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Expression and Functional Analysis of Dkk and Kremen Genes in Amphioxus (Branchiostoma belcheri)

Authors
Publication Date
Keywords
  • 文昌鱼
  • 白氏文昌鱼
  • Dkk
  • Kremen
  • Gata Factors
  • Otx
Disciplines
  • Biology

Abstract

The Cephalochordate amphioxus, widely considered as a model of vertebrate ancestor, is a key organism for understanding of the origin and evolution of vertebrate developmental mechanisms. Wnt signaling pathway is well conserved from cnidarians to vertebrates and play versatile roles during embryonic development, such as axis formation, germ layer specification and neural system development. Its ancestral function is suggested to be regulation of animal-vegetal axial polarity and germ layer segregation, while in vertebrates, it plays key roles in the dorsal-ventral axis formation at early stages and later ones in the anterior-posterior patterning and left-right asymmetry of the body. Our researches focus on the analysis of two families of Wnt regulators, Dickkopf (Dkk), Kremen in amphioxus. Secreted protein Dkk and its receptor Kremen are Wnt signaling inhibitors, which are expressed in anterior region in vertebrates and regulate anterior embryonic development. Based on vertebrate Dkk and Kremen sequences and analysis of amphioxus (Branchiostoma floridae) genome, we have cloned two Dkk family genes: BbDkk124, BbDkk3 and five Kremen genes: BbKremen-a,BbKremen-c,BbKremen-d,BbKremen-e, BbKremen-g in amphioxus (Branchiostoma belcheri). We studied their expression patterns and tested their ability to inhibit Wnt signaling in reporter gene analysis in 293T cells as well as phenotypic analysis in Xenopus embryos. Our results suggest their expression patterns are not conserved compared with vertebrate homologs. However, the function of BbDkk124 as a Wnt signaling inhibitor is conserved and when injected in Xenopus embryos, it can induce anteriorized phenotypes with big heads and short trunks. BbKremen-e and The Cephalochordate amphioxus, widely considered as a model of vertebrate ancestor, is a key organism for understanding of the origin and evolution of vertebrate developmental mechanisms. Wnt signaling pathway is well conserved from cnidarians to vertebrates and play versatile roles during embryonic development, such as axis formation, germ layer specification and neural system development. Its ancestral function is suggested to be regulation of animal-vegetal axial polarity and germ layer segregation, while in vertebrates, it plays key roles in the dorsal-ventral axis formation at early stages and later ones in the anterior-posterior patterning and left-right asymmetry of the body. Our researches focus on the analysis of two families of Wnt regulators, Dickkopf (Dkk), Kremen in amphioxus. Secreted protein Dkk and its receptor Kremen are Wnt signaling inhibitors, which are expressed in anterior region in vertebrates and regulate anterior embryonic development. Based on vertebrate Dkk and Kremen sequences and analysis of amphioxus (Branchiostoma floridae) genome, we have cloned two Dkk family genes: BbDkk124, BbDkk3 and five Kremen genes: BbKremen-a,BbKremen-c,BbKremen-d,BbKremen-e, BbKremen-g in amphioxus (Branchiostoma belcheri). We studied their expression patterns and tested their ability to inhibit Wnt signaling in reporter gene analysis in 293T cells as well as phenotypic analysis in Xenopus embryos. Our results suggest their expression patterns are not conserved compared with vertebrate homologs. However, the function of BbDkk124 as a Wnt signaling inhibitor is conserved and when injected in Xenopus embryos, it can induce anteriorized phenotypes with big heads and short trunks. BbKremen-e andBbKremen-g are functionally different in Xenopus embryos. These results provide important clue on the evolution of Dkk and Kremen functions. We also studied the amphioxus GATA factors, which are evolutionarily conserved and play crucial roles during embryonic development in both vertebrates and invertebrates. Vertebrate GATAs can be divided into two subgroups, the GATA1/2/3 and the GATA4/5/6 classes. Through genomic analysis, we have identified three GATA factors, representing the GATA1/2/3 and GATA4/5/6 subfamilies respectively, and one GATA like protein in the genome of amphioxus (Branchiostoma floridae, cephalochordata), the most basal chordate subphylum. We have cloned partial sequence of GATA123 in the amphioxus Branchiostoma belcheri (BbGATA123) and studied its expression pattern during early embryonic development. Expression of BbGATA123 is first detected in the mesendoderm during gastrulation. Interestingly, in the late neurula and early larva stages, it is expressed strongly in the cerebral vesicle and the mid gut region. Its expression is compared to Otx, a gene known crucial for the development of anterior structures. Our data suggest that GATA123, together with Otx, might play an important role in the development of amphioxus cerebral vesicle, the counterpart of the vertebrate brain.

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