this is used up. The trouble of boiling is thus reduced to a minimum, and the drops can be put upon all kinds of surfaces to test their activity. The slow growth of the modified salts can be watched for hours; and their forms are sometimes peculiar. Thus sulphate of soda often gives a single, square, flat pyramid, or a broad well-shaped prism, or occasionally small oetahedra round the edge of the drop. The pyramids and prisms change to opaque white when touched, and are apparently the 7-atom salt; the octahedra do not change, and are evidently the anhydrous salt. This fact is interesting, from its supporting the view that it is the anhydrous salt which is in solution. Or, again, a plate with drops may be dried over calcium chloride; and this sometimes modifies the results, as in the case of ammonia alum. This salt, when allowed to evaporate in air, generally forms a shining semitransparent film of greenish colour with a depression at the top, in which is often a circular opening, while inside small globular concretions of a dull, opaque, milky white colour are formed; these will remain moist inside for a couple of days or more. When touched with the normal salt, the whole drop becomes brilliant opaque white, quite dry, and apparently increases in volume, as the crust often breaks up and curls outwards.


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