The complex landscape of haematopoietic lineage commitments is encoded in the coarse-grained endogenous network

Haematopoietic lineage commitments are presented by a canonical roadmap in which haematopoietic stem cells or multipotent progenitors (MPPs) bifurcate into progenitors of more restricted lineages and ultimately mature to terminally differentiated cells. Although transcription factors playing significant roles in cell-fate commitments have been extensively studied, integrating such knowledge into the dynamic models to understand the underlying biological mechanism remains challenging. The hypothesis and modelling approach of the endogenous network has been developed previously and tested in various biological processes and is used in the present study of haematopoietic lineage commitments. The endogenous network is constructed based on the key transcription factors and their interactions that determine haematopoietic cell-fate decisions at each lineage branchpoint. We demonstrate that the process of haematopoietic lineage commitments can be reproduced from the landscape which orchestrates robust states of network dynamics and their transitions. Furthermore, some non-trivial characteristics are unveiled in the dynamical model. Our model also predicted previously under-represented regulatory interactions and heterogeneous MPP states by which distinct differentiation routes are intermediated. Moreover, network perturbations resulting in state transitions indicate the effects of ectopic gene expression on cellular reprogrammes. This study provides a predictive model to integrate experimental data and uncover the possible regulatory mechanism of haematopoietic lineage commitments.

Microtubule inhibitors enhance DNA transfection efficiency through autophagy receptor p62/SQSTM1

Ectopic gene expression is an indispensable tool in biology and medicine. However, it is often limited by the low efficiency of DNA transfection. It is known that depletion of p62/SQSTM1 enhances DNA transfection efficiency by preventing the degradation of transfected DNA. Therefore, p62 is a potential target of drugs to increase transfection efficiency. To identify drugs that enhance transfection efficiency, a non-biased high-throughput screening was applied to over 4,000 compounds from the Osaka University compound library, and their p62-dependency was evaluated. The top-scoring drugs were mostly microtubule inhibitors, such as colchicine and vinblastine, and all of them showed positive effects only in the presence of p62. To understand the mechanisms, the time of p62-dependent ubiquitination was examined using polystyrene beads that were introduced into cells as materials that mimicked transfected DNA. The microtubule inhibitors caused a delay in ubiquitination. Furthermore, the level of phosphorylated p62 at S405, which is required for ubiquitination during autophagosome formation, markedly decreased in the drug-treated cells. These results suggest that microtubule inhibitors inhibit p62-dependent autophagosome formation. Our findings provide new insights into the mechanisms of DNA transfection and also provide a solution to increase DNA transfection efficiency.

PAX6: a transcriptional guardian for glutamatergic fate in the developing neocortex

During forebrain development, the transcription factor PAX6 is highly expressed by progenitors in the dorsal telencephalon (dTel) i.e. the primitive cerebral cortex with a sharp boundary at the pallial-subpallial boundary, thereby establishing the dorso-ventral patterning of the forebrain and regulating the generation of cortical glutamatergic neurons. Strikingly, removal of Pax6led to a diversion away from the glutamatergic identity in a subset of cortical progenitors indicated by ectopic gene expression. We postulate that PAX6 confers glutamatergic fate in progenitors by preventing them from responding to signaling cues such as SHH that can induce abberant fates. In the present study, we used the transgenic mouse model with Pax6conditionally deleted in the cortex using a tamoxifen-inducible Emx1-CreER T2 transgene combined with a floxed Pax6 and an EGFP constructs. Single-cell transcriptome revealed multiple ectopic leanages in cortical progenitors with morphogen-regulated transcriptional signatures upon Pax6deletion. We also undertook a candidate approach to investigate how attenuation of signaling cues by surgical and pharmacological means using in vitroslice culture affect the magnitude of ectopic gene expression in the cortical progenitors. We demonstrated that attenuation of interneuron migration into the cortex and inhibition of SHH signaling pathway in slice culture substatially reduced aberrant gene expression in the cortical progenitors. Our findings suggest that ventral cues from vTel such as SHH possess ventralizing effect on cortical progenitors, this is consistent with a requirement for PAX6 to resist such effects in order to safeguard glutamatergic fate.

A set of shuttle plasmids for gene expression in Acinetobacter baumannii

Infections caused by the emerging opportunistic bacterial pathogen Acinetobacter baumannii are occurring at increasingly alarming rates, and such increase in incidence is further compounded by the development of wide spread multidrug-resistant strains. Yet, our understanding of its pathogenesis and biology remains limited which can be attributed in part to the scarce of tools for molecular genetic analysis of this bacterium. Plasmids based on pWH1277 originally isolated from Acinetobacter calcoaceticus are the only vehicles currently available for ectopic gene expression in Acinetobacter species, which restricts experiments that require simultaneous analysis of multiple genes. Here, we found that plasmids of the IncQ group are able to replicate in A. baumannii and can stably co-reside with derivatives of pWH1277. Furthermore, we have constructed a series of four plasmids that allow inducible expression of Flag-tagged proteins in A. baumannii by arabinose or isopropyl β-d-1-thiogalactopyranoside. Together with constructs previously developed, these plasmids will accommodate the need in genetic analysis of this increasingly important pathogen.

A modular chromosomally integrated toolkit for ectopic gene expression in Vibrio cholerae

The ability to express genes ectopically in bacteria is essential for diverse academic and industrial applications. Two major considerations when utilizing regulated promoter systems for ectopic gene expression are (1) the ability to titrate gene expression by addition of an exogenous inducer and (2) the leakiness of the promoter element in the absence of the inducer. Here, we describe a modular chromosomally integrated platform for ectopic gene expression in Vibrio cholerae. We compare the broadly used promoter elements Ptac and PBAD to versions that have an additional theophylline-responsive riboswitch (Ptac-riboswitch and PBAD-riboswitch). These constructs all exhibited unimodal titratable induction of gene expression, however, max induction varied with Ptac > PBAD > PBAD-riboswitch > Ptac-riboswitch. We also developed a sensitive reporter system to quantify promoter leakiness and show that leakiness for Ptac > Ptac-riboswitch > PBAD; while the newly developed PBAD-riboswitch exhibited no detectable leakiness. We demonstrate the utility of the tightly inducible PBAD-riboswitch construct using the dynamic activity of type IV competence pili in V. cholerae as a model system. The modular chromosomally integrated toolkit for ectopic gene expression described here should be valuable for the genetic study of V. cholerae and could be adapted for use in other species.

PAX6: a transcriptional guardian for glutamatergic fate in the developing neocortex

During forebrain development, the transcription factor PAX6 is highly expressed by progenitors in the dorsal telencephalon (dTel) i.e. the primitive cerebral cortex with a sharp boundary at the pallial-subpallial boundary, thereby establishing the dorso-ventral patterning of the forebrain and regulating the generation of cortical glutamatergic neurons. Strikingly, removal of Pax6 led to a diversion away from the glutamatergic identity in a subset of cortical progenitors indicated by ectopic gene expression. We postulate that PAX6 confers glutamatergic fate in progenitors by preventing them from responding to signaling cues such as SHH that can induce abberant fates. In the present study, we used the transgenic mouse model with Pax6 conditionally deleted in the cortex using a tamoxifen-inducible Emx1-CreER T2 transgene combined with a floxed Pax6 and an EGFP constructs. Single-cell transcriptome revealed multiple ectopic leanages in cortical progenitors with morphogen-regulated transcriptional signatures upon Pax6 deletion. We also undertook a candidate approach to investigate how attenuation of signaling cues by surgical and pharmacological means using in vitro slice culture affect the magnitude of ectopic gene expression in the cortical progenitors. We demonstrated that attenuation of interneuron migration into the cortex and inhibition of SHH signaling pathway in slice culture substatially reduced aberrant gene expression in the cortical progenitors. Our findings suggest that ventral cues from vTel such as SHH possess ventralizing effect on cortical progenitors, this is consistent with a requirement for PAX6 to resist such effects in order to safeguard glutamatergic fate.

A modular chromosomally integrated toolkit for ectopic gene expression in Vibrio cholerae

The ability to express genes ectopically in bacteria is essential for diverse academic and industrial applications. Two major considerations when utilizing regulated promoter systems for ectopic gene expression are (1) the ability to titrate gene expression by addition of an exogenous inducer and (2) the leakiness of the promoter element in the absence of the inducer. Here, we describe a modular chromosomally integrated platform for ectopic gene expression in Vibrio cholerae. We compare the broadly used promoter elements Ptac and PBAD to versions that have an additional theophylline-responsive riboswitch (Ptac-riboswitch and PBAD-riboswitch). These constructs all exhibited unimodal titratable induction of gene expression, however, max induction varied with Ptac > PBAD > PBAD-riboswitch > Ptac-riboswitch. We also developed a sensitive reporter system to quantify promoter leakiness and show that leakiness for Ptac > Ptac-riboswitch > PBAD; while the newly developed PBAD-riboswitch exhibited no detectable leakiness. We demonstrate the utility of the tightly inducible PBAD-riboswitch construct using the dynamic activity of type IV competence pili in V. cholerae as a model system. The modular chromosomally integrated toolkit for ectopic gene expression described here should be valuable for the genetic study of Vibrio cholerae and could be adapted for use in other species.