ResearchPad - Embryology https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Detection of microbial cell-free DNA in maternal and umbilical cord plasma in patients with chorioamnionitis using next generation sequencing]]> https://www.researchpad.co/product?articleinfo=N85cfbb28-a074-423a-88cd-d5e05af52830

Background

Chorioamnionitis has been linked to spontaneous preterm labor and complications such as neonatal sepsis. We hypothesized that microbial cell-free (cf) DNA would be detectable in maternal plasma in patients with chorioamnionitis and could be the basis for a non-invasive method to detect fetal exposure to microorganisms.

Objective

The purpose of this study was to determine whether next generation sequencing could detect microbial cfDNA in maternal plasma in patients with chorioamnionitis.

Study design

Maternal plasma (n = 94) and umbilical cord plasma (n = 120) were collected during delivery at gestational age 28–41 weeks. cfDNA was extracted and sequenced. Umbilical cord plasma samples with evidence of contamination were excluded. The prevalence of microorganisms previously implicated in choriomanionitis, neonatal sepsis and intra-amniotic infections, as described in the literature, were examined to determine if there was enrichment of these microorganisms in this cohort. Specific microbial cfDNA associated with chorioamnionitis was first detected in umbilical cord plasma and confirmed in the matched maternal plasma samples (n = 77 matched pairs) among 14 cases of histologically confirmed chorioamnionitis and one case of clinical chorioamnionitis; 63 paired samples were used as controls. A correlation of rank of a given microorganism across maternal plasma and matched umbilical cord plasma was used to assess whether signals found in umbilical cord plasma were also present in maternal plasma.

Results

Microbial DNA sequences associated with clinical and/or histological chorioamnionitis were enriched in maternal plasma in cases with suspected chorioamnionitis when compared to controls (12/14 microorganisms, p = 0.02). Analysis of the microbial cfDNA in umbilical cord plasma among the 1,251 microorganisms detectable with this assay identified Streptococcus mitis, Ureaplasma spp., and Mycoplasma spp. in cases of suspected chorioamnionitis. This assay also detected cfDNA from Lactobacillus spp. in controls. Comparison between maternal plasma and umbilical cord plasma confirmed these signatures were also present in maternal plasma. Unbiased analysis of microorganisms with significantly correlated signal between matched maternal plasma and umbilical cord plasma identified the above listed 3 microorganisms, all of which have previously been implicated in patients with chorioamnionitis (Mycoplasma hominis p = 0.0001; Ureaplasma parvum p = 0.002; Streptococcus mitis p = 0.007). These data show that the pathogen signal relevant for chorioamnionitis can be identified in both maternal and umbilical cord plasma.

Conclusion

This is the first report showing the detection of relevant microbial cell-free cfDNA in maternal plasma and umbilical cord plasma in patients with clinical and/or histological chorioamnionitis. These results may lead to the development of a specific assay to detect perinatal infections for targeted therapy to reduce early neonatal sepsis complications.

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<![CDATA[The SARS-CoV-2 receptor ACE2 expression of maternal-fetal interface and fetal organs by single-cell transcriptome study]]> https://www.researchpad.co/product?articleinfo=N6fc22072-15f3-4c64-b34f-34474f5cf931

The new type of pneumonia caused by the SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) has been declared as a global public health concern by WHO. As of April 3, 2020, more than 1,000,000 human infections have been diagnosed around the world, which exhibited apparent person-to-person transmission characteristics of this virus. The capacity of vertical transmission in SARS-CoV-2 remains controversial recently. Angiotensin-converting enzyme 2 (ACE2) is now confirmed as the receptor of SARS-CoV-2 and plays essential roles in human infection and transmission. In present study, we collected the online available single-cell RNA sequencing (scRNA-seq) data to evaluate the cell specific expression of ACE2 in maternal-fetal interface as well as in multiple fetal organs. Our results revealed that ACE2 was highly expressed in maternal-fetal interface cells including stromal cells and perivascular cells of decidua, and cytotrophoblast and syncytiotrophoblast in placenta. Meanwhile, ACE2 was also expressed in specific cell types of human fetal heart, liver and lung, but not in kidney. And in a study containing series fetal and post-natal mouse lung, we observed ACE2 was dynamically changed over the time, and ACE2 was extremely high in neonatal mice at post-natal day 1~3. In summary, this study revealed that the SARS-CoV-2 receptor was widely spread in specific cell types of maternal-fetal interface and fetal organs. And thus, both the vertical transmission and the placenta dysfunction/abortion caused by SARS-CoV-2 need to be further carefully investigated in clinical practice.

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<![CDATA[Comparison of DNA methylation patterns of parentally imprinted genes in placenta derived from IVF conceptions in two different culture media]]> https://www.researchpad.co/product?articleinfo=N3fcc8b8e-fc50-4b6e-acc9-d902940fabe1

Abstract

Study question

Is there a difference in DNA methylation status of imprinted genes in placentas derived from IVF conceptions where embryo culture was performed in human tubal fluid (HTF) versus G5 culture medium?

Summary answer

We found no statistically significant differences in the mean DNA methylation status of differentially methylated regions (DMRs) associated with parentally imprinted genes in placentas derived from IVF conceptions cultured in HTF versus G5 culture medium.

What is known already

Animal studies indicate that the embryo culture environment affects the DNA methylation status of the embryo. In humans, birthweight is known to be affected by the type of embryo culture medium used. The effect of embryo culture media on pregnancy, birth and child development may thus be mediated by differential methylation of parentally imprinted genes in the placenta.

Study design, size, duration

To identify differential DNA methylation of imprinted genes in human placenta derived from IVF conceptions exposed to HTF or G5 embryo culture medium, placenta samples (n = 43 for HTF, n = 54 for G5) were collected between 2010 and 2012 s as part of a multi-center randomized controlled trial in the Netherlands comparing these embryo culture media. Placenta samples from 69 naturally conceived (NC) live births were collected during 2008–2013 in the Netherlands as reference material.

Participants/materials, setting, methods

To identify differential DNA methylation of imprinted genes, we opted for an amplicon-based sequencing strategy on an Illumina MiSeq sequencing platform. DNA was isolated and 34 DMRs associated with well-defined parentally imprinted genes were amplified in a two-step PCR before sequencing using MiSeq technology. Sequencing data were analyzed in a multivariate fashion to eliminate possible confounding effects.

Main results and the role of chance

We found no statistically significant differences in the mean DNA methylation status of any of the imprinted DMRs in placentas derived from IVF conceptions cultured in HTF or G5 culture medium. We also did not observe any differences in the mean methylation status per amplicon nor in the variance in methylation per amplicon between the two culture medium

groups. A separate surrogate variable analysis also demonstrated that the IVF culture medium was not associated with the DNA methylation status of these DMRs. The mean methylation level and variance per CpG was equal between HTF and G5 placenta. Additional comparison of DNA methylation status of NC placenta samples revealed no statistically significant differences in mean amplicon and CpG methylation between G5, HTF and NC placenta; however, the number of placenta samples exhibiting outlier methylation levels was higher in IVF placenta compared to NC (P < 0.00001). Also, we were able to identify 37 CpG sites that uniquely displayed outlier methylation in G5 placentas and 32 CpG sites that uniquely displayed outlier methylation in HTF. In 8/37 (G5) and 4/32 (HTF) unique outliers CpGs, a medium-specific unique outlier could be directly correlated to outlier methylation of the entire amplicon.

Limitations, reasons for caution

Due to practical reasons, not all placentas were collected during the trial, and we collected the placentas from natural conceptions from a different cohort, potentially creating bias. We limited ourselves to the DNA methylation status of 34 imprinted DMRs, and we studied only the placenta and no other embryo-derived tissues.

Wider implications of the findings

It has often been postulated, but has yet to be rigorously tested, that imprinting mediates the effects of embryo culture conditions on pregnancy, birth and child development in humans. Since we did not detect any statistically significant effects of embryo culture conditions on methylation status of imprinted genes in the placenta, this suggests that other unexplored mechanisms may underlie these effects. The biological and clinical relevance of detected outliers with respect to methylation levels of CpGs and DMR require additional analysis in a larger sample size as well. Given the importance and the growing number of children born through IVF, research into these molecular mechanisms is urgently needed.

Study funding/competing interest(s)

This study was funded by the March of Dimes grant number #6-FY13-153. The authors have no conflicts of interest.

Trial registration number

Placental biopsies were obtained under Netherlands Trial Registry number 1979 and 1298.

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<![CDATA[The Effect of Melatonin on Mitochondrial Function and Autophagy in In Vitro Matured Oocytes of Aged Mice]]> https://www.researchpad.co/product?articleinfo=Nc76a6769-14d9-4ac6-a9fc-93fae19256cd

Objective

This study examined the in vitro effect of melatonin on the protein synthesis of mitochondria, as well as autophagy in matured oocytes of aged mice.

Materials and Methods

In this experimental study, germinal vesicles (GV) oocytes were collected from aged (with the age of six-months-old) and young mice (with age range of 6-8 weeks old) and then cultured in the in vitro culture medium (IVM) for 24 hours to each metaphase II (MII) oocytes and then supplemented with melatonin at a concentration of 10 μM. The culture medium of MII oocytes was devoid of melatonin. Afterward, the expression of the SIRT-1 and LC3 was assessed by immunocytochemistry. ATP-dependent luciferin-luciferase bioluminescence assay was employed for the measurement of the ATP contents. Intracellular reactive oxygen specious (ROS) was detected by DCFH-DA, and the total antioxidant capacity (TAC) level was determined by TAC assay.

Results

The expression of SIRT-1 and LC3, as well as the measurement of the ATP content, was significantly increased in oocytes treated with melatonin compared with the oocytes receiving no treatment. Moreover, TAC was considerably higher in melatonin-treated oocytes than oocytes receiving no treatment. On the other hand, the level of ROS was significantly decreased in oocytes treated with melatonin in comparison with the untreated oocytes. The results indicated that melatonin considerably improved the development of oocytes as well.

Conclusion

According to the data, melatonin increased mitochondrial function and autophagy via an increase in the expression of SIRT1 and LC3, as well as the ATP contents while it decreased the levels of ROS and increased TAC in oocytes derived from aged mice.

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<![CDATA[The human factor: does the operator performing the embryo transfer significantly impact the cycle outcome?]]> https://www.researchpad.co/product?articleinfo=Nab4fbc43-bcb0-41e1-a8c0-f708b1263c19

Abstract

STUDY QUESTION

Is Ongoing Pregnancy Rate (OPR) operator-dependent, and can experience improve embryo transfer efficiency?

SUMMARY ANSWER

OPR is influenced by the operators who perform the embryo transfer (ET), and experience does not assure proficiency for everyone.

WHAT IS KNOWN ALREADY

ET remains the critical step in assisted reproduction. Although many other factors such as embryo quality and uterine receptivity impact embryo implantation, the proper ET technique is clearly an operator-dependent variable and as such it should be objectively standardized.

STUDY DESIGN, SIZE, DURATION

Retrospective comparative analysis including all fresh ETs performed between January 1996 and December 2016 at the Humanitas Fertility Center after IVF—ICSI cycles.

PARTICIPANTS/MATERIALS, SETTING, METHODS

IVF/ICSI fresh ETs performed by 32 operators, 19 824 cycles in all, were analyzed. All transfers consisting of freehand insertion of a preloaded soft catheter into the uterine cavity under transabdominal ultrasound guidance were considered. Two different statistical analyses were performed. First, a logistic regression model with a random intercept for the operator was used to estimate the heterogeneity of the rate of success among operators, accounting for woman age, FSH, number of oocytes retrieved, fertilization rate, year of the procedure, number and stage of transferred embryos and operator’s experience. Second, the relationship between experience and pregnancy rate was estimated separately for each operator by logistic regression, and operator-specific results were combined and compared in a random-effects meta-analysis. In both analyses, the operator’s experience at time t was measured in terms of number of embryo transfers performed before t.

MAIN RESULTS AND THE ROLE OF CHANCE

The heterogeneity among operators was highly significant (P value <0.001) and explained 44.5% of the total variability. The odds ratio of success of the worst operator in respect to the mean was equal to 0.84. For the best operator, the odds ratio of success was equal to 1.13 in respect to the mean. Based on the meta-analysis of the relationship between operator’s experience and success rate, it resulted that, on average, the operators’ performance did not improve with additional transfers.

LIMITATIONS, REASONS FOR CAUTION

At our center, operators become independent for ET’s after performing between 30 and 50 transfers under supervision. It is also possible that other relevant factors, such as embryologists on duty for the ET, have not been included in the present analysis and this may represent a potential bias. Among these, it should be mentioned that the embryologists on duty for the ET were not taken into consideration.

WIDER IMPLICATIONS OF THE FINDINGS

Continued performance analysis and the use of a digital simulator could help operators to test their expertise over time and either correct poor performance or avoid doing transfers.

STUDY FUNDING/COMPETING INTEREST(S)

None.

TRIAL REGISTRATION NUMBER

NCT03561129.

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<![CDATA[Mutations on ent-kaurene oxidase 1 encoding gene attenuate its enzyme activity of catalyzing the reaction from ent-kaurene to ent-kaurenoic acid and lead to delayed germination in rice]]> https://www.researchpad.co/product?articleinfo=N3fbe67d9-408b-4f07-a6d8-c659dfb628bd

Rice seed germination is a critical step that determines its entire life circle, with seeds failing to germinate or pre-harvest sprouting both reduce grain yield. Nevertheless, the mechanisms underlying this complex biological event remain unclear. Previously, gibberellin has been shown to promote seed germination. In this study, a delayed seed germination rice mutant was obtained through screening of the EMS induced mutants. Besides of delayed germination, it also shows semi-dwarfism phenotype, which could be recovered by exogenous GA. Through re-sequencing on the mutant, wild-type and their F2 populations, we identified two continuous mutated sites on ent-kaurene oxidase 1 (OsKO1) gene, which result in the conversion from Thr to Met in the cytochrome P450 domain. Genetic complementary analysis and enzyme assay verified that the mutations in OsKO1 gene block the biosynthesis of GA and result in the defect phenotypes. Further analyses proved that OsKO1 could catalyze the reaction from ent-kaurene into ent-kaurenoic acid in GA biosynthesis mainly at seed germination and seedling stages, and the mutations decrease its activity to catalyze the step from ent-kaurenol to ent-kaurenoic acid in this reaction. Transcriptomic and proteomic data indicate that the defect on GA biosynthesis decreases its ability to mobilize starch and attenuate ABA signaling, therefore delay the germination process. The results provide some new insights into both GA biosynthesis and seed germination regulatory pathway in rice.

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<![CDATA[Enhanced in vivo-imaging in medaka by optimized anaesthesia, fluorescent protein selection and removal of pigmentation]]> https://www.researchpad.co/product?articleinfo=5c9902e5d5eed0c484b98849

Fish are ideally suited for in vivo-imaging due to their transparency at early stages combined with a large genetic toolbox. Key challenges to further advance imaging are fluorophore selection, immobilization of the specimen and approaches to eliminate pigmentation. We addressed all three and identified the fluorophores and anaesthesia of choice by high throughput time-lapse imaging. Our results indicate that eGFP and mCherry are the best conservative choices for in vivo-fluorescence experiments, when availability of well-established antibodies and nanobodies matters. Still, mVenusNB and mGFPmut2 delivered highest absolute fluorescence intensities in vivo. Immobilization is of key importance during extended in vivo imaging. Here, traditional approaches are outperformed by mRNA injection of α-Bungarotoxin which allows a complete and reversible, transient immobilization. In combination with fully transparent juvenile and adult fish established by the targeted inactivation of both, oca2 and pnp4a via CRISPR/Cas9-mediated gene editing in medaka we could dramatically improve the state-of-the art imaging conditions in post-embryonic fish, now enabling light-sheet microscopy of the growing retina, brain, gills and inner organs in the absence of side effects caused by anaesthetic drugs or pigmentation.

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<![CDATA[Molecular and genetic characterization of partial masculinization in embryonic ovaries grafted into male nude mice]]> https://www.researchpad.co/product?articleinfo=5c8977afd5eed0c4847d330e

In most of mammalian embryos, gonadal sex differentiation occurs inside the maternal uterus before birth. In several fetal ovarian grafting experiments using male host mice, an experimental switch from the maternal intrauterine to male-host environment gradually induces partial masculinization of the grafted ovaries even under the wild-type genotype. However, either host-derived factors causing or molecular basis underlying this masculinization of the fetal ovaries are not clear. Here, we demonstrate that ectopic appearance of SOX9-positive Sertoli cell-like cells in grafted ovaries was mediated by the testosterone derived from the male host. Neither Sox8 nor Amh activity in the ovarian tissues is essential for such ectopic appearance of SOX9-positive cells. The transcriptome analyses of the grafted ovaries during this masculinization process showed early downregulation of pro-ovarian genes such as Irx3, Nr0b1/Dax1, Emx2, and Fez1/Lzts1 by days 7–10 post-transplantation, and subsequent upregulation of several pro-testis genes, such as Bhlhe40, Egr1/2, Nr4a2, and Zc3h12c by day 20, leading to a partial sex reversal with altered expression profiles in one-third of the total numbers of the sex-dimorphic pre-granulosa and Sertoli cell-specific genes at 12.5 dpc. Our data imply that the paternal testosterone exposure is partially responsible for the sex-reversal expression profiles of certain pro-ovarian and pro-testis genes in the fetal ovaries in a temporally dependent manner.

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<![CDATA[Brain expansion promoted by polycomb-mediated anterior enhancement of a neural stem cell proliferation program]]> https://www.researchpad.co/product?articleinfo=5c7ee7d4d5eed0c4848f4f0c

During central nervous system (CNS) development, genetic programs establish neural stem cells and drive both stem and daughter cell proliferation. However, the prominent anterior expansion of the CNS implies anterior–posterior (A–P) modulation of these programs. In Drosophila, a set of neural stem cell factors acts along the entire A–P axis to establish neural stem cells. Brain expansion results from enhanced stem and daughter cell proliferation, promoted by a Polycomb Group (PcG)->Homeobox (Hox) homeotic network. But how does PcG->Hox modulate neural-stem-cell–factor activity along the A–P axis? We find that the PcG->Hox network creates an A–P expression gradient of neural stem cell factors, thereby driving a gradient of proliferation. PcG mutants can be rescued by misexpression of the neural stem cell factors or by mutation of one single Hox gene. Hence, brain expansion results from anterior enhancement of core neural-stem-cell–factor expression, mediated by PcG repression of brain Hox expression.

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<![CDATA[Furin, a transcriptional target of NKX2-5, has an essential role in heart development and function]]> https://www.researchpad.co/product?articleinfo=5c897793d5eed0c4847d307a

The homeodomain transcription factor NKX2-5 is known to be essential for both normal heart development and for heart function. But little is yet known about the identities of its downstream effectors or their function during differentiation of cardiac progenitor cells (CPCs). We have used transgenic analysis and CRISPR-mediated ablation to identify a cardiac enhancer of the Furin gene. The Furin gene, encoding a proprotein convertase, is directly repressed by NKX2-5. Deletion of Furin in CPCs is embryonic lethal, with mutant hearts showing a range of abnormalities in the outflow tract. Those defects are associated with a reduction in proliferation and premature differentiation of the CPCs. Deletion of Furin in differentiated cardiomyocytes results in viable adult mutant mice showing an elongation of the PR interval, a phenotype that is consistent with the phenotype of mice and human mutant for Nkx2-5. Our results show that Furin mediate some aspects of Nkx2-5 function in the heart.

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<![CDATA[The role of embryo contact and focal adhesions during maternal recognition of pregnancy]]> https://www.researchpad.co/product?articleinfo=5c8823f5d5eed0c484639429

Maternal recognition of pregnancy (MRP) in the mare is an unknown process. In a non-pregnant mare on day 14 post-ovulation (PO), prostaglandin F (PGF) is secreted by the endometrium causing regression of the corpus luteum. Prior to day 14, MRP must occur in order to attenuate secretion of PGF. The embryo is mobile throughout the uterus due to uterine contractions from day of entry to day 14. It is unknown what signaling is occurring. Literature stated that infusing oil or placing a glass marble into the equine uterus prolongs luteal lifespan and that in non-pregnant mares, serum exosomes contain miRNA that are targeting the focal adhesion (FA) pathway. The hypothesis of this study is embryo contact with endometrium causes a change in abundance of focal adhesion molecules (FA) in the endometrium leading to decrease in PGF secretion. Mares (n = 3/day) were utilized in a cross-over design with each mare serving as a pregnant and non-pregnant (non-mated) control on days 9 and 11 PO. Mares were randomly assigned to collection day and endometrial samples and embryos were collected on the specified day. Biopsy samples were divided into five pieces, four for culture for 24 hours and one immediately snap frozen. Endometrial biopsies for culture were placed in an incubator with one of four treatments: [1] an embryo in contact on the luminal side of the endometrium, [2] beads in contact on the luminal side of the endometrium, [3] peanut oil in contact on the luminal side of the endometrium or [4] the endometrium by itself. Biopsies and culture medium were frozen for further analysis. RNA and protein were isolated from biopsies for PCR and Western blot analysis for FA. PGF assays were performed on culture medium to determine concentration of PGF. Statistics were performed using SAS (P ≤ 0.05 indicated significance). The presence of beads on day 9 impacted samples from pregnant mares more than non-pregnant mares and had very little impact on day 11. Presence of oil decreased FA in samples from pregnant mares on day 9. On day 11, oil decreased FA abundance in samples from non-pregnant mares. Embryo contact caused multiple changes in RNA and protein abundance in endometrium from both pregnant and non-pregnant mares. The PGF secretion after 24 hours with each treatment was also determined. On day 9, there was no change in PGF secretion compared to any treatments. On day 11, presence of peanut oil increased PGF secretion in samples from non-pregnant mares. In samples from non-pregnant mares, presence of an embryo decreased PGF secretion compared to control samples from non-pregnant mares. Results revealed that while beads and peanut oil may impact abundance of FA RNA and protein in endometrial samples, it does not appear to impact PGF secretion. Conversely, embryo contact for 24 hours with endometrium from a non-pregnant mare causes a decrease in PGF secretion. These results suggest that it is not just contact of any substance/object causing attenuation of PGF secretion, but the embryo itself is necessary to decrease PGF secretion.

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<![CDATA[Swift Large-scale Examination of Directed Genome Editing]]> https://www.researchpad.co/product?articleinfo=5c8823fad5eed0c484639487

In the era of CRISPR gene editing and genetic screening, there is an increasing demand for quick and reliable nucleic acid extraction pipelines for rapid genotyping of large and diverse sample sets. Despite continuous improvements of current workflows, the handling-time and material costs per sample remain major limiting factors. Here we present a robust method for low-cost DIY-pipet tips addressing these needs; i.e. using a cellulose filter disc inserted into a regular pipet tip. These filter-in-tips allow for a rapid, stand-alone four-step genotyping workflow by simply binding the DNA contained in the primary lysate to the cellulose filter, washing it in water and eluting it directly into the buffer for the downstream application (e.g. PCR). This drastically cuts down processing time to maximum 30 seconds per sample, with the potential for parallelizing and automation. We show the ease and sensitivity of our procedure by genotyping genetically modified medaka (Oryzias latipes) and zebrafish (Danio rerio) embryos (targeted by CRISPR/Cas9 knock-out and knock-in) in a 96-well plate format. The robust isolation and detection of multiple alleles of various abundancies in a mosaic genetic background allows phenotype-genotype correlation already in the injected generation, demonstrating the reliability and sensitivity of the protocol. Our method is applicable across kingdoms to samples ranging from cells to tissues i. e. plant seedlings, adult flies, mouse cell culture and tissue as well as adult fish fin-clips.

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<![CDATA[Endothelin receptor Aa regulates proliferation and differentiation of Erb-dependent pigment progenitors in zebrafish]]> https://www.researchpad.co/product?articleinfo=5c803c68d5eed0c484ad88f6

Skin pigment patterns are important, being under strong selection for multiple roles including camouflage and UV protection. Pigment cells underlying these patterns form from adult pigment stem cells (APSCs). In zebrafish, APSCs derive from embryonic neural crest cells, but sit dormant until activated to produce pigment cells during metamorphosis. The APSCs are set-aside in an ErbB signaling dependent manner, but the mechanism maintaining quiescence until metamorphosis remains unknown. Mutants for a pigment pattern gene, parade, exhibit ectopic pigment cells localised to the ventral trunk, but also supernumerary cells restricted to the Ventral Stripe. Contrary to expectations, these melanocytes and iridophores are discrete cells, but closely apposed. We show that parade encodes Endothelin receptor Aa, expressed in the blood vessels, most prominently in the medial blood vessels, consistent with the ventral trunk phenotype. We provide evidence that neuronal fates are not affected in parade mutants, arguing against transdifferentiation of sympathetic neurons to pigment cells. We show that inhibition of BMP signaling prevents specification of sympathetic neurons, indicating conservation of this molecular mechanism with chick and mouse. However, inhibition of sympathetic neuron differentiation does not enhance the parade phenotype. Instead, we pinpoint ventral trunk-restricted proliferation of neural crest cells as an early feature of the parade phenotype. Importantly, using a chemical genetic screen for rescue of the ectopic pigment cell phenotype of parade mutants (whilst leaving the embryonic pattern untouched), we identify ErbB inhibitors as a key hit. The time-window of sensitivity to these inhibitors mirrors precisely the window defined previously as crucial for the setting aside of APSCs in the embryo, strongly implicating adult pigment stem cells as the source of the ectopic pigment cells. We propose that a novel population of APSCs exists in association with medial blood vessels, and that their quiescence is dependent upon Endothelin-dependent factors expressed by the blood vessels.

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<![CDATA[A Notch-mediated, temporal asymmetry in BMP pathway activation promotes photoreceptor subtype diversification]]> https://www.researchpad.co/product?articleinfo=5c5ca2d9d5eed0c48441ebbe

Neural progenitors produce neurons whose identities can vary as a function of the time that specification occurs. Here, we describe the heterochronic specification of two photoreceptor (PhR) subtypes in the zebrafish pineal gland. We find that accelerating PhR specification by impairing Notch signaling favors the early fate at the expense of the later fate. Using in vivo lineage tracing, we show that most pineal PhRs are born from a fate-restricted progenitor. Furthermore, sister cells derived from the division of PhR-restricted progenitors activate the bone morphogenetic protein (BMP) signaling pathway at different times after division, and this heterochrony requires Notch activity. Finally, we demonstrate that PhR identity is established as a function of when the BMP pathway is activated. We propose a novel model in which division of a progenitor with restricted potential generates sister cells with distinct identities via a temporal asymmetry in the activation of a signaling pathway.

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<![CDATA[Lens differentiation is controlled by the balance between PDGF and FGF signaling]]> https://www.researchpad.co/product?articleinfo=5c61e8e2d5eed0c48496f303

How multiple receptor tyrosine kinases coordinate cell fate determination is yet to be elucidated. We show here that the receptor for platelet-derived growth factor (PDGF) signaling recruits the p85 subunit of Phosphoinositide 3-kinase (PI3K) to regulate mammalian lens development. Activation of PI3K signaling not only prevents B-cell lymphoma 2 (BCL2)-Associated X (Bax)- and BCL2 Antagonist/Killer (Bak)-mediated apoptosis but also promotes Notch signaling to prevent premature cell differentiation. Reducing PI3K activity destabilizes the Notch intracellular domain, while the constitutive activation of Notch reverses the PI3K deficiency phenotype. In contrast, fibroblast growth factor receptors (FGFRs) recruit Fibroblast Growth Factor Receptor Substrate 2 (Frs2) and Rous sarcoma oncogene (Src) Homology Phosphatase 2 (Shp2) to activate Mitogen-Activated Protein Kinase (MAPK) signaling, which induces the Notch ligand Jagged 1 (Jag1) and promotes cell differentiation. Inactivation of Shp2 restored the proper timing of differentiation in the p85 mutant lens, demonstrating the antagonistic interaction between FGF-induced MAPK and PDGF-induced PI3K signaling. By selective activation of PI3K and MAPK, PDGF and FGF cooperate with and oppose each other to balance progenitor cell maintenance and differentiation.

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<![CDATA[Nr2f-dependent allocation of ventricular cardiomyocyte and pharyngeal muscle progenitors]]> https://www.researchpad.co/product?articleinfo=5c63393fd5eed0c484ae6300

Multiple syndromes share congenital heart and craniofacial muscle defects, indicating there is an intimate relationship between the adjacent cardiac and pharyngeal muscle (PM) progenitor fields. However, mechanisms that direct antagonistic lineage decisions of the cardiac and PM progenitors within the anterior mesoderm of vertebrates are not understood. Here, we identify that retinoic acid (RA) signaling directly promotes the expression of the transcription factor Nr2f1a within the anterior lateral plate mesoderm. Using zebrafish nr2f1a and nr2f2 mutants, we find that Nr2f1a and Nr2f2 have redundant requirements restricting ventricular cardiomyocyte (CM) number and promoting development of the posterior PMs. Cre-mediated genetic lineage tracing in nr2f1a; nr2f2 double mutants reveals that tcf21+ progenitor cells, which can give rise to ventricular CMs and PM, more frequently become ventricular CMs potentially at the expense of posterior PMs in nr2f1a; nr2f2 mutants. Our studies reveal insights into the molecular etiology that may underlie developmental syndromes that share heart, neck and facial defects as well as the phenotypic variability of congenital heart defects associated with NR2F mutations in humans.

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<![CDATA[Gamma radiation induces locus specific changes to histone modification enrichment in zebrafish and Atlantic salmon]]> https://www.researchpad.co/product?articleinfo=5c6dc9d7d5eed0c48452a2d3

Ionizing radiation is a recognized genotoxic agent, however, little is known about the role of the functional form of DNA in these processes. Post translational modifications on histone proteins control the organization of chromatin and hence control transcriptional responses that ultimately affect the phenotype. The purpose of this study was to investigate effects on chromatin caused by ionizing radiation in fish. Direct exposure of zebrafish (Danio rerio) embryos to gamma radiation (10.9 mGy/h for 3h) induced hyper-enrichment of H3K4me3 at the genes hnf4a, gmnn and vegfab. A similar relative hyper-enrichment was seen at the hnf4a loci of irradiated Atlantic salmon (Salmo salar) embryos (30 mGy/h for 10 days). At the selected genes in ovaries of adult zebrafish irradiated during gametogenesis (8.7 and 53 mGy/h for 27 days), a reduced enrichment of H3K4me3 was observed, which was correlated with reduced levels of histone H3 was observed. F1 embryos of the exposed parents showed hyper-methylation of H3K4me3, H3K9me3 and H3K27me3 on the same three loci, while these differences were almost negligible in F2 embryos. Our results from three selected loci suggest that ionizing radiation can affect chromatin structure and organization, and that these changes can be detected in F1 offspring, but not in subsequent generations.

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<![CDATA[Modeling Edar expression reveals the hidden dynamics of tooth signaling center patterning]]> https://www.researchpad.co/product?articleinfo=5c65dcd2d5eed0c484dec2fa

When patterns are set during embryogenesis, it is expected that they are straightly established rather than subsequently modified. The patterning of the three mouse molars is, however, far from straight, likely as a result of mouse evolutionary history. The first-formed tooth signaling centers, called MS and R2, disappear before driving tooth formation and are thought to be vestiges of the premolars found in mouse ancestors. Moreover, the mature signaling center of the first molar (M1) is formed from the fusion of two signaling centers (R2 and early M1). Here, we report that broad activation of Edar expression precedes its spatial restriction to tooth signaling centers. This reveals a hidden two-step patterning process for tooth signaling centers, which was modeled with a single activator–inhibitor pair subject to reaction–diffusion (RD). The study of Edar expression also unveiled successive phases of signaling center formation, erasing, recovering, and fusion. Our model, in which R2 signaling center is not intrinsically defective but erased by the broad activation preceding M1 signaling center formation, predicted the surprising rescue of R2 in Edar mutant mice, where activation is reduced. The importance of this R2–M1 interaction was confirmed by ex vivo cultures showing that R2 is capable of forming a tooth. Finally, by introducing chemotaxis as a secondary process to RD, we recapitulated in silico different conditions in which R2 and M1 centers fuse or not. In conclusion, pattern formation in the mouse molar field relies on basic mechanisms whose dynamics produce embryonic patterns that are plastic objects rather than fixed end points.

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<![CDATA[TCTP and CSN4 control cell cycle progression and development by regulating CULLIN1 neddylation in plants and animals]]> https://www.researchpad.co/product?articleinfo=5c59fee9d5eed0c4841357b5

Translationally Controlled Tumor Protein (TCTP) controls growth by regulating the G1/S transition during cell cycle progression. Our genetic interaction studies show that TCTP fulfills this role by interacting with CSN4, a subunit of the COP9 Signalosome complex, known to influence CULLIN-RING ubiquitin ligases activity by controlling CULLIN (CUL) neddylation status. In agreement with these data, downregulation of CSN4 in Arabidopsis and in tobacco cells leads to delayed G1/S transition comparable to that observed when TCTP is downregulated. Loss-of-function of AtTCTP leads to increased fraction of deneddylated CUL1, suggesting that AtTCTP interferes negatively with COP9 function. Similar defects in cell proliferation and CUL1 neddylation status were observed in Drosophila knockdown for dCSN4 or dTCTP, respectively, demonstrating a conserved mechanism between plants and animals. Together, our data show that CSN4 is the missing factor linking TCTP to the control of cell cycle progression and cell proliferation during organ development and open perspectives towards understanding TCTP’s role in organ development and disorders associated with TCTP miss-expression.

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<![CDATA[Adverse effects of paternal obesity on the motile spermatozoa quality]]> https://www.researchpad.co/product?articleinfo=5c6b2696d5eed0c484289d07

Growing evidence suggests that paternal obesity may decrease male fertility potential. During infertility treatment with intra-cytoplasmic sperm injection (ICSI), a morphologically normal motile spermatozoon is injected into a mature egg, when possible. However, sperm motility and morphology per se do not reflect the sperm molecular composition. In this study, we aimed to assess the quality of motile spermatozoa in the context of obesity by analysing their conventional and molecular characteristics as well as their ability to promote early embryonic development. A prospective study was conducted on 128 infertile men divided into three groups: 40 lean, 42 overweight, and 46 obese men. Conventional sperm parameters (concentration, motility and morphology) and sperm molecular status (chromatin composition and integrity, 5-methycytosine (5-mC) and 5-hydroxycytosine (5-hmC) contents and oxidative stress level) were analysed on raw semen and/or on motile spermatozoa selected by density gradient or swim-up techniques. Morphokinetic analysis of the embryos derived from ICSI was performed using the Embryoviewer software. Our results showed that the motile sperm-enriched fraction from obese men exhibited higher levels of retained histones (p<0.001), elevated percentage of altered chromatin integrity (p<0.001), and decreased contents of 5-hmC (p<0.001), and 5-mC (p<0.05) levels as compared to that from lean men. Importantly, there were no statistically significant correlations between these molecular parameters and the percentages of morphologically normal motile spermatozoa. Regarding embryo morphokinetics, the CC1 (p<0.05) and CC3 (p<0.05) embryonic cell cycles were significantly delayed in the cleavage embryos of the obese group as compared to the embryos of the lean group. Our data is of particular interest because, besides demonstrating the negative impacts of obesity on motile spermatozoa molecular composition, it also highlights the possible risk of disturbing early embryonic cell cycles kinetics in the context of paternal obesity.

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