International Mammalian Genome Society

The 14th International Mouse Genome Conference (2000)


I34. Nonepiblast Tissues as the Primary Site of tclw5 Expression

Yuko Karashima1, Yuji Goto1, Kuniya Abe2, Seong-Seng Tan3, Minoru Koh4, Ramaiah Nagaraja4, Karen Artzt5 and Nobuo Takagi1
1Grad. Sch. Environ. Earth Sci., Hokkaido Univ. Sapporo; 2Inst. Molec. Embryol. & Genet., Kumamoto Univ., Kumamoto; 3Howard Florey Inst., Univ. Melbourne, Parkville 4Natl. Inst. Aging, NIH, Baltimore, MD; 5Inst. Cell & Molec. Biol., Univ. Texas, Austin, TX

Of many classical t-complex lethal mutations known to date, tclw5 is especially interesting because homozygous embryos die at the gastrulation stage when cell-cell interactions play salient roles. The tclw5 lethal mutation is known to map near the H-2K region of the mouse MHC. Our recent mapping study accurately locates the tclw5 in between H-2Pb and D17Mit147 markers. In contrast to such genetic studies, embryological phenotype of the tclw5 is not well characterized.

To understand the function of tclw5 gene in the crucial developmental event, we tried to determine the primary site of tclw5 gene action employing two approaches. First, we carried out a classical histological study of homozygous embryos at E4.5 to E7.5 with semi-thin sections. Subsequently, we analyzed allocation of tclw5/tclw5 cells marked by X-linked HMG-lacZ transgene in E6.5 chimeras formed by 8-cell embryos from normal mice and those from intercrosses of tclw5 /+ animals. Homozygous embryos were smaller than their littermates by E6.5. Histological study revealed that homozygous embryos were characterized by extensive death of embryonic ectoderm cells at E6.5 though phenotypes varied considerably among embryos. The extraembryonic ectoderm and visceral endoderm were much less affected in these embryos. Examination in younger embryos suggested that the visceral endoderm in presumptive tclw5 / tclw5 embryos was different from that in other embryos in that individual cells contained vacuoles less in size and in number. Mesodermal cells were scarcely formed in homozygous embryos even at E7.5. Presumptive +/+tclw5 /tclw5 chimeras at E6.5 showed, however, an unexpected distribution of tclw5 / tclw5 cells. b-gal positive tclw5 homozygous cells were mostly restricted to the embryonic ectoderm being equally chimeric with +/+ cells. tclw5 / tclw5 cells were never found in the extraembryonic ectoderm but might be occasionally found in the extraembryonic part of the visceral endoderm. This finding suggests that it is nonepiblast rather than epiblast cells that are primarily affected by tclw5 mutation. If so, extensive damages characterizing tclw5 / tclw5 epiblast are caused by functionally abnormal nonepiblast tissues. The relative importance among three major nonepiblast tissues is not known at present. A less likely possibility would be that +/+ epiblast cells rescue tclw5 / tclw5 epiblast cells.

In addition to such embryological studies, efforts to positionally clone the tclw5 gene have been made. The entire tclw5 critical region was covered by five BAC clones and genomic sequencing of this region is now being carried out. Transgenic rescue experiment using those BAC clones is also in progress. Identification of the tclw5 gene should give new insights into molecular understanding on the early mammalian development.


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