nubbin/POU domain protein 1


cis-Regulatory Sequences and Functions

The identification of sequences that control transcription in metazoans is a major goal of genome analysis. Searching for clusters of predicted transcription factor binding sites can discover active regulatory sequences; 37 regions of the Drosophila melanogaster genome have been identified with high densities of predicted binding sites for five transcription factors involved in anterior-posterior embryonic patterning. Nine of these clusters overlapped known enhancers. This study reports the results of in vivo functional analysis of 27 remaining clusters. Transgenic flies were generated carrying each cluster attached to a basal promoter and reporter gene, and embryos were assayed for reporter gene expression. Six clusters are enhancers of adjacent genes: giant, fushi tarazu, odd-skipped, nubbin, squeeze and pdm2; three other clusters drive expression in patterns unrelated to those of neighboring genes; the remaining 18 clusters do not appear to have enhancer activity. The Drosophila pseudoobscura genome was used to compare patterns of evolution in and around the 15 positive and 18 false-positive predictions. Although conservation of primary sequence cannot distinguish true from false positives, conservation of binding-site clustering accurately discriminates functional binding-site clusters from those with no function. Conservation of binding-site clustering was incorporated into a new genome-wide enhancer screen, and several hundred new regulatory sequences, including 85 adjacent to genes with embryonic patterns, have been preducted. It is concluded that measuring conservation of sequence features closely linked to function (such as binding-site clustering) makes better use of comparative sequence data than commonly used methods that examine only sequence identity (Berman, 2004).

Each of the 37 pCRMs were assigned an identifier (of the form PCEXXXX). The first nine overlap previously known enhancers of runt, even-skipped, hairy, knirps and hunchback. To determine whether any of the remaining 28 pCRMs also function as enhancers, P-element constructs were generated containing the pCRM sequence with minimal flanking sequence on both sides fused to the eve basal promoter and a lacZ reporter gene. Since the margins of the tested sequences do not precisely correspond to the margins of the clusters, a unique identifier (of the form CEXXXX) was assigned to each tested fragment (identical CE and PCE numbers correspond to the same pCRM) (Berman, 2004).

Multiple independent transgenic fly lines were sucessfully generated for 27 of the 28 pCRMs. Transgenes containing CE8007 could not be generated. This sequence contains five copies of an approximately 358 base-pair (bp) degenerate repeat. One additional pCRM (CE8002) also contains tandem repeats. While it was possible to generate transgenes for CE8002 and assay its expression, these two tandem repeat-containing pCRMs (CE8007 and CE8002) were excluded from subsequent analyses (Berman, 2004).

The expression of these constructs was examined by in situ RNA hybridization to the lacZ transcript in embryos at different stages in at least three independent transformant lines. Nine of the 27 transgenes showed mRNA expression during embryogenesis, while the remaining 18 assayed transgenes showed no detectable expression at any stage during embryogenesis.

To identify the genes regulated by the nine pCRMs with embryonic expression, the expression patterns were examined of genes containing the pCRM in an intron and genes with promoters within 20 kb of the CRM. The embryonic microrarray and whole-mount in situ expression data available in the Berkeley Gene Expression Database were used, supplemented with additional whole-mount in situ experiments where necessary. Six of the active pCRMs drive lacZ expression in patterns that recapitulate portions of the expression of a gene adjacent to or containing the pCRM. Four of these new enhancers act in the blastoderm and two during germ-band elongation (Berman, 2004).

CE8001 is 5' of the gene for the gap transcription factor giant and recapitulates the posterior domain (65%-85% egg length measuring from the anterior end of the embryo) of gt expression in the blastoderm. CE8011 is 5' of the gene for the POU-homeobox transcription factor nubbin (nub). The CRM recapitulates the endogenous blastoderm expression pattern of nub, first detected as a broad band extending from 50% to 75% egg length. Although nub expression continues in later embryonic stages, CE8011 expression is limited to the blastoderm stage. CE8010 is 5' of the pair-rule gene odd-skipped (odd) and drives expression of two of its seven stripes: stripe 3 at 55% and stripe 6 at 75% egg length. This CRM also has the ability to drive later, more complex, patterns of expression. During stages 6 and 7, expression is detected in the procephalic ectoderm anlage and in the primordium of the posterior midgut. By stage 13, expression is also detected in the anterior cells of the midgut which will give rise to the proventriculus, the first midgut constriction, the posterior midgut and microtubule primordial as well as cells in the hindgut, all similar to portions of the pattern of wildtype Odd protein expression. CE8024 is 3' of the pair-rule gene fushi-tarazu and drives expression of two of its stripes: stripe 1 at 35% and stripe 5 at 65% egg length. CE8012 is in the third intron of POU domain protein 2 (pdm2) and appears to completely recapitulate its stage-12 expression pattern, which is limited to a subset of the developing neuroblasts and ganglion mother cells of the developing central nervous system. A similar pattern of expression was previously described for the protein product of pdm2. It is worth noting that expression of CE8012 was not detected in the blastoderm stage, whereas the endogenous gene exhibits a blastoderm expression pattern similar to nub. CE8027 is 3' of the gene for the Zn-finger transcription factor squeeze (sqz) and recapitulates the wild-type expression pattern of sqz RNA in a subset of cells in the neuroectoderm at stage 12 (Berman, 2004).

The remaining three active pCRMs cannot be easily associated with a specific gene. CE8005 drives expression in the ventral region of the embryo. It is 3' of a gene encoding a ubiquitously expressed Zn-finger containing protein (CG9650) that is maternally expressed and deposited in the embryo. This strong maternal expression potentially obscures a zygotic expression pattern. Two additional adjacent genes, CG32725 and CG1958, showed no expression in whole-mount in situ hybridization of embryos. CE8016 drives a seven-stripe expression pattern in the blastoderm. It is in the first intron of CG14502 which shows very low level expression by microarrays in the blastoderm, and has no obvious detectable pattern of expression in whole-mount in situ hybridization of embryos. This pCRM is approximately 2 kb 5' of scribbler (sbb), which is expressed maternally, possibly obscuring an early zygotic expression pattern (a few in situ images show a hint of striping). sbb is also expressed later in development in the ventral nervous system. An additional potential target, Otefin (Ote), is also expressed maternally and relatively ubiquitously through germ-band extension. All other nearby genes showed no embryonic expression in whole-mount in situ hybridization or by microarray. CE8020 drives an atypical four-stripe pattern in the blastoderm -- two stripes at 7% and 26% that are anterior to the first ftz stripe and two stripes at 39% and 87%. It is in the first intron of ome (CG32145), which is not expressed maternally and has no blastoderm expression, but is expressed late in salivary gland, trachea, hindgut and a subset of the epidermis. All other nearby genes showed no embryonic expression in whole-mount in situ hybridization or by microarray (Berman, 2004).

Transcriptional Regulation

The closely linked POU domain genes pdm-1 and pdm-2 are first expressed early during cellularization in the presumptive abdomen in a broad domain that soon resolves into two stripes. This expression pattern is regulated by the same mechanisms that define gap gene expression domains. The borders of pdm-1 expression are set by the terminal system genes torso and tailless, and the gradient morphogen encoded by hunchback. The resolution into two stripes is controlled by the gap gene knirps. Ectopic expression of pdm-1 at the cellular blastoderm stage leads to disruptions in pair rule gene expression and in anterior segmentation. The broad abdominal domain of pdm-1 protein is lacking in nanos- mutant embryos, and ectopic pdm-1 expression in nanos- embryos leads to a partial restoration of abdominal segmentation (Cockerill, 1993).

Hunchback is required to regulate early pdm-1 expression in the presumptive thoracic domain (Lloyd, 1991). UBX is sufficient to repress pdm-1 expression in abdominal segments. The effect of UBX is not direct, but is through interaction of the endoderm with Ubx-expressing mesoderm (Affolter, 1993).

Given the apparent transient overlap between Hunchback and Pdm-1 expression in the CNS, and Hb's established role as a repressor of pdm expression in the cellular blastoderm, it is likely that Hb also silences pdm expression during early NB sublineage development. Pdm-1 immunolocalizations performed on hb- embryos confirm the hypothesis that Hb functions as a repressor during CNS development. Comparisons between hb- and wild-type Pdm-1 immunostaining patterns shows that in the absence of Hb, Pdm-1 is ectopically expressed in all CNS ganglia. Transverse sections of Pdm-1 immunostained hb- embryos also revealed that early NBs that normally do not maintain Pdm-1 expression after their ectoderm delaminations fail to terminate Pdm-1 expression. The presence of ectopic Pdm-1 is also found in many early GMCs and by stage 12, Pdm-1 positive cells occupy both dorsal and inner regions of the developing ventral cord neuromeres. The expanded dorsal-ventral zone of Pdm-1 expression in hb- ventral cords indicates that many early GMCs and their progeny, normally marked by Hb expression, now ectopically maintain Pdm-1. However, ectopic Pdm-1 expression is not detected throughout the developing ganglia. After stage 11 in hb- embryos there is no Pdm-1 expression in the ventral most regions of the ventral cord ganglia. Similarly, no ectopic Pdm-1 is detected along the outer/superficial surfaces of hb- cephalic lobes after stage 11. This suggests that other mechanism(s) are regulating pdm expression in late developing sublineages (Kambadur, 1998).

To determine if Castor is a pdm repressor, Pdm-1 and Pdm-2 expression was analyzed in cas null embryos. In stage 9 and in younger embryos, no differences were detected between the cas- and wild-type expression patterns of Pdm-1 or -2. However, starting at stage 10, NBs fail to terminate expression of both Pdms. Ectopic Pdm expression is observed in most, if not all, late developing sublineages in all CNS ganglia. The sustained Pdm expression is most likely due to transcriptional derepression, since PDM-1 mRNA in situ hybridizations also reveal that its message persists in cas- late NBs. In vivo analysis of pdm-1 genomic DNA has identified the main cis-regulatory elements controlling its embryonic expression. These control elements lie within a 6.3 kb DNA fragment, flanking the 5' side of its transcribed sequence. The pdm-1 regulatory DNA contains enhancer(s) that can drive the expression of reporter genes in the same cells in which pdm-1 is normally expressed. In a cas- background, transgenes are ectopically expressed in NBs during late sublineage development. This result demonstrates that the enhancer(s) within the 6.3 kb regulatory DNA are negatively regulated by Cas. To explore the possibility that Cas may play a direct role in silencing pdm gene expression, Cas-DNA immunoprecipitation was carried out with pdm-1 promoter fragments to test for potential Cas DNA-binding sites. DNA sequence analysis of bound fragments reveals 32 potential DNA-binding sites, all sharing at least 8 out of the 10 bp with the Hb consensus sites. Cas binds to these sites. Base pair substitutions in one of the sites demonstrate that the A/T rich core sequence is essential for Cas binding. All together, the results suggest that Hb and Cas regulate pdm expression by interacting directly with their cis-regulators to deactivate controlling enhancer(s), with Hb repressing the pdm genes early and Cas silencing late in CNS development. To test if Cas can silence Pdm-1 expression outside of Cas's endogenous sublineage boundaries, the effects of misexpressing Cas were studied early in CNS development. Indicating that Cas can act as a pdm repressor outside of its normal late expression boundaries, the temporally misexpressed Cas significantly reduces Pdm-1 expression when compared to wild-type embryos stained under identical conditions (Kambadur, 1998).

In addition to its requirement for visceral mesoderm development, odd-paired is required for proper gut expression in the endodermal component of the midgut. Repression of the POU-domain gene pdm-1 in a specific region of the endoderm is dependent on opa function. pdm-1 is expressed in much of the endoderm at stage 13, but is repressed in a domain of the endoderm underlying the Ubx and dpp expression domain in the visceral mesoderm. Repression of pdm-1 in the endoderm is dependent of Ubx and dpp, and the accumulation of DPP protein in this region of the endoderm suggests that it may be the secreted factor responsible for regulating pdm-1 across germ layers. In opa mutants, pdm-1 repression does not occur. opa does not appear to contribute to pdm-1 repression through Ubx or dpp, as these genes are expressed in opa mutants, and the activation of the homeotic gene labial in the endoderm (which is dependent on Ubx/dpp signaling) occurs normally. These observations suggest that repression of pdm-1 occurs by a novel signaling pathway, independent of dpp. Since opa is not transcribed in the visceral mesoderm near the pdm-1 expression domain, interruptions in the endoderm may reflect the inability of the endodermal primordia to migrate properly along the defective visceral mesoderm, an interpretation consistent with the proposal that the visceral mesoderm is required as a substrate for endodermal migration (Affolter, 1993 and Cimbora, 1995).

A small number of major regulatory (selector) genes have been identified in animals that control the development of particular organs or complex structures. In Drosophila, the vestigial gene is required for wing formation and is able to induce wing-like outgrowths on other structures. Because ectopic expression of Vg in many imaginal discs induces the outgrowth of wing tissue, the expression of various wing patterning genes was examined to see if they are induced in ectopic growths. Vg is expressed in the entire developing wing pouch whereas Sal and SRF have specific expression patterns within this domain but are not expressed in wild-type leg discs. Targeted expression of Vg with the Gal4-UAS system induces ectopic expression of Sal and SRF in developing leg imaginal discs. Similarly, the nubbin (nub) gene (which is also expressed and required during wing development ) is ectopically induced in leg discs by Vg expression. In each case, only a subset of the cells expressing Vg activate the target gene, which suggests that additional factors control the expression pattern of each gene. In a first step toward elucidating the molecular mechanism by which Vg regulates gene expression, the response of wing-specific enhancers to ectopic Vg expression was examined. Attention was focused on both the boundary and quadrant enhancers of the vg gene and the enhancer from the SRF gene that drives expression specifically in the intervein region between veins three and four. All three enhancers are induced by ectopic Vg expression in leg and other imaginal discs. Importantly, ectopic expression of Vg in clones of cells induces the enhancers only within the clones. However, gene expression is not induced in all cells within clones nor in all clones. In addition, each individual enhancer is expressed in different regions of these discs that appear to correlate with the spatial distribution of the different signaling inputs known to be required for activation of these enhancers (Halder, 1998).

The possession of segmented appendages is a defining characteristic of the arthropods. By analyzing both loss-of-function and ectopic expression experiments, the Notch signaling pathway has been shown to play a fundamental role in the segmentation and growth of the Drosophila leg. Local activation of Notch is necessary and sufficient to promote the formation of joints between segments. This segmentation process requires the participation of the Notch ligands, Serrate and Delta, as well as Fringe. These three proteins are each expressed in the developing leg and antennal imaginal discs in a segmentally repeated pattern that is regulated downstream of the action of Wingless and Decapentaplegic. While Dl expression overlaps fngand Ser, in some cases, it appears to extend into regions of the disc where neither fng nor Ser is expressed (Rauskolb, 1999).

These studies further show that Notch activation is both necessary and sufficient to promote leg growth. Target genes regulated both positively and negatively downstream of Notch signaling have been identified that are required for normal leg development. The nubbin gene (nub) encodes a POU-domain protein that is expressed in a series of concentric rings in late discs. The strongest mutant alleles, which are not null, result in shortened and gnarled legs. Notch mutant clones cause loss of Nub expression. Conversely, ectopic expression of Nub is induced within clones of cells expressing activated Notch. These observations indicate that nub is positively regulated downstream of Notch activation in the leg. Although in most cases the influence of Notch signaling on nub appears to be autonomous, exceptions to this have been observed. These exceptions indicate that regulation of nub expression by Notch signaling may be indirect. In addition, other factors must modulate the ability of Notch to induce nub expression, because tarsal segments 1-4 do not express Nubbin, and ectopic Notch activation in this region fails to induce Nub (Rauskolb, 1999).

The development of the posterior spiracles of Drosophila may serve as a model to link patterning genes and morphogenesis. A genetic cascade of transcription factors downstream of the Hox gene Abdominal-B subdivides the primordia of the posterior spiracles into two cell populations that develop using two different morphogenetic mechanisms. The inner cells that give rise to the spiracular chamber invaginate by elongating into 'bottle-shaped' cells. The surrounding cells give rise to a protruding stigmatophore by changing their relative positions in a process similar to convergent extension. In the larvae the spiracular chamber forms a very refractile filter, the filzkorper. The opening of the spiracular chamber, the stigma, is surrounded by four sensory organs; the spiracular hairs. Clones labeling the spiracular hairs show that each one is formed by four cells related by lineage, two neural and two support cells, the typical structure of a type I external sensory organ. When the larva is buried in the semi-liquid medium on which it feeds, the stigmatophore periscopes out of the medium allowing the larva to continue breathing. The genetic cascades regulating spiracular chamber, stigmatophore, and trachea morphogenesis are different but coordinated to form a functional tracheal system. In the posterior spiracle, this coordination involves the control of the initiation of cell invagination that starts in the cells closer to the trachea primordium and spreads posteriorly. As a result, the opening of the tracheal system shifts back from the spiracular branch of the trachea into the posterior spiracle cells (Hu, 1999).

Downstream of Abd-B the cascade can be subdivided into various levels. The activation of six genes -- cut, empty spiracles (ems), nubbin (nub), klumpfuss (klu), and spalt (sal) -- does not require expression any of the other genes studied, suggesting that these six genes are at the top of the cascade under Abd-B regulation. The cut, ems, nub, and klu genes are expressed in the spiracular chamber in overlapping patterns. The sal gene is not expressed in the spiracular chamber but in the cells that surround it and will form the stigmatophore. The exclusion of sal from the spiracular chamber is partly due to repression by cut, because in cut mutants sal is expressed at low levels in the internal part of the spiracle. Downstream of these putative Abd-B targets other genes are activated. These include the transcription factors grainyhead (grh), trachealess (trh) and engrailed (en) (Hu, 1999).

The spiracle phenotypes in mutants for the early Abd-B downstream genes have been analyzed. In sal mutants the stigmatophore does not form, resulting in embryos with a normal spiraclular chamber that does not protrude. Conversely, mutations in ems and cut affect the spiracular chamber but not the stigmatophore. Mutations for ems result in a spiracular chamber that lacks a filzkorper and is not connected to the trachea. In cut mutants the filzkorper is almost completely missing, but the trachea is still connected to the spiracular chamber and the spiracular hairs are also missing. In trh mutants, where the tracheal pits do not form and there is no tracheal network, the spiracular chamber cells still invaginate, forming a filzkorper. However, this filzkorper is shorter than that of the wild type probably due to a secondary requirement of trh, which is also expressed in the spiracular chamber cells. These results show that the spiracular chamber, the stigmatophore, and the trachea develop independently of one another. No phenotypes for either klu or nub could be detected, indicting that although these genes are expressed in the spiracle, they are either redundant or their function is not required for spiracle morphogenesis (Hu, 1999).

Osa-containing Brahma chromatin remodeling complexes are required for the repression of Wingless target genes

The Wingless signaling pathway directs many developmental processes in Drosophila by regulating the expression of specific downstream target genes. The product of the trithorax group gene osa is required to repress such genes in the absence of the Wingless signal. The Wingless-regulated genes nubbin, Distal-less, and decapentaplegic and a minimal enhancer from the Ultrabithorax gene are misexpressed in osa mutants and repressed by ectopic Osa. Osa-mediated repression occurs downstream of the up-regulation of Armadillo but is sensitive both to the relative levels of activating Armadillo/Pangolin and repressing Groucho/Pangolin complexes that are present, and to the responsiveness of the promoter to Wingless. Osa functions as a component of the Brahma chromatin-remodeling complex; other components of this complex are likewise required to repress Wingless target genes. These results suggest that altering the conformation of chromatin is an important mechanism by which Wingless signaling activates gene expression (Collins, 2000).

Loss of osa can induce phenotypes similar to those caused by ectopic wg expression. Conversely, overexpression of full-length, wild-type Osa (UAS-Osa) results in dominant, gain-of-function phenotypes that often resemble those caused by loss of wg function. However, osa appears to be epistatic to wg, and loss of osa function does not induce ectopic expression of wg. Therefore, the wg gain-of-function phenotypes caused by osa loss of function are likely to result from de-repression of downstream target genes of Wg. To investigate this, the regulation of nubbin (nub) was examined. nub encodes a POU domain protein that is required for the growth and patterning of the wing and is expressed throughout the wing primordium (or wing pouch) in third-instar wing discs. wg signaling is both necessary and sufficient for the expression of nub, since ectopic expression of wg or ectopic activation of the wg pathway can induce ectopic expression of nub, whereas blocking transmission of the wg signal in the wing pouch represses the endogenous expression of nub (Collins, 2000).

nub is ectopically expressed in wing discs that are transheterozygous for null and hypomorphic alleles of osa. An activated form of Armadillo causes similar ectopic nub expression by ectopic activation of the wg pathway. Conversely, the endogenous expression of nub is reduced along the anterior/posterior (A/P) boundary when UAS-Osa is expressed there with a decapentaplegic (dpp)-Gal4 driver. A similar loss of nub expression is caused by the expression of a dominant negative form of Pangolin (Pan) that can no longer bind Arm to activate gene expression (DN-Pan). When Osa and DN-Pan are coexpressed with dpp-Gal4, they acted synergistically to cause a severe reduction in nub expression (Collins, 2000).

In addition to its role in transmitting the wg signal, Arm binds directly to cadherins and is required for the formation of adherens junctions. Over-expression of Drosophila E-Cadherin (DE-Cadherin) can sequester Arm at the plasma membrane and prevent it from participating in Wg signaling; this results in the induction of wg-like phenotypes. When DE-Cadherin (UAS-Cad) is overexpressed in the dorsal compartment of the wing disc with an apterous (ap)-Gal4 driver, dorsal expression of nub is lost and the growth of the wing pouch is reduced. Reduction of osa function in discs expressing UAS-Cad restores more normal nub expression and growth. Furthermore, the ectopic nub expression normally seen in osaeld308/osa4H discs is suppressed by the expression of UAS-Cad in the dorsal compartment. Thus, the level of nub expression is determined by the relative levels of Arm and Osa when either of these levels is reduced. To increase the levels, UAS-Osa is expressed with ap-Gal4, causing a strong reduction of nub expression in the dorsal wing pouch. Expression of DeltaArm with the same Gal4 driver causes nub to be expressed in almost the entire wing disc. The normal domain of nub expression is restored when UAS-Osa and DeltaArm are coexpressed. Taken together, these data demonstrate that Osa is required for the repression of a wg-dependent gene in vivo. Alterations in the dosage of osa can modulate the expression of wg-dependent genes even in the presence of an activated form of Arm or a dominant negative form of Pan, suggesting that Osa does not act upstream of Arm. Alterations in the level of active Pan/Arm complexes can also modulate nub expression in osa mutants; thus, lack of osa does not make Wg target genes entirely independent of Arm (Collins, 2000).

The ARID DNA-binding domain of Osa fused to the repressor domain of Engrailed (UAS-OsaRD) or the activation domain of VP-16 (UAS-OsaAD) can target these domains to genes normally regulated by osa in vivo. The ectopic expression of nub in osaeld308/osa4H wing discs can be prevented by expression of either UAS-Osa or UAS-OsaRD with ap-GAL4. This suggests that Osa functions as a repressor of transcription in the regulation of Wg target genes (Collins, 2000).

Hormone-dependent control of developmental timing through regulation of chromatin accessibility

Specification of tissue identity during development requires precise coordination of gene expression in both space and time. Spatially, master regulatory transcription factors are required to control tissue-specific gene expression programs. However, the mechanisms controlling how tissue-specific gene expression changes over time are less well understood. This study shows that hormone-induced transcription factors control temporal gene expression by regulating the accessibility of DNA regulatory elements. Using the Drosophila wing, it was demonstrated that temporal changes in gene expression are accompanied by genome-wide changes in chromatin accessibility at temporal-specific enhancers. A temporal cascade of transcription factors was uncovered following a pulse of the steroid hormone ecdysone such that different times in wing development can be defined by distinct combinations of hormone-induced transcription factors. Finally, the ecdysone-induced transcription factor E93 was shown to control temporal identity by directly regulating chromatin accessibility across the genome. Notably, it was found that E93 controls enhancer activity through three different modalities, including promoting accessibility of late-acting enhancers and decreasing accessibility of early-acting enhancers. Together, this work supports a model in which an extrinsic signal triggers an intrinsic transcription factor cascade that drives development forward in time through regulation of chromatin accessibility (Uyehara, 2017).

The importance of master transcription factors in specifying spatial identity during development suggests that they may control where other transcription factors bind in the genome. One prediction of this model is that tissues whose identities are determined by different master transcription factors would exhibit different genome-wide DNA-binding profiles. However, it was recently found that the Drosophila appendages (wings, legs, and halteres), which use different transcription factors to determine their identities, share nearly identical open chromatin profiles. Moreover, these shared open chromatin profiles change coordinately over developmental time. There are two possible explanations for these findings. Either (1) different transcription factors produce the same open chromatin profiles in different appendages or (2) transcription factors shared by each appendage control open chromatin profiles instead of the master transcription factors of appendage identity. The second model is favored for several reasons. Since the appendage master transcription factors possess different DNA-binding domains with distinct DNA-binding specificities, it is unlikely for them to bind the same sites in the genome. Supporting this expectation, ChIP for Scalloped and Homothorax, two transcription factors important for appendage identity, shows clear tissue-specific binding in both the wing and eye–antennal imaginal discs. The second model is also preferred because it provides a relatively straightforward mechanism for the observed temporal changes in open chromatin: By changing the expression of the shared temporal transcription factor over time, the open chromatin profiles that it controls would change as well. In contrast, expression of appendage master transcription factors is relatively stable over time, making it unlikely for them to be sufficient for temporal changes in open chromatin (Uyehara, 2017).

It is proposed that control of chromatin accessibility in the appendages is mediated at least in part by transcription factors downstream from ecdysone signaling. According to this model, a systemic pulse of ecdysone initiates a temporal cascade of hormone-induced transcription factor expression in each of the appendages. These are referred to as 'temporal' transcription factors. Temporal transcription factors can directly regulate the accessibility of transcriptional enhancers by opening or closing them, thereby conferring temporal specificity to their activity and driving development forward in time. Master transcription factors then bind accessible enhancers depending on their DNA-binding preferences (or other means of binding DNA) and differentially regulate the activity of these enhancers to control spatial patterns of gene expression, thus shaping the unique identities of individual appendages (Uyehara, 2017).

The experiments with E93 provide direct support for this model. In wild-type wings, thousands of changes in open chromatin occur after the large pulse of ecdysone that triggers the end of larval development. In E93 mutants, ~40% of these open chromatin changes fail to occur. Importantly, nearly three-quarters of sites that depend on E93 for accessibility correspond to temporally dynamic sites in wild-type wings. Thus, chromatin accessibility is not grossly defective across the genome; instead, defects occur specifically in sites that change in accessibility over time. This finding, combined with the large fraction of temporally dynamic sites that depend on E93 for accessibility, indicates that E93 controls a genome-wide shift in the availability of temporal-specific transcriptional enhancers. Supporting this hypothesis, temporal-specific enhancers depend on E93 for both accessibility and activity. Since it is proposed that the response to ecdysone is shared across the appendages, it is predicted that similar defects occur in appendages besides the wing. It remains to be seen whether other ecdysone-induced transcription factors besides E93 control accessibility of enhancers at different developmental times. It also remains to be seen how the temporal transcription factors work with the appendage master transcription factors to control appendage-specific enhancer activity (Uyehara, 2017).

The findings suggest that E93 controls temporal-specific gene expression through three different modalities that potentially rely on three distinct biochemical activities. The enrichment of E93 motifs and binding of E93 to temporally dynamic sites indicate that it contributes to this regulation directly. It is proposed that these combined activities drive development forward in time by turning off early-acting enhancers and simultaneously turning on late-acting enhancers (Uyehara, 2017).

First, as in the case of the tenectin tncblade enhancer, active most strongly in the interveins between the first and second and between the fourth and fifth longitudinal veins and in cells near the proximal posterior margin, E93 appears to function as a conventional activator. In the absence of E93, tncblade fails to express at high levels, but the accessibility of the enhancer does not measurably change. This suggests that binding of E93 to tncblade is required to recruit an essential coactivator. Importantly, this finding demonstrates that E93 is not solely a regulator of chromatin accessibility. E93 binds many open chromatin sites in the genome without regulating their accessibility and thus may regulate the temporal-specific activity of many other enhancers. In addition, since the tncblade enhancer opens between L3 and 24 h even in the absence of E93, there must be other factors that control its accessibility, perhaps, for example, transcription factors induced by ecdysone earlier in the temporal cascade (Uyehara, 2017).

Second, as in the case of the nubvein enhancer, E93 is required to promote chromatin accessibility. In this capacity, E93 may function as a pioneer transcription factor to open previously inaccessible chromatin. Alternatively, E93 may combine with other transcription factors, such as the wing master transcription factors, to compete nucleosomes off DNA. Testing the ability of E93 to bind nucleosomal DNA will help to discriminate between these two alternatives. In either case, it is proposed that this function of E93 is necessary to activate late-acting enhancers across the genome. Since only half of E93-dependent enhancers are directly bound by E93 at 24 h, it is also possible that E93 regulates the expression of other transcription factors that control chromatin accessibility. Alternatively, if E93 uses a “hit and run” mechanism to open these enhancers, the ChIP time point may have been too late to capture E93 binding at these sites (Uyehara, 2017).

Finally, as in the case of the broad brdisc enhancer, E93 is required to decrease chromatin accessibility. It is proposed that this function of E93 is necessary to inactivate early-acting enhancers across the genome. Current models of gene regulation do not adequately explain how sites of open chromatin are rendered inaccessible, but the ability to turn off early-acting enhancers is clearly an important requirement in developmental gene regulation. It may also be an important contributor to diseases such as cancer, which exhibits widespread changes in chromatin accessibility relative to matched normal cells. Thus, this role of E93 may represent a new functional class of transcription factor (“reverse pioneer”) or conventional transcriptional repressor activity. Additional work is required to decipher the underlying mechanisms. Notably, recent work on the temporal dynamics of iPS cell reprogramming suggest a similar role for Oct4, Sox2, and Klf4 in closing open chromatin to inactivate somatic enhancers (Chronis, 2017; Uyehara, 2017 and references therein).

Slit signaling promotes the terminal asymmetric division of neural precursor cells in the Drosophila CNS

The bipotential ganglion mother cells, or GMCs, in the Drosophila CNS asymmetrically divide to generate two distinct post-mitotic neurons. The midline repellent Slit (Sli), via its receptor Roundabout (Robo), promotes the terminal asymmetric division of GMCs. In GMC-1 of the RP2/sib lineage, Slit promotes asymmetric division by down regulating two POU proteins, Nubbin and Mitimere. The down regulation of these proteins allows the asymmetric localization of Inscuteable, leading to the asymmetric division of GMC-1. Consistent with this, over-expression of these POU genes in a late GMC-1 causes mis-localization of Insc and symmetric division of GMC-1 to generate two RP2s. Similarly, increasing the dosage of the two POU genes in sli mutant background enhances the penetrance of the RP2 lineage defects whereas reducing the dosage of the two genes reduces the penetrance of the phenotype. These results tie a cell-non-autonomous signaling pathway to the asymmetric division of precursor cells during neurogenesis (Mehta, 2001).

Since previous results tie the two POU genes, miti and nub, to the normal elaboration of the GMC-1->RP2/sib lineage, the expression of these genes was examined in sli mutant embryos. In wild type, the levels of Nub (or Miti), which are normally high in a newly formed GMC-1, are down regulated prior to the asymmetric division of GMC-1. In sli mutants the expression of Nub (or Miti) in a newly formed GMC-1 is comparable to that of wild type, but, in a late GMC-1 the level remains high compared to wild type. A brief ectopic expression of these POU genes from the hsp70 promoter prior to GMC-1 division induces GMC-1 to divide symmetrically to generate two GMC-1s; each then divides asymmetrically to generate an RP2 and a sib. If the symmetric division of GMC-1 in these mutants has anything to do with the lack of down regulation of Nub and Miti in GMC-1, ectopic expression of miti or nub should also induce GMC-1 to divide symmetrically to generate two RP2 neurons. Indeed, a brief over-expression of miti (or nub) in a late GMC-1 causes this GMC to divide symmetrically into two RP2 neurons in 27% of the hemisegments (Mehta, 2001).

The loss-of-function effects of sli on the distribution of Insc in GMC-1 (and thus the symmetrical division of GMC-1) could be due to this lack of down regulation of Miti and Nub in GMC-1. To test this possibility, the miti transgene was ectopically expressed from the hsp70 promoter. A 25-minute induction of miti was sufficient to alter the localization of Insc and the distribution of Insc in these embryos resembled the distribution of Insc in sli embryos (Mehta, 2001).

The penetrance of the symmetrical division phenotype in sim mutant is sensitive to the dosage of nub and miti genes. The penetrance of the symmetric division of GMC-1 phenotype in sli and sim mutants is ~10%, indicating a partial genetic redundancy for this pathway. Since the loss of asymmetric division of GMC-1 in sli or sim appears to be due to a failure in the down regulation of Nub and Miti, it was reasoned that the penetrance of the phenotype might be enhanced by increasing the copy numbers of these POU genes in sli or sim background. Using a duplication for nub and miti embryos were examined for the GMC-1 division phenotype. The penetrance of the phenotype in these embryos was enhanced to 42%. Similarly, halving the copy numbers of the two POU genes in sim background suppresses the phenotype to 1.4% (Mehta, 2001).

The above results indicate that the symmetrical division of GMC-1 in sli mutants is due to the up regulation of the two POU genes and that these two POU genes are the targets of Sli signaling in GMC-1; however, the partial penetrance of these phenotypes in sli mutants indicate that additional pathways also mediate this very same process and regulate the levels of the two POU proteins in GMC-1. Since the penetrance in insc mutants is also partial, additional pathways must exist to mediate the asymmetric division of GMC-1 to partially complement the loss of the Insc/Sli pathway (Mehta, 2001).

The following picture emerges from this study. The Sli-Robo signaling down regulates the levels of Nub and Miti in late GMC-1, allowing the asymmetric localization of Insc and the asymmetric division of GMC-1. The possibility is entertained that loss of sibling cells in sli mutants would mean that some projections will be duplicated, while others are eliminated. Depending upon the extent, this might have an overall bearing on the pathfinding defects in sli mutants. Since Sli signaling is conserved in vertebrates, it is possible that this signaling may regulate generation of asymmetry during vertebrate neurogenesis as well (Mehta, 2001).

Prospero, targeting pdm1/nubbin, acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells

Stem cells have the remarkable ability to give rise to both self-renewing and differentiating daughter cells. Drosophila neural stem cells segregate cell-fate determinants from the self-renewing cell to the differentiating daughter at each division. This study shows that one such determinant, the homeodomain transcription factor Prospero, regulates the choice between stem cell self-renewal and differentiation. The in vivo targets of Prospero have been identified throughout the entire genome. Prospero represses genes required for self-renewal, such as stem cell fate genes and cell-cycle genes. Surprisingly, Prospero is also required to activate genes for terminal differentiation. In the absence of Prospero, differentiating daughters revert to a stem cell-like fate: they express markers of self-renewal, exhibit increased proliferation, and fail to differentiate. These results define a blueprint for the transition from stem cell self-renewal to terminal differentiation (Choksi, 2006).

To identify sites within the Drosophila genome to which Prospero binds, use was made of an in vivo binding-site profiling technique, DamID. DamID is an established method of determining the binding sites of DNA- or chromatin-associated proteins. Target sites identified by DamID have been shown to match targets identified by chromatin immunoprecipitation (ChIP). DamID enables binding sites to be tagged in vivo and later identified on DNA microarrays. In brief, the DNA or chromatin-binding protein of interest is fused to an Escherichia coli adenine methyltransferase (Dam), and the fusion protein is expressed in vivo. The DNA-binding protein targets the fusion protein to its native binding sites, and the Dam methylates local adenine residues in the sequence GATC. The sequences near the protein-DNA interaction site are thereby marked with a unique methylation tag, over approximately 2-5 kilobase pairs (kb) from the binding site. The tagged sequences can be isolated after digestion with a methylation-sensitive restriction enzyme, such as DpnI (Choksi, 2006).

Dam was fused to the N terminus of Prospero, and transgenic flies were generated. The fusion protein is expressed from the uninduced minimal Hsp70 promoter of the UAS vector, pUAST, as high levels of expression of Dam can result in extensive nonspecific methylation and cell death. As a control for nonspecific Dam activity, animals expressing Dam alone were generated. To assess the sites to which Prospero binds during neurogenesis, genomic DNA was extracted from stage 10-11 embryos, approximately 4-7 hr after egg laying (AEL), expressing either the Dam-Prospero fusion protein or the Dam protein alone. The DNA was digested with DpnI and amplified by PCR. DNA from Dam-Prospero embryos was labeled with Cy3, and control DNA with Cy5. The samples were then cohybridized to genomic microarrays. Microarrays were designed that tile the entire euchromatic Drosophila genome. A 60 base oligonucleotide was printed for approximately every 300 bp of genomic DNA, resulting in roughly 375,000 probes on a single array (Choksi, 2006).

Log-transformed ratios from four biological replicates (two standard dye configurations plus two swapped dye configurations) were normalized and averaged. Regions of the genome with a greater than 1.4-fold log ratio (corresponding to approximately a 2.6-fold enrichment) of Dam-Prospero to the control over a minimum of four adjacent genomic probes were identified as in vivo Prospero binding sites. Using these parameters, a total of 1,602 in vivo Prospero binding sites were identified in the Drosophila genome. This work demonstrates that it is possible to map in vivo binding sites across the whole genome of a multicellular organism (Choksi, 2006).

Prospero is known to regulate the differentiation of photoreceptors in the adult eye, and recently sites have been characterized to which Prospero can bind upstream of two Rhodopsin genes, Rh5 and Rh6. A variant of the Prospero consensus sequence is found four times upstream of Rh5 and four times upstream of Rh6. Prospero was shown to bind this sequence in vitro, by band shift assay, and also by a 1-hybrid interaction assay in yeast. In addition, deletion analysis demonstrated that the consensus sequence is required for the Pros-DNA interaction both in vivo and in vitro. It was found that 67% of in vivo binding sites contain at least one Prospero binding motif. Combining in vivo binding-site data with searches for the Prospero consensus sequence reveals 1,066 distinct sites within the Drosophila genome to which Prospero binds during embryogenesis (Choksi, 2006).

A total of 730 genes have one or more of the 1,066 Prospero binding sites located within 1 kb of their transcription unit. Statistical analyses to determine GO annotation enrichment on the members of the gene list that had some associated annotation (519) was performed by using a web-based set of tools, GOToolbox. Using Biological Process (GO: 0008150) as the broadest classification, a list was generated of overrepresented classes of genes (Choksi, 2006).

The three most significant classes of genes enriched in the list of putative Prospero targets are Cell Fate Commitment, Nervous System Development, and Regulation of Transcription. Utilizing GO annotation, it was found that nearly 41% of all annotated neuroblast fate genes (11 of 27) are located near Prospero binding sites and that approximately 9% of known cell-cycle genes are near Prospero binding sites. These include the neuroblast genes achaete (ac), scute (sc), asense (ase), aPKC, and mira and the cell-cycle regulators stg and CycE. In addition, it was found that the Drosophila homolog of the mammalian B lymphoma Mo-MLV insertion region 1 (Bmi-1) gene, Posterior sex combs, is located near a Prospero binding site. Bmi-1 is a transcription factor that has been shown to regulate the self-renewal of vertebrate hematopoetic stem cells. It is concluded that Prospero is likely to regulate neuroblast identity and self-renewal genes as well as cell-cycle genes directly, repressing their expression in the GMC (Choksi, 2006).

Prospero enters the nucleus of GMCs, and its expression is maintained in glial cells but not in neurons . Therefore the list of targets was searched for genes annotated as glial development genes. Prospero binds near 45% of genes involved in gliogenesis. Among the glial genes, it was found that the master regulator of glial development, glial cells missing (gcm), and gilgamesh (gish), a gene involved in glial cell migration, are both near Prospero binding sites and are likely directly activated by Prospero in glia (Choksi, 2006).

In summary, Prospero binds near, and is likely to regulate directly, genes required for the self-renewing neural stem cell fate such as cell-cycle genes. It was also found that Prospero binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh) and to genes required for glial cell fate. The in vivo binding-site mapping experiments are supportive of a role for Prospero in regulating the fate of Drosophila neural precursors by directly controlling their mitotic potential and capacity to self-renew (Choksi, 2006).

The Drosophila ventral nerve cord develops in layers, in a manner analogous to the mammalian cortex. The deepest (most dorsal) layer of the VNC comprises the mature neurons, while the superficial layer (most ventral) is made up of the mitotically active, self-renewing neuroblasts. Neuroblast cell-fate genes and cell-cycle genes are normally expressed only in the most ventral cells, while Prospero is found in the nucleus of the more dorsally lying GMCs. If in GMCs, Prospero normally acts to repress neuroblast cell-fate genes and cell-cycle genes, then in a prospero mutant, expression of those genes should expand dorsally. Conversely, ectopically expressed Prospero should repress gene expression in the neuroblast layer.

The neuroblast genes mira, ase, and insc and the cell cycle genes CycE and stg show little or no expression in differentiated cells of wild-type stage 14 nerve cords. Expression of these neuroblast-specific genes was examined in the differentiated cells layer of prospero embryos and it was found that they are derepressed throughout the nerve cord of mutant embryos. mira, ase, insc, CycE, and stg are all ectopically expressed deep into the normally differentiated cell layer of the VNC. To check whether Prospero is sufficient to repress these genes, Prospero was expressed with the sca-GAL4 driver, forcing Prospero into the nucleus of neuroblasts. Prospero expression is sufficient to repress mira, ase, insc, CycE, and stg in the undifferentiated cell layer of the VNC. These data, combined with the Prospero binding-site data, demonstrate that Prospero is both necessary and sufficient to directly repress neuroblast genes and cell-cycle genes in differentiated cells. This direct repression of gene expression is one mechanism by which Prospero initiates the differentiation of neural stem cells (Choksi, 2006).

Having shown that Prospero directly represses genes required for neural stem cell fate, it was asked whether Prospero also directly activates GMC-specific genes. Alternatively, Prospero might regulate a second tier of transcription factors, which are themselves responsible for the GMC fate. Of the few previously characterized GMC genes, it was found that Prospero binds to eve and fushi-tarazu (ftz). In the list of targets several more GMC genes were expected to be found, but not genes involved in neuronal differentiation, since Prospero is not expressed in neurons. Surprisingly, however, it was foudn 18.8% of neuronal differentiation genes are located near Prospero binding sites (Choksi, 2006).

To determine Prospero's role in regulating these neuronal differentiation genes, in situ hybridization was carried out on prospero mutant embryos. Prospero was found to be necessary for the expression of a subset of differentiation genes, such as the adhesion molecules FasciclinI (FasI) and FasciclinII (FasII), which have roles in axon guidance and/or fasciculation. Netrin-B, a secreted protein that guides axon outgrowth, and Encore, a negative regulator of mitosis, also both require Prospero for proper expression. Therefore, in addition to directly repressing genes required for neural stem cell self-renewal, Prospero binds and activates genes that direct differentiation. These data suggest that Prospero is a binary switch between the neural stem cell fate and the terminally differentiated neuronal fate (Choksi, 2006).

To test to what extent Prospero regulates the genes to which it binds, genome-wide expression profiling was carried out on wild-type and prospero mutant embryos. While the DamID approach identifies Prospero targets in all tissues of the embryo, in this instance genes regulated by Prospero were assayed in the developing central nervous system. Small groups of neural stem cells and their progeny (on the order of 100 cells) were isolated from the ventral nerve cords of living late stage 12 embryos with a glass capillary. The cells were expelled into lysis buffer, and cDNA libraries generated by reverse transcription and PCR amplification. cDNA libraries prepared from neural cells from six wild-type and six prospero null mutant embryos were hybridized to full genome oligonucleotide microarrays, together with a common reference sample. Wild-type and prospero mutant cells were compared indirectly through the common reference (Choksi, 2006).

In the group of Prospero target genes that contain a Prospero consensus sequence within 1 kb of the transcription unit, 91 show reproducible differences in gene expression in prospero mutants. Seventy-nine percent of these genes (72) exhibit at least a 2-fold change in levels of expression. Many of the known genes involved in neuroblast fate determination and cell-cycle regulation (e.g., asense, deadpan, miranda, inscuteable, CyclinE, and string) show increased levels in a prospero mutant background, consistent with their being repressed by Prospero. Genes to which Prospero binds, but which do not contain an obvious consensus sequence, are also regulated by Prospero: CyclinA and Bazooka show elevated mRNA levels in the absence of Prospero, as does Staufen, which encodes a dsRNA binding protein that binds to both Miranda and to prospero mRNA (Choksi, 2006).

Expression of genes required for neuronal differentiation is decreased in the prospero mutant cells, consistent with Prospero being required for their transcription. These include zfh1 and Lim1, which specify neuronal subtypes, and FasI and FasII, which regulate axon fasciculation and path finding (Choksi, 2006).

The stem cell-like division of neuroblasts generates two daughters: a self-renewing neuroblast and a differentiating GMC. Prospero represses stem cell self-renewal genes and activates differentiation genes in the newly born GMC. In the absence of prospero, therefore, neuroblasts should give rise to two self-renewing neuroblast-like cells (Choksi, 2006).

The division pattern of individual neuroblasts was studied in prospero mutant embryos by labeling with the lipophilic dye, DiI. Individual cells were labeled at stage 6, and the embryos allowed to develop until stage 17. S1 or S2 neuroblasts were examined, as determined by their time of delamination. Wild-type neuroblasts generate between 2 and 32 cells, producing an average of 16.2 cells. Most of the clones exhibit extensive axonal outgrowth. In contrast, prospero mutant neuroblasts generate between 8 and 51 cells, producing an average of 31.8 cells. Moreover, prospero mutant neural clones exhibit few if any projections, and the cells are smaller in size. Thus, prospero mutant neuroblasts produce much larger clones of cells with no axonal projections, suggesting that neural cells in prospero mutants undergo extra divisions and fail to differentiate (Choksi, 2006).

Recently it was shown, in the larval brain, that clones of cells lacking Prospero or Brat undergo extensive cell division to generate undifferentiated tumors. Given that Prospero is nuclear in the GMC but not in neuroblasts, the expanded neuroblast clones in prospero mutant embryos might arise from the overproliferation of GMCs: the GMCs lacking Prospero may divide like neuroblasts in a self-renewing manner. It is also possible, however, that neuroblasts divide more frequently in prospero mutant embryos, giving rise to supernumerary GMCs that each divide only once. To distinguish between these two possibilities, the division pattern of individual GMCs was followed in prospero mutant embryos (Choksi, 2006).

S1 or S2 neuroblasts were labeled with DiI as before. After the first cell division of each neuroblast, the neuroblast was mechanically ablated, leaving its first-born GMC. All further labeled progeny derive, therefore, from the GMC. Embryos were allowed to develop until stage 17, at which time the number of cells generated by a single GMC was determined (Choksi, 2006).

To determine whether mutant GMCs are transformed to a stem cell-like state, stage 14 embryos were stained for the three neuroblast markers: Miranda (Mira), Worniu (Wor), and Deadpan (Dpn). In wild-type embryos at stage 14, the most dorsal layer of cells in the VNC consists mostly of differentiated neurons. As a result, few or none of the cells in this layer express markers of self-renewal. Mira-, Wor-, and Dpn- expressing cells are found on the midline only or in lateral neuroblasts of the differentiated cell layer of wild-type nerve cords. In contrast, a majority of cells in the differentiated cell layer of stage 14 prospero mutant embryos express all three markers: Mira is found cortically localized in most cells of the dorsal layer of prospero nerve cords; Wor is nuclear in most cells of mutant VNCs; Dpn is ectopically expressed throughout the nerve cord of prospero mutants (Choksi, 2006).

Expression of neuroblast markers in the ventral-most layer of the nerve cord (the neuroblast layer), to exclude the possibility that a general disorganization of cells within the VNC contributes to the increased number of Mira-, Wor-, and Dpn-positive cells in the dorsal layer. The number of neuroblasts in a prospero mutant embryo is normal in stage 14 embryos, as assayed by Wor, Dpn, and Mira expression. Thus, the increased expression of neuroblast markers in prospero mutants is the result of an increase in the total number of cells expressing these markers in the differentiated cell layer. It is concluded that prospero mutant neuroblasts divide to give two stem cell-like daughters. GMCs, which would normally terminate cell division and differentiate, are transformed into self-renewing neural stem cells that generate undifferentiated clones or tumors (Choksi, 2006).

Therefore, Prospero directly represses the transcription of many neuroblast genes and binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh), which regulate the timing of cell-fate specification in neuroblast progeny. Prospero maintains hb expression in the GMC, and it has been suggested that this is through regulation of another gene, seven-up (svp). Prospero not only regulates svp expression directly but also maintains hb expression directly. In addition, Prospero maintains Kr expression and is likely to act in a similar fashion on other genes of the temporal cascade. Intriguingly, Prospero regulates several of the genes that direct asymmetric neuroblast division (baz, mira, insc, aPKC). aPKC has recently been shown to be involved in maintaining the self-renewing state of neuroblasts (Choksi, 2006).

Prospero initiates the expression of genes necessary for differentiation. This is particularly surprising since prospero is transcribed only in neuroblasts, not in GMCs or neurons. Prospero mRNA and protein are then segregated to the GMC. Prospero binds near eve and ftz, which have been shown to be downstream of Prospero, as well as to genes required for terminal neuronal differentiation, including the neural-cell-adhesion molecules FasI and FasII. Prospero protein is present in GMCs and not neurons, suggesting that Prospero initiates activation of neuronal genes in the GMC. The GMC may be a transition state between the neural stem cell and the differentiated neuron, providing a window during which Prospero functions to repress stem cell-specific genes and activate genes required for differentiation. There may be few, or no, genes exclusively expressed in GMCs (Choksi, 2006).

Prospero acts in a context-dependent manner, functioning alternately to repress or activate transcription. This implies that there are cofactors and/or chromatin remodeling factors that modulate Prospero's activity. In support of this, although Prospero is necessary and sufficient to repress neuroblast genes, forcing Prospero into the nuclei of neuroblasts is not sufficient to activate all of the differentiation genes to which it binds (Choksi, 2006).

Neuroblasts decrease in size with each division throughout embryogenesis. By the end of embryogenesis, they are similar in size to neurons. A subset of these embryonic neuroblasts becomes quiescent and is reactivated during larval life: they enlarge and resume stem cell divisions to generate the adult nervous system. Neuroblasts in prospero mutant embryos divide to produce two self-renewing daughters but still divide asymmetrically with respect to size, producing a large apical neuroblast and a smaller basal neuroblast-like cell. The daughter may be too small to undergo more than three additional rounds of division during embryogenesis. prospero mutant cells eventually stop dividing, and a small number occasionally differentiate. This suggests that there is an inherent size limitation on cell division. The segregation of Brat, or an additional cell fate determinant, to the daughter cell may also limit the potential of the prospero mutant cells to keep dividing (Choksi, 2006).

The Prox family of atypical homeodomain transcription factors has been implicated in initiating the differentiation of progenitor cells in contexts as varied as the vertebrate retina, forebrain, and lymphatic system. Prospero/Prox generally regulates the transition from a multipotent, mitotically active precursor to a differentiated, postmitotic cell. In most contexts, Prox1 acts in a similar fashion to Drosophila Prospero: to stop division and initiate differentiation (Choksi, 2006).

It is proposed that Prospero/Prox is a master regulator of the differentiation of progenitor cells. Many of the vertebrate homologs of the Drosophila Prospero targets identified in this study may also be targets of Prox1 in other developmental contexts. Prospero directly regulates several genes required for cell-cycle progression, and it is possible that Prox1 will regulate a similar set of cell-cycle genes during, for example, vertebrate retinal development. In addition, numerous Prospero target genes have been identified whose orthologs may be involved in the Prox-dependent differentiation of retina, lens, and forebrain precursors (Choksi, 2006).

Precise registration of gene expression boundaries by a repressive morphogen in Drosophila

Morphogen gradients are thought to create concentration thresholds that differentially position the expression boundaries of multiple target genes. Despite intensive study, it is still unclear how the concentration profiles within gradients are spatially related to the critical patterning thresholds they generate. This study used a combination of quantitative measurements and ectopic-misexpression experiments to examine the transcriptional-repression activities of the Hunchback (Hb) protein gradient in Drosophila embryos. The results define five expression boundaries that are set primarily by differences in Hb concentration and two boundaries that are set by combinatorial mechanisms involving Hb and at least one other repressor. Hb functions as a repressive morphogen, but only within a specific range of concentrations (~40% to ~4.4% of maximum Hb concentration), within which there are at least four distinct concentration thresholds. The lower limit of the range reflects a position where the slope of the gradient becomes too shallow for resolution by specific target genes. Concentrations above the upper limit do not contribute directly to differential-repression mechanisms, but they provide a robust source that permits proper functioning of the gradient in heterozygous embryos that contain only one functional hb gene (Yu, 2008).

This study measured the relative Hb concentrations associated with the positions of seven expression boundaries and tested whether different Hb concentrations can account for the differential positioning of these boundaries along the AP axis of the Drosophila embryo. These experiments lead to the following conclusions. 1. The Hb gradient functions as a bona fide repressive morphogen for five target-gene expression boundaries, eve 3, nub, pdm2, eve 4, and kni, all of which appear to be positioned primarily, if not exclusively, by specific thresholds of Hb concentration. These boundaries move anteriorly in concert with the dynamic changes of the Hb gradient in wild-type embryos, they shift anteriorly in zygotic hb mutants, and their sensitivities to repression by ectopically expressed Hb are consistent with their relative positions in wild-type embryos. Two other boundaries, the anterior boundary of Kr and the anterior boundary of the posterior gt domain, are established by combinatorial mechanisms involving Hb and Gt, and Hb and Kr, respectively. 2. There is a specific concentration range (~40% to ~4.4% [Hb]max) that mediates the major morphogenetic activities of the Hb repression gradient. Within this range, four thresholds were detected, one at ~40% [Hb]max that sets the anterior boundary of eve 3, one at ~12% [Hb]max that sets the anterior boundaries of both nub and pdm2, one at ~8% [Hb]max that sets the anterior boundary of eve 4, and one at ~4.4% [Hb]max that sets the anterior boundary of kni. These results suggest that these five target genes are exquisitely sensitive to small changes in Hb concentration. Hb also acts as a direct repressor to position the anterior boundary of the Hox gene Ultrabithorax (Ubx), which is first activated in late cycle 14 just before the initiation of gastrulation. The anterior Ubx boundary is positioned between the eve 3 and eve 4 boundaries and thus may represent a fifth threshold within the morphogenetic range described in this study. Ventral misexpression of Hb causes a strong repression of Ubx. However, it was not possible to directly compare the sensitivity of Ubx with the other target genes because the patterns of these genes have begun to decay when Ubx is first activated (Yu, 2008).

Previous studies have identified discrete regulatory elements that recapitulate the five expression patterns within the morphogenetic range described in this study. All of these elements contain multiple Hb binding sites, and one attractive model is that differences in sensitivity are determined by the quantity and/or quality of their Hb binding sites. The more sensitive eve 4+6 enhancer seems to contain a stronger cluster of Hb binding sites than the less sensitive eve 3+7 enhancer, which is consistent with this hypothesis. However, in preliminary experiments, it was found that this simple model cannot be applied to the five target genes shown in this study to be differentially sensitive. For example, the kni expression pattern is more sensitive to Hb-mediated repression than either eve 3 or eve 4, but its enhancer sequence does not appear to have a stronger cluster of Hb binding sites than either the eve 3+7 or the eve 4+6 enhancer. Similarly, two enhancer elements have been found to be associated with the pdm locus, which contains both nub and pdm2. When tested in reporter genes, both enhancers drive patterns of gene expression similar to the endogenous nub and pdm2 patterns, but they do not appear to contain similar clusters of Hb sites (Yu, 2008).

If differential sensitivity cannot be linked to differences in the number or affinity of Hb binding sites for this set of regulatory elements, other architectural features may control the level of Hb required for repression. These features may include changes in spacing between adjacent Hb sites, or specific site orientations that affect cooperative binding. Also, specific arrangements between repressor and activator sites may influence the apparent sensitivities. Consistent with this, it has been shown that specific arrangements between Dl and Twi sites are critical for Dorsal-dependent target-gene expression in the prospective neuroectodermal region along the DV axis. A careful analysis of the enhancer elements that respond to Hb-mediated repression will be required to fully understand the molecular rules that govern differential sensitivity (Yu, 2008).

At the low end of the effective morphogenetic range, there is a ~2-fold difference between the Hb concentration at the eve 4 boundary (~8% [Hb]max) and the amount at the kni boundary (~4.4% [Hb]max). Moving farther posteriorly, from the kni boundary to the gt boundary, does not correlate with a significant drop in the relative Hb concentration (~4.4% [Hb]max to ~3.7% [Hb]max). It is proposed that the slope of the gradient in this region is too shallow for differential target-gene positioning. However, by participating in a combinatorial mechanism with Kr, the very low concentrations of Hb in this region can set the gt boundary in a more posterior position than the kni boundary. Hb and Kr both bind to the regulatory element that drives posterior expression of gt, suggesting that these interactions may be direct (Yu, 2008).

Within the morphogenetic range, the anterior-most boundary is that of eve 3, which corresponds to ~40% [Hb]max. Outside this range on the anterior side is the Kr boundary, which was previously shown to expand anteriorly in zygotic hb mutants. In these experiments, Kr appeared to be quite resistant to repression by ectopic Hb, which seemingly contradicts a previous study that showed that high levels of Hb were sufficient for repression. However, in the previous study, ectopic Hb was provided maternally, significantly before the sna-hb transgene used in this study would be activated. Together, the two studies support the idea that the Kr boundary is initially set independently by Hb, and that maintenance of the boundary requires both Hb and Gt activities. The results suggest that maintenance is mediated primarily by Gt, but that Gt is an effective repressor only in the presence of Hb. The potentiating effect of Hb on Gt-mediated repression may involve direct binding of Hb and Gt to the Kr promoter, which contains binding sites for both proteins (Yu, 2008).

One of the most important findings from this study is that the effective range of Hb's morphogenetic activity is between 40% [Hb]max and 4.4% [Hb]max. This range may seem surprising in light of the fact that Hb is expressed at much higher levels throughout the anterior half of the embryo. Previous studies suggest that anteriorly expressed Hb is required for activation of most Bcd-dependent target genes, which are expressed in a variety of anterior patterns, and that the zygotic stripe of Hb expressed at the position of PS4 is required for the activation of the Hox gene Antennapedia. It is proposed that the high level of Hb protein in anterior regions also provides a reservoir, or buffer, that ensures that the repressive gradient, with all of its thresholds, remains intact in individual embryos that vary in their absolute levels of Hb expression. Such a buffering mechanism could explain how heterozygous embryos, which contain roughly half the concentration of Hb, can nonetheless develop normally (Yu, 2008).

It is proposed further that most other morphogens will function via concentration ranges similar to the one measured in this study. The two best-studied morphogens in Drosophila are Bcd and Dorsal (Dl), both of which are viable and fertile in the heterozygous state. In embryos laid by bcd/+ females, there are dramatic shifts in the positioning of target genes in the early embryo, but the order of gene positioning remains unchanged, the embryos survive to adulthood, and the adults are fertile. Survival would not be possible if activation of a critical target gene required a threshold greater than 50% of the maximum concentration of Bcd (Yu, 2008).

Escargot maintains stemness and suppresses differentiation in Drosophila intestinal stem cells

Snail family transcription factors are expressed in various stem cell types, but their function in maintaining stem cell identity is unclear. In the adult Drosophila midgut, the Snail homolog Esg is expressed in intestinal stem cells (ISCs) and their transient undifferentiated daughters, termed enteroblasts (EB). Loss of esg in these progenitor cells causes their rapid differentiation into enterocytes (EC) or entero-endocrine cells (EE). Conversely, forced expression of Esg in intestinal progenitor cells blocks differentiation, locking ISCs in a stem cell state. Cell type-specific transcriptome analysis combined with Dam-ID binding studies identified Esg as a major repressor of differentiation genes in stem and progenitor cells. One critical target of Esg was found to be the POU-domain transcription factor, Pdm1, which is normally expressed specifically in differentiated ECs. Ectopic expression of Pdm1 in progenitor cells was sufficient to drive their differentiation into ECs. Hence, Esg is a critical stem cell determinant that maintains stemness by repressing differentiation-promoting factors, such as Pdm1 (Korzelis, 2014).

Stem cell identity is controlled by both extrinsic cues from the niche and cell-intrinsic transcriptional programs. Thus far, most studies of the Drosophila midgut have focused on the niche-derived signals that control midgut stem cell self-renewal. This study demonstrates a cell-intrinsic role for the Snail family transcription factor, Escargot, in controlling ISC self-renewal and differentiation. Loss of Esg leads to a rapid loss of all stem/progenitor cells in the midgut, due to their differentiation, whereas Esg overexpression keeps these cells permanently in an undifferentiated state. The dramatic effects of manipulating Esg levels support a central role for this Snail family member in controlling stem cell identity in the fly intestine (Korzelis, 2014).

A transcriptomics analysis indicated that Esg acts as a transcriptional repressor of a large diverse set of differentiation genes. These targets include transcription factors specific to ECs and EEs (Pdm1, Prospero) and genes used in digestion, immunity and cytoarchitectural specialization. Interestingly, one of these transcription factors, Pdm1, plays an important role in EC differentiation: ectopic expression of Pdm1 in progenitor cells was sufficient to trigger EC differentiation, partially mimicking the esg loss of function phenotype. The rapid loss of the Esg-expressing cell population upon Pdm1 overexpression suggests that Pdm1 might repress Esg expression, perhaps directly. In this case, Esg and Pdm1 together would constitute a negative feedback switch that governs EC differentiation (Korzelis, 2014).

Expression analysis also raised the possibility that Esg activates progenitor cell-specific genes in ISCs and EBs. These include the EGF signaling components Cbl, spitz, argos and Egfr as well as the Jak/Stat receptor domeless. Both EGFR and Jak/STAT pathways are crucial for ISC growth and maintenance, and receptivity to these signals is downregulated in differentiated ECs and EEs. While Snail family members are best understood as repressors, the Esg paralog Snail has been reported to function as a context-dependent transcriptional activator (Rembold, 2014), suggesting that an activating role for Esg is also plausible. The function of Esg as either an activator or repressor is likely determined by co-factors and/or other transcription factors acting on the same promoters that are expressed in the ISC and EB population. In the Drosophila embryo, Snail cooperates with Twist at distinct promoters to activate EMT gene expression during mesoderm formation (Rembold, 2014). Snail2 can bind to Sox9 to activate expression from its own promoter during chick neural crest formation. In its role as a repressor, Esg binds the co-repressor CtBP to maintain somatic Cyst stem cells and hub cells in the Drosophila male testis. Future work to unravel the complete transcriptional network within which Esg functions to maintain the stem/progenitor state should prove to be very interesting (Korzelis, 2014).

The data support a model in which Esg acts in a circuit with Delta-Notch signaling to control the switch from stem/progenitor identity to differentiated cell identities. In its simplest form, this circuit might be a bistable switch in which Esg and Notch mutually inhibited each other, with Esg being 'on' and dominant in progenitor cells and Notch signaling 'on' and dominant in their differentiated progeny, the enterocytes. However, the constant presence of a substantial population of intermediate progenitor cells, the enteroblasts (EBs), which express both Esg and Notch reporter genes, indicates that a simple bistable switch is not an accurate conception. Indeed, EBs, defined here as cells positive for both Esg and the Notch reporter Su(H)GBE-LacZ, can persist for many days in the absence of ISC division. Thus, the EB transition state is metastable. In this transition state, Notch is apparently active, but secondary downstream targets that directly affect differentiation, such as Pdm1, brush border Myosin and smooth septate junction proteins, remain repressed. Since these genes are rapidly activated following depletion of Esg, it is suggested that their repression is most likely mediated by Esg binding (Korzelis, 2014).

Two potential explanations are provided for the longevity of the EB transition state. First, it is suggested that the repression of esg transcription by Notch is indirect and that this delays esg silencing. Silencing of Esg is not likely to be mediated by the Notch-regulated transcription factor Su(H) (a transcriptional activator) but by downstream repressors that act only after enterocyte or endocrine differentiation has begun. Pdm1 in ECs and Prospero in EEs are presently the most obvious candidates. Both are specifically induced coincident with Esg silencing, in ECs and EEs, respectively, and Dam-ID assays suggest that Pros has binding sites in the esg locus. The finding that overexpression of Pdm1 caused the rapid differentiation of Esg+ stem/progenitor cells supports the notion that Pdm1 could directly repress Esg expression to control EC differentiation. Furthermore, nubbin/Pdm1 was found to restrict expression of Notch target genes in the Drosophila larval wing disc. Hence, Pdm1 likely triggers EC differentiation by downregulating both Esg and the expression of Notch target genes in the EB. Therefore, Notch is only transiently active in EBs but fully off in mature ECs with high levels of Pdm1 (Korzelis, 2014).

While a delay circuit that controls the silencing of Esg is likely, theoretically it cannot explain how Esg+ EBs can persist for such long periods during times of low gut epithelial turnover and then rapidly differentiate during gut regeneration. Hence, it is speculated that a second input signal acts in combination with Notch-dependent factor(s) to silence Esg. This second signal is likely to be a downstream effector of the growth factor signaling network that also drives ISC division and gut epithelial renewal. Of the transcriptional effectors involved in maintaining gut homeostasis, the most obvious candidate as an indirect mediator of esg repression is Stat92E, which is activated by the highly stress-dependent cytokines, Upd2 and Upd3. Tellingly, the cytokine receptor, Dome, Janus Kinase (hop) and Stat92E are all required for EB maturation into ECs. If the silencing of esg was dependent upon both Notch and Stat92E, and Delta-Notch signaling was irreversible once resolved; then, the Notch+ Esg+ EB transition state should in principle be stable in conditions of low Jak/Stat signaling, as is observed during periods of midgut quiescence. It needs to be noted, however, that ISCs and EBs maintain appreciable levels of Stat-reporter gene expression even during relative quiescence, and so, in this model, it would be Stat activity above some threshold that would combine with Notch signaling to trigger differentiation. Since Jak/Stat signaling also triggers ISC division, a surge in cytokine signaling could coordinately trigger both the differentiation of older EBs and the production of new ones in this model , thus explaining how a significant EB population is maintained even as stem cell activity waxes and wanes (Korzelis, 2014).

Snail family transcription factors have been described as regulators of epithelial-to-mesenchyme transitions (EMT) that occur during development, wound healing and cancer metastasis. In some contexts, notably metastasis, EMT is believed to accompany the acquisition of stem-like properties. Although Esg itself has not been reported to regulate EMT, its paralog in flies (Sna) and homologs in mammals (Snai1, Snai2) do promote EMT. Interestingly, RNA-seq experiments showed that not only Esg, but Snail, Worniu and the Zeb family members Zfh1 and Zfh2 were all expressed in intestinal stem cells and downregulated in ECs and EEs. Thus, these EMT-linked transcription factors may work together to affect different aspects of midgut homeostasis and ISC differentiation. Indeed, Esg-positive ISCs and EBs are morphologically more similar to mesenchymal cells than they are epithelial, whereas Esg-negative EEs and ECs have the pronounced apical-basal polarity typical of epithelial cells. Esg+ cells often make striking lateral projections, suggestive of dynamic behavior, and they have the capacity to multilayer when their differentiation is blocked or they are forced to overproliferate. Furthermore, a number of epithelial-class genes are repressed in Esg+ progenitors and activated upon EC and/or EE differentiation. These include genes encoding the apico-lateral cortical Lgl-Dlg-Scrib-Crb complex, septate junction proteins (e.g., Ssk, Cora, Mesh) and polarity factors including Par3 and Par6. Strikingly, Scrib and Ssk both have Esg-binding sites in their promoters, and their expression is highly regulated by Esg. However, some gene targets that are central to EMT in mammalian cells show opposite trends in the fly's ISC lineage. For instance, Esg+ progenitors express significant levels of integrins, and E-cadherin-typically lost during EMT-is highly upregulated specifically in ISCs and EBs. Thus, the Esg-regulated differentiation of Drosophila ISCs only partially resembles a mesenchymal-to-epithelial transition (MET) (Korzelis, 2014).

Esg's role in ISC maintenance nicely parallels the functions of other Snail family members in Drosophila and mammals. For instance, in Drosophila neuroblasts (neural stem cells), the Snail family member Worniu promotes self-renewal and represses neuronal differentiation. In mice, Snail family members have been associated with the regulation of the stem cell state in both normal and pathological conditions. For instance, mammary stem cells require the Snail family member Slug to retain their MaSC identity. Mouse Snai1 also represses the transition from the stem cell-like mitotically cycling trophoblast precursor cell to the endoreplicating trophoblast giant cell during rodent placental development. This process, which also requires a mitotic-to-endocycle switch upon differentiation, is strikingly similar to the role describe in this study for Esg in EC differentiation and its role during imaginal disc development (Korzelis, 2014).

More interesting yet, mouse Snai1 is specifically expressed and required for stem cell maintenance in the crypts of the mouse intestine and expands the stem cell population when overexpressed. However, few studies highlight the target genes responsible for the function of Snail family members in stem cell maintenance. One example is from mouse muscle progenitors (myoblasts), where Snai1 and Snai2 repress expression from MyoD target promoters and this is required to maintain their progenitor state. The work presented in this study shows that Esg affects many aspects of the differentiation process and that it can form a transcriptional switch with one of the targets it represses (Pdm1) to balance self-renewal and differentiation in this stem cell lineage. Together, these studies suggest that the function of Snail family transcription factors as repressors of differentiation genes is ancient and widespread and may be an essential component in balancing self-renewal with differentiation in diverse animal stem cell lineages (Korzelis, 2014).

Targets of Activity

PDM-1 binds to an octamer-like motif, ATTCAAAT, in the choline acetyltransferase gene of Drosophila, and presumably regulates the expression of this gene (Kitamoto, 1995). pdm-1 and pdm-2 mutations interfer with even-skipped expression in GMC4-2 and its progeny, the RP2 motor neuron (Yeo, 1995).

An investigation was carried out of the gene regulatory functions of Drosophila Sox box protein 70D (also known as Dichaete or Fish-hook), a high mobility group (HMG) Sox protein that is essential for embryonic segmentation. The Dichaete HMG domain binds to the vertebrate Sox protein consensus DNA binding sites, AACAAT and AACAAAG, and this binding induces an 85 degrees DNA bend. A heterologous yeast system has been used to show that the NH2-terminal portion of Dichaete protein can function as a transcriptional activator. The HMG and C-terminal regions may partially mask the transcriptional activation function of the N-terminal region. Dichaete directly regulates the expression of the pair rule gene even-skipped (eve) by binding to multiple sites located in downstream regulatory regions that direct formation of eve stripes 1, 4, 5, and 6. Dichaete may function along with the Drosophila POU domain proteins Pdm-1 and Pdm-2 to regulate eve transcription, since genetic interactions are detected between Dichaete and pdm mutants. In the blastoderm embryo, pdm-1 and pdm-2 are both expressed in wide posterior bands of cells that are completely contained within the Dichaete expression domain. In double Dichaete/pdm mutants there is a complete loss of eve stripe 5, and fusions between stripes 3 and 4 as well as stripes 6 and 7. This pattern of defects is never observed in mutants for only one or the other of the two genes. The downstream region contains a perfect octamer POU domain consensus binding site. Dichaete protein is expressed in a dynamic pattern throughout embryogenesis, and is present in nuclear and cytoplasmic compartments. The protein is first detected in embryos during nuclear cycle 12. At this time Dichaete is present in a wide stripe that encompasses most of the trunk domain, extending from eve stripes 2-7. It is suggested that the DNA-bending properties of Dichaete could enhance or stabilize interactions between regulatory complexes present at distant downstream eve regulatory regions and upstream regulatory complexes including those at the eve promoter. Sox proteins are known to interact with POU domain proteins in vertebrates (Ma, 1998).

The product of the Drosophila gene Serrate acts as a short-range signal during wing development to induce the organizing center at the dorsal/ventral compartment boundary, from which growth and patterning of the wing is controlled. Regulatory elements reflecting the early Serrate expression in the dorsal compartment of the wing disc have recently been confined to a genomic fragment in the 5'-upstream region of the gene (from -8 to -18 kb). This fragment, termed the dorsal wing regulator or DWR, responds to various positive and negative inputs required for the early Serrate expression. Activation and maintenance of expression in the dorsal compartment of the wing discs of second and early third instar larvae depend on apterous, as revealed by reporter gene expression in discs either lacking or ectopically expressing apterous. Transcriptional downregulation during third larval instar is mediated by hiiragi. hiiragi, which has not yet been cloned, develops a notched wing phenotype when homozygous and enhances the notched wing phenotype of SerD/+. Strikingly, in hirP1 homozygous third instar larvae the expression domain of the DWR not only persists on the dorsal wing pouch, but expands into the ventral compartment from mid-third instar onwards. hiiragi is a good candidate to be involved in the downregulation of the DWR of Ser. The lack of nubbin leads to the loss of wing structures. In discs mutant for nub expression, the DWR along the D/V boundary is upregulated and persists longer than in wild-type discs. This is in agreement with the observation that Serrate protein expression appears to be more pronounced along the dorsal wing margin in nubbin mutant discs. This regulatory element also responds to Delta signaling in a nonautonomous way to maintain Serrate expression along the dorsal margin. The results clearly show that some of the previously described transactivators of Serrate protein expression, e.g. fringe, act on elements required for later aspects of Serrate expression (Bachmann, 1998b).

In the wing wingless is expressed in a complex and dynamic pattern that is controlled by several different mechanisms. These involve the Hedgehog and Notch pathways and the nuclear proteins Pannier and U-shaped. The mechanisms that drive wingless expression in the wing hinge have been analyzed. Evidence is presented that wingless is initially activated by a secreted signal that requires the genes vestigial, rotund and nubbin. Later in development, wingless expression in the wing hinge is maintained by a different mechanism, which involves an autoregulatory loop and requires the genes homothorax and rotund. The role of wingless in patterning the wing hinge is discussed (Rodriguez, 2002).

The effects of removing wg expression in the inner ring (IR) can be observed in spade (spd) mutants. spd mutations are a type of wg allele that specifically removes wg expression from the IR, with no effects on other expression domains. In spdfg wings, the hinge region is deleted, and the wing pouch appears directly joined to more proximal cells. In these wings, both wg-expressing cells and surrounding cells are missing. It has been shown that this phenotype is not caused by cell death but is a consequence of underproliferation in this region, suggesting that one of the functions of Wg in the IR is to promote local cell proliferation (Rodriguez, 2002).

The rotund (rn) gene is a member of the Krüppel family of zinc-finger encoding genes. Among other phenotypes, rn mutations delete the wing hinge and remove wg expression from the IR. nubbin encodes a member of the POU family of transcription factors. In strong nub mutations wings are vestigial, but phenotypic analysis of weaker alleles shows that the wing hinge is deleted and the expression of wg in the IR is missing. The hinge phenotype of the triple mutant spdfg nub2; rnDelta2-2 was examined, and it is similar to the phenotype of each of them, suggesting that the main cause of the phenotype is the lack of wg expression in the IR (Rodriguez, 2002).

vestigial encodes a nuclear protein with no homology with other identified families of nuclear proteins. Based on its interaction with scalloped (sd) it has been suggested that the function of Vg is to mediate transcriptional activation by Sd. vg expression in the wing is regulated by two separate enhancers: the boundary enhancer (BE) and the quadrant enhancer (QE). The BE is activated by the Notch signaling pathway and drives vg expression at the dorsal/ventral boundary in middle/late second instar larval stage. The QE is activated by the combined action of Wg and Dpp, and drives vg expression in the rest of the wing pouch from early third instar larval stage (Rodriguez, 2002).

The expression patterns of vg, rn and nub were examined. In mature wing discs vg, rn and nub are expressed in three concentric domains, the Vg domain being the smallest one. At this stage the wing hinge is lined with several anterior/posterior folds. The boundary of vg expression coincides with the distal-most fold of the disc. The Rn domain is slightly broader and its boundary coincides with a second fold in the disc. The Nub domain contains the Rn domain and coincides with the third fold in the disc. The IR domain corresponds to the proximal-most area of the Rn domain (Rodriguez, 2002).

The expression of these genes was examined in early larval development. In middle/late second instar larvae the expression domains of vg, rn and nub in the presumptive wing pouch are slightly broader than the vg domain. The rest of the cells of the disc, those that do not express nub, express the gene teashirt (tsh). wg is expressed only in a stripe of cells that corresponds to the presumptive wing margin. In early third instar larvae, wg starts to be expressed in the IR. This expression domain corresponds to cells that express rn and nub but do not express vg. wg expression in the IR promotes the growth of the hinge and, in third instar larvae, gives rise to the expression patterns described above for vg, rn and nub. At this stage, the cells that express the wg IR enhancer are located at the limit of the domain 3 (Rn + Nub), and are several cells away from the boundary of vg expression (Rodriguez, 2002).

The results indicate that Vg is required to activate the expression of rn and nub genes in the wing disc. This activation is restricted to the cells that will take wing fate and takes place in the cell that express vg, and also in the surrounding cells, suggesting that a Vg-dependent short-range signal activates rn and nub expression. At this time, the expression of nub and tsh in the wing disc are complementary and cover the whole disc (Rodriguez, 2002).

The expression of these genes in a domain broader than the Vg domain creates a ring of cells that express rn and nub but not vg. Evidence is presented indicating that a signal from vg-expressing cells activates the wg IR enhancer in adjacent rn/nub-expressing cells. Unlike the activation of rn and nub, the activation of wg expression by the IR enhancer is repressed in cells that also express vg. So, the IR enhancer is activated only in cells that surround the Vg domain. During the development of the disc, the position of the IR moves several cells away from the Vg domain. This implies either that the Vg-dependent signaling is able to activate the IR over a long range, or that a different, Vg-independent, mechanism maintains the IR (Rodriguez, 2002).

When artificial Vg/Rn-Nub interfaces are generated experimentally, the IR enhancer is activated in rn-nub-expressing cells that abut the Vg domain. This ectopic IR is around four cells wide, indicating the active range of the signal that activates wg expression. The results indicate that at distances greater than this, a Vg-independent mechanism maintains wg expression in the IR (Rodriguez, 2002).

The Drosophila wing imaginal disc gives rise to three main regions along the proximodistal axis of the dorsal mesothoracic segment: the notum, proximal wing, and wing blade. Development of the wing blade requires the Notch and wingless signalling pathways to activate vestigial at the dorsoventral boundary. However, in the proximal wing, Wingless activates a different subset of genes, e.g., homothorax. This raises the question of how the downstream response to Wingless signalling differentiates between proximal and distal fate specification. A temporally dynamic response to Wingless signalling is shown to sequentially elaborate the proximodistal axis. In the second instar, Wingless activates genes involved in proximal wing development; later in the third instar, Wingless acts to direct the differentiation of the distal wing blade. The expression of a novel marker for proximal wing fate,Zn finger homeodomain 2 (zfh-2), is initially activated by Wingless throughout the 'wing primordium,' but later is repressed by the activity of Vestigial and Nubbin, which together define a more distal domain. Thus, activation of a distal developmental program is antagonistic to previously established proximal fate. In addition, Wingless is required early to establish proximal fate, but later when Wingless activates distal differentiation, development of proximal fate becomes independent of Wingless signalling. Since P-element insertions in the zfh-2 gene result in a revertable proximal wing deletion phenotype, it appears that zfh-2 activity is required for correct proximal wing development. These data are consistent with a model in which Wingless first establishes a proximal appendage fate over notum, then the downstream response changes to direct the differentiation of a more distal fate over proximal. Thus, the proximodistal domains are patterned in sequence and show a distal dominance (Whitworth, 2003).

The Drosophila wing imaginal disc gives rise to the structures of the dorsal mesothoracic segment. This is subdivided into three main regions: the notum, the wing blade, and the proximal wing and hinge. The wing is attached to the thorax via a complex joint comprising a small portion of the appendage, the hinge, which consists of several interlocking sclerites and plates. The wing blade tapers toward the body, forming a short, narrow region that is attached at the hinge. This region shall be referred to as the proximal wing since it is morphologically and mechanically distinct from the hinge itself. Fate mapping of the late third instar imaginal disc has determined that the central portion, the wing pouch, develops as wing blade, a ring surrounding the wing pouch develops as proximal wing and hinge, and the large dorsal territory and a narrow ventral domain form the notum and ventral pleura (Whitworth, 2003).

Previous studies have attempted to follow the development of the proximal part of the wing by analysis of genes that have some expression in the proximal region of the wing disc, e.g., wg or nub, or by the exclusion of markers for notum and wing fates, e.g., teashirt (tsh) and vg, respectively. The identification and analysis is described of a novel marker for proximal wing fate that specifically demarcates the whole of the developing proximal wing tissue, the zinc-finger homeodomain gene zfh-2. In third larval instar (L3) wing discs, Wg is expressed in a stripe along the D/V boundary, forming the wing margin, and in two concentric rings around the wing pouch. In the adult wing, expression of a wg-lacZ reporter indicates that the two rings of wg delimit the proximal wing. The inner (distal) ring runs from the medial costa, through the humeral crossvein to the alula, and the outer (proximal) ring runs from the proximal end of the proximal costa to the axillary cord. A GAL4 insertion within the zfh-2 transcription unit, MS209, (zfh-2MS209) and antisera against Zfh-2 have been used to monitor the expression of zfh-2. In both L3 wing discs and adult wings, Zfh-2 is expressed in a domain that completely overlaps the rings of Wg expression. In L3 wing discs, Zfh-2 does not extend either proximally into the notum or distally into the wing pouch. These observations indicate that, in late stages, Zfh-2 is specifically expressed throughout the developing proximal wing and therefore may be used as a useful marker for proximal wing fate (Whitworth, 2003).

Ectopic expression of Wg can induce zfh-2 only in regions outside of the wing pouch. This suggests that some factor has a repressive effect on zfh-2 in the pouch that cannot be overcome by Wg activation. Genes fundamental to wing blade development may be responsible for this repression. Since Vg expression is restricted to the presumptive wing blade and is required for wing blade development, the effects of ectopic expression of vg on the proximal wing region were examined. Using dpp-GAL4 to direct expression of vg along the A/P boundary represses zfh-2 in the proximal wing region. Endogenous wg expression, monitored with the wg-lacZ reporter, also shows complete repression at the point of intersection. Conversely, in vg1 mutant discs, the Zfh-2 expression domain is expanded into the remnant of the wing pouch and shows a greater overlap with Nub expression than in the wild type. In vg1 discs, much of the wing pouch anlagen fails to develop, and this is accompanied by complete loss of Wg expression at the wing margin; however, the two rings of Wg delimiting the proximal wing are maintained. This suggests that derepression of the zfh-2 domain into the pouch region is not caused by ectopic Wg activity (Whitworth, 2003).

Since the loss of vg does not result in complete derepression of zfh-2, it suggests that another repressor must be acting with vg. Nub is also required for wing blade development. Hypomorphic nub alleles display a severely reduced wing phenotype and a transformation of distal structures into proximal ones. nub2 discs show a complete loss of the inner ring of Wg and an expansion of Wg expression at the wing margin. In nub2 mutant discs, Zfh-2 expression is expanded into the wing pouch, along the line of the wing margin. This indicates two things: (1) that Nub normally acts to repress zfh-2 expression, and thus proximal wing fate, within the wing pouch, and (2) that ectopic zfh-2 is induced where Wg is expressed. Therefore, in an environment of reduced Nub, it can be predicted that ectopic Wg would be able to induce ectopic Zfh-2. To test this, ectopic Wg was expressed in a nub mutant background. As in the nub2 background, Zfh-2 is ectopically induced in the wing pouch along the wing margin. In addition, Zfh-2 can now be detected in the wing pouch along the line of dpp-GAL4, where high levels of Wg are ectopically expressed. This demonstrates that, in an environment of reduced Nub, Zfh-2 expression can be induced wherever Wg is expressed and is no longer restricted from the pouch. It is noted that, whereas Wg expression is expanded at the wing margin in nub discs, where ectopic Wg is induced in a nub background, endogenous Wg is expressed normally at the wing margin; however, the reason for this is unknown (Whitworth, 2003).

In nub discs, vg expression is unaffected, but vg is upregulated by high levels of ectopic Wg. Thus, it appears that the increased levels of Vg are not sufficient to repress Zfh-2 in the absence of Nub when Wg is present at high levels. However, further from the source of ectopic Wg, Zfh-2 is not induced in the nub background, and presumably here, Vg alone can repress Zfh-2. Taken together, these data suggest that zfh-2 expression is regulated by a balance between activation by Wg and repression by a combination of Nub and Vg, acting together or independently. The loss of either Nub or Vg is enough to cause only a partial derepression of zfh-2 in the wing pouch, indicating that alone neither Nub nor Vg is sufficient to completely repress proximal wing fate. However, their combined action, as is the case in the wild type, is able to completely repress zfh-2 expression in the wing pouch. Thus, these factors act to restrict zfh-2 expression to the periphery of the wing disc, thereby defining the distal limit of the proximal wing primordium (Whitworth, 2003).

Specification of motoneuron fate in Drosophila: Integration of positive and negative transcription factor inputs by a minimal eve enhancer: Pdm1 activates eve

The mechanisms that generate neuronal diversity within the Drosophila central nervous system (CNS), and in particular in the development of a single identified motoneuron called RP2, are of great interest. Expression of the homeodomain transcription factor Even-skipped (Eve) is required for RP2 to establish proper connectivity with its muscle target. The mechanisms by which eve is specifically expressed within the RP2 motoneuron lineage have been examined. Within the NB4-2 lineage, expression of eve first occurs in the precursor of RP2, called GMC4-2a. A small 500 base pair eve enhancer has been identified that mediates eve expression in GMC4-2a. Four different transcription factors (Prospero, Huckebein, Fushi tarazu, and Pdm1) are all expressed in GMC4-2a, and are required to activate eve via this minimal enhancer; one transcription factor (Klumpfuss) represses eve expression via this element. All four positively acting transcription factors act independently, regulating eve but not each other. Thus, the eve enhancer integrates multiple positive and negative transcription factor inputs to restrict eve expression to a single precursor cell (GMC4- 2a) and its RP2 motoneuron progeny (McDonald, 2003).

GMC4-2a forms at stage 9, becomes Eve+ at stage 11, and generates the Eve+ RP2/sib neurons at late stage 11. The second-born Eve-negative GMC4-2b forms at stage 10, and generates an unknown pair of neurons. The first transcription factors detected in GMC4-2a are Pros and Hkb, due to inheritance of the proteins from the neuroblast. The next transcription factors detected in GMC4-2a are Ftz and Pdm1. Ftz is first detected at stage 10, and Pdm1 is first detected at stage 11. The de novo expression of Pdm1 is distinct from its inheritance in GMCs produced by Pdm+ neuroblasts during the assignment of temporal identity. The last protein to be detected is Eve, which appears only at late stage 11. Pros, Hkb, Ftz, and Pdm1 are each expressed transiently in the RP2/sib neurons at stage 12, but by stage 16 none of these proteins is detectable in the mature RP2 neuron. It is concluded that there is a temporal sequence of transcription factor expression in GMC4-2a: first Pros and Hkb, then Ftz, then Pdm1, and that Eve is detected only after all of these proteins are present (McDonald, 2003).

GMC4-2b forms at late stage 10, never expresses Eve, and generates two unknown Eve-negative neurons. Three transcription factors that positively regulate eve expression are detected in GMC4-2b: Pros, Ftz, and Hkb. The pattern of Pdm1 expression is too complex to score at the time GMC4-2b is born. The negative regulator Klu is detected in GMC4-2b but not GMC4-2a. It is concluded that GMC4-2b expresses at least three of the four positively acting transcription factors that are required to activate eve (Pros, Ftz, Hkb), and at least one negative regulator of eve expression (Klu). The absence of eve expression is likely due to the presence of Klu, rather than the absence of a positive regulator, because klu mutants can activate eve transcription in GMC4-2b (McDonald, 2003).

The sequential expression of Pros, Hkb, Ftz, Pdm1, and Eve in GMC4-2a raises the possibility that these four transcription factors act in a linear pathway to regulate eve expression. If so, then a mutant in an early-acting gene should lead to loss of expression of all later-acting genes in the pathway. Alternatively, the four transcription factors could all act directly to activate eve transcription, with expression of eve occurring only after all transcription factors are present. In this case, mutants in one gene should have no effect on any other gene except eve. To distinguish between these two models, pros, hkb, ftz, and pdm1 mutants were examined for expression of all four transcription factors and eve. Pdm1 is detected in GMC4-2a in all mutant genotypes: Ftz is detected in GMC4-2a in all mutant genotypes: pros, hkb, and pdm1, and Hkb is detected in GMC4-2a in all mutant genotypes. Finally, Pros is observed in GMC4-2a in all mutant genotypes, as expected because Pros is transcribed and translated in neuroblasts and is asymmetrically partitioned into each GMC. Taken together, these data support the model that all four transcription factors act directly to activate eve transcription, with expression of eve occurring only after all transcription factors are present (McDonald, 2003).

To test the model that Pros, Hkb, Ftz, and Pdm1 transcription factors directly regulate eve expression, the eve cis-regulatory DNA that confers regulated expression in the NB4-2 lineage was identified. Eve is expressed in a subset of neurons in the embryonic CNS, including the aCC/pCC neurons derived from NB1-1, the U1-5 neurons derived from NB7-1, the EL neurons derived from NB 3-3, and the RP2/sib neurons derived from NB4-2. An eve cis-regulatory element [R79R92; from ~7.9 and ~9.2 kilobase pair (kb) on the eve genomic map] has been defined that accurately directs lacZ expression to the Eve+ cells within two NB lineages: GMC4-2a and its RP2 progeny and GMC1-1a and its aCC/pCC progeny. The properties of this element are examined in this study in detail. When the R79R92 eve element was truncated to ~7.9 to ~8.6 kb (R79N86), lacZ expression in RP2 and aCC was normal, whereas expression in the pCC neuron was reduced. Truncation of the eve element to ~7.9 to ~8.4 kb (R79S84) almost completely abolished expression of lacZ in pCC, although occasionally expression in pCC was observed at low levels, whereas expression in RP2 and aCC remained high. Further truncation of the left end point to ~8.0 kb (S80S84) resulted in a reduction of expression in both aCC and RP2. Addition of the region ~8.4 to ~8.6 kb to this fragment (S80N86) increased the level of expression. However, because the region ~8.4 to ~9.2 kb (S84R92) did not show any ability to activate lacZ, the region ~8.4 to ~8.6 kb is apparently insufficient on its own to direct expression, and thus serves an auxiliary function. The removal of ~8.2 to ~8.4 kb from P80N86 abolished expression (SNdeltaSC). Together with the fact that each of the fragments ~7.9 to ~8.2 kb (S79C82) and ~8.2 to ~9.2 kb (C82R92) failed to activate lacZ, this indicates that both of the regions ~7.9 to ~8.2 kb and ~8.2 to ~8.4 kb are necessary to direct expression, and that neither alone is sufficient. Consistent with this, two tandem copies of ~8.2 to ~8.4 kb failed to activate lacZ (C82S84x2), suggesting that the two regions may provide qualitatively different activities. In summary, the critical eve cis-regulatory element for the GMC4-2a and RP2 lies in a 0.5 kb fragment of genomic DNA between ~7.9 and ~8.4 kb (McDonald, 2003).

Do the genes that activate or repress eve expression in the NB4-2 lineage work through the minimal 500 bp RP2/aCC eve enhancer? Expression of R79S84-lacZ was assayed in pros, ftz, hkb, pdm1, and klu mutant embryos, and whether it was regulated identically to the endogenous eve gene was tested. ftz, pdm1, and hkb mutant embryos show loss of R79S84-lacZ in the RP2 neuron but not the aCC neuron, identical to the pattern of endogenous eve expression in these mutants. pros mutants show loss of eve-lacZ in both RP2 and aCC, identical to the pattern of endogenous eve expression in pros mutants. In embryos lacking klu, R79S84-lacZ is expressed in two cells at the RP2 position, whereas expression in aCC is normal; this matches the pattern of endogenous eve expression in klu mutant embryos. It is concluded that the R79S84 minimal eve cis-regulatory element precisely reproduces the pattern of endogenous eve expression within the NB4-2 lineage, and that transcription factors regulating eve in GMC4-2a can act through this enhancer to activate or repress eve expression (McDonald, 2003).

Expression of eve is not detected in GMC4-2b in wild-type embryos, but mutations in the klu gene result in ectopic expression of eve in GMC4-2b. Klu contains four predicted zinc fingers, one of which is highly homologous to the WT1 zinc finger domain. The consensus binding site for the WT1 zinc finger transcription factor is a ten nucleotide sequence, 5'-(C/G/T)CGTGGG( A/T)(G/T)(T/G)-3', with variable nucleotides shown in parentheses. It was reasoned that if Klu directly binds to the eve enhancer to repress expression in GMC4-2b, one or more WT1 consensus binding sites should be found in the minimal eve enhancer R79S84. Three conserved putative Klu-binding sites were found in the R79S84 sequence: site 1, GGGTGGGGAG at nucleotides ~8066 to ~8075; site 2, GCGTGGGTGA at nucleotides ~8090 to ~8099; and site 3, TCGCCCACCA at ~8262 to ~8271. Based on the fact that altering the C2, G3, G5, G6, and G7 to T or T4 to A in the WT1-consensus binding site abolished WT1 binding, nucleotide substitutions were made in the three putative Klu-binding sites. In sites 1 and 2, As were substituted for T4, G6, and G7. In site 3, which is a reversed binding site, Ts were substituted for C4, C6, and A7. These substitutions were made at all three sites; transgenic lines were constructed expressing the mutant enhancer driving lacZ (eveK123-lacZ), and the pattern of lacZ expression was examined in the CNS of wild-type embryos and embryos misexpressing Klu protein in the NB4-2 lineage (McDonald, 2003).

In wild-type embryos, the eveK123-lacZ transgene is expressed in the aCC and RP2 neurons, similar to the wild-type (R79S84) eve-lacZ transgene. However, in one or two hemisegments per embryo, an extra cell expressing eveK123-lacZ adjacent to the RP2 neuron was observed. This phenotype is very similar to wild-type (R79S84) eve-lacZ expression in klu mutant embryos, although slightly less penetrant. It is concluded that the eveK123-lacZ transgene mimics the klu mutant phenotype, and it is proposed that Klu represses eve expression via direct binding to one or more of these sites (McDonald, 2003).

To further test this hypothesis, gain of function experiments were used to test whether ectopic Klu in GMC4-2a can repress eve-lacZ expression via these sites. Expression of a wild-type (R79S84) eve-lacZ transgene was compared with a transgene containing three mutated Klu consensus binding sites (eveK123-lacZ) in embryos where Scabrous-Gal4 (Sca-Gal4) drives ectopic expression of UAS-klu in all neuroblast lineages. The wild-type (R79S84) eve-lacZ expression is partially repressed by ectopic Klu expression, but the eveK123-lacZ transgene with mutated Klu sites is repressed to a lesser extent. This difference in repression is only observed when the levels of transgene expression are lowered by raising the embryos at 18°C; when the transgenes are more strongly expressed (by raising the embryos at 23°C) no detectable repression was observed. Taken together, Klu loss of function and misexpression studies indicate that Klu acts partly, but not completely, through three predicted Klu-binding sites to repress eve expression in the NB4-2 lineage (McDonald, 2003).

In summary, hkb, ftz, pdm1, and pros are independently required to activate eve expression in GMC4-2a. This suggests that the eve enhancer is capable of integrating the input of all four of these transcription factors to activate transcription. Hb and Ind are also necessary for eve expression in GMC4-2a, but it is not known if they act directly on the eve element or via one of the four transcription factors described in this study. Putative binding sites were found for each of the positively acting transcription factors within the minimal eve element, but mutation of these sites had no effect on expression of the eve-lacZ transgene in embryos (M. Fujioka, J.A. McDonald, and C.Q. Doe, unpublished results reported in McDonald, 2003). It remains to be determined whether Pros, Hkb, Ftz, or Pdm1 activate eve transcription via direct binding to the minimal eve element, or indirectly by activating or facilitating the binding of other transcriptional activators (McDonald, 2003).

Based on functional dissection of the RP2/aCC/pCC eve element, it seems to be composed of three parts. The regions ~7.9 to ~8.2 kb and ~8.2 to ~8.4 kb are each necessary to direct the expression pattern (together they comprise the minimal element for expression in RP2 and aCC), while the region ~8.4 to ~8.6 kb enhances the level of expression. Expression in the pCC neuron is further enhanced by the region extending to ~9.2 kb. The two regions within the minimal element seem to be regulated by different factors, because two copies of ~8.2 to ~8.4 kb (increasing the number of activator binding sites within this region by twofold) could not substitute for the function of the region ~7.9 to ~8.2 kb. This is consistent with the fact that at least four factors are independently required to activate eve in RP2 neurons. How does Klu repress eve expression in GMC4-2b? Negative regulation of eve expression by Klu is due to direct binding to the eve minimal element. (1) It is shown that klu mutants exhibit similar derepression of the eve minimal element transgene and the endogenous eve gene in the NB4-2 lineage; (2) three consensus binding sites are detected for Klu in the eve minimal element (comparison of Drosophila virilis and Drosophila melanogaster shows that the three identified sites are highly conserved); (3) mutation of these sites results in ectopic expression of eve-lacZ in the NB4-2 lineage in wild-type, and (4) mutation of these sites impairs repression of eve-lacZ by ectopic Klu in the NB4-2 lineage. The predicted Klu binding sites (K123) are probably only a subset of relevant Klu binding sites, however, because mutation of the sites gives only partially penetrant phenotypes (McDonald, 2003).

Surprisingly, it was not possible to separate the GMC4-2a/ RP2 element from the GMC1-1a/aCC/pCC element. In both NB 1-1 and NB 4-2 lineages, eve is expressed in the first-born GMC and its neuronal progeny. Both first-born GMCs share expression of several transcription factors, including Pros and Ftz. However, many other transcription factors are differentially expressed, such as the GMC1-1a specific expression of Vnd and Odd-skipped, and the GMC4-2a specific expression of Hkb, Pdm1, and Ind. It is possible that one or more commonly expressed transcription factors are required for expression of eve in both GMC1-1a and GMC4-2a, such as Pros, and this is why the elements cannot be subdivided (McDonald, 2003).

Drosophila octamer elements and Pdm-1 dictate the coordinated transcription of core histone genes

This study reveals a set of divergent octamer elements in Drosophila core histone gene promoters. These elements recruit transcription factor POU-domain protein in Pdm-1, which along with co-activator dmOct-1 coactivator in S-phase (dmOCA-S), activates transcription from at least the Drosophila histone 2B (dmH2B) and 4 (dmH4) promoters in a fashion similar to the transcription of mammalian histone 2B (H2B) gene activated by octamer binding transcription factor 1 (Oct-1) and Oct-1 coactivator in S-phase (OCA-S). The expression of core histone genes in both kingdoms is coordinated; however, although the expression of mammalian histone genes involves subtype-specific transcription factors and/or co-activator(s), the expression of Drosophila core histone genes is regulated by a common module (Pdm-1/dmOCA-S) in a directly coordinated manner. Finally, dmOCA-S is recruited to the Drosophila histone locus bodies in the S-phase, marking S-phase-specific transcription activation of core histone genes (Lee, 2010).

Given that Oct-1 and Pdm-1 share similarities in their evolutionarily conserved POU-domains, as do the human OCA-S and putative dmOCA-S components, a similar trans-regulatory network is proposed in the Drosophila H2B gene activation pathway in which dmOCA-S abets the role of Pdm-1; thus, silencing individual dmOCA-S subunits may bring about phenotypes similar to those observed in Pdm-1-silenced cells. In particular, glyceraldehyde-3-phosphate dehydrogenase (p38/GAPDH) and lactate dehydrogenase (p36/LDH) were shown to be OCA-S components absolutely required for hH2B transcription (Zheng, 2003; Dai, 2008). DmGapdh1/2, dmLdh, and abnormal wing discs [Awd, the non-metastatic protein 23 in human (nm23) homolog] were subject to RNAi. The expression of these proteins was silenced to relative completion with 37 nm dsRNA doses, which was accompanied by coordinately repressed expression of dmH2B and dmH4 genes. The repressed dmH2B expression was attributed to reduced promoter activity of the dmH2B gene, for the activities of the ectopic dmH2B promoter-luciferase reporter were severely reduced upon silencing the protein expression of each of the tested dmOCA-S components (dmGapdh1/2, dmLdh, and Awd). Collectively, these results suggest an existence of a dmOCA-S co-activator in Drosophila that comprises of the above-tested proteins and functions in the dmH2B gene regulation pathway by abetting the role of Pdm-1 (Lee, 2010).

Although more than 800 million years apart in evolution, Drosophila and human share similarities in histone expression pathways, albeit with species-specific features. In Drosophila, core histone gene promoters contain multiple evolutionarily diversified Pdm-1 binding sites, contributing to the optimal histone expression​; on the other hand, a single prototype octamer ATTTGCAT mediates the hH2B transcription, and Oct-1 association with this element, which is conserved among vertebrates, is kept under strict sequence requirement. This strategy may be also employed by other Oct-1-dependent genes and their cognate coactivators. These species-specific features could imply significance for metazoan evolution (Lee, 2010).

Histone biosynthesis and DNA replication are coupled and essential for cell viability. Thus, impeding the function of Pdm-1/dmOCA-S would lead to S-phase defects and likely be detrimental to cell viability. In the case of dmGapdh and dmLdh, their roles in glycolysis and their moonlighting nuclear functions abetting the role of Pdm-1 also dictate negative consequences on the cellular ability to progress through the S-phase in a loss-of-function situation (Lee, 2010).

Efficient RNAi-mediated protein knockdown requires mRNA destruction and decay of the preexisting protein, which was realized at 72 h for Pdm-1 and 88A) with coordinately repressed expression of core histone genes; however, the cell cycle profiles were not affected or only marginally affected at this point, which was statistically insignificant. Thus, the histone expression defects at 72 h were primary defects. It was reasoned that a most likely scenario is that despite depriving cells of histone transcription at 72 h by a Pdm-1 deficiency, pre-existing histone protein levels are above a threshold that supports DNA replication. Indeed, H2B protein levels at 72 h were largely normal or slightly reduced. Beyond 72 h, however, Pdm-1-silenced cells also exhibited a prominent S-phase defect at 96 h, and cell viability defects manifested at 84 h with a more drastic viability decrease at 96 h (Lee, 2010).

The cell viability defects might be related to a mechanism coupling histone expression to S-phase progression. The more drastic histone expression defects at 84 h as compared with that at 72 h could be due to additive effects of a primary Pdm-1 loss-of-function and secondary cell viability and S-phase defects, which fed back. Histone transcription defects as a result of the Pdm-1/dmOCA-S deficiency would deprive cells of histone proteins in a long run, thus providing insufficient histone protein levels for chromatin assembly, which ultimately leads to DNA replication, S-phase, and cell viability defects. It is proposed that although histone transcription defects at 72 h were primary defects, the later-manifested cell viability and S-phase defects were secondary defects due to reduced histone protein levels as exemplified by that of H2B beyond 72 h (Lee, 2010).

Coordinated histone expression in diverse species is needed to maintain balanced expression of core histone genes, known to sustain genome integrity. In mammalian cells, eliminating OCA-S function by silencing p38/GAPDH expression led to H2B expression defect, and a lagged histone H4 expression defect manifested after a severe cell cycle arrest; eliminating H2B expression by gene deletion in yeast also led to cell cycle arrest and subsequent expression defects of other core histone genes. These observations led to a thought that coordinated histone expression was regulated through an S-phase feedback mechanism, which was later revised (Yu, 2009; Lee, 2010).

In Drosophila cells, the RNAi-mediated silencing of Pdm-1 or dmOCA-S components led to concerted and directly coordinated expression defects of all core histone genes and as primary transcription effects due to a missing Pdm-1/dmOCA-S function. In mammalian cells, the core histone genes employ distinct promoter elements and associated (co)factors, but their expression remains highly coordinated through an uncharacterized mechanism that is indirect but still does not involve S-phase feedback. Distinct histone expression coordination mechanisms in fly and mammalian cells might be of crucial relevance in metazoan evolution (Lee, 2010 and references therein).

The expression of metazoan-specific linker histone H1 gene is largely S-phase-specific, but the H1 expression output is not tightly coupled with that of core histone genes. The TATA-less Drosophila H1 gene utilizes a TRF-containing complex but not the prototype TBP-containing TFIID complex for its expression. This study found that Drosophila core histone genes, at least that of dmH2B and dmH4, contain TFIID in line with recruitment of TBP to dmH3 and dmH4 promoters and the idea that the basal transcription machineries of Drosophila core histone genes use the prototype TFIID. TRF-containing complexes have not been known to function in a cell cycle-dependent manner; it is of interest to investigate if the S-phase-specific Drosophila H1 expression is conferred upon by S-phase-specific factors that ought to be distinct from Pdm-1/dmOCA-S because the Drosophila H1 expression was not coordinately repressed with that of core histone genes in Pdm-1-deficient cells (Lee, 2010).

Mammalian histone genes are organized into Cajal bodies in a process facilitated by nuclear protein, ataxia-telangiectasia locus (NPAT), a cyclinE/cdk2 substrate that conveys the cyclinE/cdk2 signaling to histone transcription machineries. In contrast, Drosophila cells contain distinct nuclear domains dubbed HLB, which host all the histone genes and contain a cyclinE/cdk2-dependent phospho-epitope recognized by the MPM-2 monoclonal antibody in S-phase. Drosophila HLB are often in close proximity to, but never overlapped with Drosophila Cajal bodies (Lee, 2010 and references therein).

That cyclinE/cdk2 signaling is conserved from Drosophila to human, and that the HLB foci are associated with nascent histone transcripts prompted an investigation of S-phase-specific recruitment of dmOCA-S (represented by nuclear dmGapdh) to HLB. A higher percentage of HLB (~90%) foci as compared with dmOCA-S (~70%) foci in the early S-phase, i.e.,~80% of HLB foci are nuclear dmGapdh-positive, suggests that the dmOCA-S function (dmGapdh-HLB nuclear co-localization) is likely downstream of cyclinE/cdk2 signaling. The increase in the foci size and maximal degree of HLB-dmOCA-S co-localization coincided with the peak of dmH2B and dmH4 mRNA levels in the mid-S-phase, in line with the notion that the HLB foci size is proportional to histone expression levels (Lee, 2010 and references therein).

None of dmOCA-S components possesses a consensus sequence(s) for cdk; the fly cyclinE/cdk2 signaling might be conveyed to histone genes via an unidentified molecule(s) (dmNPAT), for which the phospho-epitope-containing protein recognized by MPM-2 monoclonal antibody is a potential candidate (Lee, 2010 and references therein).

Efforts to find dmNPAT have been fruitless; a dmNPAT gene might likely reside in so-far-unsequenced heterochromatic domains or possess sequences drastically divergent from vertebrate NPATs. Alternatively, an NPAT function may not have been acquired during insect evolution, necessitating histone genes to be organized into HLB and compelling cognate promoters to be regulated by diversified octamer elements and a Pdm-1/dmOCA-S module to ensure the directly coordinated expression of histone genes (Lee, 2010 and references therein).

The mechanistic aspects of co-activation by dmOCA-S seem to be similar to that of human OCA-S, in line with nuclear moonlighting transcription functions of metabolic enzyme conservation in metazoans. Conversely, some non-transcriptional functions of transcription factors or co-factors have been documented: e.g. in the cytoplasm, an isoform of co-activator OCA-B plays an essential non-transcriptional role for B-cell signaling (Lee, 2010).

Pdm-1 is a ubiquitous transcription factor for ubiquitous histone expression; the identified cell-specific roles of Pdm-1 in neuronal cell fate specification and Notch signaling might be due to cell-specific non-transcriptional functions of Pdm-1. Alternatively, cell-specific Pdm-1 phenotypes might be attributed to missing links between mutant pdm-1 alleles and alleles encoding cell-specific co-activators, especially in view of the fact that a given POU-domain is rich in separable surfaces that provide contacts for distinct ubiquitous or tissue-specific co-activators. Non-transcriptional development regulators may also interact with partners through different surfaces. Thus, it is not surprising that mutant alleles of even a ubiquitously expressed gene, be it specifying a non-transcription or transcription function, may lead to tissue-specific phenotypes (Lee, 2010).

The work toward dissecting cis- and trans-regulatory networks of the Drosophila histone transcription regulation pathway(s) will provide a new paradigm for studying transcriptional regulator changes implied in evolution and development as well as permit a broader range of questions to be asked about a cohort of genes involved in histone expression and related DNA replication-dependent transcription and its tight coupling with S-phase progression and about roles of the individual dmOCA-S components as novel S-phase-specific players in above processes in a genetically tractable organism (Lee, 2010).

JAK/STAT signaling is required for hinge growth and patterning in the Drosophila wing disc.

JAK/STAT signaling is localized to the wing hinge, but its function there is not known. The Drosophila STAT Stat92E is downstream of Homothorax and is required for hinge development by cell-autonomously regulating hinge-specific factors. Within the hinge, Stat92E activity becomes restricted to gap domain cells that lack Nubbin and Teashirt. While gap domain cells lacking Stat92E have significantly reduced proliferation, increased JAK/STAT signaling there does not expand this domain. Thus, this pathway is necessary but not sufficient for gap domain growth. Reduced Wingless (Wg) signaling dominantly inhibits Stat92E activity in the hinge. However, ectopic JAK/STAT signaling does not perturb Wg expression in the hinge. Negative interactions occur between Stat92E and the notum factor Araucan, resulting in restriction of JAK/STAT signaling from the notum. In addition, this study found that the distal factor Nub represses the ligand unpaired as well as Stat92E activity. These data suggest that distal expansion of JAK/STAT signaling is deleterious to wing blade development. Indeed, mis-expression of Unpaired within the presumptive wing blade causes small, stunted adult wings. It is concluded that JAK/STAT signaling is critical for hinge fate specification and growth of the gap domain and that its restriction to the hinge is required for proper wing development (Ayala-Camargo, 2013).

nubbin: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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