A role for Wnts in morpho-genesis and tissue polarity.

Recent studies have shown that secreted Wnt proteins control morphogenetic movements in fish and frog embryos. The analysis of Dishevelled, a cytoplasmic mediator of Wnt signalling, reveals unexpected similarity between gastrulation in vertebrates and polarization of cells in Drosophila epithelia.

Airborne lidar measurements of the soufriere eruption of 17 april 1979.

At the time of the Soufriere, St. Vincent, volcanic eruption of 17 April 1979, a NASA P-3 aircraft with an uplooking lidar (light detection and ranging) system onboard was airborne 130 kilometers east of the island. Lidar measurements of the fresh volcanic ash were made approximately 2 hours after the eruption, 120 kilometers to the northeast and east. On the evening of 18 April, the airborne lidar, on a southerly flight track, detected significant amounts of stratospheric material in layers at 16, 17, 18, and 19.5 kilometers. These data, and measurements to the north on 19 April, indicate that the volcanic plume penetrated the stratosphere to an altitude of about 20 kilometers and moved south during the first 48 hours after the eruption.

A mouse macrophage factor induces head structures and organizes a body axis in Xenopus.

Soluble peptide factors have been implicated as the agents responsible for embryonic inductions in vertebrates. Here, a protein (PIF) secreted by a mouse macrophage cell line is shown to change the developmental fate of Xenopus embryonic cells. Exposure to PIF causes presumptive ectodermal explants to form anterior neural and mesodermal tissues, including brain and eye, instead of ciliated epidermis. In addition, the induced tissues are organized into a rudimentary embryonic axis. These results suggest that PIF or a closely related molecule is involved in inducing anterior structures and organizing the frog body plan.

Wnt signaling and dorso-ventral axis specification in vertebrates.

The dorso-ventral axis is specified in vertebrates through the formation of a dorsal signaling center known as the Spemann organizer. This process depends on signal transduction by beta-catenin that can be regulated by secreted Wnt proteins. Recent discoveries of new players in this signaling pathway have narrowed down the search for the initial cues for axis specification in vertebrate embryos.

Analysis of Dishevelled signalling pathways during Xenopus development.

BACKGROUND: Recent studies have demonstrated that the Wnt, Frizzled and Notch proteins are involved in a variety of developmental processes in fly, worm, frog and mouse embryos. The Dishevelled (Dsh) protein is required for Drosophila cells to respond to Wingless, Notch and Frizzled signals, but the molecular mechanisms of its action are not well understood. Using the ability of a mutant form of the Xenopus homologue of Dsh (Xdsh) to block Wnt and Dsh signalling in a model system, this work attempts to clarify the role of the endogenous Xdsh during the early stages of vertebrate development. RESULTS: A mutant Xdsh (Xdd1) with an internal deletion of the conserved PDZ/DHR domain was constructed. Overexpression of Xdd1 mRNA in ventral blastomeres of Xenopus embryos strongly inhibited induction of secondary axes by the wild-type Xdsh and Xwnt8 mRNAs, but did not affect the axis-inducing ability of beta-catenin mRNA. These observations suggest that Xdd1 acts as a dominant-negative mutant. Dorsal expression of Xdd1 caused severe posterior truncations in the injected embryos, whereas wild-type Xdsh suppressed this phenotype. Xdd1 blocked convergent extension movements in ectodermal explants stimulated with mesoderm-inducing factors and in dorsal marginal zone explants, but did not affect mesoderm induction and differentiation. CONCLUSIONS: A vertebrate homologue of Dsh is a necessary component of Wnt signal transduction and functions upstream of beta-catenin. These findings also establish a requirement for the PDZ domain in signal transduction by Xdsh, and suggest that endogenous Xdsh controls morphogenetic movements in the embryo.

Axis determination in Xenopus involves biochemical interactions of axin, glycogen synthase kinase 3 and beta-catenin.

Signaling by the Wnt family of extracellular proteins is critical in a variety of developmental processes in which cell and tissue polarity are established [1-5]. Wnt signal transduction has been studied mostly by the genetic approach in Drosophila and Caenorhabditis elegans [1,2,5], but the biochemical mechanisms involved remain to be elucidated. The Wnt pathway also operates during axis determination in vertebrates [3,5]. Frizzled receptors transduce a signal to Dishevelled, leading to inactivation of glycogen synthase kinase 3 (GSK3) and regulation of gene expression by the complex of beta-catenin with LEF/TCF (lymphocyte enhancer factor/T-cell factor) transcription factors [3,5]. Axin is a negative regulator of Wnt signaling and dorsal axial development in vertebrates [6]. Here, we demonstrate that axin is associated with GSK3 in the Xenopus embryo and we localize the GSK3-binding domain to a short region of axin. Binding of GSK3 correlates with the ability of axin to inhibit axial development and with the axis-inducing activity of its dominant-negative form (delta RGS). We also find that wild-type axin, but not delta RGS, forms a complex with beta-catenin. Thus, axin may act as a docking station mediating negative regulation of beta-catenin by GSK3 during dorsoventral axis determination in vertebrate embryos.

Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6.

BACKGROUND: Dickkopf-1 (Dkk-1) is a head inducer secreted from the vertebrate head organizer and induces anterior development by antagonizing Wnt signaling. Although several families of secreted antagonists have been shown to inhibit Wnt signal transduction by binding to Wnt, the molecular mechanism of Dkk-1 action is unknown. The Wnt family of secreted growth factors initiates signaling via the Frizzled (Fz) receptor and its candidate coreceptor, LDL receptor-related protein 6 (LRP6), presumably through Fz-LRP6 complex formation induced by Wnt. The significance of the Fz-LRP6 complex in signal transduction remains to be established. RESULTS: We report that Dkk-1 is a high-affinity ligand for LRP6 and inhibits Wnt signaling by preventing Fz-LRP6 complex formation induced by Wnt. Dkk-1 binds neither Wnt nor Fz, nor does it affect Wnt-Fz interaction. Dkk-1 function in head induction and Wnt signaling inhibition strictly correlates with its ability to bind LRP6 and to disrupt the Fz-LRP6 association. LRP6 function and Dkk-1 inhibition appear to be specific for the Wnt/Fz beta-catenin pathway. CONCLUSIONS: Our results demonstrate that Dkk-1 is an LRP6 ligand and inhibits Wnt signaling by blocking Wnt-induced Fz-LRP6 complex formation. Our findings thus reveal a novel mechanism for Wnt signal modulation. LRP6 is a Wnt coreceptor that appears to specify Wnt/Fz signaling to the beta-catenin pathway, and Dkk-1, distinct from Wnt binding antagonists, may be a specific inhibitor for Wnt/beta-catenin signaling. Our findings suggest that Wnt-Fz-LRP6 complex formation, but not Wnt-Fz interaction, triggers Wnt/beta-catenin signaling.

Interaction of dishevelled and Xenopus axin-related protein is required for wnt signal transduction.

Signaling by the Wnt family of secreted proteins plays an important role in animal development and is often misregulated in carcinogenesis. Wnt signal transduction is controlled by the rate of degradation of beta-catenin by a complex of proteins including glycogen synthase kinase 3 (GSK3), adenomatous polyposis coli, and Axin. Dishevelled is required for Wnt signal transduction, and its activation results in stabilization of beta-catenin. However, the biochemical events underlying this process remain largely unclear. Here we show that Xenopus Dishevelled (Xdsh) interacts with a Xenopus Axin-related protein (XARP). This interaction depends on the presence of the Dishevelled-Axin (DIX) domains in both XARP and Xdsh. Moreover, the same domains are essential for signal transduction through Xdsh. Finally, our data point to a possible mechanism for signal transduction, in which Xdsh prevents beta-catenin degradation by displacing GSK3 from its complex with XARP.

Xwnt-2b is a novel axis-inducing Xenopus Wnt, which is expressed in embryonic brain.

Xwnt-2b is a novel member of the Wnt gene family and is 73-74% similar to human and mouse Wnt-2 proteins. Starting from stage 15, Xwnt-2b transcripts are localized to a non-contiguous stripe in the anterior neural plate of the Xenopus embryo. In the tailbud, Xwnt-2b is expressed along the dorsoanterior side of the prosencephalon-mesencephalon boundary. At the tadpole stages, the brain-specific expression fades, but the total amount of Xwnt-2b mRNA does not decline due to activation of its expression in non-brain areas. Microinjection of Xwnt-2b mRNA into a ventral blastomere of 4-8-cell embryos results in the formation of complete secondary body axes. These results suggest that Xwnt-2b is a member of the axis-inducing Wnts and that it is involved in brain development and in later organogenesis.

Graded amounts of Xenopus dishevelled specify discrete anteroposterior cell fates in prospective ectoderm.

Signals emitted from the prospective dorsal marginal zone (the organizer) are thought to specify neuroectodermal cell fates along the anteroposterior (AP) axis, but the mechanisms underlying this signaling event remain to be elucidated. To assess the effect of Xenopus Dishevelled (Xdsh), a proposed component of the Wnt, Notch and Frizzled signal transduction pathways, on AP axis determination, it was supplied in varying doses to presumptive ectodermal cells. Two-fold increments in levels of microinjected Xdsh mRNA revealed a gradual shift in cell fates along the AP axis. Lower doses of Xdsh mRNA activated anterior neuroectodermal markers, XAG1 and Xotx2, whereas the higher doses induced more posterior neural tissue markers such as En2, Krox20 and HoxB9. At the highest dose of Xdsh mRNA, explants contained maximal amount of HoxB9 transcripts and developed notochord and somites. When compared with Xdsh, Xwnt8 mRNA also activated anterior neuroectodermal markers, but failed to elicit mesoderm formation. Analysis of explants overexpressing Xdsh at the gastrula stage revealed activation of several organizer-specific genes which have been implicated in determination of neural tissue (Xotx2, noggin, chordin and follistatin). Whereas Goosecoid, Xlim1 and Xwnt8 were not induced in these explants, another early marginal zone marker, Xbra, was activated at the highest level of Xdsh mRNA. These observations suggest that the effects of Xdsh on AP axis specification may be mediated by combinatorial action of several early patterning genes. Increasing levels of Xdsh mRNA activate posterior markers, whereas increasing amounts of the organizer stimulate the extent of anterior development (Stewart, R.M. and Gerhart, J.C. (1990) Development 109, 363-372). These findings argue against induction of the entire organizer by Xdsh in ectodermal cells and implicate signal transduction pathways involving Xdsh in AP axis determination. Thus, different levels of a single molecule, Xdsh, can specify distinct cell states along the AP axis.

Functional and structural diversity of the human Dickkopf gene family.

Wnt proteins influence many aspects of embryonic development, and their activity is regulated by several secreted antagonists, including the Xenopus Dickkopf-1 (xDkk-1) protein. xDkk-1 inhibits Wnt activities in Xenopus embryos and may play a role in induction of head structures. Here, we characterize a family of human Dkk-related genes composed of Dkk-1, Dkk-2, Dkk-3, and Dkk-4, together with a unique Dkk-3 related protein termed Soggy (Sgy). hDkks 1-4 contain two distinct cysteine-rich domains in which the positions of 10 cysteine residues are highly conserved between family members. Sgy is a novel secreted protein related to Dkk-3 but which lacks the cysteine-rich domains. Members of the Dkk-related family display unique patterns of mRNA expression in human and mouse tissues, and are secreted when expressed in 293T cells. Furthermore, secreted hDkk-2 and hDkk-4 undergo proteolytic processing which results in cleavage of the second cysteine-rich domain from the full-length protein. Members of the human Dkk-related family differ not only in their structures and expression patterns, but also in their abilities to inhibit Wnt signaling. hDkk-1 and hDkk-4, but not hDkk-2, hDkk-3 or Sgy, suppress Wnt-induced secondary axis induction in Xenopus embryos. hDkk-1 and hDkk-4 do not block axis induction triggered either by Xenopus Dishevelled (Xdsh) or Xenopus Frizzled-8 (Xfz8), both of which function to transduce signals from Wnt ligands. Thus, hDkks 1 and 4 may inhibit Wnt activity by a mechanism upstream of Frizzled. Our findings highlight the structural and functional heterogeneity of human Dkk-related proteins.

Simultaneous electroretinograms and visually evoked potentials from adult amblyopes in response to a pattern stimulus.

Electroretinograms (ERGs) and visually evoked potentials (VEPs) were recorded simultaneously from each eye of three adult amblyopes. A spatially alternating checkerboard pattern stimulus of constant mean luminance was used to eliminate the effect of stray light on the ERG. The VEP was affected in the amblyopic eye of all subjects. In two subjects the VEP amplitude was reduced; in the third subject the amplitude was not attenuated, but the waveform of the VEP was markedly altered. Photopic ERGs recorded from the normal and amblyopic eye of each subject with an unpatterned flashing light were equal in amplitude. However, ERGs elicited by a patterned stimulus were affected in the amblyopic eye of all three subjects; the after-potential showed larger reductions in amplitude than the b-wave. These results suggest there may be some retinal involvement in human amblyopia.

Effect of stimulus orientation on the latency and amplitude of the VEP.

VEPs were recorded using 5.0 c/deg square wave gratings presented at vertical and oblique (45 deg) orientations and phase-alternated at two rates, 6 alternations/sec and 12 alternations/sec. In agreement with previous reports, VEP amplitude was smaller for obliquely oriented gratings than for vertically oriented gratings at both alternation rates. Unlike previous studies, however, the authors found that VEP latency was longer for obliquely oriented gratings at the slower (6/sec) alternation rate.

Comparison of pattern VEPs and preferential-looking behavior in 3-month-old infants.

Studies of visual acuity in human infants between 1 and 6 months of age using the visual-evoked potential (VEP) and forced-choice preferential looking (FPL) have shown that acuity is one to two octaves higher by VEP estimates than by FPL estimates. In an attempt to study these differences, the authors obtained both VEP and FPL data from 26 3-month-old infants. VEP data were obtained with gratings of 0.31, 0.62, 1.25 and 2.50 cycles/deg, which were counterphase alternated at 2 Hz. FPL data were obtained for stationary gratings using either the method of constant stimuli or a staircase procedure. Our study revealed three major findings: (1) recordable VEPs can be obtained for spatial patterns that are below threshold by behavioral measures; (2) the use of different scoring criteria that yields comparable VEP and FPL group mean acuities does not yield a significant correlation between VEP amplitude acuity and FPL acuity for individual infants, probably because of the inherent "noise" in each technique; and (3) when VEP latency rather than amplitude is used to estimate acuity, there is a significant correlation between electrophysiology and behavior.

Electrophysiological evidence for the oblique effect in human infants.

Visually-evoked potentials (VEPs) were recorded from infants between the ages of 2 and 11 months in response to 2.5 c/deg main axis and oblique square wave gratings. The oblique effect first appears at 3 months of age; some infants showed smaller VEP amplitude and/or longer VEP latency for obliquely oriented gratings. Regarding the age of onset of the oblique effect, VEP data from this study agree with the results obtained with preferential looking (PL) studies which have directly paired vertical and oblique gratings, but the current study found that fewer infants show an oblique effect by VEP than by PL.

Infant VEP and preferential looking acuity measured with phase alternating gratings.

Previously, infants' grating acuity was found to be temporally tuned, but adults' grating acuity was not. In infants, acuity was higher for gratings phase alternating at 7.5 and 14 reversals/sec than for stationary gratings and gratings alternating at 2.5 or 23 reversals/sec. Also, when preferential looking (PL) and visually evoked potential (VEP) acuity were estimated with phase alternating gratings (14 reversals/sec), the acuity difference between the two techniques was smaller than that obtained when phase alternating gratings were used to estimate VEP acuity and stationary gratings were used to estimate PL acuity. In the present study, it was determined if PL grating acuity was tuned in older children and if the smaller difference between VEP and PL acuity found when infants were tested with phase alternating gratings was independent of temporal rate. Grating acuity in infants older than 2 yr was found to be not tuned, and the smaller difference between VEP and PL grating acuity in infants when both were measured with phase-alternating gratings was not rate dependent. VEP acuity and PL acuity for phase alternating gratings developed at different rates, converging to nearly equivalent levels by 12 mo of age.

Utility of the Arden grating test in glaucoma screening: high false-positive rate in normals over 50 years of age.

The Arden grating test was administered to 64 subjects between 6 and 82 years of age without ocular pathology, to 20 glaucoma patients, and to 21 ocular hypertensives. The results show an age effect, with significantly higher scores (lower sensitivity) in normal subjects over 50 years of age. In addition, there was no significant difference in performance between age-matched normals, glaucoma patients, and ocular hypertensives on the Arden gratings. The results show a high percentage of false-positives in older normal subjects. The Arden gratings should be used cautiously when one is testing patients over 50 years of age.

Recording the contralateral PERG: effect of different electrodes.

A pattern electroretinogram (PERG) can be recorded when the eye wearing the electrode is occluded and the stimulus is viewed with the other eye. We find that this phenomenon occurs when an Arden gold foil electrode is used, but not when either an ERG-Jet lens or a scleral lens electrode is used. Therefore, a corneal-type electrode should be used in PERG recording situations where the fellow eye is not occluded.

The visual evoked potential in glaucoma and ocular hypertension: effects of check size, field size, and stimulation rate.

In order to determine the optimum stimulus conditions for the detection of optic nerve damage due to glaucoma and ocular hypertension, checkerboard pattern reversal visual evoked potentials (VEPs) were recorded from 20 glaucoma patients, 20 ocular hypertensive patients, and 20 age-matched normals. Two check sizes (12' and 48'), two field sizes (14 degrees and 28 degrees), and two alternation rates (1.9 and 7.5 alt/sec) were used. All subjects had visual acuities of 20/40 or better in each eye and equal pupils of 2 to 5 mm diameter. The largest number of VEP abnormalities were found with large checks (48') reversing at a fast rate (7.5 alt/sec). After correcting for the effects of age, visual acuity, and pupil size, 16 of 30 eyes with glaucomatous visual field defects had abnormally long VEP latencies under this condition (beyond the 99% confidence limit of the normal subjects). Nine of 40 ocular hypertensive eyes also had abnormally long latencies. Increased pattern VEP latency was significantly correlated with both the severity and location of visual field defects and the degree of cupping and pallor of the optic disc. VEP latency was not significantly related to intraocular pressure.

Cognitive processes in verbal-number production: inferences from the performance of brain-damaged subjects.

This article presents a model of the cognitive processes involved in the spoken production of verbal numbers (e.g., thirteen thousand four hundred two). On the basis of single-case studies of two brain-damaged subjects with number production deficits, we argue that verbal-number production involves the generation of a syntactic frame that constitutes a plan for the production of the appropriate sequence of words. The syntactic frame specifies each to-be-retrieved word in terms of a number-lexical class (i.e., ones, teens, or tens) and a position within that class. These class/position-within-class specifications guide the retrieval of lexical representations from a production lexicon that is partitioned into functionally distinct ones, teens, and tens classes. We conclude with a brief discussion of the rationale for, and advantages of, using patterns of impaired performance as a basis for drawing inferences about normal cognition.