BIOLOGICAL OVERVIEW

Wnt-4 is 60 kb upstream from wingless and is transcribed from the opposite strand of DNA, away from wingless. Wnt-4 was isolated from a library of genomic fragments that associate with Ultrabithorax proteins, in the hope of discovering genes regulated by homeodomain transcription factors. Expressed early in the head, and in an ectodermal segmental array, Wnt-4 is expressed in the visceral mesoderm later in embryonic development.

In most instances the targets of homeotic proteins are not known. However, Wnt-4 was isolated in a search for genes whose promoters bind homeodomain proteins; happily, investigators isolated a gene regulated by homeodomain proteins. Abdominal-A is directly required for WNT-4 induction, while Ultrabithorax is required indirectly.

Expression of Wnt-4 in the visceral mesoderm coincides with locations where two major morphological events will take place; formation of the gastric caeca (parasegment 4), and formation of the second midgut constriction (between parasegments 7 and 8). This overview will deal with the induction of the second midgut constriction. Ultrabithorax acts indirectly by first stimulating dpp production in parasegment 7. DPP then diffuses and induces Wnt-4 in parasegment 8. abdominal-A is required directly in parasegment 8 for Wnt-4 production.

Wnt-4 is inhibited in the other parasegments of the ventral mesoderm by the actions of abdominal-A, Antennapedia and Ultrabithorax, again demonstrating that Wnt-4 is downstream of homeotic genes. Wnt-4 is not the only target of DPP. DPP from parasegment 7 also induces wingless in parasegment 8. One wonders whether wingless and Wnt-4 share the same enhancer (Bienz, 1994).

Further studies show that indeed Wnt-4 and wingless developmentally interact. wingless and DWnt4 are transcribed in overlapping embryonic territories under the control of the same regulatory molecules. Co-expression and co-regulation suggest first, that the close physical linkage results from the sharing of cis-control elements and second, that the two Wnt signals cooperate in developmental patterning events. Antisense RNA experiments reveal that signaling by DWnt4 is essential for cells from the anterior compartment of each parasegment to adopt a denticled fate. It is proposed that wingless and DWnt4 achieve opposite, but complementary functions in intrasegmental cell patterning of the embryonic ectoderm (Gieseler, 1996).

Antagonist activity of DWnt-4 and wingless in the Drosophila embryonic ventral ectoderm and in heterologous Xenopus assays

To address the question of the functional relationship of wingless and DWnt-4, an examination was made of how embryonic cells respond when they are exposed, whether simultaneously or not, to the encoded Wnt signals. DWnt-4 has the capacity to antagonize Wingless signaling both in the Drosophila ventral epidermis and in a heterologous system, the Xenopus embryo. Evidence indicates that DWnt-4 inhibits the Wingless/Wnt-1 signaling pathway upstream of the activation of transcriptional targets (Gieseler, 1999).

The functional relationship between the two Wnt products was examined by comparing their ability to induce embryonic axis duplication in Xenopus. To do this, different concentrations of mRNA, encoding either Wg or DWnt-4, were injected into the vegetal region of ventral blastomeres of four-cell stage Xenopus embryos. As expected, providing WG mRNA results in the development of a secondary axis. This effect occurs in a dose-dependent manner. Five picograms of the WG message are sufficient to induce in 65% of injected embryos a fully developed supernumerary axis including cement glands, a marker of most dorso-anterior structures. In contrast, injection of as much as 6 ng of DWnt4 mRNA fails to induce embryonic axis duplication. To determine whether DWnt-4 influences the axis-inducing activity of Wg, mRNAs encoding the two Wnt molecules were co-injected into the ventral marginal zone of cleavage-stage embryos. One ng of DWnt-4 message decreases the frequency at which Wg induces a complete ectopic axis, since embryos generally develop an incomplete axis devoid of cement gland. Embryos injected with 2.4 ng of DWnt-4 mRNA never show complete axis duplication. Further increases in the amount of DWnt-4 further inhibits the phenotype induced by Wg; with 6 ng of DWnt-4 mRNA, the formation of a secondary axis is blocked in most embryos. Taken together these results indicate that DWnt-4 antagonizes in a dose-dependent manner the ability of Wg to cause axis duplication. DWnt-4 also counteracts Wnt-1 signaling upstream of the activation of target genes in the Spemann Organizer (Gieseler, 1999).

In contrast with ventral injections, directing DWnt-4 mRNA into the dorsal marginal zone of a four-cell stage Xenopus embryo inhibits formation of the endogenous axis, resulting in the disappearance of the anterior-most dorsal structures. Of the embryos injected with 6 ng of DWnt-4 mRNA, 90% fail to develop head structures including eyes and cement glands. In Xenopus, the formation of axial structures, including the head, requires beta-catenin activity, which results in the activation of Spemann Organizer genes in the dorsal territories of the early gastrula. To investigate the mechanism by which DWnt-4 inhibits dorsal patterning, the expression of chd, gsc, otx2 and Xnr3 were examined. Whole mount in situ hybridization on embryos dorsally injected at the four-cell stage and left to develop until stage 10, show that DWnt-4 does not alter the transcription patterns of the Organizer genes, nor does injection into the ventral marginal zone induce expression at ectopic sites. Thus, unlike products of the Wnt-1 class of genes, DWnt-4 does not have the capacity to influence the transcription of Organizer genes. These results indicate that the repression of dorsal axis formation by DWnt-4 presumably does not result from deficient formation of the Spemann Organizer, but from changes occurring later in dorsal development (Gieseler, 1999).

In the process of patterning the embryonic ventral ectoderm, Wg has at least two temporally distinct roles. A paracrine function of Wg is first required for the maintenance of engrailed (en) transcription in the adjacent posterior two rows of cells. This activity results in the formation and stabilization of parasegmental boundaries (cell-stabilization phase). During this time, Wg is also required for establishment of the diverse array of denticle types within the anterior half of the parasegment. Later wg function is essential for maintaining its own transcription and for specifying the cell fate that secretes naked cuticle within the posterior half of the parasegment (cell-specification phase). To determine whether DWnt-4 can modulate Wg signaling in these processes, the ventral cuticle defects resulting from DWnt-4 overexpression using the UAS/Gal4 system were examined. Several UAS-DWnt-4 transgenic lines were established and crossed with the daughterless (da) Gal4 driver that provides ubiquitous expression of UAS transgenes beginning early in embryogenesis. Embryos bearing one copy of UAS-DWnt-4 transgene show mild but consistent cuticular defects consisting of ectopic denticles within the region of naked cuticle. Posterior segments are always more affected than anterior ones and segmentation and diversity in denticle morphology are not entirely lost. Stronger phenotypes were obtained by increasing DWnt-4 expression through two copies of the UAS-DWnt-4 transgene. Under these conditions, the naked cuticle is often completely lost and replaced with denticle-decorated cuticle, especially in the posterior-most segments. The phenotype produced by ubiquitous DWnt-4 expression is opposite that of the naked cuticle mutant phenotype, in that it is produced by ectopic Wg. Providing DWnt-4 ubiquitously thus results in a phenotype that resembles that induced by loss of wg activity. When wg function is removed throughout embryogenesis, naked cuticle does not form; compartment boundaries are lost and all denticles display similar morphology. However, when wg function is removed at stage 10 in development, naked cuticle is still affected, but not completely lost, while denticle diversity is maintained. The phenotypes observed in da-Gal4-UAS-DWnt-4 embryos resemble the latter, suggesting that DWnt-4 antagonizes Wg activity during the late cell-specification phase reproducibly observed (Gieseler, 1999).

Two series of experiments were carried out to determine whether the phenotypes produced by DWnt-4 in the ventral ectoderm result from a reduction in Wg signal transduction. First, the transcription patterns of Wg target genes were examined. The expression of en marks the activity of Wg during the early phase, while the expression of wg itself marks the late phase of Wg activity through its autoregulation. In stage 9 da-Gal4-UAS-DWnt-4 embryos, expression of the two target genes is not significantly altered. Thus, wg and en presumably still function in the definition and stabilization of parasegmental boundaries; this is consistent with the previous observation that ubiquitously provided DWnt-4 does not abolish the segmental organization of the embryo. In stage 11 embryos, since the expression of wg and en is no longer interdependent and Wg is required for the maintenance of its own transcription, wg expression in the ventral ectoderm becomes severely reduced. Consistent with the stronger defects observed in posterior cuticle, a more pronounced inhibition of wg in posteriormost segments has been reproducibly observed. At the same time, the En protein distribution remains unaffected. Second, it was asked whether DWnt-4 has the ability to influence intracellular accumulation of the Armadillo (Arm)/beta-catenin protein, a key mediator of the Wg/Wnt-1 signaling. Cytoplasmic Arm is stabilized in response to signaling by Wg, allowing its translocation into the nucleus where it acts in association with the Pangolin/dTCF factor, a transcriptional effector of the pathway. Loss of wg function results in a reduction of the amount of Arm, when compared to wild type. DWnt-4 overexpression also induces a clear, though less pronounced, reduction in the levels of Arm. Thus, signaling by DWnt-4 negatively interferes with the Wg signaling pathway upstream of the Arm stabilization step (Gieseler, 1999).

Canonical Wnt signaling in the visceral muscle is required for left-right asymmetric development of the Drosophila midgut

Many animals develop left-right (LR) asymmetry in their internal organs. The mechanisms of LR asymmetric development are evolutionarily divergent, and are poorly understood in invertebrates. Drosophila has several organs that show directional and stereotypic LR asymmetry, including the embryonic gut, which is the first organ to develop LR asymmetry during Drosophila development. This study found that genes encoding components of the Wnt-signaling pathway are required for LR asymmetric development of the anterior part of the embryonic midgut (AMG). frizzled 2 and Wnt4, which encode a receptor and ligand of Wnt signaling respectively, are required for the LR asymmetric development of the AMG. arrow, an ortholog of the mammalian gene encoding low-density lipoprotein receptor-related protein 5/6, which is a co-receptor of the Wnt-signaling pathway, was also essential for LR asymmetric development of the AMG. These results are the first demonstration that Wnt signaling contributes to LR asymmetric development in invertebrates, as it does in vertebrates. The AMG consists of visceral muscle and an epithelial tube. Genetic analyses revealed that Wnt signaling in the visceral muscle but not the epithelium of the midgut is required for the AMG to develop its normal laterality. Furthermore, fz2 and Wnt4 are expressed in the visceral muscles of the midgut. Consistent with these results, it was observed that the LR asymmetric rearrangement of the visceral muscle cells, the first visible asymmetry of the developing AMG, did not occur in embryos lacking Wnt4 expression. These results also suggest that canonical Wnt/β-catenin signaling, but not non-canonical Wnt signaling, is responsible for the LR asymmetric development of the AMG. Canonical Wnt/β-catenin signaling is reported to have important roles in LR asymmetric development in zebrafish. Thus, the contribution of canonical Wnt/β-catenin signaling to LR asymmetric development may be an evolutionarily conserved feature between vertebrates and invertebrates (Kuroda, 2012).

This study found that Wnt-signaling components Wnt4 and Fz2 are required for LR asymmetric development of the AMG, although contribution of other Wnt ligands and receptors to this process could not be excluded. For example, it is known that Wnt4 binds to Fz and Fz2, and that fz and fz2 function redundantly in the segmentation of Drosophila embryos. This study found that the AMG of embryos homozygous for fz showed similar LR defects to those of fz2, although at a lower frequency. Therefore, it is possible that Fz acts redundantly as the receptor for canonical Wnt/β-catenin signaling, although the expression of fz in the midgut could not be detected by anti-Fz antibody staining. In contrast, analysis of embryos homozygous for derailed (drl) suggested that Wnt5 may not be involved in the LR asymmetric development of the AMG. Drl is a member of the RYK subfamily of receptor tyrosine kinases and is a receptor for Wnt5. The laterality of the AMG was normal in embryos homozygous for drl (Kuroda, 2012).

Wnt4 is one of the few Wnt ligands whose function has been revealed in Drosophila. This study found that Wnt4–Fz2 activates the canonical Wnt/β-catenin signaling pathway for normal LR asymmetric development of the AMG. Consistent with this finding, Wnt4 activates the canonical Wnt/β-catenin signaling pathway in salivary glands through Fz or Fz2, which is required for the glands’ proper migration. However, the Wnt4–Fz2 pathway is also known to activate non-canonical Wnt signaling in other systems. Wnt4 plays an essential role in the cell movement required for formation of the ovariolar sheath cells. In addition, Wnt4 expressed in the developing ventral lamina is required for ventral projection of the retinal axon. In both of these cases, Fz2 acts as a receptor of Wnt4, and the Wnt4–Fz2 pathway activates non-canonical Wnt signaling. Therefore, although the same combination of Wnt ligand and receptor, Wnt4–Fz2, is involved, the downstream cascades of Wnt signaling may be context-dependent, although the factors acting as molecular switches for these downstream pathways remain unknown (Kuroda, 2012).

The first indication of LR symmetric morphogenesis in the AMG is observed as the LR asymmetric rearrangement of circular visceral muscle (CVMU) cells. These rearrangements can be monitored by measuring the major axial angle of the nuclei in the CVMU cells to the midline of the AMG (Kuroda, 2012).

This study found that the LR asymmetry of the rearranged CVMU cells in the ventral AMG became bilaterally symmetric in embryos homozygous for a Wnt4 mutation. This result was consistent with the AMG’s random LR laterality in these embryos. However, unexpectedly, the CVMU cells were rearranged LR asymmetrically in the dorsal AMG in Wnt4 mutant homozygotes, even though the arrangement of these dorsal cells is bilaterally symmetric in wild-type embryos. This result suggests that Wnt signaling may counteract the LR asymmetric morphogenesis in the dorsal side of the AMG, in addition to its role in introducing a LR bias by inducing the rearrangement of CVMU cells in the ventral AMG, via the Wnt4–Fz2 pathway. In embryos homozygous for loss-of-function mutations of Wnt4, arr, or fz2, the LR asymmetric development of the posterior embryonic gut was largely normal. Thus, in wild-type embryos, the Wnt4–Fz2 signal may function to suppress the influence of the LR asymmetric morphogenic signals from the posterior midgut on the AMG (Kuroda, 2012).

The present analyses clarified the requirement for Wnt4–Fz2 signaling in the LR asymmetric morphogenesis of the AMG, but the precise molecular functions of this signal are still unclear. Because Wnt4–Fz2 activates canonical Wnt/β-catenin signaling, it will be important to identify the target genes responsible for LR asymmetric morphogenesis of the AMG (Kuroda, 2012).

cDNA clone length - 3201

Exons - two

PROTEIN STRUCTURE

Amino Acids - 389

Structural Domains

WNT-4 has a signal sequence that functions in secretion and a putative N-linked glycosylation site. There are abundant, strongly conserved cysteine residues (Graba, 1995).

Evolutionary Homologies

Extensive information on Wingless homologs and their receptors is to be found at Roel Nusse's World Wide Web Wnt Window (WWWWW).

The WNT-4 sequence is 35% identical to Wingless, 32% to WNT-2 and 33% to WNT-3. WNT-4 is the most divergent of any WNT protein identified to date (Graba, 1995).

Transcriptional Regulation

Antennapedia, Ultrabithorax and Abdominal A repress Wnt-4 in parasegments 5-6, 7 and 9-12, respectively. Both UBX and ABD-A are required to activate Wnt-4 in parasegment 8. (Graba, 1995). UBX's action is indirect. It first activates dpp in parasegment 7, and this in turn stimulates Wnt-4 in parasegment 8. ABD-A's effect is direct. Its expression in segment 8 is required for Wnt-4 induction (Graba, 1995).

Is it possible to estimate the number of target genes of the homeoproteins Eve and Ftz? Eve and Ftz have been shown to bind with similar specificities to many genes, including four genes chosen because they were thought to be unlikely targets of Eve and Ftz. Eve and Ftz bind at the highest levels to DNA fragments throughout the length of three probable target genes: eve, ftz and Ubx. However, Eve and Ftz also bind at only two- to ten-fold lower levels to four genes chosen in an attempt to find non targets: Adh, hsp70, rosy and actin 5C, suggesting that Eve and Ftz bind at significant levels to a majority of genes. The expression of these four unexpected targets is controlled by Eve and probably by the other selector homeoproteins as well. A correlation is observed between the level of DNA binding and the degree to which gene expression is regulated by Eve (Liang, 1998).

At stages 10-14, 87% of cDNAs in the 8-12 hour library are likely to be directly or indirectly regulated by Eve, Ftz, Engrailed and all of the Hox proteins. These downstream genes are each expressed in unique, segmentally repeating patterns. Some are expressed at dramatically altered levels between segments. Most vary from segment to segment in the number and position of cells in which they are most prominently expressed. This is not simply because expression follows the distribution of a particular cell type. Between segments, the majority of genes are most highly expressed in differently positioned subsets of the same cell types, indicating that these patterns cannot result solely from the action of cell-type specific transcription factors. Eve, Ftz and Engrailed establish the segmentally repeating structure of the embryo. Therefore, all genes expressed in segmentally repeated patterns by stage 11 should be downstream of these three genes. This has been experimentally confirmed for eve and ftz. The expression of all 14 segmentally expressed genes tested is altered in eve and ftz mutant embryos at stage 11. These and other downstream genes can be divided into three classes: genes expressed in strong, moderate or weak segmentally repeated patterns. 33% of cDNAs fall into the strongly repeated class. For this class, staining levels vary five fold or more between cells across a transverse section of a segment along the anterior/posterior axis of the embryo. 24% of clones belong to the moderately regulated class. These genes show two- to five-fold variations in staining across the width of a segment. Finally, the weak segmentally repeated genes vary only 1.2 to 2 fold in staining between cells across a segment. Thus, most downstream genes are expressed in all cells, but each are still subject to specific and precise control by the selector homeoproteins. The more strongly regulated genes include many developmental control genes such as Enhancer of split [E(spl)] , tramtrack, division abnormally delayed (dally), and Dwnt4. A high proportion of the moderate and weakly regulated genes are involved in essential cellular functions such as splicing (e.g. RNA helicases), translation (e.g. met tRNA synthetase), general signal transduction (e.g. G-protein beta13F) and cytoskeletal structure (e.g. alpha tubulin 84B). This raises the question of whether or not modest changes in the expression of essential enzymes and structural proteins are important for morphogenesis. It is argued that they probably are. 11% of the genes picked from the 8-12 hour cDNA library do not appear to be downstream of the selector homeoproteins. Most of these genes are expressed relatively uniformly in all cells. But even these genes show some differences in expression pattern (Liang, 1998).

The wg-like ventral expression of DWnt-4 is dependent on hh, which may be due to shared regulatory elements between the two genes. However, since the dorsal expression of wg and DWnt-4 is nonoverlapping, this aspect of DWnt-4 expression appears to be regulated differently. Since the dorsal stripes of DWnt-4 lie in between the hh and wg stripes, the effect these genes have on dorsal DWnt-4 expression was examined. In wg mutants DWnt-4 is expressed normally, indicating that its expression in the epidermis is not dependent on the activity of wg. However, in hh mutants, both dorsal and ventral expression is eliminated. The regulation of DWnt-4 by hh within the anterior half of the dorsal parasegment suggests that it acts in concert with hh to pattern these cells (Buratovich, 2000).

Btk29A promotes Wnt4 signaling in the niche to terminate germ cell proliferation in Drosophila

Btk29A is the Drosophila ortholog of the mammalian Bruton's tyrosine kinase (Btk), mutations of which in humans cause a heritable immunodeficiency disease. Btk29A mutations stabilized the proliferating cystoblast fate, leading to an ovarian tumor. This phenotype was rescued by overexpression of wild-type Btk29A and phenocopied by the interference of Wnt4-β-catenin signaling or its putative downstream nuclear protein Piwi in somatic escort cells. Btk29A and mammalian Btk directly phosphorylate tyrosine residues of β-catenin, leading to the up-regulation of its transcriptional activity. Thus, this study identified a transcriptional switch involving the kinase Btk29A/Btk and its phosphorylation target, β-catenin, which functions downstream of Wnt4 in escort cells to terminate Drosophila germ cell proliferation through up-regulation of piwi expression. This signaling mechanism likely represents a versatile developmental switch (Hamada-Kawaguchi, 2014).

Stem cell maintenance and differentiation are not entirely autonomic, but instead are under strict control by supporting cells that form the 'niche'. Recent studies in Drosophila have shown that the dynamics of Piwi and its associated piRNAs, a protein-RNA complex for gene silencing, are required in not only germ cells but also distinct niche-forming somatic cells (escort cells for germ cell development); however, their regulatory mechanisms remain largely unknown. This study identified a transcriptional switch involving the factor Bruton's tyrosine kinase (Btk) and its phosphorylation target, β-catenin, operating downstream of Wnt4 in escort cells to terminate Drosophila germ cell proliferation through modulation of piwi expression (Hamada-Kawaguchi, 2014).

Drosophila Btk29A type 2 is the ortholog of human BTK. The type 1 isoform is present and the type 2 is absent in Btk29A^ficP mutants. Germ stem cells (GSCs) and transit amplifying cystoblasts (CBs) are localized in the germarium situated at the anterior tip of an ovariole, posteriorly flanked by region 2, in which each CB divides twice and differentiates into cystocytes. The 16 cystocytes originating from a single CB remain interconnected by the fibrous structure fusome, a derivative of the spectrosome. GSCs and CBs both carry the spectrosome, a round, tubulin-enriched structure. The Btk29A mutant germarium contains significantly more germ cells than does the wild-type germanium. Although supernumerary cells were observed with spectrosomes in the Btk29A^ficP germarium, many of the excess cells appear to be cystocytes, as they were accompanied by a branched fusome structure. A large excess of cystocytes in grossly deformed ovarioles has been observed in female Drosophila that are mutant for mei-P26, a gene encoding a TRIM-NHL (tripartite motif and Ncl-1, HT2A, and Lin-41 domain) protein that binds to the argonaute protein Ago-1 for microRNA regulation. In mei-P26 mutants, an ovarian tumor 'cystocytoma' is formed because cystocytes regain the ability to self-renew after they enter the differentiation path. This suggests that mei-P26 normally terminates CB proliferation. Intriguingly, the following phenotypes of mei-P26 were recapitulated in Btk29A^ficP. First, phospho-histone H3-positive mitotic germline cells, which were restricted to the anterior tip of the wild-type germarium, were detected throughout the ovarioles. Second, the expression of Bam, a protein that induces differentiation of GSCs into CBs in the wild type, was markedly increased in CB-like GSC daughters. Third, oo18 RNA-binding protein (Orb) remained expressed in multiple cells in a cyst, contrasting to a wild-type cyst, where Orb expression becomes restricted to an oocyte (Hamada-Kawaguchi, 2014).

The reduction in mei-P26 transcription in Btk29A^ficP places mei-P26 downstream of Btk29A. Notably, mei-P26 functions cell-autonomously in germ cells. However, the almost complete rescue of germ cell defects in Btk29A^ficP was attained by overexpression of Btk29A⁺ type 2 via bab1-Gal4, which showed high levels of expression in terminal filament cells and cap cells (TF and CPC, respectively) and lower levels of expression in escort cells (EC). bab1-Gal4 was effective in inducing germ cell overproduction when used to knockdown Btk29A. hh-Gal4 with expression in the terminal filament cells and cap cells and c587-Gal4 with expression in escort cells were also used to target UAS-Btk29ARNAi expression; c587-Gal4, but not hh-Gal4, led to the overproduction of spectrosome-bearing cells, and therefore, the escort cells were considered as likely sites of Btk29A action. These observations imply that Btk29A is required in the escort cells for soma-to-germ signaling to control the switch from proliferation to differentiation in germ cells, where mei-P26 functions as a core component of the switch (Hamada-Kawaguchi, 2014).

Bone morphogenetic protein (BMP) signaling and piwi-dependent signaling compose two different pathways in the niche to control proliferation and differentiation of GSCs and their daughters. BMPs are secreted morphogens, and Piwi is an argonaute protein regulating gene expression. The Btk29A^ficP mutation abrogated piwi expression with little effect on decapentaplegic (dpp) or glass bottom boat (gbb) expression, two BMPs operating in the germarium, and the BMP downstream component Mothers against Dpp (Mad) was normally phosphorylated in Btk29A^ficP GSCs. Furthermore, somatic piwi knockdown mimicked the Btk29A^ficP ovarian phenotypes (Hamada-Kawaguchi, 2014).

Immunohistochemistry revealed that the Btk29A^ficP mutation or somatic Btk29A knockdown abrogated Piwi expression in the niche, but not in germ cells. This reduction in Piwi expression was reversed by the somatic Btk29A⁺ overexpression. Furthermore, the loss-of-function piwi allele dominantly enhanced the Btk29A mutant phenotype. Moreover, somatic overexpression of piwi⁺ in Btk29A^ficP alleviated the germ cell hypertrophy and reduced Bam expression to the normal level. It is therefore considered that Btk29A regulates the Piwi-dependent pathway in the niche to control germ cell proliferation (Hamada-Kawaguchi, 2014).

Piwi and piRNAs constitute a major transposon-silencing pathway. Somatic knockdown of Btk29A resulted in an increase in the expression of gypsy-lacZ that monitored the activity of the gypsy transposon. Also, transcript levels of the ZAM, DM412, and mdg1 transposons were significantly increased in Btk29A^ficP. It is therefore concluded that the Piwi deficiency due to the impairment of Btk29A results in derepression of transposon activities (Hamada-Kawaguchi, 2014).

Genome instability associated with transposon mobilization may lead to the activation of a DNA double-strand break (DSB) checkpoint. A mutation in DSB signaling, mnk, did not ameliorate the germ cell phenotype induced by somatic Btk29A knockdown, indicating that the germ cell hypertrophy by the Btk29A deficiency is not a consequence of the DSB checkpoint activation (Hamada-Kawaguchi, 2014).

Next, potential substrates of Btk29A in the niche were sought. Btk29A type 2 was enriched in the interface between cells where Drosophila melanogaster epithelial (DE)-cadherin and associated Arm, the β-catenin ortholog, are the major structural components. No sign were found of tyrosine phosphorylation of DE-cadherin, whereas Arm contained a high level of phosphotyrosine, which was almost entirely absent from Btk29A^ficP ovaries. However, Arm immunoprecipitated from Btk29A^ficP was strongly phosphorylated in vitro by the exposure of Arm to active Btk29A protein that had been immunoprecipitated from wild-type ovaries. These results demonstrate that Btk29A mediates the tyrosine phosphorylation of Arm in vivo (Hamada-Kawaguchi, 2014).

The anti-Arm labeling intensity of cell adhesion sites was stronger in Btk29A^ficP than in the wild type. Immunoprecipitation assays revealed that the relative amount of Arm associated with DE-cadherin was greater in Btk29A^ficP than in the wild type , suggesting that the tyrosine phosphorylation of Arm facilitates its release from the membrane to the cytoplasm, as in mammalian cells (Hamada-Kawaguchi, 2014).

Mammalian β-catenin is tyrosine-phosphorylated at residues Y86, Y142, and Y654. When transfected into mammalian Cos7 cells, Drosophila Btk29A type 2 phosphorylated all these tyrosine residues of β-catenin. Moreover, the antibodies against phosphorylated Y142 (anti-pY142) and anti-pY654 recognized Arm phosphorylated at the conserved site Y150 and Y667, respectively, in the immunoprecipitates from ovarian lysates (Hamada-Kawaguchi, 2014).

Expression of unphosphorylatable Arm-Y150F in the escort cells via c587-GAL4 or bab1-GAL4, but not hh-Gal4, induced germ cell hypertrophy, whereas another unphosphorylatable mutant, Y667F, or wild-type Arm exerted little effect. In addition, somatic arm knockdown resulted in an increase in spectrosome-containing cells, reduced piwi expression in escort cells, and increased Bam expression in germ cells. Considering these results together, it is proposed that Btk29A acts on Arm, which in turn regulates piwi in the niche (Hamada-Kawaguchi, 2014).

Arm functions in the canonical Wnt pathway. Therefore the ovaries of wg, Wnt2, Wnt4, and Wnt5 mutants were examined; the germ cell overproduction was detected only in Wnt4. Somatic knockdown of Wnt4 aided by bab1-GAL4 resulted in a reduction in the expression of Piwi, accompanied by an accumulation of germ cells carrying spectrosomes with an increase in germline Bam expression. These findings support the hypothesis that Arm in the escort cells regulates germ cell proliferation under the control of Wnt4, which was likely derived from somatic cells other than cap cells and terminal filament cells, as hh-GAL4 selective for these cells was least effective to induce germ cell overproduction when used to drive Wnt4RNAi expression (Hamada-Kawaguchi, 2014).

To evaluate the ability of Arm to activate transcription, T cell factor (TCF) reporter assays were used with Cos7 cells transiently transfected with human Btk (hBtk). The wild-type hBtk alone was sufficient to induce phosphorylation at Y142 and Y654 of β-catenin, whereas the kinase-dead hBtk (Btk-K430E) was not. Tyrosine phosphorylation of β-catenin was completely blocked by two antagonists of hBtk. Similarly, Btk29A type 2 phosphorylated Y142 and Y654 of mammalian β-catenin. Notably, the TCF reporter activity was six times as high when hBtk was transfected into Cos7 cells compared with the mock-transfected control, indicating that hBtk modulates the TCF-dependent transcriptional activation mechanism, in which Arm-β-catenin is involved as a coactivator (Hamada-Kawaguchi, 2014).

The expression of an arm-dependent Ubx-lacZ reporter was examined in the embryonic midgut. Btk29A knockdown abrogated the expression of this reporter, demonstrating that Btk29A supports Arm-dependent transcription in vivo (Hamada-Kawaguchi, 2014).

Btk29A was shown to phosphorylate Arm-β-catenin on conserved tyrosine residues, one of which (Arm-Y150) is pivotal for the niche function to prevent GSC daughters from overproliferating. Notably, most GSCs in Btk29A mutants do not express Bam (fig. S1R). This suggests that the presumptive Btk29A-Arm-Piwi pathway selectively regulates the proliferation of differentiating GSC daughters without interfering with GSC maintenance. Without Btk29A type 2, cystoblasts fail to exit the cell cycle, leading to the overproduction of germ cells, many of which are unable to complete differentiation and contribute to the genesis of an ovarian tumor (Hamada-Kawaguchi, 2014).

β-Catenin exerts multiple functions through its promiscuous binding abilities in cell-to-cell interactions and transcription. This protein plays critical roles in stem cell biology, and β-catenin malfunction results in a variety of cancers. These findings add a new dimension to the study of β-catenin by highlighting the pivotal role of the tyrosine phosphorylation of β-catenin in the control of transcription in the nucleus, in addition to the regulated control of the stability and motility of cell adhesion (Hamada-Kawaguchi, 2014).

DEVELOPMENTAL BIOLOGY

Embryonic

Transcripts of Wnt-4 are first detected by stage 8 in the ectoderm, at the anterior tip and in the trunk, where segmentally repeated stripes become visible ventrally. Shortly thereafter, expression in the head shows up in the foregut primordium and dorsal patches, presumably corresponding to cells of labral and ocular segment primordia. By germ band extension [Image], the segment polarity-like staining in the trunk intensifies, and 15 stripes are visible in parasegments 0-14. The stripes are one cell wide and overlap the ventral most wingless expressing cells.

Transcripts are present in the dorsolateral ectoderm of each parasegment in short rows of 3-4 cells, paralleling the Engrailed stripe, but located in the middle of the parasegment. An additional stripe becomes apparent at the posterior tip.

By germ band shortening, transcripts are apparent in the central nervous system and the developing gut. Transcripts are seen in foregut, hindgut and at two locations in the visceral mesoderm of the midgut. The two locations in the ventral mesoderm correspond to primordia for the gastric caeca and the second midgut constriction (Graba, 1995).

Wnt genes are often expressed in overlapping patterns, where they affect a wide array of developmental processes. To address the way in which various Wnt signals elicit distinct effects, the activities of two Wnt genes in Drosophila, DWnt-4, and wingless, were compared. These Wnt signals produce distinct responses in cells of the dorsal embryonic epidermis. Whereas wingless acts independently of hedgehog signaling in these cells, DWnt-4 requires Hh to elicit its effects. Expression of Wg signal transduction components does not mimic expression of DWnt-4, suggesting that DWnt-4 signaling proceeds through a distinct pathway. The dorsal epidermis may therefore be useful in the identification of novel Wnt signaling components (Buratovich, 2000).

DWnt-4 and wg are expressed in many of the same cells during Drosophila embryogenesis, including the ventral epidermis. However, in the cells of the dorsal epidermis each gene is expressed in distinct groups of cells. Whereas wg is expressed in the most posterior row of cells in each parasegment throughout most of embryogenesis, DWnt-4 is expressed in the anterior region of the parasegment. This expression is transient, beginning at stage 10 and fading by the end of stage 12. The wg-like ventral expression of DWnt-4 is dependent on hh, which may be due to shared regulatory elements between the two genes. However, since the dorsal expression of the two genes is nonoverlapping, this aspect of DWnt-4 expression appears to be regulated differently. Since the dorsal stripes of DWnt-4 lie in between the hh and wg stripes, the effect these genes have on dorsal DWnt-4 expression was examined. In wg mutants DWnt-4 is expressed normally, indicating that its expression in the epidermis is not dependent on the activity of wg. However, in hh mutants, both dorsal and ventral expression is eliminated. The regulation of DWnt-4 by hh within the anterior half of the dorsal parasegment suggests that it acts in concert with hh to pattern these cells (Buratovich, 2000).

Unlike the ventral epidermis, where hh and wg cooperate in the specification of naked cuticle, the dorsal epidermis is patterned through complementary activities of wg and hh. The anterior half of each parasegment consists of three cells types (1^o, 2^o, and 3^o) that are all dependent on the activity of hh. The position of the DWnt-4 expressing cells relative to the wg-expressing cells indicates that DWnt-4 is expressed in the presumptive 3^o cells. The posterior half of the parasegment consists of a single cell type, 4^o, and is dependent on wg activity. The dorsal epidermis is patterned beginning at 6 h of development, the time at which the expression of DWnt-4 is initiated in these cells. Prior to this point, between 3 h and 6 h, the expression of wg and hh are mutually dependent (Buratovich, 2000).

To determine whether DWnt-4 is able to modulate the patterning of the dorsal epidermis, and whether it mimics or otherwise regulates wg signaling in these cells, it was ubiquitously expressed using the GAL4 system. The results of ubiquitous expression of wg or DWnt-4 were compared. Ubiquitous expression of wg driven by a GAL4 insertion under the control of a daughterless enhancer (daGAL4) results in a uniform lawn of 4^o cells. Thus the hh-dependent cell types are deleted or transformed to 4^o fates. Ubiquitous expression of DWnt-4 elicits a distinct response in the hh-dependent cells, while having no effect on the wg-dependent cells. The phenotype produced by ectopic DWnt-4 is variable and dependent on levels of ectopic expression. With one copy of ectopic DWnt-4 expressed at 29^oC, 21% (23/108) of the segments exhibit a 2^o-3^o-4^o pattern, in which 1^o cells are missing and 3^o cells are expanded. In contrast, 62% of the segments exhibit either a 1^o-3^o-4^o or a 3^o-4^o pattern; it was found that 1^o and 3^o cells are difficult to distinguish. Lower levels of expression produced by rearing the flies at a lower temperature produces a higher percentage of embryos with a pattern that is more clearly 1^o-3^o-4^o along the dorsal midline, since the 2^o cell fate is still apparent laterally. Nevertheless, the 2^o-3^o-4^o phenotype shows that DWnt-4 can abolish 1^o cells, and indicates that the primary effect of DWnt-4 is to expand 3^o cells at the expense of the other two cell types (Buratovich, 2000).

These data show that cells in the anterior half of each parasegment have the ability to respond to both Wnt genes, but that each gene elicits a distinct response. Whereas Wg transforms these cells to 4^o cells or deletes them, DWnt-4 appears to modulate the specification of cell fate within the hh-dependent domain but has no effect on cell fate specification by wg. The phenotypes produced by ectopic DWnt-4 and wg therefore appear to be qualitatively distinct, in that each gene induces ectopic specification of different cell types (Buratovich, 2000).

The alteration in pattern by DWnt-4 suggests three possible interactions with hh. (1) DWnt-4 might affect the anterior half of the parasegment through modification of hh expression. However, analysis of hh transcripts following ectopic DWnt-4 expression has revealed that hh expression is not affected. (2) Since DWnt-4 expression requires hh activity, it could be a downstream effector of hh in pattern specification. (3) DWnt-4 could act in concert with hh to alter pattern. To address these possibilities, DWnt-4 was ectopically expressed in a hh temperature sensitive mutant shifted to the restrictive temperature at 6 h. Under these conditions the entire anterior half of the parasegment is missing in hh mutants. When DWnt-4 is ectopically produced in this background, anterior cell fates still fail to be specified, indicating that DWnt-4 does not simply act downstream of hh but requires hh for its activity after 6 h of development. If hh ts mutants are shifted to the restrictive temperature at 7 h, one row of 3^o denticles typically forms, while 1^o and 2^o fates are still missing. If DWnt-4 is ectopically expressed under these conditions, the number of 3^o rows increases, supporting the conclusion that DWnt-4 acts in concert with hh to specify 3^o cell fates (Buratovich, 2000).

Wnt genes encode evolutionarily conserved secreted proteins that provide critical functions during development. Although Wnt proteins share highly conserved features, they also show sequence divergence, which almost certainly contributes to the variety of their signaling activities. Wnt4 and wingless, two divergent clustered Wnt genes, can have either antagonist or distinct functions during Drosophila embryogenesis. Both genes can elicit similar cellular responses during imaginal development. Ectopic expression of Wnt4 along the anterior/posterior (A/P) boundary of imaginal discs alters morphogenesis of adult appendages. In the wing disc, Wnt4 phenocopies ectopic Wg activity by inducing notum to wing transformation, suggesting similar signaling capabilities of both molecules. In support of this, it has been demonstrated that Wnt4 can rescue wg loss-of-function phenotypes in the antenna and haltere and is able to substitute for Wg in wing field specification. In addition to effects on the axial pattern of haltere and wing, ectopic Wnt4 produces supernumerary bristles in specific regions. Wnt4, like Wg, induces extra sternopleural bristles. This may reflect an endogenous activity for Wnt4: unlike wg, it is transcribed in a dorsal domain of the leg disc that roughly corresponds to the sternopleura. Both genes are transcribed in overlapping domains in imaginal discs, suggesting that Wnt4 may cooperate with wg during limb patterning (Gieseler, 2001).

wg and Wnt4 are transcribed in overlapping patterns during embryogenesis. They also show largely similar expression profiles in third instar imaginal discs. In the wing disc, transcripts of the two genes are synthesized at the D/V compartment border, the future wing margin, and the presumptive wing hinge region. Wnt4 expression, however, is weak compared to that of wg and appears more spatially restricted. It proceeds following a cell stripe comparable in width to the wg stripe in the distal part of the future wing margin, but is more faintly detected in more proximal parts of anterior and posterior compartments. The patterns are different in the presumptive notum, where WG mRNA is found in a broad D/V stripe, whereas WNT4 transcripts faintly label a central cell domain. These cells are located beneath the columnar epithelium and, therefore, likely correspond to adepithelial cells. In antenna and leg discs, the wg domain corresponds to a ventral/anterior sector, whereas Wnt4 is expressed only in a subset of these cells. Whereas WG mRNA is detected along the entire proximal-distal axis in the leg disc, Wnt4 transcription is restricted to two segments that correspond to primordia of the tibia and a more distal segment and in a dorsally located cell cluster that presumably corresponds to the sternopleural region. The wg pattern in the haltere disc is reminiscent of that observed in the wing disc. Wnt4 is transcribed in two large stripes, which give rise to the pedicel and/or the scabellum. However, in contrast to wg, Wnt4 is not expressed in the notum and in the most distal part of the disc, which is homologous to the margin of the wing. A clear difference in expression of the two genes is also seen in the larval central nervous system, where wg is not expressed, while Wnt4 transcripts follow a segmentally repeated pattern of small clusters in the central cortex and in the optic anlagen. In summary, the transcription profile of Wnt4 in third instar imaginal discs partially overlaps that of wg but is not restricted to wg-positive cells. For example, in the wing blade, the two genes are expressed along the future margin and in the presumptive hinge region, suggesting that the encoded Wnt products may be required together for the differentiation of these structures. In the central nervous system or in the dorsal part of the leg disc, Wnt4 is expressed where wg is not, suggesting it may also function in cells that do not depend on wg (Gieseler, 2001).

The ability of Wnt4 to induce, as Wg does, additional wings indicates that the two molecules can elicit similar cellular responses. Strong support for this conclusion is provided by rescue experiments of wg loss-of-function phenotypes. Wnt4 can restore normal wing development in the absence of a functional Wg protein at the second instar. Eliminating Wg signaling during the third instar, allows wing field specification but not subsequent wing patterning and growth. Wnt4 therefore appears unable to substitute for Wg signaling during late wing development. This failure to replace Wg might be the result of ectopic expression in inappropriate domains or of distinct signaling abilities during wing growth and patterning. In support of the latter possibility, the two Wnt molecules produce different effects, depending on the developmental context. Ectopic expression of Wg, but not of DWnt4, perturbs leg, eye, or antenna development, which reveals competency to respond to Wg but not to Wnt4. In addition, Wnt4 is able to antagonize late embryonic Wg signaling in the Drosophila ventral epidermis and to block the Wg-induced body axis duplication in Xenopus. Furthermore, the two genes induce different phenotypes in the dorsal embryonic epidermis. Taken together these results strongly support a context dependence for Wnt4 activity (Gieseler, 2001).

The molecular basis underlying the ability of Wg and Wnt4 to perform antagonistic, distinct, or similar signaling activities remains to be explored. It has been proposed that the Wnt4/Wg antagonistic relationship in Drosophila embryonic ventral ectoderm and in Xenopus axis induction assay results from strong sequence divergences in the C-terminal parts of the two proteins. This has been supported by reports that C-terminal truncations in Wg and XWnt8 result in proteins with dominant negative or antagonistic functions. If this interpretation is correct, domains other than the divergent C-terminal ends in the Wnt proteins would be critical for function in other developmental contexts. Although interactions of the C-terminus with specific partners dictate activity in the ventral embryonic epidermis, the recruitment of imaginal disc-specific factors by other domains in Wg and Wnt4 would allow the proteins to exhibit similar activities during wing field specification. The existence of separated functional domains in Wnt proteins is also supported by the nature and effect of the wg^NEI mutation, where a single amino acid change affects a subset of Wg functions only (Gieseler, 2001).

An alternative model to explain antagonism versus similarity is that Wnt4 acts as a low-activity agonist of Wg. In this model, tissue-specific differences in receptor concentrations and/or differences in receptor-binding affinities would determine whether Wnt4 mimics or antagonizes Wg signaling. If, for example, receptor concentrations are limiting in the ventral epidermis, Wnt4 may act as a competitive inhibitor of Wg for receptor binding but would provide less stimulation of the pathway than would Wg itself, therefore lowering the level of Wg signaling. However, if receptor concentrations are high, Wnt4 may be able to increase signaling by binding receptors without competing with endogenous Wg. If Wnt4 interacts with Wg receptors with lower affinity than that of Wg, the tissue-specific differences in Wnt4's ability to engage the Wg signaling pathway might be explained. In particular, this may explain the inability of Wnt4 to stimulate the Wg pathway in wild type legs while stimulating the pathway, and rescuing leg defects, in the absence of Wg (Gieseler, 2001).

Wnt4 regulates the dorsoventral specificity of retinal projections in the Drosophila melanogaster visual system

In Drosophila, the axons of retinal photoreceptor cells extend to the first optic ganglion, the lamina, forming a topographic representation. DWnt4, a secreted protein of the Wnt family, is the ventral cue for the lamina. In DWnt4 mutants, ventral retinal axons misproject to the dorsal lamina. DWnt4 is normally expressed in the ventral half of the developing lamina and DWnt4 protein is detected along ventral retinal axons. Dfrizzled2 and dishevelled, respectively, encode a receptor and a signaling molecule required for Wnt signaling. Mutations in both genes caused DWnt4-like defects, and both genes are autonomously required in the retina, suggesting a direct role of DWnt4 in retinal axon guidance. In contrast, iroquois homeobox genes are the dorsal cues for the retina. Dorsal axons accumulate DWnt4 and misproject to the ventral lamina in iroquois mutants; the phenotype is suppressed in iroquois:Dfrizzled2 double mutants, suggesting that iroquois may attenuate the competence of Dfrizzled2 to respond to DWnt4 (Sato, 2005).

The spatial order of projecting neurons is preserved in the spatial order of their targets to establish the topographic maps in the nervous system. In the visual system, precise topographic mapping of photoreceptor neurons to their targets in the brain, termed retinotopic mapping, is necessary for the correct interpretation of visual information received in the retina. The Drosophila visual system includes the retina, the compound eye and the optic lobe, which is the visual center of the brain and is connected to the eye via the optic stalk. Each of the approximately 750 ommatidial units in the retina consists of eight unique types of photoreceptor neurons called R cells (R1-R8). During larval development, R cells sequentially differentiate behind the morphogenetic furrow, progressing in a posterior-to-anterior order in the third larval instar retina, and send their axons through the optic stalk to the most distal part of the optic lobe, the lamina. R1-R6 axons terminate in the lamina layer, whereas R7 and R8 axons project through the lamina to terminate in the medulla layer. Although all the retinal axons pass through the narrow optic stalk, they distribute evenly and project to their correct targets along the anteroposterior and the dorsoventral axes. Thus, R cell axon connections between the retina and the lamina (or the medulla) are precisely retinotopic in the adult. Similarly, R axon connections established during the third instar are anatomically retinotopic (Sato, 2005 and references therein).

Like the retina, the lamina must also be patterned along the dorsoventral axis so that retinal axons can project precisely to their targets. It is assumed that selector genes and genes encoding guidance molecules are asymmetrically expressed along the dorsoventral axis in the developing lamina and it was found that DWnt4, one of seven D. melanogaster Wnt family genes, is specifically expressed in the ventral half of the developing lamina during the third larval instar (Sato, 2005).

The present study investigated the involvement of DWnt4 in D. melanogaster retinotopic mapping along the dorsoventral axis. DWnt4 is normally expressed in the ventral half of the developing lamina and DWnt4 protein has been detected on the surface of ventral retinal axons. In DWnt4 mutant backgrounds, ventral axons misproject to the dorsal lamina. Conversely, ventral axons are redirected by an ectopic source of DWnt4, suggesting that DWnt4 is a ventral cue for retinal axon projections in the lamina. Furthermore, ventral axon projections are regulated by noncanonical Wnt signaling in R cells, which is most likely under the control of DWnt4. These genetic data also suggest the involvement of JNK signaling in this process. Finally, iro may attenuate the competence of Dfz2 in dorsal axons to respond to DWnt4, since dorsal-to-ventral misroutings in iro clones are significantly suppressed in iro:Dfz2 double mutant clones (Sato, 2005).

As a first step to investigating retinotopic mapping in D. melanogaster, focus was placed on the iroquois (iro) complex genes, three related homeobox genes that act as selector genes for the dorsal retina. To test if iro regulates retinotopic mapping, cells homozygous for an iro deficiency were generated and labeled with green fluorescent protein (GFP) to visualize axons using the MARCM system. Dorsal mutant R axons were occasionally observed projecting to the ventral lamina. A similar phenotype was observed by generating large iro clones using ey-flp. Both outer- and inner-photoreceptor axons visualized with ro-lacZ and ato-myc were affected by iro. Thus, iro genes seem to function as dorsal cues for the retina (Sato, 2005).

The lamina, to which the retinal axons project, must also be patterned along the dorsoventral axis. It is assumed that selector genes and genes encoding guidance molecules are asymmetrically expressed along the dorsoventral axis in the developing lamina. Attempts were made to identify genes specifically expressed in either the dorsal or the ventral lamina. It was found that DWnt4, one of seven Wnt family genes in D. melanogaster, is specifically expressed in the ventral half of the lamina during the late third instar. At this stage, the lamina expresses Dachshund (Dac) and forms a characteristic, crescent-like structure. In situ hybridization showed specific expression of DWnt4 in the ventral half of the lamina along the lamina furrow, restricted to the anterior-most two or three rows of Dac-positive cells (Sato, 2005).

Because DWnt4 is a secreted glycoprotein, the distribution of DWnt4 protein was compared with that of DWnt4 mRNA. DWnt4 localization along the lamina furrow of the ventral lamina is indistinguishable from that of DWnt4 mRNA, except for small dots found in the ventral lamina. Notably, DWnt4-positive dots are found on the surface of the anterior-most R axons, labeled by GMR-Gal4 UAS-GFP. Given that R axons sequentially project to the lamina from posterior to anterior, DWnt4 probably accumulates on the most recently arriving axons. In more apical optical sections, DWnt4 accumulation is observed between the optic stalk and the lamina. The source of DWnt4 on R axons is likely to be DWnt4 expression along the ventral lamina furrow, since DWnt4 localization is not detectable on R axons in the dorsal half of the lamina (Sato, 2005).

DWnt4 localization on ventral R axons implies its involvement in R axon guidance. This possibility was tested by investigating the DWnt4 loss-of-function phenotype. In wild-type flies, dorsal and ventral axons project to the dorsal and ventral lamina, respectively. In DWnt4 mutants, however, occasional misroutings of ventral axons toward the dorsal lamina were observed. In extreme cases, ventral axons looked as if they were about to project to ventral lamina but had abruptly reoriented toward the dorsal lamina. omb-lacZ was used to visualize the dorsal- and ventral-most axons and ventral axon misrouting within the optic stalk was noted. Together, these data suggest that DWnt4 influences ventral axon projection at various points along the path from the optic stalk to the lamina (Sato, 2005).

Frizzled family receptors and Dishevelled are required for a wide variety of Wnt signaling cascades7. Dfz2 and dsh mutant flies have ovarian defects similar to those of DWnt4, strongly suggesting that Dfz2 and Dsh are involved in DWnt4 signaling. A ventral-to-dorsal misrouting phenotype was observed in Dfz2 and dsh mutant backgrounds. The results suggest that Dfz2 and Dsh are involved in DWnt4 signaling for R axon guidance. In addition, the greater expressivity and penetrance of Dfz2 and dsh mutants suggests the involvement of other Wnt family ligands in this process. However, after examining mRNA expression of all the known D. melanogaster Wnt genes, no such genes were found acting as ventral cues in concert with DWnt4. DWnt2 is expressed just outside the lamina, but its expression is symmetric along the dorsoventral axis (Sato, 2005).

The accumulation of DWnt4 on the surface of ventral R axons implies reception of the ligand and subsequent signal activation in R cells. Consistently, Dfz2 is localized on the surface of the anterior-most R axons, but not on surrounding lamina cells. To test whether Wnt signaling autonomously regulates axon guidance, dsh homozygous clones were induced in the retina using ey-Gal4:UAS-flp18. Surprisingly, the axonal misrouting phenotype was rarely observed despite the presence of many dsh mutant clones in the retina. When the retina was entirely dsh homozygous, the same dsh allele showed axonal misrouting. It is suspected that mutant R axons project normally in the presence of surrounding wild-type axons due to axon fasciculation. If this is the case, R axons homozygous for dsh may show a misrouting phenotype in the absence of neighboring, wild-type R cells. To test this idea, GMR-hid was introduced in trans to the dsh mutant chromosome. Wild-type R cells eventually die by programmed cell death triggered by hid expression behind the furrow. In this context, a severe misrouting phenotype was observed that was ventral-to-dorsal. Notably, DWnt4 protein accumulation was observed along ventral axons that were mutant for dsh and had misprojected to the dorsal lamina. Retina-specific Dfz2 clones with GMR-hid also showing a ventral-to-dorsal misrouting phenotype. These observations are consistent with the idea that DWnt4 expressed in the lamina directly regulates R axon projections (Sato, 2005).

There are two Wnt signaling pathways: the canonical and noncanonical pathways. In the former, ß-catenin/Armadillo (Arm) and TCF/Pangolin (Pan) form a complex to activate target gene transcription. In the latter, Wnt signaling is transduced independently of Arm and Pan. Canonical Wnt signaling was manipulated using UAS-panN, which encodes a constitutive repressor form of Pan, and UAS-arm, which encodes a constitutively active form of Arm. Misrouting along the dorsoventral axis was hardly observed in either genotype. Thus it is concluded that canonical Wnt signaling plays a very minor role, if any, in dorsoventral specification of R axon guidance. The above results strongly suggest the involvement of noncanonical Wnt signaling in R cells. To confirm this idea, UAS-dshDEP, which acts as a dominant-negative mutant in noncanonical signaling, was expressed in the retina. Again a strong ventral-to-dorsal phenotype was observed (Sato, 2005).

Although the planar cell polarity (PCP) pathway is categorized as a noncanonical Wnt pathway transduced by the Fz receptor, no PCP defects were observed in DWnt4 and Dfz2 mutant retinae. In addition, the retinotopic phenotype was not observed in fz null mutant backgrounds. These results suggest that DWnt4 regulates R axon projections via a noncanonical Wnt signaling distinct from the PCP pathway. wingless (wg) is involved in the specification of the dorsal retina through the activation of iro expression. The distinct chiral forms of ommatidia in the dorsal and ventral retina reflect the dorsoventral specification of the retina and the PCP signaling. The normal iro expression and the normal ommatidial chirality suggest that axonal misroutings occur independently of the retinal dorsoventral specification in DWnt4 and Dfz2 backgrounds. Since dsh is required for PCP signaling and the specification of the dorsal retina, ommatidial chirality was disorganized and dorsal iro expression was eliminated in dsh retinae. However, the expression of Serrate (Ser), which is specific to the ventral retina in wild-type backgrounds, was not affected, suggesting that the ventral cell fate is correctly specified in dsh homozygotes. Additionally, UAS-dsh and UAS-dshDEP expression under the control of GMR-Gal4 did not affect the dorsoventral specification of the retina as visualized by iro and Ser expression. Note that GMR-Gal4 is expressed behind the morphogenetic furrow well after the dorsoventral specification at earlier stages. The data shown above suggest that dsh also regulates R axon projections independently of the dorsoventral patterning of the retina (Sato, 2005).

JNK signaling is known to act downstream of the noncanonical Wnt pathway in many developmental contexts. The involvement of JNK signaling was examined by expressing puckered (puc), which encodes a JNK phosphatase, and a dominant-negative form of JNK encoded by basket (bsk) to block JNK signaling in the retina. Defects were observed only rarely, and it was next asked whether genetic interactions exist between hemipterous (hep) encoding a JNK kinase and DWnt4 or Dfz2. In a strong hep mutant background, or in DWnt4, DWnt4 or Dfz2 heterozygous backgrounds, little or no ventral-to-dorsal misrouting was observed. However, a reduction in the dosage of DWnt4 or Dfz2 in the hep background resulted in a marked increase in the ventral-to-dorsal phenotype. These findings provide some support for the idea that JNK signaling is involved in the DWnt4/Dfz2 pathway in retinal axon guidance. Since iro expression and ommatidial chirality were normal in retinae expressing the dominant-negative form of bsk and in hep hemizygotes in combination with DWnt4/+ and Dfz2/+, the misrouting of ventral axons observed in the brain mutant for JNK signaling appears to be caused by a failure in axon guidance and independent of the dorsoventral cell specification or PCP signaling in the retina. Note that mutations in JNK pathway components alone have no PCP phenotype (Sato, 2005).

iro is thought to be the dorsal cue in the retina. The dorsal axons project to the ventral lamina in the absence of iro, perhaps because dorsal axons are attracted by ventral cues in the lamina, such as DWnt4. When iro mutant clones were generated under the control of ey-flp, dorsal axons projected to the ventral lamina in 32.4% of them, and ectopic accumulations were observed of DWnt4 on the surface of the dorsal axons misprojecting ventrally. Since Dfz2 was expressed in the dorsal axons, iro may attenuate the competence of Dfz2 in the dorsal axons to respond to DWnt4. If this is the case, simultaneous removal of Dfz2 in iro clones should suppress the iro phenotype, which was indeed observed. In iro:Dfz2 double mutant clones, 'dorsal-to-ventral' misroutings were observed in 3.5% of the cases, and the class III phenotype was no longer observed. Instead, abnormal bundles of dorsal axons were found in iro:Dfz2 clones. This might be because iro:Dfz2 axons do not respond to either dorsal or ventral cues in the lamina. Indeed, no DWnt4 accumulation was found in those abnormal bundles found in iro:Dfz2 clones. The absence of iro expression in differentiated R cells behind the morphogenetic furrow suggests indirect modulation of Dfz2-dependent Wnt signaling by iro (Sato, 2005).

It was hypothesized that three events are required for retinotopic mapping along the dorsoventral axis in D. melanogaster: (1) dorsoventral identity is specified by selector genes expressed in the retina; (2) dorsoventral identity is specified by selector genes in the lamina; (3) guidance molecules recruit R axons to their correct targets in the lamina. The results nicely fit the hypothesis. iro expressed in the dorsal retina specifies the dorsal axon identity, and DWnt4 expressed in the ventral lamina recruits ventral axon projections. The restricted expression of DWnt4 to the ventral lamina suggests there could be unidentified dorsoventral selectors in the lamina (Sato, 2005).

Different Wnt signals act through the Frizzled and RYK receptors during Drosophila salivary gland migration

Guided cell migration is necessary for the proper function and development of many tissues, one of which is the Drosophila embryonic salivary gland. Two distinct Wnt signaling pathways regulate salivary gland migration. Early in migration, the salivary gland responds to a WNT4-Frizzled signal for proper positioning within the embryo. Disruption of this signal, through mutations in Wnt4, frizzled or frizzled 2, results in misguided salivary glands that curve ventrally. Furthermore, disruption of downstream components of the canonical Wnt pathway, such as dishevelled or Tcf, also results in ventrally curved salivary glands. Analysis of a second Wnt signal, which acts through the atypical Wnt receptor Derailed, indicates a requirement for Wnt5 signaling late in salivary gland migration. WNT5 is expressed in the central nervous system and acts as a repulsive signal, needed to keep the migrating salivary gland on course. The receptor for WNT5, Derailed, is expressed in the actively migrating tip of the salivary glands. In embryos mutant for derailed or Wnt5, salivary gland migration is disrupted; the tip of the gland migrates abnormally toward the central nervous system. These results suggest that both the Wnt4-frizzled pathway and a separate Wnt5-derailed pathway are needed for proper salivary gland migration (Harris, 2007).

Salivary gland migration can be separated into three phases. In the first phase, the salivary glands invaginate into the embryo at a 45° angle, moving dorsally until they reach the visceral mesoderm. fkh, RhoGEF2 and 18 wheeler have been shown to regulate apical constriction of the salivary gland cells during this invagination process. In addition, hkb and faint sausage are needed for proper positioning of the site of invagination. No guidance cues have been identified for this first phase of migration; it may be that the patterns of constriction and cell movements at the surface of the embryo are sufficient to direct the invaginating tube (Harris, 2007).

During the second phase of migration, as the salivary gland moves posteriorly within the embryo, two guidance cues, Netrin and Slit, guide salivary gland migration along the visceral mesoderm. Netrin, which is expressed in the CNS and the visceral mesoderm, works to maintain salivary gland positioning on the visceral mesoderm. At the same time, Slit acts as a repellent from the CNS to keep the salivary glands parallel to the CNS. A third guidance signal, WNT4, which acts through FZ or FZ2 receptors, is also required in the second phase of salivary gland migration. Loss of Wnt4, fz or fz2 in the embryo results in salivary glands that are curved in a ventromedial direction. This curving affects a large portion of the salivary gland and may result from the fact that the fz and fz2 receptors, in contrast to drl, are expressed throughout the salivary gland. Furthermore, dominant-negative transgenes that disrupt the function of DSH or TCF cause the same phenotype, suggesting that transcription induced by the canonical Wnt signaling pathway is needed to maintain the proper migratory path of the salivary glands on the circular visceral mesoderm (CVM). The migration along the CVM takes more than 2 hours for completion, which would leave adequate time for a transcriptional response (Harris, 2007).

Although Wnt4 and slit are both required for the second phase of migration, and their mutants show similar, though distinguishable, phenotypes, they are thought to act independently. While most slit-mutant embryos have medially curving salivary glands, embryos lacking Wnt4 have salivary glands that curved in a distinctly different, ventromedial, direction. Embryos doubly mutant for Wnt4 and slit show predominantly one or the other phenotype and neither phenotype increases in severity. These results suggest, though they do not prove, that Wnt4 and slit act in distinct pathways (Harris, 2007).

After the entire salivary gland has invaginated, migrated posteriorly within the embryo and lies parallel to the anteroposterior axis of the embryo, the distal ends of the salivary glands come into contact with the LVM. drl and Wnt5 are required for this late phase of salivary gland positioning. Loss of either drl in the salivary gland or Wnt5 in the CNS results in the distal tip of the salivary gland being misguided to a more ventromedial position. This change in the shape of the salivary gland is seen only after the salivary glands are no longer in contact with the CVM (after stage 13). Thus it is proposed that drl is required during the third phase of salivary gland migration, as the salivary gland detaches from the CVM and contacts the LVM (Harris, 2007).

The striking expression of drl at the tip of the salivary gland makes the leading cells uniquely different from the rest of the salivary gland cells. These cells project lamellipodia upon reaching the visceral mesoderm and beginning their posterior migration. They may act to both guide and pull the rest of the gland during migration. Cells at the tip of a migrating organ are frequently specialized to guide migration. For example, the coordinated migration of the tracheal branches in Drosophila is achieved by induction of distinct tracheal cell fates within the migrating tips. This is illustrated by the fact that FGF (Branchless) signaling becomes restricted to the tips of the tracheal branches soon after they begin to extend. The migration and growth of Drosophila Malpighian tubules provide another clear example of specialized cells needed at the tip of a migrating tissue. One cell is singled out to become the tip cell, which directs the growth of the Malpighian tubules as well as organizes the mitotic response and migration of the other cells forming each tubule. In other systems, such as Dictyostelium slugs, cells at the tip of a migrating group are required and solely able to guide migration. These results establish that the leading cells of the migrating salivary glands have a specialized role to play in proper salivary gland positioning. First they are required to initiate invagination within the embryo, then they actively participate in migration along the CVM, and finally they ensure that the distal tip of the gland will remain associated with the LVM at the end of the migratory phase (Harris, 2007).

Despite the fact that it has been firmly established that Wnt5 and drl are important for the final placement of salivary glands, the signaling pathways downstream are not well defined. Because salivary-gland expression of full-length drl can rescue the drl-mutant phenotype, but drl lacking the intracellular domain cannot, it is thought that the intracellular domain of DRL is important for signaling. Similarly, misexpression of full-length drl can misguide axons in the ventral nerve cord, but misexpression of drl lacking its intracellular domain cannot (Yoshikawa, 2003). The genetic interactions found in this study between drl and Src64 support recent findings suggesting that Src64 acts downstream of drl in the ventral nerve cord. In addition, the other Drosophila Src kinase, Src42, may be required at two stages, during salivary gland migration along the CVM and downstream of WNT5-DRL signaling as the gland moves onto the longitudinal visceral mesoderm (Harris, 2007).

Another intriguing finding of this study is the involvement of the two remaining Drosophila RYKs, Drl-2 and dnt, in salivary gland development. The phenotypes of Drl-2 and dnt mutants are less penetrant than drl mutants, but they are qualitatively very similar. Furthermore, embryos doubly heterozygous for drl and Drl-2 have salivary glands that resemble those seen in drl mutant embryos. These three RYKs appear to act in a partially redundant fashion in the salivary glands, since none of the single gene mutations leads to completely penetrant phenotypes. However, no increase was seen in penetrance of the drl phenotype in embryos lacking both drl and Drl-2. In addition, it was not possible to detect transcripts for either Drl-2 or dnt in the salivary gland. While it is possible that dnt and Drl-2 are expressed at very low levels in the salivary gland, they might be acting non-autonomously (Harris, 2007).

An interesting dilemma in understanding RYK signaling is how inactive kinases propagate a signal into the cell. Recent mammalian studies have suggested that RYKs may associate with another catalytically active receptor, such as FZ or EPH, at the membrane. In the mouse, the extracellular WIF domain of RYK interacts with FZD8, and it has been proposed that the two proteins may form a ternary complex with WNT1 to initiate signaling. However, data from flies and nematodes support the argument that DRL and its C. elegans homolog LIN-18 act independently of FZ. Genetic studies of cell specification in the nematode vulva suggest that LIN-18 acts in a parallel and separate pathway from the LIN-17/FZ receptor. Similarly, reduction of fz and fz2 gene activity in flies has no effect on a DRL misexpression phenotype in the ventral nerve cord (Yoshikawa, 2003). This study has shown that double mutants for the Wnt4 and Wnt5 ligands and for the fz and drl receptors both show strong enhancements in comparison to the single mutants, reinforcing the conclusion that these two ligands are activating different pathways. In addition, the functions of these two pathways can be separated by phenotype. The Wnt4-fz/fz2 phenotype becomes evident earlier and affects a larger portion of the salivary gland than the Wnt5-drl phenotype. Taken together, these results demonstrate that there are two independent Wnt pathways regulating salivary gland positioning. The early WNT4 signal appears to activate the canonical Wnt pathway, whereas there is a later requirement for WNT5 signaling through DRL and the Src kinases (Harris, 2007).

Wnt4 is a local repulsive cue that determines synaptic target specificity

How synaptic specificity is molecularly coded in target cells is a long-standing question in neuroscience. Whereas essential roles of several target-derived attractive cues have been shown, less is known about the role of repulsion by nontarget cells. Single-cell microarray analysis was conducted of two neighboring muscles (M12 and M13) in Drosophila, that are innervated by distinct motor neurons, by directly isolating them from dissected embryos. A number of potential target cues that are differentially expressed between the two muscles, including M13-enriched Wnt4, were identifed. When the functions of Wnt4, or putative receptors Frizzled 2 and Derailed-2 or Dishevelled were inhibited, motor neurons that normally innervate M12 (MN12s) formed smaller synapses on M12 but instead formed ectopic nerve endings on M13. Conversely, ectopic expression of Wnt4 in M12 inhibits synapse formation by MN12s. These results suggest that Wnt4, via Frizzled 2, Derailed-2, and Dishevelled, generates target specificity by preventing synapse formation on a nontarget muscle. Ectopic expression of five other M13-enriched genes, including beat-IIIc and Glutactin, also inhibits synapse formation by MN12s. These results demonstrate an important role for local repulsion in regulating cell-to-cell target specificity (Inaki, 2007).

In each abdominal hemisegment of embryos and larvae of Drosophila, 37 motor neurons innervate 30 muscles in a highly stereotypic manner. Several candidate target recognition molecules that are expressed in different subsets of muscles have been identified, including Connectin, Fasciclin3, Semaphorin2, NetrinB, Toll, and Capricious. Genetic analysis of these molecules suggests that multiple cues expressed on the target muscles determine the target specificity in a combinatorial and overlapping manner. However, this issue has not been fully addressed; previous studies characterized only a small number of molecules that are expressed in different muscles (Inaki, 2007).

Toward more comprehensive understanding of the molecular basis of target specificity, genome-wide expression profiling was conducted of genes specifically expressed in two neighboring ventral muscles, M12 and M13, which are innervated by distinct motor neurons. M12 is innervated by RP5 and V (collectively called MN12s), whereas M13 is innervated by RP1 and RP4. Because these two muscles show similar morphology, run in parallel, and insert at adjacent muscle insertion sites, they are likely to share most functional characteristics other than neural connectivity. It was therefore reasoned that their subtractive expression profiling might lead to the identification of genes encoding target specificity (Inaki, 2007).

Individual M12s and M13s were collected from abdominal segments of dissected embryos during the stage of motor neuron targeting by aspiration with micropipettes. RNA was extracted from the samples of muscles, each containing 200 cells, and the RNA was amplified through two rounds of linear amplification. Affymetrix Drosophila genome chips were then hybridized with multiple samples of cRNA that was isolated and amplified in independent experiments. Comparing gene-expression profiles of M12 and M13, genes were selected that displayed differential expression consistently in two sets of hybridization experiments. This yielded a list of 96 genes predicted to be preferentially expressed in M12 and 77 genes predicted to be preferentially expressed in M13 (hereafter called M12 and M13 candidate genes) (Inaki, 2007).

A focus was placed on genes that encode putative membrane or secreted proteins with potential roles in target recognition. The predicted differential expression of this class of genes was verified by quantitative real-time RT-PCR analysis (qPCR). Twenty-five of the 34 genes examined gave concordant results with the array data, displaying at least 1.5-fold differential expression between the two muscles. RNA in situ hybridization further confirmed the preferential expression of knockout (ko) in M12, and of Wnt4, beat-IIIc, Sulf1, and CG6867 in M13. These results show specific expression of transcripts that encode a variety of candidate target-recognition molecules in these two muscles (Inaki, 2007).

The role of a prominent candidate gene, Wnt4, which encodes the secreted protein Wnt4 of the WNT family, was genetically analyzed. Wnt4 has been shown to function as an attractive guidance molecule that regulates dorsoventral specificity of photoreceptor-cell projection. Wnt4 is also known to regulate cell movement in the ovary. Wnt4 is expressed in ventral muscles M13 and M26. Much weaker expression is seen in other muscles, including M12. Therefore its function was studied in M12 and M13 targeting. In Wnt4 loss-of-function (LOF) mutant embryos, muscles and major motor nerves showed largely normal development. Specification of the Wnt4-expressing muscle, M13, also appears to be normal because the expression pattern of another M13-enriched gene, Toll, was indistinguishable from that of control. However, the innervation pattern of M12 and M13 was specifically altered. Staining with the anti-Fasciclin II monoclonal antibody 1D4, which visualizes all motor axons, revealed that the axon terminals on M12 were greatly reduced. Remarkably, this reduction of the synaptic endings on M12 was accompanied by the expansion of endings on M13. The number of Bruchpilot-positive putative active zones was also decreased in M12 and increased in M13 in Wnt4 mutants compared to control. These results are consistent with the idea that in Wnt4 mutants, MN12s formed smaller synaptic endings on M12 and instead arborized inappropriately on M13 (Inaki, 2007).

To determine whether the expansion of the M13 terminals in Wnt4 mutants is caused by the formation of ectopic arborization by MN12s, these neurons were specifically labeled with diI. DiI was applied on the M12 nerve endings and RP5 and/or V neurons, whose identities were verified by their axon trajectories and cell-body position, were retrogradely labeled. In late stage 16 wild-type embryos, RP5 neurons retained putative transient contacts on M13. In Wnt4 mutants, the length of the arborizations on M13 formed by RP5 neurons was significantly increased. Wild-type V neurons arborized exclusively on M12 and not along M13. In Wnt4 mutants, V neurons occasionally formed ectopic arborizations on M13, although the frequency is too low to be statistically significant. These results indicate that in Wnt4 mutants, MN12s formed larger or ectopic endings on M13, and further argue that Wnt4 is required to repel and/or restrict inappropriate arborization on M13 formed by MN12s (Inaki, 2007).

The LOF phenotypes suggest that Wnt4 functions in M13 to prevent synapse formation by MN12s. If so, ectopic Wnt4 expression in M12 may inhibit synapse formation by these neurons. To address this possibility, the Gal4-UAS system was used to induce forced expression of Wnt4 in muscles. Strong expression of Wnt4 was induced in all muscles by using the Gal4 driver 24B or E54. In this situation, MN12s often stalled at the edge of M12 and formed much smaller endings. Misexpression of Wnt4 did not cause targeting or pathfinding defects in other regions of the neuromusculature. Wnt4 therefore specifically inhibits synapse formation by MN12s. Next, expression of Wnt4 was induced only in M12 by using a more specific driver, 5053A-Gal4. Because 5053A-Gal4 induces a much higher level of Wnt4 expression in M12 than that of endogenous Wnt4 in M13, this experimental manipulation reverses the relative levels of Wnt4 expression in these muscles. In 5053A-Wnt4 embryos, the arborizations on M12 were greatly reduced in size, as was observed in 24B-Wnt4 embryos. In addition, unlike in 24B-Wnt4 embryos, the arborizations on M13 were enlarged. These results are consistent with the idea that the M12 motor neurons determine target specificity by detecting the relative levels of Wnt4 expressed by these two muscles. Taken together, LOF and gain-of-function (GOF) analyses indicate that differential expression of Wnt4 in M12 and M13 is critical for their targeting (Inaki, 2007).

Which receptor and signaling pathway in motor neurons mediate muscle-derived Wnt4 repulsion? Wnts bind to Frizzled (Fz) family receptors, and the receptor activation in turn activates the intracellular protein Dishevelled (Dsh). Wnt family proteins also bind to other classes of receptors, including Derailed/Ryk family members, which have been shown to transduce Wnt-mediated attraction or repulsion during axon guidance. Previous studies in the visual system and in the ovary have shown that Fz2 and Dsh are involved in Wnt4 signaling. Therefore whether Fz2 and Dsh are required for proper targeting in the neuromuscular system was investigated. Also the possible involvement of Derailed family members was also studied. When the function of Fz2 or Dsh was inhibited by expressing a dominant-negative form of these molecules, the same defects were observed in the targeting of M12 and M13 as observed in Wnt4 mutants. Similarly, LOF of derailed-2 (drl-2) causes the highly specific phenotype in the targeting of M12 and M13. These results suggest that Fz2, Dsh, and Drl-2 are involved in the signaling of Wnt4 repulsion in motor neurons. Whereas Fz2 is expressed in most or all neurons, drl-2 is expressed in subsets of neurons in the CNS. The specific expression of drl-2 may explain why Wnt4 is repulsive to only subsets of motor neurons (Inaki, 2007).

Systematic GOF analyses of the other candidate genes identified by the expression profiling was performed, and it was found that pan-muscle expression of five other genes, beat-IIIc, Glt, Lsp2, Sulf1, and CG6867, caused a reduction of MN12 nerve terminals similar to that seen when Wnt4 was misexpressed. All of these genes are normally expressed in M13 and thus, like Wnt4, may function as repulsive cues that inhibit synapse formation by MN12s. As in the case of Wnt4, misexpression of these five genes did not cause targeting or pathfinding defects in other regions of the neuromusculature. These results suggest a repulsive role for beat-IIIc, Glt, Lsp2, Sulf1, and CG6867 in specific aspects of target selection (Inaki, 2007).

Several molecules have previously been shown to function as attractive target cues that determine target specificity, including Netrins and Capricious in Drosophila, SYG-1 in C. elegans, and Sidekicks in vertebrates. However, little is known about the role of repulsion during target selection. During axon pathfinding, repulsive cues presented by surrounding tissues restrict the direction of the axons by deflecting or arresting their growth. Axons can also be guided by gradients of repulsive cues. Does repulsion also limit the choice of distinct target cells and thus mediate cell-to-cell specificity? Previous GOF analyses of semaphorin2 and Toll in Drosophila showed that they can inhibit synapse formation of specific motor neurons. However, whether such inhibition is essential for the selection of target cells is unknown. This study shows that Wnt4 is required for target recognition by MN12s. In wild-type, upon entering the M12/M13 target region, MN12s selectively innervate M12 with only a small putative transient contact on M13. In Wnt4 mutants, the target preference of these neurons is shifted to M13. This suggests that Wnt4 normally prevents MN12s from making large synapses on M13, and this Wnt4-mediated repulsion on M13 is required to lead these neurons to an alternative target, M12. Data from GOF analyses further support the notion that differential expression of Wnt4 in these two muscles is critical for target selection by MN12s. These results provide strong evidence that local repulsion plays a major role in target specificity (Inaki, 2007).

Microarray analysis identified a number of putative cell-surface or secreted proteins, in addition to Wnt4, that were differentially expressed between the two muscles. Furthermore, GOF analysis suggested that at least five of them may function, like Wnt4, as repulsive cues on M13. Some of them encode proteins with domains implicated in axon guidance and synapse formation; Beat-IIIc belongs to the Beat family of proteins with immunoglobulin motifs, and Glt belongs to a family of cell-surface proteins with cholinesterase domains. Identification of such a large number of potential cues in a single target cell is unprecedented and provides a valuable opportunity to study the mechanisms of target recognition. Future genetic analysis of these molecules, alone and in combination, may more clearly elucidate the mechanism of how these multiple target cues coordinate to determine target specificity (Inaki, 2007).

Drosophila Tey represses transcription of the repulsive cue Toll and generates neuromuscular target specificity

Little is known about the genetic program that generates synaptic specificity. This study shows that a putative transcription factor, Teyrha-Meyhra (Tey), controls target specificity, in part by repressing the expression of a repulsive cue, Toll. This study focused on two neighboring muscles, M12 and M13, which are innervated by distinct motoneurons in Drosophila. It was found that Toll, which encodes a transmembrane protein with leucine-rich repeats, is preferentially expressed in M13. In Toll mutants, motoneurons that normally innervate M12 (MN12s) form smaller synapses on M12 and instead appear to form ectopic nerve endings on M13. Conversely, ectopic expression of Toll in M12 inhibited synapse formation by MN12s. These results suggest that Toll functions in M13 to prevent synapse formation by MN12s. Tey was identified as a negative regulator of Toll expression in M12. In tey mutants, Toll is strongly upregulated in M12. Accordingly, synapse formation on M12 was inhibited. Conversely, ectopic expression of tey in M13 decreased the amount of Toll expression in M13 and changed the pattern of motor innervation to the one seen in Toll mutants. These results suggest that Tey, which contains no known transcription factor motifs, determines target specificity by repressing the expression of Toll. These results reveal a mechanism for generating synaptic specificity that relies on the negative regulation of a repulsive target cue (Inaki, 2010).

A remarkable feature of the nervous system is the precision of its circuitry. A neural circuit develops through a series of neuronal recognition events. First, neurons find their path, turn at mid-way guideposts, and fasciculate or defasciculate before reaching their final target area. Then, neurons select and form synapses with specific target cells in the target region. The final matching of pre- and post-synapses is thought to be mediated by specific cues expressed on the target cells. However, the regulation and function of such cues remain poorly understood (Inaki, 2010).

The process of neuromuscular targeting in Drosophila features highly stereotypic matchings between 37 motoneurons and 30 target muscle cells, providing a unique model system for the study of neuronal target recognition. Several target cues, including Capricious, Netrin-B and Fasciclin 3, have been identified that are expressed in specific target cells and mediate attractive interactions between the synaptic partners. It has recently been shown that target specificity is also regulated by repulsion from non-target cells. Wnt4, a member of the Wnt family of secreted glycoproteins, is expressed in muscle 13 (M13) and prevents synapse formation by motoneurons targeted to a neighboring muscle, M12. In the absence of Wnt4, motoneurons targeted to M12 form ectopic nerve endings on M13, indicating that Wnt4 repulsion on M13 is required for proper targeting of the motoneurons. In addition to Wnt4, Toll and Semaphorin II (Sema-2a - FlyBase) are known to function as negative regulators of synapse formation in this system. However, whether they have a role in target selection remains unknown (Inaki, 2010).

Another unsolved issue is how the expression of such attractive or repulsive target-recognition molecules is regulated. It is amazing that the expression of these molecules is so precisely regulated that they are present at the right time and place. It is likely that the expression of these molecules is determined as part of the differentiation program of the target cells. However, little is known about the molecules and mechanisms involved. Several transcription factors, such as S59, Krüppel and Vestigial, have been identified as being expressed in subsets of muscle cells. They are expressed from the progenitor stage, and their loss-of-function (LOF) and gain-of-function (GOF) alter the specific characteristics of the individual muscles, such as their size, shape, orientation and attachment sites to the epidermis, indicating that they function as determinants of a particular muscle fate. However, whether these transcription factors regulate the expression of target-recognition molecules and thus determine the innervation pattern is unknown (Inaki, 2010).

A comparative microarray analysis has been conducted of two neighboring target muscles, M12 and M13, that are innervated by distinct motoneurons (Inaki, 2007). By comparing the expression profile of the two muscles, attempts were made to understand the molecular mechanisms that make these muscles distinct targets for the motoneurons. From this screening, ~25 potential target-recognition molecules were identified as preferentially expressed in either muscle cell. Among them was Wnt4, mentioned above. This study reports the functional analyses of two additional genes that were identified in the screening: Toll and teyrha-meyrha (tey). Toll encodes a transmembrane protein with extracellular leucine-rich repeats, and has multiple functions in development. Toll is expressed in subsets of muscles, including 6, 7 and 15-17. Previous studies have shown that Toll inhibits synapse formation by RP3, a motoneuron targeted to muscles 6 and 7. This study shows that Toll is preferentially expressed in M13 over M12 and, like Wnt4, inhibits synapse formation by motoneurons targeted to M12. It was also shown that tey, a previously uncharacterized gene, regulates the expression of Toll in specific muscles. tey is expressed specifically in M12, where it negatively regulates Toll expression. In the absence of tey, Toll is ectopically expressed in M12 and innervation of M12 is inhibited. These results suggest that Tey regulates targeting by downregulation of the repulsive cue Toll specifically in M12. Based on these results, a mechanism is proposed for the generation of synaptic specificity that relies on negative regulation of repulsive target cues (Inaki, 2010).

Toll is preferentially expressed in M13 over M12. The size of M12 terminals was decreased in Toll mutants, with concomitant expansion of M13 terminals. This phenotype is very similar to that of Wnt4 LOF and is likely to be caused by MN12s forming ectopic synapses with M13, although it remains possible that some of the ectopic nerve endings on M13 are formed by other motoneurons. Furthermore, it was observed that the size of M12 terminals is reduced when Toll is misexpressed on the muscle. The LOF and GOF analyses suggest that Toll functions as a repulsive factor in M13 that is important for target selection by MN12s. Thus, Toll provides another example of a repulsive factor that is involved in target selection. How Toll mediates the repulsive signal to motoneurons is currently unknown. A model is that Toll functions as a ligand that is expressed in muscles and signals through receptor(s) expressed in motoneurons. However, no receptor has been identified for Toll. Toll has been shown to function as a receptor, not a ligand, in other systems, such as in dorsoventral patterning and innate immunity. Another possibility is that Toll might mediate the modification or regulation of other targeting molecules, such as Wnt4 (Inaki, 2010).

M13 expresses at least two repulsive cues, Wnt4 and Toll, that are important for the targeting of M12 and M13. It seems that these two molecules contribute to target specificity in a manner that is redundant with yet other molecules because in both single and double mutants of these genes, the connectivity is only partially disrupted. Previously, other potential repulsive cues that are expressed in M13 were identified, including Beat-IIIc and Glutactin (Inaki, 2007). Ectopic expression of these molecules in M12 inhibits synapse formation by MN12s, as observed when Toll and Wnt4 are misexpressed. Although the precise roles of these molecules remain to be verified by LOF analyses, these results suggest the possibility that a single muscle, M13, expresses a number of repulsive cues that are involved in targeting of motoneurons. This is consistent with previous hypotheses that Drosophila neuromuscular connectivity is determined by highly redundant mechanisms. It will be important to determine how the signals from multiple cues are integrated to generate the precise pattern of synaptic connections. It will also be interesting to examine whether other muscles similarly express repulsive cues to prevent inappropriate innervation. The phenotypes of Wnt4 Toll double mutants were of similar severity to those of the single mutants. This might be due to the presence of other targeting molecules, as described above. Toll and Wnt4 might also function in the same signaling pathway. For example, Toll may be involved in the regulation of Wnt4 activity through influencing its secretion, localization or protein modification. Toll and Wnt4 might also act as repellents for distinct MN12s (Inaki, 2010).

This study has shown that a novel nuclear protein, Tey, regulates the expression of Toll and is important for the determination of target specificity. tey regulates the position, orientation and attachment sites of M12. Thus, Tey seems to act as a determinant of several important properties of M12, regulating both the differentiation of the muscle itself and the specificity of nerve innervation. Expression of tey is remarkably specific, being limited within the somatic musculature to a single muscle, M12. Other, known muscle-determinant genes were expressed in broader subsets of muscles (Inaki, 2010).

Tey negatively regulates the expression of Toll in M12. In tey mutants, Toll expression is strongly upregulated in M12. This indicates that tey is required in this muscle to specifically suppress Toll expression. Consistent with this, ectopic expression of tey in M13 partially suppressed Toll expression. Toll is normally expressed in most of the other ventral muscles, including muscles 6, 7, 13-17, but not in M12, suggesting that some positive transcriptional regulator(s) higher up in the hierarchy activate Toll expression in this group of muscles and that negative regulation by Tey is required to suppress Toll expression only in M12. The regulation of Toll by Tey should be at the transcriptional level because the expression of the exogenously introduced Toll enhancer-trap lacZ reporter is affected in tey mutants or when tey is misexpressed. It remains to be determined whether Tey binds directly to the regulatory region of the Toll gene or regulates Toll transcription in an indirect manner (e.g. by regulating other transcription factors). Tey contains no known transcription factor motifs. The expression of another M13-enriched gene, Wnt4, was not affected in tey mutants or when tey was misexpressed. Unlike Toll, Wnt4 is expressed in only two ventral muscles: 13 and 30. Thus, expression of Wnt4 might be regulated in a different manner to Toll, possibly by positive transcription factors that are specifically expressed in these muscles. It will be interesting to determine how the expression of target-recognition molecules is precisely regulated by the combinatorial action of positive and negative transcription factors (Inaki, 2010).

In tey mutants or when tey is misexpressed, neuromuscular connectivity was also altered in a manner consistent with the misregulation of Toll expression. The inappropriate presence of Toll repulsion in tey LOF mutants suppressed synapse formation on M12. Conversely, suppression of Toll expression in M13 in tey GOF mutants led to changes in the innervations of M12 and M13, similar to those observed in Toll mutants. Furthermore, the effects of tey GOF were dramatically reversed when Toll was co-expressed with tey, suggesting that Toll is the major target of tey in causing the GOF phenotypes. These results suggest that Tey regulates neuromuscular connectivity by specifically repressing Toll expression in M12. As noted above, Toll is normally expressed in a number of ventral muscles, but not in M12. Furthermore, Toll is expressed in M12 in the absence of Tey suppression in tey mutants. This suggests that the default state is for Toll to be expressed in all ventral muscles, possibly by the action of positive transcription factor(s) expressed in these muscles. Tey might therefore generate target specificity by suppressing the expression of Toll in one among a group of muscle cells expressing the repulsive cue. The data thus suggest a mechanism of transcriptional control of target specificity, namely, the negative regulation of repulsive cues (Inaki, 2010).

A role for Drosophila Wnt-4 in heart development

In vertebrates, different Wnt signaling pathways are required in a temporally coordinated manner to promote cardiogenesis. In Drosophila, wingless holds an essential role in heart development. Among the known Drosophila Wnts is DWnt4, the function of which has been studied in various developmental processes except for heart development. This study re-evaluated the expression pattern of DWnt4 during embryogenesis and showed that transcripts are not restricted to the dorsal ectoderm but are also present in the cardiogenic mesoderm. Moreover, DWnt4 mRNA transcripts were detected in myocardial cells by stage 16. The heart phenotype in DWnt4 mutant embryos is characterized by various degrees of disrupted expression of different cardiac markers. Overexpression of Dwnt4 also affects heart marker expression, which can be partially rescued by simultaneous inhibition of PKC. These data reveal a role for DWnt4 in cardiogenesis, however integration of DWnt4 with other known signaling pathways that function in heart development still awaits further investigation (Tauc, 2012).

Previously published data indicate that DWnt4 expression in the visceral mesoderm is regulated by Hox genes, in particular by Ultrabithorax (Ubx) and abdominal A (abd-A). Four Hox genes, Antennapedia (Antp), Ubx, abd-A and abd-B are expressed in the Drosophila heart where they specify different regions along the anterior-posterior axis of the heart tube. Therefore, it may be that members of the Hox gene family regulate DWnt4 expression also in the heart tube. Ubx is expressed in the aorta where low levels of DWnt4 mRNA expression and the higher expression levels of Dwnt4 in the heart proper correlate with the expression of Abd-A. The strong accumulation of DWnt4 transcripts in the heart proper is detected at what appears to be the border between high-level Abd-A and Abd-B expression. Ubx and abd-A were also shown to be involved in the establishment and patterning of alary muscles that project from the dorsal vessel. There are seven pairs of alary muscles that attach the heart to the dorsal epidermis in larvae and in adult flies. The extracellular matrix marker Pericardin (Prc) is not only expressed around pericardial cells and in the basal membrane of myocardial cells but also accumulates along the alary muscles. It was noticed that in DWnt4^EMS23 mutants the number of Prc positive projections is affected. Although the phenotype was not quantified, it was observed that the number of projections varied. For example additional Prc positive projections were seen at positions different from where the seven pairs of alary muscles normally attach. Not much is known about the embryonic origin of the alary muscles and the molecules required for their development. The current data may spur investigations on the role of DWnt4 in the development of these muscles. It is intriguing to hypothesize that DWnt4 acts as a guidance cue (attractive or repulsive) for the alary muscle attachment site. A guidance function for DWnt4 has been previously described in the context of dorsoventral projections of retinal axons, of motor neuron target specificity and in salivary gland migration (Tauc, 2012).

The early expression pattern of DWnt4 in the cardiac mesoderm and in the overlying ectoderm suggested that DWnt4 could be involved in early steps of cardiogenesis such as cardiac specification and differentiation of cardiac cell types. All cardiac marker genes that were analyzed in DWnt4^EMS23 mutant embryos exhibited a range of degrees of disruption, none of which had serious detrimental effects though. Hence, in contrast to wg, DWnt4 does not appear to be essential for Drosophila cardiogenesis. Nevertheless, DWnt4 does play a role to ensure normal cardiac marker gene expression. Whereas in DWnt4 mutants a mild loss of Svp positive cells (or only Svp expression) was observed, DWnt4 overexpression resulted in the loss and increase of Svp expressing cells. It has been suggested that DWnt4 modulates cell fate specification within the Hedgehog-dependent domain and hedgehog was shown to regulate Svp expression. Hence, it is intriguing to speculate that the Svp phenotype is caused by defective Hh signaling that results from inappropriate amounts of DWnt4. Next attempts were made to investigate which components may mediate the DWnt4 signal. Immunostainings for Prc revealed two phenotypes in DWnt4 mutants. Embryos were characterized by gaps in Prc expression along the heart tube and/or by a detachment of Prc expressing cells from the Prc positive basal membrane, which indicates the detachment of pericardial cells from myocardial cells. These phenotypes are reminiscent of the phenotypes described for embryos that are mutant for the α-subunit of the heterotrimeric Go protein bkh. Gαo was shown to couple to the seven transmembrane Fz receptors and mediate Wnt signaling as well as planar polarity signaling. fz mutants, like bkh mutants, exhibit both phenotypes: gaps in Prc and a detachment of pericardial cells from myocardial cells. Of note, DWnt4 was shown to be able to bind to three Fz receptors Fz, Fz2 and Fz4. Unlike Fz2, which was shown to solely activate the arm-dependent Wg signaling pathway, Fz can also mediate a non-canonical, planar polarity signal (Tauc, 2012).

Since similar phenotypes were observed for Prc in DWnt4, fz and bkh mutants tests were performed to see whether fz and bkh may be components of the DWnt4 signaling pathway. The rationale was that if these molecules act in the same pathway, an increase of severity and/or penetrance of the phenotype in would be expected double heterozygous embryos. The results do not support such a simple linear relationship with respect to Prc expression along the heart tube. Nevertheless, changes were observed in the number of embryos showing a particular phenotype, which suggests that these factors could be genetically interacting. Due to the complexity of the data, a straight-forward interpretation is somewhat difficult at this point. Reasons for such phenotypic changes could be due to the involvement of different molecular mechanisms underlying either the gap or detachment phenotype. For example, gaps in Prc expression could result from a defective mesenchymal-epithelial transition required for proper heart morphogenesis as was shown for bkh mutants. One cause for the detachment phenotype is a misregulation of septate junction proteins present in myocardial and pericardial cells where bkh is also involved. It is feasible that DWnt4 may function as a guidance cue for the proper migration of mesodermal cells. Irregularities in mesoderm migration can also lead to heart defects similar to the ones seen in DWnt4 mutants, which was shown for example in embryos mutant for the FGF receptor heartless or for the proteoglycan syndecan (Tauc, 2012).

In addition to Prc possible genetic interactions between DWnt4 and fz or DWnt4 and bkh were examined with respect to the reduction in Odd expressing pericardial cells that weee detected in all single mutants. This analysis indeed revealed that the phenotype increased in embryos that were double heterozygous for DWnt4 and bkh compared to embryos that were single heterozygous for each factor (Tauc, 2012).

As to a possible genetic interaction between DWnt4 and fz, the data is less convincing since the phenotype was already highly penetrant in fz/+ heterozygous embryos. Multiple publications have indicated that DWnt4 acts through a non-canonical, arm-independent Wnt pathway. Therefore whether JNK, a core component of the planar polarity pathway, may be part of the DWnt4 signal transduction machinery was tested. Since ectodermal JNK signaling is essential for the morphogenetic process of dorsal closure, which impinges on normal heart development as a secondary effect, the pathway was interrupted in a tissue-specific manner. Mesodermal inhibition of JNK signaling using the dominant-negative bsk construct did not elicit a heart phenotype. Mesodermal inhibition of the canonical Wg signaling pathway using a dominant-negative construct of TCF was detrimental to heart development as expected. These results exclude a primary function for JNK in cardiogenesis and due to the lack of resemblance to the phenotypes in DWnt4 mutants, it is unlikely that JNK is a component of the DWnt4 signaling pathway in this context. Although inhibition of the canonical Wg pathway has a much more dramatic effect on cardiogenesis than lack of DWnt4, the data still leaves the option that DWnt4 may also act through the canonical pathway. Despite several publications that indicate that DWnt4 activates a non-canonical pathway, Harris and Beckendorf (2007) concluded from their data that DWnt4 acts through a canonical Wnt pathway during salivary gland migration. PKCs were shown to mediate non-canonical Wnt signaling in vertebrates and were implicated in mediating the DWnt4 signal during ovarian morphogenesis. This study performed experiments to determine whether PKCs could function in the DWnt4 signaling pathway in heart development expressing a characterized PKC pseudosubstrate that inhibits all PKCs present in Drosophila. The results show that by inhibiting PKC signaling, the amount of embryos exhibiting a reduction in Svp and Odd expressing cells was decreased after the overexpression of DWnt4. Admittedly this is an indirect indication that PKC may mediate the DWnt4 signal but this piece of data together with previously published results encourages further investigation of this possibility (Tauc, 2012).

In summary, although the definite function of DWnt4 in cardiogenesis still awaits further investigation, the data provides a good platform for subsequent analyses of DWnt4 in heart development. In particular with respect to the newly described cardiac expression pattern of DWnt4, future results can be anticipated that demonstrate a function for DWnt4 in heart tube formation and heart function (Tauc, 2012).

Wnt4 is required for ostia development in the Drosophila heart

The Drosophila ostia are valve-like structures in the heart with functional similarity to vertebrate cardiac valves. The Wnt/β-catenin signaling pathway is critical for valve development in zebrafish and mouse, but the key ligand(s) for valve induction remains unclear. This study observed high levels of Wnt4 gene expression in Drosophila ostia progenitor cells, immediately prior to morphological differentiation of these cells associated with ostia formation. This differentiation was blocked in Wnt4 mutants and in flies expressing canonical Wnt signaling pathway inhibitors but not inhibitors of the planar cell polarity pathway. High levels of Wnt4 dependent activation of a canonical Wnt signaling reporter was observed specifically in ostia progenitor cells. In vertebrate valve formation Wnt signaling is active in cells undergoing early endothelial-mesenchymal transition (EMT) and the Wnt9 homolog of Drosophila Wnt4 is expressed in valve progenitors. In demonstrating an essential role for Wnt4 in ostia development this study has identified similarities between molecular and cellular events associated with early EMT during vertebrate valve development and the differentiation and partial delamination of ostia progenitor cells in the process of ostia formation (Chen, 2016).

Evolutionarily conserved genetic pathways control heart development from Drosophila to. The linear heart tube or dorsal vessel of Drosophila, although very simple in structure and cell type composition, incorporates essential counterparts of the much more complicated mammalian heart, which also begins development as a linear tube. These include a pumping heart chamber, an aorta, an outflow tract, and ostia that are functionally analogous to vertebrate cardiac valves. The simplicity and genetic tractability of the Drosophila heart makes it a highly useful model for vertebrate heart development, facilitating the study of a large number of genes involved in congenital heart defects (CHD), the most common form of human birth defects. Although many genes have been identified that play conserved roles in cardiac morphogenesis in Drosophila and vertebrates, little is known about the genes specifying and regulating the formation of ostia, also termed inflow tracts, that function as valves to control unidirectional flow of hemolymph into the Drosophila heart. Studies in mouse and zebrafish have identified many evolutionarily conserved signaling pathways including Wnt, BMP/TGF, Notch and VEGF involved in heart valve development. The roles of these signaling pathways in Drosophila heart development after cardiac specification, however, remain largely unknown formation (Chen, 2016).

The Wnt signaling pathway plays an essential role in heart development from Drosophila to humans. In Drosophila the wingless gene, together with Dpp, is required for specification of cardiac progenitor cells in early heart development. Abolishing Wg signaling leads to absence of the dorsal vessel. Numerous studies in vertebrate models have revealed distinct positive and negative regulatory roles for Wnt signaling during different stages of heart development. In zebrafish, nuclear beta-catenin, the hallmark of activated Wnt signaling, accumulates only in valve-forming cells, and a Wnt pathway reporter was expressed specifically in the developing cardiac valves. Mutations in negative regulators of the Wnt pathway lead to increased expression of heart valve markers, whereas overexpressing negative regulators block valve formation. In mice, multiple Wnt ligands and a Wnt signaling pathway reporter are specifically expressed in developing valves. Therefore, activation of Wnt signaling can be used as a marker for valve differentiation, but the specific Wnt ligand(s) and the precise role(s) of Wnt signaling in vertebrate heart valve formation remain undefined formation (Chen, 2016).

The Drosophila dorsal vessel features both valve-like ostia that regulate hemolymph flow into the heart and specialized cardiomyocyte cells, inter-chamber valves, that divide the heart into multiple chambers. The normal differentiation of the latter cell type requires the gene pygopus (pygo), a component of Wnt signaling during Drosophila development. Pygo reportedly functions independently of Wnt signaling, however, in inter-chamber valve formation (Tang, 2014). Pygo ortholog functions in mouse heart development have not been described formation (Chen, 2016).

The genetic control of ostia formation in Drosophila remains largely unknown. The earliest marker for ostia progenitors is expression of the COUP-TFII orphan nuclear receptor Seven-up (Svp), in two out of the six cardioblasts in each hemisegment. However, svp expression alone is not sufficient to induce ostia formation since it is also expressed in anterior cardioblasts that do not differentiate into ostia, as well as in the pericardial cells throughout the heart tube formation (Chen, 2016). Discussion

This study demonstrates that Wnt4 and the canonical Wnt signaling pathway are essential for ostia formation in Drosophila. A Wnt signal reporter is highly activated in ostia progenitors and reporter activity is dependent on Wnt4. In vertebrates, Wnt signaling activity is essential for cardiac valve formation. Multiple Wnt ligands as well as a Wnt signal reporter are expressed in developing valves and Wnt reporter activity was also observed in the valve-forming region in zebrafish. Given the complexity of vertebrate valve structure and the large number of vertebrate Wnt ligands, it has proven difficult to identify the role(s) of specific Wnt ligands in activation of Wnt signaling pathway(s) during cardiac valve development. In Drosophila, by contrast, this study identified a predominant role for Wnt4 in ostia development, though wg is also expressed in ostia progenitor cells. A subtle role of Wg signaling in ostia formation formation cannot be excluded (Chen, 2016).

The discovery that a Wnt4-dependent canonical Wnt signaling pathway reporter is specifically expressed in Drosophila ostia, and that Wnt4 is required for high level Wnt signal activation in ostia suggest that the canonical Wnt signal pathway plays an evolutionarily conserved role in invertebrate ostia and vertebrate valve development. Vertebrate Wnt9b and Wnt4 are the closest homologs of Drosophila Wnt4. The fact that these Wnt ligands were found to be expressed in the valve forming region in mice (Alfieri, 2010) make them promising candidates for further studies of Wnt involvement in vertebrate cardiac valve development. Particularly striking similarities exist between the early endothelial-mesenchymal transition (EMT) associated with the initiation of vertebrate valve development in the presumptive A-V canal of the early heart tube, and the formation of ostia in the embryonic and larval heart tube of Drosophila. Early EMT is characterized by unknown Wnt ligand mediated Wnt signaling pathway activation in cells of the endothelial cell layer lining the heart tube. These cells undergo differentiation, with an initial morphological constriction phase associated with protrusion of the cell body out of the plane of endothelium (a partial delamination). This early EMT is succeeded by complete delamination and differentiation of the cells, which detach entirely from the endothelium, migrate into the underlying cardiac jelly (extracellular matrix) and proliferate, as part of the process of forming the cardiac cushion precursor of the vertebrate heart valve. In ostia formation, ostia progenitor cells expressing Wnt4 and activation of the canonical Wnt signaling pathway undergo a partial delamination from the precisely aligned row of cardioblasts, associated with differentiation into a cell displaying a constricted and elongated morphology, with cell body protrusion into the heart tube lumen. The cell layer in which this process occurs in Drosophila is developmentally analogous to the vertebrate endothelial cell layer. However, the Drosophila Tin-expressing cardioblasts adjacent to the ostia progenitors are in fact myocardial cells. Nevertheless the early EMT-like features of ostia development specifically require Wnt4 activity and do not require Wg. Further analysis of ostia formation may facilitate identification of the specific Wnt signaling components required formation (Chen, 2016).

Wnt ligands are known to play dual roles in vertebrate heart development, with Wnt signaling pathway(s) promoting cardiac specification at the early stage of cardiac mesoderm formation, but repressing myocardial cell fate at a later stage in the differentiated heart field. In Drosophila heart development, Wnt signaling activated by Wg is known to promote early stage cardiac cell specification. A previous study of Wnt4 in Drosophila heart development (Tauc, 2012) showed cardiac cell specification defects in Wnt4 mutants, using antibody staining against Eve. However, the current study did not observe the same defects in early stage 11-13 embryos, consistent with the observation that Wnt4 was not expressed in cardiac progenitor cells during stages 11 to 13. In Wnt4 mutants ostia progenitor cells do not differentiate, but retain a cardioblast cell morphology. This is consistent with a role for Wnt4 during normal development in promoting ostia cell differentiation, in opposition to a myocardial cell fate. In Drosophila, similar to vertebrates, Wnt signaling plays dual roles in heart development: promoting early stage cardiac cell specification through Wingless, and opposing the adoption of a myocardial cell fate in ostia progenitor cells through Wnt4 formation (Chen, 2016).

The Wnt signaling reporter transgene is exclusively expressed in ostia progenitor cells in the Drosophila heart. This is analogous to the Wnt pathway reporter activity described in valve progenitor cells during zebrafish and mouse heart development. Compared to the ostia-specific expression of the Wnt pathway reporter, the Drosophila wnt4 gene expression was not as exclusive. This may indicate that as yet unidentified Wnt signaling inhibitors are active in non-ostia progenitor cells. The activity of such inhibitors may explain why over expression of wnt4 in all cardioblasts did not induce the formation of ectopic ostia. Wnt pathway inhibitors have been shown to play essential roles in vertebrate heart development and valve formation, and it is reasonable to propose that Wnt inhibitors likely play evolutionarily conserved roles in Drosophila ostia development formation (Chen, 2016).

A previous study of Wnt4 in the Drosophila heart did not identify downstream Wnt signaling pathway components (Tauc, 2012). This study used multiple approaches to demonstrate that Wnt4 functions through the canonical Wnt signaling pathway. By contrast, perturbation of components of the non-canonical planar cell polarity signaling pathway did not induce defects. Using both loss-of-function and gain-of-function assays, this study showed that the canonical Wnt signal reporter expression in ostia is Wnt4-dependent: loss of Wnt4 largely abrogates Wnt reporter expression in ostia, whereas ectopic Wnt4 expands Wnt signal reporter expression to non-ostia cells formation (Chen, 2016).

The observations reported in this study illustrate the potential of Drosophila as a platform for conducting comprehensive genetic studies to elucidate the signaling pathway components and pathway interactions underlying normal cardiac valve morphogenesis as well as providing a better understanding of molecular abnormalities associated with cardiac valve defects and congenital heart disease formation (Chen, 2016).

Wg and Wnt4 provide long-range directional input to planar cell polarity orientation in Drosophila

Planar cell polarity (PCP) is cellular polarity within the plane of an epithelial tissue or organ. PCP is established through interactions of the core Frizzled (Fz)/PCP factors and, although their molecular interactions are beginning to be understood, the upstream input providing the directional bias and polarity axis remains unknown. Among core PCP genes, Fz is unique as it regulates PCP both cell-autonomously and non-autonomously, with its extracellular domain acting as a ligand for Van Gogh (Vang). This study demonstrates in Drosophila wings that Wg (Wingless) and dWnt4 (Drosophila Wnt homologue) provide instructive regulatory input for PCP axis determination, establishing polarity axes along their graded distribution and perpendicular to their expression domain borders. Loss-of-function studies reveal that Wg and dWnt4 act redundantly in PCP determination. They affect PCP by modulating the intercellular interaction between Fz and Vang, which is thought to be a key step in setting up initial polarity, thus providing directionality to the PCP process (Wu, 2013).

The data indicate that Wg/dWnt4 regulate the establishment of Fz–PCP axes by modulating the Fz–Vang intercellular interactions in a graded, dosage dependent manner. Consequently they might generate different levels of Fz–Vang interactions across a Wg/dWnt4 gradient experienced by cells. This process is reiterated across the tissue, and the directionality of Fz–Vang binding is subsequently reinforced by intracellular core PCP factor interactions. The data are consistent with a model in which Wg/dWnt4 generate a Fz ‘activity'), suggesting that both of these light sensors are necessary for light avoidance behavior.' gradient models. Accordingly, PCP axes are orientated towards the Wg/dWnt4 source, which is evident in (at least) the wing and eye. The early wing PCP axis (late larval to early pupal stages) correlates well with Wg/dWnt4 margin expression and, similarly, in the eye polarity is oriented in the dorsoventral axis towards the poles where Wg/Wnt4 are expressed. This model, relying on a Fz–Vang interaction, is also compatible with the addition of Fmi to this scenario, with intercellular (homophilic) Fmi–Fmi interactions also being required for PCP specification. As Fmi forms complexes with both Fz and Vang, the full complement of intercellular interactions includes Fz/Fmi–Fmi/Vang complexes, and these interactions would also be modulated by Wnt binding to Fz, either directly as proposed in this model or possibly by modulating the Fmi–Fmi interactions by Fmi being associated with Fz that is bound to different levels of Wg/Wnt4. In vivo, Fmi helps to enrich both Fz and Vang to the subapical junctional region, and Fmi–Fmi interactions bring Fz and Vang to close molecular proximity (Wu, 2013).

Intercellular Fmi–Fmi interactions are strong, as Fmi-expressing S2 cells form cell aggregates through homophilic Fmi interactions. The interaction between Fz and Vang is weaker, and cell–cell contacts between the two cell groups are infrequent. It was suggested that PCP signal sensing complexes include both Fmi and Fz on one cell interacting with Fmi/Vang at the surface of a neighbouring cell. Within these complexes, Fz is required for sending a polarity signal, whereas Fmi and Vang are involved in its reception, consistent with the data and model. Although it has been suggested that Fmi is capable of sensing Fz/Fmi signals in the absence of Vang, the 'Fz-sensing' capability of cells with Fmi alone (lacking Vang) is much weaker than that of cells with Vang. It will be interesting to determine if there are other PCP regulators directly involved in modifying Fmi–Fmi interactions (Wu, 2013).

How do these data relate to previous models and why was the Wg/Wnt4 requirement not observed before? Previous work attempted to address the role for the wing margin in PCP by examining either mutants affecting wing margin cells without eliminating wg/Wnt expression or in clones. Although cellular hairs near the site of wing margin loss point towards remaining wing margin areas, the effect Is considered weak. Potential effects were examined of Wnt LOF clones of Df(2L)NL, lacking wnt4, wg, wnt6 and wnt10. In contrast to the global reduction of Wg/Wnt4 through the temperature sensitive wg allele, such clones cause only mild PCP perturbations. There are several reasons why clonal loss of Wnt expression in the margin only mildly affects PCP orientation: cells can respond to Wnts from several sources/cells from remaining Wnt-expressing wing margin regions; polarization strengths (measured by nematic order) in the first few rows of cells near the margin are much weaker than those in cells further away (at 14-17 h APF) and weak PCP reorientation in cells neighbouring wing margin clones could thus reflect the initial weak polarization in these cells; and PCP orientation changes from its initial radial polarity towards the proximodistal polarity during hinge contraction morphogenesis and associated cell flow, probably leading to significant corrections of subtle defects near the margin. Similarly, PCP orientation in cells near the margin is only very weak early (at 14-16 h APF), probably because cells close to the Wnt-producing cells are exposed to saturated Wnt levels (and not a Wnt gradient), or because the presence of other organizers (directing polarity parallel to the margin) weakens the effect of Wnts. PCP in these cells is established/corrected through more local interactions during the feedback loops among neighbouring cells (Wu, 2013).

To determine the direct role for Wg/Wnts on Fz–PCP signalling, it was examined at pupal stages, as the patterning role for canonical Wg signalling is much reduced then and PCP still correlates well with Wg/Wnt4 expression. Importantly, Wnt4 does not affect expression of patterning genes through canonical signalling at larval or pupal stages, yet Wnt4 alters PCP orientation, consistent with the model that Wnt4/Wg act directly on Fz-PCP interactions. The observation that Wnt4 requires Fz to affect neighbouring cells further supports this model. It is likely that, as well as the Wg/Wnt4 input and mechanism identified in this study, both early and late PCP axes depend on further cues, provided for instance by the parallel Ft/Ds-PCP system or other morphogenetic organizers. Strikingly, such a scenario would suggest that Wg regulates PCP directionality through both PCP systems, affecting Fz-PCP interactions directly and through canonical Wg signalling transcriptionally regulating graded fj and ds expression in eyes and wings. In summary, these data provide insight into Wnt-mediated mechanisms to directly regulate long-range Fz–PCP orientation by modulating Fz–Vang/PCP interactions during tissue morphogenesis (Wu, 2013).

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date revised: 5 August 2016

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