daughterless

DEVELOPMENTAL BIOLOGY

Embryonic

In the preblastoderm, maternally supplied DA is present throughout the egg and is detected in all nuclei. By early blastoderm, DA levels decline but rapidly increase again shortly before germband extention, reaching maximal levels during stages 9-11 [Images], when most neuronal precursors form. DA protein is present in ectodermal cells as well as putative neuroblasts both during and after their delamination. Neural precursors have a somewhat higher level of DA. Every type of somatic cell expresses DA, but generally higher levels are found in salivary glands, muscles and parts of the gut (Vaessin, 1994).

Larval

The level of DA is fairly uniform in all epidermal cells of the wing disc, but is elevated in many neuronal precursors. This is true in the leg and eye disc as well.

Novel function of the class I bHLH protein Daughterless in the negative regulation of proneural gene expression in the Drosophila eye

Two types of basic helix-loop-helix (bHLH) family transcription factor have functions in neurogenesis. Class II bHLH proteins are expressed in tissue-specific patterns, whereas class I proteins are broadly expressed as general cofactors for class II proteins. The Drosophila class I factor Daughterless (Da) is upregulated by Hedgehog (Hh) and Decapentaplegic (Dpp) signalling during retinal neurogenesis. The data suggest that Da is accumulated in the cells surrounding the neuronal precursor cells to repress the proneural gene atonal (ato), thereby generating a single R8 neuron from each proneural cluster. Upregulation of Da depends on Notch signalling, and, in turn, induces the expression of the Enhancer-of-split proteins for the repression of ato. It is proposed that the dual functions of Da--as a proneural and as an anti-proneural factor--are crucial for initial neural patterning in the eye (Lim, 2008).

Da is upregulated in the furrow region. Surprisingly, however, it was found that there are two distinct patterns of Da upregulation. The first pattern is a broad, low-level upregulation in the furrow (hereafter referred to as basal level). The second pattern is a stronger expression of Da (hereafter referred to as high level) selectively in the non-neural cells surrounding the Ato-positive R8 cells between proneural clusters. Tests were perfomed to see whether this previously unrecognized pattern of expression of Da is specific by examining eye discs containing da loss-of-function (LOF) clones. Both the basal and high-level expressions of Da in the furrow were lost in the LOF clones of da³, a null allele, showing the specificity of the pattern of Da expression (Lim, 2008).

The basal level of Da upregulation overlaps with the domain of Ato expression near the furrow, where they function together to regulate neurogenesis. As the furrow progression and expression of Ato are controlled by Hh and Dpp signalling, it was reasoned that regulation of Da expression in the furrow might be linked to these signalling pathways (Lim, 2008).

To test whether Hh signalling is required for the expression of Da, Da expression was examined in hh¹ mutant eye discs in which the production of Hh ceases after the mid-third instar stage, resulting in reduced expression of Ato and arrest of furrow progression. The expression of Da was downregulated in hh¹ mutant eye discs. LOF clones of smoothened (smo), a crucial component for Hh signal transduction, were generated. Da expression was significantly reduced in smo mutant clones spanning the furrow, suggesting that Hh signalling is required for the expression of Da. However, the expression of Da was not completely eliminated in hh¹ mutant eye discs or in smo LOF clones. As Dpp signalling is partly required for the expression of Ato, whether Dpp signalling is also necessary for the expression of Da was tested by analysing LOF clones of mad (mothers against dpp), an essential factor for Dpp signalling transduction. Da expression showed little reduction in mad mutant clones, indicating that Dpp signalling by itself is not essential for Da expression. By contrast, the expression of Da was almost completely abolished in LOF clones of smo and mad double-mutant cells in the furrow region. Thus, the Hh and Dpp signalling pathways are crucial but partly redundant for the expression of Da. It was also found that loss of function of Ato reduced the level of Da expression in the furrow. Therefore, several factors, including Ato, coordinate the accumulation of Da in the furrow (Lim, 2008).

To test whether the upregulation of Da in the furrow has a function in neurogenesis, da³ LOF clones were generated and the effects of da mutation on the expression of Ato and neuronal differentiation were examined. Loss of da resulted in ectopic expansion of Ato expression in the mutant clone, suggesting that Da is crucial for repressing the expression of Ato (Lim, 2008).

Despite ectopic expression of Ato, most of the cells in da LOF mutant clones could not differentiate into photoreceptor cells, as indicated by the lack of neuronal markers such as Senseless (R8 marker) and Elav (pan-neural marker). Hence, the expression of ectopic Ato is insufficient to induce retinal differentiation in the absence of Da. However, local differentiation was occasionally detected near the posterior end of some clones. This might be due to the perdurance of Da in LOF clones, although other possibilities, such as partial non-autonomy or partial independence of photoreceptor differentiation from Da in the posterior region of the eye disc, cannot be excluded (Lim, 2008).

To support the idea that a high level of Da expression is required for the repression of Ato, a temperature-sensitive allele of da (da^ts) was examined that causes conditional partial loss of function of Da at the restrictive temperature. In da^ts mutant eye discs, Ato was expressed in several cells rather than a single R8 cell per proneural cluster. In addition, the effects of conditional expression of Da was tested by temperature shifts of heat-shock (hs)-da flies. Ato was repressed by the overexpression of Da after a longer heat shock but not after a shorter heat shock. These observations support the idea that enriched Da expression in the cells surrounding each R8 cell is required for generating a single R8 cell by the inhibition of Ato expression (Lim, 2008).

The expanded expression of Ato in da mutant clones might, in part, be due to the failure of da mutant cells to induce lateral inhibition of Ato expression. It is also possible that Da might be involved in the cell-autonomous repression of Ato expression. To test this possibility, Da was overexpressed in the dorsoventral margin of the eye disc using the optomotor blind (omb)-Gal4 driver. The overexpression of Da downregulated Ato expression in the expression domain of omb. Furthermore, the overexpression of Da in the antenna disc using the dpp-Gal4 driver resulted in Ato repression in the expression domain of dpp. Taken together, these data from LOF and overexpression analyses suggest that the high-level expression of Da is necessary and sufficient for the cell-autonomous repression of Ato during the selection of R8 (Lim, 2008).

Both Da and Notch (N) are essential for the selection of R8 by repressing Ato expression in non-R8 precursors within proneural clusters. Hence, Da might be involved in N-dependent lateral inhibition. Furthermore, the overexpression of ASC proneural factors, together with Da, can synergize with Suppressor of hairless and N to activate the expression of Enhancer-of-split (E(spl)) in cultured cells. Since E(spl) is expressed complementary to the expression of Ato in the same cells expressing a high level of Da, whether Da alone could regulate the expression of E(spl) was tested in vivo. The expression of E(spl) proteins was reduced in da³ mutant cells, showing that Da is required for the expression of E(spl) in vivo. Furthermore, the overexpression of Da with dpp-Gal4 could induce the expression of ectopic E(spl) in the dpp domain of the antenna disc. These results indicate that a high level of Da expression is necessary and sufficient for the activation of E(spl) expression (Lim, 2008).

Since E(spl) is the main mediator of N signalling, Ato repression by a high level of Da might be dependent on the expression of E(spl). To test this possibility, the MARCM method was used to generate E(spl) LOF clones in which the expression of Da is induced by tubulin (tub)-Gal4. Da overexpression in E(spl) LOF clones did not show a significant repression of Ato. Similarly, overexpression of E(spl)mδ in da LOF clones did not show noticeable repression of Ato. These data suggest that both Da and E(spl) are required for positive feedback regulation and for repression of Ato during lateral inhibition. However, it is also possible that other bHLH family genes of the E(spl) complex loci might be required, or that the overexpression of E(spl) or Da by tub-Gal4 in MARCM assays might not be strong enough to repress the expression of ato. By contrast, Da expression by dpp-Gal4 induces the expression of E(spl), even in the proximal sector of the antenna disc where Ato is not expressed. amos, the proneural gene for olfactory sensilla, is not expressed in the antenna disc at this time. Thus, a high level of Da can induce E(spl) in the absence of Ato, although Da might act with other class II proteins to promote the expression of E(spl) (Lim, 2008).

Since N signalling is activated in the same cells surrounding R8 founder neurons, whether Da expression is affected was examined by removing the function of N using a temperature-sensitive allele, N^ts. The loss of function of N at the restrictive temperature resulted in several Ato-positive cells per proneural cluster. Furthermore, the transient loss of N activity abolished the high-level of Da expression between the proneural clusters but did not eliminate the basal level of Da expression in the same cells. This suggests that N signalling is essential for the high-level upregulation of Da expression. Since the expression of da is regulated by Hh and Dpp signalling, as well as Ato, it is possible that the regulation of Da by Hh and Dpp might be mediated by Ato-dependent N signalling in the non-R8 precursor cells (Lim, 2008).

To investigate further the role of N signalling in the expression of Da, whether E(spl) proteins mediate the function of N in inducing a high level of Da expression was examined. Loss of E(spl) caused ectopic expression of Ato in E(spl) mutant clones because of the lack of N-mediated lateral inhibition. Interestingly, the high level of Da expression was suppressed, but the basal level of Da expression was still detected in E(spl) mutant clones, as seen in N^ts mutant eye discs. Thus, E(spl) is required for the high level but not for the basal level of Da expression. In contrast to da³ LOF mutant cells that fail to differentiate in spite of ectopic Ato expression, E(spl) LOF mutant cells not only expressed ectopic Ato but also differentiated into ectopic photoreceptors. Thus, the basal level of Da expression remaining in E(spl) LOF clones is sufficient for the formation of a functional complex with Ato to induce neural differentiation (Lim, 2008).

On the basis of the above observations, a model is proposed in which Da has dual functions as a proneural and as an anti-proneural factor depending on the expression level during early retinal neurogenesis . The anti-proneural function of Da proposed in this model provides an explanation for the abnormal upregulation of Ato in da mutant cells in the furrow, although the LOF experiments are also consistent with the pre-existing view that Da promotes the function of Ato. In Ato-positive neural precursors, low levels of Da expression are sufficient to form heterodimers with Ato to function as a proneural factor. In neighbouring cells, the N-E(spl) pathway further upregulates the expression of Da, which, in turn, induces more expression of E(spl). This putative feedback regulation might provide a mechanism for more effective lateral inhibition of Ato expression for the selection of R8. Interestingly, Da can form a homodimer and bind to DNA in vitro. Thus, in Ato-negative cells surrounding the R8 precursors, a high level of Da expression might enforce the formation of Da homodimers and/or heterodimers with other unknown bHLH proteins to repress the expression of ato. It would be interesting to see whether mammalian type I bHLH proteins such as E proteins might also be specifically regulated to have distinct developmental functions as seen in the case of Da (Lim, 2008).

Adult

The daughterless gene is expressed in the somatic ovary during egg chamber morphogenesis. Hypomorphic da mutant genotypes exhibit dramatic defects during oogenesis, including aberrantly defined follicles and loss of interfolicular stalks. The defects are similar to those for Notch or Delta mutant ovaries. The role of Daughterless in this case is not as a transcriptional activator of either Delta or Notch (Cummings, 1994).

During Drosophila oogenesis two distinct stem cell populations produce either germline cysts or the somatic cells that surround each cyst and separate each formed follicle. From analyzing daughterless (da) loss-of-function, overexpression and genetic interaction phenotypes, several specific requirements have been identified for da⁺ in somatic cells during follicle formation. (1) da is a critical regulator of somatic cell proliferation. (2) da is required for the complete differentiation of polar and stalk cells, and elevated da levels can even drive the convergence and extension that is characteristic of interfollicular stalks. (3) da is a genetic regulator of an early checkpoint for germline cyst progression: loss of da function inhibits normally occurring apoptosis of germline cysts at the region 2a/2b boundary of the germarium, while da overexpression leads to postmitotic cyst degradation. Collectively, these da functions govern the abundance and diversity of somatic cells as they coordinate with germline cysts to form functional follicles (Smith, 2002).

Integrating this information about da activities with several published models of early oogenesis leads to the following outline for the sequential steps required for the iterative production of follicles. (1) Signaling by Hh protein from the terminal filament creates a somatic stem cell niche at the region 2a/2b boundary. (2) Somatic stem cells produce undifferentiated mesenchymal cells that surround the germline cyst and compress it into the characteristic lens shape. (3) EGFR-mediated signaling from the germline cyst provides a continuous proliferative signal to somatic cells. (4) A second germline-to-soma signal, Delta, induces the somatic cells, located between adjacent cysts in region 2b/3, to begin differentiating as polar cells. (5) Other somatic cells, which contact only a single germline cyst, differentiate as cuboidal epithelial cells. (6) The differentiating polar cells signal to neighboring cells to refine polar cell number, recruit stalk cells and promote local somatic cell proliferation. (7) The stalk cells migrate between the polar cells associated with each cyst and converge and extend to form a single column of cells as they terminally differentiate. In this scheme, da function appears to contribute to steps 2, 3, 6 and 7 (Smith, 2002).

The initial requirement for da during follicle formation is somatic cell proliferation during steps 2 and 3. The straightforward observation that one extra copy of da⁺ results in excess somatic cell production, while da loss of function leads to an insufficient number of somatic cells for germline cyst envelopment, demonstrates a role for da in proliferation. The genetic interactions between da and every component of the EGFR pathway, which is also known to regulate somatic proliferation through mid-oogenesis, suggest that da and the EGFR pathway cooperate to control cell division. Gurken has been identified as the germline-localized ligand for this proliferative function; however, grk null ovaries have a relatively low frequency of follicle formation defects. The genetic interaction observed between da and spi implicates Spi as a second ligand for EGFR in proliferation, although this must be a somatic signal, since spi expression is restricted to somatic cells. Finally, although there is no evidence for da acting during specification of the somatic stem cells, it may control their proliferation once founded. Alternatively, da control of proliferation may be limited to the progeny of the stem cells. Either possibility is consistent with suppression of the ectopic hh phenotype in da heterozygotes and the complete epistasis of da^lyh (Smith, 2002).

Although da's role in the control of somatic proliferation is unknown, it probably involves regulation of cell cycle progression. Connections between EGFR signaling and cell cycle progression have already been established in the R2-R5 photoreceptor cells in the morphogenetic furrow of the eye, where EGFR signal transduction is required for G₂/M progression, but signal inactivity is necessary for G₁/S progression. Coincidentally, Da protein levels are high in those cells, and da function is required for their G₁/S progression. A similar connection between da and cell cycle control in the ovary is implicated by the observation that da exhibits genetic interaction phenotypes with both loss of function (rl^-) and persistently activated (rl^Sem) MAPK alleles. Loss of EGFR signaling would be expected to delay cell cycle progression at the G₂/M transition, but persistent MAPK^Sem activity, being a poor substrate for the inactivating phosphatase, would delay the cell cycle at the G₁/S transition. In either situation additionally reducing the da dose (which itself would slow G₁/S progression) would lead to the mutant phenotype observed in genetic interactions, and the higher frequency of defects with rl^Sem is consistent with both da and rl^Sem impacting on the same stage of the cell cycle (Smith, 2002).

There is probably no role for da in induction of polar cells or the differentiation of follicular epithelium (steps 4 and 5). Nevertheless, complete polar cell differentiation (step 6) depends on da and is required for several non autonomous polar cell functions. The first function of differentiating polar cells is stalk cell recruitment. Clonal analysis of fng and N has shown that the function of these genes is required in the polar cells to form interfollicular stalks. In fng^- clones in which no distinct stalk is visible, the stalk-specific enhancer trap B1-93F is expressed in a peripheral cluster of cells at the junction between two incompletely separated follicles; the same clusters are observed in N^ts1 mutant ovaries. This arrangement would result if the differentiating polar cells normally recruit stalk cells from the periphery, where a population of undifferentiated somatic cells is not in contact with any germline cyst. Like N and fng, da interferes with recruitment of stalk cells from the periphery, since similar 'stalk cell' clusters were often observed in hypomorphic mutant ovarioles. In da mutant ovaries with more extreme defects, the 'stalk cells' are actually integrated within the follicular epithelium, suggesting that there are insufficient somatic cells to complete the epithelium. Thus, when there are sufficient somatic epthelial cells to cover the germline cyst completely (weak da^- phenotype), differentiating stalk cells never touch the germline; however, when there are not enough somatic cells (strong da^- phenotype), differentiating stalk cells consequently make contact with the germline and become incorporated into the epithelium. It is proposed that a second function of differentiating polar cells is the production of a 'booster' proliferative signal to ensure sufficient somatic cells to form a stalk. A number of genetic manipulations [elevated da; ectopic N^intra, Dl, unpaired (upd) or hh, and clones of patched^- or of double mutant Protein kinaseA^- (PKA/Pka-C1) Suppressor of fused^- (Su(fu))] lead to overproduction of somatic cells within the germarium, and in every case the result is excess interfollicular cells. (To what extent these interfollicular cells organize into recognizable stalks likely reflects each genotype's impact on stalk cell differentiation.) These phenotypes implicate all of these genes in proliferation control: the N pathway (N, Dl), the JAK/STAT pathway (upd), and the hh pathway [hh, ptc, PKA, Su(fu)]. A proliferative role for the N pathway is substantiated by reconsideration of the loss-of-function phenotypes (N^ts1, fng), in which 'stalk cells' are observed within the follicular epithelium; the N pathway may be generally required for somatic proliferation, like da. However, for the JAK/STAT pathway, whose only known ligand is encoded by upd, expression of the ligand is restricted to the polar cells in the ovary. Although other studies have demonstrated roles for the JAK/STAT pathway that are limited to polar and stalk cell specification and/or differentiation during follicle formation, the effects of ectopic upd, together with the genetic interaction phenotypes between da and JAK/STAT pathway mutants, suggest that JAK/STAT is also a regulator of proliferation in the ovary, as it is elsewhere. If so, this would require expression of upd in region 2b, earlier than previously detected. Finally, complete polar cell differentiation includes refinement to two polar cells; a number of markers (FasIII, A101, fng and PZ80) show that variable numbers (4-8 between adjacent germline cysts) of polar cells form but always refine to 2 per pole by the time a follicle matures to stage 4. In a number of mutants, including da, excess polar cells often persist past stage 4, suggesting a failure in refinement (Smith, 2002).

The differentiation of stalk cells (step 7) also requires da. In weak da^- phenotypes where cells expressing stalk cell markers were seen in clusters physically isolated from the germline cyst by an epithelial layer, the 'stalk cells' did not converge and extend to form a stalk, as wild-type stalk cells would. This failure to form stalks could result from defects in recruitment of stalk cells by polar cells (step 6), in stalk cell differentiation, or both. Consistent with a proactive role for da in stalk cell differentiation, genotypes with elevated da levels occasionally formed stalk-like structures at the expense of the follicular epithelium. Indeed, Da protein levels normally remain high in the stalk and polar cells, even after they have dropped in the follicular epithelium. Additionally, stalk-like differentiation of the excess somatic cells generated by ectopic hh is da-dependent: reduced da results in more aggregation (i.e. less convergence and extension) and increased da results in more convergence and extension (i.e. less aggregation). In this context, the 'lollipop' phenotype reflects the acquisition of a stalk-like characteristic by excess somatic cells; hh-induced somatic cells that rearrange in the vitellarium to form cables running along the sides of follicles converge and extend to form a 'lollipop', when da is increased. Relatively high Da levels appear to be required to drive convergence and extension in differentiating stalk cells. This da requirement may involve transcriptional activation of stalk-specific genes, since the expression of two stalk cell markers is reduced in strong da^- phenotypes. However, reduced marker expression could result indirectly from incorporation of 'stalk cells' into the follicular epithelium, where stalk-specific gene expression may be repressed (Smith, 2002).

Successful follicle formation requires the right balance of somatic cells per germline cyst, such that ratios that are too low activate germline apoptosis to abort cyst progression in the germarium; the function of this cyst progression checkpoint was first demonstrated in nutrient-deprived flies. The mechanism for assessing the soma-to-germline ratio is completely unknown; however, the relative balance of cells is evaluated as each 16-cell cyst enters region 2b of the germarium. Thus, environmental variables such as nutrition could lead to activation of the cyst progression checkpoint either by increasing germline cyst production or retarding somatic cell production. If these two cell populations (germline and soma) have different nutritional requirements, cyst apoptosis might be activated only at nutritional extremes: at one extreme (low nutritional values), slackened somatic cell proliferation does not keep pace with normal cyst production, resulting in aborted cyst progression, while at the other extreme (high nutritional values), accelerated cyst production outpaces normal somatic cell proliferation, resulting in a similar termination of cyst progression. Only in situations in which the rates of cyst production and somatic cell proliferation are balanced (e.g., intermediate nutritional values) would activation of the cyst progression checkpoint be unnecessary. Age could affect the proliferation rate of either of these two cell populations, and the frequency of cyst apoptosis in the germarium does increase with age. Other environmental conditions that have been shown to affect egg production, such as temperature, humidity, prior anesthesia, adult crowding, mate abundance and dessication state, should be examined similarly for effects on cyst progression. Checkpoint activation is also influenced by the genetic background since the frequency of apoptosis varies among wild-type strains. How a somatic cell deficit, once detected, leads to activation of apoptosis in the germline cyst is unknown; however, evidence indicates that somatic cells are involved in the process (Smith, 2002).

Numerous observations from this analyses of da mutant phenotypes identify da as a key component in the soma's regulation of the cyst progression checkpoint. The significant reduction in cyst degradation (as viewed either by Acridine Orange staining or TUNEL) in da loss-of-function mutants indicates that the checkpoint is da-dependent, and since Da protein is absent from the germline, it is the gene's somatic dose that is critical in this process. This is consistent with the phenotype caused by moderate elevations of da (by chromosomal duplications) in which the checkpoint appears to function normally; the increased somatic cell production provided by the weaker duplication only results in longer interfollicular stalks, while the stronger duplication additionally results in more cysts surviving the checkpoint and being packaged into follicles due to the further increased production of somatic cells. Additional evidence that da normally contributes to the checkpoint comes from the analysis of the effects of higher elevations of da, which can lead to ectopic cyst degradation in the germarium. The synergistic interaction between the da dose and an environmental variable (i.e. cyst degradation in flies with elevated da levels increased with age) suggests that environmental conditions can sensitize the checkpoint to activation by da. Moreover, the apoptotic checkpoint is only activated in post-mitotic cysts, since elevated da does not lead to the degradation of still-dividing cysts, even when these slip into region 2b or 3 of the germarium. Although only post-mitotic cysts appear capable of activating the apoptosis pathway, cells in the adjacent soma are responsible for monitoring the germline cyst/somatic cell balance and sending an activating signal. The role of da in those cells could entail either positive or negative regulation. For positive regulation, da⁺ would promote the generation of a proapoptotic factor as an integral part of the checkpoint. For negative regulation, da⁺ would repress an antiapoptotic (i.e. prosurvival) factor; such a factor would normally be required for the maintenance of post-mitotic cysts in the germarium. The identification of additional genetic components of the checkpoint will help distinguish between these two models (Smith, 2002).

Effects of Mutation or Deletion

The maternal effect caused by the daughterless mutation depends on the sex of the progeny. Homozygous daughterless mothers produce only males. Their eggs cannot support female development (Cline, 1976). Daughterless is required to activate Sex lethal, the key gene for sex determination.

The requirement for da function in photoreceptor cells of the developing eye was examined in clones of mutant cells for da. Mutant cells most often give rise to a narrow anterior-posterior scar across the eye. Mutant cells never express the 22C10 antigen (see Futsch), which is expressed by differentiated photoreceptor cells R1-R8. Staining with anti-DA antibody reveals that DA protein is present within all eye disc cells, but there is elevated expression in the morphogenetic furrow. At the posterior portion of the furrow, R8 cells appear to have elevated DA levels. The anterior boundary of elevated DA levels within the furrow is adjacent to but does not overlap the posterior edge of Hairy protein expression. Hairy is a negative regulator of neuronal photoreceptor development. When da mutant patches encampass the morphogenetic furrow, apical cell constrictions are lacking, suggesting that furrow progression may stop in the absence of da function. Loss of da expression results in a absence of dividing M phase cells posterior to the furrow, including the second wave of mitosis that occurs after passing of the furrow (Brown, 1996).

The expression of the MyoD gene homolog, nautilus (nau), in the Drosophila embryo defines a subset of mesodermal cells known as the muscle 'pioneer' or 'founder' cells. These cells are thought to establish the future muscle pattern in each hemisegment. Founders appear to recruit fusion-competent mesodermal cells to establish a particular muscle fiber type. In support of this concept every somatic muscle in the embryo is associated with one or more nautilus-positive cells. However, because of the lack of known (isolated) nautilus mutations, no direct test of the founder cell hypothesis has been possible. Toxin ablation and genetic interference by double-stranded RNA (RNA interference or RNA-i) have been used to determine both the role of the nautilus-expressing cells and the nautilus gene, respectively, in embryonic muscle formation. In the absence of nautilus-expressing cells muscle formation is severely disrupted or absent. A similar phenotype is observed with the elimination of the nautilus gene product by genetic interference upon injection of nautilus double-stranded RNA (Misquitta, 1999).

The results from the injection of nautilus dsRNA point to a more general approach for the analysis of gene function during Drosophila development and suggest that the RNA interference method essentially would mimic a gene knock-out in the injected generation of Drosophila embryos. To test this idea a variety of cDNA clones were obtained representing a maternal gene expressed in the embryo (daughterless); additional genes involved in myogenesis (S59, DMEF2); homeobox genes (engrailed and S59); a gene important for gastrulation (twist), and a gene expressed in the adult eye (white). This panel of genes covers most stages of Drosophila development. twist was initiatially tested because the mutant has a clear phenotype that is easy to score when compared with wild-type larva. The injection of twist dsRNA (the complete coding region) into embryos produces a twisted larval phenotype that is indistinguishable from the original twist mutation. Similarly, injection of the first 1,200 bp of engrailed dsRNA produces the compressed dentical belt pattern characteristic of an engrailed null mutant. Daughterless mRNA is both maternally loaded and expressed zygotically, and the mutant phenotype produces very characteristic disruptions in the central nervous system (CNS) and peripheral nervous system (PNS). It has been shown previously that mex3, a maternally loaded RNA in C. elegans, can be ablated by dsRNA injection into the gonads. daughterless dsRNA (complete coding region) was injected and the characteristic neuronal phenotypes were sought by using the mAb MAB 22C10. The CNS as well as the PNS were disrupted to varying degrees in the injected embryos. The severity of the phenotype consistently shows a CNS disruption with a variable PNS pattern, possibly reflecting the fact that the CNS is formed before the PNS. This result suggests that maternally loaded as well as zygotically expressed RNA can be affected by RNA-i in Drosophila. The homeobox gene S59 marks a subset of muscle founder cells for 5 of 29 muscles in each hemisegment of the embryo corresponding to muscles 5, 18, 25, 26, and 27. Embryos with an S59 lacZ transgene marking muscles 18 and 25 were injected with S59 dsRNA (complete coding region). In this case, the S59-specific lacZ antibody-staining pattern is abolished. The total muscle pattern for embryos injected with S59 dsRNA, although disrupted, still shows the presence of poorly organized muscle groups in each hemisegment. This is unlike the almost complete absence of muscle observed with the injection of nautilus dsRNA. DMEF2, a member of the MADS domain transcription factor family, is essential for muscle formation in Drosophila. The DMEF2 / embryo has no muscle and is missing the characteristic gut constrictions found in the uninjected embryo. Injection of DMEF2 dsRNA (complete coding region) results in embryos that lack any detectable muscle and an absence of gut morphology (Misquitta, 1999).

In mutants lacking both the Achaete-Scute Complex (ASC) and atonal, coding for proteins known to establish SOP cell fate, two to three neurons of the solo-MD type remain in the dorsal region of each abdominal hemisegment. The identity of these remaining neurons are controlled by absent MD neurons and olfactory sensilla (amos). Both the ASC and Atonal physically interact with the protein Daughterless (Da). Since the solo-MD neurons that exist in ASC;ato double mutants are eliminated in da mutants, this observation implies that the proneural gene for those solo-MD neurons also encodes a transcription factor of the bHLH family (Huang, 2000).

SOP formation is very sensitive to simultaneous reduction in copy number of proneural genes and da, which encodes their common heterodimer partner. For instance, simultaneously removing one copy of the AS-C and da genes (i.e., transheterozygotes) results in adults with a proportion of missing sensory bristles. Likewise, reducing the dosage of ato and da genes results in reduction in the number of chordotonal organs and sensilla coeloconica. Although there is no specific mutation of amos, lethal chromosomal deficiencies have been investigated that delete amos (Df(2L)M36F-S5 and Df(2L)M36F-S6) for genetic interaction with da. Olfactory sensillum numbers were analyzed in flies heterozygous for an amos deficiency either alone or in combination with the loss of one copy of da (Df(2R)da^KX136/+). In flies with a single copy of each gene (abbreviated as amos^+/-:da^+/-), the number of sensilla basiconica is significantly reduced (by 30%) compared with wild-type, amos^+/-, da^+/-, or ato^+/-:da^+/- flies. This genetic interaction suggests functional cooperation between da and a bHLH gene in the amos genomic region. Given that amos is the only bHLH-encoding gene in this region, these data are consistent with the function of Amos/Da heterodimers during the formation of sensilla basiconica. Sensilla trichodea are also significantly reduced in amos^+/-:da^+/- flies, but a reduction in amos^+/- flies, when compared with wild-type was also observed. Therefore, although consistent with a requirement for amos in trichodea formation, such a requirement seems to be less sensitive to da gene dosage. Although sensillum coeloconica numbers are rather variable, amos^+/-:da^+/- flies have only slightly fewer sensilla coeloconica than flies with either mutation alone, whereas there are significantly fewer sensilla coeloconica in ato^+/-:da^+/- flies as expected. In summary, these data support a role for the chromosomal region containing amos in sensillum basiconica formation and are suggestive of a role during sensillum trichodea development (Golding, 2000).

Evidence that nervy, the Drosophila homolog of ETO/MTG8, promotes mechanosensory organ development by enhancing Notch signaling: nvy interacts with daughterless

In the imaginal tissue of developing fruit flies, achaete (ac) and scute (sc) expression defines a group of neurally-competent cells called the proneural cluster (PNC). From the PNC, a single cell, the sensory organ precursor (SOP), is selected as the adult mechanosensory organ precursor. The SOP expresses high levels of ac and sc and sends a strong Delta (Dl) signal, which activates the Notch (N) receptor in neighboring cells, preventing them from also adopting a neural fate. Previous work has determined how ac and sc expression in the PNC and SOP is regulated, but less is known about SOP-specific factors that promote SOP fate. This study describes the role of nervy (nvy), the Drosophila homolog of the mammalian proto-oncogene ETO, in mechanosensory organ formation. Nvy is specifically expressed in the SOP, where it interacts with the Ac and Sc DNA binding partner Daughterless (Da) and affects the expression of Ac and Sc targets. nvy loss- and gain-of-function experiments suggest that nvy reinforces, but is not absolutely required for, the SOP fate. A model is proposed in which nvy acts downstream of ac and sc to promote the SOP fate by transiently strengthening the Dl signal emanating from the SOP (Wildonger, 2005).

These results suggest that Nvy plays a role, albeit subtle, in the SOP's ability to send a strong Dl signal to neighboring cells. Although the data demonstrate that nvy is not required for the SOP fate, it is suggested that the ability of Nervy to increase the Dl signal sent by the SOP helps to reinforce the SOP fate. When nvy is ectopically expressed it completely inhibits the formation of mechanosensory organs. Using reagents that mark the PNC and SOP, it was found that ectopic Nvy blocks the formation of the SOP, but not the PNC. In contrast, elevating Nvy levels specifically within the SOP (using neur-Gal4) does not affect sensory organ development, indicating that ectopic Nvy blocks the formation of the SOP but does not inhibit its development once it is specified. Furthermore, ectopic Nvy does not block mechanosensory organ formation when Sens is also over-expressed, suggesting that ectopic Nvy blocks SOP formation before there are high levels of Sens in the nascent SOP. Consistent with this idea, no Sens expression is observed in the pnr domain of pnr-Gal4 UAS-nvy wing discs or in clones that ectopically express Nvy. These data suggest that ectopic Nvy interferes with SOP formation at a stage before Sens is expressed, which corresponds to when the SOP is initially specified (Wildonger, 2005).

nvy is normally expressed in the SOP shortly after Ac and Sc levels increase. Given the expression of endogenous nvy within the SOP, the following two possibilities werre considered to explain the ectopic Nvy phenotype and to gain some clues about wild type function of nvy. (1) It is possible that ectopic Nvy blocks SOP formation cell autonomously by inhibiting the expression of ac, sc, or their downstream targets (such as sens) that are necessary for SOP formation. (2) It is possible that ectopic Nvy acts cell non-autonomously by enhancing Dl signaling, resulting in the 'mutual inhibition' of cells expressing precociously high levels of nvy. A closer examination of clones that ectopically express Nvy revealed that SOPs were significantly less likely to form near the borders of Nvy expressing clones than control clones. These results suggest that Nvy is acting, at least in part, cell non-autonomously, perhaps by increasing the strength of the Dl signal (the possibility that Nvy may also act cell autonomously is discussed in the following section). As a test of this idea, Nvy was ectopically expressed in clones lacking nic, which encodes a transmembrane protein required for cleaving and activating N in response to ligand binding. Ectopic Nvy was unable to block SOP formation in nic mutant clones, demonstrating that Nvy's ability to block SOP formation requires the N signaling pathway to be intact. This finding is therefore consistent with the idea that Nvy normally enhances the level of active Dl in the SOP. Importantly, loss-of-function nvy experiments are also consistent with this proposed role for Nvy. Using two different methods to remove nvy (expressing nvy RNAi or generating clones of a nvy deficiency), it was found that PNC cells that neighbor nvy⁻ clones are more likely to adopt the SOP fate than PNC cells that neighbor wild type clones. This result is similar to what was observed when the relative amount of Dl differs between neighboring PNC cells: PNC cells that neighbor cells with less Dl are more likely to differentiate as SOPs. In contrast to the Dl experiments, however, the complete absence of nvy did not cause all PNCs to become SOPs. Keeping in mind that nvy expression is restricted to the SOP (nvy is not detectably expressed in the PNC), these data suggest that nvy is not a general regulator of Dl signaling throughout the PNC, but that nvy enhances Dl activity in the SOP when it is forming (Wildonger, 2005).

Although these experiments are consistent with the idea that nvy enhances Dl signaling in the SOP, no changes in Dl protein levels were directly detected in either nvy loss- or gain-of-function situations. There are several possible explanations for this negative result: (1) it is possible that nvy does affect Dl expression levels, but that the change is too slight or brief to distinguish with the available anti-Dl antibody; (2) nvy might not affect Dl expression, but affect its localization and/or signaling ability in a manner that cannot be detected in these experiments; (3) it is also possible that nvy does not affect Dl at all, but interacts with other factors to produce the phenotypes observed. It is suggested that experiments using VP16-Nvy help to distinguish between these possibilities. Expressing VP16-Nvy produces results opposite to those resulting from expressing Nvy: VP16-Nvy enhances E-lacZ expression, which ectopic Nvy represses, and its expression results in ectopic Sens⁺ SOPs. Based on these data and the evidence that ETO, the mammalian homolog of Nvy, acts as a transcriptional repressor, it is suggested that VP16-Nvy acts as a transcriptional activator of targets that wild type Nvy normally represses. When expressed in a PNC, VP16-Nvy strongly reduces the amount of Dl observed at the cell surface and in intracellular vesicles. This result suggests that wild type Nvy has the potential to affect Dl, although the result does not distinguish an effect on expression from an effect on protein stability or trafficking. That ectopic Nvy does not inhibit the expression of Dl-lacZ suggests that Nvy may be more likely to transcriptionally regulate a factor is involved in Dl stability or trafficking. Regardless of the mechanism, the finding that VP16-Nvy reduces Dl levels suggests that wild type Nvy has the potential to increase Dl levels, a proposal that is consistent with loss- and gain-of-function experiments (Wildonger, 2005).

The VP16-Nvy results, while consistent with the idea that Nvy affects Dl, do not explain why no change in Dl levels were detected in nvy loss- and gain-of-function experiments. Thus, it is thought that Nvy causes a small and/or transient increase in Dl activity (by affecting its expression, stability or localization). Nevertheless, no change in the amount or localization of Dl in wild type SOPs has been observed, despite genetic evidence that Dl signaling is a critical step in SOP fate determination. The lack of an observable change in Dl during wild type development, in combination with the current findings, lead to a proposal that the presumptive SOP may send a transient pulse of increased Dl signal that is sufficient to bias cell fates within the PNC. Nvy may, therefore, contribute to this transient pulse of Dl (Wildonger, 2005).

The experiments described here shed some light on the molecular activities Nvy has in the SOP. (1) Based on its ability to repress well-defined lacZ reporter genes, Nvy appears to be a transcriptional repressor, as is its mammalian homolog ETO. (2) This study shows that ectopic Nvy appears to interfere with the function (as opposed to the expression) of Ac and Sc because re-supplying Ac and Sc in pnr-Gal4 UAS-nvy flies was unable to rescue the bald phenotype. In contrast, expression of Da, a bHLH DNA binding partner for Ac and Sc, was able to partially rescue the bald phenotype of pnr-Gal4 UAS-nvy flies. Moreover, nvy and da were found to genetically interact (e.g., reducing nvy levels enhanced a da gain-of-function phenotype), and Nvy and Da were found to physically interact. These findings are consistent with a recent report showing that ETO directly interacts with HEB, a bHLH factor in the same class as Da. The domain through which ETO interacts with HEB (and other mammalian class I bHLH transcription factors) is conserved in Nvy, and HEB's ETO interaction domain is found in Da. These data lead to a proposal that Nvy, a presumptive transcriptional repressor, has the ability to function with Ac/Da and Sc/Da heterodimers to repress the transcription of some target genes. In the absence of Nvy, such as in the non-SOP cells of a PNC, Ac/Da and Sc/Da may have the potential to activate these same target genes. However, these experiments also suggest that the interaction between Nvy and Da may not be required for all of Nvy's functions because VP16-Nvy is able to lower Dl levels even in da mutant clones. One potential explanation for this Da-independent function is that Nvy may be able to directly interact with DNA. In summary, it is speculated that the Nvy–Da interaction is only required for the regulation of a subset of target genes (Wildonger, 2005).

The proposal that Nvy works with Ac/Da and Sc/Da to repress target genes may on the surface seem at odds with the suggestion that Nvy can transiently increase the levels of Dl, because it is thought that Ac/Da and Sc/Da heterodimers activate Dl expression in the SOP. However, it is not known if Dl levels are in fact directly increased by Ac/Sc. It is stressed that the timing of expression of these genes is critical to understanding how they function in vivo. Based on the wild type timing of its expression, nvy is likely to be a target of Ac/Sc in the presumptive SOP. Accordingly, there will be a window of time when Ac/Sc levels are high and Nvy levels are low in the presumptive SOP. This window of time may be sufficient for Ac/Sc to affect Dl expression and initiate the bias in favor of the SOP fate. Once Nvy levels increase, it may then work with Ac/Sc to repress the expression of some target genes, some of which may cause a further increase in Dl signaling. However, it is hypothesized that nvy's role in this process is after the bias has already been initiated (Wildonger, 2005).

In summary, it is suggested that Nvy plays a subtle but observable role in the establishment of the SOP fate. Although it is not essential for the SOP fate, it may be that Nvy helps the SOP/non-SOP bias by increasing the strength of the Dl signal sent by the SOP. Because nvy is evolutionarily conserved, both in its protein sequence and nervous system expression, it is suggested that this role, although subtle, is important for the stereotyped uniformity of mechanosensory organ development. In addition, nvy may also play a role in later stages of neurogenesis, in particular axon pathfinding. Because of Nvy's role as a transcriptional repressor, it is further suggested that Nvy increases the Dl signal indirectly, by repressing a gene (factor X) that normally inhibits Dl activity. Based on Nvy's ability to interact with Da, this hypothetical target may be repressed by Nvy in combination with Ac/Da and Sc/Da heterodimers. Interestingly, it follows that in non-SOP cells of the PNC, which express ac and sc but not nvy, this hypothetical target may continue to be expressed, helping to downregulate Dl activity in these cells and thereby further increase the SOP/non-SOP bias. Clearly, the test of this proposal requires the identification of factor X as well as a more detailed understanding of how Dl levels and activity are modulated in the SOP (Wildonger, 2005).

Acar, M., et al. (2006). Senseless physically interacts with proneural proteins and functions as a transcriptional co-activator. Development 133: 1979-1989. PubMed ID: 16624856

Alifragis, P., et al. (1997). A network of interacting transcriptional regulators involved in Drosophila neural fate specification revealed by the yeast two-hybrid system. Proc. Natl. Acad. Sci. 94(24): 13099-13104. PubMed ID: 9371806

Bardin, A. J., et al. (2010). Transcriptional control of stem cell maintenance in the Drosophila intestine. Development 137(5): 705-14. PubMed ID: 20147375

Barndt, R. J., Dai, M. and Zhuang, Y. (2000). Functions of E2A-HEB heterodimers in T-cell development revealed by a dominant negative mutation of HEB. Mol. Cell Biol. 20: 6677-6685. PubMed ID: 10958665

Brown, N. L., et al. (1996). daughterless is required for Drosophila photoreceptor cell determination, eye morphogenesis, and cell cycle progression. Dev. Biol. 179: 65-78. PubMed ID: 8873754

Buszczak, M., Paterno, S. and Spradling, A. C. (2009). Drosophila stem cells share a common requirement for the histone H2B ubiquitin protease scrawny. Science 323: 248-251. PubMed ID: 19039105

Cadigan, K. M., Jou, A. D. and Nusse, R. (2002). Wingless blocks bristle formation and morphogenetic furrow progression in the eye through repression of Daughterless. Development 129: 3393-3402. PubMed ID: 12091309

Castanon, I., et al. (2001). Dimerization partners determine the activity of the Twist bHLH protein during Drosophila mesoderm development. Development 128: 3145-3159. PubMed ID: 11688563

Caudy, M., Vaessin, H., Brand, M., Tuma, R., Jan, L.Y. and Jan, Y.N. (1988). daughterless, a gene essential for both neurogenesis and sex determination in Drosophila has sequence similarities to myc and the achaete-scute complex. Cell 55: 1061-1067. PubMed ID: 3203380

Cave, J. W., Loh, F., Surpris, J. W., Xia, L. and Caudy, M. A. (2005). A DNA transcription code for cell-specific gene activation by notch signaling. Curr. Biol. 15(2): 94-104. PubMed ID: 15668164

Cave, J. W., Xia, L. and Caudy, M. A. (2009). The Daughterless N-terminus directly mediates synergistic interactions with Notch transcription complexes via the SPS+A DNA transcription code. BMC Res. Notes 2: 65. PubMed ID: 19400956

Chandrasekaran, V. and Beckendorf, S. K. (2003). senseless is necessary for the survival of embryonic salivary glands in Drosophila. Development 130: 4719-4728. PubMed ID: 12925597

Cisse, B., et al. (2008). Transcription factor E2-2 is an essential and specific regulator of plasmacytoid dendritic cell development. Cell 135: 37-48. PubMed ID: 18854153

Cline, T.W. (1976). A sex-specific, temperature-sensitive maternal effect of the daughterless mutation of Drosophila melanogaster. Genetics 84: 723-742. PubMed ID: 827461

Corsi, A. C., et al. (2002). Characterization of a dominant negative C. elegans Twist mutant protein with implications for human Saethre-Chotzen syndrome. Development 129: 2761-2772. PubMed ID: 12015302

Cronmiller, C. and Cline, T.W. (1986). The relationship of relative gene dose to the complex phenotype of the daughterless locus of Drosophila. Dev. Genet. 7: 205-221. PubMed ID: 3453784

Cronmiller, C., Schedl, P. and Cline, T.W. (1988). Molecular characterization of the daughterless, a Drosophila sex determination gene with mutiple roles in development. Genes & Dev. 2: 1666-76. PubMed ID: 2850968

Cummings, C.A. and Cronmiller, C. (1994). The daughterless gene functions together with Notch and Delta in the control of ovarian follicle development in Drosophila. Development 120(2): 381-94. PubMed ID: 8149916

Deleuze, V., et al. (2007). TAL-1/SCL and its partners E47 and LMO2 up-regulate VE-cadherin expression in endothelial cells. Mol. Cell. Biol. 27: 2687-2697. PubMed ID: 17242194

Distefano, G. M., et al. (2012). Drosophila lilliputian is required for proneural gene expression in retinal development. Dev. Dyn. 241(3): 553-62. PubMed ID: 22275119

Frank, C. A., Baum, P. D. and Garriga, G. (2003). HLH-14 is a C. elegans Achaete-Scute protein that promotes neurogenesis through asymmetric cell division. Development 130: 6507-6518. PubMed ID: 14627726

Garcia-Bellido, A. and Santamaria, P. (1978). Developmental analysis of the achaete-scute system of Drosophila melanogaster. Genetics 91: 469-486. PubMed ID: 17248807

German, M. S., Wang, J., Chadwick, R. B. and Rutter, W. J. (2002). Synergistic activation of the insulin gene by a LIM-homeo domain protein and a basic helix-loop-helix protein: building a functional insulin minienhancer complex. Genes Dev. 6(11): 2165-76. PubMed ID: 1358758

Giebel, B., et al. (1997). Lethal of Scute requires overexpression of daughterless to elicit ectopic neuronal development during embryogenesis in Drosophila. Mech. Dev. 63 (1): 75-87. PubMed ID: 9178258

Gonzalez-Crespo, S. and Levine, M. (1993). Interactions between dorsal and helix-loop-helix proteins initiate the differentiation of the embryonic mesoderm and neuroectoderm in Drosophila. Genes Dev. 7(9): 1703-13. PubMed ID: 8370521

Goulding, S. E., zur Lage, P. and Jarman, A. P. (2000). amos, a proneural gene for Drosophila olfactory sense organs that is regulated by lozenge. Neuron 25: 69-78. PubMed ID: 10707973

Hassan, B. and Vaessin, H. (1997). Daughterless is required for the expression of cell cycle genes in peripheral nervous system precursors of Drosophila embryos. Dev. Genet. 21(2): 117-122. PubMed ID: 9332970

Hebrok, M., Fuchtbauer, A., and Fuchtbauer, E. M. (1997). Repression of muscle-specific gene activation by the murine Twist protein. Exp. Cell Res. 232(2): 295-303. PubMed ID: 9168805

Helsel, A. R., Yang, Q. E., Oatley, M. J., Lord, T., Sablitzky, F. and Oatley, J. M. (2017). ID4 levels dictate the stem cell state in mouse spermatogonia. Development. PubMed ID: 28087628

Hoshijima, K., et al. (1995). Transcriptional regulation of the Sex-lethal gene by helix-loop-helix proteins. Nucleic Acids Res. 23: 3441-3448. PubMed ID: 7567454

Huang, M.-L., Hsu, C.-H. and Chien, C.-H. (2000). The proneural gene amos promotes multiple dendritic neuron formation in the Drosophila peripheral nervous system. Neuron 25: 57-67. PubMed ID: 10707972.

Hwang, B. J. and Sternberg, P. W. (2004). A cell-specific enhancer that specifies lin-3 expression in the C. elegans anchor cell for vulval development. Development 131: 143-151. 14660442

Jafar-Nejad, H., Tien, A. C., Acar, M. and Bellen, H. J. (2006). Senseless and Daughterless confer neuronal identity to epithelial cells in the Drosophila wing margin. Development 133(9): 1683-92. PubMed ID: 16554363

Jarman, A.P., Grau, Y., Jan, L.Y. and Jan, Y.N. (1993). atonal is a proneural gene that directs chordotonal organ formation in the Drosophila peripheral nervous system. Cell 73(7): 1307-1321. PubMed ID: 8324823

Kovalick, G.E. and Beckingham, K. (1992).Calmodulin transcription is limited to the nervous system during Drosophila embryogenesis. Dev. Biol. 150(1): 33-46. PubMed ID: 15002679

Johnson, J. D., et al. (1997). Transcriptional synergy between LIM-homeodomain proteins and basic helix-loop-helix proteins: the LIM2 domain determines specificity. Mol. Cell. Biol. (7): 3488-3496. PubMed ID: 9199284

Karp, X. and Greenwald, I. (2003). Post-transcriptional regulation of the E/Daughterless ortholog HLH-2, negative feedback, and birth order bias during the AC/VU decision in C. elegans. Genes Dev. 17: 3100-3111. PubMed ID: 14701877

Kennedy, A. J., Rahn, E. J., Paulukaitis, B. S., Savell, K. E., Kordasiewicz, H. B., Wang, J., Lewis, J. W., Posey, J., Strange, S. K., Guzman-Karlsson, M. C., Phillips, S. E., Decker, K., Motley, S. T., Swayze, E. E., Ecker, D. J., Michael, T. P., Day, J. J. and Sweatt, J. D. (2016). Tcf4 regulates synaptic plasticity, DNA methylation, and memory function. Cell Rep 16: 2666-2685. PubMed ID: 27568567

Kong, Y., et al. (1997). Muscle LIM protein promotes myogenesis by enhancing the activity of MyoD. Mol. Cell. Biol. 17(8): 4750-4760. PubMed ID: 9234731

Kophengnavong, T., Michnowicz, J. E. and Blackwell, T. K. (2000). Establishment of distinct MyoD, E2A, and twist DNA binding specificities by different basic region-DNA conformations. Mol. Cell. Biol. 20(1): 261-72. PubMed ID: 10594029

Krause, M., et al. (1997). A C. elegans E/Daughterless bHLH protein marks neuronal but not striated muscle development. Development 124 (11): 2179-2189. PubMed ID: 9187144

LeBrun D. P., et al. (1997). The chimeric oncoproteins E2A-PBX1 and E2A-HLF are concentrated within spherical nuclear domains. Oncogene 15(17): 2059-2067. PubMed ID: 9366523

Lahlil, R., et al. (2004). SCL assembles a multifactorial complex that determines glycophorin A expression. Mol. Cell. Biol. 24: 1439-1452. PubMed ID: 14749362

Lécuyer, E., et al. (2002). The SCL complex regulates c-kit expression in hematopoietic cells through functional interaction with Sp1. Blood 100: 2430-2440. PubMed ID: 12239153

Lim, J., Jafar-Nejad, H., Hsu, Y. C. and Choi, K. W. (2008). Novel function of the class I bHLH protein Daughterless in the negative regulation of proneural gene expression in the Drosophila eye. EMBO Rep. 9(11): 1128-33. PubMed ID: 18758436

Lu, M., Seufert, J. and Habener, J. F. (1997). Pancreatic beta-cell-specific repression of insulin gene transcription by CCAAT/Enhancer-binding protein beta. inhibitory interactions with basic helix-loop-helix transcription factor e47. J. Biol. Chem. 272(45): 28349-28359. PubMed ID: 9353292

Markus, M., Du, Z. and Benezra, R. (2002). Enhancer-specific modulation of E protein activity, J. Biol. Chem. 277: 6469-6477. PubMed ID: 11724804

Massari, M. E., et al. (1999). A conserved motif present in a class of helix-loop-helix proteins activates transcription by direct recruitment of the SAGA complex. Mol. Cell 4: 63-73. PubMed ID: 10445028

Misquitta, L. and Paterson, B. M. (1999). Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): A role for nautilus in embryonic somatic muscle formation. Proc. Natl. Acad. Sci. 96(4): 1451-6. PubMed ID: 9990044

Nolo, R., Abbott, L. A. and Bellen, H. J. (2000), Senseless, a Zn finger transcription factor, is necessary and sufficient for sensory organ development in Drosophila. Cell. 102(3): 349-62. PubMed ID: 10975525

Oellers, N., Dehio, M. and Knust, E. (1994). bHLH protein encoded by the Enhancer of split complex of Drosophila negatively interfere with transcriptional activation mediated by proneural genes. Mol. Gen. Genet. 244(5): 465-73. PubMed ID: 8078474

Ordentlich, P., et al. (1998). Notch inhibition of E47 supports the existence of a novel signaling pathway. Mol. Cell. Biol. 18(4): 2230-2239. PubMed ID: 9528794

Portman, D. S. and Emmons, S. W. (2000). The basic helix-loop-helix transcription factors LIN-32 and HLH-2 function together in multiple steps of a C. elegans neuronal sublineage. Development 127: 5415-5426. PubMed ID: 11076762

Quong, M.W., Massari, M.E., Zwart, R. and Murre, C. (1993). A new transcriptional-activation motif restricted to a class of helix-loop-helix proteins is functionally conserved in both yeast and mammalian cells. Mol. Cell Biol. 13(2): 792-800. PubMed ID: 8423802

Ramain, P., et al. (2000). Interactions between Chip and the Achaete/Scute-Daughterless heterodimers are required for Pannier-driven proneural patterning. Mol. Cell 6: 781-790. PubMed ID: 21000488

Roberts, V.J., Steenburgen, R. and Murre, C. (1993). Localization of E2A mRNA expression in developing and adult rat tissues. Proc. Natl. Acad. Sci. 90(16): 7583-87. PubMed ID:

Romanow, W. J., Langerak, A. W., Goebel, P., Wolvers-Tettero, I. L., van Dongen, J. J., Feeney, A. J. and Murre, C. (2000). E2A and EBF act in synergy with the V(D)J recombinase to generate a diverse immunoglobulin repertoire in nonlymphoid cells. Mol. Cell 5: 343-353. PubMed ID: 10882075

Sallee, M. D., Littleford, H. E. and Greenwald, I. (2017). A bHLH code for sexually dimorphic form and function of the C. elegans somatic gonad. Curr Biol 27(12): 1853-1860 e1855. PubMed ID: 28602651

Sharma, A., Henderson, E., Gamer, L., Zhuang, Y. and Stein, R. (1997). Analysis of the role of E2A-encoded proteins in insulin gene transcription. Mol. Endocrinol. 11: 1608-1617. PubMed ID: 9328343

Shirakata, M. and Paterson, B. M. (1995). The E12 inhibitory domain prevents homodimer formation and facilitates selective heterodimerization with the MyoD family of gene regulatory factors. EMBO J 14: 1766-1772

Smith, J. E. and Cronmiller C. (2001). The Drosophila daughterless gene autoregulates and is controlled by both positive and negative cis regulation. Development 128: 4705-4714. PubMed ID: 11731451

Smith, J. E., Cummings, C. A. and Cronmiller, C. (2002). daughterless coordinates somatic cell proliferation, differentiation and germline cyst survival during follicle formation in Drosophila. Development 129: 3255-3267. PubMed ID: 12070099

Tamberg, L., Sepp, M., Timmusk, T. and Palgi, M. (2015). Introducing Pitt-Hopkins syndrome-associated mutations of TCF4 to Drosophila daughterless. Biol Open 4: 1762-1771. PubMed ID: 26621827

Thellmann, M., Hatzold, J. and Conradt, B. (2003). The Snail-like CES-1 protein of C. elegans can block the expression of the BH3-only cell-death activator gene egl-1 by antagonizing the function of bHLH proteins. Development 130: 4057-4071. PubMed ID: 12874127

Togel, M., Meyer, H., Lehmacher, C., Heinisch, J. J., Pass, G., Paululat, A. (2013). The bHLH transcription factor Hand is required for proper wing heart formation in Drosophila. Dev Biol 381: 446-459. PubMed ID: 23747982

Vaessin, H., Brand, M., Jan, L.Y. and Jan, Y.N. (1994). daughterless is essential for neuronal precursor differentiation but not for initiation of neuronal precursor formation in Drosophila embryo. Development 120: 935-945. PubMed ID: 7600969

van der Flier L. G., et al. (2009). Transcription factor achaete scute-like 2 controls intestinal stem cell fate. Cell 136: 903-912. PubMed ID: 19269367

van Doren, M., Ellis, H.M. and Posakony, J.W. (1991). The Drosophila extramachrochaetae protein antagonizes sequence-specific DNA binding by daughterless/achaete-scute protein complexes. Development 113(1): 245-55. PubMed ID: 1764999

Wadman, I. A., et al. (1997). The LIM-only protein Lmo2 is a bridging molecule assembling an erythroid, DNA-binding complex which includes the TAL1, E47, GATA-1 and Ldb1/NLI proteins. EMBO J (11):3145-3157. PubMed ID: 9214632

Wesley, C. S. and Saez, L. (2000). Analysis of Notch lacking the carboxyl terminus identified in Drosophila embryos. J. Cell Biol. 149: 683-696. PubMed ID: 10791981

Wildonger, J. and Mann, R. S. (2005). Evidence that nervy, the Drosophila homolog of ETO/MTG8, promotes mechanosensory organ development by enhancing Notch signaling. Dev. Biol. 286(2): 507-20. PubMed ID: 16168983

Younger-Shepherd, S., Vassin, H., Bier, E., Jan, L.Y. and Jan, Y.N. (1992). deadpan, an essential pan-neural gene encoding an HLH protein, acts as a denominator in Drosophila sex determination Cell 70: 911-922. PubMed ID: 1525829

Wang, L.H. and Baker, N.E. (2015). Salvador-Warts-Hippo pathway in a developmental checkpoint monitoring Helix-Loop-Helix proteins. Dev Cell 32(2):191-202. PubMed ID: 25579975

Wang, L. H. and Baker, N. E. (2018). Spatial regulation of expanded transcription in the Drosophila wing imaginal disc. PLoS One 13(7): e0201317. PubMed ID: 30063727

Wei, Q., Marchler, G., Edington, K., Karsch-Mizrachi, I. and Paterson, B. M. (2000). RNA interference demonstrates a role for nautilus in the myogenic conversion of Schneider cells by daughterless. Dev. Bio. 228: 239-255. PubMed ID: 11112327

Wong, M. C., Castanon, I. and Baylies, M. K. (2008). Daughterless dictates Twist activity in a context-dependent manner during somatic myogenesis. Dev. Biol. 317(2): 417-29. PubMed ID: 18407256

Wulbeck, C., Fromental-Ramain, C. and Campos-Ortega, J.A. (1994). The HLH domain of a zebrafish HE12 homolog can partially substitute for functions of the HLH domain of Drosophila daughterless. Mech. Dev. 46(2): 73-85. PubMed ID: 7918099

Yan, W., et al. (1997). High incidence of T-cell tumors in E2A-null mice and E2A/Id1 double-knockout mice. Mol. Cell. Biol. 17(12): 7317-7327. PubMed ID: 9372963

Yang, D., et al. (2001). Interpretation of X chromosome dose at Sex-lethal requires non-E-Box sites for the basic helix-loop-helix proteins SISB and Daughterless. Mol. Cell. Bio. 21: 1581-1592. PubMed ID: 11238895

Yang, Z., et al. (2009). MyoD and E-protein heterodimers switch rhabdomyosarcoma cells from an arrested myoblast phase to a differentiated state. Genes Dev. 23(6): 694-707. PubMed ID: 19299559

Yasugi, T., Fischer, A., Jiang, Y., Reichert, H., Knoblich, J. A. (2014). A regulatory transcriptional loop controls proliferation and differentiation in Drosophila neural stem cells. PLoS One 9: e97034. PubMed ID: 24804774

Yoon, S. J., et al. (2011). HEB and E2A function as SMAD/FOXH1 cofactors. Genes Dev. 25(15): 1654-61. PubMed ID: 21828274

Zarifi, I., et al. (2012). Essential roles of Da transactivation domains in neurogenesis and in E(spl)-mediated repression. Mol. Cell Biol. 32(22): 4534-48. PubMed ID: 22949507

Zenvirt, S., Nevo-Caspi, Y., Rencus-Lazar, S. and Segal, D. (2008). Drosophila LIM-only is a positive regulator of transcription during thoracic bristle development. Genetics 179(4): 1989-99. PubMed ID: 18689881

Zhang, J., et al. (2004). E protein silencing by the leukemogenic AML1-ETO fusion protein. Science 305: 1286-1289. PubMed ID: 15333839

Zhao, F., et al. (2001). Promotion of cell cycle progression by basic helix-loop-helix E2A. Mol. Cell. Bio.. 21: 6346-6357. PubMed ID: 11509675

date revised: 2 December 2018

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