branchless
FGF and limb patterning (part 2/2) Following amputation of a urodele limb or teleost fin, the formation of a blastema is a crucial step in facilitating subsequent
regeneration. Early caudal fin regenerative events can be separated into four stages.
(1) During the first 12 h, epidermal cells migrate to cover the stump. (2) Within the next 12 h, the wound epidermis thickens, while
fibroblasts and scleroblasts located within one or two bone segments proximal to the amputation plane lose their dense organization and
show signs of distal migration. (3) Next, these mesenchymal cells organize and proliferate to form a blastema, a mass of undifferentiated
tissue, just distal to the ray stumps. (4) During the outgrowth stage, proximal cells of the regeneration blastema differentiate to participate
in bone deposition, while distal cells divide and maintain outgrowth. Using the zebrafish caudal fin regeneration model, the hypothesis that fibroblast growth
factors initiate blastema formation from fin mesenchyme was examined. fibroblast growth factor receptor 1 (fgfr1) is
expressed in mesenchymal cells underlying the wound epidermis during blastema formation and in distal blastemal tissue
during regenerative outgrowth. fgfr1 transcripts colocalize with those of msxb and msxc, putative markers for undifferentiated,
proliferating cells. A zebrafish Fgf member, designated wfgf, is expressed in the regeneration epidermis during
outgrowth. Furthermore, a specific inhibitor of Fgfr1, applied immediately following fin amputation, blocks
blastema formation without obvious effects on wound healing. This inhibitor blocks the proliferation of blastemal cells and
the onset of msx gene transcription. Inhibition of Fgf signaling during ongoing fin regeneration prevents further outgrowth
while downregulating the established expression of blastemal msx genes and epidermal sonic hedgehog. These findings
indicate that zebrafish fin blastema formation and regenerative outgrowth require Fgf signaling (Poss, 2000).
It is proposed that following amputation and wound healing,
mesenchymal cells disorganize and begin to migrate toward the amputation plane. At the epidermal-mesenchymal junction, Fgf molecules
synthesized in the wound epidermis bind to mesenchymal Fgfr1. Signaling by Fgfr1 triggers proliferation and the induction or maintenance
of msxb and msxc expression in these cells, and a blastema forms. During later stages, Wfgf and/or other Fgfs are released from the
distalmost epidermal cells and signal through blastemal Fgfr1 to maintain msxb/c expression and cell division, which promotes outgrowth.
Meanwhile, Fgfs activate Fgfrs in basal layer epidermal cells to maintain shh transcription during outgrowth. Shh released from these cells
is thought to help direct new bone deposition by scleroblasts (Poss, 2000).
Mice homozygous for the recessive limb deformity (ld) mutation display both limb and renal defects.
The limb defects, oligodactyly and syndactyly, have been traced to improper differentiation of the
apical ectodermal ridge (AER) and shortening of the anteroposterior limb axis. The renal defects,
usually aplasia, are thought to result from failure of ureteric bud outgrowth. The ld locus gives rise
to multiple RNA isoforms encoding several different proteins (termed 'formins'). Embryonic
expression patterns of the four major ld mRNA isoforms were examined. Isoforms I, II and III (all containing a basic
amino terminus) are expressed in dorsal root ganglia, cranial ganglia and the developing kidney
(including the ureteric bud). Isoform IV (containing an acidic amino terminus) is expressed in the
notochord, the somites, the apical ectodermal ridge (AER) of the limb bud and the developing kidney, also
including the ureteric bud. Using a lacZ reporter assay in transgenic mice, it has been shown that this
differential expression of isoform IV results from distinct regulatory sequences upstream of its first
exon. These expression patterns suggest that all four isoforms may be involved in ureteric bud
outgrowth, while isoform IV may be involved in AER differentiation. To define further the
developmental consequences of the ld limb defect, the expression of a number of genes
thought to play a role in limb development was examined. Although the AERs of ld limb
buds express several AER markers, they do not express detectable levels of fibroblast growth factor 4
(Fgf-4), which has been proposed to be the AER signal to the mesoderm. Thus it is concluded that one or
more formins are necessary to initiate and/or maintain Fgf-4 production in the distal limb. Since ld limbs
form distal structures such as digits, it has been concluded that while Fgf-4 is capable of supporting distal
limb outgrowth in manipulated limbs, it is not essential for distal outgrowth in normal limb development.
In addition, ld limbs show a severe decrease in the expression of several mesodermal markers,
including Sonic hedgehog, a marker for the polarizing region, and Hoxd-12, a marker for posterior
mesoderm. It is proposed that incomplete differentiation of the AER in ld limb buds leads to reduction of
polarizing activity and defects along the anteroposterior axis (Chan, 1995).
The outgrowth of the mesoderm of the developing limb bud in response to the apical
ectodermal ridge (AER) is mediated at least in part by members of the FGF family.
Recent studies have indicated that FGFs need to interact with heparin sulfate
proteoglycans in order to bind to and activate their specific cell surface receptors.
Syndecan-3 (see Drosophila Syndecan) is an integral membrane heparin sulfate proteoglycan that is highly
expressed by the distal mesodermal cells of the chick limb bud that are undergoing
proliferation and outgrowth in response to the AER. Maintenance
of high-level syndecan-3 expression by the subridge mesoderm of the chick limb bud is
directly or indirectly dependent on the AER, since its expression is severely impaired
in the distal mesoderm of the limb buds of limbless and wingless mutant embryos,
which lack functional AERs capable of directing the outgrowth of limb mesoderm. Exogenous FGF-2 maintains a domain of high-level syndecan-3
expression in the outgrowing mesodermal cells of explants of the posterior mesoderm
of normal limb buds cultured in the absence of the AER and in the outgrowing
subapical mesoderm of explants of limbless mutant limb buds, which lack a functional
AER. These results suggest that the domain of high-level syndecan-3 expression in
the subridge mesoderm of normal limb buds is maintained by FGFs produced by the
AER. Polyclonal antibodies against a syndecan-3 fusion protein
inhibit the ability of FGF-2 to promote the proliferation and outgrowth of the posterior
subridge mesoderm of limb buds cultured in the absence of the AER. These results
suggest that syndecan-3 plays an essential role in limb outgrowth by mediating the
interaction of FGFs produced by the AER with the underlying mesoderm of the limb
bud (Dealy, 1997).
Detailed fate maps have been produced of the mesenchyme and apical ridge found in a stage 20 chick
wing bud. The fate maps of the mesenchyme show that most of the wing arises from the
posterior half of the bud. Subapical mesenchyme gives rise to digits. Cell populations
beneath the ridge in the mid apical region fan out into the anterior tip of the handplate, while
posterior cell populations extend right along the posterior margin. Subapical mesenchyme in
the leg bud behaves similarly. The absence of anterior bending of posterior cell populations
has implications when considering models of vertebrate limb evolution. The fate maps of the
apical ridge show that there is also a marked anterior expansion; cells that were in the
anterior apical ridge later become incorporated into non-ridge ectoderm along the margin of
the bud. Mesenchyme and apical ridge do not expand in concert - the apical ridge extends
more anteriorly. The fate maps were used to investigate the relationship between cell lineage
and elaboration of Hoxd-13 (Drosophila homolog: Abdominal B) and Fgf-4 domains. Hoxd-13 and Fgf-4 are initially expressed
posteriorly until about the mid-point of the early wing bud in mesenchyme and apical ridge
respectively. Later in development, the genes come to be expressed throughout most of the
handplate and apical ridge respectively. At the proximal edge of the Hoxd-13
domain, cell populations stop expressing the gene as development proceeds; no evidence was found that changes in the extent of the domains were due to initiation of gene
expression in anterior cells. Instead the changes in extent of expression fit with the fate
maps and can be attributed to the expansion and fanning out of cell populations initially
expressing the genes (Vargesson, 1997).
The limb muscles, originating from the ventrolateral portion of the somites, exhibit position-specific
morphological development through successive splitting and growth/differentiation of the muscle masses in a
region-specific manner by interacting with the limb mesenchyme and the cartilage elements. The molecular
mechanisms that provide positional cues to the muscle precursors are still unknown. The
expression patterns of Hoxa-11 and Hoxa-13 are correlated with muscle patterning of the limb bud and muscular Hox genes are activated by signals from the limb
mesenchyme. This study examines the regulatory mechanisms directing the unique expression patterns of Hoxa-11
and Hoxa-13 during limb muscle development. HOXA-11 protein is detected in both the myogenic cells
and the zeugopodal mesenchymal cells of the limb bud. The earlier expression of HOXA-11 in both the
myogenic precursor cells and the mesenchyme is dependent on the apical ectodermal ridge (AER), but later
expression is independent of the AER. HOXA-11 expression in both myogenic precursor cells and
mesenchyme is induced by fibroblast growth factor (FGF) signal, whereas hepatocyte growth
factor/scatter factor (HGF/SF) maintains HOXA-11 expression in the myogenic precursor cells, but not in
the mesenchyme. The distribution of HOXA-13 protein expression in the muscle masses is restricted to
the posterior region. HOXA-13 expression in the autopodal mesenchyme is dependent on
the AER but not on the polarizing region, whereas expression of HOXA-13 in the posterior muscle masses
is dependent on the polarizing region but not on the AER. Administration of BMP-2 at the anterior margin
of the limb bud induces ectopic HOXA-13 expression in the anterior region of the muscle masses followed
by ectopic muscle formation close to the source of exogenous BMP-2. In addition, NOGGIN/CHORDIN,
antagonists of BMP-2 and BMP-4, downregulate the expression of HOXA-13 in the posterior region of the
muscle masses and inhibit posterior muscle development. These results suggested that HOXA-13
expression in the posterior muscle masses is activated by the posteriorizing signal from the posterior
mesenchyme via BMP-2. On the contrary, the expression of HOXA-13 in the autopodal mesenchyme is
affected by neither BMP-2 nor NOGGIN/CHORDIN. Thus, mesenchymal HOXA-13 expression is
independent of BMP-2 from the polarizing region, but is under the control of as yet unidentified signals from
the AER. These results show that expression of Hox genes is regulated differently in the limb muscle
precursor and mesenchymal cells (Hashimoto, 1999).
A Japanese chick wingless mutant (Jwg) has been analyzed to elucidate the molecular mechanism underlying
wing development. The expression patterns of eleven marker genes were studied to characterize the
mutant. In Jwg mutants, expression of Fgf8, a marker gene for the apical ectodermal ridge (AER), is
delayed and shortly disappears in the wing as the AER regresses. Likewise, Shh, which is expressed in the posterior mesoderm of the normal chick limb by late stage 18, is considerably weaker in stage 19/20 mutant wing buds; Shh is expressed normally expressed in the posterior mesenchyme of the leg bud of the same mutant embryo. Fgf4 expression, which is normally induced in the posterior domain of the AER by Shh is not detected in the Jwg mutant wing bud at stage 19 and thereafter. Expressions of limb dorsoventral (DV) patterning genes such as Wnt7a and Lmx1 and mesenchymal marker genes such as Msx2 and Lh2 (a LIM homeodomain protein) are intact in nascent Jwg
limb buds. Later in development, ventral expression of dorsal
marker genes Wnt7a and Lmx1 indicate that the wing bud without the AER becomes bi-dorsal. The posterior mesoderm becomes defective, as deduced from the impaired expression patterns
of Sonic hedgehog, Msx1, and Prx1. Rescue of the wing was attempted by implanting
Fgf8-expressing cells into the Jwg wing bud. FGF8 can rescue outgrowth of the wing
bud by maintaining Shh expression. Thus, the Jwg gene seems to be involved in maintenance of the
Fgf8 expression in the wing bud. Further, it is suggested that the AER is required for maintenance of
the DV boundary and the polarizing activity of the established wing bud (Ohuchi, 1997a).
The developmental properties of the polydactylous chicken mutant, talpid(2) have been examined. Ptc, Gli1, Bmp2, Hoxd13, and Fgf4 are expressed throughout the
anteroposterior axis of the mutant limb bud, despite normal Shh expression. The expression of Gli3, Ihh, and Dhh appears to be normal, suggesting that the Shh
signaling pathway is constitutively active in talpid(2) mutants. Preaxial talpid(2) limb bud mesoderm has polarizing activity in the absence of detectable
Shh mRNA. When the postaxial talpid(2) limb bud (including all Shh-expressing cells) is removed, the preaxial cells reform a normal-shaped talpid(2) limb bud. However, a Shh-expressing region (zone of polarizing activity) does not reform; nevertheless Fgf4 expression in the apical ectodermal ridge is maintained.
Such reformed talpid(2) limb buds develop complete talpid(2) limbs. After similar treatment, normal limb buds downregulate Fgf4, the preaxial cells do not reform a normal-shaped talpid(2) limb bud,
and a truncated anteroposterior deficient limb forms. In talpid(2) limbs, distal outgrowth is independent of Shh and correlates with Fgf4, but not Fgf8, expression by
the apical ectodermal ridge. A model for talpid(2) is proposed in which leaky activation of the Shh signaling pathway occurs in the absence of Shh ligand (Caruccio, 1999).
A young tadpole of an anuran amphibian can completely regenerate an amputated limb, and it exhibits an ontogenetic
decline in the ability to regenerate its limbs. However, whether mesenchymal or epidermal tissue is responsible for this
decrease in ability remains unclear. Moreover, little is known about the molecular interactions between these two
tissues during regeneration. The results of this study show that fgf-10 expression in the limb mesenchymal cells clearly
corresponds to the regenerative capacity and that fgf-10 and fgf-8 are synergistically reexpressed in regenerating blastemas.
However, neither fgf-10 nor fgf-8 is reexpressed after amputation of a nonregenerative limb. Nevertheless, nonregenerative
epidermal tissue can reexpress fgf-8 under the influence of regenerative mesenchyme, as was demonstrated by experiments
using a recombinant limb composed of regenerative limb mesenchyme and nonregenerative limb epidermis. Taken
together, these data demonstrate that the regenerative capacity depends on mesenchymal tissue and suggest that fgf-10 is
likely to be involved in this capacity (Yokoyama, 2000).
By reciprocal transplantation experiments with regenerative and nonregenerative
Xenopus limbs, it has been demonstrated that the regenerative capacity of a Xenopus limb depends on mesenchymal tissue and it has been suggested that fgf-10 is likely to be involved in this capacity. The role of FGF-10
in regenerative capacity has been investigated by directly introducing FGF-10 protein into nonregenerative Xenopus limb stumps. Exogenously
applied FGF-10 successfully stimulates the regenerative capacity, resulting in the reinduction of all gene expressions (including shh, msx-1, and fgf-10) examined and the regeneration of well-patterned limb structures. This finding suggests that FGF-10 could be a key molecule in possible regeneration of nonregenerative limbs in higher vertebrates (Yokoyama, 2001).
A regulatory loop between the fibroblast growth factors FGF-8 and FGF-10 plays a key role in limb initiation and AER induction in vertebrate embryos. Three WNT factors signaling through beta-catenin act as key regulators of the FGF-8/FGF-10 loop. The Wnt-2b gene is expressed in the intermediate mesoderm (IM) and the lateral plate mesoderm (LPM) in the presumptive chick forelimb region. Cells expressing Wnt-2b are able to induce Fgf-10 and generate an extra limb when implanted into the flank. In the presumptive hindlimb region, another Wnt gene, Wnt-8c, controls Fgf-10 expression, and is also capable of inducing ectopic limb formation in the flank. Finally, the induction of Fgf-8 in the limb ectoderm by FGF-10 is mediated by the induction of Wnt-3a. Thus, three WNT signals mediated by beta-catenin control both limb initiation and AER induction in the vertebrate embryo (Kawakami, 2001).
Axial tissues medial to the LPM (such as the IM and somites), have been shown to produce factors that initiate limb formation, operating on cells of the LPM to maintain and restrict expression of the Fgf-10 gene. FGF proteins such as FGF-2, FGF-4, and FGF-8, expressed in the IM and the somites adjacent to the limb forming areas, are capable of inducing Fgf-10, and thus they have been postulated as the endogenous inducers of limb initiation. Once Fgf-10 expression has been consolidated and restricted to the LPM of the presumptive limb areas, FGF-10 operates on the overlying surface ectoderm to induce expression of another Fgf gene, Fgf-8. This induction is concomitant with the appearance of the AER, and expression of Fgf-8 in the AER is required for the maintenance of Fgf-10 in the nascent limb mesenchyme and the localization of Shh to the posterior margin of the limb bud. Thus, a regulatory loop between FGFs is established so that FGF-8 (and probably other FGFs) produced by the IM and/or the somites adjacent to the limb forming areas, signal to the LPM to maintain and restrict expression of Fgf-10, which, in turn, induces Fgf-8 in the overlying nascent limb ectoderm. The regulatory loop is completed by FGF-8 (and other FGFs produced in the AER) signaling back to the limb mesenchyme to maintain limb bud outgrowth (Kawakami, 2001 and references therein).
Even though in the last few years this model has constituted an excellent framework for the analysis of limb initiation and AER induction, several interesting problems still remain to be solved; from among these, three were chosen for further study: (1) conflicting reports have been published on the role of the IM in limb initiation, so that the exact contribution of the IM and the somites remains unclear; (2) the relatively long times of induction of Fgf-10 (in the LPM) by FGF-8 and of Fgf-8 (in the ectoderm) by FGF-10 clearly suggest the existence of molecular mediators of the FGF-8/FGF-10 regulatory loop; (3) although FGF-10 appears to mediate initiation of both forelimbs and hindlimbs, it is unclear whether the same upstream mechanism of regulation of Fgf-10 (i.e., induction by FGF-8) operates in both cases. Thus, it has been proposed that FGF-8 may initiate both the forelimb (coming from the IM) and the hindlimb (coming from the primitive streak and other caudal embryonic structures (Kawakami, 2001 and references therein).
The results presented here provide novel insights into all these problems. (1) A limb-inducing gene, Wnt-2b, has been identified that is expressed in the somites, the IM, and the LPM of the forelimb level. This opens the door to further molecular studies aimed at clarifying the exact role of these three tissues (and the genes expressed in them) in forelimb induction. (2) It has been demonstrated that both Wnt-2b and Wnt-8c mediate the FGF-8/FGF-10 regulatory loop that controls limb initiation. Both Wnt-2b (expressed in the forelimb area) and Wnt-8c (expressed in the hindlimb area) act through beta-catenin to control Fgf-10 in the LPM of the prospective limb territories. A beta-catenin dependent activity is a common (and necessary) requirement for both forelimb and hindlimb induction, since antagonism of beta-catenin by Axin severely interferes with early limb development. (3) Another Wnt gene, Wnt-3a, mediates the induction of Fgf-8 in the limb ectoderm by FGF-10. Thus, three Wnt genes that signal through beta-catenin act as key molecular mediators of the FGF regulatory loop that controls both limb initiation and AER induction. Of the many signaling processes of regionalization that operate in the vertebrate embryo, these results also illustrate how a unique signaling mechanism (WNT/beta-catenin), which is essential for limb induction, is triggered by two different WNT ligands at two different locations in the embryo (Kawakami, 2001).
These results allow for the proposal of an expanded model of limb initiation and AER induction in the chick embryo. WNT and FGF signaling pathways interact in a way that ensures the adequate transference of inductive signals between the different tissues involved in these crucial morphogenetic processes. Prior to limb initiation, Fgf-10 is expressed in a wide region that includes the segmental plate (SP), IM and LPM, without any specific restriction to the presumptive limb areas. At limb bud initiation, Fgf-10 expression becomes confined to the LPM of the presumptive limb bud by signals emanating from the axial structures medial to the LPM. Two members of the WNT family, WNT-2B and WNT-8C, contribute to restrict and/or maintain Fgf-10 expression at the appropriate (fore and hindlimb) levels of the LPM. Both WNT-2B and WNT-8C inductive activities are mediated by beta-catenin, whose activity is absolutely required for the maintenance of Fgf-10 expression in the presumptive limb regions. Finally, once limb initiation is underway, and after Fgf-10 expression has been restricted to the LPM that corresponds to the presumptive limb areas, FGF-10 signals to the overlying ectoderm to induce expression of Wnt-3a, which eventually will become restricted to the AER. WNT-3A then signals through beta-catenin to activate Fgf-8 expression. To complete the loop, FGF-8 signals back to the mesenchyme of the nascent limb bud, where it contributes to maintain expression of Fgf-10 and to initiate and/or maintain Shh expression (Kawakami, 2001).
The nuclear factor-kappaB (NF-kappaB) family of transcription factors is involved in proliferation, differentiation, and apoptosis in a stage- and cell-dependent manner. Recent evidence has shown that NF-kappaB activity is necessary for both chicken and mouse limb development. The NF-kappaB family member c-rel and the homeodomain gene msx-1 have partially overlapping expression patterns in the developing chick limb. In addition, inhibition of NF-kappaB activity results in a decrease in msx-1 mRNA expression. Sequence analysis of the msx-1 promoter reveals three potential kappaB-binding sites similar to the interferon-gamma (IFN-gamma) kappaB-binding site. These sites bind to c-Rel, as shown by electrophoretic mobility shift assay. Furthermore, inhibition of NF-kappaB activity significantly reduces transactivation of the msx-1 promoter in response to FGF-2/-4, known stimulators of msx-1 expression. These results suggest that NF-kappaB mediates the FGF-2/-4 signal regulation of msx-1 gene expression (Bushdid, 2001).
Loss of Twist gene function arrests the growth of the limb bud shortly after its formation. In the Twist-/-
forelimb bud, Fgf10 expression is reduced, Fgf4 is not expressed, and the domain of Fgf8 and Fgfr2 expression is altered. This is
accompanied by disruption of the expression of genes (Shh, Gli1, Gli2, Gli3, and Ptch) associated with SHH signaling in the
limb bud mesenchyme, the down-regulation of Bmp4 in the apical ectoderm, the absence of Alx3, Alx4, Pax1, and Pax3 activity in the mesenchyme, and a reduced potency of the limb bud tissues to differentiate into osteogenic and myogenic tissues. Development of the hindlimb buds in Twist-/- embryos is also retarded. The overall activity of genes involved in SHH signaling is reduced. Fgf4 and Fgf8 expression is lost or reduced in the apical ectoderm, but other genes (Fgf10, Fgfr2) involved with FGF signaling are expressed in normal patterns. Twist+/-;Gli3+/XtJ mice display more severe polydactyly than that seen in either Twist+/- or Gli3+/XtJ mice, suggesting that there is genetic interaction between Twist and Gli3 activity. Twist activity is therefore essential for the growth and differentiation of the limb bud tissues as well as regulation of tissue patterning via the modulation of SHH and FGF signal transduction. The finding of the down-regulation of the Gli genes in the Twist mutant limb mesenchyme is concordant with the observation that
the expression of a Gli-related gene (lame duck) is also altered by the loss of Twist function in the Drosophila embryo (O'Rourke, 2002).
Proximal-to-distal growth of the embryonic limbs requires Fgf10 in
the mesenchyme to activate Fgf8 in the apical ectodermal ridge (AER),
which in turn promotes mesenchymal outgrowth. The growth
arrest specific gene 1 (Gas1), coding for a GPI-anchored membrane glycoprotein, is required in the mesenchyme for
the normal regulation of Fgf10/Fgf8. Gas1 mutant limbs have defects
in the proliferation of the AER and the mesenchyme and develop with small
autopods, missing phalanges and anterior digit syndactyly. At the molecular
level, Fgf10 expression at the distal tip mesenchyme immediately
underneath the AER is preferentially affected in the mutant limb, coinciding
with the loss of Fgf8 expression in the AER. To test whether FGF10
deficiency is an underlying cause of the Gas1 mutant phenotype, a limb culture system was employed in conjunction with microinjection of
recombinant proteins. In this system, FGF10 but not FGF8 protein injected into
the mutant distal tip mesenchyme restores Fgf8 expression in the AER.
These data provide evidence that Gas1 acts to maintain high levels of
FGF10 at the tip mesenchyme and support the proposal that Fgf10
expression in this region is crucial for maintaining Fgf8 expression
in the AER (Liu, 2002).
To determine the role of fibroblast growth factor (FGF) signalling from the apical ectodermal ridge (AER), Fgf4 and Fgf8 in AER cells or their precursors were inactivated at different stages of mouse limb development. FGF4 and FGF8 regulate cell number in the nascent limb bud and are required for survival of cells located far from the AER. On the basis of the skeletal phenotypes observed, it is concluded that these functions are essential to ensure that sufficient progenitor cells are available to form the normal complement of skeletal elements, and perhaps other limb tissues. In the complete absence of both FGF4 and FGF8 activities, limb development fails. A model is presented to explain how the mutant phenotypes arise from FGF-mediated effects on limb bud size and cell survival (Sun, 2002).
Transcriptional cascades responsible for initiating the formation of vertebrate embryonic structures such as limbs are not well established. Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations along the anteroposterior axis of the embryo is not known. The T-box transcription factor TBX5 is important for normal heart and limb formation, but its role in early limb development is not well defined. Mouse embryos lacking Tbx5 do not form forelimb buds, although the patterning of the lateral plate mesoderm into the limb field is intact. Tbx5 is not essential for an early establishment of forelimb versus hindlimb identity. In the absence of Tbx5, the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression. Tbx5 directly activates the Fgf10 gene via a conserved binding site, providing a simple and direct mechanism for limb bud initiation. Lef1/Tcf1-dependent Wnt signaling is not essential for initiation of Tbx5 or Fgf10 transcription, but is required in concert with Tbx5 for maintenance of normal levels of Fgf10 expression. It is conclude that Tbx5 is not essential for the early establishment of the limb field in the lateral plate mesoderm but is a primary and direct initiator of forelimb bud formation. These data suggest common pathways for the differentiation and growth of embryonic structures downstream of T-box genes (Agarwal, 2003).
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. An additional phalange can be induced in a chick toe if sonic hedgehog protein is applied in between developing digit primordia. This study shows that formation of an additional phalange is associated with prolonged Fgf8 expression in the overlying apical ridge and that an Fgf receptor inhibitor blocks phalange 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. It is proposed 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. It is also proposed 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 (Sanz-Ezquerro, 2003).
The fact that terminal phalanges are formed by a mechanism that is completely different from that which generates proximal phalanges has not been widely appreciated, despite many observations suggesting that this is so. This study provides experimental evidence reinforcing the idea that there is a special program for making a digit tip. The fact that members of the Wnt signaling pathway, such as Wnt5a, their receptors Frizzleds (e.g., Fz4), and secreted inhibitors (e.g., FrzB) are expressed at digit tips suggests that Wnt signaling is part of this program, and the results suggest that Wnt14 may be involved. Moreover, transgenic mice expressing an inhibitor of Wnt signaling (Dickkopf) in the skin lack nails, consistent with Wnt-Wnt antagonist expression in digit tips being related to nail induction. Interestingly, regeneration of limbs in higher vertebrates is confined to digit tips. Therefore, understanding the molecular basis of tip formation could lead to development of new strategies to enhance the regenerative ability of limbs. Finally, similar mechanisms could also operate in the development of other appendages where a distal Fgf source directs outgrowth, such as genital tubercule, facial primordia, and tail. It is interesting to note that the tail tip also seems to express a special set of genes, suggesting an independent program for its formation (Sanz-Ezquerro, 2003).
In vertebrate limbs that lack webbing, the embryonic interdigit region is
removed by programmed cell death (PCD). Established models suggest that bone
morphogenetic proteins (BMPs) directly trigger such PCD, although no direct
genetic evidence exists for this. Alternatively, BMPs might indirectly affect
PCD by regulating fibroblast growth factors (FGFs), which act as cell survival
factors. The mouse BMP receptor gene Bmpr1a was inactivated
specifically in the limb bud apical ectodermal ridge (AER), a source of FGF
activity. Early inactivation completely prevents AER formation. However,
inactivation after limb bud initiation causes an upregulation of two AER-FGFs,
Fgf4 and Fgf8, and a loss of interdigital PCD leading to
webbed limbs. To determine whether excess FGF signaling inhibits interdigit
PCD in these Bmpr1a mutant limbs, double and triple
AER-specific inactivations of Bmpr1a, Fgf4 and Fgf8 were performed. Webbing
persists in AER-specific inactivations of Bmpr1a and Fgf8
owing to elevated Fgf4 expression. Inactivation of Bmpr1a,
Fgf8 and one copy of Fgf4 eliminates webbing. It is concluded that
during normal embryogenesis, BMP signaling to the AER indirectly regulates
interdigit PCD by regulating AER-FGFs, which act as survival factors for the
interdigit mesenchyme (Pajni-Underwood, 2007).
Crosstalk between the fibroblast growth factor (FGF) and Sonic Hedgehog (Shh) pathways is critical for proper patterning and growth of the developing limb bud. This study shows that FGF-dependent activation of the ETS transcription factors Etv4 and Etv5 contributes to proximal-distal limb outgrowth. Surprisingly, blockage of Etv activity in early distal mesenchyme also resulted in ectopic, anterior expansion of Shh, leading to a polydactylous phenotype. These data indicate an unexpected function for an FGF/Etv pathway in anterior-posterior patterning. FGF activity in the limb is not only responsible for maintaining posterior-specific Shh expression, but it also acts via Etvs to prevent inappropriate anterior expansion of Shh. This study identifies another level of genetic interaction between the orthogonal axes during limb development (Mao, 2009).
These data support a role for an FGF/Etv pathway in repressing Shh expression in the anterior mesenchyme, thereby contributing to the posterior restriction of a Shh-producing organizing center. Based on the temporal pattern of ectopic Shh expression seen in the limb bud after EtvEnR misexpression, it is speculated that endogenous Etv4/5 acts to repress Shh in two distinct domains, one extending from the ZPA across the distal limb bud, and a second, later domain in the anterior of the limb bud. The later timing of ectopic anterior Shh expression is consistent with the fact that in other mutants exhibiting ectopic anterior Shh activity, the anterior domain is observed at a later time in limb development than the normal domain at the posterior margin. Both of these functions of Etv4/5, in the distal and anterior limb, must occur before ~E10.5. Thus, there is an early time window, prior to Shh activation, when all distal mesenchyme is competent to activate Shh but broad activation is blocked by Fgf/Etv4/5 action. This broad competence is then lost shortly thereafter. Whether the loss of competence reflects a change in the intrinsic properties of mesenchyme, or a redundant mechanism for restricting Shh expression to the posterior margin, is unclear (Mao, 2009).
Anterior-posterior (A-P) patterning of the vertebrate limb is controlled by sonic hedgehog (SHH) signaling, and the precise restriction of Shh expression to the posterior limb bud is essential for its polarizing effect. Fibroblast growth factor (FGF) signaling, a key control of proximal-distal (P-D) limb outgrowth, is known to promote Shh expression in the posterior limb bud. This study shows that conditional knockout of the FGF-activated transcription factor genes Etv4 and Etv5 in mouse leads to ectopic Shh expression in the anterior limb bud and a preaxial polydactyly (PPD) skeletal phenotype. These unexpected results suggest that ETV4 and ETV5 act downstream of FGF signaling to inhibit Shh expression in the anterior limb bud. This finding elucidates a novel aspect of the mechanism coordinating limb development along the A-P and P-D axes (Zhang, 2009).
Normal patterning of tissues and organs requires the tight restriction of signaling molecules to well-defined organizing centers. In the limb bud, one of the main signaling centers is the zone of polarizing activity (ZPA) that controls growth and patterning through the production of sonic hedgehog (SHH). The appropriate temporal and spatial expression of Shh is crucial for normal limb bud patterning, because modifications, even if subtle, have important phenotypic consequences. However, although there is a lot of information about the factors that activate and maintain Shh expression, much less is known about the mechanisms that restrict its expression to the ZPA. This study shows that BMP activity negatively regulates Shh transcription and that a BMP-Shh negative-feedback loop serves to confine Shh expression. BMP-dependent downregulation of Shh is achieved by interfering with the FGF and Wnt signaling activities that maintain Shh expression. FGF induction of Shh requires protein synthesis and is mediated by the ERK1/2 MAPK transduction pathway. BMP gene expression in the posterior limb bud mesoderm is positively regulated by FGF signaling and finely regulated by an auto-regulatory loop. These study emphasizes the intricacy of the crosstalk between the major signaling pathways in the posterior limb bud (Bastida, 2009).
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