BIOLOGICAL OVERVIEW

Mutations in the spinster gene are characterized by an extraordinarily strong rejection behavior of female flies in response to male courtship. They are also accompanied by decreases in the viability, adult life span, and oviposition rate of the flies. In spin mutants, persistence of unnecessary cells results in the degeneration of oocytes in the ovaries and of neurons in the CNS. This cell death is preceded by reductions in programmed cell death of nurse cells in ovaries and of neurons in the pupal nervous system. The Spin protein is therefore postulated to function in mediating apoptotic signals conveyed from follicle cells to nurse cells or from glia to neurons. The accumulation of lipofuscin-like materials in spin mutant neurons further implies the possible function of the spin gene in regulating lysosomal turnover in nerve cells. The spin locus generates at least five different transcripts, with only two of these being able to rescue the spin behavioral phenotype; each encodes a protein with multiple membrane-spanning domains that are expressed in both the surface glial cells in the CNS and the follicle cells in the ovaries. Orthologs of the spin gene have also been identified in a number of species from nematodes to humans. Analysis of the spin mutant should provide new insights into neurodegenerative diseases and aging (Nakano, 2001).

spin mutant flies have a long abdominal ganglion. In an attempt at determining the cellular basis for the behavioral phenotypes of the spin mutant, the morphology of the CNS was examined and it was found that the abdominal ganglia in spin^P1 mutant flies of both sexes are longer than those of the wild type. The thoracic segment of the ventral nerve cord (VNC) appears normal, while the abdominal segment is abnormally long in the spin^P1 mutant. The ratio of the length of the posterior part over the total length of the VNC was estimated as follows: for wild-type males, 0.260 ± 0.006 (mean ±); for wild-type females, 0.265 ± 0.004 ; for spin males, 0.343 ± 0.016, and for spin females, 0.338 ± 0.010 (n = 23). The unshortened morphology of the VNC is still evident 2 weeks after eclosion in spin^P1 flies. Apart from the VNC, no gross morphological abnormalities were found in spin mutant flies (Nakano, 2001).

Programmed cell death (PCD) of the neurons in the VNC occurs shortly after pupariation and after the emergence of the adult; typically, the ventral abdominal ganglion cells die during metamorphosis and after eclosion. The abdominal portion of the VNC shortens after the first wave of PCD in the mid-pupal stage. The absence of condensation of the abdominal ganglia in the spin mutants implies that some cells in the VNC are prevented from undergoing PCD. To evaluate this possibility, the numbers of cells undergoing apoptosis between the spin heterozygous and homozygous VNCs were compared by selectively staining fragmented DNA using TUNEL. PCD in the VNC occurs in two phases, one during the first several hours after pupation and the other immediately after adult eclosion. No difference was found in the numbers of apoptotic cells between the spin heterozygote and homozygote after adult emergence. In contrast, the profile of PCD in the VNC of the spin homozygote is distinctly different from that in the spin heterozygote during the pupal stage: the number of cells undergoing PCD in heterozygotes is maximum at 6 h after puparium formation (APF) as reported for the wild type, whereas the number of apoptotic cells remains quite low at this developmental stage in homozygotes (Nakano, 2001).

The difference in the numbers of the cells undergoing apoptosis at 6 h APF between heterozygotes and homozygotes is statistically significant. No significant difference was found in the number of apoptotic cells between heterozygotes and homozygotes at 4 and 24 h APF. These observations suggest that many cells that will be eliminated at 6 h APF remain alive through the pupal stage in the spin mutant (Nakano, 2001).

spin mutations interfere with PCD in ovaries as well as in the nervous system. These cellular defects should contribute at least in part to behavioral phenotypes of spin mutant female flies, i.e., the reduction in oviposition and sexual receptivity. It is evident that some immature oocytes degenerate in spin mutant ovaries; thus, the total number of eggs available for oviposition decreases. However, this finding does not necessarily exclude the possibility that the spin mutant female flies are accompanied by some other deficits that hamper normal oviposition. It is important to note that the spin gene product is expressed in the follicle cells of ovaries and not in nurse cells or oocytes. This observation suggests that the Spin protein is required by follicle cells for inducing the PCD of nurse cells, a group of support cells which are crucial for the normal development of oocytes. The degeneration of immature oocytes in spin mutant female flies may thus result from the disturbance of the process of PCD of nurse cells as a consequence of the loss of Spin from follicle cells (Nakano, 2001).

The link between the PCD defect in the nervous system and enhanced mate refusal in spin mutant female flies is less obvious. By analogy with the effect on ovaries, it is conceivable that the spin mutations likely affect female sexual behavior through degeneration of cells in the nervous system. Indeed, neurodegeneration is widespread in the spin^P1 mutant nervous system, which accumulates lipofuscin-like materials. In the spin mutant, the neurodegenerative phenotype is observed in both sexes, but germ cell abnormality and abnormal sexual behavior are found to be female specific. It may be that changes in female sexual receptivity are the easiest parameter by which to detect the malfunctioning of the nervous system due to the accumulation of lipofuscin-like materials in neurons. Alternatively, the reduced receptivity to copulation in spin^P1 mutant females and the neurodegeneration observed in both sexes of the spin mutant flies might be unrelated. This issue remains to be resolved by future analysis of spin mutant flies (Nakano, 2001).

There are interesting parallels in the effect of spin mutations on ovaries and the nervous system. Like those in the ovaries, PCD defects in the nervous system are most pronounced in cells not expressing the spin gene product, i.e., neurons. In the nervous system, the spin gene is expressed exclusively in glial cells that play a number of different roles in the development and maintenance of the nervous system. The distribution of the observed autofluorescent material in the nervous system does not correlate with the distribution of the Spin-expressing cells; in addition, the time-dependent accumulation of lipofuscin-like materials is observed in most of the neuronal and glial cells in spin^P1 mutant flies. Thus, the spin gene product is expressed in follicle cells in ovaries and glial cells, particularly surface glial cells, in the CNS, which enwrap nurse cells and neurons, respectively. Loss of spin function of follicle cells and glial cells leads to suppression of PCD in nurse cells and neurons. These observations lead to the idea that the Spin protein functions in glial cells in the regulation of PCD of neurons, and the failure of PCD at the proper developmental timing leads to widespread neurodegeneration accompanied by accumulation of lipofuscin-like materials at a later stage (Nakano, 2001).

The most striking effect of spin mutations on neuronal PCD is found in the VNC at 6 h APF, at which time the number of cells undergoing PCD reaches a maximum in the wild type. In spin mutant pupae, the number of cells undergoing apoptosis is 60% less than that in wild-type pupae. Since no later or earlier peaks of PCD are observed in the spin mutant, the cells to be eliminated must remain there throughout the pupal and adult stages. The presence of supernumerary neurons would perturb the formation of adult neural circuits that are established during the pupal stage (Nakano, 2001).

In fact, the structure of the nervous system of spin mutants is aberrant even when it is observed at the level of gross anatomy. The abdominal segment of the VNC is remarkably longer in the mutant than in the wild type. This part of the VNC is shortened at the mid-pupal stage. The extent of PCD of neurons influences the shortening of the VNC. For example, the VNC does not shorten in the flies hemizygous for the H99 deficiency, in which three PCD genes, hid, rpr, and grim, are deleted, and thus PCD in the VNC is prevented. Furthermore, the VNCs of H99 hemizygous pupae contain autofluorescent material. These results support the idea that the long-abdominal-ganglion phenotype of spin mutants results from the reduced extent of PCD in the pupal VNC (Nakano, 2001).

In moth pupa, damage to glial cells interferes with the shortening of the VNC. The metamorphic shortening of the moth VNC cultured in vitro is accelerated by the insect steroid hormone ecdysteroid. Considering all these observations, it is postulated that the Spin protein functions in glial cells to induce PCD of neurons during metamorphosis, which is under the control of the humoral hormone ecdysteroid (Nakano, 2001 and references therein).

Widespread neurodegeneration and accumulation of lipofuscin-like materials are important features of the nervous systems of spin mutant adults. Although such neurodegeneration might be a secondary result of PCD defects, it provides a unique model to study the basis for some human diseases with similar phenotypes. Both histological and biochemical analyses indicate that accumulated materials in spin flies are very similar to lipofuscin. Lipofuscin was discovered as an aging pigment more than a century ago. However, the mechanism of lipofuscinogenesis has not yet been fully elucidated. In humans, the abnormal accumulation of lipofuscin has been reported for several neurodegenerative disorders such as the neural ceroid lipofuscinoses and Tay-Sachs disease. Both are categorized as lysosomal storage diseases. Although two human spin orthologs have been cloned and mapped to regions on chromosomes 16 and 17, these two loci do not match the mapped loci of the known disease holders. However, there are still many unidentified diseases that will probably be classified as lysosomal storage diseases; therefore, spin orthologs will be good candidate genes for these diseases (Nakano, 2001).

Interestingly, spin mutant flies exhibit a shortened life span. One Drosophila mutant, eggroll, has a very similar phenotype to that produced by the spin mutation; such mutants exhibit a shortened life span and have multilamellar structures in the CNS. These phenotypes are similar to some human neurodegenerative diseases such as Tay-Sachs disease and Niemann-Pick sphingomyelin storage diseases. Therefore, the study of the spin and eggroll mutants should provide a good tool for understanding the relationship between aging and lipofuscinogenesis (Nakano, 2001).

Spinster controls Dpp signaling during glial migration in the Drosophila eye

The development of multicellular organisms requires the well balanced and coordinated migration of many cell types. This is of particular importance within the developing nervous system, where glial cells often move long distances to reach their targets. The majority of glial cells in the peripheral nervous system of the Drosophila embryo is derived from the CNS and migrates along motor axons toward their targets. In the developing Drosophila eye, CNS-derived glial cells move outward toward the nascent photoreceptor cells, but the molecular mechanisms coupling the migration of glial cells with the growth of the eye imaginal disc are mostly unknown. This study used an enhancer trap approach to identify the gene spinster, which encodes a multipass transmembrane protein involved in endosome-lysosome trafficking, as being expressed in many glial cells. spinster mutants are characterized by glial overmigration. Genetic experiments demonstrate that Spinster modulates the activity of several signaling cascades. Within the migrating perineurial glial cells, Spinster is required to downregulate Dpp (Decapentaplegic) signaling activity, which ceases migratory abilities. In addition, Spinster affects the growth of the carpet cell, which indirectly modulates glial migration (Yuva-Aydemir, 2011).

During development of the nervous system, glia cell migration is tightly regulated in time and space. This study has demonstrated that, within the Drosophila visual system, glial migration requires the late endosomal/lysosomal protein Spinster to restrict a migratory stimulus provided by Dpp signaling. Spinster is crucially involved in the coordination of eye disc growth and glial migration (see Schematic view on spinster function). In spinster mutant eye discs, late endosomes accumulate and a glial overmigration phenotype develops. This migration phenotype can be modified by reducing the early to late endosomal transfer or late endosome-to-lysosome transfer through decreasing the levels of hrs. Moreover, the Dpp receptor Thickveins accumulates in spinster mutant cells. Thus, Spinster appears to antagonize Dpp signaling by facilitating the routing of Dpp receptors toward the lysosome (Yuva-Aydemir, 2011).

Retinal glial cells are born in the CNS and migrate onto the eye disc through the optic stalk. A reverse migratory direction is taken by the photoreceptor cell axons, which are born in the periphery and navigate toward the brain through the optic stalk. Peripheral glial cells generally follow axonal growth cones during their migration phase (Klämbt, 2009), and thus within the developing Drosophila visual system, glial cells have to be guided to the nascent photoreceptor axons by other means. In Drosophila, this is accomplished by the carpet cells, very large subperineurial cells that cover the entire eye field (Silies, 2007). The carpet cell shields the navigating axons within the stalk from the proliferating and migrating glial population. At the beginning of eye imaginal disc development, the carpet cell prevents precocious migration of glial cells onto the eye disc in a process requiring early eye patterning genes and Hedgehog signaling. Ablation of the carpet glia as well as disruption of, for example, early Hedgehog signaling result in an overshooting glial migration (Silies, 2007). As soon as the morphogenetic furrow is initiated, the carpet glial cell starts to grow and extends anteriorly. However, the carpet cell never reaches the morphogenetic furrow but stops to grow at a few cell rows behind where nascent photoreceptor cell axons are formed. Piggybacking on the carpet cell are the perineurial glial cells that migrate toward the anterior edge of the carpet cell. As soon as they come in contact with photoreceptor axons, they drop from the carpet cell and now follow the sensory axon toward the brain. Thus, two distinct phases of glial cell migration can be defined. Initially, the carpet cell prevents precocious migration, whereas in later phases of eye imaginal disc development the carpet cells provide a permissive substrate for the migrating perineurial glia (Yuva-Aydemir, 2011).

Spinster is involved in the regulation of both of these migratory phases and helps to coordinate the growth of the eye disc with the migration of glial cells. The most dramatic consequences of spinster mutants as seen in eye disc duplications are presumably attributable to effects on Wingless and Dpp within the eye disc. The broad glial overshooting phenotypes correlate with a failure of the carpet cell to prevent early-onset, precocious glial migration. The later glial overmigration phenotypes correlate with late, carpet cell-independent, defects in the perineurial glia. Thus, it is not surprising that, in contrast to panglial expression of Spinster, glial cell type-specific expression is not able to rescue the spinster overmigration phenotype (Yuva-Aydemir, 2011).

The size of the carpet glial cell is reduced in spinster mutants, and MARCM analysis as well as cell type-specific RNAi experiments indicate that spinster controls carpet cell growth cell autonomously. Directed expression of Hid or RicinA specifically in carpet cells resulted in a reduced cell size and a concomitant induction of ectopic glial cell migration (Silies, 2007). In contrast, single spinster mutant carpet cells did not lead to a glial migration defects, suggesting that the reduced carpet cell only contributes to the glial overmigration phenotype. How spinster controls the size of the carpet cells is currently unknown. Possibly, within the carpet cell, spinster acts via hedgehog signaling. Hedgehog bound to the Patched receptor is normally internalized and degraded in lysosomes. Moreover, Patched is expressed in glial cells and Hh signaling was suggested to prevent precocious glial cell migration through a yet-unknown pathway. However, mutations in different hedgehog signaling components did not suppress the spinster phenotype and inhibition of hedgehog signaling in glial cells did neither alter the morphology of the carpet glia nor did they affect glial migration. Likewise, activation of Hedgehog signaling through expression of PKA^dsRNA did not influence glial migration (Yuva-Aydemir, 2011).

Within the carpet cells, Spinster most likely does also not act through altered Dpp signaling, since no phospho-Mad expression was detected in the carpet cells. In contrast, enhanced Dpp signaling (Mad phosphorylation) was noted in the migrating perineurial glial cells. The analysis of additional mutations affecting different aspects of intracellular vesicle dynamics revealed the importance of endocytic processes in TGF-β signaling. The block of Rab5 function causes a reduction in the range of Dpp target gene activation, and Rab5 overexpression causes increased Dpp signaling range. Hrs, a component of ESCRT complex, has been shown to interact with Smad2 and participates in the recruitment of Smad2 to the activated receptor. Hrs seems to be involved in the constitutive ligand-independent receptor turnover since Tkv accumulates at the cell membrane even in the absence of Dpp. Hrs downregulation suppresses the spinster glial overmigration, possibly by inhibiting Tkv internalization and accumulation in the endosomes. Within the cell, endosome-to-lysosome transfer and subsequent lysosomal degradation also affects Dpp signaling. Enhanced degradation caused by expression of activated Rab7 reduces the Dpp signaling range, whereas the inhibition of lysosome function with chloroquine leads to an endosomal accumulation of ligands. In addition, pan-glial expression of a dominant-negative Tkv receptor, tkv^?GS or tkv^dsRNA, resulted in reduced glial migration and fewer glial cells. Similar results were obtained when put or mad expression were silenced using RNA interference, demonstrating that the regulation of Dpp signaling via controlling vesicle dynamics is required in glial cells to ensure normal proliferation and migration (Yuva-Aydemir, 2011).

Endocytosis and vesicle recycling is generally required in migrating cells to dynamically remodel their adhesive contacts and locate active signaling receptors to the front of the cell in response to extracellular signals. Integrins from the cell rear can be relocalized to the leading edge, and during border cell migration in the Drosophila ovary, spatial restriction of the receptor tyrosine kinase signaling by endocytosis ensures the localized intracellular response to guidance cues. Likewise, glial migration in the embryonic Drosophila PNS is regulated by the fine-tuning of Notch signaling by Numb-mediated endocytosis. Thus, endocytotic trafficking may affect cell migration through several pathways (Yuva-Aydemir, 2011).

In spin mutants, enhanced Dpp signaling is also observed in the eye imaginal disc and the most extreme eye disc phenotype appears as to be an induction of an ectopic morphogenetic furrow perpendicular to the normal furrow. Ectopic expression of the Dpp in the eye disc or expression of activated Tkv in the glial cells increases glial cell proliferation. In spin mutants, both increased Dpp expression and accumulation of Tkv are observed. Since spin controls glial cell proliferation cell autonomously, this is likely attributable to the accumulation of Tkv in glial cells. In addition, enhanced Mad phosphorylation is observed in spin mutants, especially in the optic stalk glia. In agreement with this, the increase in glial cell number is mostly noted in the optic stalk. Interestingly, the function of spinster in controlling glial differentiation is also required in the wrapping glia. Loss of spinster results in a reduced wrapping of axonal membranes, which can be rescued by reexpression of spinster in the wrapping glia. Thus, spinster function appears to be needed to extend cellular processes during migration and differentiation (Yuva-Aydemir, 2011).

In conclusion, Spinster affects glial migration on two different levels. Spinster regulates carpet glia differentiation, which indirectly affects early migration, and subsequently, Spinster acts in the perineurial glia to confer a general cell motility signal (Yuva-Aydemir, 2011).

GENE STRUCTURE

Genomic DNA flanking the P element in the spin^P1 mutant was cloned by plasmid rescue and subsequently used to screen genomic and cDNA libraries. Sequence analysis of the identified clones reveals that there are five different transcript forms of approximately 3 kb in length; these are produced by the alternative processing of a primary transcript. The P-element was found to be inserted 8 bp downstream of the transcription initiation site of this transcription unit in the spin^P1 mutant. Two out of five alternative splicing events resulted in the exclusion of exon 9, which contains an in-frame termination codon, thereby producing proteins containing different C-terminal amino acids (type III and type IV). A different type of splicing variation leads to the mutually exclusive utilization of exon 4 in the case of type I and type III and of exon 5 in type II and type IV transcripts. Exons 4 and 5 are found to be 262 bp long and encode amino acid sequences that are 53% identical to each other. The predicted lengths of the polypeptides encoded by the cDNAs described are 630 amino acids for type I and type II; 605 amino acids for type III and type IV, and 422 amino acids for type V proteins (Nakano, 2001).

PROTEIN STRUCTURE

Amino Acids - 422, 605 and 630

Structural Domains

A hydropathy plot analysis indicates that Spin proteins have multiple membrane-spanning domains with no cleavable signal sequence. Spin exhibits no significant homology to any of the membrane proteins of known function, such as transporters, ion channels, and receptors. A search of the sequence database has identified three C. elegans genes of unknown function (C39E9.10, C13C4, and CEF09A5) as orthologs of the Drosophila spin gene (Nakano, 2001).

The full-length coding sequence of the mouse and human spin genes were determined by sequencing cDNA clones obtained from the Expressed Sequence Tags data bank. Both mouse and human cDNAs (designated Mspin1 and Hspin1, respectively) encode proteins of 528 amino acids. The identities between Spin (type I) and Mspin1, Spin and Hspin1, and Mspin1 and Hspin1 were 41%, 42%, and 94%, respectively. Hydropathy analyses for the human, mouse, fly, and nematode proteins show that these proteins are remarkably similar to each other, thus suggesting that they share a common topological structure. The predicted cyclic-AMP- and cyclic-GMP-dependent protein kinase phosphorylation site is conserved from C. elegans to humans; therefore, this indicates that phosphorylation and/or dephosphorylation events may play an important role in Spin function (Nakano, 2001).

date revised: 2 June 2001

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