Dataset Information

Visualization and quantification of mouse prostate development by in situ hybridization.

ABSTRACT: The purpose of this study was to validate a combined in situ hybridization (ISH)/immunohistochemistry (IHC) staining method for visualizing and quantifying mouse prostatic buds. To refine animal usage in prostate development studies, we also determined whether a comparable number of prostatic buds were formed in male and female mouse urogenital sinus (UGS) explants grown in vitro in the presence of androgen. We used IHC to label UGS epithelium and ISH to label prostatic buds with one of three different prostatic bud marking riboprobes: a previously identified prostatic bud marker, NK-3 transcription factor, locus 1 (Nkx3-1), and two newly identified prostatic bud markers, wingless-related MMTV integration site 10b (Wnt10b) and ectodysplasin-A receptor (Edar). We calculated total buds formed per UGS and the proportion marked by each mRNA after male UGS development in vivo and male and female UGS development in vitro. Nkx3-1 was first to mark the prostate field during UGS development in vivo but all three mRNAs marked prostatic buds during later developmental stages. The mRNAs localized to different domains: Nkx3-1 was present along about half the prostatic bud length while Edar and Wnt10b were restricted to distal bud tips. None of the mRNAs marked all buds formed in vitro and the proportion marked was developmental stage- and gender-dependent. Nkx3-1 marked the highest proportion of prostatic buds during in vitro UGS development. Together, our results reveal that ISH staining of mouse UGS can be used to quantify prostatic bud number, Nkx3-1 is currently the best suited riboprobe for this method, and female UGSs cannot be used interchangeably with male UGSs when conducting prostate development studies in vitro. We also found that Nkx3-1, Edar, and Wnt10b mark different prostatic bud regions and are likely to be useful in future studies of regional differences in prostatic bud gene expression.

SUBMITTER: Keil KP

PROVIDER: S-EPMC3443266 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Visualization and quantification of mouse prostate development by in situ hybridization.

Keil Kimberly P KP Mehta Vatsal V Abler Lisa L LL Joshi Pinak S PS Schmitz Christopher T CT Vezina Chad M CM

Differentiation; research in biological diversity 20120813 3

The purpose of this study was to validate a combined in situ hybridization (ISH)/immunohistochemistry (IHC) staining method for visualizing and quantifying mouse prostatic buds. To refine animal usage in prostate development studies, we also determined whether a comparable number of prostatic buds were formed in male and female mouse urogenital sinus (UGS) explants grown in vitro in the presence of androgen. We used IHC to label UGS epithelium and ISH to label prostatic buds with one of three di ...[more]

PMID: 22898663

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Project description:Metagenome-assembled genomes (MAGs) of Asgardarchaeota have been recovered from a variety of habitats, broadening their environmental distribution and providing access to the genetic makeup of this archaeal lineage. The recent success in cultivating the first representative of Lokiarchaeia was a breakthrough in science at large and gave rise to new hypotheses about the evolution of eukaryotes. Despite their singular phylogenetic position at the base of the eukaryotic tree of life, the morphology of these bewildering organisms remains a mystery, except for the report of an unusual morphology with long, branching protrusions of the cultivated Lokiarchaeion strain "Candidatus Prometheoarchaeum syntrophicum" MK-D1. In order to visualize this elusive group, we applied a combination of fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH) and epifluorescence microscopy on coastal hypersaline sediment samples, using specifically designed CARD-FISH probes for Heimdallarchaeia and Lokiarchaeia lineages, and provide the first visual evidence for Heimdallarchaeia and new images of a lineage of Lokiarchaeia that is different from the cultured representative. Here, we show that while Heimdallarchaeia are characterized by a uniform cellular morphology typified by a centralized DNA localization, Lokiarchaeia display a plethora of shapes and sizes that likely reflect their broad phylogenetic diversity and ecological distribution.IMPORTANCE Asgardarchaeota are considered to be the closest relatives to modern eukaryotes. These enigmatic microbes have been mainly studied using metagenome-assembled genomes (MAGs). Only very recently, a first member of Lokiarchaeia was isolated and characterized in detail; it featured a striking morphology with long, branching protrusions. In order to visualize additional members of the phylum Asgardarchaeota, we applied a fluorescence in situ hybridization technique and epifluorescence microscopy on coastal hypersaline sediment samples, using specifically designed probes for Heimdallarchaeia and Lokiarchaeia lineages. We provide the first visual evidence for Heimdallarchaeia that are characterized by a uniform cellular morphology typified by an apparently centralized DNA localization. Further, we provide new images of a lineage of Lokiarchaeia that is different from the cultured representative and with multiple morphologies, ranging from small ovoid cells to long filaments. This diversity in observed cell shapes is likely owing to the large phylogenetic diversity within Asgardarchaeota, the vast majority of which remain uncultured.

Dataset Information

Visualization and quantification of mouse prostate development by in situ hybridization.

Publications

Visualization and quantification of mouse prostate development by in situ hybridization.

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