Gene name - castor
Synonyms - ming
Cytological map position - 83C
Function - transcription factor
Symbol - cas
Genetic map position -
Classification - zinc finger
Cellular location - nuclear
|Recent literature||Wunderlich, Z., Bragdon, M.D., Vincent, B.J.,
White, J.A., Estrada, J. and DePace, A.H. (2015). Krüppel expression levels are maintained through compensatory evolution of shadow enhancers. Cell Rep [Epub ahead of print]. PubMed ID: 26344774
Many developmental genes are controlled by shadow enhancers-pairs of enhancers that drive overlapping expression patterns. This study hypothesized that compensatory evolution can maintain the total expression of a gene, while individual shadow enhancers diverge between species. This study analyzed expression driven by orthologous pairs of shadow enhancers from Drosophila melanogaster, Drosophila yakuba, and Drosophila pseudoobscura that control expression of Krüppel, a transcription factor that patterns the anterior-posterior axis of blastoderm embryos. The expression driven by the pair of enhancers was conserved between these three species, but expression levels driven by the individual enhancers was not. Using sequence analysis and experimental perturbation, it was shown that each shadow enhancer is regulated by different transcription factors. These results support the hypothesis that compensatory evolution can occur between shadow enhancers, which has implications for mechanistic and evolutionary studies of gene regulation
|Wu, H., Manu, Jiao, R. and Ma, J. (2015). Temporal and spatial dynamics of scaling-specific features of a gene regulatory network in Drosophila. Nat Commun 6: 10031. PubMed ID: 26644070
A widely appreciated aspect of developmental robustness is pattern formation in proportion to size. But how such scaling features emerge dynamically remains poorly understood. This study generated a data set of the expression profiles of six gap genes in Drosophila melanogaster embryos that differ significantly in size. Expression patterns exhibit size-dependent dynamics both spatially and temporally. A dynamic emergence of under-scaling in the posterior was uncovered, accompanied by reduced expression levels of gap genes near the middle of large embryos. Simulation results show that a size-dependent Bicoid gradient input can lead to reduced Kruppel expression that can have long-range and dynamic effects on gap gene expression in the posterior. Thus, for emergence of scaled patterns, the entire embryo may be viewed as a single unified dynamic system where maternally derived size-dependent information interpreted locally can be propagated in space and time as governed by the dynamics of a gene regulatory network.
|Kozlov, K., Gursky, V. V., Kulakovskiy, I. V., Dymova, A. and Samsonova, M. (2015). Analysis of functional importance of binding sites in the Drosophila gap gene network model. BMC Genomics 16 Suppl 13: S7. PubMed ID: 26694511
The statistical thermodynamics based approach provides a promising framework for construction of the genotype-phenotype map in many biological systems. Among important aspects of a good model connecting the DNA sequence information with that of a molecular phenotype (gene expression) is the selection of regulatory interactions and relevant transcription factor bindings sites. As the model may predict different levels of the functional importance of specific binding sites in different genomic and regulatory contexts, it is essential to formulate and study such models under different modeling assumptions. This study elaborates a two-layer model for the Drosophila gap gene network and includes in the model a combined set of transcription factor binding sites and concentration dependent regulatory interaction between gap genes hunchback and Kruppel. The new variants of the model are more consistent in terms of gene expression predictions for various genetic constructs in comparison to previous work. The functional importance of binding sites was quantified by calculating their impact on gene expression in the model, and how these impacts correlate across all sites were calculated under different modeling assumptions. The assumption about the dual interaction between hb and Kr leads to the most consistent modeling results, but, on the other hand, may obscure existence of indirect interactions between binding sites in regulatory regions of distinct genes. The analysis confirms the previously formulated regulation concept of many weak binding sites working in concert. The model predicts a more or less uniform distribution of functionally important binding sites over the sets of experimentally characterized regulatory modules and other open chromatin domains (Kozlov, 2015).
|Averbukh, I., Lai, S. L., Doe, C. Q. and Barkai, N. (2018). A repressor-decay timer for robust temporal patterning in embryonic Drosophila neuroblast lineages. Elife 7. PubMed ID: 30526852
Biological timers synchronize patterning processes during embryonic development. In the Drosophila embryo, neural progenitors (neuroblasts; NBs) produce a sequence of unique neurons whose identities depend on the sequential expression of temporal transcription factors (TTFs), including Hb, Kr, Pdm and Cas. The stereotypy and precision of NB lineages indicate reproducible TTF timer progression. This study combines theory and experiments to define the timer mechanism. The TTF timer is commonly described as a relay of activators, but its regulatory circuit is also consistent with a repressor-decay timer, where TTF expression begins when its repressor decays. Theory shows that repressor-decay timers are more robust to parameter variations than activator-relay timers. This motivated an experimental comparison of the relative importance of the relay and decay interactions in-vivo. Comparing WT and mutant NBs at high temporal resolution, this study show that the TTF sequence progresses primarily by repressor-decay. It is suggested that need for robust performance shapes the evolutionary-selected designs of biological circuits.
It is suggested that Castor and Hunchback act in a cooperative, non-overlapping manner to control POU gene expression during Drosophila CNS development. By silencing pdm expression in early and late NB sublineages, Hb and Cas establish three pan-CNS compartments whose cellular constituents are marked by the sequential expression of Hb, Pdm, and Cas. Embryos lacking Hb function suffer multiple defects. During CNS development, hb- embryos fail to develop labial and thoracic ganglia; gaps form between the subesophageal maxillary neuromeres and the abdominal ganglia. In addition, the seventh and eighth abdominal segments are fused due to the absence of parasegment 13. Missing are the highly ordered ventral cord axon scaffolds made up of longitudinal connective and commissural fascicles. Given the apparent transient overlap between Hb and Pdm-1 expression in the CNS and Hb's established role as a repressor of pdm expression in the cellular blastoderm, it is likely that Hb also silences pdm expression during early NB sublineage development. Pdm-1 expression patterns in hb- embryos confirm the hypothesis that Hb functions as a repressor during CNS development. In the absence of Hb, Pdm-1 is ectopically expressed in all CNS ganglia (Kambadur, 1998).
Likewise, Castor represses pdm. To determine if Cas is a pdm repressor, Pdm-1 and Pdm-2 expression were analyzed in cas null embryos. In stage 9 and in younger embryos, no differences were detected between the cas- and wild-type expression patterns of Pdm-1 or -2. However, starting at stage 10, NBs fail to terminate expression of both Pdms. Ectopic Pdm expression is observed in most, if not all, late developing sublineages in all CNS ganglia. The sustained Pdm expression is most likely due to transcriptional derepression. In a cas- background transgenes bearing a pdm-1 proximal promoter fragment are ectopically expressed in NBs during late sublineage development. This result demonstrates that the enhancer(s) within the 6.3 kb regulatory DNA are negatively regulated by Cas. Binding studies reveal that Cas can bind to the same DNA sites as Hb, raising the possibility that it modulates transcriptional activities of genes also regulated by Hb. DNA sequence analysis of Cas fragments reveals 32 potential DNA-binding sites, all sharing at least 8 out of the 10 bp with the Hb consensus sites. Cas is shown to be able to bind to these sites. These results suggest that Hb and Cas regulate pdm expression by interacting directly with their cis-regulators to deactivate controlling enhancer(s), with Hb repressing the pdm genes early in CNS development, while Cas silencing acts late in CNS development (Kambadur, 1998).
The tightly choreographed NB expressions of Hb, Pdm, and Cas suggest temporally integrated processes participate in their formation. Whereas Hb is expressed early in neuroblast lineages, Cas is expressed late (see Lateral views of Drosophila CNS). The fact that NBs are found co-expressing Hb/Pdm-1 or Pdm-1/Cas, but never Hb/Cas, further suggests that at least some early NBs make a Hb->Pdm->Cas transition. However, not all NBs undergo these transitions. This is particularly evident in NBs that enter the proliferative zone during later delamination waves. For example, the first ventral cord NBs to express Cas, the S3 NB6-1s, activate Cas shortly after delaminating from the ectoderm and do not express Hb. Late developing sublineages rely on cas to both insulate cell-fate programs and to secure the expression of factors that likely play key roles in their cell-identity decisions. Cas carries out the first of these regulatory roles by selectively silencing the expression of pdm genes. It is concluded that the Zn-finger proteins Hb and Cas act in a cooperative, non-overlapping manner to control POU gene expression during Drosophila CNS development (Kambadur, 1998).
A fundamental question in developmental biology seeks to unravel the connection between cell cycle and differentiation. Do cells have to divide in order to turn on new genes? Do they have to undergo a regime of DNA synthesis? It has been found that neuroblasts require cell division to activate cas/ming expression, while single, identified neuroblasts require only cell cycle progression and not mitosis to activate even-skipped expression. Other genes, unplugged and achaete are expressed independently of cell cycle and cytokinesis (Cui, 1995). This observation also supports the late-in-development origin of cas/ming expressing cells: earlier neuroblasts would have had to go through a mitotic cycle before their neuroblast progeny could express cas/ming.
The timing mechanisms responsible for terminating cell proliferation toward the end of development remain unclear. In the Drosophila CNS, individual progenitors called neuroblasts are known to express a series of transcription factors endowing daughter neurons with different temporal identities. This study shows that Castor and Seven-Up, members of this temporal series, regulate key events in many different neuroblast lineages during late neurogenesis. First, they schedule a switch in the cell size and identity of neurons involving the targets Chinmo and Broad Complex. Second, they regulate the time at which neuroblasts undergo Prospero-dependent cell-cycle exit or Reaper/Hid/Grim-dependent apoptosis. Both types of progenitor termination require the combined action of a late phase of the temporal series and indirect feedforward via Castor targets such as Grainyhead and Dichaete. These studies identify the timing mechanism ending CNS proliferation and reveal how aging progenitors transduce bursts of transcription factors into long-lasting changes in cell proliferation and cell identity (Maurange, 2008).
Initially investigated was whether distinct temporal subsets of neurons are generated throughout the larval CNS. Chinmo and Broad Complex (Br-C), two BTB-zinc finger proteins known to be expressed in the postembryonic CNS, are distributed in complementary patterns in the central brain, thoracic, and abdominal neuromeres at the larval/prepupal transition stage at 96 hr (timings are relative to larval hatching at 0 hr). Chinmo is expressed by early-born neurons located in a deep layer, whereas Br-C marks later-born neurons in a largely nonoverlapping and more superficial layer. The deep Chinmo+ layer comprises most/all neurons born in the embryo plus an early subset of those generated postembryonically. Thoracic postembryonic neuroblasts undergo the Chinmo --> Br-C switch at ~60 hr such that they have each generated an average of 15 Chinmo+ cells expressing little or no Br-C and 39 Chinmo- Br-C+ cells by 96 hr. The Chinmo+ and Br-C+ neuronal identities can be recognized as distinct cell populations on the basis of an ~2-fold difference in cell-body volume. This equates to an average cell-body diameter for Chinmo+ neurons of 4.5 μm, compared to only 3.6 μm for Br-C+ neurons. Plotting cell diameter versus deep-to-superficial position within postembryonic neuroblast clones reveals an abrupt decrease in neuronal size at the Chinmo --> Br-C transition. Together, these results provide evidence that most, if not all, postembryonic neuroblasts sequentially generate at least two different populations of neurons. First they generate large Chinmo+ neurons and then they switch to producing smaller Br-C+ neurons (Maurange, 2008).
To begin dissecting the neuronal switching mechanism, the functions of Chinmo and Br-C were investigated, but neither factor was found to be required for the transition in cell identity and cell size. Next it was asked whether a temporal transcription factor series related to the embryonic Hb --> Kr --> Pdm --> Cas sequence might be involved. Cas is known to be expressed in the larval CNS, and this study shows that many different thoracic neuroblasts progress through a transient Cas+ phase during the 30-50 hr time window. Thoracic neuroblasts transiently express another member of the embryonic temporal series, Svp, during a somewhat later time window, from ~40 to ~60 hr. These results indicate that postembryonic Cas and Svp bursts are observed in many, but probably not all, thoracic progenitors and that their timing varies from neuroblast to neuroblast (Maurange, 2008).
To determine Svp function, thoracic neuroblast clones were generated homozygous for svpe22, an amorphic allele. In ~53% of svpe22 neuroblast clones induced in the early larva (at 12-36 hr), the Br-C+ neuronal identity is completely absent, all neurons express Chinmo, and there is no sharp decrease in neuronal size. The proportion of lineages failing to generate Br-C+ neurons rises to ~70% when clones are induced in the embryo and falls to only ~7% with late-larval (65-75 hr) induction. This is consistent with a previous finding that Svp bursts are asynchronous from neuroblast to neuroblast. The expression and clonal analyses together demonstrate that a progenitor-specific burst of Svp is required in many lineages for the switch from large Chinmo+ to small Br-C+ neurons (Maurange, 2008).
Thoracic neuroblast lineages homozygous for a strong cas allele, cas24, show no obvious defects in the Chinmo --> Br-C transition when induced at 12-36 hr. However, since Cas is expressed in many postembryonic neuroblasts before their first larval division, it can only be removed by inducing clones during embryonic neurogenesis. Such cas24 clones generate supernumerary Chinmo+ neurons and completely lack Br-C+ neurons at 96 hr, although this switching phenotype is restricted to only ~16% of thoracic neuroblasts. Constitutively expressing Cas blocks the Chinmo --> Br-C switch in a similar manner, with a frequency dependent upon whether thoracic UAS-cas clones are induced during embryogenesis (~47%), at early-larval (~10%) or at late-larval (0%) stages. This indicates that the response to Cas misexpression decreases as neuroblasts age. Together, the expression and loss- and gain-of-function analyses demonstrate that Chinmo and Br-C are negative and positive targets, respectively, of Cas and Svp. They also strongly suggest that progression through transient Cas+ and Svp+ states permits many postembryonic neuroblasts to switch from generating large to small neurons (Maurange, 2008).
To investigate whether Cas and Svp regulate neural proliferation as well as neuronal fates, the effector mechanism ending neurogenesis in the central brain and thorax was identified. In these regions, most neuroblasts cease dividing in the pupa at ~120 hr. Correspondingly, neurogenesis in all regions of the wild-type CNS ceases before the adult fly ecloses such that no adult neuroblasts are detected. In contrast to the central abdomen, blocking cell death by removing Reaper, Hid, and/or Grim (RHG) activity in the central brain and thorax does not prevent or delay pupal neuroblast disappearance. However, time-lapse movies of individual thoracic neuroblasts at ~120 hr reveal an atypical mitosis that is much slower than at ~96 hr, producing two daughters of almost equal size. This is largely accounted for by a reduction in the average diameter of neuroblasts from 10.4 μm at 96 hr to 7 μm at 120 hr, as GMC size does not vary significantly during this time window. The end of this atypical progenitor mitosis temporally correlates with reduced numbers of Mira+ cells and disappearance of the M phase marker phosphorylated-Histone H3 (PH3), indicating that it marks the terminal division of the neuroblast (Maurange, 2008).
Next whether late changes in basal complex components might underlie loss of neuroblast self-renewal was addressed. At 120 hr, it was found that Mira becomes delocalized from the cortex to the cytoplasm and nucleus of many interphase neuroblasts. In metaphase neuroblasts, Mira fails to localize to the basal side of the cortex, although it does selectively partition into one daughter during telophase. This late basal restoration resembles the 'telophase rescue' associated with several apical complex mutations. Pros, the Mira-binding transcription factor and GMC-determinant, is not detectable in the neuroblast nucleus at 96 hr, but at 120 hr a burst of Pros was observe in the nucleus of many Mira+ cells of intermediate size indicative of neuroblasts in the interphase preceding the terminal mitosis. Clones lacking Pros activity contain multiple Mira+ neuroblast-like cells. It was found that they do not respect the ~120 hr proliferation endpoint and even retain numerous dividing Mira+ progenitors into adulthood. In addition, GAL80ts induction was used to induce transiently the expression of a YFP-Pros fusion protein well before the normal ~120 hr endpoint. Under these conditions, YFP-Pros can be observed in the nucleus of neuroblasts, most Mira+ progenitors disappear prematurely, and neural proliferation ceases much earlier than normal. Together, these results provide evidence that most neuroblasts terminate activity in the pupa via a nuclear burst of Pros that induces cell-cycle exit. These progenitors are referred to as type I neuroblasts to distinguish them from the much smaller population of type II neuroblasts that terminate via RHG-dependent apoptosis (Maurange, 2008).
Whether the postembryonic pulses of Cas and Svp in type I neuroblasts are implicated in scheduling their subsequent cell-cycle exit was tested. Remarkably, it was observed that many svpe22 clones induced at early-larval stages retain a single Mira+ neuroblast at 7 days into adulthood. The persistent adult neuroblasts in a proportion of these clones also express the M phase marker, PH3, indicating that they remain engaged in the cell cycle and, accordingly, they generate approximately twice the normal number of cells by 3 days into adulthood. Furthermore, superficial last-born neurons in these over proliferating svpe22 adult clones are Chinmo+ Br-C- indicating a blocked Chinmo --> Br-C transition. Similar phenotypes are obtained in some UAS-cas and cas24 clones. This analysis demonstrates that stalling the temporal series not only inhibits the late-larval switch to Br-C+ neuronal identity but also prevents the pupal cell-cycle exit of type I neuroblasts (Maurange, 2008).
To test the regulatory relationship between Pros and the temporal series, svpe22 clones were examined at pupal stages. It was found that mutant interphase neuroblasts fail to switch on nuclear Pros at 120 hr, although svpe22 GMCs express nuclear Pros as normal. This likely accounts for why adult clones lacking Svp retain only a single neuroblast, whereas those lacking Pros contain multiple neuroblast-like progenitors. Importantly, these results demonstrate that nuclear Pros acts downstream of the temporal series in type I neuroblasts. Together, the genetic and expression analyses of Svp and Pros show that the temporal series triggers a burst of nuclear Pros in type I neuroblasts, thus inducing their cell-cycle exit (Maurange, 2008).
To determine how the temporal series is linked to the cessation of progenitor divisions, two transcription factors expressed in neuroblasts in a temporally restricted manner were examined. Dichaete (D), a member of the SoxB family, is dynamically expressed in the early embryo and is required for neuroblast formation. Consistent with the previous studies, it was observed that most or all embryonic neuroblasts progress through a transient D+ phase, but those in the lateral column of the ventral nerve cord initiate expression after their medial and intermediate counterparts. Dichaete subsequently becomes repressed in ~85% of neuroblasts during late-embryonic and postembryonic stages. Grainyhead (Grh) is first activated in neuroblasts in the late embryo and is required for regulating their mitotic activity during larval stages. Blocking early temporal series progression in the embryo, either by persistent Hb or loss of Cas activity, prevents most neuroblasts from downregulating D and also from activating Grh at late-embryonic and postembryonic stages. As forcing premature Cas expression leads to precocious D repression and Grh activation, both factors are likely to be regulated by Cas rather than by a later member of the temporal series. These results demonstrate that transient embryonic Cas activity permanently switches the expression of Grh on and D off. They also identify Grh and D as positive and negative targets, respectively, of the temporal series in many neuroblasts (Maurange, 2008).
Loss of Grh activity in thoracic neuroblasts (here defined as type I neuroblasts) leads to their reduced cell-cycle speed and disappearance during larval stages. At 96 hr, it was observed that 65% of grh370 type I neuroblasts are smaller than normal (~6.4 μm in diameter), delocalize Mira from the cortex to the cytoplasm and nucleus, and strongly express Pros in the nucleus. These events, reminiscent of the 120 hr terminal cell cycle of wild-type progenitors, show that Grh is required to prevent the premature cell-cycle exit of type I neuroblasts. Given this finding, and that Cas is required to activate Grh, the question arises as to how some neuroblasts lacking embryonic Cas activity are able to continue dividing into adulthood. However, cas24 neuroblasts persisting in adults all retain Grh, suggesting that they may derive from clones that lacked only the last of the two Cas pulses observed in some embryonic neuroblasts, perhaps retaining enough Cas activity to support Grh activation but not later progression of the temporal series (Maurange, 2008).
To determine if late temporal series inputs, after embryonic Cas, are also required to maintain the long-lasting postembryonic expression of Grh, svpe22 clones were induced in early larvae. Although type I neuroblasts in these mutant clones have a stalled temporal series, they retain postembryonic Grh expression through to adult stages. Thus, two sequential inputs from the temporal series are required for type I neuroblasts to undergo timely Pros-dependent cell-cycle exit. First, embryonic Cas activity switches on sustained Grh expression, inhibiting premature nuclear Pros and permitting continued mitotic activity. Second, a late postembryonic input, requiring Svp, counteracts this activity of Grh by triggering a pupal burst of nuclear Pros (Maurange, 2008).
Despite undergoing premature cell-cycle exit, it was noticed that grh mutant neuroblasts can still generate both Chinmo+ and Br-C+ neurons. Thus, Grh is not required for the neuronal Chinmo --> Br-C switch. Conversely, neither Chinmo nor Br-C appears to be required postembryonically for neuroblast cell-cycle exit. In summary, the properties of both neuroblasts and neurons are regulated by downstream targets of the temporal series (Maurange, 2008).
Next whether the temporal series and its targets also function in type II neuroblasts, which terminate via RHG-dependent apoptosis rather than Pros-dependent cell-cycle exit, was tested. Focus was placed on one identified type II neuroblast in the central abdomen, called dl, which undergoes apoptosis at 70-75 hr and produces only a small postembryonic lineage of ~10 neurons. It was observed that the dl neuroblast expresses bursts of Cas (~45 to ~60 hr) and Svp (~62 to ~65 hr) and sequentially generates Chinmo+ (~7 deep) and Br-C+ (~3 superficial) neurons. Loss of Cas or Svp activity, or prevention of temporal series progression in several other ways all lead to a blocked Chinmo --> Br-C transition, a failure to die at 70-75 hr, and the subsequent generation of many supernumerary progeny. These results show that the temporal series performs similar functions in type I and type II neuroblast lineages, regulating both the Chinmo --> Br-C neuronal switch and the cessation of progenitor activity. Next, the mechanism linking the temporal series to RHG-dependent death of type II neuroblasts was tested. As for type I neuroblasts, dl progenitors in cas24 clones, induced in the embryo, fail to activate Grh and repress D. Moreover, if grh activity is reduced, or if D is continuously misexpressed, dl progenitors persist long after 75 hr. Therefore, both of these early Cas-dependent events are essential for subsequent type-II neuroblast apoptosis. However, in contrast to members of the temporal series themselves, persistent misexpression of their target D does not block the Chinmo --> Br-C switch in the dl lineage. Thus, the D- Grh+ state of type I and type II neuroblasts, installed via an embryonic Cas pulse, appears to be necessary for progenitor termination but not for Chinmo --> Br-C switching. Nevertheless, dl neuroblasts stalled at a postembryonic stage in svpe22 and UAS-cas clones retain the D- Grh+ code yet still fail to undergo apoptosis. Therefore, as with type I cell-cycle exit, timely type II apoptosis requires both embryonic Cas-dependent and postembryonic Svp-dependent inputs from the temporal series (Maurange, 2008).
Finally, the regulatory relationship between the temporal series and AbdA, a Hox protein transiently expressed in postembryonic type II (but not type I) neuroblasts and required for their apoptosis was dissected. dl neuroblasts lacking postembryonic Svp activity or persistently expressing Cas still retain AbdA expression yet do not die. This suggests that AbdA is unable to kill neuroblasts unless they progress, in a Svp-dependent manner, to a late Cas- temporal state. To test this prediction directly, use was made of the previous finding that ectopic AbdA is sufficient to induce neuroblast apoptosis, albeit only within a late time window. Constitutive AbdA-induced apoptosis is efficiently suppressed by persistent Cas, not only in type II but also in type I neuroblasts. This result demonstrates that, in order to terminate, type II neuroblasts must progress to a late Cas- state, thus acquiring a D- Grh+ Cas- AbdA+ code. It also suggests that AbdA is sufficient to intercept progression of the temporal series in type I neuroblasts, inducing an early type II-like termination (Maurange, 2008).
This study has found that the Drosophila CNS contains two distinct types of self-renewing progenitors: type I neuroblasts terminate divisions by cell-cycle withdrawal and type II neuroblasts via apoptosis. Despite these different exit strategies, both progenitor types use a similar molecular timer, the temporal series, to shut down proliferation and thus prevent CNS overgrowth. These findings demonstrate that the temporal series does considerably more than just modifying neurons; it also has multiple inputs into neural proliferation. The identification and analysis of several pan-lineage targets of the temporal series also begins to shed light on the mechanism by which developmental age modifies the properties of neuroblasts and neurons. Two targets, Chinmo and Br-C, are part of a downstream pathway temporally regulating the size and identity of neurons. Two other temporal series targets, Grh and D, function in neuroblasts to regulate Prospero/RHG activity, thereby setting the time at which proliferation ends. The temporal series regulates both cell proliferation and cell identity; a feedforward mechanism is proposed for generating combinatorial transcription factor codes during progenitor aging (Maurange, 2008).
This study has found that the temporal series regulates a widespread postembryonic switch in neuronal identity. Most, if not all, type I and type II neuroblasts first generate a deep layer of large Chinmo+ neurons and then switch to producing a superficial layer of small Br-C+ neurons. Two lines of evidence argue that this postembryonic neuronal switch is likely to be regulated by a continuation of the same temporal series controlling early/late neuronal identities in the embryo. First, the postembryonic Chinmo --> Br-C neuronal switch is promoted by the transient redeployment of two known components of the embryonic temporal series, Cas and Svp. Second, this switch remains inhibitable by misexpression of the other embryonic temporal factors such as Hb. Since both Cas and Svp are expressed somewhat earlier than the neuronal-size transition, it is likely that they promote bursts of later, as yet unknown, members of the temporal series that more directly regulate Chinmo and Br-C. Although neuronal functions for both BTB zinc-finger targets have yet to be characterized, a progressive early-to-late decrease in postmitotic Chinmo levels is known to regulate the temporal identities of mushroom-body neurons. The current results now suggest that this postmitotic gradient mechanism may be linked to, rather than independent from, the temporal series (Maurange, 2008).
Type I neuroblasts in clones lacking postembryonic Cas/Svp activity or retaining an early temporal factor, fail to express nuclear Pros during pupal stages and thus continue dividing long into adulthood. These overproliferating adult clones each contain only a single neuroblast, sharply contrasting with adult clones lacking Brat or Pros, in which there are multiple neuroblast-like progenitors. Hence, manipulations of the temporal series and its progenitor targets offer the prospect of immortalizing neural precursors in a controlled manner, without disrupting their self-renewing asymmetric divisions (Maurange, 2008).
This study demonstrates that type I and type II neuroblasts must progress through at least two critical phases of the temporal series in order to acquire the D- Grh+ Cas- combinatorial transcription factor code that precedes Pros/RHG activation. The early phase corresponds to embryonic Cas activity switching neuroblasts from D+ Grh- to D- Grh+ status. The equally essential, but less well-defined, late postembryonic phase of the temporal series requires transition to a Cas- state and a late Svp burst. For type I neuroblasts, Grh and a late Cas- temporal identity are both required for timely expression of nuclear Pros and subsequent cell-cycle withdrawal. For type II neuroblasts, these two inputs are also necessary for RHG-dependent apoptosis, with the additional requirement that D must remain repressed. Although the temporal series and its targets are similarly expressed in type I and type II neuroblasts, only the latter progenitors undergo a larval burst of AbdA. This AbdA expression is likely to be the final event required to convert the D- Grh+ Cas- state, installed by the temporal series, into the D- Grh+ Cas- AbdA+ combinatorial code for RHG-dependent apoptosis. This code prevents type II neuroblasts in the abdomen from reaching the end of the temporal series and accounts for why they generate fewer progeny and terminate earlier than their type I counterparts in the central brain and thorax (Maurange, 2008).
The data in this study support an indirect feedforward model for neuroblast aging. Key to this model is the finding that, although members of the temporal series are only expressed very transiently, some of their targets can be activated or repressed in a sustained manner, as observed for Chinmo/Br-C in neurons and also for Grh/D in neuroblasts. In principle, this indirect feedforward allows aging progenitors to acquire step-wise the combinatorial transcription factor codes modulating cell-cycle speed, growth-factor dependence, competence states, and neural potential. Like Drosophila neuroblasts, isolated mammalian cortical progenitors can sequentially generate neuronal fates in the correct in vivo order. These studies suggest that it will be important to investigate whether the transcription factors controlling this process also regulate cortical proliferation and whether their targets include BTB-zinc finger, Grh, SoxB, Prox, or proapoptotic proteins. Some insect/mammalian parallels seem likely, since it is known that Sox2 downregulation and Prox1 upregulation can both promote the cell-cycle exit of certain types of vertebrate neural progenitors (Dyer, 2003, Graham, 2003). Thus, although insect and mammalian neural progenitors do not appear to use the same sequence of temporal transcription factors, at least some of the more downstream components identified in this study might be functionally conserved (Maurange, 2008).
Bases in 5' UTR - 688
Exons - four
Bases in 3' UTR - 591
There are four consecutive zinc finger repeats. The motif is Cys2-His2-Cis2-His2. Multiple PEST sequences contributing to protein turnover are present. There is an N-terminal polyglutamine sequence, an acidic region, a serine proline rich region and a polylysine sequnce (Mellerick, 1992).
Castor functions as a DNA-binding transcription factor. It contains a centrally located Zn-finger domain made-up of four consecutive C2-H2C2-H2 repeats. The second C2-H2 of each repeat closely resembles fingers of the Xenopus TFIIIA C2-H2 class. Flanking this repeat are motifs that may constitute either transcription transactivation or repression domains. UV induced protein-DNA cross-linking in vivo studies reveal that Cas binds genomic DNA. To determine if Cas is a sequence-specific DNA-binding protein, the cyclic amplification of selected targets protocol was used. After six rounds of selection/amplification, sequencing of cloned fragments revealed that all had at least one sequence motif in common and some contained two core recognition sequences. DNA fragments containing one site homologous to the consensus site produce a single prominent Cas-DNA gel-shift; a fragment with two, generates two complexes. Addition of Cas-specific antisera causes a super-shift of the Cas-DNA complex. A search of known transcription factor DNA-binding sites shows that the Cas recognition sequence is almost identical to that of the Drosophila Zn-finger protein Hunchback. The Cas consensus matches 9 out of 10 bp for the reported Hb sites. To determine if Cas binds Hb sites, gel-shift experiments using DNA fragments were carried out with exact sequence matches to Hb targets. Cas does indeed bind to these sites. The sequence-specificity of Cas-DNA binding to Hb recognition sites was further tested by competition assays and base-pair substitutions. Taken together, these experiments demonstrate that Cas can bind to the same DNA sites as Hb, raising the possibility that it modulates transcriptional activities of genes also regulated by Hb. Secondary structure predictions of the Cas finger domain indicate that only the first and third of its TFIIIA-like fingers contain alpha-helices. Interestingly, optimal alignment of Cas and Hb fingers reveals that the first and third a-helices of Cas share the highest homology with the corresponding a-helices of Hb (33% identity for the first and 27% for the third). Although speculative, their shared DNA-binding preferences may be due in part to the shared residues found in these predicted reading heads. Outside of their Zn-fingers, Hb and Cas show no obvious sequence similarities (Kambadur, 1998).
Castor is a zinc finger transcription factor that controls cell fate within neuroblast cell lineages in Drosophila melanogaster. A human castor gene (CASZ1) is structurally homologous to Drosophila castor. castor gene expression is increased when cells of neural origin as well as mesenchymal origin are induced to differentiation. CASZ1 is expressed in a number of normal tissues and exists in at least two mRNA species of 4.4 and 8.0 kb. They are named hCasz5 and hCasz11 because the predicted proteins have 5 and 11 zinc fingers, respectively. Deletion analysis of the proximal 5′-flanking sequences delineates sequences sufficient to drive transcription in cells of neural and non-neural origin. Both hCasz5 and hCasz11 localize predominantly in the nucleus, consistent with their role as Zn-finger containing transcription factor. CASZ1 is expressed in a number of human tumors and localizes to a chromosomal region frequently lost in tumors of neuroectodermal origin (Liu, 2006).
The CASTOR (CST) transcription factor was initially identified for its role in maintaining stem cell competence in the Drosophila dorsal midline. This study reports that Xenopus CST affects cardiogenesis. In CST-depleted embryos, cardiomyocytes at the ventral midline arrest and are maintained as cardiac progenitors, while cells in more dorsal regions of the heart undergo their normal program of differentiation. Cardia bifida results from failed midline differentiation, even though cardiac cell migration and initial cell fate specification occur normally. Fate mapping studies reveal that this ventral midline population of cardiomyocytes ultimately gives rise to the outer curvature of the heart; however, CST-depleted midline cells overproliferate and remain a coherent population of nonintegrated cells positioned on the outer wall of the ventricle. These midline-specific requirements for CST suggest the regulation of cardiomyocyte differentiation is regionalized along a dorsal-ventral axis and that this patterning occurs prior to heart tube formation (Christine, 2008).
date revised: 20 December 2006
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