Phenotypic Analysis of Early Neurogenesis in a Mouse Chimeric Embryo and Stem Cell-Based Neuruloid Model.

Analyzing the impact of genetic mutations on early neurogenesis of mammalian embryos in conventional mouse mutant models is laborious and time-consuming. To overcome these constraints and to fast-track the phenotypic analysis, we developed a protocol that harnesses the amenability of engineering genetic modifications in embryonic stem cells from which mid-gestation mouse chimeras and in vitro neuruloids are generated. These stem cell-based chimera and neuruloid experimental models allow phenotyping at early developmental time points of neurogenesis.

Rat epiblast-derived stem cells recapitulate the attributes of pre-gastrulation epiblast.

Iwatsuki and colleagues have generated self-renewing pluripotent stem cells from the pre-gastrulation epiblast of the rat embryo and from other cellular sources: rat embryonic stem cells (rESCs) and epiblast-like cells derived from the rESCs. These rat epiblast-derived stem cells (rEpiSCs) display germ-line competence that is characteristic of mouse formative stem cells and early signature of specification of germ layer lineages typical of primed state mouse epiblast stem cells.

Mammalian gastrulation: signalling activity and transcriptional regulation of cell lineage differentiation and germ layer formation.

The interplay of signalling input and downstream transcriptional activity is the key molecular attribute driving the differentiation of germ layer tissue and the specification of cell lineages within each germ layer during gastrulation. This review delves into the current understanding of signalling and transcriptional control of lineage development in the germ layers of mouse embryo and non-human primate embryos during gastrulation and highlights the inter-species conservation and divergence of the cellular and molecular mechanisms of germ layer development in the human embryo.

Identification and Visualization of Protein Expression in Whole Mouse Embryos by Immunofluorescence.

Mouse embryo studies are pivotal for the understanding of early development. Analysis of the spatial and temporal changes of protein expression during development of a mouse embryo allows us to identify the genetic basis of errors of development in animal disease models. Immunofluorescence is a powerful technique to study the localization and variation in expression pattern of specific proteins in cells, tissues, and organs.

Loss of Impacts Neurulation and Cranial Neural Crest Specification During Early Head Development.

The specification of anterior head tissue in the late gastrulation mouse embryo relies on signaling cues from the visceral endoderm and anterior mesendoderm (AME). Genetic loss-of-function studies have pinpointed a critical requirement of LIM homeobox 1 (LHX1) transcription factor in these tissues for the formation of the embryonic head. Transcriptome analysis of embryos with gain-of-function LHX1 activity identified the forkhead box gene, as one downstream target of LHX1 in late-gastrulation E7.

Visualization of the Cartilage and Bone Elements in the Craniofacial Structures by Alcian Blue and Alizarin Red Staining.

Craniofacial morphogenesis is underpinned by orchestrated growth and form-shaping activity of skeletal and soft tissues in the head and face. Disruptions during development can lead to dysmorphology of the skull, jaw, and the pharyngeal structures. Developmental disorders can be investigated in animal models to elucidate the molecular and cellular consequences of the morphogenetic defects.

Elucidation of Gene Expression Patterns in the Craniofacial Tissues of Mouse Embryos by Wholemount In Situ Hybridization.

Analysis of animal models allows a deeper understanding of craniofacial development in health and diseases of humans. Wholemount in situ hybridization (WISH) is an informative technique to visualize gene expression in tissues across the developmental stages of embryos. The principle of WISH is based on the complementary binding (hybridization) of the DNA/RNA probe to the target transcript.

TWIST1 and chromatin regulatory proteins interact to guide neural crest cell differentiation.

Protein interaction is critical molecular regulatory activity underlining cellular functions and precise cell fate choices. Using TWIST1 BioID-proximity-labeling and network propagation analyses, we discovered and characterized a TWIST-chromatin regulatory module (TWIST1-CRM) in the neural crest cells (NCC). Combinatorial perturbation of core members of TWIST1-CRM: TWIST1, CHD7, CHD8, and WHSC1 in cell models and mouse embryos revealed that loss of the function of the regulatory module resulted in abnormal differentiation of NCCs and compromised craniofacial tissue patterning.

Nuclear F-actin counteracts nuclear deformation and promotes fork repair during replication stress.

Filamentous actin (F-actin) provides cells with mechanical support and promotes the mobility of intracellular structures. Although F-actin is traditionally considered to be cytoplasmic, here we reveal that nuclear F-actin participates in the replication stress response. Using live and super-resolution imaging, we find that nuclear F-actin is polymerized in response to replication stress through a pathway regulated by ATR-dependent activation of mTORC1, and nucleation through IQGAP1, WASP and ARP2/3.

Critical Role for Cold Shock Protein YB-1 in Cytokinesis.

High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cancer cell lines and in immortalized fibroblasts resulted in cytokinesis failure and consequent multinucleation.

Replication stress induces mitotic death through parallel pathways regulated by WAPL and telomere deprotection.

Mitotic catastrophe is a broad descriptor encompassing unclear mechanisms of cell death. Here we investigate replication stress-driven mitotic catastrophe in human cells and identify that replication stress principally induces mitotic death signalled through two independent pathways. In p53-compromised cells we find that lethal replication stress confers WAPL-dependent centromere cohesion defects that maintain spindle assembly checkpoint-dependent mitotic arrest in the same cell cycle.

Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response.

To investigate how chromatin architecture is spatiotemporally organized at a double-strand break (DSB) repair locus, we established a biophysical method to quantify chromatin compaction at the nucleosome level during the DNA damage response (DDR). The method is based on phasor image-correlation spectroscopy of histone fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) microscopy data acquired in live cells coexpressing H2B-eGFP and H2B-mCherry. This multiplexed approach generates spatiotemporal maps of nuclear-wide chromatin compaction that, when coupled with laser microirradiation-induced DSBs, quantify the size, stability, and spacing between compact chromatin foci throughout the DDR.

Author Correction: EWS-FLI1 increases transcription to cause R-loops and block BRCA1 repair in Ewing sarcoma.

In this Letter, the sentence beginning "This work was funded…." in the Acknowledgements should have read "CPRIT (RP140105) to J.C.

Sorafenib improves alkylating therapy by blocking induced inflammation, invasion and angiogenesis in breast cancer cells.

Molecular targeted compounds are emerging as a strategy to improve classical chemotherapy. Herein, we describe that using low dose of the multikinase inhibitor sorafenib improves cyclophosphamide antitumor activity by inhibiting angiogenesis, metastasis and promoting tumor healing in MDA-MB231 xenografts and the 4T1-12B syngeneic breast cancer metastasis model. Mechanistic studies in MDA-MB231 cells revealed that alkylation upregulates inflammatory genes/proteins such as COX-2, IL8, CXCL2 and MMP1 in a MEK1/2-ERK1/2-dependent manner.

EWS-FLI1 increases transcription to cause R-loops and block BRCA1 repair in Ewing sarcoma.

Ewing sarcoma is an aggressive paediatric cancer of the bone and soft tissue. It results from a chromosomal translocation, predominantly t(11;22)(q24:q12), that fuses the N-terminal transactivation domain of the constitutively expressed EWSR1 protein with the C-terminal DNA binding domain of the rarely expressed FLI1 protein. Ewing sarcoma is highly sensitive to genotoxic agents such as etoposide, but the underlying molecular basis of this sensitivity is unclear.

Alkylating Agent-Induced NRF2 Blocks Endoplasmic Reticulum Stress-Mediated Apoptosis via Control of Glutathione Pools and Protein Thiol Homeostasis.

Alkylating agents are a commonly used cytotoxic class of anticancer drugs. Understanding the mechanisms whereby cells respond to these drugs is key to identify means to improve therapy while reducing toxicity. By integrating genome-wide gene expression profiling, protein analysis, and functional cell validation, we herein demonstrated a direct relationship between NRF2 and Endoplasmic Reticulum (ER) stress pathways in response to alkylating agents, which is coordinated by the availability of glutathione (GSH) pools.

The long lifespan of two bat species is correlated with resistance to protein oxidation and enhanced protein homeostasis.

Altered structure, and hence function, of cellular macromolecules caused by oxidation can contribute to loss of physiological function with age. Here, we tested whether the lifespan of bats, which generally live far longer than predicted by their size, could be explained by reduced protein damage relative to short-lived mice. We show significantly lower protein oxidation (carbonylation) in Mexican free-tailed bats (Tadarida brasiliensis) relative to mice, and a trend for lower oxidation in samples from cave myotis bats (Myotis velifer) relative to mice.

Protein stability and resistance to oxidative stress are determinants of longevity in the longest-living rodent, the naked mole-rat.

The widely accepted oxidative stress theory of aging postulates that aging results from accumulation of oxidative damage. Surprisingly, data from the longest-living rodent known, naked mole-rats [MRs; mass 35 g; maximum lifespan (MLSP) > 28.3 years], when compared with mice (MLSP 3.