Axial elongation of caudalized human organoids mimics aspects of neural tube development
Development (Cambridge, England)
Libby, ARG;Joy, DA;Elder, NH;Bulger, EA;Krakora, MZ;Gaylord, EA;Mendoza-Camacho, F;Butts, JC;McDevitt, TC;
PMID: 34142711 | DOI: 10.1242/dev.198275
Axial elongation of the neural tube is crucial during mammalian embryogenesis for anterior-posterior body axis establishment and subsequent spinal cord development, but these processes cannot be interrogated directly in humans as they occur post-implantation. Here, we report an organoid model of neural tube extension derived from human pluripotent stem cell (hPSC) aggregates that have been caudalized with Wnt agonism, enabling them to recapitulate aspects of the morphological and temporal gene expression patterns of neural tube development. Elongating organoids consist largely of neuroepithelial compartments and contain TBXT+SOX2+ neuro-mesodermal progenitors in addition to PAX6+NES+ neural progenitors. A critical threshold of Wnt agonism stimulated singular axial extensions while maintaining multiple cell lineages, such that organoids displayed regionalized anterior-to-posterior HOX gene expression with hindbrain (HOXB1) regions spatially distinct from brachial (HOXC6) and thoracic (HOXB9) regions. CRISPR interference-mediated silencing of TBXT, a Wnt pathway target, increased neuroepithelial compartmentalization, abrogated HOX expression and disrupted uniaxial elongation. Together, these results demonstrate the potent capacity of caudalized hPSC organoids to undergo axial elongation in a manner that can be used to dissect the cellular organization and patterning decisions that dictate early human nervous system development.
Tumor vessel co-option probed by single-cell analysis
Teuwen, LA;De Rooij, LPMH;Cuypers, A;Rohlenova, K;Dumas, SJ;García-Caballero, M;Meta, E;Amersfoort, J;Taverna, F;Becker, LM;Veiga, N;Cantelmo, AR;Geldhof, V;Conchinha, NV;Kalucka, J;Treps, L;Conradi, LC;Khan, S;Karakach, TK;Soenen, S;Vinckier, S;Schoonjans, L;Eelen, G;Van Laere, S;Dewerchin, M;Dirix, L;Mazzone, M;Luo, Y;Vermeulen, P;Carmeliet, P;
PMID: 34133923 | DOI: 10.1016/j.celrep.2021.109253
Tumor vessel co-option is poorly understood, yet it is a resistance mechanism against anti-angiogenic therapy (AAT). The heterogeneity of co-opted endothelial cells (ECs) and pericytes, co-opting cancer and myeloid cells in tumors growing via vessel co-option, has not been investigated at the single-cell level. Here, we use a murine AAT-resistant lung tumor model, in which VEGF-targeting induces vessel co-option for continued growth. Single-cell RNA sequencing (scRNA-seq) of 31,964 cells reveals, unexpectedly, a largely similar transcriptome of co-opted tumor ECs (TECs) and pericytes as their healthy counterparts. Notably, we identify cell types that might contribute to vessel co-option, i.e., an invasive cancer-cell subtype, possibly assisted by a matrix-remodeling macrophage population, and another M1-like macrophage subtype, possibly involved in keeping or rendering vascular cells quiescent.
Lipolysis drives expression of the constitutively active receptor GPR3 to induce adipose thermogenesis
Sveidahl Johansen, O;Ma, T;Hansen, JB;Markussen, LK;Schreiber, R;Reverte-Salisa, L;Dong, H;Christensen, DP;Sun, W;Gnad, T;Karavaeva, I;Nielsen, TS;Kooijman, S;Cero, C;Dmytriyeva, O;Shen, Y;Razzoli, M;O'Brien, SL;Kuipers, EN;Nielsen, CH;Orchard, W;Willemsen, N;Jespersen, NZ;Lundh, M;Sustarsic, EG;Hallgren, CM;Frost, M;McGonigle, S;Isidor, MS;Broholm, C;Pedersen, O;Hansen, JB;Grarup, N;Hansen, T;Kjær, A;Granneman, JG;Babu, MM;Calebiro, D;Nielsen, S;Rydén, M;Soccio, R;Rensen, PCN;Treebak, JT;Schwartz, TW;Emanuelli, B;Bartolomucci, A;Pfeifer, A;Zechner, R;Scheele, C;Mandrup, S;Gerhart-Hines, Z;
PMID: 34048700 | DOI: 10.1016/j.cell.2021.04.037
Thermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of β-adrenergic G protein-coupled receptors (GPCRs). Here, we uncover an alternate paradigm of GPCR-mediated adipose thermogenesis through the constitutively active receptor, GPR3. We show that the N terminus of GPR3 confers intrinsic signaling activity, resulting in continuous Gs-coupling and cAMP production without an exogenous ligand. Thus, transcriptional induction of Gpr3 represents the regulatory parallel to ligand-binding of conventional GPCRs. Consequently, increasing Gpr3 expression in thermogenic adipocytes is alone sufficient to drive energy expenditure and counteract metabolic disease in mice. Gpr3 transcription is cold-stimulated by a lipolytic signal, and dietary fat potentiates GPR3-dependent thermogenesis to amplify the response to caloric excess. Moreover, we find GPR3 to be an essential, adrenergic-independent regulator of human brown adipocytes. Taken together, our findings reveal a noncanonical mechanism of GPCR control and thermogenic activation through the lipolysis-induced expression of constitutively active GPR3.
Ventral pallidum DRD3 potentiates a pallido-habenular circuit driving accumbal dopamine release and cocaine seeking
Pribiag, H;Shin, S;Wang, EH;Sun, F;Datta, P;Okamoto, A;Guss, H;Jain, A;Wang, XY;De Freitas, B;Honma, P;Pate, S;Lilascharoen, V;Li, Y;Lim, BK;
PMID: 34048697 | DOI: 10.1016/j.neuron.2021.05.002
Drugs of abuse induce persistent remodeling of reward circuit function, a process thought to underlie the emergence of drug craving and relapse to drug use. However, how circuit-specific, drug-induced molecular and cellular plasticity can have distributed effects on the mesolimbic dopamine reward system to facilitate relapse to drug use is not fully elucidated. Here, we demonstrate that dopamine receptor D3 (DRD3)-dependent plasticity in the ventral pallidum (VP) drives potentiation of dopamine release in the nucleus accumbens during relapse to cocaine seeking after abstinence. We show that two distinct VP DRD3+ neuronal populations projecting to either the lateral habenula (LHb) or the ventral tegmental area (VTA) display different patterns of activity during drug seeking following abstinence from cocaine self-administration and that selective suppression of elevated activity or DRD3 signaling in the LHb-projecting population reduces drug seeking. Together, our results uncover how circuit-specific DRD3-mediated plasticity contributes to the process of drug relapse.
Single cell RNA-seq analysis of the flexor digitorum brevis mouse myofibers
Verma, RX;Kannan, S;Lin, BL;Fomchenko, KM;Nieuwenhuis, TO;Patil, AH;Lukban, C;Yang, X;Fox-Talbot, K;McCall, MN;Kwon, C;Kass, DA;Rosenberg, AZ;Halushka, MK;
PMID: 34001262 | DOI: 10.1186/s13395-021-00269-2
Skeletal muscle myofibers can be separated into functionally distinct cell types that differ in gene and protein expression. Current single cell expression data is generally based upon single nucleus RNA, rather than whole myofiber material. We examined if a whole-cell flow sorting approach could be applied to perform single cell RNA-seq (scRNA-seq) in a single muscle type. We performed deep, whole cell, scRNA-seq on intact and fragmented skeletal myofibers from the mouse fast-twitch flexor digitorum brevis muscle utilizing a flow-gated method of large cell isolation. We performed deep sequencing of 763 intact and fragmented myofibers. Quality control metrics across the different gates indicated only 171 of these cells were optimal, with a median read count of 239,252 and an average of 12,098 transcripts per cell. scRNA-seq identified three clusters of myofibers (a slow/fast 2A cluster and two fast 2X clusters). Comparison to a public skeletal nuclear RNA-seq dataset demonstrated a diversity in transcript abundance by method. RISH validated multiple genes across fast and slow twitch skeletal muscle types. This study introduces and validates a method to isolate intact skeletal muscle myofibers to generate deep expression patterns and expands the known repertoire of fiber-type-specific genes.
Co-occupancy identifies transcription factor co-operation for axon growth
Venkatesh, I;Mehra, V;Wang, Z;Simpson, MT;Eastwood, E;Chakraborty, A;Beine, Z;Gross, D;Cabahug, M;Olson, G;Blackmore, MG;
PMID: 33953205 | DOI: 10.1038/s41467-021-22828-3
Transcription factors (TFs) act as powerful levers to regulate neural physiology and can be targeted to improve cellular responses to injury or disease. Because TFs often depend on cooperative activity, a major challenge is to identify and deploy optimal sets. Here we developed a bioinformatics pipeline, centered on TF co-occupancy of regulatory DNA, and used it to predict factors that potentiate the effects of pro-regenerative Klf6 in vitro. High content screens of neurite outgrowth identified cooperative activity by 12 candidates, and systematic testing in a mouse model of corticospinal tract (CST) damage substantiated three novel instances of pairwise cooperation. Combined Klf6 and Nr5a2 drove the strongest growth, and transcriptional profiling of CST neurons identified Klf6/Nr5a2-responsive gene networks involved in macromolecule biosynthesis and DNA repair. These data identify TF combinations that promote enhanced CST growth, clarify the transcriptional correlates, and provide a bioinformatics approach to detect TF cooperation.
Transcriptional analysis of cystic fibrosis airways at single-cell resolution reveals altered epithelial cell states and composition
Carraro, G;Langerman, J;Sabri, S;Lorenzana, Z;Purkayastha, A;Zhang, G;Konda, B;Aros, CJ;Calvert, BA;Szymaniak, A;Wilson, E;Mulligan, M;Bhatt, P;Lu, J;Vijayaraj, P;Yao, C;Shia, DW;Lund, AJ;Israely, E;Rickabaugh, TM;Ernst, J;Mense, M;Randell, SH;Vladar, EK;Ryan, AL;Plath, K;Mahoney, JE;Stripp, BR;Gomperts, BN;
PMID: 33958799 | DOI: 10.1038/s41591-021-01332-7
Cystic fibrosis (CF) is a lethal autosomal recessive disorder that afflicts more than 70,000 people. People with CF experience multi-organ dysfunction resulting from aberrant electrolyte transport across polarized epithelia due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF-related lung disease is by far the most important determinant of morbidity and mortality. Here we report results from a multi-institute consortium in which single-cell transcriptomics were applied to define disease-related changes by comparing the proximal airway of CF donors (n = 19) undergoing transplantation for end-stage lung disease with that of previously healthy lung donors (n = 19). Disease-dependent differences observed include an overabundance of epithelial cells transitioning to specialized ciliated and secretory cell subsets coupled with an unexpected decrease in cycling basal cells. Our study yields a molecular atlas of the proximal airway epithelium that will provide insights for the development of new targeted therapies for CF airway disease.
Brain-specific lipoprotein receptors interact with astrocyte derived apolipoprotein and mediate neuron-glia lipid shuttling
Yin, J;Spillman, E;Cheng, ES;Short, J;Chen, Y;Lei, J;Gibbs, M;Rosenthal, JS;Sheng, C;Chen, YX;Veerasammy, K;Choetso, T;Abzalimov, R;Wang, B;Han, C;He, Y;Yuan, Q;
PMID: 33893307 | DOI: 10.1038/s41467-021-22751-7
Lipid shuttling between neurons and glia contributes to the development, function, and stress responses of the nervous system. To understand how a neuron acquires its lipid supply from specific lipoproteins and their receptors, we perform combined genetic, transcriptome, and biochemical analyses in the developing Drosophila larval brain. Here we report, the astrocyte-derived secreted lipocalin Glial Lazarillo (GLaz), a homolog of human Apolipoprotein D (APOD), and its neuronal receptor, the brain-specific short isoforms of Drosophila lipophorin receptor 1 (LpR1-short), cooperatively mediate neuron-glia lipid shuttling and support dendrite morphogenesis. The isoform specificity of LpR1 defines its distribution, binding partners, and ability to support proper dendrite growth and synaptic connectivity. By demonstrating physical and functional interactions between GLaz/APOD and LpR1, we elucidate molecular pathways mediating lipid trafficking in the fly brain, and provide in vivo evidence indicating isoform-specific expression of lipoprotein receptors as a key mechanism for regulating cell-type specific lipid recruitment.
MrgprC11+ sensory neurons mediate glabrous skin itch
Proceedings of the National Academy of Sciences of the United States of America
Steele, HR;Xing, Y;Zhu, Y;Hilley, HB;Lawson, K;Nho, Y;Niehoff, T;Han, L;
PMID: 33876765 | DOI: 10.1073/pnas.2022874118
Itch arising from glabrous skin (palms and soles) has attracted limited attention within the field due to the lack of methodology. This is despite glabrous itch arising from many medical conditions such as plantar and palmar psoriasis, dyshidrosis, and cholestasis. Therefore, we developed a mouse glabrous skin behavioral assay to investigate the contribution of three previously identified pruriceptive neurons in glabrous skin itch. Our results show that MrgprA3+ and MrgprD+ neurons, although key mediators for hairy skin itch, do not play important roles in glabrous skin itch, demonstrating a mechanistic difference in itch sensation between hairy and glabrous skin. We found that MrgprC11+ neurons are the major mediators for glabrous skin itch. Activation of MrgprC11+ neurons induced glabrous skin itch, while ablation of MrgprC11+ neurons reduced both acute and chronic glabrous skin itch. Our study provides insights into the mechanisms of itch and opens up new avenues for future glabrous skin itch research.
Microbiota-derived short chain fatty acids modulate microglia and promote Aβ plaque deposition
Colombo, AV;Sadler, RK;Llovera, G;Singh, V;Roth, S;Heindl, S;Sebastian Monasor, L;Verhoeven, A;Peters, F;Parhizkar, S;Kamp, F;Gomez de Aguero, M;MacPherson, AJ;Winkler, E;Herms, J;Benakis, C;Dichgans, M;Steiner, H;Giera, M;Haass, C;Tahirovic, S;Liesz, A;
PMID: 33845942 | DOI: 10.7554/eLife.59826
Previous studies have identified a crucial role of the gut microbiome in modifying Alzheimer's disease (AD) progression. However, the mechanisms of microbiome-brain interaction in AD were so far unknown. Here, we identify microbiota-derived short chain fatty acids (SCFA) as microbial metabolites which promote Aβ deposition. Germ-free (GF) AD mice exhibit a substantially reduced Aβ plaque load and markedly reduced SCFA plasma concentrations; conversely, SCFA supplementation to GF AD mice increased the Aβ plaque load to levels of conventionally colonized (specific pathogen-free [SPF]) animals and SCFA supplementation to SPF mice even further exacerbated plaque load. This was accompanied by the pronounced alterations in microglial transcriptomic profile, including upregulation of ApoE. Despite increased microglial recruitment to Aβ plaques upon SCFA supplementation, microglia contained less intracellular Aβ. Taken together, our results demonstrate that microbiota-derived SCFA are critical mediators along the gut-brain axis which promote Aβ deposition likely via modulation of the microglial phenotype.
Flexible scaling and persistence of social vocal communication
Chen, J;Markowitz, JE;Lilascharoen, V;Taylor, S;Sheurpukdi, P;Keller, JA;Jensen, JR;Lim, BK;Datta, SR;Stowers, L;
PMID: 33790464 | DOI: 10.1038/s41586-021-03403-8
Innate vocal sounds such as laughing, screaming or crying convey one's feelings to others. In many species, including humans, scaling the amplitude and duration of vocalizations is essential for effective social communication1-3. In mice, female scent triggers male mice to emit innate courtship ultrasonic vocalizations (USVs)4,5. However, whether mice flexibly scale their vocalizations and how neural circuits are structured to generate flexibility remain largely unknown. Here we identify mouse neurons from the lateral preoptic area (LPOA) that express oestrogen receptor 1 (LPOAESR1 neurons) and, when activated, elicit the complete repertoire of USV syllables emitted during natural courtship. Neural anatomy and functional data reveal a two-step, di-synaptic circuit motif in which primary long-range inhibitory LPOAESR1 neurons relieve a clamp of local periaqueductal grey (PAG) inhibition, enabling excitatory PAG USV-gating neurons to trigger vocalizations. We find that social context shapes a wide range of USV amplitudes and bout durations. This variability is absent when PAG neurons are stimulated directly; PAG-evoked vocalizations are time-locked to neural activity and stereotypically loud. By contrast, increasing the activity of LPOAESR1 neurons scales the amplitude of vocalizations, and delaying the recovery of the inhibition clamp prolongs USV bouts. Thus, the LPOA disinhibition motif contributes to flexible loudness and the duration and persistence of bouts, which are key aspects of effective vocal social communication.
Cañellas-Socias, A;Cortina, C;Hernando-Momblona, X;Palomo-Ponce, S;Mulholland, EJ;Turon, G;Mateo, L;Conti, S;Roman, O;Sevillano, M;Slebe, F;Stork, D;Caballé-Mestres, A;Berenguer-Llergo, A;Álvarez-Varela, A;Fenderico, N;Novellasdemunt, L;Jiménez-Gracia, L;Sipka, T;Bardia, L;Lorden, P;Colombelli, J;Heyn, H;Trepat, X;Tejpar, S;Sancho, E;Tauriello, DVF;Leedham, S;Attolini, CS;Batlle, E;
PMID: 36352230 | DOI: 10.1038/s41586-022-05402-9
Around 30-40% of patients with colorectal cancer (CRC) undergoing curative resection of the primary tumour will develop metastases in the subsequent years<sup>1</sup>. Therapies to prevent disease relapse remain an unmet medical need. Here we uncover the identity and features of the residual tumour cells responsible for CRC relapse. An analysis of single-cell transcriptomes of samples from patients with CRC revealed that the majority of genes associated with a poor prognosis are expressed by a unique tumour cell population that we named high-relapse cells (HRCs). We established a human-like mouse model of microsatellite-stable CRC that undergoes metastatic relapse after surgical resection of the primary tumour. Residual HRCs occult in mouse livers after primary CRC surgery gave rise to multiple cell types over time, including LGR5<sup>+</sup> stem-like tumour cells<sup>2-4</sup>, and caused overt metastatic disease. Using Emp1 (encoding epithelial membrane protein 1) as a marker gene for HRCs, we tracked and selectively eliminated this cell population. Genetic ablation of EMP1<sup>high</sup> cells prevented metastatic recurrence and mice remained disease-free after surgery. We also found that HRC-rich micrometastases were infiltrated with T cells, yet became progressively immune-excluded during outgrowth. Treatment with neoadjuvant immunotherapy eliminated residual metastatic cells and prevented mice from relapsing after surgery. Together, our findings reveal the cell-state dynamics of residual disease in CRC and anticipate that therapies targeting HRCs may help to avoid metastatic relapse.
