Byrne JA et al. (2002), From intestine to muscle: nuclear reprogramming...

XB-ART-7241

Proc Natl Acad Sci U S A 2002 Apr 30;999:6059-63. doi: 10.1073/pnas.082112099.

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From intestine to muscle: nuclear reprogramming through defective cloned embryos.

Byrne JA , Simonsson S , Gurdon JB .

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Nuclear transplantation is one of the very few ways by which the genetic content and capacity for nuclear reprogramming can be assessed in individual cells of differentiated somatic tissues. No more than 6% of the cells of differentiated tissues have thus far been shown to have nuclei that can be reprogrammed to elicit the formation of unrelated cell types. In Amphibia, about 25% of such nuclear transfers form morphologically abnormal partial blastulae that die within 24 h. We have investigated the genetic content and capacity for reprogramming of those nuclei that generate partial blastulae, using as donors the intestinal epithelium cells of feeding Xenopus larvae. We have analyzed single nuclear transplant embryos obtained directly from intestinal tissue, thereby avoiding any genetic or epigenetic changes that might accumulate during cell culture. The expression of the intestine-specific gene intestinal fatty acid binding protein is extinguished by at least 10(4) times, within a few hours of nuclear transplantation. At the same time several genes that are normally expressed only in early embryos are very strongly activated in nuclear transplant embryos, but to an unregulated extent. Remarkably, cells from intestine-derived partial blastulae, when grafted to normal host embryos, contribute to several host tissues and participate in the normal 100-fold increase in axial muscle over several months. Thus, cells of defective cloned embryos unable to survive for more than 1 day can be reprogrammed to participate in new directions of differentiation and to maintain indefinite growth, despite the abnormal expression of early genes.

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Species referenced: Xenopus
Genes referenced: dnmt1 fabp2 gsc tbxt vegt
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	Figure 1 Nuclear transfer experiment and donor cell characterization. (A) Design of nuclear transfer experiment. Arrowhead indicates region of intestine used for nuclear transfer. (B) Transverse sections of a stage-47 tadpole. (Left) Nomarski view; (Center) section in situ to show IFABP mRNA in mid-intestine; (Right) 12-101 antibody staining of muscle. (C) Whole-mount in situ hybridization for IFABP mRNA. Tail (control) (Left); intestine (Right). The dark spots in the center of the tail are pigment cells.
	Figure 2 Nuclear transfer experiment results. (A) Nuclear transfer results. (B) Photographs of whole embryos.
	Figure 3 Extinction of intestine-specific genes. RT-PCR analysis using primers for IFABP and, on the same samples, for histone H4. This analysis demonstrates extinction of intestine-specific IFABP expression after nuclear transfer; E, eye; I (1, 1/3, 1/9), whole or fractions of one stage-47 tadpole mid-intestine; IC, 5 × 104 intestinal epithelial cells; IVF, blastula from in vitro fertilization; nuclear transfer, single partial blastulae. Numbers in brackets represent cycles of PCR amplification. Strong IFABP expression is seen in intestine cells with only 17 cycles but no expression is observed in nuclear transfer embryos, even after 30 cycles. RT, omission of reverse transcriptase.
	Figure 4 Activation of early embryo genes. (A) Activation of early zygotic gene expression in single in vitro fertilization blastula (IVF) and in single partial nuclear transplant blastulae. (B) Another series of experiments in which part of each embryo was analyzed by RT-PCR (as shown). The other part of the same embryo was used to graft to hosts, as described in the text. dnmt1, DNA methyltransferase. (C) Summary of expression of the “zygotic gene Apod: histone H4” ratio in single, whole, or partial blastulae analyzed by RT-PCR. Apod, antipodean (40); gsc, goosecoid (41); Xbra, Xenopus brachyury (42).
	Figure 5 Nuclear transplant embryo development. (A) Experimental design. (B) Differentiation of grafts. In 6 experimental series, a total of 66 host embryos received grafts, and 47 of these reached the normal swimming tadpole stage 41.
	Figure 6 Growth and differentiation of muscle. (A) Mid-gastrula with graft. (B–D) Differentiated cells derived from grafts of GFP-partial nuclear transplant blastula tissue into wild-type hosts. These fluorescent photographs do not show host cells. (E) Growth of a gastrula that received a graft of cells from a GFP-partial nuclear transplant embryo. The fluorescent photografts of axial muscle show the great enlargement of muscle cells during tadpole growth. Embryos and tadpoles are shown at the same relative size (diameter and length are shown in millimeters).

References [+] :

Blau, Plasticity of the differentiated state. 1985, Pubmed