BIOLOGICAL OVERVIEW

In spite of the structural and functional similarities between gooseberry proximal and gooseberry distal, they show considerable divergence. Their exon structures have diverged, as has their regulation. gsb-n has five exons while gsb-d has two. They are each regulated by different proximal promoter regions that interact with different enhancers. gsb-d is induced by pair rule genes and is involved with wingless in an autoregulatory loop. gsb-n's role in differentiation can be carried out by gsb. gsb-n is regulated by gooseberry distal and has a more minor role in neuroblast committment. It would seem that gsb-n is redundant.

There is precedence for such overlap in function. An example is found in the working relationship between engrailed and invected. engrailed has a broader regulatory role, and invected is specialized for carrying out the tasks dictated by engrailed. A similar division of labor is found for gsb and gsb-n. Apparently gsb behaves as a director-general, leaving the more specialized tasks to gsb. What additional function might gsb-n serve that cannot be carried out by gsb? To date, the answer is unknown.

Ectopic expression of either the Drosophila gooseberry-distal or proximal gene causes alterations of cell fate in the epidermis and central nervous system

Previous studies have shown that the segment polarity locus gooseberry, which contains two closely related transcripts gooseberry-proximal and gooseberry-distal, is required for proper development in both the epidermis and the central nervous system of Drosophila. This study examines the roles of the gooseberry proteins in the process of cell fate specification by generating two fly lines in which either gooseberry-distal or gooseberry-proximal expression is under the control of an hsp70 promoter. Ectopic expression of either gooseberry protein causes cell fate transformations that are reciprocal to those of a gooseberry deletion mutant. The results suggest that the gooseberry-distal protein is required for the specification of naked cuticle in the epidermis and specific neuroblasts in the central nervous system. These roles may reflect independent functions in neuroblasts and epidermal cells or a single function in the common ectodermal precursor cells. The gooseberry proximal protein is also found in the same neuroblasts as gooseberry-distal and in the descendants of these cells (Zhang, 1994).

Both gsbd and gsbp encode putative transcription factors, each containing a homeodomain and a paired domain. In mutants deficient for both gsb genes, the naked cuticle of each epidermal segment is eliminated and replaced with a mirror-image duplication of the denticle belt. The alterations in the CNS include the deletion of the posterior commissure and the CQ neurons as well as the duplication of aCC, pCC and RP2 neurons (Patel, 1989). The regions expressing the gsb genes closely correspond to the positions where structures are eliminated in gsb mutants. In the epidermis, gsbd has been localized to the region bracketing the parasegmental boundary of each segment and, in the CNS, gsbd and gsbp are expressed in a subset of NBs and neurons in the same region (Baumgartner, 1987; Ouellette, 1992; Gutjahr, 1993; Zhang, 1994 and references therein).

In gsb mutants, the naked cuticle of each segment is eliminated and replaced by a mirror-image duplication of the denticle belts. In the cuticles of heat shocked hs-gsbd and hs-gsbp embryos, groups of denticles or entire denticle belts are replaced with naked cuticle. The severity of the phenotype correlates with the timing of the heat shock. Heat shocks from 2.75 to 3.25 hours transform complete segments while those after 4 hours can transform sections of segments. Multiple heat shocks generate embryos with completely naked cuticles. To verify that cell fates are being transformed, the Keilin’s organs, which are located at the parasegmental border in each thoracic segment, were examined. In gsb mutants Keilin’s organs are deleted, while in heat shocked hs-gsbd and hs-gsbp embryos, ectopic Keilin’s organs occasionally develop. These results indicate that the cells in the anterior portion of the segment have been respecified and that the cuticle phenotype of heat shocked hs-gsbd and hs-gsbp embryos is nearly reciprocal to that of gsb mutants (Zhang, 1994).

It was somewhat surprising that heat shocked hs-gsbp was able to give pattern transformations similar to heat shocked hs-gsbd since gsbp is not normally expressed in the epidermis at the times that the heat shocks were administered (Ouellette, 1992; Gutjahr, 1993). Therefore, the ability was examined of heat shocked hs-gsbd and hs-gsbp to rescue the phenotype of Df(2R)IIX62 embryos, which are deleted for the gsb locus. With a series of three heat shocks both hs-gsbd and hs-gsbp are able to rescue the formation of naked cuticle (Zhang, 1994).

To determine whether changes at the molecular level correlate with the cuticle phenotype of heat shocked hs-gsb (hs-gsbd or hs-gsbp) embryos, the expression of gsbd and two other segment polarity genes wg and en was examined. hs-gsb embryos given heat shocks between 2.5 and 4 hours after egg-laying, activate ectopic expression of the endogenous gsbd gene. The ectopic stripe of gsbd expression is maintained 2 hours after heat shock and is dependent on the presence of a wild-type copy of the gsb locus. Thus the ectopic gsbd expression must be due to the activation of the endogenous gsbd gene. The activation of ectopic gsbd is accompanied by the formation of an ectopic parasegmental groove flanked by inverted ectopic stripes of wg and en. Thus, it appears that the ubiquitous expression of gsb induces a mirror-image duplication of the region bracketing the parasegmental boundary. Heat shocks after 4 hours also transform the denticle belts into naked cuticle. These late heat shocks do not lead to ectopic en expression but correlate with a low level of wg expression throughout the segment (Zhang, 1994).

In addition to epidermal expression, gsbd is expressed in a subset of NBs and their progeny in the posterior portion of each segment (Gutjahr, 1993). gsbp is first expressed in NBs and is found at high levels in the progeny of these NBs (Ouellette, 1992). Later as the germ band starts to retract, low level of gsbp becomes detectable in the epidermis. Analysis of the expression patterns of gsbd and gsbp generally agrees with the results by Gutjahr (1993), who found that in the CNS gsbd is expressed in row 5, row 6 NBs and transiently in NB7-1. One exception is that gsbd was found to be continually expressed in NB7-1. gsbp is also expressed in the NBs of row 5, row 6 and 7-1, and thus gsbp appears to follow gsbd expression in the CNS. To confirm that gsbp is indeed expressed in all gsbd-positive NBs, a gsbp/lacZ line was used in which a lacZ gene is under the control of a gsbp promoter. Using double label experiments and confocal microscopy, it was found that the gsbp/lacZ construct is expressed in the exact same set of NBs and neurons as is the gsbp protein. By double labeling with anti-β-galactosidase (β-gal) and anti-gsbd antibodies, it can be seen that β-gal (representing gsbp) is expressed in gsbd-positive NBs and their progeny (Zhang, 1994).

It should be noted that the level of gsbd expression in GMCs and neurons is quite low and fades away during germ band retraction, whereas gsbp retains high levels of expression in GMCs and neurons as neurogenesis progresses. This suggests that, in the CNS, gsbd functions primarily in NBs and gsbp in GMCs and progeny neurons (Zhang, 1994).

To study the effects of heat-shock-induced ectopic gsbd expression on NB cell fate, the anti-ac antibody was used. In stage 9 wild-type embryos, four rows of NBs are present within each CNS hemisegment and the anti-ac antibody recognizes NBs in row 3 and 7. In gsb mutants, the ac protein is additionally expressed in the two NBs at the position of row 5. In heat shocked hs-gsbd embryos, ac-positive NBs are restricted to the NBs of row 7 in each CNS hemisegment and the staining of the row 3 NBs is eliminated. These reciprocal transformations suggest that, in gsb mutants, row 5 NBs are transformed into row 3 and that, in heat shocked hs-gsbd embryos, row 3 NBs are transformed into row 5 (Zhang, 1994).

This conclusion was strengthened by analyzing wg gene expression in heat shocked hs-gsbd embryos. To monitor wg expression, a wg enhancer trap line CyO/wg17en40 was used in which β-gal expression is regulated by the wg promoter. In wild-type embryos, β-gal from the wg enhancer trap is expressed only in row 5 NBs (Fig. 4C; Doe, 1992). In heat shocked hs-gsbd embryos, additional NBs anterior to row 5 NBs express β-gal (Fig. 4D), suggesting the transformation of row 3 NBs into row 5 NBs (Zhang, 1994).

Ectopic expression of the gsb genes causes CNS pattern defects reciprocal to that of a gsb mutant The effect of ectopic gsbd or gsbp on axonal pattern in the mature embryonic CNS was first examined by anti-HRP antibody staining. Heat shocks at 2.75 to 3.25 hours of development for hs-gsbd embryos and 4.5-5.0 hours for hs-gsbp embryos give the most consistent alterations to the pattern of axons. The defects in heat shocked hs-gsbd and hs-gsbp embryos are very similar, typified by the fusion of the two commissures within each segment and the elimination of the longitudinal connectives (Zhang, 1994).

In order to follow cell fate changes in heat shocked hs-gsb embryos, anti-Eve antibody staining was examined, because anti-Eve antibodies recognize subsets of neurons with different behaviors in gsb mutants: CQ neurons are deleted, aCC, pCC and RP2 neurons are duplicated, and EL neurons appear to be unaffected. Anti-Eve antibody staining of heat shocked hs-gsbp embryos reveals pattern alterations nearly reciprocal to that of gsb mutants. The Eve expression characteristic of the RP2 and EL neurons is eliminated in most segments. The eve expression expected in aCC and pCC neurons also appears to be eliminated in some segments, and there are putative duplications of the CQ neurons (Zhang, 1994).

Using anti-Eve antibody stainings, it is difficult to distinguish unambiguously the aCC and pCC neurons from the adjacent CQ neurons in heat shocked hs-gsbp or gsb mutant embryos. To resolve this problem, the A31 enhancer trap line was used. The A31 enhancer trap line has a P[lacZ] element inserted into the fasciclin II gene. In the CNS of A31 embryos, β-gal is only expressed in the aCC and pCC neurons at early stage 12. The A31 enhancer trap line was crossed into the hs-gsbp, hs-gsbd and gsb mutant backgrounds and the behavior of aCC and pCC neurons was examined by anti-β-gal antibody stainings. Two pairs of aCC and pCC neurons are in each CNS segment of wild-type embryos, and apparent duplication of aCC and pCC neurons occurs in gsb mutant embryos neurons occurs occasionally in heat shocked hs-gsbp embryos (heat shocking at 4.5 hours) and occurs much more frequently in heat shocked hs-gsbd embryos (heat shocking at 3.0 hours). Since the aCC and pCC neurons are never duplicated in heat shocked hs-gsbp embryos, the duplicated neurons are most likely CQ neurons (Zhang, 1994).

Neurons deleted in gsb mutants normally express the gsbp protein, whereas neurons duplicated in gsb mutants do not express the gsbp protein The neuronal phenotypes observed in gsb mutant and heat shocked hs-gsbp embryos suggest that gsb function is both necessary and sufficient for the cell fate specification of a subset of posterior neurons, like the CQ neurons. It is important to examine whether gsbp is indeed expressed in these neurons but not in neurons which are duplicated in gsb mutants and deleted in heat shocked hs-gsbp embryos. Again, Eve expression was used as the neuronal marker (Zhang, 1994).

Double stainings of anti-gsbp and anti-Eve antibodies in wild-type embryos show that anti-Eve-stained CQ neurons also stain with anti-gsbp antibody, while the EL neuron cluster is outside the gsbp staining region. At a more dorsal focal plane of the same embryo, the aCC and pCC neurons are stained by the anti-Eve antibody and appear to be above the gsbp-expressing neurons, while the RP2 neurons lie just anterior to the gsbp-expressing region (Zhang, 1994).

It is known that the aCC and pCC neurons are progeny from NB1-1. During neurogenesis, they migrate anteriorly to the region just dorsal to the gsbp-expressing neurons. To confirm that the aCC and pCC neurons are not labeled by anti-gsbp antibodies, a double labeling experiment was done with the A31 enhancer trap line. In the lateral view of an A31 embryo double labeled by anti Gsbp and anti-β-gal antibodies, it is clear that the aCC and pCC neurons are just outside the gsbp expression region. Thus, it is confirmed that gsbp is normally expressed in CQ neurons, but not in aCC, pCC, RP2 or EL neurons (Zhang, 1994).

gooseberry and gooseberry neuro in the central nervous system and segmentation of the Drosophila embryo

The gooseberry locus of Drosophila consists of two homologous Pax genes, gooseberry neuro (gsbn) and gooseberry (gsb). Homologous recombination was obtained of null mutants of either gene as well as a deficiency inactivating only gsbn and gsb. This analysis shows that (1) gsbn null mutants are subviable while all surviving males and most females are sterile; (2) gsb and gsbn share overlapping functions in segmentation and the CNS, in which gsbn largely, but not completely depends on the transcriptional activation by the product of gsb; (3) as a consequence, in the absence of gsbn, gsb becomes haploinsufficient for its function in the CNS, and gsbn^-/-gsb^-/+ mutants die as larvae. Such mutants display defects in the proper specification of the SNa branch of the segmental nerve, which appears intact in gsbn^-/- mutants. Lineage analysis in the embryonic CNS showed that gsbn is expressed in the entire lineage derived from NB5-4, which generates 4 or 5 motoneurons whose axons are part of the SNa branch and all of which except one also express BarH1. Analysis of gsbn^-/-gsb-/+ clones originating from NB5-4 suggests that together the genes specify the SNa fate and concomitantly repress the SNc fate in this lineage and that their products activate BarH1 transcription. Specification of the SNa fate by Gsb and Gsbn occurs mainly at the NB and GMC stage. However, the SNa mutant phenotype can be rescued by providing Gsbn as late as at the postmitotic stage. A model is proposed how selection for both genes occurred after their duplication during evolution (He, 2013).

Although many transcription factors have been identified that control the cell fates of the ISN subtypes, little is known about the factors that determine the SN subtypes. The results demonstrate that both gsb and gsbn play essential roles in the specification of SNa MNs. The fact that in gsbn^-/-gsb-/+ embryos ectopic SNc projections are induced at the expense of the normal SNa projection suggests that, in the absence of a functional gsbn gene and the presence of only one functional gsb gene, MNs of the NB5-4 lineage acquire the SNc identity. It follows that gsb and gsbn specify the SNa fate and concomitantly repress the SNc fate, thereby subdividing the SN into SNa and SNc. This hypothesis is testable, once regulators of the SNc pathway are known, and plausible, as an analogous situation may exist for the subdivision between ISN, ISNb, and ISNd. In lim3 mutants, ISNb MNs fail to innervate their normal target muscles but project ectopically in ISNd. In addition, in eve mutants - even-skipped (eve) and dHb9 display non-overlapping expression patterns in the VNC and MNs - dHb9 is derepressed in MNs that in wild-type embryos innervate dorsal muscles through ISN. Conversely, in dHb9 mutants, eve is ectopically expressed in several MNs that normally express dHb9 and innervate ventral muscles through ISNb but now fail to innervate the ventral muscles and ectopically project with ISN to the dorsal muscle field. Therefore, mutual repression between motoneuronal subtypes might be a general mechanism by which subtype identities are determined. The expression patterns of both dHb9, which is not expressed in Gsbn-positive SNa MNs, and eve in MNs seem not to be changed in gsbn^-/-gsb-/+ mutants. Hence, it remains unclear whether mutual repression mechanisms act only as switch between motoneuronal subtypes or also between ISN and SN identities (He, 2013).

Although both gsbn and gsb are involved in the specification of the SNa MNs, it appears that gsb plays a more important role, as evident from the fact that while two doses of gsb/gsbn are required to properly determine the SNa fate, one of them has to be gsb whereas gsbn is dispensable in the presence of two functional copies of gsb. As gsb is no longer expressed in the CNS after stage 13, this further implies that the fate of the SNa MNs derived from NB5-4 is specified between stages 11 and 13. It has been demonstrated that, despite the considerably diverged C-terminal moieties of the coding sequences of gsb, gsbn, and paired (prd), their proteins can still largely substitute for each other's functions and their specific developmental roles reside in the difference between their cis-regulatory regions acquired during evolution. Accordingly, the differential roles of gsb and gsbn most probably result from the dependence on gsb of gsbn transcription in SNa MNs. Once gsbn is expressed in the NB5-4 lineage, it functions redundantly with gsb to activate downstream target genes, as no SNa phenotype is observed in the absence of Gsbn. The redundancy of gsbn with gsb becomes evident only when, in addition to the homozygous deletion of gsbn, a copy of gsb is removed, which results in the SNa phenotype and in turn can be rescued by the addition of a gsbn transgene. (He, 2013).

While gsbn and gsb are the only transcription factors shown to specify the identity of SNa, at least another transcription factor is involved in the determination of the SNa pathway as at least two SNa MNs express neither gsbn nor gsb but BarH1. Since all BarH1-positive motor axons project in the SNa nerves (innervating muscles 21–24, 8 and/or 5), while the 5 Gsbn-positive MNs derived from NB5-4 innervate muscles 22–24, 5 and perhaps 8 (but not muscle 21), muscle 21 is innervated by one of the two BarH1 SNa MNs that do not express Gsbn (He, 2013).

The expression of gsbn and gsb in the NBs of rows 5 and 6 raises the question of whether the SNa phenotype observed in gsbn^-/-gsb-/+ mutants simply results from a change in NB5-4 identity. A clue to this question provides the observation that in the absence of a functional gsbn gene, no SNa phenotype is observed if two functional copies of gsb are present. Since these are expressed mainly at the NB and GMC stages, but only at low levels or not at all in MNs, it is probable that the SNa fate is specified before the MN stage. Indeed the level of gsb provided by only one gsb gene in gsbn^-/-gsb-/+ mutants is sufficient to support the wild-type SNa fate in 10% of the NB5-4 clones analyzed, while in many of the remaining clones at least one SNa MN maintains its identity and properly innervates its target muscle. However, the SNa fate can also be largely rescued in gsbn^-/-gsb-/+ mutants by gsbn at the postmitotic stage in MNs. Remarkably, all neurons of the NB5-4 lineage are motoneuronal. It is thought that these observations are best explained by a model in which NB5-4 MNs are specified during two critical periods. At the NB stage, when the gsb protein level is high, the fate of the progeny of NB5-4 is specified by gsb to be motoneuronal such that their axons will extend outside of the VNC and project through the SN. This explains why in gsbn^-/-gsb-/+ mutants the progeny of NB5-4 still are MNs although they mostly target the wrong muscles. During the second, much longer period, extending from the GMC to the MN stage, the subtype identity, SNa versus SNc, is specified. As long as levels of gsb and/or gsbn protein are sufficiently high at the GMC or MN stage, the subtype identity is properly specified. Thus, sufficient gsb protein is provided in gsbn^-/-gsb+/+ embryos during the GMC and perhaps early MN stage, whereas in gsbn^-/-gsb-/+ embryos rescued by elav-Gal4 UAS-gsbn, levels of gsbn provided at the MN stage are sufficient to properly specify the subtype identity (He, 2013).

The increase in cell number of the NB5-4 lineage in gsbn^-/-gsb-/+ mutants could result from a deregulation of the proliferation of NB5-4 and/or GMCs or from a decrease in apoptosis of the neurons, which raises the question of whether a defect in motor axons is somehow coupled to a defect in the control of cell number. In 25% of the clones that exhibit ectopic SNc projections, however, the cell number is similar to that in the wild type, in the range of 4-5. Moreover, in all three clones that show normal SNa and no ectopic SNc projection, the cell number is increased, ranging from 7 to 9. Therefore, the two phenotypes do not seem to be correlated, which suggests that the two functions in the NB5-4 lineage of gsb and gsbn are separable. Consistent with this conclusion, it has been reported that in embryos deficient for programmed cell death, NB5-4 clones consist of 10-13 neurons but display a wild type-like projection pattern (He, 2013).

For historical reasons, the strong cuticular phenotype of Df(2R)IIX62 was assumed as gold standard for a null allele of gsb despite the fact that it was soon discovered that this deficiency deletes two duplicated genes at the gsb locus, gsb and gsbn. That no gsb alleles with a strong segment-polarity phenotype like that of Df(2R)IIX62 were found was puzzling but attributed to ineffective mutagenesis screens. Accordingly, gsb alleles exhibiting weak cuticular phenotypes were assumed to be hypomorphic. The results presented in this study strongly suggest a different explanation for this long-standing problem: gsbn and gsb fulfill partially redundant functions in embryonic segmentation. They further explain why previously strong cuticular phenotypes similar to that of Df(2R)IIX62 could be obtained only by combining a gsb null allele, like the large deficiency Df(2R)SB1 that uncovers gsb but not gsbn, with a large deficiency uncovering both gsb and gsbn, thus generating gsbn^-/+gsb-/- mutants (He, 2013).

Thus, the situation for the specification of the epidermis between mid stage 9 and mid stage 11 (Li and Noll, 1993) by the segment-polarity functions of gsb and gsbn is similar to that for the determination of the SNa MNs derived from NB5-4 by their CNS functions during stages 11-13. The SNa phenotype of gsbn^-/-gsb-/+ embryos can be rescued if gsbn is provided at the MN stage. Similarly, the cuticular segment-polarity phenotype of gsb null mutants is very weak because it is rescued after stage 10 by the presence of gsbn in the epidermis. If the gsbn dose is reduced in gsb^-/- embryos, the rescue effect by gsbn is diminished and the cuticular phenotype enhanced (He, 2013).

Since it has been shown that gsbn and gsb share several overlapping functions, one should be cautious when attributing phenotypes observed in gsbn^-/+gsb-/- mutants solely to the loss of gsb functions. The two strong or null alleles of gsb generated in this study, gsb^s252 and gsb^gsbJ46, will offer the opportunity to study the functions of gsb without the discussed genetic complications (He, 2013).

A common feature of many gene pairs in Drosophila is 'enhancer sharing': two duplicated neighboring genes share the same enhancer(s) and hence are expressed in similar patterns. As a consequence, one of the two genes could be completely redundant. Indeed, this is the case for the invected gene of Drosophila at the engrailed-invected locus. By contrast, the enhancers of gsb and gsbn cannot activate the heterologous promoters properly. Moreover, the two gooseberry genes are linked in a regulatory hierarchy: transcription of gsbn partially depends on the expression of gsb. These features of the gsb locus suggest that aspects of their functional interactions differ from those of other gene pairs. The phenotype of gsbn mutants is mild compared to that of gsb mutants. Nevertheless, gsbn and gsb are still intimately linked, notably revealed by the haploinsufficiency of gsb in gsbn mutants. The detailed analysis of the SNa phenotype in gsbn^-/-gsb-/+ mutants suggests that the functional link between gsb and gsbn is established mainly through the strong requirement for full activation of gsbn by a functional gsb protein. Once gsbn is expressed, Gsb, together with Gsbn, activates their target genes in a redundant fashion, probably because of their two highly conserved DNA-binding domains, the paired-domain and the paired-type homeodomain. It appears that the gsb-gsbn hierarchy at least ensures normal development of the dorsal SNa MNs if one copy of gsb is lost as long as one functional copy of gsbn is preserved. Moreover, gsbn^-/-gsb-/+ larvae die during first instar, most probably due to defects in the CNS. Therefore, the establishment of a gsb-gsbn hierarchy avoids deleterious effects caused by the gsb haploinsufficiency (He, 2013).

The gsb locus provides an interesting paradigm of how selection for both genes may occur after duplication of their ancestral gene during evolution. A possible, though speculative, scenario explaining the selection for both genes, gsb and gsbn, could be the following. Let the general case first considered of a gene that becomes haploinsufficient for a specific function during evolution. This is a disadvantage against which evolution selects by favoring higher transcription levels in cells dependent on this haploinsufficient function. It is envisaged that such increased levels could result (1) from mutations in the enhancers that regulate transcription in the tissues subject to the haploinsufficiency, or (2) from duplication of the gene including its enhancer responsible for the haploinsufficient function. The second scenario, however, is possible only if a quadruple dose is neither lethal nor causes other disadvantages against which evolution would select. In the case of the ancestral gene of gsb/gsbn, it is argued that haploinsufficiency for a certain function, for example that required for proper development of SNa MNs, was overcome by a gene duplication and inversion, resulting in two genes that are divergently transcribed, gsbn and gsb. It is further argued that selection for both genes occurs if one of the genes is partly dependent on the other in cells that required the haploinsufficient function before gene duplication. This is conceivable if the enhancer of the ancestral gene responding to the segment-polarity gene Wg was included in the duplication. While the gsb gene remained dependent on Prd and Wg, gsbn became partly dependent on gsb that maintained Wg expression. Thus, as one duplicated gene, gsb, retained some of the original enhancers and the other, gsbn, became partially dependent on gsb in some tissues, the expression of the two genes overlapped in these tissues during development. As a consequence, gsb was no longer haploinsufficient because when one of its copies was inactivated by mutation, gsbn provided sufficient protein substituting for the missing gsb protein. This explains why gsbn, which is partly dependent on gsb, does not show an SNa phenotype when inactivated in both copies, as long as two copies of gsb remain present. This mechanism would have selected for both gsb genes after their duplication, and each genes was thus able to acquire additional enhancers and functions during evolution. Consistent with this hypothetical scenario, the haploinsufficiency of the ancestral gene is still apparent today in gsb when both copies of gsbn are inactivated by mutation. By this scenario, haploinsufficiency for a particular function of the ancestral gene can be overcome by gene duplication. However, this is not possible if twice the level of the ancestral gene product is deleterious for any of its essential functions (He, 2013).

Interestingly, a similar regulatory link may have been established during the much earlier duplication that gave rise to prd and the ancestor of gsb/gsbn. Here, the ancestral gene of gsb/gsbn may have retained most functions of its ancestor and became dependent on the activation by prd, which retained the enhancer that activated its ancestor initially. By the acquisition of additional enhancers, prd evolved as pair-rule gene activating the ancestor of gsb/gsbn in alternating segments, while other evolving pair-rule genes activated gsb/gsbn in the remaining segments. Why prd is eventually activated in a segment-polarity pattern is still a conundrum, as so far no functions of prd have been demonstrated in every other segment. A possible explanation is that prd performs in these segments an epidermal function that is redundant with that of gsb, similar to the redundancy demonstrated in this study for functions of gsb and gsbn (He, 2013).

In addition to a hierarchical regulatory link, there seems to be a structural link between the duplicated genes, which may also hold the clue to the question why their enhancers have become so selective for their cognate promoters (He, 2013).

GENE STRUCTURE

The gooseberry (gsb) locus contains two closely linked genes, gsb and gsb-n transcribed in opposite directions. They are separated by about 10 kb of a common upstream region. gooseberry-neural has five exons (Baumgartner, 1987).

PROTEIN STRUCTURE

Amino Acids - 452

Structural Domains

The two gooseberrys are structurally related to each other and to the paired (prd) gene. The structural homology between these two putative proteins and the PRD protein consists essentially of two domains forming most of the amino-terminal halves of the proteins: the PRD domain of 128 amino acids and a PRD-type homeodomain of 60 amino acids, plus an additional 18 amino acids at the amino-terminal end of the homeodomain. There is no C terminal PRD domain, as in paired (Baumgartner, 1987).

Despite the functional difference and the considerably diverged coding sequence of the two gooseberry genes, their proteins have conserved the same function. The finding that the essential difference between genes may reside in their cis-regulatory regions exemplifies an important evolutionary mechanism of how function diversifies after gene duplication (Li,1994b).

There are seven Pax genes in Drosophila and nine Pax genes known in mouse and human. Different Pax proteins use multiple combinations of the HTH motifs to recognize several types of target sites. Drosophila Paired protein can bind, in vitro exclusively through its PAI domain (the N-terminal portion of the bipartite paired domain), or through a dimer of its Homeodomain, or through cooperative interaction between PAI domain and HD. However, paired function in vivo requires the synergistic action of both the PAI domain and the HD. Pax proteins with only a PD (such as Pax-5) appear to require both PAI and RED domains, while a Pax-6 isoform and a new Pax protein Lune, may rely on the RED domain and HD. Thus Pax protein appear to recognize different target genes in vivo through various combinations of their DNA binding domains, thus expanding their recognition repertoire (Jun, 1996).

Promoter Structure

Although gsb and gsb-n are closely linked, their regulation is independent. Different non-overlapping enhancer or upstream control elements drive the specific expression of gsb and gsb-n. Specificity of these enhancers for their respective genes is indicated by their inability to activate transcription in genetically engineered combination with the heterologous promoter of the other gene (Li, 1994a).

The two Drosophia genes gooseberry (gsb) and gooseberry neuro (gsbn) are closely apposed and divergently transcribed. While gsb is a segment-polarity gene and mainly expressed in the epidermis, gsbn is expressed in the central nervous system. An intriguing question is how their transcriptional specificity arises. This study shows that different non-overlapping enhancer or upstream control elements drive the specific expression of gsb and gsbn. Specificity of these enhancers for their genes is achieved by their inability to activate transcription in combination with the heterologous promoter of the other gene. These results therefore suggest that compatibility between the enhancer and its cognate promoter is a mechanism ensuring transcriptional specificity (Li, 1994b).

Since gsb and gsbn share the same upstream sequence which includes both gsbE (the embryonic gsb enhancer) and gsbnE (the embryonic gsbn enhancer), the question arises as to why these enhancers activate only their own and not also the other gene. gsbE and gsbnE still preserve their distinct regulatory specificities in the corresponding gsb-lacZ and gsbn-lacZ fusion constructs that contain both gsbE and gsbnE. The functions of gsbE and gsbnE are easily distinguished. While gsbnE activates gsbn only in the CNS after stage 10, gsbE activates gsb mainly in the epidermis already much earlier by the successive action of its two elements, GEE and GLE. GEE, the gsb early element, begins to act on gsb during syncytial blastoderm whereas GLE, the gsb late element, takes over gsb activation after stage 10. The 9Z1 lacZ fusion construct is expressed in the epidermis before and after stage 10 in a pattern resembling that of gsb. The additional weak expression of 9Z1 in the CNS does not result from activation by gsbnE since smaller gsb-lacZ constructs that lack gsbnE still display this weak neural expression. Hence, gsbnE is inactive in 9Z1. Similarly, gsbE has no effect in 4Z1 as its expression of gsbn -lacZ remains largely restricted to the CNS and is not detected before stage 10. Therefore, the information restricting the activity of gsbE and gsbnE to their cognate genes is included in the common upstream sequence of gsb and gsbn (Li, 1994b).

This restriction could be explained in several ways. First, gsbE and gsbnE might block each other's action and thus be unable to act on their distal promoters. Second, the activities of gsbE and gsbnE might be dependent on their orientation. Third, a sequence between gsbE and gsbnE may function as a boundary element restricting their action to the gene located on the same side of the boundary. Finally, the gsbE or gsbnE enhancer might be unable to interact with and thus activate the promoter of the other gene (Li, 1994b).

To distinguish between these mechanisms, the embryonic expression patterns of the gsb-lacZ and gsbn-lacZ constructs were analysed. First, whether gsbnE can activate the gsb promoter in the absence of the gsb enhancer was analyzed, by examining the expression of 4n9Z in which gsbE has been removed from 9Z1. 4n9Z is only weakly expressed in a small set of internal cells of either neural or mesodermal origin in each segment after stage 11. This expression pattern differs dramatically from that of gsbn and is not affected by the orientation of gsbnE. Hence, these results suggest that gsbnE cannot act properly on the gsb promoter to activate the gsbn-specific CNS expression although they do not strictly eliminate the possibility of a boundary element located between gsbnE and gsbE. Similarly, gsbnE cannot function properly in combination with the hsp7O instead of the gsb promoter (Li, 1994b).

In analogous experiments, whether gsbE can activate the gsbn promoter was analyzed. When the region between gsbE and the gsbn promoter as well as the first two introns of gsbn were removed from 4Z1, the resulting 9n4Z construct is only weakly expressed in a row of epidermal and underlying neural cells in each segment after stage 11. In addition, the weak epidermal expression of 9n4Z differs from the characteristic barbell-shaped expression pattern of gsb or 9Z1. Few cells of the CNS that normally do not express gsb also express 9n4Z. These results suggest that the elimination of sequences that might block the interaction of the gsb enhancer with the gsbn promoter does not restore its activity in 9n4Z. Neither is the expression pattern of 9n4Z affected by the inversion of gsbE, indicating that orientation is not the cause of its inactivity. Similarly, if gsbE is placed in either orientation upstream of the hsp70 promoter, lacZ expression is never detected in the epidermis at any stage. However, its expression in several neuroblasts or ganglion mother cells in each segment may correspond to part of the normal gsb activity in the CNS. It is concluded that gsbE can activate neither the gsbn nor the hsp7O promoter properly. Taken together, these results clearly show that activation by the gsbE and gsbnE enhancers requires interaction with their cognate promoters (Li, 1994b).

Transcriptional Regulation

P-element-mediated transformation with the gooseberry gene has been used to demonstrate that GSB-D transactivates gsb-n and is sufficient to rescue the gooseberry cuticular phenotype in the absence of gsb-n (Gutjhar, 1993).

Patched targets gooseberry distal and gooseberry-proximal in neuroblast determination. The RP2 neuron is a motoneuron and innervates muscle number 2 of the dorsal musculature. This neuron originates along with its sibling cell from the first ganglion mother cell derived from NB4-2, and occupies the anterior commissure along with several other RP2 neurons. NB4-2 itself is formed during the second wave of neuroblast delamination in stage 9. Gooseberry and Patched participate in the Wingless-mediated specification of NB4-2 by controlling the response to the wingless signal. In gsb mutants, WG-positive NB5-3 is transformed to NB4-2 in a Wg-dependent manner, suggesting that GSB normally represses the capacity to respond to the wingless signal. In ptc mutants, gsb is ectopically expressed in normally Wg-reponsive cells, thus preventing the response to Wingless and consequently the correct specification of NB4-2 does not take place. The timing of the response to GSB suggests that the specification of neuroblast identities takes place within the neuroectoderm, prior to neuroblast delamination (Bhat, 1996).

Krüppel is coexpressed with engrailed in a subset of neurons and glia that include the medial-lateral cluster of en-expressing neurons and the dorsal channel glia cells. In Kr mutants, the medial-lateral cluster is either absent or fails to express en, but the dorsal channel glia cells are not affected. These medial-lateral cluster cells gives rise to serotoninergic neurons, and almost no neurons synthesizing serotonin remain in these mutant embryos. In Kr mutants, the number of gooseberry neural-expressing cells increases from 10% to 50%. Ectopic Kr expression leads to a strong reduction in gsb-n expressing neurons (Romani, 1996).

DEVELOPMENTAL BIOLOGY

Embryonic

The Gsb-n protein first appears during germ band extension in cells of the central nervous system. Much later it appears in epidermal stripes and in a small number of muscle cells. Like gooseberry, gsb-n is expressed in a complex pattern in the head [Image] and tail (Gutjhar, 1993).

Ectopic expression of either Gooseberry protein causes cell fate transformations that are reciprocal to those of a deletion mutant of both gooseberry genes. The Gsb protein is required for the specification of naked cuticle in the epidermis and specific neuroblasts in the central nervous system. These roles may reflect independent functions in neuroblasts and epidermal cells or a single function in the common ectodermal precursor cells. The Gsb-n protein is also found in the same neuroblasts as GSB-D and in the descendants of these cells (Zhang, 1994).

Effects of Mutation

A concerted action of Engrailed and Gooseberry-Neuro in neuroblast 6-4 is triggering the formation of embryonic posterior commissure bundles

One challenging question in neurogenesis concerns the identification of cues that trigger axonal growth and pathfinding to form stereotypic neuronal networks during the construction of a nervous system. This study shows that in Drosophila, Engrailed (En) and Gooseberry-Neuro (GsbN) act together as cofactors to build the posterior commissures (PCs), which shapes the ventral nerve cord. Indeed, these two proteins are acting together in axon growth and midline crossing, and that this concerted action occurs at early development, in neuroblasts. More precisely, their expressions in NB 6-4 are necessary and sufficient to trigger the formation of the PCs, demonstrating that segmentation genes such as En and GsbN play a crucial role in the determination of NB 6-4 in a way that will later influence growth and guidance of all the axons that form the PCs. Also, more specific function was demonstrated of GsbN in differentiated neurons, leading to fasciculations between axons, which might be required to obtain PC mature axon bundles (Colomb, 2008).

One of the most fascinating aspects of nervous system development is the establishment of stereotypic neuronal networks. An essential step in this process is the outgrowth and precise navigation of axons. Most CNS growth cones initially head straight towards the midline, and only after crossing, they change their behavior as they turn and follow specific longitudinal pathways. In Drosophila, the majority of axons cross the midline within either anterior or posterior commissures. The formation of commissures starts at stage 12 of embryonic development and involves dynamic, but reproducible interactions between: growth of the neurons, their fasciculation with other neurons to form the different bundles, apoptosis of neuronal cells, and migration of glial cells. In Drosophila, formation of posterior and anterior commissures are not believed to be related, and different cells and possibly different signals appear to be used for the guidance of the different commissures. Each neuron makes a choice as whether to cross the midline and, for those that do cross, whether to grow through the anterior or the posterior commissure, where axons are arranged in fascicles. One central issue is the identification of the intrinsic pathfinding abilities at the different steps of the neural development that are involved in the differential neuronal behavior. Whereas the process of construction of longitudinal tracts has been previously analyzed, as has the formation of ACs, little is known about the formation of the PCs (Colomb, 2008).

Obvious candidates for organizing the intrasegmental distribution of guidance cues along the antero-posterior axis are the segment polarity genes. Consistent with this assumption, embryos mutant for En/Inv and for Gsb/GsbN have severely reduced, and often missing, posterior commissures. Segment polarity genes occupy an intriguing position within the segmentation hierarchy. They are required in the epidermis to specify cell fates within each segment, and are also active both before and during the delamination of neuroblasts to generate the CNS. In particular, the specification of neuroblast identity within a given hemisegment depends upon interactions between segment polarity genes such as Engrailed and Invected with Gsb. Whereas gsb is expressed at early stage 6 and begins to be detectable when NBs start to delaminate (stage 9), GsbN is only detectable starting in stage 10 embryos and appears simultaneously to the disappearance of Gsb. En and GsbN are expressed in NBs of rows 6 and 7. Interestingly, NB 6-4 appears during the S3 wave of delamination at stage 10, just as GsbN expression begins. The axons that pioneer the first tracts will appear later, at stage 12, by which time Gsb expression is nearly completely switched off (Colomb, 2008).

This report has developed several lines of evidence for a concerted action of En and GsbN in neuroblasts. Indeed, it was shown that whereas heterozygous en/inv or gsb/gsbN deletions (respectively (Df en^X31/+) and (Df gsb^X62 /+)) show a normal architecture of the VNC, double heterozygotes (Df en^X31/Df gsb^X62) do not form PCs properly, resulting with high penetrance in loss of PCs. This result clearly indicates that En/Inv and Gsb/GsbN act together to form PCs. En has already been shown to have a major function in PC formation, comparatively to Inv. In view of the observation of physical interactions between En protein and GsbN protein, whether GsbN might be responsible for the absence of PCs in the transheterozygous (Df en^X31/Df gsb^X62) genetic background was analyzed. Using rescue experiments, it was indeed found that expression of GsbN was able to rescue the phenotype. This shows that, genetically, En and GsbN act together to build the posterior PC commissures, which are part of the VNC. There are several reasons to suspect that GsbN might act as a cofactor of En for PC formation. First, no evidence was found for a direct regulation of En on gsbN, since no En binding fragments were isolated within the GsbN locus by chromatin immunoprecipitation. This corroborates the observation that En does not bind the GsbN locus (60F region) on polytene chromosomes. Moreover, it was shown that missing PC phenotype resulting from En misexpression is not associated with a loss of gsbN function. In addition, En and GsbN proteins interact in vitro (as evidenced by GST-pull down and coIP experiments), in yeast (demonstrated using a two-hybrid assay), and in vivo in Drosophila, since they were found to bind common loci on polytene chromosomes. Together, these results support the notion that En and GsbN act as cofactors in the construction of the VNC. Interestingly, it was only possible to rescue the missing PC phenotype of transheterozygous (Df en^X31/Df gsb^X62) embryos when GsbN was restored from early stages in neuroblasts, but not in differentiated neurons. This shows that the formation of the PCs involves an early function of GsbN, which is consistent with a concerted action with En, since it has been shown that the early function of En is responsible for PC axon growth. It is known from previous studies that PCs are formed from neurons originating in rows 6 and 7 (which express both En and GsbN), as well as from neurons issued in other rows, such as row 5 (that only express GsbN). These observations strongly suggest that NBs expressing both the En and GsbN transcription factors might contain instructions for PC formation. In a first step, En/Inv and Gsb (not GsbN) were shown to be involved in NB specification. In particular expressions of En and Gsb were found to be necessary in the formation of NB 6-4. However, since gsbN is not expressed in the ventral neuroectoderm during the time of NB specification, it hence cannot play a role in neural specification at this level. Therefore, it is expected that the interaction between En and GsbN does not interfere directly in the formation and segregation of the NBs, but rather to happen after the NBs are formed. In particular, this study shows that En and GsbN are involved in the further determination of NB 6-4 to form posterior commissure. Indeed, En and GsbN functions in NB 6-4 not only influence NB 6-4 behavior, but also the behaviors of other neurons that construct the PCs, strongly suggesting that this concerted action of En and GsbN is involved in triggering formation of the PC bundles. One hypothesis is that they act together in a same complex to activate functions that are required for the development of the NBs and that will be necessary for further axon growth and pathfinding. Indeed, driving GsbN in NB 6-4 using different drivers such as eagle-Gal4 or collier-Gal4 was sufficient to rescue axonal growth and crossing of the midline of the PC formers in the transheterozygous (Df en^X31/Df gsb^X62) background (Colomb, 2008).

However, whereas axon growth and crossing of the midline seem to be rescued in both cases, separation between ACs and PCs were incomplete. One possible explanation for the fusion of the commissures, was provided by the analysis of the neuronal behavior of eagle-positive neurons. When GsbN is expressed in eagle-expressing NBs/neurons (corresponding to NB 6-4 and NB 7-3 progeny projecting through PCs, and to NB 2-4 and NB 3-3 progeny projecting through ACs), a rescue of axonal growth of PCs was observed, but it was also found that neurons projecting through ACs were fasciculating with the PCs. This suggests that the 'fuzzy' separation of ACs and PCs observed with the eagle-Gal4 driver probably resulted from abnormal axonal pathfinding in ACs. Therefore, formation of PC bundles requires at later stages, a specific function of GsbN in the neurons that is driving the fasciculation and guidance of axons forming PC commissures. This latter function of GsbN might correspond to a late function, since expression of GsbN with both early acting (in NBs, neurons, and glial cells) and late acting (in differentiated neuronal cells) Gal4 drivers was found to misroute axons that would have otherwise fasciculated to other axons at the midline. In this case too, all the axons seem to fasciculate, leading to a fuzzy separation of the commissures, sometimes collapsing at the midline. These observations support the idea that this late function of GsbN in the neurons is involved in their axonal pathfinding and in formation of fasciculations that are required to form the bundles, and that are a property of the follower neurons. En function on axonal pathfinding was found to occur early in the neuroblasts, but not in differentiated neurons. Expression of GsbN in differentiated neurons was also not able to trigger axonal growth and crossing of the midline of PC formers. Therefore, a two-step involvement of GsbN occurs in the formation of the PCs. In first, a concerted action of En and GsbN is necessary in NB 6-4 to trigger the axon growth of PC formers, whereas axonal guidance per se might rather result from independent role of En and GsbN, a specific action of GsbN on guidance occurring in differentiated neurons (Colomb, 2008).

Important questions relate to the behavior of NB 6-4 in different genetic contexts and the exact role of En and GsbN in this process. Since NB 6-4 generates both neurons and glial cells, one hypothesis is that they act together in the glial cells that are known to play a crucial role in axonal guidance. Several hypothesis could be drawn: (1) GsbN expression is needed to form NB 6-4 progeny. However, in transheterozygous (Df en^X31/Df gsb^X62) embryos, eg expressing neuronal cells were formed, but their axons were not growing. As well, it was found that glial cells were formed from NB 6-4, which does not favor this hypothesis. (2) GsbN acts directly on glial cell function. However whereas En is expressed in the glia, it was found that GsbN was not expressed in the glia, which also excludes this hypothesis. (3) En and GsbN are activating a function that will be expressed in NB 6-4 glial progeny and that is triggering axon growth and crossing of the midline, making these particular glial cells central in this process. However ectopic expression of GsbN in all the glia does not lead to abnormal architecture of the VNC, which does not favor for an indirect effect of GsbN in the glia. (4) Finally, functions activated by En and GsbN in NB 6-4 will be used in its neuronal progeny to 'show the way' of GsbN expressing neurons. The data rather favor a central role of NB 6-4 neuronal progeny in triggering the formation of the PCs. This of course does not exclude, as shown for longitudinal tracts, a crucial role between on one hand these particular neurons and the NB 6-4 glial progeny, followed by a crosstalk between these glial cells and the GsbN expressing neurons (Colomb, 2008). The molecular mechanisms involved in these processes will be particularly informative in arriving at an understanding of how neuronal axon trajectories are dictated to construct the VNC. The next challenge will be also to understand what cellular events and downstream functions are regulated in NBs by both En and GsbN to construct PC bundles, since their expression in NB 6-4 seems to be crucial to trigger the whole process of PC formation, and to understand what are the specific downstream functions regulated more specifically by GsbN to specify fasciculations between GsbN expressing neurons, a property associated to the followers (Colomb, 2008).

One way to address these questions would be to identify genes that are directly regulated by En and GsbN and that would therefore likely be misregulated in the transheterozygous (Df en^X31/Df gsb^X62) genetic background (Colomb, 2008).

Finally, the identification of direct targets of GsbN or of common direct targets of En and GsbN would allow a better understanding of the downstream functions involved in the specification and differentiation of the different neurons, which ultimately drive axon growth and axonal pathfinding (Colomb, 2008).

The observations that vertebrate homologs of En (En1 and En2) and Gsb/GsbN (Pax3 and Pax7), but also other Pax genes are required in neural fate specification, and that they are involved in axon growth, strongly suggests that the molecular mechanisms acting in Drosophila are relevant to and probably conserved in higher organisms (Colomb, 2008).

REFERENCES

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date revised: 22 May 2023

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