BIOLOGICAL OVERVIEW

Drosophila Serum response factor (SERF) has been shown to be allelic to blistered the preferred name for the gene. blistered is required for vein/intervein formation in the fly wing. Similarly, a mutation first known as pruned was isolated in a screen of Drosophila enhancer trap strains. Seen to have an affect on tracheal development, pruned also proved to be identical to blistered. blistered by any name is truely a gene with a multiple personality. In this report the gene will be referred interchangably as Serf or blistered.

Serf is expressed in the peripheral tracheal system during the late stages of the development of trachea. Many of the SERF containing nuclei belong to those tracheal cells closest to and in direct contact with the target tissues tracheated by particular branches, while cells tightly associated with the transverse connectives or with the lateral trunk do not express Serf. In flies where the chromosomal region for Serf has been deleted, defects have been observed in terminal tracheal branching. It appears that cells normally neighboring Serf-expressing cells in wild type act abnormally in Serf mutants. They do not properly stop their migration at the appropriate developmental stage, but rather appear to follow the leading cell of the branch, and eventually cluster in close proximity to the target tissue. Perhaps wild type tip cells leading outgrowing branches receive specific information upon contacting the target tissue and communicate this information to the following cells in order to arrest their migration (Affolter, 1994).

The cellular events leading to terminal branching in wild type cells begins when the terminal cell extends a cytoplasmic process. A lumen, or cavity, is present in the terminal cell, but only up to the level of the nucleus: the lumen is an early secondary tracheal branch. The cytoplasmic projection, becomes very thin as it extends along the surface of the target. The terminal cells of pruned mutants do not have long cytoplasmic projections beyond the cell body. In some cases rudimentary projections can be seen, but they never support a lumen. Thus, the absence of terminal branches in pruned mutants is a consequence of the failure of terminal cells to extend long cytoplasmic projections to their targets. The involvement of Serum response factor in the generation of cytoplasmic branches was tested by ectopically expressing the Mammalian SRF MADS box transcription factor in Drosophila. Constitutively active SRF drives formation of extra cytoplasmic projections within a single lumen. The cytoplasmic processes that form in cells expressing Mammalian SRF do not grow toward their usual targets but instead grow out in random directions, invading territories normally supplied by other tracheal branches (Guillemin, 1996).

The involvement of Serf in tracheal development recalls the involvement of Breathless, an FGF receptor homolog, in the terminal stages of tracheal development. Induction of a dominant-negative breathless construct after the tracheal branches are completed, blocks the formation of tracheoles (extensions of cellular processes by the terminal tracheal cells) demonstrating that Breathless plays an essential role in this later process, as well as in the earlier process of trachea outgrowth (Reichman-Fried, 1995). Perhaps Serf is a target of Breathless signaling. Since Breathless signaling involves the Ras pathway, perhaps Serf is a target of the Ras pathway.

Serf is expressed in the future intervein issue of the wing imaginal disc, in a complementary pattern to the EGF-R accessory gene rhomboid, and also complementary to araucan and caupolican which code for two divergent homeodomain proteins involved in establishing the prepattern for rhomboid. The genes myospheroid and inflated, coding for integrin subunits are expressed in intervein cells, while expression of the gene veinlet is restricted to the future vein tissue of the wing disc (Montagne, 1996, Sturtevant, 1993 and Gomez-Skarmeta, 1996).

In wing differentiation blistered/Serf plays a dual role in wing development. Two fully active copies of blistered/Serf are required to ensure that the formation of wing veins is limited to vein territories. In addition SERF protein is essential for proper terminal differentiation of intervein cells. One target of SERF appears to be rhomboid. rhomboid's range of expression broadens presumptive vein areas in Serf mutants to include cells normally fated to become intervein domains. A second target is likely to be myospheroid, which genetically interacts with blistered/Serf. Myospheroid is required for the tight apposition of dorsal and ventral surfaces of the wing; mutations in myospheroid result in blistered adult wings (Fristrom, 1994).

How might the roles of SERF in trachea and wing development find a common ground? In wing morphogenesis, around 18 hours after puparium formation, pairs of intervein cells on opposite dorsal and ventral surfaces undergo a process of apposition and become connected by basal extensions to form an intervein band. Later cells separate and undergo reapposition. Reapposition begins at the center of intervein regions and proceeds laterally, until by 30 hours, only vein channels remain unapposed. By 21 hours, vein cells begin to differentiate, undergoing a process of decrease in apical diameter. Vein cells become coated basally by a laminin-containing extracellular matrix, and laminin is subsequently cleared from the intervein regions. The process of formation of basal extentions, critical to the process of apposition, might be likened to the process of formation of cytoplasmic projections in tracheal terminal branching, a process involving changes in cell shape, undoubtedly based on cytoskeletal changes in the cell (Fristrom, 1994).

Serum response factor-mediated gene regulation in a Drosophila visual working memory

Navigation through the environment requires a working memory for the chosen target and path integration facilitating an approach when the target becomes temporarily hidden. Previous studies have shown that this visual orientation memory resides in the ellipsoid body, which is part of the central complex in the Drosophila brain (see Neuronal architecture of the central complex in Drosophila melanogaster in Niven's Visuomotor control: Drosophila bridges the gap). Former analysis of foraging and ignorant (ign) mutants have revealed that a hierarchical PKG and RSKII kinase signaling cascade in a subset of the ellipsoid-body ring neurons is required for this type of working memory in flies. This study shows that mutants in the ellipsoid body open (ebo) gene, which encodes the actin-binding protein Exportin 6, exhibit excessive nuclear accumulation of actin during development and in the adult brain. ebo mutants lack the orientation memory independent of the structural defect in the ellipsoid-body neuropil, and EBO activity in any type of adult ring neurons is sufficient for orientation-memory function. Moreover, genetic interaction studies revealed that nuclear actin accumulation in ebo mutants inhibits the Drosophila coactivator myocardin-related transcription factor A (dMRTF) and therefore the transcriptional activator serum response factor (dSRF). dSRF also functions in different ring neurons, suggesting that it regulates abundance of a diffusible factor that enables a working memory in ellipsoid-body ring neurons. To date, SRF has only been implicated in longer forms of memory formation like synaptic long-term potentiation and depression. This study provides the first evidence that SRF-mediated gene regulation is also required for a working memory that lasts only for a few seconds (Thran, 2013).

The central complex (CX) of the adult fly brain consists of four compartments that interconnect the protocerebral hemispheres: the protocerebral bridge, the fan-shaped body, the ellipsoid body (EB), and the ventrally located paired noduli. The analysis of several Drosophila mutant strains with structural defects in one or more neuropils of the CX has suggested that the CX represents a higher control center for locomotion and orientation behavior. These mutants walk slower than wild-type flies, have a delayed reaction to changing stimuli during flight, and show deficits in the orientation behavior toward landmarks. A major sensory input region for external stimuli, especially visual information, appears to be the ellipsoid body and the fan-shaped body, and in larger insects this includes information on the orientation of polarized light, which is used for navigation. In Drosophila, it has been shown that the protocerebral bridge is required for step-length control in walking flies and visual targeting during climbing events. Visual input is also processed in the fan-shaped body, which mediates visual pattern recognition to memorize different objects during flight control], a function that has also been attributed to the EB. The EB also holds a memory trace for the position of landmarks, as revealed in a Morris water maze-like paradigm for visual place learning in flies. In addition, the analysis of the ellipsoid body open (ebo) mutant has shown that the EB is necessary to establish a visual orientation memory for a vanishing object in walking Drosophila (Thran, 2013).

In this so-called detour paradigm, a fly approaching an attractive target is lured out of the way by a dark stripe on one side while simultaneously the original object vanishes. After the distracting stripe has also been removed, the fly is left without any visual cue. However, in over 80% of the cases, wild-type flies use idiothetic information on their past movements with respect to the original target to resume their originally intended approach. This type of working memory lasts about 4 s and must be updated during every turn the fly takes (Neuser, 2008). Analysis of two Drosophila memory mutants in ignorant (ign) and foraging (for), which encode the ribosomal-S6 kinase II (RSKII) and cGMP-dependent protein kinase (PKG), respectively, revealed that both kinases share one signaling pathway that is required in a specific type of EB ring neurons to display an orientation memory (Kuntz, 2012; Thran, 2013).

To further elucidate the structural and biochemical components that enable the EB to hold an orientation memory, the ebo mutants were genetically and molecularly analyzed. This study reports that the ebo gene encodes the actin-binding protein Exportin 6, which is required for the export of globular actin (G-actin) from the nucleus, thus preventing actin-filament formation there. This analysis of the ebo mutant confirms these findings because elevated levels of an Actin-GFP fusion protein can be found in nuclei of the mutant. Interestingly, excessive G-actin has been shown to inhibit the myocardin- related transcription factor A in vertebrates (MRTF-A), a coactivator of the transcriptional regulator serum response factor (SRF). This genetic interaction analysis of ebo with the Drosophila ortholog of MRTF, as well as rescue experiments of the dSRF mutant blistered (bs), revealed that elevated levels of nuclear actin in EB neurons and the subsequent malfunction of the dMRTF/dSRF transcription regulator complex prevent the visual orientation memory in flies (Thran, 2013).

In vertebrates, more than 200 putative target genes of SRF have been postulated, most of them involved in cytoskeleton dynamics and cell motility, but immediate early genes like c-fos have also been identified. Unfortunately, downstream targets of Drosophila dSRF in neuronal cells are yet unknown, complicating the identification of further gene products that are involved in development of the EB and a functional visual orientation memory in adult flies. Based on the current results, it is hypothesized that the effect of EBO on the transcriptional activity of the dMRTF/dSRF complex is responsible for both the developmental and behavioral phenotype (Thran, 2013).

Although transheterozygous mutants for hypomorphic bs alleles displayed no structural EB defect and the morphological defects of homozygous MrtfD7 mutants are less prominent than those of ebo mutants, it nevertheless is possible that dMRTF/dSRF-mediated gene regulation is also required in EB development. For instance, in vertebrates, transcription of profilin, an F-actin promoting factor, is activated by SRF, and the structural ebo phenotype could indicate that dSFR is also promoting transcription of the profilin encoding gene chickadee in Drosophila. Alternatively, nuclear actin in the ebo mutant could sequester profilin in the nucleus, thus reducing levels at the growth cone required in axonal outgrowth (Thran, 2013).

However, it is obvious that a memory that lasts only a few seconds cannot depend on changes in transcriptional regulation. The rescue experiments described in this study have established that EBO and dSRF can restore memory function of the respective mutant independent of the specific ring-neuron subtype. This is surprising, considering the biochemical activity of EBO and dSRF, which are definitively cell autonomous. Therefore, it is proposed that dSRF promotes gene expression that ultimately results in the production of a diffusible factor that has to be delivered to the ring neurons. Presumably, this factor feeds into pathways that enable rapid changes in synaptic transmission of the ring neurons necessary to encode an orientation memory. For instance, ring neurons might need a higher density of synaptic release sites, a highly efficient synaptic vesicle reserve pool, or elevated expression of ion channels for prolonged excitation. Similarly, a high density of dendritic neurotransmitter receptors and elevated levels of second messenger molecules at the postsynaptic site of the ring neurons could be necessary to exert their specific function in working memory formation and/or retrieval. Finding the dSRF target genes that mediate the orientation memory in flies might lead to new insights how working memories are orchestrated in general (Thran, 2013).

GENE STRUCTURE

Bases in 5' UTR - 375

Bases in 3' UTR - 482

PROTEIN STRUCTURE

Amino Acids - 450

date revised: 25 September 2023

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