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OBJECTIVES: CD4+CD8+ T cells are increased in patients with rheumatoid arthritis (RA). They are not only associated with joint erosions in established disease, but are also present in the pre-clinical stages of RA. This study aims to further investigate their expansion in the context of T cell clonality in patients with RA, as well as their responsiveness to T cell targeted treatment. METHODS: Single-cell-(sc)RNA- and scTCR-sequencing data were used to determine co-receptor expression and T cell receptor sequences to assess clonality of CD4+CD8+ T cells in RA (n=3) patients and healthy controls (n=2). Peripheral CD4+CD8+ T cells and their subpopulations were measured in patients with RA (n=53), PsA (n=52) and healthy donors (n=50) using flow cytometry. In addition, changes in CD4+CD8+ T cell frequency were prospectively followed in RA patients receiving therapy with abatacept for 12 weeks. RESULTS: We observed an increase of CD4+ T cells expressing CD8α in RA patients, both in comparison to PsA patients and to healthy controls. Clonality analysis revealed, that these CD4+CD8αlow T cells are part of large T cell clones, which cluster separately from CD4+CD8- T cell clones in the scRNA-seq gene expression analysis. Treatment with abatacept significantly reduced the frequency of peripheral CD4+CD8αlow T cells, and this was linked to reduction in disease activity. CONCLUSION: In RA, clonal expansion of CD4+ T cell clones culminates in the emergence of peripheral CD4+CD8αlow T cells, which are associated with disease activity and diminished upon abatacept treatment, and which could contribute to disease pathogenesis.
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BACKGROUND: Orbital floor fractures result in critical changes in the shape and inferior rectus muscle (IRM) position. Radiological imaging of IRM changes can be used for surgical decision making or prediction of ocular symptoms. Studies with a systematic consideration of the orbital floor defect ratio in this context are missing in the literature. Accordingly, this study on human cadavers aimed to systematically investigate the impact of the orbital floor defect ratio on changes in the IRM and the prediction of posttraumatic enophthalmos. METHODS: Seventy-two orbital floor defects were placed in cadaver specimens using piezosurgical removal. The orbital defect area (ODA), orbital floor area (OFA), position and IRM shape, and enophthalmos were measured using computed tomography (CT) scans. RESULTS: The ODA/OFA ratio correlated significantly (p < 0.001) with the shape (Spearman's rho: 0.558) and position (Spearman's rho: 0.511) of the IRM, and with enophthalmos (Spearman's rho: 0.673). Increases in the ODA/OFA ratio significantly rounded the shape of the IRM (ß: 0.667; p < 0.001) and made a lower position of the IRM more likely (OR: 1.093; p = 0.003). In addition, increases in the ODA/OFA ratio were significantly associated with the development of relevant enophthalmos (OR: 1.159; p = 0.008), adjusted for the defect localization and shape of the IRM. According to receiver operating characteristics analysis (AUC: 0.876; p < 0.001), a threshold of ODA/OFA ratio ≥ 32.691 for prediction of the risk of development of enophthalmos yielded a sensitivity of 0.809 and a specificity of 0.842. CONCLUSION: The ODA/OFA ratio is a relevant parameter in the radiological evaluation of orbital floor fractures, as it increases the risk of relevant enophthalmos, regardless of fracture localization and shape of the IRM. Therefore, changes in the shape and position of the IRM should be considered in surgical treatment planning. A better understanding of the correlates of isolated orbital floor fractures may help to develop diagnostic scores and standardize therapeutic algorithms in the future.
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Cadáver , Enoftalmia , Músculos Oculomotores , Órbita , Fraturas Orbitárias , Tomografia Computadorizada por Raios X , Humanos , Enoftalmia/etiologia , Enoftalmia/diagnóstico por imagem , Músculos Oculomotores/diagnóstico por imagem , Masculino , Fraturas Orbitárias/diagnóstico por imagem , Fraturas Orbitárias/complicações , Fraturas Orbitárias/cirurgia , Fraturas Orbitárias/patologia , Feminino , Idoso , Órbita/diagnóstico por imagem , Órbita/lesões , Pessoa de Meia-Idade , Idoso de 80 Anos ou maisRESUMO
PURPOSE: The orbital floor is frequently involved in head trauma. Current evidence on the use of reconstruction materials for orbital floor repair is inconclusive. Accordingly, this study aimed to compare the impact of polydioxanone (PDS) foil thickness on reconstruction of the orbital geometry after isolated orbital floor fractures. METHODS: Standardized isolated orbital floor fractures were symmetrically created in 11 cadaver heads that provided 22 orbits. PDS foils with thicknesses of 0.25-0.5 mm were inserted. Computed tomography (CT) scans of the native, fractured, and reconstructed orbits were obtained, and orbital volume, orbital height, and foil bending were measured. RESULTS: Orbital volume and height significantly (p < 0.01) increased after the creation of isolated orbital floor fractures and significantly (p = 0.001) decreased with overcorrection of the orbital geometry after orbital floor reconstruction with PDS 0.25 mm or PDS 0.5 mm. The orbital geometry reconstruction rate did not differ significantly with respect to foil thickness. However, compared to PDS 0.5 mm, the use of PDS 0.25 mm resulted in quantitatively higher reconstructive accuracy and a restored orbital volume that did not significantly differ from the initial volume. CONCLUSION: Orbital floors subjected to isolated fractures were successfully reconstructed using PDS regardless of foil thickness, with overcorrection of the orbital geometry. Due to its lower flexural stiffness, PDS 0.25 mm appeared to provide more accurate orbital geometry reconstruction than PDS 0.5 mm, although no significant difference in reconstructive accuracy between PDS 0.25 mm and PDS 0.5 mm was observed in this cadaveric study.
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OBJECTIVE: Gynecological sarcomas account for 3% of all gynecological malignancies and are associated with a poor prognosis. Due to the rarity and heterogeneity of gynecological sarcomas there is still no consensus on optimal therapeutic strategies. This study's objective was to describe the treatment strategies used in patients with gynecological sarcomas in the primary course of disease. METHODS: The German prospective registry for gynecological sarcoma (REGSA) is the largest registry for gynecological sarcomas in Germany, Austria and Switzerland. Primary inclusion criteria for REGSA are histological diagnosis of sarcoma of the female genital tract, sarcoma of the breast or uterine smooth muscle tumors of uncertain malignant potential (STUMP). We evaluated data of the REGSA registry on therapeutic strategies used for primary treatment from August 2015 to February 2021. RESULTS: A total of 723 patients from 120 centers were included. Data on therapeutic strategies for primary treatment were available in 605 cases. Overall, 580 (95.9%) patients underwent primary surgery, 472 (81.4%) of whom underwent only hysterectomy. Morcellation was reported in 11.4% (n=54) of all hysterectomies. A total of 42.8% (n=202) had no further surgical interventions, whereas an additional salpingo-ophorectomy was performed in 54% (n=255) of patients. An additional lymphadenectomy was performed in 12.7% (n=60), an omentectomy in 9.5% (n=45) and intestinal resection in 6.1% (n=29) of all patients. Among 448 patients with available information, 21.4% (n=96) received chemo- or targeted therapies, more commonly as single-agent treatment than as drug combinations. Information about anti-hormonal treatment was available for 423 patients, among which 42 (9.9%) received anti-hormonal treatment, 23 (54.8%) of whom with low-grade endometrial stroma sarcomas. For radiotherapy, data of 437 patients were available, among which 29 (6.6%) patients underwent radiotherapy. CONCLUSION: Our study showed that treatment of patients with gynecologic sarcomas is heterogeneous. Further trials are needed along with more information on treatment modalities, therapy response and patient-reported outcomes to implement new treatment strategies.
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Neoplasias do Endométrio , Ginecologia , Sarcoma , Neoplasias Uterinas , Humanos , Feminino , Sarcoma/epidemiologia , Sarcoma/terapia , Sarcoma/patologia , Histerectomia , Alemanha/epidemiologia , Neoplasias do Endométrio/patologia , Neoplasias Uterinas/patologia , Estudos RetrospectivosRESUMO
OBJECTIVES: CD4+CD8+ double-positive (DP) T cells are expanded in the peripheral blood of a subset of patients with RA. This study examines the clinical significance of DP T cells in RA. METHODS: In 70 RA patients, DP T cells were measured by flow cytometry. Clinical data were obtained, and hand and feet radiographs were scored according to the Sharp/van der Heijde (SvdH) method. The association between DP T cell frequency and erosive joint destruction was analysed by correlation and multiple logistic regression analysis. RESULTS: Nineteen RA patients (27.1%) displayed increased DP T cell frequencies, which correlated with age (r = 0.288, P =0.016). Expansion of DP T cells was associated with the occurrence of erosions (94,7% vs 43,1%, P <0.001), with a higher SvdH joint damage score (24.5 vs 6, P =0.008) and with more frequent use of biologic or targeted-synthetic DMARDs (68.4% vs 38%, P =0.02). In patients with non-erosive disease, DP T cell frequencies correlated with the joint space narrowing score (n = 28, r = 0.44, P =0.02). Logistic regression revealed DP T cells to be associated with erosive disease (OR 1.90, P <0.05). CONCLUSION: Expansion of DP T cells is associated with joint damage and frequent escalation of therapy, possibly suggesting a contribution to more severe RA.
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Artrite Reumatoide/sangue , Artrite Reumatoide/patologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Progressão da Doença , HumanosRESUMO
BACKGROUND/AIM: Results of Guided Bone Regeneration (GBR) primarily depend on the membrane used. The aim of this study was to compare biocompatibility of different absorbable and non-absorbable membranes by using unrestricted somatic stem cells (USSCs) as an indicator for biocompatibility. MATERIALS AND METHODS: Five absorbable membranes (Bio-Gide®, RESODONT®, GENTA-FOIL resorb®, BioMend® and BioMend® Extend™) and one non-absorbable alternative (GORE-TEX®) were colonized with USSCs. After 24 h, 3 days and 7 days, cell proliferation, cell viability, and cytotoxicity were assessed. Moreover, cell morphology was evaluated by electron microscopy. RESULTS: Significantly higher cell proliferation and cell viability rates were observed in Bio-Gide® and RESODONT® membranes. Cell toxicity was highest on GENTA-FOIL resorb® membranes. The electron microscopical assessment showed a better cell attachment on porous surfaced membranes. CONCLUSION: This study shows that USSCs can be used for assessments of biocompatibility, and that absorbable membranes with collagenous composition and porous structure tend to positively impact biocompatibility and enhance cell proliferation.
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Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Materiais Biocompatíveis , Regeneração Óssea , Membranas Artificiais , Materiais Biocompatíveis/química , Proliferação de Células , Sobrevivência CelularRESUMO
Developmental genes contribute to cancer, as reported for the homeobox gene Cdx2 playing a tumor suppressor role in the gut. In this study, we show that human colon cancers exhibiting the highest reduction in CDX2 expression belong to the serrated subtype with the worst evolution. In mice, mosaic knockout of Cdx2 in the adult intestinal epithelium induces the formation of imperfect gastric-type metaplastic lesions. The metaplastic knockout cells do not spontaneously become tumorigenic. However, they induce profound modifications of the microenvironment that facilitate the tumorigenic evolution of adjacent Cdx2-intact tumor-prone cells at the surface of the lesions through NF-κB activation, induction of inducible nitric oxide synthase, and stochastic loss of function of Apc This study presents a novel paradigm in that metaplastic cells, generally considered as precancerous, can induce tumorigenesis from neighboring nonmetaplastic cells without themselves becoming cancerous. It unveils the novel property of non-cell-autonomous tumor suppressor gene for the Cdx2 gene in the gut.
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Fator de Transcrição CDX2/genética , Carcinogênese/genética , Carcinogênese/patologia , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Animais , Ceco/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica , Heterozigoto , Humanos , Intestinos/patologia , Metaplasia , Camundongos , NF-kappa B/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Óxido Nítrico Sintase Tipo II/metabolismo , Células Estromais/metabolismo , Células Estromais/patologia , Microambiente TumoralRESUMO
On the basis of phylogenetic analyses, we uncovered a variant of the CDX2 homeobox gene, a major regulator of the development and homeostasis of the gut epithelium, also involved in cancer. This variant, miniCDX2, is generated by alternative splicing coupled to alternative translation initiation, and contains the DNA-binding homeodomain but is devoid of transactivation domain. It is predominantly expressed in crypt cells, whereas the CDX2 protein is present in crypt cells but also in differentiated villous cells. Functional studies revealed a dominant-negative effect exerted by miniCDX2 on the transcriptional activity of CDX2, and conversely similar effects regarding several transcription-independent functions of CDX2. In addition, a regulatory role played by the CDX2 and miniCDX2 homeoproteins on their pre-mRNA splicing is displayed, through interactions with splicing factors. Overexpression of miniCDX2 in the duodenal Brunner glands leads to the expansion of the territory of these glands and ultimately to brunneroma. As a whole, this study characterized a new and original variant of the CDX2 homeobox gene. The production of this variant represents not only a novel level of regulation of this gene, but also a novel way to fine-tune its biological activity through the versatile functions exerted by the truncated variant compared to the full-length homeoprotein. This study highlights the relevance of generating protein diversity through alternative splicing in the gut and its diseases.
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Fator de Transcrição CDX2/genética , Ceco/fisiologia , Mucosa Intestinal/fisiologia , Processamento Alternativo , Animais , Fator de Transcrição CDX2/metabolismo , Células CACO-2 , Ceco/metabolismo , Diferenciação Celular/genética , Genes Homeobox , Células HCT116 , Células HEK293 , Humanos , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Transgênicos , Precursores de RNA/genética , Precursores de RNA/metabolismo , TransfecçãoRESUMO
The endodermal lining of the adult gastro-intestinal tract harbours stem cells that are responsible for the day-to-day regeneration of the epithelium. Stem cells residing in the pyloric glands of the stomach and in the small intestinal crypts differ in their differentiation programme and in the gene repertoire that they express. Both types of stem cells have been shown to grow from single cells into 3D structures (organoids) in vitro. We show that single adult Lgr5-positive stem cells, isolated from small intestinal organoids, require Cdx2 to maintain their intestinal identity and are converted cell-autonomously into pyloric stem cells in the absence of this transcription factor. Clonal descendants of Cdx2(null) small intestinal stem cells enter the gastric differentiation program instead of producing intestinal derivatives. We show that the intestinal genetic programme is critically dependent on the single transcription factor encoding gene Cdx2.
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Reprogramação Celular/genética , Mucosa Gástrica/metabolismo , Proteínas de Homeodomínio/genética , Mucosa Intestinal/metabolismo , Esferoides Celulares/metabolismo , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Animais , Fator de Transcrição CDX2 , Técnicas de Cultura de Células , Diferenciação Celular/genética , Linhagem da Célula/genética , Feminino , Mucosa Gástrica/citologia , Mucosa Intestinal/citologia , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Esferoides Celulares/citologia , Células-Tronco/citologia , Fatores de Transcrição/deficiência , TranscriptomaRESUMO
AIM: The biocompatibility of human osteoblasts (HOB) and human unrestricted somatic stem cells (USSCs) with membranes (BioGide®, GORE-TEX®, GENTA-FOIL resorb®, RESODONT®, BioMend®, BioMend® Extend™) was evaluated. MATERIALS AND METHODS: After osteogenic differentiation (dexamethasone, ascorbic acid and ß-glycerolphosphate) cells were seeded on membranes. On days 1, 3 and 7, attachment, proliferation, cell vitality, cytotoxicty and cell morphology were analyzed. RESULTS: Cells on BioGide® and RESODONT® exhibited significantly higher attachment (p<0.005) and proliferation (p<0.005). On BioMend® cells showed a significantly higher attachment compared to BioMend® Extend™ (p<0.005), whereas on BioMend® Extend™ cells had significantly higher proliferation (p<0.005). The vitality of cells was significantly better on BioGide® and RESODONT® (p<0.005). There were no significant differences between USSCs and HOBs. Scanning electron microscopy confirmed these results. CONCLUSION: BioGide® and RESODONT® had the best biocompatibility and are appropriate membranes for use in stem cell-derived regeneration of bone.
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Materiais Biocompatíveis/normas , Proliferação de Células , Membranas Artificiais , Osteoblastos/citologia , Células-Tronco/citologia , Ácido Ascórbico/farmacologia , Adesão Celular , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Colágeno/normas , Dexametasona/farmacologia , Feminino , Glicerofosfatos/farmacologia , Humanos , Masculino , Teste de Materiais/métodos , Microscopia Eletrônica de Varredura , Osteoblastos/ultraestrutura , Osteogênese/efeitos dos fármacos , Politetrafluoretileno/normas , Reprodutibilidade dos Testes , Células-Tronco/ultraestruturaRESUMO
Cdx gene products regulate the extent of axial elongation from the posterior growth zone. These transcription factors sustain the emergence of trunk and tail tissues by providing a suitable niche in the axial progenitor zone, via regulation of Wnt signaling. Cdx genes are expressed in and along the complete primitive streak including its posterior part wherefrom the extraembryonic mesoderm of the allantois emerges. Cdx genes are required for the full development of the allantois and its derivatives in the placental labyrinth. The mouse germ cell lineage also originates from the proximo-posterior epiblast of the primitive streak, and is established within the extraembryonic mesoderm that generates the allantois. We asked whether the expression of Cdx genes around the newly specified PGCs is necessary for the maintenance and expansion of this population, as it is for the allantois and axial progenitors. We observed a significantly lower number of PGCs in Cdx2(null) embryos than in controls. We found that Wnt3a loss of function decreases the PGC population to the same extent as Cdx2 inactivation. Moreover, exogenous Wnt3a corrects the lower PGC number in Cdx2(null) posterior embryonic tissues cultured in vitro. Cdx2 is not expressed in PGCs themselves, and we propose that the expression of Cdx2 in posterior extraembryonic tissues contributes to the proper niche of the germ cell progenitors by stimulating canonical Wnt signaling. Since PGC residence within the posterior growth zone is a mouse-specific feature, our data suggest that mouse PGCs opportunistically became dependent on the axial progenitor niche.
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Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/citologia , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Alantoide/citologia , Alantoide/embriologia , Alantoide/metabolismo , Animais , Fator de Transcrição CDX2 , Embrião de Mamíferos/citologia , Células Germinativas/metabolismo , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Knockout , Fatores de Transcrição/metabolismoRESUMO
Mouse Cdx genes are involved in axial patterning and partial Cdx mutants exhibit posterior embryonic defects. We found that mouse embryos in which all three Cdx genes are inactivated fail to generate any axial tissue beyond the cephalic and occipital primordia. Anterior axial tissues are laid down and well patterned in Cdx null embryos, and a 3' Hox gene is initially transcribed and expressed in the hindbrain normally. Axial elongation stops abruptly at the post-occipital level in the absence of Cdx, as the posterior growth zone loses its progenitor activity. Exogenous Fgf8 rescues the posterior truncation of Cdx mutants, and the spectrum of defects of Cdx null embryos matches that resulting from loss of posterior Fgfr1 signaling. Our data argue for a main function of Cdx in enforcing trunk emergence beyond the Cdx-independent cephalo-occipital region, and for a downstream role of Fgfr1 signaling in this function. Cdx requirement for the post-head section of the axis is ancestral as it takes place in arthropods as well.
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Desenvolvimento Embrionário/fisiologia , Evolução Molecular , Animais , Fator de Transcrição CDX2 , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Somitos/citologia , Somitos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Knock out of intestinal Cdx2 produces different effects depending upon the developmental stage at which this occurs. Early in development it produces histologically ordered stomach mucosa in the midgut. Conditional inactivation of Cdx2 in adult intestinal epithelium, as well as specifically in the Lgr5-positive stem cells, of adult mice allows long-term survival of the animals but fails to produce this phenotype. Instead, the endodermal cells exhibit cell-autonomous expression of gastric genes in an intestinal setting that is not accompanied by mesodermal expression of Barx1, which is necessary for gastric morphogenesis. Cdx2-negative endodermal cells also fail to express Sox2, a marker of gastric morphogenesis. Maturation of the stem cell niche thus appears to be associated with loss of ability to express positional information cues that are required for normal stomach development. Cdx2-negative intestinal crypts produce subsurface cystic vesicles, whereas untargeted crypts hypertrophy to later replace the surface epithelium. These observations are supported by studies involving inactivation of Cdx2 in intestinal crypts cultured in vitro. This abolishes their ability to form long-term growing intestinal organoids that differentiate into intestinal phenotypes. We conclude that expression of Cdx2 is essential for differentiation of gut stem cells into any of the intestinal cell types, but they maintain a degree of cell-autonomous plasticity that allows them to switch on a variety of gastric genes.
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Endoderma/crescimento & desenvolvimento , Mucosa Intestinal/crescimento & desenvolvimento , Intestino Delgado/crescimento & desenvolvimento , Animais , Fator de Transcrição CDX2 , Diferenciação Celular/genética , Células Cultivadas , Feminino , Mucosa Gástrica/crescimento & desenvolvimento , Técnicas de Inativação de Genes , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Morfogênese/genética , Fatores de Transcrição SOXB1/biossíntese , Células-Tronco/fisiologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Decrease in Cdx dosage in an allelic series of mouse Cdx mutants leads to progressively more severe posterior vertebral defects. These defects are corrected by posterior gain of function of the Wnt effector Lef1. Precocious expression of Hox paralogous 13 genes also induces vertebral axis truncation by antagonizing Cdx function. We report here that the phenotypic similarity also applies to patterning of the caudal neural tube and uro-rectal tracts in Cdx and Wnt3a mutants, and in embryos precociously expressing Hox13 genes. Cdx2 inactivation after placentation leads to posterior defects, including incomplete uro-rectal septation. Compound mutants carrying one active Cdx2 allele in the Cdx4-null background (Cdx2/4), transgenic embryos precociously expressing Hox13 genes and a novel Wnt3a hypomorph mutant all manifest a comparable phenotype with similar uro-rectal defects. Phenotype and transcriptome analysis in early Cdx mutants, genetic rescue experiments and gene expression studies lead us to propose that Cdx transcription factors act via Wnt signaling during the laying down of uro-rectal mesoderm, and that they are operative in an early phase of these events, at the site of tissue progenitors in the posterior growth zone of the embryo. Cdx and Wnt mutations and premature Hox13 expression also cause similar neural dysmorphology, including ectopic neural structures that sometimes lead to neural tube splitting at caudal axial levels. These findings involve the Cdx genes, canonical Wnt signaling and the temporal control of posterior Hox gene expression in posterior morphogenesis in the different embryonic germ layers. They shed a new light on the etiology of the caudal dysplasia or caudal regression range of human congenital defects.
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Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Tubo Neural/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Animais , Fator de Transcrição CDX2 , Forma Celular , Feminino , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tubo Neural/citologia , Fatores de Transcrição/genética , Tretinoína/metabolismo , Proteínas Wnt/genética , Proteína Wnt3 , Proteína Wnt3ARESUMO
The Cdx (Caudal-type homeobox) group of ParaHox genes (Cdx1, Cdx2 and Cdx4 in the mouse) perform multiple functions in mammalian development. Cdx1 is concerned with axial positional information, and its deletion appears to have no important effect other than a disturbance of axial patterning. In contrast, Cdx2 is required for trophoblast differentiation, axial patterning and extension, as well as for morphological specification (i.e. patterning) of gut endoderm. Cdx4-knockout animals do not present an abnormal phenotype, but, when combined with Cdx2 haploinsufficiency, present a dramatic picture involving abnormal cloacal specification. The latter is probably due in large part to defective paraxial mesodermal development in the caudal region, but may also involve defective endodermal growth. A significant degree of redundancy is apparent between the Cdx genes with respect to caudal extension and possibly also during gut development.
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Padronização Corporal/genética , Proteínas de Homeodomínio/fisiologia , Intestinos/embriologia , Animais , Embrião de Mamíferos , Embrião não Mamífero , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Modelos BiológicosRESUMO
Hox and Cdx transcription factors regulate embryonic positional identities. Cdx mutant mice display posterior body truncations of the axial skeleton, neuraxis, and caudal urorectal structures. We show that trunk Hox genes stimulate axial extension, as they can largely rescue these Cdx mutant phenotypes. Conversely, posterior (paralog group 13) Hox genes can prematurely arrest posterior axial growth when precociously expressed. Our data suggest that the transition from trunk to tail Hox gene expression successively regulates the construction and termination of axial structures in the mouse embryo. Thus, Hox genes seem to differentially orchestrate posterior expansion of embryonic tissues during axial morphogenesis as an integral part of their function in specifying head-to-tail identity. In addition, we present evidence that Cdx and Hox transcription factors exert these effects by controlling Wnt signaling. Concomitant regulation of Cyp26a1 expression, restraining retinoic acid signaling away from the posterior growth zone, may likewise play a role in timing the trunk-tail transition.
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Padronização Corporal/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox/fisiologia , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Animais , Antineoplásicos/farmacologia , Western Blotting , Fator de Transcrição CDX2 , Sistema Enzimático do Citocromo P-450/metabolismo , Extremidades/embriologia , Perfilação da Expressão Gênica , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ácido Retinoico 4 Hidroxilase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esqueleto , Tretinoína/farmacologia , Proteínas Wnt/metabolismoRESUMO
BACKGROUND & AIMS: The Cdx2 homeobox gene exerts multiple functions including trophectoderm specification, antero-posterior patterning, and determination of intestinal identity. The aim of this study was to map genomic regions that regulate the transcription of Cdx2, with a particular interest in the gut. METHODS: Genomic fragments covering 13 kilobase (kb) of the mouse Cdx2 locus were analyzed in transgenic mice and in cell assays. RESULTS: No fragment was active in the trophectoderm. Fragments containing the first intron and extending up to -5-kb upstream of the transcription start site became active posteriorly at gastrulation and then inactive at midgestation in every tissue including the endoderm. Specific persistence of activity in the intestinal endoderm/epithelium beyond midgestation requires extending the genomic fragment up to -9 kb. We identified a 250-base pair segment around -8.5-kb binding and responding to endodermal factors, with a stimulatory effect exerted synergistically by HNF4alpha, GATA6, Tcf4, and beta-catenin. These factors were able to activate endogenous expression of Cdx2 in nonintestinal Hela cells. CONCLUSIONS: Multiple regulatory regions control the complex developmental pattern of Cdx2, including far upstream sequences required for the persistence of gene expression specifically in the gut epithelium throughout life. Cooperation between HNF4alpha, GATA6, beta-catenin, and Tcf4 contributes to the intestine-specific expression of Cdx2.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Intestinos/embriologia , Intestinos/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores Etários , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Blastocisto/citologia , Blastocisto/fisiologia , Fator de Transcrição CDX2 , Ceco/embriologia , Ceco/fisiologia , Linhagem Celular , Endoderma/embriologia , Endoderma/fisiologia , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Genômica , Células HeLa , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Intestinos/citologia , Óperon Lac , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Estômago/embriologia , Estômago/fisiologia , Fatores de Transcrição TCF/genética , Fatores de Transcrição TCF/metabolismo , Fator de Transcrição 4 , Transfecção , Trofoblastos/citologia , Trofoblastos/fisiologia , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Null mutation or haploinsufficiency of Cdx2 results in the development of heterotopic lesions with a gastric phenotype in the midgut endoderm. Conversely transgenic expression of Cdx2 in the stomach causes the endoderm to differentiate into intestinal-type mucosa. We demonstrate that the mesoderm adjacent to intestinal heterotopic areas expresses stomach specific Barx1 while the surrounding mesoderm is Barx1 negative. We conclude that the initiation of gut histodifferentiation lies in the endodermal expression of Cdx2 and that endodermal/mesodermal cross-talk involving Barx1 with appropriate feedback loops results in the development of the postnatal gut phenotype.
Assuntos
Endoderma/metabolismo , Proteínas de Homeodomínio/metabolismo , Intestinos/embriologia , Morfogênese , Estômago/embriologia , Fatores de Transcrição/metabolismo , Animais , Fator de Transcrição CDX2 , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/metabolismo , Feminino , Mucosa Gástrica/metabolismo , Proteínas HMGB/metabolismo , Proteínas de Homeodomínio/genética , Mucosa Intestinal/metabolismo , Masculino , Mesoderma/metabolismo , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Morfogênese/genética , Fatores de Transcrição SOXB1 , Fatores de Transcrição/genéticaRESUMO
The development of new therapeutic approaches against colorectal cancer requires preclinical studies in mice. In vivo imaging could greatly facilitate these trials, but the small size of the animals is a major limitation for the direct visualization of intestinal tissue. Here we report a method of in vivo imaging of the mouse intestine based on X-ray micro-computed tomography using multiple contrast agents. This method was validated in the model of non-cancerous polyp-like heteroplasia that spontaneously develops in the caecum area of Cdx2+/- mutant mice and in the model of colon adenocarcinoma induced by administration of the chemical carcinogen azoxymethane. As a simple and non-invasive method, multiple-contrast X-ray micro-computed tomography is appropriate for pre-clinical studies of intestinal diseases in living mice.
Assuntos
Colo/anormalidades , Colo/diagnóstico por imagem , Neoplasias do Colo/diagnóstico por imagem , Tomografia Computadorizada por Raios X/instrumentação , Tomografia Computadorizada por Raios X/métodos , Animais , Fator de Transcrição CDX2 , Desenho de Equipamento , Heterozigoto , Proteínas de Homeodomínio/genética , Enteropatias/diagnóstico por imagem , Masculino , Camundongos , Camundongos Knockout , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
Caudal related homeobox (Cdx) genes have so far been shown to be important for embryonic axial elongation and patterning in several vertebrate species. We have generated a targeted mutation of mouse Cdx4, the third member of this family of transcription factor encoding genes and the last one to be inactivated genetically. Cdx4-null embryos were born healthy and appeared morphologically normal. A subtle contribution of Cdx4 to anteroposterior (AP) vertebral patterning was revealed in Cdx1/Cdx4 and Cdx2/Cdx4 compound mutants. Neither Cdx4-null nor Cdx1/Cdx4 double mutants are impaired in their axial elongation, but a redundant contribution of Cdx4 in this function was unveiled when combined with a Cdx2 mutant allele. In addition, inactivation of Cdx4 combined with heterozygous loss of Cdx2 results in embryonic death around E10.5 and reveals a novel function of Cdx genes in placental ontogenesis. In a subset of Cdx2/Cdx4 compound mutants, the fully grown allantois failed to fuse with the chorion. The remaining majority of these mutants undergo successful chorio-allantois fusion but fail to properly extend their allantoic vascular network into the chorionic ectoderm and do not develop a functional placental labyrinth. We present evidence that Cdx4 plays a crucial role in the ontogenesis of the allantoic component of the placental labyrinth when one Cdx2 allele is inactivated. The axial patterning role of Cdx transcription factors thus extends posteriorly to the epiblast-derived extra-embryonic mesoderm and, consequent upon the evolution of placental mammals, is centrally involved in placental morphogenesis. The relative contribution of Cdx family members in the stepwise ontogenesis of a functional placenta is discussed, with Cdx2 playing an obligatory part, assisted by Cdx4. The possible participation of Cdx1 was not documented but cannot be ruled out until allelic combinations further decreasing Cdx dose have been analyzed. Cdx genes thus operate in a redundant way during placentogenesis, as they do during embryonic axial elongation and patterning, and independently from the previously reported early Cdx2-specific role in the trophectoderm at implantation.