gooseberry

DEVELOPMENTAL BIOLOGY

Embryonic

The Gooseberry-distal protein appears in a characteristic pattern of seven stripes at gastrulation [Images]. By convention these stripes are called the odd numbered stripes. Between the odd numbered stripes, more stripes (the even numbered ones) will later appear. The protein is initially restricted to the ectodermal and neuroectodermal germ layer, but is later detected in mesodermal and neuronal cells as well. The stripe pattern in the head and tail is more complex (Gutjahr, 1993).

The gooseberry-distal gene specifies row 5 neuroblast identity. Initially, four rows of neuroblasts form per segment (1, 3, 5, 7), while other rows develop later. gooseberry-distal is expressed in row 5 neuroblasts. By using 10 molecular markers, and by following the number and orientation of neuroblast divisions, it has been shown that lack of gooseberry-distal transforms row 5 neuroblasts into row 3 neuroblasts, whereas ubiquitous gooseberry-distal generates the reciprocal transformation. Thus, gooseberry-distal is necessary and sufficient to specify row 5 neuroblast identity autonomously. The 10 genes coordinately regulated by gooseberry-distal are prime candidates for controlling specific aspects of neuroblast identity (Skeath, 1995).

The insect brain is traditionally subdivided into the trito-, deuto- and protocerebrum. However, both the neuromeric status and the course of the borders between these regions are unclear. The Drosophila embryonic brain develops from the procephalic neurogenic region of the ectoderm, which gives rise to a bilaterally symmetrical array of about 100 neuronal precursor cells, called neuroblasts. Based on a detailed description of the spatiotemporal development of the entire population of embryonic brain neuroblasts, a comprehensive analysis was carried out of the expression of segment polarity genes (engrailed, wingless, hedgehog, gooseberry distal, mirror) and DV patterning genes (muscle segment homeobox, intermediate neuroblast defective, ventral nervous system defective) in the procephalic neuroectoderm and the neuroblast layer (until stage 11, when all neuroblasts are formed). The data provide new insight into the segmental organization of the procephalic neuroectodem and evolving brain. The expression patterns allow the drawing of clear demarcations between trito-, deuto- and protocerebrum at the level of identified neuroblasts. Furthermore, evidence is provided indicating that the protocerebrum (most anterior part of the brain) is composed of two neuromeres that belong to the ocular and labral segment, respectively. These protocerebral neuromeres are much more derived compared with the trito- and deuto-cerebrum. The labral neuromere is confined to the posterior segmental compartment. Finally, similarities in the expression of DV patterning genes between the Drosophila and vertebrate brains are discussed (Urbach, 2003).

The gooseberry (gsb) locus encodes two closely related proteins, Gsb-distal (Gsb-d) and Gsb-proximal, which are both expressed in the developing ventral nerve cord. Gsb-d is segmentally expressed at high levels in all row 5 and 6 NBs, as well as in a median row 7 NB (NB 7-1). The expression of gsb-d was analyzed during early neurogenesis in the head region; segmental expression of Gsb-d was found to be conserved in parts of the pregnathal head ectoderm and deriving NBs. Gsb-d/En double labelling shows that the gsb-d intercalary and antennal stripes are expressed anterior to the corresponding en stripes, and are partly overlapping with the en stripes. Consequently, NBs from the posterior part of the gsb-d stripe in the tritocerebrum and deutocerebrum co-express en (Td3, Dd5), and those from the anterior part co-express wg (Td4, Dd1 and Dd7; as seen in Gsb-d/Wg double labelling), resembling the situation in the ventral nerve cord. However, Dd8 and all Wg-positive protocerebral NBs do not co-express Gsb-d (except for Ppd3 which, like Ppd10, transiently expresses gsb-d during stage 10. Gsb-d can also be detected at a low level in ganglion mother cells of the respective NBs, but fades away in NBs and their progeny during germ band retraction. Expression of the protein in the brain is completely downregulated at stage 13 (Urbach, 2003).

See Chris Doe's Hyper-Neuroblast map site for information on the expression of gooseberry-distal in specific neuroblasts.

For more information on Drosophila neuroblast lineages, see Linking neuroblasts to their corresponding lineage, a site carried by Flybrain, an online atlas and database of the Drosophila nervous system.

Determination of cell fate along the anteroposterior axis of the Drosophila ventral midline

The Drosophila ventral midline has proven to be a useful model for understanding the function of central organizers during neurogenesis. The midline is similar to the vertebrate floor plate, in that it plays an essential role in cell fate determination in the lateral CNS and also, later, in axon pathfinding. Despite the importance of the midline, the specification of midline cell fates is still not well understood. This study shows that most midline cells are determined not at the precursor cell stage, but as daughter cells. After the precursors divide, a combination of repression by Wingless and activation by Hedgehog induces expression of the proneural gene lethal of scute in the most anterior midline daughter cells of the neighbouring posterior segment. Hedgehog and Lethal of scute activate Engrailed in these anterior cells. Engrailed-positive midline cells develop into ventral unpaired median (VUM) neurons and the median neuroblast (MNB). Engrailed-negative midline cells develop into unpaired median interneurons (UMI), MP1 interneurons and midline glia (Bossing, 2006).

It is likely that genes other than hedgehog and wingless are crucial for midline cell determination. In these experiments, non-Engrailed-expressing midline subsets are never transformed into Engrailed-expressing subsets, or vice versa. gooseberry-distal may be one of these genes. From the blastoderm stage, Gooseberry-distal is expressed by two midline precursors and their four daughter cells. During early embryogenesis Gooseberry-distal expression at the midline does not depend on Wingless and Hedgehog. The anterior Gooseberry-distal cells also express Wingless and most likely give rise to the UMIs. The posterior Gooseberry-distal pair also express early Engrailed and Hedgehog, and develop into the most anterior VUM neurons. At stage 10, Hedgehog activates the expression of Lethal of scute and Engrailed in midline cells posterior to the Gooseberry-distal domain. Lateral inhibition by Notch/Delta signalling selects one cell from the Lethal of scute cluster to become the MNB. The remaining cells become VUM neurons. At stage 10, the absence of Engrailed in the six midline cells anterior to the Gooseberry-distal domain defines a cell cluster that will give rise to midline glia and MP1 interneurons. Based on the expression of Odd, Delta mutants have an increased number of MP1 interneurons, up to six per segment. In Notch mutants, midline glial-specific markers are absent and the number of cells expressing a neuronal marker increases. Therefore, Notch/Delta signalling appears to determine midline glial versus MP1 interneuron cell fates in the anterior cluster. In the current model, midline cell determination takes place mainly after the division of the precursors. Although the initial determination of midline cells appears to be directed by a small number of genes, a far larger number is needed to control the differentiation of the various midline subsets. This work, and the recent identification of more than 200 genes expressed in midline cells, is the beginning of a comprehensive understanding of the differentiation of the ventral midline (Bossing, 2006).

Effects of mutation or deletion

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).

Genetic separation of the neural and cuticular patterning functions of gooseberry

In addition to their role in the specification of the epidermal pattern in each segment, several segment polarity genes, including gooseberry (gsb), specify cell fate in the Drosophila central nervous system (CNS). Analyses of the gsb CNS phenotype have been complicated by the fact that the previously available gsb mutants, all caused by cytologically visible deficiencies, have severe segmentation defects and also lack a number of additional genes. Two novel gsb mutants have been characterized that have CNS defects, due to their hypomorphic nature, but have only weak or no segmentation defects. These gsb alleles, as well as gsb rescue experiments, have allowed a determination of which aspects of the deficiency mutant phenotypes can be attributed to loss of gsb. gsb mutants lack U and CQ neurons, have duplicated RP2 neurons, and display posterior commissure defects. gsb neural defects, as well as the gsb cuticle defect, are differentially sensitive to the level of functional Gsb. One of the novel gsb alleles has been used in order to understand the genetic interactions between gsb, wingless, and patched during the patterning of the ventral neuroectoderm. In contrast to epidermal patterning, where Gsb is required to maintain wg transcription, Gsb antagonizes the Wg signal that confers neuroblast (NB) 4-2 fate. The antagonism of the Wg signal by Gsb may be a common theme to many signal transduction and cell patterning systems. A secreted signaling molecule confers a particular cell fate on adjacent cells; at the same time, the signaling molecule regulates expression of a transcription factor within the cells which secrete the signal, effectively preventing these cells from taking on the fate conferred by the signal (Duman-Scheel, 1997).

This study clarifies genetic interactions among segment polarity genes for patterning the ventral neuroectoderm. Wg specifies NB 4-2 fate and, at the same time, maintains gsb expression. In row 5, Gsb antagonizes the function of secreted Wg, preventing the row 5 cells, which secrete Wg, from taking on the NB 4-2 fate. Ptc represses the expression of wg, and consequently that of gsb, in row 4 cells. Since row 4 cells do not express gsb, they can receive the Wg signal and take on the NB 4-2 fate. Therefore, during NB 4-2 patterning, Gsb antagonizes Wg signaling. In contrast, at a later point in development, during the phase of wg-gsb autoregulation, Gsb acts to maintain wg expression, which is responsible for the specification of naked cuticle. Thus, the early genetic interactions demonstrated to occur between gsb and wg for specifying NB 4-2 are different from the previously reported epidermal patterning interactions between gsb and wg during stages 11 through 13 (Duman-Scheel, 1997).

The mechanism by which Gsb antagonizes Wg signaling in CNS development is unknown. Perhaps Gsb also antagonizes Wg signaling during epidermal patterning in a manner which has yet to be uncovered (Duman-Scheel, 1997).

The temporal aspects of the different gsb/wg interactions are very important. Previous models had proposed that during NB patterning, gsb positively regulates wg. However, such a positive interaction has not yet been demonstrated to occur before stage 11, when wg expression fades in gsb mutants. The timing of this positive wg-gsb autoregulation also relates to another problem with previous models. It has been proposed that in gsb mutants, row 5 NBs are transformed to row 3 NBs. However, gsb mutants still express Wg, a row 5 marker which is not normally expressed in row 3 cells. Therefore, analysis with NB markers can sometimes lead to inconsistent interpretations (Duman-Scheel, 1997).

The discovery that Gsb can function to repress Wg signaling could have important implications for understanding the role of Pax and Wnt genes in the patterning of the vertebrate hindbrain. Previously, researchers had proposed that Pax-2 is necessary for Wnt-1 transcription. The current results indicate, however, that gsb/Pax gene products may also antagonize the Wg/Wnt signaling functions, preventing cells which secrete the Wg/Wnt signal from taking on the fate conferred by the signal. An interesting parallel to the gsb/wg interactions described here can be found in the patterning of the wing margin. During patterning of the wing margin, Wg, secreted from the edge cells, signals adjacent marginal cells to express achaete (ac). However, the edge cells which secrete Wg do not express ac. In these edge cells, Wg regulates cut (ct) expression, and Ct blocks the Wg signal which would turn on ac expression. Thus, in both the patterning of the wing margin, as well as in the patterning of NB 4-2, a secreted signaling molecule confers a particular cell fate; at the same time, the signaling molecule regulates expression of a transcription factor within the cells which secrete the signal, effectively preventing these cells from taking on the fate conferred by the signal. Such a theme may be common to many signal transduction and cell patterning systems (Duman-Scheel, 1997).

Multiple protein functions of Paired in Drosophila development and their conservation in the Gooseberry and Pax3 homologs

The Drosophila segmentation gene paired, whose product is homologous to the Drosophila Gooseberry and mammalian Pax3 proteins, has three general functions: proper development of the larval cuticle, survival to adulthood and male fertility. Both DNA-binding domains, the conserved N-terminal paired-domain (PD) and prd-type homeodomain (HD), are required within the same molecule for all general paired functions, whereas a conserved His-Pro repeat located near its C terminus is a transactivation domain potentiating these functions. The C-terminal moiety of Paired includes two additional functional motifs: one, also present in Gooseberry and Pax3, is required for segmentation and cuticle development; the other, retained only in Gooseberry, is necessary for survival. The male fertility function, which cannot be replaced by Gooseberry and Pax3, is specified by the conserved N-terminal rather than the divergent C-terminal moiety of Paired. It is concluded that the functional diversification of paired, gooseberry and Pax3, primarily determined by variations in their enhancers, is modified by adaptations of their coding regions as a necessary consequence of their newly acquired spatiotemporal expression (Xue, 2001).

With the aid of two alleles of prd, prd-Gsb and prd-Pax3, in which the gsb and Pax3 coding regions were placed under the control of the entire prd cis-regulatory region, it has been shown that Prd activity is required in vivo during at least three distinct developmental stages to ensure proper segmentation of the larval cuticle, postembryonic viability and male fertility. In this study, a series of prd transgenes were constructed that express various versions of the Prd protein, including truncations or chimeras of Prd, Gsb and Pax3 under the control of the complete prd cis-regulatory region. All transgenes were tested for their ability to rescue any of these Prd functions. Thus, this report is the first example of a complete functional analysis of the Prd protein under natural conditions (Xue, 2001).

The presence of two DNA-binding domains, PD and HD, in Prd and some other members of the Pax gene family raises the question of whether the regulation of any of its target genes requires the binding of both or only one of its two DNA-binding domains. Both mechanisms are compatible with in vitro results. In vivo studies show that both PD and HD are absolutely required for Prd function because deletion of either or both of these domains from the prd-Gsb transgene results in the complete loss of its ability to rescue the segment-polarity gene activation, cuticular phenotype and lethality of prd mutants. Moreover, since a point mutation in the PD (i.e., prd-Gsb^P17L) eliminates all Prd functions, the DNA-binding ability of the PD is necessary for the normal functions of Prd. An analogous mutation abolishes DNA binding of the human PAX5 protein and causes Waardenburg's syndrome I when present in PAX3 (Xue, 2001).

The observation that prd-GsbdeltaP and prd-GsbdeltaH cannot complement for any function of Prd implies that the PD and HD must be present in the same Prd molecule, presumably because each Prd function requires the recognition of at least one composite DNA target site. In agreement with these findings, Prd proteins unable to bind DNA as a result of single amino acid substitutions in either the PD or HD can no longer activate the ectopic expression of Prd-target genes when expressed ubiquitously under the control of the heat-shock promoter nor will these mutant proteins perform any Prd in vivo function when expressed under the control of some of prd enhancers. In addition, a composite Prd target site has been identified in the even-skipped enhancer whose mutation in either the PD or HD binding portion dramatically reduces Prd binding activity both in vitro and in vivo. The finding that the PD and HD cannot complement in trans for any function of Prd agrees with some observations obtained with mutant transgenes in vivo, but contradicts results obtained in vitro, and in vivo when the two Prd mutant proteins are expressed under heat-shock control. Taken together, these results imply that the PD and HD of Prd may interact with their DNA targets cooperatively and that this cooperativity can occur in trans only if the proteins are produced at concentrations much higher than those occurring naturally (Xue, 2001 and references therein).

The PRD repeat, which encodes a 20-30 amino acid His-Pro repeat, was discovered in an attempt to verify predictions of the gene network hypothesis in a search for protein-coding domains of prd. The PRD repeat is found in a number of Drosophila early developmental genes, including bicoid and daughterless, but its in vivo function remained unknown. Previous experiments in cell culture systems have shown that the PRD repeat is part of a transactivation domain that is necessary to drive ectopic expression of Prd-target genes under the control of ubiquitously expressed Prd. Other studies, however, have suggested that the PRD repeat is not essential for in vivo functions of Prd. The Prd protein whose PRD repeat has been deleted in prd-PrddeltaPRD is still able to perform all in vivo functions of Prd, which implies that the PRD repeat is not absolutely required for Prd function. However, the fact that one copy of prd-PrddeltaPRD exhibits significantly reduced efficiency in its ability to rescue the lethality and male sterility of prd mutants indicates that the PRD repeat greatly facilitates these Prd functions. This conclusion is corroborated and extended by the results obtained with prd-Gsb+PRD transgenes, which demonstrate that the PRD repeat enhances the viability as well as the cuticle function of Prd. Thus, the PRD repeat is an important transactivation domain that facilitates all functions of Prd (Xue, 2001).

Previous work has demonstrated that Prd, Gsb and Pax3 proteins are, at least partially, functionally equivalent. When expressed under the control of the entire cis-regulatory region of prd, both Gsb and Pax3 can activate Prd-target genes necessary for the generation of wild-type cuticle, while Gsb is able to rescue prd mutants to adulthood. These results strongly suggested that the acquisition of cis-regulatory regions rather than the divergence of their coding regions is the primary evolutionary mechanism responsible for the functional diversification of prd, gsb and Pax3 genes. However, although Gsb and Pax3 can substitute for most Prd functions, they do so at considerably reduced efficiencies: this indicates that these proteins had to adapt their new functions for optimal performance by subsequent mutations producing the observed divergence of the Prd, Gsb and Pax3 proteins. The result of this process of adaptation has been studied by examining the functional differences between these proteins when expressed as evolutionary alleles under the same cis-regulatory region (Xue, 2001).

The results lead to the idea that, in addition to the PRD repeat, two motifs or domains are present in the C-terminal portion of Prd, on whose functions the formation of wild-type larval cuticle and survival to adulthood depend. Although no significant similarity has been found among the primary sequences of the C-terminal moieties of Prd, Gsb and Pax3, the motif required for implementing wild-type cuticle is shared by all three proteins. In contrast, the motif necessary for Prd’s viability function is retained only in Gsb, presumably as secondary or tertiary protein structure. It should be stressed that at least two independent functions of Prd are required for viability, one of which, Pax3, is able to perform even better than Gsb. However, Pax3 is unable to substitute for one of the viablity functions of Prd and even exerts a dominant-negative effect on it. In agreement with this postulate, combining the results with those obtained with two weak prd alleles encoding truncated Prd proteins, allows the motifs for the cuticle and viability functions to be mapped within the C terminus of Prd (Xue, 2001).

Although prd-Gsb rescues prd mutants to viable adults, all males are sterile. Since wild-type males transgenic for two copies of prd-Gsb are fertile, it is concluded that prd has a function required for male fertility. Moreover, since prd-Gsb includes the entire cis-regulatory region of prd, its failure to rescue male fertility must be caused by the inability of Gsb to replace this function of the Prd protein. Since Prd and Gsb share a highly conserved N-terminal portion consisting of two DNA-binding domains, the PD and HD, it seemed plausible to map this functional difference to their divergent C termini. Surprisingly, however, the protein-domain-swapping experiments indicate that the conserved N-terminal rather than the divergent C-terminal portion is the determinant for this particular function of Prd. Therefore, it is suggested that at least one specific Prd target site, recognized by Prd but not Gsb, is involved in male fertility. The male fertility function of Prd is controlled by a specific prd enhancer uncovered in prd mutants by a prd rescue construct that lacks 5 kb of the downstream regulatory region. Consistent with this interpretation, a prd transgene that expresses Prd merely under the control of this 5 kb regulatory region is able to confer fertility to prd-Gsb males mutant for prd. Males completely deficient for this fertility function of prd have no accessory glands, while accessory glands begin to form in prd mutant males rescued by prd-Gsb, but stop development at a severely reduced size. These findings are in agreement with the hypothesis that new functions evolve primarily through the acquisition of new enhancers during gene duplication and that the adaptation of the protein is secondary and a necessary consequence of its expression in the newly acquired context of this function (Xue, 2001).

These results further imply that the C-terminal portions of Prd and Gsb, though divergent in their primary sequences, are still qualitatively the same. Hence, the validity of amino acid similarity as a general measure of functional equivalence in homologous proteins can be questioned. Instead, it has been proposed that this measure of functional equivalence should be replaced by calculations of the mutual entropy between two protein sequences, a more precise statistical measure that takes into account the probability by which certain amino acids are replaced by others (Xue, 2001 and references therein).

Synaptic homeostasis is consolidated by the cell fate gene gooseberry, a Drosophila pax3/7 homolog

In a large-scale screening effort, the gene gooseberry (gsb) was identified as being necessary for synaptic homeostasis at the Drosophila neuromuscular junction. The gsb gene encodes a pair-rule transcription factor that participates in embryonic neuronal cell fate specification. This study defines a new postembryonic role for gooseberry. gsb becomes widely expressed in the postembryonic CNS, including within mature motoneurons. Loss of gsb does not alter neuromuscular growth, morphology, or the distribution of essential synaptic proteins. However, gsb function is required postembryonically for the sustained expression of synaptic homeostasis. In GluRIIA mutant animals, miniature EPSP (mEPSP) amplitudes are significantly decreased, and there is a compensatory homeostatic increase in presynaptic release that restores normal muscle excitation. Loss of gsb significantly impairs the homeostatic increase in presynaptic release in the GluRIIA mutant. Interestingly, gsb is not required for the rapid induction of synaptic homeostasis. Furthermore, gsb seems to be specifically involved in the mechanisms responsible for a homeostatic increase in presynaptic release, since it is not required for the homeostatic decrease in presynaptic release observed following an increase in mEPSP amplitude. Finally, Gsb has been shown to antagonize Wingless signaling during embryonic fate specification, and initial evidence is presented that this activity is conserved during synaptic homeostasis. Thus, gsb was identified as a gene that distinguishes between rapid induction versus sustained expression of synaptic homeostasis and distinguishes between the mechanisms responsible for homeostatic increase versus decrease in synaptic vesicle release (Marie, 2010).

This study has advanced understanding of synaptic homeostasis in several important ways. First, gsb was identified as required in postmitotic, postembryonic neurons for synaptic homeostasis at the Drosophila NMJ. Drosophila gsb is the homolog of vertebrate pax3/pax7. Thus, these data identify a new function for a conserved gene family that has been traditionally studied in the context of neuronal fate specification. Second, it was demonstrated that loss of gsb selectively disrupts the expression of synaptic homeostasis without impairing the rapid induction of synaptic homeostasis. These data suggest that genetically separable mechanisms exist for the induction versus the expression of synaptic homeostasis at the Drosophila NMJ. Third, it was demonstrated that loss of Gsb selectively disrupts the mechanisms responsible for a homeostatic increase in presynaptic release without impairing the mechanisms responsible for homeostatic decrease in presynaptic release. Therefore, these two forms of homeostatic modulation, both expressed at the Drosophila NMJ, appear to involve genetically separable mechanisms. Fourth, because gsb is a transcription factor, these data highlight the possibility that the persistent expression of synaptic homeostasis in the GluRIIA mutant is consolidated through transcription-/translation-dependent mechanisms, while the rapid induction of homeostatic signaling is independent of new protein synthesis. Thus, there may exist genetically separable phases of homeostatic signaling at the Drosophila NMJ analogous to the induction versus expression of long-term synaptic plasticity in other systems. Finally, evidence is presented to support the hypothesis that Gsb functions similarly during cell fate specification and synaptic homeostasis. According to the emerging model, Gsb may antagonize Wingless signaling in motoneurons to facilitate the consolidation of synaptic homeostasis. While there remains considerable work to prove this model, the data, in combination with prior work during embryonic cell fate specification, provide the basis for a compelling model that can be examined in greater detail in future studies (Marie, 2010).

In this study, motoneurons with decreased levels of Gsb are unable to express synaptic homeostasis in the background of a GluRIIA mutation. By contrast, the rapid, protein synthesis-independent induction of synaptic homeostasis following application of the glutamate receptor antagonist PhTx is normal. One possible explanation for this difference is that PhTx and the GluRIIA mutant cause different postsynaptic perturbations and initiate separate homeostatic signaling systems, only one of which is affected by loss of gsb. This seems unlikely, however, because previously published data indicate that PhTx primarily acts upon postsynaptic glutamate receptors including those that contain the GluRIIA receptor subunit. Furthermore, several mutations have been shown to block synaptic homeostasis both in the GluRIIA mutant and following PhTx application, demonstrating that these two perturbations share, at some level, a common molecular mechanism of homeostatic signaling. It is hypothesized, therefore, that loss of Gsb impairs a molecular process that is selectively involved in the sustained expression of synaptic homeostasis. This would be consistent with the sustained expression of synaptic homeostasis requiring new protein synthesis (Marie, 2010).

The possibility that Gsb participates specifically in the sustained expression or consolidation of synaptic homeostasis has interesting implications. In one model of homeostatic signaling, the GluRIIA mutation represents a persistent stress that induces a continuous, rapidly induced form of homeostatic compensation. In this model, the homeostatic modulation of presynaptic release is continually updated and never consolidated. An alternative model is that, once induced, the homeostatic modulation of presynaptic release is consolidated and maintained for prolonged periods of time. If this is the case, it should be possible to selectively disrupt the consolidation of synaptic homeostasis independently of the mechanisms of induction. Loss of Gsb appears to do just this. It is not possible to persistently inhibit protein synthesis during larval development. However, the demonstration that decreased Gsb disrupts synaptic homeostasis in the GluRIIA mutant suggests that transcription and translation may be involved in the mechanisms that consolidate a homeostatic change in presynaptic release (Marie, 2010).

In Drosophila, like in vertebrates, combinations of transcriptional regulators determine the fate of neurons. Indeed, transcription factors control all stages of early neuronal development and neuronal circuit formation, from the direction in which the axon initially extends from the neuronal cell body, the location of the terminal zone of the axonal arborization, and the specificity of synaptic targeting to the choice of neurotransmitter. More recently, some evidence suggests that expression of the transcription factor evenskipped during early embryogenesis could affect a motoneuron's complement of ion channels and neuron excitability. However very little is known regarding the role of transcriptional regulators in mature neurons. Recently, it was demonstrated that mild perturbation of the engrailed gene lead to mice with an adult phenotype that resembles key pathological features of Parkinson's disease. In this study, the expression of Gsb-RNAi using a postmitotic neuronal GAL4 driver leads to the conclusion that Gsb has a postmitotic activity that is essential to the maintenance of synaptic homeostasis. These data provide evidence that the transcription factors involved in embryonic development may have potent postembryonic functions that are necessary for the maintenance of stable neural function (Marie, 2010).

How do embryonic transcriptional regulators influence the expression of synaptic homeostasis? This study presents data to support a model in which Gsb function is conserved during embryonic patterning and synaptic homeostasis. Specifically, decreased wg levels rescue synaptic homeostasis in GluRIIA; gsb/+ double mutant animals. According to this model, Wg antagonizes the expression or consolidation of synaptic homeostasis, providing new insight into the activity of this potent intercellular, synaptic signaling molecule. It could be important, for example, to suppress homeostatic signaling during anatomical synaptic plasticity, a process in which Wg has been implicated. These data are strengthened by two observations. First, these data are supported by the well established embryonic activity of Gsb. Second, since partial loss of wg rescues the homeostatic defect in GluRIIA; gsb/+ double mutant animals, it is unlikely that this represents a nonspecific genetic interaction. Many additional experiments will be necessary to prove the function of wg as an antagonist of synaptic homeostasis. The data, however, take this model beyond the stage of pure speculation and suggest that this will be an important avenue of future experimental investigation (Marie, 2010).

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date revised:  15 December 2011

 

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