Genetic mosaic dissection of candidate genes in mice using mosaic analysis with double markers

Mosaic analysis with double markers (MADM) technology enables the generation of genetic mosaic tissue in mice. MADM enables concomitant fluorescent cell labeling and introduction of a mutation of a gene of interest with single-cell resolution. This protocol highlights major steps for the generation of genetic mosaic tissue and the isolation and processing of respective tissues for downstream histological analysis. For complete details on the use and execution of this protocol, please refer to Contreras et al. (2021)

The role of Langerhans cells in skin cancer growth and immune response to Imiquimod

Dendritische Zellen sind die wichtigsten antigenprozessierenden und –präsentierenden Zellen des Körpers. Die Langerhanszellen stellen eine Subpopulationen von dendritischen Zellen dar. Sie sind in der Epidermis lokalisiert und nehmen unter physiologischen Bedingungen beständig Antigen auf, migrieren zu hautdränierenden Lymphknoten und präsentieren Antigen an naive T Zellen. Während Entzündungsprozessen wird die Langerhanszell-Migration erhöht, ebenso ihre Freisetzung von Zytokinen. Dadurch werden andere Immunzell-Populationen aktiviert und zur Entzündungsstelle rekrutiert. Eine Studie in unserem Labor konnte zeigen, dass Imiquimod, ein Toll-like Rezeptor (TLR)7 Agonist, Langerhanszellen aktiviert und darüber hinaus zur Regression von Hauttumoren, wie z.B. Melanomen, führt. In humaner Therapie wird Imiquimod häufig in der erfolgreichen Behandlung von Basaliomen eingesetzt. Dennoch ist bisher nicht bekannt, welchen Beitrag die Langerhanszellen zur Wirkweise des antitumoralen Effekts von Imiquimod leisten. In meiner Diplomarbeit habe ich die Fragestellung bearbeitet, ob die Präsenz von Langerhanszellen einen Einfluss auf das Wachstum von Hautkrebs hat und ob sie wichtige Übermittler des antitumoralen Effekts von Imiquimod darstellen. Aus diesem Grund habe ich für die Studie zwei verschiedene Mausmodelle für Hautkrebs verwendet: (1) ein syngenes Melanom-Modell, wobei B16F10 Melanomzellen intradermal in LangerinDTR:EGFP Mäuse injiziert werden; und (2) Mäuse, bei welchen das Wachstum von Basaliomen durch Expression eines hyperaktiven Smoothened-Allels induziert werden kann (Rosa26SmoM2YFP Mäuse). 1. Langerhanszellen sind nicht für den antitumoralen Effekt von Imiquimod im B16F10 Melanom-Modell verantwortlich. Tumore, die mit Imiquimod behandelt werden, weisen langsamere Tumorwachstumsraten auf als unbehandelte Tumore. Dieses Resultat kann unabhängig von der Präsenz von Langerhanszellen beobachtet werden. Die Analyse der zellulären Zusammensetzung von Tumorinfiltraten zeigt, dass Langerhanszellen eine Rolle in der Rekrutierung von CD8+ T Zellen und MHCII+CD4+ Zellen in Imiquimod-behandlete Tumore spielen. Ebenso scheinen Langerhanszellen einen regulatorischen Einfluss auf die Degranulation von Mastzellen auszuüben. 2. Rosa26SmoM2YFP Mäuse wurden mit K5-CreERT und LangerinDTR:EGFP Mäusen gekreuzt, um einen Experimentenstamm (SmoM2LangerinDTR Mäuse) zu erhalten, in dem Basaliome induziert und Langerhanszellen depletiert werden können. Daher sind weitere Experimente über längere Behandlungszeiträume geplant.Dendritic cells are the main antigen processing and presenting cells in the body. One subset of dendritic cells is the Langerhans cells (LCs), which are localized in the epidermis. Under steady-state conditions they continuously migrate to skin-draining lymph nodes and present antigen to naïve T cells. Under inflammatory conditions, LC migration is enhanced and their cytokine release is increased, activating and recruiting other types of immune cells to the site of inflammation. It has been shown in our lab that Imiquimod, a Toll-like receptor (TLR)7 agonist, activates LC and can lead to the regression of skin tumours like melanoma. In human therapy it is successfully used in treatment of basal-cell carcinoma (BCC). However, the role of LC in mediation of the antitumor effect of Imiquimod is poorly understood. In my thesis I addressed the question, if the presence of LCs has any impact on skin tumour growth and if they are important mediators of the antitumor effect of Imiquimod. To address this question I employed two different mouse skin cancer models: (1) a syngeneic melanoma model, whereby B16F10 melanoma cells are injected intradermally into LangerinDTR:EGFP mice; and (2) mice carrying the SmoothenedM2 transgene (Rosa26SmoM2YFP mice) that can be induced to develop BCC. 1. LCs were not responsible for the antitumoral effect of Imiquimod in the B16F10 melanoma model. Tumour bearing mice treated with Imiquimod showed decreased tumour growth rates compared to untreated control mice. This result was obtained independent of presence of LCs. Analysis of the cellular composition of tumour infiltrates revealed a role of LCs in recruiting CD8+ T cells and MHCII+CD4+ cells to Imiquimod-treated tumours as well as a regulatory role on mast cell degranulation. 2. Rosa26SmoM2YFP mice were crossed to K5CreERT and LangerinDTR:EGFP mice to give rise to inducible BCC bearing mice which can be depleted of LCs (SmoM2LangerinDTR mice). Imiquimod application onto BCC for 2 weeks did not have any influence on tumour growth. Therefore experiments using extended treatment regimens are planned

Epigenetic cues modulating the generation of cell type diversity in the cerebral cortex

The cerebral cortex is composed of a large variety of distinct cell-types including projection neurons, interneurons and glial cells which emerge from distinct neural stem cell (NSC) lineages. The vast majority of cortical projection neurons and certain classes of glial cells are generated by radial glial progenitor cells (RGPs) in a highly orchestrated manner. Recent studies employing single cell analysis and clonal lineage tracing suggest that NSC and RGP lineage progression are regulated in a profound deterministic manner. In this review we focus on recent advances based mainly on correlative phenotypic data emerging from functional genetic studies in mice. We establish hypotheses to test in future research and outline a conceptual framework how epigenetic cues modulate the generation of cell-type diversity during cortical development. This article is protected by copyright. All rights reserved

Effects of Imiquimod on Hair Follicle Stem Cells and Hair Cycle Progression

Topical imiquimod (IMQ) application is widely used as a model for psoriasiform-like skin inflammation in mice. Although the effects on the epidermis are well characterized, it is unclear how IMQ affects hair follicles and cycling. Here we investigated how IMQ affects hair follicle stem cells and whether the timing of IMQ application influences the immune infiltrate. Our results show that IMQ application at mid and late telogen activated hair follicle stem cells leading to premature hair cycle entry (anagen), which was accompanied by massive infiltration of inflammatory macrophages and gamma delta T cells, whereas the number of the respective resident populations decreased. Interestingly, high resident macrophage numbers were present in Rag2−/− mice and were maintained after IMQ treatment explaining why IMQ-induced anagen was reduced. This could be rescued after macrophage depletion suggesting that resident macrophages inhibit whereas inflammatory infiltrating macrophages stimulate hair follicle stem cell activation. The expression of the anagen-inhibiting factor BMP-4 was reduced by IMQ treatment as well as the activating factors Wnt showing that IMQ-induced hair follicle stem cell activation occurs by a Wnt-independent mechanism involving inflammatory cytokines such as CCL2 and TNF-α. On the basis of our findings, we recommend conducting experiments with IMQ during mid and late telogen as the biggest differences in immune cell composition are observed

Generation and isolation of single cells from mouse brain with mosaic analysis with double markers-induced uniparental chromosome disomy

Mosaic analysis with double markers (MADM) technology enables concomitant fluorescent cell labeling and induction of uniparental chromosome disomy (UPD) with single-cell resolution. In UPD, imprinted genes are either overexpressed 2-fold or are not expressed. Here, the MADM platform is utilized to probe imprinting phenotypes at the transcriptional level. This protocol highlights major steps for the generation and isolation of projection neurons and astrocytes with MADM-induced UPD from mouse cerebral cortex for downstream single-cell and low-input sample RNA-sequencing experiments. For complete details on the use and execution of this protocol, please refer to Laukoter et al. (2020b)

Imprinted Cdkn1c genomic locus cell-autonomously promotes cell survival in cerebral cortex development

The cyclin-dependent kinase inhibitor p57KIP2 is encoded by the imprinted Cdkn1c locus, exhibits maternal expression, and is essential for cerebral cortex development. How Cdkn1c regulates corticogenesis is however not clear. To this end we employ Mosaic Analysis with Double Markers (MADM) technology to genetically dissect Cdkn1c gene function in corticogenesis at single cell resolution. We find that the previously described growth-inhibitory Cdkn1c function is a non-cell-autonomous one, acting on the whole organism. In contrast we reveal a growth-promoting cell-autonomous Cdkn1c function which at the mechanistic level mediates radial glial progenitor cell and nascent projection neuron survival. Strikingly, the growth-promoting function of Cdkn1c is highly dosage sensitive but not subject to genomic imprinting. Collectively, our results suggest that the Cdkn1c locus regulates cortical development through distinct cell-autonomous and non-cell-autonomous mechanisms. More generally, our study highlights the importance to probe the relative contributions of cell intrinsic gene function and tissue-wide mechanisms to the overall phenotype

Lineage tracing and clonal analysis in developing cerebral cortex using mosaic analysis with double markers (MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present

Cell-type specificity of genomic imprinting in cerebral cortex

In mammalian genomes, a subset of genes is regulated by genomic imprinting, resulting in silencing of one parental allele. Imprinting is essential for cerebral cortex development, but prevalence and functional impact in individual cells is unclear. Here, we determined allelic expression in cortical cell types and established a quantitative platform to interrogate imprinting in single cells. We created cells with uniparental chromosome disomy (UPD) containing two copies of either the maternal or the paternal chromosome; hence, imprinted genes will be 2-fold overexpressed or not expressed. By genetic labeling of UPD, we determined cellular phenotypes and transcriptional responses to deregulated imprinted gene expression at unprecedented single-cell resolution. We discovered an unexpected degree of cell-type specificity and a novel function of imprinting in the regulation of cortical astrocyte survival. More generally, our results suggest functional relevance of imprinted gene expression in glial astrocyte lineage and thus for generating cortical cell-type diversity

A genome-wide library of MADM mice for single-cell genetic mosaic analysis

Mosaic analysis with double markers (MADM) offers one approach to visualize and concomitantly manipulate genetically defined cells in mice with single-cell resolution. MADM applications include the analysis of lineage, single-cell morphology and physiology, genomic imprinting phenotypes, and dissection of cell-autonomous gene functions in vivo in health and disease. Yet, MADM can only be applied to 96% of the entire mouse genome can now be subjected to single-cell genetic mosaic analysis. Beyond a proof of principle, we apply our MADM library to systematically trace sister chromatid segregation in distinct mitotic cell lineages. We find striking chromosome-specific biases in segregation patterns, reflecting a putative mechanism for the asymmetric segregation of genetic determinants in somatic stem cell division

Correction to: EGFR/Ras-induced CCL20 production modulates the tumour microenvironment

The article ‘EGFR/Ras-induced CCL20 production modulates the tumour microenvironment’, written by Andreas Hippe, Stephan Alexander Braun, Péter Oláh, Peter Arne Gerber, Anne Schorr, Stephan Seeliger, Stephanie Holtz, Katharina Jannasch, Andor Pivarcsi, Bettina Buhren, Holger Schrumpf, Andreas Kislat, Erich Bünemann, Martin Steinhoff, Jens Fischer, Sérgio A. Lira, Petra Boukamp, Peter Hevezi, Nikolas Hendrik Stoecklein, Thomas Hoffmann, Frauke Alves, Jonathan Sleeman, Thomas Bauer, Jörg Klufa, Nicole Amberg, Maria Sibilia, Albert Zlotnik, Anja Müller- Homey and Bernhard Homey, was originally published electronically on the publisher’s internet portal on 30 June 2020 without open access. With the author(s)’ decision to opt for Open Choice the copyright of the article changed on 16 September 2021 to © The Author(s) 2021 and the article is forthwith distributed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/ licenses/by/4.0/. Open Access funding enabled and organized by Projekt DEAL