giant

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of gt at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.

GT RNA is first detected quite early in development during nuclear cycle 12. One broad anterior stripe is formed 62 to 80% egg length (measuring from the posterior). A posterior domain is formed from 2 to 33% egg length. By the middle of nuclear cycle 13, the posterior domain has become more confined (15-20% egg length). During cellularization the anterior stripe resolves into two stripes, and at the end of cellulariation a new band of expression is present, located further toward the anterior, but not extending as far as the ventral most cells. As gastrulation begins, the anterior end of the ventral furrow [Image] reaches the original anterior stripe, and the cells expressing gt become internalized. The top of the initial anterior stripe straddles the cephalic furrow (Eldon, 1991 and Kraut, 1991a).

Giant function in the short germ insect Tribolium

Segmentation is well understood in Drosophila, where all segments are determined at the blastoderm stage. In the flour beetle Tribolium castaneum, as in most insects, the posterior segments are added at later stages from a posteriorly located growth zone, suggesting that formation of these segments may rely on a different mechanism. Nevertheless, the expression and function of many segmentation genes seem conserved between Tribolium and Drosophila. The Tribolium ortholog of the abdominal gap gene giant has been cloned and characterized. As in Drosophila, Tribolium giant is expressed in two primary domains, one each in the head and trunk. Although the position of the anterior domain is conserved, the posterior domain is located at least four segments anterior to that of Drosophila. Knockdown phenotypes generated with morpholino oligonucleotides, as well as embryonic and parental RNA interference, indicate that giant is required for segment formation and identity also in Tribolium. In giant-depleted embryos, the maxillary and labial segment primordia are normally formed but assume thoracic identity. The segmentation process is disrupted only in postgnathal metamers. Unlike Drosophila, segmentation defects are not restricted to a limited domain but extend to all thoracic and abdominal segments, many of which are specified long after giant expression has ceased. These data show that giant in Tribolium does not function as in Drosophila, and suggest that posterior gap genes underwent major regulatory and functional changes during the evolution from short to long germ embryogenesis (Bucher, 2004).

Expression of Tc'giant reveals both conserved and diverged aspects. In Tribolium and in Drosophila, giant is active in the maxillary segment, and later in a highly dynamic pattern in the brain. Therefore, this expression was probably present in the last common ancestor of all holometabolous insects. Another similarity is the appearance of a second expression domain in the posterior blastoderm. However, although in Drosophila both domains appear simultaneously, the posterior domain appears later than the anterior one in Tribolium. This could simply reflect the anterior to posterior sequence of segment formation in the beetle. However, relative to emerging segment primordia, this domain is located five segments more anterior. In Drosophila, the abdominal segments A5 to A7 arise right under the posterior giant domain while in Tribolium the anlagen of segments T3 through A2 are covered by the posterior Tc'giant domain. This shift in expression must reflect a fundamental change in gene function: either the Tribolium and Drosophila giant orthologs function by different mechanisms to pattern the same segments, or alternatively, if they act through a similar short range gradient mechanism, they must specify different segments (Bucher, 2004).

The Tc'giant expression pattern also indicates three divergent interactions with other segmentation genes. (1) Since the posterior domain arises in the late blastoderm, it is probably under zygotic control. This is in contrast to the situation in Drosophila, where the maternal genes caudal and bicoid cooperate to activate posterior giant expression. (2) The posterior Tc'giant domain appears right within the Krüppel domain, and co-expression of both genes is observed in segment T3 for an extended time period. This again is in contrast to Drosophila, where strong mutual repression with Krüppel is crucial for regulation and proper function of giant. However, inhibitory interactions between Tc'Krüppel and the anterior giant domain could still be conserved, since these domains are mutually exclusive also in Tribolium. (3) Another intriguing feature is noted: maturation of all three Tc'giant stripes (maxialla, T3 and A2) occurs in identical relation to the pair-rule gene register. In fact, the split of the posterior domain into distinct stripes concurs with pair rule patterning rather than preceding it. This raises the possibility that in later stages Tc'giant may be regulated by pair rule genes. In contrast to this, Dm'giant expression precedes pair rule activation and the gene unambiguously acts on a higher hierarchical level. Evidently, it is not only the position, but also many aspects of the regulatory network involving giant that differ between Tribolium and Drosophila. Functional data confirm this, and in addition reveal that the function of the anterior domain has diverged in both insects (Bucher, 2004).

Like other Drosophila gap genes, Dm'giant functions in positioning pair-rule stripes, and its role in defining the anterior border of eve stripe 2 has been studied in much detail. Lack of giant function leads to expansion of this stripe and to concomitant loss of the labial engrailed stripe. By contrast, no defects were detected in head segmentation, and the labial engrailed stripe was unaffected in RNAi embryos. The most anterior segmentation defect observed was the deletion of the T1 engrailed stripe. The primordium of this stripe arises at a distance of one segment width to the posterior but two segment widths to the anterior Tc'giant domain. It seems more likely, therefore, that formation of this segment depends on the posterior rather than the anterior domain, which may not be involved in segmentation at all (Bucher, 2004). However, a crucial role of the anterior Tc'giant domain in homeotic specification is established by these experiments. Tc'giant RNAi larvae display a coordinated two-segment shift of all thoracic identities toward the anterior. Generally, maxillary and labial segments are fully transformed toward T1 and T2 respectively, while the T1 segment adopts T3 identity, followed by two segments with identities ranging between T3 and abdomen. The shift of several segment identities implies that Tc'giant directly or indirectly regulates several Hox genes (i.e. those required for the identities of at least maxilla, labium, T1 and T2). Homeotic function of Tc'giant is not surprising, because Drosophila gap genes are known to regulate homeotic genes, and Dm'giant, specifically, defines the anterior border of Antennapedia. However, these functions in the regulation of homeotic genes are usually not evident from Drosophila gap gene phenotypes, as evidenced by the observation that the homeotically affected regions are missing in the developed embryo because of the segmentation defects (Bucher, 2004).

The homeotic two-segment shift in Tc'giant RNAi embryos follows 'all or nothing' kinetics: A homeotic shift is never observed across one segment width. This argues against a simple mechanism where a gradient of Tc'Giant protein emanating from the anterior domain would directly position gnathal and thoracic Hox genes. In addition, partial transformation of maxilla or labium is extremely rare -- even though RNAi or morpholino knockdown experiments should produce many intermediate levels of residual gene function. Therefore, the coordinated regulation of several Hox genes by Tc'giant appears to rely on a mechanism involving tight thresholds. Interestingly, the phenotype of jaws, a mutant in the Tc'Krüppel gene displays a homeotic transformation that is the opposite of Tc'giant phenocopies. In jaws larvae, thoracic and anterior abdominal segments are transformed to alternating pairs of maxillary and labial segments, while in Tc'giant RNAi embryos, maxilla and labium are transformed to T1 and T2, respectively. This suggests that Tc'giant and Tc'Krüppel have opposing functions in regulating the same set of thoracic and gnathal Hox genes. This may indicate mutual inhibition of Krüppel and the anterior giant domain as in Drosophila (but in contrast to the posterior Tc'giant domain). In addition, the homeotic phenotypes of both Tc'Krüppel (jaws) and Tc'giant display double segmental effects, suggesting the involvement of pair-rule genes in homeotic segment specification (Bucher, 2004).

Even though the RNAi and morpholino knock down experiments may not have achieved complete inactivation of the Tc'giant gene product, segmentation phenotypes much more severe than those of Drosophila giant null-mutations were frequently obtained. In Drosophila giant mutant embryos, the loss of the posterior domain results in a fusion of the engrailed stripes corresponding to segments A5 to A7, which are the segment primordia covered by this domain. Drosophila gap genes are expressed in domains whose diffuse boundaries function as short-range morphogenetic gradients that position pair-rule stripes. Accordingly, both Drosophila giant domains regulate pair-rule stripes in this manner. However, the rather severe patterning defects observed at the pair-rule level are to some extent repaired during later stages of development, resulting in a less serious larval phenotype. By contrast, in Tribolium embryos displaying strong Tc'giant phenocopies, segmentation is disturbed in a region comprising twelve segments, ranging from T1-A9 (Bucher, 2004).

Intriguingly, the phenotype of Tc'giant knock-down larvae is not only stronger than that of Drosophila giant mutants, but it also differs in the spatial and temporal relationships between expression domain and affected segments. The posterior domain of Tc'giant appears at the posterior pole of the blastoderm embryo at a time when the primordia of the first thoracic segments are patterned in this region. By this time, cellularization has most likely occurred. Thus, if thoracic and anterior abdominal defects of Tc'giant RNAi larvae reflect a short-range regulation comparable to that of Drosophila giant, diffusion of the Tc'Giant protein across cell membranes would be required. However, the secondary Tc'giant stripes actually resemble pair-rule stripes in width and spacing, in addition to the way they arise near the growth zone. It is therefore possible that these two stripes regulate pair-rule stripes in a manner more typical of pair-rule interactions in Drosophila, i.e., by direct activation and repression within precise boundaries (Bucher, 2004).

In any case, the Drosophila paradigm cannot explain why very posterior abdominal segments require giant function in Tribolium, since these segments are formed at a large distance (spatially and temporally) from the posterior Tc'giant domain(s). The segment A9, for example, is frequently deleted in giant RNAi larvae, but arises six segments posterior to Tc'giant expression and long after expression has ceased. Tc'giant could be involved in setting up and/or starting a segmentation process in which a 'chain of induction' mechanism (involving gap or pair-rule genes) would pattern the growing abdomen. Alternatively, Tc'giant may jump-start an oscillator machinery analogous to that underlying somitogenesis in vertebrates. In both cases, loss of Tc'giant would lead to improper setup and subsequent breakdown of the machinery, which could then result in defects in distant segments. However, one could also envisage the role of Tc'giant to be a rather general one. Tc'giant expression in the early growth zone may simply be required for making a proper growth zone, and reduction of Tc'giant activity may result in aberrant behavior of the affected cells during later growth, leading to segmentation defects in an indirect way. Evidently, more data are needed to distinguish between these disparate possibilities, including data about other posterior gap genes (Bucher, 2004).

Different combinations of gap repressors for common stripes in Anopheles and Drosophila embryos

Drosophila segmentation is governed by a well-defined gene regulation network. The evolution of this network was investigated by examining the expression profiles of a complete set of segmentation genes in the early embryos of the mosquito, Anopheles gambiae. There are numerous differences in the expression profiles as compared with Drosophila. The germline determinant Oskar is expressed in both the anterior and posterior poles of Anopheles embryos but is strictly localized within the posterior plasm of Drosophila. The gap genes hunchback and giant display inverted patterns of expression in posterior regions of Anopheles embryos, while tailless exhibits an expanded pattern as compared with Drosophila. These observations suggest that the segmentation network has undergone considerable evolutionary change in the dipterans and that similar patterns of pair-rule gene expression can be obtained with different combinations of gap repressors. The evolution of separate stripe enhancers in the eve loci of different dipterans is discussed (Goltsev, 2004).

In Drosophila, different levels of the Hunchback and Knirps gap repressor gradients define the limits of eve stripes 3, 4, 6, and 7, while Giant and Kruppel establish the borders of stripes 2 and 5. In situ hybridization probes were prepared for Anopheles orthologues of all four of these gap genes, as well as a fifth gap gene, tailless. hunchback displays a broad band of expression in the anterior half of the Anopheles embryo, encompassing both the presumptive head and thorax. This pattern is similar to that observed in Drosophila, although there are a few notable deviations: (1) there is no obvious maternal expression seen in early Anopheles embryos, whereas maternal hunchback mRNAs are strongly expressed throughout early Drosophila embryos; (2) there is a significant change in the posterior staining pattern. The Drosophila gene displays a strong posterior stripe of expression that is comparable in intensity to the anterior staining pattern. In Anopheles, this staining is significantly weaker than that of the anterior domain, and the posterior pattern is shifted anteriorly into the presumptive abdomen (Goltsev, 2004).

The Kruppel and knirps staining patterns are similar in Anopheles and Drosophila embryos. In both cases, the principal sites of expression are seen in the presumptive thorax and abdomen, respectively. However, the remaining two gap genes, giant and tailless, exhibit distinctive staining patterns. In Anopheles, giant exhibits a continuous band of staining in anterior regions, whereas the Drosophila gene is excluded from the anterior pole. Moreover, there is a prominent band of staining in the presumptive abdomen of Drosophila embryos that is not seen in Anopheles. Finally, tailless is expressed in a narrow stripe in the posterior pole of Drosophila embryos, whereas Anopheles embryos display a dynamic pattern that (transiently) extends throughout the presumptive abdomen (Goltsev, 2004).

These observations document significant changes in the expression patterns of maternal determinants and gap genes in flies and mosquitoes, although the dynamic eve pattern is quite similar in the two systems. The most notable differences were seen for the gap genes hunchback and giant. Additional in situ hybridization assays were done in an effort to obtain a more comprehensive view of these changing patterns; hunchback is initially expressed in the anterior half of Anopheles embryos, with no staining detected in posterior regions. Weak posterior staining is detected by the onset of gastrulation, but expression appears to be localized within the presumptive abdomen rather than the posterior pole as seen in Drosophila. This shift was confirmed by costaining with eve. In Drosophila, the anterior hunchback pattern is lost except for a stripe of staining in the thorax, and this stripe persists along with the posterior pattern during gastrulation. In Anopheles, the early hunchback expression pattern gives way to localized expression in the presumptive serosa. Drosophila lacks a comparable staining pattern, although similar patterns have been documented in Tribolium, and mothmidges. It is conceivable that the late hunchback pattern is responsible, directly or indirectly, for the repression of eve stripes in the presumptive serosa (Goltsev, 2004).

As seen for hunchback, there is no detectable expression of giant in posterior regions of early Anopheles embryos. Weak staining appears in the posterior pole by the onset of gastrulation. This staining is clearly posterior to the hunchback pattern in the presumptive abdomen. Thus, the posterior hunchback and giant patterns are reversed in Anopheles as compared with Drosophila. The anterior giant pattern encompasses the entire anterior half of Anopheles embryos and extends into the anterior pole. The staining pattern is refined at gastrulation, including the loss of expression in the presumptive serosa and the formation of discrete bands. Nonetheless, unlike the situation in Drosophila, expression persists in the anterior pole, thereby raising the possibility that different mechanisms are used to establish the anterior border of eve stripe 2 in flies and mosquitoes (Goltsev, 2004).

The altered patterns of hunchback and giant expression in posterior regions raise the possibility that different combinations of gap repressors are used to establish eve stripes 5, 6, and 7 in Anopheles and Drosophila. It is unlikely that Giant establishes the posterior border of eve stripe 5 and that Hunchback delimits the posterior border of stripe 7, as seen in Drosophila. The expression profiles of additional gap genes were analyzed in an effort to identify potential repressors for these stripe borders. The most obvious candidates are huckebein and tailless, since both are expressed in the posterior pole of Drosophila embryos. No expression of huckebein was seen in early embryos, although strong staining appears after germband elongation (Goltsev, 2004).

The combinations of gap repressors that define the borders of eve stripes 2 to 7 are known in Drosophila. Stripes 2 and 5 are formed by the combination of Giant and Kruppel repressors, while distinctive borders for stripes 3, 4, 6, and 7 are established by the differential repression of the stripe 3/7 and stripe 4/6 enhancers in response to distinct concentrations of the Hunchback and Knirps repressor gradients. Double-staining assays provide immediate insights into the likely combination of gap repressors that are used for any given stripe. For example, the giant and Kruppel expression patterns abut the borders of eve stripes 2 and 5. Double-staining assays were done to determine the potential regulators of the Anopheles eve stripes. These experiments involved the use of digoxigenin-labeled hunchback, Kruppel, knirps, and giant hybridization probes along with an FITC-labeled eve probe. Different histochemical substrates were used to separately visualize the two patterns (Goltsev, 2004).

The anterior hunchback pattern extends through eve stripe 2 and approaches the anterior border of stripe 3. While the posterior pattern extends through stripes 6 and 7, this pattern is quite distinct from the posterior hunchback pattern seen in Drosophila, which abuts the posterior border of eve stripe 7. The anterior giant pattern extends from the anterior pole to eve stripe 2, while the posterior pattern abuts the posterior border of eve stripe 7. In Drosophila, the posterior giant pattern extends from eve stripe 5 to stripe 7. The Kruppel pattern may be somewhat narrower in Anopheles than Drosophila. It encompasses eve stripe 3 in Anopheles but includes both stripes 3 and 4 in Drosophila. Finally, knirps exhibits the same limits of expression in Anopheles as Drosophila. In both cases, the staining pattern extends from eve stripes 4 to 6. In Anopheles, the anterior knirps pattern straddles the anterior border of eve stripe 1. Some of the eve stripes are associated with the same combinations of gap repressors in flies and mosquitoes (e.g., stripes 2, 3, and possibly 4), whereas others show distinctive combinations of gap repressors (e.g., stripes 5, 6, and 7 (Goltsev, 2004).

The systematic comparison of segmentation regulatory genes in Anopheles and Drosophila suggests that the segmentation gene network has undergone considerable evolutionary change among dipterans despite highly conserved patterns of eve expression. Three particular changes in the network are discussed: the localization of maternal determinants, the formation of the anterior border of eve stripe 2, and the formation of the posterior borders of eve stripes 5, 6, and 7 (Goltsev, 2004).

In Drosophila, hunchback contains two promoters, and the maternal promoter leads to the ubiquitous distribution of hunchback mRNAs throughout early embryos. No hunchback mRNAs were detected in early Anopheles embryos. This apparent absence of maternal transcripts raises the possibility that localized Nanos products are not required for inhibiting the synthesis of Hunchback proteins in posterior regions of Anopheles embryos. In Drosophila, the embryonic lethality caused by nanos mutants can be suppressed by the removal of maternal Hunchback products. This nanos-hunchback interaction is ancient and probably operates in basal insects, and possibly basal arthropods. However, the potential absence of this interaction in Anopheles is consistent with the idea that nanos has an additional essential function. Indeed, a recent study suggests that Nanos is required for maintaining stem cell populations of germ cells in Drosophila (Goltsev, 2004).

Anopheles lacks bicoid and contains a lone Hox3 gene that is more closely related to zen and specifically expressed in the serosa. How is hunchback activated in the presumptive head and thorax in Anopheles? The homeobox gene orthodenticle can substitute for bicoid in Tribolium. However, orthodenticle does not appear to be maternally expressed in Anopheles, but instead, staining is strictly zygotic and restricted to anterior regions, similar to the pattern seen in Drosophila. Sequential patterns of orthodenticle, giant, and hunchback expression are established by differential threshold readouts of the Bicoid gradient in Drosophila. It is possible that an unknown maternal regulatory gradient emanating from the anterior pole is responsible for producing similar patterns of expression in Anopheles. It is proposed that this unknown regulatory factor may be localized to the anterior pole by Oskar. Oskar coordinates the assembly of polar granules and is essential for the localization of Nanos in the posterior plasm. It might also localize one or more unknown determinants in anterior regions of Anopheles embryos (Goltsev, 2004).

The eve stripe 2 enhancer is the most thoroughly characterized enhancer in the segmentation gene network. It can be activated throughout the anterior half of the embryo by Bicoid and Hunchback, but the Giant and Kruppel repressors delimit the pattern and establish the anterior and posterior stripe borders, respectively. Removal of the Giant repressor sites within the stripe 2 enhancer in cis or removal of the repressor in trans causes an anterior expansion of the stripe 2 pattern. However, ectopic expression does not extend to the anterior pole, suggesting that an additional anterior repressor regulates the stripe 2 enhancer. Recent studies identified Sloppy-paired as the likely anterior repressor. The limits of the giant and Kruppel expression patterns seen in Anopheles suggest that they might define the eve stripe 2 borders, just as in Drosophila. However, at the critical time when eve stripe 2 is formed in Anopheles, the giant staining pattern extends to the anterior pole, while the corresponding Drosophila gene is repressed in these regions. It is therefore possible that Giant is sufficient to form the anterior border in Anopheles and that repression by Sloppy-paired represents an innovation in Drosophila (Goltsev, 2004).

There are numerous differences in the patterns of gap gene expression in Drosophila and Anopheles. In Drosophila, the posterior stripe of hunchback expression is the source of a repressor gradient that specifies the posterior borders of eve stripes 6 and 7. Anopheles exhibits a distinct posterior staining pattern, with expression extending through stripes 6 and 7. It is therefore unlikely that Hunchback regulates these stripes as seen in Drosophila. Instead, the location of the posterior hunchback pattern suggests that it regulates the posterior border of eve stripe 5 in Anopheles. In Drosophila, this border is formed by Giant, but in Anopheles, the posterior giant expression pattern is restricted to the posterior pole where it abuts stripe 7. Thus, a combination of Kruppel and Giant defines the eve stripe 5 borders in Drosophila, whereas Kruppel and Hunchback might be used in Anopheles (Goltsev, 2004).

In Drosophila, eve stripes 6 and 7 are regulated by different concentrations of Knirps and Hunchback. Low levels of Knirps define the anterior border of stripe 7, while higher levels are needed to repress eve stripe 6. Conversely, low levels of Hunchback establish the posterior border of eve stripe 6, while higher levels regulate stripe 7. The position of the knirps expression pattern is consistent with the possibility that it defines the anterior limits of stripes 6 and 7, just as in Drosophila. However, the posterior borders of these stripes are probably not regulated by Hunchback. The expanded pattern of tailless expression seen in Anopheles might permit it to establish the posterior border of eve stripe 6 and possibly stripe 7. An alternative candidate for the posterior stripe 7 border is giant, which is expressed in a tight domain within the posterior pole. Consistent with this possibility is the observation that the posterior giant pattern comes on relatively late, and the posterior stripe 7 border is the last to form among the seven eve stripes. The reversal of the posterior hunchback and giant expression patterns, along with the expanded tailless pattern, strongly suggests that different combinations of gap repressors are used to define eve stripes 5, 6, and 7 in Drosophila and Anopheles (Goltsev, 2004).

An implication of the preceding arguments is that each of the seven eve stripes is regulated by a separate enhancer in Anopheles. Only five enhancers regulate eve in Drosophila since four of the seven stripes (3, 4, 6, and 7) are regulated by just two enhancers (3/7 and 4/6) that respond to different concentrations of the opposing Hunchback and Knirps repressor gradients. The change in the posterior hunchback pattern virtually excludes the use of this strategy in Anopheles. Thus, stripes 3 and 7 are probably regulated by separate enhancers since different combinations of gap repressors appear to define the stripe borders. Similar arguments suggest that stripes 4 and 6 are also regulated by separate enhancers (Goltsev, 2004).

Why do some enhancers generate two stripes, while others direct just one? Consider the eve stripe 2 and stripe 3/7 enhancers in Drosophila. The stripe 3/7 enhancer is activated by ubiquitous activators, including dSTAT, and the two stripes are 'carved out' by the localized Hunchback and Knirps repressors. Knirps establishes the posterior border of stripe 3 and anterior border of stripe 7, while Hunchback establishes the anterior border of stripe 3 and posterior border of stripe 7. The stripe 2 enhancer directs just a single stripe due to the localized distribution of the stripe 2 activators, particularly Bicoid. In principle, a ubiquitous activator would cause the stripe 2 enhancer to direct two stripes, stripes 2 and 5. Opposing Giant and Kruppel repressor gradients would carve out the borders of the two stripes, similar to the way in which Hunchback and Knirps regulate the stripe 3/7 and stripe 4/6 enhancers. Presumably, the eve stripe 5 enhancer directs a single stripe of expression because it is regulated by a localized activator, possibly Caudal (Goltsev, 2004).

It is suggested that ancestral dipterans contained an eve locus with separate enhancers for every stripe. Anopheles eve might represent an approximation of this ancestral locus. The consolidation of enhancers that generate multiple stripes was made possible by cross-repression of gap gene pairs. In Drosophila, there are mutually repressive interactions between Hunchback and Knirps, as well as between Giant and Kruppel. The use of these interacting gap pairs along with ubiquitous activators permits the formation of two stripes from a single enhancer. It is possible to envision two ways in which mutual cross-repression of these gap genes helps to establish the precise patterns of pair-rule gene expression: (1) it ensures that there are zones free of repressor activity on both sides of Kruppel (for the Kruppel and Giant pair) and Knirps (for the Knirps and Hunchback pair) domains; (2) it protects the patterns of pair-rule gene expression from mutations that could potentially shift the domains of gap gene expression. For example, a mutation that could shift the expression of Kruppel would simultaneously shift the expression of Giant always leaving a repressor-free zone where Eve stripes would be established. Therefore, the evolution of the eve locus depends on the changes in the preceding tier of the segmentation network: refinement in gap gene cross-regulatory interactions (Goltsev, 2004).

Finally, it is easy to imagine that certain dipterans have a single enhancer for stripes 2 and 5, rather than the separate enhancers seen in Drosophila. Perhaps, the symmetric repression of Giant and Kruppel is a relatively recent occurrence, only now creating the opportunity for consolidated expression of stripes 2 and 5 (Goltsev, 2004).

Embryonic development of the corpus cardiacum, a component of the ring gland

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).

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).

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 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).

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).

Effects of Mutation or Deletion

Mutant embryos lacking gt develop defective labial and labral head structures and are missing segments A5 through A7 [Images] (Petschek, 1987).

To identify X chromosomal genes required for salivary gland development in the Drosophila embryo, embryos hemizygous for EMS-induced lethal mutations were screened to find mutations causing gross morphological defects in salivary gland development. The parental strain carried a lac Z transgene on the second chromosome, which was specifically expressed in the salivary glands so the mutations could be unambiguously identified. Embryos from 3,383 lines were tested for salivary gland abnormalities following lacZ staining. From 63 lines exhibiting aberrant salivary gland phenotypes, 52 stable lines were established containing mutations affecting salivary gland development. From these, 39 lines could be assigned to nine complementation groups: armadillo, brinker, folded gastrulation, giant, hindsight, Notch, runt, stardust and twisted gastrulation (Lammel, 2000).

The identified X chromosomal genes with respect to their possible contributions to salivary gland development is discussed here. For mutations in giant and Notch, severe defects or the absence of the salivary glands have been described previously. In giant mutant larvae, the gnathocephalic structures are affected, which correspond to the labial segment. This is the region where most of the salivary gland anlage originates. giant is required for the expression of Sex combs reduced (Scr), the master regulator for salivary gland development. Scr expression is absent in the labial lobe in giant mutations. The small glands seen in the absence of giant may form from remaining cells of PS 2 still expressing Scr protein. Notch is involved in the formation of epidermal cells, and in its absence neural precursor cells are formed instead. As a result, in Notch mutants a coherent sheet of epidermal cells is found only in the most dorsal position, outside the neurogenic ectoderm. Only a few ventrolateral epidermal cells are left, which may fail to form a salivary gland anlage. Similar explanations may hold for mutations such as stardust, which affect other general aspects of epithelial development, since in the presence of these mutations the maintenance of coherent epidermal sheets is disrupted (Lammel, 2000 and references therein).

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date revised: 25 May 2008

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