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EVOLUTIONARY HOMOLOGS

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FGF and limb patterning (part 1/2)

The development of vertebrate limb buds is triggered in the lateral plate mesoderm by a cascade of genes, including members of the Fgf and Wnt families, as well as the transcription factor tbx5. Fgf8, which is expressed in the intermediate mesoderm, is thought to initiate forelimb formation by activating wnt2b, which then induces the expression of tbx5 in the adjacent lateral plate mesoderm. Tbx5, in turn, is required for the activation of fgf10, which relays the limb inducing signal to the overlying ectoderm. The zebrafish fgf24 gene, which belongs to the Fgf8/17/18 subfamily of Fgf ligands, acts downstream of tbx5 to activate fgf10 expression in the lateral plate mesoderm. fgf24 activity is necessary for the migration of tbx5-expressing cells to the fin bud, and for the activation of shh, but not hand2, expression in the posterior fin bud (Fischer, 2003).

Fgf-10-deficient mice (Fgf-10(-/-)) were generated to determine the role(s) of Fgf-10 in vertebrate development. Limb bud initiation was abolished in Fgf-10(-/-) mice. Strikingly, Fgf-10-/- fetuses continue to develop until birth, despite the complete absence of both forelimbs and hindlimbs. Fgf-10 is necessary for apical ectodermal ridge (AER) formation and acts epistatically upstream of Fgf-8, the earliest known AER marker in mice. Fgf-10-/- mice exhibit perinatal lethality associated with complete absence of lungs. Although tracheal development is normal, main-stem bronchial formation, as well as all subsequent pulmonary branching morphogenesis, is completely disrupted. Lack of lungs and main-stem bronchi in Fgf-10-/- mice is very reminiscent of the Drosophila mutant bnl. Both genetic and biochemical evidence indicates that Bnl is a ligand for Breathless (Btl), an Fgf receptor. Drosophila btl mutants exhibited a phenotype that was very similar to that of bnl. Mammalian Fgfr2b is highly expressed in the epithelium throughout embryonic lung development. Transgenic mice expressing a dominant-negative form of FGFR2b splice variant under control of the SP-C promotor exhibit perinatal lethality and failed to develop lungs, indicating that FGFR2b is a receptor necessary for pulmonary branching morphogenesis. Ligands of this receptor include FGF-1, FGF-7, and FGF-10, and all three have been shown to promote expansion and/or budding of endodermal cells in lung explant studies. Fgfr2b transgenic mice exhibit trachea formation and bifurcation of main-stem bronchi, unlike the Fgf-10 knockout mice, which only develop a trachea without further branching. This difference was most likely caused by spatial and temporal differences between Fgf-10 expression and SP-C promotor activity. The SP-C promotor drives transgene expression in distal lung epithelium starting at E10. In contrast, Fgf-10 is already expressed in the distal mesenchymal cells of developing respiratory tract buds at E9.5. Presumably, by E10-E10.5, the formation of the primordial bronchi has already occurred. The impaired pulmonary development observed in Fgfr2b transgenic mice, coupled with similarities in pulmonary phenotypes of Fgf-10 knockout mice and Drosophila bnl and btl mutants, suggests striking functional similarities in the signaling pathways of mammalian Fgf-10 and Drosophila bnl (Min, 1998).

Expression and mutation analyses in mice suggest that the homeobox-containing gene Engrailed (Drosophila homolog: Engrailed) is involved in dorsoventral patterning of the limb. En-1 expression is first detected in the flanking ectoderm of the trunk at stage 15; by stage 16, expression extends throughout the length of the ventral body wall. At stage 18, the anterior limit of expression is clearly demarcated at the anterior edge of the wing bud at the level of somite 15. During the initial stages of limb bud outgrowth, En-1 mRNA and protein are uniformly distributed throughout the ventral limb bud ectoderm. Limbs of En-1(-/-) mice display a double dorsal phenotype suggesting that normal expression of En-1 in the ventral ectoderm is required to establish and/or maintain ventral limb characteristics. Loss of En-1 function also results in ventral expansion of the apical ectodermal ridge (AER), suggesting that En-1 is also required for proper formation of the AER. To further investigate the role En plays in dorsoventral patterning and AER formation, the replication competent retroviral vector, RCAS, has been used to mis-express mouse En-1 in the early chick limb bud. Ectopic En-1 expression in dorsal ectoderm is sufficient to repress the endogenous expression of the dorsal ectodermal marker Wnt7a, with a resultant decrease in Lmx1 expression in underlying dorsal mesenchyme. Wnt7a appears to mediate dorsalization of underlying limb mesenchyme through induction of Lmx1, a LIM homeobox gene. The AER is disrupted morphologically and the expression patterns of the AER (ectodermal) signaling molecules Fgf-8 and Fgf-4 are altered. Consistent with recent evidence that there is a reciprocal interaction between signalling molecules in the dorsal ectoderm, AER, and the zone of polarizing activity (ZPA), loss of Wnt7a, Fgf-8 and Fgf-4 expression leads to a decrease in expression of the signalling molecule Shh in the posteriorly positioned ZPA. These results strongly support the idea that in its normal domain of expression, En-1 represses Wnt7a-mediated dorsal differentiation by limiting the expression of Wnt7a to the dorsal ectoderm. These results provide additional evidence that En-1 is involved in AER formation and suggest that En-1 may act to define ventral ectodermal identity (Logan, 1997).

Vertebrate limb formation has been known to be initiated by a factor(s) secreted from the lateral plate mesoderm. A member of the fibroblast growth factor (FGF) family, FGF10, emanates from the prospective limb mesoderm to serve as an endogenous initiator for limb bud formation. Fgf10 expression in the prospective limb mesenchyme precedes Fgf8 expression in the nascent apical ectoderm. Ectopic application of FGF10 to the chick embryonic flank can induce Fgf8 expression in the adjacent ectoderm, resulting in the formation of an additional complete limb. Expression of Fgf10 persists in the mesenchyme of the established limb bud and appears to interact with Fgf8 in the apical ectoderm and Sonic hedgehog in the zone of polarizing activity. These results suggest that FGF10 is a key mesenchymal factor involved in the initial budding as well as the continuous outgrowth of vertebrate limbs (Ohuchi, 1997b).

FGFR2 is a membrane-spanning tyrosine kinase that serves as a high affinity receptor for several members of the fibroblast growth factor (FGF) family. To explore functions of FGF/FGFR2 signals in development, FGFR2 has been mutated by deleting the entire immunoglobin-like domain III of the receptor. Murine FGFR2 is essential for chorioallantoic fusion and placenta trophoblast cell proliferation. Fgfr2 mutant embryos display two distinct defects that result in failure to form a functional placenta. About one third of the mutants fail to form the chorioallantoic fusion junction and the remaining mutants do not have the labyrinthine portion of the placenta. Consequently, all mutants die at 10-11 days of gestation. Interestingly, mutant embryos do not form limb buds. Consistent with this defect, the expression of Fgf8, an apical ectodermal factor, is absent in the mutant presumptive limb ectoderm, and the expression of Fgf10, a mesenchymally expressed limb bud initiator, is down regulated in the underlying mesoderm. These findings provide direct genetic evidence that FGF/FGFR2 signals are absolutely required for vertebrate limb induction and that an FGFR2 signal is essential for the reciprocal regulation loop between FGF8 and FGF10 during limb induction (Xu, 1998).

In an effort to define the roles of bone morphogenic proteins (BMPs) and fibroblast growth factors (FGFs) during chick limb development more closely, beads impregnated with these growth factors were implanted into chick limb buds between stages 20 and 26. Embryos were sacrificed at the time the bone chondrocyte condensations first appear (stages 27-28). Implantation of beads containing BMPs at the earlier stages (20-22) causes apoptosis to occur, in the most severe cases leading to complete limb degeneration. Application of FGF4, either in the same, or in different beads, prevents the BMP-induced apoptosis. It is argued that the apoptosis observed on removal of the AER prior to stage 23 of development is brought about by BMPs. The action of epithelial FGF in preventing BMP-mediated apoptosis in the mesenchyme would define a novel aspect of epithelial-mesenchymal interactions. Implanting the BMP4 beads into the core of the limb bud a day later (stages 25-26) causes intense chondrogenesis rather than apoptosis. FGF4 can nullify this effect and by itself causes a reduction in bone size. This is the reverse of the functional relationship these growth factors have in mouse tooth specification (where it is BMP4 that inhibits the FGF8 function), and suggests that the balance between the effects of FGFs and BMPs controls the size of the chondrocyte precursor cell pool. In this way members of these two growth factor families control the size of appendages when they are initially formed (Buckland, 1998).

Pattern in the developing limb depends on signaling by polarizing region mesenchyme cells, which are located at the posterior margin of the bud tip. In the intact bud 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. 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. 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. It is concluded that FGF4 regulation of cell-cell communication and polarizing signaling are intimately connected (Makarenkova, 1997).

It has been reported that members of the fibroblast growth factor (FGF) family can induce additional limb formation in the flank of chick embryos. The phenotype of the ectopic limb depends on the somite level at which it forms: limbs in the anterior flank resemble wings, whereas those in the posterior flank resemble legs. Ectopic limbs located in the mid-flank appear chimeric, possessing characteristics of both wings and legs; feather buds are present in the anterior halves with scales and claws in the posterior halves. To study the mechanisms underlying the chimerism of these additional limbs, chick Tbx5 and Tbx4 were cloned to use as forelimb and hindlimb markers and their expression patterns were examined in FGF-induced limb buds. Tbx5 and Tbx4 are two vertebrate T-box genes whose expression is predominantly restricted to the forelimb and hindlimb buds respectively. Tbx5 and Tbx4 are differentially expressed in the anterior and posterior halves of additional limb buds in the mid-flank, respectively, consistent with the chimeric patterns of the integument. A boundary of Tbx5/Tbx4 exists in all ectopic limbs, indicating that the additional limbs are essentially chimeric, although the degree of chimerism is dependent on the position. The boundary of Tbx5/Tbx4 expression is not fixed at a specific position within the interlimb region, but is dependent on where FGF is applied. Since the ectopic expression patterns of Tbx5/Tbx4 in the additional limbs are closely correlated with the patterns of their chimeric phenotypes, it is likely that Tbx5 and Tbx4 expression in the limb bud is involved in determination of the forelimb and hindlimb identities, respectively, in vertebrates (Ohuchi, 1998).

Signals from the apical ectodermal ridge (AER) of the developing vertebrate limb, including fibroblast growth factor-8 (FGF-8), can maintain limb mesenchymal cells in a proliferative state. CD44 has been considered to be a hyaluronan receptor. A specific CD44 splice variant is crucial for the proliferation of these mesenchymal cells. Epitopes carried by this variant colocalize temporally and spatially with FGF-8 in the AER throughout early limb development. A splice variant containing the same sequences expressed on model cells binds both FGF-4 and FGF-8 and stimulates mesenchymal cells in vitro. When applied to the AER, an antibody against a specific CD44 epitope blocks FGF presentation and inhibits limb outgrowth. Therefore, CD44 is necessary for limb development and functions in a novel growth factor presentation mechanism likely relevant in other physiological and pathological situations where a cell surface protein presents a signaling molecule to a neighboring cell. Thus CD44 is required for presentation of Fgf-8 to its receptor, rather than serving as a hyaluronan receptor (Sherman, 1998).

Fibroblast Growth Factors (FGFs) are signaling molecules that are important in patterning and growth control during vertebrate limb development. Beads soaked in FGF-1, FGF-2 and FGF-4 are able to induce additional limbs when applied to the flank of young chick embryos. However, biochemical and expression studies suggest that none of these FGFs is the endogenous signal that initiates limb development. During chick limb development, Fgf-8 transcripts are detected in the intermediate mesoderm and subsequently in the prelimb field ectoderm prior to the formation of the apical ectodermal ridge, structures required respectively for limb initiation and outgrowth. Later on, Fgf-8 expression is restricted to the ridge cells and expression disappears when the ridge regresses. Application of FGF-8 protein to the flank induces the development of additional limbs. FGF-8 can replace the apical ectodermal ridge to maintain Sonic hedgehog expression and outgrowth and patterning of the developing chick limb. Continuous and widespread misexpression of FGF-8 causes limb truncations and skeletal alterations with phocomelic or achondroplasia phenotype. Thus, FGF-8 appears to be a key signal involved in initiation, outgrowth and patterning of the developing vertebrate limb (Vogel, 1996).

Members of the fibroblast growth factor (FGF) family have been identified as signaling molecules in a variety of developmental processes, including important roles in limb bud initiation, growth and patterning. This paper reports the cloning and characterization of the chicken orthologues of fibroblast growth factor homologous factors-1 and -2 (cFHF-1/cFGF-12 and cFHF-2/cFGF-13, respectively). The FHFs lack a classical signal sequence and contain clusters of basic residues that can act as nuclear localization signals. The FHFs also differ biochemically, with affinities for heparin that are lower than those reported for other FGFs. This suggests that the FHFs may differ from other FGFs in their interactions with the extracellular matrix components. The identification of a novel, conserved isoform of FHF-2 in chickens and mammals is also described. This isoform arises by alternative splicing of the first exon of the FHF-2 gene and is predicted to encode a polypeptide with a distinct amino-terminus. Whole-mount in situ hybridization reveals restricted domains of expression of cFHF-1 and cFHF-2 in the developing neural tube, peripheral sensory ganglia and limb buds, and shows that the two cFHF-2 transcript isoforms are present in non-overlapping spatial distributions in the neural tube and adjacent structures. In the developing limbs, cFHF-1 is confined to the posterior mesoderm in an area that encompasses the zone of polarizing activity and cFHF-2 is confined to the distal anterior mesoderm in a region that largely overlaps the progress zone. Ectopic cFHF-2 expression is induced adjacent to grafts of cells expressing Sonic Hedgehog. The zone of cFHF-2 expression is expanded in talpid2 embryos, in which the expression of all known components downstream of Shh shows a loss of anterior-posterior symmetry. In the absence of the apical ectodermal ridge or in wingless or limbless mutant embryos, expression of cFHF-1 and cFHF-2 is lost from the limb bud. A role for cFHF-2 in the patterning and growth of skeletal elements is implied by the observation that engraftment of developing limb buds with QT6 cells expressing a cFHF-2 isoform that is normally expressed in the limb leads to a variety of morphological defects. A secreted version of cFHF-2 activates the ectopic expression of HoxD13, HoxD11, Fgf-4 and BMP-2, consistent with cFHF-2 playing a role in anterior-posterior patterning of the limb. Whether these FGFs interact with known FGF receptors or whether they are actually nuclear awaits further experimentation (Munoz-Sanjuan, 1999).

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. 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 induce an ectopic AER in the dorsal median ectoderm. These 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 (Yonei-Tamura, 1999).

Experiments have been carried out to investigate the role of the apical ectodermal ridge (AER) and FGF-4 on the control of cell migration during limb bud morphogenesis. By coupling vital cell labeling with ectopic implantation of FGF-4 microcarrier beads, it has been found that FGF-4 acts as a potent and specific chemoattractive agent for mesenchymal cells of the limb bud. The response to FGF-4 is dose dependent in both the number of cells stimulated to migrate and the distance migrated. The cell migration response to FGF-4 appears to be independent of the known inductive activity of FGF-4 on Shh gene expression. The role of the AER in controlling cell migration was investigated by characterizing the migration pattern of DiI-labeled subapical cells during normal limb outgrowth and following partial AER removal. Subapical cells within 75 micrometers of the AER migrate to make contact with the AER and are found intermingled with nonlabeled cells. Thus, the progress zone is dynamic, with cells constantly altering their neighbor relationships during limb outgrowth. AER removal studies show that cell migration is AER dependent and that subapical cells redirect their path of migration toward a functional AER. These studies indicate that the AER has a chemoattractive function and regulates patterns of cell migration during limb outgrowth. The results suggest that the chemoattractive activity of the AER is mediated in part by the production of FGF-4 (Li, 1999).

Fibroblast growth factors (FGFs) mediate multiple developmental signals in vertebrates. Several of these factors are expressed in limb bud structures that direct patterning of the limb. FGF4 is produced in the apical ectodermal ridge (AER) where it is hypothesized to provide mitogenic and morphogenic signals to the underlying mesenchyme that regulate normal limb development. Mutation of this gene in the germline of mice results in early embryonic lethality, preventing subsequent evaluation of Fgf4 function in the AER. A conditional mutant of Fgf4, based on site-specific Cre/loxP-mediated excision of the gene, allows a bypass of embryonic lethality and allows a direct test of the role of FGF4 during limb development in living murine embryos. This conditional mutation is designed so that concomitant with inactivation of the Fgf4 gene by excision of all Fgf4-coding sequences, a reporter gene is activated in Fgf4-expressing cells, allowing assessment of the site-specific recombination reaction. Although a large body of evidence led to a prediction that ablation of Fgf4 gene function in the AER of developing mice would result in abnormal limb outgrowth and patterning, Fgf4 conditional mutants have normal limbs. Furthermore, expression patterns of Shh, Bmp2, Fgf8 and Fgf10 are normal in the limb buds of the conditional mutants. These findings indicate that the previously proposed FGF4-SHH feedback loop is not essential for coordination of murine limb outgrowth and patterning. Some of the roles currently attributed to FGF4 during early vertebrate limb development may be performed by other AER factors in vivo. The simplest hypothesis is that another FGF compensates for the absence of FGF4 in the conditional mutants and provides a functionally equivalent signal to the underlying mesenchyme to support continued limb outgrowth and patterning. FGF1, FGF2 and FGF8 can each support limb outgrowth in the absence of the AER (Moon, 2000).

Vertebrate limbs develop in a temporal proximodistal sequence, with proximal regions specified and generated earlier than distal ones. Whereas considerable information is available on the mechanisms promoting limb growth, those involved in determining the proximodistal identity of limb parts remain largely unknown. Retinoic acid (RA) is an upstream activator of the proximal determinant genes Meis1 and Meis2. RA promotes proximalization of limb cells and endogenous RA signaling is required to maintain the proximal Meis domain in the limb. RA synthesis and signaling range, which initially span the entire lateral plate mesoderm, become restricted to proximal limb domains by the apical ectodermal ridge (AER) activity following limb initiation. Fibroblast growth factor (FGF) has been identified as the main molecule responsible for this AER activity and a model is proposed integrating the role of FGF in limb cell proliferation, with a specific function in promoting distalization through inhibition of RA production and signaling (Mercader, 2000).

The progress zone (PZ) model currently explains how limb cells acquire their PD identity in the PZ and become increasingly distalized with time. The first requirement for distalization would be sufficient cell divisions in the PZ. FGFs, as factors essential for PZ cell proliferation, are required for limb cell distalization, and different FGFs can induce the development of a complete limb from embryo flanks triggering the whole limb developmental program, including its distalization. A specific molecular mechanism by which FGFs could regulate limb distalization has not, however, been demonstrated until now. It is suggested that FGFs promote limb distalization by counteracting the RA pathway, which is essential to maintain the proximalizing Meis activity in the limb. This FGF activity is achieved by at least two different effects on the RA pathway: inhibition of RA synthesis by repressing Raldh2, and parallel direct inhibition of RA signaling, resulting in inactivation of Meis and other RA targets. As long as AER function is not affected, neither Meis activation, nor RA, inhibit limb growth during the PZ-dependent phase. The role of FGF in repressing RA/Meis pathway therefore does not appear to be related to the promotion of cell proliferation, but rather to a specific function in promoting distalization. In contrast, strong Meis activation in the AER or high RA doses applied directly beneath it, destroy the AER and lead to limb truncations. The AER thus appears to be a structure especially sensitive to RA/Meis activity. The strong expression of the RA-degrading enzyme Cyp26 in the cells delimiting the AER may preserve it from RA/Meis pathway activation during early stages of limb development. Whereas FGF signaling might be the principal and primary signal involved in Meis restriction, other diffusible molecules such as BMP and Wnt, which can also inhibit Meis expression, are likely to cooperate in this role (Mercader, 2000).

Much of what is currently known about digit morphogenesis during limb development is deduced from embryonic studies in the chick. Ex utero surgical procedures have been used to study digit morphogenesis during mouse embryogenesis. These studies reveal some similarities; however, considerable differences have been found in how the chick and the mouse autopods respond to experimentation. (1) Ectopic digit formation from interdigital cells could not be induced as a result of wounding or TGFbeta-1 application in the mouse, in contrast to what is observed in the chick. (2) FGF4, which inhibits the formation of ectopic digits in the chick, induces a digit bifurcation response in the mouse. This bifurcation response results from a reorganization of the prechondrogenic tip of the digit rudiment. The FGF4 effect on digit morphogenesis correlates with changes in the expression of a number of genes, including Msx1, Igf2, and the posterior members of the HoxD cluster. In addition, the bifurcation response is digit-specific, being restricted to digit IV. It is proposed that FGF4 is an endogenous signal essential for skeletal branching morphogenesis in the mouse. This work stresses the existence of major differences between the chick and the mouse in how digit morphogenesis is regulated and is thus consistent with the view that vertebrate digit evolution is a relatively recent event (Ngo-Muller, 2000).

The absence of an FGF4-induced distal bifurcation response in digits II or III is significant and suggests a characteristic unique to digit IV. One aspect unique to digit IV in the mouse is that it is the first autopodial condensation to appear, followed in sequence by digits III, II, V, and I. A simple explanation would be that this FGF4 bifurcation response is stage-specific; however, this hypothesis was tested and an FGF4-induced bifurcation response in other digits at later stages was not found. The unique propensity of digit IV to undergo a branching event is consistent with the idea that branching events that are important for limb patterning are spatially restricted during development. It has been proposed that the vertebrate limb skeletal pattern forms through a hierarchical sequence of chondrogenic branching and segmentation events. The evolution of the diverse morphologies of the vertebrate limb is thought to result from spatial-temporal changes in the pattern of these branching and segmentation events. The hypothetical axis from which branching events arise is called the metapterygial axis and the identification of this primitive axis has remained a mystery. It is generally thought that the metapterygial axis runs along the proximal-distal axis on the posterior side of the limb in the proximal region. However, the distal placement of this primitive axis has been variously hypothesized to run through any of the digits, between digits, or curving from posterior to anterior in congruence with the digital arch. The evidence supporting one hypothesized metapterygial axis over another is largely based on comparative limb morphologies rather than on direct experimentation. One way to interpret digit IV-specific bifurcation is to consider the data with respect to this view of sequential bifurcations of the metapterygial axis. It is proposed that the metapterygial axis can be experimentally defined based on its propensity toward chondrogenic branching events and that the bifurcation data indicate that digit IV represents the distal path of this axis in the mouse. Anatomical data placing the distal portion of the metapterygial axis in digit IV has been reported in classical studies. This model for digit bifurcation also supports the idea that the metapterygial axis is defined by the properties of the ectoderm rather than the mesenchyme. An important implication of this model is that it places FGF4 signaling in a key position to direct skeletal branching during limb morphogenesis and, as such, playing a defining role in the evolution of vertebrate digit diversity (Ngo-Muller, 2000).

During limb development, several signaling centers organize limb pattern. One of these, the apical ectodermal ridge (AER), is critical for proximodistal limb outgrowth mediated by FGFs. Signals from the underlying mesoderm, including WNTs and FGFs, regulate early steps of AER induction. Ectodermal factors, particularly En1, play a critical role in regulating morphogenesis of a mature, compact AER along the distal limb apex, from a broad ventral ectodermal precursor domain. Contribution of mesodermal factors to the morphogenesis of a mature AER is less clear. The chick T gene (Brachyury), the prototypical T-box transcription factor, is expressed in the limb bud as well as axial mesoderm and primitive streak. T is expressed in lateral plate mesoderm at the onset of limb bud formation and subsequently in the subridge mesoderm beneath the AER. Retroviral misexpression of T in chick results in anterior extension of the AER and subsequent limb phenotypes consistent with augmented AER extent and function. Analysis of markers for functional AER in mouse T-/- null mutant limb buds reveals disrupted AER morphogenesis. These data also suggest that FGF and WNT signals may operate both upstream and downstream of T. During limb induction, WNT signals maintain high Fgf10 expression in prospective limb and FGF10 activates ectodermal Wnt3a and Fgf8 expression, initiating AER formation. AER signals subsequently also maintain mesodermal Fgf10 expression. T transcripts are first clearly detected at stage 15, at the onset of Wnt3a and Fgf8 activation in the ectoderm. Both the ability of WNT3a and FGF8 to induce T expression, and the ability of T to increase subridge expression of Fgf10 early after misexpression suggest that T may be a component of the mesodermal response to developing AER signals that maintains high Fgf10 apically and thereby also maintains the forming AER, establishing a regulatory loop between ectoderm and mesoderm. Taken together, the results show that T plays a role in the regulation of AER formation, particularly maturation, and suggest that T may also be a component of the epithelial-mesenchymal regulatory loop involved in maintenance of a mature functioning AER (Liu, 2003).

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