InteractiveFly: GeneBrief

alan shepard: Biological Overview | References


Gene name - alan shepard

Synonyms -

Cytological map position - 64C8-64C11

Function - RNA-binding protein

Keywords - regulation of insulator activity in a tissue-specific manner, CNS, metamorphic development of neurons, regulation of gravitaxis

Symbol - shep

FlyBase ID: FBgn0052423

Genetic map position - chr3L:5,153,775-5,241,544

Classification - RNA recognition motif (RRM) superfamily

Cellular location - nuclear and probably cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Schachtner, L. T., Sola, I. E., Forand, D., Antonacci, S., Postovit, A. J., Mortimer, N. T., Killian, D. J. and Olesnicky, E. C. (2015). Drosophila Shep and C. elegans SUP-26 are RNA-binding proteins that play diverse roles in nervous system development. Dev Genes Evol [Epub ahead of print]. PubMed ID: 26271810
Summary:
The Caenorhabditis elegans gene sup-26 encodes a well-conserved RNA-recognition motif-containing RNA-binding protein (RBP) that functions in dendrite morphogenesis of the PVD sensory neuron. The Drosophila ortholog of sup-26, alan shepard (shep), is expressed throughout the nervous system and has been shown to regulate neuronal remodeling during metamorphosis. These studies were extended to show that sup-26 and shep are required for the development of diverse cell types within the nematode and fly nervous systems during embryonic and larval stages. Roles are described for sup-26 in regulating dendrite number and the expression of genes involved in mechanosensation within the nematode peripheral nervous system. In Drosophila, shep regulates dendrite length and branch order of nociceptive neurons, regulates the organization of neuronal clusters of the peripheral nervous system and the organization of axons within the ventral nerve cord. Taken together, these results suggest that shep/sup-26 orthologs play diverse roles in neural development across animal species. Moreover, potential roles for shep/sup-26 orthologs in the human nervous system are discussed.
Chen, D., Gu, T., Pham, T. N., Zachary, M. J. and Hewes, R. S. (2017). Regulatory mechanisms of metamorphic neuronal remodeling revealed through a genome-wide modifier screen in Drosophila melanogaster. Genetics [Epub ahead of print]. PubMed ID: 28476867
Summary:
The RNA binding factor alan shepard (shep) is an important regulator of neuronal remodeling during metamorphosis in Drosophila melanogaster, and loss of shep leads to smaller soma size and fewer neurites in a stage-dependent manner. To shed light on the mechanisms by which shep regulates neuronal remodeling, a genetic modifier screen was conducted for suppressors of shep-dependent wing expansion defects and cellular morphological defects in a set of peptidergic neurons, the bursicon neurons, that promote post-eclosion wing expansion. Out of 702 screened deficiencies that covered 86% of euchromatic genes, 24 deficiencies were isolated as candidate suppressors, and 12 of them at least partially suppressed morphological defects in shep mutant bursicon neurons. With RNAi and mutant alleles of individual genes, Daughters against dpp (Dad) and Olig family (Oli) were identified as shep suppressor genes, and both of them restored the adult cellular morphology of shep-depleted bursicon neurons. Dad encodes an inhibitory Smad protein that inhibits bone morphogenetic protein (BMP) signaling, raising the possibility that shep interacted with BMP signaling through antagonism of Dad. By manipulating expression of the BMP receptor tkv, activated BMP signaling was found to be sufficient to rescue loss-of-shep phenotypes. These findings reveal mechanisms of shep regulation during neuronal development, and they highlight a novel genetic shep interaction with the BMP signaling pathway that controls morphogenesis in mature, terminally differentiated neurons during metamorphosis.

BIOLOGICAL OVERVIEW

Chromatin insulators organize the genome into distinct transcriptional domains and contribute to cell type-specific chromatin organization. However, factors regulating tissue-specific insulator function have not yet been discovered. This study identified the RNA recognition motif-containing protein Shep as a direct interactor of two individual components of the gypsy insulator complex in Drosophila. Mutation of shep improves gypsy-dependent enhancer blocking, indicating a role as a negative regulator of insulator activity. Unlike ubiquitously expressed core gypsy insulator proteins, Shep is highly expressed in the central nervous system (CNS) with lower expression in other tissues. A novel, quantitative tissue-specific barrier assay was developed to demonstrate that Shep functions as a negative regulator of insulator activity in the CNS but not in muscle tissue. Additionally, mutation of shep alters insulator complex nuclear localization in the CNS but has no effect in other tissues. Consistent with negative regulatory activity, ChIP-seq analysis of Shep in a CNS-derived cell line indicates substantial genome-wide colocalization with a single gypsy insulator component but limited overlap with intact insulator complexes. Taken together, these data reveal a novel, tissue-specific mode of regulation of a chromatin insulator (Matzat, 2012).

Chromatin insulators are DNA-protein complexes that influence eukaryotic gene expression by organizing the genome into distinct transcriptional domains. Functionally conserved from Drosophila to humans, insulators regulate interactions between regulatory elements such as enhancers and promoters and demarcate silent and active chromatin regions. Chromatin insulators are thought to exert effects on gene expression by constraining the topology of chromatin and facilitating the formation of intra- and inter-chromosomal looping. These higher order interactions can vary between cell types, thereby facilitating tissue-specific transcriptional output (Matzat, 2012).

Drosophila harbor several distinct classes of chromatin insulators, including the well studied gypsy insulator, also known as the Suppressor of Hairy wing (Su(Hw)) insulator. The zinc-finger DNA-binding protein, Su(Hw), recognizes a particular motif, imparting specificity to the gypsy insulator. In addition to Su(Hw), the core gypsy insulator complex contains Centrosomal protein 190 (CP190), which also harbors a zinc finger domain, and the non-DNA-binding protein, Modifier of mdg4 2.2 (Mod(mdg4)2.2). These core proteins are required for gypsy insulator activity. Both CP190 and Mod(mdg4)2.2 contain broad complex, tramtrack, bric-a-brac (BTB) dimerization domains that have been suggested to mediate insulator-insulator interactions and facilitate the formation of long range insulator-mediated loops along the chromatin fiber (Matzat, 2012).

Specialized nuclear arrangement of gypsy insulator complexes correlates tightly with insulator function. The gypsy insulator proteins bind to thousands of sites throughout the genome with more than half of Su(Hw) binding sites occurring in intergenic regions and a large number of sites located within introns. Consistent with a role in boundary formation, Su(Hw) sites are positively correlated with both Lamin-associated domains and boundaries between transcriptionally active and silent chromatin. It has been shown that gypsy insulator proteins coalesce at a small number of foci in diploid nuclei, termed insulator bodies, which have been proposed to act either as hubs of higher order chromatin domains or storage sites for insulator proteins. Importantly, mutation of certain insulator components results in impaired insulator activity coincident with diffuse or smaller, more numerous insulator bodies. However, formation of insulator bodies is not sufficient for gypsy insulator activity, and a detailed mechanistic understanding of insulator bodies is still lacking. Nevertheless, the tight correlation between gypsy insulator function and insulator body localization suggests an important role for these structures. Finally, in addition to a variety of accessory proteins, a role for RNA in insulator function and insulator body organization was suggested based on RNA-dependent protein interaction with insulator complexes (Matzat, 2012).

Genome-wide studies indicate that the locations of insulator protein binding sites are mainly consistent across different cell types but that insulator-dependent looping configurations may dictate differences in gene expression. In Drosophila, it has been shown that external stimuli can alter chromatin association of CP190, possibly leading to a change in chromatin looping. Recent large-scale chromatin conformation capture (3C)-based studies have implicated insulator protein binding sites as key contact points mediating looping throughout the genome. In several studies across species, specific chromatin conformations are observed in loci that produce tissue- or cell-type specific transcripts. Whether insulators either establish tissue-specific chromatin organization or maintain configurations established via transcription is unclear. Furthermore, factors that control tissue-specific insulator-dependent chromatin organization remain unknown (Matzat, 2012).

This study identifies a CNS enriched, RNA recognition motif (RRM) containing protein, Alan Shepard (Shep), as the first tissue-specific regulator of gypsy insulator activity and insulator body localization. Shep interacts directly with Mod(mdg4)2.2 and Su(Hw) and also associates with gypsy insulator proteins in vivo. Using a novel quantitative, tissue-specific insulator assay, it was found that Shep negatively regulates gypsy insulator activity in the CNS. In addition, mutation of Shep improves compromised insulator function and insulator body formation. Finally, genome-wide localization in the CNS-derived BG3 cell line reveals enrichment of overlap between Shep and Mod(mdg4)2.2 but less frequent than expected overlap among Shep, Su(Hw) and Mod(mdg4)2.2 together. These data suggest that gypsy chromatin insulator function can be regulated in a tissue-specific manner (Matzat, 2012).

Two lines of evidence indicate that Shep affects insulator activity in a tissue-specific manner. First, insulator body localization is altered in CNS but not other tissues of shep mutants. Second, barrier activity is improved in CNS but not muscle tissue when Shep levels are reduced. Finally, genome-wide mapping of Shep and gypsy insulator proteins in BG3 cells reveals substantial overlap with individual insulator proteins but lack of three-way overlap, further supporting a role for Shep in negative regulation of insulator activity in certain tissues (Matzat, 2012).

Shep acts as a tissue-specific negative regulator of gypsy insulator function and insulator body localization. Shep localization is most enriched in the CNS at both embryonic and larval stages; however, it is also expressed at lower levels in additional tissues. Although this study has demonstrated that Shep functions in the CNS, Shep can also repress enhancer blocking activity in the wing and could possibly affect insulator activity in other tissues. For example, ubiquitous reduction of Shep levels strongly improves overall barrier activity, suggesting that tissues outside of the CNS may also harbor Shep activity. Nonetheless, Shep does not appear to function in all tissues; knockdown of Shep does not affect barrier activity in muscle tissue, no changes in insulator body localization are observed in eye or leg tissue of shep mutants, and no effect is observed for y2 enhancer blocking in pigment cells of shep mutants. Interestingly, when Shep is overexpressed in muscle tissue, reduction of barrier activity is observed, suggesting that a certain threshold of Shep protein is needed to repress insulator activity. Since Shep protein can be detected at least at low levels in all tissues tested thus far, it is unlikely that the mere presence of Shep protein is sufficient to disrupt gypsy insulator activity. It remains to be determined what other cofactors, such as proteins or RNAs, may contribute to Shep activity (Matzat, 2012).

Shep may negatively regulate insulator activity by interfering with insulator protein interactions required for their activity. ChIP-seq analyses shows that the genome-wide binding profile of Shep in CNS-derived BG3 cells overlaps substantially with that of Mod(mdg4)2.2 but not extensively with both Su(Hw) and Mod(mdg4)2.2 combined. Lack of three-way overlap is not entirely unexpected given that Shep is a negative regulator of gypsy insulator activities. Shep coimmunoprecipitation experiments copurify only a small fraction of total insulator proteins present in nuclear extracts, suggesting that Shep-insulator complexes are not abundant or not stable in vivo. Since Shep can bind either Mod(mdg4)2.2 or Su(Hw) in vitro at a 1:1 ratio, Shep binding could compete with direct interaction between Mod(mdg4)2.2 and Su(Hw) or their interactions with other factors such as CP190. Moreover, the finding that mod(mdg4) mutants are highly sensitive to Shep dosage suggests an antagonistic functional relationship between Mod(mdg4)2.2 and Shep. Specifically, Shep may negatively regulate higher order insulator-insulator complex interactions, which appear to be mediated by direct interaction between Mod(mdg4)2.2 and CP190. Insulator body localization in larval brains of shep, mod(mdg4)u1 mutants reverts back to a wildtype pattern compared to compromised mod(mdg4)u1 mutants, perhaps indicating that the normal function of Shep may be to prevent larger insulator complexes from forming in these cell types (Matzat, 2012).

The results are consistent with the possibility that Shep promotes tissue-specific chromatin configurations by modulating insulator complexes. While differential occupancy of insulator proteins at their respective binding sites may play a role in regulating certain loci, occupancy throughout the genome does not differ extensively between cell types. Therefore, alternate mechanisms to control insulator activity likely exist. Shep activity could prevent insulator-insulator contacts otherwise present in tissues that do not express shep, resulting in relief of enhancer blocking or repression by silencers. Interestingly, shep was identified as a regulator of complex behavioral traits in screens for altered sensory-motor responsiveness to gravity (Armstrong, 2006) and aggressive behavior (Edwards, 2009), suggesting the possibility that regulation of an insulator-based mechanism could exist to effect changes in neurological function (Matzat, 2012).

Given that Shep is an RRM-containing protein, RNA-binding may contribute to the ability of Shep to associate with insulator complexes in vivo. Shep RRMs are highly conserved, and lethality caused by Shep overexpression in the mod(mdg4) mutant background is not observed when the RRMs are mutated. This result suggests that Shep RRMs may be functional with respect to insulator activity. One possibility is that the specific RNA bound by Shep could affect targeting of Shep to insulator sites. Another not mutually exclusive prospect is that Shep is recruited to chromatin cotranscriptionally by binding nascent transcripts. It will be important to determine in future studies if Shep binds RNA while in complex with gypsy insulator proteins as well as the identities of Shep and insulator-associated RNA. These results point to a novel role for Shep and possibly RNA to regulate insulator activity in a tissue-specific manner (Matzat, 2012).

Neuronal remodeling during metamorphosis is regulated by the alan shepard (shep) gene in Drosophila melanogaster

Peptidergic neurons are a group of neuronal cells that synthesize and secrete peptides to regulate a variety of biological processes. To identify genes controlling the development and function of peptidergic neurons, a screen was conducted of 545 splice-trap lines and 28 loci were identified that drove expression in peptidergic neurons when crossed to a GFP reporter transgene. Among these lines, an insertion in the alan shepard (shep) gene drove expression specifically in most peptidergic neurons. shep transcripts and SHEP proteins were detected primarily and broadly in the central nervous system (CNS) in embryos, and this expression continued into the adult stage. Loss of shep resulted in late pupal lethality, reduced adult life span, wing expansion defects, uncoordinated adult locomotor activities, rejection of males by virgin females, and reduced neuropil area and reduced levels of multiple pre-synaptic markers throughout the adult CNS. Examination of the bursicon neurons in shep mutant pharate adults revealed smaller somata and fewer axonal branches and boutons, and all of these cellular phenotypes were fully rescued by expression of the most abundant wild-type shep isoform. In contrast to shep mutant animals at the pharate adult stage, shep mutant larvae displayed normal bursicon neuron morphologies. Similarly, shep mutant adults were uncoordinated and weak, while shep mutant larvae displayed largely, though not entirely, normal locomotor behavior. Thus, shep plays an important role in the metamorphic development of many neurons (Chen, 2014).

Peptidergic neurons produce small peptides, called neuropeptides, which are secreted within the nervous system to influence the activity of other neurons or into the blood to act on other tissues. Through these targets, neuropeptides regulate a wide range of processes, which include development, feeding, growth, aggression, reproduction and learning and memory. One of the first genes identified to play a specific role in the development of peptidergic neurons was dimmed (dimm), which encodes a basic helix-loop-helix transcription factor that is required for the differentiation of diverse peptidergic neurons. Dimm is a key regulator of expression of the neuropeptide biosynthetic enzyme, peptidylglycine-alphahydroxylating monooxygenase (Phantom or Phm), and it promotes the differentiation of neurosecretory properties in many neurons. Both Dimm and Phm are expressed widely and specifically in peptidergic neurons. In fact, Dimm was first identified by virtue of its pattern of peptidergic neuron expression through an enhancer-trap screen. Similar expression pattern-based strategies may be useful for identification of other factors critical for peptidergic neuron development (Chen, 2014).

This study sought to identify similar factors through a splice-trap screen for genes with peptidergic cell-specific expression patterns. 28 insertions were identified with different patterns of peptidergic cell reporter gene expression, driven by P element splicetrap insertions in specific loci. These insertions drove reporter expression in insulin-like peptide 2 (ILP2), crustacean cardioactive peptide (CCAP)/bursicon, -RFamide, Furin 1, and leucokinin (LK) cells and often caused defects typical of disrupted neuropeptide signaling. Thus, all 28 of these genes are strong candidate regulators of peptidergic cell development or function (Chen, 2014).

One of the splice-trap insertions was mapped to an exon of the alan shepard (shep) gene, and this insertion was chosen for further analysis because it displayed an expression pattern that was highly similar to Phm and Dimm. shep in situ hybridization and anti-Shep immunostaining later revealed that both the shep mRNA and Shep protein expression is enriched in most neurons, yet shep mutants displayed defects in adult eclosion and wing expansion that suggested specific disruptions in signaling by bursicon and other neuropeptides. Consistent with these behavioral phenotypes, the shep mutant bursicon neurons had smaller somata, fewer axon branches, and smaller and fewer neuroendocrine boutons, and all of these phenotypes were rescued by expression of a wild-type shep cDNA. Interestingly, pan-neuronal RNA interference to shep produced smaller CNS neuropils and defects in general locomotor behaviors, such as flipping and climbing. Most of the locomotor phenotypes were restricted to the adult stage, and the effects of shep mutations on neuronal growth were restricted to pupal development. Thus, shep regulates metamorphic growth of the bursicon neurons, and it may also serve as a general regulator of neuronal growth during metamorphic remodeling (Chen, 2014).

Anti-Shep immunostaining and additional shep reporter genes confirmed expression in peptidergic neurons, but these markers and shep in situ hybridization also revealed widespread expression in the CNS, with much lower expression in other tissues. Shep is orthologous to the c-myc single-strand binding protein, MSSP-2: Previous studies have described shep as homologous to the vertebrate genes, Rbms2/Scr3 (Armstrong, 2006) or Rbms1/Scr2/MSSP-2. Phylogenetic analysis supports the placement of Shep in the MSSP family, with the ELAV family of RNA-binding proteins being the next most closely related. In general, MSSP proteins contain RNA recognition motifs and have been found in vertebrates to bind DNA, RNA, or proteins to regulate a variety of biological processes, including DNA polymerization, gene expression, cell transformation, and apoptosis. In Drosophila, Shep interacts with the insulator proteins Mod(mdg4)2.2 and Su(Hw) to negatively regulate chromosomal insulator activities, specifically in the CNS (Matzat, 2012). These molecular insights suggest a gene regulatory mechanism by which Shep may control aspects of the metamorphic development of the bursicon neurons, as well as other neurons that contribute to the overall structure of adult brain neuropils (Chen, 2014).

The shep mutant defects in wing expansion presented an opportunity to define cellular functions of Shep in an experimentally accessible cell type, the bursicon neurons. In shep mutants, a reduction was observed in the post-pruning growth of the bursicon neurons during metamorphosis, resulting in smaller somata and less branching in the peripheral axon arbor in pharate adult animals. Interestingly, the regulation of bursicon neuron growth by shep was stage-dependent. Defects were observed in bursicon neuron soma growth and axon branching during metamorphosis in hypomorphic shep mutant animals of multiple genotypes, including shepBG00836/shepBG00836, shepExel6103/shepExel6104 and shepBG00836/shepED210. However, in each of these genotypes, the larval cellular morphologies were normal. Other behavioral defects were observed that suggested that the metamorphosis-specific actions of Shep were not limited to the bursicon neurons. For example, the most severe shep loss-of-function genotype tested was elav>shep-RNAi, Dicer-2, but elav>shep-RNAi, Dicer-2 larvae displayed normal crawling distances and self-righting behaviors, while this genotype showed lethality in the late pupal stages and severe locomotor defects in adult animals. Associated with this increase during metamorphosis in the dependence of the nervous system on shep activity, there is also a marked increase in the levels of shep expression at the onset of metamorphosis (Chen, 2014).

These results provide indirect evidence to suggest that an increase in shep expression during the pupal stage may support neuronal remodeling or other aspects of neuronal function and development in diverse neurons during metamorphosis. Although most of the larval behaviors assayed were unaffected in shep mutant animals, one behavioral phenotype in was observed in elav>shep-RNAi, Dicer-2 larvae, namely a tendency to remain in the center of the apple juice-agarose plate while making many sharp turns along the path of locomotion. Based on anti-Shep immunostaining, UAS-shep-RNAi, Dicer-2 provided a more complete knockdown of anti-Shep immunostaining in the CNS than shep RNAi without UAS-Dicer-2 or in shepBG00836 homozygotes or shepBG00836/shepED210 mutant larvae. Moreover, shep RNAi without UAS-Dicer-2 led to a greater knock-down of Shep in Western blots than shepExel6103/shepExel6104 (Matzat, 2012). Taken together with the above observation that many of the weaker shep loss-of-function genotypes had defects that were only manifest in adults, these findings suggest that shep plays a stage-dependent (largely metamorphosis-specific) role in the maintenance, function, or development of the nervous system (Chen, 2014).

The Shep expression pattern and shep mutant phenotypes reported in this study are consistent with broad actions of this protein in neuronal development and functions throughout the nervous system. Pan-neuronal loss of shep resulted in late-pupal lethality and reduced adult life span under both fed and starved conditions, as well as diverse developmental and behavioral defects, including failure to complete wing expansion, uncoordinated and weakened adult locomotion, reduced neuropil areas, and altered mating behaviors. Other groups have also shown defects in gravitaxis and reduced starvation resistance in shep mutants (Armstrong, 2006; Harbison, 2004; Chen, 2014 and references therein).

Such widespread actions may also explain the partial rescue of the mating defects by UAS-shep expression in shepBG00836/shepED210 females. Although the possibility cannot be excluded that other Shep isoforms in addition to Shep-E/G (used to create UAS-shep) str necessary to support the normal function of the post-copulatory grooming circuits, it is also possible that neurons required for female receptivity to the male may have been included in the shepBG00836 expression pattern used to drive shep rescue, whereas the neurons involved in normal post-copulatory grooming behaviors are not (Chen, 2014).

The observation of several seemingly independent behavioral defects (e.g., gravitaxis and female receptivity to mating) and reduced neuropil areas, taken together with the cellular defects described in shep-mutant bursicon neurons, suggests that Shep may have pleiotropic effects on neurite development or other processes throughout the CNS. Such pleiotropic effects of shep mutations in the CNS may be due to the loss of Shep suppression of widely distributed chromatin insulator complexes (Matzat, 2012), so as to establish altered chromatin states and gene expression, potentially in multiple signaling pathways controlling a range of developmental and physiological events. In addition, some of the adult shep loss-of-function phenotypes, such as reduced lifespan and altered mating behaviors, may reflect adult-specific (acute) effects of Shep on neuronal activity. Alternatively, the metamorphosis-specific regulation of neurite branching and cell growth in the bursicon neurons may be representative of the actions of Shep in many neuronal cell types. It will be important in future studies to distinguish among these models, as the results demonstrate that Shep is a general regulator of the postembryonic development of mature neurons (Chen, 2014).


REFERENCES

Search PubMed for articles about Drosophila Alan Shepard

Armstrong, J. D., Texada, M. J., Munjaal, R., Baker, D. A. and Beckingham, K. M. (2006). Gravitaxis in Drosophila melanogaster: a forward genetic screen. Genes Brain Behav 5: 222-239. PubMed ID: 16594976

Chen, D., Qu, C. and Hewes, R. S. (2014). Neuronal remodeling during metamorphosis is regulated by the alan shepard (shep) gene in Drosophila melanogaster. Genetics [Epub ahead of print]. PubMed ID: 24931409

Edwards, A. C., Zwarts, L., Yamamoto, A., Callaerts, P. and Mackay, T. F. (2009). Mutations in many genes affect aggressive behavior in Drosophila melanogaster. BMC Biol 7: 29. PubMed ID: 19519879

Harbison, S. T., Yamamoto, A. H., Fanara, J. J., Norga, K. K. and Mackay, T. F. (2004). Quantitative trait loci affecting starvation resistance in Drosophila melanogaster. Genetics 166: 1807-1823. PubMed ID: 15126400

Matzat, L. H., Dale, R. K., Moshkovich, N. and Lei, E. P. (2012). Tissue-specific regulation of chromatin insulator function. PLoS Genet 8: e1003069. PubMed ID: 23209434


Biological Overview

date revised: 20 July 2014

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