The Interactive Fly
Genes involved in tissue and organ development
The adult eye consists of an array of approximately 800 hexagonal ommatidia, or facets. There is also an array of sensory hairs projecting from the surface.
Each ommatidium contains eight photoreceptor cells, each associated with a rhabomere. A rhabomere is a rod-like element containing photoreceptor elements (see The Drosophila Adult Ommatidium: Illustration and explanation with Quicktime animation). Photoreceptor cells one through six of each ommatidium are placed radially around cells 7 and 8, forming an irregular trapezoid. Rhadomeres of cells seven and eight occupy a central position, cell seven above cell eight. Each ommatidium is surrounded by two primary pigment cells and these are surrounded by six secondary pigment cells, shared by adjacent ommatidia. Thus each ommatidium contains a total of 22 cells, making the total number of cells in each eye over 16,000 (Ready, 1976). From each mature cluster a bundle of eight axons runs posteriorly into a pre-optic stalk. For information on inductive relationships between the projecting axons and the innervated medula of the brain optic lobe see the brain site.
In the differentiation of the eye, R8 is the first cell population to have its fate determined. Subsequently and successively comes the differentiation of three cell pairs: R2/5, R3/4 and R1/6. Following this is the differentiation of R7 and the surrounding cone cells.
The Drosophila compound eye is specified by the concerted action of seven nuclear factors: Twin of eyeless (Toy), Eyeless (Ey), Eyes absent (Eya), Sine oculis (So), Dachshund (Dac), Eye gone (Eyg), and Optix (Opt). These factors have been called 'master control' proteins because loss-of-function mutants lack eyes and ectopic expression can direct ectopic eye development. However, inactivation of these genes does not cause the presumptive eye to change identity. Surprisingly, several of these eye specification genes are not coexpressed in the same embryonic cells -- or even in the presumptive eye. Surprisingly, the EGF Receptor and Notch signaling pathways have homeotic functions that are genetically upstream of the eye specification genes; specification occurs much later than previously thought -- not during embryonic development but in the second larval stage (Kumar, 2001).
The Egfr and Notch pathways function in the specification or determination of the eye. An ey-GAL4 driver was used to express target proteins; this element drives expression first in the eye and antenna anlagen in the embryo (by stage 11) and then in regions ahead of the furrow in just the eye imaginal disc. Egfr function was removed in this domain by expressing a dominant negative form of the receptor. Both the eye and antenna were deleted from eclosed adults indicating that both structures require Egfr signaling for their specification, determination, or survival. Under these conditions, the larval discs do not form, making analysis of later developmental phenotypes impossible. The same phenotype was obtained with dominant negative Ras indicating that this activity is Ras dependent. Wild-type and activated forms of several components of the Ras pathway were expressed in the eye anlagen using the same driver and it was found that hyperactivation of many elements leads to the homeotic transformation of the eye into a morphologically complete antenna. Homeotic transformation of the eye to antenna can also be induced by the Egfr ligand Spitz but not by two other known activators. The membrane bound version of Spitz does not induce homeotic transformations, suggesting a requirement for paracrine signaling. Wild-type and constitutively active forms of the Egfr and two other Drosophila RTKs (Breathless [Btl] and Heartless [Htl]) were expressed but only Egfr is able to induce the transformation. Expression of the constitutively active version of Egfr gives a significantly stronger phenotype than the wild-type version of Egfr, suggesting that the level of Egfr signaling is important for maintaining the balance between eye and antennal identities. The downstream elements of the pathway that can induce this transformation include Ras, Raf, and PntP1, while neither MEK, MAPK, nor PntP2 induced this effect in this assay. Aop, Tramtrack (Ttk), and BarH1/H2, each of which mediates negative feedback inhibition of Egfr signaling, delete the eye. The failure of Mek, Mapk, and PntP2 to induce this transformation reflects the existence of actual branch points in the pathway. However, it is also possible that the quantitative levels of expression of these three elements are not limiting for this signal at this time and place; indeed, their phosphorylation states may be more relevant (Kumar, 2001).
Notch and Egfr have been shown to often antagonize each other during cell fate decisions in the fly eye. Notch function was removed with a dominant negative form and results similar to the effects of Egfr signal hyperactivation were obtained. Consistent with this, when an activated form of Notch was expressed, the size of the eye was reduced and there were severe dysmorphies. Expression of dominant negative transgenes of the ligands Delta (Dl) or Serrate (Ser) also results in the eye to antenna transformation. Elevated expression levels of both Su(H) and many of the proteins of the E(spl) complex (m4, m7, m8, m8DN, malpha, mß, mgamma, and mdelta) were also expressed but no effect on either eye or antenna disc development was observed. However, homeotic eye to antenna transformations occurred when Mastermind (Mam) was expressed using a dominant negative construct. Mam is a member of the neurogenic gene group that encodes a nuclear protein of unknown function. These results suggest a Su(H) and E(spl)C independent pathway for eye and antenna disc development that involves Mam (Kumar, 2001).
The Wg and Hh pathways have been shown to regulate the size and shape of the eye. Overexpression of a transgene containing the full-length wild-type Cubitus interruptus (Ci) protein, a downstream component of Hh signaling, can mediate eye to antenna homeotic transformations. It is uncertain if these data show a positive or negative Hedgehog signal; Ci may be cleaved into the repressor form, thereby mimicking the effects on eye development seen in loss of Hedgehog signaling. Overexpression of Wg results in the near complete deletion of the eye. Downstream of Wg lie two transcription factors: Sloppy Paired 1 and 2 (Slp1, Slp2). The effect of Wg on eye specification appears to be mediated through Slp2 but not Slp1. Taken together, these observations suggest that along with Egfr and Notch, both Wg and Hh signaling function during eye and antenna disc specification (Kumar, 2001).
Do Egfr and Notch Act upstream of the eye specification genes? A molecular epistasy study was undertaken, examining the expression of some of the eye and antennal specification genes in the transforming conditions during the third larval stage (before cell types differentiate). In eye specification gene mutants (such as ey), ommatidial development is blocked, but the eye disc remains in a reduced form. Conditions that produce eye to antenna transformations, whether through hyperactivation of Egfr or downregulation of Notch signaling, show a complete replacement of the eye disc with an antenna disc. Distal-less (Dll) and Spalt-Major are normally expressed within subdomains of the antenna disc and are required for antenna development. Dll and SalM are expressed in the correct locations in the transformed antenna disc suggesting that both endogenous and transformed antenna are also both morphologically and molecularly equivalent (Kumar, 2001).
The transcription of five of the seven known eye specification genes (toy, ey, eya, so, and eyg) was examined. In transforming conditions, transcription levels of all five of the seven genes are below the levels of detection. This is consistent with both Egfr and Notch signaling acting genetically upstream to both the eye and antennal specification genes. The downregulation of ey suggests that the ey-GAL4 driver may also be downregulated via an autoregulatory mechanism. That the transformation occurs despite this may reflect a phenocritical period for the eye-antenna transformation; once the transformation has occurred the system is refractory to the loss of Egfr signaling (Kumar, 2001).
When and where are the eye and antenna specified? The seven known eye specification genes are thought to act in a genetic and biochemical complex; by pairwise tests, their products have been shown to either directly regulate each other's transcription or to interact at the protein level, or both. From the few published reports of the early expression patterns of eye specification genes and from fate mapping experiments, it has been suggested that eye versus antennal fate specification occurs during the latter stages of embryogenesis. These concepts lead to a straightforward hypothesis: at some point in the developing embryo, the seven eye specification genes' products are coexpressed in the presumptive eye and act to specify its fate. A similar event (with the action of different genes) also specifies the antenna (Kumar, 2001).
If this hypothesis is true, then three predictions should hold: (1) At some time during embryonic development, there should be two domains of expression of the eye specification genes that correspond to the future eyes, and anterior to these, should be two domains of antenna specification gene expression marking and acting to direct antenna fate. These gene products should be specific to the future structures they mark, and should not be found elsewhere. (2) The eye specification genes should be coexpressed in the same cells. This is known to be true of toy, ey, and eyg. (3) The phenocritical period for the eye to antenna transforming function of Egfr and Notch pathway signaling should be coincident with, or earlier than, the time at which the eye and antenna specification genes are first specifically coexpressed. All three of these predictions were tested (Kumar, 2001).
To test the first prediction above, embryos were collected (at 1 hr intervals from 1 to 16 hr after egg deposition, AED) and analyzed for expression of the canonical eye specification gene ey (Pax6) and the antenna specification protein Dll. Dll is first detected at 7 hr in the leg imaginal disc primordia and in several segments in the embryonic head. ey transcription in the eye imaginal disc is first detectable at 11 hr while Dll is seen in an adjacent region as well as other sites. In latter stages of embryogenesis, the eye imaginal disc invaginates and assumes a more dorsal-medial position within the embryonic head, just above the developing embryonic brain. Regions of ey expression are observed that correspond to this. Furthermore, this ey expression corresponds to domains of Escargot expression (Esg), a general imaginal disc marker. However, Dll expression is more anterior and it is not clear if these sites correspond to the presumptive antennae. It thus appears that ey is expressed in both the presumptive eye and antenna by 13 hr and remains there through the last embryonic time point observed, and that Dll is not expressed in the future antenna at any embryonic time. It is also quite clear that ey is expressed in many sites in the embryo that will never form eye (such as the segmental grooves). In short, the position of the presumptive eye or antenna during embryonic development cannot be distinguish based on the specific expression of their respective 'master control' genes—neither ey nor Dll expression are sufficient to specify the eye or the antenna; therefore, prediction 1 (above) does not hold true (Kumar, 2001).
Are the eye specification genes coexpressed during embryonic development? The expression pattern of Eya and Dac proteins and so transcription were examined at 1 hr time points (from 1 to 16 hr AED) and it was found that none of these three eye specification genes is coexpressed with ey within the presumptive eye. The fact that these genes are not expressed within the same cells during embryonic development precludes any possibility that their products act in a multiprotein complex critical for eye specification in the embryo and, thus, prediction 2 (above) does not hold true either. However, eye specification might occur later in development (Kumar, 2001).
The eye specification genes are first coexpressed in the second larval stage. In second stage larva, the eye specification gene products are completely segregated into the eye portion of eye-antennal disc, but the antennal marker Dll is evenly expressed in both the eye and antennal segments. Interestingly, the expression patterns of the eye specification genes are still not completely overlapping. For instance, toy appears to be expressed throughout the entire eye field while both eya and dac are expressed just in the posterior portions of the eye disc. In the third larval stage, the eye specification genes remain within the eye portion and Dll is now segregated to just the antennal segments (Kumar, 2001).
The phenocritical period for eye to antenna transformation is in the second larval stage. Use was made of the cold sensitivity of the GAL4 protein to determine the phenocritical period. GAL4 is a yeast protein and is fully functional at 25°C but is less active at 18°C. Flies of the ey-GAL4/UAS-SerDN genotype were raised at 18°C, shifted to 25°C for a consecutive series of 24 hr periods, and then returned to 18°C until late third instar imaginal discs could be examined. The use of the dominant negative Ser construct in this experiment effectively eliminates Notch pathway function in the developing eye. Indistinguishable transformations were observed in other experiments with constructs that either hyperactivate Egfr pathway signaling or inactivate Notch. The developing eye-antennal complex is completely normal if kept continuously at 18°C (negative control) while constant exposure to 25°C temperatures resulted in the eye to antenna transformation (positive control). These controls confirm that the cold sensitivity of GAL4 protein activity is sufficient to control the transformation (Kumar, 2001).
Temperature shifts during the embryonic and first larval stages failed to induce any effects. The eye-antennal discs are completely normal. This is consistent with the expression data suggesting that the eye is not specified during embryogenesis. A temperature shift during the first half of the second larval stage results in a reduced eye field, but no transformation to antenna. Interestingly, this phenotype is similar to that seen in ey mutant homozygotes. The eye to antenna transformation is fully induced in all cases when the temperature shift occurs during the latter half of the second larval stage. The transformed antenna expresses Dll in an identical pattern as seen in the endogenous antenna. Subsequent temperature shifts during the earliest phase of the third larval stage do not result in a complete transformation. Interestingly, loss of Notch during the next day of the third larval stage results in defects in the regulation of the morphogenetic furrow. These results clearly indicate that the phenocritical period is chiefly within the latter half of the second larval stage. This phenocritical period does not predate the expression of any of the eye specification genes, but it is coincident with their first coexpression and, thus, prediction 3 (above) holds true (Kumar, 2001).
Is the presumptive eye actually transformed into a second antenna under these conditions, or does the eye degenerate and get replaced by regrowth from elsewhere? The former interpretation (transformation) is favored because in hundreds of transformed L2 disc complexes dissected, a degenerating eye disc, or a small (presumably regrowing) antennal disc was never observed. In all cases, the transformed antenna is equal in size to the normal one. Indeed, both are somewhat larger than normal. This might suggest that a fixed number of cells that normally distribute preferentially to the eye are now equally allocated to both antennae (Kumar, 2001).
The notch pathway signals differentially in the eye and antenna primordia in the second larval stage. Loss of Notch activity during the second larval stage results in the transformation of the eye into an antenna. Thus, it is predicted that Notch signaling should be elevated in the presumptive eye versus the antenna at the critical time. Cells that are actively receiving a Notch signal upregulate Notch protein expression. Thus elevated Notch antigen expression can be used as a reporter of elevated Notch signaling. Notch and ey expression were examined in imaginal discs from first, second, and third stage larvae. Both Notch and ey are expressed throughout the entire eye-antennal disc anlagen during the first larval stage. By the second larval stage, Notch is differentially upregulated within the presumptive eye. Interestingly, Notch appears especially active along the eye margins and midline, where it is thought to regulate retinal polarity. In contrast, ey appears to be exclusively within the eye field. In the third larval stage, Notch expression is upregulated in the morphogenetic furrow, where it acts to control ommatidial spacing while ey remains upregulated ahead of the furrow (Kumar, 2001).
Therefore, Egfr signaling promotes an antennal fate while Notch signaling promotes an eye fate. This role for Notch is consistent with the observation that removal of Notch signaling can partially inhibit compound eye development. Furthermore, several of the eye and antennal specification genes (ey, toy, eya, so, eyg, salM, and Dll) are downstream of the Egfr and Notch inputs. Wg and Hh pathway signaling affect this specification. The eye specification genes form a regulatory network and the direct control of any one of these genes may affect the others. Thus, which (if any) of the known eye specification genes is a direct target of Notch or Egfr signals may require direct biochemical assays (Kumar, 2001).
While activating Egfr or blocking Notch signals transforms the eye cleanly into an antenna, the reciprocal transformation is not complete, suggesting that there may be additional positive regulators of eye fate. The reciprocal transformation experiment could not be conducted (i.e., antenna to eye switch via hyperactivation of Notch or downregulation of Egfr signaling solely within the antennal anlagen). Unlike the ey-GAL4 driver, there is not an equivalent known driver that is expressed solely with the antennal anlagen. All known antennal-determining genes are also expressed in other imaginal discs. For instance, the Dll-GAL4 driver is expressed in several places within the embryonic head and leg imaginal disc. Expression of Egfr or Notch constructs with this driver results only in embryonic lethality. It may be that the antenna can be changed to an eye via alterations of Egfr or Notch signaling provided that the appropriate tools for their missexpression are available (Kumar, 2001).
Why do homozygous mutants for eye specification genes not transform the eye into an antenna? While it may be that some alleles are not nulls (e.g., ey1), a more interesting possibility is that there may be functional redundancy in some cases -- particularly that of ey and toy. Thus, only when both genetic functions are eliminated will a true null condition exist. Just such a situation confused the phenotypic analysis of two other twin homeodomain proteins, engrailed and invected. Unfortunately, mutations of the toy gene do not yet exist (Kumar, 2001).
How do the eye specification genes function? Published genetic epistasy and biochemical interaction data suggest that the seven known eye specification genes' products interact at the transcriptional and protein levels to direct cells toward eye fate. This requires that they are expressed in the same cells. Furthermore, it has been suggested that many, if not all, of these genes are 'master regulators' of eye fate -- that is, they are both necessary and sufficient for eye specification. Many very compelling experiments have been described showing the induction of ectopic eyes through the ectopic expression of these genes alone or in synergistic combinations. It is suggested that these genes come under separate regulation by different patterning signals in early development and that there are overlapping domains. Only when all of the domains coincide (during the second larval stage) do eye specification genes specify the eye. This seems to be the simplest explanation since the eye specification genes form a very tight genetic, biochemical, and transcriptional regulatory network suggesting that they are together required for eye specification. It may be that the final coexpression of the eye specification genes' products (and the exclusion of the antennal specification factors) is the last step required to allow the morphogenetic furrow to initiate in response to the next local expression of hh and for the final specification of retinal cell types and pattern (Kumar, 2001).
In Drosophila, the eye and antenna originate from a single epithelium termed the eye-antennal imaginal disc. Illumination of the mechanisms that subdivide this epithelium into eye and antenna would enhance understanding of the mechanisms that restrict stem cell fate. This study shows that Dorsal interacting protein 3 (Dip3), a transcription factor required for eye development, alters fate determination when misexpressed in the early eye-antennal disc, and this observation has been taken advantage of to gain new insight into the mechanisms controlling the eye-antennal switch. Dip3 misexpression yields extra antennae by two distinct mechanisms: the splitting of the antennal field into multiple antennal domains (antennal duplication), and the transformation of the eye disc to an antennal fate. Antennal duplication requires Dip3-induced under proliferation of the eye disc and concurrent over proliferation of the antennal disc. While previous studies have shown that overgrowth of the antennal disc can lead to antennal duplication, these results show that overgrowth is not sufficient for antennal duplication, which may require additional signals perhaps from the eye disc. Eye-to-antennal transformation appears to result from the combination of antennal selector gene activation, eye determination gene repression, and cell cycle perturbation in the eye disc. Both antennal duplication and eye-to-antennal transformation are suppressed by the expression of genes that drive the cell cycle providing support for tight coupling of cell fate determination and cell cycle control. The finding that this transformation occurs only in the eye disc, and not in other imaginal discs, suggests a close developmental and therefore evolutionary relationship between eyes and antennae (Duong, 2008).
Dip3 is able to bind DNA in a sequence specific manner and activate transcription directly. Dip3 possesses an N-terminal MADF domain and a C-terminal BESS domain, an architecture that is conserved in at least 14 Drosophila proteins, including Adf-1 and Stonewall. The MADF domain directs sequence specific DNA binding to a site consisting of multiple trinucleotide repeats, while the BESS domain directs a variety of protein-protein interactions, including interactions with itself, with Dorsal, and with a TBP-associated factor (Bhaskar, 2002).
Antagonism between the N and EGFR signaling pathways influences developmental fate in the eye-antennal disc leading to a loss of eye tissue and the appearance of extra antennae. Although this phenotype was originally suspected to represent eye-to-antennal transformation, subsequent analysis suggests that it most likely represents antennal duplication. Specifically, the absence of the N signal leads to a failure in eye disc proliferation resulting in compensatory over-proliferation of the antennal disc and its subdivision into multiple antennae. Consistent with the idea that the extra antennae result from under-proliferation of the eye field, it was found that the phenotype was largely suppressed by over-expression of CycE to drive the cell cycle (Duong, 2008).
In this study, it was found that inhibition of eye disc growth leads to antennal duplication. But in addition, it was shown that the same treatment that leads to antennal duplication can also direct the transformation of eyes to antennae. These two phenotypes are anatomically distinct. This anatomical distinction is evident in adults: antennae resulting from antennal duplication are found anterior to the antennal foramen, while the antennae resulting from eye-to-antenna transformation are found posterior to the antennal foramen. It is also apparent in larval eye-antennal imaginal discs: antennal duplication discs exhibit multiple circular dac expression domains within a single sac of epithelium (the antennal disc), while eye-to-antennal transformation discs exhibit two or more circular dac expression domains spread over both the eye and antennal discs. The two types of discs display distinct molecular signatures as well: the antennal duplication discs exhibit duplicated Dll expression domains, while the eye discs undergoing transformation to antennae lack Dll expression (Duong, 2008).
Perhaps the most persuasive evidence that Dip3 can direct eye-to-antennal transformation is provided by the observation of eyes that are only partially transformed to antennae since is very difficult to reconcile these partial transformations with the idea of antennal duplication. In some cases, proximal antennal segments tipped with eye tissue are observed. In accord with this phenotype, some third instar larval eye discs display a central domain of Elav-positive differentiating photoreceptors surrounded by a circular dac domain (Duong, 2008).
These arguments support the idea that antennal duplication and eye-to-antennal transformation are mechanistically distinct phenomena, and the remainder of the discussion assumes this to be the case. However, the possibility that these two phenotypes are two manifestations of a single mechanism cannot be excluded. For example, the discs exhibiting duplicated Dll domains may represent complete transformations, while the discs lacking duplicated Dll domains, but containing Elav may represent partial transformations (Duong, 2008).
The data show that discs undergoing antennal duplication as a result of Dip3 expression are comprised of a severely diminished eye region and an enlarged antennal region. As shown by BrdU labeling experiments, these antennal duplication discs most likely result from suppression by Dip3 of cell proliferation in the eye field leading to overproliferation of the antennal disc. This conclusion is supported by the ability of factors that drive cell proliferation (e.g., Cyclin E) to alleviate the Dip3 misexpression defect (Duong, 2008).
Many experimental manipulations that reduce the size of the eye disc (e.g., surgical excision, induction of cell death, or suppression of cell proliferation) lead to enlargement and duplication of the antennal primordium. How might reduction of the eye field lead to antennal field over-growth? One possibility is that the eye field produces a growth inhibitory signal. Alternatively, the eye field and the antennal field may compete with each other for limited nutrients or growth factors. In support of this latter possibility, recent studies of the role of dMyc in wing development have demonstrated growth competition between groups of imaginal disc cells (Duong, 2008).
While the results imply that antennal disc overgrowth is required for antennal duplication, overgrowth is thought not to be sufficient for duplication. This conclusion derives from experiments in which an antennal disc specific driver is used to direct over-expression of CycE or Nact. This resulted in antennal overgrowth without concurrent reduction in the eye disc. In this case, antennal duplication was not observed. Thus, in addition to antennal overgrowth, antennal duplication also appears to require reduction or elimination of the eye disc. Regulatory signals from the eye disc may act to prevent antennal duplication (Duong, 2008).
The eye and antenna discs differ in several respects: (1) Specific expression of the organ-specification genes. The eye disc expresses the retinal determination gene network (RDGN) genes, including eyeless (ey), twin of eyeless (toy), eyes absent (eya), sine oculis (so), and dachshund (dac), while the antennal disc expresses Dll and hth. hth is also expressed in the eye disc but in a distinct pattern from that seen in the antennal disc. In the second instar eye disc, hth is expressed throughout the eye disc, and collaborates with ey and teashirt (tsh) to promote cell proliferation. The hth expression domain later retracts to only the anterior-most region of the eye disc. This pattern is different from the circular expression pattern observed in the antennal disc. (2) In the antennal disc, dpp is expressed in a dorsal anterior wedge and wg is expressed in a ventral anterior wedge. The intersection of Dpp and Wg signaling is required to specify the proximodistal axis in the leg and antenna. In the early eye disc, Wg and Dpp signaling may overlap. But as the disc grows in size, the wg and dpp expression domain are separated, so that there is probably no intersection between high levels of Wg and Dpp signaling. (3) Whereas the partial overlap of Dll and hth expression domains in the antennal disc is important for proximodistal axis specification, there is no Dll expression in the eye disc. Dll expression in the center of the antennal and leg discs is induced by the combination of high levels of Dpp and Wg signaling. Because there is no overlap of Dpp and Wg signaling in the eye disc, Dll is not induced (Duong, 2008).
Therefore, efficient transformation of the eye disc into an antennal disc requires at least three things: (1) repression of the eye fate pathway; (2) activation the antennal fate pathway; and (3) the intersection of Dpp and Wg signaling, mimicking the situation in the antenna and leg disc that induces proximodistal axis formation. Any one of these three conditions by itself is not sufficient. (1) Loss of the RDGN genes leads only to the loss of the eye. However, if apoptosis is blocked, or cell proliferation is induced, in the ey2 mutant (ey>p35 or ey>Nact in ey2), then Dll can be induced in the eye disc and extra antenna are formed. The induction of Dll is not ubiquitous in the eye disc, suggesting that the loss of ey does not autonomously lead to the expression of Dll and the transformation to the antennal fate. (2) Simply expressing the antennal determining genes Dll or hth in the eye disc does not change the eye fate into antennal fate. It was found that uniform expression of Dll in the eye disc (ey>Dll) resulted in mild eye reduction, whereas ey>hth completely abolished eye development. E132>Dll caused the formation of small antenna in the eye in about 46% of flies, whereas ptc>Dll and C68a>Dll induced extra antenna but not within the eye field. Therefore, although Dll and hth are important determinants for antennal identity, it is their specific spatial expression patterns that determine antennal development. (3) Creating the intersection of Wg and Dpp signaling does not change the eye into antenna. Such manipulation in the leg disc turned on vg and transdetermined the leg disc into wing disc. Therefore, the specific genes induced by Dpp and Wg signaling may depend on disc-specific factors. In the eye disc, turning on Wg signaling in the dpp expressing morphogenetic furrow only blocked furrow progression (Duong, 2008).
In this study, it was found that the ectopic expression of a single gene, Dip3, can cause eye-to-antenna transformation. Dip3 apparently satisfied all three requirements. (1) Overexpression of Dip3 repressed (non-cell-autonomously) ey and dac. The repression of ey may be due to the induction of ct. The ability of Dip3 to simultaneously repress multiple retinal determination genes is completely consistent with the many known cross-regulatory interactions between these genes. (2) ey>Dip3 turned on ct and hth. (3) By blocking cell proliferation, ey>dip3 reduced the eye field size and allowed the intersection of Dpp and Wg signaling. Furthermore, ey>Dip3 induced en, which probably created an ectopic A/P border and induced ectopic dpp/wg expression (Duong, 2008).
Interference with cell cycle progression appears to be a common link between the two phenotypes described in this study. In the case of antennal duplication, interference with eye disc growth leads to antennal disc overgrowth, which is a prerequisite for duplication. In the case of eye-to-antenna transformation, eye disc undergrowth allows the required intersection between Dpp and Wg signaling (Duong, 2008).
The observation that Dip3 misexpression can transform the eye field, but not other tissues, to an antennal fate suggests a close evolutionary relationship between the eye and the antenna. Previous studies have emphasized the homology between antennae and legs. The findings presented here that misexpression of a single transcription factor, namely Dip3, can transform eyes to antennae provides support for the notion that the eye and antenna may also, in some sense, be homologous to one another. Previous evidence in support of this idea comes from the observation that similar spatial arrangements of Wg and Dpp signaling along with a temporal cue provided by the ecdysone signal are required for the formation of the eye and the mechanosensory auditory organ. Small mechanosensory sensilla, such as Johnston's organ and the chordotonal organs (stretch receptors) are thought to represent the earliest evolving sense organs. Perhaps the eye resulted from a duplication and specialization of such a sensillum (Duong, 2008).
The eye is derived from the eye-antennal disc. The disc itself arises from approximately 20 cells of the optic primordium in the embryonic blastoderm. The disc is formed by invagination at stage 12, to produce a flattened sac of epithelium. By the third instar larva the disc contains about 2000 cells. During the middle of the third instar phase, a dorsal ventral furrow forms, advancing from posterior to anterior. The furrow, whose progression requires hedgehog function, is the site where commitment to photoreceptor fate is initiated. hh activates the expression of decapentaplegic and the proneural gene atonal in the furrow. ato expression is refined to the future R8 photoreceptor and is required for the development of this cell. The area anterior to the furrow is rich in synchronously dividing cells, but lacks a pattern. The furrow itself is caused by a shortening of cells at its center. The area posterior to the furrow shows preclusters of cells, each with a recognizable core of five cells, corresponding to cells 2, 3, 4, 5 and 8 of the photoreceptor. These photoreceptors and accessory cells are recruited to each ommatidial cluster (composed of developing photoreceptors) by waves of expression of the ligand spitz (spi) for the EGF receptor. A diagram is presented describing the progression of the morphogenetic furrow across the eye disc.
In addition to the dorso-ventral furrow described above, there is an axis of dorso-ventral symmetry. Associated with the advancing boundary of cluster formation, the area ahead of this boundary is indented along the anterior posterior axis. This groove can be seen in the early third instar disc. It is this anterior-posterior groove that structures the DV symmetry of the eye (Ready, 1976). A diagram is presented describing the specification of the eye disc primordium and the establishment of dorsal/ventral asymmetry.
Cells in the Drosophila eye are determined by inductive signalling. A model of eye development has been built that explains how simple intercellular signals could specify the diverse cell types that constitute the ommatidium. This model arises from the observation that the Drosophila homologue of the EGF receptor (EGF-R) is used reiteratively to trigger the differentiation of each of the cell types -- successive rounds of EGF-R activation recruit first the photoreceptors, then cone and finally pigment cells. It seems that a cell's identity is not determined by the specific signal that induces it, but is instead a function of the state of the cell that receives the signal. EGF-R signalling is activated by the ligand, Spitz, and inhibited by the secreted protein, Argos. Spitz is initially produced by the central cells in the ommatidium and diffuses over a small distance. Argos has a longer range, allowing it to block more distal cells from being activated by low levels of Spitz; This interplay between a short-range activator and a long-range inhibitor is termed 'remote inhibition'. Since inductive signalling is common in many organisms and its components have been conserved, it is possible that the logic of signalling may also be conserved (Freeman, 1997).
Circadian rhythms can be entrained by light to follow the daily solar cycle. In adult flies a pair of extraretinal eyelets expressing immunoreactivity to Rhodopsin 6 each contains four photoreceptors located beneath the posterior margin of the compound eye. Their axons project to the region of the pacemaker center in the brain with a trajectory resembling that of Bolwig's organ, the visual organ of the larva. A lacZ reporter line driven by an upstream fragment of the developmental gap gene Kruppel is a specific enhancer element for Bolwig's organ. Expression of immunoreactivity to the product of lacZ in Bolwig's organ persists through pupal metamorphosis and survives in the adult eyelet. It is thus demonstrated that the adult eyelet derives from the 12 photoreceptors of Bolwig's organ, which entrain circadian rhythmicity in the larva. Double labeling with anti-pigment-dispersing hormone shows that the terminals of Bolwig's nerve differentiate during metamorphosis in close temporal and spatial relationship to the ventral lateral neurons (LNv), which are essential to express circadian rhythmicity in the adult. Bolwig's organ also expresses immunoreactivity to Rhodopsin 6, which thus continues to be expressed in the adult eyelet. Action spectra of entrainment were compared in different fly strains: in flies lacking compound eyes but retaining the adult eyelet (so1), lacking both compound eyes and the adult eyelet (so1;gl60j), and retaining the adult eyelet but lacking compound eyes as well as Cryptochrome (so1;cryb). Responses to phase shifts suggest that, in the absence of compound eyes, the eyelet together with Cryptochrome mainly mediates phase delays. Thus a functional role in circadian entrainment first found in Bolwig's organ in the larva is retained in the eyelet, the adult remnant of Bolwig's organ, even in the face of metamorphic restructuring (Helfrich-Forster, 2002).
Epidermal cells of Drosophila form a variety of polarized structures during their differentiation. These polarized structures include epidermal hairs, the shafts of sensory bristles, larval denticles and the arista laterals. The arista is the terminal segment of the antenna and consists of a central core and a series of lateral extensions. The development of the arista is a complex process that involves coordinated cell shape changes, elongation of the central core, apoptosis, nuclear migration, the formation of polyploid cells and the outgrowth of the laterals. This developmental program is highly conserved in the development of the arista in the housefly (Musca domestica). Altering arista cell number in Drosophila by stimulating or inhibiting apoptosis results in an altered number of laterals. Interestingly, the increased number of laterals that result from the inhibition of apoptosis in Drosophila results in an arista whose morphology is reminiscent of the Musca arista. Both the actin and microtubule cytoskeletons have been shown to have important functions in the cellular morphogenesis of hairs and bristles. This is also the case for the formation of the arista laterals, arguing that the actin and microtubule cytoskeletons have similar functions in the morphogenesis of all of these cell types. It is concluded that the arista laterals are a valuable complementary cell type system for studying the morphogenesis of polarized cellular extensions in Drosophila (He, 2001).
The adult Drosophila arista consists of a central core that is typically around 300 mm long and 10-20 mm in diameter. Laterals extend both anteriorly and posteriorly off of the central core. There are 3-4 long laterals on the anterior side and 5-7 on the posterior side of a typical Drosophila antenna. The length of the long laterals is approximately 140 mm. In addition to the long laterals, 5-7 smaller laterals (about 20-30 mm long) are found on the dorsal side of the central core shaft. A comparative study was carried out of arista development in the larger dipteran Musca domestica . The adult housefly arista differs in several respects from the Drosophila arista. The house fly arista (over 650 mm) is approximately twice as long and the central core is shaped somewhat differently being much wider at its proximal end (diameter about 80 mm). The Musca arista contains a larger number of both long and short laterals extending off of the central core and large laterals are absent near the distal end of the arista (He, 2001).
The arista are composed of a series of long thin laterals. This seems likely to be of functional importance. Evidence suggests that the arista functions in the detection of sound. It is thought that the arista is deflected by sound vibrations and this deflection results in the movement of the third antennal segment and this is detected by sensory neurons in the second antennal segment. There are also six neurons present in the central core and these send processes distally through the hemolymph-filled lumen of the thin central core. Based on morphology it has been suggested that three of these function as thermoreceptors. For sound and temperature sensing functions, the long thin morphology of the arista seems appropriate, although further research will be necessary to obtain rigorous evidence on this point (He, 2001 and references therein).
The dramatic morphology of the arista is likely to have put constraints on the developmental mechanisms involved in its morphogenesis. The development of the epidermal arista involves a number of different types of cellular processes. The shaping of the central core appears to take place in two steps: extension and thinning. The arista is first seen as an outpocketing of cells from the third antennal segment. This outpocketing elongates over about a 12-h period to reach its maximal length. No experimental evidence has been seen for cell division in the elongating arista suggesting that cell number is constant during this process. The elongation results in a greatly reduced number of cells along the circumference at all locations along the proximal distal axis of the central core. Hence, arista elongation appears to be an example of convergent extension. The elongation of the arista differs from the eversion of the appendages such as the leg and wing in that it happens in the pupae and not the prepupae (He, 2001).
The elongation of the arista laterals starts after the central core reaches its final length. At this time nuclei start to disappear from the distal half of the arista. From time-lapse observations it is argued that this is due in large part (or entirely) to the proximal migration of nuclei. During this time period the cells become elongated at their apical surface and the extent of nuclear movement suggests that the elongation might be more extreme basally. The migration of the nuclei allows the central core of the arista to become much thinner and it is suggested that this is important for the physiological function of the arista. Consistent with this hypothesis nuclear movement is inhibited by the injection of microtubule antagonists, which results in a thickened central core. Evidence for nuclear migration is also seen during the development of the house fly arista. Starting shortly before the beginning of lateral outgrowth, apoptosis is seen in the central core. This results in a decrease in the number of cells in the arista and the data argue that this is important for arista morphogenesis. Cell number was manipulated by genetic means and it was found that increasing cell number (by decreasing apoptosis) results in the formation of ectopic laterals, while decreasing cell number (presumably by increasing apoptosis) results in a loss of laterals. How cell number alters the cellular decision to make or not make a lateral is unclear, but it is likely that there is an intercellular signaling system that allows cells to assess arista number or whether their neighbors have made a lateral. This system appears to be one that evolution has used to select for altered arista morphology. The house fly arista central core is thicker than its Drosophila homolog and it is decorated with a large number of short laterals that are reminiscent of those seen when apoptosis is inhibited in Drosophila. It is not clear why dipterans have evolved a developmental program that produces extra arista cells that are lost due to apoptosis. One possibility is that it is a consequence of the very long and thin morphology of the arista. Perhaps the extra cells are needed to allow convergent extension to produce an arista of sufficient length. The subsequent apoptosis, nuclear migration and proximal/distal elongation of the remaining cells could all function to allow the central core to achieve its long thin shape (He, 2001).
The development of the arista and in particular the laterals involves dramatic shape changes in cells. Not surprisingly the developing laterals stain very strongly for F-actin and also contain microtubules. The actin and microtubule cytoskeletons appear to have overlapping but distinct functions in the development of the laterals. Antagonism of either cytoskeleton by injecting inhibitors into pupae results in delayed initiation of lateral outgrowth, slowed lateral elongation and split laterals. These effects are similar to the effects that the same inhibitors have on the morphogenesis of hairs and bristles, which are other examples of Drosophila epidermal cells that form highly polarized outgrowths. Some differences were seen in the effects of actin versus microtubule inhibitors. The actin cytoskeleton antagonists are more potent at delaying lateral initiation and in causing split laterals. Microtubule antagonists are relatively more potent at inhibiting lateral extension and the thinning of the central core. Several of these observations are reminiscent of observations made previously on bristles and hairs. Inhibitors of actin polymerization are potent at causing split bristles and hairs, while this is a minor effect of microtubule cytoskeleton antagonists. Similarly, actin polymerization inhibitors are potent at delaying prehair and prebristle initiation, while microtubule antagonists are not. Finally, microtubule antagonists are particularly potent at inhibiting the elongation of bristles. These similarities in the effects of cytoskeleton inhibitors suggest parallel functions for the actin and microtubule cytoskeletons in the morphogenesis of all three of these polarized cells. The strikingly similar arrangement of actin bundles and microtubules in bristles and laterals also points to equivalent functions for the cytoskeleton in the morphogenesis of these cell types. These commonalties are also supported by analogous phenotypes produced in all of these cell types by mutations in some genes (e.g. tricornered, which codes for a protein kinase) (He, 2001).
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Genes involved in organ development
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