Wnt oncogene analog 2

DEVELOPMENTAL BIOLOGY

The expression patterns of Wnt2 and Wnt3 Wnt/wingless homologs are dynamic during Drosophila embryogenesis. The distribution of Wnt2 transcripts is predominantly segmented, with the additional presence of transcripts in the presumptive gonads. Transcripts of both Wnt2 and Wnt3 appear to be associated with limb primordia in the embryo and may therefore specify limb development. Wnt3 is also expressed in mesodermal and neurogenic regions. The distribution of Wnt3 transcripts in cells of the central nervous system (CNS) during Drosophila embryogenesis suggests that Wnt3 could be involved in CNS development (Russell, 1992).

Wnt2 transcripts are already present at the blastoderm stage of development. During cellularization, the highest levels of transcripts occur in the central region, between approximately 15% and 70% egg length (0% is the posterior pole). During the early stages of germ band extension (stage7), Wnt2 transcripts are visible within at least 12 columnar cells in the anterior wall of the proctodeal primordium. Wnt2 transcripts are detectable in the three thoracic and eight abdominal segments of stage 8 embryos; by stage 9/10, the RNA is restricted to discrete ectodermal patches in the dorsolateral regions of each of these segments. The dorsolateral patches are half a segment in width, but during stage 10 the patches broaden toward the ventral midline and along the anterior-posterior axis. Wnt2 transcripts occur across the anterior half of each segment and in the posterior portion of each segment. Just anterior to the parasegment boundary, there is overlap with wingless expressing cells. However, there is no overlap with the engrailed domain in the posterior portion of each segment. Thus Wnt2 exhibits a segment polarity expression pattern (Russell, 1992).

The appearance of Wnt2 transcripts in two of the gnathal segments is more or less concomitant with the broadening of the dorsolateral patches. In the maxillary and labial segments, many of the cells in which Wnt2 is transcribed lie anterior to the engrailed expression domain. Wnt2 transcripts are also present at low levels as spots in the primordial labrum, the dorsal area of the procephalic lobe, and in the proctodeum. The positions of the Wnt2 transcripts in labral, maxillary and labial segments may correspond to the positions of primordia of the larval mouth part appendages. At early stage 11, Wnt2 transcripts arise in the ventral ectoderm, forming a pattern of spots, two each for each of the thoracic and abdominal segments, arrayed in two longitudinal rows along the entire length of the germ band. The ventral thoracic spots may be within the anterior compartments of leg primordia as inferred from the pattern of Distal-less expression in the thoracic segments. It is also possible that the ventral spots in the abdominal segments are associated with precursors of the abdominal histoblasts, cell clusters that contribute to the larval epidermis, and from which the adult epidermis of the abdominal segments is derived (Russell, 1992).

During germband shortening, Wnt2 message disappears from the gnathal segments and the dorsalateral patches retract away from the ventral midline and shrink along the anterior-posterior axis such that Wnt2 transcripts are no longer detectable within cells of the wingless expression domain. By stage 14, the segmented pattern of Wnt2 transcripts is absent. Wnt2 RNA detectable in the posterior region of stage 13 and stage 14 embryos is apparently associated with the proctodeal opening. During stage 14, Wnt2 message appears in the posterior portion of the presumptive gonads, associated with either germ-line precursor cells, recruited from the pole cells, or the mesodermal cells with which they intermingle to form the embryonic gonads. Wnt2 transcripts are not detectable in pole cells prior to stage 14. From stages 15 onward, Wnt2 RNA is limited to clusters of cells in the anteriormost region of the embryo and to the posterior portion of the presumptive gonads (Russell, 1992).

To determine how Wnt2 might affect male reproductive tract development, sites of Wnt2 expression were identified by in situ hybridization. Wnt2 is expressed in embryos in all mesodermal cells of the gonad, before the mesoderm and germ cells have condensed to form a compact gonad. Wnt2 expression is limited to the posterior mesodermal cells of the gonad late in embryogenesis (Russell, 1992). This late pattern of expression is apparently maintained in the male, as Wnt2 is expressed at the posterior of the pupal gonad, in cells that will become the terminal epithelia. In the male pupal genital disc, Wnt2 is expressed in the epithelial cells at the apical tip of each developing seminal vesicle. This is a refinement of expression that was present earlier in the third instar larval disc of the male genital primordium. Wnt2 is not expressed in the genital disc of the female third instar larva, nor is it expressed during pupation in the developing oviducts, the female structures analogous to the developing seminal vesicles. Thus, Wnt2 expression occurs in sexually dimorphic patterns (Kozopas, 1998).

Effects of mutation or deletion

Flies with null mutations in Wnt2 are viable but male sterile. To determine the cause of sterility, testes were dissected from Wnt2 mutant males and examined. With variable penetrance, testes from null mutant males are much smaller than wild-type, with the most severe examples having an abnormal oblong shape, as compared to the wild-type spiral. The null mutant testes do not possess the yellow pigmentation of the testis sheath. Males with the hypomorphic allele Wnt2RJ have a less severe phenotype, in which regions of the sheath produce yellow pigment (Kozopas, 1998).

To determine whether the cell types necessary for spermatogenesis are present in the mutants, the expression of beta-galactosidase was assayed from P-element enhancer trap lines specific for cells of the testis. Expression from the germ-line-specific enhancer trap S346 clearly shows that a germ cell lineage is maintained in the mutants. All of the specialized somatic cells of the testis required for spermatogenesis are also present in the mutants. These include the hub cells, cyst cells, and terminal epithelial cells, as assayed by expression from the markers I72 and 34, respectively. The presence of these cell types, along with the observation that mutant testes occasionally possess motile sperm, suggests that the male sterility of the mutants is not attributable to a failure in the process of spermatogenesis (Kozopas, 1998 and references).

The somatic cells necessary for the structure of the testis are affected in the mutants. These cells form a sheath composed of an outer pigment cell layer, an inner muscle cell layer, and their respective basal laminae. Assaying for expression from the pigment cell marker 365 shows that the testes from null mutant adults completely lack the outermost pigment cell layer, which normally covers both the testis and the seminal vesicles. Expression from the enhancer trap L44a, which marks muscle cells of the testis sheath, reveals that the muscle cell layer is malformed or incomplete in these mutants. Cells in this morphologically aberrant tissue do have a muscle cell identity, as they express muscle myosin. The phenotype of the mutant testis muscle layer varies from a partially complete muscle layer to a tangle of muscle cells at the base of the testis (Kozopas, 1998 and references).

In order to address whether Axin inhibits only Wg, or whether it can function as a more general inhibitor of Wnt signals, an examination was carried out to see whether Axn could interfere with a different Wnt signal. Four Wnt genes have been identified in Drosophila (see The World Wide Web Wnt Window ), but mutations have been described only for two of them: wg and DWnt-2. Loss of DWnt-2 produces a muscle migration defect in the male gonads, resulting in male sterility, and a lack of the characteristic pigment cells that migrate over the male testis. Ovaries are normally not surrounded by pigment cells, but misexpression of DWnt-2 in females can induce ectopic male-specific pigment cells. This dominant phenotype of DWnt-2 misexpression was used to address whether Daxin can block ectopic pigment cell formation. Overexpression of Axn strongly reduces the frequency of DWnt-2-mediated pigment cell formation in ovaries, demonstrating that Axn can block the DWnt-2 signal in addition to the Wg signal. Overexpression of a dominant-negative Pangolin/dTCF lacking the amino-terminal Arm binding domain (DN-dTCF) also blocks the ectopic DWnt-2 induced pigment cells in the ovary (Willert, 1999).

Sequencing of the Drosophila genome has revealed that there are 'silent' homologs of many important gene family members that were not detected by classic genetic approaches. Why have so many homologs been conserved during evolution? Perhaps each one has a different but important function in every system. Perhaps each one works independently in a different part of the body. Or, perhaps some are redundant. This study takes one well known gene family and analyzes how the individual members contribute to the making of one system, the tracheae. There are seven DWnt genes in the Drosophila genome, including wingless. The wg gene helps to pattern the developing trachea but is not responsible for all Wnt functions there. Each one of the seven DWnts was tested in several ways and evidence was found that wg and DWnt2 can function in the developing trachea: when both genes are removed together, the phenotype is identical or very similar to that observed when the Wnt pathway is shut down. DWnt2 is expressed near the tracheal cells in the embryo in a pattern different from wg's, but is also transduced through the canonical Wnt pathway. The seven DWnt genes vary in their effectiveness in specific tissues, such as the tracheae; moreover, the epidermis and the tracheae respond to DWnt2 and Wg differently. It is suggested that the main advantage of retaining a number of similar genes is that it allows more subtle forms of control and more flexibility during evolution (Llimargas, 2001).

DWnt proteins bind as ligands to a family of receptor proteins -- four Frizzled (Fz) homologs in Drosophila, of which Fz and Fz2 are the most important and act through a cascade of genes [e.g., disheveled, armadillo (arm), pangolin] on the nucleus. If, therefore, Wg is the only ligand acting from the outside of the cell on the receptors, the wg^- phenotype should be identical to the phenotype when fz and fz2 are removed -- in some organs, this is so. However, in the trachea, although removal of the two receptor proteins or one of the intracellular proteins in the cascade eliminates all dorsal trunk (DT), removing only Wg leaves some DT intact. Therefore, it seems that another molecule, presumably a DWnt, acts through the canonical Wnt pathway to build DT. This study asks which DWnt is responsible (Llimargas, 2001).

Overexpression of wg or other downstream elements of the Wnt pathway in the tracheal cells results in increased DT at the expense of the ventral branch. To investigate further, each one of the seven DWnts was overexpressed locally in the embryonic trachea in a normal background. Overexpression of five DWnt genes (DWnt5, -4, -6, -8, and -10) had no detectable effects; indeed, the flies were viable, fertile, and seemed normal. This experiment suggests that the tracheae are not particularly sensitive to these five proteins. To check whether these proteins are made properly and can function, they were tested in other assays. DWnt6 and DWnt8 are able to affect tracheal development in a sensitized background. DWnt5 produces a phenotype in the ventral nerve cord when expressed with the neural specific driver 1407Gal4, in agreement with the phenotype produced by a HS-DWnt5. Moreover, protein expression is detected in the tracheae when DWnt5 is expressed in tracheal cells. DWnt4 produces ectopic denticles in the ventral epidermis when overexpressed with armGal4, and the flies die as pharate adults, showing several defects in the wings when crossed to ptcGal4. No noticeable phenotype was found when overexpressing DWnt10 in several structures, and, thus, the activity of this line awaits confirmation. However, DWnt10 together with three other DWnts (DWnt4, -6, and wg) were removed in Df(2L)RF embryos and a phenotype similar to wg^- was found, because there still was some DT. This experiment argues that at least zygotic DWnt 4, -6 and -10 do not have a significant function in the trachea under normal conditions. However, overexpression of DWnt2 locally in the tracheal cells does affect its development in a way similar to that of wg, producing an excess of DT cells and DT material at the expense of the ventral branch. These tracheae are defective; they fail to fill with air and the flies die as embryos and young larvae. This result suggests that both wg and DWnt2 act or can act in the developing trachea (Llimargas, 2001).

The tracheal placodes are specified by stage 10 in a specific part of the dorsal ectoderm and express several markers such as trachealess. The expression of DWnt2 is suggestive: it is expressed close to and dorsal to the tracheal placode by stage 10 and early stage 11 but later disappears (Llimargas, 2001).

The spalt (sal) gene (coding for a transcription factor) is expressed in the dorsal ectoderm, including some tracheal cells, during stage 10 and persists later in those tracheal cells that form the DT. sal is absolutely required for DT formation and is thus a good marker for DT cell identity. The most dorsal cells that express sal also coexpress DWnt2. The pattern of wg expression differs strikingly from that of DWnt2, although both gene products are made near the tracheal cells. In arm mutants, Sal is not expressed in tracheal cells and no DT is formed, suggesting that sal expression in tracheal cells depends on activation of the Wnt pathway. Thus, sal can be induced in the tracheal cells wherever either Wg or DWnt2 proteins are received (Llimargas, 2001).

The above results suggest that wg and DWnt2, made near the tracheal cells, together sponsor DT formation. In wg^- embryos, some DT is still formed. However, the tracheal phenotype of wg^-DWnt2^- embryos is significantly different from that of wg^- embryos: in 40%-45% of hemisegments, the DT is completely missing, and in the remaining 55%-60%, only some reduced and thin DT forms. Interestingly, in practically all hemisegments of Df(2L)RF DWnt2^- embryos, the DT is completely missing, indicating that other DWnts account for these traces of DT. Nevertheless, wg^-DWnt2^- double-mutant embryos are very similar to or indistinguishable from fz^-fz2^- embryos, suggesting that wg and DWnt2 sponsor virtually all DT formation (Llimargas, 2001).

Removal of Wg and DWnt2 proteins (in wg^-DWnt2^- embryos) eliminates detectable expression of sal in the presumptive tracheal cells of the DT, whereas in wg^- embryos, very low levels of sal still can be detected in some embryos. The early expression of sal in the dorsal ectoderm still is observed in both wg^- and wg^-DWnt2^- embryos. In wg^-DWnt2^- embryos, late kni expression in tracheal cells is normal, as is the case in arm mutants. In addition, dpp expression also is normal -- Dpp has been shown to inhibit sal expression by activating kni in tracheal cells. Thus, the lack of sal must be caused by the absence of direct or indirect stimulation by the DWnt pathway and not due to repression by the Dpp pathway (Llimargas, 2001).

Does DWnt2 act through the canonical Wnt pathway? It seems so, because the ectopic effects of DWnt2 protein are blocked in embryos that lack the arm gene. Moreover, in wg^-DWnt2^- embryos, the DT can be substantially rescued by expressing a constitutively active form of Arm in the tracheal cells. Also, the tracheal phenotype of porcupine (por) mutants is very similar to that of wg^-DWnt2^- embryos, indicating that por also might be required for DWnt2 secretion (Llimargas, 2001).

If DWnt2 sponsors at least part of DT formation, one might expect that loss of DWnt2 alone would affect trachea in some noticeable way. Surprisingly, DWnt2^- embryos and larvae have normal trachea and normal expression of sal. However, the flies have reduced viability and the males are sterile (Llimargas, 2001).

In normal embryos, the wg gene is expressed in a row of cells at the rear of the A compartment, whereas DWnt2 is expressed at the front. Wg protein spreads to make a gradient that patterns the anterior compartment. DWnt2 protein is expressed where the concentration of Wg is low or absent; that is where the tracheal placodes form and where the cuticle secretes denticles. Thus, in wg^- embryos, where there is no Wg protein and the denticles are continuous, one might expect the tracheal placodes and DWnt2 expression to form one continuous stripe and, indeed, they do (Llimargas, 2001).

This adventitious expression of DWnt2 in a broad domain in wg^- embryos could compensate at least in part for the lack of wg itself. Indeed, in these embryos, it must be mainly DWnt2 that activates some sal and determines most or all of the DT found. No change could be detected in the pattern of wg RNA or protein distribution in DWnt2 mutants (Llimargas, 2001).

The potency of DWnt2 and Wg in the tracheae was assayed: DWnt2^-wg^- double mutants were taken and each of the two missing proteins were added back in the normal pattern of expression for the wg gene. DWnt2 and Wg were found to both rescue some DT in the trachea; however, only Wg can partially rescue the various embryonic defects in morphology found in wg^- embryos. When either DWnt2 or Wg is expressed locally in the tracheal cells, each gives strong rescue, and more DT is made (Llimargas, 2001).

The DWnt2 gene was also expressed in wild-type embryos either universally and strongly (arm VP16 Gal4) or in stripes (ptcGal4), and in both cases, the tracheae are altered to the same extent as when DWnt2 is expressed in the tracheal cells alone. However, DWnt2 fails to alter the cuticle pattern, whereas wg produces a naked cuticle phenotype. This lack of effect of DWnt2 on the epidermis is remarkable since both the drivers used are strong and, when wg is driven, are more than adequate to make a naked cuticle. Interestingly, when DWnt2 is missexpressed in the eye, it also does not emulate the phenotype produced by missexpression of wg. Moreover, the effects of overexpressing DWnt2 in the ovary are stronger than when overexpressing wg. All these results argue that the tracheal cells and other tissues, including the epidermis, the eye, and the ovary are differentially sensitive to the two DWnt molecules, the trachea and the ovary being particularly responsive to DWnt2 (Llimargas, 2001).

There are several ways this difference could be achieved. Perhaps DWnt2 does not act through the canonical Wnt pathway in some tissues, such as the ectoderm or the eye. Perhaps DWnt2, on its way to the tracheal cells, could be secreted or processed differently. Perhaps the tracheal cells have something that allows efficient presentation of the ligand to the Fz receptors, or they lack a component that, in other tissues, impedes DWnt2 binding or transduction. One possibility is that glucosaminoglycans help breathless (btl, an FGF receptor expressed in tracheal cells) and are needed for Wnt signaling. Maybe Btl helps to gather or modify the heparan sulfate glucosaminoglycans, thereby altering the presentation of DWnt2 to the two receptors, Fz and Dfz2. Whatever the explanation, the tracheal cells are more responsive than other tissues to the DWnt2 signal (Llimargas, 2001).

The other DWnts were examined. DWnts5, -6, -8, and -10 were drived in the epidermis of wild-type embryos with one copy of ptcGal4; none of these affected the cuticle pattern in a noticeable way. Are these DWnts able to affect tracheal development in the wg^-DWnt2^- double mutants? Each of these five DWnts were added back to either the tracheal cells themselves or in the pattern of normal wg expression. DWnts6 and -8 are each able to rescue DT partially, whereas -4, -5 and -10 did not. Note that DWnt6 and DWnt8 are not able to produce a tracheal phenotype when expressed in tracheal cells of normal embryos, but they can do so in a sensitized background (Llimargas, 2001).

The results indicate that wg and DWnt2 make the main contribution to DT formation, as the absence of both genes completely eliminates DT in many cases. However, traces of DT still are formed in about half the hemisegments of wg^-DWnt2^- embryos, indicating that contributions of other genes might help. Also, rescue experiments show that some other DWnts are able to activate the pathway. In agreement with this result, in most Df(2L)RF DWnt2^- embryos, all DT is missing, indicating that DWnt6 and/or -4 and/or -10 can compensate weakly for the absence of wg and DWnt2. However, expression of DWnt6 and DWnt10 does not suggest that they act in tracheal development in the wild type. DWnt4 is expressed in a similar pattern to that of wg but does not seem to assist wg during embryogenesis. In addition, none of DWnt4, -6, or -10 affected tracheal development when expressed in tracheal cells of wild-type embryos. Most likely, they produce traces of DT in the wg^-DWnt2^- embryos, because those embryos offer a very sensitive test of stimulation of the Wnt pathway. It remains unclear whether these DWnts make any residual contribution to DT in the wild type (Llimargas, 2001).

However, several observations suggest that DWnt2 contributes to tracheal development in the wild-type fly. Notably, DWnt2 is expressed near the tracheal cells at the appropriate stage, and when overexpressed in tracheal cells, it mimics the effects of overexpressing wg or a constitutively activated Arm. But, most importantly, the phenotype of wg^-DWnt2^- embryos indicates that wg and DWnt2 together are responsible for virtually all DT formation. Thus, DWnt2 probably cooperates with Wg or reinforces its main action (Llimargas, 2001).

Nevertheless, DWnt2^- embryos do not show a visible tracheal phenotype, indicating, at first sight, that the gene does not normally contribute to DT formation. This lack of abnormality suggests that wg alone (or with some help from different DWnts) is sufficient to sponsor normal development in these mutant flies. Nevertheless, it remains possible that DWnt2 could act in the wild-type. There are at least two alternative hypotheses that could explain the lack of tracheal phenotype in DWnt2^- embryos (Llimargas, 2001).

(1) The loss of DWnt2 could induce compensatory changes in the amount, distribution, or activity of the other DWnts. As in the case of DWnt6, -4, and -10, the expression of DWnt5 and DWnt8 (expression is detected only in the CNS at early stages) does not suggest that they act in tracheal development, although contributions under the level of detection cannot be discarded. Moreover, although some small changes have been detected in the expression of some DWnts in wg^- and wg^-DWnt2^- embryos (e.g., the loss of DWnt5 expression in the labial segment at stage 10 as well as loss of expression in lateral clusters of the thoracic segments at stage 11), no changes have been detected in the pattern of expression that might account for any strong tracheal rescue of DWnt2^- embryos. Therefore, it is not clear how other DWnts could contribute to the complete DT formation in DWnt2^- embryos (Llimargas, 2001).

(2) It is supposed that all DWnts bind the receptor with different affinities, with Wg binding most strongly. In the wild type, the DWnts could compete, but Wg would be most effective: the contribution of DWnt2 to DT formation would be minor. However, in embryos lacking Wg, mainly DWnt2 (which is expressed in a broader domain in wg^- embryos and is not now competing with Wg) could bind and partially substitute for Wg. In the absence of DWnt2, Wg (and maybe other DWnts) would have no competition from DWnt2 and would become even more efficient, compensating for the contribution to DT formation that DWnt2 has in the wild type. Finally, in the absence of both Wg and DWnt2, other DWnts, even if they did not act in the wild type, could now bind to the unoccupied receptors and have some tiny effect on DT formation (Llimargas, 2001).

Complications of this kind may bedevil attempts to analyze the precise wild type contributions of individual members of other gene families (Llimargas, 2001).

Thus DWnt2 can act in tracheal development, whereas Wg acts in both developing epidermis and trachea. The other five DWnts do little for the trachea. As with the achaete/scute homologs (which are alike in structure and function but have different patterns of expression and, therefore, act in different places, it may be that the DWnts are preserved fundamentally because seven genes, even if they do similar things, can be regulated in a more sophisticated way than one. Perhaps, like DWnt2, they perform specialized tasks, acting locally to help Wg in ways that could not be provided by any additional regulatory control of wg itself. In the case of tracheae, this tissue can have differential sensitivity to specific homologs, a property that may allow even more intricate forms of control (Llimargas, 2001).

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date revised: 15 March 2002  

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