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 Keilinís organs, which are located at the parasegmental border in each thoracic segment, were examined. In gsb mutants Keilinís organs are deleted, while in heat shocked hs-gsbd and hs-gsbp embryos, ectopic Keilinís 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).

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).

REFERENCES

Baumgartner, S., Bopp, D., Burri, M. and Noll, M. (1987). Structure of two genes at the gooseberry locus related to the paired gene and their spatial expression during Drosophila embryogenesis. Genes & Dev 1: 1247-67. Medline abstract: 3123319

Bhat, K. M. (1996). The patched signaling pathway mediates repression of gooseberry allowing neuroblast specification by wingless during Drosophila neurogenesis. Development 122: 2921-32. Medline abstract: 8787765

Gutjahr, T., Patel, N. H., Li, X., Goodman, C. S. and Noll, N. (1993). Analysis of the gooseberry locus in Drosophila embryos: gooseberry determines the cuticular pattern and activates gooseberry neuro. Development 118: 21-31. PubMed citation; Online text

Jun, S., and Desplan, C. (1996). Cooperative interactions between paired domain and homeodomain. Development 122: 2639-50. PubMed citation; Online text

Li, X. and Noll, M., (1994a). Compatibility between enhancers and promoters determines the transcriptional specificity of gooseberry and gooseberry neuro in the Drosophila embryo. EMBO J. 13: 400-6. PubMed citation; Online text

Li, X. and Noll, M. (1994b). Evolution of distinct developmental functions of three Drosophila genes by acquisition of different cis-regulatory regions. Nature 367: 83-87. Medline abstract: 7906390

Patel, N. H., Schafer, B., Goodman, C. S. and Holmgren, R. (1989). The role of segment polarity genes during Drosophila neurogenesis. Genes Dev. 3: 890-904. Medline abstract: 2501154

Romani, S., et al. (1996). Krüppel, a Drosophila segmentation gene, participates in the specification of neurons and glial cells. Mech. Dev. 60: 95-107. Medline abstract: 9025064

Zhang, Y., Ungar, A., Fresquez, C. and Holmgren, R. (1994). Ectopic expression of either the Drosophila gooseberry-distal or proximal gene causes alterations of cell fate in the epidermis and central nervous system. Development 120: 1151-1161

date revised:  25 February 2008

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