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1.
PLoS One ; 12(10): e0186137, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29059207

RESUMO

Human malignant mesothelioma is a chemoresistant tumour that develops from mesothelial cells, commonly associated with asbestos exposure. Malignant mesothelioma incidence rates in European countries are still rising and Australia has one of the highest burdens of malignant mesothelioma on a population basis in the world. Therapy using systemic delivery of free cytotoxic agents is associated with many undesirable side effects due to non-selectivity, and is thus dose-limited which limits its therapeutic potential. Therefore, increasing the selectivity of anti-cancer agents has the potential to dramatically enhance drug efficacy and reduce toxicity. EnGeneIC Dream Vectors (EDV) are antibody-targeted nanocells which can be loaded with cytotoxic drugs and delivered to specific cancer cells via bispecific antibodies (BsAbs) which target the EDV and a cancer cell-specific receptor, simultaneously. BsAbs were designed to target doxorubicin-loaded EDVs to cancer cells via cell surface mesothelin (MSLN). Flow cytometry was used to investigate cell binding and induction of apoptosis, and confocal microscopy to visualize internalization. Mouse xenograft models were used to assess anti-tumour effects in vivo, followed by immunohistochemistry for ex vivo evaluation of proliferation and necrosis. BsAb-targeted, doxorubicin-loaded EDVs were able to bind to and internalize within mesothelioma cells in vitro via MSLN receptors and induce apoptosis. In mice xenografts, the BsAb-targeted, doxorubicin-loaded EDVs suppressed the tumour growth and also decreased cell proliferation. Thus, the use of MSLN-specific antibodies to deliver encapsulated doxorubicin can provide a novel and alternative modality for treatment of mesothelioma.


Assuntos
Proliferação de Células , Mesotelioma/patologia , Receptores de Superfície Celular/metabolismo , Animais , Humanos , Mesotelina , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto
2.
J Neurosci ; 34(39): 13033-8, 2014 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-25253850

RESUMO

The role of the p75 neurotrophin receptor (p75(NTR)) in adult cholinergic basal forebrain (cBF) neurons is unclear due to conflicting results from previous studies and to limitations of existing p75(NTR)-knock-out mouse models. In the present study we used a novel conditional knock-out line (ChAT-cre p75(in/in)) to assess the role of p75(NTR) in the cBF by eliminating p75(NTR) in choline acetyl-transferase-expressing cells. We show that the absence of p75(NTR) results in a lasting increase in cBF cell number, cell size, and cholinergic innervation to the cortex. Analysis of adult ChAT-cre p75(in/in) mice revealed that mutant animals show a similar loss of cBF neurons with age to that observed in wild-type animals, indicating that p75(NTR) does not play a significant role in mediating this age-related decline in cBF neuronal number. However, the increased cholinergic axonal innervation of the cortex, but not the hippocampus, corresponded to alterations in idiothetic but not allothetic navigation. These findings support a role for p75(NTR)-mediated regulation of cholinergic-dependent cognitive function, and suggest that the variability in previous reports of cBF neuron number may stem from limited spatial and temporal control of p75(NTR) expression in existing knock-out models.


Assuntos
Neurônios Colinérgicos/metabolismo , Prosencéfalo/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Neurônios Colinérgicos/fisiologia , Cognição , Feminino , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Prosencéfalo/citologia , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/fisiologia , Receptores de Fator de Crescimento Neural/genética , Transmissão Sináptica
3.
Dev Cell ; 29(2): 188-202, 2014 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-24780737

RESUMO

Although kidneys of equal size can vary 10-fold in nephron number at birth, discovering what regulates such variation has been hampered by a lack of quantitative parameters defining kidney development. Here we report a comprehensive, quantitative, multiscale analysis of mammalian kidney development in which we measure changes in cell number, compartment volumes, and cellular dynamics across the entirety of organogenesis, focusing on two key nephrogenic progenitor populations: the ureteric epithelium and the cap mesenchyme. In doing so, we describe a discontinuous developmental program governed by dynamic changes in interactions between these key cellular populations occurring within a previously unappreciated structurally stereotypic organ architecture. We also illustrate the application of this approach to the detection of a subtle mutant phenotype. This baseline program of kidney morphogenesis provides a framework for assessing genetic and environmental developmental perturbation and will serve as a gold standard for the analysis of other organs.


Assuntos
Rim/embriologia , Néfrons/embriologia , Ureter/embriologia , Urotélio/embriologia , Animais , Contagem de Células , Células-Tronco Embrionárias/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Rim/citologia , Rim/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Néfrons/citologia , Néfrons/fisiologia , Fenótipo , Gravidez , Ureter/citologia , Ureter/fisiologia , Urotélio/citologia , Urotélio/fisiologia
4.
Respir Res ; 14: 41, 2013 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-23560845

RESUMO

BACKGROUND: Macrophages are traditionally associated with inflammation and host defence, however a greater understanding of macrophage heterogeneity is revealing their essential roles in non-immune functions such as development, homeostasis and regeneration. In organs including the brain, kidney, mammary gland and pancreas, macrophages reside in large numbers and provide essential regulatory functions that shape organ development and maturation. However, the role of macrophages in lung development and the potential implications of macrophage modulation in the promotion of lung maturation have not yet been ascertained. METHODS: Embryonic day (E)12.5 mouse lungs were cultured as explants and macrophages associated with branching morphogenesis were visualised by wholemount immunofluorescence microscopy. Postnatal lung development and the correlation with macrophage number and phenotype were examined using Colony-stimulating factor-1 receptor-enhanced green fluorescent protein (Csf1r-EGFP) reporter mice. Structural histological examination was complemented with whole-body plethysmography assessment of postnatal lung functional maturation over time.Flow cytometry, real-time (q)PCR and immunofluorescence microscopy were performed to characterise macrophage number, phenotype and localisation in the lung during postnatal development. To assess the impact of developmental macrophage modulation, CSF-1 was administered to neonatal mice at postnatal day (P)1, 2 and 3, and lung macrophage number and phenotype were assessed at P5. EGFP transgene expression and in situ hybridisation was performed to assess CSF-1R location in the developing lung. RESULTS: Macrophages in embryonic lungs were abundant and densely located within branch points during branching morphogenesis. During postnatal development, structural and functional maturation of the lung was associated with an increase in lung macrophage number. In particular, the period of alveolarisation from P14-21 was associated with increased number of Csf1r-EGFP+ macrophages and upregulated expression of Arginase 1 (Arg1), Mannose receptor 1 (Mrc1) and Chemokine C-C motif ligand 17 (Ccl17), indicative of an M2 or tissue remodelling macrophage phenotype. Administration of CSF-1 to neonatal mice increased trophic macrophages during development and was associated with increased expression of the M2-associated gene Found in inflammatory zone (Fizz)1 and the growth regulator Insulin-like growth factor (Igf)1. The effects of CSF-1 were identified as macrophage-mediated, as the CSF-1R was found to be exclusively expressed on interstitial myeloid cells. CONCLUSIONS: This study identifies the presence of CSF-1R+ M2-polarised macrophages localising to sites of branching morphogenesis and increasing in number during the alveolarisation stage of normal lung development. Improved understanding of the role of macrophages in lung developmental regulation has clinical relevance for addressing neonatal inflammatory perturbation of development and highlights macrophage modulation as a potential intervention to promote lung development.


Assuntos
Desenvolvimento Embrionário/fisiologia , Macrófagos/citologia , Macrófagos/fisiologia , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/embriologia , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Animais , Polaridade Celular/fisiologia , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Alvéolos Pulmonares/crescimento & desenvolvimento
5.
Methods Mol Biol ; 886: 223-39, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22639265

RESUMO

Studies into the molecular basis of morphogenesis frequently begin with investigations into gene expression across time and cell type in that organ. One of the most anatomically informative approaches to such studies is the use of in situ hybridization, either of intact or histologically sectioned tissues. Here, we describe the optimization of this approach for use in the temporal and spatial analysis of gene expression in the urogenital system, from embryonic development to the postnatal period. The methods described are applicable for high throughput analysis of large gene sets. As such, ISH has become a powerful technique for gene expression profiling and is valuable for the validation of profiling analyses performed using other approaches such as microarrays.


Assuntos
Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ/métodos , RNA Mensageiro/genética , Sistema Urogenital/crescimento & desenvolvimento , Animais , Rim/embriologia , Rim/crescimento & desenvolvimento , Rim/metabolismo , Camundongos , RNA Mensageiro/isolamento & purificação , Sistema Urogenital/embriologia , Sistema Urogenital/metabolismo
6.
Development ; 139(10): 1863-73, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22510988

RESUMO

Lengthy developmental programs generate cell diversity within an organotypic framework, enabling the later physiological actions of each organ system. Cell identity, cell diversity and cell function are determined by cell type-specific transcriptional programs; consequently, transcriptional regulatory factors are useful markers of emerging cellular complexity, and their expression patterns provide insights into the regulatory mechanisms at play. We performed a comprehensive genome-scale in situ expression screen of 921 transcriptional regulators in the developing mammalian urogenital system. Focusing on the kidney, analysis of regional-specific expression patterns identified novel markers and cell types associated with development and patterning of the urinary system. Furthermore, promoter analysis of synexpressed genes predicts transcriptional control mechanisms that regulate cell differentiation. The annotated informational resource (www.gudmap.org) will facilitate functional analysis of the mammalian kidney and provides useful information for the generation of novel genetic tools to manipulate emerging cell populations.


Assuntos
Sistema Urogenital/metabolismo , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hibridização In Situ , Rim/metabolismo , Camundongos
7.
Dev Biol ; 360(1): 110-22, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-21963425

RESUMO

Nephron number in the mammalian kidney is known to vary dramatically, with postnatal renal function directly influenced by nephron complement. What determines final nephron number is poorly understood but nephron formation in the mouse kidney ceases within the first few days after birth, presumably due to the loss of all remaining nephron progenitors via epithelial differentiation. What initiates this event is not known. Indeed, whether nephron formation occurs in the same way at this time as during embryonic development has also not been examined. In this study, we investigate the key cellular compartments involved in nephron formation; the ureteric tip, cap mesenchyme and early nephrons; from postnatal day (P) 0 to 6 in the mouse. High resolution analyses of gene and protein expression indicate that loss of nephron progenitors precedes loss of ureteric tip identity, but show spatial shifts in the expression of cap mesenchyme genes during this time. In addition, cap mesenchymal volume and rate of proliferation decline prior to birth. Section-based 3D modeling and Optical Projection Tomography revealed a burst of ectopic nephron induction, with the formation of multiple (up to 5) nephrons per ureteric tip evident from P2. While the distal-proximal patterning of these nephrons occurred normally, their spatial relationship with the ureteric compartment was altered. We propose that this phase of nephron formation represents an acceleration of differentiation within the cap mesenchyme due to a displacement of signals within the nephrogenic niche.


Assuntos
Rim/crescimento & desenvolvimento , Néfrons/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Ciclina D1/genética , Ciclina D1/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imageamento Tridimensional , Rim/embriologia , Rim/fisiologia , Camundongos , Modelos Anatômicos , Modelos Biológicos , Néfrons/embriologia , Néfrons/fisiologia , Organogênese/genética , Organogênese/fisiologia , Gravidez , Tomografia Óptica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ureter/embriologia , Ureter/crescimento & desenvolvimento
8.
PLoS One ; 6(9): e24640, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21931791

RESUMO

BACKGROUND: The podocyte is a remarkable cell type, which encases the capillaries of the kidney glomerulus. Although mesodermal in origin it sends out axonal like projections that wrap around the capillaries. These extend yet finer projections, the foot processes, which interdigitate, leaving between them the slit diaphragms, through which the glomerular filtrate must pass. The podocytes are a subject of keen interest because of their key roles in kidney development and disease. METHODOLOGY/PRINCIPAL FINDINGS: In this report we identified and characterized a novel transgenic mouse line, MafB-GFP, which specifically marked the kidney podocytes from a very early stage of development. These mice were then used to facilitate the fluorescent activated cell sorting based purification of podocytes from embryos at E13.5 and E15.5, as well as adults. Microarrays were then used to globally define the gene expression states of podocytes at these different developmental stages. A remarkable picture emerged, identifying the multiple sets of genes that establish the neuronal, muscle, and phagocytic properties of podocytes. The complete combinatorial code of transcription factors that create the podocyte was characterized, and the global lists of growth factors and receptors they express were defined. CONCLUSIONS/SIGNIFICANCE: The complete molecular character of the in vivo podocyte is established for the first time. The active molecular functions and biological processes further define their unique combination of features. The results provide a resource atlas of gene expression patterns of developing and adult podocytes that will help to guide further research of these incredible cells.


Assuntos
Podócitos/metabolismo , Animais , Células Cultivadas , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos
9.
BMC Genomics ; 12: 441, 2011 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-21888672

RESUMO

BACKGROUND: The developing mouse kidney is currently the best-characterized model of organogenesis at a transcriptional level. Detailed spatial maps have been generated for gene expression profiling combined with systematic in situ screening. These studies, however, fall short of capturing the transcriptional complexity arising from each locus due to the limited scope of microarray-based technology, which is largely based on "gene-centric" models. RESULTS: To address this, the polyadenylated RNA and microRNA transcriptomes of the 15.5 dpc mouse kidney were profiled using strand-specific RNA-sequencing (RNA-Seq) to a depth sufficient to complement spatial maps from pre-existing microarray datasets. The transcriptional complexity of RNAs arising from mouse RefSeq loci was catalogued; including 3568 alternatively spliced transcripts and 532 uncharacterized alternate 3' UTRs. Antisense expressions for 60% of RefSeq genes was also detected including uncharacterized non-coding transcripts overlapping kidney progenitor markers, Six2 and Sall1, and were validated by section in situ hybridization. Analysis of genes known to be involved in kidney development, particularly during mesenchymal-to-epithelial transition, showed an enrichment of non-coding antisense transcripts extended along protein-coding RNAs. CONCLUSION: The resulting resource further refines the transcriptomic cartography of kidney organogenesis by integrating deep RNA sequencing data with locus-based information from previously published expression atlases. The added resolution of RNA-Seq has provided the basis for a transition from classical gene-centric models of kidney development towards more accurate and detailed "transcript-centric" representations, which highlights the extent of transcriptional complexity of genes that direct complex development events.


Assuntos
Rim/metabolismo , MicroRNAs/genética , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos , Transcriptoma , Processamento Alternativo , Animais , Éxons , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Rim/embriologia , Camundongos , Organogênese , RNA Antissenso/genética , Transcrição Gênica
10.
PLoS One ; 6(2): e17286, 2011 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-21386911

RESUMO

The development of the mammalian kidney is well conserved from mouse to man. Despite considerable temporal and spatial data on gene expression in mammalian kidney development, primarily in rodent species, there is a paucity of genes whose expression is absolutely specific to a given anatomical compartment and/or developmental stage, defined here as 'anchor' genes. We previously generated an atlas of gene expression in the developing mouse kidney using microarray analysis of anatomical compartments collected via laser capture microdissection. Here, this data is further analysed to identify anchor genes via stringent bioinformatic filtering followed by high resolution section in situ hybridisation performed on 200 transcripts selected as specific to one of 11 anatomical compartments within the midgestation mouse kidney. A total of 37 anchor genes were identified across 6 compartments with the early proximal tubule being the compartment richest in anchor genes. Analysis of minimal and evolutionarily conserved promoter regions of this set of 25 anchor genes identified enrichment of transcription factor binding sites for Hnf4a and Hnf1b, RbpJ (Notch signalling), PPARγ:RxRA and COUP-TF family transcription factors. This was reinforced by GO analyses which also identified these anchor genes as targets in processes including epithelial proliferation and proximal tubular function. As well as defining anchor genes, this large scale validation of gene expression identified a further 92 compartment-enriched genes able to subcompartmentalise key processes during murine renal organogenesis spatially or ontologically. This included a cohort of 13 ureteric epithelial genes revealing previously unappreciated compartmentalisation of the collecting duct system and a series of early tubule genes suggesting that segmentation into proximal tubule, loop of Henle and distal tubule does not occur until the onset of glomerular vascularisation. Overall, this study serves to illuminate previously ill-defined stages of patterning and will enable further refinement of the lineage relationships within mammalian kidney development.


Assuntos
Compartimento Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Controladores do Desenvolvimento/fisiologia , Rim/embriologia , Organogênese/genética , Animais , Análise por Conglomerados , Perfilação da Expressão Gênica , Rim/metabolismo , Camundongos , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Distribuição Tecidual/genética , Estudos de Validação como Assunto
11.
Pediatr Nephrol ; 26(9): 1395-406, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21229268

RESUMO

It has long been appreciated that the mammalian kidney arises via reciprocal interactions between an epithelial ureteric epithelium and the surrounding metanephric mesenchyme. More recently, lineage tracing has confirmed that the portion of the metanephric mesenchyme closest to the advancing ureteric tips, the cap mesenchyme, represents the progenitor population for the nephron epithelia. This Six2(+)Cited1(+) population undergoes self-renewal throughout nephrogenesis while retaining the potential to epithelialize. In contrast, the Foxd1(+) portion of the metanephric mesenchyme shows no epithelial potential, developing instead into the interstitial, perivascular, and possibly endothelial elements of the kidney. The cap mesenchyme rests within a nephrogenic niche, surrounded by the stroma and the ureteric tip. While the role of Wnt signaling in nephron induction is known, there remains a lack of clarity over the intrinsic and extrinsic regulation of cap mesenchyme specification, self-renewal, and nephron potential. It is also not known what regulates cessation of nephrogenesis, but there is no nephron generation in response to injury during the postnatal period. In this review, we will examine what is and is not known about this nephron progenitor population and discuss how an increased understanding of the regulation of this population may better explain the observed variation in final nephron number and potentially facilitate the reinitiation or prolongation of nephron formation.


Assuntos
Diferenciação Celular , Linhagem da Célula , Células-Tronco Mesenquimais/fisiologia , Néfrons/embriologia , Animais , Proliferação de Células , Transição Epitelial-Mesenquimal , Humanos , Organogênese , Nicho de Células-Tronco
12.
Dev Biol ; 344(2): 1071-87, 2010 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-20510229

RESUMO

Here we describe the first detailed catalog of gene expression in the developing lower urinary tract (LUT), including epithelial and mesenchymal portions of the developing bladder, urogenital sinus, urethra, and genital tubercle (GT) at E13 and E14. Top compartment-specific genes implicated by the microarray data were validated using whole-mount in situ hybridization (ISH) over the entire LUT. To demonstrate the potential of this resource to implicate developmentally critical features, we focused on gene expression patterns and pathways in the sexually indeterminate, androgen-independent GT. GT expression patterns reinforced the proposed similarities between development of GT, limb, and craniofacial prominences. Comparison of spatial expression patterns predicted a network of Wnt7a-associated GT-enriched epithelial genes, including Gjb2, Dsc3, Krt5, and Sostdc1. Known from other contexts, these genes are associated with normal epidermal differentiation, with disruptions in Dsc3 and Gjb2 showing palmo-plantar keratoderma in the limb. We propose that this gene network contributes to normal foreskin, scrotum, and labial development. As several of these genes are known to be regulated by, or contain cis elements responsive to retinoic acid, estrogen, or androgen, this implicates this pathway in the later androgen-dependent development of the GT.


Assuntos
Expressão Gênica , Redes Reguladoras de Genes , Sistema Urogenital/embriologia , Androgênios/genética , Animais , Diferenciação Celular/genética , Embrião de Mamíferos , Epiderme , Extremidades , Genitália Masculina/embriologia , Masculino , Camundongos , Organogênese/genética , Uretra/embriologia
13.
Pediatr Nephrol ; 25(6): 1005-16, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20049614

RESUMO

The discipline of paediatric nephrology encompasses the congenital nephritic syndromes, renal dysplasias, neonatal renal tumours, early onset cystic disease, tubulopathies and vesicoureteric reflux, all of which arise due to defects in normal kidney development. Indeed, congenital anomalies of the kidney and urinary tract (CAKUT) represent 20-30% of prenatal anomalies, occurring in 1 in 500 births. Developmental biologists have studied the anatomical and morphogenetic processes involved in kidney development for the last five decades. However, with the advent of transgenic mice, the sequencing of the genome, improvements in mutation detection and the advent of functional genomics, our understanding of the molecular basis of kidney development has grown significantly. Here we discuss how the advent of new genetic and genomics approaches has added to our understanding of kidney development and paediatric renal disease, as well as identifying areas in which we are still lacking knowledge.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Nefropatias/genética , Rim/embriologia , Animais , Criança , Expressão Gênica , Genômica , Humanos , Rim/anormalidades , Morfogênese/genética , Nefrologia/tendências , Pediatria/tendências
14.
Dev Biol ; 332(2): 273-86, 2009 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-19501082

RESUMO

While nephron formation is known to be initiated by a mesenchyme-to-epithelial transition of the cap mesenchyme to form a renal vesicle (RV), the subsequent patterning of the nephron and fusion with the ureteric component of the kidney to form a patent contiguous uriniferous tubule has not been fully characterized. Using dual section in situ hybridization (SISH)/immunohistochemistry (IHC) we have revealed distinct distal/proximal patterning of Notch, BMP and Wnt pathway components within the RV stage nephron. Quantitation of mitoses and Cyclin D1 expression indicated that cell proliferation was higher in the distal RV, reflecting the differential developmental programs of the proximal and distal populations. A small number of RV genes were also expressed in the early connecting segment of the nephron. Dual ISH/IHC combined with serial section immunofluorescence and 3D reconstruction revealed that fusion occurs between the late RV and adjacent ureteric tip via a process that involves loss of the intervening ureteric epithelial basement membrane and insertion of cells expressing RV markers into the ureteric tip. Using Six2-eGFPCre x R26R-lacZ mice, we demonstrate that these cells are derived from the cap mesenchyme and not the ureteric epithelium. Hence, both nephron patterning and patency are evident at the late renal vesicle stage.


Assuntos
Proliferação de Células , Rim/anatomia & histologia , Rim/embriologia , Mesoderma/fisiologia , Morfogênese/fisiologia , Néfrons/embriologia , Ureter , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Caderinas/genética , Caderinas/metabolismo , Calbindinas , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Epitélio/fisiologia , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Rim/fisiologia , Proteínas com Homeodomínio LIM , Laminina/genética , Laminina/metabolismo , Camundongos , Néfrons/anatomia & histologia , Néfrons/fisiologia , Gravidez , Receptores Notch/genética , Receptores Notch/metabolismo , Proteína G de Ligação ao Cálcio S100/genética , Proteína G de Ligação ao Cálcio S100/metabolismo , Fatores de Transcrição , Ureter/anatomia & histologia , Ureter/embriologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo
15.
Dev Cell ; 15(5): 781-91, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19000842

RESUMO

Kidney development is based on differential cell-type-specific expression of a vast number of genes. While multiple critical genes and pathways have been elucidated, a genome-wide analysis of gene expression within individual cellular and anatomic structures is lacking. Accomplishing this could provide significant new insights into fundamental developmental mechanisms such as mesenchymal-epithelial transition, inductive signaling, branching morphogenesis, and segmentation. We describe here a comprehensive gene expression atlas of the developing mouse kidney based on the isolation of each major compartment by either laser capture microdissection or fluorescence-activated cell sorting, followed by microarray profiling. The resulting data agree with known expression patterns and additional in situ hybridizations. This kidney atlas allows a comprehensive analysis of the progression of gene expression states during nephrogenesis, as well as discovery of potential growth factor-receptor interactions. In addition, the results provide deeper insight into the genetic regulatory mechanisms of kidney development.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Rim/embriologia , Rim/metabolismo , Animais , Embrião de Mamíferos/metabolismo , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos , Néfrons/embriologia , Néfrons/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos
16.
Histochem Cell Biol ; 130(5): 927-42, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18618131

RESUMO

The kidney is the most complex organ within the urogenital system. The adult mouse kidney contains in excess of 8,000 mature nephrons, each of which can be subdivided into a renal corpuscle and 14 distinct tubular segments. The histological complexity of this organ can make the clarification of the site of gene expression by in situ hybridisation difficult. We have defined a panel of seven antibodies capable of identifying the six stages of early nephron development, the tubular nephron segments and the components of the renal corpuscle within the embryonic and adult mouse kidney. We have analysed in detail the protein expression of Wt1, Calb1 Aqp1, Aqp2 and Umod using these antibodies. We have then coupled immunohistochemistry with RNA in situ hybridisation in order to precisely identify the expression pattern of different genes, including Wnt4, Umod and Spp1. This technique will be invaluable for examining at high resolution, the structure of both the developing and mature nephron where standard in situ hybridisation and histological techniques are insufficient. The use of this technique will enhance the expression analyses of genes which may be involved in nephron formation and the function of the mature nephron in the mouse.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Hibridização In Situ , Néfrons/química , RNA Mensageiro/análise , Animais , Aquaporina 1/análise , Aquaporina 2/análise , Calbindina 1 , Calbindinas , Proteínas de Ligação ao Cálcio/análise , Feminino , Masculino , Camundongos , Mucoproteínas/análise , Mucoproteínas/genética , Néfrons/embriologia , Néfrons/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/análise , Osteopontina/genética , Proteína G de Ligação ao Cálcio S100 , Uromodulina , Proteínas WT1/análise , Proteínas Wnt/genética , Proteína Wnt4
17.
CSH Protoc ; 2008: pdb.prot5030, 2008 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21356874

RESUMO

INTRODUCTIONSection in situ hybridization (SISH) is a high-resolution tool used to analyze gene expression patterns. This protocol utilizes the Tecan Freedom EVO150 platform to perform high-throughput SISH on paraffin sections to detect mRNA with a digoxigenin (DIG)-labeled probe. The slide is mounted and imaged before performing immunohistochemistry (IHC) on the same section. The dual reaction enables a marker of protein expression to be localized on the same section as the mRNA and facilitates more accurate annotation of the gene expression.

18.
Gene Expr Patterns ; 7(6): 680-99, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17452023

RESUMO

Cataloguing gene expression during development of the genitourinary tract will increase our understanding not only of this process but also of congenital defects and disease affecting this organ system. We have developed a high-resolution ontology with which to describe the subcompartments of the developing murine genitourinary tract. This ontology incorporates what can be defined histologically and begins to encompass other structures and cell types already identified at the molecular level. The ontology is being used to annotate in situ hybridisation data generated as part of the Genitourinary Development Molecular Anatomy Project (GUDMAP), a publicly available data resource on gene and protein expression during genitourinary development. The GUDMAP ontology encompasses Theiler stage (TS) 17-27 of development as well as the sexually mature adult. It has been written as a partonomic, text-based, hierarchical ontology that, for the embryological stages, has been developed as a high-resolution expansion of the existing Edinburgh Mouse Atlas Project (EMAP) ontology. It also includes group terms for well-characterised structural and/or functional units comprising several sub-structures, such as the nephron and juxtaglomerular complex. Each term has been assigned a unique identification number. Synonyms have been used to improve the success of query searching and maintain wherever possible existing EMAP terms relating to this organ system. We describe here the principles and structure of the ontology and provide representative diagrammatic, histological, and whole mount and section RNA in situ hybridisation images to clarify the terms used within the ontology. Visual examples of how terms appear in different specimen types are also provided.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Camundongos/genética , Sistema Urogenital/crescimento & desenvolvimento , Animais , Clitóris/crescimento & desenvolvimento , Endoderma/fisiologia , Feminino , Masculino , Mesoderma/fisiologia , Camundongos/embriologia , Camundongos/crescimento & desenvolvimento , Néfrons/embriologia , Néfrons/crescimento & desenvolvimento , Pênis/crescimento & desenvolvimento , Escroto/crescimento & desenvolvimento , Maturidade Sexual , Sistema Urogenital/anatomia & histologia
19.
Dev Dyn ; 235(6): 1709-19, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16538671

RESUMO

The term "secretome" has been defined as a set of secreted proteins (Grimmond et al. [2003] Genome Res 13:1350-1359). The term "secreted protein" encompasses all proteins exported from the cell including growth factors, extracellular proteinases, morphogens, and extracellular matrix molecules. Defining the genes encoding secreted proteins that change in expression during organogenesis, the dynamic secretome, is likely to point to key drivers of morphogenesis. Such secreted proteins are involved in the reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (MM) that occur during organogenesis of the metanephros. Some key metanephric secreted proteins have been identified, but many remain to be determined. In this study, microarray expression profiling of E10.5, E11.5, and E13.5 kidney and consensus bioinformatic analysis were used to define a dynamic secretome of early metanephric development. In situ hybridisation was used to confirm microarray results and clarify spatial expression patterns for these genes. Forty-one secreted factors were dynamically expressed between the E10.5 and E13.5 timeframe profiled, and 25 of these factors had not previously been implicated in kidney development. A text-based anatomical ontology was used to spatially annotate the expression pattern of these genes in cultured metanephric explants.


Assuntos
Rim/embriologia , Rim/metabolismo , Proteínas/genética , Proteínas/metabolismo , Animais , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Técnicas de Cultura de Tecidos
20.
Development ; 132(24): 5437-49, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16291795

RESUMO

Most studies on kidney development have considered the interaction of the metanephric mesenchyme and the ureteric bud to be the major inductive event that maintains tubular differentiation and branching morphogenesis. The mesenchyme produces Gdnf, which stimulates branching, and the ureteric bud stimulates continued growth of the mesenchyme and differentiation of nephrons from the induced mesenchyme. Null mutation of the Wt1 gene eliminates outgrowth of the ureteric bud, but Gdnf has been identified as a target of Pax2, but not of Wt1. Using a novel system for microinjecting and electroporating plasmid expression constructs into murine organ cultures, it has been demonstrated that Vegfa expression in the mesenchyme is regulated by Wt1. Previous studies had identified a population of Flk1-expressing cells in the periphery of the induced mesenchyme, and adjacent to the stalk of the ureteric bud, and that Vegfa was able to stimulate growth of kidneys in organ culture. Here it is demonstrated that signaling through Flk1 is required to maintain expression of Pax2 in the mesenchyme of the early kidney, and for Pax2 to stimulate expression of Gdnf. However, once Gdnf stimulates branching of the ureteric bud, the Flk1-dependent angioblast signal is no longer required to maintain branching morphogenesis and induction of nephrons. Thus, this work demonstrates the presence of a second set of inductive events, involving the mesenchymal and angioblast populations, whereby Wt1-stimulated expression of Vegfa elicits an as-yet-unidentified signal from the angioblasts, which is required to stimulate the expression of Pax2 and Gdnf, which in turn elicits an inductive signal from the ureteric bud.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Rim/embriologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Proteínas WT1/fisiologia , Animais , Diferenciação Celular , Eletroporação , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Rim/citologia , Rim/metabolismo , Mesoderma/citologia , Mesoderma/fisiologia , Camundongos , Microinjeções , Morfogênese , Néfrons/embriologia , Néfrons/fisiologia , Técnicas de Cultura de Órgãos , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , Ratos , Transdução de Sinais , Ureter/embriologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas WT1/biossíntese , Proteínas WT1/genética
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