Deciphering the cellular and molecular roles of cellular nucleic acid binding protein during cranial neural crest development

Abstract

Cellular nucleic acid binding protein (Cnbp) is a highly conserved single-stranded nucleic acid binding protein required for rostral head development. The use of a morpholino that inhibits Cnbp mRNA translation previously revealed a role of Cnbp in balancing neural crest cell apoptosis and proliferation in the developing zebrafish. Here, we report the use of another morpholino that specifically modifies the splicing of Cnbp pre-mRNA resulting in a reduction of full-length mRNA levels along with the generation of a novel transcript coding for an isoform that may act as dominant negative proteins. The use of this morpholino resulted in more severe phenotypes that enabled us to demonstrate that Cnbp loss-of-function adversely affects the formation and survival of craniofacial cartilaginous structures not only controlling the ratio of cell proliferation and apoptosis but also defining skeletogenic neural crest cell fate.

Beyond the Binding Site: In Vivo Identification of tbx2, smarca5 and wnt5b as Molecular Targets of CNBP during Embryonic Development

“…Embryonic development was allowed to proceed until 24-hours post-fertilization (hpf), and the expression of candidate genes was measured by quantitative real time RT-PCR (qRT-PCR). Studies were performed on 24-hpf staged embryos because (i) at this developmental stage organogenesis has already started [19] and; (ii) phenotypes of CNBP depleted embryos (CNBP morphants) can be easily distinguished in developing zebrafish [4,5].…”

“…To address this, the behavior of candidate genes was assessed in vivo in CNBP-knock down experiments in developing zebrafish embryos. Embryos at the one/two-cell stage were injected with a Morpholino (MO) that specifically blocks cnbp pre-mRNA splicing (spl-MO) or with a miss-paired MO (mis-MO) as control, as reported [5]. Embryonic development was allowed to proceed until 24-hours post-fertilization (hpf), and the expression of candidate genes was measured by quantitative real time RT-PCR (qRT-PCR).…”

“…Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) revealed a direct interaction between CNBP and the consensus-binding site found in silico, as well as candidate genes. Moreover, the role of CNBP on the expression of candidate targets was further assessed in developing zebrafish depleted of CNBP by microinjecting a specific morpholino, validated in a previous report [5]. This work represents a novel approach towards understanding the biological function and regulatory networks involving CNBP in the biology of vertebrates.…”

“…CNBP loss-of-function adversely affects the formation and survival of a subpopulation of cranial neural crest (CNC) cells, leading to a reduction in size and even loss of selected pharyngeal and craniofacial cartilaginous structures in the developing zebrafish [4,5]. CNBP is also involved in muscle human diseases, such as myotonic dystrophy type 2 (DM2, proximal myotonic myopathy, OMIM # 602668) [6] and the age-related sporadic inclusion body myositis (sIBM) [7].…”

“…CNBP, formerly ZNF9, is a highly conserved nucleic acid chaperone [1] involved in proper organization of the zebrafish, chick, and mouse forebrain [2][3][4][5]. CNBP loss-of-function adversely affects the formation and survival of a subpopulation of cranial neural crest (CNC) cells, leading to a reduction in size and even loss of selected pharyngeal and craniofacial cartilaginous structures in the developing zebrafish [4,5].…”