Impact of eIF2α phosphorylation on the translational landscape of mouse embryonic stem cells.

The integrated stress response (ISR) is critical for cell survival under stress. In response to diverse environmental cues, eIF2α becomes phosphorylated, engendering a dramatic change in mRNA translation. The activation of ISR plays a pivotal role in the early embryogenesis, but the eIF2-dependent translational landscape in pluripotent embryonic stem cells (ESCs) is largely unexplored. We employ a multi-omics approach consisting of ribosome profiling, proteomics, and metabolomics in wild-type (eIF2α+/+) and phosphorylation-deficient mutant eIF2α (eIF2αA/A) mouse ESCs (mESCs) to investigate phosphorylated (p)-eIF2α-dependent translational control of naive pluripotency. We show a transient increase in p-eIF2α in the naive epiblast layer of E4.5 embryos. Absence of eIF2α phosphorylation engenders an exit from naive pluripotency following 2i (two chemical inhibitors of MEK1/2 and GSK3α/ß) withdrawal. p-eIF2α controls translation of mRNAs encoding proteins that govern pluripotency, chromatin organization, and glutathione synthesis. Thus, p-eIF2α acts as a key regulator of the naive pluripotency gene regulatory network.

CD133/Prom1 marks proximal mouse oviduct epithelial progenitors and adult epithelial cells with a low generative capacity.

The epithelium lining the oviduct or fallopian tube consists of multiciliated and secretory cells, which support fertilization and preimplantation development, however, its homeostasis remains poorly understood. CD133/Prom1 expression has been used as a marker to identify adult stem cell populations in various organs and often associated with cancer cells that have stem-like properties. Using an antibody targeted to CD133 and a Cre recombinase-based lineage tracing strategy, we found that CD133/Prom1 expression is not associated with a stem/progenitor population in the oviduct but marked predominantly multiciliated cells with a low generative capacity. Additionally, we have shown that CD133 is disparately localised along the oviduct during neonatal development, and that Prom1 expressing secretory cells in the ampulla rapidly transitioned to multiciliated cells and progressively migrated to the ridge of epithelial folds.

ZMYM2 is essential for methylation of germline genes and active transposons in embryonic development.

ZMYM2 is a transcriptional repressor whose role in development is largely unexplored. We found that Zmym2-/- mice show embryonic lethality by E10.5. Molecular characterization of Zmym2-/- embryos revealed two distinct defects. First, they fail to undergo DNA methylation and silencing of germline gene promoters, resulting in widespread upregulation of germline genes. Second, they fail to methylate and silence the evolutionarily youngest and most active LINE element subclasses in mice. Zmym2-/- embryos show ubiquitous overexpression of LINE-1 protein as well as aberrant expression of transposon-gene fusion transcripts. ZMYM2 homes to sites of PRC1.6 and TRIM28 complex binding, mediating repression of germline genes and transposons respectively. In the absence of ZMYM2, hypermethylation of histone 3 lysine 4 occurs at target sites, creating a chromatin landscape unfavourable for establishment of DNA methylation. ZMYM2-/- human embryonic stem cells also show aberrant upregulation and demethylation of young LINE elements, indicating a conserved role in repression of active transposons. ZMYM2 is thus an important new factor in DNA methylation patterning in early embryonic development.

N-acetylneuraminate pyruvate lyase controls sialylation of muscle glycoproteins essential for muscle regeneration and function.

Deleterious variants in N-acetylneuraminate pyruvate lyase (NPL) cause skeletal myopathy and cardiac edema in humans and zebrafish, but its physiological role remains unknown. We report generation of mouse models of the disease: NplR63C, carrying the human p.Arg63Cys variant, and Npldel116 with a 116-bp exonic deletion. In both strains, NPL deficiency causes drastic increase in free sialic acid levels, reduction of skeletal muscle force and endurance, slower healing and smaller size of newly formed myofibers after cardiotoxin-induced muscle injury, increased glycolysis, partially impaired mitochondrial function, and aberrant sialylation of dystroglycan and mitochondrial LRP130 protein. NPL-catalyzed degradation of sialic acid in the muscle increases after fasting and injury and in human patient and mouse models with genetic muscle dystrophy, demonstrating that NPL is essential for muscle function and regeneration and serves as a general marker of muscle damage. Oral administration of N-acetylmannosamine rescues skeletal myopathy, as well as mitochondrial and structural abnormalities in NplR63C mice, suggesting a potential treatment for human patients.

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation.

Germline genetically engineered mouse models (G-GEMMs) have provided valuable insight into in vivo gene function in development, homeostasis, and disease. However, the time and cost associated with colony creation and maintenance are high. Recent advances in CRISPR-mediated genome editing have allowed the generation of somatic GEMMs (S-GEMMs) by directly targeting the cell/tissue/organ of interest. The oviduct, or fallopian tube in humans, is considered the tissue-of-origin of the most common ovarian cancer, high-grade serous ovarian carcinomas (HGSCs). HGSCs initiate in the region of the fallopian tube distal to the uterus, located adjacent to the ovary, but not the proximal fallopian tube. However, traditional mouse models of HGSC target the entire oviduct, and thus do not recapitulate the human condition. We present a method of DNA, RNA, or ribonucleoprotein (RNP) solution microinjection into the oviduct lumen and in vivo electroporation to target mucosal epithelial cells in restricted regions along the oviduct. There are several advantages of this method for cancer modeling, such as 1) high adaptability in targeting the area/tissue/organ and region of electroporation, 2) high flexibility in targeted cell types (cellular pliancy) when used in combination with specific promoters for Cas9 expression, 3) high flexibility in the number of electroporated cells (relatively low frequency), 4) no specific mouse line is required (immunocompetent disease modeling), 5) high flexibility in gene mutation combination, and 6) possibility of tracking electroporated cells when used in combination with a Cre reporter line. Thus, this cost-effective method recapitulates human cancer initiation.

Multiple cell types in the oviduct express the prolactin receptor.

Little is known about the physiological role of prolactin in the oviduct. Examining mRNA for all four isoforms of the prolactin receptor (PRLR) in mice by functional oviduct segment and stage of the estrous cycle, we found short form 3 (SF3) to be the most highly expressed, far exceeding the long form (LF) in highly ciliated areas such as the infundibulum, whereas in areas of low ciliation, the SF3 to LF ratio was ~1. SF2 expression was low throughout the oviduct, and SF1 was undetectable. Only in the infundibulum did PRLR ratios change with the estrous cycle. Immunofluorescent localization of SF3 and LF showed an epithelial (both mucosal and mesothelial) distribution aligned with the mRNA results. Despite the high SF3/LF ratio in densely ciliated regions, these regions responded to an acute elevation of prolactin (30 min, intraperitoneal), with LF-tyrosine phosphorylated STAT5 seen within cilia. Collectively, these results show ciliated cells are responsive to prolactin and suggest that prolactin regulates estrous cyclic changes in ciliated cell function in the infundibulum. Changes in gene expression in the infundibulum after prolonged prolactin treatment (7-day) showed prolactin-induced downregulation of genes necessary for cilium development/function, a result supporting localization of PRLRs on ciliated cells, and one further suggesting hyperprolactinemia would negatively impact ciliated cell function and therefore fertility. Flow cytometry, single-cell RNAseq, and analysis of LF-td-Tomato transgenic mice supported expression of PRLRs in at least a proportion of epithelial cells while also hinting at additional roles for prolactin in smooth muscle and other stromal cells.

Glucosamine amends CNS pathology in mucopolysaccharidosis IIIC mouse expressing misfolded HGSNAT.

The majority of mucopolysaccharidosis IIIC (MPS IIIC) patients have missense variants causing misfolding of heparan sulfate acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT), which are potentially treatable with pharmacological chaperones. To test this approach, we generated a novel HgsnatP304L mouse model expressing misfolded HGSNAT Pro304Leu variant. HgsnatP304L mice present deficits in short-term and working/spatial memory 2-4 mo earlier than previously described constitutive knockout Hgsnat-Geo mice. HgsnatP304L mice also show augmented severity of neuroimmune response, synaptic deficits, and neuronal storage of misfolded proteins and gangliosides compared with Hgsnat-Geo mice. Expression of misfolded human Pro311Leu HGSNAT protein in cultured hippocampal Hgsnat-Geo neurons further reduced levels of synaptic proteins. Memory deficits and majority of brain pathology were rescued in mice receiving HGSNAT chaperone, glucosamine. Our data for the first time demonstrate dominant-negative effects of misfolded HGSNAT Pro304Leu variant and show that they are treatable by oral administration of glucosamine. This suggests that patients affected with mutations preventing normal folding of the enzyme can benefit from chaperone therapy.

Reprogramming Mouse Oviduct Epithelial Cells Using In Vivo Electroporation and CRISPR/Cas9-Mediated Genetic Manipulation.

Advances in gene editing tools such as CRISPR/Cas9 have made precise in vivo gene editing possible, opening up avenues of research into somatic cell reprograming to study adult stem cells, homeostasis, and malignant transformation. Here we describe a method for CRISPR/Cas9 mediated in vivo gene editing, in combination with Cre-based lineage tracing via electroporation in the mouse oviduct. This method facilitates the delivery of multiple plasmids into oviduct epithelial cells, sufficient for studying homeostasis and generation of high-grade serous ovarian cancer (HGSOC) models.

Designing Genetically Engineered Mouse Models (GEMMs) Using CRISPR Mediated Genome Editing.

Genetically engineered mouse models (GEMMs) are very powerful tools to study lineage hierarchy and cellular dynamics of stem cells in vivo. Stem cell behavior in various contexts such as development, normal homeostasis and diseases have been investigated using GEMMs. The strategies to generate GEMMs have drastically changed in the last decade with the development of the CRISPR/Cas9 system for manipulation of the mammalian genome. The advantages of the CRISPR/Cas9 are its simplicity and efficiency. The bioinformatics tools available now allow us to quickly identify appropriate guide RNAs and design experimental conditions to generate the targeted mutation. In addition, the genome can be manipulated directly in the zygote which reduces the time to modify target genes compared to other technologies such as Embryonic Stem (ES) cells. Equally important is that we can manipulate the genome of any mouse background with the CRISPR/Cas9 system which omits time-consuming backcrossing processes, accelerates research and increases flexibility. Here, we will summarize basic allelic types and our standard strategies of how to generate them.

Protocol to generate mouse oviduct epithelial organoids for viral transduction and whole-mount 3D imaging.

Epithelial cells lining the oviduct/fallopian tube are essential in reproduction and have been identified as the cell-of-origin in high-grade serous ovarian carcinoma (HGSOC). This protocol describes the generation of organoids from mouse oviduct epithelial cells, providing a powerful in vitro tool to study epithelial homeostasis and malignant transformation. We also outline a protocol for whole-mount immunofluorescence and 3D confocal imaging. In addition, we describe approaches of viral transduction to investigate gene function in organoid development and epithelial cell behavior. For complete details on the use and execution of this profile, please refer to Ford et al. (2021).

Oviduct epithelial cells constitute two developmentally distinct lineages that are spatially separated along the distal-proximal axis.

Owing to technical advances in single-cell biology, the appreciation of cellular heterogeneity has increased, which has aided our understanding of organ function, homeostasis, and disease progression. The oviduct (also known as the fallopian tube) is the distalmost portion of the female reproductive tract. It is essential for reproduction and the proposed origin of high-grade serous ovarian carcinoma (HGSOC). In mammals, the oviduct is morphologically segmented along the ovary-uterus axis into four evolutionally conserved regions. It is unclear, however, if there is a diversification of epithelial cell characteristics between these regions. In this study, we identify transcriptionally distinct populations of secretory and multiciliated cells restricted to the distal and proximal regions of the oviduct. We demonstrate that distal and proximal populations are distinct lineages specified early in Müllerian duct development and are maintained separately. These results aid our understanding of epithelial development, homeostasis, and initiation of disease from the oviduct.

Modeling High-Grade Serous Ovarian Carcinoma Using a Combination of In Vivo Fallopian Tube Electroporation and CRISPR-Cas9-Mediated Genome Editing.

Ovarian cancer is the most lethal gynecologic cancer to date. High-grade serous ovarian carcinoma (HGSOC) accounts for most ovarian cancer cases, and it is most frequently diagnosed at advanced stages. Here, we developed a novel strategy to generate somatic ovarian cancer mouse models using a combination of in vivo electroporation and CRISPR-Cas9-mediated genome editing. Mutation of tumor suppressor genes associated with HGSOC in two different combinations (Brca1, Tp53, Pten with and without Lkb1) resulted in successfully generation of HGSOC, albeit with different latencies and pathophysiology. Implementing Cre lineage tracing in this system enabled visualization of peritoneal micrometastases in an immune-competent environment. In addition, these models displayed copy number alterations and phenotypes similar to human HGSOC. Because this strategy is flexible in selecting mutation combinations and targeting areas, it could prove highly useful for generating mouse models to advance the understanding and treatment of ovarian cancer. SIGNIFICANCE: This study unveils a new strategy to generate genetic mouse models of ovarian cancer with high flexibility in selecting mutation combinations and targeting areas.

Anatomical and cellular heterogeneity in the mouse oviduct-its potential roles in reproduction and preimplantation development†.

The oviduct/fallopian tube is a tube-like structure that extends from the uterus to the ovary. It is an essential reproductive organ that provides an environment for internal fertilization and preimplantation development. However, our knowledge of its regional and cellular heterogeneity is still limited. Here, we examined the anatomical complexity of mouse oviducts using modern imaging techniques and fluorescence reporter lines. We found that there are consistent coiling patterns and turning points in the coiled mouse oviduct that serve as reliable landmarks for luminal morphological regionalities. We also found previously unrecognized anatomical structures in the isthmus and uterotubal junction, which likely play roles in reproduction. Furthermore, we demarcated the ampulla-isthmus junction as a distinct region. Taken together, the oviduct mucosal epithelium has highly diverse structures with distinct epithelial cell populations, reflecting its complex functions in reproduction.

The novel aminoglycoside, ELX-02, permits CTNSW138X translational read-through and restores lysosomal cystine efflux in cystinosis.

BACKGROUND: Cystinosis is a rare disorder caused by recessive mutations of the CTNS gene. Current therapy decreases cystine accumulation, thus slowing organ deterioration without reversing renal Fanconi syndrome or preventing eventual need for a kidney transplant.15-20% of cystinosis patients harbour at least one nonsense mutation in CTNS, leading to premature end of translation of the transcript. Aminoglycosides have been shown to permit translational read-through but have high toxicity level, especially in the kidney and inner ear. ELX-02, a modified aminoglycoside, retains it read-through ability without the toxicity. METHODS AND FINDINGS: We ascertained the toxicity of ELX-02 in cells and in mice as well as the effect of ELX-02 on translational read-through of nonsense mutations in cystinotic mice and human cells. ELX-02 was not toxic in vitro or in vivo, and permitted read-through of nonsense mutations in cystinotic mice and human cells. CONCLUSIONS: ELX-02 has translational read-through activity and produces a functional CTNS protein, as evidenced by reduced cystine accumulation. This reduction is comparable to cysteamine treatment. ELX-02 accumulates in the kidney but neither cytotoxicity nor nephrotoxicity was observed.

Breakthroughs and challenges of modern developmental biology and reproductive medicine.

In recent decades we have witnessed unprecedented progress in the field of the developmental biology of mammals. Building on 20th century discoveries, we have managed to increase our understanding of the molecular and cellular mechanisms governing early mammalian embryogenesis and link them to other biological questions, such as stem cells, regeneration, cancer, or tissue and organ formation. Consequently, it has also led to a creation of a completely new branch of reproductive medicine, i.e. assisted reproductive technology (ART). In this Special Issue of The International Journal of Developmental Biology (Int. J. Dev. Biol.) we wished to review state-of-the-art research regarding early mammalian development, from fertilization up to the implantation stage, and discuss its potential meaning for practical applications, including ART. As an introduction to the issue we present a compilation of short essays written by the most renowned scientists in the field, working both in basic and clinical research. The essays are dedicated to the greatest breakthroughs and challenges of 21st century developmental biology and reproductive medicine.

Preparation of In Vitro-Transcribed RNA for Microinjection.

Perhaps the most crucial step for success in microinjection of RNAs is the preparation of the in vitro-transcribed RNA.

Microinjection of RNA into Mouse Zygotes.

This protocol describes the injection of RNA into zygotes.

Cell Polarity-Dependent Regulation of Cell Allocation and the First Lineage Specification in the Preimplantation Mouse Embryo.

During the first few days in the mouse preimplantation embryo, two types of cells, polar and apolar cells are generated from spherical totipotent blastomeres. Sequential morphogenetic events, polarization, compaction, and asymmetric division, are essential for the generation of the first distinct cell populations, polar and apolar cells, which establish the outer/inner configuration within the embryo. This leads to position-dependent activation of the Hippo signaling pathway and lineage-specific gene expression to form the trophectoderm and inner cell mass in a blastocyst. It is still unknown how each morphogenetic event is initiated and how it influences subsequent events. In this chapter, we briefly review the two classic models of mouse preimplantation development and discuss recent studies providing novel insights into the self-organizing ability of the preimplantation mouse embryo. Advances in live cell imaging and mathematical modeling contribute a greater understanding to lineage specification and cell fate commitment at the single cell level. Differential molecular and mechanistic characteristics created by the presence and absence of the apical domain in polar and apolar cells, respectively, dictate cell allocation, divisional orientation, and differential activation of the Hippo signaling pathway.

Crispr-Cas9 engineered osteogenesis imperfecta type V leads to severe skeletal deformities and perinatal lethality in mice.

Osteogenesis imperfecta (OI) type V is caused by an autosomal dominant mutation in the IFITM5 gene, also known as BRIL. The c.-14C>T mutation in the 5'UTR of BRIL creates a novel translational start site adding 5 residues (MALEP) in frame with the natural coding of BRIL. A neomorphic function has been proposed for the MALEP-BRIL but the mechanisms at play are still unknown. In order to further understand the effects of MALEP-BRIL in vivo, we generated a knockin (KI) mouse model having the exact genetic -14C>T replica of patients with OI type V. Live KI descendants were never obtained from 2 male mosaic founders. Skeletal staining with alizarin red/alcian blue and µCT imaging of KI embryos revealed striking skeletal anomalies such as hypomineralized skull, short and bent long bones, and frail and wavy ribs. Histology and histochemical labeling revealed that midshaft of long bones was filled with hypertrophic chondrocytes, lacked a defined primary ossification center with the absence of defined cortices. Gene expression monitoring at E15.5 and E17.5 showed no change in Osx but decreased Bril itself as well as other differentiated osteoblast markers (Ibsp, Bglap, Sost). However, upregulation of Ptgs2 and Nr4a3 suggested that a pro-inflammatory reaction was activated. Primary osteoblasts from KI calvaria showed delayed differentiation and mineralization, with decreased abundance of BRIL. However, the upregulation AdipoQ and Fabp4 in young cultures indicated a possible switch in fate towards adipogenesis. Altogether our data suggest that the low level expression of MALEP-BRIL in Osx+ mesenchymal progenitors blunted their further differentiation into mature osteoblasts, which may have resulted in part from an inflammatory response.