Biology subjects

Lickert, H.

Publications and source records attributed to Lickert, H..

30 records · Page 2Linked to original sources

bioRxiv2021

Sequential in vivo labeling of insulin secretory granule pools in INS-SNAP transgenic pigs

{beta}-cells produce, store and secrete insulin upon elevated blood glucose levels. Insulin secretion is a highly regulated process. The probability for insulin secretory granules to undergo fusion with the plasma membrane or being degraded is correlated with their age. However, the molecular features and stimuli connected to this behavior have not yet been fully understood. Furthermore, our understanding of {beta}-cell function is mostly derived from studies of ex vivo isolated islets and/or rodent models. To overcome this translational gap and study insulin secretory granule turnover in vivo, we have generated a transgenic pig model with the SNAP-tag fused to insulin. We demonstrate the correct targeting and processing of the tagged insulin and normal glycemic control of the pig model. Furthermore, we show specific single- and dual-color granular labeling of in vivo labeled pig pancreas. This model may provide unprecedented insights into the in vivo insulin secretory granule behavior in an animal close to humans.

cell biology↗

bioRxiv2021

Epiblast morphogenesis is controlled by selective mRNA decay triggered by LIN28A relocation

The embryonic progression from naive to primed pluripotency is accompanied by the rapid decay of pluripotency-associated mRNAs and a concomitant radical morphogenetic sequence of epiblast polarization, rosette formation and lumenogenesis. The mechanisms triggering and linking these events remain poorly understood. Guided by machine learning and metabolic RNA sequencing, we identified RNA binding proteins (RBPs), especially LIN28A, as primary mRNA decay factors. Using mRNA-RBP interactome capture, we revealed a dramatic increase in LIN28A mRNA binding during the naive-rosette-primed pluripotency transition, driven by its nucleolar-to-cytoplasmic translocation. Cytoplasmic LIN28A binds to 3UTRs of pluripotency-associated mRNAs to directly stimulate their decay and drive lumenogenesis. Accordingly, forced nuclear retention of LIN28A impeded lumenogenesis, impaired gastrulation, and caused an unforeseen embryonic multiplication. Selective mRNA decay, driven by nucleo-cytoplasmic RBP translocation, therefore acts as an intrinsic mechanism linking cell identity switches to the control of embryonic growth and morphogenesis.

developmental biology↗

bioRxiv2021

A strategy to address dissociation-induced compositional and transcriptional bias for single-cell analysis of the human mammary gland

Single-cell transcriptomics provide insights into cellular heterogeneity and lineage dynamics that are key to better understanding normal mammary gland function as well as breast cancer initiation and progression. In contrast to murine tissue, human mammary glands require laborious dissociation protocols to isolate single cells. This leads to unavoidable procedure-induced compositional and transcriptional bias. Here, we present a new strategy on how to identify and minimize systematic error by combining different tissue dissociation strategies and then directly comparing composition and transcriptome of isolated cells using single-cell RNA sequencing and flow cytometry. Depending on the tissue isolation strategy, we found dramatic differences in abundance and heterogeneity of certain stromal cells types. Moreover, we identified lineage-specific dissociation-induced gene expression changes that, if left unchecked, could lead to misinterpretation of cellular heterogeneity and, since the basal epithelial population is particularly affected by this, wrongful assignment of putative stem cell populations.

cell biology↗

bioRxiv2021

Generation of a novel Nkx6-1 Venus fusion reporter mouse line

Nkx6-1 is a member of the Nkx family of homeodomain transcription factors (TF) that regulates motor neuron development, neuron specification and pancreatic endocrine and {beta}-cell differentiation. To facilitate the isolation and tracking of Nkx6-1-expressing cells, we have generated a novel Nkx6-1 Venus Fusion (Nkx6-1-VF) reporter allele. The Nkx6-1-VF knock-in reporter is regulated by endogenous cis-regulatory elements of Nkx6-1 and the fluorescent protein fusion does not interfere with the TF function, as homozygous mice are viable and fertile. In addition, the nuclear localization of Nkx6-1-VF protein reflects the endogenous Nkx6-1 protein distribution. During embryonic pancreas development, the reporter protein marks the pancreatic ductal progenitors and the endocrine lineage, but is absent in the exocrine compartment. Moreover, the levels of Nkx6-1-VF reporter is upregulated upon {beta}-cell differentiation during the major wave of endocrinogenesis. In the adult islets of Langerhans, the reporter protein is exclusively found in insulin-secreting {beta}-cells. Importantly, the Venus reporter activities allows successful tracking of {beta}-cells in live-cell imaging and their specific isolation by flow sorting. In summary, the generation of Nkx6-1-VF reporter line provides a unique tool to study the spatio-temporal expression pattern of this TF during organ development and isolate and track Nkx6-1-expressing cells such as pancreatic {beta}-cells, but also neurons and motor neurons in health and disease.

developmental biology↗

bioRxiv2020

CellRank for directed single-cell fate mapping

Computational trajectory inference enables the reconstruction of cell-state dynamics from single-cell RNA sequencing experiments. However, trajectory inference requires that the direction of a biological process is known, largely limiting its application to differentiating systems in normal development. Here, we present CellRank (https://cellrank.org) for mapping the fate of single cells in diverse scenarios, including perturbations such as regeneration or disease, for which direction is unknown. Our approach combines the robustness of trajectory inference with directional information from RNA velocity, derived from ratios of spliced to unspliced reads. CellRank takes into account both the gradual and stochastic nature of cellular fate decisions, as well as uncertainty in RNA velocity vectors. On data from pancreas development, we show that it automatically detects initial, intermediate and terminal populations, predicts fate potentials and visualizes continuous gene expression trends along individual lineages. CellRank also predicts a novel dedifferentiation trajectory during regeneration after lung injury, which we follow up experimentally by confirming the existence of previously unknown intermediate cell states.

bioinformatics↗

bioRxiv2020

Identification and characterization of distinct murine brown adipocyte lineages

Brown adipose tissue (BAT) plays an important role in the regulation of body weight and glucose homeostasis. While increasing evidence supports white adipose tissue heterogeneity, little is known about heterogeneity within murine BAT. Using single cell RNA sequencing of the stromal vascular fraction of murine BAT and analysis of 67 brown preadipocyte and adipocyte clones we unravel heterogeneity within brown preadipocytes. Statistical analysis of gene expression profiles from these clones identifies markers distinguishing brown adipocyte lineages. We confirm the presence of distinct brown adipocyte populations in vivo using three identified markers; Eif5, Tcf25, and Bin1. Functionally, we demonstrate that loss of Bin1 enhances UCP1 expression and mitochondrial respiration, suggesting that Bin1 marks a dormant brown adipocyte type. The existence of multiple brown adipocyte lineages suggests distinct functional properties of BAT depending on its cellular composition, with potentially distinct function in thermogenesis and the regulation of whole body energy homeostasis.

cell biology↗

bioRxiv2020

Anatomical and cellular heterogeneity in the mouse oviduct-- its potential roles in reproduction and preimplantation development

The oviduct/fallopian tube is a tube-like structure that extends from the uterus to the ovary. It is an essential reproductive tissue that provides an environment for internal fertilization and preimplantation development. However, our knowledge of its regional and cellular heterogeneity is still limited. Here, we examined the anatomical complexity of mouse oviducts using modern imaging techniques and fluorescence reporter lines. We found that there are basic coiling patterns and turning points in the coiled mouse oviduct can serve as reliable landmarks for luminal morphological regionalities. We identified previously unrecognized anatomical structures in the isthmus and uterotubal junction (UTJ) that likely play important roles in reproduction. Interestingly, during ovulation, the isthmus was transiently plugged by a thick mucus, keeping the oocytes within the ampulla. Preimplantation embryos travelled along the oviduct and formed a queue within small compartments of the UTJ before uterine entry. Taken together, the oviduct luminal epithelium had highly diverse luminal structures with distinct cell populations reflecting its complex functions in reproduction.

physiology↗

bioRxiv2020

Wnt/PCP-primed intestinal stem cells directly differentiate into enteroendocrine or Paneth cells

A detailed understanding of intestinal stem cell (ISC) self-renewal and differentiation is required to better treat chronic intestinal diseases. However, different models of ISC lineage hierarchy1-6 and segregation7-12 are debated. Here we report the identification of Lgr5+ ISCs that express Flattop (Fltp), a Wnt/planar cell polarity (PCP) reporter and effector gene. Lineage labelling revealed that Wnt/PCP-activated Fltp+ ISCs are primed either towards the enteroendocrine or the Paneth cell lineage in vivo. Integration of time-resolved lineage labelling with genome-wide and targeted single-cell gene expression analysis allowed us to delineate the ISC differentiation path into enteroendocrine and Paneth cells at the molecular level. Strikingly, we found that both lineages are directly recruited from ISCs via unipotent transition states, challenging the existence of formerly predicted bi- or multipotent secretory progenitors7-12. Transitory cells that mature into Paneth cells are quiescent and express both stem cell and secretory lineage genes, indicating that these cells are the previously described Lgr5+ labelretaining cells7. Wnt/PCP-activated Lgr5+ ISCs are indistinguishable from Wnt/{beta}-catenin-activated Lgr5+ ISCs based on the expression of stem-cell signature or secretory lineagespecifying genes but possess less self-renewal activity. This suggests that lineage priming and cell-cycle exit is triggered at the post-transcriptional level by polarity cues and a switch from canonical to non-canonical Wnt/PCP signalling. Taken together, we identified the Wnt/PCP pathway as a new niche signal and polarity cue regulating stem cell fate. Active Wnt/PCP signalling represents one of the earliest events in ISC lineage priming towards the Paneth and enteroendocrine cell fate, preceding lateral inhibition and expression of secretory lineagespecifying genes. Thus, our findings provide a better understanding of the niche signals and redefine the mechanisms underlying ISC lineage hierarchy and segregation.

developmental biology↗

bioRxiv2020

Design and power analysis for multi-sample single cell genomics experiments

BackgroundThe identification of genes associated with specific experimental conditions, genotypes or phenotypes through differential expression analysis has long been the cornerstone of transcriptomic analysis. Single cell RNA-seq is revolutionizing transcriptomics and is enabling interindividual differential gene expression analysis and identification of genetic variants associated with gene expression, so called expression quantitative trait loci at cell-type resolution. Current methods for power analysis and guidance of experimental design either do not account for the specific characteristics of single cell data or are not suitable to model interindividual comparisons. ResultsHere we present a statistical framework for experimental design and power analysis of single cell differential gene expression between groups of individuals and expression quantitative trait locus analysis. The model relates sample size, number of cells per individual and sequencing depth to the power of detecting differentially expressed genes within individual cell types. Power analysis is based on data driven priors from literature or pilot experiments across a wide range of application scenarios and single cell RNA-seq platforms. Using these priors we show that, for a fixed budget, the number of cells per individual is the major determinant of power. ConclusionOur model is general and allows for systematic comparison of alternative experimental designs and can thus be used to guide experimental design to optimize power. For a wide range of applications, shallow sequencing of high numbers of cells per individual leads to higher overall power than deep sequencing of fewer cells. The model is implemented as an R package scPower.

bioinformatics↗

bioRxiv2019

Islet vascularization is regulated by primary endothelial cilia via VEGF-A dependent signaling

RationaleAccumulating evidence point to a role for primary cilia in endothelial cell function. Islet vascularization is an important determinant of islet function and glucose homeostasis. We have previously shown that {beta}-cell cilia directly regulate insulin secretion. However, it is unclear whether primary cilia are also implicated in islet vascularization and thus contribute to glucose homeostasis.\n\nObjectiveTo characterize the role of primary cilia in islet vascularization.\n\nMethods and ResultsAt four weeks, Bbs4-/- islets show markedly lower intra-islet capillary density with enlarged diameters. We transplanted islets into the anterior chamber (ACE) of mouse eyes for longitudinal and non-invasive in vivo monitoring of vascular morphology. Bbs4-/- islets exhibited significantly delayed re-vascularization and enlarged vessels during engraftment. Similar vascular phenotypes were observed in two other ciliopathy models. By shifting the relative contributions of host versus donor endothelial cells in islet revascularization, we found that primary cilia on endothelial cells is essential for this process. Electron microscopy analysis further revealed a lack of fenestration in engrafted Bbs4-/- islets, partially impairing vascular permeability and glucose delivery to {beta}-cells. Finally, we identified that Vascular endothelial cell growth factor A (VEGF-A)/VEGF receptor 2 (VEGFR2) signalling is involved in islet vascularization, islet function and vascular fenestration. In vitro silencing of two different ciliary genes in endothelial cells disrupts VEGF-A/ VEGFR2 internalization and phospho-activation of downstream signalling components. Consequently, key features of angiogenesis including proliferation, migration and tube formation are attenuated in BBS4 silenced endothelial cells.\n\nConclusionsEndothelial cell primary cilia regulate islet vascularization and vascular barrier function via VEGF-A/ VEGFR2 signaling pathway. Islet vascularization is impaired in four weeks old Bbs4-/- mice. Long-time monitoring of re-vascularization of WT and Bbs4-/- islets recapitulates the phenotype and demonstrates a role for cilia in islet vascularization and vascular barrier function. VEGF-A/ VEGFR2-dependent signalling is regulated by endothelial primary cilia.

physiology↗

bioRxiv2019

Pre-marked chromatin and transcription factor co-binding shape the pioneering activity of Foxa2.

Pioneer transcription factors (PTF) can recognize their binding sites on nucleosomal DNA and trigger chromatin opening for recruitment of other non-pioneer transcription factors. However, critical properties of PTFs are still poorly understood, such as how these transcription factors selectively recognize cell type-specific binding sites and under which conditions can they can initiate chromatin remodelling. Here we show that early endoderm binding sites of the paradigm PTF Foxa2 are epigenetically primed by low levels of active chromatin modifications in embryonic stem cells (ESC). Priming of these binding sites is supported by preferential recruitment of Foxa2 to endoderm binding sites compared to lineage-inappropriate binding sites, when ectopically expressed in ESCs. We further show that binding of Foxa2 is required for chromatin opening during endoderm differentiation. However, increased chromatin accessibility was only detected on binding sites which are synergistically bound with other endoderm transcription factors. Thus, our data suggest that binding site selection of PTFs is directed by the chromatin environment and that chromatin opening requires collaboration of PTFs with additional transcription factors.

genomics↗

bioRxiv2019

Sox17 expression in endocardium precursor cells regulates heart development in mice

The endocardium is the endothelial component of the vertebrate heart and plays a key role in heart development. Cardiac progenitor cells (CPCs) that express the homeobox gene Nkx2-5 give rise to the endocardium. Where, when, and how the endocardium segregates during embryogenesis have remained largely unknown, however. We now show that Nkx2-5+ CPCs that express the Sry-type HMG box gene Sox17 specifically differentiate into the endocardium in mouse embryos. Approximately 20% to 30% of Nkx2-5+CPCs transiently express Sox17 from embryonic day (E) 7.5 to E8.5.Although Sox17 is not essential or sufficient for endocardium fate, it can bias the fate of CPCs toward the endocardium. On the other hand, Sox17 expression in the endocardium is required for heart development. Deletion of Sox17 specifically in the mesoderm markedly impaired endocardium development with regard to cell proliferation and behavior. The proliferation of cardiomyocytes, ventricular trabeculation, and myocardium thickening were also impaired in a non-cell-autonomous manner in the Sox17 mutant, resulting in anomalous morphology of the heart, likely as a consequence of down-regulation of NOTCH signaling. Changes in gene expression profile in both the endocardium and myocardium preceded the reduction in NOTCH-related gene expression in the mutant embryos, suggesting that Sox17 expression in the endocardium regulates an unknown signal required for nurturing of the myocardium. Our results thus provide insight into differentiation of the endocardium and its role in heart development. SignificanceThe endocardium is vital for vertebrate heart development; however, the molecular mechanisms regulating fate determination and differentiation remain largely unknown. Here, we show that a part of the earliest cardiac progenitor cells (CPCs) transiently and exclusively express Sry-type HMG box gene Sox17 in the mouse embryo. Sox17-expressing CPCs specifically differentiate to the endocardium. Sox17 biases the fate of CPCs toward the endocardium, and regulates proliferation and cellular behavior cell autonomously. Conversely, Sox17 in the endocardium regulates the myocardium non-cell autonomously. Notably, Sox17 is required for the ventricular trabeculation via the NOTCH signal that is not directly induced but maintained by Sox17. This study, thus, sheds light on endocardium development.

developmental biology↗