worniu: Biological Overview | References
Gene name - worniu
Cytological map position - 35D2-35D2
Function - zinc finger transcription factor
Symbol - wor
FlyBase ID: FBgn0001983
Genetic map position - chr2L:15423293-15425585
Classification - H2C2_2: Zinc-finger double domain
Cellular location - nuclear
Snail family transcription factors are best known for regulating epithelial-mesenchymal transition (EMT). The Drosophila Snail family member Worniu is specifically transcribed in neural progenitors (neuroblasts) throughout their lifespan, and worniu mutants show defects in neuroblast delamination (a form of EMT). However, the role of Worniu in neuroblasts beyond their formation is unknown. RNA-seq was performed on worniu mutant larval neuroblasts, and reduced cell-cycle transcripts and increased neural differentiation transcripts were observed. Consistent with these genomic data, worniu mutant neuroblasts showed a striking delay in prophase/metaphase transition by live imaging and increased levels of the conserved neuronal differentiation splicing factor Elav. Reducing Elav levels significantly suppressed the worniu mutant phenotype. It is concluded that Worniu is continuously required in neuroblasts to maintain self-renewal by promoting cell-cycle progression and inhibiting premature differentiation (Lai, 2012).
Stem cells must remain proliferative without becoming tumorigenic, and must remain competent to differentiate without actually differentiating. How stem cells maintain stemness - cell survival, cell-cycle progression, and the capacity to differentiate - is a widely relevant question with clinical significance. Drosophila neural progenitors (neuroblasts) have become a good model system to study how neural stem cells self-renew and maintain stem cell identity. Larval neuroblasts undergo repeated rounds of asymmetric cell division, each time generating a smaller differentiating daughter cell and a larger self-renewing neuroblast. During neuroblast division, many proteins are asymmetrically partitioned into the neuroblast or daughter cell, where they often contribute to neuroblast self-renewal or daughter cell differentiation, but much less is known about the transcriptional program that maintains neuroblast self-renewal (Lai, 2012).
Worniu (Wor) is a zinc finger transcription factor in the 'Slug/ Snail' family, and is transcribed in neuroblasts from the time of their birth. Over 50 Snail family members have been characterized in metazoans; they can directly bind DNA, RNA, or protein and regulate a wide range of cellular functions. Snail family members are best known for inducing epithelial-mesenchymal transition (EMT) during mesoderm development and neural-crest cell formation. In Drosophila, four Snail family genes are known: wor, escargot, snail, and scratch. The genes wor, escargot, and snail are expressed in neuroectoderm during embryogenesis to trigger EMT in neuroepithelial cells and transform them into newly-delaminated neuroblasts (Ashraf, 1999). Wor, Escargot, and Snail also act redundantly to promote expression of the apical polarity gene inscuteable (insc) and the cell-cycle regulator string in newly formed embryonic neuroblasts (Ashraf, 2001; Cai, 2001; Lai, 2012 and references therein).
The only Snail family member known to be expressed continuously in neuroblasts is Wor, but its function beyond neuroblast formation has not been investigated. This study shows that Wor maintains neuroblast self-renewal via dual functions: it promotes cell-cycle progression (specifically the prophase-to-metaphase transition) and it inhibits premature differentiation (by suppressing Elav protein levels). These functions occur in neuroblasts well after their formation, highlighting the potential role of Snail family members in stem cell self-renewal (Lai, 2012).
To analyze the wor mutant phenotype, a deficiency was used that removes wor and several flanking genes, Df(2L)Exel8034, and a specific mutation within the wor gene, wor1 (Ashraf, 2004). wor1 was found to have two missense mutations, one of which alters the amino acid Pro443 to Ser in the conserved zinc finger domain and probably changes the conformation for DNA/RNA/protein binding. Because wor1/wor1 had a slightly weaker phenotype compared to wor1/Df(2L)Exel8034 due to lesser amount of Wor protein in the latter genotype, it is concluded that wor1 is a strong hypomorph. wor1/Df(2L)Exel8034 was used for all experiments described in this study (called 'wor mutants') (Lai, 2012).
Wor protein is nuclear and is predicted to be a transcription factor, so the transcriptional profile of wild-type (WT) and wor mutant neuroblasts was compared to identify biological processes that were regulated by Wor. The TU- (thiouracil-) tagging method was used to identify mRNAs that are actively transcribed in WT or wor mutant neuroblasts. TU-tagging is a spatial/ temporal intersectional method to purify nascent RNA from designated tissues during a specific developmental stage. Uracil phosphoribosyltransferase (UPRT) was expressed in larval neuroblasts using wor-gal4 (Cabernard, 2009; Lee, 2006; Miller, 2009), which produced a high level of UPRT in WT and wor mutant larval neuroblasts with some persistence into their newborn progeny. Early third instar larvae were fed 4TU for 5 hr beginning at 72 hr after larval hatching (ALH) and then thio-labeled RNA was purified and RNA-sequencing was performed; a custom computational pipeline was designed to analyze the results. Two replicates were performed from wor mutants and two from WT. An average of 5.49 million reads from WT and 5.35 million reads from wor mutants was mapped. A comparison of the averaged WT versus averaged wor data showed that wor mutants had 13.8% of genes upregulated at least 2-fold and 9.1% of genes downregulated at least 2-fold (Lai, 2012).
Genes upregulated in wor mutants were enriched for gene ontology (GO) terms linked to neuronal differentiation such as G protein coupled receptor signaling, sensory perception, serotonin receptor signaling, and synaptic transmission. In addition, a group of 'neuronal differentiation genes' was recently defined in a transcriptomic analysis of larval brains enriched for neuroblasts or neurons (Carney, 2012). 253 of the 1,100 'neuronal differentiation' genes were differentially regulated in wor mutants (>2-fold or <2-fold), with a strong bias toward being upregulated. GO analysis of the upregulated genes shows significant overrepresentation of the terms signaling, synaptic transmission, synapse organization, and neuropeptide signaling pathway categories. It is concluded that wor mutant neuroblasts aberrantly upregulate neuronal differentiation genes (Lai, 2012).
The downregulated genes were likewise analyzed. It was asked whether previously defined 'neuroblast genes' or 'cell cycle genes' are downregulated in wor mutant neuroblasts - the converse of the observed upregulation of neuronal differentiation genes. It was found that 104 of the 970 'neuroblast' genes from Carney (2012) were differentially expressed in wor mutants >2-fold or <2-fold, with a strong bias toward being downregulated. The downregulated genes had a highly significant over-representation of the GO terms cell cycle, microtubule cytoskeleton organization, cytokinesis, cell division, and chromosome segregation. Similarly, a downregulation of Drosophila genes annotated as 'cell cycle' was found: of the 586 cell cycle annotated genes (GO:0007049), 67 were differentially regulated in wor mutants versus WT, and most (74.6%) were downregulated. It is concluded that wor mutant neuroblasts fail to properly express 'neuroblast' genes including those regulating the cell cycle (Lai, 2012).
Based on transcriptomic analysis, it was predicted that wor mutant neuroblasts would show defects in neuroblast attributes (cell-cycle progression, cell polarity, and survival) and precocious neural differentiation. All of these phenotypes could lead to the smaller brain size and reduced neuroblast numbers observed in wor mutants (Ashraf, 2004). To determine whether wor mutant neuroblasts have a normal cell cycle, EdU incorporation was performed and the number of EdU+ neuroblasts was counted immediately after the pulse. In this and subsequent experiments, larval neuroblasts were identified as large (>8 mm) Dpn+ cells within the central brain; optic lobe neuroblasts were not characterized. Most WT neuroblasts were EdU+, consistent with their reported cell cycle time of 2 hr (Cabernard, 2009). In contrast, very few wor mutant neuroblasts were EdU+, indicating a cell-cycle delay between G2-M-G1. To determine if the wor mutants were delayed in mitosis, the mitotic index of WT and wor mutant brains was measured by staining for the M-phase marker phosphohistone H3 (PH3). By late third instar (96-120 hr ALH) there was a striking increase in the PH3+ neuroblasts in wor mutant compared to WT. It is concluded that third instar wor mutant neuroblasts have a delay in completing mitosis (Lai, 2012).
To determine more precisely the nature of the M-phase delay in wor mutants, live imaging of neuroblast mitosis was performed within the intact brain (see Cabernard, 2009). Third instar larval neuroblasts expressing both His2A:RFP to monitor chromosomes and Zeus:GFP to image spindle microtubules (Cabernard, 2009) were imaged. Wild-type neuroblasts showed the expected mitosis length of 20 min. In contrast, wor mutant neuroblasts showed a dramatically extended prophase and/or prometaphase. Failure in centrosomal separation and bent mitotic spindles were observed. In two cases neuroblasts were observed that 'escaped' prophase arrest, and these had a relatively normal length of anaphase. It is concluded that wor mutant neuroblasts show an arrest or delay in the prophase/metaphase transition, a stage of the cell cycle where microtubules are dramatically reorganized (see Discussion) (Lai, 2012).
Cell-cycle delays have been observed in neuroblasts lacking aPKC or Dap160 apical cortical polarity proteins (Chabu, 2008), and wor-escargot-snail triple null mutants lack apical localization of Insc in embryonic neuroblasts (Ashraf, 2001). Apical and basal polarity proteins were stained, and a failure of all proteins to be properly localized during prophase was observed; yet localization was normal by metaphase, most likely by a microtubule-dependent mechanism. It is concluded that Wor is required to establish neuroblast polarity at prophase (Lai, 2012).
wor mutants have fewer neuroblasts compared to the WT brains, which could be caused by neuroblast apoptosis or differentiation. To determine if this reduction was due to neuroblast apoptosis, a genetic sensor for caspase-mediated apoptosis was used, in which caspase activity induces nuclear localization of GFP by cleaving a membrane tether. wor mutant second instar brains were found to have multiple large GFP+ cells at the location of central brain neuroblasts, indicating an elevated level of caspase-mediated cell death. A more general cell death marker, TUNEL staining was used, and the Dpn antibody was used to unambiguously identify neuroblasts. No TUNEL+ Dpn+ neuroblasts were found in the WT brains; in contrast TUNEL+ Dpn+ neuroblasts were observed in wor mutants. RNAi depletion of the Dronc caspase gave a significant but partial rescue of the neuroblast numbers (wor1/Deficiency; wor-gal4 UAS-dronc RNAi); partial rescue is probably because wor-gal4 is only expressed in a subset of neuroblasts or because of incomplete knockdown by RNAi. It is concluded that the loss of neuroblasts seen in wor mutants is largely due to apoptosis (Lai, 2012).
Based on the transcriptomic analysis, it was predicted that wor mutant neuroblasts would show precocious neural differentiation. To determine if wor mutant neuroblasts initiate premature differentiation, well-characterized evolutionarily conserved neural differentiation marker Embryonic lethal abnormal visual system (Elav; Hu family in mammals) was stained; the Elav protein is normally only detected in mature postmitotic neurons where it promotes neuron-specific alternate splicing. Wild-type larval neuroblasts transcribe elav but have low or no Elav protein, whereas many wor mutant neuroblasts showed detectable Elav protein. It is concluded that wor mutant neuroblasts have an abnormally high level of the Elav neuronal differentiation marker, consistent with premature differentiation (Lai, 2012).
Elav is a RNA-binding protein known to promote neuronal-specific splicing of at least three direct target genes: neuroglian (nrg), erect wing (ewg), and armadillo (arm). RNA-seq reads spanning the junctions of alternatively-spliced exons of all three genes were counted, and it was found that the neural-specific, Elav-dependent splice isoforms for all three transcripts were increased in wor mutants compared to WT. Thus, the increased level of Elav in wor mutant neuroblasts appears sufficient to bias splicing toward the neuronal-specific isoforms for all three of its known target genes (Lai, 2012).
To determine the effect of increased Elav levels on the self-renewal of wor mutant neuroblasts, tests were performed to see whether wor mutant phenotypes could be rescued by reducing Elav levels. wor-gal4 was used to drive to drive UAS-elav-RNAi in larval neuroblasts, and a complete rescue of the wor mutant cell cycle phenotype and a substantial rescue of the wor mutant cell polarity phenotype were observed. Thus, the increased level of Elav protein in wor mutant neuroblasts results in most of the cell cycle and cell polarity defects. Reducing Elav levels was not able to restore normal neuroblast numbers, suggesting that it is an Elav-independent pathway. To provide an independent test for the role of Elav in neuroblast cell cycle and cell polarity, Elav levels were increased in otherwise Wt neuroblasts, and cell cycle and cell polarity phenotypes similar to wor mutants were observed, without altering neuroblast number. It is concluded that Wor keeps Elav protein levels low in neuroblasts, which is necessary for establishing neuroblast cell polarity and cell-cycle progression - both key stem cell features (Lai, 2012).
Having established that Wor is necessary to maintain neuroblast properties (proliferation, polarity, survival), it was of interest to see if ectopic Wor was sufficient to induce neuroblast attributes in GMCs or prevent neuronal differentiation. prospero gal4 was used to overexpress Wor in larval neuroblasts and their progeny (abbreviated as WorOXN hereafter). Unexpectedly, the WorOXN larval brains were smaller than WT brains, their larval neuroblasts were smaller in diameter, and the neuroblasts exhibited a severe cell cycle delay. No change was observed in the number of Dpn+ central brain neuroblasts. To determine the cause of the WorOXN phenotype, tests were performed for ectopic Prospero (Pros) protein in neuroblasts, because Pros is known to inhibit cell-cycle progression in larval neuroblasts (Lai, 2012).
Whereas both WT neuroblasts and wor mutant neuroblasts lack nuclear Pros, WorOXN neuroblasts had clearly detectable nuclear Pros. Furthermore, when Pros levels were reduced in WorOXN larvae (WorOXN; pros17/+) partial but significant rescue of the cell cycle and cell size phenotypes was found, and a slight increase in neuroblast numbers. This latter result suggests that Wor overexpression has the ability to transform GMCs/neurons into neuroblasts, but that this is usually masked by Pros-mediated cell-cycle arrest. It is concluded that overexpression of Wor does not lead to a transformation of GMC/neurons into neuroblasts, and that WT neuroblasts must precisely regulate Wor levels; too little Wor leads to Elav-induced premature differentiation, whereas too much Wor leads to Pros-induced cell-cycle arrest (Lai, 2012).
Because wor mRNA and protein are specifically detected in neuroblasts, not in neurons or glia, the brain phenotypes described in this study are most likely to be due to cell autonomous function of Wor within neuroblasts. This study has shown that Wor prevents premature differentiation of neuroblasts, a conclusion based in part on the upregulation of neuronal differentiation transcripts in wor mutant neuroblast lineages. The observed increase in neuronal differentiation transcripts is likely to be an underestimation, because wor mutant neuroblast lineages have three times fewer UPRT+ neurons than WT neuroblast lineages (due to the neuroblast cell-cycle delay in wor mutant neuroblasts). The reduced number of neurons in the wor mutant clones makes it all the more striking that neuronal differentiation transcripts were found to be upregulated in wor mutant neuroblast lineages (Lai, 2012).
A second reason it is concluded that Wor prevents premature differentiation of neuroblasts is the finding that wor mutants have increased levels of the differentiation marker Elav within neuroblasts. How does Wor normally keep Elav protein levels low in neuroblasts? Wor may repress elav at the transcriptional or post-transcriptional levels. Although no change was seen in elav transcript abundance between WT and wor mutant neuroblast lineages by RNA-seq, wor mutants have three times fewer UPRT+ neurons than wor mutants (Lai, 2012).
The extra neurons in WT should result in more elav transcripts; the fact that equal levels were seen suggests that wor mutant neuroblasts may have increased levels of elav transcription. On the other hand, Wor may repress Elav at a posttranscriptional level. Wild-type embryonic and larval neuroblasts transcribe the elav gene but little of the mRNA is translated; it is likely that elav is also posttranscriptionally regulated in larval neuroblasts, and this step could be subject to direct or indirect regulation by Wor. Thus, Wor may regulate elav at the transcriptional and/or posttranscriptional level to keep Elav protein low in neuroblasts (Lai, 2012).
How does Elav promote premature differentiation of neuroblasts? Elav may act by inducing neuronal-specific splicing of its direct targets neuroglian, erect wing, and armadillo (which was observed to be upregulated in wor mutants), or additional targets that have yet to be identified. In addition, other RNA splicing factors, many of which are up- or downregulated at least 2-fold in wor mutants, may coregulate Elav targets and/or splicing of additional pre-mRNAs. Genomic analysis of alternative splicing junction usage in wor mutants showed a profound change of global splicing events: 15.0% of all potentially alternatively-spliced exons (14,476 junctions from 3,430 genes) showed >2-fold change in wor mutants compared to WT. Because the function of different splice isoforms are so poorly understood, it can only be speculated that some or all of the upregulated splice isoforms promote neural differentiation and inhibit cell cycle in wor mutants. Neuronal differentiation seen in wor mutant neuroblasts is not complete, because wor mutant neuroblasts maintain expression of neuroblast markers such as Dpn, Ase, and Miranda. Thus, wor mutant neuroblasts have a mixed fate, in which both neuroblast and neuronal genes are expressed (Lai, 2012).
Wor is required to promote cell polarity at prophase. The defect in apical protein localization seen in wor mutants is similar to that seen in the absence of an external polarizing cue in embryonic neuroblasts, or in sgt1 mutant larval neuroblasts. It is also coincident with the prophase cell-cycle delay observed by live imaging, but the relationship between loss of polarity proteins and prophase delay is unknown. Wor is also required to prevent neuroblast apoptosis. In mammals, Snail family members are known to protect cells from apoptosis triggered by loss of survival signals. It remains unknown whether Wor acts in a similar manner; all that can be said is that Wor acts via an Elav-independent pathway to maintain neuroblast survival (Lai, 2012).
Wor is required for cell-cycle progression from prophase to metaphase. It is interesting that loss of wor causes cell-cycle delays at the precise time when the microtubule cytoskeleton is dramatically reorganized into a bipolar spindle. In addition, the RNA-seq data shows that wor mutants are depleted for 'microtubule cytoskeleton organization' annotated transcripts. Mammalian Snail family proteins confer migratory properties to epithelial cells during EMT or metastasis, which also involves a dramatic reorganization of the cytoskeleton (Barrallo-Gimeno, 2005; Cano, 2000; Nieto, 2011). Thus, Wor may have a conserved function in regulating the microtubule cytoskeleton. Because reducing Elav levels can rescue cell-cycle progression, Wor appears to regulate the microtubule cytoskeleton indirectly, via keeping Elav protein levels low. High levels of Elav in neuroblasts may induce microtubule organization characteristic of mature neurons, such as using a single centrosome to nucleate unidirectional microtubule outgrowth into the axon. Thus, neuroblasts with high levels of Elav may be unable to efficiently duplicate their centrosomes or form a bipolar mitotic spindle, leading to the observed prophase arrest phenotype (Lai, 2012).
The periphery of the fly eye contains a number of concentrically arranged cellular specializations that are induced by Wingless (Wg) signaling from the surrounding head capsule (HC). One of these is the pigment rim (PR), which is a thick layer of pigment cells that lies directly adjacent to the HC and completely circumscribes the rest of the retina. Many of the cells of the PR are derived from presumptive pigment cells that previously surrounded peripheral ommatidia that subsequently died. This study describes the Wg-elicited expression of Snail family transcription factors in the eye periphery that directs the ommatidial death and subsequent PR formation. These transcription factors are expressed only in a subset of the ommatidial cells not including the photoreceptors. Yet, the photoreceptors die and, thus, a non-autonomous death signal is released from the Snail-family-expressing cells that direct the death of the photoreceptors. In addition, Wg also elicits a similar peripheral expression of Notum, an enzyme that limits the extent of Wg signaling. Furthermore, a later requirement is described for Snail family proteins in the 2° and 3° pigment cells throughout the main body of the eye (Lim, 2006).
Wg signaling regulates the expression of Snail family genes, and a number of TCF-binding sites have been identified in the region of the three Snail genes, which is consistent with, but not proof of a direct regulation by the Wg transduction pathway. In mammalian systems, it had been shown that Snail transcription is elicited by the inhibition of glycogen synthase kinase-3 (GSK-3) which represses Snail expression by inhibiting the transcriptional activity of NFkappaB on the Snail promoter. In addition, GSK-3 can phosphorylate Snail at two consensus motifs, one for protein degradation (site I) and the other for subcellular localization (site II). Thus, in mammalian systems, Wnt signaling regulates Snail gene activity both at the level of the transcript and the protein (Lim, 2006).
The apoptotic removal of the most peripheral ring of developing ommatidia releases the surviving surrounding pigment cells to join and thicken the PR. Ectopic expression of Snail family proteins mimics the ommatidial death that is engendered by Wg expression, and loss of these proteins prevents the normal Wg-dependent removal of the peripheral ommatidia and consequently disrupts the PR. The Snail family transcription factors thus appear to direct the death of the peripheral ommatidia and development of the PR. However, within the peripheral ommatidia these proteins are expressed only in the cone cells -- they are absent from the photoreceptors (R cells) and the 1° pigment cells. They are also present in the pigment cells surrounding the ommatidia. This expression profile raises a number of points (Lim, 2006).
As the Snail family proteins are transcription factors, then the death signal is probably under their transcription control, but the molecular nature of the signal remains unknown (Lim, 2006).
As the R cells and 1° pigment cells are directed to apoptosis by the expression of Snail family proteins in other cells, then there is non-autonomous death induction. The non-autonomous initiation of death is envisaged in two possible forms. In the first model, the Snail-expressing cells sequester a survival factor that is thereby denied to other cells. Given that the cone cells express the Snail proteins but still die, this seems unlikely. The second model is that there is a factor released by Snail-expressing cells that directs the death of the ommatidial cells. The cells expressing the death factor may be the peripheral cone cells, the surrounding pigment cells or both. The second model is favored and the remainder of this discussion assumes this is correct with appropriate reservation (Lim, 2006).
The pigment cells surrounding the peripheral ommatidia are impervious to the death signal. One possibility is that the death signal is presented exclusively by the peripheral cone cells and only to the cells of the ommatidia (including themselves, and R cells and 1° cells) -- not to the surrounding pigment cells. The cone cells die before the R cells (the time of death of the 1° cells was not examined), and if the cone cells were the source of the death signal then they would probably receive the signal first. Alternatively, the pigment cells may release the death signal (secreted by themselves or the cone cells) but are programmed not to respond (Lim, 2006).
Only the cone cells of the peripheral ommatidia express Snail family proteins (and Wg and Notum) in response to Wg signaling from the HC -- the R cells and 1° cells do not. This probably represents a predisposition of the cone cells to respond to the Wg signal resulting from the selective expression of cone cell specific factors; Cut, for example, is a homeodomain transcription factor restricted to the cone cells at this stage (Lim, 2006).
The finding that Snail transcription factors promote death in Drosophila eye periphery is in contrast to their anti-apoptotic roles in other systems. For example in C. elegans, the Snail-like CES-1 (cell death specification) protein blocks death of the NSM sister cells during embryogenesis. In vertebrates, Slug (Snail2) is aberrantly upregulated by the E2F-HLF oncoprotein in some leukemias, leading to increased cell survival. Mammalian Snail has also been shown to confer resistance to cell death induced by the withdrawal of survival factors in cell cultures. However, in the fly eye a non-autonomous effect of Snail transcription family members in apoptosis is described, suggesting that a different molecular pathway is regulated from those of the autonomous examples above (Lim, 2006).
The death of the peripheral ommatidia appears to serve two functions - it removes these degenerate optical units and it supplies cells for the PR that optically insulate the entire eye. With regard to the PR, there are two sources of cells. First there is the thin layer of pigment cells that circumscribes the entire pupal eye and second there are the later cells, originally associated with the moribund ommatidia, that eventually incorporate into the existing PR to thicken it. Both aspects of PR formation appear to be under Wg signaling control. During the larval phase, the Hedgehog (Hh) morphogenetic wave sweeps the presumptive retina, triggering the ommatidial differentiation process. However, Wg is expressed in the flanking HC which inhibits the inductive mechanism. Thus, the larval retinal tissue directly adjacent to the HC does not undergo ommatidial differentiation. The 2° and 3° pigment cell fate appears to be the ground state of the retinal tissue, and thus the cells directly adjacent to the HC are destined to the pigment cell fate. Later in the pupa, Wg signaling triggers the death of the peripheral ommatidia and releases their pigment cells to join the PR and increase its thickness (Lim, 2006).
The expression of both Wg and Notum (its antagonist) by the cone cells of the peripheral ommatidia is interesting. It may suggest that high levels of Wg expression are required in the peripheral cone cells, but that the diffusion of this cone-cell derived Wg needs to be tightly contained. For example, in the model above where the death signal is provided by the peripheral cone cells, high levels of Wg may be needed to trigger sufficient levels of the apoptotic signal but any diffusion of the high levels of Wg would disturb other aspects of the peripheral patterning (Lim, 2006).
In the absence of Notum, the effects of Wg signaling spread approximately one more ommatidial row into the eye periphery. This relatively mild phenotype suggests that there could be redundant mechanisms restricting the movement of Wg gradient at the eye margin. In Drosophila wing disc, the Wg receptor Drosophila Frizzled2 (Fz2) stabilizes Wg and allows it to reach cells far from its site of synthesis. Wg signaling represses Fz2 expression, creating a gradient of decreasing Wg stability towards the D/V boundary. This might also be the case in the eye periphery, where Wg signaling, in addition to activating Notum, might also represses Fz2 to limit the extent of Wg diffusion (Lim, 2006).
Snail family gene expression in the 2° and 3° pigment cells appears to be under two different control mechanisms; in the peripheral regions it is activated by Wg signaling, but in the main body of the eye it is not. Furthermore, the genes of the Snail complex appear functionally redundant in the periphery but not in the main body of the eye. Here, the phenotypes of esg clones are as strong as those of the mutations in all three genes. This may be explained by differential regulation of the gene promoters in the two positions. For example, in the main body of the eye, Esg expression in the 2° and 3° pigment cells may activate expression of the two other genes, but in the periphery, Wg signaling directly activates each of the genes, with no cross-regulation between them. The majority of studies on the specification of the main body 2° and 3° pigment cells have focused on the mechanism of weeding out the surplus interommatidial cells which occurs between 18 hours and 36 hours APF, but little is known about their subsequent maturation. The data showed that Esg is expressed in the interommatidial pigment cells after the cell pruning mechanism, but before any sign of morphological differentiation. In the esg mutants, the 2° and 3° pigment cells do not undergo correct apical constriction, indicating that these cells are either developmentally delayed compared with their wild-type counterparts or are blocked in their maturation. If the cells are simply developmentally delayed, they should mature over time, but esg mutant clones in the adult eye show degenerate or lost 2° and 3° pigment cells. Thus, Esg appears required for the appropriate maturation/survival of the 2° and 3° pigment cells. What happens to the esg mutant pigment cells after the point when they fail to undergo apical restriction (whether they delaminate or die/degenerate in place) remains to be investigated (Lim, 2006).
The Snail family of zinc-finger transcriptional repressors is essential for morphogenetic cell movements, mesoderm formation, and neurogenesis during embryonic development. These proteins also control cell cycle, cell death, and cancer progression. In Drosophila, three members of this protein family, Snail, Escargot, and Worniu, have essential but redundant functions in asymmetric cell division of neuroblasts. In addition, Snail is critical for early mesoderm formation and Escargot is required for maintaining diploidy in wing imaginal disc cells. In this report, Worniu was demonstrated to play a role in brain development. Alleles of the l(2)35Da complementation group are mutants of worniu. The developing larvae of these mutant alleles fail to shorten their brainstems. The brain phenotype, as well as the lethality, of these mutants can be rescued by worniu transgenes. Moreover, RNAi experiments targeting the worniu transcript show the same nonshortening phenotype in larval brains. worniu is expressed in the neuroblasts of brain hemispheres and ventral ganglions. The results suggest that the loss of Worniu function within the neuroblasts ultimately causes the larval brainstem to fail to go through shortening during development (Ashraf, 2004).
Mesoderm formation in the Drosophila embryo depends on the maternal Toll signaling pathway. The Toll pathway establishes the Dorsal nuclear gradient, which regulates many zygotic genes to establish the mesodermal fate and promote the invagination of ventral cells. An important target gene of Dorsal is snail, which is required for proper mesoderm invagination. The Snail protein contains five zinc fingers and is a transcriptional repressor. However, it is not clear whether repressing target genes is a requirement for Snail to control ventral invagination. To examine such requirement, a series of genetic rescue experiments was conducted in snail mutant embryos. Snail, Worniu, and Escargot are closely related zinc-finger proteins and have equal functions during neuroblast development. However, among these three proteins, only Snail can rescue the mesoderm invagination phenotype. Moreover, the ability of various Snail mutant constructs to repress gene expression correlates with their ability to control invagination. This unique property of Snail in mesoderm formation can be attributed mostly to the CtBP co-repressor interaction motifs in the N-terminus, not to the C-terminal DNA-binding zinc fingers. Ectopic expression of Snail outside the ventral domain is not sufficient to induce cell movement even though repression of target genes still occurs. Together, the results show that the repressor function of Snail is essential for gastrulation. The repression of target genes by Snail may permit other factors in the ventral cells to positively promote mesoderm invagination (Hemavathy, 2004).
Snail, a zinc-finger transcriptional repressor, is a pan-neural protein, based on its extensive expression in neuroblasts. Previous results have demonstrated that Snail and related proteins, Worniu and Escargot, have redundant and essential functions in the nervous system. The Snail family of proteins control central nervous system development by regulating genes involved in asymmetry and cell division of neuroblasts. In mutant embryos that have the three genes deleted, the expression of inscuteable is significantly lowered, while the expression of other genes that participate in asymmetric division, including miranda, staufen and prospero, appears normal. The deletion mutants also have much reduced expression of string, suggesting that a key component that drives neuroblast cell division is abnormal. Consistent with the gene expression defects, the mutant embryos lose the asymmetric localization of Prospero RNA in neuroblasts and lose the staining of Prospero protein that is normally present in ganglion mother cells. Simultaneous expression of inscuteable and string in the snail family deletion mutant efficiently restores Prospero expression in ganglion mother cells, demonstrating that the two genes are key targets of Snail in neuroblasts. Mutation of the dCtBP co-repressor interaction motifs in the Snail protein leads to reduction of the Snail function in central nervous system. These results suggest that the members of the Snail family of proteins control both asymmetry and cell division of neuroblasts by activating, probably indirectly, the expression of inscuteable and string (Ashraf, 2001).
Both snail and worniu have extensive expression in neuroblasts, while that of escargot is transient and sparse. Furthermore, based on genetic analysis, snail and worniu have more important role than escargot in the regulation of CNS development. The expression of snail and worniu in GMCs was carefully examined. In situ hybridization has revealed that worniu RNA, in contrast to its extensive expression in neuroblasts, is present in only a small number of GMCs. Even in later staged embryos, when there should be multiple GMCs surrounding each neuroblast, the staining in no more than one small cell next to each neuroblast could be detected. The limited staining in the GMCs is probably due to the segregation of some RNA from the parental neuroblast. Once the GMC is formed, the active transcription of worniu probably ceases. The protein and RNA expression of snail was also examined. The results showed that there is also very limited expression of snail in GMCs. snail RNA-containing GMCs were rarely detected next to neuroblast. Consistent with RNA expression, antibody staining revealed that the protein is predominantly in the neuroblasts (Ashraf, 2001).
Whether the neuroblast expression of snail and worniu is regulated by proneural genes was examined. Such a result would place the snail family in the well established genetic hierarchy that controls early neuroblast differentiation. The scuteB57 deletion mutant uncovers the three pro-neural genes: achaete, scute and lethal of scute. In this mutant, the expression of worniu in neuroblasts is significantly reduced. Only a few neuroblasts within each segment exhibit staining, and the expression level is substantially lower than in the wild type. The expression of worniu is also regulated by vnd and ind, such that in these mutant embryos the whole ventral and intermediate columns of staining are missing. In the mshDelta68 mutant, no abnormal expression of worniu was detected. Previous results have shown that the neuroblast expression of snail is slightly affected in achaete-scute and vnd mutants but is not affected in a daughterless mutant. In ind and msh mutants, Snail protein expression was observed in many neuroblasts but the spatial pattern was rather disorganized. In summary, most of the proneural genes tested have profound effects on the expression of worniu, and have detectable but lesser effects on that of snail. The predominant expression of snail and worniu in neuroblasts and their regulation by proneural genes suggests that the snail family genes may have important functions within neuroblasts (Ashraf, 2001).
In mutants containing deletions that uncover escargot, worniu and snail, many early neuroblast markers are normal, but ftz expression in GMCs is abnormal. The regulation of ftz depends on Prospero, a homeodomain protein that controls GMC fate. Prospero protein and mRNA are preferentially segregated to GMCs from the neuroblast through the process of asymmetric division. Genes that are involved in asymmetric segregation of Prospero include inscuteable, miranda and staufen. The expression of these possible Snail family target genes was examined in neuroblasts (Ashraf, 2001).
Mutant embryos collected from deficiency strains that uncover the 35D1 chromosomal region, including the snail family genes were examined. In wild-type embryos, the expression of inscuteable can be detected in delaminating neuroblasts. After delamination, many neuroblasts show localization of the Inscuteable RNA. Embryos homozygous for the region 35D1 osp29 deletion, however, had significantly lower levels of the RNA and the staining was detected in a much smaller number of neuroblasts. Transgenic copies of snail, worniu or escargot efficiently rescues the expression of inscuteable RNA, demonstrating that it is the uncovering of the snail family of genes in the deletion that causes the phenotype. The rescue transgenes are under the control of the 2.8 kb snail promoter, which contains the neuroblast expression element. A 1.6 kb snail promoter construct that contains the mesoderm element but lacks the CNS element could not rescue the defect, demonstrating that expression of the transgenes within neuroblasts is essential for the function (Ashraf, 2001).
The segregation of Prospero protein into GMCs from neuroblasts is a critical event during asymmetric cell division. Since inscuteable plays a role in the segregation of prospero gene products into GMCs, whether there is Prospero protein in GMCs of mutant embryos was examined. Prospero protein staining can be easily detected in many wild type GMC nuclei. The staining is largely absent in the deletion that uncovers the snail family locus; only a few cells with the size of normal GMCs had clear nuclear staining. A band of cells along the midline also had Prospero staining, but these cells probably represent an expansion of the midline. It has been well documented that in all snail mutants there is derepression of the mid-line determinant single-minded in the blastoderm stage embryo (Ashraf, 2001).
To determine whether there are defects within GMCs in addition to the loss of Prospero, the expression of Hunchback, which is present transiently in early neuroblasts and later in many GMCs was examined. In the deletion mutant, the Hunchback protein in GMCs is also absent, while staining in cells surrounding the amnioserosa appeared normal. Transgenes of snail, worniu and escargot rescue the staining of Prospero and Hunchback, indicating that these GMC determinants are downstream of the Snail family. The results also suggest that the regulation of ftz by the Snail family is indirect, probably through an earlier event such as segregation of Prospero from neuroblast to GMC (Ashraf, 2001).
If the misregulation of inscuteable in the deletion mutant is the cause of the loss of Prospero and ftz expression in GMCs, the expression of inscuteable should correct the defects even in the absence of Snail family of proteins. A line carrying an inscuteable transgenic construct driven by the 2.8 kb snail promoter was crossed into the osp29 deletion genetic background. However, the rescue of Prospero expression in GMCs was variable and not nearly as strong as those embryos expressing the snail family transgenes. This suggests that inscuteable may not be the only important target gene of Snail. Another line of evidence supporting the idea of an additional target gene comes from the comparison of the phenotypes in osp29 and inscuteable mutant embryos. In inscuteable mutants, the Prospero crescent is formed but the mitotic spindle rotation is randomized. As a result, the Prospero protein frequently is present both in neuroblasts and GMCs. This phenotype is less severe than the almost total loss of Prospero GMC staining in osp29 deletion mutant. Therefore, it is surmised that in addition to the misregulation of inscuteable, there may be other defects that lead to the more severe phenotype in the deletion mutants (Ashraf, 2001).
One possibility that may explain the severe phenotype in snail family deletion mutants is additional defects in cell division. Neuroblasts are arrested at the G2/M transition at the embryonic cell cycle 14. After delamination, a pulse of string (which encodes a Cdc25 phosphatase homolog) expression in neuroblasts drives the cells to enter mitosis. The expression of string RNA was examined in whole-mount mutant embryos, but the result was ambiguous, owing to the dynamic, high level expression in ectoderm and other tissues, which obscures the signal in the neuroblast cell layer. Therefore tissue sectioning was used in order to better view the expression of string in neuroblasts. The sections clearly showed expression of string RNA in wild-type neuroblasts at stage 9 embryos. There are consistently three to four neuroblasts on each side of the midline that exhibit staining. This neuroblast expression appears very faint in the osp29 mutant embryos, and most sections do not show staining in neuroblasts while expression in ectoderm appears normal. The presence of wor and esg transgenes in the deletion mutant background led to accumulation of string RNA in some neuroblasts, suggesting a positive role for Snail family in regulating string expression (Ashraf, 2001).
If regulation of string is an important downstream event of Snail family of proteins, then cell division of neuroblasts should be affected in the absence of these proteins. The mitotic process was examined by staining for phosphorylated histone H3, which reveals condensed chromosomes. In wild-type embryos, although the neuroblasts do not exhibit highly synchronized mitosis, anti-phosphoH3 staining can be detected in multiple cells. In the osp29 mutant embryos, such staining is consistently reduced. The use of Prospero RNA to mark the neuroblast layer and the use of tissue sectioning has provided further support for the idea that the mutant embryos has reduced mitosis in neuroblasts (Ashraf, 2001).
The severe CNS defects are likely due to a combination of loss of inscuteable and string expression. Similar to the results obtained for inscuteable, transgenic expression of string alone has a weak and variable effect in the rescue of Prospero expression in GMCs. When both inscuteable and string are simultaneously expressed in neuroblasts of osp29 mutants using the UAS-Gal4 system, clear staining of Prospero in many cells resembling GMCs is observed. The staining is particularly apparent alongside the expanded midline, characteristic of mutant embryos with no Snail function in early mesoderm. The results support the idea that both inscuteable and string are relevant targets of the Snail family (Ashraf, 2001).
A clearly demonstrated in vivo function of Snail is transcriptional repression. The repression function is mediated through the recruitment of dCtBP (Drosophila C-terminal binding protein), which acts as a co-repressor for Snail to regulate target genes such as rhomboid, lethal of scute and single-minded. There are two conserved P-DLS-R/K motifs in Snail, as well as in Worniu and Escargot, and they have been shown to be critical for recruiting dCtBP. Mutations of these motifs abolish the repressor function of Snail in the blastoderm. To gain insight into the molecular mechanism of how Snail regulates CNS development, transgenic copies of snail, which had the dCtBP interaction motifs mutated were introduced into the osp29 deletion background. M1 contains the N-terminal motif mutation and M2 contains the C-terminal motif mutation. The expression of inscuteable and ftz was examined. The assay shows that the double mutant (M12) lost most of the ability to rescue, and M1 has lost some ability to rescue. However, M2 functioned quite efficiently, closer to that of the wild-type protein, to rescue inscuteable and ftz expression. These results demonstrate that the dCtBP interaction motifs are essential for the Snail function in the CNS, consistent with the idea that Snail acts as a repressor in neuroblasts to regulate gene expression. Thus, the activation of inscuteable and string by the Snail family may be indirect (Ashraf, 2001).
Three snail family genes -- snail, escargot and worniu -- encode related zinc finger transcription factors that mediate Drosophila central nervous system (CNS) development. Simultaneous removal of all three genes causes defective neuroblast asymmetric divisions; inscuteable transcription/translation is delayed/suppressed in the segmented CNS. Furthermore, defects in localization of cell fate determinants and orientation of the mitotic spindle in dividing neuroblasts are much stronger than those associated with inscuteable loss of function. In inscuteable neuroblasts, cell fate determinants are mislocalized during prophase and metaphase, yet during anaphase and telophase the great majority of mutant neuroblasts localize these determinants as cortical crescents overlying one of the spindle poles (Cai, 2001).
This phenomenon, known as 'telophase rescue', does not occur in the absence of the snail family genes; moreover, in contrast to inscuteable mutants, mitotic spindle orientation is completely randomized. These data provide further evidence for the existence of two distinct asymmetry-controlling mechanisms in neuroblasts both of which require snail family gene function: an inscuteable-dependent mechanism that functions throughout mitosis and an inscuteable-independent mechanism that acts during anaphase/telophase (Cai, 2001).
CNS development is abnormal in Df(2L)osp29 embryos due to deletion of Sna family proteins. Both Sna and Wor are expressed strongly in all NBs, including those in the procephalic region, during early neurogenesis. The expression of Esg is also seen in NBs and other tissues, as visualized with anti-Esg immunostaining. Expression of Esg can be detected in the midline cells as well as GMCs during embryonic development. The functions of these three genes are overlapping; the early CNS defects are detected only when all three genes are removed simultaneously. In order to test whether the defects of localization of Mir/Pros and Pon/Numb seen in Df(2L)TE35BC-3 embryos are due to the absence of the three sna family genes, the localization of Mir/Pros and Pon/Numb was examined in embryos single mutant for sna, esg or wor, a double mutant for sna/esg and deletions that removed sna/wor or esg/wor, as well as embryos double mutant for sna/esg and further subjected to wor double-stranded RNA (RNAi) treatment. In single and double mutant embryos, both Mir/Pros and Pon/Numb form normal basal crescents in mitotic NBs. Only the sna/esg double mutant embryos that have been injected with wor RNAi reproduce the phenotype found in Df(2L)TE35BC-3 embryos (Cai, 2001).
In wild-type embryos, NBs are located between the ectoderm and mesoderm. The Df(2L)TE35BC-3 embryos lack mesoderm. Therefore, it is possible that correct NB asymmetry requires signal(s) from the mesoderm, and the asymmetry defects seen in Df(2L)TE35BC-3 could be due simply to the absence of mesoderm in these embryos. This is unlikely since NB asymmetry is intact in sna embryos, which lack mesoderm and share the abnormal morphology of Df(2L)TE35BC-3 embryos. Furthermore, the partial rescue of mesoderm in Df(2L)TE35BC-3 embryos by ectopic expression of the Sna protein driven by twist-gal4 does not reverse the asymmetry defects. Thus, it is concluded that mislocalization of Mir/Pros and Pon/Numb in Df(2L)TE35BC-3 embryos is due to the absence of all three sna family genes. Based on this conclusion, Df(2L)TE35BC-3 is referred to as sna/esg/wor deficient and was used in subsequent studies (Cai, 2001).
In wild-type embryos, Baz, Insc and Pins form a complex that is localized to the apical cortex of the dividing NBs. The apical complex is required for the asymmetric distribution of cell fate determinants such as Pros and Numb to the basal cortex of NBs and coordinates the orientation of the mitotic spindle along the apical-basal axis of the NB. In embryos deficient for the sna family genes, Mir/Pros and Pon/Numb are no longer concentrated to the basal cortex of mitotic NBs, indicating defects in NB asymmetry. It is possible that the asymmetry defects seen in sna/esg/wor-deficient NBs are due to the alteration of Insc expression. Anti-Insc staining indicates that Insc protein is indeed undetectable in the segmented CNS of sna/esg/wor-deficient embryos. Although the signal intensity in the procephalic region is comparable to that in the wild-type controls, the number of cells with anti-Insc staining appears to be decreased. This altered expression of Insc in the mutant embryos suggests that the mislocalization of Mir/Pros and Pon/Numb in sna/esg/wor-deficient embryos is, at least in part, due to a lack of Insc protein expression in dividing NBs. As expected, Baz protein levels are low and undetectable in the great majority of mutant NBs. The lack of easily detectable Baz in NBs is probably due to the instability of the protein when Insc is absent since the baz mRNA levels remain unchanged in sna/esg/wor NBs. Pins protein localization is also affected in sna/esg/wor-deficient embryos (Cai, 2001).
The down-regulation of Insc protein in NBs is also dependent on the simultaneous loss of sna, esg and wor functions. Insc expression in double mutant embryos of sna/esg was similar to that of wild-type embryos. In sna/esg double mutant embryos, further removal of the third member of sna gene family, wor, with RNAi leads to the total loss of Insc protein expression. Moreover, ectopic expression of any one of the sna family genes under the control of an early neural driver sca-gal4 in sna family gene mutant embryos largely restores the Insc expression in NBs (sna 79%; esg 64% and wor 44%), further indicating that Insc expression is indeed regulated by the Sna family proteins (Cai, 2001).
insc transcript levels were examined in the sna/esg/wor-deficient embryos. In wild-type stage 9-10 embryos, insc RNA is expressed prominently in NBs of the segmented CNS and in the procephalic region. The transcript level is maintained in the segmented CNS and procephalic NBs throughout embryogenesis. In sna/esg/wor-deficient embryos, RNA in situ hybridization data indicate that the insc RNA is absent in the segmented CNS at stages 9-10 but is detectable in the procephalic NBs. This suppression of insc RNA transcription in the segmented CNS of sna/esg/wor-deficient embryos provides evidence that the Sna family proteins are essential for insc mRNA transcription during early neurogenesis. The suppression of insc transcription in the segmented CNS is transient and insc RNA can be detected, at a lower level, in late stage 11 embryos. However, Insc protein in the segmented CNS of sna/esg/wor-deficient embryos remains undetectable at late stage 11 when the insc RNA levels partially recover by an unknown mechanism. It is obvious that translation of insc RNA in late stage 11 embryos is inhibited in the segmented CNS of embryos deficient for sna/esg/wor. Although the inhibition mechanism is unknown, it is believed that the insc 5'- and/or 3'-untranslated regions (UTRs) are involved since Insc protein can be ectopically expressed in sna/esg/wor-deficient embryos from a uas-insc transgene in which the 5'- and 3'-UTRs have been partially removed. Considering that the Sna family proteins are localized to nuclei, it is unlikely that they interact directly with 5'- and/or 3'-UTRs of insc RNA. Presumably other genes regulated by the Sna family proteins mediate the observed translational effect (Cai, 2001).
The observation of delayed and decreased insc mRNA transcription and the inhibition of Insc protein synthesis in the segmented CNS of sna/esg/wor-deficient embryos suggests the dual regulation of insc expression by the Sna family proteins at both transcriptional (stage 9-10) and translational (stage 11 onwards) levels. This dual regulation mechanism is prominent in the segmented CNS but insc RNA and protein expression in the procephalic region is only partially affected in sna/esg/wor-deficient embryos. The mechanism that enables the partial restoration of insc transcription in NBs of the segmented CNS at late stage 11 in the absence of sna family gene function remains to be identified (Cai, 2001).
In insc22 mutant NBs, in which the apical complex required for correct asymmetric division is abolished, basal components such as Mir/Pros and Pon/Numb often form random crescents, sometimes broad and loose, from prophase to metaphase; however, Pros/Mir and Pon/Numb can eventually be redistributed to the 'budding site' of the future GMCs, although sometimes not as exclusively as seen in wild-type embryos, at anaphase and telophase even when the spindle is misorientated. Consequently, the great majority of all GMCs inherit, at least in part, cell fate determinants such as Pros and adopt correct GMC fate. This phenomenon, referred to as 'telophase rescue', does not occur in NBs lacking the three sna family genes. For example, in sna/esg/wor-deficient NBs, basal proteins Mir/Pros and Pon/Numb form a randomly localized crescent in dividing NBs but, unlike in insc embryos, these proteins are not redistributed at anaphase/telophase to the region of the cortex that gives rise to the GMC. Consequently, the great majority of the GMCs do not inherit the basal proteins Mir/Pros and Pon/Numb and thus lose their GMC identities. This finding explains why GMCs are not specified correctly in Df(2L)osp29 embryos (Cai, 2001).
Furthermore, it is known that the mitotic spindle in NBs rotates 90° during metaphase so that it is realigned along the apical-basal (A/B) axis of the embryos; in insc mutants, this spindle rotation during metaphase occurs only in a small proportion (~20%) of NBs; nevertheless, even some of these NBs are able to reorient spindles late in mitosis. The NB spindle orientation during anaphase or telophase was measured in wild-type and mutant embryos and they were catagorized into four equal quadrants depending on the angle that the spindle forms with the A/B axis. Based on the spindle orientation in wild-type embryos, all spindles with an angle >45° relative to the A/B axis during late mitosis are considered to be misoriented. The misoriented spindles in insc22 mutant embryos are limited; the great majority of NBs (90%) have their spindles oriented within 45° of the A/B axis, compared with 100% in wild-type NBs. In contrast to wild-type and insc NBs, in sna/esg/wor-deficient NBs, spindle orientation is completely randomized with almost equal distribution for each of the four quadrants. Moreover, a small number of NBs (10%) completely reverse their polarity, giving rise to a small apical GMC, which has never been reported in any known asymmetry mutant (Cai, 2001).
These observations indicate that removal of Insc alone has only a limited effect on NB asymmetric divisions in terms of basal protein localization and spindle orientation late in mitosis, suggesting that the Insc-dependent mechanism is not the only apparatus that controls the asymmetric divisions in NBs. It appears that an Insc-independent mechanism exists that functions in parallel to coordinate the asymmetry events at later stages (anaphase onwards) of mitosis. This Insc-independent asymmetry-controlling mechanism, which is responsible for the 'telophase rescue' phenomenon and for prevention of random spindle orientation in insc22 embryos, is destroyed upon removal of the three sna family genes. However, one might argue that the severe asymmetry defects seen in the absence of the sna family genes might be artifactual, caused by the combination of loss of insc expression and the absence of the mesoderm. This possibility is suggested because in insc/sna double mutant embryos, which lack both insc and the mesoderm, NBs exhibit phenotypes that are indistinguishable from those seen in the insc single mutant. It has therefore been concluded that in the absence of the sna family genes, both the Insc-dependent and -independent asymmetry-controlling mechanisms are destroyed, leading to asymmetry defects that are more severe than those seen in insc single mutants (Cai, 2001).
The existence of two distinct asymmetry-controlling mechanisms in wild-type NBs raises an interesting issue: how do these two mechanisms work in concert to mediate asymmetric divisions? Since embryos deficient for the sna family genes lack both mechanisms, it was reasoned that by restoring the Insc-dependent mechanism in these embryos the consequences of missing just the insc-independent mechanism could be assessed. Ectopic expression of full-length Insc protein with an early neural driver sca-gal4 in NBs of sna family gene mutant embryos shows complete rescue of the protein localization defects. The apical complex forms normally, as indicated by the formation of apical Insc as well as Pins and Baz crescents. The defects in basal protein localization are also completely rescued; Mir/Pros and Pon/Numb form tight basal crescents in mitotic NBs. These results suggest that, with respect to protein localization, Insc protein is the only component missing in the Insc-dependent asymmetry machinery, and replacement of Insc through ectopic expression is sufficient to restore wild-type localization of the apical and basal components. Furthermore, it indicates that the Insc-independent mechanism is cryptic with respect to protein localization since it is dispensable when the Insc-dependent mechanism is in place. Either mechanism alone is able to distribute basal proteins to the cortex of the future GMC 'budding site' with clear temporal and efficiency differences: the Insc-dependent mechanism localizes basal proteins starting in late prophase in the form of tight crescents, while the Insc-independent mechanism is only able to redistribute, sometimes partially, mislocalized basal proteins late in mitosis (telophase rescue) (Cai, 2001).
The spindle misorientation phenotype in sna family gene mutant embryos is also largely corrected by ectopic Insc expression. However, unlike protein localization, the rescue of mitotic spindle orientation is incomplete; the population of NBs with misoriented spindles drops from 45% to only 12%. These data suggest that both the Insc-dependent and -independent mechanisms are required for correct spindle orientation in wild-type embryos since ~10% of the mitotic spindles are misoriented in anaphase/telophase NBs defective for either mechanism. However, a complete randomization of spindle orientation is seen when both mechanisms are absent (Cai, 2001).
Thus, the underlying cause for the asymmetry defects associated with some deficiencies uncovering the 35B-D region of the genome, e.g., Df(2L)TE35BC-3, is the simultaneous loss of three members of the sna gene family: sna, esg and wor. All available lethal complementation groups uncovered by Df(2L)TE35BC-3, all deficiencies that remove only two out of the three sna family members and a sna/esg double mutant generated from recombination do not show any defects in any aspect of NB asymmetric division; only embryos double mutant for sna/esg, and further subjected to wor RNAi, reproduce the asymmetry defects seen in the deficiencies. These data indicate that the defects in sna/esg/wor-deficient embryos are caused by the simultaneous functional loss of all three sna family genes. The observation that the ectopic expression of sna, esg or wor reverses the asymmetry phenotypes in the segmented CNS of sna/esg/wor-deficient embryos further supports this conclusion. These conclusions are in agreement with an earlier study reporting that the sna family genes are required for CNS development (Cai, 2001).
It has been observed that in insc embryos, cell fate determinants such as Pros and Numb are mislocalized early during mitosis; however, in anaphase and telophase, the effect termed 'telophase rescue' causes the misplaced crescents to redistribute and overlie one spindle pole, enabling the basal cell fate determinants to segregate, exclusively or partially, to the GMCs. The insc loss-of-function alleles insc22, inscP49 and inscP72 all show telophase rescue. It has been found that essentially all NBs in insc embryos can redistribute Pros and Numb, at least partially, into GMCs. These observations suggest the existence of a second asymmetry-controlling mechanism that does not require insc functions, which operates late in mitosis to coordinate protein localization with spindle orientation. These observations explain why insc mutants have minimal effect on GMC cell fate. The Insc-independent mechanism corrects the earlier errors caused by absence of Insc during anaphase/telophase, thereby enabling cell fate determinants to be inherited by the GMC. This mechanism is apparently less efficient, as shown by the fact that in some insc NBs, normally basal components form a broad and loose crescent and are only partially sequestered into GMCs. Furthermore, the observation that mitotic spindle orientation is only mildly affected in insc NBs is also consistent with an Insc-independent compensatory mechanism (Cai, 2001).
Analysis of NB divisions in embryos deficient for the three sna family genes provides further support for the existence of an Insc-independent mechanism. In these embryos, the Insc-dependent mechanism is clearly abolished; both the transcription and the translation of insc are suppressed in the mutant NBs. In addition, telophase rescue no longer occurs; the normally basally localized components are misplaced in mitotic NBs and not redistributed to the future GMCs even at anaphase/telophase. Moreover, the spindle orientation in embryos deficient for the sna family genes becomes randomized; ~45% of NBs exhibit misoriented spindles with an angle >45° with respect to the A/B axis at anaphase/telophase, which is not seen in wild-type NBs and is at a much higher frequency than that seen in insc22 NBs. Thus, NBs deficient for the sna family genes show two defects that are not seen in insc NB: (1) the absence of telophase rescue, and (2) randomization of the spindle orientation late in mitosis. These observations indicate that both the Insc-dependent and -independent mechanisms require the sna family genes (Cai, 2001).
These two mechanisms can apparently function independently. In insc NBs, the Insc-independent mechanism functions in the absence of the Insc-dependent mechanism to correct the earlier (prophase to metaphase) asymmetry defects during anaphase/telophase. In sna/esg/wor-deficient NBs that have been forced to express Insc, the Insc-dependent mechanism can act in the absence of the Insc-independent mechanism to mediate the localization of the basal components from prophase to telophase, obviating the requirement for telophase rescue; however, although the Insc-dependent mechanism can reduce the extent of the mitotic spindle orientation defects seen in the sna/esg/wor NBs, it does not restore wild-type spindle orientation. Therefore, it appears that both mechanisms are required and act in concert to mediate mitotic spindle orientation. However, with respect to localization of the basal components, the effects of the Insc-independent mechanism are only visible when the Insc-dependent mechanism is absent (Cai, 2001).
For the Insc-dependent mechanism, three components have been identified: Baz, Insc and Pins are known to form an apically localized functional complex. The function of this complex requires the participation of all members. Insc appears to be the only component of the Insc-dependent mechanism missing in sna/esg/wor-deficient embryos since ectopic expression of Insc restores its function. Little information is available on the components of the Insc-independent mechanism. Other members of asymmetry machinery identified so far in NBs are the basal components such as Mir/Pros, Pon/Numb, Stau and pros RNA. These downstream components are controlled and coordinated by both Insc-dependent and -independent mechanisms (Cai, 2001).
In embryos deficient for the sna family genes, one of the major defects is the absence of Insc protein expression in the segmented CNS. RNA in situ hybridization indicates that the insc RNA transcripts are not detected in NBs of stage 9-10 embryos. Even in late stage 11 embryos when the insc RNA levels partially recover, Insc protein is never seen in the segmented CNS, indicating that the down-regulation of insc occurs at both the transcriptional and translational levels. In the procephalic region of these sna/esg/wor-deficient embryos, Insc expression is only partially affected. The 5'- and/or 3'-UTRs of the insc transcript appear to play an important role in the translational regulation of Insc expression. This is supported by two observations: (1) Insc protein can be detected in sna/esg/wor embryos following ectopic expression of a cDNA construct containing the complete insc coding region but with the 5'- and 3'-UTRs partially removed; (2) transcripts derived from lacZ driven by a 1.2 kb insc 5' CNS promoter sequence are not subjected to this translational repression in sna/esg/wor embryos, although their expression pattern is identical to that of Insc in the CNS. Given that the Sna family proteins are localized to nuclei, it is unlikely that they play a direct role in translational regulation. Other unknown intermediates must be involved (Cai, 2001).
By the time of neuroblast delamination, Sna is present in most of the neuroblasts that have segregated from the ectoderm. Despite the extensive expression in the neuroblasts, prior to this study, Sna had no known function in the developing nervous system. The neuroblast pattern of sna resembles that of a group of genes called pan-neural genes. One of these genes, scratch (scrt), encodes a protein that has sequence similarity to Sna in the zinc-finger domain. Mutations of scrt have no obvious phenotype except that viable escapers have morphological defects in the eyes. Furthermore, no nervous system defect can be seen in sna scrt double mutants. However, the scrt dpn double mutants exhibit some defects in nervous system development. deadpan (dpn) is another pan-neural gene that encodes a basic helix-loop-helix protein. Therefore, scrt does have a function in the central nervous system (CNS), but the function of sna, if any, in the nervous system does not overlap with that of scrt (Ashraf, 1999 and references therein).
Escargot (Esg) is another protein that contains five zinc fingers with sequences highly homologous to those of Sna. The expression of esg is rather dynamic during embryonic development. The gene is expressed in the epidermis, neuroectoderm and imaginal precursor cells. The Esg protein probably acts through the cdc2 kinase to maintain the proper cell cycle in larval imaginal disc cells; in esg mutant larvae the imaginal disc cells lose their diploidy as they re-enter the S phase without going through mitosis. Moreover, esg and sna are both expressed in the embryonic wing imaginal disc primodia and the two genes have redundant functions in this tissue; the vestigial marker gene expression in the disc is lost in esg sna double mutants. Despite a clear demonstration of the redundant requirements of sna and esg in the wing disc, the double mutant has been reported to have no significant embryonic CNS phenotype. Thus, the function of sna in nervous system development has remained a mystery (Ashraf, 1999 and references therein).
Evidence is provided that CNS expression of Snail is required for nervous system development. The neural function of snail is masked by two closely linked genes, escargot and worniu. worniu (pronounced war-niu, Chinese for 'snail') encodes a protein with a zinc-finger domain highly homologous to those of Sna and Esg; it has been identified from the Berkeley Drosophila Genome Project database. RNA in situ hybridization reveals extensive expression of worniu in the developing nervous system. wor is located between esg and sna, ~100 kb apart, in the 35D region of the second chromosome. Although not affecting expression of early neuroblast markers, the deletion of the region containing all three genes correlates with loss of expression of CNS determinants including fushi tarazu, pdm-2 and even-skipped. Transgenic expression of each of the three Snail family proteins efficiently rescues the fushi tarazu defects, and partially rescues the pdm-2 and even-skipped CNS patterns. These results demonstrate that the Snail family proteins have essential functions during embryonic CNS development, around the time of ganglion mother cell formation (Ashraf, 1999).
The putative Wor protein sequence contains a C-terminal domain with six zinc fingers that are very similar to those of Sna and Esg, even though those proteins contain only five fingers. The N-terminal halves of these proteins have rather divergent sequences, except that they all contain a conserved basic motif very close to the N-termini. The function of this motif is not known. Moreover, the proteins contain two P-DLS-K motifs. The P-DLS-K domains in Sna have been shown to interact with the Drosophila C-terminal binding protein (dCtBP) and to play important roles in transcriptional repression. Since all three Sna family proteins contain highly homologous corepressor-interacting and DNA-binding domains, and can bind to similar DNA sequences, it is possible that they bind to promoters of overlapping sets of target genes and repress transcription (Ashraf, 1999 and references therein).
While there is no maternal RNA deposition of wor, zygotic expression can be detected first at the onset of neurogenesis. At a late stage 8, WOR transcript can be observed in two small patches of cells in the dorsal head region anterior to the cephalic furrow, representing precursor cells of the developing brain. At stage 9 wor expresses in the first wave of delaminating neuroblasts along either side of the midline, as well as in cells in the head region. Later in the germ band-extended embryo, most of the neuroblasts contain WOR mRNA. This pattern greatly resembles that of sna at this stage of development, except that sna expression in some of the centrally located neuroblasts in each hemisegment is at lower levels. In later stages, wor continues to express in the brain and part of the ventral nerve cord. No expression of wor is detected in any other embryonic tissue (Ashraf, 1999).
There is no extensive expression of esg in the neuroblasts similar to that shown for wor or sna. However, it has been demonstrated that esg RNA is expressed in the ventral neuroectoderm. Careful examination of the expression reveals that esg transcript is probably present in the CNS, albeit at variable levels. Based on the expression analyses, it is hypothesized that the newly identified wor might serve a redundant function with that of sna or esg during neural development. This would explain why neither single nor double mutants of sna and esg show severe defects in the nervous system (Ashraf, 1999).
To test the hypothesis that the Sna family proteins function redundantly in the developing nervous system, the neural phenotype associated with a deletion that uncovers all three genes was examined. wor is located between esg and sna, ~100 kb apart, in the 35D region of the second chromosome. Advantage of the close proximity of these genes and the phenotypes of a deficiency mutant are examined. Since high levels of Sna and Wor are present in the neuroblasts, the expression of the proneural gene achaete, which marks a subset of early delaminating neuroblasts was examined. This expression is not affected in the osp29 deficiency mutants. The expression patterns of additional neuroblast markers including hunchback, dpn, scrt and lethal of scute also are similar in wild-type and mutant embryos. Therefore, the early waves of neuroblast delamination are normal in the absence of the Snail family proteins (Ashraf, 1999).
The CNS patterns of GMC markers ftz, pdm-2 and eve were examined. ftz is expressed in a number of midline precursor cells and extensively in GMC. In contrast to the neuroblast markers, the ftz expression is almost abolished in the mutant embryo. The pdm-2 gene is also expressed in some neuroblasts and GMC. The early neuroblast expression of pdm-2 in the mutant is nearly normal, while the expression in later staged embryos is highly defective. eve gene products are present in a number of GMC and postmitotic neurons during normal development. All the eve CNS expression is absent in homozygous osp29 deletion mutant embryos. Taken together, the deletion mutant that uncovers the three sna family genes shows severe defects in CNS development (Ashraf, 1999).
To confirm the function of these three proteins in neural development, transgenic rescue plasmids were constructed in which individual genes (esg, wor or sna) were placed under the control of a sna promoter, containing an enhancer element that directs expression in the neuroblasts. The transgenic flies obtained were then crossed with the osp29 strain and analyzed for CNS development. In the presence of any one of the three constructs the ftz expression is restored significantly. Analysis of the rescued pattern under higher magnification reveals that part of the ftz staining is clearly absent. However, more detailed analysis is required to pinpoint the exact cell lineages that are missing. Nevertheless, the results demonstrate that each of the three sna family genes can perform essential functions in the CNS in the absence of the other two. The rescue by the transgenes of the expression of pdm-2 and eve, both of which are defective in the osp29 mutant, was also examined. While all three sna family genes clearly can rescue the expression of pdm-2 , the effect is not as extensive compared with that of ftz. For eve RNA, the transgenes rescue the expression in a significant number of cells when compared with the total loss of expression in the parental osp29 mutant. The rescue of eve, again, is not as extensive as that of ftz. Later stage CNS morphology in the rescued embryos was also monitored by BP102 staining. The embryos carrying the transgenes have slightly better overall CNS axonal morphology, but they are still highly abnormal when compared with the wild type (Ashraf, 1999).
Pairwise recombination of the transgenes were constructed and a test was performed to see whether they could achieve better rescue. By staining embryos obtained from stable lines that are homozygous for two transgenes, the constructs were found to give slightly direct the expression of ftz slightly better. Meanwhile, the eve and BP102 antigen expression in the presence of two transgenes reveals only minor improvement of the axonal morphology. These results suggest that the three proteins may have some collaborative function. It is also possible that the promoter used has some limitation in driving the rescue transgenes or that there are additional genes involved for the severe CNS phenotype (Ashraf, 1999).
Increasing numbers of sna-related genes have been identified in diverse species. These proteins have been assigned to the Sna family based mostly on the similarity of the sequences in the zinc-finger domains. The expression patterns and some functional studies of the vertebrate proteins suggest a role in regulating cell movement. However, gene knock-out experiments have demonstrated that mutating a mouse Slug homolog does not lead to a detectable cell movement defect. Such a result suggests a possible redundant function provided by other genes, similar to this report. If the vertebrate homologs do have a function in controlling cell movement, it would be reminiscent of the control of cell movement during gastrulation by Drosophila Sna. However, the expression of vertebrate Sna proteins in developing CNS has not been demonstrated. A careful examination of the expression and function in the CNS is needed to reveal the importance of Sna expression. The analysis of the functions of Sna, Esg and Wor in Drosophila CNS development will certainly provide a foundation for similar analysis in other species (Ashraf, 1999 and references therein).
Search PubMed for articles about Drosophila Worniu
Ashraf, S. I., et al. (1999). The mesoderm determinant Snail collaborates with related zinc-finger proteins to control Drosophila neurogenesis. EMBO J. 18: 6426-6438. PubMed Citation: 10562554
Ashraf, S. I. and Ip, Y. T. (2001). The Snail protein family regulates neuroblast expression of inscuteable and string, genes involved in asymmetry and cell division in Drosophila. Development 128: 4757-4767. 11731456
Ashraf, S. I., Ganguly, A., Roote, J. and Ip, Y. T. (2004). Worniu, a Snail family zinc-finger protein, is required for brain development in Drosophila. Dev Dyn 231: 379-386. PubMed ID: 15366015
Barrallo-Gimeno, A. and Nieto, M. A. (2005). The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development 132: 3151-3161. PubMed ID: 15983400
Cabernard, C. and Doe, C. Q. (2009). Apical/basal spindle orientation is required for neuroblast homeostasis and neuronal differentiation in Drosophila. Dev Cell 17: 134-141. PubMed ID: 19619498
Cai, Y., Chia, W. and Yang, X. (2001). A family of Snail-related zinc finger proteins regulates two distinct and parallel mechanisms that mediate Drosophila neuroblast asymmetric divisions. EMBO J. 20: 1704-1714. 11285234
Cano, A., Perez-Moreno, M. A., Rodrigo, I., Locascio, A., Blanco, M. J., del Barrio, M. G., Portillo, F. and Nieto, M. A. (2000). The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol 2: 76-83. PubMed ID: 10655586
Carney, T. D., Miller, M. R., Robinson, K. J., Bayraktar, O. A., Osterhout, J. A. and Doe, C. Q. (2012). Functional genomics identifies neural stem cell sub-type expression profiles and genes regulating neuroblast homeostasis. Dev Biol 361: 137-146. PubMed ID: 22061480
Chabu, C. and Doe, C. Q. (2008). Dap160/intersectin binds and activates aPKC to regulate cell polarity and cell cycle progression. Development 135: 2739-2746. PubMed ID: 18614576
Hemavathy, K., Hu, X., Ashraf, S. I., Small, S. J. and Ip, Y. T. (2004). The repressor function of snail is required for Drosophila gastrulation and is not replaceable by Escargot or Worniu. Dev Biol 269: 411-420. PubMed ID: 15110709
Lai, S. L., Miller, M. R., Robinson, K. J. and Doe, C. Q. (2012). The Snail family member Worniu is continuously required in neuroblasts to prevent Elav-induced premature differentiation. Dev Cell 23: 849-857. PubMed ID: 23079601
Lee, C. Y., Robinson, K. J. and Doe, C. Q. (2006). Lgl, Pins and aPKC regulate neuroblast self-renewal versus differentiation. Nature 439: 594-598. PubMed ID: 16357871
Lim, H. Y. and Tomlinson, A. (2006). Organization of the peripheral fly eye: the roles of Snail family transcription factors in peripheral retinal apoptosis. Development 133: 3529-3537. PubMed ID: 16914498
Miller, M. R., Robinson, K. J., Cleary, M. D. and Doe, C. Q. (2009). TU-tagging: cell type-specific RNA isolation from intact complex tissues. Nat Methods 6: 439-441. PubMed ID: 19430475
Nieto, M. A. (2011). The ins and outs of the epithelial to mesenchymal transition in health and disease. Annu Rev Cell Dev Biol 27: 347-376. PubMed ID: 21740232
date revised: 10 July 2013
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