Figure 1
Figure 1. The Ncx1−/− mouse embryo provides an in vivo circulation-free environment. Ncx1 mutants were generated via insertion of LacZ reporter into exon 2 of the Ncx1 gene, thus all cells that normally express Ncx1 can be labeled with X-gal staining. (A) E9.5 embryos (WT(i), Ncx1+/−(ii), and Ncx1−/−(iii)) demonstrate that development continues well past the onset of circulation. (B) X-gal staining (∼27 sp) reveals that expression of Ncx1 is restricted to the heart through E10. Importantly, there is no expression in any putative site of hematopoietic development including the YS and PSp region nor in the hematopoietic cells themselves (see blood vessels in Ncx1+/− YS). (C) Ncx1 RT-PCR was conducted on embryonic tissues from various ages to confirm the X-gal staining. At E8.0, Ncx1 is not yet detectable in either the embryo proper or YS. At E9.0 and E11.5, Ncx1 is detected exclusively in the heart cardiomyocytes. (D) Ten-micrometer sagittal sections of hematoxylin and eosin (H&E) stained E9.25 (19 sp) embryos. Panels Di,iii are 100× magnifications of sections that best profile the structure of the PSp region (*), which is clearly present in both WT and Ncx1−/− embryos. Image Di was cut at an oblique angle compared with image Diii, leaving only a small portion of the heart and upper body in view. Image Dv is an insert that transects the hypoplastic Ncx1−/− heart. Images Dii,iv are 200× magnifications of the PSp (*) regions. Endothelial cells can be seen lining the vessels (below yellow line) and circulating blood cells (arrows) are seen in the WT embryo but are notably absent in the Ncx1−/− embryo. The images in panels A and B were viewed on a Leica MZ9.5 Stereomicroscope (1.0× Planachromatic Lens/0.20 NA)(IL-3) with DFC320 CCD camera, captured with Leica application suite (LAS) software (Leica, Bannockburn, IL). Original magnification, ×60. The images in panel D were viewed on a Zeiss Axioskop Stereomicroscope (Zeiss Plan-Neofluor 10×/0.30 NA (top) and 20×/0.50 NA (bottom)) with SPOT RTKE cooled color CCD camera, and imported into the SPOT Advanced software (Diagnostic Instruments, Sterling Heights, NJ). Original magnification, ×100.

The Ncx1−/− mouse embryo provides an in vivo circulation-free environment.Ncx1 mutants were generated via insertion of LacZ reporter into exon 2 of the Ncx1 gene, thus all cells that normally express Ncx1 can be labeled with X-gal staining. (A) E9.5 embryos (WT(i), Ncx1+/−(ii), and Ncx1−/−(iii)) demonstrate that development continues well past the onset of circulation. (B) X-gal staining (∼27 sp) reveals that expression of Ncx1 is restricted to the heart through E10. Importantly, there is no expression in any putative site of hematopoietic development including the YS and PSp region nor in the hematopoietic cells themselves (see blood vessels in Ncx1+/− YS). (C) Ncx1 RT-PCR was conducted on embryonic tissues from various ages to confirm the X-gal staining. At E8.0, Ncx1 is not yet detectable in either the embryo proper or YS. At E9.0 and E11.5, Ncx1 is detected exclusively in the heart cardiomyocytes. (D) Ten-micrometer sagittal sections of hematoxylin and eosin (H&E) stained E9.25 (19 sp) embryos. Panels Di,iii are 100× magnifications of sections that best profile the structure of the PSp region (*), which is clearly present in both WT and Ncx1−/− embryos. Image Di was cut at an oblique angle compared with image Diii, leaving only a small portion of the heart and upper body in view. Image Dv is an insert that transects the hypoplastic Ncx1−/− heart. Images Dii,iv are 200× magnifications of the PSp (*) regions. Endothelial cells can be seen lining the vessels (below yellow line) and circulating blood cells (arrows) are seen in the WT embryo but are notably absent in the Ncx1−/− embryo. The images in panels A and B were viewed on a Leica MZ9.5 Stereomicroscope (1.0× Planachromatic Lens/0.20 NA)(IL-3) with DFC320 CCD camera, captured with Leica application suite (LAS) software (Leica, Bannockburn, IL). Original magnification, ×60. The images in panel D were viewed on a Zeiss Axioskop Stereomicroscope (Zeiss Plan-Neofluor 10×/0.30 NA (top) and 20×/0.50 NA (bottom)) with SPOT RTKE cooled color CCD camera, and imported into the SPOT Advanced software (Diagnostic Instruments, Sterling Heights, NJ). Original magnification, ×100.

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