BIOLOGICAL OVERVIEW

The protein Seven-up is a so-called orphan nuclear receptor belonging to the steroid receptor superfamily that includes ligand dependent transcription factors such as the vertebrate glucocorticoid receptor, the thyroid hormone receptor and the retinoic acid receptor. Why does the term "orphan" nuclear receptor apply to SVP? For an answer, look to the interaction with ligand. For many of these transcription factors there is a cytoplasmic phase and a nuclear phase, induced by interaction with ligand, the small steroid molecules that trigger the activation of receptors. In the nuclear phase steroid receptors act as transcription factors. In the case of Seven-up, the ligand for its activation (if there is one) is currently unknown, and thus the appelation "orphan" nuclear receptor.

Seven-up is required for development of four of the eight photoreceptors that develop in each ommatidium of the eye, the R3/R4 pair and the R1/R6 pair. These photoreceptors are determined after R8 and the R2/R5 pair, but before the determination of R7. Two recent studies show the requirement for Ras signaling in seven-up function (Begemann, 1995 and Kramer, 1995). The Ras pathway is required in R7 cells to mediate between the Sevenless receptor and the proteins Pointed and Yan, but Sevenless signaling is not required for Seven-up function (Hiromi, 1993).

The activation of Ras pathway signaling on behalf of Seven-up seems to require the activity of Egf-r, the Drosophila homolog of the vertebrate epidermal growth factor receptor. Like Sevenless signaling, Egf-r activates the Ras pathway. A genetic trick has been used to provide evidence for the involvement of EGF-R. When seven-up is expressed in outer cone cells (the non-neuronal components of ommatidia), a neuronal transformation takes place, and ectopic R7 cells are induced. Suppressors of this transformation are mutants in EGF-R. This indicates that the neuronal transformation of cone cells requires EGF-R, and by inference, normal function of seven-up is similarly thought to require EGF-R (Begemann, 1995).

It is still not clear what role the Ras pathway plays in the activation of Seven-up function, nor is it clear how seven-up is activated in photoreceptor precursors. One identified target of Seven-up in cell determination is pipsqueak (Weber, 1995), but its role in cell fate determination is not well undersood. Because Seven-up is an orphan nuclear receptor, it would be of interest to see whether Seven-up function is tied to molting signals that are driven by other members of the nuclear receptor superfamily.

The Drosophila Malpighian tubules (MTs), form a simple excretory epithelium comparable in function to kidneys in vertebrates. MTs function as the insect kidney both in the larva and the adult. They consist of two pairs of blind ending tubes that are composed of a single cell-layered epithelium made up of a tightly controlled number of cells. The tubules float in the hemolymph from where they take up nitrogenous waste that is excreted as uric acid. During embryogenesis, MTs evert as four protuberances from the hindgut primordium, the proctodeum. The everting tubules grow by cell proliferation, which takes place in a few cells along the tubules and extensively in a distal proliferation domain located in the tip region of the tubules. Cell ablation experiments and studies on the pattern of cell division have shown that a single large cell at the distal end of each tubule, termed the tip cell, is decisive for controlling the proliferation of its neighboring cells. The tip cell that differentiates into a cell with neuronal characteristics during later stages of development arises by division of a tip mother cell that is selected in the tubule primordium by lateral inhibition involving the Notch signaling pathway and the transcription factor Kruppel (Kr). It has been suggested that the tip cell sends a mitogenic signal to adjacent cells in the distal proliferation zone. It has remained elusive, however, what the signal is or what its target molecules in the signal-receiving cells could be and how cell proliferation during MT morphogenesis is regulated. Seven-up is shown to be a key component that becomes induced in response to mitogenic EGF receptor signaling activity emanating from the tip cell. Seven-up (Svp) in turn is capable of regulating the transcription of cell cycle regulators (Kerber, 1998).

Two types of transcripts have been characterized at the svp locus: svp type I encodes a protein with both a DNA-binding domain and a ligand binding domain (LBD); and svp type II diverges from type I in the middle of the LBD. During MT development, both isoforms of svp are expressed in the same pattern. Their expression can first be detected in embryonic stage 10 on one side of the outgrowing tubules and, later, during the eversion, in a group of about six to eight cells in the tip region. Analysis of the MTs of amorphic svp mutants reveals a reduction of the tubule cell number, as compared to wild type. Anti-Kr antibody stainings reveal that the MT precursor cells are specified normally in svp mutants, indicating that the cause for the defect is not attributable to cell death, which might lead to a size reduction of the tubule primordium. Furthermore, tip cell determination occurs normally in the mutants. Rather, pulse labeling with BrdU, suggests that the reduction of the cell number results from a failure of proper cell divisions. In wild-type embryos, BrdU incorporation occurs asymmetrically on one side of each tubule in proliferating cells. When MT eversion begins in stage 10, the dividing cells in the distal tip region continue to incorporate BrdU extensively until the end of stage 13 when division stops. Subsequently, intense BrdU incorporation occurs in all of the tubule cells during endomitotic cycles that take place in a proximal to distal direction in the tubules. In svp mutants relatively normal BrDU incorporation is found during the initial cell divisions, but subsequently it is strongly reduced indicating a failure of DNA replication. In the later occurring endomitotic cycles, the BrdU pattern is normal again, indicating that a specific block of S phases occurs in dividing cells, but not during the endomitotic cycles. These results suggest that svp, which is expressed in the proliferation domains marked by BrdU, might be an integral component of the regulatory network that regulates division in the cells that receive the mitogenic signal from the tip cell (Kerber, 1998).

To identify the nature of the mitogenic tip cell signal a screen was carried out for genes specifically active in the tip cells. The genes rhomboid (rho) and Star (S), which encode transmembrane proteins involved in epidermal growth factor receptor (EGFR) signaling, are expressed in the tip cells and both are required for MT growth. When the tubules start to evert, rho and S are expressed in the tip mother cell; subsequently rho is strongly expressed in the tip cell and S in the tip cell and its former sister cell. An analysis of the MTs in the corresponding amorphic mutants reveals a strong decrease of cells in rho mutants and a weaker decrease in S mutants. In a rho;S double mutant, the tubules are barely detectable, indicating that rho and S activities are essential (albeit redundant) components controlling MT growth. The tubule phenotype of rho;S double mutants is very similar to that of EGFR mutants, which also show a drastic decrease in the tubule cell number. As in svp mutants, the allocation and the differentiation of the tip cells are normal in the receptor mutants, indicating that receptor activity is not required for tip cell determination and differentiation. The reduction of the tubule cell number in EGFR mutants is due to a failure of proper cell divisions. No BrdU incorporation occurs in EGFR mutants in the outbudding tubules at the time when cells divide in wild-type embryos. However, BrdU incorporation occurs again much later during the endomitotic cycles, indicating that in EGFR muants, a specific defect in DNA replication exists in cells that would normally divide (Kerber, 1998).

Rho and S process a membrane-bound form of the activating ligand of the receptor, the TGFalpha-like Spi protein, to generate the secreted form of Spi (sSpi). sSpi is then proposed to diffuse to neighboring cells, bind to the receptor, and activate target genes via the Ras/Raf signaling cassette; these include the primary target gene pointedP1 (pntP1), encoding an ETS domain transcription factor, and the secondary target gene argos (aos), encoding a negatively acting ligand of the receptor. These downstream components of the pathway are also active during tubule development. pntP1 and aos are expressed during stage 10 in six to eight cells on one side of the MTs overlapping the rho and S expression domains and later, weakly in several cells in the tip region. In amorphic aos mutants a slightly larger number of tubule cells are observed, whereas amorphic pnt mutants show a decrease of tubule cells. These results indicate that for controlling cell proliferation and cell determination, the same key components of the EGFR cascade are required (Kerber, 1998).

These findings suggest that the EGFR pathway provides the mitogenic tip cell signal that activates svp expression and regulates cell division. To test this hypothesis, svp expression was analyzed in EGFR mutants and ectopic expression studies were performed with various members of the pathway using the UAS-Gal4 system. svp is absent in mutants for the Egfr. It is still expressed, however, in amorphic pnt mutants, suggesting that Svp is a transcriptional regulator that is likely to be activated in parallel to the primary transcription factor PntP1 in the signaling cascade. If sSpi activity is provided ectopically in all of the tubule cells, the svp expression domain becomes dramatically expanded and an increase of the tubule cell number is observed. Similar, although slightly weaker effects on svp transcription and the number of tubule cells could be observed upon ubiquitous expression of other components of the EGFR pathway, like Rho, activated Ras, or Raf. Conversely, when a dominant-negative Ras allele is ectopically expressed in all of the tubule cells, svp transcription became strongly reduced. Ectopic expression of svp in an Egfr mutant background restores the tubule cell number to a considerable extent. These results provide strong evidence that svp is a downstream target gene of EGFR signaling in the tubules (Kerber, 1998).

If Svp is expressed ectopically in wild-type MTs, an increased number of tubule cells is obtained. BrdU incorporation studies indicate that this increased cell number results from extra cell divisions, indicating that svp is both necessary and sufficient to induce cell proliferation in the MTs. Analyses were carried out to further elucidate how the EGFR pathway and svp control cell proliferation, and whether these developmental regulators have an impact on components of the cell cycle machinery during MT growth. Two genes are limiting key components of the cell cycle during the period when the MT cells proliferate: string (stg), which encodes a Cdc25 phosphatase involved in the regulation of the G2/M transition, and cyclin E (cycE), which regulates the G1/S transition. In situ hybridization reveals that both genes are expressed asymmetrically in the everting tubules and subsequently in the distal proliferation zone. These expression domains match the svp expression domain. With the onset of the endomitotic cycles, a second phase of cycE expression occurs from proximal to distal in the tubules. In EGFR mutants, the transcriptional activation of stg and cycE, which occurs in the tubule proliferation domains in wild type, cannot be detected. This correlates with a strong reduction of BrdU incorporation and the dramatic reduction of the tubule cell number in Egfr mutants. During the subsequent endomitotic cycles, expression of cycE is not affected, indicating a specific function of EGFR signaling in activating early cycE expression. In svp mutants, the expression of stg and cycE is reduced (most likely reflecting that Svp is only one of the regulators that transmits the mitogenic EGFR signal); however, in MTs in which svp is ectopically expressed, stg becomes transcriptionally misexpressed in the cells that undergo extra cell divisions. Similar (although weaker) misexpression is obtained with cycE. However, extra cell divisions can only be obtained early during MT outgrowth, suggesting that other regulators limit cell proliferation during later stages of MT development (Kerber, 1998).

It is not known whether Svp, whose function has been characterized initially in the context of photoreceptor development in the eye also plays a role for cell proliferation during eye imaginal disc development. In MT there must be other factors in addition to Svp that are dependent on EGF signaling and are involved in MT growth. This is apparent from the finding that the svp mutant phenotype is less severe than that of Egfr mutants. Those predicted factors might include other steroid hormone receptors that interact with Svp as cofactors. Studies on ecdysone signaling pathways show that Svp can heterodimerize with subunits of the ecdysone receptor and regulate gene expression. Whether ecdysone-based signaling pathways also play a role in controlling cell proliferation in the MT is not known. Once cell proliferation is completed, the tubule cells elongate as a result of cell rearrangement and long thin tubes are generated with only two or three cells surrounding the lumen. An additional role of EGFR signaling during later stages of MT development cannot be excluded. This is consistent with recent results obtained with an antibody against the activated form of MAP kinase, which visualizes the activated state of receptor tyrosine kinase (RTK) signaling pathways and shows a rather uniform actived ERK pattern in all of the tubule cells. As there is no apparent tubule elongation defect in svp mutants, other downstream factors must be involved in mediating this potential aspect of EGFR signaling. In summary, these data provide a framework for further analysis of the molecular mechanisms that underlie the control of cell proliferation by developmental regulators during MT morphogenesis (Kerber, 1998).

Steroid hormone induction of temporal gene expression in Drosophila brain neuroblasts generates neuronal and glial diversity

An important question in neuroscience is how stem cells generate neuronal diversity. During Drosophila embryonic development, neural stem cells (neuroblasts) sequentially express transcription factors that generate neuronal diversity; regulation of the embryonic temporal transcription factor cascade is lineage-intrinsic. In contrast, larval neuroblasts generate longer ~50 division lineages, and currently only one mid-larval molecular transition is known: Chinmo/Imp/Lin-28+ neuroblasts transition to Syncrip+ neuroblasts. This study shows that the hormone ecdysone is required to down-regulate Chinmo/Imp and activate Syncrip, plus two late neuroblast factors, Broad and E93. Seven-up triggers Chinmo/Imp to Syncrip/Broad/E93 transition by inducing expression of the Ecdysone receptor in mid-larval neuroblasts, rendering them competent to respond to the systemic hormone ecdysone. Importantly, late temporal gene expression is essential for proper neuronal and glial cell type specification. This is the first example of hormonal regulation of temporal factor expression in Drosophila embryonic or larval neural progenitors (Syed, 2017).

This study shows that the steroid hormone ecdysone is required to trigger a major gene expression transition at mid-larval stages: central brain neuroblasts transition from Chinmo/Imp to Broad/Syncrip/E93. Furthermore, it was shown that Svp activates expression of EcR-B1 in larval neuroblasts, which gives them competence to respond to ecdysone signaling, thereby triggering this gene expression transition. Although a global reduction of ecdysone levels is likely to have pleiotropic effects on larval development, multiple experiments were performed to show that the absence or delay in late temporal factor expression following reduced ecdysone signaling is not due to general developmental delay. First, the EcR gene itself is expressed at the normal time (~56 hr) in the whole organism ecdysoneless¹ mutant, arguing strongly against a general developmental delay. Second, a type II neuroblast seven-up mutant clone shows a complete failure to express EcR and other late factors, in the background of an entirely wild type larvae; this is perhaps the strongest evidence that the phenotypes that are described are not due to a general developmental delay. Third, lineage-specific expression of EcR dominant negative leads to loss of Syncrip and E93 expression without affecting Broad expression; the normal Broad expression argues against a general developmental delay. Fourth, live imaging was used to directly measure cell cycle times, and it was found that lack of ecdysone did not slow neuroblast cell cycle times. Taken together, these data support the conclusion that ecdysone signaling acts directly on larval neuroblasts to promote an early-to-late gene expression transition (Syed, 2017).

The role of ecdysone in regulating developmental transitions during larval stages has been well studied; it can induce activation or repression of suites of genes in a concentration dependent manner. Ecdysone induces these changes through a heteromeric complex of EcR and the retinoid X receptor homolog Ultraspiracle. Ecdysone is required for termination of neuroblast proliferation at the larval/pupal transition, and is known to play a significant role in remodeling of mushroom body neurons and at neuromuscular junctions. This study adds to this list another function: to trigger a major gene expression transition in mid-larval brain neuroblasts (Syed, 2017).

Does ecdysone signaling provide an extrinsic cue that synchronizes larval neuroblast gene expression? Good coordination of late gene expression is not seen, arguing against synchronization. For example, Syncrip can be detected in many neuroblasts by 60 hr, whereas Broad appears slightly later at ~72 hr, and E93 is only detected much later at ~96 hr, by which time Broad is low. This staggered expression of ecdysone target genes is reminiscent of early and late ecdysone-inducible genes in other tissues. In addition, for any particular temporal factor there are always some neuroblasts expressing it prior to others, but not in an obvious pattern. It seems the exact time of expression can vary between neuroblasts. Whether the pattern of response is due to different neuroblast identities, or a stochastic process, remains to be determined (Syed, 2017).

It has been shown preiously that the Hunchback-Krüppel-Pdm-Castor temporal gene transitions within embryonic neuroblasts are regulated by neuroblast-intrinsic mechanisms: they can occur normally in neuroblasts isolated in culture, and the last three factors are sequentially expressed in G2-arrested neuroblasts. Similarly, optic lobe neuroblasts are likely to undergo neuroblast-intrinsic temporal transcription factor transitions, based on the observation that these neuroblasts form over many hours of development and undergo their temporal transitions asynchronously. In contrast, this study shows that ecdysone signaling triggers a mid-larval transition in gene expression in all central brain neuroblasts (both type I and type II). Although ecdysone is present at all larval stages, it triggers central brain gene expression changes only following Svp-dependent expression of EcR-B1 in neuroblasts. Interestingly, precocious expression of EcR-B1 (worniu-gal4 UAS-EcR-B1) did not result in premature activation of the late factor Broad, despite the forced expression of high EcR-B1 levels in young neuroblasts. Perhaps there is another required factor that is also temporally expressed at 56 hr. It is also noted that reduced ecdysone signaling in ecd^ts mutants or following EcRDN expression does not permanently block the Chinmo/Imp to Broad/Syncrip/E93 transition; it occurs with variable expressivity at 120-160 hr animals (pupariation is significantly delayed in these ecd^ts mutants), either due to a failure to completely eliminate ecdysone signaling or the presence of an ecdysone-independent mechanism (Syed, 2017).

A small but reproducible difference was found in the effect of reducing ecdysone levels using the biosynthetic pathway mutant ecd^ts versus expressing a dominant negative EcR in type II neuroblasts. The former genotype shows a highly penetrant failure to activate Broad in old neuroblasts, whereas the latter genotype has normal expression of Broad (despite failure to down-regulate Chinmo/Imp or activate E93). This may be due to failure of the dominant negative protein to properly repress the Broad gene. Differences between EcRDN and other methods of reducing ecdysone signaling have been noted before (Syed, 2017).

Drosophila Svp is an orphan nuclear hormone receptor with an evolutionarily conserved role in promoting a switch between temporal identity factors. In Drosophila, Svp it is required to switch off hunchback expression in embryonic neuroblasts, and in mammals the related COUP-TF1/2 factors are required to terminate early-born cortical neuron production, as well as for the neurogenic to gliogenic switch. This study showed that Svp is required for activating expression of EcR, which drives the mid-larval switch in gene expression from Chinmo/Imp to Syncrip/Broad/E93 in central brain neuroblasts. The results are supported by independent findings that svp mutant clones lack expression of Syncrip and Broad in old type II neuroblasts (Tsumin Lee, personal communication to Chris Doe). Interestingly, Svp is required for neuroblast cell cycle exit at pupal stages, but how the early larval expression of Svp leads to pupal cell cycle exit was a mystery. The current results provide a satisfying link between these findings: Svp was shown to activate expression of EcR-B1, which is required for the expression of multiple late temporal factors in larval neuroblasts. Any one of these factors could terminate neuroblast proliferation at pupal stages, thereby explaining how an early larval factor (Svp) can induce cell cycle exit five days later in pupae. It is interesting that one orphan nuclear hormone receptor (Svp) activates expression of a second nuclear hormone receptor (EcR) in neuroblasts. This motif of nuclear hormone receptors regulating each other is widely used in Drosophila, C. elegans, and vertebrates (Syed, 2017).

The position of the Svp+ neuroblasts varied among the type II neuroblast population from brain-to-brain, suggesting that Svp may be expressed in all type II neuroblasts but in a transient, asynchronous manner. This conclusion is supported by two findings: the svp-lacZ transgene, which encodes a long-lived β-galactosidase protein, can be detected in nearly all type II neuroblasts; and the finding that Svp is required for EcR expression in all type II neuroblasts, consistent with transient Svp expression in all type II neuroblasts. It is unknown what activates Svp in type II neuroblasts; its asynchronous expression is more consistent with a neuroblast-intrinsic cue, perhaps linked to the time of quiescent neuroblast re-activation, than with a lineage-extrinsic cue. It would be interesting to test whether Svp expression in type II neuroblasts can occur normally in isolated neuroblasts cultured in vitro, similar to the embryonic temporal transcription factor cascade (Syed, 2017).

Castor and its vertebrate homolog Cas-Z1 specify temporal identity in Drosophila embryonic neuroblast lineages and vertebrate retinal progenitor lineages, respectively (Mattar, 2015). Although this study shows that Cas is not required for the Chinmo/Imp to Syncrip/Broad/E93 transition, it has other functions. Cas expression in larval neuroblasts is required to establish a temporal Hedgehog gradient that ultimately triggers neuroblast cell cycle exit at pupal stages (Syed, 2017).

Drosophila embryonic neuroblasts change gene expression rapidly, often producing just one progeny in each temporal transcription factor window. In contrast, larval neuroblasts divide ~50 times over their 120 hr lineage. Mushroom body neuroblasts make just four different neuronal classes over time, whereas the AD (ALad1) neuroblast makes ~40 distinct projection neuron subtypes. These neuroblasts probably represent the extremes (one low diversity, suitable for producing Kenyon cells; one high diversity, suitable for generating distinct olfactory projection neurons). This study found that larval type II neuroblasts undergo at least seven molecularly distinct temporal windows. If it is assumed that the graded expression of Imp (high early) and Syncrip (high late) can specify fates in a concentration-dependent manner, many more temporal windows could exist (Syed, 2017).

This study illuminates how the major mid-larval gene expression transition from Chinmo/Imp to Broad/Syncrip/E93 is regulated; yet many new questions have been generated. What activates Svp expression in early larval neuroblasts - intrinsic or extrinsic factors? How do type II neuroblast temporal factors act together with Dichaete, Grainy head, and Eyeless INP temporal factors to specify neuronal identity? Do neuroblast or INP temporal factors activate the expression of a tier of 'morphogenesis transcription factors' similar to leg motor neuron lineages? What are the targets of each temporal factor described here? What types of neurons (or glia) are made during each of the seven distinct temporal factor windows, and are these neurons specified by the factors present at their birth? The identification of new candidate temporal factors in central brain neuroblasts opens up the door for addressing these and other open questions (Syed, 2017).

Seven-up-triggered temporal factor gradients diversify intermediate neural progenitors

Building a sizable, complex brain requires both cellular expansion and diversification. One mechanism to achieve these goals is production of multiple transiently amplifying intermediate neural progenitors (INPs) from a single neural stem cell. Like mammalian neural stem cells, Drosophila type II neuroblasts utilize INPs to produce neurons and glia. Within a given lineage, the consecutively born INPs produce morphologically distinct progeny, presumably due to differential inheritance of temporal factors. To uncover the underlying temporal fating mechanisms, type II neuroblasts' transcriptome was profiled across time. The results reveal opposing temporal gradients of Imp and Syp RNA-binding proteins (descending and ascending, respectively). Maintaining high Imp throughout serial INP production expands the number of neurons and glia with early temporal fate at the expense of cells with late fate. Conversely, precocious upregulation of Syp reduces the number of cells with early fate. Furthermore, this study reveals that the transcription factor Seven-up initiates progression of the Imp/Syp gradients. Interestingly, neuroblasts that maintain initial Imp/Syp levels can still yield progeny with a small range of early fates. It is therefore proposed that the Seven-up-initiated Imp/Syp gradients create coarse temporal windows within type II neuroblasts to pattern INPs, which subsequently undergo fine-tuned subtemporal patterning (Ren, 2017).

Temporal gradients of IGF-II mRNA-binding protein (Imp) and Syncrip (Syp) RNA-binding proteins have recently been described to promote early and late temporal fates, respectively, in mushroom body (MB) and antennal lobe (AL) lineages (Liu, 2015). Imp and Syp gradients oppose each other (Imp expression decreases and Syp increases over time), and they mutually inhibit each other's expression (Liu, 2015). It is unclear how the Imp-versus-Syp dominance is reversed and why the Imp-to-Syp switch occurs over different time courses in distinct NBs. Imp/Syp gradients control temporal fate in the MB by establishing a temporal gradient of Chinmo, a BTB-zinc finger nuclear transcription factor. In the AL lineages, while Imp/Syp gradients clearly promote early and late fates, it is not clear whether and how these RNA binding proteins specify all temporal fates in the rapidly changing AL lineages (Ren, 2017).

Canonical (type I) NBs produce post-mitotic neurons via budding off ganglion mother cells (GMCs), which each divides once into two neurons. By contrast, each of the 16 type II NBs (eight per brain hemisphere) first generates a series of intermediate neural progenitors (INPs). Each INP can, in turn, produce around five GMCs, thus giving rise to an INP sublineage that consists of a short sequence of neuronal and glial progeny. The complex type II pattern of neurogenesis mimics the production of neurons by mammalian neural stem cells through intermediate precursors (Ren, 2017).

Like type I NBs, type II NBs exhibit lineage identity and temporal fate. Labeling the progeny made by a type II NB (NB clone) reveals distinct lineage-characteristic morphology. Six type II NB lineages (DM1-DM6) originate from the dorsomedial posterior brain surface; the remaining two lineages (DL1 and DL2) arise from the dorsolateral posterior brain surface. An INP produces an invariant sequence of distinct sister neuron pairs, and successive INPs generate a similar, but not identical, neuronal series (Wang, 2014). These observations illustrate temporal fate diversification along both axes of NB and INP self-renewal. A cascade of three tTFs (Dichaete, Grainy head, Eyeless) governs temporal fates within the INP sublineages (Bayraktar, 2013). As to the extended axis of type II NB self-renewal, it is not clear whether a protracted tTF cascade exists or just gradients of proteins could guide the orderly derivation of variant INP sublineages (Ren, 2017).

In order to resolve the temporal fating mechanisms in type II NBs, this study profiled the transcriptome of type II NBs and uncovered 81 dynamic genes, including Imp and Syp. Imp and Syp were shown to promote early and late INP temporal fate, respectively. Two tTFs, Castor (Cas) and Seven-up (Svp), are critical for the initiation of Imp/Syp temporal gradients. Despite no progression of the Imp/Syp gradients, svp mutant clones carried INPs with multiple early fates. It is proposed that Cas/Svp-triggered Imp/Syp gradients confer coarse temporal fates to diversify INPs (Ren, 2017).

RNA-seq of type II NBs across larval development revealed 81 temporally dynamic genes, including Imp and Syp. Utilizing various neuronal classes characteristic of early or late INP sublineages, this study demonstrated that the opposing Imp/Syp gradients govern type II lineage temporal patterning. However, it is not clear whether absolute or relative levels of Imp and/or Syp confer specific temporal fates, or how a given Imp/Syp level is perceived by individual GMCs of the same INP origin. More sophisticated controls over Imp and Syp levels and finer temporal fate readouts are required to resolve these details. Nonetheless, svp mutant NBs, which maintain initial Imp/Syp levels, still undergo some limited temporal fate progression, as evidenced by the presence of various OL-elaboration neurons. It is therefore proposed that the rapidly progressing Imp/Syp gradients confer type II NBs with coarse temporal fates, which guide or permit subtemporal patterning among INPs born within a given Imp/Syp temporal window (Ren, 2017).

A brief series of Cas and Svp bursts initiates the prompt switch of Imp-versus-Syp dominance in type II NBs. Cas likely precedes Svp in its post-embryonic re-expression; however, the dynamic expression of Cas and Svp appear independently yet simultaneously controlled by an unknown temporal cue. Curiously, while Cas is dispensable, ectopic Cas delays the onset of Imp/Syp gradients. As the final NB tTF in the widely expressed embryonic cascade, Cas may be required for some embryonic INP fates that were not examined in this study. With regards to the role of Cas in Imp/Syp expression, it is hypothesized that the termination of Cas expression is important for proper Imp/Syp gradient progression. The Cas-GOF would thus expose the NBs to a prolonged Cas window. Precocious and continuous Svp expression did not accelerate or alter the Imp/Syp gradients, further implicating involvement of unknown temporal cues, not only in the regulation of dynamic Cas/Svp expressions, but also in confining the acute Svp action. It is speculated that the acute Svp signal can repress Imp as well as promote Syp and ultimately place Svp over Imp in their winner-take-all competition. Both the timing and intensity of the Cas/Svp bursts may vary among different NBs, which can potentially shape distinct Imp/Syp gradients in different neuronal lineages (Ren, 2017).

The Imp/Syp gradients in type II NBs are inherited by INPs born at distinct times and confer the INPs with their temporal identity. Each INP subsequently expresses a cascade of tTFs to assign its serially derived GMCs with distinct cell fates. Imp/Syp and their downstream effectors could interact with the INP tTFs to specify terminal temporal fates in post-mitotic cells. Multiple feedforward gene regulatory loops might also be involved to control the expression of terminal selector genes. Distinct Imp/Syp levels may specify different neuronal classes among the GMCs of the same birth order that inherit the same tTF from INPs. It is not possible to resolve such complex temporal fating mechanisms without further sophisticated single-cell lineage mapping tools (Ren, 2017).

Converging evidence indicates that conserved tTFs control neuronal diversity from Drosophila to mammalian species. Mammalian neural stem cells also show temporally patterned neurogenesis. The Ikaros family zinc finger 1, orthologs of Drosophila Hb, specify early temporal fate in both the retina and the cortex. A recent study showed that an ortholog of Drosophila castor, Casz1, promotes late neuronal fates in the mouse retina. The Chick Ovalbumin Upstream Promoter-Transcription Factors (COUP-TFI and II), mammalian orthologs of Drosophila svp, promote the temporal transition from neurogenesis to gliogenesis in neural stem cells. COUP-TFI also orchestrates the serial generation of distinct types of cortical interneurons. Loss of COUP-TFs resulted in overproduction of early-born neuronal fates, at the expense of late-born glial and interneuron fates. Thus, COUP-TFs seem to be functionally conserved to Drosophila svp (Ren, 2017).

Notably, a descending Imp gradient exists in mouse neural stem cells and governs temporal changes in stem cell properties. Given the use of INPs in both type II NB lineages and mammalian neurogenesis, it would be interesting to determine whether homologs of Imp/Syp/Chinmo play analogous roles in regulating the temporal fates of mammalian neural stem cells and whether these genes act with the conserved tTFs (Ren, 2017).

GENE STRUCTURE

There are two transcripts, types 1 and 2, differing in their 3' sequences. The transcripts are identical up to base number 1807, coding for amino acid number 452 (Mlodzik, 1990).

Bases in 3' UTR - 980 and 601 for transcripts type 1 and 2 respectively

PROTEIN STRUCTURE

date revised: 21 July 98 

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

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