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First published online 3 August 2006
doi: 10.1242/dev.02506

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Primordial germ cell deficiency in the connexin 43 knockout mouse arises from apoptosis associated with abnormal p53 activation

Laboratory of Developmental Biology, National Heart Lung and Blood Institute, National Institutes of Health, Building 50/Room 4537, 9000 Rockville Pike, Bethesda, MD 20892, USA.

View larger version (45K): [in a new window]   Fig. 1. Dye coupling of PGC cells with their neighbours. (A,B) Representative images obtained following intracellular microinjection of fluorescent dye sulforhodamine 101 into PGCs contained in hindgut explants from both Cx431 wild-type (+/+) and homozygous Cx431KO (-/-) embryos (E9.5). Both A and B comprise three images superimposed: a DIC image, green Oct4-GFP fluorescence image (PGC), and a red sulforhodamine 101 fluorescence image. (C) Dye spread was quantified by calculating total area of dye spread after 5 minutes (2 minutes' active microinjection plus 3 minutes' passive spread). PGCs in Cx431KO (-/-) explants displayed a significant reduction in their ability to pass dye into their neighbors. For this analysis, 8, 4 and 7 microelectrode impalements were carried out, respectively in four Cx431 +/+, four Cx431 +/- and five Cx431 -/- embryo explants.

View larger version (80K): [in a new window]   Fig. 2. PGC distribution in the Cx431 KO mouse embryo. The distribution of PGCs in Cx431 wild-type (+/+) and homozygous Cx431KO (-/-) mouse embryos from E8.5 to E11.5 was observed via GFP expression mediated by an Oct4-GFP transgene. No difference was seen in the overall distribution of GFP-expressing cells in the wild-type versus KO mouse embryos. However, at E11.5 there was a noticeable reduction in GFP fluorescence in the genital ridges of the KO mouse embryos. Scale bars: 100 µm.

View larger version (58K): [in a new window]   Fig. 3. Analysis of PGC migration in Cx431KO mouse embryos. (A,B) An example of how GFP-expressing PGCs were visualized and tracked in an E8.5 mouse embryo by time-lapse imaging. The migratory paths of individual PGCs are superimposed on the image of the embryo seen at the beginning of the experiment (A), and for clarity are plotted with a single origin in B. (C,D) The migratory paths of PGCs in E8.5 and E11.5 embryos were used to determine the speed (C) and directionality (D) of cell locomotion. PGCs from both homozygous and heterozygous Cx431KO embryos demonstrated significant decreases in both speed and directionality. This data entailed the analysis of a total of 72 Cx431 +/+, 74 Cx431 +/- and 66 Cx431 -/- independent cell migration tracings obtained from five embryo explants for each of the three Cx431 genotypes. Scale bar: 100 µm.

View larger version (72K): [in a new window]   Fig. 4. PGC migration along the hindgut of Cx431KO embryos.Phase contrast (A) and GFP fluorescence (B) images of an E9.5 wild-type mouse embryo demonstrate the distribution of PGCs along the hindgut. We used somites as morphological landmarks (outlined in yellow) to assess the abundance of PGCs along the hindgut. We also determined the maximal PGC migration distance by measuring the distance from the base of the allantois (arrowhead), where PGCs originate, to the migration front situated rostrally along the anterior hindgut (arrow). Scale bars: 1 mm.

View larger version (52K): [in a new window]   Fig. 5. PGC fragmentation seen by time-lapse imaging. (A) Images from a time-lapse series, showing the progressive fragmentation of a PGC seen in an E11.5 genital ridge explant from a homozygous Cx431KO mouse embryo. The images 1 through 5 are 24 minutes apart. (B) After time-lapse imaging, the genital ridges were stained with a sulforhodamine derivative of valylalanylaspartic acid fluoromethyl ketone that binds caspases 1, 3, 4, 5, 6, 7, 8 and 9 (see Cy3 panel). In the merged image, regions of co-localization of the polycaspase staining (Cy3 panel) with the PGC cell fragments (GFP panel) can be seen. (C) From analysis of the time-lapse movies, the number of apoptotic and mitotic cells was quantitated to generate an apoptotic index comprising the number of apoptotic cells normalized by the number of mitotic cells. This showed a significant increase in apoptosis in the homozygous Cx431KO genital ridges. The number of embryos used in this analysis included seven Cx431 +/+, four Cx431 +/- and five Cx431 -/- embryos. Scale bars: 25 µm.

View larger version (114K): [in a new window]   Fig. 6. PGC apoptosis and proliferation in E11.5 genital ridges. (A-C) BrdU labeling was used to assess the cell proliferation rate of PGCs in E11.5 wild-type (A) heterozygous and homozygous Cx431KO (B) embryos. BrdU immunodetection using an Alexa Fluor 546 conjugated anti-BrdU antibody (red) showed some regions of co-localization with GFP-expressing PGCs (seen as yellow). No statistical difference was seen in the proliferation rate of PGCs in wild-type versus heterozygous/homozygous Cx431KO mouse embryos (C). Note that the data are normalized to the frequency of BrdU-positive PGCs in wild-type embryos. This analysis included eight Cx431 +/+, 25 Cx431 +/- and six Cx431 -/- genital ridges. (D-F) TUNEL labeling in E11.5 genital ridges from wild-type (D) and Cx431KO (E) mouse embryos. TUNEL labeling is visualized by BrdU incorporation, detected using Alexa Fluor 546 conjugated anti-BrdU antibody (red). In the genital ridges from the KO mouse embryo, we noted many regions containing TUNEL-positive GFP-expressing PGCs (see white arrows in E). Quantitative assessments showed a significant increase in TUNEL-positive PGCs in the homozygous Cx431KO embryos (F). Note that the data are normalized to the frequency of TUNEL-positive PGCs in wild-type embryos. This analysis included 12 Cx431 +/+, 16 Cx431 +/- and 10 Cx431 -/- genital ridges. Scale bars: 100 µm.

View larger version (19K): [in a new window]   Fig. 7. Analysis of ß1-integrin function in PGC motility. (A) Treatment of wild-type PGCs with a ß1-integrin-function-blocking antibody caused a significant reduction in both the speed and directionality of PGC migration in E11.5 genital ridge explants. The migration data was obtained from the analysis of 48 cells derived from five embryo explants for both sham and function-blocking antibody treatments. (B) PGC attachment to ß1-integrin-antibody-coated dishes was used to assess ß1-integrin-mediated cell adhesion. Homozygous Cx431KO PGCs showed a marked reduction in ß1-integrin-mediated adhesion, while control assays using plates coated with anti-mouse IgG antibodies showed no difference in adhesion between the different genotypes. It should be noted that the heterozygous KO PGCs also showed some reduction in adhesion to ß1-integrin-antibody-coated dishes, but less than that observed in the homozygous KO PGCs. For assessments of ß1-integrin-mediated adhesion, assays were carried out using PGCs derived from 14 Cx431 +/+, 20 Cx431 +/- and nine Cx431 -/- embryos, while control assays using anti-mouse IgG were carried out using PCGs from three Cx431 +/+, 14 Cx431 +/- and eight Cx431 -/- embryos.

View larger version (58K): [in a new window]   Fig. 8. Abnormal p53 activation in Cx431KO PGCs. (A-C) Genital ridges from E11.5 wild-type and Cx431KO mouse embryos were whole-mount stained using antibody against activated p53 (phospho-serine 15). PGCs (green) expressing activated p53 (red) are denoted by arrowheads. Quantitative analysis (C) revealed a marked increase in the expression of activated p53 in homozygous Cx431KO PGCs. This analysis was carried out using nine Cx431 +/+, 16 Cx431 +/- and 11 Cx431 -/- explants. (D) Assessment of PGC abundance in E11.5 genital ridges obtained from embryos of mice injected with -pifithrin or with vehicle alone (DMSO). With vehicle injection, homozygous Cx431KO mouse embryos showed a significant reduction in PGC abundance. By contrast, following -pifithrin treatment there was no difference in PGC abundance in the wild-type versus heterozygous or homozygous KO embryos. This analysis included 22 Cx431 +/+, 28 Cx431 +/- and ten Cx431 -/- -pifithrin-treated embryos, and 15 Cx431 +/+, 18 Cx431 +/- and ten Cx431 -/- vehicle-treated embryos. (E) TUNEL labeling showed that following -pifithrin treatment the rate of apoptosis was similar in homozygous KO, heterozygous KO or wild-type PGCs. Note that the data are normalized to the frequency of TUNEL-positive PGCs detected in wild-type embryos. This analysis included five Cx431 +/+, 37 Cx431 +/- and 16 Cx431 -/- -pifithrin-treated genital ridges. Scale bars: 25 µm.

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