RESUMO
In the thymus, cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells support αßT cell development from lymphoid progenitors. For cTECs, expression of a specialized gene signature that includes Cxcl12, Dll4, and Psmb11 enables the cortex to support T lineage commitment and the generation and selection of CD4+CD8+ thymocytes. Although the importance of cTECs in T cell development is well defined, mechanisms that shape the cTEC compartment and regulate its functional specialization are unclear. Using a Cxcl12 DsRed reporter mouse model, we show that changes in Cxcl12 expression reveal a developmentally regulated program of cTEC heterogeneity. Although cTECs are uniformly Cxcl12 DsRed+ during neonatal stages, progression through postnatal life triggers the appearance of Cxcl12 DsRed- cTECs that continue to reside in the cortex alongside their Cxcl12 DsRed+ counterparts. This appearance of Cxcl12 DsRed- cTECs is controlled by maturation of CD4-CD8-, but not CD4+CD8+, thymocytes, demonstrating that stage-specific thymocyte cross-talk controls cTEC heterogeneity. Importantly, although fate-mapping experiments show both Cxcl12 DsRed+ and Cxcl12 DsRed- cTECs share a common Foxn1 + cell origin, RNA sequencing analysis shows Cxcl12 DsRed- cTECs no longer express Foxn1, which results in loss of the FOXN1-dependent cTEC gene signature and may explain the reduced capacity of Cxcl12 DsRed- cTECs for thymocyte interactions. In summary, our study shows that shaping of the cTEC compartment during the life course occurs via stage-specific thymocyte cross-talk, which drives loss of Foxn1 expression and its key target genes, which may then determine the functional competence of the thymic cortex.
RESUMO
In the thymus, cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells support αßT cell development from lymphoid progenitors. For cTECs, expression of a specialized gene signature that includes Cxcl12, Dll4, and Psmb11 enables the cortex to support T lineage commitment and the generation and selection of CD4+CD8+ thymocytes. Although the importance of cTECs in T cell development is well defined, mechanisms that shape the cTEC compartment and regulate its functional specialization are unclear. Using a Cxcl12DsRed reporter mouse model, we show that changes in Cxcl12 expression reveal a developmentally regulated program of cTEC heterogeneity. Although cTECs are uniformly Cxcl12DsRed+ during neonatal stages, progression through postnatal life triggers the appearance of Cxcl12DsRed- cTECs that continue to reside in the cortex alongside their Cxcl12DsRed+ counterparts. This appearance of Cxcl12DsRed- cTECs is controlled by maturation of CD4-CD8-, but not CD4+CD8+, thymocytes, demonstrating that stage-specific thymocyte cross-talk controls cTEC heterogeneity. Importantly, although fate-mapping experiments show both Cxcl12DsRed+ and Cxcl12DsRed- cTECs share a common Foxn1+ cell origin, RNA sequencing analysis shows Cxcl12DsRed- cTECs no longer express Foxn1, which results in loss of the FOXN1-dependent cTEC gene signature and may explain the reduced capacity of Cxcl12DsRed- cTECs for thymocyte interactions. In summary, our study shows that shaping of the cTEC compartment during the life course occurs via stage-specific thymocyte cross-talk, which drives loss of Foxn1 expression and its key target genes, which may then determine the functional competence of the thymic cortex.
RESUMO
An organized and dynamic cytoskeleton is required for platelet formation and function. Formins are a large family of actin regulatory proteins which are also able to regulate microtubule dynamics. There are four formin family members expressed in human and mouse megakaryocytes and platelets. We have previously shown that the actin polymerization activity of formin proteins is required for cytoskeletal dynamics and platelet spreading using a small molecule inhibitor. In the current study, we analyze transgenic mouse models deficient in two of these proteins, mDia1 and Fhod1, along with a model lacking both proteins. We demonstrate that double knockout mice display macrothrombocytopenia which is due to aberrant megakaryocyte function and a small decrease in platelet lifespan. Platelet function is unaffected by the loss of these proteins. This data indicates a critical role for formins in platelet and megakaryocyte function.
Assuntos
Plaquetas/metabolismo , Proteínas Fetais/metabolismo , Forminas/metabolismo , Microtúbulos/metabolismo , Testes de Função Plaquetária/métodos , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos KnockoutRESUMO
In the thymus, medullary thymic epithelial cells (mTEC) regulate T cell tolerance via negative selection and Foxp3(+) regulatory T cell (Treg) development, and alterations in the mTEC compartment can lead to tolerance breakdown and autoimmunity. Both the receptor activator for NF-κB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) axis and expression of the transcriptional regulator Aire are involved in the regulation of thymus medullary microenvironments. However, their impact on the mechanisms controlling mTEC homeostasis is poorly understood, as are the processes that enable the thymus medulla to support the balanced production of mTEC-dependent Foxp3(+) Treg. In this study, we have investigated the control of mTEC homeostasis and examined how this process impacts the efficacy of Foxp3(+) Treg development. Using newly generated RANK Venus reporter mice, we identify distinct RANK(+) subsets that reside within both the mTEC(hi) and mTEC(lo) compartments and that represent direct targets of OPG-mediated control. Moreover, by mapping OPG expression to a subset of Aire(+) mTEC, our data show how cis- and trans-acting mechanisms are able to control the thymus medulla by operating on multiple mTEC targets. Finally, we show that whereas the increase in mTEC availability in OPG-deficient (Tnfrsf11b(-/-)) mice impacts the intrathymic Foxp3(+) Treg pool by enhancing peripheral Treg recirculation back to the thymus, it does not alter the number of de novo Rag2pGFP(+)Foxp3(+) Treg that are generated. Collectively, our study defines patterns of RANK expression within the thymus medulla, and it shows that mTEC homeostasis is not a rate-limiting step in intrathymic Foxp3(+) Treg production.
Assuntos
Linfopoese/imunologia , Osteoprotegerina/genética , Ligante RANK/imunologia , Linfócitos T Reguladores/imunologia , Timo/metabolismo , Animais , Autoimunidade/imunologia , Células Cultivadas , Proteínas de Ligação a DNA/genética , Células Epiteliais , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Tolerância Imunológica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/imunologia , Técnicas de Cultura de Órgãos , Osteoprotegerina/biossíntese , Osteoprotegerina/imunologia , Ligante RANK/biossíntese , Transdução de Sinais/imunologia , Linfócitos T Reguladores/citologia , Timo/citologia , Timo/imunologia , Fatores de Transcrição/biossíntese , Proteína AIRERESUMO
RhoJ is a Rho GTPase expressed in endothelial cells and tumour cells, which regulates cell motility, invasion, endothelial tube formation and focal adhesion numbers. This study aimed to further delineate the molecular function of RhoJ. Using timelapse microscopy RhoJ was found to regulate focal adhesion disassembly; small interfering RNA (siRNA)-mediated knockdown of RhoJ increased focal adhesion disassembly time, whereas expression of an active mutant (daRhoJ) decreased it. Furthermore, daRhoJ co-precipitated with the GIT-PIX complex, a regulator of focal adhesion disassembly. An interaction between daRhoJ and GIT1 was confirmed using yeast two-hybrid experiments, and this depended on the Spa homology domain of GIT1. GIT1, GIT2, ß-PIX (also known as ARHGEF7) and RhoJ all colocalised in focal adhesions and depended on each other for their recruitment to focal adhesions. Functionally, the GIT-PIX complex regulated endothelial tube formation, with knockdown of both GIT1 and GIT2, or ß-PIX phenocopying RhoJ knockdown. RhoJ-knockout mice showed reduced tumour growth and diminished tumour vessel density, identifying a role for RhoJ in mediating tumour angiogenesis. These studies give new insight into the molecular function of RhoJ in regulating cell motility and tumour vessel formation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Adesões Focais/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Movimento Celular/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/metabolismo , Transdução de SinaisRESUMO
In the thymus, interactions with both cortical and medullary microenvironments regulate the development of self-tolerant conventional CD4(+) and CD8(+) αßT cells expressing a wide range of αßTCR specificities. Additionally, the cortex is also required for the development of invariant NKT (iNKT) cells, a specialized subset of T cells that expresses a restricted αßTCR repertoire and is linked to the regulation of innate and adaptive immune responses. Although the role of the cortex in this process is to enable recognition of CD1d molecules expressed by CD4(+)CD8(+) thymocyte precursors, the requirements for additional thymus microenvironments during iNKT cell development are unknown. In this study, we reveal a role for medullary thymic epithelial cells (mTECs) during iNKT cell development in the mouse thymus. This requirement for mTECs correlates with their expression of genes required for IL-15 trans-presentation, and we show that soluble IL-15/IL-15Rα complexes restore iNKT cell development in the absence of mTECs. Furthermore, mTEC development is abnormal in iNKT cell-deficient mice, and early stages in iNKT cell development trigger receptor activator for NF-κB ligand-mediated mTEC development. Collectively, our findings demonstrate that intrathymic iNKT cell development requires stepwise interactions with both the cortex and the medulla, emphasizing the importance of thymus compartmentalization in the generation of both diverse and invariant αßT cells. Moreover, the identification of a novel requirement for iNKT cells in thymus medulla development further highlights the role of both innate and adaptive immune cells in thymus medulla formation.
Assuntos
Diferenciação Celular/imunologia , Microambiente Celular/imunologia , Células Epiteliais/imunologia , Células T Matadoras Naturais/imunologia , Animais , Antígenos CD1d/genética , Antígenos CD1d/imunologia , Antígenos CD1d/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Microambiente Celular/efeitos dos fármacos , Microambiente Celular/genética , Células Epiteliais/metabolismo , Células Epiteliais/transplante , Citometria de Fluxo , Interleucina-15/administração & dosagem , Interleucina-15/genética , Interleucina-15/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Ligante RANK/imunologia , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/imunologia , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Receptores de Interleucina-15/administração & dosagem , Receptores de Interleucina-15/genética , Receptores de Interleucina-15/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timócitos/citologia , Timócitos/imunologia , Timócitos/metabolismo , Timo/citologia , Timo/imunologia , Timo/metabolismo , Fator de Transcrição RelB/genética , Fator de Transcrição RelB/imunologia , Fator de Transcrição RelB/metabolismoRESUMO
αßT cell development depends upon serial migration of thymocyte precursors through cortical and medullary microenvironments, enabling specialized stromal cells to provide important signals at specific stages of their development. Although conventional αßT cells are subject to clonal deletion in the medulla, entry into the thymus medulla also fosters αßT cell differentiation. For example, during postnatal periods, the medulla is involved in the intrathymic generation of multiple αßT cell lineages, notably the induction of Foxp3(+) regulatory T cell development and the completion of invariant NKT cell development. Although migration of conventional αßT cells to the medulla is mediated by the chemokine receptor CCR7, how other T cell subsets gain access to medullary areas during their normal development is not clear. In this study, we show that combining a panel of thymocyte maturation markers with cell surface analysis of CCR7 and CCR4 identifies distinct stages in the development of multiple αßT cell lineages in the thymus. Although Aire regulates expression of the CCR4 ligands CCL17 and CCL22, we show that CCR4 is dispensable for thymocyte migration and development in the adult thymus, demonstrating defective T cell development in Aire(-/-) mice is not because of a loss of CCR4-mediated migration. Moreover, we reveal that CCR7 controls the development of invariant NKT cells by enabling their access to IL-15 trans-presentation in the thymic medulla and influences the balance of early and late intrathymic stages of Foxp3(+) regulatory T cell development. Collectively, our data identify novel roles for CCR7 during intrathymic T cell development, highlighting its importance in enabling multiple αßT cell lineages to access the thymic medulla.
Assuntos
Diferenciação Celular/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores CCR4/fisiologia , Receptores CCR7/fisiologia , Subpopulações de Linfócitos T/imunologia , Timo/imunologia , Timo/metabolismo , Imunidade Adaptativa , Animais , Biomarcadores/análise , Linhagem da Célula/imunologia , Células Epiteliais/citologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores CCR4/deficiência , Receptores CCR7/deficiência , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Timo/citologiaRESUMO
As the primary site of T-cell development, the thymus dictates immune competency of the host. The rates of thymus function are not constant, and thymus regeneration is essential to restore new T-cell production following tissue damage from environmental factors and therapeutic interventions. Here, we show the alarmin interleukin (IL) 33 is a product of Sca1+ thymic mesenchyme both necessary and sufficient for thymus regeneration via a type 2 innate immune network. IL33 stimulates expansion of IL5-producing type 2 innate lymphoid cells (ILC2), which triggers a cellular switch in the intrathymic availability of IL4. This enables eosinophil production of IL4 to re-establish thymic mesenchyme prior to recovery of thymopoiesis-inducing epithelial compartments. Collectively, we identify a positive feedback mechanism of type 2 innate immunity that regulates the recovery of thymus function following tissue injury.
Assuntos
Alarminas , Interleucina-33 , Imunidade Inata , Interleucina-4 , LinfócitosRESUMO
The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in glucose homeostasis and food intake. GLP1R agonists (GLP1RA) are widely used in the treatment of diabetes and obesity, yet visualizing the endogenous localization, organization and dynamics of a GPCR has so far remained out of reach. In the present study, we generate mice harboring an enzyme self-label genome-edited into the endogenous Glp1r locus. We also rationally design and test various fluorescent dyes, spanning cyan to far-red wavelengths, for labeling performance in tissue. By combining these technologies, we show that endogenous GLP1R can be specifically and sensitively detected in primary tissue using multiple colors. Longitudinal analysis of GLP1R dynamics reveals heterogeneous recruitment of neighboring cell subpopulations into signaling and trafficking, with differences observed between GLP1RA classes and dual agonists. At the nanoscopic level, GLP1Rs are found to possess higher organization, undergoing GLP1RA-dependent membrane diffusion. Together, these results show the utility of enzyme self-labels for visualization and interrogation of endogenous proteins, and provide insight into the biology of a class B GPCR in primary cells and tissue.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1 , Obesidade , Camundongos , Animais , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismoRESUMO
The thymus medulla is a key site for immunoregulation and tolerance, and its functional specialisation is achieved through the complexity of medullary thymic epithelial cells (mTEC). While the importance of the medulla for thymus function is clear, the production and maintenance of mTEC diversity remains poorly understood. Here, using ontogenetic and inducible fate-mapping approaches, we identify mTEC-restricted progenitors as a cytokeratin19+ (K19+) TEC subset that emerges in the embryonic thymus. Importantly, labelling of a single cohort of K19+ TEC during embryogenesis sustains the production of multiple mTEC subsets into adulthood, including CCL21+ mTEClo, Aire+ mTEChi and thymic tuft cells. We show K19+ progenitors arise prior to the acquisition of multiple mTEC-defining features including RANK and CCL21 and are generated independently of the key mTEC regulator, Relb. In conclusion, we identify and define a multipotent mTEC progenitor that emerges during embryogenesis to support mTEC diversity into adult life.
Assuntos
Tolerância Imunológica , Queratina-19 , Timo , Animais , Camundongos , Diferenciação Celular , Células Epiteliais , Camundongos Endogâmicos C57BL , Células-TroncoRESUMO
APC is a key 'gatekeeper' gene in colorectal tumorigenesis. The high frequency of APC defects observed in colorectal cancer tissue is the result of selective growth advantage of cells with loss-of-function mutations at that locus. However, mutations may also arise due to inherent sequence instability. Defective DNA mismatch repair (MMR) and base excision repair (BER) also contribute to colorectal carcinogenesis and may compound such instability. To avoid the effect of clonal selective advantage imparted by APC mutation in cancer cells, we assessed in vitro APC mutation frequency in cell lines of lymphoid lineage to investigate the influence of defective MMR and BER. In DNA repair proficient cells, we observed substantially greater inherent sequence instability in APC gene coding sequences compared to reference sequences. Surprisingly, however, this difference was abrogated in MMR defective lines. We also found greater mutation frequency at exonic DNA sequences outwith the APC region in cells defective for either MMR or BER defects. The underlying propensity for mutation at the APC gene is intriguing, while the greater frequency of mutation in cells defective for DNA repair has relevance to understanding events leading to colorectal cancer and other malignancies.
Assuntos
Proteína da Polipose Adenomatosa do Colo/química , Proteína da Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/genética , Reparo do DNA/genética , Linfócitos/metabolismo , Mutação/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Western Blotting , Estudos de Casos e Controles , Células Cultivadas , Éxons/genética , HumanosRESUMO
Bone marrow transplantation (BMT) is a widely used therapy for blood cancers and primary immunodeficiency. Following transplant, the thymus plays a key role in immune reconstitution by generating a naive αßT cell pool from transplant-derived progenitors. While donor-derived thymopoiesis during the early post-transplant period is well studied, the ability of the thymus to synchronize T cell development with essential tolerance mechanisms is poorly understood. Using a syngeneic mouse transplant model, we analyzed T cell recovery alongside the regeneration and function of intrathymic microenvironments. We report a specific and prolonged failure in the post-transplant recovery of medullary thymic epithelial cells (mTECs). This manifests as loss of medulla-dependent tolerance mechanisms, including failures in Foxp3+ regulatory T cell development and formation of the intrathymic dendritic cell pool. In addition, defective negative selection enables escape of self-reactive conventional αßT cells that promote autoimmunity. Collectively, we show that post-transplant T cell recovery involves an uncoupling of thymopoiesis from thymic tolerance, which results in autoimmune reconstitution caused by failures in thymic medulla regeneration.
Assuntos
Autoimunidade , Microambiente Celular/imunologia , Doença Enxerto-Hospedeiro/etiologia , Tolerância Imunológica , Timo/imunologia , Animais , Transplante de Medula Óssea/efeitos adversos , Transplante de Medula Óssea/métodos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Doença Enxerto-Hospedeiro/metabolismo , Reconstituição Imune , Camundongos , Camundongos Transgênicos , Especificidade do Receptor de Antígeno de Linfócitos T , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Timo/patologiaRESUMO
Therapeutic interventions used for cancer treatment provoke thymus damage and limit the recovery of protective immunity. Here, we show that eosinophils are an essential part of an intrathymic type 2 immune network that enables thymus recovery after ablative therapy. Within hours of damage, the thymus undergoes CCR3-dependent colonization by peripheral eosinophils, which reestablishes the epithelial microenvironments that control thymopoiesis. Eosinophil regulation of thymus regeneration occurs via the concerted action of NKT cells that trigger CCL11 production via IL4 receptor signaling in thymic stroma, and ILC2 that represent an intrathymic source of IL5, a cytokine that therapeutically boosts thymus regeneration after damage. Collectively, our findings identify an intrathymic network composed of multiple innate immune cells that restores thymus function during reestablishment of the adaptive immune system.
Assuntos
Eosinófilos , Regeneração , Timo , Imunidade Adaptativa , Citocinas , Eosinófilos/imunologia , Interleucina-5/imunologia , Linfócitos , Timo/imunologiaRESUMO
Schlafen 14 (SLFN14) has recently been identified as an endoribonuclease responsible for cleaving RNA to regulate and inhibit protein synthesis. Early studies revealed that members of the SLFN family are capable of altering lineage commitment during T-cell differentiation by using cell-cycle arrest as a means of translational control by RNase activity. SLFN14 has been reported as a novel gene causing an inherited macrothrombocytopenia and bleeding in human patients; however, the role of this endoribonuclease in megakaryopoiesis and thrombopoiesis remains unknown. To investigate this, we report a CRISPR knock-in mouse model of SLFN14 K208N homologous to the K219N mutation observed in our previous patient studies. We used hematological analysis, in vitro and in vivo studies of platelet and erythrocyte function, and analysis of spleen and bone marrow progenitors. Mice homozygous for this mutation do not survive to weaning age, whereas heterozygotes exhibit microcytic erythrocytosis, hemolytic anemia, splenomegaly, and abnormal thrombus formation, as revealed by intravital microscopy, although platelet function and morphology remain unchanged. We also show that there are differences in erythroid progenitors in the spleens and bone marrow of these mice, indicative of an upregulation of erythropoiesis. This SLFN14 mutation presents distinct species-specific phenotypes, with a platelet defect reported in humans and a severe microcytic erythrocytosis in mice. Thus, we conclude that SLFN14 is a key regulator in mammalian hematopoiesis and a species-specific mediator of platelet and erythroid lineage commitment.
Assuntos
Plaquetas , Endorribonucleases/genética , Eritropoese , Animais , Linhagem da Célula/genética , Eritropoese/genética , Heterozigoto , Humanos , Camundongos , MutaçãoRESUMO
Both in vitro and in vivo studies have implicated the c-Myb transcription factor in vascular smooth muscle cell (SMC) proliferation and hematopoiesis. However, its role in differentiation and maturation of contractile, as opposed to proliferating, SMCs has not been investigated. Here we demonstrate that c-myb(-/-) embryonic stem cells (ESCs) are incapable of producing embryoid bodies (EBs) with spontaneously contracting SMCs but can differentiate into contracting cardiomyocytes unimpaired. Quantitative real-time RT-PCR revealed that whereas mesodermal differentiation was unaffected, myocardin, a critical determinant of SMC differentiation, became upregulated at day 7 in wild-type, but not in c-myb(-/-) EBs. SMC-specific genes, smooth muscle alpha-actin, SM22alpha and smooth muscle myosin heavy chain reached peak expression levels by day 15 of differentiation and were 2- to 3-fold higher in wild-type as compared with c-myb(-/-) derived EBs. Similarly, fluorescence-activated cell-sorting analysis confirmed significantly different proportions of smooth muscle alpha-actin-positive cells in wild-type (26.8+/-0.7%) versus c-myb(-/-) (12.3+/-0.4%) EBs. Temporal induction of these SMC-specific markers preceded and paralleled contractile SMC appearance and predicted the relative (in)ability of c-myb(-/-) and wild-type ESC lines to generate EBs with contracting SMCs. Importantly, data from EBs faithfully predicted a significant reduction in c-myb(-/-) cell contribution to SMC lineage in vivo, in chimeric E11.5 embryo and adult aortas relative to brain and skin chimerism, respectively. Moreover, the visceral SMC population in chimeric embryos was nearly devoid of c-myb(-/-) cells. Our data are the first to implicate c-Myb in SMC differentiation from precursor stem cell-derived populations, reinforcing its potential role in phenotypic modulation of SMCs and vascular disease.
Assuntos
Diferenciação Celular/genética , Músculo Liso Vascular/metabolismo , Proteínas Proto-Oncogênicas c-myb/fisiologia , Animais , Aorta/citologia , Aorta/embriologia , Biomarcadores/metabolismo , Linhagem da Célula , Células Cultivadas , Quimera/embriologia , Embrião de Mamíferos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Intestinos/citologia , Intestinos/embriologia , Camundongos , Camundongos Transgênicos , Contração Muscular/genética , Músculo Liso Vascular/citologia , Proteínas Nucleares/biossíntese , Proteínas Proto-Oncogênicas c-myb/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Transativadores/biossínteseRESUMO
Thymic epithelium provides an essential cellular substrate for T cell development and selection. Gradual age-associated thymic atrophy leads to a reduction in functional thymic tissue and a decline in de novo T cell generation. Development of strategies tailored toward regeneration of thymic tissue provides an important possibility to improve immune function in elderly individuals and increase the capacity for immune recovery in patients having undergone bone marrow transfer following immunoablative therapies. In this study we show that restriction of the size of the functional thymic epithelial progenitor pool affects the number of mature thymic epithelial cells. Using an embryo fusion chimera-based approach, we demonstrate a reduction in the total number of both embryonic and adult thymic epithelium, which relates to the initial size of the progenitor cell pool. The inability of thymic epithelial progenitor cells to undergo sufficient compensatory proliferation to rescue the deficit in progenitor numbers suggests that in addition to extrinsic regulation of thymus growth by provision of growth factors, intrinsic factors such as a proliferative restriction of thymic epithelial progenitors and availability of progenitor cell niches may limit thymic epithelial recovery. Collectively, our data demonstrate an important level of regulation of thymic growth and recovery at the thymic epithelial progenitor level, providing an important consideration for developing methods targeted toward inducing thymic regeneration.
Assuntos
Células Epiteliais/imunologia , Células-Tronco/imunologia , Timo/citologia , Timo/fisiologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Quimera , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/deficiência , Fatores de Transcrição Forkhead/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Mosaicismo , Regeneração/genética , Regeneração/imunologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Timo/embriologiaRESUMO
The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived ß-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo.
Assuntos
Corantes Fluorescentes , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Linhagem Celular , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Receptor do Peptídeo Semelhante ao Glucagon 1/antagonistas & inibidores , Receptor do Peptídeo Semelhante ao Glucagon 1/deficiência , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Células HEK293 , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Knockout , Modelos Moleculares , Estrutura Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Transdução de Sinais , Distribuição TecidualRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Galanin expression markedly increases in the dorsal root ganglion (DRG) after sciatic nerve axotomy and modulates pain behavior and regeneration of sensory neurons. Here, we describe transgenic mice expressing constructs with varying amounts of sequence upstream of the murine galanin gene marked by LacZ. The 20 kb region upstream of the galanin gene recapitulates the endogenous expression pattern of galanin in the embryonic and adult intact DRG and after axotomy. In contrast, 1.9 kb failed to drive LacZ expression in the intact DRG or after axotomy. However, the addition of an additional 2.7 kb of 5' flanking DNA (4.6 kb construct) restored the expression in the embryonic DRG and in the adult after axotomy. Sequence analysis of this 2.7 kb region revealed unique 18 and 23 bp regions containing overlapping putative Ets-, Stat-, and Smad-binding sites, and adjacent putative Stat- and Smad-binding sites, respectively. Deletion of the 18 and 23 bp regions from the 4.6 kb construct abolished the upregulation of LacZ expression in the DRG after axotomy but did not affect expression in the embryonic or intact adult DRG. Also, a bioinformatic analysis of the upstream regions of a number of other axotomy-responsive genes demonstrated that the close proximity of putative Ets-, Stat-, and Smad-binding sites appears to be a common motif in injury-induced upregulation in gene expression.
Assuntos
Elementos Facilitadores Genéticos/fisiologia , Galanina/genética , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica/fisiologia , Animais , Axotomia/métodos , Embrião de Mamíferos , Regulação da Expressão Gênica/genética , Óperon Lac/fisiologia , Camundongos , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Mutação/fisiologia , Estrutura Terciária de Proteína , Nervo Isquiático/metabolismo , Nervo Isquiático/cirurgia , Análise de Sequência , beta-Galactosidase/metabolismoRESUMO
BACKGROUND: Early detection of pupillary changes in patients with head injuries can alert the care team to increasing intracranial pressure. Previous research has shown inconsistencies in pupil measurement that are most likely due to the subjective nature of measuring pupils without the assistance of technology. OBJECTIVES: To evaluate nurses' abilities to assess pupil diameter accurately and detect unequal pupils. METHODS: In a 3-part study, the accuracy of critical care and neurosurgical nurses' assessments of pupils was determined. The study included assessment of drawings of eyes with an iris and pupil, examination of photographs of human eyes, and bedside examination of patients with a head injury. RESULTS: Subjective assessments of pupil diameter and symmetry were not accurate. Across all phases of the study, pupil diameters were underestimated and the rate of error increased as pupil size increased. Nurses also failed to detect anisocoria and misidentified pupil reactivity. In addition, nearly all nurses relied on subjective estimation, even when tools were available. CONCLUSIONS: Critical care and neurosurgical nurses underestimated pupil size, were unable to detect anisocoria, and incorrectly assessed pupil reactivity. Standardized use of pupil assessment tools such as a pupillometer is necessary to increase accuracy and consistency in pupil measurement and to potentially contribute to earlier detection of subtle changes in pupils. If pupillary changes are identified early, diagnostic and treatment intervention can be delivered in a more timely and effective manner.