grainy head



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 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).

Duox, Flotillin-2, and Src42A are required to activate or delimit the spread of the transcriptional response to epidermal wounds in Drosophila

The epidermis is the largest organ of the body for most animals, and the first line of defense against invading pathogens. A breach in the epidermal cell layer triggers a variety of localized responses that in favorable circumstances result in the repair of the wound. Many cellular and genetic responses must be limited to epidermal cells that are close to wounds, but how this is regulated is still poorly understood. The order and hierarchy of epidermal wound signaling factors are also still obscure. The Drosophila embryonic epidermis provides an excellent system to study genes that regulate wound healing processes. A variety of fluorescent reporters were developed that provide a visible readout of wound-dependent transcriptional activation near epidermal wound sites. A large screen for mutants that alter the activity of these wound reporters has identified seven new genes required to activate or delimit wound-induced transcriptional responses to a narrow zone of cells surrounding wound sites. Among the genes required to delimit the spread of wound responses are Drosophila Flotillin-2 and Src42A, both of which are transcriptionally activated around wound sites. Flotillin-2 and constitutively active Src42A are also sufficient, when overexpressed at high levels, to inhibit wound-induced transcription in epidermal cells. One gene required to activate epidermal wound reporters encodes Dual oxidase, an enzyme that produces hydrogen peroxide. Four biochemical treatments (a serine protease, a Src kinase inhibitor, methyl-β-cyclodextrin, and hydrogen peroxide) were found to be sufficient to globally activate epidermal wound response genes in Drosophila embryos. The epistatic relationships among the factors that induce or delimit the spread of epidermal wound signals were examined. The results define new genetic functions that interact to instruct only a limited number of cells around puncture wounds to mount a transcriptional response, mediating local repair and regeneration (Juarez, 2011).

Drosophila wound healing is an example of a regenerative process, which requires localized epidermal cytoskeletal changes, and localized wound-induced changes in epidermal transcriptional activity. This genetic screen with wound-dependent reporters has allowed identification of novel components that regulate the localized transcriptional response to wounding in epidermal cells. This research identifies seven genes that are required to either activate (Duox and ghost/stenosis) or localize (Flo-2, Src42A, wurst, varicose, and Drosophila homolog of yeast Mak3) the expression patterns of epidermal wound reporters. The number of new functions involved in the delimitation of epidermal wound response near wound sites was unexpected, but indicates that considerable genetic effort is devoted to localizing the activity of transcriptional wound responses during regeneration (Juarez, 2011).

One of the genes that limits the spread of epidermal wound reporters after clean epidermal punctures is Flo-2, as mutants of this gene show a broad expansion of epidermal wound gene activation. Drosophila Flo-2 is itself transcriptionally activated around epidermal wound sites, consistent with an evolutionarily conserved role in regeneration after wounding. In vertebrates, reggie-1/Flo-2 gene expression is activated in wounded fish optic neurons, and reggie-1/Flo-2 and reggie-2/Flo-1 morpholino knockdowns in wounded zebrafish retinal explants reduced axon outgrowth compared to controls. Flo-2 transcriptional activation around Drosophila epidermal wound sites is dependent on the grh genetic function, which is required to activate at least a few other epidermal wound response genes. Flo-2 thus appears to act in the same pathway as grh, although it may act both downstream and upstream of grh, since overexpression of Flo-2 can inhibit the activation of other grh-dependent wound response genes. In this respect, Flo-2 resembles stit receptor tyrosine kinase gene (Wang, 2009), which is both transcriptionally activated by Grh, as well as required for grh-dependent activation of other downstream wound genes. Amazingly, overexpression of Flo-2 can even inhibit the global activation of the Ddc and ple-WE1 wound reporters that are induced by the serine protease trypsin, or by hydrogen peroxide. The inhibitory function of overexpression of Flo-2 on wound induced transcription is cell non-autonomous, at least over the range of a few cell diameters, as shown by the ability of striped overexpression of Flo-2 to silence puncture or trypsin-induced gene activation throughout the epidermis (Juarez, 2011).

The only animal where Flo-2 null mutants have so far been characterized is Drosophila, where Flo-2 has been shown to regulate the spread of Wingless (Wg) and Hedgehog (Hh) signals in the wing imaginal discs. In the wing discs, both the secretion rate and the diffusion rate of these two lipid-modified morphogens were increased when Flo-2 was overexpressed, and decreased when Flo-2 and Flo-1 proteins were not expressed. Despite the reduced spread of Wg and Hh morphogen proteins in Flo-2 mutant imaginal discs, adult morphology of mutants was normal, presumably because of compensatory mechanisms that occur later in development. Whereas a reduced range of activation of wg and hh long range transcription target genes was observed in Flo-2 mutant imaginal discs, a greatly increased range of wound-induced gene activation was observed in Flo-2 mutant embryos. This apparent discrepancy could be explained if one invokes of a long-range wound-induced inhibitory signal that in wild type embryos diffuses faster and farther than a wound activating signal, and thereby functions to limit the wound response to nearby epidermal cells, and that in Flo-2 mutants this potential inhibitory signal has reduced secretion, concentration, and/or diffusion range. This notion is consistent with the cell non-autonomous effect of overexpressed Flo-2 on inhibiting wound- or trypsin-induced gene activation. A similar scheme of controlling signal spreading has been seen in the way that Mmp2 acts cell non-autonomously to limit FGF signaling during Drosophila tracheal development and branch morphogenesis. It's also possible that Flo-2 normally is required to set a global threshold that wound-induced signals must overcome in order to activate wound transcription, for example via Flo-2-dependent endocytosis/degradation of a diffusible wound signal and its receptor (perhaps the Stit RTK), and that signal strength normally surpasses the Flo-2 threshold only in the vicinity of a wound. In this model, loss of Flo-2 would result in all epidermal cells being able to exceed the wound signal threshold, and overexpression of Flo-2 would prevent any cells from exceeding the wound signal threshold. The cell non-autonomous effects of Flo-2 overexpression under this model might be explained by an increase in Flo-2-dependent endocytosis/degradation that rapidly depletes an activating signal from the extracellular space (Juarez, 2011).

Many previous studies have documented biochemical, molecular biological, and cell biological interactions between Src family kinases and Flotillins. In Drosophila, lack of Src42A and Flo-2 leads to expanded spread of wound gene activation, and overexpression of Flo-2 or activated Src42A can inhibit wound gene activation, which is consistent with an interaction between the two functions during the process of wound gene regulation. In cultured mammalian cells, Flo-2 can be phosphorylated by Src family kinases in an extracellular signal-dependent fashion. This phosphorylation is associated with changes in the normal intracellular trafficking of Flotillin-containing membrane microdomains and vesicles. Since overexpressed Flo-2 in Drosophila can act in a cell non-autonomous fashion to inhibit wound gene activation, and overexpressed Src42A acts in a cell autonomous fashion to inhibit wound gene activation, one interpretation is that Flo-2 lies genetically upstream of Src42A in the epidermal wound response. This hypothesis appears to be inconsistent with the vertebrate biochemical data indicating that Src kinases phosphorylate Flotillins to activate their diverse functions. However, an observation that is consistent with Src42A activating Flo-2 protein function, is that even when Flo-2 is overexpressed, addition of chemical inhibitors of Src family kinases to wounded embryos, results in widespread Ddc .47 or ple-WE1 wound reporter activation. One interpretation of this suggests Flo-2 protein, no matter the level of expression, is inactive in the absence of Src42A function. Complex feedback loops involving signaling proteins being regulated by a transcription factor, while the activity of the same transcription factors is regulated by the same signaling pathway, have been observed in the control of Drosophila epidermal wound gene expression and reepithelialization, so there may be similar dynamic cross-regulatory interactions between Flo-2 and Src42A in the localization of the epidermal wound response, interactions not easily captured in linear genetic pathway diagrams (Juarez, 2011).

The inhibitory effect of Src42A on wound gene activation suggests that it might antagonize a signaling cascade that leads to the epidermal wound response. A good candidate for such a signaling cascade is the RTK pathway involving the Stit kinase. Stit is a RET-family RTK that is required for robust activation of the Ddc and stit wound reporter genes in wounded embryos (Wang, 2009). Other evidence consistent with RTK pathway importance in wound gene activation is that phosphotyrosine accumulates persistently around wound sites, and that ERK kinase function is required for robust activation of the Ddc wound reporter gene. Interestingly, Src42A has been shown to act as an inhibitor of some Drosophila RTK proteins (those encoded by the torso, Egfr, and sevenless genes) in a few different tissues during Drosophila development. The Flo-2 and Src42A functions in epidermal wound localization after clean wounding are reminiscent of the role of Drosophila WntD during infectious wounding. WntD mutants show higher levels of some antimicrobial peptide genes after septic injury of adults (Juarez, 2011).

Previous evidence suggested that H2O2 and Duox could provide wound-induced inflammatory signals and antimicrobial activities. The current studies show that Duox is required to activate wound reporter genes after epidermal wounding, and that injected exogenous H2O2 is sufficient to activate widespread epidermal wound gene expression. Overexpression of either Flo-2 or Src42A.CA can inhibit the H2O2 -dependent wound reporter expression, suggesting that all of these components are in a common pathway controlling the activation of epidermal wound reporters. However, the ability of trypsin injection to activate the Ddc .47 and ple-WE1 wound reporters in Duox mutants suggests that a serine protease might act downstream of, or in parallel to, H2O2-dependent wound signals. A recent report showed that in cultured mammalian cells, a Src kinase phosphorylates and inhibits a Flo-2-associated enzyme, peroxiredoxin-1, which results in increased stability of H2O2. This is consistent with the results placing Flo-2, Src42A, and H2O2 in a common wound signaling pathway (Juarez, 2011).

Like H2O2, the injection of methyl-β-cyclodextrin (MβCD) into wounded embryos triggers a global wound response in the epidermis. MβCD strongly depletes cholesterol and other sterols from membranes and disrupt lipid rafts, but was also shown to remove sphingolipid-associated proteins such as Src-Family Kinases. The effects of MβCD, in combination with the effects of loss of Flo-2, suggests that the integrity of lipid rafts and associated proteins are required to inhibit epidermal wound signals. In cultured cells, MβCD treatments trigger a release of EGF receptors from membrane microdomains, which increases EGFR, and perhaps other RTK, signaling in a ligand-independent manner. Interestingly, in cultured keratinocytes, MβCD treatment can induce the expression of involucrin, which encodes a protein, analogous to Drosophila Ple/tyrosine hydroxylase, which is required for the formation of an epidermal barrier. Similarly, MβCD injections into Drosophila embryos might also cause an increase the levels of a wound signal produced or released from cells adjacent to the wound site, allowing more widespread transcriptional activation of wound reporter genes. The observations that overexpression of Src42A or Flo-2 can inhibit the MβCD -triggered activation of epidermal wound reporter genes suggest that high levels of these proteins might overcome lipid raft-inhibitory effects on wound signaling pathways (Juarez, 2011).

Other genes (wurst and varicose) identified in the screen have phenotypes similar to Flo-2 and Src42A mutants. wurst encodes an evolutionarily conserved trans-membrane protein, containing a heat shock cognate protein 70 binding domain and a clathrin binding motif. wurst is ubiquitously expressed in embryonic epithelial cells, strongly up-regulated during endocytosis-dependent luminal clearance, and mislocalized in mutants with endocytosis defects. wurst mutant embryos have tortuous tracheal tubes, due to a failure to properly endocytose matrix material from the tracheal lumen. varicose encodes an evolutionarily-conserved septate junction scaffolding protein, in the Membrane Associated GUanylate Kinase (MAGUK) family. varicose is expressed in epidermally-derived cells (including the hindgut and trachea) and co-localizes with the septate junction proteins, Coracle and Neurexin4. varicose mutant embryos develop permeable tracheal tubes and paracellular barrier defects in epithelia. Like wurst mutants, varicose mutants also have abnormal matrix composition in the tracheal lumen, and may also have abnormal extracellular matrix composition produced by other epidermal cells (Juarez, 2011).

Another gene (ghost), also known as stenosis) identified in this screen is required for wound reporter activation like Duox or grh. ghost encodes the Drosophila Sec24CD homolog, a coat protein of COPII vesicles in the ER/Golgi trafficking pathway. Transport of cargo from the ER to the Golgi via COPII vesicles is required to achieve normal amounts of secretion of extracellular matrix proteins into the developing Drosophila tracheae and normal apical-basal localization of membrane proteins. Presumably, similar secretion and membrane localization defects occur in non-tracheal epidermal cells, which account for the severe cuticle deposition defects in ghost (Sec24CD) mutants. It is fascinating to note that the finding that ghost (Sec24CD) is required for transcriptional activation of epidermal wound reporter genes is consistent with the finding that RNAi knockdowns of Sec24C in a planaria (Schmidtea mediterranea) interfered with normal regeneration after amputation wounds. It is possible that the ghost mutants do not secrete enough wound signals, or the protein matrix necessary for the propagation of a wound signal (Juarez, 2011).

Another gene required for the activation of wound reporters is shroud (sro). It is believed sro to be an allele in the Drosophila Fos-D isoform, and it was hypothesized that one of the Drosophila kayak/Fos transcription factors was required for the activation of some epidermal wound gene reporters. However, has been recently discovered that sro[1] and other sro point mutant alleles do not map in the kayak/Fos gene, but in an immediately adjacent transcription unit (Nm-g/sro) that encodes an enzyme in the sterol metabolic pathway that is necessary for production of ecdysone hormone. At first glance, the requirement of sro to activate some wound reporters suggested that these reporters rely on ecdysone signaling. This is possible, although deletions were tested that eliminate zygotic functions of the ecdysone receptor gene, as well as of the phantom gene (which encodes another enzyme in the ecdysone synthesis pathway), and embryos that are zygotic mutants in either gene show normal activation of the ple-WE1 wound reporter after puncture wounding (Juarez, 2011).

In summary, though this large unbiased screen, several genes were identified that add to the understanding of the complex pathways that control the signals that activate wound response transcription near puncture wounds. At the cellular level, there appears to be a correlation between genetic functions required to localize wound-induced gene activation, and cellular functions required for endocytosis and/or apical-basal polarity. For example, one function of Flo-2 is in signal-dependent endocytosis, although Flo-2 also plays other roles in vesicular trafficking. There have been many studies showing that endocytosis can regulate extracellular signaling strength and duration. For example, one study found that tagged-FGF8 showed increased accumulation, spread, and target gene activation when Rab-5-mediated endocytosis was reduced in zebrafish embryos. It is believed that further studies on wound response signaling may provide new insights into how membrane microdomains, endocytosis of membrane receptors, and the composition and organization of the extracellular matrix, regulates the transmission of wound signals (Juarez, 2011).

Hole-in-one mutant phenotypes link EGFR/ERK signaling to epithelial tissue repair in Drosophila

Epithelia act as physical barriers protecting living organisms and their organs from the surrounding environment. Simple epithelial tissues have the capacity to efficiently repair wounds through a resealing mechanism. The known molecular mechanisms underlying this process appear to be conserved in both vertebrates and invertebrates, namely the involvement of the transcription factors Grainy head (Grh) and Fos. In Drosophila, Grh and Fos lead to the activation of wound response genes required for epithelial repair. ERK is upstream of this pathway and known to be one of the first kinases to be activated upon wounding. However, it is still unclear how ERK activation contributes to a proper wound response and which molecular mechanisms regulate its activation. In a previous screen, mutants were isolated with defects in wound healing. This study describes the role of one of these genes, hole-in-one (holn1), in the wound healing process. Holn1 is a GYF domain containing protein that is required for the activation of several Grh and Fos regulated wound response genes at the wound site. Evidence is provided suggesting that Holn1 may be involved in the Ras/ERK signaling pathway, by acting downstream of ERK. Finally, it was shown that wound healing requires the function of EGFR and ERK signaling. Based on these data, it is concluded that holn1 is a novel gene required for a proper wound healing response. A model is proposed whereby Holn1 acts downstream of EGFR and ERK signaling in the Grh/Fos mediated wound closure pathway (Geiger, 2011).

Holn1 is not required for the initial rapid response to wound infliction, i.e. the formation of the actomyosin cable within minutes of wounding and the phosphorylation of ERK, which is also detectable soon after wounding. This observation is consistent with Holn1 playing an indirect role in the mechanics of wound closure by regulating the mRNA levels of genes required for this process, such as those involved in rapid and productive cable contraction. Interestingly, the actin cable was present in all the wound closure mutants isolated in the previous screen, suggesting that regulatory events downstream of cable formation dominate the wound closure process. In any case, it is clear that Holn1 is required to perform some additional function needed to sustain the closure process, as holn1 mutants take on average 1.5 times longer to close a wound compared to wild type embryos. A similar delay in wound closure was previously reported for rho1 GTPase mutants, which do not form an actin cable, but can still close small wounds, albeit 2 times slower than wild type embryos]. Aside from its possible role in the epithelial hole closure process, Holn1 could also be involved in cuticle repair. Grh and ERK activity are required for the re-establishment of the epithelial permeability barrier after injury. Thus, Holn1 might be involved in this process by regulating the ERK/Grh pathway (Geiger, 2011).

In the future, just as the holn1 mutation uncovered a connection with the EGF/Ras/ERK signaling pathway and wound healing, microarray analysis of wounded holn1 embryos would identify genes that are likely activated downstream of this wound closure pathway. Performing the same experiment using an alternative splicing array as in would further reveal if Holn1 plays a role in wound dependent splicing events (Geiger, 2011).

Combinatorial temporal patterning in progenitors expands neural diversity

Human outer subventricular zone (OSVZ) neural progenitors and Drosophila type II neuroblasts both generate intermediate neural progenitors (INPs) that populate the adult cerebral cortex or central complex, respectively. It is unknown whether INPs simply expand or also diversify neural cell types. This study shows that Drosophila INPs sequentially generate distinct neural subtypes, that INPs sequentially express Dichaete, Grainy head and Eyeless transcription factors, and that these transcription factors are required for the production of distinct neural subtypes. Moreover, parental type II neuroblasts also sequentially express transcription factors and generate different neuronal/glial progeny over time, providing a second temporal identity axis. It is concluded that neuroblast and INP temporal patterning axes act together to generate increased neural diversity within the adult central complex; OSVZ progenitors may use similar mechanisms to increase neural diversity in the human brain (Bayraktar, 2013).

Tests were carried out to determine whether D, Grh and Ey exhibit cross-regulation in INPs. wor-gal4, ase-gal80 was used to drive UAS-DRNAi in a Dichaete heterozygous background (subsequently called D RNAi, in which RNAi denotes RNA interference), which removed detectable D from INP lineages. Compared to wild type, D RNAi resulted in a significant loss of early born Grh+ Ey INPs, without altering the number of later-born Grh+ Ey+ INPs. The same result was observed in D mutant clones. By contrast, misexpression of D did not lead to ectopic Grh expression. Thus, D is necessary for the timely activation of Grh in INP lineages, although D-independent inputs also exist (Bayraktar, 2013).

To test whether Grh regulates D or Ey, R9D11-gal4 was used to drive UAS-grhRNAi in a grh heterozygous background (subsequently called grh RNAi), which significantly reduced Grh levels in middle-aged INPs. grh RNAi increased the number of D+ INPs at the expense of Ey+ INPs without altering the total number of INPs. As expected, grh RNAi did not change the numbers of D+ and Ey+ INPs in the DM1 lineage, which lacks Grh expression, nor did misexpression of Grh lead to ectopic Ey expression. It is concluded that Grh represses D and activates Ey within INP lineages (Bayraktar, 2013).

To determine whether Ey regulates D or Grh, R12E09D-gal4 UAS-FLP actin-FRT-stop-FRT-gal4 was used to drive permanent expression UAS-eyRNAi within INPs (subsequently called R12E09D) act-gal4 or INP-specific ey RNAi. It was confirmed that INP-specific ey RNAi removed Ey expression from INPs, without affecting Ey in the mushroom body or optic lobes. ey RNAi resulted in a notable increase in the number of old D-Grh+ INPs, without affecting the number of young D+ INPs. Conversely, Ey misexpression in INPs significantly reduced the number of Grh+ INPs without altering the total number of INPs. An increase was observed in D+ INPs, consistent with a regulatory hierarchy in which Ey represses Grh, which represses D. This effect was not due to ectopic Ey directly activating D because misexpression of Ey had no effect on D+ INP numbers in the DM1 lineage, which lacks Grh expression. It is concluded that Ey is necessary and sufficient to terminate the Grh expression window in INPs. A 'feedforward activation/feedback repression' model is proposed for D-to-Grh-to-Ey cross-regulation (Bayraktar, 2013).

Next, it was asked whether distinct neuronal or glial subtypes were generated during each transcription factor expression window. To determine the cell types produced by young D+ INPs or old Ey+ INPs, permanent lineage tracing was used. Cells labelled by R12E09D but not OK107ey are generated by young INPs, whereas cells labelled by OK107ey are generated by old INPs. A collection of 60 transcription factor antibodies was screened and two were found that labelled subsets of young INP progeny, and two that labelled subsets of old INP progeny. The transcription factors D and Brain-specific homeobox (Bsh) labelled sparse, non-overlapping subsets of young INP progeny, but not old INP progeny. Thus, young INPs generate Bsh+ neurons, D+ neurons, and many neurons that express neither gene. By contrast, the glial transcription factor Reverse polarity (Repo) and the neuronal transcription factor Twin of eyeless (Toy) labelled sparse, non-overlapping subsets of old INP progeny, but not young INP progeny. Additional mechanisms must restrict each marker (D, Bsh, Repo and Toy) to small subsets of young or old INP progeny; for example, each population could arise from just early or late born INPs within a type II neuroblast lineage. It is concluded that INPs sequentially express the D, Grh and Ey transcription factors, and they generate distinct neuronal and glial cell types during successive transcription factor expression windows. These data provide the first evidence in any organism that INPs undergo temporal patterning (Bayraktar, 2013).

Experiments were designed to determine whether D, Grh and Ey act as temporal identity factors that specify the identity of INP progeny born during their window of expression. First, the role of Ey in the specification of late born INP progeny was investigated. INP-specific ey RNAi resulted in the complete loss of the late born Toy+ neurons and Repo+ neuropil glia, but did not alter the number of early born D+ and Bsh+ neurons. Removal of Toy+ neurons (using toy RNAi) does not alter the number of Repo+ glia, and conversely removal of Repo+ glia (using gcm RNAi) does not alter the number of Toy+ neurons; thus Ey is independently required for the formation of both classes of late INP progeny. Conversely, permanent misexpression of Ey in early INPs increased late born Toy+ neurons and decreased early born Bsh+ neurons, consistent with Ey specifying late INP temporal identity. Unexpectedly, ectopic Ey reduced the number of late born Repo+ glia. Itis concluded that Ey is an INP temporal identity factor that promotes the independent specification of late born Toy+ neurons and Repo+ glia (Bayraktar, 2013).

Next tests were performed to see whether D and Grh specify early and mid INP temporal identity. INP-specific D RNAi led to a small but significant reduction in the number of early born Bsh+ neurons, whereas INP-specific grh RNAi severely reduced the number of early born Bsh+ neurons without impairing INP proliferation or late INP progeny. This is consistent with the Bsh+ neurons deriving from the D+ Grh+ expression window. Interestingly, misexpression of D or Grh did not increase Bsh+ neuron numbers; perhaps D/Grh co-misexpression is required to generate Bsh+ neurons. It is concluded that both D and Grh are required, but not sufficient, for the production of Bsh+ early INP progeny (Bayraktar, 2013).

The function of early or late born INP progeny in adult brain development is unknown. This study determined the role of late born INP neurons and glia in the development and function of the adult central complex (CCX), an evolutionarily conserved insect brain structure containing many type II neuroblast progeny. The CCX consists of four interconnected compartments at the protocerebrum midline: the ellipsoid body, the fan-shaped body, the bilaterally paired noduli, and the protocerebral bridge; each of these compartments is formed by a highly diverse set of neurons. First, permanent lineage tracing was used to map the contribution of late born Ey+ INP progeny to the adult CCX. Cell bodies were detected in the dorsoposterior region of the CCX, and their axonal projections extensively innervated the entire ellipsoid body, fan-shaped body, and protocerebral bridge, with much weaker labelling of the paired noduli. It is concluded that old INPs contribute neurons primarily to the ellipsoid body, fan-shaped bod and protocerebral bridge regions of the CCX. Second, INP-specific ey RNAi was used to delete the late born Toy+ neurons and Repo+ glia. Loss of late born INP progeny generated major neuroanatomical defects throughout the adult CCX: the ellipsoid body and paired noduli were no longer discernible, the fan-shaped body was enlarged, and the protocerebral bridge was fragmented. Subsets of this phenotype were observed after removal of Toy+ neurons or Repo+ glia, showing that they contribute to distinct aspects of the CCX. Previous studies have described similar or weaker morphological CCX defects in ey hypomorphs, toy mutants, and after broad glia ablation during larval stages. In addition, ey RNAi adults were found to have relatively normal locomotion, but have a significant deficit in negative geotaxis. It is concluded that Ey is a temporal identity factor that specifies late born neuron and glial identity, and that these late born neural cell types are essential for assembly of the adult central complex (Bayraktar, 2013).

Bsh+ neurons and Repo+ glia were found to be sparse within the total population of young and old INP progeny, respectively, indicating that other mechanisms must help to restrict the formation of these neural subtypes. One mechanism could be temporal patterning within type II neuroblast lineages (Bayraktar, 2013).

To determine whether type II neuroblasts change their transcriptional profiles over time, known temporal transcription factors were examined for expression in type II neuroblasts at five time points in their lineage (24, 48, 72, 96 and 120 h ALH). No type II neuroblast expression for Hunchback, Kruppel, Pdm1/2 and Broad, and Grh was expressed in all type II neuroblasts at all time points. However, three transcription factors were identified with temporal expression in type II neuroblasts. D and Castor (Cas) were specifically detected in early type II neuroblasts: 3-4 neuroblasts at 24 h ALH, 0-1 neuroblast at 48 h ALH, and none later. Although D was never detected simultaneously in all type II neuroblasts at 24 h, permanent lineage tracing with R12E09D labels all type II neuroblasts, indicating that all transiently express D. The third transcription factor, Seven up (Svp), showed a pulse of expression in a subset of type II neuroblasts at 48 h ALH, but was typically absent from younger or older type II neuroblasts. D, Cas and Svp are all detected in the anterior-most type II neuroblasts (probably corresponding to DM1-DM3), and thus at least these type II neuroblasts must sequentially express D or Cas, and Svp. It is concluded that type II neuroblasts can change gene expression over time (Bayraktar, 2013).

Next, tests were performed to determine whether type II neuroblasts produce different INPs over time. Permanently labelled clones were generated within the type II neuroblast lineages at progressively later time points. If type II neuroblasts change over time to make different INPs, early and late neuroblast clones should contain different neural subtypes. Clones were assayed for Repo+ glia and Bsh+ neurons, choosing these markers because Repo+ neuropil glia have been proposed to be born early in type II neuroblast lineages and Bsh+ neurons were positioned far from the Repo+ glia consistent with a different birth-order. Bsh+ neuron numbers began to decline only in clones induced at the latest time point, showing that they are generated late in the type II neuroblast lineage. By contrast, Repo+ glia were detected in clones induced early but not lat. This allows assigning of Repo+ glia to an 'early neuroblast, old INP' portion of the lineage, and Bsh+ neurons to a 'late neuroblast, young INP' portion of the lineage. It is concluded that type II neuroblasts undergo temporal patterning, and neuroblast temporal patterning was proposed to act together with INP temporal patterning to increase neural diversity in the adult brain (Bayraktar, 2013).

This study has shown that INPs sequentially express three transcription factors (D, Grh and then Ey), and that different neural subtypes are generated from successive transcription factor windows. It is likely that multiple GMCs are born from each of the four known INP gene expression windows; GMCs born from a particular gene expression window may have the same identity, or may be further distinguished by 'subtemporal genes' as in embryonic type I neuroblast lineages. This study also showed that each temporal factor is required for the production of a distinct temporal neural subtype. Loss of D or Grh leads to the loss of Bsh+ neurons; loss of Ey leads to loss of Toy+ neurons and Repo+ glia, although the fate of the missing cells is unknown. An unexpected finding was that Ey limits the lifespan of INPs. Mechanisms that prevent INP de-differentiation have been characterized -- loss of the translational repressor Brain tumour (Brat) or the transcription factor Earmuff (Erm) causes INPs to de-differentiate into tumorigenic type II neuroblasts, but factors that terminate normal INP proliferation have never before been identified (Bayraktar, 2013).

The D-to-Grh-to-Ey INP temporal identity factors are all used in other contexts during Drosophila development. Many embryonic neuroblasts sequentially express D and Grh. Ey is expressed in mushroom body neuroblasts, and is required for development of the adult brain mushroom body. Interestingly, mammalian orthologues of D and Ey (SOX2 and PAX6, respectively) are expressed in neural progenitors, including OSVZ progenitor, but have not been tested for a role in temporal patterning (Bayraktar, 2013).

This study has shown that there are two axes of temporal patterning within type II neuroblast lineages: both neuroblasts and INPs change over time to make different neurons and glia, thereby expanding neural diversity. It will be important to investigate whether INPs generated by OSVZ neural stem cells undergo similar temporal patterning (perhaps using SOX2 and PAX6), and whether combinatorial temporal patterning contributes to the neuronal complexity of the human neocortex (Bayraktar, 2013).

Global programmed switch in neural daughter cell proliferation mode triggered by a temporal gene cascade

During central nervous system (CNS) development, progenitors typically divide asymmetrically, renewing themselves while budding off daughter cells with more limited proliferative potential. Variation in daughter cell proliferation has a profound impact on CNS development and evolution, but the underlying mechanisms remain poorly understood. This study found that Drosophila embryonic neural progenitors (neuroblasts) undergo a programmed daughter proliferation mode switch, from generating daughters that divide once (type I) to generating neurons directly (type 0). This typeI> 0 switch is triggered by activation of Dacapo (mammalian p21CIP1/p27KIP1/p57Kip2) expression in neuroblasts. In the thoracic region, Dacapo expression is activated by the temporal cascade (castor) and the Hox gene Antennapedia. In addition, castor, Antennapedia, and the late temporal gene grainyhead act combinatorially to control the precise timing of neuroblast cell-cycle exit by repressing Cyclin E and E2f. This reveals a logical principle underlying progenitor and daughter cell proliferation control in the Drosophila CNS (Baumgardt, 2014).

Proliferation analysis of the developing Drosophila VNC reveals that most, if not all, lateral NBs initially divide in the type I proliferation mode, generating daughters that divide once. Three specific lineages, as well as many other NBs, subsequently switch to generating daughters that do not divide (type 0 mode). The full extent of the typeI>0 switch is currently difficult to precisely assess for several reasons. One such complicating issue pertains to possible developmental changes in daughter cell-cycle length over time. On this note, however, no obvious change was found in NB5-6T daughter divisions prior to the switch. In addition, if the accelerated decline in daughter division was indeed caused by a lengthening of the cell cycle rather than a typeI>0 switch, a long 'tail' of daughter divisions would be expected, perduring into St16-17. This is not the case; rather, daughter proliferation drops down to almost zero by St16-17. Similarly, no evidence was found for changes in NB cell-cycle length over time in the three specific NB lineages. Another complicating issue pertains to the fact that NBs differ in their time point of delamination, number of division rounds, and time point of switching, so that even if all NBs switched, only a fraction of the NBs would be in their type 0 window at the same time. However, these complications are more likely to lead to under- rather than overappreciation of the extent of the typeI>0 switch, and it is tempting to speculate that it may indeed involve the vast majority of NBs (Baumgardt, 2014).

The typeI>0 switch is triggered by the onset of Dap expression in NBs at precise stages of lineage progression. The mammalian Dap orthologs, p21CIP1/p27KIP1/p57Kip2, can act as inhibitors of the CycE/Cdk2 complex. By analogy, the mechanism behind the typeI>0 switch is, presumably, that type 0 daughters are prevented from entering the cell cycle by the presence of Dap at the G1/S checkpoint. The onset of Dap expression already in the NB suggests that Dap needs to be present at an early stage in newborn daughters to block their entry into S phase. These findings are also in line with the emerging role for the Cip/KIP family and cell-cycle exit in the mammalian CNS, although there has been no report of a connection to changes in daughter proliferation mode (Baumgardt, 2014).

No evidence was found for a role of pros in the type 0 mode, and, conversely, no evidence was found for a role of dap in the type I mode. The distinct roles of pros and dap in control of the type I versus 0 modes is further underscored by the expression of E2f, CycE, and Dap. In type I daughters (GMCs), E2f and CycE are rapidly repressed, by pros, and Dap is only weakly expressed at a later stage, around the time point of mitosis. The short window of E2f and CycE expression is still sufficient for the GMC to enter another cell cycle, since Dap expression is absent. As each GMC divides, the postmitotic cells (neurons/glia) are prevented from entering the cell cycle by the lack of E2f and CycE. In type 0 daughters, on the other hand, E2f and CycE expression is robust, but daughters still fail to enter the cell cycle due to the presence of high levels of Dap. These findings point to strikingly different strategies in daughter proliferation control: pros repression of E2f/CycE in type I, and Dap overriding E2f/CycE/Cdk2 in type 0 daughters (Baumgardt, 2014).

Changes in daughter cell proliferation could perhaps have been envisioned to merely result from a gradual loss of the proliferative potential of each progenitor, as a result of its undergoing many rapid cell cycles. If so, typeI>0 switches could have been predicted to occur somewhat stochastically toward the end stage of each lineage, perhaps loosely linked to the last NB division. In contrast to such simplified models, this study found that the typeI>0 switch can occur many divisions prior to NB exit and that it is programmed to occur at a precise stage during each lineage development. In the thorax, it was found that the precise timing of typeI>0 switches is controlled by the temporal gene cas and the Hox gene Antp, which are expressed at a late stage within NBs. Remarkably, in cas mutants, most, if not all, thoracic typeI>0 lineages fail to enter the type 0 mode. The primary mechanism by which cas and Antp control the switch appears to be by activating the expression of Dap, evident by the reduction of Dap in cas and Antp mutants; by the finding that cas-Antp co-misexpression triggers ectopic Dap expression; and by the finding that cas can be cross-rescued by elav>dap (Baumgardt, 2014).

The finding that the timing of the typeI>0 switch is scheduled by a temporal gene cascade points to an intriguing regulatory model where daughter cell proliferation mode switches are executed at stereotyped positions within the lineage tree by the activity of specific temporal genes. Since temporal genes also control the progression of NB competence, evident by their roles in cell fate specification, the temporal cascade can act to simultaneously control both cell fate and cell number, thereby ensuring that precise number of each neural cell subtype is produced (Baumgardt, 2014).

After a stereotyped number of divisions, each NB subtype stops proliferating. This study found that, for many NBs, this is a G1/S decision influenced by the activities of E2f, CycE, and dap. The nuclear localization of Pros was previously identified to be associated with cell-cycle exit in postembryonic NBs. However, previous studies of NB5-6T, and the current study on NB7-3A, do not indicate a general role for pros in NB cell-cycle exit in the embryonic CNS. Instead, in the thorax, the expression levels of E2f, CycE, and Dap are gradually modulated during lineage progression, by the temporal genes cas and grh as well as Antp. Because Cas, Grh, and Antp are progressively activated in thoracic NBs, this brings into view a logical model for timely NB cell-cycle exit where sequential activation of temporal and Hox genes act combinatorially to push E2f, CycE, and/or Dap to limiting levels after a determined number of divisions (Baumgardt, 2014).

For the majority of NBs in the thorax, cell-cycle exit is followed by quiescence until larval stages. In contrast, for the majority of abdominal NBs, cell-cycle exit is followed by apoptosis. However, for some NBs, such as NB7-3A, apoptosis is the functional exit mechanism. Thus, three general strategies for lineage stop are emerging: (1) cell-cycle exit > quiescence (most thoracic NBs), (2) cell-cycle exit > apoptosis (NB5-6T), and (3) lineage stop by apoptosis (NB7-3A). The balance of E2f, CycE, and Dap is involved in the first two strategies, while the balance of apoptosis gene expression presumably is at the core of the latter strategy (Baumgardt, 2014).

In addition to the type I and type 0 daughter proliferation modes described here in the embryo, recent studies of Drosophila larval CNS development have identified a third, more prolific, proliferation mode: the type II mode, identified in a small number of larval brain. Type II NBs divide asymmetrically, renewing themselves while budding of daughters that, in turn, undergo multiple rounds of proliferation before finally differentiating. This allows for the generation of very large lineages (some 500 cells) from each individual type II NB (Baumgardt, 2014).

In mammals, the most obvious equivalent of Drosophila NBs is the radial glia cell (RG), which divides asymmetrically to generate neurons and. During these RG asymmetric divisions, studies have identified several different division modes; RGs dividing asymmetrically to bud off a neuron, to bud off a daughter cell that divides once to generate two neurons, or to bud off daughter cells that themselves divide multiple times before generating neurons. Although mammalian CNS development likely will involve more complex and more elaborate lineage variations, there is, nevertheless, a striking similarity between these alternate mammalian daughter proliferation modes and the type 0, I and II modes now identified in Drosophila. Intriguingly, in line with these analogies between Drosophila and mammals, recent time-lapse studies on the developing primate cortex have revealed a global temporal switch in the proliferation profiles of daughter cells (Betizeau, 2013). It will be interesting to learn if such temporal proliferation changes are intrinsically controlled and if they are stereotypically linked to changes in neural subtype specification also in mammals (Baumgardt, 2014).

Drosophila embryonic type II neuroblasts: origin, temporal patterning, and contribution to the adult central complex

Drosophila neuroblasts are an excellent model for investigating how neuronal diversity is generated. Most brain neuroblasts generate a series of ganglion mother cells (GMCs) that each make two neurons (type I lineage), but sixteen brain neuroblasts generate a series of intermediate neural progenitors (INPs) that each produce 4-6 GMCs and 8-12 neurons (type II lineage). Thus, type II lineages are similar to primate cortical lineages, and may serve as models for understanding cortical expansion. Yet the origin of type II neuroblasts remains mysterious: do they form in the embryo or larva? If they form in the embryo, do their progeny populate the adult central complex, as do the larval type II neuroblast progeny? This study presents molecular and clonal data showing that all type II neuroblasts form in the embryo, produce INPs, and express known temporal transcription factors. Embryonic type II neuroblasts and INPs undergo quiescence, and produce embryonic-born progeny that contribute to the adult central complex. These results provide a foundation for investigating the development of the central complex, and tools for characterizing early-born neurons in central complex function (Walsh, 2017).

It has been difficult to link embryonic neuroblasts to their larval counterparts in the brain and thoracic segments owing to the period of quiescence at the embryo-larval transition, and owing to dramatic morphological changes of the CNS that occur at late embryogenesis. Recent work has revealed the embryonic origin of some larval neuroblasts: the four mushroom body neuroblasts in the central brain and about 20 neuroblasts in thoracic segments. This study used molecular markers and clonal analysis to identify all eight known type II neuroblasts in each brain lobe and show they all form during embryogenesis, perhaps the last-born central brain neuroblasts. It was not possible to identify each neuroblast individually, however, owing to their tight clustering, movements of the brain lobes, and the lack of markers for specific type II neuroblasts (Walsh, 2017).

The single previously reported embryonic type II neuroblast formed from PntP1+ neuroectodermal cells with apical constrictions called a placode. This study did not investigate this neuroectodermal origin of type II neuroblasts in much detail, but multiple type II neuroblasts were seen developing from PntP1+ neuroectoderm. In the future, it would be interesting to determine whether all type II neuroblasts arise from PntP1+ neuroectoderm or from neuroectodermal placodes. Interestingly, one distinguishing molecular attribute of type II neuroblasts is PntP1, which is not detected in type I neuroblasts. Thus, a candidate for distinguishing type I/type II neuroblast identity is EGF signaling, which can be detected in the three head placodes and is required for PntP1 expression. Clearly, there are more PntP1+ neuroectodermal cells than there are type II neuroblasts, and expression of an EGF negative regulator such as Argos might be necessary to divert some of these neuroectodermal cells away from type II neuroblast specification. The earliest steps of type II neuroblast formation represent an interesting spatial patterning question for future studies (Walsh, 2017).

Now that the embryonic type II neuroblasts have been identified, it is worth considering whether there are differences between embryonic and larval type II neuroblasts or their INP progeny. To date, molecular markers do not reveal any differences between embryonic and larval type II neuroblasts, with the exception that embryonic neuroblasts transiently express the temporal transcription factor Pdm. Interestingly, type I embryonic neuroblasts require Cas to close the Pdm expression window, whereas this study found that cas mutants do not exhibit extension of the Pdm expression window in the earliest-born type II neuroblast or de novo expression of Pdm in the later-forming neuroblasts. Are there differences between embryonic and larval INPs? Larval INPs mature over a period of 6 h and then divide four to six times with a cell cycle of about 1 h. In contrast, embryonic INPs might have a more rapid maturation because Elav+ neurons were seen within 9D11+ INP lineages by stage 14, just 3 h after the first type II neuroblast forms. This study found that INPs undergo quiescence at the embryo-larval transition, as shown by the pools of INPs at stage 16 that do not stain for the mitotic marker pH3. The fate of these quiescent INPs -- whether they resume proliferation, differentiate or die -- remains to be determined (Walsh, 2017).

Neuroblasts in the embryonic ventral nerve cord use the temporal transcription factor cascade Hb>Krüppel>Pdm>Cas>Grh to generate neural diversity. This study shows that the type II neuroblasts are among the last neuroblasts to form in the embryonic brain, and that they sequentially express only the late temporal transcription factors Pdm (in the earliest-forming neuroblast) followed by Cas and grh (in all eight type II neuroblasts). It is unknown why most type II neuroblasts skip the early Hb>Krüppel>Pdm temporal transcription factors; perhaps it is due to their late time of formation, although several earlier-forming thoracic neuroblasts also skip Hb (NB3-3), Hb>Krüppel (NB5-5), or Hb>Krüppel>Pdm. This is another interesting spatial patterning question for the future. Furthermore, misexpression of the early factors (Hb and Krüppel) would be unlikely to affect the progeny produced by type II NBs during embryogenesis, as the competence window for Hb (i.e., the stage at which neuroblasts are responsive to Hb expression) closes with the loss of Dan/Danr expression in all neuroblasts at stage 12. Thus, most embryonic type II neuroblasts form after closing of the Hb competence window and would probably be unresponsive (Walsh, 2017).

Type I neuroblasts show persistent expression of the temporal transcription factors within neurons born during each window of expression (i.e. a Hb+ neuroblast divides to produce a Hb+ GMC which makes Hb+ neurons). In contrast, this study found that type II lineages do not show persistent Cas or grh expression in INPs born during each expression window, but do contain some Cas+ neurons. Both Cas and grh transcription factors can be seen in INPs immediately adjacent to the parental neuroblast, consistent with transient perdurance from the parental neuroblast, but they are typically lacking in INPs more distant. The function of Pdm, Cas and grh in embryonic type II neuroblasts awaits identification of specific markers for neural progeny born during each expression window (Walsh, 2017).

During larval neurogenesis, virtually all INPs sequentially express the temporal transcription factors Dichaete>Grh>Ey. In contrast, embryonic INPs express only Dichaete. These data, together with the short time frame of embryogenesis, suggest that INP quiescence occurs during the Dichaete window, preventing expression of the later Grh>Ey cascade. Interestingly, INPs in the posterior cluster (presumptive DL1 and DL2 type II neuroblast progeny) completely lack Dichaete; this is similar to the DL1 and DL2 larval lineages, which also do not express Dichaete. It is possible that DL1/DL2 neuroblasts make INPs that generate identical progeny (and thus do not require an INP temporal cascade), or perhaps these two neuroblasts use a novel temporal cascade in both embryonic and larval stages (Walsh, 2017).

Larval type II neuroblasts produce many intrinsic neurons of the adult central complex. This study shows that embryonic INPs also produce neurons that contribute to the adult central complex. The data show ~54 neurons (64 minus 10 due to 'leaky' expression) born from embryonic-born INPs survive to adulthood and innervate the central complex. It is likely that this is an underestimate, however, because (1) 9D11-gal4 expression is lacking from a few INPs in the embryonic brain and (2) the time to achieve sufficient FLP protein levels to achieve immortalization could miss the earliest born neurons. The variation in immortalization of the widefield ellipsoid body neuron might represent a neuron born early in the type II lineages, thus unlabeled in a subset of embryos. Additionally, some embryonic-born neurons might perform important functions in the larval/pupal stages but die prior to eclosion (Walsh, 2017).

Further studies will be required to understand the function of neurons born from embryonic type II lineages. It remains to be experimentally determined whether some or all embryonic progeny of type II neuroblasts (1) remain functionally immature in both the larval and adult brain, but serve as pioneer neurons to guide larval-born neurons to establish the central complex, (2) remain functionally immature in the larval brain, but differentiate and function in the adult central complex, or (3) differentiate and perform a function in both the larval and adult CNS. It will be informative to ablate embryonic-born neurons selectively and determine the effect on the assembly of the larval or adult central complex, and their role in generating larval and adult behavior (Walsh, 2017).

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).

Genetic control of cuticle formation during embryonic development of Drosophila melanogaster

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, grhB37; two P-element insertions, grhs2140 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 (grhs2140/grhs2140; bnlP1/+). 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 grhFK2131 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).

New Dystrophin/Dystroglycan interactors control neuron behavior in Drosophila eye.

The Dystrophin Glycoprotein Complex (DGC) is a large multi-component complex that is well known for its function in muscle tissue. When the main components of the DGC, Dystrophin (Dys) and Dystroglycan (Dg) are affected cognitive impairment and mental retardation in addition to muscle degeneration can occur. Genetic screens have been performed using a Drosophila model for muscular dystrophy in order to find novel DGC interactors aiming to elucidate the signaling role(s) in which the complex is involved. Since the function of the DGC in the brain and nervous system has not been fully defined, this study has analyzed the DGC modifiers' function in the developing Drosophila brain and eye. Given that disruption of Dys and Dg leads to improper photoreceptor axon projections into the lamina and eye neuron elongation defects during development, the function of previously screened components and their genetic interaction with the DGC in this tissue were determined. This study first found that mutations in chif, CG34400, Nrk, Lis1, capt and Cam cause improper axon path-finding and loss of SP2353, Grh, Nrk, capt, CG34400, vimar, Lis1 and Cam cause shortened rhabdomere lengths. It was determined that Nrk, mbl, capt and Cam genetically interact with Dys and/or Dg in these processes. It is notable that most of the neuronal DGC interacting components encountered are involved in regulation of actin dynamics. These data indicate possible DGC involvement in the process of cytoskeletal remodeling in neurons. The identification of new components that interact with the DGC not only helps to dissect the mechanism of axon guidance and eye neuron differentiation but also provides a great opportunity for understanding the signaling mechanisms by which the cell surface receptor Dg communicates via Dys with the actin cytoskeleton (Marrone, 2011).

The roles that Dys and Dg play in disease have been apparent for some time since their disruption or misregulation has been closely linked to various MDs. Dg depletion results in congenital muscular dystrophy-like brain malformations associated with layering defects and aberrant neuron migration. These defects arise due to extracellular matrix protein affinity problems that influence neuronal communication and result in learning and memory defects. Similar to brain layer formation, the migration of R1-R6 growth cones into the lamina occurs in a similar manner where glia cells that migrate from progenitor regions into the lamina provide a termination cue to innervating axons. In Drosophila Dys and Dg are expressed in the CNS, PNS and visual system and both proteins are required for proper photoreceptor axon guidance and rhabdomere elongation. This work has identified novel components implicated in the process of eye-neuron development. Moreover, it was found that Nrk, Mbl, Cam and Capt genetically interact with Dys and/or Dg in visual system establishment (Marrone, 2011).

The proteins Mbl, Capt, Cam, Robo, Lis1 and Nrk have been shown previously to be associated with the nervous system, and this study has additionally found that mutations in chif, SP2353, CG34400 and vimar cause abnormal photoreceptor axon pathfinding and/or differentiation phenotypes. Lis1 has been shown to bind microtubules in the growth cone, and the human Lis1 homologue is important for neuronal migration and when mutated causes Lissencephaly, a severe neuronal migration defect characterized by a smooth cerebral surface, mental retardation and seizures. This study has found that Lis1RNAi/GMR-Gal4 mutants have abnormally formed lamina plexuses, shortened rhabdomeres, and retinal vacuoles. Chif has been shown to regulate gene expression during egg shell development and is related to a DNA replication protein in yeast. The human ortholog for SP2353 (AGRN) is involved in congenital MD development. Drosophila SP2353 is a novel agrin-like protein that contains Laminin G domains, which makes it a potential new extracellular binding partner for Dg. CG34400 encodes for a protein homologues to human DFNB31 (Deafness, autosomal recessive 31) that causes congenital hearing impairment in DFNB31 deficient people and mouse whirlin, that causes deafness in the whirler mouse. Hearing loss has been as well demonstrated in association with various forms of muscular dystrophy. Vimar has been shown to regulate mitochondrial function via an increase in citrate synthase activity (Marrone, 2011).

Mbl is a Drosophila homologue of the human gene MBNL1. Mutations of this gene cause myotonic dystrophy and are associated with the RNA toxicity of CUG expansion diseases protein. This study shows that Mbl deficiency results in similar phenotypes to Dys and Dg loss of function, and to specifically interact with Dys in axon projections which is in accord with the Dys specific interaction seen in muscle. Dys has multiple isoforms, and the variability of DMD patients to have mental impairment has been linked in part to small Dys isoform mutations, which leads to speculation that Dys is a target for Mbl mediated splicing (Marrone, 2011).

Interestingly, Mbl isoforms have been demonstrated to regulate splicing of α-actinin, which belongs to the spectrin gene superfamily that also includes dystrophins. α-actinin and Capt, the Drosophila homologue of Cyclase-associated protein (CAP) are actin-binding proteins in the growth cone. Capt was first identified in yeast and is highly conserved throughout eukaryotic evolution. The main known function of Capt is to act in the process of actin recycling by working in conjunction with Actin Depolymerization Factor (ADF a.k.a. Cofilin) to help displace Cofilin from G-actin during depolymerization. It has already been reported that ADF/Cofilin has a role in retinal elongation. The actin cytoskeleton is a major internal structure that defines the morphology of neurons, and Capt has already been shown to be required to maintain PNS neuronal dendrite homeostasis in Drosophila via kinesin-mediated transport. Additionally, Capt has been found to lead to excessive actin filament polymerization in the eye disc and to cause premature differentiation of photoreceptors. The rate of axon projection is much slower than the rate of microtubule polymerization during axonal growth, implying that depolymerization/polymerization of actin is important during pathfinding. This study has also shown that Capt interacts with Dys and is necessary for proper projection of photoreceptor axons in the developing brain, and when absent, eyes develop with abnormal rhabdomeres. Furthermore, captRNAi mutants exhibit overgrowth of photoreceptor axons, and it is believed that a possible explanation for this is improper turnover of actin (Marrone, 2011).

Importantly, proteins that can be regulated by Ca2+ to organize actin filament bundles and to promote filament turnover include α-actinin and (ADF)/Cofilin, respectively. Cam functions as an intracellular Ca2+ sensor, and when Ca2+-Cam was selectively disrupted in a subset of neurons in Drosophila embryos, stalls in axon extension and errors in growth cone guidance resulted. Actin turnover is highly regulated by Ca2+ levels, and many proteins are Ca2+-mediated to regulate motility and axon guidance. The results and those from prior studies suggest that Cam is a major functional player of Ca2+ regulation in growth cones. Since it was shown here that mutations in Cam and capt have similar phenotypes in photoreceptor axon pathfinding and rhabdomere development, it is postulated that actin dynamics is the link between these two proteins and the phenotypes described here. Due to the importance of Cam for actin dynamics, its interaction with both Dg and Dys suggests that the DGC coordinates the actin cytoskeleton in the developing eye (Marrone, 2011).

The last gene identified in this work is Nrk. Recently various kinases, channels and other enzymes have been shown to associate with the DGC, although only a few of these interactions have been confirmed in vivo. Since Nrk is a component found to interact with Dys in photoreceptor axon pathfinding, it is most likely that it functions as a receptor to sense guidance cues rather than as a molecule affecting actin cytoskeletal rearrangement. The data here hint that Dg and Nrk could be two receptors integral to transferring signals important for neuronal layering (Marrone, 2011).

It is concluded that dynamic rearrangement of the actin cytoskeleton is crucial for the central and peripheral nervous system establishment, which depends on proper neuron migration and differentiation. This process requires not only the cell autonomous regulation of neuron motility, but also the interaction between the migrating cell and its underlying substrate. This interaction is often dependent on the signaling transduced via the ECM. The DGC and other factors are believed to be mediators of actin dynamics in growing axons and during neuronal cell morphogenesis, and this study found components that interact with Dys and/or Dg in both of these activities (see The DGC coordinates actin cytoskeleton remodeling). Additionally, disruption in gene expression of these components results in the same phenotypes seen with Dys and Dg mutants in the developing and adult eye. The data lead to the conclusion that the DGC is involved in signaling to cause cytoskeletal rearrangement and actin turnover in growth cones. Since many cases of muscular dystrophies are associated with mental retardation, it is believed that it is important to understand the role of the DGC in axon migration because understanding of this process could aid in finding an adequate therapy for this aspect of the disease's physiology. Since the human brain continues to develop well after gestation, and evidence shows that nerves maintain plasticity throughout an individual's lifespan, therapies could be devised that reverse these defects after birth (Marrone, 2011).


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grainy head: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 20 April 2020  

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