division abnormally delayed and dally-like

DEVELOPMENTAL BIOLOGY

Larval

The expression of the P1 enhancer trap line, containing a P1 insertion under dally regulation, was examined through the division cycles of lamina precursor cells (LPCs) in third instar larval brains using anti-beta-gal antibody and digoxigenin-labelled DNA probes complementary to lacZ mRNA. The highest levels of beta-gal immunoreactivity are found in LPCs along the anterior segment of the lamina furrow. Cells in this region are in G2 and M phase of the first division. The lacZ mRNA shows a more limited distribution, presumably because of beta-gal protein perdurance and is restricted to the G2 and M phase domains of LPC division one. Cell cycle-dependent expression of enhancer trap insertions in this locus was obtained by staining third instar larval brains with both anti-beta-gal and anti-cyclin B antibodies. There is an overlap of expression of the enhancer trap insertion with cyclin B in several groups of dividing cells, including the inner proliferative center (IPC), suggesting that the cell cycle-restricted expression is not limited to LPCs. However, some cells of the central brain complex express the enhancer trap marker but do not show high levels of cyclin B immunoreactivity (Nakato, 1995).

Effects of Mutation or Deletion

Larvae homozygous for the P1 insertion which disrupts dally expression shows disorganization of the anterior segment of the outer proliferative center (aOPC)/lamina precursor cell (aOPC/LPC) epithelium in approximately 10% of the CNS preparations examined. Disordering of the eye and reductions or duplications of the antenna, also with low penetrance, are found in P1 homozygous adults. The low penetrance found in the P1 mutant made analysis of the cell division defect difficult. A search was carried out for more severe alleles within existing collections of P-element-induced mutants. One semi-lethal enhancer trap insertion in the region of 66D/E, sl(3)06464, shows a beta-gal-staining pattern in the larval brain like that of line P1. Homozygous sl(3)06464 adults exhibit abnormalities in several adult tissues, including reductions or complete loss of genitalia, disordering and reduction in the number of ommatidia, reductions and duplications of the antenna, and incomplete wing vein V and wing notching. P1 and sl(3)06464 enhancer trap expression patterns in other tissues (antenna, eye, leg and wing discs) and embryonic developmental stages were also coincident. The two P-element alleles are henceforth referred to as dally P1 and dally P2. Genetic complementation tests were performed with dally P1 and dally P2 alleles for the antennal phenotypes since these are easy to score and of fairly high penetrance in dally P1. For antennal defects, dally P1 and dally P2 fail to complement, supporting the conclusion that these two P-element insertions affect the same gene. To confirm that dally P1 and dally P2 are responsible for the phenotypes described above and that these phenotypes are caused by loss-of-function alleles, the P-element insert in dally P1 was mobilized. The imprecise excision class creates small deletions that can potentially effect the removal, either completely or partially, of the normal function of the targeted gene. 23 independent excision alleles failed to complement the adult phenotypes of dally P2 . These alleles, either as homozygotes or in combination with dally P2 show phenotypes with a range of expressivity and penetrance; the more severe mutants affect the same tissues and to the same degree as observed for dally P2 homozygous adults. For example, dally DP-188 shows abnormalities in the eye, antenna, genitalia and wing, similar to dally P2 (Nakato, 1995).

In the wild-type eye disc, the morphogenetic furrow (MF) serves as an anatomical marker for the assembly of the repeated sensory units, the ommatidia. As originally described, the MF is a broad indentation in the eye disc epithelium, which moves from the posterior to the anterior, marking the wave of differentiation that sweeps forward. Cells in different parts of the cell cycle occupy specific positions relative to the MF. Anti-cyclin B and propidium iodide staining were used to identify G2, and mitotic prophase, metaphase, anaphase and telophase cells in the eye disc. Cell division in the developing eye disc epithelium anterior to the MF is asynchronous as seen by the unpatterned cyclin B expression. However, an increase in the level of anti-cyclin B immunoreactivity anterior to the MF is observed, providing evidence of cell cycle synchrony beginning in G2 as cells approach the furrow. Immediately posterior to this G2 domain mitosis occurs. Mitotic cells in metaphase, anaphase and telophase are found immediately ahead of the MF. This mitotic domain reflects the cell cycle synchronization taking place just ahead of the MF. Cells complete mitosis as they enter the furrow, and become synchronized in G1 in the MF. Progression into the subsequent S phase takes place within the furrow, followed by G2. A second coordinate mitosis follows, completing the two division cycles distributed across the MF. In dally mutants, the first zone of cyclin B expression, which normally ends approximately 3-5 cell dimensions anterior to the MF, extends too far posterior, to the beginning of the MF. The M phase of this first division is also displaced toward the posterior in dally mutants. The mitotic cells found at the edge of the MF in dally P2 mutants are in earlier stages of mitosis compared to wild-type (Nakato, 1995).

The second division along the eye disc MF does take place in dally P2 mutants. The cell division defects observed for dally P2 are also found in two other dally alleles, both as homozygotes and in combination with dally P2. As is the case for the lamina, the cell division defects in the eye disc are found without an overall disruption of normal morphology. In the wild type, the neuronal marker Elav is expressed in the assembling ommatidia posterior to the MF. dally mutant discs show this pattern of Elav expression as well, indicating that the cell division defects are not secondary to a gross disruption of eye development. If dally mutations delay cell division, the time required for cells labelled in the S phase of the first division to traverse into the following G1 should be slower in dally mutants, when compared to heterozygous control larvae. In the wild-type eye disc, mitotic cells of the first division are found at the apical surface of the epithelium in front of the MF and, following M phase, nuclei migrate basally into the MF. The position of a nucleus labelled in the previous S phase can therefore be used as a measure of cell cycle progression -- the more basally located cells being further along in their progression through the cell cycle. In dally mutants 4 hours after pulse-labeling, nuclei are delayed in their descent into the furrow, as compared to labelled cells from the heterozygous control. These findings support the conclusion that the first division cycle is delayed as a consequence of compromised dally function (Nakato, 1995).

The DPP requirement for cell fate specification and cell cycle synchronization in the developing Drosophila eye was examined by determining whether cells defective for thickveins, saxophone or schnurri show abnormalities in cell division or differentiation. Clones mutant for a null allele of tkv that are anterior or posterior to the morphogenetic furrow have amounts of Cyclin B that are indistinguishable from those in surrounding cells. In contrast, tkv clones that span the MF maintain cyclin B expression in the anterior part of the furrow, even though the surrounding cells arrested in G1 have no detectable Cyclin B. Maintenance of cyclin B expression is thought to indicate a failure of cell cycle progression, as Cyclin B levels decline in M phase. Mitotic figures are not observed in clones in the anterior half of the MF. The phenotype observed in the clones is similar to defects caused by mutations in division abnormally delayed (dally), which is required for G2-M progression ahead of the furrow. Mutations in dally and dpp display genetic interactions in development of the eye, antenna, and genitalia, which suggests that dally augments Dpp function. The behavior of Dpp-receptor mutant clones supports a role for Dpp in controlling progression through G2-M as a means of synchronizing the divisions that accompany differentiation of the eye disc. Cell fate, however, is unaffected by receptor mutation, as revealed by the expression of atonal, a proneural gene required for retinal precursor cell 8 (R8) determination. Because atonal expression is maintained in tkv clones, hh must not act through dpp to induce its expression, and thus dpp mediates a subset of hh functions in the MF (Penton, 1997).

Wingless is a member of the Wnt family of growth factors, secreted proteins that control proliferation and differentiation during development. Studies in Drosophila have shown that responses to Wg require cell-surface heparan sulphate, a glycosaminoglycan component of proteoglycans. These findings suggest that a cell-surface proteoglycan is a component of a Wg/Wnt receptor complex. The protein encoded by the division abnormally delayed (dally) gene is a cell-surface, heparan-sulphate-modified proteoglycan. dally partial loss-of-function mutations compromise Wg-directed events, and disruption of dally function with RNA interference produces phenotypes comparable to those found with RNA interference of wg or frizzled/Dfz2. Ectopic expression of Dally potentiates Wg signalling without altering levels of Wg and can rescue a wg partial loss-of-function mutant. Dally, a regulator of Decapentaplegic (Dpp) signalling during post-embryonic development, has tissue-specific effects on Wg and Dpp signalling. Dally can therefore differentially influence signalling mediated by two growth factors, and may form a regulatory component of both Wg and Dpp receptor complexes (Tsuda, 1999).

In Drosophila, imaginal wing discs, Wg and Dpp, play important roles in the development of sensory organs. These secreted growth factors govern the positions of sensory bristles by regulating the expression of achaete-scute (ac-sc), genes affecting neuronal precursor cell identity. Earlier studies have shown that Dally, an integral membrane, heparan sulfate-modified proteoglycan, affects both Wg and Dpp signaling in a tissue-specific manner. dally is required for the development of specific chemosensory and mechanosensory organs in the wing and notum. dally enhancer trap is expressed at the anteroposterior and dorsoventral boundaries of the wing pouch, under the control of hh and wg, respectively. dally affects the specification of proneural clusters for dally-sensitive bristles and shows genetic interactions with either wg or dpp signaling components for distinct sensory bristles. These findings suggest that dally can differentially regulate Wg- or Dpp-directed patterning during sensory organ assembly. For pSA, a bristle on the lateral notum, dally shows genetic interactions with iroquois complex (IRO-C), a gene complex affecting ac-sc expression. Consistent with this interaction, dally mutants show markedly reduced expression of an iro::lacZ reporter. These findings establish dally as an important regulator of sensory organ formation via Wg- and Dpp-mediated specification of proneural clusters (Fujise, 2001).

dally enhancer trap expression in the wing disc was examined. This study revealed expression at the A/P and D/V boundaries in the wing pouch. At mid- to late-third larval instar, the expression of dally enhancer trap overlaps with that of ac-sc at the D/V boundary, indicating that dally is expressed at high levels in cells flanking the cut- and wg-expressing edge cells like Delta and Serrate. N signaling in cells of the D/V boundary results in the transcriptional activation of several genes, including vg, wg, ct, and members of the Enhancer of split complex, E(Spl). Like other genes affecting assembly of wing margin structures, dally expression of the D/V boundary is sensitive to N-receptor activation mediated by Dl and Ser. Blocking Wg signaling at the D/V border cells can repress dally expression, indicating that dally expression along the margin is positively regulated by Wg signaling, which is also required for the expression of proneural AS-C genes. Further studies are required, however, to determine whether N also has a direct function in the regulation of dally expression (Fujise, 2001).

dally cooperates with Wg to form the wing margin structures. Ac expression is severely decreased in dally mutants, supporting the idea that dally serves as a component of the Wg receptor complex to induce AS-C expression at the prospective wing margin. Taken together, these observations indicate that dally is a target gene of Wg signaling pathway, and at the same time, it mediates the same signaling, suggesting that dally is involved in a positive feedback loop of Wg signaling at the D/V boundary of the wing pouch. Frizzled-2, the Wg receptor, is down-regulated by Wg signaling at the D/V boundary. Dally, a putative Wg coreceptor, may also participate in the feedback circuits of Wg signaling, as has been suggested for Fz2 (Fujise, 2001).

dally is required for the development of sensory organs in the adult wing and notum. dally has been also identified as a gene that affects the development of sensory organs by a gain-of-function screen. dally mutants show reduced numbers of sensory bristles and campaniform sensilla at the wing margin. Specific macrochaetae on the notum, DC, pSA, and pPA, are affected in dally mutants. In all cases, the expression of genes specific for proneural cell differentiation is compromised in dally-sensitive bristles, indicating that dally affects sensory organ development at the step of prepatterning (Fujise, 2001).

Wg and Dpp have been shown to affect prepatterning of sensory organs by governing the expression of proneural genes, such as ac-sc. dally has been shown to affect the signaling levels of either Wg or Dpp. Therefore, an examination was made to determine whether dally affects sensory organ formation via either Wg or Dpp signaling pathways. Genetic experiments provided evidence that, in the prospective notum region of the wing disc, dally selectively influences Wg signaling to form the pPA bristle and Dpp signaling to form the pSA and DC bristles. It is particularly intriguing that, during development of DC macrochaetae, dally genetically interacts with only Dpp signaling, while the formation of these bristles requires both Wg and Dpp activities. It has been indicated that the A/P coordinates of the DC cluster are limited by Dpp signaling. In dally homozygous wing discs, the DC cluster is apparently shorter in the A/P coordinates compared with wild-type discs, suggesting that dally regulates Dpp signaling activity to limit the A/P length of the DC cluster. What are the mechanisms that can account for the selective interactions of dally and specific growth factor signaling? One obvious interpretation of genetic experiments on DC macrochaetae is that differences in dose effects between dpp and wg are responsible for the apparent specificity. It is also possible that the ligand-specificity of Dally is controlled at the cellular level through modification of heparan sulfate structures (Fujise, 2001).

Dally, Dpp, and IRO-C genetically interact with each other during the formation of the pSA macrochaete. Although interactions between Dpp signaling and IRO-C have been suggested, evidence is provided that Dpp signaling components interact with the genes of IRO-C. Ectopic Dpp signaling using a constitutively active type I receptor, tkv, leads to an ectopic induction of the pSA macrochaete, supporting the idea that Dpp signaling is required for prepatterning for this bristle. Significant reductions in the expression of iro enhancer trap is observed in dally mutant wing discs. Expression of the iro at the lateral notum region is critical for the proneural cluster formation and bristle development in this region. Taken together, these findings suggest that dally mediates Dpp signaling to control expression of the genes of IRO-C during the formation of the pSA bristle (Fujise, 2001).

Drosophila glypicans control the cell-to-cell movement of Hedgehog by a dynamin-independent process

The signalling molecule Hedgehog (Hh) functions as a morphogen to pattern a field of cells in animal development. Previous studies in Drosophila have demonstrated that Tout-velu (Ttv), a heparan sulphate polymerase, is required for Hh movement across receiving cells. However, the molecular mechanism of Ttv- mediated Hh movement is poorly defined. Dally and Dally-like (Dly), two Drosophila glypican members of the heparan sulphate proteoglycan (HSPG) family, are shown to be the substrates of Ttv and are essential for Hh movement. Embryos lacking dly activity exhibit defects in Hh distribution and its subsequent signalling. However, both Dally and Dly are involved and are functionally redundant in Hh movement during wing development. Hh movement in its receiving cells is regulated by a cell-to-cell mechanism that is independent of dynamin-mediated endocytosis. It is proposed that glypicans transfer Hh along the cell membrane to pattern a field of cells (Han, 2004).

To dissect the molecular mechanism(s) by which HSPG(s) regulates Hh signalling, attempts were made to identify specific proteoglycan(s) involved in Hh signalling during embryonic patterning. During embryogenesis, Hh and Wingless (Wg) are expressed in adjacent cells and are required for patterning of epidermis. In stage 10 embryos, Hh is expressed in two rows of cells in the posterior compartment of each parasegment, while Wg is expressed in one row of cells anterior to Hh expression cells. The expression of Hh is controlled by Engrailed (En) whose expression is maintained by Wg signalling through a paracrine regulatory loop. Hh signalling in turn is required for maintaining the expression of wg whose activity controls the production of the naked cuticles. Loss of either Hh or Wg signalling leads to a loss of naked cuticle, which is defined as segment polarity phenotype (Han, 2004 and references therein).

Disruption of dly in embryos by RNA interference (RNAi) leads to a strong segment polarity defect, suggesting that Dly is likely to be involved in Hh and/or Wingless (Wg) signalling in embryonic epidermis. To explore the potential role of Dly in Hh signalling, a number of dly mutant alleles were isolated using EMS mutagenesis. dly^A187 is a null allele and is used for further analyses. Animals zygotically mutant for dly appears to have normal cuticle patterning and survive until third instar larvae. However, homozygous mutant embryos derived from females lacking maternal dly activity (referred to as dly embryos hereafter) die with a strong segment-polarity phenotype resembling those of mutants of the segment polarity genes hh and wg. In dly embryos, both En expression and wg transcription fade by stage 10, suggesting further that dly is involved in the Hh and/or Wg pathways (Han, 2004).

To further determine whether Dly activity is required for Hh signalling in embryogenesis, Hh signalling activity was examined in dly embryos during mesoderm development. Hh and Wg signalling have distinct roles in patterning embryonic mesoderm. Hh signalling activates the expression of a mesodermal specific gene bagpipe (bap) in the anterior region of each parasegment, whereas Wg signalling inhibits bap expression in the posterior region. bap expression is diminished in the hh mutant, but is expanded to the posterior parasegment in the wg mutant. Consistent with a role of Dly in Hh signalling, it was found that bap expression was strikingly reduced in dly embryos. Together with the segment polarity phenotype, these results strongly argue that Dly is required for Hh signalling during embryogenesis (Han, 2004).

The role of Dly in Hh signalling was further examined during wing development in which Hh and Wg signalling function independently of each other. In the wing disc, Hh signalling induces the expression of its target genes in a narrow stripe of tissue in the A compartment abutting the AP boundary. Hh signalling patterns the central domain of wing blade and controls the positioning of longitudinal veins L3 and L4. The roles of Dly in Hh signalling were examined by analyzing adult wing defects using 'directed mosaic' technique. Surprisingly, no detectable phenotypes were observed in adult wings bearing dly mutant clones. It was reasoned that Hh signalling may be mediated by other HSPGs in the wing. One candidate is the glypican dally that has been shown to be involved in Wg and Dpp signalling. Because available dally alleles used previously were hypomorphic, several dally null alleles were generated by P-element mediated mutagenesis. dally⁸⁰ is a null allele and was used for analysis. However, similar to other dally alleles, homozygous dally⁸⁰animals are viable. The wing bearing dally⁸⁰ clones exhibits a partial loss of the L5 vein with a high penetrance, but no detectable defects in the central domain of wing blade. To determine whether dally and dly have overlapping roles in Hh signalling in wing development, clones mutant for both dally⁸⁰ and dly^A187 (referred as dally-dly hereafter) were generated. Interestingly, the adult wings bearing clones mutant for dally-dly show L3-L4 fusion. This phenotype is typical of loss of Hh function, suggesting that Dally and Dly play redundant roles in Hh signalling in wing development (Han, 2004).

This study demonstrates that Dly is the main HSPG involved in Hh signalling during embryogenesis, at least in epidermis and mesoderm, the two tissues that were carefully examined. Three lines of evidence strongly support this conclusion. (1) Embryos lacking both maternal and zygotic dly activities develop a strong segment polarity defect and exhibit diminished expression of En and Wg. (2) Hh can be detected as punctate particles at least one cell diameter from its producing cells and these punctate particles are absent in dly-null embryos. (3) A reduced expression of bap was observed in dly mutant embryos, a phenotype specifically attributed to the Hh signalling rather than Wg signalling defect. Previously, it was shown that the punctate particles of Hh staining are absent in ttv null embryos. The formation of such Hh staining particles, referred to as large punctate structures (LPS), requires cholesterol modification, and movement of these large punctate structures across cells is dependent on Ttv activity. The current results are consistent with these observations and suggest that Dly is the main HSPG involved in the movement of these LPS across cells. It is conceivable that the punctate particles of Hh staining that were observed may represent Hh-Dly complexes. In this regard, Dly may either prevent secreted Hh from being degraded and/or facilitate Hh movement from its expression cells to adjacent receiving cells. These two mechanisms are not mutually exclusive. In the absence of Dly function, secreted Hh is either degraded or fails to move to the adjacent cells (Han, 2004).

In addition to dly, three other HSPGs, including Dally, Dsyndecan and Trol, are also expressed in various tissues during embryogenesis. In particular, dally is expressed in epidermis and has been shown to be involved in Wg signalling. Removal of Dally activity in embryos either by dally hypomorphic mutants or by RNA interference (RNAi) generates denticle fusions. Further studies demonstrate that the cuticle defect associated with dally embryos by RNAi is weaker than that of dly. The results in this study suggest that Dly plays more profound roles in embryonic patterning than Dally. It remains to be determined whether Dally and other two Drosophila HSPGs are involved in Hh signalling in other developmental processes during embryogenesis (Han, 2004).

Dally and Dly are involved and are redundant in Hh signalling in the wing disc. Consistent with this, the GAG chains of Dally and Dly are shown to be altered in the absence of Ttv activity, suggesting that both Dally and Dly are indeed the substrates for Ttv. Redundant roles of cell membrane proteins have been demonstrated in many other signalling systems. For example, both Frizzled (Fz) and Drosophila Frizzled 2 (Fz2) are redundant receptors for Wg, although Fz2 has relative high affinity in binding to Wg protein. Dly protein is distributed throughout the entire wing disc. Previous studies have demonstrated that dally is highly expressed at the AP border. Interestingly, Dally expression at the AP border is overlapped with the ptc expression domain and is under the control of Hh signalling. It is likely that both Dally and Dly are capable of binding to Hh and facilitating the movement of the Hh protein. In the absence of one of them, another member is probably sufficient to facilitate Hh movement (Han, 2004).

dally-dly double mutant clones have relatively weaker defects in Hh signalling in the wing disc than those of the ttv and sfl mutants. One possible explanation is the perdurance of Dally and Dly proteins. Alternatively, two other HSPGs, Dsyndecan and Trol, may also participate in Hh signalling in the absence of Dally and Dly in the wing disc. These issues remain to be examined using both dsyndecan and trol null mutants (Han, 2004).

Do HSPGs act as co-receptors in Hh signal transduction? Hh is a heparin-binding protein and is likely to interact with HSPGs through their HS GAG chains. In support of this, Dly was shown to colocalize with Hh punctate particles. It is conceivable that Dally and Dly could either transfer Hh to its receptor Ptc or form a Hh-Dally/Dly-Ptc ternary complex in which Dally and Dly may function to facilitate Hh-Ptc interaction or stabilize a Hh-Ptc complex. In this regard, Dally and Dly may function both in transporting Hh protein and acting as co-receptors in Hh signalling. Consistent with this view, a recent report using RNAi in tissue culture based assays identified Dly as a new component of the Hh pathway (Lum, 2003). It was shown that Dly plays a cell-autonomous role upstream or at the level of Ptc in activating the expression of Hh responsive-reporter, suggesting a role of Dly in the delivery of Hh to Ptc (Han, 2004).

It is important to note that some of results obtained from tissue culture based assays (Lum, 2003) are not consistent with in vivo results reported in this study as well as previous studies on Ttv. Cl-8 cells were originally derived from the wing disc. However, it was found that removal of dly activity alone has no detectable effect on Hh signalling in the wing disc. This apparent discrepancy may due to several factors: (1) Hh-N, instead of Hh-Np was used as a source for Hh in their work; (2) Cl-8 cell may have altered the proteoglycan expression pattern, which can be significantly different from Hh-responding wing cells in which Dally expression is upregulated by Hh signalling; (3) it is possible that Dly may have a higher capacity than Dally to bind Hh, as in the case for Wg. In this regard, removal of Dly will probably lead to more profound effects than removal of other HSPGs on binding of Hh-N to the cell surface, perhaps in the delivery of Hh-N to Ptc (Han, 2004).

Within sfl, or ttv or dally-dly mutant clones, the posterior-most cells adjacent to wild-type cells are still capable of transducing Hh signalling. It is most likely that Hh proteins bound by Dally and Dly in wild-type cells can directly interact with Ptc located on the cell surface of the adjacent mutant cells to transduce its signalling. In support of this view, a Hh-CD2 membrane fusion protein has the ability to activate Hh signalling in its adjacent cells. Furthermore, studies on Dispatched (Disp), a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells, have shown that the first row of anterior cells adjacent to posterior Hh-producing cells have significant Hh signalling activity in disp mutant wing discx, in which Hh is retained on the cell surface of Hh producing cells. Interestingly, Hh punctate particles were observed in the posterior-most HSPG mutant cells adjacent to wild-type cells. These Hh punctate particles are most likely intracellular Hh proteins internalized through Ptc mediated endocytosis process. In this regard, HSPGs may not be required for Ptc-mediated Hh internalization (Han, 2004).

Recent biochemical studies from vertebrate cells have shown that Shh-Np is secreted from cells and can be readily detected in conditioned culture medium. It was also shown that overexpression of Disp can increase the yield of Hh protein in the culture medium. These experiments suggest that Hh can be directly secreted from Hh expressing cells. Can secreted Hh proteins freely diffuse to Hh receiving cells through extracellular spaces? To address this issue, detailed analyses for Hh signalling have been carried out in the complete absence of HS GAG using sfl and ttv or absence of glypicans using dally-dly. A narrow strip (one cell diameter in width) of sulfateless (sfl) or ttv, or dally-dly mutant cells prevents the transpassing of the Hh signal. Hh staining disappears in sfl mutant clones, except at a residual level in the posterior-most row of cells. Based on these observations, a model is favored in which Hh movement is regulated by a cell-to-cell mechanism rather than by free diffusion (Han, 2004).

The results of this study further suggest that Hh movement is independent of dynamin-mediated endocytosis, which has been shown to be involved in the transportation of morphogen molecules such as Dpp and Wg. A blockage of dynamin function does not eliminate Hh movement and its subsequent signalling; instead, it leads to a striking reduction of punctate particles of Hh staining and an accumulation of cell-surface Hh protein. Expanded Ptc expression domain is observed when dynamin-mediated endocytosis is blocked. These new findings provide compelling evidence that dynamin-mediated endocytosis is not required for Hh movement and its subsequent signalling, but is involved in Ptc-mediated internalization of the Hh protein (Han, 2004).

Several mechanisms have been proposed to explain morphogen transport across a field of cells. These mechanisms include (1) free diffusion, (2) active transport by planar transcytosis, (3) cytonemes, (4) argosomes. The results of this study suggest that Hh moves through a cell-to-cell mechanism rather than free diffusion. Furthermore, dynamin-mediated endocytosis is unlikely to be involved in Hh movement. On the basis of these findings, the following model is proposed by which the HSPGs Dally and Dly may regulate the cell-to-cell movement of the Hh protein across a field of cells. In this model, Hh is released by Disp from its producing cells and is immediately captured by the GAG chains of glypicans on the cell surface. The differential concentration of Hh proteins on the surface of producing cells and receiving cells drives the unidirectional displacement of Hh from one GAG chain to another towards more distant receiving cells. Within the same cell, the transport of Hh may be facilitated by the lateral movement of glypicans on the cell membrane. On the receiving cells, glypicans may present Hh to Ptc, which then mediates the internalization of Hh. Glypican mutant cells can not relay Hh proteins further because they lack HS GAG on the surface. However, they are able to respond to the Hh signal because Ptc may contact the Hh on the membrane of the adjacent wild-type cells. Further studies are needed to determine whether other mechanism(s) including cytonemes and argosomes are also involved in Hh movement (Han, 2004).

Glypicans shunt the Wingless signal between local signalling and further transport

The two glypicans Dally and Dally-like have been implicated in modulating the activity of Wingless, a member of the Wnt family of secreted glycoprotein. So far, the lack of null mutants has prevented a rigorous assessment of their roles. A small deletion was created in the two loci. Analysis of single and double mutant embryos suggests that both glypicans participate in normal Wingless function, although embryos lacking maternal and zygotic activity of both genes are still capable of transducing the signal from overexpressed Wingless. Genetic analysis of dally-like in wing imaginal discs leads to a model whereby, at the surface of any given cell of the epithelium, Dally-like captures Wingless but instead of presenting it to signalling receptors expressed in this cell, it passes it on to neighbouring cells, either for paracrine signalling or for further transport. In the absence of dally-like, short-range signalling is increased at the expense of long-range signalling (reported by the expression of the target gene distalless) while the reverse is caused by Dally-like overexpression. Thus, Dally-like acts as a gatekeeper, ensuring the sharing of Wingless among cells along the dorsoventral axis. This analysis suggests that the other glypican, Dally, could act as a classical co-receptor (Franch-Marro, 2005).

The fact that mutations in dally and dlp cause different phenotypes suggests that, although they both underpin Wingless function, these two glypicans could perform distinct activities. It is likely that both Dally and Dlp are able to capture Wingless at the surface of imaginal disc cells. From the point of view of a given cell in vivo, Wingless captured by Dally would be mostly destined for 'internal consumption', while Dlp-bound Wingless would be for export only. Subsequent long-range transport would occur by hopping from Dlp on one cell to Dlp on the next. Both glypicans would contribute to increasing the concentration of Wingless at the cell surface (Dally in cis and Dlp in trans). It is suggested that in the embryo too, Dlp and Dally help in the presentation and reception of Wingless, respectively. However, in this system, little Wingless transport takes place, maybe because release of Wingless from Dlp is not allowed. It is interesting that, in embryos, dlp is highly expressed in cells that secrete Wingless. Therefore, the role of Dlp would mainly be to ensure that plenty of Wingless is retained at the surface of Wingless-expressing cells thus allowing sustained short-range signalling. In both the embryonic and disc systems, the genetic redundancy between dally and dlp could be viewed as follows: reduction of capturing activity in dally mutants would be compensated by the 'presentation activity' of Dlp and vice versa. Further cell biological work will be needed to fully explore the specific activities of Dally and Dlp and also to discover how Wingless is transferred from one cell to another during transport, perhaps with the help of enzymes such as Notum/Wingful (Franch-Marro, 2005).

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date revised: 25 October 2007

 

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