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Interactions between Wnts, Fgfs and Tbx genes are involved in limb initiation and the same gene families have been implicated in mammary gland development. Here we explore how these genes act together in mammary gland initiation. We compared expression of Tbx3, the gene associated with the human condition ulnar-mammary syndrome, expression of the gene encoding the dual-specificity MAPK phosphatase Pyst1/MKP3, which is an early response to FGFR1 signalling (as judged by sensitivity to the SU5402 inhibitor), and expression of Lef1, encoding a transcription factor mediating Wnt signalling and the earliest gene so far known to be expressed in mammary gland development. We found that Tbx3 is expressed earlier than Lef1 and that Pyst1 is also expressed early but only transiently. Patterns of expression of Tbx3, Pyst1 and Lef1 in different glands suggest that the order of mammary gland initiation is 3, 4, 1, 2 and 5. Consistent with expression of Pyst1 in the mammary gland, we detected expression of Fgfr1b, Fgf8 and Fgf9 in both surface ectoderm and mammary bud epithelium, and Fgf4 and Fgf17 in mammary bud epithelium. Beads soaked in FGF-8 applied to the flank of mouse embryos, at a stage just prior to mammary bud initiation, induce expression of Pyst1 and Lef1 and maintain Tbx3 expression in flank tissue surrounding the bead. Grafting beads soaked in the FGFR1 inhibitor, SU5402, abolishes Tbx3, Pyst1 and Lef1 expression, supporting the idea that FGFR1 signalling is required for early mammary gland initiation. We also showed that blocking Wnt signalling abolishes Tbx3 expression but not Pyst1 expression. These data, taken together with previous findings, suggest a model in which Tbx3 expression is induced and maintained in early gland initiation by both Wnt and Fgf signalling through FGFR1.

Tetrapods have two pairs of limbs, each typically with five digits, each of which has a defined number of phalanges derived from an archetypal formula. Much progress has been made in understanding vertebrate limb initiation and the patterning processes that determine digit number in developing limb buds, but little is known about how phalange number is controlled. We and others previously showed that an additional phalange can be induced in a chick toe if sonic hedgehog protein is applied in between developing digit primordia. Here we show that formation of an additional phalange is associated with prolonged Fgf8 expression in the overlying apical ridge and that an Fgf Receptor inhibitor blocks its formation. The additional phalange is produced by elongation and segmentation of the penultimate phalange, suggesting that the digit tip forms when Fgf signaling ceases by a special mechanism, possibly involving Wnt signaling. Consistent with this, Fgfs inhibit tip formation whereas attenuation of Fgf signaling induces tip formation prematurely. We propose that duration of Fgf signaling from the ridge, responsible for elongation of digit primordia, coupled with a characteristic periodicity of joint formation, generates the appropriate number of phalanges in each digit. We also propose that the process that generates the digit tips is independent of that which generates more proximal phalanges. This has implications for understanding human limb congenital malformations and evolution of digit diversity.

Background: The importance of endogenous antagonists in intracellular signal transduction pathways is becoming increasingly recognized. There is evidence in cultured mammalian cells that Pyst1/MKP3, a dual specificity protein phosphatase, specifically binds to and inactivates ERK1/2 mitogen-activated protein kinases (MAPKs). High-level Pyst1/Mkp3 expression has recently been found at many sites of known FGF signaling in mouse embryos, but the significance of this association and its function are not known., Results: We have cloned chicken Pyst1/Mkp3 and show that high-level expression in neural plate correlates with active MAPK. We show that FGF signaling regulates Pyst1 expression in developing neural plate and limb bud by ablating and/or transplanting tissue sources of FGFs and by applying FGF protein or a specific FGFR inhibitor (SU5402). We further show by applying a specific MAP kinase kinase inhibitor (PD184352) that Pyst1 expression is regulated via the MAPK cascade. Overexpression of Pyst1 in chick embryos reduces levels of activated MAPK in neural plate and alters its morphology and retards limb bud outgrowth., Conclusions: Pyst1 is an inducible antagonist of FGF signaling in embryos and acts in a negative feedback loop to regulate the activity of MAPK. Our results demonstrate both the importance of MAPK signaling in neural induction and limb bud outgrowth and the critical role played by dual specificity MAP kinase phosphatases in regulating developmental outcomes in vertebrates.

More news this year about FGFs and their roles in vertebrate limb initiation; Wnt signalling is shown for the first time to be another component of the signalling cascade involved in early limb formation. Ectodermal compartments that control apical ridge formation were previously described in chick embryos and are now shown to exist in mouse embryos; Engrailed1 is expressed in the ventral ectodermal compartment but experiments in both chick and mouse show that it is not responsible for compartment specification.

We tested a diffusion gradient model for setting up overlapping domains of Hoxa gene expression in the chick limb bud. The model is based on morphogen production at the limb bud tip where the apical ridge is located and assumes that cells respond to a series of concentration thresholds. Consistent with the model, Hoxa13 gene expression rapidly switches off when the ridge is removed from stage 21/22 buds, while Hoxa11 and Hoxa10 expression is stable; Hoxa13 expression can be initiated and maintained in absence of the ridge by FGF soaked beads; the Hoxa13 domain first expands quickly and then slows up and the size is related to the dose of FGF4. Contrary to the model, addition of FGF4 to early limb buds does not activate Hoxa13 prematurely nor extend the Hoxa13 expression domain proximally. Therefore FGF4 signalling is necessary but not sufficient for Hoxa gene expression in the limb bud., (Copyright 2001 Wiley-Liss, Inc.)

SnR, twist and Fgf10 are expressed in presumptive limb territories of early chick embryos. When FGF-2/FGF-8 beads are implanted in chick flank, an ectopic limb develops and SnR is irreversibly activated as early as 1 h. Ectopic Fgf10 and twist expression are activated much later at 17 and 20 h, respectively. FGF-10 can also induce SnR, but much later, and in this case activation occurs simultaneously with that of twist and Fgf10 via the Fgf8- expressing ridge. Tbx-4 and Tbx-5 are expressed in leg and wing forming regions, respectively, in a similar pattern to SnR and twist. FGF-2 leads to ectopic expression of Tbx-4 and Tbx-5 as rapidly as ectopic expression of SnR, but the patterns of ectopic transcripts suggest that induction of SnR and Tbx gene expression occur via different pathways.

The semidominant mouse mutation hypodactyly (Hd), caused by a deletion within the Hoxa13 gene, results in reduced digits; heterozygotes lack digit I in the hindlimb and homozygotes have only one digit on each limb. We investigated expression of Shh and Fgf4 signaling molecules involved in digit specification in mutant limb buds. Shh and Fgf4 are expressed in the posterior part of the limb buds as normal but expression may be slightly prolonged. The extent of digit reduction in hypodactyly is much more severe than in the Hoxa13 deficient mouse and resembles that in the Hoxa13(-/-)/Hoxd13(-/-) double mutant mouse. We found that the pattern of Hoxd13 and Hoxd11 transcripts was not markedly different in the mutant compared with the normal limbs even though the mutant limbs are narrower. Therefore Hoxd genes are transcribed as normal in the mutant. This makes it likely that the severe digit reductions in hypodactyly are caused by interference with Hoxd13 function at the protein level. Similar interactions between mutant and normal HOX gene products have been suggested to occur in the human semidominant disorder, synpolydactyly, caused by mutations in HOXD13.

Urodele amphibians are the only vertebrates that can regenerate amputated limbs, even as adults. However, we have previously shown that amputated chick wing bud stumps can be induced to ((regenerate)) and to form a complete set of correctly-patterned skeletal elements, following implantation of beads soaked in fibroblast growth factor-4 (FGF-4). We have now performed Dil injection experiments to determine which cells contribute to FGF-4-induced chick wing bud ((regenerates)). We show that the FGF-4-induced outgrowth of the regenerating wing bud stump is comprised of mesenchyme cells that originate from a region within 200 microm of the FGF-4 bead, and that cells proximal to the bead move distally.

Pattern in the developing limb depends on signaling by polarizing region mesenchyme cells, which are located at the posterior margin of the bud tip. Here we address the underlying cellular mechanisms. We show in the intact bud that connexin 43 (Cx43) and Cx32 gap junctions are at higher density between distal posterior mesenchyme cells at the tip of the bud than between either distal anterior or proximal mesenchyme cells. These gradients disappear when the apical ectodermal ridge (AER) is removed. Fibroblast growth factor 4 (FGF4) produced by posterior AER cells controls signaling by polarizing cells. We find that FGF4 doubles gap junction density and substantially improves functional coupling between cultured posterior mesenchyme cells. FGF4 has no effect on cultured anterior mesenchyme, suggesting that any effects of FGF4 on responding anterior mesenchyme cells are not mediated by a change in gap junction density or functional communication through gap junctions. In condensing mesenchyme cells, connexin expression is not affected by FGF4. We show that posterior mesenchyme cells maintained in FGF4 under conditions that increase functional coupling maintain polarizing activity at in vivo levels. Without FGF4, polarizing activity is reduced and the signaling mechanism changes. We conclude that FGF4 regulation of cell-cell communication and polarizing signaling are intimately connected.

Bone morphogenetic protein-2 (BMP-2) has been implicated in the polarizing region signalling pathway, which specifies pattern across the antero-posterior of the developing vertebrate limb. Retinoic acid and Sonic Hedgehog (SHH) can act as polarizing signals; when applied anteriorly in the limb bud, they induce mirror-image digit duplications and ectopic Bmp-2 expression in anterior mesenchyme. In addition, the two signals can activate Fgf-4 expression in anterior ridge and HoxD expression in anterior mesenchyme. We tested the role of BMP-2 in this signalling cascade by ectopically expressing human BMP-2 (hBMP-2) at the anterior margin of the early wing bud using a replication defective retroviral vector, and found that ectopic expression of Fgf-4 was induced in the anterior part of the apical ectodermal ridge, followed later by ectopic expression of Hoxd-11 and Hoxd-13 in anterior mesenchyme. This suggests that BMP-2 is involved in regulating Fgf-4 and HoxD gene expression in the normal limb bud. Ectopically expressed hBMP-2 also induced duplication of digit 2 and bifurcation of digit 3, but could not produce the mirror-image digit duplications obtained with SHH-expressing cells. These results suggest that BMP-2 may be involved primarily in maintenance of the ridge, and in the link between patterning and outgrowth of the limb bud.

We examined systematically the ability of chick limb bud stumps to regenerate distal structures when fibroblast growth factor (FGF)-4 is applied. When amputations were made within 600 mu m of the tip and FGF-4 applied either posteriorly or both apically and posteriorly, outgrowth of stump tissues occurred and a virtually complete skeleton developed. 'Regeneration' of distal structures was correlated with reactivation of Msx-1 and Shh expression. At proximal amputation levels where FGF-4 did not lead to 'regeneration', neither Msx-1 nor Shh expression was induced. We also grafted cells from progressively more proximal levels of mouse limb buds to chick wing bud tips beneath the apical ridge and the pattern of reactivation of Msx-1 expression along the proximal distal axis of the limb buds was similar to that found in chick limb buds.

Fibroblast growth factors (FGFs) act as signals in the developing limb and can maintain proliferation of limb bud mesenchyme cells. Remarkably, beads soaked in FGF-1, FGF-2, or FGF-4 and placed in the presumptive flank of chick embryos induce formation of ectopic limb buds, which can develop into complete limbs. The entire flank can produce additional limbs, but generally wings are formed anteriorly and legs posteriorly. FGF application activates Sonic hedgehog in cells with polarizing potential to make a discrete polarizing region. Hoxd-13 is also expressed in the ectopic bud, and an apical ectodermal ridge forms. A limb bud is thus established that can generate the appropriate signals to develop into a complete limb. The additional limbs have reversed polarity. This can be explained by the distribution of cells in the flank with potential polarizing activity. The results suggest that local production of an FGF may initiate limb development.

Limb development depends on signals from the apical ectodermal ridge and underlying mesenchyme. Fibroblast growth factor (FGF) can replace the ridge and, because Fgf4 RNA is localized to the mouse posterior ridge, we proposed that FGF4 is the endogenous ridge signal. Ridge signals control limb outgrowth and maintain the zone of polarizing activity (ZPA) at the limb posterior margin, which is important in limb pattering: a ZPA graft to limb anterior mesenchyme causes cell respecification and mirror-image duplications. Sonic hedgehog (SHH) has polarizing activity, and Shh RNA co-localizes with ZPA activity, suggesting SHH is the endogenous polarizing signal. We have investigated the molecular regulation of Fgf4 and Shh expression. We report here that Fgf4 expression in the ridge can be regulated by Shh-expressing cells. Moreover, Shh expression in mesenchyme can be activated by FGF4 in combination with retinoic acid. Once induced, Shh expression can be maintained by FGF4 alone, thus establishing a positive feedback loop between ZPA and ridge.

The apical ectodermal ridge plays a central role in limb development through its interactions with the underlying mesenchyme. Removal of the AER results in cessation of limb outgrowth and leads to truncation of the limb along the proximo-distal axis. The many functions attributed to the ridge include maintenance of the progress zone mesenchyme. Here, cells are stimulated to proliferate, are maintained in an undifferentiated state, and are assigned progressively more distal positional values as the limb grows. The AER also functions to maintain the activity of the polarizing region, a region of mesenchyme which is thought to provide the primary signal for patterning along the antero-posterior axis. We have begun to explore the function of fibroblast growth factor-4 (FGF-4) during limb development. FGF-4, which encodes an efficiently secreted protein, is expressed in the AER. We have previously demonstrated that FGF-4 protein can stimulate limb mesenchyme proliferation and can induce the expression of a downstream homeobox gene, Evx-1 (homologue of the Drosophila even-skipped gene), that is normally regulated by a signal from the AER. To determine to what extent FGF-4 protein can substitute for the AER to allow normal limb outgrowth, we performed experiments on the developing chick limb in ovo. Remarkably, we find that after AER removal, the FGF-4 protein can provide all the signals required for virtually normal outgrowth and patterning of the limb.(ABSTRACT TRUNCATED AT 250 WORDS)

The apical ectodermal ridge plays a key role in limb development. We show that recombinant FGF-4 can substitute for the ridge to provide all the signals necessary for virtually complete outgrowth and patterning of the chick limb. FGF-4 stimulates proliferation of cells in the distal mesenchyme and maintains a signal from the posterior to the distal mesenchyme that appears to be required for elaboration of skeletal elements in the normal proximodistal sequence. Moreover, retinoic acid, which is capable of providing polarizing activity, can supply this signal. This suggests that polarizing activity plays a role in patterning along the proximodistal axis, in addition to its well-established role in anteroposterior patterning. Taken together, the data suggest a simple mechanism whereby FGF-4 links growth and pattern formation during limb development.

The polarizing region is a major signalling tissue involved in patterning the tissues of the vertebrate limb. The polarizing region is located at the posterior margin of the limb bud and can be recognized by its ability to induce additional digits when grafted to the anterior margin of a chick limb bud. The signal from the polarizing region operates at the tip of the bud in the progress zone, a zone of undifferentiated mesenchymal cells, maintained by interactions with the apical ectodermal ridge. A number of observations have pointed to a link between the apical ectodermal ridge and signalling by the polarizing region. To test this possibility, we removed the posterior apical ectodermal ridge of chick wing buds and assayed posterior mesenchyme for polarizing activity. When the apical ectodermal ridge is removed, there is a marked decrease in polarizing activity of posterior cells. The posterior apical ectodermal ridge is known to express FGF-4 and we show that the decrease in polarizing activity of posterior cells of wing buds that normally follows ridge removal can be prevented by implanting a FGF-4-soaked bead. Furthermore, we show that both ectoderm and FGF-4 maintain polarizing activity of limb bud cells in culture.