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We have devised a sensitive method for the isolation and structural analysis of glycosaminoglycans from two genetically tractable model organisms, the fruit fly, Drosophila melanogaster,and the nematode, Caenorhabditis elegans.We detected chondroitin/chondroitin sulfate- and heparan sulfate-derived disaccharides in both organisms. Chondroitinase digestion of glycosaminoglycans from adult Drosophilaproduced both nonsulfated and 4-O-sulfated unsaturated disaccharides, whereas only unsulfated forms were detected in C. elegans. Heparin lyases released disaccharides bearingN-,2-O-, and 6-O-sulfated species, including mono-, di-, and trisulfated forms. We observed tissue- and stage-specific differences in both chondroitin sulfate and heparan sulfate composition in Drosophila. We have also applied these methods toward the analysis of tout-velu,anEXT-related gene in Drosophilathat controls the tissue distribution of the growth factor Hedgehog. The proteins encoded by the vertebrate tumor suppressor genes EXT1and 2, show heparan sulfate co-polymerase activity, and it has been proposed that tout-veluaffects Hedgehog activity via its role in heparan sulfate biosynthesis. Analysis of total glycosaminoglycans from tout-velumutant larvae show marked reductions in heparan sulfate but not chondroitin sulfate, consistent with its proposed function as a heparan sulfate co-polymerase.

ObjectiveTo evaluate the vaccine efficacy of a fowlpox virus recombinant expressing the H7 haemagglutinin of avian influenza virus in poultry.

Objective: To describe the clinical and epidemiological features of an outbreak of a viral infection affecting humans and horses. Setting: Stables in Hendra, a suburb of Brisbane. Subjects: Affected horses and humans, and at‐risk human contacts. Results: A pregnant mare died two days after arrival from a paddock elsewhere in Brisbane. Eight to 11 days later, illness (depression, anorexia, fever, dyspnoea, ataxia, tachycardia, tachypnoea and nasal discharge) was reported among 17 other horses from the same or an adjoining stable. Fourteen horses died or were put down. Five and six days after the index mare's death, a stable‐hand and then a horse trainer, both of whom had had close contact with the sick mare's mucous secretions, developed influenza‐like illnesses. The stable‐hand recovered but the trainer developed pneumonitis, respiratory failure, renal failure and arterial thrombosis, and died from a cardiac arrest seven days after admission to hospital. A morbillivirus cultured from his kidney was identical to one isolated from the lungs of five affected horses. The two affected humans and eight other horses were seropositive for the infection, which was reproduced in healthy horses following challenge by spleen/lung homogenates from infected horses. There was no serological evidence of infection in 157 humans who had had contact with the stables or the sick horses or humans. Conclusions: A previously undescribed morbillivirus infected a probable 21 horses and two humans; one human and 14 horses died. That no further cases were detected among humans suggests that the virus was of low infectivity. The source of infection remains undetermined.

This study measures the rates of the production of NCI3and the disappearance of total residual ammonia nitrogen when chlorine reacts with ammonia nitrogen.

We have screened a collection of approximately 400 GAL4 enhancer trap lines for useful patterns of expression in the embryo, larval brain, imaginal discs, and ovary using a UAS-lacZ reporter construct. Although similar patterns of expression have previously been reported in the original P[lacZ] enhancer trap screens, these lines are useful for directing ectopic expression of genes in discrete patterns during these stages. In addition, we have identified some unique patterns of expression that have not been previously reported. Dev. Dyn. 209:310–322, 1997. © 1997 Wiley-Liss, Inc.

Transcription of the yeast GAL1 and GAL10 genes is induced by growth on galactose. Using the technique of photofootprinting in vivo, we previously documented equivalent transcription-dependent footprints within the putative "TATA" elements of both genes. To explore the functional significance of these observations, we created a 3-base-pair substitution mutation within the GAL1 promoter TATA element, which disrupted the ATATAA consensus sequence but left intact the photomodification targets. The mutation reduced galactose-induced RNA levels by a factor of 100. The mutant promoter no longer displayed the characteristic TATA sequence footprint, supporting the hypothesis that transcription activation involves the binding of a TATA box factor. We also observed a collection of transcription-correlated alterations in the modification pattern at sites between the UASG and the GAL1 TATA element, within sequences that are not required for inducible transcription. These patterns, characteristic of the induced wild-type GAL1 gene, were still galactose inducible with the TATA mutant GAl1 promoter, despite the low level of transcription from this promoter. We conclude that the GAL4-dependent protein/DNA structure responsible for the altered pattern within nonessential sequences is therefore not strictly coupled to an active TATA element or to high levels of expression. Nonetheless, the patterns probably reflect a stable protein-dependent structure that accompanies assembly of the transcription initiation complex.

The effects of preozonation on the treatment of Seine River water were evaluated in a pilot plant consisting of flocculation with basic aluminum polychloride (PAC) followed by sedimentation. Preozonation significantly increased the removals of turbidity, particle numbers and volumes, chemical oxygen demand, and UV absorbance at all treatment levels investigated (0.3–1.2 mg/L for ozone, and 15–60 mg/L for PAC), with the optimum ozone dose appearing to be about 0.8 mg/L. The favorable effects of ozone increased with pH and the particle count of the untreated water. The increases in particle removals appeared to stem partly from increases in floc size and settling velocity.

Objective: To explore the knowledge and attitudes of registered nurses employed in perinatal nursing and to determine the relationships between selected demographic variables and registered nurses' knowledge and attitudes toward perinatal substance abuse.

Decapentaplegic (Dpp) is a Drosophila member of the Transforming Growth Factor-beta (TGF-beta)/Bone Morphogenetic Protein (BMP) superfamily of growth factors. Dpp serves as a classical morphogen, where concentration gradients of this secreted factor control patterning over many cell dimensions. Regulating the level of Dpp signaling is therefore critical to its function during development. One type of molecule proposed to modulate growth factor signaling at the cell surface are integral membrane proteoglycans. We show here that division abnormally delayed (dally), a Drosophila member of the glypican family of integral membrane proteoglycans is required for normal Dpp signaling during development, affecting cellular responses to this morphogen. Ectopic expression of dally+ can alter the patterning activity of Dpp, suggesting a role for dally+ in modulating Dpp signaling strength. These findings support a role for members of the glypican family in controlling TGF-beta/BMP activity in vivo by affecting signaling at the cell surface.

To define the timing of neural crest formation, we challenged the fate of presumptive neural crest cells by grafting notochords, Sonic Hedgehog- (Shh) or Noggin-secreting cells at different stages of neurulation in chick embryos. Notochords or Shh-secreting cells are able to prevent neural crest formation at open neural plate levels, as assayed by DiI-labeling and expression of the transcription factor, Slug, suggesting that neural crest cells are not committed to their fate at this time. In contrast, the BMP signaling antagonist, Noggin, does not repress neural crest formation at the open neural plate stage, but does so if injected into the lumen of the closing neural tube. The period of Noggin sensitivity corresponds to the time when BMPs are expressed in the dorsal neural tube but are down-regulated in the non-neural ectoderm. To confirm the timing of neural crest formation, Shh or Noggin were added to neural folds at defined times in culture. Shh inhibits neural crest production at early stages (0-5 hours in culture), whereas Noggin exerts an effect on neural crest production only later (5-10 hours in culture). Our results suggest three phases of neurulation that relate to neural crest formation: (1) an initial BMP-independent phase that can be prevented by Shh-mediated signals from the notochord; (2) an intermediate BMP-dependent phase around the time of neural tube closure, when BMP-4 is expressed in the dorsal neural tube; and (3) a later pre-migratory phase which is refractory to exogenous Shh and Noggin.

The neural crest is a population of migratory cells, arising from the ectoderm, that invades many sites within the embryo and differentiate into a variety of diverse cell types. Pigment cells, most cells of the peripheral nervous system, adrenal medullary cells, and some cranial cartilage are derived from the neural crest. Despite a wealth of knowledge concerning their pathways of migration and vast array of derivatives, little is known about the formation of neural crest cells or their acquisition of positional identity. This review focuses on the origin of neural crest cells from the ectoderm and the generation of differences in neural crest cell fates along the rostrocaudal axis. In addition, we consider the role of temporal restriction in the developmental potential of premigratory neural crest cells. While evidence for the existence of multipotent stem cells is strong, some experiments also suggest that there may be heterogeneity among neural crest cell precursors, perhaps due to differences in origin, that might explain commitment events occurring early in neural crest development. Copyright 1993, 1999 Academic Press

The F1- and F2-polypeptide components of the fusion proteins and the hemagglutinin/neuraminidase proteins of the avirulent Queensland (V4) and virulent Australia-Victoria (AuV) strains of Newcastle disease virus have been isolated and subjected to extensive primary structural analysis including amino-terminal sequence analysis and fast atom bombardment-mass spectrometry mapping. Nucleotide sequence analysis was performed on the gene which encodes the V4 hemagglutinin/neuraminidase protein. Signal peptidase cleavage was found to have occurred at the Ser31-Leu32 peptide bond of the primary translation products of the fusion protein genes. Activation cleavage of the V4 fusion protein precursor generated a sequence of -Gly-Lys-Gln-Gly84 at the carboxyl terminus of the F2-polypeptide and an amino-terminal sequence of the F1-polypeptide commencing with 86Leu-Ile-Gly-. The V4 hemagglutinin/neuraminidase protein gene was found to encode a primary translation product 45 amino acids longer at the carboxyl terminus than obtainable from the corresponding gene of the AuV strain (McGinnes, L. W., and Morrison, T. G. (1986) Virus Res. 5, 343-356). However, post-translational proteolytic processing, exclusive to the primary translation product of the V4 hemagglutinin/neuraminidase protein gene, was found to have removed the last 42 residues of this carboxyl-terminal appendage.

Recent investigations have found that organic chemicals may contaminate drinking water by permeating buried plastic pipes and gasket materials. Pipe–bottle direct‐exposure experiments and microbalance experiments were conducted in order to determine the susceptibility of polybutylene (PB) piping material and gasket materials to permeation by a wide range of organic chemicals. Many lipophilic compounds tested were found to permeate to a detectable level in consumers' tap water within a month at one third of their aqueous solubilities. It was also found that PB was more permeable than low‐density polyethylene to toluene and that chlorinated hydrocarbons permeated PB faster than unchlorinated hydrocarbons. Gasoline compositions such as benzene, toluene, ethyl benzene, and xylenes appeared to be highly permeable to PB and gasket material. The organic chemicals were approximately 5 to 100 times more permeable in gasket materials than in PB.

UV light can serve as a molecular probe to identify DNA-protein interactions at nucleotide level resolution from intact yeast cells. We have used the photofootprinting technique to determine during which of three regulated states (uninduced, induced, and catabolite repressed) the transcriptional activator protein encoded by GAL4 binds to its recognition sites within the GAL1-GAL10 upstream activating sequence (UASG). GAL4 protein is bound to at least four, and probably five, related sequence blocks within UASG under both induced and uninduced states. GAL4-dependent photofootprints are lost under conditions of catabolite repression. We observed no footprint patterns unique to catabolite-repressed cells, which suggests that binding of a repressor to the UASG is not involved in this process. Photofootprints of the GAL10 TATA element are strictly correlated with transcription: uninduced, catabolite-repressed, and delta gal4 cells exhibit footprints characteristic of the inactive promoter; induced and delta gal80 cells, which express GAL10 constitutively, display footprints unique to the actively transcribed gene.

Hensen’s node in the chick embryo contains prospective notochord cells in a ▿-shaped midline region of the mesoderm, and prospective medial half somite cells in the lateral portions of the node mesoderm, whilst lateral somite cells are derived from the rostral part of the primitive streak (Selleck and Stern, Development 112, 615-626, 1991). In the present study, we have investigated the commitment of these mesoderm cells to their fates by grafting between these regions of the node and primitive streak. By challenging the cells in this way, we have attempted to discover whether prospective notochord and somite cells can change their fates. We find that the mesoderm cells In the midline ▿-shaped portion of Hensen’s node, when grafted into a different region, continue to integrate into the notochord, or form a separate notochord-like structure. By contrast, the prospective somitic cells do not appear to be committed to a somitic fate.

No abstract available for this article.

A recently identified cytokine, vascular endothelial growth factor (VEGF, vascular permeability factor) has been implicated in ovarian hyperstimulation syndrome in women undergoing assisted reproduction. We postulate that circulating and urinary VEGF values increase following gonadotrophin stimulation, in parallel with the increased ovarian vascularity. A VEGF radioimmunoassay was developed using iodinated VEGF as tracer, a goat anti-VEGF serum as antiserum and recombinant human VEGF as standard. The specificity of the assay was confirmed by comparing the reverse phase high-performance liquid chromatography (HPLC) pattern of VEGF immunoactivity in urine and urine spiked with recombinant VEGF. Urine was concentrated 5-fold prior to measurement by the radioimmunoassay. VEGF:creatinine ratios in early morning urine samples were used to monitor daily urinary VEGF concentrations based on its high correlation (r = 0.77, P < 0.001) with VEGF concentrations in 24 h urine collections. No diurnal variation in VEGF:creatinine ratios was detected. VEGF:creatinine ratios were determined daily from nine women undergoing gonadotrophin-releasing hormone (GnRH) agonist/gonadotrophin treatment. In a further 16 women, early morning urine samples were collected in the peri-ovulatory period. A significant increase (P < 0.005, n = 25) was observed in VEGF:creatinine ratios following human chorionic gonadotrophin (HCG) administration. VEGF:creatinine ratios correlated poorly (r < 0.34) with plasma oestradiol, follicle number and size. It is concluded that urinary VEGF/creatinine ratios increase following HCG stimulation.

Fate maps of chick Hensen’s node were generated using Dil and the lineage of individual cells studied by intracellular injection of lysine-rhodamine-dextran (LRD). The cell types contained within the node are organized both spatially and temporally. At the definitive primitive streak stage (Hamburger and Hamilton stage 4), Hensen’s node contains presumptive notochord cells mainly in its anterior midline and presumptive somite cells in more lateral regions. Early in development it also contains presumptive endoderm cells. At all stages studied (stages 3–9), some individual cells contribute progeny to more than one of these tissues. The somitic precursors in Hensen’s node only contribute to the medial halves of the somites. The lateral halves of the somites are derived from a separate region in the primitive streak, caudal to Hensen’s node.

The theory of pure‐Fickian diffusion of solutes through polymers is developed and explained in detail. Unique methods are presented that may be used to predict rapidly and without extensive mathematical manipulation the time in which a hydrophobic organic chemical would penetrate plastic water pipes under pure‐Fickian conditions. It is demonstrated that the results are dependent on the initial boundary conditions assumed, the difference between the external and internal activity of the permeating substance, the equilibrium partitioning of the solute between the pipe wall and the water held inside the pipe, the diffusivity of the solute in the plastic, and the pipe dimensions. Limitations to the theory are discussed in detail and example solutions are presented for four groups of organic chemicals, which are then compared qualitatively with a pipe permeation incident.

Sites of seven incidents of plastic pipe permeation were visited between July 1986 and December 1987, and samples of soil, pipe material, and pipe water were collected. In addition, a comprehensive literature review was undertaken to document other cases of permeation. This work demonstrated that the majority of permeation incidents were associated with gross soil contamination in the area surrounding the pipe. Soil contamination occurred mainly after pipe installation, suggesting that soil analysis prior to pipe installation will not significantly decrease the number of incidents. The occurrence of about half the incidents in low risk areas indicates that limiting plastic pipe use to these areas will not be effective in preventing permeation.

Polybutylene pipes (0.75 in.) buried in both water‐saturated and unsaturated soils contaminated with toluene, trichloroethylene, 1,2‐dichlorobenzene, and o‐chlorophenol were permeated to detectable levels in 1 to 150 days, depending on the organic chemical and its concentration. Comparison of these results with pipe‐bottle experiments containing no soil showed that the concentration of organic chemical in the soil pore controls the rate of organic chemical permeation through buried plastic pipes. The results also indicate that under otherwise identical conditions, plastic pipes buried in a soil of high organic carbon content will be permeated more slowly than pipe buried in a soil of low organic carbon content. Even so, soils of very high organic carbon content cannot be relied on to protect plastic pipe from permeation by organic chemicals. A method for predicting the equilibrium concentration of organic chemical in the soil pore was useful in determining whether a plastic pipe buried in contaminated soil is likely to be permeated.

The gas and liquid resistances to mass transfer were determined for two polypropylene packings placed in an experimental crossflow tower. The results were then compared with corresponding counterflow data as well as with several counterflow mass transfer correlation equations. The liquid‐side mass transfer properties of the two packings proved to he nearly identical for both types of operation, whereas Onda's countertlow gas‐side equation failed to yield accurate results with either mode of operation or packing type. Crossflow air pressure drop gradients were also evaluated, with conflicting results.

The patterning of cell types along the dorsoventral axis of the spinal cord requires a complex set of inductive signals. While the chordamesoderm is a well-known source of ventralizing signals, relatively little is known about the cues that induce dorsal cell types, including neural crest. Here, we demonstrate that juxtaposition of the non-neural and neural ectoderm is sufficient to induce the expression of dorsal markers, Wnt-1, Wnt-3a and Slug, as well as the formation of neural crest cells. In addition, the competence of neural plate to express Wnt-1 and Wnt-3a appears to be stage dependent, occurring only when neural tissue is taken from stage 8-10 embryos but not from stage 4 embryos, regardless of the age of the non-neural ectoderm. In contrast to the induction of Wnt gene expression, neural crest cell formation and Slug expression can be induced when either stage 4 or stage 8-10 neural plates are placed in contact with the non-neural ectoderm. These data suggest that the non-neural ectoderm provides a signal (or signals) that specifies dorsal cell types within the neural tube, and that the response is dependent on the competence of the neural tissue.

We have investigated the lineage and tissue interactions that result in avian neural crest cell formation from the ectoderm. Presumptive neural plate was grafted adjacent to non-neural ectoderm in whole embryo culture to examine the role of tissue interactions in ontogeny of the neural crest. Our results show that juxtaposition of non-neural ectoderm and presumptive neural plate induces the formation of neural crest cells. Quail/chick recombinations demonstrate that both the prospective neural plate and the prospective epidermis can contribute to the neural crest. When similar neural plate/epidermal confrontations are performed in tissue culture to look at the formation of neural crest derivatives, juxtaposition of epidermis with either early (stages 4-5) or later (stages 6-10) neural plate results in the generation of both melanocytes and sympathoadrenal cells. Interestingly, neural plates isolated from early stages form no neural crest cells, whereas those isolated later give rise to melanocytes but not crest-derived sympathoadrenal cells. Single cell lineage analysis was performed to determine the time at which the neural crest lineage diverges from the epidermal lineage and to elucidate the timing of neural plate/epidermis interactions during normal development. Our results from stage 8 to 10+ embryos show that the neural plate/neural crest lineage segregates from the epidermis around the time of neural tube closure, suggesting that neural induction is still underway at open neural plate stages.

Cell lineage analysis has revealed that the amniote organizer, Hensen's node, is subdivided into distinct regions, each containing a characteristic subpopulation of cells with defined fates. Here, we address the question of whether the inducing and regionalising ability of Hensen's node is associated with a specific subpopulation. Quail explants from Hensen's node are grafted into an extraembryonic site in a host chick embryo allowing host- and donor-derived cells to be distinguished. Cell-type- and region-specific markers are used to assess the fates of the mesodermal and neural cells that develop. We find that neural inducing ability is localised in the epiblast layer and the mesendoderm (deep portion) of the medial sector of the node. The deep portion of the posterolateral part of the node does not have neural inducing ability. Neural induction also correlates with the presence of particular prospective cell types in our grafts: chordamesoderm (notochord/head process), definitive (gut) endoderm or neural tissue. However, only grafts that include the epiblast layer of the node induce neural tissue expressing a complete range of anteroposterior characteristics, although prospective prechordal plate cells may also play a role in specification of the forebrain.

We have devised a genetic screen to obtain mutants affecting cell division patterning in the developing central nervous system of Drosophila. The division abnormally delayed (dally) locus was identified using a combination of "enhancer trap" and behavioral screening methods. The ordered cell cycle progression of lamina precursor cells, which generate synaptic target neurons for photoreceptors, is disrupted in dally mutants. The first of two lamina precursor cell divisions shows a delayed entry into mitosis. The second division, one that is triggered by an intercellular signal from photoreceptor axons, fails to take place. Similar to lamina precursors, cells that generate the ommatidia of the adult eye show two synchronized divisions found along the morphogenetic furrow in the eye disc and the first division cycle in dally mutants displays a delayed progression into M phase like that found in the first lamina precursor cell division. dally mutations also affect viability and produce morphological defects in several adult tissues, including the eye, antenna, wing and genitalia. Sequencing of a dally cDNA reveals a potential open reading frame of 626 amino acids with homology to a family of Glypican-related integral membrane proteoglycans. These heparan sulfate-containing proteins are attached to the external leaflet of the plasma membrane via a glycosylphosphatidylinositol linkage. Heparan sulfate proteoglycans may serve as co-receptors for a variety of secreted proteins including fibroblast growth factor, vascular endothelial growth factor, hepatocyte growth factor and members of the Wnt, TGF-beta and Hedgehog families. The cell division defects found in dally mutants implicate the Glypican group of integral membrane proteoglycans in the control of cell division during development.

We isolated from the Rauscher erythroleukemia cell line (Red 5), a subclone (Red 5-1.5), which contains erythropoietin (epo) binding sites and demonstrates an epo-dependent erythroid differentiation. One class of high affinity binding sites was detected with a Kd(± S.D.) of 0.43 ± 0.09 nMand a mean density/cell of 1200 ± 311. The cell-associated 125I-epo was displaced by nonlabeled epo but not by other hormones or factors. The 125I-epo binding to Red 5-1.5 cells was maximal within 3 h at 15 °C and 1 h at 37 °C and proportional to cell number. The addition of epo increased [3H] uridine incorporation into RNA by 6 h and [3H]thymidine incorporation into DNA by 60 h followed by 59Fe incorporation into protein, cell proliferation, and formation of hemoglobin-containing colonies. The incorporation of 59Fe into protein demonstrated a linear dose response (from 0.002 to 1.5 units of epo/ml) beginning 60 h after addition of the hormone to the cultures, and there was a dose-dependent increase (from 0.1 to 1.0 unit of epo/ml) in the formation of hemoglobin-containing colonies. We concluded that the binding of 125I-epo to Red 5-1.5 suggests the presence of specific epo receptors. The sequence of the epo-induced proliferation and differentiation events is similar to primary erythroid cultures but requires longer epo exposure. Receptor occupancy correlates with the induced biological response.

To elucidate critical steps in the transcription initiation process, we have devised a protocol for obtaining information about DNA structure and DNA–protein interactions at nucleotide level resolution from intact yeast cells. Our procedure combines the ultraviolet light 'footprinting' method developed by Becker and Wang1with the 'genomic sequencing' technique described by Church and Gilbert2. Using this approach we were able to detect the binding of GAL 4 protein at sites within the upstream activating sequence (UASG) previously mapped using other in vivo and in vitro foot-printing procedures3–5. We also observed transcription-dependent changes in sensitivity of DNA to ultraviolet-induced covalent modification at several positions between the upstream activating sequence and the transcription initiation sites of the GAL 1 and GAL 10 genes. The most prominent of these changes occurs at a common site within the putative 'TATA' boxes of the two genes. Ultraviolet modification at this site is enhanced only in transcrip-tionally active promoters.

ObjectiveTo determine the infectivity and transmissibility of Hendra virus (HeV).

This paper describes the cytodifferentiation of hind limb mesodermal cells from 12-day-old normal and brachypod (bpH) mouse embryos grown in vitro at high densities. Over a 3-day culture period normal cells underwent aggregation, nodule formation, and coalescence of nodules into large masses of cartilage. This was associated at the biochemical level with a cessation of cell division, with a concomitant increase in metachromatic matrix, and synthesis of collagen. Under the described culture conditions the collagen synthesized by 48 h cultures was predominantly of cartilage type with an αl: α2 ratio of 9:1. A change in the collagen synthetic program was observed when the entire medium was replaced after 48 h incubation with fresh, serum-free medium. Under these conditions the αl: α2 ratio was 4:1. In contrast, brachypod cells plated at the same density appeared large, flattened, and stellate. Upon aggregation, normal nodule morphology was only rarely observed. More often large, irregular clusters formed from suspended cells loosely attaching to the surface aggregates. Concomitant with the marked changes in the morphology of the mutant cells was a linear increase in DNA synthesis and the appearance of many mitotic figures. A biochemical transformation in matrix synthesis was not observed, however. After a 24 h delay, mutant matrix accumulated and stained intensely with toluidine blue. Collagen was synthesized at approximately the normal rate and was of the cartilage type with an αl: α2ratio of 9:1. When incubated in fresh, serum-free medium, the response of collagen subunit synthesis was identical to the normal cultures. In view of these results the possible manner in which brachy-podism causes developmental anomalies of the limb skeleton is suggested.

The hypoblast is generally thought to be responsible for inducing the mesoderm in the chick embryo because the primitive streak, and subsequently the embryonic axis, form according to the orientation of the hypoblast. However, some cells become specified as embryonic mesoderm very late in development, towards the end of the gastrulation period and long after the hypoblast has left the embryonic region. We argue that induction of embryonic mesoderm and of the embryonic axis are different and separable events, both in amniotes and in amphibians. We also consider the relationships between the dorsoventral and anteroposterior axes in both groups of vertebrates.