RESUMEN
Adult form and function are dependent upon the activity of specialized signaling centers that act early in development at the embryonic midline. These centers instruct the surrounding cells to adopt a positional fate and to form the patterned structures of the phylotypic embryo. Abnormalities in these processes have devastating consequences for the individual, as exemplified by holoprosencephaly in which anterior midline development fails, leading to structural defects of the brain and/or face. In the 25 years since the first association between human holoprosencephaly and the sonic hedgehog gene, a combination of human and animal genetic studies have enhanced our understanding of the genetic and embryonic causation of this congenital defect. Comparative biology has extended the holoprosencephaly network via the inclusion of gene mutations from multiple signaling pathways known to be required for anterior midline formation. It has also clarified aspects of holoprosencephaly causation, showing that it arises when a deleterious variant is present within a permissive genome, and that environmental factors, as well as embryonic stochasticity, influence the phenotypic outcome of the variant. More than two decades of research can now be distilled into a framework of embryonic and genetic causation. This framework means we are poised to move beyond our current understanding of variants in signaling pathway molecules. The challenges now at the forefront of holoprosencephaly research include deciphering how the mutation of genes involved in basic cell processes can also cause holoprosencephaly, determining the important constituents of the holoprosencephaly permissive genome, and identifying environmental compounds that promote holoprosencephaly. This article is categorized under: Congenital Diseases > Stem Cells and Development Congenital Diseases > Genetics/Genomics/Epigenetics Congenital Diseases > Molecular and Cellular Physiology Congenital Diseases > Environmental Factors.
Asunto(s)
Holoprosencefalia , Animales , Biología , Encéfalo , Proteínas Hedgehog/genética , Holoprosencefalia/genética , Humanos , Mamíferos/metabolismo , Ratones , Transducción de Señal/genéticaRESUMEN
The mechanisms of neural crest cell induction and specification are highly conserved among vertebrate model organisms, but how similar these mechanisms are in mammalian neural crest cell formation remains open to question. The zinc finger of the cerebellum 1 (ZIC1) transcription factor is considered a core component of the vertebrate gene regulatory network that specifies neural crest fate at the neural plate border. In mouse embryos, however, Zic1 mutation does not cause neural crest defects. Instead, we and others have shown that murine Zic2 and Zic5 mutate to give a neural crest phenotype. Here, we extend this knowledge by demonstrating that murine Zic3 is also required for, and co-operates with, Zic2 and Zic5 during mammalian neural crest specification. At the murine neural plate border (a region of high canonical WNT activity) ZIC2, ZIC3, and ZIC5 function as transcription factors to jointly activate the Foxd3 specifier gene. This function is promoted by SUMOylation of the ZIC proteins at a conserved lysine immediately N-terminal of the ZIC zinc finger domain. In contrast, in the lateral regions of the neurectoderm (a region of low canonical WNT activity) basal ZIC proteins act as co-repressors of WNT/TCF-mediated transcription. Our work provides a mechanism by which mammalian neural crest specification is restricted to the neural plate border. Furthermore, given that WNT signaling and SUMOylation are also features of non-mammalian neural crest specification, it suggests that mammalian neural crest induction shares broad conservation, but altered molecular detail, with chicken, zebrafish, and Xenopus neural crest induction.
Asunto(s)
Embrión de Mamíferos/embriología , Cresta Neural/metabolismo , Sumoilación , Factores de Transcripción/metabolismo , Transcripción Genética , Vía de Señalización Wnt , Animales , Embrión de Mamíferos/citología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Ratones , Ratones Transgénicos , Cresta Neural/citología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genéticaRESUMEN
In vivo function of CDK5 and Abl enzyme substrate 2 (Cables2), belonging to the Cables protein family, is unknown. Here, we found that targeted disruption of the entire Cables2 locus (Cables2d) caused growth retardation and enhanced apoptosis at the gastrulation stage and then induced embryonic lethality in mice. Comparative transcriptome analysis revealed disruption of Cables2, 50% down-regulation of Rps21 abutting on the Cables2 locus, and up-regulation of p53-target genes in Cables2d gastrulas. We further revealed the lethality phenotype in Rps21-deleted mice and unexpectedly, the exon 1-deleted Cables2 mice survived. Interestingly, chimeric mice derived from Cables2d ESCs carrying exogenous Cables2 and tetraploid wild-type embryo overcame gastrulation. These results suggest that the diminished expression of Rps21 and the completed lack of Cables2 expression are intricately involved in the embryonic lethality via the p53 pathway. This study sheds light on the importance of Cables2 locus in mouse embryonic development.
Asunto(s)
Proteínas de Ciclo Celular/genética , Gastrulación/genética , Expresión Génica , Proteínas Ribosómicas/genética , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos ICR , Fenotipo , Activación Transcripcional , Proteína p53 Supresora de Tumor/genética , Regulación hacia ArribaRESUMEN
Zinc finger of the cerebellum (Zic) proteins act as classic transcription factors to promote transcription of the Foxd3 gene during neural crest cell specification. Additionally, they can act as co-factors that bind proteins from the T-cell factor/lymphoid enhancing factor (TCF/LEF) family (TCFs) to repress WNT-ß-catenin-dependent transcription without contacting DNA. Here, we show that ZIC activity at the neural plate border is influenced by WNT-dependent SUMOylation. In the presence of high canonical WNT activity, a lysine residue within the highly conserved zinc finger N-terminally conserved (ZF-NC) domain of ZIC5 is SUMOylated, which reduces formation of the ZIC-TCF co-repressor complex and shifts the balance towards transcription factor function. The modification is crucial in vivo, as a ZIC5 SUMO-incompetent mouse strain exhibits neural crest specification defects. This work reveals the function of the ZF-NC domain within ZIC, provides in vivo validation of target protein SUMOylation and demonstrates that WNT-ß-catenin signalling directs transcription at non-TCF DNA-binding sites. Furthermore, it can explain how WNT signals convert a broad region of Zic ectodermal expression into a restricted region of neural crest cell specification.
Asunto(s)
Cresta Neural , Sumoilación , Animales , Diferenciación Celular , Ratones , Cresta Neural/metabolismo , Factores de Transcripción TCF/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
The ZIC proteins are a family of transcription regulators with a well-defined zinc finger DNA-binding domain and there is evidence that they elicit functional DNA binding at a ZIC DNA binding site. Little is known, however, regarding domains within ZIC proteins that confer trans-activation or -repression. To address this question, a new cell-based trans-activation assay system suitable for ZIC proteins in HEK293T cells was constructed. This identified two previously unannotated evolutionarily conserved regions of ZIC3 that are necessary for trans-activation. These domains are found in all Subclass A ZIC proteins, but not in the Subclass B proteins. Additionally, the Subclass B proteins fail to elicit functional binding at a multimerised ZIC DNA binding site. All ZIC proteins, however, exhibit functional binding when the ZIC DNA binding site is embedded in a multiple transcription factor locus derived from ZIC target genes in the mouse genome. This ability is due to several domains, some of which are found in all ZIC proteins, that exhibit context dependent trans-activation or -repression activity. This knowledge is valuable for assessing the likely pathogenicity of variant ZIC proteins associated with human disorders and for determining factors that influence functional transcription factor binding.
Asunto(s)
Regulación de la Expresión Génica , Genes Reporteros , Elementos de Respuesta , Factores de Transcripción , Transcripción Genética , Células HEK293 , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Expanded carrier screening (ECS) for recessive monogenic diseases requires prior knowledge of genomic variation, including DNA variants that cause disease. The composition of pathogenic variants differs greatly among human populations, but historically, research about monogenic diseases has focused mainly on people with European ancestry. By comparison, less is known about pathogenic DNA variants in people from other parts of the world. Consequently, inclusion of currently underrepresented Indigenous and other minority population groups in genomic research is essential to enable equitable outcomes in ECS and other areas of genomic medicine. Here, we discuss this issue in relation to the implementation of ECS in Australia, which is currently being evaluated as part of the national Government's Genomics Health Futures Mission. We argue that significant effort is required to build an evidence base and genomic reference data so that ECS can bring significant clinical benefit for many Aboriginal and/or Torres Strait Islander Australians. These efforts are essential steps to achieving the Australian Government's objectives and its commitment "to leveraging the benefits of genomics in the health system for all Australians." They require culturally safe, community-led research and community involvement embedded within national health and medical genomics programs to ensure that new knowledge is integrated into medicine and health services in ways that address the specific and articulated cultural and health needs of Indigenous people. Until this occurs, people who do not have European ancestry are at risk of being, in relative terms, further disadvantaged.
Asunto(s)
Metagenómica/métodos , Grupos de Población/genética , Australia , Variación Genética/genética , HumanosRESUMEN
Experiments that visualize gene expression in intact tissues or organisms are fundamental to studies of gene function. These experiments, called in situ hybridization, require the production of a riboprobe, which is a labeled antisense RNA corresponding to a particular gene. The most commonly used system for visualizing gene expression via in situ hybridization is the incorporation of a digoxigenin label into an in vitro-transcribed RNA probe. After hybridization of the riboprobe to a target mRNA, its location can be detected via a high-affinity α-digoxigenin antibody conjugated to an alkaline-phosphatase enzyme. The article describes the design and production of digoxigenin-labeled riboprobes transcribed in vitro from template DNA (either plasmid or PCR amplicon). These riboprobes are suitable for use in tissue and whole-mount in situ hybridization protocols. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Plasmid-derived riboprobes Alternate Protocol: PCR-derived riboprobes Basic Protocol 2: Riboprobe synthesis with DIG label.
Asunto(s)
Digoxigenina , Hibridación in Situ/métodos , Sondas ARN/biosíntesis , Animales , Embrión de Mamíferos , Fluoresceína , Ratones , Coloración y Etiquetado/métodosRESUMEN
Understanding RNA expression in space and time is a key initial step in dissecting gene function. The ability to visualize gene expression in whole-tissue or whole-specimen preparations, called in situ hybridization (ISH), was first developed 50 years ago. Two decades later, these protocols were adapted to establish robust methods for whole-mount ISH to murine embryos. The precise protocols vary somewhat between early-gestation and mid-gestation mouse embryos; the protocol presented here is optimal for use with post-implantation stage mouse embryos (stages 5.5-9.5 dpc). Routine uses of whole-mount ISH include documenting the wild-type expression pattern of individual genes and comparison of the expression pattern of signature genes (i.e., those that identify particular cells and tissues within an embryo) between wild-type and mutant embryos as part of a phenotyping experiment. This technique remains a mainstay of developmental biology studies and complements the massively parallel assessment of gene expression from dissociated tissues and cells via RNA-sequencing techniques. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Dissection of post-implantation (5.5-9.5 dpc) murine embryos Basic Protocol 2: Whole-mount in situ hybridization in post-implantation embryos Basic Protocol 3: Visualization of post-WMISH embryos Support Protocol 1: Creation of siliconized glass pipettes Support Protocol 2: Creation of embryo powder.
Asunto(s)
Desarrollo Embrionario , Hibridación in Situ/métodos , Animales , Embrión de Mamíferos , Expresión Génica , Edad Gestacional , RatonesRESUMEN
Quantitative reverse transcriptase PCR (RT-qPCR), a powerful and efficient means of rapidly comparing gene expression between experimental conditions, is routinely used as a phenotyping tool in developmental biology. The accurate comparison of gene expression across multiple embryonic stages requires normalisation to reference genes that have stable expression across the time points to be examined. As the embryo and its constituent tissues undergo rapid growth and differentiation during development, reference genes known to be stable across some time points cannot be assumed to be stable across all developmental stages. The immediate post-implantation events of gastrulation and patterning are characterised by a rapid expansion in cell number and increasing specialisation of cells. The optimal reference genes for comparative gene expression studies at these specific stages have not been experimentally identified. In this study, the expression of five commonly used reference genes (H2afz, Ubc, Actb, Tbp and Gapdh) was measured across murine gastrulation and patterning (6.5-9.5 dpc) and analysed with the normalisation tools geNorm, Bestkeeper and Normfinder. The results, validated by RT-qPCR analysis of two genes with well-documented expression patterns across these stages, indicated the best strategy for RT-qPCR studies spanning murine gastrulation and patterning utilises the concurrent reference genes H2afz and Ubc.
Asunto(s)
Tipificación del Cuerpo/genética , Gastrulación/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Animales , Femenino , Perfilación de la Expresión Génica , Genes del Desarrollo/genética , Ratones , Ratones Endogámicos C3H , Reproducibilidad de los Resultados , Programas InformáticosRESUMEN
The five murine Zic genes encode multifunctional transcriptional regulator proteins important for a large number of processes during embryonic development. The genes and proteins are highly conserved with respect to the orthologous human genes, an attribute evidently mirrored by functional conservation, since the murine and human genes mutate to give the same phenotypes. Each ZIC protein contains a zinc finger domain that participates in both protein-DNA and protein-protein interactions. The ZIC proteins are capable of interacting with the key transcriptional mediators of the SHH, WNT and NODAL signalling pathways as well as with components of the transcriptional machinery and chromatin-modifying complexes. It is possible that this diverse range of protein partners underlies characteristics uncovered by mutagenesis and phenotyping of the murine Zic genes. These features include redundant and unique roles for ZIC proteins, regulatory interdependencies amongst family members and pleiotropic Zic gene function. Future investigations into the complex nature of the Zic gene family activity should be facilitated by recent advances in genome engineering and functional genomics.
Asunto(s)
Familia de Multigenes/fisiología , Transducción de Señal/fisiología , Factores de Transcripción , Transcripción Genética/fisiología , Dedos de Zinc/fisiología , Animales , Ratones , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The ZIC2 transcription factor is one of the most commonly mutated genes in Holoprosencephaly (HPE) probands. HPE is a severe congenital defect of forebrain development which occurs when the cerebral hemispheres fail to separate during the early stages of organogenesis and is typically associated with mispatterning of the embryonic midline. Recent study of genotype-phenotype correlations in HPE cases has defined distinctive features of ZIC2-associated HPE presentation and genetics, revealing that ZIC2 mutation does not produce the craniofacial abnormalities generally thought to characterise HPE but leads to a range of non-forebrain phenotypes. Furthermore, the studies confirm the extent of ZIC2 allelic heterogeneity and that pathogenic variants of ZIC2 are associated with both classic and middle interhemispheric variant (MIHV) HPE which arise from defective ventral and dorsal forebrain patterning, respectively. An allelic series of mouse mutants has helped to delineate the cellular and molecular mechanisms by which one gene leads to defects in these related but distinct embryological processes.
Asunto(s)
Alelos , Heterocigoto , Holoprosencefalia , Mutación , Proteínas Nucleares , Factores de Transcripción , Animales , Holoprosencefalia/embriología , Holoprosencefalia/genética , Humanos , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The ZIC2 transcription factor is one of the genes most commonly mutated in Holoprosencephaly (HPE) probands. Studies in cultured cell lines and mice have shown a loss of ZIC2 function is the pathogenic mechanism but the molecular details of this ZIC2 requirement remain elusive. HPE arises when signals that direct morphological and fate changes in the developing brain and facial primordia are not sent or received. One critical signal is sent from the prechordal plate (PrCP) which develops beneath the ventral forebrain. An intact NODAL signal transduction pathway and functional ZIC2 are both required for PrCP establishment. We now show that ZIC2 acts downstream of the NODAL signal during PrCP development. ZIC2 physically interacts with SMAD2 and SMAD3, the receptor activated proteins that control transcription in a NODAL dependent manner. Together SMAD3 and ZIC2 regulate FOXA2 transcription in cultured cells and Zic2 also controls the foxA2 expression during Xenopus development. Variant forms of the ZIC2 protein, associated with HPE in man or mouse, are deficient in their ability to influence SMAD-dependent transcription. These findings reveal a new mechanism of NODAL signal transduction in the mammalian node and provide the first molecular explanation of how ZIC2 loss-of-function precipitates HPE.
Asunto(s)
Factor Nuclear 3-beta del Hepatocito/genética , Holoprosencefalia/genética , Proteína Nodal/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Xenopus laevis/genética , Animales , Factores de Transcripción Forkhead/biosíntesis , Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 3-beta del Hepatocito/biosíntesis , Holoprosencefalia/fisiopatología , Humanos , Masculino , Ratones , Mutación , Proteína Nodal/metabolismo , Transducción de Señal/genética , Proteína Smad2/genética , Proteína smad3/genética , Xenopus laevis/crecimiento & desarrolloRESUMEN
The cytoskeletal protein Flightless (Flii) is a negative regulator of wound healing. Upregulation of Flii is associated with impaired migration, proliferation and adhesion of both fibroblasts and keratinocytes. Importantly, Flii translocates from the cytoplasm to the nucleus in response to wounding in fibroblasts but not keratinocytes. This cell-specific nuclear translocation of Flii suggests that Flii may directly regulate gene expression in fibroblasts, providing one potential mechanism of action for Flii in the wound healing response. To determine whether the tissue-specific upregulation of Flii in fibroblasts was important for the observed inhibitory effects of Flii on wound healing, an inducible fibroblast-specific Flii overexpressing mouse model was generated. The inducible ROSA26 system allowed the overexpression of Flii in a temporal and tissue-specific manner in response to tamoxifen treatment. Wound healing in the inducible mice was impaired, with wounds at day 7 postwounding significantly larger than those from non-inducible controls. There was also reduced collagen maturation, increased myofibroblast infiltration and elevated inflammation. The impaired healing response was similar in magnitude to that observed in mice with non-tissue-specific upregulation of Flii suggesting that fibroblast-derived Flii may have an important role in the wound healing response.
Asunto(s)
Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Piel/metabolismo , Cicatrización de Heridas/genética , Animales , Antineoplásicos Hormonales/farmacología , Proteínas Portadoras , Movimiento Celular , Proliferación Celular , Células Cultivadas , Colágeno/ultraestructura , Fibroblastos/efectos de los fármacos , Fibroblastos/ultraestructura , Expresión Génica/efectos de los fármacos , Ratones , Proteínas de Microfilamentos , Modelos Animales , Recombinación Genética/efectos de los fármacos , Piel/efectos de los fármacos , Piel/lesiones , Tamoxifeno/farmacología , Transactivadores , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Cicatrización de Heridas/efectos de los fármacosRESUMEN
BACKGROUND: Murine Zic genes (Zic1-5) are expressed in the dorsal hindbrain and in periotic mesenchyme (POM) adjacent to the developing inner ear. Zic genes are involved in developmental signaling pathways in many organ systems, including the ear, although their exact roles haven't been fully elucidated. This report examines the role of Zic1, Zic2, and Zic4 during inner ear development in mouse mutants in which these Zic genes are affected. RESULTS: Zic1/Zic4 double mutants don't exhibit any apparent defects in inner ear morphology. By contrast, inner ears from Zic2(kd/kd) and Zic2(Ku/Ku) mutants have severe but variable morphological defects in endolymphatic duct/sac and semicircular canal formation and in cochlear extension in the inner ear. Analysis of otocyst patterning in the Zic2(Ku/Ku) mutants by in situ hybridization showed changes in the expression patterns of Gbx2 and Pax2. CONCLUSIONS: The experiments provide the first genetic evidence that the Zic genes are required for morphogenesis of the inner ear. Zic2 loss-of-function doesn't prevent initial otocyst patterning but leads to molecular abnormalities concomitant with morphogenesis of the endolymphatic duct. Functional hearing deficits often accompany inner ear dysmorphologies, making Zic2 a novel candidate gene for ongoing efforts to identify the genetic basis of human hearing loss.
Asunto(s)
Oído Interno/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/genética , Morfogénesis/fisiología , Fenotipo , Transducción de Señal/genética , Factores de Transcripción/genética , Animales , Proteínas de Homeodominio/metabolismo , Hibridación in Situ , Ratones , Mutación/genética , Factores de Transcripción/metabolismoRESUMEN
The first molecular herald of organ asymmetry during murine embryogenesis is found at the periphery of the node in early-somite stage embryos. Asymmetric gene expression and calcium accumulation at the node occurs in response to a left-ward flow of extracellular fluid across the node, generated by motile cilia within the pit of the node and likely sensed by immotile cilia in the periphery of the node. The ciliation of node cells is controlled by a cascade of node-restricted transcription factor activity during mid-late gastrulation. Mutation of the murine Zic2 transcription factor is associated with random cardiac situs and a loss of asymmetric gene expression at the early-somite node and in the lateral plate. Zic2 is not expressed in these regions but is transiently expressed in the mid-late gastrula node at the time of ciliogenesis. The cilia of the node are overtly abnormal in Zic2 mutant embryos being dysmorphic and short relative to wild-type littermates. The expression of the Noto, Rfx3, and Foxj1 transcription factors known to regulate ciliogenesis is greatly depleted in the mid-gastrula node of mutants, as is the expression of the Pkd1l1 gene required for cilia function. Zic2 appears to be a component of the gene regulatory network that drives ciliation of node cells during gastrulation.
Asunto(s)
Cilios/genética , Sistema de Conducción Cardíaco/embriología , Sistema de Conducción Cardíaco/metabolismo , Corazón/embriología , Organogénesis/fisiología , Factores de Transcripción/genética , Animales , Tipificación del Cuerpo/fisiología , Desarrollo Embrionario/fisiología , Endodermo/embriología , Endodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Noqueados , Morfogénesis/fisiología , Mutación , Proteína Nodal/metabolismoRESUMEN
Development of an intact epidermis is critical for maintaining the integrity of the skin. Patients with epidermolysis bullosa (EB) experience multiple erosions, which breach the epidermal barrier and lead to increased microbial colocalization of wounds, infections and sepsis. The cytoskeletal protein Flightless I (Flii) is a known regulator of both development and wound healing. Using Flii(+/-), WT and Flii(Tg/Tg) mice, we investigated the effect of altering Flii levels in embryos and adult mice on the development of the epidermal barrier and, consequently, how this affects the integrity of the skin in EB. Flii over-expression resulted in delayed formation of the epidermal barrier in embryos and decreased expression of tight junction (TJ) proteins Claudin-1 and ZO-2. Increased intercellular space and transepidermal water loss was observed in Flii(Tg)(/Tg) adult mouse skin, while Flii(Tg/Tg) keratinocytes showed altered TJ protein localization and reduced transepithelial resistance. Flii is increased in the blistered skin of patients with EB, and over-expression of Flii in experimental EBA showed impaired Claudin-1 and -4 TJ protein expression and delayed recovery of functional barrier post-blistering. Immunoprecipitation confirmed Flii associated with TJ proteins and in vivo actin assays showed that the effect of Flii on actin polymerization underpinned the impaired barrier function observed in Flii(Tg/Tg) mice. These results therefore demonstrate an important role for Flii in the development and regulation of the epidermal barrier, which may contribute to the impaired healing and skin fragility of EB patients.
Asunto(s)
Vesícula/metabolismo , Epidermis/metabolismo , Epidermólisis Ampollosa/metabolismo , Proteína Proto-Oncogénica c-fli-1/deficiencia , Proteína Proto-Oncogénica c-fli-1/metabolismo , Cicatrización de Heridas , Actinas/metabolismo , Animales , Vesícula/genética , Vesícula/patología , Células Cultivadas , Modelos Animales de Enfermedad , Impedancia Eléctrica , Epidermis/patología , Epidermólisis Ampollosa/genética , Epidermólisis Ampollosa/patología , Genotipo , Humanos , Queratinocitos/metabolismo , Queratinocitos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Permeabilidad , Fenotipo , Polimerizacion , Proteína Proto-Oncogénica c-fli-1/genética , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/metabolismo , Uniones Estrechas/patología , Factores de Tiempo , Regulación hacia Arriba , Pérdida Insensible de AguaRESUMEN
AIMS/HYPOTHESIS: Skin lesions and ulcerations are severe complications of diabetes that often result in leg amputations. In this study we investigated the function of the cytoskeletal protein flightless I (FLII) in diabetic wound healing. We hypothesised that overexpression of FLII would have a negative effect on diabetic wound closure and modulation of this protein using specific FLII-neutralising antibodies (FnAb) would enhance cellular proliferation, migration and angiogenesis within the diabetic wound. METHODS: Using a streptozotocin-induced model of diabetes we investigated the effect of altered FLII levels through Flii genetic knockdown, overexpression or treatment with FnAb on wound healing. Diabetic wounds were assessed using histology, immunohistochemistry and biochemical analysis. In vitro and in vivo assays of angiogenesis were used to assess the angiogenic response. RESULTS: FLII levels were elevated in the wounds of both diabetic mice and humans. Reduction in the level of FLII improved healing of murine diabetic wounds and promoted a robust pro-angiogenic response with significantly elevated von Willebrand factor (vWF) and vascular endothelial growth factor (VEGF)-positive endothelial cell infiltration. Diabetic mouse wounds treated intradermally with FnAb showed improved healing and a significantly increased rate of re-epithelialisation. FnAb improved the angiogenic response through enhanced formation of capillary tubes and functional neovasculature. Reducing the level of FLII led to increased numbers of mature blood vessels, increased recruitment of smooth muscle actin-α-positive cells and improved tight junction formation. CONCLUSIONS/INTERPRETATION: Reducing the level of FLII in a wound may be a potential therapeutic approach for the treatment of diabetic foot ulcers.
Asunto(s)
Proteínas del Citoesqueleto/farmacología , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/patología , Angiopatías Diabéticas/patología , Piel/patología , Cicatrización de Heridas/inmunología , Inductores de la Angiogénesis , Animales , Anticuerpos Neutralizantes/metabolismo , Proteínas Portadoras , Proliferación Celular , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Tipo 1/inmunología , Angiopatías Diabéticas/inmunología , Femenino , Humanos , Inmunohistoquímica , Inflamación , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas de Microfilamentos , Piel/lesiones , Transactivadores , Úlcera/patologíaRESUMEN
Impaired wound healing and ulceration represent a serious complication of both type 1 and type 2 diabetes. Cytoskeletal protein Flightless I (Flii) is an important inhibitor of wound repair, and reduced Flii gene expression in fibroblasts increased migration, proliferation, and adhesion. As such it has the ability to influence all phases of wound healing including inflammation, remodelling and angiogenesis. Flii has the potential to modulate inflammation through its interaction with MyD88 which it an adaptor protein for TLR4. To assess the effect of Flii on the inflammatory response of diabetic wounds, we used a murine model of streptozocin-induced diabetes and Flii genetic mice. Increased levels of Flii were detected in Flii transgenic murine wounds resulting in impaired healing which was exacerbated when diabetes was induced. When Flii levels were reduced in diabetic wounds of Flii-deficient mice, healing was improved and decreased levels of TLR4 were observed. In contrast, increasing the level of Flii in diabetic mouse wounds led to increased TLR4 and NF- κ B production. Treatment of murine diabetic wounds with neutralising antibodies to Flii led to an improvement in healing with decreased expression of TLR4. Decreasing the level of Flii in diabetic wounds may therefore reduce the inflammatory response and improve healing.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Complicaciones de la Diabetes/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Proteínas Portadoras , Proteínas del Citoesqueleto/genética , Complicaciones de la Diabetes/genética , Complicaciones de la Diabetes/patología , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Inflamación/genética , Inflamación/metabolismo , Ratones , Proteínas de Microfilamentos , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Receptor Toll-Like 4/genética , Transactivadores , Cicatrización de Heridas/genética , Heridas y Lesiones/inducido químicamenteRESUMEN
Since its development in the 1970's, whole embryo culture (WEC) has provided an important method of growing and observing murine embryos ex utero. During WEC, embryos are immersed in a combination of rat serum and cell culture media, and supplied with heat and appropriate mixtures of CO2 and oxygen that mimic growth conditions in utero. One significant factor limiting the widespread use of WEC is the perception that commercially produced rat serum is inadequate to support normal rates of embryonic growth and development. Conversely, production of serum 'in-house' is technically demanding, time-consuming and expensive. The current study aimed to identify a WEC medium comprising commercially manufactured rat serum that would produce cultured embryos of comparable standard to those grown in utero. A mixed culture medium, composed of 50% commercial rat serum and 50% F12 Ham's cell culture medium with an N-2 neuronal cell growth supplement, was shown to support both a rate of growth, and the development of a range of features comparable to that which normally occur in vivo. Furthermore, the F12 (N-2) supplemented rat serum displayed a very low propensity to induce morphological abnormalities during the culture period. The study establishes a novel method of successful WEC using readily available commercial reagents and should enable the broader use of WEC.