Novel and unexpected functions of zebrafish CCAAT box binding transcription factor (NF-Y) B subunit during cartilages development

Abstract

We used zebrafish as a model to study the biological functions of NF-YB during early development. Both RT-PCR and whole-mount in situ hybridization experiments revealed that nf-yb was a maternally inherited gene. Later, its expression was restricted in the future head cartilages as well as in the developing notochord. Embryos after injection with nf-yb-morpholino displayed reduced-head phenotypes, including smaller head (WT, length of head, L: 0.515 ± 0.019 mm, width of head, W: 0.323 ± 0.077 mm; nf-yb-morphant, L: 0.347 ± 0.037 mm; W: 0.266 ± 0.018 mm), sharpen Meckel's cartilage, loss of ceratobranchial, and enlarged angles of ceratohyal (WT: 72.6 ± 9.4°; nf-yb-morphant: 110.0 ± 32.5°). Subsequently, those abnormalities can be rescued after injection with capped nf-yb mRNA. TUNEL assay suggested that large amounts of cell apoptosis appeared in the head region of nf-yb-morphants. Staining with digoxigenin-labeled dlx2a, sox9a, runx2b and col2a1 riboprobes showed that nf-yb-morphants displayed reduced amounts of cranial neural crest cells which are required for mandibular and branchial arches formation. These observations clearly indicate that knockdown of nf-yb translation induced parts of cranial neural crest cells apoptosis, affected cartilages formation and consequently caused reduced-head phenotypes. These findings uncover a novel and unexpected role for NF-YB as a critical modulator of neural crest cell's gene expression governing embryonic cartilage growth.

Introduction

Nuclear factor Y (NF-Y) is a heteromeric transcription factor which is composed of three subunits, NF-YA, NF-YB and NF-YC that specifically binds to CCAAT motif in many eukaryotic genes [1], [2], including plant genes [3], [4] or genes expressed in specific cell types, such as bone sialoprotein (bsp), blood abo and lymphocytes trbp1 and rag1 genes [5], [6], [7], [8]. In addition, NF-Y also can regulate the gene expression during the cell cycle, such as cyclin B1, cdk2, and thymidine kinase[9], [10], [11], [12], [13], [14]. These observations indicate that NF-Y is capable of regulating widespread genes, including ubiquitously expressed and tissue-specific genes.

The embryonic expression of NF-Y has been reported in some species. In Caenorhabditis elegans, the expression of both nf-ya is restricted to the gonads and developing embryos, whereas nf-yb and nf-yc are also present in the pharyngeal bulb, in the neurons of ganglia surrounding the pharynx and in sensory organs of the head, indicating that NF-Y is not ubiquitously expressed [15]. In toad, nf-yb expression in oocyte is differential regulated by cultivation temperature [16]. These observations strongly suggest that NF-Y might possess multiple biological functions. However, the physiological roles of each subunits of NF-Y during early embryonic development and the genes that are regulated by this transcription factor complex in vivo remains largely unclear thus far.

Using an animal model where endogenous NF-Y activity can be abrogated enable us to study the physiological roles of NF-Y in vivo more precisely. Inactivation of NF-YA in primary cultures of mouse embryonic fibroblasts, leads to a block in cell proliferation and inhibition of S phase or DNA synthesis, which is followed by induction of apoptosis, suggesting that the NF-YA is essential for cell proliferation and viability [17]. Disruption of both nf-ya alleles causes early embryo lethality, which makes it difficult to investigate the physiological roles of NF-Y during early mouse development [17]. Thus, development an alternative animal model is essential.

In zebrafish, mutation of nf-yc leads to retinal lamination defects, including lack of photoreceptor and breakdown in lamination between outer and inner nuclear layers [18]. Because of the transparent embryos and rapidly embryonic development, zebrafish is a better material for studying the biological functions of NF-Y in comparison with mouse model. To establish the role of NF-YB in vivo, we carried out morpholino approach to specifically inhibit the gene translation for the NF-YB subunit in zebrafish. Through loss-of-function and rescue experiments, we show that NF-YB affects the expression of runx2b, col2a1, dlx2a and sox9a, to promote cartilage development. This is the first report to elucidate the novel functions of NF-YB in the development of the cranial neural crest, pharyngeal cartilage and bone.