Transient nuclear Prospero induces neural progenitor quiescence
Quiescent stem cells preserve the stem cell pool for activation following disease or injury . Moreover , cancer stem cells can enter remission during treatment , possibly using mechanisms similar to stem cell quiescence , and re-initiate proliferation months to years later . Thus , studying stem cell quiescence may help design clinical approaches to prevent tumor dormancy and cancer recurrence , as well as to aid in activating stem cells for tissue repair . Drosophila neuroblasts have served as a model system for identifying conserved signaling pathways that regulate stem cell proliferation and quiescence ( Ebens et al . , 1993; Datta , 1995; Britton and Edgar , 1998; Doe et al . , 1998; Egger et al . , 2008; Tsuji et al . , 2008; Sousa-Nunes et al . , 2010; Homem and Knoblich , 2012; Weng and Cohen , 2012 ) . Drosophila neuroblasts delaminate from neuroectoderm during early embryogenesis , and then they go through multiple rounds of asymmetric cell division before exiting cell cycle at the end of embryogenesis; upon cell cycle exit , neuroblasts can undergo apoptosis ( Karcavitch and Doe , 2005; Maurange and Gould , 2005; Ulvklo et al . , 2012 ) or enter quiescence ( Truman and Bate , 1988; Datta , 1995; Tsuji et al . , 2008; Chell and Brand , 2010; Sousa-Nunes et al . , 2011 ) . During neuroblast asymmetric division , the scaffolding protein Miranda ( Mira ) is partitioned into the differentiating daughter cell , called a ganglion mother cell ( GMC ) , and it carries at least three cargo proteins: the translational repressors Staufen and Brain tumor ( Brat ) and the transcriptional repressor Prospero ( Hirata et al . , 1995; Broadus and Doe , 1997; Ikeshima-Kataoka et al . , 1997; Broadus et al . , 1998; Schuldt et al . , 1998; Shen et al . , 1998; Bello et al . , 2006; Betschinger et al . , 2006; Lee et al . , 2006 ) . In the GMC , Mira appears to be degraded and its cargo is released into the cytoplasm ( Staufen , Brat ) or the nucleus ( Prospero ) . Prospero is an atypical homeodomain protein that directly binds and represses progenitor and cell cycle genes to initiate GMC and neuronal differentiation ( Li and Vaessin , 2000; Choksi et al . , 2006 ) ; the mammalian ortholog Prox1 has a similar role in repressing cell cycle gene expression ( Dyer , 2003; Foskolou et al . , 2013 ) . In addition , keeping high levels of Prospero out of the neuroblast nucleus is essential to prevent neuroblast differentiation ( Choksi et al . , 2006; Cabernard and Doe , 2009; Bayraktar et al . , 2010 ) . In this study , we show that the Prospero differentiation factor is transiently detected at low levels in the neuroblast nucleus just prior to entry into quiescence . We find that loss of Prospero prevents entry into quiescence , whereas a pulse of low level nuclear Prospero drives proliferating larval neuroblasts into quiescence . We propose a model in which low levels of Prospero repress all cell cycle and progenitor genes except deadpan , whereas high levels of Prospero additionally repress deadpan to allow neural differentiation .
To investigate the transition from a proliferating neuroblast to a quiescent neuroblast , we examined known proliferation/progenitor and differentiation marker expression during this transition . We hypothesized that loss of a progenitor marker or gain of a differentiation marker might induce neuroblast quiescence . As a model system we used the identified neuroblast 3–3 in the thoracic segments ( NB3-3T ) which reliably enters quiescence at embryonic stage 15 ( Tsuji et al . , 2008 ) . Moreover , neuroblast 3–3 in abdominal segments ( NB3-3A ) remains proliferative until the end of embryogenesis and provides an excellent internal control ( Tsuji et al . , 2008 ) . Previous work showed that the quiescent NB3-3T can be recognized by Deadpan , a basic helix-loop-helix transcription factor ( Zhu et al . , 2008; Chell and Brand , 2010 ) . The coiled-coil Miranda protein is also reported to mark quiescent neuroblasts ( Tsuji et al . , 2008 ) , which we confirm here ( Figure 1 ) , but note that mira transcription is down-regulated in quiescent neuroblasts based lack of mRNA in the stage 17 CNS in the BDGP in situ database ( Tomancak et al . , 2007 ) . Interestingly , we find that all other tested progenitor markers—Worniu , Asense , and Cyclin E ( CycE ) ( Brand et al . , 1993; Caldwell and Datta , 1998; Ashraf et al . , 2004; Lai et al . , 2012 ) —are not expressed in the quiescent NB3-3T ( Figure 1; data not shown ) , although they can be robustly detected in the proliferative NB3-3A ( Figure 1 ) . Similarly , larval quiescent neuroblasts lack expression of Worniu , Asense , CycE ( and Miranda ) , while retaining Deadpan protein and active deadpan transcription ( Figure 1—figure supplement 1 ) . However , misexpression of Worniu ( data not shown ) , Asense , or CycE ( Figure 1—figure supplement 2 ) has no effect on the timing of NB3-3T quiescence . 10 . 7554/eLife . 03363 . 003Figure 1 . Quiescent neuroblasts have a novel transcriptional profile lacking both progenitor and differentiation markers . ( A–F ) Proliferating NB3-3A contains nuclear Deadpan ( A ) , cytoplasmic Mira ( B ) , nuclear Asense ( C ) , nuclear Worniu ( D ) , and nuclear CycE ( E ) at embryonic stages 14 and 16; quantification is shown in ( F ) . Neuroblast lineages are marked by Eg-GFP ( Eg-gal4 UAS-mCD8:GFP ) , and the neuroblast is identified by Dpn ( shown in insets ) and indicated by arrows . Anterior side is up , and lateral is at right . Scale bar: 5 μm . ( G–L ) Proliferating NB3-3T expresses all above-mentioned neuroblast markers at stage 14 ( S14 ) . At stage 16 ( S16 ) , NB3-3T is quiescent and contains Deadpan ( G ) and Mira ( H ) but lacks Asense ( I ) , Worniu ( J ) and CycE ( K ) . Quantified in ( L ) . Scale bar: 5 μm . ( F and L ) Quantification; number of neuroblasts scored shown in bar . ( M ) Schematic summary of neuroblast marker profiles of proliferating neuroblast , quiescent neuroblast and neuron . DOI: http://dx . doi . org/10 . 7554/eLife . 03363 . 00310 . 7554/eLife . 03363 . 004Figure 1—figure supplement 1 . Larval quiescent neuroblasts are Deadpan-positive but lack the progenitor markers Miranda , Asense , Cyclin E , Worniu and the differentiation markers Prospero , Elav , and Repo . ( A ) The newly hatched larval ( NHL ) central brain contains five persistently proliferative neuroblasts ( the four mushroom body neuroblasts and the lateral antennal lobe neuroblast ) and ∼100 quiescent central brain neuroblasts in each lobe . ( B–E ) Proliferative neuroblasts in the NHL contain progenitor markers Deadpan ( Dpn ) , Asense ( Ase ) , and Worniu ( Wor ) but lack the differentiation markers Prospero ( Pros ) , Elav , and Repo and incorporate EdU ( top ) . Quiescent neuroblasts maintain Dpn , but lack the progenitor markers and the differentiation markers , and fail to incorporate EdU ( bottom ) . DOI: http://dx . doi . org/10 . 7554/eLife . 03363 . 00410 . 7554/eLife . 03363 . 005Figure 1—figure supplement 2 . Overexpressing Asense and Cyclin E does not change the timing of neuroblast quiescence . No significant change in the number of proliferating NB3-3T in stage 15 embryos with overexpressed Asense ( AseOXN ) or Cyclin E ( CycEOXN ) . Error bars , standard deviation; n . s . , not significant . Fly genotype: AseOXN , worniu-gal4 UAS-asense; CycEOXN , worniu-gal4 UAS-cyclin E . DOI: http://dx . doi . org/10 . 7554/eLife . 03363 . 005 We next turned to examining differentiation markers . We assayed nuclear Prospero , which marks differentiating GMCs and young neurons in the larval CNS ( Carney et al . , 2013 ) , Embryonic lethal and abnormal vision ( Elav ) , which marks all neurons ( Robinow and White , 1988 ) ; and Reversed polarity ( Repo ) , which marks all non-midline glia ( Xiong et al . , 1994 ) . We found that the mature quiescent NB3-3T did not express nuclear Prospero , Elav , or Repo ( data not shown ) , nor were these differentiation markers observed in quiescent larval brain neuroblasts ( Figure 1—figure supplement 1 ) . Interestingly , we did observe transient low level nuclear Prospero in NB3-3T just prior to its entry into quiescence ( see next section ) . We conclude that quiescent neuroblasts have a novel transcriptional profile that lacks both progenitor and differentiation markers . Here , we characterize further our observation that NB3-3T showed low level nuclear Prospero at stage 15 , concurrent with the entry into quiescence . Whereas NB3-3T showed a pulse of nuclear Prospero at stage 15 concurrent with entry into quiescence ( Figure 2A , B ) , NB3-3A did not show nuclear Prospero and did not enter quiescence ( Figure 2C , D ) . The levels of Prospero in the NB3-3T neuroblast are clearly detectable by immunofluorescent staining , but at significantly lower levels than in differentiating GMCs ( Figure 2B , arrow; Figure 2—figure supplement 1 ) . To probe the correlation of transient nuclear Prospero and neuroblast quiescence in more detail , we assayed temporal identity mutations that are known to shift the timing of NB3-3T quiescence: nubbin/pdm2 ( pdm ) mutants cause precocious NB3-3T quiescence , whereas castor ( cas ) mutants cause a delay in NB3-3T quiescence ( Tsuji et al . , 2008 ) . Strikingly , the pulse of nuclear Prospero precisely matched the timing of neuroblast quiescence: it occurred earlier in pdm mutants , and later in cas mutants ( Figure 2E , F ) . We conclude that a pulse of low level nuclear Prospero marks neuroblast entry into quiescence , suggesting a functional relationship . 10 . 7554/eLife . 03363 . 006Figure 2 . Transient low level nuclear Prospero is tightly correlated with neuroblast entry into quiescence . ( A–B ) NB3-3T shows transient nuclear Prospero ( Pros ) during entry into quiescence at stage 15 ( S15 ) ( B ) . Differentiated daughter cell shows strong nuclear Prospero and is indicated by the arrow . Neuroblast lineages are identified by Eg-GFP ( Eg-gal4 UAS-mCD8:GFP ) ( A ) , and the neuroblast is identified by Dpn and outlined by dashed lines . Summarized in schematic below ( Prospero , green ) ; quantified in ( E ) . Anterior side is up , and lateral is at right . Scale bar: 5 μm . ( C–D ) Proliferating NB3-3A does not contain nuclear Prospero . Summarized in schematic below; quantified in ( E ) . Scale bar: 5 μm . ( E ) Temporal identity factors schedule the timing of neuroblast entry into quiescence and the expression of nuclear Prospero . In cas mutant ( cas24 ) , neuroblast quiescence is delayed , as is the timing of nuclear Prospero . In the deficiency allele Df ( 2L ) ED773 which removes both nubbin/pdm2 ( pdm mutant ) , NB3-3T precociously enters quiescence , and the timing of nuclear Prospero is advanced . ( F ) Schematic of the timing of neuroblast entry into quiescence and the expression of nuclear Prospero . DOI: http://dx . doi . org/10 . 7554/eLife . 03363 . 00610 . 7554/eLife . 03363 . 007Figure 2—figure supplement 1 . Nuclear Prospero levels are lower in stage 15 NB3-3T than in differentiating GMCs . ( A ) Confocal images of a wild type NB3-3T ( blue circle , identified by Eg-GFP and Dpn ) and its adjacent GMC ( yellow circle , identified by Eg-GFP and its smaller size ) in stage 15 embryos . Scale bar: 5 μm . ( B ) Quantification of nuclear Prospero intensity in unit area in a neuroblast vs an adjacent GMC in stage 15 embryos . Prospero intensity in each confocal stack was measured with the open source software FIJI to obtain gray value and the nuclear area . The total Prospero intensity was summed from the confocal stacks , and then divided by nuclear volume to obtain the Prospero intensity per unit volume . Small triangles represent the intensity quantified from each individual neuroblast , and the large triangle is the average . Small circles represent the intensity quantified from each individual GMC , and the large circle is the average . DOI: http://dx . doi . org/10 . 7554/eLife . 03363 . 007 Having shown a strong correlation between the timing of nuclear Prospero and neuroblast entry into quiescence , we next asked if Prospero is required for neuroblast entry into quiescence and loss of nuclear Prospero can delay the timing of neuroblast quiescence . In theory , this can be done by quantifying the number of EdU+ neuroblasts in prospero null mutant embryos . The problem is that prospero mutants show de-differentiation of GMCs into proliferating neuroblasts ( Choksi et al . , 2006; Lee et al . , 2006 ) , making it necessary to distinguish the parental neuroblasts from the de-differentiated GMCs . To resolve the issue , we used the Notch signaling reporter , mγ-LacZ ( Wech et al . , 1999; Cooper et al . , 2000 ) , to identify the parental neuroblasts . During neuroblast asymmetric cell division , Miranda-Prospero and Partner of numb ( Pon ) -Numb protein complexes are independently segregated into the GMC to promote differentiation ( Doe , 2008 ) . In prospero mutants , Numb is still properly segregated to GMCs where it represses Notch activity ( Spana and Doe , 1995 ) and consequently the Notch reporter mγ-LacZ is restricted to parental neuroblasts . mγ-LacZ is transcribed only in neuroblasts; in wild type the protein perdures into neuroblast progeny , whereas in prospero mutants the protein is restricted to neuroblasts because the progeny proliferate without expressing mγ-LacZ and thus dilute out the protein ( Choksi et al . , 2006; Figure 3A , B ) . Thus , we scored for EdU incorporation in the mγ-LacZ+ parental neuroblasts . Whereas in wild type nearly all neuroblasts ceased EdU incorporation by stage 16 ( Figure 3A ) , many mγ-LacZ+ neuroblasts continued to incorporate EdU in prospero mutants at the same stage ( Figure 3B ) . This strongly supports a model in which Prospero is required for neuroblast entry into quiescence . In addition , Prospero is unique among basal cell fate determinants in regulating neuroblast quiescence: loss of function mutations in numb or brat , or the Prospero target gene string , showed no effect on the timing of neuroblast quiescence ( data not shown ) . 10 . 7554/eLife . 03363 . 008Figure 3 . Prospero is required for neuroblast quiescence . ( A–B ) Wild type and prospero mutant ( pros17 ) stage 16 embryos . Parental neuroblasts ( NB ) stain for Deadpan and mγ-LacZ , whereas ectopic ‘de-differentiated’ neuroblasts stain for Deadpan but not mγ-LacZ . Proliferating neuroblasts are marked by EdU incorporation . ( A ) In wild type , most thoracic neuroblasts are in quiescence and do not incorporate EdU . ( B ) In prospero mutants , most parental neuroblasts ( mγ-LacZ+ ) have not entered quiescence and still incorporate EdU . Ventral side is up and anterior is at left . Dashed line , boundary of neuroblast and neuron ( N ) layers . Scale bars: 20 μm . ( C–E ) NB3-3T remains proliferative in prospero mutant stage 16 embryos . ( C ) In wild type , NB3-3T is Mira+ mγ-LacZ+ EdU−; ( D ) in prospero mutants , NB3-3T is Mira+ mγ-LacZ+ EdU+ . The NB3-3T lineage was identified by Eg-GFP ( Eg-gal4 UAS-mCD8:GFP ) and the neuroblast is indicated by arrowheads . ( E ) Quantification . n . s . , not significant; ****p < 0 . 00001 . DOI: http://dx . doi . org/10 . 7554/eLife . 03363 . 008 To test further whether Prospero is required for neuroblast quiescence , we returned to the NB3-3T model system . We used mγ-LacZ , Miranda , and EdU incorporation to assay the timing of NB3-3T quiescence in wild type and prospero mutant embryos . In both wild type and prospero haplo-insufficiency embryos , the mγ-LacZ+ NB3-3T was proliferative at stage 14 and quiescent by stage 16 ( Figure 3C , quantified in Figure 3E ) . In prospero mutant embryos , the mγ-LacZ+ NB3-3T was proliferative at both stage 14 and 16 ( Figure 3D , quantified in Figure 3E ) . The results suggest that the level of Prospero required for neuroblast quiescence is lower than that in prospero haplo-insufficiency and that a low level of Prospero is sufficient to promote neuroblast quiescence ( see next section ) . We conclude that Prospero is required for neuroblast quiescence . The central brain neuroblasts are continuously proliferating during the third larval instar ( Ito and Hotta , 1992 ) , and so we used this stage to determine whether transient Prospero expression could induce neuroblast quiescence . We used the TARGET method ( McGuire et al . , 2003 ) to transiently induce Prospero expression for 2 hr , and then assayed for neuroblast quiescence 12 hr later ( Figure 4 ) . Because high levels of Prospero can induce neuroblast differentiation ( Figure 4—figure supplement 1 ) ( Choksi et al . , 2006; Cabernard and Doe , 2009; Bayraktar et al . , 2010 ) , we used Prospero levels low enough to minimize neuroblast differentiation , as shown by the persistence of most Deadpan+ neuroblasts after misexpression ( Figure 4D ) . We identified quiescent neuroblasts by their failure to incorporate EdU , lack of progenitor marker Worniu , and lack of the differentiation marker Elav—together a robust signature for quiescent neuroblasts ( see Figure 1 ) . In wild-type larval brains , we found few or no quiescent neuroblasts , as expected ( Figure 4E , F ) . In contrast , a pulse of Prospero resulted in the appearance of numerous quiescent neuroblasts ( Figure 4E , F; 12 hr timepoint ) . To rule out the possibility that these ‘quiescent neuroblasts’ were early in the differentiation pathway , we reasoned that if they were quiescent they could reactivate proliferation in a nourishing environment ( Chell and Brand , 2010; Sousa-Nunes et al . , 2010 ) whereas if they were differentiating they would never re-enter the cell cycle . Thus , we exposed larval neuroblasts to the same 2 hr pulse of Prospero , but waited 24 hr to assay neuroblasts . We found that nearly all of the neuroblasts that were quiescent at 12 hr after induction had reactivated proliferation and re-expressed progenitor markers ( Figure 4E , F; 24 hr timepoint ) . In addition , precocious low level nuclear Prospero could advance the timing of NB3-3T quiescence ( Figure 4—figure supplement 2 ) . We conclude that transient , low-level nuclear Prospero is sufficient to induce neuroblast quiescence . Interestingly , Prospero can also transiently arrest the cell cycle without inducing differentiation in embryonic longitudinal glial progenitors ( Griffiths and Hidalgo , 2004 ) , although the relative levels of Prospero have not been explored in this system . 10 . 7554/eLife . 03363 . 009Figure 4 . Prospero is sufficient to induce neuroblast quiescence . ( A–C ) Transient overexpression of nuclear Prospero in neuroblasts ( worniu-gal4 UAS-HA:prospero tub-gal80ts ) results in neuroblast quiescence ( A ) or differentiation ( B ) . Third-instar larvae were shifted from 22°C to 30°C to inactivate Gal80 , which resulted in nuclear Prospero in neuroblasts . Low levels of nuclear Prospero result in neuroblast quiescence ( Dpn+ Elav− ) ( A ) whereas high levels of nuclear Prospero suppress Deadpan and activate Elav to induce differentiation ( Dpn− Elav+ ) ( B ) . Prospero levels were quantified in ( C ) . Scale bars: 5 μm . ( C ) Quantification of Prospero levels in quiescent neuroblasts ( Dpn+ Elav− ) or differentiated neuroblasts ( Dpn− Elav+ ) . Differentiated neuroblasts were identified by their nuclear size larger than 7 μm in diameter . Prospero intensity was determined by totaling the gray value in nucleus of confocal stacks , followed by normalization to the total gray value of DNA marker 4ʹ , 6-diamidino-2-phenylindole ( DAPI ) . Number of neuroblasts quantified shown in bar . Error bars: standard deviation . ( D–F ) Larval neuroblast phenotype upon transient overexpression of Prospero . ( D ) Neuroblasts stay Deadpan+ ( Dpn+ ) and thus do not differentiate . ( E ) There is an increase of quiescent neuroblasts 12 hr after Prospero induction , as determined by the elevated percentage of EdU− ( D ) and Wor− ( E ) neuroblasts . The quiescent neuroblasts re-enter the cell cycle and express the progenitor marker Wor 24 hr after induction . Error bars: standard deviation . n . s . , not significant; *p < 0 . 05; **p < 0 . 01; ***p < 0 . 0001 . ( G ) Model . Prospero levels distinguish three progenitor fates: absent for self-renewal , low for quiescence , and high for differentiation . DOI: http://dx . doi . org/10 . 7554/eLife . 03363 . 00910 . 7554/eLife . 03363 . 010Figure 4—figure supplement 1 . Low nuclear Prospero induces neuroblast quiescence , whereas high Prospero induces neuroblast differentiation . ( A ) Two levels of Prospero were generated by increasing time of misexpression ( 4 or 12 hr ) with one copy of UAS-HA:prospero transgene . Whereas 2 hr can induce neuroblast quiescence ( Figure 4 ) , 4 hr and 12 hr can robustly induce neuroblast differentiation . ( B ) Two levels of Prospero were generated by misexpressing one vs two copies of the UAS-HA:prospero transgene for the same time ( 2 hr ) . A significant increase of the number of neuroblasts differentiated with two copies of UAS-HA:prospero transgene . DOI: http://dx . doi . org/10 . 7554/eLife . 03363 . 01010 . 7554/eLife . 03363 . 011Figure 4—figure supplement 2 . Prospero is sufficient to induce embryonic neuroblast quiescence . ( A–G ) Transient overexpression of nuclear Prospero in neuroblasts ( worniu-gal4 UAS-HA:prospero tub-gal80ts ) induces neuroblast cell cycle arrest . Embryos were shifted from 22°C to 30°C for 1 hr to inactivate Gal80 , which resulted in transient nuclear Prospero in NB3-3T at stage 14 ( A , wild type; B , transient nuclear Prospero ) . Scale bars: 2 μm . ( C–G ) Quantification of the Eg+ NB3-3T phenotype upon transient overexpression of Prospero . ( C ) Some neuroblasts have detectable nuclear Prospero . ( D ) Most neuroblasts fail to incorporate EdU . ( E–G ) There is a slight loss of Worniu+ ( E ) , Asense+ ( F ) and CycE+ ( G ) neuroblasts . DOI: http://dx . doi . org/10 . 7554/eLife . 03363 . 011 Our results confirm that high levels of Prospero can trigger differentiation and reveal that low levels of Prospero induce quiescence in the Drosophila neuroblasts . High levels of the vertebrate ortholog Prox1 can also trigger differentiation ( Dyer , 2003; Takahashi et al . , 2006; Foskolou et al . , 2013 ) ; thus it is interesting that low levels of nuclear Prospero promote neuroblast quiescence without triggering differentiation ( Figure 4—figure supplement 1 ) . Prox1 activates some target genes at high levels but represses some target genes at low levels ( Chen et al . , 2008a , 2008b ) . We suggest a speculative model for Prospero function: low levels of Prospero are sufficient to repress most progenitor-specific genes in neuroblasts ( worniu , asense , miranda , and cycE ) but do not repress the expression of deadpan ( perhaps due to lack of high affinity Prospero binding sites ) . In contrast , high levels of Prospero are sufficient to repress deadpan ( perhaps via low affinity binding sites ) and may even activate neuronal differentiation genes ( Figure 4G ) . In embryonic neuroblasts , the temporal transcription factors Pdm and Cas schedule the timing of neuroblast quiescence , but how they regulate Prospero nuclear import is unknown . In larval neuroblasts , Grh prevents accumulation of nuclear Prospero which would induce neuroblast cell cycle exit and differentiation ( Maurange et al . , 2008; Chai et al . , 2013 ) , again by an unknown mechanism . The protein Caliban is known to promote Prospero nuclear export in S2 cells ( Bi et al . , 2005 ) , but it is unknown whether Caliban has a similar role in neuroblasts or if down-regulation of Caliban levels or activity leads to increased nuclear Prospero in neuroblasts entering quiescence .
The following flies were used in this study: ( 1 ) UAS-mCD8:Gfp;eagle-gal4; ( 2 ) cas24/TM3 Sb Ubx-lacZ; ( 3 ) Df ( 2L ) ED773/CyO wg-lacZ; ( 4 ) mγ-lacZ;UAS-mCD8:Gfp; eg-gal4 pros17/TM3 Sb Ubx-lacZ; ( 5 ) UAS-HA:prospero ( attP40 ) ; ( 5 ) UAS-HA:prospero ( attP2 ) ; ( 6 ) wor-gal4;tubulin-gal80ts . Embryos with genotype UAS-HA:prospero/wor-gal4;tubulin-gal80ts/+ were collected for 4 hr at 22°C , and then cultured at 22°C for 5 days . The larvae were transferred to 30°C to inactivate Gal80 , which resulted in the activation of Gal4 to express HA:Prospero . After heat shock , the larvae were immediately dissected or moved back to 22°C to incubate for 12 or 24 hr for later dissection , followed by 2-hr EdU incorporation , fixation , and antibody staining . EdU incorporation was performed in PBS at the concentration of 200 μg/ml . Antibody staining and EdU detection were previously described ( Lai et al . , 2012 ) . Antibodies used in this study included: rabbit anti-Asense ( 1:1000; Cheng-Yu Lee , University of Michigan , Ann Arbor , MI ) , mouse anti-β-galactosidase ( 1:1000; Promega , Madison , WA ) , mouse anti-Cyclin E ( 1:50; Developmental Studies Hybridoma Bank ( DHSB ) developed under NICHD and maintained by University of Iowa ) , rabbit anti-Cyclin E ( 1:300; Santa Cruz Biotechnology , Dallas , TX ) ; rat anti-Deadpan monoclonal ( 1:50; Doe lab ) , guinea pig anti-Deadpan ( 1:1000; Jim Skeath , Washington Univ . , St . Louis , MO ) , mouse anti-Eagle ( 1:100; Doe lab ) ; rabbit anti-Eagle ( 1:500 ) ( Higashijima et al . , 1996; Freeman and Doe , 2001 ) ; rat anti-Elav ( 1:50 , DSHB ) ; chicken anti-GFP ( 1:500; Aves Labs , Tigard , OR ) , mouse anti-HA ( 1:1000; Covance , Princeton , NJ ) , chicken anti-HA ( 1:1000 , Bethyl Laboratories , Montgomery , TX ) ; guinea pig anti-Miranda ( 1:2000; Doe lab ) , mouse anti-Prospero monoclonal purified IgG ( 1:1000; Doe lab ) , and rat anti-Worniu ( 1:2; Doe lab ) . Microscopy was done using a Zeiss LSM700 or LSM710; image processing and quantification were performed with the open source software FIJI .
Stem cells can self-renew , differentiate , or enter quiescence . Understanding how stem cells switch between these states is highly relevant for stem cell-based therapeutics . Drosophila neural progenitors ( neuroblasts ) have been an excellent model for studying self-renewal and differentiation , but quiescence remains poorly understood . In this study , we show that when neuroblasts enter quiescence , the differentiation factor Prospero is transiently detected in the neuroblast nucleus , followed by the establishment of a unique molecular profile lacking most progenitor and differentiation markers . The pulse of low level nuclear Prospero precedes entry into neuroblast quiescence even when the timing of quiescence is advanced or delayed by changing temporal identity factors . Furthermore , loss of Prospero prevents entry into quiescence , whereas a pulse of low level nuclear Prospero can drive proliferating larval neuroblasts into quiescence . We propose that Prospero levels distinguish three progenitor fates: absent for self-renewal , low for quiescence , and high for differentiation .
Stem cells provide tissues in the body with a continuing source of new cells , both when the tissues are first developing and when they are growing or repairing in adulthood . A stem cell can divide to create either another stem cell , or a cell that will mature into one of many different cell types . Neuroblasts are a type of brain stem cell and can divide to create two new cells: another neuroblast that will continue to replicate itself and a cell called a ganglion mother cell that will go on to produce two mature cells for the nervous system . Moreover , when a neuroblast divides , it splits unequally , so that certain molecules end up predominantly in the ganglion mother cell—including a protein called Prospero . Once partitioned into the ganglion mother cell , the Prospero protein enters the nucleus , where it switches off ‘stem cell genes’ and switches on ‘neuron genes’ so the ganglion mother cell can form the mature neurons of the brain . Thus , neuroblasts must keep Prospero out of the nucleus to maintain stem cell properties , whereas ganglion mother cells must move Prospero into the nucleus to form neurons . Now , Lai and Doe discover a new way that the Prospero protein is used to control stem cell biology . Neuroblasts , like all stem cells , can enter periods where they go dormant or quiescent—that is , they temporarily stop generating ganglion mother cells . By analyzing which proteins are present in neuroblasts during this transition to quiescence , Lai and Doe discovered that the Prospero protein was briefly detected , at low levels , in the nucleus of the neuroblast just before it became dormant . To see whether this ‘low-level pulse’ of nuclear Prospero is linked to the cell entering a dormant state , Lai and Doe investigated two types of mutant fly in which neuroblasts become dormant either earlier or later than in normal flies . A low-level pulse of nuclear Prospero still precisely matched the start of the dormant state in both mutants . When the Prospero protein was removed altogether , the neuroblasts failed to become dormant , and instead continued dividing . Lai and Doe propose that different levels of Prospero distinguish three different fates for neuroblasts . Neuroblasts self-replicate when Prospero is kept out of the nucleus , become dormant when exposed to low level nuclear Prospero , and produce the mature cells of the brain when nuclear Prospero levels are high . Exactly how the intermediate levels of nuclear Prospero trigger the dormant state remains a question for future work , as is the question of whether the related mammalian protein , called Prox1 , has a similar function . Understanding how stem cells switch between cell division and quiescence is important for developing effective stem cell-based therapies . It could also help us understand cancer , as cancer cells go through similar periods of inactivity , during which they do not respond to many anti-tumor drugs .