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. 2013 Apr 22;8(4):e60905.
doi: 10.1371/journal.pone.0060905. Print 2013.

Detection and Organ-Specific Ablation of Neuroendocrine Cells by Synaptophysin Locus-Based BAC Cassette in Transgenic Mice

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Free PMC article

Detection and Organ-Specific Ablation of Neuroendocrine Cells by Synaptophysin Locus-Based BAC Cassette in Transgenic Mice

Chieh-Yang Cheng et al. PLoS One. .
Free PMC article

Abstract

The role of cells of the diffuse neuroendocrine system in development and maintenance of individual organs and tissues remains poorly understood. Here we identify a regulatory region sufficient for accurate in vivo expression of synaptophysin (SYP), a common marker of neuroendocrine differentiation, and report generation of Tg(Syp-EGFP(loxP)-DTA)147(Ayn) (SypELDTA) mice suitable for flexible organ-specific ablation of neuroendocrine cells. These mice express EGFP and diphtheria toxin fragment A (DTA) in SYP positive cells before and after Cre-loxP mediated recombination, respectively. As a proof of principle, we have crossed SypELDTA mice with EIIA-Cre and PB-Cre4 mice. EIIA-Cre mice express Cre recombinase in a broad range of tissues, while PB-Cre4 mice specifically express Cre recombinase in the prostate epithelium. Double transgenic EIIA-Cre; SypELDTA embryos exhibited massive cell death in SYP positive cells. At the same time, PB-Cre4; SypELDTA mice showed a substantial decrease in the number of neuroendocrine cells and associated prostate hypotrophy. As no increase in cell death and/or Cre-loxP mediated recombination was observed in non-neuroendocrine epithelium cells, these results suggest that neuroendocrine cells play an important role in prostate development. High cell type specificity of Syp locus-based cassette and versatility of generated mouse model should assure applicability of these resources to studies of neuroendocrine cell functions in various tissues and organs.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Genomic structure of the Syp gene.
(A) Location of the Syp locus on mouse, rat, and human chromosome X. Mouse Syp contains 7 exons (black boxes). The translation initiation codon, ATG, is located in the first exon. The NRSE is located within the first intron of Syp. (B) Sequence comparison of the NRSE derived from Syp across species.
Figure 2
Figure 2. Sequence comparison of upstream region of Syp across species.
Black lines and boxes represent highly conserved areas of the Syp upstream region. The position of upstream sequence is relative to the Syp transcription start site (0 bp). The assembly dates of the upstream regions are July 2007 (Mus musculus), November 2004 (Rattus norvegicus), February 2009 (Homo sapiens), October 2010 (Pan troglodytes), January 2006 (Macaca mulatta), July 2007 (Pongo pygmaeus abelii), and March 2009 (Callithrix jacchus).
Figure 3
Figure 3. BAC transgenic constructs.
(A) SypELDTA construct. The purple bar represents the EGFP probe. Restriction enzyme sites for Southern blot are indicated. (B) sSypELDTA construct. The fragment SypP-loxP-EGFP-Neo cassette-Stop-loxP-DTA-bpA from SypELDTA was cloned into pGEM-T vector. 3 kb upstream fragment of Syp was used to drive downstream gene expression.
Figure 4
Figure 4. SypELDTA transgene expression is highly specific for SYP expressing cells.
(A-E) Co-expression of EGFP (green) and SYP (red) in prostate NE cells (A), lung NE cells (B; inset: high magnification), medulla of adrenal gland (C), pancreatic islets of Langerhans (D), and brain (E) of SypELDTA transgenic mice. Yellow color (arrows) indicates co-localization of EGFP and SYP fluorescent signals. Counterstaining with DAPI, blue. Calibration bar: 18 µm (A), 37 µm (B), 50 µm (C-E), 8 µm (inset).
Figure 5
Figure 5. EIIA-Cre; SypELDTA embyros exhibit massive cell death in SYP positive cells.
(A) Design of crosses between male SypELDTA and female EIIA-Cre transgenic mice. (B-M) SYP (B, D, F, H, J, L) and cleaved Caspase-3 (C, E, G, I, K, M) expression (arrows) in serial sections of EIIA-Cre; SypELDTA (B-G) and EIIA-Cre (H-M) embryos collected on gestational day 10.5. High (D-G, J-M) magnification images of brain (D, E, J, K) and dorsal root ganglion (F, G,L, M) regions shown as rectangles in low magnification images (B, C, H, I). b, brain, g, dorsal root ganglia. ABC Elite method. Hematoxylin counterstaining. Calibration bar: 950 µm (B, C, H, I), 50 µm (D-G, J-M).
Figure 6
Figure 6. PB-Cre4; SypELDTA line 147 mice show decreased number of NE cells and prostate hypotrophy.
(A) Design of crosses between male PB-Cre4 and female SypELDTA transgenic mice resulting in male PB-Cre4; SypELDTA offspring with prostate epithelium-specific NE cell ablation. (B) Gross images of the prostate from wild-type FVB/N (FVB), SypELDTA (147), PB-Cre4; SypELDTA (AP147) mice. Calibration bar: 0.5 cm. (C, D) Detection of SYP positive NE cells (arrows) in the prostate proximal region of age-matched SypELDTA (C; n = 4) and PB-Cre4; SypELDTA (D; n = 4) mice. ABC Elite method. Hematoxylin counterstaining. Calibration bar: 50 µm. (E-G) Quantification of NE cells in the proximal regions of whole prostate (E), and in the proximal regions of ventral (F) and dorsolateral (G) lobes of prostates from age-matched SypELDTA (147; n = 4) and PB-Cre4; SypELDTA (AP147; n = 4) mice. (H) Quantification of NE cells in proximal regions of dorsolateral and ventral lobes of the prostates from SypELDTA mice (n = 4). *P<0.05. ***P<0.001. All error bars denote SD.

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