T-REX17 is a transiently expressed non-coding RNA essential for human endoderm formation

Long non-coding RNAs (lncRNAs) have emerged as fundamental regulators in various biological processes, including embryonic development and cellular differentiation. Despite much progress over the past decade, the genome-wide annotation of lncRNAs remains incomplete and many known non-coding loci are still poorly characterized. Here, we report the discovery of a previously unannotated lncRNA that is transcribed 230 kb upstream of the SOX17 gene and located within the same topologically associating domain. We termed it T-REX17 (Transcript Regulating Endoderm and activated by soX17) and show that it is induced following SOX17 activation but its expression is more tightly restricted to early definitive endoderm. Loss of T-REX17 affects crucial functions independent of SOX17 and leads to an aberrant endodermal transcriptome, signaling pathway deregulation and epithelial to mesenchymal transition defects. Consequently, cells lacking the lncRNA cannot further differentiate into more mature endodermal cell types. Taken together, our study identified and characterized T-REX17 as a transiently expressed and essential non-coding regulator in early human endoderm differentiation

Hypoxia induces an early primitive streak signature, enhancing spontaneous elongation and lineage representation in gastruloids

The cellular microenvironment, together with intrinsic regulators, shapes stem cell identity and differentiation capacity. Mammalian early embryos are exposed to hypoxia in vivo and appear to benefit from hypoxic culture in vitro. Yet, how hypoxia influences stem cell transcriptional networks and lineage choices remain poorly understood. Here, we investigated the molecular effects of acute and prolonged hypoxia on embryonic and extra-embryonic stem cells as well as the functional impact on differentiation potential. We find a temporal and cell type-specific transcriptional response including an early primitive streak signature in hypoxic embryonic stem cells mediated by HIF1α. Using a 3D gastruloid differentiation model, we show that hypoxia-induced T expression enables symmetry breaking and axial elongation in the absence of exogenous WNT activation. When combined with exogenous WNT activation, hypoxia enhances lineage representation in gastruloids, as demonstrated by highly enriched signatures of gut endoderm, notochord, neuromesodermal progenitors and somites. Our findings directly link the microenvironment to stem cell function and provide a rationale supportive of applying physiological conditions in models of embryo development

Aberrant phase separation and nucleolar dysfunction in rare genetic diseases

Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions1-3. Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus4,5. This suggests that mutations in disordered proteins may alter condensate properties and function6-8. Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans.© 2023. The Author(s)

Die Heterogenität von TRPV1 und seine Aktivierung in nozizeptiven Neuronen

The question on the number of human senses in western societies is typically answered with five. Asking people anywhere in the world, what is most annoying feeling, the majority will answer emotional, physical, or diseases related pain. Whatever reason, the answer is mostly pain. But can the perception of pain be defined as sense? The answer is yes. Pain initiating stimuli activate specialized peripheral sensory neurons from the dorsal root ganglia - the so called nociceptive neurons. One protein central for inflammatory temperature pain is the ion channel TRPV1. TRPV1 protein expression in sensory neurons is upregulated in situations of hightened pain sensitivity, suggesting a direct causal relationship between the TRPV1 expression levels and pain sensitivity. A number of processes have been identified describing the sensitization and desensitization of TRPV1 on a molecular level. But, to what extend the expression levels of TRPV1 correlates with its cellular activation kinetics on a population base has not been analyzed in detail so far. This is due to, a lack of suitable technical approaches to analyze the function and (TRPV1-) protein expression levels on a single cell base. I quantified the heterogeneity of TRPV1 expression in nociceptive neurons, with confocal and “high content screening” microscopy approaches. Antibody based expression analysis and calcium imaging identified 60.4 ± 6.5 % (immune cytochemistry) and 58.2 ± 6.8 % (capsaicin responsive cells) sensory neurons, to be TRPV1 expressing. In depth analysis of the calcium imaging experiments revealed, that submaximal capsaicin concentrations do not result in lower responses but lower numbers of capsaicin sensitive cells. Further, a correlation between TRPV1 expression and the amplitude of capsaicin induced calcium influx was clearly detectable in exogenously expressed TRPV1 transfected cell lines, but was almost absent in endogenously expressing nociceptive neurons. Pretreatment with strong sensitization inducing substances (PMA and FSK) resulted in an improvement of the correlation – nevertheless, still not explaining all the heterogeneity between expression and response. Accordingly, a sensitization signaling protein, the regulatory subunit of PKA RIIβ, correlated better with the cellular capsaicin response than TRPV1 itself. As the strong sensitization signaling did not result in a complete correlation between expression and capsaicin induced calcium influx, there must exist other mechanisms of regulation such as expression of modulators. To identify novel TRPV1 modulator and gain genetic insights into nociceptors, we developed a novel subgroup specific transcriptome analysis approach. The resulting list of up regulated genes offers the opportunity to identify novel TRPV1 and pain modulators. Furthermore, the list presents the first complete transcriptome of a subpopulation of nociceptive neurons. In collaboration with Steffen Waldherr, my results were analyzed by establishing a computational model for the cellular capsaicin sensitivity. From this model based on experimental data and literature derived values, a mechanism is suggested, which regulates the relative capsaicin sensitivity state inversely to the TRPV1 expression in sensory neurons. Thus, the TRPV1 response is surprisingly robust against changes in TRPV1 expression levels. My results question the conventional therapeutic use of TRPV1-channel blockers and suggest the need to define the cellular mechanisms how the TRPV1-mediated calcium influx-homeostasis is controlled.Die Frage nach der Anzahl menschlicher Sinne wird in westlichen Gesellschaften typischerweise mit fünf beantwortet. Fragt man Menschen irgendwo auf der Welt nach ihrem unangenehmsten Gefühl, wird die Mehrzahl entweder mit emotionalen, physischen oder krankheitsbedingten Schmerz antworten. Auf welcher Ursache auch immer die resultierende Antwort basiert, sie ist in den meisten Fällen Schmerz. Aber kann die Wahrnehmung von Schmerz als Sinn definiert werden? Die Antwort ist ja. Schmerz verursachende Stimuli aktivieren spezialisierte sensorische Neurone aus den dorsalen Hinterwurzel Ganglien, die sogenannten Nozizeptoren. Ein zentrales Protein für entzündlichen Temperaturschmerz ist der Ionenkanal TRPV1. Unter Bedingungen erhöhter Schmerzsensitivität ist die TRPV1 Proteinexpression in nozizeptiven Neuronen erhöht. Dies deutet auf eine direkte kausale Beziehung zwischen TRPV1 Expressionsmengen und Schmerzsensitivität hin. Eine Reihe von Prozessen wurde bereits identifiziert, welche die Sensitivierung und Desensitiverung von TRPV1 auf molekularer Ebene beschreiben. In welchem Ausmaß jedoch die TRPV1 Expression mit zellulären Aktivierungskinetiken korrelieren, wurde noch nicht im Detail und auf Populationsebene untersucht. Dieser Umstand beruht vor allem auf fehlenden Untersuchungsmethoden, welche erlauben zelluläre Funktionen und endogene (TRPV1-) Proteinexpression in einzelnen Zelle zu untersuchen. Mit konfokalen und “High Content Screening”- Mikroskopieverfahren quantifizierte ich die Heterogenität der TRPV1 Expression in nozizeptiven Neuronen. Antikörper basierte Expressionsanalysen als auch Calcium Imaging identifizierten 60.4 ± 6.5 % (Immunzytochemie) und 58.2 ± 6.8 % (Capsaicin sensitive Neurone) der sensorischen Neurone als TRPV1 exprimierend. Eine detaillierte Analyse der Calcium Imaging Experimente zeigte, dass submaximale Capsaicin-Konzentrationen nicht in niedrigeren Calcium Antworten resultieren sondern weniger Capsaicin sensitive Zellen zeigten. Weiterhin wurde eine klare Korrelation zwischen exogener TRPV1 Expression und der Capsaicin induzierten Calcium Einstromamplitude in TRPV1 transfizierten Zelllinien detektiert, welche in endogen expremierenden nozizeptiven Neuronen fast nicht vorhanden war. Vorbehandlungen mit starken sensitivierungs-induzierenden Substanzen (PMA und FSK) zeigten eine Verbesserung, konnten jedoch nicht die Heterogenität zwischen Expression und Antwort erklären. Dementsprechend korrelierte ein Sensitivierungs-Signalwirkungsprotein, die regulatorische Untereinheit von PKA RIIβ, besser mit der zellulären Capsaicin-Antwort als TRPV1. Da eine starke Sensitivierung nicht zu einer vollständigen Korrelation, zwischen Expression und Capsaicin induziertem Calcium Einstrom führten, müssen andere Regulationsmechanismen existieren wie beispielweise die Expression von Modulatoren. Um neue TRPV1-Modulatoren zu identifizieren und genetische Einblicke in Nozizeptoren zu erlangen, entwickelten wir subgruppen-spezifische Transkriptom-Analyseverfahren. Eine daraus resultierende Liste von hoch regulierten Genen eröffnet die Möglichkeit, neue TRPV1 und damit Schmerzmodulatoren zu identifizieren. Die Liste präsentiert weiterhin, das erste vollständige Transkriptom einer Subpopulation nozizeptiver sensorischer Neurone. In Kollaboration mit Steffen Waldherr verwendete ich meine Ergebnisse um ein computergestütztes Modell für die zelluläre Capsaicin-Sensitivität zu entwickeln. In diesem, auf experimentellen Daten und Literatur basierten Modell, wird ein Mechanismus angedeutet, welcher die relative zelluläre Capsaicin Sensitivität invers zur TRPV1-Epression in sensorischen Neuronen reguliert. Daher ist die Capsaicin-Antwort überraschend robust gegenüber Veränderungen in der TRPV1-Expression. Wenn die TRPV1 vermittelte Calciumeinströme so unempfindlich gegenüber Veränderungen der TRPV1-Expression sind, hinterfragen meine Ergebnisse das konventionelle therapeutische Konzept von TRPV1-Kanalblockern. Sie zeigen den Bedarf auf, zelluläre Mechanismen zu definieren, welche die TRPV1 vermittelte Calcium-Einstrom-Homöostase kontrollieren

Enhanced cortical neural stem cell identity through short SMAD and WNT inhibition in human cerebral organoids facilitates emergence of outer radial glial cells

Cerebral organoids exhibit broad regional heterogeneity accompanied by limited cortical cellular diversity despite the tremendous upsurge in derivation methods, suggesting inadequate patterning of early neural stem cells (NSCs). Here we show that a short and early Dual SMAD and WNT inhibition course is necessary and sufficient to establish robust and lasting cortical organoid NSC identity, efficiently suppressing non-cortical NSC fates, while other widely used methods are inconsistent in their cortical NSC-specification capacity. Accordingly, this method selectively enriches for outer radial glia NSCs, which cyto-architecturally demarcate well-defined outer sub-ventricular-like regions propagating from superiorly radially organized, apical cortical rosette NSCs. Finally, this method culminates in the emergence of molecularly distinct deep and upper cortical layer neurons, and reliably uncovers cortex-specific microcephaly defects. Thus, a short SMAD and WNT inhibition is critical for establishing a rich cortical cell repertoire that enables mirroring of fundamental molecular and cyto-architectural features of cortical development and meaningful disease modelling

T-REX17 is a transiently expressed non-coding RNA essential for human endoderm formation

Long non-coding RNAs (lncRNAs) have emerged as fundamental regulators in various biological processes, including embryonic development and cellular differentiation. Despite much progress over the past decade, the genome-wide annotation of lncRNAs remains incomplete and many known non-coding loci are still poorly characterized. Here, we report the discovery of a previously unannotated lncRNA that is transcribed 230 kb upstream of the SOX17 gene and located within the same topologically associating domain. We termed it T-REX17 (Transcript Regulating Endoderm and activated by soX17) and show that it is induced following SOX17 activation but its expression is more tightly restricted to early definitive endoderm. Loss of T-REX17 affects crucial functions independent of SOX17 and leads to an aberrant endodermal transcriptome, signaling pathway deregulation and epithelial to mesenchymal transition defects. Consequently, cells lacking the lncRNA cannot further differentiate into more mature endodermal cell types. Taken together, our study identified and characterized T-REX17 as a transiently expressed and essential non-coding regulator in early human endoderm differentiation

Dnmt1 has de novo activity targeted to transposable elements

DNA methylation plays a critical role during development, particularly in repressing retrotransposons. The mammalian methylation landscape is dependent on the combined activities of the canonical maintenance enzyme Dnmt1 and the de novo Dnmts, 3a and 3b. Here, we demonstrate that Dnmt1 displays de novo methylation activity in vitro and in vivo with specific retrotransposon targeting. We used whole-genome bisulfite and long-read Nanopore sequencing in genetically engineered methylation-depleted mouse embryonic stem cells to provide an in-depth assessment and quantification of this activity. Utilizing additional knockout lines and molecular characterization, we show that the de novo methylation activity of Dnmt1 depends on Uhrf1, and its genomic recruitment overlaps with regions that enrich for Uhrf1, Trim28 and H3K9 trimethylation. Our data demonstrate that Dnmt1 can catalyze DNA methylation in both a de novo and maintenance context, especially at retrotransposons, where this mechanism may provide additional stability for long-term repression and epigenetic propagation throughout development.ISSN:1545-9993ISSN:1545-998