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J F Fallon

Publications and source records attributed to J F Fallon.

At least 55 records · Page 3Linked to original sources
PubMed1992

Insulin improves survival but does not maintain function of cultured chick wing bud apical ectodermal ridge.

Previously we demonstrated that high levels of insulin (5 micrograms/ml) permit the survival of isolated chick apical ectodermal ridge in culture (Boutin and Fallon, Dev. Biol., 104:111-116, 1984). Here we address whether lower levels of insulin or insulin-like growth factors (IGFs) can also improve the survival of cultured apical ectodermal ridge and whether ridge function is maintained along with ridge survival. Neither IGF I nor IGF II (100 ng/ml) decreased ridge cell death; however, cell death was significantly decreased with 50 ng/ml insulin. No further improvement was obtained in the presence of both IGF I and insulin. These data suggest that insulin improved the survival of the isolated apical ectodermal ridge by binding its own receptor. To test for the maintenance of function, stage 20 ridges were cultured for 0, 6, 12, 18, or 24 hr with or without insulin (5 micrograms/ml or 5 ng/ml) and used to make recombinant limbs. Isolated ridges cultured for 12 hr or more produced fewer outgrowths and these were rarely distally complete. The medium in which the ridges had been cultured did not influence ridge activity, despite the major differences in cell survival. Recombinants made with ridges cultured with limb mesoderm for 18 hr did not yield outgrowths as often as those with freshly isolated ridges, but most of the limbs that did form were distally complete. These results suggest that the decline in function of cultured, isolated apical ectodermal ridge was not due merely to ridge cell death but rather, at least in part, to its separation from limb mesoderm.

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PubMed1992

On the measurement of cytokinetics by continuous labeling with bromodeoxyuridine with applications to chick wing buds.

The cytokinetic properties, specifically the phase-transit times, TG1, TS, and TG2+M, of chick wing bud cells were estimated using data obtained from continuous labeling of stage 20 embryos with bromodeoxyuridine (BrdUrd). The presence of BrdUrd was detected with monoclonal antibodies, and the amount of DNA in the cells was determined with propidium iodide. The fraction of cells in each cell cycle phase, the fraction of labeled cells, and the relative movement, a measure of the mean DNA content, of all labeled cells were evaluated using bivariate flow cytometry at successive times following introduction of the label. Equations are presented to describe the fraction of unlabeled cells in G2 + M, which gives a direct estimate of TG2+M; the fraction of all labeled cells, which can then be used to estimate TG1; and, finally, the relative movement, which provides an estimate of TS. Thus, the data measured in these experiments together provide estimates of the progression through the cell cycle of limb mesoderm cells.

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PubMed1992

Apical ridge dependent and independent mesodermal domains of GHox-7 and GHox-8 expression in chick limb buds.

The homeobox-containing genes GHox-7 and GHox-8 have been proposed to play fundamental roles in limb development. The expression of GHox-8, by the apical ridge cells, and GHox-7, in the subridge mesoderm, suggests the involvement of these two genes in limb outgrowth and proximo-distal pattern formation. A straightforward way to test this is to remove the apical ridge. Here we report the relationship between the mesodermal expression of GHox-7 and GHox-8 and the apical ectodermal ridge in the chick limb bud. The data from ridge removal experiments indicate that there are at least two domains of GHox-7 expression in the apical limb bud mesoderm. The posterior subridge GHox-7 domain in the progress zone requires the influence of the apical ridge for continued expression, while the anterior GHox-7 domain continues expression after ridge removal. Posterior subridge mesoderm is exquisitely sensitive to the loss of the ridge in that GHox-7 expression by these cells is reduced in only two hours and undetectable by three hours after ridge removal. It would appear that one of the ways progress zone cells respond to the apical ridge signal is by expressing GHox-7. The loss of ridge influence whether by growth at the apex or by ridge removal is followed by an unusually rapid decline in detectable GHox-7 transcripts. Maintenance of GHox-8 expression by the anterior mesoderm appears to be independent of the presence of the apical ridge.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1991

Talpid2 mutant chick limb has anteroposterior polarity and altered patterns of programmed cell death.

The talpid2(ta2) chick mutant is of interest for the study of limb pattern formation. Talpid2 is a simple Mendelian recessive lethal mutation which affects the mesoderm and results in short, spade-like, polydactylous wings and legs. Here, we describe ta2 limb development with particular attention to those aspects of ta2 which may illuminate the process of normal limb development. From the onset of budding, ta2 limb buds are significantly wider than normal buds along the anteroposterior axis. They lack the normal anterior and posterior necrotic zones and have variable development of the central opaque patch. Interdigital programmed cell death is variable and may result in development of distal phalanges without more proximal ones. Talpid2 wing vasculature is similar to that of normal wings; but ta2 legs are supplied by four large blood vessels. Feathers form regular, parallel rows, similar to normal feathers, but ta2 embryos lack the loose mesenchyme which separates the feather buds. Finally, and most significantly, ta2 wings and legs display anteroposterior polarity. Anterior and posterior limb skeletal elements can be clearly distinguished from one another within the ta2 phenotype. Our observations suggest that the ta2 mutant may be useful in analyzing programmed embryonic cell death and anteroposterior limb pattern formation.

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PubMed1991

Altered expression of the chicken homeobox-containing genes GHox-7 and GHox-8 in the limb buds of limbless mutant chick embryos.

It has been suggested that the reciprocal expression of the chicken homeobox-containing genes GHox-8 and GHox-7 by the apical ectodermal ridge and subjacent limb mesoderm might be involved in regulating the proximodistal outgrowth of the developing chick limb bud. In the present study the expression of GHox-7 and GHox-8 has been examined by in situ and dot blot hybridization in the developing limb buds of limbless mutant chick embryos. The limb buds of homozygous mutant limbless embryos form at the proper time in development (stage 17/18), but never develop an apical ectodermal ridge, fail to undergo normal elongation, and eventually degenerate. At stage 18, which is shortly following the formation of the limb bud, the expression of GHox-7 is considerably reduced (about 3-fold lower) in the mesoderm of limbless mutant limb buds compared to normal limb bud mesoderm. By stages 20 and 21, as the limb buds of limbless embryos cease outgrowth, GHox-7 expression in limbless mesoderm declines to very low levels, whereas GHox-7 expression increases in the mesoderm of normal limb buds which are undergoing outgrowth. In contrast to GHox-7, expression of GHox-8 in limbless mesoderm at stage 18 is quantitatively similar to its expression in normal limb bud mesoderm, and in limbless and normal mesoderm GHox-8 expression is highly localized in the anterior mesoderm of the limb bud. In normal limb buds, GHox-8 is also expressed in high amounts by the apical ectodermal ridge.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1989

The formation of leg or wing specific structures by leg bud cells grafted to the wing bud is influenced by proximity to the apical ridge.

When quail or chick leg bud mesoderm was grafted to a chick wing bud, toes developed from grafts placed in direct contact with the wing apical ridge. The toes were primarily derived from quail leg cells, with variable participation of host wing cells. Donor cells also integrated into wing-specific structures, such as cartilage of the wing digits and the surrounding connective tissues. In addition to forming toes, the grafted leg mesoderm expressed its leg origin by enlarging skeletal elements in the host wing. In all cases, enlargements were derived of both quail donor and chick host cells, and were not the result of the addition of mass to the host bud. Grafts placed further than 162 microns from the ridge formed neither toes nor enlargements; rather, they integrated into wing-specific structures. Under the influence of the apical ridge, the grafted leg mesoderm cells are able to maintain their leg character and to form toes and skeletal enlargements. Grafts outside the range of ridge influence (162 microns) are affected by their surroundings to integrate into wing-specific structures. The formation of leg-specific structures by leg bud mesoderm grafted to the wing bud has been used to support the principle of nonequivalence, which states that, because of their different developmental histories, wing and leg cells are restricted to form structures specific for their respective limbs. However, we have shown that leg cells can form wing-specific structures, and therefore limb cells are not restricted in their development.

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PubMed1988

Initial limb budding is independent of apical ectodermal ridge activity; evidence from a limbless mutant.

Outgrowth of normal chick limb bud mesoderm is dependent on the presence of a specialized epithelium called the apical ectodermal ridge. This ectodermal ridge is induced by the mesoderm at about the time of limb bud formation. The limbless mutation in the chick affects apical ectodermal ridge formation in the limb buds of homozygotes. The initial formation of the limb bud appears to be unaffected by the mutation but no ridge develops and further outgrowth, which is normally dependent on the ridge, does not take place. As a result, limbless chicks develop without limbs. In the present study, which utilized a pre-limb-bud recombinant technique, limbless mesoderm induced an apical ectodermal ridge in grafted normal flank ectoderm. However, at stages when normal flank ectoderm is capable of responding to ridge induction, limbless flank ectoderm did not form a ridge or promote outgrowth of a limb in response to normal presumptive wing bud mesoderm. We conclude from this that the limbless mutation affects the ability of the ectoderm to form a ridge. In addition, because the limbless ectoderm has no morphological ridge and no apparent ridge activity (i.e. it does not stabilize limb elements in stage-18 limb bud mesoderm), the limbless mutant demonstrates that the initial formation of the limb bud is independent of apical ectodermal ridge activity.

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PubMed1987

Development of the brachial lateral motor column in the wingless mutant chick embryo: motoneuron survival under varying degrees of peripheral load.

Survival of motoneurons in the lateral motor column (LMC) of the chick embryo is known to depend on the periphery. How this dependence relates to the normally occurring death of motoneurons is unknown. Analysis of the time course of LMC cell loss in the absence of varying amounts of limb musculature could help bring about an understanding of this relationship. We undertook this analysis by studying LMC development in wingless chick embryos. Grossly these embryos lack wings, but we have reported that some of them possess more than 40% of the normal volume of wing bud-derived muscles (M.E. Lanser and J.F. Fallon, Anat. Rec. 217:61-78, 1987). In the present work we compared the time course of LMC development in wingless embryos that possessed varying amounts of wing bud-derived musculature with that in normal embryos. In normal embryos little cell loss occurs from the brachial LMC prior to day 8 (15% of the total cell loss). Most of the normal cell loss occurs between 8 and 10 days (62% of the total cell loss). In the wingless LMC, anywhere from 55% to 70% of the total cell loss occurs before day 8. The death of motoneurons prior to day 8 is proportional to the amount of wing bud musculature eliminated by the mutation. Cell loss after day 8 is proportional to the amount of wing bud musculature spared by the mutation. Therefore, when the limb is missing, most motoneurons die before the major period of cell loss even begins in the normal LMC. Counts of dead cells in the LMC also support this conclusion. In addition, curves plotting the rates at which cells are lost from the brachial LMC provide a suggestion that normal cell loss is biphasic and that limb removal enhances primarily the first phase of cell loss. These data suggest that the majority of motoneurons may die for different reasons in the normal and the limb-deprived LMCs. Overall, the number of motoneurons surviving in the brachial LMC is proportional to the volume of wing bud-derived muscle present. However, as the muscle volume approaches zero, motoneuron number does not. This suggests that most, but not all, motoneurons depend on limb bud-derived muscles for survival. Finally, the decreased motoneuron number in the wingless LMC, when compared to normal after the cell death period, cannot be totally accounted for by the additional loss of cells that occurred during the cell death period in the wingless LMC.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1987

Development of wing-bud-derived muscles in normal and wingless chick embryos: a computer-assisted three-dimensional reconstruction study of muscle pattern formation in the absence of skeletal elements.

The mechanisms whereby the normal pattern of muscles within the developing chick limb bud is generated are largely unexplored. It has been proposed that the muscle pattern is established independently of the pattern for the limb skeletal elements to which the muscles normally attach (Shellswell and Wolpert: "The Pattern of Muscle and Tendon Development in the Chick Wing."In: Vertebrate Limb and Somite Morphogenesis. Cambridge University Press, Cambridge, pp. 71-86, 1977). To further examine this possibility we studied the formation of the proximal wing muscles in normal and wingless chick embryos. The muscles of the shoulder region (including the pectoralis) arise as part of the dorsal and ventral premuscle masses of the developing limb bud. These secondarily migrate out of the limb to take origin from the pectoral girdle while inserting onto the humerus (Sullivan: Aust. J. Zool., 10:458-516, 1962). With rare exceptions, wingless embryos have complete absence of wing skeletal elements, but they may possess more than 40% of the normal volume of wing-bud-derived muscles. The muscles that remain in wingless embryos are primarily shoulder muscles, and to a varying extent, the pectoralis. The question we sought to answer was whether in wingless embryos the proximal wing muscles could form a normal pattern in the absence of the humerus and distal wing skeletal elements. By examining three-dimensional reconstructions of the proximal wing region in normal and wingless embryos, we found that the initial subdivision of the dorsal and ventral premuscle masses proceeded normally in the absence of the wing skeleton. This resulted in a grossly normal pattern of proximal wing muscles despite the absence of wing skeletal elements. However, some subsequent cleavages of individual muscles within premuscle mass divisions did not occur in wingless embryos. This suggests that the skeleton may be required for this step in muscle morphogenesis to occur. We also observed that the wing-bud-derived muscles in wingless embryos were nearly always anchored to the pectoral girdle at both ends. Sometimes this resulted in muscles making abnormal tendonous fusions with other muscles derived from the opposite (i.e., dorsal or ventral) premuscle mass. Therefore, attachment to the skeleton may be important for some facet of muscle development. Finally, the supracoracoideus muscle was absent in all but one wingless embryo we examined in the present study. In that one, it was substantially reduced in volume compared to normal. absence of this muscle, the space normally occupied by the supracoracoideus was maintained beneath the pectoralis.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1987

The ability of the chick wing bud to regulate positional disparity along the anterior-posterior axis.

When wedges of wing bud tissue are added to a host wing bud so there is positional disparity between graft and host, skeletal duplications result (L. E. Iten and D. J. Murphy 1980) Dev Biol. 75, 373-385. The polarity of the duplications is predictable by the polar coordinate model, leading to the interpretation that the positional disparity caused the duplications. To determine whether positional disparity alone causes duplications, without the complication of added tissue, we rotated wedges of ectoderm and mesoderm around the proximodistal axis within the wing bud. Wedges measuring 200-800 micron along the distal edge were rotated 180 degrees at stages 20-22, reversing the anteroposterior and dorsoventral axes relative to the bud. This caused positional disparity, similar to that achieved by Iten and Murphy (1980), without the addition of tissue. We found that rotations involving no polarizing zone tissue produced normal wings or wings lacking some distal parts, as did rotations of tissue lying entirely within the polarizing zone. However, when polarizing zone mesoderm was displaced, so that polarizing and nonpolarizing tissues were juxtaposed, a majority of the operations produced polarized skeletal duplications. Our data demonstrate that positional disparity alone does not cause skeletal duplications in the chick wing bud, unless polarizing zone tissue is displaced. Further, these data demonstrate that the chick wing bud can regulate to form a normal wing skeleton in the face of large positional disparity, provided that the polarizing zone is not moved. Finally, our results may be explained by the action of the proposed polarizing morphogen on the displaced cells causing repolarization.

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PubMed1987

Posterior apical ectodermal ridge removal in the chick wing bud triggers a series of events resulting in defective anterior pattern formation.

The ability of the anterior apical ectodermal ridge to promote outgrowth in the chick wing bud when disconnected from posterior apical ridge was examined by rotating the posterior portion of the stage-19/20 to stage-21 wing bud around its anteroposterior axis. This permitted contact between the anterior and posterior mesoderm, without removing wing bud tissue. In a small but significant number of cases (10/54), anterior structures (digit 2) formed spatially isolated from posterior structures (digits 3 and 4). Thus, continuity with posterior ridge is not a prerequisite for anterior-ridge function in the wing bud. Nevertheless, posterior-ridge removal does result in anterior limb truncation. To investigate events leading to anterior truncation, we examined cell death patterns in the wing bud following posterior-ridge removal. We observed an abnormal area of necrosis along the posterior border of the wing bud at 6-12 h following posterior-ridge removal. This was followed by necrosis in the distal, anterior mesoderm at 48 h postoperatively and subsequent anterior truncation. Clearly, healthy posterior limb bud mesoderm is needed for anterior limb bud survival and development. We propose that anterior truncation is the direct result of anterior mesodermal cell death and that this may not be related to positional specification of anterior cells. In our view, cell death of anterior mesoderm, after posterior mesoderm removal, should not be used as evidence for a role in position specification by the polarizing zone during the limb bud stages of development. We suggest that the posterior mesoderm that maintains the anterior mesoderm need not be restricted to the mapped polarizing zone, but is more extensively distributed in the limb bud.

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PubMed1986

Development of the apical ectodermal ridge in the chick leg bud and a comparison with the wing bud.

Histologic examination of the leg bud of stage-18 to stage-23 chick embryos was carried out with special reference to the development of the apical ectodermal ridge. The leg bud apical ectoderm, initially an irregular columnar epithelium with an overlying simple squamous periderm, began to thicken during stage 18 and was a pseudostratified epithelium by stage 19. A notch in the base of the thickened ectoderm was seen as early as stage 19. The notch represented the cross-sectional view of a groove, which developed in the base of the ridge. As development proceeded, the ridge and its associated groove lengthened. In addition, the groove became more prominent, and irregularities were seen in its width and depth along the apex. Ectodermal cell death was not consistently seen until stage 21 at which time most of the length of the thickened ectoderm had evidence of necrosis. Development of the leg bud ridge and wing bud ridge were compared. The temporal sequence of ectodermal thickening and ridge development was very similar in both the leg and wing buds with one exception; namely, that leg bud ridge development preceded wing bud ridge development by several hours. In addition, ectodermal cell death was not evident until stage 20 in the leg bud ridge, but could be seen at late stage 18 in the wing bud apical ectoderm. However, by stage 21, cell death was associated with most of the ridge in both the wing and leg buds. Finally, with respect to the axial line, the ridge with its associated groove extended further preaxially in the leg bud than in the wing bud, making the leg bud ridge more symmetrical.

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PubMed1986

Experimental manipulation leading to induction of dorsal ectodermal ridges on normal limb buds results in a phenocopy of the Eudiplopodia chick mutant.

Elongation of chick limb buds depends on the presence of the apical ectodermal ridge which is induced by subjacent limb bud mesoderm. Recombination experiments have shown that the limb bud mesoderm loses the capacity to induce ridges by late stage 17. Moreover, in normal limb development only one ridge forms. However, in the eudiplopodia chick mutant accessory ectodermal ridges form on the dorsal surface of limb buds as late as stage 22. Tissue recombinant experiments show that the mutation affects the ectoderm, extending the time it responds to ridge induction (R.A. Fraser and U.K. Abbott (1971). J. Exp. Zool. 176, 237-248) while the mesoderm is normal. The result is polydactyly, with extra digits dorsal to the normal digits. Because eudiplopodia limb bud dorsal mesoderm can induce ridges at stage 22 but is unaffected by the gene, genetically normal dorsal limb bud mesoderm may also be able to induce ridges after stage 17. To test this possibility we grafted stages 14-18 flank ectoderm to normal limb bud dorsal mesoderm and found that mesoderm from stages 17 through 20 was able to induce a ridge and subsequently dorsal digits developed. Limbs with duplicate digits were similar to eudiplopodia limbs. In other experiments, stage 18, 19, and 20 leg bud dorsal ectoderm did not form ridges when grafted to leg bud dorsal mesoderm of the same stage, indicating a lack of response to the mesoderm. Finally, the inductive capacity of limb bud mesoderm appeared to be reduced compared to mesoderm at pre-limb bud stages. These experiments demonstrate a spatially generalized potential in limb bud dorsal mesoderm to induce ridges during the stages when the apical ridge is induced. The determination of where the ridge will form and the acquired inability of limb bud dorsal ectoderm to respond to induction by underlying mesoderm are necessary early pattern forming events which assure that a single proximodistal limb axis will form.

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PubMed1986

A method for combined gross skeletal staining and Feulgen staining of embryonic chick tissues.

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.

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PubMed1986

Survival of motoneurons in the brachial lateral motor column of limbless mutant chick embryos depends on the periphery.

Motoneuron survival in the embryonic spinal cord is influenced by the presence or absence of the developing limb bud. We have recently begun a reexamination of the relationship between limb absence and motoneuron survival in a nonsurgical limb deletion model, the limbless mutant chick embryo. As in surgically limb-deleted normal embryos, only 10% of the motoneurons that are initially produced in the limbless mutant lateral motor column (LMC) survive the embryonic period (Lanser and Fallon, 1984). We now report that, when supplied with a normal periphery (i.e., a normal limb bud), more than 40% of the motoneurons initially produced in the limbless LMC survive the embryonic period. Motoneuron cell counts in one-winged limbless embryos reveal that over 3.5 times as many motoneurons survive the cell death period in the LMC on the side with the limb than on the opposite, limbless side. This demonstrates the dependence of embryonic LMC motoneurons on the developing limb for survival and indicates that the limbless mutant is an appropriate model for studying the death and survival of LMC motoneurons during development. Using the limbless mutant to study LMC motoneuron survival eliminates the complication of possible direct surgical effects on motoneuron death. In addition, we found that a substantial effect of the wing on rescuing LMC motoneurons was exerted prior to the 6th day of embryonic development. Normally, little cell loss occurs in the brachial LMC during this time. Accordingly, motoneuron death in the limb-deprived brachial LMC, whether in surgically limb-deleted normal embryos or in genetically limbless embryos, is accelerated with respect to cell death in the normal brachial LMC.

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PubMed1985

The spatial pattern and temporal sequence in which feather germs arise in the white Leghorn chick embryo.

Feather germs arise in a specific sequence and spatio-temporal pattern within each of 10 feather areas on the White Leghorn chick embryo. The time of feather germ initiation was determined by histological and gross macroscopic analyses. Protruding feather germs are sequentially visualized in the dorsal, thigh, breast, head, humoral, ventral, wing, eye, and external auditory meatus feather areas, respectively, from stage 31- to stage 39+ [V. Hamburger and H.L. Hamilton (1951) J. Morphol. 88, 49-92]. The rate at which successive feather tracts appear was found to differ for different feather areas and was not simply due to the size of a feather area. Feather germ histogenesis was examined in the dorsal, thigh, breast, ventral, wing, and tail feather areas. The stages of feather germ histogenesis, examined on the wing feather area, are similar to those previously described for the dorsal surface. Gross and histological analyses gave different times and temporal sequences of feather germ visualization. Some feather areas were readily visualized at the time of feather germ initiation, while others showed a lag between the histological appearance of feather germs and their macroscopic visualization. Thus, macroscopic observations do not accurately reflect the pattern of histogenesis.

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PubMed1984

The effects of Janus Green B on the temporal and spatial pattern of feather germ morphogenesis.

The normal timing and appearance of feather germs was perturbed by injecting the dye Janus Green B into the amniotic fluid of chick embryos at late stage 28, prior to the first appearance of feather germs. This treatment prevented feather germ morphogenesis in some regions while elsewhere it delayed normal morphological development. The Janus Green B effect lasted for approximately 98 hours. Feather regions, which normally form epidermal placodes during the period of treatment, showed the longest delays in subsequent feather germ formation and were the most likely to remain featherless. These results suggest that the epidermal placode stage is critical for feather germ formation. Janus Green B appears to prevent feather germ morphogenesis by interfering with development prior to this critical stage. Since severely affected regions fail to recover their capacity to form feather germs, even after the period of sensitivity to the dye, a limited period of competence is suggested for feather germ formation.

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