InteractiveFly: GeneBrief

earmuff: Biological Overview | References

BIOLOGICAL OVERVIEW

To ensure normal development and maintenance of homeostasis, the extensive developmental potential of stem cells must be functionally distinguished from the limited developmental potential of transit amplifying cells. Yet the mechanisms that restrict the developmental potential of transit amplifying cells are poorly understood. This study shows that the evolutionarily conserved transcription factor dFezf/Earmuff (Erm) functions cell-autonomously to maintain the restricted developmental potential of the intermediate neural progenitors generated by type II neuroblasts in Drosophila larval brains. Although erm mutant intermediate neural progenitors are correctly specified and show normal apical-basal cortical polarity, they can dedifferentiate back into a neuroblast state, functionally indistinguishable from normal type II neuroblasts. Erm restricts the potential of intermediate neural progenitors by activating Prospero to limit proliferation and by antagonizing Notch signaling to prevent dedifferentiation. It is concluded that Erm dependence functionally distinguishes intermediate neural progenitors from neuroblasts in the Drosophila larval brain, balancing neurogenesis with stem cell maintenance (Weng, 2010).

Tissue development and homeostasis often require stem cells to transiently expand the progenitor pool by producing transit amplifying cells. Yet the developmental potential of transit amplifying cells must be tightly restricted to ensure generation of differentiated progeny and to prevent unrestrained proliferation that might lead to tumorigenesis. Transit amplifying cells are defined by their limited developmental capacity, a feature specified during fate determination. It is unknown whether an active mechanism is required to maintain restricted developmental potential in transit amplifying cells after specification. This study used intermediate neural progenitors (INPs) in developing Drosophila larval brains as a genetic model to investigate how restricted developmental potential is regulated in transit amplifying cells (Weng, 2010).

A fly larval brain hemisphere contains eight type II neuroblasts that undergo repeated asymmetric divisions to self-renew and to generate immature INPs (Bello, 2008; Boone, 2008; Bowman, 2008). Immature INPs are unstable in nature and are mitotically inactive, and they lack the expression of Deadpan (Dpn) and Asense (Ase). Immature INPs commit to the INP fate through maturation, a differentiation process necessary for specification of the INP identity. INPs express Dpn and Ase, and undergo 8-10 rounds of asymmetric divisions to self-renew and to produce ganglion mother cells (GMCs) that typically generate two neurons. While 5-6 immature INPs and 1-2 young INPs are always in direct contact with their parental neuroblasts, the older INPs become progressively displaced from their parental neuroblasts over time (Weng, 2010).

During asymmetric divisions of type II neuroblasts, the basal proteins Brain tumor and Numb are exclusively segregated into immature INPs, and function cooperatively, but nonredundantly, to ensure that immature INPs undergo maturation and commit to the INP fate. brain tumor or numb mutant type II neuroblasts generate immature INPs that fail to mature and do not commit to the INP fate. Instead, brain tumor or numb mutant immature INPs adopt their parental neuroblast fate, leading to supernumerary type II neuroblasts. Thus, brain tumor and numb specify the INP fate, and the ectopic expansion of type II neuroblasts in these mutant genetic backgrounds occurs due to failure to properly specify the INP fate. Although Brain tumor is also asymmetrically segregated into GMCs during asymmetric divisions of INPs, the mosaic clones in brain tumor mutant INPs contain only differentiated neurons. This result indicates that Brain tumor is dispensable for maintaining the restricted developmental potential of INPs. How restricted developmental potential is maintained in INPs is currently unknown (Weng, 2010).

To identify genes that regulate self-renewal of neuroblasts, a genetic screen was conducted for mutants exhibiting ectopic larval brain neuroblasts. One mutation, l(2)5138, specifically resulted in massive expansion of neuroblasts in the brain but did not affect neuroblasts on the ventral nerve cord. The l(2)5138 mutation mapped to the 22B4-7 chromosomal interval that contains the earmuff (erm) gene (Pfeiffer, 2008). The erm transcripts are first detected at embryonic stage 4-6 in the specific domain preceding formation of the embryonic brain and remain highly expressed in the brain throughout development (Chintapalli, 2007; Pfeiffer, 2008). Tbis study reports that Erm functions to restrict the developmental potential of INPs by promoting Prospero-dependent termination of proliferation and suppressing Notch-mediated dedifferentiation. By restricting their developmental potential, Erm ensures that INPs generate only differentiated neurons during Drosophila neurogenesis (Weng, 2010).

All neuroblasts in l(2)5138 homozygous mutant brains were proliferative, expressed all known neuroblast markers, and lacked neuronal and glial markers. The l(2)5138 mutation mapped to the erm gene, which encodes a homolog of the vertebrate Forebrain embryonic zinc-finger family (Fezf) transcription factors (Hashimoto, 2000; Matsuo-Takasaki, 2000). The l(2)5138 mutants contained a single A/T nucleotide change in the erm coding region, leading to the substitution of a leucine for a conserved histidine in the third C2H2 zinc-finger domain. Consistent with its predicted molecular function, ectopic expression of Erm transgenic proteins tagged with a HA epitope at the amino- or carboxyl-terminus driven by neuroblast-specific Wor-Gal4 was detected in the nuclei of neuroblasts. However, the expression of the HA-tagged Erm transgenic protein bearing the identical leucine-to-histidine substitution as in the l(2)5138 mutant was undetectable, suggesting that the mutant Erm protein is unstable. It is concluded that l(2)5138 is a mutant allele of erm (Weng, 2010).

To determine whether erm mutant brains have ectopic type I and/or type II neuroblasts, the expression pattern was examined of Ase and Prospero (Pros), which are only expressed in type I neuroblasts (Bello, 2008; Boone, 2008; Bowman, 2008). It was found that erm mutant brains contained over 20-fold more type II neuroblasts (Dpn⁺Ase^-) than wild-type brains, with no significant change in the number of type I neuroblasts (Dpn⁺Ase⁺). Next, the localization of Prospero was examined in mitotic neuroblasts in larval brains expressing GFP induced by Ase-Gal4 (Ase > GFP), which mimicked the expression pattern of the endogenous Ase protein (Bowman, 2008). In erm mutant larval brains, all mitotic type I neuroblasts (GFP⁺) showed formation of basal Prospero crescents, but none of the mitotic type II neuroblasts (GFP^-) showed the expression of Prospero. Furthermore, GFP-marked erm mutant type II neuroblast clones consistently contained multiple type II neuroblasts, whereas erm mutant type I neuroblast clones always contained single type I neuroblasts and neurons. It is concluded that erm mutant brains exhibit an abnormal expansion of type II neuroblasts (Weng, 2010).

To determine the cellular origin of ectopic type II neuroblasts in erm mutant brains, the identity of cells in the GFP-marked clones derived from wild-type or erm mutant type II neuroblasts was examined using specific cell fate markers. At 30 hr after clone induction, wild-type and erm mutant neuroblast clones appeared indistinguishable, containing single parental neuroblasts (Dpn⁺Ase^-; R10 mm) in direct contact with 5-6 immature INPs (Dpn^-Ase^-), while most of the INPs (Dpn+Ase+; R6 mm) were 1 cell or more away from the parental neuroblasts. At 48 hr after clone induction, the overall size of both wild-type and erm mutant neuroblast clones increased significantly due to an increase in cell number, reflecting continuous asymmetric divisions of the parental neuroblasts. In both wildtype and erm mutant clones, the parental neuroblasts remained surrounded by 5-6 immature INPs, while INPs and differentiated neurons (Dpn^-Ase^-Pros+) were found several cells away from the parental neuroblasts. However, erm mutant clones contained fewer INPs than the wild-type clones. Importantly, erm mutant clones consistently contained 4-6 smaller ectopic type II neuroblasts (Dpn⁺Ase^-; 6-8 mm in diameter). Thus, Erm is dispensable for both the generation and maturation of immature INPs (Weng, 2010).

Ectopic type II neuroblasts in 48 hr erm mutant clones were always several cells away from the parental neuroblasts. This result strongly suggests that ectopic type II neuroblasts in erm mutant clones likely originate from INPs and Erm likely functions in INPs. However, it was not possible to assess the spatial expression pattern of the endogenous Erm protein in larval brains due to lack of a specific antibody and low signals by fluorescent RNA in situ. Alternatively, the expression of the R9D series of Gal4 transgenes was analyzed, in which Gal4 is expressed under the control of overlapping erm promoter fragments (Pfeiffer, 2008). The expression of R9D11-Gal4 was clearly detected in INPs, but was undetectable in type II neuroblasts and immature INPs even when two copies of the UAS-mCD8-GFP transgenes were driven by two copies of R9D11-Gal4 at 32°C for 72 hr after larval hatching. Consistently, the expression of Erm-Gal4 was virtually undetectable in brain tumor mutant brains that contain thousands of type II neuroblasts and immature INPs. While the expression of UAS-erm induced by the neuroblast-specific Wor-Gal4 driver led to premature loss of type II neuroblasts, expression of UAS-erm driven by Erm-Gal4 failed to exert any effect on type II neuroblasts. Importantly, targeted expression of the fly Erm or mouse Fezf1 or Fezf2 transgenic protein driven by R9D11-Gal4 restored the function of Erm and efficiently rescued the ectopic neuroblast phenotype in erm mutant brains. Therefore, R9D11-Gal4 (Erm-Gal4) contains the enhancer element sufficient to restore the Erm function in INPs leading to suppression of ectopic type II neuroblasts in erm mutant brains (Weng, 2010).

Mutant clonal analyses and overexpression studies strongly suggest that Erm functions to suppress reversion of INPs back into a neuroblast state. This study directly tested whether INPs in erm mutant brains can dedifferentiate back into type II neuroblasts. βgal-marked lineage clones originating exclusively from INPs were induced via FRT-mediated recombination. A short pulse of flipase (FLP) expression was targeted in INPs by heat-shocking larvae carrying a UAS-flp transgene under the control of Erm- Gal4 and tub-Gal80ts at 30°C for 1 hr. At 72 hr after heat shock, INP clones in wildtype brains contained only differentiated neurons (Dpn^-Ase^-). In contrast, INP clones in erm mutant brains contained one or more type II neuroblasts as well as immature INPs, INPs, GMCs, and neurons. This result indicates that while INPs in wild-type larval brains can give rise to only neurons, INPs in erm mutant brains can dedifferentiate into type II neuroblasts that can give rise to all cell types found in a normal type II neuroblast lineage. It is concluded that Erm functions to maintain the restricted developmental potential of INPs and prevents them from dedifferentiating back into a neuroblast state (Weng, 2010).

To determine how Erm maintains the restricted developmental potential of INPs, microarray analyses was performed, and prospero mRNA was found to be drastically reduced in erm mutant brains compared to the control brains. It was confirmed that the relative level of prospero mRNA was indeed reduced by 60%-70% in erm mutant brain extracts by using real-time PCR. These data supported that Erm is necessary for proper transcription of prospero, and prompted a test to see if overexpression of Erm might be sufficient to induce ectopic Prospero expression. A short pulse of Erm expression in brain neuroblasts was induced by shifting larvae carrying a UAS-erm transgene under the control of Wor- Gal4 and tub-Gal80ts to from 25°C to 30°C. A 3.5 hr pulse of Erm expression was sufficient to induce nuclear localization of Prospero in larval brain neuroblasts. Consistent with nuclear Prospero promoting termination of neuroblast proliferation, ectopic expression of Erm induced by Wor-Gal4 resulted in decreased neuroblasts compared to wild-type brains. Thus, it is concluded that overexpression of Erm can restrict neuroblast proliferation by triggering nuclear localization of Pros (Weng, 2010).

The data suggest that Erm might restrict the developmental potential of INPs in part by limiting their proliferation by activating Prospero-dependent cell cycle exit. If so, it was predicted that overexpression of Erm should induce ectopic nuclear Prospero in INPs and overexpression of Prospero should suppress ectopic neuroblasts in erm mutant brains. In wild-type brains, 9.6% of INPs (32/325) showed nuclear localization of Prospero. However, overexpression of Erm driven by Erm-Gal4 led to nuclear localization of Prospero in 41.5% of INPs (105/253), likely restricting their proliferation potential and resulting in some parental type II neuroblasts surrounded only by differentiated neurons. Importantly, ectopic expression of Prospero induced by Erm-Gal4 efficiently suppressed ectopic neuroblasts and restored neuronal differentiation in erm mutant brains. Thus, Erm likely restricts the proliferation of INPs by promoting nuclear localization of Prospero. To confirm that Prospero indeed functions downstream of Erm to restrict the proliferation of INPs, genetic epistatic analyses were performed. Consistent with previously published results, prospero mutant type I neuroblast clones contained ectopic type I neuroblasts. In contrast, prospero mutant type II neuroblast clones exhibited accumulation of ectopic INPs while maintaining single parental neuroblasts. Furthermore, overexpression of Erm failed to suppress ectopic INPs in prospero mutant type II neuroblast clones, consistent with Prospero functioning downstream of Erm. These results indicate that blocking differentiation is not sufficient to trigger the dedifferentiation of INPs back into type II neuroblasts. Thus, Erm’s restriction on the proliferation of INPs is dependent on Prospero function, but its suppression of the dedifferentiation of INPs is independent of Prospero (Weng, 2010).

Previous studies showed that overexpression of constitutively active Notch (Notchintra) in both type I and II neuroblasts is sufficient to trigger ectopic neuroblasts. This study tested whether Erm suppresses the dedifferentiation of INPs by inhibiting Notch signaling. Indeed, knockdown of Notch function by RNAi in erm mutant brains led to a dramatic reduction in ectopic type II neuroblasts compared to erm mutant brains alone. Complementarily, ectopic expression of constitutively active Notch (Notchintra) induced by Erm-Gal4 transforms INPs into ectopic type II neuroblasts. Thus, reduced Notch function suppresses the dedifferentiation of INPs in erm mutant brains whereas ectopic activation of Notch induces the dedifferentiation of INPs. Next, whether Erm suppresses the dedifferentiation of INPs by antagonizing a Notch-activated mechanism was tested. Coexpression of Erm under the control of Erm-Gal4 is sufficient to suppress ectopic neuroblasts induced by the expression of Notchintra. Thus, it is concluded that Erm can suppress the dedifferentiation of INPs by negatively regulating a Notch-activated signaling mechanism (Weng, 2010).

This study has reported a mechanism that actively maintains the restricted developmental potential of transit amplifying cells after specification of their identity. The evolutionarily conserved transcription factor Erm/Fezf functions to maintain the restricted developmental potential of INPs by limiting their proliferation potential and suppressing their dedifferentiation capacity. Combining proper specification of the transit amplifying cell identity and active maintenance of their restricted developmental potential ensures the generation of differentiated progeny and prevents aberrant expansion of stem cells (Weng, 2010).

The lineage clones derived from single INPs in erm1/erm2 mutant brains contain dedifferentiated neuroblasts, immature INPs, INPs, GMCs, and neurons. Several mechanisms could lead to the diversity of cells within the clones. First, INPs in erm mutant brains might generate GMCs and neurons initially due to the presence of maternally deposited Erm. However, erm transcripts are undetectable in both adult male and female germlines by microarray analyses and in stage 1-3 embryos by RNA in situ. Furthermore, the erm1/erm2 allelic combination resulted in little to no zygotic Erm in the brain because the erm1 mutation likely leads to the production of an unstable Erm protein, whereas the erm2 mutation deletes the entire erm open reading frame. Additionally, the ectopic neuroblast phenotype in erm1/erm2 mutant brains can be observed as early as 36-48 hr after larval hatching. Thus, generation of GMCs and differentiated neurons by INPs in erm1/erm2 mutant brains is unlikely due to the maternal effect. Alternatively, erm may promote GMC differentiation in the type II neuroblast lineage, and in erm mutant brains, GMCs might dedifferentiate back into neuroblasts. If so, an ectopic accumulation of INPs would be predicted in similarly staged mosaic clones derived from erm mutant type II neuroblasts as compared to wild-type clones. However, 48 hr erm mutant single neuroblast clones consistently contained fewer INPs when compared to the wild-type clones. In addition, blocking GMC differentiation by removing Prospero function resulted in ectopic accumulation of INPs but did not lead to ectopic neuroblast formation. Therefore, the diversity of cells within erm mutant clones is also unlikely due to blocking GMC differentiation. The interpretation is favored that erm mutant INPs dedifferentiate into apparently normal neuroblasts that can give rise to all cell types found in a type II neuroblast lineage. Consistently, the dedifferentiated neuroblasts in erm mutant brains exhibited normal cortical polarity and proliferation potential. Furthermore, the dedifferentiated neuroblasts in erm mutant brains also lost the expression of Pros-Gal4 and Erm-Gal4 and established ectopic type II neuroblast lineages encapsulated by the cortex glial membrane.Thus, it is concluded that Erm likely restricts the developmental potential of INPs by limiting proliferation and suppressing dedifferentiation (Weng, 2010).

Although mutations in erm, brain tumor, and numb genes all lead to ectopic type II neuroblasts, the proteins appear to regulate INPs at distinct steps in the type II neuroblast lineage. Numb and Brain tumor function cooperatively, but nonredundantly, to ensure that immature INPs undergo maturation and commit to the INP fate (Boone, 2008; Bowman, 2008). While ectopic expression of Numb induces premature differentiation of type II neuroblasts and immature INPs, overexpression of Numb is not sufficient to suppress ectopic neuroblasts in brain tumor mutant brains. Thus, Numb likely promotes differentiation of immature INPs whereas Brain tumor likely prevents immature INPs, which are unstable in nature, from adopting their parental neuroblast fate. More studies will be necessary to discern whether ectopic neuroblasts in brain tumor mutant brains arise from dedifferentiation of partially differentiated immature INPs or failure of immature INPs to initiate differentiation. In contrast, immature INPs in erm mutant brains mature into functional INPs that exhibit normal cortical polarity and proliferation potential and can generate GMCs and neurons. Additionally, overexpression of Brain tumor or Numb in INPs was not sufficient to suppress ectopic neuroblasts in erm mutant brains. Finally, lineage clones derived from single INPs in erm mutant brains always contain ectopic type II neuroblasts, multiple immature INPs, INPs, GMCs, and neurons. These results indicate that Erm is dispensable for maturation of immature INPs and is not within the genetic hierarchy specifying the INP identity. Instead, Erm maintains the restricted developmental potential of INPs after specification of their identity (Weng, 2010).

Prospero encodes a homeodomain transcription factor, and nuclear Prospero has been shown to trigger cell cycle exit and GMC differentiation. In the wild-type brain, 9.6% of INPs showed nuclear Prospero and were likely undergoing differentiation. prospero mutant type II neuroblast clones showed ectopic accumulation of INPs but contained single neuroblasts, indicating that blocking differentiation is not sufficient to trigger the dedifferentiation of INPs. Thus, Prospero restricts the proliferation potential of INPs but does not suppress dedifferentiation of INPs (Weng, 2010).

While ectopic expression of Prospero in INPs can restore neuronal differentiation in erm mutant brains, targeted expression of Erm in neuroblasts or INPs was sufficient to induce rapid nuclear localization of Prospero in these cells and terminate their proliferation. In wild-type brains, Prospero is sequestered in a basal crescent by the adaptor protein Miranda in mitotic neural progenitors. Interestingly, mitotic neural progenitors including neuroblasts and INPs transiently overexpressing Erm also showed basal localization and segregation of Miranda and Prospero. As such, Erm likely restricts the proliferation potential of INPs by indirectly promoting nuclear localization of Prospero. Therefore, Prospero does not localize in the nuclei of mitotically active INPs, which express Miranda, but does localize in the nuclei of GMCs that do not express Miranda (Weng, 2010).

How does Erm suppress the dedifferentiation of INPs? The results show that reduced Notch function can efficiently suppress ectopic neuroblasts in erm mutant brains while constitutive activation of Notch signaling induced the dedifferentiation of INPs. Importantly, coexpression of Erm is sufficient to suppress the dedifferentiation of INPs triggered by expression of constitutively active Notchintra. Together, these results strongly suggest that Erm prevents the dedifferentiation of INPs by antagonizing a Notch-activated mechanism through interfering with the assembly of the Notch transcriptional activator complex or inhibiting the expression of Notch targets. Intriguingly, the amino terminus of all Fezf proteins contains an engrailed homology 1 domain. This domain can mediate direct interaction with the conserved transcriptional corepressor Groucho that can function as a corepressor of Notch signaling. Additional experiments will be needed to discern how Erm antagonizes Notch-activated dedifferentiation of INPs (Weng, 2010).

bHLH-O proteins balance the self-renewal and differentiation of Drosophila neural stem cells by regulating Earmuff expression

Balancing self-renewal and differentiation of stem cells requires differential expression of self-renewing factors in two daughter cells generated from the asymmetric division of the stem cells. In Drosophila type II neural stem cell (or neuroblast, NB) lineages, the expression of the basic helix-loop-helix-Orange (bHLH-O) family proteins, including Deadpan (Dpn) and E(spl) proteins, is required for maintaining the self-renewal and identity of type II NBs, whereas the absence of these self-renewing factors is essential for the differentiation of intermediate neural progenitors (INPs) generated from type II NBs. This study demonstrates that Dpn maintains type II NBs by suppressing the expression of Earmuff (Erm). Evidence is provided that Dpn and E(spl) proteins suppress Erm by directly binding to C-sites and N-boxes in the cis-regulatory region of erm. Conversely, the absence of bHLH-O proteins in INPs allows activation of erm and Erm-mediated maturation of INPs. The results further suggest that Pointed P1 (PntP1) mediates the dedifferentiation of INPs resulting from the loss of Erm or overexpression of Dpn or E(spl) proteins. Taken together, these findings reveal mechanisms underlying the regulation of the maintenance of type II NBs and differentiation of INPs through the differential expression of bHLH-O family proteins (Li, 2017).

This study demonstrates that similar to the canonical Notch signaling, Dpn maintains the identity and self-renewal of type II NBs at least in part by inhibiting Erm expression. Loss of Dpn leads to the ectopic activation of erm in type II NBs and that removing Erm not only prevents the transformation of dpn mutant or Dpn knockdown type II NBs into type I-like NBs but also largely inhibits their premature termination of self-renewal. The results from gel-shift assays and reporter assays provide evidence to support that Dpn and E(spl) proteins suppress Erm expression by directly binding to at least two of the three putative bHLH-O binding sites in the erm enhancer (Li, 2017).

Although Dpn and canonical Notch signaling could function through a similar mechanism, these factors do not appear to be completely functionally redundant as previously suggested. First, during early 1st instar larval stages when type II NBs are still quiescent, the maintenance of type II NBs may mainly rely on Dpn in that Notch is not activated in quiescent type II NBs, as evidenced through the findings showing that the loss of Dpn at early 1st instar larval stages leads to ectopic Erm-mediated transformation and the premature loss of type II NBs. Second, after reactivation of type II NBs, both Dpn and Notch signaling are required to suppress the ectopic Erm expression in type II NBs because both the loss of Dpn and the components of the canonical Notch signaling pathways alone lead to ectopic Erm expression in type II NBs. However, the Notch signaling likely plays a dominant role in suppressing ectopic Erm expression and maintaining type II NBs. It has been previously shown that the loss of components of the canonical Notch pathway, including E(spl) proteins, leads to ectopic Erm expression and the transformation and premature loss of type II NBs, despite the presence of Dpn in the NBs, whereas the knockdown of Dpn after the reactivation of NBs only results in the weak ectopic activation of erm but not transformation or premature loss of type II NBs. Therefore, Dpn and Notch signaling may not be completely functionally redundant in suppressing the ectopic Erm expression or maintaining type II NBs, and their functions might be dependent on developmental stages. Furthermore, a recent study reported that Klu could also bind to the R9D11 enhancer to repress the expression of Erm. Thus, type II NBs likely utilize multiple mechanisms to ensure that erm will not be prematurely activated (Li, 2017).

Previous studies suggested that all E(spl) proteins share similar DNA sequences. However, results from the present study suggest that this similarity may not always be the case. Gel-shift assays show that only members of the E(spl) family, including E(spl)mγ, mβ, mδ, m3, and m7, can bind to the bHLH-O binding sites in the erm regulatory region, whereas the other two, E(spl)m5 and m8, cannot. The difference in their DNA binding specificity is consistent with differences in the amino acid sequences of their bHLH domains and their overexpression phenotypes in type II NB lineages. Therefore, although multiple E(spl) proteins have been shown to be expressed in larval NBs and at least two of them, E(spl)mγ and m8, are activated by Notch, these E(spl) proteins may bind to different DNA sequences and regulate the expression of different target genes, which may in turn determine their functional specificity (Li, 2017).

In contrast to the maintenance of type II NBs, the maturation of imINPs requires the activation of erm by PntP1 and shutdown of Dpn expression and Notch signaling. It has previously been shown that the loss of Erm or aberrant activation of dpn or Notch signaling in imINPs both lead to the dedifferentiation of imINPs and overproliferation of type II NBs. However, the functional relationship between the activation of erm and the absence of Dpn or Notch signaling in imINPs has never been established. This study demonstrates that the absence of Dpn and Notch signaling is essential for the activation of erm and subsequent Erm-mediated maturation of INPs. First, the results show that aberrant activation of dpn or Notch signaling inhibits the activation of erm in imINPs. Second, maintaining Erm expression in imINPs largely blocks the overproliferation of type II NBs resulting from the misexpression of E(spl) or Dpn proteins, suggesting that one main reason for the dedifferentiation of imINPs caused by Dpn or E(spl) overexpression is the suppression of Erm. However, the overproliferation of type II NBs resulting from the overexpression of Nintra or Numb knockdown can only be partially suppressed by concomitant Erm expression. Therefore, in addition to functioning through the canonical pathway to activate E(spl) expression, Notch may also act through noncanonical pathways, such as the mTORC2/Akt pathway, to regulate type II NB proliferation (Li, 2017).

How does Erm promotes INP maturation and prevents the dedifferentiation of imINP? It has previously been suggested that Erm prevents the dedifferentiation of INPs by activating pros expression and attenuating the response of INPs to self-renewing factors such as Dpn and E(spl) proteins. However, two pieces of evidence argue against this notion. First, the loss of Pros only induces the overproliferation of INPs but not the dedifferentiation of imINPs into type II NBs . Second, Erm is only expressed in imINPs, which do not express Dpn or E(spl) proteins. In the present study, evidence is provided demonstrating that Erm likely promotes INP maturation in part by inhibiting the expression and/or function of PntP1. These results show that the overproliferation of type II NBs resulting from the loss of Erm or overexpression of Dpn or E(spl) proteins, which leads to suppression of Erm expression, could be significantly inhibited by knocking down PntP1. These data strongly argue that the dedifferentiation of imINPs and generation of extra type II NBs resulting from the loss of Erm is in part due to de-repression of PntP1 expression and/or function in imINPs, which is consistent with the PntP1 function in specifying type II NBs and suppressing the activation of ase. Similar to other Ets family proteins that are commonly involved in tumorigenesis, PntP1 may also activate the expression of cell cycle regulators that promote nonproliferative imINPs to enter the cell cycle and initiate unrestricted tumorigenic overproliferation. However, PntP1 may not be the only target of Erm in imINPs. As shown in a recent study, in addition to PntP1, Erm also directly inhibits the expression of Grh-O in imINPs (Janssens, 2017). Therefore, Erm likely promotes the maturation of INPs by regulating the expression/function of multiple target genes (Li, 2017).

In conclusion, this study demonstrates here that similar to Notch signaling, Dpn maintains the identity and self-renewal of type II NBs in part by inhibiting Erm expression. Whereas in imINPs, the absence of Dpn and E(spl) proteins allows PntP1-mediated activation of erm, which in turn promotes INP maturation by inhibiting the expression and/or function of PntP1 and Grh-O in imINPs. Thus, the present study elucidates the mechanistic details of the maintenance of type II NBs and maturation of INPs (Li, 2017).

Anterior CNS expansion driven by brain transcription factors

During CNS development, there is prominent expansion of the anterior region of the brain. In Drosophila, anterior CNS expansion emerges from three rostral features: (1) increased progenitor cell generation, (2) extended progenitor cell proliferation, (3) more proliferative daughters. This study finds that tailless (mouse Nr2E1/Tlx), otp/Rx/hbn (Otp/Arx/Rax) and Doc1/2/3 (Tbx2/3/6) are important for brain progenitor generation. These genes, and earmuff (FezF1/2), are also important for subsequent progenitor and/or daughter cell proliferation in the brain. Brain TF co-misexpression can drive brain-profile proliferation in the nerve cord, and can reprogram developing wing discs into brain neural progenitors. Brain TF expression is promoted by the PRC2 complex, acting to keep the brain free of anti-proliferative and repressive action of Hox homeotic genes. Hence, anterior expansion of the Drosophila CNS is mediated by brain TF driven 'super-generation' of progenitors, as well as 'hyper-proliferation' of progenitor and daughter cells, promoted by PRC2-mediated repression of Hox activity (Curt, 2019).

Detailed analysis of Drosophila CNS development has revealed that there is 'super-generation' of NBs in the B1 segment; ~160 NBs in B1 compared to 28-70 NBs/segment for each of the 18 posterior segments (B2-A10). In the ventral neurogenic regions (generating the nerve cord) a single NB delaminates from each proneural cluster. In contrast, the NB super-generation in B1 stems, at least in part, from group delamination of NBs. The specification of NB cell fate depends upon low, or no, Notch activity. In line with this notion, evidence points to reduced Notch signalling in the procephalic neuroectoderm (Curt, 2019).

Head gap genes, such as tll, were previously shown to be important for B1 NB generation, and in line with this strikingly reduced NB generation was observed in tll. Does tll intersect with Notch signalling? tll mutants show loss of expression of the proneural gene l'sc, which is negatively regulated by Notch. Recent studies furthermore reveal an intimate interplay between tll and Notch signalling in the developing Drosophila embryonic optic placodes. In addition, the C. elegans tll orthologue nhr-67 regulates both lin-12 (Notch) and lag-2 (Delta) during uterus development. Strikingly, in the mouse brain, the tll orthologue Nr2E1 (aka Tlx) was recently shown to negatively regulate the canonical Notch target gene Hes1. Against this backdrop, it is tempting to speculate that the group NB delamination normally observed in the procephalic region results, at least in part from tll repression of the Notch pathway. Indeed, tll was the only one of the four TFs that could act alone to trigger ectopic NBs in the wing disc (Curt, 2019).

Other previously identified head gap genes are oc (also known as orthodenticle: otd), buttonhead (btd) and ems. However, it was not observed that misexpression of oc or ems from elav-Gal4 efficiently drove ectopic proliferation in the nerve cord. Moreover, oc acts both in B1-B2, ems in B2-B3, being repressed from B1 by tll, and btd acts in B2-B3. Because B2 and B3 segments do not display super-generation of NBs these findings point to tll as the key head gap gene driving the super-generation of NBs specifically observed in the B1 segment (Curt, 2019).

Reduced NB generation was observed in the triple otp/Rx/hbn and Doc1/2/3 mutants. This would tentatively place them in the category of head gap genes, at least as far as being important for NB generation. However, their effects on NB generation is weaker than that observed in tll mutants. In addition, otp/Rx/hbn and Doc1/2/3 show genetic redundancy. The combination of genetic redundancy and their weaker effects on NB generation, likely explain why they were not previously categorised as head gap genes (Curt, 2019).

The connection between the brain TFs examined in this study herein and NB super-generation is not only evident from the mutant phenotypes, but also from their potent gain-of-function effects. Strikingly, it was found that brain TF co-misexpression was sufficient to generate ectopic NBs in the embryonic ectoderm and developing wing discs. A number of markers indicate that these ectopic NBs undergo normal CNS NB lineage progression, generating neurons and glia. Moreover, the ectopic expression of the brain-specific factors Rx and Hbn, the apparently higher neuron/glia ratio, the reduced GsbN expression, the generation of Dpn+/Ase- NBs (Type II-like) in both the embryonic ectoderm and wing discs, in combination suggest that brain TF co-misexpression specifically triggered reprogramming towards a B1 brain-like phenotype (Curt, 2019).

One surprising finding pertains to the clear difference between the potency of the tll,erm double and the Tetra (tll, erm, Doc2 and otp) in the embryonic ectoderm versus the wing disc, with the double being more potent in the wing disc and the Tetra more potent in the embryo. Indeed, in the wing disc the strong effect of tll,erm is suppressed by the addition of any combination of otp and Doc2. There is no obvious explanation for the different responsiveness to brain TF misexpression in the two tissues, but it may reflect the fact the embryonic neuroectoderm is already primed for the generation of NBs (Curt, 2019).

Another surprising finding pertains to the role of erm in embryonic versus larva Type II NBs. Previous studies of erm function in the larvae found that erm mutants displayed more Type II NBs. Larval MARCM clone induction and marker analysis demonstrate that this is due to de-differentiation of INPs back to type II NBs, rather than excess generation of Type II NBs in the embryo. No extra Type II or Type I NBs were found in erm mutants but rather reduced number of cells generated in the embryonic Type II lineages, showing that erm is important for lineage progression. Hence, the role of erm appears to be different in the embryonic versus larval Type II lineages (Curt, 2019).

In addition to the NB super-generation in B1, recent studies reveal that three different lineage topology mechanisms underlie the hyper-proliferation of the brain. First, the majority of NBs (136 out 160 NB) display a protracted phase of NB proliferation, and do not show evidence of switching from Type I to Type 0 daughter proliferation (Yaghmaeian Salmani, 2018). Second, the eight MBNBs, which appear to divide in the Type I mode and never enter quiescence, also generate large lineages. Third, the 16 Type II NBs progress by budding off INP daughter cells, which divide multiple times to generate daughter cells that in turn divide once, hence resulting in lineage expansion. In contrast, in the nerve cord many NBs switch from Type I to Type 0, and all halt neurogenesis by mid-embryogenesis. The Hox anti-proliferation gradient further results in a gradient of the Type I-->0 switch and NB exit along the nerve cord. The combined effects of these alternate lineage topology behaviours translate into striking differences in the average lineage size in the brain when compared to the nerve cord (Yaghmaeian Salmani, 2018) (see Mechanisms underlying the anterior expansion of the Drosophila CNS). Moreover, the three different modes of more extensive NB and daughter cell proliferation combine with the super-generation of NBs in B1 to generate many more cells in the B1 brain segment, when compared to all posterior segments (Curt, 2019).

The brain TFs examined in this study are expressed in several or all (Tll) of the three brain NB types, and are important for both NB and daughter cell proliferation. In line with this, brain TF ectopic expression, with the late neural driver elav-Gal4, drives aberrant nerve cord proliferation and blocks both the Type I-->0 daughter cell proliferation switch and NB cell cycle exit. This results in the generation of supernumerary cells, evident both by the expansion of specific lineages and an increase in overall nerve cord cell numbers. This study found that both the double and Tetra misexpression can trigger the ectopic generation of what appears to be a mix of Type I and Type II-like NBs. The mix of these two NB types may reflect that the misexpression scenario does not accurately and reproducibly recreate the temporal order of the brain TFs, with for example tll expressed prior to erm in the wild type (Curt, 2019).

The ectopic appearance of symmetrically dividing NBs in the brain TF co-misexpression nerve cords is more difficult to explain. However, since there normally are divisions of cells in the neuroectodermal layer prior to NB delamination, and given the early expression of the brain TFs (prior to NB delamination), it is tempting to speculate that brain TF co-misexpression to some extent can trigger an early neuroectodermal cell fate (Curt, 2019).

It was recently found that NB and daughter proliferation is also promoted by a set of early TFs expressed by most, if not all NBs. Strikingly, these TFs are expressed at higher levels in the brain, due to the lack of Hox expression therein, thereby contributing to the extended NB proliferation and more proliferative daughter cells observed in the brain. It will be interesting to address the possible regulatory interplay between these broadly expressed early NB factors and the brain TFs described in this study (Curt, 2019).

Gene expression studies have revealed the mutually exclusive territory of brain TF and Hox gene expression in the Drosophila CNS. In line with this notion, it was found that co-misexpression of brain TFs in the nerve cord repressed expression of the posterior Hox genes of the BX-C, and conversely that BX-C co-misexpression repressed several brain TFs; Bsh, Rx, Hbn, Tll and Doc2 (Curt, 2019).

A key 'gate-keeper' of the brain versus nerve cord territories appears to be the PRC2 epigenetic complex. Removing PRC2 function results in complete loss of the H3K27me3 repressive epigenetic mark and anterior expansion of the expression of all Hox genes. This furthermore results in repression of brain TF expression, that is Tll and Doc2, as well as Rx. Surprisingly, in spite of the many roles that PRC2 may play, this study found that transgenic brain TF co-expression could rescue the PRC2 mutant proliferation defects. Given the repressive action of BX-C Hox genes on brain TFs, this suggests that the principle role of PRC2 during early CNS development, at least regarding proliferation, is to ensure that Hox genes are prevented from being expressed in the brain, thus ensuring brain TF expression. Indeed, it was recently demonstrated that the reduced brain proliferation observed in esc mutants could also be fully rescued by the simultaneous removal of the posterior-most and most anti-proliferative Hox gene, Abd-B (Curt, 2019).

In mammals, the precise number of neural progenitors present at different axial levels during embryonic development has not yet been mapped. However, the wider expanse of the anterior embryonic neuroectoderm would suggest the generation of more progenitors anteriorly. There is also an extended phase of neurogenesis in the forebrain, when compared to the spinal cord. Dividing daughter cells (most often referred to as basal progenitors; bP) have been identified along the entire A-P axis of the mouse CNS. Intriguingly, the ratio of dividing bPs to apical progenitors (radial glial cells) was found to be higher in the telencephalon than in the hindbrain. Similarly, recent studies revealed a higher ratio of dividing cells in the outer layers than in the lumen, when comparing the developing telencephalon to the lumbo-sacral spinal cord. Albeit still limited in their scope, these studies suggest that a similar scenario is playing out along the A-P axis of mouse CNS as that observed in Drosophila, with an anteriorly extended phase of progenitor proliferation and a higher prevalence of proliferating daughter cells (Curt, 2019).

In addition to the similarities between Drosophila and mouse regarding progenitor generation, as well as progenitor and daughter cell proliferation, the genetic mechanisms controlling these events may also be conserved. Mouse orthologues of the Drosophila brain TFs examined in this study, that is Nr2E1/Tlx (Tll); Otp, Rax and Arx (Otp); Tbx2/3/6 (Doc1/2/3); and FezF1/2 (Erm), are restricted to the brain and are known to be critical for normal mouse brain development, and in several cases for promoting proliferation. Furthermore, Hox genes are not expressed in the mouse forebrain and there is a generally conserved feature of brain TFs expressed anteriorly and Hox genes posteriorly. Mutation and misexpression has revealed that Hox genes are anti-proliferative also in the vertebrate CNS. Moreover, PRC2 (EED) mouse mutants show extensive expression of Hox genes into the forebrain and reduced gene expression of for example Nr2E1, Fezf2 and Arx. This is accompanied by reduced proliferation in the telencephalon and a microcephalic brain, while the spinal cord does not appear effected (Curt, 2019).

Gene expression and phylogenetic consideration recently led to the proposal that the CNS may have evolved by 'fusion' of two separate nervous systems, the apical and basal nervous systems, present in the common ancestor. Interestingly, in arthropods for example Drosophila, the brain and nerve cord initially form in separate regions only to merge during subsequent development. Recent studies of the role of the PRC2 complex and Hox genes in controlling A-P differences in CNS proliferation, in both Drosophila and mouse, lend support for the notion of a 'fused' CNS (Yaghmaeian Salmani, 2018). This idea is further supported by recent studies of the epigenomic signature and early embryonic cell origins of the anterior versus posterior developing CNS. The findings outlined in this study, showing that brain hyperproliferation is driven not only by the lack of Hox homeotic gene expression, but also by the specific expression of highly conserved brain TFs, lend further support to the notion of a separate evolutionary origin of brain and nerve cord (Curt, 2019).

It is tempting to speculate that the possibly separate evolutionary origins of the brain and nerve cord may manifest not only as distinct modes of neurogenesis, but also be reflected by separate regulatory mechanisms. These would involve brain TFs acting anteriorly, generating an abundance of progenitors, as well as driving progenitor and daughter cell proliferation. Conversely, Hox genes would act posteriorly, counteracting progenitor generation, as well as tempering progenitor and daughter cell proliferation. In this model, PRC2 would act as a 'gate keeper', ensuring that Hox genes are restricted from the brain and thereby promoting brain TF expression. This model clearly represents an over-simplification, but may serve as a useful launching point for future comparative studies in many model systems (Curt, 2019).

Zinc finger genes Fezf1 and Fezf2 control neuronal differentiation by repressing Hes5 expression in the forebrain

Precise control of neuronal differentiation is necessary for generation of a variety of neurons in the forebrain. However, little is known about transcriptional cascades, which initiate forebrain neurogenesis. This study shows that zinc finger genes Fezf1 and Fezf2, homologs of Drosophila Earmuff, that encode transcriptional repressors, are expressed in the early neural stem (progenitor) cells and control neurogenesis in mouse dorsal telencephalon. Fezf1- and Fezf2-deficient forebrains display upregulation of Hes5 and downregulation of neurogenin 2, which is known to be negatively regulated by Hes5. FEZF1 and FEZF2 bind to and directly repress the promoter activity of Hes5. In Fezf1- and Fezf2-deficient telencephalon, the differentiation of neural stem cells into early-born cortical neurons and intermediate progenitors is impaired. Loss of Hes5 suppresses neurogenesis defects in Fezf1- and Fezf2-deficient telencephalon. These findings reveal that Fezf1 and Fezf2 control differentiation of neural stem cells by repressing Hes5 and, in turn, by derepressing neurogenin 2 in the forebrain (Shimizu, 2010).

An important question about neural development is how the differentiation of neural stem cells is precisely controlled in the forebrain. Asymmetric cell division of neural stem cells is thought to contribute to the differentiation of neural stem cells (radial glial cells) into either neurons or intermediate progenitors. Recent reports suggest that the orientation of stem cell division in the VZ might not directly control which of the two asymmetrically divided cells becomes a stem cell and which of the two becomes a differentiated cell. Although asymmetric centrosome inheritance during the asymmetric cell divisions was reported to play a role in the maintenance of the neural stem cells, it is not clear what factors determine cell fate. It is known that oscillation of Hes1 and neurogenin 2 expression in the telencephalic VZ plays an important role in maintenance of the neural stem cells and that stabilization of neurogenin 2 expression supports differentiation of the neural stem cells. However, it is still not understood what factor(s) control stabilization of neurogenin 2 expression and what factor(s) induce their differentiation. These reports imply that, besides asymmetric distribution of cell-fate determinants, extrinsic and intrinsic factors might bias the neural stem cells toward differentiation. Notch signaling plays an essential role in maintenance of the neural stem cells. Thus, regulators of Notch signaling and its downstream effectors might be involved in the decision as to whether to be a stem cell or a differentiated cell. This report demonstrates that Fezf1 and Fezf2, which are expressed in the neural stem cells at the beginning of mouse cortical development, inhibit the expression of the Notch effector Hes5 and promote differentiation of the neural stem cells. The findings suggest that Fezf1 and Fezf2 function as intrinsic factors to bias the neural stem cells toward differentiation (Shimizu, 2010).

Expression of fezf2 takes place in the radial glial cells of the telencephalic VZ of adult zebrafish (Berberoglu, 2009). fezf2 is also expressed in the neural progenitors and neurons in the pre-optic region and hypothalamus of the adult zebrafish brains (Berberoglu, 2009). In zebrafish, neurogenesis continuously takes place in adult brains. It is possible that fezf2 might control differentiation of the neural stem cells in the adult zebrafish forebrain as Fezf1 and Fezf2 do during early mouse cortical development (Shimizu, 2010).

Expression of Fezf1 or Fezf2 repressed both NOTCH1-dependent and NOTCH1-independent Hes5 promoter activity, but did not repress the Hes1 promoter or the artificial CBS-dependent promoter. Hes1 expression was not upregulated in the telencephalon of Fezf1^-/-Fezf2^-/- mice. Furthermore, FEZF1 and FEZF2 bound to the Hes5 promoter in vivo in the mouse forebrain. All of these data indicate that FEZF1 and FEZF2, rather than inhibit Notch cytoplasmic signaling, specifically bind to and directly repress the Hes5 promoter. FEZF1 and FEZF2 have an EH1 repressor motif. The data support the assertion that FEZF1 and FEZF2 function as transcriptional repressors and repress the Hes5 promoter at least during early cortical development. Hes5 deficiency suppressed neurogenesis defects in Fezf1^-/-Fezf2^-/- telencephalon, supporting the hypothesis that Fezf1 and Fezf2 suppress the expression of Hes5 and thereby control differentiation of the neural stem cells (Shimizu, 2010).

FEZF1 and FEZF2 repress only Hes5. Hes1 and Hes5 function redundantly in the maintenance of neural stem cells in the mouse central nervous system, whereas only Hes1 is reported to exhibit oscillatory expression in the neural stem cells, suggesting that Hes1 and Hes5 might have distinct roles in neurogenesis. Previous research has revealed that oscillation of Hes1 is involved in the maintenance of neural stem cells and, in the current study, it is speculated that Hes5 plays a different role in neurogenesis; specifically, it is proposed that Hes5, in contrast to Hes1, sets up the overall expression levels of Hes genes and neurogenin 2 in the forebrain. Once Fezf1 and Fezf2 expression exceeds a threshold, FEZF1 and FEZF2 might repress Hes5 expression, stabilize neurogenin 2 expression and thereby bias the neural stem cells toward differentiation (Shimizu, 2010).

The Drosophila homolog of Fezf1/2 (dFezf or Earmuff) has been shown to restrict the developmental potential of intermediate progenitors by negatively regulating Notch signaling. Although the mechanism by which dFezf represses Notch signaling is unknown, Fezf family genes function to negatively regulate Notch signaling in both vertebrates and invertebrates (Shimizu, 2010).

Fezf1 and Fezf2 function to repress the caudal diencephalon fate and their function is involved in proper rostro-caudal patterning of the forebrain (see Jeong, 2007). The prospective telencephalon domain is already smaller in Fezf1^-/-Fezf2^-/- mouse embryos than in the wild type at E9.5, before neurogenesis is initiated in the telencephalon. Therefore, the defect in rostro-caudal patterning is attributable to reduction of the telencephalon domain. In addition, Fezf2^-/- or Fezf1^-/-Fezf2^-/- telencephalon lacks layer-V subcerebral projection neurons. Hes5 deficiency did not suppress the defects in rostro-caudal patterning of the forebrain or specification of layer-V neurons in Fezf1^-/-Fezf2^-/- forebrains. Therefore, Fezf1/2-mediated downregulation of Hes5 is not involved in the rostro-caudal patterning of the forebrain and the specification of layer-V neurons. Fezf1 and/or Fezf2 probably control genes other than Hes5 to elicit these functions (Shimizu, 2010).

Fezf1^-/-Fezf2^-/- telencephalon exhibited reduced formation of early-born neurons such as SP neurons and CR cells. A birthdate analysis revealed that the reduction of SP neurons and CR cells was not due to mis-specification of these neurons to other types of neurons. The data suggest that generation of the neural stem cells into SP neurons and CR cells is impaired in Fezf1^-/-Fezf2^-/- telencephalon. This finding is consistent with a reduction of differentiated (anti-neuron-specific βIII tubulin antibody TUJ1⁺) neurons in the Fezf1^-/-Fezf2^-/- telencephalon at E10.5, when subplate (SP) neurons and Cajal-Retzius (CR) cells were born in the VZ. Hes5 deficiency rescued neurogenin 2 expression at E10.5 and the generation of SP neurons and CR cells in Fezf1^-/-Fezf2^-/- telencephalon, indicating that Fezf1- and/or Fezf2-mediated repression of Hes5 plays an important role in the generation of these early-born cortical neurons. It is reported that formation of CR cells in the choroid plexus region, near the cortical hem, is controlled by a Hes-neurogenin cascade but that the Notch signal-mediated lateral inhibition is not involved in regulation of the Hes-neurogenin cascade in the CR cell development. Fezf1 and Fezf2 are expressed in the dorsomedial telencephalon. The current data suggest that Fezf1 and Fezf2 might control the development of CR cells by regulating Hes5 and neurogenin 2 expression in the choroid plexus domain (Shimizu, 2010). Fezf1^-/-Fezf2^-/- telencephalon had normal upper-layer (layer II, III) neurons but displayed a reduction of layer-IV neurons. There are two plausible explanations for this finding: Fezf1 and Fezf2 regulate the specification of layer-IV neurons or Fezf1 and Fezf2 control the generation of layer-IV neurons. Neither Fezf1 nor Fezf2 is expressed in differentiated layer-IV neurons, but both are expressed in their progenitors (neural stem cells or intermediate progenitors). Layer-IV neurons are normally born (differentiated) from E13.5 through E15.5. Birthdate analysis indicated that Fezf1^-/-Fezf2^-/- telencephalon contained a reduced number of Rorβ-positive neurons that were born at E13.5, suggesting that Fezf1 and Fezf2 control the generation of layer-IV neurons either from the neural stem cells or the intermediate progenitors. In Fezf1^-/-Fezf2^-/- telencephalon, differentiation of the neural stem cells into the TBR2⁺ intermediate progenitors was impaired. Tbr2 is an essential regulator of the intermediate progenitors and is directly regulated by neurogenin 2. These data suggest that the gene cascade Fezf1/Fezf2 -> Hes5 -> neurogenin 2 regulates the expression of Tbr2 and controls differentiation of the neural stem cells into the intermediate progenitors. The reduction of the TBR2⁺ intermediate progenitors in the Fezf1^-/-Fezf2^-/- telencephalon might contribute to a reduction of layer-IV neurons. Consistent with this idea, Hes5 deficiency rescued the development of TBR2⁺ intermediate progenitors as well as layer-IV neurons in Fezf1^-/-Fezf2^-/- telencephalon. It is reported that TBR1⁺ layer-VI neurons are increased in Fezf2^-/- telencephalon, suggesting the transfate of layer-V to layer-VI neurons. However, they were not increased in Fezf1^-/-Fezf2^-/- telencephalon, implying that the gene cascade Fezf1/Fezf2 -> Hes5 ->neurogenin 2 controls the generation of layer-VI neurons. Future studies will clarify these issues (Shimizu, 2010).

In summary, FEZF1 and FEZF2 are transcriptional repressors that repress Hes5 expression and subsequently activate neurogenin expression. The Fezf1/Fezf2 -> Hes5 -> neurogenin 2 gene cascade controls differentiation of the neural stem cells into neurons or intermediate progenitors and contributes to the generation of a variety of neurons in the forebrain (Shimizu, 2010).

Brm-HDAC3-Erm repressor complex suppresses dedifferentiation in Drosophila type II neuroblast lineages

The control of self-renewal and differentiation of neural stem and progenitor cells is a crucial issue in stem cell and cancer biology. Drosophila type II neuroblast lineages are prone to developing impaired neuroblast homeostasis if the limited self-renewing potential of intermediate neural progenitors (INPs) is unrestrained. This study demonstrates that Drosophila SWI/SNF chromatin remodeling Brahma (Brm) complex functions cooperatively with another chromatin remodeling factor, Histone deacetylase 3 (HDAC3) to suppress the formation of ectopic type II neuroblasts. Multiple components of the Brm complex and HDAC3 physically associate with Earmuff (Erm), a type II-specific transcription factor that prevents dedifferentiation of INPs into neuroblasts. Consistently, the predicted Erm-binding motif is present in most of known binding loci of Brm. Furthermore, brm and hdac3 genetically interact with erm to prevent type II neuroblast overgrowth. Thus, the Brm-HDAC3-Erm repressor complex suppresses dedifferentiation of INPs back into type II neuroblasts (Koe, 2014).

This study reports a critical function of the Drosophila Brm remodeling complex in suppressing the formation of ectopic type II neuroblasts in larval brains. Mutants of major components of the Brm complex, including Brm and Bap55, and RNAi targeting of several Brm components formed ectopic type II neuroblasts. Therefore, the Drosophila Brm remodeling complex displays a tumor suppressor-like function in larval brains. Multiple subunits of the SWI/SNF complex are associated with various cancers. BAP47 (homologous to Snr1) is a bona fide tumor suppressor and the gene is deleted in pediatric rhabdoid tumors. Mutations in epigenetic regulators are found in approximately half of hepatocellular carcinoma and bladder cancers, and represent a significant portion of mutated genes in medulloblastoma. Drosophila Brm complex is essential for intestinal stem cell proliferation and commitment in the adult intestine. Two other chromatin remodeling factors, Iswi and Domino control germline stem cell and somatic stem cell self-renewal in the ovary (Koe, 2014).

Brm was shown to physically associate with Erm, a type II-specific transcription factor that prevents the dedifferentiation of INPs back into neuroblasts. Furthermore, Bap60 and Snr1, two other components of the Brm complex, also physically associate with Erm in a protein complex. Therefore, this study has provided the first molecular link during the regulation of type II neuroblast lineages. It is speculated that the association with Erm may provide functional specificity of the Brm remodeling complex in type II neuroblast lineages. It was also shown that brm genetically interacts with the type II-specific transcription factor erm. Ectopic neuroblast phenotype resulting from brm knockdown was dramatically enhanced by simultaneous knockdown of erm. Furthermore, brm knockdown, similar to erm^-, can be partially suppressed by loss of Notch. These functional data suggest that Erm is a co-factor of the Brm remodeling complex in type II neuroblast lineages. However, it is uncertain how the Brm-Erm protein complex functions to prevent dedifferentiation in type II neuroblast lineages (Koe, 2014).

Bioinformatic analysis has identified a 14 bp-long motif as the de novo Erm DNA-binding motif and 202 sites out of the 270 known genomic loci harboring Brm also contain the de novo Erm DNA-binding motif. As there are many genes that are potentially co-occupied by Brm and Erm, it is possible that Brm-Erm complex results in a unique configuration of the chromatin 'landscape' in INPs to prevent INP dedifferentiation into neuroblasts. Therefore, disruption of chromatin remodelers may cause widespread changes to the transcriptome, thus amplifying the effect of the single genetic mutation (Koe, 2014).

Most class I HDACs are recruited into large multi-subunit co-repressor complexes for maximal activity. HDAC1 and 2 are found in multiple co-repressor complexes, while to date HDAC3 appears to be uniquely recruited to the Silencing mediator of retinoic and thyroid receptors (SMRT)/Nuclear receptor co-repressor (N-CoR) complex. This study reports that Drosophila HDAC3 is recruited to a novel multi-subunit complex containing Brm and Erm and that this co-repressor complex prevents dedifferentiation of INPs into type II neuroblasts. The SMRT complex appears not to be important for type II neuroblasts, as knockdown of smrter that encodes a core component of the SMRT complex neither resulted in any ectopic type II neuroblasts nor enhanced the phenotype of ectopic neuroblasts by brm knockdown. This study also showed that HDAC3 dramatically enhanced the phenotype of ectopic neuroblast upon loss of brm or snr1, two core components of the Brm complex. By identifying this novel repressor complex, this study has provided a mechanistic link between transcriptional repression and histone deacetylation during the suppression of dedifferentiation. HDACs are typically recruited by oncogenic protein complexes in lymphoma and leukemia and HDAC3 inhibitors are synergistic or additive with anticancer agents for therapeutics. The finding that HDAC3 functions cooperatively with the Brm complex in suppressing suppressing dedifferentiation of INPs into neuroblasts and induces tumors in the allograph transplantation revealed an unexpected potential involvement of HDAC3 in tumor suppression in brain tissue. It will be of interest to determine whether this effect is conserved in the mammalian central nervous system and whether it occurs in tissues other than the brain (Koe, 2014).

Zinc-finger gene Fez in the olfactory sensory neurons regulates development of the olfactory bulb non-cell-autonomously

Fez is a zinc-finger gene encoding a transcriptional repressor that is expressed in the olfactory epithelium, hypothalamus, ventrolateral pallium and prethalamus at mid-gestation. To reveal its function, Fez-deficient mice were generated. The Fez-deficient mice showed several abnormalities in the olfactory system: (1) impaired axonal projection of the olfactory sensory neurons; (2) reduced size of the olfactory bulb; (3) abnormal layer formation in the olfactory bulb; and (4) aberrant rostral migration of the interneuron progenitors. Fez was not expressed in the projection neurons, interneurons or interneuron progenitors. Transgene-mediated expression of Fez in olfactory sensory neurons significantly rescued the abnormalities in olfactory axon projection and in the morphogenesis of the olfactory bulb in Fez-knockout mice. Thus, Fez is cell-autonomously required for the axon termination of olfactory sensory neurons, and Fez non-cell-autonomously controls layer formation and interneuron development in the olfactory bulb. These findings suggest that signals from olfactory sensory neurons contribute to the proper formation of the olfactory bulb (Hirata, 2006a).

Zinc-finger genes Fez and Fez-like function in the establishment of diencephalon subdivisions

Fez and Fez-like (Fezl) are zinc-finger genes that encode transcriptional repressors expressed in overlapping domains of the forebrain. By generating Fez;Fezl-deficient mice it was found that a redundant function of Fez and Fezl is required for the formation of diencephalon subdivisions. The caudal forebrain can be divided into three transverse subdivisions: prethalamus (also called ventral thalamus), thalamus (dorsal thalamus) and pretectum. Fez;Fezl-deficient mice showed a complete loss of prethalamus and a strong reduction of the thalamus at late gestation periods. Genetic marker analyses revealed that during early diencephalon patterning in Fez;Fezl-deficient mice, the rostral diencephalon (prospective prethalamus) did not form and the caudal diencephalon (prospective thalamus and pretectum) expanded rostrally. Fez;Fezl-deficient mice also displayed defects in the formation of the zona limitans intrathalamica (ZLI), which is located on the boundary between the prethalamus and thalamus. Fez and Fezl are expressed in the region rostral to the rostral limit of Irx1 expression, which marks the prospective position of the ZLI. Transgene-mediated misexpression of Fezl or Fez caudal to the ZLI repressed the caudal diencephalon fate and affected the formation of the Shh-expressing ZLI. These data indicate that Fez and Fezl repress the caudal diencephalon fate in the rostral diencephalon, and ZLI formation probably depends on Fez/Fezl-mediated formation of diencephalon subdivisions (Hirata, 2006b).

Search PubMed for articles about Drosophila Earmuff

Bello, B. C., Izergina, N., Caussinus, E. and Reichert, H. (2008). Amplification of neural stem cell proliferation by intermediate progenitor cells in Drosophila brain development. Neural Dev. 3: 5. PubMed ID: 18284664

Berberoglu M. A., Dong Z., Mueller T. and Guo S. (2009). fezf2 expression delineates cells with proliferative potential and expressing markers of neural stem cells in the adult zebrafish brain. Gene Expr. Patterns 9: 411-422. PubMed ID: 19524703

Boone, J. Q. and Doe, C. Q. (2008). Identification of Drosophila type II neuroblast lineages containing transit amplifying ganglion mother cells. Dev. Neurobiol. 68: 1185-1195. PubMed ID: 18548484

Bowman, S. K., Rolland, V., Betschinger, J., Kinsey, K. A., Emery, G. and Knoblich, J. A. (2008). The tumor suppressors Brat and Numb regulate transit amplifying neuroblast lineages in Drosophila. Dev. Cell 14: 535-546. PubMed ID: 18342578

Chintapalli, V. R., Wang, J. and Dow, J. A. T. (2007). Using FlyAtlas to identify better Drosophila models of human disease. Nat. Genet. 39: 715-720. PubMed ID: 17534367

Curt, J. R., Yaghmaeian Salmani, B. and Thor, S. (2019). Anterior CNS expansion driven by brain transcription factors. Elife 8. PubMed ID: 31271353

Hashimoto, H., Yabe, T., Hirata, T., Shimizu, T., Bae, Y., Yamanaka, Y., Hirano, T. and Hibi, M. (2000). Expression of the zinc finger gene fez-like in zebrafish forebrain. Mech. Dev. 97: 191-195. PubMed ID: 11025224

He, N., Liu, M., Hsu, J., Xue, Y., Chou, S., Burlingame, A., Krogan, N. J., Alber, T. and Zhou, Q. (2010). HIV-1 Tat and host AFF4 recruit two transcription elongation factors into a bifunctional complex for coordinated activation of HIV-1 transcription. Mol Cell 38(3): 428-438. PubMed ID: 20471948

Hirata, T., et al. (2006a). Zinc-finger gene Fez in the olfactory sensory neurons regulates development of the olfactory bulb non-cell-autonomously. Development 133(8): 1433-43. PubMed ID: 16540508

Hirata, T., et al. (2006b). Zinc-finger genes Fez and Fez-like function in the establishment of diencephalon subdivisions. Development 133(20): 3993-4004. PubMed ID: 16971467

Janssens, D. H., Hamm, D. C., Anhezini, L., Xiao, Q., Siller, K. H., Siegrist, S. E., Harrison, M. M. and Lee, C. Y. (2017). An Hdac1/Rpd3-poised circuit balances continual self-renewal and rapid restriction of developmental potential during asymmetric stem dell division. Dev Cell 40(4): 367-380 e367. PubMed ID: 28245922

Jeong J. Y., et al. (2007). Patterning the zebrafish diencephalon by the conserved zinc-finger protein Fezl. Development 134: 127-136. PubMed ID: 17164418

Koe, C. T., et al. (2014). The Brm-HDAC3-Erm repressor complex suppresses dedifferentiation in Drosophila type II neuroblast lineages. Elife 3: e01906. PubMed ID: 24618901

Li, X., Chen, R. and Zhu, S. (2017). bHLH-O proteins balance the self-renewal and differentiation of Drosophila neural stem cells by regulating Earmuff expression. Dev Biol [Epub ahead of print]. PubMed ID: 28899667

Lin, C., Smith, E. R., Takahashi, H., Lai, K. C., Martin-Brown, S., Florens, L., Washburn, M. P., Conaway, J. W., Conaway, R. C. and Shilatifard, A. (2010). AFF4, a component of the ELL/P-TEFb elongation complex and a shared subunit of MLL chimeras, can link transcription elongation to leukemia. Mol Cell 37(3): 429-437. PubMed ID: 20159561

Matsuo-Takasaki, M., Lim, J.H., Beanan, M. J., Sato, S. M., and Sargent, T. D. (2000). Cloning and expression of a novel zinc finger gene, Fez, transcribed in the forebrain of Xenopus and mouse embryos. Mech. Dev. 93: 201-204. PubMed ID: 10781957

Pfeiffer, B. D., Jenett, A., Hammonds, A. S., Ngo, T. T., Misra, S., Murphy, C., Scully, A., Carlson, J. W., Wan, K.H., Laverty, T. R., et al. (2008). Tools for neuroanatomy and neurogenetics in Drosophila. Proc. Natl. Acad. Sci. 105: 9715-9720. PubMed ID: 18621688

Shimizu, T., et al. (2010). Zinc finger genes Fezf1 and Fezf2 control neuronal differentiation by repressing Hes5 expression in the forebrain. Development 137(11): 1875-85. PubMed ID: 20431123

Weng, M., Golden, K. L. and Lee, C. Y. (2010). dFezf/Earmuff maintains the restricted developmental potential of intermediate neural progenitors in Drosophila. Dev. Cell 18(1): 126-35. PubMed ID: 20152183

Yaghmaeian Salmani, B., Monedero Cobeta, I., Rakar, J., Bauer, S., Curt, J. R., Starkenberg, A. and Thor, S. (2018). Evolutionarily conserved anterior expansion of the central nervous system promoted by a common PcG-Hox program. Development 145(7). PubMed ID: 29530878

date revised: 22 February 2022

Home page: The Interactive Fly © 2009 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.