BIOLOGICAL OVERVIEW

A conserved nuclear receptor, Tailless, is required for efficient proliferation and prolonged maintenance of mushroom body progenitors in the Drosophila brain

The intrinsic neurons of mushroom bodies (MBs), centers of olfactory learning in the Drosophila brain, are generated by a specific set of neuroblasts (Nbs) that are born in the embryonic stage and exhibit uninterrupted proliferation till the end of the pupal stage. Whereas MB provides a unique model to study proliferation of neural progenitors, the underlying mechanism that controls persistent activity of MB-Nbs is poorly understood. This study shows that Tailless (Tll), a conserved orphan nuclear receptor, is required for optimum proliferation activity and prolonged maintenance of MB-Nbs and ganglion mother cells (GMCs). Mutations of tll progressively impair cell cycle in MB-Nbs and cause premature loss of MB-Nbs in the early pupal stage. Tll is also expressed in MB-GMCs to prevent apoptosis and promote cell cycling. In addition, it was shown that ectopic expression of tll leads to brain tumors, in which Prospero, a key regulator of progenitor proliferation and differentiation, is suppressed whereas localization of molecular components involved in asymmetric Nb division is unaffected. These results as a whole uncover a distinct regulatory mechanism of self-renewal and differentiation of the MB progenitors that is different from the mechanisms found in other progenitors (Kurusu, 2009).

tll was expressed in the dividing MB-Nbs and GMCs, but not in the postmitotic neurons, through the stages of MB development. Tll expression is initially found in almost all procephalic neuroblasts, but became largely restricted to anterior cells by stage 16. Double immunostaining with an anti-Dac antibody, which labels MB neurons, confirmed that they were MB-Nbs and GMCs. In the larval stages, Tll is expressed in the MB-Nbs and GMCs as well as in lamina precursor cells. While the expression in lamina precursor cells disappears by the end of the larval stage, Tll expression in the MB progenitors is maintained during the pupal stages. In newly eclosed flies, Tll expression was found in a few GMC-like cells in the middle of the MB cell clusters, although their exact identity is unknown (Kurusu, 2009).

Several lines of evidence indicate that Tll is cell autonomously required for efficient proliferation activity MB-Nbs. BrdU labeling experiments demonstrate that DNA synthesis is partially suppressed in tll mutant Nbs in both the larval and the pupal stages. Cell cycle defects in the mutant MB-Nbs are not evident in the larval stage but confirmed by marked suppression of PH3 and Cyc B activity at 20 h APF before the disappearance of mutant Nbs. As a whole, these data suggest that Tll is required to maintain efficient cell cycle progression in MB-Nbs, particularly in the pupal stage. In contrast, although the premature loss of the mutant Nbs might be a consequence of cell cycle exit as has been suggested with other Nbs, the exact mechanism of the disappearance of mutant MB-Nbs in the early pupal stage is unknown. It is also plausible that mutant Nbs are removed by apoptosis, as is the case with mutant GMCs, although TUNEL signals for MB-Nbs were not detected at 20 h APF, shortly before their disappearance whereas cell death signals in GMCs are evident at both the larval and pupal stages (Kurusu, 2009).

Despite marginal reduction in cell division activity of MB-Nbs at the larval stage, loss of tll activity results in significant reduction of the larval MB clones. Instead, the results demonstrate that cell cycle progression is impaired in larval MB-GMCs. Moreover, the majority of the MB-GMCs are lost by cell death. The molecular mechanism underlying these GMC defects is yet to be investigated, but it is unlikely that they are mediated by altered Pros expression since Pros is co-expressed with Tll in wild-type MB-GMCs, and its expression is unaltered in mutant GMCs. In addition, the results demonstrating that neither p35 nor Diap1 rescues GMC death suggest that Tll might be involved in suppression of an unconventional cell death pathway (Kurusu, 2009).

What is the molecular function of Tll in the regulation of MB progenitors? The fact that Tll is a transcription factor localized in the nucleus suggests that Tll might specify neuronal identity of MB progenitors by regulating cell-type specific genes. However, unlike other regulatory factors that confer either spatial or temporal identity, Tll is expressed only in Nbs and GMCs, and mutant neurons exhibit wild-type like dendritic and axonal wiring patterns even in the adult stage, in which perdurance of wild-type tll activity in the mutant clones is unlikely. Rather, Tll might provide MB progenitors with cellular identity that specify a distinctive proliferation pattern, either by promoting cell cycle or by preventing apoptosis or by both in parallel. In any case, such identity cannot be determined by Tll on its own because Tll is expressed in other neuronal progenitors such as lamina precursor cells in the optic lobes. Instead, it is presumed that the proliferation identity of MB progenitors may be specified in combination with other regulatory factors such as Eyeless, which is expressed in MB-Nbs, GMCs and postmitotic neurons to control MB development (Kurusu, 2009).

In the course of MB proliferation, Tll might downregulate key regulatory genes involved in cell-cycle exit and differentiation, particularly given the fact that Tll functions mostly as a repressor in the early embryogenesis. One such candidate gene is pros. Pros is detected in MB-GMCs, but not MB-Nbs. However, loss of pros causes neither tumorous transformation of MB progenitors nor suppression of tll phenotype in pros tll double mutant clones. Moreover, Pros is not upregulated in tll mutant clones. Thus, these data argue against the involvement of pros in the regulation of MB progenitors although they do not exclude a redundant mechanism involving Pros cooperating with other factors. Alternatively, Tll could indirectly control cell cycle progression by downregulating genes that suppress progenitor division. In support of this, it is noteworthy that the mammalian homolog Tlx (NR2E1) represses a tumor suppressor gene, Pten, via consensus Tll/TLX binding sites located in the pten promoter, and thereby indirectly stimulates the expression of various cell cycle genes including Cyclin D1, p57 kip2, and p27 kip1 (Kurusu, 2009).

Studies on Drosophila neural progenitors reveal heterogeneity among the brain Nbs in terms of temporal windows of cell division, patterns of self-renewal, and susceptibility to mutations that regulate proliferation and termination of progenitors. Among the Nbs in the Drosophila brain, MB-Nbs exhibit a highly unique proliferation pattern. Most Nbs pause cell division between the late embryonic and the early first instar stages, and cease proliferation by the early pupal stage. By contrast, MB-Nbs divide continuously from the embryonic stage till the end of pupal stage, generating diverse identities of neurons by temporal order. In house cricket and moth, proliferation activity of MB-Nbs further extends beyond the pupal stage to exhibit persistent neurogenesis during adult life (Kurusu, 2009).

Although the data clearly indicate a pivotal function of Tll for persistent proliferation and maintenance of MB-Nbs, the mechanism that determines the exit from cell cycling at the end of pupal stage remains elusive. Neither extension of Tll expression beyond the end of the pupal period nor blocking cell death program, by p35 or Diap1, prolonged MB-Nb proliferation beyond the pupal stage, suggesting existence of other mechanisms that schedule the end of MB-Nb activity. In most brain Nbs, a burst of Pros in the nucleus at around 120 h after larval hatching (24 h APF) induces cell cycle exit to regulate generation of postmitotic progeny in the brain. However, no burst of nuclear Pros is detected for MB-Nbs at the end of the pupal stage when they finally exit from cell cycling, although the data demonstrate that, as is the case with other Nbs in the brain, Pros indeed has such regulatory potential in larval MBs that its overexpression results in partial loss of the MB-Nbs. Moreover, MB clones lacking pros activity, which exhibit normal growth, cease cell division by the end of the pupal stage (Kurusu, 2009).

During asymmetric cell division of Drosophila Nbs, Pros is kept inactive by tethering to the cell cortex by MIRA. At telophase of Nb cell cycle, Pros is segregated into GMC, where it enters the nucleus to trigger cell cycle exit and promote differentiation of post mitotic progeny that are generated by the division of GMC. Accordingly, nuclear Pros is expressed at high levels in postmitotic neurons and at moderate levels in GMCs. However, whereas this partition pattern of Pros in the post-embryonic brain is shared between MB and non-MB progenies, Pros seems dispensable for cell-cycle control of MB-GMCs. In non-MB lineages, loss of pros activity in GMCs leads to failure of cell-cycle exit and transforms of GMCs into Nbs. However, loss of pros activity never causes transformation of MB-GMCs although mutant MB neurons exhibit considerable dendritic defects. In contrast, Tll is expressed and required for MB-GMCs to suppress apoptosis and maintain active cell cycling. Intriguingly, whereas Pros is suppressed by Tll in non-MB progenitors, both proteins are coexpressed in MB-GMCs, clearly suggesting that, as compared to the progenitors of non-MB lineages, a different mechanism may operate in MB progenitors to control the expression of regulatory factors that are important for cell division and differentiation (Kurusu, 2009).

The brain hyperplasia produced by Tll overexpression is reminiscent of brain tumors caused by mislocalization of asymmetric determinants. Aberrant Nb divisions that disrupt the positioning of such factors generate brain tumors. Brain tissues from pins, mira, numb, or pros mutants generate tumors when transplanted in the wild-type abdomen. In double mutants of pins and lgl, mislocalization of aPKC in the basal cortex results in the generation of supernumerary Nbs at the expense of GMCs, and thus, neurons. BRAT is required for the asymmetric positioning of Pros, which in turn suppresses self-renewal of GMC and promotes cell differentiation by transcriptional control. Mutant clones of either brat or pros are highly tumorigenic, forming a large number of MIRA-positive Nbs (Kurusu, 2009).

While recapitulating the tumor phenotype, ectopic expression of Tll does not affect asymmetric localization of aPKC, PINS, and BRAT. Instead, Tll downregulates Pros in hyperplasic brains and in overexpression clones, suggesting that the tumorigenesis phenotype caused by Tll expression is mediated by Pros downregulation in GMCs. This notion is further supported by the fact that coexpression of Pros with Tll suppresses brain hyperplasia. Notably, the cis-regulatory region of pros harbors a consensus Tll binding site within 500 base pairs from the transcriptional initiation site, consistent with the idea that Tll might repress transcription of pros via direct DNA binding (Kurusu, 2009).

Recently, atypical large Nb lineages in the dorsomedial part of the larval brain have been described and designated as Posterior Asense-Negative (PAN) Nbs. Nbs of such lineages divide asymmetrically to self renew, but, unlike other Nbs, generate smaller intermediate progenitors that express Nb markers. The fact that these atypical Nbs are MIRA-positive and Pros negative raises a possibility that tumor clones induced by Tll could either correspond to or originate from them. As with other Nbs, clones of the PAN-Nb lineages accompany only a single large Nb, with their progeny arranged regularly in a columnar order. By contrast, clones generated by Tll overexpression harbor several large to intermediate-sized Nbs, exhibiting irregular morphology, which is typical of tumors. PAN-Nbs are the Nb subpopulation that exhibits overgrowth in brat mutants. However, it is also unlikely that Tll induced overgrowth originates from overgrowth of PAN Nbs, which correspond to eight Nbs in the DPM group among the ~90 Nbs per hemisphere. On the contrary, Tll induces clonal tumors not only in DPM but also in CM and BLP lineages. Indeed, Tll overgrowth phenotype is not localized to a specific location of the hemisphere, but broadly detectable in the brain including the optic lobe. Moreover, Tll overgrowth phenotype is also induced in the embryonic CNS, arguing against the involvement of larval PAN-Nbs (Kurusu, 2009).

The Drosophila Tll and the vertebrate homolog TLX (NR2E1) share high sequence similarity in the DNA binding domain. Tlx mutant mice exhibit a reduction of rhinencephalon and limbic structures with emotional and learning defects. Notably, Tlx mutant mice exhibit reduction of neuron numbers in cortical upper layers. Postnatally, TLX is localized to the adult neurogenic regions including the subgranular layer of the dentate gyrus to maintain stem cells in a proliferative and undifferentiated state. Recent behavioral studies have shown that such TLX-positive neural stem cells actually contribute to animal's spatial learning. Thus, combined with the current results, these studies highlight a functional commonality of the tll/Tlx homologs between flies and mammals, and imply an intriguing evolutionary conservation of the genetic programs underlying neural progenitor controls in crucial brain structures involved in memory and other cognitive functions (Kurusu, 2009).

Intriguingly, the mammalian pros homolog Prox1 promotes cell cycle exit and differentiation of the neural progenitors in the developing subventricular zone and the retina, the neural tissues in which Tlx functions antagonistically to control progenitor proliferation. Based on the tll GOF phenotypes in Drosophila, it is predicted that deregulation of Tlx in the developing brain may cause suppression of Prox1 and could result in severe neurological tumors in humans. On the other hand, consistent with the loss-of-function phenotypes in flies, several mutations have been identified in the regulatory regions of Tlx in humans with microcephary. Given the commonality in progenitor control, further studies of the Drosophila MB-Nbs may shed light on the molecular basis of the proliferation and differentiation of neural progenitors, and would provide important cues for understanding progenitor disorders in the human brain (Kurusu, 2009).

Tailless/TLX reverts intermediate neural progenitors to stem cells driving tumourigenesis via repression of asense/ASCL1

Understanding the sequence of events leading to cancer relies in large part upon identifying the tumour cell of origin. Glioblastoma is the most malignant brain cancer but the early stages of disease progression remain elusive. Neural lineages have been implicated as cells of origin, as have glia. Interestingly, high levels of the neural stem cell regulator TLX correlate with poor patient prognosis. This study shows that high levels of the Drosophila TLX homologue, Tailless, initiate tumourigenesis by reverting intermediate neural progenitors to a stem cell state. Strikingly, tumour formation could be blocked completely by re-expressing Asense (homologue of human ASCL1), which is a direct target of Tailless. These results predict that expression of TLX and ASCL1 should be mutually exclusive in glioblastoma, which was verified in single-cell RNA-seq of human glioblastoma samples. Counteracting high TLX is a potential therapeutic strategy for suppressing tumours originating from intermediate progenitor cells (Hakes, 2020).

The results revealed the mechanism through which high levels of the orphan nuclear receptor Tll initiate tumours in the Drosophila CNS. Tll is expressed in Type II NSCs during larval development, where it is required for Type II NSC identity and subsequent lineage progression. In the absence of Tll, the proneural transcription factor Ase is derepressed in Type II NSCs. As a consequence, transit amplifying INPs are no longer generated and the resulting NSC lineages have a lower neurogenic potential (Hakes, 2020).

In mice, TLX is expressed in NSCs during embryonic development and in adulthood. Embryonic NSCs display defects in proliferation in the absence of TLX and the loss of TLX in adults results in the loss of transit-amplifying intermediates and reduction in neurogenesis. While these effects were previously attributed to changes in the NSC cell cycle, the current results suggest a cell fate change may occur due to the loss of TLX (Hakes, 2020).

High levels of TLX in human glioblastoma are correlated with tumour aggressiveness. High level expression of TLX results in glioblastoma-like lesions derived from SVZ NSC lineages in mouse models of glioblastoma indicating that TLX can also promote glioblastoma development. However, it was not known how high TLX leads to glioblastoma, nor had the cellular origin of TLX-induced tumours been identified. TLX and its Drosophila homologue, Tll, are highly conserved proteins and this study found that both genes are able to revert INPs to NSC fate as a first step in tumour initiation. Ectopic expression of Tll was also sufficient to induce the expansion of NSCs throughout the Drosophila CNS, demonstrating the widespread vulnerability of NSC and progenitor populations to ectopic Tll expression (Hakes, 2020).

This study found that the ectopic NSCs resulting from high Tll expression are negative for Ase. Tll binds to the ase locus, suggesting that Tll directly represses ase. The absence of Ase is a hallmark of Type II NSCs. Therefore, ectopic Tll promotes a cell fate change from INP/Type I NSC to Type II NSC and thereby initiates tumourigenesis (Hakes, 2020).

The capacity of Tll to induce NSC expansion had been reported previously as part of a study showing that Tll regulates the proliferation of larval mushroom body NSCs and GMCs (Kurusu, 2009). The authors showed that overexpressing Tll resulted in ectopic NSCs, but they did not identify the origin of these tumours and argued against a role for Tll in Type II NSC fate. Tll-induced tumourigenesis could be blocked by ectopic expression of Pros (Kurusu, 2009). However, ectopic Pros results in the loss of NSCs even in wild type brains. In contrast, Type I NSC lineages appear normal after Ase misexpression in wild type brains. Furthermore, it has been reported that high levels of the human homologue of Pros, PROX1, exacerbate glioblastoma, arguing against PROX1 expression as a therapeutic strategy (Hakes, 2020).

This study found that the tumourigenic capacity of Drosophila Tll and human TLX was highly conserved. Human TLX could also induce ectopic Type II NSCs from INPs through the repression of Ase. Analysis of scRNA seq from glioblastoma revealed that TLX and ASCL1 expression is mutually exclusive. It is notable that the origin of human glioblastoma has been mapped to the SVZ. While TLX positive NSCs have been identified in both the SVZ and dentate gyrus, high levels of TLX giving rise to glioblastoma has only been shown robustly in the SVZ. Furthermore, a recent study demonstrated that low expression levels of ASCL1 correlate with glioblastoma malignancy. Ectopic expression of ASCL1 in glioblastoma stem cells was sufficient to promote neuronal differentiation. Based on these results in Drosophila, it is predicted that introducing ASCL1 would override the repressive effect of TLX, induce neuronal differentiation and reduce tumour growth, thereby providing an effective treatment (Hakes, 2020).

The results indicate that INPs are the tumour initiating cells in Type II NSC lineages expressing high levels of the orphan nuclear receptor Tll and potentially implicate intermediate progenitors as one of the cells of origin in TLX+ glioblastomas, an aggressive and lethal brain tumour. This study found that Ase is a direct target of Tll and that Ase expression not only blocks Tll-induced tumourigenesis, but also reinstates a normal neural differentiation programme (Hakes, 2020).

The Ets protein Pointed P1 represses Asense expression in type II neuroblasts by activating Tailless

Intermediate neural progenitors (INPs) boost the number and diversity of neurons generated from neural stem cells (NSCs) by undergoing transient proliferation. In the developing Drosophila brains, INPs are generated from type II neuroblasts (NBs). In order to maintain type II NB identity and their capability to produce INPs, the proneural protein Asense (Ase) needs to be silenced by the Ets transcription factor pointed P1 (PntP1), a master regulator of type II NB development. However, the molecular mechanisms underlying the PntP1-mediated suppression of Ase is still unclear. This study utilized genetic and molecular approaches to determine the transcriptional property of PntP1 and identify the direct downstream effector of PntP1 and the cis-DNA elements that mediate the suppression of ase. The results demonstrate that PntP1 directly activates the expression of the transcriptional repressor, Tailless (Tll), by binding to seven Ets-binding sites, and Tll in turn suppresses the expression of Ase in type II NBs by binding to two hexameric core half-site motifs. This study further showa that Tll provides positive feedback to maintain the expression of PntP1 and the identity of type II NBs. Thus, thus this study identifies a novel direct target of PntP1 and reveals mechanistic details of the specification and maintenance of the type II NB identity by PntP1 (Chen, 2022).

This study has dissected the molecular mechanism of how PntP1 suppresses Ase expression to specify the type II NB identity. PntP1 was demonstrated to acts as a transcriptional activator to indirectly suppress Ase expression by activating Tll, whereas Tll provides positive feedback to maintain the expression of PntP1 and the type II NB identity. The cis-elements that mediate the suppression of Ase by Tll and the activation of Tll by PntP1 were mapped. Thus, this work reveals mechanistic details of PntP1-mediated suppression of Ase expression and specification of type II NBs and identifies a novel direct target of PntP1 in type II NBs (Chen, 2022).

This study has demonstrated that PntP1 functions as a transcriptional activator by showing that the artificial chimeric repressor protein EnR-Ets antagonizes the function of endogenous PntP1 proteins when expressed in type II NB lineages and that the artificial chimeric activator proteins VP16AD-pntP1^1/2C-Ets could functionally mimic endogenous PntP1 protein when expressed in type I NBs. These results are in line with a previous in vitro study showing that PntP1 substantially activates bacterial chloramphenicol acetyltransferase (CAT) reporter expression under the control of Ets binding sites. Interestingly, the results show that the chimeric protein VP16AD-Ets is not sufficient to functionally mimic endogenous PntP1 proteins even though the Ets domain can bind to PntP1 target DNAs as demonstrated by the antagonization of PntP1's function by the EnR-Ets protein. Only when the C-terminal sub-fragment (aa. 256-511) of PntP1 is included can the chimeric activator protein functionally mimic endogenous PntP1 proteins. The Ets family proteins usually recruit additional cofactors to activate/repress target gene expression. This C-terminal fragment likely contains protein-protein interaction domains that are essential to recruit its cofactors. Since neither the pntP1^1/2C-Ets truncated protein nor the chimeric protein VP16AD-pntP1^1/2N-Ets is able to mimic wild type PntP1 protein's function, it is unlikely that this C-terminal sub-fragment contains the activation domain of PntP1 and the other N-terminal sub-fragment [pntP1^1/2N] contains the protein-protein interaction domains instead. Otherwise, with the VP16AD functioning as an activation domain and the N-terminal sub-fragment recruiting cofactors, the chimeric VP16AD-pntP1^1/2N-Ets protein would be able to functionally mimic wild type PntP1 protein. Given that a large number of Ets family transcription factors share highly conserved Ets domains but have diverse functions and activities in distinct cell types, the C-terminal sub-fragment of PntP1 may recruit cell-type-specific cofactors to regulate the expression of specific target genes in type II NB lineages, such as Erm and Tll, as it has been proposed as a general strategy for Ets family proteins to regulate the expression of tissue-specific target genes. However, the regions of PntP1 subfragments that was included in chimeric repressor/activator constructs were arbitrarily defined. The results do not tell the precise boundaries of the activation domains or potential protein-protein interaction domains. To precisely map these functional domains will require more detailed and systemic analyses in the future (Chen, 2022).

PntP1 performs diverse functions in different cell types in type II NB lineages. For example, in type II NBs, PntP1 is required to suppress Ase expression. In newly generated imINPs, PntP1 prevents premature differentiation of INPs, whereas late during imINP development, it promotes INP cell fate commitment and prevents dedifferentiation of imINPs. Therefore, as a transcriptional activator, PntP1 must activate the expression of many different target genes in type II NB lineages. However, Erm is the only direct target that has been identified previously. This study identified Tll as another direct target of PntP1 that functions primarily in type II NBs to suppress Ase expression. PntP1 is both necessary and sufficient for Tll expression. Seven putative binding sites were identified and EMSAs and ChIP-qPCR assays verified that all these sites can bind to PntP1 both in vitro and in vivo. Although ChIP-qPCR was done using type II NB-enriched DNAs isolated from Brat knockdown larval brains, it is unlikely the results are artifacts. A previous study has demonstrated that type II NB-enriched chromatin isolated from brat mutant larval brains maintains similar transcriptional status for multiple genes examined, including pntP1, as in wild type type II NBs. Furthermore, Brat mainly functions as an RNA-binding protein in the imINPs to promote degradation of mRNAs of self-renewing genes such as dpn and klu. Therefore, loss of Brat unlikely affects the binding of PntP1 to its target DNAs in type II NBs, but this possibility was not fully verified particularly because the supernumerary type II NBs in Brat knockdown brains are derived from dedifferentiation of imINPs and it has never been extensively evaluated whether the dedifferentiated type II NBs have the exact same gene expression profiles as wild type type II NBs. It might be helpful to further verify the results by single-cell ChIP using isolated wild type type II NBs. However, since the bioinformatic prediction of PntP1 binding sites was limited to the enhancer region R31F04, it is not certain whether any additional PntP1 binding sites exist outside this enhancer region and contribute the activation of tll by PntP1. In any event, the seven binding sites identified within this enhancer region are sufficient for tll to be activated by PntP1 in type II NBs as demonstrated by the specific expression of R31F04-GAL4 in type II NBs (Chen, 2022).

This work further demonstrates that Tll is the direct target of PntP1 that mediates the suppression of Ase in type II NBs by showing that simultaneous knockdown of Tll essentially blocks the suppression of Ase by misexpressed PntP1 in type I NBs. By fine mapping the cis-repressive elements in ase regulatory regions, two Tll binding sites were idnetified located at -1,292bp ~ - 1,262bp upstream of the ase TSS, consistent with a recent study showing that Tll binds to a 5-kb region upstream of the ase TSS [21]. Although the core hexameric sequences of these two binding sites are not exactly the same as the typical Tll binding hexamer 5'-AAGTCA, EMSA results demonstrate that Tll can indeed bind to these sites either as a monomer or a homodimer, the latter of which is common for orphan nuclear receptors (Chen, 2022).

In addition to PntP1 binding sites, other studies report that Suppressor of Hairless [Su(H)], a binding partner of the intracellular domain of Notch, and Zelda (Zld) also bind to the enhancer region of tll, implicating that Notch and Zld could be upstream activator of Tll. However, Su(H) and Zld are not just expressed in type II NBs but also in type I NBs. Thus, Su(H) and Zld are unlikely to be sufficient to activate Tll, but whether PntP1 acts together with Su(H) and Zld to activate Tll in type II NBs is worth further investigation (Chen, 2022).

A previous study shows that PntP1 is expressed not only in type II NBs but also strongly in imINPs. However, Tll is primarily expressed in type II NBs but only very weakly in imINPs, and ectopic expression of Tll in imINPs reverts imINPs to type II NBs. Therefore, there must be a mechanism to inhibit the activation of Tll by PntP1 in imINPs. A recent study proposed that Erm and Hamlet (Ham) function sequentially to suppresses Tll expression in imINPs based on 1) decreasing or increasing the copy number of the tll gene suppresses or enhances the supernumerary type II NB phenotype in ham erm double heterozygous mutants, respectively; and 2) overexpressing Erm or Ham in type II NBs inhibits the expression of Tll in type II NBs. However, these data could be also explained by changes in the expression of PntP1 as it is demonstrated in this study that there is a positive feedback loop between PntP1 and Tll. For example, the reduction in the Tll expression resulting from Erm overexpression in type II NBs could be due to inhibition of PntP1 by Erm in type II NB as was reported previously rather than direct inhibition of Tll by Erm. Furthermore, Erm and Ham are not expressed in the newly generated imINPs, in which Tll expression is already largely suppressed. Therefore, the suppression of Tll in the newly generated imINPs cannot be explained by the inhibition by Erm and Ham. Other mechanisms are likely involved in suppressing Tll in imINPs (Chen, 2022).

The current results not only demonstrate that PntP1 is a direct upstream activator of Tll but also show that Tll is required to maintain PntP1 expression in type II NBs. Therefore, there is a positive feedback loop between PntP1 and Tll that is essential for maintaining the type II NB identity. Considering that PntP1 misexpression induces Tll expression in all type I NBs and generation of mINPs in a subset of type I NB lineages, whereas Tll misexpression induces PntP1 expression only in a small subset of type I NB lineages and does not induce the generation of mINPs, it is thought that PntP1 functions as a master regulator of type II NB lineage development and acts upstream of Tll, which in turn suppresses Ase expression in type II NBs. Since Tll mainly functions as a transcriptional repressor as was also demonstrated in this study, it is unlikely that Tll directly activates PntP1 expression. A previous study suggests that there might be an unknown feedback signal from INPs that could be required for maintaining PntP1 expression. Thus, one potential explanation for the loss of PntP1 expression in Tll knockdown type II NBs could be the loss of INPs and their feedback signal resulting from the ectopic Ase expression in type II NBs. However, since Tll misexpression is able to induce PntP1 expression albeit only in a small subset of type I NBs, it is more likely that Tll suppresses the expression of another unknown transcriptional repressor that is normally suppressed by Tll in type II NBs. When Tll is knocked down, this unknown transcriptional repressor could be turned on in type II NBs to suppress PntP1 expression. Whereas in type I NBs, this unknown repressor may be normally expressed to suppress PntP1 expression and misexpression of Tll may relieve the suppression of PntP1 expression (Chen, 2022).

The GAL4 reporter assays also identified in the ase enhancer region a ~50-bp fragment that is sufficient for activating Ase expression in both type I and type II NBs. Earlier studies show that the Achaete-Scute (AS-C) complex proteins together with Daughterlesss (Da) activate Ase expression in NBs during the initial specification of NBs at embryonic stages by directly binding to four E-boxes in the 5'-UTR of ase. But how Ase is maintained in NBs once they are specified is not known. The current study identified a distinct enhancer region for activating/maintaining Ase expression in NBs, suggesting that factor(s) other than the AS-C proteins may be involved in activating/maintaining Ase expression in NBs after they are specified. Therefore, the lack of Ase expression in type II NBs is not because of the absence of an activation mechanism, but rather this activation mechanism is actively suppressed by Tll. Identifying the transcriptional activator(s) involved in activating/maintaining Ase expression after NBs are specified may shed a new light on the mechanisms regulating the development and maintenance of type I and type II NBs (Chen, 2022).

Patterning mechanisms diversify neuroepithelial domains in the Drosophila optic placode

The central nervous system develops from monolayered neuroepithelial sheets. In a first step patterning mechanisms subdivide the seemingly uniform epithelia into domains allowing an increase of neuronal diversity in a tightly controlled spatial and temporal manner. In Drosophila, neuroepithelial patterning of the embryonic optic placode gives rise to the larval eye primordium, consisting of two photoreceptor (PR) precursor types (primary and secondary), as well as the optic lobe primordium, which during larval and pupal stages develops into the prominent optic ganglia. This study characterize a genetic network that regulates the balance between larval eye and optic lobe precursors, as well as between primary and secondary PR precursors. In a first step the proneural factor Atonal (Ato) specifies larval eye precursors, while the orphan nuclear receptor Tailless (Tll) is crucial for the specification of optic lobe precursors. The Hedgehog and Notch signaling pathways act upstream of Ato and Tll to coordinate neural precursor specification in a timely manner. The correct spatial placement of the boundary between Ato and Tll in turn is required to control the precise number of primary and secondary PR precursors. In a second step, Notch signaling also controls a binary cell fate decision, thus, acts at the top of a cascade of transcription factor interactions to define photoreceptor subtype identity. This model serves as an example of how combinatorial action of cell extrinsic and cell intrinsic factors control neural tissue patterning (Mishra, 2018).

In the fruit fly Drosophila melanogaster, all parts of the visual system develop from an optic placode, which forms in the dorsolateral region of the embryonic head ectoderm. During embryogenesis, neuroepithelial cells of the optic placode are patterned to form two subdomains. The ventroposterior domain gives rise to the primordium of the larval eye and consists of two photoreceptor (PR) precursor types (primary and secondary precursors), whereas the dorsal domain harbors neuroepithelial precursors that generate the optic lobe of the adult visual system. The basic helix-loop-helix transcription factor Atonal (Ato) promotes PR precursor cell fate in the larval eye primordium. The orphan nuclear receptor Tailless (Tll) is confined to the optic lobe primordium and maintains non-PR cell fate. Hedgehog (Hh) and Notch (N) signaling are critical during the early phase of optic lobe patterning. The secreted Hh protein is required for the specification of various neuronal and non-neuronal cell types, while Notch acts as neurogenic factor preventing ectodermal cells from becoming neuronal precursors by a process termed lateral inhibition. In the optic placode Ato expression is promoted by Hh and the retinal determination genes sine oculis (so) and eyes absent (eya). Notch delimits the number of PR precursors and maintains a pool of non-PR precursors. Ato is initially expressed in all PR precursors in the placode and its expression gets progressively restricted to primary precursors. In a second step, primary precursors recruit secondary precursors via EGFR signaling: primary precursors express the EGFR ligand Spitz, which is required in secondary precursors to promote their survival. After this initial specification of primary and secondary PR precursors, the transcription factors Senseless (Sens), Spalt (Sal), Seven-up (Svp) and Orthodenticle (Otd) coordinate PR subtype specification. Sens and Spalt are expressed in primary PR precursors, while Svp contributes to the differentiation of secondary PR precursors. By the end of embryogenesis, primary PR precursors have fully differentiated into blue-tuned Rhodopsin5 PRs (Rh5), while secondary PR precursors have differentiated into green-tuned Rhodopsin6 PRs (Rh6). While the functional genetic interactions of transcription factors controlling PR subtype specification has been thoroughly studied, it remains unknown how the placode is initially patterned by the interplay of Hh and Notch signaling pathways. Similarly, the mechanisms of how ato and tll-expressing domains are set up to ensure the correct number of primary and secondary PR precursors as well as non-PR precursors of the optic lobe primordium remain unknown (Mishra, 2018).

This study describes the genetic mechanism of neuroepithelial patterning and acquisition of PR versus non-PR cell fate in the embryonic optic placode and provide the link to subsequent PR subtype identity specification. The non-overlapping expression patterns of ato and tll in the optic placode specifically mark domains giving rise to the larval eye precursors (marked by Ato) and the optic lobe primordium (marked by Tll). ato expression in the larval eye primordium is temporally dynamic and can be subdivided into an early ato expression domain, including all presumptive PR precursors and a late ato domain, restricted to presumptive primary PR precursors. The ato expression domain directly forms a boundary adjacent to tll expressing precursors of the optic lobe primordium. tll is both necessary and sufficient to delimit primary PR precursors by regulating ato expression. Hh signaling regulates the cell number in the optic placode and controls PR subtype specification in an ato- and sens-dependent manner. Finally, this study also shows that Notch has two temporally distinct roles in larval eye development. Initially, Notch represses ato expression by promoting tll expression and later, Notch controls the binary cell fate decision of primary versus secondary PR precursors by repressing sens expression. In summary, this study has identified a network of genetic interactions between cell-intrinsic and cell-extrinsic developmental cues patterning neuroepithelial cells of the optic placode and ensuring the timely specification of neuronal subtypes during development (Mishra, 2018).

Neurogenic placodes are transient structures that are formed by epithelial thickenings of the embryonic ectoderm and give rise to most neurons and other components of the sensory nervous system. In vertebrates, cranial placodes form essential components of the sensory organs and generate neuronal diversity in the peripheral nervous system. How neuronal diversity is generated varies from system to system, and different gene regulatory networks have been proposed for each particular type of neuron. Interestingly, some transcription factors, like Atonal, play an evolutionary conserved role during neurogenesis both in Drosophila and in vertebrates (Mishra, 2018).

Neuroepithelial patterning of the Drosophila optic placode exhibits unique segregation of larval eye and optic lobe precursors during embryogenesis. This study has identified genetic mechanisms that control early and late steps in specifying PR versus non-PR cell fate that ensure the expression of precursor cell fate determinants. During germband extension at stage 10, transcriptional regulators (so, eya, ato and tll) show complex and partially overlapping expression patterns in the optic placode. Their interactions with the Notch and Hh signaling pathways define distinct PR and non-PR domains of the larval eye and optic lobe primordium. Intriguingly, the results show a spatial organization of distinct precursor domains, supporting a new model of how the subdivision of precursor domains emerges. In agreement with previous studies initially the entire posterior ventral tip expresses Ato, defining the population of cells that give rise to PR precursors, while neuroepithelial precursors for the presumptive optic lobe are defined by Tll-expression in the anterior domain of the optic placode. Subsequently, Ato expression ceases in the ventral most cells and thus gets restricted to about four primary PR precursors that are located directly adjacent to the Tll expression domain. Hence, a few cell rows are between the primary PR precursors and the ventral most edge of the optic placode. This is in agreement with a recent observation on the transcriptional regulation of ato during larval eye formation. Thus, primary PR precursors are directly adjacent to the Tll-expressing cells while the Ato and Tll negative domain of secondary PR precursors is located at the posterior ventral most tip of the optic placode. Setting the Tll-Ato boundary is critical to define the number of putative secondary PR precursors, which can be recruited into the larval eye, probably via EGFR signaling. A model is proposed during which coordinated action of Hh, Notch and Tll restricts the initially broad expression of Ato to primary PR precursors (see Ato to primary PR precursors). Lack of Tll results in a de-repression of Ato and results in an increased number of primary PR precursors, which in turn recruit secondary PR precursors. Interestingly, while tll mutants show an increase in both primary and secondary PR precursors, the ratio between both subtypes is maintained. This notion further displays similarities of ommatidal formation in the adult eye-antennal imaginal disc, where Ato expressing R8-precursors recruit R1-R6. In the eye-antennal disc, specification of R8-precursors determines the total number of ommatida and therefore also the total number of PRs, the ratio of R8 to outer PRs however always remains the same. Thus, the initial specification of primary PR precursors defines the total number of PRs in the larval eye similarly to R8 PRs, and the ratio of founder versus recruited cells remains constant. Interestingly, the maintenance of primary versus secondary PR precursor ratio is also maintained in ptc mutants further supporting this model (Mishra, 2018).

During photoreceptor development in the eye-antennal imaginal disc hh is expressed in the posterior margin and is required for the initiation and progression of the morphogenetic furrow as well as the regulation of ato expression. During embryogenesis the loss of hh results in a complete loss of the larval eye, while increasing Hh signaling (by means of mutating ptc) generates supernumerary PRs in the larval eye. During early stages, an increase of Ato expression was found in ptc mutants suggesting that similarly to the eye-antennal disc Hh positively regulates ato expression. The observed increase of Ato-expressing cells is not due to a reduction of Tll but is likely due to increased cell proliferation in ptc mutants. Hh also controls proliferation during the formation of the Drosophila compound eye (Mishra, 2018).

During embryonic nervous system development Notch dependent lateral inhibition selects individual neuroectodermal cells to become neuroblasts. Notch represses neuroblast cell fate and promotes ectodermal cell fate. During compound eye development, Notch regulates Ato expression and acts through lateral inhibition to select Ato expressing R8 PR precursors. Similarly, during Drosophila larval eye development, Notch is required for regulating PR cell number by maintaining epithelial cell fate of the optic lobe primordium. Inhibiting Notch signaling leads to a complete transformation of the optic placode to PRs of the larval eye. In the absence of Notch signaling, Ato expression is expanded in the optic placode and as a result the total number of PRs is increased. Despite the increase of the overall PR-number the number of secondary PR precursors is significantly decreased or lost in the absence of Notch activity. In the compound eye Notch promotes R7 cell fate by repressing the R8-specific transcription factor Sens. It was also proposed that genetic interaction between Notch and Sens is required for sensory organ precursor (SOP) selection in the proneural field in a spatio-temporal manner. This study found that during PR subtype specification Notch represses Sens expression, thereby controlling the binary cell fate decision of primary versus secondary PR precursors. Therefore, in the absence of Notch signaling, Sens expression represses the secondary PR precursor fate. As a result, all PR precursors are transformed and acquire primary PR precursor identity. In conclusion, this study observed that Notch is essential for two aspects during optic placode patterning. First, Notch activity is critical for balancing neuroepithelial versus PR cell fate mediated through Tll-regulated Ato expression. Second, Notch regulates the binary cell fate decision of primary versus secondary PR precursor cell fate through the regulation of Sens expression (Mishra, 2018).

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).

Sequential activation of transcriptional repressors promotes progenitor commitment by silencing stem cell identity genes

Stem cells that indirectly generate differentiated cells through intermediate progenitors drives vertebrate brain evolution. Due to a lack of lineage information, how stem cell functionality, including the competency to generate intermediate progenitors, becomes extinguished during progenitor commitment remains unclear. Type II neuroblasts in fly larval brains divide asymmetrically to generate a neuroblast and a progeny that commits to an intermediate progenitor (INP) identity. This study identified Tailless (Tll) as a master regulator of type II neuroblast functional identity, including the competency to generate INPs. Successive expression of transcriptional repressors functions through Hdac3 to silence tll during INP commitment. Reducing repressor activity allows re-activation of Notch in INPs to ectopically induce tll expression driving supernumerary neuroblast formation. Knocking-down hdac3 function prevents downregulation of tll during INP commitment. It is proposed that continual inactivation of stem cell identity genes allows intermediate progenitors to stably commit to generating diverse differentiated cells during indirect neurogenesis (Rives-Quinto, 2020).

The expansion of outer subventricular zone (OSVZ) neural stem cells, which indirectly produce neurons by initially generating intermediate progenitors, drives the evolution of lissencephalic brains to gyrencephalic brains. Recent studies have revealed important insights into genes and cell biological changes that lead to the formation of OSVZ neural stem cells. However, the mechanisms controlling the functional identity of OSVZ neural stem cells, including the competency to generate intermediate progenitors, remain unknown. This study provided compelling evidence demonstrating that Tll is necessary and sufficient for the maintenance of an undifferentiated state and the competency to generate intermediate progenitors in type II neuroblasts. It was also shown that two sequentially activated transcriptional repressors, Erm and Ham, likely function through Hdac3 to silence tll during INP commitment ensuring normal indirect neurogenesis in larval brains. It is proposed that continual inactivation of stem cell functional identity genes by histone deacetylation allows intermediate progenitors to stably commit to generating sufficient and diverse differentiated cells during neurogenesis (Rives-Quinto, 2020).

Stem cell functional identities encompass the maintenance of an undifferentiated state and other unique functional features, such as the competency to generate intermediate progenitors. Because genetic manipulation of Notch signaling perturbs the regulation of differentiation during asymmetric stem cell division, the role of Notch in regulating other stem cell functions remains poorly understood. In the fly type II neuroblast lineage, overexpressing Notchintra or the downstream transcriptional repressors Dpn, E(spl)mγ, and Klumpfuss (Klu) induces the formation of supernumerary type II neuroblasts at the expense of generating immature INPs via the inhibition of erm activation. These results indicate that the Notch-Dpn/E(spl)mγ/Klu axis provides an evolutionarily conserved mechanism to maintain neural stem cells in an undifferentiated state. Although overexpressing Notchintra but not Dpn, E(spl)mγ, and Klu in combination in INPs is sufficient to drive supernumerary type II neuroblast formation, Notchintra overexpression is not sufficient to transform a type I neuroblast into a type II neuroblast. Thus, it is proposed that Notch functions as a general activator of genes expression in type II neuroblasts, and specific regulators of type II neuroblast functional identities must exist (Rives-Quinto, 2020).

This study strongly suggests that tll functions as a master regulator of type II neuroblast functional identities. Identical to Notch, tll is necessary for maintaining type II neuroblasts in an undifferentiated state and is sufficient to induce INP reversion into type II neuroblasts. Uniquely, high levels of Tll is sufficient to molecularly transform greater than 60% of type I neuroblasts in the ventral brain region into type II neuroblasts. Brain regionalization leads to distinct degrees of sensitivity to Tll overexpression. For example, type I neuroblasts in the dorsal-anterior region of the brain are resistant to Tll-induced lineage transformation. By contrast, Tll overexpression can transform most, if not all, type I neuroblasts in the ventral-lateral and ventral-medial regions of the brain into type II neuroblasts. High levels of Tll expression in ventral-lateral type I neuroblasts leads to accumulation of mostly supernumerary type II neuroblasts and very few Erm::V5+ immature INPs. This result phenocopies Tll overexpression driven by strong drivers in the type II neuroblast lineages, and suggests that neuroblast progeny rapidly re-acquire a neuroblast identity instead of an immature INP identity. Tll overexpression in ventral-medial type I neuroblasts leads to the formation of supernumerary type II neuroblasts interspersed with Erm::V5+ immature INPs, mimicking Tll overexpression driven by moderate drivers in the type II neuroblast lineages. It is speculated that progeny of transformed type II neuroblasts in the ventral-medial region of the brain can assume an immature INP identity and then revert into supernumerary type II neuroblasts. These data strongly support a model that Tll is a potent activator of type II neuroblast functional identities (Rives-Quinto, 2020).

A key question regarding the proposed function of Tll in regulating type II neuroblast functional identities is how it mechanistically links to genes previously shown to control these characteristics. ChIP-seq on fly embryonic nuclear extract using the Tll::GFP(Bac) transgenic protein identified hundreds of putative Tll target genes that include all previously characterized regulators of type II neuroblast functional identities. Tll binds dpn, E(spl)mγ and klu loci in embryos, suggesting that Tll likely regulates their expression. The phenotypic effects of loss- and gain-of-function of tll on type II neuroblasts mimic those of dpn, E(spl)mγ and klu. The vertebrate homolog of Tll, Tlx, has been shown to function as a transcriptional activator during neurogenesis. Thus, Tll might maintain type II neuroblasts in an undifferentiated state by promoting dpn, E(spl)mγ and klu expression. Tll also binds pntP1 and btd loci in embryos. Similar to Tll overexpression, mis-expression of PntP1 or Btd can transform type I neuroblasts in the ventral brain region into type II neuroblasts. Thus, it is plausible that tll functions through pntP1 or btd to regulate the competency to generate INPs in type II neuroblasts. These results strongly support the model that Tll is key component of the regulatory mechanism that endows type II neuroblasts with lineage-specific functional identities. Future experiments to validate the mechanistic links between tll and genes that regulate various lineage-specific functional characteristics will allow for the establishment of gene regulatory circuits that regulate type II neuroblast functional identities (Rives-Quinto, 2020).

The identification of ham as a putative regulator of INP commitment was unexpected given that a previously published study concluded that Ham functions to limit INP proliferation. Ham is the fly homolog of Prdm16 in vertebrates and has been shown to play a key role in regulating cell fate decisions in multiple stem cell lineages. Prdm16 contains two separately defined zinc-finger motifs, with each likely recognizing unique target genes. Prdm16 can also function through a variety of cofactors to activate or repress target gene expression, independent of its DNA-binding capacity. Thus, Ham can potentially inactivate stem cell functionality genes via one of several mechanisms. By using a combination of previously isolated alleles and new protein-null alleles, this study demonstrated that the N-terminal zinc-finger motif is required for Ham function in immature INPs. Based on the overexpression of a series of chimeric proteins containing the N-terminal zinc-finger motif, the data indicate that Ham prevents INP reversion to supernumerary type II neuroblasts by recognizing target genes via the N-terminal zinc-finger motif and possibly repressing their transcription. These results suggest that Ham prevents INPs from reverting to supernumerary type II neuroblasts by possibly repressing target gene transcription (Rives-Quinto, 2020).

A key question raised by this study is why two transcriptional repressors that seemingly function in a redundant manner are required to prevent INPs from reverting to supernumerary type II neuroblasts. INP commitment lasts approximately 6-8 hr following the generation of an immature INP; after this time, the immature INP transitions into an INP. erm is poised for activation in type II neuroblasts and becomes rapidly activated in the newly generated immature INP less than 90 min after its generation. As such, Erm-mediated transcriptional repression allows for the rapid inactivation of type II neuroblast functional identity genes. Because Erm expression rapidly declines in INPs when Notch signaling becomes reactivated, a second transcriptional repressor that becomes activated after Erm and whose expression is maintained throughout the life of an INP is required to continually inactivate type II neuroblast functional identity genes. Ham is an excellent candidate because it becomes expressed in immature INPs 3-4 hr after the onset of Erm expression and is detected in all INPs. Similar to Erm, Ham recognizes target genes and represses their transcription. Furthermore, ham functions synergistically with erm to prevent INP reversion to supernumerary type II neuroblasts, and overexpressed Ham can partially substitute for endogenous Erm. Thus, Erm- and Ham-mediated transcriptional repression renders type II neuroblast functional identity genes refractory to activation by Notch signaling throughout the lifespan of the INP, ensuring the generation of differentiated cell types rather than supernumerary type II neuroblasts instead (Rives-Quinto, 2020).

Genes that specify stem cell functional identity become refractory to activation during differentiation, but the mechanisms that restrict their expression are poorly understood due to a lack of lineage information. Researchers have proposed several epigenetic regulator complexes that may restrict neural stem-cell-specific gene expression in neurons. This study knocked-down the function of genes that were implicated in restricting neural stem cell gene expression during differentiation in order to identify chromatin regulators that are required to inactivate type II neuroblast functional identity genes during INP commitment. Surprisingly, this study found that only Hdac3 is required for both Erm- and Ham-mediated suppression of INP reversion to type II neuroblasts. This finding is consistent with a recent study showing that blocking apoptosis in lineage clones derived from PRC2-mutant type II neuroblasts did not lead to supernumerary neuroblast formation. The current data strongly suggest that genes specifying type II neuroblast functional identity, such as tll, are likely silenced rather than decommissioned in INPs. This result is supported by the finding that overexpressing Notch^intra, but not Notch downstream transcriptional repressors, in INPs can re-establish a type II neuroblast-like undifferentiated state. It is speculated that continual histone deacetylation is required to counter the transcriptional activator activity of endogenous Notch and silence tll in INPs. By contrast, the chromatin in the tll locus might be close and inaccessible to the Notch transcriptional activator complex in type I neuroblasts; thus, overexpressing Notch^intra cannot transform type I neuroblasts into type II neuroblasts. A key remaining question is what transcription factor is required to maintain the chromatin in the tll loci in an open state. Insights into regulation of the competency of the tll locus to respond to activated Notch signaling might improve understanding of the molecular determinants of OSVZ neural stem cells (Rives-Quinto, 2020).

Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo

Positional information in development often manifests as stripes of gene expression, but how stripes form remains incompletely understood. This study used optogenetics and live-cell biosensors to investigate the posterior brachyenteron (byn) stripe in early Drosophila embryos. This stripe depends on interpretation of an upstream ERK activity gradient and the expression of two target genes, tailless (tll) and huckebein (hkb), that exert antagonistic control over byn. High or low doses of ERK signaling were found to produce transient or sustained byn expression, respectively. Although tll transcription is always rapidly induced, hkb converts graded ERK inputs into a variable time delay. Nuclei thus interpret ERK amplitude through the relative timing of tll and hkb transcription. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop, which is sufficient to explain byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. It was further shown that 'blurring' of an all-or-none stimulus through intracellular diffusion non-locally produces a byn stripe. Overall, this study provides a blueprint for using optogenetics to dissect developmental signal interpretation in space and time (Ho, 2023).

This study has dissected the regulation of the byn stripe by combining precise optogenetic inputs in space and time with live biosensors of target gene expression. Using ectopic activation of Ras on the ventral side of wild-type embryos, high- and low-amplitude OptoSOS inputs were defined that induce distinct byn transcriptional dynamics – a pulse of expression in early NC14 versus more sustained expression – that match its endogenous responses in the posterior terminus and stripe-forming region. These conditions were then used to characterize the tll and hkb inputs that explain these byn dynamics in space and time (Ho, 2023).

This approach yielded novel insights about both the temporal and spatial interpretation of ERK inputs to pattern the byn stripe. First, differences in signal amplitude are interpreted through the timing of tll and hkb expression. The onset of tll expression is always rapid, occurring as quickly as 4 min after signaling onset, whereas there is a dose-dependent delay in the onset of hkb expression. This delay in hkb expression is a function of Ras/ERK input amplitude, not of developmental time. These data are consistent with previous observations in OptoSOS embryos that hkb RNA only accumulates to high levels in response to blue light inputs over 30 min. They also broaden the conception of the thresholds for tll and hkb expression: tll and hkb can be induced by inputs of the same amplitude, but hkb requires that the signal persist for a longer time. If the amplitude is low enough, the signal must persist longer than the developmental window allows, and hkb is never expressed. Thus, cumulative dose of ERK input (amplitude integrated over time) appears to be the relevant feature sensed by the circuit, as has been proposed for the terminal pattern as well as other systems. Integration of signal over time has similarly been shown to be important for interpretation of several morphogen pathways including Hedgehog, Wnt, Nodal and BMP. The byn circuit then processes this input through the relative timing of tll and hkb, rather than simply their presence, to determine local byn expression (Ho, 2023).

This more nuanced understanding of byn regulation resolves a conundrum of the endogenous pattern: how can the transient pulse of expression of byn in the high-ERK, Hkb-positive domain be reconciled with the presence of its inhibitor? It is shown in this study that at the high light levels which produce a comparable pulse of byn transcription, hkb transcription is delayed relative to tll and this delay is also evident in the accumulation of their protein products. Thus, there is a temporal window in which only the positive regulator is present, allowing for a pulse of byn expression, before accumulation of the repressor. The sequential appearance of Tll and Hkb was hypothesized during the initial characterizations of posterior patterning but has only now been directly shown. It is interesting to note that Tll has been characterized as a transcriptional repressor, implying that there is an intermediate node between tll and byn. However, the identity of this node and how it affects the timing of byn activation and repression remain unknown (Ho, 2023).

Improved understanding of byn regulation also explains how a byn stripe can form in conditions where tll and hkb transcription have the same spatial domain. The current study revisits these results with improved tools, in particular endogenously tagged transcriptional reporters of tll and hkb that are able to clearly resolve differences in transcriptional dynamics that were obscured by enhancer-based reporter constructs.It was found that stimulus conditions that support sustained byn can also support hkb expression in NC14, but under these conditions hkb expression is largely absent from earlier nuclear cycles. The co-expression of sustained byn with hkb under low light differs from the wild-type pattern, where the byn stripe forms in a region only expressing tll. Presumably the endogenous ERK gradient induces tll expression at even lower activity levels than optogenetic inputs. It is noted that the shortened bursting duration of sustained byn at the ectopic position (~25 min) compared with the endogenous stripe (~45 min) suggests that the late-appearing hkb under low light does ultimately repress byn in late NC14. It is also possible that the network dynamics reported in this study provide robustness to the byn circuit, allowing it to produce different outputs for even a narrow range of input strengths (Ho, 2023).

This study reveals that the tll-hkb-byn circuit can be classified as an incoherent feedforward loop with rapid activation and delayed repression, a circuit with well-characterized pulse-generation and stripe-forming properties. A unique feature of this circuit however is that the delay in hkb expression is dose-dependent, meaning that differences in signal amplitude are converted to differences in hkb dynamics and thus different byn responses (i.e. transient if hkb onset is fast, sustained if hkb onset is slow). Interestingly, similar dose-dependent delays in transcriptional onset were recently shown for Dorsal and BMP signaling targets. What is the mechanism underlying this delay in hkb onset? The dose-dependence of tll and hkb has been a longstanding open question even without the complexity of temporal dynamics. ERK signaling activates transcription of both tll and hkb through relief of the same repressor, Cic, and it is unclear why these genes would require different doses of ERK signaling. The experiments rule out a few possible explanations. Developmental time does not appear to be crucial, given that the delay in hkb transcription is observed regardless of when light is applied and both the tll and hkb loci are known to be accessible early. It is also possible to rule out interactions with other components of the anterior-posterior patterning machinery given that this study was able to produce an ectopic byn stripe rotated 90° from its endogenous counterpart. One intriguing possibility, supported by previous ChIP-seq results, is that Cic leaves the enhancers of hkb more slowly than those of tll. It is also possible that signaling-dependent chromatin changes are involved. These models will be tested in future studies (Ho, 2023).

The second major finding is that the boundary of a uniform OptoSOS input is blurred in space downstream of Ras to produce two domains from a single input – a transient byn domain within the high-ERK illuminated region and a sustained domain in the low-ERK unilluminated region. These non-local effects of a local Ras input are most likely mediated by diffusion of active intracellular components, a well-established contributor to developmental patterning in the syncytial Drosophila embryo. It remains unknown whether the endogenous terminal dpERK gradient is produced from a similar gradient of active Torso receptors, or is due to the combination of a discrete domain of Torso activity at the poles and cytoplasmic diffusion of downstream components. If the latter model is correct, the developmental rescue by an all-or-none OptoSOS input may not be an example of a simple input replacing the function of a complex one, but rather a good approximation of endogenous activation in the terminal system. A number of systems once thought to depend strictly on input concentration have similarly been shown to depend on an unexpectedly simple form of the input (Ho, 2023).

Several limitations of the optogenetic system reveal opportunities for future investigation. These experiments were performed at an ectopic position in the embryo where position-specific gene expression may influence ERK interpretation differently than at the poles. For example, the gap gene knirps has been shown to repress tll in the center of the embryo, and it was observed that the total domain of tll and byn expression was smaller under low light. Because of these positional differences in ERK sensitivity, it is not possible to make absolute comparisons about input and output strengths with the endogenous terminal pattern. In the future it will be interesting to investigate this circuit in embryos lacking other sources of positional information, preventing localized gap gene expression . Also, it is possible that the methods left some transcriptional bursts undetected, and it is not possible to distinguish whether an upper bound of ~75% transcriptionally active nuclei represents true transcriptional heterogeneity or an experimental limit of detection. These limitations could be overcome by future studies using techniques that simultaneously label the target DNA locus and measure transcription in live embryos, or advances in high-quality volumetric imaging and machine-learning approaches. Finally, many questions remain about the precise temporal relationships between ERK activation, gene transcription and protein accumulation. What is the relative influence of tll and hkb transcripts produced by early versus late nuclear cycles, and what is the delay between RNA production and protein accumulation? Combining transcriptional and protein reporters in the same embryo with mathematical models will allow these questions to be addressed (Ho, 2023).

Altogether, this work provides a blueprint for dissecting a developmental circuit with optogenetic tools to reveal new insights about network architecture. This study has manipulated amplitude, duration, timing and spatial pattern of the signal to understand the contributions of each factor to signal interpretation. This framework will be an effective strategy for dissecting other developmental circuits in the future (Ho, 2023).

Earlier discussion of Tailless function

Two transcription factors, Tailless and Huckebein, constitute the regulatory proteins that are the final products of the terminal system. "Terminal" refers to the anterior and posterior ends of the embryo. A terminal gene is one that works and affects development at the terminal ends of the embryo. tll and hkb are transcribed at these terminals, or ends of the embryo, and therefore are considered terminal genes. Early and transient expression at the posterior pole is required to establish a domain from which arise the eighth abdominal segment, telson and posterior gut.

tailless is also considered one of the gap genes, so called because gap gene mutants have gaps in their structures. In tailless mutants, these gaps are found in the head and the posterior. Therefore, tailless controls terminal genes that result in normal development of head and posterior.

Just a few nuclear cycles after activation of tailless in the tail, a brain-specific domain is initiated at the anterior; expression in this domain is maintained with complex modulations throughout embryogenesis. Expression of tailless in this domain is required to establish the most anterior region of the brain. To understand the function and regulation of these different domains of expression, a detailed description of tailless expression in brain neuroblasts is provided (Rudolph, 1997).

Transcription of tll is initiated early in the syncytial blastoderm stage (stage 4) in two symmetrical caps: these two caps depend entirely on activity of the maternally encoded terminal system. Expression in the posterior cap is required for posterior patterning of the blastoderm stage embryo. Altough expression in both caps is transient, that in the posterior cap reaches a higher level and persists longer, i.e., through most of the blastoderm stage. Expression in the posterior cap largely disappears during gastrulation and is undetectable by the end of germband extension. By the beginning of the cellular blastoderm stage, the anterior cap of expression is replaced by a horseshoe-shaped stripe that straddles the dorsal midline between 76 and 89% egg length. This stripe covers the entire brain anlage, i.e., the procephalic neuroectoderm expression in this stripe continues in domains that will become the brain. Later, TLL mRNA is present in all proneural domains of the protocerebrum; the level of transcript in a domain is highest shortly before and during the stage at which neuroblasts delaminate from that domain. During gastrulation, the horseshoe-shaped stripe of tll expression becomes tilted backwards and undergoes a process of internal differentiation into regions with different levels of expression. A group of cells in the dorsal midline gradually ceases expressing tll, resulting in a split of the horseshoe into two dorsolateral domains. Within each of these domains, a roughly triangle-shaped, anterodorsal region shows the highest level of expression and is designated HL: posteroventral to HL is a domain with a lower level of expression, designated LL. The HL domain during stages 7 and 8 covers the dorsocentral part of the protocerebral neurectoderm. From within this region, the first groups of protocerebral neuroblasts delaminate during late stage 8. Subsequently other tll expressing neuroblasts delaminate in a well defined pattern. During stage 12, tll is expressed at a high level in a new region: the primordium of the optic lobe. This structure arises by invagination of the posterior procephalic ectoderm. tll expression remains high in the optic lobe throughout late embryonic development and can also be seen in the optic lobe in the late third instar larva (Rudolph, 1997).

tailless is activated by the torso pathway. Torso, a receptor for the putative ligand Trunk, causes a phosphorylation cascade that ultimately results in the activation of transcription of tailless. Two additional factors are involved: activation and repression by Bicoid in the anterior domain and repression by Dorsal in the central domain (Liaw, 1993).

Which genes are the targets of tailless? In the anterior domain, tailless represses fushi tarazu, hunchback and deformed, as well as hedgehog, and helps to establish the borders of expression of head gap genes, like orthodenticle.

In the posterior, Tailless acts on gap genes knirps, Krüppel and giant, setting up the posterior borders of expression (Kuelskamp, 1991). POU domain genes pdm1 and pdm2 are also regulated by tailless. tailless appears to activate caudal, forkhead and hunchback in the posterior, and is implicated in the activation of the seventh stripes of even-skipped, hairy, paired and fushi tarazu . Tailless also appears to activate T-related gene, a Drosophila Brachyury homolog.

The control of cell fate in the embryonic visual system by atonal, tailless and EGFR signaling

The transcription factors encoding genes tailless (tll), atonal (ato), sine oculis (so), eyeless (ey) and eyes absent (eya), and Efgr signaling play a role in establishing the Drosophila embryonic visual system. The embryonic visual system consists of the optic lobe primordium, which, during later larval life, develops into the prominent optic lobe neuropiles, and the larval photoreceptor (Bolwig's organ). Both structures derive from a neurectodermal placode in the embryonic head. Expression of tll is normally confined to the optic lobe primordium, whereas ato appears in a subset of Bolwig's organ cells that are called Bolwig's organ founders. Phenotypic analysis of tll loss- and gain-of-function mutant embryos using specific markers for Bolwig's organ and the optic lobe, reveals that tll functions to drive cells to an optic lobe fate, as opposed to a Bolwig's organ fate. Similar experiments indicate that ato has the opposite effect, namely driving cells to a Bolwig's organ fate. Since tll and ato do not regulate one another, a model is proposed wherein tll expression restricts the ability of cells to respond to signaling arising from ato-expressing Bolwig's organ pioneers. The data further suggest that the Bolwig's organ founder cells produce Spitz (the Drosophila TGFalpha homolog) signal, which is passed to the neighboring secondary Bolwig's organ cells where it activates the Epidermal growth factor receptor signaling cascade and maintains the fate of these secondary cells. The regulators of tll expression in the embryonic visual system remain elusive: no evidence for regulation by the 'early eye genes' so, eya and ey, or by Egfr signaling is found (Daniel, 1999).

The Drosophila visual system comprises the adult compound eye, the larval eye (Bolwig's organ) and the optic lobe (a part of the brain). All of these components are recognizable as separate primordia during late stages of embryonic development. These components originate from a small, contiguous region in the dorsal head ectoderm. During the extended germband stage, the individual components of the visual system can be distinguished morphologically as well as by spatially localized expression of the homeobox gene so and the adhesion molecule Fas II. Initially centered as an unpaired, oval domain straddling the dorsal midline, the anlage of the visual system subsequently elongates in the transverse axis and narrows in the anteroposterior axis. By late gastrulation (stage 8), the anlage occupies two bilaterally symmetric stripes that are anterior and adjacent to the cephalic furrow. The domain of so expression at this stage contains two regions with a high expression level [ol_ex (the external fold of the optic lobe) and ol_in]. Only these two regions will ultimately give rise to the optic lobe and Bolwig's organ; the so-positive cells dorsal and posterior to these domains will either form part of the dorsal posterior head epidermis (dph) or undergo apoptotic cell death. During the extended germband stage, the anlage of the visual system expands further ventrally until, around stage 10, it reaches the equator (50% in the dorsoventral axis) of the embryo. Shortly thereafter, ol_in, the portion of the anlage that will give rise to most of the optic lobe and Bolwig's organ, reorganizes into a placode of high cylindrical epithelial cells that differ in shape from the surrounding more squamous cells of the head ectoderm. During stage 12, this placode starts to invaginate, forming a V-shaped structure with an anterior lip (ol_al) and a posterior lip (ol_pl). Bolwig's organ, which consists of a small cluster of sensory neurons, derives from the basal part of the posterior lip and can be recognized during stage 12 as a distinct, dome-shaped protrusion. During stage 13, invagination of the optic lobe separates it from the head ectoderm; only the cells of Bolwig's organ remain in the ectoderm. The ectodermal region ol_ex is also internalized and forms an external 'cover' of the optic lobe; many cells of this population undergo apoptosis (Daniel, 1999).

The tll gene is expressed in a dynamic pattern in the protocerebral neurectoderm. In the posterior, this region overlaps part of the anlage of the visual system, in particular that part that will give rise to the anterior lip of the optic lobe. The anterior lip of the optic lobe upregulates expression of tll during stage 12. In addition, the posterior lip of the optic lobe, does not expressed tll at an earlier stage, now switches on this gene. Expression of tll in the posterior lip is patchy, with some cells expressing the gene at a higher level than others. The Bolwig's organ primordium does not express tll. During later embryonic stages and during larval development, tll expression remains strong in the optic lobe, but is never detected in the Bolwig's organ. Also the primordium of the eye disc, which expresses tll during larval stages, is devoid of this expression during embryonic development (Daniel, 1999).

tll controls a switch between optic lobe and Bolwig's organ cell fate. Loss of zygotic tll activity results in an absence of most of the protocerebrum of the brain (Younossi-Hartenstein, 1997). In addition, the visual system of the late tll embryo shows a dramatic phenotype, namely the transformation of optic lobe into Bolwig's organ. In wild-type embryos, the neuronal marker 22C10 (see Futsch) labels 12 neurons and their axons that project towards the optic lobe. In tll mutant embryos, the number of cells in the Bolwig's organ is dramatically increased (by a factor of 2-3), while the optic lobe, marked by anti-Crumbs or a PlacZ insertion in so, is absent. Use of antibodies to FasII and Crb, which label the apical surface of the optic lobe, and not that of the Bolwig's organ, allowed an analysis of how the phenotype unfolds in the tll mutant embryo. Abnormalities first become apparent during stage 11, when FasII expression increases strongly in the domain of the anterior lip of the optic lobe placode, a region that normally ceases to express FasII. In spite of this abnormal expression, the optic lobe placode appears to invaginate normally. As a results, in stage 13 tll mutant embryos, a Crb-positive vesicle can be seen subjacent to the head epidermis. During stage 14, all cells of this aberrant vesicle activate expression of the neuronal marker 22C10 and lose Crb expression, revealing that these cells are Bolwig's organ cells. Overexpression of tll under the control of a heat-shock promoter has an effect opposite that seen in the absence of tll activity. An additional consequence of tll overexpression is that the optic lobes become located more dorsally and fused in the dorsal midline. This 'cyclops' phenotype most likely arises because the dorsomedial cells, which normally die or form part of the head epidermis, now express optic lobe markers and become an integral part of the optic lobe. The results of both loss and gain of tll expression are consistent with the interpretation that tll is required to drive cells of the anlage, which would otherwise become photoreceptor neurons of Bolwig's organ, to develop as optic lobe cells (Daniel, 1999).

atonal is expressed in and required for the development of Bolwig's organ. ato is expressed in the head in several small cell clusters, one of which is a group of three to four cells that is part of the Bolwig's organ primordium. Expression of ato in this domain begins during stage 11 and continues until stage 12. Initially, a group of 6-8 cells faintly expresses ato. By stage 12, their number has decreased to 3 cells. During this period, ato-expressing cells can be seen as a small group of cells within the dome-shaped Bolwig's organ primordium. Loss of ato function results in the absence of Bolwig's organ. Thus, similar to what has been demonstrated for the compound eye, even though only a small subset of photoreceptors actually expresses ato, lack of ato function results in absence of all photoreceptors. Since Bolwig's organ is enlarged in a tll mutant background, it was asked whether tll inhibits ato expression. The number and pattern of ato-positive cells in tll mutants is found to be normal. These results suggest that tll functions in parallel with, or downstream of ato in the development of the Bolwig's organ/optic lobe primordium (Daniel, 1999).

Epidermal growth factor receptor is activated in midline regions of the head neurectoderm, in particular in the anlage of the visual system. Moreover, increased and/or ectopic activation of Egfr results in a 'cyclops' phenotype very similar to what has been described for ectopic tll expression. Egfr signaling has been shown to be required in both chordotonal organs and compound eye for the inductive signaling triggered by ato expression. Two questions raised by these observations have been investigated: (1) is Egfr signaling required for tll expression in the optic lobe and (2) is Egfr signaling involved in the recruitment of the secondary (non-atonal-expressing) Bolwig's organ cells? The answer to both these questions is no. tll expression is unaltered when levels of Egfr signaling are manipulated, suggesting that Egfr signaling is not required for tll expression. To investigate the second question, a test was performed for the presence of Egfr-relevant mRNAs or proteins: Rhomboid mRNA, which would be expected to be present only in the signaling cells, and phosphorylated MAPK, Pointed and Argos mRNAs, which would be expected to be expressed in all cells receiving an Egfr-mediated signal. In stage 12 embryos, rho is expressed only in the small group of Bolwig's organ founder cells (the same cells expressing ato). In contrast, activated (phosphorylated) MAPK is present in a larger cluster of cells including the entire Bolwig's organ and part of the adjacent optic lobe. Consistent with this, pnt and aos, both known to be switched on in cells receiving the Spi signal, are expressed at the same stage throughout the entire Bolwig's organ primordium. These gene expression and MAPK activation patterns are consistent with the idea that the Spi signal is activated by rho in the Bolwig's organ founders and passed to the neighboring secondary Bolwig's organ cells where it activates the Egfr signaling cascade. Supporting this view, only 3-4 photoreceptor neurons are found in the Bolwig's organ of embryos lacking rho or spi; furthermore, the size of the posterior lip of the optic lobe is reduced in such embryos. The fact that absence of secondary Bolwig's organ cells in rho or spi mutant embryos can be rescued by blocking cell death in the background of a deficiency that takes out the reaper complex of genes indicates that the Spi signal is not necessary for the specification (recruitment) of secondary Bolwig's organ cells, but rather, for their maintenance (Daniel, 1999).

While the maternal patterning systems that regulate tll during its blastoderm expression have been determined, the genes required to turn on tll at a later stage in the visual system are not known. Candidates are the 'early eye genes', so, eya and ey, which encode transcription factors expressed in the embryonic visual system and in the larval eye disc in front of the morphogenetic furrow. The expression of these genes was analyzed in the visual system anlage, and tll expression was examined in embryos mutant for each of these genes. tll expression in the optic lobe does not depend on any of these three genes. It was also concluded that ey and so, which have been shown to interact with each other during eye disc determination, must act independently in embryonic visual system development, since they are expressed in those primordia in non-overlapping patterns (Daniel, 1999).

Although so is expressed initially in the entire visual system anlage, during later stages its expression becomes increasingly restricted to subsets of visual system progenitors. Thus, during stage 11, when a morphologically distinct optic lobe placode first becomes visible, the domain of so expression retreats to the posterior lip of this placode; slightly later its expression is limited to only the Bolwig's organ, where it is maintained until stage 13. eya expression is initiated during the late blastoderm stage in a trapezoidal field in the dorsomedial head region that includes the visual system anlage, as well as progenitors of the medial brain. Beginning during gastrulation (stage 6/7), the eya domain becomes divided into an anterior stripe and a narrow posterior stripe immediately anterior to the cephalic furrow that widens laterally; this posterior domain will become part of the posterior lip of the optic lobe, including Bolwig's organ. eya expression continues in the optic lobe until stage 12 and in Bolwig's organ until stage 13. Embryos that lack either so or eya exhibit defects in the portions of the visual system where these genes are expressed. In both mutants, development proceeds normally until stage 11, when the posterior lip of the optic lobe (ol_pl) would normally start to invaginate. In eya and so embryos, invagination of the optic lobe placode does not take place and differentiation markers characteristic of the lobe are not expressed. In conclusion, so and eya, although expressed coincidental with tll, are not required for its activation. ey plays no role in the embryonic visual system (Daniel, 1999).

Since tll is a nuclear receptor transcription factor, it must function to block the effect of signaling from the founder cells (which is mediated at least in part by Egfr) at the transcriptional level. In the posterior of the blastoderm stage embryo, tll has been shown to function directly as both a repressor (of Kruppel, knirps and Ubx) and as an activator (of hunchback). Additional activator effects of tll (not yet demonstrated to be direct) have been shown for brachyenteron at the posterior of the blastoderm embryo, and for the proneural gene lethal of scute in the brain. Thus, in the optic lobe, tll could repress genes that would in its absence be activated by Egfr signaling, and/or activate genes that would block receipt, or execution, of the signal (Daniel, 1999 and references).

GENE STRUCTURE

Bases in 5' UTR - 189

Bases in 3' UTR - 442

PROTEIN STRUCTURE

Structural Domains

The Tailless repressor has an N-terminal DNA binding domain and a C-terminal hormone binding domain. It is identified as a steroid receptor superfamily protein (Pignoni, 1990). Tailless is an orphan nuclear receptor, meaning that although it possesses a hormone binding domain, no hormone has been identified that can bind to this domain. Although the ligand binding domain is conserved, the effects of mutation are felt most strongly in the DNA binding domain. The ligand binding domain of Tailless and mammalian homolog contains a conserved putative dimerization domain (consisting of seven heptad repeats). There is some evidence that the protein binds DNA as a dimer (Diaz, 1996).

date revised: 1 January 2024

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