Cloning and Characterization of ifitm1 and ifitm3 Expression During Early Zebrafish Development - CORRIGENDUM.

The authors apologise for errors in the corresponding authors details given on page 1 of the article. Below is the correct information of the corresponding author and email address : 1) Wei-Wei Xue, Huan-Nan Wang, Zhi-Meng Wang, Meng-Xi Qiu, Jing Che, Feng-Jiao Deng* and Jiang-Dong Liu* 2) *All correspondence to: Feng-Jiao Deng and Jiang-Dong Liu. e-mail: fish4@whu.edu.cn 3) All authors are from the same one laboratory. The second laboratory was superfluous and should be deleted.

Cloning and characterization of ifitm1 and ifitm3 expression during early zebrafish development.

The family of interferon-inducible transmembrane proteins (IFITMs) plays a crucial role in inhibiting proliferation, promoting homotypic cell adhesion and mediating germ cell development. In the present study, the full-length cDNAs of zebrafish ifitm1 (744 bp) and ifitm3 (702 bp) were obtained by rapid amplification of cDNA ends (RACE). Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that ifitm1 mRNA was expressed in the ovary, testis, brain, muscle, liver and kidney, while ifitm3 mRNA was only detected in the ovary. Based on in situ hybridization, ifitm1 mRNA was found to be strongly expressed in the ooplasm from stage I to stage II and ifitm3 mRNA was also strongly expressed in the ooplasm from stage I to stage II, furthermore ifitm3 expression ultimately localized to the cortex region beneath the plasma membrane of stage IV oocytes. During development, ifitm1 expression was initially detected in the enveloping layer cells and deep layer cells of shield stage embryos. Then, throughout the segmentation phase (10.25-24 hours post-fertilization (hpf)), ifitm1 expression was mainly detected in the head, trunk and tail regions. Unlike ifitm1, ifitm3 expression was initially detected in sphere stage embryos and was then broadly expressed throughout the embryo from the 70% epiboly stage to 24 hpf. Interestingly, ifitm3 was also expressed in primordial germ cells (PGCs) from the bud stage to 24 hpf. This expression analysis indicates that zebrafish ifitm1 may play a critical role in early organogenesis and may perform immune or hematopoietic functions and ifitm3 might be necessary for PGC migration and the formation of female germ cells.

Identification and characterization of the pumilio-2 expressed in zebrafish embryos and adult tissues.

Pumilio proteins regulate the translation of specific proteins required for germ cell development and morphogenesis. In the present study, we have identified the pumilio-2 in zebrafish and analyze its expression in adult tissues and early embryos. Pumilio-2 codes for the full-length Pumilio-2 protein and contains a PUF-domain. When compared to the mammalian and avian Pumilio-2 proteins, zebrafish Pumilio-2 protein was found to contain an additional sequence of 24 amino acid residues within the PUF-domain. Zebrafish pumilio-2 mRNA is expressed in the ovary, testis, liver, kidney and brain but is absent in the heart and muscle as detected by RT-PCR. The results of in situ hybridization indicate that transcripts of pumilio-2 are distributed in all blastomeres from the 1-cell stage to the sphere stage and accumulate in the head and tail during the 60%-epiboly and 3-somite stages. Transcripts were also detected in the brain and neural tube of the 24 h post-fertilization (hpf) embryos. Western blot analyses indicate that the Pumilio-2 protein is strongly expressed in the ovary, testis and brain but not in other tissues. These data suggest that pumilio-2 plays an important role in the development of the zebrafish germ cells and nervous system.

Cloning and characterization of the SSB-1 and SSB-4 genes expressed in zebrafish gonads.

The protein of the gustavus (gus) gene has a typical SOCS box domain and repeats in the splA and RyR (SPRY) domains. GUS can interact with Vasa and is necessary for the specification of germ cells. We cloned two zebrafish genes, SSB-1 and SSB-4 (SPRY domain SOCS box proteins). Phylogenetic analysis shows that zebrafish SSB-1 and SSB-4 are clustered into clades of SSB-1-like and SSB-4-like genes from other species. RT-PCR analysis of tissues revealed that zebrafish SSB-1 and -4 are expressed in the ovary and testis. We investigated the spatial expression patterns of zebrafish SSB-1 and -4 in embryos from the two-cell stage to 72 h postfertilization (hpf) using whole-mount in situ hybridization. SSB-1 and -4 transcripts were present in all blastomeres during the early embryonic stages, but the genes differ in their expression pattern. SSB-4 mRNA was located in the region of the primordial germ cells in 24 and 72 hpf embryos, but SSB-1 mRNA was not detected at these stages. We hypothesize that SSB-4 plays a role in the early development of germ cells.

[18S-ITS1 sequence of rRNA in bivalves and its application in phylogenetic analysis].

Bivalves constitute a dominant and diverse group of marine animals in China, most of them are of major commercial important species, and studies of their genetic diversity are necessary for the sustainable exploitation and conservation of these bioresources. The objective of the present work is to explore the feasibility of using the ribosomal RNA as a molecular marker for studying the interspecific and intraspecific genetic variations among bivalves. The 18S-ITS1 sequences of 11 individuals at differing taxonomic levels were determined. The sequence were found to exhibit a high degree of length polymorphism among different species, ranging from 558 bp in C.farreri to 784 bp in O. rivularis, mainly resulting from the variation of ITS1, and the percent divergence ranging from 10.7% to 61.7%. The NJ (neighbor-joining) tree inferred from 18S fragment agrees with the previous study based on morphologies and chemical analysis well. The sequence variation was found to vary among 4 individuals of P. martensi (0.6% approximately 1.9%), collected from 4 geographical sites, which involved substitutions,transversion as well as insertions and deletions. All these results show that ITS1 is highly divergent among different species of bivalves and could be used in classification and distinguishing closely related species, and also could be used for molecular systematic studies at relative species, subspecies and population levels.

[Genetic diversity analysis of mitochondrial D-loop region of Chinese sucker (Myxocyprinus asiaticus)].

Chinese sucker, Myxocyprinus asiaticus, is an endemic species of China and also the only representatives of family Catostomidae in Asia. The fish was naturally distributed in Yangtze River and Mingjiang River and now few could be captured because of pollution and overexploitation. The fish has been listed in the second class of preserved animal in China. Studying and assessing its population structure is an imperative and fundamental work for making effective protection strategies. We amplified and sequenced the D-loop region of mtDNA of 8 samples. The size of the D-loop region is about 958 bp. A total of 32 variation loci were detected and the mutation rate was 0.033. All the mutation came from nucleotide substitution except one nucleotide deletion. Most of the nucleotide variations were found between the 55-365 bp region. The individual mutation rate varied from 0-1.36%, which exhibited nucleotide polymorphism to some extent among 8 samples. Compared with RAPD and other PCR-based methods, the directily sequencing of mtDNA D-loop region revealed much more genetic diversity. Meanwhile, the D-loop region of Moxostoma robustum derived from GenBank was aligned with that of Chinese sucker through CLUSTAL software. By comparison, we found that the mutation rate (0.033) of D-loop of Chinese sucker is higher than that of Moxostoma robustum (0.016).