InteractiveFly: GeneBrief

distal antenna & distal antenna-related: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - distal antenna & distal antenna-related

Synonyms - Fernandez (Dan) and Hernandez (Danr)

Cytological map position - 96C4 & 96C2

Function - presumed transcription factor

Keywords - antenna

Symbol - dan & danr

FlyBase ID: FBgn0039286 & FBgn0039283

Genetic map position -

Classification - pipsqueak domain proteins

Cellular location - nuclear

NCBI links for dan: Precomputed BLAST | Entrez Gene

NCBI links for for danr: Precomputed BLAST | Entrez Gene
Recent literature
Transcriptional profiling of olfactory system development identifies distal antenna as a regulator of subset of neuronal fates. Barish, S., Li, Q., Pan, J. W., Soeder, C., Jones, C. and Volkan, P. C. (2017). . Sci Rep 7: 40873. PubMed ID: 28102318
Drosophila uses 50 different olfactory receptor neuron (ORN) classes that are clustered within distinct sensilla subtypes to decipher their chemical environment. Each sensilla subtype houses 1-4 ORN identities that arise through asymmetric divisions of a single sensory organ precursor (SOP). Despite a number of mutational studies investigating the regulation of ORN development, a majority of the transcriptional programs that lead to the different ORN classes in the developing olfactory system are unknown. This study used transcriptional profiling across the time series of antennal development to identify novel transcriptional programs governing the differentiation of ORNs. Four critical developmental stages of the olfactory system were profiled: 3rd instar larval (prepatterning), 8 hours after puparium formation (APF, SOP selection), 40 hrs APF (neurogenesis), and adult antennae. Focus was placed on the expression profiles of olfactory receptor genes and transcription factors-the two main classes of genes that regulate the sensory identity of ORNs. Distinct clusters of genes were identified that have overlapping temporal expression profiles suggesting they have a key role during olfactory system development. The expression of the transcription factor distal antenna (dan) is highly similar to other prepatterning factors and is required for the expression of a subset of ORs.

Legs and antennae are considered to be homologous appendages. The fundamental patterning mechanisms that organize spatial pattern are conserved, yet appendages with very different morphology develop. The distal antenna (dan) and distal antenna-related (danr) genes encode novel 'pipsqueak' motif nuclear proteins that probably function as DNA binding proteins serving as sequence-specific transcription factors but may serve instead as more general chromatin modification factors. dan and danr are expressed in the presumptive distal antenna, but not in the leg imaginal disc. Ectopic expression of dan or danr causes partial transformation of distal leg structure toward antennal identity. Mutants that remove dan and danr activity cause partial transformation of antenna toward leg identity. Therefore it is suggested that dan and danr contribute to differentiation of antenna-specific characteristics. Antenna-specific expression of dan and danr depends on a regulatory hierarchy involving homothorax and Distal-less, as well as cut and spineless. It is proposed that dan and danr are effector genes that act downstream of these genes to control differentiation of distal antennal structures (Emerald, 2003).

dan and danr were identified in a large-scale modular misexpression screen of ~8500 EPg elements, as insertions that caused abnormalities in wing development when expressed under sdGal4 control and small rough eyes when expressed under eyGal4 control. Expression of dan using EPg J3-220, under control of DllGal4 causes transformation of distal leg structures toward distal antennal identity. The claws, found on the tip of the fifth tarsal segment, are transformed into the distal-most part of the antenna, the arista. In addition, there is some overall morphological abnormality of the tarsal region. Expression of danr from EPg35635 using DllGal4 causes loss of the claws but does not produce an overt transformation to arista (Emerald, 2003).

EPg J3-220 and EPg 35635 lie approximately 45 kb apart on chromosome 3R, 263 bp and 39 bp upstream of the predicted genes, CG11849 and CG13651. To verify that the predicted genes tagged by the EP-element insertions were responsible for the observed overexpression phenotypes, UAS-dan and UAS-danr transgenic strains were generated. Six independent UAS-dan transformants and five independent UAS-danr transformants were tested and found to be lethal when expressed with the DllGal4 driver. However when expressed using dppGal4, UAS-dan and UAS-danr showed distal leg to antenna transformation. Molecular markers of antennal identity were also examined in the imaginal discs. The zinc-finger protein Spalt is expressed in antenna, but not in leg discs. Ectopic expression of dan can induce limited expression of Spalt in the leg disc, consistent with the observed transformation toward antennal identity. These observations suggested a role for dan and danr in specification of the identity of distal antennal structures (Emerald, 2003).

Dan plays an important role in specification of antennal identity downstream of spineless (ss), but rescue of the spineless mutation by Dan suggests that there may be additional genes acting downstream from ss to specify antennal identity. spineless mutants lead to ectopic expression of Antennapedia and concomitant loss of Dan/Danr expression and cause a strong phenotypic transformation of distal A3 and arista to tarsus. To determine whether morphological transformation depends on loss of Dan/Danr, use was made of Gal4 to direct Dan expression in the ss mutant discs. ptcGal4 directed expression of Dan causes strong suppression of the arista-to-tarsus transformation in the ss mutant antenna. ptcGal4 is expressed in a stripe of cells adjacent to the AP boundary in the antenna region of the disc. Dan expression does not repress ectopic expression of Antp in the ptcGal4 stripe of the mutant discs. This suggests that Dan can direct antennal differentiation in the presence of Antp, and overcome the ability of Antp to cause transformation to tarsus. Remarkably, this transformation can affect the entire distal arista, even though ectopic Dan is expressed in only a subset of Antp-expressing cells. These observations suggest that Dan plays an important role in specification of antennal identity (Emerald, 2003).

Both Dan and Danr contribute to specification of antennal identity. danr single mutants produce a partial transformation of arista to tarsus. A similar, but slightly stronger phenotype results when both dan and danr are deleted. Reduced dan activity in the danems3 mutants or reduced Dan expression caused by RNA interference produces a milder version of the same phenotype (Emerald, 2003).

An additional line of evidence to indicate that both genes contribute to distal antenna identity comes from examining genetic interactions with spinelessaristapedia. ssa mutants lose Dan/Danr expression and express Antennapedia ectopically in the antenna disc. Restoring Dan expression is able to partially suppress the transformation to antenna, implicating Dan as an effector of ssa function. The consequences of removing one copy each of Dan and Danr was examined in a ss mutant background. The spineless114.4 allele shows a mild transformation of the basal capsule of the arista when heterozygous, suggesting that the reduced level of ss activity in this allele is not sufficient to support normal development. Removing one copy of danr using the danrex35 deletion in this background causes a modest increase in the size of the basal capsule and in the number of ectopic bristles. The dan danrex56 deletion causes a stronger phenotype, with the basal capsule adopting a two-segment structure with multiple bracted bristles and obvious tarsal morphology. Flies heterozygous for the dan danrex56 deletion are morphologically normal. Thus, reduction of both Dan and Danr gene dose leads to a more severe phenotype under conditions where ss activity is limiting. Even more extreme arista transformation phenotypes are observed when one copy of ss is removed in animals homozygous for the dan danrex56 deletion (Emerald, 2003).

Genetic interactions were also observed with Dll. Double heterozygotes for danr and Dll or dan danr and Dll show ectopic tarsal bristles in the basal capsule of the arista (in this case the phenotypes are similar in strength). The ss/dan danr double heterozygotes produces a more complete transformation phenotype than the dan danr homozygous mutants. This raises the possibility that there may be additional genes acting downstream from ss to specify antennal identity (Emerald, 2003).

Insect antennae develop in the absence of input from HOM-C genes. In the anterior head of Drosophila where Antennapedia-complex and Bithorax complex genes are not expressed, expression of hth and Dll overlaps and promotes antenna development. One consequence of overlapping expression of Dll and Hth is sustained expression of ss in the distal antenna. ss is expressed in the leg and antenna discs in second instar, but its expression is not maintained in the leg (Emerald, 2003).

Loss of Hth activity has been shown to cause transformation of arista to tarsus, presumably because of loss of ss. It has been suggested that uniform expression of Hth in second and early third instar antennae might be responsible for its role in specification of distal antenna identity. However, the results of this study indicate that Hth can have a non-autonomous effect on the expression of Dan in the antenna. Hth-expressing cells sort out from the distal part of the leg. Nonetheless they are able to induce Dan expression in cells that remain integrated in the distal leg. This observation is best explained by a non-autonomous induction of Dan in response to a signal from Hth-expressing cells. Responsiveness to this signal apparently requires Dll, which limits it to the distal region. These effects are presumably mediated by regulation of ss, which is required for Dan and Danr expression. These observations provide an explanation for the apparently non-autonomous role of Hth together with Dll in the distal antenna (Emerald, 2003).

ss is also required to induce Dan and Danr and to repress Antp expression. Repression of Antennapedia may be mediated in part by repression of Cut. The findings described above implicate Dan and Danr as downstream effectors of ss that promote development of distal antennal structures. Remarkably, expression of Dan or Danr under Gal4 control can restore antenna development and prevent transformation of antenna to leg in the ss mutant, even when Antp is present. A striking feature of these results is that there appears to be non-autonomous activity. Transformation is blocked in cells expressing Dan and Danr, as well as in nearby cells that do not express these proteins. The identity of the genes responsible for these non-autonomous effects in antenna specification remains to be determined. In view of recent reports of non-autonomous effects of vein/EGFR signaling in development of distal leg pattern, it will be of interest to learn if there is a link to this pathway in the non-autonomous effects of Dan and Danr (Emerald, 2003).

What are the molecular functions of Dan and Danr? The 'pipsqueak motifs' found near the N terminus of Dan and Danr are most closely related to those in the 'transposase group' of pipsqueak motif proteins, which includes the Pogo transposase and human centromere protein B (CENP-B). The pipsqueak motifs of both Pogo and CENP-B are DNA-binding domains. NMR-spectroscopy of the CENP-B pipsqueak motif demonstrates that it has a helix-turn-helix structure. Interestingly, the glutamate residue that has been transformed to a lysine in the pipsqueak motif of the danems3 allele lies at the same position as the arginine that is required for DNA binding by the Pogo pipsqueak motif. Thus, the danems3 mutation may interfere with Dan function by abrogating the ability of Dan to bind DNA, or changing its specificity. Although these data suggest that Dan/Danr bind to DNA, it remains unclear whether they act as sequence-specific transcription factors or more general chromatin modification factors. Biochemical analysis of Dan/Danr and identification of interacting proteins will be required to address this issue in detail (Emerald, 2003).

The pipsqueak-domain proteins Distal antenna and Distal antenna-related restrict Hunchback neuroblast expression and early-born neuronal identity

A fundamental question in brain development is how precursor cells generate a diverse group of neural progeny in an ordered manner. Drosophila neuroblasts sequentially express the transcription factors Hunchback (Hb), Krüppel (Kr), Pdm1/Pdm2 (Pdm) and Castor (Cas). Hb is necessary and sufficient to specify early-born temporal identity and, thus, Hb downregulation is essential for specification of later-born progeny. This study shows that distal antenna (dan) and distal antenna-related (danr), encoding pipsqueak motif DNA-binding domain protein family members, are detected in all neuroblasts during the Hb-to-Cas expression window. dan and danr were identified in a forward genetic screen of ~100 second and third chromosomal deficiency lines for mutants that had altered numbers of Even-skipped (Eve)+ early-born neurons. Dan and Danr are required for timely downregulation of Hb in neuroblasts and for limiting the number of early-born neurons. Dan and Danr function independently of Seven-up (Svp), an orphan nuclear receptor known to repress Hb expression in neuroblasts, because Dan, Danr and Svp do not regulate each other and dan danr svp triple mutants have increased early-born neurons compared with either dan danr or svp mutants. Interestingly, misexpression of Hb can induce Dan and Svp expression in neuroblasts, suggesting that Hb might activate a negative feedback loop to limit its own expression. It is concluded that Dan/Danr and Svp act in parallel pathways to limit Hb expression and allow neuroblasts to transition from making early-born neurons to late-born neurons at the proper time (Kohwi, 2011).

Dan and Danr are required to limit Hb expression in neuroblasts and restrict the number of early-born neurons generated in multiple neuroblast lineages. The orphan nuclear hormone receptor protein Svp also functions to limit Hb expression in neuroblasts, and the current data strongly suggest that Dan/Danr and Svp function in parallel pathways that are each independently required. First, the temporal expression patterns of Dan and Danr versus Svp do not suggest their coordinated activity: Dan and Danr are expressed from the time of neuroblast formation (stage 9), beyond Hb downregulation (stage 10), until the time of strong Castor expression (stage 12). By contrast, Svp protein is very transiently detected in neuroblasts only at the onset of Hb downregulation. Second, Dan/Danr and Svp are not in a linear transcriptional hierarchy: neither mutant affects expression of the other gene. Third, dan danr and svp mutants have distinct phenotypes: for example, compared with the dan danr mutant, the svp mutant has many more early-born neurons in the NB7-1 lineage, whereas it does not have any extra early-born neurons in the NB1-1 lineage. Fourth, the dan danr svp null triple mutant has the summed phenotypes of the dan danr double mutant and the svp single mutant. Fifth, misexpression of Svp, but not Dan, can repress hb transcription in neuroblasts. The fact that neither one appears to have an effect on cell fate when misexpressed in postmitotic neurons suggest that both Svp and Dan function at the level of the mitotic precursors. Taken together, it appears that Dan/Danr and Svp are each required to downregulate hb expression in neuroblasts, but do so using separate mechanisms. The data are consistent with Svp directly repressing neuroblast hb transcription (although this has not been shown) whereas Dan and Danr act more indirectly (Kohwi, 2011).

Do Dan, Danr and Svp have lineage-specific functions? Despite the widespread expression of Dan and Danr in early neuroblasts and the widespread transient expression of Svp in most neuroblasts, it is likely that each has lineage-specific functions. For example, in the NB1-1 lineage, ectopic early-born neurons are generated in dan danr mutants, but not in svp mutants. Further comparing Dan versus Danr in this lineage, it appears that Danr is more important than Dan, because the danrex35 single mutant phenocopies the dan danrex56 double mutant in the number of ectopic aCC/pCC neurons generated and the number of hemisegments affected per embryo. In contrast to the NB1-1 lineage, Dan and Danr each appear to be required for limiting the number of early-born neurons in the NB7-1 lineage, as danrex35 single mutants had a weaker phenotype than the dan danr double mutant. Additionally, there are more early-born neurons in the NB7-1 lineage in svp mutants than in dan danr mutants, highlighting their lineage-specific differences. These differences might be due to different levels or functions of each protein in distinct neuroblasts. For example, there is variability in dan and danr mRNA levels between neuroblasts, suggesting that distinct neuroblasts might have different levels of Dan and/or Danr protein (although Dan protein levels appear constant between newly formed neuroblasts), or that they express Dan and/or Danr protein for different durations. Alternatively, or in addition, the lineage-specific variation might be due to unique cofactors present in different neuroblasts. This seems likely, as Hb misexpression in all neuroblasts has varying effects within different lineages. For example, NB1-1 generates only one to three ectopic early-born neurons in response to Hb misexpression, whereas NB7-1 generates ~20 ectopic early-born neurons. Consistent with the notion that co-factors can alter the functional output of transcriptional regulators, recent evidence shows that the co-regulator CtBP forms complexes with distinct eye specification factors, including Dan and Danr, to regulate proliferation versus differentiation during eye development in Drosophila (Kohwi, 2011).

Do Dan and Danr function redundantly? This hypothesis could not be rigorously tested owing to the lack of a dan null single mutant, but the available evidence suggests that they do have redundant functions. First, they have nearly identical expression patterns. But most crucially, overexpression of Dan in the dan danr double mutant can nearly completely rescue the CNS phenotype, suggesting that high enough levels of Dan can compensate for loss of Danr. However, a danr null single mutant shows a strong phenotype in the NB1-1 lineage and a partial phenotype in the NB7-1 lineage, suggesting that endogenous levels of Dan are insufficient for normal CNS development. The most parsimonious explanation is that each protein has equivalent function, but that both genes are required to generate sufficient levels of Dan/Danr protein (Kohwi, 2011).

Hb overexpression can activate expression of both Dan and Svp. What is the significance of this activation? Previous work has shown that Hb can function both as a transcriptional activator and a repressor. Although its repressive functions are required for the neuroblast to specify early-born fates and maintain neuroblast competence, its activator functions remain elusive. One possibility is that Hb-mediated activation of Svp, and the subsequent Svp-mediated downregulation of Hb, create a negative feedback loop to ensure timely progression of the neuroblast to later temporal fates. This is not unlike what has been observed for Cas, which activates feed-forward and feed-back transcriptional cascades to regulate temporal identity in the NB5-6 lineage. By contrast, Hb activation of Dan expression might be part of the mechanism by which Hb maintains neuroblast competence, because Dan is unlikely to repress hb expression directly (Kohwi, 2011).

What might be the mechanism by which Dan and Danr function to restrict the duration of Hb expression in neuroblasts? Some clues might come from the fact that Dan and Danr are found in a subgroup of pipsqueak-domain containing nuclear proteins that have been proposed to regulate higher order chromatin structure by targeting distal DNA elements. Pipsqueak, the founding member of the family, has been shown to recruit Polycomb group complexes to specific regions of the genome to mediate gene silencing. Perhaps Dan and Danr modify chromatin structure through recruitment of chromatin remodeling complexes, which indirectly affects hb transcription by changing the accessibility of the hb locus to other transcriptional regulators. Such a function in modulating chromatin architecture might not be restricted to regulating just hb expression, but can extend to other temporal identity factors as well. Indeed, in NB7-1, the initial Hb->Cas 'competence window', during which the U1-U5 motor neurons are generated, matches nearly exactly the window of Dan and Danr expression. This raises the possibility that Dan and Danr might have a more global role in NB temporal progression by stabilizing 'transition states' between successive temporal identity factors (e.g. Hb->Kr, Kr->Pdm or Pdm->Cas). Such a function might explain the low frequency misregulation of later-born neuron numbers in several lineages (7-1, 3-1, 7-3), in addition to the extra early-born neurons phenotypes. Future experiments that address the role of Dan and Danr in later temporal identity transitions will provide a better understanding of the mechanisms that control the progression of temporal identity in neuroblasts (Kohwi, 2011).


Transcriptional Regulation

The overlap between Hth and Dll has been proposed to define antennal identity, because co-expression of the two proteins in ectopic locations can induce formation of ectopic arista structures in other discs. To ask whether Hth and Dll have a role in defining the non-overlapping expression domains of Cut and Dan/Danr, clones of cells were examined lacking hth or Dll activity in the antenna. Dan expression is lost in cells mutant for hthc1 in the region where the two expression domains overlap. This suggests that Hth activity is required for Dan expression. Likewise, clones of cells lacking Dll activity have lost Dan expression in the distal region of the disc. Many Dll mutant clones were found adjacent to the edge of the Dan domain, suggesting that loss of Dan may cause these clones to sort out proximally. Thus both Dll and Hth are required for Dan expression (Emerald, 2003).

Ectopic expression of Hth in the leg disc under dppGal4 control, induces Dan expression in distal, Dll-expressing cells. It is known that Hth-expressing cells sort out from the distal region of the disc. This is also visible in GFP labeled cells in this study. Nonetheless, dppGal4 directed expression of Hth induces Dan expression in distal cells. This raises the possibility of a non-autonomous effect of Hth expression leading to sustained Dan expression. Ectopic expression of Dll in the leg disc under dppGal4 control, induces Dan expression in proximal, Hth-expressing cells. In this case, ectopic Dan was limited to cells expressing the Gal4 driver (Emerald, 2003).

These observations indicate that the regulatory relationship between Hth, Dll and Dan (Danr) is complex. Dll and Hth are each required for Dan expression. However, it is clear that Dan is not expressed in every cell in which Hth and Dll are co-expressed in the antenna. All Dan-expressing distal antenna cells express Dll but not all express Hth. These observations point to a non-autonomous effect of Hth on Dan expression, which may explain how Hth can be required for sustained expression of Dan in distal cells where Hth is not expressed (Emerald, 2003).

ss is expressed in the distal antenna in a domain similar to that of Dan/Danr. ss mutants cause transformation of distal antenna to tarsus, suggesting a role in antennal identity. To determine whether ss regulates Dan and Danr, antenna discs were examined from spinelessaristapedia (ssa) mutants. ssa alleles appears to reduce ss activity specifically in the antenna. Dan expression was lost from the distal part of ssa mutant antennal discs. Loss of Dan expression in ssa mutant antenna discs coincides with ectopic expression of Antennapedia. These expression domains appear to be non-overlapping. Ectopic expression of Antennapedia is sufficient to cause transformation of antenna to leg. The observation of ectopic Antp in the distal part of the third antennal segment (within the Dan domain) may explain the homeotic transformation of distal A3 and arista in the ss mutant. It was next asked whether ss is sufficient to induce Dan and Danr expression in the leg disc. Ectopic expression of ss in randomly positioned clones of cells causes expression of Dan and Danr in the wing and leg discs. These observations suggest that ss defines the domain of Dan and Danr expression (Emerald, 2003).

Next, the relationship between ss and Cut was examined. Ectopic expression of ss using ptcGal4 causes ectopic expression of Dan and repression of Cut expression. Repression of Cut is stronger on one side of the disc and is associated with antenna duplication. Ectopic expression of Dan or Danr has no effect on Cut expression, suggesting that ss may act directly to regulate Cut. The ability of ss to repress Cut, contrasts with the observation that cut expression is normal in ss mutants. It is possible that there are redundant mechanisms for Cut repression, one of which is mediated by ss (Emerald, 2003).

To ask whether Cut regulates Dan and Danr, an examination was made of expression clones of cells lacking cut activity, generated using the null allele cut145 and the FLP-FRT system. cut145 mutant clones do not cause ectopic expression of Dan in proximal regions of the antenna disc, but rather show limited expansion of the Dan domain in the region where Dll is expressed. Comparable effects on Danr expression were seen in cut mutant clones. Ectopic expression of Cut in the Dan domain using Act>CD2>Gal4 causes repression of Dan (Emerald, 2003).

Taken together these results suggest that distal expression of ss limits the expression domain of Cut to the proximal antenna. ss is required for Dan and Danr expression in distal antenna. At present it is not possible to determine whether expression of Dan and Danr in response to ss is mediated directly or indirectly by repression of Cut. However, the view that it is direct is favored because removal of Cut does not cause extensive ectopic expression of Dan (Emerald, 2003).

Targets of Activity

Drosophila CtBP regulates proliferation and differentiation of eye precursors and complexes with Eyeless, Dachshund, Dan, and Danr during eye and antennal development

Specification factors regulate cell fate in part by interacting with transcriptional co-regulators like CtBP to regulate gene expression. This study demonstrates that CtBP forms a complex or complexes with the Drosophila Pax6 homolog Eyeless (Ey), and with Distal antenna (Dan), Distal antenna related (Danr), and Dachshund to promote eye and antennal specification. Phenotypic analysis together with molecular data indicate that CtBP interacts with Ey to prevent overproliferation of eye precursors. In contrast, CtBP;dan;danr triple mutant adult eyes have significantly fewer ommatidia than CtBP single or dan;danr double mutants, suggesting that the CtBP/Dan/Danr complex functions to recruit ommatidia from the eye precursor pool. Furthermore, CtBP single and to a greater extent CtBP;dan;danr triple mutants affect the establishment and maintenance of the R8 precursor, which is the founding ommatidial cell. Thus, CtBP interacts with different eye specification factors to regulate gene expression appropriate for proliferative vs. differentiative stages of eye development (Hoang, 2010).

The eyes of several CtBP loss-of-function mutant combinations have statistically significantly more ommatidia than wild-type eyes, and CtBP87De-10 clones ahead of the furrow show a statistically significant increase in the number of cells undergoing mitosis per unit area. These results suggest a role for CtBP in downregulating proliferation of eye precursors, although it cannot be currently exclude that additional processes such as apoptosis that might contribute to the increase in eye size in CtBP- mutants (Hoang, 2010).

The evidence suggests that CtBP is required to downregulate proliferation of eye precursors ahead of the morphogenetic furrow: CtBP- clones are larger and show more mitotic figures than wild type clones in the most anterior regions of the eye field. A number of factors are known to promote proliferation ahead of the furrow, and have been connected to CtBP in some way. Many of these factors can cause massive overgrowth when overexpressed. Thus, the role of CtBP may be to counteract the activity of one or more of these factors, to ensure that cells do not proliferate out of control (Hoang, 2010).

Factors that have previously been linked to CtBP and are known to regulate proliferation of eye precursors ahead of the furrow include the Wingless, Notch and JAK/STAT signaling pathways. This study has not ruled out a possible interaction between CtBP and these signaling pathways in the context of eye precursor proliferation (Hoang, 2010).

However, the combination of Hth, Tsh and Ey also regulates proliferation in ahead of the morphogenetic furrow, and this study has demonstrated that Ey and CtBP are part of a complex in eye-antennal disc cells, and that they interact genetically during eye development. Circumstantial evidence also suggests links between CtBP and Hth and Tsh. For instance, Drosophila and mouse Tsh homologs both contain a PxDLS motif, and have been shown to interact in vitro with Drosophila and mouse CtBP, respectively. In addition, the Drosophila Cdc25 homolog encoded by string, which triggers mitosis, appears to be a target of Hth, although it is not clear if it is a direct target. DamID experiments with CtBP in Kc cells have also identified string as a potential direct CtBP target. Based on current data, it is therefore proposed that CtBP interacts with the Hth/Tsh/Ey complex in eye precursors ahead of the furrow (Hoang, 2010).

CtBP87De-10 single mutant clones ahead of the furrow in the larval eye disc are larger than wild-type clones and show evidence of eye precursor overproliferation. Accordingly, adult eyes of CtBP transheterozygous combinations or of CtBP87De-10/M mosaic individuals have more ommatidia than wild-type eyes. In contrast, whereas CtBP87De-10,dan,danrex56 triple mutant clones are similar to CtBP87De-10 single mutant clones in being larger than wild-type clones ahead of the furrow, CtBP87De-10,dan,danrex56 adults have small rough eyes. This suggests either that the CtBP87De-10,dan,danrex56 triple mutant cells fail to be efficiently recruited into ommatidia and/or eventually undergo apoptosis. In support of the former hypothesis, the phenotypic analysis demonstrates that recruitment of the R8 photoreceptor, which is required to recruit all other ommatidial cells, is affected by loss of CtBP, and is more strongly affected in the CtBP,dan,danr triple mutant (Hoang, 2010).

Given the dynamic and overlapping expression patterns of the retinal determination factors, one intriguing hypothesis that fits the data is that a complex containing CtBP may have different members at different stages of eye development. For instance, a complex containing CtBP, Ey as well as possibly Tsh and Hth anterior to the furrow, might participate in maintaining a 'poised' chromatin structure with respect to eye specific genes, in which genes involved in eye differentiation are not yet expressed and the cells are kept in a proliferative state. It has been suggested that vertebrate Pax6 is a 'pioneering' factor for the lens lineage, and other 'pioneering' factors have been shown to promote a 'bivalent' state in which developmental genes are silenced, but 'poised' for activation. The down-regulation of Ey close to the furrow, and the initiation of Dan, Danr and Dac expression in the same region would be expected to change the composition of the complex containing CtBP and lead to changes in transcription that reflect the transition from proliferation to differentiation (Hoang, 2010).

At present it is not known what genes might be direct targets of the complexes containing CtBP and the eye specification factors. Some possibilities include the cell cycle regulator string, and the pre-proneural gene atonal, which is known to be regulated by Ey, So, Dan and Danr (Ey and So are direct regulators), and which plays a critical role in the transition from proliferation to differentiation of eye precursors. Thus, future work on Drosophila CtBP will shed light on the functions of this important transcriptional regulator, as well as on important transitions during development (Hoang, 2010).


Antibodies were raised to the predicted Dan and Danr proteins. Both are nuclear proteins, expressed in the eye-antenna disc. Double labeling with anti-Dan and anti-Danr shows that the two proteins are co-expressed in the antenna. Both proteins are also expressed in the brain and the eye region of the eye-antenna disc. Antibody labeling of other imaginal discs shows limited Dan expression in small groups of cells in leg and wing. These appear in the location of prominent sense organ progenitors at relatively late stages of disc development. Danr was not detected in other discs (Emerald, 2003).

To define the domain of Dan and Danr expression in the antenna, a series of double labeling experiments were performed with antibodies to other antennal proteins. Homothorax (Hth) is expressed in the presumptive head capsule and in antennal segments A1 to A3. Hth overlaps with the proximal part of the Dan domain. Distal-less (Dll) is expressed in a distal domain comprising A2, A3 and the arista. Dll overlaps the Dan domain, but extends further proximally. Cut is expressed in the proximal part of the antenna, in a domain that does not overlap expression of Dan. In optical cross-section there appears to be one row of cells with low expression of Dan at the interface between these domains. The domain of Dan/Danr expression appears to coincide with the domain in which ss transcript is expressed (Emerald, 2003). The relationship between Dan, Hth and Dll expression suggests that the Dan domain corresponds to segment A3 and the arista and that Cut is expressed in A1 and A2 as well as the head capsule. Thus, in addition to the broadly overlapping domains of Hth and Dll, the antenna is subdivided into adjacent and perhaps mutually exclusive proximal and distal domains reflected by ss, Cut and Dan/Danr expression. Although the view is favored that the reciprocal expression of Dan/Danr and Cut is likely to define the border between antenna segments A2 and A3, it is noted that it is difficult to be precise about the location of the border before overt segmentation. The possibility exists that Dan and ss expression may extend into distal A2 (Emerald, 2003).

To delimit more precisely the expression of the hern and fer (danr and dan respectively) genes in the antennal primordium, double staining was carried out of the MD634 and CES115 GAL4 lines (revealing hern and fer transcription, respectively) with genes expressed in restricted areas of mature eye-antennal discs, like Dll, hth, sal, and dac. Both lines gave the same results. Within the third antennal segment, the hern and fer expression is included within the Dll and hth domains. The hern and fer proximal limit of expression seems to coincide with that of dac, and their distal limit of strong expression in the third antennal segment with that of sal. It cannot be exclude that the GAL4 lines drive expression in a few cells from the second antennal segment (Suzanne, 2003).

Developmentally regulated subnuclear genome reorganization restricts neural progenitor competence in Drosophila

Stem and/or progenitor cells often generate distinct cell types in a stereotyped birth order and over time lose competence to specify earlier-born fates by unknown mechanisms. In Drosophila, the Hunchback transcription factor acts in neural progenitors (neuroblasts) to specify early-born neurons, in part by indirectly inducing the neuronal transcription of its target genes, including the hunchback gene. Using vivo immuno-DNA FISH the hunchback gene was found to move to the neuroblast nuclear periphery, a repressive subnuclear compartment, precisely when competence to specify early-born fate is lost and several hours and cell divisions after termination of its transcription. hunchback movement to the lamina correlated with downregulation of the neuroblast nuclear protein, Distal antenna (Dan). Either prolonging Dan expression or disrupting lamina interfered with hunchback repositioning and extended neuroblast competence. It is proposed that neuroblasts undergo a developmentally regulated subnuclear genome reorganization to permanently silence Hunchback target genes that results in loss of progenitor competence (Kohwi, 2012). .

The Drosophila embryo undergoes a reorganization of genome architecture that is gene, cell type, and developmental stage specific. As neuroblasts age, the hb genomic locus becomes repositioned to the nuclear periphery, which marks the end of the neuroblast competence window to specify early-born cell fates. Why can ectopic hb not induce early-born fates after the close of the competence window? It is proposed that hb is just one of many genes that move to the nuclear lamina at the end of the early competence window -- that a genome-wide reorganization shifts the neuroblasts into a state in which hb is unable to regulate the same targets it could during the competence window. In support of this model, misexpression of hb in the NB5-6 lineage was shown to have no effect on the activation of late-born cell fate factors, consistent with a new genome organization that is refractory to Hb-induced early-born neuronal identity (Kohwi, 2012). .

The data lead to a proposal that neural progenitors undergo a developmentally regulated reorganization of genome architecture as they age, potentially changing the palette of genes available to specify cell fate in aging progenitors. The following three-step model for neuroblast competence is proposed. (1) In the newly formed NB7-1, the hb gene is in the nuclear interior and is transcriptionally active; this is the time when early-born Hb+ U1/U2 neurons are generated. (2) After the second division of NB7-1, the transcriptional repressor Svp terminates hb transcription; however, the hb gene remains accessible in the nuclear interior where ectopic hb protein can indirectly induce hb neuronal transcription to generate extra U1/U2 neurons. (3) After the fifth division of NB7-1, Dan protein is downregulated, resulting in the movement of hb (and probably many other hb target genes) to the nuclear lamina; at this point, ectopic hb in the neuroblast can no longer induce transcription of hb, and the competence window is closed (Kohwi, 2012).

The results are consistent with growing evidence from multiple organisms that repositioning of a gene to the nuclear lamina can cause transcriptional repression. For example, forced tethering of reporter genes to Lamin can repress reporter expression , and Lamin depletion can derepress silent genes. An important difference in the current work, however, is that hb movement to the lamina occurs 3 hr after termination of hb transcription, when hb undergoes an additional level of repression to become permanently silenced. Does lamina targeting induce permanent hb gene silencing or vice versa? Depletion of the nuclear envelope protein Lamin displaces hb away from the lamina, decreases hb silencing, and increases neuroblast competence; this strongly suggests that lamina targeting is an early and essential step in permanent hb gene silencing and the loss of neuroblast competence. However, the possibility cannot be ruled out that hb positioning at the nuclear periphery might maintain, rather than establish, the permanently silenced state (Kohwi, 2012).

The results show that neuroblast cell fate specification and neuroblast competence are independently regulated temporal programs. Prolonged expression of Dan can extend the competence window but cannot induce neuronal identity. Conversely, hb can specify early-born neuronal identity but cannot extend the competence window. Importantly, coexpression of Dan and hb act synergistically: Dan extends the neuroblast competence window, and hb 'fills' this window with U1/U2 neurons, thereby specifying more early-born identity neurons than prolonging hb alone. The mechanism and function of Dan is poorly understood. Why might Dan be sufficient, but not necessary, to promote neuroblast competence? One common finding is that dan and dan related (danr) genes show weak double mutant phenotypes, but strong misexpression phenotypes, in all tissues examined. For example, dan danr double mutants can live to adulthood with weak antennal and eye defect and show no change in the neuroblast competence window. These findings are consistent with a redundant protein or pathway that can compensate for loss of Dan/ Danr. A second model arises from comparing the dan danr mutant and Dan overexpression phenotypes. dan danr double mutants have a slight delay hb transcription termination at stage 10 (distinct from permanent hb silencing at stage 12) (Kohwi, 2011) but no effect on hb gene movement to the nuclear periphery. Conversely, prolonged Dan expression has no effect on hb transcription but blocks hb gene movement and extends the competence window. Thus, during the competence window, Dan may promote a genome organization that allows timely access of the Svp transcriptional repressor to the hb locus, whereas after the competence window, Dan must be eliminated to allow a new genome organization to form. This model emphasizes the role of Dan in maintaining a genome organization in which hb and hb target genes can be efficiently regulated. A third, not mutually exclusive, model is that Dan binds DNA via its Pipsqueak domain to competitively inhibit other Pipsqueak-like factors from recruiting hb and other loci to the nuclear lamina. Indeed, the founding member of the Pipsqueak- motif family, Pipsqueak, is a GAGA-binding factor, and recent work has shown an enrichment for GAGA motifs in Lamin-associated DNA sequences. Consistent with this model, Dan protein is dispersed throughout the nucleoplasm, where it could associate with hb and other loci (Kohwi, 2012).

What is the normal function of a competence window? Competence windows could provide both flexibility and limitations on the production of neural diversity. They could serve as a substrate for natural selection by allowing variation in neuronal subtype numbers through fluctuations in the length over which a progenitor is exposed to a temporal identity cue. Conversely, a competence window could also prevent stochastic fluctuations in the expression of a temporal identity cue from generating a neuronal subtype at a completely inappropriate time (e.g., an early-born fate at the end of a progenitor lineage), thereby limiting potentially deleterious effects. Another potential function of competence windows is that successive competence windows may allow the same cell fate determinant to generate different cell types. Indeed, during spinal cord development, Olig2 first promotes neurogenesis and later induces oligodendrogenesis, and retinal progenitors are thought to progress through multiple competence states during which they can specify only limited cell fates. In support of this model, previous studies have shown that Dan has two waves of neuroblast expression, one at stages 9-late 12 and a second at stages 13-16 (Kohwi, 2011). Bimodal Dan expression may produce two competence windows in which the same temporal identity factors can access different genomic targets to generate additional neuronal diversity. For example, during the first Dan expression window, Hb, Kr, Pdm, Castor, and Svp specify U1-U5 motoneuron identities in the NB7-1 lineage; during the second Dan expression windows, Kr, Castor, and Svp are re-expressed and a different population of neurons is produced. Mammalian Ikaros, an hb homolog, is expressed in young progenitors in which it specifies early-born retinal ganglion cell (RGC) identity. As with Hb, re-expression of Ikaros in older progenitors in vivo cannot induce specification of the early-born RGCs, although Ikaros misexpression in late retinal progenitors cultured in vitro can activate RGC-specific genes. Perhaps in vitro cultured progenitors are lacking an extrinsic cue that closes the competence window, allowing Ikaros to generate more early-born neurons. Interestingly, Dan downregulation could also be regulated by an extrinsic cue, because Dan downregulation occurs nearly simultaneously in the entire neuroblast population, despite each neuroblast being at a different stage of its cell lineage. Mammalian retinal and cortical progenitor cells change competence over time, generating an ordered series of distinct neural cells. In the future, it would be interesting to determine whether the genes expressed in early-born cortical or retinal cell types are repositioned to the nuclear lamina in mammalian neural progenitors as competence to specify that these cells are lost over time. Determining the mechanisms underlying loss of competence and identifying the molecular players in this process would have important implications for understanding normal brain development, adult tissue homeostasis, and tissue repair (Kohwi, 2012).

Drosophila dany is essential for transcriptional control and nuclear architecture in spermatocytes

The terminal differentiation of adult stem cell progeny depends on transcriptional control. A dramatic change in gene expression programs accompanies the transition from proliferating spermatogonia to postmitotic spermatocytes, which prepare for meiosis and subsequent spermiogenesis. More than a thousand spermatocyte-specific genes are transcriptionally activated in early Drosophila spermatocytes. This study describes the identification and initial characterization of dany (CG30401), a gene required in spermatocytes for the large-scale change in gene expression. Similar to tMAC (a testis-specific meiotic arrest complex (see Always early)) and tTAFs (see Cannonball), the known major activators of spermatocyte-specific genes, dany has a recent evolutionary origin, but it functions independently. Like dan and danr, its primordial relatives with functions in somatic tissues, dany encodes a nuclear Psq domain protein. Dany associates preferentially with euchromatic genome regions. In dany mutant spermatocytes, activation of spermatocyte-specific genes and silencing of non-spermatocyte-specific genes are severely compromised and the chromatin no longer associates intimately with the nuclear envelope. Therefore, as suggested recently for Dan/Danr, it is proposed that Dany is essential for the coordination of change in cell type-specific expression programs and large-scale spatial chromatin reorganization (Trost, 2016).

Meiotic sex has a deep evolutionary origin in basal eukaryotes. While meiosis has generally remained well conserved, most other aspects of sexual reproduction have diverged rapidly. In animals that differentiate a male and a female gender, male-biased genes and in particular those expressed in the germline are affected by turnover and sequence divergence that is significantly faster than in other gene classes. Analyses in Drosophila melanogaster spermatocytes have provided some of the most striking evidence for rapid evolutionary dynamics even in key elements of transcriptional networks with numerous interactions (Trost, 2016).

The comparison of different tissues in adult D. melanogaster has clearly revealed that the number of genes with an expression apparently restricted to a single tissue is maximal in the case of testis. The large majority of these, as well as of the testis-biased genes, are transcribed in spermatocytes, i.e., in germline cells during a growth phase between the last mitotic division and the onset of the meiotic divisions and spermiogenesis. The transcription of more than 1000 of these genes depends on the function of the testis meiotic arrest complex (tMAC). tMAC is a testis-specific variant of the MIP/dREAM/SynMuvB complex, a widely conserved somatic transcriptional regulator. tMAC contains subunits encoded by testis-specific paralogs (aly, tomb) that are only present within the genus Drosophila. They were identified based on a characteristic loss-of-function phenotype in which the testes fill up with spermatocytes failing to enter meiotic divisions and postmeiotic differentiation. Several genes of comparably recent origin and with similar mutant phenotypes (topi, comr, achi, vis) encode proteins interacting with tMAC. A second protein complex of paramount importance for spermatocyte-specific transcription is formed by testis-specific TATA-binding protein (TBP)-associated factors (tTAFs) in the genus Drosophila. The tTAF genes [can, mia, nht, rye (Taf12L - FlyBase), sa] were also identified based on their mutant phenotype, which is slightly milder than that associated with tMAC loss (Trost, 2016).

This study describes the identification of dany, yet another gene with a recent evolutionary origin and an essential role in spermatocyte-specific gene expression. dany is most similar to the Drosophila genes distal antenna (dan) and distal antenna-related (danr). Dan and Danr have recently been implicated in the control of neuroblast competence in Drosophila embryos, where they inhibit the repositioning of crucial target genes into repressive chromatin associated with the nuclear lamina (Kohwi, et al., 2013). Similarly, Dany is required not only for cell type-specific gene expression in spermatocytes but also for normal association of chromatin with the nuclear envelope (Trost, 2016).

This identification and characterization of dany has uncovered a factor crucial for the realization of the spermatocyte-specific gene expression program in Drosophila. Loss of dany compromises the transcriptional activation of a large number of spermatocyte-specific genes and derepresses genes that are normally inactive in spermatocytes. Moreover, dany is required for normal association of spermatocyte chromatin with the nuclear periphery (Trost, 2016).

With regard to transcriptional activation, Dany is similar to tTAFs and tMAC. Mutations in tMAC genes have a severe effect on spermatocyte-specific gene. In the case of tTAF mutants, fewer genes are affected and the reduction in transcript levels is often less severe. Loss of dany has even slightly milder effects, but there are still ~1000 genes with significantly reduced transcript levels. Many of these genes are also dependent on tMAC and tTAFs (Trost, 2016).

The fact that Dany is just as important for silencing of non-spermatocyte genes as for activation of spermatocyte-specific genes indicates that this protein does not function like the activators tTAFs and tMAC, which have not been implicated in gene silencing. Several additional observations support this conclusion. Dany intracellular localization in early spermatocytes is distinct from that of the tTAF Sa. dany transcript and protein levels depend neither on Sa nor on the tMAC components Aly and Topi. Vice versa, mutations in dany affect neither transcript levels of tTAF and tMAC genes nor accumulation and intracellular localization of representative protein products (Sa and Can) (Trost, 2016).

dany orthologs cannot be detected outside the genus Drosophila, whereas the primordial dan/danr genes are present throughout the insect lineage. The testis-specific tMAC and tTAF genes are comparably young in evolutionary terms. The fact that several functionally independent factors of paramount importance for the highly complex spermatocyte-specific gene expression program have a recent origin provides further testimony of the surprising evolutionary dynamics of genes that function in the male germline. After a gene duplication event, dany has evolved to control the expression of thousands of genes in the male germline. Dany is a potent regulator; ectopic expression of dany in somatic tissues is highly toxic (Trost, 2016).

How does Dany exert its function? Dany is a nuclear protein. After ectopic expression in larval salivary glands, it binds preferentially to all euchromatic interband regions of polytene chromosomes. In maturing spermatocytes, where it is normally expressed, Dany also appears to associate preferentially with euchromatic regions within chromosome territories and not with the regions of maximal DNA staining intensity corresponding to pericentromeric heterochromatin. As the Psq motif of some other proteins, including CENPB and transposases, is involved in sequence-specific DNA binding, it is conceivable that Dany and its closest relatives Dan and Danr bind to DNA as well. The Psq domain structure of Dan as revealed by NMR is highly similar to that observed by X-ray crystallography in CENPB. However, the DNA-binding region within CENPB is not restricted to the Psq motif but includes an additional helix-turn-helix domain that is not present in Dan, Danr and Dany. Similarly, the DNA-binding region of transposases extends considerably beyond the Psq motif. By mutating three amino acid codons within the Dany Psq motif, which correspond to positions contacting bound DNA in the case of CENPB, it was possible to demonstrate the functional importance of this motif. However, DanyAAA still retains some function; it promotes spermatogenesis beyond the stage when dany null mutant spermatocytes arrest. Moreover, the intracellular localization of DanyAAA in spermatocytes appears to be normal. Additional analyses will be required to resolve whether the Psq motif of Dany indeed binds DNA. It is pointed out that the Psq motif in CENPB contacts only four base pairs. Such a limited DNA sequence specificity could not explain the observed preferential association of Dany with euchromatic regions. Additional factors will have to be identified (Trost, 2016).

Interestingly, the first phenotypic abnormalities that this study has detected in dany mutants occur in young spermatocytes soon after the onset of dany expression. Whereas chromosome territories tend to become enwrapped in the nuclear envelope when they are formed in wild-type spermatocytes during the S3 stage, dany mutant spermatocytes are devoid of such characteristic nuclear envelope deformations at the corresponding stage. Thus, Dany appears to be required for normal chromatin association with the nuclear envelope. Since Dany is not enriched at the nuclear periphery, it obviously is very unlikely to function as a factor that directly establishes physical contact between chromatin and the nuclear envelope. But the extensive chromatin reorganization, which presumably occurs in early spermatocytes when thousands of previously repressed genes become active while precursor-specific genes are inactivated, might be abnormal in the absence of dany. Thereby, chromatin properties that normally cause localization to the nuclear periphery might also be affected (Trost, 2016).

Dany's closest relatives Dan and Danr, which act partially redundantly in somatic tissues, are crucial for the control of intranuclear position and silencing of the hunchback (hb) genomic locus in neuroblasts during embryogenesis. hb relocalization to the nuclear lamina, which is correlated with hb silencing and termination of Hb response competence, depends on timely Dan downregulation (Trost, 2016).

Changes in the association of genes with the nuclear periphery have previously been implicated in the control of the spermatocyte-specific gene expression program in Drosophila. In cell types other than spermatocytes, most spermatocyte-specific genes appear to be packaged into a repressive type of heterochromatin designated 'BLACK' heterochromatin, which is associated with Lam at the nuclear envelope. In case of two representative testis-specific gene clusters (at 60D1 and 22A1), loss of Lam was shown to result in their derepression in somatic larval and cultured cells, and the normal transcriptional activation was accompanied by cluster repositioning into the nuclear interior of late spermatocytes. Immuno-FISH analyses support the notion that a repressive chromatin type that is normally associated with the nuclear periphery is impaired in dany mutants. The derepression of the eye-specific inaC gene in dany mutant spermatocytes was accompanied by delocalization away from the nuclear periphery into the interior. Extensive future work will be required to clarify the molecular basis and functional significance of these initial observations (Trost, 2016).

Even if an extensive correlation between intranuclear position and transcriptional activity were established by systematic studies, it remains to be considered whether loss of dany might affect the organization of chromatin within the nucleus indirectly. dany mutations could alter the expression program of particular chromatin regulators or proteins of the nuclear periphery. Indeed, some important chromatin regulators and nuclear envelope proteins have been shown to undergo drastic changes in expression levels during normal spermatogenesis. Su(Hw), a multi-zinc finger DNA-binding protein that can function as a transcriptional insulator and modulator of chromatin association with the nuclear lamina, and the PRC2 complex components E(z) and Su(z) are strongly downregulated in early spermatocytes, whereas Lamin C (LamC) expression is induced. However, in these particular cases, no abnormal expression program was detected in dany mutant testis by immunolabeling (Trost, 2016).

Loss of dany also results in the derepression of genes that appear to be Pc targets. Such genes are primarily within 'BLUE' heterochromatin. Pc-mediated repression of spermatocyte-specific genes has been proposed to be counteracted in spermatocytes by the combined action of tMAC and tTAFs. Although not identical, the dany mutant phenotype shares similarities with the meiotic arrest phenotype caused by loss of tMAC and tTAF function. Future work will be required to clarify the functional interactions between Dany and these major activators of spermatocyte-specific genes (Trost, 2016).

Extensive spatial intranuclear chromatin reorganization might guide many differentiation processes in complex eukaryotes. For example, hundreds of genes were reported to move towards or from the nuclear envelope during differentiation of mouse embryonic stem cells, concomitant with reduced or increased expression, respectively. Similar observations were made during adipogenic cell differentiation. This identification and initial characterization of dany should help support future progress towards a molecular understanding of the mechanisms that coordinate change in spatial genome organization and cell type-specific expression programs (Trost, 2016).


The formation of different structures in Drosophila depends on the combined activities of selector genes and signaling pathways. For instance, the antenna requires the selector gene homothorax, which distinguishes between the leg and the antenna and can specify distal antenna if expressed ectopically. Similarly, the eye is formed by a group of 'eye-specifying' genes, among them eyeless, which can direct eye development ectopically. hernandez (distal antenna related or danr) and fernandez (distal antenna or dan) are expressed in the antennal and eye primordia of the eye-antenna imaginal disc. The gene names 'Hernandez' and 'Fernandez' are an homage to twin brothers, characters in the Tintin comic-book series. The predicted proteins encoded by these two genes have 27% common amino acids and include a Pipsqueak domain. Reduced expression of either hernandez or fernandez mildly affects antenna and eye development, while the inactivation of both genes partially transforms distal antenna into leg. Ectopic expression of either of the two genes results in two different phenotypes: such expression can form distal antenna, activating genes like homothorax, spineless, and spalt, and can promote eye development and activates eyeless. Reciprocally, eyeless can induce hernandez and fernandez expression, and homothorax and spineless can activate both hernandez and fernandez when ectopically expressed. The formation of eye by these genes seems to require Notch signaling, since both the induction of ectopic eyes and the activation of eyeless by the hernandez gene are suppressed when the Notch function is compromised. These results show that the hernandez and fernandez genes are required for antennal and eye development and are also able to specify eye or antenna ectopically (Suzanne, 2003).

From a collection of P-GAL4 lines that drive expression in restricted areas of the adult cuticle, four lines were selected that specifically direct a similar expression of the yellow (y) gene: the third antennal segment, the arista, and a few bristles in the second antennal segment express the y gene. These lines, when crossed to a line carrying an UAS-lacZ reporter construct, show ß-galactosidase expression in three regions of the eye–antennal disc: in the third antennal segment, in the arista, and within the eye, in the differentiated eye and in a region just anterior to the morphogenetic furrow (Suzanne, 2003).

The insertion site of these four lines was located by inverse PCR: two lines (CES75 and MD634) are in the 5' upstream region of the predicted CG13651 transcription unit and the other two (AC116 and CES115) are in the 5' upstream region of the CG11849 transcription unit, according to the annotated Drosophila genome. The two transcription units are located in the chromosomal position 96C2-4, in the same orientation and nearly adjacent, separated by about 45 kb. The CG13651 transcription unit is 1.259 kb long, bears no introns, and produces a 1259-bp mRNA, whereas the CG11849 transcription unit is 7.175 kb long, has 3 exons, and makes a mRNA of 2232 bp. There is an EST for the CG11849 unit, indicating that this predicted gene is transcribed (Suzanne, 2003).

The CG13651 and CG11849 transcription units encode predicted proteins of 419 and 743 amino acids, respectively, that have 27% identity and 37% similarity between them. Both proteins contain similar 69-amino-acid regions, located in their N-terminal part, with 78% identity and 88% similarity between them. This region includes a previously characterized 48-amino-acid Pipsqueak (Psq) motif, present in several proteins in Drosophila and other species, including human. The Psq motif is a helix-turn-helix DNA-binding domain similar to the DNA-binding domain of prokaryotic recombinases, which, in turn, also show similarities with the homeodomain. The CG13496 predicted Drosophila gene codes for a protein with a Psq domain very similar to those of CG13651 and CG11849 genes. The CG13496 transcription unit is not expressed in the eye-antennal imaginal disc like the CG13651 and CG11849 genes (Suzanne, 2003).

In situ hybridization experiments show that, among all the imaginal discs, the CG13651 and CG11849 transcription units are only and equally expressed in the eye-antennal disc. Their expression is very similar to that observed with the P-GAL4 lines, except that, in the eye primordium, the RNA signal in the differentiated eye is very weak. The same antennal and eye expression persists in early pupal stages. Therefore, these genes present a similar sequence, expression in the eye-antennal disc, regulation, and function. These genes have been called hernandez (hern, CG13651) and fernandez (fer, CG11849), and together are referred to as the Tintin genes (Suzanne, 2003).

Since hern and fer are not expressed in the leg discs, they may be part of the mechanism that distinguishes legs from antennae. The Antennapedia (Antp) Hox gene, which is expressed in the leg but not in the antennal discs, prevents hth expression and antenna formation in the leg primordia. As expected, in Antp73b/+ flies, which show a strong transformation of antennae into legs, the expression driven by the MD634 GAL4 line is clearly reduced (Suzanne, 2003).

To characterize the function of hern and fer in normal development, the phenotype of flies without hern or fer activity was studied. One of the P-GAL4 lines, AC116, is mutant for the antennal function of the fer gene. Homozygous AC116 third instar larvae show no fer transcription in the antennal primordium but present normal fer expression in the eye primordium. AC116/Df adults show one or more bristles in the third antennal segment, normally devoid of them. To obtain more mutations in the hern and fer genes, the MD634 and CES115 P-GAL4 lines (the two closer to the hern and fer transcription units) were mobilized to isolate imprecise excisions of the transposons. ferI49-1, one w- derivative of the CES115 insertion, was isolated that in hemizygosis, shows a phenotype very similar to that described for the AC116/Df adults. PCR analysis of the mutation revealed that it is a small deletion of about 2.5 kb in the 5' upstream region of the fer transcription unit. Larvae homozygous for the ferI49-1 mutation present reduced expression of the fer RNA in the antennal primordium of mature eye-antennal discs, whereas the expression in the eye primordium is normal. It is concluded that the AC116 insertion and the ferI49-1 deletion may have affected an antennal regulatory region. This regulatory region would control only the fer gene, since in AC116 and ferI49-1 homozygous larvae, no change was detected in hern expression. No mutation has been obtained for the hern gene (279 w- derivatives analyzed), although the MD634 line used for this purpose is closer to the origin of transcription of the hern gene than the CES115 line is to the transcription unit of the fer gene (Suzanne, 2003).

The weak phenotype of the AC116 and ferI49-1 mutations suggests that there may be a partial functional redundancy between hern and fer, and that the inactivation of both genes may result in a stronger phenotype. To check this, to ascertain the phenotype of hern inactivation, and to study the effect of the absence of Tintin products in the eye, double-stranded (ds)-mediated RNA interference (RNAi) was used to inactivate hern and fer functions. The inactivation in the antennal primordium of either the hern or the fer genes with a Dll-GAL4 (MD23) driver causes a phenotype similar to that described for the AC116/Df and ferI49-1/Df adults: there is one or more bristles in the third antennal segment and the base of the arista is slightly enlarged. However, when the ds-hern and ds-fer RNAs are induced together by the Dll-GAL4 driver, a clear transformation of part of the distal antenna into leg is observed: the third antennal segment and the proximal arista are substantially enlarged and covered with bristles; those in the base of the arista bear bracts, indicating a transformation into leg.. These results indicate that both hern and fer are required to develop part of the antenna as opposed to leg and that these two genes are partially redundant in this function (Suzanne, 2003).

To test whether hern and fer are sufficient to induce eye or antennal development, they were expressed ectopically using the GAL4/UAS system. When either the hern or the fer genes are misexpressed in the leg discs with dpp-GAL4 or Dll-GAL4 (EM212) drivers, distal legs are transformed to aristae. These transformations are accompanied by the ectopic expression of hth, sal, and ss, three genes expressed in the antennal primordium but not in the distal region of mature wild-type leg disc. Clones expressing either the hern or the fer genes in the leg or wing disc have smooth borders and frequently activate the sal and hth genes cell-autonomously. In dpp-GAL4/UAS-fer or ptc-GAL4/UAS-hern leg (or wing) discs, the expression of ss is also activated. Curiously, although ss is downstream of hth in the antenna and leg, ectopic ss in the leg disc can also activate hth in a few cells (Suzanne, 2003).

The hth or ss genes, together with Dll, are sufficient to develop ectopic distal antennae when expressed in different regions of the adult. The hern or fer genes are also able to elicit this transformation in the leg and they activate hth and ss. Conversely, when high levels of the Hth or Ss products are induced in the leg discs, ectopic expression of the hern and fer genes is found. To study the interactions between these genes in normal development, the relationship between Dll, hth, ss, and hern/fer in the antennal primordium was examined. A reduction of Hth activity using a dominant negative form of hth (UAS-EN-HTH1-430) results in a decreased activity of the MD634 and AC116 GAL4 lines, which reveal hern and fer expression, respectively. Similarly, in antennal discs of a Dll strong hypomorph or a ss null mutation, the expression of hern and fer disappears. These results suggests that hth, Dll, and ss are required to maintain hern and fer expression in the antenna. By contrast, high levels of hern or fer may reduce hth expression. In dpp-GAL4/UAS-fer or dpp-GAL4/UAS-hern larvae, the expression of hth (and sal) in the third antennal segment is eliminated or strongly reduced dorsally (where levels of hern and fer are high) and does not change or is ectopically activated ventrally (where levels of hern and fer are low). Similarly, fer-expressing clones are able to downregulate hth expression in the antennal primordium. These results suggest that levels of hern and fer expression may be important for a normal antennal development (Suzanne, 2003).

hern and fer genes are required for normal eye development and form eye tissue and activate ey when ectopically expressed. To study the role of the hern and fer genes in eye development, the eye phenotype was examined when either the hern or fer genes are inactivated by RNAi or are expressed ectopically. Expression of ds-hern or ds-fer RNA in the eye primordium with a GMR-GAL4 driver causes a slightly rough eye, with some bristles irregularly positioned. Curiously, the phenotype is not increased if the ds-hern and ds-fer RNAs are induced in the same fly. Misexpression experiments also suggest that both hern and fer are involved in eye development. Thus, the expression of either hern or fer with different GAL4 drivers causes the appearance of ectopic eye tissue in the third antennal segment or rostral membrane. These transformations are accompanied by the ectopic expression of ey, although this effect may also indicate the maintenance of a previous ey expression. Conversely, the misexpression of ey activates the hern and fer genes ectopically. Both hern and fer also activate embryonic lethal abnormal vision (elav), a marker of neuronal differentiation, when ectopically expressed. The analysis of clones expressing the fer gene in the leg, eye-antennal, or wing discs shows that elav activation is strictly nonautonomous, and only occurs in some cells adjacent to some of these clones (Suzanne, 2003).

The formation of the morphogenetic furrow in the eye is limited laterally by wg signaling. hern and fer expression within the eye primordium includes the more lateral wg-expressing regions. Interestingly, both hern and fer activate wg transcription when ectopically expressed. In ptc-GAL4/UAS-hern or dpp-GAL4/UAS-fer flies, the wings show several alterations, including the appearance of marginal bristles in the middle of the wing blade. This phenotype is characteristic of ectopic wg signaling, and in fact, wg is ectopically expressed in the wing discs of these larvae. Clones expressing the fer genes in the eye-antenna, leg, or wing discs also show induction of wg, mostly within but also outside the clone. The elav gene is also induced nonautonomously by these clones. Cells ectopically expressing elav do not coincide with those expressing wg and this reproduces the wild-type situation in the eye (Suzanne, 2003).

Signaling pathways can modify the activity of selector genes and are needed for proper organ formation. N signaling, for instance, is needed for eye formation and can activate ey when ectopically activated. Moreover, N has been implicated in the decision of making eye or antenna, directing eye development, and suppressing antenna formation. Therefore, whether N signaling could alter the ey and elav expression induced by the Tintin genes was examined. The coexpression of the hern gene and a dominant negative form of the Notch receptor substantially reduces ey and eliminates elav ectopic signals. Accordingly, no ectopic eyes are formed in this genetic combination. This indicates that the effect of hern on ey expression and eye formation requires N signaling (Suzanne, 2003).

The concept of selector genes in Drosophila has evolved from a precise and restricted definition to a more loose interpretation. Selector, or selector-like genes, are now considered as those required to make a particular structure and able to form it in different positions when the gene is expressed ectopically. hern and fer fit this definition as selector genes for the distal antenna. They also can make ectopic eyes, although their requirement for eye development is not so evident as that for antenna formation (Suzanne, 2003).

The differentiation of legs or antennae depends on the activity of the hth and Antp genes. The ss gene, however, is also able to transform distal leg (and also maxillary palp and rostral membrane) into distal antenna, and the absence of ss, like that of hth, transforms antenna into leg. Although ss seems to be downstream of Dll and hth in antenna specification, ectopic ss can activate hth in some cells of the leg disc. Similarly, misexpression of ss in the rostral membrane induces Dll expression. It seems, therefore, that ss can trigger an antennal genetic program when misexpressed in certain places (Suzanne, 2003).

The fer and hern genes are both required and sufficient to make part of the distal antenna. Four different genes, hth, ss, hern, and fer, are able to form distal antenna, together with Dll, when ectopically expressed. Their mutual regulation seems to differ when misexpressed in the leg disc or when normally expressed in the antennal primordium. In the leg disc, hern or fer activates hth and ss and, reciprocally, hth and ss induce hern and fer expression. Moreover, even ss can promote hth transcription, although just in a few cells. Taken together, these results suggest that the four genes can form distal antenna by activating each other's transcription when ectopically expressed (Suzanne, 2003).

In the third antennal segment, Dll, hth, and ss are required to activate hern/fer expression. Since ss is downstream of Dll and hth in the antenna, the activation of hern/fer by Dll and hth could be mediated by ss. It is noted, however, that the levels of hern and fer may modulate hth expression. Moderately increased levels of fer can activate hth in dpp-GAL4/UAS-fer discs but, when the levels of hern or fer in the antenna are highly increased, the transcription of hth is prevented. These results suggest that the total amount of hern and fer expression may be regulated in the antennal primordium. Accordingly, in clones mutant for danr (hern), the expression of dan (fer) is upregulated. Also supporting the conclusion that levels of hern and fer have to be regulated, it was found that, in ey-GAL4/UAS-hern or ey-GAL4/UAS-fer flies, where levels of either hern or fer are highly increased in the eye–antennal disc, both the eye and the antenna disappear (Suzanne, 2003).

Several eye-specifying genes have been identified, and they fulfill two conditions: they are required to make the eye and they can form ectopic eyes when expressed in different parts of the body. The hern and fer genes probably form part of this network of 'eye-specification' genes: (1) they are expressed in the eye primordium, with higher levels of expression anterior to the morphogenetic furrow; (2) they activate ey and elav and make ectopic eyes when expressed ectopically; (3) ey also activates the hern and fer genes when ectopically expressed. hern and fer genes have also been identified as downstream of ey in eye ectopic formation (Michaut, 2003). However, the inactivation of both hern and fer genes by RNAi with the GAL4 driver does not grossly affect eye development, as do mutants in the eye-specification genes. The nonautonomous induction of elav when hern or fer are ectopically expressed reproduces the wild-type situation, in which high levels of hern and fer are observed adjacent to the differentiating, elav-expressing, photoreceptor cells. Another similarity of hern and fer with some of the 'eye-specification'genes is that ectopic eye tissue is obtained in the antennae. The eye-specification genes eya and dac also form eyes predominantly, when ectopically expressed, in this same position. This is perhaps due to ey being expressed in the antennal primordium in late embryos, thus providing a favorable genetic context for eye formation. In accordance, when either the hern or the fer gene is ectopically expressed, ectopic ey expression is detected only in the antennal disc. Eyes are also obtained in the rostral membrane when ectopically expressing the fer gene. This may be due to the absence of hth, since high levels of either hern or fer repress hth and removal of this gene in the rostral membrane forms ectopic eyes (Suzanne, 2003).

The hern and fer genes can form ectopic aristae and eye tissue, but only in a limited number of regions of the adult cuticle. This is similar to what happens with other genes making ectopic antennae (hth, ss) or eye (eye-specification genes). This is due to the particular developmental context of the region where the genes are ectopically activated (Suzanne, 2003).

Transformations are observed of third antennal segment, where hern and fer are normally transcribed, to eye tissue, in Dll-GAL4/UAS-hern or dpp-Gal4/UAS-fer flies. This suggests that the levels of Hern and Fer products may be important in inducing or maintaining ey expression and distinguishing eye from antenna. Accordingly, when Hern or Fer products are increased in the antennal primordium, the expression of hth, an inhibitor of eye development, is eliminated. It is also noted that, in the wild-type eye-antennal discs, hern and fer show higher levels of expression in the eye primordium than in the antennal one, where these genes are coexpressed with hth. However, the amount of Tintin product is not the only factor in this distinction, since, for instance, in Dll-GAL4/UAS-hern eye-antennal discs, the area of ectopic ey transcription in the antenna is smaller than the area of hern overexpression. The activity of other genes will probably contribute to the formation of either eye or antenna. Thus, the ectopic expression of either hern or fer induces wg, an inhibitor of morphogenetic furrow formation, and this probably limits the places where the eye can develop (Suzanne, 2003).

Two recent models have been proposed to explain the specification of eye and antenna within the eye-antennal disc. Both models suggest that the activation of the N signaling pathway is a key element in this process. It has been suggested that N signaling activates both ey and Dll in the eye and antennal primordia; subsequently, ey represses Dll in the eye and perhaps the hth and extradenticle genes repress ey in the antenna. In this way, the exclusive expression of ey (in the eye) and Dll and hth (in the antenna) determine eye and antenna identity, respectively. It has been proposed that the N and Egfr signaling pathways (together with the hedgehog and wg genes) are instrumental in the decisions to make eye or antenna. N signaling has been proposed to promote eye development and prevents formation of the antenna, whereas Egfr signaling does the opposite. Ectopic expression of either hern or fer in the antenna induces ectopic eyes and activates ey and elav, but the coexpression of hern and an N dominant-negative protein does not result in ectopic eyes and almost eliminates ey and elav activation. This suggests that N function impinges on hern activity to form ectopic eyes. As in other cases, the combined activity of signaling pathways and selector genes determine the specification of different structures (Suzanne, 2003).

To assess the roles of dan and danr in antenna development in detail, deletions that remove one or both genes were examined. Larvae homozygous for these deletions are viable. danrex35 is a small deletion that removes part of the Danr coding sequence. Antibody staining reveals loss of Danr protein in clones of cells mutant for danrex35. Interestingly, Dan protein is upregulated in these clones. Expression of both proteins is lost in homozygous dan danrex56 mutant clones; this loss of expression shows that ~45 kb between the two original P-element insertions have been removed. In both deletion homozygotes, the third antennal segment is reduced in size and develops ectopic bristles. In dan danrex56 animals the third antennal segment is generally smaller and more ectopic bristles are produced. In addition, the basal cylinder of the arista are enlarged and produce bracted bristles. Bracted bristles are typical of the distal leg and suggest partial transformation of antenna towards leg in the mutant tissue. A similar transformation of the basal cylinder was observed in mosaic antennae derived from ey-FLP danrex35/Minute heterozygous animals. The transformation associated with dan danrex56 homozygous clones was similar, but slightly stronger(Emerald, 2003).

Although many dan danr double mutant excisions were recovered, none was singly mutant for dan alone. To generate a dan mutant a screen was performed for EMS-induced revertants of the dan gain-of-function phenotype in the wing. One allele was recovered. Sequence analysis revealed a change of amino acid residue 45 from glutamate to lysine. This alteration lies in the conserved pipsqueak domain and affects a residue thought to be important for DNA binding of a related protein. When expressed in the leg disc under control of DllGal4, danems3 causes loss of the claws, but does not cause transformation to arista, suggesting a weaker gain of function phenotype than the wild-type protein. Thus, danems3 appears to be a hypomorphic allele that reduces but does not eliminate Dan activity. danems3 homozygotes were viable and show a mild antenna defect, including an occasional ectopic bristle in the third antennal segment. A stronger ectopic bristle phenotype was obtained when Dan activity was reduced using a Gal4 inducible construct that directs expression of a double-stranded dan RNA (DllGal4; sympUAST-dan). To verify that the inducible RNAi causes reduction of Dan protein levels, sympUAST-dan was expressed in the antenna disc using dppGal4. Dan protein levels were reduced in the RNAi-expressing cells, but Danr levels were unaffected (Emerald, 2003).


Search PubMed for articles about Drosophila distal antenna & distal antenna-related

Emerald, B. S., Curtiss, J., Mlodzik, M. and Cohen, S. M. (2003). distal antenna and distal antenna related encode nuclear proteins containing pipsqueak motifs involved in antenna development in Drosophila. Development 130: 1171-1180. 12571108

Hoang, C. Q., Burnett, M. E., Curtiss, J. (2010). Drosophila CtBP regulates proliferation and differentiation of eye precursors and complexes with Eyeless, Dachshund, Dan, and Danr during eye and antennal development. Dev. Dyn. 239: 2367-2385. PubMed Citation: 20730908

Kohwi, M., Hiebert, L. S. and Doe, C. Q. (2011). The pipsqueak-domain proteins Distal antenna and Distal antenna-related restrict Hunchback neuroblast expression and early-born neuronal identity. Development 138(9): 1727-35. PubMed Citation: 21429984

Kohwi, M., et al. (2012). Developmentally regulated subnuclear genome reorganization restricts neural progenitor competence in Drosophila. Cell 152(1-2): 97-108. PubMed ID: 23332748

Michaut, L., Flister, S., Neeb, M., White, K. P., Certa, U. and Gehring, W. J. (2003). Analysis of the eye developmental pathway in Drosophila using DNA microarrays. Proc. Natl. Acad. Sci. 100(7): 4024-9. 12655063

Siegmund, T. and Lehmann, M. (2002). The Drosophila Pipsqueak protein defines a new family of helix-turn-helix DNA-binding proteins. Dev. Genes Evol. 212: 152-157. 11976954

Suzanne, M., Estella, C., Calleja, M. and Sánchez-Herrero, E. (2003). The hernandez and fernandez genes of Drosophila specify eye and antenna. Dev. Bio. 260: 465-483. 12921746

Trost, M., Blattner, A. C., Leo, S. and Lehner, C. F. (2016). Drosophila dany is essential for transcriptional control and nuclear architecture in spermatocytes. Development 143: 2664-2676. PubMed ID: 27436041

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date revised: 21 November 2016

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