Zic3 is involved in the left-right specification of the Xenopus embryo

Establishment of left-right (L-R) asymmetry is fundamental to vertebrate development. Several genes involved in L-R asymmetry have been described. In the Xenopus embryo, Vg1/activin signals are implicated upstream of asymmetric nodal related 1 (Xnr1) and Pitx2 expression in L-R patterning. We report here that Zic3 carries the left-sided signal from the initial activin-like signal to determinative factors such as Pitx2. Overexpression of Zic3 on the right side of the embryo altered the orientation of heart and gut looping, concomitant with disturbed laterality of expression of Xnr1 and Pitx2, both of which are normally expressed in the left lateral plate mesoderm. The results indicate that Zic3 participates in the left-sided signaling upstream of Xnr1 and Pitx2. At early gastrula, Zic3 was expressed not only in presumptive neuroectoderm but also in mesoderm. Correspondingly, overexpression of Zic3 was effective in the L-R specification at the early gastrula stage, as revealed by a hormone-inducible Zic3 construct. The Zic3 expression in the mesoderm is induced by activin (beta) or Vg1, which are also involved in the left-sided signal in L-R specification. These findings suggest that an activin-like signal is a potent upstream activator of Zic3 that establishes the L-R axis. Furthermore, overexpression of the zinc-finger domain of Zic3 on the right side is sufficient to disturb the L-R axis, while overexpression of the N-terminal domain on the left side affects the laterality. These results suggest that Zic3 has at least two functionally important domains that play different roles and provide a molecular basis for human heterotaxy, which is an L-R pattern anomaly caused by a mutation in human ZIC3.     

Zic2 and Zic3 synergistically control neurulation and segmentation of paraxial mesoderm in mouse embryo

Zic family zinc-finger proteins play various roles in animal development. In mice, five Zic genes (Zic1-5) have been reported. Despite the partly overlapping expression profiles of these genes, mouse mutants for each Zic show distinct phenotypes. To uncover possible redundant roles, we characterized Zic2/Zic3 compound mutant mice. Zic2 and Zic3 are both expressed in presomitic mesoderm, forming and newly generated somites with differential spatiotemporal accentuation. Mice heterozygous for the hypomorphic Zic2 allele together with null Zic3 allele generally showed severe malformations of the axial skeleton, including asymmetric or rostro-caudally bridged vertebrae, and reduction of the number of caudal vertebral bones, that are not obvious in single mutants. These defects were preceded by perturbed somitic marker expression, and reduced paraxial mesoderm progenitors in the primitive streak. These results suggest that Zic2 and Zic3 cooperatively control the segmentation of paraxial mesoderm at multiple stages. In addition to the segmentation abnormality, the compound mutant also showed neural tube defects that ran the entire rostro-caudal extent (craniorachischisis), suggesting that neurulation is another developmental process where Zic2 and Zic3 have redundant functions.     

Genetic analysis of artificial pigmentation mutants in Porphyra yezoensis Ueda (Bangiales, Rhodophyta)

Porphyra yezoensis Ueda artificial pigmentation mutants, yel (green), fre (red‐orange) and bop (pink), obtained by treatment with /V‐methyl‐/V′‐nitro‐N‐nitrosoguanidine, were genetically analysed. The mutations associated with color phenotypes are recessive because all of the heterozygous conchocelis resembled the wild type color when they were crossed with the wild type (wt). In the reciprocal crosses of yel × wt, both parental colors and eight types of blades appeared in the F₁ gametophytic blades from the heterozygous conchocelis. Both colors segregated in the sectored F₁ blades in a 1:1 ratio, indicating that the color pheno‐type of yel resulted from a single mutation in the nuclear gene. In the reciprocal crosses of fre × wt, however, four colors and more than 40 types of blades appeared in the F₁ blades from the heterozygous conchocelis, indicating that the color phenotype of fre resulted from two mutations in different genes. In the reciprocal crosses of bop×wt, three colors and 12 types of blades were observed in the F₁ blades from the heterozygous conchocelis. Both parental colors appeared far more frequently than the third new color. These results indicated that the color phenotype of bop resulted from two closely linked mutations in different genes, and the epistasis occurred in the F₁ blades. The mutants, yel, fre and bop, differ from the spontaneous green (C‐O), the red (H‐25) and the violet (V‐O) mutants of P. yezoensis, respectively.     

Characterization of the tandem CWCH2 sequence motif: a hallmark of inter-zinc finger interactions

Background  

The C2H2 zinc finger (ZF) domain is widely conserved among eukaryotic proteins. In Zic/Gli/Zap1 C2H2 ZF proteins, the two N-terminal ZFs form a single structural unit by sharing a hydrophobic core. This structural unit defines a new motif comprised of two tryptophan side chains at the center of the hydrophobic core. Because each tryptophan residue is located between the two cysteine residues of the C2H2 motif, we have named this structure the tandem CWCH2 (tCWCH2) motif.     

Conservation and diversification of Msx protein in metazoan evolution

Msx (/msh) family genes encode homeodomain (HD) proteins that control ontogeny in many animal species. We compared the structures of Msx genes from a wide range of Metazoa (Porifera, Cnidaria, Nematoda, Arthropoda, Tardigrada, Platyhelminthes, Mollusca, Brachiopoda, Annelida, Echiura, Echinodermata, Hemichordata, and Chordata) to gain an understanding of the role of these genes in phylogeny. Exon-intron boundary analysis suggested that the position of the intron located N-terminally to the HDs was widely conserved in all the genes examined, including those of cnidarians. Amino acid (aa) sequence comparison revealed 3 new evolutionarily conserved domains, as well as very strong conservation of the HDs. Two of the three domains were associated with Groucho-like protein binding in both a vertebrate and a cnidarian Msx homolog, suggesting that the interaction between Groucho-like proteins and Msx proteins was established in eumetazoan ancestors. Pairwise comparison among the collected HDs and their C-flanking aa sequences revealed that the degree of sequence conservation varied depending on the animal taxa from which the sequences were derived. Highly conserved Msx genes were identified in the Vertebrata, Cephalochordata, Hemichordata, Echinodermata, Mollusca, Brachiopoda, and Anthozoa. The wide distribution of the conserved sequences in the animal phylogenetic tree suggested that metazoan ancestors had already acquired a set of conserved domains of the current Msx family genes. Interestingly, although strongly conserved sequences were recovered from the Vertebrata, Cephalochordata, and Anthozoa, the sequences from the Urochordata and Hydrozoa showed weak conservation. Because the Vertebrata-Cephalochordata-Urochordata and Anthozoa-Hydrozoa represent sister groups in the Chordata and Cnidaria, respectively, Msx sequence diversification may have occurred differentially in the course of evolution. We speculate that selective loss of the conserved domains in Msx family proteins contributed to the diversification of animal body organization.     

Morphological and AFLP variation of Porphyra yezoensis Ueda form, narawaensis Miura (Bangiales, Rhodophyta)

Detailed morphological observations were made on two strains of cultivated Porphyra: HG‐1 (pure line isolated from Dai‐1) and Noriken‐4 (parental strain of a pure line HG‐4). The two strains were identified as P. yezoensis f. narawaensis based on their macroscopic and microscopic features, such as long linear or oblanceolate blades up to 50 cm in maximum length, division formulae of spermatangia and zygotosporangia, shape of trichogynes and carpogonia, and the second transverse divisional plane formed at the division from c/2 to c/4 in zygotosporangia. Gametophytic blades from two completely homozygous conchocelis strains isolated in this study (HG‐1 and HG‐4) were cultured under the same conditions and compared to confirm whether the differences in their shapes are genetically determined. The shape of blades from both of conchospores and monospores was always more slender in HG‐4 than in HG‐1 at the same blade age, suggesting that the difference in the blade shape between the two pure lines is due to genetic variation. To estimate the level of genetic variation the two pure lines were subjected to amplified fragment length polymorphism fingerprint analysis. A total of 230 bands were detected in HG‐1 and HG‐4 using eight selective primer pairs, and the number of polymorphic bands was only two in HG‐1. These results indicate that the two pure lines certainly show genetic variation, which is, however, at an extremely low level. The importance of pure‐line breeding and the origin of currently cultivated Porphyra are discussed. This is the first report to identify currently cultivated Porphyra strains in Japan based on combined results of detailed morphological observations and molecular analysis.     

IP3 signaling is required for cilia formation and left-right body axis determination in Xenopus embryos

Vertebrate left–right (LR) body axis is manifested as an asymmetrical alignment of the internal organs such as the heart and the gut. It has been proposed that the process of LR determination commonly involves a cilia-driven leftward flow in the mammalian node and its equivalents (Kupffer’s vesicle in zebrafish and the gastrocoel roof plate in Xenopus). Recently, it was reported that Ca²⁺ flux regulates Kupffer’s vesicle development and is required for LR determination. As a basis of Ca²⁺ flux in many cell types, inositol 1,4,5-trisphosphate (IP₃) receptor-mediated calcium release from the endoplasmic reticulum (ER) plays important roles. However, its involvement in LR determination is poorly understood. We investigated the role of IP₃ signaling in LR determination in Xenopus embryos. Microinjection of an IP₃ receptor-function blocking antibody that can inhibit IP₃ calcium channel activity randomized the LR axis in terms of left-sided Pitx2 expression and organ laterality. In addition, an IP₃ sponge that could inhibit IP₃ signaling by binding IP₃ more strongly than the IP₃ receptor impaired LR determination. Examination of the gastrocoel roof plate revealed that the number of cilia was significantly reduced by IP₃ signal blocking. These results provide evidence that IP₃ signaling is involved in LR asymmetry formation in vertebrates.     

Photosynthesis and respiration in bladelets of Ecklonia cava Kjellman (Laminariales, Phaeophyta) in two localities with different temperature conditions

Characteristics of photosynthesis and respiration of bladelets were compared between Ecklonia cava Kjellman sporophytes growing in a warmer temperate locality (Tei, Kochi Pref., southern Japan) and in a cooler temperate locality (Nabeta, Shizuoka Pref., central Japan). Photosynthesis and respiration were measured with a differential gas-volumeter (Productmeter). In photosynthesis-light curves at 20°C, the rate of net photosynthesis was almost the same at light intensities lower than 25 μmol m⁻² s⁻¹ and the light-saturation occurred at 200–400 μmol m⁻²s⁻¹ in plants of both localities. The light-saturated net photosynthetic rates were higher in winter and spring than in summer and autumn in both plants. The optimum temperature for net photosynthesis at 400 μmol m⁻²s⁻¹ was 27°C throughout the year in the Tei plant and 25–27°C in the Nabeta plant. The decrease of net photosynthetic rates in the supraoptimal temperature range up to 29°C was sharper in winter and spring than in summer and autumn in both plants, being smaller in the Tei plant than in the Nabeta plant in all seasons. The dark respiration rate always increased with water temperature rise in both plants. No clear differences were found in the dark respiration rate between Tei and Nabeta plants except that when measured against dry weight, the Tei plant showed a slightly lower rate as compared with the Nabeta plant.     

An oligodendrocyte enhancer in a phylogenetically conserved intron region of the mammalian myelin gene Opalin

Opalin is a transmembrane protein detected specifically in mammalian oligodendrocytes. Opalin homologs are found only in mammals and not in the genome sequences of other animal classes. We first determined the nucleotide sequences of Opalin orthologs and their flanking regions derived from four prosimians, a group of primitive primates. A global comparison revealed that an evolutionarily conserved region exists in the first intron of Opalin. When the conserved domain was assayed for its enhancer activity in transgenic mice, oligodendrocyte-directed expression was observed. In an oligodendroglial cell line, Oli-neu, the conserved domain showed oligodendrocyte-directed expression. The conserved domain is composed of eight subdomains, some of which contain binding sites for Myt1 and cAMP-response element binding protein (CREB). Deletion analysis and cotransfection experiments revealed that the subdomains have critical roles in Opalin gene expression. Over-expression of Myt1, treatment of the cell with leukemia inhibitory factor (LIF), and cAMP analog (CREB activator) enhanced the expression of endogenous Opalin in Oli-neu cells and activated the oligodendrocyte enhancer. These results suggest that LIF, cAMP signaling cascades and Myt1 play significant roles in the differentiation of oligodendrocytes through their action on the Opalin oligodendrocyte enhancer.     

Interaction between Ca2+ and dipalmitoylphosphatidylcholine membranes. II. Fluorescence anisotropy study

The effect of Ca2+ on the molecular mobility in dipalmitoylphosphatidylcholine membranes was studied by steady-state and time-resolved measurements of fluorescence anisotropy. The fluorescence anisotropy decay of 1,6-diphenyl-1,3,5-hexatriene in the hydrocarbon region indicated that the free volume of molecular rotation became more restricted when the Ca2+ concentration was increased. The decrease of the molecular mobility was observed from 1 mM Ca2+, at which the number of bound Ca2+ is much less than that of the total lipid molecules. A distinct difference between Ca2+ and Mg2+ effects suggested that the change in various membrane properties was induced by the binding of these ions. From these results we propose a long-range attractive interaction between bound Ca2+ and the polar head groups of distant phosphatidylcholine molecules.