sine oculis

DEVELOPMENTAL BIOLOGY

Embryonic

Sine oculis immunoreactivity commences at the onset of the third instar larva before the morphogenetic furrow has formed, slightly later than eyes absent, the cell survival and differentiation gene. SO is found in a gradient increasing from anterior to posterior. In null mutants, the optic primordium fails to invaginate. optomotor blind transcript is present in mutants. Bolwig's organ is absent in mutants (Cheyette, 1994).

To investigate the functional roles of D-Six3 and D-Six4, two genes related to sine oculis, expression patterns were of all three genes were analyzed throughout embryogenesis by whole-mount in situ hybridization. Transcripts of both genes are first detectable in the anterior region of the early cellular blastoderm stage embryos, suggesting important roles in head development. In the case of D-Six3, expression is observed within the sharply defined circumferential stripe at approximately 85%-95% egg length (EL). This patch is somewhat wider dorsally (~6 cells) than ventrally (~4 cells). Also expression levels are higher on the dorsal side of this domain from which several head structures derive (Seo, 1999).

The D-Six4 gene is initially expressed in a dorsal patch that straddles the midline between 85%-90% EL. This patch is wider dorsally (3-4 cells) than towards its lateral edges. Hence, at the cellular blastoderm stage, D-Six4 appears to be expressed within a subregion of the D-Six3 expression domain. Interestingly, the D-Six4 expression pattern is also similar to that of so, which is expressed in a dorsal domain of the head region during the blastoderm stage. However, the area of so expression is located in a more posterior position where the primordia of the optic lobe, Bolwig's organ and eye disc are located (Seo, 1999).

Based on a projection onto the blastoderm fate map, D-Six3 is probably expressed in the region that will give rise to clypeolabrum, pharynx and the anterior part of the acron. Hence, D-Six3 and D-Six4 are apparently co-expressed in parts of the procephalic neurectoderm from which the brain originates. This conclusion is partially based on comparisons with the expression pattern of the gap gene tailless. Like D-Six3, tll is expressed within a circumferential band at 76%-89% EL from which the procephalic proneural domain is thought to be derived. In accordance with the apparent overlap (~ 5%) in expression relative to tll, the D-Six3 (and D-Six4) positive area is likely to include only an anterior part of this proneural region (Seo, 1999).

During gastrulation germ band elongation, D-Six3 continues to be expressed in the cephalic region but the pattern becomes more complex. The area and level of expression both increase dorsally, whereas ventrally, the expression fades and disappears before stage 9. After initiation of stomodeal invagination, the expression domain is separated into two major subdivisions and correlates spatiotemporally with the appearance of the clypeolabrum as a distinct part of the procephalic lobe. The clypeolabrum expression domain at the anterior end is quite uniformly stained with sharply defined borders. Further to the posterior, the labeled area, which maps mainly to the procephalic neurogenic region, has more complex features, and is connected to the clypeolabral domain dorsally. Within this procephalic region the staining is distributed along the dorsal midline and in two bilaterally paired areas, with the highest signal intensity in the domains closest to the midline (Seo, 1999).

As the germ band retracts during stage 12, expression is maintained in the clypeolabral region and in two bilateral areas associated with the newly formed supra-esophageal ganglia. Simultaneously, additional sites of expression become detectable ventrally in the maxillary and labial segments. Weaker expression is also present dorsolaterally in two small cell clusters located in the region which includes the optic lobe and dorsal ridge primordia (Seo, 1999).

By stage 13-14, when the germ band is fully retracted, parts of the clypeolabrum that express D-Six3 have invaginated through the stomodeum and contributed to the roof of the pharynx. This staining extends posteriorly towards the supra-esophageal ganglia where the expression is most conspicuous. The strongest expression is present in the medial parts of the supra-esophageal ganglia and in a patch of cells in front of the brain that probably includes the frontal ganglion and the frontal connective of the stomogastric nervous system. In addition to the clypeolabral expression, two distinct spots of staining are located ventral to the stomodeum. These apparently represent cells of head sensilla such as the terminal and labial organs that derive from stained cell clusters detected at earlier stages in the maxillary and labial segments, respectively (Seo, 1999).

During head involution the clypeolabral staining also resolves into distinct spots that may correspond to the labral sensory complex and the clypeolabral disc. Similarly, a refinement of the expression pattern occurs in the pharyngeal and supra-esophageal regions. This staining in the roof of the pharynx becomes restricted to the area including the frontal ganglion and connective. Although a row of cells just above the pharynx, in the dorsal pouch are labeled, the eye-antennal disc precursors, which are known to be located in the lateral parts probably do not express the D-Six3 gene. Expression is maintained in the dorsal pouch even after formation of the eye-antennal disc, in which staining is still not detectable at stage 18. Expression is also absent in the eye-antennal discs of first instar larvae and the transcript level is reduced in the frontal ganglia and connnective, head sensory organs of and clypeolabral derivatives. However, compatible with a possible involvement of D-Six3 in later stages of eye development, expression is detected in the eye-antennal discs of third instar larvae (Seo, 1999).

A recently reported Drosophila homeobox gene, optix, has also been shown to be expressed in restricted areas of the embryonic head during stages 5-11 (Toy, 1998). Although only the homeobox is known for optix, the 100% nucleotide identity to D-Six3 and the similarity in expression patterns suggest that the two genes are the same (Seo, 1999).

As is the case for D-Six3, expression of the D-Six4 gene splits into two bilateral domains and persists in the dorsal part of the procephalic lobe during gastrulation and germ band elongation. By stage 9-10 transcripts are also detectable in mesodermal cells along the entire germ-band. In a dorsal view the mesodermal staining appears to consist of a bilateral pair of longitudinal bands connected by a series of transverse stripes whose spacing corresponds to that of the segmental primordia. These features are consistent with the known transient segmental characteristics of the mesoderm at this embryonic stage. However, the mesodermal transcripts disappear quite rapidly and by stage 11 only the procephalic expression is detectable (Seo, 1999).

Coincident with germ-band retraction and formation of the supra-esophageal ganglion stage 12, the two domains of dorsal D-Six4 expression narrow and lengthen. During stage 13 they become associated with dorsal and medial parts of the two brain hemispheres. By stage 15, additional sites of expression are observed in the ventral cord and gonads (Seo, 1999).

Judging from these differences in expression the functional roles of D-Six3, D-Six4, and so seem quite divergent. However, the subdivision of head neurectoderm into procephalic neurectoderm and head midline cells provides a basis for identifying conserved features. Notably, the head midline neurectoderm includes the anlagen of the medial brain, the stomatogastric nervous system, optical lobe, and larval eye. Since these different head midline domains correspond well with the sites where so, D-Six3, and D-Six4 show the strongest expression during embryogenesis, one important function of the common ancestral gene was probably associated with the development of the particular neural derivatives (Seo, 1999).

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).

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).

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 possible to know if the earliest cells that express so, eya, and dac will later become cells of the eye imaginal disc. How the expression patterns described it this study 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).

Embryonic development of the Drosophila corpus cardiacum, a neuroendocrine gland with similarity to the vertebrate pituitary, is controlled by sine oculis and glass

The development of the Drosophila neuroendocrine gland, the corpus cardiacum (CC) was investigated, along with the role of regulatory genes and signaling pathways in CC morphogenesis. CC progenitors segregate from the blastoderm as part of the anterior lip of the ventral furrow. Among the early genetic determinants expressed and required in this domain are the genes giant (gt) and sine oculis (so). During the extended germ band stage, CC progenitor cells form a paired cluster of 6–8 cells sandwiched in between the inner surface of the protocerebrum and the foregut. While flanking the protocerebrum, CC progenitors are in direct contact with the neural precursors that give rise to the pars intercerebralis, the part of the brain whose neurons later innervate the CC. At this stage, the CC progenitors turn on the homeobox gene glass (gl), which is essential for the differentiation of the CC. During germ band retraction, CC progenitors increase in number and migrate posteriorly, passing underneath the brain commissure and attaching themselves to the primordia of the corpora allata (CA). During dorsal closure, the CC and CA move around the anterior aorta to become the ring gland (see Image). Signaling pathways that shape the determination and morphogenesis of the CC are decapentaplegic (dpp) and its antagonist short gastrulation (sog), as well as hedgehog (hh) and heartless (htl; a Drosophila FGFR homolog). Sog is expressed in the midventral domain from where CC progenitors originate, and these cells are completely absent in sog mutants. Dpp and hh are expressed in the anterior visceral head mesoderm and the foregut, respectively; both of these tissues flank the CC. Loss of hh and dpp results in defects in CC proliferation and migration. Htl appears in the somatic mesoderm of the head and trunk. Although mutations of htl do not cause direct effects on the early development of the CC, the later formation of the ring gland is highly abnormal due to the absence of the aorta in these mutants. Defects in the CC are also caused by mutations that severely reduce the protocerebrum, including tailless (tll), suggesting that additional signaling events exist between brain and CC progenitors. The parallels between neuroendocrine development in Drosophila and vertebrates are discussed (De Velasco, 2004).

In the larva, the ring gland forms a large and conspicuous structure located anterior to the brain and connected to the brain by a pair of tracheal branches and the paired nerve of the corpus cardiacum (NCC). Three different glands, the corpus allatum (CA; dorsally), prothoracic gland (laterally), and corpus cardiacum (CC; ventrally) form part of the ring gland. By far, most of its volume is taken up by the prothoracic gland whose cells, the source of ecdysone, grow in size and number as larval development progresses, whereas the cells of the CC remain small and do not appear to proliferate. Both the CC and CA, as well as axons innervating the ring gland, are FasII positive from the late embryonic stage onward. Labeling of the CC is stronger and starts earlier (stage 11) than that of the CA (stage 15), which makes it easy to distinguish between the two structures in the embryo. Another convenient marker of the CC is adipokinetic hormone (AKH), which is expressed exclusively in the CC from late embryonic stages onward (De Velasco, 2004).

The ring gland of the mature embryo is situated posterior to the brain hemispheres. The CC and CA occupy their positions ventral and dorsal to the aorta, respectively. The prothoracic gland cannot yet be recognized as a separate entity, possibly due to the fact that its precursors are small and few in number. Cells of the CC number around eight on each side and are arranged in a U-shape around the floor of the aorta. All cells are spindle shaped and send short processes ventromedially where they meet and form a bundle attached to the ventral wall of the aorta (subaortic processes) (De Velasco, 2004).

The homeobox gene glass (gl) is expressed in the CC from stage 10 onward. Glass-positive CC precursors first appear as two pairs of cells located between the roof of the stomodeum and the inner surface of the brain primordium. Several populations of head mesoderm cells internalized during gastrulation as part of the anterior ventral furrow form a sheet of cells covering the inner surface of the brain primordium; the CC precursors form part of this cell group. Between stages 11 and 15, CC progenitors migrate posteriorly, gradually increasing in number (3–4 cells by stage 11; 6-8 cells by stage 13). The movement of the CC precursors parallels the invagination and elongation of the esophagus. During stages 11 and 12, the primordium of the stomatogastric nervous system (SNS) appears as three invaginating pouches in the roof of the esophagus. The CC precursors maintain a position laterally adjacent to the first SNS invagination on their posterior course. By stage 15, they have passed underneath the brain commissure and join up with CA precursor cells derived from the gnathal mesoderm to form the ring gland. On their migration, posteriorly the CC precursors are always in contact with the medial surface of the developing brain. One group of neurons transiently in contact with the CC precursors is the FasII-positive P3m cluster, which becomes part of the pars intercerebralis (PI) and is likely the source of some of the NCC axons innervating the CC. This close contact between PI and CC provides the opportunity for inductive interactions between the two structures (De Velasco, 2004).

A previously undescribed population of head mesoderm cells expressing the tinman (tin) gene represents another group of cells that surround the CC precursors during their migration. The Tin-positive cells, for which the term 'cephalic vascular rudiment' (CVR; an evolutionary vestige of the cephalic aorta which forms a prominent component of the vascular system in other insects) is suggested, form a loose cluster that extends backward dorsal of the esophagus and eventually establishes contact with the Tin-positive trunk aorta. The CC precursors are initially close to the anterior (trailing) end of the CVR, but they appear to 'catch up' and lead the CVR during later stages (De Velasco, 2004).

Based on reports from other insects, it had been anticipated that the CC is derived from the foregut as part of the invaginating stomatogastric primordium. However, this is not likely to be the case in Drosophila because the CC is present in embryos mutant for forkhead (fkh) in which both esophagus and SNS are eliminated. The expression and phenotype of numerous head gap genes were subsequently investigated to determine the origin of the CC. The results of this analysis indicate strongly that the CC originates from the anterior lip of the ventral furrow (AVF). The CC is deleted in mutations in the genes sine oculis (so), giant (gt), and twist/snail (twi/sna). Each of these genes is expressed in several domains at the blastoderm stage and during gastrulation, but the AVF is the only place of overlap between the three. Furthermore, giant expression, which is particularly strong in the AVF and persists slightly longer than expression of so or twi, visualizes the AVF cells as they spread out and form the anterior part of the head mesodermal layer that lines the inner surface of the brain primordium and includes the glass-positive CC precursors (De Velasco, 2004).

Besides sine oculis, giant, and twist/snail, one more head gap gene, tailless (tll) affects CC development. Tll is expressed in the anlage of the protocerebrum and only appears faintly, if at all, in the AVF. In tll mutant embryos, the CC is absent, whereas the SNS appears normal in size. It is speculated that the effect of tll on the CC is indirect, caused by the elimination of the protocerebrum (including the PI) in tll mutants. Another head gap gene, orthodenticle (otd), is expressed similarly to tll but leaves the CC intact. Otd mutant embryos also show a reduction in size of the protocerebrum but still possess the PI contacted by the CC precursors. Taken together, these findings (which need further follow-up analysis) hint at the possibility of inductive interactions between protocerebrum and CC (De Velasco, 2004).

In the embryo, glass is expressed in the primordium of the larval eye (Bolwig's organ), a small group of protocerebral neurons, and the CC precursors. Loss of gl in the allele gl² results in the absence of both larval eye and the CC, as shown in labelings with anti-FasII and AKH probe. Interestingly, this phenotype is a dominant effect since 75% of embryos derived from crossing balanced gl parents have no CC or larval eye. In stages 11 and 12 gl mutant embryos, the gl probe still gives a signal in CC and larval eye precursors. The signal becomes patchy (first in CC precursors, slightly later in larval eye) during stage 12 and has disappeared by stage 13. This finding suggests that gl is required for CC migration and/or differentiation, and that the absence of the CC in gl mutants as assayed for by the differentiation marker AKH is caused by transformation and/or apoptosis of initially correctly specified CC precursors (De Velasco, 2004).

Two other regulatory genes that were found to play an important role in vertebrate pituitary development, are Lhx3 and goosecoid (gsc). The Drosophila homologs of both of these genes are expressed in the SNS and possibly the ring gland. To investigate the role of Lim3 and gsc during CC development, their expression and phenotype were analyzed. Lim3 appears in the precursors of the SNS at a relatively late stage (stage 11), following the complete separation of these cells from the esophagus. In addition, lim3 is expressed in several small clusters in the brain primordium. Comparison with the expression of gl makes it clear that the Lim3 expressing cells are distinct from the CC progenitors. No CC defects were found in lim3 mutant embryos. Goosecoid is expressed in the SNS and, in late stage embryos, the CC. However, no CC defects were detected in the gsc^0534lacZ allele, which does cause structural abnormalities in the SNS. It is possible that gsc plays a role in later CC differentiation (De Velasco, 2004).

Several signaling pathways, notably Shh, BMP, and BMP antagonists, Wnt and FGF, specify the fate map of the head in vertebrates and also control later morphogenetic events shaping head structures. The same signaling pathways are active at multiple stages in Drosophila head development, and the pattern of activity and requirement of these pathways in regard to CC development was therefore investigated (De Velasco, 2004). .

The first signal acting zygotically in the Drosophila head is the BMP homolog Dpp, which forms a dorsoventral gradient across the blastoderm. The homolog of the BMP antagonist Chordin, short gastrulation (Sog), is expressed in the ventral blastoderm, overlapping with the ventral furrow. Loss of sog results in the absence of the CC, while the SNS is still present, which reflects ventral origin of the CC. Sog seems to be the only signal, of those tested, required for CC determination, since mutation of all other pathways does not eliminate the CC but merely effects its size, shape, or location (De Velasco, 2004).

Following its early widespread dorsal expression, Dpp becomes more confined during gastrulation to a narrow mid-dorsal stripe and an anterior cap that corresponds to parts of the anlagen of the esophagus and epipharynx. From this domain segregates the most anterior population of head mesoderm cells that give rise to the visceral muscle of the esophagus and which maintain Dpp expression. The visceral mesoderm of the esophagus flanks both CC and SNS. Loss of Dpp causes absence of the SNS; the CC is still present and expresses AKH but does not migrate posteriorly (De Velasco, 2004).

Both Hh and Wg are expressed from gastrulation onward in a similar pattern in the developing foregut. The pattern resolves into two domains, a posterior one covering the posterior esophagus, and an anterior one overlapping with the epipharynx. The esophageal domain, which shows a higher level of expression than the anterior domain, is located posterior to the precursors of CC and SNS. No significant abnormality in CC and SNS was obvious in Hh mutants. Wingless mutants show defects in the SNS but the CC is present, if misshapen and mislocalized, in the strongly distorted head of late wg mutant embryos (De Velasco, 2004).

Activity of the MAPK signaling pathway is widespread in the Drosophila head from gastrulation onward. Beside a wide anterior and posterior domain traversing the lateral and dorsal domain of the head ectoderm, the primordia of the foregut, including the SNS, and head mesoderm show a dynamic MAPK activity. At least two RTKs, EGFR and FGFR/heartless, drive the MAPK pathway in the embryonic head. EGFR is responsible for activation in the ectoderm and foregut. Loss of EGFR causes widespread cell death in the head and the absence of the SNS. The CC is still present, although reduced in size. Activation of MAPK by Heartless (Htl) occurs in a narrow anterior domain of head mesoderm that gives rise to the dorsal pharyngeal muscles. The foregut, SNS, and CC develop rather normally in htl mutants. However, the CC shows variable defects in shape and location, which are most likely due to the absence of the aorta and CA, both of which are derivatives of the dorsal mesoderm, which is defective in htl loss of function and to which the CC is normally attached (De Velasco, 2004).

This study has identified several early acting genes functioning in the development of the corpus cardiacum; among them sine oculis, giant, and glass are essential for its development. The apparent origin of the CC from the anterior ventral furrow, rather than the SNS placode as surmised in other studies, came as a surprise. In Manduca, CC precursors seem to delaminate from the posterior part of the neurogenic foregut ectoderm that gives rise to the SNS. In Drosophila, CC precursors are also close to the SNS placode as soon as they express the marker glass. Since this marker is not expressed during the segregation of CC precursors, it could not be directly observed from which ectodermal domain of the head they derive. It is therefore still possible that they originate from the SNS placode located in the roof of the foregut primordium. However, genetic data argue strongly against this possibility. Thus, the CC is present in a mutation of fkh, which is expressed and required in the foregut primordium and which is essential for the SNS. Similarly, the CC forms normally in mutations of EGFR, which entirely eliminate the SNS. By contrast, the CC is deleted in twist;snail and giant mutations, both of which are not expressed in the SNS placode and do not affect SNS development. The apparent discrepancy between Drosophila and Manduca indicates that the CC may originate from slightly different domains in different insect groups (the distance between presumptive SNS placode and anterior ventral furrow in the blastoderm is minimal); alternatively, the Manduca CC might also delaminate from the ventral furrow and only secondarily come to lie next to the SNS precursors (De Velasco, 2004).

The proposed origin of the CC from the anterior ventral furrow, which also gives rise to most of the anterior endoderm, underlines the close relationship between endodermal and neuroendocrine lineages. Such relationship also seems to exist in vertebrates. Numerous peptide signaling factors in vertebrates are expressed in cells of the digestive tract, in particular the pancreas, and the pituitary and/or hypothalamus. Among these are cholecystokinin (CKK), as well as the glucagon-like peptide (GLP) 1. GLPs and glucagon itself are the closest vertebrate counterparts to the CC-derived insect hormone AKH. Both AKH and glucagon, besides numerous other hormones released from the neuroendocrine system, coordinately control energy metabolism and behaviors associated with food uptake and processing. It is reasonable to assume that in the simple Bilaterian ancestor, cells that carried out the food uptake and digestive activities, that is, principal cells of the digestive tract, were identical with or spatially close to those cells that regulated these activities, among them endocrine and nerve cells (De Velasco, 2004 and references therein).

The pars intercerebralis/corpora cardiaca complex of insects has been repeatedly compared to the hypothalamus-pituitary axis in vertebrates. This comparison is usually based on clear similarities between the two on a gross anatomical and functional level. Thus, in both insects and vertebrates, neurosecretory neurons located in the anteromedial brain produce peptide hormones that are transported along axons to a peripheral gland. The axons either terminate on gland cells and modulate the release of glandular hormones, or they terminate in a separate secretory part of the gland where they release their products directly into the blood. Functional similarities include a role of both insect and vertebrate neuroendocrine factors in energy metabolism, growth, water retention, and reproduction. However, to what extent do these functional similarities represent true homologies, which would imply the presence of the homologous genes in the homologous cells in the Bilaterian ancestor (De Velasco, 2004)?

The main hormone produced by the CC is adipokinetic hormone (AKH), a peptide that acts on the fat body and mobilizes lipids and carbohydrates. AKH also stimulates the nervous system and activates locomotor activity. A peripheral feedback loop controls AKH release, in that sugars (e.g., trehalose in the hemolymph) inhibit AKH release; centrally, several PI-derived neuropeptides controlling AKH secretion have been identified, among them FMRFamide, tachykinin, and crustacean cardioactive peptide. FMRFamide inhibits AKH release, whereas cardioactive peptide and tachykinins (both of which also influence contractility of the heart and visceral muscles) stimulate AKH release (De Velasco, 2004).

AKH shares common functions with the vertebrate glucagon and has some sequence similarity with the N-terminus of glucagons. However, comparison of the genes encoding AKH and glucagons, respectively, provides no clear evidence for homology of these peptides on the molecular level. Glucagon, along with two other growth factors, GLP1 and GLP2, is encoded by the proglucagon gene for which true homologs have so far only been identified among vertebrates. The arthropod AKH gene may have been traced further back to the protostome root with the recent finding of significant sequence similarity with the mollusk gene encoding the APGWamide peptides. However, no significant sequence similarity exists between proglucagon and the AKH/APWHamide genes. The expression pattern of the proglucagon and AKH/APGWamide genes is too widespread to add meaningfully to the question of common ancestry. Glucagon is produced in the endocrine pancreas, as well as the intestinal epithelium, but the GLP growth factors (and therefore the proglucagon gene) are expressed in many cells, including neurons, of the developing and mature vertebrate. AKH is expressed mainly in the corpora cardiaca but is also found in the brain of various insect species. Thus, the molecular sequence of the specific secreted products of the CC and pituitary can currently provide no support for or against the notion that both structures are homologous (De Velasco, 2004 and references therein).

The vertebrate pituitary and Drosophila CC show significant similarities during development. Precursors of both are derived from an anterior anlage; following segregation from this anlage, CC precursors contact the part of the anteromedial forebrain primordium from which they will receive innervation. Shared regulatory genes and signaling pathways add to the overall similarity. In this regard, the role of sine oculis is particularly striking. The expression pattern of so in Drosophila is fairly restricted, including the eye field, stomatogastric anlage, and anterior lip of the ventral furrow that give rise to the CC. Another gene of the sine oculis/six family, optix, is expressed in an anterior unpaired domain close to the SNS, but not the CC. In the early vertebrate embryo, six3/6 (the ortholog of optix) is specifically expressed in the eye field and the anlage of the pituitary; six1/2, orthologs of Drosophila sine oculis, are expressed in sensory placodes of the vertebrate head, but no pituitary expression has been reported yet. In both systems, a sine oculis/six gene plays an early and essential role in the specification of the CC and pituitary, respectively. In Drosophila, both CC and SNS are absent in so mutants; in vertebrate, loss of six3/6 causes severe reduction and posteriorization of the forebrain region though not mention of the pituitary effect has been described (De Velasco, 2004).

Two other regulatory genes, goosecoid and Lhx3/lim3, are relevant in the comparison of the vertebrate and Drosophila neuroendocrine systems. Gsc appears in the ventral neural tube and foregut of postgastrula mouse embryos and is required for ventral neural tube patterning.Drosophila gsc is also expressed at an early stage, but it appears exclusively in the anlage of the SNS and comes on in the CC primordium only late. Loss of gsc results in mild structural defects in the SNS and no morphologically apparent phenotype in the CC. Lhx3 is a transcription factor of the Lim family that is triggered by Shh and FGF8 in the vertebrate pituitary primordium and required for its invagination. Drosophila lim3 is expressed at a late stage in part of the SNS primordium, but not the CC primordium. As stated for gsc, no structural phenotype associated with the SNS or CC has been noted in lim3 mutants, but more careful analysis, using additional late differentiation markers for these structures, will be required to establish the role of these two genes in Drosophila neuroendocrine development (De Velasco, 2004).

glass represents a homeobox gene expressed in the eye, nervous system, and as shown in this study, the corpus cardiacum. glass, which is absolutely required for the Drosophila CC since loss of one copy of the gene causes complete absence of the CC at late embryonic stages, has vertebrate cognates but so far no eye function of these genes has been reported (De Velasco, 2004).

Several signaling pathways are expressed in similar patterns in and/or around the developing neuroendocrine system of vertebrates and Drosophila. In both, Hh/Shh is expressed posteriorly adjacent to the CC/Rathke's pouch in the primordium of the foregut/oral epithelium. Vertebrate BMP2/4 comes on in the mesenchyme surrounding the base of Rathke's pouch. Similarly, Drosophila Dpp appears in the mesoderm flanking foregut primordium, CC, and SNS. FGF8 is derived from the hypothalamus floor; the FGF receptor homolog Htl is expressed in the myogenic head mesoderm that is anteriorly adjacent to the CC/SNS complex. In vertebrates, the ventral-to-dorsal BMP gradient and dorsal-to-ventral FGF8 gradients control the differentiation of pituitary cell types; Shh is also required in the proliferation and differentiation of the pituitary primordium. The role of these signaling pathways in Drosophila is less apparent. The CC is still present in dpp, hh, or htl mutant embryos, although it exhibits abnormalities in shape and location. This may constitute an indirect effect of these genes, given their widespread role in head morphogenesis (in case of Dpp and Hh) or mesodermal migration (for Htl). It is anticipated that with the advent of additional markers for subsets of CC cell types, the role of the Hh and Dpp signaling pathways will become clearer (De Velasco, 2004).

A mutation in the Dpp antagonist Sog was the only signaling mutant analyzed in this work that was able to completely remove the CC. This is surprising, given the relatively mild phenotype of sog mutants in the primordium of the ventral nerve cord. Here, only removal of both sog and brinker (brk) together are able to suppress the appearance of most neuroblasts. However, certain domains in the ventral head (that include the precursors of the CC) may be more sensitive to a shift in balance of the Sog-Dpp antagonism. It is speculated that the loss of the CC precursors in sog mutants results from an expanded Dpp gradient, although more experiments would be required to rule out the possibility that sog (the Drosophila homolog of chordin) directly affects CC precursor fate (De Velasco, 2004).

In conclusion, this study presents evidence for a number of conserved properties in the way the progenitors of the neuroendocrine system in vertebrate and Drosophila embryos are spatially laid out and employ cassettes of signaling pathways and fate determinants. This suggests that fundamental elements of a primordial “neuroendocrine system” were already present in the Bilaterian ancestor. Current ideas on pituitary evolution are compatible with this notion. Sensory structures proposed to represent the homologs of the vertebrate pituitary are present in cephalochordates, urochordates, and hemichordates. In amphioxus, for example, these cells form the so-called Hatschek's pit, located in the roof of the pharynx in close contact with the anterior neural tube. Molecules characteristic of the vertebrate pituitary, such as GnRH and Pit-1, are found in Hatschek's pit and in the proposed homolog in urochordates. It is thought that the pituitary originated as a chemosensory structure that senses environmental cues and produced hormones controlling gametogenesis and reproductive behavior, as well as fundamental metabolic functions. Subsequently, the pituitary lost its sensory function and was taken under the control of the CNS, which was able to assimilate sensory information more efficiently. It is likely that stage one, that is, a sensory-endocrine pituitary forerunner, was present in the Bilaterian ancestor. This forerunner probably formed part of the pharynx, which would explain the conserved developmental origin in Drosophila and vertebrates. The sensory-neuroendocrine state of the pituitary homolog is still preserved in present-day protochordates. Loss of sensory function and the taking-over of pituitary control by the CNS occurred during vertebrate evolution. In arthropods or other protostomes, evidence for a sensory forerunner of the neuroendocrine gland has not yet been described; guided by situation in protochordates, one would expect to find such a structure among the sensory organs of the head (De Velasco, 2004).

Larval

SO immunoreactivity commences at the onset of the third instar larva before the morphogenetic furrow has formed, slightly later than eyes absent, the cell survival and differentiation gene. SO is found in a gradient increasing from anterior to posterior. In null mutants, the optic primordium fails to invaginate. optomotor blind transcript is present in mutants. Bolwig's organ is absent in mutants (Cheyette, 1994).

Effects of Mutation or Deletion

Mutants are embryonic lethals. Defects are apparent in the embryonic optic primordium, Bolwig's organ (the larval photoreceptor organ) and ocelli. Weak alleles produce disorganized eyes, with a reduced number of ommatidia (Cheyette, 1994). Mutation of so results in apoptosis anterior to the morphogenic furrow (Cheyette, 1994).

The somda (sine oculis-medusa) mutant is the result of a P element insertion at position 43C on the second chromosome. somda causes aberrant development of the larval photoreceptor (Bolwig's) organ and the optic lobe primordium in the embryo. Later in development, adult photoreceptors fail to project axons into the optic ganglion. Consequently optic lobe development is aborted and photoreceptor cells show age-dependent retinal degeneration. The so gene encodes a homeodomain protein expressed in the embryonic optic lobe primordium and Bolwig's organ; in the developing adult visual system of larvae, and in photoreceptor cells and optic lobes of adults. In addition, the SO product is found at several invagination sites during embryonic development: at the stomadeal invagination, the cephalic furrow, and at segmental boundaries. The mutant somda allele causes severe reduction of SO embryonic expression but maintains adult visual system expression. Ubiquitous expression of the SO gene product in 4-8-hr embryos rescues all somda mutant abnormalities, including the adult phenotypes. Thus, all deficits in adult visual system development and function result from failure to properly express the so gene during embryonic development (Serikaku, 1994).

The Drosophila yan gene encodes an ETS domain nuclear protein with a transcription repressor activity that can be downregulated through phosphorylation by mitogen-activated protein kinase (MAPK). Before photoreceptor precursor cells commit to a particular cell fate, Yan is required to maintain them in an undifferentiated state. tramtrack (ttk) mutations have been identified that act as dominant enhancers of yan. ttk synergistically interacts with yan to inhibit the R7 photoreceptor cell fate. Since ttk products are nuclear proteins with zinc-finger DNA-binding motifs, yan and ttk represent two nuclear regulators essential for the control of cellular competence for neural differentiation. Reduction of either yan or ttk activity suppresses eye phenotypes of the kinase suppressor of ras (ksr) gene mutation, which is consistent with the involvement of yan and ttk in the Ras/MAPK pathway. Based on the fact that yan acts upstream of sina and ttk acts downstream of sina, it is expected that interaction between yan and ttk does not occur through direct protein associations. A more likely scenario is that yan controls expression of downstream genes that are critical for regulating ttk expression or function. A strong candidate target gene is phyllopod, which acts downstream of yan and upstream of sina (Lai, Z.-C., 1997).

The R7 photoreceptor neuron projections form a retinotopic map in the medulla of the Drosophila optic lobe. An allele of gap1, results in the differentiation of excess R7s in the eye, whose axons invade the brain and establish functional connections. This hyperinnervation phenotype was used to explore the roles of photoreceptor-target regulation, competitive interactions, and chemoaffinity in map formation. The extra axons are supported in a wild-type brain, with all R7s from a single ommatidium sharing a single termination site; thus there is no evidence that the target regulates the size of the presynaptic population. In mosaic eyes, in which ommatidia containing extra R7s are surrounded by ommatidia lacking all R7 cells, R7 axons still target to appropriate retinotopic locations in a largely empty R7 terminal field. Axons at the edges of the projection, however, send collaterals into vacant areas of the field, suggesting they are normally restrained to share single termination sites by competitive interactions. In contrast, no sprouts are seen when the vacant sites are juxtaposed with singly innervated sites. In the third instar, R7 and R8 axons transiently display halos of filopodia that overlap adjacent terminals and provide a means to assess occupancy at adjacent sites. Finally, in sine oculis mutant larvae in which only a small number of ommatidia develop, the R7/R8 axons target to predicted dorsoventral portions of the medulla despite the absence of their neighbors, suggesting that position in the eye field determines their connectivity in the brain (Ashley, 1994).

The Drosophila homeobox gene optix is capable of inducing ectopic eyes by an eyeless-independent mechanism.

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 extraretinal eyelet of Drosophila: development, ultrastructure, and putative circadian function

Circadian rhythms can be entrained by light to follow the daily solar cycle. In adult flies a pair of extraretinal eyelets expressing immunoreactivity to Rhodopsin 6 each contains four photoreceptors located beneath the posterior margin of the compound eye. Their axons project to the region of the pacemaker center in the brain with a trajectory resembling that of Bolwig's organ, the visual organ of the larva. A lacZ reporter line driven by an upstream fragment of the developmental gap gene Kruppel is a specific enhancer element for Bolwig's organ. Expression of immunoreactivity to the product of lacZ in Bolwig's organ persists through pupal metamorphosis and survives in the adult eyelet. It is thus demonstrated that the adult eyelet derives from the 12 photoreceptors of Bolwig's organ, which entrain circadian rhythmicity in the larva. Double labeling with anti-pigment-dispersing hormone shows that the terminals of Bolwig's nerve differentiate during metamorphosis in close temporal and spatial relationship to the ventral lateral neurons (LN_v), which are essential to express circadian rhythmicity in the adult. Bolwig's organ also expresses immunoreactivity to Rhodopsin 6, which thus continues to be expressed in the adult eyelet. Action spectra of entrainment were compared in different fly strains: in flies lacking compound eyes but retaining the adult eyelet (so¹), lacking both compound eyes and the adult eyelet (so¹;gl^60j), and retaining the adult eyelet but lacking compound eyes as well as Cryptochrome (so¹;cry^b). Responses to phase shifts suggest that, in the absence of compound eyes, the eyelet together with Cryptochrome mainly mediates phase delays. Thus a functional role in circadian entrainment first found in Bolwig's organ in the larva is retained in the eyelet, the adult remnant of Bolwig's organ, even in the face of metamorphic restructuring (Helfrich-Forster, 2002).

Temporal control of glial cell migration in the Drosophila eye requires gilgamesh, hedgehog, and eye specification genes

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).

These observations raise the possibility that gish also contributes to the genetic circuitry regulating eye specification. Indeed, while ey-FLP-induced clones of gish lead to only minor defects in MF progression during third instar stage, gish mutant adult eyes are smaller and frequently contain a reduced number of ommatidia in the anterior region. These phenotypes are frequently seen in weak alleles of eye specification genes. Furthermore, double mutants of ey¹ and gish¹ , as well as so¹ and gish¹, reveal strong synergistic effects in R cell development. The glial cell migration phenotypes in double mutants is similar in severity to the single mutants. In summary, these data argue that gish acts in conjunction with eye specification genes to coordinate neuronal development and glial cell migration in the eye disc (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).

CREB binding protein functions during successive stages of eye development in Drosophila: Genetic interactions with sine oculis

During the development of the compound eye of Drosophila several signaling pathways exert both positive and inhibitory influences upon an array of nuclear transcription factors to produce a near-perfect lattice of unit eyes or ommatidia. Individual cells within the eye are exposed to many extracellular signals, express multiple surface receptors, and make use of a large complement of cell-subtype-specific DNA-binding transcription factors. Despite this enormous complexity, each cell will make the correct developmental choice and adopt the appropriate cell fate. How this process is managed remains a poorly understood paradigm. Members of the CREB binding protein (CBP)/p300 family have been shown to influence development by (1) acting as bridging molecules between the basal transcriptional machinery and specific DNA-binding transcription factors, (2) physically interacting with terminal members of signaling cascades, (3) acting as transcriptional coactivators of downstream target genes, and (4) playing a key role in chromatin remodeling. In a screen for new genes involved in eye development the Drosophila homolog of CBP has been identified as a key player in both eye specification and cell fate determination. A variety of approaches was used to define the role of CBP in eye development on a cell-by-cell basis (Kumar, 2004).

The early development of the compound eye is regulated in part by a regulatory network of genes that include the Pax genes twin of eyeless (toy), eyeless (ey), twin of eyegone (toe), and eyegone (eyg); the founding members of the Dach and Eya gene families dachshund (dac) and eyes absent (eya), and the Six class genes optix and sine oculis. Extracellular instructions from the Hh, Dpp, Egfr, Notch, and Wg signaling cascades are integrated into this network at several levels creating additional layers of complexity. A dominant allele of sine oculis, so^D, was used as the starting material for a genetic screen to isolate new genes involved in eye specification. The so^D allele appears to function as a dominant negative mutant: (1) so^D heterozygotes lack compound eyes while heterozygotes of the so³ null allele have wild-type eyes; thus so^D is a dominant mutant; (2) embryonic lethality results if the so^D mutation is placed in trans to the so³ allele (so^D/so³); (3) compound eye development is restored in so^D mutants by the addition of wild-type SO protein via UAS-so transgenes -- thus so^D has an inhibitory function. The open reading frame of the so^D mutant was sequenced and a single valine-to-aspartic acid substitution was found at amino acid 200 (V200D). This mutation occurs within the Six domain, which is implicated in both DNA-binding and protein-protein interactions with EYA. Mutations within this domain of So could negatively affect eye development by either altering its interactions with potential binding partners or causing inappropriate transcriptional regulation of downstream target genes (Kumar, 2004).

The retinal phenotypes of the eye-specific so¹ loss-of-function mutant and the so^D dominant negative allele differ slightly from one another. SO protein levels are below detection in so¹ mutant eye discs while remaining at wild-type levels in so^D discs. Similarly, the expression of several other genes that are required for eye development, such as dpp and dac, are not reduced in so^D mutants while being disrupted in so¹ mutants. Furthermore, in so¹ adults the region normally occupied by the compound eyes is replaced by surrounding head tissue. In contrast, so^D flies have a large nonpigmented and nondifferentiated field. The lack of retinal tissue in so^D adults can be traced back to a complete lack of photoreceptor differentiation during larval eye imaginal disc development as assayed by the absence of ELAV, a pan-neural protein. The presence of this nondifferentiated field in so^D adults allows for the isolation of both suppressor and enhancer mutations. Six complementation groups were discovered that suppress, and one complementation group that enhances the so^D no-eye phenotype. The enhancing locus is nej, the gene that encodes CBP in Drosophila (Kumar, 2004).

Removal of one copy of nej in a so^D background results in an eye phenotype that is now indistinguishable from so¹ loss-of-function mutants. Similar to so¹ mutants, eye imaginal discs from nej³/+; so^D/+ heterozygotes (nej³ is a null allele) are small and undergo increased levels of cell death, while adults lack the nondifferentiated field and instead contain only head tissue. Conversely, expression of CBP throughout the so^D retinal field suppresses the no-eye phenotype. Eye imaginal discs are near normal in size and contain large numbers of photoreceptor cell clusters, and adult eyes are fully pigmented although not normally patterned (Kumar, 2004).

CBP is expressed in all cells within the developing eye imaginal disc. Loss-of-function CBP mutations affect the expression of several eye specification genes within the embryonic visual system, protocerebrum, mesoderm, and the developing eye imaginal disc. Using viable loss-of-function allelic combinations, loss-of-function retinal clones, and RNAi interference, this study has demonstrated that each cell type in the developing eye, with the exception of the founder R8 photoreceptor, requires CBP for its specification. Using a 'pathway interference' approach it has been shown that CBP likely functions in the R3/R4 cell fate choice and in the specification of the R7 photoreceptor (Kumar, 2004).

The results presented here indicate a role for CBP in a myriad of developmental decisions within the developing fly retina. It remains to be determined if these effects are through repeated interactions with a small set of master regulatory proteins or with a larger set of signaling molecules and cell-subtype-specific transcription factors. It is more likely that the latter scenario will be correct. This is based on the large body of biochemical data that suggest CBP interacts with more than 100 proteins that are members of many diverse signaling cascades. Furthermore, no single gene has been shown to affect all of the processes that require the activity of CBP. Thus it is hypothesized that CBP functions as a connecting point for signaling, transcription, and chromatin remodeling during all phases of fly eye development (Kumar, 2004).

The sheer number of potential interactions mediated by CBP makes an analysis of this protein inherently difficult. To circumvent this potential problem, a pathway interference approach was used to dissect CBP function by expressing a series of truncated CBPs within the developing eye. The underlying idea behind this approach is that each protein variant will act as a protein sink and soak up a unique set of endogenous factors, thus providing insight into the processes that are affected by CBP. It also provides a first step toward understanding the role that each domain of CBP plays in the developmental process and lays the groundwork for identifying critical components using more biochemical methods. The target proteins are likely to interact with CBP at stoichiometric levels during normal development. However, by increasing the dosage of CBP, the amount of these proteins within a cell becomes limiting and loss-of-function phenotypes can be observed. This approach successfully revealed roles for CBP in the R3/R4 cell fate choice and in R7 fate specification (Kumar, 2004).

How CBP functions in any of these processes is still an unanswered question. Attempts to identify additional components of the regulatory network disrupted by expression of variant CBPs through the restoration of putative interacting and downstream factors were unsuccessful. The addition of any one single factor was insufficient to rescue the effects of any of the CBP variants. Although it is possible that none of the correct factors were tested, it is more likely that the observed phenotypes result from the loss of several proteins and adding just one is insufficient to restore normal eye development (Kumar, 2004).

How are so many developmental decisions in the developing eye regulated by CBP? On the basis of reported roles for CBP/p300 in mammalian development, CBP would appear to be the perfect candidate to act as a 'network manager' during eye development. A scenario can be envisioned in which every cell within the eye disc expresses CBP and a specific combination of transcription factors; some are present in restricted expression patterns while other are more promiscuously expressed. As signals are interpreted at the cell surface and transmitted into the nucleus, the CBP-transcription factor scaffold would interact with terminal members of signaling cascades and execute these instructions by modulating transcription of downstream target genes. Late in development this would translate into the differentiation of specific cell types -- photoreceptors, cone cells, pigment cells, and mechanosensory bristles. This is an attractive model for several reasons. (1) The uncommonly high number of described biochemical interactions suggests that CBP may act as a link between signaling pathways, specific DNA-binding proteins, and the basal transcriptional machinery. These qualities have been shown to be true in vitro. (2) It allows for individual cells to receive several common-use signals but then personalize the output. (3) The ability to interact with members of signaling pathways as well as remodel chromatin allows for very efficient transduction of extracellular instructions. This may be important for the recruitment of photoreceptors into the ommatidial cluster, a process that occurs over a relatively short period of time. This model can be extended to early events in eye specification. CBP is expressed in all cells of the eye and antennal tissues during early development, while expression of selector genes is restricted to the individual tissues. Signaling pathways that include Notch, Egfr, Hh, Dpp, and Wg are known to influence both eye and antennal development. CBP may mediate the interactions between signaling pathways and these selector genes, thereby participating in the process of subdividing the eye-antennal disc into the eye and antenna proper (Kumar, 2004).

Previous reports of CBP in the eye have focused on the role of CBP in the modulation of polyglutamine diseases and retinal degeneration. The work presented here extends these results and points to a role for CBP both in early eye determination and later in cell fate specification. The results that pertain to early eye determination are supported by the synergistic interactions between CBP and SIX, EYA, and DACH proteins observed in mammals. Furthermore, this study has demonstrated a role for CBP in the development of several photoreceptor cell subtypes including the R7 neuron. In recent years it has become increasingly clear that the molecules and mechanisms that control eye development have been preserved in both mammalian and invertebrate retinal systems. It will be interesting to elucidate the molecular and biochemical mechanisms by which CBP influences early eye specification and later photoreceptor cell fate decisions in both invertebrate and mammalian retinal systems (Kumar, 2004).

Competition among gene regulatory networks imposes order within the eye-antennal disc of Drosophila

The eye-antennal disc of Drosophila gives rise to numerous adult tissues, including the compound eyes, ocelli, antennae, maxillary palps and surrounding head capsule. The fate of each tissue is governed by the activity of unique gene regulatory networks (GRNs). The fate of the eye, for example, is controlled by a set of fourteen interlocking genes called the retinal determination (RD) network. Mutations within network members lead to replacement of the eyes with head capsule. Several studies have suggested that in these instances all retinal progenitor and precursor cells are eliminated via apoptosis and as a result the surrounding head capsule proliferates to compensate for retinal tissue loss. This model implies that the sole responsibility of the RD network is to promote the fate of the eye. This study has re-analyzed eyes absent mutant discs, and proposes an alternative model. The data suggests that in addition to promoting an eye fate the RD network simultaneously functions to actively repress GRNs that are responsible for directing antennal and head capsule fates. Compromising the RD network leads to the inappropriate expression of several head capsule selector genes such as cut, Lim1 and wingless. Instead of undergoing apoptosis, a population of mutant retinal progenitors and precursor cells adopt a head capsule fate. This transformation is accompanied by an adjustment of cell proliferation rates such that just enough head capsule is generated to produce an intact adult head. It is proposed that GRNs simultaneously promote primary fates, inhibit alternative fates and establish cell proliferation states (Weasner, 2013).

This article has used the eye-antennal disc of Drosophila to study the mechanisms by which competing gene regulatory networks (GRNs) impose regional specification upon a naive tissue. Early in development, the GRNs for the eye, antennal and head capsule fates are uniformly expressed throughout the entire epithelium. During the early/mid second instar stage, the expression and activity of these GRNs are geographically restricted by mechanisms that are poorly understood. This study has attempted to determine the mechanism by which this asymmetry is then maintained during the remainder of development. Two competing models can potentially explain the maintenance of asymmetry of GRN expression and activity in an epithelium. In the first model, individual GRNs, once segregated, function primarily to promote the primary fate of the underlying sector. This is achieved solely through internal positive transcriptional feedback loops. In the second model, GRNs not only promote the adoption of primary fates but they also inhibit the implementation of inappropriate fates. This second feature involves GRNs mutually repressing each other’s expression (Weasner, 2013).

Many well-defined mutations are known to result in replacement of the eye with head capsule tissue. Additionally, mutations in which the converse is true have also been characterized, with ectopic eyes forming within portions of the head capsule and antennae. These phenotypes provide an opportunity to elucidate the mechanisms that underlie regional specification. This study presents evidence that mutual repression of GRN expression patterns plays an important role in this process. The fate of the eye field was re-analyzed in eya and so loss-of-function mutants, and it was found that a subpopulation of retinal progenitor and precursor cells is transformed into head capsule tissue. These cells survive an apoptotic wave of developmental cell death, activate expression of head capsule genes and re-adjust their cell proliferation rates to match their newly acquired identity. These results suggest that one function of the RD network is to repress the expression of non-retinal selector genes. Although the execution of this task has been attributed to Ey, increasing evidence suggests that the repression of non-retinal selector genes is more likely to be a function of the So-Eya complex (Anderson, 2012; Wang, 2012). Currently, it is unclear whether So binds to tissue-specific enhancers of non-retinal selector genes and directly represses transcription. However, such a mechanism is possible as So consensus binding sites are found within several of these transcription units (Weasner, 2013).

If competition among GRNs is important for ensuring regional specification then what is the critical developmental window for this event to take place? Previous published reports indicate that many members of the eye, antennal and head capsule GRNs are uniformly expressed throughout the entire eye-antennal disc primordium during embryogenesis and the first larval instar. Other studies have indicated that by the late second larval instar these genes are geographically segregated within the eye-antennal disc. If a phenocritical period does exist for regional specification within the eye-antennal disc, then one would predict that the fate of the eye field could be altered during this developmental window under circumstances in which the eye GRN is compromised. Indeed, alterations in both EGF receptor and Notch signaling do indeed result in the transformation of the eye into an antenna during the second larval instar. This article demonstrates a similar phenomenon: compromising the function of the retinal determination network leads to the transformation of the eye into head capsule tissue. This transformation is initiated between 84 and 96 hours AEL as genes that are normally restricted to the developing head capsule are de-repressed within the eye field of both eya and so mutants. If one accounts for differences in the experimental temperatures at which this study and previous studies were conducted then the phenocritical windows for eye-antenna-and-eye-head capsule transformations map to very similar time periods (Weasner, 2013).

It is proposed that this model for the use of transcriptional repression by GRNs to promote regional specification is applicable to the formation of the antenna and head capsule as well because forced expression of ct within the eye field extinguishes ey expression and induces eye-antenna transformations (Anderson, 2012). Similarly, forced expression of Dip3 within the eye field is sufficient to simultaneously downregulate the RD network while ectopically activating antennal selector genes such as Distal-less (Dll) thereby resulting in eye-antennal transformations. This result indicates that the GRNs for antennal and head capsule tissue can inhibit expression of genes within the RD network. The identification of a potential phenocritical period for the eye-head capsule choice might be relevant to other tissue fate decisions as well. For instance, the critical window for the wing-notum decision has been mapped to the late second instar, and the induction of ectopic eyes (non-retinal to retinal transformation) appears in most instances to be synchronous with the specification of the normal eye. Together, these results suggest that a potential global developmental window for imaginal disc fate decisions exists and is centered around the late second larval/early third instar stage (Weasner, 2013).

In summary, it is proposed that during the earliest stages of normal eye-antennal disc development the members of multiple GRNs are expressed uniformly throughout the epithelium). As development proceeds, each GRN is geographically restricted. The maintenance of these transcriptional asymmetries is maintained by a combination of intra-GRN activation and inter-GRN repression. In situations in which the retinal determination network is compromised, the antennal and head capsule GRNs are de-repressed within the eye field. The choice of which developmental pathway is to be activated depends heavily upon the type of genetic manipulation. Likewise, when an individual retinal determination gene such as ey is forcibly expressed in a non-retinal tissue it simultaneously activates the rest of the RD network while repressing expression of the endogenous GRN. As a result, an ectopic eye is generated (Weasner, 2013).

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date revised: 10 April 2014 

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