Coordination of cell proliferation and anterior-posterior axis establishment in the mouse embryo

During development, the growth of the embryo must be coupled to its patterning to ensure correct and timely morphogenesis. In the mouse embryo, migration of the anterior visceral endoderm (AVE) to the prospective anterior establishes the anterior-posterior (A-P) axis. By analysing the distribution of cells in S phase, M phase and G2 from the time just prior to the migration of the AVE until 18 hours after its movement, we show that there is no evidence for differential proliferation along the A-P axis of the mouse embryo. Rather, we have identified that as AVE movements are being initiated, the epiblast proliferates at a much higher rate than the visceral endoderm. We show that these high levels of proliferation in the epiblast are dependent on Nodal signalling and are required for A-P establishment, as blocking cell division in the epiblast inhibits AVE migration. Interestingly, inhibition of migration by blocking proliferation can be rescued by Dkk1. This suggests that the high levels of epiblast proliferation function to move the prospective AVE away from signals that are inhibitory to its migration. The finding that initiation of AVE movements requires a certain level of proliferation in the epiblast provides a mechanism whereby A-P axis development is coordinated with embryonic growth.     

Progressive Myoclonic Epilepsy-Associated Gene KCTD7 is a Regulator of Potassium Conductance in Neurons

The potassium channel tetramerization domain-containing protein 7 (KCTD7) was named after the structural homology of its predicted N-terminal broad complex, tramtrack and bric à brac/poxvirus and zinc finger domain with the T1 domain of the Kv potassium channel, but its expression profile and cellular function are still largely unknown. We have recently reported a homozygous nonsense mutation of KCTD7 in patients with a novel form of autosomal recessive progressive myoclonic epilepsy. Here, we show that KCTD7 expression hyperpolarizes the cell membrane and reduces the excitability of transfected neurons in patch clamp experiments. We found the expression of KCTD7 in the hippocampal and Purkinje cells of the murine brain, an expression profile consistent with our patients’ phenotype. The effect on the plasma membrane resting potential is possibly mediated by Cullin-3, as we demonstrated direct molecular interaction of KCTD7 with Cullin-3 in co-immunoprecipitation assays. Our data link progressive myoclonic epilepsy to an inherited defect of the neuron plasma membrane’s resting potential in the brain.     

The HrpN effector of Erwinia amylovora, which is involved in type III translocation, contributes directly or indirectly to callose elicitation on apple leaves

Erwinia amylovora is responsible for fire blight of apple and pear trees. Its pathogenicity depends on a type III secretion system (T3SS) mediating the translocation of effectors into the plant cell. The DspA/E effector suppresses callose deposition on apple leaves. We found that E. amylovora and Pseudomonas syringae DC3000 tts mutants or peptide flg22 do not trigger callose deposition as strongly as the dspA/E mutant on apple leaves. This suggests that, on apple leaves, callose deposition is poorly elicited by pathogen-associated molecular patterns (PAMPs) such as flg22 or other PAMPs harbored by tts mutants and is mainly elicited by injected effectors or by the T3SS itself. Callose elicitation partly depends on HrpW because an hrpW-dspA/E mutant elicits lower callose deposition than a dspA/E mutant. Furthermore, an hrpN-dspA/E mutant does not trigger callose deposition, indicating that HrpN is required to trigger this plant defense reaction. We showed that HrpN plays a general role in the translocation process. Thus, the HrpN requirement for callose deposition may be explained by its role in translocation: HrpN could be involved in the translocation of other effectors inducing callose deposition. Furthermore, HrpN may also directly contribute to the elicitation process because we showed that purified HrpN induces callose deposition.     

Protective effect of ε-viniferin on β-amyloid peptide aggregation investigated by electrospray ionization mass spectrometry

Abnormal β-amyloid peptide accumulation and aggregation is considered to be responsible for the formation and cerebral deposition of senile plaques in the brains of patients with Alzheimer's disease (AD). Inhibition of the formation of β-amyloid (Aβ) fibrils would be an attractive therapeutic target for the treatment of AD. Resveratrol and its derivatives exhibit a broad range of pharmacological properties such as protection against cardiovascular diseases and cancers, as well as promoting antiaging effects. We reported previously that ε-viniferin glucoside (VG), a resveratrol-derived dimer, strongly inhibits Aβ (25-35) fibril formation in vitro. In this study, we investigated the effects of VG on the aggregation of the full-length peptides (Aβ (1-40) and Aβ (1-42)) and on the β-amyloid-induced toxicity in PC12 cells. VG inhibited Aβ cytotoxicity and the non-covalent complex between VG and Aβ was observed by electrospray ionization mass spectrometry.     

Mannosylated dextran nanoparticles: a pH-sensitive system engineered for immunomodulation through mannose targeting

Biotherapeutic delivery is a rapidly growing field in need of new materials that are easy to modify, are biocompatible, and provide for triggered release of their encapsulated cargo. Herein, we report on a particulate system made of a polysaccharide-based pH-sensitive material that can be efficiently modified to display mannose-based ligands of cell-surface receptors. These ligands are beneficial for antigen delivery, as they enhance internalization and activation of APCs, and are thus capable of modulating immune responses. When compared to unmodified particles or particles modified with a nonspecific sugar residue used in the delivery of antigens to dendritic cells (DCs), the mannosylated particles exhibited enhanced antigen presentation in the context of major histocompatibility complex (MHC) class I molecules. This represents the first demonstration of a mannosylated particulate system that enables enhanced MHC I antigen presentation by DCs in vitro. Our readily functionalized pH-sensitive material may also open new avenues in the development of optimally modulated vaccine delivery systems.     

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders ^1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid ^3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS ⁵. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate. Download video file.^{(37M, mov)}     

Correct patterning of the primitive streak requires the anterior visceral endoderm

Anterior-posterior axis specification in the mouse requires signalling from a specialised extra-embryonic tissue called the anterior visceral endoderm (AVE). AVE precursors are induced at the distal tip of the embryo and move to the prospective anterior. Embryological and genetic analysis has demonstrated that the AVE is required for anterior patterning and for correctly positioning the site of primitive streak formation by inhibiting Nodal activity. We have carried out a genetic ablation of the Hex-expressing cells of the AVE (Hex-AVE) by knocking the Diphtheria toxin subunit A into the Hex locus in an inducible manner. Using this model we have identified that, in addition to its requirement in the anterior of the embryo, the Hex-AVE sub-population has a novel role between 5.5 and 6.5dpc in patterning the primitive streak. Embryos lacking the Hex-AVE display delayed initiation of primitive streak formation and miss-patterning of the anterior primitive streak. We demonstrate that in the absence of the Hex-AVE the restriction of Bmp2 expression to the proximal visceral endoderm is also defective and expression of Wnt3 and Nodal is not correctly restricted to the posterior epiblast. These results, coupled with the observation that reducing Nodal signalling in Hex-AVE ablated embryos increases the frequency of phenotypes observed, suggests that these primitive streak patterning defects are due to defective Nodal signalling. Together, our experiments demonstrate that the AVE is not only required for anterior patterning, but also that specific sub-populations of this tissue are required to pattern the posterior of the embryo.