Shaping a Morphogen Gradient for Positional Precision

Morphogen gradients, which provide positional information to cells in a developing tissue, could in principle adopt any nonuniform profile. To our knowledge, how the profile of a morphogen gradient affects positional precision has not been well studied experimentally. Here, we compare the positional precision provided by the Drosophila morphogenetic protein Bicoid (Bcd) in wild-type (wt) embryos with embryos lacking an interacting cofactor. The Bcd gradient in the latter case exhibits decreased positional precision around mid-embryo compared with its wt counterpart. The domain boundary of Hunchback (Hb), a target activated by Bcd, becomes more variable in mutant embryos. By considering embryo-to-embryo, internal, and measurement fluctuations, we dissect mathematically the relevant sources of fluctuations that contribute to the error in positional information. Using this approach, we show that the defect in Hb boundary positioning in mutant embryos is directly reflective of an altered Bcd gradient profile with increasing flatness toward mid-embryo. Furthermore, we find that noise in the Bcd input signal is dominated by internal fluctuations but, due to time and spatial averaging, the spatial precision of the Hb boundary is primarily affected by embryo-to-embryo variations. Our results demonstrate that the positional information provided by the wt Bcd gradient profile is highly precise and necessary for patterning precision.     

When are cellular oscillators sufficient for sequential segmentation?

Sequential segmentation during embryogenesis involves the generation of a repeated pattern along the embryo, which is concurrently undergoing axial elongation by cell division. Most mathematical models of sequential segmentation involve inherent cellular oscillators, acting as a segmentation clock. The cellular oscillation is assumed to be governed by the cell's physiological age or by its interaction with an external morphogen gradient. Here, we address the issue of when cellular oscillators alone are sufficient for predicting segmentation, and when a morphogen gradient is required. The key to resolving this issue lies in how cells determine positional information in the model - this is directly related to the distribution of cell divisions responsible for axial elongation. Mathematical models demonstrate that if axial elongation occurs through cell divisions restricted to the posterior end of the unsegmented region, a cell can obtain its positional information from its physiological age, and therefore cellular oscillators will suffice. Alternatively, if axial elongation occurs through cell divisions distributed throughout the unsegmented region, then positional information can be obtained through another mechanism, such as a morphogen gradient. Two alternative ways to establish a morphogen gradient in tissue with distributed cell divisions are presented - one with diffusion and the other without diffusion. Our model produces segment polarity and a distribution of segment size from the anterior-to-posterior ends, as observed in some systems. Furthermore, the model predicts segment deletions when there is an interruption in cell division, just as seen in heat shock experiments, as well as the growth and final shrinkage of the presomitic mesoderm during somitogenesis.     

Genes that control dorsoventral polarity affect gene expression along the anteroposterior axis of the Drosophila embryo

At least 13 genes control the establishment of dorsoventral polarity in the Drosophila embryo and more than 30 genes control the anteroposterior pattern of body segments. Each group of genes is thought to control pattern formation along one body axis, independently of the other group. We have used the expression of the fushi tarazu (ftz) segmentation gene as a positional marker to investigate the relationship between the dorsoventral and anteroposterior axes. The ftz gene is normally expressed in seven transverse stripes. Changes in the striped pattern in embryos mutant for other genes (or progeny of females homozygous for maternal-effect mutations) can reveal alterations of cell fate resulting from such mutations. We show that in the absence of any of ten maternal-effect dorsoventral polarity gene functions, the characteristic stripes of ftz protein are altered. Normally there is a difference between ftz stripe spacing on the dorsal and ventral sides of the embryo; in dorsalized mutant embryos the ftz stripes appear to be altered so that dorsal-type spacing occurs on all sides of the embryo. These results indicate that cells respond to dorsoventral positional information in establishing early patterns of gene expression along the anteroposterior axis and that there may be more significant interactions between the different axes of positional information than previously determined.     

LiCl disrupts axial development in mouse but does not act through the beta-catenin/Lef-1 pathway

Chimera and cell marking studies suggest that axial determination in mouse embryos occurs at postimplantation stages. In contrast, Xenopus laevis axes are determined early due to the asymmetric distribution of maternally derived factors in the one-cell zygote. In our earlier study we used lithium chloride (LiCl) to perturb development of mouse axes. Here we investigate whether the lithium induced axial defects in mouse are being mediated by the beta-catenin/Lef-1 pathway as in Xenopus laevis. In lithium treated embryos we did not observe any changes in the amount or localization of beta-catenin protein. Furthermore, the lack of Lef-1 mRNA in treated and untreated embryos indicates the LiCl induced axial defects in the mouse are not mediated by the beta-catenin/Lef-1 pathway.     

Lithium-induced teratogenesis in frog embryos prevented by a polyphosphoinositide cycle intermediate or a diacylglycerol analog

Microinjection of LiCl into prospective ventral blastomeres of the 32-cell Xenopus embryo gives rise to duplication of dorsoanterior structures such as the notochord, neural tube, eyes, and cement gland. We report here that this teratogenic effect of Li+ is prevented by coinjection of equimolar myo-inositol, an intermediate of the polyphosphoinositide cycle. In contrast, epi-inositol, a nonbiological positional isomer of inositol not employed in this cycle, is ineffective at rescuing Li+-injected embryos. Treatment of embryos at stage 7 with the tumor promoter, phorbol myristate acetate (an analog of the polyphosphoinositide cycle-derived second messenger, diacylglycerol), also prevents dorsoanterior duplication of Li+ embryos, while the nontransforming analog, phorbol myristate acetate-4-O-methyl ether, is without effect. Both of these rescuing agents are without obvious effects on development when administered alone (i.e., without Li+). Li+-selective microelectrode measurements demonstrate that intracellular Li+ levels are identical when Li+ is injected with or without myo-inositol. Clonal analysis shows that blastomeres injected with Li+ plus myo-inositol make a normal contribution of progeny to the later embryo. Because Li+ is a well-established inhibitor of the polyphosphoinositide cycle and can thereby have profound effects on cellular myo-inositol and diacylglycerol levels, these observations concerning inositol-mediated rescue suggest a role for altered polyphosphoinositide cycle activity in lithium-induced teratogenesis.     

Repatterning in amphibian limb regeneration: A model for study of genetic and epigenetic control of organ regeneration

Limb regeneration is an excellent model for understanding organ reconstruction along PD, AP and DV axes. Re-expression of genes involved in axial pattern formation is essential for complete limb regeneration. The cellular positional information in the limb blastema has been thought to be a key factor for appropriate gene re-expression. Recently, it has been suggested that epigenetic mechanisms have an essential role in development and regeneration processes. In this review, we discuss how epigenetic mechanisms may be involved in the maintenance of positional information and the regulation of gene re-expression during limb regeneration.     

Discontinuities of pattern and rules for regeneration in limbs of crayfish

The most caudal limb in crayfish, the uropod, has two rami, the exopodite and the endopodite. Results of earlier experiments (J.E. Mittenthal et al. (1985) W. Roux's Arch. Dev. Biol. 194, 121-130) indicated that ramus morphogenetic fields in the two rami are equivalent and tandem. Thus the proximal (inner) junction of the rami, where intersegmental membrane separates them from each other and from the coxa, is analogous to a boundary between segments of the body or of a leg. In this region a discontinuity in the positional information carried by the ramus fields might occur. To characterize the morphogenetic fields in the region near this junction we have exchanged the medial and lateral margins of the two rami, performing the four possible grafting operations of this kind. While an experimentally generated discontinuity between the lateral margin of the exopodite and the medial margin of the endopodite (outer-to-outer junction) triggers intercalation of supernumerary rami, a discontinuity of pattern between the medial margin of the exopodite and the lateral margin of the endopodite (inner-to-inner junction) is stable despite the absence of intervening intersegmental membrane. Where intercalation does occur, it can proceed in either direction along the margin of a supernumerary ramus. These results suggest that there is no discontinuity of positional value at the boundary between the rami. The results of all of our experiments on the uropod indicate that a conjunction of separate proximodistal, dorsoventral, and mediolateral component fields may give positional information for generating the uropod. Intercalation restores the continuity of pattern in the proximodistal field. In the mediolateral field a discontinuity of pattern may result from a preferred polarity of intercalation: Outer cells may be competent to generate inner cells, but not vice versa. According to this hypothesis the two rami have tandem ramus fields but mirror-symmetric polarity of competence. Alternatively, intercalation may eliminate a mediolateral discontinuity only if the mismatch of mediolateral positional values at the discontinuity exceeds a threshold. The threshold criterion may be a weighted sum of limb field and ramus field positional values.     

The effect of somite manipulation on the development of motoneuron projection patterns in the embryonic chick hindlimb

Although the formation of motoneuron projections to individual muscles in the embryonic chick hindlimb has been shown to involve the specific recognition of environmental cues, the source of these cues and their mode of acquisition are not known. I show in the accompanying paper (C. Lance-Jones, 1988, Dev. Biol. 126, 394-407) that there is a correlation between the segmental level of origin of motoneurons and the somitic level of origin of the muscle cells of their targets in the chick hindlimb. These data are compatible with the hypothesis that the developmental basis for specific recognition is a positional one. Motoneurons and myogenic cells may be uniquely labeled in accord with their axial level of origin early in development and subsequently matched on the basis of these labels. To test this hypothesis, I have assessed motoneuron projection patterns in the embryonic chick hindlimb after somitic tissue manipulations. In one series of embryos, somitic mesoderm at levels 26-29 or 27-29 was reversed about the anteroposterior axis prior to myogenic cell migration and axon outgrowth. Since previous studies have shown that cells migrate from the somites in accord with their position and that somites 26-29 populate anterior thigh musculature, this operation will have reversed the somitic level of origin of anterior thigh muscles. Retrograde HRP labeling of projections to anterior thigh muscles at stage (st) 30 and st 35-38 showed that motoneuron projections were largely normal. This finding suggests that limb muscle cells or their source, the somites, do not contain the cues responsible for specific recognition prior to myogenic cell migration and axon outgrowth. To confirm that specific guidance cues were still intact after somitic mesoderm reversal, I also assessed motoneuron projections in embryos where somitic tissue plus adjacent spinal cord segments at levels 26-29 were reversed in a similar manner. Analyses of the distribution of retrogradely labeled motoneurons in reversed cord segments at st 35-36 indicated that motoneuron projections were reversed. This finding suggests that motoneurons have altered their course to project to correct targets despite the altered somitic origin of their targets and, thus, that specific guidance cues were intact. I conclude that if cues governing target or pathway choice are encoded positionally then they must be associated with other embryonic tissues such as the connective tissues or that guidance cues are acquired by myogenic cells after the onset of migration and motoneuron specification.     

Fasciculation and guidance of spinal motor axons in the absence of FGFR2 signaling

During development, fibroblast growth factors (FGF) are essential for early patterning events along the anterior-posterior axis, conferring positional identity to spinal motor neurons by activation of different Hox codes. In the periphery, signaling through one of four fibroblast growth factor receptors supports the development of the skeleton, as well as induction and maintenance of extremities. In previous studies, FGF receptor 2 (FGFR2) was found to interact with axon bound molecules involved in axon fasciculation and extension, thus rendering this receptor an interesting candidate for the promotion of proper peripheral innervation. However, while the involvement of FGFR2 in limb bud induction has been extensively studied, its role during axon elongation and formation of distinct nervous projections has not been addressed so far. We show here that motor neurons in the spinal cord express FGFR2 and other family members during the establishment of motor connections to the forelimb and axial musculature. Employing a conditional genetic approach to selectively ablate FGFR2 from motor neurons we found that the patterning of motor columns and the expression patterns of other FGF receptors and Sema3A in the motor columns of mutant embryos are not altered. In the absence of FGFR2 signaling, pathfinding of motor axons is intact, and also fasciculation, distal advancement of motor nerves and gross morphology and positioning of axonal projections are not altered. Our findings therefore show that FGFR2 is not required cell-autonomously in motor neurons during the formation of initial motor projections towards limb and axial musculature.     

Role of cell contact in the specification process of pigment founder cells in the sea urchin embryo

Effects of LiCl on the specification process of pigment founder cells were examined in the sea urchin Hemicentrotus pulcherrimus. If embryos were treated with 30 mM LiCl during 4-7 or 7-10 hours postfertilization, pigment cells increased significantly. Aphidicolin treatment indicated that this increase was due to the increase in the pigment founder cells. Interestingly, if the embryos were treated sequentially with LiCl and Ca2+-free seawater during 4-7 and 7-10 hr, respectively, they differentiated only about the same number of pigment cells as control embryos. Further, the increase was scarcely discerned when the embryos were treated with LiCl in the absence of Ca2+ during 7-10 hr. These results suggested that effect of LiCl would be ascribed to the increase in cell adhesiveness. In fact, LiCl-treated embryos were more difficult to be dissociated into single cells. Cell electrophoresis showed that the amount of the negative cell surface charges decreased considerably in LiCl-treated embryos. It was also found that the number of pigment cells seldom exceeded 100, even if embryos were exposed to a higher concentration of LiCl. This suggested that only a subpopulation of the descendants of veg2 blastomeres received the inductive signal emanated from the micromere progeny.     

Parallel waves of inductive signaling and mesenchyme maturation regulate differentiation of the chick mesonephros

The mesonephros is a linear kidney that, in chicken embryos, stretches between the axial levels of the 15th to the 30th somites. Mesonephros differentiation proceeds from anterior to posterior and is dependent on signals from the nephric duct, which migrates from anterior to posterior through the mesonephric region. If migration of the nephric duct is blocked, markers of tubule differentiation, including Lhx1 and Wnt4, are not activated posterior to the blockade. However, activation and maintenance of the early mesonephric mesenchyme markers Osr1, Eya1 and Pax2 proceeds normally in an anterior-to-posterior wave, indicating that these genes are not dependent on inductive signals from the duct. The expression of Lhx1 and Wnt4 can be rescued in duct-blocked embryos by supplying a source of canonical Wnt signaling, although epithelial structures are not obtained, suggesting that the duct may express other tubule-inducing signals in addition to Wnts. In the absence of the nephric duct, anterior mesonephric mesenchyme adjacent to somites exhibits greater competence to initiate tubular differentiation in response to Wnt signaling than more posterior mesonephric mesenchyme adjacent to unsegmented paraxial mesoderm. It is proposed that mesonephric tubule differentiation is regulated by two independent parallel waves, one of inductive signaling from the nephric duct and the other of competence of the mesonephric mesenchyme to undergo tubular differentiation, both of which travel from anterior to posterior in parallel with the formation of new somites.     

Extra cytoproct mutant in Paramecium tetraurelia: morphogenetical analysis of proters and opisthes.

To account for the differences between proters and opisthes with regard to extra cytoproct morphogenesis in Paramecium tetraurelia, two hypotheses have been proposed and tested. (i) The differences may be a result of different actions of proter-macronucleus and opisthe-macronucleus. This hypothesis has been tested by cytoplasmically connecting the proter with the opisthe in the form of chains, some of which have only one macronucleus per chain. However, the connected proters and opisthes remain different in extra cytoproct morphogenesis, thus arguing against the hypothesis. (ii) The differences may be a result of differences between the proter and the opisthe with regard to the development of their posterior-ventral cortex: proters have a newly-developed posterior-ventral cortex whereas opisthes receive the posterior-ventral cortex from the pre-fission mother animal. This hypothesis has been tested by surgically producing an opisthe with a newly-regenerated posterior cortex. Such opisthes, however, remain different from proters in extra cytoproct morphogenesis. Thus no direct support for the second hypothesis is obtained. Also, proter-opisthe difference in morphogenesis may be understood in terms of Wolpert's positional information hypothesis, by assuming that the anterior and posterior ends of a dividing animal serve as reference points for establishing a gradient and that positional information before separation of the two daughter animals leads to differences in extra cytoproct morphogenesis between them after separation.     

The rat leukocyte-type 12-lipoxygenase exhibits an intrinsic hepoxilin A3 synthase activity

Hepoxilins are biologically relevant eicosanoids formed via the 12-lipoxygenase pathway of the arachidonic acid cascade. Although these eicosanoids exhibit a myriad of biological activities, their biosynthetic mechanism has not been investigated in detail. We examined the arachidonic acid metabolism of RINm5F rat insulinoma cells and found that they constitutively express a leukocyte-type 12S-lipoxygenase. Moreover, we observed that RINm5F cells exhibit an active hepoxilin A(3) synthase that converts exogenous 12S-HpETE (12S-5Z,8-Z,10E,14Z-12-hydro(pero)xy-eicosa-5,8,10,14-tetraenoic acid) or arachidonic acid predominantly to hepoxilin A(3). 12S-lipoxygenase and hepoxilin A(3) synthase activities were co-localized in the cytosol; immunoprecipitation with an anti-12S-lipoxygenase antibody co-precipitated the two catalytic activities. These data suggested that hepoxilin A(3) synthase activity may be considered an intrinsic catalytic property of the leukocyte-type 12S-lipoxygenase. To test this hypothesis we cloned the leukocyte-type 12S-LOX from RINm5F cells, expressed it in Pichia pastoris, and found that the recombinant enzyme exhibited both 12S-lipoxygenase and hepoxilin A(3) synthase activities. The recombinant human platelet-type 12S-lipoxygenase and the porcine leukocyte-type 12S-lipoxygenase also exhibited hepoxilin A(3) synthase activity. In contrast, the native rabbit reticulocyte-type 15S-lipoxygenase did not convert 12S-HpETE to hepoxilin isomers. These data suggest that the positional specificity of lipoxygenases may be crucial for this catalytic function. This hypothesis was confirmed by site-directed mutagenesis studies that altered the positional specificity of the rat leukocyte-type 12S- and the rabbit reticulocyte-type 15-lipoxygenase. In summary, it may be concluded that naturally occurring 12S-lipoxygenases exhibit an intrinsic hepoxilin A(3) synthase activity that is minimal in lipoxygenase isoforms with different positional specificity.     

Hensen's node induces neural tissue in Xenopus ectoderm. Implications for the action of the organizer in neural induction.

The development of the vertebrate nervous system is initiated in amphibia by inductive interactions between ectoderm and a region of the embryo called the organizer. The organizer tissue in the dorsal lip of the blastopore of Xenopus and Hensen's node in chick embryos have similar neural inducing properties when transplanted into ectopic sites in their respective embryos. To begin to determine the nature of the inducing signals of the organizer and whether they are conserved across species we have examined the ability of Hensen's node to induce neural tissue in Xenopus ectoderm. We show that Hensen's node induces large amounts of neural tissue in Xenopus ectoderm. Neural induction proceeds in the absence of mesodermal differentiation and is accompanied by tissue movements which may reflect notoplate induction. The competence of the ectoderm to respond to Hensen's node extends much later in development than that to activin-A or to induction by vegetal cells, and parallels the extended competence to neural induction by axial mesoderm. The actions of activin-A and Hensen's node are further distinguished by their effects on lithium-treated ectoderm. These results suggest that neural induction can occur efficiently in response to inducing signals from organizer tissue arrested at a stage prior to gastrulation, and that such early interactions in the blastula may be an important component of neural induction in vertebrate embryos.     

Suppression of positional errors in biological development

Cells in developing embryos behave according to their positions in the organism, and therefore seem to be receiving 'positional information'. A widespread view of the mechanism for this is that each cell responds locally to the concentration level of some extracellular chemical which is distributed in a spatial gradient. For molecules conveying and receiving the positional signal, concentrations are likely to be low enough that, per individual cell, only a few thousand molecules may be involved. Fluctuations to be expected in these numbers (Poisson distribution) could readily lead to errors up to a few percent of embryo length in the reading of position. This is an intolerable level of error for some developmental pattern-forming events. Embryos must have means of suppressing such errors. We maintain that this requires communication between cells, and illustrate this by using the reaction part of two well-known Turing-type reaction-diffusion models as the local gradient reader. We show that switching on diffusion in these models leads to adequate suppression of positional errors.     

The development and evolution of hydrozoan polyp and colony form

Hydrozoans represent an extremely diverse group of mostly colonial forms. Despite this tremendous diversity, many of the morphological differences between hydrozoan species can be attributed to simple changes in the relative position of regions/structures along the axes of the polyp and the stolon or hydrocaulus from which polyps bud. Many genes have been implicated in the specification of positional information along the axis of the polyp. Knowledge from these studies in Hydra, and from comparative studies in Hydractinia polyp polymorphs, suggests that evolutionary changes in the regulation of axial patterning genes may be a prominent mechanism underlying hydrozoan evolution. Despite the paucity of interspecies comparative expression information, hypotheses can be formulated about the role of developmental regulatory genes in hydrozoan evolution from information available from Hydra.     

Positional information and patterning revisited

The concept of positional information proposes that cells acquire positional values as in a coordinate system, which they interpret by developing in particular ways to give rise to spatial patterns. Some of the best evidence for positional information comes from regeneration experiments, and the patterning of the leg and antenna in Drosophila and the vertebrate limb. Central problems are how positional information is set up, how it is recorded, and then how it is interpreted by the cells. A number of models have been proposed for the setting up of positional gradients, and most are based on diffusion of a morphogen and its interactions with extracellular molecules. It is argued that diffusion may not be reliable mechanism. There are also mechanisms based on timing. There is no good evidence for the quantitative aspects of any of the gradients and details how they are set up. The way in which a signalling gradient regulates differential gene expression in a concentration-dependent manner also raises several mechanistic issues.