Initiation of dorso-ventral axis during chick limb development

We analysed spatio-temporal expression of dorso-ventral genes - Wnt-7a, En-1, Lmx-1 and Fgf-8 - during both normal and ectopic limb formation following fibroblast growth factor (FGF) application to the flank. We confirm that Wnt-7a is the first of these genes to be expressed in dorsal ectoderm in limb-forming regions. We also noticed patterns and kinetics of gene expression specific to chick that could account for differences observed in ridge formation between chick and mouse. We find that Wnt-7a expression, in dorsal ectoderm, is rapidly and locally induced by FGF application. In contrast, ectopic induction of Lmx-1 expression, in dorsal mesoderm, is much slower, occurs first at a distance from the FGF-2 bead and seems initially independent of direct Wnt-7a signalling during FGF-2 limb induction. Finally, we show that there is no contribution to extra-limb mesoderm from normal limb mesoderm and confirm that flank cells give rise to the extra limb. Furthermore, we suggest that an inhibitor present in the flank normally prevents Lmx-1 expression in this region and restricts its expression to limb-forming regions.     

FGF7 and FGF10 directly induce the apical ectodermal ridge in chick embryos.

During vertebrate limb development, the apical ectodermal ridge (AER) plays a vital role in both limb initiation and distal outgrowth of the limb bud. In the early chick embryo the prelimb bud mesoderm induces the AER in the overlying ectoderm. However, the direct inducer of the AER remains unknown. Here we report that FGF7 and FGF10, members of the fibroblast growth factor family, are the best candidates for the direct inducer of the AER. FGF7 induces an ectopic AER in the flank ectoderm of the chick embryo in a different manner from FGF1, -2, and -4 and activates the expression of Fgf8, an AER marker gene, in a cultured flank ectoderm without the mesoderm. Remarkably, FGF7 and FGF10 applied in the back induced an ectopic AER in the dorsal median ectoderm. Our results suggest that FGF7 and FGF10 directly induce the AER in the ectoderm both of the flank and of the dorsal midline and that these two regions have the competence for AER induction. Formation of the AER of the dorsal median ectoderm in the chick embryo is likely to appear as a vestige of the dorsal fin of the ancestors.     

Fibroblast growth factor-induced gene expression and cartilage pattern formation in chick limb bud recombinants

To clarify the roles of fibroblast growth factors (FGF) in limb cartilage pattern formation, the effects of various FGF on recombinant limbs that were composed of dissociated and reaggregated mesoderm and ectodermal jackets were examined. Fibroblast growth factor-soaked beads were inserted just under the apical ectodermal ridge (AER) of recombinant limbs and the recombinant limbs were grafted and allowed to develop. Control recombinant limbs without FGF beads formed one or two cartilage elements. Recombinants with FGF-4 beads formed up to five cartilage elements, which were aligned along the anteroposterior (AP) axis. Each cartilage element showed digit-like segmentation. In contrast, recombinants with FGF-2 beads showed formation of multiple thick and unsegmented cartilage rods, which elongated inside and outside the AP plane from the distal end of the recombinants. Recombinants with FGF-8 beads formed a truncated cartilage pattern and recombinants with FGF-10 beads formed a cartilage pattern similar to that of the control recombinants. The expression of the Fgf-8, Msx-1 and Hoxa-13 genes in the developing recombinant limbs were examined. FGF-4 induced extension of the length of the Fgf-8-positive epidermis, or AER, along the AP axis 5 days after grafting, at which time the digits are specified. FGF-2 induced expansion of the Msx-1-positive area, first in the proximal direction and then along the dorsoventral axis. The functions of these FGF in recombinant and normal limb patterning are discussed in this paper.     

Msx1 expressing mesoderm is important for the apical ectodermal ridge (AER)-signal transfer in chick limb development

The apical ectodermal ridge (AER) is a specialized thickening of the distal limb ectoderm, and its signals are known to support limb morphogenesis. The expression of a homeobox gene, Msx1 , in the distal limb mesoderm depends on signals from the AER. In the present paper it is reported that Msx1 expression in the distal mesoderm is necessary for the transfer of AER signals in chick limb buds. Interruption of AER-mesoderm interaction by insertion of a thick filter led to the inhibition of pattern specification in the mesoderm just under the filter. In such cases, the expression of Msx1 disappeared in the mesoderm under the filter, suggesting that AER is able to signal over short ranges. In advanced limb buds, Msx1 is also expressed in the proximal mesoderm under the anterior ectoderm. However, it was found that a grafted antero-proximal mesoderm shows no inhibitory effects on pattern specification of the host mesoderm, as is the case with the distal mesoderm. On the other hand, grafted mesoderms without potent Msx1 re-expression, even underneath AER, disturbed normal limb development. In such cases, the expression of Msx1 disappeared in the mesoderm under the grafts, whereas Fgf-8 expression was maintained in the AER above the graft. These results indicate that the expression of Msx1 in the mesoderm is important for the transfer of AER signals.     

Beta-catenin-dependent Wnt signaling in apical ectodermal ridge induction and FGF8 expression in normal and limbless mutant chick limbs

The fibroblast growth factor (FGF) and beta-catenin-dependent Wnt signaling pathways are key regulators of vertebrate limb development. FGF10 induces expression of Wnt3a, which regulates the formation and FGF8 expression of the apical ectodermal ridge (AER). In amelic limbless limbs, an AER fails to form and FGF8 is not expressed, despite expression of FGF10. It has been found that Wnt3a is initially expressed in limbless ectoderm, although subsequently is drastically reduced. In addition, changes in the expression pattern or level of several Frizzled receptors, Axin, Lef1/Tcf1 and beta-catenin have been found in limbless limbs. Notably, while normal wing buds respond to LiCl-stimulated activation of beta-catenin-dependent signaling by forming ectopic, FGF8-expressing AER, LiCl was unable to induce an AER in limbless wing buds. The results of this study suggest that the limbless gene is required for beta-catenin-dependent Wnt signaling in limb ectoderm leading to FGF8 expression and AER formation.     

Gap junction signalling mediated through connexin-43 is required for chick limb development

During chick limb development the gap junction protein Connexin-43 (Cx43) is expressed in discrete spatially restricted domains in the apical ectodermal ridge (AER) and mesenchyme of the zone of polarising activity. Antisense oligonucleotides (ODNs) were used to investigate the role of Connexin-43 (Cx43) in the development of the chick limb bud. We have used unmodified ODNs in Pluronic F-127 gel, which is liquid at low temperature but sets at room temperature and so remains situated at the point of application. As a mild surfactant, the gel increases antisense ODN penetration and supplies ODNs to the embryo continually for 12-18 h. We have shown a strong decrease in Cx43 protein expression after application of specific antisense oligonucleotides but the abundance of a closely related protein, Connexin-32 (Cx32), was not affected. Application of antisense Cx43 ODNs at stages 8-15 HH before limb outgrowth resulted in dramatic limb phenotypes. About 40% of treated embryos exhibited defects such as truncation of the limb bud, fragmentation into two or more domains, or complete splitting of the limb bud into two or three branches. Molecular analysis of antisense treated embryos failed to detect Shh or Bmp-2 in anterior structures and suggested that extra lobes seen in nicked and split limbs were not a result of establishment of new signalling centres as found after the application of FGF to the flank. However, examination of markers for the AER showed a number of abnormalities. In severely truncated specimens we were unable to detect the expression of either Fgf-4 or Fgf-8. In both nicked and split limbs the expression of these genes was discontinuous. Down-regulation of Cx43 after the antisense application could be comparable to AER removal and results in distal truncation of the limb bud. Taken together these data suggest the existence of a feedback loop between the FGFs and signalling mediated by Cx43.     

Transient Inhibition of FGFR2b-Ligands Signaling Leads to Irreversible Loss of Cellular β-Catenin Organization and Signaling in AER during Mouse Limb Development

The vertebrate limbs develop through coordinated series of inductive, growth and patterning events. Fibroblast Growth Factor receptor 2b (FGFR2b) signaling controls the induction of the Apical Ectodermal Ridge (AER) but its putative roles in limb outgrowth and patterning, as well as in AER morphology and cell behavior have remained unclear. We have investigated these roles through graded and reversible expression of soluble dominant-negative FGFR2b molecules at various times during mouse limb development, using a doxycycline/transactivator/tet(O)-responsive system. Transient attenuation (≤24 hours) of FGFR2b-ligands signaling at E8.5, prior to limb bud induction, leads mostly to the loss or truncation of proximal skeletal elements with less severe impact on distal elements. Attenuation from E9.5 onwards, however, has an irreversible effect on the stability of the AER, resulting in a progressive loss of distal limb skeletal elements. The primary consequences of FGFR2b-ligands attenuation is a transient loss of cell adhesion and down-regulation of P63, β1-integrin and E-cadherin, and a permanent loss of cellular β-catenin organization and WNT signaling within the AER. Combined, these effects lead to the progressive transformation of the AER cells from pluristratified to squamous epithelial-like cells within 24 hours of doxycycline administration. These findings show that FGFR2b-ligands signaling has critical stage-specific roles in maintaining the AER during limb development.     

Wnt5a participates in distal lung morphogenesis

Operational parallels in overall mechanisms of three-dimensional patterning of vertebrate organs are becoming increasingly apparent. Many key mediators, such as FGFs, BMPs, and sonic hedgehog, participate in organization of a number of organs, including the lungs, which exhibit a defined proximodistal (P-D) polarity. Recently, Wnt5a a member of the wingless family of signaling molecules involved in cell proliferation, differentiation, and organogenesis, was shown to underlie the outgrowth and P-D morphogenesis of the vertebrate limb. In the current study, we show that Wnt5a is expressed in the mouse lung and plays an important role in lung distal morphogenesis. Analysis of the mutant phenotype in mice carrying a targeted disruption of the Wnt5a locus shows distinct abnormalities in distal lung morphogenesis as manifested by distinct truncation of the trachea and overexpansion of the distal respiratory airways. In the face of deleted WNT5a activity, both epithelial and mesenchymal cell compartments of the Wnt5a(-/-) lungs exhibit increased cell proliferation. The overall architecture of the mutant lungs is characterized by overexpansion of the distal airways and inhibition of lung maturation as reflected by persistence of thickened intersaccular interstitium. Absence of WNT5a activity in the mutant lungs leads to increased expression of Fgf-10, Bmp4, Shh, and its receptor Ptc, raising the possibility that WNT5a, FGF-10, BMP4, and SHH signaling pathways are functionally interactive.     

FGF10 can induce Fgf8 expression concomitantly with En1 and R-fng expression in chick limb ectoderm, independent of its dorsoventral specification

The limb bud has a thickened epithelium at the dorsal-ventral boundary, the apical ectodermal ridge (AER), which sustains limb outgrowth and patterning. A secreted molecule fibroblast growth factor (FGF)10 is involved in inducing Fgf8 expression in the prospective AER and mutual interaction between mesenchymal FGF10 and FGF8 in the AER is essential for limb outgrowth. A secreted factor Wnt7a and a homeobox protein Lmx1 are involved in the dorsal patterning of the limb, whereas a homeobox protein Engrailed 1 (En1) is involved in the dorsal-ventral patterning as well as AER formation. Radical fringe (R-fng), a vertebrate homolog of Drosophila fringe was also found to elaborate AER formation in chicks. However, little is known about the molecular interactions between these factors during AER formation. The present study clarified the relationship between FGF10, Wnt7a, Lmx1, R-fng and En1 during limb development using a foil-barrier insertion experiment. It was found that a foil-barrier inserted into the chick prospective wing mesenchyme lateral to the mesonephric duct blocks AER induction. This experiment was expanded by implanting Fgf10-expressing cells lateral to the barrier and examined whether FGF10 could rescue the expression of the limb-patterning genes reported in AER formation. It was found that FGF10 is sufficient to induce Fgf8 expression in the ectoderm of the foil-inserted limb bud, concomitantly with R-fng and En1 expression. However, FGF10 could not rescue the expression of the dorsal marker genes, Wnt7a or Lmx1. Thus, it is suggested that epithelial factors of En1 and R-fng can induce Fgf8 expression in the limb ectoderm in cooperation with a mesenchymal factor FGF10. Some factor(s) other than FGF10, possibly from the paraxial structures medial to the limb mesoderm, is responsible for the initial dorsal-ventral specification of the limb bud.     

Bmp4 in limb bud mesoderm regulates digit pattern by controlling AER development

In the developing limb, Bmp4 is expressed in the apical ectodermal ridge (AER) and underlying mesoderm. Insight into the function of Bmp4 in limb development has been hampered by the early embryonic lethality of Bmp4 null embryos. We directly investigated Bmp4 using a conditional null allele of Bmp4 and the Prx1(cre) transgene to inactivate Bmp4 in limb bud mesoderm. The limb bud mesoderm of Prx1(cre);Bmp4 mutants was defective in production of Bmp4 but still competent to respond to Bmp signaling. Prx1(cre);Bmp4 mutant embryos had defective digit patterning including hindlimb preaxial polydactyly with posterior digit transformations. The Prx1(cre);Bmp4 mutants also had postaxial polydactyly with digit five duplications. Bmp4 mutant limbs had delayed induction and maturation of the AER that resulted in expanded Shh signaling. Moreover, the AER persisted longer in the Bmp4 mutant limb buds exposing the forming digits to prolonged Fgf8 signaling. Our data show that Bmp4 in limb mesoderm regulates AER induction and maturation and implicate signaling from the AER in regulation of digit number and identity.     

Limb bud and flank mesoderm have distinct "physical phenotypes" that may contribute to limb budding

Limb bud outgrowth in chicken embryos is initiated during the third day of development by Fibroblast Growth Factor 8 (FGF8) produced by the newly formed apical ectodermal ridge (AER). One of the earliest effects of this induction is a change in the properties of the limb field mesoderm leading to bulging of the limb buds from the body wall. Heintzelman et al. [Heintzelman, K.F., Phillips, H.M., Davis, G.S., 1978. Liquid-tissue behavior and differential cohesiveness during chick limb budding. J. Embryol. Exp. Morphol. 47, 1–15.] suggested that budding of the limbs is caused by a higher liquid-like cohesivity of limb bud tissue compared with flank. We sought additional evidence relevant to this hypothesis by performing direct measurements of the effective surface tension, a measure of relative tissue cohesivity, of 4-day embryonic chicken wing and leg bud mesenchymal tissue, and adjacent flank mesoderm. As predicted, the two types of limb tissues were 1.5- to 2-fold more cohesive than the flank tissue. These differences paralleled cell number and volume density differences: 4-day limb buds had 2- to 2.5-fold as many cells per unit area of tissue as surrounding flank, a difference also seen at 3 days, when limb budding begins. Exposure of flank tissue to exogenous FGF8 for 24 h increased its cell number and raised its cohesivity to limb-like values. Four-day flank tissue exhibited a novel and unique active rebound response to compression, which was suppressed by the drug latrunculin and therefore dependent on an intact actin cytoskeleton. Correspondingly, flank at this stage expressed high levels of α-smooth muscle actin (SMA) mRNA and protein and a dense network of microfilaments. Treatment of flank with FGF8 eliminated the rebound response. We term material properties of tissues, such as cohesivity and mechanical excitability, the “physical phenotype”, and propose that changes thereof are driving forces of morphogenesis. Our results indicate that two independent aspects of the physical phenotype of flank mesoderm can be converted to a limb-like state in response to treatment with FGF8. The higher tissue cohesivity induced by this effect will cause the incipient limb bud to phase separate from the surrounding flank, while the active mechanical response of the flank could help ensure that the limb bud bulges out from, rather than becoming engulfed by, this less cohesive tissue.