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Sex determination is remarkably variable among animals with examples of environmental sex determination, male heterogametic (XX/XY) and female heterogametic (ZZ/ZW) chromosomal sex determination, and other genetic mechanisms. The cephalochordate amphioxus occupies a key phylogenetic position as a basal chordate and outgroup to vertebrates, but its sex determination mechanism is unknown. During the course of generating Nodal mutants with transcription activator-like effector nucleases (TALENs) in amphioxus Branchiostoma floridae, serendipitously, we generated three mutant strains that reveal the sex determination mechanism of this animal. In one mutant strain, all heterozygous mutant offspring over three generations were female and all wild-type descendants were male. This pattern suggests the Nodal allele targeted is on a female-specific W chromosome. A second mutant showed the same W-linked inheritance pattern, with a female heterozygote passing the mutation only to daughters. In a third mutant strain, both male and female offspring could be heterozygous, but a female heterozygote passed the mutation only to sons. This pattern is consistent with the targeted allele being on a Z chromosome. We found an indel polymorphism linked to a Nodal allele present in most females, but no males in our cultured population. Together, these results indicate that Nodal is sex chromosome-linked in B. floridae, and that B. floridae has a ZZ/ZW sex chromosome system.

The organizer is an essential signaling center required for axial formation during vertebrate embryonic development. In the basal chordate amphioxus, the dorsal blastopore lip of the gastrula has been proposed to be homologous to the vertebrate organizer. Lefty is one of the first genes to be expressed in the organizer. The present results show that Lefty overexpression abolishes the organizer; the embryos were severely ventralized and posteriorized, and failed to develop anterior and dorsal structures. In Lefty knockouts the organizer is enlarged, and anterior and dorsal structures are expanded. Different from Lefty morphants in vertebrates, amphioxus Lefty mutants also exhibited left-right defects. Inhibition of Nodal with SB505124 partially rescued the effects of Lefty loss-of-function on morphology. In addition, while SB505124 treatment blocked Lefty expression in the cleavage stages of amphioxus embryos, activation of Nodal signaling with Activin protein induced ectopic Lefty expression at these stages. These results show that the interplay between Lefty and Nodal signaling plays an essential role in the specification of the amphioxus organizer and axes.

Background Left-right (LR) asymmetry is an essential feature of bilateral animals. Studies in vertebrates show that LR asymmetry formation comprises three major steps: symmetry breaking, asymmetric gene expression, and LR morphogenesis. Although much progress has been made in the first two events, mechanisms underlying asymmetric morphogenesis remain largely unknown due to the complex developmental processes deployed by vertebrate organs. Results We here addressed this question by studying Pitx gene function in the basal chordate amphioxus whose asymmetric organogenesis, unlike that in vertebrates, occurs essentially in situ and does not rely on cell migration. Pitx null mutation in amphioxus causes loss of all left-sided organs and incomplete ectopic formation of all right-sided organs on the left side, whereas Pitx partial loss-of-function leads to milder phenotypes with only some LR organs lost or ectopically formed. At the N1 to N3 stages, Pitx expression is gradually expanded from the dorsal anterior domain to surrounding regions. This leads to activation of genes like Lhx3 and/or Prop1 and Pit, which are essential for left-side organs, and downregulation of genes like Hex and/or Nkx2.1 and FoxE4, which are required for right-side organs to form ectopically on the left side. In Pitx mutants, the left-side expressed genes are not activated, while the right-side genes fail to decrease expression on the left side. In contrast, in embryos overexpressing Pitx genes, the left-side genes are induced ectopically on the right side, and the right-side genes are inhibited. Several Pitx binding sites are identified in the upstream sequences of the left-side and right-side genes which are essential for activation of the former and repression of the latter by Pitx. Conclusions Our results demonstrate that (1) Pitx is a major (although not the only) determinant of asymmetric morphogenesis in amphioxus, (2) the development of different LR organs have distinct requirements for Pitx activity, and (3) Pitx controls amphioxus LR morphogenesis probably through inducing left-side organs and inhibiting right-side organs directly. These findings show much more dependence of LR organogenesis on Pitx in amphioxus than in vertebrates. They also provide insight into the molecular developmental mechanism of some vertebrate LR organs like the lungs and atria, since they show a right-isomerism phenotype in Pitx2 knockout mice like right-sided organs in Pitx mutant amphioxus. Our results also explain why some organs like the adenohypophysis are asymmetrically located in amphioxus but symmetrically positioned in vertebrates.

To find out the recessive factors of soil degradation threatening the healthy development of Weibei apple orchards, we examined soil compaction status and its inducing factors. This study could provide a theoretical basis for apple orchard scientific management. We evalua-ted the changes of soil bulk density and compaction with the depth of soil layer in 0-60 cm in four apple orchards with various planting period, includinglt;10 years (4-6 years), 10-20 years (14-16 years) and ＞20 years (24-26 years). The location and degradation degree of soil compaction in orchard were investigated. Through analyzing the number of soil aggregates and the stability, soil clay and organic matter contents, we tried to find the reasons for the internal compacting of soil in Weibei orchards. The results showed that soil bulk density and compactness in the 0-60 cm soil layer significantly increased with increasing planting years and soil depth. With the 20 cm soil layer as a boundary, soil of Weibei orchards in different planting years showed obvious variation characteristics of loose in upper and compact in lower. The above indicators in soil layer above 20 cm basically met the normal requirements of apple trees, whereas soil layer below 20 cm exceeded the threshold for healthy growth of apple trees. The main reasons for soil compaction below the subsurface layer were poor soil aggregation, the lack of soil organic matter, less human disturbance during fruit planting, and the movement of scattered clay particles to the lower layer. With increasing years of fruit planting, soil compaction became more severe.本研究通过分析渭北旱塬苹果园土壤的紧实化现状及其诱导因素,找出影响当地苹果园健康发展的土壤退化隐性因素,为果园科学管理提供理论依据。分别选取种植年限＜10年(4～6年)、10～20年(14～16年)和＞20年(24～26年)的苹果园各4个,分析0～60 cm土层土壤容重和紧实度随土层深度的变化规律,探明果园土壤内部紧实化发生的部位和退化程度,同时,通过分析土壤团聚体数量及其稳定性、土壤黏粒和有机质含量,揭示引起渭北果园土壤内部紧实化的原因。结果表明: 渭北果园0～60 cm土层土壤容重和紧实度均随植果年限和土层深度的增加而显著增大。以20 cm土层为界,渭北各园龄段苹果园土壤具有明显的“上松下实”变异特征,20 cm以上土层上述各指标基本满足苹果树的正常生长需求,20 cm以下土层土壤则已超出了苹果树健康生长的阈值。造成渭北苹果园亚表层以下土壤紧实化的原因主要是土壤团聚作用差、有机质含量低,加之植果期间人为扰动少,土壤中分散的黏粒会向下层移动。此外,随着植果年限的增加,土壤紧实化过程更加明显。.

The cephalochordate amphioxus is a promising animal model for studying the origin of vertebrate due to its key phylogenetic position among chordates. Although transcription activator-like effector nucleases (TALENs) has been adopted in amphioxus genome editing, its labor intensive construction of the TALEN proteins limits its usage in many laboratories. We here report an application of the CRISPR/Cas9 system, a more amenable genome editing method, in this group of animals. Our data show that while co-injection of Cas9 mRNAs and sgRNAs into amphioxus unfertilized eggs causes no detectable mutations at targeted loci, injections of Cas9 mRNAs and sgRNAs at two cell stage, or of Cas9 protein and sgRNAs before fertilization can executes efficient disruptions of targeted genes. Among the nine tested sgRNAs (targeting five genes) co-injected with Cas9 protein, seven introduced mutations at efficiency ranging from 18.4% to 90% and four caused specific phenotypes in the injected embryos. We also demonstrate that monomerization of sgRNAs via thermal treatment or integration of sgRNAs into a longer backbone could increase mutation efficacies. Our study will not only promote application of genome editing method in amphioxus research, but also provide valuable experiences for other organisms in which the CRISPR/Cas9 system has not been successfully applied.

Cephalochordate amphioxus contain two Brachyury genes (AmphiBra1 and AmphiBra2). Using probes from the highly conserved coding regions, a summation of their expression profiles in amphioxus embryos have been investigated by several previous studies. However, their respective expression patterns have not been determined up to date. We here address this issue using both qRT-PCR and in situ hybridization methods (with probes from the divergent untranslated regions). qRT-PCR detected a very low maternal expression for AmphiBra2, but not for AmphiBra1. Zygotic expression of both genes are activated around early gastrula stage and change in a similar pattern at subsequent stages. However, compared to AmphiBra1, the expression level of AmphiBra2 is much higher in all examined stages of embryos; in some extreme cases an over fifty-times difference is observed. In situ hybridization and embryonic sections reveal that while AmphiBra2 is highly expressed in the blastopore, the tail bud and the notochord, AmphiBra1 is weakly transcribed only in the notochord. Our results show that the two Brachyury genes, resulted from a lineage-specific duplication in amphioxus, have evolved different embryonic expression profiles.

It is a long-term challenge to undertake reliable transcriptomic research under different circumstances of genome availability. Here, we newly developed a genome-free computational method to aid accurate transcriptome assembly, using the amphioxus as the example. Via integrating ten next generation sequencing (NGS) transcriptome datasets and one third-generation sequencing (TGS) dataset, we built a sequence library of non-redundant expressed transcripts for the amphioxus. The library consisted of overall 91,915 distinct transcripts, 51,549 protein-coding transcripts, and 16,923 novel extragenic transcripts. This substantially improved current amphioxus genome annotation by expanding the distinct gene number from 21,954 to 38,777. We consolidated the library significantly outperformed the genome, as well as de novo method, in transcriptome assembly from multiple aspects. For convenience, we curated the Integrative Transcript Library database of the amphioxus (http://www.bio-add.org/InTrans/). In summary, this work provides a practical solution for most organisms to alleviate the heavy dependence on good quality genome in transcriptome research. It also ensures the amphioxus transcriptome research grounding on reliable data.

In vertebrate embryos, Hedgehog (Hh) is expressed in some anterior basal plate domains and by notochord and floorplate cells, and ventral neural cells are patterned by the activities of Hh-regulated transcription factors. Hh signalling is antagonized by signals from the dorsal neural tube and loss of Hh leads to loss of ventral patterning as dorsal pattern expands. These mechanisms are critical for producing the neurons that implement motor responses to sensory inputs but understanding how they evolved has been hindered by lack of insight from commonly studied invertebrates where nervous system morphology and genetic mechanisms are non-conserved with vertebrates. The invertebrate chordate amphioxus, which expresses Hh in its notochord and floorplate, provides a window into the prevertebrate condition. We examined amphioxus neural development by manipulating Hh and downstream genes involved in neural pattern and cell identity. We show that Hh signalling regulates the differentiation of some neurons in amphioxus, including a subset of motor neurons. This demonstrates some conservation of mechanism between vertebrates and amphioxus. However, other aspects of neural patterning differ between the lineages. We suggest the complexity of Hh-dependent neural patterning in vertebrates evolved in a step-wise manner. Alongside other previously described regulatory changes, initial recruitment of Hh along the length of the axis occurred in an ancestor to the chordates to regulate the differentiation of a subset of neurons. This was followed, in the vertebrate lineage, by additional changes to the downstream gene regulatory network of transcription factors, giving Hh a broader role in dorsal-ventral neural patterning.

Zinc(II) phthalocyanine (ZnPc) is a promising photosensitizer for PDT but suffers from aggregation in a physiological aqueous environment. In this paper, a class of biocompatible polymeric nanoparticles (NPs) was prepared to encapsulate ZnPc molecules. Mostly because of the planar structure, ZnPc molecules were difficult to be encapsulated into the polymeric NPs unless further coated with a thick poly-L-lysine (PLL) layer. The PLL shell endowed the NPs with good biocompatibility, efficient cellular uptake, and potential bioconjugation. The degree of aggregation (DOA) of ZnPc molecules in PLL-NPs was thoroughly investigated based on self-defined relative DOA, and a loading capacity of 4 wt% was deduced as the turning point for aggravating aggregation. Similarly, the optimal loading capacity of ZnPc was determined to be 4% according to the 1O2 generation rate, demonstrating the feasibility of the DOA approach. Polymers with large rigid units (PVK and PFO) were also utilized to relieve the aggregation of ZnPc in NPs. Taking advantage of the optimized ZnPc-loaded NPs, high PDT efficacy was demonstrated in HepG2 cells and in tumor-bearing mice as well. Both high in vitro and in vivo PDT efficacy and biocompatibility are demonstrated. Aside from affording a class of efficient biocompatible nanophotosensitizers, this work is also instructive to design other types of ZnPc-based nanocarriers, in which aggregation should be well considered.

With the globalization of supply chains, intermodal operations have become a key player in transportation. In real life, the consumer’s demands are often uncertain. The recent literature is rich of papers that either emphasizes on uncertain customer demands or intermodal transportation planning. In this paper, we combine these two fields in order to identify new potentials for saving cost and improving the efficiency of intermodal logistics network. A two stage stochastic programming model was proposed to decide the terminal location and transportation routes under uncertain customer demands. And the AP data set is used to test the presented model. The result is compared with the traditional counterparts, which shows superiority in solution quantity under uncertain environment.

Cilia rotation-driven nodal flow is critical for the left-right (L-R) break in symmetry in most vertebrates. However, the mechanism by which the flow signal is translated to asymmetric gene expression has been insufficiently addressed. Here, we show that Hh signalling is asymmetrically activated (L

This study was conducted to assess the effects of fertilizing regime and orchard planting age on soil calcium contents and stocks in the apple orchards on the Loess Plateau. The apple orchards in Luochuan County, one of the best regions for apple plantation in the world, were selec-ted in this study. The contents of calcium carbonate,water-soluble calcium and exchangeable cal-cium at 0-100 cm soil layer under different fertilizing regimes and various planting ages were mea-sured, their stocks were calculated and their variation features were analyzed. The results showed that soil in the apple orchards in the study region was characterized by the decline in calcium contents. The decline was more serious in apple orchards with long-term application of chemical fertili-zer than in those with combined application of chemical fertilizer and farmyard manure. The average contents of calcium carbonate, water-soluble calcium and exchangeable calcium at 0-100 cm soil layer in apple orchards with long-term application of chemical fertilizer decreased by 38.8%, 25.4% and 5.6% respectively than those in the apple orchards with long-term application of both chemical fertilizer and farmyard manure. The stocks of calcium carbonate, water-soluble calcium and exchangeable calcium decreased by 36.4%, 26.0% and 4.3%, respectively. The decline of soil cal-cium was aggravated with the increase of orchard planting age. The contents of calcium carbonate, water-soluble calcium and exchangeable calcium at 0-100 cm soil layer in orchards of more than 25 years of planting age decreased by 48.8%, 69.4% and 39.5% respectively, compared with orchards of less than 10 years of planting age, and the stocks decreased by 40.8%, 64.1% and 33.0%, respectively. These results indicated that either long-term application of chemical fertilizer or long-term plantation of apple trees obviously depleted soil calcium carbonate, water-soluble calcium and exchangeable calcium. Therefore, it was recommended that application of chemical fertilizer and farmyard manure should be combined to mitigate soil calcium decline, and calcium management should be strengthened in apple orchards of more than 25 years of planting age. The fertilizing regime was a driving factor of soil calcium decline which had a significant temporal (orchard planting age) and spatial (soil depth) effect.为明确黄土高原苹果产区施肥方式和园龄对土壤钙素含量和钙素贮量的影响,本研究以位于世界苹果优生区的陕西省洛川县苹果园为研究对象,分别研究不同施肥方式和不同园龄苹果园0～100 cm土层土壤碳酸钙、水溶性钙和交换性钙含量及其贮量的变化特征.结果表明: 洛川县苹果园土壤钙素递减式退化现象严重,长期大量单施化肥苹果园土壤钙素退化现象明显大于化肥与农家有机肥配施苹果园,单施化肥苹果园比化肥与农家有机肥配施苹果园0～100 cm土层土壤碳酸钙、水溶性钙和交换性钙平均含量分别减少38.8%、25.4%和5.6%,3种形态土壤钙素贮量依次减少36.4%、26.0%和4.3%;苹果园土壤钙素退化程度随园龄增加不断加剧,园龄25年苹果园比园龄≤10年苹果园0～100 cm土层土壤碳酸钙、水溶性钙和交换性钙平均含量分别减少48.8%、69.4%和39.5%,3种形态土壤钙素贮量分别减少40.8%、64.1%和33.0%.长期大量单施化肥和长期种植苹果树均对土壤碳酸钙、水溶性钙、交换性钙有明显的耗竭作用,钙素递减式退化特征明显,化肥与农家有机肥配施能够有效减缓土壤钙素退化,对于园龄25年的高龄苹果园应加强土壤钙素管理.施肥方式是苹果园土壤钙素递减的驱动因素,钙素递减呈现出明显的时(园龄)空(土层深度)效应.

Sensing of intracellular singlet oxygen (1O2) is required in order to optimize photodynamic therapy (PDT). An optical nanoprobe is reported here for the optical determination of intracellular 1O2. The probe consists of a porous particle core doped with the commercial 1O2 probe 1,3-diphenylisobenzofuran (DPBF) and a layer of poly-L-lysine. The nanoparticle probes have a particle size of ~80 nm in diameter, exhibit good biocompatibility, improved photostability and high sensitivity for 1O2 in both absorbance (peak at 420 nm) and fluorescence (with excitation/emission peaks at 405/458 nm). Nanoprobes doped with 20% of DPBF are best suited even though they suffer from concentration quenching of fluorescence. In comparison with the commercial fluorescent 1O2 probe SOSG, 20%-doped DPBF-NPs (aged) shows higher sensitivity for 1O2 generated at an early stage. The best nanoprobes were used to real-time monitor the PDT-triggered generation of 1O2 inside live cells, and the generation rate is found to depend on the supply of intracellular oxygen.

Star-like amphiphilic triblock copolymers were rationally designed and synthesized by combining two sequential atom-transfer radical polymerization reactions with a click reaction. Subsequently, a family of uniform magnetic/plasmonic core/shell nanoparticles was crafted by capitalizing on these triblock copolymers as nanoreactors. The diameter of the magnetic core and the thickness of the plasmonic shell could be independently and accurately controlled by varying the molecular weights (i.e., the chain lengths) of the inner and intermediate blocks of the star-like triblock copolymers, respectively. The surface plasmonic absorption of core/shell nanoparticles with different core diameters and shell thicknesses was systematically studied and theoretically modeled. This robust strategy provides easy access to a large variety of multifunctional nanoparticles with large lattice mismatches for use in optics, optoelectronics, catalysis, or bioimaging.

The Wnt/β-catenin transcriptional activation complex requires the adapter protein Pygopus (Pygo), which links the basal transcription machine to β-catenin to regulate the transcription of target genes. Previous studies revealed that the Pygo genes were broadly involved in the embryonic development of Drosophila, Xenopus and mouse, however, their functions in the embryonic development of amphioxus and zebrafish remain to be investigated. Here, we cloned and characterized one Pygo gene from amphioxus Branchiostoma belcheri (BbPygo) and two from zebrafish Danio rerio (DrPygo1 and DrPygo2). The spatial–temporal expression profiles showed that all these genes were maternally expressed. BbPygo expression was distributed ubiquitously in the amphioxus embryos before the gastrula stage and became restricted in the pharyngeal region in late stages. Similar to BbPygo, DrPygo2 was also expressed throughout whole embryos before the early gastrula stage, but limited in the pharyngeal region and central nervous system (CNS) in subsequent developing stages. Further knockdown of the DrPygo2 resulted in developmental defects of the body axis, CNS and cartilage segments in zebrafish. In contrast, the expression signal of the DrPygo1 gene was not detectable using the in situ hybridization method, which is consistent with its extremely low expression level detected by RT-qPCR. The expression patterns and function investigations revealed in this study suggest that the three Pygo genes probably function differentially though all of them show a conserved maternal expression pattern during the early embryonic stages of amphioxus and zebrafish.

To meet the practical demands of mobile Ad Hoc network on quality of service, we proposed an improved wireless on-demand routing protocol on the basis of traditional AODV. We integrate multi-path routing into route discovery process and arrange data originated from source to be transmitted over several different paths concurrently, and defined the path level to evaluate the path’s transmission ability based on its available bandwidth. Numerical simulation results demonstrate the proposed routing protocol outperforms AODV in end-to-end delay, packet delivery rate and network lifetime.

The pharynx is a major characteristic of chordates. Compared with vertebrates, amphioxus has an advantage for the study of pharynx development, as embryos lack neural crest, and the pharynx is mainly derived from endoderm cells. The Pax1/9 subfamily genes have essential roles in vertebrate pharyngeal patterning, but it is not known if the Pax1/9 gene has similar functions in amphioxus pharynx development. To answer this question, we examined the Pax1/9 gene expression pattern in amphioxus embryos at different developmental stages, and observed morphological changes following Pax1/9 knockdown. RT-qPCR analysis indicated that Pax1/9 expression was initiated during early neurula stage and rapidly peaked during mid-neurula stage. Furthermore, in situ hybridization analysis showed that Pax1/9 transcripts were localized exclusively in the most endodermal region of the developing pharynx in early neurula stage embryos; however, Pax1/9 expression was strikingly down-regulated in the region where gill slits would form from the fusion of endoderm and ectoderm in subsequent developmental stages and was maintained in the border regions between adjacent gill slits. Knockdown of Pax1/9 function using both morpholino and siRNA approaches led to embryonic defects in the first three gill slits, and fusion of the first two gill slits. Moreover, the expression levels of the pharyngeal marker genes Six1/2 and Tbx1/10 were reduced in Pax1/9 knockdown embryos. From these observations, we concluded that the Pax1/9 gene has an important role in the initial differentiation of amphioxus pharyngeal endoderm and in the formation of gill slits, most likely via modulation of Six1/2 and Tbx1/10 expression.

The systematic position of the amphioxus species with dextral gonads distributed in the southeast Pacific and Indian Oceans has remained to be clarified due to the adoption of different names by different researchers. Mitochondrial (mt) DNA is generally considered to be a powerful molecular marker in taxonomic studies. For a reliable systematics of the amphioxi collected from the South China Sea, we sequenced the complete mtDNA from a single specimen and compared it with those of the other eight amphioxus species. The present mtDNA genome contains 13 protein-coding genes, 22 tRNA genes, and two rRNA genes, with the same gene order as those in Branchiostoma and Epigonichthys, which, however, is different from that in Asymmetron. Based on our morphological data (including measurements of some characters) and the features of the mt genome, together with the distribution records of the dextral-gonad amphioxi, we conclude that the present mtDNA sequence most likely represents that of E. cultellus.

Luminescent perovskite quantum dots (QDs) had attracted great attention by virtue of the merits of color-tunable and narrow-band emissions. However, sofar reported perovskite QDs suffered from instability more or less. In this work, a type of silica-coated orthorhombic CH3NH3PbBr3 QDs (SiO2-QDs) with greatly improved stability was reported. The SiO2-QDs were one-pot synthesized using a reprecipitation-encapsulation method assisted with an amine functional silane, which not conly controlled the crystallization of QDs, but also encapsulated QDs with a silica layer simultaneously. More interestingly, the in situ encapsulation of silica shell induced the presence of orthorhombic perovskite that was thought to be unstable at room temperature. This is the first report of orthorhombic CH3NH3PbBr3 QDs, as far as we are concerned. The orthorhombic SiO2-QDs exhibited narrow-band green luminescence with a quantum yield of 78%, and a high production yield of ∼70wt%. Moreover, stability of SiO2-QDs was considerably improved due to silica-coating. White-LEDs were also successfully fabricated with the green SiO2-QDs and a red commercial phosphors using a noncontact configuration. These results demonstrated that the orthorhombic SiO2-QDs held great promise for high-performance display or lighting technology.

With the development of international trade, intermodal operations have become more and more important in goods transportation, in order to show the effect of carbon emission tax on a marine-land intermodal supply chain, this paper use dynamic game theory to get a sub-game Nash equilibrium, and the result of this paper show that the carbon emission tax will lead to sea transport and land transport to reduce the carbon emission of every goods, But the carbon tax is not necessarily guaranteed to reduce the total amount of carbon emissions. meanwhile, one part’s reduction of carbon emission may incentive the other part to reduce carbon emission.

Correct patterning of left-right asymmetry is an important process during the embryonic development of bilaterians. It is known that Hh signaling plays a role in LR asymmetry development of mouse, chicken and sea urchin embryos by regulating Nodal expression. In this study, we reported a novel regulating way of Hh gene in the LR asymmetry development of amphioxus embryos. Our results revealed that Hh−/− embryos abolished Cerburs (Cer) transcription and led to bilaterally symmetric expression of Nodal, Lefty and Pitx. As a consequence, Hh−/− mutants duplicated its left-side structures and lost right-side characters displaying an abnormal bilaterally symmetric body plan. Those morphological LR defects and disturbed gene expressions in Hh−/− embryos could be precisely rescued by Hh mRNA injection. Our results indicate that Hh gene takes part in amphioxus LR patterning by controlling Cer gene expression. Curiously, however, upregulation of Hh signal failed to alter Cer expression pattern and LR morphology in amphioxus embryos, showing that Hh might not provide asymmetric cue for Cer expression. Besides, the observation also found that Hh gene is required for mouth opening in amphioxus, hinting a homologous relationship between amphioxus mouth and vertebrate mouth.

Many bilaterally symmetrical animals develop genetically programmed left-right asymmetries. In vertebrates, this process is under the control of Nodal signaling, which is restricted to the left side by Nodal antagonists Cerberus and Lefty. Amphioxus, the earliest diverging chordate lineage, has profound left-right asymmetry as a larva. We show that Cerberus, Nodal, Lefty, and their target transcription factor Pitx are sequentially activated in amphioxus embryos. We then address their function by transcription activator-like effector nucleases (TALEN)-based knockout and heat-shock promoter (HSP)-driven overexpression. Knockout of Cerberus leads to ectopic right-sided expression of Nodal, Lefty, and Pitx, whereas overexpression of Cerberus represses their left-sided expression. Overexpression of Nodal in turn represses Cerberus and activates Lefty and Pitx ectopically on the right side. We also show Lefty represses Nodal, whereas Pitx activates Nodal These data combine in a model in which Cerberus determines whether the left-sided gene expression cassette is activated or repressed. These regulatory steps are essential for normal left-right asymmetry to develop, as when they are disrupted embryos may instead form two phenotypic left sides or two phenotypic right sides. Our study shows the regulatory cassette controlling left-right asymmetry was in place in the ancestor of amphioxus and vertebrates. This includes the Nodal inhibitors Cerberus and Lefty, both of which operate in feedback loops with Nodal and combine to establish asymmetric Pitx expression. Cerberus and Lefty are missing from most invertebrate lineages, marking this mechanism as an innovation in the lineage leading to modern chordates.

The Wnt signaling pathway plays crucial roles in the embryonic patterning of all metazoans. Recent studies on Wnt genes in amphioxus have shed important insights into the evolution of the vertebrate Wnt gene family and their functions. Nevertheless, the potential roles of Wnt family receptors encoded by Frizzled ( Fz ) genes in amphioxus embryonic development remain to be investigated. In the present study, we identified four amphioxus Fz genes— AmphiFz1 / 2 / 7 , AmphiFz4 , AmphiFz5 / 8, and AmphiFz9 / 10 —and analyzed their expression patterns during amphioxus embryogenesis. We found that these four Fz genes were maternally expressed and might be involved in early animal-vegetal axis establishment. The AmphiFz1 / 2 / 7 transcripts were detected in the central dorsal neural plate, mesoderm, the Hatschek’s pit, and rim of the mouth, whereas those of AmphiFz4 were detected in the mesoderm, pharyngeal endoderm, and entire gut region. AmphiFz5 / 8 was exclusively expressed in the anterior-most region, whereas AmphiFz9 / 10 was expressed in the neural plate, somites, and tail bud. The dynamic and diverse expression patterns of amphioxus Fz genes suggest that these genes are not only associated with early embryonic axis establishment but also are involved in the development of several organs in amphioxus.

In addition to protein coding sequence, the organism genome contains a significant amount of regulatory DNA. Comparative genomics reveals that the organism genomes of vertebrates, tunicate, cephalochordate, flies, and nematodes contain cis-regulatory elements with highly conserved non-coding elements (CNEs). CNEs that cluster around trans-dev genes are part of core gene regulatory networks (GRNs), and usually, they can act as transcriptional enhancers. In this review, we described the identification of CNEs and summarized their key properties across the metazoans, and then discussed the evolution of CNEs after large-scale genome duplication events and the role of CNEs in the evolution of ani-mal body plan.

Endogenous green fluorescent proteins (GFPs) have recently been found in amphioxus, where different characteristics of light production in different development stages and between different individuals have manifested. We identified the GFP genes in an amphioxus species Branchiostoma belcheri and monitored real time fluorescence signals of GFPs during different developmental stages to provide an insight on the GFP function in amphioxus. We found there are at least 12 endogenous GFP genes in amphioxus genome, and fluorescent expression changes in body position during different developmental stages. Additionally, GFP expression after metamorphosis development differed significantly among different amphioxus individuals, suggesting that there are multiple GFP homologous genes responsible for fluorescent expression. Expression levels of these genes varied significantly during different developmental stages, indicating that different GFP genes may have their unique functions in the development of amphioxus during specific phases of growth.

Vertebrate Pax1 gene is a member of Pax gene family and encodes a transcription factor associated with crucial roles in the development of pharyngeal pouch, scletrotome and limb bud. In zebrafish, the genome contains two Pax1 paralogs, DrPax1a and DrPax1b, which share high sequence similarity with other Pax1 genes. To elucidate the function of zebrafish DrPax1b gene, we first examined the gene expression pattern and found that it was mainly expressed in the endodermal pharyngeal pouch, caudal somites, notochord, and fin bud. Then, we performed knockdown experiments using antisense morpholino oligonucleotides, which lead to the defects in the vertebral column, tail, pharyngeal skeleton, and pectoral fin. Additionally, we also found that the mouse MmPax1 mRNA, but not the amphioxus AmphiPax1/9 mRNA, could rescue the MO-induced defects. Furthermore, sequence alignment revealed that the N-terminal region of vertebrate Pax1 and amphioxus Pax1/9 were highly conserved, whereas their C-terminal regions were relatively divergent. However, the chimeric Am(N)Dr(C)Pax1, Mm(N)Dr(C)Pax1 and Dr(N)Mm(C)Pax1 mRNA could partially rescue the defects, while the Dr(N)Am(C)Pax1 mRNA could not. In conclusion, our data demonstrate a conserved function of DrPax1b in the development of the vertebral column, pectoral fin and pharyngeal skeleton formation in zebrafish and also provide critical insight into the functional evolution of Pax1 gene by changing its C-terminal sequence.

Amphioxus is a promising laboratorial model animal for studying the evolutionary and developmental mechanisms that appeared during the invertebrate-chordate to vertebrate transition. However, the main drawback for the use of amphioxus as a model organism is the lack of well-developed technical approaches. Conditional gene expression, as performed with thermal control, is a very useful strategy in gene function studies. To make this method possible in amphioxus studies, here we report the isolation and characterization of an amphioxus Hsp70 gene (Hsp70a) and its promoter in Chinese amphioxus (Branchiostoma belcheri). Hsp70a showed very low expression at normal temperatures but was robustly induced in animals upon heat shock. The basal cis-acting elements (CAAT and TATA), as well as four heat shock elements (HSEs), were found within the regulatory region (-1031 to -11 upstream from the start codon), but surprisingly most of the elements were located in the 5'UTR region (-252 to -10). Reporter constructs, including sequences from both the transcription start site (TSS) and ATG were tested for transient expression in EPC cells and microinjected zebrafish embryos. Results suggested that the 5'UTR region, which includes a TATA box at -92bp, a CAAT box at -152bp, and three HSE elements (-212 to -106), represents the core hsp promoter sequence of the B. belcheri Hsp70a gene. Therefore in this study we identified an effectively thermo-inducible promoter in amphioxus that could be used for the establishment of a conditional gene expression system in which the target gene can be regulated in a temporal- or tissue-specific way in amphioxus.

Suicide in college students has become an important public health issue in China. The aim of this study was to identify the differences between suicide attempters and suicide ideators based on a cross-sectional survey. Our results indicate that although female gender, positive screening for psychiatric illness, positive family history of suicide, elevated overall impulsivity, and elevated motor impulsivity were correlated with suicidal ideation, only positive family history of suicide and high motor impulsivity could differentiate suicide attempters from suicidal ideators. Future research with a longitudinal and prospective study design should be conducted to confirm these findings.

The Wnt/β-catenin signaling pathway plays a crucial role in the embryonic development of metazoans. Although the pathway has been studied extensively in many model animals, its function in amphioxus, the most primitive chordate, remains largely uncharacterized. To obtain basic data for functional analysis, we identified and isolated seven genes (Lrp5/6, Dvl, APC, CkIα, CkIδ, Gsk3β, and Gro) of the Wnt/β-catenin signaling pathway from the amphioxus (Branchiostoma floridae) genome. Phylogenetic analysis revealed that amphioxus had fewer members of each gene family than that found in vertebrates. Whole-mount in situ hybridization showed that the genes were maternally expressed and broadly distributed throughout the whole embryo at the cleavage and blastula stages. Among them, Dvl was expressed asymmetrically towards the animal pole, while the others were evenly distributed in all blastomeres. At the mid-gastrula stage, the genes were specifically expressed in the primitive endomesoderm, but displayed different patterns. When the embryo developed into the neurula stage, the gene expressions were mainly detected in either paraxial somites or the tail bud. With the development of the embryo, the expression levels further decreased gradually and remained only in some pharyngeal regions or the tail bud at the larva stage. Our results suggest that the Wnt/β-catenin pathway might be involved in amphioxus somite formation and posterior growth, but not in endomesoderm specification.

A novel titanium dioxide (TiO2) film comprising both nanotubes and nanopaticles was fabricated by an anodization process of the modified titanium. The local electric field at the anodized surface was simulated and its influence on the morphology of the TiO2 film was discussed. The results show that the electric field strength is enhanced by the covering. The growth rate of TiO2 increases with the assist of the local electric field. However, TiO2 dissolution is hindered since the local electric field prevents [TiF6]6− from diffusing. It means that the balance condition for the formation of nanotubes is broken, and TiO2 nanoparticles are formed. Moreover, the crystal structure of the TiO2 film was confirmed using X-ray diffraction and Raman analysis. The anatase is a main phase for the proposed film.

The amphioxus is a promising animal model for evolutionary-developmental studies due to its key position on the animal phylogenetic tree. In the present study, we reported a genetically modified amphioxus strain on the Hedgehog (Hh) gene locus using the TALEN method. The result showed that our TALEN pair injection could bring about 34% mutations in the amphioxus Hh coding region. Further analysis on the F(0) gametic DNA revealed that the mutations had entered into gametes. So, we paired one F(0) male carrying an 8 bp deletion with a wild-type (WT) female, and carefully nursed the F(1) embryos up to adulthood. We then screened F(1) individually via analyzing their genomic DNA from a tiny tail tip, and obtained eight heterozygous mutants from the F(1) offspring. Moreover, our observation on the F(2) embryos generated by mating F(1) mutants also revealed that about 25% of early larvae developed aberrantly with head and tail curving ventrally, agenesis of the mesoblastic tissue under their anterior notochord, and no mouth opening. With the larva growth, deformities (such as twist of head and tail, mouth absent, ventrally localized endostyle and gill slits) became more severe, and eventually those malformed larvae died due to no food intake. Genetic analysis showed that all these deformed embryos were homozygous mutants and the ratio of Hh hetorozygotes vs WT agreed with Mondel's law. WT amphioxus larvae are asymmetric with the mouth on the left and gill slits on the right side. However, the homozygous mutant larvae became left-right symmetric with the gill slits on the ventral side, indicating a conserved role of Hedgehog signaling in establishing the left-right embryonic axis.

Amphioxus occupies a transitional position between invertebrates and vertebrates and is therefore a promising laboratory model animal. As a powerful molecular marker in population genetics studies, microsatellite DNA is required for both the investigation of genetic diversity of a wild amphioxus population and strain breeding of laboratory cultured amphioxus. In the present study, we constructed a microsatellite-enriched DNA library of amphioxus Branchiostoma belcheri to develop this molecular marker. After screening the library and combining it with the data obtained from a public database, we successfully developed 21 pair of primers that could effectively amplify the microsatellite loci of amphioxus. Of those primer pairs, seven were in deviation of Hardy–Weinberg equilibrium, mostly caused by null alleles. The others had no linkage disequilibrium, indicating they are applicable for the genetic studies. Using those primer pairs, we further detected the genetic diversity of the wild amphioxus p...

A series of signal transduction pathways have been found to regulate the polarity establishment and formation of animal primary body axis. Among them, Wnt signaling pathway is extremely conserved and several key components in the pathway have been identified in the demosponge lineage. This implies that it is one of the earliest pathways involved in the ancestral metazoan axis development and might play an important role in specification and development of posterior and ventral fate of animal axis. Recently, with the establishment of functional experiments in vitro, the body plan formation has been found to be affected, in varying degrees, by many genes in the Wnt signaling pathway, such as members of wnt gene family, maternal gene beta-catenin and some transcription factor encoding genes. In this review, we analyzed the evolutionary origin of the wnt gene family involved in development of metazoan body plans, and then made a brief review on the roles of canonical Wnt/beta-catenin signaling in the polarity establishment and formation of primary body axis in diverse deuterostomes including sea urchin, amphioxus, zebrafish, frog, and mouse.

As one of the closest living invertebrate relatives of vertebrates, amphioxus (subphylum Cephalochordata) occupies a key position in animal evolution and is becoming the best available proxy and model animal for studying the last common ancestor of all chordates, especially vertebrates. As long-term continuous culturing of amphioxus in laboratory became reliable, for pushing this animal to be a more successful model system, whole-genome sequencing of one or more species derived from this branch will be another urgent issue needed to address. In the present study, we described the construction and characterization of a bacterial artificial chromosome (BAC) library, using a single individual of Chinese amphioxus (Branchiostoma belcheri). High quality genomic DNA extracted from the spermary was partially digested with EcoRand EcoRmethylase. Desirable DNA fragments were isolated by pulsed field gel electrophoresis (PFGE), ligated to linearized and phosphorylased carrier pCC1BAC, and then transformed to Escherichia coli EPI300. The constructed library consists of 44 706 clones with the average insert fragment size around 80 kb as estimated by PFGE. The representation of the library is about 9 equivalents to the amhioxus genome. These results indicate that the BAC library will be useful for functional genomic studies and facilitate the whole-genome sequencing of Chinese amphioxus.

National Natural Science Foundation of China [30830023]; National High Technology Research and Development Program of China (863 Program) [2008AA092602]; Ministry of Education, China [20070384041]

The Pax gene family encodes a group of transcription factors whose evolution has accompanied the major morphological and functional innovations of vertebrate species. The evolutionary conservation throughout diverse lineages of metazoan and the functional importance in development rendered Pax family an ideal system to address the relationship inside Chordata phylum. In the present study, we sequenced and annotated four genomic regions containing Chinese amphioxus (Branchiostoma belcheri) Pax genes, and retrieved homologous sequences from public database. In comparison with vertebrate homologues, the predicted amphioxus Pax proteins display high sequence conservation. Evidences from the molecular phylogenetic studies and gene organization analyses supports cephalochordates have a much closer relationship to vertebrates than that between tunicates and vertebrates, contrasting to urochordate relatives hypothesis proposed by several latest studies. Analysis of phylogenetic topology derived from concatenated subfamily datasets uncovered a potential statistical bias of supermatrix approach. Furthermore, we deduced an evolutionary scenario of Pax gene family. This scenario provided a plausible explanation for the origin and dynamics of the Pax gene members.

Green fluorescent protein (GFP) has been widely used as a molecular marker in modern biological research. Before the recent report of one GFP gene in Branchiostoma floridae, GFP family members were cloned only from other two groups of species: Cnidaria and Copepoda. Here we describe the complete GFP gene repertoire of B. floridae which includes 13 functional genes and 2 pseudogenes, representing the largest GFP family found so far. Coupling with nine other GFP sequences from another two species of genus Branchiostoma and the sequences from Cnidaria and Copepoda, we made a deep-level phylogenetic analysis for GFP genes in cephalochordates and found: 1) GFP genes have experienced a divergent evolution in cephalochordates; 2) all amphioxus GFP genes form four main clades on the tree which had diverged before the radiation of the last common ancestor of all extant cephalochordates; 3) GFP genes in amphioxus shared a common ancestor with that in Copepoda rather than being derived from horizontal gene transfer, which indicates that our ancestor was derived from a fluorescent organism and lost this ability after its separation from Cephalochordata, and also makes GFP a rare gene which has a rather unusual evolutionary path. In addition, we also provided evidence indicating that GFP genes have evolved divergent functions by specializing their expression profile, and different fluorescent spectra by changing their emission peaks. These findings spark two interesting issues: what are GFP in vivo functions in cephalochordates and why they are lost in other examined deuterostomes?

Amphioxus has an important evolutionary position as a result of their phylogenetic position relative to vertebrates. Understanding their chromosomes would provide key points in the study of evolutionary biology and comparative genomics. The difficulty in preparing amphioxus chromosomes currently provides a significant hurdle in this research. In the current study, we describe an improved method for metaphase preparation from amphioxus embryos and methodology for preparing metaphase spreads from regenerative somatic cells. Chromosomes of two amphioxus species from Xiamen waters in China are also observed. The diploid chromosome number was found to be 40 in Branchiostoma belcheri , while B. japonicum has 36, confirming the two are distinct species from cytotaxonomic viewpoint.

Interferon (IFN)-inducible GTPases currently include four families of proteins: myxovirus resistant proteins (Mxs), guanylate-binding proteins (GBPs), immunity-related GTPase proteins (IRGs), and very large inducible GTPase proteins (VLIGs). They are all under conserved regulation by IFNs in humans and mice and play a critical role in preventing microbial infections. However, differences between vertebrates are poorly characterized, and their evolutionary origins have not been studied in detail. In this study, we performed comparative genomic analysis of the four families in 18 representative animals that yielded several unexpected results. Firstly, we found that Mx, GBP, and IRG protein families arose before the divergence of chordate subphyla, but VLIG emerged solely in vertebrates. Secondly, IRG, GBP, and VLIG families have experienced a high rate of gene gain and loss during the evolution, with the GBP family being lost entirely in two pufferfish and VLIG family lost in primates and carnivores. Thirdly, the regulation of these genes by IFNs is highly conserved throughout vertebrates although the VLIG protein sequences in fish have lost the first 870 amino acid residues. Finally, amphioxus IFN-inducible GTPase genes are all highly expressed in immune-related organs such as gill, liver, and intestine and are upregulated after challenge with PolyI:C and pathogens, although no IFNs or their receptors were detected in the current amphioxus genome database. These results suggest that IFN-inducible GTPase genes play conserved immune functions both in vertebrates and in cephalochordates.

C-type lectins found in many animals are non-enzymatic proteins and able to bind with mono- and oligosaccharides in a Ca2+-dependent fashion. Here, we report the cloning of two C-type lectins named BML-1 and BML-2 from the venom gland of Bungarus multicinctus, and expression of their mature peptides with 135 and 137 amino acids as inclusion bodies. Recombinant BML-1 and BML-2 proteins with 135 amino acids formed monomers, and those with 137 amino acids formed homodimers and monomers and both of them displayed certain hemagglutinating activity to rabbit erythrocytes. The results of Western blotting and immuno-affinity chromatography demonstrated that C-type lectins in B. multicinctus formed dimers in physiological conditions, and their molecular weight is lower than previous predictions. This is the first report of the cloning of the BML-2 gene from the venom gland of B. multicinctus, as well as an investigation of its confirmation and biological functions.

The lancelet is considered to be a promising laboratory model animal. To establish laboratory colonies of lancelet, we collected parental lancelets of Branchiostoma belcheri and B. japonicum (previously named as B. belcheri tsingtauense) with fully developed gonads from Xiamen Rare Marine Creature Conservation Areas (Fujian, China) on dates just before their spawning in the field in 2005. Those parental lancelets spawned spontaneously in the laboratory and produced thousands upon thousands of fertilized eggs. After carefully hatching and maintaining for almost 1 year, we successfully obtained about 500 first generation (F1) adults of B. belcheri and 3,300 of B. japonicum. Part of those F1 lancelets ripened and spontaneously spawned in 2006, and several thousands of second generation (F2) individuals of both species were produced. The young F2 lancelets are growing in good condition and some of B. japonicum initiated gonad development in December, 2006. Our experience emphasizes that cleanness of settlement substratum and sufficient food supply are important factors for long-term culture of lancelets in the laboratory.

Amphioxus is an emerging model organism for evolutionary developmental (Evo-Dev) studies owing to its key phylogenetic position in chordates. However, the rare supply of living embryonic materials is a major drawback for using amphioxus as a laboratorial model animal. Although the problem has been partially resolved in several recent reports, the spawning of amphioxus still remains unpredictable to some extent. In the present study, we reported an accurate method to distinguish spawning-ready and non-spawning-ready individuals of amphioxus Branchiostoma belcheri. In comparison with non-spawning-ready amphioxus, all spawning-ready individuals display following features several hours before their spawning: 1) for both males and females, the interstices between two adjacent gonads are obvious and relatively wide; and 2) the connections among eggs are loose and the crannies appear in each individual ovary of females. These morphological features were also observed in B. japonicum, indicating their conservation among different lancelet species. Based on this observable criterion, we made predictions on the spawning of about 600 ripe B. belcheri individuals and acquired an accuracy of 86.7% for females and 80.4% for males. In addition, we found that advancing or delaying onset of darkness has no detectable effect on the timing of spawning of B. belcheri. Our study makes amphioxus spawning more amenable for our experiments and will greatly facilitate its utilization as a laboratorial model animal.