grainy head

DEVELOPMENTAL BIOLOGY

Embryonic

Zygotic GRH protein is detected in nuclei of ectodermal derivatives. Expression of grh is first seen in stage 11 in both the epidermis and CNS (Bray, 1989).

During Drosophila embryonic CNS development, the sequential neuroblast (NB) expression of four proteins, Hunchback (Hb), Pou-homeodomain proteins 1 and 2 (referred to collectively as Pdm), and Castor (Cas), identifies a transcription factor network regulating the temporal development of all ganglia. The Zn-finger proteins Hb and Cas, acting as repressors, confine Pdm expression to a narrow intermediate temporal window; this results in the generation of three panneural domains whose cellular constituents are marked by expression of Hb, Pdm, or Cas. Seeking to identify the cellular mechanisms that generate these expression compartments, the lineage development of isolated NBs in culture were studied. The Hb, Pdm, and Cas expression domains are generated by transitions in NB gene expression that are followed by gene product perdurance within sequentially produced sublineages. These results also indicate that following Cas expression, many CNS NBs continue their asymmetric divisions and generate additional progeny, which can be identified by the expression of the bHLH transcription factor Grainyhead (Gh). Gh appears to be a terminal embryonic CNS lineage marker. Taken together, these studies indicate that once NBs initiate lineage development, no additional signaling between NBs and the neuroectoderm and/or mesoderm is required to trigger the temporal progression of Hb followed by Pdm and then Cas, and subsequently Gh expression during NB outgrowth (Brody, 2000).

Underpinning the formation of NB lineages are spatially and temporally regulated transcription factor networks that play pivotal roles in establishing the unique cellular identities of NBs and their progeny. Prior to NB delamination, during the initial specification of NBs, two spatially regulated transcription factor networks subdivide the ventral neuroectoderm along its anterior/posterior (A/P) x axis and dorsal/ventral (D/V) y axis. Later, during NB lineage development, at least one additional network, acting over several hours, gives rise to sequentially formed multilayered basal (inner or dorsal) to apical (outer or ventral) neuronal subpopulations. Along the basal/apical z axis, neuronal subpopulations in all ganglia can be identified by their expression of the transcription factors Hb, Pdm and Cas. Hb marks a deeper, basally distributed population of neurons that are born early, Cas marks a superficial, apically distributed population of neurons that are born late, and Pdm marks an intermediate population arrayed between the Hb- and the Cas-expressing cells. Both genetic and molecular analysis indicates that two Zn-finger proteins, Hb and Cas, act as repressors to silence pdm expression. By restricting pdm expression primarily to intermediate-born neuronal precursors these structurally different Zn-finger proteins help establish three pan-CNS neural subpopulations whose cellular constituents are marked by the expression of Hb, Pdm, or Cas (Brody, 2000).

Triple-immunolabeling studies have revealed that many of the overnight NB clones contain a subset of cells that do not contain detectable levels of Hb, Pdm-1, or Cas. In many of these in vitro lineages the putative NB is also unstained. The bHLH transcription factor Gh is known to be expressed in CNS NBs but only after stage 14. In view of the late onset of Gh expression in NBs and the triple-staining results identifying cells in o/n clones that do not express Hb, Pdm-1, or Cas, it was hypothesized that these negative cells may represent an additional late NB expression window marked by Gh expression. To test this hypothesis, the spatial/temporal expression dynamics of Gh were compared to other members of the z axis network. Similar to its late activation during in vivo development, Gh expression was observed only in overnight cultures; when more than one Gh-positive cell was detected in a clone they were consistently found clustered together. Two-thirds of the Cas+ clones had at least one Gh+ cell and the average number of Gh+ cells in all clones was 2.3. Approximately 2/3 of the Gh+ clones also contained Hb-immunopositive cells. While no Hb-Gh coexpressing cells were observed, approximately 20% of the Gh+ cells also expressed Cas. Given the late onset of Gh expression in both the embryo and the cultured NB clones and the overlapping Cas and Gh expression, it is likely that Gh marks a fourth temporal window for NB transcription factor expression. In addition, because there was an average of more than one cell in an o/n clone that was immunopositive for Gh, it is likely that Gh is also expressed/maintained in a sublineage(s) born after the one marked by Cas expression (Brody, 2000).

The following model for the origin of the layer sublineages marked by these transcription factors has been suggested. As each NB divides, generating a succession of GMCs, it undergoes multiple transitions in transcription factor expression. In succession, the NBs express Hb, Pdm, Cas, and Gh. The first progeny generated by the early S1 and S2 NBs express Hb, and the presence of Hb protein persists in their neural progeny. These early S1 and S2 NBs go on to activate the expression of the Pdms that, like Hb, persist in neural sublineages generated during this temporal window. Subsequently Cas is activated in NBs, represses Pdm transcription, and likewise persists in neural sublineages. After Cas expression, a fourth neural subpopulation, generated by dividing NBs, expresses Gh. This Gh subpopulation most likely represents the terminal sublineage of the embryonic NB. The data also reveal that not all NBs generate cells that occupy all four layers, a result that reflects the unique set of lineages, generated by each NB. Most likely, each NB has a preprogrammed time of delamination, but the timing of transitions is synchronized in a global fashion. The model further suggests that late delaminating NBs can be distinguished from early NBs by their inability to activate Hb. Although Hb is activated shortly after the S1s and S2s have delaminated, Hb is never seen in the proliferative zone during late delaminations (Brody, 2000).

Temporal transcription factors and their targets schedule the end of neural proliferation in Drosophila

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 svp^e22, an amorphic allele. In ~53% of svp^e22 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, GAL80^ts 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 svp^e22 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 svp^e22 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, svp^e22 clones were examined at pupal stages. It was found that mutant interphase neuroblasts fail to switch on nuclear Pros at 120 hr, although svp^e22 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, svp^e22 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 svp^e22 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).

Effects of mutation or deletion

The major developmental defect of grh mutants is alteration to head skeleton and cuticular structures. The [Image] becomes "grainy," hence the gene's sobriquet (Bray, 1991).

The embryonic cuticle of Drosophila is deposited by the epidermal epithelium during stage 16 of development. This tough, waterproof layer is essential for maintaining the structural integrity of the larval body. Mutations in a set of genes required for proper deposition and/or morphogenesis of the cuticle have been characterized. Zygotic disruption of any one of these genes results in embryonic lethality. Mutant embryos are hyperactive within the eggshell, resulting in a high proportion being reversed within the eggshell (the 'retroactive' phenotype), and all show poor cuticle integrity when embryos are mechanically devitellinized. This last property results in embryonic cuticle preparations that appear grossly inflated compared to wild-type cuticles (the 'blimp' phenotype). One of these genes, krotzkopf verkehrt (kkv), encodes the Drosophila chitin synthase enzyme and a closely linked gene, knickkopf (knk), encodes a novel protein that shows genetic interaction with the Drosophila E-cadherin, shotgun. Two other known mutants, grainy head (grh) and retroactive (rtv), show the blimp phenotype when devitellinized, and a new mutation, called zeppelin (zep), is described that shows the blimp phenotype but does not produce defects in the head cuticle as the other mutations do (Ostrowski, 2002).

The cuticle defects, particularly the disruption of the head skeleton, are most severe in kkv and grh mutants. All alleles of kkv, both those isolated previously and those identified in this screen, produce similar phenotypes. When removed from the vitelline membrane, kkv and grh mutant embryos are very flaccid and are not motile although they are able to contract their body wall muscles. All three alleles of knk and the one available allele of rtv produce milder defects in the head skeleton and denticle belts. When removed from the vitelline membrane they are more robust than the kkv and grh mutants, and they are motile but die within hours after removal from the eggshell. The head skeleton and denticle belts of zep mutants are almost wild type and these embryos are sometimes able to hatch on their own, although they die at roughly the same stage as the knk and rtv mutants. The degree of cuticle expansion can vary among cuticle preparations due to uncontrollable differences in the mechanical devitellinization process. However, the severity of head defects, flaccidity, and motility are consistent within the alleles of each complementation group. Thus the phenotypic effects of the blimp class mutations can be ranked from most to least severe: kkv, grh > rtv, knk > zep (Ostrowski, 2002).

The identification of kkv as a chitin synthase and the ability of a chitin synthesis inhibitor to phenocopy kkv shows that disrupting synthesis or deposition of chitin alone can account for the blimp phenotype. However, it is believed that two of the blimp class genes, knk and zep, may function in the epidermis prior to cuticle deposition because both interact genetically with mutations in the Drosophila E-cadherin, encoded by shotgun (Ostrowski, 2002).

grainy head affects head skeleton and embryonic cuticle. grh encodes a GATA family transcription factor and activates the transcription of a number of genes during development, one of which is Dopa-decarboxylase (Ddc). This enzyme is synthesized in the cuticle-secreting layer of cells at the end of embryogenesis; the dopamine produced undergoes further metabolism and oxidation to produce quinones that crosslink cuticular proteins. Thus, loss of grh function would result in weakening of the cuticle indirectly through its failure to activate Ddc expression (Ostrowski, 2002).

Grainy head controls apical membrane growth and tube elongation during tracheal development

Epithelial organogenesis involves concerted movements and growth of distinct subcellular compartments. Apical membrane enlargement is critical for lumenal elongation of the Drosophila airways, and is independently controlled by the transcription factor Grainy head. Apical membrane overgrowth in grainy head mutants generates branches that are too long and tortuous without affecting epithelial integrity, whereas Grainy head overexpression limits lumenal growth. The chemoattractant Branchless/FGF induces tube outgrowth -- it upregulates Grainy head activity post-translationally, thereby controlling apical membrane expansion to attain its key role in branching. A two-step model for FGF in branching is favored: first, induction of cell movement and apical membrane growth, and second, activation of Grainy head to limit lumen elongation, ensuring that branches reach and attain their characteristic lengths (Hemphälä, 2003).

A characteristic feature of transporting and secretory tubular organs, such as lung, kidney and many glands, is the structural and functional compartmentalisation of their epithelium. Tubulogenesis and branching rely on extensive cell rearrangements and an immense increase of apical lumenal surface, yet in many cases the epithelium remains intact and functional during development. Thus, the driving forces for cell movement, shape changes and growth must act in the context of prefixed distinct subcellular compartments, and they must be highly co-ordinated with cell adhesion. Although the molecular determinants of epithelial cell architecture are becoming increasingly clear, the regulation of the different subcellular compartments during epithelial tissue morphogenesis remains largely unknown. Epithelial cell movement and morphogenesis are commonly induced and guided by secreted factors from the surrounding tissues. How then are these morphogenetic cues integrated to regulate the dynamic cell behaviours that underlie epithelial tube formation and organ growth (Hemphälä, 2003)?

The development of the Drosophila trachea, a complex network of epithelial airways that supplies oxygen to the entire animal, provides a well-defined system for the analysis of regulatory mechanisms that control cell migration and branching. The tracheal system arises from 20 independent sacks of approximately 80 cells each that undergo a distinct sequential program of branch sprouting, directed branch outgrowth and branch fusion. Initially, the actions of at least three independent signals, TGFß-like (Decapentaplegic; Dpp), Wingless (Wg) and EGF, subdivide the cells in each tracheal placode into branch-specific groups. Subsequent branch sprouting and outgrowth occurs without cell division as cells migrate towards localized sources of Branchless (Bnl), an attractant signal of the FGF family. Primary branch growth entails the initial extension of cytoplasmic processes towards the Bnl source, followed by movement of the cell body and a concomitant increase in apical cell surface to promote lumenal extension. The characteristic lengths and diameters of the newly formed branches of the larval trachea are stereotyped and become specified during distinct developmental intervals (Hemphälä, 2003).

Bnl is the key morphogen co-ordinating branching that acts via the receptor tyrosine kinase Breathless (Btl) and the adaptor protein Dof/Stumps. This pathway leads to phosphorylation and activation of MAPK, which in turn may alter the activity of regulatory proteins to control cell behavior. During primary branching, actin-rich basal extensions are sent by the tracheal cells towards the sources of Bnl, a process that is likely to involve cytoskeletal modulation by the Rho family GTPases. Bnl signalling is also required for the expression of cell-fate determining genes in specific subsets of tracheal cells in each primary branch. Analysis of these genes has identified key components of the patterning and guidance of the unicellular secondary and terminal branches. However, the role of Bnl in the movement of the cell bodies and the growth of the branch lumen remains unknown (Hemphälä, 2003).

The mechanisms that control the elongation of tracheal tubes have been investigated. Mutations have been characterized in three genes that affect branch growth, resulting in abnormally long tubes. Mutations in fasII and Atpalpha alter cell adhesion and the basolateral cell domains, causing aberrations in cell shapes, excessive tubular elongation and sporadic lumenal dilations and breaks. In contrast, the transcription factor Grainy head (Grh) is required to specifically control tube elongation. Both loss of function and overexpression of grh indicate that it is required to limit lumenal growth and control tubular length. Grh selectively affects the growth of the apical cell membrane, arguing that different genetic programs regulate distinct sub-cellular domains during branching morphogenesis. Grh is uniformly expressed in the trachea, but its activity is modulated by Bnl/Btl signalling and Grh counteracts the activity of Bnl induced branch growth. Thus, through its regulation of Grh, Bnl regulates epithelial apical membrane growth to accommodate its role in branching morphogenesis (Hemphälä, 2003).

Grh is expressed in a number of epithelial structures, including the embryonic epidermis where it has been suggested to be involved in the formation of the cuticular layer that covers the apical surface of epidermal tissues. Early descriptions of grh mutants also have revealed a tracheal defect, which led to an investigation of the expression and phenotype of grh in the trachea (Hemphälä, 2003).

Nuclear Grh is detected in all tracheal cells, appearing first at stage 11, just after they have invaginated from the epidermis, and persisting throughout embryogenesis. To investigate its function, an antibody that specifically stains the tracheal lumen (mAb2A12) and several cell fate markers were used to analyse the tracheal phenotype of three strong loss-of-function grh alleles (one EMS allele, grh^B37; two P-element insertions, grh^s2140 and grh⁰⁶⁸⁵). None of the grh mutations affect the patterning, outgrowth and connection of branches or the expression of terminal cell markers (DSRF) and fusion cell markers (fusion-3). It is only when primary and secondary branching is completed (during stage 16), that grh mutant embryos begin to display tubular irregularities. The first signs of a defect are that the dorsal trunk (main airway) appears convoluted and elongated compared to the wild type. This phenotype subsequently becomes exaggerated, and is also seen in additional branches, including the lateral trunk, transverse connectives and ganglionic branches. These convoluted branches represent an overgrowth in tracheal tube length, as indicated by an increase of 40% in the tube length of grh mutants. Despite this substantial increase in tubular lengths, the tubular continuity is not affected in grh mutant embryos. Grh is therefore required for the restriction or maintenance of tubular length (Hemphälä, 2003).

The tracheal phenotypes produced by alterations in Grh levels imply that Grh activity must be carefully controlled during branching morphogenesis to ensure branch extension at the right stage and to the right extent. Consequently, tracheal Grh activity is likely to be modulated during branching morphogenesis. To assay the in vivo activity of Grh, carrying a transgene with four high-affinity Grh response elements (GBE-lacZ) were used. GBE-lacZ expression is detected in all tissues where Grh is expressed, is absent in grh mutants, and becomes activated upon ectopic Grh expression. It is thus representative of Grh transcriptional activity in vivo. During tracheal development GBE-lacZ is expressed in all tracheal cells after invagination, and requires Grh for its expression. However, GBE-lacZ expression is not uniform: it becomes temporarily enhanced in the fusion and terminal cells during branching. Since Grh itself appears to be uniform in all tracheal cells, the enhanced expression of GBE-lacZ indicates that the activity of Grh is regulated post-translationally during branching (Hemphälä, 2003).

One possible mechanism for regulation of Grh activity is through Bnl signalling, which is instrumental in the formation and extension of all tracheal branches. Initially, it was established that apical cell surface growth is an intrinsic component of Bnl-induced tube extension, by combining alleles of grh and bnl. This revealed that a subset of the branch outgrowth defects seen in embryos that carry only one copy of the bnl gene are partially rescued by a reduction in grh function (grh^s2140/grh^s2140; bnl^P1/+). Thus, in embryos heterozygous for bnl, 40% of the ganglionic branches fail to reach the CNS, whereas the simultaneous removal of grh restores this phenotype so that 78% of the branches now enter the CNS. These data therefore show that Grh-mediated modulation of the apical cell surface has an active inhibitory role on Bnl-induced branch extension (Hemphälä, 2003).

In order to analyse whether tracheal Grh activity could be targeted by Bnl/Btl signal transduction, GBE-lacZ expression was analyzed in embryos with altered levels of Bnl and Btl activity. When Bnl is ectopically expressed in all tracheal cells, GBE-lacZ expression becomes significantly upregulated, although the levels of Grh protein are not altered. This suggests that Bnl controls Grh activity post-translationally, and surprisingly, upregulates the expression of this artificial Grh target. Nevertheless, the effects of Btl appear specific since with more limited Bnl expression using the Term-Gal4 driver, GBE-lacZ expression becomes enhanced specifically in the cells that respond to Bnl by ectopically expressing the terminal marker DSRF. Similar enhancement of GBE-lacZ expression is evident upon tracheal expression of an activated form of the Btl receptor itself (UASBtl-Tor). In all instances the augmented GBE-lacZ expression is dependent on Grh, since embryos that express ectopic Bnl or the activated form of Btl, but lack Grh activity, do not express GBE-lacZ. Furthermore, ectopic activation of Dpp, another signalling pathway that promotes the growth of dorsal and ganglionic branches during tracheal development, has no effect on GBE-lacZ, indicating that the effects on GBE-lacZ are specific for Bnl/Btl (Hemphälä, 2003).

Whether Bnl signalling is a prerequisite for the transcriptional activity of Grh was tested by analysing the levels of GBE-lacZ expression in mutants for bnl, btl or pointed (pnt). Tracheal GBE-lacZ expression is both reduced and uniform in bnl and btl mutant embryos, but is unchanged in pnt embryos that lack the activity of a downstream transcriptional effector of the ETS family. Since Grh is a substrate for activated MAPK (ERK2) in vitro, its activity could be modulated directly during branching by Bnl-induced phosphorylation. This would account for the fact that GBE-lacZ expression is affected by mutations in bnl and btl, but not by mutations in the nuclear effector pnt (Hemphälä, 2003).

The apparent upregulation of Grh activity by Bnl signalling and the fact that Grh and Bnl exert opposing effects on branch extension suggests that there are two possible models of Grh activity. The first assumes a two-step process, where upregulation of Grh activity represents a second function of Bnl to prevent excessive tube extension. Alternatively, the Bnl signalling augments some aspects of Grh function (e.g. activation of GBE-lacZ) but inhibits others (e.g. the restriction of apical membrane growth) allowing for branch extension. These two models are discussed below (Hemphälä, 2003).

It is concluded that Bnl signalling converts Grh to a more potent activator of its GBE-lacZ target. Since Grh becomes phosphorylated by MAPK in vitro, and MAPK is a downstream effector of Btl signal transduction, the alteration in Grh activity may be brought about by MAPK-mediated phosphorylation of the Grh protein (Hemphälä, 2003).

Currently, two ways of explaining the biological consequence of the regulation of Grh have been suggested. In the first model, the regulation of Grh by Bnl increases its activity, and thereby delimits lumen growth. This invokes a hierarchical two step function for Bnl in which it first promotes branching and tube elongation and it then activates Grh to halt excess apical surface growth and establish a functional lumen. In this model active restriction of morphogenetic processes is required to achieve stereotyped tube dimensions and is an intrinsic part of the program that induces branching morphogenesis. In the second model, regulation by Bnl has differential consequences on Grh, activating some functions (like the one necessary for GBE-lacZ expression) and inactivating others, necessary for inhibiting apical membrane growth. In this model, high levels of Btl signalling would temporarily inactivate Grh, in order to allow for apical membrane expansion during the process of branch extension. Both models are consistent with the genetic interactions, which indicate an antagonistic relationship between grh and bnl, and add the control of apical membrane growth to the repertoire of cellular activities regulated by FGF signalling during morphogenesis (Hemphälä, 2003).

Of the two models, the former, where Btl coordinates branching through a sequence of activities, is currently favored since this model is consistent with the activation of the GBE-lacZ reporter. It can also be well integrated with the apical overgrowth phenotype of grh mutants, which becomes apparent first in the branches that have reached their final length and only after the completion of branch elongation at stage 16. If Grh were acting to restrict membrane growth continuously, the grh mutant phenotype would be expected to appear at earlier stages. A two step model could also explain the inhibiting effect on tube elongation that is seen upon expression of activated forms of Btl receptors in all tracheal cells of wild-type embryos (Hemphälä, 2003).

Since restriction of apical membrane growth depends on Grh-mediated alterations in transcriptional activity, the induction of apical membrane expansion upon branch elongation may also rely on changes in gene expression. The nuclear factor Ribbon (Rib) is required for branch elongation, and may act as an activator of apical membrane growth. In rib mutants, the extension of basal cytoplasmic processes towards the Bnl source appears normal, but the movement of the cell body fails and the apical membrane does not expand, causing a tracheal phenotype that is reminiscent of that seen with ectopic Grh expression. It is thus conceivable that a balance between Rib and Grh activity determines the extent of apical membrane growth and is coordinated by Bnl through direct modulation of Grh, and perhaps also of the Rib protein. Such a regulation of apical cell surface size by signals deriving from the target tissue could coordinate branch elongation, and would provide an elegant allometric control of organ size depending on the signal strength, size and respiratory demand of the target tissue (Hemphälä, 2003).

Apart from its tracheal expression, Grh is found in the embryonic epidermis and all primary epithelial tissues. The epidermal expression of grh is also essential because grh mutant embryos show a 'blimp' phenotype, where the embryonic cuticle stretches to a much greater extent than the wild-type cuticle upon removal of the vitelline membrane. It is found that the epidermal cells in grh embryos also show an abnormal apical membrane expansion. This is associated with the production of an enlarged cuticle that lines the apical cell surface. Grh may therefore have a common biological function in the epithelial tissues where it is expressed, being required to regulate apical cell membrane growth. Grh protein is continuously expressed in epithelial tissues during larval life, a period of extensive organ growth to accommodate the dramatic increase in animal size. Thus, Grh is likely to be required not only for organogenesis, but also for the continuous modulations in organ size and shape that occurs throughout the animals life. However, the temporal and spatial control of Grh activity must be accomplished through distinct mechanisms in different tissues, since Bnl signalling does not operate in the epidermis (Hemphälä, 2003).

Grh belongs to a small family of transcription factors that is found only in higher eukaryotes. The specific, but basic function of Grh in the regulation of epithelial apical cell membrane growth raises intriguing questions as to its functional conservation in higher organisms. Two mammalian Grh homologs (MGR and BOM) have been recently identified (Wilanowski, 2002). Like Grh, MGR and BOM form dimers and MGR interacts specifically with Grh DNA binding sites in vitro. Intriguingly, these mammalian homologs display similar expression patterns to that of Grh. During mouse development MGR is expressed predominantly in the epidermis, and BOM is expressed in the epidermis as well as in several internal tubular organs including the kidney and lung. Thus the biological function of Grh may be conserved in its murine homologs. Given the functional conservation of FGF signalling in tracheal and lung morphogenesis, it will be of great interest to test whether the mammalian homologs of Grh participate in the growth of the lung and to investigate their functional relationship with FGF signalling (Hemphälä, 2003).

grainy head is essential for the function of the frizzled pathway in the Drosophila wing

The Drosophila wing is covered by an array of distally pointing hairs. This tissue planar polarity is regulated by the frizzled pathway. The function of the grainy head transcription factor is essential for the function of the frizzled pathway. grainy head mutant cells fail to localize planar polarity proteins at either the proximal or distal sides of wing cells and produce multiple hairs of abnormal polarity. Levels of the Starry night protein are strongly reduced in grainy head mutants in both larval wing discs and pupal wings, which is sufficient to account for much of the polarity phenotype. In addition, grh has frizzled pathway independent functions during the development of the adult cuticle (Lee, 2004).

grh function is required for several different processes during the differentiation of the adult Drosophila epidermis. These include the function of the fz dependent tissue polarity pathway, pigmentation, the timing of differentiation, epidermal hair morphogenesis and wing vein/blade specification. The Grh protein was originally isolated by virtue of its ability to bind to DNA in a sequence specific manner and to regulate the expression of target genes. These and later experiments led to the conclusion that grh functions as a transcription factor for development specific gene regulation. Experiments on vertebrate homologs of grh also suggest a similar cellular function. It is likely that it serves a similar function in the development of the adult epidermis (Lee, 2004).

The analysis of grh function in regulating gene expression appears complex. The first studies on grh argued that it acted as a positive regulator of Ddc and Ubx expression. Curiously, although Grh was isolated by virtue of its ability to bind to a sequence essential for the neuronal activation of Ddc, grh mutations alter the epidermal and not neuronal expression of Ddc. More recently it was found that grh positively regulates tll expression and negatively regulates ventral dpp expression (Lee, 2004).

The function of the grh transcription factor is shown in this study to be required for the function of the fz pathway in the wing. In the absence of grh function the Fz, Dsh and Vang proteins fail to accumulate apically and the levels of the Stan protein are dramatically decreased. Furthermore, Stan levels are increased in cells with two versus one copy of grh. Thus, stan expression is directly related to grh dose suggesting that stan might be a direct target of Grh. The direct relationship between stan expression and grh dose is seen in both pupal wing cells where Stan is localized assymetrically and in third instar wing disc cells where it is evenly distributed. Thus, it is concluded that the decreased levels of Stan protein in grh cells is not due to a failure of assymetric localization. Grh does not affect Stan stability; stan expression from the endogenous stan gene is altered, consistent with Grh having an important role in promoting stan transcription. It is suspected that this could be due to a direct interaction of Grh protein with stan genomic DNA. stan does not appear to be highly enriched in putative Grh binding sites but this may be a reflection of the variability in identified Grh binding sites not providing an ideal consensus site. It is also concluded that the decreased level of Stan protein is neither the cause or effect of the the delay in hair morphogenesis in grh cells. Thus far, all of the proteins that localize assymetrically are co-required for the asymmetric localization of the others, however only Stan is required for the apical accumulation of all of the other proteins. The alterations in tissue polarity protein localization seen in grh mutant cells could be explained entirely by the effect of grh on Stan expression. It remains possible however, that grh could be important for the expression of several or all members of the tissue polarity group. These experiments did not allow the assessment of possible changes in Fz or Vang levels due to altered expression of these genes, since the localization was examined of proteins produced from transgenes that did not utilize the normal promoters. Decreased levels of Dsh were not seen by antibody staining, however the staining background was relatively high in these experiments which could have hidden a modest effect on Dsh levels. The finding that Arm cortical localization is not altered in grh clone cells indicates that apical-basal polarity is not altered and suggests that gross cellular physiology is not altered in grh clones (Lee, 2004).

While it is possible that the grh mutant planar polarity phenotype could be due solely to a lack of stan expression in grh mutant cells, this may not be the case since there are a number of differences between the phenotypes of grh and stan clones. For example, the multiple hair phenotype of grh is much stronger than stan. There is also a difference in the non-autonomy of grh and stan clones. For mutations in both of these genes the domineering nonautonomy of clones is much weaker than that of fz or Vang. However, the weak domineering nonautonomy is seen much more frequently with grh than stan clones, suggesting that grh mutations alter the expression of additional tissue polarity genes or other cellular genes that interact with the planar polarity system. Genetic screens for enhancers or suppressors of the dominant negative grh^FK2131 allele could be useful in identifying such genes (Lee, 2004).

grh has both fz pathway dependent and independent functions during wing development. Epistasis experiments showed that the ectopic wing vein, cuticle pigmentation, disturbed marginal bristle row and extreme multiple hair cell phenotypes of grh mutations are not altered in a null fz, in or mwh mutants. Thus, it is quite likely that some of the target genes whose transcription is altered by grh mutations are not part of the fz pathway (Lee, 2004).

grh cells are often dramatically delayed in hair morphogenesis. This is not seen in cells mutant for fz or stan and hence is unlikely to be an indirect consequence of a failure of stan expression or in the inactivation of the frizzled pathway. The time course of pupal development is controlled by ecdysone and it is possible that grh functions as part of the ecdysone cascade. The delay in hair morphogenesis could be due to a failure to induce the expression of genes such as kojak, where a loss of function results in a similar delay (Lee, 2004).

The grh multiple hair cell phenotype differs from that of downstream members of the fz pathway such as inturned, in not showing the typical fz/in abnormal polarity pattern and in the hairs being much more erect. The identity of the targets responsible for this phenotype are unkown. The grh hair phenotype is somewhat reminiscent of that seen with mutations in genes such as Rho kinase or crinkled (myosin VII) suggesting these or related genes as possible targets (Lee, 2004 and references therein).

The transcription of the Ddc gene has previously been shown to be regulated by grh and Ddc activity is required for melanization. Is ddc likely to be the target gene whose altered expression leads to the lowered pigmentation of grh clone cells? This is certainly possible but it seems unlikely to be the entire story. Ddcts2 flies raised at the restrictive condition have more profound pigmentation defects than grh clones. However, clones of ddc null alleles typically have a less severe pigmentation phenotype than grh clones due to partial rescue of the pigmentation phenotype by neighboring cells (i.e. ddc displays submissive cell non-autonomy). Based on these observations it is argued that grh must have other targets that contribute to the decreased pigmentation (Lee, 2004).

The data reported in this paper argue that grh has multiple functions during the development of the adult epidermis. In this context it is not clear to what extent grh functions in a permissive fashion to promote the expression of developmentally important genes and/or to promote changes in gene expression that are associated with the differentiation of the adult cuticle. The data are consistent with grh functioning in both ways. The requirement for grh for the expression of stan was seen at multiple stages consistent with grh having a permissive role. The effects on the timing of hair morphogenesis are consistent with, but do not demand an instructive role (Lee, 2004).

Genetic modifier screens reveal new components that interact with the Drosophila dystroglycan-dystrophin complex

The Dystroglycan-Dystrophin (Dg-Dys) complex has a capacity to transmit information from the extracellular matrix to the cytoskeleton inside the cell. It is proposed that this interaction is under tight regulation; however the signaling/regulatory components of Dg-Dys complex remain elusive. Understanding the regulation of the complex is critical since defects in this complex cause muscular dystrophy in humans. To reveal new regulators of the Dg-Dys complex, genetic interaction screens to identify modifiers of Dg and Dys mutants in Drosophila wing veins. These mutant screens revealed that the Dg-Dys complex interacts with genes involved in muscle function and components of Notch, TGF-β and EGFR signaling pathways. In addition, components of pathways that are required for cellular and/or axonal migration through cytoskeletal regulation, such as Semaphorin-Plexin, Frazzled-Netrin and Slit-Robo pathways show interactions with Dys and/or Dg. These data suggest that the Dg-Dys complex and the other pathways regulating extracellular information transfer to the cytoskeletal dynamics are more intercalated than previously thought (Kucherenko, 2008).

At the basal side of follicle epithelium, actin filaments exhibit a planar cell polarity that is perpendicular to the long axis, the AP axis, of the egg chamber. In Dg follicle cell clones the basal actin array is disrupted non-cell-autonomously. Integrins and the receptor tyrosine phosphatase Lar are also involved in basal actin orientation. It is unclear whether Dg and the other genes involved in basal actin polarity act together with the canonical planar cell polarity pathway or function independently of this pathway. Interestingly, strong interactions were found between the DGC and grainy head (grh) a transcription factor which is required for several different processes during the differentiation including the function of the frizzled dependent tissue polarity pathway, epidermal hair morphogenesis and wing vein specification. In the absence of grh function the Fz, Dsh and Vang proteins fail to accumulate apically and the levels of Stan (or Flamingo) protein are dramatically decreased. The interactions seen with stan (Fla) and wg in wing veins supports the hypothesis that Dg might act together with the frizzled-dependent tissue polarity pathway in coordinating the polarity of cells in epithelial sheets (Kucherenko, 2008).

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date revised: 22 December 2011   

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