Table of contents

WNT homologs and limb patterning

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 or 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, 1997).

Expression and mutation analyses in mice suggest that the homeobox-containing gene 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).

Classical embryological experiments have demonstrated that dorsal-ventral patterning of the vertebrate limb is dependent on ectodermal signals. One such factor is Wnt-7a, a member of the Wnt family of secreted proteins, which is expressed in the dorsal ectoderm. Loss of Wnt-7a results in the appearance of ventral characteristics in the dorsal half of the distal limb. Conversely, En-1, a homeodomain transcription factor, is expressed exclusively in the ventral ectoderm, where it represses Wnt-7a. En-1 mutants have dorsal characteristics in the ventral half of the distal limb. Experiments in the chick suggest that the dorsalizing activity of Wnt-7a in the mesenchyme is mediated through the regulation of the LIM-homeodomain transcription factor Lmx-1. The relationship between Wnt-7a, En-1 and Lmx-1b, a mouse homolog of chick Lmx-1, is examined in the patterning of the mammalian limb. Wnt-7a is required for Lmx-1b expression in distal limb mesenchyme; Lmx-1b activation in the ventral mesenchyme of En-1 mutants requires Wnt-7a. Consistent with Lmx-1b playing a primary role in dorsalization of the limb, a direct correlation is found between regions of the anterior distal limb in which Lmx-lb is misregulated during limb development and the localization of dorsal-ventral patterning defects in Wnt-7a and En-1 mutant adults. Thus, ectopic Wnt-7a expression and Lmx-1b activation underlie the dorsalized En-1 phenotype, although this analysis also reveals a Wnt-7a-independent activity for En-1 in the repression of pigmentation in the ventral epidermis. Ectopic expression of Wnt-7a in the ventral limb ectoderm of En-1 mutants results in the formation of a second, ventral apical ectodermal ridge (AER) at the junction between Wnt-7a-expressing and nonexpressing ectoderm. Unlike the normal AER, ectopic AER formation is dependent upon Wnt-7a activity, indicating that distinct genetic mechanisms may be involved in primary and secondary AER formation (Cygan, 1997).

It appears that the interaction between Wingless and Apterous in limb compartmentalization is evolutionarily conserved. During vertebrate limb development, the ectoderm directs the dorsoventral patterning of the underlying mesoderm. To define the molecular events involved in this process, an analysis has been made of the function of WNT7a, a secreted factor expressed in the dorsal ectoderm, and LMX1, a LIM homeodomain transcription factor expressed in the dorsal mesenchyme. Ectopic expression of Wnt7a is sufficient to induce and maintain Lmx1 expression in limb mesenchyme, both in vivo and in vitro. Ectopic expression of Lmx1 in the ventral mesenchyme is sufficient to generate double-dorsal limbs. Thus, the dorsalization of limb mesoderm appears to involve the WNT7a-mediated induction of Lmx1 in limb mesenchymal cells (Riddle, 1995).

The positional cues that govern the fate of cells along the dorsoventral axis of the developing vertebrate limb are established in the mesoderm before outgrowth of limb buds. apterous, a Drosophila LIM/-homeodomain gene expressed in the dorsal compartment of the wing disc, specifies dorsal cell fate. A vertebrate LIM-homeodomain containing gene, Chick Lmx1 (C-Lmx1), is expressed in the presumptive dorsal limb mesoderm and is restricted thereafter to the dorsal mesoderm of the developing chick bud. C-Lmx1 expression is regulated by the overlying ectoderm where Wnt7a messenger RNA is localized. Wnt7a, required for normal development of the dorsoventral axis in mouse limbs, can induce ectopic expression of C-Lmx1 in ventral mesoderm. Misexpression of C-Lmx1 during limb outgrowth causes ventral to dorsal transformations of limb mesoderm. This paper proposes that C-Lmx1 specifies dorsal cell fate during chick limb development (Vogel, 1995).

Mouse engrailed-1, expressed in embryonic ventral limb ectoderm, is essential for ventral limb patterning. Loss of En-1 results in dorsal transformations of ventral paw structures, and in subtle alterations along the proximal-distal limb axis. EN-1, a murine homolog of Drosophila Engrailed, seems to act in part by repressing dorsal differentiation induced by Wnt-7a, and is essential for proper formation of the apical epidermal ridge (Loomis, 1996).

The apical ectodermal ridge (AER), a rim of thickened ectodermal cells at the interface between the dorsal and ventral domains of the limb bud, is required for limb outgrowth and patterning. The limbs of En1 mutant mice display dorsal-ventral and proximal-distal abnormalities, the latter being reflected in the appearance of a broadened AER and formation of ectopic ventral digits. A detailed genetic analysis of wild-type, En1 and Wnt7a mutant limb buds during AER development has delineated a role for En1 in normal AER formation. These studies support previous suggestions that AER maturation involves the compression of an early broad ventral domain of limb ectoderm into a narrow rim at the tip and show that En1 plays a critical role in the compaction phase. Loss of En1 leads to a delay in the distal shift and stratification of cells in the ventral half of the AER. At later stages, this often leads to development of a secondary ventral AER, which can promote formation of an ectopic digit. The second AER forms at the juxtaposition of the ventral border of the broadened mutant AER and the distal border of an ectopic Lmx1b expression domain. Analysis of En1/Wnt7a double mutants demonstrates that the dorsalizing gene Wnt7a is required for the formation of the ectopic AERs in En1 mutants and for ectopic expression of Lmx1b in the ventral mesenchyme (Loomis, 1998).

En1 is not required for the process of AER induction or the initial stages of AER formation. Loss of En1 function has no apparent effect on the early thickening of the pre-AER ventral ectoderm or on the initial induction of AER marker genes, such as Dlx2 and FGF8. Cells of the dorsal AER of En1 mutants, like those of wild type limbs, do not express Wnt7a and they stratify and initiate Fgf4 expression, an AER differentiation marker. In contrast, differentiation of the ventral portion of the AER, where En1 is normally expressed, appears to be delayed and abnormal in En1 mutants. At a time when a mature AER is apparent in wild-type limbs, the ventral portion of the En1 mutant AER remains extended with the anterior region being much broader than the posterior. A model is suggested whereby, in En1 mutants, ectopic ventral Wnt7a and/or Lmx1b expression leads to the transformation of ventral cells in the broadened AER to a more dorsal phenotype. This leads to induction of a second zone of compaction ventrally, which in some cases goes on to form an autonomous secondary AER. It is proposed that in normal AER development, cells which give rise to the mouse AER initially overlie much of the presumptive limb mesoderm, and that sequential, convergent morphogenetic movements are required for normal ridge formation. A first wave of lateral morphogenetic movements results in the compaction of the AER precursor cells onto the ventral ectodermal thickening. A second wave compresses this domain to the distal 1/3 of the ventral limb, and a final wave constricts the cells into the densely packed AER. In En1 mutants the final wave is markedly inhibited and a secondary compaction process is initiated at the ventroproximal border of the widened mutant AER. The normal final wave of ectodermal movements resembles the closing of a zipper. This process initiates posteriorly and proceeds anteriorly, bringing the ventral domain of the wild-type AER into close proximity to the dorsal domain, which is anchored at the D-V interface. In En1 mutant limbs, the anatomically ventral AER cells take on dorsal characteristics, initiating a secondary AER (Loomis, 1998).

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.

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).

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).

A tight loop between members of the fibroblast growth factor and the Wnt families plays a key role in the initiation of vertebrate limb development. Tbx5 and Tbx4 are directly involved in this process. When dominant-negative forms of these Tbx genes were misexpressed in the chick prospective limb fields, a limbless phenotype arises with repression of both Wnt and Fgf genes. By contrast, when Tbx5 and Tbx4 are misexpressed in the flank an additional wing-like and an additional leg-like limbs are induced, respectively. This additional limb formation is accompanied by the induction of both Wnt and Fgf genes. These results highlight the pivotal roles of Tbx5 and Tbx4 during limb initiation, specification of forelimb/hindlimb and evolution of tetrapod limbs, placing Tbx genes at the center of a highly conserved genetic program (Takeuchi, 2003).

The data reveal that Tbx5 and Tbx4 specifically regulate Wnt2b and Wnt8c (see Drosophila Wnt8), respectively, to initiate limb outgrowth in the early stages of development. In the later stages, Tbx5 and Tbx4 exert different actions to form distinct forelimb and hindlimb structures, respectively. These indicate that these genes play distinct roles with distinct specificity. Nonetheless, Tbx5 and Tbx4 are derived from the same ancestral gene. During evolution, these genes have diversified their biological functions to regulate different Wnt genes and make different limb structures. This is related to the observation that EnTbx5 and EnTbx4 (dominant negative proteins) failed to repress Wnt8c in the leg and Wnt2b in the wing. As expected, misexpression of EnTbx5 in the leg and EnTbx4 in the wing does not affect limb development. This suggests that Tbx5 and Tbx4 have acquired different target specificities during evolution (Takeuchi, 2003).

The epithelial b variant of Fgfr2 is active in the entire surface ectoderm of the early embryo, and later in the limb ectoderm and AER, where it is required for limb outgrowth. Since limb buds do not form in the absence of Fgfr2, chimera analysis was used to investigate the mechanism of action of this receptor in limb development. ES cells homozygous for a loss-of-function mutation of Fgfr2 that carry a ß-galactosidase reporter were aggregated with normal pre-implantation embryos. Chimeras with a high proportion of mutant cells do not form limbs, whereas those with a moderate proportion form limb buds with a lobular structure and a discontinuous AER. Where present, the AER do not contain mutant cells, although mutant cells localize to the adjacent surface ectoderm and limb mesenchyme. In the underlying mesenchyme of AER-free areas, cell proliferation is reduced, and transcription of Shh and Msx1 is diminished. En1 expression in the ventral ectoderm is discontinuous and exhibits ectopic dorsal localization, whereas Wnt7a expression is diminished in the dorsal ectoderm but remains confined to that site. En1 and Wnt7a are not expressed in non-chimeric Fgfr2-null mutant embryos, revealing that they are downstream of Fgfr2. In late gestation chimeras, defects presented in all three limb segments as bone duplications, bone loss or ectopic outgrowths. It is suggested that Fgfr2 is required for AER differentiation, as well as for En1 and Wnt7a expression. This receptor also mediates signals from the limb mesenchyme to the limb ectoderm throughout limb development, affecting the position and morphogenesis of precursor cells in the dorsal and ventral limb ectoderm, and AER (Gorivodsky, 2003).

The cellular and molecular bases allowing tissue regeneration are not well understood. By performing gain- and loss-of-function experiments of specific members of the Wnt pathway during appendage regeneration, it has been demonstrated that this pathway is not only necessary for regeneration to occur, but it is also able to promote regeneration in axolotl, Xenopus, and zebrafish. Furthermore, it has been shown that changes in the spatiotemporal distribution of β-catenin in the developing chick embryo elicit apical ectodermal ridge and limb regeneration in an organism previously thought not to regenerate. The detailed mechanism by which Wnt overexpression in ectodermal cells adjacent to the amputation entails both AER regeneration in the chick, as well as AEC formation in the axolotl/zebrafish/Xenopus, remains to be elucidated. Perhaps, and more importantly, some of the results discussed in this paper -- specifically (1) the observations that changes in Wnt and BMP activities during limb/fin regeneration-limb development induced alterations in the formation of the AEC-AER that are related to spatiotemporal deregulation of p63, and (2) the accomplishment of AER regeneration and subsequent limb development in an embryo not previously shown to have this capability -- support the notion that variations in the concentration and/or spatiotemporal distribution of molecules involved in tissue generation during embryogenesis may be the raw material upon which evolution has granted some animals the ability to regenerate. Understanding the mechanisms responsible for the deployment and fine-tuning of developmental regulators might constitute the basis for inducing tissue regeneration in adult nonregenerating animals (Kawakami, 2006).

The vertebrate limb is a classical model for understanding patterning of three-dimensional structures during embryonic development. Although decades of research have elucidated the tissue and molecular interactions within the limb bud required for patterning and morphogenesis of the limb, the cellular and molecular events that shape the limb bud itself have remained largely unknown. This study shows that the mesenchymal cells of the early limb bud are not disorganized within the ectoderm as previously thought but are instead highly organized and polarized. Using time-lapse video microscopy, it was demonstrated that cells move and divide according to this orientation. The combination of oriented cell divisions and movements drives the proximal-distal elongation of the limb bud necessary to set the stage for subsequent morphogenesis. These cellular events are regulated by the combined activities of the WNT and FGF pathways. WNT5A/JNK is necessary for the proper orientation of cell movements and cell division. In contrast, the FGF/MAPK signaling pathway, emanating from the apical ectodermal ridge, does not regulate cell orientation in the limb bud but instead establishes a gradient of cell velocity enabling continuous rearrangement of the cells at the distal tip of the limb. Together, these data shed light on the cellular basis of vertebrate limb bud morphogenesis and uncover new layers to the sequential signaling pathways acting during vertebrate limb development (Gros, 2010).

WNTs and urogenital development

The vertebrate urogenital system forms due to inductive interactions between the Wolffian duct, its derivative the ureteric bud, and their adjacent mesenchymes. These establish epithelial primordia within the mesonephric (embryonic) and metanephric (adult) kidneys and the Müllerian duct, the anlage of much of the female reproductive tract. Wnt9b is expressed in the inductive epithelia and is essential for the development of mesonephric and metanephric tubules and caudal extension of the Müllerian duct. Wnt9b is required for the earliest inductive response in metanephric mesenchyme. Further, Wnt9b-expressing cells can functionally substitute for the ureteric bud in these interactions. Wnt9b acts upstream of another Wnt, Wnt4, in this process, and the data implicate canonical Wnt signaling as one of the major pathways in the organization of the mammalian urogenital system. Together these findings suggest that Wnt9b is a common organizing signal regulating diverse components of the mammalian urogenital system (Carroll, 2005).

WNTs and gonadal development

Genes previously implicated in mammalian sexual development have either a male- or female-specific role. The signaling molecule WNT4 has been shown to be important in female sexual development. Lack of Wnt4 gives rise to masculinization of the XX gonad, and WNT4 inhibits endothelial and steroidogenic cell migration into the developing ovary. Wnt4 also has a function in the male gonad. Sertoli cell differentiation is compromised in Wnt4 mutant testes and this defect occurs downstream of the testis-determining gene Sry but upstream of Sox9 and Dhh, two early Sertoli cell markers. Genetic analysis shows that this phenotype is primarily due to the action of WNT4 within the early genital ridge. Analysis of different markers identifies the most striking difference in the genital ridge at early stages of its development between wild-type and Wnt4 mutant embryos to be a significant increase of steroidogenic cells in the Wnt4 -/- gonad. These results identify WNT4 as a new factor involved in the mammalian testis determination pathway and show that genes can have a specific but distinct role in both male and female gonad development (Jeay-Ward, 2004).

WNT homologs and external genitalia

Coordinated growth and differentiation of the genital tubercle (GT), an embryonic anlage of external genitalia, generates the proximodistally elongated structure suitable for copulation, erection, uresis and ejaculation. Despite recent progress in molecular embryology, few attempts have been made to elucidate the molecular developmental processes of external genitalia formation. Bone morphogenetic protein genes (Bmp genes) and their antagonists are spatiotemporally expressed during GT development. Exogenously applied BMP increases apoptosis of GT and inhibits its outgrowth. The distal urethral epithelium (DUE), distal epithelia marked by the Fgf8 expression, may control the initial GT outgrowth. Exogenously applied BMP4 downregulates the expression of Fgf8 and Wnt5a, concomitant with increased apoptosis and decreases cell proliferation of the GT mesenchyme. Furthermore, noggin mutants and Bmpr1a conditional mutant mice display hypoplasia and hyperplasia of the external genitalia respectively. noggin mutant mice exhibited downregulation of Wnt5a and Fgf8 expression with decreased cell proliferation. Consistent with such findings, Wnt5a mutant mice display GT agenesis with decreased cell proliferation. By contrast, Bmpr1a mutant mice display decreased apoptosis and augmented Fgf8 expression in the DUE associated with GT hyperplasia. These results suggest that some of the Bmp genes could negatively affect proximodistally oriented outgrowth of GT with regulatory functions on cell proliferation and apoptosis. The DUE region can be marked only until 14.0 dpc (days post coitum) in mouse development, while GT outgrowth continues thereafter. Possible signaling crosstalk among the whole distal GT regions were also investigated (Suzuki, 2003).

Wnts and uterine development

Epithelial-mesenchymal interactions play a crucial role in the correct patterning of the mammalian female reproductive tract (FRT). Three members of the Wnt family of growth factors are expressed at high levels in the developing FRT in the mouse embryo. The expression of Wnt genes is maintained in the adult FRT, although levels fluctuate during estrous. Wnt4 is required for Müllerian duct initiation, whereas Wnt7a is required for subsequent differentiation. Wnt5a is required for posterior growth of the FRT. It has been demonstrated, using grafting techniques, that the mutant FRT has the potential to form the posterior compartments of the FRT. Postnatally, Wnt5a plays a crucial role in the generation of uterine glands and is required for cellular and molecular responses to exogenous estrogens. Wnt5a participates in a regulatory loop with other FRT patterning genes including Wnt7a, Hoxa10 and Hoxa11. Data presented provide a mechanistic basis for how uterine stroma mediates both developmental and estrogen-mediated changes in the epithelium and demonstrate that Wnt5a is a key component in this process. The similarities of the Wnt5a and Wnt7a mutant FRT phenotypes to those described for the Hoxa11 and Hoxa13 mutant FRT phenotypes reveal a mechanism whereby Wnt and Hox genes cooperate to pattern the FRT along the anteroposterior axis (Mericskay, 2004).

WNT homologs and placentation defects

Wnt2 is expressed in the early heart field of 7.5-8.5 dpc (days post-coitum) mouse embryos, making Wnt2 a potentially useful gene marker for the early stages of heart development. No heart defects, however, are observed in Wnt2 knockouts. Expression is also detected in the allantois from 8.0 dpc and at later stages in the placenta and umbilicus. Mice deficient in Wnt2, generated by gene targeting, display runting and approximately 50% died perinatally. Histological analysis reveals alterations in the size and structure of placentas from these mice from 14.5 dpc. The placental defects are associated primarily with the labyrinthine zone and included oedema and tissue disruption and accumulation of maternal blood in large pools. There is also an apparent decrease in the number of foetal capillaries and an increase in the amount of fibrinoid material in the Wnt2 mutant placentas. These results suggest that Wnt2 is required for the proper vascularisation of the mouse placenta and the placental defects in Wnt2-deficient mice result in a reduction in birthweight and perinatal lethality (Monkley, 1996).

Wnt signaling and stem cells

Hematopoietic stem cells (HSCs) have the ability to renew themselves and to give rise to all lineages of the blood; however, the signals that regulate HSC self-renewal remain unclear. The Wnt signalling pathway has an important role in this process. Overexpression of activated ß-catenin expands the pool of HSCs in long-term cultures by both phenotype and function. Furthermore, HSCs in their normal microenvironment activate a LEF-1/TCF reporter, which indicates that HCSs respond to Wnt signalling in vivo. To demonstrate the physiological significance of this pathway for HSC proliferation it was shown that the ectopic expression of axin or a frizzled ligand-binding domain, inhibitors of the Wnt signalling pathway, leads to inhibition of HSC growth in vitro and reduced reconstitution in vivo. Furthermore, activation of Wnt signalling in HSCs induces increased expression of HoxB4 and Notch1, genes previously implicated in self-renewal of HSCs. It is concluded that the Wnt signalling pathway is critical for normal HSC homeostasis in vitro and in vivo; this study provides insight into a potential molecular hierarchy of regulation of HSC development (Reya, 2003).

The observation that CD45+ stem cells injected into the circulation participate in muscle regeneration raised the question of whether CD45+ stem cells resident in muscle play a physiological role during regeneration. CD45+ cells cultured from uninjured muscle are uniformly nonmyogenic. However, CD45+ cells purified from regenerating muscle readily give rise to determined myoblasts. The number of CD45+ cells in muscle rapidly expands following injury, and a high proportion enter the cell cycle. Investigation of candidate pathways involved in embryonic myogenesis reveal that Wnt signaling is sufficient to induce the myogenic specification of muscle-derived CD45+ stem cells. Moreover, injection of the Wnt antagonists sFRP2/3 into regenerating muscle markedly reduces CD45+ stem cell proliferation and myogenic specification. These data therefore suggest that mobilization of resident CD45+ stem cells is an important factor in regeneration after injury and highlight the Wnt pathway as a potential therapeutic target for degenerative neuromuscular disease (Polesskaya, 2003).

Canonical Wnt signaling ameliorates aging of intestinal stem cells

Although intestinal homeostasis is maintained by intestinal stem cells (ISCs), regeneration is impaired upon aging. This study first uncover changes in intestinal architecture, cell number, and cell composition upon aging. Second, a decline was identified in the regenerative capacity of ISCs upon aging because of a decline in canonical Wnt signaling (see Drosophila Wingless) in ISCs. Changes in expression of Wnts are found in stem cells themselves and in their niche, including Paneth cells and mesenchyme. Third, reactivating canonical Wnt signaling enhances the function of both murine and human ISCs and, thus, ameliorates aging-associated phenotypes of ISCs in an organoid assay. These data demonstrate a role for impaired Wnt signaling in physiological aging of ISCs and further identify potential therapeutic avenues to improve ISC regenerative potential upon aging (Nalapareddy, 2017).

Wnt homologs and organ development

Development of the metanephric kidney requires the concerted interaction of two tissues, the epithelium of the ureteric duct and the metanephric mesenchyme. Signals from the ureter induce the metanephric mesenchyme to condense and proliferate around the ureter tip, reciprocal signals from the mesenchyme induce the ureter tip to grow and to branch. Wnt genes encode secreted glycoproteins, which are candidate mediators of these signaling events. There are three Wnt genes with specific, non-overlapping expression patterns in the metanephric kidney, Wnt-4, Wnt-7b and Wnt-11. Wnt-4 is expressed in the condensing mesenchyme and the comma- and S-shaped bodies. Wnt-7b is expressed in the collecting duct epithelium from 13.5 days post coitum onward. Wnt-11 is first expressed in the nephric duct adjacent to the metanephric blastema prior to the outgrowth of the ureteric bud. Wnt-11 expression in Danforth's short-tail mice suggests that signaling from the mesenchyme may regulate Wnt-11 activation. During metanephric development, Wnt-11 expression is confined to the tips of the branching ureter. Maintenance of this expression is independent of Wnt-4 signaling and mature mesenchymal elements in the kidney. Moreover, Wnt-11 expression is maintained in recombinants between ureter and lung mesenchyme suggesting that branching morphogenesis and maintenance of Wnt-11 expression are independent of metanephric mesenchyme-specific factors. Interference with proteoglycan synthesis by using chlorate to interfer with the sulphation of polysaccharides leads to loss of Wnt-11 expression in the ureter tip. It suggested that Wnt-11 acts as an autocrine factor within the ureter epithelium and that its expression is regulated at least in part by proteoglycans. In Drosophila, wingless is expressed in the Malpighian tubules and is required for proliferation in the morphogenesis of this organ. Wnt-11 is likely to play an important role in regulating development of the metanephric kidney, possibly in the branching morphogenesis of the ureter epithelium. Thus there may be an evolutionarily conserved function of Wnt genes in the ductal epithelium during development of excretory structures (Kispert, 1996).

Elongation and branching of epithelial ducts is a crucial event during the development of the mammary gland. Branching morphogenesis of the mouse mammary epithelial TAC-2 cell line was used as an assay to examine the role of Wnt, HGF, TGF-beta, and the Notch receptors in branching morphogenesis. Wnt-1 induces the elongation and branching of epithelial tubules, like HGF and TGF-beta2, and strongly cooperates with either HGF or TGF-beta2 in this activity. Wnt-1 displays morphogenetic activity in TAC-2 cells as it induces branching even under conditions that normally promote cyst formation. The Notch4(int-3) mammary oncoprotein, an activated form of the Notch4 receptor, inhibits the branching morphogenesis normally induced by HGF and TGF-beta2. The minimal domain within the Notch4(int-3) protein required to inhibit morphogenesis consists of the CBF-1 interaction domain and the cdc10 repeat domain. Coexpression of Wnt-1 and Notch4(int-3) demonstrates that Wnt-1 can overcome the Notch-mediated inhibition of branching morphogenesis. These data suggest that Wnt and Notch signaling may play opposite roles in mammary gland development, a finding consistent with the convergence of the wingless and Notch signaling pathways found in Drosophila (Uyttendaele, 1998).

The murine female reproductive tract differentiates along the anteroposterior axis during postnatal development. This process is marked by the emergence of distinct cell types in the oviduct, uterus, cervix and vagina and is dependent on specific mesenchymal-epithelial interactions as demonstrated by earlier heterografting experiments. Members of the Wnt family of signaling molecules have been recently identified in this system and an early functional role in reproductive tract development has been demonstrated. Mice were generated using ES-mediated homologous recombination for the Wnt-7a gene. Since Wnt-7a is expressed in the female reproductive tract, the developmental consequences of lack of Wnt-7a in the female reproductive tract was examined. The oviduct is found to lack a clear demarcation from the anterior uterus, and it acquires several cellular and molecular characteristics of the uterine horn. The uterus acquires cellular and molecular characteristics that represent an intermediate state between normal uterus and vagina. Normal vaginas have a stratified epithelium and normal uteri have a simple columnar epithelium, however, mutant uteri have stratified epithelium. Additionally, Wnt-7a mutant uteri do not form glands. The changes observed in the oviduct and uterus are accompanied by a postnatal loss of hoxa-10 and hoxa-11 expression, revealing that Wnt-7a is not required for early hoxa gene expression, but is required for maintenance of expression. These clustered hox genes have been shown to play a role in anteroposterior patterning in the female reproductive tract. In addition to this global posterior shift in the female reproductive tract, the uterine smooth muscle was found to be disorganized, indicating development along the radial axis is affected. Changes in the boundaries and levels of other Wnt genes are detectable at birth, prior to changes in morphologies. These results suggest that a mechanism exists whereby Wnt-7a signaling from the epithelium maintains the molecular and morphological boundaries of distinct cellular populations along the anteroposterior and radial axes of the female reproductive tract (Miller, 1998).

An important feature of mammalian development is the generation of sexually dimorphic reproductive tracts from the Mullerian and Wolffian ducts. In females, Mullerian ducts develop into the oviduct, uterus, cervix and upper vagina, whereas Wolffian ducts regress. In males, testosterone promotes differentiation of Wolffian ducts into the epididymis, vas deferens and seminal vesicle. The Sertoli cells of the testes produce Mullerian-inhibiting substance, which stimulates Mullerian duct regression in males. The receptor for Mullerian-inhibiting substance expressed by mesenchymal cells underlying the Mullerian duct which are thought to mediate regression of the duct. Mutations that inactivate either Mullerian-inhibiting substance or its receptor allow development of the female reproductive tract in males. These pseudohermaphrodites are frequently infertile because sperm passage is blocked by the presence of the female reproductive system. Male mice lacking the signaling molecule Wnt-7a fail to undergo regression of the Mullerian duct as a result of the absence of the receptor for Mullerian-inhibiting substance. Wnt7a-deficient females are infertile because of abnormal development of the oviduct and uterus, both of which are Mullerian duct derivatives. Therefore, it is proposed that signaling by Wnt-7a allows sexually dimorphic development of the Mullerian ducts (Parr, 1998).

In the mammalian embryo, both sexes are initially morphologically indistinguishable: specific hormones are required for sex-specific development. Mullerian inhibiting substance and testosterone secreted by the differentiating embryonic testes results in the loss of female (Mullerian) or promotion of male (Wolffian) reproductive duct development, respectively. The signaling molecule Wnt-4 is crucial for female sexual development. At birth, sexual development in males with a mutation in Wnt-4 appears to be normal; however, Wnt-4-mutant females are masculinized: the Mullerian duct is absent while the Wolffian duct continues to develop. Wnt-4 is initially required in both sexes for formation of the Mullerian duct. Subsequently, Wnt-4 in the developing ovary appears to suppress the development of Leydig cells; consequently, Wnt-4-mutant females ectopically activate testosterone biosynthesis. Wnt-4 may also be required for maintenance of the female germ line. Thus, the establishment of sexual dimorphism is under the control of both local and systemic signals (Vainio 1999).

Female reproductive hormones control mammary gland morphogenesis. In the absence of the progesterone receptor (PR) from the mammary epithelium, ductal side-branching fails to occur. This defect can be overcome by ectopic expression of the protooncogene Wnt-1. Transplantation of mammary epithelia from Wnt-4-/- mice shows that Wnt-4 has an essential role in side-branching early in pregnancy. PR and Wnt-4 mRNAs colocalize to the luminal compartment of the ductal epithelium. Progesterone induces Wnt-4 in mammary epithelial cells and is required for increased Wnt-4 expression during pregnancy. Although Wnt-4 is the only Wnt gene directly induced by progesterone, it is not unique in its ability to trigger side-branching, since late in pregnancy, the ductal epithelium of Wnt-4-/- mutants shows normal side-branching. It is speculated that this compensation is due to the expression of other Wnt proteins later in pregnancy, consistent with the notion that various Wnt proteins trigger similar biochemical responses and that their different biological functions are due to differences in their patterns of expression. It is concluded that Wnt signaling is essential in mediating progesterone function during mammary gland morphogenesis (Brisken, 2000).

During mammalian development, the Cdx1 homeobox gene exhibits an early period of expression when the embryonic body axis is established, and a later period where expression is restricted to the embryonic intestinal endoderm. Cdx1 expression is maintained throughout adulthood in the proliferative cell compartment of the continuously renewed intestinal epithelium, the crypts. In this study, evidence in vitro and in vivo is provided that Cdx1 is a direct transcriptional target of the Wnt/beta-catenin signaling pathway. Upon Wnt stimulation, expression of Cdx1 can be induced in mouse embryonic stem (ES) cells as well as in undifferentiated rat embryonic endoderm. Tcf4-deficient mouse embryos show abrogation of Cdx1 protein in the small intestinal epithelium, making Tcf4 the likely candidate to transduce Wnt signal in this part of gut. The promoter region of the Cdx1 gene contains several Tcf-binding motifs, and these bind Tcf/Lef1/beta-catenin complexes and mediate beta-catenin-dependent transactivation. The transcriptional regulation of the homeobox gene Cdx1 in the intestinal epithelium by Wnt/beta-catenin signaling underlines the importance of this signaling pathway in mammalian endoderm development (Lickert, 2000).

The signalling molecule WNT4 has been associated with sex reversal phenotypes in mammals. The role of WNT4 in gonad development is shown to pattern the sex-specific vasculature and to regulate steroidogenic cell recruitment. Vascular formation and steroid production in the mammalian gonad occur in a sex-specific manner. During testis development, endothelial cells migrate from the mesonephros into the gonad to form a coelomic blood vessel. Leydig cells differentiate and produce steroid hormones a day later. Neither of these events occurs in the XX gonad. WNT4 represses mesonephric endothelial and steroidogenic cell migration in the XX gonad, preventing the formation of a male-specific coelomic blood vessel and the production of steroids. In the XY gonad, Wnt4 expression is downregulated after sex determination. Transgenic misexpression of Wnt4 in the embryonic testis does not inhibit coelomic vessel formation but vascular pattern is affected. Leydig cell differentiation is not affected in these transgenic animals and the data implies that Wnt4 does not regulate steroidogenic cell differentiation but represses the migration of steroidogenic adrenal precursors into the gonad. These studies provide a model for understanding how the same signalling molecule can act on two different cell types to coordinate sex development (Jeays-Ward, 2003).

To assess the critical role of Wnt signals in intestinal crypts, transgenic mice were generated ectopically expressing Dickkopf1 (Dkk1), a secreted Wnt inhibitor. Epithelial proliferation is greatly reduced coincidentally with the loss of crypts. Although enterocyte differentiation appears unaffected, secretory cell lineages are largely absent. Disrupted intestinal homeostasis is reflected by an absence of nuclear ß-catenin, inhibition of c-myc expression, and subsequent up-regulation of p21CIP1/WAF1. Thus, these data are the first to establish a direct requirement for Wnt ligands in driving proliferation in the intestinal epithelium, and also define an unexpected role for Wnts in controlling secretory cell differentiation (Pinto, 2003).

Oncogenic targets of Wnts

WISP-1 (Wnt-1 induced secreted protein 1) is a member of the CCN family of growth factors. This study identifies WISP-1 as a beta-catenin-regulated gene that can contribute to tumorigenesis. The promoter of WISP-1 was cloned and shown to be activated by both Wnt-1 and beta-catenin expression. TCF/LEF sites play a minor role, whereas the CREB site played an important role in this transcriptional activation. WISP-1 demonstrates oncogenic activities; overexpression of WISP-1 in normal rat kidney fibroblast cells (NRK-49F) induces morphological transformation, accelerates cell growth, and enhances saturation density. Although these cells did not acquire anchorage-independent growth in soft agar, they readily form tumors in nude mice, suggesting that appropriate cellular attachment is important for signaling oncogenic events downstream of WISP-1. Taken together, these data suggest that beta-catenin is able to regulate downstream events through multiple factors, in addition to the TCF/LEF family members. The heterogeneity of genetic elements regulated by Wnt-1 and beta-catenin also suggests the functional diversity of the Wnt-signaling pathway (Xu, 2000).

Wnt signaling plays a critical role in embryonic development, and genetic aberrations in this network have been broadly implicated in colorectal cancer. This study found that the Wnt receptor Frizzled2 (Fzd2; see Drosophila Frizzled) and its ligands Wnt5a/b are elevated in metastatic liver, lung, colon, and breast cancer cell lines and in high-grade tumors and that their expression correlates with markers of epithelial-mesenchymal transition (EMT). Pharmacologic and genetic perturbations reveal that Fzd2 drives EMT and cell migration through a previously unrecognized, noncanonical pathway that includes Fyn and Stat3 (see Drosophila Src42A and Stat92E). A gene signature regulated by this pathway predicts metastasis and overall survival in patients. An antibody was developed to Fzd2 that reduces cell migration and invasion and inhibits tumor growth and metastasis in xenografts. It is proposed that targeting this pathway could provide benefit for patients with tumors expressing high levels of Fzd2 and Wnt5a/b (Gujral, 2014).

A Wnt/ß-Catenin --> Pitx2 pathway controls the turnover of Pitx2 and other unstable mRNAs

The Wnt/β-catenin pathway rapidly induces the transcription of the cell-type-restricted transcription factor Pitx2 that is required for effective cell-specific proliferation activating growth-regulating genes. Pitx2 mRNA displays a rapid turnover rate and activation of the Wnt/β-catenin pathway stabilizes Pitx2 mRNA as well as other unstable mRNAs, including c-Jun, Cyclin D1, and Cyclin D2, encoded by critical transcriptional target genes of the same pathway. The data indicate that Pitx2 mRNA stabilization is due to a reduced interaction of Pitx2 3'UTR with the destabilizing AU-rich element (ARE) binding proteins (BPs) KSRP and TTP as well as to an increased interaction with a stabilizing ARE-BP, HuR [ELAV-like 1 (Hu antigen R)]. Pitx2 itself is a mediator of Wnt/β-catenin-induced mRNA stabilization. These previous and present data support the hypothesis that a single pathway can coordinately regulate sequential transcriptional and posttranscriptional events leading to an integrated functional gene regulatory network (Briata, 2003).

Many tissue-restricted transcription factors mediate crucial steps during development, functioning as the distal targets of different classes of regulatory signaling pathways. One of these, the Wnt signaling cascade, controls organogenesis by inducing a wide range of responses from cell proliferation to cell fate determination and terminal differentiation. Extracellular Wnt signals activate the cytoplasmic protein Dishevelled (Dvl) that, in turn, inhibits the constitutive proteasomal destruction of β-catenin. As a result, β-catenin accumulates in the nucleus, associates with TCF/LEF transcription factors, and TCF/LEF target genes become transiently activated (Briata, 2003).

Pitx2 gene, which encodes a transcription factor belonging to the bicoid family and exerts a crucial role during mammalian development, is a LEF1 target gene. A Wnt/Dvl/β-catenin->Pitx2 pathway has been described that mediates cell-type-specific proliferation during cardiac outflow tract and pituitary gland development. Once induced by Wnt signaling, Pitx2 is required for cell-type-specific proliferation and directly activates specific growth-regulating genes, such as Cyclin D1, Cyclin D2, and c-Myc. The rapid induction of Pitx2 by Wnt as well as the observation that the expression of Pitx2 is tightly regulated in time and space during development, led to the hypothesis that additional levels at which Pitx2 expression can be modulated might exist (Briata, 2003 and references therein).

It is a rising concept that most genes are regulated by multiple mechanisms, the sum of which dictates the unique expression pattern of a gene under certain conditions. Several examples suggest that gene transcription and mRNA degradation rates are coordinately regulated to allow temporal modulation of gene expression. The rate of mRNA turnover not only determines the rate of disappearance of mRNA but also its induction. mRNAs with short half-lives respond to changes in transcription more rapidly than those that are relatively stable, thus contributing to rapid changes in the pattern of cellular gene expression in response to changing environmental or developmental cues. Inherently unstable mRNAs include those encoding oncoproteins, cytokines, and cell cycle-regulated proteins. Furthermore, during early Drosophila development, several genes undergo dramatic changes in abundance and in spatial distribution. To achieve these rapid changes, multiple regulatory mechanisms exist that include both transcriptional control and regulation of mRNA processing (Briata, 2003 and references therein).

Rapid degradation of mRNAs requires at least three components, (1) an instability element, such as the adenylate/uridylate-rich element (ARE) located in the 3'-untranslated region (UTR); (2) certain ARE binding proteins (ARE-BPs), and (3) an enzyme, the exosome. RNA decay is compromised by the removal of any of these components. AREs have been recognized as potent destabilizing elements in a wide variety of short-lived mRNAs. AREs are grouped into three classes according to their sequence features and RNA decay characteristics. Class I AREs contain 1 to 3 scattered copies of the pentanucleotide AUUUA embedded within a U-rich region, and are found in the c-Fos and c-Myc mRNAs. Class II AREs contain multiple overlapping copies of the AUUUA motif, and are found in cytokine mRNAs. Class III AREs, such as the one in c-Jun mRNA, lack the hallmark AUUUA pentanucleotide but present U-rich sequences. Some ARE-BPs have been proven to possess destabilizing activity on ARE-RNAs (TTP, BRF1, KSRP), while another (HuR) has been demonstrated to stabilize target transcripts. AUF1 has a dual role in ARE-mediated mRNA decay, functioning either as a destabilizing or a stabilizing factor depending on the cell type. The described ARE-BPs are required for regulation of class I and II ARE-RNAs. However, it is unclear whether the same ARE-BPs are involved in the control of class III ARE-RNAs. The exosome is a multisubunit particle, containing nine 3'-to-5' exoribonucleases and some ARE-BPs, that rapidly degrade ARE-RNAs (Briata, 2003 and references therein).

This study reports an unexpected mechanism controlling Pitx2 gene expression. Pitx2 mRNA is rapidly degraded due to the presence of destabilizing elements in both its coding sequence and 3'UTR. Activation of the Wnt/β-catenin pathway in pituitary cells induces a strong stabilization of Pitx2 mRNA as well as of Cyclins D1 and D2, and c-Jun mRNAs, which are known to be transcriptional targets of the Wnt pathway. Pitx2 mRNA stabilization correlates with a change in the pattern of interaction of both destabilizing and stabilizing ARE-BPs with Pitx2 3'UTR, with Pitx2 itself modulating the turnover of unstable mRNAs subsequent to Wnt/β-catenin activation (Briata, 2003).

A model is proposed that integrates a view of how Wnt signaling can regulate the expression of target genes in pituitary-derived cells. Wnt activation rapidly induces, through LEF1, the transcription of Pitx2 and of additional target genes, including c-Jun, Cyclin D1, and Cyclin D2, through either TCF/LEF or Pitx2. Wnt activation regulates the expression of the same target genes affecting their mRNA turnover rates. Once induced, Pitx2 itself plays a central role in the stabilization of its own transcript and in the turnover control of other unstable transcripts (Briata, 2003).

The results suggest a direct role of Pitx2 in controlling HuR function: (1) LiCl treatment of αT3-1 cells strongly increases cytoplasmic levels of Pitx2; (2) Pitx2 interacts with HuR; (3) a Pitx2 mutant unable to exert cell-type-specific proliferation control does not interact with HuR and does not reconstitute RNA stabilization in αPitx2-immunodepleted S100. Furthermore, this mutant blocks LiCl effect on mRNA stability functioning as a Pitx2 dominant negative in αT3-1 cells. (4) Neither recombinant nor endogenous Pitx2 is able to directly interact with ARE mRNA, ruling out the possibility of an RNA binding-dependent function of Pitx2 in mRNA stabilization. It is tempting to speculate that Pitx2 plays a role in modulating the cytoplasmic concentration of HuR and, consequently, its in vitro and in vivo binding activity to ARE-mRNAs. However, on the basis of the data, it is suggested that Pitx2 regulates additional events that control mRNA stability. Pitx2 immunodepletion from LiCl-treated S100 removes less than 50% of HuR present in the extracts while it causes a complete destabilization of Pitx2 3'UTR RNA. Conversely, complete HuR immunodepletion from LiCl-treated S100 (that removes less than 50% of Pitx2) does not completely destabilize Pitx2 3'UTR RNA. Altogether these results suggest that HuR is not the only target of Pitx2-mediated mRNA stabilization (Briata, 2003).

Regulating a rate-limiting step is an efficient way to control the overall rate of a multistep process. However, there is a limit to the level of regulation that can be achieved by controlling a single step; there is no point in increasing the rate of one step if another soon becomes rate limiting. Therefore, eukaryotes have developed methods to regulate the expression of their proteins at multiple levels in a coordinated fashion. The mechanisms used largely depend on the level of regulation required for proper gene function and are selected through evolution. Genes whose expression must be rapidly and tightly controlled tend to be quickly transcribed and translated, and their mRNAs and proteins have short half-lives. These data add a further level to the definition of a multistep regulation by the Wnt/β-catenin pathway that enhances the expression of selected genes by, at least, two independent and coordinated mechanisms. In a sense, these events convert Pitx2 from a rapidly induced gene to a more stable regulator of cell-type-specific gene function (Briata, 2003).

Table of contents

wingless continued: Biological Overview | Transcriptional regulation |Targets of Activity | Protein Interactions | mRNA Transport | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.