Functional properties of rat brain sodium channels lacking the beta 1 or beta 2 subunit

The sodium channel purified from rat brain is a heterotrimeric complex of alpha (Mr 260,000), beta 1 (Mr 36,000), and beta 2 (Mr 33,000) subunits. alpha and beta 2 are attached by disulfide bonds. Removal of beta 1 subunits by incubation in 1.0 M MgCl2 followed by reconstitution into phospholipid vesicles yielded a preparation of alpha beta 2 which did not bind [3H]saxitoxin, mediate veratridine-activated 22Na+ influx, or bind the 125I-labeled alpha-scorpion toxin from Leiurus quinquestriatus (LqTx). In contrast, removal of beta 2 subunits by reduction of disulfide bonds with 1.5 mM dithiothreitol followed by reconstitution into phospholipid vesicles yielded a preparation of alpha beta 1 that retained full sodium channel function. Alpha beta 1 bound [3H]saxitoxin with a KD of 4.1 nM at 36 degrees C. It mediated veratridine-activated 22Na+ influx at a comparable initial rate as intact sodium channels with a K0.5 for veratridine of 46 microM. Tetracaine and tetrodotoxin blocked 22Na+ influx. Like intact sodium channels, alpha beta 1 bound 125I-LqTx in a voltage-dependent manner with a KD of approximately 6 nM at a membrane potential of -60 mV and was specifically covalently labeled by azidonitrobenzoyl 125I-LqTx. When incorporated into planar phospholipid bilayers, alpha beta 1 formed batrachotoxin-activated sodium channels of 24 pS whose voltage-dependent activation was characterized by V50 = -110 mV and an apparent gating charge of 3.3 +/- 0.3. These results indicate that beta 2 subunits are not required for the function of purified and reconstituted sodium channels while a complex of alpha and beta 1 subunits is both necessary and sufficient for channel function in the purified state.     

Expression of Notch signaling pathway genes in mouse embryos lacking beta4galactosyltransferase-1

A requirement for beta4galactosyltransferase-1 (beta4GalT-1) activity in the modulation of Notch signaling by the glycosyltransferase Fringe was previously identified in a mammalian co-culture assay. Notch signaling is necessary for the formation of somites in mammals. We therefore investigated the expression of eleven Notch pathway and somitogenic genes in E9.5 mouse embryos lacking beta4GalT-1. Four of these genes were altered in expression pattern or expression level. The Notch target genes Hes5 and Mesp2 were affected to some degree in all mutant embryos. The Notch ligand genes Dll1 and Dll3 were reduced or altered in expression in a significant proportion of mutants. While there were no differences in the number or morphology of somites in E9.5 B4galt1 null embryos, the number of lumbar vertebrae in mutant embryos differed from control littermates (P < or = 0.01). The subtlety of the in vivo phenotype may be due to redundancy since several B4galt genes related to B4galt1 are expressed during embryogenesis.     

Otx1l, Otx2 and Irx1b establish and position the ZLI in the diencephalon

The thalamic complex is the major sensory relay station in the vertebrate brain and comprises three developmental subregions: the prethalamus, the thalamus and an intervening boundary region - the zona limitans intrathalamica (ZLI). Shh signalling from the ZLI confers regional identity of the flanking subregions of the ZLI, making it an important local signalling centre for regional differentiation of the diencephalon. However, our understanding of the mechanisms responsible for positioning the ZLI along the neural axis is poor. Here we show that, before ZLI formation, both Otx1l and Otx2 (collectively referred to as Otx1l/2) are expressed in spatially restricted domains. Formation of both the ZLI and the Irx1b-positive thalamus require Otx1l/2; embryos impaired in Otx1l/2 function fail to form these areas, and, instead, the adjacent pretectum and, to a lesser extent, the prethalamus expand into the mis-specified area. Conditional expression of Otx2 in these morphant embryos cell-autonomously rescues the formation of the ZLI at its correct location. Furthermore, absence of thalamic Irx1b expression, in the presence of normal Otx1l/2 function, leads to a substantial caudal broadening of the ZLI by transformation of thalamic precursors. We therefore propose that the ZLI is induced within the competence area established by Otx1l/2, and is posteriorly restricted by Irx1b.     

Expression of a homeobox gene product in normal and mutant zebrafish embryos: evolution of the tetrapod body plan

An antibody was used to detect antigens in zebrafish that appear to be homologous to the frog homeodomain-containing protein XlHbox 1. These antigens show a restricted expression in the anteroposterior axis and an anteroposterior gradient in the pectoral fin bud, consistent with the distribution of XlHbox 1 protein in frog and mouse embryos. In the somitic mesoderm, a sharp anterior limit of expression coincides exactly with the boundary between somites 4 and 5, and the protein level fades out posteriorly. A similar, graded expression of the antigen is seen within the series of Rohon-Beard sensory neurons of the CNS. We also immunostained the mutant spt-1 ('spadetail'), in which the trunk mesoderm is greatly depleted and disorganized in the region of XlHbox 1 expression. The defects stem from misdirected cell movements during gastrulation, but nervertheless, newly recruited cells that partially refill the trunk mesoderm express the antigen within the normal span of the anteroposterior axis. This finding suggests that the mutation does not delete positional information required for activation of the XlHbox 1 gene.     

Otx1 and Otx2 in the development and evolution of the mammalian brain.

In the last decade, a number of genes related to the induction, specification and regionalization of the brain were isolated and their functional properties currently are being dissected. Among these, Otx1 and Otx2 play a pivotal role in several processes of brain morphogenesis. Findings from several groups now confirm the importance of Otx2 in the early specification of neuroectoderm destined to become fore-midbrain, the existence of an Otx gene dosage-dependent mechanism in patterning the developing brain, and the involvement of Otx1 in corticogenesis. Some of these properties appear particularly fascinating when considered in evolutionary terms and highlight the central role of Otx genes in the establishment of the genetic program defining the complexity of a vertebrate brain. This review deals with the major aspects related to the roles played by Otx1 and Otx2 in the development and evolution of the mammalian brain.     

Selective binding of ligands to beta 1, beta 2 or chimeric beta 1/beta 2-adrenergic receptors involves multiple subsites.

The molecular basis of ligand binding selectivity to beta-adrenergic receptor subtypes was investigated by designing chimeric beta 1/beta 2-adrenergic receptors. These molecules consisted of a set of reciprocal constructions, obtained by the exchange between the wild-type receptor genes of one to three unmodified transmembrane regions, together with their extracellular flanking regions. Eight different chimeras were expressed in Escherichia coli and studied with selective beta-adrenergic ligands. The evaluation of the relative effect of each chimeric exchange on ligand binding affinity was based on the analysis of delta G values, calculated from the equilibrium binding constants, as a function of the number of substituted beta 2-adrenergic receptor transmembrane domains. The data showed that the contribution of each exchanged region to subtype selectivity varies with each ligand; moreover, while several regions are critical for the pharmacological selectivity of all ligands, others are involved in the selectivity of only some compounds. The selectivity displayed by beta-adrenergic compounds towards beta 1 or beta 2 receptor subtypes thus results from a particular combination of interactions between each ligand and each of the subsites, variably distributed over the seven transmembrane regions of the receptor; these subsites are presumably defined by the individual structural properties of the ligands.     

Effects of the infusion of non-selective beta-, and selective beta1- or beta2-adrenergic agonists, on body fat mobilisation in underfed or overfed non-pregnant heifers.

The aim of this study was to analyse the effects of the intravenous infusion of a non-selective beta-agonist (beta-A) (isoproterenol, ISO), and selective beta1-A (dobutamine, D) or beta2-A (terbutaline, T) on body fat mobilisation in non-pregnant heifers, in a 2 x 2 crossover design with two treatments (underfeeding or overfeeding) and two periods. The effect of the duration of submission to each energy level on basal and ISO-induced fat mobilisation was also studied. ISO had a high and significant lipolytic effect whatever the energy level. Nevertheless, the response area of non-esterified fatty acids (NEFA) to ISO in underfed cows was 1.7 times greater than that in overfed cows. T had a slight but significant lipolytic effect on NEFA plasma response in the underfed group. D had no lipolytic effect. Basal and ISO-stimulated plasma NEFA levels were similar after 1 or 4 weeks of underfeeding.     

Induction of ApL3 expression by trehalose complements the starch-deficient Arabidopsis mutant adg2-1 lacking ApL1, the large subunit of ADP-glucose pyrophosphorylase

The disaccharide trehalose has strong effects on plant metabolism and development. In Arabidopsis seedlings, growth on trehalose-containing medium leads to an inhibition of root elongation, an accumulation of starch in the shoots, an increased activity of ADP-Glc pyrophosphorylase (AGPase), and an induction of the expression of the AGPase gene, ApL3 (A. Wingler, T. Fritzius, A. Wiemken, T. Boller, R.A. Aeschbacher [2000] Plant Physiol 124: 105-114). We used Arabidopsis mutants deficient in starch synthesis to examine whether the primary effect of trehalose was to affect carbohydrate allocation by the induction of AGPase in the photosynthetic tissue. In a mutant lacking the large AGPase subunit, ApL1, (adg2-1 mutant) growth on trehalose restored AGPase activity and led to a strong accumulation of starch in the shoots. In contrast, starch synthesis could not be induced in a mutant lacking the small AGPase subunit, ApS, (adg1-1 mutant) or in a mutant lacking plastidic phosphoglucomutase (pgm1-1 mutant). These results indicate that ApL3 can substitute for ApL1 in the AGPase complex. In addition, root elongation in the mutants, especially in the adg1-1 mutant, was partially resistant to trehalose, suggesting that the induction of ApL3 expression and the resulting accumulation of starch in the shoots were partially responsible for the effects of trehalose on the growth of wild-type plants.     

Otx1 and Otx2 activities are required for the normal development of the mouse inner ear

The Otx1 and Otx2 genes are two murine orthologues of the Orthodenticle (Otd) gene in Drosophila. In the developing mouse embryo, both Otx genes are expressed in the rostral head region and in certain sense organs such as the inner ear. Previous studies have shown that mice lacking Otx1 display abnormal patterning of the brain, whereas embryos lacking Otx2 develop without heads. In this study, we examined, at different developmental stages, the inner ears of mice lacking both Otx1 and Otx2 genes. In wild-type inner ears, Otx1, but not Otx2, was expressed in the lateral canal and ampulla, as well as part of the utricle. Ventral to the mid-level of the presumptive utricle, Otx1 and Otx2 were co-expressed, in regions such as the saccule and cochlea. Paint-filled membranous labyrinths of Otx1-/- mutants showed an absence of the lateral semicircular canal, lateral ampulla, utriculosaccular duct and cochleosaccular duct, and a poorly defined hook (the proximal part) of the cochlea. Defects in the shape of the saccule and cochlea were variable in Otx1-/- mice and were much more severe in an Otx1-/-;Otx2(+/)- background. Histological and in situ hybridization experiments of both Otx1-/- and Otx1-/-;Otx2(+/)- mutants revealed that the lateral crista was absent. In addition, the maculae of the utricle and saccule were partially fused. In mutant mice in which both copies of the Otx1 gene were replaced with a human Otx2 cDNA (hOtx2(1)/ hOtx2(1)), most of the defects associated with Otx1-/- mutants were rescued. However, within the inner ear, hOtx2 expression failed to rescue the lateral canal and ampulla phenotypes, and only variable rescues were observed in regions where both Otx1 and Otx2 are normally expressed. These results suggest that both Otx genes play important and differing roles in the morphogenesis of the mouse inner ear and the development of its sensory organs.     

The origin of the turtle body plan: evidence from fossils and embryos

The origin of the unique body plan of turtles has long been one of the most intriguing mysteries in evolutionary morphology. Discoveries of several new stem-turtles, together with insights from recent studies on the development of the shell in extant turtles, have provided crucial new information concerning this subject. It is now possible to develop a comprehensive scenario for the sequence of evolutionary changes leading to the formation of the turtle body plan within a phylogenetic framework and evaluate it in light of the ontogenetic development of the shell in extant turtles. The fossil record demonstrates that the evolution of the turtle shell took place over millions of years and involved a number of steps.     

Circulating integrins: alpha 5 beta 1, alpha 6 beta 4 and Mac-1, but not alpha 3 beta 1, alpha 4 beta 1 or LFA-1.

The alpha 5 beta 1, alpha 6 beta 4 and Mac-1 integrins all participate in the endocytotic cycle. By contrast, alpha 3 beta 1, alpha 4 beta 1 and LFA-1 do so much more slowly, or not at all, in the cell lines examined. This indicates that the alpha-chains appear to determine whether an integrin cycles or not, and that alpha 5 beta 1, alpha 6 beta 4 and Mac-1 can be brought to the leading edge of a moving cell by endocytosis and recycling.     

Differential expression of TGF beta 1, beta 2 and beta 3 genes during mouse embryogenesis

We have examined by Northern analysis and in situ hybridisation the expression of TGF beta 1, beta 2 and beta 3 during mouse embryogenesis. TGF beta 1 is expressed predominantly in the mesodermal components of the embryo e.g. the hematopoietic cells of both fetal liver and the hemopoietic islands of the yolk sac, the mesenchymal tissues of several internal organs and in ossifying bone tissues. The strongest TGF beta 2 signals were found in early facial mesenchyme and in some endodermal and ectodermal epithelial cell layers e.g., lung and cochlea epithelia. TGF beta 3 was strongest in prevertebral tissue, in some mesothelia and in lung epithelia. All three isoforms were expressed in bone tissues but showed distinct patterns of expression both spatially and temporally. In the root sheath of the whisker follicle, TGF beta 1, beta 2 and beta 3 were expressed simultaneously. We discuss the implication of these results in regard to known regulatory elements of the TGF beta genes and their receptors.     

Molecular markers comparing the extremely simple body plan of dicyemids to that of lophotrochozoans: insight from the expression patterns of Hox, Otx, and brachyury

SUMMARY Because of their extremely simple body plan, dicyemids have long been the subject of phylogenetic controversy, regarding whether their body plan reflects their primitiveness or a degeneration from complex metazoans. Several studies have argued that the simple body plan of dicyemids are likely secondarily derived from higher lophotrochozoan animals, as a result of their endoparasitic, or endosymbiotic, lifestyle in the cephalopod kidney. To clarify the evolution of their simple body plan, we investigated the developmental expression patterns of three important regulatory genes, the central type Hox gene ( DoxC ), otx , and brachyury homologs in the dicyemid mesozoa, Dicyema orientale. DoxC was expressed in the trunk and tail of the asexually developing vermiform embryo, with clear anterior boundaries. Do-otx was expressed in the vegetal pole cells of the developing infusoriform embryos, suggesting that the invagination in infusoriform embryo is homologous to the gastrulation of other metazoans. Do-bra is expressed in the presumptive ventral cells, which are ventral to the opening of the urn cavity. The expression of Do-bra suggests that the urn cavity opening of the infusoriform embryo is comparable to the stomodium of trochophore larvae. These gene expression patterns provide molecular clues to trace the evolutionary history of degeneration in the dicyemid embryogenesis and life cycle from those of ancestral lophotrochozoan animals.