Superresolution microscopy localizes endogenous Dvl2 to Wnt signaling-responsive biomolecular condensates.

During organismal development, homeostasis, and disease, Dishevelled (Dvl) proteins act as key signaling factors in beta-catenin-dependent and beta-catenin-independent Wnt pathways. While their importance for signal transmission has been genetically demonstrated in many organisms, our mechanistic understanding is still limited. Previous studies using overexpressed proteins showed Dvl localization to large, punctate-like cytoplasmic structures that are dependent on its DIX domain. To study Dvl's role in Wnt signaling, we genome engineered an endogenously expressed Dvl2 protein tagged with an mEos3.2 fluorescent protein for superresolution imaging. First, we demonstrate the functionality and specificity of the fusion protein in beta-catenin-dependent and beta-catenin-independent signaling using multiple independent assays. We performed live-cell imaging of Dvl2 to analyze the dynamic formation of the supramolecular cytoplasmic Dvl2_mEos3.2 condensates. While overexpression of Dvl2_mEos3.2 mimics the previously reported formation of abundant large "puncta," supramolecular condensate formation at physiological protein levels is only observed in a subset of cells with approximately one per cell. We show that, in these condensates, Dvl2 colocalizes with Wnt pathway components at gamma-tubulin and CEP164-positive centrosomal structures and that the localization of Dvl2 to these condensates is Wnt dependent. Single-molecule localization microscopy using photoactivated localization microscopy (PALM) of mEos3.2 in combination with DNA-PAINT demonstrates the organization and repetitive patterns of these condensates in a cell cycle-dependent manner. Our results indicate that the localization of Dvl2 in supramolecular condensates is coordinated dynamically and dependent on cell state and Wnt signaling levels. Our study highlights the formation of endogenous and physiologically regulated biomolecular condensates in the Wnt pathways at single-molecule resolution.

A novel method to efficiently differentiate human osteoclasts from blood-derived monocytes.

BACKGROUND: Osteoclasts are the tissue-specific macrophage population of the bone and unique in their bone-resorbing activity. Hence, they are fundamental for bone physiology in health and disease. However, efficient protocols for the isolation and study of primary human osteoclasts are scarce. In this study, we aimed to establish a protocol, which enables the efficient differentiation of functional human osteoclasts from monocytes. RESULTS: Human monocytes were isolated through a double-density gradient from donor blood. Compared to standard differentiation schemes in polystyrene cell culture dishes, the yield of multinuclear osteoclasts was significantly increased upon initial differentiation of monocytes to macrophages in fluorinated ethylene propylene (FEP) Teflon bags. This initial differentiation phase was then followed by the development of terminal osteoclasts by addition of Receptor Activator of NF-κB Ligand (RANKL). High concentrations of RANKL and Macrophage colony-stimulating factor (M-CSF) as well as an intermediate cell density further supported efficient cell differentiation. The generated cells were highly positive for CD45, CD14 as well as the osteoclast markers CD51/ITGAV and Cathepsin K/CTSK, thus identifying them as osteoclasts. The bone resorption of the osteoclasts was significantly increased when the cells were differentiated from macrophages derived from Teflon bags compared to macrophages derived from conventional cell culture plates. CONCLUSION: Our study has established a novel protocol for the isolation of primary human osteoclasts that improves osteoclastogenesis in comparison to the conventionally used cultivation approach.

Extracellular vesicle-associated tyrosine kinase-like orphan receptors ROR1 and ROR2 promote breast cancer progression.

BACKGROUND: Extracellular vesicles (EVs) harbor a plethora of different biomolecules, which they can transport across cells. In cancer, tumor-derived EVs thereby support the creation of a favorable tumor microenvironment. So far, EV uptake and cargo delivery into target cells have been regarded as the main mechanisms for the pro-tumoral function of EVs. To test this hypothesis, we investigated the fate of the oncogenic transmembrane Wnt tyrosine kinase-like orphan receptor 1 and 2 (ROR1, ROR2) delivered via distinct EV subpopulations to breast cancer cells and aimed to unravel their impact on tumor progression. METHODS: EVs were isolated by differential ultracentrifugation from cell culture supernatant as well as plasma samples from healthy individuals (n = 27) and breast cancer patients (n = 41). EVs were thoroughly characterized by electron microscopy, nanoparticle tracking analysis, immunoblot, and flow cytometry. ROR transfer to target cells was observed using microscopy-based assays and biodistribution experiments were conducted in syngeneic mice. EV impact on cancer cell migration and invasion was tested in functional assays. RESULTS: We observed that the supernatant of ROR-overexpressing cells was sufficient for transferring the receptors to ROR-negative cells. Analyzing the secretome of the ROR-overexpressing cells, we detected a high enrichment of ROR1/2 on large and small EVs, but not on large oncosomes. Interestingly, the majority of ROR-positive EVs remained attached to the target cell surface after 24 h of stimulation and was quickly removed by treatment with trypsin. Nonetheless, ROR-positive EVs increased migration and invasion of breast cancer cells, even after chemically inhibiting EV uptake, in dependence of RhoA downstream signaling. In vivo, ROR-depleted EVs tended to distribute less into organs prone for the formation of breast cancer metastases. ROR-positive EVs were also significantly elevated in the plasma of breast cancer patients and allowed to separate them from healthy controls. CONCLUSIONS: The oncogenic Wnt receptors ROR1/2 are transferred via EVs to the surface of ROR-negative cancer cells, in which they induce an aggressive phenotype supporting tumor progression. Video Abstract.

Obstacles and opportunities for reducing dwelling size to shrink the environmental footprint of housing: tenants' residential preferences and housing choice.

The environmental footprint of housing is greatly influenced by the size of a dwelling. Housing size is the result of households' dwelling selections; accordingly, it is critical to consider residential preferences and choices to inform efforts towards housing sustainability. This study aimed to understand tenants' preferences for and choices of housing size as one amongst several dwelling characteristics and identify obstacles and opportunities for reducing size in the light of promoting sustainable housing. We employed logistic regression models to analyse a survey with 878 Swiss tenants, and our results identify preference for large dwellings as a major obstacle for reducing dwelling size among affluent tenants. Conversely, tenants with lower income might be forced to move to a smaller dwelling due to financial constraints or attribute higher importance to the financial benefit of lower rents. However, financial disincentives along with substantial non-monetary costs of moving, such as the disruption of local bonds and the difficulty of finding a satisfactory dwelling, can outweigh the benefits of moving to a smaller dwelling. To overcome such obstacles, we suggest offering incentives and other facilitating measures for downsizing moves as well as ensuring an adequate supply of smaller dwellings capable of providing high living quality. We highlight the potential of studying housing functions to conceptualize dwellings fulfilling these requirements.

PCR-assisted impedimetric biosensor for colibactin-encoding pks genomic island detection in E. coli samples.

A fast PCR-assisted impedimetric biosensor was developed for the selective detection of the clbN gene from the polyketide synthase (pks) genomic island in real Escherichia coli samples. This genomic island is responsible for the production of colibactin, a harmful genotoxin that has been associated with colorectal cancer. The experimental protocol consisted of immobilizing the designated forward primer onto an Au electrode surface to create the sensing probe, followed by PCR temperature cycling in blank, positive, and negative DNA controls. Target DNA identification was possible by monitoring changes in the system's charge transfer resistance values (Rct) before and after PCR treatment through electrochemical impedance spectroscopy (EIS) analysis. Custom-made, flexible gold electrodes were fabricated using chemical etching optical lithography. A PCR cycle study determined the optimum conditions to be at 6 cycles providing fast results while maintaining a good sensitivity. EIS data for the DNA recognition process demonstrated the successful distinction between target interaction resulting in an increase in resistance to charge transfer (Rct) percentage change of 176% for the positive DNA control vs. 21% and 20% for the negative and non-DNA-containing controls, respectively. Results showed effective fabrication of a fast, PCR-based electrochemical biosensor for the detection of pks genomic island with a calculated limit of detection of 17 ng/µL.

Ovary cold storage and shipment affect oocyte yield and cleavage rate of cat immature vitrified oocytes.

In feline species, cooled transport of ovaries can be employed without detrimental effects to retrieve immature oocytes intended for in vitro embryo production purposes. Indeed, this is the most common way to collect gametes from gonads of wild, valuable animals after they die or are castrated far from specialized laboratories. However, fresh retrieved gametes are generally used, and their cryosensitivity is not known. This study employed ovariectomy-derived domestic cat gonads as a model for wild felids, and aimed to compare the yield and developmental competence of Cryotop-vitrified oocytes (VOs) collected and cryopreserved right after ovary excision (In loco-VOs) or after 24 h cooled transport of ovaries (Shipped-VOs). The number of collected oocytes was higher in In loco-VOs than in Shipped-VOs (mean ± SD: 8 ± 3.36 vs 5.6 ± 3.1, p = 0.05). In vitro embryo production resulted in similar maturation (35% for both vitrified groups, p = 1) and fertilization rates (In loco-VOs: 29.1%; Shipped-VOs: 22.2%; p = 0.295), but showed a difference in cleavage (In loco-VOs: 25.6%; Shipped-VOs: 14.5%; p = 0.0495). No differences were found in further embryo development. Taken together, results suggested that delayed oocyte vitrification after cooled transport of organs was feasible and allowed embryo development. However, the number of collected oocytes and the cleavage rate of matured oocytes were higher when oocyte vitrification was performed without delay after ovary excision, and this should be considered in gamete conservation programs for endangered felids.

Metabolomic Profiling of Blood-Derived Microvesicles in Breast Cancer Patients.

Malignant cells differ from benign ones in their metabolome and it is largely unknown whether this difference is reflected in the metabolic profile of their microvesicles (MV), which are secreted into the blood of cancer patients. Here, they are present together with MV from the various blood and endothelial cells. Harvesting MV from 78 breast cancer patients (BC) and 30 controls, we characterized the whole blood MV metabolome using targeted and untargeted mass spectrometry. Especially (lyso)-phosphatidylcholines and sphingomyelins were detected in a relevant abundance. Eight metabolites showed a significant discriminatory power between BC and controls. High concentrations of lysoPCaC26:0 and PCaaC38:5 were associated with shorter overall survival. Comparing BC subtype-specific metabolome profiles, 24 metabolites were differentially expressed between luminal A and luminal B. Pathway analysis revealed alterations in the glycerophospholipid metabolism for the whole cancer cohort and in the ether lipid metabolism for the molecular subtype luminal B. Although this mixture of blood-derived MV contains only a minor number of tumor MV, a combination of metabolites was identified that distinguished between BC and controls as well as between molecular subtypes, and was predictive for overall survival. This suggests that these metabolites represent promising biomarkers and, moreover, that they may be functionally relevant for tumor progression.

Tenants' residential mobility in Switzerland: the role of housing functions.

The interaction between residential preferences and dwellings is a complex system whose function thus far remains insufficiently explored. In this paper, we investigate housing functions as orchestrators of households' residential mobility in the context of Swiss rental housing. We propose a theoretical multi-step model and use survey data from 878 Swiss tenants to inspect the model's linkages. From the statistical analysis, we firstly observe that tenants' residential satisfaction is more likely to increase when the gap between ideal housing functions and those actually fulfilled by the current dwelling decreases. Secondly, results show that the effectiveness of an event (e.g. a job opportunity) in triggering the move is significantly related to both residential satisfaction and the functions the dwelling fulfils prior to the trigger. Thirdly, findings show that these trigger events can be grouped into three types: radical change, problem-solving and opportunity. With a medium effect size, a radical change was found to bring about the strongest change in housing functions between past and current dwellings. Lastly, in line with the hypothesis that residential preferences vary over the life course, socio-demographic characteristics and tenancy types are found to be significant explanatory variables for households' ideal housing functions. By disentangling the complexity of the housing system, the proposed multi-step model can be used to integrate households' preferences with supply-side constraints in agent-based model simulations, thereby contributing to fostering the provision of quality housing, i.e. dwellings able to meet the needs of current and future occupants.

LEF1 supports metastatic brain colonization by regulating glutathione metabolism and increasing ROS resistance in breast cancer.

More than half of all brain metastases show infiltrating rather than displacing growth at the macro-metastasis/organ parenchyma interface (MMPI), a finding associated with shorter survival. The lymphoid enhancer-binding factor-1 (LEF1) is an epithelial-mesenchymal transition (EMT) transcription factor that is commonly overexpressed in brain-colonizing cancer cells. Here, we overexpressed LEF1 in an in vivo breast cancer brain colonization model. It shortened survival, albeit without engaging EMT at the MMPI. By differential proteome analysis, we identified a novel function of LEF1 as a regulator of the glutathione (GSH) system, the principal cellular redox buffer. LEF1 overexpression also conferred resistance against therapeutic GSH depletion during brain colonization and improved management of intracellular ROS. We conclude that besides EMT, LEF1 facilitates metastasis by improving the antioxidative capacity of epithelial breast cancer cells, in particular during colonization of the brain parenchyma.

Microvolume Screening of Extraction and Phase Behavior in a Liquid-Liquid Microsystem.

Spontaneous formation of a third immiscible phase during liquid-liquid solvent extraction presents an enormous technical challenge for industry. Insight from current empirical investigations is greatly limited by the lack of methodologies that simultaneously report the progress of the extraction, third-phase onset time, and chemical and physical nature. The microfluidic strategy presented here answers this challenge by supporting an optically transparent submicroliter organic-phase film in a micropillar array surrounded by the aqueous phase. To demonstrate, we used 1 M Cyanex 572 in Shellsol D70 (organic phase) to extract Yb3+ and Dy3+ from a pH 2 aqueous phase. Real-time optical tracking confirmed that the visual onset of third-phase formation is consistent with the cessation of extraction (at the loading limit). Spectroscopic analysis of the solid-like third phase was carried out successfully. The new analytical approach offers a step change in speed and efficiency for reagent development, process control, and fundamental studies of complex phase behavior in reactive multiphase systems.

Microvesicles in Cancer: Small Size, Large Potential.

Extracellular vesicles (EV) are secreted by all cell types in a tumor and its microenvironment (TME), playing an essential role in intercellular communication and the establishment of a TME favorable for tumor invasion and metastasis. They encompass a variety of vesicle populations, among them the well-known endosomal-derived small exosomes (Exo), but also larger vesicles (diameter > 100 nm) that are shed directly from the plasma membrane, the so-called microvesicles (MV). Increasing evidence suggests that MV, although biologically different, share the tumor-promoting features of Exo in the TME. Due to their larger size, they can be readily harvested from patients' blood and characterized by routine methods such as conventional flow cytometry, exploiting the plethora of molecules expressed on their surface. In this review, we summarize the current knowledge about the biology and the composition of MV, as well as their role within the TME. We highlight not only the challenges and potential of MV as novel biomarkers for cancer, but also discuss their possible use for therapeutic intervention.

GeoFarmer: A monitoring and feedback system for agricultural development projects.

Farmers can manage their crops and farms better if they can communicate their experiences, both positive and negative, with each other and with experts. Digital agriculture using internet communication technology (ICT) may facilitate the sharing of experiences between farmers themselves and with experts and others interested in agriculture. ICT approaches in agriculture are, however, still out of the reach of many farmers. The reasons are lack of connectivity, missing capacity building and poor usability of ICT applications. We decided to tackle this problem through cost-effective, easy to use ICT approaches, based on infrastructure and services currently available to small-scale producers in developing areas. Working through a participatory design approach, we developed and tested a novel technology. GeoFarmer provides near real-time, two-way data flows that support processes of co-innovation in agricultural development projects. It can be used as a cost-effective ICT-based platform to monitor agricultural production systems with interactive feedback between the users, within pre-defined geographical domains. We tested GeoFarmer in four geographic domains associated with ongoing agricultural development projects in East and West Africa and Latin America. We demonstrate that GeoFarmer is a cost-effective means of providing and sharing opportune indicators of on-farm performance. It is a potentially useful tool that farmers and agricultural practitioners can use to manage their crops and farms better, reduce risk, increase productivity and improve their livelihoods.

[Quality characteristics in 24-hour care from the perspective of those affected - Results of a pilot study in Austria]. / Qualitätsmerkmale in der 24 h-Betreuung aus der Perspektive der Betroffenen - Ergebnisse einer Pilotbefragungsstudie in Österreich.

Quality characteristics in 24-hour care from the perspective of those affected. Results of a pilot study in Austria Abstract. Background: Of the almost 460 000 recipients of care allowance in Austria, 80 % are cared for at home. In 2018, more than 32 000 people were cared for at home by a total of almost 65 000 24-hour caregivers. Isolated work situations in combination with complex illnesses of the clients, often inadequate training and a lack of language skills of the personal carers have a challenging effect on the quality of care. Aim: The aim of the pilot study is to describe quality characteristics in 24-hour personal care from the perspective of the actors involved. The research question relates firstly to the individual experience and secondly to the desired quality of care. Methods: The survey method used is an individual survey along a guideline; the summarising content analysis has been chosen as the evaluation method. Results: A total of 32 interviews were conducted (14 relatives, 1 client, 8 caregivers and 9 DGKP). In the analysis process, it was possible to map all the aspects mentioned using the developed main category "field of action-oriented quality characteristics". These relate to comprehensive everyday care, specialist nursing and medical activities, individual care, language skills and communication, as well as initial and continuing training and interface management. Conclusions: The development of binding quality criteria together with continuous external support is essential to ensure sustainable quality of care. This could be taken over by the established social associations.

Oriented Cell Division in the C. elegans Embryo Is Coordinated by G-Protein Signaling Dependent on the Adhesion GPCR LAT-1.

Orientation of spindles and cell division planes during development of many species ensures that correct cell-cell contacts are established, which is vital for proper tissue formation. This is a tightly regulated process involving a complex interplay of various signals. The molecular mechanisms underlying several of these pathways are still incompletely understood. Here, we identify the signaling cascade of the C. elegans latrophilin homolog LAT-1, an essential player in the coordination of anterior-posterior spindle orientation during the fourth round of embryonic cell division. We show that the receptor mediates a G protein-signaling pathway revealing that G-protein signaling in oriented cell division is not solely GPCR-independent. Genetic analyses showed that through the interaction with a Gs protein LAT-1 elevates intracellular cyclic AMP (cAMP) levels in the C. elegans embryo. Stimulation of this G-protein cascade in lat-1 null mutant nematodes is sufficient to orient spindles and cell division planes in the embryo in the correct direction. Finally, we demonstrate that LAT-1 is activated by an intramolecular agonist to trigger this cascade. Our data support a model in which a novel, GPCR-dependent G protein-signaling cascade mediated by LAT-1 controls alignment of cell division planes in an anterior-posterior direction via a metabotropic Gs-protein/adenylyl cyclase pathway by regulating intracellular cAMP levels.

Directed Growth of Orthorhombic Crystals in a Micropillar Array.

We report directed growth of orthorhombic crystals of potassium permanganate in spatial confinement of a micropillar array. The solution is introduced by spontaneous wicking to give a well-defined film (thickness 10-15 µm; volume ∼600 nL) and is connected to a reservoir (several microliters) that continuously "feeds" the evaporating film. When the film is supersaturated, crystals nucleate and preferentially grow in specific directions guided by one of several possible linear paths through the pillar lattice. Crystals that do not initially conform are stopped at an obstructing pillar, branch into another permitted direction, or spontaneously rotate to align with a path and continue to grow. Microspectroscopy is able to track the concentration of solute in a small region of interest (70 × 100 µm2) near to growing crystals, revealing that the solute concentration initially increases linearly beyond the solubility limit. Crystal growth near the region of interest resulted in a sharp decrease in the local solute concentration (which rapidly returns the concentration to the solubility limit), consistent with estimated diffusion time scales (<1 s for a 50 µm length scale). The ability to simultaneously track solute concentration and control crystal orientation in nanoliter samples will provide new insight into microscale dynamics of microscale crystallization.

Inactivation of Patched1 in mice leads to development of gastrointestinal stromal-like tumors that express Pdgfrα but not kit.

BACKGROUND & AIMS: A fraction of gastrointestinal stromal tumor (GIST) cells overexpress the platelet-derived growth factor receptor (PDGFR)A, although most overexpress KIT. It is not known if this is because these receptor tyrosine kinases have complementary oncogenic potential, or because of heterogeneity in the cellular origin of GIST. Little also is known about why Hedgehog (HH) signaling is activated in some GIST. HH binds to and inactivates the receptor protein patched homolog (PTCH). METHODS: Ptch was conditionally inactivated in mice (to achieve constitutive HH signaling) using a Cre recombinase regulated by the lysozyme M promoter. Cre-expressing cells were traced using R26R-LacZ reporter mice. Tumors were characterized by in situ hybridization, immunohistochemistry, immunoblot, and quantitative reverse-transcriptase polymerase chain reaction analyses. Cell transformation was assessed by soft agar assay. RESULTS: Loss of Ptch from lysozyme M-expressing cells resulted in the development of tumors of GIST-like localization and histology; these were reduced when mice were given imatinib, a drug that targets KIT and PDGFRA. The Hh signaling pathway was activated in the tumor cells, and Pdgfrα, but not Kit, was overexpressed and activated. Lineage tracing revealed that Cre-expressing intestinal cells were Kit-negative. These cells sometimes expressed Pdgfrα and were located near Kit-positive interstitial cells of Cajal. In contrast to KIT, activation of PDGFRA increased anchorage-independent proliferation and was required for tumor formation in mice by cells with activated HH signaling. CONCLUSIONS: Inactivation of Ptch in mice leads to formation of GIST-like tumors that express Pdgfrα, but not Kit. Activation of Pdgfrα signaling appears to facilitate tumorigenesis.

PD-L1 on large extracellular vesicles is a predictive biomarker for therapy response in tissue PD-L1-low and -negative patients with non-small cell lung cancer.

Immunotherapy has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC). High expression of tissue PD-L1 (tPD-L1) is currently the only approved biomarker for predicting treatment response. However, even tPD-L1 low (1-49%) and absent (<1%) patients might benefit from immunotherapy but, to date, there is no reliable biomarker, that can predict response in this particular patient subgroup. This study aimed to test whether tumour-associated extracellular vesicles (EVs) could fill this gap. Using NSCLC cell lines, we identified a panel of tumour-related antigens that were enriched on large EVs (lEVs) compared to smaller EVs. The levels of lEVs carrying these antigens were significantly elevated in plasma of NSCLC patients (n = 108) and discriminated them from controls (n = 77). Among the tested antigens, we focused on programmed cell death ligand 1 (PD-L1), which is a well-known direct target for immunotherapy. In plasma lEVs, PD-L1 was mainly found on a population of CD45- /CD62P+ lEVs and thus seemed to be associated with platelet-derived vesicles. Patients with high baseline levels of PD-L1+ lEVs in blood showed a significantly better response to immunotherapy and prolonged survival. This was particularly true in the subgroup of NSCLC patients with low or absent tPD-L1 expression, thus identifying PD-L1-positive lEVs in plasma as a novel predictive and prognostic marker for immunotherapy.

Carcinoma cells misuse the host tissue damage response to invade the brain.

The metastatic colonization of the brain by carcinoma cells is still barely understood, in particular when considering interactions with the host tissue. The colonization comes with a substantial destruction of the surrounding host tissue. This leads to activation of damage responses by resident innate immune cells to protect, repair, and organize the wound healing, but may distract from tumoricidal actions. We recently demonstrated that microglia, innate immune cells of the CNS, assist carcinoma cell invasion. Here we report that this is a fatal side effect of a physiological damage response of the brain tissue. In a brain slice coculture model, contact with both benign and malignant epithelial cells induced a response by microglia and astrocytes comparable to that seen at the interface of human cerebral metastases. While the glial damage response intended to protect the brain from intrusion of benign epithelial cells by inducing apoptosis, it proved ineffective against various malignant cell types. They did not undergo apoptosis and actually exploited the local tissue reaction to invade instead. Gene expression and functional analyses revealed that the C-X-C chemokine receptor type 4 (CXCR4) and WNT signaling were involved in this process. Furthermore, CXCR4-regulated microglia were recruited to sites of brain injury in a zebrafish model and CXCR4 was expressed in human stroke patients, suggesting a conserved role in damage responses to various types of brain injuries. Together, our findings point to a detrimental misuse of the glial damage response program by carcinoma cells resistant to glia-induced apoptosis.

T cell development critically depends on prethymic stromal patched expression.

We recently described that T cell specification in mice deficient in the Hedgehog (Hh) receptor Patched (Ptch) is blocked at the level of the common lymphoid progenitor in the bone marrow (BM). Adoptive transfer of wild-type BM in Ptch-deficient mice provides evidence that T cell development strictly depends on Ptch expression in the nonhematopoietic compartment. Transplantation experiments using BM deficient in the glucocorticoid receptor exclude any involvement of the stress hormone corticosterone in our model. Using cell-type-specific knockout mice, we show that T cell development is independent of T cell-intrinsic Ptch expression. Furthermore, Ptch expression by the thymus stroma is dispensable, as revealed by fetal thymus organ culture and thymus transplantation. In contrast, analysis of the earliest thymic progenitors in Ptch-deficient mice indicated that Ptch is required for the development or supply of thymic homing progenitors that give rise to earliest thymic progenitors. Collectively, our findings identified Ptch as an exclusive T cell-extrinsic factor necessary for proper development of T cells at their prethymic stage. This observation may be important for current considerations using Hh inhibitors upstream of Ptch in diseases accompanied by aberrant Hh signaling.