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1.
Cell Syst ; 15(5): 411-424.e9, 2024 May 15.
Article in English | MEDLINE | ID: mdl-38754365

ABSTRACT

The snapshot nature of single-cell transcriptomics presents a challenge for studying the dynamics of cell fate decisions. Metabolic labeling and splicing can provide temporal information at single-cell level, but current methods have limitations. Here, we present a framework that overcomes these limitations: experimentally, we developed sci-FATE2, an optimized method for metabolic labeling with increased data quality, which we used to profile 45,000 embryonic stem (ES) cells differentiating into neural tube identities. Computationally, we developed a two-stage framework for dynamical modeling: VelvetVAE, a variational autoencoder (VAE) for velocity inference that outperforms all other tools tested, and VelvetSDE, a neural stochastic differential equation (nSDE) framework for simulating trajectory distributions. These recapitulate underlying dataset distributions and capture features such as decision boundaries between alternative fates and fate-specific gene expression. These methods recast single-cell analyses from descriptions of observed data to models of the dynamics that generated them, providing a framework for investigating developmental fate decisions.


Subject(s)
Cell Differentiation , Single-Cell Analysis , Transcriptome , Single-Cell Analysis/methods , Cell Differentiation/genetics , Transcriptome/genetics , Animals , Mice , Gene Expression Profiling/methods , Embryonic Stem Cells , Humans
2.
Nat Commun ; 12(1): 6926, 2021 12 03.
Article in English | MEDLINE | ID: mdl-34862376

ABSTRACT

Animals are essential genetic tools in scientific research and global resources in agriculture. In both arenas, a single sex is often required in surplus. The ethical and financial burden of producing and culling animals of the undesired sex is considerable. Using the mouse as a model, we develop a synthetic lethal, bicomponent CRISPR-Cas9 strategy that produces male- or female-only litters with one hundred percent efficiency. Strikingly, we observe a degree of litter size compensation relative to control matings, indicating that our system has the potential to increase the yield of the desired sex in comparison to standard breeding designs. The bicomponent system can also be repurposed to generate postnatal sex-specific phenotypes. Our approach, harnessing the technological applications of CRISPR-Cas9, may be applicable to other vertebrate species, and provides strides towards ethical improvements for laboratory research and agriculture.


Subject(s)
CRISPR-Cas Systems/genetics , Gene Editing/methods , Sex Determination Processes/genetics , Animal Husbandry , Animals , Female , Litter Size/genetics , Male , Mice , Mice, Transgenic , Models, Animal , Pregnancy , Selective Breeding , Synthetic Lethal Mutations
3.
Curr Biol ; 28(22): R1313-R1324, 2018 11 19.
Article in English | MEDLINE | ID: mdl-30458153

ABSTRACT

Fundamental differences exist between males and females, encompassing anatomy, physiology, behaviour, and genetics. Such differences undoubtedly play a part in the well documented, yet poorly understood, disparity in disease susceptibility between the sexes. Although traditionally attributed to gonadal sex hormone effects, recent work has begun to shed more light on the contribution of genetics - and in particular the sex chromosomes - to these sexual dimorphisms. Here, we explore the accumulating evidence for a significant genetic component to mammalian sexual dimorphism through the paradigm of sex chromosome evolution. The differences between the extant X and Y chromosomes, at both a sequence and regulatory level, arose across 166 million years. A functional result of these differences is cell autonomous sexual dimorphism. By understanding the process that changed a pair of homologous ancestral autosomes into the extant mammalian X and Y, we believe it easier to consider the mechanisms that may contribute to hormone-independent male-female differences. We highlight key roles for genes with homologues present on both sex chromosomes, where the X-linked copy escapes X chromosome inactivation. Finally, we summarise current experimental paradigms and suggest areas for developments to further increase our understanding of cell autonomous sexual dimorphism in the context of health and disease.


Subject(s)
Mammals/genetics , Sex Characteristics , Sex Chromosomes/genetics , Animals , Biological Evolution , Evolution, Molecular , Female , Humans , Male , Sex Factors , X Chromosome/genetics , Y Chromosome/genetics
4.
Brain ; 136(Pt 4): 1067-82, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23518709

ABSTRACT

Axonal degeneration is a major cause of permanent disability in the inflammatory demyelinating disease multiple sclerosis, but no therapies are known to be effective in axonal protection. Sodium channel blocking agents can provide effective protection of axons in the white matter in experimental models of multiple sclerosis, but the mechanism of action (directly on axons or indirectly via immune modulation) remains uncertain. Here we have examined the efficacy of two sodium channel blocking agents to protect white matter axons in two forms of experimental autoimmune encephalomyelitis, a common model of multiple sclerosis. Safinamide is currently in phase III development for use in Parkinson's disease based on its inhibition of monoamine oxidase B, but the drug is also a potent state-dependent inhibitor of sodium channels. Safinamide provided significant protection against neurological deficit and axonal degeneration in experimental autoimmune encephalomyelitis, even when administration was delayed until after the onset of neurological deficit. Protection of axons was associated with a significant reduction in the activation of microglia/macrophages within the central nervous system. To clarify which property of safinamide was likely to be involved in the suppression of the innate immune cells, the action of safinamide on microglia/macrophages was compared with that of the classical sodium channel blocking agent, flecainide, which has no recognized monoamine oxidase B activity, and which has previously been shown to protect the white matter in experimental autoimmune encephalomyelitis. Flecainide was also potent in suppressing microglial activation in experimental autoimmune encephalomyelitis. To distinguish whether the suppression of microglia was an indirect consequence of the reduction in axonal damage, or possibly instrumental in the axonal protection, the action of safinamide was examined in separate experiments in vitro. In cultured primary rat microglial cells activated by lipopolysaccharide, safinamide potently suppressed microglial superoxide production and enhanced the production of the anti-oxidant glutathione. The findings show that safinamide is effective in protecting axons from degeneration in experimental autoimmune encephalomyelitis, and that this effect is likely to involve a direct effect on microglia that can result in a less activated phenotype. Together, this work highlights the potential of safinamide as an effective neuroprotective agent in multiple sclerosis, and implicates microglia in the protective mechanism.


Subject(s)
Alanine/analogs & derivatives , Benzylamines/therapeutic use , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Flecainide/therapeutic use , Microglia/drug effects , Multiple Sclerosis/drug therapy , Multiple Sclerosis/pathology , Neuroprotective Agents/therapeutic use , Alanine/administration & dosage , Alanine/therapeutic use , Animals , Benzylamines/administration & dosage , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/chemically induced , Flecainide/administration & dosage , Male , Microglia/metabolism , Multiple Sclerosis/metabolism , Neuroprotective Agents/administration & dosage , Rats , Sodium Channel Blockers/therapeutic use
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