Ion flow regulates left–right asymmetry in sea urchin development

The degree of conservation among phyla of early mechanisms that pattern the left–right (LR) axis is poorly understood. Larvae of sea urchins exhibit consistently oriented LR asymmetry. The main part of the adult rudiment is formed from the left coelomic sac of larvae, the left hydrocoel. Although this left preference is conserved among all echinoderm larvae, its mechanism is largely not understood. Using two marker genes, HpNot and HpFoxFQ-like, which are asymmetrically expressed during larval development of the sea urchin Hemicentrotus pulcherrimus, we examined in this study the possibility that the recently discovered ion flux mechanism controls asymmetry in sea urchins as it does in several vertebrate species. Several ion-transporter inhibitors were screened for the ability to alter the expression of the asymmetric marker genes. Blockers of the H⁺/K⁺-ATPase (omeprazole, lansoprazole and SCH28080), as well as a calcium ionophore (A23187), significantly altered the normal sidedness of asymmetric gene expression. Exposure to omeprazole disrupted the consistent asymmetry of adult rudiment formation in larvae. Immuno-detection revealed that H⁺/K⁺-ATPase-like antigens in sea urchin embryos were present through blastula stage and exhibited a striking asymmetry being present in a single blastomere in 32-cell embryos. These results suggest that, as in vertebrates, endogenous spatially-regulated early transport of H⁺ and/or K⁺, and also of Ca²⁺, functions in the establishment of LR asymmetry in sea urchin development.Electronic Supplementary Material Supplementary material is available for this article at     

Asymmetries in H+/K+-ATPase and cell membrane potentials comprise a very early step in left-right patterning

A pharmacological screen identified the H+ and K+ ATPase transporter as obligatory for normal orientation of the left-right body axis in Xenopus. Maternal H+/K+-ATPase mRNA is symmetrically expressed in the 1-cell Xenopus embryo but becomes localized during the first two cell divisions, demonstrating that asymmetry is generated within two hours postfertilization. Although H+/K+-ATPase subunit mRNAs are symmetrically localized in chick embryos, an endogenous H+/K+-ATPase-dependent difference in membrane voltage potential exists between the left and right sides of the primitive streak. In both species, pharmacologic or genetic perturbation of endogenous H+/K+-ATPase randomized the sided pattern of asymmetrically expressed genes and induced organ heterotaxia. Thus, LR asymmetry determination depends on a very early differential ion flux created by H+/K+-ATPase activity.     

Serotonin signaling is a very early step in patterning of the left-right axis in chick and frog embryos

BACKGROUND: Consistent left-right (LR) asymmetry is a fascinating problem in developmental and evolutionary biology. Conservation of early LR patterning steps among vertebrates as well as involvement of nonprotein small-molecule messengers are very poorly understood. Serotonin (5-HT) is a key neurotransmitter with crucial roles in physiology and cognition. We tested the hypothesis that LR patterning required prenervous serotonin signaling and characterized the 5-HT pathway in chick and frog embryos. RESULTS: A pharmacological screen implicated endogenous signaling through receptors R3 and R4 and the activity of monoamine oxidase (MAO) in the establishment of correct sidedness of asymmetric gene expression and of the viscera in Xenopus embryos. HPLC and immunohistochemistry analysis indicates that Xenopus eggs contain a maternal supply of serotonin that is progressively degraded during cleavage stages. Serotonin's dynamic localization in frog embryos requires gap junctional communication and H,K-ATPase function. Microinjection of loss- and gain-of-function constructs into the right ventral blastomere randomizes asymmetry. In chick embryos, R3 and R4 activity is upstream of the asymmetry of Sonic hedgehog expression. MAO is asymmetrically expressed in the node. CONCLUSIONS: Serotonin is present in very early chick and frog embryos. 5-HT pathway function is required for normal asymmetry and is upstream of asymmetric gene expression. The microinjection data reveal asymmetry existing in frog embryos by the 4-cell stage and suggest novel intracellular 5-HT mechanisms. These functional and localization data identify a novel role for the neurotransmitter serotonin and implicate prenervous serotonergic signaling as an obligate aspect of very early left-right patterning conserved to two vertebrate species.     

H,K-ATPase protein localization and Kir4.1 function reveal concordance of three axes during early determination of left-right asymmetry

Consistent laterality is a fascinating problem, and study of the Xenopus embryo has led to molecular characterization of extremely early steps in left-right patterning: bioelectrical signals produced by ion pumps functioning upstream of asymmetric gene expression. Here, we reveal a number of novel aspects of the H+/K+-ATPase module in chick and frog embryos. Maternal H+/K+-ATPase subunits are asymmetrically localized along the left-right, dorso-ventral, and animal-vegetal axes during the first cleavage stages, in a process dependent on cytoskeletal organization. Using a reporter domain fused to molecular motors, we show that the cytoskeleton of the early frog embryo can provide asymmetric, directional information for subcellular transport along all three axes. Moreover, we show that the Kir4.1 potassium channel, while symmetrically expressed in a dynamic fashion during early cleavages, is required for normal LR asymmetry of frog embryos. Thus, Kir4.1 is an ideal candidate for the K+ ion exit path needed to allow the electroneutral H+/K+-ATPase to generate voltage gradients. In the chick embryo, we show that H+/K+-ATPase and Kir4.1 are expressed in the primitive streak, and that the known requirement for H+/K+-ATPase function in chick asymmetry does not function through effects on the circumferential expression pattern of Connexin43. These data provide details crucial for the mechanistic modeling of the physiological events linking subcellular processes to large-scale patterning and suggest a model where the early cytoskeleton sets up asymmetric ion flux along the left-right axis as a system of planar polarity functioning orthogonal to the apical-basal polarity of the early blastomeres.     

Expression profiles of the organic acid metabolism-associated genes during rat liver regeneration

In this study, 55 of the organic acid metabolism-involved genes were primarily confirmed to be associated with liver regeneration (LR) by bioinformatics and gene expression profiling analysis. Number of the initially and totally expressed genes occurring in initiation phase of LR, G₀/G₁, cell proliferation, cell differentiation and liver tissue structure-function reconstruction were 21, 5, 33, 1 and 40, 20, 174, 44, respectively, illustrating that genes were initially expressed mainly in initiation stage, and worked in different phases. 151 times up-regulation and 114 times down-regulation as well as 14 types of expression patterns showed the diversification and complication of genes expression changes. It is inferred from the above gene expression changes and patterns that acetate biosynthesis enhanced at forepart, propionate biosynthesis at forepart, prophase and early metaphase, pyruvate biosynthesis at forepart, metaphase and anaphase, succinate biosynthesis at forepart and anaphase; malate biosynthesis in metaphase and N-acetylneuraminate biosynthesis at 36, 66 and 96 h. Whereas, carnitine biosynthsis attenuates at forepart and prophase, enhancement at middle metaphase; isocitrate in the forepart, quinolinate at forepart and early metaphase, creatine at early metaphase and fumarate at anaphase perform the restrained biosynthesis, respectively; catabolisms of propionate and pyruvate were depressed in metaphase.     

Asymmetric expression of antivin/lefty1 in the early chick embryo

Mammalian lefty and zebrafish antivin, highly related to lefty, are shown to be expressed asymmetrically and involved in the specification of the left body side of early embryos. We isolated a chick homologue of the antivin/lefty1 cDNA and studied its expression pattern during early chick development. We found that antivin/lefty1 is expressed asymmetrically on the left side of the prospective floorplate, notochord and lateral plate mesoderm of the chick embryo.     

左右不对称信号分子Pitx2

同型框基因Pitx2在鸡、小鼠和爪蟾胚胎中不对称地表达在左侧板中胚层和衍生器官(如心脏、肠等)中. 转录因子Pitx2看来是Shh和Nodal等信号分子的下游效应子. Pitx2的错误表达足以产生器官逆位和身体旋转逆向,人类若有Pitx2表达缺陷就可能导致Rieger综合征. Pitx2看来是脊椎动物介导左右不对称的关键且保守的信号分子.     

细胞外基质相关基因在大鼠肝再生中表达模式分析

细胞外基质具有维持细胞极性、调节细胞粘附、增殖、组织器官形态、发生、分化等功能。为了进一步在基因转录水平了解细胞外基质在大鼠肝再生中变化和作用, 用搜集网站资料和查阅相关论文等方法获得细胞外基质基因, 用Rat Genome 230 2.0芯片检测它们在大鼠再生肝中表达情况, 用真、假手术比较方法确定肝再生相关基因。初步证实上述97个基因与肝再生相关。其中, 肝再生启动(部分肝切除(parital hepatectomy, PH)后0.5~4 h)、G0/G1过渡(PH后4~6 h)、细胞增殖(PH后6~66 h)、细胞分化和组织结构功能重建(PH后72~168 h)等4个阶段起始表达的基因数为49、19、73、5, 基因总表达的次数为84、51、369、144, 表明相关基因主要在肝再生启动阶段起始表达, 在不同阶段发挥作用。它们表达的相似性分为均上调、上调占优势、均下调、下调占优势、上调和下调相近等5类, 涉及38、21、21、10和7个基因, 共上调411次, 下调186次, 分为24种表达模式, 表明肝再生中细胞生理生化活动具有阶段性、多样性和复杂性。根据细胞外基质相关基因在肝再生中表达变化推测, 肝再生前期纤粘连蛋白形成相关基因表达增强, 肝再生中期胶原形成相关基因表达增强。     

QTL analysis of seed germination and pre-emergence growth at extreme temperatures in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

Enhancing the knowledge on the genetic basis of germination and heterotrophic growth at extreme temperatures is of major importance for improving crop establishment. A quantitative trait loci (QTL) analysis was carried out at sub- and supra-optimal temperatures at these early stages in the model Legume Medicago truncatula. On the basis of an ecophysiological model framework, two populations of recombinant inbred lines were chosen for the contrasting behaviours of parental lines: LR5 at sub-optimal temperatures (5 or 10°C) and LR4 at a supra-optimal temperature (20°C). Seed masses were measured in all lines. For LR5, germination rates and hypocotyl growth were measured by hand, whereas for LR4, imbibition and germination rates as well as early embryonic axis growth were measured using an automated image capture and analysis device. QTLs were found for all traits. The phenotyping framework we defined for measuring variables, distinguished stages and enabled identification of distinct QTLs for seed mass (chromosomes 1, 5, 7 and 8), imbibition (chromosome 4), germination (chromosomes 3, 5, 7 and 8) and heterotrophic growth (chromosomes 1, 2, 3 and 8). The three QTL identified for hypocotyl length at sub-optimal temperature explained the largest part of the phenotypic variation (60% together). One digenic interaction was found for hypocotyl width at sub-optimal temperature and the loci involved were linked to additive QTLs for hypocotyl elongation at low temperature. Together with working on a model plant, this approach facilitated the identification of genes specific to each stage that could provide reliable markers for assisting selection and improving crop establishment. With this aim in view, an initial set of putative candidate genes was identified in the light of the role of abscissic acid/gibberellin balance in regulating germination at high temperatures (e.g. ABI4, ABI5), the molecular cascade in response to cold stress (e.g. CBF1, ICE1) and hypotheses on changes in cell elongation (e.g. GASA1, AtEXPA11) with changes in temperatures based on studies at the whole plant scale.     

RLF,a cytochrome b5‐like heme/steroid binding domain protein,controls lateral root formation independently of ARF7/19‐mediated auxin signaling in Arabidopsis thaliana

Lateral root (LR) formation is important for the establishment of root architecture in higher plants. Recent studies have revealed that LR formation is regulated by an auxin signaling pathway that depends on auxin response factors ARF7 and ARF19, and auxin/indole‐3‐acetic acid (Aux/IAA) proteins including SOLITARY‐ROOT (SLR)/IAA14. To understand the molecular mechanisms of LR formation, we isolated a recessive mutant rlf (reduced lateral root formation) in Arabidopsis thaliana. The rlf‐1 mutant showed reduction of not only emerged LRs but also LR primordia. Analyses using cell‐cycle markers indicated that the rlf‐1 mutation inhibits the first pericycle cell divisions involved in LR initiation. The rlf‐1 mutation did not affect auxin‐induced root growth inhibition but did affect LR formation over a wide range of auxin concentrations. However, the rlf‐1 mutation had almost no effect on auxin‐inducible expression of LATERAL ORGAN BOUNDARIES‐DOMAIN16/ASYMMETRIC LEAVES2‐LIKE18 (LBD16/ASL18) and LBD29/ASL16 genes, which are downstream targets of ARF7/19 for LR formation. These results indicate that ARF7/19‐mediated auxin signaling is not blocked by the rlf‐1 mutation. We found that the RLF gene encodes At5g09680, a protein with a cytochrome b₅‐like heme/steroid binding domain. RLF is ubiquitously expressed in almost all organs, and the protein localizes in the cytosol. These results, together with analysis of the genetic interaction between the rlf‐1 and arf7/19 mutations, indicate that RLF is a cytosolic protein that positively controls the early cell divisions involved in LR initiation, independent of ARF7/19‐mediated auxin signaling.