|Budget Amount *help
¥7,200,000 (Direct Cost: ¥7,200,000)
Fiscal Year 1997: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1996: ¥5,200,000 (Direct Cost: ¥5,200,000)
Chick limb development has been extenisvely studied as a model system for pattern formation. Recently, a retrovirus system with RCAS vector has been developed to misexpress a cDNA in chick embryos. Although RCAS vectoris very useful, the size of insert in the vector is restricted to be within 1.6kbp. Thus, it is very difficult to misexpress large cDNAs. To improve this problem, we have tried to develop a new retroivrus system. However, it has not succeded yet. Instead, recently electroporation method has been shown to introduce cDNAs in chick embryos in ovo. Thus, we have also examined the method. We found that efficiency is high in neural tissues, but not in limb mesenchyme. We are now still trying to know conditions for electroporation of cDNAs in limb.
A limb initiation factor has been searched by many investigators. Recently, we reported that members of the fibroblast growth factor (FGF) family can induce addional limb formation in the flank of chick embryos. FGF thus might be a com
ponent of the endogenous signaling cascade of limb initiation. In addition to functional studies, examination of gene expression patters is pivotal to elucidate molecular mechanisms underlying in vivo developmental phenomena. Since we found that FGF10 is expressed in the prospective limb mesoderm and to induce an ectopic limb in the flank, FGF10 is most likely to be a limb-forming factor in the lateral plate mesoderm so far. We also have studied the mechanisms for fore- and hindlimb specification with the FGF-induced ectopic limb. Ectopic limbs located in the mid-flank appear chimeric ; feather buds are present in the anterior halves, while scales and claws are formed in the posterior halves. Expression patterns of Tbx5 (a forelimb marker) and Tbx4 (a hindlimb marker) genes verified the chimerism : Tbx5 aand 4 were expressed in the anterior and posterior halves of additional limb buds, respectively. This implies that Tbx5 and 4 genes are involved in determination of the forelimb and hindlimb identities, respectively. Less