wingless
See the embryonic expression pattern of wingless at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
Wingless shows a pattern of 16 regularly spaced bands in the extended germ band stage. These bands coincide with the posterior aspect of each parasegment (Rijsewijk, 1987)
wingless and DWnt4 are
physically clustered and transcribed in overlapping embryonic territories under the
control of the same regulatory molecules. Co-expression and co-regulation suggest
that the close physical linkage results from the sharing of cis-control elements.
Their proximity also suggests that these 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 has been
suggested that wingless and DWnt4 achieve opposite, but complementary functions in
intrasegmental cell patterning of the embryonic ectoderm (Gieseler, 1996).
Wingless is also expressed in head segments. Based on the expression pattern of the segment polarity genes engrailed and wingless during the embryonic development of the larval head, it has been found that the head of Drosophila consists of remnants of seven segments (4 pregnathal and 3 gnathal) [Images] all of which contribute cells to
neuromeres in the central nervous system (Schmidt-Ott, 1992)
The proneural genes achaete and scute and the segment polarity genes wingless and engrailed each have limited expression in only a few identifiable and stereotyped clusters of the head. For example, sc appears exclusively in a small part of the protocerebral domain, followed by transient expression in one to two protocerebral neuroblasts. wg is expressed in a total of three patches and engrailed is expressed in domains that are posterior and ventral to the adjacent wg domains. en is expressed in one patch in both the protocerebrum and the deuterocerebrum (Younossi-Hartenstein, 1996).
Wingless acts non-autonomously to specify the fate of a specific neuronal precursor, NB4-2. Gooseberry and Patched participate in the Wingless-mediated specification of NB4-2 by controlling the response to the wingless signal. Patched targets gooseberry distal and gooseberry-proximal in neuroblast determination. The RP2 neuron is a motoneuron and innervates muscle number 2 of the dorsal musculature. This neuron originates along with its sibling cell from the first ganglion mother cell derived from NB4-2, and occupies the anterior commissure along with several other RP2 neurons. NB4-2 itself is formed during the second wave of neuroblast delamination in stage 9. In gsb mutants, WG-positive NB5-3 is transformed to NB4-2 in a Wg-dependent manner, suggesting that GSB normally represses the capacity to respond to the wingless signal. In ptc mutants, gsb is ectopically expressed in normally Wg-reponsive cells, thus preventing the response to Wingless and consequently the correct specification of NB4-2 does not take place. The timing of the response to GSB suggests that the specification of neuroblast identities takes place within the neuroectoderm, prior to neuroblast delamination (Bhat, 1996).
During neurogenesis, the transmembrane protein Patched promotes a wingless-mediated specification of a neuronal precursor cell, NB4-2. Wg, secreted by row 5 cells promotes wingless expression in adjacent row 4 cells; Wg in turn represses gooseberry. Novel interactions of these genes with engrailed and invected
during neurogenesis have been uncovered. While in row 4 cells Ptc represses gsb and wg, in
row 5 cells en/inv relieve Ptc repression of gsb by a non-autonomous mechanism that does
not involve hedgehog. The non-autonomous mechanism originates in Row 6/7 cells where en/inv engender hedgehog and another unknown secreted signal which acts in turn on adjacent row 5 cells to heighten wingless, and consequently, the expression of gooseberry. This differential regulation of gsb leads to the specification of
NB5-3 and NB4-2 identities to two distinct neuroblasts. The row 5, NB5-3, neuroblasts are specified by high levels of gsb, expressed autonomously in row 5. The fate of row 4, NB4.2, requires an absence of gooseberry, assured by Patched repression and Wingless signaling from adjacent row 5 cells. The uncoupling of the ptc-gsb
regulatory circuit by hedgehog and the unknown secreted signal from row 6/7 cells enables gsb to promote Wg expression in row 5 cells. These results
suggest that the en/inv->ptc->gsb->wg pathway uncovered here and the hh->wg are distinct
pathways that function to maintain the wild-type level of Wg. These results also indicate that Hh is
not the only ligand for Ptc and similarly, that Ptc is not the only receptor for Hh (Bhat, 1997).
Pattern formation in developing animals requires that cells exchange signals mediated by secreted proteins. How these signals spread still remains unclear. It is generally assumed that they reach their target site either by diffusion or active
transport. An alternative mode of transport for Wingless is described here. In embryos of Drosophila, the wingless gene is transcribed in narrow stripes of cells abutting the source of Hedgehog protein. These cells or their progeny are free to roam toward the anterior. As they do so, they no longer receive the Hedgehog signal and stop transcribing wg. However, the cells leaving the expression domain retain inherited Wg protein in secretory vesicles and carry it forward over a distance of up to four cell diameters. Experiments using a membrane-tethered form of Wg show that this mechanism is sufficient to account for the normal range of Wg. Nevertheless, evidence exists that Wg can also reach distant target cells independent of protein inheritance, possibly by restricted diffusion. It is suggested that both transport mechanisms operate in wild-type embryos (Pfeiffer, 2000).
In the ventral abdominal epidermis of Drosophila embryos, Wg signaling specifies bald cuticle by repressing the transcription of shavenbaby, a gene required for the formation of hair-like protrusions called denticles. As expected for a secreted product, the Wg protein is distributed more widely than its mRNA. Transcription of wg is in single-cell-wide stripes whereas the protein is detected over 3-4 cell diameters. Cuticle preparations from larvae carrying a lacZ reporter under the control of the wg promoter (wg-lacZ) show that bald cuticle is made by cells located as much as four cell diameters away from the wg expression domain (Pfeiffer, 2000).
Much of the Wg protein is detected in intracellular vesicles. Vesicles outside the domain of transcription are assumed to contain Wg that has been taken up by non-expressing cells. In fact, internalization of Wg has been proposed to be required for its transport: according to the model of planar transcytosis, internalized Wg is subsequently re-released into the extracellular space and hence presented to more distant cells. Of course, internalization of Wg could also constitute the first step towards degradation in lysosomes. An alternative to the planar transcytosis model of transport is that Wg diffuses in the extracellular space, possibly interacting with membrane-associated glycoproteins (Pfeiffer, 2000).
Irrespective of the transport mechanism, one would expect that, if the Wg protein were artificially tethered to the membrane of secreting cells and, hence, prevented from being released into the extracellular space, its range would be reduced and the area of bald cuticle would narrow. This was tested by expressing membrane-tethered Wg in a wg null mutant with a wg-GAL4 driver. Surprisingly, wg mutants rescued by membrane-tethered Wg could hardly be distinguished from wild-type embryos. In particular, the bands of naked cuticle were as wide as in the wild type, suggesting that membrane-tethered Wg can act as far as four cell diameters away.
Two trivial explanations could account for the rescue. One is that membrane-tethered Wg might be leaky. Tethering Wg to the cell membrane was achieved by fusing it to the transmembrane protein Neurotactin (Nrt). Rescue could be explained if this fusion protein were cleaved, releasing active Wg into the extracellular space. There is, however, no indication of cleavage from Western blots. Moreover, functional assays confirm that Wg remains attached to expressing cells (Pfeiffer, 2000).
During normal development, en-expressing cells do not cross into the anterior compartment where wg is expressed. However, wg-expressing cells and their progeny may be free to roam in the anterior direction. This suggests an alternative explanation for the 'long-range action' of tethered Wingless: it could be carried toward the anterior by moving cells and their progeny. To test the feasibility of such a mechanism, the progeny of single cells were tracked, and marked at the time when wg expression commences. Single cells were marked by photoactivating caged rhodamine with the ultraviolet (UV) laser beam of a confocal microscope. To have a spatial landmark at later developmental times, this experiment was performed with embryos expressing the green fluorescent protein (GFP) in the posterior compartment. The progeny of marked cells were identified in live embryos at late stage 11 (after three mitoses) and mapped relative to the domain of en expression. Although no clones crossed the parasegment boundary, those located just in front of the en domain spanned several cell diameters (up to five) in the anteroposterior direction. Since the parasegment boundary is a clonal boundary, it imposes directionality to this spread, resulting in the net movement of wg-expressing cells towards the anterior. Importantly, clonal spread covers a broad area of the ectoderm and can account for the range of tethered Wg in these rescue experiments (Pfeiffer, 2000).
If, as proposed, Wg is carried by moving cells and their progeny, a stable non-secreted protein should also be transported towards the anterior. This prediction was tested using a Gal4-responsive transgene encoding nuclear-targeted beta-galactosidase (beta-gal), which is relatively stable. It was asked and determined whether this product was carried forward in the ventral epidermis. Indeed, in embryos carrying wg-GAL4 and tethered Wg, beta-gal is detected in stripes 3-4 cells wide in front of the parasegment boundary. This is substantially wider than the GAL4 RNA stripes. In hatched larvae of the same genotype, beta-gal activity is detected within similarly wide bands of cells occupying the middle of the bald regions. It is suggested that beta-gal made by wg-expressing cells is retained even when cells move away from the source of Hedgehog and therefore shut off the wg promoter. Since cells could move only towards the anterior of the parasegment boundary, beta-gal appears to spread in the anterior direction. Note that such spreading could not have occurred through cellular extensions such as cytonemes because the beta-gal product is nuclear in this experiment. Thus, a non-secreted protein can spread by being passed on to the progeny of expressing cells. As expected then, when driven by wg-GAL4, tethered Wg is detected in stripes that are similar in width to the stripes of Wg protein in wild-type embryos (3-4 cells wide). It is suggested that, like nuclear beta-gal, tethered Wg driven by wg-GAL4 is retained by cells as they spread toward the anterior and this accounts for its range of action (Pfeiffer, 2000).
The Wg-containing vesicles found at the anterior of the transcription domain are within cells that descended from wg-expressing cells. These vesicles are therefore not necessarily endocytic. They could equally contain unsecreted protein inherited from past expression. No good immunological markers are available in Drosophila to distinguish endocytic vesicles from secretory ones. To label the secretory pathway, flies were made that express GFP fused to the signal peptide of Wg (UAS-GFPsecr). When this fusion is expressed with the en-Gal4 driver, fluorescence is detected in bright intracellular dots within the en domain as well as weakly throughout the perivitelline space. The punctate fluorescence in expressing cells most probably represents GFP transiting through the secretory pathway and thus identifies secretory vesicles. In live embryos expressing GFPsecr under the control of wg-GAL4, vesicular staining is detected in stripes 3-4 cells wide at stage 11. Thus, secretory vesicles are present several cell diameters beyond the domain of expression (Pfeiffer, 2000).
These embryos were fixed and stained with anti-Wg antibody. Although much GFP fluorescence is lost upon fixation, extensive colocalization between Wg protein and the remaining GFP signal is detected, even outside the wg expression domain. This suggests that many Wg-containing vesicles are secretory as opposed to endocytic (although endocytic vesicles may exist as well). The presence of Wg-containing vesicles at the anterior of the Wg expression domain is often taken as evidence for transport from cell to cell. The results presented here show that this assumption must be revised. It also shows that, during normal development, cell spreading contributes significantly to the anterior movement of endogenous Wg protein.
In conclusion, as cells proliferate and spread, they can retain the Wg signal and thus affect target cells some distance away from the site of wg transcription. It is important to note that Wg can also spread independent of cell movement, possibly by restricted diffusion. It has been possible to uncouple the two mechanisms of Wg movement and, thus, show that either is sufficient to ensure a normal range of action. Presumably, both contribute during wild-type development although their relative importance cannot yet be assessed. Interestingly, the parasegment boundary allows cells to carry Wg only toward the anterior and this adds to other mechanisms ensuring an asymmetric range of Wg in Drosophila embryos. Without this border, cells carrying Wg could wander towards the posterior and disrupt segment polarity (Pfeiffer, 2000).
Asymmetric mRNA localization targets proteins to their cytoplasmic site of function. The
mechanism of apical localization of wingless and pair-rule transcripts in the Drosophila blastoderm embryo has been elucidated by
directly visualizing intermediates along the entire path of transcript movement. After release from their site of
transcription, mRNAs diffuse within the nucleus and are exported to all parts of the cytoplasm, regardless of
their cytoplasmic destinations. Endogenous and injected apical RNAs assemble selectively into cytoplasmic
particles that are transported apically along microtubules. Cytoplasmic dynein is required for correct localization
of endogenous transcripts and apical movement of injected RNA particles. It is proposed that dynein-dependent movement of RNA particles is a widely deployed mechanism for mRNA localization (Wilkie, 2001).
To study the mechanism of apical localization, whether actin and/or MTs are necessary for localization of injected mRNA was tested by preinjecting cytoskeletal inhibitors 10 min before injecting the RNA. It was found that preinjection of Cytochalasin B, at concentrations that disrupt the organization of actin filaments, has no affect on Runt mRNA localization. However, a similar disruption of nuclear position has been observed in the cortical cytoplasm. In contrast, preinjection of colcemid, which destabilizes blastoderm MTs, disrupts runt, wingless, and fushi tarazu RNA localization almost entirely. It is concluded that an intact MT cytoskeleton is required for apical localization of injected RNA and that actin does not play a major role in the process. However, some minor role for actin in apical localization of RNA cannot be excluded (Wilkie, 2001).
Whether the localization of injected RNA occurs by minus end directed MT-dependent motor movement was tested by preinjecting embryos with antibodies against Drosophila cytoplasmic dynein heavy chain (dhc). Two independently raised monoclonal antibodies against dhc are each sufficient to inhibit RUN, FTZ, and WG mRNA apical localization in most, or all, embryos. Either one, the anti-dynein antibody or the colcemid injections, is sufficient to cause apical RNA to partly diffuse away from the site of injection in a similar manner to embryos injected with HB RNA alone. Injected apical RNA does not diffuse in the absence of anti-dynein antibodies or Colcemid preinjections. These results suggest that apical RNA is tethered to MTs by dynein and that dynein is required for the transport of RNA particles (Wilkie, 2001).
To further test the involvement of cytoplasmic dynein in apical transcript localization, RNA was injected into mutant cytoplasmic dynein heavy chain (Dhc64C) embryos. A marked reduction was found in the speed of movement of injected apical targeted RNAs in dynein mutants. Cytoplasmic dynein is essential for many cellular processes, so strong mutations in Dhc64C are homozygous lethal in Drosophila and cannot be studied at the blastoderm stage. Instead hypomorphic allelic combinations of Dhc64C, which are viable in trans due to intragenic complementation, were used. In two different allelic combinations of Dhc64C, injected RNA particles move at speeds 60% to 70% slower than they do in wild-type. Staining Dhc64C mutant embryos with anti-tubulin antibodies showsthat MT distribution is indistinguishable from wild-type, indicating that the reduced speed of localization is not due indirectly to a disruption of the MTs. Instead, the reduction in speed is likely to show a direct requirement for dynein in particle transport (Wilkie, 2001).
To test whether cytoplasmic dynein is also required for apical localization of endogenous transcripts, the effects of Dhc64C hypomorphic mutants and anti-dhc antibodies on the apical localization of endogenous FTZ transcripts was tested by in situ hybridization. As expected, hypomorphic Dhc64C mutants show no detectable effects on FTZ apical mRNA localization since injected RNA localizes correctly, but more slowly. In contrast, injection of anti-dhc antibody disrupts endogenous FTZ localization, leading to unlocalized stripes of ftz mRNA 20–30 min after injection. Given that FTZ mRNA has a half-life of 6 min in the blastoderm, the FTZ transcripts observed are likely to have been synthesized after the injection. It is concluded that endogenous apical mRNA localization is also dynein dependent (Wilkie, 2001).
Dynactin is a protein complex that is involved in coordinating the activities of cytoplasmic dynein, and is thought to be required for most forms of dynein-based transport. To test whether dynactin is also required for apical RNA localization, a large excess of p50/dynamitin is preinjected into embryos 10 min before injecting apically targeted RNA. p50/dynamitin causes a significant reduction in the speed of RNA particle movement. p50/dynamitin is a subunit of dynactin whose overexpression is a widely used method of disrupting the dynactin complex and demonstrating conclusively dynein-dependent motility. Dynactin is required for some cargo binding and for dynein processivity. It is concluded that apical transcript localization in the blastoderm embryo occurs by cytoplasmic dynein- and dynactin-mediated transport along MTs toward their minus ends (Wilkie, 2001).
It is thought that export and localization of apical mRNA in the blastoderm embryo can be divided into six distinct steps. (1) During or after completion of transcription and processing, transcripts are assembled into particles, which contain various hnRNPs and export factors, some of which may form part of the cytoplasmic localization machinery. (2) mRNA particles diffuse freely after release from the site of transcription and processing until they reach nuclear pore complexes (NPCs) on the nuclear periphery. (3) mRNA particles are exported through NPCs in all parts of the nuclear envelope. (4) The composition of the particles probably changes during export from the nucleus and in the cytoplasm to recruit dynein, dynactin, and associated proteins. (5) Particles attach to MTs and are actively transported to the apical cytoplasm. (6) Particle movement arrests in the apical cytoplasm, where they may associate with other particles and become anchored (Wilkie, 2001).
The first three steps of apical localization are thought to be common to most mRNAs, because they are essential universal processes in eukaryotic cells. However, the last three steps of the localization pathway are likely to vary among different kinds of transcripts, since the key determinant in sorting different mRNAs to their correct cytoplasmic destinations is presumably RNP particle composition in the cytoplasm. It is possible that some components required for cytoplasmic sorting are preassembled in the nucleus, as suggested by studies showing that the localization of injected FTZ mRNA depends on preincubation with the hnRNPA1 protein Squid. Indeed, a requirement for hnRNPs has also been shown for GRK mRNA localization in the oocyte, for myelin basic protein mRNA in rat oligodendrocytes, and for Vg1 transcripts in Xenopus oocytes. However, the data in this study show that injected protein-free apical RNA assembles in the cytoplasm into particles that localize correctly, arguing that all the factors needed to assemble competent localization particles can also be recruited in the cytoplasm (Wilkie, 2001).
Many developing and adult tissues are comprised of polarized epithelia. Proteins that are asymmetrically
distributed in these cells are thought to be localized by protein trafficking. The distribution and function of the signaling protein Wingless is predetermined by the subcellular localization of its mRNA. High-resolution in situ hybridization reveals apical transcript localization in the majority of tissues examined. This localization is mediated by two independently acting elements in the 3' UTR. Replacement of these elements with either non-localizing or basolaterally localizing elements yields proteins with altered intracellular and extracellular distributions and reduced signaling activities. This novel aspect of the wingless signaling pathway is conserved and may prove to be a mechanism used commonly for establishing epithelial cell polarity (Simmonds, 2001).
Whereas wg transcripts are enriched apically, transcripts encoded by a lacZ reporter gene, expressed in the same cells under control of the wg promoter are distributed uniformly throughout the cytoplasm. It is concluded that this localization is a transcript-specific and not a cell-specific property. Indeed, apical localization of wg transcripts is also observed in most other polarized cells (Simmonds, 2001).
Different portions of the wg transcript were tested for their ability to confer apical localization in vivo to a nonlocalized lacZ transcript. Transgenic constructs with the wg 5' UTR and/or the wg ORF, fused either 5' (in-frame) or 3' to the lacZ sequence, yield uniformly distributed transcripts. However, fusion of the wg 3' UTR to lacZ results in apical transcript localization. Thus, the wg 3' UTR is both necessary and sufficient for apical transcript localization (Simmonds, 2001).
To map the specific sequences responsible for apical localization within the 1098 nt wg 3' UTR, deletions were introduced into the lacZ-wg 3' UTR reporter, and the deleted reporters were tested for their ability to confer apical transcript localization in transgenic embryos. These deletions defined two wg localization elements (WLEs), each of which is sufficient to confer apical transcript localization. WLE1 is located between nucleotides 60-178 and WLE2 is located between nucleotides 670-780. These elements may function differently as localization conferred by WLE2 is more closely associated with the apical cortex than that conferred by WLE1. Differences in function are also suggested by the lack of apparent similarity in sequence or predicted secondary structure (Simmonds, 2001).
To examine the effect of transcript localization on Wg signaling, constructs expressing wg transcripts that localize to different parts of the cell were made. The three constructs that were made differ only in their 3' UTRs. The first uses the endogenous wg 3' UTR, the second a 3' UTR derived from the SV40 small t antigen gene, and the third a 3' UTR derived from the partner of paired (ppa) gene. Each of the transgenes was placed under the control of a GAL4-dependent promoter and the vectors introduced into embryos to obtain transgenic flies. Transcripts containing the wg 3' UTR are localized apically while transcripts containing the SV40 3' UTR are uniformly distributed. In contrast, transcripts containing the ppa 3' UTR are localized basally. The distribution of the ppa-tagged transcript is not as tightly localized to the basal side of the cell as full-length wg transcripts are to the apical surface. Rather, the two distributions appear to be complementary (Simmonds, 2001).
Prior to comparing the signaling activities of the proteins made from these three transgenes, Western blot analysis was used to select transgenic lines that express equivalent levels of protein. Expression of the transgenes was induced by crossing the UAS-wg flies to ptc-GAL4 flies. These express GAL4 in the majority of ectodermal cells. Two matched sets of transgenic lines were selected: a 'low'-expressing set and a 'high'-expressing set. Quantitation of the protein levels expressed by each of the fly lines in these sets shows that, when the endogenous Wg contribution is subtracted, the high lines express about six times the levels of the low lines. Based on the spatial differences between endogenous wg and ptc-GAL4-driven wg expression patterns, it is estimated that the high lines express about half the levels of endogenous Wg protein on a per cell basis (Simmonds, 2001).
Semi-quantitative RT-PCR analysis of the transgenic transcripts shows that transcription levels are also equivalent for each of the three lines in each of the matched sets. Initial levels of protein and RNA were also observed to be approximately equal when visualized in situ by immunocytochemistry and in situ hybridization. It is concluded that the 3' UTR swaps have little effect on the synthesis and stability of wg transcripts and protein. Posttranslational modifications also appear to be the same for each protein, since each lane on the Western blot contains a similar set of bands equivalent in number, mobility, and relative intensity (Simmonds, 2001).
Wg facilitates its own expression via both autocrine and paracrine signaling pathways. To test whether the localization of wg transcripts affects these activities, pulses of wg construct expression were induced by crossing the high set of UAS-wg transgenic flies to a heat shock-Gal4 line and subjecting 3- to 5-hr-old embryos to a 30 min heat shock. Protein levels were assessed by Western blot analysis. Immediately following the heat pulse, each of the matched transgenic lines produced the same amount of protein. This was about three times the amount of endogenous Wg expressed in heat-shocked controls. In the apical transcript line, these levels rose about 5-fold higher during the next half hour, and subsequently remained at a high level. This increase in expression levels is due to the spatial expansion and intensification of endogenous Wg stripes. In the line with uniform wg transcript distribution, autoregulation also occurs but with slower kinetics. In contrast, the basal transcript line shows no further increase in Wg expression levels 30 min after the heat pulse, and by 60 min, expression levels are similar to those seen in the heat shock control. Transcript levels for each of the lines and each time point were also measured using RT-PCR and NIH image, and it was confirmed that, as with the ptc-GAL4-driven expression, each of the transgene mRNAs is expressed and turned over at equivalent rates. Thus, it is concluded that apical transcript localization is important for Wg autoregulation (Simmonds, 2001).
In order to test the signaling activities of the differentially localized
transcripts in a more comprehensive fashion, each of the constructs of the high- and low-expressing matched sets was tested for their ability to rescue wg-dependent segmental patterning. This was accomplished by recombining the two sets of wg-expressing transgenic lines into a wg null mutant background and crossing these lines to flies that express GAL4 under wg promoter control. As expected, the apically localized transcript of the high apical line is capable of restoring much of the naked cuticle that is missing in wg mutant embryos. The incomplete nature of this rescue is most likely due to suboptimal levels of expression as compared to the endogenous wg gene (about 50%). In comparison, the high uniform construct yields significantly reduced rescuing activity and the high basolateral line very little rescuing activity. The low set of lines shows a similar trend, but with substantially lower degrees of rescue. It is concluded that transcript localization within apical cytoplasm is essential for robust signaling activity (Simmonds, 2001).
In order to help understand how transcript localization affects protein function, an examination was carried out to see whether differences in protein distribution, in and around wg-expressing cells, could be detected. Expression of the three transgenes in the high matched set was driven using a wg-GAL4 driver, and Wg distributions were observed in a wg null background. The single-cell-wide wg stripes serve as a point source from which diffusion of the protein, laterally and apically/basally, can be readily observed (Simmonds, 2001).
Most of the Wg protein detected in wild-type embryos is observed to be enriched in the apical cytoplasm of wg-expressing cells. Although the majority of this signal is diffuse, brightly staining punctate bodies are also observed. Similar punctae are also found in cells nearby. In the wg-expressing cells, these punctate bodies are thought to represent both endocytic and exocytic vesicles (Simmonds, 2001).
Wg expressed from the apically localized transgene transcript is distributed much the same as in the wild-type control. However, protein expressed from the uniformly distributed transcript shows clear differences in distribution. Although it still appears to be somewhat enriched in the apical cytoplasm of wg-expressing cells, there is less of the protein in these cells and more extending laterally into the middle of the segment. The difference in distribution of protein translated from the basal transcript is even more striking. There is little detectable enrichment within the wg-expressing cells, and more of the protein extends laterally across the segment. Interestingly, this extracellular protein still appears to be apically enriched. It is concluded that protein synthesized basally is secreted more efficiently, diffuses more rapidly within the extracellular matrix, or is less effectively endocytosed (Simmonds, 2001).
Transcripts encoded by the Drosophila virilis wg gene have been examined to see if apical transcript localization is conserved in this species. Despite an estimated evolutionary divergence of about 60 million years, and the tendency of 3' UTRs to diverge rapidly in sequence, this transcript is also localized apically. Indeed, the D. virilis wg 3' UTR is functional in D. melanogaster, and elements with sequence similarity to the two wg localization elements, WLE1 and WLE2, exist in similar positions within the 3' UTR. Although conserved between species, the sequences and predicted secondary structures of WLE1 and WLE2 bear no resemblance to one another. Their sequences also fail to show significant homology to other sequences in the database, including those of other localized transcripts. Taken together with the observation that wg transcripts colocalize to the same particles as other localized transcripts, it is surmised that transcript recognition is mediated either by transcript-specific adaptors or by common adaptors that recognize similar secondary structures (Simmonds, 2001).
This functional conservation of wg localization elements in Drosophila further substantiates the importance of wg transcript localization and suggests that this step in the pathway may be conserved in other organisms. Indeed, a number of vertebrate wnt transcripts have been shown to be localized within oocytes. For example, transcripts encoded by the Xwnt5 and Xwnt11 genes of Xenopus are localized in the oocyte vegetal pole while those of X-Wnt8b are localized to the animal pole. In Ascidians, the maternally expressed HrWnt-5 transcript is localized to the posterior of early embryos (Simmonds, 2001 and references therein).
Establishment of segmental pattern in the Drosophila syncytial
blastoderm embryo depends on pair-rule transcriptional regulators. mRNA
transcripts of pair-rule genes localise to the apical cytoplasm of the
blastoderm via a selective dynein-based transport system and signals within
their 3'-untranslated regions. However, the functional and evolutionary
significance of this process remains unknown. Subcellular
localisation of mRNAs from multiple dipteran species has been analyzed both in situ and by injection into Drosophila embryos. Transcript localisation was assayed in four species that can be cultured in the laboratory. Two of them, Episyrphus (Syrphidae) and Megaselia (Phoridae), are cyclorrhaphan flies (i.e. higher dipterans) but, unlike Drosophila, belong to basal branches of this taxon; the other two, Coboldia (Scatopsidae) and Clogmia (Psychodidae), belong to different branches of lower Diptera. Although localisation
of wingless transcripts is conserved in Diptera, localisation of
even-skipped and hairy pair-rule transcripts is
evolutionarily labile and correlates with taxon-specific changes in
positioning of nuclei. In Drosophila localised pair-rule
transcripts target their proteins in close proximity to the nuclei and
increase the reliability of the segmentation process by augmenting gene
activity. These data suggest that mRNA localisation signals in pair-rule
transcripts affect nuclear protein uptake and thereby adjust gene activity to
a variety of dipteran blastoderm cytoarchitectures (Bullock, 2004).
Apical localisation of pair-rule mRNAs in Drosophila syncytial
blastoderm embryos was first noted 20 years ago, but the developmental and
evolutionary significance of this process has remained unclear. Apical pair-rule mRNA localisation is conserved in cyclorrhaphan species that
diverged over 145 million years ago, indicating that this process has a
significant developmental role under natural conditions. Likewise, the
widespread maintenance of wg transcript localisation in Diptera
supports the importance of this process on a phylogenetic scale, even though,
in Drosophila, wg appears to be less sensitive than pair-rule genes
to a reduction in endogenous transcript localisation (Bullock, 2004).
The Rho small GTPase has been implicated in many cellular processes, including actin cytoskeletal regulation and transcriptional activation. The molecular mechanisms underlying Rho function in many of these processes are not yet clear. In Drosophila, reduction of maternal Rho1 compromises signaling pathways consistent with defects in membrane trafficking events. These mutants fail to maintain expression of the segment polarity genes engrailed (en), wingless (wg), and hedgehog (hh), contributing to a segmentation phenotype. Formation of the Wg protein gradient involves the internalization of Wg into vesicles. The number of these Wg-containing vesicles is reduced in maternal Rho1 mutants, suggesting a defect in endocytosis. Consistent with this, stripes of cytoplasmic β-catenin that accumulate in response to Wg signaling are narrower in these mutants relative to wild type. Additionally, the amount of extracellular Wg protein is reduced in maternal Rho1 mutants, indicating a defect in secretion. Signaling pathways downregulated by endocytosis, such as the epidermal growth factor receptor (EGFR) and Torso pathways, are hyperactivated in maternal Rho1 mutants, consistent with a general role for Rho1 in regulating signaling events governing proper patterning during Drosophila development (Magie, 2005).
The data indicate that a number of signaling pathways important during early development in Drosophila are compromised in maternal Rho1 mutants. The observation that secretion of Wg protein is aberrant in these mutants together with the endocytosis defects observed in S2R+ cells treated with Rho1 dsRNA and in maternal Rho1 embryos indicates that Rho1 plays a general role in membrane trafficking processes in the early embryo. The biochemical mechanisms through which Rho proteins affect membrane trafficking are currently unclear. One possibility is that the function of Rho1 in this process is a byproduct of its regulation of the actin cytoskeleton. In yeast, there is evidence that the actin cytoskeleton is important in endocytosis, as mutations in actin and some actin-binding proteins inhibit endocytosis. In addition, yeast Rho1 has been shown to be involved in endocytosis of the α-receptor. In mammalian cells, treatment with pharmacological agents that perturb actin structure can affect endocytosis in a cell-type-specific way. In polarized epithelial cells, for example, treatment with the actin-depolymerizing drug cytochalasin D inhibits endocytosis specifically at the apical, but not the basolateral surface. RhoA has also been implicated in endocytosis in polarized epithelial cells. In Drosophila, Rho1 has clear roles in actin cytoskeletal regulation during oogenesis and embryogenesis, consistent with the notion that Rho1 may be acting primarily through its effects on the actin cytoskeleton (Magie, 2005).
The observation that the segmentation phenotype in maternal Rho1 mutants is the result of general defects in membrane trafficking processes (both secretion and endocytosis) and not a primary effect on transcriptional activation has important implications for the interpretation of data linking Rho to disparate cellular processes. While current data cannot exclude the possibility that Rho directly acts in transcriptional activation or through many disparate mechanistic pathways, data are accumulating that suggest Rho may act primarily as a regulator of the actin cytoskeleton and other functions it has been linked to are indirect effects. For instance, the ability of Rho to influence transcriptional activation through the serum response factor (SRF), as well as affect cell cycle progression, is due to its direct effects on actin cytoskeletal regulation. Identifying the molecular mechanisms underlying each of Rho's activities will be crucial to determining whether Rho1 has direct effects on a number of pathways or has a small number of primary functions that indirectly affect other functions. Investigations of Rho GTPase function in genetically amenable model organisms are providing a diversity of developmental contexts in which to examine all aspects of Rho biology, and the ability to examine specific, loss-of-function phenotypes will continue to aid identification of the mechanisms underlying Rho function (Magie, 2005).
Trichomes are cytoplasmic extrusions of epidermal cells. The molecular
mechanisms that govern the differentiation of trichome-producing cells are
conserved across species as distantly related as mice and flies. Several
signaling pathways converge onto the regulation of a conserved target gene,
shavenbaby (svb, ovo), which, in turn, stimulates trichome
formation. The Drosophila ventral epidermis consists of the segmental
alternation of two cell types that produce either naked cuticle or trichomes
called denticles. The binary choice to produce naked cuticle or denticles is
affected by the transcriptional regulation of svb, which is
sufficient to cell-autonomously direct denticle formation. The expression of
svb is regulated by the opposing gradients of two signaling molecules
- the epidermal growth factor receptor (Egfr) ligand Spitz (Spi), which
activates svb expression, and Wingless (Wg), which represses it. It
has remained unclear how these opposing signals are integrated to establish a
distinct domain of svb expression. This study shows that the expression of the
high mobility group (HMG)-domain protein SoxNeuro (SoxN) is activated by Spi,
and repressed by Wg, signaling. SoxN is necessary and sufficient to
cell-autonomously direct the expression of svb. The closely related
protein Dichaete is co-regulated with SoxN and has a partially redundant
function in the activation of svb expression. In addition, SoxN and Dichaete function upstream of Wg and antagonize Wg pathway
activity. This suggests that the expression of svb in a discreet
domain is resolved at the level of SoxN and Dichaete (Overton, 2007).
In the embryonic ventral epidermis of Drosophila, two alternative
cell fates are specified: smooth cells and trichome-producing cells. These
binary cell fates are distinguished by the expression of svb, the
most-downstream effector of epidermal morphogenesis. svb is necessary
and sufficient to cell-autonomously direct trichome formation. The
expression of svb is regulated by the opposing gradients of two
signaling molecules: Spi, which activates, and Wg, which represses,
svb expression. svb is expressed in segmentally reiterated,
epidermal stripes, which invariantly encompass six rows of cells. This raises
the question of how is opposing extrinsic information integrated to establish
a distinct domain of svb expression with a sharp posterior
border (Overton, 2007)?
This study demonstrates that the HMG-domain proteins SoxN and
Dichaete represent a molecular link between the expression of svb and
the upstream Der- and Wg-signaling cascades. SoxN and Dichaete
are expressed in the ventral epidermis at the time when epidermal cell fates
are specified. The late phase of SoxN and Dichaete expression is stimulated by
Der- and repressed by Wg-pathway activity. These regulatory mechanisms result
in the expression of SoxN and Dichaete in those six rows of cells within each
abdominal segment that differentiate to produce trichomes. SoxN and, to a
lesser extent, Dichaete, are necessary and sufficient to activate the
expression of svb. Furthermore, these results show that the
well-described repression of svb by Wg is due to the repression of
SoxN, which, in turn, results in the loss of svb activation.
Likewise, the Spi-mediated activation of svb expression relies on the
activation of SoxN, which, in turn, activates svb. This indicates
that the competition of Der- and Wg-pathway activities for the specification
of trichome-producing versus smooth cell fates is resolved at the level of
SoxN and Dichaete (Overton, 2007).
These results do not provide much insight into the issue of how opposing
extrinsic information is integrated such that a sharp posterior border of
svb expression is achieved. Instead, they raise the question of how
is a sharp posterior border of SoxN and Dichaete expression
established/maintained? The findings suggest that this is achieved by a
combination of negative- and positive-feedback loops. (1) Evidence is provided that SoxN and Dichaete negatively regulate Wg pathway
activity. This negative-feedback loop provides a likely mechanism for the
establishment and maintenance of a sharp posterior border of SoxN and Dichaete
expression. The issue arises of how robust this system might be in the face of
fluctuating levels of Wg pathway activity. The efficiency with which SoxN and
Dichaete restrict Wg pathway activity will crucially rely on the levels of
SoxN and Dichaete protein. In this context, it is noteworthy that the levels
of SoxN protein, but not Dichaete, are several-fold higher in the two
posterior-most rows of the SoxN stripe compared with the anterior four rows. The regulatory mechanisms that underlie the different levels of SoxN expression are currently unclear. (2) Evidence is provided that the maintenance of SoxN and Dichaete expression is supported by a positive-feedback loop: svb, the
expression of which is activated by SoxN and Dichaete, is itself required for
the maintenance of SoxN and Dichaete expression. Together,
these mechanisms contribute to an invariant read-out of cell identity from
opposing Der- and Wg-pathway activities (Overton, 2007).
In Drosophila, SoxN and Dichaete are necessary and sufficient to
activate the expression of svb, which in turn directly regulates the
expression of genes involved in trichome morphogenesis.
Is a function in hair formation of the Sox proteins conserved in other
species, including vertebrates? A previous study has shown that the mouse Sox9
protein is required for the differentiation of hair-producing epidermal cells
and acts genetically downstream of sonic hedgehog pathway activity
(Vidal, 2005). This
study did not address whether Sox9 regulates the expression of movo1
(Ovol1), the mouse ortholog of svb.
Nevertheless, the demonstrated roles of SoxN, Dichaete and Sox9 raise the
exciting question of do Sox proteins have an essential function in the
activation of an epidermal differentiation program that is conserved across
species as distantly related as mice and flies (Overton, 2007).
The insect brain is traditionally subdivided into the trito-, deuto- and protocerebrum. However, both the neuromeric status and the course of the borders between these regions are unclear. The Drosophila embryonic brain develops from the procephalic neurogenic region of the ectoderm, which gives rise to a bilaterally symmetrical array of about 100 neuronal precursor cells, called neuroblasts. Based on a detailed description of the spatiotemporal development of the entire population of embryonic brain neuroblasts, a comprehensive analysis was carried out of the expression of segment polarity genes (engrailed, wingless, hedgehog, gooseberry distal, mirror) and DV patterning genes (muscle segment homeobox, intermediate neuroblast defective, ventral nervous system defective) in the procephalic neuroectoderm and the neuroblast layer (until stage 11, when all neuroblasts are formed). The data provide new insight into the segmental organization of the procephalic neuroectodem and evolving brain. The expression patterns allow the drawing of clear demarcations between trito-, deuto- and protocerebrum at the level of identified neuroblasts. Furthermore, evidence is provided indicating that the protocerebrum (most anterior part of the brain) is composed of two neuromeres that belong to the ocular and labral segment, respectively. These protocerebral neuromeres are much more derived compared with the trito- and deuto-cerebrum. The labral neuromere is confined to the posterior segmental compartment. Finally, similarities in the expression of DV patterning genes between the Drosophila and vertebrate brains are discussed (Urbach, 2003).
In the trunk neuroectoderm, segment-polarity genes are expressed in
stereotypical segmental stripes, and in NBs that delaminate from these
domains, subdividing each neuromere along the AP axis. In the
pregnathal head region the expression domains of segment polarity genes are less obvious, but analysis of engrailed and wingless expression in the head peripheral ectoderm, and of PNS
mutant phenotypes, support the existence of four pregnathal segments in
Drosophila: the intercalary, antennal, ocular and labral segments (from posterior to anterior). However, the identity and organization of brain structures
deriving from these segments is still obscure. In order to obtain evidence concerning the number and extent of the brain neuromeres, and to map the position of their boundaries, the expression of segment polarity genes, including wingless, hedgehog, gooseberry-distal, engrailed, invected and mirror, was analyzed. The spatiotemporal pattern of their expression was traced in the neuroectoderm and in the NB-layer until stage 11,
when all brain NBs are formed. The data
show that segmental expression is retained for most of the investigated segment polarity genes in both the developing head ectoderm (procephalon) and brain NBs, providing landmarks for the definition of segmental domains within the developing brain NB pattern (Urbach, 2003).
engrailed (en) expression domains in the trunk define the posterior segmental compartments, from which NBs of row 6 and 7 and NB1-2 derive. In the pregnathal head en expression was found as follows: from late stage 8 in the posterior ectoderm of the antennal segment (en antennal stripe; en as) from which four deutocerebral NBs (Dv8, Dd5, Dd9, Dd13) delaminate; from stage 9 in a small ectodermal domain in the posterior part of the ocular segment, the en head spot (en hs), from which two protocerebral NBs (Ppd5, Ppd8) evolve; and from stage 10 in an ectodermal stripe in the posterior intercalary segment (en intercalary stripe; en is), which gives rise to three to four tritocerebral NBs (Tv4, Tv5, Td3, Td5). Furthermore, from stage 11 onwards, En is weakly detected in the anteriormost ectoderm of the procephalon corresponding to the region of the 'anterior dorsal hemispheres' (en dh). About 10 weakly En-positive NBs were identified that delaminate from the en dh. Thus all four pregnathal head segments contribute to the early embryonic brain. The spatial distribution of the En-positive NBs closely corresponds to the en domains of their origin in the ectoderm. This suggests they demarcate the posterior borders of the respective brain neuromeres (Urbach, 2003).
In the trunk, hedgehog (hh) matches en expression. This is also the case for the intercalary segment in the pregnathal head ectoderm. By contrast, the En-positive antennal stripe and head spot are only subfractions of the large hh-lacZ domain, which, between stages 9 and 10, encompasses the antennal segment and the posterior part of the ocular segment. All NBs delaminating from this domain express hh-lacZ. From stage 10 onwards, en expressing NBs maintain a strong hh-lacZ signal, whereas hh-lacZ subsequently diminishes in the neuroectoderm and in NBs between the en antennal stripe and head spot. Additionally, hh-lacZ-expressing NBs positioned dorsally to the en/hh-lacZ-co-expressing Ppd5 and Ppd8 (both NBs demarcating part of the posterior border of the ocular neuromere), appear to prolong the boundary between the deuto- and proto-cerebrum in the dorsal direction (Urbach, 2003).
From late stage 8 onwards, Wingless (Wg) protein is expressed in a neuroectodermal domain spanning a broad area of the ocular and the anterior antennal segment (and in the invaginating foregut). This becomes clearer in En/Wg double labelling at stage 9, revealing that the en hs is localized within this Wg domain. At that stage, Wg is already detectable in about 4-5 protocerebral NBs (Pcd6, Pcd15, Pcd7, Ppd3), derived from the region with strongest Wg expression (which later corresponds to the wg head blob). Furthermore, Wg is faintly expressed in the deutocerebral Dd7 emerging from the antennal part of the Wg domain, which corresponds to the later wg antennal stripe. By stage 10, when the wg head blob is clearly distinguishable from the wg antennal stripe, about 10-12 Wg-positive NBs have emerged from this domain. In addition, a small, spot-like wg domain was found in the intercalary segment from which a single NB (Td4) delaminates. Thus, all three wg domains, the intercalary, antennal and ocular (head blob), contribute to the anlage of the brain. From late stage 9 an additional wg domain is visible in the ectodermal anlage of the clypeolabrum, which is the wg counterpart to the En/Inv-positive region in the 'dorsal hemispheres'. Upon double labelling for either asense or deadpan (both are general markers for neural precursor cells) and wg, in embryos between stage 9 and 11 no NB emerging from the wg labral spot could be detected. By stage 11 the number of wg expressing NBs originating from the ocular head blob has increased to about 16-20, which is more than 25% of the total number of identified protocerebral NBs. Three Wg-positive NBs are identified in the deutocerebrum and one in the tritocerebrum (Urbach, 2003).
wingless function is specifically required for heart development. A
temperature-sensitive mutation of wg has been used to inactivate wg function during precise
developmental time periods. Elimination of wg function for a short time period after gastrulation
results in the selective loss of heart precursors, without significantly affecting the formation of the
body wall or visceral muscles, although some pattern defects are observed. This developmental
requirement of wg for cardiac organogenesis is distinct from its function in segmentation and
neurogenesis (Wu, 1995).
The embryonic dorsal vessel in Drosophila possesses anteroposterior polarity and is subdivided into two chamber-like
portions, the aorta in the anterior and the heart in the posterior. The heart portion features a wider bore as compared with
the aorta and develops inflow valves (ostia) that allow the pumping of hemolymph from posterior toward the anterior. Homeotic selector genes provide positional information that determines the anteroposterior
subdivision of the dorsal vessel. Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B
(Abd-B) are expressed in distinct domains along the anteroposterior axis within the dorsal vessel, and, in particular, the
domain of abd-A expression in cardioblasts and pericardial cells coincides with the heart portion. Evidence is provided that
loss of abd-A function causes a transformation of the heart into aorta, whereas ectopic expression of abd-A in more anterior
cardioblasts causes the aorta to assume heart-like features. These observations suggest that the spatially restricted
expression and activity of abd-A determine heart identities in cells of the posterior portion of the dorsal vessel. Abd-B, which at earlier stages is expressed posteriorly to the cardiogenic mesoderm, represses cardiogenesis. In light of the developmental and morphological similarities between the Drosophila dorsal vessel and the primitive heart tube
in early vertebrate embryos, these data suggest that Hox genes may also provide important anteroposterior cues during
chamber specification in the developing vertebrate heart (Lo, 2002).
Since abd-A expression coincides with the heart portion
of the dorsal vessel, tests were made to see whether it acts to specify
the cardioblasts in which it is expressed to eventually form
the heart. In order to distinguish aorta cardioblasts from
heart cardioblasts, two different molecular markers were
utilized. The first marker was the pattern of ß-Gal derived
from the tinCdelta5-lacZ transgene, where the expression of a
lacZ gene is controlled by an internally deleted tinman
cardiac enhancer element, tinCdelta5.
This element drives ß-Gal expression in all the cardioblasts
of the aorta, whereas in the heart it is only expressed in
three segmentally-spaced double pairs of cardioblasts. These particular cardioblasts correspond to the
svp cardioblasts of the heart. The second marker
is wingless (wg), which is expressed in these same three
double pairs of svp cardioblasts within the heart of the late
embryonic dorsal vessel (Lo, 2002).
In abd-A null mutant embryos, the pattern of tinCdelta5-lacZ-derived ß-Gal is continuous in the heart as well as in
the aorta of the dorsal vessel. In addition,
it appears that the width of the heart is now the same as
that of the aorta when compared with a wildtype embryonic
dorsal vessel. Similarly, the late
expression of Wg in the svp cardioblasts of the heart is not
detectable in these mutant embryos. The alterations
in the pattern of these two markers strongly suggest
that heart cardioblasts have not been specified in the
posterior of the dorsal vessel of abd-A null mutant embryos
and that these posterior cardioblasts have been transformed
instead into aorta cardioblasts. This would indicate that
abd-A is necessary for the specification of heart cardioblasts
in the posterior portion of the dorsal vessel where it is
normally expressed (Lo, 2002).
The pattern of Wg expression in three segmentally repeated
double pairs of cardioblasts within the late stage
heart is strongly reminiscent of the pattern
of svp expression, which suggests that these are the
heart svp cardioblasts. Double antibody staining for Wg and
ß-Gal in the dorsal vessel of svp-lacZ embryos clearly
confirmed that the heart svp cardioblasts express the Wg
protein. Since the heart svp cardioblasts eventually
form the ostia (inflow valves) of the larval heart and since
Wg is a developmentally significant signaling molecule, the regulation of Wg expression in the heart svp cardioblasts during late embryogenesis was more closely examined (Lo, 2002).
Wg expression in heart cardioblasts is dependent on abd-A. Since these
Wg-expressing cardioblasts correspond to svp cardioblasts,
whether Wg expression is also dependent on svp
function was also tested. In homozygous null svpAE127 mutant embryos,
there is no detectable Wg expression in the heart cardioblasts
that are marked by svp-lacZ. Therefore, the
Wg expression seen in the heart svp cardioblasts of late
embryonic dorsal vessels requires both abd-A and svp
function. Accordingly, ectopic expression of SvpI in the
cardioblasts of the entire dorsal vessel results in wg expression
in all cardioblasts of the heart, and ectopic
expression of both SvpI and Abd-A in the whole dorsal
vessel causes Wg expression in the majority of the cardioblasts of the entire dorsal vessel. These results
demonstrate that the combination of abd-A and svp is both
necessary and sufficient to activate wg expression in cardioblasts
during late dorsal vessel development (Lo, 2002).
Since Wg expression in heart svp cardioblasts initiates toward the end of embryogenesis (stage 16), tests were made to see whether this expression could also be detected in the dorsal vessel during later larval stages when
the corresponding cells have formed the ostia. Because of
high levels of unspecific background staining with Wg
antibodies in larval preparations, wg expression in the
dorsal vessel of third instar larvae was indirectly monitored
by anti-ß-Gal staining of dissected wg-lacZ animals. Moderate
levels of wg-lacZ-derived ß-Gal can indeed be detected
in the ostia of the heart, although stronger levels are now
present in four separated patches in the aorta that correspond
to Tin-negative svp cardioblasts. While this
pattern of expression differs from that seen in the late
embryonic dorsal vessel, it is clear that wg is expressed
differentially and in a temporally regulated manner within
the heart and aorta, respectively, of late stage embryos and
third instar larvae. These observations suggest a yet undefined role for the signaling molecule in larval dorsal vessel
development and/or functioning (Lo, 2002).
The target genes of abd-A that are required for generating
functional ostia and for the other heart cells to adopt their
characteristic morphology are not yet known. Based on its
ostia-specific expression in late stage embryos, wg is a
candidate target of abd-A that may function either in an
autocrine fashion during ostia differentiation or in a paracrine
fashion during the differentiation of the adjacent heart
cardioblasts. The activation of the wg gene in the svp cells
of the aorta during third instar also precedes ostia formation,
in this case of the adult ostia, from these cells. Hence, there is a strong correlation between the initiation of wg expression in svp cardioblasts and their subsequent differentiation into functional
ostia (Lo, 2002).
The Drosophila lymph gland is a hematopoietic organ and, together with prospective vascular cells (cardioblasts) and excretory cells (pericardial nephrocytes), arises from the cardiogenic mesoderm. Clonal analysis provided evidence for a hemangioblast that can give rise to two daughter cells: one that differentiates into heart or aorta and another that differentiates into blood. In addition, the GATA factor gene pannier (pnr) and the homeobox gene tinman (tin), which are controlled by the convergence of Decapentaplegic (Dpp), fibroblast growth factor (FGF), Wingless (Wg) and Notch signaling, are required for the development of all cardiogenic mesoderm, including the lymph gland. An essential genetic switch differentiates between the blood or nephrocyte and vascular lineages involves the Notch pathway. Further specification occurs through specific expression of the GATA factor Serpent (Srp) in the lymph-gland primordium. These findings suggest that there is a close parallel between the molecular mechanisms functioning in the Drosophila cardiogenic mesoderm and those functioning in the mammalian aorta-gonadal-mesonephros mesoderm (Mandal, 2004).
Blood and vascular cells in the vertebrate embryo are thought to derive from oligopotent progenitor cells, called hemangioblasts, that arise in the yolk sac and in the aorta-gonadal-mesonephros (AGM) mesenchyme. A close relationship between blood and vascular progenitors is well established, but in vivo evidence that a single cell can divide to produce a blood cell and an endothelial cell is lacking in vertebrate systems. Similarly, the molecular mechanism that distinguishes between the two lineages is not well understood. To address these issues in a simple, genetically amenable system, the genetic control of hematopoiesis was analyzed in Drosophila. The results show that there are close lineage relationships between hematopoietic and vascular cells, similar to those present in the AGM of mammalian systems. Evidence is provided for conserved cassettes of transcription factors and signaling cascades that limit the pool of hemangioblastic cells and promote the blood versus vascular fate (Mandal, 2004).
In the mature Drosophila embryo, the lymph gland is formed by a paired cluster of ~20 cells flanking the aorta. The aorta and heart represent a contractile tube lined by a layer of myoepithelial vascular cells called cardioblasts. The cells flanking the aorta and heart posterior to the lymph gland are the pericardial cells, which function as excretory cells (nephrocytes). Lymph gland progenitors express the prohemocyte marker Srp and ultrastructurally resemble prohemocytes that develop at an earlier stage from the head mesoderm. Monitoring expression of the zinc-finger protein Odd-skipped (Odd) shows that the lymph gland originates from the dorsal thoracic mesoderm. Odd is expressed in segmental clusters in the dorsal mesoderm of segments T1-A6. The three thoracic Odd-positive clusters coalesce to form the lymph gland, whereas the abdominal clusters formed the pericardial nephrocytes (Mandal, 2004).
Lymph-gland progenitors, cardioblasts and pericardial cells are closely related by lineage. Labeled 'flipout' (FLP/FRT) clones were induced in embryos aged 3-4 h such that the clones contained only 2-4 cells. Of the two-cell clones, ~50% contained cardioblast and lymph-gland cells; the other clones comprised either cardioblasts or lymph-gland cells alone. Mixed clones were recovered at the late third larval stage. The finding of mixed clones indicates that the cardiogenic mesoderm of D. melanogaster contains oligopotent progenitors that, up to the final division, can give rise both to Srp-positive blood-cell progenitors that form the lymph gland and to vascular cells (Mandal, 2004).
The cardiogenic mesoderm forms part of the dorsal mesoderm, which requires the homeobox protein Tin and the GATA factor Pnr. In embryos with mutations in tin or pnr, the lymph gland was absent. Maintenance of Tin expression in the dorsal mesoderm requires the activity of at least two signaling pathways regulated by Dpp (the Drosophila homolog of transforming growth factor-ß) and Heartless (Htl; one of the D. melanogaster homologs of the FGF receptor); the dependence of cardioblast and pericardial nephrocyte development on these signaling pathways has been documented. Lymph-gland progenitors did not develop in loss-of-function dpp and htl mutants (Mandal, 2004).
Between 6 h and 8 h of development, the dorsal mesoderm splits into the cardiogenic mesoderm and the visceral mesoderm. The cardiogenic mesoderm is regulated positively by Wg and negatively by Notch. Lack of Wg signaling results in the absence of all cardiogenic lineages including lymph gland. Notch signaling has the opposite effect and restricts cardiogenic mesodermal fate. Notch is active in the dorsal mesoderm from 6 h to 10 h of development. Eliminating Notch during the first half of this interval by raising embryos homozygous with respect to the temperature-sensitive allele Nts1 at the restrictive temperature resulted in substantially more cardioblasts, pericardial cells and lymph-gland progenitors (Mandal, 2004).
Lymph-gland progenitors, cardioblasts and pericardial nephrocytes are specified in the cardiogenic mesoderm around the phase of germband retraction 8-10 h after fertilization. At this stage, Tin, which was initially expressed in the whole cardiogenic mesoderm, becomes restricted to a narrow medial compartment containing the cardioblasts. Pnr follows the same restriction. Cells located at a more lateral level in the cardiogenic mesoderm give rise to lymph-gland progenitors (in the thoracic domain) and pericardial nephrocytes (in the abdominal domain) and activate the gene odd. Slightly later, Srp is expressed in lymph-gland progenitors. As reported for the early hemocytes derived from the embryonic head, srp is centrally involved in lymph-gland specification. In srp-null embryos, Odd-expressing cells still formed a lymph gland−shaped cluster flanking the aorta, but these cells also express the pericardial marker pericardin (Prc), suggesting that they lose some aspects of hemocyte precursor identity or gain properties of nephrocytes. As a countercorrelate, ectopic expression of Srp in the whole cardiogenic mesoderm directed by mef2-Gal4 induces pericardial cells to adopt lymph-gland fate (Mandal, 2004).
Downregulation of tin and pnr in cells in the lateral domain of the cardiogenic mesoderm is essential for lymph-gland specification. Ectopic expression of tin or pnr by twist-Gal4 (or mef2-Gal4) causes a marked reduction in the number of lymph-gland and pericardial cells. The antagonistic effect of tin on lymph-gland progenitors resembles its earlier role in the head mesoderm that gives rise to the larval blood cells; here too, ectopic expression of tin causes a reduction in the number of hemocytes (Mandal, 2004).
Inhibiting tin and upregulating odd and srp requires input from the Notch signaling pathway. A function of Notch at 6-8 h in specification of the cardiogenic mesoderm is described. Reducing Notch function between 8 h and 10 h causes an increase in the number of cardioblasts and a concomitant loss of pericardial and lymph-gland cells. Overexpressing an activated Notch construct causes a marked increase in lymph-gland size. This late requirement for Notch signaling is separable from the earlier role of Notch in restricting the overall size of the cardiogenic mesoderm. Thus, the sum total of cardioblasts and pericardial or lymph-gland cells in Nts1 embryos shifts between 8 h and 10 h and does not differ substantially from that in wild type, whereas a combined effect on cell number and cell fate is seen in embryos with a Notch deletion. In these embryos, the cardiogenic mesoderm is hyperplasic and develops as cardioblasts at the expense of lymph-gland progenitors and pericardial nephrocytes. The dual role of Notch in restricting the numbers of a pluripotent progenitor pool and in distinguishing between the progeny of these progenitors is reminiscent of the function of Notch in sense-organ development (Mandal, 2004).
Lymph-gland formation is restricted to the thoracic region by positional cues that are provided by expression of the homeobox proteins of the Antennapedia and Bithorax complex. Specifically, Ultrabithorax (Ubx), which is expressed in segments A2-A5 of the cardiogenic mesoderm, inhibits lymph-gland formation. Loss of Ubx results in the expansion of the lymph-gland fate into the abdominal segments. Conversely, overexpression of Ubx driven by mef2-Gal4 causes the transformation of lymph-gland progenitors into pericardial nephrocytes (Mandal, 2004).
These findings are suggestive of a model of lymph-gland development in Drosophila that is similar to mammalian hematopoiesis. Lymph-gland progenitors develop as part of the cardiogenic mesoderm that also gives rise to the vascular cells (aorta and heart) and to excretory cells. Similarly, progenitor cells of the blood, aorta and excretory system are closely related both molecularly and developmentally in mammals, where they form part of the AGM. Specification of the cardiogenic mesoderm requires the input of FGF and Wg signaling, as in vertebrate hematopoiesis, where the AGM region is induced in response to several converging signaling pathways including FGF, BMP and Wnt (Mandal, 2004).
The cardiogenic mesoderm in Drosophila evolves from the dorsal mesoderm and requires input from the Htl, Dpp, Wg and Notch (N) signaling pathways. The cardiogenic mesoderm then differentiates into lymph gland, vascular cells (cardioblasts) and excretory cells (pericardial nephrocytes). A subpopulation of cardioblasts and lymph-gland cells is derived from one progenitor (hemangioblast; HB). Essential for the differentiation of the cardiogenic mesoderm is the Notch-Delta (Dl)-dependent restriction of Tin and Pnr to cardioblasts and the expression of Srp in the lymph gland. In vertebrates, similar cell types are derived from a mesodermal domain called the AGM, which also requires the input of FGF, BMP and Wnt signaling. A subset of AGM-derived cells has been proposed to constitute hemangioblasts, which produce blood progenitors and endothelial cells (Mandal, 2004).
These findings show that in Drosophila, the cardiovascular and blood-cell lineages are differentiated by an antagonistic relationship between Tin or Pnr expression in the cardioblasts and Srp expression in the lymph-gland progenitors. In vertebrates, GATA factors also have a pivotal role in specifying different lineages among blood-cell progenitors, although not much is known about what differentiates between blood progenitors as a group and endothelial progenitors. The results indicate that this step is driven by input from the Notch signaling pathway. In the thoracic cardiogenic mesoderm, Notch antagonizes tin and pnr expression and aortic cardioblast formation, and promotes srp expression and the development of lymph-gland progenitors. In vertebrates, Notch signaling is also involved in both blood and vascular development. The role of Notch during AGM morphogenesis remains to be investigated (Mandal, 2004).
Cardioblasts and lymph-gland cells can arise from the division of a single cardiogenic mesodermal cell, which should be called a hemangioblast. A previous study induced clones in the cardiogenic mesoderm but used only Tin as a marker. This study also yielded mixed two-cell clones comprising a cardioblast and a nonlabeled cell, which, in light of the current findings, must be interpreted as a lymph-gland cell. Hemangioblasts have been proposed in vertebrates, although the definitive experiment in which a precursor is marked and its lineage is tracked has not been done. Blast colony-forming cells that give rise to both lineages in vitro and common markers that belong to both cell types in vivo have been identified, but direct evidence for the existence of a common precursor has not yet been found. This study, using genetic analysis of two-cell clones, establishes the existence of such a population in Drosophila. On the basis of these results, and given the conservation of the signaling and transcriptional components described here, the prediction is that many cells of the AGM in vertebrates may give rise to only blood or only vascular cells, but a number of intermixed hemangioblasts may give rise to mixed lineages. Future genetic screens aimed at finding components in early lymph-gland development will probably identify additional pathways and strategies important for vertebrate hematopoiesis (Mandal, 2004).
The stomatogastric nervous system (SNS) of Drosophila is a simply organized neural circuitry that innervates the anterior enteric system. Unlike the central and the peripheral nervous systems, the SNS derives from a compact epithelial anlage in which three invagination centers, each giving rise to an invagination fold headed by a tip cell, are generated. Tip cell selection involves lateral inhibition, a process in which Wingless (Wg) activity adjusts the range of Notch signaling. RTK signaling mediated by the Epidermal growth factor receptor plays a key role in two consecutive steps during early SNS development. Like Wg, Egfr signaling participates in adjusting the range of Notch-dependent lateral inhibition during tip cell selection. Subsequently, tip cells secrete the Egfr ligand Spitz and trigger local RTK signaling, which initiates morphogenetic movements resulting in the tip cell-directed invaginations within the SNS anlage (González-Gaitán, 2000).
In order to investigate the role of RTK signaling in SNS development, lack-of-function mutants of the Egfr ligand Spitz were examined. In spitz mutants, the formation of the four SNS ganglia is strongly impaired. The SNS anlage, however, forms normally. In addition, the expression domain of wg and proneural AS-C genes is indistinguishable from a wild-type SNS anlage. At the stage when the three ac-expressing cells were singled-out within the wild-type SNS anlage, only one ac positive cell is found in spitz mutants. The same phenotype has been observed in wg mutants or mutants lacking an integral component of the wg pathway. Since no altered wg pattern was found in the spitz mutant SNS anlage, Spitz-dependent RTK signaling may act in parallel or in combination with wg to adjust the proper range of Notch-dependent lateral inhibition. In contrast to wg mutants, however, no invagination fold is observed. This observation indicates that the singled-out ac-expressing cell of spitz mutants has lost the ability to function as a tip cell and possibly fails to induce morphogenetic movements within the SNS anlage (González-Gaitán, 2000).
spitz, like other genes encoding components of the Egfr signaling pathway such as Egfr, Ras, Raf and the cascade of MAP kinases, is ubiquitously expressed. Local activation of Egfr signaling requires the transmembrane protein Star, which is necessary for the secretion of Spitz. Star is expressed in restricted patterns corresponding to the Spitz secreting cells. In the SNS anlage, it was noted that Star becomes restricted to the three tip cells and is maintained in these cells when invagination takes place. As in spitz mutants, the Star mutant SNS anlage is established normally; only one ac-expressing cell is selected and no invagination occurs. Consistently, Star mutants fail to develop the proper set of SNS ganglia and the associated nerves. These observations suggest that tip cells are a Star-dependent source of Spitz activity that triggers Egfr-dependent RTK signaling in the neighboring cells within the SNS anlage. This conclusion is supported by the finding that phosphorylated MAPK, a cellular marker for RTK signaling activity, is indeed activated in cells of the invagination folds, whereas phosphorylated MAPK does not appear in the Star mutant or in the spitz mutant SNS anlage (González-Gaitán, 2000).
To examine whether activated Spitz is sufficient to induce cell movements within the SNS anlage, use was made of the GAL4/UAS system to misexpress secreted Spitz in an ectopic pattern. This was achieved through the expression of activated Spitz from a UAS promotor driven transgenethat was activated by Gal4 under the control of the actin promotor. Under the conditions applied, scattered UAS-dependent transgene expression is observed throughout the early embryo, including the SNS anlage. When activated Spitz is expressed in such a pattern, a variable number of supernumerary infoldings within the SNS anlagen are observed, indicating that activated Spitz is sufficient to initiate cell movements. This result, in conjunction with the observation that the invaginated cells express phosphorylated MAPK, provides evidence that tip cell-derived activated Spitz triggers RTK signaling to initiate the invagination process. This proposal was tested by blocking Egfr signaling in the anterior most region of the SNS anlage that gives rise to the first invagination fold. For this, a GAL4 driver (SNS1-Gal4) was used that causes UAS-dependent gene expression in the corresponding region of the SNS anlage. SNS1-Gal4-mediated expression of a dominant-negative Egfr mutant form from a UAS-controlled transgene causes a specific suppression of the anterior most invagination fold without affecting the others (González-Gaitán, 2000).
The results demonstrate that RTK signaling participates in the selection of tip-cell-dependent invagination centers in the SNS anlage and is subsequently required to initiate morphogenetic movements resulting in invagination folds. This study does not focus on how RTK signaling ties into the wg-modulated Notch signaling process previously shown to be necessary for the selection of the three SNS invagination centers. The data indicate, however, that RTK signaling acts either in parallel or in combination with wg signaling to adjust the proper range of Notch-dependent lateral inhibition. Although in both wg and Egfr signaling mutants, only one ac-expressing cell is singled-out, the selected cells differ with respect to whether they function as tip cells or not. In wg mutants, the single cell causes an invagination, whereas in Egfr signaling mutants, the selected cell fails to provide this feature of SNS invagination centers. The results, therefore, consistently argue that tip cell-derived Spitz triggers local RTK signaling and thereby initiates the formation of invagination folds each headed by the Spitz-secreting tip cell. Thus, Egfr-dependent RTK signaling in Drosophila does not only participate in cell fate decisions and cell proliferation, but also triggers morphogenetic movements within an epithelium, as has been recently demonstrated for fibroblast growth factor (FGF) signaling. It will be interesting to see whether the role of the EGF pathway in cell migration differs at the cellular level from cell migration events triggered by activated FGF receptors (González-Gaitán, 2000).
Many epithelial cells are polarized along the plane of the epithelium, a property termed planar cell polarity. The Drosophila wing and eye imaginal discs are the premier models of this process. Many proteins required for polarity establishment and its translation into cytoskeletal polarity were identified from studies of those tissues. More recently, several vertebrate tissues have been shown to exhibit planar cell polarity. Striking similarities and differences have been observed when different tissues exhibiting planar cell polarity are compared. This study describe a new tissue exhibiting planar cell polarity -- the denticles, hair-like projections of the Drosophila embryonic epidermis. the changes in the actin cytoskeleton that underlie denticle development are described in real time, and this is compared with the localization of microtubules, revealing new aspects of cytoskeletal dynamics that may have more general applicability. An initial characterization is presented of the localization of several actin regulators during denticle development. Several core planar cell polarity proteins are asymmetrically localized during the process. Finally, roles for the canonical Wingless and Hedgehog pathways and for core planar cell polarity proteins in denticle polarity are described (Price, 2006).
Among the hallmarks of PCP in structures as diverse as Drosophila wing hairs to stereocilia in the mammalian ear is polarization of the actin cytoskeleton. The polarized actin cytoskeleton underlying wing hair polarity has been described and defects in polarization in fz and dsh mutants have been documented. Microtubules (MTs) are also polarized in developing wing hairs, and disruption of either actin or MTs disrupts wing hair formation. The data suggest that basic features of cytoskeletal polarity in pupal wing hairs are also seen in denticles. Denticles, like wing hairs, arise from polarized actin accumulations – in denticles this occurs along the posterior cell margin. Further, like wing hairs, denticles all elongate in the same direction. The less detailed analysis of dorsal hairs suggests that they also arise from polarized actin accumulations, but these are more complex; different cell rows accumulate actin either along the anterior or posterior cell margin (Price, 2006).
The effect of Wg and Hh on denticle development is mediated in part by their regional activation of the Shaven-baby transcription factor (Ovo), which is necessary and sufficient for cells to generate actin-based denticles. Therefore genes that are targets of Shaven-baby are likely to be triggers for actin accumulation and cytoskeletal rearrangements. Wg and Hh signaling may also trigger polarization of cellular machinery that is not typically thought to be involved in PCP – e.g. the polarity of Arm that was observed. It will be useful in the future to examine whether proteins polarized during germband extension, such as Bazooka, are also polarized during denticle formation. Mutations in both hh and wg also affected the normal changes in cell shape accompanying denticle formation – rather than elongating along the dorsal-ventral axis, cells remain columnar. A similar failure of cells to polarize during dorsal closure is observed in wg mutants. These effects may reflect alterations in cell polarization or cytoskeletal regulation. It will be of interest to determine whether changes in cell shape are coupled to the establishment of cytoskeletal polarity (Price, 2006).
Thus far the analysis of actin in wild-type and mutant pupal wings has been restricted to snapshots in fixed tissue. This was extended by examining F-actin in developing denticles in real time, revealing features of polarization that have not been noted previously; these features may be shared with wing hairs or other polarized structures. The initial cytoskeletal change observed was actin accumulation all across the apical surface of the cell. This actin gradually 'condenses', becoming more restricted to the posterior cell margin and forming distinct condensations, which then brighten and sometimes merge. They then elongate, all in the posterior direction. It will be interesting to learn whether the dynamic aspects of condensation involve de novo actin polymerization and/or collection of preexisting actin filaments (Price, 2006).
It is only in late condensations that enrichment was seen of any of the actin regulators that were examined. Arp3 and Dia are weakly enriched in late condensations, with enrichment increasing as denticles elongate, and Ena is enriched even later. Of course, the localization of these actin regulators to developing denticles does not by itself demonstrate that they play an important role there, but it is consistent with the possibility that they have a role in actin remodeling associated with denticle elongation. To test this hypothesis, genetic analyses will be necessary. This presents significant obstacles, since Arp2/3 and Dia are required for much earlier events (syncytial stages and cellularization), while maternal Ena plays a role in oogenesis, complicating analysis of loss-of-function mutants. Surprisingly, none of these actin regulators localizes in an informative fashion during the initial formation of actin condensations (though APC2 localizes there during this time). Thus additional regulators functioning during early denticle development need to be identified. Studies of cytoskeletal regulation in the larger adult sensory bristles may guide this. EM studies, the use of cytoskeletal inhibitors, and FRAP, which has proved informative in studies of wing hairs and bristles, may reveal how actin in denticles is assembled. Finally, it will be important to study in denticles additional actin regulators that regulate bristle development (Price, 2006).
What signals regulate denticle polarity? As examples of PCP have proliferated, understanding of the signals that instruct cells about their orientation in epithelial sheets has evolved. Certain features are shared in many, if not all, tissues. Fz receptors play a key role. Other core polarity proteins including Dsh, Fmi, Van Gogh/Strabismus and Prickle act in many if not all places. The current data extend this analysis to the denticles. Intriguing differences were found between the phenotypes of loss of Wg or Hh signaling, in which polarity was severely altered or abolished and loss of proteins that play dedicated roles in PCP, such as embryos null for either fz or stbm, which exhibit more subtle defects. A strong polarity bias was retained in these latter mutants, with cells in the posterior denticle rows correctly polarized and only cells in the anterior two rows making frequent mistakes. Interestingly, occasional mistakes are also observed in wild-type embryos (albeit at much lower frequency) and these are also restricted to the anterior most rows. This is in strong contrast to the effects of these mutants in the wing disc, where they globally disrupt polarity (Price, 2006).
One possible reason for this difference is the different scales of the tissues. The embryonic segment is only 12 cells across, while the wing disc encompasses hundreds of cells. Many core polarity proteins help mediate a feedback loop that amplifies an initially small difference in signal strength between the two sides of a wing cell. Perhaps the small scale of the embryonic segment makes this reinforcement less essential. It is also intriguing that the polarity is most sensitive to disruption in the anterior two denticle rows. If signal emanated from the posterior, signal strength might be lower in the anteriormost cells, rendering the reinforcement process more important. The lower frequency of defects in pk1 mutants may also reflect the reduced role of the feedback loop, but this is subject to the caveat that pk is a complex locus with different mutations having different consequences. Future work will be needed to test these possibilities (Price, 2006).
Significant questions also remain about the signal(s) activating Fz receptors during PCP. Wnts were initial candidates, since Fz proteins are Wnt receptors. In vertebrates, this may be the case – Wnt11 regulates convergent extension and Wnt proteins can regulate PCP in the inner ear. By contrast, Drosophila Wnt proteins may not play a direct role. The Wg expression pattern in the eye and wing discs is not consistent with a role as the PCP ligand. Detailed studies of PCP in the eye and abdomen are most consistent with the idea that neither Wg nor other Wnt proteins are polarizing signals, but suggest that Wg regulates production of a secondary signal [dubbed `X'). Recent work suggests that Fj, Ds and Fat may be this elusive signal, with Drosophila Wg acting as an indirect cue of polarity. In fact, one cannot rule out the possibility Wnt11's role in vertebrate convergent extension is also indirect (Price, 2006).
Roles were found for Wg, Dsh and Arm in establishing denticle polarity. At face value, Arm's role is surprising, since the current view is that the Wg pathway diverges at Dsh, with a non-canonical branch (see Eisenmann's Wnt Signaling) mediating PCP and the canonical pathway playing no role in this. However, the data do not imply that Arm is required in denticle PCP per se. Wg acts in a paracrine feedback loop to maintain its own expression. In embryos maternally and zygotically mutant for arm alleles that cannot transduce Wg, Wg expression is lost by late stage 9. Thus, even though Arm is not in the non-canonical pathway, loss of Arm could still disrupt PCP indirectly due to the loss of Wg expression (Price, 2006).
While the data demonstrate that Wg is required for denticle PCP, two things suggest its role is indirect. wg mutants retain segmental periodicity in denticle orientation, suggesting that polarity is not totally disrupted, while in hh mutants there is no segmental periodicity. Second, when Wg signaling was reduced but did not eliminated, many cells retained normal polarity and there was segmental periodicity to which cells lost polarity or exhibited polarity reversals. This is consistent with the idea that Wg regulates production of another ligand. In fact, Wg's role may be even more indirect – given the more dramatic effect of hh, Wg's primary role in polarity may be to maintain Hh expression (this is also consistent with a requirement for canonical pathway components like Arm). Global activation of Hh signaling in the ptc mutant also disrupts polarity. Hh thus remains a possible directional cue. In the abdomen, Hh also plays an important role in polarity, but it does not seem to be the directional cue either but rather regulates its production; this may also be the case in the embryo. Thus the precise roles for canonical Wg and Hh signaling in denticle polarization must be addressed by future experiments. If neither Wnts nor Hh are directional signals, what is? Data from the eye, wing and abdomen suggest roles for Ds, Fj, Fat and Fmi but details differ in different tissues. It thus will also be useful to examine Ds, Fj and Fat's roles in embryonic PCP (Price, 2006).
Cellular interaction between the proximal and distal domains of the limb plays key roles in proximal-distal patterning. In Drosophila, these domains are established in the embryonic leg imaginal disc as a proximal domain expressing escargot, surrounding the Distal-less expressing distal domain in a circular pattern. The leg imaginal disc is derived from the limb primordium that also gives rise to the wing imaginal disc. Essential roles of Wingless in patterning the leg imaginal disc are described. (1) Wingless signaling is essential for the recruitment of dorsal-proximal, distal, and ventral-proximal leg cells. Wingless requirement in the proximal leg domain appears to be unique to the embryo, since it has previously been shown that Wingless signal transduction is not active in the proximal leg domain in larvae. (2) Downregulation of Wingless signaling in wing disc is essential for its development, suggesting that Wg activity must be downregulated to separate wing and leg discs. In addition, evidence is provided that Dll restricts expression of a proximal leg-specific gene expression. It is proposed that those embryo-specific functions of Wingless signaling reflect its multiple roles in restricting competence of ectodermal cells to adopt the fate of thoracic appendages (Kubota, 2003).
At embryonic stage 11, the early expression of D11 expression marks the entire limb primordium that gives rise to both wing and leg discs. After separation of wing and leg discs at stage 12, Dll expression becomes restricted to the center of the leg disc. Double labeling of stage 15 leg discs reveals that there is still a significant number of cells that coexpress Dll and the proximal leg marker Esg, suggesting that expression of Dll and Esg is not a strictly exclusive event. Rather, the result suggests that those marker genes respond differentially to inductive signals in the leg primordium (Kubota, 2003).
In the leg disc, Hth defines the trunk and proximal cell identities, and its expression is excluded in the distal leg domain in the larval stage. Double labeling with antibodies against Esg and Hth reveal that the Esg expression overlaps with Hth expression. Esg is used as a marker uniquely labeling the distinct cell identity of the proximal leg domain in the trunk region (Kubota, 2003).
The expression domain of wg and the position of wing and leg primordia were compared. Wg expression in the trunk ectoderm starts as stripes along the anterior side of the compartment boundaries. At early stage 11, most of the limb primordia marked with Dll protein expression overlap with wg stripes, as revealed by the wg-lacZ reporter. At late stage 11, wg-lacZ stripes break up into dorsal patches and ventromedial stripes. By late stage 12, expression of Dll protein becomes limited to a group of cells partially overlapping the dorsal edge of ventromedial wg stripes. The ventromedial wg stripe also overlaps with proximal leg cells that are labeled with anti-Esg at stage 15. The ventral half of proximal leg cells is nearly completely included within the ventral wg stripes. The dorsal half of leg cells is also located adjacent to, but not included in, the dorsal edge of the wg stripes. On the other hand, a reciprocal relationship between wg expression and wing primordia was observed. When wing primordia are first recognizable at stage 12 as cells expressing Vestigial (Vg), they do not overlap with the stripe of wg. Dorsal cell migration further separates wing primordia from the source of Wg at stage 15. The absence of Wg expression near wing primordia suggests that Wg does not play a positive role in wing disc development (Kubota, 2003).
Wg has been shown to be required for the induction of the thoracic limb primordium and other imaginal discs. To investigate a late role of Wg signaling, the functions of intracellular signal transducers of Wg were studied. The Drosophila homolog of ß-catenin encoded by armadillo (arm) plays dual roles, one as a mediator of Wg signaling by regulating transcription of various target genes, and the other as a component of Cadherin-dependent cell adhesion. Two alleles of arm were analyzed, one being null allele armYD35, and the other armH8.6, which is specifically defective in Wg signaling. Since both armYD35 and armH8.6 show the same phenotype, the function of Arm in Wg signaling, but not in cell adhesion, is required for leg disc development (Kubota, 2003).
To confirm whether Wg signaling is required cell autonomously for leg disc development, the dominant-negative forms of Drosophila TCF (DTCFdeltaN) or Drosophila axin (Daxin) were expressed in the limb primordia. The Dll-Gal4 driver, which is turned on in the limb primordium at stage 11 and continues to be active in leg and wing discs, was used. In Dll-GAL4 embryos carrying UAS-DTCFdeltaN or UAS-Daxin, the overall size of leg discs was reduced. Expression of Esg was preferentially reduced in the dorsal side. The drastic reduction of Dll mRNA and protein in distal leg cells in the armH8.6 mutants as well as in DTCFdeltaN- and Daxin-expressing embryos demonstrate that Wg signaling is required for both proximal and distal leg cells. In arm mutants, Hth-expressing cells expand to the distal domain. This observation suggests that, upon loss of Wg signaling, prospective leg disc cells lose their identity and adopt the fate of trunk ectoderm. However, disc-specific reduction of Wg signaling does not affect wing disc formation, although the Dll-Gal4 driver is active in the wing primordium. It is concluded that late function of Wg signaling promotes formation of the leg disc with a higher requirement in the proximal domain, but is dispensable for wing disc formation (Kubota, 2003).
Cell fate maintenance of proximal leg requires continuous signaling by Wg. The requirement for arm and wg is higher in the proximal leg domain. arm mutations nearly eliminate all Esg expression, but leave some Dll-positive cells. wgts is a hypomorph at the restrictive temperature and leaves distal leg cells nearly intact while significantly affecting proximal leg cells, especially those at the dorsal side of the disc. Dorsal cells are far from the source of Wg and are first to lose identity upon reduction of Wg activity. Since Esg expression in ventral proximal cells overlaps with the wg stripe, it is proposed that the localized expression of Wg and its range of diffusion are major determinants of the site of proximal cell formation. It is likely that dorsal-proximal cells require a higher level of Wg to be produced to reach their position (Kubota, 2003).
Dll expression is initially found in the entire limb primordia and becomes restricted to the edge of the Wg stripe that becomes the center of the leg disc. One candidate for an additional factor that places Dll in this position is Dpp, is expressed in stripes abutting the Wg stripe; Dpp is known to be required for distal leg development (Kubota, 2003).
Finally, the center of the embryonic leg disc is devoid of the expression of proximal cell markers Esg and Hth, marking the distal leg domain. Separation of the proximal and distal leg domains is a slow process, taking several hours to complete. One model for the mechanism regulating this separation process is that proximal gene expression is downregulated by a distal gene, as shown by ectopic Dll expression repressing Esg expression. Since expression of Esg is also regulated by positive input from Wg signaling, Esg expression does not necessary mirror the absence of Dll. In support of this idea, Dll is known to repress proximal genes in larval leg discs. The second possibility is a restriction of proximal cell movement into the distal domain. Cells in the Hth-expressing proximal domain in the larval leg disc have distinct cell-adhesive properties from those in the Dll-expressing distal domain, and by extension, cells with high levels of Dll or Hth may not mix well in the embryo as well. Since Hth is widely expressed in the embryonic ectoderm, Dll-expressing cells may be forced to localize at the center of the leg disc (Kubota, 2003).
This study tested a distal organizer model. The model proposes that a distal organizer placed in the field of a developing appendage signals surrounding cells to acquire proximal cell fate. If this signal acts in all directions, circular arrangement of proximal cells can be achieved. This study demonstrates that dorsal and ventral halves of proximal leg cells have different requirements for Wg. The way Wg acts to organize proximal cell differentiation is not consistent with the distal organizer model. Rather, the results support a second model where dorsoproximal, distal, and ventral-proximal cells are specified separately and assembled to form a circular pattern (Kubota, 2003).
The preferential loss of proximal leg cells upon partial loss of Wg signaling is very similar to the phenotype of EGFR mutant embryos, suggesting that both Wg and EGFR contribute to differentiation of proximal leg cells. A difference in the temporal requirement for the two signals was noted, however. EGFR signaling is activated transiently at the time of disc specification, and the requirement of its activity is limited to a short period around this stage. It has been proposed that EGFR acts within limb primordial cells to promote leg development. This study shows that Wg is persistently expressed at the ventral part of the leg disc and maintains the fate of proximal and distal leg cells. These findings indicate that the leg disc development is initiated by transient activation of EGFR, and its cell fate is maintained by the persistent activity of Wg (Kubota, 2003).
Wg and EGFR signaling are not active in wing discs and are not required for wing formation after stage 11. However, ectopic activation of either of the signals in the limb primordium suppresses the wing disc development, suggesting that downregulation of Wg and EGFR signals have a permissive role in the wing development. These signals are reactivated in postembryonic stages to organize the wing disc. The downregulation of Wg and EGFR signals in a prospective wing disc is accomplished by two mechanisms, one by limiting activation of the two signals to the ventral side of the embryo, and the other by allowing wing primordium to migrate to the dorsal direction away from the source of the inhibitory signals. Thus, cell migration serves as a novel mechanism to restrict the effect of diffusible signaling molecules (Kubota, 2003).
Although the analogous pattern of Wg and Dpp expression plays essential roles in PD patterning in embryonic and larval leg development, significant differences are noted. In embryonic leg discs, expression of both proximal and distal leg markers is lost in mutants of Wg signaling or Dpp signaling. Therefore, Wg and Dpp contribute to both proximal and distal leg development in the embryo. In the larvae, reduction of Wg and Dpp expression due to the loss of hh function causes a loss of the distal domain, but no effect on the proximal gene expression was observed, suggesting that Wg and Dpp play little or no role in the development of proximal domain. The inability of Wg or Dpp to participate in the proximal leg patterning in the larvae is due to, at least in part, the function of Hth to block activation of target genes for Wg and Dpp. In the embryo, however, Hth does not block expression of esg, a target gene for Wg, as demonstrated by coexpression of Esg and Hth. Therefore, proximal domains of embryonic and larval leg discs are different in the way Hth regulates target genes for Wg. This difference may reflect distinct stages of leg development in the embryo, where proximal leg and epidermal cells are continuous, as defined by Hth expression, and in the larvae, where they are separated by the peripodial membrane (Kubota, 2003).
The complementary pattern of Wg and Dpp expression in the larval leg disc is maintained by mutual repression. No evidence for mutual repression of Wg and Dpp was observed in embryonic leg discs. Perhaps the complementary expression pattern of Wg and Dpp in the embryonic leg disc is under the control of the mechanism regulating the global dorsoventral pattern of the embryo (Kubota, 2003).