RESUMO
The extra-embryonic yolk sac contains adjacent layers of mesoderm and visceral endoderm. The mesodermal layer serves as the first site of embryonic hematopoiesis, while the visceral endoderm provides a means of exchanging nutrients and waste until the development of the chorioallantoic placenta. While defects in chorioallantoic fusion and yolk sac hematopoiesis have been described in Cdx mutant mouse models, little is known about the gene targets and molecular mechanisms through which Cdx members regulate these processes. To this end, we used RNA-seq to examine Cdx-dependent gene expression changes in the yolk sac. We find that loss of Cdx function impacts the expression of genes involved in yolk sac hematopoiesis, as previously described, as well as novel Cdx2 target genes. In addition, we observed Cdx-dependent changes in PRC2 subunit expression accompanied by altered H3K27me3 deposition at a subset of Cdx target genes as early as E7.5 in the embryo proper. This study identifies additional Cdx target genes and provides further evidence for Cdx-dependent epigenetic regulation of gene expression in the early embryo, and that this regulation is required to maintain gene expression programs in the extra-embryonic yolk sac at later developmental stages.
Assuntos
Fator de Transcrição CDX2/genética , Desenvolvimento Embrionário/genética , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Complexo Repressor Polycomb 2/genética , Animais , Endoderma/embriologia , Feminino , Expressão Gênica , Técnicas de Inativação de Genes/métodos , Hematopoese/genética , Masculino , Mesoderma/embriologia , Camundongos , Camundongos Knockout , Gravidez , RNA-Seq/métodos , Transcrição Gênica/genética , Saco Vitelino/embriologiaRESUMO
The primordia of the post-otic mouse embryo forms largely from a bipotential cell population containing neuromesodermal progenitors (NMP) which reside in the tail bud and contribute to the elaboration of the major body axis after gastrulation. The mechanisms by which the NMP population is both maintained and subsequently directed down mesodermal and neural lineages is incompletely understood. The vertebrate transcription factor Cdx2, is essential for axial elongation and has been implicated in maintaining the NMP niche and in specification of NMP derivatives. To better understand the role of the Cdx family in axial elongation, we employed a conditional mutant allele which evokes total loss of Cdx function, and enriched for tail bud progenitors through the use of a Pax2-GFP transgenic reporter. Using this approach, we identified 349 Cdx-dependent genes by RNA sequencing (RNA-seq). From these, Gene Ontology and chromatin immunoprecipitation analysis further revealed a number of putative direct Cdx candidate target genes implicated in axial elongation, including Sp8, Isl1, Evx1, Zic3 and Nr2f1. Additional analysis of available single-cell RNA-seq data from mouse tail buds revealed the co-expression of Sp8, Isl1, Evx1 and Zic3 with Cdx2 in putative NMP cells, while Nr2f1 was excluded from this population. These findings identify a number of novel Cdx targets and provide further insight into the critical roles for Cdx in elaborating the post-otic embryo.
Assuntos
Padronização Corporal/genética , Fator de Transcrição CDX2/metabolismo , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Proteínas de Homeodomínio/metabolismo , Transdução de Sinais/genética , Animais , Fator de Transcrição CDX2/genética , Fator I de Transcrição COUP/genética , Imunoprecipitação da Cromatina/métodos , Proteínas de Ligação a DNA/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas com Homeodomínio LIM/genética , Masculino , Mesoderma/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , RNA-Seq/métodos , Células-Tronco/metabolismo , Fatores de Transcrição/genéticaRESUMO
The intestine-specific transcription factor Cdx2 is essential for intestinal homeostasis and has been implicated in the pathogenesis of disorders including inflammatory bowel disease. However, the mechanism by which Cdx2 influences intestinal disease is not clear. Here, we present evidence supporting a novel Cdx2-TRIM31-NLRP3 (NLR family, pyrin domain containing 3) signaling pathway, which may represent a mechanistic means by which Cdx2 impacts intestinal inflammation. We found that conditional loss of Cdx function resulted in an increase in proinflammatory cytokines, including tumor necrosis factor alpha, interleukin (IL)-1ß, and IL-6, in the mouse colon. We further show that TRIM31, which encodes a suppressor of NLRP3 (a central component of the NLRP3 inflammasome complex) is a novel Cdx2 target gene and is attenuated in the colon of Cdx conditional mutants. Consistent with this, we found that attenuation of TRIM31 in Cdx mutant intestine occurs concomitant with elevated levels of NLRP3 and an increase in inflammasome products. We demonstrate that specific inhibition of NLRP3 activity significantly reduced IL-1ß and IL-6 levels and extended the life span of Cdx conditional mutants, reflecting the therapeutic potential of targeting NLRP3. Tumor necrosis factor-alpha levels were also induced independent of NLRP3, potentially via elevated activity of the proinflammatory NF-κB signaling pathway in Cdx mutants. Finally, in silico analysis of ulcerative colitis patients revealed attenuation of CDX2 and TRIM31 expression coincident with enhanced expression of proinflammatory cytokines. We conclude that the novel Cdx2-TRIM31-NLRP3 signaling pathway promotes proinflammatory cytokine expression, and its inhibition may have therapeutic potential in human intestinal diseases.
Assuntos
Fator de Transcrição CDX2 , Inflamassomos , Doenças Inflamatórias Intestinais , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Animais , Humanos , Camundongos , Fator de Transcrição CDX2/genética , Fator de Transcrição CDX2/metabolismo , Citocinas/metabolismo , Homeostase , Inflamassomos/genética , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/genética , Intestinos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Doenças Inflamatórias Intestinais/metabolismoRESUMO
Murine cardiac and hematopoietic progenitors are derived from Mesp1+ mesoderm. Cdx function impacts both yolk sac hematopoiesis and cardiogenesis in zebrafish, suggesting that Cdx family members regulate early mesoderm cell fate decisions. We found that Cdx2 occupies a number of transcription factor loci during embryogenesis, including key regulators of both cardiac and blood development, and that Cdx function is required for normal expression of the cardiogenic transcription factors Nkx2-5 and Tbx5 Furthermore, Cdx and Brg1, an ATPase subunit of the SWI/SNF chromatin remodeling complex, co-occupy a number of loci, suggesting that Cdx family members regulate target gene expression through alterations in chromatin architecture. Consistent with this, we demonstrate loss of Brg1 occupancy and altered chromatin structure at several cardiogenic genes in Cdx-null mutants. Finally, we provide evidence for an onset of Cdx2 expression at E6.5 coinciding with egression of cardiac progenitors from the primitive streak. Together, these findings suggest that Cdx functions in multi-potential mesoderm to direct early cell fate decisions through transcriptional regulation of several novel target genes, and provide further insight into a potential epigenetic mechanism by which Cdx influences target gene expression.
Assuntos
Fator de Transcrição CDX2/metabolismo , Animais , Fator de Transcrição CDX2/genética , Imunoprecipitação da Cromatina , Feminino , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Masculino , Camundongos , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismoRESUMO
The Cdx transcription factors play essential roles in primitive hematopoiesis in the zebrafish where they exert their effects, in part, through regulation of hox genes. Defects in hematopoiesis have also been reported in Cdx mutant murine embryonic stem cell models, however, to date no mouse model reflecting the zebrafish Cdx mutant hematopoietic phenotype has been described. This is likely due, in part, to functional redundancy among Cdx members and the early lethality of Cdx2 null mutants. To circumvent these limitations, we used Cre-mediated conditional deletion to assess the impact of concomitant loss of Cdx1 and Cdx2 on murine primitive hematopoiesis. We found that Cdx1/Cdx2 double mutants exhibited defects in primitive hematopoiesis and yolk sac vasculature concomitant with reduced expression of several genes encoding hematopoietic transcription factors including Scl/Tal1. Chromatin immunoprecipitation analysis revealed that Scl was occupied by Cdx2 in vivo, and Cdx mutant hematopoietic yolk sac differentiation defects could be rescued by expression of exogenous Scl. These findings demonstrate critical roles for Cdx members in murine primitive hematopoiesis upstream of Scl.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fator de Transcrição CDX2/genética , Hematopoese/genética , Proteínas de Homeodomínio/genética , Neovascularização Fisiológica/genética , Proteínas Proto-Oncogênicas/biossíntese , Saco Vitelino/irrigação sanguínea , Animais , Diferenciação Celular/genética , Imunoprecipitação da Cromatina , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Knockout , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Saco Vitelino/embriologiaRESUMO
The packaging of genomic DNA into nucleosomes creates a barrier to transcription that can be relieved through ATP-dependent chromatin remodeling via complexes such as the switch-sucrose non-fermentable (SWI-SNF) chromatin remodeling complex. The SWI-SNF complex remodels chromatin via conformational or positional changes of nucleosomes, thereby altering the access of transcriptional machinery to target genes. The SWI-SNF complex has limited ability to bind to sequence-specific elements, and, therefore, its recruitment to target loci is believed to require interaction with DNA-associated transcription factors. The Cdx family of homeodomain transcript ion factors (Cdx1, Cdx2, and Cdx4) are essential for a number of developmental programs in the mouse. Cdx1 and Cdx2 also regulate intestinal homeostasis throughout life. Although a number of Cdx target genes have been identified, the basis by which Cdx members impact their transcription is poorly understood. We have found that Cdx members interact with the SWI-SNF complex and make direct contact with Brg1, a catalytic member of SWI-SNF. Both Cdx2 and Brg1 co-occupy a number of Cdx target genes, and both factors are necessary for transcriptional regulation of such targets. Finally, Cdx2 and Brg1 occupancy occurs coincident with chromatin remodeling at some of these loci. Taken together, our findings suggest that Cdx transcription factors regulate target gene expression, in part, through recruitment of Brg1-associated SWI-SNF chromatin remodeling activity.
Assuntos
Fator de Transcrição CDX2/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Mapas de Interação de ProteínasRESUMO
Although many regulatory networks involved in defining definitive endoderm have been identified, the mechanisms through which these networks interact to pattern the endoderm are less well understood. To explore the mechanisms involved in midgut patterning, we dissected the transcriptional regulatory elements of nephrocan (Nepn), the earliest known midgut specific gene in mice. We observed that Nepn expression is dramatically reduced in Sox17(-/-) and Raldh2(-/-) embryos compared with wild-type embryos. We further show that Nepn is directly regulated by Sox17 and the retinoic acid (RA) receptor via two enhancer elements located upstream of the gene. Moreover, Nepn expression is modulated by Activin signaling, with high levels inhibiting and low levels enhancing RA-dependent expression. In Foxh1(-/-) embryos in which Nodal signaling is reduced, the Nepn expression domain is expanded into the anterior gut region, confirming that Nodal signaling can modulate its expression in vivo. Together, Sox17 is required for Nepn expression in the definitive endoderm, while RA signaling restricts expression to the midgut region. A balance of Nodal/Activin signaling regulates the anterior boundary of the midgut expression domain.
Assuntos
Padronização Corporal/fisiologia , Endoderma/fisiologia , Trato Gastrointestinal/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Redes Reguladoras de Genes/fisiologia , Glicoproteínas/metabolismo , Transdução de Sinais/fisiologia , Ativinas/metabolismo , Aldeído Oxirredutases/metabolismo , Animais , Ensaio de Desvio de Mobilidade Eletroforética , Redes Reguladoras de Genes/genética , Vetores Genéticos/genética , Proteínas HMGB/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Luciferases , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição SOXF/metabolismoRESUMO
Retinoic acid is required for diverse ontogenic processes and as such identification of the genes and pathways affected by retinoic acid is critical to understanding these pleiotropic effects. The presomitic mesoderm of the E8.5 mouse embryo is composed of undifferentiated cells that are depleted of retinoic acid, yet are competent to respond to the retinoid signal. We have exploited these properties to use this tissue to identify novel retinoic acid-responsive genes, including candidate target genes, by treating E8.5 embryos with retinoic acid and assessing changes in gene expression in the presomitic mesoderm by microarray analysis. This exercise yielded a cohort of genes that were differentially expressed in response to exogenous retinoic acid exposure. Among these were a number of previously characterized retinoic acid targets, validating this approach. In addition, we recovered a number of novel candidate target genes which were confirmed as retinoic acid-responsive by independent analysis. Chromatin immunoprecipitation assays revealed retinoic acid receptor occupancy of the promoters of certain of these genes. We further confirmed direct retinoic acid regulation of the F11r gene, a new RA target, using tissue culture models. Our results reveal a significant number of potential RA targets implicated in embryonic development and offer a novel in vivo system for better understanding of retinoid-dependent transcription.
Assuntos
Moléculas de Adesão Celular/metabolismo , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Mesoderma/metabolismo , Receptores de Superfície Celular/metabolismo , Tretinoína/metabolismo , Animais , Moléculas de Adesão Celular/genética , Imunoprecipitação da Cromatina , Primers do DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Hibridização In Situ , Camundongos , Análise em Microsséries , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tretinoína/farmacologiaRESUMO
In humans, colorectal cancer is often initiated through APC loss of function, which leads to crypt hyperplasia and polyposis driven by unrestricted canonical Wnt signaling. Such polyps typically arise in the colorectal region and are at risk of transforming to invasive adenocarcinomas. Although colorectal cancer is the third most common cause of cancer-related death worldwide, the processes impacting initiation, transformation, and invasion are incompletely understood. Murine APC(Min/+) mutants are often used to model colorectal cancers; however, they develop nonmetastatic tumors confined largely to the small intestine and are thus not entirely representative of the human disease. APC(Min/+) alleles can collaborate with mutations impacting other pathways to recapitulate some aspects of human colorectal cancer. To this end, we assessed APC(Min/+)-induced polyposis following somatic loss of the homeodomain transcription factor Cdx2, alone or with a Cdx1 null allele, in the adult gastrointestinal tract. APC(Min/+)-Cdx2 mutants recapitulated several aspects of human colorectal cancer, including an invasive phenotype. Notably, the concomitant loss of Cdx1 led to a significant increase in the incidence of tumors in the distal colon, relative to APC(Min/+)-Cdx2 offspring, demonstrating a previously unrecognized role for this transcription factor in colorectal tumorigenesis. These findings underscore previously unrecognized roles for Cdx members in intestinal tumorigenesis.
Assuntos
Neoplasias Colorretais/metabolismo , Proteínas de Homeodomínio/metabolismo , Neoplasias Experimentais/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Fator de Transcrição CDX2 , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Knockout , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/imunologiaRESUMO
The vertebrate Cdx genes (Cdx1, Cdx2 and Cdx4) encode homeodomain transcription factors with well-established roles in anteroposterior patterning. To circumvent the peri-implantation lethality inherent to Cdx2 loss of function, we previously used the Cre-loxP system to ablate Cdx2 at post-implantation stages and confirmed a crucial role for Cdx2 function in events related to axial extension. As considerable data suggest that the Cdx family members functionally overlap, we extended this analysis to assess the consequence of concomitant loss of both Cdx1 and Cdx2. Here, we report that Cdx1-Cdx2 double mutants exhibit a severely truncated anteroposterior axis. In addition, these double mutants exhibit fused somites, a widened mediolateral axis and craniorachischisis, a severe form of neural tube defect in which early neurulation fails and the neural tube remains open. These defects are typically associated with deficits in planar cell polarity (PCP) signaling in vertebrates. Consistent with this, we found that expression of Ptk7, which encodes a gene involved in PCP, is markedly reduced in Cdx1-Cdx2 double mutants, and is a candidate Cdx target. Genetic interaction between Cdx mutants and a mutant allele of Scrib, a gene involved in PCP signaling, is suggestive of a role for Cdx signaling in the PCP pathway. These findings illustrate a novel and pivotal role for Cdx function upstream of Ptk7 and neural tube closure in vertebrates.
Assuntos
Polaridade Celular/genética , Proteínas de Homeodomínio/metabolismo , Tubo Neural/embriologia , Tubo Neural/metabolismo , Neurulação/genética , Receptores Proteína Tirosina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Animais , Padronização Corporal/genética , Fator de Transcrição CDX2 , Imunoprecipitação da Cromatina , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Transgênicos , Receptores Proteína Tirosina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Fatores de Transcrição/genéticaRESUMO
The homeodomain transcription factors Cdx1 and Cdx2 are expressed in the intestinal epithelium from early development, with expression persisting throughout the life of the animal. While our understanding of the function of Cdx members in intestinal development has advanced significantly, their roles in the adult intestine is relatively poorly understood. In the present study, we found that ablation of Cdx2 in the adult small intestine severely impacted villus morphology, proliferation and intestinal gene expression patterns, resulting in the demise of the animal. Long-term loss of Cdx2 in a chimeric model resulted in loss of all differentiated intestinal cell types and partial conversion of the mucosa to a gastric-like epithelium. Concomitant loss of Cdx1 did not exacerbate any of these phenotypes. Loss of Cdx2 in the colon was associated with a shift to a cecum-like epithelial morphology and gain of cecum-associated genes which was more pronounced with subsequent loss of Cdx1. These findings suggest that Cdx2 is essential for differentiation of the small intestinal epithelium, and that both Cdx1 and Cdx2 contribute to homeostasis of the colon.
Assuntos
Colo/metabolismo , Proteínas de Homeodomínio/metabolismo , Intestino Delgado/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fator de Transcrição CDX2 , Diferenciação Celular , Proliferação de Células , Colo/citologia , Colo/crescimento & desenvolvimento , Deleção de Genes , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Mucosa Intestinal/crescimento & desenvolvimento , Mucosa Intestinal/metabolismo , Intestino Delgado/citologia , Intestino Delgado/crescimento & desenvolvimento , Camundongos , Fatores de Transcrição/genéticaRESUMO
Vertebrate Cdx genes encode homeodomain transcription factors related to caudal in Drosophila. The murine Cdx homologues Cdx1, Cdx2 and Cdx4 play important roles in anterior-posterior patterning of the embryonic axis and the intestine, as well as axial elongation. While our understanding of the ontogenic programs requiring Cdx function has advanced considerably, the molecular bases underlying these functions are less well understood. In this regard, Cdx1-Cdx2 conditional mutants exhibit abnormal somite formation, while loss of Cdx1-Cdx2 in the intestinal epithelium results in a shift in differentiation toward the Goblet cell lineage. The aim of the present study was to identify the Cdx-dependent mechanisms impacting on these events. Consistent with prior work implicating Notch signaling in these pathways, we found that expression of the Notch ligand Dll1 was reduced in Cdx mutants in both the intestinal epithelium and paraxial mesoderm. Cdx members occupied the Dll1 promoter both in vivo and in vitro, while genetic analysis indicated interaction between Cdx and Dll1 pathways in both somitogenesis and Goblet cell differentiation. These findings suggest that Cdx members operate upstream of Dll1 to convey different functions in two distinct lineages.
Assuntos
Padronização Corporal/genética , Diferenciação Celular/genética , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mucosa Intestinal/embriologia , Somitos/embriologia , Fatores de Transcrição/metabolismo , Animais , Padronização Corporal/fisiologia , Fator de Transcrição CDX2 , Proteínas de Ligação ao Cálcio , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Células Caliciformes/citologia , Técnicas Histológicas , Proteínas de Homeodomínio/genética , Hibridização In Situ , Mesoderma/embriologia , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Somitos/metabolismo , Fatores de Transcrição/genéticaRESUMO
Cdx and Hox gene families encode homeodomain-containing transcription factors involved in anterior-posterior vertebral patterning. Although Cdx proteins are direct transcriptional regulators of Hox gene expression, both Hox and Cdx proteins are known to interact with other homeodomain transcription factors, leading us to speculate that Cdx and Hox proteins may also interact physically. In testing this, we found that that Cdx1 is indeed capable of associating with a subset of Hox proteins. This interaction is localized to the homeodomain region of both classes of proteins, is reliant on specific arginine residues in helix I of the Hox homeodomain, and is further modulated by N-terminal Hox sequences. More promiscuous interactions were seen with Hox proteins expressed in vivo, suggestive of bridging factors or post-translational modifications. Finally, we demonstrate that this interaction modulates Cdx-Hox transcriptional activity on a Hox-responsive element. This study is the first example of Cdx-Hox protein interactions and suggests that such complexes may modulate Hox and/or Cdx function.
Assuntos
Proteínas de Homeodomínio/metabolismo , Animais , Células COS , Chlorocebus aethiops , Imunoprecipitação da Cromatina , CamundongosRESUMO
The homeodomain transcription factors Cdx1, Cdx2 and Cdx4 play essential roles in anteroposterior vertebral patterning through regulation of Hox gene expression. Cdx2 is also expressed in the trophectoderm commencing at E3.5 and plays an essential role in implantation, thus precluding assessment of the cognate-null phenotype at later stages. Cdx2 homozygous null embryos generated by tetraploid aggregation exhibit an axial truncation indicative of a role for Cdx2 in elaborating the posterior embryo through unknown mechanisms. To better understand such roles, we developed a conditional Cdx2 floxed allele in mice and effected temporal inactivation at post-implantation stages using a tamoxifen-inducible Cre. This approach yielded embryos that were devoid of detectable Cdx2 protein and exhibited the axial truncation phenotype predicted from previous studies. This phenotype was associated with attenuated expression of genes encoding several key players in axial elongation, including Fgf8, T, Wnt3a and Cyp26a1, and we present data suggesting that T, Wnt3a and Cyp26a1 are direct Cdx2 targets. We propose a model wherein Cdx2 functions as an integrator of caudalizing information by coordinating axial elongation and somite patterning through Hox-independent and -dependent pathways, respectively.
Assuntos
Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Alelos , Animais , Fator de Transcrição CDX2 , Sistema Enzimático do Citocromo P-450/genética , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Fator 8 de Crescimento de Fibroblasto/genética , Proteínas de Homeodomínio/antagonistas & inibidores , Camundongos , Mutação , Ácido Retinoico 4 Hidroxilase , Transdução de Sinais/genética , Coluna Vertebral/embriologia , Tamoxifeno/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Proteínas Wnt/genética , Proteína Wnt3 , Proteína Wnt3ARESUMO
Cdx1, Cdx2 and Cdx4 encode homeodomain transcription factors that are involved in vertebral anterior-posterior (AP) patterning. Cdx1 and Cdx2 are also expressed in the intestinal epithelium during development, suggesting a role in this tissue. Intestinal defects have not been reported in Cdx1 null mutants, while Cdx2 null mutants die at embryonic day 3.5 (E3.5), thus precluding assessment of the null phenotype at later stages. To circumvent this latter shortcoming, we have used a conditional Cre-lox strategy to inactivate Cdx2 in the intestinal epithelium. Using this approach, we found that ablation of Cdx2 at E13.5 led to a transformation of the small intestine to a pyloric stomach-like identity, although the molecular nature of the underlying mesenchyme remained unchanged. Further analysis of Cdx1-Cdx2 double mutants suggests that Cdx1 does not play a critical role in the development of the small intestine, at least after E13.5.
Assuntos
Padronização Corporal/fisiologia , Proteínas de Homeodomínio/fisiologia , Mucosa Intestinal/embriologia , Fatores de Transcrição/fisiologia , Animais , Western Blotting , Fator de Transcrição CDX2 , Ciclo Celular/fisiologia , Hibridização In Situ , Mucosa Intestinal/citologia , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The intestinal epithelium is a unique tissue, serving both as a barrier against pathogens and to conduct the end digestion and adsorption of nutrients. As regards the former, the intestinal epithelium contains a diverse repertoire of immune cells, including a variety of resident lymphocytes, macrophages and dendritic cells. These cells serve a number of roles including mitigation of infection and to stimulate regeneration in response to damage. The transcription factor Cdx2, and to a lesser extent Cdx1, plays essential roles in intestinal homeostasis, and acts as a context-dependent tumour suppressor in colorectal cancer. Deletion of Cdx2 from the murine intestinal epithelium leads to macrophage infiltration resulting in a chronic inflammatory response. However the mechanisms by which Cdx2 loss evokes this response are poorly understood. To better understand this relationship, we used a conditional mouse model lacking all intestinal Cdx function to identify potential target genes which may contribute to this inflammatory phenotype. One such candidate encodes the histocompatability complex protein H2-T3, which functions to regulate intestinal iCD8α lymphocyte activity. We found that Cdx2 occupies the H3-T3 promoter in vivo and directly regulates its expression via a Cdx response element. Loss of Cdx function leads to a rapid and pronounced attenuation of H2-T3, followed by a decrease in iCD8α cell number, an increase in macrophage infiltration and activation of pro-inflammatory cascades. These findings suggest a previously unrecognized role for Cdx in intestinal homeostasis through H2-T3-dependent regulation of iCD8α cells.
Assuntos
Antígenos CD8/imunologia , Fator de Transcrição CDX2/fisiologia , Proteínas de Homeodomínio/fisiologia , Intestinos/imunologia , Linfócitos/imunologia , Macrófagos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Diferenciação Celular , Intestinos/patologia , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , Elementos de RespostaRESUMO
The majority of colorectal cancers harbor loss-of-function mutations in APC, a negative regulator of canonical Wnt signaling, leading to intestinal polyps that are predisposed to malignant progression. Comparable murine APC alleles also evoke intestinal polyps, which are typically confined to the small intestine and proximal colon, but do not progress to carcinoma in the absence of additional mutations. The Cdx transcription factors Cdx1 and Cdx2 are essential for homeostasis of the intestinal epithelium, and loss of Cdx2 has been associated with more aggressive subtypes of colorectal cancer in the human population. Consistent with this, concomitant loss of Cdx1 and Cdx2 in a murine APC mutant background leads to an increase in polyps throughout the intestinal tract. These polyps also exhibit a villous phenotype associated with the loss of EphrinB1. However, the basis for these outcomes is poorly understood. To further explore this, we modeled Cdx2 loss in SW480 colorectal cancer cells. We found that Cdx2 impacted Notch signaling in SW480 cells, and that EphrinB1 is a Notch target gene. As EphrinB1 loss also leads to a villus tumor phenotype, these findings evoke a mechanism by which Cdx2 impacts colorectal cancer via Notch-dependent EphrinB1 signaling.
Assuntos
Fator de Transcrição CDX2/genética , Efrina-B1/genética , Regulação da Expressão Gênica , Mucosa Intestinal/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Biomarcadores , Fator de Transcrição CDX2/metabolismo , Linhagem Celular Tumoral , Humanos , Mucosa Intestinal/patologia , Modelos Biológicos , Regiões Promotoras GenéticasRESUMO
Clonality is, at present, the only means by which the self-renewal potential of a given stem cell can be determined. To assess the clonality of human embryonic stem cells (hESC), a protocol involving seeding wells at low cell densities is commonly used to surmount poor cloning efficiencies. However, factors influencing the accuracy of such an assay have not been fully elucidated. Using clonogenic assays together with time-lapse microscopy, numerical analyses, and regulated gene expression strategies, we found that individual and collective cell movements are inherent properties of hESCs and that they markedly impact the accuracy of clonogenic assays. Analyses of cell motility using mean-square displacement and paired migration correlation indicated that cell movements become more straight-line or ballistic and less random-walk as separation distance decreases. Such motility-induced reaggregation (rather than a true clone) occurs approximately 70% of the time if the distance between two hESCs is <6.4 mum, and is not observed if the distance is >150 mum. Furthermore, newly formed small hESC colonies have a predisposition toward the formation of larger colonies through asymmetric colony expansion and movement, which would not accurately reflect self-renewal and proliferative activity of a true hESC clone. Notably, inhibition of Rho-associated kinase markedly upregulated hESC migration and reaggregation, producing considerable numbers of false-positive colonies. Conversely, E-cadherin upregulation significantly augmented hESC clonogenicity via improved survival of single hESCs without influencing cell motility. This work reveals that individual cell movement, asymmetric colony expansion, Rho-associated kinase, and E-cadherin all work together to influence hESC clonogenicity, and provides additional guidance for improvement of clonogenic assays in the analysis of hESC self-renewal.
Assuntos
Caderinas/metabolismo , Movimento Celular , Proliferação de Células , Células-Tronco Embrionárias/fisiologia , Quinases Associadas a rho/metabolismo , Algoritmos , Caderinas/genética , Técnicas de Cultura de Células/métodos , Linhagem Celular , Sobrevivência Celular/fisiologia , Simulação por Computador , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/enzimologia , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Microscopia de Fluorescência , Modelos Biológicos , Fatores de Tempo , Gravação em Vídeo , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genéticaRESUMO
The Cdx transcription factors regulate anterior-posterior (AP) vertebral patterning, at least in part, through direct regulation of Hox gene expression. Analysis of allelic series of Cdx mutant mice suggests functional overlap between these family members. However, the lack of a Cdx2 null mutant makes these analyses incomplete. Moreover, Hox proteins are sometimes redundant, making it difficult to discern whether Cdx members regulate identical Hox target genes in a redundant manner, or whether they regulate separate Hox genes which then converge on events related to vertebral patterning. To more directly assess this question, we developed a "knock in" model whereby Cdx2 was substituted for Cdx1. Consistent with functional redundancy Cdx2 "knock-in" mice exhibited perfect complementation of the Cdx1-null phenotype, as evidenced by the lack of skeletal defects or altered expression of Hox genes typically impacted by Cdx1 loss-of-function. It has been proposed that vertebral AP patterning is reliant on a gradient of the sum total of Cdx proteins, a posit that is consistent with functional redundancy between Cdx family members. To further assess this, we generated a gain-of-function model using BAC transgenesis to alter Cdx1 dosage. Cdx1 BAC transgenic mice overexpressed Cdx1 mRNA and protein, and fully complemented the Cdx1 null allele. However, gain of Cdx1 dosage via this BAC transgene in an otherwise wild type background had no discernible effects on vertebral patterning or Hox gene expression, suggesting that a moderate alteration in the Cdx protein gradient is of no consequence.
Assuntos
Padronização Corporal/fisiologia , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fator de Transcrição CDX2 , Embrião de Mamíferos/metabolismo , Proteínas de Homeodomínio/genética , Hibridização In Situ , Camundongos , Camundongos Knockout , Fatores de Transcrição/genéticaRESUMO
The SLP-76 family of immune cell-specific adaptors is composed of three distinct members named SLP-76, Blnk, and Clnk. They have been implicated in the signaling pathways coupled to immunoreceptors such as the antigen receptors and Fc receptors. Previous studies using gene-targeted mice and deficient cell lines showed that SLP-76 plays a central role in T-cell development and activation. Moreover, it is essential for normal mast cell and platelet activation. In contrast, Blnk is necessary for B-cell development and activation. While the precise function of Clnk is not known, it was reported that Clnk is selectively expressed in mast cells, natural killer (NK) cells, and previously activated T-cells. Moreover, ectopic expression of Clnk was shown to rescue T-cell receptor-mediated signal transduction in an SLP-76-deficient T-cell line, suggesting that, like its relatives, Clnk is involved in the positive regulation of immunoreceptor signaling. Stimulatory effects of Clnk on immunoreceptor signaling were also reported to occur in transfected B-cell and basophil leukemia cell lines. Herein, we attempted to address the physiological role of Clnk in immune cells by the generation of Clnk-deficient mice. The results of our studies demonstrated that Clnk is dispensable for normal differentiation and function of T cells, mast cells, and NK cells. Hence, unlike its relatives, Clnk is not essential for normal immune functions.