טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentTsabar Nir
SubjectA Functional Screen to Identify Genes Involved in Neural
Crest Induction
DepartmentDepartment of Medicine
Supervisor Professor Dale Frank


Abstract

A genetic screen in the frog Xenopus laevis aimed at finding genes involved in neural crest induction. A cDNA library was constructed and validated to contain various novel Xenopus genes. Transcribed RNA was injected to one side of the embryos, and embryo development was monitored. Different phenotypes were found, although no novel neural crest induction. A pigment dislocation phenotype led to the cloning of XDel1 partial sequence (D2).

Injection of D2 after fertilization dissociated pigment from the cell membrane to form central aggregates. In contrast, immature oocytes had only a week dislocation response. The D2 sequence contains only XDel1's C-terminal discoidin domain. D2-eGFP chimeric protein located near the cell membrane. XDel1 mRNA expression was found to be diffuse and continuous from early oocyte to tail-bud stages, and enriched in the cement gland. Ectopic chimeric full-length-eGFP protein also localized in the cement gland as well as around the vitelline membrane. Various deletion or full length constructs or antisense oligonucleotides didn't induce any developmental phenotype.

            Del1 sequence analysis and literature review suggests a combined binding capability to RGD receptors, sulfated-glycans and phosphatydil-serine rich membranes and that the secreted XDel1 protein may be internalized to the cytosol.

            In conclusion, expression of XDel1 begins before fertilization. XDel1 binding capability suggests internalization to the cytosol. Pigment granules gain the ability to react to the cytosolic XDel1 protein between oocytes maturation and fertilization. Participation of XDel in fertilization is thus possible. Expression in the cement gland may imply an additional role in the hatching process.