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. Detecting the antigens with their specific antibodies labeled with fluorescent probes allows visualization of proteins at the cellular level. Here, we provide the optimized protocol of immunostaining whole mouse embryos at embryonic stages E7.5 to E11.5.

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. The bound probe can then be visualized by an enzymatic color reaction to delineate the expression pattern of transcripts within a tissue. Here we describe an optimized method to perform in situ hybridization in mouse embryos.

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. A first step in determining the disruption in the development of the head and face is to analyze the phenotypic features of the skeletal tissues. Examination of the anatomy of bones and cartilage over time and space will identify structural defects of head structures and guide follow-up analysis of the molecular and cellular attributes associated with the defects. Here we describe a protocol to simultaneously visualize the cartilage and bone elements by Alcian blue and Alizarin red staining, respectively, of wholemount specimens in mouse models.

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. Following NCC delamination, low level of TWIST1-CRM activity is instrumental to stabilize the early NCC signatures and migratory potential by repressing the neural stem cell programs. High level of TWIST1 module activity at later phases commits the cells to the ectomesenchyme. Our study further revealed the functional interdependency of TWIST1 and potential neurocristopathy factors in NCC development.

Shaping the head and face during development relies on a complex ballet of molecular signals that orchestrates the movement and specialization of various groups of cells. In animals with a backbone for example, neural crest cells (NCCs for short) can march long distances from the developing spine to become some of the tissues that form the skull and cartilage but also the pigment cells and nervous system. NCCs mature into specific cell types thanks to a complex array of factors which trigger a precise sequence of binary fate decisions at the right time and place. Amongst these factors, the protein TWIST1 can set up a cascade of genetic events that control how NCCs will ultimately form tissues in the head. To do so, the TWIST1 protein interacts with many other molecular actors, many of which are still unknown. To find some of these partners, Fan et al. studied TWIST1 in the NCCs of mice and cells grown in the lab. The experiments showed that TWIST1 interacted with CHD7, CHD8 and WHSC1, three proteins that help to switch genes on and off, and which contribute to NCCs moving across the head during development. Further work by Fan et al. then revealed that together, these molecular actors are critical for NCCs to form cells that will form facial bones and cartilage, as opposed to becoming neurons. This result helps to show that there is a trade-off between NCCs forming the face or being part of the nervous system. One in three babies born with a birth defect shows anomalies of the head and face: understanding the exact mechanisms by which NCCs contribute to these structures may help to better predict risks for parents, or to develop new approaches for treatment.

Loss of Foxd4 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, Foxd4, as one downstream target of LHX1 in late-gastrulation E7.75 embryos. Our analysis of single-cell RNA-seq data show Foxd4 is co-expressed with Lhx1 and Foxa2 in the anterior midline tissue of E7.75 mouse embryos, and in the anterior neuroectoderm (ANE) at E8.25 alongside head organizer genes Otx2 and Hesx1. To study the role of Foxd4 during early development we used CRISPR-Cas9 gene editing in mouse embryonic stem cells (mESCs) to generate bi-allelic frameshift mutations in the coding sequence of Foxd4. In an in vitro model of the anterior neural tissues derived from Foxd4-loss of function (LOF) mESCs and extraembryonic endoderm cells, expression of head organizer genes as well as Zic1 and Zic2 was reduced, pointing to a need for FOXD4 in regulating early neuroectoderm development. Mid-gestation mouse chimeras harbouring Foxd4-LOF mESCs displayed craniofacial malformations and neural tube closure defects. Furthermore, our in vitro data showed a loss of FOXD4 impacts the expression of cranial neural crest markers Twist1 and Sox9. Our findings have demonstrated that FOXD4 is essential in the AME and later in the ANE for rostral neural tube closure and neural crest specification during head development.

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. During replication stress, nuclear F-actin increases the nuclear volume and sphericity to counteract nuclear deformation. Furthermore, F-actin and myosin II promote the mobility of stressed-replication foci to the nuclear periphery through increasingly diffusive motion and directed movements along the nuclear actin filaments. These actin functions promote replication stress repair and suppress chromosome and mitotic abnormalities. Moreover, we find that nuclear F-actin is polymerized in vivo in xenograft tumours after treatment with replication-stress-inducing chemotherapeutic agents, indicating that this pathway has a role in human disease.

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. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging we found that YB-1 was essential for orchestrating the spatio-temporal distribution of the microtubules, ß-actin and the chromosome passenger complex (CPC) to define the cleavage plane. We show that phosphorylation at six serine residues was essential for cytokinesis, of which novel sites were identified using mass spectrometry. Using atomistic modelling we show how phosphorylation at multiple sites alters YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining precisely how YB-1 regulates cell division.

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. Mitotic arrest then drives cohesion fatigue and triggers mitotic death through a primary pathway of BAX/BAK-dependent apoptosis. Simultaneously, a secondary mitotic death pathway is engaged through non-canonical telomere deprotection, regulated by TRF2, Aurora B and ATM. Additionally, we find that suppressing mitotic death in replication stressed cells results in distinct cellular outcomes depending upon how cell death is averted. These data demonstrate how replication stress-induced mitotic catastrophe signals cell death with implications for cancer treatment and cancer genome evolution.

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. Using this technology, we identify that ataxia-telangiectasia mutated (ATM) and RNF8 regulate rapid chromatin decompaction at DSBs and formation of compact chromatin foci surrounding the repair locus. This chromatin architecture serves to demarcate the repair locus from the surrounding nuclear environment and modulate 53BP1 mobility.

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.R." rather than "CPRIT (RP150445) to J.C.R." This error has been corrected online.

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. These proteins enrich the secretome of cancer cells, stimulating cell invasion and angiogenesis via autocrine and paracrine mechanisms. Sorafenib inhibits MEK1/2-ERK1/2 pathway thereby decreasing inflammatory genes and mitigating cell invasion and angiogenesis at basal and alkylation-induced conditions whereas NRF2 and ER stress pathways involved in alkylation survival are not affected. In non-invasive/non-angiogenic breast cancer cells (SKBR3 and MCF7), alkylation did not elicit inflammatory responses with the only sorafenib effect being ERK1/2-independent ROS-dependent cytotoxicity when using higher drug concentrations. In summary, our data show that alkylating agents may elicit inflammatory responses that seems to contribute to malignant progression in specific breast cancer cells. Identifying and targeting drivers of this phenotype may offer opportunities to optimize combined drug regimens between classical chemotherapeutics and targeted agents.

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. Here we show that Ewing sarcoma cells display alterations in regulation of damage-induced transcription, accumulation of R-loops and increased replication stress. In addition, homologous recombination is impaired in Ewing sarcoma owing to an enriched interaction between BRCA1 and the elongating transcription machinery. Finally, we uncover a role for EWSR1 in the transcriptional response to damage, suppressing R-loops and promoting homologous recombination. Our findings improve the current understanding of EWSR1 function, elucidate the mechanistic basis of the sensitivity of Ewing sarcoma to chemotherapy (including PARP1 inhibitors) and highlight a class of BRCA-deficient-like tumours.

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. GSH is essential for both drug detoxification and protein thiol homeostasis within the ER, thus inhibiting ER stress induction and promoting survival, an effect independent of its antioxidant role. NRF2 accumulation induced by alkylating agents resulted in increased GSH synthesis via GCLC/GCLM enzyme, and interfering with this NRF2 response by either NRF2 knockdown or GCLC/GCLM inhibition with buthionine sulfoximine caused accumulation of damaged proteins within the ER, leading to PERK-dependent apoptosis. Conversely, upregulation of NRF2, through KEAP1 depletion or NRF2-myc overexpression, or increasing GSH levels with N-acetylcysteine or glutathione-ethyl-ester, decreased ER stress and abrogated alkylating agents-induced cell death. Based on these results, we identified a subset of lung and head-and-neck carcinomas with mutations in either KEAP1 or NRF2/NFE2L2 genes that correlate with NRF2 target overexpression and poor survival. In KEAP1-mutant cancer cells, NRF2 knockdown and GSH depletion increased cell sensitivity via ER stress induction in a mechanism specific to alkylating drugs. Overall, we show that the NRF2-GSH influence on ER homeostasis implicates defects in NRF2-GSH or ER stress machineries as affecting alkylating therapy toxicity. Mol Cancer Ther; 15(12); 3000-14. ©2016 AACR.