Hensen's node,but not other biological signallers,can induce supernumerary digits in the developing chick limb bud

Summary The purpose of this study was to determine whether the organizer regions of early avian and amphibian embryos could induce supernumerary (SN) wing structures to develop when they were grafted to a slit in the anterior side of stage 19–23 chick wing buds. Supernumerary digits developed in 43% of the wings that received anterior grafts of Hensen's node from stage 4–6 quail or chick embryos; in addition, 16% of the wings had rods of SN cartilage, but not recognizable SN digits. The grafted quail tissue did not contribute to the SN structures. When tissue anterior or lateral to Hensen's node or lateral pieces of the area pellucida caudal to Hensen's node were grafted to anterior slits, the wings usually developed normally. No SN structures developed when Hensen's nodes were grafted to posterior slits in chick wing buds. Wings developed normally when pieces of the dorsal lip of the blastopore from stage 10–11.5 frog (Xenopus laevis and Rana pipiens) embryos were grafted to anterior slits. No SN digits developed when other tissues that have limb-inducing activity in adult urodele amphibians [chick otic vesicle, frog (Rana pipiens) lung and kidney] or that can act as heteroinductors in neural induction (rat kidney, lung, submaxillary gland and urinary bladder; mouse liver and submaxillary gland) were grafted to anterior slits in chick wing buds. SN digits also failed to develop following preaxial grafts of chick optic vesicles. These results suggest that although the anteroposterior polarity of the chick wing bud can be influenced by factors other than the ZPA (e.g., Hensen's node, retinoids), the wing is not so labile that it can respond to a wide variety of inductively-active tissues.     

The wings ofBombyx mori develop from larval discs exhibiting an early differentiated state: a preliminary report

Lepidopteran insects present a complex organization of appendages which develop by various mechanisms. In the mulberry silkworm,Bombyx mori a pair of meso- and meta-thoracic discs located on either side in the larvae gives rise to the corresponding fore- and hind-wings of the adult. These discs do not experience massive cell rearrangements during metamorphosis and display the adult wing vein pattern. We have analysed wing development inB. mori by two approaches, viz., expression of patterning genes in larval wing discs, and regulatory capacities of larval discs following explantation or perturbation. Expression of Nubbin is seen all over the presumptive wing blade domains unlike inDrosophila, where it is confined to the hinge and the wing pouch. Excision of meso- and meta-thoracic discs during the larval stages resulted in emergence of adult moths lacking the corresponding wings without any loss of thoracic tissues suggesting independent origin of wing and thoracic primordia. The expression of wingless and distal-less along the dorsal/ventral margin in wing discs correlated well with their expression profile in adultDrosophila wings. Partially excised wing discs did not showin situ regeneration or duplication suggesting their early differentiation. The presence of adult wing vein patterns discernible in larval wing discs and the patterns of marker gene expression as well as the inability of these discs to regulate growth suggested that wing differentiation is achieved early inB. mori. The timings of morphogenetic events are different and the wing discs behave like presumptive wing buds opening out as wing blades inB. mori unlike evagination of only the pouch region as wing blades seen inDrosophila.     

Morphological and histological examination of polyphenic wing formation in the pea aphid <Emphasis Type="Italic">Acyrthosiphon pisum</Emphasis> (Hemiptera,Hexapoda)

Aphids display divergent adult phenotypes, depending on environmental conditions experienced during their embyonic and nymphal stages in their complex life cycles. The plastic developmental mode is an extreme case of phenotypic plasticity, so-called “polyphenism”, in which discrete multiple phenotypes are produced based on a single genome. For example, winged and wingless adult females are derived from a single genotype. However, the developmental mechanisms producing these polyphenic traits according to the extrinsic stimuli, such as density conditions, still remain unknown. In this study, to analyze the developmental processes underlying the wing polyphenism, we extensively observed and compared wing development in the winged and wingless individuals in parthenogenetic generations of the aphid Acyrthosiphon pisum (Harris), using scanning electron microscopy and histological sectioning. At the first-instar stage, the wing primordia were observed both in the future winged (W) and wingless (WL) nymphs. Developmental differences can be seen from the second-instar stage, when wing primordia degenerate in the WL nymphs, while they develop and become more thickened in the W nymphs, suggesting that the developmental programs should be launched prior to this stage. Furthermore, during the third- to fifth-instar stages, wing buds and flight muscles were well developed in the W nymphs, while wing primordia completely disappeared in the WL ones. In addition, the observation on the detailed developmental process of wing primordia during the third-instar W nymphs showed that the wing buds become swollen especially at the basal part, even during the intermolt period. This was caused by the development of wing epithelia under the cuticle of this instar nymph. Actually on the surface of the cuticle of wing-bud bases, there were numerous furrows, which gradually expand during the intermolt period. The similar situation was also observed at the forth-instar nymphs, in which the wings are formed in the complicated manner inside the wing pads. Furthermore, the developmental process of flight muscles was also described in detail. These dynamic developmental differences between the wing morphs should be regulated under the gene expression cascades that switch according to environmental stimuli.     

The effects of local application of retinoids to different positions along the proximo-distal axis of embryonic chick wings

Summary Two retinoids, all-trans-retinoic acid and a synthetic analog, TTNPB, were locally applied to different positions along the proximo-distal axis of embryonic chick wing buds using controlled release carriers. Truncations or limbs with duplicated structures across the antero-posterior axis develop after retinoid application to distal positions in buds from stage 20–24 embryos. Phocomelic limbs develop when the retinoids are applied more proximally to buds of stage 23–24 embryos. Duplications of the pattern of structures along the proximo-distal axis never occur.Using TTNPB that is relatively stable, the amount of retinoid in the wing tissue when phocomelia is induced was measured. There is twice as much retinoid per cell in the proximal half of the bud as in the distal half of the bud. The concentration of TTNPB in proximal tissue is estimated to be three times higher than in distal tissue in which pattern formation and cartilage morphogenesis are relatively normal.At early stages in the development of phocomelia, the shape of the bud changes and the indentation that marks the elbow does not arise. Neither retinoid-induced cell killing nor effects on the pattern of programmed cell death were detected.The induction of phocomelia by retinoids appears to be based on effects on proximal cells, whereas retinoids produce pattern changes by acting on distal cells. Furthermore, compared with pattern changes, higher concentrations of retinoid in the bud tissue are required to produce phocomelia.     

Comparative studies on alate wing formation in two related species of rotten-wood termites: <Emphasis Type="Italic">Hodotermopsis sjostedti</Emphasis> and <Emphasis Type="Italic">Zootermopsis nevadensis</Emphasis> (Isoptera,Termopsidae)

Summary Termite colonies are comprised of several types of castes that differentiate throughout postembryonic development. In termopsid termites (family Termopsidae), alates are normally differentiated from apterous instars by two nymphal instar stages and three moulting events. Here, we report that of the rotten-wood termite Hodotermopsis sjostedti. There is only a single nymphal instar between the pseudergate and alate stages. During the annual alate production season in June/July, we observed some nymphs with small wing buds which were dorsally swollen. Those nymphal individuals subsequently moulted into alates through only a single moult. We examined their histology and internal morphology and observed that the folding pattern of the newly formed wings was very different from that seen in second stage nymphs of the closely related species Zootermopsis nevadensis. The newly formed wings of H. sjostedti are formed inside the relatively smaller wing buds and therefore must be folded in a complicated manner. Our observations revealed that the tips of the folded wings were elongated and bent, such that they overlapped in the median plane. We suggest that heterochronic evolutionary change accounts for the compression of nymphal development into a single instar. We also suggest that this probably occurred at either the individual or colony level in this species.Received 18 August 2003; revised 11 December 2003; accepted 8 January 2004.     

A comparative study of myogenic cell invasion of the avian wing and leg bud.

The borders of myogenic cell invasion of avian wing and leg buds were determined using the interspecific grafting technique between quail and chick embryos. Distal parts of quail limb buds were grafted ectopically into the coelomic cavity of chick embryos. The presence or absence of skeletal muscle was investigated in histological sections of the reincubated grafts. A comparison between the borders of myogenic cell invasion of the wing and leg buds showed that the differences in the position of the distal most muscles in the adult avian limbs could be a consequence of the cranio-caudal sequence of development.     

The influence of morphogene Wg on the formation of an ectopic eye in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Due to the ectopic expression of the ey gene in the wing imaginal disc under the action of the 1096-Gal4 driver, a part of the wing disc cells change their fate and become eye cells. Ectopic eyes are induced in definite regions of the wing disc and form a stable pattern on the wing of an adult fly. Here, we have shown that the ectopic expression of Wg inhibits the formation of ectopic eyes, and conversely the expression of Wg is reduced in the sites of ectopic Ey expression. Experiments with overexpression of the vesicular traffic protein Hrs capable of inhibiting the Wg signaling agree with the notion on antagonism of Wg and Ey in ectopic eyes. Our results confirm that the processes of formation of normal and ectopic eyes are principally similar with regard to genetic control.     

Wing and haltere venation in larvae of the Austrosimulium (Austrosimulium) australense group from New Zealand (Diptera: Simuliidae)

The venation pattern found in the developing wing and haltere buds of larvae is compared with the adult venation in the Austrosimulium (Austro-simulium) australense group of Simuliidae.     

Distribution of Retinoids Applied Exogenously to Chick Limb Buds: an Autoradiographic Analysis

An autoradiographic analysis was undertaken to examine the localization of retinoids applied exogenously to chick limb buds. Ion-exchange beads (AGI-X2) containing a tritium-labeled synthetic retinoid, Am80, were implanted to various regions of chick wing buds. This synthetic retinoid is known to induce a duplicated limb pattern as retinoic acid (RA) does. One to 24 hours after the application, wing buds were fixed, sectioned, and prepared for autoradiography. Heavy labeling was observed in the peripheral region of the wing mesoderm, but no gradient along the antero-posterior axis was found.
These results suggest that the peripheral region of the limb bud may be important for the morphogenetic function of RA. Tissue-bound retinoids may not form an antero-posterior concentration gradient when retinoids are added to the anterior margin of the chick limb bud.     

Impact of a gall-inducing aphid on Pistacia atlantica Desf. trees

The impact of gall-inducing aphids on shoot development was analyzed in 900 shoots from 20 pistachio trees, Pistacia atlantica Desf. (Anacardiaceae): 600 in which the axillary—lateral buds were galled by Slavum wertheimae HRL during the previous growth season, and 300 ungalled shoots. Although P. atlantica is a compensating tree, and the aphids do not attack the apical buds, further development of shoots from the apical buds was stopped in 62% of the galled shoots, while only 8.7% of nongalled shoots stopped their growth. Further development was stopped more often on shoots carrying two or more galls than on shoots supporting only one gall. To assess the hypothesis that bud destruction by the aphids explains this pattern, a field experiment was conducted in 140 shoots, distributed across seven trees. One, two or three axillary buds from five shoots of each tree were removed for each treatment, and five other shoots were marked as controls. Only 14 shoots (10%) of the 140 did not develop. The growth of the other shoots was not very different among the treatments. The colonization of the apical shoots, which developed on previously treated shoots, by three other galling aphid species was monitored. Removing lateral buds considerably reduced the establishment of Geoica sp. galls (70% of them colonized control shoots), but weakly influenced Forda riccobonii (Stefani). It also contributed only 5% of the total variance of the distribution of Smynthurodes betae West. The different results of the survey and the experiment show that the impact of S. wertheimae galls on the future growth of shoots from apical buds is more complex than the simple physical destruction of the axillary buds. Handling editor: Graham Stone     

Restoring avian wing digits

The precise identification of the digits of the avian wing is of importance in evolutionary studies. If the digits are numbered two, three and four, this has been taken to suggest that birds are not descended directly from dinosaurs. If the digits are numbered one, two and three, dinosaur origins become more plausible. Studies of the development of the avian wing have failed to resolve this dilemma. However, in some instances, it is possible to deduce information about evolutionary morphologies by manipulating development experimentally. We grafted beads loaded with fibroblast growth factor 4 into the distal tip of chick wing buds at times when the apical ectodermal ridge is regressing. The consequence was that the cartilage structure conventionally labelled ''element 5'' increased dramatically in size and acquired a digit-like morphology in some instances. Corresponding changes in soft tissue morphology were also observed. We conclude that it may be possible to resolve the issue of avian digit homology by the induction of experimental atavisms of this kind.     

Bud structure and resprouting in coppiced stools of Salix viminalis L., S. eriocephala Michx., and S. amygdaloides Anders

Fast-growing willows are cultivated as coppice in short rotation biomass plantations. The production and sustainability of the system is based on the ability of trees to resprout after repeated harvesting. The large variation in coppicing ability is due to plant genotypic differences in structure and physiology as well as environmental factors. Morphological and structural prerequisites for resprouting were compared in two shrubby willows with high coppicing ability, S. viminalis and S. eriocephala, and one tree-formed species, S. amygdaloides, with low coppicing ability. The initiation and development of buds and the resprouting pattern of coppiced stools were compared. All buds were axillary in origin and showed the same principal structure consisting of one main shoot primordium and two lateral primordia. In S. viminalis and S. eriocephala the lateral buds contained several leaf primordia and sprouted shortly after the main bud. In S. amygdaloides further development of lateral buds was inhibited after formation of two budscales, and leaf primordia were not formed until the buds were forced to sprout. The number of sprouts developing after coppicing were correlated to the structure and number of buds and their position on the stools. Self-thinning rate was high and many shoots originating from lateral buds died. Most buds were located above ground on the remaining basal portions of harvested stems. No adventitious buds were found on the stools. Significantly different bud differentiation pattern and frequent sylleptic sprouting resulted in lower coppice response in S. amygdaloides compared to S. viminalis and S. eriocephala.     

Normal development of the chick wing following removal of the polarizing zone.

The negative results of assays for polarizing activity along the posterior border of the chick wing 24 and 48 hours after removal of the polarizing zone demonstrate that this zone is not regenerated following removal. These results, and the fact that normal wing development can occur after polarizing zone removal from stages 15 through 24 wing buds, indicate that during these stages the polarizing zone has no direct role in normal development of the limb bud. It is speculated that the polarizing zone is effective only during limb induction and that after this time it exists in latent or residual form.     

Onset of troponin synthesis in the chick wing bud.

Indirect antibody labeling techniques were used to determine when cells in the chick embryo wing bud begin to synthesize troponin. Frozen sections of stage 22 through stage 27 wing buds were treated with antibodies to the troponin complex and fluorescein-labeled antiimmunoglobulin. Cells producing detectable quantities of troponin were found first in late stage 24 or early stage 25 wing buds; all wing buds stage 25 and older contained labeled cells. Cells synthesizing troponin were initially localized in the muscle-forming areas of the wing bud nearest to the body wall. As the wing bud developed, cells located in more distal areas of the wing bud became labeled with fluorescent antibody, and the number of cells engaged in troponin synthesis increased in all areas. At all stages in which labeling occurred, some cells contained fluorescent cross-striations. When placed in the context of recent studies on the appearance of myofibrillar proteins, these results indicate that myogenic cells in the chick limb bud begin to synthesize large quantities of troponin at approximately the same time as the other muscle contractile proteins.     

Rapid in vitro propagation of Aloe barbadensis Mill

Axillary bud development and adventitious bud formation was obtained with decapitated shoot explants of Aloe barbadensis Mill. Maximal bud growth and rooting of shoots was obtained on a modified medium of Murashige and Skoog supplemented with 5 M IBA. More adventitious and axillary buds developed on nutrient media supplemented with IBA than with NAA. Axillary buds but not adventitious buds developed with IAA in the medium. Morphogenesis was inhibited by 2,4-D. Kinetin, benzyladenine and thidiazuron were toxic to the explants and did not stimulate the development of axillary of adventitious buds. The optimal temperature for bud growth and development was 25°C. Axillary bud growth and the formation of adventitious buds was slowed down at 10°C and totally inhibited by 30°C. The optimal sucrose concentration was 3% with the inhibition of bud growth and development by higher sucrose levels.     

Pseudomonas syringae pv. syringae as One of the Factors Affecting the Ice Nucleation of Grapevine Buds in Controlled Conditions

Ice nucleation in grapevine buds (cv. Pinot noir) was studied in freezing chamber in container grown plants. The ice nucleation of individual 278 buds at different stages of their development (between stage B and stage E according to Baggiolini scale) was measured by differential thermal analysis. Treatments were applied to plants to modify the Pseudomonas syringae pv. syringae content of buds and alter their water potential. Based on the results it was pointed out that (i) the ice nucleation temperatures of buds were generally normally distributed, (ii) the probability of bud nucleation at a given temperature was, to a certain extent, dependent on the amount of Pseudomonas present, (iii) the modification of water potential following an artificial rain led to a signification increase of the probability of bud nucleation and (iv) the nucleation probability did not increase significantly with the bud development between stage B and stage E.     

Pigment patterns in neural crest chimeras constructed from quail and guinea fowl embryos

The pattern of pigmentation in bird embryos is determined by the spatial organization of melanocyte differentiation. Some of the results from recent, neural crest transplantation experiments support a model based on a prepattern in the feathers; others could be interpreted in terms of a nonspecific pattern resulting from a failure of the crest cells to read the positional values in another species. To distinguish between these possibilities, the crucial test is to construct chimeras from two species with different pigment patterns. We have examined the wing plumage of quail and guinea fowl embryos. The quail has a characteristic pattern of pigmented and unpigmented feather papillae, whereas the guinea fowl shows uniform pigmentation. Chimeras were constructed by grafting wing buds isotopically between embryos. The wing buds were transplanted before they had become invaded by neural crest cells. Quail wing buds grafted to the guinea fowl developed, in most cases, a pigment pattern resembling that of the quail and not that of the guinea fowl. A few cases became uniformly pigmented and appeared to represent nonspecific patterns. The reciprocal grafts (guinea fowl wing buds grafted to the quail) became pigmented all over. We found evidence that the timing of melanocyte differentiation is controlled by cues in the feather papillae. Some cases developed a severe inflammatory response. The model which best accounts for these findings--and which can account for inconsistencies in previous reports--is the following. A prepattern is present in the feathers and this can control the differentiation of melanoblasts, even if they come from a different species. The local cues which constitute the prepattern are not positional values. In some chimeras melanoblasts fail to respond to the prepattern and so a nonspecific pattern of uniform pigmentation is produced.