clift/eyes absent

DEVELOPMENTAL BIOLOGY

Embryonic

clift/eyes absent is expressed within the mesoderm from 4 to 6 hours of development as the ventrally derived invaginated mesoderm spreads along the ectoderm. clift is also expressed in a segmentally repeated pattern in the ectoderm and in a complex pattern in the head. By 6.5 hours, clift expression is lost in most mesodermal cells. However, by seven hours, two distinct patterns emerge: a segmental repeat of 12 lateral cells (muscle progenitors) and an additional and separate expression in parasegments 10, 11 and 12 of three clusters (9-12 cells each) that serve as somatic gonad precursor cells (SGP). Clift positive cells develop just ventral of the visceral mesoderm underneath ectodermal cells expressing wingless. As the germ band retracts, however, clift expression restricts to the posterior SGP cells in each segment. By 11 hours, clift expression is further restricted to the posterior-most 10-12 SGP cells in the female gonad, while it is lost entirely in the male (Boyle, 1997).

Eya as a marker for somatic gonad: Sex-specific apoptosis regulates sexual dimorphism in the Drosophila embryonic gonad

Sexually dimorphic development of the gonad is essential for germ cell development and sexual reproduction. The Drosophila embryonic gonad is already sexually dimorphic at the time of initial gonad formation. Male-specific somatic gonadal precursors (msSGPs) contribute only to the testis and express a Drosophila homolog of Sox9 (Sox100B), a gene essential for testis formation in humans. The msSGPs are specified in both males and females, but are recruited into only the developing testis. In females, these cells are eliminated via programmed cell death dependent on the sex determination regulatory gene doublesex. This work furthers the hypotheses that a conserved pathway controls gonad sexual dimorphism in diverse species and that sex-specific cell recruitment and programmed cell death are common mechanisms for creating sexual dimorphism (DeFalco, 2003).

To investigate when sexual dimorphism is first manifested in the somatic gonad, expression of SGP markers were examined in embryos whose sex could be unambiguously identified, at a developmental stage (stage 15) soon after gonad coalescence has occurred. Analysis of Eya expression reveals anti-Eya immunoreactivity throughout the female somatic gonad, though Eya expression is somewhat stronger in the posterior. In males, anti-Eya immunoreactivity is also found throughout the somatic gonad. However, the expression at the posterior of the gonad is much more intense than in females, as there appears to be a cluster of Eya-expressing cells at the posterior of the male gonad that is not present in females. In blind experiments, the sex of an embryo could be accurately identified by the Eya expression pattern in the gonad. Thus, sexual dimorphism is already apparent in the somatic gonad soon after initial gonad formation. A sex-specific expression pattern is also observed with Wnt-2 at this stage. As is observed with Eya, Wnt-2 is expressed in the SGPs of the female gonad, but its expression is greatly increased at the posterior of the male gonad. The SGP marker bluetail (see Galloni, 1993) exhibits a similar sex-specific pattern as Eya; however, the SGP marker 68-77 is expressed equally in both sexes (see below). Thus, the somatic gonad is sexually dimorphic by stage 15, but only a subset of SGP markers reveals this sexual dimorphism (DeFalco, 2003).

During Drosophila embryogenesis, Sox100B is expressed in a number of cell types, including the gonad (Loh, 2000). Since Sox100B is closely related to Sox9 (an important sex determination factor in humans and mice), whether Sox100B expression is sexually dimorphic in Drosophila was tested. Interestingly, it was found that after gonad coalescence (stage 15), Sox100B expression in the gonad is male-specific. Sox100B immunoreactivity is not observed in the coalesced female gonad, whereas it is detected in a posterior cluster of SGPs in the male gonad. While this expression pattern is seen in most wild-type backgrounds (including Canton-S and faf-lacZ), in certain 'wild-type' lines, such as w¹¹¹⁸, a few Sox100B-positive cells are observed in the posterior of the coalesced female gonad (however, this is still clearly distinguishable from the number of Sox100B-positive cells in the male). Unlike Eya and Wnt-2, Sox100B is not expressed in all SGPs, since it is usually absent from female gonads and from the anterior region of the male gonad and does not colocalize with the SGP marker 68-77. Sox100B expression appears restricted to the posterior cluster of SGPs that is observed only in the male gonad. Thus, like Sox9 expression in vertebrates, Sox100B exhibits a male-specific pattern of expression in the Drosophila embryonic gonad, suggesting that it may indeed be an ortholog of Sox9 (DeFalco, 2003).

After having identified sexually dimorphic markers of the embryonic gonad, these markers were used to investigate how sexual dimorphism is established. It was asked whether proper gonad formation is necessary for the establishment of sexual dimorphism by examining Sox100B expression in fear-of-intimacy (foi) mutant embryos. In foi mutants, germ cells migrate and associate normally with the SGPs, but these two cell types fail to coalesce into a round and compact gonad. Despite the failure of gonad coalescence, a cluster of Sox100B-expressing cells was still observed at the posterior of the male gonad, while no Sox100B-expressing cells are observed in the female at this stage. Whether the presence of germ cells is necessary for the establishment of sexual dimorphism in the embryonic gonad was examined. Embryos that lack germ cells due to a hypomorphic mutation in oskar, a gene required for germ cell formation, were examined. Other aspects of embryonic development occur normally in these embryos, including the formation and coalescence of the SGPs. Agametic gonads show identical sexual dimorphism to wild-type embryos. Sox100B is coexpressed with Eya in the cluster of somatic cells in the posterior of the male gonad, but Sox100B expression is not observed in the female gonad. Thus, sexual dimorphism of the embryonic somatic gonad does not require proper gonad morphogenesis or the presence of germ cells (DeFalco, 2003).

The posterior cluster of Eya and Sox100B coexpressing cells could result from sex-specific differences in gene expression within the cells of the gonad. Alternatively, it could reflect a difference in gonad morphology, in which these cells are only present in males and not in females. To distinguish between these possibilities, the morphology of the male and female coalesced (stage 15) gonad was examined, using approaches that do not depend on cell-type-specific SGP markers. First, a CD8-GFP fusion protein was expressed broadly in the mesoderm. The fusion of the extracellular and transmembrane regions of mouse CD8 with GFP allows for visualization of cell and tissue morphology. A cluster of mesodermal cells is consistantly observed attached to the posterior of the male gonad that is not observed in the female. In blind experiments, the sex of the embryo can be predicted based on the presence of this posterior cluster of cells. Male and female gonads were also examined by transmission electron microscopy (TEM). Male and female embryos were first sorted using an X chromosome-linked GFP expression construct and then processed separately for TEM. In this analysis, a cluster of cells that is not present in the female gonad was consistently at the posterior of the male gonad. Both the size and morphology of these cells indicate that they are somatic cells rather than germ cells. Thus, the observed sexual dimorphism reflects a change in gonad morphology, not just a change in gene expression. Since the additional cells at the posterior of the male gonad express at least some markers in common with SGPs (e.g., Eya), these cells are referred to as male-specific SGPs (msSGPs) (DeFalco, 2003).

Since no sex-specific differences were observed in SGP proliferation in the gonad, it seems unlikely that the SGPs are dividing to produce the msSGPs. Therefore, Sox100B was used as a marker for the msSGPs to determine where and when these cells are first specified. At stages prior to gonad coalescence (stages 12 and 13), a cluster of Eya/Sox100B double-immunopositive cells is observed posterior and ventral to the developing clusters of SGPs, which express Eya alone. Interestingly, this cluster of Eya/Sox100B double-positive cells is initially observed in both males and females and appears identical, although Eya expression may be somewhat lower in the female cluster. During stage 13, as the SGPs and germ cells associate closely along PS 10-12, the Eya/Sox100B double-positive cells move toward the gonad in both sexes. In males, these cells join the posterior of the coalescing gonad. In contrast, these cells do not join the gonad in females, and only Eya-positive, Sox100B-negative cells are found in the coalesced gonad. It is concluded that the Eya/Sox100B double-positive cells are the msSGPs and that they form separately from the SGPs. These cells are initially specified in both males and females and move anteriorly to join the gonad in males. In females, these cells do not form part of the gonad, as judged by the above morphological analysis, and are no longer detected using available markers (DeFalco, 2003).

Since the msSGPs develop separately from the SGPs, it was of interest to address where the msSGPs arise and what controls their specification. By marking the anterior of each parasegment using an antibody against Engrailed, it was determined that the msSGPs are specified in PS13. This observation is consistent with these cells arising posterior to the SGPs, which form in PS 10, 11, and 12. Other Sox100B expression is observed in nongonadal tissues. Whether, like the SGPs, the msSGPs are specified in the dorsolateral domain of the mesoderm was also addressed. Mesodermal cell types that form in this region, such as the SGPs and the fat body, require the homeodomain proteins Tinman and Zfh-1 for their specification. However, in embryos double-mutant for tinman and zfh-1, the msSGPs are still specified, even though the SGPs fail to develop. Thus, msSGPs do not arise from the dorsolateral domain, consistent with the fact that the msSGPs are first observed in a position ventral to the SGPs. The msSGPs also differ from the SGPs in terms of their requirements for the homeotic gene abd-A. SGP specification absolutely requires abd-A, while msSGPs are still present in these mutants. Thus, despite the fact that the msSGPs and the SGPs share expression of some molecular markers such as Eya and Wnt-2, their specification is under independent control (DeFalco, 2003).

Since the msSGPs express both Eya and Sox100B, the requirements for each of these genes in msSGP specification was investigated. In eya mutants, Sox100B-positive cells are still observed posterior to the germ cells at early stages, in a position where the msSGPs normally develop. Since the SGPs are not maintained in these mutants, the germ cells disperse and the gonad does not coalesce. Therefore, it is impossible to tell if the msSGPs would join the posterior of the male gonad in eya mutants. However, initial msSGP specification does not require eya. Similarly, in a deletion that removes the Sox100B locus, a large cluster of Eya-positive cells was still observed at the posterior of the male gonad that does not appear in females. Thus, the initial development of the msSGPs does not require Sox100B. Expression of Eya and Sox100B are mutually independent and are likely to be downstream of factors controlling initial msSGP specification (DeFalco, 2003).

Since the msSGPs are initially specified in both males and females, a determination was made of how these cells receive information about their sexual identity that allows them to behave differently in the two sexes. tra plays a key role in the sex determination pathway in Drosophila and is required to promote female differentiation in somatic tissues. tra mutant gonads were examined to test if tra function is required for gonad sexual dimorphism (XX embryos are masculinized by mutations in tra). Sox100B-immunopositive cells are observed in the posterior somatic gonad of both XX and XY tra mutant embryos in a manner comparable to wild-type males. Analysis of the Sox100B expression pattern in the gonad reveals that there are no differences between XX and XY tra mutants, or between either of these genotypes and wild-type males. Conversely, when Transformer is expressed in XY embryos (UAS-tra^F, tubulin-GAL4), Sox100B-immunopositive cells are no longer observe in these gonads, and they now appear similar to wild-type females (DeFalco, 2003).

In most somatic tissues, the principle sex determination factor downstream of tra is dsx. Unlike tra, dsx is required for both the male and female differentiation pathway, since both XX and XY dsx mutant adults show an intersexual phenotype. However, in the somatic gonad, dsx mutant XY embryos are indistinguishable from wild-type males and show no change in Sox100B expression. Thus, unlike in most somatic tissues, this early characteristic of male development does not require dsx. In XX embryos that are mutant for dsx, a completely masculinized phenotype is observed, in which Sox100B expression in the gonad is similar to a wild-type male. When a dominant allele of dsx, dsx^D, is used to express Dsx^M (dsx^D/dsx) in XX embryos, these gonads are no more masculinized than dsx null XX gonads. Therefore, while Dsx^F is required for the proper female phenotype in XX gonads, it appears that the male Sox100B expression pattern is the 'default' state in the absence of dsx function (DeFalco, 2003).

Since the msSGPs join the posterior of the male gonad but are no longer detected in the female, the basis for the sexually dimorphic behavior of these cells was investigated. In the female, these cells could turn off Sox100B and Eya and contribute to some other tissue, or they might be eliminated altogether. To test this latter hypothesis, whether msSGPs are eliminated by sex-specific programmed cell death in the female was addressed. Since programmed cell death occurs in a caspase-dependent manner, the gonad phenotype was examined in embryos in which caspase activity was inhibited by expressing the baculovirus p35 protein in the mesoderm. In these embryos, XX gonads now appear masculinized; Sox100B-positive cells (msSGPs) persist and join the posterior of female gonads, and coexpress Eya, as in wild-type male embryos. There are not as many Sox100B-positive cells in females as in males, suggesting that p35 may not be completely suppressing cell death. The presence of such cells in the female gonad does not appear to drastically affect ovary formation or oogenesis, since embryos develop into fertile adult females (DeFalco, 2003).

To investigate how programmed cell death might be controlled in the msSGPs, the genes of the H99 region (head involution defective [hid], reaper [rpr], and grim), which are regulators of apoptosis in Drosophila, were examined. A small deletion (DfH99) removes all three of these genes and blocks most programmed cell death in the Drosophila embryo. In DfH99 mutants, an equivalent cluster of Sox100B-positive cells is observed in both males and females. Again, these posterior cells are also Eya positive. Furthermore, XX embryos mutant for hid alone also contain Sox100B-positive cells in the posterior of the gonad, although the posterior cluster of cells is slightly smaller than in the male. It is concluded that the msSGPs are normally eliminated from females through sex-specific programmed cell death, controlled by hid and possibly also other genes of the H99 region. However, if cell death is blocked in females, these cells can continue to exhibit the normal male behavior of the msSGPs, including proper marker expression and recruitment into the gonad. Therefore, the decision whether or not to undergo apoptosis is likely the crucial event leading to the sexually dimorphic development of these cells at this stage (DeFalco, 2003).

It is concluded that proper information from the sex determination pathway is required to control the sexually dimorphic behavior of the msSGPs. The female phenotype in the embryonic gonad is dependent on both tra and dsx. Interestingly, it seems that the male phenotype is the default state; in the absence of any tra or dsx function, msSGPs in both XX and XY embryos behave as in wild-type males. This is a different situation than in most other tissues, in which dsx is required in both sexes to promote proper sexual differentiation. In particular, while no role is found for Dsx^M in this process, Dsx^F is positively required either to establish the female fate in the posterior somatic gonad or to repress the male fate. This role for Dsx^F in msSGP development is analogous to its role in the genital disc, in which Dsx^F is required to block recruitment of btl-expressing cells into the disc; in both cases, dsx female function serves to repress incorporation of a male-specific cell type. Since the msSGPs are initially specified in a sex-independent manner, this may account for the fact that the persistence of these cells (the male phenotype) is the default state. It will be of interest in the future to address the role of the msSGPs in testis development, and how genes such as dsx, eya, and Sox100B act in this process (DeFalco, 2003).

Although sex determination schemes vary widely in the animal kingdom, there is evidence that the molecular and cellular pathways used to control sexual dimorphism may be conserved, even between vertebrates and invertebrates. One example is Sox9, which has been implicated as an ancestral sex-determining gene in vertebrates given its male-specific gonad expression in diverse species such as human, mouse, turtle, and chicken. A potential Drosophila ortholog of Sox9, Sox100B, is expressed in a male-specific manner in the embryonic somatic gonad. The manner of Sox100B expression is reminiscent of that in the mouse; Sox9 is initially expressed in both sexes, but is maintained and upregulated in the male gonad. It will be very interesting to compare the role that Sox100B plays in the development of the Drosophila testis to the one played by Sox9 in vertebrates (DeFalco, 2003).

Molecular conservation is also observed amongst the members of the Dsx/Mab-3 Related Transcription Factor (DMRT) family. DMRT family members have been shown to be essential for sex-specific development in Drosophila (Dsx), C. elegans (mab-3), medaka fish (DMY), and mice (DMRT1) and have been implicated in human sex reversal. This study demonstrates that dsx is essential for proper sex-specific development of the msSGPs. Thus, increasing evidence indicates that DMRT family members are also conserved regulators of sexual dimorphism (DeFalco, 2003).

D-Six-4 and its cofactor Eyes absent play a key role in patterning cell identities deriving from the Drosophila mesoderm

Patterning of the Drosophila embryonic mesoderm requires the regulation of cell type-specific factors in response to dorsoventral and anteroposterior axis information. For the dorsoventral axis, the homeodomain gene, tinman, is a key patterning mediator for dorsal mesodermal fates like the heart. However, equivalent mediators for more ventral fates are unknown. This study shows that Six4, which encodes a Six family transcription factor, is required for the appropriate development of most cell types deriving from the non-dorsal mesoderm: the fat body, somatic cells of the gonad, and a specific subset of somatic muscles. Misexpression analysis suggests that Six4 and its likely cofactor, Eyes absent, are sufficient to impose these fates on other mesodermal cells. At stage 10, the mesodermal expression patterns of Six4 and tin are complementary, being restricted to the dorsal and non-dorsal regions respectively. These data suggest that Six4 is a key mesodermal patterning mediator at this stage that regulates a variety of cell-type-specific factors and hence plays an equivalent role to tin. At stage 9, however, Six4 and tin are both expressed pan-mesodermally. At this stage, tin function is required for full Six4 expression. This may explain the known requirement for tin in some non-dorsal cell types (Clark, 2006).

A fundamental question in developmental biology concerns the means by which uncommitted cells become specified to form a diversity of tissues according to their spatial location. In general, it is clear that a relatively small number of signaling and transcription factors are expressed in response to positional information, and in turn, these act combinatorially to regulate the expression of more specialized cell type regulatory factors. There is much interest in understanding the combinatorial regulation of cell type factors, particularly through computational analysis of their cis-regulatory regions. This is hampered, however, by an incomplete understanding of the identity and function of the upstream regulatory mediators themselves (Clark, 2006).

The specification of the mesoderm in Drosophila provides a tractable model system in which to study how the expression of cell type regulators is patterned within a large group of cells that are initially identical. A diverse range of organs derives from the Drosophila mesoderm, including the heart, the somatic and visceral muscles, the fat body and the somatic component of the gonad. For parasegments 4-12, an approximate fate map can be constructed outlining the mesodermal regions that give rise to these organs. Transplantation experiments show that fate determination is dependent on cell position, and therefore, patterning the mesoderm requires positional information. This is provided in part by inductive signaling from the overlying ectoderm, which results in the establishment of specific expression patterns for mesodermal transcription factors (Clark, 2006).

Along the anteroposterior axis, the parasegmental mesoderm is divided into two domains that correspond to the action of the pair rule genes, even skipped (eve) and sloppy paired (slp). The eve domain includes the cells underlying ectodermal stripes of hedgehog (hh) and engrailed (en) expression, and these genes participate in the development of the tissues that derive from this region. The action of hh is antagonized by that of wingless (wg), which signals to cells of the slp domain leading to body wall muscle and heart development. In the slp domain, twist (twi) is expressed at a high level and contributes to the development of the somatic muscles, while Notch signaling modulates twi to low levels in the eve domain (Clark, 2006 and references therein).

In the dorsoventral axis, the homeodomain transcription factor, Tinman (Tin), plays a central role in establishing dorsal mesoderm fates. In the dorsal region, ectodermal Decapentaplegic (Dpp) signaling maintains the expression of tin, which is lost from the remainder of the mesoderm following gastrulation. Tin and Dpp combine with factors involved in anteroposterior patterning to establish the primordia of the various dorsal mesodermal organs. For example, in the dorsal slp domain, Tin cooperates with Wg to activate specific sets of target genes, leading to heart and dorsal muscle development. Conversely, the visceral mesoderm is formed in the dorsal eve domain through the activation of bagpipe by Tin and Dpp and its repression by wg/slp. Apart from its dorsal function, tin also has a poorly understood role in the development of more ventral mesodermal fates (Clark, 2006 and references therein).

Outside the dorsal domain, it has been suggested that the non-dorsal mesoderm is divided into ventral and dorsolateral domains. This was based on the response of fat body cells to Wg signaling, although it is not clear whether this distinction has a genetic basis. The dorsolateral domain contains cells with dual fat body/somatic gonadal precursors (SGP) competence, although normally only those cells in parasegments 10-12 take on an SGP fate. Apart from the role of Tin dorsally, the patterning of mesodermal fates in the dorsoventral axis is poorly understood. Nevertheless, recently it has been demonstrated that Pox meso exhibits an early function, partially redundant with the function of lethal of scute, in demarcating the 'Poxm competence domain', a domain of competence for ventral and lateral muscle development and for the determination of at least some adult muscle precursor cells. A major unanswered question is whether there are factors in the non-dorsal mesoderm that perform functions complementary to those of Tin in the dorsal region. A candidate for such a factor is the Six family homeodomain protein, Six4. In mouse, Six1, Six4, and Six5 genes are coexpressed during myogenesis, while Six1 and Six4 at least are required during mesoderm development. In human, reduction of SIX5 expression may underlie some of the abnormalities associated with Type 1 Myotonic Dystrophy (DM1). In Drosophila, the sole Six4/Six5 homologue, Six4, is the only Six homeoprotein expressed in the early mesoderm, and its mutation disrupts gonad and muscle development (Clark, 2006).

Evidence that Six4 is a key mesodermal patterning factor and is necessary for the correct development of various cell types deriving from the non-dorsal mesoderm, including fat body, SGP, and somatic muscles. Correspondingly, at stages 10/11, Six4 is expressed in non-dorsal mesoderm in a complementary pattern to tin. Moreover, with its cofactor Eyes absent (Eya), Six4 is sufficient to drive the specification of certain non-dorsal fates. In addition, these results clarify the function of tin in ventral mesodermal cells: it is proposed that earlier in development (at stages 8/9), part of tin's function ventrally is to initiate expression of Six4 (Clark, 2006).

Using a GFP reporter gene construct (referred to as Six4-III-GFP), an enhancer was identified within the Six4 third intron that activates GFP in a pattern corresponding closely to the mesodermal expression of Six4 RNA. At stage 9, Six4-III-GFP is coexpressed with D-Mef2 in a broad mesodermal domain. Subsequently, by stage 10, GFP expression becomes largely restricted ventrally, although some perduring protein remains in the dorsal region. At this point, lateral/ventral Six4-III-GFP expression is complementary to the dorsal expression of Tin (although the two are coexpressed earlier). Once restricted, the dorsal limit of Six4-III-GFP expression coincides with that of serpent (srp) protein, which marks the dorsolateral fat body cells. The levels of Six4 mRNA and Six4-III-GFP expression are modulated in the anteroposterior axis of the segment, being stronger in the slp domain (between the dorsolateral Srp clusters of the eve domain). This anteroposterior modulation of Six4 expression resembles that of twi, raising the possibility that different levels of protein have different functional consequences (Clark, 2006).

At stage 10, inductive dpp signaling from the dorsal ectoderm acts to maintain tin expression, thereby driving the dorsal restriction of Tin. Conversely, the ventral restriction of Six4 may depend on an inhibitory effect of dpp signaling. Consistent with this, misexpression of dpp throughout the mesoderm reduces expression of Six4 RNA to a low level. Thus, it is suggested that dpp signaling acts to establish two, non-overlapping spatial domains of gene expression in the mesoderm: a dorsal domain expressing tin and a ventral and lateral domain in which Six4 is expressed. Six4 is therefore a candidate for the counterpart of tin in patterning more ventral mesodermal fates (Clark, 2006).

Six4 is a key factor for the development of a variety of tissues that originate from the non dorsal mesoderm. It is required for the SGPs, fat body precursors and specific lateral and ventral muscles and is likely to be a competence factor or patterning mediator, acting to regulate a variety of key tissue and cell identity genes, such as srp for the fat body and ladybird for the segment border muscle founder cells. Different target genes would be regulated in different locations by the combinatorial action of Six4 and other factors involved in dorsoventral and anteroposterior axis patterning. Six4 may play additional roles later in gonad development, since its expression is maintained in SGPs throughout embryogenesis, whereas it is expressed transiently in most of the mesoderm (Clark, 2006).

Defective Six4 function results partly in failure of cell fate maintenance and/or cell survival. This is a common mutant phenotype of members of the Six and Eya gene families. Strikingly, however, expression of Six4 with its cofactor, Eya, throughout the mesoderm causes the expansion of Six4-dependent cell types (fat body and SGPs) with the concomitant disruption of other mesodermal derivatives, including the cardioblasts and visceral mesoderm. This supports an active role for Six4 in initial patterning of cell fates. It is possible, therefore, that maintenance/survival phenotypes are a secondary effect of defects in the initial establishment of cell identity (Clark, 2006).

Specific aspects of muscle cell identity are affected in Six4 mutant embryos. The phenotype is variable, but the external lateral and some ventral muscles are consistently disrupted. When Six4 and Eya are misexpressed/overexpressed in the mesoderm, an aberrant but regular muscle pattern is formed, suggesting that they have a patterning role, as opposed to a function in differentiation or myoblast fusion. It is likely that Six4 participates in the activation of certain muscle identity genes in founder myoblasts. Expression of the SBM identity gene, ladybird, requires Six4, while misexpression of Six4 and eya specifically in founder cells (using a dumbfounded-Gal4 driver) results in a muscle phenotype indistinguishable from that of embryos misexpressing these genes throughout the mesoderm (Clark, 2006).

The relationship between Six4 and tin is complex, partly because it changes over time and also because tin has functions in the ventral and lateral mesoderm that have remained obscure. The best characterized functions of tin concern the dorsal mesoderm, reflected in its restricted dorsal expression at stage 10/11. At this time, Six4 expression is complementary to that of tin, and there are no discernable effects on dorsal mesoderm structures in Six4 mutants. It is proposed that these two genes play complementary roles in their respective domains, promoting the development of specific cell types in conjunction with additional patterning factors. Despite their complementary expression patterns at this stage, there is no evidence that tin and Six4 are mutually antagonistic: although Tin can act as a repressor as well as an activator, there is no significant expansion of Six4 expression along the dorsoventral axis in a tin mutant and vice versa (unpublished data). It is more likely that, like tin, Six4 is directly regulated by dpp signaling (Clark, 2006).

In addition to dorsal mesoderm defects, tin mutant embryos show SGP, fat body and specific lateral and ventral muscle defects that presumably depend on its early pan-mesodermal expression. At least one tin function appears to depend on its regulation of Six4 in the early mesoderm before their mutually exclusive refinement of expression. Like Six4, tin is required for correct SGP development: a reduced number of SGPs appear at stage 11, and the number diminishes further until stage 13 when germ cell migration defects become apparent. However, the ventral expression of tin is lost before the SGPs are apparent, suggesting that another factor mediates its function in SGP development. Six4 may be this factor, since initially the two genes are transiently coexpressed broadly in the mesoderm, and Six4 expression is partly dependent on tin function. At this stage, Tin could be a direct transcriptional activator of Six4, since there are a number of sequences in the third intron that match the core E-box of the canonical Tin binding site (ACAAGTGG) (Clark, 2006).

The pattern of lateral and ventral muscle defects in embryos lacking Tin is different from that of Six4 mutants. Muscles affected by tin include LL1, LO1, VL3, VL4, and VT1, which do not require Six4 or Eya. Conversely, muscles that are severely affected by Six4 mutation appear normal in tin mutants, including VA3, the SBM, and the external lateral muscles LT1, LT2, LT3, and LT4. Based on these findings, it is proposed that muscles fall into at least three categories. The visceral, cardiac, and dorsal somatic muscles all require tin function directly through persistent dorsal tin expression. A second group, comprising a subset of ventral and lateral muscles, requires tin function via its transient pan-mesodermal expression, either directly or perhaps through unknown patterning mediators. A third group, a different subset of lateral and ventral muscles, is dependent on Six4/eya function and is not affected in tin single mutants, presumably because the reduced Six4 expression in these embryos is sufficient for their patterning. Muscles in this last category resemble the fat body precursors in their functional requirements, being dependent on an early, partially redundant function of tin and zfh-1, which is necessary to initiate Six4 expression in most parasegments. Confirmation of this model awaits a comprehensive characterization of muscle identity gene expression in founder cells in tin and Six4 mutant embryos (Clark, 2006).

The role of Six4 in mesoderm patterning appears to be conserved in other organisms. Expression of human SIX5 is reduced in Type I Myotonic Dystrophy, which may suggest a role in myogenesis since the most severe forms of this condition display muscle developmental defects (Harper, 1989). The murine orthologues, Six4 and Six5, are both expressed during myogenesis, although their precise roles are not yet established as single gene knock-out models have no clear muscle defects, perhaps owing to compensatory interactions. Six4 mutation, however, strongly exacerbates the muscle loss of mice mutant for the more divergent homologue, Six1. It is striking in particular that hypaxial progenitors (which contribute to limb muscles) lose their identity in Six1 Six4 double mutant mice. These muscle progenitors require the function of an lb homologue, Lbx1, and there is evidence that Lbx1 may be a target of Six/Six4. Thus, it appears that the function of Six4/5 genes might be conserved to a high degree (Clark, 2006).

The requirement for Six4 in diverse cell types, linked by their location of origin during mesoderm patterning, may represent a primordial state. The C. elegans homologue (unc-39) is also required for a number of mesodermal cell types. Although knowledge of Six4 and Six5 function is incomplete, it is notable that Six1 is required for the development of diverse organs such as muscle, kidney, and otic vesicle. It is interesting to note that Lbx1 regulation may be achieved by the combinatorial action of Six1/4 and Hox genes, which would thus behave as patterning factors in a similar way to Six4. The current studies suggest that diverse roles of SIX genes in vertebrate organogenesis as apparent cell- or tissue-type regulators may have their evolutionary origins in a general primordial developmental patterning mechanism, part of which may be preserved more clearly in the role of Six4 in mesoderm development in Drosophila (Clark, 2006).

Larval

The Eya protein first becomes detectable in cells of the eye portion of the eye-antennal disc during the second larval instar; the expression is graded, being stronger in cells in the posterior and at the edges of the eye portion of the disc, than in cells in the anterior and central region. This staining pattern persists to the third larval instar. As the morphogenetic furrow forms, the protein stays on in a graded manner anterior to it, with the strongest expression just anterior to the furrow. Protein expression persists in cells as the furrow passes. Posterior to the furrow, the expression is patterned, reflecting the array of developing neural clusters. Expression is apparent in the ocellar progenitor cells within the eye disc. The gene does not appear to be expressed in the embryonic eye primordia or in eye discs during the first larval instar (Bonini, 1993).

The eyes absent gene plays an essential role in the events that lead to formation of the Drosophila eye; without expression of eya in retinal progenitor cells, they undergo programmed cell death just prior to the morphogenetic furrow, leading to an eyeless or reduced eye phenotype. Among the available alleles of eya, there are certain ones that result in loss only of the compound eyes. These alleles function as nulls for the early eye activity of the gene. However, others show loss of both eyes and ocelli and others lead to loss of eyes, ocelli and female fertility. Analysis of the embryonic lethal phenotype indicates that mutant alleles show defects in head morphogenesis. Mutant embryos have relatively normal, albeit somewhat disorganized, development of the peripheral and central nervous systems. The most dramatic defects are seen in the anterior, with the failure of invagination of the developing brain into the head. In the mutant, the brain appears to be roughly normal size, but is positioned anomalously at the dorsal surface of the embryo. Mutant animals display a deep dorsal cleft, presumably where the cephalic furrow initially forms. The lateral nerves of the PNS are somewhat disrupted and peripheral nerves of the ninth abdominal segment are located in a slightly more dorsal position than normal. In the anterior, the antennomaxillary complex is more lateral than normal, and the nerves of the epiphysis and hypophysis are positioned aberrantly to the anterior, rather than being internal to the head. These anomalies in positioning of varous nerves and brain would appear to reflect failure of proper morphogenetic movements involved in head involution. The structure of the cephalopharyngeal skeleton in the head is abnormal, being broad, fused, and distorted. (Bonini, 1998).

Detailed studies at the subcellular level indicate that the Eya protein is localized to the nucleoplasm, suggesting a role in control of nuclear events. Eya is excluded from the nucleolus, and not present to any extent on the chromatin (Bonini, 1998).

The eya gene shows expression and roles in tissues other than the eye, including subsets of cells of the adult visual system, brain, and ovary, as well as an elaborate expression pattern in the embryo. In the developing eye, Eya protein is found in the nuclei of cells anterior to the furrow. Following the differentiation events at the furrow, the protein persists in undifferentiated cells at the basal region of the disc and in the developing cone cells at the apical region of the disc. The developing photoreceptor cells gradually shut off expression of Eya. In pupal stages Eya protein is strongest in cells identifiable by position as cone cell procursors, as well as in progenitors of the pigment cells. In the adult eye, the Eya protein is expressed in the outermost layers of the retina; most, if not all, cone and pigment cell nuclei are labeled but not the photoreceptor cells. In the third-instar larval eye imaginal disc, both EYA mRNA and Eya protein are also expressed in progenitor cells of the ocelli, which occupy a lateral position in the disc: each disc gives rise to one lateral adult ocellus, plus half of the central one. About 30-40 cells of each adult ocellus also express Eya protein (Bonini, 1998).

Eya protein is expressed within the adult brain in a set of bilaterally symmetric cell clusters, each containing some 50-100 cells. In serial sections through the brain, seven or eight pairs can be identified. These cell clusters do not appear to coincide with previously identified clusters of cells defined by anatomical methods. Thus, rather than identifying anatomical subsets of cells, these clusters would appear to define functional subsets of brain cells. What functions these centers serve, and whether they are visual or otherwise, remain to be determined. During imaginal development, Eya is expressed in the lamina precursor cells, and in cells of the developing brain and ventral cord. Eya does not highlight the developing lamina of the adult brain, indicating that at least parts of the brain expression are transient in nature between the imaginal and adult brain (Bonini, 1998).

The adult female ovary shows expression of both mRNA and Eya protein in the follicle cells, from the time these cells form an epithelial monolayer around the germline cysts (region 2b of the germarium) to stage 10 of the ovariole, when the follicle cells commence migration over the developing oocyte. This correlates with the fact that select eya allelic combinations are female sterile; in extreme allelic cominations, ovarian development is arrested very early, at the germarium stage. These data suggest that a lack of follicle cells or inappropriate follice cell development contributes to the sterile phenotype (Bonini, 1998).

The sole EYA transcript expressed during the embryonic stages is type II, while both type I and type II cDNAs are expressed in eye progenitors. Gene expression begins zygotically at the onset of the cellular blastoderm, in a dorsal anterior crescent at 76-86% of egg length, extending to a ventral point about 3/4 of the way toward the ventral midline. This expression pattern persists through early gastrulation. During gastrulation, the expression pattern broadens anteriorly to cover a wider domain in the dorsal head, overlapping the area destined to give rise to the brain; expression is now seen also in a new region along the dorsal part of the cephalic furrow. During extended germ band stages, the head expression pattern develops into an elaboprate mask along dorsal and lateral regions. This expression partially overlaps the procephalic neuroblasts that will form the brain, extends along the anterior lip of the cephalic furrow from the dorsal midline, and extends ventrally to encompass part of the invaginating optic lobes. More anteriorly, expression is seen along part of the clypeolabrum, near the stomadeal opening. At this time, expression is also seen in the mesoderm along the length of the embryo. In late stage 11 and early stage 12 when germ band shortening begins, expression persists in the head, whereas the mesodermal expression is rescinded. A segmentally reiterated expression pattern begins, with time of onset proceeding from anterior to posterior. Internally, the germ cell primordia express the gene. Upon germ band retraction, expression in the head persists, in part associated with the supraesophageal ganglion, and internal to the clypeolabrum at the most anterior. The reiterated segmental expression appears associated with the segmental furrows and lateral ventral nerve cord. Expression also occurs along the peripheral edges of the anal pads, and in the pharyngeal muscles. In later stages, expression is prominent in three bilaterally symmetric clusters in the brain lobes and in cells along the anterior portion of the ventral cord. The segmental expression pattern is distinct along the segmental grooves (Bonini, 1998).

Despite multiple roles at multiple stages of development of the fly, both the type I and type II forms of the protein, when expressed ectopically during larval development, can direct eye formation (Bonini, 1998).

Eye specification in Drosophila is thought be controlled by a set of seven nuclear factors that includes the Pax6 homolog, Eyeless. This group of genes is conserved throughout evolution and has been repeatedly recruited for eye specification. Several of these genes are expressed within the developing eyes of vertebrates and mutations in several mouse and human orthologs are the underlying causes of retinal disease syndromes. Ectopic expression in Drosophila of any one of these genes is capable of inducing retinal development, while loss-of-function mutations delete the developing eye. These nuclear factors comprise a complex regulatory network and it is thought that their combined activities are required for the formation of the eye. The expression patterns of four eye specification genes [eyeless (ey), sine oculis (so), eyes absent (eya), and dachshund (dac)] were examined throughout all time points of embryogenesis; only eyeless is expressed within the embryonic eye anlagen. This is consistent with a recently proposed model in which the eye primordium acquires its competence to become retinal tissue over several time points of development. The expression of Ey was compared with that of a putative antennal specifying gene, Distal-less (Dll). The expression patterns described here are quite intriguing and raise the possibility that these genes have even earlier and wide ranging roles in establishing the head and visual field (Kumar, 2001).

Ey directs the transcription of eya by binding to regulatory regions within the eya promoter. Ectopic expression of eya was not however observed to induce eye transcription. It is therefore possible to see cells that are Eya positive and Ey negative, but any cell that is Ey positive should also be Eya positive. To determine if this is indeed the case, the expression of ey-lacZ was compared with that of eya, with the expectation that all cells (especially the eye imaginal disc primordia) that express ey-lacZ would also express eya. Surprisingly, eya expression begins even earlier than that of dac and is obviously also independent of ey regulation (Kumar, 2001).

Eya protein is first seen at approximately 2 h AED at which time the embryo is still in the synctial blastoderm time point and can be seen as a band of cells that runs along the dorsal surface of the embryo. By approximately 4 h AED this band is transformed into a crown that extends more laterally. As is the case with dac, eya begins to be expressed in subsets of cells within the embryonic brain by approximately 7 h AED, but these cells are distinct from those that express dac. Unlike dac, eya is not expressed within the ventral nerve cord, but rather is found in a small clustering of cells within the segmental grooves of the embryo. From the onset of ey-lacZ expression at approximately 11 h AED through the end of embryogenesis, eya is not expressed within the eye imaginal disc. Eya protein is first detected in the eye imaginal disc during the first larval instar (Kumar, 2001).

Recently it has been shown that the patterning genes hedgehog (hh) and decapentaplegic (dpp) are required for the specification in the eye. In an interesting model it has been proposed that Hh signals to Eya which then in turn induces (directly or indirectly) the transcription of both so and dac. This would then suggest that during embryogenesis all three proteins should have overlapping expression patterns during the allocation of the eye disc. The expression of a so-lacZ transgene was compared with that of dac. Interestingly, while the onset of expression of both genes are first detected at approximately 4 h AED, their expression patterns abut each other and are not overlapping. While dac is expressed in two clusters of dorsal medially located cells, so-lacZ expression is seen in a broad swathe of cells that extends from one lateral surface to another. Its expression appears to be delimited by the more-anterior domain of dac expression and the cephalic furrow. By approximately 5 h AED there is a cluster of cells along the lateral margins just anterior to the cephalic furrow in which both so-lacZ and dac are co-expressed. However, the vast majority of so-lacZ and dac expression is non-overlapping. Not unlike eya, so-lacZ is expressed in a subset of cells within the developing brain but is not expressed in the ventral nerve cord. There is considerable overlap between the dac and so-lacZ expression patterns within the developing brain lobes. In the segmental grooves so-lacZ expression can be seen much like that of eya. At approximately 11-14 h AED there is no expression of so-lacZ within the developing eye imaginal disc (Kumar, 2001).

Eya can form a complex with So in vitro. Within the eye imaginal disc of second and third instar larvae, both genes are expressed in overlapping, although not completely exact, patterns. It has also been shown that so and eya act synergistically in promoting ectopic eye development. The expression of so-lacZ was compared with that of eya. Eya protein is first detected in the cellular blastoderm at approximately 2 h AED, while so-lacZ expression is not seen until approximately 4 h AED. The dorsal expression of both genes overlaps considerably. There is also considerable overlap in their expression patterns within the developing brain lobes. By the end of embryogenesis so-lacZ expression is severely reduced. Interestingly, the patterns of expression of so-lacZ and eya within the segmental grooves are not overlapping. However, the degree of overlap between the patterns within the embryo and the late second and third eye imaginal discs furthers supports the in vitro biochemical evidence that these genes do interact (Kumar, 2001).

These results have several implications for current thinking on how the Drosophila eye is specified. It has been shown that the ectopic expression of each of the eye specification genes (with the exception of so) is sufficient to induce the formation of ectopic eyes. What prevents the induction of retinal tissue throughout the embryo? It is argued here that expression of all eye specification genes are required for eye determination. Within the embryo no region is found in which all these factors are present. It is not until the second larval instar that all genes are expressed within the same tissue. Within the embryo, positive or repressive mechanisms must be in place to prevent the eye specification genes from being co-expressed. For example, ey is capable of directly inducing the transcription of both so and eya within the mature eye imaginal disc, but within the eye anlagen these genes are not expressed, although Ey protein is present. The nature of this regulatory mechanism is unknown (Kumar, 2001).

It is still unclear as to why only three eye specification genes (toy, ey, and eyg) are expressed within the embryonic eye primordium. Is the expression of these three genes within the eye primodium a priming step for the eventual specification of the eye or is it simply a step that distinguishes one disc from another? Since Ey protein has been shown to directly bind to the so and eya promoters, there must be an inhibitory signal within the eye disc that prevents the transcription of these genes from being induced. This repression is first released for eya transcription because it is localized to the first instar eye disc. The inhibition upon the remaining genes is released during the second larval instar. Unraveling this mystery will certainly require extensive molecular and biochemical analysis on embryonic and early larval eye discs (Kumar, 2001).

Another lingering question focuses on the fates of the cells that are derived from the initial expression of so, eya, and dac. All three of these genes are expressed very early; for instance eya is expressed in a cluster of cells at the cellular blastoderm time point. Do these cells contribute to the formation of the visual field? Are these three proteins committing cells to adopt an eye imaginal disc fate, an event that will occur much later in embryogenesis? Such questions can only be addressed by precise single cell fate mapping experiments. Only by labeling a single cell and tracing its progeny will it be known if the earliest cells that express so, eya, and dac will later become cells of the eye imaginal disc. How the expression patterns described here correlate with the genetic, molecular, and biochemical interactions of the eye specification genes is an interesting problem that will undoubtedly require the identification of additional instructive and inhibitory signals (Kumar, 2001).

Finally, are the earliest expression patterns of these eye specification genes homologous between vertebrates and invertebrates? This is certainly a much more difficult question to answer. A decade ago this question would be easily answered with a resounding 'no'. Now as more molecular and physiological similarities between the visual systems of vertebrates and invertebrates are being discovered, the answer to this developmental question may not be as easily or as negatively answered. It would be truly remarkable if a common developmental history underlies the use of identical molecules to create the different types of eyes seen throughout the animal kingdom. The key to such questions may lie in the precise fate mapping of individual cells that express each of the genes responsible for eye specification (Kumar, 2001).

Oogenesis

Throughout Drosophila oogenesis, specialized somatic follicle cells perform crucial functions in egg chamber formation and in signaling between somatic and germline cells. In the ovary, at least three types of somatic follicle cells, polar cells, stalk cells and main body epithelial follicle cells, can be distinguished when egg chambers bud from the germarium. Although specification of these three somatic cell types is important for normal oogenesis and subsequent embryogenesis, the molecular basis for establishment of their cell fates is not completely understood. Studies reveal the gene eyes absent (eya) to be a key repressor of polar cell fate. Eya is a nuclear protein that is normally excluded from polar and stalk cells, and the absence of Eya is sufficient to cause epithelial follicle cells to develop as polar cells. Furthermore, ectopic expression of Eya is capable of suppressing normal polar cell fate and compromising the normal functions of polar cells, such as promotion of border cell migration. Finally, it has been shown that ectopic Hedgehog signaling, which is known to cause ectopic polar cell formation, does so by repressing eya expression in epithelial follicle cells (Bai, 2002).

Drosophila oogenesis provides an excellent system with which to study the mechanisms underlying specification of different cell fates. The Drosophila ovary is made up of germline cells and somatic follicle cells. Germline and somatic stem cells can be found at the anterior end of the ovary in a structure called the germarium. Germline stem cells divide asymmetrically and produce cystoblasts, which undergo four rounds of incomplete cell division and give rise to 16-cell germline cysts. One of the cyst cells becomes the oocyte and the remaining 15 cells differentiate as nurse cells. In the germarium, somatic follicle cells surround the 16-cell cysts. As the nascent egg chamber buds off from the germarium, at least three types of somatic cells can be distinguished by their morphologies and locations: polar cells, stalk cells and epithelial follicle cells. Polar cells are pairs of specialized follicle cells at each pole of the egg chamber, whereas the five to eight stalk cells separate adjacent egg chambers. Stalk and polar cells may descend from a common precursor. They differentiate and cease division soon after egg chambers form. The remaining somatic follicle cells, referred to here as epithelial follicle cells, proliferate until stage 6 of oogenesis and form a continuous epithelium around the sixteen germ cells. Subsequently, further differentiation of epithelial follicle cells occurs (Bai, 2002).

In wild-type egg chambers, the anterior polar cells recruit four to eight follicle cells to surround them and become migratory border cells at early stage 9. They migrate through the nurse cell cluster during stage 9 and arrive at the border between the oocyte and the nurse cells at stage 10. Ectopic HH signaling, e.g., caused by loss of cos2, results in the formation of ectopic polar cells and recruitment of extra border cells, which frequently migrate. In the course of a genetic screen for mutations on the left arm of the second chromosome (2L) that affect border cells in mosaic clones, one ethyl methanesulfonate (EMS)-induced mutation, 54C2, was identified that causes extra clusters of border cells to form. Border cells were marked by ß-galactosidase expression from an enhancer trap line, PZ6356. In wild-type egg chambers, there was only one cluster of border cells. By contrast, in egg chambers containing 54C2 mutant clones, either multiple clusters of migrating border cells or a single abnormally large cluster was observed. This phenotype resembled that of egg chambers containing cos2 or ptc mutant clones (Bai, 2002).

The extra border cell clusters found in cos2 or ptc mutant egg chambers result from overproduction of polar cells. Polar cells can be detected by staining with an antibody against Fasciclin III, or by expression of ß-galactosidase from the enhancer trap line A101 (neuralized-lacZ), which is a marker for mature polar cells. FAS3 is a homophilic cell adhesion molecule that accumulates to the highest levels in immature follicle cells in the germarium and at the interface between the two polar cells from stage 3 to stage 10A of oogenesis in wild-type egg chambers. There are two pairs of polar cells in wild-type egg chambers, one pair located at the anterior pole of the egg chamber and another at the posterior. In 54C2 mutant egg chambers, the extra border cell clusters that formed contained extra polar cells. In addition, ectopic polar cells were observed in early stage egg chambers and were found in many positions throughout the follicle epithelium in egg chambers containing mutant clones (Bai, 2002).

The gene mutated in the 54C2 line was identified based on deficiency and meiotic recombination mapping. 54C2 was found to reside in the 26D-27A region. Mutations in one known gene in this region, eya, failed to complement the 54C2 mutation with respect to lethality, whereas all other mutations in this region complemented. In addition, two independent eya alleles, cli^E11 and cli^D1, caused ovarian phenotypes in mosaic clones that are similar to those of 54C2, including ectopic polar cells and overproduction of border cells. The phenotype of 54C2 in other tissues also resembled that of eya. Therefore, it was concluded that 54C2 is a new allele of the eya gene (Bai, 2002).

Since loss of eya in follicle cells leads to ectopic polar cells in the ovary, it was postulated that expression of Eya might normally be repressed in the polar cells. Alternatively, Eya might be repressed via a post-translational modification in polar cells. To distinguish between these possibilities, the expression pattern of the Eya protein was examined in the ovary. Egg chambers were double stained with antibodies against Eya and anti-ß-galactosidase antibodies in order to identify either polar cells, in the A101 enhancer trap line, or stalk cells in the enhancer trap line 93F. The earliest expression of Eya was observed in follicle cells in region 2b of the germarium. Eya continues to be expressed in all follicle cells with the exception of polar and stalk cells until late stage 8. After stage 8, Eya protein is restricted to the anterior follicle cells, including border cells, squamous cells and centripetal cells. Eya is not expressed detectably in the germ cells of any stage. Thus, the absence of Eya protein in the polar cells is consistent with a role as a repressor of polar cell fate (Bai, 2002).

Thus, the data demonstrate that eya is required to suppress polar cell fate in the epithelial follicle cells. The evidence for this is that Eya protein is absent from polar cells in wild-type egg chambers as soon as the polar cells express markers such as A101. Furthermore, loss of Eya can transform other epithelial follicle cells into polar cells in a cell autonomous fashion. Finally, ectopic expression of eya is capable of suppressing normal polar cell fate and compromising the normal functions of polar cells, such as promotion of border cell migration (Bai, 2002).

Eya protein is first lost in the cells along the border between regions 2b and 3 of the germarium. Those cells are likely to be the cells that separate cysts and may be the early progeny of the polar/stalk cell precursors. Therefore, loss of Eya expression appears to be the earliest available marker for this lineage (Bai, 2002).

Although Eya is normally missing from both polar cells and stalk cells, the expression of the mature stalk cell marker 93F was never observed in eya mosaic clones. Hence, loss of eya transforms the epithelial follicle cells only into polar cells, not into stalk cells. However, mis-expression of eya early in oogenesis leads to the absence of stalk cells and generates compound egg chambers, sometimes containing normal pairs of polar cells. It seems that the formation of compound egg chambers depends more directly on the loss of stalk cells than on the loss of polar cells. This was also the case when Eya expression was forced exclusively in the polar cells. Although the function of polar cells was compromised, the stalk cells still formed and compound egg chambers were not observed. Further evidence for the crucial role of stalk cells in separating egg chambers is the compound egg chamber phenotype resulting from loss of so, a gene that is only expressed in the stalk cells. Therefore, repression of Eya appears to be required for stalk cell formation, which is in turn essential to separate egg compartments (Bai, 2002).

Why does loss of eya lead only to ectopic polar cells, not to stalk cells in the epithelial follicle layer? One possible reason is that the stalk cells, as opposed to polar cells and epithelial follicle cells, normally form in the absence of direct contact with germline cells. Thus, signals from the germline might prevent stalk cell fate in cells that directly contact the germline (Bai, 2002).

One germline signal that is known to play a role in polar cell specification is Delta, which signals from the germline to Notch in the soma to control the differentiation of polar cells. Epithelial follicle cells do not respond to Delta in the same way, presumably because, unlike polar cells, they do not express fringe. fringe encodes a glucosyltransferase that potentiates the ability of the Notch receptor to be activated by its ligand, Delta. Mutation of either Notch or fringe leads to the disappearance of polar cells. As a result, Eya-negative cells are not found in the follicles. Mis-expression of either Fng or activated Notch produces ectopic polar cells only at the poles of the egg chamber, whereas loss of Eya can cause polar cells to form throughout the follicle epithelium. Thus Notch signaling appears to be necessary, but not sufficient to repress Eya expression and leads to polar cell formation. Surprisingly, activated Notch also can produce ectopic polar cells cell-nonautonomously at the poles of the egg chamber. The reason for this could be that activated Notch signaling might activate the expression of Delta, which, in turn, can activate Notch signaling in the adjacent cells (Bai, 2002).

Another signaling pathway that impinges on polar and stalk cell fates is the JAK/STAT pathway. The ligand for this pathway, Unpaired (Upd), is expressed specifically in polar cells. The ligand interacts with a receptor, which in turn activates the tyrosine kinase known as Hopscotch (Hop). Hop activity results in phosphorylation and nuclear translocation of the transcription factor STAT92E. In the ovary, Upd secreted from polar cells functions to suppress polar cell fate in stalk cells. It has been proposed that N signaling specifies a pool of cells competent to become polar and stalk cells and the Upd/JAK/STAT pathway distinguishes polar versus stalk fates. Thus, whatever signal normally represses Eya in the polar/stalk lineage presumably acts prior to Upd/JAK/STAT since Eya repression occurs in both polar and stalk cells, possibly in the common precursor cell. The observation that forcing Eya expression in polar cells under the control of upd-GAL4 can repress polar cell fate suggests that this fate remains malleable for some time after its normal specification. The relatively low penetrance of this phenotype (~30%) might be due to the late expression of upd-GAL4 relative to the normal timing of Eya repression (Bai, 2002).

Loss of Eya results in the production of ectopic polar cells virtually anywhere in the egg chamber. At first glance, this phenotype looks very similar to that of ectopic activation of the HH pathway, either by overexpression of Hh or by loss of the negative regulators Ptc, Pka or Cos2. Indeed the ectopic polar cells that form in ptc, Pka or cos2 mutant clones lack Eya. However, ectopic Hh signaling has additional effects besides ectopic polar cell formation, whereas loss of Eya does not. Several different cell types are observed in the ptc, Pka or cos2 mutant clones. There are Eya-positive but Fas3-negative cells, which may correspond to differentiated epithelial follicle cells. There are also cells expressing both Eya and Fas3, which could be immature, undifferentiated precursor cells. Finally, there are the Eya-negative but Fas3-postive polar cells. In this study, it has been shown that the production of ectopic polar cells caused by ectopic activation of the Hh pathway occurs by repression of Eya (Bai, 2002).

But what is the normal relationship between Hh signaling and polar cell formation, and why does excessive Hh signaling generate ectopic polar cells as well as other cell types? To address these questions, the normal role of Hh signaling in the ovary has to be considered. Expression of Hh protein has been observed only in the terminal filament and cap cells at the extreme anterior tip of the germarium. The normal function of Hh appears to be to regulate somatic stem cell fate and proliferation. Loss of Hh signaling in somatic stem cells results in the loss of stem cell fate. Conversely, overexpression of Hh leads to overproduction of stem-cells. Despite the fact that ectopic expression of Hh leads to ectopic polar cells, Hh signaling does not appear to specify polar cell fate normally. The best direct evidence for that is that smo mutant cells, which cannot transduce Hh signals, are still capable of generating normal polar cells at normal positions. In addition, normal polar cells can develop in the absence of ci (Bai, 2002).

Why, then, does ectopic Hh signaling produce ectopic polar cells? It has been argued that excessive Hh signaling might maintain follicle cells, and the polar/stalk cell lineage in particular, in a precursor state for an abnormally long period of time. Thus, delayed specification of polar cells would permit more proliferation than usual in this lineage. This model might explain the presence of extra polar cells at the two poles of the egg chamber, where the polar cells normally reside. However, it does not explain the presence of ectopic polar cells elsewhere in the egg chamber, or why there are three different cell types present in the ptc, Pka and cos2 mutant clones. Based on the normal role of Hh in regulating proliferation and maintenance of stem cells and their immediate progeny, the prefollicle cells, it is proposed that ectopic Hh signaling might cause ectopic prefollicle cell fates within the epithelial follicle layer of early egg chambers. As these cells undergo further proliferation, and then differentiation, they produce the various follicle cell types observed in the ptc, Pka and cos2 clones. Additional, as yet unknown, signals might determine which specific fates the differentiating cells adopt. However, the normal mechanisms that function to coordinate follicle cell fates spatially are obviously lacking in the mutant clones, since the three types of cells appear in random locations relative to each other. This provides an explanation for how ectopic Hh signaling might produce polar cells all over the egg chamber, rather than only at the two poles of the egg chamber, where the polar/stalk precursors normally reside (Bai, 2002).

Ectopic Hh signaling produces numerous effects in the Drosophila ovary, which include regulating proliferation of somatic cells as well as specification of polar cells. Both of these effects appear to be achieved through the cell autonomous action of Ci. This raises the question of how different effects are elicited by the same signal. The data presented here indicate that ectopic Hh activates polar cell fate by repressing eya expression, the function of which is required to repress polar cell fate. Since loss of eya does not mimic ectopic Hedgehog in causing extra proliferation, it is not yet clear what factors act downstream of ectopic Hh to affect proliferation (Bai, 2002).

The relationship between Eya and Ci is not a simple linear one. Although Eya expression is repressed by Ci^AC, mutations in eya also alter the balance between Ci^AC and Ci^R, without affecting overall ci expression. Ci^AC is upregulated in eya mutant follicle cells. In addition, some of the ectopic polar cells in eya mosaic egg chambers express ptc-lacZ, which is an indicator for activation of Ci. Thus, there appears to be mutual repression between Ci^AC and Eya. One place in the mammalian embryo where a similar relationship between Ci and Eya might exist is in patterning the eye field. Hh is normally expressed at the midline where it represses eye development. In the absence of Hh, a single cyclopic eye forms at the midline. The three mammalian homologs of Eya are all expressed in the eye primordium and therefore it may be that the antagonism between Hh and Eya revealed in this study is also employed in the mammalian embryo to repress midline eye development (Bai, 2002).

It is clear that the effect of the ectopic Hh signaling on the specification of the polar cell fate is through the repression of Eya. What still remains unknown is the spatially localized signal that normally represses Eya expression in polar and stalk cells. Since Notch signaling is necessary, but not sufficient, to define polar cells, it is likely that there is an additional, spatially localized signal required for specifying polar cell fate. Clearly, Eya is a key regulator that represses polar and stalk cell fates. Whatever the regulatory signal that normally specifies polar cell fate, it must regulate Eya expression to determine a polar versus non-polar cell fate in the follicular epithelium (Bai, 2002).

Effects of Mutation or Deletion

In eya mutants, progenitor cells divide normally. The amount of cell division is similar to that in the region anterior to the furrow in normal eye discs at the same stage. However cells in mutant eye discs undergo programmed cell death anterior to (prior to passing of) the morphogenetic furrow, rather than proceeding along the normal developmental pathway to retinal differentiation. A low level of cell death normally occurs at this stage, suggesting that eya activity influences the distribution of cells between differentiation and death. In eye discs of larvae expressing intermediate and severe allele combinations of eya, where very reduced numbers of ommatidia form, the clusters develop in the posterior-most region of the eye disc. Eye discs of larvae expressing intermediate allele combinations show cell division both anterior to the furrow and in a band posterior to the furrow, reflecting aspects of pattern formation seen in normal eye discs. In allelic combinations producing a sever phenotype, no furrow is seen, no clusters differentiate, and cell death increases dramatically. (Bonini, 1993).

Sine oculis and Eyes absent have been found to form a complex and to regulate multiple steps in Drosophila eye development. So and Eya (1) regulate common steps in eye development including cell proliferation, patterning, and neuronal development; (2) they synergize in inducing ectopic eyes and (3) interact in yeast and in vitro through evolutionarily conserved domains. Clones of so and of eya mutant cells overproliferate and fail to differentiate into neurons. Mutant clones retain their epithelial organization and lead to abnormal folding of the disc. The previously reported cell death phenotype is a secondary result of cell overgrowth. It is concluded that both so and eya play a role in controlling proliferation in the eye primordium of the eye-antennal disc and may therefore contribute to regulating the size of the disc (Pignoni, 1997).

Morphogenetic furrow (MF) initiation does not occur in so and eya mutant clones. decapentaplegic expression is not detected in mutant so and eya clones. Development of eye tissue in the posterior and lateral regions but not the anterior region of so and eya mutant discs is rescued by ectopic expression of the respective genes. Rescue is restricted to the most posterior region of the mutant discs, leading to the conclusion that so and eyaare required during MF formation. so and eya are also required for neuronal development. Homozygous mutant cells were produced selectively posterior to the MF. These mutant clones result in absence of a significant number of photoreceptors, precisely those that are known to be born late under normal conditions, corresponding to the time of so and eya mutant clone induction (Pignoni, 1997).

Ectopic expression of so has little or no effect on antennal, wing, or leg disc development, while ectopic eya expression often causes mild growth alterations resulting in extra folds in the epithelium and, rarely, formation of small ectopic ommatidial arrays in the antennal disc. Coexpression of so and eya leads to a dramatic increase in the development of ectopic eye tissue in antennal discs. These ommatidial arrays lead to adult eye structures. Ectopic so/eya induce eyeless expression in the antennal disc. When expressed in eyeless mutant discs, ectopic so/eya produces growth alterations, but ectopic eyes are not observed (Pignoni, 1997).

In clift mutants, germ cells scatter throughout posterior regions of the embryo and germ cells do not coalesce into a gonad. One clift mutant, which acts as a strong allele, nevertheless produces a transcript. Use of this mutant allowed an examination of SGP cell development in the absence of clift function. Shortly after SGP cells are specified in cli mutants there is a reduction in the number of cli expressing SGP cells, such that by germ band retraction, few SGP cells remain. The few cells that remain fail to complete their migration to the position of gonad formation (Boyle, 1997).

In eyes absent (eya) mutants, eye progenitor cells undergo cell death early in development. Whereas the phenotype of eya1 is limited to the eye, other mutations are lethal. Genetic and molecular analysis reveals that mutations in one region of the gene cause embryonic lethality, whereas mutations throughout the gene cause defects in eye development. Mosaic analysis indicates that the eya requirement is cell autonomous. In eye-specific mutants, expression in the eye disc is lacking while embryonic expression is normal. Both the type I and type II transcripts are expressed in the developing eye, and expression of either can rescue the eye phenotype. The normal embryo shows selective expression of type II transcript. The requirement for eya defined by the eye-specific mutant alleles is not only intrinsic to eye progenitor cells, but also occurs after the completion of embryogenesis. These data indicate a specific requirement for eya function in eye progenitor cells that is normally fulfilled by both transcripts. The fine map of the eya eye and embryonic phenotypes reveals that disruptions at any location within the gene have similar effects on eye development, but that disruptions in the 3' region preferentially affect the ability of animals to survive embryogenesis. These results suggest that the embryonic function of the eya gene, encoded by the type II transcription unit and the cis-regulatory regions necessary for embryonic expression, are located distal to the XI breakpoint, 7 kb upstream from sequences shared by type I and type II transcripts. It is suggested that the regulatory sequences important for expression in the eye disc affect both transcrpts similarly, while those critical for embryonic expression are preferentially associated with the type II transcriptions start site, located 11 kb upstream of the type I start site (Leiserson, 1998).

The receptor tyrosine kinase (RTK) signaling pathway is used reiteratively during the development of all multicellular organisms. While the core RTK/Ras/MAPK signaling cassette has been studied extensively, little is known about the nature of the downstream targets of the pathway or how these effectors regulate the specificity of cellular responses. Drosophila yan is one of a few downstream components identified to date, functioning as an antagonist of the RTK/Ras/MAPK pathway. Ectopic expression of a constitutively active protein (yan^ACT) inhibits the differentiation of multiple cell types. In an effort to identify new genes functioning downstream in the Ras/MAPK/yan pathway, a genetic screen was performed to isolate dominant modifiers of the rough eye phenotype associated with eye-specific expression of yan^ACT. Approximately 190,000 mutagenized flies were screened, and 260 enhancers and 90 suppressors were obtained. Among the previously known genes recovered are four RTK pathway components [rolled (MAPK), son-of-sevenless, Star, and pointed], and two genes (eyes absent and string) that have not been implicated previously in RTK signaling events. Mutations in five previously uncharacterized genes were also recovered. One of these, split ends, has been characterized molecularly and is shown to encode a member of the RRM family of RNA-binding proteins (Rebay, 2000).

eyes absent (eya may be relevant to the RTK pathway, basis of these genetic tests. eya encodes a novel nuclear protein of unknown function that functions in a hierarchy of 'master eye regulatory genes' that are required to specify and promote differentiation of eye tissue. However, on the basis of expression pattern and phenotypes, it is possible that eya plays additional roles in development independent of its role in determining competence to become eye tissue. One possibility is that eya could be directly complexed with yan, and could direct its transcriptional repressor activity in certain tissues. However, preliminary yeast two-hybrid experiments have failed to indicate Yan-Eya protein-protein interactions. An alternate possibility to be investigated is transcriptional regulation of eya by Yan. Given the genetic interactions observed between eya and yan^ACT, it will be interesting to investigate the possible role of eya in RTK/yan-mediated signaling events in the embryo and developing eye. It could be that in order to differentiate as eye tissue, a developing cell must receive both a 'general' differentiation signal from the RTK pathway and a more specific eye fate specification signal (Rebay, 2000 and references therein).

The eyes absent gene is critical for normal eye development in Drosophila and is highly conserved to vertebrates. To define regions of the gene critical for eye function, the mutations in the four viable eya alleles have been defined. Two of these mutations are eye specific and undergo transvection with other mutations in the gene. These are deletion mutations that remove regulatory sequences critical for eye cell expression of the gene. Two other viable alleles cause a reduced eye phenotype and affect the function of the gene in additional tissues, such as the ocelli. These mutations are insertion mutations of different transposable elements within the 5' UTR of the transcript. Detailed analysis of one of these reveals that the transposable element has become subject to regulation by eye enhancer sequences of the eya gene, disrupting normal expression of Eya in the eye. More extensive analysis of the deletion region in the eye-specific alleles indicates that the deleted region defines an enhancer that activates gene expression in eye progenitor cells. This enhancer is responsive to ectopic expression of the eyeless gene. This analysis has defined a critical regulatory region required for proper eye expression of the eya gene (Zimmerman, 2000).

The eya¹ and eya² alleles are highly selective for loss of the eye. Moreover, they complement all functions of the eya gene except the eye phenotype. Detailed analysis of the expression pattern of eya reveals that these alleles are normal for eya gene expression, except in the eye where they show complete loss of eya expression in progenitor cells for the compound eye, with normal ocellar expression. These alleles show interallelic complementation with other eya alleles, including the eya⁴ allele. This complementation reflects transvection, also known as pairing-dependent complementation. Transvection reflects interactions between the chromosomal homologs, such that two alleles with different types of mutations can partially complement, leading to restoration of gene function. One model for transvection is that one class of mutations defines a regulatory element required for proper gene expression, whereas the complementing class is in the transcription unit. The eya¹ and eya² alleles, being highly specific for loss of gene function in the eye, are candidates to define regions of the eya gene critical for eye progenitor cell expression (Zimmerman, 2000).

Two alleles, both spontaneous alleles that were independently isolated, have deletions within the same region of the eya gene. The eya¹ allele defines an approximately 1.5-kb deletion, which was not analyzed further, whereas the eya² allele is a small 322-bp deletion. The region deleted in eya² activates gene expression in eye progenitor cells, indicating that this is a DNA element that is both necessary and sufficient for expression in eye progenitor cells. The expression pattern in the eye reflects that seen of the Eya protein, being broadly expressed within the entire eye field. Further analysis of the expression pattern revealed that indeed the element appears to be selective for eye progenitor cells, failing to be expressed in other tissues where the eya gene is expressed (Zimmerman, 2000).

A test was performed to see whether this element displays a response to directed expression of the ey gene, which has been shown to direct ectopic eye formation and Eya expression. This regulatory domain of eya indeed responds to ectopic ey expression, consistent with previous studies of Eya protein. This suggests that the enhancer reflects aspects of the normal regulatory pattern of the eya gene in eye formation. No evidence has been found that this region responds to ectopic expression of the eya gene itself, however, evidence does suggest that it is not a domain involved in a potential autoregulatory loop. Within this region are several intriguing protein binding elements, including potential ETS binding domains and a Sine oculus binding site; their possible significance awaits further analysis (Zimmerman, 2000).

A Sine oculis/Eyes absent complex regulates multiple steps in eye development and functions within the context of a network of genes to specify eye tissue identity. Ectopic expression of so alone does not induce ectopic eyes, and ectopic expression of eya alone induces ectopic eyes just in the antenna at low frequency (10%), but coexpression of so and eya leads to an increase in the induction of ectopic eyes in the antenna both in frequency (76%) and size. This synergistic effect is probably due to the capability of So and Eya to form a protein complex. The domains required for complex formation are the evolutionarily conserved Six and Eya domains. Since Optix has a Six domain as well, a test was performed to see whether Optix and Eya also synergize and enhance ectopic eye induction. UAS-eya;UAS-Optix was crossed to dpp ^blink-GAL4 and the frequency of induction of ectopic eyes was examined. Optix can induce ectopic eyes (22%) but so cannot (0%); so has a synergistic function with eya (0% and 10% individually, to 60% when coexpressed), but coexpression of Optix and eya does not lead to an increase in frequency (20%) nor in size of ectopic eyes. Therefore, although Optix has a Six domain, no synergistic interaction with Eya has been demonstrated (Seimiya, 2000).

The eyes absent gene is critical to eye formation in Drosophila; upon loss of eya function, eye progenitor cells die by programmed cell death. Moreover, ectopic eya expression directs eye formation, and eya functionally synergizes in vivo and physically interacts in vitro with two other proteins of eye development, Sine oculis and Dachshund. The Eya protein sequence, while highly conserved to vertebrates, is novel. To define amino acids critical to the function of the Eya protein, eya alleles have been sequenced. Loss of the entire Eya Domain is null for eya activity, but alleles with truncations within the Eya Domain display partial function. The molecular genetic analysis was extended to interactions within the Eya Domain. This analysis has revealed regions of special importance to interaction with Sine Oculis or Dachshund. Select eya missense mutations within the Eya Domain diminish the interactions with Sine Oculis or Dachshund. Taken together, these data suggest that the conserved Eya Domain is critical for eya activity and may have functional subregions within it (Bui, 2000b).

Mutants with premature terminations within the Eya Domain have been found. Surprisingly, none of these alleles appears to be fully embryonic lethal; rather, all retain some embryonic activity. These alleles leave intact the first part of the Eya protein, EF1, which may be of special importance for interactions with So/Six proteins. Thus, rather than the entire Eya Domain being essential for all aspects of Eya activity, there may be regions within the Eya Domain with critical roles at particular developmental times or in specific tissues. Interestingly, So and other Six gene family members in the fly show strong expression or function in the embryo in locations that may overlap with part of the Eya expression pattern. Potentially, interactions with So or other Six genes may be important in the embryonic function of Eya (Bui, 2000b).

A detailed analysis was performed of the Eya Domain, dividing the domain into three regions, two of which display some selective interactions. On the basis of the missense mutations found within the protein and conservation of the protein with other species, the Eya Domain was subdivided into three parts. The first region, EF1, shows selective interaction (albeit weak) with So -- an interaction that is disrupted by a point mutation in the EF1 Domain. The second domain, EF2, shows interaction with Dac; this interaction, too, is selectively interfered with by a point mutation in the EF2 Domain. The Dac protein has two domains of high conservation with vertebrate proteins: an N-terminal domain called Dachbox-N and a C-terminal domain called Dachbox-C. These domains are predicted to have structural homology with the Ski/Sno family of genes, with Dachbox-C having a potential role in dimerization. Dachbox-C interacts with the Eya-conserved Domain, with the EF2 subdomain perhaps being of special importance. No selective interaction by these studies has been revealed with the third domain of Eya, although this domain is highly conserved in vertebrates, and also highly conserved in the nematode C. elegans. C. elegans appears to have a dac homolog as well as an eya homolog; it will be of interest to determine whether the nematode Dac homolog interacts with the C. elegans Eya homolog. One point mutation found within EF3 appears critical for the function of the Eya Domain: a G (glycine) to E (glutamate) mutation at residue 723 in eya^IR3. This mutation appears to be more severe than those with premature termination within the Eya Domain, perhaps indicating that this mutation generates an unstable form of the protein subject to degradation. Overall, interactions between the mutant forms of the Eya Domain and the EF subdomains with the So and Dac proteins were considerably weaker than with the wild-type Eya Domain. This may suggest that, despite some evidence for selective interactions, both Eya Domain mutations -- either due to a requirement for additional protein domains or to a three-dimensional structure conferred by the smaller region in the context of the entire Eya domain -- may disrupt interactions to some degree with both protein partners in vivo. This might contribute to the weaker activity of these Eya mutant forms in vivo in restoring eye development to eya mutants (Bui, 2000b).

This analysis of the mutations in the Eya Domain was extended to the situation in vivo by generating transgenics expressing the selective point mutants that disrupt interactions with So and Dac in the yeast two-hybrid system. Although this has failed to provide evidence in support of a special functional relevance of the Dac interaction (both mutant forms appeared to interact similarly in ectopic eye formation upon coexpression with Dac) evidence has been found supporting the importance of the So interaction. These data indicate that the Eya^E11 mutant form shows a diminished ability to synergize with So upon coexpression. This supports the hypothesis that the eya^E11 mutation within the Eya Domain disrupts interactions in vivo with so (and/or possibly with other Six homologs) that are critical for the function in eye formation. The Eya^E7 mutant form, which shows a disrupted Dac interaction, still supports ectopic eye formation, although at decreased penetrance compared to normal Eya. dac null mutations frequently show some degree of eye development, suggesting that dac may be partially redundant in eye formation. Therefore, even if interaction with Dac in vivo were disrupted by the eya^E7 mutation, eye formation might still occur due to compensation by such mechanisms. Nevertheless, this eya allele also shows a dominant reduced-eye phenotype when coexpressed with so -- this is a new property not observed with the wild-type Eya protein. The eya^E7 mutation may generate a protein with some dominant-negative property in eye formation. The data that So and Dac may interact, in part, differentially within the conserved domain of Eya supports the idea that the three proteins have the potential to interact in a single complex in vivo. Such a hypothesis, however, is complicated by other data indicating that the molecular activity of Eya-So coexpression in eye formation is at least in part distinct from that of Dac or Eya-Dac coexpression: whereas Dac, and Eya coexpression with Dac, activate an eya enhancer, Eya alone or Eya with So fails to activate enhancer activity, despite ectopic eye formation (Bui, 2000b).

In the Drosophila visual system, photoreceptor neurons (R cells) extend axons towards glial cells located at the posterior edge of the eye disc. In gilgamesh (gish) mutants, glial cells invade anterior regions of the eye disc prior to R cell differentiation and R cell axons extend anteriorly along these cells. gish encodes casein kinase Igamma. gish, sine oculis, eyeless, and hedgehog (hh) act in the posterior region of the eye disc to prevent precocious glial cell migration. Targeted expression of Hh in this region rescues the gish phenotype, though the glial cells do not require the canonical Hh signaling pathway to respond. It is proposed that the spatiotemporal control of glial cell migration plays a critical role in determining the directionality of R cell axon outgrowth (Hummel, 2002).

A set of genes encoding nuclear proteins [e.g., eyeless (ey), eyes absent (eya), sine oculis (so) and secreted factors such as hedgehog (hh)] regulates the initiation of neuronal differentiation in the posterior region of the eye disc. The effect of loss-of-function mutations in these genes on glial cell migration was tested. As in gish mutants, glial cells migrate precociously out of the optic stalk in a hh temperature-sensitive mutation incubated at the nonpermissive temperature during first and second instar. This is an early function of hh, since ectopic glial cells are not observed in hh¹; in this allele, the posterior eye field develops normally, but anterior progression of the MF is inhibited. A similar early onset glial cell migration defect is observed in eye-specific alleles of so and ey. In contrast, glial cells did not migrate out from the optic stalk in an eye-specific allele of eya, raising the possibility that eya is required to activate glial cell migration. Since glial cells migrate out of the stalk precociously in eya/gish double mutants, the production of an eya-dependent signal is not necessary to promote anterior migration. Hence, in contrast to their role in R cell development, eye specification genes ey and so seem to function independent of eya to control the onset of glial cell migration (Hummel, 2002).

A somatic role for eyes absent and sine oculis in spermatocyte development

Interactions between the soma and the germline are a conserved feature of spermatogenesis throughout the animal kingdom. The transcription factors eyes absent (eya) and sine oculis (so) are each required in the somatic cyst cells of the testis for proper Drosophila spermatocyte development. eya mutant testes exhibit degenerating young spermatocytes. Mosaic analysis reveals a somatic requirement for both eya and so, in that neither gene is required in the germline for spermatocyte development. Immunolocalization analysis supports this somatic role, since both proteins are localized within cyst cell nuclei as spermatocytes differentiate from amplifying spermatogonia. Using antibodies against known cyst cell markers, it has been demonstrated that cysts of degenerating spermatocytes in eya mutant testes are encysted, ruling out a role for eya in cyst cell viability. A genetic interaction has been uncovered between eya and so in the testis, suggesting that, as in the eye, eya and so may form a transcription complex responsible for the activation of target genes involved in cyst cell differentiation and spermatocyte development (Fabrizio, 2003).

While this study has uncovered a somatic role for eya and so in spermatocyte development, no spermatocyte defect was found prior to cell death. Amplifying gonial cells in eya^3cs/Df testes appear morphologically indistinguishable from wild-type gonia and exhibit branched fusomes, indicating that spermatogonia in eya mutant testes undergo amplification as in wild-type. Moreover, spermatogonia in eya mutant testes express cytoplasmic Bam protein, a faithful marker of amplifying gonia in wild-type testes. Taken together, these data suggest that spermatogonial differentiation is unaffected in eya mutant testes. However, it should be noted that eya^3cs/Df testes are not null for eya function. Thus, the residual Eya protein might prevent earlier defects. To address this issue, degenerating spermatocytes from testis containing unmarked eya^IIE clones were examined. In 4 of 8 degenerating cysts, 16 spermatocytes were counted. This confirms that depletion of eya does not necessarily affect gonial development, though it leads to spermatocyte degeneration. Four other degenerating cysts contain less than 16 spermatocytes, yet all contain greater than 10. Either all degenerating cells within these cysts could not be counted or some spermatocytes fully degenerated before the testes were harvested. In either event, since all degenerating cysts progressed pass the third mitotic division, depletion of eya does not appear to perturb germline development prior to spermatocyte differentiation (Fabrizio, 2003).

Since in a clonal analysis, cysts of degenerating spermatocytes are very young, an early requirement for both eya and so during spermatocyte development is inferred. This finding is distinct from earlier work on both the spermatocyte arrest class of genes and genes required for meiotic entry. In both classes of mutants, spermatocytes form and mature, as judged by morphology and marker analysis, but fail to initiate meiosis. Given that cysts of degenerating spermatocytes in presumed eya^null cyst cell clones are small and adjacent to the spermatogonia-to-spermatocyte transition zone, clonal analysis suggests that spermatocytes degenerate prior to maturation. Thus, eya and so may act temporally prior to or in parallel to the spermatocyte arrest and meiotic entry pathways. Indeed, further support for this proposition comes from the observation that Eya expression in cyst cells is normal in mutants such as spermatocyte arrest, cannonball, and always early (Fabrizio, 2003).

Proper spermatocyte development requires Eya/So function in the somatic cyst cells. One formal possibility is that removal of Eya/So function from the cyst cells leads to cyst cell death, subsequently resulting in spermatocyte degeneration. However, the data indicate that degenerating spermatocytes are encysted, suggesting that Eya/So is involved in gene regulation within the soma. Putative targets of Eya/So in the cyst cells are unknown, but two major possibilities exist. Their interaction might serve to promote spermatocyte development indirectly, by promoting cyst cell differentiation, or directly, by regulating a signal to the germline. Given that cyst cells progress through developmental stages marked by differential gene expression, the first possibility is favored. The target genes of Eya/So within the soma that promote cyst cell differentiation are unknown. In addition, since the somatic cells are essential for continued spermatocyte development and viability, the signal(s) from the somatic cyst cells to the germline may be among Eya/So targets. Thus, Eya and So represent a useful starting point for elucidating players involved in further cyst cell as well as spermatocyte development (Fabrizio, 2003).

Interestingly, previous studies have uncovered a somatic role for eya in the gonad prior to its expression in testis cyst cells. During gonadogenesis in Drosophila, eya functions independently of so and is required in the somatic gonadal precursor (SGP) cells, which are the precursors of both the hub and the cyst cells of the adult testis. Thus, eya is required independently of so during the initial development of the somatic cells of the testis, and only later in maturing cyst cells. Therefore, as in eye development, where eya functions with different partners at several time points within the same tissue, eya is redeployed after gonad formation to function synergistically with so during spermatogenesis (Fabrizio, 2003).

Taken together, the observed interaction between eya and so during spermatocyte development is consistent with other developmental pathways that do not involve the canonical members of the eye-specification cascade. Moreover, a genetic interaction does not necessarily dictate a physical interaction and eya and so might be functioning in separate, but parallel pathways in the cyst cells. Thus, future experiments will aim to identify other players in this potentially unique signaling cascade (Fabrizio, 2003).

Independent roles of the dachshund and eyes absent genes in BMP signaling, axon pathfinding and neuronal specification

In the Drosophila nerve cord, a subset of neurons expresses the neuropeptide FMRFamide related (Fmrf). Fmrf expression is controlled by a combinatorial code of intrinsic factors and an extrinsic BMP signal. However, this previously identified code does not fully explain the regulation of Fmrf. The Dachshund (Dac) and Eyes Absent (Eya) transcription co-factors participate in this combinatorial code. Previous studies have revealed an intimate link between Dac and Eya during eye development. Here, by analyzing their function in neurons with multiple phenotypic markers, it is demonstrated that they play independent roles in neuronal specification, even within single cells. dac is required for high-level Fmrf expression, and acts potently, together with apterous and BMP signaling, to trigger Fmrf expression ectopically, even in motoneurons. By contrast, eya regulates Fmrf expression by controlling both axon pathfinding and BMP signaling, but cannot trigger Fmrf ectopically. Thus, dac and eya perform entirely different functions in a single cell type to ultimately regulate a single phenotypic outcome (Miguel-Aliaga, 2004).

Phenotypic and transcriptional synergy between So, Dac and Eya during development and in vitro has been well documented. By contrast, the current results indicate that these genes can act independently in the embryonic nervous system to specify neuronal identity. This is the case even when they are coexpressed in the same neuron; while no evidence of so expression was found in the ap-cluster, dac and eya functioned together with the previously identified ap/sqz/BMP combinatorial code to activate Fmrf expression in Tv neurons. However, eya controls additional aspects of Tv neuronal identity, such as axon pathfinding and the ability to respond to a BMP signal. Furthermore, the expression of Dac, but not Eya, So or Ap, in a large number of interneurons has suggested that Dac has additional, independent functions in postmitotic neurons (Miguel-Aliaga, 2004).

The molecular mechanisms underlying transcriptional synergy between So (Six), Eya and Dac (Dach) have proven to be quite complex. In most cases examined, So/Six binds DNA and Dac/Dach and Eya regulate its activity. These biochemical models would not appear to explain the current observations fully. In these studies, Dac appears to act as a potent activator of Fmrf expression but to rely on Eya for activating Fmrf expression only within ap-neurons; when dac and ap are co-misexpressed in all neurons there is widespread ectopic Fmrf expression without any ectopic Eya expression. Why Eya is required in the ap-neurons for both endogenous and ectopic Fmrf expression, but not for ectopic Fmrf expression outside ap-neurons, is currently unclear (Miguel-Aliaga, 2004).

The current findings illustrate the fact that regulators acting within a postmitotic neuron can act together in a combinatorial fashion to specify one aspect of neuronal identity (Fmrf expression, in this case). However, some of these regulators can simultaneously function in combinatorial sub-codes to control other aspects of neuronal identity; the additional roles of ap and eya in Tv axon pathfinding may be one such example. In abdominal hemisegments, Ap is expressed in the two vAp and the single dAp neurons. Normally, the axons of these neurons join a common ipsilateral longitudinal fascicle running the length of the VNC. Previous studies have revealed that ap is important for proper ap-axon fasciculation as well as for their avoidance of the midline. Eya is not expressed in vAp neurons, and the results indicate that it specifically controls dAp pathfinding. The eya mutant phenotype only partially phenocopies the ap phenotype, since eya affects midline crossing but not fasciculation; once dAp neurons have aberrantly crossed the midline they join the contralateral ap-fascicle. Neither the ap nor the eya mutant phenotypes are due to any apparent crossregulation between these two genes. Surprisingly, these findings indicated that different genetic mechanisms underlie the indistinguishable, ap-dependent axon pathfinding of dAp and vAp neurons; dAp axons crucially depend upon eya to avoid crossing the midline, whereas vAp axons neither express eya nor depend upon it (Miguel-Aliaga, 2004).

Together with previous findings these results indicate that Fmrf expression is triggered by the combinatorial action of ap, sqz, dimm, dac, eya and BMP signaling. However, with the exception of BMP signaling, none of these factors are absolutely necessary for endogenous Fmrf expression - in all mutants, expression of Fmrf is not lost from all Tv neurons. Similarly, although misexpression of a partial code can lead to ectopic Fmrf expression, its expression levels are consistently weaker than those seen in Tv neurons. Thus, it appears that a partial code is sufficient for some level of Fmrf expression: the ectopic expression of Fmrf in BMP-positive RP neurons (cells that do not express sqz, eya or dimm) in response to dac and ap is one such example. However, the complete code (ap/sqz/dimm/dac/eya/BMP) appears to be necessary for wild-type (high) levels of expression, as seen in the Tv neurons. It is possible that the simultaneous misexpression of all these factors would lead to robust ectopic Fmrf expression in all neurons. Due to obvious technical limitations, this idea has not been tested (Miguel-Aliaga, 2004).

Multiple signal transduction inputs/outputs appear to revolve around Eya: (1) phosphorylation of Eya by the Ras/MAPK pathway has been found to regulate Eya activity and synergy with So; (2) the transcriptional activity of Eya itself depends upon an intrinsic tyrosine phosphatase activity that is also required for ectopic eye induction in Drosophila. The target(s) of Eya phosphatase activity are currently unknown. (3) It is found that Eya regulates the BMP pathway in Tv neurons and pMad cannot be reactivated in eya mutants even by cell-autonomous introduction of the BMP ligand Gbb. A probable explanation for this result is that eya regulates the expression or activity of the BMP type receptors Wit, Tkv or Sax. When the BMP pathway is dominantly activated by the use of activated type I receptors, nuclear pMad is restored. However, this still does not reactivate Fmrf expression, indicating that Eya additionally plays important roles downstream of pMad activation. One interpretation of these findings is that Eya acts directly on the Fmrf gene. However, it is also tempting to speculate that Eya may act to modulate pMad activity directly. There are several reasons for this proposal. It is known that several other kinase pathways, such as MAPK, can phosphorylate Smad proteins on residues other than those phosphorylated by TGFß/BMP type I receptors. The in-vivo roles of such modifications are unclear, but in-vitro evidence points to both repression and activation of Smad activity. Nevertheless, given its nuclear localization and phosphatase activity, it is possible that Eya acts to de-phosphorylate inhibitory residues in pMad. A regulatory circuitry between MAPK (and other kinases), Eya and the TGFß/BMP pathway is an intriguing possibility. Moreover, recent studies reveal that vertebrate orthologs of Dac can physically interact with the Smad complex, thereby affecting TGF-ß signaling. Together with these previous findings, the current results point to a model wherein Eya and Dac play central roles in integrating input from, and controlling the activity of, multiple signal transduction networks. Determination of the precise mechanisms by which Eya and Dac orchestrate these events should enhance understanding of how both intrinsic and extrinsic signals intersect to affect cellular differentiation (Miguel-Aliaga, 2004).

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date revised: 10 August 2009
 

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