Gene name - spitz
Cytological map position - 37E2-38A1
Function - ligand for Torpedo/EGR
Keywords - spitz group
Symbol - spi
Genetic map position - 2-
Classification - EGF-repeat - TGF-alpha homolog
Cellular location - surface transmembrane and secreted
|Recent literature||Patel, P. H., Dutta, D. and Edgar, B. A. (2015). Niche appropriation by Drosophila intestinal stem cell tumours. Nat Cell Biol 17: 1182-1192. PubMed ID: 26237646
Mutations that inhibit differentiation in stem cell lineages are a common early step in cancer development, but precisely how a loss of differentiation initiates tumorigenesis is unclear. This study investigated Drosophila intestinal stem cell (ISC) tumours generated by suppressing Notch (N) signalling, which blocks differentiation. Notch-defective ISCs require stress-induced divisions for tumour initiation and an autocrine EGFR ligand, Spitz, during early tumour growth. On achieving a critical mass these tumours displace surrounding enterocytes, competing with them for basement membrane space and causing their detachment, extrusion and apoptosis. This loss of epithelial integrity induces JNK and Yki/YAP activity in enterocytes and, consequently, their expression of stress-dependent cytokines (Upd2, Upd3). These paracrine signals, normally used within the stem cell niche to trigger regeneration, propel tumour growth without the need for secondary mutations in growth signalling pathways. The appropriation of niche signalling by differentiation-defective stem cells may be a common mechanism of early tumorigenesis.
The spitz group was defined on the basis of phenotypic similarities between mutants and genetic interactions. Spitz group mutants show defects in the cuticular structures derived from the ventro-lateral blastoderm, and defects in the ventral cord known as the CNS. Four of the six genes in the spitz group are pointed, rhomboid, Star and spitz itself (Mayer, 1988). The common element unifying members of the spitz group is association with signaling involving the EGF-R, either as ligands, cofactors or targets.
Spitz is a transmembrane protein with a potential cleavage site. This means Spitz could act either as a secreted ligand or as a cell bound ligand. The protein has a single epidermal growth factor repeat and is homologous to TGF-alpha, reflecting the same evolutionary affinity as Gurken. Like Gurken, Spitz is a ligand of the Epidermal growth factor receptor (EGF-R), also known as Torpedo or DER. The function of Drosophila epidermal growth factor is modulated by several other candidate ligands, each of which (Spi, Vein, and Argos) are structurally unrelated except within the EGF domain.
spitz expression is especially complex, reflecting its involvement in a variety of differentiation processes. Since EGF-R is expressed almost ubiquitously, localized expression of Spitz gives rise to patterning of EGF-R signaling. In embryos mutated for Egf-r/torpedo or spitz, the ventral midline ectoderm is reduced, producing defects in Fasciclin III and orthodenticle expression. When spitz is ubiquitously expressed, the embryo becomes ventralized due to expansion of the ventral midline.
Spitz interacts with rhomboid and Star, both of which are thought to modulate EGF-R/Torpedo signaling. Once Spitz is secreted, Rhomboid and Star become redundant for the induction of ventral ectodermal fates. This suggests that these proteins facilitate processing of the Spitz precursor (Schweitzer, 1995).
Spitz is involved in the differentiation of the eye. spitz is expressed ahead of the morphogenic furrow, the advancing line of cells becoming differentiated into photoreceptors (see Progression of the morphogenetic furrow across the eye disc). It reaches its highest levels of concentration in the furrow, while Egf-r/torpedo is expressed behind the furrow. Thus the only place where Spitz and EGF-R levels are high is near the furrow itself, implying that both ligand and receptor act in concert near the furrow. spitz is required in the specification of the first photoreceptor cell of each facet; without spitz, later cells do not arise. Spitz function is required for the recruitment of R2/R5 photoreceptors. R2 and R5, in turn, secrete Spitz, which increases the local Spitz concentration, and may help with the recruitment of R3/R4 (Tio, 1994 and 1997).
Intercellular signaling through the EGF receptor (EGFR) patterns the Drosophila egg. The TGF alpha-like ligand Gurken signals from the oocyte to the receptor in the overlying somatic follicle cells. In the dorsal follicle cells, this initial paracrine signaling event triggers an autocrine amplification by two other EGFR ligands: Spitz and Vein. Spitz becomes an effective ligand only in the presence of the multitransmembrane domain protein Rhomboid. Consequent high-level EGFR activation leads to localized expression of the diffusible inhibitor Argos, which alters the profile of signaling. This sequential activation, amplification, and local inhibition of the EGFR forms an autoregulatory cascade that leads to the splitting in two of an initial single peak of signaling, thereby patterning the egg (Wasserman, 1998).
Although Gurken is the only ligand previously reported to activate the Egfr during oogenesis, the requirement for Rhomboid in the follicle cells of the egg chamber led to an examination of the necessity for Spitz. Complete loss of Spitz function causes embryonic lethality, but spitz hypomorphs are viable. Adult females with reduced spitz lay eggs with a significant loss of the most dorsal tissue, implying that Spitz is indeed required for normal development of the egg. This phenotype was quantified by measuring the gap between the dorsal appendages: Their mean separation was found to be reduced in spitz hypomorphs; furthermore, in 20% of the mutant eggs the dorsal appendages were fused at the point of attachment, a phenotype never seen in wild-type eggs. Spitz could be required in the oocyte or in the somatic follicle cells that surround it. To examine this, germline clones of spitz null mutations were generated; in these, the oocyte is mutant but the follicle cells are wild type. This causes no defects, either in patterning the egg or in the viability or patterning of the embryos derived from such eggs. There is no requirement for the Egfr in the oocyte, only in the follicle cells. There is therefore an essential function for the Egfr ligand Spitz in dorsal-anterior patterning of the egg, and it is required only in the somatic follicle cells, where the receptor is also needed (Wasserman, 1998).
In other tissues Rhomboid appears to activate Spitz/Egfr signaling, leading to the suspicion that Rhomboid might mediate autocrine Spitz signaling in the follicle cells. Consistent with this idea, the phenotype caused by loss of Spitz from the follicle cells is similar to that caused by loss of Rhomboid. Expression of antisense rhomboid causes loss of dorsal tissue and fusion of the appendages in eggs from heat-shocked females expressing HS-as-rho. Unmarked follicle cell clones of a rhomboid null mutation also give fused appendage phenotypes; as with spitz clones, these range from mild to severe fusions. Like Spitz and the Egfr, Rhomboid is not needed in the oocyte, implying that it, too, is only required in the follicle cells (Wasserman, 1998).
spitz is expressed uniformly in all follicle cells (region 2 of the germarium) from very early in oogenesis through stage 13, when egg patterning is complete. No transcript can be detected in the oocyte, consistent with a lack of Spitz requirement. This expression domain coincides exactly with that of the Egfr itself. spitz expression in follicle cells is unaffected by Egfr signaling; in egg chambers from gurken or Egfr mutant mothers, spitz expression is unaltered. The same is true of egg chambers from fs(1)K10 mothers, which have ectopic Egfr signaling. These expression data show that Gurken/Egfr signaling does not affect Spitz transcription, implying that the dependence of Spitz signaling on prior Gurken signaling must be posttranscriptional. In contrast, rhomboid is expressed in a dynamic pattern in follicle cells and is dependent on EGFR signaling. At stages 9-10a of oogenesis, rhomboid is expressed in a central patch of the dorsal-anterior follicle cells: this resolves to a stripe of cells on either side of the dorsal midline by stage 10b. In the absence of Egfr signaling, rhomboid expression is lost and, conversely, it is ectopically expressed in fs(1)K10 egg chambers. These expression profiles of spitz and rhomboid are consistent with Gurken signaling from the oocyte activating the expression of rhomboid in the follicle cells. This may in turn allow Spitz to become an autocrine ligand in the follicle cells and thus establish an autocrine amplification of the initial paracrine signal. The expression of the neuregulin-like Egfr ligand vein was also examined. It is also expressed in two stripes of follicle cells at stage 10b. Interestingly, vein expression is dependent on Egfr signaling: it is ectopically expressed in fs(1)K10 eggs and absent from gurken null eggs, establishing another potentially important feedback mechanism. This suggests that the autocrine amplification of Egfr signaling also involves Vein, although in this case the feedback occurs by direct transcriptional activation of the ligand (Wasserman, 1998). vein expression has also been found to be dependent on Egfr signaling during embryogenesis (T. Volk, personal communication to Wasserman, 1998).
The expression of the secreted Egfr inhibitor, Argos, is dependent on Egfr signaling in many tissues. Consistent with this, argos is expressed in the dorsal-anterior follicle cells at the time when Egfr signaling occurs. At stage 11 the RNA is detectable in a single, T-shaped group of cells centered on the dorsal midline, and by stage 13, argos, like rhomboid and vein, is found in two groups of cells: one on either side of the midline. As elsewhere, argos expression is dependent on Egfr activation: in gurken mutant egg chambers it is lost, and it is ectopically expressed in fs(1)K10 egg chambers. Is argos expression dependent on Spitz amplification of Egfr signaling? An examination was performed to see if Spitz contributes to a signaling threshold required to induce argos expression. argos expression is normal in eggs from mothers with reduced Ras1, but when Spitz is halved, dorsal-anterior argos expression is abolished in most egg chambers. Therefore, there is indeed a threshold of Egfr signaling required to switch on argos, and both Gurken and Spitz participate in reaching this threshold (Wasserman, 1998).
The initial expression of argos at the dorsal midline led to the speculation that it might cause a reduction of Egfr signaling near the midline, thereby splitting the single signaling peak in two. The resulting twin peaks of Egfr activation would then specify the location of the dorsal appendages. A prediction of this model is that loss of Argos should remove inhibition of the Egfr at the midline and produce a single peak of signaling, leading to the formation of a fused appendage phenotype. The eggs from females with hypomorphic argos mutations were examined. A significant proportion of these eggs have a partially or, in the most severe cases, fully fused phenotype. The same fused appendage phenotype is observed in follicle cell clones of an argos null mutation. These data imply that there is a requirement for Argos in eggshell patterning and that, as with Spitz, Rhomboid, and the Egfr, this requirement is confined to the follicle cells (Wasserman, 1998).
It is proposed that Argos modifies the initial Egfr activation profile in the follicle cells, producing twin peaks of activity displaced from the midline. These specify the position of the dorsal appendages. Direct evidence for a transition from one to two peaks of signaling was obtained with an antibody that recognises only the activated, diphosphorylated form of MAP kinase, a key member of the signal transduction pathway downstream of the receptor. At stages 9-10, there is a single domain of activated MAP kinase in the follicle cells, centered on the dorsal midline. By stage 11, two domains, are observed: one on each side of the dorsal midline. From their position, these cells correspond to the cells that will form the dorsal appendages. In Egfr hypomorphs, which have a fused appendage phenotype, the single peak of activated MAP kinase does not split in two. These results clearly demonstrate that Egfr signaling does indeed evolve from a single peak into twin peaks of activation. This is supported by examining the expression pattern of known Egfr target genes in the follicle cells. By stage 11 these targets (pointed, rhomboid, argos, vein, and Broad) are expressed in two dorsal anterior domains, one on each side of the midline. This is taken as additional evidence for twin peaks of Egfr activation. Earlier, pointed, rhomboid, and argos are all also detectable in a single peak at the dorsal midline (Wasserman, 1998).
Egfr signaling specifies the dorsoventral axis and patterns the eggshell. It is suggested that these two functions are controlled by temporally separate phases of Egfr activation. When amplification and splitting of Egfr signaling do not occur, eggs have only a single, fused appendage. Surprisingly, larvae emerge from these eggs at the frequency predicted by Mendelian principles, and those that emerge have no apparent dorsoventral defects. When follicle cell clones of a spitz null are induced, the hatching rate of eggs with fused appendages os 82% of the predicted number. Similarly, all of the predicted number of eggs with a single fused appendage hatch from mutant females. The same is true of eggs with fused appendages caused by follicle cell clones of argos null mutations. Therefore, disruption of the amplifying and splitting process does not perturb dorsoventral axis specification, implying that the initial Gurken signal to the Egfr is sufficient to specify the axis. The subsequent cascade of amplification and splitting then patterns the eggshell (Wasserman, 1998).
What are the biological roles of the EGF domains of the related EGF ligands? The EGF domain contains a series of six cysteines, which form three disulfide bonds to generate a looped structure, and a number of other highly conserved residues that are known to be required for binding and activating members of the vertebrate ErbB receptor family. The EGF domains of Vn and Spi are not highly related (38% conserved) but have more sequence conservation with each other than with Aos. Additionally, the length of the predicted B loop that forms from the region between cysteines 3 and 4 is significantly longer in Aos than in the activating ligands (Schnepp, 1998).
Chimeric molecules were created by exchanging the EGF domain of Vn for those of either Spi or Aos. The activity of these chimeras was compared with the native factors in vitro and in vivo. Secreted Spi (sSpi, the active form of Spi) and Aos increase or decrease, respectively, the level of Egfr tyrosine phosphorylation in Drosophila S2-DER tissue-culture cells. The Vn:Vn EGF chimera, which serves as a control for the effect of the additional residues introduced during construction of the chimeras, behaves like native Vn. In contrast, possession of the Spi-EGF domain converts Vn into a stronger Egfr activator. The Vn:Aos EGF chimera behaves as an inhibitor, rather than an activator and caused a reduction in Egfr activation resulting from the ligand-independent activation of Egfr. These results show that the properties of Vn are changed when its EGF domain is swapped with that of Spi or Aos so that the chimeras behave like the factors from which the EGF domain is derived (Schnepp, 1998).
In the embryo, ectopic activation of the DER pathway by sSpi, using the Gal4-UAS system, causes an expansion of ventral cell fates that can be monitored by expression of the ventral cell marker orthodenticle (otd). Ectopic expression of native Vn causes no change in the expression of otd. The Vn:Vn EGF chimera causes a very mild expansion of otd expression. This slight effect could be the result of higher expression of the transgene. In contrast, ectopic expression of the Vn:Spi EGF chimera causes a dramatic expansion of otd expression that is similar to that seen with ectopic expression of sSpi. In the wing, ectopic activation of the DER pathway is characterized by the appearance of extra veins. Ectopic expression of native Vn in pupal interveins produces a mild or moderate extra-vein phenotype, whereas ectopic expression of sSpi causes a strong extra-vein phenotype. A direct role for Vn in normal vein development has been demonstrated; such a role has not been demonstrated for sSpi but is likely to take place. Ectopic expression of the Vn:Vn EGF chimera gives extra-vein phenotypes similar to those seen after ectopic expression of native Vn. In contrast, ectopic expression of the Vn:Spi EGF chimera produces a strong extra-vein phenotype like that seen following ectopic expression of sSpi. In the eye, ectopic activation of the Egfr pathway is characterized by loss of ommatidia, over-recruitment of cell types, and blistering. Ectopic expression of native Vn posterior to the morphogenetic furrow in the eye disc has no effect on the adult eye phenotype; in contrast, ectopic expression of sSpi and the Vn:Spi EGF chimera produces small disorganized eyes with blisters. Surprisingly, ectopic expression of the Vn:Vn EGF chimera also showed a strong eye phenotype. This result suggests that regions outside the EGF domain can affect the activity of a factor because the manipulation used to create the chimeras changed 4 residues flanking the EGF domain. These in vivo data corroborate the biochemical data that Vn is a less potent activator of Egfr than sSpi. The EGF domain is a key feature that differentiates Vn and sSpi because Vn can be converted into a more potent Egfr activator if its EGF domain is swapped with that of Spi. The ability to differentially regulate signaling, depending on whether Vn or sSpi is utilized, may be one mechanism by which DER elicits specific cell responses during development (Schnepp, 1998).
To test whether Vn can be converted into an inhibitor by swapping its EGF domain with that of Aos, the effects of ectopic expression of Vn, Aos, and the Vn:Aos EGF chimera were compared in larval wing and eye discs. Native Vn produces an extra-vein phenotype when expressed ectopically in larval wing discs, as expected for an activator of Egfr signaling. In the wing, ectopic suppression of the Egfr pathway is characterized by vein loss; ectopic expression of native Aos or the Vn:Aos EGF chimera results in vein loss. The vein loss phenotype associated with ectopic expression of Vn:Aos EGF is not as severe as that caused by native Aos. In the eye, reduction in activity of the Egfr pathway is characterized by loss of cell types and fusion of ommatidia. There is no observable effect on adult eye phenotype following ectopic expression of native Vn in eye discs, but ectopic expression of the Vn:Aos EGF chimera produces a rough eye phenotype with fused lenses similar to, but not as severe as, that produced by ectopic expression of native Aos. These results show that the EGF domain is a key determinant responsible for the difference between Vn and Aos and that the EGF swap is sufficient to convert an Egfr activator into an inhibitor. The Vn:Aos EGF chimera is apparently not as potent an inhibitor as native Aos in the eye or the wing, suggesting that other regions of the proteins (Vn and/or Aos) may play modulating roles (Schnepp, 1998).
Neuroepithelial cell proliferation must be carefully balanced with the transition to neuroblast (neural stem cell) to control neurogenesis. This study shows that loss of the Drosophila microRNA mir-8 (the homolog of vertebrate miR-200 family) results in both excess proliferation and ectopic neuroblast transition. Unexpectedly, mir-8 is expressed in a subpopulation of optic-lobe-associated cortex glia that extend processes that ensheath the neuroepithelium, suggesting that glia cells communicate with the neuroepithelium. Evidence is provided that miR-8-positive glia express Spitz, a transforming growth factor α (TGF-α)-like ligand that triggers epidermal growth factor receptor (EGFR) activation to promote neuroepithelial proliferation and neuroblast formation. Further, these experiments suggest that miR-8 ensures both a correct glial architecture and the spatiotemporal control of Spitz protein synthesis via direct binding to Spitz 3' UTR. Together, these results establish glial-derived cues as key regulatory elements in the control of neuroepithelial cell proliferation and the neuroblast transition (Morante, 2013).
This analysis reveals that the production by an optic-lobe-associated cortex glia of the EGFR ligand Spitz is critical for coordination of neuroepithelial proliferation and the spatiotemporal emergence of neuroblasts. External signaling from the surrounding microenvironment is a common mechanism for the regulation of stem cell number and behavior in mature tissues, but the need of a niche microenvironment during early neurogenesis was unknown (Morante, 2013).
Flies deficient for the microRNA mir-8 exhibit brain degeneration and behavioral defects. Strikingly, the microRNA is expressed in cortex glial cells lying underneath the blood-brain-barrier (subperineurial) glial layer. These glia are of large size and produce long protrusions that ensheath the developing optic lobe neuroepithelium and can be distinguished by their selective expression of the EGFR ligand spitz as well as its modulators aos and rho (Morante, 2013).
Genetic manipulation of mir-8, spi, or aos, in this glial cell population unveiled cell nonautonomous influences of these glia on the development of the underlying neuroepithelium. Similar glial signaling to neuroblasts in the larval Drosophila brain has been demonstrated for a class of neuroblasts that remain quiescent until nutrient-responsive satellite and cortical glia reactivate their proliferation. This study identifies another population of glia cells that sustain growth of neuroepithelial cells and the neuroepithelial-neuroblast transition via a mir-8-Spitz axis (Morante, 2013).
Glial-mediated regulation of the neuroepithelium is reminiscent to the roles of mammalian astrocytes that are known to potently stimulate neurogenesis in cell culture and a component of endogenous neural stem cell niche in adult mammalian neurogenesis. Additionally, EGFR is also implicated in glial cell proliferation in Drosophila and human glioma often exhibits elevated EGFR signaling (Morante, 2013).
The notion that mir-8-Spitz-positive cortex glia constitute an anatomically and functionally distinct population of surface-associated glia cells is strongly supported by the finding that RNAi knockdown of spi in subperineurial glia using moody- Gal4 has no effect on neuroepithelial development. Spitz protein is converted to its active form by the Rhomboid protease, which is also expressed by miR-8 glia. The extracellular factor Aos limits Spitz spreading and signaling level that may influence the effects of Spitz-EGFR in the responding neuroepithelium (e.g., sustaining proliferation and preventing premature or ectopic neuroblast formation). The posttranscriptional silencing of spi mRNA by the miR-8 binding to a sequence in its 3' UTR provides another layer of regulation to fine-tune the timing, localization and/or amount of Spitz protein translation. Moreover, the distinctive architecture of miR-8-Spitz-positive cortex glia appears to be regulated by endoreplication regulators dup/ Cdt1 and the microRNA miR-8. Importantly, expression of a spitz transgene lacking its 3' UTR (and hence unable of regulating by miR-8) fully rescued the undergrowth defect caused by mir-8 overexpression in the glia cells. Therefore, it is suggested that cortex glia employ a coordinated strategy that is mediated by miR-8 to ensure that: (1) the glia establish a correct architecture to provide a continuous layer of cortex glia cells that extend long processes to the neuroepithelium; and (2) correct local (or temporal) control of Spitz protein synthesis. Given the expression of rho and aos is directly induced by EGFR signaling in other context, a feed-back signaling via EGFR may also occur in miR-8-Spitz positive glial cells, thereby contributing to the fine-tuning of Spitz protein activation and secretion (Morante, 2013).
A niche typically refers to a confined anatomical location where adult stem cells reside and provides the signals required to sustain stem cell function and number. Niches are usually composed of supporting cells that make physical contact with the stem cells and act locally. The optic-lobe-associated miR-8-Spitz-positive cortex glia appear to represent a niche that contributes signals for the growth and morphogenesis of the neuroepithelium and that constitutes a functionally distinct population of that of the bloodbrain- barrier glial cells (Morante, 2013).
Extrinsic signaling in the coordination of neuroepithelial proliferation in the developing mammalian forebrain of Foxc1 mutant mice has also been suggested. In these mice, the meninges are reduced or absent, resulting in an expansion of the neuroepithelium due to the predominance of symmetric divisions. The meninges are a source of the retinoic acid required for the transition of neuroepithelial cells into radial glia and neurons (Siegenthaler, 2009). Furthermore, meningeal cells secrete and organize the pial basement membrane, a thin sheet of extracellular matrix that covers the brain and that is enriched in a variety of growth factors. Rupture of the basement membrane in the developing brain causes type II lissencephaly, generating ectopic precursor clusters and cortical heterotopias due to impaired attachment of the radial glia to the basement membrane, resulting in a general laminar disorganization. These defects are reminiscent to those of disrupted surface glia cells described in this study (Morante, 2013).
In summary these findings suggest that neuroepithelial proliferation and the onset of neuroblasts in the developing optic lobe neuroepithelium are largely influenced by extrinsic cues via a miR-8-dependent mechanism in the overlying glia. The reprogramming of neuroepithelial cells into neural stem cells (neuroblasts) is associated with dramatic morphological and molecular changes, including the loss of epithelial determinants DE-Cadherin, Crumbs and PatJ, and enhanced expression of the Snail-family zinc-finger transcriptional repressor Worniu. These changes are strikingly reminiscent of the events that drive the epithelial-to-mesenchymal transition (EMT), which confers a stem-like character in mammalian epithelial cells and in cancer cells and which are regulated by the miR-200 family. Indeed, downregulation of human mir-200 genes in epithelial normal and cancer cells promotes EMT and the acquisition of 'stemness' effects that are presumed to be cell-autonomous. These findings demonstrate that miR-8 can exert its effects non-cell- autonomously, opening the possibility that microRNA of the miR-200 family may play similar roles in stem cell fate niches and/or microenvironmental regulation of metastasis (Morante, 2013).
There are numerous 5' alternative exons, all of them non-coding. Each cDNA uses one or two of these resulting in 4 characterized cDNAs. The coding region is contained within a single exon (Rutledge, 1992).
Bases in 5' UTR - variable, depending on alternative promoter usage
Exons - two or three
Bases in 3' UTR - 396
spitz encodes a putative 26-kD, EGF-like transmembrane protein with structural similarity to TGF-alpha. There is a single EGF domain in the extracellular region and a dibasic signal located between the EGF and the TM domain that could serve as a cleavage site. (Rutledge, 1992).
The EGF-like domain in Vein is 43 amino acids long and has the six invariant cysteines and highly conserved glycine and arginine residues characteristic of the motif. The cysteines are thought to form disulfide bonds, thereby producing a looped structure. The EGF motif in VN is between 30% and 44% identical to other EGF-like ligands. It shares 37% identity with Spitz and 33% identity with Gurken. Amino-terminal to the EGF domain of VN is an Ig-like domain of the C2 type that includes nonimmunological proteins (Schnepp, 1996).
date revised: 15 May 98
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