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J F Fallon

Publications and source records attributed to J F Fallon.

At least 37 records · Page 2Linked to original sources
PubMed1996

Surgical removal of limb bud Sonic hedgehog results in posterior skeletal defects.

Using Sonic Hedgehog (Shh) as a marker for polarizing region cells we have repeated the experiments of MacCabe et al. (1973) and Fallon and Crosby (1975) in an attempt to reexamine the question of a continuous role for the polarizing region during limb development. We report that the earlier experiments probably left Shh-expressing cells after surgery. Our results show that Shh-expressing cells do not regenerate and complete removal of the polarizing region results in truncations along the anteroposterior (A-P) axis; further, A-P patterning cannot be restored when a bead soaked in FGF is implanted in the limb bud mesenchyme to maintain outgrowth after extirpation of the polarizing region. However, in order to reproducibly remove all Shh-positive cells, it is possible that cells with posterior limb skeletal fate also must be removed. Therefore, microsurgical approaches do not permit an unequivocal answer to the question raised in this and the earlier papers and it remains a reasonable possibility that at least up to stage 20-21 the polarizing region plays a continuous role in patterning of the limb bud during its development.

Animals↗
PubMed1996

The limb field mesoderm determines initial limb bud anteroposterior asymmetry and budding independent of sonic hedgehog or apical ectodermal gene expressions.

We have analyzed the pattern of expression of several genes implicated in limb initiation and outgrowth using limbless chicken embryos. We demonstrate that the expressions of the apical ridge associated genes, Fgf-8, Fgf-4, Bmp-2 and Bmp-4, are undetectable in limbless limb bud ectoderm; however, FGF2 protein is present in the limb bud ectoderm. Shh expression is undetectable in limbless limb bud mesoderm. Nevertheless, limbless limb bud mesoderm shows polarization manifested by the asymmetric expression of Hoxd-11, -12 and -13, Wnt-5a and Bmp-4 genes. The posterior limbless limb bud mesoderm, although not actually expressing Shh, is competent to express it if supplied with exogenous FGF or transplanted to a normal apical ridge environment, providing further evidence of mesodermal asymmetry. Exogenous FGF applied to limbless limb buds permits further growth and determination of recognizable skeletal elements, without the development of an apical ridge. However, the cells competent to express Shh do so at reduced levels; nevertheless, Bmp-2 is then rapidly expressed in the posterior limbless mesoderm. limbless limb buds appear as bi-dorsal structures, as the entire limb bud ectoderm expresses Wnt-7a, a marker for dorsal limb bud ectoderm; the ectoderm fails to express En-1, a marker of ventral ectoderm. As expected, C-Lmx1, which is downstream of Wnt-7a, is expressed in the entire limbless limb bud mesoderm. We conclude that anteroposterior polarity is established in the initial limb bud prior to Shh expression, apical ridge gene expression or dorsal-ventral asymmetry. We propose that the initial pattern of gene expressions in the emergent limb bud is established by axial influences on the limb field. These permit the bud to emerge with asymmetric gene expression before Shh and the apical ridge appear. We report that expression of Fgf-8 by the limb ectoderm is not required for the initiation of the limb bud. The gene expressions in the pre-ridge limb bud mesoderm, as in the limb bud itself, are unstable without stimulation from the apical ridge and the polarizing region (Shh) after budding is initiated. We propose that the defect in limbless limb buds is the lack of a dorsal-ventral interface in the limb bud ectoderm where the apical ridge induction signal would be received and an apical ridge formed. These observations provide evidence for the hypothesis that the dorsal-ventral ectoderm interface is a precondition for apical ridge formation.

Animals↗
PubMed1995

Limb-patterning activity and restricted posterior localization of the amino-terminal product of Sonic hedgehog cleavage.

BACKGROUND: Sonic hedgehog (Shh), a vertebrate homolog of the Drosophila segment polarity gene hedgehog (hh), has been implicated in patterning of the developing chick limb. Such a role is suggested by the restricted expression of Shh along the posterior limb bud margin, and by the observation that heterologous cells expressing Shh have limb-polarizing activity resembling that of cells from the polarizing region of the posterior limb bud margin. It has not been demonstrated, however, that the Sonic hedgehog protein (SHH) alone is sufficient for limb patterning. SHH has been shown to undergo autoproteolytic cleavage in vitro, yielding two smaller products. It is of interest, therefore, to determine whether processing of SHH occurs in the developing limb and how such processing influences the function of SHH. RESULTS: We demonstrate that SHH is proteolytically processed in developing chick limbs. Grafts of cells expressing SHH protein variants that correspond to individual cleavage products demonstrate that the ability to induce patterned gene expression and to impose morphological pattern upon the limb bud is limited to the amino-terminal product (SHH-N) of SHH proteolytic cleavage. We also demonstrate that bacterially synthesized and purified SHH-N, released from implanted beads, is sufficient for limb-patterning activity. Finally, we show that the endogenous amino-terminal cleavage product is tightly localized to the posterior margin of the limb bud. CONCLUSIONS: Our data show that, of the two cleavage products resulting from SHH autoproteolysis, SHH-N expressed in grafted heterologous cells or supplied in purified form is sufficient to impose pattern upon the developing limb. Moreover, the restricted localization of the endogenous amino-terminal SHH cleavage product to the posterior border of the chick limb bud makes it unlikely that its patterning activity results from it being distributed in a broad gradient across the antero-posterior axis. More consistent with the observed localization is a model in which the amino-terminal SHH cleavage product exerts its patterning effects by local induction in or near the polarizing region, initiating a cascade of gene expression that ultimately extends across the developing limb.

Animals↗
PubMed1995

Differential expression of retinoic acid receptor-beta isoforms during chick limb ontogeny.

Retinoids influence both morphogenetic events and differentiation during development of the vertebrate limb. These effects are mediated through nuclear retinoid receptors, which modulate target gene expression. We report here the cloning and characterization of three promoter- and splicing-variants of the retinoic acid receptor-beta (RAR-beta) from chick. These receptor isoforms are independently expressed during limb development. RAR beta 2 but not RAR beta 1 transcripts are enriched three-fold in the posterior limb bud, reflecting the increased RA concentrations in this region. RAR beta 1 transcripts are initially present throughout the limb bud mesenchyme and ectoderm, then become restricted within perichondrial regions and loose connective tissue of the limb. RAR beta 1 expression closely overlaps that of NCAM (neural cell adhesion molecule) and tenascin in non-neuronal tissues. RAR beta 2 transcripts are present within a subset of those limb tissues which express RAR beta 1. In the early limb bud RAR beta 2 transcripts are detected in proximal limb mesenchyme and in the initial mesenchymal condensate. In older limbs RAR beta 2 mRNAs are abundant in cells lateral to the digit cartilage. Neither RAR beta 1 nor RAR beta 2 transcripts are associated specifically with regions of limb cell death. The differential expression and regulation of RAR beta isoforms suggests these variants may have different roles in limb development.

Amino Acid Sequence↗
PubMed1995

FGF-2 mRNA and its antisense message are expressed in a developmentally specific manner in the chick limb bud and mesonephros.

FGF-2 protein is present in the ectoderm and mesoderm of the developing chick limb bud. Its importance has been shown by the ability of ectopically applied FGF-2 to replace the apical ectodermal ridge, allowing complete outgrowth and subsequent pattern formation of the limb bud. The first goal of this study was to determine whether FGF-2 mRNA was present in the same ectodermal and mesodermal regions of the chick embryo as FGF-2 protein. FGF-2 also has an antisense message that is convergently transcribed from the opposite DNA strand (Kimelman and Kirschner [1989] Cell 59:687-696; Volk et al. [1989] EMBO J. 8:2983-2988). The second goal was to demonstrate the expression and distribution of the antisense message. Using RNAse protection assays we detected a full length protected fragment that corresponds to chick embryo FGF-2 mRNA, and a partially protected fragment that corresponds to the antisense message. We used in situ hybridization to show that FGF-2 mRNA was present in the ectoderm and subjacent mesoderm of the chick wing bud. FGF-2 mRNA was also present in body ectoderm and undifferentiated mesoderm throughout the embryo, and in muscle cells, dorsal neural tube, and mesonephros. In situ hybridization also revealed evidence for the presence of the natural antisense message in the embryo in most, but not all, of the same regions as the FGF-2 mRNA. FGF-2 mRNA and its antisense message colocalized in undifferentiated limb mesoderm; however, antisense message was not detected in differentiated muscle or cartilage. It is important to note that FGF-2 mRNA was always present in the mesonephros but that the antisense message was never observed in the mesonephros, thereby providing an internal control for non-specific signal. Although little is known about its function, Kimelman and Kirschner ([1989] Cell 59: 687-696) proposed that the antisense message may increase turnover of FGF-2 mRNA. When we compared the in situ hybridization data of both mRNAs with levels of FGF-2 protein (Savage et al. [1994] Dev. Dyn. 198:159-170), interesting tissue specific patterns emerged that support this hypothesis.

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PubMed1995

Changes in the expression of fibroblast growth factor receptors mark distinct stages of chondrogenesis in vitro and during chick limb skeletal patterning.

Members of the fibroblast growth factor (FGF) family of growth factors are key regulators of limb skeletal patterning and growth. Abnormal expression of FGFs or mutations in their receptors (fgfrs) result in skeletal disorders. Here we show that changes in the expression of fgfrs are intrinsic properties of differentiating cartilage. In mesenchymal micromass cultures differentiating into cartilage, as in ovo, fgfr 1 mRNA was found predominantly in undifferentiated, proliferating mesenchyme, fgfr 2 in precartilage cell aggregates, and fgfr 3 in differentiating cartilage nodules. Thus, our data suggest that switches in the expression of fgfr 1, 2, and 3 mRNAs are associated with phases of cartilage patterning both in vitro and in ovo, and mark distinct stages in the development of the limb skeleton.

Alternative Splicing↗
PubMed1994

FGF-2: apical ectodermal ridge growth signal for chick limb development.

The apical ectodermal ridge permits growth and elongation of amniote limb buds; removal causes rapid changes in mesodermal gene expression, patterned cell death, and truncation of the limb. Ectopic fibroblast growth factor (FGF)-2 supplied to the chick apical bud mesoderm after ridge removal will sustain normal gene expression and cell viability, and allow relatively normal limb development. A bioassay for FGFs demonstrated that FGF-2 was the only detectable FGF in chick limb bud extracts. By distribution and bioactivity, FGF-2 is the prime candidate for the chick limb bud apical ridge growth signal.

Animals↗
PubMed1994

Recombinant limbs as a model to study homeobox gene regulation during limb development.

Recombinant limb buds constructed of an ectodermal jacket containing dissociated anterior wing bud mesoderm cells form limb-like structures lacking anteroposterior polarity. Polarity can be restored by implanting a small piece of polarizing region into the recombinant limb. We used recombinant limbs to study expression of Msx1 and Msx2 and 5' Hoxd genes in limb development. Detectable mesodermal expression of Msx1 and Msx2 was lost during preparation of the recombinants. When expression was restored both genes were expressed in a symmetrical fashion in the apical mesoderm whether or not the recombinant was polarized. The normal anterior pattern of Msx1 and Msx2 was never reestablished. The roles Msx1 and Msx2 play in limb patterning are at present not clear. In nonpolarized recombinant limbs the 5' Hoxd genes were sequentially and uniformly expressed in the subridge mesoderm across the anteroposterior axis. They exhibited nested domains of expression showing exclusively distal restriction. In polarized recombinants the 5' Hoxd genes were expressed progressively in an asymmetric, nested fashion adjacent to the implanted polarizing region; there was also a distal mesodermal region further from the implant that did not express the most 5' Hoxd genes. Both of these observations are similar to the normal limb. Our results demonstrate that the polarizing region is not necessary for the activation or maintenance of 5' Hoxd gene expression but that its presence is required in order for the normal pattern to be established. We propose that the expression of the Hoxd genes is an intrinsic property of rapidly growing limb cells and that their expression is related to the limb bud proximodistal outgrowth. Our data also support the influence of the polarizing region upon Hoxd gene expression in two ways. First there is a short-range action responsible for the normal asymmetric expression and second a long-range effect suppressing the expression of 5'-Hoxd genes by the anterior cells.

Animals↗
PubMed1994

Formation of extra digits in the interdigital spaces of the chick leg bud is not preceded by changes in the expression of the Msx and Hoxd genes.

By in situ hybridization we studied the expression patterns of Msx and Hoxd genes during the late development of the chick leg autopodium (foot) and compared them to patterns during the experimental development of interdigital extra digits. Extra digits are induced in the third interdigital space after various experimental manipulations, such as transient isolation of the interdigit, or removal of the interdigital marginal ectoderm and mesoderm. Msx1 and Msx2 are normally expressed in the interdigital tissue programmed to die. Our experiment changes the fate of the interdigital tissue from cell death to chondrogenesis and provides a good model for studying Msx involvement in defining areas of programmed cell death. Among the proposed roles of Hoxd genes is their involvement in the specification of digit identity early in development. The induction of extra digits allows us to examine whether this new morphogenetic commitment of the interdigital tissues involves changes in the domains of expression of Hoxd genes. Our results show that extra digits develop without a previous modification of the normal pattern of expression of Msx or Hoxd genes. This observation does not support the correlation between the expression of Msx genes and programmed cell death and suggests a role for these genes in maintaining the interdigital tissue in an undifferentiated state. Our results show that an increased number of digits can be formed without modifications in the pattern of expression of the 5'-located Hoxd genes and suggest the existence of latent or residual digit organization mechanisms past the time when digits are normally determined, independent of Hoxd gene expression.

Animals↗
PubMed1994

Products, genetic linkage and limb patterning activity of a murine hedgehog gene.

The hedgehog (hh) segmentation gene of Drosophila melanogaster encodes a secreted signaling protein that functions in the patterning of larval and adult structures. Using low stringency hybridization and degenerate PCR primers, we have isolated complete or partial hh-like sequences from a range of invertebrate species including other insects, leech and sea urchin. We have also isolated three mouse and two human DNA fragments encoding distinct hh-like sequences. Our studies have focused upon Hhg-1, a mouse gene encoding a protein with 46% amino acid identity to hh. The Hhg-1 gene, which corresponds to the previously described vhh-1 or sonic class, is expressed in the notochord, ventral neural tube, lung bud, hindgut and posterior margin of the limb bud in developing mouse embryos. By segregation analysis the Hhg-1 gene has been localized to a region in proximal chromosome 5, where two mutations affecting mouse limb development previously have been mapped. In Drosophila embryos, ubiquitous expression of the Hhg-1 gene yields effects upon gene expression and cuticle pattern similar to those observed for the Drosophila hh gene. We also find that cultured quail cells transfected with a Hhg-1 expression construct can induce digit duplications when grafted to anterior or mid-distal but not posterior borders within the developing chick limb; more proximal limb element duplications are induced exclusively by mid-distal grafts. Both in transgenic Drosophila embryos and in transfected quail cells, the Hhg-1 protein product is cleaved to yield two stable fragments from a single larger precursor. The significance of Hhg-1 genetic linkage, patterning activity and proteolytic processing in Drosophila and chick embryos is discussed.

Animals↗
PubMed1993

Distribution of FGF-2 suggests it has a role in chick limb bud growth.

We developed and characterized antibodies specific for FGF-2 and used them to locate FGF-2 during chick embryo development. A series of micrographs demonstrated the progression of FGF-2 staining during development of the different tissues and organs. FGF-2 was present in the ectoderm covering the entire embryo, muscle cells, nervous system, neural crest cells, and mesonephros. FGF-2 was also present in the limb from initiation of budding through differentiation. The limb ectoderm and subjacent mesoderm showed the strongest immunostaining, with lower levels in the center of the bud. However, the distribution of FGF-2 positive cells in the mesoderm was not homogeneous. This heterogeneity was not due to cell cycle specific distribution of FGF-2 protein, as flow cytometric analysis showed that FGF-2-positive cells were distributed throughout the cell cycle. However, the amount of anti-FGF-2 fluorescence varied most during G1, consistent with the possibility that FGF-2 is low after M phase and increases during G1. A bioassay was used to demonstrate FGF-2 levels in the wing ectoderm were approximately 2.7-fold greater than in the mesoderm. We propose that the location of FGF-2 in the embryo is consistent with a role in epithelial-mesenchymal interactions; in the limb bud it may prevent differentiation and permit limb outgrowth and subsequent expression of patterning events.

Amino Acid Sequence↗
PubMed1993

Retroviral expression of FGF-2 (bFGF) affects patterning in chick limb bud.

To investigate the role of fibroblast growth factor-2 (basic fibroblast growth factor) in chick limb development, we constructed a replication-defective spleen necrosis virus to ectopically express fibroblast growth factor-2 in stage 20-22 chick limb bud. Because infecting cells in vivo proved to be inefficient, limb bud cells were dissociated, infected in vitro, and then grafted back into host limbs. This procedure caused duplications of anterior skeletal elements, including proximal humerus, distal radius, and digits 2 and 3. Eighty-nine percent of host wings receiving infected grafts at their anterior borders had duplications of one or more of these elements. The frequency of duplication declined dramatically when infected cells were grafted to progressively more posterior sites of host limb buds, and grafting to the posterior border had no effect at all. Several techniques were used to determine the role of infected tissue in forming skeletal duplications. First, staining with an fibroblast growth factor-2 specific monoclonal antibody showed higher than endogenous levels of fibroblast growth factor-2 expression associated with extra elements. Second, the host/donor composition of duplicated elements was determined by simultaneously infecting donor cells with viruses encoding fibroblast growth factor-2 or beta-galactosidase; donor tissue was then visualized by X-gal staining. Patterns of ectopic fibroblast growth factor-2 expression and X-gal staining confirmed the presence of infected donor tissue near duplicated structures, but the duplicated skeletal elements themselves showed very little staining. Similar results were obtained in duplications caused by infected quail wing bud cells grafted to the chick wing bud. These observations suggest that fibroblast growth factor-2-expressing donor tissue induced host tissue to form normally patterned extra elements. In support of this conclusion, implanting beads containing fibroblast growth factor-2 caused partial duplications of digit 2. These data provide the first direct evidence that fibroblast growth factor-2 plays a role in patterning in the limb bud.

Animals↗
PubMed1992

The talpid2 chick limb has weak polarizing activity and can respond to retinoic acid and polarizing zone signal.

The talpid2 (ta2) chick mutant has wide, polydactylous wings and legs. Talpid2 limb cartilages have abnormal morphology and a very subtle anteroposterior polarity. Specifically, posterior ta2 limb structures are identifiable, while more anterior cartilages are less distinctive. Here, we investigate the development of anteroposterior limb pattern in the ta2 embryo. We show that ta2 posterior limb bud mesoderm is capable of respecifying the anteroposterior axis of a normal wing. However, the average duplication obtained after grafting a ta2 polarizing region was significantly less than the average duplication formed after a graft of normal wing bud polarizing zone. Thus, polarizing activity appears to be weak in ta2. Grafts of normal polarizing zone to the posterior edge of ta2 wing buds had no effect on the ta2 phenotype. This result suggests that a weakly functioning polarizing signal does not account for the altered anteroposterior polarity in ta2 limbs, and that normal polarizing zone activity is not sufficient for formation of normal limb bud cartilages. We demonstrated that transmission of a polarizing signal through the ta2 limb mesoderm was normal. In addition, ta2 anterior border mesoderm had no polarizing activity. We also assessed the ability of ta2 limb bud mesoderm to respond to a polarizing signal. Either normal polarizing zone tissue or a bead containing retinoic acid was placed at the anterior edge of ta2 wing buds at stages 18-23. Both polarizing zone and retinoic acid caused respecification of the ta2 wing anteroposterior axis. The result was that a ta2 ulna replaced the radius, and the most posterior digit was duplicated anteriorly. Limb cartilages with normal morphology never formed. When a bead containing retinoic acid was placed at the posterior edge of ta2 wing buds, there was no effect on anteroposterior pattern. However, beads with retinoic acid always caused a reduction in the number of ta2 wing digits which formed, whether the beads were placed at the anterior or posterior edge of the developing ta2 limb.

Animals↗
PubMed1992

Leg bud mesoderm retains morphogenetic potential to express limb-like characteristics ("limbness") in collagen gel culture.

Recent in situ hybridization studies have correlated expression of potential regulatory genes with pattern formation in limb bud mesoderm (Tabin: Cell 66:199-217, 1991); however, the mechanism(s) controlling their expression in mesoderm and their relevance to the establishment of a limb morphogenetic pattern remain unknown. One likely candidate for regulating patterning events in limb mesoderm is the apical ectodermal ridge, as its removal in ovo results in a graded truncation of limb skeletal elements in the proximal-distal axis dependent upon the time of excision (Rowe and Fallon: J Embryol Exp Morph 68:1-7, 1982). In the present study, we investigate whether the hypothetical imprint of ridge ectoderm is retained in cultured mesoderm. Specifically, we sought to determine if a subpopulation of limb mesoderm that forms in collagen gel culture (Markwald et al: Anat Rec 226:91-107, 1990), retains any expression of "limbness" in the absence of limb ectoderm as characterized by the formation of a predictable number and distribution of limb-like chondrogenic elements in comparison to the temporal and spatial relationships of the in situ proximal, hindlimb skeletal structures. Accordingly, explants of undissociated mesoderm from stage 18-22 chicken leg buds were cultured without ectoderm on collagen gel lattices and the central subpopulation of mesoderm was examined morphologically. We show that this central subset of mesoderm will form chondrogenic cells which were not expressed uniformly throughout the subset, but rather distinct nodules or elements of cartilage were elaborated. Moreover, the number of elements expressed by the central subset increased with the age of the mesoderm at the time of explantation; spatially and temporally, the sequence of elements that formed always proceeded from the proximal, anterior margin of the subset to its distal, posterior border. The shapes of the initial elements (designated I and II) resembled the forms of in situ proximal skeletal structures (girdle and femur-like), whereas more distal elements (III-V) were often fused and without structural similarity to in situ skeletal structures. When cultures were established from the posterior mesoderm of stage 19/20 or 21 mesoblasts, the frequency of element I formation was reduced approximately one-half, whereas formation of more distal elements was unaffected. Conversely, element formation from the central subset established from isolated anterior mesoderm was virtually identical to intact mesoblasts, indicating a capacity to regulate for the loss of mesoderm as occurs in situ (Hampé: Archs Anat Microsc Morph Exp 48:345-378, 1959).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Talpid2 limb bud mesoderm does not express GHox-8 and has an altered expression pattern of GHox-7.

We have studied the expression patterns of the chick homeobox-containing genes, GHox-7 and GHox-8, in the talpid2 (ta2) chick mutant whose limbs have abnormal pattern. These studies provide new insight into how homeobox gene expression and limb patterning may be related. This is the first study demonstrating a natural change in GHox-7 and GHox-8 along the anteroposterior axis. While GHox-7 is expressed asymmetrically in normal limb buds, it is expressed at a uniform level across the anteroposterior axis of ta2 limb buds. GHox-8 is expressed in anterior mesoderm of normal limb buds, but is undetectable in ta2 limb bud mesoderm. These data are consistent with the subtle anteroposterior polarity in ta2 limbs, and allow us to propose that ta2 limb buds lack anterior positional information, but have a narrow range of posterior positional values. We suggest that in normal limb buds GHox-8 may establish the anterior limb bud boundary. Furthermore, we point out that coexpression of GHox-7 and GHox-8 in normal anterior limb bud mesoderm can be correlated with the reduced apical ridge maintenance activity of this tissue, while the lack of coexpression in ta2 limb buds is correlated with the strong ridge maintenance activity in the mutant's anterior limb bud mesoderm. Last, ta2 limbs contain no dying cells in their anterior and posterior border mesoderm; nevertheless, they express GHox-7 in these regions. These data challenge the proposal that this gene determines cell death.

Animals↗