Regulation Of Development Of Gold Fish, Carassius Auratus By Mitogen-Activated Protein Kinases

Mitogen-activated protein kinases (MAPKs), a family of special protein kinases, exist in most cells and play significant roles in signal transduction from extra-cellular circumstances to nuclei, which mediate various cellular processes such as proliferation, differentiation, transformation and apoptosis. Through Western blot analysis,immunoprecipitation-linked kinase assays,and microinjection of the dominant negative mutant RAF,MEK and ERK, the expression patterns of MAPKs and the function of the ERK signaling pathway on development of gold fish have been investigated and the results are summarized below:(1),The three types of MAPKs are differentially expressed in lens epithelium, lens fiber, retina, heart, brain and muscle of gold fish with distinct tissue specificity. Among three types of MAPKs, the expression and activities of the extracellular signal-regulated kinases (ERK1/2) are the most abundant in all of the six tissues examined. The expression of c-jun N-terminal kinase 1 (JNK1) is only observed in lens fiber, retina and brain tissues. In contrast, expression of JNK2 is detected in retina, heart, brain and muscle with minor expression in lens epithelium. The expression of p38 is detected in retina, heart, brain and muscle. However, its activity is absent in all the tissues of gold fish examined.(2),The whole length open reading frame (ORF) of gold fish ERK2 gene has been cloned. It is 1100 bp long and encodes a protein of 369 amino acid residues. A decreased homology can be obtained of gold fish ERK2 gene to those of zebrafish, mouse, rat, human, dog, chicken, bovine and Xenopus laevis, among which zebrafish is the highest while Xenopus laevis the lowest. However, at the amino acid level, the highest homology is of gold fish ERK2 to zebrafish, and to Xenopus laevis the second high, while the lowest to mouse or rat, despite an 89.9% high score can still be drawn to both of these rodents. These data greatly contribute to further exploring the regulatory mechanism of ERK and the understanding of involved transcription factors in the development process of gold fish.(3),It has been analyzed of the two major MAP kinases of ERK2 and JNK1 in nine different gold fish tissues in ten gold fish developing stages at the mRNA level. Experimental results show that in different early embryonic developing stages of gold fish, the two genes of ERK and JNK are highly expressed, which demonstrates a time-dependent pattern. Meanwhile, ERK and JNK are widely expressed in different tissues of adult gold fish, and an apparent tissue-specific difference can be observed among all these tissues, among which ovary expression is the highest. All these results indicate that ERK and JNK are two important protein kinases in gold fish.(4),Microinjection of dominant negative mutant ERK( DNM-ERK) into gold fish zygotes apparently blocks the ERK signaling pathway of the injected embryos as reflected by the attenuated or abolished ERK activity at different stages of development. When the ERK signaling pathway is blocked by DNM-ERK, the development of gold fish is severely interrupted as shown by the appearance of abnormal phenotypes of stunted embryos and abnormal phenotypes which include abnormal shape of ocular lens, asymmetric pigment, reduced somite number, curled tails, reduced heartbeat, unconspicuous movement of muscles and high lethality rate, etc.These results indicate ERKs play an impotant role in vertebrate embryo development.(5),The DNMJNK gene has been microinjected into zygote of gold fish to explore its affection on development after blocking the JNK signaling pathway. Experimental results display an absolutely necessary role of JNK in gold fish development, for the inhibition of JNK signaling pathway can obstruct the formations of many gold fish organs, especially eye, nervous tube and muscle. The present experiment demonstrates that JNK plays an important leading role in the early embryonic developing period of vertebrate, and this firstly confirms that JNK serves a significant function in vertebrate eye development by the method of functional absence.