Effects of external environmental and internal genetic factors on Sonic Hedgehog pathway in zebrafish embryonic development
外在環境與內在基因因素對斑馬魚胚胎發育中 Sonic Hedgehog 途徑之影響
Student thesis: Master's Thesis
In the course of embryonic development, an embryo employs a variety of mechanisms to gain size and complexity. While the developing process is precisely programmed, it may be susceptible to undesirable disturbances, both genetically and environmentally. The hedgehog (Hh) signaling pathway plays a crucial role in determining cell fate and patterning during development, and any perturbation to the pathway may result in developmental defects. Exposure of vertebrate embryos to ethanol, a well-known environmental teratogen, was discovered to give rise to cyclopia. Meanwhile, symptoms arisen from sonic hedgehog (SHH) mutations were observed to resemble those of fetal alcohol syndrome (FAS). To estimate the effects of ethanol on shh expression, 2.4% ethanol served as an inducing agent of cyclopia and any craniofacial malformations in zebrafish embryos. While comparing with the control groups, only half of the zebrafish embryos exposed to ethanol grew normally, regardless of their age. For the 24-hpf ethanol treated embryos, 18.30% and 25.11% were scored craniofacial malformations and type A cyclopia, respectively, indicating that ethanol effectively disturbed normal craniofacial development, especially midline development of the eye field. As the embryos grew older from 24 hpf to the fifth day, other deformities like reduction of eye size, narrowing of eye distance, and more severe types of cyclopia began to dominate, suggesting there might be a slight shift in the targets of ethanol. Besides, ethanol exposure was observed to result in a decrease of shh expression levels in telencephalon and presumptive hypothalamus of zebrafish embryos. In view of the influences of both external and internal factors in signal transduction, other than Hh itself, I attempted to look into one of the other components in the pathway. Costal2 (Cos2) has been identified in Drosophila as a negative regulator of the Hh pathway. Recently, a Cos2 homolog was cloned in zebrafish by degenerate PCR and was annotated as kinesin superfamily protein 7 (KIF7). In this project, gene-specific primers were used to amplify zebrafish KIF7 (namely ZfKIF7 in this project). The isolated clone consists of a 99-bp 5’ untranslated region (UTR), a 4092-bp open reading frame (ORF) encoding 1364 amino acids and a 3’UTR of 155 bp. Both RT-PCR and whole mount in situ hybridization demonstrated that ZfKIF7 was strongly expressed in developing zebrafish embryos. Furthermore the expression profile was rather broad throughout the body. At 48 hpf, the majority of ZfKIF7 mRNA was localized at the mid-hindbrain boundary (MHB) and ventral hindbrain. Upon further development and growth, expression of ZfKIF7 mRNA faded gradually and was hardly detected in 76- and 92-hpf juveniles, as well as in adult tissues, thus suggesting that ZfKIF7 may be important in early embryonic development and of lesser significance at later stages of development. Furthermore, the zebrafish kinesin superfamily protein member 4 (ZfKIF4), being itself resembling cos2, was identified during the course of cloning ZfKIF7. KIF4 was originally recognized as the hypothetical protein MGC66125 (Accession BC060673) comprising 206 bp of 5’ UTR, a 3747-bp ORF encoding 1248 amino acids and a 3’UTR with 1541 bp. ZfKIF4 was strongly and ubiquitously expressed in early developing zebrafish embryos; whereas only low levels of ZfKIF4 mRNA were observed in adult brain, muscle and testis. In 48-hpf embryos, ZfKIF4 expression was localized at the eyes, MHB, and along the cleavage furrow of the hindbrain. Such observations illustrated that expression of ZfKIF4 may be regulated by developmental stage and possibly related to zebrafish embryonic neurodevelopment. At 72 hpf, ZfKIF4 expression was restricted to the retinal ganglion cell layer (GCL), where the vast majority of neurons originate to the eyes, the MHB and along the hindbrain cleavage furrow, where numerous mitotic cells are situated. It was suggested, therefore, that ZfKIF4 may participate in mitosis as its homologs in other organisms do. Since mitosis is required for growth and development of all cell types, it was not surprising to detect ubiquitous expression of ZfKIF4 at early embryonic stages and to see such a wide expression pattern throughout the whole body of zebrafish embryos.
- Animal models, Neurogenetics, Zebra danio, Embryos