Large neutral amino acid levels tune perinatal neuronal excitability and survival.

Little is known about the critical metabolic changes that neural cells have to undergo during development and how temporary shifts in this program can influence brain circuitries and behavior. Inspired by the discovery that mutations in SLC7A5, a transporter of metabolically essential large neutral amino acids (LNAAs), lead to autism, we employed metabolomic profiling to study the metabolic states of the cerebral cortex across different developmental stages. We found that the forebrain undergoes significant metabolic remodeling throughout development, with certain groups of metabolites showing stage-specific changes, but what are the consequences of perturbing this metabolic program? By manipulating Slc7a5 expression in neural cells, we found that the metabolism of LNAAs and lipids are interconnected in the cortex. Deletion of Slc7a5 in neurons affects the postnatal metabolic state, leading to a shift in lipid metabolism. Additionally, it causes stage- and cell-type-specific alterations in neuronal activity patterns, resulting in a long-term circuit dysfunction.

EGFR/Ras-induced CCL20 production modulates the tumour microenvironment.

BACKGROUND: The activation of the EGFR/Ras-signalling pathway in tumour cells induces a distinct chemokine repertoire, which in turn modulates the tumour microenvironment. METHODS: The effects of EGFR/Ras on the expression and translation of CCL20 were analysed in a large set of epithelial cancer cell lines and tumour tissues by RT-qPCR and ELISA in vitro. CCL20 production was verified by immunohistochemistry in different tumour tissues and correlated with clinical data. The effects of CCL20 on endothelial cell migration and tumour-associated vascularisation were comprehensively analysed with chemotaxis assays in vitro and in CCR6-deficient mice in vivo. RESULTS: Tumours facilitate progression by the EGFR/Ras-induced production of CCL20. Expression of the chemokine CCL20 in tumours correlates with advanced tumour stage, increased lymph node metastasis and decreased survival in patients. Microvascular endothelial cells abundantly express the specific CCL20 receptor CCR6. CCR6 signalling in endothelial cells induces angiogenesis. CCR6-deficient mice show significantly decreased tumour growth and tumour-associated vascularisation. The observed phenotype is dependent on CCR6 deficiency in stromal cells but not within the immune system. CONCLUSION: We propose that the chemokine axis CCL20-CCR6 represents a novel and promising target to interfere with the tumour microenvironment, and opens an innovative multimodal strategy for cancer therapy.

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 lineages. The vast majority of cortical projection neurons and certain classes of glial cells are generated by radial glial progenitor cells in a highly orchestrated manner. Recent studies employing single cell analysis and clonal lineage tracing suggest that neural stem cell and radial glial progenitor 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.

EGFR in Tumor-Associated Myeloid Cells Promotes Development of Colorectal Cancer in Mice and Associates With Outcomes of Patients.

BACKGROUND & AIMS: Inhibitors of the epidermal growth factor receptor (EGFR) are the first-line therapy for patients with metastatic colorectal tumors without RAS mutations. However, EGFR inhibitors are ineffective in these patients, and tumor level of EGFR does not associate with response to therapy. We screened human colorectal tumors for EGFR-positive myeloid cells and investigated their association with patient outcome. We also performed studies in mice to evaluate how EGFR expression in tumor cells and myeloid cells contributes to development of colitis-associated cancer and ApcMin-dependent intestinal tumorigenesis. METHODS: We performed immunohistochemical and immunofluorescent analyses of 116 colorectal tumor biopsies to determine levels of EGFR in tumor and stroma; we also collected information on tumor stage and patient features and outcomes. We used the Mann-Whitney U and Kruskal-Wallis tests to correlate tumor levels of EGFR with tumor stage, and the Kaplan-Meier method to estimate patients' median survival time. We performed experiments in mice lacking EGFR in intestinal epithelial cells (Villin-Cre; Egfrf/f and Villin-CreERT2; Egfrf/f mice) or myeloid cells (LysM-Cre; Egfrf/f mice) on a mixed background. These mice were bred with ApcMin/+ mice; colitis-associated cancer and colitis were induced by administration of dextran sodium sulfate (DSS), with or without azoxymethane (AOM), respectively. Villin-CreERT2 was activated in developed tumors by administration of tamoxifen to mice. Littermates that expressed full-length EGFR were used as controls. Intestinal tissues were collected; severity of colitis, numbers and size of tumors, and intestinal barrier integrity were assessed by histologic, immunohistochemical, quantitative reverse transcription polymerase chain reaction, and flow cytometry analyses. RESULTS: We detected EGFR in myeloid cells in the stroma of human colorectal tumors; myeloid cell expression of EGFR associated with tumor metastasis and shorter patient survival time. Mice with deletion of EGFR from myeloid cells formed significantly fewer and smaller tumors than the respective EGFR-expressing controls in an ApcMin/+ background as well as after administration of AOM and DSS. Deletion of EGFR from intestinal epithelial cells did not affect tumor growth. Furthermore, tamoxifen-induced deletion of EGFR from epithelial cells of established intestinal tumors in mice given AOM and DSS did not reduce tumor size. EGFR signaling in myeloid cells promoted activation of STAT3 and expression of survivin in intestinal tumor cells. Mice with deletion of EGFR from myeloid cells developed more severe colitis after DSS administration, characterized by increased intestinal inflammation and intestinal barrier disruption, than control mice or mice with deletion of EGFR from intestinal epithelial cells. EGFR-deficient myeloid cells in the colon of DSS-treated LysM-Cre; Egfrf/f mice had reduced expression of interleukin 6 (IL6), and epithelial STAT3 activation was reduced compared with controls. Administration of recombinant IL6 to LysM-Cre; Egfrf/f mice given DSS protected them from weight loss and restored epithelial proliferation and STAT3 activation, compared with administration of DSS alone to these mice. CONCLUSIONS: Increased expression of EGFR in myeloid cells from the colorectal tumor stroma associates with tumor progression and reduced survival time of patients with metastatic colorectal cancer. Deletion of EGFR from myeloid cells, but not intestinal epithelial cells, protects mice from colitis-induced intestinal cancer and ApcMin-dependent intestinal tumorigenesis. Myeloid cell expression of EGFR increases activation of STAT3 and expression of survivin in intestinal epithelial cells and expression of IL6 in colon tissues. These findings indicate that expression of EGFR by myeloid cells of the colorectal tumor stroma, rather than the cancer cells themselves, contributes to tumor development.

The late endosomal adaptor molecule p14 (LAMTOR2) represents a novel regulator of Langerhans cell homeostasis.

Langerhans cells (LCs) are dendritic cells (DCs) residing in epithelia, where they critically regulate immunity and tolerance. The p14 adaptor molecule is part of the late endosomal/LAMTOR (lysosomal adaptor and mitogen-activated protein kinase and mammalian target of rapamycin [mTOR] activator/regulator) complex, thereby contributing to the signal transduction of the extracellular signaling-regulated kinase (ERK) and the mTOR cascade. Furthermore, p14 represents an important regulator for endosomal sorting processes within the cell. Mutated, dysfunctional p14 leads to a human immunodeficiency disorder with endosomal/lysosomal defects in immune cells. Because p14 participates in the regulation of endosomal trafficking, growth factor signaling, and cell proliferation, we investigated the role of p14 in mouse DCs/LCs using a conditional knockout mouse model. p14-deficient animals displayed a virtually complete loss of LCs in the epidermis early after birth due to impaired proliferation and increased apoptosis of LCs. Repopulation analysis after application of contact sensitizer leads to the recruitment of a transient LC population, predominantly consisting of short-term LCs. The underlying molecular mechanism involves the p14-mediated disruption of the LAMTOR complex which results in the malfunction of both ERK and mTOR signal pathways. Hence, we conclude that p14 acts as a novel and essential regulator of LC homeostasis in vivo.

Protocol for sorting cells from mouse brains labeled with mosaic analysis with double markers by flow cytometry.

Mosaic analysis with double markers (MADM) technology enables the generation of genetic mosaic tissue in mice and high-resolution phenotyping at the individual cell level. Here, we present a protocol for isolating MADM-labeled cells with high yield for downstream molecular analyses using fluorescence-activated cell sorting (FACS). We describe steps for generating MADM-labeled mice, perfusion, single-cell suspension, and debris removal. We then detail procedures for cell sorting by FACS and downstream analysis. This protocol is suitable for embryonic to adult mice. For complete details on the use and execution of this protocol, please refer to Contreras et al. (2021).1.

Imaging brain tissue architecture across millimeter to nanometer scales.

Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanometer synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS uses fixation-compatible extracellular labeling and optical imaging, including stimulated emission depletion or expansion microscopy, to comprehensively delineate cellular structures. It enables three-dimensional reconstruction of single synapses and mapping of synaptic connectivity by identification and analysis of putative synaptic cleft regions. Applying CATS to the mouse hippocampal mossy fiber circuitry, we reconstructed and quantified the synaptic input and output structure of identified neurons. We furthermore demonstrate applicability to clinically derived human tissue samples, including formalin-fixed paraffin-embedded routine diagnostic specimens, for visualizing the cellular architecture of brain tissue in health and disease.

Tissue-wide genetic and cellular landscape shapes the execution of sequential PRC2 functions in neural stem cell lineage progression.

The generation of a correctly sized cerebral cortex with all-embracing neuronal and glial cell-type diversity critically depends on faithful radial glial progenitor (RGP) cell proliferation/differentiation programs. Temporal RGP lineage progression is regulated by Polycomb repressive complex 2 (PRC2), and loss of PRC2 activity results in severe neurogenesis defects and microcephaly. How PRC2-dependent gene expression instructs RGP lineage progression is unknown. Here, we use mosaic analysis with double markers (MADM)-based single-cell technology and demonstrate that PRC2 is not cell-autonomously required in neurogenic RGPs but rather acts at the global tissue-wide level. Conversely, cortical astrocyte production and maturation is cell-autonomously controlled by PRC2-dependent transcriptional regulation. We thus reveal highly distinct and sequential PRC2 functions in RGP lineage progression that are dependent on complex interplays between intrinsic and tissue-wide properties. In a broader context, our results imply a critical role for the genetic and cellular niche environment in neural stem cell behavior.

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).

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 <25% of all mouse genes on select chromosomes to date. To overcome this limitation, we generate transgenic mice with knocked-in MADM cassettes near the centromeres of all 19 autosomes and validate their use across organs. With this resource, >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.

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.

EGFR Controls Hair Shaft Differentiation in a p53-Independent Manner.

Epidermal growth factor receptor (EGFR) signaling controls skin development and homeostasis in mice and humans, and its deficiency causes severe skin inflammation, which might affect epidermal stem cell behavior. Here, we describe the inflammation-independent effects of EGFR deficiency during skin morphogenesis and in adult hair follicle stem cells. Expression and alternative splicing analysis of RNA sequencing data from interfollicular epidermis and outer root sheath indicate that EGFR controls genes involved in epidermal differentiation and also in centrosome function, DNA damage, cell cycle, and apoptosis. Genetic experiments employing p53 deletion in EGFR-deficient epidermis reveal that EGFR signaling exhibits p53-dependent functions in proliferative epidermal compartments, as well as p53-independent functions in differentiated hair shaft keratinocytes. Loss of EGFR leads to absence of LEF1 protein specifically in the innermost epithelial hair layers, resulting in disorganization of medulla cells. Thus, our results uncover important spatial and temporal features of cell-autonomous EGFR functions in the epidermis.

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.

Mouse models of nonmelanoma skin cancer.

The skin is the largest organ of the mammalian body, made up of multiple layers, which include the epidermis, dermis, and subcutis (Alam and Ratner, N Engl J Med 344(13):975-983, 2001). The human interfollicular epidermis can be subdivided into five different layers: (1) stratum basale, (2) stratum spinosum, (3) stratum granulosum, (4) stratum lucidum, and (5) stratum corneum, all originating from basal keratinocytes by differentiation (Hameetman et al., BMC cancer 13:58, 2013; Ramirez et al., Differentiation 58(1):53-64, 1994). The epidermis is also able to generate different appendages: hair follicles (HF) and their associated sebaceous glands (Sibilia et al., Cell 102(2):211-220, 2000) as well as sweat glands (Luetteke et al., Genes Dev 8(4):399-413, 1994). The skin has important functions in several biological processes like environmental barrier, tissue regeneration, hair cycling, and wound repair. During these processes, stem cells from the interfollicular epidermis and from the hair follicle bulge are activated to renew the epidermis or hair. The epidermis and hair undergo continuous homeostatic regeneration and mutations, upon mutations which disturb the balance of homeostatic regeneration of epidermis and hair and lead to enhanced proliferation of keratinocytes, development of skin cancer is developed. Tumors that arise in the skin are mainly of three types: malignant melanoma, arising from melanocytes, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC), the latter two both arising from keratinocytes or hair follicle cells. In this chapter, we will describe some genetically engineered mouse models (GEMM) that aim at modeling human BCC and SCC and their respective precancerous lesions. We will describe the experimental approaches used in our laboratory to analyze tumor-bearing mice focusing on methods necessary for the induction of tumor growth as well as for the molecular and histological analysis of tumor tissue.

Specific roles for dendritic cell subsets during initiation and progression of psoriasis.

Several subtypes of APCs are found in psoriasis patients, but their involvement in disease pathogenesis is poorly understood. Here, we investigated the contribution of Langerhans cells (LCs) and plasmacytoid DCs (pDCs) in psoriasis. In human psoriatic lesions and in a psoriasis mouse model (DKO* mice), LCs are severely reduced, whereas pDCs are increased. Depletion of pDCs in DKO* mice prior to psoriasis induction resulted in a milder phenotype, whereas depletion during active disease had no effect. In contrast, while depletion of Langerin-expressing APCs before disease onset had no effect, depletion from diseased mice aggravated psoriasis symptoms. Disease aggravation was due to the absence of LCs, but not other Langerin-expressing APCs. LCs derived from DKO* mice produced increased IL-10 levels, suggesting an immunosuppressive function. Moreover, IL-23 production was high in psoriatic mice and further increased in the absence of LCs. Conversely, pDC depletion resulted in reduced IL-23 production, and therapeutic inhibition of IL-23R signaling ameliorated disease symptoms. Therefore, LCs have an anti-inflammatory role during active psoriatic disease, while pDCs exert an instigatory function during disease initiation.