Dorsoventral Asymmetry Research Articles

Mosaic analyses reveal the function ofDrosophila Rasin embryonic dorsoventral patterning and dorsal follicle cell morphogenesis

In Drosophila melanogaster, the Ras signal transduction pathway is the primary effector of receptor tyrosine kinases, which govern diverse developmental programs. During oogenesis, epidermal growth factor receptor signaling through the Ras pathway patterns the somatic follicular epithelium, establishing the dorsoventral asymmetry of eggshell and embryo. Analysis of follicle cell clones homozygous for a null allele of Ras demonstrates that Ras is required cell-autonomously to repress pipe transcription, the critical first step in embryonic dorsoventral patterning. The effects of aberrant pipe expression in Ras mosaic egg chambers can be ameliorated, however, by post-pipe patterning events, which salvage normal dorsoventral polarity in most embryos derived from egg chambers with dorsal Ras clones. The patterned follicular epithelium also determines the final shape of the eggshell, including the dorsal respiratory appendages, which are formed by the migration of two dorsolateral follicle cell populations. Confocal analyses of mosaic egg chambers demonstrate that Ras is required both cell- and non cell-autonomously for morphogenetic behaviors characteristic of dorsal follicle cell migration, and reveal a novel, Ras-dependent pattern of basal E-cadherin localization in dorsal midline follicle cells.

Role of DIVARICATA in the control of dorsoventral asymmetry in Antirrhinum flowers.

Dorsoventral asymmetry of the Antirrhinum corolla depends on expression of the CYC and DICH genes in dorsal petals. One role of these genes is to inhibit DIVARICATA (DIV), a determinant of ventral identity. Therefore, in cyc;dich double mutants ventral identity spreads all around the flower. We show that DIV encodes a protein belonging to the MYB family of transcription factors. Early on in corolla development, DIV affects specifically the growth of ventral and lateral petals but is transcribed in all petals. Analysis of a closely related gene suggests that the lack of effect on dorsal petals is not due to redundancy. More likely, therefore, DIV is regulated posttranscriptionally through a mechanism that depends on CYC and DICH. Later on, DIV affects growth and cell types and is transcribed mostly in a single layer of cells of ventral and lateral petals. This late pattern may itself depend on DIV activity because it fails to be established in a transcribed but inactive div mutant and, conversely, spreads all around the flower in cyc;dich double mutants.

Floral Ontogeny of Salpiglossis (Solanaceae) and the Oblique Gynoecium

AMPORNPAN, L. AND J. E. ARMSTRONG. (Faculty of Science, Srinakharinwirot University, Bangkok, Thailand, and Campus Box 4120-Biological Sciences, Illinois State University, Normal, IL 61790-4120). Floral ontogeny of Salpiglossis (Solanaceae) and the oblique gynoecium. J. Torrey Bot. Soc. 129:85-95, 2002.-Inflorescence development in Salpiglossis consists of repeated modular units consisting of two floral bracts on an axis terminating in a flower. The calyx is initiated in a helix with the first primordium appearing on the abaxial side of the floral apex. Primordia of the corolla, androecium, and gynoecium arise in three successive whorls. Zygomorphy first becomes manifest with the slightly delayed initiation of a staminodial primordium. Growth of the corolla and androecium show a dorso-ventral asymmetry that also results in zygomorphy. During early development the median plane of many flowers is on the axillary plane. The median plane of other flowers is oriented 10-15? off the axillary plane, but this is less than half the expected 36?. The off-median orientation does not produce the oblique gynoecial orientation described in the literature as a defining feature of Solanaceae. The zygomorphic flowers of Salpiglossis have a gynoecium that is on the median plane and not oblique. Further the flowers are not obliquely oriented unless via pedicel rotation prior to anthesis. The floral orientation of Solanaceae, as exemplified by Salpiglossis, has the staminode and a calyx lobe in the abaxial position, which is a synapomorphy with respect to other Asteridae.

Ancient asymmetries in the evolution of flowers

Dorsoventral asymmetry in flowers is thought to have evolved many times independently as a specialized adaptation to animal pollinators [1, 2]. To understand how such a complex trait could have arisen repeatedly, we have compared the expression of a gene controlling dorsoventral asymmetry in Antirrhinum with its counterpart in Arabidopsis, a distantly related species with radially symmetrical flowers. We found that the Arabidopsis gene is expressed asymmetrically in floral meristems, even though they are destined to form symmetrical flowers. This suggests that, although the flowers of the common ancestor were probably radially symmetrical, they may have had an incipient asymmetry, evident at the level of early gene activity, which could have been recruited many times during evolution to generate asymmetric flowers.

The Handlebars gene is required with Phantastica for dorsoventral asymmetry of organs and for stem cell activity in Antirrhinum.

In angiosperms, individual lateral organs and whole flowers may develop asymmetrically along their dorsoventral axes. Dorsoventral asymmetry of Antirrhinum leaves requires activity of the Phantastica gene and other factors acting redundantly with it. We describe the effects of a mutation in the Handlebars gene, identified as an enhancer of the phantastica mutant phenotype. Genetic analysis suggests that Handlebars functions redundantly with Phantastica to promote dorsal fate in lateral organs and to maintain activity of stem cells within shoot apical meristems. Handlebars appears dispensable in vegetative development but is needed for asymmetry of petals along the dorsoventral axis of the flower as a whole. This suggests that common mechanisms may control dorsoventral asymmetry in lateral organ primordia and in floral meristems.

Identification of RALDH-3, a novel retinaldehyde dehydrogenase, expressed in the ventral region of the retina

In the developing retina, a retinoic acid (RA) gradient along the dorso-ventral axis is believed to be a prerequisite for the establishment of dorso-ventral asymmetry. This RA gradient is thought to result from the asymmetrical distribution of RA-generating aldehyde dehydrogenases along the dorso-ventral axis. Here, we identified a novel aldehyde dehydrogenase specifically expressed in the chick ventral retina, using restriction landmark cDNA scanning (RLCS). Since this molecule showed enzymatic activity to produce RA from retinaldehyde, we designated it retinaldehyde dehydrogenase 3 (RALDH-3). Structural similarity suggested that RALDH-3 is the orthologue of human aldehyde dehydrogenase 6. We also isolated RALDH-1 which is expressed in the chick dorsal retina and implicated in RA formation. Raldh-3 was preferentially expressed first in the surface ectoderm overlying the ventral portion of the prospective eye region and then in the ventral retina, earlier than Raldh-1 in chick and mouse embryos. High level expression of Raldh-3 was also observed in the nasal region. In addition, we found that Pax6 mutants are devoid of Raldh-3 expression. These results suggested that Raldh-3 is the key enzyme in the formation of an RA gradient along the dorso-ventral axis during the early eye development, and also in the development of the olfactory system.

Regulative specification of ectoderm in skeleton disrupted sea urchin embryos treated with monoclonal antibody to Pl-nectin.

Pl-nectin is a glycoprotein first discovered in the extracellular matrix (ECM) of Paracentrotus lividus sea urchin embryo, apically located on ectoderm and endoderm cells. The molecule has been described as functioning as an adhesive substrate for embryonic cells and its contact to ectoderm cells is essential for correct skeletogenesis. The present study was undertaken to elucidate the biochemical characteristics of Pl-nectin and to extend knowledge on its in vivo biological function. Here it is shown that the binding of mesenchyme blastula cells to Pl-nectin-coated substrates was calcium dependent, and reached its optimum at 10 mM Ca2+. Perturbation studies using monoclonal antibody (McAb) to Pl-nectin, which prevent ectoderm cell-Pl-nectin contact, show that dorsoventral axis formation and ectoderm differentiation were retarded. At later stages, embryos recovered and, even if growth and patterning of the skeleton was greatly affected, the establishment of dorsoventral asymmetry was reached. Similarly, the expression of specific ectoderm and endoderm territorial markers was achieved, although occurring with some delay. Endoderm differentiation and patterning was not obviously affected. These results suggest that both endoderm and ectoderm cells have regulative capacities and differentiation of territories is restored after a lag period. On the contrary, failure of inductive differentiation of the skeleton cannot be rescued, even though the ectoderm has recovered.

Retinal ganglion cells with NADPH-diaphorase activity in the chick form a regular mosaic with a strong dorsoventral asymmetry that can be modelled by a minimal spacing rule

We have identified a class of retinal ganglion cells in the chick retina that can be labelled by NADPH-diaphorase histochemistry. These cells have a remarkable topographic distribution, being restricted to the dorsal hemiretina, and form a highly regular mosaic, as revealed by the analysis of nearest neighbour distribution and Delaunay triangulation. Autocorrelation analysis of the mosaic of NADPH-diaphorase-positive retinal ganglion cells shows that the mosaic spatial organization could be generated with the single constraint that two elements cannot be closer than a given minimal distance (d(min)), which we confirmed by computer simulations. In contrast with what has been observed in other mosaics, here d(min) varies with cell density. However, the observed variation of the exclusion area is consistent with an original assembly of the mosaic with a constant d(min) (as is the case in other mosaics), followed by differential expansion of the retina during development.

Graded interference with FGF signalling reveals its dorsoventral asymmetry at the mid-hindbrain boundary.

Signalling by fibroblast growth factors (FGFs) at the mid-hindbrain boundary (MHB) is of central importance for anteroposterior neural patterning from the isthmic organiser. Graded suppression of FGF signalling by increasing amounts of a dominant negative FGF receptor provides evidence that in addition to anteroposterior patterning, FGF signalling is also involved in patterning along the dorsoventral axis at the MHB. FGF signalling at the MHB is required for the activation of the HH target gene spalt at the MHB. Our results indicate that FGF signalling mediates the competence of the MHB to activate spalt in response to SHH. This interdependence of the two signalling pathways is also found in the outbudding optic vesicle where HH requires functional FGF signalling to activate spalt in the proximal eye region.

Gene silencing and toadflax

Cubas, P., Vincent, C. and Coen, E. (1999) An epigenetic mutation responsible for natural variation in floral symmetry, Nature 401, 157–161

Predictability of dorso-ventral asymmetry in the cleavage stage zebrafish embryo: an analysis using lithium sensitivity as a dorso-ventral marker

The dorso-ventral axis in zebrafish first becomes apparent at gastrulation, when the future ventral side appears thinner than the dorsal side. The exact time of establishment of the dorso-ventral axis is not known. We show here that the dorso-ventral axis is specified as early as the 32 cell stage. Using lithium as a marker for dorso-ventral asymmetry, we show that lithium-sensitivity is a characteristic of future ventral cell, but not future dorsal cells, and that there is an asymmetric lithium-sensitivity along the long axis of the 32 cell stage embryo. Consequently, the dorso-ventral axis corresponds to the long axis of the embryo. Because the effect of lithium treatment is short-lived, the dorso-ventral axis must be specified in zebrafish already at the 32 cell stage.

An epigenetic mutation responsible for natural variation in floral symmetry.

Although there have been many molecular studies of morphological mutants generated in the laboratory, it is unclear how these are related to mutants in natural populations, where the constraints of natural selection and breeding structure are quite different. Here we characterize a naturally occurring mutant of Linaria vulgaris, originally described more than 250 years ago by Linnaeus, in which the fundamental symmetry of the flower is changed from bilateral to radial. We show that the mutant carries a defect in Lcyc, a homologue of the cycloidea gene which controls dorsoventral asymmetry in Antirrhinum. The Lcyc gene is extensively methylated and transcriptionally silent in the mutant. This modification is heritable and co-segregates with the mutant phenotype. Occasionally the mutant reverts phenotypically during somatic development, correlating with demethylation of Lcyc and restoration of gene expression. It is surprising that the first natural morphological mutant to be characterized should trace to methylation, given the rarity of this mutational mechanism in the laboratory. This indicates that epigenetic mutations may play a more significant role in evolution than has hitherto been suspected.

Dorsoventral asymmetry of colour discrimination in bees

Colour discrimination performance of honeybees was examined by training bees to a two-coloured disc presented on a vertical plane. Access to the food reward was through the centre of the disc. In one experiment, the upper half of the disc was yellow, and the lower half blue. In another experiment, this was vice versa. In either case, the learned disc was tested against each of a series of ten discs whose colour differed in either the upper or the lower half. A comparison between the results obtained in the two experiments reveals that colour discrimination is significantly better in the lower half of the frontal eye region than it is in the upper half. The results, similar to earlier results obtained in pattern discrimination tasks, cannot be explained by peripheral eye-region-specific specializations. It is proposed that the functional significance of the lower frontal visual field is based on more central neural mechanisms that might constitute an adaptation to the forager's natural needs.

From cortical rotation to organizer gene expression: toward a molecular explanation of axis specification in Xenopus.

After fertilization of Xenopus eggs, the cortex rotates relative to the cytoplasm, resulting in the formation of a cytoplasmic and transplantable dorsal-determining activity opposite the sperm entry point. This activity induces the dorsal expression of regulatory genes, which in turn establishes the Spemann organizer at the start of gastrulation. There has been considerable debate as to whether Vg1, or components of the Wnt-1 signaling pathway, normally function as this early dorsal determinant. Experiments now support the hypothesis that beta-catenin, a component of the Wnt pathway, provides the initial dorsoventral polarity to the embryo, and that Vg1 functions at a subsequent step in development. Specifically, beta-catenin is required for formation of the endogenous axes, and it is expressed at greater levels in dorsal cells during the early cleavage stages. Moreover, on the dorsal side of the embryo, complexes of beta-catenin and Tcf-3 directly bind the promoter of the dorsal regulatory genes siamois and twin and facilitate their expression, thereby contributing to the subsequent formation of the Spemann organizer. On the ventral side of the embryo, Tcf-3 likely works in the absence of beta-catenin as a transcriptional repressor of siamois. These and other data are considered in the context of how the initial polarization of the fertilized egg by the localized accumulation of beta-catenin establishes a range of subsequent dorsoventral asymmetries in the embryo.

From cortical rotation to organizer gene expression: toward a molecular explanation of axis specification in Xenopus

After fertilization of Xenopus eggs, the cortex rotates relative to the cytoplasm, resulting in the formation of a cytoplasmic and transplantable dorsal-determining activity opposite the sperm entry point. This activity induces the dorsal expression of regulatory genes, which in turn establishes the Spemann organizer at the start of gastrulation. There has been considerable debate as to whether Vg1, or components of the Wnt-1 signaling pathway, normally function as this early dorsal determinant. Experiments now support the hypothesis that β-catenin, a component of the Wnt pathway, provides the initial dorsoventral polarity to the embryo, and that Vg1 functions at a subsequent step in development. Specifically, β-catenin is required for formation of the endogenous axes, and it is expressed at greater levels in dorsal cells during the early cleavage stages. Moreover, on the dorsal side of the embryo, complexes of β-catenin and Tcf-3 directly bind the promoter of the dorsal regulatory genes siamois and twin and facilitate their expression, thereby contributing to the subsequent formation of the Spemann organizer. On the ventral side of the embryo, Tcf-3 likely works in the absence of β-catenin as a transcriptional repressor of siamois. These and other data are considered in the context of how the initial polarization of the fertilized egg by the localized accumulation of β-catenin establishes a range of subsequent dorsoventral asymmetries in the embryo. BioEssays 20:536–545, 1998.© 1998 John Wiley & Sons Inc.

Nested expression and sequential downregulation of the Xenopus caudal genes along the anterior-posterior axis

Expression of the Xenopus Xcad-1 and Xcad-2 genes initiates during early gastrulation exhibiting a dorsoventral asymmetry in their domains of transcription. At mid-gastrulation the ventral preference becomes stronger and the caudal genes take up a posterior localization in their expression, which they will maintain until their downregulation along the dorsal midline. Comparison of the three Xenopus caudal genes revealed a temporal and spatial nested set of expression patterns. The transcription of the caudal genes is sequentially downregulated with the one expressed most caudally (Xcad-2) being shut down first, this sequence is most evident along the dorsal midline. This pattern of expression suggests a role for the caudal genes as posterior determinants along the anteroposterior axis. In chicken, mouse, man and Xenopus three members of the caudal family have been identified in the genome. Even though in Xenopus the Xcad-3 gene has been previously described, in order to obtain a better insight on the role of the caudal genes a comparative study of all three frog genes was performed.

The global pattern of cytochrome oxidase stripes in visual area V2 of the macaque monkey.

In primate visual area V2, histochemical staining for cytochrome oxidase (CO) reveals a tripartite pattern of densely labeled thick and thin stripes separated by pale interstripes. This modularity is believed to be related to functionally distinct processing streams that course through the hierarchy of visual areas. Here, we studied the overall pattern of CO stripes in V2 of the macaque monkey, using tissue that had been physically unfolded and flattened prior to histological sectioning. CO stripes were identified on the basis of their physical dimensions and on their differential immunoreactivity for the monoclonal antibody Cat-301. We observed several distinctive features of compartmental organization in V2. The most prominent was a dorso-ventral asymmetry in the stripe pattern, occurring in the majority of cases studied. In dorsal V2, most stripes measure approximately 10 mm in length and run roughly orthogonal to both the posterior and anterior borders of V2. In contrast, many stripes in ventral V2 have a curved or oblique trajectory, and some extend up to 20 mm in length. Stripes following a curved trajectory often become nearly parallel to the anterior border of V2. These differences imply an asymmetry in how the visual field maps onto dorsal versus ventral stripes. Occasionally, thin stripes fail to alternate with thick stripes but instead occur next to one other, separated only by interstripes. In three most complete reconstructions, we found that unfolded V2 is approximately 110 mm in length, approximately 900 mm2 in surface area, and that it contains approximately 28 complete sets of stripes (one thick, one thin and two interstripes), yielding an average of approximately 4 mm per set of stripes. The maximum width of ventral V2 (13-14 mm) exceeds that of dorsal V2 (10 mm), and there is a consistent narrowing of V2 in the region of foveal representation (3-5 mm).

Pattern Formation injanus-Mutant Zebrafish Embryos

Mechanisms that underlie the formation of the vertebrate body appear to be highly conserved between amphibia and teleosts. For teleosts, however, mesoderm induction and the establishment of dorsoventral polarity are poorly understood. In this study, we present an analysis of early pattern formation in the zebrafish maternal-effect mutationjanus.This mutation frequently results in a separation of the cleavage stage blastoderm into two halves that undergo separate development until fusion occurs at the end of gastrulation. Here, we employjanus-mutant embryos to analyze the mechanisms of mesoderm formation and ventral specification in a teleost. Analysis of the expression of the panmesodermal markerno tailinjanus-mutant embryos indicates that mesoderm induction depends on a marginal position. In an analysis of ventral specification, we show that the early expression of the ventral markerGATA-2is confined to the area on both blastodermal halves opposite the dorsal shield region. Since, injanus-mutant embryos, the dorsal position is random with respect to the division plane bisecting the two blastodermal halves, a variety of dorsoventral asymmetries arise within individual embryos. In one constellation, the dorsal position is localized to the plane of bisection and two ventral positions develop at opposite ends of the blastodermal halves. Hence, ventral fates can be specified at any position around the blastodermal margins and are excluded from the dorsal position. The diblastodermic system of thejanus-mutant embryo allows for the study of the interactions of dorsal and ventral determinants in varying spatial arrangements. We have studied pattern formation in dorsal half-blastoderms that contain the entire shield region but only a reduced ventrolateral marginal zone. As assessed by the presence of the most ventral cell type, blood, ventral specification within a dorsal half-blastoderm is not suppressed.

Establishment of the dorso-ventral axis in Xenopus embryos is presaged by early asymmetries in beta-catenin that are modulated by the Wnt signaling pathway.

Eggs of Xenopus laevis undergo a postfertilization cortical rotation that specifies the position of the dorso-ventral axis and activates a transplantable dorsal-determining activity in dorsal blastomeres by the 32-cell stage. There have heretofore been no reported dorso-ventral asymmetries in endogenous signaling proteins that may be involved in this dorsal-determining activity during early cleavage stages. We focused on beta-catenin as a candidate for an asymmetrically localized dorsal-determining factor since it is both necessary and sufficient for dorsal axis formation. We report that beta-catenin displays greater cytoplasmic accumulation on the future dorsal side of the Xenopus embryo by the two-cell stage. This asymmetry persists and increases through early cleavage stages, with beta-catenin accumulating in dorsal but not ventral nuclei by the 16- to 32-cell stages. We then investigated which potential signaling factors and pathways are capable of modulating the steady-state levels of endogenous beta-catenin. Steady-state levels and nuclear accumulation of beta-catenin increased in response to ectopic Xenopus Wnt-8 (Xwnt-8) and to the inhibition of glycogen synthase kinase-3, whereas neither Xwnt-5A, BVg1, nor noggin increased beta-catenin levels before the mid-blastula stage. As greater levels and nuclear accumulation of beta-catenin on the future dorsal side of the embryo correlate with the induction of specific dorsal genes, our data suggest that early asymmetries in beta-catenin presage and may specify dorso-ventral differences in gene expression and cell fate. Our data further support the hypothesis that these dorso-ventral differences in beta-catenin arise in response to the postfertilization activation of a signaling pathway that involves Xenopus glycogen synthase kinase-3.

Locomotor design of dolphin vertebral columns: bending mechanics and morphology of Delphinus delphis.

The primary skeletal structure used by dolphins to generate the dorsoventral bending characteristic of cetacean swimming is the vertebral column. In the vertebral column of the saddleback dolphin Delphinus delphis, we characterize the static and dynamic mechanical properties of the intervertebral joints, describe regional variation and dorsoventral asymmetries in mechanical performance, and investigate how the mechanical properties are correlated with vertebral morphologies. Using a bending machine that applies an external load (N m) to a single intervertebral segment, we measured the resulting angular deformation (rad) of the segment in both dorsal extension and ventral flexion. Intervertebral segments from the thoracic, lumbar and caudal regions of the vertebral column were tested from five individuals. Using quasi-static bending tests, we measured the initial (low-strain) bending stiffness (N m rad-1) as a function of segment position, direction of bending (extension and flexion) and sequential cutting of intervertebral ligaments. We found that initial bending stiffness was significantly greater in the lumbar region than in adjacent thoracic and caudal regions, and all joints were stiffer in extension than is predicted (r2 = 0.554) by the length and width of the intervertebral disc and the length of the cranial vertebral body in the segment. Stiffness in flexion is predicted (r2 = 0.400) by the width of the nucleus pulposus, the length of the caudal vertebral body in the segment and the height of the transverse processes from the ventral surface of the vertebral body. We also performed dynamic bending tests on intervertebral segments from the lumbo-caudal joint and the joint between caudal vertebrae 7 and 8. Dynamic bending stiffness (N m rad-1) increases with increasing bending amplitude and is independent of bending frequency. Damping coefficient (kg m2 rad-2 s-1) decreases with increasing bending amplitude and frequency. Resilience (% energy return) increases from approximately 20% at low bending amplitudes (+/-0.6 degree) to approximately 50% at high bending amplitudes (+/-2.9 degrees). Based on these findings, the dolphin's vertebral column has the mechanical capacity to help control the body's locomotor reconfigurations, to store elastic energy and to dampen oscillations.