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High resolution 4-dimension imaging of metanephric embryonic kidney morphogenesis.

ABSTRACT: High-resolution three-dimensional imaging of fixed embryonic kidney tissues has advanced considerably in the past decade. Here we developed a new process for imaging whole metanephric organ culture at cell resolution in three dimensions over time. This technique combines the use of the newly available generation of infrared-optimized long working distance, high numerical aperture objectives and multiphoton fluorescence microscopy with a new system for vital staining of metanephric organ cultures with bodipy ceramide. This allows all cells in the organ culture to be visualized over time, enabling detailed observation of tissue morphogenesis. Thus, our method offers a powerful new approach for visualizing and understanding early events in renal development and for extending observations made in genetically manipulated models.

SUBMITTER: Clendenon SG

PROVIDER: S-EPMC3658135 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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High resolution 4-dimension imaging of metanephric embryonic kidney morphogenesis.

Clendenon Sherry G SG Ward Heather H HH Dunn Kenneth W KW Bacallao Robert R

Kidney international 20130116 4

High-resolution three-dimensional imaging of fixed embryonic kidney tissues has advanced considerably in the past decade. Here we developed a new process for imaging whole metanephric organ culture at cell resolution in three dimensions over time. This technique combines the use of the newly available generation of infrared-optimized long working distance, high numerical aperture objectives and multiphoton fluorescence microscopy with a new system for vital staining of metanephric organ cultures ...[more]

PMID: 23325081

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Project description:Metanephric adenoma (MA) of the kidney is a rare, indolent tumor that may be difficult to differentiate from other small renal masses (SRMs). Genetic alterations associated with MA remain largely unknown.We aimed at defining genetic events in MA of the kidney and determining their influence in the management of this disease.Multiplexed mass spectrometric genotyping was performed on 29 MA cases after tumor DNA extraction. We also conducted a mutational screen in an additional 129 renal neoplasms. Immunohistochemistry was performed on the MA cases to assess molecular markers of signaling pathway activation. Patients' baseline characteristics, as well as follow-up data, were captured.We used descriptive statistics for baseline clinical characteristics and incidence of mutations. The Wilcoxon rank-sum test was used to correlate patient characteristics with mutational status.We identified the v-raf murine sarcoma viral oncogene homolog B1 (BRAF) V600E mutation in 26 of 29 MA cases. These results were validated in all cases using the commercially available BRAF Pyro Kit (QIAGEN). In contrast, BRAF mutations were rare in the other 129 non-MA renal neoplasms that were screened. We detected a BRAF mutation (V600E) in only one papillary renal cell carcinoma case. In all MA tumors, we documented expression of phosphorylated mitogen-activated protein kinase and phosphorylated extracellular signal-regulated kinase, accompanied by immunoreactivity for p16 (INK4a). All patients were treated with a partial or radical nephrectomy, and after a median follow-up of 26.5 mo, there were no local or distant recurrences. Limitations include the retrospective nature of this study.BRAF V600E mutations are present in approximately 90% of all MA cases, serving as a potential valuable diagnostic tool in the differential diagnosis of SRMs undergoing a percutaneous biopsy. The presence of BRAF V600E and mitogen-activated protein kinase activation in a largely benign tumor supports the necessity for secondary events (e.g., p16 loss) in BRAF-driven oncogenesis.

Project description:The vertebrate peripheral nervous system contains sensory neurons that arise from ectodermal placodes. Placodal cells ingress to move inside the head to form sensory neurons of the cranial ganglia. To date, however, the process of placodal cell ingression and underlying cellular behavior are poorly understood as studies have relied upon static analyses on fixed tissues. Visualizing placodal cell behavior requires an ability to distinguish the surface ectoderm from the underlying mesenchyme. This necessitates high resolution imaging along the z-plane which is difficult to accomplish in whole embryos. To address this issue, we have developed an imaging system using cranial slices that allows direct visualization of placode formation.We demonstrate an effective imaging assay for capturing placode development at single cell resolution using chick embryonic tissue ex vivo. This provides the first time-lapse imaging of mitoses in the trigeminal placodal ectoderm, ingression, and intercellular contacts of placodal cells. Cell divisions with varied orientations were found in the placodal ectoderm all along the apical-basal axis. Placodal cells initially have short cytoplasmic processes during ingression as young neurons and mature over time to elaborate long axonal processes in the mesenchyme. Interestingly, the time-lapse imaging data reveal that these delaminating placodal neurons begin ingression early on from within the ectoderm, where they start to move and continue on to exit as individual or strings of neurons through common openings on the basal side of the epithelium. Furthermore, dynamic intercellular contacts are abundant among the delaminating placodal neurons, between these and the already delaminated cells, as well as among cells in the forming ganglion.This new imaging assay provides a powerful method to analyze directly development of placode-derived sensory neurons and subsequent ganglia formation for the first time in amniotes. Viewing placode development in a head cross-section provides a vantage point from which it is possible to study comprehensive events in placode formation, from differentiation, cell ingression to ganglion assembly. Understanding how placodal neurons form may reveal a new mechanism of neurogenesis distinct from that in the central nervous system and provide new insight into how cells acquire motility from a stationary epithelial cell type.

Dataset Information

High resolution 4-dimension imaging of metanephric embryonic kidney morphogenesis.

Publications

High resolution 4-dimension imaging of metanephric embryonic kidney morphogenesis.

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