RESUMEN
Sexual reproduction and meiotic sex are deeply rooted in the eukaryotic tree of life, but mechanisms determining sex or mating types are extremely varied and are only well characterized in a few model organisms1. In malaria parasites, sexual reproduction coincides with transmission to the vector host. Sex determination is non-genetic, with each haploid parasite capable of producing either a male or a female gametocyte in the human host2. The hierarchy of events and molecular mechanisms that trigger sex determination and maintenance of sexual identity are yet to be elucidated. Here we show that the male development 1 (md1) gene is both necessary and sufficient for male fate determination in the human malaria parasite Plasmodium falciparum. We show that Md1 has a dual function stemming from two separate domains: in sex determination through its N terminus and in male development from its conserved C-terminal LOTUS/OST-HTH domain. We further identify a bistable switch at the md1 locus, which is coupled with sex determination and ensures that the male-determining gene is not expressed in the female lineage. We describe one of only a few known non-genetic mechanisms of sex determination in a eukaryote and highlight Md1 as a potential target for interventions that block malaria transmission.
Asunto(s)
Regulación de la Expresión Génica , Malaria Falciparum , Malaria , Parásitos , Procesos de Determinación del Sexo , Transcripción Genética , Animales , Humanos , Malaria/parasitología , Malaria Falciparum/parasitología , Parásitos/genética , Plasmodium falciparum/genética , Reproducción , Masculino , Femenino , Procesos de Determinación del Sexo/genética , Caracteres SexualesRESUMEN
Human neural progenitors derived from pluripotent stem cells develop into electrophysiologically active neurons at heterogeneous rates, which can confound disease-relevant discoveries in neurology and psychiatry. By combining patch clamping, morphological and transcriptome analysis on single-human neurons in vitro, we defined a continuum of poor to highly functional electrophysiological states of differentiated neurons. The strong correlations between action potentials, synaptic activity, dendritic complexity and gene expression highlight the importance of methods for isolating functionally comparable neurons for in vitro investigations of brain disorders. Although whole-cell electrophysiology is the gold standard for functional evaluation, it often lacks the scalability required for disease modeling studies. Here, we demonstrate a multimodal machine-learning strategy to identify new molecular features that predict the physiological states of single neurons, independently of the time spent in vitro. As further proof of concept, we selected one of the potential neurophysiological biomarkers identified in this study-GDAP1L1-to isolate highly functional live human neurons in vitro.
Asunto(s)
Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Potenciales de Acción/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Electrofisiología , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Aprendizaje Automático , Neuronas/metabolismo , Técnicas de Placa-Clamp , Células Madre Pluripotentes , ARNRESUMEN
The brain's serotonergic system centrally regulates several physiological processes and its dysfunction has been implicated in the pathophysiology of several neuropsychiatric disorders. While in the past our understanding of serotonergic neurotransmission has come mainly from mouse models, the development of pluripotent stem cell and induced fibroblast-to-neuron (iN) transdifferentiation technologies has revolutionized our ability to generate human neurons in vitro. Utilizing these techniques and a novel lentiviral reporter for serotonergic neurons, we identified and overexpressed key transcription factors to successfully generate human serotonergic neurons. We found that overexpressing the transcription factors NKX2.2, FEV, GATA2 and LMX1B in combination with ASCL1 and NGN2 directly and efficiently generated serotonergic neurons from human fibroblasts. Induced serotonergic neurons (iSNs) showed increased expression of specific serotonergic genes that are known to be expressed in raphe nuclei. iSNs displayed spontaneous action potentials, released serotonin in vitro and functionally responded to selective serotonin reuptake inhibitors (SSRIs). Here, we demonstrate the efficient generation of functional human serotonergic neurons from human fibroblasts as a novel tool for studying human serotonergic neurotransmission in health and disease.
Asunto(s)
Técnicas Citológicas/métodos , Fibroblastos/fisiología , Neuronas Serotoninérgicas/fisiología , Animales , Astrocitos/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular , Transdiferenciación Celular/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/metabolismo , Vectores Genéticos , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Células Madre Embrionarias Humanas/fisiología , Humanos , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Lentivirus/genética , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma , Proteínas de Pez CebraRESUMEN
In anesthetized and immobilized domestic cats, we have studied the effects of brief reversible inactivation (by cooling to 10 degrees C) of the ipsilateral or contralateral postero-temporal visual (PTV) cortices on: 1) the magnitude of spike-responses of neurons in striate cortex (cytoarchitectonic area 17, area V1) to optimized sine-wave modulated contrast-luminosity gratings confined to the classical receptive fields (CRFs) and 2) the relative strengths of modulation of CRF-induced spike-responses by gratings extending into the extra-classical receptive field (ECRF). Consistent with our previous reports (Bardy et al., 2006; Huang et al., 2007), inactivation of ipsilateral PTV cortex (presumed homologue of primate infero-temporal cortex) resulted in significant reversible changes (almost all substantial reductions) in the magnitude of spike-responses to CRF-confined stimuli in about half of the V1 neurones. Similarly, in half of the present sample, inactivation of ipsilateral PTV cortex resulted in significant reversible changes (in over 70% of cases, reduction) in the relative strength of ECRF modulation of the CRF-induced spike-responses. By contrast, despite the fact that receptive fields of all V1 cells tested were located within 5 degrees of representation of the zero vertical meridian, inactivation of contralateral PTV cortex only rarely resulted in significant (yet invariably small) changes in the magnitude of spike-responses to CRF-confined stimuli or significant (again invariably small) changes in the relative strength of ECRF modulation of spike-responses. Thus, the ipsilateral, but not contralateral, 'higher-order' visual cortical areas make significant contribution not only to the magnitude of CRF-induced spike-responses but also to the relative strengths of ECRF-induced modulation of the spike-responses of V1 neurons. Therefore, the feedback signals originating from the ipsilateral higher-order cortical areas appear to make an important contribution to contextual modulation of responses of neurons in the primary visual cortices.