RESUMEN
MYT1L is an autism spectrum disorder (ASD)-associated transcription factor that is expressed in virtually all neurons throughout life. How MYT1L mutations cause neurological phenotypes and whether they can be targeted remains enigmatic. Here, we examine the effects of MYT1L deficiency in human neurons and mice. Mutant mice exhibit neurodevelopmental delays with thinner cortices, behavioural phenotypes, and gene expression changes that resemble those of ASD patients. MYT1L target genes, including WNT and NOTCH, are activated upon MYT1L depletion and their chemical inhibition can rescue delayed neurogenesis in vitro. MYT1L deficiency also causes upregulation of the main cardiac sodium channel, SCN5A, and neuronal hyperactivity, which could be restored by shRNA-mediated knockdown of SCN5A or MYT1L overexpression in postmitotic neurons. Acute application of the sodium channel blocker, lamotrigine, also rescued electrophysiological defects in vitro and behaviour phenotypes in vivo. Hence, MYT1L mutation causes both developmental and postmitotic neurological defects. However, acute intervention can normalise resulting electrophysiological and behavioural phenotypes in adulthood.
Asunto(s)
Trastorno del Espectro Autista , Animales , Humanos , Ratones , Trastorno del Espectro Autista/tratamiento farmacológico , Trastorno del Espectro Autista/genética , Trastorno Autístico/tratamiento farmacológico , Trastorno Autístico/genética , Haploinsuficiencia/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Fenotipo , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Glioblastoma (GBM) is an aggressive tumor that frequently exhibits gain of chromosome 7, loss of chromosome 10, and aberrantly activated receptor tyrosine kinase signaling pathways. Previously, we identified Mesenchyme Homeobox 2 (MEOX2), a gene located on chromosome 7, as an upregulated transcription factor in GBM. Overexpressed transcription factors can be involved in driving GBM. Here, we aimed to address the role of MEOX2 in GBM. METHODS: Patient-derived GBM tumorspheres were used to constitutively knockdown or overexpress MEOX2 and subjected to in vitro assays including western blot to assess ERK phosphorylation. Cerebral organoid models were used to investigate the role of MEOX2 in growth initiation. Intracranial mouse implantation models were used to assess the tumorigenic potential of MEOX2. RNA-sequencing, ACT-seq, and CUT&Tag were used to identify MEOX2 target genes. RESULTS: MEOX2 enhanced ERK signaling through a feed-forward mechanism. We identified Ser155 as a putative ERK-dependent phosphorylation site upstream of the homeobox-domain of MEOX2. S155A substitution had a major effect on MEOX2 protein levels and altered its subnuclear localization. MEOX2 overexpression cooperated with p53 and PTEN loss in cerebral organoid models of human malignant gliomas to induce cell proliferation. Using high-throughput genomics, we identified putative transcriptional target genes of MEOX2 in patient-derived GBM tumorsphere models and a fresh frozen GBM tumor. CONCLUSIONS: We identified MEOX2 as an oncogenic transcription regulator in GBM. MEOX2 increases proliferation in cerebral organoid models of GBM and feeds into ERK signaling that represents a core signaling pathway in GBM.
Asunto(s)
Glioblastoma , Glioma , Ratones , Animales , Humanos , Genes Homeobox , Proteínas de Homeodominio/genética , Glioma/genética , Glioblastoma/patología , Proliferación Celular , Factores de Transcripción/genética , Carcinogénesis/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión GénicaRESUMEN
This protocol provides a detailed description of how to fabricate and use the dual-flow-RootChip (dfRootChip), a novel microfluidic platform for investigating root nutrition, root-microbe interactions and signaling and development in controlled asymmetric conditions. The dfRootChip was developed primarily to investigate how plants roots interact with their environment by simulating environmental heterogeneity. The goal of this protocol is to provide a detailed resource for researchers in the biological sciences wishing to employ the dfRootChip in particular, or microfluidic devices in general, in their laboratory.
RESUMEN
Roots grow in highly dynamic and heterogeneous environments. Biological activity as well as uneven nutrient availability or localized stress factors result in diverse microenvironments. Plants adapt their root morphology in response to changing environmental conditions, yet it remains largely unknown to what extent developmental adaptations are based on systemic or cell-autonomous responses. We present the dual-flow-RootChip, a microfluidic platform for asymmetric perfusion of Arabidopsis roots to investigate root-environment interactions under simulated environmental heterogeneity. Applications range from investigating physiology, root hair development and calcium signalling upon selective exposure to environmental stresses to tracing molecular uptake, performing selective drug treatments and localized inoculations with microbes. Using the dual-flow-RootChip, we revealed cell-autonomous adaption of root hair development under asymmetric phosphate (Pi) perfusion, with unexpected repression in root hair growth on the side exposed to low Pi and rapid tip-growth upregulation when Pi concentrations increased. The asymmetric root environment further resulted in an asymmetric gene expression of RSL4, a key transcriptional regulator of root hair growth. Our findings demonstrate that roots possess the capability to locally adapt to heterogeneous conditions in their environment at the physiological and transcriptional levels. Being able to generate asymmetric microenvironments for roots will help further elucidate decision-making processes in root-environment interactions.