The zinc-finger protein CNBP is required for craniofacial development in Zebrafish

WEINER, A.; ARMAS, P.; ALLENDE, M.; BECKER, T.; CALCATERRA, N. B.

Los Cocos, Córdoba, Argentina.

Simposio; 1st International Workshop “Latest concepts in Developmental Biology; 2006

Society of Developmental Biology

Cellular nucleic acid binding protein (CNBP) is a single-stranded nucleic acid binding protein with nucleic acid chaperone activity. It is a small protein made of seven CCHC zinc knuckles and an RGG box between the first and two zinc knuckles. Although CNBP is strikingly conserved among vertebrates, its biological function has not been defined yet. Cellular nucleic acid binding protein (CNBP) is a single-stranded nucleic acid binding protein with nucleic acid chaperone activity. It is a small protein made of seven CCHC zinc knuckles and an RGG box between the first and two zinc knuckles. Although CNBP is strikingly conserved among vertebrates, its biological function has not been defined yet. Cellular nucleic acid binding protein (CNBP) is a single-stranded nucleic acid binding protein with nucleic acid chaperone activity. It is a small protein made of seven CCHC zinc knuckles and an RGG box between the first and two zinc knuckles. Although CNBP is strikingly conserved among vertebrates, its biological function has not been defined yet. In this work we analyzed zebrafish CNBP gene general organization, developmental expression pattern and in vivo function. The CNBP gene is organized in five exons and four introns, being noticeable the absence of a predicted canonical promoter region. Putative promoter regions were analyzed by embryo microinjection of mRNA carrying different DNA fragments fused to the reporter gene EGFP. A region within the first intron was identified as responsible for specific developmental expression. Expression analysis by in situ hybridization showed homogeneous CNBP-mRNA distribution in embryos up to 20 hours post-fertilization (hpf). Thereafter, the expression was specifically detected in eyes, tectum, myotomes, and skeletal system. After 48 hpf, expression in liver, pectoral fins, and retina was also observed. The protein showed a differential subcellular localization pattern. CNBP is cytoplasmic in pre-MBT blastomeres and becomes nuclear at post-MBT developmental stages. The in vivo CNBP function was studied by transient modification of the embryonic protein level by means of Morpholino or specific anti-CNBP antibody embryo microinjection. Lower levels of CNBP induced severe abnormalities in the midbrain/hindbrain border and craniofacial structures formation. In situ hybridization analysis of key tissue marker genes did not show significant differences between treated and control embryos. However, c-myc, foxD3, and col2A1 expression was clearly reduced in treated embryos. Experiments using anti-H3 and anti-PCNA antibodies, as well as Br-dU labeling assays showed higher cell proliferation levels in cephalic regions of affected embryos than those observed in control ones. Finally, TUNEL assay showed higher level of apoptosis in the same regions. Based on these results, we propose that CNBP is involved in neural crest specification and differentiation and cell proliferation/apoptosis control during zebrafish embryonic development.