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Isolation and live imaging of enteric progenitors based on Sox10-Histone2BVenus transgene expression.

ABSTRACT: To facilitate dynamic imaging of neural crest (NC) lineages and discrimination of individual cells in the enteric nervous system (ENS) where close juxtaposition often complicates viewing, we generated a mouse BAC transgenic line that drives a Histone2BVenus (H2BVenus) reporter from Sox10 regulatory regions. This strategy does not alter the endogenous Sox10 locus and thus facilitates analysis of normal NC development. Our Sox10-H2BVenus BAC transgene exhibits temporal, spatial, and cell-type specific expression that reflects endogenous Sox10 patterns. Individual cells exhibiting nuclear-localized fluorescence of the H2BVenus reporter are readily visualized in both fixed and living tissue and are amenable to isolation by fluorescence activated cell sorting (FACS). FACS-isolated H2BVenus+ enteric NC-derived progenitors (ENPs) exhibit multipotency, readily form neurospheres, self-renew in vitro and express a variety of stem cell genes. Dynamic live imaging as H2BVenus+ ENPs migrate down the fetal gut reveals cell fragmentation suggesting that apoptosis occurs at a low frequency during normal development of the ENS. Confocal imaging both during population of the fetal intestine and in postnatal gut muscle strips revealed differential expression between individual cells consistent with down-regulation of the transgene as progression towards non-glial fates occurs. The expression of the Sox10-H2BVenus transgene in multiple regions of the peripheral nervous system will facilitate future studies of NC lineage segregation as this tool is expressed in early NC progenitors and maintained in enteric glia.

SUBMITTER: Corpening JC

PROVIDER: S-EPMC3212811 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Isolation and live imaging of enteric progenitors based on Sox10-Histone2BVenus transgene expression.

Corpening Jennifer C JC Deal Karen K KK Cantrell V Ashley VA Skelton Stephanie B SB Buehler Dennis P DP Southard-Smith E Michelle EM

Genesis (New York, N.Y. : 2000) 20110621 7

To facilitate dynamic imaging of neural crest (NC) lineages and discrimination of individual cells in the enteric nervous system (ENS) where close juxtaposition often complicates viewing, we generated a mouse BAC transgenic line that drives a Histone2BVenus (H2BVenus) reporter from Sox10 regulatory regions. This strategy does not alter the endogenous Sox10 locus and thus facilitates analysis of normal NC development. Our Sox10-H2BVenus BAC transgene exhibits temporal, spatial, and cell-type spec ...[more]

PMID: 21504042

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Project description:BackgroundPlacental dysfunction underlies numerous complications of pregnancy. A major obstacle to understanding the roles of potential mediators of placental pathology has been the absence of suitable methods for tissue-specific gene manipulation and sensitive assays for studying gene functions in the placentas of intact animals. We describe a sensitive and noninvasive method of repetitively tracking placenta-specific gene expression throughout pregnancy using lentivirus-mediated transduction of optical reporter genes in mouse blastocysts.Methodology/principal findingsZona-free blastocysts were incubated with lentivirus expressing firefly luciferase (Fluc) and Tomato fluorescent fusion protein for trophectoderm-specific infection and transplanted into day 3 pseudopregnant recipients (GD3). Animals were examined for Fluc expression by live bioluminescence imaging (BLI) at different points during pregnancy, and the placentas were examined for tomato expression in different cell types on GD18. In another set of experiments, blastocysts with maximum photon fluxes in the range of 2.0E+4 to 6.0E+4 p/s/cm(2)/sr were transferred. Fluc expression was detectable in all surrogate dams by day 5 of pregnancy by live imaging, and the signal increased dramatically thereafter each day until GD12, reaching a peak at GD16 and maintaining that level through GD18. All of the placentas, but none of the fetuses, analyzed on GD18 by BLI showed different degrees of Fluc expression. However, only placentas of dams transferred with selected blastocysts showed uniform photon distribution with no significant variability of photon intensity among placentas of the same litter. Tomato expression in the placentas was limited to only trophoblast cell lineages.Conclusions/significanceThese results, for the first time, demonstrate the feasibility of selecting lentivirally-transduced blastocysts for uniform gene expression in all placentas of the same litter and early detection and quantitative analysis of gene expression throughout pregnancy by live BLI. This method may be useful for a wide range of applications involving trophoblast-specific gene manipulations in utero.

Dataset Information

Isolation and live imaging of enteric progenitors based on Sox10-Histone2BVenus transgene expression.

Publications

Isolation and live imaging of enteric progenitors based on Sox10-Histone2BVenus transgene expression.

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