BIOLOGICAL OVERVIEW

gooseberry-distal is a segment polarity gene induced by pair rule genes. gooseberry is primarily involved in a hedgehog-independent wingless autoregulatory loop. In other words, gooseberry acts to establish the maintanence of the crucial wingless signal, on which the segmental structure of the fly depends. In return, wingless acts to regulate gsb. gsb also regulates the synthesis of its sister gene, gooseberry-proximal. They function as a team in neurogenesis to specify and differentiate a subset of neurons (Gutjahr, 1993 and Li, 1993a).

Despite considerable divergence in their coding sequences, the two gooseberry genes and 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).

The pathway regulating gsb in determination of neural fate is worth examining in detail because of the complexity of the interactions. During neurogenesis, the transmembrane protein Patched promotes a wingless-mediated specification of a neuronal precursor cell, NB4-2. Wg, secreted by row 5 cells promotes wingless expression in adjacent row 4 cells; Wg in turn represses gooseberry. Novel interactions of these genes with engrailed and invected during neurogenesis have been uncovered. While in row 4 cells Ptc represses gsb and wg, in row 5 cells en/inv relieve Ptc repression of gsb by a non-autonomous mechanism that does not involve hedgehog. The non-autonomous mechanism originates in Row 6/7 cells where en/inv engender hedgehog and another unknown secreted signal which acts in turn on adjacent row 5 cells to heighten wingless, and consequently, the expression of gooseberry. This differential regulation of gsb leads to the specification of NB5-3 and NB4-2 identities to two distinct neuroblasts. The row 5, NB5-3, neuroblasts are specified by high levels of gsb, expressed autonomously in row 5. The fate of row 4, NB4.2, requires an absence of gooseberry, assured by Patched repression and Wingless signaling from adjacent row 5 cells. The uncoupling of the ptc-gsb regulatory circuit by hedgehog and the unknown secreted signal from row 6/7 cells enables gsb to promote Wg expression in row 5 cells (Bhat, 1997).

Mechanisms have been studied leading to the fate specification of a set of late delaminating neuroblasts, NB 6-4 and NB 7-3, both of which arise from the engrailed (en) expression domain, with NB 6-4 delaminating first. No evidence is found for a direct role of hedgehog in the process of NB 7-3 specification. NB 7-3 normally requires Hh only for maintenance of Wg expression, which in turn leads to En maintenance. Evidence is presented to show that the interplay of the segmentation genes naked cuticle (nkd) and gooseberry (gsb), both of which are targets of wingless (wg) activity, leads to differential commitment to NB 6-4 and NB 7-3 cell fate. In the absence of either nkd or gsb, one NB fate is replaced by the other. However, the temporal sequence of delamination is maintained, suggesting that formation and specification of these two NBs are under independent control (Deshpande, 2001).

In the En domain Wg plays a role both in NB formation and NB specification. The homeodomain transcription factor En is a prerequisite for the formation of the NBs 6-4 and 7-3, because in its absence both NBs fail to form. Since Wg signaling is necessary for maintaining En expression, it is also essential for the formation of these two NBs. Hh is co-expressed in the En domain and En maintains Hh expression in rows 6 and 7, and Hh in turn is essential for Wg expression in row 5, thereby constituting a maintenance loop. Thus, for late NBs in row 6 and 7, the expression of En is crucial and Hh is required to maintain En expression via Wg. However, for the separate specification of NB 6-4 and NB 7-3, differential regulation of two Wg targets, nkd and gsb, is essential (Deshpande, 2001).

Wg is a diffusible molecule expressed in row 5 and acts on neighboring rows, which include rows 6 and 7. However, row 6 differs from row 7 because it expresses gsb, which is, as stated above, a target of Wg signaling. The fact that row 7 does not express gsb, despite being under the influence of Wg raises the question of how this differential regulation is brought about. In this work it is shown that Nkd is essential for this regulation. Nkd is a negative regulator of the Wg signal transduction pathway, itself being a target of this pathway. In the absence of Nkd, Gsb is derepressed, owing to Wg hyperactivity in row 7, leading to the generation of an ectopic NB 6-4 like fate. Thus, the distinct identities of NB 6-4 and NB 7-3 are brought about by the interplay of Gsb and Nkd. For NB 6-4 specification, Gsb is an essential factor. In the absence of Gsb NB 6-4 fails to be specified and instead takes the identity of NB 7-3 fate. Conversely, for NB 7-3 specification, a Gsb-free environment, which is created by the activity of Nkd, is essential. In summary, NB 6-4 needs the expression of Gsb and En, whereas NB 7-3 needs En but the absence of Gsb (Deshpande, 2001).

However, the fact that gsb as well as nkd are targets of Wg signaling makes it difficult to explain why gsb is repressed by nkd only in the posterior region of the En stripe. The posterior En domain is further away from the Wg source than the anterior En domain and therefore should receive a lower signaling input when compared with the anterior region. As a consequence, this should lead to higher Nkd activity in the anterior En cells, leading to a stronger Gsb repression in this region -- the opposite of what was observed. A careful analysis of the expression pattern on the transcriptional level does not give any obvious clues to solve this apparent paradox. During early germ band extension (stage 8-9) nkd transcription is nearly ubiquitous with higher RNA levels in the two to four cell rows posterior to the En stripe. At late phase of germ band extension, nkd expression is most abundant anterior to the En stripe and lower just posterior to the En-stripe. No significant difference between the anterior and posterior En domain could be detected. One explanation for the differential regulation of gsb could be that, owing to earlier pair rule gene activity of paired, the level of Gsb protein at the time of NB 6-4 delamination in the anterior En region is high enough to override repression by Nkd activity. Alternatively, a direct differential regulation of the two Wg targets that is due to the different levels of Wg signaling could be responsible for the observed regulatory differences. It could be that the regulation is such that the amount of Wg signaling within the En stripe causes a relatively homogenous level of nkd expression in this region. At the same time, the transcriptional activation of gsb could be more sensitive to Wg signaling levels, resulting in a very strong activation, especially near to the Wg-expressing cells. As a result, the relatively low Nkd activity in the whole En stripe might be able to inhibit gsb expression in the region of low gsb activation only: the posterior En domain. A hint that a differential regulation of Wg targets indeed exists comes from the Wg-dependent En regulation: it seems that a lower Nkd activity is sufficient to repress gsb but not to inhibit en expression. This conclusion was drawn from the finding that overexpression of nkd within the En stripe using an EnGal4 driver line leads to a selective repression of gsb with no obvious effect on en expression itself. Clearly, additional work has to be carried out to clarify these points (Deshpande, 2001).

Besides row 6 neuroectoderm, row 3 neuroectoderm also has the potential to generate an ectopic NB 7-3. It has been shown previously that in embryos mutant for ptc, neuroectodermal cells in the area of row 3 begin to express En and additional serotonergic neurons can be found in these mutant embryos, which suggests the presence of an ectopic NB 7-3 like fate. Additionally, when En is ubiquitously expressed, only row 3 has the ability to give rise to an ectopic NB 7-3 fate. In all cases, this occurs at the cost of row 3 NBs such as NB 3-3. It is thought that this might reflect that row 3 neuroectoderm, which is right in the middle of the segment, represents something like a 'ground state' in the neuroectoderm: in this area neither Hh nor Wg signaling may take place. Therefore the decision to specify late row 3 or late row 7 NBs seems to be only dependent on the absence or presence of En, respectively (Deshpande, 2001).

Previous work has indicated that genes expressed in proneural clusters are involved in specifying the individual fates of NBs that develop from these clusters. The finding that NB 6-4 and NB 7-3 can be mutually transformed while the sequence of birth does not change suggests that the mechanism for the timing of late NB delamination is independent from mechanisms that regulate NB identity. This might be reminiscent of early NBs. Initiation of S1 NB formation requires the activity of proneural genes that have been shown to be dependent on pair-rule genes. The identity of the NBs delaminating from these clusters, however, is dictated by the activity of segment polarity genes. Thus, the control of proneural gene expression that enables NB formation and the control of segmentation genes conferring NB identity occurs in parallel. At later stages, pair-rule gene expression vanishes and can no longer be responsible for NB formation. How is NB formation regulated in the following segregation waves? One possibility is that after the first segregation wave, NB formation and identity are more tightly linked; the finding that specific NBs like NB 4-2 are sometimes not transformed but missing in wg mutant embryos seems to support this idea. However, the finding that the transformed NB 6-4 and NB 7-3 are delaminating according to the 'old identity' shows that, at least in these cases, NB formation and specification is independent. The results favour the idea that the timing of the formation of proneural clusters within the neuroectoderm is generally independent of the segment polarity genes investigated here. This does not exclude permissive functions, such as those of En, which enable the proneural cluster formation as such. According to this hypothesis, intrinsic or extrinsic factors present in the position of the proneural cluster at the time of delamination govern the identities of the NBs. This might be not only true for the positional regulation of NB identity but also for the determination of NB identity along the temporal axis. Indeed, heterochronic transplantation experiments strongly support the possibility that one or more extrinsic factors exist that lead to stage specific NB identities. It will be a challenge for the future to identify these factors, and to investigate whether similar mechanisms exist in higher organisms (Deshpande, 2001).

GENE STRUCTURE

The gooseberry (gsb) locus contains two closely linked genes, gsb (often referred to as goosberry distal (gsb-d) and gooseberry-neuro, transcribed in opposite directions. They are separated by about 10kb of a common upstream region.

Bases in 5' UTR - 130

Exons - two

PROTEIN STRUCTURE

Amino Acids - 427

Structural Domains

Both gooseberry genes are structurally related to one another and to the paired (prd) gene. The two proteins have considerably divergent coding sequences. The structural homology between these two proteins and the Paired 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 its amino-terminal end (Baumgartner, 1987). Unlike Paired, there is no C-terminal PRD domain.

date revised: 3 July 97 

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