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.     

Position of origin of donor posterior chick wing bud tissue transplanted to an anterior host site determines the extra structures formed

The formation of supernumerary limb structures was studied by juxtaposing normally nonadjacent embryonic chick limb bud tissue. Different “wedges” (ectodern and mesoderm) of posterior donor right wing bud (stage 21) were transplanted to a slit made in stage 20–23 host right wing buds. Donor posterior tissue was transplanted to an anterior position in a host wing bud or, as a control, to the same position as its position of origin. Transplanting different wedges of posterior tissue to the same anterior host position results in wings with supernumerary structures, and different extra structures form depending on the position of origin of the donor tissue. The identification of extra limb structures formed was based on the skeletal and integumentary patterns of resulting wings and the pattern of muscles as seen in serial sections of resulting limbs. The results of experiments presented here are considered in light of current models that have been used to describe the formation of supernumerary limb structures by the embryonic chick limb bud.     

用红外方法对蝇翅视动行为的探索

本文报告了利用红外装置对蝇翅视动行为实验研究的初步结果及其分析:1.在红外探测器探测到的信号中找到了一个能反映蝇翅拍动幅度的参数.2.双侧、单侧刺激域的宽度及刺激域的高度对视动反应发生几率在一定范围内正相关,当超过一阈值(即饱和阈值)后,即出现稳定的视动反应,它们的饱和阈值分别为60°,30°,40°刺激条纹的亮度生有类似情况.刺激条纹的运动速度在一定范围内对视动反应无影响.3.当刺激没有达到饱和时,蝇翅出现断续的典型的视动反应,即“0-1波动反应”.4.单侧条纹由前向后运动时,蝇翅出现典型反应,而条纹从后向前运动时,不出现典型的视动反应或反应很弱.双侧刺激时,条纹向前运动几乎不诱发反应;条纹向后运动诱发明显的蝇翅视动反应,且蝇翅平面的方向在拍动过程中发生变化.     

Preservation of the ability of dissociated quail wing bud mesoderm to elicit a position-related differentiative response

Previous studies showed that grafting wedges of fresh or cultured anterior quail wing mesoderm into posterior slits in chick wing buds resulted in the formation of supernumerary cartilage in a high percentage of cases. When anterior quail mesoderm, which had been dissociated into single cells and pelleted by centrifugation, was grafted into posterior slits of host chick wing buds, supernumerary rods or nodules of cartilage formed in 74.3% of the cases. Few supernumerary skeletal structures formed following control operations in which pelleted dissociated anterior or posterior mesoderm was grafted into homologous locations in host chick wing buds. When pelleted, dissociated anterior mesoderm was cultured in vitro for 1 or 2 days prior to being implanted in posterior locations, the incidence of supernumerary cartilage formation increased to 95.5% and 93.8%, respectively. The incidence of supernumerary cartilage formation following control orthotopic grafts of cultured mesoderm was 11.8% for 1-day and 31% for 2-day cultured anterior mesoderm; for 1- and 2-day cultured posterior mesoderm, the incidence of supernumerary cartilage formation was 20% and 41.7%, respectively. Longer-term culture resulted in a substantial decrease in the percentage of supernumerary cartilage after anterior to posterior grafts and an increase in the incidence of supernumerary cartilage from control grafts. The results demonstrate that quail anterior wing bud mesodermal cells do not need to maintain constant contact with one another in order to retain the ability to form or stimulate the formation of supernumerary cartilage after being grafted into a posterior location in a host wing bud. This ability is retained when the pelleted dissociated mesoderm is cultured in vitro outside the limb field for at least 1 to 2 days.     

A test of positional properties of avian wing-bud mesoderm

Supernumerary wing structures are readily produced by grafting pieces of wing-bud mesoderm into different locations of host wing buds, but the mechanism underlying their formation remains obscure. The major aim of this study was to examine the ability of posterior quail wing-bud mesoderm, cultured in vitro long enough to lose ZPA (zone of polarizing activity) activity, to stimulate or participate in the formation of supernumerary structures when grafted into anterior slits of host chick wing buds. Small pieces of anterior and posterior quail wing-bud mesoderm (HH stages 21-23) were placed in in vitro culture for up to 3 days. After 2 days, ZPA activity of cultured mesoderm was lost. After the grafting of 2- to 3-day cultured anterior quail wing-bud mesoderm into posterior slits of host chick wing-buds, a consistently high percentage (70%-90%) of grafts result in formation of supernumerary cartilage; in this experiment, however, only a low percentage of grafts resulted in supernumerary cartilage when 2- to 3-day cultured posterior mesoderm was grafted into anterior slits. Taken with controls, these results show that positional differences exist between cultured anterior and posterior wing-bud mesoderm. Serial-section analysis of numerous operated wings has shown several patterns of contribution to supernumerary structures by cells of graft and host. Single supernumerary digits induced by grafts of ZPA mesoderm into anterior slits were normally composed entirely of host cells, but graft cells regularly contributed to skeletal elements of more complex supernumerary structures. Cartilage rods produced by anterior-to-posterior grafts were composed mostly of graft cells, but cartilage nodules and the bases of some rods were often mosaics of chick and quail cells. The results support the proposition that mesodermal cells of the quail wing-bud possess a form of anteroposterior positional memory, but its nature and the means by which the memory of grafted cells interacts with host mesoderm are still not clear.     

The in vitro maintenance of the apical ectodermal ridge of the chick embryo wing bud: an assay for polarizing activity.

We have devised an in vitro bioassay for limb bud polarizing activity in the chick embryo. This assay has proven to be a relatively quick and effective test for a morphogenetic factor asymmetrically distributed in the limb bud which is capable of maintaining or thickening the apical ectodermal ridge.A small section of the preaxial border of the chick embryo wing bud was cultured alone, with tissue from the posterior border, mid-dorsal or anterior corner of a second donor wing, or from the flank. The tissue from the preaxial border (responding tissue) consisted of mesoderm with overlying ectoderm and apical ectodermal ridge. When the responding tissue was cultured alone, with flank, or with anterior corner limb tissue, the apical ectodermal ridge flattened in 24–36 hr and many macrophages appeared in the underlying mesoderm. When cultured with posterior border limb tissue however, the apical ridge of the responding tissue remained thickened for up to 48 hr., and no macrophages appear in the underlying mesoderm. The behavior of responding tissue was intermediate between these two extremes when cultured with mid-dorsal limb tissue. The morphogenetic activity assayed by this procedure thus seems to be present as a gradient in the wing bud, with activity decreasing from posterior to anterior. Contact with the responding tissue is not required to enable posterior border tissue to elicit ridge thickening and inhibit the cell death.     

Essential role of Duox in stabilization of Drosophila wing

NADPH oxidase produces reactive oxygen species (ROS). Drosophila melanogaster has two homologs of NADPH oxidase, dNox and dDuox, with functions that remain unclear in vivo. To clarify these functions, two independent transgenic fly lines expressing dsRNA targeted for different portions of dDuox mRNA were used. In both flies, en-GAL4> UAS-dDuoxIR(976-1145) and en-GAL4> UAS-dDuoxIR(370-518), in which dDuox was knocked down selectively in the posterior area of the wing disc, the posterior compartment of the adult wings became paler and more fragile with wing veins that were indistinct by comparison with the anterior one. Fluorescence staining of the en-GAL4> UAS-dDuoxIR(976-1145) adult wings revealed that the ROS concentration in the posterior compartment was significantly lower than that in the anterior compartment. Moreover, in these flies, the posterior compartment of the wing imaginal disc showed a greater number of apoptotic cells detected by immunostaining with anti-cleaved caspase-3 antibody than those in the anterior compartment. Respective knockdown of tyrosine hydroxylase or dopa-decarboxylase showed paler wing blades in the posterior compartment similar to the phenotype of dDuox-knockdown files. Along with this observation, analysis of the catecholic and dityrosine components in the wings of adult flies proved that dDuox plays important roles in the stabilization of the cuticle structure of the wings via tyrosine cross-linking, the sclerotization and melanization processes possibly through ROS production. These dDuox-knockdown fly lines would be useful tools for further studying dDuox functions during the development of Drosophila.     

The mouse polydactylous mutation,luxate (lx), causes anterior shift of the anteroposterior border in the developing hindlimb bud

Pattern formation along the anterior-posterior axis of the vertebrate limb is established upon activation of Sonic Hedgehog (SHH) in the zone of polarizing activity (ZPA). Since many mouse mutants with preaxial polydactyly show ectopic expression of Shh at the anterior margin of the limb buds, it has been thought to be a primary defect caused by these mutations. We show here that the mouse mutation luxate (lx) exhibits dose-dependent reduction in the size of the Fgf8 expression domain in the ectoderm from the initial stage of limb development. This aberration was independent of Fgf10 expression in the limb mesenchyme. Shh was induced in the mesenchyme underlying the posterior end of the Fgf8 expression domain, indicating an anterior shift of Shh expression in lx hindlimb buds. Prior to the ectopic induction of Shh, the expression domains of genes downstream from Shh, namely dHAND, Gli1, Ptc and Gre, which are normally expressed in posterior mesenchyme of limb buds, expanded anteriorly on the lx hindlimb buds. Conversely, the expression domains of anterior mesenchymal markers such as Gli3and Alx4 decreased in size. Thus, ectopic Shh is not a primary defect of the lx mutation. Rather, our results indicate that the lx mutation affects the positioning of the anteroposterior border in developing hindlimb buds.     

Retinoic acid respecifies limb bud cells in vitro.

Retinoic acid (RA) is known to have dramatic effects on limb pattern formation and has been shown to exert its effects on limbs by converting anterior limb bud cells into cells with posterior positional properties. In this study we find that dissociated posterior limb bud cells from chick and mouse embryos cultured at high density (micromass cultures) are able to stimulate the formation of supernumerary digits when grafted into developing wing buds and that the positional identity of both chick and mouse limb bud cells can be maintained for finite periods of time in vitro. Furthermore, using this assay system we have tested whether anterior cells from mouse and chick limb buds can be converted into cells with posterior identity by exposure to RA in vitro. We find that anterior limb bud cells acquire posterior properties after culture in the presence of RA.     

Control of the Expression of AV-1 Protein, a Position-Specific Antigen, by ZPA (Zone of Polarizing Activity) Factor in the Chick Limb Bud

AV-1 protein is a molecule which shows position-specific expression during chick limb development, and is expected to have some important roles in limb pattern formation. In this study, to examine whether the ZPA (Zone of polarizing activity) effects the expression of the AV-1 protein, we have removed or grafted the ZPA in chick limb buds and observed AV-1 expression. Anterior halves of the limb buds which lack a ZPA were used as hosts. In such anterior halves, AV-1 expression was initially observed in distal mesodermal cells including the cut surface. These anterior halves were combined with ZPA fragments, anterior fragments, posterior half limb buds, or left to develop alone, and the distribution of AV-1 expression was examined. The results of these experiments show that AV-1 expression requires the ZPA, and that expression occurs in the distal mesodermal cells certain distance from the ZPA.     

Role of the chicken homeobox-containing genes GHox-4.6 and GHox-8 in the specification of positional identities during the development of normal and polydactylous chick limb buds.

During early stages of normal chick limb development, the homeobox-containing (HOX) gene GHox-4.6 is expressed throughout the posterior mesoderm of the wing bud from which most of the skeletal elements including the digits will develop, whereas GHox-8 is expressed in the anterior limb bud mesoderm which will not give rise to skeletal elements. In the present study, we have examined the expression of GHox-4.6 and GHox-8 in the wing buds of two polydactylous mutant chick embryos, diplopodia-5 and talpid2, from which supernumerary digits develop from anterior limb mesoderm, and have also examined the expression of these genes in response to polarizing zone grafts and retinoic acid-coated bead implants which induce the formation of supernumerary digits from anterior limb mesoderm. We have found that the formation of supernumerary digits from the anterior mesoderm in mutant and experimentally induced polydactylous limb buds is preceded by the ectopic expression of GHox-4.6 in the anterior mesoderm and the coincident suppression of GHox-8 expression in the anterior mesoderm. These observations suggest that the anterior mesoderm of the polydactylous limb buds is "posteriorized" and support the suggestion that GHox-8 and GHox-4.6, respectively, are involved in specifying the anterior non-skeletal and posterior digit-forming regions of the limb bud. Although the anterior mesodermal domain of GHox-8 expression is severely impaired in the mutant and experimentally induced polydactylous limb buds, this gene is expressed by the prolonged, thickened apical ectodermal ridges of the polydactylous limb buds that extend along the distal anterior as well as the distal posterior mesoderm.(ABSTRACT TRUNCATED AT 250 WORDS)     

The preservation of the ability of cultured quail wing bud mesoderm to elicit a position-related differentiative response

A previous study showed that grafting wedges of fresh anterior quail wing mesoderm into posterior slits of chick wing buds resulted in the formation of rods and nodules of cartilage in a high percentage of cases (B. Carlson, 1983, Dev. Biol. 101, 97-105). The purpose of the present study was to determine if a similar response could be elicited by grafting pieces of mesoderm that had been cultured in vitro. When pieces of 1-day cultured anterior mesoderm from stage 17-24 donors were grafted into standard posterior slits of chick wing buds, the percentages of supernumerary structures differed little from those which formed after the grafting of pieces of fresh mesoderm. In a time series, grafts of stage 22-23 anterior mesoderm which had been cultured for 1-4 days retained the ability to form cartilage after being grafted into posterior locations. A time series showed that the duration of this retention was longer in cultured mesoderm than it was in mesoderm that remains in the donor wing bud.     

The formation of supernumerary structures after grafting anterior quail wing bud mesoderm of various ages into chick wing buds

Wedges of anterior quail mesoderm grafted into posterior slits in the wing buds of chick embryo hosts result in the formation of rods and nodules of supernumerary cartilage in a high percentage of cases. Identifiable digits do not form unless the ectoderm is allowed to remain on the grafts. Control experiments have shown that wedges of anterior or posterior wing mesoderm placed into homologous locations of host wing buds produce few or no supernumerary skeletal structures. Anterior-to-posterior grafts of stage 17 mesoderm evoke a 71.4% incidence of supernumerary cartilage. This percentage increases to 100% with stage 22 donor mesoderm. The percentage of supernumerary structures formed declines markedly with donor mesoderm of stages 24-30. By stages 35-36, only 10% of the grafts result in the formation of supernumerary structures. The period of decline coincides with the onset of overt cytodifferentiation within the donor mesoderm.     

Temporal pattern of posterior positional identity in mouse limb buds

This study describes the temporal pattern of posterior positional identity in mouse limb bud cells. To do this wedges of tissue from the posterior edge of mouse limb buds at various stages (limb stages: Wanek et al., 1989b. J. Exp. Zool. 249, 41-49) were grafted to the anterior edge of a host chick embryo wing bud. Grafts of mouse posterior cells are able to induce the formation of supernumerary digits every time when they are taken from buds from stage 3 through stage 6. At stage 7, the frequency declines and by stage 8 the chick cells no longer respond. The results indicate a change in tissue properties at stage 7, which progresses by stage 8 to the point at which posterior positional identity is no longer detectable by this assay. These temporal changes in this aspect of limb pattern formation can be used as an additional criterion to guide the identification of genes involved in the specification of posterior positional identity.     

The role of the zone of polarizing activity in controlling the differentiation of the apical mesenchyme of the chick wing-bud: histochemical thechniques in the analysis of a developmental problem

Summary Following excision of the posterior half of the three-day chick wing bud, the anterior half which normally forms humerus (part), radius and digit 2, forms only a single skeletal element (humerus, of humerus fused with reduced radius). If part of the zone of polarizing activity is included in the remaining anterior part of the wing bud, a normal wing with normal skeleton forms. Excision of the anterior half of the chick wing-bud results in the posterior half forming humerus (part), ulna and digits 3–5. This is confirmed as the normal prospective fate of the posterior half by chimeric quail-chick wing-buds in which Feulgen staining of the nucleolus-associated heterochromatin of quail cells enables their contribution to the resultant skeleton to be identified. Beginning at 18 h alter posterior half amputation, the anterior distal mesenchyme becomes necrotic and the apical e todernal ridge regresses. By contrast, following anterior half amputation, posterior halves develop no more cell death than control wing-buds.Anterior half regression is characterized by cell fragmentation and phagocytosis. First, in both the apical ectodermal ridge and distal mesenchyme cells, acid phosphatase-rich autophagic bodies appear, the cells then becoming autolytic (with diffuse acid phosphatase activity) and fragmenting. Neighbouring cells phagocytose the dead cell fragments, the mesenchyme cells forming large non-professional macrophages containing many acid phosphatase-rich vacuoles.These experiments show that for survival and differentiation, the anterior and distal mesenchyme of the wing bud requires a factor from the posterior part, thus suggesting that the zone of polarizing activity controlsantero-posterior differentiation in the normal wing.     

Ultrabithorax gene expression in Drosophila imaginal discs and larval nervous system

Using a monoclonal antibody and image-processing procedures, the patterns of expression of the Ultrabithorax (Ubx) gene product have been characterized in Drosophila larvae. As reported previously, the metathoracic imaginal discs stain most intensely with anti-Ubx, with some mesothoracic and no prothoracic expression detectable. In the metathoracic discs, the greatest modulation in anti-Ubx staining is along the proximodistal axis. Ubx is generally expressed at higher levels in the posterior regions of metathoracic discs, although relatively high anterior expression is found in some areas. Expression in the mature wing disc is confined to the squamous peripodial membrane cells; in younger wings, Ubx expression fills the posterior half of the peripodial side of the disc. The mesothoracic leg stains with a pattern that is qualitatively similar (but not identical) to that of the metathoracic leg; Ubx is expressed in some anterior regions of the mesothoracic leg, in parasegment 4. Double staining with anti-Ubx and anti-engrailed reveals that discontinuities in Ubx expression that have been suggested to correspond to compartment borders do not coincide with the compartment boundaries in some cases. In the larval ventral ganglion, Ubx expression is greatest in parasegments 5 and 6, as in the embryonic nervous system.