Single-cell roadmap of human gonadal development

Nature

2022 Jul 01

Garcia-Alonso, L;Lorenzi, V;Mazzeo, CI;Alves-Lopes, JP;Roberts, K;Sancho-Serra, C;Engelbert, J;Marečková, M;Gruhn, WH;Botting, RA;Li, T;Crespo, B;van Dongen, S;Kiselev, VY;Prigmore, E;Herbert, M;Moffett, A;Chédotal, A;Bayraktar, OA;Surani, A;Haniffa, M;Vento-Tormo, R;
PMID: 35794482 | DOI: 10.1038/s41586-022-04918-4

Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal-mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.

TGFβ1 Induces Senescence and Attenuated VEGF Production in Retinal Pericytes

Biomedicines

2022 Jun 14

Avramovic, D;Archaimbault, SA;Kemble, AM;Gruener, S;Lazendic, M;Westenskow, PD;
PMID: 35740425 | DOI: 10.3390/biomedicines10061404

Diabetic retinopathy (DR) is a microvascular disease of the retina and a serious complication of type I and type II diabetes mellitus. DR affects working-age populations and can cause permanent vision loss if left untreated. The standard of care for proliferative DR is inhibiting VEGF. However, the mechanisms that induce excessive VEGF production in the retina remain elusive, although some evidence links elevated VEGF in the diabetic retina with local and systemic TGFβ1 upexpression. Here, we present evidence from animal models of disease suggesting that excessive TGFβ1 production in the early DR is correlated with VEGF mRNA and protein production by senescent pericytes and other retinal cells. Collectively, these results confirm that TGFβ1 is strongly implicated in the vascular complications of DR.

Targeted ablation of Lgr5-expressing intestinal stem cells in diphtheria toxin receptor-based mouse and organoid models

STAR protocols

2022 Jun 17

Lim, HYG;Yada, S;Barker, N;
PMID: 35620071 | DOI: 10.1016/j.xpro.2022.101411

Intestinal cells marked by Lgr5 function as tissue-resident stem cells that sustain the homeostatic replenishment of the epithelium. By incorporating a diphtheria toxin receptor (DTR) cassette linked to the Lgr5 coding region, native Lgr5-expressing cells are susceptible to ablation upon DT administration in vivo. A similar strategy can be used for Lgr5-expressing cells within organoids established from DTR models. Together, these in vivo and in vitro approaches will facilitate dissection of the roles of Lgr5-expressing cells residing in different tissue compartments. For complete details on the use and execution of this protocol, please refer to Tan et al. (2021).

Stromal Hedgehog signalling is downregulated in colon cancer and its restoration restrains tumour growth

Nat Commun.

2016 Aug 05

Gerling M, Büller NV, Kirn LM, Joost S, Frings O, Englert B, Bergström Å, Kuiper RV, Blaas L, Wielenga MC, Almer S, Kühl AA, Fredlund E, van den Brink GR, Toftgård R.
PMID: 27492255 | DOI: 10.1038/ncomms12321

A role for Hedgehog (Hh) signalling in the development of colorectal cancer (CRC) has been proposed. In CRC and other solid tumours, Hh ligands are upregulated; however, a specific Hh antagonist provided no benefit in a clinical trial. Here we use Hh reporter mice to show that downstream Hh activity is unexpectedly diminished in a mouse model of colitis-associated colon cancer, and that downstream Hh signalling is restricted to the stroma. Functionally, stroma-specific Hh activation in mice markedly reduces the tumour load and blocks progression of advanced neoplasms, partly via the modulation of BMP signalling and restriction of the colonic stem cell signature. By contrast, attenuated Hh signalling accelerates colonic tumourigenesis. In human CRC, downstream Hh activity is similarly reduced and canonical Hh signalling remains predominantly paracrine. Our results suggest that diminished downstream Hh signalling enhances CRC development, and that stromal Hh activation can act as a colonic tumour suppressor.

Lgr5-expressing chief cells drive epithelial regeneration and cancer in the oxyntic stomach.

Nat Cell Biol.

2017 Jun 05

Leushacke M, Tan SH, Wong A, Swathi Y, Hajamohideen A, Tan LT, Goh J, Wong E, Denil SLIJ, Murakami K, Barker N.
PMID: 28581476 | DOI: 10.1038/ncb3541

The daily renewal of the corpus epithelium is fuelled by adult stem cells residing within tubular glands, but the identity of these stem cells remains controversial. Lgr5 marks homeostatic stem cells and 'reserve' stem cells in multiple tissues. Here, we report Lgr5 expression in a subpopulation of chief cells in mouse and human corpus glands. Using a non-variegated Lgr5-2A-CreERT2 mouse model, we show by lineage tracing that Lgr5-expressing chief cells do not behave as corpus stem cells during homeostasis, but are recruited to function as stem cells to effect epithelial renewal following injury by activating Wnt signalling. Ablation of Lgr5+ cells severely impairs epithelial homeostasis in the corpus, indicating an essential role for these Lgr5+ cells in maintaining the homeostatic stem cell pool. We additionally define Lgr5+ chief cells as a major cell-of-origin of gastric cancer. These findings reveal clinically relevant insights into homeostasis, repair and cancer in the corpus.

FOXO1 reduces tumorsphere formation capacity and has crosstalk with LGR5 signaling in gastric cancer cells

Biochemical and Biophysical Research Communications

2017 Sep 29

Choi Y, Park J, Ko YS, Kim Y, Pyo JS, Jange BG, Kim MA, Leef JS, Chang MS, Lee BL.
PMID: - | DOI: 10.1016/j.bbrc.2017.09.163

Gastric cancer (GC) is a major of cause of cancer-related death and is characterized by its heterogeneity and molecular complexity. FOXO1 is a transcription factor that plays a key role in GC growth and metastasis. However, the implication of FOXO1 in GC cell stemness has been elusive. This study, for the first time, demonstrates that FOXO1 regulates GC cell stemness in association with LGR5. FOXO1 expression was significantly lower in GC tumorsphere cells than in adherent GC cells. FOXO1 silencing and overexpression promoted and inhibited the tumorsphere formation capacity of GC cells, respectively. Additionally, there was an inverse correlation between FOXO1 and GC stem cell marker LGR5 in human GC specimens. Further in vitro and in vivo experiments showed that negative crosstalk between these two molecules exists and that LGR5 silencing reversed the FOXO1 shRNA-induced tumorsphere formation even without FOXO1 restoration. Taken together, our results suggest that FOXO1 inhibits the self-renewal capacity of GC cells through interaction with LGR5. Thus, FOXO1/LGR5 signaling pathway may provide a novel targeted therapy for GC.

Biocompatible, Purified VEGF-A mRNA Improves Cardiac Function after Intracardiac Injection One Week Post-Myocardial Infarction in Swine

Molecular Therapy: Methods & Clinical Development

2018 Apr 10

Carlsson L, Clarke JC, Yen C, Gregoire F, Albery T, Billger M, Egnell AC, Gan LM, Jennbacken K, Johansson E, Linhardt G, Martinsson S, Sadiq MW, Witman N, Wang QD, Chen CH, Wang YP, Lin S, Ticho B, Hsieh P, Chien KR, Fritsche-Danielson R.
PMID: - | DOI: 10.1016/j.omtm.2018.04.003

Messenger RNA (mRNA) can direct dose-dependent protein expression in cardiac muscle without genome integration, but to date has not been shown to improve cardiac function in a safe, clinically applicable way. Herein, we report that a purified and optimized mRNA in a biocompatible citrate-saline formulation is tissue specific, long-acting, and does not stimulate an immune response. In small and large animal, permanent occlusion myocardial infarction models VEGF-A 165 mRNA improves systolic ventricular function and limits myocardial damage. Following a single administration a week post infarction in mini-pigs, left ventricular ejection fraction, inotropy, and ventricular compliance improved, border zone arteriolar and capillary density increased, and myocardial fibrosis decreased at two months post-treatment. Purified VEGF-A mRNA establishes the feasibility of improving cardiac function in the sub-acute therapeutic window and may represent a new class of therapies for ischemic injury.

L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

Nat Cancer

2020 Jan 13

Karuna Ganesh, Harihar Basnet, Yasemin Kaygusuz, Ashley M. Laughney, Lan He, Roshan Sharma, Kevin P. O�Rourke, Vincent P. Reuter, Yun-Han Huang, Mesruh Turkekul, Ekrem Emrah Er, Ignas Masilionis, Katia Manova-Todorova, Martin R. Weiser, Leonard B. Saltz, Julio Garcia-Aguilar, Richard Koche, Scott W. Lowe, Dana Pe�er, Jinru Shia & Joan Massagu�
| DOI: 10.1038/s43018-019-0006-x

Metastasis-initiating cells with stem-like properties drive cancer lethality, yet their origins and relationship to primary-tumor-initiating stem cells are not known. We show that L1CAM+ cells in human colorectal cancer (CRC) have metastasis-initiating capacity, and we define their relationship to tissue regeneration. L1CAM is not expressed in the homeostatic intestinal epithelium, but is induced and required for epithelial regeneration following colitis and in CRC organoid growth. By using human tissues and mouse models, we show that L1CAM is dispensable for adenoma initiation but required for orthotopic carcinoma propagation, liver metastatic colonization and chemoresistance. L1CAMhigh cells partially overlap with LGR5high stem-like cells in human CRC organoids. Disruption of intercellular epithelial contacts causes E-cadherin�REST transcriptional derepression of L1CAM, switching chemoresistant CRC progenitors from an L1CAMlow to an L1CAMhigh state. Thus, L1CAM dependency emerges in regenerative intestinal cells when epithelial integrity is lost, a phenotype of wound healing deployed in metastasis-initiating cells.

Local coordination between intracortical bone remodeling and vascular development in human juvenile bone

Bone

2023 May 05

Andreasen, CM;El-Masri, BM;MacDonald, B;Laursen, KS;Nielsen, MH;Thomsen, JS;Delaisse, JM;Andersen, TL;
PMID: 37150243 | DOI: 10.1016/j.bone.2023.116787

Although failure to establish a vascular network has been associated with many skeletal disorders, little is known about what drives development of vasculature in the intracortical bone compartments. Here, we show that intracortical bone resorption events are coordinated with development of the vasculature. We investigated the prevalence of vascular structures at different remodeling stages as well as their 3D organization using proximal femoral cortical bone from 5 girls and 6 boys (aged 6-15 years). A 2D analysis revealed that non-quiescent intracortical pores contained more vascular structures than quiescent pores (p < 0.0001). Type 2 pores, i.e., remodeling of existing pores, had a higher density of vascular structures than type 1 pores, i.e., de novo created pores (p < 0.05). Furthermore, pores at the eroded-formative remodeling stage, had more vascular structures than pores at any other remodeling stage (p < 0.05). A 3D reconstruction of an intracortical remodeling event showed that osteoclasts in the advancing tip of the cutting cone as well as preosteoclasts in the lumen expressed vascular endothelial growth factor-A (VEGFA), while VEGFA-receptors 1 and 2 mainly were expressed in endothelial cells in the adjacent vasculature. Consequently, we propose that the progression of the vascular network in intracortical remodeling events is driven by osteoclasts expressing VEGFA. Moreover, the vasculature is continuously reconfigured according to the demands of the remodeling events at the surrounding bone surfaces.

Cell-matrix interface regulates dormancy in human colon cancer stem cells

Nature

2022 Jul 07

Ohta, Y;Fujii, M;Takahashi, S;Takano, A;Nanki, K;Matano, M;Hanyu, H;Saito, M;Shimokawa, M;Nishikori, S;Hatano, Y;Ishii, R;Sawada, K;Machinaga, A;Ikeda, W;Imamura, T;Sato, T;
PMID: 35798028 | DOI: 10.1038/s41586-022-05043-y

Cancer relapse after chemotherapy remains a main cause of cancer-related death. Although the relapse is thought to result from the propagation of resident cancer stem cells (CSCs)1, a lack of experimental platforms that enable prospective analysis of CSC dynamics with sufficient spatiotemporal resolution has hindered testing of this hypothesis. Here, we develop a live genetic lineage-tracing system that allows longitudinal tracking of individual cells in xenotransplanted human colorectal cancer organoids and identify LGR5+ CSCs that display a dormant behavior in a chemo-naive state. Dormant LGR5+ cells are marked by p27 expression, and intravital imaging directly demonstrates the persistence of LGR5+p27+ cells during chemotherapy, followed by clonal expansion. Transcriptome analysis reveals an upregulation of COL17A1, a cell adhesion molecule that strengthens hemidesmosome, in dormant LGR5+p27+ cells. COL17A1-knockout organoids lose the dormant LGR5+p27+ subpopulation and become sensitive to chemotherapy, suggesting a role of cell-matrix interface in dormancy maintenance. Chemotherapy disrupts COL17A1 and breaks the dormancy in LGR5+p27+ cells through FAK-YAP activation. Abrogation of YAP signaling restrains chemo-resistant cells from exiting dormancy and delays tumor regrowth, highlighting the therapeutic potential of YAP inhibition in preventing cancer relapse. These results offer a viable therapeutic approach to overcome refractoriness of human colorectal cancer to conventional chemotherapy.

Opposing effects of Wnt/β-catenin signaling on epithelial and mesenchymal cell fate in the developing cochlea

Development (Cambridge, England)

2021 Jun 01

Billings, SE;Myers, NM;Quiruz, L;Cheng, AG;
PMID: 34061174 | DOI: 10.1242/dev.199091

During embryonic development, the otic epithelium and surrounding periotic mesenchymal cells originate from distinct lineages and coordinate to form the mammalian cochlea. Epithelial sensory precursors within the cochlear duct first undergo terminal mitosis before differentiating into sensory and non-sensory cells. In parallel, periotic mesenchymal cells differentiate to shape the lateral wall, modiolus and pericochlear spaces. Previously, Wnt activation was shown to promote proliferation and differentiation of both otic epithelial and mesenchymal cells. Here, we fate-mapped Wnt-responsive epithelial and mesenchymal cells in mice and found that Wnt activation resulted in opposing cell fates. In the post-mitotic cochlear epithelium, Wnt activation via β-catenin stabilization induced clusters of proliferative cells that dedifferentiated and lost epithelial characteristics. In contrast, Wnt-activated periotic mesenchyme formed ectopic pericochlear spaces and cell clusters showing a loss of mesenchymal and gain of epithelial features. Finally, clonal analyses via multi-colored fate-mapping showed that Wnt-activated epithelial cells proliferated and formed clonal colonies, whereas Wnt-activated mesenchymal cells assembled as aggregates of mitotically quiescent cells. Together, we show that Wnt activation drives transition between epithelial and mesenchymal states in a cell type-dependent manner.

Single-cell sequencing reveals suppressive transcriptional programs regulated by MIS/AMH in neonatal ovaries

Proceedings of the National Academy of Sciences of the United States of America

2021 May 18

Meinsohn, MC;Saatcioglu, HD;Wei, L;Li, Y;Horn, H;Chauvin, M;Kano, M;Nguyen, NMP;Nagykery, N;Kashiwagi, A;Samore, WR;Wang, D;Oliva, E;Gao, G;Morris, ME;Donahoe, PK;Pépin, D;
PMID: 33980714 | DOI: 10.1073/pnas.2100920118

Müllerian inhibiting substance (MIS/AMH), produced by granulosa cells of growing follicles, is an important regulator of folliculogenesis and follicle development. Treatment with exogenous MIS in mice suppresses follicle development and prevents ovulation. To investigate the mechanisms by which MIS inhibits follicle development, we performed single-cell RNA sequencing of whole neonatal ovaries treated with MIS at birth and analyzed at postnatal day 6, coinciding with the first wave of follicle growth. We identified distinct transcriptional signatures associated with MIS responses in the ovarian cell types. MIS treatment inhibited proliferation in granulosa, surface epithelial, and stromal cell types of the ovary and elicited a unique signature of quiescence in granulosa cells. In addition to decreasing the number of growing preantral follicles, we found that MIS treatment uncoupled the maturation of germ cells and granulosa cells. In conclusion, MIS suppressed neonatal follicle development by inhibiting proliferation, imposing a quiescent cell state, and preventing granulosa cell differentiation.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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