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A complete cDNA of a novel zebrafish gene named onecut has been isolated; this gene encodes a protein of 446 amino acids with a Cut domain (73 amino acid residues) and a homeodomain. The Cut domain of zebrafish Onecut is highly similar to those in mammalian hepatocyte nuclear factor-6 and Drosophila Onecut, sharing 90 and 88% amino acid identity, respectively. The expression of zebrafish onecut is restricted to neuronal cells, being first detected in trigeminal ganglia neurons at the end of gastrulation. By the 1-somite stage, onecut expression has begun in primary neurons of the lateral stripes in the neural plate, and appeared in neuronal cells of the medial stripes at the 2-somite stage. By the 4-somite stage, onecut expression expanded to the intermediate stripes and to subsets of neuronal cells in the midbrain and hindbrain. Subsequently, onecut expression intensified in the lateral region of midbrain and hindbrain, yet no onecut-positive cells were seen in the telencephalon. By 24hpf, onecut transcripts remained abundant in the spinal cord but were no longer detectable in differentiated Rohon-Beard sensory neurons. The expression of onecut was greatly increased in the neural mutant mindbomb, while being decreased in narrowminded.

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During skeletal development, both osteogenic and chondrogenic programs are initiated from multipotent mesenchymal cells, requiring a number of signaling molecules, transcription factors, and downstream effectors to orchestrate the sophisticated process. Col10a1, an important downstream effector gene, has been identified as a marker for maturing chondrocytes in higher vertebrates, such as mammals and birds. In zebrafish, this gene has been shown to be expressed in both osteoblasts and chondrocytes, but no study has reported its role in osteoblast development. To initially delineate the osteogenic program from chondrogenic lineage development, we used the zebrafish col10a1 promoter to establish a transgenic zebrafish expressing a GFP reporter specifically in osteoblast-specific bone structures that do not involve cartilaginous programs. A construct harboring a ~2.2-kb promoter region was found to be sufficient to drive the reporter gene in osteoblast-specific bone structures within the endogenous col10a1 expression domain, confirming that separable cis-acting elements exist for distinct cell type-specific expression of col10a1 during zebrafish skeletal development. The ~2.2-kb col10a1:GFP transgenic zebrafish marking only bone structures derived from osteoblasts will undoubtedly be an invaluable tool for identifying and characterizing molecular events driving osteoblast development in zebrafish, which may further provide a differential mechanism where col10a1 is involved in the development of chondrocytes undergoing maturation in other vertebrate systems.

Estrogen-related receptor alpha (ESRRa) regulates a number of cellular processes including development of bone and muscles. However, direct evidence regarding its involvement in cartilage development remains elusive. In this report, we establish an in vivo role of Esrra in cartilage development during embryogenesis in zebrafish. Gene expression analysis indicates that esrra is expressed in developing pharyngeal arches where genes necessary for cartilage development are also expressed. Loss of function analysis shows that knockdown of esrra impairs expression of genes including sox9, col2a1, sox5, sox6, runx2 and col10a1 thus induces abnormally formed cartilage in pharyngeal arches. Importantly, we identify putative ESRRa binding elements in upstream regions of sox9 to which ESRRa can directly bind, indicating that Esrra may directly regulate sox9 expression. Accordingly, ectopic expression of sox9 rescues defective formation of cartilage induced by the knockdown of esrra. Taken together, our results indicate for the first time that ESRRa is essential for cartilage development by regulating sox9 expression during vertebrate development.

Background: Chitinase 3 like 1 protein (Chi3L1) is expressed in several cancers, and a few evidences suggest that the secreted Chi3L1 contributes to tumor development. However, the molecular mechanisms of intracellular Chi3L1 are unknown in the lung tumor development. Methods: In the present study, we generated Chi3L1 knockout mice (Chi3L1 KO(−/−) ) using CRISPR/Cas9 system to investigate the role of Chi3L1 on lung tumorigenesis. Results:We established lung metastasis induced by i.v. injections of B16F10 in Chi3L1 KO(−/−) . The lung tumor nodules were significantly reduced in Chi3L1 KO(−/−) and protein levels of p53, p21, BAX, and cleaved-caspase 3 were significantly increased in Chi3L1 KO(−/−) , while protein levels of cyclin E1, CDK2, and phsphorylation of STAT3 were decreased in Chi3L1 KO(−/−) . Allograft mice inoculated with B16F10 also suppressed tumor growth and increased p53 and its target proteins including p21 and BAX. In addition, knockdown of Chi3L1 in lung cancer cells inhibited lung cancer cell growth and upregulated p53 expression with p21 and BAX, and a decrease in phosphorylation of STAT3. Furthermore, we found that intracellular Chi3L1 physically interacted and colocalized with p53 to inhibit its protein stability and transcriptional activity for target genes related with cell cycle arrest and apoptosis. In lung tumor patient, we clinically found that Chi3L1 expression was upregulated with a decrease in p53 expression, as well as we validated that intracellular Chi3L1 was colocalized, reversely expressed, and physically interacted with p53, which results in suppression of the expression and function of p53 in lung tumor patient.Conclusions: Our studies suggest that intracellular Chi3L1 plays a critical role in the lung tumorigenesis by regulating its novel target protein, p53 in both an in vitro and in vivo system.

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