giant


DEVELOPMENTAL BIOLOGY

Embryonic

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).

Mechanisms of gap gene expression canalization in the Drosophila blastoderm

Extensive variation in early gap gene expression in the Drosophila blastoderm is reduced over time because of gap gene cross regulation. This phenomenon is a manifestation of canalization, the ability of an organism to produce a consistent phenotype despite variations in genotype or environment. The canalization of gap gene expression can be understood as arising from the actions of attractors in the gap gene dynamical system. In order to better understand the processes of developmental robustness and canalization in the early Drosophila embryo, this study investigated the dynamical effects of varying spatial profiles of Bicoid protein concentration on the formation of the expression border of the gap gene hunchback. At several positions on the anterior-posterior axis of the embryo, attractors and their basins of attraction were analyzed in a dynamical model describing expression of four gap genes with the Bicoid concentration profile accounted as a given input in the model equations. This model was tested against a family of Bicoid gradients obtained from individual embryos. These gradients were normalized by two independent methods, which are based on distinct biological hypotheses and provide different magnitudes for Bicoid spatial variability. It was shown how the border formation is dictated by the biological initial conditions (the concentration gradient of maternal Hunchback protein) being attracted to specific attracting sets in a local vicinity of the border. Different types of these attracting sets (point attractors or one dimensional attracting manifolds) define several possible mechanisms of border formation. The hunchback border formation is associated with intersection of the spatial gradient of the maternal Hunchback protein and a boundary between the attraction basins of two different point attractors. How the positional variability for hunchback is related to the corresponding variability of the basin boundaries was demonstrated. The observed reduction in variability of the hunchback gene expression can be accounted for by specific geometrical properties of the basin boundaries. The mechanisms of gap gene expression canalization in early Drosophila embryos were clarified. These mechanisms were specified in the case of hunchback in well defined terms of the dynamical system theory (Gursky, 2011).

This study presents the dynamical analysis of the simplified model of the gap gene network on the ensemble of early Drosophila embryos. The main goal was to decode the mechanistic basis of the gap gene border formation and stability under the Bcd morphogen variance. The hb border formation mechanisms were described in terms of attracting sets and their attraction basins calculated in the nuclei surrounding the border position (Gursky, 2011).

The results reveal that the border formation can be associated with the event of intersection between a boundary separating the attraction basins of two di®erent point attractors and the initial Hb profile presenting the input from the maternally expressed hb gene. Attracting sets of another type, the unstable manifolds of saddle equilibria, actively participate in the adjustment of the border position. They do so by attracting the solution trajectories in the nuclei surrounding this position. The model predicts that these attracting manifolds can be involved in the border formation for some Bcd profiles (Gursky, 2011).

The hb border correctly forms in the model by the onset of gastrulation for all individual Bcd profiles. For about a half of these profiles, however, the Kr and Gt patterns in the solutions exhibit defects in the anterior part of the spatial domain (solution classes II and III). It turns out that the hb border formation mechanism involving the attracting manifolds is mostly associated with these cases. This may lead to the conclusion about restricted applicability of this mechanism in the case of hb expression. However, this mechanism exists and plays an important role for the gap domain borders in a posterior part of the embryo, where the domains form and vary in time under the control of an unstable manifold. To analyze canalization for the posterior borders, the variation for external inputs from Cad and Tll should be taken into account, where these transcription factors are among the key regulators, and a modified model should be considered including an input from the terminal gene huckebein (Gursky, 2011).

As previously reported, the model exhibits a significant filtration (canalization) of the Bcd positional variability at the level of hb border formation. The results show how this filtration stems from the stable behavior of the attraction basin boundaries. Has been shown that the mutual regulatory repression between the gap genes accounts for the observed variance reduction, thus presenting a buffering mechanism for canalization. This buffering mechanism was translated to the level of attractors and their attraction basins. As the hb border position is well encoded by the intersection between the initial Hb profile and corresponding attraction basin boundaries, the stability of hb border predicted by the model can be explained by inspecting the geometrical properties of these attraction basins (Gursky, 2011).

From this inspection, the following two mechanisms responsible for the observed robustness can be elucidated. First, the initial Hb profile is a monotonously decreasing function of A/P position, while the basin boundary to be crossed is a monotonously increasing one, i.e., these curves have opposite dependencies on the A/P position. This purely geometrical fact evidently prescribes a smaller variation of the intersection point when the basin boundary is changing due to the variance of Bcd concentration, as opposed to the case if the curves would jointly rise or jointly fall along the A/P axis (Gursky, 2011).

The second mechanism is associated with the specific nonlinear form of the response curve. The gap gene cross regulation of hb bends the response line exhibited in absence of this regulation. This bending e®ectively reduces the Hb positional variance by about half. In terms of attractors, this bending is controlled by the fact that a basin boundary responsible for the hb border formation does not change monotonously, but oscillate in the state space with the changing Bcd profile (Gursky, 2011).

The results show that the full range of the hb positional variance is broken down into two almost equal parts, the anterior and posterior ones. These parts are associated with two families of the Bcd individual profiles (Family I and Family II, respectively) and two di®erent mechanisms of hb border formation. The Bcd profiles from Family I lead to the hb border formation as a switch from a hb/ON attractor in a hb-expressing nucleus to a hb/OFF attractor in a hb-nonexpressing nucleus, while for Family II the border forms with the help of an attracting invariant manifold in a hb-nonexpressing nucleus. Since the difference between the two families is in the amplitude of the Bcd profiles, it is concluded that Bcd profiles of high amplitude canalize by a dynamical mechanism different from those of lower amplitude. Each dynamical mechanism provides only half of the full variance for the hb border, but in two adjacent spatial domains. Therefore, the change of the dynamical mechanism that happens with rising Bcd amplitude e®ectively doubles the variance (Gursky, 2011).

The hb border positions from the more posterior range are placed posterior to the spatial position of a bifurcation annihilating attractor A3. This bifurcation position delimits the anterior and posterior dynamical regimes in the model. Therefore, the Bcd profiles from the second family shift the hb border to the posterior dynamical regime, which is characterized by an active role of an attracting invariant manifold in the pattern formation (Gursky, 2011).

The results indicate that the posterior range of hb positional variation is almost equal to the anterior one only due to smaller variation of the Bcd profiles in Family II compared to Family I. This suggests that the solutions in the anterior and posterior dynamical regimes have quite different sensitivity rates to variation of the Bcd concentration. For Family I, the standard deviation for the hb border position is 2.6 times less than for the Bcd threshold position, while it is only 1.4 times less in the case of Family II. This difference can be explained by an observation that Bcd profiles of higher amplitude correspond to the linear part of the response curve, and this is a consequence of specific regulatory interactions in the gap gene circuit as explained further (Gursky, 2011).

The model was used (1) to study the canalization mechanisms based on the assessment that the model provides one of the best spatio-temporal precision for the description of gap gene expression. This model is an approximation to a more general model of gene regulation, which should be grounded on the statistical-mechanical formalism. One possible limitation is the linear approximation for the argument of the nonlinear regulation function g. The canalization mechanisms described in terms of attractors and attraction basins generally depend on the structure of the model that predicts these attracting states. Therefore, an important direction for future investigations should be verification of the proposed mechanisms in a phase space of a more general model (Gursky, 2011).

The nonlinear nature of the Bcd readout by the gap gene circuit is clearly represented in a specific nonlinear form of the response curve showing the Bcd dependence of the hb border position in the model. The nonlinear part of the curve can be explained by the regulatory actions on hb from the other gap genes. In particular, a regulatory analysis in the full model revealed that the regulatory interactions between hb, gt, and Kr underlie the folding part of the response curve. The gap gene cross-regulation also participate in the linear parts of the response curve by tuning the incline of these parts (Gursky, 2011).

It was previously pointed out that the gt and Kr expression borders in the anterior part of the A/P axis show large variation in the model in response to Bcd variation because the model is missing some regulators in this part. For example, these gt and Kr borders are absent in the solutions from class III. This fact raises doubts on the specific folding part that the response curve exhibits in the middle range of the Bcd concentration values. On the other hand, the folding part exists only for the Bcd profiles associated with the solutions from class I, with all expression borders formed correctly, which means that an essential portion of the artificial variation of the gt and Kr borders can be excluded from the consideration without affecting the folding form of the curve (Gursky, 2011).

The model was investigated on the ensemble of Bcd profiles normalized by the alternative method, which provided lower Bcd variance. One used this method as an artificial limit case, in which the ensemble possessing minimal Bcd variance was dealt with, and it was applied for the crosschecking purposes (Gursky, 2011).

No essential discrepancy was found in the mechanisms of hb border formation and canalization for the two normalization methods. A distinct bifurcation structure in the model with the new parameter values does not lead to changes in the solutions during the biologically important time. The model preserves an attracting invariant manifold related to the posterior dynamical regime. The same border formation mechanisms appear except the one associated with the attractor/manifold transition. It is important that, even though the second family of Bcd profiles does not appear in the alternative normalization case, the invariant manifolds still play their role in adjusting the border position. The model also demonstrates an essentially nonlinear response curve for the hb border. Therefore, the conclusions formulated above are robust with respect to the choice of the normalization method, and, in more general terms, they should be valid for different estimates of the actual Bcd variance (Gursky, 2011).

This correspondence can be explained by the fact that the parameters A and l obtained for the alternatively normalized Bcd profiles form a subset in similar parameters obtained in the case of the basic normalization method. Roughly speaking, the alternatively normalized Bcd profiles can be associated with Family I. In particular, this means that the Bcd data rescaled according to the alternative algorithm support the conclusion formulated above about different dynamical mechanisms of canalization for Bcd profiles of different amplitude (Gursky, 2011).

There is an important issue concerning the comparison of the Bcd variance filtration rates. The calculations reveal that, for the basic normalization method, the Hb positional variation of 1.3%EL in the model output follows from the Bcd positional variation of 4.5%EL, thus implying that more than 70% of the positional variance has been filtrated. The same calculations for the alternative normalization method give the filtration rate of approximately 60%. Therefore, the filtration still happens in the model even if Bcd profiles are normalize according to the precisionist hypothesis. This result is quite expected since the reported dynamical mechanisms underlying the processing of the Bcd variation in the model are valid irrespective of the absolute variation range. Whatever actual variation the Bcd morphogen exhibits, the nonlinear model response translates it to a smaller variation of the target gene patterns (Gursky, 2011).

It is concluded that the formation of hb border is coded by the intersection between the maternal Hb gradient and a boundary between attraction basins in the gap gene dynamical system. Small positional variance for hb border can be explained by the geometrical properties of this basin boundary and its nonmonotonic dependence on the Bcd concentration. Main features of the phase portraits underlying the canalization mechanisms do not depend on the normalization method for Bcd (Gursky, 2011).

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).

The Drosophila gap gene giant has an anterior segment identity function mediated through disconnected and teashirt

The C2H2 zinc-finger-containing transcription factors encoded by the disconnected (disco) and teashirt (tsh) genes contribute to the regionalization of the Drosophila embryo by establishing fields in which specific Homeotic complex (Hom-C) proteins can function. In Drosophila embryos, disco and the paralogous disco-related (disco-r) are expressed throughout most of the epidermis of the head segments, but only in small patches in the trunk segments. Conversely, tsh is expressed extensively in the trunk segments, with little or no accumulation in the head segments. Little is known about the regulation of these genes; for example, what limits their expression to these domains? This study reports the regulatory effects of gap genes on the spatial expression of disco, disco-r, and tsh during Drosophila embryogenesis. The data shed new light on how mutations in giant (gt) affect patterning within the anterior gt domain, demonstrating homeotic function in this domain. However, the homeosis does not occur through altered expression of the Hom-C genes but through changes in the regulation of disco and tsh (Sanders, 2008).

disco and disco-r, referred to together as the disco genes, and teashirt (tsh) are differentially expressed in the embryonic head and trunk segments and are therefore markers for head and trunk segment types. In the head segments the disco genes are required for the proper development of the larval mouthpart structures, while in the trunk segments, these genes are necessary for development of the Keilin's organs, small thoracic sensory structures and some peripheral neurons. By contrast, tsh is necessary for proper development of most of the ventral trunk epidermis. Both the disco genes and tsh are also members of the proximal-distal patterning network. The disco and tsh genes encode C2H2 zinc-finger transcription factors that are expressed early in embryonic development with precise, nearly reciprocal expression patterns in the trunk and head segments, but not much is known as to how these patterns are established. What is known is that ectopically expressing tsh in the head segments converts the expression of the disco genes to a trunk-like pattern. The Spalt major (Salm) protein represses tsh expression in the posterior labial segment, but otherwise little is known regarding the regulation of disco and tsh—in particular, what factors distinguish the head and trunk modes of expression. The gap genes are logical candidates for this role (Sanders, 2008).

Patterning the Drosophila embryo involves initial establishment of the axes, regionalization of the embryo, definition of the segments and their polarity, and the specification of unique identities to each segment. The early acting components of this genetic cascade include both maternally and zygotically expressed genes that set in motion the segmentation and segment identity processes. The gap genes are among the earliest zygotic factors involved in these processes. Regulated by maternal morphogens in the blastoderm, and by one another, these genes act via overlapping gradients to divide the embryo into broad regions and to regulate the expression of downstream segmentation genes (Sanders, 2008).

Comparative studies in other insects have revealed significant conservation in the function of many segmentation genes, but less clear is the functional conservation of the gap genes between insect species. Earlier studies -- one in Tribolium castaneum, examining a giant (gt) homolog (Tc'giant), and one in Oncopeltus fasciatus, examining a hunchback (hb) homolog (Of'hb) -- conclude that the function of these gap genes is one of segmentation and segment identity, differing somewhat from the segmentation function characterized in Drosophila. This difference is likely due to the differential patterning of long vs. short germ-band insect embryos. The results presented in this study indicate that Drosophila gt, in fact, has an embryonic segment identity role similar to that observed in Tc'giant. Surprisingly, this identity function arises not only through changes in homeotic (Hom-C) gene expression, but also from the regulation of disco and tsh. This, in conjunction with other gap genes, defines the position of the embryonic head and trunk segment types (Sanders, 2008).

To explore the regulation of the disco gene during embryogenesis, disco mRNA accumulation was studied in homozygous gap mutant embryos. disco is normally expressed in the clypeolabrum, the optic lobe region, the antennal segment, the gnathal segments (mandibular, maxillary, and labial), the embryonic leg primordia, and transiently in similar positions in the abdominal segments and the proctodeum. Five homozygous gap gene mutants exhibited altered disco mRNA distribution -- hunchback (hb), Krüppel (Kr), giant (gt), tailless (tll), and caudal (cad). Of these, hb, Kr, and gt affected the gnathal/thoracic disco expression domains. disco-r mRNA accumulation was examined in hb12, Kr2, gtQ292, and gtX11 mutant embryos. Alterations in disco-r expression mirrored those of disco. Because the effects on disco and disco-r mRNA accumulation appeared to be identical, the remaining studies focused on the regulation of disco. It is noted that gt mutations had a particularly interesting effect, indicating a central role for gt in disco regulation and possibly head-trunk boundary formation. Therefore, this study concentrated on gt. Indeed, the effects of hb and Kr could be interpreted through their known cross-regulation of gt (Sanders, 2008).

Two significant conclusions are drawn from this study: (1) In its anterior expression domain, gt acts in both segment identity and segmentation roles, and these two roles are functionally separable; and (2) the distinction between the gnathal and trunk segment types is determined by the gap genes and is reflected by the head and trunk expression patterns of disco and tsh, which appear to be regulated by a series of repressive interactions (Sanders, 2008).

The assertion that gt acts in both segment identity and segmentation is based upon the following observations:

  1. The dorsal cuticle of gt embryos displayed ectopic dorsal hair development anterior to the first thoracic segment, and the ventral cuticle of some gtQ292 individuals displayed first thoracic-like denticle belts in the labial segment. This ventral transformation was masked in some gt mutant alleles (i.e., gtX11).
  2. In many gt mutant embryos, the reduction or loss of En protein from the posterior labial segment likely prevented the formation of the labial/first thoracic segmental groove, resulting in the fusion of the two segments and, consequently, the presence of only one organizer for denticle development in the fused segment.
  3. In the labial segment of gt mutant embryos, the expression of the homeotic genes Scr, Antp, disco, and tsh recapitulated their expression in the first thoracic segment, indicating a transformation to a first thoracic identity.
  4. In gt mutant embryos, expression of the gnathal-specific Hom-C gene pb is significantly reduced in the labial segment, indicating a loss of gnathal identity. Such a separation of segmentation and segment identity functions as this study describes for the anterior domain of different gt alleles has been observed in the posterior gt domain.

Initial characterizations of the gt mutant phenotype, based on SEM studies, described a fusion between the labial, first thoracic, and second thoracic segment, which, later in development, resolved such that the first and second thoracic segments separated, but the labial segment remained fused with the thoracic segment. The loss of the third (labial) En stripe has been described as indicating the deletion of the labial posterior compartment, and it was suggested that this may be the extent of the 'gap' phenotype in the anterior gt domain. The current examination of gtQ292 embryos revealed clear indications of a homeotic transformation of the labial segment to a first thoracic identity. The presence of ectopic hairs in the dorsal cuticle of gtX11 mutants has been noted, this observation was not related to a change in segment identity (Sanders, 2008).

En-expressing cells are the first to regress during the formation of the segment groove. There is an absolute requirement for En expression in cells adjacent to the developing groove. Thus, in gt mutant embryos, the amount of En accumulation retained in the posterior labial compartment likely determines the extent of segmentation that will occur between the labial and first thoracic segments. Embryos hemizygous for gtX11 almost completely lose the third En stripe, coinciding with the virtually complete fusion between the labial and first thoracic segments in these individuals. Consequently, a duplication of the first thoracic segment was never observed in embryos of this genotype. This lack of ventral denticle duplication in gtX11 embryos follows when considering the loss of En in the posterior labial segment. Since more of the labial En stripe remains in gtQ292 embryos, a segment border can form. Interestingly, both gtX11 and gtQ292 develop ectopic dorsal hairs anterior to the first thoracic segment. En staining revealed that at least a portion of the dorsal ridge fuses (or never properly separates) from the dorsal labial segment, creating a segment that resembles the first thoracic segment. It is likely that the ectopic dorsal hairs arise from the dorsal ridge, which has been transformed toward dorsal first thoracic identity (Sanders, 2008).

The case for a gt segment identity function is strengthened by the alterations in the homeotic genes expressed in the gnathal and thoracic regions. In all gtQ292mutants examined, the labial segment expressed Scr, Antp, and tsh. This combination of segment identity factors is normally found in the first thoracic segment. Further, the labial segment shows significant reduction or alteration in pb and disco expression, both markers of gnathal identity (Sanders, 2008).

There are two potential explanations for the differential effects on En accumulation and the ventral cuticle phenotype in the gt alleles that were examined. First, the available gtQ292 stock may, over time, have acquired second site suppressors responsible for the occasional persistence of En accumulation in the posterior labial segment. However, when this allele was crossed into a different genetic background, the presence of En accumulation and ventral cuticle transformation was still observed. If it is a second site suppressor of the gt mutation, then it must lie on the X chromosome carrying the gtQ292 allele. A second possibility is that the gtQ292 allele is a strong hypomorph, rather than an amorphic allele, and the residual Gt function is sufficient in some individuals to allow the labial En segmentation process to proceed, although the segment identity process remains faulty. Regardless of which explanation proves to be true, it appears that the anterior gt domain regulates embryonic patterning at two different levels -- segmentation and segment identity -- and that these two processes are functionally separable from one another (Sanders, 2008).

This conclusion is not without precedent. Early reports suggested a possible homeotic function in addition to the segmentation function of Gt in its posterior expression domain. Homeotic transformations and segmentation defects are observed in the mutant phenotypes of other gap genes. For example, in hb mutants, the loss of mid-abdominal segmentation is accompanied by mirror image duplications. The current results are significant, as they are the first to definitively demonstrate a segment identity role of the anterior gt domain (Sanders, 2008).

A recent study characterized the expression and function of a gt homolog in Tribolium (Tc'gt). As in Drosophila, Tc'gt mRNA is expressed in two primary domains -- one in the anterior of the embryo, overlying the gnathal region, and a second in the region of the third thoracic segment to the second abdominal segment. Although the anterior Tc'gt domain is similarly placed as compared to Drosophila, the posterior domain is shifted forward approximately five segments. RNA interference and morpholinos were used to knock down the expression of Tc'gt to explore its function. Interestingly, it was found that Tc'gt has a role in the identity specification of the maxillary and labial segments, but did not have a role in segmentation. The maxillary and labial segments were transformed to a first and second thoracic identity, respectively, while all three thoracic segments exhibit a third thoracic identity. There was no loss of the gnathal Tc'engrailed (Tc'En) stripes, although thoracic and abdominal Tc'En accumulation was affected to varying degrees in different embryos. Although the region affected by the loss of Tc'gt function is broader than the transformed region observed in Drosophila gtQ292 mutants, the nature of the homeotic change is quite similar. A gnathal segment(s) is transformed to a thoracic identity, and this identity change is separate from the segmentation process. The segment identity function for gt may have been present in the last common ancestor of the holometabolous insects, and the segmentation role of the anterior gt might have been acquired separately to accommodate the long germ-band mode of development (Sanders, 2008).

In the head, disco is expressed in most cells of the segmental epidermis, while there is little or no expression of tsh. By contrast, tsh is expressed throughout most of the trunk segmental epidermis, while disco is limited to the limb primordia. The genetic studies presented in this study demonstrate that the difference between head and trunk expression patterns, and therefore segment types, is dependent upon the gap genes, and particularly, on gt (Sanders, 2008).

In gt mutant embryos, both disco and tsh expression are altered reciprocally. disco expression is severely reduced in the labial segment and in fact is altered such that the remaining disco mRNA resembled the embryonic limb primordia expression observed in the thoracic segments. There is a concomitant expansion of tsh expression into the labial segment. It has been demonstrated that UAS-driven ectopic tsh expression in the gnathal segments reduces and alters disco expression such that it mimics the expression pattern of the thoracic segments (Robertson, 2004). Similarly, in gt mutants, it is the expansion of tsh expression into the labial segment that is responsible for the changes in disco expression. When both gt and tsh are absent, disco expression in the labial lobe recovers significantly, and the overall morphology of the labial segment and adjoining dorsal ridge is notably improved (Sanders, 2008).

The results may support a direct role for gt in the regulation of tsh. Although previous work demonstrated the requirement of Antp for appropriate tsh expression in the thoracic segments, and the loss of gt results in ectopic Antp protein in the labial segment, Antp is not required for ectopic tsh activation in the labial and maxillary segments of gt mutants. It is likely that gt directly limits the anterior expression of both tsh and Antp. Gt functions as a short-range repressor and has been shown to bind with high affinity to the CD1 sequence (TAT GAC GCA AGA) derived from the Kr regulatory region. There is a sequence ~0.5 kb upstream of the transcription start site of tsh that is similar to the CD1 sequence (TAT GAA GGA AGG), differing by only three bases. Although it remains to be investigated as to whether the Gt protein can bind to this sequence, the similarity in sequence to a known in vivo Gt-binding site supports direct repression of tsh by Gt (Sanders, 2008).

The results outline a model for the positioning of the gnathal/trunk boundary in the Drosophila embryo, involving a network of repressive factors. gt is a key player in this model. The anterior domain of gt is limited by its interactions with the zygotic gap genes hb and Kr, both of which act as repressors of gt expression. Gt in turn limits tsh expression, preventing expression in the labial segment. tsh expression is further limited by the expression of salm in the anlagen of the maxillary and labial segments. However, the results demonstrate that salm alone is insufficient for repressing tsh in the posterior labial segment in embryos lacking gt function. In the trunk segments, Tsh limits disco expression to only the embryonic appendage primordial, so that, lacking Tsh, disco expression is expanded through much of the gnathal segments (Sanders, 2008).

Questions remain regarding the activation of tsh and disco. disco mRNA accumulates in the cellular blastoderm prior to gastrulation, implying the involvement of maternal factors or early acting gap genes. However, none of the gap mutants that were tested affected the initiation of the initial anterior disco domain. disco was significantly affected by the loss of maternal bcd. This suggests that bcd and/or maternal hb may play a role in the initial activation of the anterior disco domain, after which Tsh acts to limit the disco to the gnathal region. tsh expression initiates prior to gastrulation, first with a central stripe that resolves to form a striped pattern reminiscent of the pair-rule genes. Again, none of the gap genes that were examined eliminated tsh expression. Although Kr is expressed in the central region of the embryo, where tsh is first transcribed, it is not the activator of tsh. Early tsh may respond to maternal factors and/or a combination of gap gene products in a concentration-specific manner, which would account for the inability to detect a single activator in the gap mutant studies. Finally, although several instances were found where Tsh represses disco, there is no evidence that the reverse is true. What leads to the repression of tsh and concomitant maintenance of disco in the maxillary segment of gt mutant embryos is unclear at this time (Sanders, 2008).

The Drosophila gap gene giant regulates ecdysone production through specification of the PTTH-producing neurons

In Drosophila, hypomorphic mutations in the gap gene giant (gt) have long been known to affect ecdysone titers resulting in developmental delay and the production of large (giant) larvae, pupae and adults. However, the mechanism by which gt regulates ecdysone production has remained elusive. This study shows that hypomorphic gt mutations lead to ecdysone deficiency and developmental delay by affecting the specification of a pair of bilaterally symmetric neurons (PG neurons) located in the cerebral labrum portion of the brain that produce prothoracicotropic hormone (PTTH). The gt1 hypomorphic mutation leads to random loss of PTTH production in one or more of the 4 PG neurons in the larval brain. In cases where PTTH production is lost in all four PG neurons, delayed development and giant larvae are produced. Since immunostaining shows no evidence for Gt expression in the PG neurons once PTTH production is detectable, it is unlikely that Gt directly regulates PTTH expression. Instead, it was found that innervation of the prothoracic gland by the PG neurons is absent in gt hypomorphic larvae that do not express PTTH. In addition, PG neuron axon fasciculation is abnormal in many gt hypomorphic larvae. Since several other anteriorly expressed gap genes such as tailless and orthodenticle have previously been found to affect the fate of the cerebral labrum, a region of the brain that gives rise to the neuroendocrine cells that innervate the ring gland, it is concluded that gt likely controls ecdysone production indirectly by contributing the peptidergic phenotype of the PTTH-producing neurons in the embryo (Ghosh, 2010).

In many insects, the regulation of ecdysone production in larvae involves two major components: a pair of bilaterally symmetric neurons (PG neurons) and the prothoracic gland, the endocrine organ that actually produces and secretes ecdysone. In Drosophila, the PG neurons directly innervate the prothoracic gland and induce production and secretion of ecdysone by releasing an adenotropic peptide hormone called prothoracicotropic hormone (PTTH). PTTH signals through the receptor tyrosine kinase Torso to activate a RAS/ERK cascade that ultimately stimulates transcription of ecdysone biosynthetic enzymes. Intriguingly, elimination of PTTH signaling delays the rise in ecdysone titer and the onset of pupation by approximately 5 days resulting in large pupae and adults, similar to those produced by gt hypomorphs. The similarity in phenotype between gt hypomorphs and loss of PTTH signaling prompted an investigation of whether gt in some way controls PTTH signaling. This paper reports that rather than directly regulating PTTH production in the PG neurons, gt indirectly controls PTTH and subsequent ecdysone production by influencing the development of the PTTH-producing PG neurons (Ghosh, 2010).

In the absence of any evidence supporting a role for Gt in regulating ptth transcription, attempts were made to determine if loss of Gt affects the specification of PG neurons. Besides ptth, the only other described marker for PG neuron fate is the Feb211-Gal4 enhancer trap line that contains an insertion into an unknown gene on chromosome 3. Analysis of expression from this enhancer line in gt1 mutant larvae revealed a similar stochastic loss of GFP expression in different numbers of PG neurons as seen for ptth expression itself. The all or none response observed for both ptth and Feb211-Gal4 expression in gt hypomorphs is consistent with a stochastic loss of PG neurons in these mutants (Ghosh, 2010).

The gt1 mutation has been shown to be associated with two spontaneous insertions, one near the 5' region of the gene and the other in the 3' region. It was predicted that these insertions likely affect gt expression levels during embryogenesis, and altered gt expression may affect the specification of different neuron subtypes within the brain including precursors that give rise to the PG neurons. To examine this issue in more detail, attempts were made to determine if Gt expression is reduced or if fewer cells express Gt in gt1 mutant animals compared to wild type embryos. Under identical staining and exposure conditions, Gt staining intensity in the control embryo is stronger compared to the gt1 embryo. The primary staining is in an anterior medial position that is anatomically close to or overlapping with the pars intercerebralis (PI) and pars lateralis (PL) region of the brain that gives rise to a number of neurosecretory cells including several neurons that innervate the corpus cardaicum and corpus allatum, two other portions of the ring gland. The PI placode derives from neuroepithilium that expresses tailless and orthodenticle, two anteriorly expressed gap genes. The exact origin of the PG neurons has not been established, but they may be derived from two other placodes that reside more posterior to the PI region (see de Velasco, 2007). Interestingly, a prominent cluster of approximately 5 bilateral posterior midline neurons is noted that express Gt in stage 13 embryos. In equivalently staged gt1 mutant embryos, the number of cells in this cluster that express Gt is reduced to two to four cells. Similarly, staining of a cluster of three cells positioned anteriorly on the midline axis is also dramatically reduced in the gt1 sample (Ghosh, 2010).

These results suggest that the specification of multiple neuron subtypes in the brain is likely affected in the gt1 mutant animals. Since no lineage tracers are available to directly determine if the PG neurons are derived from earlier precursors that express Gt, an indirect assay was used to determine if PG neurons are mis-specified in gt1 hypomorphs. Previous axon tracing experiments have revealed that the PG neurons are the only neurons that innervate the prothoracic gland. To determine if gt affects the specification of PG neurons, cysteine string protein (Csp) distribution was examined on ring gland cells. Csp is enriched in synaptic boutons. Csp co-localizes with PTTH in axon terminals and boutons on the surface of wildtype prothoracic gland cells as well as in gt1 mutant larvae that still show PTTH expression. In contrast, developmentally delayed gt1 larvae in which PTTH expression is absent from all 4 PG neurons, no Csp-containing boutons are observed on prothoracic gland cells. In these same larvae however, Csp-containing axons and boutons are still seen within the corpus cardiacum and corpus allatum, two regions of the ring gland that are innervated by different sets of neurons H, yellow arrowheads. It is concluded that loss of Gt affects the development of the PG neurons since its absence leads to a loss of prothoracic gland innervation. At this point, it cannot be distinguished if Gt directly affects the specification of PG neurons, or if it affects PG neuron development in a cell non-autonomous manner, perhaps by affecting cell-cell interactions during early cortex development. Nevertheless, these experiments add gt to the list of anteriorly expressed gap genes that affect the specification of the proto-cerebrum (Ghosh, 2010).

In Manduca sexta PTTH is believed to have a tropic effect on the larval prothoracic gland as it has been shown to induce general protein synthesis. Similar to Manduca sexta, prothoracic gland cells in Drosophila are mitotically quiescent during larval stages. Nevertheless, the gland cells exhibit substantial growth during the three larval stages and this growth is characterized by the formation of polytene chromosomes and an increase in size of the gland cells. It was observed that gt1 mutant larvae exhibiting unilateral innervation of the prothoracic glands consistently produced an asymmetrically sized gland in which the innervated portion was significantly larger than the non-innervated side. Measuring the diameter of DAPI stained nuclei revealed that cells on the non-innervated side contained nuclei that are significantly smaller compared to the innervated side. This difference was consistently observed in all samples that failed to innervate one of the prothoracic glands indicating that DNA synthesis is likely reduced in absence of prothoracic gland innervation. Curiously, when both sides lacked innervation, the ring gland did not appear substantially smaller than wild type. However these glands are from developmentally delayed larvae in which the extra growth time likely enables them to 'catch up' to the wildtype in terms of prothoracic gland size. Ultimately it will be necessary to examine PTTH null mutants to prove that PTTH, and not some other factor, is the tropic signal secreted from the PG neurons. However, the recent finding that PTTH signals through the Drosophila receptor tyrosine kinase (RTK) Torso is certainly consistent with the idea that PTTH is the tropic factor since the Torso signal is transduced through the canonical Ras-Raf-ERK pathway (Rewitz, 2009) which is known to regulate cell proliferation in many systems (Ghosh, 2010).

In addition to the absence of prothoracic gland innervation in many gt1 hypomorphic larvae, it was noted that there is an enhanced frequency of axon misrouting in gt mutant larvae that still show ptth-HA expression in one or more of their PG neurons. In wild type larvae, the polarized PG neurons in the left lobe of the brain send out their axons from the cell body across the central axis of the CNS to the right brain lobe. There the axon forms a loop with a left hand twist and then projects anteriorly to innervate the prothoracic gland cells on the right half of the ring gland. Similarly the neurons in the right lobe extend their axons into the left lobe and innervate the left half of the ring gland. This innervation pattern is most clearly revealed in gt1 mutant larvae retaining one pair of the bilateral PG neurons. For example, in a gt1 mutant larva that retains the right side set of PG neurons, there is innervation only within the left prothoracic gland. In wild type larvae, the axon tracts from each pair of PG neurons are almost parallel to each other at the base of the ring gland and rarely exhibit cross (only 1 out of 23 CNSs from wt controls showed branching). However, in the gt1 CNSs containing one or two PG neurons in only one brain lobe, the axons are often seen branching at the base of the ring gland and innervating both prothoracic glands. Interestingly similar branching events were observed in gt1 samples that have all four PG neurons. This suggests that the cross innervations are not likely to be caused by a mechanism that tries to compensate for the lack of innervation on one side of the prothoracic gland. Consistent with this view, it was found that in certain cases such branching events caused excessive innervation of one of the prothoracic glands at the cost of the other. Approximately 24% of gt1 CNSs that retained at least one PG neurons showed cross innervation events with clear branching at the base of the ring gland (Ghosh, 2010).

These results suggest that gt is required not only for correct specification of the PG neurons, but also influences the projection of PG neurites to their target tissue. At present, it is not possible distinguish if these axon guidance defects represent reduction in the expression of intrinsic factors within the PG neurons that respond to guidance cues or whether Gt not only affects the specification of the PG neurons themselves, but also surrounding neurons that might provide guidance cues. Ultimately, lineage tracing experiments will be required to determine which neurons are descendent from Gt-expressing cells in order to address these issues (Ghosh, 2010).


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giant: Biological Overview | Evolutionary homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 25 March 2015

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