prdm1a Regulates sox10 and islet1 in the development of neural crest and Rohon‐Beard sensory neurons

The PR domain containing 1a, with ZNF domain factor, gene (prdm1a) plays an integral role in the development of a number of different cell types during vertebrate embryogenesis, including neural crest cells, Rohon‐Beard (RB) sensory neurons and the cranial neural crest‐derived craniofacial skeletal elements. To better understand how Prdm1a regulates the development of various cell types in zebrafish, we performed a microarray analysis comparing wild type and prdm1a mutant embryos and identified a number of genes with altered expression in the absence of prdm1a. Rescue analysis determined that two of these, sox10 and islet1, lie downstream of Prdm1a in the development of neural crest cells and RB neurons, respectively. In addition, we identified a number of other novel downstream targets of Prdm1a that may be important for the development of diverse tissues during zebrafish embryogenesis. genesis 48:656–666, 2010. © 2010 Wiley‐Liss, Inc.     

Molecular cloning and gene expression of the prox1a and prox1b genes in the medaka,Oryzias latipes

Prox1 is a prospero-related homeobox gene. Prox1 is expressed in various internal organs and is related to those differentiations. Small fishes such as the zebrafish and the medaka are useful model animals in the clarification of the mechanism of development. The zebrafish prox1 is also identified, and it contributes to clarifying the function of prox1. However, it is necessary to note that many genes are duplicated in teleost fishes. In this study, we identified the orthologs of the mammalian prox1 gene in the medaka. The gene was also duplicated in the medaka, and we named it prox1a and prox1b. In silico analysis from the perspective of synteny indicated that medaka prox1a was similar to the prox1 gene of other vertebrates. Medaka prox1a was expressed in all internal organs that we have examined by RT-PCR. In contrast, medaka prox1b expression was limited to the brain, heart, liver, kidney, thymus, gill, testis, and ovary. This suggests that the two prox1 genes do not have a complementary relationship. In addition, we examined their expression patterns during embryonic development using whole-mount in situ hybridization. The expression pattern of prox1a showed a pattern similar to that of zebrafish prox1. In contrast, medaka prox1b was expressed asymmetrically in part of the central nervous system, especially strongly in the right side of the habenula.     

Characterisation and differential expression during development of a duplicate Disabled-1 (Dab1) gene from zebrafish

We identified a new duplicated Dab1 gene (drDab1b) spanning around 25 kb of genomic DNA in zebrafish. Located in zebrafish chromosome 2, it is composed of 11 encoding exons and shows high sequence similarity to other Dab1 genes, including drDab1a, a zebrafish Dab1 gene previously characterised. drDab1b encodes by alternative splicing at least five different isoforms. Both drDab1a and drDab1b show differential gene expression levels in distinct adult tissues and during development. drDab1b is expressed in peripheral tissues (gills, heart, intestine, muscle), the immune system (blood, liver) and the central nervous system (CNS), whereas drDab1a is only expressed in gills, muscle and the CNS, suggesting a division of functions for two Dab1 genes in zebrafish adult tissues. RT-PCR analysis also reveals that both drDab1 genes show distinct developmental-specific expression patterns throughout development. drDab1b expression was higher than that of drDab1a, suggesting a major role of drDab1b in comparison with drDab1a during development and in different adult tissues. In addition, new putative Dab1 (a and/or b) from different teleost species were identified in silico and predicted protein products are compared with the previously characterised Dab1, demonstrating that the Dab1b group is more ancestral than their paralogue, the Dab1a group.     

青鳉prdm14的原核表达、多克隆抗体制备及其应用

青鳉(Oryzias latipes)是研究遗传发育和细胞多能性的重要模式鱼类, 为探究prdm14同源基因的潜在作用, 实验将青鳉prmd14经原核表达后制备了兔抗Prdm14多克隆抗体。首先, 将prdm14基因的部分编码区连接到pET32a质粒中, 构建重组表达载体pET32a-prdm14?600。随后将重组载体转化至大肠杆菌(Escherichia coli)Rosetta(DE3), 经异丙基-β-d-硫代半乳糖苷(Isopropyl-β-d-thiogalactoside, IPTG)诱导表达, 获得分子量为60 kD的Prdm14重组蛋白。接着大量诱导蛋白表达并切胶纯化, 免疫家兔(Oryctolagus cuniculus), 6周后获得阳性抗体, 最后通过ELISA和Western blot检测抗体效价及其特异性。结果显示, 在37℃、0.6 mmol/L IPTG、诱导3h的条件下, 可获得Prdm14重组蛋白的高效表达; 制备的兔抗青鳉Prdm14多克隆抗体能够特异性识别青鳉组织中表达的Prdm14蛋白以及在HepG2细胞中过表达的青鳉Prdm14: EGFP融合蛋白。综上所述, 研究首次制备了一种能有效识别青鳉Prdm14的多克隆抗体, 该抗体的获得为后续研究prdm14基因在鱼类多能性干细胞中的作用提供了有力工具。     

The HMGB protein gene family in zebrafish: Evolution and embryonic expression patterns

The High-Mobility Group Box (HMGB) proteins are highly abundant proteins with both nuclear and extracellular roles in key biological processes. In mammals, three family members are present: HMGB1, HMGB2 and HMGB3. We characterized the HMGB family in zebrafish and report a detailed phylogenetic analysis of HMGB proteins. The B1, B2, and B3 subfamilies are present in cartilaginous fish, bony fish, and tetrapods, while jawless fish sequences emerge as basal to the gene family expansion. Two co-orthologs of each mammalian HMGB gene are present in zebrafish. All six zebrafish hmgb genes are maternally expressed, but huge differences in expression levels exist during embryonic development. The hmgb2a/hmgb2b genes are the most highly expressed, while hmgb3b is expressed at the lowest level. Remarkably, hmgb3 genes are not present in fugu, medaka, Tetraodon and stickleback. Our analysis highlights substantial overlaps, but also subtle differences and specificities in the expression patterns of the zebrafish hmgb genes.     

A zebrafish gene trap line expresses GFP recapturing expression pattern of foxj1b

Foxj1 has been found to play an important role in cilia formation and function in vertebrates. The zebrafish or Xenopus genome expresses two Foxjl genes, foxjlalFoxJ1 and foxjlb/FoxJ1.2. In this study, we have generated a zebrafish transgenic line T2BGSZIO by Tol2 transposon-based gene trapping approach. T2BGSZ10 transgenic fish carry an insertion of the transposon genome into the first intron of thefoxj1b locus. This insertion results in GFP expression in the forebrain, otic vesicles, floorplate, pronephric ducts and other domains during embryogenesis, which recaptures the expression pattern offoxj1b. Although normal expression offoxj1b is dramatically reduced, T2BGSZ10 homozygous embryos develop normally and grow to adulthood without detectable defects, which may be due to the incomplete interruption of foxjlb expression. Nevertheless, this transgenic line may serve as a useful model for dynamic observation of GFP-labeled tissues and organs and for isolation of GFP-labeled cells.     

inka1b expression in the head mesoderm is dispensable for facial cartilage development

Inka box actin regulator 1 (Inka1) is a novel protein identified in Xenopus and is found in vertebrates. While Inka1 is required for facial skeletal development in Xenopus and zebrafish, it is dispensable in mice despite its conserved expression in the cranial neural crest, indicating that Inka1 function in facial skeletal development is not conserved among vertebrates. Zebrafish bears two paralogs of inka1 (inka1a and inka1b) in the genome, with the biological roles of inka1b barely known. Here, we analyzed the expression and function of inka1b during facial skeletal development in zebrafish. inka1b was expressed sequentially in the head mesoderm adjacent to the pharyngeal pouches essential for facial skeletal development at the stage of arch segmentation. However, a loss-of-function mutation in inka1b displayed normal head development, including the pouches and facial cartilages. The normal head of inka1b mutant fish was unlikely a result of the genetic redundancy of inka1b with inka1a, given the distinct expression of inka1a and inka1b in the cranial neural crest and head mesoderm, respectively, during craniofacial development. Our findings suggest that the inka1b expression in the head mesoderm might not be essential for head development in zebrafish.     

sept8a and sept8b mRNA expression in the developing and adult zebrafish

Septins are highly conserved GTP-binding proteins involved in numerous cellular processes. Despite a growing awareness of their roles in the cell biology, development and signal transmission in nervous systems, comparably little is known about precise septin expression. Here, we use the well-established model organism zebrafish (Danio rerio) to unravel the expression of sept8a and sept8b, with special focus on the CNS. We performed whole mount RNA in situ hybridization on zebrafish 1–4 dpf in combination with serial sectioning of epon-embedded samples as well as on brain sections of adult zebrafish to obtain precise histological mapping of gene expression. Our results show a common expression of both genes at embryonic stages, whereas sept8a is mainly restricted to the gill arches and sept8b to specific brain structures at later stages. Brains of adult zebrafish reveal a large spatial overlap of sept8a and sept8b expression with few regions uniquely expressing sept8a or sept8b. Our results indicate a neuronal expression of both genes, and additionally suggest expression of sept8b in glial cells. Altogether, this study provides a first detailed insight into the expression of sept8a and sept8b in zebrafish and contributes to a more comprehensive understanding of septin biology in vertebrate model systems.     

Twist controls skeletal development and dorsoventral patterning by regulating runx2 in zebrafish

Background

Twist1a and twist1b are the principal components of twists that negatively regulate a number of cellular signaling events. Expression of runx2 and downstream targets is essential for skeletal development and ventral organizer formation and specification in early vertebrate embryos, but what controls ventral activity of maternal runx2 and how twists function in zebrafish embryogenesis still remain unclear.

Methodology/Principal Findings

By studying the loss of twist induced by injection of morpholino-oligonucleotide in zebrafish, we found that twist1a and twist1b, but not twist2 or twist3, were required for proper skeletal development and dorsoventral patterning in early embryos. Overexpression of twist1a or twist1b following mRNA injection resulted in deteriorated skeletal development and formation of typical dorsalized embryos, whereas knockdown of twist1a and twist1b led to the formation of abnormal embryos with enhanced skeletal formation and typical ventralized patterning. Overexpression of twist1a or twist1b decreased the expression of runx2b, whereas twist1a and twist1b knockdown increased runx2b expression. We have further demonstrated that phenotypes induced by twist1a and twist1b knockdown were rescued by runx2b knockdown.

Conclusions/Significance

Together, these results suggest that twist1a and twist1b control skeletal development and dorsoventral patterning by regulating runx2b in zebrafish and provide potential targets for the treatment of diseases or syndromes associated with decreased skeletal development.