RNA In Embryos Research Articles

Screening of NIAS World Rice Core Collection for Seeds with Long Longevity as Useful Potential Breeding Materials Focusing on the Stability of Embryonic RNAs.

Seed longevity is a crucial trait for the seed industry and genetic resource preservation. To develop excellent cultivars with extended seed lifespans, it is important to understand the mechanism of keeping seed germinability long term and to find useful genetic resources as prospective breeding materials. This study was conducted to identify the best cultivars with a high and stable seed longevity trait in the germplasm of rice (Oryza sativa L.) and to analyze the correlation between seed longevity and embryonic RNA integrity. Seeds from 69 cultivars of the world rice core collection selected by the NIAS in Japan were harvested in different years and subjected to long-term storage or controlled deterioration treatment (CDT). The long-term storage (4 °C, RH under 35%, 10 years) was performed on seeds harvested in 2010 and 2013. The seeds harvested in 2016 and 2019 were used for CDT (36 °C, RH of 80%, 40 days). Seed longevity and embryonic RNA integrity were estimated by a decrease in the germination percentage and RNA integrity number (RIN) after long-term storage or CDT. The RIN value was obtained by the electrophoresis of the total RNA extracted from the seed embryos. Seeds of "Vandaran (indica)", "Tupa 729 (japonica)", and "Badari Dhan (indica)" consistently showed higher seed longevity and embryonic RNA integrity both under long-term storage and CDT conditions regardless of the harvest year. A strong correlation (R2 = 0.93) was observed between the germination percentages and RIN values of the seeds after the long-term storage or CDT among nine cultivars selected based on differences in their seed longevity. The study findings revealed the relationship between rice seed longevity and embryo RNA stability and suggested potential breeding materials including both japonica and indica cultivars for improving rice seed longevity.

Human trophectoderm becomes multi-layered by internalization at the polar region

To implant in the uterus, mammalian embryos form blastocysts comprising trophectoderm (TE) surrounding an inner cell mass (ICM), confined to the polar region by the expanding blastocoel. The mode of implantation varies between species. Murine embryos maintain a single layered TE until they implant in the characteristic thick deciduum, whereas human blastocysts attach via polar TE directly to the uterine wall. Using immunofluorescence (IF) of rapidly isolated ICMs, blockade of RNA and protein synthesis in whole embryos, or 3D visualization of immunostained embryos, we provide evidence of multi-layering in human polar TE before implantation. This may be required for rapid uterine invasion to secure the developing human embryo and initiate formation of the placenta. Using sequential fluorescent labeling, we demonstrate that the majority of inner TE in human blastocysts arises from existing outer cells, with no evidence of conversion from the ICM in the context of the intact embryo.

3D exploration of gene expression in chicken embryos through combined RNA fluorescence in situ hybridization, immunofluorescence, and clearing

BackgroundFine characterization of gene expression patterns is crucial to understand many aspects of embryonic development. The chicken embryo is a well-established and valuable animal model for developmental biology. The period spanning from the third to sixth embryonic days (E3 to E6) is critical for many organ developments. Hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA-FISH) enables multiplex RNA detection in thick samples including embryos of various animal models. However, its use is limited by tissue opacity.ResultsWe optimized HCR RNA-FISH protocol to efficiently label RNAs in whole mount chicken embryos from E3.5 to E5.5 and adapted it to ethyl cinnamate (ECi) tissue clearing. We show that light sheet imaging of HCR RNA-FISH after ECi clearing allows RNA expression analysis within embryonic tissues with good sensitivity and spatial resolution. Finally, whole mount immunofluorescence can be performed after HCR RNA-FISH enabling as exemplified to assay complex spatial relationships between axons and their environment or to monitor GFP electroporated neurons.ConclusionsWe could extend the use of HCR RNA-FISH to older chick embryos by optimizing HCR RNA-FISH and combining it with tissue clearing and 3D imaging. The integration of immunostaining makes possible to combine gene expression with classical cell markers, to correlate expressions with morphological differentiation and to depict gene expressions in gain or loss of function contexts. Altogether, this combined procedure further extends the potential of HCR RNA-FISH technique for chicken embryology.

HCR spectral imaging: 10-plex, quantitative, high-resolution RNA and protein imaging in highly autofluorescent samples.

Signal amplification based on the mechanism of hybridization chain reaction (HCR) provides a unified framework for multiplex, quantitative, high-resolution imaging of RNA and protein targets in highly autofluorescent samples. With conventional bandpass imaging, multiplexing is typically limited to four or five targets owing to the difficulty in separating signals generated by fluorophores with overlapping spectra. Spectral imaging has offered the conceptual promise of higher levels of multiplexing, but it has been challenging to realize this potential in highly autofluorescent samples, including whole-mount vertebrate embryos. Here, we demonstrate robust HCR spectral imaging with linear unmixing, enabling simultaneous imaging of ten RNA and/or protein targets in whole-mount zebrafish embryos and mouse brain sections. Further, we demonstrate that the amplified and unmixed signal in each of the ten channels is quantitative, enabling accurate and precise relative quantitation of RNA and/or protein targets with subcellular resolution, and RNA absolute quantitation with single-molecule resolution, in the anatomical context of highly autofluorescent samples.

NLRP7 participates in the human subcortical maternal complex and its variants cause female infertility characterized by early embryo arrest.

Successful human reproduction requires normal oocyte maturation, fertilization, and early embryo development. Early embryo arrest is a common phenomenon leading to female infertility, but the genetic basis is largely unknown. NLR family pyrin domain-containing 7 (NLRP7) is a member of the NLRP subfamily. Previous studies have shown that variants of NLRP7 are one of the crucial causes of female recurrent hydatidiform mole, but whether NLRP7 variants can directly affect early embryo development is unclear. We performed whole-exome sequencing in patients who experienced early embryo arrest, and five heterozygous variants (c.251G > A, c.1258G > A, c.1441G > A, c. 2227G > A, c.2323C > T) of NLRP7 were identified in affected individuals. Plasmids of NLRP7 and subcortical maternal complex components were overexpressed in 293T cells, and Co-IP experiments showed that NLRP7 interacted with NLRP5, TLE6, PADI6, NLRP2, KHDC3L, OOEP, and ZBED3. Injecting complementary RNAs in mouse oocytes and early embryos showed that NLRP7 variants influenced the oocyte quality and some of the variants significantly affected early embryo development. These findings contribute to our understanding of the role of NLRP7 in human early embryo development and provide a new genetic marker for clinical early embryo arrest patients. KEY MESSAGES: Five heterozygous variants of NLRP7 (c.1441G > A; 2227G > A; c.251G > A; c.1258G > A; c.2323C > T) were identified in five infertile patients who experienced early embryo arrest. NLRP7 is a component of human subcortical maternal complex. NLRP7 variants lead to poor quality of oocytes and early embryo development arrest. This study provides a new genetic marker for clinical early embryo arrest patients.

Preimplantation embryo gene expression: 56 years of discovery, and counting.

The biology of preimplantation embryo gene expression began 56 years ago with studies of the effects of protein synthesis inhibition and discovery of changes in embryo metabolism and related enzyme activities. The field accelerated rapidly with the emergence of embryo culture systems and progressively evolving methodologies that have allowed early questions to be re-addressed in new ways and in greater detail, leading to deeper understanding and progressively more targeted studies to discover ever more fine details. The advent of technologies for assisted reproduction, preimplantation genetic testing, stem cell manipulations, artificial gametes, and genetic manipulation, particularly in experimental animal models and livestock species, has further elevated the desire to understand preimplantation development in greater detail. The questions that drove enquiry from the earliest years of the field remain drivers of enquiry today. Our understanding of the crucial roles of oocyte-expressed RNA and proteins in early embryos, temporal patterns of embryonic gene expression, and mechanisms controlling embryonic gene expression has increased exponentially over the past five and a half decades as new analytical methods emerged. This review combines early and recent discoveries on gene regulation and expression in mature oocytes and preimplantation stage embryos to provide a comprehensive understanding of preimplantation embryo biology and to anticipate exciting future advances that will build upon and extend what has been discovered so far.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo.

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

Long-Term Storage Does Not Affect the Expression Profiles of mRNA and Long Non-Coding RNA in Vitrified-Warmed Human Embryos.

Although vitrification has been widely applied in assisted reproductive technology, it is unknown whether storage time has any impact on the mRNA and lncRNA expression profiles in human embryos. Eleven women (aged 23–35 years) who had undergone in vitro fertilization treatment were recruited for this study. The transcriptomes of 3 fresh eight-cell embryos and 8 surviving vitrified-warmed eight-cell embryos (4 embryos were cryostored for 3 years, and the others were cryostored for 8 years) were analyzed through single-cell RNA-Seq. No differentially expressed mRNAs or lncRNAs were identified between the 3-years group and 8-years group. A total of 128 mRNAs and 365 lncRNAs were differentially expressed in the 8 vitrified-warmed embryos compared with the fresh embryos. The vitrification-warming impact was moderate, and it was mainly related to the pathways of metabolism, stress response, apoptosis, cell cycle, cell adhesion, and signaling for TFG-β and Hippo. The analysis of target mRNAs suggested that lncRNAs might contribute to the regulation of mRNAs after vitrification-warming. Our findings indicated that long-term storage after vitrification does not affect the mRNA and lncRNA expression profiles in human embryos, however, the procedure of vitrification-warming would lead to minor alteration of transcriptome.

Pak2 reduction induces a failure of early embryonic development in mice

BackgroundThe quality of the early embryo is vital to embryonic development and implantation. As a highly conserved serine/threonine kinase, p21-activated kinase 2 (Pak2) participates in diverse biologic processes, especially in cytoskeleton remodeling and cell apoptosis. In mice, Pak2 knock out and endothelial depletion of Pak2 showed embryonic lethality. However, the role of Pak2 in preimplantation embryos remains unelucidated.MethodsIn the present work, Pak2 was reduced using a specific small interfering RNA in early mouse embryos, validating the unique roles of Pak2 in spindle assembly and DNA repair during mice early embryonic development. We also employed immunoblotting, immunostaining, in vitro fertilization (IVF) and image quantification analyses to test the Pak2 knockdown on the embryonic development progression, spindle assembly, chromosome alignment, oxidative stress, DNA lesions and blastocyst cell apoptosis. Areas in chromatin with γH2AX were detected by immunofluorescence microscopy and serve as a biomarker of DNA damages.ResultsWe found that Pak2 knockdown significantly reduced blastocyst formation of early embryos. In addition, Pak2 reduction led to dramatically increased abnormal spindle assembly and chromosomal aberrations in the embryos. We noted the overproduction of reactive oxygen species (ROS) with Pak2 knockdown in embryos. In response to DNA double strand breaks (DSBs), the histone protein H2AX is specifically phosphorylated at serine139 to generate γH2AX, which is used to quantitative DSBs. In this research, Pak2 knockdown also resulted in the accumulation of phosphorylated γH2AX, indicative of increased embryonic DNA damage. Commensurate with this, a significantly augmented rate of blastocyst cell apoptosis was detected in Pak2-KD embryos compared to their controls.ConclusionsCollectively, our data suggest that Pak2 may serve as an important regulator of spindle assembly and DNA repair, and thus participate in the development of early mouse embryos.

Loss of iqgap1 (IQ Motif Containing GTPase Activating Protein 1) in Zebrafish Leads to Intracerebellar Hemorrhage, Morphological Abnormalities and Activates Hematopoietic Response

Defects in multiple cell signaling molecules lead to disruptions of vascular integrity given the need for fine-tuned regulation of the cell adhesion complexes. These genetic defects have been linked to the development of intracerebral hemorrhage (ICH). There is genetic evidence in humans for ICH due to some genetic variants, while other variants have been identified in preclinical animal models. Signaling adaptor proteins play a crucial role in cell signaling by promoting interactions between effector proteins and even enabling integration of different pathways. IQGAP1 is a conserved signaling adaptor known for its roles in cell adhesion, cancer and for other cell biological effects. We engineered a zebrafish null mutant in the zebrafish iqgap1 gene by introducing an 11-bp deletion using a CRISPR/Cas9 genome editing method and characterized its phenotype. Homozygous mutants exhibit severe brain hemorrhage and morphological abnormalities, which are ultimately lethal, in about 30-40% of cases, whereas the other embryos survive to adulthood. We visualized the expression pattern of iqgap1 relative to the established fli1a vascular marker and found that iqgap1 strongly overlapped with fli1a expression, but was expressed much more broadly in tissues, such as muscle, branchial arches, and the caudal hematopoietic tissue (equivalent to the mammalian fetal liver).Critically, iqgap1 exhibited co-localization with fli1a in the blood vessels of the central nervous system, whose disruption is likely responsible for the brain hemorrhage. Whole embryo RNA sequencing-based comparison of hemorrhage-positive iqgap1-/- embryo pools with wild-type embryos at 52 hours post-fertilization (hpf) shortly after the onset of hemorrhage identified approximately 800 differentially regulated genes. The most striking feature of this dataset was up-regulation of hematopoietic markers especially those of erythrocytes, neutrophils, mast cells and HSPCs (hematopoietic stem and progenitor cells), but not macrophages. We have confirmed by in situ hybridization with marker gene probes that erythrocyte and neutrophil production is up-regulated most strongly in iqgap1-/- embryos undergoing some level of hemorrhage. By contrast, fli1a endothelial and stem cell marker was downregulated. This animal model provides a compelling genotype-phenotype correlation, implicating IQGAP1 as a new player in vascular disorders such as ICH and identifying a previously unrecognized relationship between IQGAP and regulation of hematopoiesis. Furthermore, this model is now poised to identify ameliorating and exacerbating modifier lesions and potential therapeutic agents that restore normal vascular integrity and prevent ICH. DisclosuresRobitaille: Novartis: Consultancy. Berman: Oxford Immune Algorithmics: Membership on an entity's Board of Directors or advisory committees.

Effects of high-molecular-weight polyvinyl chloride on Xenopus laevis adults and embryos: the mRNA expression profiles of Myf5, Esr1, Bmp4, Pax6, and Hsp70 genes during early embryonic development.

Microplastics and associated adverse effects have been on the global agenda in recent years. Because of its importance as a model organism for studies on developmental biology, Xenopus laevis has been chosen as the study animal in in vitro teratogenesis studies. FETAX test uses early-stage embryos of X. laevis to measure the potential of substances to cause mortality, malformation, and growth inhibition in developing embryos. The aim of this study was to examine the effects of high molecular weight polyvinyl chloride (HMW-PVC) on parental X. laevis frogs and their embryos using the FETAX test. To this purpose, a HMW-PVC dose of 1% of body weight/twice each week was provided to frogs by oral gavage throughout 6 weeks. After the procedure, oocytes and sperms of HMW-PVC-exposed frogs were fertilized and FETAX was applied to selected embryos. After the completion of a 96-h incubation period, tadpoles were examined, their live/dead status were determined, their lengths were measured, and their anomalies were photographed. Besides, excised organs of the parental frogs were referred to histopathology examination. On the other hand, the mRNA expression levels of Hsp70, Myf5, Bmp4, Pax6, and Esr1 genes were determined by applying real-time quantitative PCR method to cDNA which was synthesized from the total RNA of embryos. The results showed that treatment with HMW-PVC dose of 1% of body weight/twice each week caused malformations and decreased viability. Hsp70 and Pax6 gene expression levels significantly decreased in all assay groups, as compared with controls. Lung and intestine tissues showed normal appearance in histopatological examination. Further research is required to explain the whole effects of HMW-PVC exposure on X. laevis embryos.

The biologicalfunctionofIGF2BPsandtheirroleintumorigenesis.

The insulin-like growth factor-2 mRNA-binding proteins (IGF2BPs) pertain to a highly conservative RNA-binding family that works as a post-transcriptional fine-tuner for target transcripts. Emergingevidence suggests that IGF2BPs regulate RNA processing and metabolism, including stability, translation, and localization, and are involved in various cellular functions and pathophysiologies. In this review, we summarize the roles and molecular mechanisms of IGF2BPs in cancer development and progression. We mainly discuss the functional relevance of IGF2BPs in embryo development, neurogenesis, metabolism, RNA processing, and tumorigenesis. Understanding IGF2BPs role in tumor progression will provide new insight into cancer pathophysiology.

Dynamics of small non-coding RNAs in bovine scNT embryos through the maternal-to-embryonic transition†.

The efficiency of somatic cell nuclear transfer (scNT) for production of viable offspring is relatively low as compared to in vitro fertilization (IVF), presumably due to deficiencies in epigenetic reprogramming of the donor cell genome. Such defects may also involve the population of small non-coding RNAs (sncRNAs), which are important during early embryonic development. The objective of this study was to examine dynamic changes in relative abundance of sncRNAs during the maternal-to-embryonic transition (MET) in bovine embryos produced by scNT as compared to IVF by using RNA sequencing. When comparing populations of miRNA in scNT versus IVF embryos, only miR-2340, miR-345, and miR34a were differentially expressed in morulae, though many more miRNAs were differentially expressed when comparing across developmental stages. Also of interest, distinct populations of piwi-interacting like RNAs (pilRNAs) were identified in bovine embryos prior to and during embryonic genome activation (EGA) as compared bovine embryos post-EGA and differentiated cells. Overall, sncRNA sequencing analysis of preimplantation embryos revealed largely similar profiles of sncRNAs for IVF and scNT embryos at the 2-cell, 8-cell, morula, and blastocyst stages of development. However, these sncRNA profiles, including miRNA, piRNA, and tRNA fragments, were notably distinct prior to and after completion of the MET.

Nuclear m6A reader YTHDC1 regulates the scaffold function of LINE1 RNA in mouse ESCs and early embryos

N6-methyladenosine (m6A) on chromosome-associated regulatory RNAs (carRNAs), including repeat RNAs, plays important roles in tuning the chromatin state and transcription, but the intrinsic mechanism remains unclear. Here, we report that YTHDC1 plays indispensable roles in the self-renewal and differentiation potency of mouse embryonic stem cells (ESCs), which highly depends on the m6A-binding ability. Ythdc1 is required for sufficient rRNA synthesis and repression of the 2-cell (2C) transcriptional program in ESCs, which recapitulates the transcriptome regulation by the LINE1 scaffold. Detailed analyses revealed that YTHDC1 recognizes m6A on LINE1 RNAs in the nucleus and regulates the formation of the LINE1-NCL partnership and the chromatin recruitment of KAP1. Moreover, the establishment of H3K9me3 on 2C-related retrotransposons is interrupted in Ythdc1-depleted ESCs and inner cell mass (ICM) cells, which consequently increases the transcriptional activities. Our study reveals a role of m6A in regulating the RNA scaffold, providing a new model for the RNA-chromatin cross-talk.

Gene knockdown via electroporation of short hairpin RNAs in embryos of the marine hydroid Hydractinia symbiolongicarpus

Analyzing gene function in a broad range of research organisms is crucial for understanding the biological functions of genes and their evolution. Recent studies have shown that short hairpin RNAs (shRNAs) can induce gene-specific knockdowns in two cnidarian species. We have developed a detailed, straightforward, and scalable method to deliver shRNAs into fertilized eggs of the hydrozoan cnidarian Hydractinia symbiolongicarpus via electroporation, yielding effective gene-targeted knockdowns that can last throughout embryogenesis. Our electroporation protocol allows for the transfection of shRNAs into hundreds of fertilized H. symbiolongicarpus eggs simultaneously with minimal embryo death and no long-term harmful consequences on the developing animals. We show RT-qPCR and detailed phenotypic evidence of our method successfully inducing effective knockdowns of an exogenous gene (eGFP) and an endogenous gene (Nanos2), as well as knockdown confirmation by RT-qPCR of two other endogenous genes. We also provide visual confirmation of successful shRNA transfection inside embryos through electroporation. Our detailed protocol for electroporation of shRNAs in H. symbiolongicarpus embryos constitutes an important experimental resource for the hydrozoan community while also serving as a successful model for the development of similar methods for interrogating gene function in other marine invertebrates.

Single C-to-T substitution using engineered APOBEC3G-nCas9 base editors with minimum genome- and transcriptome-wide off-target effects.

Cytosine base editors (CBEs) enable efficient cytidine-to-thymidine (C-to-T) substitutions at targeted loci without double-stranded breaks. However, current CBEs edit all Cs within their activity windows, generating undesired bystander mutations. In the most challenging circumstance, when a bystander C is adjacent to the targeted C, existing base editors fail to discriminate them and edit both Cs. To improve the precision of CBE, we identified and engineered the human APOBEC3G (A3G) deaminase; when fused to the Cas9 nickase, the resulting A3G-BEs exhibit selective editing of the second C in the 5'-CC-3' motif in human cells. Our A3G-BEs could install a single disease-associated C-to-T substitution with high precision. The percentage of perfectly modified alleles is more than 6000-fold for disease correction and more than 600-fold for disease modeling compared with BE4max. On the basis of the two-cell embryo injection method and RNA sequencing analysis, our A3G-BEs showed minimum genome- and transcriptome-wide off-target effects, achieving high targeting fidelity.

Variants in miRNA regulome and their association with the risk of nonsyndromic orofacial clefts.

Aim: To investigate the associations between single nucleotide polymorphisms(SNPs) in miRNA regulome and nonsyndromic orofacial clefts. Materials & methods: The associations were evaluated by logistic regression model in stage I (504 cases and 455 controls) and stage II (1500 cases and 1386 controls). Functional experiments including luciferase activity assay, cell apoptosis and proliferation, serum miRNA expression, and mouse embryo RNA sequencing were performed. Results: Rs3830766 in the enhancer of hsa-miR-4260 was associated with cleft lip only (CLO) and enhancer activity. Hsa-miR-4260 expression decreased in the serum of CLO. Overexpression of miR-4260 inhibited cell proliferation and promoted cell apoptosis. UBB was the target gene of hsa-miR-4260. Conclusion: Rs3830766 in the hsa-miR-4260 enhancer that can interact with UBB was relevant to CLO.

Long Non-Coding RNA and mRNA Profiling in Early-Stage Bovine Embryos Treated with Glutathione.

We measured differential expression profiles of genes and long non-coding RNA (lncRNA) using RNA sequencing in bovine embryos with or without glutathione (GSH) treatment. Bovine embryos fertilized in vitro were treated with GSH to blastocyst. Embryos at the 8-16-cell and morula stages were collected, with embryos without GSH treatment as the control. RNA was isolated, amplified, and sequenced. Differentially expressed genes (DEGs) and lncRNAs (DElncRNAs) were identified and bioinformatic analyses carried out. Transcript levels were confirmed using quantitative RT-PCR. A total of 4100 DEGs were identified, of which 3952 were in GSH-treated morulae and 884 in untreated morulae. More gene ontology (GO) terms were associated with GSH treatment than with control conditions. KEGG analysis showed that glutathione metabolism, citrate cycle, and metabolic pathways involving glycine, serine, and threonine were observed only in GSH-treated embryos. Among 4273 DElncRNAs identified, 59 were potentially important in GSH-treated embryo development, including 14 involved in glutathione metabolism. The 59 DElncRNAs co-expressed with protein-coding mRNAs involved similar GO terms and pathways as the DEGs. This appears to be the first comprehensive profiling of DEGs and DElncRNAs in bovine embryos fertilized in vitro with or without GSH, and the first systematic screen of potential lncRNAs in bovine embryos.

Natural size variation among embryos leads to the corresponding scaling in gene expression

Xenopus laevis frogs from laboratory stocks normally lay eggs exhibiting extensive size variability. We find that these initial size differences subsequently affect the size of the embryos prior to the onset of growth, and the size of tadpoles during the growth period. Even though these tadpoles differ in size, their tissues, organs, and structures always seem to be properly proportioned, i.e. they display static allometry. Initial axial patterning events in Xenopus occur in a spherical embryo, allowing easy documentation of their size-dependent features. We examined the size distribution of early Xenopus laevis embryos and measured diameters that differed by about 38% with a median of about 1.43 ​mm. This range of embryo sizes corresponds to about a 1.9-fold difference in surface area and a 2.6-fold difference in volume. We examined the relationship between embryo size and gene expression and observed a significant correlation between diameter and RNA content during gastrula stages. In addition, we investigated the expression levels of genes that pattern the mesoderm, induce the nervous system and mediate the progression of ectodermal cells to neural precursors in large and small embryos. We found that most of these factors were expressed at levels that scaled with the different embryo sizes and total embryo RNA content. In agreement with the changes in transcript levels, the expression domains in larger embryos increased proportionally with the increase in surface area, maintaining their relative expression domain size in relation to the total size of the embryo. Thus, our study identified a mechanism for adapting gene expression domains to embryo size by adjusting the transcript levels of the genes regulating mesoderm induction and patterning. In the neural plate, besides the scaling of the expression domains, we observed similar cell sizes and cell densities in small and large embryos suggesting that additional cell divisions took place in large embryos to compensate for the increased size. Our results show in detail the size variability among Xenopus laevis embryos and the transcriptional adaptation to scale gene expression with size. The observations further support the involvement of BMP/ADMP signaling in the scaling process.

Whole embryo culture, transcriptomics and RNA interference identify TBX1 and FGF11 as novel regulators of limb development in the mouse

Identifying genes involved in vertebrate developmental processes and characterizing this involvement are daunting tasks, especially in the mouse where viviparity complicates investigations. Attempting to devise a streamlined approach for this type of study we focused on limb development. We cultured E10.5 and E12.5 embryos and performed transcriptional profiling to track molecular changes in the forelimb bud over a 6-hour time-window. The expression of certain genes was found to diverge rapidly from its normal path, possibly reflecting the activation of a stress-induced response. Others, however, maintained for up to 3 hours dynamic expression profiles similar to those seen in utero. Some of these resilient genes were known regulators of limb development. The implication of the others in this process was either unsuspected or unsubstantiated. The localized knockdown of two such genes, Fgf11 and Tbx1, hampered forelimb bud development, providing evidence of their implication. These results show that combining embryo culture, transcriptome analysis and RNA interference could speed up the identification of genes involved in a variety of developmental processes, and the validation of their implication.