Nörotrofin reseptörü ile ilişkili ölüm bölgesi proteininin Wnt/ β - katenın sinyal iletiminin düzenlenmesindeki rolünün zebrabalığı embriyonik modeli ve CRISPR/Cas9 knockout modeli ile araştırılması

Wnt/β-catenin signaling pathway plays essential roles in many biological processes including cell proliferation, migration, differentiation and apoptosis during development and adulthood. Many regulatory molecules of the pathway were revealed by researchers over the years. Yet, the whole mechanism is a puzzle with missing pieces, waiting to be solved. In this study, we identified neurotrophin receptor-associated death domain (nradd), a homolog of p75 neurotrophin receptor (p75NTR), as a negative feedback regulator of Wnt/β-catenin signaling in zebrafish embryos. We revealed that nradd overexpression significantly suppresses Wnt8-mediated/β-catenin signaling which controls mesoderm and neuroectoderm patterning during gastrulation. Also, its overexpression suppresses the expression of Wnt-target genes: Lymphoid Enhancer Binding Factor 1(Lef1), Sp5 transcription factor-like (Sp5l), and conductin (Axin2) and can rescue the effect of high Wnt8-mediated/β-catenin signaling activity, loss of forebrain and eyes. In addition, we investigated nradd F0 knockout model generated by CRISPR/Cas9 gene-editing. To observe nradd loss-of-function efficiently in F0 embryos, we injected Cas9 protein with 4 guide RNAs targeting nradd gene to one-celled embryos. Our approach is significantly suppressed nradd expression in F0 crispants. In crispants, Wnt-target genes' expressions are significantly upregulated, supporting the negative regulator role of Nradd in Wnt/β-catenin signaling. Moreover, we also analyzed the apoptosis level in crispants to evaluate the defined apoptotic function of nradd by previous studies. Interestingly, we observed an increased apoptosis level in crispant embryos at 24 hours-post-fertilization (hpf), confirmed by increased expression level of pro-apoptotic genes and increased immunoreactivity of cleaved-caspase-3 antibody. In summary, Nradd acts as a negative feedback modifier of Wnt/β-catenin signaling and a regulator of cell death during embryonic development. As the pathway modifiers serve as attractive drug targets especially in targeted therapies for cancer, further characterization of Nradd at the molecular level will significantly contribute to both elucidations of as yet unknown pathway mechanisms and the discovery of novel drugs targeting the pathway.